GB2541463A - Novel methods - Google Patents

Abstract

A method for identifying a subject having breast cancer, or at risk of developing breact cancer comprising determining function and/or expression of and/or nucleotide sequence variation within one or more of the genes identified in Tables 1 or 4. Also claimed is a method for diagnosing metaststic breast cancer by determining function and/or expression of and/or nucleotide sequence variation within one or more of the genes identified in Tables 7 or 9 a method for identifying a lead candidate with efficacy in the treatment of breast cancer and a method of treating breast cancer in a subject who has been identified as having breast cancer.

Description

NOVEL METHODS

Field of Invention

The present invention relates to methods for the diagnosis, prognosis and treatment of breast cancer.

Background

Breast cancer (BC) represents the highest incident tumour for females in developed economies such as Europe, United States and Saudi Arabia. BC is a complex heterogeneous disease, encompassing a diverse spectrum of different subtypes with distinct histological, biological, and clinical outcomes. Traditional well-established classification systems and the currently used prognostic/predictive factors are insufficient to reflect the biological and clinical heterogeneity of BC. Tumours of similar clinico-pathological features show different behaviour in terms of outcome and response to specific therapy. Up to one-third of lymph node-negative BC patients, classified as being within the good prognostic group, have been reported to develop recurrence later in their disease course [1]. An equivalent proportion of node-positive patients, assigned into the poor prognosis group, remain free of distant metastases thus underpinning the challenging situation regarding BC prognostication [2], Approximately 60% of patients with BC present as node negative, 80% as oestrogen receptor positive and more than 40% as grade 2 tumours. Independent studies have investigated the genetic profile of ER+ve/luminal breast tumours (the most common molecular subtype) and the impact of genetic makeup on the biologic heterogeneity of these tumours [3] [4] [5]. While heterogeneity of triple negative BC [6] and HER2 enriched BC [7] have been explored there is however, a requirement to identify new candidates with relevance to tumour behaviour (diagnostic/prognostic value) and therapeutic responses that are predicated upon specific genomic signatures.

Several genes have been identified as significant molecular contributors to the development and progression of BC including ER, HER2, PIK3CA and AKT1 yet the ability to identify driver mutations remains challenging [8], In a concerted effort to explore the heterogeneity of BC, microarray expression studies involving several thousand tumour cases have pointed towards the existence of intrinsic subtypes [9], thus, underpinning why BC is regarded as a histologically, biologically, and clinically heterogeneous disease.

Molecular heterogeneity of tumours represents a significant challenge to the clinical management of patients. This has been highlighted in renal tumours of a single patient where RNA expression studies, immunohistochemistry and ploidy comparisons were undertaken in primary and distant metastases. Good and poor prognoses signatures were obtained from the same tumour samples suggesting the existence of genetic variation both intra- (spatial) and inter- (temporal) humoral heterogeneity [10]. The dynamic nature of cancer mutational landscapes has also been observed in non-small cell lung cancer (NSCLC). Somatic alterations within tumour genomes were shown to be non-static and branched chain evolutionary processes could be detected. Driver mutations could be identified that were critical to the initial stages of tumour development and critical for the early stages of cancer progression. As the tumour evolved and new molecular pathways became activated, the earlier key genetic players were hypothesised to play less of a critical role. This study suggested that tumour evolution may be not only exhibit heterogeneity in the mutational profile intra- and inter-tumourally, but also with respect to the relative importance of specific pathways that are exploited by cancers during their life cycle. Consequently, the ability to target key gene pathways therapeutically would appear to be not only contingent upon the specific mutation/pathway involved but also the timing in the tumour life-cycle is also a critical factor [11], The latter will have specific ramifications with respect to response patterns to a given therapeutic regimen.

Routine pathology necessitates sampling of tumour tissue in order make assessment regarding the histological grade (tumour differentiation) and aggressiveness (invasive behaviour with respect to surrounding tissue). In order to derive this information consistently between samples that would represent the ideal scenario and a single sample would suffice and be genetically representative of the tumour as whole. Such an idealised scenario would facilitate informed decisions to be taken regarding the clinical management of the patient in question. Yates et al have undertaken a combination of whole genome sequencing combined with targeted re-sequencing to explore genomic variation using 50 individual BC patients (comprising 303 samples). Although mutations were noted in genes commonly associated with BC (e.g. PIK3CA, TP53, PTEN, BRCA2 and MYC), importantly no temporal order was noted and 13/50 tumours with therapeutically targetable mutations were sub-clonal in nature [12], Interestingly, while there was sub-clonal heterogeneity, these authors noted that driver mutations associated with chemotherapy and metastatic potential did arise within detectable sub-clones of ancestral lesions. Consequently, sub-clonal variation needs to be taken into consideration when considering therapeutic options for the treatment of BC.

Molecular heterogeneity in BC has been shown to not only impact growth promoting regulatory networks directly but also to abrogate loci that are important in suppressing tumour proliferation. The progesterone receptor (PR) is a downstream target for the oestrogen receptor (ERa) and binding to ERa nuclear receptor binding sites located within the DNA promoter of PR augments gene activation. Recent studies suggest that PR is capable of regulating ERa activity directly through protein-protein interaction (through the formation of ER-PR receptor heterodimers) that results in an altered ERa mediated gene expression pattern. Such attenuation of ERa activity and its differential gene expression patterns results a favourable clinical outcome. Copy number variations resulting in the loss of PR are frequent events in BC and may therefore provide a rationale for developing improved therapeutic strategies implementing the combined use of agonists for PR and ERa antagonists [13].

The studies highlighted above would support the contention that molecular heterogeneity in BC is dynamic and potentially key driver mutations may be detected throughout the course of tumour development. A firm characterisation is therefore required of primary breast tumours and their metastatic variants in order to identify potential pathways that are crucial for progression. Pathway activation/repression may vary in a temporal/spatial manner but when contextualised (e.g. a specific panel of gene mutations occurring at a specific time point in the evolutionary cycle) then therapeutic strategies may be achievable. Exome sequencing has the potential to rapidly screen coding regions of the human genome for variants that can impact protein function. Such a strategy could provide the foundation for novel insight into the biological pathways that are important for disease development and tumour dissemination. The mapping and characterisation of molecular changes within the genomes of BC patients may facilitate the identification of key loci and their associated pathways linked to phenotypes of clinical interest. Such alterations in DNA sequence may include, for example, single point mutations/polymorphisms (SNP), INDEL (insertion/deletion), inversions, copy number variations and chromosomal translocations. Characterisation studies that explore the biological changes occurring in tumour cells at temporal/spatial levels may provide essential information regarding biological variation integral to tumour development, progression and ultimately therapeutic response patterns.

There are several areas where novel genetic signatures (biomarkers) might be exploited in order to improve the clinical management of cancer patients:

Genetic variants indicative of aggressive disease phenotypes: Variant profiling could assist in the triaging patients into groups that may or may not require aggressive therapeutic intervention and even the possibility that the disease is likely to exhibit recurrence. The ability to effectively prognosticate response patterns would provide the foundation for the development of companion diagnostic assays. Such tests may facilitate in the pre-selection of patients towards suitable treatments and appropriate forms of clinical management specifically predicated to a particular genetic profile. These patients would also be monitored as potentially high risk for disease recurrence.

Drug targets: Identification of germline/somatic mutations that are linked to poor prognosis (e.g. pathways that may promote metastasis or increase the rate at which therapeutic resistance emerges) could provide a platform to identify targets linked with these detrimental phenotypes. They may also indicate patients at risk of minimal disseminated disease (MDD) and thus would warrant that they remain under close clinical observation. Characterising mutated genes and their associated biological pathways that are intrinsically linked with unfavourable outcomes would facilitate the development of strategies (and potentially novel pharmaceutical compounds) with the ability to control disease progression (e.g. circumventing drug resistance).

Molecular variation: Clinical differences at the intra- and inter-patient level are primarily caused by the genetic composition of an individual patient tumour. Complexity is increased further through chromosomal instability which leads to unregulated changes in the primary cancer genome promoting the creation of sub-clones with variable genomic (mutational) profiles. The consequence of such a dynamic genotype would translated into a variable phenotype (e.g. metastatic potential; therapeutic resistance/response and dosing levels). Concordant/discordant variants between BC cases may therefore provide information regarding genic loci that are important in the development, progression and therapeutic response of BC.

Genomic screening technologies have emerged that are capable of exploring variant signatures in patients’ tumour samples. These range from either a re-sequencing of specific gene combinations (e.g. panels of tumour suppressor genes) to whole genome sequencing. Exome sequencing represents another powerful category of these targeted re-sequencing methodologies and focuses upon the specific profiling of the exonic regions of genes. These include protein coding, 573’ UTR (un-translated regions) and micro-RNAs as part of epigenetic control mechanisms. Computational algorithms are a fundamental requisite of the discovery pipeline and are necessitated in order to expedite the identification of variants of interest given the massive amounts of data that are generated in sequencing experiments. In combination with gene annotation, filtering and in-silico prediction of variant effects on protein function, the methodology provides a powerful screening tool to identify candidates that may be of biological and/or clinical significance. Variants may for example occur in positions that result in the loss of a translational start/stop site, code for rare amino acids, introduce frame-shift mutations or impact in a detrimental manner splice donor/acceptor sites. Other variant impacts include synonymous amino acid changes which are believed to be low impact variant changes to those that can result in the production of in-frame deletions not affect catalytic domains or overall protein conformation but may modulate protein activity (moderate impact variants). Therefore, screening exercises play a pivotal role in identifying variant changes. With appropriate validation studies, the role of genes that may be potentially important molecular components for the development, progression and even therapeutic response patterns of cancers can be elicited.

The present invention seeks to provide new methods for the diagnosis, prognosis and treatment of breast cancer.

Summary of Invention A first aspect of the invention provides an in vitro method for identifying a subject having breast cancer, or at risk of developing the same, the method comprising the steps of: (a) providing a sample of cells from a subject to be tested; (b) testing the cells to determine the function and/or expression of, and/or nucleotide sequence variation within, one or more of the genes identified in Table 1 wherein modulation of the function and/or expression of the one or more of the genes identified in Table 1 compared to a reference value, and/or the presence of a variant sequence in one or more of the genes identified in Table 1, is indicative of the subject having breast cancer, or being at risk of developing the same. TABLE 1

Genes modulated in primary breast tumours (A) Genes carrying INsertions/DELetions (INDELs) _ MSS51 _PPP1R9B_ (B) Genes carrying single nucleotide polymorphisms (SNPs)

FAM173B

In the above table (and those below), genes are identified by reference to an abbreviation (‘Gene ID’); the full name of the gene can be obtained from recognised gene databases (such as GeneCards®).

By “breast cancer” we include all malignancies of the breast tissue, including both carcinomas and sarcomas.

Carcinomas are cancers that arise from the epithelial component of the breast. The epithelial component consists of the cells that line the ducts, lobules and terminal ducts; under normal conditions, these epithelial cells are responsible for making milk. Carcinomas comprise the vast majority of all breast cancers, and will be further discussed below. Sarcomas are rare cancers that arise from the stromal (connective tissue) components of the breast. These stromal component cells include myofibroblasts, stromal connective/supportive tissues, and blood vessel cells, and cancers arising from these "supportive" cells include phyllodes tumours and angiosarcoma and others. Sarcomas account for less than 1% of primary breast cancers.

Within the large group of carcinomas, there are many different types of breast cancer. The first major division is between in situ and invasive carcinoma. In situ carcinoma is "pre-invasive" carcinoma that has not yet invaded the breast tissue. These in situ cancer cells grow inside of the pre-existing normal lobules or ducts. In situ carcinoma has significant potential to become invasive cancer, and that is why it must be adequately treated to prevent the patient from developing invasive cancer. Invasive cancers have cancer cells that infiltrate outside of the normal breast lobules and ducts to grow into the breast connective tissue. Invasive carcinomas have the potential to spread to other sites of the body, such as lymph nodes or other organs, in the form of metastases.

Approximately 80% of breast carcinomas are invasive ductal carcinoma, followed by invasive lobular carcinomas which account for approximately 10-15% of cases. Invasive ductal carcinomas and invasive lobular carcinomas have distinct pathologic features. Specifically, classical lobular carcinomas grow as single cells arranged individually, in single file, or in sheets, and they have different molecular and genetic aberrations that distinguish them from ductal carcinomas. Ductal and lobular carcinomas may have different prognoses and treatment options, depending upon all of the other features of the particular cancer.

The remaining cases of invasive carcinoma are comprised of other special types of breast cancer that are characterized by unique pathologic findings. These special types include colloid (mucinous), medullary, micropapillary, papillary, and tubular carcinomas. It is important to distinguish between these various subtypes, because they can have different prognoses and treatment implications.

Breast cancers may be classified according to several different grading systems, including:

Histopathology. Breast cancer is usually classified primarily by its histological appearance. Most breast cancers are derived from the epithelium lining the ducts or lobules, and these cancers are classified as ductal or lobular carcinoma. Carcinoma in situ is growth of cancerous or precancerous cells within a particular tissue compartment such as the mammary duct without invasion of the surrounding tissue. In contrast, invasive carcinoma does not confine itself to the initial tissue compartment.

Grade. Grading compares the appearance of the breast cancer cells to the appearance of normal breast tissue. Normal cells in an organ like the breast become differentiated, meaning that they take on specific shapes and forms that reflect their function as part of that organ. Cancerous cells lose that differentiation. In cancer, the cells that would normally line up in an orderly way to make up the milk ducts become disorganized. Cell division becomes uncontrolled. Cell nuclei become less uniform. Pathologists describe cells as well differentiated (low grade), moderately differentiated (intermediate grade), and poorly differentiated (high grade) as the cells progressively lose the features seen in normal breast cells. Poorly differentiated cancers (the ones whose tissue is least like normal breast tissue) have the worst prognosis.

Stage. Breast cancer staging using the TNM system is based on the size of the tumour (T), whether or not the tumour has spread to the lymph nodes (N) in the armpits, and whether the tumour has metastasised (M) (i.e. spread to a more distant part of the body). Larger size, nodal spread, and metastasis have a larger stage number and a worse prognosis.

The main stages are:

Stage 0 is a pre-cancerous or marker condition, either ductal carcinoma in situ (DCIS) or lobular carcinoma in situ (LCIS);

Stages 1-3 are within the breast or regional lymph nodes; and Stage 4 is 'metastatic' cancer that has the least favourable prognosis.

Where available, imaging studies may be employed as part of the staging process in select cases to look for signs of metastatic cancer. However, in cases of breast cancer with low risk for metastasis, the risks associated with PET scans, CT scans, or bone scans outweigh the possible benefits, as these procedures expose the patient to a substantial amount of potentially dangerous ionizing radiation. Receptor status. Breast cancer cells have receptors on their surface and in their cytoplasm and nucleus. Chemical messengers such as hormones bind to receptors, and this causes changes in the cell differentiation/proliferation. Breast cancer cells may or may not have three important receptors: estrogen receptor (ER), progesterone receptor (PR), and HER2. ER+ cancer cells (that is, cancer cells that have estrogen receptors) depend on estrogen for their growth, so they can be treated with drugs to block estrogen effects (e.g. tamoxifen), and generally have a better prognosis. Untreated, HER2+ breast cancers are generally more aggressive than HER2- breast cancers,[81 ][82] but HER2+ cancer cells respond to drugs such as the monoclonal antibody trastuzumab (in combination with conventional chemotherapy), and this has improved the prognosis significantly.[83] Cells that do not have any of these three receptor types (estrogen receptors, progesterone receptors, or HER2) are called triple-negative, although they frequently do express receptors for other hormones, such as androgen receptor and prolactin receptor.

In one embodiment, the breast cancer is selected from the group consisting of invasive ductal carcinomas and invasive lobular carcinomas.

In a further embodiment, the breast cancer may manifest itself solely as a primary tumour (i.e. in the absence of any metastases).

By “modulation” of the genes identified in Table 1, we include that the function and/or expression of the gene(s) may be upregulated or inhibited relative to a reference value (e.g. in comparable cells from a healthy subject). Thus, by "reference value” we include comparable measures of the function and/or expression of said gene(s) in cells from a subject who does not have breast cancer. Such measures of gene function and/or expression may include direct measurements of protein function and/or amount and/or mRNA amount, as well as indirect measures such as nucleotide sequence information for the mRNA and/or genomic DNA associated with said gene(s).

It will be appreciated by persons skilled in the art that the function and/or expression of the gene(s) may be determined at the level of protein function and/or mRNA expression and/or DNA sequence. For example, the function and/or expression of the gene(s) may be determined by analysis of the genomic DNA to identify sequence variants that impact negatively on the expression and/or function of the gene(s), e.g. the presence of insertions/deletions (INDELs) and/or single nucleotide polymorphisms (SNPs) that result in abrogation of gene expression.

It will be understood by persons skilled in the art that the subject to be tested may be any species of mammal; typically, however, the subject is human.

In the methods of the first aspect of the invention, the initial step comprises the provision of a sample of protein and/or mRNA and/or DNA from a subject to be tested; typically, the sample contains cells from the subject to be tested that are suspected of being tumour cells.

In one embodiment, the sample of cells in step (a) is from a tumour biopsy sample.

In an alternative embodiment, the sample in step (a) is from a blood sample.

In a further embodiment, the sample in step (a) is from urine or saliva.

Typically, the sample of cells from the subject to be tested is processed in some manner prior to undertaking step (b), with the nature of the processing being dependent upon the type of testing to be performed (e.g. genomic sequencing, proteome analysis, etc.).

In one embodiment, DNA is purified from the sample of cells for analysis in step (b). For example, genomic DNA may be prepared from the sample, as described in Youssif et at., 2009, Genes Chromosomes Cancer 48(100):1018-1026 (the disclosures of which are incorporated herein by reference). Sequencing of part or all of the genomic DNA (e.g. whole genome sequencing, exome sequencing, sequencing of a panel of selected genes, analysis of selected single nucleotide polymorphisms [SNPs], etc.) may then be conducted using methods known in the art, for example as described in Barnes, 2010, Methods Mol. Biol. 628:1-20 (the disclosures of which are incorporated herein by reference). For example, analysis of the DNA may comprise or consist of exome sequencing (e.g. using next-generation sequencers, such as those produced by lllumina and Life Technologies), PCR-based methods or hybridisation-based methods.

In an alternative embodiment, mRNA is purified from the sample of cells for analysis in step (b), e.g. as described in Analysing Gene Expression, A Handbook of Methods: Possibilities and Pitfalls (2003), edited by Lorkowski &. Cullen, Wiley (see Chapters 2 to 4 therein) (the disclosures of which are incorporated herein by reference). For example, analysis of the mRNA may comprise or consist of sequencing (e.g. using next-generation sequencers, such as those produced by lllumina and Life Technologies), PCR-based methods or hybridisation-based methods.

In a further embodiment, proteins are purified from the sample of cells for analysis in step (b), e.g. as described in Analysing Gene Expression, A Handbook of Methods: Possibilities and Pitfalls (2003), edited by Lorkowski &. Cullen, Wiley (see Chapter 5 therein) (the disclosures of which are incorporated herein by reference). For example, analysis of the protein(s) produced by the cells may comprise or consist of antibody-based methods to detect the presence of the protein products of the genes of Table 1.

One preferred embodiment of the methods of the invention comprises testing a DNA sample from the subject to determine the presence of a variant sequence in the coding region and/or promoter region of one or more of the genes identified in Table 1, wherein the presence of a variant sequence in the coding region and/or promoter region of one or more of the genes identified in Table 1 is indicative of the subject having breast cancer, or being at risk of developing the same. For example, step (b) may comprises testing DNA from the subject to determine the presence of a variant sequence in at least two of the genes identified in Table 1, for example at least three of the genes identified in Table 1.

By “sequence variant” or “variant sequence" we include a difference in nucleotide sequence relative to a corresponding reference sequence (e.g. the corresponding sequence in normal, healthy cells). The nature of the sequence variation may be an insertion of one or more nucleotides at a given location within the gene, a deletion of one or more nucleotides at a given location within the gene, and/or a substitution of one or more nucleotides at a given location within the gene, wherein the sequence variation results in modulation of the function and/or expression of the gene. It will be appreciated by persons skilled in the art that such modulation of gene function/expression may arise from sequence variation in the coding region of the gene and/or in the promoter region of said gene.

In one embodiment, the variant sequence is detrimental to the function and/or expression of the gene. For example, the variant sequence may inhibit (in whole or in part) expression of the gene, such that level of its protein product within the tumour cells is reduced. Alternatively, the variant sequence may result in production of a modified protein product of the gene having reduced or no biological activity (for example, the protein may be a truncated version of the gene product with reduced or absent any biological activity).

In an alternative embodiment, the variant sequence may enhance the function and/or expression of the gene. For example, the variant sequence may be a deletion that removes a phosphorylation site from the gene product or an amino acid substitution that confers enhanced activity.

Advantageously, step (b) comprises exome sequencing (for example, using a MiSeq DNA sequencer from lllumina).

Alternatively, step (b) may comprise the use of a PCR-based assay to assess sequence variation within the genes.

Analysis of DNA sequence variation may be undertaken using a bioinfomnatics approach, such as those described in Bioinformatics and Computational Biology Solutions Using R and Bioconductor (2005), edited by Gentleman, Carey, Huber, Irizarry, Dudoit, Springer (the disclosures of which are incorporated herein by reference) and Analysing Gene Expression, A Handbook of Methods: Possibilities and Pitfalls (2003), edited by Lorkowski &. Cullen, Wiley (see Chapter 7 therein) (the disclosures of which are incorporated herein by reference).

Conveniently, step (b) comprises the use of an algorithm to detect sequence variation within the genes (for example, ‘SNPeffect’ may be used for phenotyping human single nucleotide polymorphisms, insertions and deletions; see Baets et al., 2012, Nucleic Acids Res. 40(1):D935-9, the disclosures of which are incorporated herein by reference).

In one embodiment, step (b) comprises testing for the presence of insertions or deletions (“INDELs”) in the coding region and/or the promoter region of one or more of the genes identified in Table 1(a). For example, step (b) may comprise testing DNA from the subject to determine the presence of an INDEL in the coding region and/or the promoter region of all of the genes identified in Table 1 (a).

Thus, step (b) may comprise testing DNA from the subject to determine the presence of an INDEL in one or more genes selected from the group consisting of MSS51, GP6, PPP1R9B and PRUNE2 (see Example below).

In a specific embodiment, however, step (b) does not consist solely of testing DNA from the subject to determine the presence of a variant sequence in PPP1R9B.

Preferably, the INDELs are classified as ‘high impact’, suggestive of a greater likelihood of them having a functional effect on the gene.

Examples of high impact INDELs are identified in Table 2 below. TABLE 2

High Impact INDELs in concordant genes in primary breast tumours

* "exonic" = INDEL predicted to result in exonic frameshift

Thus, step (b) may comprise testing DNA from the subject to determine the presence of all of the INDELS identified in Table 2.

Additionally, or alternatively, step (b) may comprise testing DNA from the subject to determine the presence of a SNP in the coding region and/or the promoter region of a gene identified in Table 1(b).

For example, step (b) may comprise testing DNA from the subject to determine the presence of a SNP in one or more genes selected from the groups consisting of FAM173B, OBSCN, ACVR1C and CAP1.

Preferably, the SNPs are classified as ‘high impact’, suggestive of a greater likelihood of them having a functional effect on the gene.

Examples of high impact SNPs are identified in Table 3 below. TABLE 3

High Impact SNPs in concordant genes in primary breast tumours

* Predicted to result in gain of new stop codon

Thus, step (b) may comprise testing DNA from the subject to determine the presence of all of the SNPs identified in Table 3.

Advantageously, step (b) comprises testing DNA from the subject to determine the presence of a variant sequence in all of the genes identified in Table 1. For example, step (b) may comprise testing DNA from the subject to determine the presence of all of the INDELs identified in Table 2 and all of the SNPs identified in Table 3.

In one preferred embodiment, step (b) comprises testing the cells to determine the function and/or expression of, and/or nucleotide sequence variation within, one or more of the genes identified in Table 4, wherein modulation of the function and/or expression of the one or more of the genes identified in Table 4 compared to a reference value {e.g. in comparable cells from a healthy subject), and/or the presence of a variant sequence in one or more of the genes identified in Table 4, is indicative of the subject having breast cancer, or being at risk of developing the same. TABLE 4

Additional genes with diagnostic value in primary breast tumours

Thus, step (b) may comprise testing DNA from the subject to determine the presence of a variant sequence in the coding region and/or promoter region of one or more of the genes identified in Table 4 (for example, using exome sequence or PCR-based assays). The variant sequences may be detrimental to the function of the gene or the product thereof.

In one embodiment, step (b) comprises testing for the presence of INDELs in the coding region and/or the promoter region of one or more of the genes identified in Table 4(a). For example, step (b) may comprise testing DNA from the subject to determine the presence of an INDEL in the coding region of all of the genes identified in Table 4(a).

Preferably, the INDELs are classified as ‘moderate impact’, suggestive of a possibility of them having a functional effect on the gene.

Examples of moderate impact INDELs are identified in Table 5 below. TABLE 5

Moderate Impact INDELs associated with primary breast tumours

* Predicted to result in disruptive in frame deletion ** Predicted to result in disruptive inframe insertion

Thus, step (b) may comprise testing DNA from the subject to determine the presence of all of the INDELS identified in Table 5.

In one embodiment, step (b) comprises testing DNA from the subject to determine the presence of a SNP in the coding region and/or the promoter region of one or more of the genes identified in Table 4(b), for example at least two, three, four, five, ten, twenty, thirty, forty, fifty, sixty, seventy, eighty, ninety, one-hundred or more of the genes identified in Table 4(b).

For example, step (b) may comprise testing DNA from the subject to determine the presence of a SNP in all of the genes identified in Table 4(b).

Preferably, the SNPs are classified as 'moderate impact’, suggestive of a possibility of them having a functional effect on the gene.

Examples of moderate impact SNPs are identified in Table 6 below (each of which results in a missense variant). TABLE 6

Moderate Impact SNPs associated with primary breast tumours

Thus, step (b) may comprise testing DNA from the subject to determine the presence of all of the SNPs identified in Table 6.

Advantageously, step (b) comprises testing DNA from the subject to determine the presence of a variant sequence in all of the genes identified in Table 4. For example, step (b) may comprise testing DNA from the subject to determine the presence of all of the INDELs identified in Table 5 and all of the SNPs identified in Table 6. A second, related aspect of the invention provides an in vitro method for monitoring disease progression in a subject with breast cancer comprising: (a) providing a first sample of cells from a subject to be tested; and (b) testing the cells provided in step (a) to determine the function and/or expression of, and/or nucleotide sequence variation within, one or more of the genes identified in Table 1 and/or Table 4; (c) providing a further sample of cells, obtained subsequently, from a subject to be tested; and (d) testing the cells provided in step (c) to determine the function and/or expression of, and/or nucleotide sequence variation within, one or more of the genes identified in Table 1 and/or Table 4 wherein modulation of the function and/or expression of, and/or nucleotide sequence within, the one or more of the genes identified in Table 1 and/or Table 4 in the further sample of cells compared to the function and/or expression of, and/or nucleotide sequence within, said genes in the first sample of cells is indicative of disease progression in the period between collection of the cell samples.

It will be appreciated by persons skilled in the art that such temporal monitoring of gene function/expression not only provides means for monitoring treatment progression but also enables the efficacy of a treatment regime to be monitored.

In one embodiment, step (b) is performed as defined above in relation to the first aspect of the invention. A third, related aspect of the invention provides a method for treating a subject with breast cancer, or at risk of developing the same, comprising: (a) identifying the subject as having breast cancer, or at risk of developing the same, using a method according to the first aspect of the invention; and (b) administering to the subject a medicament with efficacy in the treatment of the breast cancer and/or surgically removing the tumour from said subject.

In one embodiment, the breast cancer is selected from the group consisting of invasive ductal carcinomas and invasive lobular carcinomas.

It will be appreciated by persons skilled in the art that subjects identified as having breast cancer may be treated with any medicament known to have efficacy in the treatment of the breast cancer, including conventional anti-cancer drugs (such as chemotherapeutic agents, immunotherapeutic agents and radiotherapeutic agents).

Cytotoxic chemotherapeutic agents useful as anticancer agents include: alkylating agents including nitrogen mustards such as mechlorethamine (HN2), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin) and chlorambucil; ethylenimines and methylmelamines such as hexamethylmelamine, thiotepa; alkyl sulphonates such as busulfane; nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU) and streptozocin (streptozotocin); and triazenes such as decarbazine (DTIC; dimethyltriazenoimidazole-carboxamide); Antimetabolites including folic acid analogues such as methotrexate (amethopterin); pyrimidine analogues such as fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR) and cytarabine (cytosine arabinoside); and purine analogues and related inhibitors such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG) and pentostatin (2’-deoxycoformycin). Natural Products including vinca alkaloids such as vinblastine (VLB) and vincristine; epipodophyllotoxins such as etoposide and teniposide; antibiotics such as dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin) and mitomycin (mitomycin C); enzymes such as L-asparaginase; and biological response modifiers such as interferon alphenomes. Miscellaneous agents including platinum coordination complexes such as cisplatin (cis-DDP) and carboplatin; anthracenedione such as mitoxantrone and anthracycline; substituted urea such as hydroxyurea; methyl hydrazine derivative such as procarbazine (N-methylhydrazine, MIH); and adrenocortical suppressant such as mitotane (o,p’-DDD) and aminoglutethimide; taxol and analogues/derivatives; and hormone agonists/antagonists such as flutamide and tamoxifen.

Such chemotherapeutic agents may be given alone or in combinations. For example, common chemotherapy regimens include: • AT: Adriamycin and Taxotere • AC ± T: Adriamycin and Cytoxan, with or without Taxol or Taxotere • CMF: Cytoxan, methotrexate, and fluorouracil • CEF: Cytoxan, Ellence, and fluorouracil • FAC: fluorouracil, Adriamycin, and Cytoxan • CAF: Cytoxan, Adriamycin, and fluorouracil (The FAC and CAF regimens use the same medicines but use different doses and frequencies) • TAC: Taxotere, Adriamycin, and Cytoxan • GET: Gemzar, Ellence, and Taxol

Depending on the characteristics of the breast cancer, a targeted immunotherapeutic agent such as Herceptin® (trastuzumab) may be administered, either alone or in combination with one or more chemotherapeutic agents. For example, a “TCH” regimen may be administered, which includes Taxotere, Herceptin, and carboplatin.

Certain radioactive atoms may also be cytotoxic if delivered in sufficient doses. Thus, the cytotoxic medicament may comprise a radioactive atom which, in use, delivers a sufficient quantity of radioactivity to the target site so as to be cytotoxic. Suitable radioactive atoms include phosphorus-32, iodine-125, iodine-131, indium-111, rhenium-186, rhenium-188 or yttrium-90, or any other isotope which emits enough energy to destroy neighbouring cells, organelles or nucleic acid.

The treatment regime will be selected based on a number of factors, notably the type of tumour, the stage of the disease and the age of the patient.

Additionally, or alternatively, the subject may undergo surgery to remove the tumour(s).

The subject may also receive radiotherapy, often upon completion of a course of chemotherapy or surgical removal of the tumour(s).

It will be appreciated that the methods of the third aspect of the invention may further comprise step (c) of providing a second sample of cells obtained from the subject after the commencement of treatment, testing said cells to determine the function and/or expression of, and/or nucleotide sequence variation within, one or more of the genes identified in Table 1 and/or 4, and comparing the results of said testing with the results obtained in step (a).

In one embodiment, the method further comprises maintaining or modifying the treatment of the subject in order to optimise its therapeutic efficacy.

Advantageously, step (c) is repeated one or more times in order to monitor the therapeutic efficacy of the treatment received by the subject over a period of time, e.g. until the subject is considered no longer to be at risk from the tumour(s). A fourth aspect of the invention provides a method for identifying a lead candidate with efficacy in the treatment or prevention of breast cancer comprising: (a) providing a compound to be tested; and (b) determining in vitro whether said test compound modulates the function and/or expression of one or more of the genes identified in Table 1 and/or 4, wherein the test compound is identified as a lead candidate with efficacy in the treatment or prevention of breast cancer if it modulates the function and/or expression of one or more of the genes identified in Table 1 and/or 4.

By “lead candidate” we include pharmaceutical and/or biopharmaceutical agents which have potential efficacy in the treatment of breast cancer, and thus represent a promising candidate for drug development and optimisation.

Conveniently, the method comprises the use of a high-throughput screening assay.

Step (b) may be performed using methods well-known in the art for assessing the function and/or expression of genes and the products thereof.

In one embodiment, step (b) is performed as described above in relation to the methods of the first aspect of the invention.

In another embodiment, the method further comprises step (c) of testing the lead candidate for efficacy in an in vivo model of said breast cancer.

In addition to providing methods for the diagnosis and treatment of subjects with primary breast tumours, the invention also provides methods for the diagnosis and prognosis of subjects with metastatic breast cancer.

Thus, a fifth aspect of the invention provides an in vitro method for the diagnosis of a subject with metastatic breast cancer, or prognosis for said subject, comprising: (a) providing a sample of cells from a subject to be tested; (b) testing the cells to determine the function and/or expression of, and/or nucleotide sequence variation within, a gene identified in Table 7 wherein modulation of the function and/or expression of the gene identified in Table 7 compared to a reference value, and/or the presence of a variant sequence in the gene identified in Table 7, is indicative of the subject having metastatic breast cancer, or of developing the same. TABLE 7

Genes with diagnostic and/or prognostic value in metastatic breast cancer

By “metastatic” we mean that the breast cancer is characterised by cancerous cells spreading from the site of the primary tumour within the breast to other areas of the body, such as the lymph nodes, lungs and liver (also referred to as “Stage IV breast cancer” or “advanced breast cancer”).

By “prognosis” we include the prediction of disease outcome (e.g. life expectancy) in a subject with breast cancer. For example, the method may be used to identify subjects with an increased risk of death from the cancer within a defined period, e.g. within one year of the prognosis. Such knowledge can be invaluable in terms of deciding upon treatment strategies and the level of follow-up monitoring of patients during and after completion of treatment.

In one embodiment, the breast cancer is selected from the group consisting of invasive ductal carcinomas and invasive lobular carcinomas.

Thus, the method allows the identification of patients with breast cancer in need of an aggressive treatment regime (for example, high doses and/or combination therapies) and/or regular monitoring of treatment efficacy/disease progression.

It will be appreciated by persons skilled in the art that the subject to be tested may be any species of mammal; typically, however, the subject is human.

In the methods of the fifth aspect of the invention, the initial step comprises the provision of a sample of protein and/or mRNA and/or DNA from a subject to be tested; typically, the sample contains cells from the subject to be tested that are suspected of being tumour cells.

In one embodiment, the sample of cells in step (a) is from a tumour biopsy sample.

In an alternative embodiment, the sample in step (a) is from a blood sample.

In a further embodiment, the sample in step (a) is from urine or saliva.

Typically, the sample of cells from the subject to be tested is processed in some manner prior to undertaking step (b), with the nature of the processing being dependent upon the type of testing to be performed (e.g. genomic sequencing, proteome analysis, etc.).

In one embodiment, DNA is purified from the sample of cells for analysis in step (b).

In an alternative embodiment, mRNA is purified from the sample of cells for analysis in step (b).

In a further embodiment, proteins are purified from the sample of cells for analysis in step (b). A preferred embodiment of the methods of the fifth aspect of the invention comprises testing a DNA sample from the subject to determine the presence of a variant sequence in the coding region and/or promoter region of a gene identified in Table 7, wherein the presence of a variant sequence in the coding region and/or promoter region of the gene in Table 7 is indicative of the subject having metastatic breast cancer, or being at risk of developing the same.

By “variant sequence” we include a difference in nucleotide sequence relative to a corresponding reference sequence (e.g. the corresponding sequence in normal, healthy cells). The nature of the sequence variation may be an insertion of one or more nucleotides at a given location within the gene, a deletion of one or more nucleotides at a given location within the gene, and/or a substitution of one or more nucleotides at a given location within the gene, wherein the sequence variation results in modulation of the function or expression of the gene. It will be appreciated by persons skilled in the art that such modulation of gene function/expression may arise from sequence variation in the coding region of the gene and/or it promoter.

In one embodiment, the variant sequence is detrimental to the function and/or expression of the gene. For example, the variant sequence may inhibit (in whole or in part) expression of the gene, such that levels of its protein product within the tumour cells are reduced. Alternatively, the variant sequence may result in production of a modified protein product of the gene having reduced or no biological activity (for example, the protein may be truncated).

In an alternative embodiment, the variant sequence may enhance the function and/or expression of the gene. For example, the variant sequence may be a deletion that removes a phosphorylation site from the gene product or an amino acid substitution that confers enhanced activity.

Advantageously, step (b) comprises exome sequencing (for example, using a MiSeq DNA sequencer from lllumina).

Alternatively, step (b) may comprise the use of a PCR-based assay.

Conveniently, step (b) comprises a bioinformatics approach, such as those described in Analysing Gene Expression, A Handbook of Methods: Possibilities and Pitfalls (2003), edited by Lorkowski &. Cullen, Wiley (see Chapter 7 therein) (the disclosures of which are incorporated herein by reference).

Thus, step (b) may comprise the use of an algorithm (for example, ‘SNPeffect’ for phenotyping human single nucleotide polymorphisms, insertions and deletions; see Baets et al., 2012, Nucleic Acids Res. 40(1):D935-9, the disclosures of which are incorporated herein by reference).

In one embodiment, step (b) comprises testing DNA from the subject to determine the presence of a SNP in the coding region and/or the promoter region of a gene identified in Table 7.

Preferably, the SNPs are classified as 'high impact’, suggestive of a greater likelihood of them having a functional effect on the gene.

Examples of high impact SNPs are identified in Table 8 below. TABLE 8

High Impact SNPs associated with metastatic breast cancer

* Predicted to generate new stop codon

Thus, step (b) may comprise testing DNA from the subject to determine the presence of both of the SNPs identified in Table 8.

In one preferred embodiment, step (b) comprises testing the cells to determine the function and/or expression and/or nucleotide sequence variation of one or more of the genes identified in Table 9, wherein modulation of the function and/or expression of the one or more of the genes identified in Table 9 compared to a reference value and/or the presence of a variant sequence in one or more of the genes identified in Table 9 is indicative of the subject having a metastatic breast cancer, or the prognosis for said subject. TABLE 9

Additional genes with diagnostic and/or prognostic value in metastatic breast cancer

Thus, step (b) may comprise testing DNA from the subject to determine the presence of a variant sequence in the coding region and/or promoter region of one or more of the genes identified in Table 9 (for example, using exome sequencing or PCR-based assays). The variant sequences may be detrimental to the function of the gene or the product thereof.

In one embodiment, step (b) comprises testing for the presence of an INDEL in the coding region and/or the promoter region of a gene identified in Table 9(a).

Preferably, the INDELs are classified as ‘moderate impact’, suggestive of a possibility of them having a functional effect on the gene.

Examples of moderate impact INDELs are identified in Table 10 below. TABLE 10

Moderate Impact INDELs associated with metastatic breast cancer

* Predicted to result in a disruptive inframe deletion

Thus, step (b) may comprise testing DNA from the subject to determine the presence of both of the INDELS identified in Table 10.

In one embodiment, step (b) comprises testing DNA from the subject to determine the presence of a SNP in the coding region and/or the promoter region of one or more of the genes identified in Table 9(b), for example at least two, three, four, five, ten, twenty, thirty, forty or more of the genes identified in Table 9(b).

For example, step (b) may comprise testing DNA from the subject to determine the presence of a SNP in all of the genes identified in Table 9(b).

Preferably, the SNPs are classified as ‘moderate impact’, suggestive of a possibility of them having a functional effect on the gene.

Examples of moderate impact SNPs are identified in Table 11 below (each of which is predicted to be a missense mutation). TABLE 11

Moderate Impact SNPs associated with metastatic breast cancer

Thus, step (b) may comprise testing DNA from the subject to determine the presence of all of the SNPs identified in Table 11.

Advantageously, step (b) comprises testing DNA from the subject to determine the presence of a variant sequence in all of the genes identified in Table 9. For example, step (b) may comprise testing DNA from the subject to determine the presence of all of the INDELs identified in Table 10 and all of the SNPs identified in Table 11. A sixth, related aspect of the invention provides an in vitro method for monitoring disease progression in a subject with metastatic breast cancer comprising (a) providing a first sample of cells from a subject to be tested; and (b) testing the cells provided in step (a) to determine the function and/or expression of, and/or nucleotide sequence variation within, one or more of the genes identified in Table 7 and/or Table 9; (c) providing a further sample of cells, obtained subsequently, from a subject to be tested; and (d) testing the cells provided in step (c) to determine the function and/or expression of, and/or nucleotide sequence variation within, one or more of the genes identified in Table 7 and/or Table 9 wherein modulation of the function and/or expression of the one or more of the genes identified in Table 7 and/or Table 9 in the further sample of cells compared to the function and/or expression of said genes in the first sample of cells is indicative of disease progression in the period between collection of the cell samples.

It will be appreciated by persons skilled in the art that such temporal monitoring of gene function/expression not only provides means for monitoring treatment progression but also enables the efficacy of a treatment regime to be monitored.

In one embodiment, step (b) is performed as defined above in relation to the fifth aspect of the invention. A seventh, related aspect of the invention provides a method for treating a subject with metastatic breast cancer, or at risk of developing the same, comprising: (a) identifying the subject as having breast cancer, or at risk of developing the same, using a method according to the fifth aspect of the invention; and (b) administering to the subject a medicament with efficacy in the treatment of the breast cancer and/or surgically removing the tumour from said subject.

In one embodiment, the breast cancer is selected from the group consisting of invasive ductal carcinomas and invasive lobular carcinomas.

It will be appreciated by persons skilled in the art that subjects identified as having breast cancer may be treated with any medicament known to have efficacy in the treatment of the metastatic breast cancer, including conventional anti-cancer drugs (such as chemotherapeutic agents, immunotherapeutic agents and radiotherapeutic agents; see above).

Additionally, or alternatively, the subject may undergo surgery to remove the tumour(s).

The subject may also receive radiotherapy, often upon completion of a course of chemotherapy or surgical removal of the tumour(s).

In one embodiment of the seventh aspect of the invention, the method further comprises step (c) of providing a second sample of cells obtained from the subject after the commencement of treatment, testing said cells to determine the function and/or expression of, and/or nucleotide sequence variation within, one or more of the genes identified in Table 7 and/or 9, and comparing the results of said testing with the results obtained in step (a).

In one embodiment, the method further comprises maintaining or modifying the treatment of the subject in order to optimise its therapeutic efficacy.

Advantageously, step (c) is repeated in order to monitor the therapeutic efficacy of the treatment received by the subject.

An eighth aspect of the invention provides a method for identifying a lead candidate with efficacy in the treatment or prevention of metastatic breast cancer comprising: (a) providing a compound to be tested; and (b) determining in vitro whether said test compound modulates the function and/or expression of one or more of the genes identified in Table 7 and/or 9, wherein the test compound is identified as a lead candidate with efficacy in the treatment or prevention of metastatic breast cancer if it modulates the function and/or expression of one or more of the genes identified in Table 7 and/or 9.

Conveniently, the method comprises the use of a high-throughput screening assay.

Step (b) may be performed using methods well-known in the art for assessing the function and/or expression of genes and the products thereof.

In one embodiment, step (b) is performed as described above in relation to the methods of the fifth aspect of the invention.

In another embodiment, the method further comprises step (c) of testing the lead candidate for efficacy in an in vivo model of said metastatic breast cancer.

Preferences and options for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features and parameters of the invention.

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Preferred, non-limiting examples which embody certain aspects of the invention will now be described, with reference to the following figure:

Figure 1: General representation of the GATK pipeline used for variant discovery as recommended by the Broad Institute

EXAMPLE

Exome Sequencing to Explore Temporal and Spatial Mutational Evolutionary Changes in Invasive Breast Cancer Specimens

This study provides data regarding the temporal and spatial analyses of BC tumour samples derived from the same patient. Using exome sequencing combined with bioinformatics, sequence variants (INDELs and SNPs) have been identified that are concordant between primary tumour samples or their corresponding lymph node metastasis but absent from patient matched control (non-tumour) samples. Variants were screened for presence or absence at a specific chromosomal location. It was rationalised that such an approach may provide a method to analyse a subset of changes that could potentially be important in the tumorigenic process by virtue of whether they remain concordant or discordant between samples. The presence or absence of particular variants may therefore provide evidence that a subset of genes have a key role in the process of BC development and/or progression. Such an approach would not consider parochial issues such as molecular heterogeneity within a given patient per se, nor would the methodology identify SNP/INDEL present in the same gene but occurring elsewhere in its exonic structure that could also have greater, equal or less impact upon protein function. DNA sequencing and bioinformatic analyses of BC tumours have identified variants that are predicted to contain either high or moderate impact effects upon protein function.

Two classes of genes were explored in this study of breast cancer patients: (a) Gene variants that are consistent amongst primary tumour samples but absent from control samples. These may therefore provide information regarding genes that are important molecular components in the development of BC. and (b) Gene variants that are consistent amongst lymph node metastases but absent from control samples. These genes may therefore be important for the progression of BC during the metastatic process.

Such gene variants represent provide novel therapeutic targets.

Methods

Exome library preparation

Genomic DNA (gDNA) was prepared from formalin fixed paraffin embedded (FFPE) sections using the Biostic Tissue DNA isolation kit (Mo Bio) as per manufacturer’s instructions. Exome libraries were constructed using the Nextera Rapid Capture kits (lllumina) as per manufacturer’s instructions. 50 ng of gDNA was utilised to construct DNA exomes for each sample. DNA quantitation was undertaken using a Qubit fluorimeter and size distribution of insert was assayed using a Bioanalyser 2100 with high sensitivity DNA kits. DNA sequencing A MiSeq DNA sequencing instrument (lllumina) was used for sequencing the exome libraries. Libraries were barcoded and multiplexed and run a minimum of 11x times in order to generate sufficient coverage. Sequencing reactions were carried out using 2 x 75 paired end cycles (+1 cycle to each forward and reverse read to allow for phasing/pre-phasing). Fastq files were generated and used for downstream variant analyses.

Bioinformatic analyses

Fastq files were unzipped and concatenated from multiple runs to produce a single fastq file. Adapters were automatically trimmed by the sequencing instrument. Pre-processing of sequences occurred as followed: base quality scores (less than Q30) and the first 14 base pairs of the 5’ were removed from forward and reverse reads using the Trim Galore wrapper and Cutadapt (v1.7.1) in order to minimise the risk of sequence bias. FastQC (vO. 11.2) was used for general quality control of sequences and to confirm adapter removal/quality trimming. Alignments to the reference human genome (hg19) were conducted using BWA MEM (version 0.7.15) with 32 parallel threads. Optical duplicates were marked using Picard Tools (Broad Institute) and sequence metrics collected using the command “Collectmultiplemetrics”. Samtools “Flagstat” command was used to collect basic statistics regarding forward and reverse read alignment success as well as the percentage of reads properly paired.

The Genome Analysis Tool Kit (GATK version 3.0.0) was used for INDEL realignment, base quality score recalibration, raw variant calling and hard filtering to identify high quality variants (SNP/INDEL) for downstream analyses (refer to Figure 1 for the “Best Practice” set of guidelines recommended by the Broad Institute) [14]. INDEL and SNP were corrected for multi-allelic sites and left normalised using BCFTOOLS prior to annotation (e.g. Annovar (version June 2015)). Comparative analyses were carried out using GATK SelectVariant functions for concordance/discordance between vcf files of interest. SnpEff (v4.1) was exploited to determine predicted in siiico impacts upon protein function of candidate genes.

Identification of concordant SNP and INDEL between BC tumour samples

Three independent sets of patient tumour samples were sequenced and processed computationally using the pipeline described above (patient samples ['Ml Number’] 4470, 4616 and 5029; see Tables A and B). To identify concordant SNP and INDEL patient 4616 was utilised as the baseline discovery tumour sample set. Sequential processing was undertaken for each tumour (primary and lymph node) with a single vcf file being produced for each sample, vcf files were then tested for concordance for all variants. SNP and INDEL were treated independently of each other. The product of the first comparison was then used as the baseline for concordance testing with the next sample. This process was iterated until all BC tumour samples (primary tumours and lymph node) were tested for SNP and INDEL concordance. This produced a single vcf file containing concordant SNP and INDEL for: a) Patient 4616 primary breast cancer b) Patient 4616 lymph node metastases

Variants concordant to the control sample were filtered out and analysed for high and medium impact variants (as determined in-silico using SnpEff version 4.1). Variants of potential interest were then screened against patient samples 4470 and 5029 to examine the variant distribution profile.

Table A: Clinico-pathological data for the patients used in this study

Table B: FFPE samples used in primary gene identification and validation studies with percentage tumour cellularity. Origin of tumour samples (normal, primary tumour or lymph node metastases is provided).

Results

Primary breast cancer (concordant variants across primary breast tumour samples but discordant against control non-tumour samples) (a) INDELs A total of 3 distinct genes were identified as having concordant INDEL with potential high impact effect changes to protein structure (Table C). Two distinct genes were identified as having moderate potential impacts upon protein function (Table D).

Concordant high impact effect INDELs between primary BC tumour samples identified for patient 4616 were subsequently validated in two other patients, samples 4470 and 5029 (Table E).

Several genes identified in this cohort had been previously associated as having differential expression in tumours. The following represented selected examples: 1) PPP1R9B (High impact INDEL): PPP1R9B (Spinophilin) is a regulator of protein phosphatase 1 and putative tumour suppressor gene. Low expression of Spinophilin has been associated with poor prognosis in 162 colon adenocarcinoma patients. In-vitro studies using colorectal cancer cell lines and shRNA knockdown experiments have indicated that low expression of Spinophilin results in an increase in cancer stem cell phenotypic properties and enhanced anchorage independent growth/colony formation suggesting that knockdown of this gene may promote tumour formation and metastatic potential. Cell lines with reduced Spinophilin expression also exhibited increased resistance to 5-flourouracil (a commonly used chemotherapeutic agent used in the treatment of CRC) indicating that this tumour suppressor may have multiple downstream effects capable of enhancing the survival of cancer cells [15]. Schwarzenbacher et al have explored the expression levels of Spinophilin in BC and suggest that Spinophilin expression was significantly lower in basal-like BC and an independent prognostic factor for poor prognosis in BC patients. In-vitro modelling studies in which Spinophilin levels were reduced and phenotypically manifested in an increase in anchorage independent growth and cellular proliferation. In-vivo studies also corroborated clinical data suggesting that low Spinophilin levels enhanced metastatic potential [16]. The genomic region 17q21 is often associated with microsatellite instability and loss of heterozygosity in cancer. This region contains several putative tumor suppressor genes, including BRCA1, NM23 in addition to Spinophilin.

An analysis was undertaken to explore whether the INDEL was also present in the lymph node samples for patient 4616 as well as other primary/metastatic samples of patient 4470 and 5029. Table D presents the location of the INDEL for all BC tumours for all three patients. Interestingly two high impact INDEL could be detected at locations 48227384 and 48227403 on chromosome 17. The control sample did not contain either INDEL although a non-effect INDEL could be identified in the UTR of this control sample. Overall 70% of tumour samples (for all three patients 4616, 4470 and 5029) had concordant INDEL at the same location in primary and/or lymph node metastases. This may suggest that spinophilin may have an integral role in BC initiation and progression in the patients analysed within this study. 2) MSS51 (High Impact INDEL): MSS51 has been shown to regulate the translation of mitochondrial proteins [17] [18]. Aberrations in mitochondrial function may play a potential role in the tumorigenesis, mutagenesis and therapeutic response patterns to chemotherapeutic agents [19] [20], 3) PODXL (Moderate Impact INDEL): Podocalyxin is a sialomucin that is implicated in the processes of cell adhesion and migration. Although protein function is not believed to be abrogated through the introduction of a moderate impact INDEL, PODXL stability and/or function may be potentially altered. High PODXL expression has been associated with BC growth and metastasis and has therefore been suggested to be a potential therapeutic target for biologies such as monoclonal antibodies [21]. It has also been shown to be a poor prognostic marker in CRC [22], 4) PRUNE2 (Moderate Impact INDEL): PRUNE2 has been reported to be a potential tumour suppressor gene that controls the growth of prostate cancer cells. Low expression of PRUNE2 resulted in an increase in cellular proliferation and has been suggested as a potential therapeutic target for the treatment of prostate cancer [23],

Given that several tumour suppressor genes have been identified as being mutated in other cancer types these is strong a possibility that these same genes may have potential roles in suppressing/augmenting BC development and progression. Several genes listed that have not historically been associated with BC and thus may therefore represent novel candidates for diagnostic, prognostic and/or therapeutic targets.

Table C: Concordant high impact effect INDEL between primary BC tumour samples for patient 4616. Variant impacts were determined using the in-silico modelling algorithm SnpEff

Table D: Concordant moderate impact effect INDEL between primary BC tumour samples for patient 4616. Variant impacts were determined using the in-silico modelling algorithm SnpEff

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(b) SNPs A total of 111 distinct genes were identified as having concordant SNP but absent in control samples. 1 SNP was noted to have a potential high impact effect change to protein structure (Table F). 110 SNP had moderate impact non-synonymous changes that could potentially impact protein stability and/or structure function (Table G). The following represent selected examples: 1) FAM173B (High Impact SNP): FAM173B (JS-2) has been reported to have significantly lower expression in late stage CRC tumours compared to early stage tumours or benign counterparts. This may suggest a potential tumour suppressor role for this gene [24], Interestingly this high impact SNP was identified in all primary tumours of patient 4616 and associated lymph node metastases. The SNP was not however detected in patient 4470 or 5029. 2) OBSCN (Moderate Impact SNP): Obscurins are encoded by a single OBSCN gene and are giant cytoskeletal proteins with structural and regulatory roles. OBSCN is often mutated in multiple cancer types. OBSCN has been shown to be down-regulated in BC compared to non-tumour tissue and that loss of expression is associated with epithelial to mesenchymal phenotypic transition resulting in enhanced tumorigenesis and invasiveness in-vitro/in-vivo [25] [26]. Loss of OBSCN has been associated with BC survival through reduced apoptosis [27], 3) ACVR1C\ ACVR1C is a type I receptor for the ΤΘΡβ family of signalling molecules. A low expression ACVR1C has been associated with poor prognosis in gall bladder tumours with a suggestion that this gene may be a potential tumour suppressor [28] , Interestingly this is in contrast to findings with malignant mesothelioma in which high expression of ACVR1C was associated with SMAD2/3 signalling pathway activation. Inhibition of this pathway reduced tumour growth and therefore suggested that Activins may have tumour promoting activity in this type of cancer [29] , 4) CAP1: Adenylate cyclase-associated protein 1 (CAP1) regulates actin filaments and the Ras/cAMP pathway. CAP1 has been implicated to play a role in promoting cell cycle progression in epithelial ovarian cancer cells and down-regulating its activity may therefore provide a novel therapeutic target [30]. 71% of breast cancers assayed by immunohistochemistry were found to have high expression of CAP1 and reducing expression levels in BC cells were found to negatively impact both proliferation and migration [31].

These data presented would suggest that high impact SNP variants may potentially abrogate protein function for proteins previously established as putative tumour suppressor genes in other tumour types (Table E). These genes may play concordant roles in BC driven pathways. Moderate impact SNP may have the ability to attenuate protein activity and several genes previously linked to cancer development, progression and therapeutic response patterns have been shown to have moderate impact SNP (Table F). These genes may play similar roles in the development of BC and its progression towards more aggressive phenotypes e.g. metastasis. Many genes listed do not have prior association to BC development and potentially represent novel diagnostic, prognostic and/or therapeutic targets. Moderate impact SNP could also have downstream impact upon protein stability therefore modulating turnover rates. This may translate ultimately to a reduction or increase in protein accumulation in tumour cells.

Table F: Concordant high impact effect SNP occurring between primary BC tumour samples for patient 4616 but absent from control non-tumour samples. Variant impacts were determined using the in-silico modelling algorithm SnpEff

Table G: Concordant moderate impact effect SNP occurring between primary BC tumour samples for patient 4616 but absent from control non-tumour samples. Variant impacts were determined using the in-silico modelling algorithm SnpEff

Lymph node metastases (concordant variants across BC lymph node metastatic samples but discordant against control non-tumour samples)

(a) INDEL

No high impact INDELs were noted as concordant across samples although one gene (PODXL) was identified as having concordant INDEL with moderate impact effect changes to protein structure (Table H). This INDEL was common not only to the primary tumours but was also maintained in metastatic variants and may implicate a potential role in BC development and progression. Appropriate validation studies are needed to corroborate this suggestion.

Table H: Concordant moderate impact effect INDEL occurring across BC lymph node metastatic samples but discordant against control non-tumour samples (patient 4616). Variant impacts were determined using the in-silico modelling algorithm SnpEff

(b) SNP

HIGH IMPACT SNP FAM173B high impact SNP was detected in all metastatic lymph node samples but discordant with control tissue samples (Table I). This SNP was also concordant amongst primary tumour samples and may potentially indicate its importance in BC development for this patient.

Table I: Concordant high impact effect SNP occurring across BC lymph node metastatic samples but discordant against control non-tumour samples (patient 4616). Variant impacts were determined using the in-silico modelling algorithm SnpEff

MODERATE IMPACT SNP A total of 42 moderate impact effect SNP have been identified as being concordant amongst the metastatic samples but discordant to control tissue variants (Table J). Several genes have been implicated in tumour development and progression. The following represent selected examples: 1) ACVR1C: ACVR1C was identified as being concordant between all lymph node metastatic samples but discordant with control. Interestingly this gene was also identified as being variant amongst the primary tumour samples. There may be the possibility that this gene/gene variant may be important in the tumorigenic process due to its consistency in primary and metastatic samples. 2) BAIAP3: BAIAP3 is actively involved in exocytosis and its overexpression in desmoplastic small round cell tumour has been shown to promote tumorigenesis [32], The gene is a target of p53 and has been shown to be an inhibitor of angiogenesis within the brain [33], 3) CUT A. CIITA is a transcription factor that is required for MHC class II expression. It has been shown that overexpression of CIITA in melanoma leads to a reduction of T-cell immune response against tumour cells [34], The gene has recently been implicated as a prognostic biomarker for drug resistance in BC patients [35], MHC class II expression in ERa +ve tumour has been shown to be negatively regulated in BC through a suppressive pathway impacting IFN-γ signalling. Such control may therefore provide an escape mechanism in which breast tumours are capable of subverting anti-tumour responses by the immune system [36]. 4) NOP14: NOP14 has been shown to be poorly expressed in BC and its metastatic variants and is associated with a reduction in survival rates [37], Reduced expression of NOP14 has also been associated with poor prognosis in ovarian cancer patients [38].

Table J: Concordant moderate impact effect SNP occurring across BC lymph node metastatic samples but discordant against control non-tumour samples (patient 4616). Variant impacts were determined using the in-silico modelling algorithm SnpEff

Forty-one moderate impact SNPs have been identified as being concordant amongst the lymph node metastases and discordant with the non-tumour control variant population. Several genes have been suggested to be potential candidate for the development of novel therapeutics. The other loci listed above may also be novel players linked to BC development/progression and thus could form the basis of new and improved diagnostics, prognostics and therapeutics.

Discussion

Personalised molecular medicine is predicated upon tailoring specific genomic signatures to clinical management protocols and therapeutic modalities capable of translating into maximum patient benefit. High throughput DNA sequencing is providing a powerful screening technology for genetic variants of clinical importance. The data presented within this study has explored exonic variation in a cohort of BC samples from three patients with primary and metastatic BC. The methodology has exploited the use of computational algorithms to identify SNP/INDEL that are concordant amongst several samples of either primary tumours or their corresponding metastases but discordant to variants present in control tissue. Through a series of filtering steps, a panel of high quality SNP and INDEL were then identified for downstream analyses of biological/clinical relevance. Variant concordance was also explored in independent patient samples to confirm whether SNP/INDEL were either unique to the patient used to identify the variants or whether the same mutational landscape could be observed in other patients. This may provide corroborative data as to whether the same pathways are potentially exploited in other BC patient tumours.

Variants identified included tumour suppressor genes including Spinophilin which is located in the same chromosomal region linked to other tumour suppressor genes including BRCA1. Genes linked to chemotherapeutic response, cell cycle control, immune modulation and cellular adhesion/stem cell phenotypic control were also identified as having potential variants that could potentially impact protein function. Some loci have been previously reported as key molecular players in the development of cancer including BC. There are however several novel genes that have been elicited in this study as being novel potential players in BC biology.

In summary, these gene panels form the basis of diagnostic/prognostic companion diagnostic assays for BC (e.g. MDD/tumour recurrence) and provide the foundation for developing novel therapeutic agents.

References 1. Cole B.F, et al: Polychemotherapy for early breast cancer: an overview of the randomised clinical trials with quality-adjusted survival analysis. The

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Claims (95)

1. An in vitro method for identifying a subject having breast cancer, or at risk of developing the same, the method comprising the steps of: (a) providing a sample of cells from a subject to be tested; (b) testing the cells to determine therein the function and/or expression of, and/or nucleotide sequence variation within, one or more of the genes identified in Table 1 wherein modulation of the function and/or expression of the one or more of the genes identified in Table 1 compared to a reference value, and/or the presence of a variant sequence within one or more of the genes identified in Table 1, is indicative of the subject having breast cancer, or being at risk of developing the same.

2. An in vitro method according to Claim 1 wherein the breast cancer is selected from the group consisting of invasive ductal carcinomas and invasive lobular carcinomas.

3. An in vitro method according to Claim 1 or 2 wherein the sample in step (a) is from a tumour biopsy sample.

4. An in vitro method according to Claim 1 or 2 wherein the sample in step (a) is from a blood sample.

5. An in vitro method according to Claim 1 or 2 wherein the sample in step (a) is from a urine or saliva sample.

6. An in vitro method according to any one of the preceding claims wherein DNA is purified from the sample of cells for analysis in step (b).

7. An in vitro method according to any one of the preceding claims wherein mRNA is purified from the sample of cells for analysis in step (b).

8. An in vitro method according to any one of the preceding claims wherein proteins are purified from the sample of cells for analysis in step (b).

9. An in vitro method according to any one of the preceding claims comprising testing a DNA sample from the subject to determine the presence of a variant sequence in the coding region and/or promoter region of one or more of the genes identified in Table 1, wherein the presence of a variant sequence in the coding region and/or promoter region of one or more of the genes identified in Table 1 is indicative of the subject having breast cancer, or being at risk of developing the same.

10. An in vitro method according to Claim 9 wherein the variant sequence is detrimental to the function of the gene or the product thereof.

11. An in vitro method according to any one of the preceding claims wherein step (b) comprises exome sequencing.

12. An in vitro method according to any one of the preceding claims wherein step (b) comprises the use of a PCR assay.

13. An in vitro method according to any one of the preceding claims wherein step (b) comprises a bioinformatics analysis.

14. An in vitro method according to any one of the preceding claims wherein step (b) comprises the use of an algorithm.

15. An in vitro method according to any one of the preceding claims wherein step (b) comprises testing DNA from the subject to determine the presence of an INDEL in the coding region and/or the promoter region of one or more of the genes identified in Table 1(a), for example at least two of the genes identified in Table 1(a).

16. An in vitro method according to any one of the preceding claims wherein step (b) comprises testing DNA from the subject to determine the presence of an INDEL in one or more genes selected from the groups consisting of MSS51, GP6, PPP1R9B and PRUNE2.

17. An in vitro method according to Claim 15 or 16 wherein step (b) comprises testing DNA from the subject to determine the presence of an INDEL in all of the genes identified in Table 1(a).

18. An in vitro method according to any one of Claims 15 to 17 wherein the INDEL is classified as high-impact.

19. An in vitro method according to Claim 18 wherein the INDEL is selected from the group of INDELS identified in Table 2.

20. An in vitro method according to Claim 19 wherein step (b) comprises testing DNA from the subject to determine the presence of all of the INDELS identified in Table 2.

21. An in vitro method according to any one of Claims 9 to 20 wherein step (b) comprises testing DNA from the subject to determine the presence of a SNP in the coding region and/or the promoter region of a gene identified in Table 1(b).

22. An in vitro method according to Claim 21 wherein step (b) comprises testing DNA from the subject to determine the presence of a SNP in one or more genes selected from the groups consisting of FAM173B, OBSCN, ACVR1C and CAP1.

23. An in vitro method according to Claim 21 or 22 wherein the SNP is classified as high-impact.

24. An in vitro method according to any one of Claims 21 to 23 wherein the SNP is selected from the group of SNPs identified in Table 3.

25. An in vitro method according to Claim 24 wherein step (b) comprises testing DNA from the subject to determine the presence of all of the SNPs identified in Table 3.

26. An in vitro method according to any one of the preceding claims wherein step (b) comprises testing DNA from the subject to determine the presence of a variant sequence in all of the genes identified in Table 1.

27. An in vitro method according to Claim 26 wherein step (b) comprises testing DNA from the subject to determine the presence of all of the INDELs identified in Table 2 and all of the SNPs identified in Table 3.

28. An in vitro method according to any one of the preceding claims wherein step (b) comprises testing the cells to determine the function and/or expression of, and/or nucleotide sequence variation within, one or more of the genes identified in Table 4, wherein modulation of the function and/or expression of the one or more of the genes identified in Table 4 compared to a reference value, and/or the presence of a variant sequence within one or more of the genes identified in Table 4, is indicative of the subject having breast cancer.

29. An in vitro method according to Claim 28 wherein step (b) comprises testing DNA from the subject to determine the presence of a variant sequence in the coding region and/or promoter region of one or more of the genes identified in Table 4.

30. An in vitro method according to Claim 29 wherein the variant sequence is detrimental to the function of the gene or the product thereof.

31. An in vitro method according to any one of Claims 28 to 30 wherein step (b) comprises testing for the presence of INDELs in the coding region and/or the promoter region of one or more of the genes identified in Table 4(a).

32. An in vitro method according to Claim 31 wherein step (b) comprises testing DNA from the subject to determine the presence of an INDEL in all of the genes identified in Table 4(a).

33. An in vitro method according to Claim 31 or 32 wherein the INDEL is classified as moderate-impact.

34. An in vitro method according to Claim 33 wherein the INDEL is selected from the group of INDELS identified in Table 5.

35. An in vitro method according to Claim 34 wherein step (b) comprises testing DNA from the subject to determine the presence of all of the INDELS identified in Table 5.

36. An in vitro method according to any one of Claims 28 to 35 wherein step (b) comprises testing DNA from the subject to determine the presence of a SNP in the coding region and/or the promoter region of one or more of the genes identified in Table 4(b), for example at least two, three, four, five, ten, twenty, thirty, forty, fifty, sixty, seventy, eighty, ninety, one-hundred or more of the genes identified in Table 4(b).

37. An in vitro method according to Claim 36 wherein the SNP is classified as moderate-impact.

38. An in vitro method according to Claim 37 wherein the SNP is selected from the group of SNPs identified in Table 6.

39. An in vitro method according to Claim 38 wherein step (b) comprises testing DNA from the subject to determine the presence of all of the SNPs identified in Table 6.

40. An in vitro method according to any one of the preceding claims wherein step (b) comprises testing DNA from the subject to determine the presence of a variant sequence in all of the genes identified in Table 4.

41. An in vitro method according to Claim 40 wherein step (b) comprises testing DNA from the subject to determine the presence of all of the INDELs identified in Table 5 and all of the SNPs identified in Table 6.

42. An in vitro method for monitoring disease progression in a subject with breast cancer comprising (a) providing a first sample of cells from a subject to be tested; and (b) testing the cells provided in step (a) to determine the function and/or expression of, and/or nucleotide sequence variation within, one or more of the genes identified in Table 1 and/or Table 4; (c) providing a further sample of cells, obtained subsequently, from a subject to be tested; and (d) testing the cells provided in step (c) to determine the function and/or expression of, and/or nucleotide sequence variation within, one or more of the genes identified in Table 1 and/or Table 4 wherein modulation of the function and/or expression of the one or more of the genes identified in Table 1 and/or Table 4 in the further sample of cells compared to the function and/or expression of said genes in the first sample of cells is indicative of disease progression in the period between collection of the cell samples.

43. An in vitro method according to Claim 42 wherein step (b) and step (d) are performed as defined in any one of Claims 9 to 41.

44. A method for treating a subject with breast cancer, or at risk of developing the same, comprising: (a) identifying the subject as having breast cancer, or at risk of developing the same, using a method according to any one of Claims 1 to 41; and (b) administering to the subject a medicament with efficacy in the treatment of the breast cancer and/or surgically removing the tumour from said subject.

45. An in vitro method according to Claim 44 wherein the breast cancer is selected from the group consisting of invasive ductal carcinomas and invasive lobular carcinomas.

46. A method according to Claim 44 or 45 further comprising step (c) of providing a second sample of cells obtained from the subject after the commencement of treatment, testing said cells to determine the function and/or expression of, and/or nucleotide sequence variation within, one or more of the genes identified in Table 1 and/or 4, and comparing the results of said testing with the results obtained in step (a).

47. A method according to Claim 46 further comprising maintaining or modifying the treatment of the subject in order to optimise its therapeutic efficacy.

48. A method according to Claim 46 or 47 comprising repeating step (c) in order to monitor the therapeutic efficacy of the treatment received by the subject.

49. A method for identifying a lead candidate with efficacy in the treatment or prevention of breast cancer comprising: (a) providing a compound to be tested; and (b) determining in vitro whether said test compound modulates the function and/or expression of one or more of the genes identified in Table 1 and/or 4, wherein the test compound is identified as a lead candidate with efficacy in the treatment or prevention of breast cancer if it modulates the function and/or expression of one or more of the genes identified in Table 1 and/or 4.

50. A method according to Claim 49 wherein step (b) is performed as defined in any one of Claims 9 to 41.

51. A method according to Claim 49 or 50 further comprising step (c) of testing the lead candidate for efficacy in an in vivo model of breast cancer.

52. An in vitro method for the diagnosis of a subject with metastatic breast cancer, or prognosis for said subject, comprising: (a) providing a sample of cells from a subject to be tested; (b) testing the cells to determine the function and/or expression of, and/or nucleotide sequence variation within, a gene identified in Table 7 wherein modulation of the function and/or expression of the gene identified in Table 7 compared to a reference value and/or the presence of a variant sequence in the gene identified in Table 7 is indicative of the subject having a metastatic breast cancer, or the prognosis for said subject.

53. An in vitro method according to Claim 52 wherein the breast cancer is selected from the group consisting of invasive ductal carcinomas and invasive lobular carcinomas.

54. An in vitro method according to Claim 52 or 53 wherein the sample of cells in step (a) is from a tumour biopsy sample.

55. An in vitro method according to Claim 52 or 53 wherein the sample in step (a) is from a blood sample.

56. An in vitro method according to Claim 52 or 53 wherein the sample in step (a) is from a urine or saliva sample.

57. An in vitro method according to any one of Claims 52 to 56 wherein DNA is purified from the sample of cells for analysis in step (b).

58. An in vitro method according to any one of Claims 52 to 57 wherein mRNA is purified from the sample of cells for analysis in step (b).

59. An in vitro method according to any one of Claims 52 to 58 wherein proteins are purified from the sample of cells for analysis in step (b).

60. An in vitro method according to any one of Claims 52 to 59 comprising testing a DNA sample from the subject to determine the presence of a variant sequence in the coding region and/or promoter region of a gene identified in Table 7, wherein the presence of a variant sequence in the coding region and/or promoter region of the gene identified in Table 7 is indicative of the subject having a metastatic breast cancer, or prognosis for said subject.

61. An in vitro method according to Claim 60 wherein the variant sequence is detrimental to the function of the gene or the product thereof.

62. An in vitro method according to any one of Claims 52 to 61 wherein step (b) comprises exome sequencing.

63. An in vitro method according to any one of Claims 52 to 62 wherein step (b) comprises the use of a PCR assay.

64. An in vitro method according to any one of Claims 52 to 63 wherein step (b) comprises a bioinformatics analysis.

65. An in vitro method according to any one of Claims 52 to 64 wherein step (b) comprises the use of an algorithm.

66. An in vitro method according to any one of Claims 52 to 65 wherein step (b) comprises testing DNA from the subject to determine the presence of a SNP in the coding region and/or the promoter region of a gene identified in Table 7.

67. An in vitro method according to Claim 66 wherein the SNP is classified as high-impact.

68. An in vitro method according to Claim 66 or 67 wherein the SNP is selected from the group of SNPs identified in Table 8.

69. An in vitro method according to Claim 68 wherein step (b) comprises testing DNA from the subject to determine the presence of all of the SNPs identified in Table 8.

70. An in vitro method according to any one of the preceding claims wherein step (b) comprises testing the cells to determine the function and/or expression of, and/or nucleotide sequence variation within, one or more of the genes identified in Table 9, wherein modulation of the function and/or expression of the one or more of the genes identified in Table 9 compared to a reference value, and/or the presence of a variant sequence in one or more of the genes identified in Table 9, is indicative of the subject having a metastatic breast cancer, or the prognosis for said subject.

71. An in vitro method according to Claim 70 wherein step (b) comprises testing DNA from the subject to determine the presence of a variant sequence in the coding region and/or promoter region of one or more of the genes identified in Table 7.

72. An in vitro method according to Claim 71 wherein the variant sequence is detrimental to the function of the gene or the product thereof.

73. An in vitro method according to any one of Claims 70 to 72 wherein step (b) comprises testing for the presence of an INDEL in the coding region and/or the promoter region of one or more of the genes identified in Table 9(a).

74. An in vitro method according to Claim 73 wherein the INDEL is classified as moderate-impact.

75. An in vitro method according to Claim 74 wherein the INDEL is selected from the group of INDELS identified in Table 10.

76. An in vitro method according to Claim 75 wherein step (b) comprises testing DNA from the subject to determine the presence of all of the INDELS identified in Table 10.

77. An in vitro method according to any one of Claims 70 to 76 wherein step (b) comprises testing DNA from the subject to determine the presence of a SNP in the coding region and/or the promoter region of one or more of the genes identified in Table 9(b), for example at least two, three, four, five, ten, twenty, thirty, forty or more of the genes identified in Table 9(b).

78. An in vitro method according to Claim 77 wherein step (b) comprises testing DNA from the subject to determine the presence of a SNP in one or more genes selected from the groups consisting of ACVR1C, BAIAP3, CIITA and NOP14.

79. An in vitro method according to Claim 77 or 78 wherein step (b) comprises testing DNA from the subject to determine the presence of a SNP in the coding region and/or the promoter region of all of the genes identified in Table 9(b).

80. An in vitro method according to any one of Claims 77 to 79 wherein the SNP is classified as moderate-impact.

81. An in vitro method according to Claim 80 wherein the SNP is selected from the group of SNPs identified in Table 11.

82. An in vitro method according to Claim 81 wherein step (b) comprises testing DNA from the subject to determine the presence of all of the SNPs identified in Table 11.

83. An in vitro method according to any one of Claims 70 to 82 wherein step (b) comprises testing DNA from the subject to determine the presence of a variant sequence in all of the genes identified in Table 7.

84. An in vitro method according to Claim 83 wherein step (b) comprises testing DNA from the subject to determine the presence of all of the INDELs identified in Table 10 and all of the SNPs identified in Table 11.

85. An in vitro method for monitoring disease progression in a subject with metastatic breast cancer comprising (a) providing a first sample of cells from a subject to be tested; and (b) testing the cells provided in step (a) to determine the function and/or expression of, and/or nucleotide sequence variation within, one or more of the genes identified in Table 7 and/or Table 9; (c) providing a further sample of cells, obtained subsequently, from a subject to be tested; and (d) testing the cells provided in step (c) to determine the function and/or expression of, and/or nucleotide sequence variation within, one or more of the genes identified in Table 7 and/or Table 9 wherein modulation of the function and/or expression of the one or more of the genes identified in Table 7 and/or Table 9 in the further sample of cells compared to the function and/or expression of said genes in the first sample of cells is indicative of disease progression in the period between collection of the cell samples.

86. An in vitro method according to Claim 85 wherein step (b) and step (d) are performed as defined in any one of Claims 60 to 84.

87. A method for treating a subject with metastatic breast cancer, or at risk of developing the same, comprising: (a) identifying the subject as having breast cancer, or at risk of developing the same, using a method according to any one of Claims 52 to 84; and (b) administering to the subject a medicament with efficacy in the treatment of the breast cancer and/or surgically removing the tumour from said subject.

88. An in vitro method according to Claim 87 wherein the breast cancer is selected from the group consisting of invasive ductal carcinomas and invasive lobular carcinomas.

89. A method according to Claim 87 or 88 further comprising step (c) of providing a second sample of cells obtained from the subject after the commencement of treatment, testing said cells to determine the function and/or expression of, and/or nucleotide sequence variation within, one or more of the genes identified in Table 7 and/or 9, and comparing the results of said testing with the results obtained in step (a).

90. A method according to Claim 89 further comprising maintaining or modifying the treatment of the subject in order to optimise its therapeutic efficacy.

91. A method according to Claim 89 or 90 comprising repeating step (c) in order to monitor the therapeutic efficacy of the treatment received by the subject.

92. A method for identifying a lead candidate with efficacy in the treatment or prevention of breast cancer comprising: (a) providing a compound to be tested; and (b) determining in vitro whether said test compound modulates the function and/or expression of one or more of the genes identified in Table 7 and/or 9, wherein the test compound is identified as a lead candidate with efficacy in the treatment or prevention of breast cancer if it modulates the function and/or expression of one or more of the genes identified in Table 7 and/or 9.

93. A method according to Claim 92 wherein step (b) is performed as defined in any one of Claims 60 to 84.

94. A method according to Claim 92 or 93 further comprising step (c) of testing the lead candidate for efficacy in an in vivo model of breast cancer.

95. A method for the diagnosis, prognosis or treatment of a subject with breast cancer substantially as described herein with reference to the Examples.