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Vannuffel: Incyte: Consultancy. Soverini:Incyte: Consultancy.

Chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL) are, respectively, a myeloproliferative and a lymphoproliferative neoplasm that can be characterized by the chimeric fusion oncogene BCR-ABL1. Tyrosine Kinase Inhibitors (TKI) are the standard therapy for patients with CML/ALL. However, mutations of the BCR-ABL1kinase domain constitute a major cause of treatment failure in CML and ALL receiving TKI therapy. While 2nd and 3rd generation TKI have proven their efficacy against mutated BCR-ABL1-mediated clonal expansion, the presence of compound mutations can produce high level of resistance to these TKIs. Even the last addition to the TKI armamentarium, ponatinib, remains ineffective against some BCR-ABL1compound mutations (Zabriskie, M.S., et al., BCR-ABL1 Compound Mutations Combining Key Kinase Domain Positions Confer Clinical Resistance to Ponatinib in Ph Chromosome-Positive Leukemia. Cancer Cell, 2014. 26(3):p.428-442). Therefore, the distinction between compound (different mutations present on 1 unique malignant clone) and polyclonal mutations (different mutations present on 2 or more different clones) is of great clinical importance in order to select the most suitable treatment and to estimate outcomes.

Vannuffel: ARIAD Pharmaceuticals: Research Funding.

Numerous studies have shown that the presence, number and type of chromosomal aberrations represent an independent predictor of prognosis in B-cell chronic lymphocytic leukemia (CLL). Consequently, cytogenetic analysis is routinely performed in this disease. However, CLL lymphocytes have a poor mitotic index, generating only 40–50% of abnormal karyotypes. The rate of detection can be increased to 80% by interphase FISH analysis. Since some aberrations, i.e. those not involving the classical regions (13q, 12, 11q, 17p, and 6q), can escape FISH detection, there has been great interest in improved culturing methods with an immunostimulatory CpG oligonucleotide (CpG). We performed a multicentric cytogenetic study to assess the impact of 2 different culturing procedures on the detection of clonal abnormalities in 159 consecutive unselected cases with CLL referred to our centers for routine analysis from October 2007 to July 2008. Dual 72 hours cultures of bone marrow or peripheral blood were set up with the addition of either a conventional B cell mitogen (TPA) or CpG and interleukin-2 (IL2). Cytogenetic analysis was performed on both cultures. FISH analysis using a CLL probe panel analyzing 1–6 regions (13q, centromere 12, 11q, 17p, 6q and 14q32) was also applied on uncultured material, on CpG and/or on TPA culture in 146 cases. Quality of banding and proliferation capacity were assessed in 38 cases. The quality was good in 17 (CpG) and 8 (TPA), intermediate in 12 (CpG) and 18 (TPA), and poor in 9 (CpG) and 12 (TPA) cases. The mitotic index was slightly higher in CpG cultures: <15 mitoses/slide were seen in 12 (CpG) and 15 (TPA) cases, 15–20 mitoses/slide in 13 (CpG) and 14 (TPA) cases, and >20 mitoses/slide in 13 (CpG) and 9 (TPA) cases, respectively. Clonal abnormalities were identified in 89 cases (56%). In 56 cases, the aberrant clone was detected in both cultures. Of these, the percentage of aberrant metaphases was similar in both cultures in 13, higher in CpG culture in 32 and higher in TPA culture in 11 cases. In 29 and 4 additional cases, a clonal abnormality was detected only in CpG or TPA culture, respectively. Thus, the percentage of abnormal karyotypes with CpG and TPA was 53 and 38%, respectively (p=0,005). “Typical” aberrations, including del(13q), +12, del(11q), del(17p) and del(6q), were detected in 16, 26, 15, 9, and 7 (CpG) cases, and in 10, 20, 16, 8, and 3 (TPA) cases, respectively. Translocations (balanced and unbalanced) were observed in 46 (CpG) and 27 (TPA) cases, whereas aberrations involving 14q32 were seen in 5 (CpG) and 4 (TPA) cases, respectively. Interphase FISH detected del(13q), +12, del(11q), del(17p), del(6q) and del(14q) in 53, 1, 5, 0, 0 and 1 cases, respectively, in which cytogenetic analysis was either normal or abnormal but did not show the specific change. Conversely, FISH did not detect del(13q), +12, del(11q), del(17p), del(6q) and del(14q) in 1, 1, 4, 1, 2 and 0 cases in which the specific change was visible at karyotypic level. FISH was performed on both CpG and TPA cultures in 9 selected cases harbouring an aberration for which a specific commercial probe was available (+12, n=3; del(13q), n=4; del(11q), n=1; and del(17p), n=1) and showing different percentages of aberrant cells in both cultures. In 3 of these cases, fresh uncultured material was also analyzed. The highest percentage of abnormal nuclei was observed in TPA culture in all cases. In the 3 uncultured samples the percentage of aberrant nuclei was similar to the CpG culture. In conclusion, our results confirm an increased detection rate of abnormalities in CLL by using CpG/IL2 stimulation. Interphase FISH can further increase the detection rate of recurrent abnormalities. However, neither cytogenetics nor FISH detected all aberrations, demonstrating the complementary nature of these techniques and the necessity of performing both.

Numerous studies have shown that the presence, number and type of chromosomal aberrations represent an independent predictor of prognosis in B-cell chronic lymphocytic leukemia (CLL). Consequently, cytogenetic analysis is routinely performed in this disease. However, CLL lymphocytes have a poor mitotic index, generating only 40–50% of abnormal karyotypes. The rate of detection can be increased to 80% by interphase FISH analysis. Since some aberrations, i.e. those not involving the classical regions (13q, 12, 11q, 17p, and 6q), can escape FISH detection, there has been great interest in improved culturing methods with an immunostimulatory CpG oligonucleotide (CpG). We performed a multicentric cytogenetic study to assess the impact of 2 different culturing procedures on the detection of clonal abnormalities in 159 consecutive unselected cases with CLL referred to our centers for routine analysis from October 2007 to July 2008. Dual 72 hours cultures of bone marrow or peripheral blood were set up with the addition of either a conventional B cell mitogen (TPA) or CpG and interleukin-2 (IL2). Cytogenetic analysis was performed on both cultures. FISH analysis using a CLL probe panel analyzing 1–6 regions (13q, centromere 12, 11q, 17p, 6q and 14q32) was also applied on uncultured material, on CpG and/or on TPA culture in 146 cases. Quality of banding and proliferation capacity were assessed in 38 cases. The quality was good in 17 (CpG) and 8 (TPA), intermediate in 12 (CpG) and 18 (TPA), and poor in 9 (CpG) and 12 (TPA) cases. The mitotic index was slightly higher in CpG cultures: <15 mitoses/slide were seen in 12 (CpG) and 15 (TPA) cases, 15–20 mitoses/slide in 13 (CpG) and 14 (TPA) cases, and >20 mitoses/slide in 13 (CpG) and 9 (TPA) cases, respectively. Clonal abnormalities were identified in 89 cases (56%). In 56 cases, the aberrant clone was detected in both cultures. Of these, the percentage of aberrant metaphases was similar in both cultures in 13, higher in CpG culture in 32 and higher in TPA culture in 11 cases. In 29 and 4 additional cases, a clonal abnormality was detected only in CpG or TPA culture, respectively. Thus, the percentage of abnormal karyotypes with CpG and TPA was 53 and 38%, respectively (p=0,005). “Typical” aberrations, including del(13q), +12, del(11q), del(17p) and del(6q), were detected in 16, 26, 15, 9, and 7 (CpG) cases, and in 10, 20, 16, 8, and 3 (TPA) cases, respectively. Translocations (balanced and unbalanced) were observed in 46 (CpG) and 27 (TPA) cases, whereas aberrations involving 14q32 were seen in 5 (CpG) and 4 (TPA) cases, respectively. Interphase FISH detected del(13q), +12, del(11q), del(17p), del(6q) and del(14q) in 53, 1, 5, 0, 0 and 1 cases, respectively, in which cytogenetic analysis was either normal or abnormal but did not show the specific change. Conversely, FISH did not detect del(13q), +12, del(11q), del(17p), del(6q) and del(14q) in 1, 1, 4, 1, 2 and 0 cases in which the specific change was visible at karyotypic level. FISH was performed on both CpG and TPA cultures in 9 selected cases harbouring an aberration for which a specific commercial probe was available (+12, n=3; del(13q), n=4; del(11q), n=1; and del(17p), n=1) and showing different percentages of aberrant cells in both cultures. In 3 of these cases, fresh uncultured material was also analyzed. The highest percentage of abnormal nuclei was observed in TPA culture in all cases. In the 3 uncultured samples the percentage of aberrant nuclei was similar to the CpG culture. In conclusion, our results confirm an increased detection rate of abnormalities in CLL by using CpG/IL2 stimulation. Interphase FISH can further increase the detection rate of recurrent abnormalities. However, neither cytogenetics nor FISH detected all aberrations, demonstrating the complementary nature of these techniques and the necessity of performing both.