Identification of Genetic and Epigenetic Factors for Patient-Tailored Treatment In

Identification of genetic and epigenetic factors for patient-tailored treatment in myelodysplastic syndromes and acute leukemias.

Abstract of the project

High-risk myelodysplastic syndromes (MDS) are heterogeneous malignant pathologieswith a globally unfavorable prognosis. In these diseases, there is a strong need to develop new diagnostic and therapeutic options,based on a better understanding of the biology and the molecular pathogenesis of the diseases and their relationship tothe characteristics of the host.We plan to characterize genomic copy-number alterations in acute myeloid leukemia (AML) and MDS by high-resolution single nucleotide polymorphism(SNP) arrays to identify genome-wide cryptic genomic aberrations , which may have diagnostic or prognostic significance.This technology facilitates studies of acquired genetic imbalances, large and cryptic aberrations and common deletedregions (CDR's), and reveals areas of loss of heterozygosity (LOH) and uniparental disomy (UPD).DNA methylation and histone acetylation, are universally recognized to participate in carcinogenesis and represent potentialtargets of therapeutic intervention. In the last years, treatment using the DNA methyl-transferase inhibitor 5-Azacitidine(5-Aza) has been successful in MDS and AML patients, inducing prolonged survival and delayed AML evolution, with acceptableside effects, when compared to standard supportive care. Although these biologic drugs are in principle very welltargeted, there is no valuable parameter, either disease- or patient-associated, predicting response. Based on previously published data and preliminary findings, we plan to study gene methylation by selected or complete profiling to identify the geneor gene groups mostly predictive of treatment response. Recently, heterozygous deletions of regions and genes potentially involved in MDS genesis have been identified, includingASXL1, TET2 and UTX. TET2 and ASXL1 function is linked to regulation of transcriptionenhancing histone acetylation and contributing to epigenetic regulation. Given their epigenetic function, we will studymutations of TET2 and ASXL1 as markers of response in patients who have undergone hypomethylating treatment.

To identify patient-related factors in important for disease outcome, we will study interindividual differences in drug pharmacokineticsand pharmacodynamics related to drug exposure or to response to potential toxic drugs and to adjust the drug dosebased on genetic variations, we will use the Affymetrix Drug Metabolizing Enzymes and Transporters (DMET) genotypingplatform, which scans 1,936 variants in 225 genes related to drug metabolism and disposition.This will hopefully lead to the development of a diagnostic methylation-kit and a pharmacogenomic kit to be included in newMDS and AML treatment protocols, resulting into a more rationale patient-oriented pharmacological approach.In particular, we aimed to molecularly characterize patients with MDS and AML with a blast cell count of 20-30% treated with 5-Azacitidine.

State of the art

Acute myeloid leukemia (AML) is a heterogeneous group of clonal myeloid malignanciesthatpredominantly affect middle-aged and elderly adults. They are all characterized by an arrest of maturationalong with uncontrollable proliferation of hematopoieticprogenitor cells.In general, the prognosis of patients with AML is currently based upon the presence or absence of cytogenetic abnormalities and is divided into favorable, intermediate and unfavorable subgroups(1). However, approximately 40% of AML patients have no identifiable cytogenetic abnormality by using modern cytogenetic and fluorescence in-situ hybridization (FISH) methods. As an adjunct to cytogenetic studies,small subcytogenetic amplifications and deletionscan be identified with the use of genomicmethods, such as single-nucleotide-polymorphism(SNP) array and array CGH platforms. However, these techniques remain investigational,and studies suggest that thereare few recurrent acquired copy-number alterationsin each AML genome(2-3). Gene-expressionprofiling has identified patients with knownchromosomal lesions and genetic mutations andsubgroups of patients with normal cytogeneticprofiles who have variable clinical outcomes(4).Candidate-gene resequencingstudies have also identified recurrentmutations in several genes — for example, genesencoding FMS-related tyrosine kinase 3 (FLT3), nucleophosmin 1 (NPM1), Mixed Lineage Leukemia (MLL),CCAAT/enhancer binding protein α (CEPBα) which may identify good and poor risk normal cytogenetic (NC-AML)subgroups.Recently, next-generation sequencing (NGS) platforms have evolved to provide an accurate and comprehensive means for the detection of molecular mutations. Massively parallel pyrosequencing in picoliter-sized wells is an innovative technique that allows the detection of low-abundance oncogene aberrations in complex research samples, even with low tumor content. Recently, in a whole genome sequencing the leukemic genome of a patient with de novo cytogenetically normal AML with minimal maturation(5) a mutation at codon 132 of cytosolic isocitrate dehydrogenase 1 (IDH1) was found and thereafter confirmed in 15 of 187 additional AML genomes. All these mutations were heterozygous and strongly associated with normal cytogenetic status.In the last few months, several studies have been published on somatic mutations of IDH1 and IDH2 in AML(6-11). They are found mainly, although not exclusively, in cytogenetically normal AML, in which they are – with a few exceptions - mutually exclusive;in the vast majority of somatic mutations of IDH1 and IDH2 involve residues R132 of IDH1, and R140 or R172 of IDH2; the prognostic significance of somatic mutations of IDH1 and IDH2 in AML is currently under investigation, but the available evidence indicates that they may be associated with an intermediate to high genetic risk. In addition, mutations in CBL, TET2and EZH2 have also been identified (12-14). Genetic and epigenetic alterations are implicated in biology and progression of high risk MDS and AML. Both single genemutations and methylation and acetylation of specific promoter regions cooperate with major genetic alterations, such as abnormalkariotype and chromosome deletions, to define the pathogenesis, the characteristics and the prognosis of these diseasesand therefore influence the nature and intensity of treatment.

Finally, the individual genetic background of the host significantly contributes to the outcome of the disease and is part ofpharmacogenomics. Focus is the genetic variation of drug-metabolizing enzymes, targets and transporters, and how these geneticvariations interact to produce specific drug-related phenotypes. Single Nucleotide Polymorphism (SNP) may also influencethe ability of the cell to repair and survive DNA damage, thus influencing treatment response.

Objectives

The main aim of this project is to perform a molecular genetic screeningand epigenetic changesin a cohort of adult AML with a low percentage of blast cells (20-30%) and MDS patients. Furthermore, we sought to correlate the specific mutations with clinical outcome and to investigate mutational patterns associated with relapse.

Plan of investigation

This project will be developed in 4 tasks.

Task 1. Analysis of genomic copy-number alterations by SNP array.Clonal chromosomal abnormalities are a common occurrence in MDSand AML, being detected in 40%-70% of cases of de novo MDS,and in up to 95% of cases of therapy related MDS (Haase et al, 2007). However, due to the poor in vitro growth of the dysplasticclone, it is often difficult to obtain useful cytogenetic data from MDS patients. The advent of high-resolution SNPmicroarrays has provided an opportunity to identify genome-wide cytogenetically cryptic genomic aberrations in MDS patients,which may prove to be of diagnostic or prognostic significance. Furthermore, these aberrations may help identify genesthat lead to progression. This technology facilitates studies of acquired genetic imbalances, reveals areas of loss of heterozygosity(LOH) and uniparental disomy (UPD). The readily available Affymetrix® Genome-Wide Human SNP Array 6.0contains more than 906,600 SNPs and more than 946,000 probes for the detection of copy number aberrations. The array alsocontains 202,000 probes targeting 5,677 known regions of copy number variation from the Toronto Database of Genomic Variants.These regions resolve into 3,182 distinct, non overlapping segments, each interrogated with an average of 61 probes.In addition to the interrogation of these regions of known copy number polymorphism, more than 744,000 probes were chosen,evenly spaced along the genome, to enable the detection of novel copy number variation. The median inter-marker distancetaken over all 1.8 million SNP and copy number markers combined is less than 700 bases.

We aim to use SNP array technology to identify:

- large and cryptic aberrations and common deleted regions (CDR's) such as in cases with monosomy 7;

- copy neutral regions of homozygosity (UPD) and novel association with gene aberrations.

Task 2. High-Resolution Pharmacogenetic Profiles of Genes involved in drug absorption, distribution, metabolism and elimination.To explore functionally relevant interactions among genetic variations and drug disposition and response, we will use the AffymetrixDrug Metabolizing Enzymes and Transporters (DMET) genotyping platform, which offers the ability to scan 1,936variants in 225 genes related to drug metabolism and disposition. The DMET Plus Panel features markers in all FDA-validatedgenes and covers more than 90 percent of the current drug absorption, distribution, metabolism and excretion(ADME) Core markers as defined by the PharmaADME group. The DMET array interrogates several types of markers, includingcopy-number variations, insertions/deletions, biallelic and triallelic SNPs. While quality testing of genotyping arrays isusually accompanied by less than 100% representation due to inappropriate SNP detection, or gene homology, the DMETplatform is quite comprehensive; often it includes more variants than were previously investigated in other studies. Recently,Affymetrix has added additional content relevant to drug ADME, and a tool to identify haplotypes amongst 779 polymorphismsin a core set of 61 genes identified by the PharmaADME consortium to be of high-importance in drug metabolism (Sissunget al, 2010). We recently received the certification from Affymetrix to performsuch experiments. This method will help to identify:

- genetic variation and interindividual differences in drug pharmacokinetics and pharmacodynamics;

- useful biomarkers related to drug exposure or to response to potential toxic drugs and to adjust the drug dose basedon genetic variations.

Task 3. Epigenetic Changes. Two distinct alterations of the normal DNA methylation pattern occur in cancer: a globalhypomethylation associated to genomic instability, and gene-specific promoter hypermethylation, which leads to silencing oftumor suppressor genes.In the last years, treatment using the DNA methyl-transferase (DNMT) inhibitor 5-Azacitidine (5-Aza) at the standard doseof 75 mg/sqm/day for 7 days has been successful in MDS patients, inducing prolonged survival and delayed AML evolution,with acceptable side effects, when compared to standard supportive care (Silverman et al 2002, Kornblith et al 2002, Sorianoet al 2007). The Italian Gimema group has recently tested the tolerability and efficacy of the combination of 5-Aza with a histone-deacetylase inhibitor, valproic acid (VPA) in higher risk MDS patients (Voso et al 2009).Studies on global methylation have confirmed hypomethylation due to 5-Aza, but this does not seem to correlate with treatmentresponse (Soriano et al 2007, Blum et al 2007, Fandy et al 2009, Follo et al 2009, Jiang et al 2009). Importantly, it hasbeen observed that during the transition from MDS to AML, DNA methylation profiles significantly change with increasedhypermethylation of either tumor suppressors or differentiation genes. However, there is not a general consensus on those geneswhose methylation profile correlates with the progression of MDS to AML. Therefore genome wide definition of the methylomeof MDS vs AML patients with low blast cell count (20-30%) would be of great help to identify those DNA regions with typical altered methylation profiles.

Task 4. Mutations of critical genes. Recently, next-generation sequencing (NGS) platforms have evolved to provide an accurate and comprehensive means for the detection of molecular mutations. Massively parallel pyrosequencing in picoliter-sized wells is an innovative technique that allows the detection of low-abundance oncogene aberrations in complex research samples, even with low tumor content. Recently, in a whole genome sequencing the leukemic genome of a patient with de novo cytogenetically normal AML with minimal maturation(5) a mutation at codon 132 of cytosolic isocitrate dehydrogenase 1 (IDH1) was found and thereafter confirmed in 15 of 187 additional AML genomes. All these mutations were heterozygous and strongly associated with normal cytogenetic status.In the last few months, several studies have been published on somatic mutations of IDH1 and IDH2 in AML(6-11). They are found mainly, although not exclusively, in cytogenetically normal AML, in which they are – with a few exceptions - mutually exclusive;in the vast majority of somatic mutations of IDH1 and IDH2 involve residues R132 of IDH1, and R140 or R172 of IDH2; the prognostic significance of somatic mutations of IDH1 and IDH2 in AML is currently under investigation, but the available evidence indicates that they may be associated with an intermediate to high genetic risk. In addition, mutations in CBL, TET2and EZH2 have also been identified (12-14).An aim of this project is to perform a molecular screening of five genes (CBL, IDH1, IDH2, TET2 and EZH2) in a cohort of adult AML with a blast cell count of 20-30% and MDS patientsby using a next-generation sequencing platform (GS Junior, Roche, Penzberg, Germany).This novelapproach allows the detection oflow-abundance oncogene aberrations in complex samples, even with low tumor content. For each gene different amplicons will be produced in order to cover all the known mutational hotspot regions. Each amplicon will have a size of around 350 bp. DNA will be extracted from blast cells and diluted to 20 ng/μl for subsequent PCR amplification. PCR products will be purified using immunomagnetic beads (Beckman Coulter) and quantifiedusing the Quant-iT PicoGreen kit (Invitrogen, Carlsbad, CA). For each patient, all amplicons will be pooled in an equimolar ratio to generate one singlelibrary. Thereafter next-generation pyrosequencing using GS FLX System will be used for amplicon sequencingaccording to the manufacturer’s recommendation. The GS Junior System produces 400 base-pair average read lengths and Generate 35 million high-quality filter-passed basesin a 10-hour sequencing run (100,000 shotgun reads,80,000 amplicon reads). Sequencing results will be analyzed by GS Amplicon Variant Analyzer software version 2.0.01(Roche). For the detection of variances, filters will be set to display sequencevariances occurring in more than 3% of bidirectional reads per amplicon in atleast one patient.

Duration of the study

The study will be conducted in approximately two years.

References

1.Mrozek K, Heinonen K, de la Chapelle A, Bloomfield CD. Clinical significance of cytogenetics in acute myeloid leukemia. Semin Oncol 1997 Feb; 24(1): 17-31.

2.Bullinger L, Kronke J, Schon C, Radtke I, Urlbauer K, Botzenhardt U, et al. Identification of acquired copy number alterations and uniparental disomies in cytogenetically normal acute myeloid leukemia using high-resolution single-nucleotide polymorphism analysis. Leukemia 2010 Feb; 24(2): 438-449.

3.Walter MJ, Payton JE, Ries RE, Shannon WD, Deshmukh H, Zhao Y, et al. Acquired copy number alterations in adult acute myeloid leukemia genomes. Proc Natl Acad Sci U S A 2009 Aug 4; 106(31): 12950-12955.

4.Bullinger L, Dohner K, Bair E, Frohling S, Schlenk RF, Tibshirani R, et al. Use of gene-expression profiling to identify prognostic subclasses in adult acute myeloid leukemia. N Engl J Med 2004 Apr 15; 350(16): 1605-1616.

5.Mardis ER, Ding L, Dooling DJ, Larson DE, McLellan MD, Chen K, et al. Recurring mutations found by sequencing an acute myeloid leukemia genome. N Engl J Med 2009 Sep 10; 361(11): 1058-1066.

6.Schnittger S, Haferlach C, Ulke M, Alpermann T, Kern W, Haferlach T. IDH1 mutations are detected in 6.6% of 1414 AML patients and are associated with intermediate risk karyotype and unfavorable prognosis in adults younger than 60 years and unmutated NPM1 status. Blood 2010 Aug 30.

7.Thol F, Damm F, Wagner K, Gohring G, Schlegelberger B, Hoelzer D, et al. Prognostic impact of IDH2 mutations in cytogenetically normal acute myeloid leukemia. Blood 2010 Jul 29; 116(4): 614-616.

8.Paschka P, Schlenk RF, Gaidzik VI, Habdank M, Kronke J, Bullinger L, et al. IDH1 and IDH2 mutations are frequent genetic alterations in acute myeloid leukemia and confer adverse prognosis in cytogenetically normal acute myeloid leukemia with NPM1 mutation without FLT3 internal tandem duplication. J Clin Oncol 2010 Aug 1; 28(22): 3636-3643.

9.Noordermeer SM, Tonnissen E, Vissers I, Van Der Heijden A, Van De Locht LT, Deutz-Terlouw PP, et al. Rapid identification of IDH1 and IDH2 mutations in acute myeloid leukaemia using high resolution melting curve analysis. Br J Haematol 2010 Oct 19.

10.Marcucci G, Maharry K, Wu YZ, Radmacher MD, Mrozek K, Margeson D, et al. IDH1 and IDH2 gene mutations identify novel molecular subsets within de novo cytogenetically normal acute myeloid leukemia: a Cancer and Leukemia Group B study. J Clin Oncol 2010 May 10; 28(14): 2348-2355.

11.Green A, Beer P. Somatic mutations of IDH1 and IDH2 in the leukemic transformation of myeloproliferative neoplasms. N Engl J Med 2010 Jan 28; 362(4): 369-370.

12.Bacher U, Haferlach C, Schnittger S, Kohlmann A, Kern W, Haferlach T. Mutations of the TET2 and CBL genes: novel molecular markers in myeloid malignancies. Ann Hematol 2010 Jul; 89(7): 643-652.

13.Nibourel O, Kosmider O, Cheok M, Boissel N, Renneville A, Philippe N, et al. Incidence and prognostic value of TET2 alterations in de novo acute myeloid leukemia achieving complete remission. Blood 2010 Aug 19; 116(7): 1132-1135

14.Makishima H, Jankowska AM, Tiu RV, Szpurka H, Sugimoto Y, Hu Z, et al. Novel homo- and hemizygous mutations in EZH2 in myeloid malignancies. Leukemia 2010 Oct; 24(10): 1799-1804.