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STAT Pathway upon Dasatinib Induced Apoptosis for CML Cell Model K562
Abstracts 312 Normalization of B2-microglobulin During the First Year of Treatment is Predictive for Prolonged Progression-free and Overall Survival in CLL Treated with FCR or Ibrutinib-based Regimens Philip A. Thompson, Michael J. Keating, Jan A. Burger, Nitin Jain, Alessandra Ferrajoli, Zeev Estrov, Susan M. O’Brien, William G. Wierda Department of Leukemia, University of Texas MD Anderson Cancer Center, Houston, United States
Introduction: In CLL, b2-microglobulin (B2M) level correlates with disease stage and tumor burden. High level at diagnosis is independently associated with shorter time to first therapy and when treatment is required, with poorer event-free and overall survival. However, the prognostic and predictive significance of changes in B2M during treatment have not been reported. Methods: We analyzed 198 treatment-naïve (TN) patients treated with fludarabine, cyclophosphamide and rituximab (FCR), 83 treated with ibrutinib-based regimens (66/83 relapsed/refractory) and 36 TN patients treated with idelalisib plus rituximab (IdR) to determine whether changes in B2M would be predictive of outcome. Results: B2M over time Median B2M at baseline was higher in ibrutinib-treated patients compared to FCR or IdR (4.1 vs 3.6 vs 3.65 mg/L, respectively p¼0.02) and there was a higher incidence of high-risk baseline patient characteristics; 35/83 (42%) ibrutinib-treated patients had del(17p) compared to 11/198 (6%) FCR patients and 7/36 (19%) IdR patients. Normalization of B2M by one year after treatment initiation was more frequent in ibrutinib-treated patients compared to FCR-treated patients or IdRtreated patients (63% vs 48% vs 16%, p <0.001) and median time to normalization of B2M was shorter (median 5.4 vs 8.3 vs 22.1mo, respectively). Survival analysis Median follow-up was 23.8 months (mo), range 5.5-45.1 for Ibrutinib-treated patients, 41.5mo (14.469.1) for FCR-treated patients and 34.2mo (4.5-40.8) for IdRtreated patients. Landmark analysis from the time of the lowest B2M level obtained during the 1st yr of treatment was performed; normalization of B2M was associated with superior progression-free (PFS) and overall survival (OS) in FCR- and ibrutinib-treated patients, independent of baseline B2M and other baseline prognostic features. On multivariable analysis (MVA) of ibrutinib-treated patients, fludarabine-refractory disease [HR 3.27(1.2-8.8), p¼0.019] and normalization of B2M [HR 0.3 (0.1-0.9), p¼0.029] were independently associated with PFS. These did not retain significance in MVA for OS. In FCR-treated patients, on MVA, normalization of B2M was strongly associated with improved survival [HR 0.13 (0.03-0.58), p¼0.008]. 78 of 98 patients who achieved MRD-negative CR with FCR normalized their B2M. These patients had significantly longer PFS (p¼0.048) and survival (p¼0.012) than those who did not normalize B2M. Conclusions: Normalization of B2M within 1yr of commencing treatment for CLL is an independent predictor of PFS and OS. These results require prospective confirmation.
Chronic Myelogenous Leukemia
401 Investigating The Role of JAK/STAT Pathway upon Dasatinib Induced Apoptosis for CML Cell Model K562 Ceyda Tunakan Dalgiç,1 Burçin Tezcanli Kaymaz,2 Ays¸egül Dalmizrak,2 Melda Cömert Özkan,3 Buket Kosova,2 Fahri S¸ahin,3 Güray Saydam3 1
Ege University Medical Faculty Department of Internal Medicine; Ege University Medical Faculty Department of Medical Biology; 3Ege University Medical Faculty Department of Haematology, _Izmir, Turkey 2
Aim: STAT5A and STAT5B genes; members of JAK-STAT signaling pathway; are nuclear transcription factors that are responsible for activating the genes that exhibit increased expression in hemato- logical malignancies and signal transduction. Dasatinib prevents the gained imatinib resistance, responsible for MDR genes’ overexpression and BCR-ABL kinase region mutations by activating signalling ways of SRC kinase family (LYN, HCK) and by inhibiting BCR-ABL, SRC kinase family (SRC, LCK, YES, FYN), c-KIT,EPHA2,PDGFR kinases.Investigating the apoptotic case of leukemic cells and evaluating the transcriptional changes of STAT5A and STAT5B following dasatinib treatment on CML model cell line K562 was aimed. Methods: The cytotoxic effective dose IC50 of dasatinib upon K562 cells was determined via XTT method. The apoptotic case of the cells was assessed spectrophotometrically by ‘Cell Death Detecton Kit’ after applying of IC50 dose between 24e96 hours. Target genes’ expression levels were also determined by real time qRT-PCR following dasatinib treatment for the same time interval. Protein expression analyses were performed by Western Blot analysis according to ‘WesternBreeze Chromogenic Kit-Anti-Rabbit’ kit manuel instructions. Statistical analyses were done by GraphPad prism software with a significance of p<0.05. Results: The IC50 dose of dasatinib was determined as 3.3 nM for 48th hour. When we compared the apoptosis rate of dasatinib treated/untreated cells’; a 4.5 fold apoptosis induction was assessed at 96th hour in dasatinib applied group (p<0.0001). When we compared target genes’ mRNA expression levels, while STAT5A expression was decreased 1.5 fold (by %33.2 inhibition) at 96th hour (p¼0.02), STAT5B exhibited a 4.47 fold downregulation (by %77.6 inhibition) for the same hour (p<0.001). STAT5A and STAT5B protein expression levels were significantly supressed at 96th hour and these protein expression results were in the same line with mRNA expression results. Conclusion: One possible reason of dasatinib induced leukemic cell apoptosis might be due to significant decrease in STAT5A and STAT5B expression levels that are transcription factors and exhibit upregulated expression in leukemia. Therefore, STAT5A and STAT5B are important moleculer targets in the research of CML pathogenesis.
Clinical Lymphoma, Myeloma & Leukemia June 2015
- S209
Abstracts Table Review of the Literature
Period
Patients (median age, range)
Disease Phase at Conditioning
Donor Type/ Stem Cell Source
Conditioning
OS
EFS/LFS
TRM/NRM
Shimoni et al1
2005-2006
n¼21 (45 y, 16-59)
CP1¼5, Adv¼16
MSD¼7 MUD¼13 Haplo¼1 BM¼NA PB¼NA
MAC¼14 RIC¼7
64% (2y)
46% (2y)
7% (2y)
Luo et al2
June 2005October 2007
n¼28 (26 y, 17-49)
Cp1¼28
RIC¼28
81% (3y)
67% (3y)
15% (3 y)
Saussele of et al3
March 2003November 2008
n¼84 (37 y, 16-62)
Cp1¼56, Adv¼28
MSD¼13 MUD¼15 BM¼7 PB¼7 MSD¼30 MUD¼54 BM¼20 PB¼64
MAC¼57 RIC¼11 Others¼ 16
91% (3y)
88% (3y)
8% (3y)
n¼335 (37 y, 18-58) n¼1.716 (40 y, 1-68)
CP¼229, Adv¼106 CP1¼1.084, Adv¼542
MSD¼335 BM¼335 MSD¼773 MUD¼932 BM¼640 PB¼1.069
MAC¼335
71% (3y)
69% (3y)
14% {3 y)
MAC¼724 RIC¼147 Others¼800
70% (5y)
41% (5y)
28% (5y)
MSD¼34 MUD¼5 BM¼35 PB¼4 MSD¼13 MUD¼14 BM¼4 PB¼23 CB¼3
MAC¼38 Others¼1
71% (1 y) 57% (3 y) 54% (5 y)
74% (1y) 63% (3y) 49% (5y)
24% (1y) 32% (3y) 35% (5y)
MAC¼24 RIC¼6
76% (1 y) 71% (3 y) 71% (5 y)
76% (1y) 66% (3y) 58% (5y)
21% (1y) 21% (3y) 21% (5y)
Copelan of et al4
1978-1998
Bacher et al5
1998-2004
Group A
January 1989December 2001
n¼39 (35 y, 16-53)
CP¼32, Adv¼7
Group B
January 2002December 2013
n¼30 (42 y, 15-64)
CP¼13, Adv¼17
402 A retrospective study of the allogeneic hematopoietic stem cell transplantation in Ph+ leukemia patients: the 25 years’ experience of Gustave Roussy Cancer Center A.N. Chamseddine, A. Granier, S. Wittnebel, C. Willekens, S. De Botton, J.H. Bourhis Medicine Department, Hematology and Hematopoietic Stem Cell Transplantation Unit, GUSTAVE ROUSSY Cancer Campus, Villejuif, France
Context: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for Philadelphia-positive (Ph+) chronic and acute leukemia, but its outcome is not well described in the tyrosine kinase inhibitor (TKI) era. Objectives: We compared the outcome of Ph+ leukemia patients who received a first alloHSCT in our center in pre-TKI era (Group A) and in TKI era (Group B). The primary endpoint was to compare OS, LFS, RI and NRM of both groups. The secondary endpoint was to underline in the TKI era the impact of pre-transplant minimal residual disease (MRD) on outcome. Design: We screened the registry of the ProMISe (Project Manager Internet Server) which is the central data
S210
-
Clinical Lymphoma, Myeloma & Leukemia June 2015
management system used by the EBMT. 69 patients were included. Group A (n¼39) represented patients treated in pre-TKI era from January 1989 till December 2001 and Group B (n¼30) represented those treated in TKI era from January 2002 till December 2013. The median follow-up was 61.7 months (range 1.1-240.1). Additional analysis was performed in Group B patients for whom detailed TKIs and MRD data were collected. Setting: Gustave Roussy Cancer Center which is a private non-profit center, in the department of HSCT, at Villejuif, France. Results: The OS, LFS, RI and NRM in Groups A and B were respectively at 3 years (p > 0.05): 57% (95% CI,42%-73%) and 71% (95% CI,54%88%), 63% (95% CI,48%-79%) and 66% (95% CI,48%-85%), 5% (95% CI,1%-20%) and 16% (95% CI,7%-40%), 32% (95% CI,20%-50%) and 21% (95% CI,10%-43%). In Group B, the OS, LFS and NRM between patients having a pre-transplant MRD value > 0.01 and those having a value < 0.01 were respectively at 3 years ( p < 0.05): 46% (95% CI,19%73%) and 94% (95% CI,81%-100%), 43% (95% CI,14%-72%) and 83% (95% CI,61%-100%), 38% (95% CI,19%-76%) and 6% (95% CI,0%42%). Conclusion: Our data, although not statistically significant, suggest better outcomes for Ph+ leukemia patients undergoing alloHSCT in the TKI-era. In addition we statistically highlight the importance of obtaining low pre-transplant MRD burden. The latter appears as a factor of favorable post-transplant outcome.
Introduction: In CLL, b2-microglobulin (B2M) level correlates with disease stage and tumor burden. High level at diagnosis is independently associated with shorter time to first therapy and when treatment is required, with poorer event-free and overall survival. However, the prognostic and predictive significance of changes in B2M during treatment have not been reported. Methods: We analyzed 198 treatment-naïve (TN) patients treated with fludarabine, cyclophosphamide and rituximab (FCR), 83 treated with ibrutinib-based regimens (66/83 relapsed/refractory) and 36 TN patients treated with idelalisib plus rituximab (IdR) to determine whether changes in B2M would be predictive of outcome. Results: B2M over time Median B2M at baseline was higher in ibrutinib-treated patients compared to FCR or IdR (4.1 vs 3.6 vs 3.65 mg/L, respectively p¼0.02) and there was a higher incidence of high-risk baseline patient characteristics; 35/83 (42%) ibrutinib-treated patients had del(17p) compared to 11/198 (6%) FCR patients and 7/36 (19%) IdR patients. Normalization of B2M by one year after treatment initiation was more frequent in ibrutinib-treated patients compared to FCR-treated patients or IdRtreated patients (63% vs 48% vs 16%, p <0.001) and median time to normalization of B2M was shorter (median 5.4 vs 8.3 vs 22.1mo, respectively). Survival analysis Median follow-up was 23.8 months (mo), range 5.5-45.1 for Ibrutinib-treated patients, 41.5mo (14.469.1) for FCR-treated patients and 34.2mo (4.5-40.8) for IdRtreated patients. Landmark analysis from the time of the lowest B2M level obtained during the 1st yr of treatment was performed; normalization of B2M was associated with superior progression-free (PFS) and overall survival (OS) in FCR- and ibrutinib-treated patients, independent of baseline B2M and other baseline prognostic features. On multivariable analysis (MVA) of ibrutinib-treated patients, fludarabine-refractory disease [HR 3.27(1.2-8.8), p¼0.019] and normalization of B2M [HR 0.3 (0.1-0.9), p¼0.029] were independently associated with PFS. These did not retain significance in MVA for OS. In FCR-treated patients, on MVA, normalization of B2M was strongly associated with improved survival [HR 0.13 (0.03-0.58), p¼0.008]. 78 of 98 patients who achieved MRD-negative CR with FCR normalized their B2M. These patients had significantly longer PFS (p¼0.048) and survival (p¼0.012) than those who did not normalize B2M. Conclusions: Normalization of B2M within 1yr of commencing treatment for CLL is an independent predictor of PFS and OS. These results require prospective confirmation.
Chronic Myelogenous Leukemia
401 Investigating The Role of JAK/STAT Pathway upon Dasatinib Induced Apoptosis for CML Cell Model K562 Ceyda Tunakan Dalgiç,1 Burçin Tezcanli Kaymaz,2 Ays¸egül Dalmizrak,2 Melda Cömert Özkan,3 Buket Kosova,2 Fahri S¸ahin,3 Güray Saydam3 1
Ege University Medical Faculty Department of Internal Medicine; Ege University Medical Faculty Department of Medical Biology; 3Ege University Medical Faculty Department of Haematology, _Izmir, Turkey 2
Aim: STAT5A and STAT5B genes; members of JAK-STAT signaling pathway; are nuclear transcription factors that are responsible for activating the genes that exhibit increased expression in hemato- logical malignancies and signal transduction. Dasatinib prevents the gained imatinib resistance, responsible for MDR genes’ overexpression and BCR-ABL kinase region mutations by activating signalling ways of SRC kinase family (LYN, HCK) and by inhibiting BCR-ABL, SRC kinase family (SRC, LCK, YES, FYN), c-KIT,EPHA2,PDGFR kinases.Investigating the apoptotic case of leukemic cells and evaluating the transcriptional changes of STAT5A and STAT5B following dasatinib treatment on CML model cell line K562 was aimed. Methods: The cytotoxic effective dose IC50 of dasatinib upon K562 cells was determined via XTT method. The apoptotic case of the cells was assessed spectrophotometrically by ‘Cell Death Detecton Kit’ after applying of IC50 dose between 24e96 hours. Target genes’ expression levels were also determined by real time qRT-PCR following dasatinib treatment for the same time interval. Protein expression analyses were performed by Western Blot analysis according to ‘WesternBreeze Chromogenic Kit-Anti-Rabbit’ kit manuel instructions. Statistical analyses were done by GraphPad prism software with a significance of p<0.05. Results: The IC50 dose of dasatinib was determined as 3.3 nM for 48th hour. When we compared the apoptosis rate of dasatinib treated/untreated cells’; a 4.5 fold apoptosis induction was assessed at 96th hour in dasatinib applied group (p<0.0001). When we compared target genes’ mRNA expression levels, while STAT5A expression was decreased 1.5 fold (by %33.2 inhibition) at 96th hour (p¼0.02), STAT5B exhibited a 4.47 fold downregulation (by %77.6 inhibition) for the same hour (p<0.001). STAT5A and STAT5B protein expression levels were significantly supressed at 96th hour and these protein expression results were in the same line with mRNA expression results. Conclusion: One possible reason of dasatinib induced leukemic cell apoptosis might be due to significant decrease in STAT5A and STAT5B expression levels that are transcription factors and exhibit upregulated expression in leukemia. Therefore, STAT5A and STAT5B are important moleculer targets in the research of CML pathogenesis.
Clinical Lymphoma, Myeloma & Leukemia June 2015
- S209
Abstracts Table Review of the Literature
Period
Patients (median age, range)
Disease Phase at Conditioning
Donor Type/ Stem Cell Source
Conditioning
OS
EFS/LFS
TRM/NRM
Shimoni et al1
2005-2006
n¼21 (45 y, 16-59)
CP1¼5, Adv¼16
MSD¼7 MUD¼13 Haplo¼1 BM¼NA PB¼NA
MAC¼14 RIC¼7
64% (2y)
46% (2y)
7% (2y)
Luo et al2
June 2005October 2007
n¼28 (26 y, 17-49)
Cp1¼28
RIC¼28
81% (3y)
67% (3y)
15% (3 y)
Saussele of et al3
March 2003November 2008
n¼84 (37 y, 16-62)
Cp1¼56, Adv¼28
MSD¼13 MUD¼15 BM¼7 PB¼7 MSD¼30 MUD¼54 BM¼20 PB¼64
MAC¼57 RIC¼11 Others¼ 16
91% (3y)
88% (3y)
8% (3y)
n¼335 (37 y, 18-58) n¼1.716 (40 y, 1-68)
CP¼229, Adv¼106 CP1¼1.084, Adv¼542
MSD¼335 BM¼335 MSD¼773 MUD¼932 BM¼640 PB¼1.069
MAC¼335
71% (3y)
69% (3y)
14% {3 y)
MAC¼724 RIC¼147 Others¼800
70% (5y)
41% (5y)
28% (5y)
MSD¼34 MUD¼5 BM¼35 PB¼4 MSD¼13 MUD¼14 BM¼4 PB¼23 CB¼3
MAC¼38 Others¼1
71% (1 y) 57% (3 y) 54% (5 y)
74% (1y) 63% (3y) 49% (5y)
24% (1y) 32% (3y) 35% (5y)
MAC¼24 RIC¼6
76% (1 y) 71% (3 y) 71% (5 y)
76% (1y) 66% (3y) 58% (5y)
21% (1y) 21% (3y) 21% (5y)
Copelan of et al4
1978-1998
Bacher et al5
1998-2004
Group A
January 1989December 2001
n¼39 (35 y, 16-53)
CP¼32, Adv¼7
Group B
January 2002December 2013
n¼30 (42 y, 15-64)
CP¼13, Adv¼17
402 A retrospective study of the allogeneic hematopoietic stem cell transplantation in Ph+ leukemia patients: the 25 years’ experience of Gustave Roussy Cancer Center A.N. Chamseddine, A. Granier, S. Wittnebel, C. Willekens, S. De Botton, J.H. Bourhis Medicine Department, Hematology and Hematopoietic Stem Cell Transplantation Unit, GUSTAVE ROUSSY Cancer Campus, Villejuif, France
Context: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for Philadelphia-positive (Ph+) chronic and acute leukemia, but its outcome is not well described in the tyrosine kinase inhibitor (TKI) era. Objectives: We compared the outcome of Ph+ leukemia patients who received a first alloHSCT in our center in pre-TKI era (Group A) and in TKI era (Group B). The primary endpoint was to compare OS, LFS, RI and NRM of both groups. The secondary endpoint was to underline in the TKI era the impact of pre-transplant minimal residual disease (MRD) on outcome. Design: We screened the registry of the ProMISe (Project Manager Internet Server) which is the central data
S210
-
Clinical Lymphoma, Myeloma & Leukemia June 2015
management system used by the EBMT. 69 patients were included. Group A (n¼39) represented patients treated in pre-TKI era from January 1989 till December 2001 and Group B (n¼30) represented those treated in TKI era from January 2002 till December 2013. The median follow-up was 61.7 months (range 1.1-240.1). Additional analysis was performed in Group B patients for whom detailed TKIs and MRD data were collected. Setting: Gustave Roussy Cancer Center which is a private non-profit center, in the department of HSCT, at Villejuif, France. Results: The OS, LFS, RI and NRM in Groups A and B were respectively at 3 years (p > 0.05): 57% (95% CI,42%-73%) and 71% (95% CI,54%88%), 63% (95% CI,48%-79%) and 66% (95% CI,48%-85%), 5% (95% CI,1%-20%) and 16% (95% CI,7%-40%), 32% (95% CI,20%-50%) and 21% (95% CI,10%-43%). In Group B, the OS, LFS and NRM between patients having a pre-transplant MRD value > 0.01 and those having a value < 0.01 were respectively at 3 years ( p < 0.05): 46% (95% CI,19%73%) and 94% (95% CI,81%-100%), 43% (95% CI,14%-72%) and 83% (95% CI,61%-100%), 38% (95% CI,19%-76%) and 6% (95% CI,0%42%). Conclusion: Our data, although not statistically significant, suggest better outcomes for Ph+ leukemia patients undergoing alloHSCT in the TKI-era. In addition we statistically highlight the importance of obtaining low pre-transplant MRD burden. The latter appears as a factor of favorable post-transplant outcome.