Annacarolina da Silva

Dual loss of the SWI/SNF complex ATPases SMARCA4/BRG1 and SMARCA2/BRM is highly sensitive and specific for small cell carcinoma of the ovary, hypercalcaemic type.

Small cell carcinoma of the ovary, hypercalcaemic type (SCCOHT) is a lethal and sometimes familial ovarian tumour of young women and children. We and others recently discovered that over 90% of SCCOHTs harbour inactivating mutations in the chromatin remodelling gene SMARCA4 with concomitant loss of its encoded protein SMARCA4 (BRG1), one of two mutually exclusive ATPases of the SWI/SNF chromatin remodelling complex. To determine the specificity of SMARCA4 loss for SCCOHT, we examined the expression of SMARCA4 by immunohistochemistry in more than 3000 primary gynaecological tumours. Among ovarian tumours, it was only absent in clear cell carcinoma (15 of 360, 4%). In the uterus, it was absent in endometrial stromal sarcomas (4 of 52, 8%) and high‐grade endometrioid carcinomas (2 of 338, 1%). Recent studies have shown that SMARCA2 (BRM), the other mutually exclusive ATPase of the SWI/SNF complex, is necessary for survival of tumour cells lacking SMARCA4. Therefore, we examined SMARCA2 expression and discovered that all SMARCA4‐negative SCCOHTs also lacked SMARCA2 protein by IHC, including the SCCOHT cell lines BIN67 and SCCOHT1. Among ovarian tumours, the SMARCA4/SMARCA2 dual loss phenotype appears completely specific for SCCOHT. SMARCA2 loss was not due to mutation but rather from an absence of mRNA expression, which was restored by treatment with the histone deacetylase inhibitor trichostatin A. Re‐expression of SMARCA4 or SMARCA2 inhibited the growth of BIN67 and SCCOHT1 cell lines. Our results indicate that SMARCA4 loss, either alone or with SMARCA2, is highly sensitive and specific for SCCOHT and that restoration of either SWI/SNF ATPase can inhibit the growth of SCCOHT cell lines.

A survey of DICER1 hotspot mutations in ovarian and testicular sex cord-stromal tumors

Sertoli–Leydig cell tumors are characterized by the presence of somatic DICER1 hotspot mutations. In this study, we sought to define the association between DICER1 hotspot mutations and different morphologic subtypes of ovarian Sertoli–Leydig cell tumors. Furthermore, we aimed to assess whether DICER1 hotspot mutations occur in other ovarian sex cord-stromal tumors, testicular sex cord-stromal tumors, or other female genital tract tumors with rhabdomyosarcomatous differentiation. We subjected a series of ovarian Sertoli–Leydig cell tumors (n=32), Sertoli cell tumors (n=5) and gynandroblastomas (n=5), testicular sex cord-stromal tumors (n=15) and a diverse group of female genital tract tumors with rhabdomyosarcomatous morphology (n=10) to DICER1 hotspot mutation analysis using Sanger sequencing. We also tested two gynandroblastomas for the presence of FOXL2 hotspot mutations (p.C134W; c.402C>G). Twenty of 32 (63%) Sertoli–Leydig cell tumors harbored a DICER1 hotspot mutation, of which 80% had the p.E1705K mutation. No association was found between DICER1 mutation status and the presence of heterologous or retiform differentiation in Sertoli–Leydig cell tumors. DICER1 mutations were found at similar frequencies in gynandroblastoma (2/5; 40%) and ovarian Sertoli cell tumors (5/8; 63%; P>0.1), and all mutated tumors harbored a p.E1705K mutation. DICER1 hotspot mutations were also identified in a single cervical rhabdomyosarcoma and in the rhabdomyosarcomatous component of a uterine carcinosarcoma. No DICER1 mutations were detected in testicular sex cord-stromal tumors. Two DICER1 wild-type gynandroblastomas harbored a p.C134W FOXL2 hotspot mutation in both tumor components. In this study we confirmed that DICER1 hotspot mutations occur in over half of ovarian Sertoli–Leydig cell tumors, and are unrelated to tumor differentiation. We also widened the spectrum of ovarian sex cord-stromal tumors with sertoliform differentiation, in which DICER1 mutations are known to occur, to include Sertoli cell tumors and gynandroblastomas. Our results suggest that DICER1 mutations may not have a role in testicular sex cord-stromal tumorigenesis.

Loss of SMARCA4 Expression Is Both Sensitive and Specific for the Diagnosis of Small Cell Carcinoma of Ovary, Hypercalcemic Type.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare ovarian neoplasm that occurs in young women and has a poor prognosis. The histologic diagnosis of SCCOHT can be challenging due to its rarity and relatively nonspecific histologic features, which range from the classic, first-described small cell morphology to a pattern in which there are large cells with abundant eosinophilic cytoplasm. Many entities can be in the differential diagnosis and to date, immunohistochemical stains have shown no distinctive profile and have been of limited aid. SMARCA4 (also known as BRG1) mutations have recently been reported at high frequency in these tumors. SMARCA4 is an important component of the SWI/SNF complex that regulates gene expression through alteration of nucleosome conformation. Studies to date have suggested that immunohistochemical loss of expression of SMARCA4 is associated with the presence of a SMARCA4 mutation in most cases. In this study, the sensitivity and specificity of the immunohistochemical loss of SMARCA4 expression for the diagnosis of SCCOHT is examined in the context of the differential diagnosis with other primary or metastatic ovarian tumors. All but one of the SCCOHT showed loss of SMARCA4 expression (16/17; 94%), while of 279 other tumors tested, only two tumors (one clear cell carcinoma and one ovarian melanoma) showed loss of SMARCA4 expression. We conclude that SMARCA4 immunohistochemistry is highly sensitive and specific for a diagnosis of SCCOHT and is of clinical utility in the differential diagnosis of poorly differentiated ovarian tumors.

Tumor LINE-1 methylation level and colorectal cancer location in relation to patient survival

Colorectal tumors arise with genomic and epigenomic alterations through interactions between neoplastic cells, immune cells, and microbiota that vary along the proximal to distal axis of colorectum. Long interspersed nucleotide element-1 (LINE-1) hypomethylation in colorectal cancer has been associated with worse clinical outcome. Utilizing 1,317 colon and rectal carcinoma cases in two U.S.-nationwide prospective cohort studies, we examined patient survival according to LINE-1 methylation level stratified by tumor location. Cox proportional hazards model was used to assess a statistical interaction between LINE-1 methylation level and tumor location in colorectal cancer-specific mortality analysis, controlling for potential confounders including microsatellite instability, CpG island methylator phenotype, and KRAS, BRAF, and PIK3CA mutations. A statistically significant interaction was found between LINE-1 methylation level and tumor location in colorectal cancer-specific mortality analysis (Pinteraction = 0.011). The association of LINE-1 hypomethylation with higher colorectal cancer-specific mortality was stronger in proximal colon cancers (multivariable hazard ratio [HR], 1.66; 95% confidence interval [CI], 1.21 to 2.28) than in distal colon cancers (multivariable HR, 1.18; 95% CI, 0.81 to 1.72) or rectal cancers (multivariable HR, 0.87; 95% CI, 0.57 to 1.34). Our data suggest the interactive effect of LINE-1 methylation level and colorectal cancer location on clinical outcome.

MicroRNA let-7, T Cells, and Patient Survival in Colorectal Cancer

The population-based data presented in this study support a possible role for microRNA let-7a in the suppression of antitumor immunity in colorectal cancer patients. This may have implications for expanding the benefit of immunotherapy through targeting microRNAs. Experimental evidence suggests that the let-7 family of noncoding RNAs suppresses adaptive immune responses, contributing to immune evasion by the tumor. We hypothesized that the amount of let-7a and let-7b expression in colorectal carcinoma might be associated with limited T-lymphocyte infiltrates in the tumor microenvironment and worse clinical outcome. Utilizing the molecular pathological epidemiology resources of 795 rectal and colon cancers in two U.S.-nationwide prospective cohort studies, we measured tumor-associated let-7a and let-7b expression levels by quantitative reverse-transcription PCR, and CD3+, CD8+, CD45RO (PTPRC)+, and FOXP3+ cell densities by tumor tissue microarray immunohistochemistry and computer-assisted image analysis. Logistic regression analysis and Cox proportional hazards regression were used to assess associations of let-7a (and let-7b) expression (quartile predictor variables) with T-cell densities (binary outcome variables) and mortality, respectively, controlling for tumor molecular features, including microsatellite instability, CpG island methylator phenotype, LINE-1 methylation, and KRAS, BRAF, and PIK3CA mutations. Compared with cases in the lowest quartile of let-7a expression, those in the highest quartile were associated with lower densities of CD3+ [multivariate odds ratio (OR), 0.40; 95% confidence interval (CI), 0.23–0.67; Ptrend = 0.003] and CD45RO+ cells (multivariate OR, 0.31; 95% CI, 0.17–0.58; Ptrend = 0.0004), and higher colorectal cancer-specific mortality (multivariate hazard ratio, 1.82; 95% CI, 1.42–3.13; Ptrend = 0.001). In contrast, let-7b expression was not significantly associated with T-cell density or colorectal cancer prognosis. Our data support the role of let-7a in suppressing antitumor immunity in colorectal cancer and suggest let-7a as a potential target of immunotherapy. Cancer Immunol Res; 4(11); 927–35. ©2016 AACR.

Real-time Genomic Characterization of Advanced Pancreatic Cancer to Enable Precision Medicine

Clinically relevant subtypes exist for pancreatic ductal adenocarcinoma (PDAC), but molecular characterization is not yet standard in clinical care. We implemented a biopsy protocol to perform time-sensitive whole-exome sequencing and RNA sequencing for patients with advanced PDAC. Therapeutically relevant genomic alterations were identified in 48% (34/71) and pathogenic/likely pathogenic germline alterations in 18% (13/71) of patients. Overall, 30% (21/71) of enrolled patients experienced a change in clinical management as a result of genomic data. Twenty-six patients had germline and/or somatic alterations in DNA-damage repair genes, and 5 additional patients had mutational signatures of homologous recombination deficiency but no identified causal genomic alteration. Two patients had oncogenic in-frame BRAF deletions, and we report the first clinical evidence that this alteration confers sensitivity to MAPK pathway inhibition. Moreover, we identified tumor/stroma gene expression signatures with clinical relevance. Collectively, these data demonstrate the feasibility and value of real-time genomic characterization of advanced PDAC.Significance: Molecular analyses of metastatic PDAC tumors are challenging due to the heterogeneous cellular composition of biopsy specimens and rapid progression of the disease. Using an integrated multidisciplinary biopsy program, we demonstrate that real-time genomic characterization of advanced PDAC can identify clinically relevant alterations that inform management of this difficult disease. Cancer Discov; 8(9); 1096-111. ©2018 AACR.See related commentary by Collisson, p. 1062This article is highlighted in the In This Issue feature, p. 1047.

Calcium intake and risk of colorectal cancer according to expression status of calcium-sensing receptor (CASR).

Objective Although evidence suggests an inverse association between calcium intake and the risk of colorectal cancer, the mechanisms remain unclear. The calcium-sensing receptor (CASR) is expressed abundantly in normal colonic epithelium and may influence carcinogenesis. We hypothesized that calcium intake might be associated with lower risk of CASR-positive, but not CASR-negative, colorectal cancer. Design We assessed tumour CASR protein expression using immunohistochemistry in 779 incident colon and rectal cancer cases that developed among 136 249 individuals in the Nurses’ Health Study and Health Professionals Follow-Up Study. Duplication method Cox proportional hazards regression analysis was used to assess associations of calcium intake with incidence of colorectal adenocarcinoma subtypes by CASR status. Results Total calcium intake was inversely associated with the risk of developing colorectal cancer (ptrend=0.01, comparing ≥1200 vs <600 mg/day: multivariable HR=0.75, 95% CI 0.60 to 0.95). For the same comparison, higher total calcium intake was associated with a lower risk of CASR-positive tumours (ptrend=0.003, multivariable HR=0.67, 95% CI 0.51 to 0.86) but not with CASR-negative tumours (ptrend=0.67, multivariable HR=1.15, 95% CI 0.75 to 1.78; pheterogeneity=0.06 between the CASR subtypes). The stronger inverse associations of calcium intake with CASR-positive but not CASR-negative tumours generally appeared consistent regardless of sex, tumour location and source of calcium. Conclusions Our molecular pathological epidemiology data suggest a causal relationship between higher calcium intake and lower colorectal cancer risk, and a potential role of CASR in mediating antineoplastic effect of calcium.

Tumor PDCD1LG2 (PD-L2) Expression and the Lymphocytic Reaction to Colorectal Cancer

Colorectal tumor PDCD1LG2 (PD-L2) expression was inversely associated with Crohns-like lymphoid reactions, which are a key adaptive antitumor response in colorectal carcinogenesis. This population data can inform the development of immunotherapy strategies targeting immune checkpoint mechanisms. Expression of the immune checkpoint ligand CD274 (programmed cell death 1 ligand 1, PD-L1, from gene CD274) contributes to suppression of antitumor T cell–mediated immune response in various tumor types. However, the role of PDCD1LG2 (PD-L2, CD273, from gene PDCD1LG2) in the tumor microenvironment remains unclear. We hypothesized that tumor PDCD1LG2 expression might be inversely associated with lymphocytic reactions to colorectal cancer. We examined tumor PDCD1LG2 expression by IHC in 823 colon and rectal carcinoma cases within two U.S.-nationwide cohort studies and categorized tumors into quartiles according to the percentage of PDCD1LG2–expressing carcinoma cells. We conducted multivariable ordinal logistic regression analysis to assess the associations of tumor PDCD1LG2 expression with Crohn-like lymphoid reaction, peritumoral lymphocytic reaction, intratumoral periglandular reaction, or tumor-infiltrating lymphocytes, controlling for potential confounders, including microsatellite instability, CpG island methylator phenotype, long-interspersed nucleotide element-1 methylation, and KRAS, BRAF, and PIK3CA mutations. Tumor PDCD1LG2 expression was inversely associated with Crohn-like lymphoid reaction (Ptrend = 0.0003). For a unit increase in the three-tiered ordinal categories of Crohn-like lymphoid reaction, a multivariable OR in the highest (vs. lowest) quartile of the percentage of PDCD1LG2–expressing tumor cells was 0.38 (95% confidence interval, 0.22–0.67). Tumor PDCD1LG2 expression was not associated with peritumoral lymphocytic reaction, intratumoral periglandular reaction, tumor-infiltrating lymphocytes, or patient survival (Ptrend > 0.13). Thus, tumor PDCD1LG2 expression is inversely associated with Crohn-like lymphoid reaction to colorectal cancer, suggesting a possible role of PDCD1LG2-expressing tumor cells in inhibiting the development of tertiary lymphoid tissues during colorectal carcinogenesis. Cancer Immunol Res; 5(11); 1046–55. ©2017 AACR.

Association of Alterations in Main Driver Genes With Outcomes of Patients With Resected Pancreatic Ductal Adenocarcinoma

Importance Although patients with resected pancreatic adenocarcinoma are at high risk for disease recurrence, few biomarkers are available to inform patient outcomes. Objective To evaluate the alterations of the 4 main driver genes in pancreatic adenocarcinoma and patient outcomes after cancer resection. Design, Setting, and Participants This study analyzed protein expression and DNA alterations for the KRAS, CDKN2A, SMAD4, and TP53 genes by immunohistochemistry and next-generation sequencing in formalin-fixed, paraffin-embedded tumors in 356 patients with resected pancreatic adenocarcinoma who were treated at the Dana-Farber/Brigham and Women’s Cancer Center (October 26, 2002, to May 21, 2012), University of Rochester Medical Center (March 1, 2006, to November 1, 2013), or Stanford Cancer Institute (September 26, 1995, to May 22, 2013). Associations of driver gene alterations with disease-free survival (DFS) and overall survival (OS) were evaluated using Cox proportional hazards regression with estimation of hazard ratios (HRs) and 95% CIs and adjustment for age, sex, tumor characteristics, institution, and perioperative treatment. Data were collected September 9, 2012, to June 28, 2016, and analyzed December 17, 2016, to March 14, 2017. Main Outcomes and Measures The DFS and OS among patients with resected pancreatic adenocarcinoma. Results Of the 356 patients studied, 191 (53.7%) were men and 165 (46.3%) were women, with a median (interquartile range [IQR]) age of 67 (59.0-73.5) years. Patients with KRAS mutant tumors had worse DFS (median [IQR], 12.3 [6.7 -27.2] months) and OS (20.3 [11.3-38.3] months) compared with patients with KRAS wild-type tumors (DFS, 16.2 [8.9-30.5] months; OS, 38.6 [16.6-63.1] months) and had 5-year OS of 13.0% vs 30.2%. Particularly poor outcomes were identified in patients with KRAS G12D-mutant tumors, who had a median (IQR) OS of 15.3 (9.8-32.7) months. Patients whose tumors lacked CDKN2A expression had worse DFS (median, 11.5 [IQR, 6.2-24.5] months) and OS (19.7 [10.9-37.1] months) compared with patients who had intact CDKN2A (DFS, 14.8 [8.2-30.5] months; OS, 24.6 [14.1-44.6] months). The molecular status of SMAD4 was not associated with DFS or OS, whereas TP53 status was associated only with shorter DFS (HR, 1.33; 95% CI, 1.02-1.75; P = .04). Patients had worse DFS and OS if they had a greater number of altered driver genes. Compared with patients with 0 to 2 altered genes, those with 4 altered genes had worse DFS (HR, 1.79 [95% CI, 1.24-2.59; P = .002]) and OS (HR, 1.38 [95% CI, 0.98-1.94; P = .06]). Five-year OS was 18.4% for patients with 0 to 2 gene alterations, 14.1% for those with 3 alterations, and 8.2% for those with 4 alterations. Conclusions and Relevance Patient outcomes are associated with alterations of the 4 main driver genes in resected pancreatic adenocarcinoma.

Concomitant detection of hematological neoplasm and carcinoma: report on seven cases.

CONTEXT The presence of multiple neoplasms in one patient is an uncommon event. Its occurrence ranges from 1.2% to 4.5% of cancer patients in autopsy and clinical studies. In the present article, cases of synchronous diagnoses of carcinoma and lymphoid neoplasms are reported. The intention of this report was to alert clinicians and pathologists to the possibility of the existence of concomitant neoplasms, in order to prevent inaccurate or delayed diagnosis and staging. CASES Seven patients (four female and three male) with a median age of 61.4 years were diagnosed as having concomitant epithelial and hematological neoplasms. DISCUSSION AND CONCLUSION Lymph nodes should be carefully examined when searching for metastases, because of the possibility of a second hematological malignancy. Whenever uncommon suspicious morphological features are seen in such neoplasms, an immunohistochemical analysis is essential.