Fluorescence In Situ Hybridization Testing Research Articles

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.

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.

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.

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.

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.

Preoperative fluorescence in situ hybridization analysis as a predictor of tumor recurrence in patients with non-muscle invasive bladder cancer: a bi-institutional study

BackgroundNon-muscle invasive bladder cancer (NMIBC) is known for its elevated recurrence rate, necessitating an enhancement in the current risk stratification for recurrence. The urine-based fluorescence in situ hybridization (FISH) assay has emerged as a noninvasive auxiliary tool for detecting bladder cancer. The aim of this study was to explore the potential relationship between the preoperative FISH assay and recurrence, and to develop a FISH-clinical nomogram for predicting the recurrence-free survival (RFS) in NMIBC patients.MethodsIn total, 332 eligible patients were enrolled from two hospitals. The SYSMH cohort was randomly assigned to the training set (n = 168) and the validation set I (n = 72) at a ratio of 7:3, while the SYSUTH cohort was allocated to the validation set II (n = 92). The correlation between the preoperative FISH assay and recurrence was determined through the Cox regression analysis. The least absolute shrinkage and selection operator (LASSO) Cox regression algorithm was used for model construction. The performance of the model was assessed by its discrimination, calibration, and clinical usefulness.ResultsWe uncovered that chromosome 7 aneuploidy, p16 locus loss, number of the positive FISH sites, and the FISH test result were significantly associated with tumor recurrence. Then, a FISH-clinical nomogram incorporating the FISH test result, T stage, associated CIS, tumor grade, and tumor status was developed. It showed favorable calibration and discrimination with a C-index of 0.683 (95%CI, 0.611–0.756) in the training set, which was confirmed in the validation set I and validation set II with C-indexes of 0.665 (95%CI, 0.565–0.765) and 0.778 (95%CI, 0.665–0.891), respectively. Decision curve analysis revealed the clinical usefulness of the nomogram. Moreover, our proposed nomogram significantly outperformed the guideline-recommended EORTC and CUETO scoring models.ConclusionOur study confirmed the prognostic value of the preoperative FISH assay and proposed a FISH-clinical nomogram to predict RFS in NMIBC patients. Our nomogram can serve as a more precise tool for recurrence risk stratification, which may optimize disease management in bladder cancer and improve patient prognosis.

Loss of Chromosome 3q Is a Prognostic Marker in Fusion-Negative Rhabdomyosarcoma.

Soft tissue sarcomas (STS) are rare mesenchymal neoplasms that frequently show complex chromosomal aberrations such as amplifications or deletions of DNA sequences or even whole chromosomes. We recently found that gain of chromosome (chr) 8 is associated with worse overall survival (OS) in STS as a group. We therefore aimed to investigate the overall copy number profile of rhabdomyosarcoma (RMS) to evaluate for prognostic signatures. Fluorescence in situ hybridization (FISH) testing was performed on a cohort of STS to assess for chr8 gain. Copy number variation (CNV) data from the National Cancer Institute were analyzed to assess for prognostically significant CNV aberrations in FOXO1 fusion-negative (FN)- versus fusion-positive (FP)-RMS. FISH testing was performed on a cohort of FN-RMS to assess for chr3q loss and correlate with outcomes. Chr8 gain is a highly prevalent CNV in embryonal RMS and shows slightly improved prognosis. Meanwhile, loss of chr3q was associated with worse outcome in FN-RMS compared with FP-RMS. The pathogenesis of STS including FN-RMS remains poorly understood, emphasizing the need for new therapeutic advances and adequate risk stratification. Our data demonstrate that loss of chr3q is associated with poor OS in FN-RMS, supporting it as an important tool for risk stratification.

Evaluation of the diagnostic efficiency of voided urine fluorescence in situ hybridization for predicting the pathology of preoperative "low-risk" upper tract urothelial carcinoma.

To evaluate the clinical utility of voided urine fluorescence in situ hybridization (FISH) for predicting the pathology of preoperative "low-risk" upper tract urothelial carcinoma (UTUC). Information of patients preoperatively diagnosed with "low-risk" UTUC receiving radical nephroureterectomy (RNU) between May 2014 and October 2019 were retrospectively collected. All of the patients accepted the FISH test and then were divided into two groups according to the results of FISH. The diagnostic value of FISH was assessed through the receiver operating characteristics (ROC) curve and area under the curve. Logistic regression analysis was applied to examine FISH as a predictive factor of tumor final stage and grade of preoperative "low-risk" UTUC. In total, 129 patients were included. Of them, 70 (54.2%) were marked with positive FISH result. The difference at final pathology in tumor stage and tumor grade between these two groups of FISH (-) and FISH (+) had significantly statistical significance (p<0.001). Regarding to the tumor stage at final pathology, the sensitivity, specificity, positive predictive value and negative predictive value of FISH were 70.7 (58.9-80.3)68.5 (54.3-80.1)75.7 (63.7-84.8) and 62.7 (49.1-74.7), respectively. Regarding to the tumor grade at final pathology, the sensitivity, specificity, positive predictive value and negative predictive value of FISH were 64.7 (53.5-74.6), 65.9 (50.0-79.1), 78.6 (66.8-87.1) and 49.1 (36.5-62.3), respectively. The results of logistic regression analysis indicated that FISH could predict the pathologic characteristics of preoperative "low-risk" UTUC independently. FISH was qualified with relatively high diagnostic estimates for predicting tumor stage and grade of preoperative "low-risk" UTUC, and could be an independent predictive factor in clinical practice. For preoperative "low-risk" UTUC patients but with positive FISH result, choosing nephron-sparing surgery may require special caution.

HER-2 overexpression in female genital tract clear cell carcinomas: Evaluation of different scoring guidelines, clinicopathological features and prognostic impact

BackgroundClear cell carcinoma (CCC) is a rare high-grade adenocarcinoma associated with poor response to platinum-based chemotherapy agents in the female genital tract. Human epidermal growth factor receptor 2 (HER2) overexpression is routinely used as a biomarker for targeted therapy in breast and gastric carcinomas, but its role in CCC remains unclear. MethodsIn this study, HER2 overexpression was evaluated by immunohistochemistry (IHC) using College of American Pathologists (CAP) HER2 scoring guidelines for breast and endometrial serous carcinoma (ESC) on tissue microarray blocks. In equivocal and positive cases, fluorescence in situ hybridization (FISH) was performed. IHC score 3, and all amplified cases on FISH test were considered positive. ResultsThirty-six cases of ovarian (OCCC), 36 endometrial (ECCC), and 2 cervical CCC were included. According to ESC and breast scoring guidelines, 20 % and 15.1 % of ECCC and 14.7 % and 6 % of OCCC were HER2 positive, respectively. Both cases of cervical CCC were negative. Scoring based on breast carcinoma guideline showed higher concordance (100 %) with gene amplification results, in comparison with ESC guideline (82.7 %). On multivariate survival analysis, HER2 positive ECCC and OCCC (based on ESC scoring methods) had significantly lower overall and disease-free survivals (OS, DFS) (P < 0.05). ConclusionHER2 immunoscoring based on ESC guideline can yield a higher sensitivity with relevant clinical and prognostic features in OCCC and ECCC. HER2 can be considered a potential biomarker for targeted therapy and future clinical trials.

Paediatric Spitzoid Neoplasms: 10-Year Retrospective Study Characterizing Histological, Clinical, Dermoscopic Presentation and FISH Test Results.

Spitzoid lesions are a wide tumour class comprising Spitz nevus (SN), atypical Spitz tumour (AST) and Spitz melanoma (SM). We conducted a single-centre-based retrospective survey on all histologically diagnosed spitzoid lesions of paediatric patients (1-18 years) of the last 10 years (2012-2022). Histopathological reports and electronic records of patients were used to retrieve relevant data regarding patients' features, clinical and dermatoscopical aspects of lesions when recorded, and FISH tests when present. Of 255 lesions, 82% were histologically benign, 17% atypical, 1% malignant. Clinically, 100% of SM were large (≥6 mm) and raised; AST were mainly large (63%), raised (98%), pink (95%). Small (≤5 mm), pigmented, flat lesions correlated with benign histology (respectively 90%, 97%, 98% SN) (p < 0.0001). Dermatoscopical patterns were analysed in 100 patients: starburst pattern correlated with benign histology (26% SN (p = 0.004)), while multicomponent pattern correlated with atypical/malignant lesions (56% AST, 50% SM (p = 0.0052)). Eighty-five lesions were subjected to fluorescence in situ hybridization (FISH): 34 (71% AST; 29% SN) were FISH-positive; 51 (63% SN; 37% AST) were FISH-negative (p = 0.0038). This study confirmed predominant benign histology (82%) of paediatric spitzoid lesions, thus detecting 17% AST and 1% SM, highlighting the need for caution in handling spitzoid lesions. Until AST are considered potentially malignant proliferations and no reliable criteria are identified to distinguish them, the authors suggest a prudent approach, especially in children.

Round-robin testing for LMO2 and MYC as immunohistochemical markers to screen MYC rearrangements in aggressive large B-cell lymphoma.

Aggressive large B-cell lymphomas (aLBCL) include a heterogeneous group of lymphomas with diverse biological features. One of the approaches to the diagnosis of aLBCL is based on the identification of MYC rearrangements (MYC-R), in addition to BCL2 and BCL6 rearrangements by genetic techniques, mainly fluorescent in situ hybridization (FISH). Because of the low incidence of MYC-R, the identification of useful immunohistochemistry markers to select cases for MYC FISH testing may be useful in daily practice. In a previous work, we identified a strong association between the profile CD10 positive/LMO2 negative expression and the presence of MYC-R in aLBCL and obtained good intralaboratory reproducibility. In this study, we wanted to evaluate external reproducibility. To evaluate whether LMO2 can be a reproducible marker between observers 50 aLBCL cases were circulated among 7 hematopathologists of 5 hospitals. Fleiss' kappa index for LMO2 and MYC were 0.87 and 0.70, respectively, indicating high agreement between observers. In addition, during 2021-2022, the enrolled centers included LMO2 in their diagnostic panels to evaluate prospectively the utility of the marker, and 213 cases were analyzed. Comparing LMO2 with MYC, the group of CD10 positive cases showed higher specificity (86% vs 79%), positive predictive value (66% vs 58%), likelihood positive value (5.47 vs 3.78), and accuracy (83% vs 79%), whereas the negative predictive values remained similar (90% vs 91%). These findings place LMO2 as a useful and reproducible marker to screen MYC-R in aLBCL.

HER2 Status Assessment in Endometrial Serous Carcinoma: Comparative Analysis of Two Proposed Testing and Interpretation Algorithms.

HER2 status is now routinely assessed in endometrial serous carcinoma (ESC) due to the reported predictive value of HER2 protein overexpression and/or gene amplification. Herein the authors compare 2 proposed testing and interpretation guidelines for HER2 in ESC. Forty-three consecutive cases of ESC that had been dually tested by both HER2 immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) were interpreted using 2 sets of guidelines. Guideline set 1 (GS1) is the 2018 American Society of Clinical Oncology/College of American Pathologists guidelines for breast cancer. Guideline set 2 (GS2) is a recent proposal that is a slight modification of the enrollment criteria for the clinical trial (NCT01367002) that demonstrated a survival benefit for anti-HER2 therapy in ESC. By IHC, GS1 and GS2, respectively classified 39.5% (17/43) and 28% (12/43) of ESC as HER2-negative, 37.2% (16/43) and 53.4% (23/43) as HER2 equivocal, and 23.2% (10/43) and 18.6% (8/43) as HER2-positive (P > 0.05 for all). IHC and FISH were highly concordant at the extremes using either set of guidelines, as no cases were found to be IHC3+/FISH-negative or IHC 0-1+/FISH-positive. GS1 and GS2 were comparable regarding the proportion of IHC equivocal cases that were HER2 amplified by FISH (19% vs 23% respectively; [P = 0.71]). GS1 and GS2 displayed 98% (42/43) concordance regarding the final (IHC and/or FISH-based) classification of tumors as being HER2-positive or negative, and the same 13 cases were ultimately classified as HER2 amplified using either GS1 or GS2. One "discordant" case was classified as HER2-positive using GS2 but HER2-negative using GS1 (HER2 IHC score 2+ using both guidelines, HER2:CEP17 signal ratio of 3, HER2 signal number of 3.4). Six (14%) of the 43 cases (FISH Groups: 2, 3, and 4) would require IHC results to interpret the FISH findings using GS1. Because GS1 requires that the HER2 IHC staining be observed within a homogeneous and contiguous invasive cell population, and this is not a requirement in GS2, GS2 may be better suited for ESC given its frequently heterogeneous staining pattern. Additional studies may be required on the optimal interpretation of problematic dual-probe FISH scenarios in GS2 and the necessity for IHC correlation in such scenarios. Using either set of guidelines, our findings support a reflex testing strategy of restricting FISH testing to cases that are IHC equivocal.

NTRK Gene Fusions in Non-Small-Cell Lung Cancer: Real-World Screening Data of 1068 Unselected Patients.

(1) Background: The main objectives of our study are (i) to determine the prevalence of NTRK (neurotrophic tyrosine kinase) fusions in a routine diagnostic setting in NSCLC (non-small cell lung cancer) and (ii) to investigate the feasibility of screening approaches including immunohistochemistry (IHC) as a first-line test accompanied by fluorescence in situ hybridization (FISH) and RNA-(ribonucleic acid-)based next-generation sequencing (RNA-NGS). (2) Methods: A total of 1068 unselected consecutive patients with NSCLC were screened in two scenarios, either with initial IHC followed by RNA-NGS (n = 973) or direct FISH testing (n = 95). (3) Results: One hundred and thirty-three patients (14.8%) were IHC positive; consecutive RNA-NGS testing revealed two patients (0.2%) with NTRK fusions (NTRK1-EPS15 (epidermal growth factor receptor pathway substrate 15) and NTRK1-SQSTM1 (sequestosome 1)). Positive RNA-NGS was confirmed by FISH, and NTRK-positive patients benefited from targeted treatment. All patients with direct FISH testing were negative. RNA-NGS- or FISH-positive results were mutually exclusive with alterations in EGFR (epidermal growth factor receptor), ALK (anaplastic lymphoma kinase), ROS1 (ROS proto-oncogene 1), BRAF (proto-oncogene B-Raf), RET (rearranged during transfection) or KRAS (kirsten rat sarcoma viral oncogene). Excluding patients with one of these alterations raised the prevalence of NTRK-fusion positivity among panTrk-(tropomyosin receptor kinase-) IHC positive samples to 30.5%. (4) Conclusions: NTRK fusion-positive lung cancers are exceedingly rare and account for less than 1% of patients in unselected all-comer populations. Both RNA-NGS and FISH are suitable to determine clinically relevant NTRK fusions in a real-world setting. We suggest including panTrk-IHC in a diagnostic workflow followed by RNA-NGS. Excluding patients with concurrent molecular alterations to EGFR/ALK/ROS1/BRAF/RET or KRAS might narrow the target population.