HLA-A+ tertiary lymphoid structures with reactivated tumor infiltrating lymphocytes are associated with a positive immunotherapy response in esophageal squamous cell carcinoma.

BACKGROUND: Immune checkpoint blockade (ICB) therapy provides remarkable clinical benefits for multiple cancer types. However, the overall response rate to ICB therapy remains low in esophageal squamous cell carcinoma (ESCC). This study aimed to identify biomarkers of ICB therapy for ESCC and interrogate its potential clinical relevance. METHODS: We investigated gene expression in 42 treatment-naïve ESCC tumor tissues and identified differentially expressed genes, tumor-infiltrating lymphocytes and immune-related genes signatures associated with differential immunotherapy responses. We systematically assessed the tumor microenvironment using the NanoString GeoMx digital spatial profiler, single-cell RNA-seq and multiplex immunohistochemistry in ESCC. Finally, we evaluated the associations between HLA-A-positive tertiary lymphoid structures (TLSs) and patients' responses to ICB in 60 ESCC patients. RESULTS: Tumor infiltrating B lymphocytes and several immune-related gene signatures, such as the antigen presenting machinery (APM) signature, are significantly elevated in ICB treatment responders. Multiplex immunohistochemistry identified the presence of HLA-A+ TLSs and showed that TLS-resident cells increasingly express HLA-A as TLSs mature. Most TLS-resident HLA-A+ cells are tumor-infiltrating T (TIL-T) or tumor-infiltrating B (TIL-B) lymphocytes. Digital spatial profiling of spatially distinct TIL-T lymphocytes and single-cell RNA-seq data from 60 ESCC tumor tissues revealed that CXCL13-expressing exhausted TIL-Ts inside TLSs are reactivated with elevated expression of the APM signature as TLSs mature. Finally, we demonstrated that HLA-A+ TLSs and their major cellular components, TIL-Ts and TIL-Bs, are associated with a clinical benefit from ICB treatment for ESCC. CONCLUSIONS: HLA-A+ TLSs are present in ESCC tumor tissues. TLS-resident TIL-Ts with elevated expression of the APM signature may be reactivated. HLA-A+ TLSs and their major cellular components, TIL-Ts and TIL-Bs, may serve as biomarkers for ICB-treated ESCC patients.

Identification of Optimal Parameters for Assessing Lymph Node Status of Patients with Esophageal Squamous Cell Carcinoma After Neoadjuvant Chemoradiotherapy.

BACKGROUND: This study aimed to compare the prognostic discrimination power of pretreatment pathologic N stage (prepN), lymph node tumor regression grade (LNTRG), and posttreatment pathologic N (ypN) category for esophageal squamous cell carcinoma (ESCC) patients who received neoadjuvant chemoradiotherapy (nCRT) plus surgery. METHODS: The study reviewed 187 ESCC patients from two medical centers who underwent nCRT plus surgery. Pathologic LNTRG was defined by the proportion of viable tumor area within the tumor bed in lymph nodes (LNs). An average LNTRG then was calculated by averaging the tumor regression grade (TRG) score of all resected LNs. Lymph nodes containing regression changes or vital tumor cells were used for interpretation of the prepN stage, which reflects the estimated number of originally involved LNs. RESULTS: The ypN, prepN, and LNTRG categories had significant prognostic stratification power (p < 0.001, log-rank test). Multivariable cox regression showed that all three categories were independent prognostic factors of disease-free survival (DFS) (p < 0.05). The LNTRG category showed a better prognostic value for DFS prediction than the ypN and prepN categories (Akaike information criterion [AIC]: LNTRG [933.69], ypN [937.56], prepN [937.45]). Additionally, the superior predictive capacity of the LNTRG category was demonstrated by decision curve analysis. Similar results were discovered for patients with remaining diseased LNs. CONCLUSIONS: The three staging categories had prognostic relevance for DFS, with the LNTRG category seeming to have better prognostic indication power. Comprehensive consideration of the ypN status, prepN status, and LN regression may allow for better prognostic stratification of patients.

Development and Validation of PET/CT-Based Nomogram for Preoperative Prediction of Lymph Node Status in Esophageal Squamous Cell Carcinoma.

PURPOSE: This study was conducted to predict the lymph node status and survival of esophageal squamous cell carcinoma before treatment by PET-CT-related parameters. METHODS: From January 2013 to July 2018, patients with pathologically diagnosed ESCC at our hospital were retrospectively enrolled. Completed esophagectomy and two- or three-field lymph node dissections were conducted. Those with neoadjuvant therapy were excluded. The first 65% of patients in each year were regarded as the training set and the last 35% as the test set. Nomogram was constructed by the "rms" package. Five-year, overall survival was analyzed based on the best cutoff value of risk score determined by the "survivalROC" package. RESULTS: Ultimately, 311 patients were included with 209 in the training set and 102 in the test set. The positive rate of the lymph node in the training set was 36.8% and that in the test set was 32.4%. The C-index of the training set was 0.763 and the test set was 0.766. The decision curve analysis showed that it was superior to the previous methods based on lymph node uptake or long/short axis diameter or axial ratio. Risk score > 0.20 was significantly associated with 5-year, overall survival (p = 0.0015) in all patients. CONCLUSIONS: The nomogram constructed from PET-CT parameters including primary tumor metabolic length and thickness can accurately predict the risk of lymph node metastasis in ESCC. The risk score calculated by our model accurately predicts the patient's 5-year overall survival.

The efficacy of additional surgical resection after endoscopic resection in pT1b esophageal squamous cell carcinoma: A multi-institutional retrospective study in China.

BACKGROUND: pT1b esophageal squamous cell carcinoma (ESCC) patients treated by endoscopic resection (ER) required additional treatment with surgical resection (SR) or chemoradiotherapy (CRT) according to 2020 Japan Gastroenterological Endoscopy Society (JGES) guideline. Given the evidences for this recommendation were largely based on small-size studies, our study collected 166 cases of ER-treated pT1b patients in order to investigate the efficacy of additional SR as compared to ER-alone treatment. METHODS: A multi-institutional retrospective study in China was conducted. The pT1b ESCC treated by ER + SR (n = 42) and ER-alone (n = 124) from 2007 to 2018 were recruited. Meanwhile, patients with positive lymphovascular invasion (LVI(+)) and/or with positive vertical margin (VM(+)) were put into high-risk group, and those with both VM(-) and LVI(-) were selected into low-risk group. The clinicopathological parameters, lymph node metastasis (LNM), and survival between ER + SR and ER-alone groups were analyzed. RESULTS: In high-risk group, concurrent LNM revealed in surgically resected specimens accounted for 52.6% cases in ER + SR group. After surgical removal, the incidence of post-resection LNM dropped down to 5.6%. However, in low-risk group, patients with ER + SR treatment did not exhibit any concurrent LNM in surgically resected specimens, and the incidence of their overall LNM was similar to that in ER-alone group (0% vs. 2.8%, p = 1.000). More importantly, these cases demonstrated significantly shorter overall survival (OS) than that in ER-alone group (81.8% and 100.0%, respectively, at 3 years; log-Rank: P = 0.010). CONCLUSIONS: For ER-treated pT1b patients in high-risk group, additional SR is strongly recommended. However, for those in low-risk group, additional SR does not generate much benefit for clearance of LNM, but brings harm to shorten their OS. Therefore, additional SR is not recommended for ER-treated pT1b patient in low-risk group.

Prognostic value of visual residual tumour cells (VRTC) for patients with esophageal squamous cell carcinomas after neoadjuvant therapy followed by surgery.

BACKGROUND: We assessed visual residual tumour cells (VRTC) with both Becker's tumour regression grading (TRG) system and Japanese TRG system in esophageal squamous cell carcinoma (ESCC) patients treated with neoadjuvant therapy followed by surgery. METHODS: We compared Becker system and Japanese system in 175 ESCC patients treated between 2009 and 2015. RESULTS: According to Becker system, the 5-year DFS/DSS rates were 70.0%/89.3, 53.8%/56.7, 43.0%/49.0, and 42.4%/39.1% for TRG 1a (VRTC 0), TRG 1b (1-10%), TRG 2 (11-50%), and TRG 3 (> 50%). According to Japanese system, the rates were 38.8%/34.1, 49.5%/58.7, 50.2%/49.0 and 70.0%/89.3% for Grade 0-1a (VRTC> 66.6%), Grade 1b (33.3-66.6%), Grade 2 (1-33.3%) and Grade 3 (0). TRG according to two systems significantly discriminate the patients' prognosis. TRG according to Becker system (HR 2.662, 95% CI 1.151-6.157), and lymph node metastasis (HR 2.567, 95% CI 1.442-4.570) were independent parameters of DSS. CONCLUSIONS: Both Becker and Japanese system had their advantage in risk stratification of these ESCC patients. It was speculated that dividing 1-10% VRTC into a group might contribute to independently prognostic significance of Becker's TRG system. Therefore, in addition to TRG of different systems, the percentage of VRTC might be recommended in the pathologic report, which could make the results more comparable among different researches, and more understandable for oncologists in the clinical practice.

Unclassified mesenchymal sarcoma with NTRK1-KHDRBS1 gene fusion: a case report of long-term tumor-free survival with crizotinib treatment.

BACKGROUND: Mesenchymal sarcomas are tumors that originate from mesenchymal tissue. Most mesenchymal sarcomas can be accurately classified, but some are unclassifiable in clinical practice. Molecular detection methods enable patients to benefit from molecular-targeted therapies for many cancers, including lung, breast, and bowel cancers. Further, even unclassified tumors can have therapeutic targets. NTRK gene fusions are sporadic genetic alterations that occur across tumor entities. If NTRK gene fusions are detected, TRK inhibitors can be used regardless of the tumor entity. CASE PRESENTATION: This report describes a case with an unclassifiable mesenchymal sarcoma carrying a neurotrophic tyrosine receptor kinase NTRK1-KHDRBS1 gene fusion that was diagnosed and treated at multiple hospitals. Diagnostic work-up included pathological and immunohistochemical analysis, which excluded angiosarcoma, dendritic cell sarcoma, and pseudomyogenic hemangioendothelioma. The patient achieved a long-term survival without tumor relapse after treatment with crizotinib. CONCLUSIONS: This case will be of significant interest to pathologists because, despite the tumor being unclassified, a molecular target was identified. Although the FDA does not currently approve crizotinib for treatment of patients harboring NTRK gene fusions, this case provides new insights for diagnosis and treatment of mesenchymal sarcomas with NTRK1 gene translocations. Similar to ALKomas, which can be successfully treated using NTRK molecular-targeted therapy, tumors with NTRK gene translocations can be classified as NTRKomas, even when they occur at different organ sites, and with varying histological morphologies, and immunophenotypes.

Mouse double minute 2 amplification in oesophageal squamous cell carcinoma is associated with better outcome.

AIMS: This study aimed to determine the frequency of mouse double minute 2 (MDM2) amplification in oesophageal squamous cell carcinomas (ESCC) and to clarify its prognostic significance. METHODS AND RESULTS: We investigated MDM2 amplification on tissue microarrays using fluorescence in-situ hybridisation and analysed its correlations with clinicopathological features and outcomes in 515 Chinese ESCC patients. MDM2 amplifications were found in 37 of 515 ESCC patients (7.2%). They were significantly negatively correlated with tumour size (P = 0.045), disease progression (P = 0.002) and death (P = 0.003). Univariate analysis showed that the following clinicopathological factors were associated with disease-free survival (DFS) and overall survival (OS): differentiation (P = 0.025 for DFS and P = 0.061 for OS), vessel invasion (P = 0.001 and P = 0.002), nerve invasion (P = 0.009 and P = 0.001), clinical stage (P < 0.001 and P < 0.001) and MDM2 amplification (P = 0.012 and P = 0.014). Multivariate Cox regression analysis showed that MDM2 amplification was an independent prognostic factor for improved outcomes (P = 0.023 for DFS, P = 0.027 for OS) and the clinical stage was an independent prognostic factor for poor outcomes (P < 0.001). When survival analyses were conducted at different clinical stages, MDM2 amplification was associated with longer DFS and OS in stages I-II ESCC (P = 0.003 for DFS and P = 0.003 for OS), but there was no significant survival difference in stages III-IVa ESCC. CONCLUSIONS: MDM2 amplification was significantly correlated with an improved patient outcome, especially in stage I and II disease, and was verified as an independent prognostic factor in our patients. Therefore, MDM2 amplification may be a potential biomarker for risk stratification of the lower stages of ESCC.

The prognostic role of c-MYC amplification in schistosomiasis-associated colorectal cancer.

OBJECTIVE: The purpose of this study was to explore the prognostic role of c-MYC amplification in colorectal cancer, particularly in schistosomiasis-associated colorectal cancer. METHODS: Three hundred and fifty four cases of colorectal cancer, which were from Qingpu Branch of Zhongshan Hospital affiliated to Fudan University, were retrospectively analyzed in a tissue microarray (TMA) format, with fluorescence in situ hybridization (FISH) assay and immunohistochemistry (IHC). RESULTS: c-MYC gene amplification was found in 14.1% (50 out of 354) of patients with colorectal cancer and was correlated with old age (P = 0.028), positive lymph node metastasis (P = 0.004) and advanced stage tumors (P = 0.002). The overexpression of c-MYC was closely associated with the amplification status (P = 0.023). Kaplan-Meier survival curves for overall survival (OS) showed a statistically significant difference for patients with c-MYC amplification in full cohort of colorectal cancer, stage III-IV set and patients with lymph node metastasis (P = 0.002, 0.034, 0.012, respectively). Further analysis found c-MYC amplification associated with poorer survival in the subgroup of colorectal cancer with schistosomiasis (CRC-S, P < 0.001), but not in colorectal cancer without schistosomiasis (CRC-NS, P = 0.155). By multivariate analysis, c-MYC amplification was an independent poor-prognostic factor in CRC-S set (P = 0.046). CONCLUSIONS: Our study firstly found c-MYC amplification could predict poor prognosis in schistosomiasis-associated colorectal cancer, but not in colorectal cancer without schistosomiasis.

Increased MET gene copy number negatively affects the survival of esophageal squamous cell carcinoma patients.

BACKGROUNDS: Since Mesenchymal epithelial transition (MET) amplification has been regarded as a potential treatment target, the knowledge of its prevalence and prognostic importance is crucial. However, its clinical pathologic characteristics are not well known in esophageal squamous cell carcinoma (ESCC). METHODS: We investigated MET gene status with fluorescence in situ hybridization (FISH) assay in 495 ESCC cases using tissue microarrays. Prognostic significance as well as correlations with various clinicopathological parameters was evaluated. RESULTS: Among 495 patients, 28 (5.7%) cases were MET FISH positive, including 5 cases (1%) with true gene amplification. There were no statistically significant associations between MET FISH-positivity and clinicopathologic characteristics. A significantly poorer prognosis was observed in 28 patients with MET FISH-positivity (disease free survival/DFS, P < 0.001 and overall survival/OS, P = 0.001). Multivariate analysis revealed MET FISH-positivity was an independent prognostic factor for DFS (hazard ratio/HR, 1.953; 95% confidence interval/CI, 1.271-2.999; P = 0.002) and OS (HR, 1.926; 95% CI, 1.243-2.983; P = 0.003). MET FISH-positivity was associated with DFS (P = 0.022 and 0.020) and OS (P = 0.046 and 0.024) both in stage I-II ESCC and in stage III-IVa ESCC. No statistical significance (DFS, P = 0.492 and OS, P = 0.344) was detected between stage I-II ESCC with MET FISH-positivity and stage III-IVa ESCC with FISH-negativity. CONCLUSIONS: Increased MET gene copy number is an independent prognostic factor in ESCC, and ESCC might have potentially been up-staged by increased MET gene copy number. The results indicate that increased MET gene copy number is a very promising parameter, in clinical therapy and follow-up plans.

HER2 assessment in locally advanced gastric cancer: comparing the results obtained with the use of two primary tumour blocks versus those obtained with the use of all primary tumour blocks.

AIMS: HER2 is currently the only biomarker used to select eligible patients with advanced gastric cancer (GC) for targeted therapy. The aims of this study were to verify the value of dual-block HER2 assessment and to explore whether increasing the block number is more beneficial by carrying out a randomized prospective cohort study in which dual-block and all-block HER2 assessment were compared in resected specimens of GC. METHODS AND RESULTS: Five hundred and forty-nine resected GC specimens were randomly enrolled into two cohorts: a dual-block group (n = 274) with two primary tumour blocks tested, and an all-block group (n = 275) with all primary tumour blocks tested. Immunohistochemical staining of HER2 was performed. For HER2-equivocal (2+) cases, fluorescence in-situ hybridization (FISH) was performed. As compared with single-block assessment, dual-block assessment increased the HER2 immunohistochemistry (IHC)-positive (3+) rate. The rate with dual-block assessment (11.3%) was significantly higher than that with block 1 assessment (8.8%) (P = 0.016) and block 2 assessment (9.1%) (P = 0.031). Similarly, all-block assessment demonstrated a higher HER2 3+ rate (12.4%) than single-block assessment (block 1, 6.5%; block 2, 6.2%; block 3, 7.2%; block 4, 8.7%) (P < 0.05). HER2 3+ rates of all-block and dual-block assessments showed no significant difference (P = 0.703). After IHC and FISH results had been combined, the HER2-positive rate with all-block assessment (13.5%) was slightly higher than that with dual-block assessment (12.0%), although the difference was not statistically significant (P = 0.62). CONCLUSIONS: Dual-block immunohistochemical assessment is an effective, practical and economic approach that is suitable for the preliminary screening of HER2. We recommend that dual-block HER2 assessment be routinely performed on resected specimens of GC. All-block assessment can be a supplement to dual-block assessment if necessary.

A retrospective study of endoscopic resection for 368 patients with early esophageal squamous cell carcinoma or precancerous lesions.

BACKGROUND: To retrospectively investigate the clinicopathological features and prognosis of early esophageal squamous cell neoplasm (ESCN) treated with endoscopic resection (ER), especially, to compare the prognosis in patients with sm2 cancer and non-sm2 cancer. METHODS: From 2007 to 2013, 368 patients were included in our analysis. RESULTS: The patients were 252 (68.5 %) men and 116 (31.5 %) women with a median age of 61 (range 16-84 years) years. Hyperplasia, mild dysplasia, moderate dysplasia, severe dysplasia, m1, m2, m3, sm1, and sm2 were diagnosed in 47 (12.8 %), 27 (7.3 %), 34 (9.2 %), 61 (16.6 %), 54 (14.7 %), 38 (10.3 %), 63 (17.1 %), 12 (3.3 %), and 32 (8.7 %) cases. The mean (range) follow-up time was 29 (0-84) months. The cumulative overall 1-, 3-, and 5-year metachronous esophageal lesion rates were 4.1, 12.9, and 32.6 %. The incidence of lymph node or distant metastasis was 1.54 % in m3, 6.25 % in sm2, and 0 in other subgroups. The overall 1-, 3-, and 5-year survival rates were 99.5, 97.3, and 87.5 %. There was significant difference between sm2 and non-sm2 patients in metastatic rate (P = 0.021); however, no difference existed between m3 patients and sm2 patients (P = 0.252). The difference of metachronous esophageal lesion (P = 0.401) and survival (P = 0.634) between sm2 and non-sm2 patients was not obvious. CONCLUSIONS: Our study showed that ER was an effective and relatively safe treatment for superficial ESCN. ER is still appropriate in select sm2 patients. To monitor the second primary cancer in sm2 is necessary during the follow-up.

PIK3C2A is a gene-specific target of microRNA-518a-5p in imatinib mesylate-resistant gastrointestinal stromal tumor.

Imatinib mesylate resistance occurs in some patients with gastrointestinal stromal tumors (GISTs) during the course of treatment. In this study, we investigated the relationship between microRNAs (miRNAs) and imatinib-resistant GISTs, and the effect of miR-518a-5p on PIK3C2A in imatinib-resistant GISTs. A total of 20 matched-pair GIST samples from imatinib-resistant patients were included in the study. Each of the paired tumor specimens were from the same patient who had surgical removal of GISTs preimatinib and postimatinib treatment. Seven pairs of tissues were resected for microarray analysis, and the remaining 13 pairs were utilized for miRNAs analysis. Target genes were selected based on bioinformatics from multiple biological databases. Luciferase reporter assays were used to confirm the binding of miR-518a-5p to PIK3C2A 3'UTR. GIST882R-NC, 882R-miR-518a-5p-OE, and 882R-miR-518a-5p-KD cell lines were constructed using lentiviral vectors. miR-518a-5p and PIK3C2A expression in 882R-NC, 882R-OE, and 882R-KD cells was assessed by real-time PCR and western blotting. A cell counting kit was used to detect the influence of miR-518a-5p to cell proliferation. TUNEL staining was applied to detect the influence of miR-518a-5p to cell apoptosis. Microarray analysis showed that miR-518a-5p was downregulated in imatinib-resistant GISTs, and the expression of miR-518a-5p was confirmed with good concordance between real-time PCR and miRNA microarray results. Luciferase reporter assays indicated that miR-518a-5p bound to the PIK3C2A 3'UTR. Compared with 882R-OE, PIK3C2A expression was significantly increased in 882R-KD cells. MiR-518a-5p reduced 882R proliferation and promoted 882R apoptosis. In conclusion, PIK3C2A is a gene-specific target of miR-518a-5p in imatinib mesylate-resistant GISTs. Low expression of miR-518a-5p is likely to upregulate PIK3C2A and affect the cellular response to the drug, causing resistance to imatinib in GISTs.

Prognostic impact and potential interaction of EGFR and c-Met in the progression of esophageal squamous cell carcinoma.

This study is to examine EGFR and c-Met variation in precancerous lesion, early esophageal squamous cell carcinoma (ESCC), and advanced ESCC and to explore their prognostic significance. EGFR and c-Met were detected by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Of 158 endoscopy resection (ER) specimens, c-Met high expression and FISH positive were 44.9 and 12.6 %, respectively. EGFR high expression and FISH positive were 2.5 and 19.6 %, respectively. Of 84 surgical specimens, c-Met high expression and FISH positive were 50 and 8.3 %, respectively. EGFR high expression and FISH positive were 7.1 and 28.5 %, respectively. A significant correlation was observed between c-Met and EGFR FISH positive both in ER (P < 0.001) and surgical specimens (P = 0.029). Patients with EGFR high expression had poorer disease-free survival (DFS) and overall survival (OS) (P = 0.031 and P = 0.013) in c-Met high-expression group but not in c-Met low-expression group (P = 0.301 and P = 0.439). C-Met FISH positive did not represent a statistically significant adverse prognosis until 24 months later (P = 0.027 and 0.048). EGFR and c-Met might be involved in the tumorigenesis and development of ESCC. EGFR high expression has different prognostic significance in patients with differing c-Met expression status. C-Met FISH positive represent delayed prognostic factor.

The prognostic value of EGFR overexpression and amplification in Esophageal squamous cell Carcinoma.

BACKGROUND: In view of the prominent role in cancer cell biology and alteration in substantial numbers of ESCC, defining EGFR molecular characteristics relevant to patient prognosis is of great importance. Therefore, we analyzed the protein expression and gene copy variation of the epithelial growth factor receptor (EGFR) in Chinese esophageal squamous cell carcinoma (ESCC) and explored the possible associations with various features of the tumors and survival of the patients. METHODS: Sections were made from tissue microarray composed of 96 ESCC, and examined for EGFR expression by means of immunohistochemistry (IHC) and for EGFR gene amplification by means of fluorescence in situ hybridization (FISH). The results of IHC were evaluated with six different reported scoring systems. Correlation with clinical features and survival was evaluated using chi-square test and Kaplan-Meier analysis. RESULTS: EGFR overexpression according to scoring system 1 significantly correlated with advanced lymph node involvement (P = 0.046), patient disease specific free survival (DFS) (P = 0.006) and overall survival (OS) (P = 0.007). No such association was observed using other 5 scoring systems (P > 0.05 ). EGFR amplification was associated with lymph node metastasis (P = 0.028), but not correlated with DFS and OS until 20 months. CONCLUSIONS: EGFR IHC overexpression evaluated by scoring system 1 might be suitable to be used in predicting patients survival in ESCC. EGFR gene amplification showed delayed prognostic information after 20 months.

Establishment and characterization of esophageal squamous cell carcinoma patient-derived xenograft mouse models for preclinical drug discovery.

The purpose of this study was to establish and characterize patient-derived esophageal squamous cell carcinoma xenograft (PDECX) mice for utilization in antitumor drug discovery. A total of 96 esophageal squamous cell carcinoma (ESCC) tissues from Chinese patients were transplanted subcutaneously into immunodeficient mice. Histology, EGFR, K-ras, B-raf, and PIK3CA mutations, and HER2 gene amplifications were analyzed in both patient tumors and mouse xenograft tissues using immunohistochemistry, mutant-enriched liquid chip sequencing and fluorescence in situ hybridization assays, respectively. Furthermore, in vivo efficacy studies using five PDECX mice harboring a variety of genetic aberrations were performed using the chemotherapy agents 5-fluorouracil (5-FU) and cisplatin. Thirty-seven PDECX mouse models were successfully established in immunodeficient mice. Pathological analysis revealed similar histological architecture and degrees of differentiation between patient ESCC and xenografted tumors. No mutations were identified in EGFR, K-ras, and B-raf genes in either xenograft models or patient ESCC tissues. In contrast, PIK3CA gene mutations were detected in 12.5% (12/96) ESCC patients and 18.9% (7/37) PDECX models. Interestingly, patient ESCC tissues exhibiting HER2 overexpression or gene amplification were unable to survive in immunodeficient mice. Further analysis showed that PDECX models carrying HER2 2+ expression had no response to 5-FU/cisplatin, compared with HER2-negative models. In conclusion, a panel of PDECX mouse models, which include PIK3CA mutant and HER2-positive models, was established and characterized thus mimicking the current clinical genetic setting of esophageal carcinoma. The sensitivity of HER2-negative ESCC models to chemotherapy supports stratification approaches in the treatment of esophageal carcinoma patients and warrants further investigation of the impact of PI3KCA on treatment response.

Abnormal p16 expression and prognostic significance in esophageal squamous cell carcinoma.

BACKGROUND: The purpose of this study was to analyze p16 expression status and evaluate whether abnormal p16 expression was associated with prognosis in a large-scale esophageal squamous cell carcinoma (ESCC) cohort of patients. METHODS: We retrospectively evaluated p16 expression status of 525 ESCC samples using immunohistochemistry. Associations between abnormal p16 expression and survival were analyzed. RESULTS: P16 negative, focal expression and overexpression were found in 87.6%, 6.9% and 5.5% of ESCC patients. No significant association was observed between abnormal p16 expression and age, sex, tumor site and location, differentiation, vessel and nerve invasion, T stage and lymph node metastasis. In all patients, the survival of p16 focal expression group tended to be better compared with negative group (disease free survival/DFS P=0.040 and overall survival/OS P=0.052) and overexpression group (DFS P=0.201 and OS P=0.258), and there was no survival difference between negative group and overexpression group. The multivariate analysis for OS and DFS found that only clinical stage was a significantly independent prognostic factor (P<0.001). When patients were divided into I-II stage (n=290) and III-IVa stage (n=235), the survival of focal expression group was better compared with negative group (DFS P=0.015 and OS P=0.019), and tended to be better compared with overexpression group (DFS P=0.405 and OS P=0.432) in I-II stage ESCC, which was not found in III-IVa stage ESCC. CONCLUSION: P16 overexpression or negative expression tend to be associated with unfavorable outcomes, especially in I-II stage ESCC. Our study will help to identify a subgroup of ESCC patients with excellent prognosis after surgical therapy.

Comprehensive Proteogenomic Profiling Reveals the Molecular Characteristics of Colorectal Cancer at Distinct Stages of Progression.

Colorectal cancer (CRC) is the second most common malignant tumor world-wide. Analysis of the changes that occur during CRC progression could provide insights into the molecular mechanisms driving CRC development and identify improved treatment strategies. Here, we performed an integrated multi-omics analysis of 435 trace-tumor-samples from 148 colorectal cancer (CRC) patients, covering non-tumor (NT), intraepithelial neoplasia (IEN), infiltration (IFT), and advanced-stage CRC (A-CRC) phases. Proteogenomics analyses demonstrated that KRAS and BRAF mutations were mutually exclusive and elevated oxidation phosphorylation in the IEN phase. Chr17q loss and chr20q gain were also mutually exclusive, occurred predominantly in the IEN and IFT phases, respectively, and impacted the cell cycle. Mutation of TP53 was frequent in the A-CRC phase and associated with tumor microenvironment, including increased extracellular matrix rigidity and stromal infiltration. Analysis of the profiles of CRC based on CMS and CRIS classifications revealed the progression paths of each subtype and indicated that microsatellite instability was associated with specific subtype classifications. Additional comparison of molecular characteristics of CRC based on location showed that ANKRD22 amplification by chr10q23.31 gain enhanced glycolysis in the right-sided CRC. The AOM/DSS-induced CRC carcinogenesis mouse model in mice indicated that DDX5 deletion due to chr17q loss promoted CRC development, consistent with the findings from the patient samples. Collectively, this study provides an informative resource for understanding the driving events of different stages of CRC and identifying the potential therapeutic targets.

Deep learning on tertiary lymphoid structures in hematoxylin-eosin predicts cancer prognosis and immunotherapy response.

Tertiary lymphoid structures (TLSs) have been associated with favorable immunotherapy responses and prognosis in various cancers. Despite their significance, their quantification using multiplex immunohistochemistry (mIHC) staining of T and B lymphocytes remains labor-intensive, limiting its clinical utility. To address this challenge, we curated a dataset from matched mIHC and H&E whole-slide images (WSIs) and developed a deep learning model for automated segmentation of TLSs. The model achieved Dice coefficients of 0.91 on the internal test set and 0.866 on the external validation set, along with intersection over union (IoU) scores of 0.819 and 0.787, respectively. The TLS ratio, defined as the segmented TLS area over the total tissue area, correlated with B lymphocyte levels and the expression of CXCL13, a chemokine associated with TLS formation, in 6140 patients spanning 16 tumor types from The Cancer Genome Atlas (TCGA). The prognostic models for overall survival indicated that the inclusion of the TLS ratio with TNM staging significantly enhanced the models' discriminative ability, outperforming the traditional models that solely incorporated TNM staging, in 10 out of 15 TCGA tumor types. Furthermore, when applied to biopsied treatment-naïve tumor samples, higher TLS ratios predicted a positive immunotherapy response across multiple cohorts, including specific therapies for esophageal squamous cell carcinoma, non-small cell lung cancer, and stomach adenocarcinoma. In conclusion, our deep learning-based approach offers an automated and reproducible method for TLS segmentation and quantification, highlighting its potential in predicting immunotherapy response and informing cancer prognosis.