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Background: Comprehensive tumor analysis by Next Generation Sequencing (NGS) is imperative for targeted and immunotherapy related biomarkers’ analysis in metastatic breast cancer enabling personalization of cancer treatment in these patients. In parallel with targeted therapies, immunotherapies are also evolving, revolutionizing cancer therapy. Thus, biomarkers such as. Microsatellite Instability (MSI), and Tumor Mutational Burden (TMB) are commonly employed in NGS analysis due to their correlation to Immune Checkpoint inhibitors treatment.Methods: In the present study, tumor molecular profile analysis was performed in 73 metastatic breast cancer patients using a broad NGS assay, which analyzes more than 500 unique cancer related genes and is able to detect relevant single nucleotide variations (SNVs), indels, copy number variations (CNVs) and gene fusions, in addition to TMB and MS) simultaneously. Results: An actionable alteration was detected in 79.45% of the patients. 35.62% of them had at least one alteration with an on label treatment associated and 10.96% received information related to an off-label treatment (Tiers 1A.1 and 2C.1 respectively). Additionally, 31.51% of the tumors harbored an alteration that could be used for clinical trial inclusion (Table 1). The most prevalent altered gene in these patients was the PIK3CA gene, with 27.40% mutation rate. Moreover, in 11 cases (15.07%) the mutation detected was in a gene involved in the Homologous recombination (HR) pathway, with evidence of response to PARP inhibitors. BRCA1/2 genes were as expected the most prominent HR mutated genes (5 out of 11 HR mutations), while the remaining HR mutations involved other HR genes (ATM, BLM, CHEK2, FANCM, RAD50). 24.66% of the tumors had a TMB value >10muts/MB and thus were eligible for ICI treatment. The addition of TMB to targeted biomarkers’ analysis increased the number of patients with an on-label treatment recommendation by 8.22%. Conclusions: Tumor molecular profile analysis using NGS, in real world samples, is a first-tier method for metastatic breast cancer and provides important information for decision making in the treatment of such patients. Importantly, simultaneous analysis for targeted therapy and immunotherapy biomarkers could lead to better tumor characterization and offered actionable information in 9 out of 10 patients tested. However, appropriate interpretation of results from such analysis is essential for implementation in clinical practice and accurate refinement of the treatment strategy.. Table 1.Biomarker's summary in the 73 patients included in the studyPercentage of patients with Tier1 variantsPercentage of patients with 2C.1 variantsPercentage of patients with 2C.2/2D variantsPercentage of patients with Tier 3 variantsPercentage of patients with TMB>10muts/MBMEDIAN TMBPercentage of MSI-High patients35.62% (26/73)10.96% (8/73)12.33% (9/73)0% (0/73)24.66% (18/73)5.050% (0/73) Citation Format: Eirini Papadopoulou, Nikolaos Tsoulos, Vasiliki Metaxa-Mariatou, Aikaterini Tsantikidi, Georgios Kapetsis, Chrysiida Florou-Chatzigiannidou, Sonia Maravelaki, Evgenia Bourkoula, Dimitrios Fotiou, Georgios Tsaousis, Nikolaos Touroutoglou, Dimitrios Trafalis, Ioannis Boukovinas, Ioannis Varthalitis, Zacharenia Saridaki, Charalampos Zoublios, Eleni Galani, George Papatsibas, Christos Papadimitriou, Tania Zlatintsi, Polixenia Iorga, Bülent Orhan, Sualp Tansan, Tahsin Özatlı, George Nasioulas. Comprehensive tumor analysis by NGS in metastatic breast cancer patients [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-01.

Background Classification of splicing variants (SVs) in genes associated with hereditary cancer is often challenging. The aim of this study was to investigate the occurrence of SVs in hereditary cancer genes and the clinical utility of RNA analysis. Material and methods 1518 individuals were tested for cancer predisposition, using a Next Generation Sequencing (NGS) panel of 36 genes. Splicing variant analysis was performed using RT-PCR and Sanger Sequencing. Results In total, 34 different SVs were identified, 53% of which were classified as pathogenic or likely pathogenic. The remaining 16 variants were initially classified as Variant of Uncertain Significance (VUS). RNA analysis was performed for 3 novel variants. Conclusion The RNA analysis assisted in the reclassification of 20% of splicing variants from VUS to pathogenic. RNA analysis is essential in the case of uncharacterized splicing variants, for proper classification and personalized management of these patients.

zeta-chain associated protein 70 and CD38 combined predict the time to first treatment in patients with chronic lymphocytic leukemia BACKGROUND. xi-Chain associated protein (ZAP)-70 has been proposed as a surrogate marker for immunoglobulin heavy-chain variable region (IgVH) mutation in chronic lymphocytic leukemia (CLL), but it is still not clear whether it is an independent prognostic factor. METHODS. The authors evaluated ZAP-70 expression by flow cytometry in 201 untreated patients and correlated ZAP-70 levels with CD38 expression, genetic abnormalities detected by fluorescence in situ hybridization (FISH), and the time from diagnosis to first treatment. RESULTS. Fifty-seven patients (28%) were positive for ZAP-70 (>= 20%). Positive ZAP-70 status was associated with advanced disease stage, atypical morphology, CD38-positive status, trisomy 12, del(6q), or no detectable abnormalities; negative ZAP-70 status was correlated with del(13q) as a sole abnormality. The treatment-free interval (TFI) was 17.7 months for ZAP-70-positive patients and 44.6 months for ZAP-70-negative patients (P = 7%, and the absence of del(13q) as a sole abnormality as independent factors for short TFI. Excluding FISH, ZAP-70 status acquired independent prognostic value along with CD38 status. The authors proposed a risk model that combines ZAP-70 and CD38 to identify patients who are likely to progress. When both markets were positive, the TFI was 12 months; when both were negative, the median TFI was 54 months; a median TFI of 26 months was observed in patients who had discordant results (P < 0.00001). CONCLUSIONS. The current findings suggested that both ZAP-70 and CD38 should be tested prospectively in all patients with early-stage CLL. (c) 2005 American Cancer Society.

One of the most intriguing features of chronic lymphocytic leukemia (CLL) is its clinical heterogeneity. The mutational status of immunoglobulin heavy chain variable region (IgVH) genes is one of the most powerful predictors of overall survival (OS) and progression-free survival (PFS). The expression of the tyrosine kinase ZAP-70 in CLL cells has been proposed as a surrogate marker for the mutational status of IgVH genes. Recent reports suggest that ZAP-70 over-expression is an independent prognostic factor for shorter OS and PFS. There is little information about correlation between ZAP-70 expression and other clinical and biological features in CLL. The aims of this study are 1) to evaluate prospectively ZAP-70 expression in a series of CLL patients; 2) to correlate this with clinical stage, lymphocyte morphology, CD38 expression and chromosomal abnormalities detected by FISH and 3) to analyze the independent prognostic value of ZAP-70 in predicting time to first treatment (treatment free interval, TFI). ZAP-70 expression was analyzed using four-colour flow cytometry (as described by Crespo et al, NEJM2003; 348:1764–75). 201 previously untreated CLL patients were evaluated at diagnosis (32%) or during the disease course. Amongst them, 143 patients were entered in the LRF CLL4 trial. Clinical stage at the time of the study was A in 92 patients (A progressive in 46), B in 63 and C in 46. 28% were ZAP-70 positive (+) (cut-off ≥ 20% of CD5/CD19+ cells). ZAP-70 positivity was associated with prevalence of stage B/C at diagnosis (p=0.004), CLL with >10% prolymphocytes (CLL/PL) or atypical morphology (p