CN116621946B

CN116621946B - Application of polypeptide circ1946-109aa as esophageal squamous carcinoma prognosis marker

Info

Publication number: CN116621946B
Application number: CN202310677725.3A
Authority: CN
Inventors: 范丽媛; 田辉; 孙亮; 李冕; 徐晓英
Original assignee: Qilu Hospital of Shandong University
Current assignee: Qilu Hospital of Shandong University
Priority date: 2023-05-31
Filing date: 2023-06-07
Publication date: 2024-02-20
Anticipated expiration: 2043-06-07
Also published as: CN116621946A

Abstract

The invention relates to application of a polypeptide circ1946-109aa as an esophageal squamous carcinoma prognosis marker. The research of the invention finds that hsa_circ_0001946 can code for a polypeptide with the length of 109aa, and the polypeptide is named circ1946-109aa. The polypeptide can be used as a marker for prognosis of esophageal squamous carcinoma patients. Meanwhile, the expression of immune checkpoint molecules B7-H3 on the surface of esophageal squamous carcinoma cells and the proliferation and migration capacity of a cell line are obviously reduced after the circ1946-109aa is overexpressed, so that the circ1946-109aa has the potential of being applied to clinical esophageal squamous carcinoma RNA immunotherapy.

Description

Application of polypeptide circ1946-109aa as esophageal squamous carcinoma prognosis marker

Technical Field

The invention belongs to the technical field of esophageal squamous carcinoma markers, and particularly relates to a polypeptide circ1946-109aa, a fusion protein of the polypeptide, a pharmaceutical composition containing the circ1946-109aa and application of the polypeptide as an esophageal squamous carcinoma prognosis marker and an immune checkpoint regulator.

Background

The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.

Esophageal squamous cell carcinoma is a malignant tumor with a pathological type of squamous cells, belongs to one of malignant tumors common in digestive systems, and accounts for 90% of people suffering from esophageal cancer. The most common clinical symptoms of esophageal squamous carcinoma are progressively aggravated dysphagia, and as the condition progresses, the patient also develops clinical symptoms associated with metastases, which in severe cases can be life-threatening. At present, the research on molecular mechanisms of the occurrence and the development of esophageal squamous carcinoma is relatively lagged, and no effective screening method exists.

The prior researches prove that the circRNA is closely related to the aspects of growth and development of organisms, stress response and occurrence and development of diseases, and the circRNA has related reports as a marker of esophageal squamous cell carcinoma. There is still a large unknown portion of the biological function of circRNA at present, and it is generally thought that circRNA may regulate protein transcription by means of miRNA "sponges", regulatory RNA binding proteins or RNA polymerases. With the verification of the coding potential and the research of functions of the circRNA, part of the circRNA coding products are also proved to exert certain physiological activities. At present, the effect of the coding circRNA and the coding product thereof on tumors such as glioma and the like is initially explored, and the coding product of the circRNA can interfere the biological behaviors such as proliferation, metastasis and the like of tumor cells and influence the occurrence and development of the tumors. However, the role of the encoded product from circRNA in esophageal squamous carcinoma has not been reported.

B7-H3, also known as CD276, is an important immune checkpoint that protects tumor cells from attack and killing by human T cells, and has become a novel target for immunotherapy, but its role in esophageal squamous cell carcinoma is yet to be studied further. In addition, the previous research focuses on the T cell inhibition function and downstream regulation mechanism of B7-H3, and the work on the aspect of the upstream regulation mechanism of B7-H3 is relatively deficient at home and abroad.

Disclosure of Invention

According to the research of the invention, hsa_circ_0001946 can encode and obtain a polypeptide sequence with the length of 109aa, and the invention verifies the true existence of the polypeptide sequence through experimental methods such as mass spectrum detection, western blot, ribosome association analysis and the like. As hsa_circ_0001946 is related to the expression of immune checkpoint molecules B7-H3, the invention researches the relativity of the polypeptide and B7-H3, and proves that the polypeptide can reduce the expression of B7-H3 at RNA level so as to regulate and control the immune escape of esophageal squamous cell carcinoma, on the other hand, cell scratch and transwell experiments show that over-expression of the polypeptide is favorable for inhibiting the metastasis of esophageal squamous cell carcinoma and can be used as a potential therapeutic target.

Based on the research results, the invention provides the following technical scheme:

in a first aspect, there is provided a polypeptide having the sequence:

(1) A polypeptide having an amino acid sequence as shown in SEQ ID NO. 1;

(2) And a derivative polypeptide formed by adding, substituting or deleting one or more amino acids to the amino acid sequence shown in SEQ ID NO. 1, wherein the derivative polypeptide still has the same or basically the same function as the polypeptide.

In one embodiment of the verification of the invention, the hsa_circ_0001946 encoded polypeptide has an amino acid sequence shown in SEQ ID NO. 1 and is named circ1946-109aa; proved by verification, the circ1946-109aa is expressed in a patient with esophageal squamous carcinoma and can be used as a prognosis marker of esophageal squamous carcinoma. Furthermore, it has been found by the present invention that circ1946-109aa is capable of inhibiting the expression of immune checkpoint B7-H3. B7-H3 is expressed in cancer cells and bone marrow cells, including T cells and B cells, monocytes/macrophages and dendritic cells, and has dual role as a co-stimulatory/co-inhibitory immune checkpoint molecule, closely related to tumor growth, metastasis, recurrence and poor prognosis. The findings provide application of circ1946-109aa as an immunopoint tumor inhibitor and an esophageal squamous carcinoma therapeutic active ingredient.

In the above aspect (2), the one or more may be 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5 or 1 to 3, and the addition, deletion or substitution may occur at the N-terminal and/or C-terminal or sequence of the amino acid sequence shown in SEQ ID NO. 1; further, the sequence of the derivative polypeptide has 90% or more similarity with the amino acid sequence shown in SEQ ID NO. 1; still further, the similarity is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%; the amino acid sequences may be aligned for similarity using methods commonly used in the art, such as the Blast method. The derivative polypeptide still has the same or basically the same functions as circ1946-109aa, and the functions mainly comprise functions of inhibiting B7-H3 expression, regulating immune escape of esophageal squamous cell carcinoma, inhibiting metastasis of esophageal squamous cell carcinoma and the like.

In addition, the derivative polypeptide also broadly comprises a polypeptide product formed by chemically or genetically modifying circ1946-109aa; modifications common in the art include streptavidin, modifications of various molecules (e.g., biotin, radioisotopes, fluorescers, enzymes, cytotoxic substances, antineoplastic agents, etc.), and the like; also included are immobilized products of circ1946-109aa, such as products employing physical adsorption, carriers, resin cross-linked materials, and the like to immobilize or modify the polypeptides described above.

In a second aspect, there is provided a fusion protein comprising a polypeptide as set out in the first aspect, and further comprising an additional effector fragment.

Such other effector fragments may exert effects such as extending in vivo half-life, improving solubility, or enhancing the ability to penetrate biological barriers;

further, the fragment for extending the in vivo half-life is one of, but not limited to, serum albumin or a fragment thereof, polyethylene glycol, a domain that binds serum albumin (e.g., a single domain antibody against serum albumin), polyethylene glycol-liposome complex.

Further, the effector fragment for improving the permeability of biological barrier, such as a transmembrane peptide, is one of H7R8, HIV-I TAT (48-60), TAT (47-57), TAT (48-57) and R9-TAT.

In other preferred embodiments, a connecting peptide is further provided between the polypeptide and the effector fragment, wherein the connecting peptide is a flexible polypeptide chain composed of alanine (a) and/or serine (S) and/or glycine (G), and the length of the connecting peptide may be 3 to 30 amino acids, more preferably 3 to 9, 9 to 12, 12 to 16, 16 to 20, etc.

In a third aspect, there is provided a nucleic acid material encoding a polypeptide according to the first aspect, a fusion protein according to the second aspect.

In view of codon degeneracy, the nucleic acid material is any nucleic acid encoding the polypeptides and fusion proteins described above, including DNA forms, including cDNA, genomic DNA, or synthetic DNA, which may be single-stranded or double-stranded, and which may be either encoding or non-encoding, in addition to hsa_circ_ 0001946. Methods for isolating such nucleic acid materials should be known to those skilled in the art, e.g., by automated DNA synthesis and/or recombinant DNA techniques, etc., and may be isolated from suitable natural sources.

In a fourth aspect, there is provided a construct comprising the nucleic acid substance of the third aspect.

The construction of the constructs is conventional to the skilled person, e.g. by means of in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, etc., and more particularly by insertion of the isolated polynucleotide into the multiple cloning site of the expression vector. Expression vectors in the present invention generally refer to various commercially available expression vectors and the like well known in the art, and may be, for example, bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus, or other vectors.

In a fifth aspect, there is provided a pharmaceutical composition comprising a polypeptide according to the first aspect or a fusion protein according to the second aspect, and a pharmaceutically acceptable excipient.

The pharmaceutically acceptable excipients, the dosages of which should be harmless to the subject, may be of the specific classes: buffers, antioxidants, preservatives, bactericides, hydrophilic polymers, carbohydrates, chelating agents, tonicity adjusting agents, surfactants, salt forming counterions, metal complexes and/or nonionic surfactants and the like.

In a sixth aspect, there is provided the use of a polypeptide according to the first aspect as an esophageal squamous carcinoma immune checkpoint modulator.

Specific forms of the above application include, but are not limited to, the following forms:

(1) Application of a detection reagent of circ1946-109aa in preparing esophageal squamous carcinoma prognosis products;

(2) Use of the polypeptide of the first aspect, the fusion protein of the second aspect or the pharmaceutical composition of the fifth aspect for the preparation of a medicament for the treatment of a B7-H3 over-expressed disease;

(3) A method of treating esophageal squamous carcinoma, the method comprising administering to a subject in need of treatment a therapeutic dose of the polypeptide of the first aspect, the fusion protein of the second aspect, or the pharmaceutical composition of the fifth aspect.

In the above (1), the variety of the esophageal squamous carcinoma prognosis judging product includes a reagent and an instrument, and a specific example is a kit.

In the above (2), the B7-H3 over-expression disease is a tumor, which is one of melanoma, leukemia, breast cancer, prostate cancer, and digestive tract cancer, and an example of the present invention is esophageal squamous cell carcinoma.

The beneficial effects of the above technical scheme are that:

at present, research on the coding function of the circRNA is still blank in the field, and there are a plurality of unknown parts on the correlation between the coding product of the circRNA and the activity of the circRNA, so that the identification of the coding product of the circRNA is still difficult. The verification of the invention firstly proves that hsa_circ_0001946 can code novel protein circ1946-109aa, and the research result still has higher innovation. The findings further enrich research data of the coding function and the disease regulation function of the circRNA, provide a feasible esophageal squamous cell carcinoma therapeutic active ingredient and have important application prospects.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.

FIG. 1 is a schematic diagram of the ORF encoding the protein hsa_circ_0001946 described in example 1;

FIG. 2 is the results of the mass spectrometry detection described in example 1;

FIG. 3 is a circular 1946-109aa electrophoresis strip expressed by the vector described in example 1;

FIG. 4 is the overall survival curve analysis results described in example 2;

FIG. 5 is the result of the cell proliferation assay described in example 2;

FIG. 5A shows the proliferation of K30 cells transfected with T0 and T1 plasmids;

FIG. 5B shows the proliferation of K30 cells transfected with T3 and T4 plasmids;

FIG. 5C shows the proliferation of K510 cells transfected with T0 and T1 plasmids;

FIG. 5D shows the proliferation of K510 cells transfected with T3 and T4 plasmids;

FIG. 6 is the result of the cell scratch test described in example 2;

FIG. 6A shows the results of a K30 cell streak assay for the transfection of T0, T1, T3, T4 plasmids, respectively;

FIG. 6B shows the results of a K510 cell streak assay for the transfection of T0, T1, T3, T4 plasmids, respectively;

FIG. 7 is the result of the Transwell assay described in example 2;

FIG. 7A is a graph showing the results of K30 cell invasion experiments by transfecting T0, T1, T3, and T4 plasmids, respectively;

FIG. 7B is a graph showing the results of K510 cell invasion experiments by respectively transfecting T0, T1, T3 and T4 plasmids;

FIG. 8 shows the correlation results of circ1946-109aa and B7-H3 as described in example 3.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.

In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.

Example 1circ1946-109aa protein verification

Verification of the authenticity of the Presence of the circ1946-109aa protein

The sequence of hsa_circ_0001946 contains an Open Reading Frame (ORF), and this example speculates that it may have protein coding capacity (as shown in fig. 1), and in order to verify the presence of the hsa_circ_0001946 encoded product, this example translates the corresponding polypeptide according to the ORF sequence to 109 amino acids in length: MSSNNFKVFHQIQIFQLIHVFQKNLCLPPNPSLPVNLVLPEKSRSSSQSVSSRKK SRSSSQSVSSRKKSRSSSQSVSSRKIYVFHQIQVFQSIHIFRKKSRSSSQYMSS, shown as SEQ ID NO. 1, designated circ1946-109aa, the specific information of this novel protein is shown in the following table:

to verify whether such circ1946-109aa is actually present, this example first performed TMT-tagged quantitative proteome sequencing and small peptide nonstandard proteome sequencing on esophageal squamous carcinoma cell lines (K30, K140, K150, K180, K510, K200, TE1, colo680N, EC8712, TE 10) and searched for the above protein sequences by pFIND software. Raw data generated by mass spectrometry were processed using pFind 3.2.0 and databases were searched using the target protein sequences. Search parameter setting: the precursor mass tolerance was 20ppm and the secondary debris mass tolerance was 0.02Da. Oxidation (methionine) (+ 15.9949 Da) and acetylation (protein N-terminus) (+ 42.0106 Da) as variable modifications, ureido (cysteine) (+ 57.0215 Da) as fixed modifications, and cleavage allowing three trypsin misses. The single peptide only considers the peptide with the length of at least 6 amino acids, so that the False Discovery Rate (FDR) is controlled to be within 1 percent, at least one specific peptide is needed for identifying the protein, and the detection result is shown in figure 2.

Then, hsa_circ_0001946 related T series vectors (circ 1946 overexpressing plasmid empty vector (T0), circ1946 overexpressing plasmid (T1), circ1946-3 x Flag tag plasmid (T2), circ1946-ORF overexpressing plasmid empty vector (T3), circ1946-ORF overexpressing plasmid (T4)) were constructed, and the presence of the authenticity of circ1946-109aa was determined by Western Blot (WB) detection of Flag tag proteins.

The construction and identification flow of the over-expression plasmid is as follows:

t1 is constructed as follows:

the hsa_circ_0001946 (sequence source, circ Bank ID: hsa_circCDR 1_001) full-length 1485bp sequence is amplified by PCR, ecoRI and BamHI are used for double digestion and connected into pLC5-ciR, positive clones are screened, sequencing primers are added, a sequencing peak diagram is normal, no impurity peak or overlapping band exists, and sequence comparison is identical, so that hsa_circ_0001946 is successfully inserted into pLC5-ciR, and the over-expression vector is successfully constructed.

Primer sequences are shown in the following table:

t2 is constructed as follows:

the hsa_circ_0001946_011-3xFlag|1563nt full length 1563bp sequence was amplified by PCR and the In-Fusion clone was ligated into pLC5-ciR. T2 construction vector PLL2212 hsa_circ_0001946_011 inserts the 3xFlag expression vector. The sequencing peak diagram is normal, no impurity peak or overlapping band exists, and the sequence comparison is identical, which shows that the target sequence is successfully inserted into pLC5-ciR, and the carrier is successfully constructed.

The primer sequences were as follows:

t4 is constructed as follows:

the circ1946-109aa is combined with a 3xFlag tag to serve as a target sequence, a Kozak sequence (GCCACC) is added before ATG, the full-length sequence is 414bp, the full-length sequence is seamlessly cloned to the PCDH-CMV-MCS-EF1a-GFP-puro vector, the sequencing peak diagram is normal, no impurity peak or overlapping bands exist, the sequence comparison is identical, and the target sequence is successfully inserted into the PCDH-CMV-MCS-EF1a-GFP-puro, and the vector construction is successful.

The primer sequences were as follows:

TABLE 1T series vector and PCR, WB detection results

Note that: circ1946 is hsa_circ_0001946, 109aa is circ1946-109aa, and the ORF is the ORF encoding circ1946-109aa; "+" indicates endogenous expression, "-" indicates endogenous no expression, "+.f" indicates high expression.

T0 is an empty vector, and the construction method is different from that of T1 in that T0 is not inserted into a target sequence. Similarly, T3 was used as an empty vector, and T3 was not inserted into the target sequence compared to the construction of T4. Based on the results of Table 1, it can be seen that circ1946-109aa is actually present as the encoded product of hsa_circ_ 0001946. The results show that circ1946-109aa practically has corresponding expression in esophageal squamous carcinoma cell lines.

2. Preparation of circ1946-109aa specific antibodies

Using recombinant protein as antigen, a circ1946-109aa specific antibody can be prepared as follows:

the ORF sequence is constructed to pET-28a-SUMO carrier expression protein by means of total gene synthesis, and the sequencing identification is correct. After 16 hours of automatic induction culture, the protein is obtained by purifying and identifying the broken bacteria. Separating and obtaining 2 experimental Japanese big ear white rabbits immunized by the recombinant protein, and taking the three-immune serum to obtain the antibody after affinity purification.

Example 2circ1946-109aa verification of esophageal squamous carcinoma correlation

1. Kaplan-Meier (K-M) survival curve analysis

113 esophageal squamous carcinoma tumor tissues are collected, a microarray tissue chip is prepared, the circle 1946-109aa is dyed and read by using an immunohistochemical dyeing method, and the dyed tissue slice is observed and analyzed under a fluorescence microscope.

The Kaplan-Meier (K-M) curve was used for Overall Survival (OS) analysis. The R language is the primary mapping and statistics tool. As shown in FIG. 4, circ1946-109aa was expressed poorly in esophageal squamous carcinoma patients, and overall survival was shorter, demonstrating the ability of circ1946-109aa to serve as a prognostic marker for esophageal squamous carcinoma.

2. Cell proliferation, scratch and Transwell experiments

The effect of circ1946-109aa on the activity, migration and invasiveness of esophageal squamous carcinoma cell lines (K30 and K510) was evaluated using cell proliferation, scoring, transwell experiments, and the specific procedures were as follows:

(1) Cell proliferation assay:

cell counting kit-8 (CCK-8) was used to determine cell viability. T-series plasmids (table 1) of K30 and K510 were inoculated into 96-well plates and stably cultured for 24, 48, 72 hours, CCK8 solution was added to each well of the plates and incubated, and absorbance at 450nm was measured with a microplate reader, and the results are shown in fig. 5.

(2) Cell scratch assay:

scratch tests were used to assess the migration ability of cells. Cells were stably cultured overnight by plating appropriate numbers of T-series plasmids (table 1) into 6-well plates and streaked perpendicular to the cell plane until the cells grew out of the plates. After the scratch was finished, 0 point of the experiment was counted, and then taken out for photographing at 24 hours and 48 hours, respectively. As a result, as shown in FIG. 6, the T4 plasmid transfected cell line showed significantly less number of cells at the streak than the other experimental groups, demonstrating that overexpression of circ1946-109aa inhibited the migration ability of tumor cells.

(3) Transwell experiments:

the Transwell model was used to assess the invasive capacity of cells. A suitable amount of the T-series plasmid (Table 1) stable cell suspension was plated into the transwell upper chamber, 500uL of 20% serum medium was added to the 24-well plate lower chamber, and no air bubbles were present between the lower culture broth and the upper chamber, millions of times. Placing the culture plate into an incubator for continuous culture for 12-48h, sucking and discarding the culture solution of the upper chamber and the lower chamber, washing twice by PBS, soaking the lower surface of the culture plate into methanol solution for fixation, staining with crystal violet or trypan blue, performing microscopic examination, calculating the cell number of the lower surface of the PET film, calculating 5 fields in the middle and four sides, taking an average value, and reducing the invasion capacity of esophageal squamous carcinoma cells over expressing circ1946-109aa as shown in figure 7.

According to the above figures 5, 6 and 7, it is shown that the overexpression of circ1946-109aa can inhibit proliferation, migration and invasion of esophageal squamous carcinoma cells.

Example 3 verification of the correlation of circ1946-109aa with the expression of the immune checkpoint molecule B7-H3

The method comprises the following steps of transfecting circ1946-109aa over-expression plasmids into esophageal squamous carcinoma cell lines (K30, K510 and TE-1), using empty vector transfected cells as a control, extracting cellular RNA, and detecting the change of the expression quantity of B7-H3 by using qRT-PCR technology:

(1) RNA extraction

Collecting esophageal squamous carcinoma cells, adding Trizol, and standing at room temperature for 5min to allow full lysis; adding chloroform according to 200ul chloroform/mL Trizol, shaking vigorously for 15s, and standing at room temperature for 5min; centrifuging at 4deg.C for 15min at 12,000Xg; sucking the upper water phase and transferring the upper water phase into another centrifuge tube; adding isopropanol according to 0.5mL isopropanol/mL Trizol, mixing uniformly, and standing at room temperature for 10min; centrifuging at 4deg.C for 10min at 12,000Xg, and discarding supernatant; adding 75% ethanol into 1mL of 75% ethanol/mL Trizol, gently shaking a centrifuge tube, and suspending and precipitating; centrifuging at 4deg.C for 5min at 7,500Xg, and discarding supernatant as much as possible; airing at room temperature for 5-10 min, adding 20 mu L DEPC water to dissolve the precipitate.

(2) RNA quality and integrity detection

OD value was measured to quantify RNA, record A _260/280 Ratio and RNA concentration. 1% agarose gel electrophoresisAfter electrophoresis, the sample is observed under an ultraviolet lamp, photographed and stored.

(3) Reverse transcription

(1) Preparing first strand cDNA synthesis reaction liquid

Note that: gently beating and mixing by a pipette.

(2) The first strand cDNA synthesis reaction was performed under the following conditions

* If the template has a complex secondary structure or a high GC region, the reaction temperature can be increased to 55℃to help increase the yield.

(4) qPCR experiment

(1) Preparation of qPCR reaction system

cDNA (1:10 dilution) 2. Mu.L

Upstream primer (10. Mu.M) 0.4. Mu.L

Downstream primer (10. Mu.M) 0.4. Mu.L 2X SYBR Green PCR Master Mix. Mu.L

ddH ₂ O up to 20μL

(2) qPCR reaction program settings

(5) qPCR primers

As shown in FIG. 8, the reduced content of B7-H3 (i.e., CD276 in FIG. 8) in the circ1946-109aa over-expression group suggests that circ1946-109aa is capable of inhibiting the expression of immune checkpoint B7-H3.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A polypeptide is characterized in that the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1.

2. A nucleic acid encoding the polypeptide of claim 1.

3. A pharmaceutical composition comprising the polypeptide of claim 1, further comprising a pharmaceutically acceptable excipient.

4. Use of the polypeptide of claim 1 for the preparation of a prognosis product for esophageal squamous carcinoma.

5. Use of the polypeptide of claim 1 or the pharmaceutical composition of claim 3 for the preparation of a medicament for esophageal squamous carcinoma.