Break-apart Fluorescence In Situ Hybridization Research Articles

Renal epithelioid angiomyolipomas overexpress TFE3 and the TFE3-regulated gene TRIM63 in the absence of TFE3 rearrangement.

Angiomyolipoma (AML) is a neoplasm within the perivascular epithelioid cell tumor family that occurs somewhat frequently in the kidney. Most are indolent and discovered incidentally, with rare tumors demonstrating malignant clinical behavior. A small subset of renal AMLs withepithelioid features are associated with aggressive behavior, and may demonstrate morphologic overlap with renal cell carcinomas (e.g., clear cell renal cell carcinoma (RCC), TFE3-rearranged RCC). Prior studies of spindle cell and epithelioid AMLs have identified rare examples with underlying TFE3 gene fusions. TFE3 protein expression (demonstrated by immunohistochemistry) with no evidence of concurrent TFE3 rearrangements has been reported previously in 4/24 AMLs (17%) (Argani et al. Am J Surg Pathol 34:1395-1406, 2010). Currently, the relationship between TFE3 protein expression, TFE3 fusions, and expression of TFE3-mediated genes remains incompletely understood in renal epithelioid AMLs. We sought to explore these relationships using TFE3 break-apart fluorescence in situ hybridization (FISH) and TRIM63 RNA in situ hybridization (ISH) on epithelioid AMLs with moderate to strong TFE3 expression by immunohistochemistry. RNA sequencing (fusion panel) was performed on two cases with negative FISH results to assess for FISH-cryptic gene fusions. The series comprised five epithelioid AMLs from four patients (three women, one man) aged 13 to 76years. Allwere considered positive for TFE3by immunohistochemistry (2 + /3 + expression). TRIM63 ISH was performed on four specimens from three patients, yielding positive results in 3/3 tumors(100%) that were successfully analyzed. TFE3 break-apart FISH was performed on all samples, demonstrating a TFE3 rearrangement in only 1/4 tumors (25%). RNA sequencing demonstrated the absence of productive TFE3 gene fusions in three tumors with negative break-apart TFE3 FISH results. This study demonstrates that renal epithelioid AMLs overexpress TFE3 and TFE3-mediated genes (TRIM63) even in the absence of TFE3 rearrangements. This finding could be explained byfunctional upregulation of TFE3secondary to activation of themammalian target of rapamycin complex 1 (mTORC1). Expression of TFE3 and TRIM63 in this tumor type represents a potential pitfall, given the morphologic and immunophenotypic overlap between epithelioid AML and TFE3-alteredrenal cell carcinoma.

ZMIZ1::ABL1 Fusion: An Uncommon Molecular Event With Clinical Implications in Pediatric Cancer.

Pediatric B-cell acute lymphoblastic leukemia is genetically and phenotypically heterogeneous, with a genetic landscape including chromosomal translocations that disrupt ABL proto-oncogene 1, non-receptor tyrosine kinase (ABL1). To characterize an uncommon chromosomal translocation in acute leukemia. Genetic testing, including karyotype and fluorescence in situ hybridization (FISH) analysis, was used to determine the underlying genetic aberration driving the disorder and to guide disease classification and risk stratification. More-detailed testing using RNA sequencing was performed, based on the results from these assays. Three-dimensional molecular modeling was used to visualize the impact of aberrant fused transcripts identified by transcriptome profiling. Karyotype analysis of the bone marrow demonstrated a complex karyotype with, most notably, a t(9;10)(q34.1;q22) translocation. ABL1 break-apart probe FISH findings supported ABL1 disruption. Bone marrow transcriptome analysis revealed mutant ZMIZ1::ABL1 (ZMIZ1, zinc finger MIZ-type containing 1) fusion transcripts as a consequence of t(9;10)(q34.1;q22). Three-dimensional modeling of the mutant ZMIZ1::ABL1 fusion protein confirmed an altered ABL1 protein structure compared to that of the wild type, suggesting a constitutively active conformation. The t(9;10) translocation resulting in ZMIZ1::ABL1 fusion transcripts is an uncommon form of BCR::ABL1-like (BCR, BCR activator of RhoGEF and GTPase) acute lymphoblastic leukemia. Although the karyotype was complex, identifying the t(9;10)(q34.1;q22) translocation, ABL1 disruption, and ZMIZ1::ABL1 transcript enabled effective ABL1-targeted treatment. Our data support the use of tyrosine kinase inhibitors to treat ZMIZ1::ABL1-derived B-cell acute lymphoblastic leukemia.

RET Fusion Testing in Patients With NSCLC: The RETING Study

IntroductionRET inhibitors with impressive overall response rates are now available for patients with NSCLC, yet the identification of RET fusions remains a difficult challenge. Most guidelines encourage the upfront use of next-generation sequencing (NGS), or alternatively, fluorescence in situ hybridization (FISH) or reverse transcriptase-polymerase chain reaction (RT-PCR) when NGS is not possible or available. Taken together, the suboptimal performance of single-analyte assays to detect RET fusions, although consistent with the notion of encouraging universal NGS, is currently widening some of the clinical practice gaps in the implementation of predictive biomarkers in patients with advanced NSCLC. MethodsThis situation prompted us to evaluate several RET assays in a large multicenter cohort of RET fusion–positive NSCLC (n = 38) to obtain real-world data. In addition to RNA-based NGS (the criterion standard method), all positive specimens underwent break-apart RET FISH with two different assays and were also tested by an RT-PCR assay. ResultsThe most common RET partners were KIF5B (78.9%), followed by CCDC6 (15.8%). The two RET NGS-positive but FISH-negative samples contained a KIF5B(15)-RET(12) fusion. The three RET fusions not identified with RT-PCR were AKAP13(35)-RET(12), KIF5B(24)-RET(9) and KIF5B(24)-RET(11). All three false-negative RT-PCR cases were FISH-positive, exhibited a typical break-apart pattern, and contained a very high number of positive tumor cells with both FISH assays. Signet ring cells, psammoma bodies, and pleomorphic features were frequently observed (in 34.2%, 39.5%, and 39.5% of tumors, respectively). ConclusionsIn-depth knowledge of the advantages and disadvantages of the different RET testing methodologies could help clinical and molecular tumor boards implement and maintain sensible algorithms for the rapid and effective detection of RET fusions in patients with NSCLC. The likelihood of RET false-negative results with both FISH and RT-PCR reinforces the need for upfront NGS in patients with NSCLC.

Clinicopathological and molecular features of metaplastic thymoma

Objective: To investigate the clinicopathological features, and molecular genetic alterations of metaplastic thymoma (MT). Methods: A total of ten MT cases, diagnosed from 2011 to 2021, were selected from the Department of Pathology of Jinling Hospital, Nanjing University Medical School, Nanjing, China for clinicopathological and immunohistochemical (IHC) examination and clinical follow-up. Fluorescence in situ hybridization (FISH), next-generation sequencing (NGS), and YAP1 C-terminus (YAP1-CT) IHC were performed to detect YAP1::MAML2 fusions. Results: There were four males and six females, ranging in age from 29 to 60 years (mean 50 years, median 54 years). Microscopically, all tumors showed a typical biphasic morphology consisting of epithelial components and gradually or abruptly transitioning spindle cell components. The two components were present in varying proportions in different cases. Immunophenotypically, the epithelial cells were diffusely positive for CKpan, CK5/6 and p63. The spindle cells were diffusely positive for vimentin and focally positive for EMA. TdT was negative in the background lymphocytes. Ki-67 proliferation index was less than 5%. YAP1 and MAML2 break-apart FISH analyses showed that all ten cases had narrow split signals with a distance of nearly 2 signal diameters and may be considered false-negative. Using YAP1::MAML2 fusion FISH assays, abnormal fusion signals were observed in all the ten cases. NGS demonstrated YAP1::MAML2 fusions in all eight cases with adequate nucleic acids; in two cases the fusions were detected by DNA sequencing and in eight cases by RNA sequencing. All ten cases of MT demonstrated loss of YAP1 C-terminal expression in epithelioid cells. Conclusions: MT is a rare and low-grade thymic tumor characterized by a biphasic pattern and YAP1::MAML2 fusions. Break-apart FISH assays may sometimes show false-negative results due to the proximity of YAP1 and MAML2, while YAP1 C-terminal IHC is a highly sensitive and specific marker for MT. Loss of YAP1 C-terminal expression can also be used to screen YAP1::MAML2 fusions for possible MT cases.

Deep and Durable Cytogenetic and Molecular Responses with Pemigatinib in Myeloid/Lymphoid Neoplasms with Fibroblast Growth Factor Receptor 1 Rearrangement: The Fight-203 Study

Introduction:Myeloid/lymphoid neoplasmswith fibroblast growth factor receptor 1 rearrangement (MLN FGFR1) are aggressive hematologic neoplasms caused by various reciprocal translocations involving chromosome band 8p11, resulting in tyrosine kinase fusion genes (eg, ZMYM2::FGFR1 from t[8;13][p11;q12] or BCR::FGFR1 from t[8;22][p11;q11]) and constitutive FGFR1 activationand downstream signaling. Pemigatinib is a potent and selective oral inhibitor of FGFR1-3 approved for the treatment of adults with relapsed or refractory MLN FGFR1. Efficacy and safety data for 41 patients with MLN FGFR1 in the ongoing FIGHT-203 study of pemigatinib were previously reported. Median daily pemigatinib dose was 9.5 mg; treatment-emergent adverse events led to dose interruptions in 66% of patients. Rates of complete response (CR) among the 38 patients evaluable for clinical response and rates of complete cytogenetic response (CCyR) for the 40 patients evaluable for cytogenetic response were 74% and 70%, respectively. CR and CCyR occurred in >80% of patients with chronic phase disease and in >50% of patients with blast phase disease. Here we present an analysis of centralized fluorescence in situ hybridization (FISH) evaluation of bone marrow (BM) cells and serial characterization of levels of FGFR1 fusion transcripts in this same cohort. Methods: FIGHT-203 (NCT03011372) is an ongoing open-label, multicenter, phase 2 study evaluating pemigatinib in patients ≥18 years old with MLN FGFR1 and ≥1 prior therapy. The starting dose was pemigatinib 13.5 mg daily (2 weeks on, 1 week off). Following protocol amendments, patients without prior therapy were eligible to enroll, and the dosing regimen was changed to a continuous schedule. Patients were censored for cytogenetic and molecular follow-up at the time of transplantation. CCyR was defined as 0 metaphases positive for the respective reciprocal translocation in ≥20 evaluated BM metaphases on local and/or central karyotyping or 0 cells (or not exceeding the lower level of detection of the probe) with the FGFR1 rearrangement in ≥200 BM cells on local and/or central break-apart FISH assays (central FISH prioritized). As part of the translational research plan, fusion partner genes were identified with next-generation sequencing (NGS; FusionPlex, Archer, Boulder, CO, USA) and targeted PCR profiling of RNA extracted from whole blood; droplet digital PCR (ddPCR) assays were developed to monitor the frequencies of specific FGFR1 fusion transcripts in RNA extracted from whole blood samples (estimated limit of detection, 0.05 copies/μL). Results: As of June 30, 2021, 39 of 41 enrolled and treated patients were evaluable for translational analyses. Among the 39 patients with whole blood samples, FGFR1 fusion partner genes identified on translational testing were ZMYM2 (n=17), BCR (n=11), FGFR1OP (n=4), TPR (n=3), TRIM24 (n=2), and C14orf93, CCDC6, CNTRL and ETV6 (n=1 each). Two of 39 patients had 2 fusions detected ( ZMYM2 and BCR, n=1; ZMYM2 and TPR, n=1). C14orf93, CCDC6 and ETV6 have not been previously identified as FGFR1 fusion partner genes. Central FISH assessment of BM cells confirmed FGFR1-aberrantcells were reduced to undetectable levels in responding patients but rebounded in some patients during periods of treatment interruption (Figure A). Serial ddPCR of whole blood samples demonstrated marked reductions in FGFR1 fusion transcripts for the 34 patients with ≥3 measures while on treatment (Figure B). Among patients with CR, 81.0% had a >2-log reduction and 47.6% had a >3-log reduction in detectable FGFR1 fusion transcripts. Smaller reductions in FGFR1 fusion transcripts were observed in patients with stable disease. Changes in FGFR1 fusion transcripts correlated with FGFR1 FISH results (Pearson coefficient = 0.81) and reflected changes in treatment intensity. Conclusion:Several new FGFR1 fusion partner genes, including C14orf93, CCDC6, and ETV6 were identified with NGS and PCR profiling. Pemigatinib treatment resulted in marked decreases in the percentages of cells with the FGFR1 rearrangement on FISH, as well as 2-3 log reductions in FGFR1 fusion transcripts in patients with MLN FGFR1. Further investigations are ongoing to determine the relationship between pemigatinib dose intensity and the depth and durability of molecular response.

Comprehensive Molecular Characterization of Polymorphous Adenocarcinoma, Cribriform Subtype: Identifying Novel Fusions and Fusion Partners

Polymorphous adenocarcinoma (PAC) is a common, usually low-grade salivary gland carcinoma. While conventional PACs are most associated with PRKD1 p.E710D hotspot mutations, the cribriform subtype is often associated with gene fusions in PRKD1, PRKD2, or PRKD3. These fusions have been primarily identified by fluorescence in situ hybridization (FISH) analysis, with a minority evaluated by next-generation sequencing (NGS). Many of the reported fusions were detected by break-apart FISH probes and therefore have unknown partners or were negative by FISH altogether. In this study, we aimed to further characterize the fusions associated with PAC with NGS. Fifty-four PACs (exclusively cribriform and mixed/intermediate types to enrich the study for fusion-positive cases) were identified and subjected to NGS. Fifty-one cases were successfully sequenced, 28 of which demonstrated gene fusions involving PRKD1, PRKD2, or PRKD3. There were 10 cases with the PRKD1 p.E710D mutation. We identified a diverse group of fusion partners, including 13 novel partners, 3 of which were recurrent. The most common partners for the PRKD genes were ARID1A and ARID1B. The wide variety of involved genes is unlike in other salivary gland malignancies and warrants a broader strategy of sequencing for molecular confirmation for particularly challenging cases, as our NGS study shows.

Loss of YAP1 C-terminus expression as an ancillary marker for metaplastic thymoma: a potential pitfall in detecting YAP1::MAML2 gene rearrangement.

Metaplastic thymoma is a rare thymic tumour characterized by Yes Associated Protein 1 (YAP1) and Mastermind Like Transcriptional Coactivator 2 (MAML2) gene fusions resulting from an intrachromosomal inversion of chromosome 11. Immunohistochemistry with an antibody directed against the C-terminus of YAP1 has shown loss of expression inYAP1-rearranged vascular neoplasms, poromas, and porocarcinomas. This study aimed to validate ananti-YAP1 C-terminal antibody as an ancillary immunohistochemical marker for the diagnosis of metaplastic thymoma. Ten metaplastic thymomas were selected for the current study. Fluorescence in situ hybridization (FISH), next-generation sequencing (NGS), and reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed to detect YAP1::MAML2 fusions. We then performed immunohistochemistry to detect YAP1 C-terminus expression in 10 metaplastic thymomas, 50 conventional thymomas (10 each of type A thymoma, type AB thymoma, type B1 thymoma, type B2 thymoma, and type B3 thymoma) and seven thymic carcinomas. All 10 cases showed narrow split signals with a distance of nearly two signal diameters and sometimes had false-negative results in YAP1 and MAML2 break-apart FISH (BA-FISH). Abnormal colocalized signals of the YAP1::MAML2 fusion were observed in all 10 cases using fusion FISH (F-FISH) assays. Eight of 10 cases with adequate nucleic acids were successfully sequenced and all showed YAP1::MAML2 fusions; in two cases the fusions were detected by both DNA and RNA sequencing and in six cases by RNA sequencing only. YAP1::MAML2 fusion transcripts were identified in four cases by RT-PCR. Metaplastic thymoma showed loss of YAP1 C-terminus expression in all 10 (100%) cases. All other thymic neoplasms showed retained YAP1 C-terminus expression. YAP1 C-terminus immunohistochemistry is a highly sensitive and specific ancillary marker that distinguishes metaplastic thymoma from its mimics. BA-FISH assays could not effectively detect YAP1::MAML2 fusions due to the proximity of the two genes. Loss of YAP1 C-terminus expression is a reliable surrogate for the detection of YAP1::MAML2 fusions in metaplastic thymoma.

Cryptic KMT2A/MLLT10 fusion detected by next-generation sequencing in a case of pediatric acute megakaryoblastic leukemia

KMT2A (11q23.3) gene rearrangements are found in acute leukemia and are associated with a poor or intermediate prognosis. MLLT10 is the fourth most common gene fusion partner for KMT2A. A reciprocal translocation t(10;11) is insufficient to produce an in-frame KMT2A/MLLT10 fusion, because the genes involved in the rearrangement have opposite transcriptional orientations. In order to bring KMT2A and MLLT10 into juxtaposition, complex rearrangements are required. Until now, conventional chromosome, fluorescence in situ hybridization (FISH), and reverse transcriptase-polymerase chain reaction (RT-PCR) studies have been used to detect KMT2A/MLLT10 fusions. However, conventional studies have limitations, such as poor and inconsistent resolution, when compared to next-generation sequencing (NGS). In this study, we report a pediatric patient with acute megakaryoblastic leukemia, in whom the cryptic KMT2A/MLLT10 fusion was not detected by KMT2A break-apart probe FISH and chromosome analysis, but detected by NGS. In this patient, NGS showed cryptic insertion of MLLT10 exons 9-24 into intron 9 of KMT2A, resulting in a KMT2A/MLLT10 fusion. Therefore, NGS is a valuable complementary option for the evaluation of structural aberrations, especially those with a cryptic size.

Elaboration of NTRK-rearranged colorectal cancer: Integration of immunoreactivity pattern, cytogenetic identity, and rearrangement variant

Fused information from protein status, DNA breakage, and transcripts are still limited because of the low rate of activated-NTRK in colorectal cancer (CRC). In total, 104 archived CRC tissue samples with dMMR were analyzed using immunohistochemistry (IHC), polymerase chain reaction (PCR), and pyrosequencing to mine the NTRK-enriched CRC group, and then subjected to NTRK fusion detection using pan-tyrosine kinase IHC, fluorescence in situ hybridization (FISH), and DNA-/RNA-based next generation sequencing (NGS) assays. Of the 15 NTRK-enriched CRCs, eight NTRK fusions (53.3%, 8/15), including two TPM3(e7)-NTRK1(e10), one TPM3(e5)-NTRK1(e11), one LMNA(e10)-NTRK1(e10), two EML4(e2)-NTRK3(e14), and two ETV6(e5)-NTRK3(e15) fusions, were identified. There was no immunoreactivity for ETV6-NTRK3 fusion. In addition to cytoplasmic staining found in six specimens, membrane positive (TPM3-NTRK1 fusion) and nuclear positive (LMNA-NTRK1 fusion) were also observed in two of them. Atypical FISH-positive types were observed in four cases. Unlike IHC, NTRK-rearranged tumors appeared homogeneous on FISH. ETV6-NTRK3 may be missed in pan-TRK IHC screening for CRC. Regarding break-apart FISH, NTRK detection is difficult because of the diversity of signal patterns. Further research is warranted to identify the characteristics of NTRK-fusion CRCs.

Optimising biomarkers for accurate ependymoma diagnosis, prognostication and stratification within International Clinical Trials: A BIOMECA study.

Accurate identification of brain tumour molecular subgroups is increasingly important. We aimed to establish the most accurate and reproducible ependymoma subgroup biomarker detection techniques, across 147 cases from International Society of Pediatric Oncology (SIOP) Ependymoma II trial participants, enrolled in the pan-European "Biomarkers of Ependymoma in Children and Adolescents (BIOMECA)" study. Across six European BIOMECA laboratories we evaluated epigenetic profiling (DNA methylation array); immunohistochemistry (IHC) for nuclear p65-RELA, H3K27me3, and Tenascin-C; copy number analysis via FISH and MLPA (1q, CDKN2A), and MIP and DNA methylation array (genome-wide copy number evaluation); analysis of ZFTA- and YAP1-fusions by RT-PCR and sequencing, Nanostring and break-apart FISH. DNA Methylation profiling classified 65.3% (n=96/147) of cases as EPN-PFA and 15% (n=22/147) as ST-ZFTA fusion-positive. Immunohistochemical loss of H3K27me3 was a reproducible and accurate surrogate marker for EPN-PFA (sensitivity 99-100% across three centres). IHC for p65-RELA, FISH, and RNA-based analyses effectively identified ZFTA- and YAP1- fused supratentorial ependymomas. Detection of 1q gain using FISH exhibited only 57% inter-centre concordance and low sensitivity and specificity whilst MIP, MLPA and DNA methylation-based approaches demonstrated greater accuracy. We confirm, in a prospective trial cohort, that H3K27me3 immunohistochemistry is a robust EPN-PFA biomarker. Tenascin-C should be abandoned as a PFA marker. DNA methylation and MIP arrays are effective tools for copy number analysis of 1q gain, 6q and CDKN2A loss whilst FISH is inadequate. Fusion detection was successful, but rare novel fusions need more extensive technologies. Finally, we propose test sets to guide future diagnostic approaches.

Integrated Genomic DNA/RNA Profiling vs Fluorescence in Situ Hybridization in the Detection of MYC and BCL2 (and BCL6) Rearrangements in Large B-Cell Lymphomas: Updates Amid the New WHO Classification of Lymphoid Neoplasms.

Large B-cell lymphomas (LBCLs) are a heterogeneous group of lymphoid neoplasms whose molecular and cytogenetic profile has predictive and prognostic implications. The concept of double-hit lymphomas (DHLs) was recently updated in the fifth edition of the World Health Organization classification, with the exclusion of MYC and BCL6 rearranged tumors from the group. Now, DHLs are referred to as diffuse large B-cell lymphoma/high-grade B-cell lymphoma with MYC and BCL2 rearrangements. Fluorescence in situ hybridization (FISH) is the current gold standard for detecting rearrangements in LBCLs, but comprehensive genomic profiling (CGP) has recently been suggested to be at least as accurate as FISH in classifying these neoplasms and providing additional genetic information. We analyzed a cohort of 131 patients in whom FISH and CGP studies were performed as part of our normal clinical workflow and compared the effectiveness of FISH and CGP in detecting these clinically relevant rearrangements. Our findings are in agreement with our previously published study, which analyzed a cohort of 69 patients, supporting our hypothesis that the best approach to maximize detection of DHLs while limiting waste seems to be a combination of CGP and MYC break-apart FISH testing, the latter to capture the presence of non-IGH::MYC events. Our study supports the combined use of FISH and GCP rather than either method alone to better detect MYC and BCL2 (and BCL6) gene rearrangements.

Cryptic COL1A1-PDGFB fusion in dermatofibrosarcoma protuberans: a clinicopathological and genetic analysis

Objective: To investigate the clinicopathological and cytogenetic features of cryptic COL1A1-PDGFB fusion dermatofibrosarcoma protuberans (CC-DFSP). Methods: Three cases of CC-DFSP diagnosed in West China Hospital, Sichuan University, Chengdu, China from January 2021 to September 2021 were studied. Immunohistochemistry for CD34 and other markers, fluorescence in situ hybridization (FISH) for PDGFB, COL1A1-PDGFB and COL1A1, next-generation sequencing (NGS), reverse-transcriptase polymerase chain reaction (RT-PCR) and Sanger sequencing were performed. Results: There were three cases of CC-DFSP, including two females and one male. The patients were 29, 44 and 32 years old, respectively. The sites were abdominal wall, caruncle and scapula. Microscopically, they were poorly circumscribed. The spindle cells of the tumors infiltrated into the whole dermis or subcutaneous tissues, typically arranging in a storiform pattern. Immunohistochemically, the neoplastic cells exhibited diffuse CD34 expression, but were negative for S-100, SMA, and Myogenin. Loss of H3K27me3 was not observed in the tumor cells. The Ki-67 index was 10%-15%. The 3 cases were all negative for PDGFB rearrangement and COL1A1-PDGFB fusion, whereas showing unbalanced rearrangement for COL1A1. Case 1 showed a COL1A1 (exon 31)-PDGFB (exon 2) fusion using NGS, which was further validated through RT-PCR and Sanger sequencing. All patients underwent extended surgical resection. Except for case 3 with recurrence 2 years after surgical resection, the other 2 cases showed no recurrence or metastasis during the follow-up. Conclusions: FISH has shown its validity for detecting PDGFB rearrangement and COL1A1-PDGFB fusion and widely applied in clinical detection. However, for cases with negative routine FISH screening that were highly suspicious for DFSPs, supplementary NGS or at least COL1A1 break-apart FISH screening could be helpful to identify cryptic COL1A1-PDGFB fusions or other variant fusions.

Unilateral Primary Breast Dermatofibrosarcoma Protuberans with Negative COL1A1-PDGFB Fusion on Fluorescence In Situ Hybridization

Abstract Introduction/Objective Dermatofibrosarcoma protuberans (DFSP) is a superficial and locally aggressive cutaneous soft tissue sarcoma. In most patients, DFSP arises from the trunk and proximal extremities with breast being an uncommon location. Although they rarely metastasize (<5%), DFSP variants tend to recur locally. Most molecularly characterized cases have been defined by the translocation t(17;22)(q21.3;q13.1), which yields a fusion of the COL1A1 and PDGFB genes. In routine practice, dual fusion or break-apart fluorescence in situ hybridization (FISH) for the COL1A1-PDGFB fusion gene is the most widely used screening method for DFSP. However, in ~4% of typical DFSP cases, routine FISH screening is negative. In some of the DFSP cases negative for COL1A1-PDGFB fusion gene by FISH, RNA sequencing has identified two novel gene fusions, COL6A3-PDGFD and EMILIN2-PDGFD, with the former having a predilection for the female breast. Methods/Case Report We present a 32-year-old woman who underwent a biopsy for a 3.4 cm echogenic firm mass in her right breast. Histology showed a cytologically-uniform spindle cell neoplasm with fibroblastic morphology. The neoplastic cells displayed a storiform growth pattern and honeycomb-like infiltration of the adipose tissue. Immunohistochemically, the neoplastic cells expressed CD34 and vimentin and were negative for AE1/AE3, p63, S100, CD31, SMA, β-catenin, and STAT6. PDGFB break-apart FISH was negative. Based on the histologic and immunohistochemical findings, a diagnosis of DFSP was rendered. FISH for PDFGD rearrangements or RNA sequencing targeting PDGFD was not performed at this time. Results (if a Case Study enter NA) NA. Conclusion Awareness is warranted for the rare variant of DSFP with a predilection for female breast, which is characterized by a novel gene fusion not detectable by routine FISH used for DFSP screening.

23. Clinical and diagnostic challenges of atypical findings during fluorescence in situ hybridization (FISH) testing: CBFB breakapart FISH as an example

Fluorescence in situ hybridization (FISH) has been widely used as a diagnostic and/or prognostic tool in the field of clinical laboratory diagnosis, especially in field of hematologic malignancies. However, atypical findings that are less or not encountered during an assay validation can pose diagnostic challenges. In this presentation, we'd like to share our experience using CBFB breakapart FISH as an example. From January 1, 2000 to May 31, 2021, a total of 2809 CBFB Breakapart FISH tests were performed in 1629 cases with acute myeloid leukemia (AML) in our institute, and 16.1% (n=262), 75.7% (n=1234) and 8.2% (n=133) cases were reported as positive, normal and abnormal respectively.

Categorization of cutaneous epithelioid angiomatous nodule as epithelioid hemangioma or angiolymphoid hyperplasia with eosinophilia: Clinicopathologic, immunohistochemical, and molecular analyses of seven lesions.

The status of cutaneous epithelioid angiomatous nodule (CEAN) as a distinct entity remains controversial. This study investigated the relationship between CEAN and epithelioid hemangioma/angiolymphoid hyperplasia with eosinophilia (ALHE). Data of seven lesions with CEAN features from four cases (Cases 1-4: 61-year-old, 76-year-old, 53-year-old, and 21-year-old men, respectively) were investigated. Cases 1 and 2 showed multiple lesions in the head and neck region, but Cases 3 and 4 showed solitary lesions on the back and scalp, respectively. Moreover, the histopathologic findings of the lesions of Cases 1 and 2 were consistent with those of conventional epithelioid hemangioma or classic cutaneous ALHE. Diffuse immunoexpression of FOSB was observed in Cases 1 and 2, but FOSB split signals were absent in break-apart fluorescence in situ hybridization (FISH). In contrast, the histopathologic findings of the lesions of Cases 3 and 4 were consistent with those of cellular-type epithelioid hemangiomas. Diffuse immunoreactivity for c-FOS was observed in Cases 3 and 4, and split signals of FOS were present in break-apart FISH in Case 3. This study showed that the seven tumors with CEAN features could be reclassified under the epithelioid hemangioma/ALHE group, although the small sample size is a limitation.

Identification of novel SSX1 fusions in synovial sarcoma

Synovial sarcoma is characterized by variable epithelial differentiation and specific SS18-SSX gene fusions. The diagnosis is primarily based on phenotype, but fusion gene detection is increasingly being considered indispensable, with SS18 break-apart fluorescence in situ hybridization (FISH) being favored in many laboratories. However, SS18 FISH assay produces negative or atypical results in a minority of cases, leaving uncertainties in diagnosis and management. Here, we analyzed this challenging subset of SS18 FISH-negative/atypical synovial sarcoma using RNA sequencing and monoclonal antibodies that recognize SS18-SSX and the SSX C-terminus. Among 99 synovial sarcoma cases that were previously subjected to SS18 break-apart FISH, eight cases were reported as negative and three cases were indeterminate, owing to atypical signal patterns. Three of these 11 tumors (two monophasic and one biphasic) harbored novel EWSR1-SSX1 fusions, were negative for SS18-SSX staining, and were positive for SSX C-terminus staining. One monophasic tumor harbored a novel MN1-SSX1 fusion, and showed negative SS18-SSX expression and positive SSX C-terminus staining. Another monophasic tumor carried an SS18L1-SSX1 fusion, and was weakly positive for SS18-SSX, while SMARCB1 expression was reduced. The presence of these novel and/or rare fusions was confirmed using RT-PCR and Sanger sequencing. EWSR1-SSX1 was further validated by EWSR1 FISH assay. The remaining six tumors (five monophasic and one biphasic) showed strong SS18-SSX expression, and RNA sequencing successfully performed in three cases identified canonical SS18-SSX2 fusions. Based on a DNA methylation-based unsupervised clustering, the tumors with EWSR1-SSX1 and SS18L1-SSX1 clustered with synovial sarcoma, while the MN1-SSX1-positive tumor was not co-clustered despite classic histology and immunoprofile. In summary, we discovered novel and rare SSX1 fusions to non-SS18 genes in synovial sarcoma. The expanded genetic landscape carries significant diagnostic implications and advances our understanding of the oncogenic mechanism.

Molecular Characterization of Dermatofibrosarcoma Protuberans: The Clinicopathologic Significance of Uncommon Fusion Gene Rearrangements and Their Diagnostic Importance in the Exclusively Subcutaneous and Circumscribed Lesions.

The clinicopathologic relevance of various gene rearrangements underlying dermatofibrosarcoma protuberans (DFSP) remains insufficiently characterized. In 188 DFSPs, we determined PDGFB, COL1A1, PDGFD, COL6A3, and EMILIN2 rearrangements by fluorescence in situ hybridization (FISH). The clinicopathologic significance of rearrangement types and factors related to recurrence and metastasis were statistically analyzed. In all, classic PDGFB rearrangement, cryptic COL1A1-PDGFB fusion, and PDGFD rearrangement were identified in 172 (91.4%), 8 (4.3%), and 8 (4.3%: 4 COL6A3-PDFGD, 4 EMILIN2-PDGFD) cases, respectively. In an index DFSP harboring the cryptic fusion, the COL1A1-PDGFB transcript was confirmed by both RNA sequencing and reverse transcription-polymerase chain reaction. In comparison with cases harboring classic PDGFB rearrangement, cryptic PDGFB-rearranged DFSPs usually exhibited higher 5'-COL1A1 copy numbers. In a combined reappraisal of published and current cases, COL6A3-PDGFD-positive DFSPs (n=16) predominated in females (n=14, 88%) and torso (n=14, 88%), especially the breast (n=7, 44%); EMILIN2-PDGFD-positive DFSPs (n=6) preferentially demonstrated near exclusively subcutaneous growth (n=5, 83%) and fibrosarcomatous transformation (n=5, 83%). In our cohort, local recurrence was related to fibrosarcomatous variant (P=0.029, odds ratio=3.478) and head and neck location (P=0.046, odds ratio=3.508). Distant metastasis only occurred in the fibrosarcomatous variant (9/73, 12.3%) but not in other cases. In conclusion, 8.6% of DFSPs are negative for PDGFB break-apart FISH, which, especially those with challenging subcutaneous and circumscribed manifestation, require complementary diagnosis by FISH assays targeting COL1A1 and PDGFD. The types of fusion gene rearrangements, head and neck location, and fibrosarcomatous transformation may account for clinicopathologic and prognostic variations in DFSPs and warrant future independent validation.

Myeloid/Lymphoid Neoplasm with Eosinophilia and a Novel RUFY1-Pdgfrb Rearrangement

▪Myeloid/lymphoid neoplasms associated with eosinophilia (MLN-Eo) and tyrosine kinase fusion genes include rearrangements of PDGFRB with over 30 different partner genes, most commonly ETV6-PDGFRB. Rearrangements are evident by cytogenetic analysis and fluorescence in situ hybridization (FISH) break-apart probes. Herein, we describe a novel rearrangement partner for PDGFRB.A 25-year-old man with a medical history significant for gout and hypertension presented to medical care after several days of progressive dyspnea, chest pain, fatigue, epistaxis, and rash. Physical exam revealed palpable cervical, axillary, and inguinal lymphadenopathy and massive splenomegaly. By CT scan the spleen measured 25 cm and diffuse lymphadenopathy was identified in the chest, abdomen and pelvis with the largest lymph node mass measuring 8.9 x 2.5 cm. Transthoracic echocardiogram showed normal heart function and no evidence of myocardial injury. The patient subsequently developed respiratory failure requiring intubation and mechanical ventilation, shock requiring vasopressors, and renal failure requiring renal replacement therapy. Initial laboratory evaluation demonstrated a leukocytosis to 83.3 x 10 9/L with neutrophilic predominance with left shift and eosinophilia (10% of WBCs, absolute eosinophil count of 8.2 x 10 9/L); hemoglobin of 8.3 g/dL and hematocrit of 25%; and thrombocytopenia to 50 x 10 9/L. Ferritin was 660 ng/mL, lactate dehydrogenase 384 U/L, and uric acid 10.4 mg/dL. Secondary causes of eosinophilia including infection, immunodeficiency, atopy, medications, and rheumatologic disease were ruled out. The patient was positive for strongyloides IgG antibodies and received ivermectin.Core biopsy of a lymph node was non-diagnostic and could not repeated safely due to progressive severe thrombocytopenia. Skin biopsy demonstrated a perivascular leukocytoclasis, but no abnormal B or T cell population. Bone marrow aspirate and biopsy revealed a hypercellular marrow (90%) with mild trilineage dysplasia, granulocytic hyperplasia, moderate reticulin fibrosis, and slightly increased mast cells. Blasts were not increased and no hemophagocytic histiocytes were identified. Eosinophils accounted for 6.9% of myeloid cells. Flow cytometry did not reveal an abnormal B or T lymphoid, blast or myeloid population.Peripheral blood flow cytometry did not reveal an abnormal B or T-cell population and T-cell rearrangement studies showed an oligoclonal population. No clonal B cell population was identified.FISH, which identified a rearrangement of PDGFRB in 28% of cells, confirmed the diagnosis. Karyotype analysis revealed an abnormal male karyotype with a paracentric inversion of 5q with breakpoints at 5q32 and 5q35. FusionPlex® Solid Tumor Panel (Clinical Genomics Laboratory, University of Washington SOM) identified a novel fusion between RUFY1 and PDGFRB. RUFY1 is expressed in most tissues and encodes a protein that contains a RUN domain and a FYVE-type zinc finger domain and plays a role in early endosomal trafficking (Figure). We are currently demonstrating the fusion is oncogenic and sensitive to tyrosine kinase inhibition in Ba/F3 cells. The patient's remarkable clinical response, however, supports imatinib sensitivity and is in keeping with other published reports of MLN-Eo with PDGFRB rearrangements. Initial treatment included high dose steroids for 7 days and hydroxyurea briefly. Imatinib 400 mg daily was started, then decreased to 200 mg daily due to gastrointestinal toxicity. Follow-up is limited (8 weeks). However, the patient has recovered clinically. Six weeks after starting imatinib he had renal recovery and has achieved a complete hematologic response except for mild anemia due to resolving acute kidney injury. Splenomegaly and peripheral lymphadenopathy have resolved. PDGFRB FISH has declined to 9%.Inversions of chromosome 5 have very rarely been described in association with eosinophilia, but only one report has specifically identified a rearrangement of PDGFRB. This report described a novel rearrangement involving exons 12 to 23 of PDGFRB at 5q32 and the 5′ (UTR) of G3BP1 at 5q33.1. Notably, this specific rearrangement was undetectable by PDGFRB break-apart FISH. These reports and ours highlight the clinical relevance of NGS in identifying novel rearrangement partners. Furthermore, patient-specific ddPCR assays can be designed for sensitive monitoring. [Display omitted] DisclosuresOehler: BMS: Consultancy; OncLive: Honoraria; Pfizer: Research Funding; Takeda: Consultancy; Blueprint Medicines: Consultancy.

CBFB Break-Apart FISH Testing: An Analysis of 1629 AML Cases with a Focus on Atypical Findings and Their Implications in Clinical Diagnosis and Management.

Simple SummaryThe inv(16)/t(16;16) AML is a disease that is considered relatively easy and straightforward to be diagnosed in the clinical laboratories. Up to date, CBFB FISH and/or CBFB-MYH11 RT-PCR are still the major diagnostic assays utilized in the clinical laboratories. However, incidental CBFB FISH findings and their implication in clinical laboratory diagnostics and management, especially in the era of next-generation sequencing (NGS)-based methods, have not been systemically investigated. In this study, we systemically studied over 1600 AML cases tested with CBFB FISH. Over 5% of cases with a confirmed CBFB rearrangement were challenging, including those with discrepant FISH and RT-PCR results and/or atypical FISH findings. Meanwhile, atypical FISH findings usually indicate additional chromosome 16 aberrations (AC16As) overlooked by other methods including RT-PCR and almost all NGS-based methods if following the published parameters. The information revealed in this study will be useful for further workup and interpreting atypical CBFB FISH findings and confirmation of inv(16)/t(16;16) AML diagnosis and related treatment, as well as selection of samples to better validate NGS-based new diagnostic methods.Fluorescence in situ hybridization (FISH) is a confirmatory test to establish a diagnosis of inv(16)/t(16;16) AML. However, incidental findings and their clinical diagnostic implication have not been systemically studied. We studied 1629 CBFB FISH cases performed in our institution, 262 (16.1%), 1234 (75.7%), and 133 (8.2%) were reported as positive, normal, and abnormal, respectively. The last included CBFB copy number changes (n = 120) and atypical findings such as 3′CBFB deletion (n = 11), 5′CBFB deletion (n = 1), and 5′CBFB gain (n = 1). Correlating with CBFB-MYH11 RT-PCR results, totally 271 CBFB rearrangement cases were identified, including five with discrepancies between FISH and RT-PCR due to new partner genes (n = 3), insertion (n = 1), or rare CBFB-MYH11 variant (n = 1) and eight with 3′CBFB deletion. All cases with atypical findings and/or discrepancies presented clinical diagnostic challenges. Correlating FISH signal patterns and karyotypes, additional chromosome 16 aberrations (AC16As) show impacts on the re-definition of a complex karyotype and prognostic prediction. The CBFB rearrangement but not all AC16As will be detected by NGS-based methods. Therefore, FISH testing is currently still needed to provide a quick and straightforward confirmatory inv(16)/t(16;16) AML diagnosis and additional information related to clinical management.

BRAF Rearrangements and BRAF V600E Mutations Are Seen in a Subset of Pancreatic Carcinomas With Acinar Differentiation.

Comprehensive genomic profiling has demonstrated that approximately 20% of pancreatic carcinomas with acinar differentiation harbor potentially targetable BRAF fusions that activate the MAPK pathway. To validate the above finding by BRAF break-apart fluorescence in situ hybridization (FISH) in a large series of pure acinar cell carcinomas (ACCs), evaluate tumors for the presence of BRAF V600E mutations, and compare clinicopathologic features of tumors with BRAF rearrangements with those without. Thirty cases of pure ACC and 6 cases of mixed acinar-neuroendocrine carcinoma (ACC-NEC) were retrieved. A break-apart FISH probe was used to detect BRAF rearrangements. Immunohistochemistry for BRAF V600E was performed. BRAF rearrangements by FISH were found in 6 of 36 cases (17%), 5 of which were pure ACC and 1 was a mixed ACC-NEC. Follow-up was available in 29 of 36 cases (81%). The median survival was 22 months for BRAF-rearranged cases and 16 months for BRAF-intact cases; the 2-year overall survival was 50% for BRAF-rearranged cases and 35% for BRAF-intact cases. No significant clinicopathologic differences were identified in cases with BRAF rearrangement compared with those without BRAF rearrangement. BRAF V600E mutation was identified in 2 of 34 cases (6%), both of which were pure ACC and were BRAF-intact by FISH. This study supports the finding that BRAF rearrangements are present in approximately 20% of cases and identified BRAF V600E mutations in approximately 5% of cases. These cases may benefit from targeted therapy.