GS Junior Research Articles

Analysis of mixtures using next generation sequencing of mitochondrial DNA hypervariable regions.

AimTo apply massively parallel and clonal sequencing (next generation sequencing or NGS) to the analysis of forensic mixed samples.MethodsA duplex polymerase chain reaction (PCR) assay targeting the mitochondrial DNA (mtDNA) hypervariable regions I/II (HVI/HVII) was developed for NGS analysis on the Roche 454 GS Junior instrument. Eight sets of multiplex identifier-tagged 454 fusion primers were used in a combinatorial approach for amplification and deep sequencing of up to 64 samples in parallel.ResultsThis assay was shown to be highly sensitive for sequencing limited DNA amounts ( ~ 100 mtDNA copies) and analyzing contrived and biological mixtures with low level variants ( ~ 1%) as well as “complex” mixtures (≥3 contributors). PCR artifact “hybrid” sequences generated by jumping PCR or template switching were observed at a low level (<2%) in the analysis of mixed samples but could be eliminated by reducing the PCR cycle number.ConclusionThis study demonstrates the power of NGS technologies targeting the mtDNA HVI/HVII regions for analysis of challenging forensic samples, such as mixtures and specimens with limited DNA.

WS17.2 Identification of CF mutations in deep intronic regions: Design of antisense oligonucleotides for a targeted therapeutic approach

Considering that the extensive study of the CFTR gene classically performed in molecular diagnosis does not detect all disease-causing mutations, we previously developed an approach for a complete resequencing of the CFTR locus to search for mutations deeply located in introns (Bonini et al. Genet Med, 2015). After identifying candidate intronic mutations, we now aim to restore full-length CFTR transcripts by using antisense oligonucleotides also named Target Site Blockers (TSB). DNA from 15 patients with only one CF mutation were collected through a national collaborative study. Enrichments of the CFTR locus (250 kb) by long-range PCR or targeted capture were sequenced using 454 GS Junior and MiSeq Illumina platforms. TSB (Exiqon) were specifically designed for the deleterious variants. Out of 200 variations detected, our in-house pipeline pointed out 10 intronic variations, per DNA in average. In silico analysis tools predicted that 13 variants (for the 15 patients) had a deleterious effect on CFTR splicing by promoting inclusion of pseudoexons. TSB were tested whether the splicing defect was confirmed. For instance, TSB used on bronchial and primary nasal cells transfected with minigene constructs, significantly restored the full-length CFTR transcript for the well-known mutation c.1680–886A>G and a new identified c.1680–883A>G. Two new mutations in introns 18 and 23 are being tested. Finally, our massively parallel sequencing strategy lead to the identification of new CF-causing mutations in introns. Our findings also demonstrate the efficiency of antisense oligonucleotides for a targeted therapeutic for cystic fibrosis. Work supported by Vaincre La Mucoviscidose.

Abstract P4-01-09: Exploring the intra-patient PIK3CA mutational heterogeneity of circulating tumour cells by massive parallel sequencing in patients with metastatic hormone receptor-positive breast cancer

Abstract Introduction: Circulating tumour cells (CTCs) are a real-time reflection of the ad hoc relevant subpopulation in patients with progressive disease. The study comprises the clinical application of a semi-automated methodology to determine the PIK3CA mutational status at a single cell level. Methods: Using CellSearch and DEPArray, we purified single (n=172) and groups (n=93, ranging 5–120 cells) of CTCs from peripheral blood in 18 patients with metastatic ER+/PR+/HER2- breast cancer, of whom archival primary tumour (PT) material was available. Isolation of WBCs served as internal negative control. Recovered cells were subjected to whole-genome-amplification (WGA). During CTC blood draw, an extra SST tube was filled for isolation of circulating cell-free DNA (cfDNA) from serum as comparator. All samples from different compartments underwent gene mutation analysis via targeted amplification of exons 9 and 20 of the PIK3CA gene and downstream 454 massive parallel sequencing (MPS) on the GS Junior system. Results: WGA of single and group CTC samples had a success rate of 83±22 % and 96±8%, respectively. MPS resulted in an average throughput of 135.996±24.423 high quality reads with a normal-distributed mean fold coverage depth of 1257±703 and 1494±897 reads for exon 9 and 20, respectively. PIK3CA mutations were present at a relatively high frequency in archival PT (20/29 (68,9%)) and showed poor and moderate agreement with cfDNA (n=24; 33,3% disparity; kappa=0,03) and CTCs (n=14; 11,28% disparity; kappa=0,329), respectively. Comparison of CTCs and temporally matched cfDNA samples revealed substantial agreement (n=14; 14,3% disparity; kappa=0,536). A concordant PIK3CA status across all compartments (PT, cfDNA and CTCs) was observed in 11/15 (73,3%) samples. At the used sequencing depth, cfDNA failed to the detect PIK3CA mutations in 2 cases (13,3%), which were present in the respective PT and corresponding CTCs. CTCs failed to detect the mutant PIK3CA gene in one case (6,7%), which was present in PT and cfDNA. Gain of mutation was observed in 2/15 patients (13,3%), with a wild-type PT and mutant cfDNA and CTCs at progression. A wild-type PIK3CA sequence in recovered WBCs of all patients (n=15) indicates a high specificity and tumorigenic nature of the picked up variants. In depth intra-patient analysis of mutant CTCs on a single cell level reveals PIK3CA mutational heterogeneity with the presence of both mutant and wild-type CTCs (average %MT/%WT ratio of 50/50). Additionally, unique double-mutated CTCs were detected in 5/15 (30%) cases as well. Conclusions: We report the frequent occurrence of PIK3CA hotspot mutations in metastatic HR+ breast cancer. Intra-patient mutational heterogeneity was observed in the single CTC samples of the same patient. Thereby, this study provides evidence of both concordance and discordance of the PIK3CA genotype in the intravascular compartment (down to the single cell level) with comparison to the temporally unmatched PT. The study presents the utilization of a liquid biopsy, thereby paving the way towards the application of a more personalized medicine in the management of patients with metastatic cancer. Citation Format: Bram De Laere, Dieter JE Peeters, Salgado Roberto, Vermeulen B Peter, Van Dam A Peter, Dirix Y Luc, Van Laere J Steven. Exploring the intra-patient PIK3CA mutational heterogeneity of circulating tumour cells by massive parallel sequencing in patients with metastatic hormone receptor-positive breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-09.

Massively parallel sequencing fails to detect minor resistant subclones in tissue samples prior to tyrosine kinase inhibitor therapy.

BackgroundPersonalised medicine and targeted therapy have revolutionised cancer treatment. However, most patients develop drug resistance and relapse after showing an initial treatment response. Two theories have been postulated; either secondary resistance mutations develop de novo during therapy by mutagenesis or they are present in minor subclones prior to therapy. In this study, these two theories were evaluated in gastrointestinal stromal tumours (GISTs) where most patients develop secondary resistance mutations in the KIT gene during therapy with tyrosine kinase inhibitors.MethodsWe used a cohort of 33 formalin-fixed, paraffin embedded (FFPE) primary GISTs and their corresponding recurrent tumours with known mutational status. The primary tumours were analysed for the secondary mutations of the recurrences, which had been identified previously. The primary tumours were resected prior to tyrosine kinase inhibitor therapy. Three ultrasensitive, massively parallel sequencing approaches on the GS Junior (Roche, Mannheim, Germany) and the MiSeqTM (Illumina, San Diego, CA, USA) were applied. Additionally, nine fresh-frozen samples resected prior to therapy were analysed for the most common secondary resistance mutations.ResultsWith a sensitivity level of down to 0.02%, no pre-existing resistant subclones with secondary KIT mutations were detected in primary GISTs. The sensitivity level varied for individual secondary mutations and was limited by sequencing artefacts on both systems. Artificial T > C substitutions at the position of the exon 13 p.V654A mutation, in particular, led to a lower sensitivity, independent from the source of the material. Fresh-frozen samples showed the same range of artificially mutated allele frequencies as the FFPE material.ConclusionsAlthough we achieved a sufficiently high level of sensitivity, neither in the primary FFPE nor in the fresh-frozen GISTs we were able to detect pre-existing resistant subclones of the corresponding known secondary resistance mutations of the recurrent tumours. This supports the theory that secondary KIT resistance mutations develop under treatment by “de novo” mutagenesis. Alternatively, the detection limit of two mutated clones in 10,000 wild-type clones might not have been high enough or heterogeneous tissue samples, per se, might not be suitable for the detection of very small subpopulations of mutated cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1311-0) contains supplementary material, which is available to authorized users.

P1288 : Budd chiari syndrome (BCS) in France from a large national cohort

increased in recent years. This increase may be due to factors such as high HCV viral load in blood and semen, sex with risk of mucosal damage, a higher number of sexual partners, presence of concomitant ulcerative sexually transmitted diseases and the use of recreational drugs. The aim of our study was to investigate the dynamics of HCV transmission in an outbreak of acute hepatitis C in HIV-infected MSM in Barcelona. Methods: Between 2008 and 2013, 113 cases of acute hepatitis C in HIV-infected MSM were diagnosed in the Infectious Diseases Unit, Hospital Clinic, Barcelona. Phylogenetic analysis of the HCV NS5B gene was performed in a total of 70 patients. Viral RNA was extracted from serum samples collected from each patient at the time of diagnosis. Massive sequencing was performed using the Roche 454 GS Junior platform. To define possible transmission networks, phylogenetic trees and multidimensional scaling maps were constructed from genetic distance matrices (Da). Results: At the time of diagnosis of acute hepatitis C, 53 of the 70 (76%) patients included in the study were receiving antiretroviral therapy. HIV viral load was undetectable in 48 patients (69%) and the mean CD4 cell count was 923 cells /ul. HCV viral load was 6.37 log IU/mL (range 3.73–6.99). Thirty-five of 53 (66%) patients treated with pegIFN and ribavirin achieved a sustained virological response. The prevalence of HCV genotypes was: 4d 51% (n =36), 1a 40% (n =28), 1b 7% (n =5) and 3a 1% (n =1). Phylogenetic analysis showed the existence of at least 13 monophyletic groups: 5 of genotype 1a, 2 of genotype 1b and 6 genotype 4d. Molecular analysis showed that the genetic distances between genotype 4d viruses (Da 5.42) were significantly lower than those of the subtypes 1a (Da 18.50, p < 2.2×10−16) and 1b (Da 15.25, p < 1.1×10−6). This result may suggest the existence of a single source of infection for genotype 4d and different sources for subtypes 1a and 1b. Conclusions: HCV infection spreads rapidly among HIV-infected MSM through a local network in Barcelona. The implementation of public health campaigns and preventive measures, as well as treatment interventions with the new direct-acting antivirals will allow the development of strategies to reduce the HCV transmission of HCV within these high-risk groups.

LBP04: APPLICATION OF SINGLE MOLECULE REAL-TIME (SMRT) SEQUENCING TECHNOLOGY FOR THE FIELD 4 LEVEL GENOTYPING OF CLASSICAL HLA LOCI

Aim Super high resolution-single molecule-sequence-based typing (SS-SBT) is an HLA DNA typing method for the field 4 level of allelic resolution to efficiently detect new and null alleles without phase ambiguity by combination of long ranged PCR amplification and next generation sequencing (NGS) technologies. We previously reported the development and application of the SS-SBT method such as long ranged PCR system from the promoter-enhancer region to 3’UTR for 11 classical HLA loci, A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1 and DPB1 using NGS platforms such as Ion PGM, GS Junior and MiSeq. However, HLA alleles could not be assigned at the field 4 level in some HLA loci such as HLA-DQA1, -DPA1 and -DPB1, because SNP and indel densities to separate both of the phases were much lower than in other HLA loci. In this respect, third generation sequencers such as single molecule, real-time (SMRT®) DNA sequencer PacBio RS II (2.5∼10 kb) are expected to solve this current genotyping problem to improve the SS-SBT method. Here we report an accuracy of genotyping results obtained from SMRT® technology. Method Ten genomic DNAs (TU1∼TU10) obtained from Japanese were provided for long ranged PCR amplification using eight HLA loci specific primer pairs (A, B, C, DRB1, DQA1, DQB1, DPA1 and DPB1). Consensus sequences were obtained via HlaTools/Long Amplicon analysis such as overlapping, clustering of filtered subreads, phasing and removing PCR artifacts. Results HLA sequences obtained from 10 pooled amplicon samples applied to SMRT® technology with HlaTools/Long Amplicon analysis were compared to published reference sequences for consensus alleles. A total of 153 amplicon sequences were compared, 117 with the field 4 level sequences and 36 with the field 3 level sequences. At the field 3 level 100% of sequences were identical to reference ones while ∼70% (80/117) of sequences were identical at the field 4 level. Error sequences were mostly localized to intronic single- and di-nucleotide homopolymer regions. Conclusion The SMRT® technology is useful for highly accurate DNA typing for all the classical HLA loci.

LBP25: TYPE 1 DIABETES: AROUND THE WORLD WITH HLA

Aims HLA association with type 1 diabetes (T1D) is well established. HLA alleles and haplotype combinations vary among populations; however, the vast majority of T1D association studies have focused on subjects of European descent. Even the Type 1 Diabetes Genetics Consortium collection is predominantly comprised of European samples. The aim of this study is to determine HLA association with T1D in understudied populations around the globe, with a particular focus on developing countries, to elucidate differences in HLA-associated T1D susceptibility among populations and increase predictive power for T1D for all. Methods In collaboration with the Life for a Child program, we are collecting 100 blood samples from T1D patients and 200 saliva samples from unrelated controls from each of six countries, including Azerbaijan, Pakistan, Bangladesh, Mali, Sudan, and Haiti. For 469 samples collected to date, DNA was extracted and HLA DRB1 genes were sequenced with exon-based NGS technology on the Roche 454 GS Junior platform. Association analyzes were performed by gene counting, followed by chi-square analysis. Results To date, we have received 20 patients and 209 controls from Azerbaijan, 20 patients and 110 controls from Pakistan, and 110 controls from Bangladesh; DRB1 was genotyped for all samples. In preliminary analyzes, for Azerbaijan, DRB1 ∗ 03:01 (OR = 8.22; p = 1.35 × 10 - 9 ) and DRB1 ∗ 04:02 (OR = 6.93; p = 1.39 × 10 - 7 ) are significantly predisposing for T1D, while both DRB1 ∗ 15:01 and DRB1 ∗ 15:02 appear protective, but do not yet reach statistical significance. For Pakistan, the DRB1 ∗ 03:01 association is even stronger (OR = 10.37; p = 1.26 × 10 - 9 ), and DRB1 ∗ 15:02 protection is nearly significant (OR = 0.00, p = 0.063); however, no T1D association was observed for any DRB1 ∗ 04 allele. Conclusion These data illustrate the differences in HLA associated T1D susceptibility among populations. Additional differences will be revealed as we continue to receive and genotype samples. These differences are relevant for understanding the basic biology of HLA-associated T1D susceptibility, for prediction and differential diagnosis of T1D, and for eventual development of precision medicine-based treatment strategies for all individuals, regardless of ethnic heritage.

Aplicación de la secuenciación masiva de nueva generación al diagnóstico molecular de la hipercolesterolemia familiar

IntroductionFamilial hypercholesterolemia is an autosomal dominant disorder that causes increased levels of cholesterol associated with low density lipoproteins in plasma. The presence of altered lipid metabolism in these patients increases their level of risk of suffering from a cardiovascular disease. The certainty of having this genetic disorder by making a timely and precise molecular diagnostic is crucial for the appropriate treatment and the reduction of the disease morbidity-mortality. The aim of this work is to describe the applicability of next generation sequencing technology to the genetic diagnosis of familial hypercholesterolemia and compare this novel method with the conventional Sanger sequencing method. Material and methodsA next generation sequencing commercial panel (SEQPRO Lipo, Progenika) was used to analyze 110 peripheral venous blood samples from patients with familial hypercholesterolemia. This enables the assessment of mutations in genes associated with the disease (e.g. LDLR, APOB, PCSK9 and LDLRAP1) with the GS JUNIOR 454 technology (Roche). ResultsApplication of next generation sequencing enables the sequencing of the genes involved in the familial hypercholesterolemias, described so far, in groups of 20 patients simultaneously. Using this novel technology, a total of 35 mutations were detected in the 110 analysed samples, with 94.29% being located in the LDLR gene. Mutations were confirmed by Sanger sequencing. ConclusionNext generation sequencing enables a quick genetic diagnosis and a more efficient molecular analysis of all genes described so far to be involved in familial hypercholesterolemia, with similar reliability to that of conventional Sanger sequencing.

Mutations and Long-Term Outcome of 217 Young Patients with Essential Thrombocythemia or Early Primary Myelofibrosis

IntroductionYoung adults with Essential Thrombocythemia (ET) or early Primary Myelofibrosis (early-PMF) are a category of patients projected to a prolonged survival but also to an extended utilization of medical resources. Mutations, including those in the calreticulin (CALR) gene, have been reported to affect main clinical features and outcome in large cohorts of patients with Ph-negative MPNs. However, no data are available on mutational status and long-term outcome in young MPN patients.MethodsA clinic-pathologic database of ET patients followed in 5 Italian Hematology Centers was created. A total of 217 WHO-diagnosed ET or early-PMF patients ≤ 40 years at diagnosis was retrieved from the general database of 2635 patients. All bone marrow biopsies were reviewed at local institution. Baseline clinical/molecular characteristics and outcome measures (thrombosis, hemorrhages, secondary MF and AL, second neoplasia, death, overall and event-free survival) were evaluated. JAK2V617F allele-burden was assessed in granulocyte DNA by using ipsogen JAK2 MutaQuant Kit (qPCR). CALR mutations were identified by next generation sequencing (NGS) approach on GS Junior (Roche-454 platform); MPL mutations were evaluated by using ipsogen MPLW515L/K MutaScreen Kit.ResultsOverall, 197 WHO-defined ET and 20 early-PMF (age range: 16-40, median 34) were included in the study. Mutational frequencies were 61% for JAK2, 25% for CALR, 1% for MPL and 13% for triple negative. Baseline clinical characteristics and use of antiplatelet/cytoreductive therapies were comparable in ET and early-PMF, although frequency of triple negative was higher in the early-PMF cohort.Compared to the JAK2 positive population, both CALR and triple-negative patients showed higher platelet count and lower hemoglobin and hematocrit levels (Table 1).Median follow-up was 10.2 years (range: 0.5-37.5). During follow-up, 19 (9,6%) ET and 3 (15%) early-PMF patients experienced a total of 31 thrombotic (arterial: 38%) and 12 hemorrhagic events, with an incidence rate of 0.91% and 0.39% patients/yr, respectively. The cumulative incidence of thrombosis was 0,14% and 0,24% at 15 and at 20 years, respectively.Overall, 10 patients (4,6%) and 1 (0,4%) patients evolved to MF and AL, respectively; 10 developed a second neoplasia. The cumulative incidence of disease progression into MF/AL was 0,03% and 0,13% at 15 and at 20 years, respectively. At last contact, 6 (2,7%) patients had died, at a median age of 61 years (20-71), for an overall survival of 98% at 15 years. Causes of death were related to the myeloproliferative neoplasm (disease evolution or thrombotic complications) in all patients but one. Event-free survival was similar in the ET/early-PMF cohorts considering both every event separately and all together.In univariate analysis, male sex (p=0.003), previous thrombosis (p=0.001), splenomegaly (p=0.037), JAK2V617F (p=0.019) were associated with increased thrombotic risk; in multivariate Cox analysis, only previous thrombosis and male sex remained significant (p=0.012). Baseline splenomegaly was the only predictive factor for subsequent hemorrhages (p=0.017). Abnormal karyotype was associated with secondary MF (p=0.013) and also with second neoplasia (p<0.001). Together with JAK2V617F positivity and leukocytosis >11x109/L, abnormal karyotype was also associated with worse survival in univariate analysis. However, in multivariate analysis only JAK2V617F mutation remained as negative predictor of survival (p=0.019). Also, multivariable analysis confirmed JAK2 mutation and splenomegaly as independent risk factors for cumulative events.ConclusionsWith the limitations due to the low number of early-PMF, the outcome of young adults with early-PMF and true ET seemed to be comparable. The correlation of abnormal karyotype with MF transformation and second neoplasia suggests the need for an accurate cytogenetic analysis at diagnosis. Mutational status did influence disease phenotype, in terms of baseline characteristics and prognosis. Indeed, JAK2 mutational status confirmed a negative prognostic role for thrombosis and survival, while event-free survival was significantly better in triple-negative patients. Notably, causes of death were mostly related to the hematological malignancy, pointing out the substantial impact that this generally indolent disease may acquire in young adults. [Display omitted] DisclosuresLatagliata:Novartis: Consultancy; Bristol Myers-Squibb: Consultancy; Celgene: Consultancy; Shire: Consultancy. Cavo:Janssen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; BMS: Consultancy, Honoraria; Millenium: Consultancy, Honoraria; Onyx: Honoraria.

Backtracking BCR-ABL1 Mutants in Philadelphia-Positive Acute Lymphoblastic Leukemia Patients Relapsing on Tyrosine Kinase Inhibitors with Deep Sequencing: Implications for Routine Mutation Testing

Background: With the release of benchtop next-generation sequencers allowing to conjugate assay sensitivity and throughput with affordability, it was envisioned that NGS could soon replace conventional methods in the routine diagnostic identification of mutations relevant for diagnostication, prognostication and therapeutic tailoring. Having recently implemented and optimized a strategy for BCR-ABL1 kinase domain (KD) mutation screening based on the Roche-454 NGS technology, we decided to take advantage of its unique capability to capture the complexity and the clonal relationships of major and minor mutated populations present in a sample to focus on Philadelphia-positive (Ph+) acute lymphoblastic leukemias (ALL), where BCR-ABL1 KD mutations frequently arise and undermine the long-term efficacy of tyrosine kinase inhibitor (TKI)-based therapies.Aims: Next generation amplicon-based deep sequencing (DS) was used i) to study the dynamics of expansion of BCR-ABL1 KD mutations in relation to BCR-ABL1 transcript levels in Ph+ ALL patients who relapse on TKIs and ii) to assess whether emerging mutations can be detected earlier than with conventional Sanger sequencing (SS).Methods: This retrospective analysis was conducted on 35 Ph+ ALL patients who relapsed and acquired BCR-ABL1 KD mutations while receiving TKI-based therapy (imatinib, dasatinib, nilotinib) either first-line (Group A; n=10) or for recurrent disease (Group B; n=25). All the patients had been referred to our laboratory for minimal residual disease (MRD) follow-up monitoring by real-time reverse transcription-polymerase chain reaction and for BCR-ABL KD mutation analysis by SS in case of MRD positivity. To reconstruct the dynamics of mutation emergence, longitudinal re-analysis of bone marrow samples (n=113) from the time of hematologic relapse back to diagnosis (Group A) or back to TKI start (Group B) was performed on a Roche GS Junior instrument. Median time lapse between samples was 6 weeks (range, 4-15 weeks). Runs were designed to achieve high sequencing depth, allowing reliable detection of sequence variants at least down to 1% abundance.Results: DS was found to provide a more accurate picture of BCR-ABL1 KD mutation status, both in terms of presence/absence of mutations and in terms of clonal complexity. DS indeed detected low level mutations, alone or in addition to SS-detectable mutations, in 14/35 samples from patients in Group A and 24/72 samples from patients in Group B. In all the cases with two or more mutations, DS uncovered polyclonal mixtures of single and compound mutants, with up to five distinct mutant subclones detectable. With very few exceptions, compound mutants were found to represent the dominant clone. Patients in Group B, who had received two or even three lines of TKI therapy, generally displayed greater complexity than patients in Group A.TKI-resistant mutants were found to undergo rapid selection in Ph+ ALL patients. In 13/35 (37%) patients, however, DS would have allowed to detect emerging mutations earlier than SS actually did and before hematologic relapse – including a Y253H mutation in a sample collected at diagnosis from a patient who relapsed eight weeks later.Detection of emerging TKI-resistant mutations by DS was accompanied by total BCR-ABL1/ABL1 transcript increase from half a logarithm to two logarithms, or preceded by a shift from undetectable to detectable BCR-ABL1 transcripts. Even low MRD levels were found to hide low level TKI-resistant mutants.Conclusions: The enhanced sensitivity as well as the possibility to identify compound mutations point to next generation amplicon-based deep sequencing as an ideal alternative to conventional sequencing for BCR-ABL1 KD mutation screening of Ph+ ALL patients receiving TKI based therapies. Cost is not a concern even for small/medium scale laboratories, considering that many samples and many genes can be pooled and analyzed simultaneously. A turnaround time of 3-4 days is generally feasible, irrespective of chemistries and instruments, allowing DS results to be practically integrated in the clinical decision algorithm. Further, prospective evaluation of DS for BCR-ABL1 KD sequence surveillance in the framework of a clinical trial of TKI therapy and MRD monitoring of Ph+ ALL patients is warranted.Supported by: European LeukemiaNet, AIL, AIRC, FP7 NGS-PTL project, Progetto Regione-Università 2010-12 (L. Bolondi). DisclosuresSoverini:Ariad: Consultancy; Bristol-Meyers Squibb: Consultancy; Novartis: Consultancy. Baccarani:Bristol-Myers Squibb: Consultancy, Honoraria, Speakers Bureau.

The Prognostic Significance of TP53 Mutations for Overall Outcome in Lower-Risk MDS Patients

BACKGROUND AND AIMSRecent results have indicated that the TP53 mutations had a negative prognostic relevance for lower-risk myelodysplastic syndrome (MDS) patients but the relations between the presence of mutations and treatment has not been clarified yet. Therefore, we analyzed the prevalence of TP53 mutations in lower-risk MDS patients and determined the proportion of TP53 mutated bone marrow cells during the disease. We further observed mutual relation of treatment and mutated clones and defined impact of mutations on survival of patients.METHODSTP53 mutations were analyzed in DNA from bone marrow of 141 low risk or intermediate-1 risk MDS patients included into the cohort according to the International Prognostic Scoring System using primers designed within the IRON-II study research consortium (Roche Applied Science) on the GS Junior system. If mutations were detected, we searched for mutations at different timepoints of the disease and overall 210 samples were examined for mutations. Ten of the 16 patients with TP53 mutation received lenalidomide and/or 5-azacytidine. Mean coverage of sequenced exons was about 800-fold allowing an approximate detection sensitivity of 2% mutational burden.RESULTSThe overall incidence of TP53 mutations was 11.3 % (16/141), three patients harbored two mutated clones. The mutation frequency in patients with and without del(5q) was 21% (13/62) and 3.8% (3/79), respectively. During the observation period, 37 patients (26.2%) died, the data obtained from remaining patients were censored either at the last date they were known to be alive (94 patients - 66.7%) or at the time of stem cell transplantation (HSCT) (10 patients - 7.1%). The median overall survival was 164.2 months in TP53 unmutated group and 84.5 in mutated group respectively calculated by Kaplan-Meier Survival Curves and the Log-Rank Test (P=0.18). Mutations were detectable in eight of the sixteen available patients (50%) at the time of diagnosis and remaining patients (50 %) displayed mutations in an average 35.1 months (range 12-69 months) from diagnosis.Three patients with mutational burden below 5% are alive more than 3 years without signs of disease progression and three patients with clone size above 40% died. The mutated clone disappeared after bone marrow transplantation in one patient. The proportion of TP53 mutated cells decreased radically after successful treatment with azacytidine in two patients. The clone size increased in four patients treated with lenalidomide and all of them died. Two patients with mutated clone sizes below 20% are still under observation.CONCLUSIONSWe confirmed the higher frequency of TP53 mutations in lower- risk MDS with del(5q) in a large cohort of patients. Patients with the TP53 mutations had shorter survival compared to those without mutation. The mutations were detected in a half of the patients at the time of diagnosis and approximately three years after diagnosis in remaining patients. The presence of TP53 mutation as an adverse prognostic factor should be routinely investigated in lower risk MDS patients at the time of the diagnosis as well as repeatedly in the course of the disease.Supported by grant (NT/13899, NT/14377, NT/14539) and the project for conceptual development of research organization (00023736) from the Ministry of Health of the Czech Republic. DisclosuresNo relevant conflicts of interest to declare.

Refinement of IKZF1 Genomic Status in Pediatric Philadelphia Positive Acute Lymphoblastic Leukemia

Background:IKZF1 deletions are the most frequent secondary genetic alterations with prognostic impact in pediatric Philadelphia-positive B-cell precursor acute lymphoblastic leukemia (Ph+ BCP-ALL). Studies of IKZF1 status have mainly focused on deletions of the gene whereas very little is known about other genetic alterations in the IKZF1 locus.Aim: Using an amplicon deep sequencing approach to investigate IKZF1 mutations occurrence in pediatric Ph+ BCP-ALL reported as IKZF1wildtype (WT) for deletions by MLPA analysis.Methods: Six European centres participated in this study with a total of 98 patients.The 454 GS Junior system (Roche) was used to sequence the coding regions of IKZF1, Exon1 - Exon7 (Mullighan C. et al, 2009). An average depth of 250 reads (minimum of 102 reads, maximum of 853 reads) per amplicon was achieved. Quality control analysis excluded variants coverage of <50 reads in both forward and reverse directions. Variant analyses were performed using Amplicon Variant Analyzer (AVA) software (Roche). Variants at intronic regions or in homopolymeric stretches were excluded from further analysis, and a cut-off ≥10% mutated reads was applied to define mutations. Finally, variants described as SNPs or silent mutations were excluded.Results: In total, 882 amplicons were screened obtaining 337 variants. Of these, 14 variants corresponding to mutations with predicted deleterious impact on the function of IKZF1 passed the filters of our analysis pipeline. Twelve out of 98 (12%) IKZF1 non-deleted patients were mutated and 2 of them carried double mutations. Both cases showed different mutant allele frequencies of the 2 mutations, suggesting the presence of leukemic sub-populations with different IKZF1 status. Mutations can be subdivided in 2 categories: 5 miss-sense point mutations localized at the DNA binding domain with a dominant negative effect and 7 frameshift mutations leading to haploinsufficiency and impairing also the dimerization activity of IKZF1. The 5 point mutations were located in exon4 and 4 out of 5 were localized in the coding region of zinc finger 2 (ZF2), essential for DNA-protein interaction. Three patients carried the same c.698A>G substitution, that caused the change of Asparagine 159 into Serine. The 7 frameshift mutations (2 deletions, 4 insertions and 1 InDel) were identified all along the IKZF1locus. These nucleotide variations caused a shift in the reading frame and a consequent formation of premature stop codons depleting the C-terminal dimerization domain.Six out of 12 patients carrying IKZF1 mutations experienced an event (either relapse or death in CCR): 6 patients were treated in the pre-TKI (tyrosine kinase inhibitor) era and had 5 events while the remaining 6 patients received imatinib and showed a more favourable outcome with only one patient who died in CCR. This trend is in line with the findings recently reported by Van der Veer et al. (2014). In this study on IKZF1 deletions were identified in 126 of 191 patients, while 65 were defined IKZF1 WT on the basis of deletions analysis alone. Our study revealed that 6 of these 65 IKZF1 WT patients carried IKZF1 mutations (3 in the pre-TKI and 3 in the EsPhALL cohort). The outcome of mutated patients and deleted patients taken together (IKZF1 aberrant) is consistent with results reported in the Van der Veer study, both for the pre-TKI cohort [(4-year disease free survival (DFS) was 28.1% [SE 6.4] for IKZF1 aberrant vs 64.3% [SE 9.7] for IKZF1 WT, P=0.0036); Van der Veer study (30.0% [SE 6.8] for IKZF1 deleted vs 57.5% [SE 9.4] for IKZF1 WT, P=0.013)] and for the EsPhALL cohort [(4-year DSF was 55.7% [SE 6.8] for IKZF1 aberrant vs 59.8% [SE 11.6] for IKZF1 WT (P=0.348); Van der Veer study (4-year DFS was 53.6 [SE 7.0] for IKZF1 deleted vs 63.1 [SE 11.0] for IKZF1WT, P=0.1676].Conclusion: This work revealed IKZF1 mutations in >10% of Ph+ BCP-ALL pediatric patients previously classified as IKZF1 WT on the basis of deletions analysis. Our results, together with data of IKZF1 deletions, highlight a 70-75% incidence of IKZF1 aberrations in pediatric Ph+ patients. Exon4 carried 9 of 14 aberrations: 7 point mutations and 2 deletions, suggesting a hotspot region for somatic mutations acquisition. The outcome of mutated patients is in line with the data previously published on the impact of IKZF1 deletions in pre and post TKI cohorts, suggesting a similar impact of mutations and deletions on clinical outcome of pediatric Ph+ patients. DisclosuresNo relevant conflicts of interest to declare.

The Fas Ligand Inhibitor APG101 in Transfusion Dependent Patients with Low Risk MDS: Interim Results from a Phase I Study

Introduction: Myelodysplastic syndromes (MDS) are heterogeneous diseases characterized by clonal, ineffective hematopoiesis resulting in peripheral cytopenia and an increased risk of progression to acute myeloid leukemia. Particularly in low risk MDS a pro-apoptotic milieu has been found with increased levels of apoptosis-promoting factors such as tumor necrosis factor and fas ligand (CD95 ligand). This is thought to be a major pathomechanism in low risk MDS resulting in increased apoptosis of medullary erythroid progenitors leading to peripheral cytopenia. APG101 is a fusion protein consisting of the extracellular domain of human CD95 and the Fc domain of human IgG1. APG101 binds to CD95 ligand expressed on effector cells as well as to functionally active ligand in solution, thus blocking the interaction between CD95 and its ligand. Here, we report on interim results of a phase I study in transfusion dependent low risk MDS patients treated with APG101.Methods: A total of 15 patients have been enrolled by July 30th 2014. All patients gave their written informed consent. Patients were treated for 12 weeks with weekly intravenous infusions of 100 mg or 400 mg APG101, respectively. Thereafter the treatment was discontinued and the patients were followed for another 6 months. Bone marrow biopsies were taken prior to the first APG101 dosing (baseline), at the end of treatment (EOT, week 12), at week 25 (follow up) and at week 37 (follow up). Methylcellulose assays on isolated CD34+ cells from the bone marrow aspirates at the different time points were performed. In addition, bone marrow samples at each time point were subjected to amplicon deep sequencing to detect gene mutations prior to APG101 dosing and to follow the mutational allele burden during and after treatment. This was done by sequencing of genomic DNA from ficollized mononuclear bone marrow cells on a Roche 454 GS Junior system.Results: At data cut-off, 5 patients (3 male) had completed the study. Median age was 77 (range 65-78). WPSS categories were low in 2 patients and intermediate in 3 patients, respectively. According to the exclusion criteria none of the patients showed a medullary blast count ≥ 5%. Two patients received 400 mg weekly while 3 patients received 100 mg APG101 weekly, respectively. All patients stayed on treatment as scheduled and no adverse events were reported so far. Careful evaluation of bone marrow smears as well as immune phenotypic analysis of bone marrow aspirates showed no increase in bone marrow blasts at any time point. Moreover, quantification of the allele burden of pre-existing mutations (mutations of ASXL1 and SF3B1 in one patient and DNMT3A in the other patient) showed no significant expansion of the mutated clones. Ex vivo differentiation analyses revealed an increase in granulocytic colony forming as well as CFU-E/BFU-E forming capacity. With regard to efficacy, 4 of 5 patients showed a decrease in transfusion frequencies, which so far did not fulfill the IWG 2006 criteria for hematologic improvement (HI).Conclusions: APG101 is a well tolerable compound in transfusion-dependent low risk MDS patients, particularly in elderly patients. None of the patients discontinued treatment due to toxicity. Monitoring did not reveal any signs of progress i.e. increase in medullary blasts in any of the patients. Deep amplicon sequencing did not show any signs of expansion of preexisting clones during therapy, i.e. APG101 did not provide malignant clones with a survival benefit. Although no major or minor HI (IWG 2006) was seen in 5 analyzed patients, the majority of patients showed a slight reduction in transfusion need which makes APG101 an interesting target for further investigation. DisclosuresKunz:Apogenix GmbH: Employment. Fricke:Apogenix GmbH: Employment. Nolte:Apogenix GmbH: Consultancy, Research Funding.

Hidden DNA Copy Number Alterations and Mutations in IKZF1, TP53, CRLF2 and JAK2 Genes Are Associated with a Poor Prognosis in B-Progenitor Acute Lymphoblastic Leukemia

Background: In B-progenitor acute lymphoblastic leukemia (B-ALL) the identification of additional genetic alterations associated with treatment failure is still a challenge.Aims: 1.To identify genomic gains and losses in B-ALL at the time of diagnosis and to correlate these abnormalities with the genetic characteristics and the patients outcome. 2.To assess the prevalence and prognostic impact of genetic lesions in IKZF1, TP53, CRLF2, IL7R, PAX5, JAK2 and LEF1 genes in B-ALL.Methods: A total of 215 B-ALL patients were eligible for this study. 115 (53.5%) had less than <18 years. In all cases oligonucleotide microarrays (aCGH) were carried out. For genetic gain and losses analysis, the NimbleGen CGH 12x135K array (Roche) was used. The data analysis was performed with GISTIC and aCGHweb software. In 118 of these cases (65 children and 53 adults), an integrative analysis of aCGH and NGS was performed to identify genetic lesions in genes associated with B-ALL: JAK2 (Exon 12-Exon 16), PAX5 (E2-E3), LEF1 (E2-E3), CRLF2 (E6), IL7R (E5), IKZF1 (E2-E8) and TP53 (E4-E11). The 454 GS Junior system (Roche) was used. The variant analysis was performed using Amplicon Variant Analyzer (AVA-Roche 454) and Sequence Pilot (JSI Medical Systems) software.Results: DNA copy number aberrations (CNAs) were observed in 96.5% of cases. Gains on chromosomes 4 (10%), 6 (10%), 10 (12%), 14 (13%), 21 (31%) and X (18%) were more common in children, whereas losses in 7p (13%) and 9p21 (24%) were frequent in adults. In children, gains on chromosomes 6, 10, 14q, 17, 18, 21 and X were associated with longer overall survival (OS) while loss on 7p and 17p were associated with shorter OS in adults (Table 1).The integrative analysis of aCGH and NGS showed that 60% of patients carried at least one alteration (deletion and/or mutation) in the seven genes analyzed. Focal deletions were common in IKZF1, CRLF2, PAX5, and LEF1 genes while broad deletions were more frequent in TP53, JAK2 and IL7R. Forty-one percent of patients harbored IKZF1 alterations (IKZF1+), 22.9% in PAX5, 18.6% in JAK2 and 11% had TP53 or CRLF2 alterations. LEF1 and IL7R genomic lesions were only present in 4.2% and 2.5% of the B-ALL, respectively. JAK2 and CRLF2 mutations were associated (p=0.01). Moreover abnormalities in IKZF1+ were associated with alterations in JAK2 (p=0.004), TP53 (p=0.04) and PAX5 (p=0.03).The presence of alterations was most frequent in high-risk (HR) B-ALL (38% vs 18%, p=0.02), although 8% low-risk (LR) childhood patients showed genetic alterations. Fourteen B-ALL patients carried mutations in TP53, CRLF2 and/or JAK2 genes: all of them were Ph-, and 11 were classified as HR. Of note, 3 cases were children. In the HR group the presence of mutation in TP53, CRLF2 and/or JAK2 was related to a shorter OS (3-year OS: 56% vs. 36%; P=0.034) and event-free survival (3-year EFS: 56% vs. 11%; P<0.002) (Figure 1), due to an increased cumulative incidence of relapse (3-year CIR: 38% vs. 88%; P<0.004).The presence of IKZF1 alterations stratified HR-Ph- B-ALL cases. Thus HR-Ph- patients with normal IKZF1 were associated with longer EFS compared to HR-Ph- B-ALL IKZF1+ (3-year EFS: 60% vs. 33%; P=0.005) (Figure 2). The presence of IKZF1 deletion was associated with shorter EFS (89% vs. 67%, P= 0.04) and increased CIR (3-year CIR: 12% vs. 33%; P=0.05) in children.Summary/Conclusions: CNAs are frequent in B-ALL and are associated with genetic subtype, age and overall survival. The integrative analysis by aCGH and NGS techniques demonstrated that alterations in IKZF1 gene and mutations in TP53, CRLF2 and/or JAK2 genes could stratify high-risk Ph- B-ALL patients.Subvention:FP7/2007-13, Nº306242-NGS-PTL; HUS272U13, JCyL,Consejería de Educación; BIO/SA31/13 Gerencia Regional de Salud, SACYL, SpainTable 1DNA copy number aberrations (CNAs) associated with overall survival in 215 B-ALL patientsAssociationCNAnMedian (months)P valueShort overall survival7p-ChildrenAdults1961396150.0517p-ChildrenAdults12576940.029+19ChildrenAdults351421Not reached100.00122q+ChildrenAdults261214Not reached7<0.0001Longoverall survival+6ChildrenAdults26197Not reached7<0.0001+10ChildrenAdults25196Not reached70.001+14qChildrenAdults26188Not reached4<0.0001+17ChildrenAdults24177Not reached600.012+18ChildrenAdults25187Not reachedNot reached<0.000121q+ChildrenAdults473116Not reached8<0.0001XChildrenAdults522725Not reached9<0.0001 [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clinical Impact of TP53 Mutations in Patients with MDS and Isolated Deletion 5(q) Treated with Lenalidomid: Results from the German Prospective Le-Mon-5 Trial

Introduction: MDS with isolated deletion 5(q) accounts for approximately 5% of all MDS cases. A recent retrospective analysis has found a cumulative progression rate of 18% after 5 years in patients with MDS deletion 5(q) without an increased medullary blast count[1]. Retrospective analyses have indicated that mutations in TP53 have an adverse impact on the clinical course of affected patients and their response to Len treatment. Here we report on the results of the German multi-center, prospective Le-Mon-5 trial that investigated the safety and efficacy of lenalidomide (Len) in patients with MDS and isolated deletion 5(q).Methods: Le-Mon-5 is a trial of Len in patients with MDS with isolated 5q abnormality, a blast count of < 5% in the bone marrow, a platelet count > 50.000/µl and absolute neutrophil counts of > 500/µl in the peripheral blood. Patients were treated with the standard dose of 10 mg Len for 21 days q28 days. The primary endpoint was safety. Secondary endpoints included response according to IWG criteria (2000), time to response, duration of transfusion independency and incidence and time to AML-transformation, respectively. All patients gave written informed consent to the trial including additional molecular genetic analyses. Central cytologic, cytogenetic and histologic review was performed. Mutational analysis of TP53 was done by next generation sequencing (NGS). For generation of PCR amplicon libraries TP53 primer plate assays were used and technically validated within the IRON-II (Interlaboratory Robustness Of Next generation sequencing) study network. Amplicon sequencing of TP53 was performed on a Roche 454 GS Junior system. Mean coverage of sequenced exons was about 800-fold allowing an approximate detection sensitivity of 2% mutational burden.Results: A total of 91 patients were enrolled into the trial. Of those, 71 patients (male, n=13) were analyzed for TP53 mutations. Median age was 71 years (range 40-88 years). TP53 mutations prior to treatment initiation with Len were found in 7 patients (10%). There was no difference between the TP53 mutated (TP53mut) versus TP53 wildtype patients (TP53WT) with regard to age, IPSS risk, hemoglobin levels, absolute neutrophil counts and platelet counts at baseline. Transfusion independence was achieved in a significantly lower proportion of patients in the TP53mut group versus TP53WT group (43% vs. 62%, p=0.036). Moreover, median survival was significantly shorter in the TP53mut group as compared to TP53WT group (533 days vs. not reached, p=0.0002). No difference was seen with regard to cytologic and cytogenetic response. Data on evolution into AML are currently being collected. Of 2 patients we had follow-up samples available. Both patients showed no difference with regard to the mutation frequency after a follow-up of 4-17 months on Len treatment (27% and 51%, respectively), although one of the patients achieved a complete cytogenetic response during Len treatment.Conclusions: Using the NGS technique on a routine basis, we achieved high quality runs with a high mean coverage of analyzed exons of TP53. Presence of TP53 mutations adversely affected response to Len with regard to transfusion independence. Moreover, TP53mut patients had a shorter overall survival as compared to TP53WT patients underlining the prognostic relevance of TP53 mutations in this patient cohort. Therefore, mutation analysis of TP53 might guide treatment decisions in the future, e.g. consideration of combination regimens. Since the TP53 clone obviously prevails during Len treatment, careful monitoring for signs of transformation should be performed. DisclosuresPlatzbecker:Celgene Corp.: Honoraria, Research Funding. Götze:Celgene Corp, Novartis Pharma: Honoraria. Schlenk:Celgene Corp.: Research Funding, Speakers Bureau. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Bug:Celgene: Honoraria, Research Funding. Germing:Celgene Corp.: Honoraria, Research Funding. Nolte:Celgene Corp., Novartis Pharma: Honoraria, Research Funding.

Detection of TP53 Mutations in Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML). a Comparison Between a Functional Method (FASAY) and Next Generation Sequencing (NGS)

BackgroundTP53 mutations inactivating p53 protein, often associated with loss of the remaining TP53 allele through 17p deletion, are major prognostic factors in many hematological malignancies, including CLL, myeloma, AML and MDS. In AML and MDS, they are usually associated with complex karyotype (including del 17p) and very poor prognosis (Blood 1991, 78(7):1652-7 , Bejar, NEJM 2011), including after allogeneic SCT (Middeke JM, Blood 2014) but they are also seen in lower risk MDS with isolated del 5q, where they confer resistance to Lenalidomide (Jadersten, JCO 2011). The advent of Next Generation Sequencing (NGS) techniques has improved the detection of such mutations, by allowing the identification of small mutated clones. Other detection methods may prove interesting, especially functional methods like FASAY ( Functional Assay of Separated Allele in Yeast) , an easy and sensitive method that detects TP53 mutations by assessing the p53 function as transcription factor (Flaman et al, PNAS 1995). We compared the detection of TP53 mutations in MDS and AML by FASAY and NGS approaches.MethodsThe 84 patients analyzed included 10 AML, 10 higher risk MDS, and 64 lower risk MDS with del 5q. RNA and DNA were extracted from marrow mononuclear cells. TP53 mutations were detected on RNA by FASAY where, after amplification of the TP53 mRNA, the PCR product is co transfected with an open gap repair plasmid leading, by homologous recombination, to p53 protein expression in the yeast. The yeast strain used is dependent on p53 functionality for growth and color and detection of more than 10% of small red yeast colonies defines a non-functional FASAY result. All non-functional FASAY were confirmed by the split versions of the test and TP53 defects were characterized by Sanger sequencing. The detection limit is around 10% in our hands (Manie E, Cancer Res 2009). In parallel, TP53 mutations were detected on DNA by NGS using the IRON II plate design and pyrosequencing on a GS Junior System (Roche). (Kohlmann, Leukemia 2011).FASAY (+Sanger sequencing) and NGS were performed in two different labs.ResultsBy FASAY, 47 patients (56%) had a functional p53 and 37 cases (44%) a non-functional p53 and a mutation was confirmed by Sanger in all non functional cases.By NGS analysis, no TP53 mutation was found in 47 cases (56%) and a mutation was detected in 37 cases (44%).In the 37 mutated cases by NGS, the median proportion of mutated allele was 35% (range 3 to 99%), including a median of 72%, 35%, 25 % in AML, higher risk MDS and lower risk MDS with del 5q, respectively. The mutated clone size was lower than 10% in only 2 patients who both had lower risk MDS with del 5q (3 and 6%, respectively).A perfect correlation between FASAY and NGS was found in 80 (95.5%) cases. The 4 discordant cases included a mutation detected only by FASAY in 2 cases, and only by NGS in 2 cases. Undetected mutations by NGS were insertions of intronic sequences (intron 9) not explored by the technique used. These insertions resulted in non-functional protein well detected by FASAY which analyses the global cDNA sequence including splicing defects. Undetected mutations by FASAY were mutations in which the percentage of mutated alleles was less than 10% (3% and 6 % respectively).Finally, while the cost of NGS analysis for TP53 mutation is around 200 euros when performed alone (and around 2000 euros when combined to analysis of the 30 main other genes involved in MDS and AML), the cost of the FASAY technique is around 20 euros (prices including reagents only).ConclusionThe FASAY technique is a cheap method, that in spite of a sensitivity of only 10%, was able to detect 98% of TP53 mutations detected by NGS. In fact those mutations appear to involve generally relatively large clones in MDS and AML. FASAY could also detect 2 atypical intronic mutations overlooked by NGS. Demonstrating in such difficult cases that the resulting p53 protein is non functional and therefore probably has pathophysiological significance, is an advantage of FASAY .The combination of the 2 methods, and especially the combination of DNA and RNA analysis, may be useful in such cases. DisclosuresNo relevant conflicts of interest to declare.

In Chronic Myeloid Leukemia Patients on 2nd-Line Tyrosine Kinase Inhibitor Therapy, Deep Sequencing at the Time of Warning May Allow Sensitive Detection of Emerging BCR-ABL1 Mutants

▪Background and Aims: Next generation amplicon-based deep sequencing (DS) on the Roche, Illumina or Ion Torrent instruments is becoming accessible to a wider and wider number of diagnostic laboratories. Although conventional sequencing is still the gold standard, DS has been hailed by many as the future of diagnostic BCR-ABL1 kinase domain (KD) mutation screening. BCR-ABL1 KD mutations are infrequent in newly diagnosed chronic myeloid leukemia (CML) patients (pts) receiving 1st-line TKI therapy, but remain a challenge in relapsed pts, who usually display a greater genetic instability. Indeed, pts already harboring BCR-ABL1 KD mutations have a higher likelihood of developing additional, dasatinib (DAS)- or nilotinib (NIL)-resistant mutations – which is defined as a 'failure’ by the 2013 European LeukemiaNet (ELN) recommendations. Taking advantage of a next-generation amplicon sequencing design and protocol set up and validated in the framework of the IRON-II international study, we aimed to assess whether DS may allow a larger window of detection of emerging BCR-ABL1 KD mutants predicting for an impending relapse.Methods: among the imatinib (IM)-resistant CML pts who switched to 2nd-line TKI therapy and were referred to our laboratory for routine BCR-ABL1 transcript level monitoring and KD mutation screening by conventional sequencing, 51 acquired DAS- or NIL-resistant mutations after a median of 9 months (range, 3-27 months) of therapy and had leftover cDNA available at previous timepoints. To reconstruct the dynamics of mutation emergence, resequencing on a Roche GS Junior instrument was performed from the time of failure and mutation detection by conventional sequencing backwards. Runs were designed to achieve high sequencing depth, allowing reliable detection of variants down to 1% abundance. BCR-ABL1/ABL1%IS transcript levels and/or cytogenetic response, whichever available, were used to define whether the patient had an 'optimal response’, 'warning’ or 'failure’ at the time of first mutation detection by DS.Results: baseline mutation status, as assessed by conventional sequencing, was available for all the 51 CML pts included in this retrospective study; 29/51 pts were positive for BCR-ABL1 KD mutations, with switch to NIL or DAS selected accordingly. Twenty-six pts were later found to have acquired DAS-resistant mutations (T315I, n=13; F317L/V, n=10; V299L, n=3) and 25 pts were later found to have acquired NIL-resistant mutations (T315I, n=4; F359V/I/C, n=7; Y253H, n=6; E255K, n=9; one patient acquired two mutations). DS was able to backtrack the DAS- or NIL-resistant mutations to the previous sample(s) in 23/51 (45%) pts. Median mutation burden at the time of first detection by DS was 5% (range, 1-17%); median interval between detection by DS and detection by conventional sequencing was 3 months (range, 3-9 months). In 5 cases, the mutations were traceable at baseline; in the remaining cases, correlation with response at the time mutations were first detected by DS revealed a 'warning’ according to the 2013 ELN definitions of response to 2nd-line therapy in 13 cases; an 'optimal response’ in one case; a 'failure’ in 4 cases. As a control, we used DS to explore BCR-ABL1 KD mutation status in 10 randomly selected pts with 'warning’ at various timepoints, that later turned into optimal responses; no DAS- or NIL-resistant mutations were detected.Conclusions: the 2011 ELN recommendations for mutation analysis suggest BCR-ABL1 KD to be screened by conventional sequencing in case of 'failure’ of 2nd-line TKI therapy – according to the provisional definitions available at the time. Earlier detection of emerging BCR-ABL1 KD mutations allows a greater leeway in tackling drug resistance and enhancing therapeutic efficacy. Data presented herein indicate that:1) DS may reliably pick TKI-resistant mutations earlier than conventional sequencing in a proportion of pts, and that2) the recently introduced definitions of ‘warning’ may provide a rational trigger, besides ‘failure’, for DS-based BCR-ABL1 KD mutation screening in CML pts on 2nd-line TKI therapy.A prospective cost-benefits evaluation of using DS in this and other settings is warranted, and will contribute useful information to the revision of the ELN recommendations for BCR-ABL1 KD mutation analysis.Supported by: European LeukemiaNet, AIL, AIRC, FP7 NGS-PTL project, Progetto Regione-Università 2010-12 (L. Bolondi). DisclosuresSoverini:Novartis: Consultancy; Bristol-Meyers Squibb: Consultancy; Ariad: Consultancy. Castagnetti:Novartis Farma: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy. Gugliotta:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria. Bonifacio:Amgen Inc.: Consultancy. Rosti:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy. Baccarani:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Ariad: Consultancy; Pfizer: Consultancy. Martinelli:NOVARTIS: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Ultra-Deep Sequencing Strategy Is a Precious Tool to Find Small Clones Harbouring FLT3 Mutations in AML Patients

Background: FLT3 internal tandem duplication (ITD) is frequently detected in AML patients and is an independent predictor of unfavourable outcome, while secondary point mutations in the FLT3 tyrosine kinase domain (KD) are common causes of acquired clinical resistance to FLT3 inhibitors, such as AC220 and Sorafenib.Technologies allowing massively parallel, ultra-deep sequencing (UDS) are currently being evaluated in diagnostic settings since they may conjugate throughputness, sensitivity and accurate quantification of mutated clones.Aim: Since recent whole genome sequencing studies have suggested that FLT3 ITD may evolve from small subclones undetectable at diagnosis by routine PCR, we tested the ability of an UDS strategy for FLT3 mutation screening to highlight small clones harbouring ITD mutations. Furthermore we evaluated if an UDS strategy could highlight in AML patients treated with FLT3 inhibitors emerging clones harbouring critical mutations, anticipating the development of drug-resistance.Methods: 886 AML patients were analyzed in Seràgnoli Institute of Bologna between 2002 and 2013 for a panel of genetic alterations, including FLT3. For our purpose, we retrospectively analyzed five AML (four CN-AML and one t(3;3) AML) who were found negative for FLT3 ITD- at diagnosis by conventional PCR and Sanger Sequencing, but were then found FLT3 ITD+ during follow-up at relapse or disease progression and ten AML (five FLT3+ and five FLT3-) treated with the FLT3 inhibitor AC220.In order to reconstruct the dynamic of mutation emergence, we performed a longitudinal re-analysis of RNA samples with UDS on a Roche GS Junior. UDS achieved a lower detection limit between 0,1% and 1%, depending on the relative number of sequence reads per sample obtained in each run.Results:886 AML patients were analyzed for a panel of genetic alterations including FLT3 ITD and TKD and 239 of 886 (27%) were FLT3+. In particular 256 were cytogenetically normal (CN) AML and of these 66 (25,8%) were FLT3+ and 192 were FLT3-.UDS strategy revealed that all the CN-AML analyzed already carried at diagnosis a small clone FLT3 ITD+ (allelic ratio 0.2-2%), that increased over time during follow-up, while in the t(3;3) AML the ITD clone emerged only at disease progression. In the three CN patients treated with chemotherapy the ITD+ was a minor clone until complete remission (CR), while after relapse the ITD+ clone expanded; one of these patients carried two rare ITD clones at diagnosis and only one became dominant at relapse, likely through loss of heterozygosity (LOH) of the mutated allele. In one CN patient who was treated only with FLT3 inhibitor AC220 the allelic load of the mutated clone increased over time before treatment and then followed the dynamic of the disease (regression at complete remission). For the five AML FLT3- treated with AC220 we didn’t find any novel FLT3 mutation after treatment, while for the five AML FLT3-ITD+ analyzed, we were able to follow the allelic load of the FLT3-ITD+ clone during treatment and the appearance in two patients of novel TKD mutations after treatment that are able to confer drug resistance (allelic ratio 2-55%).Conclusions: The high sensitivity of UDS technology allows detection of emergence of mutated clones earlier than conventional methods: this is a precious tool to find small clones FLT3 ITD+ that may evolve over time and worsen the prognosis of otherwise good prognosis CN-AML patients and to better calibrate therapy for these patients. Furthermore the prognostic value of determining the presence of FLT3-ITD by UDS is stronger than conventional methods because of the possibility to determine the ratio of mutated versus wild-type allele, the length and the size of insertion within a single analysis. In the setting of FLT3 inhibitors, UDS gives advantage in monitoring MRD by determining exactly the allelic load of mutated FLT3 ITD clones before and after treatment and high sensitivity in highlighting emerging mutated clones during treatment that may confer resistance, giving possibility to switch eventually to other inhibitors before coming out of overt clinical resistance to therapy. For monitoring patients treated with FLT3 inhibitors with UDS we will go on by screening AML treated with Sorafenib, to follow the emergence of any novel critical mutation during treatment.Acknowledgments: ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. DisclosuresMartinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

Ultra-Deep Sequencing (UDS) Allows More Sensitive Detection of the D816V and Other Kit Gene Mutations in Systemic Mastocytosis

Objectives and background: According to the World Health Organization (WHO) classification, the diagnosis of Systemic Mastocytosis (SM) relies on bone marrow (BM) examination and is based on a major and four minor criteria. The somatic 'autoactivating’ point mutation D816V in the KIT receptor gene is one of the minor criteria, founded in the great majority of patients (90%) and it plays a central role in the pathogenesis of the disease. Indolent Systemic Mastocytosis (ISM) is the most common variant of SM, characterized by a very low MC burden and associated with very different clinical pictures. A highly sensitive diagnostic methods for D816V detection are required to assure an appropriate diagnosis and to reduce false-negative results. The recent development of “ultra-deep amplicon sequencing” (UDS) technologies has opened the way to a more accurate characterization of molecular aberrations with higher sensitivity of screening for known and unknown mutations. Our aims were: i) to set-up and optimize a UDS-based mutation screening strategy of the KIT gene on the Roche GS Junior Instrument; ii) to test the sensitivity of our UDS assay to detect the D816V mutation; iii) to investigate the presence of additional KIT mutations in SM.Methods: We decided to take advantage of a next generation sequencing approach to perform an UDS KIT gene mutation analysis on 20 bone marrow (BM) samples from patients whit ISM that were negative for the D816V mutation by Sanger Sequencing which has a sensitivity of 20%. Fusion primers were designed to generate ten partially overlapping amplicon covering the whole KIT transcript (exons 1-21) by RT-PCR. To determine the lower detection limit of our UDS-assay, serial dilutions of the HMC-1 cell line (harboring the D816V mutation) into an unmutated K562 cell line in ratios such as to simulate the following mutation loads were sequenced: 50%, 37.5%, 25%, 12.5%, 5%; 2.5%, 1.25%, 0.5%, 0.25%.Results and significance: UDS of cell line dilutions showed a high accuracy of D816V mutation detection and linearity of mutation calling over the entire range down to 0.25%. The UDS technology allowed to detected the D816V mutation, below the lower detection limit of Sanger Sequencing, with an abundance from 0.5% to 11%, in 12/20 ISM patients. Two additional sequence variations were detected in a large proportion of patients. These two variations included a 3bp in-frame deletion in exon 15 (GenBank X06182.1: c.2164_2166delAGC; p.S715del) found in 11/20 patients and a 12bp in frame-deletion in exon 9 in all patients, whit an abundance ranging from 83% to 97% (GenBank X06182.1: c.1550_1561delGTAACAACAAAG; p.G510_K513del). Previously published studies indicate that the KIT Gly-Asn-Asn-Lys510-513+/- alternatively spliced located immediately downstream to the extracellular KIT domain and KIT Ser715+/-, an interkinase KIT domain, are expressed in normal human hematopoietic cell, leukemic cell lines, acute myeloid leukemia blast and GISTs and represent rather a splice variant of KIT transcript. Interestingly our results showed the presence of the transmembrane domain M541L (GenBank X06182.1: c.1642A>C; p.Met541Leu) KIT-activating mutation in exon 10, with an abundance of 50%, in addition to D816V, in 2/20 ISM. This mutation is known to retain sensitivity to imatinib mesylate.Conclusions: Our preliminary results suggest that our-UDS based KIT gene mutation screening assay might be a reliable and sensitive alternative to conventional sequencing methods for the detection of the D816V. We are now planning to investigate whether the greater sensitivity of UDS allows to detect the D816V mutation in peripheral blood mononuclear cells from patients with a suspected clonal mast cell disorder. These results could represent a starting point to plan other extensive studies to better understand the exact role of KIT receptor alterations in SM.Supported by ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. DisclosuresCavo:Celgene: Consultancy, Honoraria, Speakers Bureau. Martinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

DNA storage under high temperature conditions does not affect performance in human leukocyte antigen genotyping via next-generation sequencing (DNA integrity maintained in extreme conditions).

Stable dry-state storage of DNA is desirable to minimize required storage space and to reduce electrical and shipping costs. DNA purified from various commercially available dry-state stabilization matrices has been used successfully in downstream molecular applications (e.g., quantitative polymerase chain reaction [qPCR], microarray, and sequence-based genotyping). However, standard DNA storage conditions still include freezing of DNA eluted in aqueous buffers or nuclease-free water. Broad implementation of dry-state, long-term DNA storage requires enhancement of such dry-state DNA stabilization products to control for temperature fluctuations at specimen collection, transit, and storage. This study tested the integrity of genomic DNA subjected to long-term storage on GenTegra(™) DNA stabilization matrices (GenTegra LLC, Pleasanton, CA) at extreme conditions, as defined by a 4-year storage period at ambient temperature with an initial incubation for 7 months at 37°C, 56°C, or ambient temperature. Subsequently, purified DNA performance and integrity were measured by qPCR and next-generation sequencing (NGS)-based human leokocyte antigen (HLA) genotyping. High molecular weight genomic DNA samples were recovered from the GenTegra product matrix and exhibited integrity comparable to a highly characterized commercial standard under assessment by qPCR. Samples were genotyped for classical HLA loci using next generation sequencing-based methodolgy on the Roche 454 GS Junior instrument. Amplification efficiency, sequence coverage, and sequence quality were all comparable with those produced from a cell line DNA sequenced as a control. No significant differences were observed in the mean, median, or mode quality scores between samples and controls (p≥0.4). Next generation HLA genotyping was chosen to test the integrity of GenTegra-treated genomic DNA due to the requirment for long sequence reads to genotype the highly polymorphic classical HLA genes. Experimental results demonstrate the efficacy of the GenTegra product as a suitable genomic DNA preservation tool for collection and long-term biobanking of DNA at fluctuating and high temperatures.