Myxoid pleomorphic liposarcoma (MPLPS), a rare, aggressive liposarcoma subtype, is thought to occur chiefly in adolescents with a mediastinal predilection. MPLPS lack DDIT3 rearrangements or MDM2 amplification; a small number have recently been shown to harbor widespread copy-neutral loss of heterozygosity (cnLOH). We studied a large series of MPLPS and compared them to other sarcomas, particularly other LPS. Available slides/blocks for cases coded as "MPLPS" were retrieved (2008-2026). We also searched our single nucleotide polymorphism (SNP) assay records for tumors demonstrating 1) high cnLOH frequency (>30%), and 2) "liposarcoma" or "pleomorphic sarcoma" diagnosis. Non-mesenchymal tumors were excluded. Twenty-four MPLPS cases were identified, occurring in 10 females (42%) and 14 males (58%), ranging from 5-85 years of age (median 46 years). Involved anatomical locations included the mediastinum/thorax (n=12), trunk (n=3), head and neck (n=3), various intra-abdominal sites (n=3), retroperitoneum/pelvis (n=2) and extremities (n=1). Two patients had clinical features of Li-Fraumeni syndrome or a germline TP53 mutation. All tumors displayed characteristic features of MPLPS, including areas resembling conventional myxoid liposarcoma and hypercellular, pleomorphic liposarcoma-like foci. SNP testing (n=22) demonstrated cnLOH of >50% in 19 cases (86%), often with pseudohyperdiploidy. One tumor showed cnLOH of 38%; two otherwise typical tumors did not have widespread cnLOH. Other recurrent alterations included gains of chromosomes 1/1q, 6- 8, and 18-21 and loss of chromosome 14. Specific losses/gains involving the RB1 and TP53 loci were seen in 12 and 2 cases, respectively. Clinical follow-up (n=22; median 16 months; range 1-75 months) showed 9 patients dead of disease (median survival 12 months), 6 patients alive with disease, and 6 patients alive without disease. Local recurrences and distant metastases were seen in 6 and 8 patients, respectively. We conclude that the age range and anatomical distribution of MPLPS are considerably wider than has been previously appreciated, including tumors arising in the elderly and various non-mediastinal locations. Although most MPLPS harbor widespread cnLOH, rare otherwise-typical tumors do not. MPLPS are aggressive sarcomas with poor prognosis.

Context.—:One objective of the College of American Pathologists/American College of Medical Genetics Cytogenetics Committee is to ensure the accurate detection and clinical interpretation of abnormalities observed by fluorescence in situ hybridization (FISH) analysis when evaluating hematologic neoplasms. Objective.—:To report a 20-year performance summary (2005-2024) of FISH challenges focusing on hematologic neoplasms. Design.—:A retrospective review was performed from 2005 through 2024 to identify FISH challenges specifically addressing hematologic neoplasms. The overall performance of participants was evaluated to identify potential recurring errors of clinical relevance. Results.—:A total of 124 hematologic neoplasm FISH challenges from 2005 to 2024 were provided to participating laboratories, including 45 paper challenges (results and/or images were provided for interpretation purposes), 43 liquid challenges (bone marrow aspirate or peripheral blood), and 36 formalin-fixed paraffin-embedded (FFPE) tissue challenges. Of the 124 challenges, 120 (97%) exceeded the required 80% consensus for grading purposes. Of the 4 failed challenges (3%), 2 (1 liquid, 1 paper) involved the interpretation of atypical FISH signal patterns; 1 (paper) involved the interpretation of FISH signal patterns correlating involvement by myeloid versus lymphoid interphase nuclei; and 1 (liquid) likely involved a specimen processing or handling error that resulted in discordant participant responses. All 36 FFPE tissue challenges exceeded the 80% consensus for grading purposes. Conclusions.—:This 20-year retrospective review demonstrates that clinical cytogenomic laboratories have been and continue to be highly proficient in the detection and interpretation of FISH abnormalities associated with hematologic neoplasms.

Despite advances in molecular genetics that have helped elucidate the pathogenesis of many soft tissue neoplasms, subsets of fibromyxoid tumors remain difficult to subclassify because of their nonspecific morphologic and immunohistochemical features, and lack of discrete molecular alterations. We report 7 cases of a distinctive fibromyxoid soft tissue neoplasm characterized at the cytogenomic level by massive loss of heterozygosity, resulting in a near-haploid or pseudohyperdiploid cytogenome. The tumors occurred in superficial and deep soft tissue locations (mesentery, mediastinum, thigh, pelvis, leg, orbit, and head) of 5 males and 2 females (median age, 45 years). They ranged in size from 2.6 to 14 cm (median, 5 cm) and were composed of small, bland spindled cells in a variably vascularized, fibromyxoid stroma with abundant wiry collagen. Mitotic activity was low, and necrosis was absent in all but 1 case, which demonstrated foci of infarct-type necrosis. One tumor showed infiltrative growth into surrounding tissue while all others were circumscribed and noninfiltrative. Immunohistochemistry demonstrated CD34 (5/5) and desmin (3/5) expression; S100 protein, SOX10, MUC4, and GLUT1 were negative, among others. All tumors demonstrated massive loss of heterozygosity with copy number gain and retained heterozygosity of selected chromosomes, including chromosomes 8 and 19, evaluated with OncoScan single-nucleotide polymorphism array. Biallelic inactivation of NF1 was seen in 2 cases, including 1 in a patient with neurofibromatosis type 1. Three patients showed possible evidence of stable locally recurrent/residual disease following incomplete resection while all other patients were free of disease (median follow-up, 17 months). The tumors presented in this study are essentially identical to those of a very recently reported series of 5 cases, strongly suggesting that these collectively represent a novel entity, which we propose terming distinctive near-haploid fibromyxoid neoplasm.

CONTEXT.—:The joint College of American Pathologists/American College of Medical Genetics and Genomics Cytogenetics Committee works to ensure competency and proficiency of clinical cytogenetics testing laboratories through proficiency testing programs for various clinical tests offered by such laboratories, including the evaluation of constitutional abnormalities. OBJECTIVE.—:To review and analyze 20 years of constitutional chromosome analysis proficiency testing results (2003-2022), primarily utilizing G-banded karyograms. DESIGN.—:A retrospective review of results from 2003 through 2022 was performed, identifying challenges addressing constitutional disorders. The chromosomal abnormalities and overall performance were evaluated. RESULTS.—:A total of 184 cases from 161 proficiency testing challenges were administered from 2003 through 2022. Challenges consisted of metaphase images and accompanying clinical history for evaluation of numerical and/or structural abnormalities. Of the 184 cases, only 2 (1%) failed to reach an 80% grading consensus for recognition of the abnormality. Both cases illustrated the limitations of correctly characterizing some chromosomal abnormalities, including recombinant chromosomal abnormalities and isochromosome identification. In addition, 2 cases failed to reach a consensus for nomenclature reporting: 1 with an isochromosome and another with a duplication. CONCLUSIONS.—:This 20-year review illustrates the high rate of competency and proficiency of cytogenetic laboratories in the correct identification of constitutional chromosome abnormalities.

Myxoid liposarcoma (MLS) accounts for 20%-30% of all liposarcomas, with most cases harboring the fusion gene FUS::DDIT3, while approximately 5% exhibit the EWSR1::DDIT3 fusion. We report the case of a 26-year-old male patient with a right upper arm mass. The tumor displayed the classic histological features of MLS, including small spindle/ovoid cells, variable univacuolated lipoblasts, and a prominent myxoid stroma with delicate arborizing vasculature. Despite these characteristic features, fluorescence in situ hybridization (FISH) revealed no apparent rearrangement of the DDIT3 locus. Next-generation sequencing (NGS) identified a novel fusion transcript in which SMARCA2 exon 4 was fused in-frame with DDIT3 exon 2. Chromosomal microarray analysis demonstrated the unbalanced nature of the rearrangement, with partial deletions of 0.243 and 0.176 Mb flanking the centromeric end of the DDIT3 locus on 12q13.3 (which also included GLI) and disrupting the SMARCA2 locus on 9p24, respectively. The resultant chimeric fusion protein is predicted to lack the SMARCA2 DNA-binding domains while retaining the DDIT3 leucine zipper dimerization domain. These findings indicate an unusual and complex rearrangement, leading to the recruitment of a novel DDIT3 partner gene. Moreover, they emphasize that the functional aspects of myxoid liposarcoma fusion genes depend on the retention of the key DDIT3 domain. Finally, this case illustrates how classic morphology can appropriately trigger reflex molecular analyses, which may, in turn, uncover novel fusion genes or other molecular alterations.

An amplicon-based targeted next-generation sequencing (NGS) assay for the detection of gene fusions in sarcomas was developed, validated, and implemented. This assay can detect fusions in targeted regions of 138 genes and BCOR internal tandem duplications. This study reviews our experience with testing on the first 652 patients analyzed. Gene fusions were detected in 238 (36.5%) of 652 cases, including 83 distinct fusions in the 238 fusion-positive cases, 10 of which had not been previously described. Among the 238 fusion-positive cases, the results assisted in establishing a diagnosis for 137 (58%) cases, confirmed a suspected diagnosis in 66 (28%) cases, changed a suspected diagnosis in 25 (10%) cases, and were novel fusions with unknown clinical significance in 10 (4%) cases. Twenty-six cases had gene fusions (ALK, ROS1, NTRK1, NTRK3, and COL1A1::PDGFB) for which there are targetable therapies. BCOR internal tandem duplications were identified in 6 (1.2%) of 485 patients. Among the 138 genes in the panel, 66 were involved in one or more fusions, and 72 were not involved in any fusions. There was little overlap between the genes involved as 5'-partners (31 different genes) and 3'-partners (37 different genes). This study shows the clinical utility of a next-generation sequencing gene fusion detection assay for the diagnosis and treatment of sarcomas.

Molecularly defined renal cell carcinomas include TFE3-rearranged renal cell carcinoma (TFE3-RCC) and TFEB-altered renal cell carcinoma (TFEB-RCC). There is significant morphologic and immunophenotypic overlap between these entities and common renal tumors, such that molecular testing is often required to make the diagnosis. Herein, we reviewed our reference laboratory experience pertaining to TFE3 and TFEB FISH testing, targeted next generation RNA sequencing (NGS), and GPNMB immunohistochemistry (IHC). Most FISH testing (2963/3543, 83.6%) was performed on renal tumors. TFE3 FISH showed rearrangements in 449 of 2467 specimens (18.2%), including 281 (of 1887, 14.9%) renal tumors. TFEB FISH identified an abnormality in 107 of 1076 (9.9%) renal tumors, including 52 (of 107, 48.6%) rearrangements, 41 (of 107, 38.3%) amplifications, or 14 (of 107, 13.1%) with both rearrangements and amplifications. More specifically, TFE3-rearranged, TFEB-rearranged, TFEB-amplified, and TFEB-rearranged/amplified renal tumors occurred in females in 54%, 69.6%, 39.1%, and 40% of cases, respectively. The pediatric and young adult population (aged ≤21 years) included 44 (of 121, 36.3%) TFE3-RCC and 9 (of 50, 18%) TFEB-rearranged RCC. TFE3-RCC fusion partners included RBM10, NONO, ASPSCR1, FUBP1, SFPQ, MAPK1IP1L, and PRCC. TFEB-rearranged RCC fusion partners SYNRG and BYSL were identified. Diffuse GPNMB expression was seen in 92% of TFE3-RCC (24/26; median H-score 275), 100% of TFEB-rearranged RCC (19/19; median H-score 300), and 100% of TFEB-amplified RCC (17/17; 240). Finally, our cohort included 5 eosinophilic TFEB-amplified RCCs with non-focal keratin 20 expression. This large series of TFE3-RCC and TFEB-RCC provides population data regarding these rare tumors and demonstrates the clinical value of targeted FISH strategies. Our results suggest that GPNMB IHC is an effective screen for TFE3-RCC and TFEB-RCC. Additionally, we report a RCC harboring a novel SYNRG::TFEB fusion.

Fluorescence in situ hybridization (FISH) using a break-apart probe (BAP) design is a rapid, clinically useful method for targeted evaluation of gene rearrangements in formalin-fixed, paraffin-embedded tumors. Although clinically validated BAP FISH assays usually yield unequivocal positive or negative results, rare tumors yield equivocal FISH results. This study had two aims: to summarize typical and atypical BAP FISH results on 56,584 formalin-fixed, paraffin-embedded solid tumors over approximately one decade of clinical testing; and to investigate the clinical utility of RNA sequencing (RNA-seq) for tumors with equivocal FISH results. Of 8586 (15.2%) cases with abnormal FISH results reported, 748 (8.7%) were equivocal. RNA-seq was performed on 113 tumors, and oncogenic fusions involving the gene of interest were detected in 46 of 113 tumors (40.7%). Of the 106 tumors with equivocal FISH results, RNA-seq detected a fusion involving the expected gene target in 37 of 62 (59.7%) tumors with isolated probe signals corresponding to the active side of the gene region but only 4 of 44 (9.1%) tumors with other atypical signal patterns. This study provides a useful framework for categorizing atypical BAP FISH results and demonstrates the clinical utility of follow-up RNA-seq testing on tumors with equivocal FISH results.

OBJECTIVES:Sarcomatoid carcinoma of the prostate (SCP) is a rare neoplasm known for its diagnostic difficulties and aggressive clinical course. Given the paucity of literature on its molecular landscape, we aimed to investigate a cohort of SCP, using a multi-modal approach. METHODS:Our surgical pathology archive was queried for patients diagnosed with SCP (2006-2022), followed by re-review of archived slides. For each case, a panel of immunohistochemical stains (including programmed death-ligand 1 [PD-L1] clones SP142, SP263, and 22C3) was performed on a representative block. Fluorescence in situ hybridization (FISH) was used to evaluate chromosomes 10 (including PTEN) and 17 (including TP53). All cases were evaluated using a next-generation sequencing (NGS) panel. RESULTS:Eight patients were included. Three (37.5 %) had a prior history of acinar adenocarcinoma, while a concomitant adenocarcinoma was present in five patients (62.5 %). The median duration of follow-up was 20.5 months. Seven patients (87.5 %) presented with or developed systemic metastases during follow-up. At last follow-up, 6 patients (75 %) were dead of disease. Three of the 7 cases (42.9 %) assessed for PD-L1 expression showed some staining. The most common pathogenic alterations identified by NGS involved TP53 (n = 5), followed by APC, BRCA2, CHECK2, CTNNB1, and RB1 (n = 1, each). On FISH testing, copy number changes involving chromosome 10 and 17 were found in 80 % and 60 % of the cases, respectively. CONCLUSIONS:This study sheds light on the molecular landscape of SCP, which may be valuable to elucidate the prognostic and therapeutic implications for this uncommon disease.

Context.-The joint College of American Pathologists/ American College of Medical Genetics and Genomics Cytogenetics Committee works to ensure competency and proficiency of clinical cytogenetics testing laboratories through proficiency testing programs for various clinical tests offered by such laboratories, including the evaluation of constitutional abnormalities. Objective.-To review and analyze 20 years of constitutional chromosome analysis proficiency testing results (2003- 2022), primarily utilizing G-banded karyograms. Design.-A retrospective review of results from 2003 through 2022 was performed, identifying challenges addressing constitutional disorders. The chromosomal abnormalities and overall performance were evaluated. Results.-A total of 184 cases from 161 proficiency testing challenges were administered from 2003 through 2022. Challenges consisted of metaphase images and accompanying clinical history for evaluation of numerical and/ or structural abnormalities. Of the 184 cases, only 2 (1%) failed to reach an 80% grading consensus for recognition of the abnormality. Both cases illustrated the limitations of correctly characterizing some chromosomal abnormalities, including recombinant chromosomal abnormalities and isochromosome identification. In addition, 2 cases failed to reach a consensus for nomenclature reporting: 1 with an isochromosome and another with a duplication. Conclusions.-This 20-year review illustrates the high rate of competency and proficiency of cytogenetic laboratories in the correct identification of constitutional chromosome abnormalities.

Background: The joint College of American Pathologists/American College of Medical Genetics and Genomics Cytogenetics Committee works to ensure the competency and proficiency of clinical cytogenetic testing laboratories through proficiency testing (PT) programs for various clinical tests offered by such laboratories, including the evaluation of cytogenetic abnormalities in solid tumors. Methods: Review and analyze 25 years (1999-2023) of solid tumor chromosome analysis PT results, utilizing G-banded karyograms. A retrospective review of results from 1999 to 2023 was performed, identifying the challenges addressing solid tumors. The chromosomal abnormalities and overall performance were evaluated. Results: A total of 21 solid tumor challenges were administered during the period 1999-2018. No solid tumor challenges were administered during the period 2019-2023. Challenges consisted of metaphase images and accompanying clinical history for the evaluation of numerical and/or structural abnormalities. All 21 cases reached 80% grading consensus for abnormality recognition. However, five cases (24%) failed to reach consensus for nomenclature reporting by participating laboratories. These cases illustrate errors in reporting chromosomal abnormalities, including whole-arm translocations and those involving sex chromosomes. In addition, they highlight the challenges with differentiation of terminal and interstitial deletions, difficulties in identifying correct breakpoints, and omission of brackets in neoplastic cases. Conclusions: This comprehensive 25-year review demonstrates the exceptional proficiency of cytogenetic laboratories in accurately identifying chromosome abnormalities in solid tumors, while also highlighting the challenges of reporting specific types of chromosomal abnormalities.

Introduction Recent studies suggest that expansile cribriform morphology contributes to adverse oncologic outcomes. Among specific tumor suppressor gene alterations associated with cribriform morphology, PTEN and CHD1 are known to confer an aggressive behavior. However, alteration of both genes among patients with expansile cribriform Gleason pattern 4 prostate cancer (PCa) and their correlation with oncologic outcomes have not been extensively studied. Herein, we sought to determine the impact of expansile cribriform morphology and somatic PTEN and CHD1 gene loss on oncologic outcomes for patients undergoing radical;prostatectomy (RP) for grade groups 2-5 PCa. Methods We reviewed the medical records from patients who underwent RP for Gleason score 7 or higher PCa from 2000 to 2002 at our institution. Experienced GU pathologists re reviewed all pathology specimens to verify Gleason scores and percentages of each Gleason pattern. Disagreements were solved by consensus. Gleason pattern 4 was characterized as poorly formed/fused glands, small uniform cribriform glands, expansile cribriform glands, mucinous glands, or glands with glomerulations. Tissue microarrays (TMAs) were constructed with quadruplicate 0.6 mm cores containing the Gleason pattern 4 from each case. Fluorescence in situ hybridization (FISH) using probes detecting PTEN and CHD1 was performed on TMAs (Figure). The association of expansile cribriform glands with each gene loss was assessed by Cox regression analyses. The association of Gleason 4 histological patterns and gene loss with biochemical recurrence (BCR), systemic progression (SP), and death from prostate cancer was assessed using Cox proportional hazard regression models. Results A total of 409 and 262 patients were included in the FISH and survival;analyses, respectively. Median follow-up from RP to death or last follow up for the entire cohort was 15.1 years (IQR: 11.5 – 16.3). Expansile cribriform morphology was significantly associated with worse BCR (p<0.0001), SP (p<0.0001), and death from PCa (p<0.001) compared to other Gleason pattern 4 morphologies (Table). Only PTEN gene loss (as opposed to CHD1) was associated with worse oncologic outcomes compared to patients without any gene loss (Table). Overall, CHD1 and PTEN gene loss were identified in 18.3% and 24.7% of patients with any Gleason pattern 4. Expansile cribriform Gleason pattern 4 was more commonly associated with CHD1 gene loss compared to other Gleason pattern 4 morphologies (p=0.004). On the other hand, PTEN gene loss was not preferentially found among expansile cribriform morphology compared to other Gleason 4 patterns (p=0.08). Conclusions Expansile cribriform Gleason 4 differs from other Gleason 4 patterns. Morphologically, these glands are large with less open/ill formed lumens and irregular sprawling edges. Genetically, CHD1 loss is commonly identified within these glands. More importantly, patients with expansile cribriform glands and PTEN gene loss seem to have worse oncologic outcomes. Pending external validation, these data suggest that an expansile cribriform architecture could be more appropriately classified as Gleason pattern 5.

Leiomyosarcoma with adipocytic differentiation or lipoleiomyosarcoma is an uncommon sarcoma of the female genital tract with only a few individual reports in the literature. We therefore performed a morphologic, immunohistochemical, MDM2 gene amplification and RNA and DNA sequencing analysis of a series of gynecologic lipoleiomyosarcoma to better define the clinicopathologic spectrum. Six tumors from 6 patients were identified and classified as spindled lipoleiomyosarcoma (n = 2), mixed spindled and myxoid lipoleiomyosarcoma (n = 1), epithelioid lipoleiomyosarcoma (n = 1) and mixed spindled and epithelioid lipoleiomyosarcoma (n = 2). Patient age ranged from 41 to 64 years (mean: 49; median: 50). Primary location included uterine corpus (3), uterine corpus/cervix (2) and broad ligament (1). Tumor size ranged from 4.5 to 22 cm (mean: 11.2; median: 9.8). Four patients had metastasis at presentation or subsequently developed recurrent or distant disease. Patient status was known for 5: 2 dead of disease, 2 alive with disease and 1 alive without evidence of disease. Immunohistochemical expression of smooth muscle markers, ER, PR and WT-1 showed patterns similar to non-adipocytic gynecologic leiomyosarcomas. MDM2 amplification fluorescence in situ hybridization performed on 2 tumors was negative in 1 and equivocal in 1. Sequencing studies performed on 3 tumors found TP53 mutations in 3, with 1 tumor also having an ATRX alteration. No gene fusions were identified. Although lipoleiomyosarcomas have a diverse morphologic spectrum, our findings suggest the smooth muscle component shares morphologic and immunohistochemical features with female genital tract non-adipocytic leiomyosarcomas. Lipoleiomyosarcomas also have genetic alterations associated with non-adipocytic gynecologic leiomyosarcomas.

AimsThe majority of dermatofibrosarcoma protuberans (DFSP) harbour PDGFB or PDGFD rearrangements. We encountered ALK expression/rearrangement in a PDGFB/D‐negative CD34‐positive spindle cell neoplasm with features similar to DFSP, prompting evaluation of ALK‐rearrangements in DFSP and plaque‐like CD34‐positive dermal fibroma (P‐LDF).Methods and ResultsWe searched the archives of academic institutions for cases previously coded as DFSP and P‐LDF. NGS‐naïve or PDGFB‐negative DFSP were screened for ALK (clone D5F3) expression by immunohistochemistry. NGS or ALK FISH was performed on ALK‐positive cases. Methylome profiling studies were performed and compared with conventional DFSP. One case of “DFSP” and two “P‐LDF” with ALK expression were identified from the archives, while four cases were detected prospectively. These seven cases (6F:1M; 8 months to 76 years) arose in the dermis of the arm (two), scalp, eyelid, thigh, abdomen, and shoulder and ranged from 0.4 to 4.2 cm. Tumours were composed of spindled cells and displayed a storiform growth pattern. Cytologic atypia was absent, and mitotic figures were scarce (0–2/10 HPFs, high power fields). The lesional cells were diffusely positive for CD34 and ALK and negative for S100 protein. By NGS (n = 5), ALK fusion partners included DCTN1 (2), PLEKHH2, and CLIP2 in DFSP‐like cases and FLNA in P‐LDF‐like lesions. ALK FISH was positive in one (of two) cases previously labelled P‐LDF. Methylome profiling of two (of three) ALK‐rearranged DFSP‐like tumours showed clustering with conventional DFSP in the UMAP dimension reduction plot. To date, no tumour has recurred (n = 2; 26, 27 months).ConclusionWe describe a cohort of novel ALK‐rearranged tumours with morphologic features similar to DFSP.