- Email: [email protected]
4.15 FISH for CLL – A More Sensitive Scoring Method
Abstracts Abstract 4.13: Survival Curves According to Risk Groups as Defined by Prognostic Score
Cumulative Survival
1.0
0.8
Low risk
0.6
Intermediate risk
0.4 High risk 0.2 p < 0.0001 Very high risk 0.0 0
12
24
36
48
60 72 84 96 108 120 132 144 156 Time (months)
cells and therefore could promote mistargeted mutations. To examine if AID function might be relevant in clinical disease, cytogenetic aberrations determined by FISH at 17p13.1 (n ⫽ 39), 11q22.3 (n ⫽ 39), 12-CEN (n ⫽ 41), 13q34 (n ⫽ 32), 11q13 (n ⫽ 22), 14q32 (n ⫽ 23), and 6q23.3 (n ⫽ 20) were counted in a subset of our initial CLL patient cohort. 13q14.3 aberrations were excluded from this count, because this sole abnormality correlates with favorable clinical course. AID⫹ CLL patients had significantly more cytogenetic aberrations (n ⫽ 21, average aberrations ⫽ 1) than AID- CLL patients (n ⫽ 22, average aberrations ⫽ 0) (P ⫽ 0.0009). These data support the notion that mistargeted AID action can promote mutations resulting in cytogenetic aberrations in CLL. While our initial CLL patient cohort (n ⫽ 126), from whom AID expression was measured by nested PCR, had clinical outcome (TFT n ⫽ 113, OS n ⫽ 112) and IGHV mutation (n ⫽ 121) data, only a fraction of patients had CD38 (n ⫽ 42), ZAP-70 (n ⫽ 32) and cytogenetic (n ⫽ 43) measurements making multivariate analyses impossible. Therefore, multivariate analyses were performed on a second cohort of patients (n ⫽ 100), from whom we determined AID expression by quantitative real-time PCR and had information on clinical outcome (TFT n ⫽ 100), IGHV mutation (n ⫽ 97), CD38 (n ⫽ 100) and ZAP-70 (n ⫽ 98) levels, and cytogenetics (n ⫽ 91). In this second group, TFT was significant for all prognostic factors by univariate analyses (AID expression – P ⫽ 0.0071, IGHV mutation – P ⫽ 0.0005, CD38 – P ⫽ 0.0236, ZAP-70 – P ⫽ 0.0022, cytogenetic aberrations excluding 13q14.3 – P ⫽ 0.0289). Using a backward selection procedure for multivariate analyses, only AID expression, ZAP-70 level and IGHV mutation status remained as independent prognostic factors. Because cytogenetic aberrations and CD38 expression drop out as independent prognostic factors, AID function may be linked to these. In conclusion, AID expression is an independent risk factor for aggressive CLL disease. As a possible explanation for this observation, we
present data consistent with AID function promoting mistargeted mutations leading to cytogenetic aberrations and poorer prognosis.
4.15 FISH for CLL – A More Sensitive Scoring Method Stephanie A. Smoley,1 Patricia T. Greipp,1 Clive S. Zent,2 Neil E. Kay,2 Daniel L. Van Dyke1 1
Cytogenetics, Mayo Clinic, Rochester, MN; 2Hematology, Mayo Clinic, Rochester, MN
Cytogenetic abnormalities are prognostically important in CLL. Mayo Clinic CLL FISH analyses are derived from direct preparations of buffy coat samples not purified or sorted for lymphocytes. Consecutive qualifying nuclei are scored for each FISH probe (deletion 6q, 11q, 13q, 17p, ⫹12 & IGH/CCND1). Based on the premise that CLL B-cells typically present as perfectly round lymphocytes, we found1 that the % abnormal nuclei in only the affected cell type of CLL patients is significantly higher than that of the general cell population. In 79 of 87 patients (91%), the % abnormal nuclei was greater in round vs unselected nuclei (mean difference 24%; range 1.5 - 57%). By univariate regression analysis, round cells (p ⫽ 0.004) had a better correlation with % B-cells by flow cytometry than either consecutive unselected nuclei (p ⫽ 0.046) or the lobed (non-round) cells (p ⫽ 0.40). We proposed that scoring consecutive round cells would improve detection of an abnormal CLL FISH pattern in patients, especially those with low B-cell counts. The next experiment was designed to test whether an abnormality found at follow-up in a patient with normal FISH at diagnosis would have been detected by scoring round nuclei only. Methods: After IRB approval, we identified 28 patients with a normal CLL FISH study at diagnosis and
Clinical Lymphoma, Myeloma & Leukemia Supplement October 2011
S227
Abstracts abnormal FISH in a follow-up study. Original specimens (slide or fixed cell pellet) were re-scored for % abnormal nuclei among 200 consecutive round nuclei. Scores were compared to the original clinical FISH analyses (200 consecutive nuclei not selected for nuclear morphology). Results: After re-scoring by the round cell approach, 12 originally normal samples remained normal while 16 (57%) were abnormal. The average number of days between the diagnostic and follow-up sample was 826 for those that remained normal and 601 for those abnormal by scoring round cells only. In one case, ⫹12 had originally been scored in 0.5% but round cell approach showed 10% ⫹12. Another 2 previously normal cases with the unusual combination of 11q- and 17p- became abnormal by the round cell approach. Among previously normal samples, 3 were abnormal for multiple FISH probes by the round cell approach and 13 were abnormal for 1 FISH probe. Discussion: The present experiment shows, as in our past study, that abnormal CLL FISH patterns are confined to round nuclei. Thus, utilization of the round cell approach for scoring FISH in CLL increases the sensitivity to detect a genetic defect. We believe restricting CLL FISH panel scoring to the leukemic B-cell population will be a powerful adjunct to cytogenetic analysis for CLL patients with low lymphocyte counts, including those in clinical remission or with minimal residual disease.
Reference
For this study, the samples from 2 patients taken before and after the therapy administration were also analyzed by NGS sequencer ‘GS Junior’ (Roche). Results: Both FASAY and FISH did not show any TP53 abnormality in samples at diagnosis in both patients. Patient A was subsequently treated (FC, R-CHOP, CHOP, A) and 22 months from diagnosis mutation I195T was identified by FASAY. Later on, this mutation was eliminated by peripheral blood stem cell transplantation, but it was displaced by a more aggressive variant R248W (hot-spot, DNA-contact mutation) detected at the time of relapse (this mutation was accompanied by 17p-). Using NGS, we were able to detect mutation I195T in the sample at diagnosis (2.6% of mutated DNA) and mutation R248W in the sample taken before the transplantation (3.6% of mutated DNA). Patient B developed an alteration R273C (hot-spot, DNA-contact mutation) accompanied by del(17p) after the treatment by FCR (follow-up 25 months). Again, using NGS we were able to detect this mutation in the diagnostic sample (0.24% of mutated DNA). Discussion: CLL patients may harbor a minor proportion of p53-mutated clones, which are at risk to be selected by the therapy. The NGS technology is able to detect such clones. The crucial point, which remains to be solved, is to understand which mutations might be the subject of this selection. Supported by NS9858-4/2009, NS10439-3/2009, NS10448-3/ 2009 (IGA MH, CZ), and MSM0021622430 (MEYS, CZ).
1. ASH 2010, #3606.
4.17 4.16 Identification of Minor Proportion p53 Mutations Selected Later by Therapy Jitka Malcikova,1,2 Katerina S. Kozubik,1,2 Martin Trbusek,1,2 Nikola Tom,1,2 Jana Smardova,3 Boris Tichy,1,2 Petr Kuglik,4 Yvona Brychtova,1 Michael Doubek,1 Jiri Mayer,1 Sarka Pospisilova1,2 1
Department of Internal Medicine - Hematooncology, University
Hospital Brno; 2Central European Institute of Technology, Masaryk 3
University; Department of Pathology, University Hospital Brno; 4
S228
Department of Medical Genetics, University Hospital Brno and
A Novel Approach to Minimal Residual Disease Assessment in Chronic Lymphocytic Leukaemia Utilizing the Tumour Specific Antigen CD160 Timothy Farren,1,2 Fengting Liu,2 Marion Macey,1,2 Michael Jenner,1 Archie Prentice,3 Amit Nathwani,4 Samir G Agrawal1,2 1
Division of Haemato⫺Oncology and Immunophenotyping
Laboratory, Department of Haematology, Barts and The London NHS Trust; 2Centre for Haemato⫺Oncology, Barts Cancer Institute, Queen Mary University of London, London, U.K; 3Department of Haematology, The Royal Free Hospital, London, UK; 4UCL Cancer
Institute of Experimental Biology, Masaryk University
Institute, University College London, London, UK
Background: Abnormalities of the clinically important tumor suppressor p53 arise during the course of chronic lymphocytic leukemia (CLL) and significantly contribute to disease aggressiveness. Several reports have shown the association between therapy administration and clonal selection of p53 defects. According to the p53 mutation profile in untreated and treated CLL patients, we have suggested earlier that treatment selects p53 mutations rather than directly induces them de novo. The aim of this study was to verify whether p53 mutations, which were later selected by therapy, are detectable in samples taken before treatment, at the time when the patient is declared as p53 wild-type by routinely used techniques. Therefore, we used the next-generation sequencing (NGS), variant deep sequencing, with an extremely high sensitivity under 1% of mutated DNA. Methods: The patients were routinely examined by the p53 yeast functional analysis FASAY (sensitivity of mutation detection 10%) and by I-FISH detecting del(17p) (sensitivity 5%).
Introduction: With the advent of highly effective chemo⫺immunotherapy regimens, the treatment of chronic lymphocytic leukaemia (CLL) is no longer simple palliation but aims to achieve a minimal residual disease (MRD)-negative remission as this confers a longer progression free survival. Historically, monitoring dual CD5/ CD19 expression and light chain (LC) clonality was the mainstay of MRD evaluation in CLL. However, this approach is limited by the presence of normal B1-cells (CD5⫹CD19⫹) and the inability to demonstrate LC restriction with very low B-cell numbers. More recently, an international standardized approach (ISA), involving 8 monoclonal antibodies (mAbs) in a 5 test system, was published with a detection accuracy of 95.7% for a threshold of 0.01%.1 CD160 is an activatory NK cell receptor, also expressed on a subset of T-cells. CD160 is not expressed on normal B-lymphocytes, regardless of tissue location or stage of maturity, but is expressed on malignant B-cells in ⬎ 98% of cases of CLL and hairy cell leukaemia.2 This
Clinical Lymphoma, Myeloma & Leukemia Supplement October 2011
Cumulative Survival
1.0
0.8
Low risk
0.6
Intermediate risk
0.4 High risk 0.2 p < 0.0001 Very high risk 0.0 0
12
24
36
48
60 72 84 96 108 120 132 144 156 Time (months)
cells and therefore could promote mistargeted mutations. To examine if AID function might be relevant in clinical disease, cytogenetic aberrations determined by FISH at 17p13.1 (n ⫽ 39), 11q22.3 (n ⫽ 39), 12-CEN (n ⫽ 41), 13q34 (n ⫽ 32), 11q13 (n ⫽ 22), 14q32 (n ⫽ 23), and 6q23.3 (n ⫽ 20) were counted in a subset of our initial CLL patient cohort. 13q14.3 aberrations were excluded from this count, because this sole abnormality correlates with favorable clinical course. AID⫹ CLL patients had significantly more cytogenetic aberrations (n ⫽ 21, average aberrations ⫽ 1) than AID- CLL patients (n ⫽ 22, average aberrations ⫽ 0) (P ⫽ 0.0009). These data support the notion that mistargeted AID action can promote mutations resulting in cytogenetic aberrations in CLL. While our initial CLL patient cohort (n ⫽ 126), from whom AID expression was measured by nested PCR, had clinical outcome (TFT n ⫽ 113, OS n ⫽ 112) and IGHV mutation (n ⫽ 121) data, only a fraction of patients had CD38 (n ⫽ 42), ZAP-70 (n ⫽ 32) and cytogenetic (n ⫽ 43) measurements making multivariate analyses impossible. Therefore, multivariate analyses were performed on a second cohort of patients (n ⫽ 100), from whom we determined AID expression by quantitative real-time PCR and had information on clinical outcome (TFT n ⫽ 100), IGHV mutation (n ⫽ 97), CD38 (n ⫽ 100) and ZAP-70 (n ⫽ 98) levels, and cytogenetics (n ⫽ 91). In this second group, TFT was significant for all prognostic factors by univariate analyses (AID expression – P ⫽ 0.0071, IGHV mutation – P ⫽ 0.0005, CD38 – P ⫽ 0.0236, ZAP-70 – P ⫽ 0.0022, cytogenetic aberrations excluding 13q14.3 – P ⫽ 0.0289). Using a backward selection procedure for multivariate analyses, only AID expression, ZAP-70 level and IGHV mutation status remained as independent prognostic factors. Because cytogenetic aberrations and CD38 expression drop out as independent prognostic factors, AID function may be linked to these. In conclusion, AID expression is an independent risk factor for aggressive CLL disease. As a possible explanation for this observation, we
present data consistent with AID function promoting mistargeted mutations leading to cytogenetic aberrations and poorer prognosis.
4.15 FISH for CLL – A More Sensitive Scoring Method Stephanie A. Smoley,1 Patricia T. Greipp,1 Clive S. Zent,2 Neil E. Kay,2 Daniel L. Van Dyke1 1
Cytogenetics, Mayo Clinic, Rochester, MN; 2Hematology, Mayo Clinic, Rochester, MN
Cytogenetic abnormalities are prognostically important in CLL. Mayo Clinic CLL FISH analyses are derived from direct preparations of buffy coat samples not purified or sorted for lymphocytes. Consecutive qualifying nuclei are scored for each FISH probe (deletion 6q, 11q, 13q, 17p, ⫹12 & IGH/CCND1). Based on the premise that CLL B-cells typically present as perfectly round lymphocytes, we found1 that the % abnormal nuclei in only the affected cell type of CLL patients is significantly higher than that of the general cell population. In 79 of 87 patients (91%), the % abnormal nuclei was greater in round vs unselected nuclei (mean difference 24%; range 1.5 - 57%). By univariate regression analysis, round cells (p ⫽ 0.004) had a better correlation with % B-cells by flow cytometry than either consecutive unselected nuclei (p ⫽ 0.046) or the lobed (non-round) cells (p ⫽ 0.40). We proposed that scoring consecutive round cells would improve detection of an abnormal CLL FISH pattern in patients, especially those with low B-cell counts. The next experiment was designed to test whether an abnormality found at follow-up in a patient with normal FISH at diagnosis would have been detected by scoring round nuclei only. Methods: After IRB approval, we identified 28 patients with a normal CLL FISH study at diagnosis and
Clinical Lymphoma, Myeloma & Leukemia Supplement October 2011
S227
Abstracts abnormal FISH in a follow-up study. Original specimens (slide or fixed cell pellet) were re-scored for % abnormal nuclei among 200 consecutive round nuclei. Scores were compared to the original clinical FISH analyses (200 consecutive nuclei not selected for nuclear morphology). Results: After re-scoring by the round cell approach, 12 originally normal samples remained normal while 16 (57%) were abnormal. The average number of days between the diagnostic and follow-up sample was 826 for those that remained normal and 601 for those abnormal by scoring round cells only. In one case, ⫹12 had originally been scored in 0.5% but round cell approach showed 10% ⫹12. Another 2 previously normal cases with the unusual combination of 11q- and 17p- became abnormal by the round cell approach. Among previously normal samples, 3 were abnormal for multiple FISH probes by the round cell approach and 13 were abnormal for 1 FISH probe. Discussion: The present experiment shows, as in our past study, that abnormal CLL FISH patterns are confined to round nuclei. Thus, utilization of the round cell approach for scoring FISH in CLL increases the sensitivity to detect a genetic defect. We believe restricting CLL FISH panel scoring to the leukemic B-cell population will be a powerful adjunct to cytogenetic analysis for CLL patients with low lymphocyte counts, including those in clinical remission or with minimal residual disease.
Reference
For this study, the samples from 2 patients taken before and after the therapy administration were also analyzed by NGS sequencer ‘GS Junior’ (Roche). Results: Both FASAY and FISH did not show any TP53 abnormality in samples at diagnosis in both patients. Patient A was subsequently treated (FC, R-CHOP, CHOP, A) and 22 months from diagnosis mutation I195T was identified by FASAY. Later on, this mutation was eliminated by peripheral blood stem cell transplantation, but it was displaced by a more aggressive variant R248W (hot-spot, DNA-contact mutation) detected at the time of relapse (this mutation was accompanied by 17p-). Using NGS, we were able to detect mutation I195T in the sample at diagnosis (2.6% of mutated DNA) and mutation R248W in the sample taken before the transplantation (3.6% of mutated DNA). Patient B developed an alteration R273C (hot-spot, DNA-contact mutation) accompanied by del(17p) after the treatment by FCR (follow-up 25 months). Again, using NGS we were able to detect this mutation in the diagnostic sample (0.24% of mutated DNA). Discussion: CLL patients may harbor a minor proportion of p53-mutated clones, which are at risk to be selected by the therapy. The NGS technology is able to detect such clones. The crucial point, which remains to be solved, is to understand which mutations might be the subject of this selection. Supported by NS9858-4/2009, NS10439-3/2009, NS10448-3/ 2009 (IGA MH, CZ), and MSM0021622430 (MEYS, CZ).
1. ASH 2010, #3606.
4.17 4.16 Identification of Minor Proportion p53 Mutations Selected Later by Therapy Jitka Malcikova,1,2 Katerina S. Kozubik,1,2 Martin Trbusek,1,2 Nikola Tom,1,2 Jana Smardova,3 Boris Tichy,1,2 Petr Kuglik,4 Yvona Brychtova,1 Michael Doubek,1 Jiri Mayer,1 Sarka Pospisilova1,2 1
Department of Internal Medicine - Hematooncology, University
Hospital Brno; 2Central European Institute of Technology, Masaryk 3
University; Department of Pathology, University Hospital Brno; 4
S228
Department of Medical Genetics, University Hospital Brno and
A Novel Approach to Minimal Residual Disease Assessment in Chronic Lymphocytic Leukaemia Utilizing the Tumour Specific Antigen CD160 Timothy Farren,1,2 Fengting Liu,2 Marion Macey,1,2 Michael Jenner,1 Archie Prentice,3 Amit Nathwani,4 Samir G Agrawal1,2 1
Division of Haemato⫺Oncology and Immunophenotyping
Laboratory, Department of Haematology, Barts and The London NHS Trust; 2Centre for Haemato⫺Oncology, Barts Cancer Institute, Queen Mary University of London, London, U.K; 3Department of Haematology, The Royal Free Hospital, London, UK; 4UCL Cancer
Institute of Experimental Biology, Masaryk University
Institute, University College London, London, UK
Background: Abnormalities of the clinically important tumor suppressor p53 arise during the course of chronic lymphocytic leukemia (CLL) and significantly contribute to disease aggressiveness. Several reports have shown the association between therapy administration and clonal selection of p53 defects. According to the p53 mutation profile in untreated and treated CLL patients, we have suggested earlier that treatment selects p53 mutations rather than directly induces them de novo. The aim of this study was to verify whether p53 mutations, which were later selected by therapy, are detectable in samples taken before treatment, at the time when the patient is declared as p53 wild-type by routinely used techniques. Therefore, we used the next-generation sequencing (NGS), variant deep sequencing, with an extremely high sensitivity under 1% of mutated DNA. Methods: The patients were routinely examined by the p53 yeast functional analysis FASAY (sensitivity of mutation detection 10%) and by I-FISH detecting del(17p) (sensitivity 5%).
Introduction: With the advent of highly effective chemo⫺immunotherapy regimens, the treatment of chronic lymphocytic leukaemia (CLL) is no longer simple palliation but aims to achieve a minimal residual disease (MRD)-negative remission as this confers a longer progression free survival. Historically, monitoring dual CD5/ CD19 expression and light chain (LC) clonality was the mainstay of MRD evaluation in CLL. However, this approach is limited by the presence of normal B1-cells (CD5⫹CD19⫹) and the inability to demonstrate LC restriction with very low B-cell numbers. More recently, an international standardized approach (ISA), involving 8 monoclonal antibodies (mAbs) in a 5 test system, was published with a detection accuracy of 95.7% for a threshold of 0.01%.1 CD160 is an activatory NK cell receptor, also expressed on a subset of T-cells. CD160 is not expressed on normal B-lymphocytes, regardless of tissue location or stage of maturity, but is expressed on malignant B-cells in ⬎ 98% of cases of CLL and hairy cell leukaemia.2 This
Clinical Lymphoma, Myeloma & Leukemia Supplement October 2011