Homozygous deletion of the DSG3 terminal exon associated with acantholytic blistering of the oral and laryngeal mucosa.

We report a novel homozygous 49.6 kb deletion of chromosome 18q12.1 involving the last exon of DSG3 in dizygotic twins with phenotype consistent with acantholytic blistering of the oral and laryngeal mucosa (ABOLM). The twin siblings presented predominantly with friability of the laryngeal and respiratory mucosa. This is only the second report in the literature of this unusual autosomal recessive blistering disorder. The diagnosis explains the mucosal phenotype of a pemphigus-like disorder without evidence of autoimmune dysfunction. The exclusion of an autoimmune basis has management implications. The deletion also involved the DSG2 gene, which is associated with arrhythmogenic right ventricular dysplasia (ARVD). The affected siblings and heterozygous parents do not show any cardiac phenotype at this time. Functional studies would further clarify how deletions resulting in loss of function of DSG3 may cause the reported phenotypes of DSG3-related ABOLM.

Erythrophagocytosis is not a reproducible finding in liver biopsies, and is not associated with clinical diagnosis of hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a rare disease with high mortality. Liver involvement is common (based on elevated liver function tests) with most patients demonstrating acute hepatitis. Liver biopsies are frequently obtained in the setting of suspected HLH for the purpose of identification of erythrophagocytosis, and if present, this finding is thought to suggest or support the diagnosis of HLH. However, there are problems with this approach; in particular, we do not know whether this finding is reproducible or whether it is specific to HLH. Therefore, we conducted a multi-institutional study in which experienced liver pathologists reviewed images taken from liver biopsies from patients with normal liver, acute hepatitis, possible HLH, and clinical HLH to determine if there was agreement about the presence or absence of erythrophagocytosis, and to ascertain whether the finding corresponds to a clinical diagnosis of HLH. Twelve liver pathologists reviewed 141 images in isolation (i.e., no clinical information or diagnosis provided). These came from 32 patients (five normal, 17 acute hepatitis, six HLH, four possible HLH). The pathologists classified each image as negative, equivocal, or positive for erythrophagocytosis. Kappa was .08 (no agreement) for case-level and 0.1 for image-level (1.4% agreement, based on two images which were universally considered negative). There was no difference in the proportion of pathologists who diagnosed erythrophagocytosis among those with different diagnoses at case or image-level (p = 0.82 and p = 0.82, respectively). Thus, erythrophagocytosis is an entirely unreliable histologic parameter in liver, as it is irreproducible and not demonstrably associated with a clinical disease (namely, HLH). Unless and until more reliable guidelines can be established, pathologists should refrain from commenting on the presence or absence of erythrophagocytosis in liver biopsy.

A Therapeutically Targetable TAZ-TEAD2 Pathway Drives the Growth of Hepatocellular Carcinoma via ANLN and KIF23.

BACKGROUND & AIMS: Despite recent progress, long-term survival remains low for hepatocellular carcinoma (HCC). The most effective HCC therapies target the tumor immune microenvironment (TIME), and there are almost no therapies that directly target tumor cells. Here, we investigated the regulation and function of tumor cell-expressed Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in HCC. METHODS: HCC was induced in mice by Sleeping Beauty-mediated expression of MET, CTNNB1-S45Y, or TAZ-S89A, or by diethylnitrosamine plus CCl4. Hepatocellular TAZ and YAP were deleted in floxed mice via adeno-associated virus serotype 8-mediated expression of Cre. TAZ target genes were identified from RNA sequencing, confirmed by chromatin immunoprecipitation, and evaluated in a clustered regularly interspaced short palindromic repeats interference (CRISPRi) screen. TEA domain transcription factors (TEADs), anillin (ANLN), Kif23, and programmed cell death protein ligand 1 were knocked down by guide RNAs in dead clustered regularly interspaced short palindromic repeats-associated protein 9 (dCas9) knock-in mice. RESULTS: YAP and TAZ were up-regulated in murine and human HCC, but only deletion of TAZ consistently decreased HCC growth and mortality. Conversely, overexpression of activated TAZ was sufficient to trigger HCC. TAZ expression in HCC was regulated by cholesterol synthesis, as demonstrated by pharmacologic or genetic inhibition of 3-hydroxy-3-methylglutaryl- coenzyme A reductase (HMGCR), farnesyl pyrophosphate synthase, farnesyl-diphosphate farnesyltransferase 1 (FDFT1), or sterol regulatory element-binding protein 2 (SREBP2). TAZ- and MET/CTNNB1-S45Y-driven HCC required the expression of TEAD2 and, to a lesser extent, TEAD4. Accordingly, TEAD2 displayed the most profound effect on survival in patients with HCC. TAZ and TEAD2 promoted HCC via increased tumor cell proliferation, mediated by TAZ target genes ANLN and kinesin family member 23 (KIF23). Therapeutic targeting of HCC, using pan-TEAD inhibitors or the combination of a statin with sorafenib or anti-programmed cell death protein 1, decreased tumor growth. CONCLUSIONS: Our results suggest the cholesterol-TAZ-TEAD2-ANLN/KIF23 pathway as a mediator of HCC proliferation and tumor cell-intrinsic therapeutic target that could be synergistically combined with TIME-targeted therapies.

Validation of a non-oncogene encoded vulnerability to exportin 1 inhibition in pediatric renal tumors.

BACKGROUND: Malignant rhabdoid tumors (MRTs) and Wilms' tumors (WTs) are rare and aggressive renal tumors of infants and young children comprising ∼5% of all pediatric cancers. MRTs are among the most genomically stable cancers, and although WTs are genomically heterogeneous, both generally lack therapeutically targetable genetic mutations. METHODS: Comparative protein activity analysis of MRTs (n = 68) and WTs (n = 132) across TCGA and TARGET cohorts, using metaVIPER, revealed elevated exportin 1 (XPO1) inferred activity. In vitro studies were performed on a panel of MRT and WT cell lines to evaluate effects on proliferation and cell-cycle progression following treatment with the selective XPO1 inhibitor selinexor. In vivo anti-tumor activity was assessed in patient-derived xenograft (PDX) models of MRTs and WTs. FINDINGS: metaVIPER analysis identified markedly aberrant activation of XPO1 in MRTs and WTs compared with other tumor types. All MRT and most WT cell lines demonstrated baseline, aberrant XPO1 activity with in vitro sensitivity to selinexor via cell-cycle arrest and induction of apoptosis. In vivo, XPO1 inhibitors significantly abrogated tumor growth in PDX models, inducing effective disease control with sustained treatment. Corroborating human relevance, we present a case report of a child with multiply relapsed WTs with prolonged disease control on selinexor. CONCLUSIONS: We report on a novel systems-biology-based comparative framework to identify non-genetically encoded vulnerabilities in genomically quiescent pediatric cancers. These results have provided preclinical rationale for investigation of XPO1 inhibitors in an upcoming investigator-initiated clinical trial of selinexor in children with MRTs and WTs and offer opportunities for exploration of inferred XPO1 activity as a potential predictive biomarker for response. FUNDING: This work was funded by CureSearch for Children's Cancer, Alan B. Slifka Foundation, NIH (U01 CA217858, S10 OD012351, and S10 OD021764), Michael's Miracle Cure, Hyundai Hope on Wheels, Cannonball Kids Cancer, Conquer Cancer the ASCO Foundation, Cycle for Survival, Paulie Strong Foundation, and the Grayson Fund.

Neoadjuvant chemoradiation alters the immune microenvironment in pancreatic ductal adenocarcinoma.

Patients with pancreatic ductal adenocarcinoma (PDAC) have a grim prognosis despite complete surgical resection and intense systemic therapies. While immunotherapies have been beneficial with many different types of solid tumors, they have almost uniformly failed in the treatment of PDAC. Understanding how therapies affect the tumor immune microenvironment (TIME) can provide insights for the development of strategies to treat PDAC. We used quantitative multiplexed immunofluorescence (qmIF) quantitative spatial analysis (qSA), and immunogenomic (IG) analysis to analyze formalin-fixed paraffin embedded (FFPE) primary tumor specimens from 44 patients with PDAC including 18 treated with neoadjuvant chemoradiation (CRT) and 26 patients receiving no treatment (NT) and compared them with tissues from 40 treatment-naïve melanoma patients. We find that relative to NT tumors, CD3+ T cell infiltration was increased in CRT treated tumors (p = .0006), including increases in CD3+CD8+ cytotoxic T cells (CTLs, p = .0079), CD3+CD4+FOXP3- T helper cells (Th, p = .0010), and CD3+CD4+FOXP3+ regulatory T cells (Tregs, p = .0089) with no difference in CD68+ macrophages. IG analysis from micro-dissected tissues indicated overexpression of genes involved in antigen presentation, T cell activation, and inflammation in CRT treated tumors. Among treated patients, a higher ratio of Tregs to total T cells was associated with shorter survival time (p = .0121). Despite comparable levels of infiltrating T cells in CRT PDACs to melanoma, PDACs displayed distinct spatial profiles with less T cell clustering as defined by nearest neighbor analysis (p < .001). These findings demonstrate that, while CRT can achieve high T cell densities in PDAC compared to melanoma, phenotype and spatial organization of T cells may limit benefit of T cell infiltration in this immunotherapy-resistant tumor.

In situ hybridisation for albumin RNA in paediatric liver cancers compared with common immunohistochemical markers.

AIMS: In situ hybridisation (ISH) for albumin mRNA is a sensitive marker of primary liver tumours in adults. However, paediatric tumours, such as hepatoblastoma (HB) and fibrolamellar hepatocellular carcinoma (FLC), have not been tested thoroughly and may require ancillary tests to diagnose with confidence. We aim to determine if albumin ISH is useful in the pathological evaluation of these malignancies and to compare it to commonly used immunohistochemical markers HepPar 1 (HEPA) and arginase-1 (ARG). METHODS: Tissue microarrays of 26 HB and 10 FLC were constructed. Controls included 4 embryonal undifferentiated sarcomas of the liver, 51 neuroblastomas and 64 Wilms tumours. We evaluated a commercially available RNA ISH to detect albumin mRNA. Immunohistochemistry for HEPA and ARG was performed in the usual fashion. RESULTS: Twenty-six of 26 HB showed positive staining by albumin ISH including 14 fetal, 8 embryonal and 4 mixed variants. All 10 FLC were diffusely positive. The sensitivity and specificity of albumin ISH were 100% for HB and FLC. ARG had 100% sensitivity and specificity for HB (26 of 26 cases) and FLC (9 of 9). HEPA stained 22 of 26 HB (85% sensitivity, 99.2% specificity) and 7 of 9 FLC (78% sensitivity, 99.1% specificity). CONCLUSION: Albumin RNA ISH is a useful test to determine hepatocytic origin in HB and FLC. ARG was equally sensitive and easy to interpret, while HEPA was inferior to both in HB and FLC.

Detection of STRN-ALK fusion in thyroid nodules with indeterminate cytopathology facilitates papillary thyroid cancer diagnosis.

Thyroid cancer is the most common endocrine malignancy. Approximately 70% of cases of papillary thyroid carcinoma and 50% of poorly differentiated and anaplastic thyroid carcinoma harbor well-characterized driver mutations and chromosomal rearrangements that drive tumorigenesis. Molecular profiling has been helpful in identifying and informing follow-up strategies in tumors with more aggressive trajectories. Here, we report a case of papillary thyroid cancer (PTC) discovered in a patient with thyroid nodules with relatively benign ultrasound and fine needle aspiration (FNA) findings. Molecular testing in this patient identified a rare STRN-ALK fusion in two thyroid nodules with indeterminate and/or benign cytology. This led to the patient undergoing a thyroid lobectomy and a subsequent confirmation of papillary thyroid carcinoma upon resection. The report highlights the role of comprehensive molecular testing in thyroid lesions of indeterminate cytology.

Hepatic pathology in patients dying of COVID-19: a series of 40 cases including clinical, histologic, and virologic data.

The novel coronavirus SARS-CoV-2 (coronavirus disease 19, or COVID-19) primarily causes pulmonary injury, but has been implicated to cause hepatic injury, both by serum markers and histologic evaluation. The histologic pattern of injury has not been completely described. Studies quantifying viral load in the liver are lacking. Here we report the clinical and histologic findings related to the liver in 40 patients who died of complications of COVID-19. A subset of liver tissue blocks were subjected to polymerase chain reaction (PCR) for viral ribonucleic acid (RNA). Peak levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were elevated; median ALT peak 68 U/l (normal up to 46 U/l) and median AST peak 102 U/l (normal up to 37 U/l). Macrovesicular steatosis was the most common finding, involving 30 patients (75%). Mild lobular necroinflammation and portal inflammation were present in 20 cases each (50%). Vascular pathology, including sinusoidal microthrombi, was infrequent, seen in six cases (15%). PCR of liver tissue was positive in 11 of 20 patients tested (55%). In conclusion, we found patients dying of COVID-19 had biochemical evidence of hepatitis (of variable severity) and demonstrated histologic findings of macrovesicular steatosis and mild acute hepatitis (lobular necroinflammation) and mild portal inflammation. We also identified viral RNA in a sizeable subset of liver tissue samples.

Malignant Rhabdoid Tumor, an Aggressive Tumor Often Misclassified as Small Cell Variant of Hepatoblastoma.

The clinical management of pediatric liver tumors involves stratification into risk groups. One previously defined, high-risk group of hepatoblastomas is the small cell undifferentiated variant. In light of molecular studies showing SMARCB1 deletion in these tumors, it is now recognized that most small cell, undifferentiated liver tumors represent an aggressive unrelated tumor-the malignant rhabdoid tumor (MRT). SMARCB1 is a member of the chromatin remodeling SWI/SNF complex and encodes the INI1 protein. The histologic diagnosis of MRT is currently based on INI1 negative immunoreactivity and the presence of rhabdoid morphology. INI1-negative small cell liver tumors lacking classic rhabdoid morphology are often misclassified as small cell undifferentiated hepatoblastomas (SCUD-HB), according to the current classification. Pediatric liver tumors diagnosed between 2003-2017 as SCUD-HB (four cases) or MRT (two cases) were identified from the Columbia University Pathology Department Archives. All tumors were associated with normal or low serum alpha fetoprotein levels, and showed an absence of immunohistochemical staining of hepatocellular markers (Hep-par1, Arginase) and loss of INI1 staining. Two cases were initially diagnosed as MRT, one with prominent rhabdoid morphology, the other with predominant small cell morphology. The remaining four cases with small cell morphology were classified as SCUD-HB. Ancillary molecular studies confirmed the loss of SMARCB1, supporting the diagnosis of MRT in all cases, proving morphology an unreliable criterion. It is critical to eliminate the term INI1-negative hepatoblastoma from the current classification scheme, and classify INI1-negative tumors as MRT, particularly since high-risk HB-chemotherapy regimens are not effective for treating MRT.

HER2 Heterogeneity in Gastroesophageal Cancer Detected by Testing Biopsy and Resection Specimens.

CONTEXT: - In advanced gastric, esophageal, and gastroesophageal junction adenocarcinomas (GE-GEJ-AC) that overexpress ERBB2 (erb-b2 receptor tyrosine kinase 2 or HER2), anti-HER2 monoclonal antibody therapy confers survival benefit. To select patients for treatment, HER2 expression and gene amplification are evaluated by immunohistochemistry (IHC) and in situ hybridization. OBJECTIVE: - To determine whether GE-GEJ-AC tested for HER2 on biopsy specimens of a primary tumor show different IHC scores and/or HER2 amplification by in situ hybridization in matched resection specimens, potentially changing therapy eligibility. DESIGN: - Immunohistochemistry and silver in situ hybridization were performed in biopsy and/or resection specimens from 100 patients. HER2 testing was performed in matched resection and biopsy specimens of 15 cases to determine whether GE-GEJ-AC with IHC scores of 0, 1+, and 2+ in biopsy and resection specimens had different IHC and silver in situ hybridization results. RESULTS: - The IHC 3+ cases showed HER2 amplification in 4 of 5 cases (80%), and IHC scores of 0, 1+, and 2+ showed 3.5%, 14.3%, and 23.5% HER2 amplification by silver in situ hybridization. Among the 15 paired biopsy and resection specimens, 9 (60%) had at least pT2 stage GE-GEJ-AC with HER2 IHC scores of 0, 1+, or 2+ in the biopsy, and 2 of those 9 cases (22%) had IHC 3+ and HER2 amplification by silver in situ hybridization on the resection specimen. CONCLUSIONS: - Our data suggest that HER2 testing should be repeated on resection specimens of GE-GEJ-AC with HER2 IHC scores of negative (0 and 1+) or equivocal (2+) and in situ hybridization amplification negative biopsy specimen results to evaluate for HER2 heterogeneity when patients are being considered for anti-HER2 therapy.

PD-L1 expression in non-small cell lung carcinoma: Comparison among cytology, small biopsy, and surgical resection specimens.

BACKGROUND: One immunotherapeutic agent for patients with advanced non-small cell lung carcinoma, pembrolizumab, has a companion immunohistochemistry (IHC)-based assay that predicts response by quantifying programmed death-ligand 1 (PD-L1) expression. The current study assessed the feasibility of quantifying PD-L1 expression using cytologic non-small cell lung carcinoma specimens and compared the results with those from small biopsy and surgical resection specimens. METHODS: PD-L1 expression was quantified using the IHC-based 22C3 pharmDx assay, with "positivity" defined as staining in ≥50% viable tumor cells; ≥ 100 tumor cells were required for test adequacy. For cytology specimens, IHC was performed on cell block sections. RESULTS: A total of 214 specimens were collected from 188 patients, 206 of which (96%) were found to be adequately cellular, including 36 of 40 cytology (90%) and 69 of 72 small biopsy (96%) specimens. There was no significant difference noted with regard to the feasibility of PD-L1 IHC on small biopsy specimens compared with surgical resection specimens (P = .99), or between the percentage of PD-L1-positive cytology and histology (including surgical resection and histologic small biopsy) specimens (P = .083). PD-L1 expression was found to be concordant among samples from 21 of 23 patients from whom > 1 specimen was collected (91%). There also was no significant difference observed with regard to rates of PD-L1 positivity when comparing age, sex, diagnosis, and specimen site. CONCLUSIONS: Quantification of PD-L1 expression is feasible on cytology specimens, and the results are comparable to those obtained from surgical resection and small biopsy specimens, including in matched specimens and using a single predictive IHC marker. Future studies will be necessary to determine the comparative value of other antibodies and their ability to predict response to immunotherapy. Cancer Cytopathol 2017;125:896-907. © 2017 American Cancer Society.

Inactivation of the tumor suppressor WTX in a subset of pediatric tumors.

WTX is a tumor suppressor gene expressed during embryonic development and inactivated in 20-30% of cases of Wilms tumor, the most common pediatric kidney cancer. WTX has been implicated in several cellular processes including Wnt signaling, WT1 transcription, NRF2 degradation, and p53 function. Given that WTX is widely expressed during embryonic development and has been recently shown to regulate mesenchymal precursor cells in several organs, we tested for the potential involvement of WTX in a panel of pediatric tumors and adult sarcomas. A total of 353 tumors were screened for WTX deletions by fluorescence in situ hybridization (FISH). Discrete somatic WTX deletions were identified in two cases, one hepatoblastoma and one embryonal rhabdomyosarcoma, and confirmed by array comparative genomic hybridization. Direct sequencing of the full WTX open reading frame in 24 hepatoblastomas and 21 embryonal rhabdomyosarcomas did not identify additional mutations in these tumor types. The presence of WTX mRNA was confirmed in hepatoblastomas and embryonal rhabdomyosarcomas without WTX deletions by RNA-in situ hybridization. Notably, tumors with evidence of WTX inactivation, Wilms tumor, hepatoblastoma and rhabdomyosarcoma, are primitive tumors that resemble undifferentiated precursor cells and are linked to overgrowth syndromes. These results indicate that WTX inactivation occurs in a wider variety of tumor types than previously appreciated and point to shared pathogenic mechanisms between a subset of pediatric malignancies.

Mosaic amplification of multiple receptor tyrosine kinase genes in glioblastoma.

Tumor heterogeneity has been implicated in tumor growth and progression as well as resistance to therapy. We present an example of genetic heterogeneity in human malignant brain tumors in which multiple closely related driver genes are amplified and activated simultaneously in adjacent intermingled cells. We have observed up to three different receptor tyrosine kinases (EGFR, MET, PDGFRA) amplified in single tumors in different cells in a mutually exclusive fashion. Each subpopulation was actively dividing, and the genetic changes resulted in protein production, and coexisting subpopulations shared common early genetic mutations indicating their derivation from a single precursor cell. The stable coexistence of different clones within the same tumor will have important clinical implications for tumor resistance to targeted therapies.

Diagnosis of hydatidiform moles by polymorphic deletion probe fluorescence in situ hybridization.

Because products of conception often contain maternal and villous tissues, the determination of maternal and villous genotypes based on genetic polymorphisms can help discern maternal and paternal chromosomal contribution and aid in the diagnosis of hydatidiform moles. Polymorphic deletion probe (PDP) fluorescence in situ hybridization (FISH) probes based on copy number variants are highly polymorphic and allow in situ determination of genetic identity. By using three informative PDPs on chromosomes 2p, 4q, and 8p, we compared maternal with villous genotypes and determined the ploidy of villous tissue. PDP FISH was performed on 13 complete moles, 13 partial moles, 13 nonmolar abortions, and an equivocal hydropic abortion. PDP FISH permitted definitive diagnosis of complete moles in five of 13 cases for which maternal and villous genotypes were mutually exclusive. A complete mole was highly suspected when all three PDP loci showed homozygous villous genotypes. The diagnosis of a complete mole by PDP FISH yielded a theoretical test sensitivity of 87.5%, specificity of 91.8%, an observed test sensitivity of 100%, and specificity of 92.3%. Triploidy was observed in all partial moles, in which diandric triploidy was confirmed in six cases. In the equivocal hydropic abortion, PDP FISH combined with p57 immunofluorescence revealed placental androgenetic/biparental mosaicism. PDP FISH can be used in clinical practice and research studies to subclassify hydatidiform moles and evaluate unusual products of conception.

Glioblastoma recurrence after cediranib therapy in patients: lack of "rebound" revascularization as mode of escape.

Recurrent glioblastomas (rGBM) invariably relapse after initial response to anti-VEGF therapy. There are 2 prevailing hypotheses on how these tumors escape antiangiogenic therapy: switch to VEGF-independent angiogenic pathways and vessel co-option. However, direct evidence in rGBM patients is lacking. Thus, we compared molecular, cellular, and vascular parameters in autopsy tissues from 5 rGBM patients who had been treated with the pan-VEGF receptor tyrosine kinase inhibitor cediranib versus 7 patients who received no therapy or chemoradiation but no antiangiogenic agents. After cediranib treatment, endothelial proliferation and glomeruloid vessels were decreased, and vessel diameters and perimeters were reduced to levels comparable to the unaffected contralateral brain hemisphere. In addition, tumor endothelial cells expressed molecular markers specific to the blood-brain barrier, indicative of a lack of revascularization despite the discontinuation of therapy. Surprisingly, in cediranib-treated GBM, cellular density in the central area of the tumor was lower than in control cases and gradually decreased toward the infiltrating edge, indicative of a change in growth pattern of rGBMs after cediranib treatment, unlike that after chemoradiation. Finally, cediranib-treated GBMs showed high levels of PDGF-C (platelet-derived growth factor C) and c-Met expression and infiltration by myeloid cells, which may potentially contribute to resistance to anti-VEGF therapy. In summary, we show that rGBMs switch their growth pattern after anti-VEGF therapy--characterized by lower tumor cellularity in the central area, decreased pseudopalisading necrosis, and blood vessels with normal molecular expression and morphology--without a second wave of angiogenesis.