Graham, Rondell P

AimsMucinous cystic neoplasms (MCN) are defined by the presence of an ovarian‐type stroma (OTS). Once ovarian stroma has become a requirement for the diagnosis of MCNs, studies using this criterion have disclosed that MCNs are seen almost exclusively (97%) in women. The occurrence of MCNs (with ovarian stroma) in men is exceedingly rare and raises questions about the origin of OTS and the role of hormones in tumourigenesis. This study aims to investigate the clinical and histopathological features of pancreatic or hepatic MCNs in men.Methods and resultsWe examined the MCN cases in men and compared them with age‐matched women. We further investigated MCN in premenopausal and postmenopausal women to explore the impact of hormonal status.The stromal cellularity tended to be lower in men as compared to women; however, limited numbers prevented statistical significance. Clinical presentation, tumour location, body mass index and cyst complexity were similar between men and age‐matched women, similarly in pre‐ and postmenopausal woman groups. All MCN cases, regardless of gender or hormonal status, showed OTS with positivity for oestrogen receptor (ER), androgen receptor (AR) and SF‐1 immunostains. Additionally, the presence of AR and ER‐beta in lining epithelial cells, in addition to the stromal cells, raised the probability for a potential role for local hormonal signalling in the pathogenesis of these tumours.ConclusionsMCNs in men had overlapping histopathologic and immunohistochemical profiles with those in women, regardless of menopausal status.

ABSTRACT EPHA2 is a receptor tyrosine kinase highly expressed in many cancers. By analyzing cancer patient databases for mutations in the EPHA2 coding sequence, we found that cholangiocarcinoma (a hepatobiliary cancer with dismal prognosis) exhibits a uniquely high incidence of EPHA2 mutations. To deUine the functional signiUicance of these mutations, we generated representative EPHA2 mutants and monitored major receptor autophosphorylation sites as indicative of kinase activity-dependent signal transduction (known as forward signaling). We found that missense mutations in the ligand-binding domain abrogate ligand binding and ligand-induced EPHA2 tyrosine phosphorylation, while most missense mutations in the kinase domain abrogate kinase activity. We detected less pronounced effects of missense mutations in other domains, which vary depending on the phosphosite, suggesting that these EPHA2 mutations might differentially affect (or bias) different downstream signaling pathways. Other EPHA2 mutations introduce early stop codons and encode receptor truncated forms lacking all or part of the kinase domain. We also found that an EPHA2 secreted truncated form, a transmembrane truncated form, and a full-length kinase inactive mutant can inhibit tyrosine phosphorylation of co-expressed EPHA2 wild-type. Taken together, these data suggest that mutations interfering with EPHA2 forward signaling facilitate cholangiocarcinoma development. We indeed obtained evidence that an EPHA2 kinase inactive mutant, but not EPHA2 wild-type, can induce proliferative masses consistent with well differentiated cholangiocarcinoma in a validated mouse model of cholangiocarcinogenesis. Taken together, our Uindings suggest that EPHA2 is a driver gene in cholangiocarcinoma and that its forward signaling has tumor suppressor activity.

Abstract Purpose: Digital proteomic profiling was performed to identify spatial context in relationship to patient response and survival after anti–PD-1 therapy in metastatic colorectal cancer. Experimental Design: Primary colorectal cancers with deficient mismatch repair from patients treated with anti–PD-1 antibodies were analyzed (N = 30) using digital spatial profiling (GeoMx nCounter). At the invasive margin, 71 proteins were profiled in 10 regions of interest/slide that were segmented into 3 compartments labeled with pan-cytokeratin (epithelia), CD45 (stromal cells), and SYTO13 (nuclei). In an independent cohort (n = 13), digital spatial profiling data and single-cell transcriptomic data were analyzed. Differential protein abundance, after Benjamini–Hochberg correction, was examined by clinical response and progression-free survival (PFS) using multivariable Cox regression. Results: Protein abundance varied significantly between epithelial and stromal compartments. Nonresponders to anti–PD-1 showed higher fibronectin and smooth muscle actin abundance in the epithelial compartment that was associated with significantly shorter PFS (adjusted HR: 6.49 and 4.52, respectively; P < 0.05). In CD45+ stroma, increased expression of proteins related to T cells (CD3 and CD4), NK cells (CD56), antigen presentation (CD40), immune activation (CD27, ICOS), and apoptosis (GZMA) were found in responders (vs nonresponders) to anti–PD-1; each marker was significantly associated with longer patient PFS (0.02 < adjusted HR < 0.17; P < 0.05). In a separate cohort, consistent results by compartment were found for fibronectin and CD56. Gene expression data revealed that fibronectin and smooth muscle actin were primarily derived from cancer-associated fibroblasts. Conclusions: Spatially resolved protein profiles within microenvironments of deficient mismatch repair colorectal cancers can influence patient response and survival after anti–PD-1, highlighting their potential clinical significance.

AimsWe compiled two cohorts of colorectal adenosquamous carcinoma (ASC) to describe its histologic and molecular aspects using modern parameters to compare them with literature reports using meta‐analysis of cohorts and individual case series.Methods and ResultsWe identified 53 colorectal ASC from 19 North American academic medical centres, in addition to national database reports on 94 Dutch cases. We analysed available clinical, histologic, and immunohistochemical features and patient outcome. ASC comprised 0.02% of colorectal cancers in the Dutch database. The median cohort patient ages at resection were 65 and 69 years (North American and Dutch cohorts, respectively), with a roughly equal male:female ratio. The squamous component represented between 5% and 95% of each tumour, with a median of 50%. Tumour‐infiltrating lymphocytes (TILs) were generally low (66%), and tumour budding was often Bd1 (64%). Most cases were pT3 (55%) or pT4 (40%), with nodal metastases in more than half (58%). Twenty‐three cases (43%) metastasized distantly, most commonly to the liver. Mismatch repair (MMR) deficiency was identified in 34% of the cases. Follow‐up was available for 48 patients; 13 (27%) had recurrent disease and 29 (60%) died. A total of 31 patients progressed, with median time to progression of 18 months. Available data for the Dutch cohort revealed largely similar findings, as did review of cases in the literature.ConclusionColorectal ASC usually presents at an advanced stage. Despite high rates of MMR deficiency and low tumour budding, TILs were generally low, and there is a high recurrence rate and poor prognosis.

Aims. To assess the utility of C4d immunohistochemistry for esophageal pemphigus vulgaris. Methods and results. We searched for patients with a history of esophageal pemphigus vulgaris who had esophageal biopsies for routine hematoxylin and eosin (H&E) staining. A total of 8 biopsies from 7 patients were available. We also identified 18 non-pemphigus esophageal biopsies for controls. C4d immunohistochemistry was performed on each biopsy. Five of 6 (83%) biopsies with classic pemphigus vulgaris histologic findings were positive for intercellular staining at the basal layer. The negative biopsy was in a patient that had recently received high-dose corticosteroid treatment for a flare. Two biopsies with atypical histologic features for pemphigus vulgaris had negative C4d staining but positive direct immunofluorescence (DIF) studies. Various nonspecific C4d staining patterns were observed in the controls, but none showed the intercellular staining pattern that was observed in pemphigus vulgaris. Conclusions. Suprabasal clefting with acantholysis and “tombstone effect” are described histologic features of pemphigus vulgaris on H&E. However, procedural artifact may mimic these findings. Currently, the gold standard for pemphigus vulgaris is DIF, which is not always available because it cannot routinely be performed on formalin-fixed paraffin embedded tissue. Our study shows that C4d immunohistochemistry may be a useful adjunct in evaluating esophageal pemphigus vulgaris.

Abstract Aberrant activation of GLI transcription factors has been implicated in the pathogenesis of different tumor types including pancreatic ductal adenocarcinoma. However, the mechanistic link with established drivers of this disease remains in part elusive. In this study, using a new genetically engineered mouse model overexpressing constitutively active mouse form of GLI2 and a combination of genome-wide assays, we provide evidence of a novel mechanism underlying the interplay between KRAS, a major driver of pancreatic ductal adenocarcinoma development, and GLI2 to control oncogenic gene expression. These mice, also expressing KrasG12D, show significantly reduced median survival rate and accelerated tumorigenesis compared with the KrasG12D only expressing mice. Analysis of the mechanism using RNA sequencing demonstrate higher levels of GLI2 targets, particularly tumor growth–promoting genes, including Ccnd1, N-Myc, and Bcl2, in KrasG12D mutant cells. Furthermore, chromatin immunoprecipitation sequencing studies showed that in these cells KrasG12D increases the levels of trimethylation of lysine 4 of the histone 3 (H3K4me3) at the promoter of GLI2 targets without affecting significantly the levels of other major active chromatin marks. Importantly, Gli2 knockdown reduces H3K4me3 enrichment and gene expression induced by mutant Kras. In summary, we demonstrate that Gli2 plays a significant role in pancreatic carcinogenesis by acting as a downstream effector of KrasG12D to control gene expression.

Abstract Deep learning may detect biologically important signals embedded in tumor morphologic features that confer distinct prognoses. Tumor morphologic features were quantified to enhance patient risk stratification within DNA mismatch repair (MMR) groups using deep learning. Using a quantitative segmentation algorithm (QuantCRC) that identifies 15 distinct morphologic features, we analyzed 402 resected stage III colon carcinomas [191 deficient (d)-MMR; 189 proficient (p)-MMR] from participants in a phase III trial of FOLFOX-based adjuvant chemotherapy. Results were validated in an independent cohort (176 d-MMR; 1,094 p-MMR). Association of morphologic features with clinicopathologic variables, MMR, KRAS, BRAFV600E, and time-to-recurrence (TTR) was determined. Multivariable Cox proportional hazards models were developed to predict TTR. Tumor morphologic features differed significantly by MMR status. Cancers with p-MMR had more immature desmoplastic stroma. Tumors with d-MMR had increased inflammatory stroma, epithelial tumor-infiltrating lymphocytes (TIL), high-grade histology, mucin, and signet ring cells. Stromal subtype did not differ by BRAFV600E or KRAS status. In p-MMR tumors, multivariable analysis identified tumor-stroma ratio (TSR) as the strongest feature associated with TTR [HRadj 2.02; 95% confidence interval (CI), 1.14–3.57; P = 0.018; 3-year recurrence: 40.2% vs. 20.4%; Q1 vs. Q2–4]. Among d-MMR tumors, extent of inflammatory stroma (continuous HRadj 0.98; 95% CI, 0.96–0.99; P = 0.028; 3-year recurrence: 13.3% vs. 33.4%, Q4 vs. Q1) and N stage were the most robust prognostically. Association of TSR with TTR was independently validated. In conclusion, QuantCRC can quantify morphologic differences within MMR groups in routine tumor sections to determine their relative contributions to patient prognosis, and may elucidate relevant pathophysiologic mechanisms driving prognosis. Significance: A deep learning algorithm can quantify tumor morphologic features that may reflect underlying mechanisms driving prognosis within MMR groups. TSR was the most robust morphologic feature associated with TTR in p-MMR colon cancers. Extent of inflammatory stroma and N stage were the strongest prognostic features in d-MMR tumors. TIL density was not independently prognostic in either MMR group.

Abstract Purpose: There is a need to improve current risk stratification of stage II colorectal cancer to better inform risk of recurrence and guide adjuvant chemotherapy. We sought to examine whether integration of QuantCRC, a digital pathology biomarker utilizing hematoxylin and eosin–stained slides, provides improved risk stratification over current American Society of Clinical Oncology (ASCO) guidelines. Experimental Design: ASCO and QuantCRC-integrated schemes were applied to a cohort of 398 mismatch-repair proficient (MMRP) stage II colorectal cancers from three large academic medical centers. The ASCO stage II scheme was taken from recent guidelines. The QuantCRC-integrated scheme utilized pT3 versus pT4 and a QuantCRC-derived risk classification. Evaluation of recurrence-free survival (RFS) according to these risk schemes was compared using the log-rank test and HR. Results: Integration of QuantCRC provides improved risk stratification compared with the ASCO scheme for stage II MMRP colorectal cancers. The QuantCRC-integrated scheme placed more stage II tumors in the low-risk group compared with the ASCO scheme (62.5% vs. 42.2%) without compromising excellent 3-year RFS. The QuantCRC-integrated scheme provided larger HR for both intermediate-risk (2.27; 95% CI, 1.32–3.91; P = 0.003) and high-risk (3.27; 95% CI, 1.42–7.55; P = 0.006) groups compared with ASCO intermediate-risk (1.58; 95% CI, 0.87–2.87; P = 0.1) and high-risk (2.24; 95% CI, 1.09–4.62; P = 0.03) groups. The QuantCRC-integrated risk groups remained prognostic in the subgroup of patients that did not receive any adjuvant chemotherapy. Conclusions: Incorporation of QuantCRC into risk stratification provides a powerful predictor of RFS that has potential to guide subsequent treatment and surveillance for stage II MMRP colorectal cancers.

Abstract Purpose: Targeting the programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) interaction has led to durable responses in fewer than half of patients with mismatch repair–deficient (MMR-d) advanced colorectal cancers. Immune contexture, including spatial distribution of immune cells in the tumor microenvironment (TME), may predict immunotherapy outcome. Experimental Design: Immune contexture and spatial distribution, including cell-to-cell distance measurements, were analyzed by multiplex immunofluorescence (mIF) in primary colorectal cancers with d-MMR (N = 33) from patients treated with anti–PD-1 antibodies. By digital image analysis, density, ratio, intensity, and spatial distribution of PD-L1, PD-1, CD8, CD3, CD68, LAG3, TGFβR2, MHC-I, CD14, B2M, and pan-cytokeratin were computed. Feature selection was performed by regularized Cox regression with LASSO, and a proportional hazards model was fitted to predict progression-free survival (PFS). Results: For predicting survival among patients with MMR-d advanced colorectal cancer receiving PD-1 blockade, cell-to-cell distance measurements, but not cell densities or ratios, achieved statistical significance univariately. By multivariable feature selection, only mean number of PD-1+ cells within 10 μm of a PD-L1+ cell was significantly predictive of PFS. Dichotomization of this variable revealed that those with high versus low values had significantly prolonged PFS [median not reached (>83 months) vs. 8.5 months (95% confidence interval (95% CI), 4.7–NR)] with a median PFS of 28.4 months for all patients [adjusted HR (HRadj) = 0.14; 95% CI, 0.04–0.56; P = 0.005]. Expression of PD-1 was observed on CD8+ T cells; PD-L1 on CD3+ and CD8+ T lymphocytes, macrophages (CD68+), and tumor cells. Conclusions: In d-MMR colorectal cancers, PD-1+ to PD-L1+ receptor to ligand proximity is a potential predictive biomarker for the effectiveness of PD-1 blockade.

Abstract A major hurdle to the application of precision oncology in pancreatic cancer is the lack of molecular stratification approaches and targeted therapy for defined molecular subtypes. In this work, we sought to gain further insight and identify molecular and epigenetic signatures of the Basal-like A pancreatic ductal adenocarcinoma (PDAC) subgroup that can be applied to clinical samples for patient stratification and/or therapy monitoring. We generated and integrated global gene expression and epigenome mapping data from patient-derived xenograft models to identify subtype-specific enhancer regions that were validated in patient-derived samples. In addition, complementary nascent transcription and chromatin topology (HiChIP) analyses revealed a Basal-like A subtype-specific transcribed enhancer program in PDAC characterized by enhancer RNA (eRNA) production that is associated with more frequent chromatin interactions and subtype-specific gene activation. Importantly, we successfully confirmed the validity of eRNA detection as a possible histologic approach for PDAC patient stratification by performing RNA-ISH analyses for subtype-specific eRNAs on pathologic tissue samples. Thus, this study provides proof-of-concept that subtype-specific epigenetic changes relevant for PDAC progression can be detected at a single-cell level in complex, heterogeneous, primary tumor material. Implications: Subtype-specific enhancer activity analysis via detection of eRNAs on a single-cell level in patient material can be used as a potential tool for treatment stratification.