Remodeling the tumor-immune microenvironment by anti-CTLA4 blockade enhanced subsequent anti-PD-1 efficacy in advanced nasopharyngeal carcinoma.

Sequential immunotherapy has shown certain advantages in malignancy. Here, we aim to evaluate the efficacy of sequential anti-CTLA-4 and anti-PD-1 treatment for recurrent or metastatic nasopharyngeal carcinoma patients (R/M NPC). We retrospectively analysis 2 phase I trial of ipilimumab and camrelizumab in Chinese R/M NPC patients. These patients were initially treated with ipilimumab, a CTLA4 blockade, followed by anti-PD-1 treatment. We observed a durable tumor remission in these patients (mPFS: 12.3 months; mDoR: 20.9 months). Multimodal investigations of biopsy samples disclosed remodeling of tumor-immune microenvironment triggered by ipilimumab. In responders, we found increased tumoral PD-L1/PD-L2 expression and T-cell infiltration after ipilimumab treatment, accompanied by reduced stroma and malignant cell components. In contrast, non-responders exhibited increased B-cell infiltration and increased peripheral CD19 + B cells, suggesting a defective transition from memory B cells to plasma cells. This study proposes that sequential therapy can potentially enhance treatment efficacy in chemotherapy-resistant NPC patients and provides insights into how preexisting anti-CTLA4 blockade can influence subsequent anti-PD-1 efficacy by remodeling the TME. Additionally, our results highlight the need for therapeutic strategies targeting naïve/memory B cells.

Validation of a one-step genomics-based molecular classifier for endometrial carcinoma in a large Chinese population.

OBJECTIVE: The aim of this study is to delineate the molecular classification features within Chinese endometrial cancer (EC) patients and to evaluate the concurrence between two widely employed methods for diagnosing EC molecular subtypes. METHODS: This retrospective observational cohort study encompassed 479 cases of EC for analysis. Utilizing next-generation sequencing (NGS) panels targeting POLE, TP53, and microsatellite instability (MSI) status, four subtypes [POLE ultramutated (POLE mut), MMR-deficient (MMRd), p53 abnormal (p53abn), and no specific molecular profile (NSMP)] were classified. Immunohistochemistry (IHC) was employed to ascertain the expression of p53 and MMR proteins. RESULTS: Among the 479 patients, the distribution of EC subtypes was as follows: 28 (5.85%) POLE mut, 67 (13.99%) MMRd, 60 (12.53%) p53abn, and 324 (67.64%) NSMP. When compared to published findings on EC subtypes in the Caucasian population, our real-world data on Chinese ECs revealed a notably higher proportion of NSMP/CNL (copy number low). The evaluation of MSI/MMR status through NGS-based and IHC-based methods displayed substantial concordance (Kappa = 0.91). Slight discordance between the two techniques in identifying p53 abnormalities (Kappa = 0.83) might stem from TP53 truncating mutations, cytoplasmic p53 expression, null TP53 mutants, and well-documented challenges in interpreting p53 IHC. CONCLUSIONS: Chinese ECs exhibit distinctive molecular attributes. For accurate molecular subtyping of Chinese ECs, additional molecular markers that align with the Chinese population's characteristics should be incorporated into existing classifiers. The study's outcomes underscore a strong agreement between NGS and IHC in TP53/p53 detection and MSI assessment.

COL19A1 is a predictive biomarker for the responsiveness of esophageal squamous cell carcinoma patients to immune checkpoint therapy.

BACKGROUND: The use of neoadjuvant immunotherapy plus chemotherapy has revolutionized the management of esophageal squamous cell carcinoma (ESCC) patients. Nevertheless, patients who would maximally benefit from these therapies have not been identified. METHODS: We collected postoperative specimens from 103 ESCC patients, of which 66 patients comprised a retrospective cohort and 37 comprised a prospective cohort. Patient specimens were subjected to applied multi-omics analysis to uncover the mechanistic basis for patient responsiveness to cancer immunotherapy. The tumor microenvironment characteristics of these patient specimens was explored and identified by multiplex immunofluorescence and immunohistochemistry. RESULTS: Results demonstrated high COL19A1 expression to be a novel biomarker for successful immunotherapy (COL19A1high , odds ratio [95% confidence interval]: 0.31 [0.10-0.97], p = 0.044). Compared with COL19A1low patients, COL19A1high patients benefited more from neoadjuvant immunotherapy (p < 0.01), obtained better major pathological remissions (63.3%, p < 0.01), with a trend toward better recurrence-free survival (p = 0.013), and overall survival (p = 0.056). Moreover, analysis of an immune-activation subtype of patients demonstrated increased B cell infiltration to be associated with favorable patient survival and a better response to neoadjuvant immunotherapy plus chemotherapy. CONCLUSIONS: The findings of this study provide insight into the optimal design of individual treatments for ESCC patients.

Application of NGS molecular classification in the diagnosis of endometrial carcinoma: A supplement to traditional pathological diagnosis.

OBJECTIVE: This study aims to demonstrate the advantages of NGS molecular classification in EC diagnosis and to assess whether molecular classification could be performed on curettage specimens and its concordance with subsequent hysterectomy specimens. METHODS: 80 patients with hysterectomy specimens and 35/80 patients with paired curettage specimens were stratified as POLE mut, MSI-H, TP53 wt, or TP53 abn group by NGS panel. Histotype, tumor grade, IHC results, and other pathological details were taken from original pathological reports. RESULTS: The correlation analysis of 80 patients with hysterectomy specimens between NGS molecular classification and clinicopathological characters displayed that the POLE mut group was associated with EEC (87.5%) and TP53 abn subtype was correlated to a later stage (Stage II-IV, 47.6%), G3 (76.2%), serous histology (61.9%) and myometrial invasion ≥50% (47.6%). A favorable concordance (31/32, 96.9%) was shown in MSI analysis and MMR IHC results, and the agreement rate of p53 IHC and TP53 mutation was 81.5% (53/65). Compared with the p53 IHC abnormal group, the TP53 mutation group had a higher correlation with high-risk factors. A high level of concordance (31/35, 88.0%) of NGS molecular classification was achieved between curettage specimens and hysterectomy specimens while grade and histotype (including unclassified group) from curettage specimens and hysterectomy specimens showed only moderate levels of agreement, 54.3% (19/35) and 68.6% (24/35), respectively. CONCLUSION: NGS molecular classification achieved on curettage samples showed high concordance with the final hysterectomy specimens, demonstrating superior to the conventional pathological assessment of grade and histotype and potential utilization in clinical practice.

PLEK2 and IFI6, representing mesenchymal and immune-suppressive microenvironment, predicts resistance to neoadjuvant immunotherapy in esophageal squamous cell carcinoma.

BACKGROUND: Immunotherapy has largely improved clinical outcome of patients with esophageal squamous cell carcinoma (ESCC). However, a proportion of patients still fail to benefit. Thus, biomarkers predicting therapeutic resistance and underlying mechanism needs to be investigated. METHODS: Transcriptomic profiling was applied in FFPE tissues from 103 ESCC patients, including surgical samples from 66 treatment-naïve patients with long-term follow-up, and endoscopic biopsies from 37 local advanced ESCC cases receiving neoadjuvant immunotherapy plus chemotherapy. Unsupervised clustering indicated an aggressive phenotype with mesenchymal character in 66 treatment-naïve samples. Univariant logistic regression was applied to identify candidate biomarkers potentially predicted resistance to neoadjuvant immunotherapy within the range of mesenchymal phenotype enriched genes. These biomarkers were further validated by immunohistochemistry. Putative mechanisms mediating immunotherapy resistance, as indicated by microenvironment and immune cell infiltration, were evaluated by transcriptomic data, and validated by multiplex immunofluorescence. RESULTS: PLEK2 and IFI6, highly expressed in mesenchymal phenotype, were identified as novel biomarkers relating to non-MPR in neoadjuvant immunotherapy cohort [PLEK2high, OR (95% CI): 2.15 (1.07-4.33), P = 0.032; IFI6high, OR (95% CI): 2.21 (1.16-4.23), P = 0.016). PLEK2high and IFI6 high ESCC patients (versus low expressed patients) further exhibit higher chance of non-major pathological remissions (90%, P = 0.004) in neoadjuvant immunotherapy cohort and high mortality (78.9%, P = 0.05), poor prognosis in retrospective cohort. PLEK2high/IFI6high ESCC recapitulated mesenchymal phenotype, characterized by extracellular matrix composition and matrix remodeling. In addition, PLEK2high or IFI6high ESCC displayed an immune-unfavored microenvironment, represented by positive correlating with regulatory T cells, Helper 2 T cell as well as less infiltration of B cells, effector T cells and mast cells. CONCLUSIONS: PLEK2 and IFI6 was discovered of first time to identify a distinct ESCC subpopulation cannot be benefited from neoadjuvant immunotherapy and present a poor survival, which putatively associated with mesenchymal and immune-suppressive microenvironment.

Genomic Scar Score: A robust model predicting homologous recombination deficiency based on genomic instability.

OBJECTIVE: To develop a novel machine learning-based algorithm called the Genomic Scar Score (GSS) for predicting homologous recombination deficiency (HRD) events. DESIGN: Method development study. SETTING: AmoyDx Medical Laboratory and Jiangsu Cancer Hospital. POPULATION OR SAMPLE: A cohort of individuals with ovarian or breast cancer (n = 377) were collected from the AmoyDx Medical Laboratory. Another cohort of patients with ovarian cancer treated with PARP inhibitors (n = 58) was enrolled in the Jiangsu Cancer Hospital. METHODS: We used linear support vector machines to build a Genomic Scar (GS) model to predict HRD events, and Kaplan-Meier analyses were performed by comparing the progression-free survival (PFS) of patients in different groups using a two-sided log-rank test. MAIN OUTCOME MEASURES: The performance of the GS model and the result of clinical validation. RESULTS: The GS model displayed more than 97.0% sensitivity to detect BRCA-deficient events, and the GS model identified patients that could benefit from poly(ADP-ribose) polymerase inhibitors (PARPi), as the GS score (GSS)-positive group had a longer progression-free survival (PFS) (9.4 versus 4.4 months; hazard ratio [HR] = 0.54, P < 0.001) than the GSS-negative group after PARPi treatment. Meanwhile, the GSS showed high concordance among different NGS panels, which implied the robustness of the GS model. CONCLUSIONS: The GS was a robust model to predict HRD and had broad clinical applications in predicting which patients will respond favourably to PARPi treatment.

Intratumor heterogeneity of driver mutations and TMB distribution in 30 early-stage LUAD patients with multiple lesions.

Background: Differentiating multiple pulmonary lesions as multiple primary lung cancer (MLC) or intra-pulmonary metastasis (IPM) is critical. Lung cancer also has a high genetic heterogeneity, which influenced the treatment strategy. Genetic information may aid in tracing lineage information on multiple lung lesions. This study applied comprehensive genomic profiling to decipher the intrinsic genetics of multiple lung lesions. Methods: Sixty-six lung adenocarcinomas (LUAD) tumor lesions (FFEP) archived from 30 patients were included in this study. The 508 cancer-related genes were evaluated by targeted next-generation sequencing (MGI-seq 2000). Results: The study included a total of 30 LUADs (66 samples). The majority of tumors demonstrated intra-tumoral heterogeneity. Two hundred twenty-four mutations were detected by sequencing the 66 samples. We investigated the driver gene mutations of NSCLC patients with multiple lesions. EGFR was the most frequently (48/198) mutated driver gene. The codons in EGFR mainly affected by mutations were p.L858R (18/66 [27.3%]) and exon 19del (8/66 [12.1%]). In addition, additional driver genes were found, including TP53, BRAF, ERBB2, MET, and PIK3CA. We also found that the inter-component heterogeneity of different lesions and more than two different mutation types of EGFR were detected in seven patients with two lesions (P3, P10, P24, P25, P28, P29, and P30). The TMB values of different lesions in each patient were different in 26 patients (except P4, P5, P14, and P30). Conclusions: Comprehensive genomic profiling should be applied to distinguishing the nature of multiple lung lesions irrespective of radiologic and histologic diagnoses.

Short-term dynamics of circulating tumor DNA predicting efficacy of sintilimab plus docetaxel in second-line treatment of advanced NSCLC: biomarker analysis from a single-arm, phase 2 trial.

OBJECTIVE: Robust biomarker predicting efficacy of immunotherapy is limited. Circulating tumor DNA (ctDNA) sought to effectively monitor therapeutic response as well as disease progression. This study aims to investigate predictive role of ctDNA short-term dynamic change (6 weeks postimmunotherapy) in a single-arm, phase 2 trial of sintilimab plus docetaxel for previously treated advanced non-small cell lung cancer (NSCLC) patients. METHODS: A total of 33 patients with advanced NSCLC with disease progression during or after any first-line treatment were prospectively enrolled between 2019 and 2020. Patients received sintilimab (200 mg, day 1, every 3 weeks) plus docetaxel (75 mg/m2, day 3, every 3 weeks) for 4-6 cycles, followed by maintenance therapy with sintilimab (200 mg, day 1, every 3 weeks) until disease progression or unacceptable toxic effects. Blood samples were prospectively collected at baseline, and after 2 cycles of treatment (6 weeks post-treatment). All samples were subjected to targeted next-generation sequencing with a panel of 448 cancer-related genes. The landscape of high-frequency genomic profile of baseline and 6th week was described. Major molecular characteristics in preselected genes of interest associated with response to second-line chemoimmunotherapy were analyzed. The curative effects and prognosis of patients were evaluated. RESULTS: Patients with ctDNA clearance at 6th week had decreased tumor volume, while most patients with positive ctDNA at 6th-week experienced an increase in tumor volume. Positive 6th-week ctDNA was associated with significantly shorter progression-free survival (PFS) (91 vs NR days; p<0.0001) and overall survival (47 vs 467 days; p =0.0039). Clearance of clonal mutations and none new clonal formation at 6th week were associated with longer PFS (mPFS 89 vs 266 days, p =0.003). ctDNA clearance at 6th week was an independent risk factor for progression or death (HR=100 (95% CI 4.10 to 2503.00), p=0.005). CONCLUSION: ctDNA status and ctDNA mutation clearance putatively serve as predictive biomarkers for sintilimab combined with docetaxel chemotherapy in pretreated advanced NSCLC patients.

Detecting ALK Rearrangement with RT-PCR: A Reliable Approach Compared with Next-Generation Sequencing in Patients with NSCLC.

BACKGROUND: Precise detection of anaplastic lymphoma kinase (ALK) rearrangement guides the application of ALK-targeted tyrosine kinase inhibitors (ALK-TKIs) in patients with non-small-cell lung cancer (NSCLC). Next-generation sequencing (NGS) has been widely used in clinics, but DNA-based NGS used to detect fusion genes has delivered false-negative results. However, fusion genes can be successfully detected at the transcription level and with higher sensitivity using RNA-based reverse transcription polymerase chain reaction (RT-PCR). OBJECTIVE: This study compared the performance of RT-PCR and NGS in the detection of echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion in Chinese patients with NSCLC. METHODS: Formalin-fixed paraffin-embedded tissues from 153 patients who were pathologically diagnosed as having NSCLC were collected from November 2017 to October 2019. Both DNA/RNA-based NGS and RNA-based RT-PCR were used to detect EML4-ALK fusion. For samples with discordant ALK status results, fluorescence in situ hybridization (FISH) or Sanger sequencing was used to further confirm the ALK status. RESULTS: In total, 124 samples were successfully analyzed using both NGS and RT-PCR. For 118 samples, results were consistent between NGS and RT-PCR, with 25 reported as ALK fusion positive and 93 as ALK fusion negative, achieving a concordance rate of 95.16%. Among the six samples with disconcordant results, five were positive using RT-PCR but negative using NGS, and one was positive using NGS but negative using RT-PCR. Four of six cases with disconcordant results (three RT-PCR positive and one NGS positive) were successfully validated using either FISH or Sanger sequencing. CONCLUSIONS: Compared with NGS, RT-PCR appears to be a reliable method of detecting EML4-ALK fusion in patients with NSCLC.

Unique Profile of Driver Gene Mutations in Patients With Non-Small-Cell Lung Cancer in Qujing City, Yunnan Province, Southwest China.

OBJECTIVE: Qujing City, Yunnan Province, China, has a high incidence of lung cancer and related mortality. The etiology of NSCLC in Qujing area and distribution of associated molecular aberrations has not been fully elucidated. This study aimed to reveal the profile of driver gene mutations in patients with non-small-cell lung cancer (NSCLC) in Qujing and explore their relationships with clinicopathological characteristics. METHODS: In this study, the mutation profiles of NSCLC driver genes, including EGFR, ALK, ROS1, KRAS, BRAF, RET, MET, HER2, NRAS, and PIK3CA, were investigated in patients with NSCLC from Qujing and compared with those from other regions in Yunnan Province. The associations between molecular mutations and clinicopathological characteristics were further analyzed. RESULTS: A distinct profile of driver gene mutations was discovered in patients with NSCLC from Qujing. Interestingly, a higher proportion of EGFR compound mutations, including G719X + S768I (19.65% vs 3.38%, P < 0.0001) and G719X + L861Q (21.10% vs 2.82%, P < 0.0001), was observed in patients with NSCLC in Qujing compared with patients in non-Qujing area, besides significantly different distributions of EGFR (46.01% vs. 51.07%, P = 0.0125), ALK (3.17% vs. 6.97%, P = 0.0012), ROS1 (0.5% vs. 2.02%, P = 0.0113), and KRAS (23.02% vs. 7.85%, P < 0.0001). Further, EGFR compound mutations were more likely associated with the occupation of patients (living/working in rural areas, e.g., farmers). Moreover, KRAS G12C was the dominant subtype (51.11% vs 25.00%, P = 0.0275) among patients with NSCLC having KRAS mutations in Qujing. CONCLUSIONS: Patients with NSCLC in Qujing displayed a unique profile of driver gene mutations, especially a higher prevalence of EGFR compound mutations and dominant KRAS G12C subtype, in this study, indicating a peculiar etiology of NSCLC in Qujing. Therefore, a different paradigm of therapeutic strategy might need to be considered for patients with NSCLC in Qujing.

Genomic Profiling Comparison of Germline BRCA and Non-BRCA Carriers Reveals CCNE1 Amplification as a Risk Factor for Non-BRCA Carriers in Patients With Triple-Negative Breast Cancer.

Background: Differences in genomic profiling and immunity-associated parameters between germline BRCA and non-BRCA carriers in TNBC with high tumor burden remain unexplored. This study aimed to compare the differences and explore potential prognostic predictors and therapeutic targets. Methods: The study cohort included 21 consecutive TNBC cases with germline BRCA1/2 mutations and 54 non-BRCA carriers with a tumor size ≥ 2 cm and/or ≥1 affected lymph nodes. Differences in clinicopathological characteristics and genomic profiles were analyzed through next-generation sequencing. Univariate Kaplan-Meier analysis and Cox regression model were applied to survival analysis. Immunohistochemistry was used to confirm the consistency between CCNE1 amplification and cyclin E1 protein overexpression. Results: The cohort included 16 and five patients with germline BRCA1 and BRCA2 mutations, respectively. Patients with germline BRCA1/2 mutations were diagnosed at a significantly younger age and were more likely to have a family history of breast and/or ovarian cancer. Six non-BRCA carriers (11.11%) carried germline mutations in other cancer susceptibility genes, including five mutations in five homologous recombination repair (HRR) pathway genes (9.26%) and one mutation in MSH3 (1.85%). Somatic mutations in HRR pathway genes were found in 22.22 and 14.29% of the non-BRCA and BRCA carriers, respectively. PIK3CA missense mutation (p = 0.046) and CCNE1 amplification (p = 0.2) were found only in the non-BRCA carriers. The median tumor mutation burden (TMB) was 4.1 Muts/Mb, whereas none of the cases had high microsatellite instability (MSI). BRCA status did not affect disease-free survival (DFS, p = 0.15) or overall survival (OS, p = 0.52). CCNE1 amplification was an independent risk factor for DFS in non-BRCA carriers with TNBC (HR 13.07, 95% CI 2.47-69.24, p = 0.003). Consistency between CCNE1 amplification and cyclin E1 protein overexpression was confirmed with an AUC of 0.967 for cyclin E1 signal intensity. Conclusions: We found differences in genetic alterations between germline BRCA and non-BRCA carriers with TNBC and a high tumor burden. TMB and MSI may not be suitable predictors of TNBC for immune checkpoint inhibitors. Notably, CCNE1 amplification is a novel potential prognostic marker and therapeutic target for non-BRCA carriers with TNBC. Cyclin E1 may be used instead of CCNE1 to improve clinical applicability.

Concomitant Mutations in EGFR 19Del/L858R Mutation and Their Association with Response to EGFR-TKIs in NSCLC Patients.

OBJECTIVE: Differences in efficacy of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) have been observed between non-small cell lung cancer (NSCLC) patients with 19 exon deletion (19Del) and L858R mutation. We explored whether the total number or pattern of concomitant mutations of 19Del and L858R may explain their different sensitivities. PATIENTS AND METHODS: This study contained the mutational profiles of EGFR-mutated NSCLC patients from two cohorts: Guangzhou (G1) and database (G2). Concomitant mutation status and EGFR-TKI response information were retrieved. RESULTS: A total of 403 patients covered 283 genes in the G1 and 803 patients with a different gene set in the G2 were included. Similar prevalence of total concomitant mutation number was observed in both G1 (19Del 32.48% vs L858R 30.45%; P=0.68) and G2 (19Del 74.9% vs L858R 73.2%; P=0.65) cohorts. Only HGF/c-Met pathway same more related to L858R mutation. EGFR-TKI response information was recorded for 134 patients in the G2 cohort. 19Del showed a higher objective response (OR) rate compared with L858R, regardless of concomitant mutations. Compared to patients with OR, non-OR patients had more concomitant mutations, both in 19Del (53.8% vs 83.3%; P=0.021) and L858R (51.4% vs 77.8%; P=0.029). In particular, total concomitant mutations (OR=0.27; P=0.03), sensitive EGFR mutations (OR=2.21; P=0.04), and T790M (OR=0.244; P=0.02) significantly affected the TKI response. CONCLUSION: Concomitant mutations were widespread in 19Del and L858R and were associated with poorer OR to EGFR-TKIs. However, 19Del and L858R had similar numbers and patterns of concomitant mutations, which might not explain the different sensitivity to EGFR-TKI.

Periostin Is Expressed by Pericytes and Is Crucial for Angiogenesis in Glioma.

The expression of the matricellular protein periostin has been associated with glioma progression. In previous work we found an association of periostin with glioma angiogenesis. Here, we screen gliomas for POSTN expression and identify the cells that express periostin in human gliomas. In addition, we study the role of periostin in an in vitro model for angiogenesis. The expression of periostin was investigated by RT-PCR and by immunohistochemistry. In addition, we used double labeling and in situ RNA techniques to identify the expressing cells. To investigate the function of periostin, we silenced POSTN in a 3D in vitro angiogenesis model. Periostin expression was elevated in pilocytic astrocytoma and glioblastoma, but not in grade II/III astrocytomas and oligodendrogliomas. The expression of periostin colocalized with PDGFRß+ cells, but not with OLIG2+/SOX2+ glioma stem cells. Silencing of periostin in pericytes in coculture experiments resulted in attenuation of the numbers and the length of the vessels formation and in a decrease in endothelial junction formation. We conclude that pericytes are the main source of periostin in human gliomas and that periostin plays an essential role in the growth and branching of blood vessels. Therefore, periostin should be explored as a novel target for developing anti-angiogenic therapy for glioma.

Integrating Histologic and Genomic Characteristics to Predict Tumor Mutation Burden of Early-Stage Non-Small-Cell Lung Cancer.

Tumor mutation burden (TMB) serves as an effective biomarker predicting efficacy of mono-immunotherapy for non-small cell lung cancer (NSCLC). Establishing a precise TMB predicting model is essential to select which populations are likely to respond to immunotherapy or prognosis and to maximize the benefits of treatment. In this study, available Formalin-fixed paraffin embedded tumor tissues were collected from 499 patients with NSCLC. Targeted sequencing of 636 cancer related genes was performed, and TMB was calculated. Distribution of TMB was significantly (p < 0.001) correlated with sex, clinical features (pathological/histological subtype, pathological stage, lymph node metastasis, and lympho-vascular invasion). It was also significantly (p < 0.001) associated with mutations in genes like TP53, EGFR, PIK3CA, KRAS, EPHA3, TSHZ3, FAT3, NAV3, KEAP1, NFE2L2, PTPRD, LRRK2, STK11, NF1, KMT2D, and GRIN2A. No significant correlations were found between TMB and age, neuro-invasion (p = 0.125), and tumor location (p = 0.696). Patients with KRAS p.G12 mutations and FAT3 missense mutations were associated (p < 0.001) with TMB. TP53 mutations also influence TMB distribution (P < 0.001). TMB was reversely related to EGFR mutations (P < 0.001) but did not differ by mutation types. According to multivariate logistic regression model, genomic parameters could effectively construct model predicting TMB, which may be improved by introducing clinical information. Our study demonstrates that genomic together with clinical features yielded a better reliable model predicting TMB-high status. A simplified model consisting of less than 20 genes and couples of clinical parameters were sought to be useful to provide TMB status with less cost and waiting time.

Prevalence of hereditary breast and ovarian cancer (HBOC) predisposition gene mutations among 882 HBOC high-risk Chinese individuals.

Identification of deleterious variants in hereditary breast and ovarian cancer (HBOC) susceptibility genes allows for increased clinical surveillance and early detection, and could predict the response to poly (ADP-ribose) polymerase (PARP) inhibitor in patients with advanced ovarian carcinomas. To determine the prevalence and clinical prediction factors for HBOC syndrome, 882 selected individuals underwent multigene panel testing for HBOC risk assessment during the period from January 2015 to March 2018. Overall, 176 deleterious mutations were observed in 19.50% (n = 172) of individuals. Twenty-six of 176 mutations could not be retrieved in related public databases and were considered to be novel. Among patients with ovarian cancer, 115 deleterious mutations were identified in 429 patients (48.6%) with significant enrichment for a family history of breast or ovarian cancer syndrome (P < .05). In the breast cancer subgroup, 31 deleterious mutations were identified in 261 patients. Besides BRCA1 (8; 25.8%) and BRCA2 (11; 35.5%), the most frequently occurring genes, an additional 12 deleterious mutations (38.7%) were found in seven other susceptibility genes. Higher mutation incidence (57.9%) was observed in subjects with histories of breast and ovarian cancer. Our results highlighted the genetic heterogeneity of HBOC and the efficiency of a multigene panel in carrying out risk assessment.

Incidence of metastatic disease and survival among patients with newly diagnosed primary CNS tumors in the United States from 2004-2013.

Background: Population-based estimates of the incidence and prognosis of metastatic disease at the initial diagnosis of primary central nervous system (CNS) tumors are currently lacking. Methods: A total of 43,455 patients diagnosed with a primary CNS tumor were enrolled to evaluate metastatic rates utilizing the data from the Surveillance, Epidemiology, and End Results (SEER) program. We used multivariate logistic regression to analyze the risk factors associated with the presence of metastasis at the first visit of patients with metastatic medulloblastoma (MB), atypical teratoid/rhabdoid tumor (ATRT), glioblastoma multiforme (GBM), or pilocytic astrocytoma (PA). Hazard ratios (HRs) and 95% confidence intervals (CIs) for cancer-specific death (CSD) of patients with these four CNS tumors were analyzed using multivariate Cox regression. Results: In patients with primary CNS embryonal tumors, the metastatic rates of patients with MB and ATRT were 14.51% and 19.25%, respectively. The metastatic rate for MB patients aged 0 to 18 years was 16.69%. In the patients with glioma, the metastatic rates of patients with PA and GBM were 1.55% and 1.39%, respectively. On multivariate logistic regression among patients with glioma, GBM (vs PA; OR, 2.12; 95% CI, 1.37 to 3.30; P=0.001) was associated with greater odds of having metastatic disease at diagnosis. On multivariate logistic regression among patients with GBM, MB, or ATRT, MB (vs GBM; OR, 4.66; 95% CI, 2.81 to 7.72; P<0.001) and ATRT (vs GBM; OR, 5.65; 95% CI, 3.27 to 9.75; P<0.001) were associated with greater odds of having metastatic disease at diagnosis. In the multivariate Cox proportional hazards model for CSD among patients with metastatic GBM or MB at diagnosis, gross total resection/total lobectomy (vs partial resection/partial lobectomy) was not related to a decreased or an increased risk of CSD. In patients with metastatic ATRT, compared to no surgery, gross total resection/total lobectomy or partial resection/partial lobectomy was not associated with a decreased risk of CSD. Conclusions: The findings in this study provide a population-based estimate of the incidence and prognosis of metastatic disease at the initial diagnosis of primary CNS tumors. These survival outcomes are relevant because they will help to prioritize future research directions to improve the treatment strategies of these metastatic CNS tumors.

CMTM4 regulates angiogenesis by promoting cell surface recycling of VE-cadherin to endothelial adherens junctions.

Vascular endothelial (VE) cadherin is a key component of endothelial adherens junctions (AJs) and plays an important role in maintaining vascular integrity. Endocytosis of VE-cadherin regulates junctional strength and a decrease of surface VE-cadherin reduces vascular stability. However, disruption of AJs is also a requirement for vascular sprouting. Identifying novel regulators of endothelial endocytosis could enhance our understanding of angiogenesis. Here, we evaluated the angiogenic potential of (CKLF-like MARVEL transmembrane domain 4) CMTM4 and assessed in which molecular pathway CMTM4 is involved during angiogenesis. Using a 3D vascular assay composed of GFP-labeled HUVECs and dsRED-labeled pericytes, we demonstrated in vitro that siRNA-mediated CMTM4 silencing impairs vascular sprouting. In vivo, CMTM4 silencing by morpholino injection in zebrafish larvae inhibits intersomitic vessel growth. Intracellular staining revealed that CMTM4 colocalizes with Rab4+ and Rab7+ vesicles, both markers of the endocytic trafficking pathway. CMTM4 colocalizes with both membrane-bound and internalized VE-cadherin. Adenovirus-mediated CMTM4 overexpression enhances the endothelial endocytic pathway, in particular the rapid recycling pathway, shown by an increase in early endosomal antigen-1 positive (EEA1+), Rab4+, Rab11+ , and Rab7+ vesicles. CMTM4 overexpression enhances membrane-bound VE-cadherin internalization, whereas CMTM4 knockdown decreases internalization of VE-cadherin. CMTM4 overexpression promotes endothelial barrier function, shown by an increase in recovery of transendothelial electrical resistance (TEER) after thrombin stimulation. We have identified in this study a novel regulatory function for CMTM4 in angiogenesis. CMTM4 plays an important role in the turnover of membrane-bound VE-cadherin at AJs, mediating endothelial barrier function and controlling vascular sprouting.

Comparative proteomic analysis of cat eye syndrome critical region protein 1- function in tumor-associated macrophages and immune response regulation of glial tumors.

INTRODUCTION: Tumor associated macrophages (TAMs) promote tumor development, angiogenesis and distal metastasis. In previous studies, we showed that Cat Eye Syndrome Critical Region Protein 1 (CECR1) is expressed by M2-like TAMs in human glioma samples. CECR1 promoted M2 TAMs differentiation and affected glioma cell proliferation and migration. Here we investigated the proteomic profile of TAMs expressing CECR1 in absence or presence of glioma cells. RESULTS: CECR1 siRNA transfection upregulated 67 proteins in THP-1-derived Macrophages (MQs). Pathway annotation mapped this set to 3 major pathways relevant for MQ function, including 'MHC-I antigen presentation', 'phagosome maturation' and 'endocytosis'. Co-culture of siCECR1 THP-1-derived MQs with U87 glioma cells attenuated the changes observed on protein and mRNA level in response to MQ CECR1 silencing. SiCECR1 in U87 co-cultured MQs was associated with an IL-10low, IL-12high M1-like phenotype. In U87 co-culture conditions, SiCECR1 also downregulated S20 proteasome complex proteins PSMA5, PSMA7, PSMC6 and PSMD8. This protein profile was linked to a low proliferation rate of siCECR1 MQs. Overlap analysis identified S100A9 and PLAU as CECR1-related proteins that were significantly correlated with expression of CECR1 and macrophage lineage markers in three large public GBM datasets. CONCLUSION: This study reports the molecular pathways and key molecules that are mediated by CECR1 function in THP- 1-derived MQs and TAMs in glioma. METHODS: PMA-treated THP-1 cells (MQs) were siRNA transfected for CECR1 in vitro, with or without stimulation of the primary glioma cell line U87. Lysates were analyzed by (nano)LC-MS. Significant altered protein levels were identified (P < 0.05), followed by pathway annotation.

Deciphering the Epitranscriptome in Cancer.

Technological and methodological advancements have recently revolutionized our understanding of widespread epitranscriptome including RNA modifications and editing. N6-methyladenosine (m6A) represents the most prevalent internal modification in mammalian RNAs. Adenosine to inosine (A-to-I) RNA editing is an important mechanism underlying RNA generation and protein diversity through the post-transcriptional modification of single nucleotides in RNA sequences. In this review, we attempt to summarize its functional importance in various fundamental bioprocesses of m6A and A-to-I editing. We also highlight some of the key findings that have helped shape our understanding of epitranscriptome in tumorigenesis, tumor progression, and metastasis. Finally, we discuss conceivable targets and future directions of m6A and A-to-I editing in cancer therapeutics.

Risk factors stratifying malignancy of nodules in contralateral thyroid lobe in patients with pre-operative ultrasound indicated unilateral papillary thyroid carcinoma: A retrospective analysis from single centre.

OBJECTIVE: Papillary thyroid carcinoma (PTC) is the most common thyroid carcinoma with a favourable clinical outcome. For unilateral PTC patients with thyroid nodules in the contralateral lobes, the necessity of total thyroidectomy (TT) is still in doubt. In this study, we aimed to define clinical factors that could be indicators for malignancy in nodules in the contralateral thyroid lobe, which could aid the clinician in selecting the appropriate operation approach. DESIGN, PATIENTS AND MEASUREMENTS: This is a retrospective study from January 2014 to December 2016 conducted in Shanghai Ruijin Hospital. A total of 1442 cases with unilateral PTC and ultrasonographically benign nodules in the contralateral lobe who underwent TT at a single institution were enrolled. All patients underwent pre-operative ultrasonography (US), and all the cases were confirmed by board-certified pathologists. Clinicopathological features such as age, gender, tumour location, tumour size, TgAb and TPOAb levels, capsular invasion, multifocality, central lymph node metastases and BRAF mutation were examined to evaluate the rate of malignancy in the contralateral thyroid nodules. RESULTS: In total, 47% of patients (677 cases)were confirmed to have malignancy in the contralateral lobe. Univariant analysis indicated that capsular invasion, Hashimoto's thyroiditis, multifocal loci, central lymph node metastases as well as BRAF mutation predicted a high incidence of occult contralateral carcinoma. Multivariant analysis showed capsular invasion, multifocal ipsilateral thyroid lobe, central lymph node metastases as well as BRAF mutation can serve as independent predictors for malignancy in the contralateral thyroid lobe. CONCLUSIONS: Malignancy in the contralateral lobe was found in 47% of patients. This finding was associated with multifocal primary carcinomas involvement, capsular invasion, Hashimoto's thyroiditis history, central lymph node metastases and BRAF mutation, which should therefore be taken into consideration when planning therapeutic strategy for the patients.