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PDF file - 171KB, Supplementary Fig. S2. NUT is translocated to the NSD3 locus in 1221 cell line as demonstrated by FISH on metaphase spread.

PDF file - 304KB, Supplementary Fig. S4. Among several ET-interacting proteins, only NSD3 is required for the blockade of differentiation in NMC. (A) Quantitative RT-PCR of NSD3, JMJD6, GLTSCR1, and ATAD5 levels 24 h following transfection of siRNAs. (B) Immunoblots of TC-797 lysates 120h following transfection with siRNAs.

PDF file - 213KB, Supplementary Fig. S3. Wild type NSD3 is required for the blockade of differentiation in BRD4-NUT-expressing NMC cells, 8645 and PER-403.

PDF file - 140KB, Supplementary Fig. S1. Karyotype of the 1221 NSD3-NUT-positive cell line.

Purpose: Clear cell sarcoma (CCS) usually arises in the lower extremities of young adults and is typically associated with a t(12;22) translocation resulting in the fusion of EWS (EWSR1) with ATF1, a gene encoding a member of the cyclic AMP–responsive element binding protein (CREB) family of transcription factors. CCS arising in the gastrointestinal tract is rare and its pathologic and molecular features are not well defined.Experimental Design: We report a novel variant fusion of EWS to CREB1, a gene at 2q32 encoding another CREB family member highly related to ATF1, detected in three women with gastrointestinal CCS. All three cases contained an identical EWS-CREB1 fusion transcript that was shown by reverse transcription-PCR. In two of the cases tested, EWS gene rearrangement was also confirmed by fluorescence in situ hybridization and the EWS-CREB1 genomic junction fragments were isolated by long-range DNA PCR.Results: Morphologically, all three tumors lacked melanin pigmentation. By immunohistochemistry, there was a strong and diffuse S100 protein reactivity, whereas all melanocytic markers were negative. Ultrastructurally, two of the cases lacked melanosomes. The melanocyte-specific transcript of MITF was absent in two cases, and only weakly expressed in the third case. The Affymetrix gene expression data available in one case showed lower expression of the melanocytic genes MITF, TYR, and TYRP1, compared with four EWS-ATF1-positive CCSs of non-gastrointestinal origin.Conclusions:EWS-CREB1 may define a novel subset of CCS that occurs preferentially in the gastrointestinal tract and shows little or no melanocytic differentiation. Thus, evidence of melanocytic lineage or differentiation is not a necessary feature of sarcomas with gene fusions involving CREB family members.

Purpose: The disease course of chronic lymphocytic leukemia (CLL) varies significantly within cytogenetic groups. We hypothesized that high-resolution genomic analysis of CLL would identify additional recurrent abnormalities associated with short time-to-first therapy (TTFT).Experimental Design: We undertook high-resolution genomic analysis of 161 prospectively enrolled CLLs using Affymetrix 6.0 SNP arrays, and integrated analysis of this data set with gene expression profiles.Results: Copy number analysis (CNA) of nonprogressive CLL reveals a stable genotype, with a median of only 1 somatic CNA per sample. Progressive CLL with 13q deletion was associated with additional somatic CNAs, and a greater number of CNAs was predictive of TTFT. We identified other recurrent CNAs associated with short TTFT: 8q24 amplification focused on the cancer susceptibility locus near MYC in 3.7%; 3q26 amplifications focused on PIK3CA in 5.6%; and 8p deletions in 5% of patients. Sequencing of MYC further identified somatic mutations in two CLLs. We determined which catalytic subunits of phosphoinositide 3-kinase (PI3K) were in active complex with the p85 regulatory subunit and showed enrichment for the α subunit in three CLLs carrying PIK3CA amplification.Conclusions: Our findings implicate amplifications of 3q26 focused on PIK3CA and 8q24 focused on MYC in CLL. Clin Cancer Res; 18(14); 3791–802. ©2012 AACR.

Supplementary Figure 1 from EWS-CREB1: A Recurrent Variant Fusion in Clear Cell Sarcoma—Association with Gastrointestinal Location and Absence of Melanocytic Differentiation

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Low-grade fibromyxoid sarcoma (LGFMS) is a malignancy with propensity for late relapse that principally affects deep soft tissues of the extremities and trunk. Its occurrence in the lower female genital tract is rare, and thus it may not be always considered in the differential diagnosis. We describe the salient features of 7 vulvovaginal LGFMS identified in the authors' consultation files. Clinical information was obtained from referring pathologists. Archival slides were reviewed, and immunohistochemistry and fluorescence in situ hybridization were performed in cases with available material. Median age was 40 years (range, 34 to 58 y). Primary sites included vulva (n=6) and vagina (n=1). Tumors were 1.2 to 8.7 cm (median, 5.0 cm) in size and grossly circumscribed with firm to focally gelatinous cut surfaces. Microscopically, 5/7 had infiltrative edges. All tumors showed fibrous and myxoid areas, with lobulated myxoid foci in 5/7, comprising storiform, patternless, or (less often) fascicular arrangement of spindled to stellate cells with bland, slender to ovoid nuclei. In all cases, mitoses were1/2.4 mm 2 , and necrosis was absent. Capillary "arcades" were seen in 3/7. Margins were positive in 3/6. Immunohistochemistry showed positive epithelial membrane antigen in 4/6 and MUC4 in 5/6. Fluorescence in situ hybridization detected FUS rearrangement in 5/7. Both tumors without FUS rearrangement were also negative for EWSR1 rearrangement. All 5 patients with available follow-up were alive and disease-free 10 to 150 months (median, 57 mo) after diagnosis. However, a review of vulvovaginal/pelvic LGFMS previously reported shows recurrences as late as 45 years after initial diagnosis. Pathologists need to be aware that LGFMS can arise in the vulvovaginal region. Tumor lobulation, capillary arcades, and positive MUC4 are helpful features distinguishing LGFMS from other bland myxoid spindle cell neoplasms in the lower female genital tract. Molecular testing can be useful in challenging cases. Complete excision is feasible for most vulvovaginal LGFMS. Long-term surveillance is required as local and/or distant spread can occur decades after diagnosis.

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.

BCR-ABL1 kinase inhibitors have improved the prognosis of Philadelphia-chromosome-positive (Ph+)-acute lymphoblastic leukemia (ALL). Ph-like (or BCR-ABL1-like) ALL does not express BCR-ABL1 but commonly harbors other genomic alterations of signaling molecules that may be amenable to therapy. Here, we report a case with a NUP214-ABL1 fusion detected at relapse by multiplexed, targeted RNA sequencing. It had escaped conventional molecular work-up at diagnosis, including cytogenetic analysis and fluorescence in situ hybridization for ABL1 rearrangements. The patient had responded poorly to initial multi-agent chemotherapy and inotuzumab immunotherapy at relapse before the fusion was revealed. The addition of dasatinib targeting NUP214-ABL1 to inotuzumab resulted in complete molecular remission, but recurrence occurred rapidly with dasatinib alone. However, deep molecular remission was recaptured with a combination of blinatumomab and ponatinib, so he could proceed to allotransplantation. This case illustrates that next-generation sequencing approaches designed to discover cryptic gene fusions can benefit patients with Ph-like ALL.

A subset of clinically benign uterine polyps shows atypical morphologic features worrisome for, but not diagnostic of, Mullerian adenosarcoma. We report clinicopathologic data for 63 polyps from 58 women with atypical morphologic features including abnormal architecture, abnormal periglandular stroma, stromal atypia, and mitoses >2 per 10 hpf. Four (11%) of 36 women with follow-up tissue sampling had residual/recurrent atypical polyp. Twelve (27%) of 44 women underwent hysterectomy subsequent to a diagnosis of atypical polyp. No patient developed adenosarcoma over median follow-up of 150 months. Twenty-one primary atypical polyps underwent molecular profiling. Five (24%) harbored chr 12q13-15 gain or amplification, 9/20 (45%) harbored chr 6q25.1 gain, and 7/21 (33%) had no significant copy number alterations. Gains of chr 1q, chr 8p12, and chr 10q11.21-23, amplifications of chr 12q24.12-13, chr 15p24.1-26.1, and chr 18q21.33, and loss of chr 7 and chr 11q21 were each seen in a single polyp. Mean tumor mutational burden was 3.1 (range, 0.76-8.365) mutations/Mb. Pathogenic point mutations were identified in 12/20 (60%) primary atypical polyps. We propose the term "atypical uterine polyps" for these lesions, which show biologic overlap with early Mullerian adenosarcoma but lack molecular alterations characteristic of clinically aggressive adenosarcoma and appear to follow a benign clinical course. Conservative management with close clinical follow-up and repeat sampling can be considered for these lesions, when clinically appropriate.

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.

The classification of myeloid neoplasms and acute leukemias was last updated in 2016 within a collaboration between the World Health Organization (WHO), the Society for Hematopathology, and the European Association for Haematopathology. This collaboration was primarily based on input from a clinical advisory committees (CACs) composed of pathologists, hematologists, oncologists, geneticists, and bioinformaticians from around the world. The recent advances in our understanding of the biology of hematologic malignancies, the experience with the use of the 2016 WHO classification in clinical practice, and the results of clinical trials have indicated the need for further revising and updating the classification. As a continuation of this CAC-based process, the authors, a group with expertise in the clinical, pathologic, and genetic aspects of these disorders, developed the International Consensus Classification (ICC) of myeloid neoplasms and acute leukemias. Using a multiparameter approach, the main objective of the consensus process was the definition of real disease entities, including the introduction of new entities and refined criteria for existing diagnostic categories, based on accumulated data. The ICC is aimed at facilitating diagnosis and prognostication of these neoplasms, improving treatment of affected patients, and allowing the design of innovative clinical trials.

Recurrent chromosomal rearrangements are a hallmark of hematologic malignancies and play critical roles in pathogenesis. The TP53 analog TP63 is rearranged in 5-10% of diverse subtypes of both aggressive T- and B-cell lymphomas. Patients with TP63-rearranged lymphomas have dismal outcomes, with 5-year overall survival rates between 0-17%, depending on cohorts. The function and mechanisms of TP63 rearrangements and TP63 fusion proteins in tumorigenesis are poorly understood. As a result, attempts to treat these patients to date have been largely empiric. Thus, there is an urgent need to understand how TP63 fusions contribute to tumorigenesis and to translate the findings into novel therapeutic options for these patients. Here, we demonstrated that TP63 fusions are essential for the propagation of T-cell lymphomas (TCLs). Knockdown of TP63 fusions with specific shRNAs in TCL cell lines harboring TP63 fusions suppressed both cell growth in vitro and tumor growth in vivo. Retroviral expression of TBL1XR1-TP63, the most common TP63 fusion, conferred cytokine independence in Ba/F3 cells, consistent with its role as an oncogene. To investigate the role of TP63 fusions in T- and B-cell lymphomagenesis, we engineered a CAG-Loxp-Stop-Loxp-TBL1XR1-TP63 conditional knock-in mouse model and crossed with hCD2-Cre mice. This results in expression beginning during early lymphoid development. As observed in patients, transgenic mice developed multiple subtypes of both T- and B-cell lymphoma. To define the effects and mechanisms of TP63 fusions within T cells, we performed CRISPR scanning, transcriptomic, epigenomic, and proteomic analyses. Our data showed that domains within both the N-terminal TBL1XR1 and C-terminal TP63 portions contribute to the function of this fusion. We found that the N-terminal component of TP63 fusions interacts with components of the NCOR/SMRT complex. At the same time, the C-terminal portion of TP63 (which recapitulates the deltaN-p63 isoform expressed in some carcinomas) interacts with the enhancer modifier KMT2D and its complex members. TBL1XR1-TP63 binds to a novel distal enhancer to drive MYC expression, and thus upregulates the expression of the histone H3K27 methylase EZH2. Finally, we assessed whether EZH2 is a vulnerability of TP63-rearranged lymphomas. We found that knockdown of EZH2 in TP63-rearranged lines significantly impaired cell growth, as did treatment with the EZH2 and 1 dual inhibitor valemetostat. Valemetostat, which is now being tested in patients with lymphoma, counteracted the oncogenic effects of TP63 fusions in multiple preclinical models in vivo. Together, our results identify the TP63 fusion as a highly unique oncogenic driver in lymphomagenesis capable of recruiting multiple epigenetic modifier complexes and inducing a targetable dependence on EZH2. Citation Format: Gongwei Wu, Noriaki Yoshida, Jihe Liu, Xiaoyang Zhang, Yuan Xiong, Tayla Heavican-Foral, Huiyun Liu, Geoffrey Nelson, Lu Yang, Renee Chen, Katherine Donovan, Marcus Jones, Mikhail Roshal, Yanming Zhang, Ran Xu, Ajit Nirmal, Salvia Jain, Catharine Leahy, Kristen Jones, Kristen Stevenson, Natasha Galasso, Nivetha Ganesan, Tiffany Chang, Wen-Chao Wu, Abner Louissaint, Lydie Debaize, Hojong Yoon, Paola Dal Cin, Wing Chan Chan, Shannan Ho Sui Ho Sui, Samuel Ng, Andrew Feldman, Steven M. Horwitz, Mathew Meyerson, Karen Adelman, Eric Fischer, Chun-Wei Chen, David Weinstock, Myles Brown. TP63 fusions drive enhancer rewiring, lymphomagenesis, and dependence on EZH2. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5755.

Pseudosarcomatous myofibroblastic proliferation is a descriptive term that designates a group of clinically indolent genitourinary lesions that most commonly arise in the urinary bladder. Given that pseudosarcomatous myofibroblastic proliferation may show morphologic overlap with inflammatory myofibroblastic tumor, the relationship, if any, between the two entities has been unclear. Moreover, pseudosarcomatous myofibroblastic proliferations are known to be positive for ALK immunohistochemistry in a subset of cases, although an inconsistent association with ALK rearrangement (ranging from 0 to 60%) has been reported. The objectives of this study were to determine the frequency of ALK rearrangement and to identify fusion partners using fluorescence in situ hybridization (FISH) and targeted RNA sequencing studies in a contemporary series of 30 pseudosarcomatous myofibroblastic proliferations of the urinary bladder, as well as to investigate ROS1 status by immunohistochemistry. ALK immunohistochemistry was positive in 70% (21/30) of pseudosarcomatous myofibroblastic proliferations; ROS1 immunohistochemistry was consistently negative (0/28). ALK rearrangements were detected by FISH in 86% (18/21) of cases, correlating with ALK immunohistochemical positivity in all but 3 cases. Of eight cases confirmed to be ALK rearranged by FISH, targeted RNA-sequencing detected FN1-ALK fusions in seven (88%) cases, which involved exons 20-26 of FN1 (5') and exon 18-19 of ALK (3'). In conclusion, ALK rearrangements are frequent in pseudosarcomatous myofibroblastic proliferations, typically involving exon 19, and FN1 appears to be a consistent fusion partner. Given the significant clinicopathologic differences between inflammatory myofibroblastic tumor and pseudosarcomatous myofibroblastic proliferation, our findings provide further support for classification of pseudosarcomatous myofibroblastic proliferation as a distinct clinicopathologic entity, and propose the alternate terminology "pseudosarcomatous myofibroblastic neoplasm of the genitourinary tract."

Aims Renal epithelial neoplasms (RENs) can be difficult to subclassify, owing to overlapping morphological features. Carbonic anhydrase 9 (CA9) is a common biomarker for clear cell renal cell carcinoma (CCRCC); however, the sensitivity and specificity across REN subtypes are less clear. The aim of this study was to investigate CA9 expression in RENs, especially those in the differential diagnosis with CCRCC and less common entities, to determine its reliability as a diagnostic biomarker. Methods and results CA9 immunostaining was performed on 262 RENs, including 119 CCRCCs and 143 non-CCRCC. Immunostaining was evaluated as negative (0%), rare (1+, 1-10%), focal (2+, 11-50%), or diffuse (3+, >50%). CCRCCs were 3+ CA9-positive in 93% of cases; 4% were CA9-negative. Sixty-seven percent of papillary renal cell carcinomas (RCCs) were 1+/2+ CA9-positive, whereas 33% were CA9-negative. Chromophobe RCCs were nearly always CA9-negative (93%), with 7% showing rare cell reactivity. Clear cell tubulopapillary RCCs (CCTPRCCs) were consistently 3+ CA9-positive, but with a cup-like staining pattern. Fifty-three percent of Xp11.2 RCCs were CA9-negative; however, 6% were 3+ CA9-positive and 12% were 2+ CA9-positive. Two of eight fumarate hydratase-deficient RCCs were 3+ CA9-positive. A small subset of the remaining RCCs showed rare to focal CA9 expression. All oncocytomas and eosinophilic solid and cystic RCCs were CA9-negative. Conclusions Overall, diffuse CA9 expression was identified in nearly all CCRCCs and in all CCTPRCCs (high sensitivity); however, CA9 was not entirely specific. At least focal CA9 expression can been seen in a subset of many RCCs, and such findings should be taken into consideration with other morphological, immunophenotypic and clinical findings.

Key Points A novel PCM1-FGFR1 gene rearrangement was identified in a patient with a myeloid neoplasm with eosinophilia. Futibatinib, an oral selective small molecule inhibitor of FGFR1-4, resulted in a durable complete hematologic and cytogenetic remission.

Loss of the Y chromosome (LOY) is one of the most common somatic genomic alterations in hematopoietic cells in men. However, due to the high prevalence of LOY as the sole cytogenetic finding in the healthy older population, differentiating isolated LOY associated with clonal hematologic processes from aging-associated mosaicism can be difficult in the absence of definitive morphological features of disease. In the past, various investigators have proposed that a high percentage of metaphases with LOY is more likely to represent expansion of a clonal myeloid disease-associated population. It is unknown whether the proportion of metaphases with LOY is associated with the incidence of myeloid neoplasia-associated genomic alterations. To address this question, we identified marrow samples with LOY as isolated cytogenetic finding and used targeted next generation sequencing-based molecular analysis to identify common myeloid neoplasia-associated somatic mutations. Among 73 patients with median age of 75 years (range 29-90), the percentage of metaphases with LOY was

Localized pleural mesothelioma is a rare solitary circumscribed pleural tumor that is microscopically similar to diffuse malignant pleural mesothelioma. However, the molecular characteristics and nosologic relationship with its diffuse counterpart remain unknown. In a consecutive cohort of 1110 patients with pleural mesotheliomas diagnosed in 2005-2018, we identified six (0.5%) patients diagnosed with localized pleural mesotheliomas. We gathered clinical history, evaluated the histopathology, and in select cases performed karyotypic analysis and targeted next-generation sequencing. The cohort included three women and three men (median age 63; range 28-76), often presenting incidentally during radiologic evaluation for unrelated conditions. Neoadjuvant chemotherapy was administered in two patients. All tumors (median size 5.0 cm; range 2.7-13.5 cm) demonstrated gross circumscription (with microscopic invasion into lung, soft tissue, and/or rib in four cases), mesothelioma histology (four biphasic and two epithelioid types), and mesothelial immunophenotype. Of four patients with at least 6-month follow-up, three were alive (up to 8.9 years). Genomic characterization identified several subgroups: (1) BAP1 mutations with deletions of CDKN2A and NF2 in two tumors; (2) TRAF7 mutations in two tumors, including one harboring trisomies of chromosomes 3, 5, 7, and X; and (3) genomic near-haploidization, characterized by extensive loss of heterozygosity sparing chromosomes 5 and 7. Localized pleural mesotheliomas appear genetically heterogeneous and include BAP1-mutated, TRAF7-mutated, and near-haploid subgroups. While the BAP1-mutated subgroup is similar to diffuse malignant pleural mesotheliomas, the TRAF7-mutated subgroup overlaps genetically with adenomatoid tumors and well-differentiated papillary mesotheliomas, in which recurrent TRAF7 mutations have been described. Genomic near-haploidization, identified recently in a subset of diffuse malignant pleural mesotheliomas, suggests a novel mechanism in the pathogenesis of both localized pleural mesothelioma and diffuse malignant pleural mesothelioma. Our findings describe distinctive genetic features of localized pleural mesothelioma, with both similarities to and differences from diffuse malignant pleural mesothelioma.

B-lymphoblastic leukemia/lymphoma (B-ALL) is the most common pediatric malignancy and the most commonly diagnosed adult lymphoblastic leukemia. Recent advances have broadened the spectrum of B-ALL, with DUX4 gene fusions implicated in a subclass occurring in adolescents and young adults and harboring a favorable prognosis. DUX4 fusions have been challenging to identify. We aimed to determine whether expression of the DUX4 oncoprotein, as detected by targeted immunohistochemistry, might serve as a surrogate for molecular detection of DUX4 fusions in B-ALL. A cohort of investigational B-ALLs was generated with enrichment for DUX4 fusions by the inclusion of cases with characteristic demographic features and immunophenotypic properties. B-ALLs with mutually exclusive cytogenetics were collected. Immunohistochemical staining by a monoclonal antibody raised against the N-terminus of the DUX4 protein was performed. N-DUX4 immunohistochemistry demonstrated strong, crisp nuclear staining in blasts of seven investigational cases, six of which had nucleic acid material available for molecular evaluation. Five of these cases demonstrated RNA-seq DUX4-fusion positivity. One N-DUX4 immunohistochemistry positive case lacked a definitive DUX4-fusion by RNA-seq, though demonstrated a gene expression profile characteristic of DUX4-rearranged B-ALLs, a CD2+ immunophenotype, and a lack of staining by C-terminus DUX4 antibody immunohistochemistry. At least 83.3% [5/6] positive predictive value. N-DUX4 immunohistochemistry was negative in blasts of three RNA-seq DUX4-fusion-negative cases (3/3; 100% negative predictive value). B-ALLs with mutually exclusive cytogenetic profiles were all N-DUX4 negative (0/10, specificity 100%). N-DUX4 immunohistochemistry is reliable for the distinction of DUX4-rearranged B-ALLs from other B-ALLs. We recommend its use for subclassification of B-ALLs in adolescents and young adults and in B-ALLs that remain "not otherwise specified."

A subset of clinically benign uterine polyps shows atypical morphologic features worrisome for, but not diagnostic of, Mullerian adenosarcoma. We report clinicopathologic data for 63 polyps from 58 women with atypical morphologic features including abnormal architecture, abnormal periglandular stroma, stromal atypia, and mitoses2 per 10 hpf. Four (11%) of 36 women with follow-up tissue sampling had residual/recurrent atypical polyp. Twelve (27%) of 44 women underwent hysterectomy subsequent to a diagnosis of atypical polyp. No patient developed adenosarcoma over median follow-up of 150 months. Twenty-one primary atypical polyps underwent molecular profiling. Five (24%) harbored chr 12q13-15 gain or amplification, 9/20 (45%) harbored chr 6q25.1 gain, and 7/21 (33%) had no significant copy number alterations. Gains of chr 1q, chr 8p12, and chr 10q11.21-23, amplifications of chr 12q24.12-13, chr 15p24.1-26.1, and chr 18q21.33, and loss of chr 7 and chr 11q21 were each seen in a single polyp. Mean tumor mutational burden was 3.1 (range, 0.76-8.365) mutations/Mb. Pathogenic point mutations were identified in 12/20 (60%) primary atypical polyps. We propose the term "atypical uterine polyps" for these lesions, which show biologic overlap with early Mullerian adenosarcoma but lack molecular alterations characteristic of clinically aggressive adenosarcoma and appear to follow a benign clinical course. Conservative management with close clinical follow-up and repeat sampling can be considered for these lesions, when clinically appropriate.

Acute myeloid leukemia (AML) with t(4;12)(q12;p13) translocation is rare and often associated with an aggressive clinical course and poor prognosis. Previous reports based on fluorescence in situ hybridization (FISH) analysis have suggested that ETV6::PDGFRA fusions are present in these patients, despite the absence of eosinophilia, which is typically found in other hematopoietic malignancies with PDGFRA-containing fusions. We first detected an ETV6-SCFD2 fusion by targeted RNA sequencing in a patient with t(4;12)(q12;p13) who had been diagnosed with an ETV6-PDGFRA fusion by FISH analysis but failed to respond to imatinib. We then retrospectively identified 4 additional patients with AML and t(4;12)(q12;p13) with apparent ETV6-PDGFRA fusions using chromosome and FISH analysis and applied targeted RNA sequencing to archival material. We again detected rearrangements between ETV6 and non-PDGFRA 4q12 genes, including SCFD2, CHIC2, and GSX2. None of the 3 patients who received imatinib based on the incorrect assumption of an ETV6-PDGFRA fusion responded. Our findings highlight the importance of using a sequencing-based assay to confirm the presence of targetable gene fusions, particularly in genomic regions, such as 4q12, with many clinically relevant genes that are too close to resolve by chromosome or FISH analysis. Finally, combining our data and review of the literature, we show that sequence-confirmed ETV6-PDGFRA fusions are typically found in eosinophilic disorders (3/3 cases), and patients with t(4;12)(q12;p13) without eosinophilia are found to have other 4q12 partners on sequencing (17/17 cases).

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.

Comprehensive characterization of somatic genomic alterations has led to fundamental shifts in our understanding of tumor biology. In clinical practice, these studies can lead to modifications of diagnosis and/or specific treatment implications, fulfilling the promise of personalized medicine. Herein, we describe a 78-yr-old woman under surveillance for long-standing untreated chronic lymphocytic leukemia (CLL). Molecular studies from a peripheral blood specimen revealed a TP53 p.V157F mutation, whereas karyotype and fluorescence in situ hybridization (FISH) identified a 17p deletion, trisomy 12, and no evidence of IGH-CCND1 rearrangement. Positron emission tomography-computed tomography scan identified multistation intra-abdominal lymphadenopathy and a pulmonary nodule, and subsequent pulmonary wedge resection confirmed the presence of a concurrent lung adenocarcinoma. Targeted next-generation sequencing of the lung tumor identified an EGFR in-frame exon 19 deletion, two TP53 mutations (p.P152Q, p.V157F), and, unexpectedly, a IGH-CCND1 rearrangement. Follow-up immunohistochemistry (IHC) studies demonstrated a cyclin D1–positive lymphoid aggregate within the lung adenocarcinoma. The presence of the TP53 p.V157F mutation in the lung resection, detection of an IGH-CCND1 rearrangement, and cyclin D1 positivity by IHC led to revision of the patient's hematologic diagnosis and confirmed the extranodal presence of mantle cell lymphoma within the lung mass, thus representing a “tumor in tumor.” Manual review of the sequencing data suggested the IGH-CCND1 rearrangement occurred via an insertional event, whose size precluded detection by original FISH studies. Thus, routine imaging for this patient's known hematologic malignancy led to detection of an unexpected solid tumor, whose subsequent precision medicine studies in the solid tumor redefined the original hematological diagnosis.

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 Mullerian 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.

Pseudosarcomatous myofibroblastic proliferation is a descriptive term that designates a group of clinically indolent genitourinary lesions that most commonly arise in the urinary bladder. Given that pseudosarcomatous myofibroblastic proliferation may show morphologic overlap with inflammatory myofibroblastic tumor, the relationship, if any, between the two entities has been unclear. Moreover, pseudosarcomatous myofibroblastic proliferations are known to be positive for ALK immunohistochemistry in a subset of cases, although an inconsistent association with ALK rearrangement (ranging from 0 to 60%) has been reported. The objectives of this study were to determine the frequency of ALK rearrangement and to identify fusion partners using fluorescence in situ hybridization (FISH) and targeted RNA sequencing studies in a contemporary series of 30 pseudosarcomatous myofibroblastic proliferations of the urinary bladder, as well as to investigate ROS1 status by immunohistochemistry. ALK immunohistochemistry was positive in 70% (21/30) of pseudosarcomatous myofibroblastic proliferations; ROS1 immunohistochemistry was consistently negative (0/28). ALK rearrangements were detected by FISH in 86% (18/21) of cases, correlating with ALK immunohistochemical positivity in all but 3 cases. Of eight cases confirmed to be ALK rearranged by FISH, targeted RNA-sequencing detected FN1-ALK fusions in seven (88%) cases, which involved exons 20-26 of FN1 (5') and exon 18-19 of ALK (3'). In conclusion, ALK rearrangements are frequent in pseudosarcomatous myofibroblastic proliferations, typically involving exon 19, and FN1 appears to be a consistent fusion partner. Given the significant clinicopathologic differences between inflammatory myofibroblastic tumor and pseudosarcomatous myofibroblastic proliferation, our findings provide further support for classification of pseudosarcomatous myofibroblastic proliferation as a distinct clinicopathologic entity, and propose the alternate terminology "pseudosarcomatous myofibroblastic neoplasm of the genitourinary tract."

Aims Tumours of the female genital tract with a combination of malignant Mullerian and germ cell or trophoblastic tumour (MMGC/T) components are usually diagnosed in postmenopausal women, and pursue an aggressive clinical course characterised by poor response to therapy and early relapses. These clinical features suggest that MMGC/T are somatic in origin, but objective molecular data to support this interpretation are lacking. This study evaluates the molecular features of nine MMGC/T, including seven tumours containing yolk sac tumour (YST), one tumour containing choriocarcinoma and one tumour containing epithelioid trophoblastic tumour. The objectives were to: (i) investigate whether MMGC/T show a distinct genetic profile and (ii) explore the relationship between the different histological components. Methods and results Next-generation sequencing of paired samples demonstrated that the mutational profile of the Mullerian and non-Mullerian components of the tumour were almost identical in all cases. Moreover, the driver mutations identified were those expected in the specific subtype of Mullerian component present in each case. In contrast, variants expected in postpubertal germ cell tumours and gestational trophoblastic tumours were not identified, and FISH for i(12p) was negative in all cases tested. In this study, mismatch repair-proficient MMGC/T (eight of nine) were characterised by a complex copy-number variant profile, including numerous focal, regional, arm-level and chromosome-level events. Conclusions Comparison of paired samples supports that the YST and trophoblastic tumour components of MMGC/T have a somatic origin and often show numerous copy-number variants, suggestive of underlying genomic instability.

Chromosome 12p gains are typically present in postpubertal male patients with testicular malignant germ cell tumors, including most teratomas, and absent in pure ovarian teratomas, both mature and immature. We sought to evaluate the clinicopathologic features and chromosome 12p status of pediatric patients with sacrococcygeal teratomas (SCTs) using the institutional databases of 2 tertiary medical centers. Seven mature teratomas (3 pure, 2 with yolk sac tumor, 1 with medulloepithelioma, and 1 with ependymoma) and 3 immature teratomas (2 pure: grade 2 and grade 3 and 1 mixed: grade 3 with yolk sac tumor) were identified. All patients underwent surgery and 2 received adjuvant chemotherapy. Fluorescence in situ hybridization analysis was performed to elucidate chromosome 12p gains, including isochromosome 12p. All 10 tumors analyzed lacked 12p gains regardless of the components. No patient had evidence of disease at their most recent interval follow-up (mean: 30, range: 7–91 months), irrespective of margin status or of receiving chemotherapy. Overall, our study suggests an absence of chromosome 12p abnormalities in clinically nonaggressive SCTs. Additional data are required to confirm these findings before definitive patient care recommendations can be made.

AimsCytogenetic and molecular aberrations are the strongest factors in determining outcome in acute myeloid leukaemia (AML). AML with complex karyotype confers a particularly poor prognosis and is associated with morphologic dysplasia. Flow cytometric immunophenotyping (FCI) has been investigated in defining dysplasia within myelodysplastic syndromes, but little is known about immunophenotypic dysplasia in AML and correlation with genetic abnormalities. This study aimed to explore differences in antigen expression by FCI in AML with complex karyotype (AML-CK) and AML with complex karyotype and TP53 mutations (AML-TP53) compared with AML with normal karyotype (AML-NK).MethodsTwenty-five cases of AML-CK, 13 of which had abnormalities of TP53, were compared with 83 cases of AML-NK using FCI.ResultsOur findings demonstrated brighter expression of CD34 with decreased CD33 and aberrant expression of CD5 in blasts of AML-CK, while AML-TP53 blasts exhibited brighter expression of CD13. Granulocytes in AML-CK exhibited brighter expression of CD5, CD7, CD10 and CD14, with brighter CD3 also seen in AML-TP53.ConclusionsOur results suggest that immunophenotypic dysplasia correlates with complex karyotype and TP53 mutation, including increased expression of T-cell antigens.