Sukov, William R.

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.

ABSTRACT 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.

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.

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.

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.

AbstractBackgroundMeningiomas are the most common primary central nervous system (CNS) tumor in adults and CNS World Health Organization grade 2 (atypical) meningiomas show an intermediate risk of recurrence/progression. Molecular parameters are needed to better inform management following gross total resection (GTR).MethodsWe performed comprehensive genomic analysis of tumor tissue from 63 patients who underwent radiologically confirmed GTR of a primary grade 2 meningioma, including a CLIA-certified target next-generation sequencing panel (n = 61), chromosomal microarray (n = 63), genome-wide methylation profiling (n = 62), H3K27me3 immunohistochemistry (n = 62), and RNA-sequencing (n = 19). Genomic features were correlated with long-term clinical outcomes (median follow-up: 10 years) using Cox proportional hazards regression modeling and published molecular prognostic signatures were evaluated.ResultsThe presence of specific copy number variants (CNVs), including -1p, -10q, -7p, and -4p, was the strongest predictor of decreased recurrence-free survival (RFS) within our cohort (P < .05). NF2 mutations were frequent (51%) but did not show a significant association with RFS. DNA methylation-based classification assigned tumors to DKFZ Heidelberg benign (52%) or intermediate (47%) meningioma subclasses and was not associated with RFS. H3K27 trimethylation (H3K27me3) was unequivocally lost in 4 tumors, insufficient for RFS analysis. Application of published integrated histologic/molecular grading systems did not improve prediction of recurrence risk over the presence of -1p or -10q alone.ConclusionsCNVs are strong predictors of RFS in grade 2 meningiomas following GTR. Our study supports incorporation of CNV profiling into clinical evaluation to better guide postoperative patient management, which can be readily implemented using existing, clinically validated technologies.

Abstract Atypical meningiomas (CNS WHO grade 2) show an intermediate risk of recurrence/progression, and molecular parameters informing management following gross total resection (GTR) remain to be established. We performed comprehensive genomic analysis in 63 patients who underwent GTR of an atypical meningioma. Median age was 59.8 years, 41 patients (65.1%) were female, and 8/62 patients (12.9%) underwent radiation therapy following resection. Follow-up was available for all patients (median 10 years), with recurrence/progression in 17 (5-year PFS: 84.2%; 10-year PFS: 71.0%). Tumors were evaluated by next generation sequencing (n=61), chromosomal microarray (n=63), DNA methylation profiling (n=62), and H3K27me3 immunohistochemistry (n=62). Next generation sequencing using a CLIA-certified 50-gene Neuro-Oncology specific panel identified pathogenic alterations in NF2 (n=31; 50.8%), SMARCB1 (n=5; 8.2%), AKT1 (n=2; 3.3%), TERT (n=1, 1.6%), SMO (n=1), SUFU (n=1), TP53 (n=1), and PTEN (n=1). There was trend toward decreased PFS in patients with NF2 alterations (p=0.078) which was not statistically significant. Copy number variants (CNVs) were identified using OncoScan (ThermoFisher). The most frequent CNVs were loss -22 (n=45; 71.4%), -1p (n=30, 47.6%), -14 (n=21, 33.3%), -6q (n=18, 28.6%), -10q (n=15, 23.8%), -X (n=13, 20.6%), and -18 (n=11, 17.4%). CNVs associated with decreased PFS included -1p (p=0.015), -7p (p=0.007), -10q (p=0.0007), and -18 (p=0.0376). DNA methylation profiling was performed using the Illumina MethylationEPIC array (n=62) and evaluated using the DKFZ classifier (v12.3). Based on the highest score, tumors matched to benign (n=32; 51.6%) or intermediate (n=29; 46.7%) meningioma subclasses or showed non-contributory profiles (n=1; 1.6%). Methylation class (benign vs. intermediate) showed no association with PFS (p=0.96). H3K27 trimethylation (H3K27me3) was unequivocally lost in 4 (6.5%) tumors, which was insufficient for PFS analysis. Overall, our study supports CNV profiling of atypical meningiomas following GTR, which can be implemented using existing, clinically validated technologies. Molecular predictors could guide use of radiotherapy in these patients.

Abstract Purpose: Selective FGFR inhibitors are effective against cholangiocarcinomas that harbor gene alterations in FGFR2. Clinical trials suggest that expression of wild-type FGFR mRNA can predict sensitivity to FGFR inhibitors, but this biomarker has not been well characterized in cholangiocarcinoma. This study explores the prevalence of FGFR mRNA overexpression in cholangiocarcinoma, its role in predicting sensitivity to FGFR inhibitors, and its association with immune markers. Experimental Design: Tissue microarrays of intrahepatic (ICC) and extrahepatic cholangiocarcinomas (ECC) resected between 2004 and 2015 were used to evaluate FGFR1–4 mRNA expression levels by RNA in situ hybridization (ISH). Expression levels of FGFR2 mRNA were correlated with FGFR2 fusion status and with patient outcomes. Immune markers expression was assessed by IHC and CSF1 and CSF1 receptor expression were examined by RNA ISH. Results: Among 94 patients with resected cholangiocarcinoma, the majority had ICC (77%). FGFR2 fusions were identified in 23% of ICCs and 5% of ECCs. High levels of FGFR mRNA in FGFR2 fusion–negative ICC/ECC were seen for: FGFR1 (ICC/ECC: 15%/0%), FGFR2 (ICC/ECC: 57%/0%), FGFR3 (ICC/ECC: 53%/18%), and FGFR4 (ICC/ECC: 32%/0%). Overall, 62% of fusion-negative cholangiocarcinomas showed high levels of FGFR mRNA. In patients with advanced FGFR2 fusion–positive ICC, high levels of FGFR2 mRNA did not correlate with clinical benefit. FGFR2 fusion–positive tumors showed a paucity of PD-L1 on tumor cells. Conclusions: FGFR mRNA overexpression occurs frequently in cholangiocarcinoma in the absence of genetic alterations in FGFR. This study identifies a molecular subpopulation in cholangiocarcinoma for which further investigation of FGFR inhibitors is merited outside currently approved indications.

Abstract The diagnosis of acute promyelocytic leukemia (APL) relies on the identification of PML::RARA fusion. While the majority of APL cases harbor a typical t(15;17)(q24;q21), atypical genetic mechanisms leading to the oncogenic PML::RARA fusion have been reported yet their frequency and scope remain poorly characterized. We assessed the genetic findings of 831 cases with APL investigated with concurrent chromosome banding analysis and dual‐color dual‐fusion fluorescence in situ hybridization (D‐FISH) analysis at our institution over an 18.5‐year timeframe. Seven hundred twenty‐three (87%) cases had a typical balanced t(15;17) with both testing modalities. Atypical karyotypic results including complex translocations, unbalanced rearrangements and insertional events occurred in 50 (6%) cases, while 6 (0.7%) cases were cryptic by conventional chromosome studies despite PML::RARA fusion by D‐FISH evaluation. Atypical FISH patterns were observed in 48 (6%) cases despite apparently balanced t(15;17) on chromosome banding analysis. Two hundred fifty (30%) cases displayed additional chromosome abnormalities of which trisomy/tetrasomy 8 (37%), del(7q)/add(7q) (12%), and del(9q) (7%) were most frequent. Complex and very complex karyotypes were observed in 81 (10%) and 34 (4%) cases, respectively. In addition, 4 (0.5%) cases presented as an apparently doubled, near‐tetraploid stemline clone. This report provides the largest appraisal of cytogenetic findings in APL with conventional chromosome and PML::RARA D‐FISH analysis. By characterizing the frequency and breadth of typical and atypical results through the lens of these cytogenetic testing modalities, this study serves as a pragmatic source of information for those involved in the investigation of APL in both the clinical and research laboratory settings.

Background: The morphologic distinction between thymic carcinomas and thymomas, specifically types B3, A, and occasionally micronodular thymomas with lymphoid stroma (MNTLS) can be challenging, as has also been shown in interobserver reproducibility studies. Since thymic carcinomas have a worse prognosis than thymomas, the diagnosis is important for patient management and treatment. This study aimed to identify a panel of immunohistochemical (IHC) markers that aid in the distinction between thymomas and thymic carcinomas in routine practice. Materials and Method: Thymic carcinomas, type A and B3 thymomas, and MNTLS were identified in an institutional database of thymic epithelial tumors (TET) (1963–2021). IHC was performed using antibodies against TdT, Glut-1, CD5, CD117, BAP1, and mTAP. Percent tumor cell staining was recorded (Glut-1, CD5, CD117); loss of expression (BAP1, mTAP) was considered if essentially all tumor cells were negative; TdT was recorded as thymocytes present or absent (including rare thymocytes). Results: 81 specimens included 44 thymomas (25 type A, 11 type B3, 8 MNTLS) and 37 thymic carcinomas (including 24 squamous cell carcinomas). Using BAP1, mTAP, CD117 (cut-off, 10%), and TdT, 88.9% of thymic carcinomas (95.7% of squamous cell carcinomas) and 77.8% of thymomas could be predicted. Glut-1 expression was not found to be useful in that distinction. All tumors that expressed CD5 in ≥50% of tumor cells also expressed CD117 in ≥10% of tumor cells. In four carcinomas with homozygous deletion of CDKN2A, mTAP expression was lost in two squamous cell carcinomas and in a subset of tumor cells of an adenocarcinoma and was preserved in a lymphoepithelial carcinoma. Conclusion: A panel of immunostains including BAP1, mTAP, CD117 (using a cut-off of 10% tumor cell expression), and TdT can be useful in the distinction between thymomas and thymic carcinomas, with only a minority of cases being inconclusive.