Median Mutation Load Research Articles

Molecular Characterization of Diffuse Large B-Cell Lymphoma Associated to Hepatitis C Virus Infection

BACKGROUND. HCV-positive DLBCL has distinct clinical and pathologic characteristics compared to its negative counterpart: patients (pts) are usually older with more frequent splenic and extranodal involvement and elevated LDH. Differently from its clinical hallmarks, the molecular landscape of this pathological entity has been scarcely outlined.METHODS. In this bicentric study, we investigated the clinical and molecular features and outcome of 54 pts with HCV-positive DLBCL. Targeted next generation sequencing (NGS) was performed on DNA extracted from formalin-fixed paraffin-embedded tissue biopsies. A core panel probes covering coding exons from 184 genes frequently mutated in mature B cell neoplasms was designed using IDT tool and libraries were prepared using Illumina DNA-prep-with enrichment. Sequencing was performed on Illumina HiSeq 2500. Cluster analysis was performed using LymphGen tool. We also applied fluorescence in situ hybridization (FISH) for MYC, BCL2 and BCL6.RESULTS. Median age was 71 (33-84; IQR: 61.9-77). Stage was III/IV in 34 pts (63%). Extranodal sites were involved in 21 pts (38%), spleen in 20 pts (37%). LDH was higher than the upper limit in 40 pts (74%). R-IPI was good for 2 pts (4%), intermediate for 24 pts (44%), poor for 28 pts (52%). HPS score was intermediate or high in 33 of 44 assessed pts (75%). A histological low-grade component was identified in 15 pts (27%). Hans algorithm differentiated pts almost equally in GCB (26/50, 52%) and non-GCB (24/50, 48%) subtype. HCV-RNA was detectable in 52 pts (96%) and quantifiable in 43 pts (79%). Of 29 pts assessed, genotype was 1 in 9 (31%), 2 in 16 (55%), 3 in 4 pts (14%). Among 37 pts whose data were available, 11 pts (30%) received direct antiviral agents, 7 pts (19%) received interferon-containing regimen, 19 pts (51%) were not treated for HCV. Twenty-seven pts (50%) received rituximab-enriched protocols, 23 pts (43%) were treated with chemotherapy alone, 1 pt (2%) with surgery alone, 3 pts (5%) were lost to follow up. With a median follow up of 7.7 years (yrs) (IQR: 4.6-10.6), 5-yrs overall survival (OS) (95%CI) was 49.3% (34.1-62.8%) and 5-yrs progression free survival (PFS) (95%CI) was 39.5% (25.5-53.3%). Median OS and PFS were 4.9 and 3.1 yrs, respectively. FISH analysis showed lack of BCL2 (0/19) and MYC translocations (0/15). BCL6 fusions were found in 76% of pts (16/21).NGS showed mutations in 154 of the 184 analyzed genes. The informativity of the panel was 100% with all pts presenting at least one oncogenic variant. Gene mutation frequencies are presented in Fig. 1. The median mutation load (MML) was 13 mutated genes per case (2-32; IQR: 9-16). Most frequently mutated genes were the epigenetic regulators KMT2D, mutated in 23 pts (42.6%), and SETD1B, mutated in 17 pts (31.5%). FAS, PM1 and RERE were mutated in 15 pts each (27.8%). TBL1XR1, BCL11A and SGK1 were mutated in 14 (26%), 13 (24%) and 12 pts (22%), respectively. Considering genes in their specific pathway, 94% of pts harbored mutations in genes involved in epigenetic regulation (MML: 3; range 1-7; IQR: 1.25-4), 90% of pts in apoptosis-related genes (MML: 2; 1-7; IQR: 1-3) and 77% of pts in genes belonging to BCR/NFkB signaling pathway (MML: 2; 1-7; IQR: 1-3). Of note, 56% of pts carried mutations in genes related to immune regulation (MML: 1.7; 1-5; IQR: 1-2) and 25% of pts had mutations within the NOTCH pathway (MML: 1.2; range 1-2). Via the LymphGen 1.0 tool, we classified 26 pts (48%) into 4 genetic clusters: BN2 (11/26, 42%), ST2 (8/26, 31%), MCD (4/26, 15%), EZB (3/26, 12%). Twenty-eight pts (52%) were classified as “others”. Among those belonging to BN2 cluster, 7 pts (64%) had a histologically confirmed transformed DLBCL. No significant differences in terms of OS and PFS were identified according to cluster subgroups.CONCLUSIONS. The prevalence of the BN2 cluster and enrichment of mutations of genes involved in NOTCH pathway seem to indicate a preferential marginal-zone origin in HCV-positive DLBCL. In addition, our data confirm the absence of BCL2 translocation in this subset of DLBCL and show a high prevalence of mutated genes within the epigenetic and immune regulation pathways in HCV-positive DLBCL, pointing out their compelling role in the pathogenesis and suggesting potential implications for molecularly targeted therapies. [Display omitted] DisclosuresPassamonti: Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Arcaini: Celgene: Speakers Bureau; Gilead Sciences: Research Funding; Bayer, Celgene, Gilead Sciences, Roche, Sandoz, Janssen-Cilag, VERASTEM: Consultancy; Celgene, Roche, Janssen-Cilag, Gilead: Other: Travel expenses.

PS1038 FLT3 TESTING IN THE RYDAPT ERA

Background:The approval of Rydapt (midostaurin) heralds the first targeted therapy in acute myeloid leukaemia (AML). Accurate detection of both FLT3 internal tandem duplications (ITD) and tyrosine kinase domain (TKD) variants is required to ensure that patients correctly receive the therapy. Additionally, recent European LeukaemiaNet AML guidelines now require the testing of a number of genetic markers including FLT3 to risk stratify patients.Aims:With the increased importance of FLT3 testing for the diagnosis, prognosis and treatment of AML, the aims of this study were to assess methodological variation and the quality of laboratory testing of FLT3 ITD variants.Methods:The United Kingdom National External Quality Assessment Service for Leucocyte Immunophenotyping (UK NEQAS LI), has been providing external quality assessment (EQA) to laboratories undertaking FLT3 ITD testing by molecular genetic methods since 2011, and became IS0 17043 accredited in 2014. The programme currently has 130 active participants in 36 countries globally. Two lyophilised patient donated or cell line based samples are shipped to laboratories 3 times per annum and participants are asked to test the samples with their in‐house techniques, reporting both qualitative (ITD present/absent) and quantitative (mutation load/allelic ratio) data along with a number of methodological details.Results:Forty samples (including samples both positive and negative for the FLT3 ITD) have been issued to laboratories, in 20 separate distributions, since 2011. Positive samples have featured ITD sizes ranging from 30–126 bp, with mutation loads ranging from 10–90%.The most popular analysis methods used by participants were capillary electrophoresis (70.8%) and agarose gel electrophoresis (20.0%), with a small number of participant using next generation sequencing (NGS), melt curve analysis, acrylamide gel electrophoresis, microfluidic electrophoresis and Sanger sequencing. The majority of laboratories use laboratory developed (in‐house) tests (85.8%), with the remaining participants using commercially available assays.Concordance rates ranged from 83.3% to 100% (average 97.5%). Higher error rates were associated with low mutation load samples and larger ITDs (126 bp). High error rates were associated with gel electrophoresis and NGS based methods. Quantitatively, all laboratories were able to distinguish a high (median mutation load = 92%) and low (median mutation load = 11.7%) mutation load sample with a high degree of concordance (interquartile range = 2.5% and 3.7%, respectively), but variation in calculation methods used were identified.Summary/Conclusion:The quality of FLT3 testing was generally high, although a higher error rate was associated with low mutation load samples and large ITDs when participants used agarose gel or NGS based techniques. Gel electrophoresis is an inherently variable, outdated method and its use should not be encouraged. Standard NGS analysis software has difficulty in detecting large ITDs, as large insertions do not retain sufficient homology in the regions flanking the insertion to permit accurate alignment. Pair‐end based analysis approaches have been developed that can reliably detect larger insertions. The high degree of concordance seen in quantitative data was reassuring but could be improved further by standardising calculation methods.

Incorporation of Clinical Information to Decipher Driver Mutations in Myeloproliferative Neoplasms

Introduction: Mutations in the epigenetic regulators are commonly found in myeloid disorders including Myeloproliferative Neoplasm (MPN). Primary myelofibrosis, dysplastic changes and severity of the disease were associated with the mutation load. Most of the studies had a limited number of targeted genes and included a mixture of JAK2 positive and negative disease. The objective of this study is to assess the impact of mutations in the epigenetic regulators on the presentation of patients with JAK2 V617F positive MPN.Methods: We retrieved the clinical and laboratory information on 61 consecutive eligible patients. Mutation analysis of the entire coding regions of ASXL1, ASXL2, CBL, CEBPA, CSF3R, DNMT3A, EZH2, IDH2, TET1 and TET2 genes was performed using next generation sequencing (NGS; Ion PGM Sequencer; Thermo Fisher ScientificÒ). The library was constructed and the templates were prepared using the PGM tool. The variants were annotated using the ClinVar database and the prediction from the Scale-Invariant Feature Transform (SIFT) and or Polymorphism Phenotyping (Polyphen) algorithms. Alignment, variant filtering and annotation were performed using Ion Torrent Suite. Standard descriptive and analytical statistics were used as appropriate to describe and compare different groups. The MPN subtype, bleeding, thrombosis, hemoglobin, platelet count, White Cell Count (WBC), Lactate Dehydrogenase (LDH) and erythropoietin level were compared for each candidate variant. An alpha threshold of 0.05 was used with no adjustment for multiple comparisons as the analyses were considered exploratory. All statistics were performed using R program. Variants were selected for further experimentation based on their frequency and association with the clinical information at diagnosis.Results: Sixty-one patients were included (Polycythemia Vera: 29, Essential Thrombocythemia: 21, Primary Myelofibrosis: 9, MPN unclassifiable: 2) with a median age of 62 years (Interquartile Range [IQR]: 44 - 70). Male to female ratio was 35:26. The median hemoglobin, WBC, platelet count, LDH and erythropoietin were 14.6 g/dL (IQR: 12.8 - 16.8), 11.5 *109/L (IQR: 11.5 - 14.4), 507 *109/L (IQR: 391 - 779), 265 mU/mL (IQR: 231 - 409) and 1.2 mU/mL (IQR: 1.0 - 4.8) respectively. At presentation, 54% had splenomegaly, 23% had an arterial or venous thrombosis, and 5% had bleeding. Sixty-three variants were found in the samples tested. The median mutation load was 13 variants (Range: 11-14). Patients with higher than the median mutation load had higher mean erythropoietin (7.8 vs. 0.9 g/dL; p = 0.02016). ASXL1 p.Leu815Pro variant was found in all patients. Only three variants were found in the ClinVar database. Seven variants were predicted to be pathogenic (ASXL1: 1, EZH2: 1, IHD1: 1, TET1: 1 and TET2: 3). Patients with TET2: p.Leu1721Trp variant had 6.4 higher odds of bleeding (p = 0.04345). Patients with TET2: p.His1778Arg variant had a lower WBC (9.1 vs. 13.9 *109/L; p = 0.01699) and LDH (213 vs. 348 mU/mL; p = 0.0006528) while those with TET1: p.Lle1123Met variant had a higher WBC (13.5 vs. 8.3 *109/L; p = 0.02756). None of the remaining comparisons were statistically significant.Conclusions: Incorporation of clinical information facilitates the prioritization of variants from DNA sequencing experiments. In MPN, we recommend ASXL1: p.Leu815Pro (expressed in all patients), TET2: p.Leu1721Trp (associated with bleeding), TET2: p.His1778Arg (correlated with the WBC and LDH) and TET1: p.Lle1123Met (correlated with LDH) for further functional experimentation. DisclosuresNo relevant conflicts of interest to declare.

ATM/RB1 mutations predict shorter overall survival in urothelial cancer.

BackgroundMutations of DNA repair genes, e.g. ATM/RB1, are frequently found in urothelial cancer (UC) and have been associated with better response to cisplatin-based chemotherapy. Further external validation of the prognostic value of ATM/RB1 mutations in UC can inform clinical decision making and trial designs.ResultsIn the discovery dataset, ATM/RB1 mutations were present in 24% of patients and were associated with shorter OS (adjusted HR 2.67, 95% CI, 1.45–4.92, p = 0.002). There was a higher mutation load in patients carrying ATM/RB1 mutations (median mutation load: 6.7 versus 5.5 per Mb, p = 0.072). In the validation dataset, ATM/RB1 mutations were present in 22.2% of patients and were non-significantly associated with shorter OS (adjusted HR 1.87, 95% CI, 0.97–3.59, p = 0.06) and higher mutation load (median mutation load: 8.1 versus 7.2 per Mb, p = 0.126).Materials and MethodsExome sequencing data of 130 bladder UC patients from The Cancer Genome Atlas (TCGA) dataset were analyzed as a discovery cohort to determine the prognostic value of ATM/RB1 mutations. Results were validated in an independent cohort of 81 advanced UC patients. Cox proportional hazard regression analysis was performed to calculate the hazard ratio (HR) and 95% confidence interval (CI) to compare overall survival (OS).ConclusionsATM/RB1 mutations may be a biomarker of poor prognosis in unselected UC patients and may correlate with higher mutational load. Further studies are required to determine factors that can further stratify prognosis and evaluate predictive role of ATM/RB1 mutation status to immunotherapy and platinum-based chemotherapy.

Comprehensive genomic profiling of parathyroid carcinoma.

6088 Background: Parathyroid carcinoma (PC) is a rare endocrine malignancy, which can cause life-threatening hypercalcemia. Initial surgery is often noncurative, and adjunctive radiotherapy and previous chemotherapies have not been shown to be effective. Previous studies identified recurring mutations in CDC73 and PRUNE2in a limited number of patients. We queried whether comprehensive genomic profiling (CGP) would have potential to discover novel targets of therapy. Methods: DNA was extracted from 40 microns of FFPE sections from 13 consecutive cases of relapsed/metastatic PC. CGP was performed on hybridization-captured, adaptor ligation based libraries to a mean coverage depth of 672x for up to 315 cancer-related genes plus 37 introns from 14 genes frequently rearranged in cancer. Genomic alterations (GA) included base substitutions (SUB), INDELs, copy number alterations (CNA) and fusions/rearrangements. Clinically relevant GA (CRGA) were defined as GA linked to drugs on the market or under evaluation in mechanism driven clinical trials. Results: Total of 13 specimens were identified from 7 male and 6 female patients. The mean age of the patients in this study was 54 years (range 38 to 76 years). All (100%) cases were Stage IV at the time of CGP. Tumor mutation burden was generally low - median mutation load per mega base was 1.8. There were 58 total GA (4.5 GA/sample) and 10 CRGA (0.8 CRGA/sample). The most frequent GA were non-CRGA mutations in TP53 (31%) and CDC73 (31%). MEN1 mutations were identified in 23% of cases. Frequent alterations in genes controlling cell cycle progression at G1 including CDKN1B, CDKN2A, CDKN2B and CDKN2C were identified (30%). The most frequent CRGA involved PTEN (23%), NF1 (23%) and KDR (15%). No alterations in BRAF or RETwere identified. A patient with KDR mutation treated with cabozantinib experienced > 50% drop in PTH level and radiographic partial response in 3 months. Conclusions: CGP identified previously unreported TP53 mutations in PCs and potentially actionable genomic alterations including PTEN, NF1 and KDR. Clinical benefit and response observed in a patient treated with VEGFR targeted therapy suggest that patients with this rare tumor may be candidates for targeted therapies.

ATM/RB1 mutations to predict shorter overall survival (OS) in bladder cancer.

4547 Background: DNA repair defect plays an important role in tumorigenesis, progression and treatment outcomes of urothelial cancer. Somatic mutations of ATM/RB1 genes are frequently found in urothelial cancer and have been associated with a better response to cisplatin-based neoadjuvant chemotherapy. However, their prognostic value overall in urothelial cancer have not been determined. Methods: Exome sequencing data of 130 urothelial bladder cancer patients (pts) from The Cancer Genome Atlas (TCGA) dataset were analyzed as a discovery cohort to determine the prognostic value of ATM and RB1 mutations. Results from discovery dataset were further validated by an independent cohort of 79 advanced urothelial cancer pts who received comprehensive genomic sequencing for urothelial cancer with FoundationOne. OS was measured from time of initial diagnosis and Cox proportional hazard regression analysis was performed to calculate the hazard ratio (HR) and 95% confidence interval (CI). Results: In the discovery dataset, somatic mutations of ATM/RB1 genes were present in 24% of pts and were associated with significantly shorter OS [all stages: adjusted HR = 2.67, 95% CI, 1.45–4.92, P = 0.002; stage II-III only: adjHR = 2.76, 95% CI, 1.23–6.20, P = 0.014]. There was high mutation load in pts carrying ATM/RB1 mutations (median mutation count: 196 versus 160, P = 0.09). In the validation (stage IV) dataset, ATM/RB1 mutations were present in 31.7% of pts and tended to associate with shorter OS (adjHR = 1.97, 95% CI, 0.89–4.40, P = 0.094) and higher mutation load (median mutation load: 8.1 versus 7.2 per Mb, P = 0.136), although statistical significance was not reached. Conclusions: These results suggest that ATM/RB1 mutations may be considered as a poor prognostic biomarker in unselected urothelial cancer pts and may correlate with higher mutational load. Further studies are required to determine patient characteristics that can further stratify prognosis based on ATM/RB1 mutation status, and evaluate the potential predictive role of ATM/RB1 mutation status in response to immunotherapy.

ATM/RB1 mutations to predict shorter overall survival (OS) in bladder cancer.

393 Background: DNA repair defect plays an important role in tumorigenesis, progression and treatment outcomes of urothelial cancer. Somatic mutations of ATM/RB1 genes are frequently found in urothelial cancer and have been associated with a better response to cisplatin-based neoadjuvant chemotherapy. However, their prognostic value overall in urothelial cancer have not been determined. Methods: Exome sequencing data of 130 urothelial bladder cancer patients (pts) from The Cancer Genome Atlas (TCGA) dataset were analyzed as a discovery cohort to determine the prognostic value of ATM and RB1 mutations. Results from discovery dataset were further validated by an independent cohort of 79 advanced urothelial cancer pts who received comprehensive genomic sequencing for urothelial cancer with FoundationOne. OS was measured from time of initial diagnosis and Cox proportional hazard regression analysis was performed to calculate the hazard ratio (HR) and 95% confidence interval (CI). Results: In the discovery dataset, somatic mutations of ATM/RB1 genes were present in 24% of pts and were associated with significantly shorter OS [all stages: adjusted HR = 2.67, 95% CI, 1.45–4.92, P = 0.002; stage II-III only: adjHR = 2.76, 95% CI, 1.23–6.20, P = 0.014]. There was high mutation load in pts carrying ATM/RB1 mutations (median mutation count: 196 versus 160, P = 0.09). In the validation (stage IV) dataset, ATM/RB1 mutations were present in 31.7% of pts and tended to associate with shorter OS (adjHR = 1.97, 95% CI, 0.89–4.40, P = 0.094) and higher mutation load (median mutation load: 8.1 versus 7.2 per Mb, P = 0.136), although statistical significance was not reached. Conclusions: These results suggest that ATM/RB1 mutations may be considered as a poor prognostic biomarker in unselected urothelial cancer pts and may correlate with higher mutational load. Further studies are required to determine patient characteristics that can further stratify prognosis based on ATM/RB1 mutation status, and evaluate the potential predictive role of ATM/RB1 mutation status in response to immunotherapy.

In Chronic Lymphocytic Leukemia the Gain of the Short Arm of Chromosome 2 Is Highly Associated with an Unmutated IGHV status, 11q/ATM Deletion, SF3B1 Mutation and a Complex Karyotype

Background: CLL is characterized by a distinct pattern of translocations, genomic gains and losses and molecular mutations. The most frequent abnormalities such as trisomy 12 and deletions of 6q, 11q, 13q and 17p have been intensively studied. However, data on less frequent recurrent abnormalities such as the partial gain of the short arm of chromosome 2 is lacking.Aims: a) Determine the frequency of 2p gain in CLL, b) Characterize the size and the commonly gained region, c) Analyze the spectrum of additional cytogenetic abnormalities and molecular mutations, and d) Evaluate the prognostic impact.Patients and Methods: Chromosome banding analysis (CBA) revealed a gain of 2p in 113 out of 5564 (2%) CLL cases. In 72 cases with sufficient material genomic array analysis (SurePrint G3 ISCA CGH+SNP Microarray, Agilent, Waldbronn, Germany) and determination of the mutation status of TP53, SF3B1 and IGHV were performed.Results:76% of patients with gain of 2p were male. The median WBC count was 33,700/µL (range: 5,900 - 228,000). Median age was 66 years (range: 29 - 87).The gain of 2p always encompassed the 2p telomere (2pter) while the centromeric border of the 2p gain varied between 2p21 and the centromere of chromosome 2 (2p10) (genomic positions 45,859,076 to 92,297,003).The gain of 2p was the sole chromosomal abnormality in only 8/72 cases (11%) and was accompanied by one, two or more than two additional aberrations in 10, 20, and 34 cases. In total 209 chromosome abnormalities were observed in addition to the 2p gain (median per patient: 2, range: 0-16). Of these only 21 were balanced while 188 were unbalanced abnormalities leading to gain or loss of chromosomal material. Gain of 2p was most frequently accompanied by deletions in 13q (total: 74%, homozygous: 11%), 11q (56%), 18p (18%), and 6q (13%) and gains of 8q (11%). 17p deletions were present in 6% of cases.In 49 cases (68%) the gain of 2p was present in the main clone while it was present in a subclone only in 23 cases (32%). The gain of 2p material was due to a duplication in the short arm of chromosome 2 in 10 cases, while a gain of an isochromosome 2p was present in 3 cases. In the remaining cases material of the short arm of chromosome 2p was attached to a variety of different partner chromosomes. The most frequent acceptor chromosome was chromosome 18 (n=13; 18%).In two cases (2%) 2 IGH rearrangements were observed of which one was mutated and the other unmutated. The IGHV status was unmutated (IGHV-U) in 66 (92%) and mutated in only 4 cases (6%). Three of these 4 cases with mutated IGHV showed only a low mutation rate (sequence homology to germline 97-97.9%). Stereotyped B-cell receptors were present in 14 cases (19%). SF3B1 mutations were observed in 21 cases (29%) with a median mutation load (ML) of 39% (range: 10-51%). TP53 mutations were detected in 8 (11%) cases (median ML: 60%, range: 13-100%). In 2 patients with a TP53 mutation a TP53 deletion was present and in 3 cases a copy neutral loss of heterozygosity (CN-LOH) of 17p was detected leading to TP53 wild-type loss in these 5 cases. TP53 mutations were less frequent in cases harboring the gain of 2p as the sole abnormality (3% vs 21%, p=0.02)The prognostic impact of 2p gain was evaluated in an unselected cohort of 1381 CLL cases with available follow up data (median follow up: 5.1 years) including 22 cases with 2p gain. The frequency of IGHV-Ustatus, SF3B1 mutations and 11q/ATM deletions was significantly higher in CLL with 2p gain compared to cases without (for all p<0.05). In univariate Cox regression analysis gain of 2p was significantly associated with shorter overall survival (OS) (relative risk (RR): 2.1; p=0.05). 5 year OS was 69% in CLL with 2p gain compared to 85% in cases without 2p gain (p=0.05). However, in multivariate analysis only IGHV-U, mutations in SF3B1 and TP53 and TP53/17p deletion were independently associated with shorter OS, while gain of 2p and 11q/ATM deletion were not. 2p gain was associated with shorter time to treatment (TTT) (RR: 2.0; p=0.02). In multivariate analysis only IGHV-U, SF3B1 mutation and 11q/ATM and TP53/17p deletion were independently associated with shorter TTT, while gain of 2p and TP53 mutations were not.Conclusions:CLL with gain of 2p is highly associated with an unmutated IGHV status (92%), a high frequency of 11q/ATM deletion (56%), 13q deletion (74%), SF3B1 mutation (29%) and a complex karyotype (47%). Data suggest that gain of 2p is a later event in CLL pathogenesis and might be a marker of progression. DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Jeromin:MLL Munich Leukemia Laboratory: Employment. Stengel:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Abstract 3576: Broad analysis of recurrent somatic mutations in cancer reveals a common novel non-coding mutation in the promoter of PMS2 associated with greatly increased tumor mutation load

Abstract Purpose Tumor mutation load is an emerging prognostic and diagnostic marker for many cancers. Sensitivity to immunotherapeutic agents, which stimulate an antitumor immune response by selectively inhibiting immunosuppressive cell surface ligands, is known to correlate with high mutation load in the tumor. Numerous somatic and germline defects can cause genomic instability, including alterations affecting the mismatch repair pathway, DNA polymerases, and cell cycle regulators. We describe here the discovery of previously unreported mutations in the promoter of the PMS2 gene that are associated with significantly increased tumor mutation load. Methods Comprehensive genomic profiling by hybridization capture of exonic regions from either 236 or 315 cancer-related genes and select introns from 19 genes commonly rearranged in cancer was used to characterize more than 60,000 clinical FFPE cancer specimens. At least 50 ng of extracted DNA was analyzed per sample and the constructed libraries were sequenced to high, uniform median coverage (&amp;gt;500x). Samples were assessed for base substitutions, short insertions and deletions, copy number alterations and gene fusions/rearrangements. Mutation load is assessed as the number of somatic coding point mutations per megabase of targeted territory. Results Mutation load from targeted cancer gene analysis recapitulates previous results evaluating whole exome and whole genome mutation load in tumors and cell lines. A novel mutation hotspot was identified in the promoter of PMS2, which codes for the PMS2 protein, a dimerization partner of MLH1 and integral to the DNA mismatch repair complex. Promoter mutations were found in 7.5% of melanoma specimens (n = 101/1348) and 17% of skin squamous cell carcinomas (n = 30/175). In both diseases, PMS2 promoter mutations are the most significant genomic correlate of high mutation load. In melanoma, PMS2 promoter mutant specimens have 4x the median mutation load of the general melanoma population. Skin squamous cell carcinomas show a 2.5-fold increase. Functional characterization is underway to support the hypothesis that these mutations lead to modified PMS2 transcriptional activity, which is known to cause hypermutation. Conclusions The growing corpus of cancer genome data continues to enable novel discoveries in cancer biology. Non-coding and regulatory mutations have not been the target of focused study, and our findings extend the small set of regulatory mutations thought to affect tumor development. Our discovery highlights the power of large-scale genomic analysis to uncover additional disease mechanisms, and furthers our understanding of how cells maintain genome integrity. With the documented association of mutation load and immunotherapy sensitivity, investigation into the response of PMS2 promoter mutant tumors is warranted. Citation Format: Zachary R. Chalmers, Franklin W. Huang, Laurie M. Gay, Siraj M. Ali, Roman Yelensky, Juliann Chmielecki, Jeffery S. Ross, Vincent A. Miller, Philip J. Stephens, Levi A. Garraway, Garrett M. Frampton. Broad analysis of recurrent somatic mutations in cancer reveals a common novel non-coding mutation in the promoter of PMS2 associated with greatly increased tumor mutation load. [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 3576.

Comparison of Genetic and Epigenetic Alterations of Primary Tumors and Matched Plasma Samples in Patients with Colorectal Cancer

BackgroundAlthough recent advances in circulating DNA analysis allow the prediction of tumor genomes by noninvasive means, some challenges remain, which limit the widespread introduction of cfDNA in cancer diagnostics. We analyzed the status of the two best characterized colorectal cancer (CRC) genetic and epigenetic alterations in a cohort of CRC patients, and then compared the degree to which the two patterns move from tissue to plasma in order to improve our understanding of biology modulating the concordance between tissues and plasma methylation and mutation profiles.MethodsPlasma and tumor tissues were collected from 85 patients (69±14 years, 56 males). KRAS and SEPT9 status was assessed by allele refractory mutation system quantitative PCR and quantitative methylation-specific PCR, respectively. Six of the most common point mutations at codon 12 and 13 were investigated for KRAS analysis.Results KRAS mutations and SEPT9 promoter methylation were present in 34% (29/85) and in 82% (70/85) of primary tumor tissue samples. Both genetic and epigenetic analyses of cfDNA revealed a high overall concordance and specificity compared with tumor-tissue analyses. Patients presenting with both genetic and epigenetic alterations in tissue specimens (31.8%, 27/85) were considered for further analyses. The median methylation rates in tumour tissues and plasma samples were 64.5% (12.2–99.8%) and 14.5% (0–45.5%), respectively. The median KRAS mutation load (for matched mutations) was 33.6% (1.8–86.3%) in tissues and 2.9% (0–17.3) in plasma samples. The plasma/tissue (p/t) ratio of SEPT9 methylation rate was significantly higher than the p/t ratio of KRAS mutation load, especially in early stage cancers (p=0.0108).ConclusionThe results of this study show a discrepant rate of epigenetic vs. genetic alterations moving from tissue to plasma. Many factors could affect mutation cfDNA analysis, including both presence of tumor clonal heterogeneity and strict compartmentalization of KRAS mutation profile. The present study highlights the importance of considering the nature of the alteration when analyzing tumor-derived cfDNA.

Ultra-Deep Sequencing Leads to Earlier and More Sensitive Detection of the TKI Resistance Mutation p.T315I in CML

▪Background: Chronic myeloid leukemia (CML) cells can acquire resistance to tyrosine kinase inhibitors (TKI) that in ~40% of cases is due to acquisition of mutations in the ABL1 kinase domain of the BCR-ABL1 transcript. The p.T315I (c.944C>T) mutation (mut) mediates resistance to most BCR-ABL1 TKIs (Imatinib, Dasatinib, Nilotinib and Bosutinib), whereas sensitivity to ponatinib has been demonstrated. Patients with p.T315Imut show a rapid increase in malignant cell burden and can progress to blast crisis. An earlier detection of the p.T315Imut may allow TKI treatment intervention ahead of disease progression. However, the sensitivity of conventional Sanger sequencing for detection of mutations is not less than 10-20%.Aim: To study the dynamics of evolution and progression of the p.T315Imut using ultra-deep sequencing (UDS) in comparison with Sanger sequencing.Patients and Methods: We selected 18 CML patients with high p.T315Imut levels originally detected by Sanger sequencing for routine diagnostics. Subsequently, we backtracked prior blood samples of all patients for a mean period of eight months (2-15 months) before detection of p.T315Imut by Sanger sequencing, analyzing 3-7 time points per patient. Patients (4 female and 14 male) had a median age of 60 years (18-84 years) and received treatment as follows: only Imatinib (n=3), only Nilotinib (n=3), only Dasatinib (n=1), treated with two prior (n=6) or three prior TKIs (n=5) by the time of p.T315Imut detection by Sanger sequencing. For more sensitive mutation detection, we amplified the BCR-ABL1 fusion transcript and designed two sequencing amplicons (550 bp and 575 bp) for UDS with the XL+ Kit for extended read length (Roche/454, Branford, CT). A minimal read coverage of 1,000 per base was reached. Our backtracking study by UDS was performed on samples sent in at intervals of approximately 3 months.Results: To prove high sensitivity of UDS with the 454 XL+ protocol we performed dilution experiments for three sequence variants and replicated sequencing experiments with low level mutations. The detection limit was at 1-2% mutation level and thus is 10-fold better than the sensitivity reached by Sanger sequencing. At the time point of initial routine diagnosis of p.T315Imut the median mutation load was 87.5% (30-100%) by Sanger sequencing and very similar by UDS (median: 84%; range: 40-99%; R2=0.7). In 6/18 patients backtracking identified a sample with a low p.T315I mutation level of <5% (1.9-13.6 months, median: 3.2 months) before a mutation load of >10% (Sanger sequencing detection level) was reached. Thus, in 33.3% of all cases a small, early clone of CML with p.T315Imut was identified. At subsequent time points, all 6 patients experienced a strong increase of the p.T315Imut level (>50%), which represents the very fast expansion of the mutated clone. In a second subset of 10 patients, the p.T315Imut load was already >30% when first detected by UDS. The median interval to the last p.T315I negative time point was 2.4 months (0.9-3.5) and no sample between the p.T315I negativity and high positivity was available. This subset confirms the fast outgrowth of the p.T315Imut positive clone. The p.T315Imut load had a median increase of 0.9% (0.2-3.1%) per day, when calculated as average increase from the last negative sample to the time point with maximum mutation load. The other 2 patients had high p.T315Imut levels (>40%) for our entire monitoring period.At the time of p.T315I detection by UDS, we observed eight patients with additional resistance mutations. The accumulation of mutations in one clone results in an extremely resistant CML. This was detected in one patient, where a p.T253H clone (Imatinib and Dasatinib resistant) gained the p.T315Imut. This clone expanded to 73% within 79 days. In contrast, we identified five cases with multiple CML clones carrying different mutations. However, the p.T315Imut clone was able to overgrow up to six other resistant clones.Conclusions: We showed: 1) the p.T315Imut rapidly increases upon occurrence, supporting the relevance of regular mutation monitoring in CML patients, when resistance to TKIs is suspected. 2) that small p.T315Imut clones in the 1-2% range can be sensitively detected by UDS in 33% of all samples if sampling intervals are within the 3 months range. 3) earlier detection of the p.T315Imut by UDS is a potentially valid method to allow a prompt change of TKIs before clonal expansion of the p.T315Imut cells. DisclosuresBaer:MLL Munich Leukemia Laboratory: Employment; ARIAD Pharmaceuticals: Research Funding. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Mariathas:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership; ARIAD Pharmaceuticals: Research Funding.

In CML Patients with Good Response to TKIs Other Gene Mutations Are Frequently (37%) Present in Addition to Philadelphia Negative, Cytogenetically Aberrant Clones but Are Rare (4%) in Cases with MMR and Normal Karyotype

Background: In chronic myeloid leukemia (CML) clonal chromosome aberrations in metaphases not carrying a t(9;22)(q34;q11) have been described during treatment with tyrosine kinase inhibitors (TKI), so-called Philadelphia-negative (Ph-) clones. Very rarely transformation to MDS was observed in patients carrying such Ph- clones but mainly restricted to patients harboring -7. Overall, the clinical significance of this phenomenon remains obscure.Aim: 1) Analyze in a large cohort of TKI-treated CML patients who developed Ph- clones the presence and occurrence of molecular mutations over time. 2) Evaluate whether molecular mutations are also present in CML patients who were at least in major molecular remission (MMR) and presented with a normal karyotype.Patients and Methods: First Cohort: 51 CML patients (pts, 23 males, 28 females; median age: 60 yrs, range: 37-84 yrs) with response to TKI (imatinib only: n=32, nilotinib only: n=2, imatinib and dasatinib or nilotinib: n=11, all three TKIs: n=6) who developed Ph- clones. Cytogenetics in these pts were as follows: +8 sole (n=24), -Y (n=8), -7 sole (n=4), +9 (n=2), other trisomies (n=4), 9 had other aberrations including some with combinations of two different clones (n=4). In median these abnormalities were present in 30% (range 8-100%) of analyzed metaphases. BCR-ABL1 levels at the time point of analysis were between 0 and 3.8 (median: 0.023) according to international scale. Second Cohort: 50 CML pts (24 males, 26 females; median age: 56 yrs, range: 21-83 yrs), who were at least in MMR and without development of any cytogenetic aberration after 3 years of imatinib treatment. Median time from start of therapy to analysis was 2.6 years (range 3 months to 14 yrs).All cases were analyzed with a pan-myeloid gene panel of 29 genes: ASXL1, BCOR, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3 (TKD), IDH1, IDH2, JAK2, KIT, KRAS, MLL-PTD, NOTCH1, NPM1, NRAS, PRPF40B, PTPN11, SF1, SF3A1, SF3B1, SRSF2, TET2, TP53, U2AF1, U2AF2 and ZRSR2. Either complete coding genes or hotspots were first amplified by a microdroplet-based assay (RainDance, Billerica, MA) and subsequently sequenced with a MiSeq instrument (Illumina, San Diego, CA). In addition, RUNX1 was sequenced on the 454 NGS platform (454 Life Sciences, Branford, CT).Results: In the first cohort 28 mutations were found in 19 patients, as 5 patients had 2 and 2 patients even 3 mutations.Thus,in 19/51 pts (37.3%) ≥1 mutation was detected. Median mutation load was 11.5% (range: 2-56%). In detail, mutations in the following genes were detected: ASXL1 (n=9), DNMT3A (n=7), RUNX1 (n=3), NRAS (n=2), TET2 (n=2) and one each in CBL, EZH2, IHD1, PRPF40B, and TP53. Subsequently, these mutations were evaluated in samples from earlier or later time points (18 pts with a total of 235 samples, range: 3-20 samples/pt). In 12 cases a sample from diagnosis of CML was available. In 2 cases a CBL and an ASXL1 mutation were already detectable at low levels, 1.4% and 2%, respectively, at the time of diagnosis and later increased with decreasing BCR-ABL1 levels. In all other 10 cases the mutations were not detectable at diagnosis and were for the first time detectable during TKI treatment (in median after 24 months after diagnosis, range 2-73 months). In the remaining 6 cases date of occurrence could not be determined by backtracking as all earlier samples available were positive for the respective mutation. However, the over time mutation levels were inversely related to BCR-ABL1 expression indicating the presence in independent clones.Within the second cohort with cases in MMR that remained cytogenetically normal only in 2 of the 50 pts (4%) mutations were detected. In one patient a DNMT3A mutation was detected that could be monitored for 8 years with constant low mutation load (3-6%). This was not detectable at diagnosis and occurred after 6 months on imatinib. Very similarly, in the second case a TET2 mutation was first detected after 6 months on imatinib with a mutation load of 2% that very slowly increased to 7% within 8 years.Conclusions: 1) In CML patients that develop Ph- clones other mutations occur in 37.3%. 2) In contrast, in randomly selected CML pts with MMR that are cytogenetically normal, molecular mutations can be detected in only 4%. 3) The clinical importance of molecular mutations in CML in MMR remains unclear. 4) However, these results implicate that chromosomal aberrations are an indicator for genomic instability, also at the molecular level. DisclosuresSchnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Tyrosine Kinase Inhibitor Treated CML Patients Harboring Philadelphia-Negative Cytogenetically Aberrant Clones Show Molecular Mutations In 31% Of Cases Not Present At Diagnosis: A High-Throughput Amplicon Sequencing Study Of 29 Genes

BackgroundIn chronic myeloid leukemia (CML), clonal chromosome aberrations in metaphases not carrying a t(9;22)(q34;q11) have been described during treatment with tyrosine kinase inhibitors (TKIs), so-called Philadelphia-negative (Ph-) clones. The most frequent abnormalities in these Ph- clones reported are -7, +8 and -Y. This pattern of cytogenetic aberrations is comparable to alterations found in MDS. In the majority of patients no clear evidence of a secondary hematologic malignancy is observed. However, few cases especially with -7 were reported to evolve to MDS or even AML. Aim1) Analyze whether TKI-treated CML patients who developed Ph- clones also harbour molecular mutations. 2) Evaluate whether these mutations were already present at diagnosis of CML. Patients and MethodsWe selected 42 patients (pts) treated with TKIs (Imatinib: n=27, Nilotinib: n=2, and 13 pts who were treated with 2 or 3 different TKIs) who developed Ph- clones. Median time from start of therapy to analysis was 3.2 years (range 8.9 months-13.5 years). 23 cases were male and 19 female; median age was 63.3 years (range: 34.1-83.9 years). Cytogenetics in pts with Ph- clones were as follows: +8 sole (n=18), -Y (n=7), -7 sole (n=2), +9 (n=2), an 10 cases with various abnormalities. Additional three cases had two different clones: 1) -7 and +8; 2) +8 and +Y, 3) -Y and +Y. Median clone size was 37.5% (range 10-100%) of all metaphases. BCR-ABL1 levels at the time point of analysis were between 0 and 4.5 (median: 0.019) according to international scale. All cases were analyzed with a pan-myeloid gene panel of 29 genes: ASXL1, BCOR, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3 (TKD), IDH1, IDH2, JAK2, KIT, KRAS, MLL-PTD, NOTCH1, NPM1, NRAS, PRPF40B, PTPN11, RUNX1, SF1, SF3A1, SF3B1, SRSF2, TET2, TP53, U2AF1, U2AF2 and ZRSR2. Either complete coding genes or hotspots were first amplified by a microdroplet-based assay (RainDance, Lexington, MA) and subsequently sequenced with a MiSeq instrument (Illumina, San Diego, CA). RUNX1 was sequenced on the 454 Life Sciences NGS platform and the MLL-PTD was analyzed by quantitative real time PCR. Median coverage per amplicon was 2215 reads (range 100-24,716). The lower limit of detection was set at a cut-off of 1.5%. ResultsIn total, in 13/42 pts (31.0%) one (n=9) or two (n=4) of the analyzed genes were mutated. Median mutation load was 15% (range: 3-50%). In detail, mutations in the following genes were detected: ASXL1 (n=5), DNMT3A (n=4), NRAS (n=2) and each one in BCOR, CBL, MLL-PTD, RUNX1, TET2 and ZRSR2. 4 pts had combinations of: 2 ASXL1mut, 2 NRASmut, BCORmut and RUNX1mut, or ASXL1mut with MLL-PTD, respectively. Subsequently, the mutations were tracked as far as serially collected samples were available (12 pts with a total of 125 samples, range: 3-20 samples/pt) from earlier or later time points. In 6 cases it clearly could be shown that the mutations were not present at diagnosis and arose 5 months, 7 months, 14 months, 15 months, 5 years, and 7 years after start of TKI therapy, respectively. In all these cases the mutation loads increased in parallel to decreasing BCR-ABL1 levels. In one of these cases one mutation was found 3 years and a second 11 years after start of therapy. Importantly, in three of these cases the molecular mutations were detectable before the cytogenetic aberration in the Ph- clone. In six further cases mutations were present with similar levels in several samples but due to lack of samples from initial diagnosis no estimation of the time to their occurrence was possible. In one of these cases the BCR-ABL1 load increased again during therapy and the DNMT3A mutation decreased showing that the BCR-ABL+ clone and the DNMT3Amut clone were competitive clones. Conclusions1) For the first time we show that more than 30% of CML patients treated with TKI who develop Ph- clones as defined by cytogenetic abnormalities, also harbour molecular mutations. 2) For most of the cases it was shown that these mutations arose during treatment. 3) In some cases the molecular mutations were detectable before the cytogenetic aberrations. This may be regarded as further proof of the malignant character of these Ph- clones. However, none of the analyzed cases, so far, developed a secondary disease, i.e. MDS or AML, thus the relevance of these findings is still unclear and needs further attention. Disclosures:Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kuznia:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Albuquerque:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership; Novartis: Research Funding. Hochhaus:Novartis: Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Analysis For Loss Of 13q Heterozygosity Using STR Or SNP Analysis Can Replace Analysis Of FLT3-ITD To Detect Prognostically Adverse AML

BackgroundFLT3-ITD has been established as an adverse prognostic factor in acute myeloid leukemia (AML). However, it has been repeatedly shown that not the presence of FLT3-ITD by itself confers the unfavorable effect, but the excess load of the FLT3-ITD in comparison to the FLT3 wild-type (wt) allele, which occurs by loss of heterozygosity (LOH) due to somatic isodisomy of chromosome 13q. AimAs now analysis of FLT3-ITD in many countries is restricted by patent claims we aimed at developing a novel assay for LOH detection on 13q as a substitute for FLT3-ITD to identify patients (pts) with a higher risk for shorter survival. Patients and Methods1) First an intensively treated AML cohort with normal karyotype (NK) (n=453) was selected in which the FLT3-ITD/wt ratio was assessed by a standard procedure (fragment length analysis by gene scan). 2) In a second step, a subcohort (cohort 2) of 233/453 pts was analyzed for LOH in 13q by fragment analysis of polymorphic single tandem repeat (STR) markers flanking the FLT3 gene as previously described (Whitman et al., JCO 2001). 3) A further subset of 91 pts from cohort 2 was subjected to a proof-of-principle study of applying next generation amplicon sequencing (NGS) for detection of LOH using a combination of Access Arrays (Fluidigm, South San Francisco, CA) and the 250 bp paired-end read chemistry on a MiSeq instrument (Illumina, San Diego, CA). In total, 41 amplicons were designed covering 67 different known SNPs (single nucleotide polymorphisms), with 43 SNPs flanking the FLT3 gene in the telomeric and the centromeric direction (genomic position: chr13:26,755,356-38,231,018), 22 SNPs being located in FLT3 intronic regions and one SNP each in exons 2 and 6. The achieved median coverage per SNP was 6,143 reads (range 58 - 30,311). Results1) In the control cohort of 453 NK-AML (FLT3-ITD+: n=162, 35.8%) the adverse impact of high FLT3-ITD burden was reproduced. According to FLT3-ITD/FLT3wt a ratio of <0.5 was detected in n=60 (13.2%), between 0.5 and <1 in 69 (15.2%) and ≥1 in 33 (7.3%) cases. Survival in patients (pts) with a FLT3-ITD load <0.5 was not different from those with FLT3-wt (54% 3-year survival for both, p=n.s.). In contrast, the outcome in pts with a load of >1 (LOH) was significantly shorter as compared to all others (EFS: 3.6 vs 14.4 months, p<0.001, respectively). These results clearly show that 13q-LOH outperforms the presence of FLT3-ITD per se as a prognostic marker. 2) Therefore, we next performed the STR analysis in a subcohort of 233 pts (FLT3-ITD+ load >1: n=83, FLT3-ITD+ load ≤1: n=105, FLT3wt: n=45). The median mutation load was 0.80 (range: 0.03-181.7). In all patients at least one informative STR marker was identified: 1: n=6 pts, 2: n=23 pts, 3: n=60 pts, 4: n=77 pts, 5: n=67 pts). STR allele ratios were similar for FLT3wt and FLT3-ITD with burden of ≤1. A threshold of a STR aIlelic ratio ≥70% allows identification of 81/83 samples with LOH (positive detection: 97.6%). Median EFS was 4.7 months in the 13q LOH group compared to 14.4 months in all others (p=0.001), clearly demonstrating that STR analysis using five 13q-specific markers can be used as a powerful tool to generate prognostic data. 3) As the gene scan procedure was time consuming, we next tested whether an NGS-based high-throughput SNP analysis can detect the LOH cases with the same precision. In total, 110 different SNPs were detected in this cohort (43 more than the anticipated 67, as many pts had additional rare SNPs). Complete homozygous SNP constellations were regarded as not informative. A median of 67 SNPs/pts (range: 44-80) were detected with a median informative allele constellation of 51/pts (range: 27-70). The allelic SNP load was calculated from the mean allelic load of all informative SNPs and then correlated to the FLT3-ITD load as calculated by gene scan. Across the total subset of 91 pts a very good correlation was detected (Spearman, R=0.807, p<0.001). This was even better when only the cases with LOH, as assessed by the other two methods, were regarded (n=48, R=0.916, p<0.001). In total, 47/48 of the LOH pts were identified by this method showing a positive detection rate of 97.9%. Conclusions1) Not the presence of FLT3-ITD per se but a high FLT3-ITD load due to LOH of 13q has prognostically adverse impact. 2) Assessment of LOH can be done either by STR analysis or even more accessible by quantification of SNP allelic ratios using NGS amplicon deep-sequencing with a positive detection rate of 97.9%. Disclosures:Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Weber:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Clinical Relevance of Concomitant Gene Mutations in CEBPA Double-Mutated Acute Myeloid Leukemia.

Abstract 2400 Background:CEBPA mutations occur in 5–14% of patients with acute myeloid leukemia (AML). A difference in clinical outcome between single- (sm) and double-mutated (dm) cases has been reported, whereupon dm cases were shown to be associated with longer overall (OS) and event-free survival (EFS). Aims:1. Determine the frequency and clinical impact of CEBPA sm and dm in a large AML cohort. 2. Evaluate the spectrum of additional molecular mutations in CEBPA dm AML and their prognostic influence. Patients and Methods:First, we investigated CEBPA mutations in a cohort of 997 AML cases (462 female, 535 male; median age: 66.8 years) by 454 deep-sequencing (454 Life Sciences, Branford, CT). The cohort included: t(15;17)(q22;q12) n=33; t(8;21)(q22;q22) n=39; inv(16)(p13q22) n=31; normal karyotype (NK) n=447; complex karyotype (CK) (≥4 abnormalities) n=116; other abnormalities n=331. Second, we investigated an additional cohort of 111 AML dm CEBPA cases for mutations in ASXL1, DNMT3A, FLT3-ITD, FLT3-TKD, GATA2, IDH1/2, KRAS, MLL-PTD, NPM1, NRAS, RUNX1, TET2, TP53, and WT1 using 454 sequencing, Sanger sequencing, conventional PCR and melting curve analyses. This cohort was composed of 60 female and 51 male cases; median age: 62.3 years; 76 cases showed NK, 19 aberrant karyotype (n=4 n.a.). Survival data was available in 90/111 (81.1%) cases. Results:1. In total, CEBPA mutations were detected in 75/997 (7.5%) of cases (t(15;17)(q22;q12) n=2/33; NK n=52/447; CK n=1/116; other abnormalities n=20/331). Of the 75 patients with CEPBA mutations 31 (41.3%) were sm, while 44 (58.7%) were dm. Patients with dm CEPBA showed better outcome compared to sm cases (OS at 3 yrs: 78.9% vs 38.5%, P=0.014; EFS after 3 yrs: 53.9% vs 36.6%, P=0.108). OS and EFS of CEBPA sm cases were comparable to CEPBA wt cases (OS at 3 yrs: 38.5% and 43.6%, P=0.689, EFS at 3 yrs: 36.6% and 29.4%, P=0.678). OS of CEBPA dm cases was comparable to patients with t(15;17)(q22;q12) (OS after 3 yrs: 78.9% vs 86.1%, P=0.597).2. In the cohort of 111 patients we detected 227 CEBPA mutations. In 106 (95.5%) cases two mutations, and in 5 (4.5%) cases three mutations were detected. The median mutation load was 42% (range: 2–98%). The majority of mutations were frame-shift (n=135) and in-frame (n=66). Further, missense (n=19) and nonsense (n=7) mutations were observed. Most cases showed one N- and one C-terminal mutation (92/111, 82.8%), 10 (9.0%) cases harbored two N-terminal mutations, and 4 (3.6%) cases showed two C-terminal mutations. In addition, two cases showed one N- and two C-terminal mutations, two cases two N- and one C-terminal mutations, and one case harbored three N-terminal mutations.In 92/111 (82.9%) cases we observed at least one additional mutation (mean: 1.6 mutations; range: 1–4): TET2 39/109 (35.8%), ASXL1 20/111 (18.0%), GATA2 20/111 (18.0%), WT1 14/111 (12.6%), DNMT3A 11/109 (10.1%), IDH1/2 9/111 (8.1%) (IDH1 n=2, IDH2 n=7), NRAS 9/111 (8.1%), RUNX1 7/111 (6.3%), FLT3-ITD 7/111 (6.3%), KRAS 4/109 (3.7%), NPM1 3/111 (2.7%), FLT3-TKD 2/110 (1.8%), MLL-PTD 1/111 (1.0%), and TP53 1/110 (1.0%). With respect to clinical outcome we observed no differences in OS for concomitant mutations in DNMT3A, FLT3-ITD, IDH1/2, NRAS, TET2 and WT1. Cases with additional GATA2 mutations showed longer survival than wt cases (OS at 3 yrs: 100% versus 73.4%, P=0.026, EFS at 3 yrs: 67.5% versus 48.5%, P=0.137). In contrast, cases harboring additional ASXL1 or RUNX1 mutations were associated with worse outcome (ASXL1: OS at 3 yrs: 32.8% versus 85.7%, P<0.001, EFS at 3 yrs: 0% versus 57.9%, P=0.002; RUNX1: OS at 3 yrs: 0% versus 81.8%, P=0.001, EFS at 3 yeaers: 0% versus 53.8%, P=0.003). Since mutations in ASXL1 and RUNX1 frequently occurred concomitantly, they were grouped together (n=21). When then separating cases into (1) GATA2 mut, (2) GATA2 wt, ASXL1wt, RUNX1wt and (3) ASXL1mut and/or RUNX1mut we observed 3 distinct survival curves (OS at 3 yrs: 100% vs 81.2% vs 32.8, P<0.001, EFS at 3 yrs: 67.5% and 55.3% and 0%, P=0.005). No statistical analysis was performed for FLT3-TKD, KRAS, MLL-PTD, NPM1, TP53 due to the low number of mutations. Conclusions:1. In CEBPA dm cases a high frequency of concomitant mutations (82.9%) was observed. 2. Most common mutated genes were TET2 (35.8%), ASXL1 (18.0%), GATA2 (18.0%), WT1 (12.6%), DNMT3A (10.1%), and RUNX1 (6.3%). 3. In CEBPA dm cases GATA2 mutations were associated with longer OS, whereas OS was poor in ASXL1 and/or RUNX1 mutated cases. Disclosures:Grossmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Stopp:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Comprehensive Cytogenetic and Molecular Genetic Characterization of T-PLL Identifies for the First Time BCOR Mutations in a Lymphatic Disease.

Abstract 2539 Introduction:T-cell prolymphocytic leukemia (T-PLL) is a rare mature post-thymic T-cell neoplasm with aggressive clinical course and a median overall survival of less than one year. Due to the rareness of this disease (∼2% of cases of mature lymphocytic leukemias in adults) only few cases with cytogenetic and molecular genetic aberrations have been reported so far. However, almost 75% of T-PLL cases are reported to harbor chromosome 14 abnormalities involving the TCRA/D locus resulting in the aberrant activation of the proto-oncogenes TCL1A or MTCP1. Aim:Perform a comprehensive cytogenetic and molecular characterization of T-PLL. Patient and Methods:The cohort comprised 36 T-PLL cases diagnosed between 10/2005 and 07/2012 (23 male, 13 female patients). Median age was 71.0 yrs (range: 26.8–82.8 yrs). According to the WHO classification all T-PLL cases were characterized using immunophenotyping and cytomorphology. Patients were further investigated using chromosome banding analysis (CBA) (n=30), FISH for deletions of ATM (n=30), TP53 (n=29), and 13q (n=26), and CGH arrays (n=3, Human CGH 6×630K Whole-Genome Tiling Array, Roche NimbleGen, Madison, WI). Further, mutation analyses for BCOR and TP53 were performed using amplicon sequencing (Roche 454, Branford, CT). Results:Aberrant karyotypes were observed by CBA in 25/30 cases (83.3%). However, the 5 remaining cases with normal karyotype were due to insufficient proliferation of the T-PLL clone as gains, losses and rearrangements were detected in these 5 cases using FISH analyses. In more detail, combined CBA and FISH data revealed in 20/30 (66.7%) cases an inv(14)(q11q32)/t(14;14)(q11;q32)/TCRA/D-TCL1A (n=18) or t(X;14)(q27;q11)/TCRA/D-MTCP1 (n=2). Further, a gain of chromosome 8q and concomitant loss of 8p was observed in 13/30 (43.3%) cases. In addition, in 10/25 (40.0%) cases a 6q deletion and in 7/25 (28.0%) an 12p deletion were observed. Based on FISH data, deletions were detected of ATM in 19/30 (63.3%) cases, TP53 in 7/29 (24.1%), and 13q in 9/26 (34.6%) cases.In addition, 3 cases were studied using array CGH. Hereby, an intragenic deletion in the BCOR gene was observed in one patient. BCOR is a BCL6 corepressor and located on chromosome Xp11.4. BCOR mutations were recently described in cases with AML. BCOR mutation frequency was determined at 3.8% in AML with normal karyotype and mutations were associated with shorter overall and event-free survival. The deletion in BCOR identified in one of our cases and the TP53 deletions in 7 T-PLL cases prompted us to screen 35 cases for molecular mutations in these two genes. Overall, BCOR mutations were detected in 5/35 (14.3%) patients and TP53 mutations in 4/35 (11.4%) cases. In total, 7 missense, one frame-shift and one nonsense mutations were found. Median mutation load was 90.0% for BCOR (range: 38–100%) and 80.0% (59–87%) for TP53.Next, we performed correlation analyses between mutations in BCOR and TP53, rearrangements involving chromosome 14, deletions of ATM, TP53, 6q, 12p, and 13q and gain of 8q. Here, BCOR was not associated with any of these parameters. In contrast, TP53 mutations were accompanied in all 4 cases by TP53 deletions, while only 3/24 TP53 wild-type cases harbored a TP53 deletion (P=0.002). In addition, only one of 4 TP53 mutated cases harbored a chromosome 14 rearrangement while 18/25 (72%) TP53 wild-type cases did (P=0.105). In line with this result, TP53 deletions were also negatively associated with chromosome 14 rearrangements (2/7 vs 17/22, P=0.030). Further, all cases with a gain of 8q harbored a 14q rearrangement (13/13 vs 8/18 without gain of 8q, P=0.001). Conclusions:1. CBA, FISH and mutation analysis of TP53 and BCOR revealed genetic abnormalities in all 36 analyzed cases. 2. The most frequent abnormality involved the TCRA/D locus (14q11) (20/30; 66.7%) activating the proto-oncogenes TCL1A on chromosome 14q32 (90.0%) or MTCP1 on chromosome Xq28 (10.0%). 3. Deletions were detected for ATM (63.3%), TP53 (24.1%), 6q (40.0%), 13q (34.6%), 12p (28.0%), and a gain was detected for the long arm of chromosome 8 (43.3%). 3. In addition to the detection of TP53 mutations in 11.4%, BCOR mutations were observed for the first time in a lymphatic malignancy with a mutation frequency of 14.3%. 4. The prognostic relevance of such cytogenetic and molecular genetic aberrations has to be determined in T-PLL, given that in myeloid malignancies both BCOR and TP53 are associated with shorter OS. Disclosures:Grossmann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Recurrent ATM and BIRC3 Mutations in Patients with Chronic Lymphocytic Leukemia (CLL) and Deletion 11q22-q23

Abstract 1771 Introduction:Deletion of the long arm of chromosome 11 (11q22-q23) is one of the most common chromosome aberrations (∼12%) in CLL and is associated with rapid disease progression and shorter overall survival (OS). The commonly deleted region contains the genes ATM and BIRC3. ATM (chromosome 11q22.3) plays a central role in activating TP53, DNA recombination processes, and cell cycle control. ATMmut were described to occur in ∼35% of CLL patients with 11q deletions. The apoptosis inhibitor BIRC3 is located on chromosome band 11q22.2, 6.0 Mb proximal of ATM. BIRC3mut have recently been described with a mutation frequency of 4% in CLL (Rossi D, Blood 2012). Aims:1. Determine the frequency of deletions and mutations of ATM and BIRC3 in CLL with 11q deletion. 2. Analyze the association of ATM and BIRC3 mutations with ATM and BIRC3 deletions. 3. Characterization of the landscape and prognostic impact of ATM and BIRC3 mutations. Patient and Methods:The investigated cohort comprised 60 de novo CLL cases harboring del(11q) identified by chromosome banding analysis. The cohort comprised 47 males and 13 females, median age was 68.9 years. Survival data was available in 38 cases. For sensitive mutation detection of the complete coding region of ATM (3056 amino acids) and BIRC3 (604 amino acids) an amplicon deep-sequencing approach (454 Roche Life Sciences, Branford, CT) was developed. Patients were further characterized for mutation status of TP53 (n=60), SF3B1 (n=59), and IGHV (n=60). Additionally, patients were investigated by interphase FISH for ATM (n=60; MetaSystems, Altlussheim, Germany) and BIRC3 (n=39; BAC RP11–177O8, BlueGnome, Cambridge, UK) deletions. Results:Based on FISH results, all 60 cases showed an entire gene deletion of ATM. Of those cases investigated for BIRC3del, 34/39 (87.2%) cases showed a deletion, thus 5/39 (12.8%) carried an ATMdel only. Mutational screening of the ATM and BIRC3 genes identified 24 ATMmut in 19/60 (31.7%) patients and 3 BIRC3mut in 3/60 (5.0%) cases. Additional mutations were detected in TP53 (3/58; 5.2%), and the spliceosome machinery gene SF3B1 (10/59, 16.9%). 49/60 (81.7%) cases were IGHV unmutated, whereas 11/60 (18.3%) cases were IGHV mutated.The majority of ATMmut were found to be missense mutations (n=15) followed by nonsense (n=4), frame-shift (n=4), and in-frame (n=1) variants. In more detail, only one mutation occurred in two distinct patients (p.Ile2888Thr). Most cases showed only one mutation (n=15, 78.9%), whereas 3 cases (15.8%) showed two and one case (5.3%) three mutations. Mutations were spread across the entire coding region (exon 3–59). The median mutational burden as assessed by deep-sequencing read counts was 38.5%, ranging from 4.0–89.5%.For BIRC3 3 deletions (1–4 bp) were observed resulting in 2 frame-shift and 1 splice-site mutation. The median mutation load was 32.5% (range: 20.0%-92.0%). In addition, two cases with BIRC3mut showed also a BIRC3del (n=1 data not available). Interestingly, all of these 3 cases with BIRC3mut were wild-type in the ATM gene.To further address the role of ATMmut association analyses with respect to age, gender, white blood cell count, haemoglobin level, and platelet count were performed, however, no correlations were observed. Further, ATMmut were not correlated with IGHV (3/11 vs 16/49, P=1.00), TP53 (0/3 vs 19/55, P=0.544) or SF3B1 (3/10 vs 15/49, P=1.00) mutation status. However, the presence of ATMmut in CLL with del(11q) was associated with shorter OS (76.2% vs 95.0% at 3 years), although this difference was not significant. However, when comparing OS of ATMmut cases to cases without del(11q) (n=1,245) we detected a trend towards a shorter OS (76.2% vs 94.2% at 3 years, P=0.149) in contrast to cases with del(11q), but without ATMmut in comparison to cases with no del(11q) (95.0% vs 94.2%, P=0.731). Conclusions:1. BIRC3 was mutated in only 5.0%, whereas ATM was mutated in 31.7% of patients with del(11q). 2. No mutation hotspot regions were observed, thus analyses of the whole genes are mandatory. 3. ATMmut were associated with a trend to shorter OS in CLL with del(11q). In comparison to cases without del(11q), cases with ATMmut showed shorter OS, indicating a more relevant role of ATMmut on prognosis versus cases with a del(11q) only. 4. In 38 cases (63.3%) with del(11q) neither ATM nor BIRC3 mutations were detected suggesting that further genomic alterations which are not yet identified play a role in CLL with del(11q). Disclosures:Grossmann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Weissmann:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Kienast:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Acquisition of TP53 Mutations in Chronic Lymphocytic Leukemia (CLL) Patients During the Course of Disease Is Associated with an Unmutated IGHV Status and Mutations in XPO1 and SF3B1

Abstract 1768 Background:In chronic lymphocytic leukemia (CLL), an association between TP53 mutations (TP53mut) and a poor prognosis has been reported. Two hypotheses are discussed regarding the occurrence of TP53mut during the course of disease: 1. TP53mut are already present at first diagnosis in a small subclone, which selectively grow despite treatment, and 2. TP53mut occur de novo during disease progression. Aim:Investigation of the acquisition of TP53mut in previously TP53wt CLL cases during the course of disease. Patient and Methods:The discovery cohort included 20 cases, which were selected based on negativity for TP53mut at first diagnosis and acquisition of TP53mut during follow-up (7 female, 13 male patients; median age: 60.7 yrs, range: 41.3–76.6 yrs). 16/20 patients were treated according to various regimens (including fludarabine and rituximab). TP53 analyses of the discovery cohort were performed by DHPLC and subsequent direct Sanger sequencing and, additionally, by 454 deep-sequencing analyses (454 Life Sciences, Branford, CT) enabling a higher sensitivity. Further, a comprehensive molecular characterization included in addition to TP53 the following markers: IGHV mutation status, NOTCH1, FBXW7, SF3B1, XPO1, and MYD88. All markers had been analyzed both at initial diagnosis and at the first follow-up time point that revealed an acquired TP53mut. A second cohort (n=326) was used for comparison of the mutation frequencies of IGHV, NOTCH1, FBXW7, SF3B1, XPO1, and MYD88. Results:Next-generation sequencing confirmed 18/20 cases as wild-type at first diagnosis (median coverage: 733-fold; range: 378-1, 369; sensitivity: <2.0%). However, in 2/20 cases deep-sequencing revealed a low-level clone of TP53 aberration (4.0% and 7.0%) that had not been detected by DHPLC and Sanger sequencing. Both patients with a detectable TP53mut at first diagnosis were excluded from further analyses.New TP53mut occurred at a median of 39.0 months after first diagnosis (range: 13.7–68.4 months). The patients harbored between 1 and 4 TP53mut (mean: 1.3) with a median mutation load of 18.0% (range: 2.0–93.0%). Of note, 15/18 patients had received therapy prior to the acquisition of the TP53mut. In addition, 13/17 (76.5%; n=1 no data available) cases showed a concomitant TP53 deletion detected by fluorescence in situ hybridization. Only 1/10 of these cases showed a TP53 deletion already at first diagnosis (no data available: n=3).Mutation frequencies of the NOTCH1 PEST domain, MYD88, FBXW7, XPO1, SF3B1 and the IGHV mutation status were compared between the discovery cohort and an independent cohort of 326 newly diagnosed CLL cases. Mutation frequencies in NOTCH1 PEST domain (2/18, 11.1% vs 45/326, 13.8%), MYD88 (1/18, 5.6% vs 6/326, 1.8%), and FBXW7 (0/18, 0% vs 8/326, 2.5%) were comparable between both cohorts, while mutations in XPO1 (4/18; 22.2% vs 14/326; 4.3%, P=0.010) and in SF3B1 (4/18; 22.2% vs 27/326 8.3%, P=0.067) were observed at significantly higher frequencies in the discovery cohort. Also an unmutated IGHV status was significantly more frequent in the discovery cohort compared to the unselected comparison cohort (16/18, 88.9% vs 196/326, 60.1%; P=0.01). Of note, in one case with a MYD88 mut and another case with a SF3B1 mut, these alterations were detected at first diagnosis, but disappeared during course of disease. In contrast, an XPO1 mutation was acquired during the course of disease in parallel to the TP53mut. In the remaining cases with SF3B1 (n=3), XPO1 (n=3), and NOTCH1 (n=2), mutations were observed both at first diagnosis and follow-up time point that revealed an acquired TP53mut. Conclusions:1. A small fraction of CLL patients (2/20) harbored a subclone with TP53 mutations at first diagnosis which escaped detection by conventional laboratory assays (DHPLC and Sanger sequencing) and were only identified retrospectively by using a more sensitive next-generation sequencing assay. These subclones increased in size during the course of disease. 2. A subset of CLL patients acquired TP53mut during course of their disease. Thus, repeated TP53 mutation analyses are necessary for optimal treatment decisions. 3. An unmutated IGHV status and mutations in XPO1 and SF3B1 were more frequent in CLL patients who acquired TP53 mutations during the course of disease, suggesting that these are potential risk factors for the acquisition of TP53 mutations during disease progression. Disclosures:Grossmann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Weissmann:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Boeck:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.