George Koutsodontis

Greek BRCA1 and BRCA2 mutation spectrum: two BRCA1 mutations account for half the carriers found among high-risk breast/ovarian cancer patients

Abstract127 Greek breast/ovarian cancer families were screened for germline BRCA1/2 mutations by dHPLC followed by direct sequencing. Our results indicated 16 and 5 breast/ovarian cancer families bearing deleterious mutations in the BRCA1 and BRCA2 genes, respectively. Two novel BRCA2 germline mutations (G4X and 3783del10) are reported here for the first time. Subsequent compilation of our present findings with previously reported mutation data reveals that in a total of 287 Greek breast/ovarian cancer families, 46 and 13 carry a deleterious mutation in BRCA1 and BRCA2, respectively. It should be noted that two BRCA1 mutations, 5382insC and G1738R, both located in exon 20, account for 46% of the families found to carry a mutation. Based on our mutation analysis results, we propose here a hierarchical, cost-effective BRCA1/2 mutation screening protocol for individuals of Greek ethnic origin. The suggested protocol can impact on the clinical management of breast-ovarian cancer families on a national healthcare system level.

Prognostic significance of PD-L1 expression on circulating tumor cells in patients with head and neck squamous cell carcinoma

Background Successful application of programmed death 1 (PD1) checkpoint inhibitors in the clinic may ultimately benefit from appropriate patient selection based upon predictive biomarkers. Molecular characterization of circulating tumor cells (CTC) is crucial for the investigation of molecular-targeted therapies while predictive biomarkers for response to PD1 checkpoint inhibitors are lacking. We sought to assess whether overexpression of PD-L1 in CTCs could be detected at baseline and at different timepoints during treatment in a prospective cohort of head and neck squamous cell carcinoma (HNSCC) patients and used to predict clinical outcome after treatment with curative intent. Patients and methods We developed a highly sensitive, specific and robust RT-qPCR assay for PD-L1 mRNA expression in EpCAM(+) CTCs. In a prospective cohort of 113 locally advanced HNSCC patients treated with curative intent we evaluated PD-L1 expression in the EpCAM(+) CTC fraction at baseline, after 2 cycles of induction chemotherapy (week 6) and at the end of concurrent chemoradiotherapy (week 15). Results PD-L1 overexpression was found in 24/94 (25.5%) patients at baseline, 8/34 (23.5%) after induction chemotherapy and 12/54 (22.2%) patients at the end of treatment. Patients with CTCs overexpressing PD-L1 at end of treatment had shorter progression-free survival (P = 0.001) and overall survival (P < 0.001). Multivariate analysis revealed that PD-L1 overexpression at end of treatment was independent prognostic factor for progression-free survival and overall survival. The absence of PD-L1 overexpression at the end of treatment was strongly associated with complete response with an odds ratio = 16.00 (95% CI = 2.76-92.72, P = 0.002). Conclusions We demonstrate that detection of CTCs overexpressing PD-L1 is feasible and may provide important prognostic information in HNSCC. Our results suggest that adjuvant PD1 inhibitors deserve evaluation in HNSCC patients in whom PD-L1(+) CTCs are detected at the end of curative treatment.

Identification of two novel mutations in the RET proto-oncogene in the same family.

BACKGROUND Activating germline mutations of the RET gene cause multiple endocrine neoplasia type 2 and familial medullary thyroid carcinoma (FMTC), conditions that are inherited in an autosomal dominant manner. In addition, somatic RET mutations have been identified in a variable proportion (about 30-70%) of sporadic (nonfamilial) MTC cases. METHODS We describe a Greek family with two novel likely pathogenic sequence variants of the RET gene. The first is a C to T transition at position 2458 (c.2458C>T) that causes an arginine to cysteine substitution (p.R820C) in exon 14 in the intracellular region of the kinase. This sequence variant was identified in an apparently healthy woman who had a recently deceased sister with confirmed aggressive MTC (age of onset 37 years). To assess the pathogenicity of this novel missense sequence variant, screening was performed on all available relatives: her two sons, the mother, and a second sister, including an MTC tumor sample from the deceased sister of the proband. At the time of the investigation, no clinical symptoms suggestive of multiple endocrine neoplasia type 2 or MTC were present in any of the individuals screened. RESULTS The c.2458C>T transition was found in one son, the living sister, and the mother. Interestingly, it was not present in the tumor sample from the deceased sister. Instead, an in-frame deletion of 54 nt in exon 10 resulting in a protein missing 18 amino acids from I590 to G608 (c.1766_1819del 54) was found. Both genetic alterations were present in heterozygous state. CONCLUSIONS These data suggest that the novel in-frame deletion was the disease-causing mutation in the deceased sister. The effect of the 2458C>T mutation on the activity of the kinase is under investigation.

Evaluation of the impact of tumor HPV status on outcome in patients with locally advanced unresectable head and neck squamous cell carcinoma (HNSCC) receiving cisplatin, 5-fluorouracil with or without docetaxel: a subset analysis of EORTC 24971 study

Background EORTC 24971 was a phase III trial demonstrating superiority of induction regimen TPF (docetaxel, cisplatin, 5-fluorouracil) over PF (cisplatin/5-fluorouracil), in terms of progression-free (PFS) and overall survival (OS) in locoregionally advanced unresectable head and neck squamous cell carcinomas. We conducted a retrospective analysis of prospectively collected data aiming to evaluate whether only HPV(-) patients (pts) benefit from adding docetaxel to PF, in which case deintensifying induction treatment in HPV(+) pts could be considered. Patients and methods Pretherapy tumor biopsies (blocks or slides) were assessed for high-risk HPV by p16 immunohistochemistry, PCR and quantitative PCR. HPV-DNA+ and/or p16+ tumors were subjected to in situ hybridization (ISH) and HPV E6 oncogene expression qRT-PCR analysis. Primary and secondary objectives were to evaluate the value of HPV/p16 status as predictive factor of treatment benefit in terms of PFS and OS. The predictive effect was analyzed based on the model used in the primary analysis of the study with the addition of a treatment by marker interaction term and tested at two-sided 5% significance level. Results Of 358, 119 pts had available tumor samples and 58 of them had oropharyngeal cancer. Median follow-up was 8.7 years. Sixteen of 119 (14%) evaluable samples were p16+ and 20 of 79 (25%) evaluable tumors were HPV-DNA+. 13 of 40 pts (33%) assessed with HPV-DNA ISH and 12 of 28 pts (43%) assessed for HPV E6 mRNA were positive. The preplanned analysis showed no statistical evidence of predictive value of HPV/p16 status for PFS (P = 0.287) or OS (P = 0.118). Conclusions The incidence of HPV positivity was low in the subset of EORTC 24971 pts analyzed. In this analysis only powered to detect a large treatment by marker interaction, there was no statistical evidence that treatment effect found overall was different in magnitude in HPV(+) or HPV(-) pts. These results do not justify selection of TPF versus PF according to HPV status.

Abstract 3108: PD-L1 expressing circulating tumor cells (CTCs) in patients with head and neck squamous cell carcinoma (HNSCC)

Background: Predictive biomarkers for response to anti-PD1 therapy are lacking. Because therapy with checkpoint inhibitors is cost intensive, noninvasive tools for prediction of responders are of major interest. Methods: The “Liquid Biopsy’’in head and neck squamous cell carcinoma (HNSCC) project involved the isolation of Circulating Tumor Cells (CTC) from patients with HNSCC at baseline, at different time points during treatment and at relapse. Herein, we assessed gene expression of PD-L1 on CTCs in a prospective fashion in a cohort of locally advanced inoperable HNSCC patients treated with curative intent (n = 61), in a second cohort of recurrent/metastatic HNSCC patients (n = 18) and in 20 healthy individuals, used as normal controls. For the quantification of PD-L1mRNA in CTCs, we developed a highly sensitive, specific, and robust RT-qPCR assay that was firstly analytically validated prior to its application in HNSCC patients. Results: In patients with locally advanced disease, 54/61(88.5%) samples were evaluable for CTCs at baseline. Twenty four samples were obtained after induction chemotherapy (IC) and 34(55.7%) at the end of concurrent chemo-radiation. At baseline 22/54(40.7%) pts were found to be positive for PD-L1 overexpression, at the post-IC samples 12/24(50%) patients were positive for PD-L1overexpressionand at the end of treatment, 11/34 (32.4%) patients were positive for PD-L1 overexpression. Patients with PD-L1 positive CTCs at the end of treatment had shorter progression-free survival (PFS) (p = 0.011) and overall survival (OS) (p = 0.004). Multivariate analysis showed that PD-L1 overexpression in patients at the end of treatment was independent prognostic factor of PFS (HR = 2422.4; p = 0.014) and OS (HR = 32.23; p = 0.014). Five R/M patients were found to be positive for PD-L1 out of the 18 (27.8%) at baseline. Conclusions: We demonstrate for the first time that detection of PD-L1+ CTCs at the end of treatment in patients with locally advanced disease is associated with shorter PFS and OS. Serial PD-L1 expression assessment has potential to select and monitor pts for PD-1 checkpoint inhibitors. These data support testing of PD-1 inhibitors in the adjuvant setting in patients with locally advanced HNSCC in whom PD-L1 positive CTCs are detected at the end of treatment. Citation Format: Areti Strati, George Koutsodontis, Ilias Angelidis, Clarence Sasaki, Margaritis Avgeris, Amanda Psyrri, Evi S. Lianidou. PD-L1 expressing circulating tumor cells (CTCs) in patients with head and neck squamous cell carcinoma (HNSCC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3108.

Screening for TSC1 and TSC2 mutations using NGS in Greek children with tuberous sclerosis syndrome

Tuberous Sclerosis Complex (TSC) is a rare neurocutaneous syndrome inherited by an autosomal dominant manner. The disorder is commonly manifested by the presence of multiple benign tumors located in numerous tissues, including the brain, heart, skin and kidneys. Seizures, autism, developmental and behavioral delay, as well as non-neurological phenotypic findings, are suggestive of TSC. The identification of one pathogenic mutation in either the TSC1 or TSC2 genes is considered to be an independent diagnostic criterion. In our study, seventeen Greek patients, 2yo on average, were analyzed for the presence of pathogenic germline mutations in the aforementioned loci by Next-Generation Sequencing. A TSC1/2 gene panel was designed for the molecular diagnosis of the disease. Patients underwent initial diagnosis based on their clinical symptoms, most frequently involving the presence of skin lesions and/or epilepsy. Only one case was familial. Sixteen different genetic alterations were identified in TSC1 and TSC2 genes in fifteen patients, giving a 88% detection rate by employing NGS technology. Overall, most pathogenic mutations (11/15) identified were located in the TSC2 gene with exon 41 being the most frequent. With respect to genotype-phenotype association, no patient TSC1 (+) developed SEGA or renal cysts. No significant differences were observed between different types of TSC2 mutations and any clinical feature. Sequencing also revealed 18 different SNPs across the TSC1 and 20 across the TSC2 genes. This is the first registry of the genetic profile of TSC patients in Greece using a custom-made gene panel as molecular diagnostic tool.

Abstract 1718: RT-qPCR gene expression analysis of CTCs isolated through an epitope-independent enrichment microfluidic device in patients with head and neck squamous cell carcinoma

Background: Molecular characterization of circulating tumor cells (CTCs) is very challenging since these cells are rare, and the amount of available sample for their analysis is very limited. Moreover, CTC are highly heterogeneous and enrichment technologies based on EpCAM expression present the risk of missing EpCAM-negative CTCs. The Parsortix system (ANGLE plc, UK), is a novel microfluidic technology platform designed for marker-independent capture of CTCs. In this study we used for the first time the Parsortix system to isolate CTCs from patients with Head and Neck Squamous Cell carcinoma (HNSCC), and proceeded to downstream molecular characterization through RT-qPCR gene expression analysis. Methods: Peripheral blood samples (10 mL) from head and neck squamous cell carcinoma (HNSCC) patients (n=19) and healthy donors used as a control group (n=10) were used for the isolation of CTCs using the Parsortix device. Enriched CTCs were harvested in Trizol reagent, followed by extraction of total RNA and cDNA synthesis. RT-qPCR was performed in the LightCycler (Roche) for the following gene targets: PD-L1, VIM, TWIST, EGFR, and B2M (used as a reference gene). The expression levels of PD-L1, VIM, TWIST and EGFR were normalized using the 2 -ΔΔCt approach in respect to the expression of B2M. Results: All samples analyzed were of excellent RNA quality as this was evaluated by B2M expression. According to our results, PD-L1 overexpression was detected in 5/19(26.3%) samples, VIM was overexpressed in 3/19 (15.7%) and TWIST-1 in 1/19 (5.3%) sample, while EGFR expression was not detected in any patient (0/19, 0%). These are preliminary results and these percentages may change, since the number of samples that we are analyzing is continuously increasing. Conclusions: This preliminary study is showing for the first time that RT-qPCR can be successfully used for the molecular characterization of CTCs isolated by the label-free Parsortix microfluidic device in HNSCC. Overexpression of individualized immunotherapy important biomarkers such as PD-L1 in CTCs of HNSCC patients could be of significant clinical importance for the selection and follow up of these patients. Citation Format: Martha Zavridou, Areti Strati, George Koutsodontis, Amanda Psyrri, Evi Lianidou. RT-qPCR gene expression analysis of CTCs isolated through an epitope-independent enrichment microfluidic device in patients with head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1718. doi:10.1158/1538-7445.AM2017-1718

Abstract 1730:ESR1methylation in circulating tumor cells, ctDNA and primary tumors of breast cancer patients

Background: Estrogen receptor (ER) is an important prognostic biomarker in breast cancer. Epigenetic silencing of ESR1 could be of important clinical significance especially for its potential impact on endocrine treatment efficacy. Liquid biopsy provides real-time monitoring of tumor evolution and response to therapy through analysis of CTCs and ctDNA. Our group has evaluated for the first time epigenetic silencing of tumor and metastasis suppressor genes in CTCs and corresponding ctDNA. In this study, we evaluated for the first time ESR1 methylation in CTCs, paired ctDNA and primary tumors of breast cancer patients. Methods: We developed and validated a highly sensitive and specific real-time MSP assay for ESR1 methylation. We further applied the developed assay in sodium bisulfite (SB) treated DNA samples from: a) FFPEs from 40 patients with operable breast cancer, 25 patients with metastasis, 30 mammoplasties and 15 fibroadenomas, b) EpCAM+ immunomagnetically isolated CTCs fractions, from 74 early breast cancer patients, 48 patients with metastasis and 30 healthy donors, c) CellSearch® cartridges from 36 early breast cancer patients, 22 patients with metastasis, d) ctDNA isolated from plasma of matched samples and 54 healthy donors as a control group. Results: By using this highly specific and sensitive assay (sensitivity 0.1%) we detected methylation of ESR1 in: a) FFPEs: 16/40(40%) early breast cancer patients, 9/25(36%) patients with verified metastasis, 7/30(23.3%) mammoplasties and 5/15(33.3%) fibroadenomas. A statistically significant negative correlation was observed between ESR1 methylation status and ER protein expression (56/65 samples, 86%, p Conclusions: ER expression and ESR1 methylation were found 100% inversely correlated in primary tissues. The EpCAM+ CTC fraction of patients with breast cancer was found methylated for ESR1. Interestingly, ESR1 methylation was detected exclusively in CTC+ samples as analyzed from CellSearch® cartridges but in none of CTC- samples. In paired plasma samples, ESR1 methylation showed a high concordance (p Citation Format: Sophia Mastoraki, Areti Strati, Eleni Tzanikou, Eleni Politaki, George Koutsodontis, Loukas Kaklamanis, Nikolaos Malamos, Amanda Psyrri, Vassilis Georgoulias, Evi Lianidou. ESR1 methylation in circulating tumor cells, ctDNA and primary tumors of breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1730. doi:10.1158/1538-7445.AM2017-1730