Molecular endoscopy with next-generation sequencing improves diagnosis of cholangiocarcinoma in patients with extrahepatic biliary strictures.

The accuracy of current endoscopic modalities for diagnosing cholangiocarcinoma in primary sclerosing cholangitis (PSC) is suboptimal. To evaluate the comparative effectiveness of endoscopic retrograde cholangiopancreatography (ERCP)-based modalities, independently or in combination, for the diagnosis of cholangiocarcinoma in patients with PSC-induced biliary strictures. Searches of PubMed, EMBASE, Web of Science and the Cochrane Library databases were performed through December 2015. Measured outcomes included sensitivity and specificity of all diagnostic modalities independently or in combination. A bivariate model was used to compute the pooled sensitivity and specificity, and to plot the summary receiver operating characteristics curve with summary point and corresponding 95% confidence interval (95% CI). A logistic regression model was used to impute the incremental performance of combining two diagnostic tests. Twenty-one studies met inclusion criteria: 13 on bile duct brushing for cytology, 7 on fluorescence insitu hybridisation (FISH), 2 on probe-based confocal laser endomicroscopy, and 4 on single-operator cholangioscopy with targeted biopsies. Single-operator cholangioscopy with targeted biopsies was the most accurate diagnostic modality at 96% (95% CI, 94-97%). The pooled sensitivity and specificity of single-operator cholangioscopy for diagnosis of cholangiocarcinoma in patients with PSC was 65% (95% CI, 35-87%) and 97% (95% CI, 87-99%), respectively. The pooled diagnostic odds ratio to detect cholangiocarcinoma was 59 (95% CI, 10-341). Single-operator cholangioscopy with targeted biopsies appears to be the most accurate ERCP-based modality for diagnosing cholangiocarcinoma in primary sclerosing cholangitis. However, future large, well-designed comparative diagnostic studies are warranted to validate these findings.

Advances in the diagnosis of cholangiocarcinoma in patients with primary sclerosing cholangitis

Targeted deep sequencing of myeloma in a multi-institutional clinical workflow

Introduction: Fluorescence in situ hybridization (FISH) is the gold standard for evaluating ERBB2 amplification in breast cancer (BC) when HER2 status is equivocal by immunohistochemistry, per ASCO guidelines (15–20% of cases). Next-generation sequencing (NGS) can confirm the HER2 status of BC samples and, in turn, help reduce the incidence of equivocal samples. Here, we examined the correlation between FISH and NGS in annotating HER2 status of BC samples and evaluated the clinical utility of NGS as a support tool for improving analysis outcomes. Methods: The BostonGene internal breast cancer (total n=358, FISH annotation=124) and TCGA-BRCA (total n=972, FISH annotation n=285) cohorts were assessed together for ERBB2 expression via RNA sequencing and ERBB2 mutation and amplification via DNA sequencing. High ERBB2 gene expression was set at >8.5 log2(TPM+1)1. For NGS, Gain was defined as an increase of one gene copy relative to sample ploidy, while Amplification referred to an increase of two or more gene copies compared to sample ploidy. Samples were assessed for FISH positivity (+) per ASCO/CAP guidelines. Results: There was substantial agreement between FISH and NGS analysis for both cohorts combined (Cohen’s kappa=0.57). All FISH+ samples had significantly higher ERBB2 expression (n=71, median (med)=8.8) than all FISH- samples (n=338, med=6.6, U-statistics (stats), p<0.001). Among the FISH+ samples, 53% (n=37) had high ERBB2 expression. Samples with amplification as shown by NGS had significantly higher ERBB2 expression (n=35, med=9.6) than non-amplified samples (n=335, med=6.6, U-stats, p<0.001). High ERBB2 expression was observed in 31% (n=23 of 74) of samples with ERBB2 gain or amplification. FISH+ samples with ERBB2 gain had significantly higher ERBB2 expression (n=14, med=10.5) than FISH- samples (n=13, med=7.9, U-stats, p<0.001). NGS also detected 23 unique ERBB2 mutations, among which 18 were classified as missense, 4 as frameshift, and 9 as gain-of-function. Interestingly, 20 non-amplified samples with ERBB2 mutation exhibited significantly higher ERBB2 expression (med=7.4) than non-amplified samples with wildtype ERBB2 (n=999, med=6.9, U-stats, p=0.004). Conclusion: Our study demonstrated substantial agreement between FISH and NGS (Cohen’s kappa=0.57) in evaluating HER2 status in BC, highlighting NGS as a viable support tool for assessing HER2 status, particularly in equivocal cases. First, NGS reliably detected significantly higher ERBB2 expression in FISH+ samples than in FISH- samples. Next, it detected ERBB2 mutations in non-amplified samples that were not covered by FISH. Since these mutations may be associated with increased ERBB2 expression, they are clinically relevant and warrant further study. Last but not least, NGS detected focal ERBB2 amplifications and gains at a higher resolution than FISH. While associated with varied FISH results, these events consistently concurred with higher ERBB2 expression, reflecting the heterogeneous nature of HER2 amplification in BC that FISH does not take into account. The ability of NGS to identify these nuanced genomic alterations yields valuable insights into the tumor biology and potential resistance mechanisms of BC that are crucial for designing personalized treatment strategies and improving patient outcomes. Citation Format: Viktor Smirnov, Polina Turova, Vladimir Kushnarev, Gleb Khegai, Sheila T. Yong, Anna Butusova, Nikita Kotlov, Konstantin Chernyshov. Evaluating the Correlation Between FISH and NGS in Assessing ERBB2 Alterations in Breast Cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-07-11.

PGT-A, a new dance for two couples: NGS with FISH and trophectoderm cells with blastocelic fluid

Accessing Genomic Alternations in Chronic Lymphocytic Leukemia Using an NGS-Based Comprehensive Genomic Profiling Assay

e14021 Background: Homozygous deletion of CDKN2A (Homdel) has been included in the WHO CNS5 classification as a grade 4 definition in IDH mutated gliomas without the 1p-/19q-codeletion ( IDH mu-noncodel). Hemizygous CDKN2A deletion (Hemdel) has also been reported to be associated with poor prognosis. The identification methods for CDKN2A deletion include next generation sequencing (NGS), methylation array, fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC). However, at present, there is no gold standard threshold for FISH to judge the homozygous and hemizygous deletion. Methods: We collected formalin fixed paraffin-embedded (FFPE) tissue samples from 99 patients with adult-type diffuse glioma and all samples were tested using DNA-based NGS profiling (Simceredx). In addition, CDKN2A status was also detected by FISH and IHC (P16 and MTAP). Based on the CDKN2A status determined by NGS, the FISH were evaluated using ROC curves, including Area Under Curve (AUC), sensitivity, specificity, and cut-off values of Homdel and Non-Homdel, and deletion (Homdel and Hemdel) and non-deletion. The consistency of CDKN2A status identified by NGS, FISH IHC-P16 and IHC-MTAP was compared by Kappa test and Cramer V coefficient. Results: In 99 enrolled patients, NGS results showed 49 patients with CDKN2A Homdel, 18 patients with CDKN2A Hemdel, and 32 patients with CDKN2A Normal. There were no significant differences in age, gender, location of occurrence, IDH mutation status, or receiving chemoradiotherapy among the three groups. There were statistically significant differences in cancer subtype (p value=0.015) and grade (p value=0.010) among the three groups: oligodendroglioma and 2-3 grade was more likely to be normal in CDKN2A and grade 4 significantly predisposed to be Homdel. The ROC curve determines that cutoff of Homdel and Non- Homdel by FISH is 23%, and greater than 23% is Homdel. The ROC curve showed that the AUC of FISH method using 23% cutoff is 0.958, and the sensitivity and specificity were 85.11% and 100%, respectively. The consistency between FISH and NGS was 0.854. For the inconsistent samples, all NGS results were Homdel, and all FISH were deletion except one. Compared the results with FISH, we found that the results of NGS and IHC-P16 were more consistent. The ROC curve determines that the cutoff of deletion and non- deletion by FISH is 39%, and greater than 39% is deletion. The ROC curve showed that the AUC of FISH method with 39% cutoff is 0.962 and the sensitivity and specificity were 95.31% and 90.62%, respectively. Consistency comparison of the four methods showed that the consistency between FISH and NGS in determining Homdel and Non-Homdel, and deletion and non-deletion were the highest, 0.841 and 0.86, respectively. Conclusions: In our study, four methods were used to determine the status of CDKN2A, and results showed that the consistency between FISH and NGS was the highest.

In our review, most techniques (except G-banding) appeared to have good sensitivity (few false negatives) for detection of 1p/19q codeletions in glioma against both FISH and PCR-based LOH as a reference standard. However, we judged the certainty of the evidence low or very low for all the tests. There are possible differences in specificity, with both NGS and SNP array having high specificity (fewer false positives) for 1p/19q codeletion when considered against FISH as the reference standard. The economic analysis should be interpreted with caution due to the small number of studies.

Comparison of fluorescence in situ hybridization and cytology for the accurate detection of malignant biliary strictures, with emphasis on unusual results.

Diagnostic Role of Serum CA 19-9 for Cholangiocarcinoma in Patients With Primary Sclerosing Cholangitis

P1.02-025 Evaluation of NGS and RT-PCR Methods for ALK Assessment in European NSCLC Patients: Results from the ETOP Lungscape Project

Identification of targetable molecular changes is essential for selecting appropriate treatment in patients with advanced lung adenocarcinoma. Methods: In this study, a Sanger sequencing plus Fluorescence In Situ Hybridization (FISH) sequential approach was compared with a Next-Generation Sequencing (NGS)-based approach for the detection of actionable genomic mutations in an experimental cohort (EC) of 117 patients with advanced lung adenocarcinoma. Its applicability was assessed in small biopsies and cytology specimens previously tested for epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) mutational status, comparing the molecular changes identified and the impact on clinical outcomes. Subsequently, an NGS-based approach was applied and tested in an implementation cohort (IC) in clinical practice. Using Sanger and FISH, patients were classified as EGFR-mutated (n = 22, 18.8%), ALK-mutated (n = 9, 7.7%), and unclassifiable (UC) (n = 86, 73.5%). Retesting the EC with NGS led to the identification of at least one gene variant in 56 (47.9%) patients, totaling 68 variants among all samples. Still, in the EC, combining NGS plus FISH for ALK, patients were classified as 23 (19.7%) EGFR; 20 (17.1%) KRAS; five (4.3%) B-Raf proto-oncogene (BRAF); one (0.9%) Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2); one (0.9%) STK11; one (0.9%) TP53, and nine (7.7%) ALK mutated. Only 57 (48.7%) remained genomically UC, reducing the UC rate by 24.8%. Fourteen (12.0%) patients presented synchronous alterations. Concordance between NGS and Sanger for EGFR status was very high (κ = 0.972; 99.1%). In the IC, a combined DNA and RNA NGS panel was used in 123 patients. Genomic variants were found in 79 (64.2%). In addition, eight (6.3%) EML4-ALK, four (3.1%), KIF5B-RET, four (3.1%) CD74-ROS1, one (0.8%) TPM3-NTRK translocations and three (2.4%) exon 14 skipping MET Proto-Oncogene (MET) mutations were detected, and 36% were treatable alterations. Conclusions: This study supports the use of NGS as the first-line test for genomic profiling of patients with advanced lung adenocarcinoma.

Purpose: Despite the low prevalence of oncogenic neurotrophin receptor kinase (NTRK) fusion among most solid tumors, the encouraging results of tropomyosin receptor kinase (Trk) inhibitors such as larotrectinib and entrectinib have prompted the screening of eligible patients. Here, we aimed to report the prevalence of NTRK fusion in the Chinese solid tumor patients and to compare NTRK fusion evaluation with targeted next-generation sequencing (NGS), fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Methods: A total of 25976 tumor samples from Chinese solid tumor patients were collected between January 2017 and June 2020. All samples were assessed with targeted NGS in a CLIA-certified laboratory. NTRK genes fusions were confirmed by FISH (ZytoLight SPEC NTRK1/2/3 Dual Color Break Apart Probe) and/or pan-TRK IHC (clone EPR17341). Results: In total, 47/25976 (0.2%) patients harbored one or more NTRK fusions. NTRK fusions were found in 15 cancer types including thyroid (1/92, 1.1%), sarcoma (3/547, 0.5%), neuroendocrine (1/183, 0.5%), GIST (1/265, 0.4%), endometrium (1/274, 0.4%), esophagus (1/293, 0.3%), colorectum (14/4548, 0.3%), head neck (1/375, 0.3%), lung (16/8950, 0.2%), ovary (1/625, 0.2%), breast (1/706, 0.1%), biliary tract (2/1530, 0.1%), liver (2/2282, 0.1%), stomach (1/1505, 0.1%), and others (1/3801, 0.03%). Among these 47 patients, 11 were further confirmed by both FISH and IHC and 3 were confirmed only by IHC. 36.4% (4/11) of the patients had completely concordant NTRK status (NGS+/FISH+/IHC+). 45.5% (5/11) of the patients had consistency between NGS and FISH (NGS+/FISH+). 71.4% (10/14) of the patients had consistency between NGS and IHC (NGS+/IHC+). Moreover, there were two novel fusions includingTEX10-NTRK2 (NGS+/FISH+/IHC+) in head and neck cancer and PTPN22-NTRK1 (NGS+/IHC+) in ovarian cancer, which is the first case of ovarian cancer with NTRK fusion. Conclusions: NGS was effectively help to screen patients with novel or reported NTRK fusion, who may benefit from NTRK inhibitor therapy. And NGS showed good consistency with IHC. Further prospective study is needed to compare the impact of the NTRK status identified by NGS and IHC on NTRK inhibitors efficacy. Citation Format: Yuan Peng, Hui Chen, Xiaochen Zhao, Zhijun Han, Yuezong Bai. The capability of NTRK fusion detection by next-generation sequencing/FISH/IHC in 25976 solid tumor patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2285.

Addition of chromosomal microarray and next generation sequencing to FISH and classical cytogenetics enhances genomic profiling of myeloid malignancies

3 - Addition of Chromosomal Microarray and Next Generation Sequencing to FISH and Classical Cytogenetics Enhances Genomic Profiling of Myeloid Malignancies