DEAR EDITOR, Esophageal squamous cell carcinoma(ESCC)is one of the most common fatal malignancies worldwide and is especially common in East Asian regions,including China.Screening lymph node metastasis(LNM)-related biomarkers and elucidating the mechan-ism could provide promising therapeutic targets and help ESCC patients to select reasonable individual therapies.Recent studies have shown that extracellular vesicles(EVs)secreted by tumors contain miRNAs and that miRNAs in EVs can rebuild the tumor microenvironment(TME),thereby providing favorable conditions for tumor metastasis.However,whether specific EVs can be derived from ESCC cells and what affects EVs-mediated commu-nication between ESCC cells and recipient cells in the process of LNM have not been investigated.

Background:Previous observations have demonstrated that the response to neoadjuvant chemoradiotherapy (nCRT) is highly variable in patients with locally advanced rectal cancer (LARC). Recent studies focusing on the intratumoral microbiota of colorectal cancer have revealed its role in oncogenesis and tumor progression. However, limited research has focused on the influence of intratumoral microbiota on the nCRT of LARC. Methods:We explored the microbial profiles in the tumor microenvironment of LARC using RNA-seq data from a published European cohort. Microbial signatures were characterized in pathological complete response (pCR) and non-pCR groups. Multi-omics analysis was performed between intratumor microbiomes and transcriptomes. Results:Microbial α and β diversity were significantly different in pCR and non-pCR groups. Twelve differential microbes were discovered between the pCR and non-pCR groups, six of which were related to subclusters of cancer-associated fibroblasts (CAFs) associated with extracellular matrix formation. A microbial risk score based on the relative abundance of seven differential microbes had predictive value for the nCRT response (AUC = 0.820, p < 0.001). Conclusion:Our study presents intratumoral microbes as potential independent predictive markers for the response of nCRT to LARC and demonstrates the underlying mechanism by which the interaction between intratumoral microbes and CAFs mediates the response to nCRT.

Additional file 3: Table S2. Upregulated genes in the animal model of OSCC tumorigenesis.

BACKGROUND:Oral squamous cell carcinoma (OSCC) is one of the common cancers worldwide. The lack of specific biomarkers and therapeutic targets leads to delayed diagnosis and hence the poor prognosis of OSCC patients. Thus, it is urgent to identify effective biomarkers and therapeutic targets for OSCC.METHODS:We established the golden hamster carcinogenic model of OSCC induced by 7,12-dimethylbenz(a) anthrancene (DMBA) and used mRNA microarrays to detect the differentially expressed genes (DEGs). DEGs were validated in OSCC clinical tissue microarrays using immunohistochemistry method. Whole transcriptome sequencing was performed to obtain an overview of biological functions of Lsm12. PCR assay and sequencing were employed to investigate the alternative splicing of genes regulated by Lsm12. Cell proliferation, colony formation, Transwell migration and invasion assay and in vivo tumor formation assay were performed to investigate the roles of Lsm12 and two transcript variants of USO1 in OSCC cells.RESULTS:Lsm12 was identified to be significantly up-regulated in the animal model of OSCC tumorigenesis, which was validated in the clinical OSCC samples. In the paired normal tissues, Lsm12 staining was negative (91%, 92/101) or weak, while in OSCC tissues, positive rate is 100% and strong staining spread over the whole tissues in 93 (93/101, 92%) cases. Lsm12 overexpression significantly promoted OSCC cell growth, colony formation, migration and invasion abilities, while Lsm12 knockdown showed the opposite trends on these phenotypes and obviously inhibited the tumor formation in vivo. Furthermore, Lsm12 overexpression caused the inclusion of USO1 exon 15 and Lsm12 knockdown induced exon 15 skipping. Exon 15-retained USO1 significantly promoted the malignant phenotypes of OSCC cells when compared with the exon 15-deleted USO1.CONCLUSIONS:We identified Lsm12, a novel tumorigenesis-related gene, as an important regulator involved in OSCC tumorigenesis. Lsm12 is a novel RNA-splicing related gene and can regulate the alternative splicing of USO1 exon 15 which was associated closely with OSCC carcinogenesis. Our findings thus provide that Lsm12 might be a potent biomarker and potential therapeutic target for OSCC.

Background The treatment strategies for T1 oesophageal squamous cell carcinoma (ESCC) patients with or without lymph node metastasis (LNM) are different. Given the advantages of the minimally invasive, sensitive and real-time detection, liquid biopsy has become an important cancer diagnostic and prognostic tool. Methods MiRNA array and small-RNA sequencing were performed. Then, 222 formalin-fixed and paraffin-embedded tumour samples and 229 pretreatment serum samples from T1 ESCC patients were used to verify and evaluate the results. Results We demonstrated that serum miR-20b-5p could predict LNM in T1 ESCC patients. The AUC for serum miR-20b-5p was higher (0.827) than those for lymphovascular invasion (LVI) (0.751, P = 0.2128), invasion depth (0.662, P = 0.0027) and tumour differentiation grade (0.634, P = 0.0019). A nomogram for predicting LNM with three independent significant predictors (miR-20b-5p, LVI and invasion depth) was constructed with a concordance index of 0.931. Serum miR-20b-5p was also significantly correlated with disease-free survival ( P < 0.001). An algorithm of improved T1 ESCC treatment strategy after biopsy and/or after endoscopic resection based on serum miR-20b-5p level was constructed. Conclusions This study suggests that serum miR-20b-5p is a potential biomarker for predicting LNM and can be helpful for precise clinical decision-making strategies and improve treatment outcomes for T1 ESCC patients.

Additional file 5: Table S4. Alternative splicing of genes induced by Lsm12 knockdown.

Objective: To evaluate the association of p53 protein expression with clinicopathological features and prognosis in esophageal spindle cell carcinoma. Methods: A total of 4 439 esophageal squamous cell carcinoma (ESCC) patients who underwent radical esophagectomy without neoadjuvant therapy between May 2010 and May 2019 were included. The HE slides and clinicopathological parameters were reviewed. Among these, there were 63 cases of esophageal spindle cell carcinoma; p53 protein expression was evaluated by immunohistochemistry (IHC) and its correlation with clinicopathological parameters and patients' outcome was analyzed. Results: The 63 esophageal spindle cell carcinoma accounted for 1.4% (63/4 439) of all ESCC. Of the 63 patients there were 55 males and 8 females, male to female ratio was 7∶1. The p53 protein mutation expression rate was 77.8% (49/63), including 14 cases with wild-type expression, 22 with nonsense mutation expression, and 27 with missense mutation expression. The concordance rate of p53 protein expression between carcinoma components and spindle cell components was 100%. Survival analysis showed that p53 protein mutation expression was significantly correlated with overall survival (OS, P=0.044), patients with p53 protein mutation expression had poorer OS. Conclusion: p53 protein expression is highly concordant in the squamous cell carcinoma components and spindle cell components of esophageal spindle cell carcinoma; its mutation expression is associated with poor outcome of the patients.

Patient survival remains poor even after diagnosis in lung cancer cases, and the molecular events resulting from lung cancer progression remain unclear. Raman spectroscopy could be used to noninvasively and accurately reveal the biochemical properties of biological tissues on the basis of their pathological status. This study aimed at probing biomolecular changes in lung cancer, using Raman spectroscopy as a potential diagnostic tool. Herein, biochemical alterations were evident in the Raman spectra (region of 600-1800 cm(-1)) in normal and cancerous lung tissues. The levels of saturated and unsaturated lipids and the protein-to-lipid, nucleic acid-to-lipid, and protein-to-nucleic acid ratios were significantly altered among malignant tissues compared to normal lung tissues. These biochemical alterations in tissues during neoplastic transformation have profound implications in not only the biochemical landscape of lung cancer progression but also cytopathological classification. Based on this spectroscopic approach, classification methods including k-nearest neighbour (kNN) and support vector machine (SVM) were successfully applied to cytopathologically diagnose lung cancer with an accuracy approaching 99%. The present results indicate that Raman spectroscopy is an excellent tool to biochemically interrogate and diagnose lung cancer.

目的 研究结直肠癌人工智能病理诊断模型构建过程中,病理医师对数字切片癌组织的人工标注在不同扫描仪构建的全切片图像(WSI)中准确迁移的方法.方法 在本研究中,我们提出了一种基于图像配准的标注迁移方法,在来自不同扫描仪的WSI之间建立仿射映射.通过多分辨率最小化两个WSI缩略图之间的互信息来估计最佳仿射映射参数,以避免和改变扫描仪特定特性的影响,减少计算时间.我们使用了181张结直肠癌病理切片,使用两个品牌的扫描仪获得相应的WSI,对上述标注迁移方法进行测试.结果 181张HE切片的扫描结果表明,同一张切片由不同扫描仪构建的WSI在颜色、位置、大小等属性上都有不同的表现.使用我们提出的标注迁移方法,其中179张图像的人工标注成功地在不同扫描仪构建的WSI中迁移,其中125对使用单个CPU核心的计算时间不到1分钟.结论 我们提出了一种快速、准确的全自动的标注迁移方法,用于在不同扫描仪构建的WSI之间传递人工标注.在准备深度学习训练数据过程中,既可以避免病理医师对新图像的重新标注,也可以避免病理医师之间在标注上的差异.

Importance:Programmed cell death 1 (PD-1) antibodies have shown substantial survival benefit in patients with advanced non-small cell lung cancer (NSCLC). Toripalimab is a promising and practicable PD-1 antibody; however, its performance in NSCLC has not been established. Objectives:To assess the safety, antitumor activity, and pharmacokinetics of toripalimab in patients with advanced NSCLC and to evaluate the utility of JS311, a novel PD ligand 1 (PD-L1) immunohistochemistry (IHC) assay. Design, Setting, and Participants:This single-arm open-label phase 1 trial enrolled 41 patients with advanced NSCLC that had progressed after at least 3 lines of therapy between September 21, 2017, and June 5, 2018, with a median (interquartile range) follow-up of 14.9 (3.2-22.5) months and included a cohort study comparing JS311 with other PD-L1 IHC assays that included 280 NSCLC specimens collected from January 1, 2016, to May 21, 2018. Data collection was conducted from September 21, 2017, to September 27, 2019, and analysis was conducted from September 27, 2019, to December 30, 2019. Exposure:Enrolled patients were administered a single dose of toripalimab, under 2 manufacturing processes and scales (200 L and 500 L), for safety and pharmacokinetic analysis within 28 days, followed by subsequent multidose infusions every 2 weeks. PD-L1 expression was determined by IHC with JS311, comparing its results with results from 22C3, 28-8, and SP263 simultaneously. Main Outcomes and Measures:Progression-free survival (PFS) and overall survival (OS), estimated by Kaplan-Meier curves, and continuous variables compared by t test or Mann-Whitney test. Correlations between PD-L1 IHC antibodies were evaluated by Spearman correlation test. Results:A total of 41 patients (29 [70.7%] men) with a median (interquartile range) age of 59 (53 to 63) years who experienced disease progression following chemotherapy were included. The most common treatment-related adverse events were rash (6 [14.6%]), increased amylase level (5 [12.2%]), and increased aspartate aminotransferase level (5 [12.2%]). In 35 patients included in the pharmacokinetic analysis, drug exposure and area under curve after 1 dose was similar under both manufacturing processes and scales (mean [SD] for 200-L group: 12 465.28 [4128.17] hour × μg/mL; for 500-L group: 12 331.42 [2472.58] hour × μg/ml). In 28 patients included in the response and survival analysis, the median PFS and OS were 2.8 (95% CI, 2.7 to 4.6) months and 13.8 months (95% CI, 10.0 months to not reached), respectively. Stratified by PD-L1 tumor proportion score of at least 50%, 1% to 49%, and less 1%, median PFS rates were 11.2 months (95% CI, 2.3 months to not evaluable), 2.3 (95% CI, 1.7 to 2.7) months, and 2.8 (95% CI, 2.7 to 4.6) months, respectively. A total of 4 anti-PD-L1 IHC antibodies were compared during PD-L1 staining, using 280 NSCLC specimens. The consistency rates between the 4 antibodies were 80.8% to 89.5% (ρ, 0.619 to 0.790) and 93.3% to 95.5% (ρ, 0.691 to 0.773), with PD-L1 tumor proportion scores of 1% and 50% as cut points, respectively. Conclusions and Relevance:In this study, toripalimab exhibited encouraging antitumor activity and manageable safety profiles among patients with heavily treated NSCLC. The novel PD-L1 IHC antibody JS311 was highly consistent with previously verified PD-L1 IHC assays.

[This corrects the article DOI: 10.1371/journal.pgen.1007888.].

Background: Recently, copy number alteration (CNA) of 9p24.1 were demonstrated in 10% of diffuse large b-cell lymphoma (DLBCL), with gene expression and mutation profiles that were similar to those of primary mediastinal large B-cell lymphoma(PMBCL). However, their CNA-based profile and clinical impact still remain unclear. Methods: Multiplex ligation-dependent probe amplification were employed to investigate the prevalence of JAK2/PD-L2 amplification in DLBCL and their CNA-based pattern of driver genes. The clinical outcome and characteristics were also analyzed. Results: Using unsupervised hierarchical clustering, a small group of DLBCL (10.5%, 8/76) was clustered together with PMBCL as Cluster_2, demonstrating amplification of JAK2 (100%,8/8) and PD-L2 (75.0%,6/8). This subgroups of DLBCL demonstrated significant higher expression of PD-L1 than those with MYD88 L265P mutation(p=0.024). And they exhibited dismal OS and PFS as compared with DLBCL_others(p=0.003 and 0.001, respectively), which is similar to DLBCL with MYD88 L265P mutation. Conclusions: DLBCL with amplification of JAK2/PD-L2 exhibits CNA pattern that is similar to PMBCL, and demonstrates unfavorable clinical outcome that resembles those with MYD88 L265P mutation. It is essential to identify this subgroup of DLBCL who may acquire more benefits from the JAK2 and PD-L1 signaling inhibition.

[This corrects the article DOI: 10.21037/jtd.2018.09.22.].

Objective: To investigate the factors for recurrence of laryngeal carcinoma, 95 cases of laryngeal carcinoma and precancerous lesions were retrospectively reviewed according to protein expression of carcinoma markers (including p53, c-Myc, PTEN, survivin, p21 and p27 proteins) and clinicopathologic factors. Methods: Ninety-five patients with laryngeal carcinoma and vocal cord squamous epithelial precancerous lesions were collected in Cancer Hospital Chinese Academy of Medical Sciences from January 1999 to December 2015. Three years after R0 resection, recurrence occurred in 41 of the 95 patients. Immunohistochemical methods were used to detect the expression of p53, c-Myc, PTEN, survivin, p21 and p27. Results: There was significant difference between the recurrence group and the nonrecurrence group in the expression of c-Myc, survivin and PTEN (P<0. 05); there was no significant difference in the expression of p21, p27 and p53 ( P>0. 05). Recurrence of laryngeal carcinoma after operation was correlated to pathological differentiation of tumor, and was not statistically related to age, gender and alcohol consumption and smoking. The worse the differentiation of tumor was, the higher the recurrence rate was. Conclusion: Factors for recurrence of laryngeal carcinoma are complex. The expression of c-Myc, survivin and PTEN is related to postoperative recurrence of laryngeal cancer, which may be used as an indicator of postoperative recurrence of laryngeal cancer.

Objective To investigate the association between clinical pathological factors and lymph node metastases in T1 stage rectal carcinoma. Methods We retrospectively reviewed the 251 consecutive T1 rectal carcinoma patients who had undergone radical colectomy with lymph node dissection in a single institution. The histopathological factors were reviewed. The correlation between these factors and lymph node metastasis, tumor recurrence and survival were analyzed. Results A total of 251 consecutive patients with T1 rectal carcinoma were included in this study. Lymph node metastasis occurred in 11.2% (28/251) of patients. The 3, 5 and 10 years overall survival was 98.6%, 96.8% and 94.9%, respectively, for all patients. The 3, 5 and 10 years overall survival of patients with or without lymph node metastasis were 100%, 95.6% and 90.9% or 99%, 96.9% and 95.4%, respectively. There was no statistical difference between the two group in overall survival. Univariate analysis showed that each of the following histopathological factors had a significant influence on lymph node metastasis, which are patients′ age (P=0.05); adenomatous background (P<0.01), tumor differentiation (P<0.01), cribriform structure (P=0.03), PDC (P=0.02), tumor budding (P=0.01), lymphvascular invasion (P<0.01), submucosa venous invasion (P<0.01) and glandular pattern at the submucosal invasive front (P=0.04). Multivariate analysis showed that age (P=0.02), no adenomatous background (P<0.01), tumor differentiation (P=0.04) and submucosa venous invasion (P=0.02) were significantly associated with lymph node metastasis. We also found that open-type glandular pattern is also correlated with gross plat-type (P=0.03); no adenoma background (P=0.03), complete disruption of the muscularis mucosa (P=0.05), high grade tumor budding (P<0.001) and tumor necrosis (P<0.001). Conclusion In the present study, we not only verified the effectiveness of those classical pathological factors, but proposed the cribriform structure, open-type glandular pattern at the submucosal invasive front in predicting lymph node metastasis in T1 stage rectal carcinoma. Key words: Rectal neoplasms; Neoplasm metastasis; Prognosis; T1 stage; Recurrence; Risk factor

Peripheral T-cell lymphoma not otherwise specified (PTCL-NOS) is an aggressive lymphoma associated with a poor outcome. To date, the factor consistently associated with prognosis is the International Prognostic Index (IPI) score; however, it is considered that the IPI score cannot be beneficial for guiding potential targeted therapies. New scoring systems have recently been developed. The aim of the present study was to observe the expression of NME/NM23 nucleoside diphosphate kinase 1 (nm23), nuclear DNA topoisomerase 2-α (TOP2A), multiple myeloma oncogene-1 (MUM-1) and vascular endothelial growth factor (VEGF), and evaluate their prognostic value in PTCL-NOS. A retrospective analysis of 124 cases of PTCL-NOS showed that 70/122 (57.4%) cases were positive for nm23, 71/122 (58.2%) for TOP2A, 30/119 (25.2%) for MUM-1 and 64/122 (52.5%) for VEGF. Of note, 50/122 cases concurrently expressed nm23, TOP2A and VEGF. The univariate analysis results revealed that the nm23 (P=0.012), TOP2A (P=0.002) and VEGF (P=0.008) expression had a negative prognostic effect in patients with PTCL-NOS, while the MUM-1 expression did not have a significant prognostic value (P=0.918). In addition, the concurrent expression of nm23, TOP2A and VEGF was significantly associated with a worse prognosis (P=0.002). However, in multivariate Cox regression analysis, the concurrent expression of nm23, TOP2A and VEGF tended to predict a worse prognosis, however the P-value was borderline (hazard ratio, 1.495; 95% confidence interval, 0.993-2.250; P=0.054). It is speculated that there may be an association among the expression of nm23, TOP2A and VEGF, and that their expression may serve as a promising prognostic factor for PTCL-NOS.

During the last several years, a growing number of studies have shown that microRNAs (miRNAs) participate in cancer metastasis. Brain metastasis (BM) is a frequent complication of lung adenocarcinoma (LAD), and the incidence of locally advanced LAD with BM can be as high as 30-50%. This study was performed to identify the miRNA expression patterns of LAD with BM and to determine the biological role that miRNAs play in tumorigenesis. To this end, we conducted microarray and quantitative PCR analyses to evaluate BM-related miRNAs independently validated from a total of 155 patients with LAD. A series of in vivo and in vitro assays were also conducted to verify the impact of miRNAs on BM. We found significantly increased expression of miR-423-5p, and BM was predicted in non-small cell lung cancer when compared to LAD without BM. We next examined the function of miR-423-5p and discovered that it significantly promoted colony formation, cell motility, migration, and invasion in vitro. We computationally and experimentally confirmed that metastasis suppressor 1 (MTSS1) was a direct miR-423-5p target. Through a combination of image, histological, and molecular analyses, we found that miR-423-5p overexpression significantly increased tumor burden, local invasion, and distant BM. The level of MTSS1 expression was inversely correlated with miR-423-5p upregulation in the LAD specimens and was associated with survival of patients with BM. MiR-423-5p promoted BM in LAD and inhibited MTSS1 expression. Together, these results show that MiR-423-5p has the potential to be a marker of BM and/or a therapeutic target in LAD.

Peripheral T cell and natural killer cell lymphomas (PT/NKCLs) are rare malignant tumors of lymphoid tissue. The incidence varies by geographical region and race. We reclassified 313 cases of PT/NKCLs based on the 4th edition of the World Health Organization (WHO) classification to demonstrate the distribution of each histologic type of PT/NKCLs in Chinese populations. In our series, extranodal NK/T-cell lymphoma (ENKT) was the most common (37.1%) type, followed by peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) (31.3%) and angioimmunoblastic T-cell lymphoma (AITL) (12.8%). Also, we investigated the expression level of nm23, VEGF, TOP2A, and MUM-1 in all 313 cases. The positive rate of nm23, VEGF, TOP2A, and MUM-1 expression was more than 50% in most histologic types. Among the five common types, the expression rate of nm23 (34.5%) and TOP2A (27.6%) in ENKT were the lowest (P<0.05). VEGF expression was also lowest in ENKT (26.7%), but it was much higher in PTCL-NOS (54.1%), AITL (70.0%), and ALCL-ALK- (56.5%) and ALCL-ALK+ (42.9%). The difference of VEGF expression between ENKT and PTCL-NOS and AITL was significant (P<0.05). Fourteen of 23 (60.9%) cases of ALCL-ALK- and 9 of 14 (64.3%) cases of ALCL-ALK+ were positive for MUM-1, which was much higher than in ENKT (19.8%), PTCL-NOS (26.5%) and AITL (35.0%) (P<0.05). Although the significance of their expression in PT/NKCLs is not clear, we suggested that they may be novel tumor markers for developing targeted therapy in the future.

Lynch syndrome is the most common form of hereditary CRC, accounting for approximately 2~3% of population-based CRC. Individuals with Lynch syndrome have an increased risk for extracolonic cancer and second primary colorectal cancer. It has been dedicated that Lynch syndrome is an autosomal dominant cancer predisposition syndrome caused by germline mutations in the DNA mismatch repair genes (MMR)—MLH1, MSH2, MSH6 and PMS2, and EPCAM mutation is also found associated with MSH2 deficiency recently. Even though clinical diagnostic criteria (Amsterdan I/II, revised Bethesda) have been released decades ago, molecular testing of MMR genes is still the golden standard of Lynch syndrome diagnosis. Colorectal cancer patients less than 70y, are recommended to get into the procedure of Lynch syndrome screening, starting with microsatellite instability and/or immunohistochemical analysis on the tumor specimen followed by germline genetic testing and possibly further studies, such as MLPA. Notably, Testing on BRAF mutation is necessary, when the expression of MLH1 is lost, in order to exclude the sporadic colorectal cancer. Individuals with identified germline mutations need further screening for their immediate relatives.

食管梭形细胞癌是食管鳞状细胞癌的一种特殊亚型,发生率较低,镜下具有双相性的特点,上皮成分常为高中分化的鳞状细胞癌或原位癌,梭形细胞成分常为高级别肉瘤样成分.肿瘤通常呈息肉样的生长方式,分期较早,目前研究普遍认为食管梭形细胞癌侵袭性低,淋巴结转移率低,生存率高,预后较好,但由于食管梭形细胞癌病例发生率较低,目前多为个案报道,关于食管梭形细胞癌的流行病学、临床特征、双相性的组织来源及预后尚不十分清楚.本文综述了食管梭形细胞癌的流行病学、临床特征、分子病理学特征及预后特点.