Deletions of Immunoglobulin heavy chain and T cell receptor gene regions are uniquely associated with lymphoid blast transformation of chronic myeloid leukemia

The BCR/ABL1 fusion, housed in the Philadelphia chromosome and resulting from t(9;22)(q34;q11) or variants, is the hallmark of chronic myeloid leukemia (CML) and the most frequent recurrent genetic aberration in B lymphoid leukemia in adults [1, 2]. The BCR/ABL1 fusion gene encodes a constitutively active tyrosine kinase that is necessary and thought sufficient to drive malignant transformation. The natural history of the disease follows progression from a relatively benign chronic phase, through accelerated phase into terminal blast crisis. This course of events can be diverted or aborted by nearly curative therapies, such as tyrosine kinase inhibitors, transplantation and occasionally interferon [3] The staging of CML is based on clinical and pathologic features, including bone marrow and peripheral blood count, cytogenetic evolution and response to therapy rather than biological criteria. Although at present the molecular mechanisms that contribute to the development of the blast crisis are largely unknown, it is well established that the Ph bearing cells acquire additional genetic changes during disease progression [3, 4]. There is evidence that the BCR/ABL1 fusion product not only regulates numerous proteins involved in apoptosis, proliferation and cell-cell interactions but also is a driving force behind the acquisition of additional genomic changes through regulation of the nucleotide excision/mismatch repair systems and promoting unfaithful restoration of double strand breaks [5]. This activity could lead to the formation of mutations within the BCR/ABL1 kinase and accumulation of additional genetic lesions, including point mutations, gene amplifications, genome loss and chromosome translocations that are believed to drive the malignant process [6, 7]. Mutations or abnormal expression of CDKN2A/2B, EVI-1, RB, MYC and p53 a mong others have been reported in CML (reviewed in Radich [3]). Gene expression profiling studies have identified a relatively large number of additional genes that are differentially expressed in patients at advanced stages of the disease but no consistent pattern has as yet been established (for review see Guintas-Cardama, 2009 [4]). While recurrent genome gains at a chromosomal level, such as +8, +19, +Ph and/or iso17q, are associated with disease progression, the occurrence of translocations is infrequent[8]. The identification of cryptic deletions at der(9)t(9;22) fuelled expectations that submicroscopic lesions may mark disease evolution [9]. Indeed genome wide searches using array comparative genomic hybridization (aCGH) by us and others clearly demonstrated the accumulation of genome imbalances in advanced stages of the disease [10, 11]. As the neoplastic transformation is occurring early in a haematopoietic stem cell, the resulting blast crisis (BC) in CML could be myeloid (BCM), lymphoid (BCL), or indeed mixed (BCmix). Lymphoid BC occurs in about a third of the CML patients [12] . Previous studies have shown that the BCL is characterized by the loss of IKZF1 in the majority of the BCR/ABL1 positive ALL cases and loss of CDKN2A/B in approximately half of the CML/BCL patients [13]. These losses were rarely present in non-BCL patients [14].

Here we report an analysis by oligonucleotide array comparative genomic hybridization (aCGH) showing the detection of universal deletions within both the immunoglobulin heavy chain and T cell receptor loci exclusively present in lymphoid blast crisis CML. These deletions are accompanied in a sub-set of cases by concomitant genome loss of regions within chromosomes 7 and 9, including the IKZF1 and CDKN2A genes, which appear to be associated with variety of structural chromosome changes - from cryptic interstitial deletions to unbalanced rearrangements resulting in short arm or whole chromosome loss.

The fluorescence ratio (FR) standard deviation (SD) for each of the 92 samples analysed with the 105 K array, ranged from 0.057 - 0.187 with a median value of 0.092. In a preliminary analysis of recurrent events, we detected a set of 1,262 loci in the BC cohort with a FR outside a threshold of ±4 SD that were seen in two adjacent probes in at least 3 samples. 410 loci corresponded to known genes after correction for copy number variations (CNV) identified by Redon et al [15]. These genes were found to be associated with Focal adhesion, MAPK signalling and Cell cycle control and their distribution across the genome is shown in Additional file 1: Figure S1.

(i) Significance Analysis of Microarrays (SAM) identifies concomitant genome losses associated with lymphoid blast transformation

We chose SAM to carry out a 2-class analysis based on myeloid and lymphoid BC CML for 74 samples - 60 CML and 14 control samples (Additional file 2: Table S1). Altogether 435 loci were found with a FDR = 0. We plotted the "expected" versus "actual" values of delta, the metric that quantifies the difference between the two classes and selected for further detailed consideration the top 22 that displayed an enhanced value of delta (Additional file 3: Figure S2). The heat map in Figure 1 illustrates the results of a hierarchical cluster analysis of these probes. The complete set of the 435 significant probes with reference to location, genome assignment and scores are given in the supplementary data (Additional file 4: Table S2). Among the known genes covered by these genome loci are IKZF1, HOXA7, CDKN2A/2B, MLLT3, IFNA/B, RNF38, PAX5, JMJD2C and PDCD1LG2.

The heat map (Figure 1) shows a dramatic demarcation between lymphoid/mixed lineage and the remaining samples where the green coloured columns indicate the consistent genome loss. The top 22 most significant loci are restricted to four chromosome regions, namely 7p12-14, 9p21-24, 14q11.2 and 14q32.33. Their deletion status in the 10 BCL and 2 BCmix samples is shown in Figure 2, where the probes are grouped by their genomic location. None of these probes are consistently deleted in any of the other samples studied. The probes fall into two main groups. The primary group members, highlighted in colour in Figure 2, are nearly always deleted. These include probes covering the regions of IGH, TCR and IKZF1 genes. The gains and losses for all cases in the IGH region are summarised in Figure 3. The locus at chr14:105.41 shows a 100% loss unique to the BCL class (in blue) whereas the loci at chr14:105.61 and chr14:105.62 display either gains or losses typical of CNVs. A region mapping to parts of the T-cell receptor gamma chain complex region (TCR G) extending from chr7:38.27 to chr7:38.32 on chromosome 7p14.1 is also frequently deleted (Figure 4). There are five array probes in this region, three of which are deleted in 11/12 samples (Figure 2). There are three probes in the 105 K arrays covering the IKZF1 (Ikaros) gene, all of which are deleted in 9/12 samples (Figure 2). The remaining probes make up the second group, which represents the full concomitant loss of loci within the 7p12-14, 9p13-24.1 and 14q11.2 regions shared by 7 of the 12 BCL samples (Figure 1 and Figure 2).

(ii) Verification of the 105 K array results

Both FISH and aCGH on high-resolution custom oligonucleotide arrays were used to verify of the 105 K array results. FISH analysis of the TCR and IGH data was considered inappropriate due to the small size of the loci involved (2 - 50 k). All FISH tests for deletions of IKZF1, CDKN2A, MLLT3, PAX5, JMJD2C and PDCD1LG2 on cases where chromosome preparation were available were positive. Furthermore, we identified complex clonal heterogeneity as summarized in Figure 5. We applied aCGH at ~1 k resolution to the TCR, IGH and IKZF1 loci on 40 samples, 10 from each group of BCL, BCM, CP and controls (Figure 6 and Additional File 5: Figure S3 and Additional file 6: Figure S4). The copy number aberrations (CNA) identified by the 105 K arrays were confirmed and in particular, the deletions of TCR and IGH were shown to be remarkably consistent. These deletions along with the IKZF1 loss were confirmed to be exclusively present in the BCL samples as indicated by the whole genome study. Furthermore, in case 163, a deletion of 119 Kb covering only exon 3 of the IKZF1 region was revealed thus increasing the involvement of this gene to 10/12 BCL samples (Figure 6).

A comparison of the location of the IGH probes found as significant for the BCL samples with their corresponding VDJ segments within the IGH@ locus (IMGT database) is shown in Additional file 7: Figure S5. The probe at chr14:105.41 is located between the IGH J1P and IGH D1-26 loci, while the chr14:105.48 probe - maps between IGH V4-4 and IGH V(II)-1-1. This suggests that the observed deletions in the BCL samples are likely to result from an early VD joining that precedes any rearrangements the IGH@ region (Additional file 7: Figure S5). Likewise, the probes found deleted within the TCRG were located at the V and J segments and could reflect a recurrent rearrangement. There are three probes in the 105 K array covering the TCR A/D region and they actually map within the TCR D sequences as follows: probe chr14:21.98 is located between TRDD2 and TRDD3, chr14:21.99 is located between TRDJ1 and TRDJ4 and chr14:22.00 is located between TRDC and TRDV3 (Additional file 8: Figure S6).

(iv) Evolution of the concomitant genome loss in BCL

FISH analysis was employed to address the discrepancies between the G banding and CGH data with main emphasis on chromosomes 7 and 9. The FISH results obtained in five cases, for which chromosome preparations were available, are summarized in Figure 5. Genome losses were confirmed in all cases studied as illustrated by the results for case 164 (Figure 7). Multiple Ph positive cell clones differing in the scope and composition of the 7p/9p regional loss were identified in 4 of the 5 cases studied ranging in complexity (Additional file 10: Figure S7). Therefore it is unlikely that the concomitant deletions in these cases result from a single event. Instead, a spectrum of consecutive aberrations probably leads to the formation of the concomitant loss.

Although the frequency and size of the copy number aberrations varied greatly, samples from both accelerated and blast stage CML showed substantially larger numbers of genome imbalances than chronic phase. These aberration frequencies obtained at a resolution of 33 K by oligonucleotide aCGH are broadly in agreement with earlier investigations of CML samples using BAC [10, 11] and SNP array CGH [14].

Altogether 435 loci were identified as significantly different in lymphoid and myeloid BC samples. All loci were located on the short arms of chromosomes 7 & 9, with the exception of 4 from chromosomes 14, 2 from chromosome 2 and a single chromosome 22 probe. Most of the probes mapped to known genes and a number were in regions associated with immune response. We show that deletion of certain regions within TCR (alpha/delta and gamma) (11/12) and IGH (12/12), are almost universal while being accompanied by deletions within IKZF1 (10/12) and, to a lesser extent, CDKN2A and sequences within several other genes in the short arm regions of chromosomes 7 and 9 (7/12). These genome losses were not seen in any of 31 CP, 6 accelerated and 12 BCM samples or controls. Furthermore, they were present in a BCL sample (Case 199) when DNA from the patient's CP was used as reference for the aCGH analysis, thus proving their secondary origin.

A recent genome wide single nucleotide polymorphism (SNP) array analysis of Ph positive samples - 43 ALL (paediatrics and adults) and 23 CML (11 CP, 9 BCM and 3 BCL) [14] - revealed the presence of three common deletions affecting the IKZF1, PAX5 and CDKN2A loci. These deletions, confirmed by FISH and qPCR, also detected in 2 out of 3 CML BCL samples, were not always co-existent. How the reduced activity of IKZF1 and PAX5 genes, that play a key role in regulating B lineage commitment, collaborates with BCR/ABL1 to induce Ph(+) ALL remains unclear [14, 16]. The frequency of the genome loss described by Mullighan et al.,[14] in the Ph(+) ALL, is in overall agreement with our findings in CML lymphoid blast crisis. In particular, CDKN2A deletions are 53.5% in Ph(+)ALL . 58.3% in CML/BCL and PAX5 loss is seen in 51.% of Ph(+)ALL vs. 58.3% in CML/BCL, while deletions of IKZNF1 are seen in 20 of 22 adults with Ph(+)ALL (90.9%) [14] and our aCGH analysis at 1 K resolution detected loss in 10/12 CML/BCL samples (83.3%). In contrast with the Ph(+) ALL results, we found that the loss of IKZF1 always precedes the deletions of CDKN2A and that losses of RNF38 were always accompanied by loss of the 9p13-p23.1 region that houses, among others, CDKN2A, IFNA, RNF38, PAX5 and MLLT3. In addition, we demonstrated by FISH the presence of clonal evolution leading to the concomitant 7p/9p loss as seen in less than half of patients (5/12 samples) (see typical aCGH profiles in Additional file 10: Figure S7). Importantly, the cell clone bearing the concomitant loss in the studied cases was dominant and hence, detectable by aCGH.

No imbalances of IGH and/or TCR loci were reported in Ph(+) ALL study by Mullighan et al [14] either alone or in combination with IKZF1, CDKN2A or PAX5. Substantial parts of the IGH and TCR regions are known to show copy number variations (CNVs) in disease free individuals http://projects.tcag.ca/variation, but whereas CNVs are characterised by amplifications and deletions of a specific locus within the sample population, we found a set of probes in the IGH and TCR regions to be universally deleted in exclusively lymphoid BC samples, thus showing features typical of secondary genomic aberrations. That they are not somatic aberrations is illustrated by Case 199, where identical features emerged when the BCL sample was hybridised against patient's DNA from the CP. These deletions characteristic of lymphoid BC were also identified in the 2 samples with mixed immunophenotype (Figure 1).

There are two possible explanations for the origin of these deletions. Either they represent genome losses associated with cross lineage rearrangements known to occur in both ALL and AML, or they represent a bona fide event with oncogenic potential. IGH/TCR rearrangement patterns are widely used to monitor minimal residual disease (MDR) in a range of haematological malignancies, the levels of which significantly correlate with clinical outcome [17]. The immunophenotype data for five of our 12 CML/BC samples that carry concomitant 7p/9p loss (cases 184, 192, 193, 195 & 200, Additional file 12: Table S4) are consistent with early B cell origin of the blast cells (TdT+, CD10+, CD19+, CD22+, CD7 neg, so it could be that the recurrent loss of the TCR G/D region in B cells with legitimate IGH rearrangements represents the lineage infidelity as reported previously in blast phase CML [18–21]. However, the similarity of the deletions within both immunoglobulin genes in BCL does not comply with the notion that IGH and TCR configurations are unique (clonal by origin and patient specific). The common deleted regions could indicate a preferential VDJ rearrangement in the clone that ultimately becomes dominant in the BCL patients. Such preferential VDJ usage has been reported before with a preference for the V_H4 family segment in about 35% of children with B-ALL [22]. In our case the deletions in IGH and TCR loci possibly reflect the clonal origin of the malignancy and the stage at which the additional chromosomal aberrations that we observed were initiated. Further work needs to be done to relate these possibilities to the waterfall of changes that characterise blast crisis.

So, how does the loss of a relatively small ~100 Kb region from IGH and TCR contribute to the development of the lymphoid blast transformation? RAG1 and RAG2 (RAG) are the key components of the V(D)J recombinase machinery that catalyses the somatic gene rearrangements of antigen receptor genes during lymphocyte development. B cell progenitors undergo a development program involving ordered IGH gene rearrangements conducted by the RAG enzyme system. Recent evidence suggests that BCR/ABL1 induces expression of activation-induced cytidine deaminase (AID), found exclusively in early B cells. It participates in the class switch recombination and has been shown to lead to single strand breaks in IGH regions in Ph(+)ALL [23]. Therefore, it would appear that the deregulation of AID silencing by transcriptional or epigenetic factors is the key event in compromising the V(D)J recombination/RAG impairment. This could lead to the TCR rearrangements in B cells, which have already undergone IGH rearrangements and are expressing the CD10, CD19 and CD22 antigens. Thus genome losses identified in the BCL samples represent the consequence of cross lineage rearrangement i.e. DJ joining occurring in early B cells and aberrant incomplete recombination in the TCR complex.

This view is supported by Mullighan et al [14] who showed that the mechanism responsible for IKZF1 deletions in Ph(+) ALL appears to involve aberrant RAG mediated recombination as heptamer signal sequences were found internal to the deletion breakpoints. Similarly, CDKN2A loss was linked to RAG activity in lymphoid leukemia [24, 25]. Furthermore, the impairment of the RAG system in Ph positive cells could be a direct consequence of the genetic damage caused by BCR/ABL1 [5, 23] either through inflicting direct damage via reactive oxygen species or by compromised DNA repair [4, 7]. It is also possible that the reduced/abolished expression of c-ABL [26] could further contribute to the impaired V(D)J recombination [27]. In summary, a chain of events initiated by BCR/ABL1 impairs the RAG enzyme system. The compromised V(D)J recombinase machinery is thus instrumental in creating, by means of cross lineage rearrangements, clonal populations of B-cell progenitors carrying IGH and TCR deletions.

We have observed a signature of deletions that is uniquely present in our lymphoid samples. The top 22 most significant loci are restricted to four chromosome regions, namely 7p12-14, 9p21-24, 14q11.2 and 14q32.33. Their common appearance points at an event that is orchestrating the cascade of aberrations leading to the lymphoid BC. Given the high frequency of IGH and TCRG loci deletions we speculate that the evolution into lymphoid blast crisis is initiated by abnormal RAG mediated NHEJ. It appears we are edging nearer to identifying the key aberrations responsible for blast transformation at least in a proportion of the CML patients.