Systematic identification of anticancer drug targets reveals a nucleus-to-mitochondria ROS-sensing pathway.

Multiple anticancer drugs have been proposed to cause cell death, in part, by increasing the steady-state levels of cellular reactive oxygen species (ROS). However, for most of these drugs, exactly how the resultant ROS function and are sensed is poorly understood. It remains unclear which proteins the ROS modify and their roles in drug sensitivity/resistance. To answer these questions, we examined 11 anticancer drugs with an integrated proteogenomic approach identifying not only many unique targets but also shared ones-including ribosomal components, suggesting common mechanisms by which drugs regulate translation. We focus on CHK1 that we find is a nuclear H2O2 sensor that launches a cellular program to dampen ROS. CHK1 phosphorylates the mitochondrial DNA-binding protein SSBP1 to prevent its mitochondrial localization, which in turn decreases nuclear H2O2. Our results reveal a druggable nucleus-to-mitochondria ROS-sensing pathway-required to resolve nuclear H2O2 accumulation and mediate resistance to platinum-based agents in ovarian cancers.

Extrauterine epithelioid trophoblastic tumour and its somatic carcinoma mimics: short tandem repeat genotyping meets the diagnostic challenges.

AIMS: While epithelioid trophoblastic tumour (ETT) primarily arises from the uterus, cases have been increasingly documented at extrauterine sites, originating from an ectopic gestation or presenting as a metastatic tumour, leading to the major differential diagnosis of somatic carcinoma with trophoblastic differentiation. The precise separation of a gestational trophoblastic tumour from its somatic carcinoma mimics is highly relevant and crucial for patient management and prognosis. METHODS AND RESULTS: We summarise the clinicopathological and molecular features of four challenging epithelioid malignancies presenting at extrauterine sites, with ETT as the main differential diagnosis. All four tumours demonstrated histological and immunohistochemical features overlapping between a somatic carcinoma and an ETT, combined with inconclusive clinical and imaging findings. Serum beta-hCG elevation was documented in two cases. Short tandem repeat (STR) genotyping was performed and was informative in all cases. The presence of a unique paternal allelic pattern in the tumour tissue confirmed the diagnosis of ETT in two cases with an initial consideration of either somatic carcinoma or suspicion of a gestational trophoblastic tumour. The presence of matching genetic profile with the patient's paired normal tissue was seen in two other cases (both initially considered as ETT), confirming their somatic origin, including one metastatic triple-negative breast carcinoma and one primary lung carcinoma. CONCLUSIONS: Diagnostic separation of ETT at an extrauterine site from its somatic carcinoma mimics can be difficult at the histological and immunohistochemical levels. STR genotyping offers a robust ancillary tool that precisely separates ETT from somatic carcinomas with trophoblastic differentiation.

Midline brain hamartomatous lesions in fibrodysplasia ossificans progressiva with ACVR1 mutations.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder characterized by extensive heterotopic ossification of soft tissue structures leading to severe limitations in movement. FOP is caused by a germline mutation in the activating receptor type IA (ACVR1) gene. Worrisome is the fact that up to a third of diffuse intrinsic pontine gliomas (DIPG) also harbor the same point mutation in ACVR1. Radiological reports of central nervous system (CNS) involvement by FOP have described brainstem masses; however, the literature on the histopathology or pathogenesis of these lesions is scant. Here we present detailed neuropathologic findings of a brainstem mass in a patient with FOP and suggest that the tumor is hamartomatous in nature. This report, along with a literature review of radiographic and laboratory data, offers support for the idea that the ACVR1 mutation may incite CNS proliferation, predominantly in the brainstem, but is probably not an oncologic driver. These lesions may be seen at autopsy and are likely noncontributory to death.

Uterine PEComas: correlation between melanocytic marker expression and TSC alterations/TFE3 fusions.

Uterine PEComas often present a diagnostic challenge as they share morphological and immunohistochemical features with smooth muscle tumors. Herein we evaluated a series of 19 uterine PEComas to compare the degree of melanocytic marker expression with their molecular profile. Patients ranged from 32-77 (median 48) years, with six tumors classified as malignant based on the modified gynecologic-specific prognostic algorithm. All patients with malignant PEComas were alive with disease or dead  of disease at last follow-up, while all those of uncertain malignant potential were alive and well (median follow-up, 47 months).Seventeen of 19 (89%) PEComas harbored either a TSC1 or TSC2 alteration. One of the two remaining tumors showed a TFE3 rearrangement, but the other lacked alterations in all genes evaluated. All showed at least focal (usually strong) positivity for HMB-45, with 15/19 (79%) having >50% expression, while the tumor lacking TSC or TFE3 alterations was strongly positive in 10% of cells. Melan-A and MiTF were each positive in 15/19 (79%) tumors, but staining extent and intensity were much more variable than HMB-45. Five of six (83%) malignant PEComas also harbored alterations in TP53, ATRX, or RB1, findings not identified in any tumors of uncertain malignant potential. One malignant PEComa was microsatellite-unstable/mismatch repair protein-deficient.In summary, TSC alterations/TFE3 fusions and diffuse (>50%) HMB-45 expression are characteristic of uterine PEComas. In morphologically ambiguous mesenchymal neoplasms with myomelanocytic differentiation, especially those with metastatic or recurrent disease, next-generation sequencing is recommended to evaluate for TSC alterations; as such, patients can be eligible for targeted therapy.

Low-grade Endometrial Stromal Sarcoma With Sex Cord-like Differentiation and PHF1-JAZF1 Fusion With Deletions: A Diagnostic Pitfall of JAZF1 FISH.

The molecular knowledge of endometrial stromal neoplasms has been rapidly increasing and is considered complementary to morphologic and immunohistochemical findings for better categorization of these tumors. The most common molecular alteration observed in low-grade endometrial stromal sarcomas is the JAZF1-SUZ12 fusion, whereas, low-grade endometrial stromal sarcoma with sex cord-like differentiation have been shown more commonly to have fusions involving PHF1. Herein, we present a low-grade endometrial stromal sarcoma with sex cord-like differentiation with a fluorescence in situ hybridization showing the apparent loss of one copy of JAZF1 5' and 3' signals, rather than the expected "break-apart" pattern seen in the setting of a JAZF1 fusion. The case was then further evaluated by chromosome microarray and RNA fusion analysis. Overall, the molecular findings supported a PHF1-JAZF1 fusion with deletions right before and after the JAZF1 locus, impairing probe binding and resulting in the unusual "deletion" pattern observed in the JAZF1 fluorescence in situ hybridization, which would not intuitively suggest a fusion involving JAZF1. This case illustrates the importance of integration of morphological and molecular findings as well as the limitations of fluorescence in situ hybridization in detecting fusions, particularly in the setting of more complex chromosomal alterations even though the fusion partners are well-known.

Locally Recurrent Secretory Carcinoma of the Breast with NTRK3 Gene Fusion.

Enhanced understanding of the molecular events underlying oncogenesis has led to the development of "tumor-agnostic" treatment strategies, which aim to target a tumor's genomic profile regardless of its anatomic site of origin. A classic example is the translocation resulting in an ETV6-NTRK3 gene fusion, a characteristic driver of a histologically diverse array of cancers. The chimeric ETV6-NTRK3 fusion protein elicits constitutive activation of the tropomyosin receptor kinase (TRK) C protein, leading to increased cell survival, growth, and proliferation. Two TRK inhibitors, larotrectinib and entrectinib, are currently approved for use in the metastatic setting for the treatment of advanced solid tumors harboring NTRK fusions. Here we report a rare case of recurrent secretory carcinoma of the breast (SCB) with NTRK3 gene fusion. Whereas most cases of SCB represent slow-growing tumors with favorable outcomes, the case detailed here is the first to the authors' knowledge of recurrence within 1 year of surgery. We review the molecular findings and potential clinical significance. KEY POINTS: The translocation resulting in the ETV6-NTRK3 gene fusion is a known oncogenic driver characteristic of secretory carcinoma of the breast (SCB). Whereas most cases of SCB represent slow-growing tumors with favorable outcomes, the case here with ETV6-NTRK3 gene fusion had local recurrence within 1 year of surgery. Two tropomyosin receptor kinase (TRK) inhibitors, larotrectinib and entrectinib, are approved to treat NTRK fusion-positive tumors, demonstrating sustained high overall response rates in the metastatic setting. Approval of TRK inhibitors necessitates optimization of NTRK fusion detection assays, including detection with liquid biopsies.

Reproducibility of scoring criteria for HER2 immunohistochemistry in endometrial serous carcinoma: a multi-institutional interobserver agreement study.

Targeted anti-human epidermal growth factor receptor 2 (HER2) therapy has recently been proven to improve progression-free and overall survival of patients with advanced stage or recurrent endometrial serous carcinoma. To date, no specific pathology HER2 testing or scoring guidelines exist for endometrial cancer. However, based on evidence from the recent successful clinical trial and comprehensive pre-trial pathologic studies, a new set of HER2 scoring criteria have been proposed for endometrial serous carcinoma-distinct from the existing breast and gastric cancer-specific criteria. We present the first study assessing interobserver agreement of HER2 scores using the proposed serous endometrial cancer-specific scoring system. A digitally scanned set of 40 HER2-immunostained slides of endometrial serous carcinoma were sent to seven gynecologic pathologists, who independently assigned HER2 scores for each slide following a brief tutorial. Follow-up fluorescent in situ hybridization (FISH) for HER2 gene amplification was performed on cases with interobserver disagreement when a 2+ HER2 score was assigned by at least one observer. Complete agreement of HER2 scores among all 7 observers was achieved on 15 cases, and all but one case had an agreement by at least 4 observers. The overall agreement was 72.3% (kappa 0.60), 77.5% (kappa 0.65), and 83.3% (kappa 0.65), using four (0 to 3+ ), three (0/1+ , 2+ , 3+ ), or two (0/1+ , 2/3+ ) HER2 scoring categories, respectively. Based on the combination of HER2 immunostaining scores and FISH, the interobserver disagreement may have potentially resulted in a clinically significant difference in HER2 status only in three tumors. We conclude, that the proposed serous endometrial cancer-specific HER2 scoring criteria are reproducible among gynecologic pathologists with moderate to substantial interobserver agreement rates comparable to those of previously reported in breast and gastric carcinomas. Our findings significantly strengthen the foundation for establishing endometrial cancer-specific HER2 scoring guidelines in the future.

Embryonal rhabdomyosarcoma of the uterine corpus: a clinicopathological and molecular analysis of 21 cases highlighting a frequent association with DICER1 mutations.

Herein we evaluated a series of 21 embryonal rhabdomyosarcomas of the uterine corpus (ucERMS), a rare neoplasm, to characterize their morphology, genomics, and behavior. Patients ranged from 27 to 73 (median 52) years and tumors from 4 to 15 (median 9) cm, with extrauterine disease noted in two. Follow-up (median 16 months) was available for 14/21 patients; nine were alive and well, four died of disease, and one died from other causes. Most tumors (16/21) showed predominantly classic morphology, comprised of alternating hyper- and hypocellular areas of primitive small cells and differentiating rhabdomyoblasts in a loose myxoid/edematous stroma. A cambium layer was noted in all; seven had heterologous elements (six with fetal-type cartilage) and eight displayed focal anaplasia. The remaining five neoplasms showed only a minor component (≤20%) of classic morphology, with anaplasia noted in four and tumor cell necrosis in three. The most frequent mutations detected were in DICER1 (14/21), TP53 (7/20), PI3K/AKT/mTOR pathway (7/20), and KRAS/NRAS (5/20). Copy-number alterations were present in 10/19 tumors. Overall, 8/14 DICER1-associated ucERMS showed concurrent loss of function and hotspot mutations in DICER1, which is a feature more likely to be seen in tumors associated with DICER1 syndrome. Germline data were available for two patients, both DICER1 wild type (one with concurrent loss of function and hotspot alterations). DICER1-associated ucERMS were more likely to show a classic histological appearance including heterologous elements than DICER1-independent tumors. No differences in survival were noted between the two groups, but both patients with extrauterine disease at diagnosis and two with recurrences died from disease. As no patients had a known personal or family history of DICER1 syndrome, we favor most DICER1-associated ucERMS to be sporadic.

Computational Prediction of Position Effects of Apparently Balanced Human Chromosomal Rearrangements.

Interpretation of variants of uncertain significance, especially chromosomal rearrangements in non-coding regions of the human genome, remains one of the biggest challenges in modern molecular diagnosis. To improve our understanding and interpretation of such variants, we used high-resolution three-dimensional chromosomal structural data and transcriptional regulatory information to predict position effects and their association with pathogenic phenotypes in 17 subjects with apparently balanced chromosomal abnormalities. We found that the rearrangements predict disruption of long-range chromatin interactions between several enhancers and genes whose annotated clinical features are strongly associated with the subjects' phenotypes. We confirm gene-expression changes for a couple of candidate genes to exemplify the utility of our analysis of position effect. These results highlight the important interplay between chromosomal structure and disease and demonstrate the need to utilize chromatin conformational data for the prediction of position effects in the clinical interpretation of non-coding chromosomal rearrangements.

Molecular and clinicopathologic characterization of intravenous leiomyomatosis.

Intravenous leiomyomatosis (IVL) is an unusual uterine smooth muscle proliferation that can be associated with aggressive clinical behavior despite a histologically benign appearance. It has some overlapping molecular characteristics with both uterine leiomyoma and leiomyosarcoma based on limited genetic data. In this study, we assessed the clinical and morphological characteristics of 28 IVL and their correlation with molecular features and protein expression, using array comparative genomic hybridization (aCGH) and Cyclin D1, p16, phosphorylated-Rb, SMARCB1, SOX10, CAIX, SDHB and FH immunohistochemistry. The most common morphologies were cellular (n = 15), usual (n = 11), and vascular (n = 5; including 3 cellular IVL showing both vascular and cellular features). Among the immunohistochemical findings, the most striking was that all IVL showed differential expression of either p16 or Cyclin D1 in comparison to surrounding nonneoplastic tissue. Cytoplasmic phosphorylated-Rb was present in all but one IVL with hyalinization. SMARCB1, FH, and SDHB were retained; S0X10 and CAIX were not expressed. The most common genetic alterations involved 1p (39%), 22q (36%), 2q (29%), 1q (25%), 13q (21%), and 14q (21%). Hierarchical clustering analysis of recurrent aberrations revealed three molecular groups: Groups 1 (29%) and 2 (18%) with associated del(22q), and Group 3 (18%) with del(10q). The remaining IVL had nonspecific or no alterations by aCGH. Genomic index scores were calculated for all cases and showed no significant difference between the 14 IVL associated with aggressive clinical behavior (extrauterine extension or recurrence) and those without (median scores 5.15 vs 3.5). Among the 5 IVL associated with recurrence, 4 had a vascular morphology and 3 had alterations of 8q. Recurrent chromosome alterations detected herein overlap with those observed in the spectrum of uterine smooth muscle tumors and involve genes implicated in mesenchymal tumors at different sites with distinct morphological features.

Pediatric liver transplant following near catastrophic head bleed: Lessons learned.

Evaluation for liver transplant candidacy is a multidisciplinary effort that involves all aspects of clinical care including social work, nutrition, and a multitude of medical specialties. The prognosis of a pretransplant clinical condition is integrated into the decision to list a patient. Herein, we report a successful liver transplant and recovery of a 3-month-old male following a large right hemispheric subdural hematoma related to acute coagulopathy secondary to undiagnosed end-stage liver disease. On presentation with jaundice, lethargy, and unequal pupils, a CT scan was obtained which demonstrated a large right subdural hematoma with herniation. Once his coagulopathy was corrected, he went for decompressive craniectomy. He survived with medically controlled seizures and improving L-sided neglect and extremity weakness. Six weeks later, given his continued neurologic recovery and worsening liver function, the decision was made to list him for liver transplantation. One month later, he underwent orthotopic liver transplant. His post-operative hospital course was complicated by DVTs and heparin-induced thrombocytopenia, but no neurologic decline, and he was eventually discharged from the hospital on post-op day 26. Three years later, he has a well-functioning allograft and no clinically evident neurologic deficits. The prognosis following pediatric neurologic trauma remains somewhat unclear as recovery and neurologic examinations can be influenced by numerous extrinsic factors. This is one of the first reports of near full neurologic recovery of a pediatric liver transplant recipient following a large subdural hematoma with herniation.

Structural Chromosomal Rearrangements Require Nucleotide-Level Resolution: Lessons from Next-Generation Sequencing in Prenatal Diagnosis.

In this exciting era of "next-gen cytogenetics," integrating genomic sequencing into the prenatal diagnostic setting is possible within an actionable time frame and can provide precise delineation of balanced chromosomal rearrangements at the nucleotide level. Given the increased risk of congenital abnormalities in newborns with de novo balanced chromosomal rearrangements, comprehensive interpretation of breakpoints could substantially improve prediction of phenotypic outcomes and support perinatal medical care. Herein, we present and evaluate sequencing results of balanced chromosomal rearrangements in ten prenatal subjects with respect to the location of regulatory chromatin domains (topologically associated domains [TADs]). The genomic material from all subjects was interpreted to be "normal" by microarray analyses, and their rearrangements would not have been detected by cell-free DNA (cfDNA) screening. The findings of our systematic approach correlate with phenotypes of both pregnancies with untoward outcomes (5/10) and with healthy newborns (3/10). Two pregnancies, one with a chromosomal aberration predicted to be of unknown clinical significance and another one predicted to be likely benign, were terminated prior to phenotype-genotype correlation (2/10). We demonstrate that the clinical interpretation of structural rearrangements should not be limited to interruption, deletion, or duplication of specific genes and should also incorporate regulatory domains of the human genome with critical ramifications for the control of gene expression. As detailed in this study, our molecular approach to both detecting and interpreting the breakpoints of structural rearrangements yields unparalleled information in comparison to other commonly used first-tier diagnostic methods, such as non-invasive cfDNA screening and microarray analysis, to provide improved genetic counseling for phenotypic outcome in the prenatal setting.

Describing sequencing results of structural chromosome rearrangements with a suggested next-generation cytogenetic nomenclature.

With recent rapid advances in genomic technologies, precise delineation of structural chromosome rearrangements at the nucleotide level is becoming increasingly feasible. In this era of "next-generation cytogenetics" (i.e., an integration of traditional cytogenetic techniques and next-generation sequencing), a consensus nomenclature is essential for accurate communication and data sharing. Currently, nomenclature for describing the sequencing data of these aberrations is lacking. Herein, we present a system called Next-Gen Cytogenetic Nomenclature, which is concordant with the International System for Human Cytogenetic Nomenclature (2013). This system starts with the alignment of rearrangement sequences by BLAT or BLAST (alignment tools) and arrives at a concise and detailed description of chromosomal changes. To facilitate usage and implementation of this nomenclature, we are developing a program designated BLA(S)T Output Sequence Tool of Nomenclature (BOSToN), a demonstrative version of which is accessible online. A standardized characterization of structural chromosomal rearrangements is essential both for research analyses and for application in the clinical setting.

Implication of LRRC4C and DPP6 in neurodevelopmental disorders.

We performed whole-genome sequencing on an individual from a family with variable psychiatric phenotypes that had a sensory processing disorder, apraxia, and autism. The proband harbored a maternally inherited balanced translocation (46,XY,t(11;14)(p12;p12)mat) that disrupted LRRC4C, a member of the highly specialized netrin G family of axon guidance molecules. The proband also inherited a paternally derived chromosomal inversion that disrupted DPP6, a potassium channel interacting protein. Copy Number (CN) analysis in 14,077 cases with neurodevelopmental disorders and 8,960 control subjects revealed that 60% of cases with exonic deletions in LRRC4C had a second clinically recognizable syndrome associated with variable clinical phenotypes, including 16p11.2, 1q44, and 2q33.1 CN syndromes, suggesting LRRC4C deletion variants may be modifiers of neurodevelopmental disorders. In vitro, functional assessments modeling patient deletions in LRRC4C suggest a negative regulatory role of these exons found in the untranslated region of LRRC4C, which has a single, terminal coding exon. These data suggest that the proband's autism may be due to the inheritance of disruptions in both DPP6 and LRRC4C, and may highlight the importance of the netrin G family and potassium channel interacting molecules in neurodevelopmental disorders. © 2016 Wiley Periodicals, Inc.

Intravenous leiomyomatosis: an unusual intermediate between benign and malignant uterine smooth muscle tumors.

Intravenous leiomyomatosis is an unusual smooth muscle neoplasm with quasi-malignant intravascular growth but a histologically banal appearance. Herein, we report expression and molecular cytogenetic analyses of a series of 12 intravenous leiomyomatosis cases to better understand the pathogenesis of intravenous leiomyomatosis. All cases were analyzed for the expression of HMGA2, MDM2, and CDK4 proteins by immunohistochemistry based on our previous finding of der(14)t(12;14)(q14.3;q24) in intravenous leiomyomatosis. Seven of 12 (58%) intravenous leiomyomatosis cases expressed HMGA2, and none expressed MDM2 or CDK4. Colocalization of hybridization signals for probes from the HMGA2 locus (12q14.3) and from 14q24 by interphase fluorescence in situ hybridization (FISH) was detected in a mean of 89.2% of nuclei in HMGA2-positive cases by immunohistochemistry, but in only 12.4% of nuclei in negative cases, indicating an association of HMGA2 expression and this chromosomal rearrangement (P=8.24 × 10(-10)). Four HMGA2-positive cases had greater than two HMGA2 hybridization signals per cell. No cases showed loss of a hybridization signal by interphase FISH for the frequently deleted region of 7q22 in uterine leiomyomata. One intravenous leiomyomatosis case analyzed by array comparative genomic hybridization revealed complex copy number variations. Finally, expression profiling was performed on three intravenous leiomyomatosis cases. Interestingly, hierarchical cluster analysis of the expression profiles revealed segregation of the intravenous leiomyomatosis cases with leiomyosarcoma rather than with myometrium, uterine leiomyoma of the usual histological type, or plexiform leiomyoma. These findings suggest that intravenous leiomyomatosis cases share some molecular cytogenetic characteristics with uterine leiomyoma, and expression profiles similar to that of leiomyosarcoma cases, further supporting their intermediate, quasi-malignant behavior.

Fibroids: Genotype and Phenotype.

Fibroids represent a major public health care problem as the most prevalent pelvic tumors in women of reproductive age and as the leading cause of gynecologic surgeries in the United States. The recent advances in the genomic technologies including genome-wide association studies and high-throughput sequencing provide insight into their pathogenesis and molecular classification. Understanding the molecular basis of fibroids may facilitate development of effective targeted treatment options of this very common disease.