Multifocal endometriotic lesions associated with cancer are clonal and carry a high mutation burden.

Endometriosis is a significant risk factor for clear cell and endometrioid ovarian cancers and is often found contiguous with these cancers. Using whole-genome shotgun sequencing of seven clear cell ovarian carcinomas (CCC) and targeted sequencing in synchronous endometriosis, we have investigated how this carcinoma may evolve from endometriosis. In every case we observed multiple tumour-associated somatic mutations in at least one concurrent endometriotic lesion. ARID1A and PIK3CA mutations appeared consistently in concurrent endometriosis when present in the primary CCC. In several cases, one or more endometriotic lesions carried the near-complete complement of somatic mutations present in the index CCC tumour. Ancestral mutations were detected in both tumour-adjacent and -distant endometriotic lesions, regardless of any cytological atypia. These findings provide objective evidence that multifocal benign endometriotic lesions are clonally related and that CCCs arising in these patients progress from endometriotic lesions that may already carry sufficient cancer-associated mutations to be considered neoplasms themselves, albeit with low malignant potential. We speculate that genomically distinct classes of endometriosis exist and that ovarian endometriosis with high mutational burden represents one class at high risk for malignant transformation.

Mismatch repair status may predict response to adjuvant chemotherapy in resectable pancreatic ductal adenocarcinoma.

Deficiencies in DNA mismatch repair have been associated with inferior response to 5-FU in colorectal cancer. Pancreatic ductal adenocarcinoma is similarly treated with pyrimidine analogs, yet the predictive value of mismatch repair status for response to these agents has not been examined in this malignancy. A tissue microarray with associated clinical outcome, comprising 254 resected pancreatic ductal adenocarcinoma patients was stained for four mismatch repair proteins (MLH1, MSH2, MSH6 and PMS2). Mismatch repair deficiency and proficiency was determined by the absence or presence of uniform nuclear staining in tumor cells, respectively. Cases identified as mismatch repair deficient on the tissue microarray were confirmed by immunohistochemistry on whole slide sections. Of the 265 cases, 78 (29%) received adjuvant treatment with a pyrimidine analog and 41 (15%) showed a mismatch repair-deficient immunoprofile. Multivariable disease-specific survival in the mismatch repair-proficient cohort demonstrated that adjuvant chemotherapy, regional lymph-node status, gender, and the presence of tumor budding were significant independent prognostic variables (P≤0.04); however, none of the eight clinico-pathologic covariates examined in the mismatch repair-deficient cohort were of independent prognostic significance. Univariable assessment of disease-specific survival revealed an almost identical survival profile for both treated and untreated patients with a mismatch repair-deficient profile, while treatment in the mismatch repair-proficient cohort conferred a greater than 10-month median disease-specific survival advantage over their untreated counterparts (P=0.0018). In this cohort, adjuvant chemotherapy with a pyrimidine analog conferred no survival advantage to mismatch repair-deficient pancreatic ductal adenocarcinoma patients. Mismatch repair immunoprofiling is a feasible predictive marker in pancreatic ductal adenocarcinoma patients, and further prospective evaluation of this finding is warranted.

Retrospective review using targeted deep sequencing reveals mutational differences between gastroesophageal junction and gastric carcinomas.

BACKGROUND: Adenocarcinomas of both the gastroesophageal junction and stomach are molecularly complex, but differ with respect to epidemiology, etiology and survival. There are few data directly comparing the frequencies of single nucleotide mutations in cancer-related genes between the two sites. Sequencing of targeted gene panels may be useful in uncovering multiple genomic aberrations using a single test. METHODS: DNA from 92 gastroesophageal junction and 75 gastric adenocarcinoma resection specimens was extracted from formalin-fixed paraffin-embedded tissue. Targeted deep sequencing of 46 cancer-related genes was performed through emulsion PCR followed by semiconductor-based sequencing. Gastroesophageal junction and gastric carcinomas were contrasted with respect to mutational profiles, immunohistochemistry and in situ hybridization, as well as corresponding clinicopathologic data. RESULTS: Gastroesophageal junction carcinomas were associated with younger age, more frequent intestinal-type histology, more frequent p53 overexpression, and worse disease-free survival on multivariable analysis. Among all cases, 145 mutations were detected in 31 genes. TP53 mutations were the most common abnormality detected, and were more common in gastroesophageal junction carcinomas (42% vs. 27%, p = 0.036). Mutations in the Wnt pathway components APC and CTNNB1 were more common among gastric carcinomas (16% vs. 3%, p = 0.006), and gastric carcinomas were more likely to have ≥3 driver mutations detected (11% vs. 2%, p = 0.044). Twenty percent of cases had potentially actionable mutations identified. R132H and R132C missense mutations in the IDH1 gene were observed, and are the first reported mutations of their kind in gastric carcinoma. CONCLUSIONS: Panel sequencing of routine pathology material can yield mutational information on several driver genes, including some for which targeted therapies are available. Differing rates of mutations and clinicopathologic differences support a distinction between adenocarcinomas that arise in the gastroesophageal junction and those that arise in the stomach proper.

Venous invasion in oesophageal adenocarcinoma: enhanced detection using elastic stain and association with adverse histological features and clinical outcomes.

AIMS: In oesophageal adenocarcinoma, detection rates of venous invasion using haematoxylin and eosin (H&E) and elastic stains have not been compared. The aims of this study were to investigate whether or not elastic stains facilitate the detection of venous invasion, and to determine the prognostic significance of venous invasion following review with elastic stains. METHODS AND RESULTS: One hundred and three resection specimens containing oesophageal adenocarcinoma, all reported originally as negative for venous invasion, were examined for the presence of venous invasion using H&E and subsequently Movat pentachrome stains. Venous invasion was detected in eight cases with H&E and an additional 66 cases using Movat pentachrome; overall, 72% of cases contained venous invasion. Venous invasion was associated with advanced stage, tumour size, lymphatic and perineural invasion and subsequent distant metastases. Venous invasion, stage, size, grade, lymphatic invasion and perineural invasion were prognostically significant on univariate analysis. Only tumour stage was independently prognostic. Two of eight patients with venous invasion but no other indication for adjuvant treatment died of recurrent disease. CONCLUSIONS: Elastic stains improve detection of venous invasion significantly in oesophageal adenocarcinoma. Venous invasion is associated with multiple adverse clinicopathological features. Its identification may facilitate the stratification of patients at risk for visceral metastases and disease-related death.

Feasibility and Performance of Elastin Trichrome as a Primary Stain in Colorectal Cancer Resection Specimens: Results of an Interobserver Variability Study.

Venous invasion (VI) is a powerful prognostic factor in colorectal cancer (CRC) that is widely underreported. The ability of elastin stains to improve VI detection is now recognized in several international CRC pathology protocols. However, concerns related to the cost and time required to perform and evaluate these stains in addition to routine hematoxylin and eosin (H&E) stains remains a barrier to their wider use. We therefore sought to determine whether an elastin trichrome (ET) stain could be used as a "stand-alone" stain in CRC resections, by comparing the sensitivity, accuracy, and reproducibility of detection of CAP-mandated prognostic factors using ET and H&E stains. Representative H&E- and ET-stained slides from 50 CRC resections, including a representative mix of stages and prognostic factors, were used to generate 2 study sets. Each case was represented by H&E slides in 1 study set and by corresponding ET slides from the same blocks in the other study set. Ten observers (3 academic gastrointestinal [GI] pathologists, 4 community pathologists, 3 fellows) evaluated each study set for CAP-mandated prognostic factors. ET outperformed H&E in the assessment of VI with respect to detection rates (50% vs. 28.6%; P<0.0001), accuracy (82% vs. 59%, P<0.0001), and reproducibility (k=0.554 vs. 0.394). No significant differences between ET and H&E were observed for other features evaluated. In a poststudy survey, most observers considered the ease and speed of assessment at least equivalent for ET and H&E for most prognostic factors, and felt that ET would be feasible as a stand-alone stain in practice. If validated by others, our findings support the use of ET, rather than H&E, as the primary stain for the evaluation of CRC resections.

A structured latent model for ovarian carcinoma subtyping from histopathology slides.

Accurate subtyping of ovarian carcinomas is an increasingly critical and often challenging diagnostic process. This work focuses on the development of an automatic classification model for ovarian carcinoma subtyping. Specifically, we present a novel clinically inspired contextual model for histopathology image subtyping of ovarian carcinomas. A whole slide image is modelled using a collection of tissue patches extracted at multiple magnifications. An efficient and effective feature learning strategy is used for feature representation of a tissue patch. The locations of salient, discriminative tissue regions are treated as latent variables allowing the model to explicitly ignore portions of the large tissue section that are unimportant for classification. These latent variables are considered in a structured formulation to model the contextual information represented from the multi-magnification analysis of tissues. A novel, structured latent support vector machine formulation is defined and used to combine information from multiple magnifications while simultaneously operating within the latent variable framework. The structural and contextual nature of our method addresses the challenges of intra-class variation and pathologists' workload, which are prevalent in histopathology image classification. Extensive experiments on a dataset of 133 patients demonstrate the efficacy and accuracy of the proposed method against state-of-the-art approaches for histopathology image classification. We achieve an average multi-class classification accuracy of 90%, outperforming existing works while obtaining substantial agreement with six clinicians tested on the same dataset.

Genomic consequences of aberrant DNA repair mechanisms stratify ovarian cancer histotypes.

We studied the whole-genome point mutation and structural variation patterns of 133 tumors (59 high-grade serous (HGSC), 35 clear cell (CCOC), 29 endometrioid (ENOC), and 10 adult granulosa cell (GCT)) as a substrate for class discovery in ovarian cancer. Ab initio clustering of integrated point mutation and structural variation signatures identified seven subgroups both between and within histotypes. Prevalence of foldback inversions identified a prognostically significant HGSC group associated with inferior survival. This finding was recapitulated in two independent cohorts (n = 576 cases), transcending BRCA1 and BRCA2 mutation and gene expression features of HGSC. CCOC cancers grouped according to APOBEC deamination (26%) and age-related mutational signatures (40%). ENOCs were divided by cases with microsatellite instability (28%), with a distinct mismatch-repair mutation signature. Taken together, our work establishes the potency of the somatic genome, reflective of diverse DNA repair deficiencies, to stratify ovarian cancers into distinct biological strata within the major histotypes.

Clinically-inspired automatic classification of ovarian carcinoma subtypes.

CONTEXT: It has been shown that ovarian carcinoma subtypes are distinct pathologic entities with differing prognostic and therapeutic implications. Histotyping by pathologists has good reproducibility, but occasional cases are challenging and require immunohistochemistry and subspecialty consultation. Motivated by the need for more accurate and reproducible diagnoses and to facilitate pathologists' workflow, we propose an automatic framework for ovarian carcinoma classification. MATERIALS AND METHODS: Our method is inspired by pathologists' workflow. We analyse imaged tissues at two magnification levels and extract clinically-inspired color, texture, and segmentation-based shape descriptors using image-processing methods. We propose a carefully designed machine learning technique composed of four modules: A dissimilarity matrix, dimensionality reduction, feature selection and a support vector machine classifier to separate the five ovarian carcinoma subtypes using the extracted features. RESULTS: This paper presents the details of our implementation and its validation on a clinically derived dataset of eighty high-resolution histopathology images. The proposed system achieved a multiclass classification accuracy of 95.0% when classifying unseen tissues. Assessment of the classifier's confusion (confusion matrix) between the five different ovarian carcinoma subtypes agrees with clinician's confusion and reflects the difficulty in diagnosing endometrioid and serous carcinomas. CONCLUSIONS: Our results from this first study highlight the difficulty of ovarian carcinoma diagnosis which originate from the intrinsic class-imbalance observed among subtypes and suggest that the automatic analysis of ovarian carcinoma subtypes could be valuable to clinician's diagnostic procedure by providing a second opinion.

Synchronous Endometrial and Ovarian Carcinomas: Evidence of Clonality.

Many women with ovarian endometrioid carcinoma present with concurrent endometrial carcinoma. Organ-confined and low-grade synchronous endometrial and ovarian tumors (SEOs) clinically behave as independent primary tumors rather than a single advanced-stage carcinoma. We used 18 SEOs to investigate the ancestral relationship between the endometrial and ovarian components. Based on both targeted and exome sequencing, 17 of 18 patient cases of simultaneous cancer of the endometrium and ovary from our series showed evidence of a clonal relationship, ie, primary tumor and metastasis. Eleven patient cases fulfilled clinicopathological criteria that would lead to classification as independent endometrial and ovarian primary carcinomas, including being of FIGO stage T1a/1A, with organ-restricted growth and without surface involvement; 10 of 11 of these cases showed evidence of clonality. Our observations suggest that the disseminating cells amongst SEOs are restricted to physically accessible and microenvironment-compatible sites yet remain indolent, without the capacity for further dissemination.

Gastrointestinal Endoscopic Ultrasound-Guided Fine-Needle Aspiration Biopsy Specimens: Adequate Diagnostic Yield and Accuracy Can Be Achieved without On-Site Evaluation.

BACKGROUND: Endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNA) is the preferred method for biopsying the gastrointestinal tract, and rapid on-site cytological evaluation is considered standard practice. Our institution does not perform on-site evaluation; this study analyzes our overall diagnostic yield, accuracy, and incidence of nondiagnostic cases to determine the validity of this strategy. DESIGN: Data encompassing clinical information, procedural records, and cytological assessment were analyzed for gastrointestinal EUS-FNA procedures (n = 85) performed at Vancouver General Hospital from January 2012 to January 2013. We compared our results with those of studies that had on-site evaluation and studies that did not have on-site evaluation. RESULTS: Eighty-five biopsies were performed in 78 patients, from sites that included the pancreas, the stomach, the duodenum, lymph nodes, and retroperitoneal masses. Malignancies were diagnosed in 45 (53%) biopsies, while 24 (29%) encompassed benign entities. Suspicious and atypical results were recorded in 8 (9%) and 6 (7%) cases, respectively. Only 2 (2%) cases received a cytological diagnosis of 'nondiagnostic'. Our overall accuracy was 72%, our diagnostic yield was 98%, and our nondiagnostic rate was 2%. Our results did not significantly differ from those of studies that did have on-site evaluation. CONCLUSION: Our study highlights that adequate diagnostic accuracy can be achieved without on-site evaluation.

Tumor budding is an independent adverse prognostic factor in pancreatic ductal adenocarcinoma.

Tumor budding is a well-established adverse prognostic factor in colorectal cancer. However, the significance and diagnostic reproducibility of budding in pancreatic carcinoma requires further study. We aimed to assess the prognostic significance of tumor budding in pancreatic ductal adenocarcinoma, determine its relationship with other clinicopathologic features, and assess interobserver variability in its diagnosis. Tumor budding was assessed in 192 archival cases of pancreatic ductal adenocarcinoma using hematoxylin and eosin (H&E) sections; tumor buds were defined as single cells or nonglandular clusters composed of <5 cells. The presence of budding was determined through assessment of all tumor-containing slides, and associations with clinicopathologic features and outcomes were analyzed. Six gastrointestinal pathologists participated in an interobserver variability study of 120 images of consecutive tumor slides stained with H&E and cytokeratin. Budding was present in 168 of 192 cases and was associated with decreased overall survival (P=0.001). On multivariable analysis, tumor budding was prognostically significantly independent of stage, grade, tumor size, nodal status, lymphovascular invasion, and perineural invasion. There was substantial agreement among pathologists in assessing the presence of tumor budding using both H&E (K=0.63) and cytokeratin (K=0.63) stains. The presence of tumor budding is an independent adverse prognostic factor in pancreatic ductal carcinoma. The assessment of budding with H&E is reliable and could be used to better risk stratify patients with pancreatic ductal adenocarcinoma.

Personalized oncogenomics: clinical experience with malignant peritoneal mesothelioma using whole genome sequencing.

Peritoneal mesothelioma is a rare and sometimes lethal malignancy that presents a clinical challenge for both diagnosis and management. Recent studies have led to a better understanding of the molecular biology of peritoneal mesothelioma. Translation of the emerging data into better treatments and outcome is needed. From two patients with peritoneal mesothelioma, we derived whole genome sequences, RNA expression profiles, and targeted deep sequencing data. Molecular data were made available for translation into a clinical treatment plan. Treatment responses and outcomes were later examined in the context of molecular findings. Molecular studies presented here provide the first reported whole genome sequences of peritoneal mesothelioma. Mutations in known mesothelioma-related genes NF2, CDKN2A, LATS2, amongst others, were identified. Activation of MET-related signaling pathways was demonstrated in both cases. A hypermutated phenotype was observed in one case (434 vs. 18 single nucleotide variants) and was associated with a favourable outcome despite sarcomatoid histology and multifocal disease. This study represents the first report of whole genome analyses of peritoneal mesothelioma, a key step in the understanding and treatment of this disease.

Hereditary Diffuse Gastric Cancer Syndrome: CDH1 Mutations and Beyond.

IMPORTANCE: E-cadherin (CDH1) is a cancer predisposition gene mutated in families meeting clinically defined hereditary diffuse gastric cancer (HDGC). Reliable estimates of cancer risk and spectrum in germline mutation carriers are essential for management. For families without CDH1 mutations, genetic-based risk stratification has not been possible, resulting in limited clinical options. OBJECTIVES: To derive accurate estimates of gastric and breast cancer risks in CDH1 mutation carriers and determine if germline mutations in other genes are associated with HDGC. DESIGN, SETTING, AND PARTICIPANTS: Testing for CDH1 germline mutations was performed on 183 index cases meeting clinical criteria for HDGC. Penetrance was derived from 75 mutation-positive families from within this and other cohorts, comprising 3858 probands (353 with gastric cancer and 89 with breast cancer). Germline DNA from 144 HDGC probands lacking CDH1 mutations was screened using multiplexed targeted sequencing for 55 cancer-associated genes. MAIN OUTCOMES AND MEASURES: Accurate estimates of gastric and breast cancer risks in CDH1 mutation carriers and the relative contribution of other cancer predisposition genes in familial gastric cancers. RESULTS: Thirty-one distinct pathogenic CDH1 mutations (14 novel) were identified in 34 of 183 index cases (19%). By the age of 80 years, the cumulative incidence of gastric cancer was 70% (95% CI, 59%-80%) for males and 56% (95% CI, 44%-69%) for females, and the risk of breast cancer for females was 42% (95% CI, 23%-68%). In CDH1 mutation-negative index cases, candidate mutations were identified in 16 of 144 probands (11%), including mutations within genes of high and moderate penetrance: CTNNA1, BRCA2, STK11, SDHB, PRSS1, ATM, MSR1, and PALB2. CONCLUSIONS AND RELEVANCE: This is the largest reported series of CDH1 mutation carriers, providing more precise estimates of age-associated risks of gastric and breast cancer that will improve counseling of unaffected carriers. In HDGC families lacking CDH1 mutations, testing of CTNNA1 and other tumor suppressor genes should be considered. Clinically defined HDGC families can harbor mutations in genes (ie, BRCA2) with different clinical ramifications from CDH1. Therefore, we propose that HDGC syndrome may be best defined by mutations in CDH1 and closely related genes, rather than through clinical criteria that capture families with heterogeneous susceptibility profiles.

HER2/neu testing in gastric cancer by immunohistochemistry: assessment of interlaboratory variation.

CONTEXT: Immunohistochemical (IHC) testing for HER2/neu is becoming the standard of care for guiding adjuvant treatment of gastric carcinoma with trastuzumab. OBJECTIVE: To assess interlaboratory variation in IHC staining and interpretation across multiple laboratories. DESIGN: A tissue microarray consisting of 45 cores from 28 gastric cancers was distributed to 37 laboratories for HER2/neu assessment. The IHC results were compared against expert scores at an academic institution and correlated with in situ hybridization results from the originating specimen. Interlaboratory agreement was calculated using Cohen κ statistic. RESULTS: The survey demonstrated several variations in IHC methods, including the primary antibodies in use. There was excellent agreement among laboratories in HER2/neu(+) (IHC 3(+)) cases (κ = 0.80 ± 0.01) and very good agreement among laboratories in HER2/neu(-) (IHC 0 or 1(+)) cases (κ = 0.58 ± 0.01). Less agreement was observed among laboratories when scoring equivocal (IHC 2(+)) cases (κ = 0.22 ± 0.01). Sensitivity and specificity of HER2/neu IHC were 99% and 100%, respectively, when measured against expert review and consensus score as a reference standard. CONCLUSIONS: There is substantial interlaboratory agreement in the interpretation of HER2/neu IHC despite variability in protocols. Although HER2/neu IHC is a highly sensitive and specific test, primary antibody selection may significantly affect IHC results. Furthermore, gastric tumors require a unique scoring system and expertise in interpretation. Intratumoral heterogeneity has a significant effect on HER2/neu scoring by IHC. Ongoing quality assurance exercises among laboratories will help ensure optimized HER2/neu testing.

Interobserver variability in assessing dysplasia and architecture in colorectal adenomas: a multicentre Canadian study.

AIMS: Following the introduction of colorectal cancer screening programmes throughout Canada, it became necessary to standardise the diagnosis of colorectal adenomas. Canadian guidelines for standardised reporting of adenomas were developed in 2011. The aims of the present study were (a) to assess interobserver variability in the classification of dysplasia and architecture in adenomas and (b) to determine if interobserver variability could be improved by the adoption of criteria specified in the national guidelines. METHODS: An a priori power analysis was used to determine an adequate number of cases and participants. Twelve pathologists independently classified 40 whole-slide images of adenomas according to architecture and dysplasia grade. Following a wash-out period, participants were provided with the national guidelines and asked to reclassify the study set. RESULTS: At baseline, there was moderate interobserver agreement for architecture (K=0.4700; 95% CI 0.4427 to 0.4972) and dysplasia grade (K=0.5680; 95% CI 0.5299 to 0.6062). Following distribution of the guidelines, there was improved interobserver agreement in assessing architecture (K=0.5403; 95% CI 0.5133 to 0.5674)). For dysplasia grade, overall interobserver agreement remained moderate but decreased significantly (K=0.4833; 95% CI 0.4452 to 0.5215). Half of the cases contained high-grade dysplasia (HGD). Two pathologists diagnosed HGD in ≥75% of cases. CONCLUSIONS: The improvement in interobserver agreement in classifying adenoma architecture suggests that national guidelines can be useful in disseminating knowledge, however, the variability in the diagnosis of HGD, even following guideline review suggests the need for ongoing knowledge-transfer exercises.

ARID1A/BAF250a as a prognostic marker for gastric carcinoma: a study of 2 cohorts.

ARID1A/BAF250a has been recently implicated as a tumor suppressor in gastric cancer. We sought to clarify the clinical significance of BAF250a/ARID1A in relation to other clinical parameters and relevant biomarkers in gastric carcinoma. Cases from 2 separate cohorts of patients with gastric carcinoma from Vancouver (n = 173) and Toronto (n = 80) were selected for the construction of tissue microarrays, which were used to assess the immunohistochemical status of BAF250a (anti-ARID1A), mismatch repair proteins and p53, as well as in situ hybridization for HER2 amplification and Epstein-Barr virus infection. The Toronto cohort contained a higher proportion of early stage cases (P = .019) and a smaller proportion of cases from the proximal stomach (P < .001). Overall, immunohistochemical loss of BAF250a was observed in 22.5% of gastric adenocarcinomas from the Vancouver group and 20% from Toronto. In both cohorts, loss of BAF250a was positively associated with loss of mismatch repair protein expression (P < .0001 and P = .035, respectively). Loss of BAF250a expression was independently associated with poor overall survival in the Toronto cohort (P = .0015), whereas no significant association with survival was observed in the Vancouver cohort. BAF250a loss was not significantly associated with any additional clinical parameters in either cohort. HER2 amplification was confirmed as a negative prognostic factor in both cohorts. These findings suggest that ARID1A/BAF250a may be of prognostic significance in a subset of patients with early stage gastric cancer and that pathological assessment should increasingly use a multimarker approach.

A rare case of carcinoma cuniculatum of the penis in a 55-year-old.

Carcinoma cuniculatum of the penis is an extremely rare variant of squamous cell carcinoma characterized by an endophytic deeply branching and burrowing growth pattern. One documented case series demonstrated afflicted patients ranging in age from 73-83 years with the tumour located on the glans penis, coronal sulcus or foreskin. We report a case of a 55-year-old with disease located on the ventral aspect of the shaft of the penis. The tumour was invasive into the deep dermal connective tissue, comparatively superficial to all previous documented cases. He subsequently underwent a partial penectomy. The case is discussed with a brief review of the literature.