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Significance: Triple negative breast carcinoma (TNBC), characterized by lack of estrogen (ER) and progesterone receptors (PR) and absence of Her2/neu (Her2) receptor amplification, typically follows an aggressive course that includes high relapse rates, rapid progression, and poor outcome. Advances in treatment have been limited by molecular heterogeneity within TNBCs and lack of effective molecular targets. Although a subset of TNBC associated with tumor infiltrating lymphocytes (TILS) is more immunogenic than other breast cancers, response to immunotherapy has been variable and reported in only 10-40% of cases. Identification of prognostic immune biomarkers will identify novel therapeutic targets and facilitate appropriate patient selection for therapy. Aim: To analyze the immune gene expression profile in TNBC and identify a prognostic marker. Methods: Targeted mRNA sequencing was performed on formalin fixed paraffin embedded whole sections from 30 treatment naïve TNBC with TILs (15 cases with a favorable outcome (recurrence free overall survival (OS) ≥5yrs) and 15 with unfavorable outcome (never disease free or dead of disease in < 5yrs) using the HTG Edge Seq Assay (HTG Molecular Diagnostics, Inc. Tucson AZ) to analyze the differential expression of 1392 immune related genes in their Precision Immuno-Oncology Panel. The top 40 up- and down- regulated DE genes with adjusted p-value of < 0.001 were then validated against the TNBC mRNA profiles in The Cancer Genome Atlas (TCGA) database. LAG3 was one of four genes that showed significant differential expression in both data sets. We used immunohistochemistry (IHC) (Rabbit monoclonal AntiLAG3 antibody, Abcam plc.) to analyze LAG3 expression in whole sections from 57 consecutive TNBCs where tumor excision had been the first line treatment. All patients were female, age 29-89 years (median 55 yrs). The infiltrating ductal carcinomas varied from 0.6 to 9.0 cm (median 2.6 cm). All tumors were ER-, PR-, Her2- with a median Ki67 of 42%. 26 patients had metastatic carcinoma in 1-17 lymph nodes (median 2). Two patients had metastatic disease at presentation. Disease free survival (DFS) varied from 0 to 114.8 months (median 81.4 mos.), and overall survival (OS) varied from 7.6 to 114.8 months (median 81.5 mos.). LAG3 expression was categorized as negative (0), low (1+) and high (2+). The student t-test was used to correlate LAG3 expression with DFS and OS. Results: Differential expression analysis from our data set yielded 222 statistically significant differentially expressed genes. Validation against the TCGA TNBC data set revealed 4 common genes, one of which is LAG3. Only TILS showed LAG3 expression by IHC: 10 cases no expression, 17 low expression and 29 high expression. Median DFS and OS in TNBC with no LAG3 expression was 32.3 and 55.2 months, in TNBC with low expression 83.3 and 81.1 and those with high expression was 82 and 82.1 month. Median DFS and OS in LAG3 negative cases was significantly different from those in LAG3 positive cases (p=0.038, p=0.013) and between LAG3 0 and LAG3 2+ positive cases (p=0.002, p=0.0018). No difference in either median DFS or OS was seen when LAG3 1+ and LAG3 2+ cases were compared. Conclusions: 1. LAG3 is a prognostic marker for TNBCs. 2. LAG3 is expressed only on TILS and shows heterogenous expression. 3. TNBC with no LAG3 expression had a median survival of 32 months, suggesting they might benefit from more aggressive therapy than TNBC that express LAG3. 4. Confirmation of these results requires a larger TNBC cohort. Citation Format: Shikha Bose, Yizhou Wang, V Krishnan Ramanujan, Ann E. Walts. LAG3+ Tumor Infiltrating Lymphocytes Predict Outcome in Treatment Naïve Triple Negative Breast Carcinoma [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-09.

Purpose: To elucidate molecular pathways contributing to metastatic cancer progression and poor clinical outcome in serous ovarian cancer.Experimental Design: Poor survival signatures from three different serous ovarian cancer datasets were compared and a common set of genes was identified. The predictive value of this gene signature was validated in independent datasets. The expression of the signature genes was evaluated in primary, metastatic, and/or recurrent cancers using quantitative PCR and in situ hybridization. Alterations in gene expression by TGF-β1 and functional consequences of loss of COL11A1 were evaluated using pharmacologic and knockdown approaches, respectively.Results: We identified and validated a 10-gene signature (AEBP1, COL11A1, COL5A1, COL6A2, LOX, POSTN, SNAI2, THBS2, TIMP3, and VCAN) that is associated with poor overall survival (OS) in patients with high-grade serous ovarian cancer. The signature genes encode extracellular matrix proteins involved in collagen remodeling. Expression of the signature genes is regulated by TGF-β1 signaling and is enriched in metastases in comparison with primary ovarian tumors. We demonstrate that levels of COL11A1, one of the signature genes, continuously increase during ovarian cancer disease progression, with the highest expression in recurrent metastases. Knockdown of COL11A1 decreases in vitro cell migration, invasion, and tumor progression in mice.Conclusion: Our findings suggest that collagen-remodeling genes regulated by TGF-β1 signaling promote metastasis and contribute to poor OS in patients with serous ovarian cancer. Our 10-gene signature has both predictive value and biologic relevance and thus may be useful as a therapeutic target. Clin Cancer Res; 20(3); 711–23. ©2013 AACR.

PDF file 831K, Supplementary Figures. Supplementary Fig. 1. A schematic diagram of the methods, datasets and patient samples used in this study. Supplementary Fig. 2. Correlogram of the 10 poor outcome. signature genes. Supplementary Fig. 3. Validation of the 10-gene signature - Kaplan-Meyer plot. Supplementary Fig. 4. Real-time PCR of poor outcome signature genes in normal, primary, and metastatic ovarian cancer samples. Supplementary Fig. 5. Increase in COL11A1 expression in recurrent tumors in comparison to primary ovarian tumors. Supplementary Fig. 6. Confinement of COL11A1 expression to intra- and peri-tumoral stroma - immunohistochemical detection. Supplementary Fig. 7. Knockdown of COL11A1 in the A2780 ovarian cancer cell line - qPCR and Western blot

XLS file 139K, Supplementary Tables. Supplementary Table 1A-C. Poor prognosis genes identified from 3 different datasets (A) TCGA, (B) GSE26712, (C) Karlan. Supplementary Table 2. Functional annotation of poor prognosis genes in three datasets. Supplementary Table 3. PCR primer sequences. Supplementary Table 4. Genes and corresponding probes used for Oncomine analysis of expression in primary tumors and metastases. Supplementary Table 5.Matched primary, metastatic and recurrent tumor samples

FOXC2 is a forkhead family transcription factor that plays a critical role in specifying mesenchymal cell fate during embryogenesis. FOXC2 expression is associated with increased metastasis and poor survival in various solid malignancies. Using in vitro and in vivo assays in mouse ovarian cancer cell lines, we confirmed the previously reported mechanisms by which FOXC2 could promote cancer growth, metastasis, and drug resistance, including epithelial-mesenchymal transition, stem cell-like differentiation, and resistance to anoikis. In addition, we showed that FOXC2 expression is associated with vasculogenic mimicry in mouse and human ovarian cancers. FOXC2 overexpression increased the ability of human ovarian cancer cells to form vascular-like structures in vitro, while inhibition of FOXC2 had the opposite effect. Thus, we present a novel mechanism by which FOXC2 might contribute to cancer aggressiveness and poor patient survival.

Sialylated HEG1 has been reported as a highly specific and sensitive mesothelioma marker but a comprehensive evaluation of its expression in carcinomas in different organs, various sarcomas and reactive mesothelial proliferations has not been reported. The aim of this study was to evaluate the clinical applicability of HEG1 as a marker in the diagnosis of mesothelioma. HEG1 immunoreactivity was evaluated in whole sections of 122 mesotheliomas, 75 pulmonary carcinomas, 55 other carcinomas, 16 mesenchymal tumors, and 24 reactive mesothelial proliferations and in tissue microarrays containing 70 epithelioid (EM), 36 biphasic (BM), and 2 sarcomatoid mesotheliomas (SM). In whole sections and tissue microarrays, respectively, membranous HEG1 was expressed in 93.0% and 85.5% of EM, 81.3% and 69.4% of BM, 0% and 0% of SM. HEG1 was not expressed in pulmonary adenocarcinomas. HEG1 was expressed as cytoplasmic immunoreactivity in pulmonary squamous cell carcinomas (21.7%). Membranous HEG1 staining was seen in ovarian carcinomas (66.7%), thyroid carcinomas (100%), reactive conditions (16.7%), and mesenchymal tumors (18.8%). The sensitivity of membranous HEG1 expression to distinguish EM/BM from all carcinomas was 88.8%. The specificity for the differential diagnosis between EM/BM and all carcinomas and pulmonary carcinomas was 92.3% and 98.7%, respectively.

Malignant peritoneal mesothelioma historically carried a grim prognosis, but outcomes have improved substantially in recent decades. The prognostic significance of clinical, morphologic, and immunophenotypic features remains ill-defined. This multi-institutional cohort comprises 225 malignant peritoneal mesotheliomas, which were assessed for 21 clinical, morphologic, and immunohistochemical parameters. For epithelioid mesotheliomas, combining nuclear pleomorphism and mitotic index yielded a composite nuclear grade, using a previously standardized grading system. Correlation of clinical, morphologic, and immunohistochemical parameters with overall and disease-free survival was examined by univariate and multivariate analyses. On univariate analysis, longer overall survival was significantly associated with diagnosis after 2000 (P = 0.0001), age

Aims To gather the best available evidence regarding Ki-67% values in large-cell neuroendocrine carcinoma (LCNEC) and determine whether certain cut-off values could serve as a prognostic feature in LCNEC. Methods and results Aperio ScanScope AT Turbo, eSlide Manager and ImageScope software (Leica Biosystems) were used to measure Ki-67% in 77 resected LCNEC diagnosed by World Health Organisation (WHO) criteria. Cases were stratified into six classes by 10% Ki-67 increments. Using the Kaplan-Meier method, overall (OS) and disease-free survivals (DFS) were compared by AJCC stage, by six Ki-67% classes and with Ki-67% cut-points ≥20% and ≥40%. Tumours were from 0.9 to 11.5 cm and pathological stages 1-3. The system measured Ki-67% positivity using 4072-44 533 tumour nuclei per case (mean 16610 ± 8039). Ki-67% ranged from 1 to 64% (mean = 26%; median = 26%). Only 16 (21%) tumours had Ki-67% ≥40%. OS ranged from 1 to 298 months (median follow-up = 25 months). DFS ranged from 1 to 276 months (median follow-up = 9 months). OS and DFS differed across AJCC stage (overall log-rank P = 0.038 and P = 0.037). However, neither OS nor DFS significantly correlated with Ki-67% when six or two classes were used with either ≥20% Ki-67 or ≥40% Ki-67 as cut-point. A literature review identified 14 reports meeting our inclusion criteria with ≥10 LCNEC. Reported Ki-67% ranged from 2% to 100%. Problems contributing to variability in Ki-67% measurements are discussed. Conclusion Our findings caution against a blanket use of 20%, 40% or other Ki-67% cut-points for LCNEC diagnosis or prognostication.

Localized malignant mesotheliomas (LMM) is an uncommon and poorly recognized neoplasm. Its pathologic diagnosis is often surprising in patients with serosal/subserosal based localized tumors that are clinically suspicious for metastatic lesions or primary sarcomas. Once a tumor is diagnosed as "mesothelioma", LMM is often mistaken for diffuse malignant mesothelioma (DMM). Best currently available evidence about LMM was collected from the literature and cases diagnosed by members of the International Mesothelioma Panel (IMP). One hundred and one (101) LMM have been reported in the English literature. Patients had localized tumors with identical histopathologic features to DMM. Patients ranged in age from 6 to 82 years; 75% were men. Most (82%) of the tumors were intrathoracic. Others presented as intrahepatic, mesenteric, gastric, pancreatic, umbilical, splenic, and abdominal wall lesions. Tumors varied in size from 0.6 to 15 cm. Most patients underwent surgical resection and/or chemotherapy or radiation therapy. Median survival in a subset of patients was 29 months. Seventy two additional LMM from IMP institutions ranged in age from 28 to 95 years; 58.3% were men. Sixty tumors (83.3%) were intrathoracic, others presented in intraabdominal sites. Tumors varied in size from 1.2 to 19 cm. Median survival for 51 cases was 134 months. Best evidence was used to formulate guidelines for the diagnosis of LMM. It is important to distinguish LMM from DMM as their treatment and prognosis is different. A multidisciplinary approach is needed for the diagnosis of LMM as it shows identical histopathology and immunophenotype to DMM.

Fine needle aspiration biopsy (FNAB) and ultrasonography are the most common modalities for the diagnosis and follow up of thyroid nodules. FNAB is able to distinguish benign from malignant nodules with high sensitivity and specificity; however, 20% to 30% of nodules are diagnosed as indeterminate with a risk of malignancy varying from 10% to 75% based on the 2017 revision of the Bethesda System for Reporting Thyroid Cytopathology. Molecular tests are being increasingly used to triage this group of nodules. Several molecular tests are commercially available and newer upgrades are being developed to either "rule in" or "rule out" malignancy with greater accuracy. The Afirma gene expression classifier and its recent upgrade (the Afirma gene sequencing classifier), Thryoseq v2, a next generation sequencing test and its recent upgrade (the v3), RosettaGX Reveal based on microRNA alterations, and ThyGenX/ThyraMIR, a combination test, are currently on the market. Familiarity with these tests, their performance, and postvalidation publications will enable appropriate test selection and improve triage of patients for appropriate therapy. The underlying rate of malignancy at different institutions and the interobserver variability in cytologic and histologic diagnosis of thyroid lesions are important factors that impact the performance of the various molecular tests.

It is uncertain whether thymic neuroendocrine tumors (NET) associated with Cushing's syndrome (CS) produce corticotropin-releasing hormone (CRH) and adrenocorticotropin hormone (ACTH) and whether the thymus contains ACTH and/or CRH cells that could originate NET. The clinicopathologic features of 5 typical (TC) and 6 atypical carcinoids (ATC), 10 additional non-neoplastic thymi, 6 adrenal glands with bilateral nodular hyperplasia and 8 adrenal cortical adenomas were reviewed. Representative slides were immunostained for ACTH and CRH. Four (36.4%) of the 11 patients had CS. The incidence of Masaoka stage IV was higher (p 0.0001) in patients with ATC than TC. Only 2 (18.1%) of the 11 patients were alive at follow-up. Ten NET were CRH immunoreactive and 6 were ACTH immunoreactive. Thymic NET with CS exhibited stronger immunoreactivity for ACTH and CRH than those without CS. Non-neoplastic thymi exhibited scattered ACTH and CRH immunoreactive cells. Normal adrenal cortex and glands with bilateral nodular hyperplasia showed diffuse CRH immunoreactivity while adrenal adenomas showed no or only focal CRH immunoreactivity. Literature review showed no association between thymic NET and adrenal adenomas. The thymus contains CRH and ACTH immunoreactive cells that are probably the origin of thymic NET. Neoplasms associated with CS exhibit strong immunoreactivity for both hormones, suggesting that CRH probably plays a role in the pathogenesis of CS. As adrenals with bilateral nodular hyperplasia exhibit diffuse CRH immunoreactivity and adrenal cortical adenomas either lack this finding or show few immunoreactive cells, this marker may be useful to distinguish these lesions.

Objectives Numerous studies on malignant mesothelioma (MM) highlight the prognostic importance of histologic subtype, nuclear grade, and necrosis. This study compares these parameters in paired biopsy and resection specimens of pleural MM. Methods Histologic subtype, percentage of epithelioid morphology, nuclear grade, and the presence or absence of necrosis were compared in 429 paired biopsies and resection specimens of pleural MM from 19 institutions. Results Histologic subtype was concordant in 81% of cases (κ = 0.58). When compared with resection specimens, epithelioid morphology at biopsy had a positive predictive value (PPV) of 78.9% and a negative predictive value (NPV) of 93.5%; sarcomatoid morphology showed high PPV (92.9%) and NPV (99.3%), and biphasic morphology PPV was 89.7% and NPV was 79.7%. Agreement of the percentage of epithelioid morphology was fair (κ = 0.27). Nuclear grade and necrosis were concordant in 75% (κ = 0.59) and 81% (κ = 0.53) of cases, respectively. Nuclear grade showed moderate (κ = 0.53) and substantial (κ = 0.67) agreement from patients with and without neoadjuvant therapy, respectively, and necrosis showed moderate (κ = 0.47 and κ = 0.60) agreement, respectively, in the same subsets of paired specimens. Conclusions Paired biopsy-resection specimens from pleural MM show overall moderate agreement in pathologic parameters. These findings may help guide postbiopsy management and triage of patients with MM.

Diffuse malignant mesothelioma (MM) is an incurable tumour of the serosal membranes, which is often caused by exposure to asbestos and commonly diagnosed at advanced stage. Malignant mesothelioma in situ (MMIS) is now included as diagnostic category by the World Health Organization (WHO). However, our international survey of 34 pulmonary pathologists with an interest in MM diagnosis highlights inconsistency regarding how the diagnosis is being made by experts, despite published guidelines. Whilst the WHO restricts the diagnosis to surgical samples, the very concept has implication for cytological diagnosis, which is already regarded as controversial in itself by some. MMIS is currently only applicable as precursor to MM with an epithelioid component, and raises the possibility for different molecular pathways for different histological MM subtypes. The clinical implications of MMIS at this stage are uncertain, but aggressive therapies are being initiated in some instances. Based on the results of the survey we here present a critical appraisal of the concept, its clinical and conceptual implications and provide practice suggestions for diagnosis. A low threshold for ancillary testing is suggested. The designations of 'malignant mesothelioma, cannot exclude MMIS' or 'atypical mesothelial proliferation with molecular indicators of malignancy, so-called MMIS' could be used on cytology samples, adding 'no evidence of invasion in sample provided' for surgical samples. Clinical and radiological correlation are integral to diagnosis and best done at multidisciplinary meetings. Finally, collaborative studies are required to improve our understanding of MMIS. ispartof: PATHOLOGY vol:53 issue:4 pages:446-453 ispartof: location:England status: published

3-D histology has become an attractive technique providing insights into morphology of histologic specimens. However, existing techniques in generating 3-D views from a stack of whole slide images are scarce or suffer from poor co-registration performance when displaying diagnostically important areas at sub-cellular resolution. Our team developed a new scale-invariant feature transform (SIFT)-based workflow to co-register histology images and facilitate 3-D visualization of micro-structures important in histopathology of lung adenocarcinoma. The co-registration accuracy and visualization capacity of the workflow were tested by digitally perturbing the staining coloration seven times. The perturbation slightly affected the co-registration but overall the co-registration errors remained very small when compared to those published to date. The workflow yielded accurate visualizations of expert-selected regions permitting confident 3-D evaluation of the clusters. Our workflow could support the evaluation of histologically complex tumors such as lung adenocarcinomas that are currently routinely viewed by pathologists in 2-D on slides, but could benefit from 3-D visualization.

Background Approximately 25% of thyroid nodule fine‐needle aspirates (FNAs) have cytology that is indeterminate for malignant disease. Accurate risk stratification of these FNAs with ancillary testing would reduce unnecessary thyroid surgery. Methods We evaluated the performance of an ancillary multiplatform test (MPTX) that has three diagnostic categories (negative, moderate, and positive). MPTX includes the combination of a mutation panel (ThyGeNEXT®) and a microRNA risk classifier (ThyraMIR®). A blinded, multicenter study was performed using consensus histopathology diagnosis among three pathologists to validate test performance. Results Unanimous consensus diagnosis was reached in 197 subjects with indeterminate thyroid nodules; 36% had disease. MPTX had 95% sensitivity (95% CI,86%‐99%) and 90% specificity (95% CI,84%‐95%) for disease in prevalence adjusted nodules with Bethesda III and IV cytology. Negative MPTX results ruledout disease with 97% negative predictive value (NPV; 95% CI,91%‐99%) at a 30% disease prevalence, while positive MPTX results ruledin high risk disease with 75% positive predictive value (PPV; 95% CI,60%‐86%). Such results are expected in four out of five Bethesda III and IV nodules tested, including RAS positive nodules in which the microRNA classifier was useful in rulingin disease. 90% of mutation panel false positives were due to analytically verified RAS mutations detected in benign adenomas. Moderate MPTX results had a moderate rate of disease (39%, 95% CI,23%‐54%), primarily due to RAS mutations, wherein the possibility of disease could not be excluded. Conclusions Our results emphasize that decisions for surgery should not solely be based on RAS or RAS‐like mutations. MPTX informs management decisions while accounting for these challenges.

Survival data from 225 patients with resected pulmonary typical carcinoids were analyzed with Kaplan-Meier statistics (K-M) and "deep learning" methods to illustrate the difference between establishing "correlations" and "prognostications". Cases were stratified into G1 and G2 classes using a ≤5% Ki-67% cut-point. Overall survival, number of patients at risk and 95% confidence intervals (CI) were estimated for the two classes. Seven neural network models (NN) were developed with GMDH Shell 3.8.2 and Statgraphics Centurion 18.1 software, using variable prior probabilities and different numbers of training vs testing cases. The NNs used age, sex, and pTNM, G1 and G2 as input neurons and "alive" and "dead" as output neurons. Areas under the curve (AUC) and other performance measures were evaluated for all models. Log-rank test showed a significant difference in overall survival between G1 and G2 (p 0.001). However, 95% CI estimates showed considerable variability in survival at different time intervals. Including the number of patients at risk at different time intervals showed that most G2 patients had been censored by 100 weeks. The NN models provided variable "prognostications", with AUC ranging from 0.5 to 1 and variability in the sensitivity, specificity, and other performance measures. The results illustrate the limitations of survival statistics and NNs in predicting the prognosis of individual patients. The need for pathologists not to overinterpret the finding of significant correlations as "prognostic" or "predictive" for individual patients is discussed.

When diagnosing and reporting lung adenocarcinoma (LAC), pathologists currently include an assessment of histologic tumor growth patterns because the predominant growth pattern has been reported to impact prognosis. However, the subjective nature of manual slide evaluation contributes to suboptimal inter-pathologist variability in tumor growth pattern assessment. We applied a deep learning approach to identify and automatically delineate areas of four tumor growth patterns (solid, acinar, micropapillary, and cribriform) and non-tumor areas in whole slide images (WSI) from resected LAC specimens. We trained a DenseNet model using patches from 109 slides collected at two institutions. The model was tested using 56 WSIs including 20 that were collected at a third institution. Using the same slide set, the concordance between the DenseNet model and an experienced pathologist (blinded to the DenseNet results) in determining the predominant tumor growth pattern was substantial (kappa score = 0.603). Using a subset of 36 test slides that were manually annotated for tumor growth patterns, we also measured the F1-score for each growth pattern: 0.95 (solid), 0.78 (acinar), 0.76 (micropapillary), 0.28 (cribriform) and 0.97 (non-tumor). Our results suggest that DenseNet assessment of WSIs with solid, acinar, and micropapillary predominant tumor growth is more robust than for the WSIs with predominant cribriform growth which are less frequently encountered.

Background Molecular tests are increasingly used to triage cytologically indeterminate thyroid nodules for surgery and/or follow-up. We retrospectively compared the performance of the Afirma Gene Expression Classifier (AGEC) with that of the more recently developed RosettaGX® Reveal™ miRNA Classifier (Reveal) in a cohort of Bethesda III-V thyroid FNAs with surgical follow-up. Design Eighty-one samples (54 Bethesda III, 26 Bethesda IV, 1 Bethesda V) with available AGEC (74 AGEC-SUSP and 7 AGEC-BENIGN) and surgical pathology results were studied from three academic centers. Reveal was performed in a blinded fashion. Results The final diagnoses were benign/NIFTP (n = 63) and malignant (n = 18). The overall "correct" rate was 64.2% for Reveal and 28.4% for AGEC (P = 1.4e-6). The specificity of Reveal was 60.3%, compared with 9.5% for AGEC (P = 2.1e-9). Among the 18 malignant cases, 77.8% and 94.4% were correctly classified as suspicious by Reveal and AGEC, respectively (P = 0.2). In the FLUS and the FN group, the specificity of AGEC was lower than the specificity of Reveal. Whether the 7 NIFTP in our study were considered benign or malignant, specificity and PPV of Reveal were higher than those of AGEC. Reveal also outperformed AGEC in correctly classifying the 26 benign Hurthle lesions studied (P = 7.6e-5). Conclusion Reveal outperformed AGEC in this cohort, whether NIFTP is considered benign or malignant, and in Hurthle lesions. Reveal has the potential to reduce the number of unnecessary resections in patients with indeterminate thyroid cytology. Based on our findings and the practical advantages offered by Reveal methodology, large prospective studies are warranted. Diagn. Cytopathol.

Data from pathology reports of estrogen receptor positive (ER+) breast cancers with Ki67 < 14% (luminal-A; n = 128) and Ki67 ≥ 14% (luminal-B; n = 100) were entered into the automatic recurrence score (RS) calculator accessible at the University of Pittsburgh Department of Pathology website. Using RS obtained with Magee equations #1 and #3, an average modified Magee recurrence score (AMM RS) was calculated for each tumor. The AMM RS and the Oncotype DX RS (Onco RS) were compared per tumor and associated with follow-up. AMM RS and Onco RS agreed in 64.9% (148 of 228) breast cancers (70.3% luminal-A and 58% luminal-B). There was only one two-step (low risk by Onco/high risk by AMM) RS disagreement. This luminal-B patient is alive without recurrence and free of tumor at 46 months postdiagnosis. Low-risk/intermediate-risk disagreements comprised 94.7% (36 of 38) and 69% (29 of 42) of the RS disagreements in the luminal-A and luminal-B groups, respectively (P = 0.004). In luminal-A, there were only two intermediate/high-risk disagreements; both high-risk ratings were by Onco RS. In luminal-B, there were 12 intermediate/high-risk disagreements; 11 of the high-risk ratings were by Onco RS. 100% (3 of 3) luminal-A tumors and 75% (6 of 8) luminal-B tumors that were high risk by AMM RS were also high risk by Onco RS. Eight tumors recurred and/or metastasized. AMM RS and Onco RS disagreed in only two of these eight tumors. The high percentage of tumors scored as intermediate risk (50% by AMM RS and 39% by Onco RS) is a major limitation of both scoring algorithms.

Malignant mesothelioma (MM) is an uncommon tumor that can be difficult to diagnose.- To provide updated, practical guidelines for the pathologic diagnosis of MM.- Pathologists involved in the International Mesothelioma Interest Group and others with an interest and expertise in the field contributed to this update. Reference material included up-to-date, peer-reviewed publications and textbooks.- There was discussion and consensus opinion regarding guidelines for (1) distinguishing benign from malignant mesothelial proliferations (both epithelioid and spindle cell lesions), (2) cytologic diagnosis of MM, (3) recognition of the key histologic features of pleural and peritoneal MM, (4) use of histochemical and immunohistochemical stains in the diagnosis and differential diagnosis of MM, (5) differentiating epithelioid MM from various carcinomas (lung, breast, ovarian, and colonic adenocarcinomas, and squamous cell and renal cell carcinomas), (6) diagnosis of sarcomatoid MM, (7) use of molecular markers in the diagnosis of MM, (8) electron microscopy in the diagnosis of MM, and (9) some caveats and pitfalls in the diagnosis of MM. Immunohistochemical panels are integral to the diagnosis of MM, but the exact makeup of panels employed is dependent on the differential diagnosis and on the antibodies available in a given laboratory. Depending on the morphology, immunohistochemical panels should contain both positive and negative markers for mesothelial differentiation and for lesions considered in the differential diagnosis. Immunohistochemical markers should have either sensitivity or specificity greater than 80% for the lesions in question. Interpretation of positivity generally should take into account the localization of the stain (eg, nuclear versus cytoplasmic) and the percentage of cells staining (10% is suggested for cytoplasmic and membranous markers). Selected molecular markers are now being used to distinguish benign from malignant mesothelial proliferations. These guidelines are meant to be a practical diagnostic reference for the pathologist; however, some new pathologic predictors of prognosis and response to therapy are also included.

Although Ki-67 labeling index (Ki-67%) is not a diagnostic or grading criterion in the World Health Organization classification of pulmonary carcinoid tumor, oncologists often request this test. A survey was administered at a North American Society for Neuroendocrine Tumors meeting to understand how Ki-67% is used in oncologic practices. A systematic literature review was performed to gather best evidence regarding the use of Ki-67%. Consecutive pulmonary carcinoids were stratified into pulmonary typical carcinoids with Ki-67%

Median Arcuate Ligament Syndrome (MALS) is a rare entity characterized by severe post-prandial epigastric pain, nausea, vomiting, and/or weight loss. Symptoms have been attributed to vascular compression (celiac artery compression syndrome, CACS), but it remains controversial whether they could be secondary to neural compression. Literature review identified rare description of pathologic findings in surgery journals. The clinico-pathologic findings of four MALS patients who underwent robotic or laparoscopic surgery in our hospital are described. All our patients were female with a median age of 32.5 (range 25–55 years), and a median BMI of 23.5 kg/m2. They presented with chronic often post-prandial abdominal pain (4/4), nausea (3/4), emesis (2/4), anorexia (1/4), and weight loss (1/4). Two patients had a history of Crohn's disease. At intraoperative exploration, the celiac artery and adjacent nerves and ganglia were encased and partially compressed by fibrotic tissue in each patient. In each case laparoscopic excision of fibrotic tissue, celiac plexus and ligament division and was performed; celiac plexus nerve block was also performed in one patient. After surgical intervention, symptoms improved in three of the patients whose specimens show periganglionic and perineural fibrosis with proliferation of small nerve fibers. Our findings support neurogenic compression as a contributing factor in the development of pain and other MALS symptoms, and favor the use of MALS rather than CACS as diagnostic terminology. To further study the pathogenesis of this unusual syndrome, surgeons should submit all tissues excised during MALS procedures for histopathologic examination.

Distinguishing between multiple lung primary tumors and intrapulmonary metastases is imperative for accurate staging. The American Joint Committee on Cancer (AJCC) criteria are routinely used for this purpose but can yield equivocal conclusions. This study evaluated whether next-generation sequencing (NGS) using the 50-gene AmpliSeq Cancer Hotspot Panel version 2 can help facilitate this distinction. NGS was performed on known primary-metastatic pairs (8 patients) and multiple lung adenocarcinomas (11 patients). Primary-metastatic pairs had high mutational concordance. Seven pairs shared mutations, and 1 was concordant for having no mutations. Driver mutations in KRAS (n = 4), EGFR (n = 2), and BRAF (n = 1) were always concordant. Multiple lung tumors from 3 patients were completely concordant and predicted by NGS to be intrapulmonary metastases, whereas 8 had completely discordant mutations and were predicted to be independent primary tumors. The NGS prediction correlated with the AJCC (eighth edition) prediction in all patients for whom the latter was unequivocal (8 of 11). Furthermore, it separated patients by overall survival. Patients with predicted multiple independent primary tumors by NGS had better survival than those with distant metastases (P = 0.016, log-rank test), whereas those with predicted intrapulmonary metastases had no difference (P = 0.527). With further validation, the 50-gene panel has the potential to serve as an adjunct to the AJCC criteria.

Context.— Tumor spread through alveolar spaces (STAS) has been correlated with unfavorable prognosis in lung adenocarcinomas treated with sublobar resection, but it is unknown whether STAS can be reliably identified in frozen section (FS) to help stratify patients for lobectomy or sublobar resection. Objective.— To evaluate STAS in FS. Design.— Tumor spread through alveolar spaces was evaluated in hematoxylin-eosin–stained FS, FS control slides, and all additional slides with lung tissue adjacent to tumor (AdLT) from 48 pT1–2 adenocarcinomas operated on using video-assisted thoracotomy (n = 25) or open thoracotomy (n = 23). The samples included lobectomies (n = 27) and sublobar resections (n = 21). The STAS incidences were compared by FS versus FS control versus AdLT, video-assisted thoracotomy versus open thoracotomy, and lobectomy versus sublobar resection. Sensitivity, specificity, and positive and negative predictive values of STAS+ findings were calculated. The literature was queried for best evidence regarding incidence and predictive value of STAS in FS. Results.— Tumor spread through alveolar spaces positivity was identified in 46 of 48 cases (95.8%), including 23 FS (47.9%), 32 FS control (66.7%), and 43 AdLT (89.6%). The STAS incidence was significantly higher in AdLT than in FS or FS control. Only 2 of the 25 cases that were STAS− in FS were true negatives. Frozen section sensitivity to detect STAS positivity was 50%, with a 100% positive predictive value and 8% negative predictive value. Systematic literature review identified no evidence regarding STAS identification in FS. Conclusions.— The sensitivity and negative predictive value of FS for STAS detection are unacceptably low. There are insufficient data to support intraoperative detection of STAS as a useful predictive feature to help stratify patients for lobectomy or sublobar resections.

The checkpoint protein programmed cell death ligand-1 protein (PD-L1) binds to its receptor (PD-1) activating the PD-L1/PD-1 pathway, an important therapeutic target. There is limited information regarding PD-L1 and PD-1 expression in thymic lesions. Sections from nonneoplastic thymi (n = 20), thymomas World Health Organization types A, AB, B1, B2, and B3 (n = 38) and thymic squamous cell carcinoma (n = 8) were stained for PD-L1 (clone SP142; Spring BioScience), PD-1 (MRQ22; Cell Marque), CD4 (clone SPO32; Cell Marque), and CD8 (JCB117; Ventana). Immunoreactivity for each antibody was classified as focal or diffuse and scored as follows: 0, negative; 1%-5%, 1+; 6%-20%, 2+; and >20%, 3+. The proportions of cases expressing PD-L1, PD-1, CD4, and C8 at score ≥1+ were compared by diagnosis, using χ2 statistics. PD-L1 was expressed in 90% of nonneoplastic thymi, 92% of thymomas, and 50% of carcinomas, with significantly higher scores (P < .01) in B2 and B3 thymomas and carcinomas than in AB and B1 thymomas; PD-L1 was diffuse in most B2 and B3 thymomas and focal in carcinomas. PD-1 was focally expressed, and mostly with scores 1+, in 55% of nonneoplastic thymi, 63% of thymomas, and 37.5% of carcinomas. CD4+ and CD8+ cells were diffusely distributed with scores 3+ in all lesions other than B3 thymomas and carcinomas. The latter showed CD4+ cells mostly at the interface between neoplastic cells and stroma. PD-L1 and PD-1 are not expressed in similar locations and cellular proportions in thymic lesions, raising a question as to whether the PD-L1/PD-1 pathway is an actionable therapeutic target in these lesions.

Objective To report the response to progestin therapy in young women with endometrial complex atypical hyperplasia (CAH) or FIGO grade 1 endometrial adenocarcinoma (FIGO 1 EAC) based on clinicopathologic features, including abnormal DNA mismatch repair (MMR) by immunohistochemistry (IHC). Design Consecutive case series. Setting Olive View-UCLA Medical Center in Sylmar, CA, USA, and Cedars-Sinai Medical Center in Los Angeles, CA, USA. Population Women ≤55 years old with CAH or FIGO 1 EAC. Methods Response to progestin therapy in 84 consecutive patients was assessed based on clinicopathologic factors, including age, body mass index (BMI), initial histology, and IHC staining for MMR proteins. Main outcome measures Rates of abnormal MMR protein expression and response to progestin therapy were determined. Results Six (7%) patients had abnormal IHC staining, of whom five (83%) had FIGO 1 EAC at initial diagnosis. Following progestin treatment, none of the endometrial lesions in patients with abnormal IHC for MMR proteins had resolution of hyperplasia or malignancy, in contrast to 41 (53%) with normal staining (P = 0.028). Age ≤40 years and initial lesion (CAH versus FIGO 1 EAC) were predictors of response to progestin; BMI was not. Conclusions In this cohort, 7% of women ≤55 years of age with CAH or FIGO 1 EAC had loss of MMR proteins by IHC. These patients had a higher incidence of invasive cancer and a lower incidence of resolution with progestin therapy. Tweetable abstract Abnormal MMR protein expression predicts poor response to progestins in young women with CAH or FIGO 1 EAC.

"Personalized medicine" has been proposed as a new paradigm for patient care that, based on the integration of genomics and other "omics" data with clinical and other multidisciplinary information, promises early disease detection, improved outcomes and reduced side effects to therapies. Pathologists have become important participants in this new approach as the guardians of tissues and experts in the performance of molecular and other laboratory tests. Large amounts of new laboratory data in multiple neoplasms and other entities are being reported but there has been limited discussion about how best to evaluate the clinical significance of this information and how to integrate it into currently available diagnostic and therapeutic modalities. This article introduces a variety of epistemological problems presented by the "personalized medicine" paradigm and briefly discusses various topics that will be evaluated in further detail in future articles of this new series on Evidence-Based Pathology.

During the diagnostic workup of lung adenocarcinomas (LAC), pathologists evaluate distinct histological tumor growth patterns. The percentage of each pattern on multiple slides bears prognostic significance. To assist with the quantification of growth patterns, we constructed a pipeline equipped with a convolutional neural network (CNN) and soft-voting as the decision function to recognize solid, micropapillary, acinar, and cribriform growth patterns, and non-tumor areas. Slides of primary LAC were obtained from Cedars-Sinai Medical Center (CSMC), the Military Institute of Medicine in Warsaw and the TCGA portal. Several CNN models trained with 19,924 image tiles extracted from 78 slides (MIMW and CSMC) were evaluated on 128 test slides from the three sites by F1-score and accuracy using manual tumor annotations by pathologist. The best CNN yielded F1-scores of 0.91 (solid), 0.76 (micropapillary), 0.74 (acinar), 0.6 (cribriform), and 0.96 (non-tumor) respectively. The overall accuracy of distinguishing the five tissue classes was 89.24%. Slide-based accuracy in the CSMC set (88.5%) was significantly better (p

Patch-based image classification approaches are frequently employed in digital pathology applications with the goal to automatically delineate diagnostically important regions in digital slides. However, patches into which a slide is partitioned are often classified singly and sequentially without additional context. To address this issue, we tested a contextual classification of image patches with soft voting applied to a multi-class classification problem. The context comprised five or nine overlapping patches. The classification is performed using convolutional neural networks (CNNs) trained to recognize four histologically distinct growth patterns of lung adenocarcioma and non-tumor areas. Classification with soft voting outperformed sequential classification of patches yielding higher whole slide classification accuracy. The F1-scores for the four tumor growth patterns improved by 3% and 4.9% when the context consisted of five and nine neighboring patches, respectively. We conclude that the context can improve classification performance of areas sharing the same histological features. Soft voting is a non-trainable approach and therefore straightforward to implement. However, it is computationally more expensive than the classical single patch-based approach.

Whether or not autoimmune thyroid disease influences the progression of differentiated thyroid cancer (DTC) remains controversial. Findings of previous studies are influenced by lead time bias and/or procedure bias selection. These biases can be reduced by studying a single-institution patient population that underwent a similar extent of surgical resection.From a cohort of 660 patients with DTC who underwent thyroidectomy, we retrospectively studied 357 patients who underwent total thyroidectomy and central compartment node dissection (CCND) for DTC between 2003 and 2013.Forty-one percent (140/345) of study patients had lymphocytic thyroiditis (LT), and 30% (91/301) had serum positive for thyroglobulin antibody (TgAb). LT was reported in 78% of the TgAb-positive cases. Sixty percent (213/357) of cases had metastatic thyroid carcinoma in 1 or more neck lymph nodes (55% [198/357] central compartment, and 22% [77/356] lateral compartment). Patients with LT had fewer metastatic cervical lymph nodes than those with no LT (2.7 ± 4.7 vs 3.5 ± 4.8, respectively, P = .0285). Patients with positive TgAb and thyroiditis had a larger number of benign cervical lymph nodes removed than those with negative TgAb or no LT. No significant difference was observed in age, tumor size, multifocality, extrathyroidal extension, vascular invasion, or frequency of cervical lymph node metastasis between TgAb-negative and -positive cases or between cases with and without LT.Lymphocytic thyroiditis is associated with fewer central neck compartment metastatic lymph nodes and a larger number of excised reactive benign cervical lymph nodes. Whether this association indicates a protective role of thyroid autoimmunity in lymph node spreading remains unclear.CCND = central compartment node dissection DTC = differentiated thyroid cancer HT = Hashimoto thyroiditis LT = lymphocytic thyroiditis TgAb = thyroglobulin antibody TPO = thyroid peroxidase.

Osteosarcoma is a highly malignant tumor that contains a small subpopulation of tumor-propagating cells (also known as tumor-initiating cells) characterized by drug resistance and high metastatic potential. The molecular mechanism by which tumor-propagating cells promote tumor growth is poorly understood. Here, we report that the transcription factor forkhead box C2 (FOXC2) is frequently expressed in human osteosarcomas and is important in maintaining osteosarcoma cells in a stem-like state. In osteosarcoma cell lines, we show that anoikis conditions stimulate FOXC2 expression. Downregulation of FOXC2 decreases anchorage-independent growth and invasion in vitro and lung metastasis in vivo, while overexpression of FOXC2 increases tumor propagation in vivo. In osteosarcoma cell lines, we demonstrate that high levels of FOXC2 are associated with and required for the expression of osteosarcoma tumor-propagating cell markers. In FOXC2 knockdown cell lines, we show that CXCR4, a downstream target of FOXC2, can restore osteosarcoma cell invasiveness and metastasis to the lung.

Background Loss of BAP1 by immunohistochemistry (IHC) and CDKN2A(p16) deletion by fluorescence in situ hybridization (FISH) have been proposed to distinguish malignant mesothelioma (MM) from atypical reactive mesothelial proliferations (ARMP) in effusions but it is uncertain whether both tests are needed routinely. Methods Paraffin embedded blocks from 67 effusions (32 MM, 35 ARMP) were evaluated with BAP1 IHC. 38 of them (17 MM, 21 ARMP) were also analyzed with CDKN2A (p16) FISH. Criteria for MM were absence of BAP1 nuclear staining in >50% of atypical mesothelial cells in the presence of a positive internal control, and/or CDKN2A(p16) homozygous or hemizygous deletion pattern in >15% or >41.5% of atypical mesothelial cells, respectively. Sensitivity (SS), specificity (SP), positive, and negative predictive values (PPV, NPV) for MM were calculated. Results 17 of 32 MM were correctly diagnosed by IHC (SS 53.1%, SP 85.7%, PPV 77.3%, NPV 64.3% for MM). In the subset of 38 cases studied by IHC and FISH, the SS, SP, PPV, and NPV of IHC for MM were 41.2, 81.0, 63.6, and 63.0%, respectively. 7 of 17 MM were diagnosed by FISH (SS 41.2%, SP 100%, PPV 100%, NPV 67.7% for MM). Conclusion BAP1 IHC and CDKN2A(p16) FISH yield similar sensitivities for MM in paired samples but FISH has a higher specificity. To diagnose effusions with atypical mesothelial cells, use of both tests is optimal. However, BAP1 IHC alone may be sufficient to diagnose MM when clinical, imaging, and cytological findings strongly suggest MM. Diagn. Cytopathol. 2016. © 2016 Wiley Periodicals, Inc. Diagn. Cytopathol. 2016;44:599–606. © 2016 Wiley Periodicals, Inc.

Background Mesothelioma patients often present with serosal effusions, which are ideal for cytopathological diagnoses. However, the morphological overlap between malignant and benign mesothelial proliferation can make a conclusive cytological diagnosis of mesothelioma elusive because immunohistochemical staining does not discriminate definitively between the two in this setting. p16 is deleted in up to 80% of pleural mesotheliomas. The aim of this study was to establish the correlation between the p16 deletion status of the cell block with that of its corresponding tumor using fluorescence in situ hybridization (FISH) analysis for individual patient tumors. Methods Twenty-two biopsies and 24 corresponding cell blocks, containing serosal effusions with atypical mesothelial cells from 22 patients with histologically confirmed pleural mesotheliomas, were analyzed with p16 FISH. Seventeen cell blocks consisting of serosal effusions with reactive mesothelial cells from nonmesothelioma cases were also analyzed. Combined immunofluorescence and FISH were also performed. Results Seventeen of the 22 mesothelioma patients (77.3%) showed homozygous deletions of p16 in the tumor tissue and in the atypical mesothelial cells from the cell blocks. p16 FISH followed by immunofluorescence with EMA was helpful towards identifying the mesothelioma cells in the cell blocks. Conclusion We confirmed that the p16 FISH results obtained from the cell blocks are as reliable as those from the tissue sections. Cell block analysis is recommended for patients with serosal effusions of unknown origins with the following methods: immunohistochemistry should be performed to validate the mesothelial origin, and p16 FISH should be performed to confirm malignancy. Diagn. Cytopathol. 2016;44:591-598. © 2016 Wiley Periodicals, Inc.

Expression of the homeodomain transcription factor HOXB13 has been demonstrated in several malignancies but its role in tumorigenesis remains elusive. We observed high levels of HOXB13 in poorly differentiated pediatric tumors including a highly aggressive childhood neoplasm - malignant rhabdoid tumor. In a xenograft model of rhabdoid tumor, knockout of HOXB13 diminished tumor growth while partial knockdown of HOXB13 promoted differentiation of tumor cells into bone. These results suggest that HOXB13 enhances rhabdoid malignancy by interfering with mesenchymal stem cell differentiation. Consistent with this hypothesis, overexpression of HOXB13 in mesenchymal progenitor cells inhibited adipogenic, myogenic, and osteogenic differentiation. Mechanistically, we demonstrated that HOXB13 binds to super-enhancer regions regulating genes involved in differentiation and tumorigenesis.

Kappa statistics have been widely used in the pathology literature to compare interobserver diagnostic variability (IOV) among different pathologists but there has been limited discussion about the clinical significance of kappa scores. Five representative and recent pathology papers were queried using clinically relevant specific questions to learn how IOV was evaluated and how the clinical applicability of results was interpreted. The papers supported our anecdotal impression that pathologists usually assess IOV using Cohen's or Fleiss' kappa statistics and interpret the results using some variation of the scale proposed by Landis and Koch. The papers did not cite or propose specific guidelines to comment on the clinical applicability of results. The solutions proposed to decrease IOV included the development of better diagnostic criteria and additional educational efforts, but the possibility that the entities themselves represented a continuum of morphologic findings rather than distinct diagnostic categories was not considered in any of the studies. A dataset from a previous study of IOV reported by Thunnissen et al. was recalculated to estimate percent agreement among 19 international lung pathologists for the diagnosis of 74 challenging lung neuroendocrine neoplasms. Kappa scores and diagnostic sensitivity, specificity, positive and negative predictive values were calculated using the majority consensus diagnosis for each case as the gold reference diagnosis for that case. Diagnostic specificity estimates among multiple pathologists were > 90%, although kappa scores were considerably more variable. We explain why kappa scores are of limited clinical applicability in pathology and propose the use of positive and negative percent agreement and diagnostic specificity against a gold reference diagnosis to evaluate IOV among two and multiple raters, respectively.

Anal intraepithelial neoplasia (AIN) is a precursor to invasive anal squamous cell carcinoma. Histologic evaluation is hampered by intra- and interobserver variability. Various biomarkers have been investigated to improve the accuracy and reproducibility of diagnosis and grading, but interpretation can be challenging. ProEx™ C is an antibody cocktail for proteins upregulated in cervical intraepithelial neoplasia. This study investigated ProEx™ C's role alone and with p16 and Ki-67 in the diagnosis and grading of AIN. Sixty-seven anal tissue samples (22 AIN I, 25 AIN II/III, and 20 non-dysplastic) were stained for ProEx™ C, Ki-67, and p16. Staining patterns were recorded and correlated with morphologic diagnoses. Considering AIN II/III vs I, full-thickness ProEx™ C staining was more frequent in AIN II/III (p = 0.0373), and showed the highest sensitivity of the biomarkers. In combination with Ki-67, sensitivity was lower, but specificity for AIN II/III rose to 83%. For differentiating non-dysplasia from AIN I, negative ProEx™ C staining correlated with non-dysplasia (p < 0.0001) and had the highest sensitivity (90%). In combination with Ki-67, sensitivity dropped to 80%, but specificity was high (96%). ProEx™ C is useful for diagnosing and grading AIN, performing as well or better than other markers at identifying AIN II/III and non-dysplastic epithelium.