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To examine the diagnostic yield of trio exome sequencing in fetuses with multiple structural defects with no pathogenic findings in cytogenetic and microarray analyses.We recruited 51 fetuses with two or more defects, non-immune fetal hydrops or fetal akinesia deformation syndrome|or fetal akinesia deformation sequence (FADS). Trio exome sequencing was performed on DNA from chorionic villi samples and parental blood. Detection of genomic variation and prioritization of clinically relevant variants was performed according to in-house standard operating procedures.Median maternal and gestational age was 32.0 years and 21.0 weeks, respectively. Forty-three (84.3%) fetuses had two or more affected organ systems. The remaining fetuses had isolated fetal hydrops or FADS. In total, the exome analysis established the genetic cause for the clinical abnormalities in 22 (43.1%, 95% CI 29.4%-57.8%) pregnancies.In fetuses with multiple defects, hydrops or FADS and normal standard genetic results, trio exome sequencing has the potential to identify genetic anomalies in more than 40% of cases.

BackgroundCleidocranial dysplasia (CCD) is a rare skeletal dysplasia with significant clinical variability. Patients with CCD typically present with delayed closure of fontanels and cranial sutures, dental anomalies, clavicular hypoplasia or aplasia and short stature. Runt-related transcription factor 2 (RUNX2)is currently the only known disease-causing gene for CCD, but several studies have suggested locus heterogeneity.MethodsThe cohort consists of eight subjects from five unrelated families partially identified through GeneMatcher. Exome or genome sequencing was applied and in two subjects the effect of the variant was investigated at RNA level.ResultsIn each subject a heterozygous pathogenic variant inCBFBwas detected, whereas no genomic alteration involvingRUNX2was found. ThreeCBFBvariants (one splice site alteration, one nonsense variant, one 2 bp duplication) were shown to result in a premature stop codon. A large intragenic deletion was found to delete exon 4, without affectingCBFBexpression. The effect of a second splice site variant could not be determined but most likely results in a shortened or absent protein. Affected individuals showed similarities withRUNX2-related CCD, including dental and clavicular abnormalities. Normal stature and neurocognitive problems were however distinguishing features.CBFBencodes the core-binding factor β subunit, which can interact with all RUNX proteins (RUNX1, RUNX2, RUNX3) to form heterodimeric transcription factors. This may explain the phenotypic differences betweenCBFB-related andRUNX2-related CCD.ConclusionWe confirm the previously suggested locus heterogeneity for CCD by identifying five pathogenic variants inCBFBin a cohort of eight individuals with clinical and radiographic features reminiscent of CCD.

Purpose: We sought to investigate the clinical response to MET inhibition in patients diagnosed with structural MET alterations and to characterize their functional relevance in cellular models.Experimental Design: Patients were selected for treatment with crizotinib upon results of hybrid capture–based next-generation sequencing. To confirm the clinical observations, we analyzed cellular models that express these MET kinase alterations.Results: Three individual patients were identified to harbor alterations within the MET receptor. Two patients showed genomic rearrangements, leading to a gene fusion of KIF5B or STARD3NL and MET. One patient diagnosed with an EML4-ALK rearrangement developed a MET kinase domain duplication as a resistance mechanism to ceritinib. All 3 patients showed a partial response to crizotinib that effectively inhibits MET and ALK among other kinases. The results were further confirmed using orthogonal cellular models.Conclusions: Crizotinib leads to a clinical response in patients with MET rearrangements. Our functional analyses together with the clinical data suggest that these structural alterations may represent actionable targets in lung cancer patients. Clin Cancer Res; 24(6); 1337–43. ©2017 AACR.

SOX4 is a transcription factor with pleiotropic functions required for different developmental processes, such as corticogenesis. As with all SOX proteins, it contains a conserved high mobility group (HMG) and exerts its function via interaction with other transcription factors, such as POU3F2. Recently, pathogenic SOX4 variants have been identified in several patients who had clinical features overlapping with Coffin–Siris syndrome. In this study, we identified three novel variants in unrelated patients with intellectual disability, two of which were de novo (c.79G>T, p.Glu27*; c.182G>A p.Arg61Gln) and one inherited (c.355C>T, p.His119Tyr). All three variants affected the HMG box and were suspected to influence SOX4 function. We investigated the effects of these variants on transcriptional activation by co-expressing either wildtype (wt) or mutant SOX4 with its co-activator POU3F2 and measuring their activity in reporter assays. All variants abolished SOX4 activity. While our experiments provide further support for the pathogenicity of SOX4 loss-of-function (LOF) variants as a cause of syndromic intellectual disability (ID), our results also indicate incomplete penetrance associated with one variant. These findings will improve classification of novel, putatively pathogenic SOX4 variants.

Ayme-Gripp syndrome is a multisystemic disorder caused by a heterozygous variation in the MAF gene (OMIM*177075). Key features are congenital cataracts, sensorineural hearing loss, and a characteristic facial appearance. In a proportion of individuals, pericardial effusion or pericarditis has been reported as part of the phenotypic spectrum. In the present case, a large persistent cytokine-enriched pericardial effusion was the main pre- and postnatal symptom that led to the clinical and later molecular diagnosis of Ayme-Gripp syndrome. In the postnatal course, the typical Ayme-Gripp syndrome-associated features bilateral cataracts and hearing loss were diagnosed. We propose that activating dominant variants in the cytokine-modulating transcription factor c-MAF causes cytokine-enriched pericardial effusions possibly representing a key feature of Ayme-Gripp syndrome.

Transformer2 proteins (Tra2α and Tra2β) control splicing patterns in human cells, and no human phenotypes have been associated with germline variants in these genes. The aim of this work was to associate germline variants in the TRA2B gene to a novel neurodevelopmental disorder.Twelve individuals from 11 unrelated families who had predicted loss-of-function monoallelic variants, mostly de novo, were recruited. RNA-seq and Western blot analyses of Tra2β-1 and Tra2β-3 isoforms from patient-derived cells were performed. Tra2β1-GFP, Tra2β3-GFP and CHEK1 exon 3 plasmids were transfected into HEK-293 cells.All variants clustered in the 5' part of TRA2B, upstream of an alternative translation start site responsible for the expression of non-canonical Tra2β-3 isoform. All of affected individuals presented intellectual disability and/or developmental delay, frequently associated with infantile spasms, microcephaly, brain anomalies, autism spectrum disorder, feeding difficulties, and short stature. Experimental studies showed that these variants decreased the expression of the canonical Tra2β-1 isoform, while they increased the expression of the Tra2β-3 isoform, which is shorter and lacks the N-terminal RS1 domain. Increased expression of Tra2β-3-GFP were shown to interfere with the incorporation of CHEK1 exon 3 into its mature transcript, normally incorporated by Tra2β-1.Predicted loss-of-function variants clustered in the 5' portion of TRA2B cause a new neurodevelopmental syndrome through an apparently dominant negative disease mechanism involving the use of an alternative translation start site and the overexpression of a shorter, repressive Tra2β protein.

Cleidocranial dysplasia (CCD) is a rare skeletal dysplasia with significant clinical variability. Patients with CCD typically present with delayed closure of fontanels and cranial sutures, dental anomalies, clavicular hypoplasia or aplasia and short stature. Runt-related transcription factor 2 (The cohort consists of eight subjects from five unrelated families partially identified through GeneMatcher. Exome or genome sequencing was applied and in two subjects the effect of the variant was investigated at RNA level.In each subject a heterozygous pathogenic variant inWe confirm the previously suggested locus heterogeneity for CCD by identifying five pathogenic variants in

Purpose: We sought to investigate the clinical response to MET inhibition in patients diagnosed with structural MET alterations and to characterize their functional relevance in cellular models. Experimental Design: Patients were selected for treatment with crizotinib upon results of hybrid capture–based next-generation sequencing. To confirm the clinical observations, we analyzed cellular models that express these MET kinase alterations. Results: Three individual patients were identified to harbor alterations within the MET receptor. Two patients showed genomic rearrangements, leading to a gene fusion of KIF5B or STARD3NL and MET. One patient diagnosed with an EML4-ALK rearrangement developed a MET kinase domain duplication as a resistance mechanism to ceritinib. All 3 patients showed a partial response to crizotinib that effectively inhibits MET and ALK among other kinases. The results were further confirmed using orthogonal cellular models. Conclusions: Crizotinib leads to a clinical response in patients with MET rearrangements. Our functional analyses together with the clinical data suggest that these structural alterations may represent actionable targets in lung cancer patients. Clin Cancer Res; 24(6); 1337–43. ©2017 AACR.

Background Lorlatinib, an ALK/ROS1 inhibitor, showed activity in ROS1+ NSCLC pretreated with crizotinib. However, molecular events predictive for tumor response during lorlatinib treatment are largely unknown. Methods The PFROST trial included ROS1+ NSCLCs refractory to crizotinib. Eligible patients received lorlatinib 100 mg daily until disease progression. Primary end point was response rate (RR). For all patients, pre-lorlatinib tumor tissue or blood sample collection was mandatory. At lorlatinib failure liquid biopsy was recommended. The samples were then run with the NEOliquid assay, specifically designed for liquid biopsies, or NEOselect, a panel optimized for formalin-fixed paraffin-embedded (FFPE) tumor tissue, covering 39 cancer related genes. Results From June 2017 to April 2019, 22 ROS1+ crizotinib refractory lung adenocarcinoma patients were included in 10 Institutions. Median age was 56 years (range 39-82); male/female: 8/14; ECOG PS 0 (N = 8; 36.4%), PS1 (N = 14; 63,6%); The majority had brain metastases at baseline (N = 15; 68.1%), were never smokers (N = 13; 59.1%) and received lorlatinib as third-line therapy (N = 16; 72.7%). In all cases crizotinib was the last therapy before lorlatinib. Accrual is completed and 13 patients are still receiving therapy. A total of 18 patients were evaluable for response and 7 had confirmed complete (N = 1) or partial (N = 6) responses for an overall RR of 38.8%. In 4 patients, response to therapy was not yet evaluated. A total of 10 tissue biopsies and 20 blood samples obtained after crizotinib and before lorlatinib therapy were collected. For 7 samples analyses are ongoing. Among responders, no patient harbored a secondary ROS1 mutation. Conversely, no response was observed among patients with secondary ROS1 mutations (N = 1 ROS1S1861I, N = 1 ROS1 V2054A, N = 3 ROS1G2032R). All patients harboring the ROS1G2032R mutation rapidly progressed and maintained this aberration in liquid biopsy at the time of of lorlatinib failure. Conclusions In our study lorlatinib confirmed its efficacy in crizotinib resistant ROS1+ NSCLC. Molecular profile of refractory patients suggests reduced efficacy in individuals harbouring crizotinib-induced secondary ROS1 mutations. Clinical trial identification EudraCT Number: 2016-001259-34. Legal entity responsible for the study Fondazione Ricerca Traslazionale. Funding Has not received any funding. Disclosure L. Landi: Advisory / Consultancy: Pzifer; Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: BMS; Advisory / Consultancy: MSD; Advisory / Consultancy: Boehringer Ingelheim. M. Tiseo: Advisory / Consultancy, Speaker Bureau / Expert testimony: AstraZeneca; Advisory / Consultancy, Speaker Bureau / Expert testimony: Pfizer; Advisory / Consultancy, Speaker Bureau / Expert testimony: Eli Lilly; Advisory / Consultancy, Speaker Bureau / Expert testimony: BMS; Advisory / Consultancy, Speaker Bureau / Expert testimony: Novartis; Advisory / Consultancy, Speaker Bureau / Expert testimony: Roche; Advisory / Consultancy, Speaker Bureau / Expert testimony: MSD; Advisory / Consultancy, Speaker Bureau / Expert testimony: Boehringer Ingelheim; Advisory / Consultancy, Speaker Bureau / Expert testimony: Otsuka; Advisory / Consultancy, Speaker Bureau / Expert testimony: Takeda; Advisory / Consultancy, Speaker Bureau / Expert testimony: Pierre Fabre. D.L. Cortinovis: Advisory / Consultancy, Speaker Bureau / Expert testimony: AstraZeneca; Advisory / Consultancy, Speaker Bureau / Expert testimony: BMS; Advisory / Consultancy, Speaker Bureau / Expert testimony: Roche; Advisory / Consultancy, Speaker Bureau / Expert testimony: MSD; Advisory / Consultancy, Speaker Bureau / Expert testimony: Boehringer Ingelheim; Advisory / Consultancy, Speaker Bureau / Expert testimony: Amgen. A. Delmonte: Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Roche; Advisory / Consultancy: MSD; Advisory / Consultancy: Boehringer Ingelheim. D. Galetta: Advisory / Consultancy, Speaker Bureau / Expert testimony: AstraZeneca; Advisory / Consultancy, Speaker Bureau / Expert testimony: Eli Lilly; Advisory / Consultancy, Speaker Bureau / Expert testimony: BMS; Advisory / Consultancy, Speaker Bureau / Expert testimony: Roche; Advisory / Consultancy, Speaker Bureau / Expert testimony: MSD; Advisory / Consultancy, Speaker Bureau / Expert testimony: Boehringer Ingelheim. R. Chiari: Advisory / Consultancy: Pfizer; Advisory / Consultancy, Speaker Bureau / Expert testimony: AstraZeneca; Advisory / Consultancy: Boehringer Ingelheim; Speaker Bureau / Expert testimony: Takeda; Speaker Bureau / Expert testimony: Roche; Speaker Bureau / Expert testimony: BMS; Speaker Bureau / Expert testimony: Novartis. L. Crino: Advisory / Consultancy: AbbVie; Advisory / Consultancy: Pfizer; Advisory / Consultancy: Novartis; Advisory / Consultancy: Bristol; Advisory / Consultancy: Roche; Advisory / Consultancy: AstraZeneca. F. Cappuzzo: Advisory / Consultancy, Speaker Bureau / Expert testimony: Roche; Advisory / Consultancy, Speaker Bureau / Expert testimony: Pfizer; Advisory / Consultancy, Speaker Bureau / Expert testimony: AstraZeneca; Advisory / Consultancy, Speaker Bureau / Expert testimony: Takeda; Advisory / Consultancy, Speaker Bureau / Expert testimony: Eli Lilly; Advisory / Consultancy, Speaker Bureau / Expert testimony: BMS; Advisory / Consultancy, Speaker Bureau / Expert testimony: MSD; Advisory / Consultancy, Speaker Bureau / Expert testimony: Boehringer Ingelheim. All other authors have declared no conflicts of interest.

Immune evasion of tumors poses a major challenge for immunotherapy. For human papillomavirus (HPV)-induced malignancies, multiple immune evasion mechanisms have been described, including altered expression of antigen processing machinery (APM) components. These changes can directly influence epitope presentation and thus T-cell responses against tumor cells. To date, the APM had not been studied systematically in a large array of HPV+ tumor samples. Therefore in this study, systematic expression analysis of the APM was performed on the mRNA and protein level in a comprehensive collection of HPV16+ cell lines. Subsequently, HPV+ cervical tissue samples were examined by immunohistochemistry. ERAP1 (endoplasmic reticulum aminopeptidase 1) was the only APM component consistently altered – namely overexpressed – in HPV16+ tumor cell lines. ERAP1 was also found to be overexpressed in cervical intraepithelial neoplasia and cervical cancer samples; expression levels were increasing with disease stage. On the functional level, the influence of ERAP1 expression levels on HPV16 E7-derived epitope presentation was investigated by mass spectrometry and in cytotoxicity assays with HPV16-specific T-cell lines. ERAP1 overexpression did not cause a complete destruction of any of the HPV epitopes analyzed, however, an influence of ERAP1 overexpression on the presentation levels of certain HPV epitopes could be demonstrated by HPV16-specific CD8+ T-cells. These showed enhanced killing toward HPV16+ CaSki cells whose ERAP1 expression had been attenuated to normal levels. ERAP1 overexpression may thus represent a novel immune evasion mechanism in HPV-induced malignancies, in cases when presentation of clinically relevant epitopes is reduced by overactivity of this peptidase.

Introduction Detection of somatic genomic alterations in the plasma of patients with cancer ("liquid biopsy") are increasingly being used in the clinic. However, the concordance of alterations identified in liquid biopsies with those detected in cancer specimens is not routinely being determined. Methods We sought to systematically compare alterations found by a massively parallel sequencing liquid biopsy assay covering 39 genes (NEOliquid [NEO New Oncology GmbH, Koln, Germany]) with those identified through routine diagnostic testing in a certified central pathology laboratory in a cohort of patients with nonsquamous NSCLC. NEOliquid is based on enrichment of the genomic territory of interest by hybrid capture and is thus capable of detecting point mutations, small insertions and deletions, copy number alterations, and gene rearrangements/fusions in a single assay. Results In a cohort of 82 patients with matched blood/tissue samples, the concordance between NEOliquid and tissue-based routine testing was 98%, the sensitivity of NEOliquid was higher than 70%, and the specificity was 100%. Discordant cases included those with insufficient amounts of circulaating tumor DNA in plasma and cases in which known driver mutations (e.g., isocitrate dehydrogenase (NADP(+)), 1 systolic gene [IDH1] R132H, kinesin family member 5B gene [KIF5b–ret proto-oncogene [RET], or MNNG HOS Transforming gene [MET] exon 14) were found in the plasma but were not interrogated by routine tissue analyses. Conclusions In summary, NEOliquid offers accurate and reliable detection of clinically relevant driver alterations in plasma of patients with cancer.

Introduction: Old age and unfavorable cytogenetics are well-established adverse prognostic factors for outcome in patients (pts) with AML. Recently, high-throughput sequencing techniques have identified multiple somatically acquired driver mutations (muts) with a strong prognostic impact in AML. We explored the relative contribution of baseline molecular genetics versus a response-based sequential treatment approach on response and overall survival (OS) at two years in pts >60 years (y) with newly diagnosed AML treated within the multicenter RAS-AZIC study of the OSHO. Patients and methods: In the RAS-AZIC trial, 112 pts received priming with Azacitidine (AZA) followed by a day (d) 15 bone marrow (BM) blast count-adapted sequential treatment with AZA or intensive chemotherapy (IC). Pts with d15 blasts ≥45% received IC. If the count was Baseline BMs of 77 pts who signed the translational informed consent were analyzed by NGS using the NEOmyeloid panel of NEO New Oncology (Siemens Healthineers Company, Germany). This hybrid capture-based assay comprises 43 genes and detects point muts, small insertions and deletions in 30 genes as well as gene fusions in 18 genes. Molecular results were correlated to outcome data. Results: Median age was 70y. Secondary AML was present in 39%. Genetic risk was int-II and adverse in 25% and 27% respectively. Response on d90 was 67% (CR/CRi 58%, PR 9%). A median of 3 muts per pt were found. Mutations in DNA methylation-related-, spliceosome complex-, and chromatin-modifying-genes were 62%, 39%, and 30% respectively. Overall, muts in DNMT3A (34%), ASXL1 (25%), RUNX1 (24%), SRSF2 (23%), and TET2 (23%) were the most frequently detected (fig. 1). As expected, 72% of SRSF2 muts were associated with ASXL1 and/or RUNX1 muts. CR/CRi in the favorable and int-I 2010 ELN genetic risk groups was 81% and 57.5% in int-II and adverse risk pts. With the exception of muts in spliceosome complex-related genes, the mutational status (including the FLT3 status) seemed to have no impact on response in this cohort (table 1). In the presence of splicing factor gene muts a trend towards an inferior response of 53% compared to 77% in pts without such a mut was observed. High risk muts such as ASXL1, RUNX1, and TP53 muts which were included in the 2017 ELN adverse risk stratification were present in 20/32 (63%) of pts with favorable and int-I genetic risk. Yet, only 5/32 (16%) did not achieve a response. Similarly, CR/CRi was achieved in 7/9 (78%) pts with TP53 mut which was associated with an adverse karyotype in 8 pts. Likewise, OS tended to be similar across all molecular classes with the exception of KMT2A gene fusions with their well-known negative impact on outcome. The presence of such a fusion (n=4) was associated with a median survival time of only 8 compared to 16 months in pts without a detectable KMT2A fusion. These KMT2A gene fusions also correlated with a shorter EFS and RFS. The presence of myeloid transcription factor gene muts such as in RUNX1 (24%) and BCOR (13%) tended to be associated with an inferior EFS, while cohesin complex- (STAG2 8%), tumour suppressor-, and DNA-methylation-related gene muts with a negative RFS (table 1). Conclusions: This explorative analysis with a limited number of patients describes the pattern of genetic mutations in elderly AML treated with a response-based sequential approach that integrates epigenetic and chemotherapeutic treatments and their association with outcome. Whether such a response-based treatment mitigates the negative impact of (some) high risk mutations on outcome needs to be addressed in a much larger trial. Disclosures Jaekel: Celgene: Other: travel grant. Siebolts:Merck: Honoraria; Boehringer Ingelheim: Honoraria; MSD: Honoraria; Pfizer: Honoraria; AstraZeneca: Honoraria; Roche: Honoraria; Novartis: Honoraria; BMS: Honoraria. Pazaitis:Novartis: Consultancy. Niederwieser:Daichii: Speakers Bureau; Cellectis: Consultancy. Hänel:Amgen: Honoraria; Roche: Honoraria; Novartis: Honoraria; Takeda: Other: advisory board; Celgene: Other: advisory board. Maschmeyer:Gilead, Janssen Cilag, Astra Zeneca; BMS, Merk-Serono: Honoraria. Heukamp:NEO New Oncology GmbH: Employment. Menon:NEO New Oncology GmbH: Employment. Bertrand:NEO New Oncology GmbH: Employment. Wickenhauser:BMS: Honoraria; Merck-Serono: Honoraria; MSD: Honoraria; Böhringer: Honoraria; Novartis: Honoraria; Takeda: Honoraria; Roche: Honoraria. Brosteanu:University Leipzig: Employment. Al-Ali:Celgene: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; CTI: Honoraria.

Structural rearrangements that activate receptor kinases account for an ever-increasing pool of druggable targets in lung adenocarcinoma patients. Among these tumors RET kinase fusions and MET alterations represent two genetically distinct groups that share a common lack of clinically effective strategies. While RET rearranged tumors show a limited susceptibility to currently available RET inhibitors, MET rearranged tumors have not been yet fully appreciated as a relevant group that may benefit from treatment with MET targeted drugs. Using systematic molecular profiling of genetically engineered RET rearranged in vitro and models and patient-derived in vivo models we identified the type II kinase inhibitors ponatinib and AD80 as the most potent drugs. While both inhibitors are effective against gatekeeper mutant RET we identified a novel resistance mutation RETI788N that triggers a selective resistance against AD80 and other RET inhibitors but retains susceptibility to ponatinib. Furthermore, we studied the clinical and preclinical activity of MET targeted drugs. We specifically characterized genomic rearrangements of KIF5B-MET and STARD3NL-MET in cellular models that were found in two distinct LADC. In parallel we identified and characterized a MET kinase domain duplication that developed in an EML4-ALK rearranged positive tumor as a resistance mechanism to ceritinib. All three patients showed a partial response to crizotinib that effectively inhibits MET and ALK in these tumors. Thus, our molecular characterization of drug-target engagement in genetically defined models may further enhance the clinical efficacy of kinase inhibitors in lung tumors driven by rare oncogenic kinase alterations. Citation Format: Dennis Plenker, Carina Lorenz, Miriam Bertrand, Richard Riedel, Joop de Langen, Johannes Brägelmann, Reinhard Büttner, Jürgen Wolf, Roman K. Thomas, Johannes Heuckmann, Martin L. Sos. Targeting structural RET and MET kinase alterations in lung adenocarcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1920.

5555 Background: Surgical outcome is an important prognostic factor in advanced high-grade serous ovarian cancer (HGOC). Intra-operative determination of residual disease may be prone to subjective bias. Methods: We prospectively collected and analyzed tumor tissue and plasma from 21 pts with FIGO IIIC-IV HGOC. All tumor specimen showed loss of TP53 in immunohistochemistry. Tumor DNA from tissue and peri-operative cell-free DNA at baseline (BL) and at d(ays) 1, d4 and d10 were subjected to comprehensive hybrid-capture based next-generation sequencing (NEO New Oncology GmbH, Cologne, Germany). Results: The initial cohort comprised 10 pts without (TR>0) and 11 pts with complete resection TR0, with a total of 43 somatic genomic alterations. TP53 was mutated in 20/21 (95.2%) of tissue samples and in 15/21 (71.4%) corresponding plasma samples at BL. Therefore, TP53 mutations were used as a molecular marker of circulating tumor DNA levels post-surgery. In the remaining 5 BL cases the TP53 mutations were either not present in plasma (N= 3) or rested below the detection limit (N= 2). In d1, d4 and d10 post-surgical samples, the sample specific TP53 mutations were detected in 12 out of 15, 10 out of 14 and in 7 out of 13 cases, respectively. TP53variant allele frequency (VAF) did not differ among TR0 and TR>0 at BL (mean= 1.91 versus 1.73, Mann-Whitney test p = 0.86) whereas it was significantly lower for TR0 at d1 (mean= 0.06 versus 2,06; p value= 0.002), d4 (mean= 0.07 versus 1.8; p= 0.04) and d10 (mean VAF= 0 versus 1.04; p= 0.008). Five out of 9 TR>0 cases showed at least one increase in VAF between d0 and any additional time-point. In 8 out of 9 mutations remained detectable at d10 (VAF: 0.076 – 3.26). By contrast, TR0 cases showed consistent reduction in VAF throughout the follow-up period which maximized at d10. Conclusions: Liquid biopsycan efficiently detect somatic mutations in the cfDNA of pts with HGOC following debulking surgery. Variation of the TP53 VAF in sequential post-surgical samples appears to be restricted to TR>0 cases, whereas in TR0 pts VAF progressively decreases. Liquid biopsy may hold promise as a tool for the objective determination of residual disease after debulking surgery.

e23146 Background: The importance and clinical use of liquid biopsies to identify somatic, targetable alterations in the plasma of cancer patients is steadily increasing. However, their concordance with alterations identified in cancer specimens tested in routine diagnostics is not being monitored. In a cohort of non-squamous cell lung cancer patients, our aim was to systematically compare alterations found by a massively parallel sequencing liquid biopsy assay covering 39 cancer related genes (NEOliquid) with alterations identified by routine diagnostics in a certified central pathology laboratory. Methods: Routine Diagnostics: DNA mutational analysis was performed using cobas or Sanger-Sequencing. Rearrangements were identified using immunohistochemistry or FISH. NEOliquid assay: Cell-free DNA from plasma was subjected to hybrid-capture based next-generation sequencing to detect point mutations, small insertions and deletions, copy number alterations and genomic translocations in 39 clinically relevant genes in a single assay. To evaluate and compare the performance of liquid biopsies, we applied the NEOliquid tests on blood samples of 82 non-squamous NSCLC patients and correlated them with results of routine diagnostics of matched tissue samples. Results: Routine diagnostics for lung cancer related oncogenes (EGFR, ALK, ROS1, KRAS, BRAF) identified a total of 50 somatic alterations of which 37 were point mutations, 11 InDels and 2 gene fusions. NEOliquid analysis of the corresponding plasma sample revealed a total of 34 alterations, including 21 targetable driver alterations. Overall, the concordance of mutation calls between routine diagnostic testing and NEOliquid was 97.6%, with a sensitivity of > 70% and a specificity of 100%. Importantly, NEOliquid identified additional clinically relevant genomic alterations, not covered by routine testing. Conclusions: We have shown high concordance of genomic alterations identified from liquid biopsies and tumor tissue specimens. The excellent specificity of actionable alterations identified by NEOliquid offers analytical performance for routine diagnostic use.

The protein tyrosine kinase Ephrin type-B receptor 3 (EPHB3) counteracts tumor-cell dissemination by regulating intercellular adhesion and repulsion and acts as tumor/invasion suppressor in colorectal cancer. This protective mechanism frequently collapses at the adenoma–carcinoma transition due to EPHB3 transcriptional silencing. Here, we identify a transcriptional enhancer at the EPHB3 gene that integrates input from the intestinal stem-cell regulator achaete-scute family basic helix-loop-helix transcription factor 2 (ASCL2), Wnt/β-catenin, MAP kinase, and Notch signaling. EPHB3 enhancer activity is highly variable in colorectal carcinoma cells and precisely reflects EPHB3 expression states, suggesting that enhancer dysfunction underlies EPHB3 silencing. Interestingly, low Notch activity parallels reduced EPHB3 expression in colorectal carcinoma cell lines and poorly differentiated tumor-tissue specimens. Restoring Notch activity reestablished enhancer function and EPHB3 expression. Although essential for intestinal stem-cell maintenance and adenoma formation, Notch activity seems dispensable in colorectal carcinomas. Notch activation even promoted growth arrest and apoptosis of colorectal carcinoma cells, attenuated their self-renewal capacity in vitro, and blocked tumor growth in vivo. Higher levels of Notch activity also correlated with longer disease-free survival of colorectal cancer patients. In summary, our results uncover enhancer decommissioning as a mechanism for transcriptional silencing of the EPHB3 tumor suppressor and argue for an antitumorigenic function of Notch signaling in advanced colorectal cancer.