Malic Enzyme 1 Absence in Synovial Sarcoma Shifts Antioxidant System Dependence and Increases Sensitivity to Ferroptosis Induction with ACXT-3102.

PURPOSE: To investigate the metabolism of synovial sarcoma (SS) and elucidate the effect of malic enzyme 1 absence on SS redox homeostasis. EXPERIMENTAL DESIGN: ME1 expression was measured in SS clinical samples, SS cell lines, and tumors from an SS mouse model. The effect of ME1 absence on glucose metabolism was evaluated utilizing Seahorse assays, metabolomics, and C13 tracings. The impact of ME1 absence on SS redox homeostasis was evaluated by metabolomics, cell death assays with inhibitors of antioxidant systems, and measurements of intracellular reactive oxygen species (ROS). The susceptibility of ME1-null SS to ferroptosis induction was interrogated in vitro and in vivo. RESULTS: ME1 absence in SS was confirmed in clinical samples, SS cell lines, and an SS tumor model. Investigation of SS glucose metabolism revealed that ME1-null cells exhibit higher rates of glycolysis and higher flux of glucose into the pentose phosphate pathway (PPP), which is necessary to produce NADPH. Evaluation of cellular redox homeostasis demonstrated that ME1 absence shifts dependence from the glutathione system to the thioredoxin system. Concomitantly, ME1 absence drives the accumulation of ROS and labile iron. ROS and iron accumulation enhances the susceptibility of ME1-null cells to ferroptosis induction with inhibitors of xCT (erastin and ACXT-3102). In vivo xenograft models of ME1-null SS demonstrate significantly increased tumor response to ACXT-3102 compared with ME1-expressing controls. CONCLUSIONS: These findings demonstrate the translational potential of targeting redox homeostasis in ME1-null cancers and establish the preclinical rationale for a phase I trial of ACXT-3102 in SS patients. See related commentary by Subbiah and Gan, p. 3408.

Placental Tissue Destruction and Insufficiency From COVID-19 Causes Stillbirth and Neonatal Death From Hypoxic-Ischemic Injury.

CONTEXT.­: Perinatal death is an increasingly important problem as the coronavirus disease 2019 (COVID-19) pandemic continues, but the mechanism of death has been unclear. OBJECTIVE.­: To evaluate the role of the placenta in causing stillbirth and neonatal death following maternal infection with COVID-19 and confirmed placental positivity for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). DESIGN.­: Case-based retrospective clinicopathologic analysis by a multinational group of 44 perinatal specialists from 12 countries of placental and autopsy pathology findings from 64 stillborns and 4 neonatal deaths having placentas testing positive for SARS-CoV-2 following delivery to mothers with COVID-19. RESULTS.­: Of the 3 findings constituting SARS-CoV-2 placentitis, all 68 placentas had increased fibrin deposition and villous trophoblast necrosis and 66 had chronic histiocytic intervillositis. Sixty-three placentas had massive perivillous fibrin deposition. Severe destructive placental disease from SARS-CoV-2 placentitis averaged 77.7% tissue involvement. Other findings included multiple intervillous thrombi (37%; 25 of 68) and chronic villitis (32%; 22 of 68). The majority (19; 63%) of the 30 autopsies revealed no significant fetal abnormalities except for intrauterine hypoxia and asphyxia. Among all 68 cases, SARS-CoV-2 was detected from a body specimen in 16 of 28 cases tested, most frequently from nasopharyngeal swabs. Four autopsied stillborns had SARS-CoV-2 identified in internal organs. CONCLUSIONS.­: The pathology abnormalities composing SARS-CoV-2 placentitis cause widespread and severe placental destruction resulting in placental malperfusion and insufficiency. In these cases, intrauterine and perinatal death likely results directly from placental insufficiency and fetal hypoxic-ischemic injury. There was no evidence that SARS-CoV-2 involvement of the fetus had a role in causing these deaths.

Imaging Mass Spectrometry Is an Accurate Tool in Differentiating Clear Cell Renal Cell Carcinoma and Chromophobe Renal Cell Carcinoma: A Proof-of-concept Study.

Clear cell renal cell carcinoma (ccRCC) and chromophobe renal cell carcinoma (chRCC) are relatively common tumors that can have significant risk for mortality. Treatment and prognostication in renal cell carcinoma (RCC) are dependent upon correct histologic typing. ccRCC and chRCC are generally straightforward to diagnose based on histomorphology alone. However, high-grade ccRCC and chRCC can sometimes resemble each other morphologically, particularly in small biopsies. Multiple immunostains and/or colloidal iron stain are sometimes required to differentiate the two. Imaging mass spectrometry (IMS) allows simultaneous spatial mapping of thousands of biomarkers, using formalin-fixed paraffin-embedded tissue sections. In this study, we evaluate the ability of IMS to differentiate between World Health Organization/International Society for Urological Pathology grade 3 ccRCC and chRCC. IMS spectra from a training set of 14 ccRCC and 13 chRCC were evaluated via support vector machine algorithm with a linear kernel for machine learning, building a classification model. The classification model was applied to a separate validation set of 6 ccRCC and 6 chRCC, with 19 to 20, 150-µm diameter tumor foci in each case sampled by IMS. Most evaluated tumor foci were classified correctly as ccRCC versus chRCC (99% accuracy, kappa=0.98), demonstrating that IMS is an accurate tool in differentiating high-grade ccRCC and chRCC.

CD4+ T cells induce rejection of urothelial tumors after immune checkpoint blockade.

Immune checkpoint blockade (ICB) provides clinical benefit to a minority of patients with urothelial carcinoma (UC). The role of CD4+ T cells in ICB-induced antitumor activity is not well defined; however, CD4+ T cells are speculated to play a supportive role in the development of CD8+ T cells that kill tumor cells after recognition of tumor antigens presented by MHC class I. To investigate the mechanisms of ICB-induced activity against UC, we developed mouse organoid-based transplantable models that have histologic and genetic similarity to human bladder cancer. We found that ICB can induce tumor rejection and protective immunity with these systems in a manner dependent on CD4+ T cells but not reliant on CD8+ T cells. Evaluation of tumor infiltrates and draining lymph nodes after ICB revealed expansion of IFN-γ-producing CD4+ T cells. Tumor cells in this system express MHC class I, MHC class II, and the IFN-γ receptor (Ifngr1), but none were necessary for ICB-induced tumor rejection. IFN-γ neutralization blocked ICB activity, and, in mice depleted of CD4+ T cells, IFN-γ ectopically expressed in the tumor microenvironment was sufficient to inhibit growth of tumors in which the epithelial compartment lacked Ifngr1. Our findings suggest unappreciated CD4+ T cell-dependent mechanisms of ICB activity, principally mediated through IFN-γ effects on the microenvironment.

Prostate Cancer Pathology: Recent Updates and Controversies.

Prostate cancer is common, and recent efforts in clinical management have focused on identifying patients who could be candidates from less aggressive management or who could benefit from more aggressive therapy. As prostate cancer histology, especially Gleason score, plays a critical role in predicting patient outcomes, attempts have been made to refine histologic classification and reporting in prostate cancer to facilitate patient risk stratification. This review discusses recent updates in prostate cancer grading and reporting.

Acute graft-versus-host disease following lung transplantation in a patient with a novel TERT mutation.

Familial pulmonary fibrosis is associated with loss-of-function mutations in telomerase reverse transcriptase (TERT) and short telomeres. Interstitial lung diseases have become the leading indication for lung transplantation in the USA, and recent data indicate that pathogenic mutations in telomerase may cause unfavourable outcomes following lung transplantation. Although a rare occurrence, solid organ transplant recipients who develop acute graft-versus-host disease (GVHD) have very poor survival. This case report describes the detection of a novel mutation in TERT in a patient who had lung transplantation for familial pulmonary fibrosis and died from complications of acute GVHD.

Lymphoepithelioma-like carcinoma of the ureter: a rare presentation, synchronous with conventional urothelial carcinoma.

Lymphoepithelioma-like carcinoma (LELC) is a rare finding in the upper urinary tract. The presenting clinical findings mimic those of other more common upper-tract tumors, such as urothelial carcinoma. Preoperative imaging has not been shown to reliably predict the diagnosis of LELC. This tumor can be misdiagnosed as a reactive inflammatory lesion or lymphoma if the proper immunohistochemical stains for cytokeratin are not used.

Mycotic Aneurysm after Bacillus Calmette-Guérin Treatment: Case Report and Review of the Literature.

Background. Intravesicular Bacillus Calmette-Guérin (BCG) is an effective adjunctive therapy for superficial bladder cancer that has been shown to delay recurrence and progression of disease. Serious side effects are relatively rare but are difficult to diagnosis and commonly overlooked. Case Presentation. We report the case of a patient who was found to have mycotic aortic aneurysms secondary to treatment with BCG after a prolonged course with multiple intervening hospitalizations. Conclusion. Through this report, we discuss our present understanding of BCG infection following treatment and review the literature regarding this particular rare manifestation.

A new model of multi-visceral and bone metastatic prostate cancer with perivascular niche targeting by a novel endothelial specific adenoviral vector.

While modern therapies for metastatic prostate cancer (PCa) have improved survival they are associated with an increasingly prevalent entity, aggressive variant PCa (AVPCa), lacking androgen receptor (AR) expression, enriched for cancer stem cells (CSCs), and evidencing epithelial-mesenchymal plasticity with a varying extent of neuroendocrine transdifferentiation. Parallel work revealed that endothelial cells (ECs) create a perivascular CSC niche mediated by juxtacrine and membrane tethered signaling. There is increasing interest in pharmacological metastatic niche targeting, however, targeted access has been impossible. Here, we discovered that the Gleason 7 derived, androgen receptor negative, IGR-CaP1 cell line possessed some but not all of the molecular features of AVPCa. Intracardiac injection into NOD/SCID/IL2Rg -/- (NSG) mice produced a completely penetrant bone, liver, adrenal, and brain metastatic phenotype; noninvasively and histologically detectable at 2 weeks, and necessitating sacrifice 4-5 weeks post injection. Bone metastases were osteoblastic, and osteolytic. IGR-CaP1 cells expressed the neuroendocrine marker synaptophysin, near equivalent levels of vimentin and e-cadherin, all of the EMT transcription factors, and activation of NOTCH and WNT pathways. In parallel, we created a new triple-targeted adenoviral vector containing a fiber knob RGD peptide, a hexon mutation, and an EC specific ROBO4 promoter (Ad.RGD.H5/3.ROBO4). This vector was expressed in metastatic microvessels tightly juxtaposed to IGR-CaP1 cells in bone and visceral niches. Thus, the combination of IGR-CaP1 cells and NSG mice produces a completely penetrant metastatic PCa model emulating end-stage human disease. In addition, the metastatic niche access provided by our novel Ad vector could be therapeutically leveraged for future disease control or cure.

Postmenopausal Invasive Endometriosis Requiring Supralevator Pelvic Exenteration.

BACKGROUND: Endometriosis is rare in postmenopausal women. We report a case of invasive pelvic endometriosis in a postmenopausal woman requiring a supralevator pelvic exenteration for palliation of symptoms and for tissue diagnosis. CASE: A 65-year-old woman with a history of total abdominal hysterectomy and bilateral salpingo-oophorectomy for endometriosis at age 43 years presented with acute vaginal bleeding, hematuria, and a recently diagnosed pelvic mass. Biopsies revealed endometriosis, and she underwent supralevator pelvic exenteration with vaginectomy, end colostomy, ileal conduit, and coagulation of endometriotic implants. Pathologic examination showed invasive endometriosis and no evidence of malignancy. CONCLUSION: Endometriosis should remain on the differential diagnosis for pelvic mass in a postmenopausal woman, although suspicion for malignancy should remain high.

Occult Specimen Contamination in Routine Clinical Next-Generation Sequencing Testing.

OBJECTIVES: To evaluate the extent of human-to-human specimen contamination in clinical next-generation sequencing (NGS) data. METHODS: Using haplotype analysis to detect specimen admixture, with orthogonal validation by short tandem repeat analysis, we determined the rate of clinically significant (>5%) DNA contamination in clinical NGS data from 296 consecutive cases. Haplotype analysis was performed using read haplotypes at common, closely spaced single-nucleotide polymorphisms in low linkage disequilibrium in the population, which were present in regions targeted by the clinical assay. Percent admixture was estimated based on frequencies of the read haplotypes at loci that showed evidence for contamination. RESULTS: We identified nine (3%) cases with at least 5% DNA admixture. Three cases were bone marrow transplant patients known to be chimeric. Six admixed cases were incidents of contamination, and the rate of contamination was strongly correlated with DNA yield from the tissue specimen. CONCLUSIONS: Human-human specimen contamination occurs in clinical NGS testing. Tools for detecting contamination in NGS sequence data should be integrated into clinical bioinformatics pipelines, especially as laboratories trend toward using smaller amounts of input DNA and reporting lower frequency variants. This study provides one estimate of the rate of clinically significant human-human specimen contamination in clinical NGS testing.

Cervical spinal cord dimensions and clinical outcomes in adults with klippel-feil syndrome: a comparison with matched controls.

Study Design Retrospective case-control study. Objectives To confirm the fact that spinal cord dimensions are smaller in adults with Klippel-Feil syndrome (KFS) than in pediatric patients with KFS and to compare the clinical characteristics and outcomes of neurologic complications in patients with KFS with matched controls. Methods We performed an independent 1:2 case-control retrospective radiographic and chart review of a consecutive series of adults with KFS who underwent surgical intervention. The control group consisted of consecutive non-KFS surgical patients. Patients were matched in 1:2 case-control manner. Their charts were reviewed and the clinical characteristics were compared. Axial T2-weighted magnetic resonance imaging (MRI) was used to measure the anteroposterior and mediolateral axial spinal cord and spinal canal at the operative levels and measurements were compared. Results A total of 22 patients with KFS and 44 controls were identified. The KFS group had a tendency of more myeloradiculopathy, and the control group had a tendency toward more radiculopathy. Both tendencies, however, were not significantly different. MRIs of 10 patients from the KFS group and 22 controls were available. There was no difference in the area of both spinal cord and canal at the operative levels. Conclusion Contrary to the finding in previous reports on pediatric patients, there were no differences between KFS and well-matched control groups in terms of age of onset, presentation, revision rate, complication rate, surgical outcome, and cross-sectional spinal cord and canal dimensions at the operative level.

Copy number variants in clinical next-generation sequencing data can define the relationship between simultaneous tumors in an individual patient.

Targeted next-generation sequencing (NGS) cancer panels have become a popular method for the identification of clinically predictive mutations in cancer. Such methods typically detect single nucleotide variants (SNVs) and small insertions/deletions (indels) in known cancer genes and can provide further information regarding diagnosis in challenging surgical pathology cases, as well as identify therapeutic targets and prognostically significant mutations. However, in addition to SNVs and indels, other mutation classes, including copy number variants (CNVs) and translocations, can be simultaneously detected from targeted NGS data. Here, as proof of methods, we present clinical data which demonstrate that targeted NGS panels can separate synchronous liver tumors based on CNV status, in the absence of distinct SNVs and indels. Such CNV-based analysis can be performed without additional cost using existing targeted cancer panel data and publically available software.

Detection of viral pathogens in high grade gliomas from unmapped next-generation sequencing data.

Viral pathogens have been implicated in the development of certain cancers including human papillomavirus (HPV) in squamous cell carcinoma and Epstein-Barr virus (EBV) in Burkitt's lymphoma. The significance of viral pathogens in brain tumors is controversial, and human cytomegalovirus (HCMV) has been associated with glioblastoma (GBM) in some but not all studies, making the role of HCMV unclear. In this study we sought to determine if viral pathogen sequences could be identified in an unbiased manner from previously discarded, unmapped, non-human, next-generation sequencing (NGS) reads obtained from targeted oncology, panel-based sequencing of high grade gliomas (HGGs), including GBMs. Twenty one sequential HGG cases were analyzed by a targeted NGS clinical oncology panel containing 151 genes using DNA obtained from formalin-fixed, paraffin-embedded (FFPE) tissue. Sequencing reads that did not map to the human genome (average of 38,000 non-human reads/case (1.9%)) were filtered and low quality reads removed. Extracted high quality reads were then sequentially aligned to the National Center for Biotechnology Information (NCBI) non-redundant nucleotide (nt and nr) databases. Aligned reads were classified based on NCBI taxonomy database and all eukaryotic viral sequences were further classified into viral families. Two viral sequences (both herpesviruses), EBV and Roseolovirus were detected in 5/21 (24%) cases and in 1/21 (5%) cases, respectively. None of the cases had detectable HCMV. Of the five HGG cases with detectable EBV DNA, four had additional material for EBV in situ hybridization (ISH), all of which were negative for expressed viral sequence. Overall, a similar discovery approach using unmapped non-human NGS reads could be used to discover viral sequences in other cancer types.

Diagnostic utility of targeted next-generation sequencing in problematic cases.

Targeted next-generation sequencing (NGS) provides predictive and prognostic information in the routine care of patients with cancer. However, with increasing knowledge of the biological basis of cancer, NGS of the same gene sets can also provide diagnostic information in challenging cases, on the basis of identification of both known and novel variants, including single-nucleotide variants, insertions and deletions, copy number alterations, and translocations. Here, we present 3 clinical cases in which targeted NGS of hybrid-capture-enriched DNA from formalin-fixed, paraffin-embedded tumor samples provided unique and clinically important diagnostic and/or staging information in 3 different challenging clinical scenarios. In the first patient, NGS played a key role in both diagnosis and staging in a patient with multiple tumors of the same histologic type. The second case demonstrates the ability of NGS to clarify the tumor tissue type in a single mass involving multiple organs, and thereby guide appropriate chemotherapy. The third case illustrates that information regarding susceptibility to targeted therapeutics can also clarify the original histologic diagnosis.

Ovarian complete hydatidiform mole: case study with molecular analysis and review of the literature.

Ectopic complete molar pregnancy in the ovary is an exceptionally rare event. Here we present a case of ovarian complete hydatidiform mole in a 20-year-old gravida 2 para 1 woman. At presentation, the patient underwent excision of a hemorrhagic left ovarian cyst, with routine sections demonstrating a hemorrhagic corpus luteum with a single microscopic focus of detached atypical trophoblast, without chorionic villi. Subsequent left salpingo-oophorectomy for persistently elevated human chorionic gonadotropin led to a final diagnosis of complete hydatidiform mole arising in the ovary. The fallopian tube was unremarkable. Zygosity was determined using short tandem repeat analysis, confirming the diagnosis of monospermic complete mole. In the clinical setting of a markedly elevated human chorionic gonadotropin level and an ovarian mass, histopathologic examination is critical in distinguishing ectopic pregnancy from choriocarcinoma. Short tandem repeat analysis can be a useful adjunct to histologic diagnosis in challenging cases.

Comparison of clinical targeted next-generation sequence data from formalin-fixed and fresh-frozen tissue specimens.

Next-generation sequencing (NGS) has emerged as a powerful technique for the detection of genetic variants in the clinical laboratory. NGS can be performed using DNA from FFPE tissue, but it is unknown whether such specimens are truly equivalent to unfixed tissue for NGS applications. To address this question, we performed hybridization-capture enrichment and multiplexed Illumina NGS for 27 cancer-related genes using DNA from 16 paired fresh-frozen and routine FFPE lung adenocarcinoma specimens and conducted extensive comparisons between the sequence data from each sample type. This analysis revealed small but detectable differences between FFPE and frozen samples. Compared with frozen samples, NGS data from FFPE samples had smaller library insert sizes, greater coverage variability, and an increase in C to T transitions that was most pronounced at CpG dinucleotides, suggesting interplay between DNA methylation and formalin-induced changes; however, the error rate, library complexity, enrichment performance, and coverage statistics were not significantly different. Comparison of base calls between paired samples demonstrated concordances of >99.99%, with 96.8% agreement in the single-nucleotide variants detected and >98% accuracy of NGS data when compared with genotypes from an orthogonal single-nucleotide polymorphism array platform. This study demonstrates that routine processing of FFPE samples has a detectable but negligible effect on NGS data and that these samples can be a reliable substrate for clinical NGS testing.