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Jianghong Guo, Jing Li, Jiang Chang, Li Wang, Yanfeng Xi Department of Pathology, Cancer Hospital Affiliated to Shanxi Medical University, Shanxi Province Cancer Hospital, Shanxi Hospital Affiliated to Cancer Hospital, Chinese Academy of Medical Sciences, Taiyuan, People’s Republic of ChinaCorrespondence: Yanfeng Xi, Email xiyanfeng1998@163.comObjective: To explore the clinical value of the combined screening of the methylation statuses of the RPRM, SDC2, and TCF4 genes in plasma of gastric cancer patients.Methods: Differential expressed genes (DEGs) were selected from the Gene Expression Omnibus database, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed using DAVID, and a protein-protein interaction network was constructed. Hub genes were obtained using Cytoscape. Screening results combined with literature reports identified three genes (RPRM, SDC2, and TCF4). Further analysis was done using biopsies collected through gastroscopy at Shanxi Cancer Hospital from January 8, 2020 to February 22, 2021. The patients were divided into two groups: gastric adenocarcinoma group, and control group which are not gastric adenocarcinoma according to pathological or gastroscopic results. The methylation statuses of the three genes in peripheral blood plasma were detected by fluorescence polymerase chain reaction, and the relationships between the positive rates of the three combined genes with pathology and/or gastroscopy results were analyzed. The clinical value of the combined detection of the three genes was evaluated according to these indicators. The diagnostic specificity and sensitivity of this detective method were analyzed.Results: A total of 197 DEGs were identified and 12 hub genes were obtained. The enriched functions and pathways of DEGs include regulation of cell proliferation, extracellular space, cytokine activity, and pathways in cancer. The combination of RPRM, SDC2, and TCF4 gene methylation had a specificity of 93.39% and sensitivity of 80.33%. The combined positive rate of RPRM, SDC2, and TCF4 gene methylation in patients with gastric adenocarcinoma was significantly higher compared with those without gastric adenocarcinoma (χ2=151.179, P< 0.05).Conclusion: Combined detection of RPRM, SDC2, and TCF4 gene methylation in peripheral blood plasma maybe helpful in screening for gastric adenocarcinoma, and maybe a complementary method to gastroscopy and serum tumor markers.Keywords: gastric cancer, RPRM, SDC2, TCF4, methylation

Purpose: Cell-free circulating tumor DNA (ctDNA) in plasma enables rapid and repeat testing of actionable mutations. Next-generation sequencing (NGS) is an attractive platform for multiplex sequencing capabilities compared to traditional methods such as PCR. The purpose of this study is to evaluate the value of the NGS-based ctDNA assay and to identify the genomic alteration profile of ctDNA in real-world Chinese non-small cell lung (NSCLC) patients. Methods: In total, 294 Chinese patients with pathological diagnosis of Phase III-IV NSCLC were enrolled. 3-4mL peripheral blood was collected and NGS-based analysis was carried out using a 20-gene panel. The analytical sensitivity and specificity of ctDNA NGS-based assay was validated using droplet digital PCR (ddPCR). Results: We have tested 570 sites from 286 samples using ddPCR, which included 108 positive sites and 462 negative sites from NGS results, and the concordance rate was 99.8% (418/419) for single-nucleotide variants (SNVs) and 96.7% (146/151) for insertions and deletions (InDels). The most frequent genes were TP53 (32%), EGFR (31.97%), KRAS (6.46%), PIK3CA (4.76%), and MET (4.08%). Exon 19 deletion (19del) was the most common alteration in EGFRand G12C was the most common alteration in KRAS. Furthermore, the detection rate of TP53was higher in the male and patients with squamous cell carcinoma. We also found the prevalence of TP53 in L858R was higher than in 19del (61.29% vs. 40%; p=0.1115). Conclusion: The results indicate that the results of NGS-based ctDNA assay are highly consistent with ddPCR. In Chinese NSCLC patients, TP53 mutation was more frequently associated with male and squamous cell carcinoma. The prevalence of concomitant mutations in L858R may be different from that in 19del.

Objectives Magee equation 3 (ME3) is predictive of the pathologic complete response (pCR) to neoadjuvant chemotherapy (NAC) in patients with hormone receptor (HR)–positive, human epidermal growth factor receptor 2 (HER2)–negative breast cancer but with insufficient predictive performance. This study was designed to improve predictive ability by combining ME3 with additional clinicopathologic markers. Methods We retrospectively enrolled 460 patients with HR-positive/HER2-negative breast cancer from 2 centers. We obtained baseline characteristics, the ME3 score, and the number of stromal tumor-infiltrating lymphocytes (sTILs). After performing a logistic regression analysis, a predictive nomogram was built and validated externally. Results ME3 score (adjusted odds ratio [OR], 1.14 [95% confidence interval (CI), 1.10-1.17]; P < .001) and TILs (adjusted OR, 5.21 [95% CI, 3.33-8.14]; P < .001) were independently correlated with pCR. The nomogram (named ME3+) was established using ME3 and sTILs, and it demonstrated an area under the curve of 0.816 and 0.862 in internal and external validation, respectively, outperforming the ME3 score alone. sTILs and ME3 scores were also found to be positively correlated across the entire cohort (P < .001). Conclusions The combination of sTILs and ME3 score potentially shows better performance for predicting pCR than ME3 alone. Larger validations are required for widespread application of ME3+ nomogram in NAC settings for HR-positive/HER2-negative breast cancer.

Jing Li,1 Peng Li,2 Hong Su,1 Haonan Feng,3 Zhongyuan Bai,4 Yanfeng Xi1 1Department of Pathology, Cancer Hospital Affiliated to Shanxi Medical University, Shanxi Province Cancer Hospital, Shanxi Hospital Affiliated to Cancer Hospital, Chinese Academy of Medical Sciences, Taiyuan, Shanxi, 030013, People’s Republic of China; 2Department of Breast Surgery, Cancer Hospital Affiliated to Shanxi Medical University, Shanxi Province Cancer Hospital, Shanxi Hospital Affiliated to Cancer Hospital, Chinese Academy of Medical Sciences, Taiyuan, Shanxi, 030013, People’s Republic of China; 3Department of Pathology, Chengdu Second People’s Hospital, Chengdu, Sichuan, 610000, People’s Republic of China; 4Department of Pathology, Shanxi Medical University, Taiyuan, Shanxi, 030001, People’s Republic of ChinaCorrespondence: Yanfeng Xi, Department of Pathology, Cancer Hospital Affiliated to Shanxi Medical University, Shanxi Province Cancer Hospital, Shanxi Hospital Affiliated to Cancer Hospital, Chinese Academy of Medical Sciences, 3 Workers’ New Village Street, Xing Hua Ling, Taiyuan, Shanxi, 030013, People’s Republic of China, Email xiyanfeng1998@163.comObjective: To study the relationship between cyclin-dependent protein kinase 6 (CDK6) expression in diffuse large B-cell lymphoma (DLBCL) and the clinical biological behavior and prognosis.Methods: Data mining was performed using the Oncomine and The Cancer Genome Atlas (TCGA) databases to analyze the expression level of CDK6 in DLBCL. CDK6 alterations in DLBCL and related functional networks were analyzed with c-BioPortal and the Gene Set Enrichment Analysis was performed by using DAVID and FunRich software. In addition, screening for differential gene expression of CDK6 was done and enriched by using LinkedOmics. Finally, formalin-fixed and paraffin-embedded (FFPE) tissue samples from 102 patients with DLBCL were collected from the Department of Pathology, Shanxi Cancer Hospital (Taiyuan, Shanxi, China) from January 2015 through December 2020. All cases had complete clinical course records. Thirty cases of lymph node reactive hyperplasia tissues were used as controls. The expression of CDK6 in DLBCL tissues was detected by qRT‑PCR and immunohistochemistry.Results: Bioinformatics analysis: The data showed that mRNA expression level and DNA copy number variations (CNVs) of CDK6 were significantly higher in DLBCL as compared to normal tissue (P ˂ 0.05). Based on C-BioPortal analysis, we speculated that amplification was the most common copy of CDK6 CNV in DLBCL. Through Gene Ontology (GO) analysis of these genes, it was found that the proteins were mainly located in the nucleus and cytoplasm. The biological interaction network of CDK6 alterations were found to participate primarily in the G1-S phase of the process. Analysis of LinkedOmics mRNA sequencing data showed that three genes were positively correlated with CDK6 expression: PSMD1, C2orf29 and ASB1. Through experimental verification, we found that CDK6 was overexpressed in DLBCL, and the expression of CDK6 mRNA and protein in DLBCL were positively correlated with Ann Arbor staging and IPI score (P< 0.05), and negatively correlated with overall survival (P< 0.001).Conclusion: Data mining results and experiments revealed and confirmed multi-level evidence for the importance of CDK6 in DLBCL; hence, CDK6 may be a potential marker in DLBCL. Thus, our study will perhaps lay the foundation for further research on the role of CDK6 in the genesis and development of DLBCL.Keywords: cyclin-dependent protein kinase 6, diffuse large B-cell lymphoma, bioinformatics analysis, prognosis

Background: Gene expression profiling (GEP) is considered as gold standard for cell-of-origin (COO) classification of diffuse large B-cell lymphoma (DLBCL). However, the high dimensionality of GEP limits its application in clinical practice. Methods: In this study, we aim to develop a parsimonious model based on GEP to accurately predict COO subtype of DLBCL for clinical applications. We first proposed a variable important measure to solve the instability of penalized regression methods in high-dimensional settings. Then this strategy was applied to six penalized methods to identify a small gene subset for DLBCL classification. Using a training dataset of 350 DLBCL patients, we developed a six-gene model for DLBCL classification. For training and validation datasets, model evaluations showed the six-gene model performed well in terms of discrimination, calibration and clinical usefulness. Subgroups of patients characterized by these six genes showed significantly different prognosis. Furthermore, model comparisons demonstrated that the six-gene model outperformed models constructed by typical penalized regression methods. Results: In this study, we applied the above strategies for DLBCL classification analysis. Finally, six genes were identified as an optimal gene subset for both subtype classification and survival prediction in DLBCL. The predictive and prognostic performances of those six genes were further validated in the external dataset. What’s more, taking simplicity and predictability of clinical models into consideration, we found that the six-gene model outperformed the typically penalized regression models. Conclusions: In conclusion, the six genes had considerable clinical usefulness in DLBCL classification and prognosis. The genes order based on their importance provided a priority for further functional and targeted drug research.

Zhenwen Chen,1,2,&ast; Ning Zhao,1,&ast; Qi Wang,1 Yanfeng Xi,3 Xiaoai Tian,2 Huiwen Wu,1 Yirong Xu1,2 1Department of Pathology, Fenyang College of Shanxi Medical University, Fenyang, 032200, Shanxi Province, People’s Republic of China; 2Department of Pathology, Shanxi Fenyang Hospital, Fenyang, Shanxi Province, People’s Republic of China; 3Department of Pathology, Shanxi Cancer Hospital (Shanxi Institute of Oncology), Taiyuan, Shanxi Province, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Huiwen Wu; Yirong XuDepartment of Pathology, Fenyang College of Shanxi Medical University, No. 16 Xueyuan Road, Fenyang, 032200, Shanxi Province, People’s Republic of ChinaTel +86 151 3588 2981Email yirongxu@163.comPurpose: To investigate PD-L1 protein expression and gene amplification in lung squamous cell carcinoma (LUSC) and analyse their correlation with the clinicopathological characteristics and prognosis of LUSC patients.Patients and Methods: Tissue samples from 164 LUSC patients were collected. PD-L1 protein was detected by immunochemistry (IHC), and PD-L1 gene amplification was investigated by fluorescence in situ hybridization in LUSC patients.Results: The positive expression rate of PD-L1 in LUSC was 47.6% (78/164), and the amplification rate of PD-L1 was 6.7% (11/164); both rates were higher than those of paratumor tissue. Both PD-L1 positive expression and gene amplification were correlated with clinical stage and lymph node metastasis (P< 0.05). PD-L1 protein expression, PD-L1 gene amplification, late stage, lymph node metastasis and distant metastasis were significantly correlated with the prognosis of patients. Among these factors, late stage, lymph node metastasis, PD-L1 protein expression and PD-L1 gene amplification were independent prognostic factors for LUSC.Conclusion: Positive PD-L1 protein expression and gene amplification are involved in the malignant progression and metastasis of LUSC. Both PD-L1 protein expression and gene amplification are associated with poor prognosis.Keywords: PD-L1 protein expression, PD-L1 gene amplification, lung squamous cell carcinoma, prognosis

Objective The aim of this study is to evaluate the clinicopathological features and the treatment of the Burkitt-like lymphoma with 11q aberration. Methods We reported two patients with Burkitt-like lymphoma with 11q aberration: a 56-year-old man with AIDS (case 1) and a 37-year-old woman (case 2) without AIDS. The biopsy of cervical lymph nodes showed Burkitt-like morphologic and immunophenotypic features. But both of them lack MYC rearrangement and carry an 11q-arm aberration with proximal gains and/or telomeric losses. The diagnosis was confirmed by pathological morphology, immunohistochemistry, and fluorescence in situ hybridization. Result After a cycle of R-CTOEP (rituximab, cyclophosphamide, pirarubicin, vincristine, and prednisone) chemotherapy, case 1 refused to chemotherapy and radiotherapy and was followed up for 34 months without recurrence and new focus. Case 2 received R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) for two cycles and achieved PR (partial response). Then, the patient in case 2 received EPOCH (etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin) for three cycles, and the right cervical mass disappeared. She achieved complete response and was followed up for 16 months without recurrence and new focus. Conclusion Burkitt-like lymphoma with 11q abnormalities resembles Burkitt lymphoma morphologically but lacks MYC rearrangement and may have a better prognosis.

Esophageal squamous cell carcinoma (ESCC) is a poor-prognosis cancer type with limited understanding of its molecular etiology. Using 508 ESCC genomes, we identified five novel significantly mutated genes and uncovered mutational signature clusters associated with metastasis and patients' outcomes. Several functional assays implicated that NFE2L2 may act as a tumor suppressor in ESCC and that mutations in NFE2L2 probably impaired its tumor-suppressive function, or even conferred oncogenic activities. Additionally, we found that the NFE2L2 mutations were significantly associated with worse prognosis of ESCC. We also identified potential noncoding driver mutations including hotspot mutations in the promoter region of SLC35E2 that were correlated with worse survival. Approximately 5.9% and 15.2% of patients had high tumor mutation burden or actionable mutations, respectively, and may benefit from immunotherapy or targeted therapies. We found clinically relevant coding and noncoding genomic alterations and revealed three major subtypes that robustly predicted patients' outcomes. Collectively, we report the largest dataset of genomic profiling of ESCC useful for developing ESCC-specific biomarkers for diagnosis and treatment.

Integrated molecular analysis of human cancer has yielded molecular classification for precise management of cancer patients. Here, we analyzed the whole genomic, epigenomic, transcriptomic, and proteomic data of 155 esophageal squamous cell carcinomas (ESCCs). Multi-omics analysis led to the classification of ESCCs into four subtypes: cell cycle pathway activation, NRF2 oncogenic activation, immune suppression (IS), and immune modulation (IM). IS and IM cases were highly immune infiltrated but differed in the type and distribution of immune cells. IM cases showed better response to immune checkpoint blockade therapy than other subtypes in a clinical trial. We further developed a classifier with 28 features to identify the IM subtype, which predicted anti-PD-1 therapy response with 85.7% sensitivity and 90% specificity. These results emphasize the clinical value of unbiased molecular classification based on multi-omics data and have the potential to further improve the understanding and treatment of ESCC.

To investigate the potential value of radiomics features based on preoperative multiparameter MRI in predicting disease-free survival (DFS) in patients with local advanced rectal cancer (LARC).We identified 234 patients with LARC who underwent preoperative MRI, including T2-weighted, diffusion kurtosis imaging, and contrast enhanced T1-weighted. All patients were randomly divided into the training (n = 164) and validation (n = 70) cohorts. 414 features were extracted from the tumor from above sequences and the radiomics signature was then generated, mainly based on feature stability and Cox proportional hazards model. Two models, integrating pre- and postoperative variables, were constructed to validate the radiomics signatures for DFS estimation.The radiomics signature, composed of six DFS-related features, was significantly associated with DFS in the training and validation cohorts (both p0.001). The radiomics signature and MR-defined extramural venous invasion (mrEMVI) were identified as the independent predictor of DFS both in the pre- and postoperative models. In both cohorts, the two radiomics-based models exhibited better prediction performance (C-index ≥0.77, all p0.05) than the corresponding clinical models, with positive net reclassification improvement and lower Akaike information criterion (AIC). Decision curve analysis also confirmed their clinical usefulness. The radiomics-based models could categorize LARC patients into high- and low-risk groups with distinct profiles of DFS (all p0.05).The proposed radiomics models with pre- and postoperative features have the potential to predict DFS, and may provide valuable guidance for the future individualized management in patients with LARC.

Nonresponse, or acquired resistance to immune checkpoint inhibitors in colorectal cancer (CRC) highlight the importance of finding potential tolerance mechanisms. Low expression of major histocompatibility complex, class I (MHC-I) on the cell surface of the tumor is one of the main mechanisms of tumor escape from T-cell recognition and destruction. In this study, we demonstrated that a high level of calnexin (CANX) in the tumors is positively correlated with the overall survival in colorectal cancer patients. CANX is a chaperone protein involved in the folding and assembly of MHC-I molecules. Using miRNA target prediction databases and luciferase assays, we identified miR-148a-3p as a potential regulator of CANX. Inhibition of miR-148a-3p restores surface levels of MHC-I and significantly enhanced the effects of CD8+ T-cell-mediated immune attack in vitro and in vivo by promoting CANX expression. These results reveal that miR-148a-3p can function as a tumor promotor in CRC by targeting the CANX/MHC-I axis, which provides a rationale for immunotherapy through targeting the miR-148a-3p/CANX/MHC-I pathway in patients with CRC.

Nonresponse, or acquired resistance to immune checkpoint inhibitors in colorectal cancer (CRC) highlight the importance of finding potential tolerance mechanisms. Low expression of major histocompatibility complex, class I (MHC-I) on the cell surface of the tumor is one of the main mechanisms of tumor escape from T-cell recognition and destruction. In this study, we demonstrated that a high level of calnexin (CANX) in the tumors is positively correlated with the overall survival in colorectal cancer patients. CANX is a chaperone protein involved in the folding and assembly of MHC-I molecules. Using miRNA target prediction databases and luciferase assays, we identified miR-148a-3p as a potential regulator of CANX. Inhibition of miR-148a-3p restores surface levels of MHC-I and significantly enhanced the effects of CD8

Diffuse large B-cell lymphoma (DLBCL) has been reported to have a significant association with the hepatitis B virus (HBV) infection. However, there has been no experimental evidence to determine whether the components of the hepatitis B virus are expressed in lymphoid cells. In this study, we used immunohistochemical methods to explore whether the antigens of hepatitis B virus are expressed in DLBCL lymphoma cells in HBsAg-positive DLBCL patients (HBsAg + DLBCL). HBx antigen was detected in 48.9% of HBsAg + DLBCL patients, and the expression rate of the Pre-S2 antigen was 57.2%. HBx expression was significantly associated with high-level expression of c-Myc, while the Pre-S2 antigen was not. In this study, we demonstrated that HBx antigen and Pre-S2 antigen could be detected in lymphoma cells, and HBx expression was related to c-Myc expression. Our findings provide a strong basis for further study of the HBV-infected DLBCL and molecular mechanism underlying the lymphomagenesis.

Triple negative breast cancer (TNBC) is characterized by aggressive malignant tumor, poor prognosis and lack of targeted treatment. Several studies have established that macrophages are closely associated with the progression of TNBC. Through immunohistochemical analysis, we found that the infiltration of macrophage in TNBC tissue was more than that in non-triple negative breast cancer (nTNBC) tissue. Furthermore, the conditioned medium (CM) of MDA-MB-231 and HCC1937, the TNBC cell lines, had significant migration-promoted effect on macrophages. However, the macrophages migration-promoted ability of nTNBC cell line MCF-7 was weaker than that of MDA-MB-231 and HCC1937 cells. Mechanistically, MDA-MB-231 and HCC1937 cells secreted more macrophage colony-stimulating factor (M-CSF) than MCF-7, which is the main inducer of macrophage migration, and the secreted M-CSF promoted the increase in actin and the elongation of pseudopodia. When M-CSF was neutralized by antibody, the elongation of macrophage pseudopodia was disappeared and the migration was inhibited. In vivo, there were more macrophages in tumors induced by MDA-MB-231 than MCF-7 did. Therefore, M-CSF specially secreted by TNBC was the important cause of macrophages aggregation in TNBC, which further promoted the aggressiveness of TNBC.

Objective This study aims to screen the MSI detection loci suitable for the East Asian colorectal cancer patients. and explore its intratumoral heterogeneity. Methods A total of 271 pathological tissues specimens of colorectal cancer were collected. The MSI status was detected using different PCR reagent kits with different detection loci. Then, the results were compared with the immunohistochemical (IHC) staining results. Microdissection of pathological tissues specimens detected to be MSI‐H was performed to examine whether there was intratumoral heterogeneity of MSI status. Results Thirty‐nine out of 271 cases were dMMR. dMMR occurred mostly in patients with right‐hemi colon cancer (P

This study aims to understand the clinical features, treatment, and prognosis of patients with male breast cancer (MBC) in Shanxi province of China from 2007 to 2016. Data for 77 patients with MBC were collected for analysis. Immunohistochemistry, pathological results, and other data such as demographic characteristics (age, marital status, smoking history, drinking history, and family history of cancer) as well as clinical data were investigated by retrieving information from the patients’ medical records. A total of 12,404 patients were diagnosed with breast cancer between 2007 and 2016, and 77 were patients with MBC among them. The median diagnosis age of patients with MBC was 62 years (range, 24–84 years). The most common complaint was a painless lump in the breast, accounting for 68.8% of the patients, and the main pathological type in MBC was infiltrating ductal carcinoma (66.2%). In terms of hormone receptors, 80.5% (62/77) of patients with MBC were estrogen receptor positive, 75.3% (58/77) of patients were progesterone receptor positive, and only 6.5% (5/77) of patients were HER2 overexpressing. The multivariant Cox proportional hazards regression analysis showed that M stage is an independent prognostic factor (p=0.018, HR=18.791, 95% CI 1.663 to 212.6). The epidemiological and clinical features of Chinese MBC are similar to that of other countries. As the Chinese public have limited knowledge of MBC, it is necessary to increase awareness among them about it. Further research with a large sample size is required for better understanding of the risks associated with MBC.

Background: Cancers of unknown primary (CUP) are a type of metastatic cancer. However, their primary anatomical site of origin cannot be clinically determined using routine history inquiries, laboratory tests, endoscopy, and imaging. CUP account for ~3-5% of cancers. Empirical chemotherapy (paclitaxel, carboplatin, etc.) is generally used, although the curative effect is poor. Finding the primary site of origin for CUP patients is of great significance for clinical treatment. Method: Our study employed 10,001 pathological images and 9,775 gene detection data points based on whole-exome sequencing (WES) or YUANSU®(OrigiMed, Shanghai, China) for primary cancer within a database containing 32 common cancers. We applied machine learning algorithms (autoML, Transformers, attention) and constructed two diagnostic models. The models diagnosed by identifying pathological images (Model 1) and genomic data (Model 2). Accuracy for the two models was verified. Result: Both models were evaluated using top-k differential diagnosis accuracy, in other words how often the ground truth label was found for k highest confidence predictions for the model. The pathological model (Model 1) achieved a top-3 accuracy of 83.38% and a top-5 accuracy of 90.36%. Using the same methodology, genomic model (Model 2) results were 87.5% and 92.2%, respectively. Conclusion: Using deep learning, we developed diagnostic models for CUP based on pathological images and genomic data. Accuracy for both the pathological image model (Model 1) and the genomic data model (Model 2) was not satisfactory. To improve diagnostic accuracy, further studies on developing a diagnostic model that combines pathological imaging and genomic data are ongoing.. Citation Format: Yanan Wang, Guanjun Zhang, Xi Liu, Yanfeng Xi, Pan Wang, Yuman Zhang, Xing Li. Genomics and pathology based deep learning to predict cancers of unknown primary [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5045.

e15565 Background: As we all know that patients with advanced colorectal cancer (CRC) are often diagnosed with liver-only metastases (LM), pulmonary-only metastases (PM) and widespread metastasis (WM). Various methods have also been applied to molecular mutation detection in CRC, However, the differences in molecular mutation profiles between the different metastatic lesions are unclear. Methods: In the period of September 2019 to January 2022, 376 LM, 111 PM and 116 WM were included in this retrospective analysis. The average age of patients with LM, PM and WM were respectively 61, 63 and 63 (range, 21-86, 39-95, 29-83). The proportion of male patients accounted for about 60% of LM (246/376), PM (69/111) and WM (74/116) . In this study, we retrospectively analyzed the single nucleotide variants (SNV), copy number variation (CNV) and fusion variants, detected in patients with different metastatic lesions by using next-generation sequencing (NGS). Pearson’s chi-square test was used to test differences in mutation frequency. P-values < 0.05 were considered significant. SPSS 26.0 was used for the statistical analyses. Results: In LM, PM and WM analyzed cohorts, SNV was detected in 370, 108 and 114 patients. CNV was detected in 225, 58 and 60 patients, and fusions were detected in 25,7 and 9 patients. In LM, PM and WM analyzed cohorts, the genes with the highest mutation frequency were APC (11.60%, 8.73%, 6.40%), TP53 (8.90%, 7.70%, 5.29%), KRAS (5.90%, 6.67%, 5.03%) . In addition, the SMAD4 mutation frequency was 2.13%, 0.82% and 1.04%, respectively. APC and SMAD4 mutation frequency was higher in LM than PM ( P= 0.012, P= 0.007) and WM ( P < 0.0001, P= 0.008). 1026, 221 and 281 CNV were detected in LM, PM and WM, respectively. The genes with the highest mutation frequency were MYC (43%, 7.24%, 4.63%), MET (5.95%, 7.69%, 5.34%), EGFR (5.36%, 7.24%, 4.63%) and FLT3 (4.39%, 4.98%, 6.05%) in LM, PM and WM. The SMAD4 mutation frequency were 0.58% and 1.07% in LM and WM, which was not detected in PM. No significant difference was found in those CNVs. No characteristic fusions were found in the total analyzed cohorts. Conclusions: The SNV and CNV molecular mutation profiles were similar in LM, PM and WM. However, the genes with higher mutation probability show higher mutation frequency in LM. Compared with PM, SNV of SMAD4 displays a higher mutation frequency in LM. It has been previously reported that LM may display poorer overall survival (OS) compared with PM. Considering the effect of SMAD4 on the prognosis of CRC, this study may provide a possible analysis of the poor prognosis of LM patients. However, there is still a need for more clinical research to support these results.

Background:Anaplastic carcinoma mural nodules presenting in ovarian mucinous cystic tumors are very rare. Here, we reported clinicopathological, immunohistochemical and molecular features of 3 such cases, and reviewed the related literature.Case presentation:The expression of pan-cytokeratin (CK) in the mural nodules of all three patients supported the diagnosis of mural nodules of anaplastic carcinoma. Immunohistochemical staining showed wild-type expression of p53 in the mural nodules and mucinous epithelium of Cases 2 and 3, while Case 1 was negative for the p53 mutation. The synchronous expression of p53 in epithelia and mural nodules suggested that mural nodules might be homologous with mucinous adenocarcinoma and might be the result of dedifferentiation of mucinous adenocarcinoma. In the sarcomatoid region of Case 1, p53 was wild-type in spindle cells and multinucleated giant cells in the background. In Case 3, a broad-based serrated adenoma of the appendix was also found. Therefore, exons of tumor-related genes were detected by high-throughput next-generation sequencing (NGS). Missense mutations of PIK3CA and PTEN were found, but no germline mutations were detected.Conclusions:In young patients with sarcomalike mural nodule (SLMNs) morphology, pathological analysis is recommended to avoid overlooking the existence of malignant mural nodules. Serrated lesions occurred in the appendix and ovarian mucinous tumor simultaneously, but no germline mutations were detected by NGS, indicating this was a sporadic case.

Background The pre-stored memory T cells in organ transplant patient carry a high risk of allograft rejection. The current study aimed to determine whether the allogenic response of adoptively transferred memory T cells in mice was suppressed by vitamin D3 monotherapy alone or in combination with monoclonal antibody treatment. Methods Prior to vascularized heterotopic heart transplantation, naive C57BL/6 mice were primed with memory T cells. Recipient mice were administered vitamin D3 alone or in combination with monoclonal antibodies (anti-CD40L/ anti-LFA-1). Memory T cells and CD4+ forkhead box P3+ T cells in recipient spleens were measured using flow cytometry. Additionally, the expression of cytokines was measured by ELISA and quantitative PCR. Inflammatory factors in the grafts were identified by hematoxylin and eosin staining. Results Vitamin D3 in conjunction with anti-CD40L/ anti-LFA-1 antibodies were administered according to the median survival time from 6.5 to 80 days. The results revealed that grafts were protected through the prevention of inflammatory cell infiltration. Combined treatment decreased the mRNA levels of IL-2, IFN-γ and IL-10 and increased the mRNA levels of IL-4, Foxp3 and TGF-β in the allograft. Rejection was suppressed by a reduction of CD4+CD44high CD62L+ and CD8+ CD44high CD62L+ memory T cells, the induction of regulatory T cells in the recipient spleen and a reduction of serum IL-2, IFN-γ and IL-10 levels. Conclusion Vitamin D3 efficiently protected allografts from memory T-cell allo-responses when combined with anti-CD40L/anti-LFA-1 antibodies therapy.

Introduction: Lung cancer is the leading cause of cancer-related morbidity and mortality in the People’s Republic of China. Targeted therapies for patients with lung cancer, which depend on accurate identification of actionable genomic alteration, have improved survival compared with previously available treatments. However, data on the types of molecular testing often used in the People’s Republic of China, and how they have changed over time, are scarce. We explored the overall landscape of molecular testing of lung cancer in mainland People’s Republic of China in the past decade. Methods: We distributed a stratified random sampling survey of molecular testing to 49 hospitals from members of the Molecular Pathology Collaboration Group of Chinese Anti-Cancer Association which was weighted by the numbers of lung cancer cases in seven different geographic regions in mainland People’s Republic of China from 2010 to 2019. The questionnaire contained four parts for all respondents. The questionnaire ascertained the use of approved in vitro diagnostic (IVD) devices published by the Center for Medical Device Evaluation, National Medical Products Administration of the People’s Republic of China. Results: A total of 226,227 NSCLC specimens were tested from 2010 to 2019 in the selected hospitals. The annual number of initiated molecular tests increased over time (p < 0.0001), with an average annual growth rate of 31.8%. A notable increase in the number of molecular tests occurred during 2014 and 2016, which coincided with the approval of the National Medical Products Administration to IVD devices. For the diagnosis of molecular subtypes, EGFR mutation testing was first conducted in year 2007, followed by ALK translocation testing in 2010 and ROS1 in 2011. For other rare genetic variations in NSCLC, BRAF mutation testing was first launched in 2012, MET exon 14 skipping mutation in 2014, HER2 exon 20 mutations in 2017, and RET translocation in 2015. A markedly uneven distribution was also observed in the geography of leading units with the largest number of leading units located in east People’s Republic of China (34.7%, 17 of 49) and the smallest number located in northwest People’s Republic of China (6.1%, 3 of 49). The growth trends we observed illustrate the progress and increasing capability of molecular testing of lung cancer achieved in mainland People’s Republic of China in the decade from 2010. Conclusions: In the decade 2010 to 2019, progress and increased capability of molecular testing of lung cancer were achieved in mainland People’s Republic of China. Further efforts should address the clinical application of next-generation sequencing technology, rare genomic aberrations, and the balance between novel genomic testing techniques and the approval of IVD products.

For patients with diffuse large B-cell lymphoma (DLBCL), survival at 24 months is a milestone for long-term survival. The purpose of this study was to develop a multigene risk score (MGRS) to refine the International Prognostic Index (IPI) model to identify patients with DLBCL at high risk of death within 24 months. Using a robust statistical strategy, we built a MGRS incorporating nine mRNAs and two lncRNAs. Stratification and multivariable Cox regression analysis confirmed the MGRS as an independent risk factor. A nomogram based on IPI+MGRS model was constructed and its calibration plot showed close agreement between predicted 2-year survival rate and observed rate. The 2-year AUC was bigger with the IPI+MGRS model (ΔAUC=0.162; 95%CI 0.1295–0.1903) than with the IPI model, and the IPI+MGRS model more accurately predicted the prognostic risk of DLBCL. The 2-year survival decision curve revealed the IPI+MGRS model was more useful clinically than the IPI model. Functional enrichment analysis showed that the MGRS correlated with cell cycle, DNA replication and repair. The results were validated using an independent external dataset. In conclusion, we successfully developed an integrated mRNA–lncRNA signature to refine the IPI model for predicting long-term survival of patients with DLBCL.