To elucidate whether tyrosine kinase inhibitor (TKI) resistance in chronic myeloid leukemia is associated with characteristic genomic alterations, we analyzed DNA samples from 45 TKI-resistant chronic myeloid leukemia patients with 250K single nucleotide polymorphism arrays. From 20 patients, matched serial samples of pretreatment and TKI resistance time points were available. Eleven of the 45 TKI-resistant patients had mutations of BCR-ABL1, including 2 T315I mutations. Besides known TKI resistance-associated genomic lesions, such as duplication of the BCR-ABL1 gene (n = 8) and trisomy 8 (n = 3), recurrent submicroscopic alterations, including acquired uniparental disomy, were detectable on chromosomes 1, 8, 9, 17, 19, and 22. On chromosome 22, newly acquired and recurrent deletions of the IGLC1 locus were detected in 3 patients, who had previously presented with lymphoid or myeloid blast crisis. This may support a hypothesis of TKI-induced selection of subclones differentiating into immature B-cell progenitors as a mechanism of disease progression and evasion of TKI sensitivity.

Chronic myeloid leukemia (CML) patients can develop secondary resistance in the course of treatment with tyrosine kinase inhibitors (TKIs), such as imatinib, nilotinib, or dasatinib.1,2  The main known causes for resistance are mutations or overexpression of the BCR-ABL1 fusion protein, reduced bioavailability of the drugs, and activation of compensatory molecular pathways.2,3  CML cells exhibit increased genomic instability, which could lead to genomic lesions harboring additional mechanisms of resistance. Use of high-density single nucleotide polymorphism (SNP) arrays in combination with a new computational algorithm termed “molecular allelokaryotyping” allows robust and detailed detection of cryptic micro-deletions, micro-amplifications, and loss of heterozygosity (LOH), including acquired uniparental disomy.4,5  We performed a genomic DNA profiling of 45 TKI-resistant CML patients with 250K SNP arrays to elucidate genomic alterations, which could be associated with TKI resistance.

Patients and DNA samples

We studied 45 cases of TKI-resistant CML. From 25 patients, DNA was extracted upon development of clinical resistance to imatinib, dasatinib, or nilotinib. From 20 patients, sequential DNA samples were available for comparison between diagnosis and development of resistance against at least 1 of the aforementioned TKIs. All patients were confirmed positive for BCR-ABL1 by polymerase chain reaction (PCR) and fluorescence in situ hybridization. Eleven resistant patient samples had BCR-ABL1 mutations, including 2 T315I mutations. The anonymized DNA samples were obtained from patients referred to the Munich Leukemia Laboratory, Munich, Germany. Genomic DNA was isolated from mononuclear cells from bone marrow aspirates or leukemic peripheral blood. The acquisition and analysis of DNA samples from the patients were with approval of the ethical committee of all participating institutions. A detailed summary of patient information is given in supplemental Table 1 (available on the Blood website; see the Supplemental Materials link at the top of the online article).

High-density SNP array analysis

High-quality genomic DNA was processed according to the genomic mapping 250K NspI protocol and hybridized to 250K NspI SNP arrays according to the manufacturer's instructions (Affymetrix). Data analysis of deletions, amplifications, and uniparental disomy was carried out using the CNAG software with nonmatched references as previously described.5,6  The SNP array data are publicly available at the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/projects/geo/) under accession number GSE18964.

Mutation analysis and validation of SNP array results

Mutation analysis of candidate genes was carried out by standard genomic PCR of all exons and subsequent direct sequencing of the amplified and purified PCR products with the BigDye Terminator, Version 3.1 Cycle Sequencing Kit (Applied Biosystems). Validation of copy number results and uniparental disomy (UPD) were carried out as previously described,7  and results are shown in supplemental Figures 1 and 2.

After exclusion of genomic copy number polymorphisms by comparison of the data with recorded copy number polymorphisms in the UCSC Genome Browser (http://genome.ucsc.edu/) databases, a total of 36 deletions (Table 1), 29 duplications (Table 2), and 9 regions of copy number neutral LOH (Table 3) were identified by SNP array analysis.

Table 1

Deletions found in the TKI-resistant CML samples

IDChromosomeStarting positionEnding positionLength, MbCandidate genesTreatmentDisease status
1p36 8206962 9061724 0.85 RERE, ENO-1 Imatinib, DA, nilotinib, dasatinib Myeloid blast crisis 
40-R* 2p14 67344057 67586748 0.24 ETAA1 Imatinib Myeloid blast crisis 
40-R* 2p14 67726052 67862441 0.13 No gene Imatinib Myeloid blast crisis 
40-R* 3q26 182773194 183414855 0.64 SOX2 Imatinib Myeloid blast crisis 
45-R* 3q13 107395968 113108377 5.71  Imatinib Unknown 
34 6p22 21716958 22106421 0.39 FLJ22536 HU, ICT, imatinib, dasatinib Lymphoid blast crisis 
45-R* 6p12 47456409 54815576 7.35  Imatinib Unknown 
40-R* 8p 180568 28696690 28.51 INTS9 Imatinib Myeloid blast crisis 
45-R 8q12 56513192 56568360 0.05 SBF1B1, homo del Imatinib Unknown 
34 9p 30910 130700428 130.70 Del der Chromosome 9 HU, ICT, imatinib, dasatinib Lymphoid blast crisis 
12 9q34 128779763 130659964 1.88 ABL1-PHYHD1 Imatinib Unknown 
32-R 9q34 128779763 130643119 1.86 ABL1-PHYHD1 Imatinib CML in hematologic remission 
41-R 9q34 128779763 130760738 1.98 ABL1-PHYHD1 Imatinib Unknown 
44-R 9q34 129888844 130775671 0.88 ABL1-GPR107 Imatinib CML in hematologic remission 
43-R 9q34 130889873 131275779 0.38 LAMC3, AIF1L, NUP214 and more Imatinib Unknown 
40-R8 9q33 117671985 119985986 2.31 DBC1 Imatinib Myeloid blast crisis 
33-R 10q21 67879503 67935343 0.06 CTNNA3 DA, imatinib Myeloid blast crisis 
45-R* 11q 89538451 92629460 3.09  Imatinib Unknown 
34 12p12 14971302 17224159 2.25 ERP27, RERG, PTPRO + other genes HU, ICT, imatinib, dasatinib Lymphoid blast crisis 
34 12p12 25334604 25429100 0.09 No gene HU, ICT, imatinib, dasatinib Lymphoid blast crisis 
40-R* 12p11 28549306 28659885 0.11 CCDC91 Imatinib Myeloid blast crisis 
35 14q31 85977359 86198515 0.22 No gene HU + IFN, imatinib CML 
34 15q15 38796960 41889601 3.09 RAD51-MFAP1 HU, ICT, imatinib, dasatinib Lymphoid blast crisis 
40-R* 16q 45065445 88690776 43.62  Imatinib Myeloid blast crisis 
27 17p 18901 22029237 22.01 p53 Imatinib Unknown 
40-R* 17p 18901 21346948 21.32 p53 Imatinib Myeloid blast crisis 
46-R* 17p 18901 18857962 18.83 p53 Unknown CML 
34 17q12 32663038 33785091 1.12 ACACA-SOCS7 HU, ICT, imatinib, dasatinib Lymphoid blast crisis 
22q11 20724157 20832986 0.11 IGLC1 Imatinib, DA, nilotinib, dasatinib Myeloid blast crisis 
22-R* 22q11 20724157 20832986 0.11 IGLC1 Imatinib Lymphoid blast crisis 
34 22q11 20685204 20859240 0.17 IGLC1 HU, ICT, imatinib, dasatinib Lymphoid blast crisis 
12 22q11 21970272 23413582 1.44 BCR-GGT1 Imatinib Unknown 
41-R 22q11 21947838 23157101 1.20 BCR, ADORA2A Imatinib Unknown 
44-R 22q11 21939318 27992558 6.05 BCR, CHEK2 Imatinib CML in hematologic remission 
32-R 22q11 22353181 23209777 0.79 VPREB3 Imatinib CML in hematologic remission 
43-R 22q11 22860184 23556124 0.69 CABIN1-PIWIL3 Imatinib Unknown 
IDChromosomeStarting positionEnding positionLength, MbCandidate genesTreatmentDisease status
1p36 8206962 9061724 0.85 RERE, ENO-1 Imatinib, DA, nilotinib, dasatinib Myeloid blast crisis 
40-R* 2p14 67344057 67586748 0.24 ETAA1 Imatinib Myeloid blast crisis 
40-R* 2p14 67726052 67862441 0.13 No gene Imatinib Myeloid blast crisis 
40-R* 3q26 182773194 183414855 0.64 SOX2 Imatinib Myeloid blast crisis 
45-R* 3q13 107395968 113108377 5.71  Imatinib Unknown 
34 6p22 21716958 22106421 0.39 FLJ22536 HU, ICT, imatinib, dasatinib Lymphoid blast crisis 
45-R* 6p12 47456409 54815576 7.35  Imatinib Unknown 
40-R* 8p 180568 28696690 28.51 INTS9 Imatinib Myeloid blast crisis 
45-R 8q12 56513192 56568360 0.05 SBF1B1, homo del Imatinib Unknown 
34 9p 30910 130700428 130.70 Del der Chromosome 9 HU, ICT, imatinib, dasatinib Lymphoid blast crisis 
12 9q34 128779763 130659964 1.88 ABL1-PHYHD1 Imatinib Unknown 
32-R 9q34 128779763 130643119 1.86 ABL1-PHYHD1 Imatinib CML in hematologic remission 
41-R 9q34 128779763 130760738 1.98 ABL1-PHYHD1 Imatinib Unknown 
44-R 9q34 129888844 130775671 0.88 ABL1-GPR107 Imatinib CML in hematologic remission 
43-R 9q34 130889873 131275779 0.38 LAMC3, AIF1L, NUP214 and more Imatinib Unknown 
40-R8 9q33 117671985 119985986 2.31 DBC1 Imatinib Myeloid blast crisis 
33-R 10q21 67879503 67935343 0.06 CTNNA3 DA, imatinib Myeloid blast crisis 
45-R* 11q 89538451 92629460 3.09  Imatinib Unknown 
34 12p12 14971302 17224159 2.25 ERP27, RERG, PTPRO + other genes HU, ICT, imatinib, dasatinib Lymphoid blast crisis 
34 12p12 25334604 25429100 0.09 No gene HU, ICT, imatinib, dasatinib Lymphoid blast crisis 
40-R* 12p11 28549306 28659885 0.11 CCDC91 Imatinib Myeloid blast crisis 
35 14q31 85977359 86198515 0.22 No gene HU + IFN, imatinib CML 
34 15q15 38796960 41889601 3.09 RAD51-MFAP1 HU, ICT, imatinib, dasatinib Lymphoid blast crisis 
40-R* 16q 45065445 88690776 43.62  Imatinib Myeloid blast crisis 
27 17p 18901 22029237 22.01 p53 Imatinib Unknown 
40-R* 17p 18901 21346948 21.32 p53 Imatinib Myeloid blast crisis 
46-R* 17p 18901 18857962 18.83 p53 Unknown CML 
34 17q12 32663038 33785091 1.12 ACACA-SOCS7 HU, ICT, imatinib, dasatinib Lymphoid blast crisis 
22q11 20724157 20832986 0.11 IGLC1 Imatinib, DA, nilotinib, dasatinib Myeloid blast crisis 
22-R* 22q11 20724157 20832986 0.11 IGLC1 Imatinib Lymphoid blast crisis 
34 22q11 20685204 20859240 0.17 IGLC1 HU, ICT, imatinib, dasatinib Lymphoid blast crisis 
12 22q11 21970272 23413582 1.44 BCR-GGT1 Imatinib Unknown 
41-R 22q11 21947838 23157101 1.20 BCR, ADORA2A Imatinib Unknown 
44-R 22q11 21939318 27992558 6.05 BCR, CHEK2 Imatinib CML in hematologic remission 
32-R 22q11 22353181 23209777 0.79 VPREB3 Imatinib CML in hematologic remission 
43-R 22q11 22860184 23556124 0.69 CABIN1-PIWIL3 Imatinib Unknown 

Copy number alterations (CNAs) in TKI-resistant samples without a matched diagnostic sample are shown with plain numbers in the column “ID.” CNAs in TKI-resistant samples with a matched diagnostic sample with the CNAs being present in both the diagnostic sample and the resistant sample are shown with numbers + “R” in the column “ID.” CNAs which were newly acquired in matched resistant samples compared to the diagnostic samples are shown with numbers + “R*” in the column “ID.”

ID indicates patient ID; Starting position, start position of genomic lesion; Ending position, end position of genomic lesion; HU, hydroxyurea; DA, daunorubicin plus cytarabine; ICT, induction chemotherapy; and IFN, interferon-α.

Table 2

Duplications present in TKI-resistant CML samples

IDChromosomeStarting positionEnding positionLength, MbCandidate genesTreatmentDisease status
27 2p21 42500507 43164500 0.66 KCNG3, MTA3, OXER1, HAAO Imatinib Unknown 
16 2q36 227668515 227890738 0.22 RHBDD1, COL4A4, COL4A3 HU, imatinib CML 
25 2q37 231049480 231135251 0.09 SP100 HU + IFN, imatinib Unknown 
11 8p-q 180568 146263538 146.1 Trisomy 8 Imatinib CML in hematologic remission 
33-R 8p-q 180568 146263538 146.1 Trisomy 8 DA, imatinib Myeloid blast crisis 
42-R* 8p-q 180568 146263538 146.1 Trisomy 8 HU, imatinib CML in acceleration 
38-R 8q24 138371297 139210288 0.84 Hypothetical protein FLJ45872 HU, imatinib CML in hematologic remission 
45-R 8q11 53916301 146263538 92.34  Imatinib Unknown 
27 9q34 130686824 138303776 7.62 Duplication ABL1 Imatinib Unknown 
16 9q34 130686824 138303776 7.62 Duplication ABL1 HU, imatinib CML 
24 9q34 130641293 138303776 7.66 Duplication ABL1 Imatinib Unknown 
9q34 130737915 138303776 7.57 Duplication ABL1 Imatinib CML 
33-R* 9q34 130737915 138303776 7.57 Duplication ABL1 DA, imatinib Myeloid blast crisis 
40-R* 9q34 130659964 138303776 7.64 Duplication ABL1 Imatinib Myeloid blast crisis 
41-R 10p11 35,083,411 35,252,155 0.16 PARD3 Imatinib Unknown 
10 11q24 122000841 122217796 0.22 STS-1 / CBL interacting protein Imatinib, dasatinib, HU CML in hematologic remission 
4-R 15q21 48682473 48832856 0.15 TRPM7, SPPL2A Imatinib CML in hematologic remission 
42-R* 17q 33318471 47862514 14.54 STAT3, STAT5A/B HU, imatinib CML in acceleration 
45-R* 17q 37042201 78599918 41.55 STAT3, STAT5A/B Imatinib Unknown 
46-R* 17p-q 18857962 78599918 59.74 STAT3, STAT5A/B Unknown CML 
21-R 20p13 1658272 1861481 0.2 SIRPA Imatinib Unknown 
27 22q11 14441016 21947838 7.51 Duplication BCR Imatinib Unknown 
16 22q11 14441016 21947838 7.51 Duplication BCR HU, imatinib CML 
24 22q11 14441016 21947838 7.51 Duplication BCR Imatinib Unknown 
22q11 14441016 21947838 7.51 Duplication BCR Imatinib CML 
33-R* 22q11 14441016 21947838 7.51 Duplication BCR DA, imatinib Myeloid blast crisis 
40-R* 22q11 14441016 21960478 7.51 Duplication BCR Imatinib Myeloid blast crisis 
34 22q11 14441016 21947838 7.51 Duplication BCR HU, ICT, imatinib, dasatinib Lymphoid blast crisis 
33-R* 154824264 154 Trisomy X DA, imatinib Myeloid blast crisis 
IDChromosomeStarting positionEnding positionLength, MbCandidate genesTreatmentDisease status
27 2p21 42500507 43164500 0.66 KCNG3, MTA3, OXER1, HAAO Imatinib Unknown 
16 2q36 227668515 227890738 0.22 RHBDD1, COL4A4, COL4A3 HU, imatinib CML 
25 2q37 231049480 231135251 0.09 SP100 HU + IFN, imatinib Unknown 
11 8p-q 180568 146263538 146.1 Trisomy 8 Imatinib CML in hematologic remission 
33-R 8p-q 180568 146263538 146.1 Trisomy 8 DA, imatinib Myeloid blast crisis 
42-R* 8p-q 180568 146263538 146.1 Trisomy 8 HU, imatinib CML in acceleration 
38-R 8q24 138371297 139210288 0.84 Hypothetical protein FLJ45872 HU, imatinib CML in hematologic remission 
45-R 8q11 53916301 146263538 92.34  Imatinib Unknown 
27 9q34 130686824 138303776 7.62 Duplication ABL1 Imatinib Unknown 
16 9q34 130686824 138303776 7.62 Duplication ABL1 HU, imatinib CML 
24 9q34 130641293 138303776 7.66 Duplication ABL1 Imatinib Unknown 
9q34 130737915 138303776 7.57 Duplication ABL1 Imatinib CML 
33-R* 9q34 130737915 138303776 7.57 Duplication ABL1 DA, imatinib Myeloid blast crisis 
40-R* 9q34 130659964 138303776 7.64 Duplication ABL1 Imatinib Myeloid blast crisis 
41-R 10p11 35,083,411 35,252,155 0.16 PARD3 Imatinib Unknown 
10 11q24 122000841 122217796 0.22 STS-1 / CBL interacting protein Imatinib, dasatinib, HU CML in hematologic remission 
4-R 15q21 48682473 48832856 0.15 TRPM7, SPPL2A Imatinib CML in hematologic remission 
42-R* 17q 33318471 47862514 14.54 STAT3, STAT5A/B HU, imatinib CML in acceleration 
45-R* 17q 37042201 78599918 41.55 STAT3, STAT5A/B Imatinib Unknown 
46-R* 17p-q 18857962 78599918 59.74 STAT3, STAT5A/B Unknown CML 
21-R 20p13 1658272 1861481 0.2 SIRPA Imatinib Unknown 
27 22q11 14441016 21947838 7.51 Duplication BCR Imatinib Unknown 
16 22q11 14441016 21947838 7.51 Duplication BCR HU, imatinib CML 
24 22q11 14441016 21947838 7.51 Duplication BCR Imatinib Unknown 
22q11 14441016 21947838 7.51 Duplication BCR Imatinib CML 
33-R* 22q11 14441016 21947838 7.51 Duplication BCR DA, imatinib Myeloid blast crisis 
40-R* 22q11 14441016 21960478 7.51 Duplication BCR Imatinib Myeloid blast crisis 
34 22q11 14441016 21947838 7.51 Duplication BCR HU, ICT, imatinib, dasatinib Lymphoid blast crisis 
33-R* 154824264 154 Trisomy X DA, imatinib Myeloid blast crisis 

Copy number alterations (CNAs) in TKI-resistant samples without a matched diagnostic sample are shown with plain numbers in the column “ID.” CNAs in TKI-resistant samples with a matched diagnostic sample with the CNAs being present in both the diagnostic sample and the resistant sample are shown with numbers + “R” in the column “ID.” CNAs which were newly acquired in matched resistant samples compared to the diagnostic samples are shown with numbers + “R*” in the column “ID.”

ID indicates patient ID; Starting position, start position of genomic lesion; Ending position, end position of genomic lesion; HU, hydroxyurea; DA, daunorubicin plus cytarabine; ICT, induction chemotherapy; and IFN, interferon-α.

Table 3

Copy number neutral LOH detected in resistant CML samples

IDChromosomeStarting positionEnding positionLength, MbCandidate genesTreatmentDisease status
41-R 1p36 825,852 18193698 17.36 RERE, ENO-1 Imatinib Unknown 
43-R 1p36 18,323,554 52881039 34.55 RERE, ENO-1 Imatinib Unknown 
2q 182983286 242712341 59.73  Imatinib CML 
31 3q 123593611 136239458 12.65  HU, imatinib CML in hematologic remission 
36-R 5p 29256280 35121357 5.87  HU, imatinib CML in hematologic remission 
41-R 6q 109232515 124242464 15.01 FYN Imatinib Unknown 
36-R 6q 139970070 153644361 13.67  HU, imatinib CML in hematologic remission 
41-R 11p15 8895510 19285890 10.39  Imatinib Unknown 
42-R* 17q 23581904 78599918 55.01 STAT3, STAT5A/B HU, imatinib CML in acceleration 
27 19q13 58448005 63731511 5.28 BIRC8, AURKC Imatinib Unknown 
10 19q13 38496239 63700378 25.2 BIRC8, AURKC Imatinib, dasatinib, HU CML in hematologic remission 
IDChromosomeStarting positionEnding positionLength, MbCandidate genesTreatmentDisease status
41-R 1p36 825,852 18193698 17.36 RERE, ENO-1 Imatinib Unknown 
43-R 1p36 18,323,554 52881039 34.55 RERE, ENO-1 Imatinib Unknown 
2q 182983286 242712341 59.73  Imatinib CML 
31 3q 123593611 136239458 12.65  HU, imatinib CML in hematologic remission 
36-R 5p 29256280 35121357 5.87  HU, imatinib CML in hematologic remission 
41-R 6q 109232515 124242464 15.01 FYN Imatinib Unknown 
36-R 6q 139970070 153644361 13.67  HU, imatinib CML in hematologic remission 
41-R 11p15 8895510 19285890 10.39  Imatinib Unknown 
42-R* 17q 23581904 78599918 55.01 STAT3, STAT5A/B HU, imatinib CML in acceleration 
27 19q13 58448005 63731511 5.28 BIRC8, AURKC Imatinib Unknown 
10 19q13 38496239 63700378 25.2 BIRC8, AURKC Imatinib, dasatinib, HU CML in hematologic remission 

Copy number alterations (CNAs) in TKI-resistant samples without a matched diagnostic sample are shown with plain numbers in the column “ID.” CNAs in TKI-resistant samples with a matched diagnostic sample with the CNAs being present in both the diagnostic sample and the resistant sample are shown with numbers + “R” in the column “ID.” CNAs which were newly acquired in matched resistant samples compared to the diagnostic samples are shown with numbers + “R*” in the column “ID.”

ID indicates patient ID; Starting position, start position of genomic lesion; Ending position, end position of genomic lesion; HU, hydroxyurea; DA, daunorubicin plus cytarabine; ICT, induction chemotherapy; and IFN, interferon-α.

Recurrent lesions were detected on chromosomes 1, 8, 9, 17, 19, and 22. On chromosome 1p36, 1 resistant sample displayed a submicroscopic deletion (sample 2) and 2 serial samples from other persons showed a common region of UPD (samples 41, 41-R and 43 and 43-R; Tables 1,3; supplemental Figure 3). These lesions of UPD were present in both the diagnostic sample and the TKI-resistant sample. Therefore, they are probably not associated with TKI resistance but confirm previous reports of frequent LOH in this region in CML,8  which may play a role in the initial development of CML. In search for possible mutations, we sequenced the candidate genes arginine-glutamic acid dipeptide (RE) repeats (RERE) and enolase 1 (ENO-1), which were contained in the microdeletion in sample 2. However, apart from a 12-bp insertion in exon 20 of the RERE gene (supplemental Figure 3), which probably can be interpreted as a rare polymorphism or genomic abnormality, we found no gene disrupting mutations in the remaining alleles of these genes.

Chromosome 17 was most heavily affected by secondary genomic alterations on development of TKI resistance. Four of the 20 serial samples showed newly acquired genomic alterations on chromosome 17. Changes composed either large deletions of chromosome 17p or large duplications or UPD of chromosome 17q (Tables 1,Table 23; supplemental Figure 4). Genomic disruptions occurring on chromosome 17 are one of the most common known changes arising during disease progression.9  In some cases, deletions of chromosome 17p have been found to contain inactivating mutations of p53.10  Of note, in samples 42-R and 45-R, the breakpoints leading to duplication of chromosome 17q lie in close proximity to the STAT3 and STAT5A/B genes.

On chromosome 19q, 2 patients displayed a common region of acquired UPD (19q13.32-19q13.43; Table 3).

On chromosomes 9 and 22, deletions flanking the ABL1 and BCR genes were found in 5 patients. These are deletions of the reciprocal ABL1-BCR fusion product, which are known to occur in up to 10% to 17% of CML cases and have an effect on prognosis of patients treated with either hydroxyurea or interferon-α11  but not with imatinib.12  These deletions have been characterized with conventional cytogenetic methods13  showing that the size of the deletions affects prognosis. However, a tumor suppressor gene has not been identified. In 3 of our 5 patients, the deletions on chromosome 9 spanned a common 1.9-Mb region centromeric to ABL1. One of the genes in the vicinity to the common breakpoint in this deleted region was protein phosphatase 2A activator, regulatory subunit 4 (PPP2R4), an activator of protein phosphatase 2A (PP2A), which was recently shown to be suppressed in imatinib-resistant CML.14 PPP2R4 therefore appeared as a candidate tumor suppressor gene, and we sequenced all exons of this gene in the patients with 9q deletions. However, no alteration from the reference sequence was detected in the remaining allele. On chromosome 22q11, the boundaries of the reciprocal deletions were heterogeneous; and in 2 cases, they began clearly telomeric to the BCR gene (Table 1). These SNP array results can be explained by duplication and insertion of the BCR-ABL1 fusion gene in a situation of a deleted reciprocal ABL1-BCR fusion product as evidenced by cytogenetic analysis (supplemental Table 1). Because the deletions of the reciprocal ABL1-BCR fusions are already detectable in the diagnostic samples, they are probably not associated with secondary resistance against TKIs.

Another accumulation of common lesions detectable on chromosome 22q11 were heterozygous deletions in the immunoglobulin lambda constant 1 (IGLC1) locus. These deletions were detectable in 2 resistant samples without a paired diagnostic sample and in 1 serial resistant sample on development of TKI resistance (Table 1; supplemental Figure 5). Therefore, the acquisition of these deletions is probably associated with TKI resistance or disease progression. Deletions of the IGLC1 locus are occasionally observed in B-cell acute lymphoblastic leukemia,15  resulting from λ light chain rearrangements. Prior investigators have shown that lymphoid blast crisis of CML displays a similar genomic profile to de novo Philadelphia chromosome-positive acute lymphoblastic leukemia.16  Furthermore, recent data suggested that transition from chronic phase CML to lymphoid blast crisis and drug resistance involved activation of the lymphoid transcriptional programs, such as up-regulation of the lymphoid transcription factor PAX5 and the activation-induced cytidine deaminase (AID).17  This uncovered a causative role of AID in the acquisition of BCR-ABL1 mutations and increased genomic instability in the progression of CML. Most importantly, imatinib treatment of PAX5-transduced CML cells led to the selection and outgrowth of CD19+ CML subclones, which showed evidence of de novo immunoglobulin rearrangement indicating RAG1/RAG2 activity. Therefore, finding IGLC1 deletions on development of TKI resistance in our CML samples corroborates these findings and fits well with the clinical observation of lymphoid blast crisis, and interestingly, also myeloid blast crisis with concomitant TKI resistance in these patients.

In conclusion, our high-density SNP array analysis identified new submicroscopic genomic lesions in 26 of 45 TKI-resistant CML patients. The resulting mean of 1.68 copy number alterations per TKI-resistant patient is slightly higher than in SNP array data from chronic phase CML samples demonstrated by Mullighan et al16,18  and less than the frequency of genomic lesions detected by higher-density SNP arrays carried out by Khorashad et al.19  We did not observe a new unequivocal recurrent genomic lesion associated with TKI resistance. Nevertheless, in individual cases, our data identified interesting candidate genes in the context of TKI resistance. Moreover, the observation of acquired IGLC1 deletions on TKI resistance corroborates recent findings17  of a causative role of B-lymphoid transcriptional programs in the disease progression and acquisition of resistance against TKI therapy.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

This work was supported by the Parker Hughes Fund and National Institutes of Health grants (5R01CA026038-31). D.N. was supported by a research grant from the Deutsche Forschungsgemeinschaft (DFG, NO 817/1-1). H.P.K. holds the Mark Goodson Chair in Oncology Research at Cedars-Sinai Medical Center and is a member of the Jonsson Cancer Center and the Molecular Biology Institute of University of California–Los Angeles.

National Institutes of Health

Contribution: D.N. designed research, performed SNP array analysis, and wrote the paper; S.O., M.K., and N.K. performed SNP array analysis; M. Müschen, R.P., N.H.T., and W.-K.H. analyzed data and edited the paper; A.M., V.N., H.S.K., M.-S.C., S.K., and M. Mossner performed mutation analyses; A.K., T.W., T.H., and C.H. designed research and acquired patient samples; and H.P.K. designed research, performed data analysis, and edited the paper.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Seishi Ogawa, Department of Regeneration Medicine for Hematopoiesis, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 Japan; e-mail: sogawa-tky@umin.ac.jp; and Daniel Nowak, Division of Hematology and Oncology, Cedars-Sinai Medical Center, University of California–Los Angeles School of Medicine, 8700 Beverly Blvd, Los Angeles, CA 90048; e-mail: Daniel.nowak@medma.uni-heidelberg.de.

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Author notes

*

D.N. and S.O. contributed equally to this study.

C.H. and H.P.K. contributed equally to this study.