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Additional genomic lesions in adult Ph+ ALL
possibility of a cure was allogeneic stem cell transplanta- tion (HSCT), when feasible.6,7 The introduction of tyrosine kinase inhibitors, administered with low doses or without chemotherapy during induction, followed by consolida- tion chemotherapy and HSCT has markedly improved the management and outcome of adult Ph+ ALL patients, with survival rates at 5 years now approaching 50%.8-17
Different biological features - the type of fusion tran- script (i.e. p190 or p210),18 the persistence and/or reappear- ance of minimal residual disease (MRD),19,20 additional genomic deletions (particularly IKZF1, and to a lesser extent CDKN2A/B and PAX521-24) - and the presence of mutations at relapse are associated with a worse outcome.25-27 However, a broad and refined biological algo- rithm that could help to optimize treatment strategies and define better whether some patients could be spared intensive treatment, including HSCT, has so far not been proposed.
To this end, in the present study we investigated copy number aberrations (CNA) in 116 newly diagnosed adult Ph+ ALL patients to identify additional molecular lesions with the aim of improving patients’ stratification and management.
Methods
Experimental strategy
Bone marrow and/or peripheral blood samples from 116 patients (Table 1) with newly diagnosed Ph+ ALL enrolled in four GIMEMA (Gruppo Italiano Malattie EMatologiche dell'Adulto) trials were analyzed (Online Supplementary Table S1). The study was car- ried out in four phases (Online Supplementary Figure S1): (i) CNA analysis of 116 samples by Cytoscan; (ii) multiplex ligation-depen- dent probe amplification analysis; (iii) validation of MEF2C dele- tions by digital droplet (dd) polymerase chain reaction (PCR); and (iv) MEF2C and KRAS mutational screening.
This study was approved in the context of an Associazione Italiana per la Ricerca sul Cancro (AIRC) project (10007) with Institutional Review Board number 2182/16.06.2011.
Copy number aberration analysis
CNA were analyzed using CytoScan® HD Arrays (Affymetrix, Santa Clara, CA, USA) and Chromosome Analysis Suite (ChAS) software. Germline material from five paired samples was also evaluated. Recurrent deletions were validated with the Salsa MLPA P335 ALL-IKZF1 kit (MRC-Holland, Amsterdam, the Netherlands)28,29 (Online Supplementary Data). Statistical analyses on clinical correlates are described in the Online Supplementary Data.
Digital droplet polymerase chain reaction assays
MEF2C deletions were validated by ddPCR using the QX200TM Droplet DigitalTM PCR System (BioRad, Hercules, CA, USA) and QuantaSoft Analysis Pro software according to the manufacturer's instructions (Online Supplementary Data).
Mutational screening
Sanger sequencing of PCR products for MEF2C and KRAS exons (Online Supplementary Table S2) was performed with the ABI-Prism 3500 sequencer (Applied Biosystem, Life Technologies, Foster City, CA, USA) (Online Supplementary Data).
Results
Copy number aberration analysis
CNA analysis revealed 7.8 aberrations/patient (range: 0- 28), the majority being losses (88%) with only 12% gains, both spanning from whole chromosomes to focal lesions;22,23,30-32 no differences were recorded among trials (Figure 1A).
Gross chromosomal lesions were found in 42% of cases: the majority were losses of chromosome 7 (18.1%), followed by monosomy of chromosome 9 or 9p deletion (9%) and gain of 1q (7.7%) (Figure 1B, Online Supplementary Table S3). Smaller deletions - limited to one to three genes and defined as focal - were found in 56% of cases.
The most frequently deleted region involved the 7p12 cytoband comprising IKZF122,23,33,34 which was detected in 97 cases (84%).
Table 1. Patients’ clinical features. Gender (male/female)
Age, years (range)
Median white cell count x 109/L (range) Median hemoglobin g/dL (range) Median platelet count x 109/L (range) Fusion transcript (p190/p210/p190-210)¥ Complete molecular response* yes/no
Patients (n=116)
55/61
51.1 (18.9-89) 25.4 (1.7-597) 9.6 (4-16.3) 50 (4-333) 70/29/16 17/99
Multiplex ligation-dependent probe amplification con- firmed IKZF1, PAX5, CDKN2A, BTG1, EBF1, ETV6 and RB1 lesions, and allowed evaluation of IKZF1 isoforms. These isoforms were grouped into four classes:24,36 wild- type, dominant-negative (comprising Δ4-7 cases, 29.8%), haploinsufficient (including all cases harboring a deletion that involves exon 2 - i.e. Δ2-7, Δ2-8, Δ2-3, Δ1-3 - or the whole gene, 57.7%) and miscellaneous (remaining cases, 11.3%).
Identification of novel lesions
CNA analysis highlighted additional genomic lesions (Table 2, Online Supplementary Table S4). We focused in particular on MEF2C and KRAS deletions since these had prognostic significance (see below). MEF2C deletions were detected in 21 patients (18.1%) and differed in size. According to the length of intron 1-2 losses, deletions were grouped into two categories. One category - detect- ed in 14 cases (67% of MEF2C deleted cases) - was char- acterized by a larger minimal common region (6.2 Kb) involving introns 1-2 and exon 2 (the first codifying exon),
PAX5 was deleted in 43 patients (36.2%), while 37 (31.9%) had deletions of CDKN2A/B. MLLT3, BTG1, BTLA, CD200 and RB1 were deleted in 30, 27, 21, 17 and 16 cases, respectively (25.9%, 23%, 18.1%, 17.2%, 14.6%, and 13.8%) (Figure 1C).
IKZF1 deletions (ΔIKZF1) occurred together with CDKN2A/B and/or PAX5 deletions in 45/97 cases (46.4%) and are defined as ΔIKZF1+CDKN2A and/or PAX5 (Figure 1D): this subset displayed similar lesions to those recently described by Stanulla and colleagues.35 With regard to potential interactions, we found a significant association between IKZF1 and PAX5 deletions (P=0.01), but not with CDKN2A.
*As per protocol definition; ¥information missing for one patient.
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