Characterization and Prognostic Impact of Multiple Productive IGHV Rearrangements in CLL

Abstract Background: In chronic lymphocytic leukemia (CLL) one of the strongest prognostic factors is IGHV mutational status. Infrequently, patients present not only with a single IGHV rearrangement but with multiple productive rearrangements. In about 2% of all CLL patients analyzed on cDNA level multiple rearrangements display the same mutational status and are categorized accordingly following ERIC recommendations. In another 1% rearrangements with discordant IGHV mutational status are detected and preclude a definite risk assignment. Only limited data exist on these rare subgroups. Aim: To characterize treatment-naive CLL patients with multiple productive IGHV rearrangements and determine the impact on prognosis. Patients and Methods: Out of 8,016 treatment-naive CLL patients between 2005 and 2015 and with data on IGHV mutational status we identified 204 (3%) with multiple productive rearrangements. IGHV mutational status was analyzed on cDNA and in all cases according to ERIC recommendations. IGHV mutated status (M) was defined by sequence identity <98% and unmutated status (U) by ≥98%. Chromosome banding analysis was available in 102 cases and interphase FISH with probes for 17p13, 13q14, 11q22 and centromeric region of chromosome 12 in 191. Male:female ratio was 3:1 and median age 68 years (range: 38-89). Additionally, data on SF3B1 and TP53 mutations was present in all cases. Follow-up data on time to first treatment (TTT) and overall survival (OS) was available in 105 cases with a median follow-up of 4 years. For statistical comparison we used a cohort of 1,262 untreated CLL patients with single IGHV rearrangement (median age: 67 years; range: 30-91, median follow-up: 6 years). Results: Out of 204 patients with multiple, productive rearrangements 199 (98%) presented with two and 5 patients (2%) with three IGHV rearrangements. Concordant IGHV mutated status (MM) was present in 120 cases (59%), whereas concordant unmutated status (UU) was seen in 34 patients (17%). In 50 cases (25%) a mixed IGHV status (UM) was detected. We analyzed frequencies of complex karyotype by CBA, biclonality according to immunophenotype (concurrent kappa restricted and lambda restricted subpopulations) and/or CBA, TP53 disruption (TP53mut and/or del(17p)), SF3B1mut, del(11q), trisomy 12, and del(13q). Overall, a higher frequency of biclonality was detected in patients with multiple vs. single IGHV rearrangements (16% vs. 1%, p<0.001). However, association to neither MM, UU nor UM existed. MM presented with molecular and cytogenetic characteristics similar to M. Correspondingly, UU showed similar frequencies of mutations and aberrations to U, except for higher frequency of trisomy 12 in UU vs. U (42% vs. 19%, p=0.003). Interestingly, UM presented with characteristics similar to U and UU. UM was associated with TP53 disruption vs. M (16% vs. 5%, p=0.003) and vs. MM (5%, p=0.035) as well as with SF3B1mut vs. M (16% vs. 5%, p=0.008). Furthermore, UM cases showed high frequency of del(11q) vs. M (29% vs. 3%, p<0.001) and vs. MM (1%, p<0.001) and less frequently del(13q) sole vs. M (41% vs. 60%, p=0.011) and MM (41% vs. 69%, p=0.001). No significantly differences in TTT were observed between MM and M (median: 13 vs. 14 years) and between UU and U (6 vs. 4 years), respectively. However, the difference between MM vs. UU (p=0.022) and M vs. U (p<0.001) was significant. The UM subgroup presented with a TTT (median: 4 years) similar to U and UU, whereas it was significantly shorter vs. M (p=0.003) and MM (p=0.006), respectively. A similar picture emerged for survival. 5-year OS of MM was not different vs. M (94% vs. 90%) but vs. U (78%, p=0.001). The statistical analysis of OS in UU was hampered by low case numbers. UM presented again with similar 5-year OS vs. U (81% vs. 78%, n.s.) and significantly worse OS vs. M (90%, p=0.049) and vs. MM (94%, p=0.014). Conclusions: (1) Patients with multiple productive IGHV rearrangements and concordant IGHV status show similar prognosis and characteristics to patients with single rearrangement with the respective IGHV status. (2) Cases with mixed IGHV status show similar prognosis to patients with IGHV unmutated status and accordingly are characterized by high frequencies of adverse prognostic factors like TP53 disruption, SF3B1mut, and del(11q), whereas del(13q) sole is less frequent. Disclosures Jeromin: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Dicker:Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Download Full-text

Frequency and Prognostic Impact of CD8 Expression In 5,523 Patients with Chronic Lymphocytic Leukemia (CLL) In Relation to Chromosomal Aberrations, IGHV Mutational Status and ZAP-70 Expression.

Blood ◽

10.1182/blood.v116.21.4616.4616 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4616-4616

Author(s):

Wolfgang Kern ◽

Claudia Haferlach ◽

Susanne Schnittger ◽

Torsten Haferlach

Keyword(s):

Chromosomal Aberration ◽

Chromosomal Aberrations ◽

Lymphocytic Leukemia ◽

Prognostic Impact ◽

Employment Equity ◽

Mutational Status ◽

Trisomy 12 ◽

Equity Ownership ◽

Wbc Count

Abstract Abstract 4616 Chronic lymphocytic leukemia (CLL) is an indolent lymphoma with largely heterogeneous clinical course. Identifying patients which benefit from early therapy is one of the most important issues in the management of CLL. Besides patient-specific characteristics and applicability of different treatment approaches the decision on therapy is based on conventional prognostic parameters as well as on chromosomal aberrations, IgVH mutational status and expression of ZAP-70, which all have been shown to be independently associated with outcome. The expression of CD8 has been rarely observed in CLL and its prognostic impact is unclear yet. In order to clarify the frequency of CD8 expression, its correlation to established prognostic parameters as well as its prognostic impact we analyzed a total of 5,523 patients with CLL by multiparameter flow cytometry including the expression of CD8 between August 2005 and August 2010. Fluorescence in situ hybridization (FISH) applying a standard set of probes for the detection of del(6q), del(11q22.3) (ATM), trisomy 12, del(13q14) (D13S25, D13S319), and del(17p13) (TP53) was performed in 3,407 patients. IGHV mutational status was determined and evaluable in 2,845 patients. Clinical follow-up data was available in 1,021 patients (median follow-up: 21.3 months). 61 patients (1.1%) showed an expression of CD8 (antibody clone B9.11, Immunotech, France) as compared to isotype used as negative control. CD8+ vs. CD8- cases did not differ in age (mean±SD: 70.3±9.7 vs. 68.3±10.4 years, n.s.) and the male/female ratio was 1.11 vs. 1.61 (n.s.). There were no significant differences in WBC count (mean±SD, 31.7±33.9 vs. 36.1±53.1 × 10e9/l) and hemoglobin level (Hb, mean±SD, 13.6±2.0 vs. 13.2±2.1 g/dl), while platelet counts were higher in CD8+ vs. CD8- cases (229±72 vs. 194±99 × 10e9/l, p=0.021). In CD8+ vs. CD8- cases chromosomal aberrations were detected by FISH analysis with the following frequencies: del(6q), 5.9% vs. 3.1%, n.s.; del(11q22.3), 17.1% vs. 10.7% (n.s.); trisomy 12, 20.0% vs. 14.5% (n.s.); del(13q14), 66.7% vs. 59.8% (n.s.); del(13q14) as sole chromosomal aberration, 41.2% vs. 45.5% (n.s.); del(17q13), 2.9% vs. 5.7% (n.s.). The IGHV status was mutated in 79.3% vs. 60.4% (p=0.054) in CD8+ vs. CD8- cases. Patients with CD8 expression had a significantly shorter time to therapy (TTT) as compared to CD8- patients (median TTT, 12.0 vs. 77.1 months, p=0.008). The following parameters showed a significant relation to a shorter TTT in univariate Cox analyses: CD8 positivity, p=0.011, relative risk (RR)=2.87; higher WBC count, p=0.008, RR=1.01 per 10 × 10e9 increase; del(11q22.3), p<0.001, RR=1.97; del(17p13), p=0.005, RR=1.72; higher % of CLL cells with ZAP-70 expression (antibody clone SBZAP, Immunotech, France), p=0.011, RR=1.04 per 10% increase. Parameters significantly related to a longer TTT in univariate Cox analyses were: higher Hb level, p<0.001, RR=0.86 per 1 g/dl; higher platelet count, p=0.021, RR=0.98 per 10 × 10e9 increase; and del(13q14) as sole chromosomal aberration, p<0.001, RR=0.58. Multivariate analysis identified two parameters to be independently related to a shorter TTT: higher ZAP-70 expression (p=0.002) and CD8 positivity (p=0.036), while a higher Hb level (p<0.001) and del(13q14) as sole chromosomal aberration (p=0.011) were identified to be independently related to a longer TTT. This data supports the further evaluation of the prognostic impact of CD8 expression in CLL in order to define its role in identifying the most appropriate timepoint for therapy and the most appropriate treatment modality for patients with CLL. Disclosures: Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Download Full-text

Risk Stratification In Chronic Lymphocytic Leukemia Patients With IGHV3-21 Gene Usage According To Presence Of Stereotypy and Mutations In SF3B1

Blood ◽

10.1182/blood.v122.21.4114.4114 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4114-4114

Author(s):

Sabine Jeromin ◽

Frank Dicker ◽

Katharina Bayer ◽

Sandra Weissmann ◽

Christiane Eder ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Risk Stratification ◽

Lymphocytic Leukemia ◽

Prognostic Impact ◽

Employment Equity ◽

Mutation Status ◽

Mutational Status ◽

Trisomy 12 ◽

Equity Ownership

Abstract Introduction Chronic Lymphocytic Leukemia (CLL) patients with monoclonal IGHV3-21 gene rearrangements have been described to have adverse prognosis independent of mutational status. Heterogeneous data exists whether only patients with a stereotyped motif in the junctional region (designated as subset #2, Stamatopoulos K. et al., Blood 2007) suffer from worse prognosis. Furthermore, it was recently suggested that co-occurrence of subset #2 and mutations (mut) in SF3B1 are indicative of a shorter time to treatment (TTT). Aims 1. Determine the prognostic impact of IGHV3-21 and subset #2 rearrangements. 2. Evaluate the association with SF3B1mut and its prognostic impact. Patients and Methods IGHV3-21 positive (n=213) and independently 1,094 unselected CLL patients without prior treatment were analyzed. The whole cohort comprised 63.9% (835/1,307) males and 36.1% (472/1,307) females with a median age of 66.8 years (range: 27.5 – 90.5 years). In all cases IGHV mutation status was analyzed. IGHV unmutated (unmut) status was present in 38.6% (504/1,307) and mutated status in 61.4% (803/1,307). Stereotypy of IGHV3-21 was classified according to published criteria (Agathangelidis A. et al., Blood 2012). SF3B1 was analyzed in all and TP53 in 1,262 cases for mutations. For all patients data on immunophenotype was available. Cases were further analyzed by FISH using probes for del(17p) (n=1,305), del(11q) (n=1,303), trisomy 12 (n=1,303) and del(13q) (n=1,305). Clinical follow-up data was available in 1,040 patients with a median follow-up of 4.4 years (IGHV3-21: n=160, 4.2 years). Results Of 213 IGHV3-21 positive patients, 111 (52.1%) cases were classified as subset #2 B-cell receptor. The frequency of IGHVmut was significantly higher in subset #2 vs. non-subset #2 (78/111, 70.3% vs. 49/102, 48.0%, p=0.001). IGHV3-21 was highly associated with SF3B1mut (52/213, 24.4% vs. 92/1,094, 8.4%, p<0.001), which were particularly frequent in subset #2 cases (38/111, 34.2% vs. 14/102, 13.7%, p=0.001). Furthermore, IGHV3-21 was associated with del(11q) (35/210, 16.7% vs. 122/1,093, 11.2%, p=0.028) and was rare in patients with trisomy 12 (8/210, 3.8% vs. 168/1,093, 15.4%, p<0.001). Accordingly, del(11q) was particularly frequent in subset #2 patients (25/110, 22.7% vs. 10/100, 10.0%, p=0.016), whereas trisomy 12 (1/110, 0.9% vs. 7/100, 7.0%, p=0.029) and del(17p) (1/111, 0.9% vs. 8/101, 7.9%, p=0.015) were nearly absent. Kaplan-Meier analysis revealed no significant difference in TTT between IGHV3-21mut vs. unmutated cases. However, IGHV3-21mut cases had slightly longer TTT compared to IGHVunmut (5.3 years vs. 3.4 years, p=0.039). Taking stereotypy into account, subset #2 patients showed nearly identical TTT compared to IGHVunmut patients (3.5 vs. 3.4 years). Further stratification according to IGHV mutational status presented mutated non-subset #2 patients with a similar TTT compared to IGHVmut cases (9.2 vs. 10.2 years), whereas all other subgroups assorted together with IGHVunmut (Fig. 1A). Additionally, there was a trend to a shorter TTT in subset #2 in combination with SF3B1mut vs. SF3B1wt (1.2 vs. 4.4 years, p=0.056) (Fig. 1B). In univariate Cox regression analysis, following parameters were analyzed and showed significant impact on TTT: IGHVmut (p<0.001, HR 0.33), IGHV3-21 (p=0.002, HR 1.51), subset #2 (p=0.005, HR 2.04), SF3B1mut (p<0.001, HR 2.06). A multivariate analysis including IGHV3-21, IGHVmut and SF3B1mut revealed independent impact on TTT only for the latter two parameters: IGHVmut (p<0.001, HR 0.35) and SF3B1mut (p=0.001, HR 1.59). In contrast, analyzing subset #2, IGHVmut and SF3B1mut in a multivariate model, only subset #2 (p=0.011, HR 1.93) and SF3B1mut (p=0.023, HR 1.82) retained their prognostic effect, whereas IGHV mutational status had no independent impact. Conclusions 1. Our data suggests to prognostically stratify IGHV3-21 patients according to the presence of stereotypy, since only subset #2 patients showed shorter TTT, whereas mutated non-subset #2 cases had a TTT similar to IGHVmut cases. 2. Mutation status of SF3B1 further refines the risk stratification of subset #2 patients, as co-occurrence of subset #2 with SF3B1mut leads to shorter TTT compared to subset #2/SF3B1wt cases. Disclosures: Jeromin: MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Bayer:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Download Full-text

Patients with TP53 Disruption and IGHV Mutated Status Show Indolent Clinical Course: A Study on 1,327 Treatment-Naive CLL Cases

Blood ◽

10.1182/blood.v128.22.4378.4378 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4378-4378

Author(s):

Sabine Jeromin ◽

Claudia Haferlach ◽

Frank Dicker ◽

Torsten Haferlach ◽

Wolfgang Kern

Keyword(s):

Disease Course ◽

Complex Karyotype ◽

Employment Equity ◽

Mutational Status ◽

Treatment Naïve ◽

Trisomy 12 ◽

Binet Stage ◽

Equity Ownership ◽

Indolent Disease

Abstract Background: Treatment-naive CLL patients displaying TP53 disruption (TP53 mutation and/or del(17p)) show heterogeneity in their clinical course. A few studies have proposed a mutated IGHV status and Rai 0 as potential factors for an indolent disease course in these cases. Aim: To determine the prognostic impact of TP53 disruption in a treatment-naive CLL cohort and the factors associated with an indolent disease course. Patients and Methods: 1,327 CLL patients at diagnosis or without prior treatment were screened both for TP53 mutations (mut) and del(17p). IGHV mutational status was analyzed in all cases. IGHV mutated status (IGHV-M) was defined by sequence identity <98%. Chromosome banding analysis and interphase FISH with probes for 13q14, 11q22 and centromeric region of chromosome 12 were performed in all cases. Additionally, data on mutations in SF3B1 (n=1,183) and NOTCH1 (n=967) was present. Male:female ratio was 2:1 and median age was 67 years (range: 30 - 91 years). Data on Binet stage was available in 711 cases. Follow-up data on time to first treatment (TTT) and overall survival (OS) was available in 1,123 (TP53 disruption: 84) and 1,143 (TP53 disruption: 88) cases, respectively, with a median follow-up of 6 years, each. Results: Overall in 7% (97/1,327) of patients a TP53 disruption was present. TP53mut was detected in 89 (7%) of cases and del(17p) in 51 (4%). Three different combinations were determined: TP53mut with del(17p) in 43 (44%) cases, TP53mut sole in 46 (48%) patients and del(17p) sole in 8 (8%) cases. TP53 disruption was associated with IGHV-U vs. IGHV-M (12% vs. 5%, p<0.001), NOTCH1mut vs. NOTCH1wt (14% vs. 8%, p=0.027), complex karyotype (≥ 3 abnormalities) vs. non-complex karyotype (27% vs. 6%, p<0.001), and Binet B/C vs. A (10% vs. 5%, p=0.010). In contrast, TP53 disruption was rare in del(11q) vs. others (3% vs. 8%, p=0.046). Next, we analyzed patients with TP53 disruption for differences in TTT and OS according to IGHV mutational status, SF3B1mut, NOTCH1mut, del(11q), trisomy 12, del(13q), complex karyotype and Binet stage. We detected a significantly longer TTT in IGHV-M cases (median: 10 vs. 2 years, p<0.001) and in patients without trisomy 12 (median: 6 vs. 2 years, p=0.046). A better 5-year OS was seen in cases with IGHV-M (76% vs. 57%, p=0.006) and in patients with Binet A vs. B/C (80% vs. 46%, p=0.049). Interestingly, in TP53 disruption/IGHV-M patients as compared to IGHV-M without TP53 disruption no significant difference in TTT was detected. In contrast, TP53 disruption/IGHV-M cases showed an intermediate 5-year OS comparable to IGHV-U without TP53 disruption, whereas patients with IGHV-U/TP53 disruption showed the most adverse OS (see Table). A cox regression analysis in TP53 disrupted cases including all the above mentioned factors showed significant impact only for IGHV-M both on TTT (HR: 0.24, p<0.001) and OS (HR: 0.38, p=0.008). Therefore, we further focused on the characterization of patients with TP53 disruption/IGHV-M vs. TP53 disruption/IGHV-U. TP53 disruption/IGHV-M showed higher frequencies of del(13q) (71% vs. 45%, p=0.013), whereas del(17p) (39% vs. 63%, p=0.025) and trisomy 12 (2% vs. 23%, p=0.003) were less frequent. NOTCH1mut were mutually exclusive of TP53 disruption/IGHV-M (0% vs. 36%, p<0.001). Thus, within the cohort of TP53 disruption cases the subgroup with IGHV-M is characterized by the occurrence of adverse prognostic factors to a lesser extent. Conclusions: Treatment-naive patients with TP53 disruption and IGHV-M show a more indolent disease course: (1) TTT was not significantly altered in comparison to IGHV-M cases without TP53 disruption; (2) for OS an additive effect of IGHV-U and TP53 disruption was detected with patients presenting with both factors showing the most adverse OS. Thus, in treatment-naive CLL patients TP53 disruption should always be evaluated on the background of IGHV mutational status and as recommended only at progression and before treatment is required. Disclosures Jeromin: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Dicker:Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Download Full-text

Flow Cytometric Identification of Biclonal Disease Among 5,523 Patients with Chronic Lymphocytic Leukemia and Its Genetic Characterization

Blood ◽

10.1182/blood.v116.21.3608.3608 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3608-3608

Author(s):

Wolfgang Kern ◽

Susanne Schnittger ◽

Frank Dicker ◽

Torsten Haferlach ◽

Claudia Haferlach

Keyword(s):

Light Chain ◽

Chromosomal Aberrations ◽

Molecular Genetic ◽

Lymphocytic Leukemia ◽

Fish Analysis ◽

Employment Equity ◽

Mutational Status ◽

Trisomy 12 ◽

Genetic Level ◽

Equity Ownership

Abstract Abstract 3608 Chronic lymphocytic leukemia (CLL) is an indolent disease with largely heterogeneous clinical course. While the diagnosis is made based on the characteristic immunophenotype as determined by multiparameter flow cytometry (MFC) another aspect of major clinical importance is the estimation of the prognosis which includes the analysis of chromosomal aberrations, the IGHV mutational status as well as the expression of CD38 and ZAP-70. In general, the methods used to determine these parameters are applied in the assumption of analyzing one homogeneous leukemic population and are evaluated accordingly. The potential presence of subpopulations and even subclones may not always be considered adequately in this regard. We identified 76 out of 5,523 patients (1.4%) in whom MFC identified biclonal disease based on the presence of both kappa- and lambda-light chain restricted leukemic subpopulations. In 45 of these cases fluorescence in situ hybridization (FISH) analysis was performed applying a standard set of probes for the detection of del(6q), del(11q22.3) (ATM), trisomy 12, del(13q14) (D13S25, D13S319), and del(17p13) (TP53). In 17 of the 76 patients a chromosome banding analysis (CBA) was performed and the IGHV mutational status was determined in 38 of 76 patients. The patients` ages ranged from 47.7 to 88.0 years (median, 71.3 years), 50 patients were male. The median WBC count amounted to 19 ×10e9/l (range, 0.6–237 ×10e9/l). In most cases the kappa-light chain restricted subpopulation was larger than the lambda-light chain restricted one. The median values and ranges for the respective percentages of subpopulations amounted to: kappa, 24%, 1%-88%; lambda, 12%, 1%-85%. The respective peripheral blood concentrations amounted to: kappa, 3.68 ×10e9/l, 0.12–142 ×10e9/l; lambda, 2.37 ×10e9/l, 0.01–128 ×10e9/l. The median ratio kappa population/lambda population amounted to 1.9 (range, 0.05–76). FISH analysis identified del(6q) in 2/44 (4.5%) cases, del(11q) in 2/44 (4.5%), trisomy 12 in 7/44 (15.9%), del(13q) in 28/45 (62.2%), del(13q) as sole aberration detected by FISH in 23/43 (53.5%), and del(17p) in 1/45 (2.2%). In three cases more than one aberration was detected by FISH: two cases with del(11q) and del(13q) and one case with trisomy 12 and del(13q). While in two of these three cases the size-ratios of the respective subpopulations were similar in MFC and FISH analysis (1.7:1 vs. 4.3:1 and 2.0:1 vs. 1.5:1) this was not true for the third case (15.5:1 vs. 1.1:1). The further two cases could be considered in line with both methods detecting independent clones. In the latter case the chromosomal aberrations were present in 54% and 59% of the cells and the subpopulations detected by MFC amounted to 62% and 4%. Thus, both chromosomal aberrations must be considered to coexist in one population and not related to the two subpopulations detected by MFC. Overall, however, no clear-cut conclusions can be drawn from FISH results regarding the presence of independent subpopulations and therefore we next focused on the results of CBA. Within the 17 patients analyzed by CBA 12 cases showed an aberrant karyotype (70.6%). In four of these cases more than one clone was identified by differences in the chromosomal aberrations, respectively. In three cases chromosomal evolution was suggested by shared aberrations in both clones and additional aberrations in one of both clones only, respectively. Conversely, in the fourth case two completely different aberration patterns were observed. In 14 out of the 38 patients (36.8%) in whom an IGHV mutational analysis was performed two independent clones were identified by the presence of two different B-cell receptor rearrangements. The presence of biclonal disease had no impact on the clinical outcome of the patients as assessed by time to therapy and overall survival. This data indicates that subpopulations can be identified in a significant number of patients with CLL based on the immunophenotype as well as on the cytogenetic and molecular genetic level. These subpopulations at least in part must be considered as subclones with differing genetic background. These subclones may be associated with differing clinical courses. This data therefore suggests to vigorously screen patients with CLL for subpopulations by MFC and to comprehensively characterize positive cases cytogenetically by CBA and FISH analysis as well as on the molecular genetic level. Disclosures: Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Dicker:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Download Full-text

ASXL1 exon 12 Mutations Are Frequent in AML with Intermediate Risk Karyotype and Are Independently Associated with An Extremely Poor Outcome

Blood ◽

10.1182/blood.v118.21.416.416 ◽

2011 ◽

Vol 118 (21) ◽

pp. 416-416

Author(s):

Susanne Schnittger ◽

Christiane Eder ◽

Tamara Alpermann ◽

Annette Fasan ◽

Vera Grossmann ◽

...

Keyword(s):

De Novo ◽

Strong Association ◽

Multivariable Analysis ◽

Adverse Impact ◽

Prognostic Impact ◽

Intermediate Risk ◽

Employment Equity ◽

Mutational Status ◽

Equity Ownership ◽

The Impact

Abstract Abstract 416 Introduction: ASXL1 mutations have recently been described in a number of different myeloid malignancies. Data on frequency, association with other markers and outcome in AML are rare. Aim: The aim of this study was to evaluate ASXL1 mutations (ASXL1mut) in AML with intermediate risk karyotype for frequency, association with other mutations and impact on outcome. Methods: We analyzed 476 cases with intermediate risk de novo AML for ASXL1 mutations by direct Sanger sequencing of exon 12. Other mutations were analyzed as described previously and were available in part of the patients (NPM1: n=474, FLT3-ITD: n=473, FLT3-TKD: n=407, MLL-PTD: n=474, CEBPA: n=447, RUNX1: n=150, WT1: n=384, IDH1: n=464 and IDH2: n=444, TET2: n=109, NRAS: n=191; KRAS: n=110, DNMT3A: n=83). 397 cases had a normal karyotype (NK) and 79 had intermediate risk aberrant cytogenetics (according to MRC). Female/male ratio was 221/255 and age ranged from 18.5–100.4 y (median: 66.4). Results: Overall, in 70/476 patients (14.7%) ASXL1mut were detected. In detail, the most frequent mutation was p.G646WfsX12 (n=36) followed by p.E635RfsX15 (n=9), and p.Y591X (n=2). The remaining 21 mutations were non-recurrent consisting of 2 frameshift, 13 nonsense and 6 missense mutations. All mutations were detected with a mutation/wildtype load of 40–50% and none of the cases had more than one ASXL1mut. ASXL1mut were more frequent in males than in females (56/255, 22.0% vs 14/221, 6.3%, p=0.001) and were associated with higher median age (72.4 yrs vs 64.1 yrs, p<0.001). In detail, in the cohort > 65 yrs 21.7% (n=55/254) and in those <65 yrs only 6.8% (n=15/222) were ASXL1mut (p<0.001). With respect to morphology ASXL1mut were more frequent in AML without maturation than in all others (37.5% vs 14.3%, p=0.022). In 242 cases immunophenotyping data was available and cases with ASXL1mut (n=34) had a higher expression of CD13 (mean±SD, 55±23% vs. 43±25%, p=0.012), CD34 (46±32% vs. 24±26%, p<0.001), CD133 (29±27% vs. 16±23%, p=0.006) and HLA-DR (42±25% vs. 30±24%, p=0.009) as well as a lower expression of CD33 (66±21% vs. 77±21%, p=0.005) and thus had a more immature immunophenotype as compared to ASXL1wt. There was no association with leukocyte or platelet counts. With regard to cytogenetics ASXL1mut were more frequent in those with aberrant karyotype than in NK (20/79, 25.3% vs 50/397, 12.6%, p=0.008). Generally, ASXL1mut were observed together with all other molecular mutations but there was a strong correlation to RUNX1mut (n=18/43, 41.9% vs 19/107, 17.8% in RUNX1wt, p=0.003) and a negative correlation with NPM1mut (n=9/274; 3.3% vs. n=61/200, 30.5% in NPM1wt, p<0.001) and DNMT3Amut (1/26, 3.8% vs. 19/55 in DNMT3A, 34.5%, p=0.002). Patients with ASXL1mut had a shorter overall survival (OS) (median: 11.2 vs 38.8 months, p<0.001) and event free survival (EFS) (median: 9.0 vs 23.9 months, p<0.001). In detail, this adverse impact could be shown for both NK (OS: median: 10.9 vs 38.3 months, p<0.001; EFS: 9.8 vs. 26.5 months, p<0.001) and intermediate risk aberrant cytogenetics (OS: median: 8.6 vs 38.8 months, p<0.001; EFS: 5.3 vs 21.5 months, p=0.011), separately. Although the ASXL1mut were much more frequent in the elderly and compared to the ASXL1wt had a shorter OS (median: 7.0 vs 16.3 months, p=0.002) an adverse effect on survival could also be shown in the cohort <65 yrs (median OS: 11.6 vs 47.3 months, p<0.001 and median EFS: 9.3 vs 34.5 months, p<0.001). Because of the high coincidence of the two mutations the impact of ASXL1mut in dependence of RUNX1 status was analyzed. In the RUNX1mut (n=43) the ASXL1mut (n=18) still had an adverse impact on EFS (median: 5.3 vs 15.6 months, p=0.010) and a trend for shorter OS (10.7 vs. 20.5 months, p=0.079). In a multivariable analysis ASXL1 is an unfavourable factor for OS independent of age and RUNX1 mutational status (p=0.026, RR: 2.0). Conclusions:ASXL1 mutations belong to the most frequent mutations in intermediate risk AML. There is a strong association with male sex, high age, immature phenotype and RUNX1mut. Still, ASXL1mut retained its independent very poor prognostic impact. Although the number of known molecular markers in AML is continuously increasing and selection of the most import markers for diagnostic work-up seems challenging this data indicates that ASXL1 is one of the most prominent candidates. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Eder:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Download Full-text

Clinical Impact of Clonal and Subclonal TP53, SF3B1, BIRC3, and ATM Mutations in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v126.23.4138.4138 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4138-4138

Author(s):

Ferran Nadeu ◽

Julio Delgado ◽

Cristina Royo ◽

Tycho Bauman ◽

Tatjana Stankovic ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Allele Frequency ◽

Lymphocytic Leukemia ◽

Molecular Characteristics ◽

Prognostic Impact ◽

Driver Genes ◽

Germline Variants ◽

Tp53 Mutations ◽

Mutational Status ◽

The Impact

Abstract Genomic studies have provided a complete profile of somatic mutations in chronic lymphocytic leukemia (CLL). These comprehensive approaches have revealed a relatively large number of mutated genes, the adverse prognostic value of some of which has been demonstrated in a number of reports. Recent studies have shown the clinical relevance of TP53 mutations at very low allele frequency. The presence and prognostic impact of minor mutated clones of other CLL driver genes and their clonal dynamics in the evolution of the disease is not well known. The goal of this study was to explore the presence of clonal and subclonal mutations of TP53, SF3B1, BIRC3, and ATM using an ultra-deep next-generation sequencing (NGS) strategy, to define the evolution of these subclones in different time-points of the disease, and to determine their influence in the outcome of the patients. Samples from 363 untreated CLL cases were included in this study. Copy number alterations were investigated by high density SNP-arrays or by quantitative PCR in 341 and 16 cases, respectively. Targeted ultra-deep NGS of TP53 (exons 4-10), ATM (exons 2-63), BIRC3 (exons 2-9), and SF3B1 (exons 14-16 and 18), including splicing sites, was performed using the Access-Array system (Fluidigm) and sequenced in a MiSeq equipment (Illumina). This methodology combined with a robust bioinformatic analysis based on well-known available tools allowed the identification of mutations down to 0.3% of variant allele frequency (VAF). Results obtained were fully verified by orthogonal techniques. Twelve per cent of VAF was used as threshold for the classification of clonal or subclonal mutations since 12% was the cut-off for detection of mutations by Sanger sequencing. Deletions of 11q comprising ATM or BIRC3 were found in 7% of the cases and were associated with mutations of the other ATM allele in 19/26 (73%) cases and BIRC3 in 3/23 (13%). Deletions of 17p were found in 19 (5%) cases and co-existed with TP53 mutations in 15 (79%) of them. Regarding the mutational status of the studied genes, TP53 mutations were present in 11.6% of patients (7.2% clonal, 4.4% subclonal), ATM mutations in 10% (7% clonal, 1% subclonal, 2% germline mutations considered pathogenic), SF3B1 mutations in 12% (7% clonal, 5% subclonal), and BIRC3 mutations in 4% (2% clonal, 2% subclonal). These subclonal mutations had similar molecular characteristics to their respective high-allele frequency mutations supporting a comparable pathogenic effect. In this regard, clonal and subclonal SF3B1 mutations were associated with shorter time to first treatment (TTT) independently of IGHV mutations. Clonal and subclonal TP53 mutations predicted for shorter overall survival (OS) together with the IGHV mutational status, although the impact of isolated TP53 mutations (i.e. without 17p deletion) on OS was not so evident, as has been the case in other studies. In addition, the outcome of patients with clonal and subclonal BIRC3 mutations showed a similar significant shorter OS. Regarding ATM, the effect of isolated subclonal ATM mutations could not be evaluated because of their low number, but ATM mutations as a whole had a significant impact on TTT even in the absence of 11q deletions. This study also reinforces the need to study the germline of the patients to fully characterize the ATM mutations observed in the tumors. Of note, germline variants previously described as pathogenic were associated with 11q deletions, confirming the hypothesis already suggested that these germline variants may influence disease progression through loss of the otherallele. Clonal dynamics was examined in longitudinal samples of 45 CLL patients. We confirmed the expansion of most TP53 mutated clones after therapy. However, both TP53 and SF3B1 mutations expanded also before any therapy in some patients, indicating that progressive dynamics of these clones is not only dependent on therapy selection. On the contrary, small ATM mutated clones seemed to be more stable. Although the number of cases is limited, we observed that clonal evolution in longitudinal samples had an unfavorable impact on OS. In conclusion, this study shows the presence of a high number of subclonal mutations of different driver genes in CLL and provides insights on the impact of these mutations on the outcome of the patients. These findings suggest that the characterization of the subclonal architecture may be relevant for a better management of CLL patients. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Lack of Prognostic Significance of the Conventional and Novel Prognostic Markers in Trisomy 12 Chronic Lymphocytic Leukemia (CLL)

Blood ◽

10.1182/blood.v128.22.4354.4354 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4354-4354

Author(s):

Valter Gattei ◽

Riccardo Bomben ◽

Michele Dal Bo ◽

Antonella Zucchetto ◽

Francesca Rossi ◽

...

Keyword(s):

Flow Cytometry ◽

Research Funding ◽

Prognostic Markers ◽

Lymphocytic Leukemia ◽

Cytogenetic Abnormality ◽

Mutational Status ◽

Ighv Gene ◽

Trisomy 12 ◽

Gene Status

Abstract Background. Trisomy 12 (tris12) is a recurrent cytogenetic abnormality in chronic lymphocytic leukemia (CLL), occurring in approximately 15-20% of cases, often as the unique cytogenetic alteration, that is usually considered a clonal driver lesion occurring early in CLL evolution. In the Dohner hierarchical categorization, tris12 CLL are identified as having an intermediate prognostic risk, although recent reports suggest a more complex and heterogeneous clinical behavior. Compared to CLL lacking this cytogenetic abnormality, tris12 CLL show more atypical morphology and immunophenotype, more frequent expression of the negative prognostic markers CD49d and CD38, and presence of NOTCH1 mutations and an unmutated (UM) IGHV gene status. The increased fraction of tris12 CLL carrying adverse prognostic features is in contrast to the intermediate clinical behavior associated with most tris12 CLL cases. Aim. To perform a comprehensive evaluation of the clinical impact of the major genetic, immunogenetic and immunophenotypic prognostic markers in tris12 CLL. Methods. The study was based on a multicenter series of tris12 CLL defined according to Dohner (n=283, including 73 cases also bearing del13q), and a comparison group (control) of 553 cases with either del13q (n=308) or without any cytogenetic abnormality (no del17p, del11q, tris12, del13q, n=245). Median follow-up of patients in the tris12 and control groups were 4 years (range 0-22) and 7 years (range 0-28), with 54% and 57% treated patients, and 18% and 15% deaths, respectively. Patient characterization included modified Rai stage, CD49d (CD49dhigh, ≥30% positive cells by flow cytometry), CD38 (CD38high, ≥30% positive cells by flow cytometry) and ZAP-70 (ZAP-70high, ≥20% positive cells by flow cytometry) expression, and IGHV mutational status (mutated, M, or UM according to the 2% cutoff). TP53, BIRC3, NOTCH1 andSF3B1 mutations were screened either at diagnosis or before therapy by NGS with at least 1000X coverage and 1% of sensitivity. Groups were compared by chi-square test; overall survival (OS) was computed from diagnosis to death or censored at last observation, and analyzed by Cox regression analysis. Results. Comparing the tris12 and the control groups, median age was 64 years (range 30-92) vs 66 years (range 33-92), male gender 55% vs 56% (p=0.86), the modified Rai stage was early in 52% vs 54%, intermediate in 41% vs 42% and advanced in 7% vs 4% (p=0.20). As previously reported, tris12 CLL were characterized by a higher prevalence of cases expressing CD49d (85% vs 31%) and CD38 (62% vs 17%; all p<0.0001), and of UM IGHV cases (55% vs 25%, p<0.0001). Analysis of recurrent mutations highlighted a higher prevalence of NOTCH1 mutations (26% vs 8%, p<0.0001) and of BIRC3 mutations (21% vs 1%, p<0.0001) in tris12 vs control group CLL. Conversely, no differences were found in the fraction of cases with TP53 mutations (3% vs 4%, p=0.38) or SF3B1 mutations (7% vs 7%, p=0.89), and in cases expressing ZAP-70 (62% vs 52%, p=0.09). The impact of these features on OS was tested by univariate analysis: in tris12 CLL, only the UM IGHV gene status predicted shorter OS (HR=2.37, p=0.0063), while none of the other characteristics reaching statistical significance as OS predictors (CD49d HR=1.36, p=0.36; CD38 HR=0.42, p=0.052; ZAP-70 HR=3.12, p=0.07; TP53 HR=2.33, p=0.25; NOTCH1 HR=1.40, p=0.22; SF3B1 HR=2.05, p=0.17; BIRC3 HR=1.22, p=0.61). On the other hand, in the control cohort, a significantly higher HR was found for CD49d (HR 3.11, p<0.0001) and CD38 (HR 3.45, p<0.0001) expression, TP53 (HR 2.88, p=0.0026), NOTCH1 (HR 3.57, p<0.0001), and SF3B1 (HR 2.57, p=0.0038) mutations, as well as for the UM IGHV gene status (HR=2.81, p<0.0001), but not for ZAP-70 expression and BIRC3 mutations (HR=1.74 and HR=1.91, p=0.15 and p=0.37, respectively). Conclusions. Mutational status of IGHV genes was the sole prognostic factor able to stratify OS in tris12 CLL. Despite the high frequency of NOTCH1 and BIRC3 mutations, as well as of CD49d and CD38 overexpression, these markers failed to convey a prognostic risk in tris12 CLL. The lack of a significant clinical impact for TP53 and SF3B1 mutations might be partly explained by the low number of mutated cases combined with a relative short follow up in our tris12 cohort. These findings are in keeping with the hypothesis of a different patho-biological mechanism occurring in tris12 CLL, which however remains to be fully elucidated. Disclosures D'Arena: Janssen-Cilag: Honoraria. Rossi:Gilead: Honoraria, Research Funding; Abbvie: Honoraria; Janseen: Honoraria. Gaidano:Janssen: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; Morphosys: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Shanafelt:Genentech: Research Funding; Janssen: Research Funding; Celgene: Research Funding; GlaxoSmithkKine: Research Funding; Pharmacyclics: Research Funding; Cephalon: Research Funding; Hospira: Research Funding.

Download Full-text

Lymphocyte Doubling Time As A Key Prognostic Factor To Predict Time To First Treatment In Early-Stage Chronic Lymphocytic Leukemia

Frontiers in Oncology ◽

10.3389/fonc.2021.684621 ◽

2021 ◽

Vol 11 ◽

Author(s):

Fortunato Morabito ◽

Giovanni Tripepi ◽

Riccardo Moia ◽

Anna Grazia Recchia ◽

Paola Boggione ◽

...

Keyword(s):

Prognostic Factors ◽

Chronic Lymphocytic Leukemia ◽

Doubling Time ◽

Lymphocytic Leukemia ◽

Prognostic Impact ◽

Newly Diagnosed ◽

Prognostic Role ◽

Mutational Status ◽

Binet Stage ◽

Time To First Treatment

The prognostic role of lymphocyte doubling time (LDT) in chronic lymphocytic leukemia (CLL) was recognized more than three decades ago when the neoplastic clone’s biology was almost unknown. LDT was defined as the time needed for the peripheral blood lymphocyte count to double the of the initial observed value. Herein, the LDT prognostic value for time to first treatment (TTFT) was explored in our prospective O-CLL cohort and validated in in two additional CLL cohorts. Specifically, newly diagnosed Binet stage A CLL patients from 40 Italian Institutions, representative of the whole country, were prospectively enrolled into the O-CLL1-GISL protocol (clinicaltrial.gov identifier: NCT00917540). Two independent cohorts of newly diagnosed CLL patients recruited respectively at the Division of Hematology in Novara, Italy, and at the Hospital Clinic in Barcelona, Spain, were utilized as validation cohorts. In the training cohort, TTFT of patients with LDT >12 months was significantly longer related to those with a shorter LDT. At Cox multivariate regression model, LDT ≤ 12 months maintained a significant independent relationship with shorter TTFT along with IGHV unmutated (IGHVunmut) status, 11q and 17p deletions, elevated β2M, Rai stage I-II, and NOTCH1 mutations. Based on these statistics, two regression models were constructed including the same prognostic factors with or without the LDT. The model with the LTD provided a significantly better data fitting (χ2 = 8.25, P=0.0041). The risk prediction developed including LDT had better prognostic accuracy than those without LDT. Moreover, the Harrell’C index for the scores including LDT were higher than those without LDT, although the accepted 0.70 threshold exceeded in both cases. These findings were also confirmed when the same analysis was carried out according to TTFT’s explained variation. When data were further analyzed based on the combination between LDT and IGHV mutational status in the training and validation cohorts, IGHVunmut and LDT>12months group showed a predominant prognostic role over IGHVmut LTD ≤ 12 months (P=0.006) in the O-CLL validation cohort. However, this predominance was of borden-line significance (P=0.06) in the Barcelona group, while the significant prognostic impact was definitely lost in the Novara group. Overall, in this study, we demonstrated that LDT could be re-utilized together with the more sophisticated prognostic factors to manage the follow-up plans for Binet stage A CLL patients.

Download Full-text

Phase 1/2 Study of Venetoclax with Low-Dose Cytarabine in Treatment-Naive, Elderly Patients with Acute Myeloid Leukemia Unfit for Intensive Chemotherapy: 1-Year Outcomes

Blood ◽

10.1182/blood.v130.suppl_1.890.890 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 890-890

Author(s):

Andrew Wei ◽

Stephen A. Strickland ◽

Gail J. Roboz ◽

Jing-Zhou Hou ◽

Walter Fiedler ◽

...

Keyword(s):

Myeloid Leukemia ◽

Research Funding ◽

Low Dose ◽

Intensive Chemotherapy ◽

Employment Equity ◽

Advisory Committees ◽

Treatment Naïve ◽

Equity Ownership ◽

Low Dose Cytarabine

Abstract Background: Older patients with acute myeloid leukemia (AML) who are ineligible for intensive chemotherapy are unlikely to achieve remission with available therapy and have unacceptably short survival. Venetoclax (VEN) is a small molecule inhibitor of BCL-2 that achieved remission rates of >60% combined with low-dose cytarabine (LDAC). Presented are long-term outcomes, including 1-year overall survival (OS) and biomarker analyses. Methods: This phase 1b/2, open-label study (NCT02287233) evaluates the safety and preliminary efficacy of orally administered VEN combined with LDAC in patients ≥65 years with previously untreated AML (except for hydroxyurea). Patients were ineligible for intensive chemotherapy because of comorbidity or other factors and had an ECOG performance score of 0-2, with adequate hepatic and renal function. Exclusion criteria were acute promyelocytic leukemia, active CNS involvement with AML, concominant use of moderate or strong CYP3A inhibitors, or prior treament with cytarabine for a preexisting myeloid disorder. Prior treatment for myelodysplastic syndrome (MDS) was allowed. In cycle 1, VEN was started at 50 mg/day PO and increased over a 5-day ramp-up to reach the designated cohort dose of 600 or 800 mg/day on day 6, which was continued through day 28. In subsequent cycles, the desingated dose of VEN 600 or 800 mg/day was administered on days 1-28. LDAC 20 mg/m2/day SQ was given on days 1-10 of each cycle. Preliminary efficacy was assessed as the overall response rate (ORR, which included complete remission [CR], CR with incomplete blood count recovery [CRi], and partial remission [PR]). Adverse events (AEs) and laboratory values were monitored. Exploratory analysis of biomarkers (eg, cytogenetics, molecular markers) was performed to identify potential predictors of clinical outcomes. Results: Data cutoff was May 30, 2017. All 71 patients were enrolled ≥1 year prior (46 [65%] male; median age, 74 years [range, 66-87 years]): 10 received VEN 800 mg and 61 received VEN 600 mg, the recommended phase 2 dose. Thirty-three patients (47%) had a history of antecedent hematologic disorder (AHD), most commonly MDS. Among 61 patients given VEN 600 mg, median time on VEN treatment was 6 months (range, <1 to 21 months). Thirty-eight (62%) of these patients achieved CR/CRi with a median duration of CR/CRi of 14.9 months (95% CI, 5.6 months to not reached [NR]; Figure). Best responses were 26% CR, 36% CRi, and 2% PR. Median OS was 11.4 months (95% CI, 5.7-15.7 months); the observed 12-month OS was 46% (95% CI, 33-58%). Only 1 patient has subsequently undergone bone marrow transplantation. Treatment-emergent grade 3/4 AEs (in ≥20% of 61 patients) were thrombocytopenia (59%), neutropenia (46%), febrile neutropenia (36%), anemia (28%), and decreased WBC count (26%). One case (2%) of tumor lysis syndrome occurred. Serious AEs (in ≥3 of 61 patients) were febrile neutropenia (20%), malignant neoplasm progression (13%), lung infection/pneumonia (13%), and sepsis (7%). The 30-day mortality rate was 3%; causes of death were disease progression (n=1) and lung infection (n=1). Common recurrent mutations in 53 patients who received VEN 600 mg are shown in the Table. All patients with an NPM1 mutation (including 3 with a co-mutation in FLT3-ITD) achieved CR/CRi. Patients with DNMT3A, FLT3-ITD, and SRSF2 mutations had CR/CRi rates of ≥75%, whereas those with TP53 mutations had the lowest CR/CRi rates of 44%. For patients with CR/CRi, median OS was 18.4 months (95% CI, 13.5 months to NR). The 12-month OS rate for patients in the 600-mg VEN cohort who achieved CR/CRi was 70.4% from Kaplan-Meier estimates, with 11 deaths. Among 19 patients who received study treatment ≥12 months, 17 remain alive. The longest, ongoing, disease-free follow-up after treatment completion is 12 months. Conclusions: The safety profile of VEN 600 mg/day plus LDAC was acceptable for elderly patients with treatment-naive AML who were ineligible for intensive chemotherapy. After ≥1 year of follow-up, the observed median OS was 11.4 months. This cohort included 44% (27/61) of patients with AHDs. Corelations of specified AML mutations with response and duration should be confirmed in later trials. Due to the observced CR/CRi rate of 62%, extended duration of response, and encouraging OS in a cohort of patients with particularly poor-risk features, the 600-mg dose of VEN combined with LDAC is being tested in an ongoing phase 3 study. Figure Figure. Disclosures Wei: AbbVie, Celgene, Novartis, Amgen, Servier: Honoraria; AbbVie, Celgene, Servier: Research Funding; AbbVie, Celgene, Novartis, Amgen, Servier: Membership on an entity's Board of Directors or advisory committees. Strickland: Boehringer-Ingelheim: Consultancy, Research Funding; Sunesis: Consultancy, Research Funding; Novartis: Consultancy; Tolero: Consultancy; Astellas: Consultancy; CTI BioPharma: Consultancy; Baxalta: Consultancy. Roboz: AbbVie, Agios, Amgen, Amphivena, Array Biopharma Inc., Astex, AstraZeneca, Celator, Celgene, Clovis Oncology, CTI BioPharma, Genoptix, Immune Pharmaceuticals, Janssen Pharmaceuticals, Juno, MedImmune, MEI Pharma, Novartis, Onconova, Pfizer, Roche Pharmace: Consultancy; Cellectis: Research Funding. Hou: Teva Oncology, Seattle Genetics: Speakers Bureau. Fiedler: Amgen, Pfizer: Research Funding; Amgen, Gilead, GSO, Teva, Jazz Pharmaceuticals: Other: Support for meeting attendance; Amgen: Patents & Royalties; Amgen, ARIAD/Incyte: Membership on an entity's Board of Directors or advisory committees. Lin: Jazz Pharmaceuticals: Consultancy. Walter: ADC Therapeutics: Research Funding; Aptevo Therapeutics: Research Funding. Chyla: Abbvie: Employment, Equity Ownership. Popovic: AbbVie: Employment, Equity Ownership. Fakouhi: AbbVie: Employment, Equity Ownership. Shah: AbbVie: Employment, Equity Ownership. Dunbar: AbbVie: Employment, Equity Ownership. Xu: AbbVie: Employment, Equity Ownership. Mabry: AbbVie: Employment, Equity Ownership. Hayslip: AbbVie: Employment, Equity Ownership.

Download Full-text

Sensitivity of Ibrutinib Exposed Chronic Lymphocytic Leukemia B-Cells to Inhibition of Axl Receptor Tyrosine Kinase

Blood ◽

10.1182/blood.v128.22.2020.2020 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2020-2020

Author(s):

Sutapa Sinha ◽

Justin C Boysen ◽

Kari G. Chaffee ◽

Brian F Kabat ◽

Susan L. Slager ◽

...

Keyword(s):

B Cells ◽

Board Of Directors ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Employment Equity ◽

Advisory Committees ◽

Mutation Status ◽

Axl Receptor Tyrosine Kinase ◽

Equity Ownership

Abstract Introduction: The use of B-cell receptor (BCR) signal inhibitors-based therapies (e.g., Ibrutinib) for B-chronic lymphocytic leukemia (CLL) was initiated just a few years ago but has rapidly escalated due to their clinical efficacy and relative ease of use. However newer therapeutic approaches are needed due to multiple issues including the continued need to improve complete responses and reduce toxicity profiles. To that end our group has discovered a novel membrane target in the ubiquitous presence of Axl receptor tyrosine kinase (Axl RTK) on CLL B-cells and has reported that the Axl RTK inhibitor TP-0903 is able to induce apoptosis of CLL B-cells at nanomolar doses (Sinha, Clin Cancer Res, 2015). Given this we assessed if TP-0903 would be effective in the induction of apoptosis of leukemic B-cells from CLL patients who are currently on Ibrutinib therapy or whom have relapsed while on Ibrutinib treatment. Methods: Relapsed/refractory CLL patients (n=22) who were placed on Ibrutinib for progressive disease provided blood samples at a median of 3.2 months after Ibrutinib therapy initiation for these studies. We also obtained sequential samples on 8 patients from initial start of ibrutinib therapy and then over a 6 month follow-up period. CLL B-cells from these blood samples were subject to Ficoll separation, purified by using a Rosette Sep B-cell enrichment kit and then studied by flow cytometry to determine Axl RTK expression levels by flow cytometric analysis. Purified CLL B-cells (CD19+/CD5+) were cultured with TP-0903 in vitroat increasing doses (0.01µM - 0.50µM) for 24 hours and the LD50 dose was determined. In addition, 3 CLL patients who had been on Ibrutinib therapy and had a documented relapse were studied in similar fashion using TP-0903. LD50-sensitivity was measured. "LD50-sensitivity" was defined as an LD50 ≤0.50µM and "insensitive" was defined as an LD50 dose >0.50µM. CLL prognostic factors (e.g., FISH, IGHV mutation status, Rai stage, CD38, and CD49d) were evaluated at the time of ibrutinib treatment. Differences in factors between sensitive and insensitive cases were computed using the Kruskal-Wallis test for continuous variables and Chi-square test for categorical variables. Results: Twenty-two CLL patients (5 female, 17 male) were included in the analysis. Fourteen (64%) patients were found to be TP-0903 LD50-sensitive. Axl expression on CLL B-cells for this cohort was heterogeneous with a median of CD19+/CD5+ cells positive for Axl at 69.9% (range of 2.7-91.3%). The sensitive subjects tended to be younger with a median age at Ibrutinib treatment initiation of 62 vs 75.5 years (p=0.004). There were no significant differences in gender, FISH, IGHV mutation status, CD38, CD49d, or Rai stage between the sensitive and insensitive LD50 groups. There were no significant differences in relation to median Axl expression on CLL B-cells (sensitive: 72.6%, range: 2.7-91.3%; insensitive: 41.5%, range: 16.5-83.1%; p=0.35). The median number of treatments prior to initiation of ibrutinib did not differ between sensitivity groups (sensitive: 2.53, range: 8-10; insensitive: 43.5, range 12-20; p=0.2833). Association for ZAP70+ CLL B-cells tended to have more apoptosis induction by TP-0903 (sensitive: 84.6% ZAP70+; insensitive: 42.9% ZAP70+; p=0.052). In 8 CLL patients that were studied sequentially while on Ibrutinib continued to express Axl or increased their Axl expression (n=2) over a 3-6 month follow-up period. Three CLL patients who had relapsed on Ibrutinib were sensitive to TP-0903 with LD50 values of ≤0.50µM. Summary: Here we find that CLL B-cells from over 60% of relapsed CLL patients on Ibrutinib therapy were highly sensitive to the high-affinity Axl inhibitor TP-0903 with induction of apoptosis at nanomolar doses (≤0.50µM). The sensitivity of CLL B-cells to TP-0903 appears to be independent of Axl expression levels and of the known CLL prognostic factors but more evident for younger patients and for ZAP70+ expression status. Given this level of activity for apoptosis induction of CLL B-cells by TP-0903 encourages the further testing of this drug in clinical trials for CLL patients. Disclosures Parikh: Pharmacyclics: Honoraria, Research Funding. Shanafelt:Pharmacyclics: Research Funding; Janssen: Research Funding; Genentech: Research Funding; GlaxoSmithKline: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Hospira: Research Funding. Warner:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Bearss:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Kay:Pharmacyclics: Research Funding; Tolero Pharmaceuticals: Research Funding; Acerta: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Morpho-Sys: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Download Full-text