Gene detection method and kit for detecting gene variance of tumor tissue and variance from synchronization radiation to evaluate therapeutic

Abstract

The present invention provides a gene detection method and kit for detecting tumor tissue gene variation and changes before and after synchronized radiotherapy to perform therapeutic response, recurrence and survival assessment of esophageal cancer patients. The invention develops an esophageal cancer NGS analysis kit. According to 402 variation sites containing 35 genes frequently occurring in esophageal squamous epithelial cell carcinoma tissue cells, 62 specific site variation of tissues before and after esophageal squamous epithelial cell carcinoma CCRT is analyzed, and it is expected that a new prediction marker can be found. The potential markers are combined into an esophageal cancer detection kit, and the kit has extremely high value for improving the prognosis of esophageal cancer.

Description

Technical Field

[0001] The present invention relates to a gene detection method and kit for detecting gene mutations in tumor tissues and synchronizing changes before and after chemoradiotherapy to evaluate the treatment response, recurrence, and survival of esophageal cancer patients. Background Art

[0002] Esophageal cancer is a fatal disease. Even for patients detected at an early stage, there is still a high risk of in-situ recurrence and distant metastasis. Esophageal cancer is mainly divided into esophageal squamous cell carcinoma (ESCC) and adenocarcinoma. The standard treatment for locally advanced esophageal cancer is neoadjuvant concurrent chemoradiotherapy (CCRT) before surgery, with or without surgical resection. Esophageal squamous cell carcinoma patients who respond well to CCRT have a better survival period. Under other treatment methods, the pathological complete remission rate is only 10% to 40%. Nevertheless, the prognosis of esophageal cancer is still poor. Even with combined therapies, the 5-year survival rate is still less than 20%, and more than 50% of patients have in-situ recurrence or distant metastasis within two to three years. The tumour, node and metastasis stage (TNM stage) is considered the golden standard for predicting clinical outcomes and guiding treatment strategies. Therefore, identifying and using multiple reliable markers for chemoradiotherapy response and prognosis to provide more complete treatment information for esophageal cancer patients is what researchers in this field are working on.

[0003] As mentioned above, esophageal cancer is a fatal disease with a high risk of recurrence. Especially for esophageal squamous cell carcinoma patients, if recurrence occurs, the median survival is generally only 8 months. Therefore, developing a more effective in vitro tumor tissue gene detection kit for evaluating the response and prognosis of preoperative concurrent chemoradiotherapy (CCRT) in esophageal cancer patients and understanding the gene mutation situation before and after treatment will be very helpful for improving the treatment effect of esophageal cancer. Summary of the Invention

[0004] In view of the above, an object of the present invention is to provide a gene detection method for detecting gene mutations in tumor tissues and synchronizing changes before and after chemoradiotherapy to evaluate the treatment response, recurrence, and survival of esophageal cancer patients, comprising: (a) obtaining an in vitro tumor tissue of an esophageal cancer patient, and extracting genomic DNA from the in vitro tumor tissue to obtain at least one genomic DNA sample; and (b) establishing an amplicon library from the at least one genomic DNA sample and performing next generation sequencing (NGS) to obtain at least one genomic DNA data, thereby evaluating the preoperative chemoradiotherapy response and prognosis of the esophageal cancer patient; wherein the at least one genomic DNA data includes a gene locus related to the chemoradiotherapy response and a gene locus related to the prognosis of the esophageal cancer patient. When the esophageal cancer patient carrying the gene locus related to the chemoradiotherapy response or the gene locus related to the prognosis of the esophageal cancer patient cannot achieve a 5-year progression-free survival (PFS), it indicates a poor preoperative chemoradiotherapy response or prognosis; wherein the gene locus related to the prognosis of the esophageal cancer patient includes the Mucin 17 (MUC17) gene locus, the p.Asp2397His locus, the p.His2381Asp locus, the p.Pro3360His locus, the p.Thr2382Ala locus, the p.Thr2411Ser locus, the p.Thr3355Ser locus, and the p.Val3353Ala locus of the Mucin4 (MUC4) gene, the USH2A gene locus, and the myosin, heavy chain 4 (MYH4) gene locus; wherein the MUC17 gene locus is selected from the group consisting of the p.Thr2702Val locus of the MUC17 gene, the p.Thr3355Ser locus of the MUC17 gene, the p.Leu2712Val locus of the MUC17 gene, the p.Asn2706Ser locus of the MUC17 gene, the p.Leu2703_Leu2704delinsProVal locus of the MUC17 gene, the p.Pro2716Ala locus of the MUC17 gene, and combinations thereof.

[0005] In an embodiment of the present invention, the gene locus related to the preoperative chemoradiotherapy response is selected from the group consisting of the p.Pro1319Ser locus of the MUC17 gene, the p.Arg2159Gly locus of the MUC17 gene, the p.Gly1307Ser locus of the MUC17 gene, the p.Val1309Met locus of the MUC17 gene, and combinations thereof.

[0006] In one embodiment of the present invention, the esophageal cancer is esophageal squamous cell carcinoma.

[0007] In one embodiment of the present invention, the prognosis includes recurrence and death.

[0008] In one embodiment of the present invention, the variation of the p.Pro1319Ser site in the pre-treatment tissue shows that the risk of partial response to preoperative concurrent chemoradiotherapy in esophageal cancer patients is 6.22 times that of those without variation.

[0009] In one embodiment of the present invention, the variation of the p.Thr2702Val site in the pre-treatment tissue shows a 3.32-fold recurrence risk compared to those without variation.

[0010] In one embodiment of the present invention, the variation of the p.Thr2702Val site in the post-treatment tissue shows a 3.21-fold recurrence risk compared to those without variation.

[0011] In one embodiment of the present invention, in esophageal cancer patients compared before and after CCRT, the gene variations include increases and decreases. An increase means there is a variation in the post-treatment tissue but no variation in the pre-treatment tissue, and a decrease means there is a variation in the pre-treatment tissue but no variation is seen in the post-treatment tissue.

[0012] In one embodiment of the present invention, the combination of the p.Pro1319Ser site of the MUC17 gene and the p.Arg2159Gly site of the MUC17 gene in the pre-treatment tissue shows that the risk of partial response to preoperative concurrent chemoradiotherapy in esophageal cancer patients is 7 times that of those without variation.

[0013] In one embodiment of the present invention, the combination of the p.Pro1319Ser site of the MUC17 gene and the p.Gly1307Ser site of the MUC17 gene in the pre-treatment tissue shows that the risk of partial response to preoperative concurrent chemoradiotherapy in esophageal cancer patients is 6.03 times that of those without variation.

[0014] In one embodiment of the present invention, the combination of the p.Pro1319Ser site of the MUC17 gene and the p.Val1309Met site of the MUC17 gene in the pre-treatment tissue shows that the risk of partial response to preoperative concurrent chemoradiotherapy in esophageal cancer patients is 6.35 times that of those without variation.

[0015] Another object of the present invention is to provide an in vitro tumor tissue gene detection kit for evaluating the response and prognosis of preoperative concurrent chemoradiotherapy (CCRT) in esophageal cancer patients, which is established by the method as described above.

[0016] Another object of the present invention is to provide a gene detection kit for detecting gene mutations in tumor tissues and synchronizing changes before and after chemoradiotherapy to evaluate the treatment response, recurrence, and survival of esophageal cancer patients, comprising: (a) obtaining in vitro tumor tissues from esophageal cancer patients before and after CCRT treatment, and extracting genomic DNA from the in vitro tumor tissues to obtain at least one genomic DNA sample; and (b) establishing an amplicon library from the at least one genomic DNA sample and performing next generation sequencing (NGS) to obtain at least one genomic DNA data, thereby evaluating the changes in gene locus mutations before and after chemoradiotherapy in esophageal cancer patients; wherein the at least one genomic DNA data contains a gene locus related to chemoradiotherapy response, and the changes in gene locus mutations of the esophageal cancer patient carrying the gene locus related to chemoradiotherapy response before and after chemoradiotherapy response are statistically significant; wherein the gene locus related to chemoradiotherapy response related to treatment response is selected from the group consisting of: the p.Glu1523Lys locus of the EP300 gene, the p.Glu5905Asp locus of the SYNE1 gene, the p.Asp2397His locus of the MUC4 gene, the p.Ala2409Val locus of the MUC4 gene, the p.Glu5905Asp locus of the SYNE1 gene, the p.Ala2409Val locus of the MUC4 gene, and combinations thereof; the gene locus related to chemoradiotherapy response related to recurrence is selected from the group consisting of: the p.Glu5905Asp locus of the SYNE1 gene, the p.Pro3360His locus of the MUC4 gene, the p.Thr2382Ala locus of the MUC4 gene, the p.Ala2390Thr locus of the MUC4 gene, the p.Leu2712Val locus of the MUC17 gene, and combinations thereof; the gene locus related to chemoradiotherapy response related to survival is selected from the group consisting of: the p.Asp2397His locus of the MUC4 gene, the p.Pro3360His locus of the MUC4 gene, the p.Ala2390Thr locus of the MUC4 gene, and combinations thereof.

[0017] In an embodiment of the present invention, compared with before CCRT, the gene mutation situation of the esophageal cancer patient includes increase and decrease. Increase means that there is a mutation in the tissue after treatment and no mutation before treatment, and decrease means that there is a mutation in the tissue before treatment and no mutation is seen after treatment.

[0018] In summary, the present invention has developed a next-generation sequencing (NGS) panel for esophageal cancer (especially esophageal squamous cell carcinoma). Therefore, 402 variant sites of 35 genes that often occur in esophageal squamous cell carcinoma tissue cells were first analyzed, and specific site variations in 62 pairs of esophageal squamous cell carcinoma tissues before and after CCRT were analyzed to find new predictive markers. It was also found that the variations at some sites changed before and after CCRT, and the change patterns were significantly correlated with treatment response, recurrence, and survival. The present invention combined these potential markers into an esophageal cancer detection panel, which has extremely high value for improving the prognosis of esophageal cancer.

[0019] The following will further illustrate the implementation manners of the present invention. The following listed embodiments are used to clarify the present invention and are not used to limit the scope of the present invention. Any person skilled in this art can make some modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to what is defined by the following claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Receiver operating characteristic (ROC) curves showing the combined risk changes are used to distinguish patients with esophageal squamous cell carcinoma (ESCC) who have a complete or partial response to concurrent chemoradiotherapy (CCRT). AUC represents the area under the ROC curve.

[0021] Figure 2A and Figure 2B ROC curves showing the risk genotypes of tissues before CCRT are used to distinguish Figure 2A () patients with disease recurrence or non-recurrence; Figure 2B () dead or surviving ESCC patients. AUC represents the area under the ROC curve.

[0022] Figure 3A and Figure 3B Kaplan-Meier estimates of the progression-free survival (PFS, A) and overall survival (OS, B) of patients with unfavorable genotypes (positive) or without (negative) in tissues before CCRT are shown. Unfavorable genotypes: MUC17 p.Thr2702Val (variant) and MUC4 p.Thr3355Ser (wild type); MST represents the median survival time.

[0023] Figure 4Show the overall changes in tissue-site mutations before and after CCRT. There were 1,814 total mutation sites before CCRT and 1,754 total mutation sites after CCRT, and the difference in the amount of mutations after and before was -60.

[0024] Figure 5 Show the ranking of the number of increased site mutations after CCRT (≥1), that is, the total increase in gene site mutations after CCRT was greater than 1 sample pair. The genes where the top 10 mutants with the highest increase frequencies were located were USH2A, MUC4 (2), MUC17 (3), EP300, LRP2, SYNE1, and TP53.

[0025] Figure 6 Show the ranking of the number of decreased site mutations after CCRT (≥1), that is, the decrease in gene site mutations after CCRT was greater than 1 sample pair. The genes where the top 10 mutants with the highest increase frequencies were located were MUC4 (4), MUC17 (4), ZFHX4, and TP53.

[0026] Figure 7 Show that the decrease in MUC17 p.Leu2712Val mutation after CCRT was significantly correlated with the risk of shorter PFS. The decrease in mutation here refers to the presence of mutation in the tissue before treatment and the absence of mutation in the tissue after treatment.

[0027] Figures 8 - 11 Show that the unchanged mutation amounts of 4 MUC17 sites after CCRT, including MUC17 p.Leu2712Val, MUC17 p.Leu2703_Leu2704delinsProVa, MUC17 p.Asn2706Ser, and MUC17 p.Pro2716Ala, were significantly correlated with better overall survival (OS) time.

[0028] Figure 12 Show that the decrease in the mutation of MUC4 p.Pro3360His site after CCRT was significantly correlated with longer overall survival (OS) time. The decrease in mutation here refers to the presence of mutation in the tissue before treatment and the absence of mutation in the tissue after treatment. Detailed implementation methods

[0029] Definitions

[0030] The numerical values used in this article are approximate values. All experimental data are expressed within the range of ±20%, preferably within the range of ±10%, and most preferably within the range of ±5%.

[0031] Unless otherwise specified in the text, the terms "a", "the", and similar terms used in this specification (especially in the following claims) should be understood to include both singular and plural forms.

[0032] The present invention is further illustrated by the following examples. These examples are provided for illustration only and are not intended to limit the scope of protection of the present invention. The scope of protection of the present invention is as defined by the scope of the claims.

[0033] Example 1. Patient characteristic analysis

[0034] The study population of this example is described as follows. Approved by the hospital ethics committee, a total of 62 patients with locally advanced esophageal squamous cell carcinoma (ESCC) (T3N0-1M0 or T1-3N1M0) who received neoadjuvant (preoperative) concurrent chemoradiotherapy (CCRT) followed by esophagectomy were included in Mackay Memorial Hospital, Taiwan, China, and a retrospective study was conducted. The specified radiation doses for large-volume tumors and metastatic lymph nodes or subclinical mucosal / submucosal diseases and regional lymph pools were 48 Gy and 43.2 Gy, respectively. All patients received 24 sessions of intensity-modulated radiotherapy and were concurrently treated with cisplatin chemotherapy.

[0035] Among these patients, 22 (35.5%) had a complete response to neoadjuvant CCRT (defined as the tumor regression grade (pathological complete response, pCR)), and the remaining enrolled patients (N = 40, 64.5%) had only a partial response (PR) to the treatment. Information on demographic and clinical data was obtained from the medical records and clinical databases of the Department of Surgery, Pathology, and Laboratory Medicine of Mackay Memorial Hospital.

[0036] Formalin-fixed paraffin-embedded (FFPE) pathological esophageal tumor tissue specimens were collected from endoscopic biopsies before CCRT treatment. FFPE tissues for pathological response evaluation and genomic analysis were collected during esophagectomy.

[0037] The characteristic analysis of the patients is shown in Table 1. Among the 62 patients, 58 (93.5%) were male, 38 (61.3%) were in the T3 clinical tumor stage (cT), 32 patients were in the N0 or N1 clinical lymph node (cN) stage, and the resulting clinical stage (cStage) was stage 2 and stage 3, accounting for 29% and 71.0%, respectively. The distribution of these clinical or demographic variables was not significantly different between the partial response (PR) group and the pathological complete response (pCR) group (see Table 1).

[0038] Table 1

[0039]

[0040]

[0041] Example 2. Gene variants related to CCRT reaction

[0042] The experimental procedure for analyzing gene variants by next generation sequencing (NGS) used in this example is as follows. The gene variant analysis of ESCC tissues by NGS was carried out in the laboratory of LIHPAO Life Science Co. The workflow includes steps of genomic DNA extraction, library preparation, sequencing, and data analysis. Each step is described in the reference (Tang P, Tan C, Pang Q, Chi CW, Wang Y, Yuan Z, Huang YC, Chen YJ. Combination of 35-Gene Mutation Profile and Radiotherapy Dosimetry Predicts the Therapeutic Outcome of Definitive Chemoradiation in Patients With Esophageal Squamous Cell Carcinoma. Front Oncol. 2021 Aug 27;11:729418) and is briefly described as follows.

[0043] The operating procedure for genomic DNA extraction is as follows. For each sample, 5×5 mm 2 and 5-μm thick FFPE (formalin-fixed paraffin-embedded) sections were cut from the block. According to the manufacturer's instructions, genomic DNA was extracted using a DNA sample preparation kit (Roche, Basel, Switzerland). The isolated DNA was quantified using a Qubit dsDNA HS Assay kit (ThermoFisher Scientific, Waltham, MA, USA). The DNA concentration and integrity were analyzed using a DNF-474 high-sensitivity NGS fragment analysis kit (AATI, Ankeny, IA, USA) and a Fragment Analyzer automated CE system.

[0044] The operating procedures for amplicon library construction and NGS are as follows. An amplicon library was constructed using 10 ng of DNA from FFPE tissue samples with the IonAmpliSeqTM Library Kit 2.0 (Thermo Fisher Scientific) and the Ion XpressTM Barcode Adapters Kit (Thermo Fisher Scientific). The amplicon library was purified with Agencourt AMPure XP reagent (Beckman Coulter, Brea, CA, USA) and washed with 70% ethanol on a DynaMagTM-2 Magnet (Thermo Fisher Scientific). Quality control of the amplicon library was established using the IonLibrary TaqMan Quantitation Kit in conjunction with the 7500Fast and 7500 Real-Time PCR Systems (Thermo Fisher Scientific). The esophageal squamous cell carcinoma (ESCC) detection panel contains 402 gene variants, 159 amplicons, covering 35 genes, including ABCA13, DNAH5, FBXW7, FAT1, FAT3, GPR98, EP300, DMD, KDM6A, CSMD3, CDKN2A, KMT2D, MUC4, MUC17, MUC2, MUC16, MYH4, TNN, HMCN1, USH2A, LRP1B, XIRP2, LRP2, NFE2L2, NOTCH1, TTN, FSIP2, SI, PIK3CA, RB1, TP53, ZFHX4, TRIO, SYNE1, and PCLO. The IonOneTouch TM 2 system was used to clone and amplify the quantified library on ion sphere particles by emulsion polymerase chain reaction. Next, the ion sphere particles were enriched in the Ion OneTouch TM ES instrument (Thermo Fisher Scientific). Finally, the enriched ion sphere particles were loaded onto a 316 chip and sequenced on an Ion Torrent PGM system (Ion Torrent, Paisley, UK).

[0045] DNA sequencing data based on the Personal Genome Machine was generated using Torrent Suite software (Thermo Fisher Scientific). Variant calling and annotation were performed using Ion-Reporter v5.1.0. Variants with an average coverage of ≥1500 reads and a variant allele frequency of ≥5% were reported.

[0046] The statistical analysis used in this example is described below. Pearson’s Chi-square test or Fisher’s exact test was used to analyze the demographic, clinical characteristics, and variant genotype distribution between subgroups with different clinical outcomes (including treatment response, recurrence, and mortality) if any of the cell counts in the contingency table was less than 5. Univariate or multivariate logistic regression was used to evaluate the odds ratio (OR) for partial response (PR) to CCRT. The hazard ratio (HR) obtained from Cox regression analysis was used to describe the relative risk of recurrence or death. Data are presented as mean and 95% confidence interval (CI) for regression analysis. The correlation between variant genotype and overall survival (OS) and progression-free survival (PFS) was also analyzed using the Kaplan-Meier survival function and compared using the log-rank test. The linear relationship between genotype variables was analyzed by collinearity diagnostic using the variance inflation factor (VIF). All statistical analyses were performed using IBM SPSS Statistics for Windows version 25.0 (IBM Corp., Armonk, N.Y., USA). A P-value ≤0.05 was considered statistically significant.

[0047] Table 2 shows that 19 variants were detected from tumor tissues before the CCRT showed significant (P≤0.05) or borderline significant (0.05<P<0.10) association with the CCRT response by Pearson chi-square test or Fisher's exact test. Among these gene variants, 14 variants were located within the coding region of the Mucin 17 (MUC17) gene. Univariate logistic regression further revealed 11 variants, including FAT1 p.Asn2678Asp, MUC17 p.Ala1322Thr, MUC17 p.Arg2159Gly, MUC17 p.Asn2706Ser, MUC17 p.Gly1307Ser, MUC17 p.Leu2712Val, MUC17 p.Pro1319Ser, MUC17 p.Pro1321Thr, p.Thr2702Val, MUC17 p.Thr2721Ile, and MUC17 p.Val1309Met, which were significantly associated with an increased risk of partial response.

[0048] Table 2

[0049]

[0050]

[0051] Among these variants, MUC17 p.Pro1319Ser showed the most significant correlation, with a 6.22-fold increased risk of partial response (OR[95%CI] = 6.22[1.96 - 19.78], p = 0.002, Table 3).

[0052] Table 3

[0053]

[0054]

[0055] In this example, the collinearity and collinearity diagnosis of the unfavorable variants of the CCRT response within the MUC17 gene were analyzed by examining the variance inflation factor (VIF). Three variants with major unfavorable variants, including MUC17 p.Arg2159Gly, p.Gly1307Ser, and p.Val1309Met, showed a high degree of genetic collinearity (Table 4).

[0056] Table 4

[0057]

[0058] Each of these three variants was combined with p.Pro1319. Patients carrying at least one variant (positive) showed a significant increase in the partial response group compared to patients carrying no variants (negative), especially for MUC17 p.Pro1319Ser and p.Arg2159Gly (PR vs. pCR, 80% and 31.8%, p < 0.001, Table 5).

[0059] Table 5

[0060]

[0061] Univariate logistic regression also demonstrated a significant effect of MUC17 p.Pro1319Ser and p.Arg2159Gly in increasing the risk of partial response by 7.00-fold (OR [95% CI] = 7.00 [3.07 - 15.94], p < 0.001, Table 6).

[0062] Table 6

[0063] Variant Unfavorable genotype OR (95% CI) p - value * MUC17 p.Pro1319Ser + p.Arg2159Gly Negative 1 Positive 7.00(3.07-15.94) <0.001 MUC17 p.Pro1319Ser + p.Gly1307Ser Negative 1 Positive 6.03(2.69-13.52) <0.001 MUC17 p.Pro1319Ser + p.Val1309Met Negative 1 Positive 6.35(2.81-14.39) <0.001

[0064] Figure 1 Receiver operating characteristic (ROC) curves showing the change in combined risk were used to distinguish ESCC patients with complete or partial response to CCRT. AUC represents the area under the ROC curve.

[0065] The ROC curves showed that the positivity of MUC17 p.Pro1319Ser and p.Arg2159Gly both had good ability to predict the response to CCRT (AUC = 0.718, Figure 1 )

[0066] Example 3. Prognosis-related gene variants

[0067] This example further analyzed the correlation between gene variants and prognosis, including tumor recurrence and death of patients, that is, poor prognosis. Table 7 shows that 24 and 16 variants detected from tumor tissues before and after CCRT respectively showed significant or borderline significant correlations with the CCRT response.

[0068] Table 7

[0069]

[0070] *Pearson chi-square test or Fisher's exact test

[0071] Most variants are located in the coding regions of the MUC17 and MUC4 genes. Univariate logistic regression further showed that 15 variants in the pre-CCRT tissues, including MUC17 p.Asn2706Ser, MUC17 p.Leu2712Val, MUC17 p.Thr2702Val, MUC17 p.Thr2721Ile, MUC17 p.Asn2706Ser, MUC17 p.Leu2703_Leu2704delinsProVa, MUC17 p.Pro2716Ala, MUC17 p.Thr2721Ile, MUC4 p.Asp2397His, MUC4 p.His2381Asp, MUC4 p.Pro3360His, MUC4 p.Thr2382Ala, MUC4 p.Thr2411Ser, MUC4 p.Thr3355Ser, and MUC4 p.Val3353Ala, were significantly associated with an increased risk of disease recurrence (see Table 8). In the tissue analysis after CCRT, 10 gene variants such as EP300, MUC17, MUC4, and USH2A significantly increased the recurrence risk of patients (Table 8). Among these variants, MUC17 p.Thr2702Val showed the most obvious adverse effect on the prognosis of tissues before and after CCRT. Compared with patients without gene variants, the recurrence risks of patients with gene variants increased by 3.32-fold and 3.21-fold, respectively (before CCRT, OR [95% CI] = 3.32 (1.84 - 6.00), p < 0.001; after CCRT, OR [95% CI] = 3.21 (1.80 - 5.76), p < 0.001, Table 8). Meanwhile, the variant of MUC4 p.Thr3355Ser showed an obvious effect in reducing the recurrence risk (before CCRT, OR [95% CI] = 0.39 (0.21 - 0.69), p = 0.002; after CCRT, OR [95% CI] = 0.45 (0.26 - 0.80), p = 0.006, Table 8).

[0072] Table 8

[0073]

[0074]

[0075] Similar results were also found when analyzing the survival rate of patients by univariate logistic regression. A total of 13 and 17 variants were significantly associated with an increased risk of patient death detected from tissues before and after CCRT, respectively. Most variants were also within the coding regions of the MUC17 and MUC4 genes. MUC17 p.Thr2702Val detected from tissues before and after CCRT was also the main variant related to patient survival (tissue before CCRT, OR [95% CI] = 4.22 (2.20 - 8.11), p < 0.001; tissue after CCRT, OR [95% CI] = 4.47 (2.35 - 8.49), p < 0.001, Table 9). The variant of MUC4 p.Thr3355Ser was also shown to be associated with a reduced risk of death (before CCRT, OR [95% CI] = 0.40 (0.21 - 0.74), p = 0.004; after CCRT, OR [95% CI] = 0.53 (0.29 - 0.97), p = 0.039, Table 9). Notably, the variant MYH4 p.Gln1210fs within the MYH4 gene detected from tissues before and after CCRT was also shown to be significantly associated with a reduced risk of patient death (before CCRT, OR [95% CI] = 0.38 (0.17 - 0.87), p = 0.022; after CCRT, OR [95% CI] = 0.38 (0.18 - 0.79), p = 0.010, Table 9).

[0076] Table 9

[0077]

[0078]

[0079] In this example, collinearity diagnosis was used to analyze the collinearity of adverse variants in the prognosis response within the MUC17 and MUC4 genes. None of the variants showed a high degree of gene collinearity (results not presented). In this example, the variant of MUC17 p.Thr2702Val and the wild type of MUC4 p.Thr3355Ser were defined as adverse genotypes. Patients carrying at least one adverse genotype (positive) had 100% sensitivity for predicting disease recurrence in tumor tissues before and after CCRT (P < 0.001 respectively, Table 10 and Table 11, Fisher's exact test).

[0080] Table 10

[0081]

[0082] Table 11

[0083]

[0084]

[0085] Univariate Cox regression further showed that the recurrence risk of patients with gene mutations detected before and after CCRT was 4.57-fold and 3.43-fold higher, respectively, compared with those without detection (before CCRT, OR [95% CI]=4.57 (2.31-9.01), p<0.001, Table 12; after CCRT, OR [95% CI]=3.43 (1.90-6.17), p<0.001, Table 13).

[0086] Table 12

[0087]

[0088] Table 13

[0089]

[0090] The mortality rates of patients with unfavorable genotypes of MUC17 p.Thr2702Val and / or MUC4 p.Thr3355Ser in pre- and post-operative tissues were 92.1% and 90.9%, respectively (P values <0.001 and 0.003, respectively, Table 15).

[0091] Table 14

[0092]

[0093] Table 15

[0094]

[0095] Univariate Cox regression showed that the mortality risk of patients with unfavorable genotypes of MUC17 p.Thr2702Val and / or MUC4 p.Thr3355Ser in fresh (before CCRT) or post-treatment tissues was as high as 4.98-fold and 3.55-fold, respectively, compared with those without (before CCRT, OR [95% CI]=4.98 (2.34-10.58), p<0.001, Table 16; after CCRT, OR [95% CI]=3.55 (1.86-6.79), p<0.001, Table 17).

[0096] Table 16

[0097]

[0098] Table 17

[0099]

[0100] Figure 2A and Figure 2BThe ROC curve showing the risk genotypes of the tissue before CCRT was used to distinguish ( Figure 2A ) between disease recurrence and non-recurrence; ( Figure 2B ) between death and survival in ESCC patients. AUC represents the area under the ROC curve. The ROC curve showed that either positive for the unfavorable genotype of MUC17 p.Thr2702Val and MUC4 p.Thr3355Ser had good predictive ability for tumor recurrence (AUC = 0.873, Figure 2A ), and fair discriminative ability for death (AUC = 0.787, Figure 2B ).

[0101] Patients with or without the favorable genotype showed a strong significant difference in the distribution of progression-free survival (PFS) and overall survival (OS) (by log-rank test, p < 0.001, respectively).

[0102] Figure 3A and Figure 3B Showing Kaplan-Meier estimates of the progression-free survival (PFS, A) and overall survival (OS, B) of patients with unfavorable genotype (positive) or without (negative) in the tissue before CCRT. Unfavorable genotypes: MUC17 p.Thr2702Val (variant) and MUC4 p.Thr3355Ser (wild type); MST represents the median survival time. The median PFS times of the negative and positive groups were 35.7 and 10.2 months, respectively ( Figure 3A ). At the same time, none of the carriers of all unfavorable genotypes could achieve a 5-year progression-free survival, while the 5-year progression-free survival probability (PFS probability) of negative patients was 41.0% ( Figure 3A ). For the overall survival analysis, patients without any unfavorable genotypes could achieve a median overall survival of 68.4 months and a long-term survival with an overall survival probability (OS probability) of 57.2%. In contrast, those with unfavorable genotypes had a median overall survival of only 18 months. And only 3.1% showed long-term survival ( Figure 3B ).

[0103] Example 4. Comprehensive comparison of variant changes before and after CCRT and analysis of clinical significance

[0104] This example explores the comprehensive comparison of variant changes before and after CCRT and the analysis of clinical significance. Figure 4 Showing an overview of tissue-site variant changes before and after CCRT, where there were 1814 total variant sites before CCRT and 1754 total variant sites after CCRT, and the difference in the amount of variation after and before was -60.

[0105] Figure 5 Show the ranking of the number of increased site mutations after CCRT (≥1), that is, the total increase in gene site mutations after CCRT is greater than 1 sample pair. The genes where the top 10 mutants with the highest increase frequencies are located are USH2A, MUC4 (2), MUC17 (3), EP300, LRP2, SYNE1, and TP53.

[0106] Figure 6 Show the ranking of the number of decreased site mutations after CCRT (≥1), that is, the decrease in gene site mutations after CCRT is greater than 1 sample pair. The genes where the top 10 mutants with the highest increase frequencies are located are MUC4 (4), MUC17 (4), ZFHX4, and TP53.

[0107] Tables 18 and 19 show the correlation analysis between site mutation changes after CCRT and CCRT response. Among them, Table 18 uses chi-square analysis, and Table 19 uses univariate regression analysis. There are 3 types of change patterns: -1 means before: there is a mutation; after: there is no mutation; 0 means no change before and after; +1 means before: there is no mutation; after: there is a mutation. The results of the chi-square analysis show that different change patterns of EP300p.Glu1523Lys, SYNE1p.Glu5905Asp, MUC4p.Asp2397His, and MUC4p.Ala2409Val are significantly correlated with the CCRT treatment response (P values are 0.019, 0.049, 0.018, and 0.049 respectively, Table 18). However, in the regression analysis, only SYNE1p.Glu5905Asp, MUC4p.Asp2397His, and MUC4p.Ala2409Val show a marginally significant effect (Table 19).

[0108] Table 18

[0109]

[0110] Table 19

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[0112]

[0113] Tables 20 and 21 show the correlation analysis between site variation changes and recurrence after CCRT. Among them, Table 20 was analyzed by chi-square test, and Table 21 was analyzed by univariate regression analysis. The results of the chi-square test showed that different change patterns of SYNE1 p.Glu5905Asp, MUC4 p.Pro3360His, MUC4 p.Thr2382Ala, and MUC4 p.Ala2390Thr were significantly correlated with tumor recurrence (P values were 0.035, 0.011, 0.030, and 0.035 respectively, Table 20). In the regression analysis, significant correlations were found between the pre- and post-variation changes of MUC17 p.Thr2721Ile and MUC4 p.Pro3360His and recurrence (P values were <0.001 and 0.043 respectively) (Table 21).

[0114] Table 20

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[0116]

[0117] Table 21

[0118]

[0119]

[0120] Figure 7 It was shown that a significant correlation existed between the reduction of MUC17 p.Leu2712Val variation and the risk of shorter PFS after CCRT. The reduction of variation refers to the presence of variation in the pre-treatment tissue and the absence of variation in the post-treatment tissue. Tables 22 and 23 show the correlation analysis between site variation changes and survival after CCRT. Among them, Table 22 was analyzed by chi-square test, and Table 23 was analyzed by univariate regression analysis. The results of the chi-square test showed that different change patterns of MUC4 p.Asp2397His, MUC4 p.Pro3360His, and MUC4 p.Ala2390Thr were significantly correlated with the death of patients (P values were 0.014, 0.015, and 0.014 respectively, Table 22). In the regression analysis, significant correlations were found between the pre- and post-variation changes of multiple sites and death (Table 23).

[0121] Table 22

[0122]

[0123]

[0124] Table 23

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[0126] Figures 8 - 11The unchanged variation amounts of 4 MUC17 loci after CCRT, including MUC17p.Leu2712Val, MUC17p.Leu2703_Leu2704delinsProVa, MUC17p.Asn2706Ser and MUC17p.Pro2716Ala, were significantly correlated with better overall survival (OS) time.

[0127] Figure 12 The reduced variation of the MUC4p.Pro3360His locus after CCRT was significantly correlated with longer overall survival (OS) time, where the reduced variation means that there was variation in the pre-treatment tissue but no variation in the post-treatment tissue.

[0128] In summary, the present invention has developed a next-generation sequencing (NGS) analysis kit for esophageal cancer (especially esophageal squamous cell carcinoma). For 402 variant sites of 35 genes that often occur in esophageal squamous cell carcinoma tissue cells, specific site variations of 62 pairs of esophageal squamous cell carcinoma tissues before and after CCRT were analyzed to find new predictive markers, and it was found that the variations at some sites would change before and after CCRT treatment, and the change patterns were significantly correlated with treatment response, recurrence and survival. The present invention combines these potential markers into an esophageal cancer detection kit, which has extremely high value for improving the prognosis of esophageal cancer.

[0129] The above is only illustrative and not restrictive. Any equivalent modification or change without departing from the spirit and scope of the present invention shall be included in the scope defined by the claims.

[0130]

Symbol Explanation

[0131] None.

Claims

1. A genetic testing method for detecting gene mutations in tumor tissue and changes before and after concurrent chemoradiotherapy to evaluate the treatment response, recurrence and survival of esophageal cancer patients, characterized in that: Include: Obtaining an in vitro tumor tissue from an esophageal cancer patient, and extracting genomic DNA from the in vitro tumor tissue to obtain at least one genomic DNA sample; as well as An amplicon library is established by using the at least one genomic DNA sample, and next generation sequencing (NGS) is performed to obtain at least one genomic DNA data, thereby evaluating the preoperative concurrent chemoradiotherapy response and prognosis of esophageal cancer patients; wherein the at least one genomic DNA data report contains a gene locus related to the response to preoperative synchronous chemoradiotherapy and a gene locus related to the prognosis of esophageal cancer patients; when the esophageal cancer patient carrying the gene locus related to the response to preoperative synchronous chemoradiotherapy or the gene locus related to the prognosis of esophageal cancer patients cannot achieve a 5-year progression-free survival (PFS), it indicates that the response to preoperative synchronous chemoradiotherapy or the prognosis is poor; The gene loci related to the prognosis of esophageal cancer patients include the mucin 17 (MUC17) gene locus, the p.Asp2397His site, the p.His2381Asp site, the p.Pro3360His site, the p.Thr2382Ala site, the p.Thr2411Ser site, the p.Thr3355Ser site and the p.Val3353Ala site of the mucin 4 (MUC4) gene, the USH2A gene locus and the myosin heavy chain 4 (MYH4) gene locus; The MUC17 gene locus is selected from the following group: the p.Thr2702Val site of the MUC17 gene, the p.Thr3355Ser site of the MUC17 gene, the p.Leu2712Val site of the MUC17 gene, the p.Asn2706Ser site of the MUC17 gene, the p.Leu2703_Leu2704delinsProVal site of the MUC17 gene, the p.Pro2716Ala site of the MUC17 gene, and combinations thereof.

2. The method according to claim 1, characterized in that The gene loci associated with preoperative concurrent chemoradiotherapy response are selected from the following group: p.Pro1319Ser site of MUC17 gene, p.Arg2159Gly site of MUC17 gene, p.Gly1307Ser site of MUC17 gene, p.Val1309Met site of MUC17 gene, and combinations thereof.

3. The method according to claim 1, characterized in that The esophageal cancer is esophageal squamous cell carcinoma (ESCC).

4. The method according to claim 1, characterized in that: The prognosis includes recurrence and death.

5. The method according to claim 2, characterized in that: The p.Pro1319Ser site mutation in pre-treatment tissue showed that the risk of partial response to preoperative concurrent chemoradiotherapy in patients with esophageal cancer was 6.22 times that of those without mutation.

6. The method according to claim 1, characterized in that The p.Thr2702Val site mutation in pre-treatment tissues showed a 3.32-fold risk of recurrence compared to the non-mutated tissues.

7. The method according to claim 1, characterized in that The p.Thr2702Val site mutation showed a 3.21-fold higher risk of recurrence than the non-mutated group in the post-treatment group.

8. The method according to claim 1, characterized in that The genetic mutations of the esophageal cancer patients after CCRT compared with those before CCRT include increases and decreases. Increase refers to mutations in the tissue after treatment and no mutations before treatment, and decrease refers to mutations in the tissue before treatment and no mutations after treatment.

9. The method according to claim 2, characterized in that: The combination of the p.Pro1319Ser site of the MUC17 gene and the p.Arg2159Gly site of the MUC17 gene in pre-treatment tissue mutations showed that the risk of partial response to preoperative concurrent chemoradiotherapy in patients with esophageal cancer was 7 times that of those without mutations.

10. The method according to claim 2, characterized in that The combination of the p.Pro1319Ser site of the MUC17 gene and the p.Gly1307Ser site of the MUC17 gene in pre-treatment tissue mutations showed that the risk of partial response to preoperative concurrent chemoradiotherapy in patients with esophageal cancer was 6.03 times that of those without mutations.

11. The method according to claim 2, characterized in that The combination of the p.Pro1319Ser site of the MUC17 gene and the p.Val1309Met site of the MUC17 gene in pre-treatment tissue mutations showed that the risk of partial response to preoperative concurrent chemoradiotherapy in patients with esophageal cancer was 6.35 times that of those without mutations.

12. A gene detection kit for detecting gene mutations in tumor tissues and changes before and after concurrent chemoradiotherapy to evaluate the treatment response, recurrence and survival of esophageal cancer patients, characterized in that: The method according to any one of claims 1 to 11 is used to establish the present invention.

13. A method for evaluating the correlation between changes in gene loci and treatment response and prognosis of esophageal cancer patients before and after concurrent chemoradiotherapy (CCRT) using in vitro tumor tissue, characterized in that: Include: Obtaining in vitro tumor tissue from an esophageal cancer patient before and after CCRT treatment, and extracting genomic DNA from the in vitro tumor tissue to obtain at least one genomic DNA sample; as well as An amplicon library is established by using the at least one genomic DNA sample, and next generation sequencing (NGS) is performed to obtain at least one genomic DNA data, thereby evaluating the changes in gene locus variation before and after the response to concurrent chemoradiotherapy in esophageal cancer patients; wherein the at least one genomic DNA data contains a gene locus associated with synchronous chemoradiotherapy response, and the change in gene locus variation of the esophageal cancer patient carrying the gene locus associated with synchronous chemoradiotherapy response before and after synchronous chemoradiotherapy response is statistically significant; The gene loci associated with the treatment response and the concurrent chemoradiotherapy response are selected from the group consisting of: p.Glu1523Lys site of EP300 gene, p.Glu5905Asp site of SYNE1 gene, p.Asp2397His site of MUC4 gene, p.Ala2409Val site of MUC4 gene, p.Glu5905Asp site of SYNE1 gene, p.Ala2409Val site of MUC4 gene, and combinations thereof; The gene loci associated with concurrent chemoradiotherapy response and associated with recurrence are selected from the group consisting of: p.Glu5905Asp site of SYNE1 gene, p.Pro3360His site of MUC4 gene, p.Thr2382Ala site of MUC4 gene, p.Ala2390Thr site of MUC4 gene, p.Leu2712Val site of MUC17 gene, and combinations thereof; The gene loci associated with survival and concurrent chemoradiotherapy response are selected from the group consisting of: p.Asp2397His site of MUC4 gene, p.Pro3360His site of MUC4 gene, p.Ala2390Thr site of MUC4 gene, and combinations thereof.

14. The method according to claim 13, characterized in that The genetic mutations of the esophageal cancer patients after CCRT compared with those before CCRT include increases and decreases. Increase refers to mutations in the tissue after treatment and no mutations before treatment, and decrease refers to mutations in the tissue before treatment and no mutations after treatment.

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