Research autopsy programmes in oncology: shared experience from 14 centres across the world.

While there is a great clinical need to understand the biology of metastatic cancer in order to treat it more effectively, research is hampered by limited sample availability. Research autopsy programmes can crucially advance the field through synchronous, extensive, and high-volume sample collection. However, it remains an underused strategy in translational research. Via an extensive questionnaire, we collected information on the study design, enrolment strategy, study conduct, sample and data management, and challenges and opportunities of research autopsy programmes in oncology worldwide. Fourteen programmes participated in this study. Eight programmes operated 24 h/7 days, resulting in a lower median postmortem interval (time between death and start of the autopsy, 4 h) compared with those operating during working hours (9 h). Most programmes (n = 10) succeeded in collecting all samples within a median of 12 h after death. A large number of tumour sites were sampled during each autopsy (median 15.5 per patient). The median number of samples collected per patient was 58, including different processing methods for tumour samples but also non-tumour tissues and liquid biopsies. Unique biological insights derived from these samples included metastatic progression, treatment resistance, disease heterogeneity, tumour dormancy, interactions with the tumour micro-environment, and tumour representation in liquid biopsies. Tumour patient-derived xenograft (PDX) or organoid (PDO) models were additionally established, allowing for drug discovery and treatment sensitivity assays. Apart from the opportunities and achievements, we also present the challenges related with postmortem sample collections and strategies to overcome them, based on the shared experience of these 14 programmes. Through this work, we hope to increase the transparency of postmortem tissue donation, to encourage and aid the creation of new programmes, and to foster collaborations on these unique sample collections. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Cytopathologic features of epithelioid hemangioendothelioma including touch imprints for rapid on-site evaluation.

Objectives: Epithelioid hemangioendothelioma (EHE) is a vascular tumor of intermediate malignant potential, which presents as infiltrative lesions involving multiple organs. We reviewed our institutional experience with the cytologic diagnosis of this neoplasm including the performance of rapid on-site evaluation (ROSE). Material and Methods: From our institutional database, we identified 29 cytology specimens, obtained between 2012 and 2020, from 21 patients with biopsy confirmation of EHE. ROSE and final diagnosis were compared. All cytology slides were reviewed, and selected cytologic features were recorded. Results: The cohort included 29 specimens comprising 17 (59%) from liver, 6 (21%) from lung, 2 (7%) from lymph node, and 4 (14%) from other sites. At ROSE, 8/27 (30%) were reported inadequate, yet on review, all cases contained scattered cells typical of EHE in the touch imprint air-dried slides including two cases reported with a final diagnosis of non-diagnostic. All cases contained epithelioid and plasmacytoid cells with ovoid nuclei, fine chromatin, delicate (or biphasic) cytoplasm, and scattered cells with delicate, elongated cytoplasmic tails. The majority 26/29 (90%) of cases had multi-nucleated and multi-lobated nuclei. Intracytoplasmic lumens/blister cells were in 17/29 (59%), and a subset had erythrocytes therein (4/29, 14%). Metachromatic fibromyxoid or fibrotic stroma fragments were commonly seen (23/29, 79%). Mitoses and necrosis were absent in all cases. Of 11 tested cases, WWTR1::CAMTA1 and YAP1::TFE3 fusions were detected in nine and two cases, respectively. Conclusion: EHE has distinctive cytologic features which are often under-recognized during ROSE.

Tissue-specific features of the T cell repertoire after allogeneic hematopoietic cell transplantation in human and mouse.

T cells are the central drivers of many inflammatory diseases, but the repertoire of tissue-resident T cells at sites of pathology in human organs remains poorly understood. We examined the site-specificity of T cell receptor (TCR) repertoires across tissues (5 to 18 tissues per patient) in prospectively collected autopsies of patients with and without graft-versus-host disease (GVHD), a potentially lethal tissue-targeting complication of allogeneic hematopoietic cell transplantation, and in mouse models of GVHD. Anatomic similarity between tissues was a key determinant of TCR repertoire composition within patients, independent of disease or transplant status. The T cells recovered from peripheral blood and spleens in patients and mice captured a limited portion of the TCR repertoire detected in tissues. Whereas few T cell clones were shared across patients, motif-based clustering revealed shared repertoire signatures across patients in a tissue-specific fashion. T cells at disease sites had a tissue-resident phenotype and were of donor origin based on single-cell chimerism analysis. These data demonstrate the complex composition of T cell populations that persist in human tissues at the end stage of an inflammatory disorder after lymphocyte-directed therapy. These findings also underscore the importance of studying T cell in tissues rather than blood for tissue-based pathologies and suggest the tissue-specific nature of both the endogenous and posttransplant T cell landscape.

Cancer cells co-evolve with retrotransposons to mitigate viral mimicry.

Overexpression of repetitive elements is an emerging hallmark of human cancers 1 . Diverse repeats can mimic viruses by replicating within the cancer genome through retrotransposition, or presenting pathogen-associated molecular patterns (PAMPs) to the pattern recognition receptors (PRRs) of the innate immune system 2-5 . Yet, how specific repeats affect tumor evolution and shape the tumor immune microenvironment (TME) in a pro- or anti-tumorigenic manner remains poorly defined. Here, we integrate whole genome and total transcriptome data from a unique autopsy cohort of multiregional samples collected in pancreatic ductal adenocarcinoma (PDAC) patients, into a comprehensive evolutionary analysis. We find that more recently evolved S hort I nterspersed N uclear E lements (SINE), a family of retrotransposable repeats, are more likely to form immunostimulatory double-strand RNAs (dsRNAs). Consequently, younger SINEs are strongly co-regulated with RIG-I like receptor associated type-I interferon genes but anti-correlated with pro-tumorigenic macrophage infiltration. We discover that immunostimulatory SINE expression in tumors is regulated by either L ong I nterspersed N uclear E lements 1 (LINE1/L1) mobility or ADAR1 activity in a TP53 mutation dependent manner. Moreover, L1 retrotransposition activity tracks with tumor evolution and is associated with TP53 mutation status. Altogether, our results suggest pancreatic tumors actively evolve to modulate immunogenic SINE stress and induce pro-tumorigenic inflammation. Our integrative, evolutionary analysis therefore illustrates, for the first time, how dark matter genomic repeats enable tumors to co-evolve with the TME by actively regulating viral mimicry to their selective advantage.

Interobserver Reproducibility in Assessing Eosinophilic Cells in Ovarian Serous Borderline Tumors to Predict BRAF Mutational Status.

Ovarian serous borderline tumors (SBTs) harboring the BRAFV600E mutation are associated with decreased risk of progression to low-grade serous carcinoma, and often prominently feature tumor cells with abundant eosinophilic cytoplasm. Since eosinophilic cells (ECs) may be a marker of the underlying genetic driver, we proposed morphologic criteria and evaluated the interobserver reproducibility for assessing this histologic feature. Following the completion of an online training module, representative tumor slides from 40 SBTs ( BRAFV600E -mutated, n=18, BRAF -wildtype, n=22) were independently reviewed by 5 pathologists. For each case, reviewers provided a semiquantitative assessment of the extent of ECs (0: absent, 1: <10%, 2: 10%-50%, or 3: >50%, of tumor area). Interobserver reproducibility for estimating the extent of ECs was moderate (κ=0.41). Applying a cut-off score of ≥2, the median sensitivity and specificity for predicting BRAFV600E mutation were 67% and 95%, respectively. With a cut-off score of ≥1, median sensitivity and specificity were 100% and 82%, respectively. Morphologic mimics of ECs, including tumor cells with tufting or hobnail change and detached cell clusters in micropapillary SBTs, were possible contributing factors for discordant interobserver interpretations. BRAFV600E immunohistochemistry showed diffuse staining in BRAF -mutated tumors, including those with few ECs. In conclusion, the finding of extensive ECs in SBT is highly specific for BRAFV600E mutation. However, in some BRAF -mutated SBTs, ECs may be focal and/or difficult to distinguish from other tumor cells with overlapping cytologic features. The morphologic finding of definitive ECs, even when scarce, should therefore prompt consideration for BRAFV 600E mutation testing.

Landscape of chromatin remodeling gene alterations in endometrial carcinoma.

OBJECTIVE: Chromatin remodeling genes (CRGs) encode components of epigenetic regulatory mechanisms and alterations in these genes have been identified in several tumor types, including gynecologic cancers. In this study, we sought to investigate the prevalence and clinicopathological associations of CRG alterations in endometrial carcinoma (EC). METHODS: We performed a retrospective analysis of 660 ECs sequenced using a clinical massively parallel sequencing assay targeting up to 468 genes, including 25 CRGs, and defined the presence of somatic CRG alterations. Clinicopathologic features were obtained for all cases. Immunohistochemical interrogation of ARID1A and PTEN proteins was performed in a subset of samples. RESULTS: Of the 660 ECs sequenced, 438 (66.4%) harbored CRG alterations covered by our panel. The most commonly altered CRG was ARID1A (46%), followed by CTCF (21%), KMT2D (18%), KMT2B (17%), BCOR (16%), ARID1B (12%) and SMARCA4 (11%). We found that ARID1A genetic alterations were preferentially bi-allelic and often corresponded to altered ARID1A protein expression in ECs. We further observed that ARID1A alterations were often subclonal when compared to PTEN alterations, which were primarily clonal in ECs harboring both mutations. Finally, CRG alterations were associated with an increased likelihood of myometrial and lymphovascular invasion in endometrioid ECs. CONCLUSION: CRG alterations are common in EC and are associated with clinicopathologic features and likely play a crucial role in EC.

Evaluation of Women With a Positive Urine Cytology and no Demonstrable Disease in the Urinary Tract.

Urinary cytology is indispensable both for the evaluation of gross hematuria and surveillance of patients with urothelial neoplasms. A positive urine cytology usually indicates the presence of urothelial carcinoma somewhere in the urinary tract. However, in women, it may also signal urothelial carcinoma involvement of the lower gynecologic tract or be the presenting sign for a primary cancer of the lower gynecologic tract or rectum. Guidelines for the evaluation of women with a positive cytology and normal urinary tract are lacking. We present a review of the current literature with case scenarios to bring clinicians attention to this diagnostic dilemma.

Cytologic features of gynecologic germ cell tumors and carcinomas exhibiting germ cell tumor differentiation.

BACKGROUND: In this study, the authors sought to describe the cytologic features of primary gynecologic germ cell tumors and carcinomas exhibiting germ cell differentiation because little information currently exists. METHODS: An institutional database search was performed to identify histologically confirmed gynecologic germ cell tumors and carcinomas with germ cell tumor differentiation. Available cytologic material was reviewed by three observers, and morphologic features were recorded in addition to patient age at original diagnosis, primary tumor site, site(s) from which the examined cytologic material was obtained, and the type of examined cytologic preparations. RESULTS: In total, 15 cytologic specimens from 12 women (aged 19-82 years) were identified and included touch preparations of core biopsies from various sites (n = 6), fine-needle biopsies (n = 2), pelvic washings (n = 1), ascitic fluids (n = 4), pelvic cyst fluid (n = 1), and endometrial aspirate (n = 1). Of the 12 patients, seven had primary gynecologic germ cell tumors, four had gynecologic (ovarian and endometrial) tumors exhibiting somatic yolk sac tumor-like differentiation, and the remaining patient had an intestinal-type adenocarcinoma arising within an ovarian teratoma. There was morphologic overlap among many of the cases, although cytoplasmic vacuolation/granular cytoplasm was seen in 75% of primary yolk sac tumors or carcinomas with yolk sac tumor differentiation, and dense/squamoid cytoplasm was seen in 100% of teratomatous elements that were sampled. CONCLUSIONS: Germ cell tumors and somatic neoplasms exhibiting germ cell tumor differentiation occurring in adult women share some cytologic features and may be difficult to distinguish from one another, although some tumor types showed characteristic cytomorphologic findings.

Human Papillomavirus 42 Drives Digital Papillary Adenocarcinoma and Elicits a Germ Cell-like Program Conserved in HPV-Positive Cancers.

The skin is exposed to viral pathogens, but whether they contribute to the oncogenesis of skin cancers has not been systematically explored. Here we investigated 19 skin tumor types by analyzing off-target reads from commonly available next-generation sequencing data for viral pathogens. We identified human papillomavirus 42 (HPV42) in 96% (n = 45/47) of digital papillary adenocarcinoma (DPA), an aggressive cancer occurring on the fingers and toes. We show that HPV42, so far considered a nononcogenic, "low-risk" HPV, recapitulates the molecular hallmarks of oncogenic, "high-risk" HPVs. Using machine learning, we find that HPV-driven transformation elicits a germ cell-like transcriptional program conserved throughout all HPV-driven cancers (DPA, cervical carcinoma, and head and neck cancer). We further show that this germ cell-like transcriptional program, even when reduced to the top two genes (CDKN2A and SYCP2), serves as a fingerprint of oncogenic HPVs with implications for early detection, diagnosis, and therapy of all HPV-driven cancers. SIGNIFICANCE: We identify HPV42 as a uniform driver of DPA and add a new member to the short list of tumorigenic viruses in humans. We discover that all oncogenic HPVs evoke a germ cell-like transcriptional program with important implications for detecting, diagnosing, and treating all HPV-driven cancers. See related commentary by Starrett et al., p. 17. This article is highlighted in the In This Issue feature, p. 1.

Gastric-type adenocarcinoma of the cervix: Clinical outcomes and genomic drivers.

OBJECTIVES: Gastric-type endocervical adenocarcinoma (GEA) is a rare form of cervical cancer not associated with human papilloma virus (HPV) infection. We summarize our experience with GEA at a large cancer center. METHODS: Clinical and demographic information on all patients diagnosed with GEA between June 1, 2002 and July 1, 2019 was obtained retrospectively from clinical charts. Kaplan-Meier survival analysis was performed to describe progression-free survival (PFS) and overall survival (OS). Tumors from a subset of patients underwent next generation sequencing (NGS) analysis. RESULTS: A total of 70 women with GEA were identified, including 43 who received initial treatment at our institution: of these 4 (9%) underwent surgery alone, 15 (35%) underwent surgery followed by adjuvant therapy, 10 (23%) were treated with definitive concurrent chemoradiation (CCRT), 7 (16%) with chemotherapy alone, and 3 (7%) with neoadjuvant CCRT and hysterectomy with or without chemotherapy. One-third (n = 14) of patients experienced disease progression, of whom 86% (n = 12) had prior CCRT. The median PFS and OS for patients with stage I GEA were 107 months (95% CI 14.8-199.2 months) and 111 months (95% CI 17-205.1 months) respectively, compared to 17 months (95% CI 5.6-28.4 months) and 33 months (95% CI 28.2-37.8 months) for patients with stages II-IV, respectively. On NGS, 4 patients (14%) had ERBB2 alterations, including 2 patients who received trastuzumab. CONCLUSIONS: GEA is an aggressive form of cervical cancer with poor PFS and OS when diagnosed at stage II or later. Further investigation is needed to identify the optimal management approach for this rare subtype.

Genetic and Methylation Analysis of CTNNB1 in Benign and Malignant Melanocytic Lesions.

Melanocytic neoplasms have been genetically characterized in detail during the last decade. Recurrent CTNNB1 exon 3 mutations have been recognized in the distinct group of melanocytic tumors showing deep penetrating nevus-like morphology. In addition, they have been identified in 1-2% of advanced melanoma. Performing a detailed genetic analysis of difficult-to-classify nevi and melanomas with CTNNB1 mutations, we found that benign tumors (nevi) show characteristic morphological, genetic and epigenetic traits, which distinguish them from other nevi and melanoma. Malignant CTNNB1-mutant tumors (melanomas) demonstrated a different genetic profile, instead grouping clearly with other non-CTNNB1 melanomas in methylation assays. To further evaluate the role of CTNNB1 mutations in melanoma, we assessed a large cohort of clinically sequenced melanomas, identifying 38 tumors with CTNNB1 exon 3 mutations, including recurrent S45 (n = 13, 34%), G34 (n = 5, 13%), and S27 (n = 5, 13%) mutations. Locations and histological subtype of CTNNB1-mutated melanoma varied; none were reported as showing deep penetrating nevus-like morphology. The most frequent concurrent activating mutations were BRAF V600 (n = 21, 55%) and NRAS Q61 (n = 13, 34%). In our cohort, four of seven (58%) and one of nine (11%) patients treated with targeted therapy (BRAF and MEK Inhibitors) or immune-checkpoint therapy, respectively, showed disease control (partial response or stable disease). In summary, CTNNB1 mutations are associated with a unique melanocytic tumor type in benign tumors (nevi), which can be applied in a diagnostic setting. In advanced disease, no clear characteristics distinguishing CTNNB1-mutant from other melanomas were observed; however, studies of larger, optimally prospective, cohorts are warranted.

Molecular Subclasses of Clear Cell Ovarian Carcinoma and Their Impact on Disease Behavior and Outcomes.

PURPOSE: To identify molecular subclasses of clear cell ovarian carcinoma (CCOC) and assess their impact on clinical presentation and outcomes. EXPERIMENTAL DESIGN: We profiled 421 primary CCOCs that passed quality control using a targeted deep sequencing panel of 163 putative CCOC driver genes and whole transcriptome sequencing of 211 of these tumors. Molecularly defined subgroups were identified and tested for association with clinical characteristics and overall survival. RESULTS: We detected a putative somatic driver mutation in at least one candidate gene in 95% (401/421) of CCOC tumors including ARID1A (in 49% of tumors), PIK3CA (49%), TERT (20%), and TP53 (16%). Clustering of cancer driver mutations and RNA expression converged upon two distinct subclasses of CCOC. The first was dominated by ARID1A-mutated tumors with enriched expression of canonical CCOC genes and markers of platinum resistance; the second was largely comprised of tumors with TP53 mutations and enriched for the expression of genes involved in extracellular matrix organization and mesenchymal differentiation. Compared with the ARID1A-mutated group, women with TP53-mutated tumors were more likely to have advanced-stage disease, no antecedent history of endometriosis, and poorer survival, driven by their advanced stage at presentation. In women with ARID1A-mutated tumors, there was a trend toward a lower rate of response to first-line platinum-based therapy. CONCLUSIONS: Our study suggests that CCOC consists of two distinct molecular subclasses with distinct clinical presentation and outcomes, with potential relevance to both traditional and experimental therapy responsiveness. See related commentary by Lheureux, p. 4838.

Genetic and methylation profiles distinguish benign, malignant and spitzoid melanocytic tumors.

Accurate classification of melanocytic tumors is important for prognostic evaluation, treatment and follow-up protocols of patients. The majority of melanocytic proliferations can be classified solely based on clinical and pathological criteria, however in select cases a definitive diagnostic assessment remains challenging and additional diagnostic biomarkers would be advantageous. We analyzed melanomas, nevi, Spitz nevi and atypical spitzoid tumors using parallel sequencing (exons of 611 genes and 507 gene translocation analysis) and methylation arrays (850k Illumina EPIC). By combining detailed genetic and epigenetic analysis with reference-based and reference-free DNA methylome deconvolution we compared Spitz nevi to nevi and melanoma and assessed the potential for these methods in classifying challenging spitzoid tumors. Results were correlated with clinical and histologic features. Spitz nevi were found to cluster independently of nevi and melanoma and demonstrated a different mutation profile. Multiple copy number alterations and TERT promoter mutations were identified only in melanomas. Genome-wide methylation in Spitz nevi was comparable to benign nevi while the Leukocytes UnMethylation for Purity (LUMP) algorithm in Spitz nevi was comparable to melanoma. Histologically difficult to classify Spitz tumor cases were assessed which, based on methylation arrays, clustered between Spitz nevi and melanoma and in terms of genetic profile or copy number variations demonstrated worrisome features suggesting a malignant neoplasm. Comprehensive sequencing and methylation analysis verify Spitz nevi as an independent melanocytic entity distinct from both nevi and melanoma. Combined genetic and methylation assays can offer additional insights in diagnosing difficult to classify Spitzoid tumors.

Treatment of ovarian clear cell carcinoma with immune checkpoint blockade: a case series.

BACKGROUND: Although immune checkpoint blockade has demonstrated limited effectiveness against ovarian cancer, subset analyses from completed trials suggest possible superior efficacy in the clear cell carcinoma subtype. OBJECTIVE: To describe the outcomes of patients with ovarian clear cell carcinoma treated with immune checkpoint blockade. METHODS: This was a single-institution, retrospective case series of patients with ovarian clear cell carcinoma treated with a programmed cell death protein 1 (PD-1) or programmed death-ligand 1 (PD-L1) inhibitor with or without concomitant cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibition between January 2016 and June 2021. Demographic variables, tumor microenvironment, molecular data, and clinical outcomes were examined. Time to treatment failure was defined as the number of days between start of treatment and next line of treatment or death. RESULTS: A total of 16 eligible patients were analyzed. The median treatment duration was 56 days (range 14-574); median time to treatment failure was 99 days (range 27-1568). The reason for discontinuation was disease progression in 88% of cases. Four patients (25%) experienced durable clinical benefit (time to treatment failure ≥180 days). One patient was treated twice with combined immune checkpoint blockade and experienced a complete response each time. All 12 patients who underwent clinical tumor-normal molecular profiling had microsatellite-stable disease, and all but one had low tumor mutation burden. Multiplex immunofluorescence analysis available from pre-treatment biopsies of two patients with clinical benefit demonstrated abundant tumor-infiltrating lymphocytes expressing PD-1. CONCLUSION: Our study suggests a potential role for immune checkpoint blockade in patients with clear cell carcinoma of the ovary. Identification of genetic and microenvironmental biomarkers predictive of response will be key to guide therapy.

Recurrent WWTR1 S89W mutations and Hippo pathway deregulation in clear cell carcinomas of the cervix.

Clear cell carcinoma (CCC) of the cervix (cCCC) is a rare and aggressive type of human papillomavirus (HPV)-negative cervical cancer with limited effective treatment options for recurrent or metastatic disease. Historically, CCCs of the lower genital tract were associated with in utero diethylstilbestrol exposure; however, the genetic landscape of sporadic cCCCs remains unknown. Here we sought to define the molecular underpinning of cCCCs. Using a combination of whole-exome, targeted capture, and RNA-sequencing, we identified pathogenic genetic alterations in the Hippo signaling pathway in 50% (10/20) of cCCCs, including recurrent WWTR1 S89W somatic mutations in 40% (4/10) of the cases harboring mutations in the Hippo pathway. Irrespective of the presence or absence of Hippo pathway genetic alterations, however, all primary cCCCs analyzed in this study (n = 20) harbored features of Hippo pathway deregulation at the transcriptomic and protein levels. In vitro functional analysis revealed that expression of the WWTR1 S89W mutation leads to reduced binding of TAZ to 14-3-3, promoting constitutive nuclear translocation of TAZ and Hippo pathway repression. WWTR1 S89W expression was found to lead to acquisition of oncogenic behavior, including increased proliferation, migration, and colony formation in vitro as well as increased tumorigenesis in vivo, which could be reversed by targeted inhibition of the TAZ/YAP1 complex with verteporfin. Finally, xenografts expressing WWTR1 S89W displayed a shift in tumor phenotype, becoming more infiltrative as well as less differentiated, and were found to be composed of cells with conspicuous cytoplasmic clearing as compared to controls. Our results demonstrate that Hippo pathway alterations are likely drivers of cCCCs and likely contribute to the clear cell phenotype. Therapies targeting this pathway may constitute a new class of treatment for these rare, aggressive tumors. © 2022 The Pathological Society of Great Britain and Ireland.

Cytologic features of sex cord-stromal tumors in women.

BACKGROUND: Gynecologic sex cord-stromal tumors (SCSTs) arise from sex cords of the embryonic gonad and may display malignant behavior. We describe the cytomorphologic features of SCSTs in females, including adult and juvenile granulosa cell tumors (AGCTs and JGCTs), Sertoli-Leydig cell tumors (SLCTs), and steroid cell tumors (SCTs). METHODS: We retrieved available cytology slides from females with a histologic diagnosis of sex cord-stromal tumor between 2009 and 2020 from institutional archives and reviewed their cytoarchitectural features. RESULTS: There were 25, 2, 2, and 1 cytology specimens from 19, 2, 2, and 1 patients (aged 7-90 years, median 57 years) with AGCT, JGCT, SLCT, and SCT, respectively. Features common to all SCSTs included 3-dimensional groups, rosettes, rare papillary fragments, abundant single cells and naked nuclei. Rosettes and a streaming appearance of cell groups were only seen in AGCTs, which also rarely featured eosinophilic hyaline globules and metachromatic stroma. AGCTs exhibited high nuclear:cytoplasmic (N:C) ratios, with mild nuclear pleomorphism, uniform nuclei with finely granular chromatin, nuclear grooves and small nucleoli; in contrast, other SCSTs lacked rosettes and nuclear grooves and had generally lower N:C ratios, greater nuclear pleomorphism, coarse chromatin and more abundant cytoplasm. Mitotic figures, necrosis, and inflammation were rarely identified. CONCLUSIONS: AGCTs show cytomorphologic features that are distinct from those of other SCSTs. Careful evaluation of the cytological features and ancillary studies (eg, immunochemistry for FOXL2, inhibin and calretinin, or sequencing for FOXL2 mutations) can aid in the accurate diagnosis of these tumors.

TSC2-mutant uterine sarcomas with JAZF1-SUZ12 fusions demonstrate hybrid features of endometrial stromal sarcoma and PEComa and are responsive to mTOR inhibition.

Uterine perivascular epithelioid cell tumor (PEComa) is a rare mesenchymal neoplasm that occasionally shares morphologic and immunohistochemical overlap with low- and high-grade endometrial stromal sarcoma (LGESS and HGESS). In this study, we sought to characterize the clinical, morphologic, genetic, and epigenetic features of five uterine sarcomas that display histologic features of LGESS, HGESS, and PEComa. All tumors demonstrated epithelioid cells often associated with a low-grade spindled component resembling LGESS, with both regions expressing CD10, ER, PR, variable HMB45, and Melan-A immunoreactivity, and strong cathepsin K and pS6 expression. Targeted massively parallel sequencing analysis revealed the presence of somatic TSC2 mutations in all five cases, of which four harbored concurrent or consecutive JAZF1-SUZ12 gene fusions. Unsupervised hierarchical clustering analysis of methylation profiles of TSC2-mutant uterine sarcomas (n = 4), LGESS (n = 10), and HGESS (n = 12) demonstrated two clusters consisting of (1) all LGESS and TSC2-mutant uterine sarcomas and (2) all HGESS. KEGG pathway analysis detected methylation differences in genes involved in PI3K/AKT, calcium, and Rap1 signaling. TSC2-mutant uterine sarcomas were responsive to hormone suppression, and mTOR inhibition demonstrated clinical benefit in four patients with these neoplasms. Our results suggest that these tumors represent histologically distinctive LGESS with TSC2 mutations. TSC2 mutations and JAZF1-SUZ12 fusion may help diagnose these tumors and possibly direct effective treatment.

TERT promoter mutations are associated with longer progression-free and overall survival in patients with BRAF-mutant melanoma receiving BRAF and MEK inhibitor therapy.

BACKGROUND: Around 50% of cutaneous melanomas harbour therapeutically targetable BRAF V600 mutations. Reliable clinical biomarkers predicting duration of response to BRAF-targeted therapies are still lacking. Recent in vitro studies demonstrated that BRAF-MEK inhibitor therapy response is associated with tumour TERT promoter mutation status. We assessed this potential association in a clinical setting. METHODS: The study cohort comprised 232 patients with metastatic or unresectable BRAF V600-mutated melanoma receiving combined BRAF/MEK inhibitor treatment, including a single-centre retrospective discovery cohort (N = 120) and a prospectively collected multicenter validation cohort (N = 112). Patients were excluded if they received BRAF or MEK inhibitors in an adjuvant setting, as monotherapy, or in combination with immunotherapy. Kaplan-Meier and univariate/multivariate Cox regression analyses were performed as appropriate. RESULTS: median age at first diagnosis was 54 years (range 16-84 years). The majority of patients were men 147/232 (63.4%). Most tumours harboured TERT promoter mutations (72%, N = 167). A survival advantage was observed in both progression-free survival (PFS) and overall survival (OS) for patients with TERT promoter-mutant versus wild-type tumours in both the discovery cohort (mPFS of 9.6 months [N = 87] vs 5.0 months [N = 33]; hazard ratio [HR] = 0.56 [95% confidence interval {CI} 0.33-0.96] and mOS of 33.6 months vs 15.0 months; HR = 0.47 [95%CI 0.32-0.70]) as well as the validation cohort (mPFS of 7.3 months [N = 80] vs 5.8 months [N = 32]; HR = 0.67 [95%CI 0.41-1.10] and mOS of 51.1 months vs 15.0 months; HR = 0.33 [95%CI 0.18-0.63]). In the pooled cohort of TERT promoter-mutant (N = 167) versus wild-type (N = 65) tumours, respectively, PFS was 8.9 versus 5.5 months, (HR = 0.62; 95%CI 0.45-0.87; P = 0.004), and OS was 33.6 versus 17.0 months, (HR = 0.51; 95%CI 0.35-0.75, P = 0.0001). CONCLUSIONS: In patients with melanoma receiving BRAF/MEK-targeted therapies, TERT promoter mutations are associated with longer survival. If validated in larger studies, TERT promoter mutation status should be included as a predictive biomarker in treatment algorithms for advanced melanoma.

NF1-mutated melanomas reveal distinct clinical characteristics depending on tumour origin and respond favourably to immune checkpoint inhibitors.

BACKGROUND: NF1-mutated tumours represent a small subset (10-15%) of melanomas, not sufficiently analysed in large clinical cohorts. This study investigated the largest multicentre collection of NF1-mutated melanomas to date. METHODS: This study analysed a multicentre tumour tissue sample cohort from 266 patients with NF1-mutated melanoma. Targeted next-generation sequencing of the TERT promoter and 29 relevant melanoma genes was performed. Survival was compared with NF1 wild-type cohorts from the Tissue Registry in Melanoma project (n = 432). RESULTS: Most NF1-mutated melanoma arose in the head-and-neck region of patients >60 years. NF1 alterations were frequently inactivating, primarily non-sense, less frequently truncating mutations. Non-inactivating NF1 mutations more frequently co-occurred with activating BRAF and RAS mutations. NF1-mutated tumours had higher numbers of gene mutations and UV signature C>T and CC>TT transitions than BRAF, RAS and triple wild-type melanomas. NF1-mutated acral and mucosal melanomas harboured a different mutation signature and were frequent in women (69% and 83%, respectively), differing from non-acral cutaneous NF1-mutated melanomas (men 73%, women 27%). Overall survival in stage IV disease was comparable for patients with NF1-mutated or wild-type melanoma. However, in patients receiving first-line immune checkpoint inhibitor treatment, better median overall survival (mOS) was observed for NF1-mutated than wild-type tumours (mOS = not reached vs mOS = 25.82, p = 0.0154, n = 80 and 432, respectively). CONCLUSIONS: Cutaneous, acral and mucosal NF1-mutated melanomas vary in clinical and genetic characteristics and demonstrate a favourable outcome on immune checkpoint inhibition therapy.

Characterization, isolation, and in vitro culture of leptomeningeal fibroblasts.

Meninges, or the membranous coverings of the brain and spinal cord, play host to dozens of morbid pathologies. In this study we provide a method to isolate the leptomeningeal cell layer, identify leptomeninges in histologic slides, and maintain leptomeningeal fibroblasts in in vitro culture. Using an array of transcriptomic, histological, and cytometric analyses, we identified ICAM1 and SLC38A2 as two novel markers of leptomeningeal cells in vivo and in vitro. Our results confirm the fibroblastoid nature of leptomeningeal cells and their ability to form a sheet-like layer that covers the brain and spine parenchyma. These findings will enable researchers in central nervous system barriers to describe leptomeningeal cell functions in health and disease.