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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 Tumor molecular profile analysis by Next Generation Sequencing technology is currently widely applied in clinical practice and has enabled the detection of predictive biomarkers of response to targeted treatment. In parallel with targeted therapies, immunotherapies are also evolving, revolutionizing cancer therapy, with Programmed Death-ligand 1 (PD-L1), Microsatellite instability (MSI), and Tumor Mutational Burden (TMB) analysis being the biomarkers employed most commonly. Methods In the present study, tumor molecular profile analysis was performed using a 161 gene NGS panel, containing the majority of clinically significant genes for cancer treatment selection. A variety of tumor types have been analyzed, including aggressive and hard to treat cancers such as pancreatic cancer. Besides, the clinical utility of immunotherapy biomarkers (TMB, MSI, PD-L1), was also studied. Results Molecular profile analysis was conducted in 610 cancer patients, while in 393 of them a at least one biomarker for immunotherapy response was requested. An actionable alteration was detected in 77.87% of the patients. 54.75% of them received information related to on-label or off-label treatment (Tiers 1A.1, 1A.2, 2B, and 2C.1) and 21.31% received a variant that could be used for clinical trial inclusion. The addition to immunotherapy biomarker to targeted biomarkers’ analysis in 191 cases increased the number of patients with an on-label treatment recommendation by 22.92%, while an option for on-label or off-label treatment was provided in 71.35% of the cases. Conclusions Tumor molecular profile analysis using NGS is a first-tier method for a variety of tumor types and provides important information for decision making in the treatment of cancer patients. Importantly, simultaneous analysis for targeted therapy and immunotherapy biomarkers could lead to better tumor characterization and offer actionable information in the majority of patients. Furthermore, our data suggest that one in two patients may be eligible for on-label ICI treatment based on biomarker analysis. However, appropriate interpretation of results from such analysis is essential for implementation in clinical practice and accurate refinement of treatment strategy.

Additional file 1: Table S1. Title: Genomic Regions and fusions analyzed with the 24 and 50 gene panels. Additional file 2: Table S2. Title: Gene alterations detected by the 161 gene panel. Description: The frequency of the different mutation types (SNVs, indels, CNV, fusions) for each gene is reported. Additional file 3: Table S3. Title: Alterations identified by the 161 gene panel in the 610 patients analyzed. Description: Genomic alterations and level of evidence of the variants detected in the 610 patients analyzed. The tumor type, age of diagnosis and gender are also reported. Additional file 4: Table S4. Title: Biomarker's summary in the 610 patients included in the study. Description: Patients' categorization based on TIER classification of their most clinically significant variant and immunotherapy biomarkers’ results are reported. Additional file 5: Figure S1. Title and description: Pancreatic cancer patients' categorization based on TIER classification of their most clinically significant variant. A. Pancreatic cancer patients' categorization based on TIER classification of their most clinically significant variant. Patients were categorized in the following categories: No Biomarker: Patients with no biomarker available, 1B: Patients harboring biomarkers with strong evidence of correlation to treatment, 2C.1 KRAS: Patients with a single finding in the KRAS gene, 2C.1: Patients with biomarkers related to off-label treatment. B. Percentage of patients with On-label and off-label mutations identified and the type of alterations detected. Genes of the homologous recombination complex are labeled in blue. Additional file 6: Figure S2. Title and description: Lung cancer patients' categorization based on TIER classification. A. Lung cancer patients' categorization based on TIER classification of their most clinically significant variant. The following categories were used:, 1A.1: Patients with biomarkers related to on-label treatment, 1B: Patients harboring biomarkers with strong evidence of correlation to treatment, 2C.1: Patients with biomarkers related to off-label treatment, 1A.2R; 2C.1: Patients harboring a KRAS mutation related to resistance to treatment plus an off-label, 1A.2R: Patients harboring a KRAS mutation related to EGFR TKIs resistance, 2.C.2: Patients B. % of patients with On-label and off-label mutations identified and the type of alterations detected. Genes of the homologous recombination complex are labeled in blue. Additional file 7: Figure S3. Title and description: Breast cancer patients' categorization based on the TIER classification. A. Patients' categorization based on TIER classification of their most clinically significant variant. The following categories were used: No Biomarker: Patients with no biomarker available, 1A.1: Patients with biomarkers related to on-label treatment, 2C.1: Patients with biomarkers related to off-label treatment, 2C.2: Patients with biomarkers related to clinical trials, 2D: Patients with biomarkers with preclinical evidence. B. Percentage of patients with On-label and off-label mutations identified and the type of alterations detected. Genes of the homologous recombination complex are labeled in blue. Additional file 8: Figure S4. Title and description: Colorectal cancer patients' categorization based on TIER classification. A. Patients' categorization based on TIER classification of their most clinically significant variant. Patients were categorized in the following categories: No Mutation, 1A.1: Patients with no mutation in KRAS/NRAS genes with or without other variations, 1A.2: Patients with biomarkers included in professional guidelines, 1A.1R: Patients harboring either KRAS or NRAS mutations related to resistance to treatment. B. % of patients with on-label and off-label mutations identified and the type of alterations detected. Additional file 9: Table S5. Title and description: Alterations that would have been detected if two hotspot panels of 24 and 50 genes respectively, had been used in the 610 patients analyzed. Additional file 10: Table S6. Title and description: Simulation results of the alterations that would have been identified if the gene set of the 161 gene NGS panel was used in the PCAWG samples. Additional file 11: Table S7. Title and description: Simulation results of the alterations that would have been identified if the gene set of the 514 gene NGS panel was used in the PCAWG samples. Additional file 12: Table S8. Title and Description: TMB, PD-L1 and MSI results in the 395 patients with at least one immunotherapy biomarker requested. Additional file 13: Table S9. Title and Description: Patients with suspicious hereditary pathogenic findings detected in tumor tissue.

Background: Tumor molecular profile is of great importance for the detection of biomarkers of response to targeted treatment due to the increased availability, with concomitant reduction of cost, of Next Generation Sequencing technology (NGS). In parallel to targeted therapies’, immunotherapies are also evolving, revolutionizing cancer therapy, with Programmed Death-ligand 1 (PD-L1), Microsatellite Instability (MSI), and Tumor Mutational Burden (TMB) analysis being the biomarkers employed most commonly.Methods: In the present study, a 161 gene NGS panel, containing the majority of clinically significant genes for cancer treatment selection, was used for tumor molecular profile analysis. A variety of tumor types have been analyzed, including aggressive and hard to treat cancers such as pancreatic cancer. Besides, the clinical utility of immunotherapy biomarkers (TMB, MSI, PD-L1), was also studied.Results: Molecular profile analysis was conducted in 610 cancer patients, while in 393 of them a at least one biomarker for immunotherapy response was requested. At least one actionable alteration was detected in 77.70% of the patients. 54.59% of them received information related to on-label or off-label treatment (Tiers 1A.1, 1A.2, 2B, and 2C.1) and 21.31% received a variant that could be used for clinical trial inclusion. The addition to immunotherapy biomarker to targeted biomarkers’ analysis in 191 cases increased the number of patients with an on-label treatment recommendation by 22.40%, while an option for on-label or off-label treatment was provided in 71.35% of the cases. Conclusions: Tumor molecular profile analysis by NGS is a first-tier methodology for a variety of tumor types, which provides critical information for treatment decision making in cancer patients. Importantly, simultaneous analysis for targeted therapy and immunotherapy biomarkers could lead to a better tumor characterization and provide actionable information in the majority of patients. Moreover, our results indicate that one in two patients is eligible for ICI treatment based on the biomarkers’ analysis. However, when these analyses are performed, the challenge is their implementation in clinical practice. Multidisciplinary patient management is critical to the refinement of the strategy incorporating such information.

Gastrointestinal malignancies are among the leading causes of cancer-related deaths worldwide. Like all human malignancies they are characterized by accumulation of mutations which lead to inactivation of tumor suppressor genes or activation of oncogenes. Advances in Molecular Biology techniques have allowed for more accurate analysis of tumors' genetic profiling using new breakthrough technologies such as next generation sequencing (NGS), leading to the development of targeted therapeutical approaches based upon biomarker-selection. During the last 10 years tremendous advances in the development of targeted therapies for patients with advanced cancer have been made, thus various targeted agents, associated with predictive biomarkers, have been developed or are in development for the treatment of patients with gastrointestinal cancer patients. This review summarizes the advances in the field of molecular biomarkers in tumors of the gastrointestinal tract, with focus on the available NGS platforms that enable comprehensive tumor molecular profile analysis.

BackgroundAnalysis of circulating tumor nucleic acids in plasma of Non-Small Cell Lung Cancer (NSCLC) patients is the most widespread and documented form of "liquid biopsy" and provides real-time information on the molecular profile of the tumor without an invasive tissue biopsy.MethodsLiquid biopsy analysis was requested by the referral physician in 121 NSCLC patients at diagnosis and was performed using a sensitive Next Generation Sequencing assay. Additionally, a comparative analysis of NSCLC patients at relapse following EGFR Tyrosine Kinase Inhibitor (TKIs) treatment was performed in 50 patients by both the cobas and NGS platforms.ResultsAt least one mutation was identified in almost 49% of the cases by the NGS approach in NSCLC patients analyzed at diagnosis. In 36 cases with paired tissue available a high concordance of 86.11% was observed for clinically relevant mutations, with a Positive Predictive Value (PPV) of 88.89%. Furthermore, a concordance rate of 82% between cobas and the NGS approach for the EGFR sensitizing mutations (in exons 18, 19, 21) was observed in patients with acquired resistance to EGFR TKIs, while this concordance was 94% for the p.T790M mutation, with NGS being able to detect this mutation in three 3 additional patients.ConclusionsThis study indicates the feasibility of circulating tumor nucleic acids (ctNA) analysis as a tumor biopsy surrogate in clinical practice for NSCLC personalized treatment decision making. The use of new sensitive NGS techniques can reliably detect tumor-derived mutations in liquid biopsy and provide clinically relevant information both before and after targeted treatment in patients with NSCLC. Thus, it could aid physicians in treatment decision making in clinical practice.

It has been reported that certain patients with non-small-cell lung cancer (NSCLC) that harbor activating somatic mutations within the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene may be effectively treated using targeted therapy. The use of EGFR inhibitors in patient therapy has been demonstrated to improve response and survival rates; therefore, it was suggested that clinical screening for EGFR mutations should be performed for all patients. Numerous clinicopathological factors have been associated with EGFR and Kirsten-rat sarcoma oncogene homolog (KRAS) mutational status including gender, smoking history and histology. In addition, it was reported that EGFR mutation frequency in NSCLC patients was ethnicity-dependent, with an incidence rate of ~30% in Asian populations and ~15% in Caucasian populations. However, limited data has been reported on intra-ethnic differences throughout Europe. The present study aimed to investigate the frequency and spectrum of EGFR mutations in 1,472 Greek NSCLC patients. In addition, KRAS mutation analysis was performed in patients with known smoking history in order to determine the correlation of type and mutation frequency with smoking. High-resolution melting curve (HRM) analysis followed by Sanger sequencing was used to identify mutations in exons 18-21 of the EGFR gene and in exon 2 of the KRAS gene. A sensitive next-generation sequencing (NGS) technology was also employed to classify samples with equivocal results. The use of sensitive mutation detection techniques in a large study population of Greek NSCLC patients in routine diagnostic practice revealed an overall EGFR mutation frequency of 15.83%. This mutation frequency was comparable to that previously reported in other European populations. Of note, there was a 99.8% concordance between the HRM method and Sanger sequencing. NGS was found to be the most sensitive method. In addition, female non-smokers demonstrated a high prevalence of EGFR mutations. Furthermore, KRAS mutation analysis in patients with a known smoking history revealed no difference in mutation frequency according to smoking status; however, a different mutation spectrum was observed.

Non‑small cell lung cancer (NSCLC) is the most common type of lung cancer and a tumor with a broad spectrum of targeted therapies already available or in clinical trials. Thus, molecular characterization of the tumor using next generation sequencing (NGS) technology, has become a key tool for facilitating treatment decisions and the clinical management of NSCLC patients. The performance of a custom 23 gene multiplex amplification hot spot panel, based on Ion AmpliSeq™ technology, was evaluated for the analysis of tumor DNA extracted from formalin-fixed and paraffin-embedded (FFPE) tissues. Furthermore, the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel was used for fusion RNA transcript analysis. The mutation spectrum of the tumors was determined in a cohort of 502 patients with NSCLC using the aforementioned targeted gene panels. The panel used for tumor DNA analysis in this study exhibited high rates (100%) of sensitivity, specificity and reproducibility at a mutation allelic frequency of 3%. At least one DNA mutation was detected in 374 patients (74.5%) and an RNA fusion was identified in 16 patients, (3.2%). In total, alterations in a cancer-driver gene were identified (including point mutations, gene rearrangements and MET amplifications) in 77.6% of the tumors tested. Among the NSCLC patients, 23% presented a mutation in a gene associated with approved or emerging targeted therapy. More specifically, 13.5% (68/502) presented a mutation in a gene with approved targeted therapy (EGFR, ALK, ROS1) and 9.4% (47/502) had an alteration in a gene related to emerging targeted therapies (ERBB2, BRAF, MET and RET). Furthermore, 51.6% of the patients had a mutation in a gene that could be related to an off label therapy or indicative for access to a clinical trial. Thus, the targeted NGS panel used in this study is a reliable approach for tumor molecular profiling and can be applied in personalized treatment decision making for NSCLC patients.

Germline mutations in the BRCA1 and BRCA2 genes are associated with hereditary predisposition to breast and ovarian cancer. Sensitive and accurate detection of BRCA1 and BRCA2 mutations is crucial for personalized clinical management of individuals affected by breast or ovarian cancer, and for the identification of at-risk healthy relatives. We performed molecular analysis of the BRCA1 and BRCA2 genes in 898 Greek families, using Sanger sequencing or Next Generation Sequencing for the detection of small insertion/deletion frameshift, nonsynonymous, truncating and splice-site alterations and MLPA for the detection of large genomic rearrangements. In total, a pathogenic mutation was identified in 12.9% of 898 families analyzed. Of the 116 mutations identified in total 9% were novel and 14.7% were large genomic rearrangements. Our results indicate that different types of mutational events in the BRCA1 and BRCA2 genes are responsible for the hereditary component of breast/ovarian cancer in the Greek population. Therefore the methodology used in the analysis of Greek patients must be able to detect both point and small frameshift mutations in addition to large genomic rearrangements across the entire coding region of the two genes.

e23193 Background: Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and a tumor with broad spectrum of targeted therapies already available or in clinical trials. Thus, molecular characterization of the tumor using the Next Generation Sequencing (NGS) technology, has become a key tool for NSCLC patients’ treatment decision and clinical management. Methods: The performance of a custom 23 gene multiplex amplification hot spot panel, based on Ion AmpliSeq Technology, was evaluated for the analysis of tumor DNA extracted from FFPE (Formalin Fixed Parraffin Embedded) tissue. Furthermore the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel was used for fusion RNA transcript analysis. Tumors’ mutation spectrum was determined in a cohort of 502 patients with NSCLC using the aforementioned targeted gene panels. Results: The panel used for tumor DNA analysis in this study exhibit high rates (100%) of sensitivity, specificity and reproducibility at a mutation frequency of 3%. At least one DNA mutation was detected in 374 patients (74.5%) and an RNA fusion was identified in 16 patients, (3.2%). In total, alterations in a cancer driver gene were identified (including point mutations, gene rearrangements and MET amplifications) in 77.6% of the tumors tested. Among the NSCLC patients, 13.5% (68/502) presented a mutation in a gene with approved targeted therapy (EGFR, ALK ROS1) and 9.4% had an alteration in a gene related to emerging targeted therapies according the NCCN guidelines. These alterations include ERBB2, BRAF and MET mutations, MET amplification and RET rearrangements. The remaining 51.6% of the patients had a mutation in a gene that could be related to an off label therapy or give them access to a clinical trial. Conclusions: Thus the NGS panel validated is a reliable approach of clinical applicability for tumor molecular profile detection in NSCLC patients.

It has been reported that certain patients with non-small-cell lung cancer (NSCLC) that harbor activating somatic mutations within the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene may be effectively treated using targeted therapy. The use of EGFR inhibitors in patient therapy has been demonstrated to improve response and survival rates; therefore, it was suggested that clinical screening for EGFR mutations should be performed for all patients. Numerous clinicopathological factors have been associated with EGFR and Kirsten-rat sarcoma oncogene homolog (KRAS) mutational status including gender, smoking history and histology. In addition, it was reported that EGFR mutation frequency in NSCLC patients was ethnicity-dependent, with an incidence rate of ~30% in Asian populations and ~15% in Caucasian populations. However, limited data has been reported on intra-ethnic differences throughout Europe. The present study aimed to investigate the frequency and spectrum of EGFR mutations in 1,472 Greek NSCLC patients. In addition, KRAS mutation analysis was performed in patients with known smoking history in order to determine the correlation of type and mutation frequency with smoking. High-resolution melting curve (HRM) analysis followed by Sanger sequencing was used to identify mutations in exons 18–21 of the EGFR gene and in exon 2 of the KRAS gene. A sensitive next-generation sequencing (NGS) technology was also employed to classify samples with equivocal results. The use of sensitive mutation detection techniques in a large study population of Greek NSCLC patients in routine diagnostic practice revealed an overall EGFR mutation frequency of 15.83%. This mutation frequency was comparable to that previously reported in other European populations. Of note, there was a 99.8% concordance between the HRM method and Sanger sequencing. NGS was found to be the most sensitive method. In addition, female non-smokers demonstrated a high prevalence of EGFR mutations. Furthermore, KRAS mutation analysis in patients with a known smoking history revealed no difference in mutation frequency according to smoking status; however, a different mutation spectrum was observed.

e22178 Background: NSCLC patients harboring activating somatic mutations within the tyrosine kinase (TK) domain of the epidermal growth factor receptor (EGFR) significantly benefit from EGFR target...

e12536 Background: Approximately 5-10% of breast cancer cases are hereditary. Germline mutations in the BRCA1 and BRCA2genes are responsible for 80% of inherited breast cancer cases. Carriers of su...

It has been proposed that differentiation between M. bovis and M. tuberculosis is possible by using a PCR assay for the 500bp fragment present only in the M. bovis genome.Forty clinical samples and 16 clinical isolates from the Department of Microbiology, as well as 4 clinical isolates obtained from another laboratory, were tested for the purpose of this study. As controls we tested 2 M. bovis (M. bovis BCG Pasteur TMC1011 and M. bovis BCG Copenhagen), 1 H37Rv M. tuberculosis strain, 2 M. avium (ATCC15765 and ATCC1975, respectively) and 1 M. paratuberculosis (ATCC19698) strains.None of the mtp40- negative clinical isolates amplified the 500bp fragment, whereas 4 out of 17 mtp40-positive clinical isolates scored positive for the 500bp fragment. All clinical isolates scored positive for IS6110, mtp40, the pncA and oxyR PCR's. All but one of the clinical isolates amplified the 500bp fragment. Sequence analysis of the pncA and oxyR PCR products revealed the presence of nucleotide C at position 169 and G at position 285 respectively, suggesting M. tuberculosis as the causative agent.Our data suggest that the 500bp PCR fragment is present not only in M. bovis but also in M. tuberculosis.

Background: The levonorgestrel intrauterine system (LNG-IUS) is a contraceptive device that is used for treatment of menorrhagia. The system induces inter-menstrual bleeding within the first few months after insertion. We hypothesized that this bleeding might be associated with a change in vascular development. Methods: A randomized, controlled study was undertaken on 48 women. Results: Hysterectomy specimens were obtained and immunocytochemistry was carried out with antibodies to CD31, {alpha}-smooth muscle actin and myosin. Stereological measurement of blood vessels was also undertaken. Most vessels appeared normal, including the arterioles. Large thin-walled vessels were present in the superficial endometrium of the treated group but were almost completely absent in the controls. The distribution of cytoskeletal markers revealed well-formed basal arterioles with more widespread expression in the superficial stroma than was found in untreated tissue. The volume fraction of blood vessels (P = 0.0001), the number of vessel cross-sections per unit area (P = 0.0003) and the cross-sectional diameters of the largest vascular lumens (P = 0.0001) were significantly increased following treatment with LNG-IUS. However, there was no difference in the median values of vessel diameter or the vascular surface density. Conclusion: These findings suggest that the LNG has a localized effect on some vessels within the superficial endometrium.