Non-hotspot Mutations Research Articles

Abstract 3564: Survival-based grouping of genetic variants: A novel statistical framework with an application to TP53 mutations

Abstract Advancements in genotyping and sequencing techniques, coupled with the growing magnitude of genome databases, have led to comprehensive investigations into the impact of genetic variants on human diseases, including cancers. It is commonly acknowledged that genetic variants have homogeneous effects on clinical outcomes, i.e., variants exhibit similar effects within a certain group but are distinct from those in other groups. However, such grouping information is typically unknown, and identification of the underlying genetic variation groups is crucial for understanding their functional consequences, facilitating patient management, and improving risk prediction. To resolve this challenge, we introduce a novel statistical framework called Survival-based Clustering of Predictors (SCP) in Cox regression to group genetic variants based on patient survival outcomes. Built upon a penalized Cox regression model, SCP considers different genetic variants, as well as additional patient-specific features if needed, as survival predictors and then searches for homogeneity among the coefficients for individual variants to recover the underlying grouping structure. Focusing on TP53, the most frequently mutated gene in cancer that has resulted in a wide range of functions and clinical outcomes, we apply SCP to group TP53 germline mutations with the age of cancer diagnosis as the time-to-event. Using datasets from four Li-Fraumeni syndrome (LFS) cohorts at MD Anderson, NCI, and DFCI, we obtained 75 recurring TP53 germline mutations from 513 patients and clustered these mutations into high-, medium-, and low-risk groups. Hotspot mutations such as R175H, R248Q, G245S, and R273C are in the high-risk group as supported in many TP53 studies, while several non-hotspot mutations such as R290H, V218G, and G244D also exhibit strong positive effects and are grouped together with hotspot mutations. Overall, mutations in all three risk groups, obtained through applying SCP, are highly consistent with a yeast functional assay-based approach for grouping TP53 mutations, supporting the utility of SCP for survival-based grouping of genetic variants. The contribution of our study is three-fold. Statistically, our method fills a critical gap in the survival analysis literature, offering a novel statistical framework for clustering predictors in Cox regression based on survival outcomes. Clinically, we provide a timely solution to the patient outcome-based grouping of TP53 mutations, facilitating clinical management of TP53 mutation carriers. Biologically, we bring fresh insights into knowledge for the most frequently observed genetic variants in cancer, providing new hypotheses to TP53-related biological research. Citation Format: Xiaoqian Liu, Haoming Shi, Emilie Montellier, Pierre Hainaut, Wenyi Wang. Survival-based grouping of genetic variants: A novel statistical framework with an application to TP53 mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3564.

Structural insights into non-hotspot KRAS mutations and their potential as targets for effective cancer therapies

Kirsten rat sarcoma virus protein (KRAS) is a protein that plays a central role in signal transduction using extracellular signal regulated kinase (ERK) and mitogen activated protein kinase (MAPK) cellular signaling pathway. KRAS is a frequently mutated oncogene and plays a pivotal role in tumor initiation and progression. Hotspot mutations on codon 12, 13 and 61 in KRAS are well-known for their role in drug resistance and non-hotspot mutations also play a significant part in contributing to resistance mechanisms. The understanding of how these non-hotspot mutations might affect the signal transduction of KRAS and their contribution towards drug resistance is understudied. Here we provide structural insights into the interaction of non-hotspot KRAS mutants with GTP (the native ligand) using a molecular docking and molecular dynamics simulation approach. Extensive molecular docking and simulation studies suggest that non-hotspot mutations (E31D and E63K) show stable interaction with native ligand using all five trajectories, as evidenced by root mean square of distance (RMSD), root mean square of fluctuation (RMSF), radius of gyration (RoG), coulomb short-range energy and MMGBSA analysis. These results suggest that non-hotspot mutations do not undermine the oncogenic nature of KRAS. This observation is consistent with previous findings where overexpressing E31D and E63K mutations share phenotypic features with G12D and G13D transfected cells, including increased proliferative capacity, actin cytoskeleton organization, and migration rates. We further test whether FDA-approved KRAS inhibitors sotorasib and adagrasib successfully inhibit the E31D and E63K mutants. Results suggest that these two non-hotspot mutants can be inhibited by both drugs with following trend of structural stability (E31D > E63K > wild-KRAS > Q61H > G12C). Based on sharp coherence in trajectories between wild KRAS and non-hotspot mutants, it is suggested that these novel mutants do not contribute to drug resistance mechanism. Overall, we provide a comprehensive understanding of the impact of non-hotspot mutations on KRAS and their potential as targets for effective cancer therapies. Communicated by Ramaswamy H. Sarma

Genomic Landscape of Ibrutinib- and/or Acalabrutinib-intolerant Patients with B-cell Malignancies Treated with Zanubrutinib in a Phase 2 Study

Introduction:Bruton tyrosine kinase inhibitors (BTKi) have shown remarkable utility in the treatment of B-cell malignancies; yet some patients have experienced toxicities to the BTKis, ibrutinib and acalabrutinib, which lead to dose reduction or treatment discontinuation. Zanubrutinib is a potent and selective next-generation BTKi. BGB-3111-215 (NCT04116437) is an ongoing, phase 2 study of the safety and efficacy of zanubrutinib monotherapy in patients with chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), Waldenström macroglobulinemia (WM), mantle cell lymphoma (MCL), or marginal zone lymphoma (MZL) who discontinued ibrutinib, acalabrutinib, or acalabrutinib and ibrutinib because of intolerance. Methods:Because the genomic profile of patients with B-cell malignancies who are intolerant to ibrutinib and/or acalabrutinib has not been extensively studied, mutations were assessed using a 106 gene-targeted next-generation sequencing panel (PredicineHEME TM, Predicine) on baseline and disease progression samples. Samples were sequenced to a median depth of >20,000 reads, with a validated sensitivity of 0.25% mutant allele frequency for all genomic regions, and 0.1% for mutational hotspots. Variant allele frequency (VAF) <0.1% for hotspot mutations and <0.25% for non-hotspot mutations were excluded from analysis; germline and CHIP mutations were also excluded from analysis. The association between gene mutations and progression-free survival (PFS, defined as time from the date of first zanubrutinib dose to date of first PD or death) was quantified by the log rank test and the hazard ratio and summarized by the Kaplan-Meier method. Results:As of June 6, 2022, baseline peripheral blood samples were collected from 71 (95.9%) of the 74 patients enrolled in the study. There were 46 patients with CLL, 11 with WM, eight with SLL, three with MCL, and three with MZL. A total of 56 patients were intolerant to ibrutinib only; 15 were intolerant to acalabrutinib or acalabrutinib and ibrutinib. At baseline, high mutation frequencies were observed in TP53, SF3B1, ATM, and NOTCH1 across disease indications ( Figure). The majority of TP53 mutations were missense mutations and located in the DNA binding domain. Nineteen different mutations were identified in ATM and were distributed across the whole gene with a majority clustered in the 3' regions encoding the FAT and PI3 kinase domains. All mutations (n=21) in SF3B1 were missense and 38% (n=8/21) of the mutations occurred at p.K700E/c.A2098G. In NOTCH1, the most common mutation (n=8/14, 57.1%) was a two base pair deletion (c.7541_7542delCT) in the PEST domain of exon 34. In patients with CLL/SLL with TP53, SF3B1, or ATM mutations at baseline, shorter progression-free survival was observed compared to patients without these mutations, suggesting that mutations in DNA damage response and RNA splicing/metabolism pathways are associated with less favorable prognosis. BTK mutations were detected in 4.2% (n=3/71) of baseline samples and in 57.1% (n=4/7) of samples available at disease progression (PD). All BTK mutations were at the C481 BTKi binding site. Two patients with baseline BTK mutations had PD and increased VAF of BTK mutants were detected for both patients at PD. A third patient died due to illness unrelated to treatment. Two patients acquired new BTKC481 mutations at PD. Conclusion: This is the first study to describe the genomic landscape of patients with B-cell malignancies who were intolerant to ibrutinib and/or acalabrutinib. Here we show that the gene mutational profile of these patients at baseline or at/after disease progression is comparable with patients with relapse/refractory disease who tolerate ibrutinib and, consistent with other studies, patients with mutations in TP53, SF3B1 or ATM genes had less favorable prognosis on BTKi. Further, intolerant patients who progressed on zanubrutinib acquired new BTK mutations and/or had an increase in the frequency of BTK mutations. These data contribute to the growing understanding of the mutational profiles of patients with B-cell malignancies, particularly those who were intolerant to BTKi, and further explores the association between mutations and response to subsequent zanubrutinib treatment. This study may provide supporting information to inform treatment guidelines for patients who become intolerant to BTKi.

Novel KIF5C Gene Mutation Leading to Frontal Pachygyria: A Rare Case Report and a Review of the Literature

Abstract Lissencephaly or smooth brain can be classified based on the site of predominant cortical involvement. Identification of “gene-imaging pattern” associations facilitates specific analysis in some genes. KIF5C, a motor kinesin has been implicated in cortical dysplasia with eight reported cases to date. The Glu237 residue represents a hotspot being substituted in six out of seven patients. A uniform pattern of neuroimaging with a predominant frontal and mesio-frontal pachygyria has been observed in patients with mutations in the hotspot residue. Two different de novo disease-causing variants (nonhotspot mutations) were detected to be causative in the remaining two patients, resulting in posterior predominant pachygyria. This has led to the hypothesis of a mutation-specific imaging pattern, in KIF5C-associated lissencephaly. We hereby present a female with a novel nonhotspot mutation in the KIF5C gene. Using whole exome sequencing, a novel de novo missense mutation c.265T>C (p.Ser89Pro) of KIF5C was identified. Neuroimaging revealed a predominant frontal pachygyria, which is akin to the pattern observed with the Glu237 hotspot residue of KIF5C. We also compared the phenotype between patients with and without involvement of the hotspot residue and were able to show that no major differences exist between both groups. We expand the currently known narrow KIF5C mutation spectrum and challenge the notion of a typical pattern of “mutation-specific” imaging abnormality.

Somatic mutation spectrum of a Chinese cohort of pediatrics with vascular malformations

BackgroundSomatic mutations of cancer driver genes are found to be responsible for vascular malformations with clinical manifestations ranging from cutaneous birthmarks to life-threatening systemic anomalies. Till now, only a limited number of cases and mutations were reported in Chinese population. The purpose of this study was to describe the somatic mutation spectrum of a cohort of Chinese pediatrics with vascular malformations.MethodsPediatrics diagnosed with various vascular malformations were collected between May 2019 and October 2020 from Beijing Children’s Hospital. Genomic DNA of skin lesion of each patient was extracted and sequenced by whole-exome sequencing to identify pathogenic somatic mutations. Mutations with variant allele frequency less than 5% were validated by ultra-deep sequencing.ResultsA total of 67 pediatrics (33 males, 34 females, age range: 0.1–14.8 years) were analyzed. Exome sequencing identified somatic mutations of corresponding genes in 53 patients, yielding a molecular diagnosis rate of 79.1%. Among 29 PIK3CA mutations, 17 were well-known hotspot p.E542K, p.E545K and p.H1047R/L. Non-hotspot mutations were prevalent in patients with PIK3CA-related overgrowth spectrum, accounting for 50.0% (11/22) of detected mutations. The hotspot GNAQ p.R183Q and TEK p.L914F mutations were responsible for the majority of port-wine stain/Sturge–Weber syndrome and venous malformation, respectively. In addition, we identified a novel AKT1 p.Q79K mutation in Proteus syndrome and MAP3K3 p.E387D mutation in verrucous venous malformation.ConclusionsThe somatic mutation spectrum of vascular malformations in Chinese population is similar to that reported in other populations, but non-hotspot PIK3CA mutations may also be prevalent. Molecular diagnosis may help the clinical diagnosis, treatment and management of these pediatric patients with vascular malformations.

Unmatched Whole-Genome Sequencing As a Clinical Tool for Hematological Neoplasms with Significant Utility in Cases with Tumor-in-Normal Contamination

Introduction For patients with acute leukemias, mapping of clinical biomarkers is critical for disease classification, risk stratification and treatment decisions. At diagnosis, molecular profiling is primarily based on cytogenetic analysis, SNP-arrays and increasingly complemented with targeted assays to include Fluorescent In Situ Hybridization (FISH), and DNA/RNA panel assays. Recently, Duncavage et al. showed that for acute myeloid leukemia, whole genome sequencing (WGS) can replace cytogenetics and detect the hotspot mutations in the ELN2017 guidelines (Duncavage et al., 2021). However, the 2022 revision of disease classification and risk stratification led to the incorporation of additional events characterized by a diverse spectrum of non-hotspot mutations (Khoury et al. 2022, Bataller et al. 2022). To this end, the diagnostic utility of WGS for comprehensive characterization of relevant biomarkers across hematological neoplasms is not established. A further limitation for matched WGS (mWGS) is the requirement of a normal sample at diagnosis, as sources of control tissue can be contaminated with tumor cells. Methods To resolve this unmet clinical need, we developed a computational framework for the analysis of WGS data without the use of a normal germline control (uWGS). We applied this uWGS in 2 independent cohorts of patients (Table 1). In cohort 1 of 49 patients, representative of a broad range of hematological neoplasms, uWGS findings were compared against standard-of-care molecular assays to include cytogenetics, FISH, SNP-arrays, targeted-gene panels and RNA sequencing for findings of clinical relevance (disease classification, risk stratification, therapy informing) as annotated by OncoKB (Chakravarty et al. 2017). To further evaluate whether an uWGS can detect the same spectrum of acquired mutations that a matched WGS workflow (mWGS), we compared findings for 52 B-ALL patients in cohort 2 with mWGS data, and we estimated the tumor-in-normal contamination (TiN) with the algorithm deTiN (Taylor-Weiner et al., 2018). Results In Cohort 1, uWGS detected 100% (24/24) of highly confident findings (cancer cell fraction >20) of clinical relevance described by standard-of-care assays (0.49 average, 0-3 range) (Fig.1). Moreover, uWGS identified 9 mutations in 8 patients that were not detected by standard-of-care. These include 2 disease-defining P2RY8::CRLF2 in B-ALL, 2 canonical t(7;9)(q34;q34.3) NOTCH1-intergenic in T-ALL, a TCF3 D561V point mutation and a IGH::MYC event in Burkitt lymphoma, 2 focal deletions in CDKN2A and IKZF1 in B-ALL, and a focal NF1 deletion in JMML. Importantly, 5/8 of these cases did not have a clinically relevant alteration identified by standard-of-care. In Cohort 2, we evaluated what proportion of relevant events identified by mWGS can be detected by uWGS. Putative biomarkers had been identified by mWGS in 88% of patients (46/52). These included delineations of aberrant karyotypes where chromosome banding failed (5/52), newly described fusion genes (i.e. UBTF::ATXN7L3, EP300::ZNF384, in 27/52) and recurrent gene mutations (i.e. PAX5 P80R, ZEB2 H1038R, in 14/52). As a result, uWGS workflow captured 100% of biomarkers identified in the mWGS (5/5 ploidy, 27/27 fusion, 14/14 coding). Remarkably, evaluation of TiN levels reveals that 4 driver events had been missed in the mWGS workflow, affecting genetic alterations across variant classes. For example, a focal BTG1 loss detected by uWGS was not called by mWGS. Overall, 25% of B-ALL cases had evidence of TiN >2% (14/55, ranges 2-57%), resulting in lower sensitivity of detection of copy number variants (CNVs) in mWGS. When looking into the whole spectrum of CNVs detected only by uWGS, we found lower levels of precision in mWGS that were associated with evidence of TiN (median 0.45 vs 0.64, p-value 0.03). Additionally, the other 3 drivers missed by mWGS were indels in CHEK2, JAK1 and TCF3. Conclusions We have developed and validated an uWGS workflow for the detection of clinically relevant alterations in leukemia across variant classes to include CNVs, translocations and point mutations. We demonstrated that for hematological neoplasms, uWGS rescues events that can be missed by mWGS workflows owing to TiN, which shows how uWGS enables the detection of clinically relevant biomarkers, and the opportunity to discover new clinical findings using a single test and a single biopsy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

96P Use of computational algorithms to predict mutation effect in clinical setting

Non-hotspot mutations in oncogenes are frequently identified during complex molecular profiling. Lack of functional or clinical data complicates interpretation of such mutations as driver or passenger event. Computational algorithms may be used to define the role of previously not annotated mutation in cancerogenesis and therapy decision.

Distinct mutational landscapes characterize melanomas metastatic to different anatomical sites.

9562 Background: Despite revolutionary advances in systemic therapies for melanoma, many patients with metastatic disease have limited treatment options and some sites of disease remain particularly challenging to control, such as brain and liver metastases. We sought to define anatomical site-specific mutational profiles of melanoma metastases from which potentially novel personalized therapeutic opportunities may be developed. Methods: Targeted exome genomic profiling was performed via the FoundationOneCDx platform and known or likely pathogenic variants retained for analysis. PD-L1 immunohistochemistry was performed with the Dako 22C3 assay with PD-L1+ defined as tumor proportion score ≥1. Tumor mutational burden (TMB)-high was defined as TMB ≥10mut/Mb. UV signature was calculated using established algorithms. Aberrations in 23 genes potentially actionable in melanoma ( BARD1, BRAF, BRCA1/2, CDKN2A, DDX3X, FANCC, HRAS, IDH1, KIT, KRAS, MAP2K1, NF1, NRAS, PALB2, PIK3CA, PPP6C, PTEN, RAC1, RAD51C/D, RB1, TP53) were compared across metastatic sites including skin, lymph node, lung, liver, and brain. Results: A total of 4918 cutaneous-type melanoma tumors was evaluated, including 2854 skin lesions (primary/metastasis) and metastases from lymph nodes (n = 858), liver (n = 194), lung (n = 342), and brain (n = 200). The commonly mutated genes were CDKN2A (2220/4918, 45.1%), BRAF (2148/4918, 43.7%), NRAS(1347/4918, 27.4%), NF1 (1038/4918, 21.1%), TP53 (1234/4918, 25.1%) and PTEN (1038/4918, 13.5%). Compared with skin lesions, metastases to the lung were enriched for variants affecting NF1 (OR 2.57, p = 8.99e-13), TP53 (OR 1.65, p = 3.04e-04) and depleted in NRAS (OR 0.49, p = 9.00e-06) and BRAF (OR 0.70, p = 1.13e-02). Lung metastases were associated with higher prevalence of UV signatures, PD-L1 positivity and high TMB. PTEN variants were enriched in both brain (OR 3.00, p = 2.38e-08) and small intestine (OR3.46, p = 6.63e-3) metastases. Brain metastases were also enriched for CDKN2A variants (OR 1.56, p = 4.50e-02). Lymph node metastases had lower rates of UV signatures, NF1 variants, or high TMB, but were associated with PD-L1 positivity. Positional analysis of variants revealed that several, including NRAS, MAP2K1, KRAS, HRAS, IDH1, RAC1, GNA11 and GNAQ were highly restricted to hotspot loci, without definite variation by metastatic site. Conversely, variants affecting CDKN2A, PTEN, TP53 and NF1 were distributed widely across the gene, with high levels of non-hotspot mutations observed in skin lesions, suggesting that such non-hotspot subclones contribute less to distant metastatic potential and are lost during disease evolution. Conclusions: We observed distinct and organ site-specific mutational patterns in patients with metastatic melanoma. These data raise the possibility of metastatic phenotype-directed therapy to improve the personalization and outcomes of treatment for this disease.

Abstract 788: Differences in somatic TP53 mutation type in breast tumors by race and receptor status

Abstract Somatic driver mutations in TP53 are associated with triple negative breast cancer (TNBC) and poorer outcomes. African American women with breast cancer are more likely to have TNBC and TP53 driver mutations. Missense driver mutations in TP53 can be loss-of function (LOF) or gain-of-function (GOF). Additionally, some variants show dominant negative (DNE) activity. There are a number of hot-spot TP53 mutations, many occurring at CpG sites. Given the differences in TNBC status and TP53 driver mutation frequency by race, we wanted to determine if there are differences in TP53 mutation type by race. From multiple databases and a literature review, we identified 98 studies with patient race and TP53 mutation details. We compiled TP53 mutations from 2964 breast tumors from these 98 studies. The cohort included 715 (24.1%) Asian, 258 (8.7%) African (AA), and 1931 (65.1%) European (EA) ancestry individuals. Additionally, 60 patients (2%) had Hispanic or Latina ethnicity. Of the somatic TP53 mutations, 939 (31.7%) were GOF, 1739 (58.7%) were LOF, while the remaining 286 (9.6%) were not able to be classified by existing literature. With respect to DNE activity, 1246 (42%) showed DNE, 1190 (40.1%) showed no DNE, and 528 (17.8%) were unknown DNE status. CpG hotspots accounted for 613 (20.6%) mutations. Using Fisher's Exact test, we did not identify significant associations between any of the functional categories and overall race. However, 35.8% of tumors from EA individuals had GOF mutations compared to 29% in AA individuals (p=0.04). Mutations with DNE activity were positively associated with TNBC (OR=1.37, p=0.03) and negative estrogen receptor (ER) status (OR=1.38; p=0.005). Patients with hotspot mutations were slightly younger (mean age 53.61) compared to those with non-hotspot mutations (mean age 55.04), and this association neared significance (p=0.065). Larger cohorts are needed, in particular the AA cohort, to further elucidate some of these borderline findings. In summary, ER negative and TNBC breast tumors are more likely to have DNE TP53 mutations which could reflect biological processes. Citation Format: Nijole Pollock, Johnny Ramroop, Joseph McElroy, Amanda E. Toland. Differences in somatic TP53 mutation type in breast tumors by race and receptor status [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 788.

Identification of Predictive ERBB Mutations by Leveraging Publicly Available Cell Line Databases

Although targeted therapies can be effective for a subgroup of patients, identification of individuals who benefit from the treatments is challenging. At the same time, the predictive significance of the majority of the thousands of mutations observed in the cancer tissues remains unknown. Here, we describe the identification of novel predictive biomarkers for ERBB-targeted tyrosine kinase inhibitors (TKIs) by leveraging the genetic and drug screening data available in the public cell line databases: Cancer Cell Line Encyclopedia, Genomics of Drug Sensitivity in Cancer, and Cancer Therapeutics Response Portal. We assessed the potential of 412 ERBB mutations in 296 cell lines to predict responses to 10 different ERBB-targeted TKIs. Seventy-six ERBB mutations were identified that were associated with ERBB TKI sensitivity comparable with non-small cell lung cancer cell lines harboring the well-established predictive EGFR L858R mutation or exon 19 deletions. Fourteen (18.4%) of these mutations were classified as oncogenic by the cBioPortal database, whereas 62 (81.6%) were regarded as novel potentially predictive mutations. Of the nine functionally validated novel mutations, EGFR Y1069C and ERBB2 E936K were transforming in Ba/F3 cells and demonstrated enhanced signaling activity. Mechanistically, the EGFR Y1069C mutation disrupted the binding of the ubiquitin ligase c-CBL to EGFR, whereas the ERBB2 E936K mutation selectively enhanced the activity of ERBB heterodimers. These findings indicate that integrating data from publicly available cell line databases can be used to identify novel, predictive nonhotspot mutations, potentially expanding the patient population benefiting from existing cancer therapies.

Abstract 4152: Preclinical evaluation of PRKC fusions and GNA11/GNAQ mutations as genetic drivers of PKC activation in non-MUM indications to support a phase 1/2 basket trial of IDE196

Abstract Background: IDE196 is a potent and selective PKC inhibitor targeting the novel (δ, ϵ, η, θ) and classical (α, β) PKC isoforms that has demonstrated preliminary clinical activity in patients with metastatic uveal melanoma (MUM). IDE196 is being further evaluated for safety and efficacy as a monotherapy in a Phase1/2 basket trial in non-MUM patients whose tumors harbor GNAQ/GNA11 hotspot mutations (Clinicialtrials.gov: NCT03947385). Patients with solid tumors harboring PRKC fusions or GNAQ/11 non-hotspot mutations may be eligible after preclinical validation of the activity of these fusions and mutations in non-MUM indications. PRKC gene fusions exist at very low frequencies with various gene partners across multiple indications. Hotspot mutations in GNAQ/GNA11 (codons Q209 and R183) constitutively activate the PKC pathway in 90% of MUM patients and may also occur in non-MUM indications such as colorectal cancer (CRC) and cutaneous melanoma. Furthermore, there are additional non-hotspot mutations found in these GTPases across various non-MUM indications. To help identify patients that may benefit from IDE196 in this basket trial, we assessed the sensitivity of non-MUM cell lines that have PRKC gene fusions or GNAQ/11 hotspot/non-hotspot mutations to IDE196. Methods The most prevalent fusions in patient samples listed in TCGA are the HIF1A-PRKCH (n=20) and RP11-47I22.3-PRKCH (n=3) fusions, therefore cell lines containing PRKCH fusions listed in CCLE were prioritized for cell viability screening using short (3-day) and long-term (10-day) assays after IDE196 treatment. For PD analyses, cells were treated for 3hrs with IDE196. Expression of the fusions and pharmacodynamic modulation of phospho-MARCKS was assessed using western blotting (WB). RNASeq data was used to determine fusion structure, size and whether the fusion was in-frame. Results 8/10 PRKCH fusion lines were highly resistant to IDE196 (IC50>10μM, including HIF1A-PRKCH and RP11-47I22.3-PRKCH fusion lines), whereas 2/10 showed sensitivity at high doses in the long-term viability assay (IC50≥1μM, lines with PRKCH-SPTB or VWA2-PRKCH fusions). PD assessment showed that the PKC substrate, pMARCKS, was decreased in all cell lines by IDE196 at doses similar to those seen in the MUM cell line, 92.1 (≥0.3μM). Fusion protein expression was not detectable by WB consistent with literature suggesting that PKC fusions are unstable (1). In summary, cell lines with clinically prevalent PRKCH fusions were insensitive to IDE196 despite robust PD modulation. Assessment of cell lines harboring GNAQ/GNA11 hotspot/non-hotspot mutations for IDE196 sensitivity is ongoing. (1) An-Angela N. Van, Timothy R. Baffi, Maya T. Kunkel, Corina E. Antal and Alexandra C. Newton, FASEB J. (2018); vol 32, suppl.no. 1, abstract 687.6. Citation Format: Marie Wagle, Nandini Ravindran, Carol O'Brien, Christian Frey, Julie Hambleton, Mark R. Lackner, Zineb Mounir. Preclinical evaluation of PRKC fusions and GNA11/GNAQ mutations as genetic drivers of PKC activation in non-MUM indications to support a phase 1/2 basket trial of IDE196 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4152.

Molecular Barcode Sequencing of the Whole Ligand Binding Domain of the ESR1 Gene in Cell-Free DNA from Patients with Metastatic Breast Cancer

ESR1 mutations in breast cancer are known as one of the mechanisms of resistance to aromatase inhibitors. These mutations often occur in the hotspot regions in the ligand binding domain (LBD), but comprehensive mutational analysis has shown that mutations are observed throughout the whole LBD. We previously developed a molecular barcode sequencing (MB-NGS) technique to detect ESR1 hotspot mutations in plasma with high sensitivity. In this study, we have developed a multiplex MB-NGS assay that covers the whole LBD of ESR1. The assay demonstrated that the background errors in the plasma DNA of 10 healthy controls were below 0.1%; thus, the limit of detection was set at 0.1%. We analyzed the plasma DNA of 54 patients with estrogen receptor–positive metastatic breast cancer. Seventeen mutations were detected in 13 patients (24%), with variant allele frequencies ranging from 0.13% to 10.67%, including six rare mutations with a variant allele frequency <1.0% and a novel nonhotspot mutation (A312V). Three patients had double mutations located in the same amplicons, and it was revealed that the double mutations were located in different alleles. ESR1 hotspot mutations were associated with a longer duration of aromatase inhibitor treatment under metastatic conditions and to liver metastasis. The multiplex MB-NGS assay is useful for the sensitive and comprehensive detection of mutations throughout the whole LBD of ESR1. Our assay can be applied to any specific target region of interest using tailor-made primers and can result in minimized sequencing volume and cost.

Abstract P4-10-15: PIK3CA mutational analysis using cell-free DNA next-generation sequencing detects activating mutations that may be missed with targeted hot-spot testing

Abstract Background: The FDA recently approved alpelisib, in combination with fulvestrant, for HR+/HER2- PIK3CA-mutated advanced/metastatic breast cancer after trials demonstrated improved clinical outcomes with this targeted combination. The companion diagnostic, the Qiagen Therascreen, is a PCR-based kit detecting 11 single nucleotide variant (SNV) mutations limited to 3 exons in the PIK3CA gene. However, other functionally significant mutations outside these hotspots, including activating SNVs and indels, have been reported, suggesting they confer PI3K dependence and, therefore, sensitivity to PI3K inhibitors. We explored the prevalence and spectrum of PIK3CA mutations that can be identified with more comprehensive testing methods. Methods: We queried a large commercial laboratory database of clinical genomic test results from Guardant360 (Guardant Health, Inc) plasma cell-free DNA (cfDNA) next-generation sequencing (NGS) analysis of 74 genes detecting SNVs, indels, copy number amplifications, and fusions. This assay includes full exonic sequencing of PIK3CA. Clinical genomic results from patients with a diagnosis of advanced breast cancer who had at least one genomic alteration detected by Guardant360 between 11/25/2016 - 6/8/2019 were retrospectively analyzed. Results: 6940 eligible samples from 5549 unique patients with advanced breast cancer were identified; some patients had samples submitted at multiple timepoints. Excluding duplicate mutations from serial sampling, a total of 2761 nonsynonymous PIK3CA SNVs were identified in 2095 unique patients (38%); 2435 of these 2761 (88%) detected in 1982 patients (36%) were predicted to be activating. 353/1982 (18%) mutation positive patients had &amp;gt;1 PIK3CA activating mutation detected. Among the 2435 activating SNVs, 626 (26%) were located outside the hotspots covered by the companion diagnostic test. These 626 occurrences included over 70 unique activating mutations, with 16 unique mutations observed in &amp;gt;10 patients each. The most common non-hotspot mutations included E726K (117 patients), N345K (83 patients), and E453K (48 patients). Additional analysis was performed to assess for PIK3CA indels; 118 PIK3CA indels were identified in 118/5549 unique patients (2%). Predicted activating indels were identified across the PIK3CA gene, including the C2 and kinase domains and in the linking region between the adapter binding and Ras-binding domains. Conclusions: PIK3CA mutation analysis with PCR-based hotspot testing is limited to only the most common mutations and will miss as many as one-quarter of alterations that could potentially be targeted with alpelisib, an FDA-approved PI3Ka inhibitor. While additional data may be needed to determine the clinical response from targeting these alterations, molecular data and case reports suggest that these less common PIK3CA mutations are viable targets for a PI3K inhibitor. Comprehensive NGS, including plasma-based cfDNA testing, should be considered to identify the full spectrum of patients who may respond to PI3K targeted therapies. Citation Format: Lesli A Kiedrowski, Dejan Juric, Aaron I Hardin, Kristin S Price, Rebecca J Nagy, Carlos L Arteaga, Joyce O'Shaughnessy, Aditya Bardia, Massimo Cristofanilli, Richard B Lanman. PIK3CA mutational analysis using cell-free DNA next-generation sequencing detects activating mutations that may be missed with targeted hot-spot testing [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-10-15.

An analysis of mutational signatures of synonymous mutations across 15 cancer types

BackgroundSynonymous mutations have been identified to play important roles in cancer development, although they do not modify the protein sequences. However, relatively little research has specifically delineated the functionality of synonymous mutations in cancer.ResultsWe investigated the nucleotide-based and amino acid-based features of synonymous mutations across 15 cancer types from The Cancer Genome Atlas (TCGA), and revealed novel driver candidates by identifying hotspot mutations. Firstly, synonymous mutations were analyzed between TCGA and 1000 Genomes Project at nucleotide and amino acid levels. We found that C:G → T:A transitions were the most frequent single-base substitutions, and leucine underwent the largest number of synonymous mutations in TCGA due to prevalent C → T transition, which induced the transformation between optimal and non-optimal codons. Next, 97 synonymous hotspot mutations in 86 genes were nominated as candidate drivers with potential cancer risk by considering the mutational rates across different sequence contexts. We observed that non-CpG-island GC transition sequence context was positively selected across most of cancer types, and different sequence contexts under which hotspot mutations occur could be significance for genetic differences and functional features. We also found that the hotspots were more conserved than neutral mutations of hotspot-mutation-containing-genes and frequently happened at leucine. In addition, we mapped hotspots, neutral and non-hotspot mutations of hotspot-mutation-containing-genes to their respective protein domains and found ion transport domain was the most frequent one, which could mediate the cell interaction and had relevant implication for tumor therapy. And the signatures of synonymous hotspots were qualitatively similar with those of harmful missense variants.ConclusionsWe illustrated the preferences of cancer associated synonymous mutations, especially hotspots, and laid the groundwork for understanding the synonymous mutations act as drivers in cancer.

BCOR Mutation and TLS-ERG Expression in Acute Myeloid Leukemia with Monoclonal Immunoglobulinemia

Acute myeloid leukemia(AML) originates from the abnormal clonal proliferation of myeloblast which often combined with clinical symptoms. Cytogenetic and molecular abnormalities are frequent in AML patience. To date, the driver genes for leukemia remain largely undiscovered. Monoclonal immunoglobulinemia is a group of diseases caused by excessive proliferation of plasma cells or immunoglobulin-producing lymphoid plasma cells and B lymphocytes. It can develop into malignant plasma cell disease. Herein, we report a AML patient was concomitant with monoclonal immunoglobulinemia, the patient was also accompanied by BCOR mutation and TLS-ERG fusion gene. A 55-year-old married female was admitted into our hospital due to repeated edema for 3 weeks. On admission, peripheral blood counts: PLT142×10^9/L, HB77g/L↓, WBC35.2×10^9/L.Bone marrow examination showed the mononuclear cell system proliferated actively, and the primitive infantile monocytes accounted for 86%. Cell morphology suggested M5b(Figure1A ). Fusion gene screening in bone marrow revealed that TLS-ERG expression. Immunophenotype of bone marrow cell:Abnormal myeloid primitive cells accounted for 96.39% of the nuclear cells,expressCD33, CD13, CD123, CD34, CD9, MPO(Figure 1D). Karyotype analysis of bone marrow cells showed in Figure 1B. Thus, AML was diagnosed. Next-generation DNA sequencing technology showed that BCOR (51.7%),PLCG1(49.9%),DIS3(48.4%),BRAF(51.6%), JAK2(45.1%) ,JAK3(49.0%) were mutated. Meanwhile, we found that Peripheral blood immunofixation electrophoresis showed that Gamma region is seen with a monoclonal light chain lambda component((Figure 1C.).Then, the patient underwent one cycle of IA(Idabisine hydrochloride 10mg d1-4, cytarabine 0.075g q12h d1-7). Twenty-five after chemotherapy onset, bone marrow examination showed that primitive and immature monocytes accounted for 3%. Chromosome become normal. Minimal residual disease(MRD):0.01%. The disease reached complete remission(CR). Peripheral blood immunofixation electrophoresis turned negative. Fusion gene detection showed that TLS-ERG turned negative. BCOR mutation was not detected by Next-generation DNA sequencing. Mutations of PLCG1,DIS3,BRAF,JAK2,JAK3 still exist. Monoclonal immunoglobulinemia and AML are both clonal diseases, but originated from different clones. This case has both malignant clones of granulocyte stem cell and malignant clones of B line, so it is worthy of discussion. By comparing CR before and after we found that while the patient's M protein turned negative, the TLS-ERG fusion gene and BCOR gene mutation also disappeared. The TLS-ERG fusion gene is formed by the rearrangement of TLS and ERG genes on chromosomes 16 and 21. The current study holds that the expression of this fusion gene indicates rapid disease progression and poor prognosis. BCOR mutations can be found in AML and often coincide with DNMT3 gene mutations, suggesting it may affect the occurrence of leukemia through epigenetics. BCOR is a newly discovered corepressor of BCL-6, which can play a supporting role when BCOR combines with DNA; when BCOR is overexpressed, it can enhance the inhibition of BCL-6. BCL-6 is highly expressed in tumor cells,it encodes transcriptional repressors which are required for the formation of germinal center and may affect apoptosis. We thinked that the monoclonal immunoglobulinemia of this patient may caused by the BCOR abnormal expression which increased the inhibitory effect of BCL-6 and affect the apoptosis of B cells, and B cells continue to secrete immunoglobulin. BCOR mutations are associated with poor prognosis. The patient with TLS-ERG fusion gene which is a poor prognosis gene.However, the BCOR gene mutation site is a non-hot spot mutation which has few clinical studies. Whether the BCOR gene mutation results in the combination of the two diseases requires further study. Acknowledgment:The research was supported by fundings of the public technology research projects of Yiwu,China (2016-S-05), the key medical discipline of Yiwu,China(Hematology,2018-2020),and the academician workstation of the Fourth Affiliated Hospital of Zhejiang University School of Medicine. Correspondence to: Dr Jian Huang, Department of Hematology, The Fourth Affiliated Hospital of Zhejiang University School of Medicine. N1 Shangcheng Road. Yiwu, Zhejiang, Peoples R China. Email: househuang@zju.edu.cn Figure 1 Disclosures No relevant conflicts of interest to declare.

Abstract 3387: Comprehensive detection and analysis of mutant-SF3B1 splice pattern

Abstract Aberrant alternative splicing is emerging as a frequent and important process in cancer. Splice aberrations are mainly associated with missense mutations within genes encoding splice factors involved in 3′-splice site recognition. SF3B1, U2AF1, SRSF2 and ZRSR2 are the most frequently mutated genes encoding splicing factors in tumors. Interestingly, these mutations are mutually exclusive and each leads to a distinct aberrant splice pattern. Based on RNA-seq analyses of 74 primary tumors of uveal melanoma, we previously defined the aberrant splice pattern of SF3B1 hotspot mutations. In order to further characterize the catalogue of pathogenic mutations in SF3B1 that induce the aberrant splice pattern, we used a Sequence Bloom Tree (SBT) constructed from RNA-seq data for a total of 11,350 different tumors from the TCGA (Collaborative project with Seven Bridges Cancer Genome Cloud) to rapidly screen for samples consistent with the aberrant splice pattern. Based on this analysis, 75 tumors from different tissues of origin presented with the mutant-SF3B1 pattern, including 70 tumors harboring SF3B1 mutations: 48 cases of the hotspot mutations (21 tumors with SF3B1R625, 6 with SF3B1K666 and 21 with SF3B1K700) and 22 cases harbored non-hotspot SF3B1 mutations. No mutation or deletion in SF3B1 was detected in 5 tumors. Taking into account that the threshold of scores defining the significant presence of the aberrant splice pattern varies between tumor types, we proceeded to an in cellulo validation of a set of newly described mutations of SF3B1 and confirmed the induction of the aberrant splice pattern. Overall, our study highlights the potential of splice patterns as a biomarker in absence of detectable splice related mutations and sheds light on an efficient, large-scale computational approach to screen clinical cases for splice patterns. Citation Format: Samar Alsafadi, Dorine Bellanger, Michele Cornella, Erik Lehnert, Sergio Roman-Roman, Marc-Henri Stern, Tatiana Popova. Comprehensive detection and analysis of mutant-SF3B1 splice pattern [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3387.

Uncommon mutational profiles of metastatic colorectal cancer detected during routine genotyping using next generation sequencing

RAS genotyping is mandatory to predict anti-EGFR monoclonal antibodies (mAbs) therapy resistance and BRAF genotyping is a relevant prognosis marker in patients with metastatic colorectal cancer. Although the role of hotspot mutations is well defined, the impact of uncommon mutations is still unknown. In this study, we aimed to discuss the potential utility of detecting uncommon RAS and BRAF mutation profiles with next-generation sequencing. A total of 779 FFPE samples from patients with metastatic colorectal cancer with valid NGS results were screened and 22 uncommon mutational profiles of KRAS, NRAS and BRAF genes were selected. In silico prediction of mutation impact was then assessed by 2 predictive scores and a structural protein modelling. Three samples carry a single KRAS non-hotspot mutation, one a single NRAS non-hotspot mutation, four a single BRAF non-hotspot mutation and fourteen carry several mutations. This in silico study shows that some non-hotspot RAS mutations seem to behave like hotspot mutations and warrant further examination to assess whether they should confer a resistance to anti-EGFR mAbs therapy for patients bearing these non-hotspot RAS mutations. For BRAF gene, non-V600E mutations may characterise a novel subtype of mCRC with better prognosis, potentially implying a modification of therapeutic strategy.

Several genotypes, one phenotype: PIK3CA/AKT1 mutation-negative hidradenoma papilliferum show genetic lesions in other components of the signalling network

About 60-70% of hidradenoma papilliferum (HP), a benign tumour of the anogenital region, were recently described to harbour mutations in major driver genes of the PI3K/AKT/MAPK-signalling pathways. However, the underlying genetic defects of the non-mutant cases are still unknown. Using a 409 gene panel, we employed targeted next generation sequencing to investigate the mutational landscape in a cohort of seven PI3K/AKT-negative cases and five cases with known hotspot mutations in either PIK3CA or AKT1. In total, we identified 29 mutations in 22 of 409 genes. The four cases with PIK3CA hotspot mutations carried no or only few additional mutations. The AKT1 hotspot mutated case harboured additional mutations in four genes (SYNE1, ADAMTS20, EP400 and CASC5). At least two of these genes are involved in or contribute to the PI3K/AKT-pathway. In the seven non-hotspot mutated cases we observed 18 mutations. Each case carried at least one mutation in a gene contributing to or involved in PI3K/AKT-signalling. Affected genes were PIK3CA (n=1, non-hotspot mutation), PIK3R1 (n=3), SYNE1, AR, IL6ST, PDGFRB, KMT2C, AR, BTK, DST, KAT6A, BRD3, RNF213, USP9X, ADGRB3, MAGI1, and IL7R (each gene mutated once). The identified PIK3CA and PIK3R1 mutations lead to constitutive activated PI3K/AKT-signalling. In conclusion, we demonstrate the genetic basis of HP in all cases. Our data suggest that tumourigenic alterations in the PI3K/AKT-pathway are indispensable in HP and establish a homogenous morphomolecular entity with a functionally converging and selecting tumourigenic mechanism.

Abstract P5-11-09: Feasibility of tracking plasma DNA mutation kinetics in estrogen receptor positive metastatic breast cancer using a novel digital PCR amplicon sequencing assay

Abstract Estrogen Receptor 1 (ESR1) gene mutations in estrogen receptor positive (ER+) metastatic breast cancer (MBC) are associated with clinical resistance to endocrine therapy. A highly sensitive digital PCR (dPCR) assay was developed to evaluate the baseline abundance and kinetics of hotspot ESR1 mutations in plasma circulating tumor DNA (ctDNA) from 58 ER+ MBC patients who had progressed on at least one line of prior endocrine therapy. Consistent with prior reports, we demonstrate that detection of hotspot ESR1 mutations in plasma ctDNA is associated with shorter progression-free survival. However, after patients initiated a new course of systemic therapy, changes in plasma ESR1 mutation abundance did not correlate with the duration of disease control. To investigate whether mutational heterogeneity may account for this lack of correlation, we developed a customized dPCR multiplexed amplicon next-generation sequencing (NGS) assay to detect plasma ctDNA mutations in the complete coding sequence of ESR1 and TP53, as well as hotspot regions in PIK3CA. Assay validation revealed a 79% sensitivity and 100% specificity for detecting plasma ctDNA mutations with a lower limit of detecting a 1.6% mutant allele fraction. The NGS assay revealed non-hotspot ESR1 mutations in plasma DNA from 14 of 31 (45%) patients. We also observed dynamic changes in plasma ctDNA mutant allele fraction of PIK3CA and TP53 during systemic therapy. Changes in the cumulative mutant allele fraction of ESR1, PIK3CA, and TP53 in plasma ctDNA correlated with duration of clinical treatment response in a limited subset of patients (n=8) for whom NGS was performed at two or more time points. Thus, mutational heterogeneity limits the clinical utility of monitoring individual hotspot mutations in ESR1 over time in ER+ MBC patients. However, a plasma ctDNA NGS assay targeting ESR1, PIK3CA, and TP53 may be useful as an early predictor of response to systemic therapy. Citation Format: Kumar S, Lindsay D, Chen B, Garrett AL, Tan XM, Anders CK, Carey LA, Gupta GP. Feasibility of tracking plasma DNA mutation kinetics in estrogen receptor positive metastatic breast cancer using a novel digital PCR amplicon sequencing assay [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-11-09.

Abstract CT033: Safety and early efficacy signals for COTI-2, an orally available small molecule targeting p53, in a phase I trial of recurrent gynecologic cancer

Abstract Background: Mutated p53 tumor suppressor protein is involved in &amp;gt;50% of all malignancies; however, only recently have candidate drugs designed to target p53 mutations entered clinical trials. COTI-2, an orally bioavailable "third-generation" thiosemicarbazone, was shown to restore the structure and function of mutant p53 proteins and induced growth inhibition in a number of p53 mutant cell lines. Thus, a three-part phase I dose escalation trial of COTI-2 was initiated with Part 1 reported herein. Methods: A multicenter, open label, 3+3 dose escalation trial enrolling patients with recurrent gynecological cancers with adequate performance status and bone marrow function. Patients could have unlimited prior therapies. Patients received COTI-2 5x/week orally over a 28-day cycle. Disease response was assessed by RECIST 1.1 every 8 weeks. Results: Twenty-four patients received study treatment: 19 ovarian, 3 cervical, and 2 endometrial cancers. Median age was 60 years (45-73 yrs) and patients had received a median of 5 prior chemotherapy regimens. After 4 dose cohorts (0.25, 0.5, 1.0, and 1.7 mg/kg), the Recommended phase II Dose (R2PD) for gynecologic cancer was 1.0 mg/kg. Dose-limiting toxicities (DLT) were encountered at 1.0 mg/kg (G3 abdominal pain, G3 sensory peripheral neuropathy, n=1) and 1.7 mg/kg, (G3 neuralgia n=1, G3 myalgia n=1). All DLTs resolved off treatment. Most common adverse events (&amp;gt; 10%) were nausea (67%), vomiting (67%), fatigue (54%), abdominal pain (46%), constipation (29%), anemia (29%), dyspnea (29%), anorexia (29%), urinary tract infection (21%), and hypokalemia (20%), myalgia (16%), diarrhea (16%), pyrexia (17%), peripheral neuropathy (17%), increased creatinine (13%), and weight loss (13%). Pharmacokinetic assessment showed oral administration of COTI-2 yielded a Tmax between 15-90 minutes and a half-life of 8-10 hours. Fifteen patients were evaluable for efficacy, having received at least one cycle of treatment. Of these, 13 had ovarian, 1 had cervical, and 1 had endometrial cancer. Eight patients had received one or more bevacizumab combinations, and two had received immune checkpoint inhibitors. Following COTI-2 treatment, 10 patients showed signs of possible activity: 1 patient had stable disease, 4 had stable target lesions, and 5 had stable non-target lesions. Twelve patients had comprehensive genomic profiling by next generation sequencing: 3 had wild-type p53 and 9 had either hotspot or non-hotspot mutations. Conclusions: COTI-2 was deemed generally safe and well-tolerated. Part 2 of the study is currently underway in HNSCC patients, with additional data expected in 2018, and Part 3 will follow with expansion to combination treatment of ovarian and HNSCC patients. Clinical trial information: NCT02433626. Citation Format: Shannon N. Westin, Wilberto Nieves-Neira, Christian Lynam, Kowthar Y. Salim, Alison D. Silva, Richard T. Ho, Gordon B. Mills, Robert L. Coleman, Filip Janku, Daniela Matei. Safety and early efficacy signals for COTI-2, an orally available small molecule targeting p53, in a phase I trial of recurrent gynecologic cancer [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 CT033.