Primary Cutaneous NUT Carcinoma: Clinicopathologic and Genetic Study of 4 Cases.

WHO Classification of Skin Tumors, fifth edition (2023) has newly described primary cutaneous NUT carcinoma; however, information on this cancer type remains scarce. Herein, we performed clinicopathologic and genetic analyses of 4 cases. Four elderly women (median age 77 y, range: 68 to 82 y) were included. The median tumor size was 12.5 (10 to 40 mm). Tumors were located on the scalp, temple, thigh, and palm. Two (50%) patients presented with regional lymph node metastases. Neither distant metastasis nor mortality was observed during patient follow-up of 10.5 (3 to 15) months. Sanger, panel DNA and whole-exome RNA sequencing revealed BRD3::NUTM1 (n=2) and BRD4::NUTM1 (n=2) fusions. Histology of BRD3-rearranged tumors revealed an epidermal connection, relatively small tumor nests, and ductal or intracytoplasmic luminal formation, whereas that of BRD4-rearranged tumors revealed large solid nests comprising discohesive tumor cells. NUT, cytokeratins, p63, EMA, TRPS1, c-MYB, CD56, and INSM1 were immunoexpressed to varying degrees in all (100%) tumors. Furthermore, diffuse SOX10 expression was common (3/4, 75%). The literature review of five previously described cases revealed women predominance, no recurrence, frequent BRD3::NUTM1 fusions, and histology of ductoglandular structures. Our study findings and literature suggest elderly women predominance, relatively frequent BRD3::NUTM1 fusions, histopathologic ductoglandular differentiation, absence of abrupt keratinisation, and a characteristic immunoprofile in primary cutaneous NUT carcinoma, unlike in that of other organ. No distant metastasis or disease-associated mortality was seen in all cases with limited follow-up.

Loss of p16 Immunoexpression and Deletions of CDKN2A in the Progression of Extramammary Paget Disease: An Immunohistochemical and Genetic Study of 24 Invasive/Metastatic Cases.

ABSTRACT: Information regarding the genetic alterations in extramammary Paget disease (EMPD) is scarce. This study investigated the significance of CDKN2A and MTAP alterations in EMPD progression using immunohistochemistry and panel DNA sequencing. In total, 24 invasive/metastatic EMPD cases were included in this study. The immunoexpression of p16 and MTAP in the primary in situ, primary invasive, and metastatic tumor components was evaluated. Panel DNA sequencing was performed for metastatic tumor components in 5 of the 24 cases. Immunoexpression of p16 in the in situ tumor component was at least partially preserved in all 19 tested cases (100%). By contrast, the invasive tumor component was diffusely or partially lost in 18 (81.8%) of 22 tested cases. Regarding the foci of lymph node metastasis, 13 (81.2%) of the 16 patients showed a significant loss of p16 expression. Loss of MTAP immunoexpression was observed less frequently compared with the loss of p16 expression. CDKN2A homozygous deletions were confirmed in all 5 tested cases by sequencing, whereas MTAP deletions were detected in only 2 cases. In conclusion, p16 expression loss and CDKN2A deletions can be frequently seen in invasive/metastatic cases of EMPD.

Recurrent GATA3 P409Afs*99 Frameshift Extension Mutations in Sweat-gland Carcinoma With Neuroendocrine Differentiation.

Sweat-gland carcinoma with neuroendocrine differentiation (SCAND) was recently proposed as a new cutaneous adnexal neoplasm with neuroendocrine differentiation; however, its genetics are not well known. Herein, we performed clinicopathologic and genetic analyses of 13 SCAND cases and 5 control cases of endocrine mucin-producing sweat gland carcinoma (EMPSGC). The SCAND group included 11 males and 2 females with a median age of 68 years (range, 50 to 80 y). All SCAND lesions occurred in the ventral trunk or genital area. Of the 13 SCAND cases, 9 and 5 exhibited lymph node and distant metastases, respectively. Three (23.1%) patients with SCAND died of the disease. In contrast, neither metastasis nor mortality was confirmed in the EMPSGC cases. Immunoexpression of the androgen receptor, c-Myb, and MUC2 was limited in SCAND, whereas EMPSGC frequently expressed these immunomarkers. GATA3 P409Afs*99 extension mutations were detected in 7 (53.8%) of the 13 SCAND cases, using Sanger or panel sequencing. All 7 SCAND cases with GATA3 mutations were located in the genital, inguinal, or lower abdominal regions, whereas 5 of the other 6 SCAND cases were located in the anterior upper to mid-trunk. No GATA3 mutations were detected in the EMPSGC cases (0/5, 0%). These clinicopathologic and genetic findings support SCAND as a tumor entity distinguishable from EMPSGC. In addition, the characteristic frameshift extension mutations in GATA3 contribute to the establishment of the tumor-type concept of SCAND.

Clonal Origin and Lineage Ambiguity in Mixed Neuroendocrine Carcinoma of the Uterine Cervix.

Small-cell neuroendocrine carcinoma (SCNEC) of the cervix is a rare disease characterized by a high incidence of mixed tumors with other types of cancer. The mechanism underlying this mixed phenotype is not well understood. This study established a panel of organoid lines from patients with SCNEC of the cervix and ultimately focused on one line, which retained a mixed tumor phenotype, both in vitro and in vivo. Histologically, both organoids and xenograft tumors showed distinct differentiation into either SCNEC or adenocarcinoma in some regions and ambiguous differentiation in others. Tracking single cells indicated the existence of cells with bipotential differentiation toward SCNEC and adenocarcinomas. Single-cell transcriptional analysis identified three distinct clusters: SCNEC-like, adenocarcinoma-like, and a cluster lacking specific differentiation markers. The expression of neuroendocrine markers was enriched in the SCNEC-like cluster but not exclusively. Human papillomavirus 18 E6 was enriched in the SCNEC-like cluster, which showed higher proliferation and lower levels of the p53 pathway. After treatment with anticancer drugs, the expression of adenocarcinoma markers increased, whereas that of SCNEC decreased. Using a reporter system for keratin 19 expression, changes in the differentiation of each cell were shown to be associated with the shift in differentiation induced by drug treatment. These data suggest that mixed SCNEC/cervical tumors have a clonal origin and are characterized by an ambiguous and flexible differentiation state.

KMT2A-rearranged sarcoma with unusual fusion gene CBX6::KMT2A::PYGO1.

Recently, rare sarcomas harboring KMT2A rearrangements have been reported. They occur in relatively young individuals, exhibit a sclerosing epithelioid fibrosarcoma-like morphology, and often have an aggressive prognosis. YAP1::KMT2A::YAP1 is the most common fusion gene, followed by VIM::KMT2A. We report the case of a 47-year-old man with a spindle cell tumor arising from the subcutaneous tissue of the right anterior chest. The tumor harbored an unusual novel fusion gene, CBX6::KMT2A::PYGO1. Histologically, the tumor consisted of proliferating spindle-shaped cells with uniform nuclei, which varied in cell density and the amount of intervening collagen fibers. After 2 years and 8 months without postoperative treatment, the patient showed no recurrence or metastasis. Although highly likely irreproducible, tumors with the CBX6::KMT2A::PYGO1 fusion gene were morphologically somewhat different from those containing the YAP1::KMT2A::YAP1. This suggests that KMT2A rearrangements with fusion gene partners different from YAP1 result in purely spindle-shaped cell tumors that produce collagen fibers.

A Method for Extending Target Regions of Genomic Profiling by Combining a Custom Probe Pool with a Commercial Targeted Panel.

BACKGROUND: Next-generation sequencing (NGS)-based genomic profiling is becoming widespread in determining treatment policies for patients with tumors. Commercially available gene panels for pan-tumor targets comprise hundreds of tumor-related genes but frequently lack genes of interest in specific tumor types. In this study, we demonstrate a method for extending target regions of genomic profiling by combining a custom probe pool with a commercial targeted panel. METHODS: We used TruSight Oncology 500 (TSO500) as a commercial targeted panel and a custom probe pool designed for all exons of the SMARCA2 gene. Sequencing libraries of custom targets were constructed using a portion of the TSO500 library solution before the hybridization-capture process. After hybridization capture, both libraries were combined and sequenced using a next-generation sequencer. RESULTS: Sequencing results showed that >96.8% and 100% of the target exons were covered at a depth of over 100× using the TSO500 and custom panels, respectively. The custom panels had slightly better median exon coverage than the TSO500. The combined libraries of the custom and TSO500 panels showed a mapped read ratio close to the mixing ratio. Analysis of mutation-free regions showed similar accuracies between the TSO500 and custom panels regarding variant calling. CONCLUSIONS: Our devised method easily and affordably extends the targets beyond a ready-made panel. This method provides a valuable solution until the widespread adoption of whole-exome sequencing, which is costly for large target sizes.

Secondary leiomyosarcoma of the nasal cavity in a treated patient with possible hereditary retinoblastoma with germline reciprocal translocation of RB1 and DMXL1 and somatic TP53 mutation: A case report.

Retinoblastoma is a common primary intraocular malignant tumor that affects infants and young children. Radiation therapy for hereditary retinoblastoma increases the risk of secondary malignancy. The present report discusses the case of a retinoblastoma survivor who developed secondary leiomyosarcoma 42 years after receiving radiation therapy. The retinoblastoma of the patient was unilateral, and the patient had no family history of the disease. RNA and DNA panel sequencing of the leiomyosarcoma tissue was performed to elucidate the molecular mechanism of this secondary malignancy. The RNA panel sequencing detected a germline reciprocal translocation of RB1 and DMXL1, leading to a diagnosis of possible hereditary retinoblastoma. Furthermore, it detected a somatic fusion gene (RAD51-KNL1). The DNA panel sequencing identified various germline or somatic variants, including a somatic splice acceptor site mutation of TP53. We hypothesized that the molecular mechanism of the secondary malignancy of this patient was the combination of a germline reciprocal translocation of RB1 and DMXL1 and the accumulation of various somatic mutations containing the splice acceptor site mutation of TP53, which ultimately led to the development of a secondary leiomyosarcoma. Further prospective investigations are necessary to fully understand the role of reciprocal translocation of RB1 and DMXL1 or other mutations in the tumorigenesis of second malignancies in patients with hereditary retinoblastoma.

KDM2B-Rearranged Soft Tissue Sarcomas Expand the Concept of BCOR-Associated Sarcoma.

Sarcomas with BCOR genetic alterations (BCOR-associated sarcomas) represent a recently recognized family of soft tissue and bone tumors characterized by BCOR fusion, BCOR internal tandem duplication, or YWHAE::NUTM2B fusion. Histologically, the tumors demonstrate oval to spindle cell proliferation in a variably vascular stroma and overexpression of BCOR and SATB2. Herein, we describe 3 soft tissue sarcomas with KDM2B fusions that phenotypically and epigenetically match BCOR-associated sarcomas. The cases included 1 infant, 1 adolescent, and 1 older patient. All tumors showed histologic findings indistinguishable from those of BCOR-associated sarcomas and were originally diagnosed as such based on the phenotype. However, none of the tumors had BCOR or YWHAE genetic alterations. Instead, targeted RNA sequencing identified in-frame KDM2B::NUTM2B, KDM2B::CREBBP, and KDM2B::DUX4 fusions. KDM2B fusions were validated using reverse-transcription PCR, Sanger sequencing, and in situ hybridization assays. Genome-wide DNA methylation analysis matched all 3 tumors with BCOR-associated sarcomas using the Deutsches Krebsforschungszentrum (DKFZ) classifier and t-distributed stochastic neighbor embedding analysis. One localized tumor showed a flat genome-wide copy number profile, and the patient remained disease-free after treatment. The other tumors showed multiple copy number alterations, including MDM2/CDK4 amplification and/or CDKN2A/B loss, and both tumors metastasized, leading to the patient's death in one of the cases. When tested using KDM2B immunohistochemistry, all 3 KDM2B-rearranged sarcomas showed diffuse strong staining, and all 13 sarcomas with BCOR genetic alterations also demonstrated diffuse, strong, or weak staining. By contrast, among 72 mimicking tumors, only a subset of synovial sarcomas showed focal or diffuse weak KDM2B expression. In conclusion, our study suggests that KDM2B-rearranged soft tissue sarcomas belong to the BCOR-associated sarcoma family and expand its molecular spectrum. This may be related to the known molecular relationship between KDM2B and BCOR in the polycomb repressive complex 1.1. Immunohistochemical analysis of KDM2B is a potentially valuable diagnostic tool for BCOR-associated sarcomas, including those with KDM2B rearrangement.

Clinicopathologic and genetic characterization of invasive melanoma with BRAF V600K mutation: A study of 16 cases.

BACKGROUND: The clinicopathologic and genetic features of cutaneous melanoma with a BRAF V600K mutation are not well-known. We aimed to evaluate these characteristics in comparison with those associated with BRAF V600E. METHODS: Real-time polymerase chain reaction (PCR) and/or the MassARRAY® system were used to detect BRAF V600K in 16 invasive melanomas and confirm BRAF V600E in another 60 cases. Immunohistochemistry and panel next-generation sequencing were used to evaluate protein expression and tumor mutation burden, respectively. RESULTS: The median age of melanoma patients harboring the BRAF V600K mutation (72.5 years) was higher than those with the BRAF V600E (58.5 years). The two groups also differed in sex (13/16 [81.3%] male in the V600K group vs. 23/60 [38.3%] in V600E) and in the frequency of scalp involvement (8/16 [50.0%] in V600K vs. 1/60 [1.6%] in V600E). The clinical appearance was similar to a superficial spreading melanoma. Histopathologically, non-nested lentiginous intraepidermal spread and subtle solar elastosis were observed. One patient (1/13, 7.7%) had a pre-existing intradermal nevus. Diffuse PRAME immunoexpression was seen in only one (14.3%) of seven tested cases. Loss of p16 expression was observed in all 12 cases (100%) analyzed. The tumor mutation burden was 8 and 6 mutations/Mb in the two tested cases. CONCLUSIONS: Melanoma carrying the BRAF V600K mutation showed the predominance on the scalp of elderly men, lentiginous intraepidermal growth, subtle solar elastosis, possible existence of intradermal nevus component, frequent loss of p16 immunoexpression, limited immunoreactivity for PRAME, and intermediate tumor mutation burden.

Analytical Performance of a Highly Sensitive System to Detect Gene Variants Using Next-Generation Sequencing for Lung Cancer Companion Diagnostics.

The recent increase in the number of molecular targeted agents for lung cancer has led to the demand for the simultaneous testing of multiple genes. Although gene panels using next-generation sequencing (NGS) are ideal, conventional panels require a high tumor content, and biopsy samples often do not meet this requirement. We developed a new NGS panel, called compact panel, characterized by high sensitivity, with detection limits for mutations of 0.14%, 0.20%, 0.48%, 0.24%, and 0.20% for EGFR exon 19 deletion, L858R, T790M, BRAF V600E, and KRAS G12C, respectively. Mutation detection also had a high quantitative ability, with correlation coefficients ranging from 0.966 to 0.992. The threshold for fusion detection was 1%. The panel exhibited good concordance with the approved tests. The identity rates were as follows: EGFR positive, 100% (95% confidence interval, 95.5-100); EGFR negative, 90.9 (82.2-96.3); BRAF positive, 100 (59.0-100); BRAF negative, 100 (94.9-100); KRAS G12C positive, 100 (92.7-100); KRAS G12C negative, 100 (93.0-100); ALK positive, 96.7 (83.8-99.9); ALK negative, 98.4 (97.2-99.2); ROS1 positive, 100 (66.4-100); ROS1 negative, 99.0 (94.6-100); MET positive, 98.0 (89.0-99.9); MET negative 100 (92.8-100); RET positive, 93.8 (69.8-100); RET negative, 100 (94.9-100). The analytical performance showed that the panel could handle various types of biopsy samples obtained by routine clinical practice without requiring strict pathological monitoring, as in the case of conventional NGS panels.

Utility of Comprehensive Genomic Profiling Tests for Patients with Incurable Pancreatic Cancer in Clinical Practice.

Although comprehensive genomic profiling (CGP) tests have been covered under the Japanese national health insurance program since 2018, the utility and issues of CGP tests have not been clarified. We retrospectively reviewed 115 patients with incurable pancreatic cancer (IPC) who underwent CGP tests in a Japanese cancer referral center from November 2019 to August 2021. We evaluated the results of CGP tests, treatments based on CGP tests, and survival time. Eight cases (6.9%) were diagnosed as tumor mutation burden-high (TMB-H) and/or microsatellite instability-high (MSI-H). The gene mutation rates of KRAS/TP53/CDKN2A/SMAD4 were 93.0/83.0/53.0/25.2%, respectively. Twenty-five patients (21.7%) had homologous recombination deficiency (HRD)-related genetic mutations. Four patients (3.5%) having TMB-H and/or MSI-H were treated with pembrolizumab, and only two patients (1.7%) participated in the clinical trials. Patient characteristics were not significantly different between patients with and without HRD-related gene mutations. The median OS was significantly longer in the HRD (+) group than in the HRD (-) group (749 days vs. 519 days, p = 0.047). In multivariate analysis, HRD-related gene mutation was an independent prognostic factor associated with favorable OS. CGP tests for patients with IPC have the potential utility of detecting HRD-related gene mutations as prognostic factors as well as a therapeutic search.

Cutaneous syncytial myoepithelioma with folliculocentric growth and EWSR1 gene rearrangement: A case report.

Cutaneous syncytial myoepithelioma is a tumor type that was initially reported in 2013 as a syncytial variant of cutaneous myoepithelioma characterized by intradermal nodular proliferation of oval to spindle-shaped tumor cells in solid and syncytial patterns. Fusion of genes Ewing sarcoma breakpoint region 1 / EWS RNA binding protein 1 (EWSR1) and pre-B cell leukemia homeobox 3 (PBX3) is found in approximately 90% of the cases. We report a case of cutaneous syncytial myoepithelioma with diagnostic difficulty due to folliculocentric morphology and atypical immunohistochemical results, including diffuse positivity of α-smooth muscle actin and claudin 4 and negative immunoreactions for epithelial membrane antigen and S100 protein. In the present case, fluorescence in situ hybridization study demonstrated EWSR1 rearrangement. We further provide a discussion of differential diagnoses with a review of relevant literature.

Prevalence and characteristics of immune checkpoint inhibitor-related myocardial damage: A prospective observational study.

An increasing number of patients with cancer are being treated with immune checkpoint inhibitors. Consequently, the incidence of immune checkpoint inhibitor-related myocarditis has been increasing. Nonetheless, the diagnostic criteria for the immune checkpoint inhibitor-related myocarditis have not been sufficiently established. Therefore, the real-world incidence or prevalence of immune checkpoint inhibitor-related myocardial damage remains unknown. This was a single-center cohort study that included 100 patients admitted for immune checkpoint inhibitor therapy for any type of cancer. The patients underwent monthly measurement of cardiac troponin I and N-terminal pro-brain natriuretic peptide levels with electrocardiography. Additionally, echocardiography was performed every 3 months. Our protocol was continued until 6 months after the initiation of immune checkpoint inhibitors. We defined immune checkpoint inhibitor-related myocardial damage as an increase in cardiac troponin I levels by >0.026 ng/mL and/or a decrease in the left ventricular ejection fraction by >10% to <53% on echocardiography. The mean patient age was 64 years; 71% were men. The most commonly used immune checkpoint inhibitor was nivolumab (47%), followed by pembrolizumab (29%). Overall, 5% of patients received combination therapy. Among 100 patients, 10 (10%) were diagnosed with immune checkpoint inhibitor-related myocardial damage. Among them, five patients underwent endomyocardial biopsy. Of these patients, four were histopathologically observed to have lymphocyte infiltration in their myocardium. In conclusion, serial cardiac troponin I measurement during immune checkpoint inhibitor treatment could help detect early-phase myocardial damage. The prevalence of myocardial damage was much higher than previously expected.

Clinical application of comprehensive genomic profiling panel to thoracic malignancies: A single-center retrospective study.

BACKGROUND: The usefulness of comprehensive genomic profiling (CGP) panels for thoracic malignancies after completion of the standard treatment is unclear. METHODS: The results of CGP panels for malignant thoracic diseases performed at our hospital between December 2019 and June 2022 were collected. We examined whether CGP panel results led to new treatment, correlated with the effectiveness of immune checkpoint inhibitors (ICIs), or revealed secondary findings related to hereditary tumors. RESULTS: A total of 60 patients were enrolled, of which 52 (86.6%) had lung cancer. In six (10%) patients, the panel results led to treatment with insurance-listed molecular-targeted agents; four patients had EGFR mutations not detected by the real-time polymerase chain reaction assay and two had MET ex.14 skipping mutations. In small-cell lung cancer, the tumor mutation burden was high in 4/6 (66.7%) patients and pembrolizumab was available. Another MET ex.14 skipping mutation was detected in two cases with EGFR-tyrosine kinase inhibitor resistance. ICI efficacy was ≤1 year in patients with STK-11, KEAP1, and NEF2L2 mutations. A BRCA2 mutation with a high probability of germline mutation was detected in one patient. A thymic carcinoma with no detectable oncogenic mutation responded to second-line treatment with Tegafur-Gimeracil-Oteracil Potassium (TS-1) for ≥9 years. CONCLUSIONS: CGP panels are useful in thoracic malignancies, especially lung cancer, because they can detect overlooked driver mutations and genetic alterations. We believe that the significance of conducting a CGP panel prior to treatment may also exist, as it may lead to the prediction of ICI treatment efficacy.

BRAF Immunoexpression Can Be Intralesionally Heterogeneous but BRAF V600E Mutation Status Is Intralesionally Homogeneous and Interlesionally Concordant in Melanoma: A Study of 140 Lesions From 98 Patients.

ABSTRACT: This study sought to confirm the homogeneity of BRAF V600E mutation status in melanoma. BRAF immunohistochemistry was performed on 102 lesions from 60 patients of melanoma with BRAF V600E mutation and 38 negative-control melanoma lesions from 38 patients, both of which were confirmed by real-time PCR or the MassARRAY System. In the positive-control lesions, 9 lesions from 7 patients with preceding BRAF-inhibitor therapy were included. Of the 102 BRAF-mutant lesions, 101 (99.0%) showed diffuse BRAF immunoexpression, but 39 (38.2%) of them showed various heterogeneous intensities. The heterogeneous intensity of immunostaining was due to necrosis (n = 10), minimal or clear cytoplasm (n = 5), tissue crush (n = 8), insufficient fixation (n = 24), or technical error (n = 4). Only 1 lesion (1.0%) with nondiffuse immunoexpression harbored 80% weakly BRAF-positive tumor area and 20% BRAF-negative area with tissue damage. Sanger sequencing performed on the weak or negative regions in 7 lesions revealed BRAF V600E mutation in all the tested lesions. By contrast, all 38 negative-control lesions demonstrated no BRAF immunoexpression. This study demonstrated intralesional homogeneity and interlesional concordance for BRAF V600E mutation status and intralesional frequent heterogeneity for BRAF immunoexpression. The abovementioned 5 phenomena caused substantial reduction in BRAF immunostaining intensity. In 9 lesions within this study, BRAF immunoexpression and BRAF V600E point mutation status were not affected by preceding BRAF inhibitor therapy. Our data would also support the position that it does not matter whether we select primary or metastatic samples for BRAF mutation analysis.