Comprehensive clinicopathological, molecular, and methylation analysis of mesenchymal tumors with NTRK and other kinase gene aberrations.

Alterations in kinase genes such as NTRK1/2/3, RET, and BRAF underlie infantile fibrosarcoma (IFS), the emerging entity 'NTRK-rearranged spindle cell neoplasms' included in the latest WHO classification, and a growing set of tumors with overlapping clinical and pathological features. In this study, we conducted a comprehensive clinicopathological and molecular analysis of 22 cases of IFS and other kinase gene-altered spindle cell neoplasms affecting both pediatric and adult patients. Follow-up periods for 16 patients ranged in length from 10 to 130 months (mean 38 months). Six patients were treated with targeted therapy, achieving a partial or complete response in five cases. Overall, three cases recurred and one metastasized. Eight patients were free of disease, five were alive with disease, and two patients died. All cases showed previously reported morphological patterns. Based on the cellularity and level of atypia, cases were divided into three morphological grade groups. S100 protein and CD34 were at least focally positive in 12/22 and 14/22 cases, respectively. Novel PWWP2A::RET, NUMA1::RET, ITSN1::RAF1, and CAPZA2::MET fusions, which we report herein in mesenchymal tumors for the first time, were detected by RNA sequencing. Additionally, the first uterine case with BRAF and EGFR mutations and CD34 and S100 co-expression is described. DNA sequencing performed in 13 cases uncovered very rare additional genetic aberrations. The CNV profiles showed that high-grade tumors demonstrate a significantly higher percentage of copy number gains and losses across the genome compared with low- and intermediate-grade tumors. Unsupervised clustering of the tumors' methylation profiles revealed that in 8/9 cases, the methylation profiles clustered with the IFS methylation class, irrespective of their clinicopathological or molecular features. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Molecular pathology in diagnosis and prognostication of head and neck tumors.

Classification of head and neck tumors has evolved in recent decades including a widespread application of molecular testing in tumors of the salivary glands, sinonasal tract, oropharynx, nasopharynx, and soft tissue. Availability of new molecular techniques allowed for the definition of multiple novel tumor types unique to head and neck sites. Moreover, the expanding spectrum of immunohistochemical markers facilitates a rapid identification of diagnostic molecular abnormalities. As such, it is currently possible for head and neck pathologists to benefit from a molecularly defined classifications, while making diagnoses that are still based largely on histopathology and immunohistochemistry. This review highlights some principal molecular alterations in head and neck neoplasms presently available to assist pathologists in the practice of diagnosis, prognostication and prediction of response to treatment.

Spindle cell rhabdomyosarcomas: With TFCP2 rearrangements, and novel EWSR1::ZBTB41 and PLOD2::RBM6 gene fusions. A study of five cases and review of the literature.

AIMS: Spindle-cell/sclerosing rhabdomyosarcomas (SS-RMS) are clinically and genetically heterogeneous. They include three well-defined molecular subtypes, of which those with EWSR1/FUS::TFCP2 rearrangements were described only recently. This study aimed to evaluate five new cases of SS-RMS and to perform a clinicopathological and statistical analysis of all TFCP2-rearranged SS-RMS described in the English literature to more comprehensively characterize this rare tumour type. METHODS AND RESULTS: Cases were retrospectively selected and studied by immunohistochemistry, fluorescence in situ hybridization with EWSR1/FUS and TFCP2 break-apart probes, next-generation sequencing (Archer FusionPlex Sarcoma kit and TruSight RNA Pan-Cancer Panel). The PubMed database was searched for relevant peer-reviewed English reports. Five cases of SS-RMS were found. Three cases were TFCP2 rearranged SS-RMS, having FUSex6::TFCP2ex2 gene fusion in two cases and triple gene fusion EWSR1ex5::TFCP2ex2, VAX2ex2::ALKex2 and VAX2intron2::ALKex2 in one case. Two cases showed rhabdomyoblastic differentiation and spindle-round cell/sclerosing morphology, but were characterized by novel genetic fusions including EWSR1ex8::ZBTB41ex7 and PLOD2ex8::RBM6ex7, respectively. In the statistical analysis of all published cases, CDKN2A or ALK alterations, the use of standard chemotherapy and age at presentation in the range of 18-24 years were negatively correlated to overall survival. CONCLUSION: EWSR1/FUS::TFCP2-rearranged SS-RMS is a rare rhabdomyosarcoma subtype, affecting predominantly young adults with average age at presentation 34 years (median 29.5 years; age range 7-86 years), with a predilection for craniofacial bones, rapid clinical course with frequent bone and lung metastases, and poor prognosis (3-year overall survival rate 28%).

EWSR1::POU2AF3(COLCA2) Sarcoma: An Aggressive, Polyphenotypic Sarcoma With a Head and Neck Predilection.

EWSR1::POU2AF3 (COLCA2) sarcomas are a recently identified group of undifferentiated round/spindle cell neoplasms with a predilection for the head and neck region. Herein, we report our experience with 8 cases, occurring in 5 men and 3 women (age range, 37-74 years; median, 60 years). Tumors involved the head/neck (4 cases), and one each the thigh, thoracic wall, fibula, and lung. Seven patients received multimodal therapy; 1 patient was treated only with surgery. Clinical follow-up (8 patients; range, 4-122 months; median, 32 months) showed 5 patients with metastases (often multifocal, with a latency ranging from 7 to 119 months), and 3 of them also with local recurrence. The median local recurrence-free and metastasis-free survival rates were 24 months and 29 months, respectively. Of the 8 patients, 1 died of an unknown cause, 4 were alive with metastatic disease, 1 was alive with unresectable local disease, and 2 were without disease. The tumors were composed of 2 morphologic subgroups: (1) relatively bland tumors consisting of spindled to stellate cells with varying cellularity and fibromyxoid stroma (2 cases) and (2) overtly malignant tumors composed of nests of "neuroendocrine-appearing" round cells surrounded by spindled cells (6 cases). Individual cases in the second group showed glandular, osteogenic, or rhabdomyoblastic differentiation. Immunohistochemical results included CD56 (4/4 cases), GFAP (5/8), SATB2 (4/6), keratin (AE1/AE3) (5/8), and S100 protein (4/7). RNA sequencing identified EWSR1::POU2AF3 gene fusion in all cases. EWSR1 gene rearrangement was confirmed by fluorescence in situ hybridization in 5 cases. Our findings confirm the head/neck predilection and aggressive clinical behavior of EWSR1::POU2AF3 sarcomas and widen the morphologic spectrum of these rare lesions to include relatively bland spindle cell tumors and tumors with divergent differentiation.

MITF::CREM-rearranged tumor: a novel group of cutaneous tumors with melanocytic differentiation.

Cutaneous tumors with melanocytic differentiation represent a broad group of neoplasms of both melanocytic and non-melanocytic origin. Besides traditional members such as clear-cell sarcoma (CCS) and PEComa, the latter group has recently expanded to also include MITF::CREM fusion-associated tumors, but the available data are limited. Herein, we present a third case of this rare neoplasm which occurred in the temporal region in a 1-year-old girl. It was an infiltratively growing polypoid dermal-based lesion lacking an intraepidermal component. It consisted of cellular solid sheets or small nests of epithelioid to spindled cells with a predominantly eosinophilic and much less commonly clear cytoplasm. The nuclei had round to ovoid shape and exhibited moderate to high-grade atypia and prominent nucleoli. The mitotic activity was 11 mitoses per 10 high-power fields, and atypical mitotic figures were present. Immunohistochemically, the tumor was strongly positive with S100 protein, SOX10, and MITF, while HMB45, tyrosinase, and Melan A were negative. Extensive molecular analysis revealed only MITF::CREM gene fusion. There had no evidence of disease 9 months after the diagnosis. These tumors need to be distinguished from malignant tumors with melanocytic differentiation, primarily from melanoma. However, additional cases still need to be studied to precisely define their biological potential and establish their nosologic status.

High-grade non-intestinal type sinonasal adenocarcinoma with ETV6::NTRK3 fusion, distinct from secretory carcinoma by immunoprofile and morphology.

We report 2 cases of high-grade sinonasal adenocarcinoma with a distinct morphological and immunohistochemical phenotype. Albeit histologically different from secretory carcinoma of the salivary glands, both tumors presented here share an ETV6::NTRK3 fusion. The highly cellular tumors were composed of solid and dense cribriform nests, often with comedo-like necroses in the center, and minor areas with papillary, microcystic, and trabecular formations without secretions, mostly located at the periphery of the lesion. The cells displayed high-grade features, with enlarged, crowded, and often vesicular nuclei with conspicuous nucleoli and brisk mitotic activity. The tumor cells were immunonegative for mammaglobin while showing immunopositivity for p40/p63, S100, SOX10, and GATA3, as well as for cytokeratins 7, 18, and 19. For the first time, we describe 2 cases of primary high-grade non-intestinal type adenocarcinomas of the nasal cavity, distinct from secretory carcinoma by morphology and immunoprofile, harboring the ETV6::NTRK3 fusion.

Biphenotypic sinonasal sarcoma with PAX3::MAML3 fusion transforming into high-grade rhabdomyosarcoma: report of an emerging rare phenomenon.

We report a case of a 67-year-old male patient with a sinonasal tumor that showed areas of classic biphenotypic sinonasal sarcoma (BSNS) which in some sections sharply transitioned into high-grade rhabdomyosarcoma. Immunohistochemically, the conventional BSNS parts showed S100 protein, SMA, PAX7, and focal MyoD1 expression, whereas desmin and myogenin were negative. In contrast, the cells in high-grade areas expressed desmin, MyoD1, myogenin, and PAX7, while being negative for S100 protein and SMA. Using the Archer FusionPlex assay, the classical PAX3::MAML3 gene fusion was detected. FISH for PAX3 and MAML3 confirmed a break of these genes in both components. Despite aggressive therapy, the tumor progression resulted in the patient's death. The herein presented case, together with 2 previously published cases of BSNS with high-grade transformation, helps to better understand this novel phenomenon. Although the risk for such transformation appears low, it has important clinical and diagnostic implications which are discussed.

Clear cell mesotheliomas with inactivating VHL mutations and near-haploid genomic features.

Clear cell mesothelioma is uncommon and shows predominance of clear cells with resemblance to clear cell carcinomas. Clinicopathologic and molecular descriptions of clear cell mesothelioma remained limited. In this study, we identified an index patient with clear cell mesothelioma, confirmed by immunohistochemical and ultrastructural studies. Targeted next-generation sequencing revealed the presence of an inactivating VHL mutation. We then systematically searched for VHL-mutant mesotheliomas in a comprehensive genomic profiling database of 1532 mesotheliomas. Collectively, we identified a cohort of four VHL-mutant clear cell mesotheliomas, including three peritoneal and one pleural tumors from three females and one male, with age range of 47-68 (median 63) years. Histologically, each tumor showed a microcystic to tubulopapillary architecture with prominent clear cells. By next-generation DNA sequencing, each of the four clear cell mesotheliomas harbored inactivating VHL mutations, while lacking other alterations typical of mesotheliomas such as BAP1, NF2, SETD2, CDKN2A, CDKN2B, TP53, and PTEN. By using low-pass whole genome sequencing on the index case and targeted next-generation sequencing on the remaining three cases, we identified extensive loss of heterozygosity throughout the genome but consistently sparing chromosomes 5, 7, and 20, characteristic of genomic near-haploidization. In summary, clear cell mesotheliomas were characterized by inactivating VHL mutations and genomic near-haploidization and appeared to represent a distinct clinicopathologic and molecular category of mesotheliomas. Our findings implicate VHL in the pathogenesis of a subset of mesotheliomas, particularly those with clear cell morphology.

A minority of cases of acinic cell carcinoma of the salivary glands are driven by an NR4A2 rearrangement: the diagnostic utility of the assessment of NR4A2 and NR4A3 alterations in salivary gland tumors.

Acinic cell carcinoma (AciCC) is a common salivary gland malignancy, typically composed of neoplastic acinic cells with zymogen granules. The vast majority of cases are driven by a t(4;9)(q13;q31) leading to enhancer hijacking and upregulation of the NR4A3 gene. However, a minority of cases do not display NR4A3 overexpression on immunohistochemical examination and are negative for the rearrangement involving the NR4A3 gene when tested by FISH. Such cases overexpress NR4A2, and the protein product is detectable by immunohistochemistry. In this study, we aimed to assess the utility of NR4A2 and NR4A3 immunohistochemistry in the differential diagnosis of salivary gland tumors. Eighty-five cases of classic low-grade ACiCC, as well as 36 cases with high-grade transformation (HGT) and 7 high-grade AciCC cases were included in the analysis. NR4A3 was at least focally positive in 105/128 (82%) cases. Out of the 23 cases that were immunohistochemically negative for NR4A3, 6 displayed nuclear immunopositivity with the NR4A2 antibody. The NR4A3 rearrangement was confirmed by FISH in 38/52 (73%) cases. In addition, this is the first report of an NR4A2 rearrangement being detected by FISH in 2 AciCC cases that were negative for the NR4A3 rearrangement. Our analysis confirms that the majority of AciCC, including high-grade cases and cases with HGT, are immunopositive for NR4A3, and suggests that NR4A3 immunohistochemistry is a powerful tool in the differential diagnosis of salivary gland tumors. However, its utility is limited in sub-optimally fixed samples which often display weaker and focal positivity. Our study also indicates that in a minority of cases, AciCC might be negative for NR4A3 immunostaining, because the pathogenic genetic event in these cases is instead a rearrangement involving the NR4A2 gene.

Effect of green cardamom on the expression of genes implicated in obesity and diabetes among obese women with polycystic ovary syndrome: a double blind randomized controlled trial.

BACKGROUND: Polycystic ovary syndrome (PCOS) is an endocrine disease in which related to obesity, metabolic disorders and is considered as one of the main causes of infertility in women. This trial was investigated the effects of green cardamom on the expression of genes implicated in obesity and diabetes among obese women with PCOS. METHODS: One hundred ninety-four PCOS women were randomly divided two groups: intervention (n = 99; 3 g/day green cardamom) and control groups (n = 95). All of them were given low calorie diet. Anthropometric, glycemic and androgen hormones were assessed before and after 16-week intervention. The reverse transcription-polymerase chain reaction (RT-PCR) method was used to measure fat mass and obesity-associated (FTO), peroxisome proliferative activating receptor- (PPAR-), carnitine palmitoyltransferase 1A (CPT1A), acetyl-CoA carboxylase beta (ACAB), leptin receptor (LEPR), ghrelin, and lamin A/C (LAMIN) genes expression in each group. RESULTS: Anthropometric indices were significantly decreased after intervention in both two studied groups. Glycemic indices and androgen hormones were significantly improved in the intervention group compared to the control group. The expression levels of FTO, CPT1A, LEPR, and LAMIN were significantly downregulated compared to control group (P < 0.001), as well as, PPAR-y was significantly upregulated in the intervention group after intervention with green cardamom compared to control group (P < 0.001). CONCLUSION: This current study showed that the administration of green cardamom is a beneficial approach for improving anthropometric, glycemic, and androgen hormones, as well as obesity and diabetes genes expression in PCOS women under the low-calorie diet. TRIAL REGISTRATION: This trial was registered with the Iranian Clinical Trials Registry (registration number: IRCT20200608047697N1). 1 August, 2020; https://www.irct.ir/trial/48748.

The significance of the fusion partner gene genomic neighborhood analysis in translocation-defined tumors.

INTRODUCTION: This study presents a novel molecular parameter potentially co-defining tumor biology-the total tumor suppressor gene (TSG) count at chromosomal loci harboring genes rearranged in fusion-defined tumors. It belongs to the family of molecular parameters created using a black-box approach. METHOD: It is based on a public curated Texas TSG database. Its data are regrouped based on individual genes loci using another public database (Genecards). The total TSG count for NTRK (NTRK1; OMIM: 191315; NTRK2; OMIM: 600456; NTRK3; OMIM: 191316), NRG1 (OMIM: 142445), and RET (OMIM: 164761) rearranged tumors in patients treated with a theranostic approach is calculated using the results of recently published studies. RESULTS: Altogether 138 loci containing at least three TSGs are identified. These include 21 "extremely hot" spots, with 10 to 28 TSGs mapping to a given locus. However, the study falls short of finding a correlation between tumor regression or patient survival and the TSG count owing to a low number of cases meeting the study criteria. CONCLUSION: The total TSG count alone cannot predict the biology of translocation-defined tumors. The addition of other parameters, including microsatellite instability (MSI), tumor mutation burden (TMB), homologous recombination repair deficiency (HRD), and copy number heterogeneity (CNH), might be helpful. Thus a multi-modal data integration is advocated. We believe that large scale studies should evaluate the significance and value of the total TSG count.

Clear Cell Neoplasms of Salivary Glands: A Diagnostic Challenge.

This review focuses on the heterogenous group of clear cell neoplasms of salivary glands and attempts to identify major differential diagnostic features. Within the head and neck region, clear cells are found most commonly in salivary gland tumors, but may also be seen in tumors of squamous or odontogenic epithelial origin, primary or metastatic carcinomas, benign or malignant melanocytic lesions, or benign or malignant mesenchymal tumors. Clear cells occur fairly commonly among a wide variety of salivary gland neoplasms, but mostly they constitute only a minor component of the tumor cell population. Clear cells represent a major diagnostic feature in two salivary gland neoplasms, epithelial-myoepithelial carcinoma and hyalinizing clear cell carcinoma. In addition, salivary gland neoplasms composed predominantly of clear cells could also include clear cell variants of other salivary neoplasms, such as mucoepidermoid carcinoma and myoepithelial carcinoma, but their tumor type-specific histologic features may only be available in limited nonclear cell areas of the tumor. Diagnosing predominantly clear cell salivary gland tumors is difficult because the immunoprofiles and morphologic features may overlap and the same tumor entity may also have a wide range of other histologic presentations. Many salivary gland tumors are characterized by tumor type-specific genomic alterations, particularly gene fusions of the ETV6 gene in secretory carcinoma, the MYB and MYBL1 genes in adenoid cystic carcinoma, the MAML2 gene in mucoepidermoid carcinoma, the EWSR1 gene in hyalinizing clear cell carcinoma, and others. Thus, along with conventional histopathologic examination and immunoprofiling, molecular and genetic tests may be important in the diagnosis of salivary gland clear cell tumors by demonstrating genetic alterations specific to them.

Misleading Morphologic and Phenotypic Features (Transdifferentiation) in Solitary Fibrous Tumor of the Head and Neck: Report of 3 Cases and Review of the Literature.

Solitary fibrous tumor (SFT) is a rare fibroblastic neoplasm with potentially malignant behavior that may develop in any anatomic site and may involve the head and neck (H&N) region as well. Although typical SFT has a relatively characteristic morphology, its morphologic spectrum is extraordinarily broad and also includes rare cases with dedifferentiation or transdifferentiation which result in aberrant morphologic and/or immunohistochemical features. However, since virtually all cases are molecularly characterized by NAB2::STAT6 gene fusions, molecular genetic methods or STAT6 immunohistochemistry can be effectively used in confirming the diagnosis. Herein, we report 3 diagnostically challenging H&N SFT cases with an unusual morphology and/or phenotypes closely mimicking other well-known H&N entities. The tumors originated in the oral minor salivary glands, the base of the tongue, and sinonasal tract and closely resembled hyalinizing clear cell carcinoma of the salivary gland, adenocarcinoma not otherwise specified and biphenotypic sinonasal sarcoma, respectively. All cases were positive for cytokeratins, variably expressed S100 protein, showed diffuse nuclear STAT6 positivity, and harbored NAB2::STAT6 gene fusions.

Green cardamom plus low-calorie diet can decrease the expression of inflammatory genes among obese women with polycystic ovary syndrome: a double-blind randomized clinical trial.

PURPOSE: Polycystic ovary syndrome (PCOS) is an endocrine disorder and a common cause of infertility among women that is associated with low-grade inflammation. Therefore, the current randomized, double-blind, placebo-controlled clinical trial was conducted to assess the effects of green cardamom supplementation on the serum level of inflammatory markers and their gene expression among obese women with PCOS. METHODS: We included 194 obese women with PCOS and administered low-calorie diet to all of them. These subjects were randomly divided into two groups including the intervention group with 3 g/day green cardamom (n = 99) and the placebo group (n = 95). Anthropometric indices, androgen hormones, and inflammatory factors [tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), and C-reactive protein (CRP)] were assessed before and after the 4-month intervention. TNF-α, IL-6, and CRP gene expression levels were measured using reverse transcription-polymerase chain reaction (RT-PCR) method. RESULTS: Anthropometric indices were improved in both studied groups (P < 0.001). Among androgen hormones, luteinizing hormone, androstenedione, and dehydroepiandrosterone were significantly decreased (P < 0.001), and follicle-stimulating hormone was significantly increased (P < 0.001) in the green cardamom group. Our findings showed that TNF-α, IL-6, and CRP serum levels were significantly decreased after the intervention with green cardamom plus low-calorie diet (P < 0.001). In addition, the expression levels of TNF-α and CRP genes were significantly decreased in the intervention group (P < 0.001). CONCLUSIONS: The present study supports the beneficial anti-inflammatory effect of green cardamom on the inflammatory state in PCOS women. LEVEL OF EVIDENCE: Level I: randomized clinical trial. Trial registration This trial was registered with the Iranian Clinical Trials Registry (registration number: IRCT20200608047697N1). 1 August, 2020; https://www.irct.ir/trial/48748 .

Mitochondrial A12308G alteration in tRNA(Leu(CUN)) in colorectal cancer samples.

BACKGROUND: Colorectal cancer is the third most common type of cancer in men and women and the second leading cause of cancer-related deaths in the United States and UK. Colorectal cancer is strongly related to age, with almost three-quarters of cases occurring in people aged 65 or over. Pre-symptomatic screening is one of the most powerful tools for preventing colorectal cancer. Recently, the use of mitochondrial tRNA genes mutation or polymorphism patterns as a biomarker is rapidly expanding in different cancers because tRNA genes perform several functions including processing and translation which are essential components of mitochondrial protein synthesis. The aim of the present study was to find out the association of mitochondrial A12308G alteration in tRNA(Leu(CUN)) in colorectal cancer and its usage as a new biomarker screening test. METHODS: A tumor tissues from 30 patients who had colorectal cancer were selected randomly. The A12308G alteration in tRNALeu (CUN) was screened in the 30 colorectal tumor tissues. For comparison, 100 blood samples of healthy controls using PCR-sequencing methods were selected and the following results were found. RESULT: The A12308G, a polymorphic mutation in V-loop tRNA(Leu(CUN)), was found in 6 Colorectal tumor tissues and 3 healthy controls. A statistical significant difference was found between cases and control regarding the association of the A12308G mutation with the colorectal tumor (P < 0.05). CONCLUSIONS: The A12308G, a polymorphic mutation in V-loop tRNA(Leu(CUN)), could be considered as pathogenic mutation in combination with mitochondrial external conditions and other mitochondrial genes in developing different diseases especially cancers and could be used as one of the diagnostic tool. Also it seems that maybe there is relevance between A12308G mutation and other mutations that it can cause various phenotypes.

T4216C mutation in NADH dehydrogenase I gene is associated with recurrent pregnancy loss.

Several genetic factors are involved with recurrent pregnancy loss (RPL). However, few attempts have been made to associate mitochondrial DNA (mtDNA) variations with RPL. Therefore, we investigated the possible effect of the T4216C mutation in the mitochondrial NADH dehydrogenase I (ND1) gene of 33 women with RPL and 100 controls, using polymerase chain reaction amplification and DNA sequence analysis. Our results showed a statistically significant association of the T4216C mutation (p <  0.05) between patients and controls, which are 30% and 11%, respectively. In conclusion, more research is essentially needed to understand the effect and role of the T4216C mutation in the progress of RPL, which may vary among individuals and different ethnic groups.