Fluorescence In Situ Hybridization Testing Research Articles

Prognostic implications and diagnostic significance of TFE3 rearrangement in renal cell carcinoma

ObjectivesTo investigate the impact of TFE3 rearrangement, analyzing clinicopathological features that influence renal cell carcinoma (RCC) recurrence, and clarify the role of immunohistochemistry (IHC) staining in diagnosis.MethodsWe screened patients diagnosed of clear cell RCC (ccRCC), fluorescence in situ hybridization (FISH) was performed on all TFE3 positive IHC tumors. Clinicopathological and survival features were collected for analysis.ResultsOut of 695 patients treated for renal tumors, 478 (68.7%) were ccRCC and 22 were suspected of TFE3 rearrangement based on IHC. Subsequent testing revealed 8 (1.15%) were positive in the FISH test (TFE3-rearranged-RCC) and 14 (2.01%) tested negative. No significant differences were noted in general characteristics among the three groups, except for age, TFE3-rearranged-RCC were younger than ccRCC (median age, 49 vs. 58 years, p=0.02). TFE3-rearranged-RCC exhibited a significant higher recurrence rate compared to ccRCC (50% vs 18.8%) and multivariate analysis revealed that TFE3 rearrangement, along with tumor size and metastasis, was an independent prognostic factor for recurrence (HR=4.6; 95% CI 1.1-21.2; p=0.05). Survival analysis demonstrated a significant shorter PFS (progression-free survival) for TFE3-rearranged-RCC compared to ccRCC.ConclusionsTFE3 rearrangement is an independent prognostic factor for recurrence and contributes to a worse PFS, suggesting the necessity of careful follow-up. Diagnosis should be confirmed using FISH due to low specificity of IHC. Further studies are needed to confirm TFE3 IHC staining as a prognostic factor.

Diagnostic Accuracy Performance of Fluorescence In Situ Hybridization (FISH) for Biliary Strictures: A Systematic Review and Meta-Analysis

Background and Aims: This systematic review and meta-analysis aims to compare the performance of UroVysion® FISH based on the different definitions of a positive result used in published literature with the goal of determining the optimal FISH definition for detecting pancreaticobiliary malignancy. Methods: A systematic literature search identified studies from database inception to Sept 2024 that evaluated the diagnostic performance of FISH in determining malignancy among patients with biliary strictures. All thresholds for positive FISH, as defined by the individual study, were included in this review. Subgroup analysis was performed based on the definitions of positive FISH as follows: (1) polysomy only; (2) polysomy, tetrasomy, or trisomy; and (3) polysomy or 9p deletion. Results: Eighteen studies comprising 2516 FISH specimens were analyzed, including 1133 (45.0%) with malignancy. Using a threshold for positivity as defined in individual studies, the overall sensitivity of FISH was 57.6% (95% confidence interval [CI], 49.4–65.4%), and the overall specificity was 87.8% (95% CI, 79.2–93.2%). Subgroup analysis showed that polysomy as the threshold for positive FISH yielded a sensitivity of 49.4% (95% CI, 43.2–55.5%), with an increased specificity of 96.2% (95% CI, 92.7–98.1%), while polysomy + tetrasomy/trisomy as positive FISH resulted in an increased sensitivity of 64.3% (95% CI 55.4–72.2%) but a decreased specificity of 78.9% (95% CI 64.4–88.5%). The addition of 9p deletion to polysomy as the criteria for a positive test resulted in a non-significant increase in sensitivity (54.7% (95% CI 42.4–66.5%) while maintaining specificity (95.1% (95% CI 84.0–98.6%). Conclusions: Based on these findings, polysomy only or polysomy/9p deletion should be considered as the criterion for defining a positive FISH test to improve diagnostic sensitivity while maintaining high specificity.

ROS1 Translocation Detection in NSCLC: A Comparative Study of PGDx and FISH

Abstract In the United States, lung cancer is the leading cause of cancer-related deaths. In recent decades, the advent of molecularly-targeted therapies in the management of non-small-cell lung cancer (NSCLC) has led to significant improvements in patient outcomes. As such, optimum care depends on timely molecular testing. The mutational spectrum associated with NSCLC, however, is complex and riddled with various point mutations, amplifications, and rearrangements/fusions across multiple genes. Comprehensive testing of NSCLC has historically required multiple testing modalities to detect all clinically-relevant molecular targets. At our institution, a combination of immunohistochemistry, fluorescence in situ hybridization (FISH), and sequencing-based tests are performed on all NSCLCs. Though fragmented, this approach ensures testing for all current targetable genetic alterations is performed. Unfortunately, this style of testing is often incompatible with the limited amount of tumor tissue acquired by biopsy. In 2021, our institution implemented PGDx elio tissue complete (PGDx), introducing redundancy in ROS1 translocation testing, previously assessed by FISH through send-out testing to Mayo Clinic. The objective of this study was to perform a retrospective analysis of our molecular testing experience for NSCLC, with a spotlight on the nuances of ROS1 testing. Since its implementation at our institution, PGDx has been performed on 172 cases of NSCLC. As part of our NSCLC reflex testing agreement, ROS1 translocation FISH testing was also attempted on all cases. Of the 172 cases, one clinically actionable ROS1 translocation was identified by PGDx (0.5%). Interestingly, FISH testing for this case was equivocal. The remaining cases were negative for ROS1 translocations. Notably, FISH failed in six cases (3.5%) due to insufficient tissue. Three additional cases (1.7%) had minimal tumor tissue available for FISH testing (less than 300 cells), providing suboptimal results. Altogether, PGDx was successfully performed on all samples and, in the 166 cases where FISH was successful, there was no discordance between orthogonal tests. This supports the validity and reliability of PGDx in identifying clinically relevant rearrangements/fusions in ROS1. In this study, we demonstrate that PGDx can effectively replace FISH testing for ROS1 translocations in NSCLC. In addition to being technically equivalent to FISH for ROS1 with comparable turnaround times, PGDx adds substantial benefits. Replacing FISH testing with PGDx improves the diagnostic workflow, improves tissue utilization, and eliminates the costs associated with send-out orders. The cost of Mayo ROS1 FISH testing averages $520 per sample and this amount is billed to patient insurance, though reimbursements vary. Further, PGDx can detect the ROS1 gene fusion partners, a feature not possible through routine FISH testing in NSCLC. This added clinical information reduces the ambiguity of equivocal FISH results, exemplified by our study. By implementing this change, our institution will optimize both diagnostic practices and resource utilization in the clinical setting.

Molecular Classification of Large B-Cell Lymphoma and High-Grade B-Cell Lymphoma Cases and Association with Outcomes in Morocco

Background: High-grade B-cell lymphoma with c-MYC and BCL2 and/or BCL6 rearrangements (HGBL-DHL/THL) is a recently identified category in the most recent World Health Organization (WHO) classification. For all tumors displaying the appearance of diffuse large B-cell lymphoma (DLBCL) or high-grade B-cell lymphoma (HGBL), it is necessary to perform fluorescence in situ hybridization (FISH) in order to achieve an accurate diagnosis. The findings of FISH and immunohistochemistry (IHC) examinations from 50 DLBCL/HGBL samples obtained from Hassan II University Hospital in Fez/Morocco are reported. Methods: This retrospective study included 50 patients diagnosed with DLBCL/HGBL over a period of nine years (2013–2022) and treated with RCHOP chemotherapy protocol. All patients underwent a histological study followed by an immunohistochemical study to confirm the diagnosis and to classify patients according to cell of origin into non-GCB and GCB subtypes; then, a cytogenetic study using FISH was performed to classify patients according to the presence or absence of rearrangements in the c-MYC, BCL2 and BCL6 genes. A comparison was made between the molecular subtypes of DLBCL/HGBL in relation to clinicopathological features and outcomes. Results: Among the 50 cases studied in our population, we found 5 cases of HGBL with DLBCL morphology and 45 cases of DLBCL, which consisted of 13 cases (28.89%) of GCB subtype and 32 cases (71.11%) of non-GCB subtype based on the immunohistochemistry Hans algorithm. After FISH testing of all cases, we found three cases of double-hit lymphoma (DHL) and one case of triple-hit lymphoma (THL). Thus, HGBL-DHL/THL accounted for 8% of the cases. Furthermore, two cases were detected with only one rearrangement in the BCL2 gene and one case harboring a rearrangement in the BCL6 gene. DHL and THL patients and patients with a single rearrangement (BCL2 or BCL6) have a worse prognosis than patients with no rearrangement. Conclusions: DHL and THL are an aggressive entity of HGBL with poorer outcomes in comparison to DLBCL/HGBL NOS. First-line treatment with the RCHOP chemotherapy protocol may not be effective for all aggressive DLBCL cases. More targeted treatment is crucial for better patient outcomes.

Interim efficacy of a multicenter cohort study for China Net Childhood Lymphoma mature B-cell lymphoma 2017 regimen in the treatment of pediatric High-grade-B cell lymphoma

Objective: To analyze the mid-term efficacy of the China Net Childhood Lymphoma mature B-cell lymphoma 2017 (CNCL-B-NHL-2017) regimen in treating children with high-grade B-cell lymphoma (HGBL). Methods: Clinical and pathological data of HGBL children aged≤18 years admitted to 16 hospitals of the Chinese Children's Lymphoma Collaborative Group (CNCL) from May 2017 to April 2021 were collected retrospectively. They were divided in to high-grade B-cell lymphoma with double hit/triple hit (HGBL-DH/TH) group and high-grade B-cell lymphoma non-specified (HGBL-NOS) group, according to the 2016 version of the World Health Organization (WHO) Hematopoietic and Lymphoid Tissues Cancer Classification. Both groups of patients were treated with stratified chemotherapy by risk according to the CNCL-B-NHL-2017 scheme. The deadline for follow-up was December 31, 2023. All the patients were examined by chromosome fluorescence in situ hybridization (FISH), and the rearrangement of genes MYC, BCL-2 and BCL-6 was confirmed. The clinical and pathological characteristics of patients at disease onset were analyzed, and the therapeutic effects of patients in different clinical stages and risk groups were compared. Survival analysis was drawn by Kaplan Meier method, the log-rank test was used to compare the differences in the cumulative survival rate between different groups, and multivariate Cox regression model was used to identify the prognostic factors. Results: A total of 62 patients were included, with an onset age [M(Q1, Q3)] of 7 (4, 11) years, including 48 males and 14 females. There were 11 (17.7%) patients in stageⅡ, 33(53.2%)patients in stage Ⅲ and 18(29.1%)patients in stage Ⅳ. FISH testing showed that 4 cases (6.5%) were HGBL-DH and 3 (4.8%) were HGBL-TH. The remaining 55 cases (88.7%) were HGBL-NOS, with 18 cases accompanied by MYC rearrangement. There were 7 cases in the HGBL-DH/TH group and 55 cases in the HGBL-NOS group. Thirteen cases (20.9%) were treated with the B1 regimen, 3 cases (4.8%) with B2 regimen, 37 cases (59.6%) with C1 regimen, and 9 cases (14.7%) with the C2 regimen. Forty-eight cases (77.4%) received rituximab therapy at the same time. Five cases (8.0%) progressed during treatment. The follow-up time [M(Q1, Q3)] was 43.5 (36.1, 53.7) months. The complete remission rate was 91.9% (57/62). The 3 year overall survival rate was 93.5% and event-free survival (EFS) rate was 91.9%. The 3-year overall survival rate in the HGBL-NOS group was higher than that in the HGBL-DH/TH group (96.3% vs 71.4%, P=0.011). The 3-year EFS rate of the HGBL-NOS group was higher than that of the HGBL-DH/TH group (94.5% vs 71.4%, P=0.037). In the HGBL-NOS subgroup, the overall survival rate of children with MYC rearrangement was lower (100% vs 88.9%,P=0.039). Multivariate Cox regression analysis showed that central invasion (HR=6.05, 95%CI: 1.96-38.13, P=0.046) was a risk factor for overall survival. Conclusion: CNCL-B-NHL-2017 regimen shows significant effects in the treatment of pediatric HGBL, with a good prognosis.

Economic Impact of Whole Genome Sequencing and Whole Transcriptome Sequencing Versus Routine Diagnostic Molecular Testing to Stratify Patients with B-Cell Acute Lymphoblastic Leukemia

Whole genome and whole transcriptome sequencing (WGTS) can accurately distinguish B-cell acute lymphoblastic leukemia (B-ALL) genomic subtypes. However, whether this is economically viable remains unclear. This study compared the direct costs and molecular subtype classification yield using different testing strategies for WGTS in adolescent and young adult/adult patients with B-ALL. These approaches were: (1) combined BCR::ABL1 by fluorescence in situ hybridization (FISH) +WGTS for all patients; and (2) sequential BCR::ABL1 FISH+WGTS contingent on initial BCR::ABL1 FISH test outcome. The cost of routine diagnostic testing was estimated using Medicare or hospital fees, and the additional cost of WGTS was evaluated from the health care provider perspective using time-driven activity-based costing with resource identification elicited from experts. Molecular subtype classification yield data were derived from literature sources. Parameter uncertainty was assessed through deterministic sensitivity analysis; additional scenario analyses were performed. The total per patient cost of WGTS was $4319 (all costs reported in US dollars); consumables accounted for 74% of the overall cost, primarily driven by sequencing-related consumables. The incremental cost per additional patient categorized into molecular subtype was $8498 for combined BCR::ABL1 FISH+WGTS for all patients and $5656 for initial BCR::ABL1 FISH+WGTS for select patients compared with routine diagnostic testing. A reduction in the consumable costs of WGTS or an increase in the yield of molecular subtype classification is favorable.

Performance of MYC, BCL2, and BCL6 break-apart FISH in small biopsies with large B-cell lymphoma: a retrospective Cytopathology Hematopathology Interinstitutional Consortium study.

Fluorescence in situ hybridization (FISH) is an essential ancillary study used to identify clinically aggressive subsets of large B-cell lymphomas that have MYC, BCL2, or BCL6 rearrangements. Small-volume biopsies such as fine needle aspiration biopsy (FNAB) and core needle biopsy (CNB) are increasingly used to diagnose lymphoma and obtain material for ancillary studies such as FISH. However, the performance of FISH in small biopsies has not been thoroughly evaluated or compared to surgical biopsies. We describe the results of MYC, BCL2, and BCL6 FISH in a series of 222 biopsy specimens, including FNAB with cell blocks, CNBs, and surgical excisional or incisional biopsies from 208 unique patients aggregated from 6 academic medical centers. A subset of patients had FNAB followed by a surgical biopsy (either CNB or excisional biopsy) obtained from the same or contiguous anatomic site as part of the same clinical workup; FISH results were compared for these paired specimens. FISH had a low hybridization failure rate of around 1% across all specimen types. FISH identified concurrent MYC and BCL2 rearrangements in 20 of 197 (10%) specimens and concurrent MYC and BCL6 rearrangements in 3 of 182 (1.6%) specimens. The paired FNAB and surgical biopsy specimens did not show any discrepancies for MYC or BCL2 FISH; of the 17 patients with 34 paired cytology and surgical specimens, only 2 of the 49 FISH probes compared (4% of all comparisons) showed any discrepancy and both were at the BCL6 locus. One discrepancy was due to necrosis of the CNB specimen causing a false negative BCL6 FISH result when compared to the FNAB cell block that demonstrated a BCL6 rearrangement. FISH showed a similar hybridization failure rate in all biopsy types. Ultimately, MYC, BCL2, or BCL6 FISH showed 96% concordance when compared across paired cytology and surgical specimens, suggesting FNAB with cell block is equivalent to other biopsy alternatives for evaluation of DLBCL or HGBCL FISH testing.

Abstract PO4-25-11: Inconclusive Her2 by immunohistochemistry compared to in situ hybridization in breast cancer: a cross-sectional study

Abstract Introduction: Breast cancer stands as the most common malignancy diagnosed among women worldwide, with the Her2-positive subtype accounting for approximately 15% of the cases. The introduction of HER-targeted therapies has changed the natural history of this subtype, however, evaluating the Her2 status using immunohistochemistry (IHC), can yield inconclusive results in a significant percentage of cases, which requires the use of in situ hybridization techniques. The study of such cases has not been thoroughly explored, particularly in the brazilian population, and increasing interest in this subgroup of patients arises as the "Her2 low" category gathers evidence of clinical applicability. The present study aimed to assess the final Her2 status of samples with indeterminate IHC results and compare these findings with current literature. Methods: We conducted a cross-sectional study at the breast unit service of a private health network in the state of Minas Gerais, Brazil (Rede Mater Dei de Saúde). Breast tissue samples obtained over the past 5 years were analyzed, and specimens with inconclusive IHC results for Her2 were included in the study. Subsequently, the final Her2 status was determined through fluorescence in situ hybridization (FISH) test, or reanalysis in cases in which a new sample (surgical specimen) was obtained. Results: Among the initially identified 73 samples with indeterminate IHC results for Her2, a total of 15 patients were excluded due to lack of follow-up within the breast unit service. The final cohort consisted of 58 women aged between 28 and 71 years. Notably, approximately 91% of the patients exhibited positive hormone receptor tumors, whereas 9% displayed negative hormone expression. After reviewing the medical records, approximately 36% of the patients were classified as having a positive final Her2 status, while 62% had a negative final status. One patient had an inconclusive FISH result. Conclusion: The present study revealed that approximately 10% of the breast tissue samples included in the study yielded inconclusive results for Her2 by immunohistochemistry. Following FISH testing, the majority of these patients were reclassified into the negative final Her2 status category. The classification of most patients to what is known as the emerging "Her2 low" category, aligns with international publications, and highlights the clinical relevance of exploring this subset of breast cancer that gains relevance as advances provide prospects for targeted therapies. Table 1. Summarized results for the final Her2 status Absolute and percentage values for the final HER2 status in the study cohort Citation Format: Felipe Marcondes de Oliveira Coelho, Renata Capanema Saliba Franco, Enaldo Melo de Lima, Marco Antonio Dias, Anna Dias Salvador, Waldeir José de Almeida Junior, Julia Wanderley Rennó, José Tadeu Campos de Avelar. Inconclusive Her2 by immunohistochemistry compared to in situ hybridization in breast cancer: a cross-sectional study [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-25-11.

Multiple Fluorescences in Situ Hybridization Status in Excreted Urine Improve Diagnostic Efficacy for Upper Urinary Tract Urothelial Carcinomas.

To evaluate the diagnostic accuracy of single and multiple fluorescence in situ hybridization (FISH) tests for upper urinary tract cancer (UTUC), we analyzed the diagnostic efficacy of FISH in patients with UTUC and the difference between it and the Tumor Node Metastasis (TNM) stage and grade of the tumor. Patients treated for UTUC at our institution between 2011 and 2021 who had not been previously diagnosed with UTUC were included. Patients were divided into single, two, and multiple (three times or four times) FISH groups based on the number of FISH tests performed on different samples from the same patient, and the diagnostic efficiency of single, two, and multiple FISH tests for muscle-invasive tumors and highgrade tumors were assessed. We included a total of 207 patients with UTUC, and when compared to single FISH, the sensitivity of multiple and double FISH for the diagnosis of UTUC increased from 62% to 76% and 78%, respectively. It went from 67% to 78% and 80% for muscle-invasive UTUC (> = pT2) and from 71% to 79% and 81% for the highest- grade UTUC. Multiple FISH improves the diagnostic efficacy of UTUC and helps to differentiate aggressive tumors.

The challenge of diagnosing and classifying eosinophilia and eosinophil disorders: A review.

Eosinophilia is a feature of multiple conditions, both hematologic and non-hematologic, and may be associated with organ damage. The pathogenesis of eosinophilia can follow two distinct pathways. Primary eosinophilia is caused by a cell-intrinsic mechanism originating from clonal expansion of eosinophils through acquisition of a somatic mutation, such as FIP1L1-PDGFRA. In recent years, great progress has been made in the field of pathogenesis and molecularly targeted therapy of neoplastic eosinophilia. The diagnostic procedure should include, among other things, morphologic analysis of blood and bone marrow samples, cytogenetics and fluorescence in situ-hybridization tests to detect evidence of an acute or chronic myeloid or lymphoid disorder. Secondary eosinophilia follows a cell-extrinsic mechanism as a response to exogenous cytokines. In most clinical cases, peripheral blood eosinophilia is reactive and typically associated with non-hematological disorders such as infections, allergic conditions, connective tissue disorders, vasculitis, malignancy, or endocrinopathies. Nonetheless, the cause of most cases of hypereosinophilic syndrome remains unknown. In this article, we present a short review focused on differential diagnosis of eosinophilia and eosinophilic disorders. The diagnosis of eosinophilia is a challenge for physicians; thus this review may be useful in clinical practice.

MDM2 FISH testing criteria in adipose tissue tumors with mature adipocytic morphology-A resection case-based study

The current criteria for utilizing MDM2 Fluorescence In Situ Hybridization (FISH) in adipose-derived tumors were first introduced in 2015 and have been widely adopted. However, these criteria may fail to identify some atypical lipomatous tumors / well-differentiated liposarcoma (ALT/WDL) with mature adipocytic morphology in clinical practice, possibly due to the fact that the existing criteria are primarily based on biopsy cases. Hence, a criterion based on resection cases is needed. In this study, we included 87 adipose tissue tumors with mature adipocytic morphology which were first resected, as well as 9 consultation cases and 25 recurrent resection cases. The final diagnosis was based on MDM2 amplification status. Among the 87 first-time resection cohort, MDM2 FISH amplification was observed in only 2 (5%) of the 39 superficial cases. Marginal infiltration was significantly different in both the MDM2 FISH negative and positive groups (p<0.05). Of the 37 intramuscular tumors, 17 (46%) showed MDM2 FISH amplification. The MDM2 amplification positive group had a larger tumor size than MDM2 amplification negative group (p=0.042). Tumors of larger size (≥11cm) were highly correlated with MDM2 amplification (p=0.003), but still, 35.3% of the MDM2 amplification-positive cases had tumor sizes less than 11cm. Eight (66.7%) out of twelve retroperitoneal/ pelvic cases were MDM2 FISH positive. Among the 25 recurrent cases, twenty (80%) of them had MDM2 FISH amplification. In conclusion, we recommend MDM2 FISH for: 1. superficial cases with marginal infiltration based on adequate margin sampling; 2. all intramuscular tumors, retroperitoneal/pelvic tumors and recurrent tumors, both in resection cases and biopsy cases.

Prioritization of Fluorescence In Situ Hybridization (FISH) Probes for Differentiating Primary Sites of Neuroendocrine Tumors with Machine Learning.

Determining neuroendocrine tumor (NET) primary sites is pivotal for patient care as pancreatic NETs (pNETs) and small bowel NETs (sbNETs) have distinct treatment approaches. The diagnostic power and prioritization of fluorescence in situ hybridization (FISH) assay biomarkers for establishing primary sites has not been thoroughly investigated using machine learning (ML) techniques. We trained ML models on FISH assay metrics from 85 sbNET and 59 pNET samples for primary site prediction. Exploring multiple methods for imputing missing data, the impute-by-median dataset coupled with a support vector machine model achieved the highest classification accuracy of 93.1% on a held-out test set, with the top importance variables originating from the ERBB2 FISH probe. Due to the greater interpretability of decision tree (DT) models, we fit DT models to ten dataset splits, achieving optimal performance with k-nearest neighbor (KNN) imputed data and a transformation to single categorical biomarker probe variables, with a mean accuracy of 81.4%, on held-out test sets. ERBB2 and MET variables ranked as top-performing features in 9 of 10 DT models and the full dataset model. These findings offer probabilistic guidance for FISH testing, emphasizing the prioritization of the ERBB2, SMAD4, and CDKN2A FISH probes in diagnosing NET primary sites.

Ossifying Fibromyxoid Tumor with Atypical Features: A Case Report and Review of Literature

Abstract Introduction/Objective Ossifying fibromyxoid tumor (OFMT) is a soft tissue neoplasm usually behaving in a benign fashion, however, it can rarely show atypical or malignant features. It most frequently occurs within the extremities and trunk. Although OFMTs are translocation-associated tumors, and very commonly associated with recurrent gene rearrangements involving the PHF1 gene, their morphologic and immunophenotypic profile can overlap with other soft tissue tumors posing a diagnostic challenge. Methods/Case Report Herein we present a case of a 23-year-old woman who had an excision of a subcutaneous shoulder mass. Grossly, the 5.3 cm mass had a thin translucent capsule and a pink, soft-cut surface with scattered calcifications. Microscopic examination revealed a well-circumscribed lesion composed of plump spindle cells with uniform oval nuclei arranged in short fascicles within a mildly collagenous background. There was prominent hemangiopericytoma-like vasculature and foci of ossification. The tumor cells contained moderate cytoplasm and showed a mild degree of nuclear atypia. Mitotic activity was up to 6 per 10 high-power fields. Immunohistochemical stains showed tumor cells are positive for Desmin, SMA, AE1/AE3 (patchy), TLE, and SATB2, while negative for CD31, CD34, S-100, C-kit, DOG-1, CD117, HMB-45, Melan-A, ALK-1, SOX10, Inhibin, and STAT6. Ki-67 proliferation index was low (1-2%). Fluorescence in situ hybridization (FISH) testing was negative for SYT and MDM2 gene amplification. FISH testing for PHF1 gene (using experimental probes) showed a break apart signal in a subset (~ 25%) of tumor cells, which is equivocal but insufficient to establish clonal interchromosomal translocation involving PHF1. Results (if a Case Study enter NA) NA Conclusion There is a histologic spectrum and morphologic overlap of OFMTs with several other soft tissue neoplasms. OFMT with atypical features (increased cellularity and mitotic activity), as in this case, may harbor the unpredictable biologic potential for recurrence and metastasis. Additional studies are required to discover a morphologic, immunohistochemical, and possibly molecular pattern that will help to differentiate OFMT subtypes.

Outcomes of Patients with Small Lymphocytic Lymphoma (SLL) Receiving First Line Treatment

INTRODUCTION Chronic lymphocytic leukemia (CLL) and SLL represent a single entity in current classifications per the WHO-HAEM5 and ICC. Both are characterized by malignant B cells with an identical immunophenotype, but they are distinguished by the predominant site of disease burden, such as peripheral blood for CLL and nodal compartments for SLL. Most clinical trials and retrospective studies involving CLL/SLL do not separate out SLL patients. In this study, we report reasons for treatment initiation and outcomes in a cohort of SLL patients seen at our institution. METHODS Using the Mayo Clinic CLL Database, we retrospectively identified patients with SLL who received first line therapy between 12/1995 and 2/2023. Patients were classified as having SLL if they met at least one of the following criteria for diagnosis: 1) presence of lymphadenopathy or splenomegaly (either by exam or scans) and an absolute clonal B cell count of &amp;lt; 5 x 109/L; or 2) presence of lymphadenopathy or splenomegaly (either by exam or scans) and an absolute lymphocyte count of &amp;lt; 11 x 109/L (if absolute clonal B cell count was not available). Although the presence of anemia (hemoglobin &amp;lt; 11 g/dL) and thrombocytopenia (platelets &amp;lt; 100 x 109/L) are considered as exclusion criteria for a diagnosis of SLL, patients were included if these cytopenia were due to autoimmune hemolytic anemia (AIHA) or immune thrombocytopenia (ITP) with &amp;lt; 50% bone marrow involvement by SLL (bone marrow biopsy confirmed). Patients treated for SLL with extranodal involvement were also included. Indications for SLL treatment were abstracted by chart review and included one of the following: 1) symptomatic or progressive lymphadenopathy and/or splenomegaly, 2) autoimmune cytopenias, 3) extranodal/direct organ infiltration; and 4) other (e.g., cryoglobulinemia, paraneoplastic, non-infiltrative renal disease such as minimal change disease). Overall survival (OS) was defined as the time from treatment start until date of death or last known to be alive. Treatment-free survival (TFS) was defined as the time from treatment start until the earliest of date of next treatment, or death. Kaplan-Meier method was used to display OS and TFS. Cox proportional hazards regression models were used to estimate associations of factors with time-to-event outcomes. RESULTS We identified 182 patients with SLL who received first line treatment. The median age at treatment start was 66 years, and 132 (73%) were male. Unmutated IGHV was identified in 60/82 (73%) patients and peripheral blood fluorescent in situ hybridization (FISH) assessed pre-treatment start showed del13q in 10%, trisomy 12 in 43%, del11q in 7%, del17p in 8%, and no abnormalities in 33%. TP53 disruption (del17p and/or TP53 mutation) was present in 7/89 (8%) patients prior to first therapy. The median follow-up after diagnosis was 6.9 years. The indications for treatment were grouped as follows: symptomatic or progressive lymphadenopathy/splenomegaly (n=130), extranodal/direct organ infiltration (n=30), AIHA/ITP (n=10), and other (n=12). For the entire cohort, the median TFS was 2.9 years and the median OS was 12.3 years. The median TFS by the treatment indication groups were 3.3, 5.1, 1.6, and 5.3 years, respectively (Figure 1). The median OS by treatment indication groups were 11.1, 10.5, 8.8, and 19.9 years, respectively. Among the 160 patients with symptomatic or progressive lymphadenopathy/splenomegaly or extranodal disease, the treatments administered include: mAb alone (n=35), chemotherapy alone (n=33), chemoimmunotherapy (n=57), targeted agent (n=33; Bruton tyrosine kinase inhibitor [BTKi] based n=28; venetoclax-based n=3, BTKi + venetoclax n=2), and other (n=2). The median TFS by treatment groups were 2.4, 2.0, 3.8, and 6.0 years (Figure 2), respectively (excluding the small “other” subgroup of 2 patients). The median OS for these groups were 9.5, 9.7, 15.0, and not evaluable, respectively. Only older age at treatment start predicted for shorter TFS (HR 1.02; 95%CI 1.002-1.038; p=0.03) and shorter OS (HR 1.05; 95%CI 1.03-1.07; p&amp;lt;0.01). CONCLUSIONS In this study of patients with SLL who received therapy, the most frequent indications for treatment included symptomatic lymphadenopathy and/or splenomegaly, followed by extranodal disease. Trisomy 12 was the most frequently detected abnormality on peripheral blood FISH testing. Treatment with novel agents achieved durable TFS and OS.

Clinical and Molecular Features of Patients with Double/Triple Hit Large B-Cell Lymphoma

Background: High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) is a recently defined category in the latest revision of the World Health Organization (WHO) classification, which comprises approximately 5-15% of newly diagnosed DLBCL. This subtype of lymphoma poses numerous challenges from diagnosis to treatment of patients. On the one hand, diagnosis of HGBL-DH/TH requires the accomplishment of fluorescent in-situ hybridization (FISH) to identify the translocations of MYC(8q24) and BCL2(18q21) or/and BCL6(3q27), with no consensus being reached regarding the most applicable population for FISH tests due to high cost and low prevalence. On the other hand, HGBL-DH/TH displays aggressive features, including a high incidence of advanced disease at diagnosis, and inferior efficacy to standard-induced chemical immunotherapy with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Despite several treatment strategies including intensified protocols and hematopoietic cell transplantation (HCT) have been explored, there is no standard regimen to improve outcomes for HGBL patients, and participation in clinical trials is encouraged. Aims: This study aims to investigate the clinical characteristics and prognostic outcomes of HGBL-DH/TH patients, and performed genomic and transcriptomic analyses to link the oncogenic mutations and tumor microenvironment alterations to different DLBCL subgroups. Method:In this study, 107 patients with newly diagnosed HGBL-DH/TH were analyzed. Histological diagnoses were established according to the WHO classification. These HGBL-DH/THs were termed DHL-BCL2 (MYC and BCL2 translocation), DHL-BCL6 (MYC and BCL6 translocation), and THL in our study. DNA sequencing was carried out on 95 patients, for detection of genetic aberrations. Results: Patients with DHL-BCL2 (n=44) and THL (n=15) have similar clinical features, including elevated serum lactate dehydrogenase (LDH), increased proportion of double expression (DE) as well as increased prevalence of non-GCB subtype compared to those with DHL-BCL6 (n=48). With the median follow-up time of 19.3 months (2.5-114.2 months), patients in the DHL-BCL2 and THL group had dismal survival (2-year PFS: 49.3% and 34.2%; 2-year OS: 64.0% and 55.6%) when compared with those in the DHL-BCL6 group (2-year PFS: 63.5%; 2-year OS: 89.3%) (Figure A). Moreover, intensive chemoimmunotherapy or combination with novel targeted agents seemed to improve outcomes in patients with DHL/THL compared with those who received a standard R-CHOP regime. The use of autologous stem cell transplant (ASCT) did not yield a significant improvement in survival for patients who achieved remission after receiving first-line R-DA-EDOCH therapy. However, it is worth noting that there was a trend towards prolonging progression-free survival (PFS) and overall survival (OS) in patients who achieve complete remission (CR) through RCHOP-based treatment and undergo ASCT (Figure B). Compared to DHL-BCL6, the primary genetic lesions in DHL-BCL2 and THL were alterations associating epigenetic regulators like EZH2 and CREBBP, while the primary genetic lesions in DHL-BCL6 were alterations associating cellular differentiation and transcription factors like BTG2, PTPN6, CD70, and BCL6. Correlatively, the EZB genotype was more frequently observed in patients with DHL-BCL2 and THL, while the BN2 genotype was more frequently observed in patients with DHL-BCL6. Conclusion: DHL-BCL2/THL exhibits similar clinical characteristics, prognostic outcomes, and molecular features that set it apart from DHL-BCL6. This implies the need for testing novel agents or therapeutic strategies to expedite treatment development for patients with DHL-BCL2/THL.

Are There More HER2 FISH in the Sea? An Institution’s Experience in Identifying HER2 Positivity Using Fluorescent In Situ Hybridization in Patients with HER2 Negative Immunohistochemistry

BackgroundApproximately 20% of breast cancers express HER2-positive receptors in the USA. HER2 receptor immunohistochemistry (IHC) staining with equivocal (2+) results commonly undergoes fluorescence in-situ hybridization (FISH) for further classification. Current guidelines do not recommend routine FISH testing in IHC-negative (0 or 1+) cases. This study investigates an institution that performs both IHC and FISH testing on all cases to identify the true HER2-positive rate.Patients and MethodsA retrospective chart review from 2015 to 2021 was conducted at an institution where both HER2 IHC and FISH testing were performed at the time of diagnosis for all invasive breast cancers. The rate of true HER2-positive patients was determined, and patient and tumor characteristics were further explored.ResultsA total of 1835 invasive breast cancer cases were primarily treated at this institution. A total of 289 cases were HER2 positive on IHC and FISH testing (15.7%). An additional 38 cases were identified as HER2 negative on IHC, but reclassified as HER2 positive on reflex FISH testing. Total HER2 positive cases increased from 289 (15.7%) to 327 cases (17.8%) with reflex FISH testing.ConclusionsThe additional HER2-positive cases after completing FISH testing on IHC-negative tumors suggests there may be a role for routine FISH testing in addition to standard IHC staining to determine HER2 status for breast cancer. The ethical, prognostic and even benefits of a correct diagnosis outweigh the added expense of FISH testing.

Optical Genome Mapping Allows Detection and Characterization of Cytogenetically Cryptic Oncogenic Fusions in Pediatric Acute Myeloid Leukemia

Background: Abnormal gene fusions are predominant oncogenic drivers in pediatric acute myeloid leukemia (AML) and are important diagnostic and prognostic markers. However, several oncogenic fusions specific for or enriched in pediatric AML (like CBFA2T3::GLIS2, NUP98::NSD1, ETV6::MNX1 and others) are not detectable by conventional cytogenetic analysis (cytogenetically cryptic) and may not be included in fluorescence in situ hybridization (FISH) or molecular fusion panels designed for adult patients. Optical genome mapping (OGM) is a powerful new method for genome-wide, high-resolution detection of copy number abnormalities and balanced chromosome rearrangements. This study determined the frequency of cryptic oncogenic fusions in an institutional cohort of pediatric AML cases and evaluated OGM as a method for their detection and characterization. Methods: Retrospective review of genetic testing results was performed for 93 consecutive de novo or relapsed pediatric AML cases tested at the Children's Hospital Los Angeles (CHLA) Center for Personalized Medicine between March 2016 and July 2023. Genetic testing was performed as part of clinical care following standard procedures and consisted of karyotype analysis, FISH testing, chromosomal microarray (CMA) analysis, CHLA custom sequencing panel (OncoKids®) (June 2017 onwards) and RNA-Seq fusion assay (March 2022 onwards). Cases with normal or inconclusive karyotype in which an oncogenic fusion was detected by another clinical assay were selected for OGM testing. High molecular weight DNA from peripheral blood or bone marrow samples was labeled, processed and imaged in a Saphyr System and analyzed in the Bionano Access software following the manufacturer's recommendations (BionanoGenomics, San Diego CA). Results: Out of 93 pediatric AML cases included in the study, a gene fusion was identified as the primary oncogenic driver in 54 (58%) and a DNA sequence variant in 11 (12%). In 28 cases (30%), the primary genetic abnormality remained undetermined after comprehensive genetic testing. In 17/54 (31%) fusion-driven cases [17/93 (18%) overall cases], the oncogenic fusion remained undetected by karyotype analysis (cryptic). OGM was completed in 13/17 fusion positive cases with non-informative karyotype; it could not be performed in 4 cases due to lack of samples (n=2) or poor DNA quality (n=2), and is still in progress for a subset of 28 unknown cases with available material. OGM analysis successfully revealed all cryptic fusions previously detected by FISH, CMA, OncoKids® and/or RNA-Seq, including NUP98::NSD1 (n=3), KMT2A::MLLT10 (n=3), KMT2A::MLLT3 (n=2)and one case each of CBFA2T3::GLIS2, NUP98::KDM5A, DEK::NUP214, MYB::GATA1 and ETV6::MNX1. Chromosomal mechanism and structure of cryptic fusions were resolved by OGM and included: intrinsically cryptic fusions due to chromosome location of partner genes ( NUP98::NSD1, n=4; CBFA2T3::GLIS2, NUP98::KDM5A and ETV6::MNX1, n=1 each); complex rearrangements due to incompatible gene orientation ( KMT2A::MLLT10 n=2; MYB::GATA1, n=1); three-way rearrangements involving an additional chromosome ( KMT2A::MLLT3 n=2) and small insertions ( KMT2A::MLLT10 and DEK::NUP214, n=1 each). OGM also matched the performance of chromosomal microarray (CMA) analysis in detection of copy number variants. Conclusions: We confirm the significant contribution of cryptic chromosome rearrangements to pathogenesis of pediatric AML, and highlight the importance of tailored testing approaches that effectively detect highly heterogeneous fusion drivers. Our preliminary results show potential of OGM to identify clinically significant cryptic fusions in pediatric AML, and to thus markedly increase diagnostic yield compared to karyotype analysis. As a single assay that detects abnormalities currently interrogated by conventional cytogenetics, FISH and CMA, OGM may allow streamlined and more cost-effective genetic evaluation for pediatric AML.

High Deletion Burden Identified By Whole Genome Sequencing Is Associated with Enhanced Risk in del17p CLL Patients

Presence of deletion17p (del17p) in frontline (1L) and relapsed/refractory (R/R) CLL has been associated with shorter time to treatment initiation, early relapse after therapy and inferior overall survival (OS) (Mato et.al., Haemotologica 2022). However, there exists notable clinical heterogeneity in terms of outcome within this group (Tam et.al., Blood 2009, Stephens et.al; AJH 2015). Previous reports in 1L and R/R CLL have identified percentage of del17p nuclei by fluorescence in situ hybridization (FISH), having TP53 mutations on the second allele, copy number alterations and complex karyotype (CK) as additional risk factors within the del17p population (Yu et al., CCR 2017; Baliakas et al., Blood 2019). Here we profile a cohort of wait and watch (W&amp;W, n=14), 1L treated (n=36) and R/R CLL (n=3) patients by whole genome sequencing (WGS) and RNA sequencing (RNAseq) to gain insights into genomic factors and gene expression pathways contributing to the high-risk nature of del17p across the spectrum of CLL patients. Using the Mayo Clinic CLL Database, we identified 53 patients with del17p (identified by FISH testing in routine clinical practice at diagnosis) who had stored peripheral blood mononuclear cells (PBMCs) available. RNA and DNA from matched peripheral blood (tumor) and DNA from CD19- (germline) samples at diagnosis (n=50) and relapse (n=3) was extracted using Qiagen RNeasy (RNA) and Purene (DNA) kits and subjected to RNAseq and WGS respectively. Additional FISH assays (del11q, trisomy 12, del13q) and CK by chromosomal banding analysis (CBA) were performed. Baseline clinical characteristics, time to first treatment, types of treatment, and survival were abstracted from the CLL Database. The Mayo Clinic IRB approved this study. Copy number analysis identified deletions in 3p, 4p, 8p, 9p, 9q and gains in 2p, 8q, 15q and 17q chromosomal lesions occurring in patients. We explored the association of clinical and genetic factors, and genomic instability signatures with survival to identify high-risk features in del17p patients. Both total number of deletions by WGS and number of chromosomal aberrations by CBA as a continuous variable were significantly associated with unfavorable OS in the entire cohort (n=53) in a univariate analysis (Hazard ratio, HR= 2.29, p= 0.00054 and HR= 2.33, p=0.0054 respectively). However, there was no significant association of number of gained genes by WGS or aneuploidy scores with survival (HR=0.74, p=0.37). In patients with CK data (n=41), deletion burden, but not gains, were highly correlated to chromosomal aberrations by CBA. Using cutpointr package for identifying deletion threshold corresponding to CK≥ 3, we defined patients with deletion burden ≥ 514 as enhanced-high risk (EHR; median OS = 4.83 years) compared to the group with high-risk (HR; median OS=9.94 years) (p=0.025). There was no significant difference in number of deleted genes between WW and treated patients. The EHR group is enriched in patients with high clonal cell fraction of del17p by FISH, unmutated IGHV and CK≥ 3. The EHR group remains significant in a multi-variable analysis with IGHV-mutation status, ZAP70, and age (HR: 5.16, 95% CI: 1.04 - 25.6, p=0.04). Mutational analysis identified 35 known CLL driver mutations with TP53 (86%), ATM (15%), NOTCH1(13%), MGA (13%), CHD2 and MED12(11%) as the top mutated genes. Total number of driver genes mutated was not significantly different between EHR and HR subgroups, but the EHR subset was significantly enriched with mutations in NOTCH1 (21%), and MGA (21%) as compared to no mutations in the HR group in these genes. Deleted genes (≥ 514) was prognostic in bi-allelic TP53 aberration patients as well (p=0.021) Differential expression gene-set enrichment analysis indicated a significant enrichment of DNA repair, cellular response to DNA damage and downregulation of inflammatory responses in the EHR group. CK is a known prognostic marker which includes both chromosomal losses and gains. In this small cohort of del17p we were able to show the prognostic value of deletions only, but not gains, as captured by WGS. Using genome wide sequencing, we identify increasing genomic deletions as a feature of enhanced-high risk del17p. While deletion burden cut-offs identified here are specific to our research method and require further validation in additional independent cohorts, EHR subgroup remained significant after adjusting for other known prognostic variables .

Impact of 1q21 Gain and Amplification on Daratumumab-Treated Multiple Myeloma Patients: Real-World Data from the Australia-New Zealand and Asia-Pacific Myeloma and Related Diseases Registries

Introduction: Gain/amplification of 1q21 (1q21+) is found in 40% of patients (pts) with newly diagnosed Multiple Myeloma (MM). There is increasing evidence that presence of gain of 1q21 [gain(1q21), 3 copies] or amplification of 1q21 [amp(1q21), ≥4 copies] are independent poor prognostic factors. A recent post-hoc analysis of the ICARIA-MM and IKEMA studies showed that the addition of monoclonal anti-CD38 antibody isatuximab in combination with pomalidomide-dexamethasone or carfilzomib-dexamethasone abrogated the negative impact of 1q21 on progression-free survival (PFS). However, the impact of 1q21+ has not been consistently assessed in daratumumab (Dara) exposed pts. Methods: We identified MM pts treated with Dara between Jan 2020 and Jan 2023 from the Australia-New Zealand and Asia-Pacific Myeloma and Related Diseases Registries. Pts who had interphase fluorescence in situ hybridization (FISH) testing were included. High-risk cytogenetics (HRCyto) was defined as the presence of t(4;14), t(14;16) or del(17p) by FISH, and standard risk cytogenetics (SRCyto) as pts without 1q21+ and HRCyto. Pt groups analysed were: (1) without 1q21+ vs with 1q21+ (2) SRCyto vs isolated 1q21+ vs HRCyto without 1q21+ vs HRCyto with 1q21+. Subgroup analyses performed include: (3) gain(1q21) vs amp(1q21). Overall survival (OS) and PFS from commencement of Dara were estimated using the Kaplan-Meier method. Comparisons of PFS and OS were performed using the log-rank test. Categorical data were compared using a chi-square test and continuous variables with a rank-sum test. Results: Of 654 pts who received Dara, 451 had interphase FISH testing performed and were included. There were 132 pts with 1q21+ [gain(1q): 23, amp(1q):14, gain/amp status unknown: 95] and 319 without. Median follow up was 15 months (95% CI 14-18). Median prior lines of therapy was 1 (IQR 1-2). Dara-based regimens used were: Dara monotherapy (17.7%), Dara-bortezomib-dex (54.6%), Dara-lenalidomide-dex (11.3%), Dara-pomalidomide-dex (DPd) (9.3%), Dara-carfilzomib-dex (DKd) (2.5%) and Dara-bortezomib-lenalidomide-dex (4.6%). There were no differences in age, gender, ECOG, line of therapy in which Dara was used, or rate of autologous stem cell transplantation between pts with 1q+ vs pts without (Table 1). Pts with 1q21+ had higher rates of other HRCyto (37.1% vs 20.4%; p=0.005) and ISS Stage 3 disease (44.1% vs 29.3%; p=0.005) vs those without 1q21+. There was no significant difference in overall response rate (65.9% vs 71.6%; p=0.32). Through univariate analyses, PFS for 1q21+ pts was significantly shorter than those without 1q21+ [median PFS 8.2 vs 18.4 months (HR 1.7, 95% CI 1.3-2.3; p&amp;lt;0.001)] (Figure 1). This difference was still significant when adjusted for ISS, the presence of other HRCyto and country of treatment (HR 1.61, 95% CI 1.17-2.22; p=0.004). Pts with 1q21+ with or without HRCyto, and those with HRCyto and no 1q21+ had inferior PFS vs SRCtyo (median PFS 7.7 vs 8.9 vs 8.5 vs 30.3 months; p&amp;lt;0.001) (Figure 2). Of the pts where 1q21+ copy number was available, OS was significantly poorer in pts with amp(1q) vs those with gain(1q) (median OS 22.6 vs 46.9 months; p=0.035). There was also a trend towards poorer PFS in pts with amp(1q) (median PFS 4.9 vs 10.5 months; p=0.096) Conclusions: 1q21+ often co-exists with other HRCyto abnormalities. However, it remains an independent negative prognostic marker. The use of Dara does not appear to abrogate the negative impact of 1q21+ on PFS. Amp(1q21) potentially confers an even poorer outcome. This finding differs from an earlier retrospective study (Parrondo et al, Blood 2022), although &amp;lt;10% of patients in our study received DKd and DPd combinations. As such, more data on Dara in combination with carfilzomib and pomalidomide is needed.

UroVysion™ fluorescence in situ hybridization (FISH) possibly has a high positive rate in carcinoma of non-urothelial lineages.

Background: Positive UroVysion™ fluorescence in situ hybridization (FISH) is generally considered as urothelial carcinoma (UC). We clarify if UroVysion™ FISH can be positive in carcinoma of non-urothelial lineages (CNUL), and verify the consistency of urine FISH and histological FISH in CNUL. Methods: All CNUL subjects detected by urine FISH assay due to haematuria from Tongji Hospital were screened. Meanwhile, 2 glandular cystitis and 2 urothelial carcinoma were served as negative or positive control. Paraffin-embedded tissue sections of all subjects were sent to the pathology department for histological FISH detection. Results: A total of 27 patients were included in this study, including 9 with adenocarcinomas, 11 with squamous cell carcinomas, and 7 with other tumour types. The overall positive rate in urine FISH was 64.00% (16/25) in patients with CNUL, 77.78% (7/9) in those with adenocarcinoma and 54.55% (6/11) in those with squamous carcinoma. There was a significant difference in the GLP p16 gene deletion rate between UC and CNUL (100% vs. 8.00%, p = 0.017). Histological FISH results showed that the histological results of 19 patients were consistent with their urine FISH results, and only one patient with stage Ⅲa urachal carcinoma had inconsistent histological FISH results (positive) and urine FISH (negative) results. Conclusion: We demonstrated for the first time the application value of FISH in CNUL on urine samples. Positive urine FISH tests indicate not only UC, but also CNUL. UroVysion™ FISH possibly has a high positive rate in CNUL. CNUL and UC have different genetic changes shown by FISH.