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.

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.

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.

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.

Immunohistochemical evaluation of nuclear 5-hydroxymethylcytosine (5-hmC) accurately distinguishes malignant pleural mesothelioma from benign mesothelial proliferations.

Accurate distinction of benign mesothelial proliferations from malignant mesothelioma remains a diagnostic challenge. Sequential use of BAP1 immunohistochemistry and CDKN2A fluorescence in situ hybridization is specific for diagnosis of mesothelioma, but fluorescence in situ hybridization is both costly and time-consuming. Early data indicate that mesothelioma shows extensive loss of nuclear 5-hydroxymethylcytosine (5-hmC). We studied 49 cases of mesothelioma (17 epithelioid mesothelioma, 22 biphasic mesothelioma, and 10 sarcomatoid mesothelioma) and 23 benign mesothelial proliferations using a 5-hmC single immunohistochemical stain, CAM5.2/5-hmC double immunohistochemical stain, and BAP1 immunohistochemistry. Estimations of extent of 5-hmC loss were made using the 5-hmC single stain and CAM5.2/5-hmC double stain, and extent of nuclear 5-hmC loss was definitively quantitated in at least 1000 cells per case. Mean nuclear 5-hmC loss in mesothelioma (84%) was significantly greater than in benign mesothelial proliferations (4%) (p < 0.0001). Using 5-hmC loss in > 50% of tumor nuclei to define the diagnosis of mesothelioma, 5-hmC immunohistochemistry showed sensitivity of 92% and specificity of 100%. An immunopanel including 5-hmC and BAP1 immunohistochemistry achieved sensitivity of 98% and specificity of 100%. Extensive nuclear 5-hmC loss is sensitive and specific for mesothelioma in the differential diagnosis with benign mesothelial proliferations. In challenging mesothelial lesions, immunohistochemical studies showing either extensive 5-hmC loss or BAP1 loss indicate a diagnosis of mesothelioma, precluding the need for CDKN2A fluorescence in situ hybridization in a considerable number of cases.

Nuclear grade and necrosis predict prognosis in malignant epithelioid pleural mesothelioma: a multi-institutional study.

A recently described nuclear grading system predicted survival in patients with epithelioid malignant pleural mesothelioma. The current study was undertaken to validate the grading system and to identify additional prognostic factors. We analyzed cases of epithelioid malignant pleural mesothelioma from 17 institutions across the globe from 1998 to 2014. Nuclear grade was computed combining nuclear atypia and mitotic count into a grade of I-III using the published system. Nuclear grade was assessed by one pathologist for three institutions, the remaining were scored independently. The presence or absence of necrosis and predominant growth pattern were also evaluated. Two additional scoring systems were evaluated, one combining nuclear grade and necrosis and the other mitotic count and necrosis. Median overall survival was the primary endpoint. A total of 776 cases were identified including 301 (39%) nuclear grade I tumors, 354 (45%) grade II tumors and 121 (16%) grade III tumors. The overall survival was 16 months, and correlated independently with age (P=0.006), sex (0.015), necrosis (0.030), mitotic count (0.001), nuclear atypia (0.009), nuclear grade (<0.0001), and mitosis and necrosis score (<0.0001). The addition of necrosis to nuclear grade further stratified overall survival, allowing classification of epithelioid malignant pleural mesothelioma into four distinct prognostic groups: nuclear grade I tumors without necrosis (29 months), nuclear grade I tumors with necrosis and grade II tumors without necrosis (16 months), nuclear grade II tumors with necrosis (10 months) and nuclear grade III tumors (8 months). The mitosis-necrosis score stratified patients by survival, but not as well as the combination of necrosis and nuclear grade. This study confirms that nuclear grade predicts survival in epithelioid malignant pleural mesothelioma, identifies necrosis as factor that further stratifies overall survival, and validates the grading system across multiple institutions and among both biopsy and resection specimens. An alternative scoring system, the mitosis-necrosis score is also proposed.

An Ovarian Adenocarcinoma With Combined Low-grade Serous and Mesonephric Morphologies Suggests a Müllerian Origin for Some Mesonephric Carcinomas.

Mesonephric carcinomas are rare adenocarcinomas of the female genital tract that occur most commonly in the uterine cervix. They are classically thought to arise from benign mesonephric remnants, and are rarely reported at other sites in the gynecologic tract. Here we present an interesting biphenotypic ovarian adenocarcinoma with intimately associated but distinct components of both low-grade serous carcinoma and mesonephric-like carcinoma. A serous borderline tumor was present adjacent to the invasive carcinoma, and no benign mesonephric precursors were identified. Numerous invasive peritoneal metastases were present, including multiple metastases with both low-grade serous and mesonephric-like elements. Consistent with recent reports, foci of mesonephric-like carcinoma were morphologically and immunohistochemically identical to classic mesonephric carcinoma of the cervix. On molecular analysis, the serous borderline tumor, primary and metastatic low-grade serous carcinoma, and primary and metastatic mesonephric-like carcinoma each harbored a shared NRAS p.Q61R hotspot mutation, shared gains in chromosome 1q and 18p, and shared losses in chromosomes 1p, 18q, and 22. These shared molecular features indicate a clonal relationship between all morphologic elements of this ovarian adenocarcinoma, suggesting that at least some mesonephric carcinomas may arise from Müllerian precursors.

Epigenetic regulation of SMARCB1 By miR-206, -381 and -671-5p is evident in a variety of SMARCB1 immunonegative soft tissue sarcomas, while miR-765 appears specific for epithelioid sarcoma. A miRNA study of 223 soft tissue sarcomas.

Complete/partial loss of SMARCB1 nuclear-immunopositivity is characteristic of a certain subset of soft tissue sarcomas (STSs). Our previous work showed that oncomiRs-206,-381, and 671-5p could silence the SMARCB1 mRNA and protein expression and that they display significant overexpression in epithelioid sarcomas (ESs). MiR-765 was overexpressed too, but functionally was inactive in the silencing. In the current work, using quantitative PCR, we conducted a miRNA study of 51 ESs, 20 rhabdoid tumors (RTs), 20 synovial sarcomas (SSs), 15 malignant peripheral nerve sheath tumors (MPNSTs), 11 myoepithelial carcinomas (MECs), and 10 extraskeletal myxoid chondrosarcomas (EMCSs) with complete/partial loss of SMARCB1 nuclear immunostain, in contrast to controls (SMARCB1-immunopositive) of 96 STSs, 13 melanomas and 10 sarcomatoid carcinomas. The SMARCB1 genetic status of ESs was determined by MLPA and FISH. A subset of ESs (5/51) showed biallelic deletion of SMARCB1 with no overexpression of any miRNA, suggesting these tumors could be the counterpart of pediatric RT, at least genetically. Another subset (5/51) was genetically either intact or monoallelic deleted with at least threefold overexpression of one of miR-206,-381,-671-5p, suggesting epigenetic regulation only. 39/51 ESs had a biallelic deletion (>20% by FISH and/or by MLPA) but with overexpressed miR-206,-381, and 671-5p, suggesting intratumoral heterogeneity, i.e., both genetic and epigenetic regulation. At least threefold overexpression of one of miR-206,-381, and 671-5p was detected in all MPNSTs, EMCSs, SSs and 7 MCs. Except for ESs, four SSs and one MPNST, there was no event above threefold overexpression of miR-765 among all 195 tested tumors. Our results suggest a general role of miR-206,-381, and 671-5p in SMARCB1 gene silencing of ES, MC, EMCS, MPNST and SS. In the future, miR-765 could possibly be a diagnostic tool for ES because of its 97% specificity and 80% sensitivity. © 2016 Wiley Periodicals, Inc.

Melanocytic tumors with intraepidermal melanophages: a report of five cases with review of 231 archived cutaneous melanocytic tumors.

Dermal melanophages are frequently encountered in both benign melanocytic nevi and malignant melanoma. In contrast, intraepidermal melanophages (IEM) are under-recognized in melanocytic lesions and their biologic significance is not understood. Herein, we report the clinical and histopathologic features of five melanocytic lesions featuring IEM encountered prospectively in our dermatopathology practice at the University of Chicago. Two hundred and thirty-one (231) archived skin primary melanocytic proliferations were also investigated retrospectively in a de-identified, archival teaching set collection. Nineteen of 231 of the archived cases were positive for IEM. Among the total 24 IEM-positive cases (5 prospective and 19 archived cases), 13 were categorized as Spitz nevi (p < 0.0001) and 3 as atypical Spitz tumors (p = 0.0152). Fourteen of 24 cases with IEM also exhibited intracorneal melanocytes (p < 0.0001). IEM are evidently not rare, especially in spitzoid melanocytic neoplasms. IEM in our series were significantly correlated with intracorneal melanocytosis, possibly indicating an association between IEM and suprabasal melanocytosis and/or transepidermal elimination of melanocytes.

SMARCB1 expression in epithelioid sarcoma is regulated by miR-206, miR-381, and miR-671-5p on Both mRNA and protein levels.

Proximal type epithelioid sarcoma shares similarities with malignant rhabdoid tumor, including the lack of nuclear immunoreactivity of SMARCB1. Biallelic mutation of SMARCB1 has been convincingly established as the cause of loss of protein expression in rhabdoid tumor, but the cause in epithelioid sarcoma remains unknown. In our previous work, we demonstrated that DNA hypermethylation and post-translational modification mechanisms were not involved. In this current work, we explored the hypothesis that miRNAs regulate SMARCB1 gene expression in epithelioid sarcomas. In silico target prediction analysis revealed eight candidate miRNAs, and quantitative PCR-in 32 formalin-fixed, paraffin-embedded tumor samples comprising 30 epithelioid sarcomas and two malignant rhabdoid tumors-demonstrated significant (P < 0.001) overexpression of four miRNAs in epithelioid sarcomas: miR-206, miR-381, miR-671-5p, and miR-765. Two human tumors (fibrosarcoma and colon adenocarcinoma) and a normal cell line (human dermal fibroblast) with retained SMARCB1 expression were cultured for miRNA transient transfection (electroporation) experiments. SMARCB1 mRNA expression was analyzed by quantitative real-time PCR and immunostaining of SMARCB1 was performed to examine the effect of miRNAs transfections on both RNA and protein levels. Only three of the overexpressed miRNAs (miR-206, miR-381, and miR-671-5p) could silence the SMARCB1 mRNA expression in cell cultures; most effectively miR-206. Transfection of miR-206, miR-381, miR-671-5p, and some combination of them also eliminated SMARCB1 nuclear staining, demonstrating a strong effect on not only mRNA but also protein levels. Our results suggest loss of SMARCB1 protein expression in epithelioid sarcoma is due to the epigenetic mechanism of gene silencing by oncomiRs.

ZFP36-FOSB fusion defines a subset of epithelioid hemangioma with atypical features.

Epithelioid hemangioma (EH) is a benign neoplasm with distinctive vasoformative features, which occasionally shows increased cellularity, cytologic atypia, and/or loco-regional aggressive growth, resulting in challenging differential diagnosis from malignant vascular neoplasms. Based on two intraosseous EH index cases with worrisome histologic features, such as the presence of necrosis, RNA sequencing was applied for possible fusion gene discovery and potential subclassification of a novel atypical EH subset. A ZFP36-FOSB fusion was detected in one case, while a WWTR1-FOSB chimeric transcript in the other, both were further validated by fluorescence in situ hybridization (FISH) and reverse transcription polymerase chain reaction (RT-PCR). These abnormalities were then screened by FISH in 44 EH from different locations with seven additional EH revealing FOSB gene rearrangements, all except one being fused to ZFP36. Interestingly, 4/6 penile EH studied showed FOSB abnormalities. Although certain atypical histologic features were observed in the FOSB-rearranged EH, including solid growth, increased cellularity, mild to moderate nuclear pleomorphism, and necrosis in 3/9 cases, no overt sarcomatous areas were discerned to objectively separate the lesions from the fusion-negative EH. No patient has developed recurrence to date, but the follow-up was relatively limited and short to draw definitive conclusions regarding behavior. Although FOSB-rearranged EH do not show significant morphologic overlap with SERPINE1-FOSB fusion-positive pseudomyogenic hemangioendothelioma, FOSB oncogenic activation is emerging as an important event in these benign and intermediate groups of vascular tumors.

Evaluation of clonal origin of malignant mesothelioma.

BACKGROUND: The hypothesis that most cancers are of monoclonal origin is often accepted as a fact in the scientific community. This dogma arose decades ago, primarily from the study of hematopoietic malignancies and sarcomas, which originate as monoclonal tumors. The possible clonal origin of malignant mesothelioma (MM) has not been investigated. Asbestos inhalation induces a chronic inflammatory response at sites of fiber deposition that may lead to malignant transformation after 30-50 years latency. As many mesothelial cells are simultaneously exposed to asbestos fibers and to asbestos-induced inflammation, it may be possible that more than one cell undergoes malignant transformation during the process that gives rise to MM, and result in a polyclonal malignancy. METHODS AND RESULTS: To investigate the clonality patterns of MM, we used the HUMARA (Human Androgen Receptor) assay to examine 16 biopsies from 14 women MM patients. Out of 16 samples, one was non-informative due to skewed Lyonization in its normal adjacent tissue. Fourteen out of the 15 informative samples revealed two electrophoretically distinct methylated HUMARA alleles, the Corrected Allele Ratio (CR) calculated on the allele peak areas indicating polyclonal origin MM. CONCLUSIONS: Our results show that MM originate as polyclonal tumors and suggest that the carcinogenic "field effect" of mineral fibers leads to several premalignant clones that give rise to these polyclonal malignancies.

Novel YAP1-TFE3 fusion defines a distinct subset of epithelioid hemangioendothelioma.

Conventional epithelioid hemangioendotheliomas (EHE) have a distinctive morphologic appearance and are characterized by a recurrent t(1;3) translocation, resulting in a WWTR1-CAMTA1 fusion gene. We have recently encountered a fusion-negative subset characterized by a somewhat different morphology, including focally well-formed vasoformative features, which was further investigated for recurrent genetic abnormalities. Based on a case showing strong transcription factor E3 (TFE3) immunoreactivity, fluorescence in situ hybridization (FISH) analysis for TFE3 gene rearrangement was applied to the index case as well as to nine additional cases, selected through negative WWTR1-CAMTA1 screening. A control group, including 18 epithelioid hemangiomas, nine pseudomyogenic HE, and three epithelioid angiosarcomas, was also tested. TFE3 gene rearrangement was identified in 10 patients, with equal gender distribution and a mean age of 30 years old. The lesions were located in somatic soft tissue in six cases, lung in three and one in bone. One case with available frozen tissue was tested by RNA sequencing and FusionSeq data analysis to detect novel fusions. A YAP1-TFE3 fusion was thus detected, which was further validated by FISH and reverse transcription polymerase chain reaction (RT-PCR). YAP1 gene rearrangements were then confirmed in seven of the remaining nine TFE3-rearranged EHEs by FISH. No TFE3 structural abnormalities were detected in any of the controls. The TFE3-rearranged EHEs showed similar morphologic features with at least focally, well-formed vascular channels, in addition to a variably solid architecture. All tumors expressed endothelial markers, as well as strong nuclear TFE3. In summary, we are reporting a novel subset of EHE occurring in young adults, showing a distinct phenotype and YAP1-TFE3 fusions.

Histologic Features of Mycobacterial Spindle Cell Pseudotumors: A Multi-institutional Clinicopathologic Analysis of 14 Cases.

Mycobacterial spindle cell pseudotumors (MSPs) are a rare and diagnostically challenging manifestation of non-tuberculous mycobacterial (NTM) infections. Proper recognition of these pseudotumors is important because they are treatable and benign. In this study, we evaluated the morphologic patterns of MSPs to improve their pathologic identification. Clinical and morphologic features of 14 MSPs were analyzed. Histologic factors evaluated included the architectural growth pattern of spindled or epithelioid macrophages, granulomas and their location within the lesion, neutrophilic microabscesses, multinucleated giant cells, necrosis, and effacement of background tissue. The composition of inflammatory infiltrates, organism density by acid-fast staining, and stromal changes were also assessed. In addition, 8 of 14 cases underwent molecular microbiology identification by a clinical amplicon-sequencing assay for non-tuberculous mycobacteria. MSP sites included 2 bowel, 10 lymph nodes, 1 liver, and 1 extremity. Cases with available clinical history (n=10) all occurred in immunocompromised patients. All demonstrated effacement of normal structures with spindled cells arranged in a storiform or fascicular architectural pattern. In addition, all cases showed lymphocytic inflammation, with prominent concurrent neutrophilic inflammation in 50% (7/14) of cases. Other morphologic findings included foamy histiocytes (64%, 9/14), peripherally situated granulomas (21%, 3/14), and neutrophilic microabscesses (21%, 3/14). All tested cases were positive for NTM by PCR methods. Mycobacterium avium was the most commonly isolated pathogen (6/8). Mycobacterial spindle cell pseudotumors show predominantly spindled morphology that may be mistaken as a neoplasm. Surgical pathologists who evaluate lymph nodes, soft tissue, and gastrointestinal tissues should be aware of this spindled tumefactive phenomenon in the setting of immunocompromised patients. Recognition of key morphologic features of neutrophilic inflammation, peripheral granulomas, or foamy histiocytes within a spindled lesion can help guide the pathologist to a correct diagnosis of an inflammatory process secondary to infection rather than a spindle cell neoplasm. Accurate diagnosis to facilitate appropriate antimicrobial and/or surgical therapy requires a comprehensive evaluation combining clinical, histopathologic, and microbiological findings.

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.

Expression profiling of 519 kinase genes in matched malignant peripheral nerve sheath tumor/plexiform neurofibroma samples is discriminatory and identifies mitotic regulators BUB1B, PBK and NEK2 as overexpressed with transformation.

About 50% of all malignant peripheral nerve sheath tumors (MPNSTs) arise as neurofibromatosis type 1 associated lesions. In those patients malignant peripheral nerve sheath tumors are thought to arise through malignant transformation of a preexisting plexiform neurofibroma. The molecular changes associated with this transformation are still poorly understood. We sought to test the hypothesis that dysregulation of expression of kinases contributes to this malignant transformation. We analyzed expression of all 519 kinase genes in the human genome using the nanostring nCounter system. Twelve cases of malignant peripheral nerve sheath tumor arising in a background of preexisting plexiform neurofibroma were included. Both components were separately sampled. Statistical analysis compared global changes in expression levels as well as changes observed in the pairwise comparison of samples taken from the same surgical specimen. Immunohistochemical studies were performed on tissue array slides to confirm expression of selected proteins. The expression pattern of kinase genes can separate malignant peripheral nerve sheath tumors and preexisting plexiform neurofibromas. The majority of kinase genes is downregulated rather than overexpressed with malignant transformation. The patterns of expression changes are complex without simple recurring alteration. Pathway analysis demonstrates that differentially expressed kinases are enriched for kinases involved in the direct regulation of mitosis, and several of these show increased expression in malignant peripheral nerve sheath tumors. Immunohistochemical studies for the mitotic regulators BUB1B, PBK and NEK2 confirm higher expression levels at the protein level. These results suggest that the malignant transformation of plexiform neurofibroma is associated with distinct changes in the expression of kinase genes. The patterns of these changes are complex and heterogeneous. There is no single unifying alteration. Kinases involved in mitotic regulation are particularly enriched in the pool of differentially expressed kinases. Some of these are overexpressed and are therefore possible targets for kinase inhibitors.

SMARCB1 protein and mRNA loss is not caused by promoter and histone hypermethylation in epithelioid sarcoma.

About 10% of epithelioid sarcomas have biallelic mutation of the SMARCB1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily b, member 1) gene resulting in a lack of this nuclear protein. It has been suggested that SMARCB1 may be silenced by epigenetic changes in the remaining 90% of tumors. Thus, we hypothesized that the promoter of SMARCB1 is hypermethylated. We also examined SMARCB1 mRNA level to determine if a post-translational change was possible. Thirty-six cases of epithelioid sarcomas were studied. Immunohistochemistry and mutation analysis of the SMARCB1 gene were performed to select appropriate cases. Methylation status was assessed by methylation-specific PCR. Laser capture microdissection of tumor cells followed by real-time PCR was applied to examine the expression of SMARCB1 mRNA. Of 36 epithelioid sarcomas, 31 (86%) displayed a lack of SMARCB1 nuclear protein. In all, 4 (13%) of 31 SMARCB1-negative cases harbored biallelic deletion while 9 (33%) cases showed single-allelic deletion. One (4%) frameshift deletion of exon 3 and one point mutation of exon 7 were also found. In 16 (59%) cases, both alleles were intact. Altogether, 25/31 (81%) SMARCB1-negative cases had at least one intact allele. None of these cases demonstrated promoter hypermethylation. Low levels of SMARCB1 mRNA were found in all cases with tumor tissue extracted RNA (because of the minimal normal cell contamination) but no mRNA could be detected in laser dissected cases (containing only tumor cells). Enhancer of zeste homolog 2 (EZH2) overexpression was not characteristic of epithelioid sarcoma. Thus, loss of SMARCB1 expression in epithelioid sarcoma is caused neither by DNA hypermethylation nor by post-translational modifications. Most likely it is the microRNA destruction of SMARCB1 mRNA but further investigations are needed to elucidate this issue.

Programmed necrosis induced by asbestos in human mesothelial cells causes high-mobility group box 1 protein release and resultant inflammation.

Asbestos carcinogenesis has been linked to the release of cytokines and mutagenic reactive oxygen species (ROS) from inflammatory cells. Asbestos is cytotoxic to human mesothelial cells (HM), which appears counterintuitive for a carcinogen. We show that asbestos-induced HM cell death is a regulated form of necrosis that links to carcinogenesis. Asbestos-exposed HM activate poly(ADP-ribose) polymerase, secrete H(2)O(2), deplete ATP, and translocate high-mobility group box 1 protein (HMGB1) from the nucleus to the cytoplasm, and into the extracellular space. The release of HMGB1 induces macrophages to secrete TNF-alpha, which protects HM from asbestos-induced cell death and triggers a chronic inflammatory response; both favor HM transformation. In both mice and hamsters injected with asbestos, HMGB1 was specifically detected in the nuclei, cytoplasm, and extracellular space of mesothelial and inflammatory cells around asbestos deposits. TNF-alpha was coexpressed in the same areas. HMGB1 levels in asbestos-exposed individuals were significantly higher than in nonexposed controls (P < 0.0001). Our findings identify the release of HMGB1 as a critical initial step in the pathogenesis of asbestos-related disease, and provide mechanistic links between asbestos-induced cell death, chronic inflammation, and carcinogenesis. Chemopreventive approaches aimed at inhibiting the chronic inflammatory response, and especially blocking HMGB1, may decrease the risk of malignant mesothelioma among asbestos-exposed cohorts.

Hereditary hemochromatosis restores the virulence of plague vaccine strains.

Nonpigmented Yersinia pestis (pgm) strains are defective in scavenging host iron and have been used in live-attenuated vaccines to combat plague epidemics. Recently, a Y. pestis pgm strain was isolated from a researcher with hereditary hemochromatosis who died from laboratory-acquired plague. We used hemojuvelin-knockout (Hjv(-/-)) mice to examine whether iron-storage disease restores the virulence defects of nonpigmented Y. pestis. Unlike wild-type mice, Hjv(-/-) mice developed lethal plague when challenged with Y. pestis pgm strains. Immunization of Hjv(-/-) mice with a subunit vaccine that blocks Y. pestis type III secretion generated protection against plague. Thus, individuals with hereditary hemochromatosis may be protected with subunit vaccines but should not be exposed to live-attenuated plague vaccines.