Targeted RNA Sequencing Highlights a Diverse Genomic and Morphologic Landscape in Low-grade Endometrial Stromal Sarcoma, Including Novel Fusion Genes.

Low-grade endometrial stromal sarcoma (LGESS) represents a morphologically and genetically heterogenous mesenchymal neoplasm. Previous work has shown that approximately half of LGESS are characterized by JAZF1::SUZ12 gene fusions, while a smaller proportion involves rearrangement of other genes. However, a subset of cases has no known genetic abnormalities. To better characterize the genomic landscape of LGESS, we interrogated a cohort with targeted RNA sequencing (RNA-Seq). Cases previously diagnosed as low-grade endometrial stromal neoplasia (n=51) were identified and re-reviewed for morphology and subjected to RNA-Seq, of which 47 were successfully sequenced. The median patient age was 49 years (range: 19 to 85). The most commonly detected fusions were JAZF1::SUZ12 (n=26, 55%) and BRD8::PHF1 (n=3, 6%). In addition to the usual/typical LGESS morphology, some JAZF1::SUZ12 fusion tumors showed other morphologies, including fibrous, smooth muscle, sex-cord differentiation, and myxoid change. Novel translocations were identified in 2 cases: MEAF6::PTGR2 and HCFC1::PHF1 . Ten tumors (21%) had no identifiable fusion, despite a similar morphology and immunophenotype to fusion-positive cases. This suggests that a subset of cases may be attributable to fusion products among genes that are not covered by the assay, or perhaps altogether different molecular mechanisms. In all, these findings confirm that RNA-Seq is a potentially useful ancillary test in the diagnosis of endometrial stromal neoplasms and highlight their diverse morphology.

NR4A3 Expression Is Consistently Absent in Acinic Cell Carcinomas of the Breast: A Potential Nosologic Shift.

Acinic cell carcinoma (AciCC) is a tumor that is recognized in both the breast and salivary glands. Recently, the recurrent genomic rearrangement, t(4;9)(q13;q31) was identified in salivary AciCC that results in constitutive upregulation of the nuclear transcription factor NR4A3, which can be detected by immunohistochemistry. In this study, we sought to evaluate NR4A3 expression in breast AciCC using immunohistochemistry. Strong and diffuse nuclear staining was considered a positive result. Sixteen AciCCs were studied, including 8 pure AciCCs and 8 AciCCs admixed with other types (invasive carcinoma of no special type in 5 cases and metaplastic carcinoma in 3 cases). All 16 AciCCs showed negative results for NR4A3 expression. Four cases with available material were evaluated for rearrangements of the NR4A3 gene by fluorescence in situ hybridization and no rearrangements were observed. Whole-genome sequencing of 1 AciCC revealed a TP53 splice-site mutation, high levels of genomic instability, and genomic features of homologous recombination DNA repair defects; a structural variant analysis of this case did not reveal the presence of a t(4;9) rearrangement. We conclude that breast AciCCs consistently lack NR4A3 rearrangement or overexpression, unlike most of the salivary AciCCs, and that consistent with previous results, breast AciCCs are associated with genomic alterations more similar to those seen in triple-negative breast carcinomas than salivary gland AciCCs. These results suggest that unlike other salivary gland-like tumors that occur in the breast, the molecular underpinnings of the salivary gland and breast AciCCs are different and that the salivary gland and breast AciCCs likely represent distinct entities.

Myelodysplastic/myeloproliferative neoplasms-unclassifiable with isolated isochromosome 17q represents a distinct clinico-biologic subset: a multi-institutional collaborative study from the Bone Marrow Pathology Group.

Classification of myeloid neoplasms with isolated isochromosome i(17q) [17p deletion with inherent monoallelic TP53 loss plus 17q duplication] is controversial. Most cases fall within the WHO unclassifiable myelodysplastic/myeloproliferative neoplasms (MDS/MPN-U) category. The uniformly dismal outcomes warrant better understanding of this entity. We undertook a multi-institutional retrospective study of 92 adult MDS/MPN-U cases from eight institutions. Twenty-nine (32%) patients had isolated i(17q) [MDS/MPN-i(17q)]. Compared to MDS/MPN without i(17q), MDS/MPN-i(17q) patients were significantly younger, had lower platelet and absolute neutrophil counts, and higher frequency of splenomegaly and circulating blasts. MDS/MPN-i(17q) cases showed frequent bilobed neutrophils (75% vs. 23%; P = 0.03), hypolobated megakaryocytes (62% vs. 20%; P = 0.06), and a higher frequency of SETBP1 (69% vs. 5%; P = 0.002) and SRSF2 (63% vs. 5%; P = 0.006) mutations that were frequently co-existent (44% vs. 0%; P = 0.01). TP53 mutations were rare. The mutation profile of MDS/MPN-U-i(17q) was similar to other myeloid neoplasms with i(17q) including atypical chronic myeloid leukemia, chronic myelomonocytic leukemia, myelodysplastic/myeloproliferative neoplasm with ring sideroblasts and thrombocytosis, myelodysplastic syndrome and acute myeloid leukemia, with frequent concomitant SETBP1/SRSF2 mutations observed across all the diagnostic entities. Over a median follow-up of 52 months, patients with MDS/MPN-i(17q) showed a shorter median overall survival (11 vs. 28 months; P < 0.001). The presence of i(17q) retained independent poor prognostic value in multivariable Cox-regression analysis [HR 3.686 (1.17-11.6); P = 0.026] along with splenomegaly. We suggest that MDS/MPN-i(17q) warrants recognition as a distinct subtype within the MDS/MPN-U category based on its unique clinico-biologic features and uniformly poor prognosis.

Re-evaluating tumors of purported specialized prostatic stromal origin reveals molecular heterogeneity, including non-recurring gene fusions characteristic of uterine and soft tissue sarcoma subtypes.

Tumors of purported specialized prostatic stromal origin comprise prostatic stromal sarcomas (PSS) and stromal tumors of uncertain malignant potential (STUMP). Prior studies have described their clinicopathologic characteristics, but the molecular features remain incompletely understood. Moreover, these neoplasms are morphologically heterogeneous and the lack of specific adjunctive markers of prostatic stromal lineage make precise definition more difficult, leading some to question whether they represent a specific tumor type. In this study, we used next-generation DNA and RNA sequencing to profile 25 primary prostatic mesenchymal neoplasms of possible specialized prostatic stromal origin, including cases originally diagnosed as PSS (11) and STUMP (14). Morphologically, the series comprised 20 cases with solid architecture (11 PSS and 9 STUMP) and 5 cases with phyllodes-like growth pattern (all STUMP). Combined DNA and RNA sequencing results demonstrated that 19/22 (86%) cases that underwent successful sequencing (either DNA or RNA) harbored pathogenic somatic variants. Except for TP53 alterations (6 cases), ATRX mutations (2 cases), and a few copy number variants (-13q, -14q, -16q and +8/8p), the findings were largely nonrecurrent. Eight gene rearrangements were found, and 4 (NAB2-STAT6, JAZF1-SUZ12, TPM3-NTRK1 and BCOR-MAML3) were useful for reclassification of the cases as specific entities. The present study shows that mesenchymal neoplasms of the prostate are morphologically and molecularly heterogeneous and include neoplasms that harbor genetic aberrations seen in specific mesenchymal tumors arising in other anatomic sites, including soft tissue and the uterus. These data suggest that tumors of purported specialized prostatic stromal origin may perhaps not represent a single diagnostic entity or specific disease group and that alternative diagnoses should be carefully considered.

Synovial Sarcoma of the Female Genital Tract: A Protean Mimic of Müllerian Neoplasia.

Synovial sarcoma most commonly occurs in the extremities but has rarely been described in the female genital tract. In this series, we describe the clinical, morphologic, immunohistochemical, and molecular features of 7 cases of vulvovaginal synovial sarcoma (vulva, n=6; vagina, n=1). We emphasize their wide morphologic spectrum, which can overlap significantly with other more common tumors at these sites, as highlighted by 2 cases initially diagnosed as other entities (endometrioid carcinoma and malignant peripheral nerve sheath tumor). The average patient age was 41 (range: 23 to 62) years and tumor size ranged from 0.8 to 7 cm. Histologically, the tumors were biphasic (n=6) and monophasic (n=1). All cases were confirmed with fluorescence in situ hybridization or sequencing, and 5/5 cases were positive for the novel immunohistochemical markers SSX and SS18-SSX. In 3 cases with follow-up, 2 patients died of disease and 1 was alive with no evidence of disease. Previously described cases arising in the female genital tract are also reviewed. Vulvovaginal monophasic synovial sarcoma raises a broad differential diagnosis, including smooth muscle tumors, spindled carcinomas, and melanoma. Biphasic synovial sarcoma may mimic Müllerian carcinosarcoma, endometrioid carcinoma with spindled, corded, and hyalinized elements, and mesonephric-like adenocarcinoma. Awareness that synovial sarcoma can occur in the female genital tract with a wide variety of histologic appearances is critical for correctly diagnosing this rare entity. In particular, synovial sarcoma should be considered for any deeply situated "adenocarcinoma" in the vulva, with attention to subtle spindle cell differentiation.

Integrated single-cell genetic and transcriptional analysis suggests novel drivers of chronic lymphocytic leukemia.

Intra-tumoral genetic heterogeneity has been characterized across cancers by genome sequencing of bulk tumors, including chronic lymphocytic leukemia (CLL). In order to more accurately identify subclones, define phylogenetic relationships, and probe genotype-phenotype relationships, we developed methods for targeted mutation detection in DNA and RNA isolated from thousands of single cells from five CLL samples. By clearly resolving phylogenic relationships, we uncovered mutated LCP1 and WNK1 as novel CLL drivers, supported by functional evidence demonstrating their impact on CLL pathways. Integrative analysis of somatic mutations with transcriptional states prompts the idea that convergent evolution generates phenotypically similar cells in distinct genetic branches, thus creating a cohesive expression profile in each CLL sample despite the presence of genetic heterogeneity. Our study highlights the potential for single-cell RNA-based targeted analysis to sensitively determine transcriptional and mutational profiles of individual cancer cells, leading to increased understanding of driving events in malignancy.

An Unusual Case of YWHAE-NUTM2A/B Endometrial Stromal Sarcoma With Confinement to the Endometrium and Lack of High-Grade Morphology.

Endometrial stromal sarcoma (ESS) characterized by YWHAE-NUTM2A/B genetic fusion is a recently recognized entity that is classified as a high-grade (HG) ESS in the 2014 World Health Organization Classification. These are myoinvasive neoplasms and typically contain a monomorphous HG round-cell cyclinD1-positive component with or without an accompanying low-grade (LG) component that is only focally positive/negative for cyclinD1. We report a case of YWHAE-NUTM2A/B ESS in a 46-yr-old woman that showed a number of unusual histologic features, including being entirely confined to the endometrium with no myoinvasion or lymphovascular space invasion. The initial hysteroscopic biopsy showed a cyclinD1-positive classic LG ESS-like component which merged with a smaller cyclinD1 negative/focally positive fibroblastic component with no HG areas. YWHAE-NUTM2A/B genetic fusion was shown by real-time quantitative polymerase chain reaction and Sanger sequencing. In the subsequent hysterectomy specimen, the tumor was entirely confined to the endometrium and was largely composed of cellular and classic LG ESS-like areas (80%) which were strongly and diffusely positive for cyclinD1 and a focal fibroblastic component (20%) which was largely cyclinD1 negative. Despite the cellular areas showing mild nuclear enlargement, the entire tumor had a very low mitotic and proliferation index and showed strong and diffuse positivity for estrogen and progesterone receptors. The patient remains alive and well with no evidence of disease 14 mo following diagnosis. To our knowledge, this is the first reported case of YWHAE-NUTM2A/B ESS that is confined to the endometrium and which exhibits entirely LG morphology.

Reporting of Diagnostic Cytogenetic Results.

This appendix, developed by the staff at the Center for Advanced Molecular Diagnostics in the Department of Pathology at the Brigham and Women's Hospital, includes a comprehensive list of current "macros" or standardized statements used to facilitate reporting of cytogenetic results. These are provided as a useful reference for other laboratories. The statements are organized under the general categories of constitutional or acquired abnormalities and subdivided into analysis type (GTG-banding, FISH, or chromosomal microarray). Multi-specimen usage macros are included that can be applied to two or more specimen types.