Guidelines for Pathologic Diagnosis of Mesothelioma.

CONTEXT.­: Mesothelioma is an uncommon tumor that can be difficult to diagnose. OBJECTIVE.­: To provide updated, practical guidelines for the pathologic diagnosis of mesothelioma. DATA SOURCES.­: Pathologists involved in the International Mesothelioma Interest Group and others with expertise in mesothelioma contributed to this update. Reference material includes peer-reviewed publications and textbooks. CONCLUSIONS.­: There was consensus opinion regarding guidelines for (1) histomorphologic diagnosis of mesothelial tumors, including distinction of epithelioid, biphasic, and sarcomatoid mesothelioma; recognition of morphologic variants and patterns; and recognition of common morphologic pitfalls; (2) molecular pathogenesis of mesothelioma; (3) application of immunohistochemical markers to establish mesothelial lineage and distinguish mesothelioma from common morphologic differentials; (4) application of ancillary studies to distinguish benign from malignant mesothelial proliferations, including BAP1 and MTAP immunostains; novel immunomarkers such as Merlin and p53; fluorescence in situ hybridization (FISH) for homozygous deletion of CDKN2A; and novel molecular assays; (5) practical recommendations for routine reporting of mesothelioma, including grading epithelioid mesothelioma and other prognostic parameters; (6) diagnosis of mesothelioma in situ; (7) cytologic diagnosis of mesothelioma, including use of immunostains and molecular assays; and (8) features of nonmalignant peritoneal mesothelial lesions.

Mesothelioma in situ of the peritoneum: report of three cases and review of the literature.

AIM: Diagnosis of mesothelioma in situ (MIS) is historically controversial and, until recently, specific features defining the entity have not been well characterized. Most reported cases of MIS occurred in the pleura; peritoneal MIS is very rare. This study investigates the morphologic features and results of ancillary testing in peritoneal MIS. METHODS: We present three patients with peritoneal MIS, as defined by a single layer of mesothelial cells with loss of nuclear BRCA-1-associated protein-1 (BAP1) immunostaining and without evidence of invasive tumour by microscopic evaluation, imaging, or direct examination of the peritoneum. Histology and immunostains were reviewed by three expert thoracic pathologists with multidisciplinary input. Next-generation sequencing (NGS) was performed in all three cases. A literature review was conducted to characterize this rare precursor lesion. RESULTS: BAP1 was lost in all three lesions, while methylthioadenosine phosphorylase (MTAP) was retained in two (not performed in the third). NGS revealed BAP1 pathogenic alterations in all three cases as well as mutations of SMO, ERCC3, TET2, and U2AF1. Progression to invasive mesothelioma occurred in one patient at 13 months postdiagnosis (case 1). One patient was diagnosed at age 24 and was later found to harbour a BAP1 germline mutation (case 3). CONCLUSION: This work describes the histologic features and clinicopathologic characteristics of peritoneal MIS in three cases, highlights BAP1 somatic and germline mutations in peritoneal MIS, and strengthens the importance of ancillary studies (including immunohistochemical and molecular studies) in the diagnosis of MIS.

Medical and Surgical Care of Patients With Mesothelioma and Their Relatives Carrying Germline BAP1 Mutations.

The most common malignancies that develop in carriers of BAP1 germline mutations include diffuse malignant mesothelioma, uveal and cutaneous melanoma, renal cell carcinoma, and less frequently, breast cancer, several types of skin carcinomas, and other tumor types. Mesotheliomas in these patients are significantly less aggressive, and patients require a multidisciplinary approach that involves genetic counseling, medical genetics, pathology, surgical, medical, and radiation oncology expertise. Some BAP1 carriers have asymptomatic mesothelioma that can be followed by close clinical observation without apparent adverse outcomes: they may survive many years without therapy. Others may grow aggressively but very often respond to therapy. Detecting BAP1 germline mutations has, therefore, substantial medical, social, and economic impact. Close monitoring of these patients and their relatives is expected to result in prolonged life expectancy, improved quality of life, and being cost-effective. The co-authors of this paper are those who have published the vast majority of cases of mesothelioma occurring in patients carrying inactivating germline BAP1 mutations and who have studied the families affected by the BAP1 cancer syndrome for many years. This paper reports our experience. It is intended to be a source of information for all physicians who care for patients carrying germline BAP1 mutations. We discuss the clinical presentation, diagnostic and treatment challenges, and our recommendations of how to best care for these patients and their family members, including the potential economic and psychosocial impact.

Epigenetic Dysregulation of 5-hydroxymethylcytosine in Well-Differentiated Pancreatic Neuroendocrine Tumors.

Dysregulation of epigenetic mechanisms, reflected by loss of expression of 5-hydroxymethylcytosine (5-hmC) is being increasingly recognized as a marker of aggressive behavior in several neoplasms; however, the role of such epigenetic modifiers in pancreatic neuroendocrine tumors (PanNETs) has not been studied. Annotated cohort of 60 PanNETs was evaluated for 5-hmC expression using immunohistochemistry. Univariable and multivariable analyses were performed. To determine intratumor heterogeneity of 5-hmC expression, 26 additional synchronous metastatic deposits of PanNETs from 8 patients were evaluated for 5-hmC expression. 5-hmC level showed significant association with the presence of distant metastases (P=0.02), female sex (P=0.04), and Ki-67 proliferation index (P=0.002). A multivariate model created using the stepwise logistic regression analysis showed the presence of nodal metastases (odds ratio=6.15), lymphovascular invasion (odds ratio=4.07) and lack of 5-hmC expression (odds ratio=5.34) were predictive of the risk of distant metastasis in PanNETs with a c-statistic of 0.845. Epigenetic intratumoral heterogeneity of 5-hmC expression was seen in 37.5% cases (3/8). Our work provides evidence that epigenetic regulators are involved in the pathobiology of PanNETs and immunohistochemical analysis of 5-hmC may be able to refine prognostic evaluation of these tumors.

Primary Desmoplastic Small Round Cell Tumor of the Femur: Case Report and Review of a Rare Intraosseous Malignancy.

Background: Desmoplastic small round cell tumors (DSRCT) are malignant neoplasms of young males arising most commonly in the abdominopelvic cavity, with a subset originating from extra-abdominal soft tissues. As either primary or metastatic lesions, they are rare in intraosseous sites. Case Presentation: We describe the fifth report of primary DSRCT of bone. A healthy 18-year old male presented with a blastic, 17 cm lesion within the left distal femur, suspicious for osteosarcoma or Ewing sarcoma. Subsequent biopsy revealed nests of small round blue cells infiltrating through a desmoplastic stroma. These cells were diffusely positive for epithelial markers, with paranuclear staining for desmin and focal reactivity with NSE. Break-apart FISH revealed a rearrangement in EWSR1, and RNA fusion panel confirmed WT1 as its partner in the pathognomonic t(11;22)(p13;q12) rearrangement. PET/CT showed widespread metastatic disease to visceral and bony sites. Conclusions: Due to their rarity as well as clinicopathologic and immunomorphologic overlap, primary intraosseous DSRCT can create diagnostic challenges with the more frequently encountered tumors of bone.

Pembrolizumab Plus Ipilimumab Following Anti-PD-1/L1 Failure in Melanoma.

PURPOSE: Combination of antiprogrammed cell death protein-1 (PD-1) plus anti-cytotoxic T-cell lymphocyte-4 (anti-CTLA-4) immunotherapy shows greater response rates (RRs) than anti-PD-1 antibody alone in melanoma, but RR after initial anti-PD-1 and programmed death ligand-1 (PD-L1) antibody progression awaits robust investigation. Anti-CTLA-4 antibody alone after anti-PD-1/L1 antibody progression has a historical RR of 13%. We report the results of the first prospective clinical trial evaluating ipilimumab 1 mg/kg plus pembrolizumab following progression on anti-PD-1 immunotherapy. METHODS: Patients with advanced melanoma who had progressed on anti-PD-1/L1 antibody as immediate prior therapy (including non-anti-CTLA-4 antibody combinations) were eligible. Patients received pembrolizumab 200 mg plus ipilimumab 1 mg/kg once every 3 weeks for four doses, followed by pembrolizumab monotherapy. The primary end point was RR by irRECIST. After 35 patients, the trial met the primary end point and was expanded to enroll a total of 70 patients to better estimate the RR. RESULTS: Prior treatments included 60 on anti-PD-1 antibody alone and 10 on anti-PD-1/L1 antibody-based combinations. Thirteen patients had progressed in the adjuvant setting. The median length of prior treatment with anti-PD-1/L1 antibody was 4.8 months. Response assessments included five complete and 15 partial responses, making the irRECIST RR 29% among the entire trial population. The median progression-free survival was 5.0 months, and the median overall survival was 24.7 months. The median duration of response was 16.6 months. There was no difference in median time on prior anti-PD1/L1 or time to PD1 + CTLA4 initiation between responders and nonresponders. Grade 3-4 drug-related adverse events occurred in 27% of patients. Responses occurred in PD-L1-negative, non-T-cell-inflamed, and intermediate tumor phenotypes. CONCLUSION: To our knowledge, this is the first prospective study in melanoma of pembrolizumab plus low-dose ipilimumab after anti-PD-1/L1 immunotherapy failure, demonstrating significant antitumor activity and tolerability.

A series of COVID-19 autopsies with clinical and pathologic comparisons to both seasonal and pandemic influenza.

Autopsies of patients who have died from COVID-19 have been crucial in delineating patterns of injury associated with SARS-CoV-2 infection. Despite their utility, comprehensive autopsy studies are somewhat lacking relative to the global burden of disease, and very few comprehensive studies contextualize the findings to other fatal viral infections. We developed a novel autopsy protocol in order to perform postmortem examinations on victims of COVID-19 and herein describe detailed clinical information, gross findings, and histologic features observed in the first 16 complete COVID-19 autopsies. We also critically evaluated the role of ancillary studies used to establish a diagnosis of COVID-19 at autopsy, including immunohistochemistry (IHC), in situ hybridization (ISH), and electron microscopy (EM). IHC and ISH targeting SARS-CoV-2 were comparable in terms of the location and number of infected cells in lung tissue; however, nonspecific staining of bacteria was seen occasionally with IHC. EM was unrevealing in blindly sampled tissues. We then compared the clinical and histologic features present in this series to six archival cases of fatal seasonal influenza and six archival cases of pandemic influenza from the fourth wave of the 'Spanish Flu' in the winter of 1920. In addition to routine histology, the inflammatory infiltrates in the lungs of COVID-19 and seasonal influenza victims were compared using quantitative IHC. Our results demonstrate that the clinical and histologic features of COVID-19 are similar to those seen in fatal cases of influenza, and the two diseases tend to overlap histologically. There was no significant difference in the composition of the inflammatory infiltrate in COVID-19 and influenza at sites of acute lung injury at the time of autopsy. Our study underscores the relatively nonspecific clinical features and pathologic changes shared between severe cases of COVID-19 and influenza, while also providing important caveats to ancillary methods of viral detection.

Comparison of Nuclear Grade, Necrosis, and Histologic Subtype Between Biopsy and Resection in Pleural Malignant Mesothelioma: An International Multi-Institutional Analysis.

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.

The concept of mesothelioma in situ, with consideration of its potential impact on cytology diagnosis.

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.

A Distinctive Adnexal (Usually Paratubal) Neoplasm Often Associated With Peutz-Jeghers Syndrome and Characterized by STK11 Alterations (STK11 Adnexal Tumor): A Report of 22 Cases.

We describe 22 examples of a novel, usually paratubal, adnexal tumor associated with Peutz-Jeghers syndrome in nearly 50% of cases that harbored STK11 alterations in all tested (n=21). The patients ranged from 17 to 66 years (median=39 y) and the tumors from 4.5 to 25.5 cm (median=11 cm). Most (n=18) were paratubal, with metastases noted in 11/22 (50%) and recurrences in 12/15 (80%). Morphologically, they were characterized by interanastomosing cords and trabeculae of predominantly epithelioid cells, set in a variably prominent myxoid to focally edematous stroma, that often merged to form tubular, cystic, cribriform, and microacinar formations, reminiscent of salivary gland-type tumors. The tumor cells were uniformly atypical, often with prominent nucleoli and a variable mitotic index (median=9/10 HPFs). The tumors were usually positive to a variable extent for epithelial (CAM5.2, AE1/AE3, cytokeratin 7), sex cord (calretinin, inhibin, WT1), and mesothelial (calretinin, D2-40) markers, as well as hormone receptors. PAX8, SF1, and GATA-3 were rarely positive, while claudin-4, FOXL2, and TTF-1 were consistently negative. All sequenced tumors (n=21) harbored alterations in STK11, often with a loss of heterozygosity event. There were no other recurrently mutated genes. Recurrent copy number alterations included loss of 1p and 11q, and gain of 1q, 15q, and 15p. Despite an extensive morphologic, immunohistochemical, and molecular evaluation, we are unable to determine with certainty the histogenesis of this unique tumor. Wolffian, sex cord stromal, epithelial, and mesothelial origins were considered. We propose the term STK11 adnexal tumor to describe this novel entity and emphasize the importance of genetic counseling in these patients as a significant number of neoplasms occur in association with Peutz-Jeghers syndrome.

Malignant peritoneal mesothelioma: prognostic significance of clinical and pathologic parameters and validation of a nuclear-grading system in a multi-institutional series of 225 cases.

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 <60 years (P = 0.0001), ECOG performance status 0 or 1 (P = 0.01), absence of radiographic lymph-node metastasis (P = 0.04), cytoreduction surgery (P < 0.0001), hyperthermic intraperitoneal chemotherapy (P = 0.0001), peritoneal carcinomatosis index <27 (P = 0.01), absence of necrosis (P = 0.007), and epithelioid histotype (P < 0.0001). Among epithelioid malignant mesotheliomas only, longer overall survival was further associated with female sex (P = 0.03), tubulopapillary architecture (P = 0.005), low nuclear pleomorphism (P < 0.0001), low mitotic index (P = 0.0007), and low composite nuclear grade (P < 0.0001). On multivariate analyses, the low composite nuclear grade was independently associated with longer overall and disease-free survival (P < 0.0001). Our data further clarify the interactions of clinical and pathologic features in peritoneal mesothelioma prognosis and validate the prognostic significance of a standardized nuclear-grading system in epithelioid malignant mesothelioma of the peritoneum.

A Descriptive and Quantitative Immunohistochemical Study Demonstrating a Spectrum of Platelet Recruitment Patterns Across Pulmonary Infections Including COVID-19.

OBJECTIVES: Pulmonary platelet deposition and microangiopathy are increasingly recognized components of coronavirus disease 2019 (COVID-19) infection. Thrombosis is a known component of sepsis and disseminated intravascular coagulation. We sought to compare the level of platelet deposition in the pulmonary vasculature in cases of confirmed COVID-19 infection to other lung injuries and infections. METHODS: Immunohistochemistry was performed on 27 autopsy cases and 2 surgical pathology cases targeting CD61. Multiple cases of normal lung, diffuse alveolar damage, COVID-19, influenza, and bacterial and fungal infections, as well as one case of pulmonary emboli, were included. The levels of CD61 staining were compared quantitatively in the autopsy cases, and patterns of staining were described. RESULTS: Nearly all specimens exhibited an increase in CD61 staining relative to control lung tissue. The area of CD61 staining in COVID-19 infection was higher than influenza but still comparable to many other infectious diseases. Cases of aspiration pneumonia, Staphylococcus aureus infection, and blastomycosis exhibited the highest levels of CD61 staining. CONCLUSIONS: Platelet deposition is a phenomenon common to many pulmonary insults. A spectrum of staining patterns was observed, suggestive of pathogen-specific mechanisms of platelet deposition. Further study into the mechanisms driving platelet deposition in pulmonary injuries and infections is warranted.

Improved Survival Associated with Local Tumor Response Following Multisite Radiotherapy and Pembrolizumab: Secondary Analysis of a Phase I Trial.

PURPOSE: Multisite stereotactic body radiotherapy followed by pembrolizumab (SBRT+P) has demonstrated safety in advanced solid tumors (ASTs). However, no studies have examined the relationships between irradiated tumor response, SBRT-induced tumor gene expression, and overall survival (OS). PATIENTS AND METHODS: Patients with AST received SBRT (30-50 Gy in 3-5 fractions) to two to four metastases followed by pembrolizumab (200 mg i.v. every 3 weeks). SBRT was prescribed to a maximum tumor volume of 65 mL. Small metastases received the complete prescribed coverage (complete-Rx), while larger metastases received partial coverage (partial-Rx). Treated metastasis control (TMC) was defined as a lack of progression for an irradiated metastasis. Landmark analysis was used to assess the relationship between TMC and OS. Thirty-five biopsies were obtained from 24 patients: 19 pre-SBRT and 16 post-SBRT (11 matched) prior to pembrolizumab and were analyzed via RNA microarray. RESULTS: Sixty-eight patients (139 metastases) were enrolled with a median follow-up of 10.4 months. One-year TMC was 89.5% with no difference between complete-Rx or partial-Rx. On multivariable analysis, TMC was independently associated with a reduced risk for death (HR, 0.36; 95% confidence interval, 0.17-0.75; P = 0.006). SBRT increased expression of innate and adaptive immune genes and concomitantly decreased expression of cell cycle and DNA repair genes in the irradiated tumors. Elevated post-SBRT expression of DNASE1 correlated with increased expression of cytolytic T-cell genes and irradiated tumor response. CONCLUSIONS: In the context of SBRT+P, TMC independently correlates with OS. SBRT impacts intratumoral immune gene expression associated with TMC. Randomized trials are needed to validate these findings.

Biological Mechanisms and Clinical Significance of BAP1 Mutations in Human Cancer.

Among more than 200 BAP1-mutant families affected by the "BAP1 cancer syndrome," nearly all individuals inheriting a BAP1 mutant allele developed one or more malignancies during their lifetime, mostly uveal and cutaneous melanoma, mesothelioma, and clear-cell renal cell carcinoma. These cancer types are also those that, when they occur sporadically, are more likely to carry somatic biallelic BAP1 mutations. Mechanistic studies revealed that the tumor suppressor function of BAP1 is linked to its dual activity in the nucleus, where it is implicated in a variety of processes including DNA repair and transcription, and in the cytoplasm, where it regulates cell death and mitochondrial metabolism. BAP1 activity in tumor suppression is cell type- and context-dependent. BAP1 has emerged as a critical tumor suppressor across multiple cancer types, predisposing to tumor development when mutated in the germline as well as somatically. Moreover, BAP1 has emerged as a key regulator of gene-environment interaction.This article is highlighted in the In This Issue feature, p. 1079.

Uterine Tumor Resembling Ovarian Sex Cord Stromal Tumor (UTROSCT): A Series of 3 Cases With Extensive Rhabdoid Differentiation, Malignant Behavior, and ESR1-NCOA2 Fusions.

ESR1 and GREB1 fusions have recently been described in uterine tumor resembling ovarian sex cord tumor (UTROSCT). Thus far, recurrences have been documented in a subset of those harboring GREB1 fusions, but not in those with ESR1 rearrangements. Here we describe the clinicopathologic features of 3 recurrent UTROSCTs with striking rhabdoid morphology (an unusual feature of these tumors overall) and ESR1-NCOA2 fusions. The patients were 32, 37, and 54 years at initial diagnosis and first recurrence occurred at 7, 9, and 32 years. The primary tumors (available in two cases) were centered in the myometrium and showed infiltrative borders. They predominantly grew in sheets and cords, but also had a pseudopapillary appearance. Cells were uniformly epithelioid with eccentric nuclei, prominent nucleoli, abundant eosinophilic globular/glassy (rhabdoid) cytoplasm, and infrequent mitoses (≤4/10 high-power fields [HPFs]). Recurrences were morphologically identical to the primary tumors, but demonstrated brisk mitotic activity (≥16/10 HPFs). The third tumor (with only recurrences available) had multiple patterns, including diffuse, corded, trabecular, and a focal retiform growth. Rhabdoid cells were conspicuous, but only comprised ~50% of the tumor, and mitoses numbered up to 2/10 HPFs. All tumors were strongly and diffusely positive for WT1, CAM5.2, ER, and PR, but negative for inhibin. Diffuse calretinin and desmin expression, as well as focal melan-A positivity, was noted in one tumor, but was negative in the others. In all 3 tumors, INI-1 and BRG-1 were retained, and ESR1-NCOA2 fusions were detected by targeted RNA sequencing. This study is the first to highlight an association between UTROSCTs with extensive rhabdoid differentiation, ESR1-NCOA2 fusions, and aggressive behavior. UTROSCTs are considered neoplasms of uncertain malignant potential, but have a benign course in most cases. Thus, it is important to be aware of these specific features and recommend long-term follow-up due to their propensity for late recurrences.

Application of immunohistochemistry in diagnosis and management of malignant mesothelioma.

Immunohistochemistry plays an indispensable role in accurate diagnosis of malignant mesothelioma, particularly in morphologically challenging cases and in biopsy and cytology specimens, where tumor architecture is difficult or impossible to evaluate. Application of a targeted panel of mesothelial- and epithelial-specific markers permits correct identification of tumor lineage in the vast majority of cases. An immunopanel including two mesothelial markers (calretinin, CK5/6, WT-1, or D2-40) and two epithelial markers (MOC-31 and claudin-4) offers good sensitivity and specificity, with adjustments as appropriate for the differential diagnosis. Once mesothelial lineage is established, malignancy-specific studies can help verify a diagnosis of malignant mesothelioma. BAP1 loss, CDKN2A homozygous deletion, and MTAP loss are highly specific markers of malignancy in a mesothelial lesion, and they attain acceptable diagnostic sensitivity when applied as a diagnostic panel. Novel markers of malignancy, such as 5-hmC loss and increased EZH2 expression, are promising, but have not yet achieved widespread clinical adoption. Some diagnostic markers also have prognostic significance, and PD-L1 immunohistochemistry may predict tumor response to immunotherapy. Application and interpretation of these immnuomarkers should always be guided by clinical history, radiographic findings, and above all histomorphology.

Cooperation of genes in HPV16 E6/E7-dependent cervicovaginal carcinogenesis trackable by endoscopy and independent of exogenous estrogens or carcinogens.

Human papillomavirus (HPV) infection is necessary but insufficient for progression of epithelial cells from dysplasia to carcinoma-in situ (CIS) to invasive cancer. The combination of mutant cellular and viral oncogenes that regulate progression of cervical cancer (CC) remains unclear. Using combinations of HPV16 E6/E7 (E+), mutant Kras (mKras) (K+) and/or loss of Pten (P-/-), we generated autochthonous models of CC without exogenous estrogen, carcinogen or promoters. Furthermore, intravaginal instillation of adenoCre virus enabled focal activation of the oncogenes/inactivation of the tumor suppressor gene. In P+/+ mice, E6/E7 alone (P+/+E+K-) failed to cause premalignant changes, while mKras alone (P+/+E-K+) caused persistent mucosal abnormalities in about one-third of mice, but no cancers. To develop cancer, P+/+ mice needed both E6/E7 and mKras expression. Longitudinal endoscopies of P+/+E+K+ mice predicted carcinoma development by detection of mucosal lesions, found on an average of 23 weeks prior to death, unlike longitudinal quantitative PCRs of vaginal lavage samples from the same mice. Endoscopy revealed that individual mice differed widely in the time required for mucosal lesions to appear after adenoCre and in the time required for these lesions to progress to cancer. These cancers developed in the transition zone that extends, unlike in women, from the murine cervix to the distal vagina. The P-/-E+K+ genotype led to precipitous cancer development within a few weeks and E6/E7-independent cancer development occurred in the P-/-E-K+ genotype. In the P-/-E+K- genotype, mice only developed CIS. Thus, distinct combinations of viral and cellular oncogenes are involved in distinct steps in cervical carcinogenesis.

MTAP immunohistochemistry is an accurate and reproducible surrogate for CDKN2A fluorescence in situ hybridization in diagnosis of malignant pleural mesothelioma.

Ancillary studies facilitate accurate diagnosis of morphologically challenging mesothelial proliferations. The current diagnostic algorithm proceeds from BAP1 immunohistochemistry to CDKN2A fluorescence in situ hybridization. While MTAP immunohistochemistry has recently shown promise as a surrogate for CDKN2A fluorescence in situ hybridization, it has been examined in only a few single-institution studies. Furthermore, there are no published reports on interobserver agreement or interlaboratory reproducibility for MTAP immunohistochemistry. We performed MTAP immunohistochemistry on 20 benign mesothelial lesions and 99 malignant mesotheliomas from five mesothelioma centers in four countries, and each MTAP stain was independently interpreted by four pathologists. CDKN2A fluorescence in situ hybridization data were available for a subset of cases, and a subset of cases was subjected in MTAP immunohistochemistry in multiple laboratories to assess interlaboratory reproducibility. Interobserver agreement in MTAP immunostain interpretation was excellent for all mesothelial lesions (kappa: 0.85) and for malignant mesothelioma cases only (kappa: 0.82). Interlaboratory reproducibility was also excellent (kappa values for paired protocols: 0.77-0.89). MTAP loss by immunohistochemistry was 78% sensitive and 96% specific for CDKN2A homozygous deletion. MTAP immunohistochemistry is a reliable surrogate for CDKN2A fluorescence in situ hybridization in diagnosis of malignant mesothelioma. Interobserver agreement is excellent for interpretation of MTAP staining, and protocols performed in different laboratories yield concordant MTAP staining results. Rare cases with immunohistochemical MTAP loss may retain normal CDKN2A copy number, and the MTAP staining results should be correlated with clinicopathologic findings and other ancillary studies.

Loss of microfibril-associated protein 5 (MFAP5) expression in colon cancer stroma.

MFAP5, a 25-kD microfibril-associated glycoprotein that is involved in elastic microfibril assembly, has been demonstrated to be significantly downregulated in tumor stroma by previous gene expression study. The aim of this study was to confirm the reduced expression of MFAP5 in colonic tumor stroma using immunohistochemistry and to explore the utility of MFAP5 as a marker to facilitate diagnosing an invasive component versus pseudoinvasion in colon polyps. In all 19 colon cancer resection cases evaluated, while there was intact MFAP5 immunoreactivity in the uninvolved normal connective tissue, there was marked reduction of MFAP5 immunoreactivity in the desmoplastic stroma surrounding the invasive component. The difference in MFAP5 expression levels was most pronounced within the tumor, while a more heterogeneous expression pattern was observed at the tumor invasive front. Reduction of MFAP5 staining was also observed in the stroma around mucin pools in 6 out of 9 sections from mucinous adenocarcinomas and in areas with high-grade dysplasia. For the polypectomy cases, intact expression of MFAP5 was seen in the stroma surrounding the displaced adenomatous glands in 9 out of 12 polyps with pseudoinvasion. Loss of expression of MFAP5 was observed in the stroma surrounding small foci of invasive adenocarcinoma in 8 of 10 malignant polyps. MFAP5 is a useful marker that may help distinguish normal connective tissue from stroma within invasive colonic adenocarcinoma. MFAP5 may facilitate the distinction between pseudoinvasion and true invasive cancer in colonic adenomatous polyps with a sensitivity of 80% (confidence interval 44-96%) and a specificity of 75% (confidence interval 43-93%) in this small cohort.

Female adnexal tumors of probable Wolffian origin: morphological, immunohistochemical, and molecular analysis of 15 cases.

Female adnexal tumors of probable Wolffian origin are rare and present a diagnostic challenge due to their morphological and immunohistochemical overlap with more common ovarian and broad ligament entities. We evaluated the morphological, immunohistochemical, and molecular features of 15 tumors of probable Wolffian origin. Patients ranged from 32 to 69 (mean 47) years and tumors from 1.8 to 30 (mean 10) cm. All except one arose in para-adnexal soft tissues. Follow-up was available for six patients, five of whom were alive and well, while the sixth, who had extra-adnexal disease at diagnosis, died from unrelated causes. The following patterns were noted: tubular (all tumors), solid 11/15 (73%), sieve-like 7/15 (47%), and reticular 1/15 (7%). A myxoid background was present in 3/15 (20%) of tumors and eosinophilic luminal secretions in 11/15 (73%). Most tumors (12/15, 80%) had low-grade nuclear atypia, while three showed foci with scattered high-grade atypia. Mitotic index ranged from 0 to 17 (mean 4) per ten high-power fields. Tumors were positive for pankeratin and negative for TTF-1. EMA, GATA3, and PAX8 were positive in 2/10 (20%; focal), 3/15 (20%; focal), and 1/15 (7%; focal) of tumors, respectively. CD10, SF-1, calretinin, inhibin, ER, PR, cytokeratin 7, and WT1 were variably expressed. Pathogenic mutations were rare and included STK11 (n = 3), APC (n = 1), and MBD4 (n = 1). Copy number variations were detected in the three tumors with STK11 mutations and a myxoid background. These data demonstrate that female adnexal tumors of probable Wolffian origin are morphologically and immunohistochemically diverse, but infrequently harbor pathogenic mutations. However, their lack of mutations in contrast to their mimickers may be a valuable tool in diagnostically difficult cases.