Whole-Transcriptome Sequencing of a Chronic Myeloid Leukemia (CML) Patient at Diagnosis and at the Time of Progression to Blast Crisis (BC).

Abstract 4259

Philadelphia-chromosome positive (Ph+) CML is generally regarded as a quite genetically heterogeneous disease and therapy with Bcr-Abl tyrosine kinase inhibitors results in high response rates. Nevertheless, resistance may develop, especially in high Sokal risk patients, and progression to blast crisis still represents a major concern. The biological bases underlying Sokal risk, as well as the determinants of disease progression remain largely unknown. Several high-throughput technologies have been recently developed that allow to perform genome-wide studies with unprecedented informativity and resolution. For the first time in CML, we have integrated three such technologies – massively-parallel sequencing, gene expression profiling (GEP) by microarrays and high-resolution karyotyping by SNP-arrays – for a deeper characterization of a tyrosine kinase inhibitor-resistant patient at diagnosis and at the time of progression to BC.

A 62-year-old female was diagnosed with Philadelphia-chromosome positive (Ph+), BCR-ABL (p210)-positive CML, high risk according to both Sokal (2.4) and Euro (1704.39) scores. At presentation, no additional chromosomal abnormalities (ACA) were detected. The patient received first-line therapy with nilotinib 800 mg/d, achieved a major molecular response (MMR; Bcr-Abl transcript levels, 0.1% according to the International Scale as assessed by real-time quantitative PCR) after 3 months, but suddenly progressed to lymphoid BC after 6 months from diagnosis and died of her disease one month thereafter. At the time of progression, a T315I mutation was identified by conventional direct sequencing of the BCR-ABL kinase domain (KD); no ACAs were detected by chromosome banding analysis. After having obtained written informed consent from her next of kin, the samples collected at diagnosis, at the time of remission (MMR) and at the time of progression to BC were used for RNA and DNA extraction. Poly(A) RNA was then obtained and used to prepare double-stranded cDNA libraries for paired-end sequencing on an Illumina/Solexa Genome Analyzer. The number of 75bp-long sequence reads obtained was 40,193,384 (corresponding to 3.01 billion bases), 35,592,588 (2.7 billion bases), and 32,867,700 (2.5 billion bases) for diagnosis, remission and progression samples, respectively. The open-source software MAQ (http://maq.sourceforge.net) was used for read alignment and mapping against the human reference genome (hg18, NCBI build 36.1) and for subsequent single nucleotide variant (SNV) calling. Aligned reads were 31,886,732 (16,566,482 with no mismatches and 15,320,250 with at least one mismatch), 27,722,967 (16,199,728 with no mismatches and 11,523,239 with at least one mismatch) and 25,563,423 (14,315,149 with no mismatches and 11,248,274 with at least one mismatch) for diagnosis, remission and relapse, respectively. Comparison of the SNVs identified in the diagnosis and relapse samples with the SNVs identified in the remission sample was crucial to rule out all the inherited sequence variants non-specific of Ph+ cells. Nonaligned reads were further analyzed for the discovery of small insertions/deletions (indels) and novel or aberrantly spliced transcripts. In parallel, high-resolution (<1kb) genome wide copy number alteration (CNA) and loss of heterozigosity (LOH) analyses were performed on genomic DNA using the Genome-Wide Human SNP Arrays 6.0 (Affymetrix) and GEP was performed on RNA using the GeneChip Human Genome U133 Plus 2.0 Arrays (Affymetrix). MAQ's predictions are currently being confirmed by conventional direct sequencing. The validated results will be presented and correlated with those of GEP and SNP-arrays.

Although this study focuses - at present - on a single CML case, it first offers a comprehensive overview of the complexity of the Ph+ cell genome and transcriptome of a high-risk patient both at the time of diagnosis and at the time of TKI-resistance and progression to BC. Genome-wide integrated approaches like this might provide novel insights and fill the gaps in our knowledge of the pathogenesis of CML and of the mechanisms of disease progression and Abl KD mutation outgrowth. For this reason, whole-transcriptome sequencing of additional CML cases has already been planned. Supported by European LeukemiaNet, AIL, AIRC, PRIN, Fondazione del Monte di Bologna e Ravenna.