BCR-ABL Positive Chronic Myeloid Leukemia Research Articles

Concomitant BCR-ABL and JAK2 V617F in a Patient with Myeloproliferative Neoplasm: A Case Report

Myeloproliferative neoplasm (MPN) is a clonal proliferation of the haematopoietic stem cells leading to activated tyrosine kinase signaling activity. Myeloproliferative neoplasm is classified into BCR-ABL positive chronic myeloid leukemia (CML) and BCR-ABL negative MPN which harbors JAK2 V617F mutation in most cases. The genetic combination of BCR-ABL and JAK2 V617F mutation is rare with estimated frequency of 0.4% based on recent study. Herein, we reported a case of a man diagnosed with CML detected by fluorescence in-situ hybdridisation (FISH) showing atypical BCR-ABL fusion pattern in 29% nucleated cells (cut-off levels ≥5% for positive signals) in the presence of JAK2 V617F mutation. However, the BCR-ABL transcript was not detected by the conventional reverse transcriptase-polymerase chain reaction (RT-PCR) method which was specific for major fragments. Interestingly, complete hematological remission was not achieved despite initiation of tyrosine kinase inhibitor (Imatinib). In conclusion, it is imperative to scrutinise CML cases for concomitant JAK2 V617F mutation especially patients with atypical clinical or laboratory findings. Therefore, close monitoring with clinical and ancillary technique especially FISH and molecular methods such as DNA sequencing were crucial to help achieve complete hematological, cytogenetic and deep molecular response alongside targeted therapy.

A Ten-Year Experience of Treating Chronic Myeloid Leukemia in Rural Rwanda: Outcomes and Insights for a Changing Landscape.

In describing our ten-year experience with treating chronic myeloid leukemia (CML) as part of the Glivec Patient Assistance Program (GIPAP) in rural Rwanda, we evaluate (1) patient characteristics and treatment outcomes, (2) resource-adapted management strategies, and (3) the impact of diagnostic capacity development. We retrospectively reviewed all patients with BCR-ABL-positive CML enrolled in this GIPAP program between 2009 and 2018. Clinical data were analyzed using descriptive statistics, Kaplan-Meier methods, proportional hazards regression, and the Kruskal-Wallis test. One hundred twenty-four patients were included. The median age at diagnosis was 34 (range 8-81) years. On imatinib, 91% achieved complete hematologic response (CHR) after a median of 49 days. Seven (6%) and 12 (11%) patients had primary and secondary imatinib resistance, respectively. The 3-year overall survival was 80% (95% CI, 72 to 87) for the cohort, with superior survival in imatinib responders compared with those with primary and secondary resistance. The median time from imatinib initiation to CHR was 59 versus 38 days (P = .040) before and after in-country diagnostic testing, whereas the median time to diagnosis (P = .056) and imatinib initiation (P = .170) was not significantly different. Coupling molecular diagnostics with affordable access to imatinib within a comprehensive cancer care delivery program is a successful long-term strategy to treat CML in resource-constrained settings. Our patients are younger and have higher rates of imatinib resistance compared with historic cohorts in high-income countries. High imatinib resistance rates highlight the need for access to molecular monitoring, resistance testing, and second-generation tyrosine kinase inhibitors, as well as systems to support drug adherence. Hematologic response is an accurate resource-adapted predictor of survival in this setting. Local diagnostic capacity development has allowed for continuous, timely CML care delivery in Rwanda.

Repositioning antispasmodic drug Papaverine for the treatment of chronic myeloid leukemia.

Papaverine is a benzylisoquinoline alkaloid from the plant Papaver somniferum (Opium poppy). It is approved as an antispasmodic drug by the US FDA and is also reported to have anti-cancer properties. Here, Papaverine's activity in chronic myeloid leukemia (CML) is explored using Saccharomyces cerevisiae, mammalian cancer cell lines, and in silico studies. The sensitivity of wild-type and mutant (anti-oxidant defense, apoptosis) strains of S. cerevisiaeto the drug Papaverine was tested by colony formation, spot assays, and AO/EB staining. In vitro cytotoxic effect was investigated on HCT15 (colon), A549 (lung), HeLa (cervical), and K562 (Bcr-Abl positive CML), and RAW 264.7 cell lines; cell cycle, mitochondrial membrane potential, ROS detection analyzed in K562 cells using flow cytometry and apoptotic markers, Bcr-Abl signaling pathways examined by western blotting. Molecular docking and molecular dynamics simulation of Papaverine against the target Bcr-Abl were also carried out. Investigation in S. cerevisiae evidenced Papaverine induces ROS-mediated apoptosis. Subsequent in vitro examination showed that CML cell line K562 was more sensitive to the drug Papaverine. Papaverine induces ROS generation, promotes apoptosis, and inhibits Bcr-Abl downstream signaling. Papaverine acts synergistically with the drug Imatinib. Furthermore, the docking and molecular dynamic simulation studies supported that Papaverine binds to the allosteric site of Bcr-Abl. The data presented here have added support to the concept of polypharmacology of existing drugs and natural compounds to interact with more than one target. This study provides a proof-of-concept for repositioning Papaverine as an anti-CML drug.

Isolated central nervous system blast crisis in chronic myeloid leukemia - Report of two cases

AbstractExtramedullary blast crisis (BC) presenting as the central nervous system (CNS) involvement in chronic myeloid leukemia (CML) is rare and usually accompanies systemic involvement. Imatinib has shown efficacy in treating patients with CML in accelerated or blastic phases. However, CNS penetration of Imatinib is poor due to the P-glycoprotein mediated efflux mechanisms. Therefore, patients on long-term Imatinib therapy with hematological and cytogenetic remission may rarely present with CNS BC. Isolated CNS BC is uncommon and the literature is limited to only a few case reports. Here, we present two cases of isolated CNS BC in patients with BCR ABL positive CML on treatment.

"All Our Wisdom is Stored in the Trees" - Degrading BCR-ABL with Berberis Vulgaris.

Treating BCR-ABL-positive chronic myeloid leukemia remains impeded by the development of clinical resistance to imatinib. It has been demonstrated that berberine, a plant alkaloid, has activity against imatinib-resistant BCR-ABL mutants by inducing autophagic degradation of BCR-ABL, thereby preventing the acquisition of drug-resistant mutations.See related article by Yin et al., p. 4040.

BCR-ABL Gene Transcript Types of Patients with Chronic Myelogenous Leukemia in Yogyakarta, Indonesia.

Background:The aim of this study was analyzing the BCR-ABL transcript types of patients with chronic myeloid leukemia (CML) in Dr Sardjito General Hospital, Yogyakarta, Indonesia. This study is very relevant because the data concerning BCR-ABL gene transcript types is very limited in Indonesia. Furthermore, it is important for patient’s management, particularly in defining the tyrosine kinase inhibitors (TKIs) therapy and monitoring after therapy. The introduction of TKIs has become a major advance in the management of patients with CML, especially in the chronic phase (CML-CP), in which most patients are diagnosed. Methods:One hundred eighty five (185) of 370 recruited patients were included in this study (2010–2014). RNA samples were isolated from mononuclear cells of peripheral blood of the subjects taken at primary diagnosis. Detection of BCR-ABL gene transcript types was done using multiplex reverse transcriptase PCR (multiplex RT-PCR) and/or nested PCR following the cDNA synthesis. When the first PCR set failed to amplify the BCR-ABL gene, RT-conventional PCR and/or nested PCR would be applied. The proportion of each transcript type was calculated among the BCR-ABL positive CML patients. Results:Approximately 99% (183/185) of CML patients are BCR-ABL positive, with the most common type is major b3a2 (136/183; 74.3%), followed by major b2a2 (41/183; 22.4%). Two samples (1.1%) showed co-expression of b3a2 and b2a2; 1 sample showed co-expression of b3a2 and fragment at 500bp; and 3 samples showed uncommon fragments. Conclusion:Ninety nine percent (99%) of CML patients in Yogyakarta, Indonesia are BCR-ABL positive, with 74.3% have b3a2 transcript, 22.4% have b2a2 trascript, 1.1% have co-expression of b3a2 and b2a2 transcript, and the rest (2.2%) have uncommon bands that still need to be confirmed.

Ten Years of Treating Chronic Myeloid Leukemia in Rural Rwanda: Feasible Approaches for Low-Resource Settings

Objective: To describe the characteristics and outcomes of chronic myeloid leukemia (CML) patients treated through the Glivec Patient Assistance Program (GIPAP) in Rwanda. Methods: We retrospectively reviewed all BCR-ABL positive CML patients treated with imatinib at two rural hospitals between 2009 and 2018. Clinical data were analyzed using descriptive statistics, Kaplan-Meier method, and proportional hazards regression. Results: One hundred twenty-four patients were included; median age 35 (range: 9-81), 60% male, 88% without comorbidities. All patients presented with symptomatic disease, median duration 11 months (IQR: 4-24) and median WBC count 225.5 x 109/L (IQR: 117.7-308.5). At diagnosis, 71% were in chronic phase. On imatinib, 91% achieved complete hematologic remission (CHR); 67% within 90 days. Of those who achieved CHR, 51 (45%) lost it at some point and, of which, 36 (71%) regained CHR. At four years, overall survival was 79% (95% CI:69%-86%) for the entire cohort; 93% (95% CI: 79%-98%) for 53 patients who remained in CHR, 89% (95% CI: 69%-96%) for 34 patients who lost but regained CHR, 19% (95% CI: 1%-54%) for 12 patients who lost and never regained CHR. Never achieving CHR was associated with increased mortality (HR: 40.89; 95% CI: 9.88-169.32; p<0.001) as was losing CHR without regaining it (HR 15.73; 95% CI: 4.14-59.81; p<0.001) compared to never losing CHR. Conclusions: Here we demonstrate the feasibility of coupling molecular diagnostics with chronic targeted therapy in a rural low-resource setting through health systems strengthening and international collaboration. Our CML patients are younger, slower to achieve CHR with imatinib, and have higher rates of primary and early hematologic resistance than historical cohorts, suggesting biological differences that warrant further investigation. It is uncertain what role non-adherence to medication influences these data. Without molecular response assessment, hematologic response is an accurate marker of disease control and survival. Our imatinib failure rates highlight the need for access to resistance testing and second generation tyrosine kinase inhibitors, as well as adherence rates. Disclosures No relevant conflicts of interest to declare.

Has MET met its match?

The last decade has seen a revolution in both our understanding of the genomic landscape of a wide range of malignancies and the recognition that a spectrum of tumors harbors dominant oncogenic alterations leading to a unique dependence of these tumors to the presence/activity of the oncogene, called oncogene addiction. In tumors where the activity of such oncogenic gene products can be successfully inhibited pharmacologically, responses can be dramatic. Leading examples of such aberrations include BCR-ABL positive chronic myeloid leukemia, KIT-mutated gastrointestinal stromal tumor (GIST) sarcomas and epidermal growth factor receptor (EGFR)-mutated lung adenocarcinomas, and in patients with these tumors, the treatment landscape has been completely redrawn. With the advent of high-throughput genomics technology and through major international collaborative efforts, such as The Cancer Genome Atlas (TCGA), comprehensive information is now available of genomic alterations in many common and less common malignancies leading some to question whether the information to be learned about highly actionable alterations is reaching saturation. It is with this background that we will review recent major findings, including the manuscript of Frampton et al. (1), leading to a breakthrough in our understanding of the oncogenic role and long-proposed but elusive actionability of the MET tyrosine kinase.

Treatment of Chronic Myeloid Leukemia in Rural Rwanda: Promising Early Outcomes.

PurposeThe burden of cancer is rising in low- and middle-income countries, yet cancer treatment requires resources that are often not available in these settings. Although management of chronic myeloid leukemia (CML) has been described in low- and middle-income countries, few programs involve patients treated in rural settings. We describe characteristics and early outcomes of patients treated for CML at rural district hospitals in Rwanda.MethodsWe conducted a retrospective review of patients with confirmed BCR-ABL–positive CML who were enrolled between July 1, 2009 and June 30, 2014. Types of data included patient demographics, diagnostic work up, treatment, clinical examination, laboratory testing, and death.ResultsForty-three patients were included, with a maximum follow-up of 58 months. Of 31 patients who were imatinib-naïve at enrollment, 54.8% were men and the median age at diagnosis was 36.9 years (interquartile range: 29-42 years). Approximately two-thirds of patients (67.7%) were on the national public insurance scheme. The imatinib dose was reduced for 16 patients and discontinued for five. Thirty-two of the 43 patients continued to have normal blood counts at last follow-up. Four patients have died and four are lost to follow-up.ConclusionOur experience indicates that CML can be effectively managed in a resource-constrained rural setting, despite limited availability of on-site diagnostic resources or specialty oncology personnel. The importance of model public-private partnerships as a strategy to bring high-cost, life-saving treatment to people who do not have the ability to pay is also highlighted.

Gene expression profile of circulating CD34+ cells and granulocytes in chronic myeloid leukemia

PurposeWe compared the gene expression profile of peripheral blood CD34+ cells and granulocytes in subjects with chronic myeloid leukemia (CML), with the accent on signaling pathways affected by BCR–ABL oncogene. MethodsThe microarray analyses have been performed in circulating CD34+ cells and granulocytes from peripheral blood of 7 subjects with CML and 7 healthy donors. All studied BCR–ABL positive CML patients were in chronic phase, with a mean value of 2012±SD of CD34+cells/μl in peripheral blood. ResultsThe gene expression profile was more prominent in CML CD34+ cells (3553 genes) compared to granulocytes (2701 genes). The 41 and 39 genes were significantly upregulated in CML CD34+ cells (HINT1, TXN, SERBP1) and granulocytes, respectively. BCR–ABL oncogene activated PI3K/AKT and MAPK signaling through significant upregulation of PTPN11, CDK4/6, and MYC and reduction of E2F1, KRAS, and NFKBIA gene expression in CD34+ cells. Among genes linked to the inhibition of cellular proliferation by BCR–ABL inhibitor Imatinib, the FOS and STAT1 demonstrated significantly decreased expression in CML. ConclusionThe presence of BCR–ABL fusion gene doubled the expression quantity of genes involved in the regulation of cell cycle, proliferation and apoptosis of CD34+ cells. These results determined the modified genes in PI3K/AKT and MAPK signaling of CML subjects.

Survival of BCR-ABL positive and negative chronic myeloid leukemia: A propensity matched analysis of the Surveillance, Epidemiology, and End Results database.

e18053 Background: BCR-ABL negative chronic myeloid leukemia (CML) is uncommon without any previous largescale studies. Because of the genetic differences between BCR/ABL negative and positive CML,...

Effect of Imatinib on Proliferation, Apoptosis and Expression of Platelet Derived Growth Factor Receptor By Mesenchymal Stem Cells

IntroductionImatinib Mesylate (IM) is a tyrosine kinase inhibitor (TKI), which targets Platelet Derived Growth Factor Receptor (PDGF-R), c-kit and BCR-ABL and is used in the treatment of Gastrointestinal Stromal Tumors (GIST) and Chronic Myeloid Leukemia (CML). Patients using Imatinib for a long time display abnormalities in bone metabolism (Vandyke et al. J Clin Endocrinol Metab 2013). In vitro, IM affects osteoblastic differentiation through inhibition of PDGF/PDGF-Rb signaling, as well as osteoclast function/differentiation through modulation of PDGF/PDGF-Ra signaling (Berman et al. Leuk Res 2013). Conflicting reports have shown differential effects on in vivo bone marrow density. Confounding factors include patient age (pediatric ve adult), type of bone (osteochondral vs trabecular), duration and dose of IM treatment. Mesenchymal Stem Cells (MSCs) are cells with great regenerative potential and differentiate into adipogenic, osteogenic cells and multiple other cell lineages (Bianco et al. Cell Stem Cell 2008). MSCs express high cell surface levels of PDGF-Rb and intermediate levels of PDGF-Ra and are therefore likely to be affected by IM treatment. Here, we wanted to assess the effect of IM on proliferation and apoptosis of MSCs as well as on surface expression of PDGF-Rb and PDGF-Ra.MethodsHealthy human bone marrow MSCs were isolated using Ficoll and plastic adherence and cultured up till passage 3. Proliferation assays were performed using Real Time Cell Analysis (XCELLigence, Roche) to determine optimal in vitro doses of IM (Novartis). IM was used at doses from 2,5 uM to 20 uM and found to be optimal at 5 uM. PDGF-Rb (CD140b) and PDGF-Ra (CD140a) surface expression were measured using monoclonal antibodies and read using a FACSARIA (Becton Dickinson). Apoptosis was assessed using Annexin-V and Propidium Iodide. Adipogenic and osteogenic differentiation was evaluated after 21 days in differentiation media with or without IM, using spectrophotometric quantitation of levels of Oil Red O (Adipogenic differentiation) and Calcium Phosphate (Osteogenic differentiation).ResultsIM inhibited proliferation of MSCs in a dose-dependent fashion, with doses > 5 uM resulting in severe suppression of proliferation. Inhibition of proliferation by IM could be overcome by increasing cell densities of MSCs, but not by addition of PDGF-BB. Co-treatment with IM and PDGF-BB resulted in more pronounced suppression of MSC proliferation. Treatment with IM resulted in a decrease of cell surface expression of both PDGF-Rb from 97,4±2,36% to 77,3±0,13% (n=3, p<0.02) and PDGF-Ra from 18,4% to 6,4% (n=1). Addition of PDGF-BB resulted in a further decrease in cell surface expression of PDGF-Rb, but had no effect on expression of PDGF-Ra. IM increased apoptosis levels (Annexin-V positive cells) about twofold. Addition of 5 or 10 ng/mL PDGF could completely abrogate this effect. Treatment of K562, a BCR-ABL positive CML cell line, with 5 uM IM suppressed proliferation of K562 four-fold, but had no obvious effect on levels of apoptosis. Treatment of MSCs with IM during differentiation revealed no clear effect on adipogenesis, but did increase osteogenic differentiation, as measured as an increase in Calcium-Phosphate.ConclusionsImatinib Mesylate inhibits PDGF/PDGF-R signaling through interference with tyrosine kinases. Here, we found that 5 uM IM not only affects MSC proliferation through inhibition of PDGF-receptor signaling, but also through downregulation of PDGF-Rb and PDGF-Ra cell surface expression. This dose is very close to the maximal plasma concentration of 4.6 uM observed in patients (Druker et al. New England J Med 2001). Addition of PDGF-BB enhanced the effects of IM on suppression of MSC proliferation, likely through further downregulation of surface PDGF-Rb expression, thus decreasing PDGF/PDGF-R signaling. PDGF signaling has been implicated in regulation of invasiveness of cancers and TKI have been used in the treatment of several types of cancer. Whether or not combination treatment of IM with PDGF-BB similarly affects proliferation/invasiveness of BCR-ABL+ CML cells, and PDGF-Ra positive GIST cells, remains to be investigated. Prolonged treatment with IM in patients has been shown to affect bone remodeling and bone densities. Our current results suggest that treatment with IM impacts BM-resident MSCs, supporting increased osteogenesis. DisclosuresNo relevant conflicts of interest to declare.

Silencing of suppressor of cytokine signaling-3 due to methylation results in phosphorylation of STAT3 in imatinib resistant BCR-ABL positive chronic myeloid leukemia cells.

Silencing due to methylation of suppressor of cytokine signaling-3 (SOCS-3), a negative regulator gene for the JAK/STAT signaling pathway has been reported to play important roles in leukemogenesis. Imatinib mesylate is a tyrosine kinase inhibitor that specifically targets the BCR-ABL protein and induces hematological remission in patients with chronic myeloid leukemia (CML). Unfortunately, the majority of CML patients treated with imatinib develop resistance under prolonged therapy. We here investigated the methylation profile of SOCS-3 gene and its downstream effects in a BCR-ABL positive CML cells resistant to imatinib. BCR-ABL positive CML cells resistant to imatinib (K562-R) were developed by overexposure of K562 cell lines to the drug. Cytotoxicity was determined by MTS assays and IC50 values calculated. Apoptosis assays were performed using annexin V-FITC binding assays and analyzed by flow cytometry. Methylation profiles were investigated using methylation specific PCR and sequencing analysis of SOCS-1 and SOCS-3 genes. Gene expression was assessed by quantitative real-time PCR, and protein expression and phosphorylation of STAT1, 2 and 3 were examined by Western blotting. The IC50 for imatinib on K562 was 362 nM compared to 3,952 nM for K562-R (p=0.001). Percentage of apoptotic cells in K562 increased upto 50% by increasing the concentration of imatinib, in contrast to only 20% in K562-R (p<0.001). A change from non-methylation of the SOCS-3 gene in K562 to complete methylation in K562-R was observed. Gene expression revealed down- regulation of both SOCS-1 and SOCS-3 genes in resistant cells. STAT3 was phosphorylated in K562-R but not K562. Development of cells resistant to imatinib is feasible by overexposure of the drug to the cells. Activation of STAT3 protein leads to uncontrolled cell proliferation in imatinib resistant BCR-ABL due to DNA methylation of the SOCS-3 gene. Thus SOCS-3 provides a suitable candidate for mechanisms underlying the development of imatinib resistant in CML patients.

Detection of Jak2 V617f Mutation, Secondary to the Presence of Bcr-Abl1 Translocation in a Patient with Chronic Myeloid Leukemia: Report of a Case and Review of the Literature

The myeloproliferative neoplasms are classified in four major diseases: Chronic Myeloid Leukemia, Polycythemia Vera, Primary Myelofibrosis and Essential Thrombocythemia. The JAK2 V617F mutation is found in 95% of Polycythemia Vera, and 50% of Essential Thrombocythemia and Primary Myelofibrosis patients. It was thought that the JAK2 V617F mutation and BCR-ABL1 translocation were mutually exclusive; but now a few cases have been reported with both alterations. We report a rare case with the presence of JAK2 V617F mutation, secondary to a diagnosis of BCR-ABL positive chronic myeloid leukemia. The patient was initially diagnosed as chronic myeloid leukemia and was BCR-ABL1 positive, so he started to receive Imatinib. He responded well to the therapy for three years, but after this time the patient had a hematological relapse still with no detectable copies of BCR-ABL1. For this reason, we thought of the possible development of another genetic alteration. Because the patient had a very high platelet count, we decided to look for the JAK2 V617F mutation, which result was positive. This case is just one of the few that have been reported worldwide that have a coexistence of these two genetic alterations: the BCR-ABL1 transcript and JAK2 V617F mutation in chronic myeloproliferative syndromes. This is the first case in the Central American population, found in our series of a total of 168 patients with Philadelphia positive chronic myeloid leukemia.

Elderly CML Patients On Early Applied High Dose Imatinib Achieve Molecular Remissions As Fast As Younger Patients In Contrast To Patients On Standard Dose Imatinib: A Subanalysis Of The German CML-Study IV

IntroductionThe outcome of elderly patients with chronic myeloid leukemia (CML) treated with imatinib has been studied in several trials. However, there are no reports on the effects of different imatinib dosages in older vs. younger CML patients. MethodsTo evaluate the efficacy of imatinib in the elderly, we analyzed data from the German CML-Study IV, a randomized 5-arm trial designed to optimize imatinib therapy alone or in combination. There was no upper age limit for inclusion. Patients with BCR-ABL positive CML in chronic phase randomized to imatinib 400 mg/d (IM400) or imatinib 800 mg/d (IM800) were compared, stratified according to median age at diagnosis in western populations ≥ 65 years vs. < 65 years, regarding effectively administered imatinib dose, time to hematologic, cytogenetic and molecular remissions, adverse events (AEs), rates of progression to accelerated phase (AP) and blast crisis (BC), survival, and causes of death. The full 800 mg dose was given after a 6 weeks run-in period with imatinib 400 mg/d to avoid excessive cytopenias. The dose could then be reduced according to tolerability for maximum patients' compliance. ResultsFrom July 2002 through March 2012, 1,551 patients were randomized, 828 of these to IM400 or IM800. Median age of these patients was 52 years (IM400: 53 years; IM800: 51 years). 784 patients were evaluable for follow-up (IM400: 382; IM800: 402). 193 patients were ≥ 65 years, 591 < 65 years. 110 patients (29%) on IM400 and 83 (21%) on IM800 were ≥ 65 years. Median observation time on IM400 was 63.0 months in the elderly and 67.6 months in the younger group, on IM800 50.9 months in the elderly and 50.1 months in the younger group. The median dose per day was lower for elderly patients on IM800 (421 mg/d for patients ≥ 65 years vs. 556 mg/d for patients < 65 years), with the highest median dose in the first year (466mg/d for patients ≥ 65 years vs. 630mg/d for patients < 65 years). The median dose for patients on IM400 was 400 mg/d for both age groups. There was no difference between age groups in achieving a complete hematologic remission or a complete cytogenetic remission, neither if IM400 and IM800 were combined, nor in an analysis according to treatment groups. Elderly patients on IM400 achieved major molecular remission (MMR) and deep molecular remission (MR4) significantly later than younger patients (18.1 vs. 15.9 months, p=0.013; 54.4 vs. 33.3 months, p=0.012, respectively) whereas no difference was detected for patients on IM800 (11.9 vs. 10.5 months; 24.2 vs. 26.1 months, respectively). Imatinib was well tolerated in elderly patients with only few WHO grade 3-4 AEs being more frequent in the elderly than in younger patients (dermatologic AEs on IM400: 5.4 vs. 0.4%; infections on IM800: 8.3 vs. 2.5%). There were no significant differences between age groups in probabilities of progression to AP or BC neither if IM400 and IM800 were combined, nor in an analysis according to treatment groups. Five-year age-adjusted relative survival for elderly patients was comparable to that of younger patients. ConclusionWe could demonstrate that elderly patients achieved molecular remissions significantly later when treated with standard dose imatinib but not when treated with higher imatinib dosages. As the safety profile of IM800 in senior patients was favorable too we conclude, that the optimal dose for elderly patients could be higher than 400 mg/d. Disclosures:Müller:Ariad: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Hochhaus:Pfizer: Consultancy; ARIAD: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding, Travel Other. Hehlmann:BMS: Consultancy, Research Funding; Novartis: Research Funding. Saussele:BMS: Honoraria, Research Funding, Travel, Travel Other; Pfizer: Honoraria; Novartis: Honoraria, Research Funding, Travel Other.

The Pattern Of Cytogenetic Aberrations In CML BC Stratified For Treatment

IntroductionThe impact of the type of therapy on cytogenetic evolution in chronic myeloid leukemia (CML) regarding the occurrence of additional cytogenetic aberrations (ACA) at the time point of blast crisis (BC) may be critical. The aim of this analysis was to elucidate whether patients (pts) treated with imatinib (IM) had ACA less frequently than pts treated with BU and other therapies used prior to IM as hydroxyurea (HU) and interferon alpha (IFN). We comparatively analyze the BC karyotype of CML pts treated in consecutive trials of the German CML Study Group (Studies I, II and IV) to answer the following question: Does CML therapy influence the occurrence or even induce ACA or do these alterations rather reflect the natural history and the biology of the disease and are independent of therapy? Materials and methodsCytogenetic data of 157 pts with Philadelphia chromosome and BCR-ABL positive CML in BC were analyzed from a total of 2,380 pts randomized to CML study I (BU vs. HU vs. IFN, recruitment 1983 – 1991), CML study II (IFN + HU vs. HU, recruitment 1991 – 1994), and CML study IV (IM 400 mg vs. IM 800 mg vs. IM 400 mg +IFN vs. IM 400 mg + AraC vs. IM 400 mg after IFN failure recruitment 2002 – 2012). Cytogenetic analysis was reported according to ISCN 2005. ACA were divided into major route (+8, i(17)(q10), +19, +der(22)t(9;22)(q34;q11)) and minor route alterations (reciprocal translocations other than the t(9;22)(q34;q11), e.g. t(1;21), t(2;16), t(3;12), t(4;6), t(5;8), t(15;20) (Fabarius et al., Blood 2011).Confirmatory testing of pairwise comparisons of therapies with regard to their frequency of major ACAs was performed using two-sided chi-square test. To keep the level of significance at 0.05 despite multiple testing, a priori hypotheses were hierarchically ordered: First, frequency of major route ACA of pts on IM was compared with that on BU, then, with HU and with IFN. Next, the comparisons of IFN vs. BU and IFN vs. HU were planned. Cumulative incidences were estimated under consideration of death before BC as a competing risk. Results115 of 188 pts randomized to BU (CML study I), 117 of 194 pts randomized to HU (CML study I only) and 159 of 360 randomized to IFN-based therapy (CML studies I+II) progressed to BC. Eight-year cumulative incidence probability of BC was 0.63 [95%-confidence interval (CI): 0.56; 0.69], 0.60 [95%-CI: 0.53; 0.66], and 0.49 [95%-CI: 0.43; 0.54] in pts randomized to BU, HU, and IFN-based therapy, respectively and 0.06 [95%-CI: 0.04; 0.07] in pts on IM (CML-study IV). Three-year survival probabilities after BC were 0.009 [95%-CI: 0.001; 0.043] with BU, 0.017 [95%-CI: 0.003; 0.055] with HU, 0.013 [95%-CI: 0.003; 0.042] with IFN, and 0.252 [95%-CI: 0.157; 0.368] with IM.Cytogenetic data at BC with banding analysis were available from 21 pts on BU, 31 on HU, 56 on IFN and 49 on IM. 81% of pts treated with BU, 52% with HU, 38% with IFN and 55% with IM showed major route ACA. All other pts had minor route ACA or translocation t(9;22)(q34;q11) and variant translocation (t(v;22)) without ACA (Table 1). The difference in major route ACA between BU and IM was significant (p = 0.04, two-sided chi-square test). There was no statistically significant difference in ACA between pts on HU and IFN in comparison to IM. According to the testing order, further comparative testing was not possible. However, the differences of induction of major route ACA between HU and BU and IFN and BU were even more pronounced than the difference between IM and BU. The most frequently observed major route ACA was trisomy 8 in all studies and therapy arms.Table 1Overview of cytogenetic analysis of BC ptsTotal BC ptsPts with BC cytogeneticsMajor route ACA n (%)Minor route ACA n (%)t(9;22)(q34;q11) or t(v;22) without ACA n (%)BU1152117 (81)1 (5)3 (14)HU1173116 (52)7 (23)8 (26)IFN1595621 (38)6 (11)29 (52)IM734927 (55)13 (27)9 (18)total464157812749 ConclusionsThe type of cytogenetic aberrations in CML BC after different therapies is comparable. The characteristic major route ACA after various therapies points to a CML BC-related chromosomal pattern rather than a therapy-induced effect. Pts treated with IM showed a significantly lower rate of major route ACA than BU. IM not only reduces the frequency of BC and increases survival probabilities but appears to moderately change the biology of BC as compared to BU Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Hehlmann:BMS: Consultancy, Research Funding; Novartis: Research Funding. Hochhaus:Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding, Travel Other. Müller:Novartis: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding; Ariad: Honoraria. Kolb:Pierre Fabre, Therakos: Honoraria; Kolb Consulting UG: Consultancy, Equity Ownership. Saussele:BMS: Honoraria, Research Funding, Travel, Travel Other; Pfizer: Honoraria; Novartis: Honoraria, Research Funding, Travel Other.

Poor Survival Outcomes In Myelodysplastic Syndrome Patients With Non-t(6;9) and Non-Inv(3) Balanced Chromosomal Rearrangements Are Influenced By SRSF2 Mutations

Cytogenetics is an important predictor of overall outcomes in patients with myeloid malignancies including myelodysplastic syndromes (MDS). Traditional metaphase cytogenetics can detect chromosomal abnormalities in 45-50% of MDS patients. Unbalanced chromosomal abnormalities including deletions, monosomies and others are the predominant lesions commonly encountered. The prognostic value of most of these unbalanced chromosomal changes is well established specifically -7/-7q, -5/-5q, and +8. However, the role played by balanced chromosomal rearrangements (BCR) specifically balanced translocations and inversions in MDS is less established. Most cases especially in the absence of poor prognostic chromosomal karyotypes are assigned by default to the intermediate risk group. The importance of BCR especially translocations cannot be underscored since novel fusion proteins may form during such chromosomal events that can be subsequently targeted similarly as in the prototypical disease bcr-abl positive chronic myeloid leukemia. We hypothesized that BCR are commonly found in MDS and may confer important prognostic and therapeutic impact. We studied a total of 303 MDS patients seen at the Cleveland Clinic between the years 2002-2011. There were 197 males and 106 females. The median age of the cohort is 69 years (range: 19-92). The median follow-up time is 17 months. Cytogenetic, hematologic and survival data were collected from individual patients. Responses were assessed using the International working group criteria for MDS. Categorical data were analyzed using X2 test and survival outcomes were analyzed using Kaplan-Meier method. A p-value of <0.05 was considered statistically significant. Sanger sequencing for genes relevant in MDS pathophysiology including TET2, CBL, DNMT3A, NRAS, KRAS, TP53, SF3B1, U2AF1, SRSF2, ASXL1, RUNX1, JAK2, IDH1/2 were performed according to previously published methods. Balanced rearrangements with known poor prognostic significance specifically t(6;9) and inv(3) were excluded from the analysis. A total of 23 patients with BCR (8%) were identified. The most commonly affected chromosomes are chromosomes 1 and 7 with primarily translocations including 2 cases each of t(7;8) and t(1;7); while inversions involved various chromosomes including 2, 4, 9, 10, and 11. To understand the prognostic significance of these BCR, we compared the survival outcomes of patients with BCR with those of patients with specific chromosomal defects prognostically defined using the IPSS. Patients with BCR have worse OS similar to patients with poor risk cytogenetics as defined by IPSS (BCR=7 mos vs [Good risk=23 mos, Int=15 mos, vs. poor=5 mos], p=.0013). We previously reported the prognostic importance of single nucleotide polymorphism array analysis (SNP-A) lesions in MDS. New SNP-A and acquired somatic uniparental disomy abnormalities did not alter the prognosis of patients with BCR (SNP-A: new lesions= 6.7 mos vs. no lesions= 6.7 mos; p=.57, UPD: new lesions=4.6 mos vs. no lesions=7.7 mos, p=.19). In the advent of molecular genetics, specific molecular mutations affect outcomes in MDS and related cancers. We investigated the frequency and the role played by specific molecular mutations in the outcomes of patients with BCR. Among detected somatic mutations (TET2 (9%), ASXL1 (17%), JAK2 (13%), TP53 (9%) and SRSF2 (9%)), only patient harboring mutations in the RNA splicing gene SRSF2 conferred worse outcomes in this group (MUT= 1 mo vs. WT= 9 mos, p=.001). Therapeutically, 3 patients underwent allogeneic hematopoietic cell transplant (Allo-HCT), 11 received pharmacologic therapies (Induction chemotherapy [IC]=3; hypomethylating therapy [HMT]=11) and 5 with supportive treatments. All patients who received Allo-HCT achieved a complete remission and remain alive while 2 patients who received IC and 7 patients who received HMT did not respond to treatment. In conclusion, BCR are not uncommon in MDS. Interestingly, even when excluding known poor-risk balanced translocations like t(6;9) and inv(3), they still conferred a poor survival outcome in MDS patients. RNA splicing gene mutations specifically SRSF2 is a driver of worse outcomes within this group. Patients with BCR have better outcomes when managed with allo-HCT compared to HMT and IC. Disclosures:No relevant conflicts of interest to declare.

Comorbidity, Measured By The Charlson Index, Has No Negative Impact On Remission In Patients With Chronic Myeloid Leukemia: Results Of The Randomized CML-Study IV

▪ BackgroundFive-year overall survival (OS) of chronic myeloid leukemia (CML) patients treated with imatinib exceeds 90%. With many tyrosine kinase inhibitors (TKI) available as treatment options for CML, the influence of TKI therapy on OS is difficult to define. Comorbidities can complicate randomized trials. Their influence on OS in CML has not been studied so far. AimsWe sought to evaluate the influence of comorbidities at diagnosis of CML on remission rates and OS of patients with Philadelphia and/or BCR-ABL positive chronic-phase CML. The CML-Study IV, a randomized five-arm trial designed to optimize imatinib therapy alone or in combination, used very few exclusion criteria as compared to other studies which typically excluded patients with severe illnesses. MethodsThe age-adjusted Charlson Comorbidity Index (CCI) is the most extensively studied comorbidity index (Charlson ME et al., 1987) and has been validated for long-term studies. The score weighs a) the severity of comorbidities (e.g. one point is allocated to myocardial infarction and diabetes, two points to non-active malignancies) and b) the age of patients (with one point for each decade above 40 years). The CCI at diagnosis was calculated for each randomized patient. For the analyses, patients were grouped into CCI 2, 3-4, 5-6, and ≥7. Performance status was measured by the Karnofsky Score (KS) and patients were grouped into 50-80, >80-<100, and 100. Correlation analyses were performed by the chi-square test. Survival probabilities were calculated by Kaplan-Meier curves. Calculating cumulative incidences, the competing risks progression and/or death were considered. Cox models were estimated for the multivariate analysis to analyse the prognostic influence of the candidate factors age, sex, leukocytes, hemoglobin, EUTOS score, KS, and CCI on OS. Results1551 patients were randomized from 2002 to 2012, 1524 patients were evaluable. Median follow-up time was 67.5 months. Additional to CML, 521 index comorbidities were reported in 1519 patients resulting in the following CCI groups: i) CCI 2: 589 patients, ii) CCI 3 or 4: 599 patients, iii) CCI 5 or 6: 229 patients, and iv) CCI ≥ 7: 102 patients. Median value of the CCI was 3 (range: 2-12). The distribution of the CCI groups was not different between treatment arms. Most common comorbidities were diabetes (n=106), non-active cancer (n=102), chronic pulmonary disease (n=74), renal insufficiency (n=47), myocardial infarction (n=38), cerebrovascular disease (n=29), congestive heart failure (n=28), and peripheral vascular disease (n=28).Between patients with CCI 2, 3-4, 5-6, and ≥7 no significant differences in remission rates were found neither for time to complete cytogenetic remission (CCR) nor for time to major molecular remission (MMR). Median times to CCR were 12.9, 12.6, 13.7, and 13.1 months and to MMR 17.5, 15.9, 16.5, and 18.1 months, respectively. No differences were observed between the CCI groups for the cumulative incidences of progression. As expected, significant differences in OS according to CCI at diagnosis were observed (s. Fig. 1, p<0.001). Probabilities of OS at 8 years for patients with CCI 2, 3-4, 5-6, and ≥7 were 93.6%, 89.4%, 78.7%, and 45.2%. [Display omitted] We found a correlation between CCI and KS (p<0.001). In multivariate analysis CCI (p<0.001), KS (p=0.022), and EUTOS Score (p=0.012) were significant predictors of OS. Hazard ratios for the CCI group 3-4, 5-6, >7 (each vs. 2), were 1.695 (95%-confidence interval, CI 1.066-2.695), 3.231 (CI 1.942-5.376) and 6.495 (CI 3.817-11.111), respectively. Separating the CCI into an age-related part and a comorbidity-related part, the comorbidity-related part was still an important risk factor (Wald test, p=0.002). ConclusionsComorbidities of CML-patients do not seem to have an impact on the success of imatinib treatment. In CML-Study IV, even patients with a considerable comorbidity benefitted from imatinib as the chances to achieve MMR and CCR did not differ from those of healthier CML-patients. Our data also indicate that OS alone is not any more an appropriate measure for the effectiveness of a specific treatment for CML, as TKI have reduced the CML-related lethality to too low levels. Adjusting for comorbidity is essential for a valid comparison and interpretation of OS observed with different TKIs in CML-patients. Disclosures:Saussele:Pfizer: Honoraria; BMS: Honoraria, Research Funding, Travel, Travel Other; Novartis: Honoraria, Research Funding, Travel Other. Hehlmann:BMS: Consultancy, Research Funding; Novartis: Research Funding. Hochhaus:Novartis: Consultancy, Honoraria, Research Funding, Travel Other; BMS: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Ariad: Consultancy, Honoraria. Müller:Ariad: Honoraria; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau.

The Impact Of Balanced and Unbalanced Karyotypes On Prognosis Of CML: From Chronic Phase To Blast Crisis

IntroductionCurrent evidence indicates that acquired genetic instability in chronic myeloid leukemia (CML) as a consequence of the balanced reciprocal translocation t(9;22)(q34;q11) or the variant translocation t(v;22) and the resulting BCR-ABL fusion causes the continuous acquisition of additional chromosomal aberrations (ACA) and mutations and thereby progression to accelerated phase and blast crisis (BC). At least 10% of patients in chronic phase (CP) CML show ACA already at diagnosis and more than 80% of patients acquire ACA during the transformation process into BC. Therefore, alterations at diagnosis as well as acquisition of chromosomal changes during treatment are considered as a poor prognostic factor.Differences in progression-free survival (PFS) and overall survival (OS) have been detected depending on the type of ACA. Patients with major route ACA (+8, i(17)(q10), +19, +der(22)t(9;22)(q34;q11)) and with other alterations like -X, del(1)(q21), del(5)(q11q14), +10, -21 at diagnosis resulting in an unbalanced karyotype have a worse outcome. Patients with minor route ACA (for example reciprocal translocations other than the t(9;22)(q34;q11) (e.g. t(1;21), t(2;16), t(3;12), t(4;6), t(5;8), t(15;20)) resulting in a balanced karyotype show no differences in OS and PFS compared to patients with the standard translocation, a variant translocation or the loss of the Y chromosome (Fabarius et al., Blood 2011). Here we compare the type of chromosomal changes (i.e. balanced vs. unbalanced karyotypes) during the course of the disease from CP to BC aiming to provide a valid parameter for future risk stratification. Patients and MethodsClinical and cytogenetic data available from 1,346 out of 1,524 patients at diagnosis (40% females vs. 60% males; median age 53 years (range, 16-88)) with Philadelphia and BCR-ABL positive CP CML included until March 2012 in the German CML-Study IV (a randomized 5-arm trial to optimize imatinib therapy) were investigated. ACA were comparatively analyzed in CP and in BC. ResultsAt diagnosis 1,174/1,346 patients (87%) had the standard t(9;22)(q34;q11) only and 75 patients (6%) had a variant t(v;22). Ninety-seven patients (7%) had additional cytogenetic aberrations. Of these, 44 patients (3%) lacked the Y chromosome (-Y) and 53 patients (4%) had ACA. Regarding the patients with ACA thirty-six of the 53 patients (68%) had an unbalanced karyotype and 17/53 patients (32%) a balanced karyotype.During the course of the disease 73 patients (out of 1,524 patients) developed a BC during the observation time (5%). Cytogenetic data were available in 52 patients with BC (21 patients with BC had no cytogenetic analysis). Three patients had a normal male or female karyotype after stem cell transplantation. Nine patients showed the translocation t(9;22)(q34;q11) or a variant translocation t(v;22) (six and three patients, respectively) only and in 40 patients ACA could be observed in BC (40/49 (82%)). Out of these 40 patients with ACA, 90% showed an unbalanced karyotype whereas only 10% of patients had a balanced karyotype. No male patient in BC showed the loss of the Y chromosome pointing to a minor effect of this numerical alteration on disease progression. ConclusionWe conclude that patients with CML and unbalanced karyotype at diagnosis are under higher risk to develop CML BC compared to patients with balanced karyotypes or compared to patients without ACA. In BC, 90% of CML patients showed unbalanced karyotypes (only 68% of CML patients at diagnosis have unbalanced karyotypes) supporting the hypothesis that the imbalance of chromosomal material is a hallmark of disease progression, representing the natural history of the disease from CP to BC and indicating therefore a strong prognostic impact. Consequently, different therapeutic options (such as intensive therapy or stem cell transplantation) should be considered for patients with unbalanced karyotypes in CP CML at diagnosis. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Hehlmann:BMS: Consultancy, Research Funding; Novartis: Research Funding. Hochhaus:Novartis: Consultancy, Honoraria, Research Funding, travel Other; BMS: Consultancy, Honoraria, Research Funding; Pfizer : Consultancy, Honoraria; Ariad : Consultancy, Honoraria. Müller:Ariad: Honoraria; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau. Saussele:Pfizer: Honoraria; BMS: Honoraria, Research Funding, Travel, Travel Other; Novartis: Honoraria, Research Funding, Travel Other.

Treatment Response and Predictors of Response Among Patients with Chronic Myeloid Leukemia: A Retrospective Medical Record Review

AbstractAbstract 4450 Objective:To assess treatment responses and predictors of response among patients with chronic myeloid leukemia (CML) in Australia, Canada, and South Korea. Methods:Oncologists and hematologists abstracted data from medical records of patients diagnosed with CML between 1/1/2005 and 9/30/2010 via a web-based data collection tool. Patients included were at least 18 years old; had chronic phase, Philadelphia chromosome and/or BCR-ABL positive CML at the time of diagnosis; received imatinib as first-line therapy; and were not enrolled in a clinical trial between the date of CML diagnosis and the end of available chart data. A subset of patients received second- and/or third-line therapy with dasatinib or nilotinib. We assessed rates of complete hematological response (CHR), complete cytogenetic response (CCyR), and complete or major molecular response (MMR) at 3, 6, 12, and 18 months. Multivariable logistic regression models were fitted to assess predictors of response (CHR, CCyR, and MMR) to 1stline therapy with imatinib. The independent and control variables incorporated into the models were age, gender, employment status, Charlson comorbidity index (CCI) score, time to initiation of therapy, imatinib dose at treatment initiation, and presence of treatment-related side effects. Follow-up was truncated if the patient had a change in dose. Only those still in chronic phase at the time of imatinib initiation who had non-missing data on the variables of interest (n=387) were included in the regression analyses. Results:Ninety-three physicians (31 in Australia, 28 in Canada, and 34 in S. Korea) provided data on 609 patients (203 in Australia, 199 in Canada, and 207 in S. Korea). The average patient age was 57 years, and 59% of the patients were male. At 3 months after imatinib initiation, CHR was attained by 67%, 83%, and 86% of patients in Australia, S. Korea, and Canada respectively. Over 85% of all patients maintained CHR at 6, 12, and 18 months. Cytogenetics were not commonly evaluated at 3 and 6 months in the three countries studied. At month 12, 48% of patients achieved CCyR (42% in Australia and Canada, 60% in S. Korea), and a similar proportion of patients had CCyR at 18 months. Overall, MMR was achieved by 59% of patients at month 12, increasing to 70% at month 18. Multivariable regressions showed that receipt of previous treatment (odds ratio [OR]=1.88, 95% CI=1.08–3.25; reference [ref]: no previous treatment), and low Sokal score (OR=2.04, 95% CI=1.13–3.69; ref: intermediate) were associated with a greater likelihood of CHR at 3 months while initial dose > 400mg (OR=0.37 95% CI=0.13–1.03; ref: initial dose <= 400 mg), CCI score of 2 or higher (OR=0.23, 95% CI=0.11–0.51; ref: CCI score 0), and presence of treatment-related side effects (OR=0.41, 95% CI=0.18–0.95; ref: no side effects) predicted a lower likelihood of CHR at 3 months. High Sokal score (OR=0.39, 95% CI=0.18–0.87; ref: intermediate) was found to be associated with a lower likelihood of achieving CCyR at 12 months while the presence of treatment-related side effects (OR=1.86, 95% CI=1.04 – 3.33; ref: no side effects) was found to be associated with a higher probability of achieving CCyR at 12 months. Low Sokal score (OR=1.85, 95% CI=1.10–3.10; ref: intermediate) was the only significant prognostic indicator that was associated with a greater likelihood of MMR at 18 months while age >65 years was the only significant adverse prognostic indicator of MMR by 18 months (OR=0.42, 95% CI=0.20–0.86; ref: age 45–65). Conclusions:The favorable prognostic indicators of response among patients with chronic phase CML included previous treatment, low Sokal score, lower initial imatinib dose (< 400 mg), and no comorbidities. Better understanding of predictors of response may enable physicians to better tailor therapy to optimize patient outcomes. Disclosures:Whiteley:Pfizer Inc: Employment, Equity Ownership. Mitra:Pfizer Inc: Research Funding. Iyer:Pfizer Inc: Employment. Candrilli:Pfizer Inc: Research Funding. Kaye:Pfizer Inc: Research Funding.