CN103547683A - The KRAS variant and tumor biology - Google Patents

Abstract

The disclosure provides methods for identifying a subject at risk of developing cancer, predicting the onset of cancer, and predicting a subject's response to chemotherapy/treatment by determining the presence or absence of a SNP in the KRAS oncogene, known as the KRAS variant.

Description

KRAS sudden change and oncobiology

Related application

The application requires the provisional application USSN61/454 proposing on March 21st, 2011,765; The USSN61/454 that on March 21st, 2011 proposes, 767; With the USSN61/454 proposing on March 21st, 2011,769 right of priority, the full content in above-mentioned document is all done to be as a wholely introduced into as a reference in the present invention.

To put forward the mode of stating, include in

Take " 34592-515001WOST25.txt " be the content of the text that name is submitted to, text file created on March 16th, 2012, file size 32.2KB, reference is incorporated herein by reference.

Government supports

The enforcement of part in the present invention, has United States Government and encourages (CTSA) according to clinical with conversion science, the support that grant number is UL1RR024139, and wherein CTSA is provided by branch of NIH country's resources for research center.

Shi of the present invention United States Government completes under supporting, part completes under supporting, obtain the grant number RO1CA131301-01A1 that National Cancer Institute provides, the grant number CA124484 that NIH provides (K08), the grant number RO1CA122728 that NIH provides, the grant number RO1CA74415 that NIH provides, the support of the grant number RC4CA153828 that National Cancer Institute and the Director-General's Office of NIH provide.

Government enjoys certain right in the present invention.

Invention field

The present invention relates generally to cancer, healthy reproduction and biology field.The invention provides by determining the existence of genetic marker or not existing, diagnosis and prediction main body suffer from the method for cancer.In addition, the invention provides by determining the existence of genetic marker or not existing, determine the method for patient to the reflection for the treatment of.

Background technology

The heterogeneity of the reaction of change risks and assumptions, patient treatment and result reflection cancer.Although it is highly non-homogeneous that predicted gene is expressed mark, several modules are as DNA rectification of defects, and immunne response mark or epithelial cell-mesenchymal cell changes conventionally and many Tumor-assaciateds.Therefore, in the art, be necessary to identify the promotor of these transcription modules, become a kind of promising method of finding specificity personalized treatment.

Summary of the invention

The research proposing in the present invention, the problem that relates to core is the effect of miRNAs in cancer: interference miRNAs regulation and control cause oncogene or tumor suppressor gene affects the development of cancered risk, tumour, and treats response.MiRNAs may directly or indirectly regulate and control to cause oncogene or tumor suppressor gene.For instance, KRAS transgenation, single nucleotide polymorphism (SNP) is positioned at the let-7 paratope 6 (LCS6) of KRAS gene 3 ' UTR, by the KRAS that regulates interference of miRNA let-7 family.In this case, KRAS gene let-7 mediation regulates and is blocked; Yet, also have the second-order effect of KRAS transgenation.The blocking-up of the interactive upstream of let-7/KRAS continues the factor downstream and sends abnormal signal.In addition signal pathway and the composition of non-classical RAS approach is affected.The existence of KRAS variant, has increased vasculogenesis, survival (even under anoxia condition), has shifted, and the resistance to Common Chemotherapy medicine.In addition, the epigenetic of cancer cell changes, and as the change of tumor suppressor gene promoter methylation and cell cycle gene, has affected suddenly change development, survival and the treatment of positive cancer cells of KRAS and has replied.Finally, the cell consequence of KRAS transgenation is irrelevant with other sudden changes of KRAS, comprise, for instance, the sudden change of KRAS gene coding region.For many cancer cells, the generation of KRAS transgenation and other KRAS transgenation be mutually to repel.Unlike KRAS sudden change, KRAS transgenation is a kind of germ line mutation.Therefore, KRAS genetic mutation is a kind of heredity biomarker of cellular biology of tumor.

KRAS transgenation sudden change cause KRAS genetic expression increase and/or high abundance KRAS, cause miRNAs let-7 family to be expressed and reduce.KRAS sudden change also affects transcription factor, and miRNAs but not the expression level of the miRNAs of let-7 family.For instance, KRAS sudden change and miR-23 and the increase of miR-27 expression level statistically significance are associated, and miR-23 and miR-27 target angiogenesis inhibitor gene are as Sprouty2 and Sema6A.Therefore, the weak effect of traditional chemotherapeutics and resistance may be derived from KRAS transgenation and by RAS path, drive the ability of activating cells propagation, and by vasculogenesis path, blood and nutrient supply tumour, promote the existence of cancer cells in tumour.In the face of having a kind of two abnormal paths of common incitant, some chemotherapeutics activity may be not enough to the development to anticancer.Carry the interior RAS of tumour of KRAS sudden change and the disturbance of other path tumour and between cancer cells and tumor type, have high homology (as mammary cancer and ovarian cancer).

KRAS transgenation is not good relevant with various cancer clinical therapeutic efficacies, and these cancers include, but not limited to colorectal carcinoma, ovarian cancer, incidence cancer, lung cancer.Evidence suggests, KRAS transgenation determines the response of a patient to treatment.If KRAS mutation carriers is to standard chemotherapeutic agents resistance, patient's result is poor so.Data acknowledgement in this paper, KRAS transgenation is to traditional chemotherapeutics resistance, and the susceptibility increase to monoclonal antibody therapy sometimes.For instance, when Cetuximab is during as unique methods for the treatment of administration, KRAS sudden change has increased the susceptibility of patient to Cetuximab, target KRAS path (EGFR) upstream regulatory factor.Therefore, the generation of KRAS transgenation may show, the medicine of selectively targeted KRAS upstream region of gene will be successful, yet traditional chemotherapeutics of targeting cell-cycle check point may be invalid, and cell cycle check point is the downstream of KRAS gene.Equally, KRAS transgenation has resistance to platinum-based chemotherapy.Platinum class medicament is cross-linked DNA molecular, to prevent DNA replication dna, finally causes apoptosis.Yet DNA replication dna occurs in KRAS activation downstream, and therefore, may be invalid, especially raises but not RAS gene according to data presentation signal path.

The new discovery of these KRAS oncobiologies provided herein has important clinical value, because chemotherapy is very arduous as a kind of methods for the treatment of to patient.The side effect of chemotherapeutics, has not only increased patient's discomfort, and has brought physical function system concurrency disease.For example, the chemotherapeutics that kills cancer cells also may damage or weaken patient's heart.Therefore, KRAS transgenation is a kind of biomarker of determining the resistance of known chemotherapeutics or susceptibility.If patient is KRAS, sudden change is positive, and doctor may select a best treatment plan so, or at least avoids futile treatment.

In the present invention, term main body and patient can Alternates.

The invention provides a kind of method of vasculogenesis risk increase of predicting tumors, comprise that (a) detects the sudden change in people KRAS gene let-7 paratope LCS6 in first patient sample, wherein said sudden change is a kind of single nucleotide polymorphism (SNP), comprise that LCS6 site 4 place's uridylics (U) or thymus pyrimidine (T) change to guanine (G), and (b) determine miRNA expression level, miRNA selects the group that in free second clinical samples, miR-23 and miR-27 form, wherein contrast with control group, the existence suddenling change in described (a) and (b) increase of middle miRNA expression level show that angiogenesis inhibitor genetic transcription gene silencing increases, thereby prediction of tumor forms risk and increases.In first and second clinical samples, extract from same patient.In addition, first may comprise identical fluid, tissue or biopsy with second clinical samples.Preferably, second clinical samples extract from or be derived from tumour or with the nonneoplastic tissue of tumour physical contact in region (for example,, around tumour).For instance, angiogenesis inhibitor gene can be Sprouty2 or Sema6A.Tumour may comprise a kind of cancer cells, and cancer cells is derived from the cancer that acquired immune deficiency syndrome (AIDS) is relevant, breast cancer, digestion/GI cancer, anus cancer, appendix cancer, cholangiocellular carcinoma, colorectal carcinoma, large bowel cancer, esophagus cancer, carcinoma of gallbladder, islet cell tumor, Pancreatic Neuroendocrine Tumors, liver cancer, cancer of pancreas, the rectum cancer, carcinoma of small intestine, cancer of the stomach, endocrine system cancer, adrenocortical carcinoma, parathyroid carcinoma, pheochromocytoma, pituitary tumor, thyroid carcinoma, cancer eye, intraocular melanoma, retinoblastoma, bladder cancer, kidney (nephrocyte) cancer, penile cancer, prostate cancer, transitional cell renal plevis and carcinoma of ureter, carcinoma of testis, urethral carcinoma, the nephroblastoma, other children's tumor of kidney, germinocarcinoma, central nervous system cancer, extracranial germ cell knurl, Extragonadal germ cell tumor, ovarian germ cell tumors, gynecological tumor, cervical cancer, carcinoma of endometrium, gestational trophoblastic tumor, ovarian cancer, sarcoma of uterus, carcinoma of vagina, carcinoma vulvae, head and neck cancer, hypopharyngeal cancer, laryngocarcinoma, lip and oral carcinoma, invisible primary transitivity squamous neck cancer, oral carcinoma, nasopharyngeal carcinoma, oropharynx cancer, nasal sinus and CARCINOMA OF THE NASAL CAVITY, pharynx cancer, salivary-gland carcinoma, laryngocarcinoma, skeletal muscle cancer, osteocarcinoma, Ewing's sarcoma, gastrointestinal stromal tumors (GIST), osteosarcoma, malignant fibrous histiocytoma of bone, rhabdosarcoma, soft tissue sarcoma, sarcoma of uterus, neural system cancer, cerebral tumor, astrocytoma, brain stem glioma, central nervous system atypia monster sample/rhabdoid tumor, the Embryo tumour of central nervous system, central nervous system gonioma, craniopharyngioma, ependymoma, medulloblastoma, tumor of spinal cord, primitive neuroectodermal tumor and pinealoblastoma on curtain, neuroblastoma, respiratory system carcinoma, chest cancer, nonsmall-cell lung cancer, small cell lung cancer, malignant mesothe, thymoma, thymic carcinoma, skin carcinoma, Kaposi's sarcoma, melanoma, Merkel cell carcinoma.Alternatively, or additionally, tumour or cancer are metastatic.

The invention provides a kind of survival of cancer cell increase under anoxia condition or method of propagation predicted, comprise that (a) detects the sudden change in people KRAS gene let-7 paratope LCS6 in first patient sample, wherein said sudden change is the uridylic (U) in a kind of LCS6 of being included in site 4 or the single nucleotide polymorphism (SNP) that thymus pyrimidine (T) changes guanine (G) into, and (b) determine the expression level of miR-210miRNA in second clinical samples, wherein contrast with control group, the increase that under the increase prediction anoxia condition of the middle existence suddenling change of described (a) and (b) middle miRNA expression level, cancer cell is survived or bred.In first and second clinical samples, extract from same position patient.In addition, first may comprise identical fluid, tissue or biopsy with second clinical samples.Cancer cells may stem from the cancer that acquired immune deficiency syndrome (AIDS) is relevant, breast cancer, digestion/GI cancer, anus cancer, appendix cancer, cholangiocellular carcinoma, colorectal carcinoma, large bowel cancer, esophagus cancer, carcinoma of gallbladder, islet cell tumor, Pancreatic Neuroendocrine Tumors, liver cancer, cancer of pancreas, the rectum cancer, carcinoma of small intestine, cancer of the stomach, endocrine system cancer, adrenocortical carcinoma, parathyroid carcinoma, pheochromocytoma, pituitary tumor, thyroid carcinoma, cancer eye, intraocular melanoma, retinoblastoma, bladder cancer, kidney renal cell carcinoma, penile cancer, prostate cancer, transition cell renal plevis and carcinoma of ureter, carcinoma of testis, urethral carcinoma, the nephroblastoma, other children's tumor of kidney, germinocarcinoma, central nerve neuroma, extracranial germ cell knurl, Extragonadal germ cell tumor, ovarian germ cell tumors, gynecological tumor, cervical cancer, carcinoma of endometrium, gestational trophoblastic tumor, ovarian cancer, sarcoma of uterus, carcinoma of vagina, carcinoma vulvae, incidence cancer, hypopharyngeal cancer, laryngocarcinoma, lip and oral carcinoma, invisible primary transitivity squamous neck cancer, oral carcinoma, nasopharyngeal carcinoma, oropharynx cancer, nasal sinus and CARCINOMA OF THE NASAL CAVITY, pharynx cancer, salivary-gland carcinoma, laryngocarcinoma, skeletal muscle cancer, osteocarcinoma, Ewing's sarcoma, gastrointestinal stromal tumor (GIST), osteosarcoma, malignant fibrous histiocytoma of bone, rhabdosarcoma, soft tissue sarcoma, sarcoma of uterus, neural cancer, cerebral tumor, astrocytoma, brain stem glioma, central nervous system atypia monster sample/rhabdoid tumor, central nervous system embryo's tumour, central nervous system gonioma, craniopharyngioma, ependymoma, medulloblastoma, tumor of spinal cord, primitive neuroectodermal tumor and pinealoblastoma on curtain, neuroblastoma, respiratory system carcinoma, chest cancer, nonsmall-cell lung cancer, small cell lung cancer, malignant mesothe, thymoma, thymic carcinoma, skin carcinoma, Kaposi's sarcoma, melanoma, Merkel cell carcinoma.

The invention provides a kind of method of predicting that cancer cell survival or propagation increase, comprise that (a) detects the sudden change in the let-7 paratope LCS6 of people KRAS gene in first patient sample, wherein said sudden change is the uridylic (U) in a kind of LCS6 of being included in site 4 or the single nucleotide polymorphism (SNP) that thymus pyrimidine (T) changes guanine (G) into, and (b) determine the methylation state of tumor suppressor gene promotor in second clinical samples, wherein contrast with control group, in described (a), the existence of sudden change and promotor (b) methylate increases survival or the propagation of predicting tumors cell.In first and second clinical samples, extract from same position patient.In addition, first can comprise identical fluid, tissue or biopsy with second clinical samples.Optionally, tumor suppressor gene is Notchl.Survival may comprise maintenance tumorigenesis potential.Cancer cells may stem from the cancer that acquired immune deficiency syndrome (AIDS) is relevant, breast cancer, digestive system cancer/gi tract, anus cancer, appendix cancer, cholangiocellular carcinoma, colorectal carcinoma, large bowel cancer, esophagus cancer, carcinoma of gallbladder, islet cell tumor, Pancreatic Neuroendocrine Tumors, liver cancer, cancer of pancreas, the rectum cancer, carcinoma of small intestine, cancer of the stomach, endocrine system cancer, adrenocortical carcinoma, parathyroid carcinoma, pheochromocytoma, pituitary tumor, thyroid carcinoma, cancer eye, intraocular melanoma, retinoblastoma, bladder cancer, kidney renal cell carcinoma, penile cancer, prostate cancer, transition cell renal plevis and carcinoma of ureter, carcinoma of testis, urethral carcinoma, the nephroblastoma, other children's tumor of kidney, germinocarcinoma, central nerve neuroma, extracranial germ cell knurl, Extragonadal germ cell tumor, ovarian germ cell tumors, gynecological tumor, cervical cancer, carcinoma of endometrium, gestational trophoblastic tumor, ovarian cancer, sarcoma of uterus, carcinoma of vagina, carcinoma vulvae, incidence cancer, hypopharyngeal cancer, laryngocarcinoma, lip and oral carcinoma, invisible primary transitivity squamous neck cancer, oral carcinoma, nasopharyngeal carcinoma, oropharynx cancer, nasal sinus and CARCINOMA OF THE NASAL CAVITY, pharynx cancer, salivary-gland carcinoma, laryngocarcinoma, skeletal muscle cancer, osteocarcinoma, Ewing's sarcoma gastrointestinal stromal tumor (GIST), osteosarcoma, malignant fibrous histiocytoma of bone, rhabdosarcoma, soft tissue sarcoma, sarcoma of uterus, neural cancer, cerebral tumor, astrocytoma, brain stem glioma, central nervous system atypia monster sample/rhabdoid tumor, central nervous system embryo's tumour, central nervous system gonioma, craniopharyngioma, ependymoma, medulloblastoma, tumor of spinal cord, primitive neuroectodermal tumor and pinealoblastoma on curtain, neuroblastoma, respiratory system carcinoma, chest cancer, nonsmall-cell lung cancer, small cell lung cancer, malignant mesothe, thymoma, thymic carcinoma, skin carcinoma, Kaposi's sarcoma, melanoma, Merkel cell carcinoma.Optionally, described cancer cells is cancer stem cell.

Mammary cancer

The invention provides for identifying the patient's that may develop aggressiveness and excessive risk mammary cancer risk method, and the method for predicting these form pathogenesis of breast carcinomas.Data provided herein have formed first disclosure and have driven the genome mutation of breast cancer tumour development machine-processed, it is characterized in that lacking estrogen receptor or progesterone expression.In preferred embodiments, the mammary cancer of aggressiveness and excessive risk form is three negative breast cancer, and it is further characterized in that and lacks human epidermal growth factor receptor 2's (HER2) genetic transcription or protein expression.

The invention provides a kind of method that identification may develop the patient of estrogen receptor (ER) and progesterone receptor (PR) feminine gender (ER/PR is negative) mammary cancer risk, comprise and detect the sudden change in people KRAS gene let-7 paratope LCS6 in clinical samples, wherein said sudden change is the uridylic (U) in a kind of LCS6 of being included in site 4 or the single nucleotide polymorphism (SNP) that thymus pyrimidine (T) changes guanine (G) into, the existence of wherein said sudden change, showing has the more risk that develops ER/PR negative breast cancer.

The invention provides a kind of to there being the patient of development mammary cancer risk to predict the method for its estrogen receptor (ER) and progesterone receptor (PR) negative (ER/PR is negative) pathogenesis of breast carcinoma, comprise the sudden change in the let-7 paratope LCS6 that detects people KRAS gene in clinical samples, wherein said sudden change is a kind of single nucleotide polymorphism (SNP), comprise that LCS6 site 4 place's uridylics (U) or thymus pyrimidine (T) change to guanine (G), the existence of wherein said sudden change, shows the more early morbidity of ER/PR negative breast cancer.

In a preferred embodiment of method described herein, ER/PR negative breast cancer is also that HER2 is negative, therefore, is a kind of three negative breast cancer (TNBC).Three negative breast cancer (TNBC) may be substrate or luminal type or tumour.Some aspect of these methods, three negative breast cancer (TNBC) are a kind of substrate tumours, express the protein of a kind of transcripton or EGF-R ELISA (EGFR) or CK5/6 (CK5/6) genes encoding.In other side, ER/PR negative breast cancer or ER/PR/HER2 negative breast cancer are further characterized in that low expression or negative mammary cancer 1 (BRCA1) gene of expressing.

Main body (patient) is Pre-menopausal Women preferably; Yet main body can be any age.Or in addition, main body is less than 51 years old, yet main body optionally, is less than 100 years old, 90 years old, 80 years old, 70 years old, 60 years old, 50 years old, 40 years old, 30 years old, 20 years old, or therebetween any age.

Large bowel cancer

The invention provides a kind of prediction and suffer from the method for large bowel cancer (CRC) main body, comprise and detect the sudden change in people KRAS gene let-7 paratope LCS6 in clinical samples, wherein said sudden change is the uridylic (U) in a kind of LCS6 of being included in site 4 or the single nucleotide polymorphism (SNP) that thymus pyrimidine (T) changes guanine (G) into, wherein, with control group contrast, the existence of described KRAS transgenation shows that survival rate increases.In an aspect of present method, described detecting step further comprises microsatellite instability (MSI) analysis.KRAS transgenation is the independent tag thing of colorectal cancer cells and patient's survival; Yet microsatellite instability (MSI) analysis can be used as secondary analysis.For example, although MSI (predicts good molecular marker CRC patient, the patient who suffers from MSI tumour is considered to a good prediction), determine KRAS transgenation state, show to suffer from MSI tumour, but KRAS transgenation feminine gender (or, the prediction of individual body large intestine cancer in other words, wild-type) is poor.Therefore, the invention provides a kind of prediction clinical effectiveness, or the superior method of large bowel cancer prediction, especially during PATIENTS WITH LARGE BOWEL cancer stage layering.

Especially in the embodiment in the method, large bowel cancer (CRC) is commitment large bowel cancer.Preferably, large bowel cancer (CRC) is 1 phase or 2 phases.

Test main body can have the sudden change of KRAS gene secondary, and KRAS transgenation is once sudden change.

Test subject or control group main body BRAF gene carry one or more sudden changes.Alternatively, or additionally, test subject or control group main body may have hyper-methylation RASSF1A promotor.

Control group main body is not carried KRAS transgenation (for example, control group main body is wild-type KRAS transgenation sudden change).Yet control group main body may suffer from large bowel cancer, may be maybe individual without cancer.In addition, control group main body KRAS gene may have secondary sudden change, is not KRAS transgenation.

In aspect some of the method, patient's survival rate be an overall survival (for example, some embodiment, include, but are not limited to, from cancer development, or diagnosis is until main body is died from cancer (death), enters the catabasis, or physician declared main body is cured or all tumour cells are disposed, calculate survival rate), 5 years survival rates or the annual rate of depositing.Calculate shorter lifetime, for example, from cancer development or cancer diagnosis, calculate, until a determinacy time, as 1 year or 5 years.

Treatment of ovarian cancer is replied

The invention provides (EOC) patient's that suffers from ovarian epithelial carcinoma Forecasting Methodology, in addition, provide and by prediction patient, platinum-based chemotherapy has been replied the method for optimizing treatment.Method described herein and the interior let-7miRNA of data identification KRAS gene 3 ' non-translational region (UTR) are in conjunction with position specific gene group sudden change (being referred to as KRAS variant).

The invention provides a kind of method of suffering from epithelial ovarian cancer (EOC) patient prediction, comprise and detect the sudden change in people KRAS gene let-7 paratope LCS6 in clinical samples, wherein said sudden change is a kind of single nucleotide polymorphism (SNP), comprise that LCS6 site 4 uridylics (U) or thymus pyrimidine (T) change to guanine (G), wherein, while contrasting with control group, the existence of described KRAS transgenation shows that survival rate reduces.

Although the method can be applied to the women of patient and institute's has age, in some embodiment of this method, test subject is after menopause or 52 years old or 52 years old above the elderly.Control group main body comprises that healthy individuals and those suffer from epithelial ovarian cancer (EOC), but does not carry the women of KRAS transgenation.In addition, according to as test subject same year birth women, or as the women's of the same generation of test patient expection existence, control group main body is average national level.In a preferred embodiment, control value does not comprise that those carry the individuality of KRAS transgenation.In aspect some of the method, survival be overall survival (for example, some embodiment, include, but are not limited to, from cancer development, or diagnosis is until main body is died from cancer (death), enters the catabasis, or physician declared main body is cured or all tumour cells are disposed, calculate survival rate), 5 years survival rates or the annual rate of depositing.Calculate shorter lifetime, for example, from cancer development or cancer diagnosis, calculate, until the determinacy time, as 1 year or 5 years.

The present invention also provides the method for a kind of prediction epithelial ovarian cancer (EOC) cell platinum-based chemotherapy response, comprise and detect the sudden change in people KRAS gene let-7 paratope LCS6 in clinical samples, wherein said sudden change is the uridylic (U) in a kind of LCS6 of being included in site 4 or the single nucleotide polymorphism (SNP) that thymus pyrimidine (T) changes guanine (G) into, and the existence of wherein said sudden change shows platinum-based chemotherapy resistance.Ovarian cancer cell can be evaluated in vitro or in vitro.When the external assessment of ovarian cancer cell, this cell is taken from main body.Main body can be any age, yet in a preferred embodiment, patient can be postclimacteric, or at least 52 years old.Or, in same embodiment, patient at least 30 years old, 35 years old, 40 years old, 45 years old, 50 years old, 55 years old, 60 years old, 65 years old, 70 years old, 75 years old, or any age of between.Aspect other of this method, due to secondary medical condition or medical treatment, after patient is not menopause, a similar hormonal readiness has been proposed.Exemplary, but non-limiting climacterium hormone level comprise oestrogenic hormon and the progestogen of decline level, for example, main body blood or urine specimen assessment are determined.Bring out the exemplary of hormonal readiness in climacteric but nonrestrictive secondary medical condition is ovary surgical excision operation (ovariectomy at least one times, be also referred to as operation menopause), cervical cancer, uterus carcinoma or ovarian cancer must be carried out uterectomy (especially, if hysterectomized in conjunction with uterine tube and one or both sides oophorectomize).Exemplary but nonrestrictive, the secondary medical condition of bringing out hormonal readiness in climacteric is chemotherapy and estrogen antagonist treatment.

When ovarian cancer cell is assessed in vitro, cellular segregation, copy, or from BG1, CAOV3 or IGR-OV1 cell strain.These cell strains are non-limiting examples of Ovarian Cancer Cells.Ovarian cancer cell carries out separation, copies, or from the cell strain of any ovarian cancer, includes, but not limited to these and carry KRAS transgenation, harmful BRCA1 transgenation, harmful BRCA2 transgenation, or the cell strain of its arbitrary combination.Harmful BRCA1 or BRCA2 transgenation are to increase its carrier by developing into the sudden change of risk or the possibility of cancer, in preferred embodiments, develop into mammary cancer or ovarian cancer.Harmful BRCA1 or BRCA2 transgenation are also to increase the sudden change (for example, experience cancer is fallen ill in early days) that little carrier of its age develops into risk or the possibility of cancer, and in preferred embodiments, cancer is mammary cancer or ovarian cancer.

Method described herein, preferred platinum-based chemotherapy is carboplatin or taxol, however platinum-based chemotherapy comprises all chemotherapeutics, platinum or platinum salt are used for the treatment of or preventing cancer.In some aspect of these methods, platinum-based chemotherapy is assisting therapy.Therefore, method as herein described prediction patient adopt as monotherapy or with the response of the platinum-based chemotherapy of other known anticancer agent or technology (for example, for instance, radiotherapy and surgical operation) combination therapy.

The treatment response of large bowel cancer

The invention provides the Forecasting Methodology of suffer from large bowel cancer (CRC) or Metastatic Colorectal Cancer (mCRC), in addition, provide by prediction patient and carried out independent mab treatment, or separately mab treatment associational cells toxicity chemotherapy response and optimize the method for the treatment of.Method described herein and data identification KRAS gene 3 ' non-translational region (UTR) let-7miRNA binding site specific gene group sudden change, be referred to as KRAS sudden change.

The invention provides a kind of method of suffering from the test subject prediction of early stage large bowel cancer (CRC), comprise and detect people KRAS gene let-7 paratope LCS6 sudden change in clinical samples, wherein said sudden change is the uridylic (U) in a kind of LCS6 of being included in site 4 or the single nucleotide polymorphism (SNP) that thymus pyrimidine (T) changes guanine (G) into, wherein with control group main body or suffer from advanced CRC and (comprise, for instance, III phase, IV phase, and Metastatic Colorectal Cancer) main body contrast, the existence of described sudden change shows that survival rate increases.

The invention provides a kind of method of suffering from advanced CRC (CRC) patient prediction, comprise and detect people KRAS gene let-7 paratope LCS6 sudden change in clinical samples, wherein said sudden change is a kind of single nucleotide polymorphism (SNP), comprise that LCS6 site 4 uridylics (U) or thymus pyrimidine (T) change to guanine (G), wherein with control group main body or suffer from the main body contrast of early stage large bowel cancer, the existence of described KRAS-sudden change shows that survival rate reduces.Advanced CRC comprises, for instance, and III phase, IV phase, and Metastatic Colorectal Cancer.

The invention provides and a kind ofly predict that cancer cells carries out the method for the response of monoclonal antibody single therapy, comprise and detect people KRAS gene let-7 paratope LCS6 sudden change in clinical samples, wherein said sudden change is the uridylic (U) in a kind of LCS6 of being included in site 4 or the single nucleotide polymorphism (SNP) that thymus pyrimidine (T) changes guanine (G) into, the existence of wherein said sudden change, shows the susceptibility to monoclonal antibody single therapy.In some embodiment of present method, cancer cells is large bowel cancer (CRC) cell.Cancer cells can be evaluated in vitro or in vitro.A nonrestrictive embodiment of monoclonal antibody single therapy is Cetuximab.

The invention provides the method for a kind of predicting tumors cell to the response of chemotherapy combined mab treatment, comprise and detect people KRAS gene let-7 paratope LCS6 sudden change in clinical samples, wherein said sudden change is the uridylic (U) in a kind of LCS6 of being included in site 4 or the single nucleotide polymorphism (SNP) that thymus pyrimidine (T) changes guanine (G) into, the existence of wherein said sudden change, shows the resistance to combination therapy.In some embodiment of present method, cancer cells is large bowel cancer (CRC) cell.Cancer cells can be evaluated in vitro or in vitro.A non-limiting example of monoclonal antibody single therapy is Cetuximab.Chemotherapy may be cytotoxic agent.A non-limiting example of cytotoxic agent is irinotecan.In certain embodiments, the main body of using chemotherapeutic (for example, irinotecan) treatment to carry KRAS transgenation causes KRAS transgenation to be expressed to be increased.KRAS transgenation contacts after irinotecan with not mutated cancer cells, during comparison report genetic expression, finds that wild-type 3 ' UTR reporter gene does not change.Yet, find that wild-type 3 ' UTR reporter gene expression has significance,statistical to increase (Figure 24 A and Figure 24 B).These data show, irinotecan contacts to activate the allelic mode of KRAS transgenation and changes cell background.

Although the method goes for the patient of all age brackets, in some embodiment of this method, test subject newborn infant, children, adult, or older (65 years old or more than).Main body may be premenopausal or postclimacteric (age 52 years old or more than).

Control group or contrast main body comprise that Healthy People and those suffer from large bowel cancer, but do not carry the individuality of KRAS transgenation.In addition, according to as test subject same year birth women, or as the women's of the same generation of test subject expectation survival rate, control group main body is average national level.In a preferred embodiment, control value does not comprise that those carry the individuality of KRAS transgenation.In aspect some of the method, survival be overall survival (for example, some embodiment, include, but are not limited to, from cancer development, or diagnosis is until main body is died from cancer (death), enters the catabasis, or physician declared main body is cured or all tumour cells are disposed, calculate survival rate), 5 years survival rates or annual depositing the phase.Calculate shorter lifetime, for example, from cancer development or cancer diagnosis, calculate, until a determinacy time, as 1 year or 5 years.

The present invention also provides the method for a kind of forecast colorectal cancer (CRC) cell to mab treatment response, comprise and detect people KRAS gene let-7 paratope LCS6 sudden change in clinical samples, wherein said sudden change is the uridylic (U) in a kind of LCS6 of being included in site 4 or the single nucleotide polymorphism (SNP) that thymus pyrimidine (T) changes guanine (G) into, the existence of wherein said sudden change, showing increases the susceptibility of mab treatment.Colorectal cancer cells can be evaluated in vitro or in vitro.Mab treatment can be Cetuximab.

The present invention also provides the method for a kind of forecast colorectal cancer (CRC) cell to the response of cytotoxicity chemotherapy, comprise and detect people KRAS gene let-7 paratope LCS6 sudden change in clinical samples, wherein said sudden change is a kind of single nucleotide polymorphism (SNP), comprise that LCS6 site 4 uridylics (U) or thymus pyrimidine (T) change to guanine (G), the existence of wherein said sudden change, represents cytotoxicity chemotherapy resistance.In some embodiment of this method, large bowel cancer is evaluated in vitro or in vitro.Cytotoxicity chemotherapy may be irinotecan chemotherapy.In an embodiment of this method, cytotoxicity chemotherapy is a kind of combination therapy, comprises mab treatment.Mab treatment may be Cetuximab.

When the external assessment of colorectal cancer cells, this cell is taken from patient.Patient can be any age bracket.In some embodiment of this method, patient at least 30 years old, 35 years old, 40 years old, 45 years old, 50 years old, 55 years old, 60 years old, 65 years old, 70 years old, 75 years old, or any age between the two.

When the external assessment of colorectal cancer cells, cell may be separated, copy, or take from the cell strain of having set up, and comprises colon or colorectal cancer cell lines in NCI-60 culture plate.Colorectal cancer cells can be separated, copy, or take from any colon or colorectal cancer cell lines, comprises, but be not limited to, and carries KRAS transgenation, separately or with secondary sudden change in KRAS gene or other genes or sudden change is combined in addition cell strain.

For the method, preferred monoclonal antibody single therapy is Cetuximab, yet monoclonal antibody single therapy comprises any being used for the treatment of or the monoclonal antibody of preventing cancer.Preferably, described monoclonal antibody is partially or completely people's or humanized.For this method, preferred chemotherapy is cytotoxicity chemotherapy, and as irinotecan, yet chemotherapy comprises any being used for the treatment of or the chemotherapeutics of preventing cancer.In aspect some of this method, chemotherapy or cytotoxicity chemotherapy are a kind of assisting therapy.Therefore, the method prediction patient adopts as monotherapy or for example, with chemotherapeutics or be used for the treatment of or the response of the monoclonal antibody (, radiotherapy and surgical operation) of other known technology combination therapy of preventing cancer.

Accompanying drawing explanation

Figure 1A-B is a pair of chart, describes the KRAS sudden change distribution in (≤51 years old) women (B) mammary cancer hypotype before all women (A) of study group 2 and menopause.Data are the patient's number that is diagnosed as mammary cancer hypotype case load/tested K RAS sudden change.* the every other hypotype of P=0.044vs.The every other hypotype of P=0.033vs.

Fig. 2 A-B is a pair of case line chart, describes BRCA1 genetic expression in three negative breast cancer KRAS positive gene mutations and KRAS transgenation feminine gender.Y-axis is arbitrary unit.(A) BRCA1 gene probe 1, p=006.(B) BRCA1 gene probe 2, p=0.01.

Fig. 3 is a series of case line chart, describes microRNA let-7 family in the negative case of three negative breast cancer KRAS positive gene mutations and KRAS transgenation and expresses.Y-axis is arbitrary unit.

Fig. 4 is hotspot graph, shows the three Breast Cancer Patients with Negative Axillary KRAS transgenation difference expression genes that LIMMA model analysis obtains.50 significance genes are used to carry out cluster; Cluster P<0.0001.KRAS transgenation sample is Dark grey; Wild-type sample is light grey.White has unknown KRAS mutation status.

Fig. 5 is the chart of describing ER/PR+ and ER/PR-premenopausal breast cancer patients patient KRAS transgenation.

Fig. 6 is a series of case line charts of describing the gene expression profile that three Breast Cancer Patients with Negative Axillary tumour KRAS transgenations are relevant.

Fig. 7 is a graphic representation, shows that 52 years old above total survival rate of Postmenopausal Ovaries cancer patient significance of KRAS mutation forecasting is poor.Adopt Kaplan-Meier to analyze, contrast has the overall survival of the ovarian cancer patients (n=59) of KRAS transgenation and ovarian cancer patients (n=220) without KRAS transgenation.Log-rank assay is 52 years old above KRAS positive gene mutation epithelial ovarian carcinoma patients significance poor (P=0.0399).

Fig. 8 shows KRAS transgenation and the chart that after new adjuvant chemotherapy, undesirable surgical cytoreduction is relevant.Carry KRAS transgenation (n=26) or without the surgical cytoreduction after contrast new adjuvant chemotherapy in the ovarian cancer patients (n=116) of KRAS transgenation (n=90).Through χ ²analyze, KRAS transgenation patient more may make undesirable surgical cytoreduction have larger residual disease (RD) (P=0.044) than not mutated patient's significance.

Fig. 9 A is 50 differential expression candidate gene signatures in KRAS transgenation (KV) three negative breast cancer tumours (TNBC KRAS signature), and KRAS transgenation ovarian epithelial carcinoma sample reveals more balloon score than not mutated schedule of samples.

Fig. 9 B is to the relevant gene label of the addicted tumour of KRAS gene (KRAS habituation label), at KRAS transgenation epithelial ovarian tumor, raises.

Fig. 9 C is front 20 the gene differential expression labels of KRAS transgenation epithelial ovarian tumor, reflects reanalysing of carboplatin sensitivity and carboplatin resistance ovarian epithelial carcinoma cytogene differential expression.

Fig. 9 D is the hotspot graph of anterior difference expression gene between KRAS transgenation (Dark grey) and not mutated (light gray) tumor sample.Color key is depicted blueness (0 to 5) to white (approximately 5), is transferred to the difference expression gene spectrum of redness (5 to 10) by white.The colored version of this hotspot graph, referring to Ratner ES, et al.Oncogene, (5December2011), 1-8; Its content is integrated with herein by citation).

Figure 10 shows the KRAS transgenation chart relevant with carboplatin/Paclitaxel Chemotherapy resistance with cell strain carboplatin.Carry KRAS transgenation (BG1) cell strain and without KRAS transgenation (CAOV3) cell strain through chemotherapeutic treatment, half-inhibition concentration (IC50) is Y-axis, X-axis is chemotherapeutic agents.Higher IC50 represents chemotherapeutic agents resistance.The strain of BG1=KRAS transgenation/BRCA gene wild-type cell; CAOV3=is not mutated/strain of BRCA wild-type cell; IGR-OV1=KRAS-sudden change/BRCA1 mutant clone.Error line is Relative Error.

Figure 11 A is the chart that shows that KRAS transgenation cell strain cell survival rate reduces, BG1 (* p<0.001), and to not mutated cell strain, CAOV3, without impact.Carry KRAS sudden change (BG1) and without the cell strain of KRAS sudden change (CAOV3), carry out siRNA/miRNA combination therapy, selective binding variant allelotrope.

Figure 11 B shows that the minimizing of BG1 (right side) KRAS protein expression reduces consistent chart with cell survival rate, on not impact of CAOV3 (left side).The cell strain that carries (BG1) KRAS sudden change and suddenly change without (CAOV3) KRAS, carries out siRNA/miRNA combination therapy, selective binding mutation allele.Different siRNA mark by numeral.

Figure 12 is a chart, and with not mutated cell strain (CAOV3) contrast, the cell strain of describing to carry KRAS transgenation (BG-1 and IGROV1) has significance lower level let-7b.Statistical study is carried out in single factor Anovea and the check of Tukey ' s multiple comparisons.

Figure 13 A-B schematic representation KRAS-mutant nucleotide sequence and not mutated sequence alignment.Panel A describes the not mutated sequence of KRAS gene.Panel B describes the exemplary sudden change siRNA oligonucleotide of target KRAS transgenation sequence.In two Panel, band underscore sequence description let-7 binding site.In two Panel, the Nucleotide being framed refers to wild-type (not mutated) Nucleotide (A) or KRAS transgenation single nucleotide polymorphism (SNP) (B).SiRNA demonstration originates in its 3 ' end.

Figure 14 is KRAS transgenation and the Kaplan-Meier curve of specific cause of disease lifetime in all carcinoma stage.

Figure 15 A is the Kaplan-Meier curve of early stage (I phase and II phase) large bowel cancer KRAS transgenation and specific cause of disease lifetime.

Figure 15 B is the Kaplan-Meier curve of the KRAS transgenation of III phase large bowel cancer and specific cause of disease lifetime.

Figure 15 C is the Kaplan-Meier curve of the KRAS transgenation of IV phase large bowel cancer and specific cause of disease lifetime.

Figure 16 A is the Kaplan-Meier curve of early stage (I phase and II phase) large bowel cancer KRAS transgenation, KRAS sudden change and specific cause of disease lifetime, P=0.875.

Figure 16 B is the Kaplan-Meier curve of III phase large bowel cancer KRAS transgenation, KRAS sudden change and specific cause of disease lifetime.

Figure 16 C is the Kaplan-Meier curve of IV phase large bowel cancer KRAS transgenation, KRAS sudden change and specific cause of disease lifetime.

Figure 17 is the Kaplan-Meier curve of early stage (I phase and II phase) large bowel cancer KRAS transgenation, microsatellite instability state and specific cause of disease lifetime.

Figure 18 A is a graphic representation, describe anti-epidermal growth factor receptor monoclonal antibody single therapy or with the meta Progression free survival phase of combined chemotherapy as the patient KRAS LCS6 genotypic state of rescuing.

Figure 18 B is a graphic representation, describe anti-epidermal growth factor receptor monoclonal antibody single therapy or with combined chemotherapy as the patient KRAS LCS6 genotypic state meta Overall survival of rescuing.

Figure 19 A is a graphic representation, describes the meta Progression free survival phase of all patient KRAS LCS6 genotypic states of anti-epidermal growth factor receptor monoclonal antibody single therapy.

Figure 19 B is a graphic representation, describes anti-epidermal growth factor receptor monoclonal antibody base combined chemotherapy as the meta Progression free survival phase of all patient KRAS LCS6 genotypic states of rescuing.

Figure 19 C is the graphic representation of describing according to the meta Progression free survival phase of all KRAS carriers of mutation treatment types.

Figure 19 D is the graphic representation of describing according to the meta Progression free survival phase of had or not KRAS mutation carriers treatment type.

Figure 20 A is a graphic representation, describes the meta Progression free survival phase as two (KRAS and BRAF) wild-type patient group KRAS LCS6 genotypic states of rescuing according to anti-epidermal growth factor receptor monoclonal antibody single therapy.

Figure 20 B is a graphic representation, describes the meta Progression free survival phase as two (KRAS and BRAF) wild-type patient group KRAS LCS6 genotypic states of rescuing according to anti-epidermal growth factor receptor monoclonal antibody base combined chemotherapy.

Figure 20 C is a graphic representation, describes according to the meta progresson free survival rate of two (KRAS and BRAF) wild-type KRAS mutation carriers treatment types.

Figure 20 D is a graphic representation, describes according to the meta progresson free survival rate of the non-KRAS carriers of mutation treatment of two (KRAS and BRAF) wild-types type.

Figure 21 A is a graphic representation, the total survival rate of meta of the genotypic state of KRAS LCS6 in all patients that rescue according to anti-epidermal growth factor receptor monoclonal antibody single therapy.

Figure 21 B is a graphic representation, the total survival rate of meta of the genotypic state of KRAS LCS6 in all patients that describe to rescue according to the combined chemotherapy based on anti-epidermal growth factor receptor monoclonal antibody.

Figure 21 C is the graphic representation of describing according to the total survival rate of meta of all KRAS carriers of mutation treatment types.

Figure 21 D is the graphic representation of describing according to the total survival rate of meta of the treatment type of had or not KRAS mutation carriers.

Figure 22 A is a graphic representation, describes the total survival rate of meta as two (KRAS and BRAF) wild-type patient group KRAS LCS6 genotypic states of rescuing according to anti-epidermal growth factor receptor monoclonal antibody single therapy.

Figure 22 B is a graphic representation, describes the total survival rate of meta as KRAS LCS6 genotypic state in two (KRAS and BRAF) the wild-type patient groups that rescue according to anti-epidermal growth factor receptor monoclonal antibody single therapy.

Figure 22 C is a graphic representation, describes according to the total survival rate of meta of two (KRAS and BRAF) wild-type KRAS mutation carriers treatment types.

Figure 22 D is a graphic representation, describes according to the total survival rate of meta of the non-KRAS mutation carriers treatment of two (KRAS and BRAF) wild-types type.

Figure 23 A is a graphic representation, describes according to the meta Progression free survival phase of KRAS and BRAF sudden change KRAS mutation carriers treatment type.

Figure 23 B is a graphic representation, describes to suddenly change meta Progression free survival phase of non-KRAS mutation carriers treatment type according to KRAS and BRAF.

Figure 23 C is a graphic representation, describes the suddenly change total survival rate of meta of non-KRAS mutation carriers treatment type according to KRAS and BRAF.

Figure 23 D is a graphic representation, describes the suddenly change total survival rate of meta of non-KRAS carriers of mutation treatment type according to KRAS and BRAF.

Figure 24 A is a graphic representation, describes after chemotherapeutics irinotecan normalization method luciferase expression in wild-type KRAS and KRAS sudden change cancer cells.

Figure 24 A is a graphic representation, has described when cancer cell adopts irinotecan, and multiple returns the relation function with (being expressed as KRAS genic mutation type/KRAS wild-type) irinotecan concentration.

Embodiment

In KRAS proto-oncogene (rs61764370) 3 ' UTR relevant to cancer, the functional variant of let-7microRNA paratope is previously by definite (international patent application no PCT/US2008/065302, its full content is integrated with herein by citation).This paper describes the research of sudden change and oncobiology dependency.

Mammary cancer

Breast tumor is divided into ER (oestrogenic hormon) and/or PR (progestogen) receptor positive, HER2 (Her2/neu/ERBB2) amplification, three negative tumours (for example, ER/PR is negative and HER2 feminine gender) (

t, et al.Proc Natl Acad Sci USA2001; 98:10869-74).Genetic expression and receptor assay, further mammary cancer is divided into four biological subgroups: luminal A tumour (ER-and/or PR-receptor positive, HER-2 is negative), luminal B (ER-and/or PR-receptor positive, HER2 is positive), HER-2 positive (HER2-is positive, and ER/PR is negative) and substrate sample tumour (ER/PR/HER2-is negative, is also referred to as three negative breast cancer (TNBC)) (

t, et al.Proc Natl Acad Sci USA2001; 98:10869-74).

Contrast other hypotypes, three negative breast cancer (TNBC) are the most aggressive subclasses, have the 5 years specific cause of disease (Ha ty BG et al.J Clin Oncol2006 lifetime; 24:5652-57).Transcriptional expression profile research in the recent period shows that three cloudy breast cancer tumours are heterogeneous, and these tumours can be divided into two large subgroups; The negative tumour of ER/PR/HER2 (three) of expressing EGFR or Keratin sulfate (CK) 5/6, therefore, is called as ' substrate sample ', does not express the negative tumour of ER/PR/HER2 (three) of EGFR or CK5/6.Substrate sample three feminine genders (TN) tumour is characterised in that, than non-substrate sample form and low expression BRCA1 (mammary cancer) age of onset Zao (low age); Basaloid cells phenotype is (Rakha EA and Ellis IO.Pathology2009 common in BRCA1 mutation carriers; 41:40-47).Abnormal luminal type precursor cell group (it may be that ER is positive) is target (Lim E, the et al.Nat Med2009 of the substrate sample tumour conversion that BRCA-1 is relevant; 15:907-13).Although it is height homology that predicted gene is expressed mark, several modules, as DNA rectification of defects, immunne response mark, or epithelial cell-mesenchymal cell changes conventionally at these tumour subsets (Bild AH, et al.Breast Cancer Res2009; 11:R55).The promotor of identifying these transcription modules is to find a kind of method of specificity, personalized treatment.

Three negative breast cancer phenotypes and young dependency of falling ill, and known risk or reproductive factors shortage dependency (Yang XR, et al.Cancer Epidemiol Biomarkers Prev2007; 16:439-43), show that this cancer development has genetic risk (Bauer KR et al.Cancer2007; 109:1721-28).Before the present invention, the genetic marker that does not exist this risk to increase.Although BRCA1 transgenation often with three negative Tumor-assaciateds, these sudden changes are rare, account for and suffer from three Breast Cancer Patients with Negative Axillaries only 10% to 15%, depend on ethnic background and family's medical history (Young SR, et al.BMC Cancer2009; 9:86; Nanda R, et al.JAMA2005; 294:1925-33).

The frequency distribution of KRAS transgenation is determined in research provided herein in routine proved by pathology patient with breast cancer and 457 control groups (study group 1) from Connecticut, USA 415, and carry known estrogen receptor (ER), progesterone receptor (PR), HER2 state 690 Irishwomans and and 360 collators (study group 2) in determine the dependency of this sudden change and mammary cancer hypotype.Study group 1 and 2 140 three negative breast cancer women patients of data acquisition and 113 collators, assessment KRAS transgenation and three negative breast cancer risks, and the dependency of the full genome mRNA of three Breast Cancer Patients with Negative Axillaries and specificity miRNA expression.

Although the frequency distribution of the 1KRAS of study group transgenation is indifference in all genotype individualities, contrast before 201 menopause in collator 27 examples (13%) (P=0.015), suffer from 8 examples (33%) in 24 pre-menopausal womens of the negative cancer of ER/PR-and there is KRAS sudden change.In study group 2, contrast in 478 routine luminal A mammary cancer and account for 64 examples (13%), in 87 routine luminal B mammary cancer, account for 13 examples (15%), in the positive subgroups of 35 routine HER2, account for 2 examples (6%), the KRAS significance enrichment in three negative breast cancer women (accounting for 19 examples [21%] in 90 examples) (p value=0.044) that suddenlys change.The multiplicity that merges study group shows, KRAS transgenation relevant to pre-menopausal women three negative breast cancer (odds ratio 2.307,95%CI1261-4.219, P=0.0067).Three negative breast cancer oncogene expression analysis show, KRAS positive gene mutation tumour significance change genetic expression, and enrichment luminal type precursor and BRCA1 lack label.The level that MiRNA analyzes let-7miRNA kind in hint KRAS transgenation tumour reduces.

KRAS transgenation is a kind of genetic marker that three negative breast cancer occur pre-menopausal women.Cytogene and miRNA express molecule and the biological layering that label starts three Breast Cancer Patients with Negative Axillaries.

Large bowel cancer

KRAS transgenation is a kind of early stage large bowel cancer (CRC) predicting marker.In addition, tumor inhibitor lethal-7 (let-7) miRNA of higher levels of KRAS gene carcinogenic protein and lower level is induced in KRAS transgenation.In 409 examples early stage (I phase and II phase), 182 routine III phases and the 69 routine IV phase cases of large-scale, perspective Dutch cohort study (NLCS), the impact of KRAS transgenation is studied.The early stage patient that carries KRAS sudden change has a better prediction, and especially those also carry other KRAS sudden change.This discovery and microsatellite instability or other predictive factorses are irrelevant.In addition, the data of 1, the 886 routine sub-queue member by Dutch cohort study (NLCS), KRAS transgenation is also studied the impact of rectum cancer risk.G allelotrope (for example, KRAS mutation allele) is uncorrelated with the possibility of the development rectum cancer, but G allelotrope is enriched in advanced rectal cancer, and this shows that it may predict the more disease in late period.Because this study population is only untreated crowd's analysis, these results provide a neodoxy of learning about carrying the natural biology of KRAS transgenation large bowel cancer.

Due to digital proof given in this article, KRAS transgenation is a kind of new large bowel cancer (CRC) biomarker that instructs the decision-making of commitment patient treatment.Carrying the KRAS early stage large bowel cancer case of suddenling change has better result, yet, in the terminal stage of a disease, no longer include better result.For early stage patient, KRAS transgenation genotype is combined at least one KRAS sudden change is also the predicting marker of the better healing result that will consider for the treatment of decision-making.

Although the innovation of diagnosis and treatment, large bowel cancer (CRC) remains the second largest reason of the Western countries cancer mortality.Tumor lympha knot transfer system (TNM) is the tool master that information of forecasting is provided at present.Tumor lympha knot transfer system is that the two poles of the earth (early stage and advanced CRC) prediction is had to high predicted, but has low predictability for the intermediate stage.According to current guilding principle, do not give early stage patient adjuvant chemotherapy (for example, the TNM of T1-3-N0-M0 ，An International Union Against Cancer).In early days in patient's group (for example, five year survival rate T1-3-N0-M0) is greater than 70%.However, the early stage patient of 20%-30% (I phase and II phase) will be died from large bowel cancer in 5 years, if these patients make a definite diagnosis in advance, carry out corresponding treatment, cause the problem that can these death be avoided.Before this, numerous research is delivered, and claims the predicted impact of molecular marker.Contrary with asserting of these previously reports, these results of study are inconsistent.Therefore, before methods described herein form, the problem of molecular changes impact prediction be not still resolved (Smits KM, et al.Pharmacogenomics.2008; 9 (12): 1903-16).

MicroRNAs (miRNA), has been confirmed as the important factor that cancer occurs and develops.Evidence suggests, single miRNA can regulate a lot of mRNAs (Paranjape T, et al.Gut.2009 simultaneously; 58 (11): 1546-54).In addition, miRNA can play tumor suppressor gene and the effect (Johnson SM, the et al.Cell.2005 that cause oncogene; 120 (5): 635-47).MiRNAs lethal-7 family (let-7) is in first found miRNA family.Expression at many cancer Zhong， let-7 miRNA of family is changed.For instance, in lung cancer, the low expression of let-7 (Calin GA, et al.Proc Natl Acad Sci U S is A.2004; 101 (9): 2999-3004; Takamizawa J, et al.Cancer Res.2004; 64 (11): 3753-6), let-7 overexpression suppresses cell in-vitro growth (Takamizawa J, et al.Cancer Res.2004; 64 (11): 3753-6) and suppress cell growth in vivo (Kumar MS, et al.Proc Natl Acad Sci U S A.2008; 105 (10): 3903-8; Esquela-Kerscher A, et al.Cell Cycle.2008; 7 (6): 759-64), this prompting let-7miRNA may play effect (Johnson SM, the et al.Cell.2005 of tumor inhibitor; 120 (5): 635-47).

In the contrast of colon cancer cell Zhong，Yu Fei cancer beside organism, in tumor tissues, the expression significance of let-7 reduces (AkaoY, et al.Biol Pharm Bull.2006; 29 (5): 903-6).In addition, let-7 expresses increase, and after the transfection of let-7a-1miRNA precursor cell, RAS expresses and declines, and this prompting let-7 participates in regulating growth (AkaoY, the et al.Biol Pharm Bull.2006 of colon cancer cell; 29 (5): 903-6).

In conjunction with the complementary element of said target mrna 3 ' non-translational region (UTR), MiRNA can express by controlling gene.Behind KRAS gene mRNA 3 '-UTR specificity site, let-7 induction RAS lowers.KRAS transgenation affects let-7 mediation regulation and control KRAS genetic expression.With wild-type contrast, the level that causes higher KRAS gene level and lower let-7 of sudden change G allelotrope (for example, KRAS sudden change).In moderate smoker, G-allelotrope carrier has the risk that increases lung cancer, increases the risk (particularly postclimacteric women) of ovarian cancer, the danger suffering from breast cancer increases (especially, three negative breast cancer hypotypes), reduce the survival of oral carcinoma, but do not reduce lung cancer existence.The large bowel cancer of KRAS/BRAF sudden change, G allelotrope carrier (KRAS mutation carriers) shows that later stage large bowel cancer survival reduces, and the difference of Cetuximab is replied, and confirms the effect of KRAS transgenation in colorectal carcinoma.Because the KRAS mutator gene type in early days effect in large bowel cancer is unresolved, experiment given here and data, having assessed Dutch cohort study (NLCS) 409 examples, early stage (I phase and II phase tumor lympha are carried down and are moved; T1-4, N0, M0), the impact of 182 routine III phases (T1-4, N1, M0) and (T1-4, N0-1, M1) the large bowel cancer case prediction of 69 routine IV phases.Utilize the data of 1,886 routine sub-queue member of Dutch cohort study (NLCS), KRAS mutator gene type is also assessed the impact of risk of colorectal cancer.

This result of study shows, the prediction of early stage (I phase and the II phase) large bowel cancer of KRAS gene 3 ' UTR LCS6T>G sudden change impact.KRAS transgenation is present in 16.4% case, and only in world population, 6% is found (Chin LJ, et al.Cancer Res2008; 68:8535-40), in 12% to 15% European, be found (Ratner E, et al.Cancer Res2010; 70:6509-15).In case, find that late the increase of KRAS sudden change (G-allelotrope) frequency (is respectively patient early stage 14%, the III phase 19.2%, with the IV phase 21.4%), there is comparability (Graziano F, et al.Pharmacogenomics J2010 with the III phase frequency of previous report; 10:458-64).In 18% sub-queue member, find G allelotrope (KRAS sudden change).Find that KRAS transgenation and Advanced Colon Cancer have significance,statistical dependency between increasing, and provide the valuable opinion of learning about KRAS mutation carriers colorectal carcinoma natural biology.In addition, find that the early stage large bowel cancer case survival significance,statistical that carries KRAS sudden change increases, in the patient of KRAS transgenation, the commitment patient who carries G allelotrope (KRAS sudden change) does not die from large bowel cancer.The significance,statistical increase of carrying the early stage large bowel cancer case survival of KRAS sudden change has nothing to do with other predictive factors, as Tumor Differentiation or dislocation.Because T4 tumour is rare in case in early days, because observe the reason of worse result, the IIb phase case of the higher frequency in KRAS wild-type is excluded.Although result shows, the three phases cases prediction of KRAS transgenation (G allelotrope) and KRAS sudden change is poor, in III phase or IV phase, does not find significance,statistical impact.In addition KRAS transgenation, (G allelotrope) is studied the impact of risk of colorectal cancer.Find that early stage risk of colorectal cancer reduces, but on the not impact of the risk of advanced CRC, this shows that G allelotrope (KRAS sudden change) is irrelevant with the more high likelihood of development large bowel cancer.

In former research, KRAS suddenlys change and predicts the poor dependency that has.Yet, inconsistent about the result of this theme, and, it be unclear that (Smits KM, et al.Pharmacogenomics2008 with the clinical meaning of these results; 9:1903-16).KRAS sudden change is different from KRAS transgenation, and this is geneogenous sudden change, therefore, the development of tumour, biology and prediction is had to different impacts.

It is the interest that causes people that KRAS sudden change improves related discovery with early stage large bowel cancer survival rate.Study and show before this, cell aging can be caused by the overexpression of carcinogenic Ras, may contribute to precancerous lesion or innocent tumour growth to stop (Mooi WJ and Peeper DS.N Engl J Med2006; 355:1037-46).In human cancer, tumor cell senescence is in the news.Before cancer, adenoma of colon also shows old and feeble feature (Collado M and Serrano M.Nat Rev Cancer2010; 10:51-7).The aging of oncogene induction may only play a role in precancerous lesion.Yet the physiological level of KRAS gene can be induced old and feeble (Vicent S, et al.J Clin Invest2010 in the situation that there is no the transcription factor nephroblastoma 1 (WT1); 120:3940-52).If the expression of the patients with lung cancer WT1 genes involved of KRAS gene high expression reduces, they have a good prediction (Vicent S, et al.J Clin Invest2010; 120:3940-52).In a word, these results mean that other molecule factors can participate in the decision of cell fate, after the aging that causes oncogene induction can occur in other heredity or epigenetic target unconventionality expression.The aging that causes oncogene induction also can play a role at large bowel cancer: in KRAS gene-LCS6 genotype, may cause late tumor, or infantile tumour, infantile tumour has the better prediction of another heredity (epigenetic) mark based on being affected.

The two has all participated in Ras signal path, carries methylated early stage (I phase and II phase) the better result of case-finding of KRAS sudden change and BRAF sudden change or RASSF1A.The aging that BRAF is relevant was reported and was occurred in (Michaloglou C, et al.Nature2005 in melanoma in the past; 436:720-4), but be not also proved the effect that RASSF1A may bring into play in the aging that causes oncogene induction.In study population, it is uncommon that KRAS transgenation and BRAF sudden change and/or RASSF1A hyper-methylation exist simultaneously, therefore, does not reach statistical significance as described in the text.By these heredity (epigenetic) event in conjunction with time, KRAS transgenation (G-allelotrope) and KRAS, BRAF or RASSF1A alternate combinations strengthen the better result of patient more.Therefore, due to KRAS sudden change (G allelotrope) Ras overexpression, in conjunction with Ras pathway gene (epigenetic) genetic mutation, can induce the aging of early stage large bowel cancer, thus impact survival.For late case, increasing molecular pathway is affected, impact prediction.

MiRNA let-7 family confirms, contrast wild-type (13), and in the situation that KRAS transgenation exists, tumor growth suppresses, and let-7 expresses reduction, and KRAS level increases.Therefore, the patient that KRAS transgenation is carried in expection has poor prediction, as shown, for example, oral carcinoma (Christensen BC, et al.Carcinogenesis2009; 30:1003-7).For large bowel cancer, there are two report research KRAS genotype to the impact of patient treatment effect (Graziano F, et al.Pharmacogenomics J2010; 10:458-64; Zhang w et al.Ann Oncol2011; 22:104-9).First report, the a small set of irinotecan resistance patient who carries KRAS transgenation adopts irinotecan and Cetuximab treatment, survival rate variance, and the dependency (Graziano F, the et al.Pharmacogenomics J2010 that have KRAS sudden change and do not have BRAF to suddenly change; 10:458-64), however due to patient's untreated, these researchs are found that it(?) may not can in this research be repeated.Second report, Metastatic Colorectal Cancer adopts separately Cetuximab treatment to have better response, and carrying KRAS transgenation does not have the survival of patients phase of KRAS sudden change long, but response not statistically significant (Zhang w et al.Ann Oncol2011; 22:104-9).Better, although contrast without significance,statistical, this possible explanation is that IV phase patient organizes small scale in data acknowledgement IV phase KRAS mutation carriers prediction in this paper.The assessment KRAS of reproduction organization cell genotype is used in other researchs, yet tumour DNA assessment KRAS genotype is used in research described herein.The genotype of healthy tissues and tumor tissues is identical being confirmed with KRAS transgenation.

Contrast advanced CRC, early stage and advanced CRC seems discordant result and has proposed some problems about various cancers stage tumour origin and progress, and whether early stage large bowel cancer can develop by the different paths of molecule.KRAS transgenation is more common in terminal illness, yet the patient that KRAS sudden change is carried in early diagnosis seems a more favourable result.Therefore, these Notes of Key Datas, with late case contrast, the different biological of commitment.Even if early stage KRAS wild-type patient has a microsatellite instability tumour, they predict poor discovery, may show, these patients will benefit from other assisting therapy.Need further research, comprise randomized clinical trial, whether to assess these, predict that poor early stage patient will benefit from other assisting therapy.Before finding biomarker and method as herein described, microsatellite instability has been considered to predict good mark (Boland CR and Goel A.Gastroenterology2010; Yet 138:2073-87.e3), the better result of data acknowledgement KRAS mutation allele carrier of this research and microsatellite instability state are irrelevant.

The impact analysis of the early stage large bowel cancer case KRAS sudden change of mentioning in literary composition, confirms that early stage G allelotrope (KRAS sudden change) carrier who carries KRAS sudden change has a better result.In this research, crowd is only common untreated crowd, and for the first time, in this research, collected data provide the valuable opinion of learning carrying the natural biology of the early stage large bowel cancer of KRAS transgenation.Therefore, the evidence proposing is herein that first points out that KRAS mutator gene type is the possible predicting marker of early stage large bowel cancer, can be used for the good PATIENTS WITH LARGE BOWEL of identification prediction.

Treatment is replied

Ovarian cancer

Ovarian epithelial carcinoma (EOC) is second modal female pelvic cavity reproductive organ cancer of the U.S., and in the Western countries, this type of mortality ratio is the highest.This is the 5th major cause of American Women's cancer mortality, has every year 13,850 women to die from this disease.Although there is multiple new methods for the treatment of, the high mortality of ovarian epithelial carcinoma remains unchanged for many years, only has 30-39% (Parmar MK, et al. (2003) .Lancet361:2099-2106) 5 year lifetime.

The standard chemotherapy regimen of the treatment ovarian epithelial carcinoma adopting is at present carboplatin and taxol (Pfisterer J, et al. (2006) .J Clin Oncol24:4699-4707), based on perspective random test (Herzog T and Pothuri B. (2006) .Nat Clin Pract Oncol3:604-611; Esquela-Kerscher A and Slack F. (2006) .Nat Rev Cancer6:259-269; Iorio M, et al. (2007) .Cancer Res67:8699-8707).Although some patient has resistance to platinum-based chemotherapy at first, (be referred to as " platinum class resistance '), treat recurrence in 6 months, this is the first-line treatment to all epithelial ovarian carcinoma patients.Understand better the basic biology difference of epithelial ovarian tumor, can explain that in epithelial ovarian carcinoma patients, platinum resistance will be selected a more rational methods for the treatment of (Parmar MK, et al. (2003) .Lancet361:2099-2106; Pfisterer J, et al. (2006) .J Clin Oncol24:4699-4707; Herzog T and Pothuri B. (2006) .Nat Clin Pract Oncol3:604-611).

MicroRNAs (miRNA) is 22 Nucleotide non-coding RNAs of a class, unconventionality expression in the cancer of nearly all type, and they can be used as oncogene or the tumor suppressor gene that a class is new.Ovarian epithelial carcinoma, except distinguishing (Iorio M, et al. (2007) .Cancer Res67:8699-8707 normal ovarian tissue from pernicious ovary tissue; Zhang L, et al. (2008) .Proc Natl Acad Sci USA105:7004-7009), miRNA expression pattern shows that morbidity is important (.Int J Biochem Cell Biol42:1262-1272 to ovarian epithelial carcinoma; Van Jaarsveld M, et al. (2010) .Int J Biochem Cell Biol42:1282-1290), change (Eitan R with epithelial ovarian carcinoma patients result, et al. (2009) .Gynecol Oncol114:253-259) and treatment reply relevant (Lu L, et al. (2011) .Gynecol Oncol122:366-371).MiRNA differential expression is (Eitan R, et al. (2009) .Gynecol Oncol114:253-259 relevant to platinum resistance epithelial ovarian cancer chemotherapy also; Lu L, et al. (2011) .Gynecol Oncol122:366-371; Chen K, et al. (2008) .Carcinogenesis29:1306-1311).

Other opinion of the importance of miRNAs in cancer comes from the discovery of genetic single nucleotide polymorphism (SNP), disturb encoding sequence (the Chin LJ of miRNA, et al. (2008) .Cancer Res68:8535-8540) and binding site (Chen K, et al. (2008) .Carcinogenesis29:1306-1311 of miRNA proto-oncogene 3 ' non-translational region (3 ' UTRs); Chin LJ, et al. (2008).The embodiment of this functional sudden change is rs61764370, is referred to as KRAS transgenation, the let-7miRNA paratope KRAS gene 3 ' UTR in being located at.Once reported association between rs61764370 and the risk of epithelium ovarian cancer (EOC) (referring to, international patent application no PCT/US2008/065302 and international patent application no PCT/US2010/023412, its full content is incorporated in herein separately by citation).In addition, the method and the embodiment that provide prove, this sudden change is a kind of biomarker of ovarian epithelial carcinoma (EOC) clinical effectiveness and chemotherapy resistance.Evidence is supported the effect of tumour KRAS sudden change continuous function, and the cognation between malignant tumour biology and cancer poor outcome.

Assessed herein in harmful BRCA sudden change existence and non-existent situation, KRAS transgenation is as the potentiality of large bowel cancer result biomarker.And the response of research platinum class new adjuvant chemotherapy confirms the potential cause that KRAS transgenation PATIENTS WITH LARGE BOWEL result changes, and has assessed platinum class resistance and has assessed large bowel cancer oncogene and expressed.These data show, directly the acquired KRAS transgenation of target function may reduce Growth of Cells and survival in the epithelial ovarian JEG-3 with this pathology.

KRAS transgenation is that the postmenopausal women (more than 52 years old) who suffers from ovarian epithelial carcinoma predicts poor biomarker.KRAS suddenly change the prediction of relevant ovarian cancer bad be due to, at least part of cognation due to KRAS transgenation and platinum-based chemotherapy resistance, worse response based on platinum class new adjuvant chemotherapy, the epithelial ovarian carcinoma patients that carries KRAS transgenation of assisting therapy has the platinum class resistance that significance,statistical increases.

Biology difference between KRAS transgenation ovarian epithelial carcinoma and not mutated epithelial ovarian tumor obtains gene expression data support, shows KRAS habituation and AKT-mediation platinum class resistance in the ovarian epithelial carcinoma of KRAS transgenation-be associated.With not mutated cell strain contrast, further confirm to carry the external platinum class of the Ovarian Cancer Cells resistance of KRAS transgenation.By this sudden change of direct target, the relevant ovarian epithelial carcinoma of KRAS sudden change shows the evidence of KRAS sudden change reproduction pathology continuous dependence, causes significantly suppressing tumor growth and cell survival in KRAS transgenation epithelial ovarian JEG-3 and not mutated epithelial ovarian JEG-3.

The KRAS sudden change cognation not good to postmenopausal women's survival rate may be due to the fundamental biological knowledge relevant with this sudden change.Support the hypothesis of cognation reflection fundamental biological knowledge, KRAS sudden change is associated with Postmenopausal Ovaries cancer (Ratner E, et al. (2010) .Cancer Res15:6509-6515), nearly 59 years old of average diagnosis of age.Relative survival rate is with change of age, being diagnosed as mortality ratio within the elderly woman 5 years of ovarian epithelial carcinoma may be the twice of young woman, what further support this hypothesis is that postclimacteric women may have the different tumour of biology (ACS (2010) .Cancer facts & figures2010.Cancer Facts & Figures.ACS:Atlanta, GA, PP1-56).In addition, KRAS transgenation has been proved to be a kind of biomarker that pre-menopausal women (age <52 year) is suffered from three cloudy mammary cancer risks.Therefore, the miRNA that in the effect of KRAS transgenation in suffering from cancer risk and different tissues, biology may depend in response to physiological condition expresses change, as menopause.The risk that KRAS transgenation women suffers from breast cancer may be first, then, may have the risk that develops into Postmenopausal Ovaries cancer.

KRAS sudden change can not predict that one group of epithelial ovarian carcinoma patients that carries known harmful BRCA transgenation predicts poor discovery, and possible explanation is for because BRCA transgenation is associated with platinum class susceptibility.May there is KRAS transgenation and cause or aggravate platinum medicine resistance in the consequence that BRCA transgenation is relevant to platinum class susceptibility.This may be the young patient of studying herein, may have the harmful BRCA transgenation that has no document.Or in addition, the young patient providing in research, also may suffer from the ovarian cancer that is more common in young woman of other hypotypes, as borderline tumor, causes these patient's mistaken diagnosis.Although data provided herein have been carried out clinical note widely, do not obtain our all epithelial ovarian carcinoma patients BRCA states, although pathological replacement can be used, tumor tissues cannot obtain examination again.A nearest research fails to find the bad cognation of KRAS sudden change and prediction and for the resistance of the ovarian epithelial treatment of ovarian cancer of genome-wide association study, this research has very limited clinical information, for example, as the factor of BRCA state and the survival of ovarian cancer specificity cannot obtain, also be not included in its analysis (Pharoah P, et al. (2011) .Clin Cancer Res17:3742-3750).

In two kinds of dissimilar KRAS transgenation related neoplasms, find similar gene false demonstration, show these tumours, no matter tissue-derived, when occurring, tumour uses similar path.The necrocytosis that in the epithelial ovarian JEG-3 that directly target KRAS transgenation is relevant, KRAS transgenation pathology causes significance to strengthen, and reduce KRAS level.These discoveries show, KRAS transgenation tumour relies on continuous severe single, non-coding reproduction pathology.Although by measuring oncogene, express, and the sudden change of definite tumour, significance is made great efforts customization cancer therapy always, even if having, seldom has germinal mutation, has previously been proved to be the important target of cancer therapy.

According to data provided in this article, determine that KRAS transgenation important in ovarian cancer is a kind of functional oncogene sudden change, KRAS sudden change impels the ovarian tumor hypotype being associated with it.These find that there is the prediction that helps improve ovarian cancer patients.

Large bowel cancer

Two kinds of monoclonal antibodies of targeting epidermal growth factor receptor (anti-EGFR moAbs) are incorporated in Metastatic Colorectal Cancer (mCRC) clinical practice, Cetuximab and Victibix, as single therapy or with chemotherapy, combine, offer treatment front gentle clinical benefit of patient (Cunningham D, et al.N Engl J Med2004; 351 (4): 337-345; Saltz LB, et al.J Clin Oncol2004; 22 (7): 1201-1208; Saltz LB, et al.N Engl J Med2000; 343 (13): 905-914; Van CE, et al.J Clin Oncol2007; 25 (13): 1658-1664).But, it becomes obviously very soon, and its effect only limits to a group patient.Nonrandom retrospective study (Amado RG et al.J Clin Oncol2008; 26 (10): 1626-1634; De RW, et al.Ann Oncol2008; 19 (3): 508-515; Lievre A, et al.Cancer Res2006; 66 (8): 3992-3995; Lievre A, et al.JClin Oncol2008; 26 (3): 374-379; Moroni M, et al.Lancet Oncol2005; 6 (5): 279-286; Sartore-Bianchi A, al.J Clin Oncol2007; 25 (22): 3238-3245), perspective random test retrospective analysis (BokemeYer C, et al.J Clin Oncol2009; 27 (5): 663-671; Douillard J et al.AnnOncol supp.2009; Karapetis CS, et al.N Engl J Med2008; 359 (17): 1757-1765; Tol J, et al.N Engl J Med2009; 360 (6): 563-572; Van Cutsem E, et al.N Engl J Med2009; 360 (14): 1408-1417), the research of large European Union shows (De RW, et al.Lancet Oncol2010; 11 (8): 753-762), monoclonal antibody against EGFR treatment resistance has been predicted in the existence of tumour KRAS sudden change, and short relevant to predicted difference, lifetime.The anti-EGFR monoclonal antibody treatment of mandatory startup KRAS mutation status has had a lot of years, problem is not resolved, because, the wild blastomogenic Metastatic Colorectal Cancer patient of KRAS that suffers from of about 50-65% does not benefit from these treatments, this means, there is (De RW, et al.Ann Oncol2008 in other hereditary factor or the sensitivity of resistance; 19 (3): 508-515; Allegra CJ, et al.J Clin Oncol2009; 27 (12): 2091-2096; De RW, al.Lancet Oncol2010; 11 (8): 753-762; RooCk WD, et al.Lancet Oncol2010).More and more evidence shows, monoclonal antibody against EGFR resistance (De RW, et al.Lancet Oncol2010 are given in BRAF V600E sudden change; 11 (8): 753-762; Di NF, et al.J Clin Oncol2008; 26 (35): 5705-5712; Laurent-Puig P, et al.J Clin Oncol2009; 27 (35): 5924-5930; Saridaki Z, et al.IPLoS One2011; 6 (1): e15980; Souglakos J, et al.Br J Cancer2009; 101 (3): 465-472), yet, although and imperfectly understand, PIK3CA sudden change tumour seems not have or seldom benefit from such controlling; Treat (De RW, et al.Lancet Oncol2010; 11 (8): 753-762; Prenen H, et al.Clin Cancer Res2009; 15 (9): 3184-3188; Sartore-Bianchi A, et al.Cancer Res2009; 69 (5): 1851-1857; Jhawer M, et al.Cancer Res2008; 68 (6): 1953-1961; Ogino S, et al.J Clin Oncol2009; 27 (9): 1477-1484).

Except tumour hereditary feature, there is increasing evidence to show, patient's sexual cell genome also may play a role giving anti-EGFR mab treatment resistance or susceptibility.Support this concept, Fc γ RIIa and Fc γ RIIIa, EGF-R ELISA, Urogastron, cyclin D1 and COX-2 genes encoding polymorphism, with monotherapy and relevant with the Cetuximab treatment Metastatic Colorectal Cancer patient's of combined chemotherapy administration result.

MicroRNAs (miRNAs) is a class height homology, endogenic, noncoding small RNA molecular, and length is 18-25 Nucleotide, in conjunction with target mRNA3 '-non-translational region (UTR) part paratope negative regulator gene, expresses.After Dicer and the processing of Drosha enzyme RNase III endonuclease, ripe miRNA can suppress mRNA to said target mrna by the silencing complex of guiding RNA induction and translate.MiRNA regulates some to participate in basic bioprocess, as the gene of cell proliferation, cytodifferentiation and apoptosis, no matter is in cancer occurs and develops, to play key player as oncogene or tumor suppressor gene.Although, confirmed so far more than 700 miRNA sequence in human genome, this numeral is estimated to double.In addition, each miRNA can be simultaneously by regulating many miRNA to control hundreds of genes.

MiRNA is vital in conjunction with regulation process and the protein expression subsequently of mRNA gene pairs mRNA level, and this adjusting can be subject to miRNA target SNP (SNP) impact (SNPs).These single nucleotide polymorphism (SNP) can create wrong binding site or the correct binding site of cancellation (elimination), cause miRNA to regulate resistance, reflection can be in Human diseases, as the another kind of genetic mutation playing a role in cancer (or to some disease, as cancer increases risk).Emerging research mainly concentrates on the system gene group evaluation of these binding sites and detects function and the biological dependency of single nucleotide polymorphism (SNP), and this is the important molecule mark in personalized medicine rapid growth field.Occur that such single nucleotide polymorphism (SNP) not only affects genetic expression, but also affect oncobiology and drug reaction and resistance.

MiRNA Lethal-7 family (let-7) is found first in many cancers, and its differential expression detected.KRAS proto-oncogene is the direct effect target spot of let-7miRNA family, and or rather, once in conjunction with some site of KRAS gene mRNA 3 ' non-translational region (3 '-UTR), let-7 induction KRAS gene is lowered.

KRAS transgenation be a kind of functional single nucleotide polymorphism (SNP) (SNP), occur in the complementary site (LCS) of KRAS gene 3 '-UTR mRNA let-7.Described single nucleotide polymorphism (SNP) (rs61764370) is replaced into T G base, be found to change ripe let-7 in conjunction with the ability of KRAS gene mRNA, and cause KRAS gene cancer protein vivoexpression to increase, and causing the interior let-7miRNA level of body lower, this may be due to negative feedback loop.Consistent with the carcinogenic character of KRAS gene, KRAS transgenation (also referred to as G allelotrope) shows that having increased moderate smoker suffers from the risk of nonsmall-cell lung cancer (NSCLC), increase the risk that develops three negative breast cancer, increased the risk having ovarian cancer in a group women.In addition the frequency that detects KRAS mutation allele in the row BRCA1 of ， squad carrier increases.And KRAS mutation carriers (allelotrope G-), suffers from head and neck cancer, but is not to suffer from nonsmall-cell lung cancer, demonstrate overall survival and reduce.The survival rate that significance,statistical is poor and platinum resistance are found in the ovarian cancer patients of carrying KRAS transgenation (G allelotrope).Evidence has also proved function and the clinical significance of KRAS transgenation (being also referred to as KRAS gene 3 '-UTR LCS6SNP) together.

The treatment of the anti-EGFR monoclonal antibody of Metastatic Colorectal Cancer target arranges the date, KRAS transgenation small portion crowd and there is contradiction and two researchs of the result of conflict in assess (Graziano F, et al.Pharmacogenomics J2010; 10 (5): 458-464; Zhang W, et al.Ann Oncol2011; 22 (1): 104-109).Carry (Graziano F, et al.Pharmacogenomics J2010 in KRAS and BRAF wild-type allele patient crowd's first research; 10 (5): 458-464), treatment is succoured in the combination therapy of irinotecan-Cetuximab, and KRAS transgenation (G-allelotrope) carrier all shows has even worse Progression free survival phase (PFS) of significance,statistical and Overall survival (OS).On the contrary, (Zhang w, et al.Ann Oncol2011 in second research; 22 (1): 104-109), wherein patient accepts Cetuximab list medicine relief treatment, KRAS transgenation (G-allelotrope) carrier demonstrates longer Progression free survival phase (PFS) and Overall survival (OS), has one better objective efficient (ORR).Although these researchs seem contrary result, these patient treatments are not identical, and in fact, irinotecan chemotherapy adds the not good reaction that Cetuximab has also been found to predict KRAS transgenation (G-allelotrope) carrier (Winder T, et al.J.Clin.Oncol.[27 (15S Suppl)] .2009.Abstract).Evidence suggests, unlike tumor complicated KRAS protein mutation, KRAS transgenation (G-allelotrope) carrier is carried out to the impact of combination therapy otherness to the response of Cetuximab (Winder T, et al.J.Clin.Oncol.[27 (15SSuppl)] .2009.Abstract).This dynamic adjustments miRNA in disease is combined the data consistent of position variant.

In this research, KRAS transgenation, and other molecular markers, as KRAS and BRAF mutation status, in a series of 559 the Metastatic Colorectal Cancer patients that accept anti-EGFR monoclonal antibody single therapy or monoclonal antibody and chemotherapy combined treatment, evaluate.Given data are clear and definite herein, and prediction mab treatment is replied the effect of middle KRAS transgenation.In this patient's queue, and in cell strain, the positive response of monoclonal antibody single therapy has been predicted in KRAS transgenation (G allelotrope), and cytotoxicity chemotherapy is without any extra benefit.

In this paper studies confirm that, the meta Progression free survival phase statistically significant property improvement (a pair of wild-type patient improves the trend of Overall survival) of suffering from all KRAS carriers of mutation of transitivity colorectal carcinoma, KRAS carriers of mutation is accepted anti-EGFR monoclonal antibody single therapy.In addition, with respect to the non-KRAS carriers of mutation in all queues of anti-epidermal growth factor receptor monoclonal antibody response investigations, comprise KRAS or RAF sudden change patient, find that these patient's significance,statistical predictions are good.It is not depend on adding of chemotherapy that this prediction improves, and in fact, and except anti-EGFR mab treatment, carrier demonstrates chemotherapy there is no effect in KRAS transgenation (G allelotrope).This is contrary with non-KRAS sudden change patient, it show in all queues that significance is benefited from chemotherapy and adds anti-EGFR monoclonal antibody, and adding to the same level KRAS mutation allele carrier who accepts anti-EGFR monoclonal antibody single therapy of chemotherapy brought prediction.Cell strain research shows, contrasts not mutated cell strain, and KRAS transgenation cell strain lacks the effect same benefiting in combination therapy.These results of study show, the very first time, suffer from the KRAS transgenation allelotrope patient of transitivity colorectal carcinoma, can, perhaps the toxicity of chemotherapy should be avoided, sometimes or even fatal impact, and significant anti-EGFR monoclonal antibody single therapy can be carried out separately.

The baseline morbidity of comparison report, mainly originates from European a group patient and shows KRAS gene mutation frequency rising 19.5%.In 6% world population, find KRAS sudden change, in healthy white people, its frequency has been estimated to rise more than 10%.In addition, suffer from Patients with Non-small-cell Lung, it is nearly 20% that the morbidity of KRAS transgenation significantly increases, and given prominence to the cognation that risk increases.The people such as Graziano (Pharmacogenomics J2010; 10 (5): 458-464) in white man's Metastatic Colorectal Cancer patient group with European descent of research, KRAS transgenation (G allelotrope) (mixing TG and GG genotype) is found in 25% patient, however (Ann Oncol2011 in the more heterogeneous crowd of people's researchs such as Zhang; 22 (1): 104-109), the frequency of KRAS sudden change is 15.3%.Data provided herein do not find that KRAS mutation allele is the risk of suffering from colorectal carcinoma, although IV phase patient enrichment KRAS sudden change.In a word, evidence shows, although the risk of the colorectal carcinoma of KRAS sudden change (G allelotrope) not all type, the possibility of it and later stage of development and transitivity colorectal carcinoma is relevant.KRAS mutation forecasting during employing Cetuximab single therapy, early stage colorectal carcinoma and the prediction of transitivity colorectal cancer patients are good.Yet KRAS transgenation (G allelotrope) may be relevant with transitivity advancing of disease in colorectal carcinoma, this is general fatal.

Report before contrast is observed according to the different distributions of the KRAS mutator gene type of KRAS and BRAF mutation status in about this research of Metastatic Colorectal Cancer patient crowd.In this research, KRAS genotype is evenly distribute between KRAS wild-type and sudden change group, and still, in BRAF sudden change group, the frequency of KRAS sudden change is that significance,statistical increases, and for example, compares high twice with wild-type.At the later stage of large bowel cancer canceration, KRAS transgenation allelotrope may mediate low differentiation and have more aggressive clone's selection, and this clone carries BRAF sudden change.In addition, under the existence of KRAS transgenation, selection pressure may be conducive to KRAS or BRAF sudden change development, and this depends on accepts specific therapy.When carrying out Cetuximab treatment, no matter patient has had KRAS or BRAF transgenation, KRAS sudden change (G allelotrope) patient has different predictions, the potentiality of pointing out these groups need to reappraise Cetuximab treatment.

When analyzing existence result according to treatment, adopt in the whole and a pair of wild-type patient group of anti-EGFR monoclonal antibody single therapy, KRAS mutator gene type carrier has the longer Progression free survival phase of significance,statistical (P=0.019 and P=0.039 respectively).Although, in the patient group of whole single therapy, contrast wild-type carrier 28.85 weeks, KRAS mutator gene type carrier has 45 weeks longer Overall survivals (OS), however this species diversity does not reach significance,statistical.In two (KRAS and BRAF) the wild-type patient groups of KRAS mutation carriers, observe the trend (55.43 and 35.71 weeks) of the longer Overall survival of significance,statistical (P=0.087) (OS).

Contrast has LCS6 wild type gene type (P=0.037, log-rank check) 12 weeks Progression free survival phases of patient, the KRAS sudden change patient with the genotypic Cetuximab/irinotecan of KRAS transgenation (G-allelotrope), shows 6.4 weeks worse Progression free survival phases of significance (PFS).In our analysis, anti-EGFR monoclonal antibody is in basic combined chemotherapy group, people use respectively various medicaments treatment, between KRAS sudden change and wild type gene type carrier, in arbitrary crowd, find that Progression free survival phase (PFS) and Overall survival (OS) are without statistical significant difference., when they accept respectively chemotherapy and single therapy, there is the trend of poorer lifetime (23 weeks with 28 weeks) in the KRAS carriers of mutation with KRAS or RAF sudden change.These results show jointly, and it is disadvantageous that KRAS carriers of mutation adopts the treatment of chemotherapy combined based on anti-EGFR monoclonal antibody.

In other cancers, an important step of large bowel cancer development, is miRNA imbalance.In the past few years, miRNA has been brought to the arena theatre that molecular weight tumor is learned, and has greatly changed us and treat and understand the mode of gene regulating.KRAS transgenation is that the miRNA relevant to risk of cancer is in conjunction with first single nucleotide polymorphism (SNP) in position.Data in this paper show, the patient who carries KRAS mutation allele has more biology otherness than not mutated or LCS6 wild-type patient.Add by chemotherapy in the situation without income, carrying the genotypic patient of KRAS transgenation allelotrope has high probability to benefit from anti-EGFR monoclonal antibody single therapy and better macro-forecast.Due to the overexpression of KRAS sudden change tumor inducing KRAS path, upstream suppresses this path may make these tumours responsive especially.This mechanism demonstrates denies that KRAS transgenation tumour mab treatment lacks curative effect, yet likely KRAS transgenation can not brought out high-caliber independent KRAS path signal as KRAS transgenation tumour.

KRAS sudden change tumour can not add monoclonal antibody single therapy and acquire benefit from cell toxicity medicament treatment.Because miRNA let-7 family regulates KRAS sudden change, because chemotherapy reduces let-7 level, allow higher KRAS genetic expression (especially under KRAS transgenation exists), use the treatment plan of chemotherapy may increase this allelic activation, thereby remove the ability that upstream monoclonal antibody therapy overcomes KRAS Pathway Activation.The Potential feasibility of the functional sudden change of 3 ' UTR (comprising KRAS transgenation), predicting tumors biological modification and the response to treatment, obtain the better risk score of patient.

MicroRNA

MicroRNAs (miRNAs) is the little non-coding RNA that a class is new, there is said target mrna 3 ' non-translational region (UTR), and the base pairing regulate gene expression of sequence in 5 ' non-translational region (UTR) and encoding sequence district, cause cracking and/or the translation of mRNA to suppress (He L, et al.Nature2005; 435:828-33; Esquela-Kerscher A.and Slack FJ.Nat Rev Cancer2006; 6:259-69).In each routine cancer of research, MiRNAs is regulated and controled by mistake so far, comprise, but be not limited to, mammary cancer and large bowel cancer, in tumour initiator cell, find that some miRNA changes (especially reducing let-7), this shows that low let-7 makes these cell self refreshes and propagation (Yu F, et al.Cell2007; 131:1109-23), and increase cancered risk.

Because miRNA plays the effect of overall gene regulating, miRNA genetic mutation increases relevant to risk of cancer.Evidence rapid growth, disturbs miRNA encoding sequence (Ho man A, et al.Cancer Res2009; The polymorphism of 69:5970-77) or 3 ' UTR miRNA binding site is the strong predictor of risk of cancer, includes, but not limited to mammary cancer and large bowel cancer (Pongsavee M, et al.Genet Test Mol Biomarkers2009; 13:307-17; Tchatchou S, et al.Carcinogenesis2009; 30:59-64).Yet, the miRNA-that does not have previously to have found change polymorphism and three negative breast cancer (TNBC) or with tumour in the gene and/or the miRNA expression that change relevant.

Determined recently a kind of new germline polymorphism (rs61764370) (the international patent application no PCT/US2008/065302 in the complementary site of KRAS proto-oncogene 3 ' UTR let-7miRNA, its full content is incorporated in herein by reference), be referred to as " LCS6-SNP ' or ' KRAS transgenation '.

KRAS gene regulating relevant (Chin L, et al.Cancer Res2008 that KRAS sudden change changes to lower concentration let-7 in tumour and lung cancer; 68:8535-40).In addition, cancer specific result poor in head and neck cancer (Christensen BC, et al.Carcinogenesis2009 have been predicted in KRAS transgenation; 30:1003-07), also predicted drug responses (Graziano F, the et al.Pharmacogenomics J2010 changing in colorectal carcinoma; 10:458-64; Zhang W, et al.Ann Oncol2011; 22:104-09).KRAS transgenation is also enriched in ovarian cancer, is the most common (Ratner E, the et al.Cancer Res2010 of being associated with the patient of hereditary breast cancer and ovarian cancer (HBOC) family; 70:6509-15).The effect of KRAS transgenation in risk of cancer and oncobiology has further been assessed in research provided herein.

Data presentation provided herein, for instance, KRAS gene 3 ' UTR germline polymorphism, is called as " KRAS transgenation ", is that pre-menopausal women develops a kind of genetic marker that three negative breast cancer risks increase.Because study group's 1 small scale can only be assessed in having known ER and PR state patient, dependency is verified in the more massive case-control group of full receptor status.The most important thing is, data acknowledgement and the patient's contrast that there is no KRAS transgenation, the tumour with three negative breast cancer (TNBC) patient of KRAS transgenation has different gene expression patterns, confirm that KRAS transgenation drives specificity path, this specificity path affects as everyone knows oncobiology and revises tumor development.Therefore, KRAS transgenation can be significant biological subgroup by staging, in future anticipation prediction and guiding treatment decision-making.

In three cloudy breast cancer tumours, the let-7 expression minimizing discovery relevant to KRAS transgenation is important clinically.By NFKB, KRAS Overexpression can be induced lin-28, let-7 negative regulation, therefore, reduces let-7 and expresses (Iliopoulos D, et al.Cell2009; 139:1-14; Meylan E, et al.Nature2009; 462:104-08; Barbie D, et al.Nature2009; 462:108-12).This points out Liao，Yu malignant tissue, final, the potential mechanism that in the relevant tumour of KRAS transgenation, let-7 reduces.In addition let-7 regulation and control mammary gland sample stem cells hyperplasia (Yu F, YaoH, Zhu P, et al.Cell2007; 131:1109-23), and regulate and control the low expression of let-7 or lower concentration and make this group cell expansion, thereby increased, carry the risk that the women of KRAS transgenation suffers from breast cancer.KRAS transgenation only and the dependency of pre-menopausal women three negative breast cancer risks pointed out KRAS transgenation and the hormone Meaningful Interaction between exposing.

Although the breast cancer tumour of the BRCA1 of carrying transgenations more than half (TNBC) develops into three negative hypotypes (Atchlev DP, et al.J Clin oncol2008; 26:4282-88), BRCA1 transgenation is rare, therefore, accounts for greatly 10-15% (Young SR, the et al.BMC Cancer2009 of all three cloudy mammary cancer cases; 9:86; Nanda R, et al.JAMA2005; 294:1925-33).In first three cloudy patient with breast cancer of more than 23% menopause, find KRAS transgenation, in the negative BRCA1 mutation carriers of ER/PR of the BRCA mutator gene carrier of these queues or youth, there is no obvious significance enrichment (miRNA expression map, the public can login WWW.appliedbiosystems.com/absite/us/en/home/applications-technol ogies/real-time-pcr/mirna-profiling.html (accessed Jan1,2008)).KRAS transgenation is expressed label with BRCA1 transgenation genoid and is associated, and shows no matter be by sudden change or other mechanism, likely increases the carcinogenic risk under the low expression existence of KRAS transgenation, KRAS gene high expression and BRCA1.

The external regulation and control KRAS of KRAS transgenation impact genetic expression, and cause higher KRAS concentration (Chin L, et al.Cancer Res2008; 68:8535-40).It is the important upstream regulation of MAPK path that KRAS causes oncogene, and its overexpression can cause Raf/MEK/MAPK signal pathway activated to increase, thereby impels the generation of tumour.Provided herein studies confirm that, the patient with KRAS transgenation and three negative breast cancer demonstrates MAPK signal pathway activated (Table X).In breast cancer cell, MAPK superactivation reduces ER expression, causes ER-negative phenotype (Atchley D P, et al.J Clin Oncol2008; 26:4282-88), consistent with our discovery, more low estrogen signal correction in the negative tumour of KRAS transgenation and these histologies ER.MAPK activation is treated insensitivity relevant (Oh AS, et al.Mol Endocrinol2001 with growth and the estrogen antagonist of non-estrogen-dependent type tumour; 15:1344-59), may be that KRAS transgenation drives the breast cancer tumour mechanism that just other mammary cancer hypotype does not develop.

KRAS transgenation is that certain cancers is predicted poor biomarker, comprises head and neck cancer (Christensen BC, et al.Carcinogenesis2009; 30:1003-07).KRAS transgenation is also not good biomarker (Graziano F, the et al.Pharmacogenomics J2010 of target treatment of colon cancer combined chemotherapy reaction; 10:458-64).KRAS positive gene mutation three Breast Cancer Patients with Negative Axillaries have luminal type precursor blood vessel label, and in label, the differential expression of vasculogenesis and transitivity mark confirms, KRAS sudden change tumour is aggressiveness subgroup three negative breast cancer.

Provided herein studies confirm that, KRAS transgenation and Tumor-assaciated, tumour keeps unique gene expression pattern.Although work is carried out, these datas provide for the valuable opinion that transforms committed step and path and women's tumor development.These are all to understand the very significant step of carcinobiology mechanism that function miRNA destroys polymorphism, and function miRNA destroys the risk of cancer genetic marker that is different from previous discovery in polymorphism function.

KRAS sudden change

The present invention be based on, part is based on unexpected discovery, there is KRAS gene 3 ' non-translational region (UTR) single nucleotide polymorphism (SNP), be referred to as " LCS6SNP " herein ' or " KRAS transgenation ", the response of individual cancer stricken risk and individual treatment cancer predicted.KRAS transgenation is positioned at LCS6, and wild-type and mutant nucleotide sequence are provided below.

KRAS sudden change can be represented by one or more following sequences.For instance, KRAS transgenation can be by GenBank accession number rs61764370 and sequence GTCTCGAACTCCTGACCTCAAGTGATGCACCCACCTTGGCCTCATAAACCTG (SEQ ID NO:22, wherein single nucleotide polymorphism (SNP) marks with runic underscore) definition.

The three-type-person RAS gene being formed by HRAS, KRAS and NRAS.Each gene comprises the complementary site of miRNA in a plurality of its mRNA transcript 3 ' UTR.Specifically, every kind of people RAS gene comprises the complementary site (LCSs) of a plurality of let-7.MicroRNAs let-7 family (miRNAs) comprises the important overall genetic regulator of controlling the expression of lung cancer oncogene in conjunction with its target messenger RNA(mRNA) s (mRNAs) 3 ' UTRs (non-coding region).

Particularly, term " let-7 paratope " is in order to describe any region in conjunction with miRNAs let-7 family member's gene or genetic transcription thing.In addition, this term comprise with the gene of let-7 family miRNA sequence complementation or genetic transcription thing in these sequences.The threshold value of combination between two sequences described in term " complementation ", and wherein the most of Nucleotide in each sequence can the interior most nucleoside acid of another sequence of trans combination.

People KRAS3 ' UTR comprises 8 LCS1, respectively called after LCS1-LCS8.Following sequence, thymus pyrimidine (T) can be substituted by uridylic (U).LCS1 comprises sequence GACAGUGGAAGUUUUUUUUUCCUCG (SEQ ID NO:1).LCS2 comprises sequence A UUAGUGUCAUCUUGCCUC (SEQ ID NO:2).LC S3 comprises sequence A AUGCCCUACAUCUUAUUUUCCUCA (SEQ ID NO:3).LCS4 comprises sequence GGUUCAAGCGAUUCUCGUGCCUCG (SEQ ID NO:4).LCS5 comprises sequence

GGCUGGUCCGAACUCCUGACCUCA(SEQ?ID?NO：5)。LCS6 comprises sequence GAUUCACCCACCUUGGCCUCA (SEQ ID NO:6).LCS7 comprises sequence GGGUGUUAAGACUUGACACAGUACCUCG (SEQ ID NO:7).LCS8 comprises sequence

AGUGCUUAUGAGGGGAUAUUUAGGCCUC(SEQ?ID?NO：8)。

People KRAS gene has two wild-type forms, by transcript a and b, is encoded, and the sequence number that hereinafter provided is provided: 9 and 10.The sequence of everyone KRAS transcript, contains LCS6SNP, as the sequence number hereinafter being provided: 11 and 12.

People KRAS gene, transcript variant a, by following mRNA sequence encoding (NCBI accession number: NM_033360 and SEQ ID NO:9) (non-coding region show with runic, LCS6 lines out below):

People KRAS, transcript variant b, by following mRNA sequence encoding (NCBI accession number: NM_004985 and SEQ ID NO:10) (non-coding region show with runic, LCS6 lines out below):

People KRAS, transcript variant a, comprises LCS6SNP, by following mRNA sequence encoding (SEQ ID NO:11) (non-coding region shows with runic, and LCS6 lines out below, and SNP capitalizes out):

People KRAS, transcript variant b, comprises LCS6SNP, by following mRNA sequence encoding (SEQ ID NO:12) (non-coding region shows with runic, and LCS6 lines out below, and SNP capitalizes out):

KRAS sudden change is the result that the 4th G of LCS6SEQ ID NO:6 is substituted by U.KRAS sudden change comprises sequence GAUGCACCCACCUUGGCCUCA (SNP emphasizes with runic) (SEQ ID NO:13).

KRAS sudden change causes KRAS to express change by upsetting KRAS gene miRNA regulation and control.International application no is in PCT/US08/65302 (WO2008/151004), to have further described discriminating and the sign of KRAS transgenation, and its full content is incorporated in herein by reference.

The miRNAs of Let-7 family

The expression of carrying the miRNA of let-7 family in the cell of KRAS sudden change improves.What is interesting is, the miRNA of let-7 family is in conjunction with LCS, and wherein KRAS sudden change is positioned at this.The existence of KRAS sudden change disturbs let-7 in conjunction with KRAS.By disturbing, KRAS transgenation or induction let-7 combination, or many or few tightly in conjunction with KRAS LCS6.It is found that, compare with wild-type cell, the cell that contains KRAS transgenation has the KRAS gene mRNA of lower level, has the KRAS albumen that level increases.Therefore,, although do not wish to be bound by theory, in cell, the existence of KRAS transgenation, may disturb let-7 in conjunction with the translation of ability the arrestin matter of KRAS, thereby make the higher level of KRAS gene protein.

In triple negative breast cancer, the existence of KRAS transgenation is also associated with the let-7miRNA of significance lower level.For example, let-7miRNA expresses and reduces 2 times of (2X), 3 times (3X), 4 times (4X), 5 times (5X), 6 times (6X), 7 times (7X), 8 times (8X), 9 times (9X), 10 times (10X), 20 times (20X), 50 times (50X), 100 times (100X), 200 times (200X), 500 times (500X), 1000 times (1000X), or any multiple of between.Alternatively, or in addition, in contrast to and do not suffer from the interior let-7miRNA expression level of cell that three negative breast cancer main bodys obtain with it, suffers from the interior let-7miRNA expression of the cell minimizing that three negative breast cancer main bodys obtain with it, statistical significant difference (for example normal cell or control cells) is presented as that p value is less than 0.05 between the two, and preferably, p value is less than 0.01, or most preferably, p value is less than 0.001.Let-7miRNA expression level from suffer from the cell that three negative breast cancer main bodys obtain with it, also can with standard level contrast known in the art.In addition, also can suffer from mammary cancer, or, especially between the influenced cell of the main body of three negative breast cancer and unaffected cell, comparing the level that let-7 expresses, wherein said unaffected cell is as internal contrast group.

Exemplary let-7miRNA includes, but not limited to let-7a (let-7a-1, let-7a-2, let-7a-3), let-7b, let-7c, let-7d, let-7e, let-7f (let-7f-1 and let-7f-2), let-7g and let-7i.For following sequence, thymus pyrimidine (T) can be substituted by uridylic (U).Let-7a comprises sequence UUGAUAUGUUGGAUGAUGGAGU (SEQ ID NO:14).Let-7b comprises sequence UUGGUGUGUUGGAUGAUGGAGU (SEQ ID NO:15).Let-7c comprises sequence UUGGUAUGUUGGAUGAUGGAGU (SEQ ID NO:16).Let-7d comprises sequence UGAUACGUUGGAUGAUGGAGA (SEQ ID NO:17).Let-7e comprises sequence UAUAUGUUGGAGGAUGGAGU (SEQ ID NO:18).Let-7f comprises sequence UUGAUAUGUUAGAUGAUGGAGU (SEQ ID NO:19).Let-7g comprises sequence GACAUGUUUGAUGAUGGAGU (SEQ ID NO:20).Let-7i comprises sequence UGUCGUGUUUGUUGAUGGAGU (SEQ ID NO:21).

Other let-7 family member's sequence, the public can login miRBase (www.mirbase.org).

Methods for the treatment of

The identification of KRAS sudden change sudden change shows that the danger of three negative breast cancer development increases.In the context of the present invention, " risk " relates to the probability that an event can occur during a specified time, can mean " definitely " risk or " relatively " risk of main body.Absolute risk can contrast to be measured queue correlation time after actual observation and measures, or the index value of the effective historical queue of contrast statistics measures, and the relevant time period is followed in historical queue.The absolute risk of comparing low risk queue or average crowd's risk, relative risk refers to the ratio of main body absolute risk, it can be assessed and be changed by clinical risk factors.Odds ratio, the ratio for the Positive events of a given test result with respect to negative events, also need not change (ratio is general formula P/ (1-P), and wherein p is probability of occurrence, is (1-p) according to the probability that there is no event) conventionally.

" risk assessment ", or " risk assessment " comprises the possibility that probabilistic forecasting, probability or event or morbid state may occur in the context of the present invention, this events incidence or the conversion from a morbid state to another state, for example, transformation from from primary tumo(u)r to metastatic tumo(u)r or develop into metastatic risk, or the risk changing from former transitivity event to secondary transferring sexual behavior part, or from developing the primary tumo(u)r of a type to the risk of the dissimilar one or more primary tumo(u)rs of development.Risk assessment can also comprise the clinical parameter of predict future, traditional experiment chamber Hazard Factor value, or the index of other cancer, the absolute or relative terms relevant with previous tested crowd.

Than the individuality that does not carry KRAS sudden change, " risk increases " is used for describing and carries the individual development of KRAS sudden change or the probability increase of developing cancer.In certain embodiments, may to develop or suffer from cancer be not carry individual 1.5X, 2X, 2.5X, 3X, 3.5X, 4X, 4.5X, 5X, 5.5X, 6X, 6.5X, 7X, 7.5X, 8X, 8.5X, 9X, 9.5X, 10X, 20X, 30X, 40X, 50X, 60X, 70X, 80X, 90X or the 100X of KRAS sudden change to KRAS mutation carriers.

Predict and poorly (for example refer to development aggressiveness, excessive risk, the cancer serious or form of succession, three negative breast cancer) individual survival probability, or the probability of aggressiveness, excessive risk, the cancer development serious or form of succession or the individual survival of deterioration, is less than benign form cancer development or the individual probability of surviving of deterioration more.

Predict to be poorly also used for describing that a not too gratifying recovery, decubation are long, have more invasive or high risk treatment plan, or the recurrence of cancer or the probability of transfer increase.For instance, three negative breast cancer or its transfer are the poorest closely related with the prediction of mammary cancer hypotype, cause main body prediction poor.

Term main body, patient, individuality are used alternatingly in specification sheets.Main body preferred mammal.Mammals can be people, non-human primate, mouse, rat, dog, cat, horse or ox, but is not limited to these embodiment.Main body is the male sex or women.Main body may previously be diagnosed as and suffer from cancer, the cancer of particular type (for example, mammary cancer), or a hypotype of cancer three negative breast cancer of a hypotype of mammary cancer (for example, as).Main body shows one or more risk of cancer factors, the cancer of a particular type (for example, mammary cancer), or a hypotype of cancer three negative breast cancer of a hypotype of mammary cancer (for example, as).Alternatively, main body does not show Hazard Factor of cancer, a kind of cancer of particular type (for example, mammary cancer), or a kind of cancer hypotype three negative breast cancer of mammary cancer hypotype (for example, as).

Mammary cancer, comprises three negative breast cancer, and Hazard Factor include, but not limited to exist KRAS transgenation; Women, aging, obesity, shortage fertility or lactation, hormonal readiness are higher, smoking, be exposed to radiation, individual mammary cancer medical history, family history of breast cancer, particularly the variation of breast (for example, the variation of those and fibrous capsule disease-related, comprise, but be not limited to atypical hyperplasia and lobular carcinoma in situ).Exemplary sfgd. for three negative breast cancer development comprises; but be not limited to; often take exercise; avoid ambient induced factor (as smoking, drink, high-fat diet is causeed fat, occupational radioactive exposure); select breast-feeding child; for those the most serious risks, preventative bilateral mastectomy.May there are one or more Hazard Factor in main body of the present invention, risk factors may further be eased or change through sfgd..

Method as herein described obtains sample with it from main body.Sample can be any tissue or liquid, wherein comprises nucleic acid.Multiple embodiments comprises, but be not limited to, paraffin embeds tissue, frozen tissue, fine needle for operation paracentesis, and mammary cell (comprising the cell that conduit, leaflet or reticular tissue obtain), lymphoglandula (comprising outpost or axillary gland), chest or abdominal muscles or reticular tissue, organ (any latent lesion position that comprises potential metastatic cell, as brain, liver, kidney, stomach, intestines, marrow, pancreas, colon or lung).Other embodiment comprises fluid sample, as blood, blood plasma, serum, lymph liquid, ascites, slurries and urine.

SNP classifying method

KRAS transgenation is the single nucleotide polymorphism (SNP) of a kind of mankind of occurring in KRAS gene 3 ' UTR.Linkage disequilibrium (LD) refers at two or more different SNP site allelotrope inherits, and its frequency is greater than each allelotrope of appointment crowd and occurs separately frequency (for example, substituting Nucleotide).First allelic frequency of the expected frequence of two allelotrope symbiosis of independent inheritance is multiplied by second allelic frequency.Under expected frequence, the allelotrope of symbiosis is considered to " linkage equilibrium ".In contrast, linkage disequilibrium refers to any nonrandom genetic association between the allelotrope in two or more different SNP sites, and this is normally due to the physical proximity in two sites along item chromosome.During linkage disequilibrium, two or more single nucleotide polymorphism (SNP) website may occur, and on given karyomit(e), intimate physical is approaching each other, therefore, it is not separated that allelotrope in these SNP sites tends to many generations, and the specific Nucleotide (allelotrope) of a SNP site shows nonrandom associated near the specific nucleotide (allelotrope) of different SNP site.Therefore the information that, SNP Genotyping can provide with in linkage disequilibrium, another SNP Genotyping is identical.

For example, for screening the object of the individuality (prediction or risk) of heredopathia, if it is useful that a specific SNP site is found to screen disease, those skilled in the art will recognize that other SNP site of this SNP site linkage disequilibrium is also by Effective selection illness.Between two or more single nucleotide polymorphism (SNP), may run into linkage disequilibrium in various degree, consequently, some single nucleotide polymorphism (SNP) for example, than other more closely related (, stronger linkage disequilibrium).In addition, along the physical distance of chromosomal linkage disequilibrium, between genomic different zones, be different, the physically-isolated degree therefore occurring between the required two or more SNP site of LD is also variant between genome different zones.

Screening application, polymorphism is also useful (for example, SNPs and/or haplotype), is not actual (causing a disease) polymorphism of causing a disease, but has the linkage disequilibrium of this Disease-causing gene polymorphism.In this case, prediction has the genotype of the polymorphism in the linkage disequilibrium of Disease-causing gene polymorphism, and prediction is subject to the polymorphic sex genotype of Disease-causing gene (as, disease).Therefore, in Disease-causing gene polymorphism linkage disequilibrium, polymorphic mark is effective mark, when actual Disease-causing gene polymorphism is when being unknown, is particularly useful.

Human genome linkage disequilibrium is at Wall et al., " Haplotype blocks and linkage disequilibrium in the human genome ", Nat Rev Genet.2003August; 4 (8): 587-97; Gamer et al., " On selecting markers for association studies:patterns of linkage disequilibrium between two and three diallelic loci ", Genet Epidemiol.2003January; 24 (1): 57-67; Ardlie et al., " Patterns of linkage disequilibrium in the human genome ", Nat Rev Genet.2002April; 3 (4): 299-309 (erratum in Nat Rev Genet2002July; 3 (7): 566); With Remm et al., " High-density genotyping and linkage disequilibrium in the human genome using chromosome22as a model "; Curr Opin Chem Biol.2002February; 6 (1): in 24-30., comment on.

Triage techniques of the present invention, can adopt various method, to determine whether test subject has single nucleotide polymorphism (SNP), or reduce related SNP pattern with risk increase or the risk of the detectable feature of development, determining whether that main body suffers from detectable feature, can detected characteristics be the result of specific polymorphism/sudden change, comprises, for instance, analyze the method for haplotype, family's research, monosperm DNA analysis or the somatic cell hybrid of individual chromosome.The feature of using disclosure diagnostic method to analyze may be any feature being detected that pathology and disease are observed conventionally.

Determine that specific nucleotide (being allelotrope) is present in each of one or more SNP position, as at SEQ ID NO:11, SNP position in disclosed nucleic acid molecule, is called as SNP gene type in 12,13 or 22.The method of SNP gene type provided by the invention, for example, for various diseases screening, or susceptibility is definite, or determines a kind of response of form of therapy, prediction, or genomic mapping or SNP association analysis etc.

By means commonly known in the art, nucleic acid samples can carry out gene type, for example, to determine that allelotrope appears at any given gene region (, SNP position).Adjacent sequence can be used for designing the detection reagent of SNP, oligonucleotide probe for example, and it can optionally be implemented with kit form.Exemplary SNP methods of genotyping is at Chen et al., and " Single nucleotide polymorphism genotyping:biochemistry, protocol, cost and throughput ", Pharmacogenomics is J.2003; 3 (2): 77-96; Kwok et al., " Detection of single nucleotide polymorphisms ", Curr Issues Mol.Biol.2003April; 5 (2): 43-60; Shi, " Technologies for individual genotyping:detection of genetic polymorphisms in drug targets and disease genes ", Am J Pharmacogenomics.2002; 2 (3): 197-205; And Kwok, " Methods for genotyping single nucleotide polymorphisms ", Annu Rev Genomics Hum Genet2001; In 2:235-58, be described.High-throughput SNP classifying method exemplary technique is at Marnellos, " High-throughput SNP analysis for genetic association studies ", Curr Opin Drug Discov Devel.2003May; 6 (3): in 317-21, be described.Common SNP methods of genotyping comprises, but be not limited to, TaqMan analyzes, molecular beacons detection, nucleic acid array, allele-specific primers extension method, allele-specific round pcr, array primer extension, uniform primer extension analysis, the primer extension detecting by mass spectroscopy, tetra-sodium order-checking, the genetic analysis of the multiple sequence of primer extension, rolling circle amplification ligation, evenly ligation, OLA (U.S. Patent number: 4, 988, 167), the genetic analysis of multiple ligation sequence, restriction fragment length polymorphism, single-basic extension marker detection and intrusion detection technique.This method can be combined use with testing mechanism, for example, for instance, the testing mechanism of luminous or chemiluminescence detector, fluoroscopic examination, time resolved fluorescence detection, FRET (fluorescence resonance energy transfer), fluorescence polarization, mass spectroscopy and electro-detection.

For detection of the several different methods of gene pleiomorphism, include, but not limited to cutting reagent guard method and be used for detecting base mismatch in RNA/RNA or RNA/DNA two strands (Myers et al., Science230:1242 (1985); Cotton et al., PNAS85:4397 (1988); And Saleeba et al., Meth.Enzymol.217:286-295 (1992)), the electrophoretic mobility comparison of saltant type and wild-type nucleic acid molecule (Orita et al., PNAS86:2766 (1989); Cotton et al., Mutat.Res.285:125-144 (1993); With Hayashi et al., Genet.Anal.Tech.Appl.9:73-79 (1992)), motion (Myers et al., Nature313:495 (1985)) with polymorphic or wild-type fragment in the polyacrylamide gel that adopts denaturing gradient gel electrophoresis (DGGE) check to contain denaturing agent gradient.The series jump of specific position also can be assessed by ribonuclease protection assay, as rnase and SI protection or chemical cracking method.

In a preferred embodiment, use TaqMan method to carry out SNP gene type, this is also referred to as 5 ' nuclease assay method (U.S. Patent number 5,210,015 and 5,538,848).TaqMan method detects the accumulation of specific amplified production during PCR.TaqMan method is utilized the oligonucleotide probe of fluorescence report group and quenching group mark.Reporter group is excited by suitable wavelength illumination, by FRET (fluorescence resonance energy transfer) (FRET) method, shifts energy to the quenching group in same probe.When being connected to probe, the reporter group exciting does not transmit.Quenching group approaches the fluorescence that the reporter group in complete probe keeps reporter group to reduce.Reporter group and quenching group may be respectively 5 ' and 3 ' least significant end, or vice versa.Alternative, reporter group may be 5 ' or 3 ' least significant end, and quenching group is attached on an inner nucleotide, or vice versa.In another embodiment, reporter group and quenching group can be connected to each other on the inner nucleotide of a segment distance, and the fluorescence of reporter group is reduced.

In PCR process, the active cracking probe of 5 ' nucleic acid of archaeal dna polymerase, thus reporter group is separated with quenching group, cause thus the fluorescence of reporter group to increase.The accumulation of the increase direct-detection pcr amplification product of the fluorescence by Surveillance group.If when probe hybridization to increase in PCR process containing in the template of target single nucleotide polymorphism (SNP), probe between archaeal dna polymerase cracking reporter group and quenching group, only have when specific SNP allelotrope existence, probe design is used for hybridizing to target SNP site.

Use SNP and relevant nucleic acid sequence information provided in this article can determine at an easy rate preferred TaqMan primer and probe sequence.Some computer programs, as Primer Express (Applied biosystems, Foster city, California), can be used for obtaining rapidly best primer/probe groups.For those skilled in the art, be apparent, be effectively for detection of the primer of single nucleotide polymorphism of the present invention (SNP) and probe in the predicted detection of various diseases that comprises cancer, can easily join in test kit.The present invention also comprises the modification of the Taqman method that this area is known, as used molecular beacon probe (U.S. Patent number 5,118,801 and 5,312,728), and other mutant form (U.S. Patent number 5,866,336 and 6,117,635).

Use unmatched detection technique, also can determine the identification of polymorphism, include, but are not limited to use ribose probe (Winter et al., Proc.Natl.Acad Sci.USA82:7575,1985; Meyers et al., Science230:1242,1985) and the protein of identification Nucleotide mispairing, as the ribonuclease protecting method of intestinal bacteria mutS albumen (Modrich, P.Ann.Rev.Genet.25:229-253,1991).Alternatively, single-strand conformation polymorphism analysis (SSCP) (Orita et al., Genomics5:874-879,1989; Humphries et al., in Molecular Diagnosis of Genetic Diseases, R.Elles, ed., pp.321-340,1996), or denaturing gradient gel electrophoresis (DGGE) (Wartell et al., Nuci.Acids Res.18:2699-2706,1990; Sheffield et al., Proc.Natl.Acad.Sci.USA86:232-236,1989), can identify mutation allele.

Polymerase-mediated primer extension method also can be used for identifying polymorphism.In patent and scientific literature, describe several such methods, comprised method (WO92/15712) and the ligase enzyme/polymerase-mediated heredity position analysis (U.S. Patent number 5,679,524) of " hereditary position is analyzed ".Methods involving is disclosed in WO9I/02087, WO90/09455, WO95/17676, U.S. Patent number 5,302,509 and 5,945,283.By mass spectroscopy, the expansion primer that contains polymorphism can be detected, as U.S. Patent number 5,605, described in 798.Another kind of primer extension method is allele-specific PCR (Ruano et al., Nucl.Acids Res.17:8392,1989; Ruano et al., Nucl.Acids Res.19,6877-6882,1991; WO93/22456; Turki et al., J Clin.Invest.95:1635-1641,1995).In addition, as described in people such as Wallace (WO89/10414), is used allele-specific primers collection, simultaneously a plurality of polymorphic sites of a plurality of regional studies of amplification of nucleic acid.

The another kind of preferred method of KRAS transgenation gene type be in OLA, use two oligonucleotide probes (referring to, for example, U.S. Patent number 4,988,617).In this method, a probe hybridization has the segment of the target nucleic acid in SNP site to its 3 ' least significant end.Second probe hybridization to the adjacent segment of target nucleic acids molecule directly to 3 ' hold the first probe.Two juxtaposition probe hybridizations are to target nucleic acids molecule, if having between first probe 3 ' least significant end Nucleotide in SNP site perfect complementaryly, under linking agent exists as ligase enzyme, link together.If any not mating, connection will can not occur.After reaction, this linking probe and target nucleic acids molecular separation, the indicator that linking probe exists as SNP is detected.

Following patent, patent application, the international patent application of having announced, by citation, be all incorporated in herein, provide the extraneous information of the technology of carrying out all kinds OLA: U.S. Patent number 6,027,889,6,268,148,5494810,5830711 and 6054564 have described the OLA strategy of carrying out SNP detection; WO97/31256 and WO00/56927 have described the OLA strategy that uses general array to carry out SNP detection, and within wherein zipcode sequence can be introduced in hybridization probe, products therefrom, or amplified production, hybridize to common zip code array; U.S. Patent application US01/17329 (sequence number 09/584,905) has described OLA (or LDR), then carries out PCR reaction, and wherein zipcode joins in OLA probe, and electrophoresis or general zipcode array are read definite pcr amplification product; U. S. application 60/427,818,60/445,636 and 60/445,494 describe SNPlex method and use OLA, the multiple sample multidigit point SNP of round pcr detects software subsequently, wherein zipcodes includes in OLA probe, and the PCR product of amplification and the hybridization of zipchute reagent, read definite single nucleotide polymorphism (SNP) from the electrophoresis of zipchute.In some embodiments, PCR (or another kind of method of nucleic acid amplification) carries out OLA before.In other embodiments, before OLA, carry out PCR (or another kind of method of nucleic acid amplification).

The another kind of method of SNP gene type is based on mass spectrum.Mass spectroscopy is utilized in 4 Nucleotide of DNA the unique qualities of each.Single nucleotide polymorphism (SNP) can have the difference of quality of the allelic nucleic acid of alternative single nucleotide polymorphism (SNP) and clear and definite genotype by mass spectroscopy measurement.MALDI-TOF (substance assistant laser desorpted ionized-flight time) mass-spectrometric technique is preferred Accurate Measurement molecular mass very, as single nucleotide polymorphism (SNP).Developed based on mass spectrographic multiple snp analysis method.The preferred method based on mass spectrographic SNP gene type, comprises primer extension analysis, also can combine additive method and use, as traditional gel form and gel chip.

Generally, primer extension analysis relates to design and primer annealing to the sub-upstream of template pcr amplification (5 ') of target SNP position.Dideoxy nucleotide triphosphoric acid (ddNTPs) and/or deoxynucleoside triphosphate (dNTPs) are mixed to join and (for example contain template, the nucleic acid molecule that contains SNP, conventionally be amplified, for example, pass through PCR), in the reaction mixture of primer and archaeal dna polymerase.Primer extension terminates in first location in this template, one of ddNTPs in the complementary mixing of Nucleotide herein.Primer can be directly for example, in abutting connection with (, the Nucleotide of primer 3 ' end hybridizes to the Nucleotide that adjoins target spot SNP position), or two or more Nucleotide of removing for SNP position.If primer is several Nucleotide of removing from target spot SNP position, unique restriction is, template sequence between primer 3 ' end and single nucleotide polymorphism (SNP) position can not comprise the Nucleotide of same type, as the Nucleotide being detected, or this can cause extending the early stopping of crossing of primer.Alternatively, if all four ddNTPs do not have dNTPs, join separately in reaction mixture, primer will only extend a Nucleotide, corresponding to target spot SNP position.In this case, primer is for example designed to, in conjunction with the upstream of a Nucleotide of SNP position (, the Nucleotide of primer 3 ' end hybridizes to target spot SNP site 5 ' side Nucleotide, this Nucleotide next-door neighbour target spot SNP position).It is preferred extending a Nucleotide, because it reduces the total mass of extending primer to greatest extent, thereby has improved the mass discrepancy resolving power between alternative SNP Nucleotide.In addition, the ddNTPs of quality status stamp is applied in the ddNTPs that substitutes unmodified in primer extension reaction.This can increase these ddNTPs and extend of poor quality between primers, thereby higher sensitivity and accuracy is provided, and keys in heterozygosis base position and is particularly useful.Quality status stamp is also alleviated the needs of intensive sample preparation program, reduces the resolving power of mass spectrograph necessity.

The primer extending, then carries out purifying, and by MALDI-TOF analytical reagent composition, to determine the identity of target spot SNP position Nucleotide.In a kind of analytical procedure, the product of primer extension reaction, in conjunction with photoabsorption crystal, forms matrix.Then, energy source laser bombardment matrix, makes nucleic acid molecule ionization desorb become gas phase.Then, ionized molecule spirt tof tube, and accelerate under pipe towards detector.The time of ionization (as laser pulse) and molecule and detector collision is the flight time of this molecule.Flight time is accurately relevant to the mass-to-charge ratio (m/z) of ionized molecule.Compared with the little m/z ion speed lower along pipe than m/z larger soon, therefore, compared with light ion, before compared with heavy ion, arrive detector.Then, the flight time convert to accordingly, high precision m/z.In this manner, can be based on quality fine difference and corresponding flight time difference identification form nucleotide polymorphisms (SNP), nucleic acid molecule has different IPs thuja acid in single base position.Wish is understood primer extension assay and in conjunction with MALDI-TOF mass spectrograph, is used for the more information of SNP gene type, refer to, for example, Wise et al., A standard protocol for single nucleotide primer extension in the human genome using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry ", Rapid Commun Mass Spectrom.2003; 17 (11): 1195-202.

Following reference further provides the speech of describing based on mass spectrographic SNP methods of genotyping breath: Bocker, ' ' SNP and mutation discovery using base-specific cleavage and MALDI-TOF mass spectrometry ", Bioinformatics.2003July; 19Suppl1:144-153; Storm et al., " MALDI-TOF mass spectrometry-based SNP genotyping ", Methods Mol.Biol.2003; 212:241-62; Jurinke et al., " The use of MassARRAY technology for high throughput genotyping ", Adv Biochem Eng Biotechnol.2002; 77:57-74; With Jurinke et al., " Automated genotyping using the DNA MassArray technology ", Methods Mol.Biol.2002; 187:179-92..

Single nucleotide polymorphism (SNP) also can be evaluated by DNA direct Sequencing.Various automatic sequencing programs can be utilized ((1995) Biotechniques19:448), comprise by mass spectrograph check order (referring to, for example, PCT International Publication WO94/16101; Cohen et al., Adv.Chromatogr.36:127-162 (1996); With Griffin et al., Appl.Biochem.Biotechnol.38:147-159 (1993)).Nucleotide sequence of the present invention, makes those skilled in the art be easily such automatic sequencing programdesign sequencing primer.Commercial instrument ，Ru Applied biosystems 377,3100,3700,3730 and 3730.times.1DNA analyser (Foster City, California) are that this area automatic sequencing is normally used.

The additive method that can be used for KRAS transgenation gene type, comprises single strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE) (Myers et al., Nature313:495 (1985)).SSCP change to determine base difference by strand PCR product electrophoretic migration, as Orita et al., described in Proc.Nat.Acad.Strand PCR product can be by adding thermogenesis, or otherwise make double-stranded PCR product sex change produce.Collapsible or the forming section of single-chain nucleic acid depends on the secondary structure of base sequence.The different electrophoretic mobilities of single-stranded amplification product are relevant with the base sequence difference of SNP position.Sequence based on different relies on stability and intrinsic meltbility and the denaturing gradient gel electrophoresis migration model respective differences opposite sex of polymorphic dna, DGGE distinguishes the allelotrope (Erlich of single nucleotide polymorphism (SNP), ed., PCR Technology, Principles and Applications for DNA Amplification, W.H.Freeman and Co, New York, 1992, Chapter7).

Development based on ribozyme cleavage site or loss, sequence-specific ribozyme (U.S. Patent number 5,498,531) also can be for marking to single nucleotide polymorphism (SNP).By nuclease cracking digestion detection or by the difference of melt temperature, preferred matching sequence can distinguish over not matching sequence.If SNP affects restriction enzyme cleavage site, can change by restriction endonuclease map, and determine SNP by the respective change of gel electrophoresis definite kernel acid fragment length.

SNP gene type can comprise the following steps, for instance, from human subject's collection of biological sample (for example, tissue samples, cell, body fluid, secretory product, Deng), from sample cellular segregation, (for example go out nucleic acid, genomic dna, mRNA or both have), one or more primer contact nucleic acid, specific hybrid is to the region of the nucleic acid being separated that contains target spot SNP, there is target nucleic acid area hybridization and amplification, determine SNP position Nucleotide, or, in some test, detect the existence of amplified production or do not have (design detection, if specific SNP allelotrope exists or does not exist, to only there is hybridization and/or amplification).In some is analyzed, amplified production size detected, the length of contrast check sample; For instance, contrast normal genotype, the variation of amplified production size can detect deletion and insertion.

Embodiment

1: three negative breast cancer (TNBC) KRAS of embodiment transgenation

Study population

In this case control study and genetic analysis, assess four queuing datas (table 1).In order to assess the individuality of KRAS transgenation genotype frequency distribution ，Dui Yale University's breast cancer research (study group), assess.Yale University's breast cancer research (study group 1) individuality is participated in the mammary cancer case comparative study at Connecticut, USA, obtains institutional review board approval (Ho man A, the et al.Cancer Res2009 of Yale University; 69:5970-77).In brief, patient age in 30-80 year and easily occur, the mammary cancer of proved by pathology do not have cancer history (except nonmelanoma skin cancer).All cases have been set up ER and PR state, but HER2 Status unknown and can not obtaining.Contrast main body or from Yale-New Haven hospital (New Haven, CT, USA) or U.S. Tolland county, CT, USA collection.Yale-New Haven hospital contrast main body has been accepted mammary cancer related surgical, proved by pathology benign breast disease.Tolland county contrast main body can be by random digit dialing (individual age <65 year) or definite by health care financial management file (>=65 years old).From all participants, obtain the data of informed consent and cancer family history, childbearing history, demographic factor and blood sample.Between nineteen ninety to 1999 year, 415 patients and 457 DNA samples that contrast main body obtains from this research.

Table 1 study group

TNBC=tri-negative breast cancer, ER=estrogen receptor, and PR=progesterone receptor

In order to determine the cognation of KRAS transgenation and receptor status and mammary cancer hypotype, the diagnosis with complete receptor status and Subtypes suffers from 690 Irishwoman's queues of mammary cancer and assesses.From patient (study group 2) histology of this queue, make a definite diagnosis and suffer from after the ethics approval of Ethics Committee of mammary cancer ，Jing Galway university hospital (Galway, Ireland), from Ireland, western recruitment comes.From all cases, obtain detailed family history, the blood sample of informed consent and mammary cancer or ovarian cancer.The mammary cancer in 710 routine all stages and histological type, got rid of preinvasive carcinoma.Use normal structure pathological analysis and immune group chemistry, set up ER, PR and the HER2 state of all samples, HER2 is positive, and fluorescence in situ hybridization confirms.These sample classifications are luminal A, luminal B, HER2 or three negative breast cancer receptor status (table 2).In 710 patients, 690 patients have complete information, and assess in this research.In this queue 360 contrast main body is the healthy women from areal, is mainly the age to surpass 60 years old, without any the self-report personal history of cancer, without the family history of mammary cancer or ovarian cancer.Case group and control group are mainly recruited from year July in July, 2006 to 2010.

Table 2. subtype acceptor state

To determine whether that the risk of KRAS mutation forecasting three negative breast cancer development increases, three Breast Cancer Patients with Negative Axillary queues and from the contrast main body (study group 3) of Yale University, three Breast Cancer Patients with Negative Axillaries and from the contrast main body of study group 2, with the contrast main body of study group 1, carry out Macro or mass analysis.The patient of study group 3 receives treatment in Yale-New Haven hospital or Bridgeport hospital (Bridgeport, Connecticut, the U.S.).After the approval of Yale's manpower survey council, tissue or saliva sample are taken from 156 routine patients.The 140 routine patients that diagnosed at 1990-2007 provide complete data, and these data are included in this research.Before any treatment of gene and miRNA expression analysis, 130 example three negative breast cancer have available tumor sample, and 78 people have also carried out KRAS mutator gene phenotypic analysis.113 contrast main bodys of this queue are healthy womens, there is no cancer case history, and Yale-New Haven hospital provides, and gets rid of nonmelanoma skin cancer, between 2000 to 2007, is recruited.From all cases and contrast main body, clinical data, age, race, family history have been obtained.Table 3 is summed up the essential information of these three queues.

Three independently explanations of mammary cancer case-contrast queue that utilize in this research of table 3..

In order to assess the dependency of BRCA transgenation in the negative tumour of KRAS transgenation and ER, the known KRAS transgenation state with the BRCA1 mutation carriers of mammary cancer and the Rotterdam crowd of our former research is analyzed.Rotterdam crowd is described to (Hollestelle A, et al.Breast Cancer Res Treat2010; Published online July30.DOI:10.1007/s10549-010-1080-z), but, briefly, this crowd comprises the Dutch patient of the BRCA1 transgenation of suffering from mammary cancer and being recorded, and Erasmus University determines by Rotterdam's policlinic (Rotterdam, NED) investigation.

Step

KRAS transgenation gene type assay: use custom TaqMan SNP genotype tests, the DNA of all samples carries out KRAS transgenation gene type.Sudden change G allelotrope heterozygosis or the sample isozygotying are considered to KRAS positive gene mutation (Chin L, et al.Cancer Res2008; 68:8535-40).

Gene expression analysis: the 78 routine patients from Yale University's three negative queues measure full genome mRNA genetic expression, to KRAS, sudden change is tested.Use the total RNA of RecoverAll total separate nucleic acid test kit (Applied biosystems) to extract self-organization sample, hybridize to full genome DASL and analyze (HumanRef-8 version 3 .0， Illumina company, San Diego, California, the U.S.).Use Bioconductor/R software lumi software package to carry out data pre-treatment and statistical study.Merge and process together three full genome DASL and analyze the gene expression data that operation obtains.There is the sample of the detection probes that is less than 30% and be less than the probe detecting in 10% sample, before quantile-normalization, being dropped.Remaining 74 samples and 18345 probes after filtering.

MicroRNA analyzes: use Multiplex RT and TaqMan low density array mankind's miRNA culture plate real-time PCR systems (Applied biosystems), according to the agreement of manufacturers, (miRNA express spectra, the public can login to carry out microRNA analysis

Www.appliedbiosystems.com/absite/us/en/home/applications-technol ogies/real-time-pcr/mirna-profiling.html (addressable after 1 day January in 2008).MiRNAs expression level checks.

Statistical study

The genotype of all cases and control group distributes and is used to test Hardy-Weinberg balance, and is considered in equilibrium state.Non-condition logistic returns the relative risk that is used to estimation and each genotypic correlation.Adjust contrast main body age (continuously) and mational origin (white man, Black people, Hispanic or other race).Study population is divided into menopausal states (by age≤51 year old or assess in >51 year), through Multivariate logistic regression analysis, independent risk estimation ER and PR state, three horizontal outcome variables are encoded to, 0 is control group, 1 for suffering from the case of the tumour of the ER positive and/or the PR positive, and 2 is the case of the negative tumour of ER/PR.Use Wald χ ²verification test interaction, with respect to ER/PR disease-negative, compares each genotypic parameter estirmation in the ER positive and/or PR positive diseases case.

According to mammary cancer hypotype, 2 patients of study group carry out layering, use GraphPad Prism4 software to carry out χ ²check, calculates p value, odds ratio (Ors) and 95% credibility interval (CI).Due to low frequency KRAS transgenation, dominant models is used to all genetic association analyses.

Between study group, χ ²check or two-sided Fisher rigorous examination match stop variable (for example, mational origin, stage and research website), t check is continuous variable (as the age) relatively.Use Unconditional logistic regression model, and binary result variable, odds ratio (Ors) and 95% credibility interval (95%CI) of control group and three negative breast cancer case group KRAS sudden change calculated.Binary result variable is encoded to control group and case group, has the variable that the logistic Regression Analysis of this binary result variable comprises, as KRAS transgenation state, age, race and research website.The age-based component layers of crowd, patient age carries out independent logistic regression analysis in 51 years old or young (group menopause before) or age over 51 years old (menopause group).With SAS version, 9.1.3 carries out statistical study.

Measure Channel activates, and corresponding to the expression label of previously having announced and expression, collects principle component analysis as axle.PCA is for separating of biotechnology information source in gene expression data collection.The corresponding RNA quality of each composition, the biology of the structure of batch processing effect and contribution probe is explained (be express spectra probe, express spectra has the absolute high projection value of specified ingredients).Gene expression label is provided as list of genes, and at specified conditions, it is expressed and changes.These labels are valuable especially noise datas, because they need a plurality of probes to coordinate differential expression, conventionally in 100 orders.Because mRNA extracts and fixes from formalin, paraffin embedding (FFPE) piece, reaches 20 years, with stamp methods analytical data collection, is valid (Kibriya M, et al.BMC Genomics2010; 11:622).Pearson's correlation calculations S label mark between these gene expression profiles of each sample of label vector sum of gene contribution.Pairing Kolmogorov-Smirnov check is used for analyzing the dependency of KRAS transgenation and the middle KRAS transgenation of wild-type sample and the result that label is described separately.Adopt linear model to assess gene expression difference, consider the skew of batch processing simultaneously.The R of employing LIMMA software package carries out model-fitting and the Empirical Bayes error of multiple variation and adjusts (Smyth GK.Limma:linear models for microarray data.In:Gentleman R, et al, eds.Bioinformatics and computational biology solutions using R and bioconductor.New York, USA:Springer, 2005:397-420).

8 batches of 46 miRNA and 2 internal references are analyzed miRNA and are expressed.Use geometrical mean, all expression sample miRNA are expressed to stdn: lower than 35 all after dates (CT<35), if baseline fluorescent value detected, differentiate the miRNA being expressed, deduct the geometric mean cycle life of the miRNA of all expression.MiRNA more than 2/3rds is not expressed all samples, removes miRNA, all remaining threshold cycle (C _t) value normalization method.

The genotypic frequency distribution of KRAS transgenation does not have significant difference between case and control group, and study group's 1 control group carries out gene type (table 1 and table 4).Yet, KRAS sudden change and the mammary cancer significant correlation (table 4) with the front patient of menopause of the negative tumour of ER/PR.Postclimacteric women does not observe this dependency.In contrast to 27 (13%) and 44 in 201 contrast main bodys and suffer from ER and/or PR and be positive 4 (9%) in the pre-menopausal women of cancer, suffer from 24 pre-menopausal womens of the negative cancer of ER/PR 8 and there is KRAS and suddenly change (33%) (Fig. 5).Therefore, KRAS transgenation may be a kind of genetic marker for the risk increase of pre-menopausal women development receptor negative mammary cancer.

The dependency of table 4.KRAS transgenation and the ER/PR positive and ER/PR negative breast cancer.

In study group 2,478 women suffer from luminal A type mammary cancer, suffer from luminal Type B mammary cancer for 87, suffer from three negative breast cancer for 90, and 35 are suffered from HER2 positive breast cancer.In 690 mammary cancer cases of this queue, 98 (14%) carry KRAS transgenation, but between mammary cancer hypotype, morbidity changes: contrast 64 examples (13%) in 478 routine luminal A, 13 examples (15%) in 87 routine luminal B, with 2 examples (6%) in the positive subgroup of 35 routine HER2, KRAS Mutation Statistics significance is enriched in (19 examples [21%] in 90 examples) (P=0.044, Fig. 1) in three negative breast cancer women.Also notice that the age does not surpass the dependency (P=0.033, Fig. 1) of three negative breast cancer in 51 years old women.

Contrast three negative breast cancer cases (n=1160) of 2 groups, 3 groups of all three queues and control group, between case group, the morbidity of KRAS transgenation finds that there is statistical significant difference, or the morbidity of KRAS transgenation between control group finds that there is statistical significant difference (table 5). Study group 1 and 3 control group have more non-pink toes than study group 2, and multivariate analysis assessment KRAS transgenation and non-pink toes suffer from the dependency of three negative breast cancer.After controlling age, race, research website, KRAS transgenation does not predict that all women in multivariate analysis develop the danger of three negative breast cancer and increase (table 6 and table 7).Yet the danger increase that in the merging group of KRAS transgenation and multivariate analysis, 361 pre-menopausal womens develop three negative breast cancer has statistically significant dependency (table 6, table 8 and table 9).

Table 5. is used chi square test classified variable, for example, as race and t check continuous variable (, the age), three cloudy mammary cancer case groups (A) of Irish queue and Yale's queue and control group (B) demography variable.

A. three cloudy mammary cancer case groups

B. control group

930 collators of the 1-3 of Biao6. study group Macro or mass analysis contrast, the KRAS transgenation dependency of 230 three Breast Cancer Patients with Negative Axillaries.

Logistic regression model has been adjusted age, race, menopausal state and research website.G/G phenotype occurs in the case group and control group that is less than 5%, with the phenotype combination of G/T.

Table 7. adopts chi square test classified variable, and as race, t checks continuous variable (for example age), the demography variable of all age bracket three cloudy mammary cancer case groups and control group.

Demography

Table 8. adopts chi square test classified variable, and as race, t checks continuous variable (for example, the age), the demography variable of first three cloudy mammary cancer case group of menopause and control group.

Demography

The dependency of KRAS transgenation and three negative breast cancer in 51 years old case group of the Irish queue of table 9. and Yale's queue and control group.

Ireland queue *

* all control group single factor analysis

Yale's queue

Multiplicity, controls race and age

Because the sudden change of BRCA1 gene coded sequence is associated with suffering from three negative breast cancer risks, and because KRAS sudden change is enriched in (Hollestelle A, et al.Breast Cancer Res Treat2010 in mammary cancer BRCA1 mutation carriers; Published online July30.DOI:10.1007/s10549-010-1080-z), determine whether that first three heavy negative breast cancer of menopause and the KRAS dependency of suddenling change is only the dependency due to itself and BRCA1 mutation carriers.In 36 patients that suffer from three negative breast cancer of queue 2 and 3 BRCA genetic test, 25 (69%) be BRCA negative and 11 (31%) be the BRCA positive.In these patients, in contrast to 3 (27%) women of the BRCA positive, the negative women of 8 examples (32%) BRCA-has KRAS transgenation.These discoveries show, KRAS transgenation to without one group of BRCA sudden change independently three Breast Cancer Patients with Negative Axillaries be relevant.

Dependency (Hollestelle A, et al.Breast Cancer Res Treat2010 in discovery Rotterdam crowd queue between KRAS transgenation state and ER or PR negative status; Published online July30.DOI:10.1007/s10549-010-1080-z; Kibriya M, et al.BMC Genomics2010; 11:622), yet, menopausal state do not consider these research in.For result of study as herein described, compare 268 BRCA1-transgenation-carrier of Rotterdam's queue, before the menopause of 126 ER/PR negative breast cancer, in BRCA1-mutation carriers, do not observe KRAS sudden change enrichment (218%vs235%, p=0.95).Therefore, the dependency of the KRAS sudden change of first three heavy negative breast cancer of menopause is irrelevant with BRCA1 transgenation dependency.

For potential biotic interactions between the BRCA1 genetic expression change of further assessment KRAS sudden change and triple negative breast cancer, from definite BRCA1 gene expression dose (table 1) in the triple negative tumours of 74 example of study group 3.Compare with KRAS transgenation-feminine gender three negative tumours, KRAS transgenation patient confirm BRCA1 express statistically significance reduce (probe 1:p=0.06[ILMN_2311089] and probe 2:p=0.01[ILMN_1738027], Fig. 2).In addition, KRAS transgenation confirms have statistically significant dependency (P=0.04) (van ' t Veer LJ, et al.Nature2002 with the gene expression label of BRCA1 activity decreased; 415:530-36).Data provided herein are pointed out, although KRAS sudden change is not limited to three Breast Cancer Patients with Negative Axillaries with known BRCA1 sudden change, the biological interaction between KRAS transgenation, BRCA1 genetic expression or functional change and the development of three negative breast cancer may exist.

In the patient of study group 3, in three negative breast cancer tumours of KRAS positive gene mutation signal path and KRAS transgenation feminine gender three negative breast cancer tumours in signal path contrast.Although KRAS gene miRNA analyzes and not press KRAS transgenation change of state, data and other publications are consistent (Chin L, et al.Cancer Res2008 about miRNA in conjunction with the effect of KRAS gene 3 '-UTR; 68:8535-40; Johnson SM, et al.Cell2005; 120:635-47).In the tumour of carrying KRAS transgenation, find that NRAS sudden change increases (Croonquist PA, et al.Blood2003; 102:2581-92) increase (Creighton CJ, et al.Cancer Res2006 with MAP-kinase activation label; 66:3903-11) (table 10).Data show, KRAS sudden change has changed the genetic expression of typical RAS path.In addition, data provide the first individual interior research evidence, and KRAS transgenation causes downstream gene expression in tumour associated with it to continuously change.

The dependency of three Breast Cancer Patients with Negative Axillary tumour inner gateway labels of table 10.KRAS transgenation and positive KRAS transgenation.

Owing to carrying the concentration of let-7miRNA in the lung cancer tumour of KRAS sudden change, be changed, in the three negative breast cancer tumours of carrying KRAS sudden change, check let-7 concentration.Data acknowledgement all let-7miRNA family members (Fig. 3) of lower concentration in KRAS transgenation related neoplasms.

How integrated with the known expression label of three negative breast cancer in order to determine KRAS sudden change, the known label of differential expression in this type of tumour is assessed.KRAS transgenation tumour has several features of some three feminine genders and substrate template oncobiology, comprises that the main component inner estrogen signal that comes from expression group reduces (p=0.04).In addition, KRAS transgenation tumour has luminal type precursor cell label (P=0.04), is candidate's precursor cell (Lim E, the et al.Nat Med2009 of substrate sample mammary cancer; 15:907-13) (table 10 and Fig. 6).In luminal type precursor cell and BRCA transgenation class label, difference regulates the mark (for example α 5 integrins, DUSP6 and aurora kinase B) (table 11) of cell adhesion, tissue invasion and attack, propagation and vasculogenesis.This discovery is consistent with the slight enrichment of functional annotation, with KRAS transgenation and the contrast of nonmutationed data set, according to difference expression gene in linear model, in 3 genes in 41 genes of wound healing, observe (P=0.02), in 151 genes that glycan is expressed, in 3 genes, observe (P=0.05), in 148 genes that MEK activates, in 4 genes, observe (P=0.009) (Fig. 4, table 12 and table 13).

Table 11.LIMMA analyzes the interior KRAS sudden change difference expression gene of luminal type precursor cell label and the BRCA sudden change label of three Breast Cancer Patients with Negative Axillaries.

Table 12. is analyzed through LIMMA, and the label that is derived from document is enriched in three Breast Cancer Patients with Negative Axillaries, and these labels have the gene of the KRAS transgenation that is defined as differential expression.

Abbreviation: p.adj:FDR-adjusts p-value, number gene in label on MAXG:Illumina gene chip, diff.Exp: the number gene in this signature of expressing that finds differences.Positive 50 difference expression gene lists of three negative cancer patientss of KRAS sudden change of table 13.LIMMA Analysis deterrmination

embodiment 2:KRAS transgenation is in the morbidity of various tumor cell strains

Materials and methods

Gene type .NCI-60 cell strain culture plate DNA is from NCI ' s development treatment plan.As previously mentioned, carry out Taqman gene type and determine whether that KRAS mutation allele exists, (Bussey KJ, et al.Mol Cancer Ther2006; 5:853-67).Under standard conditions, culturing cell is (referring to dtp.cancer.gov/branches/btb/ivclsp.html; Monks A, et al.J Natl Cancer Inst1991; 83:757-66), stored frozen maximum 20 generations.Use Qiagen QIAamp DNA blood extraction agent box Program extraction DNA (cat.51192).

Statistical study .KRAS transgenation allelotrope data are carried out numerical coding, and 1 represents that KRAS transgenation allelotrope exists and 0 expression does not exist.This pattern is used as comparative analysis " seed " (Paull KD, et al.J Natl Cancer Inst1989; 81:1088-9248), detect the existing NCI-60 data set of NCI-DTP lane database.Dependency comprises, for instance, miRNA measures and DNA methylation measurement.Positive correlation shows, for instance, the cell strain that carries mutation allele often has the miRNA/mRNA of high expression level more or the DNA methylation of vast scale more.On the contrary, negative correlation shows that the cell strain that carries mutation allele often has the given miRNA/mRNA of low expression or the lower per-cent DNA methylation of appointment gene.These data sets can or be downloaded in dtp.cancer.gov inquiry.

The existence of KRAS transgenation is a genetic marker of forecasting risk and oncobiology and kinds cancer treatment response.The existence of KRAS sudden change causes KRAS gene 3 ' UTR controlling changing.The biological significance of KRAS transgenation in tumour cell has been illustrated in this research.Data interpretation provided herein the example molecule path that existed by KRAS transgenation to affect.For while analyst coverage cancer types widely, use comprehensive NCI-60 culture plate (Blower PE, the et al.Mol Cancer Ther2007 of tumor cell line; 6:1483-91; Liu H, et al.Mol Cancer Ther2010; 9:1080-91).Research different kinds of molecules parameter, to determine which molecular events relevant to existing of KRAS transgenation in these tumor cell lines (Kundu, S.T.et al.2012Jan15.Cell Cycle11:2,361-366).

7 in NCI-60 culture plate in 60 cell strains have KRAS mutation allele (table 14).Based on KRAS gene coding region dominant mutation, exist (KRAS transgenation) or KRAS transgenation to exist, when cell strain NCI-60 culture plate is classified, determined that the sudden change of KRAS gene activation exists, 7 cell strains that contain KRAS transgenation are negative.Equally, carry the cell strain shortage KRAS transgenation allelotrope of KRAS gene coded sequence sudden change.Therefore, the existence of KRAS genes encoding sudden change or KRAS mutation allele or be mutually to repel in these cell strains.In addition,, because this mutual exclusiveness occurs in the cell strain that comes from broad variety cancer, this mutual exclusion is not specific to a specific organization type.On the contrary, this mutual repulsion is the common feature of these JEG-3, no matter its source.These results show, (for example, KRAS sudden change occurs or the sudden change of KRAS genes encoding occurs) occurs separately one of these two events, are enough to affect the generation of the tumour of these cancer types.These results also show to have comparability in the KRAS expression rising function of KRAS transgenation existence induction in the caused KRAS activation levels of standard code series jump and 3 ' UTR.Patients with Non-small-cell Lung is also found this mutual exclusiveness (Chin LJ, the et al.Cancer Res2008 of the sudden change of KRAS genes encoding and KRAS sudden change; 68:8535-40) and in ovarian cancer patients (Ratner E, Cancer Res20l0; 70:6509-15), but be not to find (ZhangW, et al.Ann Oncol2011 in colorectal cancer patients; 22:484-5; Zhang W, et al.Ann Oncol2011; 22:104-9).

The strain of table 14.NCI-60 culture plate inner cell, has KRAS transgenation allelotrope or KRAS gene coded sequence function mutation.

Determine whether to carry the allelic cell strain of KRAS transgenation and show that miRNA guards change in expressing, miRNA express spectra contrast with the strain of NCI-60 culture plate remaining cell, the miRNA express spectra that 7 cell strains that comprise KRAS mutation allele generate carries out statistical study (Blower PE, et al.Mol Cancer Ther2007; 6:1483-91; Gaur A, et al.Cancer Res2007; 67:2456-68).The allelic existence of KRAS transgenation shows, increases and has statistically significant positive correlation (table 15) with miR-23, miR-27 and miR-210 expression.MiR-23 and miR-27 express malignant development (Zhou Q, the et al.Proc Natl Acad Sci USA20l1 of same cluster and vasculogenesis and transfer; 108:8287-92).For instance, miR-23 and miR-27 are enriched in vascular endothelial cell and height vascular tissue.In addition in, have the expression of Sprouty2 and the Sema6A of angiogenesis inhibitor function by minimizing, miR-23 and miR-27 promote the requisite signal path of vasculogenesis.The function of blocking-up miR-23 or miR-27, the kapillary causing formation and transfer reduce, retinal vessel minimizing in postpartum (Zhou Q, et al.Proc Natl Acad Sci USA2011 in response extracorporeal blood vessel endothelial cell growth factor (ECGF) and body; 108:8287-92).KRAS transgenation and miR-23, miR-27 express the statistically significant positive correlation increasing, and show, due to miR-23 and the rising of miR-27 level, to have the allelic tumour of KRAS transgenation and easily occur Growth of Cells and transfer.

The cell strain microRNAs that table 15. has KRAS mutation allele has significance,statistical expression increase.

In the expression of miR-210 and cell KRAS mutation allele to have significance,statistical associated.MiR-210 is the mark of chronic hypoxia.In addition, miR-210 is compared with difference correlation with the propagation of mammary cancer and melanoma tumour and transfer and prediction.MIR-210 be HIF albumen directly transcribe target.Under anoxia condition, the level of the living necessities miR-210 of tumour cell raises.The expression of MiR-210 direct regulation and control MNT, the cell cycle, needed MYC antagonist was stagnated under anoxia condition.Therefore, hypoxia in tumor cells stress condition under, the increase of miR-210 level promotes cell cycle arrest to cancel.Because it is relevant to KRAS sudden change existence that miR-210 expresses increase, the tumour cell that contains KRAS sudden change is survived and breeds under anoxia condition.

Data acknowledgement provided in this article, KRAS sudden change contributes to or starts abnormal signal path, and abnormal signal path is controlled the expression (comprise, for instance, miR-23, miR-27 and miR-210) of several miRNAs.Disturbing signal path, regulation and control miRNA expresses, for example miR-23, miR-27 and miR-210, cause tumor proliferation and transfer startup, develop, maintain or strengthen.

Promoter methylation is by a kind of mechanism of genetic expression silence in many cancers, because the methylation state of gene promoter changes, causes genetic expression to reduce.Particularly, DNA methylation is that CpG dinucleotides methylates and the epigenetic-effect that causes, conventionally at the promoter region of gene.The molecule of transcribing due to the obstruction mediated gene that methylates approaches promotor, and promoter methylation causes gene silencing.Different cancers shows different methylation patterns, and consequently gene expression profile changes.Therefore, determine whether to exist the change of DNA methylation pattern in the tumor cell line with KRAS transgenation, the methylation state of these cell strains and the contrast of non-KRAS transgenation cell strain (Ehrich M, et al.Proc Natl Acad Sci USA2008 in NCI-60 culture plate; 105:4844-9).The allelic existence of KRAS transgenation demonstrates and is permitted the positive correlation of polygenic promoter methylation increase significance,statistical, comprises, for instance, Notchl gene, cyclinD3 and CNBP (also referred to as ZNF9) (table 16).

The gene of significance,statistical promotor supermethylation in table 16.KRAS positive gene mutation cell strain.

The effect that in cancer, Notchl expresses is various.In many tumours, Notchl Overexpression or activation drive tumor development and transfer.For instance, Notchl activation causes breast cancer cell invasion and migrate attribute to increase.Or Notchl gene overexpression inducing mouse lung adenoma under MYC background, causes adenocarcinoma of lung to form.Therefore, evidence shows, Notchl can be used as a kind of oncogene that causes.In contrast, Notchl also can play the function of tumor suppressor gene.For instance, determined Notchl gene inhibition sudden change in head and neck squamous cell cancer.The exhaustion of mouse skin cutin Notchl causes chemical carcinogen or Ras to cause the tumour that oncogene causes to be increased.In the positive cervical cancer of human papillomavirus (HPV), compare with healthy tissues, Notchl expresses decline.In the positive cervical cancer of human papillomavirus (HPV) and the overexpression (Zage PE, et al.Pediatr Blood Cancer2011) of the activation Notchl gene in neuroblastoma cell, cause growth-inhibiting.Combine consideration, evidence demonstration Notchl gene is lacked of proper care in many cancers, in some cases, may play the function of tumor suppression undetermined.Because methylating of Notch1 gene promoter increases in KRAS positive gene mutation cancer cells, Notch1 expresses and reduces in carrying the allelic cell of KRAS transgenation, therefore, by suppressing Notch1 tumor inhibition effect, KRAS transgenation cell strain may bring out or maintain its tumorigenesis potential.

The member of cyclin D3Shi cyclin family, cyclin is that cell cycle G1/S conversion is needed.In KRAS mutant clone, the promoter methylation of cyclin D3 increases, and this shows to suppress cyclin D3 and transcribes.Therefore, evidence shows, two exemplary scheme, and wherein cyclin D3 is not that these cell strains conversion phenotypes are required, or retardance cyclin D3 transcription inhibition factor that methylates of cyclin D3 promotor.

In contrast to Notch1 and cyclin D3, nucleus binding-protein (CNBP), also referred to as ZNF9, with the generation of cancer or develop uncorrelated.Yet CNBP/ZNF9 is the part in conjunction with the title complex of MYC gene promoter.When MYC expresses imbalance, MYC promotes generation and the progress of cancer.KRAS sudden change contributes to the mechanism of KRAS transgenation cell cancer progression it be unclear that with the dependency of ZNF9 methylation state.

The genetic expression and the interior remaining cell pnca gene spectrum of NCI-60 culture plate that have allelic 7 cell strains of KRAS transgenation compare, and determine that the specificity of these cell strain genetic expressions changes.As shown in Table 17, it is Glutathione S transferase theta1 (GSTT1) that expression rising has to the existence of KRAS transgenation in cell strain the gene that significance,statistical is relevant.The member of the glutathione s-transferase family of GSTT1 genes encoding people II phase detoxication enzyme, by these compounds and gsh group conjugation, detoxify complicated meta-bolites, xenobiotics and medicine, thus it is water-soluble that they are had more, and easily discharges extracellular.Kinds cancer is relevant to theta1 hypotype.For instance, GSTT1 expression increases relevant to pernicious bladder cancer statistically significant.In other different tumor types, because toxic metabolite product accumulation or accumulation increase, thereby cause the danger of canceration to increase and predicted difference, GSTT1 is inoperative or lack because of genetic polymorphism.

Table 17. has the gene of the higher mrna expression of KRAS gene masculine cell strain significance,statistical.

Mitogen activated protein kinase 3 (MAPK3) is a member of map kinase family.In addition, the expression increase of mitogen activated protein kinase 3 (MAPK3) is relevant to KRAS transgenation in tumour cell.MAPK3 transducer cell epimatrix signal, regulates cell internal procedure, as cell proliferation and differentiation.For instance, phosphorylation MAPK3 expression increases relevant with transitivity medulloblastoma to aggressiveness large bowel cancer tumour.In KRAS positive gene mutation cancer cells, KRAS gene level improves increases relevant to MAPK3mRNA.At least partly, MAPK3 expresses increases these intracellular cell proliferations of induction and tumour progression growth.Equally, the expression of another MAPK (MAP2K4) increases in KRAS positive gene mutation express spectra.In addition, KRAS and MAPK (MAPK3 and/or MAP2K4) may contribute to Synergistic interaction, induction or enhancing cell proliferation and/or tumor development between the interior KRAS of KRAS transgenation cancer cells and MAPK signal.

Synaptotagmin-12 express to increase and middle alpha-globulin inhibitions-H1 expresses the positive correlation that exists of KRAS transgenation in increase and tumor cell line.Under normal operation, in cynapse transmittance process, the transportation of synaptotagmin regulation and control Ca-dependent film.Although also do not have at present evidence prove synaptotagmin-12 to participate in cancer, synaptotagmin-13rd, synaptotagmin-12 kinsfolk, synaptotagmin-13 overexpression suppresses the cellular transformation phenotype from Liver Tumors cell strain.The overexpression of synaptotagmin-12 in KRAS positive gene mutation tumor cell line, shows to relate to the protein-bonded new path imbalance of cynapse in cancer cells.Middle α-(sphaeroprotein inhibition)-H1 is the heavy chain of blood plasma serpin.In function, α-(sphaeroprotein inhibition)-H1 in the middle of the stability of extracellular matrix needs.Although the effect of α-(sphaeroprotein inhibition)-H1 is not still developed in the middle of in cancer, nearest evidence shows, in various solid tumors in the middle of α-(sphaeroprotein inhibition)-H1 expression or loss or constrained, comprise, for instance, lung cancer, colorectal carcinoma and mammary cancer.

example 3:KRAS sudden change and the response (ovarian cancer) of patient to treatment

Materials and methods

Overall survival is analyzed queue.Complete clinical data, is diagnosed as the DNA of the women with epithelial ovarian cancer that there is no known BRCA transgenation, comprises the following San Ge mechanism from the approval of international examination board.Institute's protocols having when medical, should expect accumulation patient, to avoid selection bias.Reference has shown the previously detailed description to these patients: (1) Turin, Italy #1 group (n=197) (Lu L, et al. (2007) .Cancer Res67:10117-10122), (2) Italy Brescia, #2 group (n=59) (Ratner E, and New Haven hospital of (3) Yale University (YNHH) group (n=198) et al. (2010) .Cancer Res15:6509-6515).Yale patient is collected in two clinical trials of Yale University School of Medicine in advance, and between 2000 and 2009, first visit is diagnosed as epithelial ovarian carcinoma patients (table 18).

The clinicopathologic features that table 18. Overall survival is analyzed.

From following two collects the BRCA sudden change ovarian epithelial carcinoma case of record, this case has known results: (1) YNHH group (n=17) and (2) U.S. wish city DKFZ (n=62) (table 19).

The clinicopathologic features of table 19.BRCA sudden change epithelial ovarian carcinoma patients.

* not from wish city patient to histology information

* not from wish city patient to histology information

Because not all first phase ovarian cancer patients is all accepted adjuvant chemotherapy, when subage information is not during from first phase tumour patient, these patients are excluded outside analyzing.Otherwise 1B phase and 1C phase tumour comprise the 2-4 phase.In order to reduce to greatest extent borderline tumor, surprisingly list in, the not clear tumour of classification is got rid of outside this is analyzed.For the women of Neoadjuvant Chemotherapy for Treatment, the pathological diagnosis date is considered to the Start Date for the treatment of.For adjuvant chemotherapy treatment women, date of surgery is considered to the Start Date for the treatment of.79 routine patients of the totally 386 routine patients of wild-type BRCA or not test b RCA transgenation and the BRCA transgenation of record meet above-mentioned parameter, are included in two survival analysises.

New adjuvant chemotherapy queue.Determined that women and Equal Opportunities Commission accept the protocol groups (n=125) (table 20) that platinum class is the international examination of the YNHH of surgical cytoreduction between 1996 and 2010 Board of Directors approval after basic new adjuvant chemotherapy.Due to tumor load, concerning the long-pending operation of subtracting of the best, tumour is too large, and this queue patient accepts chemotherapy as main treatment.Patient accepts surgical cytoreduction after chemotherapy and extra assisting therapy.The patient who only carries out the combined therapy of four cycles or more multiply periodic new auxiliary platiniferous is included into this analysis group (n=116).After ideal cytoreductive surgery is defined as operation, remaining residual is measured <1 centimetre, and undesirable surgical cytoreduction has been defined as >=1 centimetre of operation residual measurement.In Yale University, same group of surgeon performs the operation to women, avoids the bias causing as the surgical skills that affects residual factor.

Table 20. is accepted the patient's of new adjuvant chemotherapy clinicopathologic features.

Platinum drug resistance analysis patient.Platinum resistance is defined as platiniferous adjuvant chemotherapy and is accomplished to Progression free survival phase <6 month of recurrence date.From the women of Italian #1 group, Italian #2 group and YNHH patient's group (n=291), obtain during Progression free survival every.Table 21 has been described these patients' clinicopathologic features.

[01] clinicopathologic features of table 21. platinum drug resistance analysis.

The detection of KRAS sudden change.The method of use standard is DNA isolation from tumour, blood or saliva.KRAS sudden change can not obtain from somatocyte, does not need loss of heterozygosity，LOH (Chin LJ, et al. (2008) .Cancer Res68:8535-8540) yet; Therefore, blood and saliva, for instance, be not suitable for test, do not consider tested tissue, and result is identical.Use is carried out the detection of KRAS mutation allele to the specific primer of KRAS transgenation and TaqMan probe (Applied Biosystems, Inc., Foster city, California, the U.S.) to all samples.YNHH group is carried out gene type analysis, except COH sample is carried out to gene type, carries out gene type in its equipment.Population less than 3% carries two copy KRAS sudden changes (Chin LJ, et al. (2008) .Cancer Res68:8535-8540).Therefore the patient who, carries the KRAS mutation allele of at least one copy is categorized as KRAS mutation carriers.

Carry and do not carry the gene expression analysis of the ovarian epithelial carcinoma of KRAS sudden change.Affymetrix GeneChip Human Genome U133Plus2.0platform (Affymetrix company, Santa Clara, California, U.S.) depict the genetic expression of the fresh food frozen tumor sample (9 not mutated and 7 KRAS sudden changes) of taking from 16 routine patients.All samples is all from III phase or high-grade serosity epithelial ovarian tumor of IV phase.MAS5 algorithm process image, removes probe, and at least 75% sample is judged as and does not have probe.Intensity level carries out logarithmic transformation and fractile normalizing.In the LIMMA of R statistical software software package, carry out, adopt linear model and Empirical Bayes error appropriateness, the sample that more than 52 years old patient obtains is with it carried out to gene expression difference assessment (n=6 not mutated and 4 KRAS transgenations), (R Foundation for Statistical Computing, the public can login www.r-project.org) (Smyth G. (2005) .Limma.in Gentleman R, et al. (eds) Bioinformatics and Computational Biology Solutions using R and Bioconductor.Springer:New York, pp.397-420).

Use stamp methods to assess KRAS in these group data and suddenly change and announce the dependency of result, to reduce cross-platform impact (Paranjape T, et al. (2011) .Lancet Oncol12:377-386).Briefly, according to the Pearson's correlation calculations label mark between these gene expression profiles of each sample of label vector sum of gene contribution.

Pairing Kolmogorov-Smirnov check is used for analyzing the difference between KRAS transgenation and not mutated ovarian epithelial carcinoma sample label mark.Except as otherwise noted, from the list of genes of publication separately as label vector.Data (Mol Cancer Ther4:1605-1616) in the people such as Peters research are from genetic expression storehouse (GSE1926), and reanalyse, and generate the label of between platinum sensitivity and resistance sample 50 significant difference expressing genes.

Chemosensitivity and cell viability analysis.High-throughput CellTiter-Blue cell viability detects the activity of determining the medicine being used singly or in combination.For these, detect, utilize accurate XS liquid treatment workstation (BIO-TEK instrument company, Wei Nusiji, vermont, the U.S.), 1.2 * 10 ³individual cell is seeded in each hole of 384 orifice plates, and allow at 37 ℃, 5%CO ₂overnight incubation under atmosphere.Use liquid treatment workstation, all medicines in medium according to 2:3 or the serial dilution of 1:2 ratio, at the appointed time in, 5 μ l diluents join in suitable hole.Four to copy hole concentrated for every kind of medicine, and other four control wells add the contrast diluent without medicine.When medicine culture cycle finishes, 5 μ l CellTiter-Blue reagent (Promega company, Madison, the state of Wisconsin, the U.S.) join in each hole.By residue viable cell, become the ability of resorufin to assess cell viability resazurin biological reducing.Use Synergy4 microplate reader (Bio-Tek Instruments Inc.) to measure the fluorescence (579nm Ex/584nm Em) of resorufin.Fluorescence data is transferred to Microsoft Excel (Microsoft), with respect to 4 repetitive cell holes that do not add medicine, calculates survival rate per-cent.Use XLfit version 5.2 (ID Business Solutions Ltd), S type balance model returns, and determines IC50.Growth/the survival rate that is defined as IC50 reduces by 50% required drug level.All experiments are all carried out at least 3 times.

Target KRAS sudden change.Sequences of small interfering RNAs, is designed to target KRAS transgenation sequence, corresponding to so-called " Seed Sequences ", at the different positions of 6 Nucleotide of 5 ' end of siRNA guiding chain, places single nucleotide polymorphism (SNP).Carry out Blast search to reduce cross reaction.In some siRNA sequence, to introduce DNA Nucleotide and optimize heat energy feature, guiding chain is preferably incorporated in argonaute effect mixture, or introduces the specificity that DNA Nucleotide increases sudden change.

The siRNA guiding chain row following (small letter=RNA, the capitalization=DNA that in experiment, use; GS=guiding chain, PS=complementary strand):

2-l?GS?ugcaucacuugaggucaggag(SEQ?ID?NO：23)

2-l?PS?ccugaccucaagugaugcacc(SEQ?ID?NO：24)

2-3GS?TGCATCACuugaggucaggag(SEQ?ID?NO：25)(passenger?strand?same?as2-1)

3-2GS?ucaucacuugaggucaggagu(SEQ?ID?NO：26)

3-2PS?uccugaccucaagugaugcac(SEQ?ID?NO：27)

Negative control group used is purchased from Qiagen company (Valencia, California, the U.S.) (AllStars negative control siRNA).Use Dual-Luciferase detects, and that determines these sequences knocks out effect and KRAS transgenation specificity (referring to wO/2009/155100, its content is incorporated in herein by citation).Under oligonucleotide combination standard condition, anneal, then use standard agreement transfectional cell.Adopt mtt assay to measure cell survival rate, experiment is carried out four parts, and all row repeat 4 independent experiments.72 hours collecting cell lysates after transfection, as previously mentioned, are used KRAS specific probe to measure KRAS gene protein level (Chin LJ, et al. (2008) .Cancer Res68:8535-8540).

Statistics.In order to assess the significance of population biometric variables, χ ²the definite check of check or bilateral Fisher ' s is for classified variable.T check is for continuous variable, as the age.Adopt Kaplan-Meier method, the total survival time (Kaplan E and Meier P. (1958) .J Am Stat Assoc53:457-481) that compares KRAS transgenation and wild-type patient, survival curve statistical significance is determined (Mantel N. (1966) .Cancer Chemother Rep50:163-170) by log-rank check.Cox proportional hazards regression models (Cox D. (1972) .J R Stat Soc34:187-220) is used to assess KRAS transgenation, population and predictive factors to the impact of overall survival (as the age, by stages, pathological grading and histology).Multivariate logistic regression analysis (Cox D. (1970). bit is. Mei Xiuyin according to one's analysis, London) for determining KRAS sudden change, other populations and the impact of predictive factors on undesirable surgical cytoreduction probability.Multivariate logistic regression analysis (Cox D. (1970). bit is. Mei Xiuyin according to one's analysis, London) impact on platinum resistance probability for assessment of KRAS transgenation and other predictive factors.Adopt SAS9.1.3 (SAS Institute Inc., Cary, NC, USA) and R2.12.1 (R basis statistical computation) to carry out all statistical study.

Data and result

KRAS sudden change with after tested harmful BRCA sudden change is negative or not after tested the dependency of 454 routine epithelial ovarian cancer patient's Overall survivals of harmful BRCA sudden change assess.While considering whole queue, Kaplan-meier survival analysis shows, KRAS sudden change does not predict that lifetime is poor.Due to KRAS transgenation and Postmenopausal Ovaries cancer the most closely related (Chin LJ, et al. (2008) .Cancer Res68:8535-8540), to evaluating 52 years old above women's (n=279) lifetime.Surpass 52 years old and comprise and within 52 years old, be considered to a suitable alternative menopausal state.Kaplan-Meier survival analysis, in contrast to nonmutationed epithelial ovarian cancer patient's group (n=220, Fig. 7, logrank checks P=0.0399, non-KRAS transgenation median survival interval is 60 months, KRAS transgenation median survival interval 34 months), after menopause, the epithelial ovarian cancer patient of KRAS transgenation organizes and significantly reduces (n=59) lifetime.When other variable comprise the age, by stages, pathological grading, histology and therapeutic community, and KRAS transgenation state is included in multivariate Cox proportional hazards regression models, KRAS transgenation is to suffer from the significance,statistical predictor (table 22) that epithelial ovarian cancer postmenopausal women Overall survival reduces; The risk ratio of KRAS transgenation is 1.67 (95% credibility intervals: 1.09-2.57, P=0.019).

Table 22.KRAS transgenation and Postmenopausal Ovaries cancer patient organize survival time reduction relevant (>52 year) (n=279).

KRAS transgenation is assessed with the dependency of the lifetime of EOC patient's group (n=79) of the harmful BRCA1 of carrying of independent queue or BRCA2 sudden change.The EOC patient who carries BRCA transgenation than the EOC patient's significance,statistical that there is no BRCA transgenation young (52.7vs60.8 year, P<0.0001).In addition, through multiplicity, control the age, by stages, pathological grading and histology, do not compare with there is no the EOC patient of BRCA transgenation, the EOC patient who carries BRCA transgenation has the longer median survival interval of significance, and (within 120 months, vs52 is individual month, P=0.0036).In multiplicity, adopt multivariate Cox proportional hazards regression models, carrying BRCA transgenation and tool is with or without and there is no lifetime between the EOC patient of KRAS transgenation significant difference (table 23, risk ratio=0.75 of KRAS transgenation, 95% fiducial interval: 0.21-2.72, P=0.66).In this research, only has few patient evaluation KRAS transgenation to carrying the impact of lifetime in EOC patient after the menopause of harmful BRCA transgenation.

Table 23. has KRAS transgenation and the Overall survival (n=79) of EOC patient's group of harmful BRCA transgenation.

HR: the danger ratio that the analysis of polynary Cox Proportional hazards obtains;

CI: fiducial interval;

Research comprises: New Haven hospital of Yale University, wish city.

In order to explain that the survival time of KRAS positive gene mutation EOC patient after menopause reduces, KRAS positive gene mutation is assessed with the dependency that platinum-based chemotherapy responds.Platinum-based chemotherapy is the standard First-line chemotherapy of EOC treatment.First, to having all women of EOC, assess, these women carry out platiniferous new adjuvant chemotherapy in Yale-New Haven hospital (YNHH), then by surgical cytoreduction (n=116).Postoperative (surgical cytoreduction) residual is used as the substitute marker of patient to chemotherapy response.Determine, be compared to not mutated patient's group (n=90) of only having 3.33%, the patient (n=26) of 15.4% KRAS transgenation has carried out undesirable surgical cytoreduction (Postoperative Residual focus is 41 centimetres) (Fig. 8, P=0.044).In multivariate logistic regression analysis model, controlled the age, by stages, pathological grading and histology, KRAS transgenation also to new adjuvant chemotherapy and operation after the relevant (table 24 of undesirable surgical cytoreduction significance, odds ratio=9.36,95% fiducial interval: 1.34-65.22, P=0.024).

Undesirable surgical cytoreduction group (n=116) after table 24.KRAS transgenation prediction new adjuvant chemotherapy.

1.OR:logistic regression model obtains odds ratio mutually

2.CI: fiducial interval

3. multifactor: adjust the age, by stages, cycle life before pathological grading and histological type, scheme and operation

In order to determine whether that in KRAS transgenation EOC patient, the not good reason of new auxiliary platinum-based chemotherapy reaction is due to resistance to platinum-based chemotherapy, to carrying out platinum-based chemotherapy assisting therapy, do not record BRCA transgenation, all EOC patient's platinum resistances with available response data are assessed (n=291).Having determined, obviously more likely there is platinum resistance (be defined as in this embodiment and accept palindromia in 6 months after platinum-based chemotherapy) (16.67vs7.56%, P=0.034) than non-KRAS sudden change EOC patient in KRAS positive gene mutation EOC patient.Logistic regression analysis, control residual after surgical cytoreduction, by stages, histology, age and pathological grading, KRAS transgenation is the has age EOC patient of institute platinum class resistance significance predictor (table 25, odds ratio=3.18,95% fiducial interval: 1.31-7.72, P=0.0106).

Table 25.KRAS transgenation is relevant to platinum class resistance.

To extracting a small group ovarian cancer patients of Fresh Frozen tissue, carry out gene expression research (Brescia queue), between 7 slurries EOC samples with KRAS transgenation and 9 slurries EOC samples (n=16) without KRAS sudden change, compare.In this queue, EOC patient (n=10) after the menopause over 52 years old, (Fig. 9 a) for raising in the relevant EOC of KRAS transgenation to the KRAS relevant three cloudy breast cancer related gene labels of sudden change (Paraniape T, et al. (2011) .Lancet Oncol12:377-86) of previously having found.Being similar to previous TNBC analyzes, find the overexpression of the downstream passages that in EOC KRAS sudden change tumour, KRAS is relevant, this consistent with ' KRAS habituation ' (Singh A, et al. (2009) .Cancer Cell15:489-500) (Fig. 9 b).

Use identification platinum resistance and responsive previous Gene Expression Data Analysis (the Peters D indicating, et al. (2005) .Mol Cancer Ther4:1605-1616), can determine with respect to not mutated EOC sample, KRAS transgenation EOC sample has a lower carboplatin susceptibility label (Fig. 9 c).The investigation result that often participates in platinum resistance with AKT signal pathway activated is consistent, can determine that AKT3 raises one of transcripton (Fig. 9 d) in KRAS transgenation EOC tumour the most significantly.

Although tumor sample provides miRNA expression data, expression (CAOV3) contrast of let-7b in the expression (BG-1 and IGROV1) with let-7b miRNA in two kinds of cell strains of KRAS sudden change and non-KRAS mutant clone.Let-7b miRNA expresses at KRAS sudden change-positive lung cancer tumour (Chin LJ, et al. (2008) .Cancer Res68:8535-8540) and in three negative breast cancer tumours (Paranjape T, et al. (2011) .Lancet Oncol12:377-386) change.

Determine let-7b significance,statistical lower (Figure 12) in KRAS mutant cell.

In order to confirm that KRAS transgenation exists lower drug susceptibility to change, and carries and do not carry the EOC cell strain of KRAS sudden change for testing the susceptibility of different chemotherapeutics.For instance, the cell strain (IGR-OV1) of the cell strain of KRAS positive gene mutation/BRCA gene wild-type (BG1), not mutated/BRCA wild-type cell strain (CAOV3) and KRAS positive gene mutation/BRCA1 sudden change is tested.Definite, with respect to CAOV3, there is no the cell strain of KRAS sudden change, KRAS transgenation system, BG1, has significance,statistical resistance to carboplatin (P<0.04) and carboplatin/paclitaxel plus chemotherapy (P<0.0001).By contrast, in contrast to IGROV3, carry the cell strain of KRAS transgenation and harmful BRCA1 transgenation, IGROV1, does not have resistance (Figure 10) to these preparations.The corresponding clinical effectiveness that these results are relevant with platinum resistance to having confirmed KRAS sudden change is consistent, but does not have harmful BRCA transgenation.

In addition the medicine that, the patient of carboplatin/Paclitaxel Chemotherapy failure is carried out to second line treatment is assessed.These the second line medicines comprise Zorubicin, topotecan, gemcitabine.With respect to CAOV3 (not mutated cell strain), KRAS mutant clone, BG1, has remarkable resistance (table 26) to each in these medicines.

The chemosensitivity of table 26.KRAS transgenation cell strain (BG1) and not mutated cell strain (CAOV3).

Because the continuation of KRAS signal in the relevant tumour of given herein data acknowledgement KRAS transgenation is used, the impact of direct target KRAS transgenation is evaluated.The similar mixture of siRNA (siRNA)/miRNA is designed to the allelotrope directly changing in conjunction with KRAS sudden change transcripton, but not in conjunction with non--KRAS transgenation transcripton, (Figure 13).Determine, the oligonucleotide of these targets of transfection KRAS sudden change is double-stranded, cause the BG1 cell strain inner cell survival time significance,statistical that carries KRAS transgenation to reduce (P<0.001), but (Figure 11 a) or not impact in SKOV3, two not mutated EOC cell strains at CAOV3.After this result and treatment in BG1, but not in CAOV3 (Figure 11 b) or not in SKOV3, it is consistent that the appropriateness of immunoblotting tested K RAS protein level declines.

example 4: predict the biological markers KRAS sudden change of early stage large bowel cancer (CRC)

Materials and methods

Study population.Until 1994, originate in 1986, between 55 years old to 69 years old 120, in 852b Healthy People, study Dutch diet and cancer cohort study (NLCS) and determine 925 example morbidity CRC cases (ICD-O:153.0-154.1).Holland cancer register office (NCR) and PALGA, national organization's pathology and cytopathology register office, combine and determine accident carninomatosis example (Van den Brandt PA, et al.Int J Epidemiol.1990; 19 (3): 553-8).Describe elsewhere Dutch diet and cancer cohort study (Van den Brandt PA, et al.J Clin Epidemiol.1990 in detail; 43 (3): 285-95).The 815 routine large bowel cancers that may link up with PALGA and paraffin embedding tumor tissues are collected in whole Dutch 54 pathology register office.The good quality DNA extraction of sufficient amount is in 734 routine case (90%) (Brink M, et al.Carcinogenesis.2003; 24 (4): 703-10).At baseline values, the sub-queues of 5000 Healthy Peoples of stochastic sampling in whole queue, estimate the person-time of queue risk by the life state of biennial follow-up period.1,886 human oral swab DNA carries out KRAS gene mutation typing.

Data gathering.Can by Dutch cancer register office obtain tumor-localizing, by stages, differentiation degree, date of the onset and diagnose the information for the treatment of in latter 3 months.Until the life state in May, 2005 retrieves from central bureau of statistics's family tree and population register office, city, and can from all 734 routine cases, obtain.By the interlock of Dutch statistics bureau, retrieve the cause of death.The death that CRC is relevant is defined as the death that colon, rectum, rectum, gi tract (non-specific) or metastatic liver cancer cause.In the situation of enteron aisle (non-designated) or hepatic metastases, the information of Dutch cancer register office (NCR) and PALGA is used for eliminating the possibility of another main cancer cause of the death.

DNA extraction and KRAS transgenation are determined.Each tumor tissue's 5 μ m section adopts phenodin and eosin dyeing, by pathologist, is revised.Five sections is 20 μ m, removes paraffin, according to manufacturer specification, uses Puregene

dNA separating kit extracts DNA (Gentra system).In brief, cell pyrolysis liquid and Proteinase K (20 mg/ml, Qiagen company) add in tissue, and are incubated overnight at 55 ℃.At 37 ℃, after 72 hours, extract DNA, except deproteinize, the 2-propyl alcohol precipitation DNA with 100%.Finally, DNA aquation in aquation damping fluid.TaqMan PCR detects the separated DNA of amplification, aims at and differentiates the interior T of let-7 paratope 6 (LCS6) or G allelotrope (being respectively wild-type and mutation allele) (applying biological science) in KRAS gene 3 ' UTR.Although tumour DNA is used to assess genotype, confirm well that the genotype of healthy tissues and tumor tissues is identical with KRAS mutation allele carrier (Chin LJ, et al.Cancer Res.2008; 68 (20): 8535-40).

As previously mentioned, nido polymerase chain reaction (PCR) and direct Sequencing (KRAS), and restriction fragment length polymorphism (BRAF) assessment KRAS and BRAF sudden change (Brink M, et al.Carcinogenesis.2003; 24 (4): 703-10; De Vogel S, et al.Carcinogenesis.2008; 29 (9): 1765-73).By using sodium bisulfite chemically modified genomic dna and methylation status of PTEN promoter (MSP) (de Vogel Set al.Carcinogenesis.2008; 29 (9): 1765-73; 26.Herman J G, et al.Proc Natl Acad Sci U S is A.1996; 93 (18): 9821-6; Derks S, et al.Cell Oncol.2004; 26 (5-6): RASSF1A, O that 291-9) assessment Weisenberger proposes ⁶the promoter methylation of-MGMT, CHFR and CIMP mark (Weisenberger DJ, et al.Nat Genet.2006; 38 (7): 787-93).As previously mentioned, use BAT-26, BAT-25, NR-21, NR-22, NR-24 to determine MSI state (Suraweera N, et al.Gastroenterology.2002; 123 (6): 1804-11).When main research terminal, establish when blind, for example, CRC associated death, carries out all detections and analyzes.

Statistical study.Until CRC associated death or follow-up period termination, cause-specific survival rate is defined as the time of cancer diagnosis.Kaplan-Meier curve and Log-Rank check are used to assess the impact of KRAS sudden change on cause-specific survival rate.Use the Cox proportional hazard model assessment HR and the corresponding 95%CI that adjust underlying factor.If these factors known predictive factors that is CRC, affects the dangerous probability of rough estimate and surpass 10%, be considered to possible Confounding Factor.Age (continuously) when Confounding Factor comprises diagnosis, sex, Tumor Differentiation degree (well differentiated, moderate differentiation, poorly differentiated, not differentiation), position (near-end, far-end, sigmoid colon and rectum).Use Schoenfeld residual sum log (log) hazards plots test Proportional hazards hypothesis.After this point, the relevant cause of the death of CRC is impossible, and survival analysis is only limited to 10 years after diagnosis.Use Cox proportional hazard model assessment sickness rate than (RR) and 95%CI.Use the sandwich variance estimated value of robust Huber-White evaluation criteria error, consider that queue sampling introducing is from additive variance.All analyses are all used statistical package STATA10.0 to complete.

Data and result

Patient in this research is male sex's (55.6%) often, diagnosis infantile tumour (62.0%) or near-end or far-end tumour (65.3%; Table 27).Between follow-up period, 41.4% patient dies from CRC.KRAS gene-LCS6 (P=0.160 that suddenlys change in 14.0% commitment (I phase and II phase), III phase of 19.2% and 21.4% IV phase patient, detected; P _trend=0.060).The patient of KRAS sudden change is more often diagnosed as the terminal illness (patient of KRAS sudden change: the patient of wild-type=47.5%:36.9%, P=0.046).The sex of wild-type and KRAS mutation carriers, age, diagnosis, differentiation degree, tumor locus, microsatellite instability or KRAS sudden change (table 27), BRAF (P=0.640) or RASSF1A promoter CpG island methylate and between (P=0.423), do not find other statistical significant differences.As expected, the patient of III phase or IV phase disease often dies from CRC (P<0.001), often has a kind of low differentiation tumor (P<0.001)., in early stage patient, late stage patient often has a kind of near-end (P=0.036) or microsatellite stability tumour (P=0.047).

Table 27.1986 year and the total crowd, KRAS transgenation and the wild-type carrier that between 1994, in Dutch diet and cancer cohort study, comprise, early stage and late period CRC case baseline characteristic.

With respect to other stage G allelotrope (KRAS transgenation) carrier, IV phase G allelotrope (KRAS transgenation) carrier may be more women (66.7%; P=0.097), and more may there is near-end tumour (71.4%; P=0.004), (table 28).

Table 28. is according to KRAS mutation status, commitment, III phase, IV phase patient's baseline characteristic and characterization of molecules.

KRAS transgenation is well relevant lifetime to early stage CRC.(log-rank checks, P=0.864) (Figure 14) during the Kaplan-Meier of total crowd's KRAS sudden change and specific cause of disease lifetime analyzes, not observe statistical significant difference.

Due to the cancer staging depending on lifetime, analyze and carry out stage layering.With respect to wild-type case, early stage G allelotrope (KRAS sudden change) carrier demonstrates significance,statistical better lifetime (log-rank check, P=0.038; Figure 15 A).Late case is not observed this species diversity (Figure 1B and Tu C; Log-rank check, III, IV phase case is respectively P=0.775 and P=0.875).

KRAS/BRAF mutation status improves the association between KRAS sudden change and existence.Figure 16 A has shown that the Kaplan-Meier of early stage (I phase and II phase) the CRC case of carrying KRAS sudden change and KRAS sudden change analyzes.Due to CRC, there is 20G-allelotrope (KRAS sudden change) carrier of KRAS sudden change there is no death.KRAS wild-type patient has poor lifetime, if especially they have KRAS sudden change (log-rank check, P=0.043; Compare in the KRAS mutant allele that there is no KRAS sudden change, log-rank check has the KRAS mutant allele carrier of KRAS sudden change, P=0.017).This finds with T by stages irrelevant; Carry 115 routine KRAS wild-type cases of KRAS sudden change, only have 5 examples (4%) to be diagnosed as the high-risk IIb phase (T4N0M0).In G-allelotrope (KRAS mutation allele) carrier, do not have patient to be diagnosed as the IIb phase.For patients with terminal, do not find survival rate difference (Figure 16 B and Figure 16 C, log-rank check, III rank P=0.989, IV rank P=0.535)).III phase patient result shows, KRAS wild-type patient's the prediction of carrying KRAS transgenation is the poorest.Subgroup analysis shows, the main better result of finding early stage KRAS carriers of mutation in subordinate phase case.Due to patient's limited amount, T layer analysis is by stages impossible.

The CRC that carries the allelic BRAF sudden change of G shows similarly better result, although be not that (log-rank check P=0.166), may be carried out KRAS transgenation and KRAS sudden change (9 patients) due to small number of patients to significance,statistical.Similarly, with respect to there is no the methylated wild-type carrier of RASSF1A, the G-allelotrope carrier (KRAS mutation allele) with abnormal RASSF1A promoter methylation, participate in the another kind of gene of Ras path, there is better prediction, although (log-rank checks, P=0.062) more not have significance,statistical.The analysis combining with KRAS, BRAF and RASSFlA state shows, early stage G allelotrope (KRAS sudden change) carrier with KRAS, BRAF or other change of RASSF1A provide good prediction (log-rank check, P=0.026).On the contrary, when adding while not relating to Ras path as the gene methylation state of MGMT or CHFR, do not observe difference lifetime (MGMT:log-rank check, P=0.220; CHFR:log-rank check, P=0.118).

KRAS transgenation is irrelevant with other predictive factorses in conjunction with impact lifetime of KRAS transgenation state.In multivariate analysis, compare with wild-type, early stage (the HR0.46 with G allelotrope (KRAS sudden change), 95%CI:0.18-1.14), III phase (HR0.98,95%CI:0.55-1.74) or the specific cause of disease of IV phase case (HR0.42,95%CI:0.17-1.06) all do not find statistical significant difference lifetime, although early stage and IV phase G-allelotrope (KRAS sudden change) carrier confirms raising lifetime (table 29).

Dangerous probability and the 95%CI of specific cause of disease mortality ratio, clinicopathologic features and KRAS sudden change in 734 routine CRC of the Dutch diet of table 29. and cancer cohort study.

Carrier has good prediction to have the early stage G allelotrope (KRAS sudden change) of KRAS sudden change, because these patients do not die from CRC.On the contrary, there is the early stage (HR0.77 that the KRAS gene of KRAS transgenation does not suddenly change, 95%CI:0.30-1.97), III phase (HR0.95,95%CI:0.44-2.05) or between IV phase case (HR0.35,95%CI:0.11-1.13), do not find lifetime of statistical significant difference.Yet III phase G-allelotrope (KRAS sudden change) carrier with KRAS sudden change presents the poor (HR1.52 of prediction; 95%CI:0.66-3.54), although contrast does not have significance,statistical.Although Dutch guide does not advise that the patient who is diagnosed as CRC carries out assisting therapy in Dutch diet and cancer cohort study, the Proportion of patients of accepting assisting therapy is very low.In case, 9% accepts adjuvant chemotherapy in early days.For late period more, 31% III phase and 19% IV phase patient accept adjuvant chemotherapy.The patient who gets rid of adjuvant chemotherapy treatment does not change our conclusion.In fact, the difference that the patient of eliminating adjuvant chemotherapy treatment has improved between early stage and III phase G-allelotrope (KRAS sudden change) carrier with KRAS transgenation is (early stage: without CRC associated death; The III phase: HR2.36,95%CI:0.99-5.67), show that III phase G-allelotrope (KRAS sudden change) carrier has an even worse natural history.Yet this analysis is based on the little patient of quantity size.

The existence impact of KRAS transgenation and microsatellite instability (MSI) are irrelevant.Before biomarker provided in this article and method, MSI is unique molecule of large intestine cancer predicting marker.Therefore, the impact of KRAS mutator gene type in MSl layering patient crowd is studied.Get rid of the good patient with microsatellite instability tumour of prediction, do not change conclusion provided herein; Microsatellite instability and the microsatellite stability case with KRAS sudden change have good prediction.On the contrary, the patient with KRAS wild-type predicts poor, even if they have a microsatellite instability tumour (log-rank check, P=0.036) (Figure 17).Due to patient's limited amount, other layer analysis of microsatellite instability Gender, tumor-localizing or differentiation degree is impossible.

Late period, the risk of CRC was uncorrelated with KRAS sudden change.In order to study the allelic possibility of KRAS transgenation of easy trouble CRC in late period, the cognation between KRAS genotype and CRC onset risk is studied.18% sub-queue member has been found KRAS transgenation (G-allelotrope).For CRC, when carrying KRAS transgenation (G-allelotrope), find the Risk Reduction (RR0.68,95%CI:0.49-0.94) of the early stage CRC of development (I phase or II phase).The risk of later stage of development CRC (III phase or IV phase) is not subject to the genotypic impact of KRAS (the RR III phase: 1.02,95%CI:0.68-1.53; The RR IV phase: 1.15,95%CI:0.63-2.09).

embodiment 5:KRAS sudden change, Metastatic Colorectal Cancer patient's prediction and treatment response

Materials and methods

Patient's feature.559 Metastatic Colorectal Cancer patients altogether, hospital of 300Li Univ Louvain accepts anti-EGFR monoclonal antibody single therapy and monoclonal antibody in conjunction with the treatment of chemotherapy, it is basic rescue combined chemotherapy (De RW, et al.Lancet Oncol2010 that 148 examples are accepted Cetuximab in Paris Descartes university; 11 (8): 753-762), before 111 examples, announced (Zhang W, al.Ann Oncol2011; 22 (1): the Metastatic Colorectal Cancer patient (Zhang W, the al.Ann Oncol2011 that 104-109) after 5-FU, irinotecan and the failure of oxaliplatin Regimen Chemotherapy, accept Cetuximab single therapy; 22 (1): 104-109; Lenz HJ, et al.J Clin Oncol2006; 24 (30): 4914-4921) provide tissue, and be suitable for KRAS transgenation polymorphism analysis.Above-mentioned patient crowd's KRAS and BRAF transgenation state are disclosed (De RW, et al.Lancet Oncol2010; 11 (8): 753-762; Zhang W, al.Ann Oncol2011; 22 (1): 104-109).Characterization of molecules above-mentioned is relevant to ORR, PFS and OS.Enter 559 routine Metastatic Colorectal Cancer patients of this research, because the DNA of other Molecular Detection exhausts, in 512 patients, determined KRAS gene 3 '-UTR LCS6 sudden change.

Genetic analysis.As previously described, the formalin of use scalpel blade eye anatomy patient sample is fixed, paraffin-embedded healthy tissues, DNA isolation (De RW, etal.Lancet Oncol2010; 11 (8): 753-762; Zhang W, al.Ann Oncol2011; 22 (1): 104-109).As previously mentioned, DNA amplification (Hollestelle A, et al.Breast Cancer Res Treat2010), specialized designs customization TaqMan genotype tests (Applied biosystems, Foster city, California) identification has KRAS transgenation T or sudden change G allelotrope (rs61764370): the 5 '-GCCAGGCTGGTCTCGAA-3 ' (SEQ ID NO:28) of upstream primer, downstream primer: 5 '-CTGAATAAATGAGTTCTGCAAAACAGGT T-3 ' (SEQ ID NO:29), VIC reporter probe: 5 '-CTCAAGTGATTCACCCA C-3 ' (SEQ ID NO:30), FAM reporter probe: 5 '-CAAGTGATTCACCCAC-3 ' (SEQ ID No:31).Determine as previously mentioned KRAS and BRAF mutation status (De RW, et al.Lancet Oncol2010; 11 (8): 753-762; Zhang W, al.Ann Oncol2011; 22 (1): 104-109).

Cell strain research.To thering is cell strain (HCC2998) of KRAS transgenation (G-allelotrope) and being studied without allelic cell strain with without the cell strain of KRAS obtained sudden change (HT-29), assess the impact of independent chemotherapeutic treatment, or the impact for the treatment of in conjunction with Cetuximab.Cell strain is used respectively Cetuximab (100nM) treatment, or treats, and uses irinotecan diluent (1mg/ml-100mg/ml) treatment.Inoculating cell, makes treated with medicaments 24 hours after inoculation, medium is changed after 24 hours in exposure, then uses MTT to detect, existence scoring 48 hours.

Statistical study.Hardy-Weinberg balance test genotype distributes, χ ²check P=0.8.Homozygote due to low-frequency KRAS mutation allele, the heterozygote of clinical samples or KRAS mutation allele (TG) or homozygote (GG), be considered to the LCS6 positive (KRAS transgenation or G allelotrope), enter and analyze as at least one KRAS sudden change (G allelotrope) genotypic group.Measure as previously mentioned PFS and OS (De RW, et al.Lancet Oncol2010; 11 (8): 753-762; Zhang W, al.Ann Oncol2011; 22 (1): 104-109)

Two tail Fisher rigorous examination are for comparing the ratio between wild-type (wt) TT genotype carrier and at least one G allelotrope genotype (TG and GG) carrier.Adopt Kaplan-Meier method assessment PFS and OS, use log-rank verification test itself and genotypic dependency.Contingency table and Fisher rigorous examination are determined genotype and objective curative effect.The impact that may cause for fully excavating KRAS sudden change, whole Metastatic Colorectal Cancer crowd, analyzes in the patient (two wild-type crowd) that in KRAS and BRAF gene, nothing is suddenlyd change and in KRAS sudden change crowd.Significance level is set as two-tailed test P value <0.05.All statistical tests all adopt statistical package SPSS the 13rd edition to complete.

Result

The KRAS LCS6 of whole patient's queue.In these 512 Metastatic Colorectal Cancer patients, there are 403 wild-type LCS6TT genotype carrier (72%), 102 allelic carrier of heterozygote KRAS transgenation TG (18%), with 7 homozygote KRAS sudden change GG allelotrope (1.3%), 109 genotypic carrier of at least one G allelotrope (19.5%).In 184 routine patients (33%), find the interior KRAS transgenation of codon 12,13 and 61, BRAF V600E is found in 29 patients (5.3%).All patients accept the rescue based on anti-EGF acceptor monoclonal antibody, and 169 accept single therapy and 377 in conjunction with chemotherapeutic treatment.Between the sex and age of KRAS gene wild-type and the diagnosis of KRAS carriers of mutation, found without statistical significant difference.559 routine patients' feature (De RW, et al.Lancet Oncol2010 have been announced before this; 11 (8): 753-762; Zhang W, al.Ann Oncol2011; 22 (1): 104-109).

Shown in table 30, between KRAS and the patient of BRAF sudden change, the genotypic distribution of KRAS is different.Especially, the genotypic per-cent of at least one G mutation allele is uniformly distributed (each 20%) in KRAS gene wild-type and sudden change group, with respect to wild-type group (20%), in BRAF V600E sudden change group, KRAS transgenation (G allelotrope) has increased twice (40%), has statistical significant difference (Fisher rigorous examination P=0.030).

Table 30. is according to Metastatic Colorectal Cancer patient queue KRAS and BRAF mutation status, KRAS gene 3 '-genotypic distribution of UTR LCS6.

Abbreviation: 3 '-UTR LCS6, Let-7 paratope 3 ' non-translational region, WT, wild-type

Whole patient's queue result and lifetime are analyzed.In whole queue, PFS and OS information and LCS6 gene type patient (n=510 and n=503 respectively), between LCS6 wild-type TT genotype carrier and LCS6G sudden change (KRAS sudden change) genotype carrier, meta PFS and OS (Figure 18 A and Figure 18 B) do not detect significant difference.Equally, the difference in PFS and OS is not observed in two wild-types (KRAS and BRAF) or KRAS sudden change patient queue.In addition in whole group and two wild-type patient's queue, between KRAS transgenation and wild-type carrier, do not observe, the significant correlation (table 31) of response group (n=483) and fash group (n=359).

Table 31. is analyzed according to the result of other clinical variables of KRAS genotype and all patients group and lifetime.

Abbreviation: 3 '-UTR LCS6, Let-7 paratope 3 ' non-translational region; WT, wild-type; PFS, Progression free survival rate; OS, Overall survival; CR, replys completely; PR, part is replied; SD, stable disease; PD, progression of disease.

The relevant Progression free survival phase for the treatment of is analyzed.Analyze respectively the patient who accepts monoclonal antibody single therapy and monoclonal antibody combination therapy.Evaluate in 501 patients of LCS6SNP gene type and treatment administration, 160 (32%) accepts anti-EGF acceptor monoclonal antibody single therapy.In the patient of single therapy, have 128 (80%) for the genotypic carrier of LCS6 wild-type TT, 32 (20%) are the carrier of LCS6G mutator gene type.There are 341 (68%) patients to accept multiple chemotherapy combination.In the patient of combination therapy, 266 (78%) is the genotypic carrier of LCS6 wild-type TT, and 75 (22%) is the carrier of at least one G mutator gene type of LCS6.

Full single therapy patient crowd's meta PFS is 10.43 weeks (95%CI:7.73-13.12 week), observe statistical significant difference (P=0.019, log-rank check), LCS6 wild-type TT genotype carrier, 7.85 weeks (95%CI:3.897-11.817 week), LCS6G (KRAS sudden change) the genotype carrier that suddenlys change, 16.86 weeks (95%CI:10.2-23.51 week) (Figure 19 A).The patient group's of whole combination therapy meta PFS is 18 weeks (95%CI:15.87-20.12 week), observe without statistical significant difference (P=0.760, log-rank check), LCS6 wild-type TT genotype carrier, 18.43 weeks (95%CI:16.16-20.69 week), LCS6G mutator gene type carrier, 18 weeks (95%CI:9.97-26.02 week) (Figure 19 B).Accept the KRAS sudden change patient's of mab treatment PFS[16.86 week, (95%CI:8.55-25.18 week)] with the PFS[18 week of accepting the KRAS sudden change patient of combination therapy, (95%CI:13.37-22.64 week)] between there is no significant difference (p=0.291, log-rank check) (Figure 19 C), but not increasing chemotherapy, KRAS sudden change patient has remarkable benefit [P<0.0001, log-rank check], monoclonal antibody single therapy PFS is 7.86 weeks), (95%CI:3.9-11.82 week), combination therapy PFS is 19.29 weeks, (95%CI:17-21.58 week) (Figure 19 D).It should be noted that between the KRAS sudden change patient PFS of single therapy and the non-KRAS sudden change patient PFS of conjoint therapy treatment and there is no significant difference.

In two (KRAS and BRAF) wild-type patient group, single therapy patient meta PFS is 12 weeks (95%CI:8.38-15.61 week), again observe statistical significant difference (P=0.039, log-rank check), LCS6 wild-type TT genotype carrier, 10.43 weeks (95%CI:6.74-14.11 week), LCS6G mutator gene type carrier, 18 weeks (95%CI:5.16-30.83 week) (Figure 20 A).In two wild-type patient groups, combination therapy patient's meta PFS is 28.71 weeks (95%CI:24.98-32.43 week), LCS6 wild-type TT genotype carrier, 28.3 weeks (95%CI:24.15-32.45 week), LCS6G mutator gene type carrier, 28.85 weeks (95%CI:14.82-42.87 week) (Figure 20 B), observes without statistical significant difference (P=0.39, log-rank check).Accept the LCS6 sudden change patient's of monoclonal antibody single therapy PFS[23 week, (95%CI:9.5-36.5 week)] with the PFS[28 week of accepting the LCS6 sudden change patient of combination therapy, (95%CI:14.83-42.87 week)] between there is no significant difference (P=0.096, log-rank check) (Figure 20 C), but not increasing chemotherapy, LCS6 patient has remarkable benefit [P<0.0001, log-rank check], monoclonal antibody single therapy PFS is 10.43 weeks, (95%CI:6.75-14.15 week), combination therapy PFS is 28.71 weeks, (95%CI:24.8-32.6 week) (Figure 20 D).Between the non-KRAS sudden change patient PFS of KRAS sudden change (allelotrope) patient PFS of single therapy and conjoint therapy treatment, there is no significant difference.

The relevant Overall survival analysis for the treatment of.The patient group's of whole single therapy the meta OS phase is 33.14 weeks (95%CI:26.70-39.57 week), LCS6 wild-type TT genotype carrier is 28.85 weeks (95%CI:22.53-35.18 week), LCS6G mutator gene type carrier is 45 weeks (95%CI:35.02-54.97 week), observe between the two without statistical significant difference (P=0.139, log-rank check) (Figure 21 A).Patient group's meta OS phase of whole combination therapy is 44 weeks (95%CI:40.11-47.88 week), LCS6 wild-type TT genotype carrier is 44 weeks (95%CI:40.06-47.93 week), at least one G mutator gene type carrier of LCS6 is 43 weeks (95%CI:29.8-56.2 week), observe between the two without statistical significant difference (P=0.759, log-rank check) (Figure 21 B).Again, the KRAS that accepts monoclonal antibody single therapy suddenlys change patient's OS[45 week (95%CI:35-55 week)] all with the OS[43 that accepts combination therapy KRAS sudden change patient, (95%CI:29.8-56.2 week)] between do not have significance to improve (P=0.574, log-rank check) (Figure 21 C), and KRAS sudden change patient increases the helpful [P<0.0001 of chemotherapy, log-rank check], monoclonal antibody single therapy OS is 28.86 weeks, (95%CI:22.53-35.18 week), combination therapy OS44 week, (95%CI:40-47.93 week) (Figure 21 D).Again, between the LCS6G carriers of mutation OS of single therapy and the non-KRAS carriers of mutation OS of conjoint therapy treatment, there is no significant difference.

In two (KRAS and BRAF) wild-type patient group, single therapy patient's meta OS is 37 weeks (95%CI:30.82-43.17 week), 35.71 weeks (95%CI:32.03-39.4 week) of LCS6 wild-type TT genotype carrier and at least one G mutator gene type carrier of LCS6 observe the trend (p=0.087, log-rank check) (Figure 22 A) of statistical significant difference between 55.43 weeks (95%CI:36.98-73.87 week).In two wild-type patient groups, combination therapy patient's meta OS is 55 weeks (95%CI:48.3-61.7 week), 57 weeks (95%CI:49.4-64.6 week) of LCS6 wild-type TT genotype carrier and at least one G mutator gene type carrier of LCS6 observe between 54 weeks (95%CI:45.46-62.53 week) without statistical significant difference (P=0.649, log-rank check) (Figure 22 B).Accept KRAS transgenation (G allelotrope) patient's of monoclonal antibody single therapy OS[55.43 week, (95%CI:37-73.87 week)] and to accept KRAS transgenation (G allelotrope) patient's the OS[54 of combination therapy all, (95%CI:45.47-62.54 week)] between do not have significance to improve (P=0.705, log-rank check) (Figure 22 C), but not increasing chemotherapy, KRAS sudden change patient has remarkable benefit [P<0.0001, log-rank check], monoclonal antibody single therapy OS is 35.71 weeks (95%CI:32-39.4 week), combination therapy OS is 57 weeks (95%CI:49.4-64.6 week) (Figure 22 D).Between two wild-type patient KRAS mutation carriers of single therapy and the non-LCS6 carrier of combination therapy, there is no significant difference.

LCS6 sports KRAS and BRAF sudden change patient's prediction.In KRAS and BRAF sudden change patient group, carry out observing in the patient of anti-epidermal growth factor receptor monoclonal antibody single therapy and combination chemotherapy PFS and OS without statistical significant difference (data do not show) simultaneously.Between KRAS sudden change and non-KRAS sudden change patient's meta PFS, be identical, accept the KRAS sudden change patient's of monoclonal antibody single therapy PFS[6 week, (95%CI:0-13.25 week)] with the PFS[12 week of accepting the KRAS sudden change patient of combination therapy, (95%CI:6.45-17.56 week)] between do not have significance to improve (P=0.641, log-rank check) (Figure 23 A).The non-KRAS sudden change patient's of monoclonal antibody single therapy PFS[6 week, (95%CI:4.46-7.53 week)] with PFS[12 week of the non-KRAS sudden change patient of combination therapy, (95%CI9.72-14.28 week)] there is significance to improve [P<0.0001, log-rank check] (Figure 23 B).For OS, accept KRAS transgenation (G allelotrope) patient [28.43 weeks of monoclonal antibody list medicine, (95%CI:9.47-47.39 week)] with KRAS transgenation (G allelotrope) patient of combination therapy [23 weeks, (95%CI:10.8-35.19 week)] OS between there is no significant difference (p=0.303, log-rank check) (Figure 23 C), and there is non-KRAS transgenation patient [P=0.002, log-rank check, between the OS21.29 week of monoclonal antibody list medicine (95%CI:15-27.55 week) and combination therapy OS31 week (95%CI:25.65-36.34 week), there is no significant difference (Figure 23 D).

KRAS sudden change and response.Evaluate in response and the whole crowd of KRAS mutator gene somatotype for 483,147 (30.4%) once accepted EGF acceptor monoclonal antibody as monotherapy, accepted multiple chemotherapy combined treatment (69.6%) for 336.In single therapy group, 123 patients (83.6%) are nonresponder (SD and PD), 104 LCS6 wild-types and 19 LCS6 sudden change (KRAS sudden change) carrier, 24 (16.4%) is responder (PR and CR), 13 LCS6 wild-types and 11 LCS6 sudden change (KRAS sudden change) carrier.In effective and invalid group, wild-type and KRAS mutator gene type carrier have observed statistical significant difference (Fisher rigorous examination P=0.002) between distributing.In combined chemotherapy group, 252 (75%) patients are nonresponder's (stable disease and progression of disease), and 84 (25%) is responder's (part is replied and replied completely).Between wild-type and KRAS mutator gene type carrier, observe without statistical significant difference, be respectively 197 vs.55 position nonresponders and 66 vs.18 position responders (Fisher rigorous examination P=1).

In 270 two (KRAS and BRAF) wild-type crowds, 90 (33.3%) once accepted anti-EGF acceptor monoclonal antibody as monotherapy, and 180 (66.6%) accepts multiple chemotherapy combined treatment.In single therapy group, 71 (78.8%) is nonresponder's (stable disease and progression of disease), 60 LCS6 wild-types, 11 LCS6 sudden change (KRAS transgenation) carrier, 19 (21.2%) is responder's (part is replied and replied completely), 10 LCS6 wild-types, 9 LCS6 sudden change (KRAS sudden change) carrier.In effective and invalid group, wild-type and KRAS mutator gene type carrier observe statistical significant difference (Fisher ' s rigorous examination P=0.010) between distributing.In combined chemotherapy group, 102 (56.6%) patients are nonresponder's (stable disease and progression of disease), and 78 (43.4%) is responder's (part is replied and replied completely).Between wild-type and KRAS mutator gene type carrier, observe without statistical significant difference, be respectively 81 vs.21 position nonresponders, 62 vs.16 position responders (Fisher ' s rigorous examination P=1).

The cell strain research of the effect of monoclonal antibody single therapy and combination therapy and LCS6 sudden change.For determining the response of KRAS transgenation (G allelotrope) prediction monoclonal antibody single therapy, without any extra cell toxicity medicament treatment benefit, carry with the colon cancer cell line single therapy of LCS6G sudden change and the impact of conjoint therapy and evaluate.It is found that, than cell toxicity medicament treatment, in non-KRAS transgenation cell strain, in cell toxicity medicament treatment, increase Cetuximab, radiation and irinotecan chemotherapy, increase necrocytosis.On the contrary, in carrying the cell strain of KRAS transgenation (G allelotrope), in cell toxicity medicament treatment, increasing Cetuximab does not have extra cell to kill, and when adding Cetuximab, under radiation event, cell survival rate is higher.These results of study are consistent with finding in our body, and KRAS transgenation (G allelotrope) carrier's Cetuximab associational cells cytotoxic drug is treated without any benefit.

Other embodiment

Although the present invention is described together with its detailed description, description is above intended to illustrate, rather than limits the scope of the invention, by the scope definition of appended claims.Other aspects, advantage and modification are all within the scope of following claim.

The patent of mentioning herein and scientific literature offer those skilled in the art's knowledge.All United States Patent (USP)s of quoting herein and announcement or unpub U.S. Patent application are incorporated herein by reference.Foreign patent and the patent application of all announcements of quoting are herein incorporated herein by reference.The gene pool of the accession number of quoting herein (GENBANK) and NCBI submit to and introduce for your guidance.Every other announcement reference, file, manuscript and the scientific literature quoted are herein incorporated herein by reference.

Although the present invention specifically illustrates and is described with reference to its preferred embodiment, be apparent, however, to one skilled in the art that, under the condition of the scope of the invention, can carry out to it the form made and the various changes in details herein not deviating from appending claims and comprise.

Claims (49)

1. one kind has estrogen receptor of making (ER) and progesterone receptor (PR) negative (ER/PR feminine gender) for identification and develops into the main body of mammary cancer risk or patient's method, comprise the sudden change in the let-7 paratope LCS6 that detects people KRAS in clinical samples, wherein said sudden change is the uridylic (U) at 4 places, a kind of LCS6 of being included in site or the single nucleotide polymorphism (SNP) that thymus pyrimidine (T) changes guanine (G) into, and the existence of wherein said sudden change shows to have the increase risk that ER/PR feminine gender is developed into mammary cancer.

2. one kind for developing into the main body of mammary cancer risk or patient predicts that estrogen receptor (ER) and progesterone receptor (PR) negative (ER/PR is negative) develop into the method for the morbidity of mammary cancer having, comprise the sudden change in the let-7 paratope LCS6 that detects people KRAS in clinical samples, wherein said sudden change is the uridylic (U) at 4 places, a kind of LCS6 of comprising site or the single nucleotide polymorphism (SNP) that thymus pyrimidine (T) changes guanine (G) into, and the existence of wherein said sudden change shows to have the more early morbidity that ER/PR feminine gender is developed into mammary cancer.

3. method according to claim 2, wherein ER/PR negative breast cancer is also that HER2 is negative, thus it is a kind of three negative breast cancer (TNBC).

4. method according to claim 3, wherein three negative breast cancer (TNBC) are a kind of substrate tumour or luminal type tumour.

5. method according to claim 4, wherein three negative breast cancer (TNBC) are a kind of substrate tumours, and it expresses the protein of a kind of transcripton or EGF-R ELISA (EGFR) or CK5/6 (CK5/6) genes encoding.

6. according to the method described in claim 1,2 or 3, wherein mammary cancer is further characterized in that low expression or negative mammary cancer 1 (BRCA1) gene of expressing.

7. according to the method described in claim 1,2 or 3, wherein main body or patient are Pre-menopausal Womens.

8. according to the method described in claim 1,2 or 3, wherein main body or patient's age is 51 years old or younger.

9. one kind to suffering from the main body of epithelial ovarian cancer (EOC) or the method that patient predicts, comprise the sudden change in the let-7 paratope LCS6 that detects people KRAS in clinical samples, wherein said sudden change is the uridylic (U) in a kind of LCS6 of being included in site 4 or the single nucleotide polymorphism (SNP) that thymus pyrimidine (T) changes guanine (G) into, wherein, when contrasting with control group, the existence of sudden change shows that survival rate reduces.

10. method according to claim 9, wherein main body or patient for after menopause, 52 years old or at least 52 years old.

11. methods according to claim 9, wherein control group does not carry sudden change.

12. methods according to claim 1, wherein survival rate is total survival rate, the survival rate of 5 years or the survival rate of a year.

13. 1 kinds of methods that response is predicted to epithelial ovarian cancer (EOC) cell platinum-based chemotherapy, comprise the sudden change in the let-7 paratope LCS6 that detects people KRAS in clinical samples, wherein said sudden change is the uridylic (U) in a kind of LCS6 of being included in site 4 or the single nucleotide polymorphism (SNP) that thymus pyrimidine (T) changes guanine (G) into, and the existence of wherein said sudden change shows platinum-based chemotherapy resistance.

14. methods according to claim 13, wherein ovarian cancer cell is evaluated in vitro or in vitro.

15. methods according to claim 14, wherein main body be after menopause, 52 years old or at least 52 years old, main body ovarian cancer cell is evaluated in vitro.

16. methods according to claim 14, wherein ovarian cancer cell is assessed in vitro, wherein ovarian cancer cell separated, copy, or from BG1, CAOV3 or IGR-OV1 cell strain.

17. methods according to claim 13, wherein platinum-based chemotherapy is carboplatin or taxol.

18. methods according to claim 13, wherein platinum-based chemotherapy is assisting therapy.

19. 1 kinds of methods of suffering from the test subject prediction of large bowel cancer (CRC), comprise and detect people KRAS gene let-7 paratope LCS6 sudden change in clinical samples, wherein said sudden change is the uridylic (U) in a kind of LCS6 of being included in site 4 or the single nucleotide polymorphism (SNP) that thymus pyrimidine (T) changes guanine (G) into, and the existence of wherein said sudden change shows that survival rate increases.

20. methods according to claim 19, wherein detecting step further comprises microsatellite instability (MSI) analysis

21. methods according to claim 19, wherein large bowel cancer (CRC) is commitment large bowel cancer.

22. methods according to claim 19, wherein large bowel cancer (CRC) is 1 phase or 2 phase large bowel cancers.

23. methods according to claim 19, wherein control group main body is not carried KRAS transgenation.

24. methods according to claim 23, wherein control group main body KRAS gene has secondary sudden change.

25. methods according to claim 19, wherein main body or patient KRAS gene have secondary sudden change.

26. methods according to claim 19, wherein main body or control group main body BRAF gene carry one or more sudden changes.

27. methods according to claim 19, wherein main body or control group main body have hyper-methylation RASSFlA promotor.

28. methods according to claim 19, wherein survival rate is overall survival rate, five year survival rate or the annual rate of depositing.

Predict that cancer cells carries out the method for the response of monoclonal antibody single therapy for 29. 1 kinds, comprise and detect people KRAS gene let-7 paratope LCS6 sudden change in clinical samples, wherein said sudden change is the uridylic (U) in a kind of LCS6 of being included in site 4 or the single nucleotide polymorphism (SNP) that thymus pyrimidine (T) changes guanine (G) into, the existence of wherein said sudden change, shows the susceptibility to monoclonal antibody single therapy.

30. methods according to claim 29, wherein cancer cells is large bowel cancer (CRC) cell.

31. methods according to claim 29, wherein cancer cells is evaluated in vitro or in vitro.

32. methods according to claim 29, wherein monoclonal antibody single therapy is Cetuximab.

33. 1 kinds of predicting tumors cells method to the response of chemotherapy combined mab treatment, comprise and detect people KRAS gene let-7 paratope LCS6 sudden change in clinical samples, wherein said sudden change is the uridylic (U) in a kind of LCS6 of being included in site 4 or the single nucleotide polymorphism (SNP) that thymus pyrimidine (T) changes guanine (G) into, and the existence of wherein said sudden change shows the resistance to combination therapy.

34. methods according to claim 33, wherein cancer cells is large bowel cancer (CRC) cell.

35. methods according to claim 33, wherein cancer cells is evaluated in vitro or in vitro.

36. methods according to claim 33, wherein monoclonal antibody single therapy is Cetuximab.

37. methods according to claim 33, wherein chemotherapy is a kind of cytotoxic agent.

38. according to the method described in claim 37, and wherein cytotoxic agent is irinotecan.

The method that the vasculogenesis risk of 39. 1 kinds of predicting tumors increases, comprises

(a) detect the sudden change in people KRAS gene let-7 paratope LCS6 in first patient sample, wherein said sudden change is the uridylic (U) in a kind of LCS6 of being included in site 4 or the single nucleotide polymorphism (SNP) that thymus pyrimidine (T) changes guanine (G) into;

(b) determine miRNA expression level, miRNA selects the group that in free second clinical samples, miR-23 and miR-27 form;

Wherein, with control group contrast, the existence suddenling change in described (a) and (b) increase of middle miRNA expression level show that angiogenesis inhibitor genetic transcription gene silencing increases, thereby prediction of tumor forms risk increase.

40. according to the method described in claim 39, and wherein angiogenesis inhibitor gene is Sprouty2 or Sema6A.

41. according to the method described in claim 39 or 40, and wherein tumour comprises a kind of cancer cells, and this cancer cells is derived from the cancer that acquired immune deficiency syndrome (AIDS) is relevant, breast cancer, digestion/GI cancer, anus cancer, appendix cancer, cholangiocellular carcinoma, colorectal carcinoma, large bowel cancer, esophagus cancer, carcinoma of gallbladder, islet cell tumor, Pancreatic Neuroendocrine Tumors, liver cancer, cancer of pancreas, the rectum cancer, carcinoma of small intestine, cancer of the stomach, endocrine system cancer, adrenocortical carcinoma, parathyroid carcinoma, pheochromocytoma, pituitary tumor, thyroid carcinoma, cancer eye, intraocular melanoma, retinoblastoma, bladder cancer, kidney (nephrocyte) cancer, penile cancer, prostate cancer, transitional cell renal plevis and carcinoma of ureter, carcinoma of testis, urethral carcinoma, the nephroblastoma, other children's tumor of kidney, germinocarcinoma, central nervous system cancer, extracranial germ cell knurl, Extragonadal germ cell tumor, ovarian germ cell tumors, gynecological tumor, cervical cancer, carcinoma of endometrium, gestational trophoblastic tumor, ovarian cancer, sarcoma of uterus, carcinoma of vagina, carcinoma vulvae, head and neck cancer, hypopharyngeal cancer, laryngocarcinoma, lip and oral carcinoma, invisible primary transitivity squamous neck cancer, oral carcinoma, nasopharyngeal carcinoma, oropharynx cancer, nasal sinus and CARCINOMA OF THE NASAL CAVITY, pharynx cancer, salivary-gland carcinoma, laryngocarcinoma, skeletal muscle cancer, osteocarcinoma, Ewing's sarcoma, gastrointestinal stromal tumors (GIST), osteosarcoma, malignant fibrous histiocytoma of bone, rhabdosarcoma, soft tissue sarcoma, sarcoma of uterus, neural system cancer, cerebral tumor, astrocytoma, brain stem glioma, central nervous system atypia monster sample/rhabdoid tumor, the Embryo tumour of central nervous system, central nervous system gonioma, craniopharyngioma, ependymoma, medulloblastoma, tumor of spinal cord, primitive neuroectodermal tumor and pinealoblastoma on curtain, neuroblastoma, respiratory system carcinoma, chest cancer, nonsmall-cell lung cancer, small cell lung cancer, malignant mesothe, thymoma, thymic carcinoma, skin carcinoma, Kaposi's sarcoma, melanoma, Merkel cell carcinoma.

42. according to the method described in claim 39 or 41, and wherein tumour is metastatic.

43. 1 kinds of methods of predicting that under anoxia condition, cancer cell is survived or propagation increases, comprise

(a) detect the sudden change in people KRAS gene let-7 paratope LCS6 in first patient sample, wherein said sudden change is the uridylic (U) in a kind of LCS6 of being included in site 4 or the single nucleotide polymorphism (SNP) that thymus pyrimidine (T) changes guanine (G) into, and

(b) determine the expression level of miR-210miRNA in second clinical samples,

Wherein contrast with control group, the increase that under anoxia condition, cancer cell is survived or bred is predicted in the middle existence suddenling change of described (a) and (b) increase of middle miRNA expression level.

44. according to the method described in claim 43, and wherein cancer cells stems from the cancer that acquired immune deficiency syndrome (AIDS) is relevant, breast cancer, digestion/GI cancer, anus cancer, appendix cancer, cholangiocellular carcinoma, colorectal carcinoma, large bowel cancer, esophagus cancer, carcinoma of gallbladder, islet cell tumor, Pancreatic Neuroendocrine Tumors, liver cancer, cancer of pancreas, the rectum cancer, carcinoma of small intestine, cancer of the stomach, endocrine system cancer, adrenocortical carcinoma, parathyroid carcinoma, pheochromocytoma, pituitary tumor, thyroid carcinoma, cancer eye, intraocular melanoma, retinoblastoma, bladder cancer, kidney renal cell carcinoma, penile cancer, prostate cancer, transition cell renal plevis and carcinoma of ureter, carcinoma of testis, urethral carcinoma, the nephroblastoma, other children's tumor of kidney, germinocarcinoma, central nerve neuroma, extracranial germ cell knurl, Extragonadal germ cell tumor, ovarian germ cell tumors, gynecological tumor, cervical cancer, carcinoma of endometrium, gestational trophoblastic tumor, ovarian cancer, sarcoma of uterus, carcinoma of vagina, carcinoma vulvae, incidence cancer, hypopharyngeal cancer, laryngocarcinoma, lip and oral carcinoma, invisible primary transitivity squamous neck cancer, oral carcinoma, nasopharyngeal carcinoma, oropharynx cancer, nasal sinus and CARCINOMA OF THE NASAL CAVITY, pharynx cancer, salivary-gland carcinoma, laryngocarcinoma, skeletal muscle cancer, osteocarcinoma, Ewing's sarcoma, gastrointestinal stromal tumor (GIST), osteosarcoma, malignant fibrous histiocytoma of bone, rhabdosarcoma, soft tissue sarcoma, sarcoma of uterus, neural cancer, cerebral tumor, astrocytoma, brain stem glioma, central nervous system atypia monster sample/rhabdoid tumor, central nervous system embryo's tumour, central nervous system gonioma, craniopharyngioma, ependymoma, medulloblastoma, tumor of spinal cord, primitive neuroectodermal tumor and pinealoblastoma on curtain, neuroblastoma, respiratory system carcinoma, chest cancer, nonsmall-cell lung cancer, small cell lung cancer, malignant mesothe, thymoma, thymic carcinoma, skin carcinoma, Kaposi's sarcoma, melanoma, Merkel cell carcinoma.

45. 1 kinds of methods of predicting that cancer cells survival or propagation increase, comprise

(a) sudden change in the let-7 paratope LCS6 of people KRAS gene in detection first patient sample, wherein said sudden change is the uridylic (U) in a kind of LCS6 of being included in site 4 or the single nucleotide polymorphism (SNP) that thymus pyrimidine (T) changes guanine (G) into, and

(b) determine the methylation state of tumor suppressor gene promotor in second clinical samples,

Wherein, with control group contrast, in described (a), the existence of sudden change and promotor (b) methylate increases survival or the propagation of predicting tumors cell.

46. according to the method described in claim 45, and wherein tumor suppressor gene is Notchl.

47. according to the method described in claim 45, and wherein cancer cells stems from the cancer that acquired immune deficiency syndrome (AIDS) is relevant, breast cancer, digestive system cancer/gi tract, anus cancer, appendix cancer, cholangiocellular carcinoma, colorectal carcinoma, large bowel cancer, esophagus cancer, carcinoma of gallbladder, islet cell tumor, Pancreatic Neuroendocrine Tumors, liver cancer, cancer of pancreas, the rectum cancer, carcinoma of small intestine, cancer of the stomach, endocrine system cancer, adrenocortical carcinoma, parathyroid carcinoma, pheochromocytoma, pituitary tumor, thyroid carcinoma, cancer eye, intraocular melanoma, retinoblastoma, bladder cancer, kidney renal cell carcinoma, penile cancer, prostate cancer, transition cell renal plevis and carcinoma of ureter, carcinoma of testis, urethral carcinoma, the nephroblastoma, other children's tumor of kidney, germinocarcinoma, central nerve neuroma, extracranial germ cell knurl, Extragonadal germ cell tumor, ovarian germ cell tumors, gynecological tumor, cervical cancer, carcinoma of endometrium, gestational trophoblastic tumor, ovarian cancer, sarcoma of uterus, carcinoma of vagina, carcinoma vulvae, incidence cancer, hypopharyngeal cancer, laryngocarcinoma, lip and oral carcinoma, invisible primary transitivity squamous neck cancer, oral carcinoma, nasopharyngeal carcinoma, oropharynx cancer, nasal sinus and CARCINOMA OF THE NASAL CAVITY, pharynx cancer, salivary-gland carcinoma, laryngocarcinoma, skeletal muscle cancer, osteocarcinoma, Ewing's sarcoma gastrointestinal stromal tumor (GIST), osteosarcoma, malignant fibrous histiocytoma of bone, rhabdosarcoma, soft tissue sarcoma, sarcoma of uterus, neural cancer, cerebral tumor, astrocytoma, brain stem glioma, central nervous system atypia monster sample/rhabdoid tumor, central nervous system embryo's tumour, central nervous system gonioma, craniopharyngioma, ependymoma, medulloblastoma, tumor of spinal cord, primitive neuroectodermal tumor and pinealoblastoma on curtain, neuroblastoma, respiratory system carcinoma, chest cancer, nonsmall-cell lung cancer, small cell lung cancer, malignant mesothe, thymoma, thymic carcinoma, skin carcinoma, Kaposi's sarcoma, melanoma, Merkel cell carcinoma.

48. according to the method described in claim 45, and wherein survival comprises maintenance tumorigenesis potential.

49. according to the method described in claim 45 or 48, and wherein cancer cells is cancer stem cell.

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