Untreated Chronic Phase Research Articles

Using viral diversity to identify HIV-1 variants under HLA-dependent selection in a systematic viral genome-wide screen.

The pathogenesis of HIV-1 infection is governed by a highly dynamic, time-dependent interaction between the host and the viral genome. In this study, we developed a novel systematic approach to assess the host-virus interaction, using average pairwise viral diversity as a proxy for time since infection, and applied this method to nearly whole viral genome sequences (n = 4,464), human leukocyte antigen (HLA) genotyping data (n = 1,044), and viral RNA load (VL) measurements during the untreated chronic phase (n = 829) of Swiss HIV Cohort Study participants. Our systematic genome-wide screen revealed for 98 HLA/viral-variant pairs a signature of immune-driven selection in the form of an HLA-dependent effect of infection time on the presence of HIV amino acid variants. Of these pairs, 12 were found to have an effect on VL. Furthermore, 28/58 pairs were validated by time-to-event analyses and 48/92 by computational HLA-epitope predictions. Our diversity-based approach allows a powerful and systematic investigation of the interaction between the virus and cellular immunity, revealing a notable subset of such interaction effects. From an evolutionary perspective, these observations underscore the complexity of HLA-mediated selection pressures on the virus that shape viral evolution and pathogenesis.

Tyrosol Derivatives, Bearing 3,5-Disubstituted Isoxazole and 1,4-Disubstituted Triazole, as Potential Antileukemia Agents by Promoting Apoptosis.

In the present study, we assess tyrosol derivatives bearing 3,5-disubstituted isoxazoles and 1,4-disubstituted triazoles for their ability to inhibit the proliferation of K562 cells derived from leukemia as well as primary chronic myeloid leukemia (CML) cells obtained from the peripheral blood of 15 CML patients including 10 patients with untreated chronic phase and 5 patients with resistance against imatinib or multiple TKI. Our results showed that most derivatives displayed significant anti-proliferative activity against K562 cells in a dose-dependent manner. Among them, compounds 3d and 4a exhibited greater potent anticancer activity with respective IC50 values of 16 and 18 µg/mL (45 µM and 61 µM). Interestingly, compound 3d inhibited CML cell proliferation not only in newly diagnosed but also in imatinib-resistant patients. We demonstrated that the anti-proliferative effect of this compound is mediated by a pro-apoptotic activity by promoting oxidative stress and modulating the activity of the Akt, p38 MAPK and Erk 1/2 pathways. In conclusion, our data highlight the potential of this class of derivative as a novel promising therapeutic agent for CML therapy.

Knockdown (KD) of Mir-126 Expression Enhances Tyrosine Kinase Inhibitor (TKI)-Mediated Targeting of Chronic Myelogenous Leukemia (CML) Stem Cells

BCR-ABL tyrosine kinase inhibitors (TKI), although highly effective in inducing remission and improving survival in CML patients, fail to eliminate leukemia stem cells (LSC), which remain a potential source of relapse. The long-term goal of our research is to improve the understanding of molecular mechanisms regulating LSC growth and develop effective mechanism-based therapeutic strategies to target LSC in CML. MicroRNAs (miRNAs) are short non-coding RNAs that regulate translation of target protein-coding messenger RNAs (mRNAs) and levels of the corresponding encoded proteins. Among distinct miRNAs, miR-126-3p (miR-126) is expressed in normal hematopoietic stem cells (HSCs) and early hematopoietic progenitor cells (HPCs) and plays a pivotal role in restraining cell-cycle progression of HSC in vitro and in vivo (Cell Stem Cell, 2012). We previously reported that miR-126 expression varies among AML patients; higher level of miR-126 associated with poor outcome, a LSC-associated gene expression signature and increased LSC quiescence; and miR-126 down-regulation decreased LSC self-renewal activity in serial transplant experiments (Leukemia, 2015). Here, we report that the more primitive LSK (Lin-Sca-1+Kit+) from SCL-tTA-BCR/ABL mouse model of CML had higher miR-126 expression compared to more differentiated subpopulations, i.e. common myeloid progenitors (CMP) (3.2 fold) and granulocyte-macrophage progenitors (GMP) (6.4 fold). Among LSK subpopulations, LSC (Flt3-CD150+CD48- LSK) demonstrated the highest miR-126 levels (P<0.01). Furthermore, quiescent (Hoechst-Pyronin-) LSC demonstrated higher miR-126 expression (2.6 fold, p<0.01), generated significantly higher long-term engraftment of donor CML cells in recipient mice (58.6±4.4% vs 33±3.4% in BM at 16 wks after transplanted with 100 LSC/mouse, p=0.003) and enhanced leukemogenic capacity (5 out of 9 mice receiving quiescent LSC developed CML within 14 wks after transplantation vs. none of 10 mice receiving proliferating LSC) compared with proliferating (Hoechst+/-Pyronin+) LSC. Human primary untreated chronic phase (CP) CML CD34+CD38- primitive progenitors also showed higher miR-126 expression compared with CD34+CD38+ committed progenitors. KD of miR-126 in LSK cells of CML mouse model and in primary CML CD34+CD38- cells by using either miRZip-126-3p anti-miR lentivirus (System Biosciences) or a novel, myeloid cell-specific CpG-miR-126 oligonucleotide (ODN) inhibitor increased LSC cell-cycle entry as demonstrated by increased Ki67 staining (p<0.05) and EdU labeling (p<0.05), but affected apoptosis only slightly compared to scramble-treated controls. More importantly, miR-126 KD either by lentivirus or CpG-miR-126 ODN inhibitor combined with Nilotinib (NIL) significantly increased apoptosis of LSC compared with NIL alone (34% vs 19%; p=0.01 for lentivirus KD; and 40% vs 35%; p=0.03 for CpG-miR-126 inhibitor KD), resulting in significant reduction of cell growth and colony forming cell (CFC) frequency in methylcellulose progenitor assays. Conversely, enforced miR-126 expression by miR-126 precursor lentivirus (System Biosciences) impaired cell-cycle entry (28% vs 33%; p=0.05) and led to significantly reduced apoptosis (27% vs 19%; P=0.01) in NIL (5µM) treated CML CD34+CD38- cells compared to cells treated with control lentivirus and NIL. We further investigated the effect of miR-126 KD on growth of primary CML LSC and therapeutic response in vivo. CP CML CD34+ cells were cultured with CpG-scrambled RNA or CpG-miR-126 ODN inhibitor (500nM), in the presence or absence of NIL (5µM) for 4 days, and then transplanted into irradiated NSG mice. While the experiment is still ongoing, we have already observed a significantly reduced engraftment in blood at 4 wks in recipient mice receiving cells treated with CpG-miR-126 inhibitor and NIL compared with those receiving cells treated with CpG-scrambled RNA and NIL (0.16±0.03% vs 0.4±0.04%; P=0.01). Final results of this in vivo experiment and in vivo treatment of SCLtTA/BCR-ABL mice with CpG-miR-126 inhibitor and NIL will be reported at the meeting. Altogether, these observations suggest that miR-126 play a role in CML LSC homeostasis and down-regulation of miR-126 may decrease CML LSC quiescence, increase LSC proliferation and in turn enhance their sensitivity to TKI. miR-126 therefore may represent a novel therapeutic target in CML. DisclosuresStein:Amgen: Speakers Bureau. Snyder:BMS: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Forman:Mustang: Research Funding; Amgen: Consultancy.

Reassessment of HIV-1 Acute Phase Infectivity: Accounting for Heterogeneity and Study Design with Simulated Cohorts

BackgroundThe infectivity of the HIV-1 acute phase has been directly measured only once, from a retrospectively identified cohort of serodiscordant heterosexual couples in Rakai, Uganda. Analyses of this cohort underlie the widespread view that the acute phase is highly infectious, even more so than would be predicted from its elevated viral load, and that transmission occurring shortly after infection may therefore compromise interventions that rely on diagnosis and treatment, such as antiretroviral treatment as prevention (TasP). Here, we re-estimate the duration and relative infectivity of the acute phase, while accounting for several possible sources of bias in published estimates, including the retrospective cohort exclusion criteria and unmeasured heterogeneity in risk.Methods and FindingsWe estimated acute phase infectivity using two approaches. First, we combined viral load trajectories and viral load-infectivity relationships to estimate infectivity trajectories over the course of infection, under the assumption that elevated acute phase infectivity is caused by elevated viral load alone. Second, we estimated the relative hazard of transmission during the acute phase versus the chronic phase (RHacute) and the acute phase duration (d acute) by fitting a couples transmission model to the Rakai retrospective cohort using approximate Bayesian computation. Our model fit the data well and accounted for characteristics overlooked by previous analyses, including individual heterogeneity in infectiousness and susceptibility and the retrospective cohort's exclusion of couples that were recorded as serodiscordant only once before being censored by loss to follow-up, couple dissolution, or study termination. Finally, we replicated two highly cited analyses of the Rakai data on simulated data to identify biases underlying the discrepancies between previous estimates and our own.From the Rakai data, we estimated RHacute = 5.3 (95% credibility interval [95% CrI]: 0.79–57) and d acute = 1.7 mo (95% CrI: 0.55–6.8). The wide credibility intervals reflect an inability to distinguish a long, mildly infectious acute phase from a short, highly infectious acute phase, given the 10-mo Rakai observation intervals. The total additional risk, measured as excess hazard-months attributable to the acute phase (EHMacute) can be estimated more precisely: EHMacute = (RHacute - 1) × d acute, and should be interpreted with respect to the 120 hazard-months generated by a constant untreated chronic phase infectivity over 10 y of infection. From the Rakai data, we estimated that EHMacute = 8.4 (95% CrI: -0.27 to 64). This estimate is considerably lower than previously published estimates, and consistent with our independent estimate from viral load trajectories, 5.6 (95% confidence interval: 3.3–9.1). We found that previous overestimates likely stemmed from failure to account for risk heterogeneity and bias resulting from the retrospective cohort study design.Our results reflect the interaction between the retrospective cohort exclusion criteria and high (47%) rates of censorship amongst incident serodiscordant couples in the Rakai study due to loss to follow-up, couple dissolution, or study termination. We estimated excess physiological infectivity during the acute phase from couples data, but not the proportion of transmission attributable to the acute phase, which would require data on the broader population's sexual network structure.ConclusionsPrevious EHMacute estimates relying on the Rakai retrospective cohort data range from 31 to 141. Our results indicate that these are substantial overestimates of HIV-1 acute phase infectivity, biased by unmodeled heterogeneity in transmission rates between couples and by inconsistent censoring. Elevated acute phase infectivity is therefore less likely to undermine TasP interventions than previously thought. Heterogeneity in infectiousness and susceptibility may still play an important role in intervention success and deserves attention in future analyses

Chronic myelogenous leukemia, version 1.2015.

Chronic myelogenous leukemia (CML) is usually diagnosed in the chronic phase. Untreated chronic phase CML will eventually progress to advanced phase (accelerated or blast phase) CML. Tyrosine kinase inhibitors (TKIs) have been shown to induce favorable response rates in patients with accelerated and blast phase CML. The addition of TKIs to chemotherapy has also been associated with improved outcomes in patients with blast phase CML. Allogeneic hematopoietic stem cell transplant remains a potentially curative option for patients with advanced phase CML, although treatment with a course of TKIs will be beneficial as a bridge to transplant. This manuscript discusses the recommendations outlined in the NCCN Guidelines for the diagnosis and management of patients with advanced phase CML.

Analysis of the clinico-hematological relevance of the breakpoint location within M-BCR in chronic myeloid leukemia

The Philadelphia chromosome (Ph) derives from the balanced translocation between chromosomes 9 and 22. This chromosomal translocation results in the fusion between the 5' part of the BCR gene, normally located on chromosome 22, and the 3' part of the ABL gene on chromosome 9 giving origin to a BCR-ABL fusion gene which is transcribed and then translated into a hybrid protein. In general, three breakpoint cluster regions in the BCR gene have been described: major (M-BCR), minor (m-BCR) and micro (μ-BCR). Three main variants of the BCR-ABL gene have been described depending on the length of the sequence of the BCR gene included that encode for the P190, P210, P230 proteins. Most patients (95 %) were found to have P210 protein that resulted from rearrangement in the M-BCR region in the BCR gene and thus gives rise to b2a2 or b3a2 variants. The incidence of one or other rearrangement in chronic myeloid leukemia (CML) patients varies in different reported series. These two variants are associated with distinct clinical types of human leukemias. In this study, we report the frequencies of M-BCR-ABL fusion transcripts in 44 CML patients and we review the data on the correlations between the type of M-BCR/ABL variant and the corresponding sex, age and biological features. Forty-four untreated chronic phase CML patients were studied. The type of BCR-ABL fusion transcripts was determined by reverse transcriptase polymerase chain reaction (RT-PCR). More than half of them showed b3a2 fusion transcript (64 %), while (36 %) showed b2a2 transcript. No patients coexpressed b3a2/b2a2. Correlation between biological data demonstrated that: (a) M-BCR rearrangements were not associated with the sex of the patients. (b) Patients with b3a2 rearrangements were older than patients with b2a2 transcripts. (c) M-BCR rearrangements were influenced neither by the white blood count (WBC) nor with hemoglobin levels. However, platelet level is more elevated in patients with b3a2 transcript (681.2/L vs. 207/L; P = 0.001). In conclusion, we observed significant correlations between age, platelet level and M-BCR-ABL transcript, these observations deserve further investigations.

Utility values for chronic myelogenous leukemia chronic phase health states from the general public in the United Kingdom

This study estimated time trade-off preference values associated with the four chronic myelogenous leukemia (CML) chronic phase-related health states (i.e. untreated, hematologic response, cytogenetic response and molecular response) among members of the general public in the UK (n = 241). All four health states were associated with decreases in preference values from full health. The molecular response to treatment was the most preferred health state (mean utility of 0.94). The second-most preferred health state was cytogenetic response followed by hematologic response (mean utilities were 0.89 and 0.80, respectively). The least preferred health state was untreated chronic phase CML (mean utility of 0.72). The utility values for each state were significantly different from one another (p < 0.001). This study demonstrated and quantified the impact that more robust treatment responses have on the health-related quality of life of patients with chronic phase CML.

Correlation between the Type of BCR-ABL Transcripts and Blood Cell Counts in Chronic Myeloid Leukemia—A Possible Influence of MDR1 Gene Expression

The impact of BCR-ABL mRNA type (b3a2 vs. b2a2) on chronic myeloid leukemia (CML) phenotype is still a subject of controversies. We searched for a correlation between the BCR-ABL transcripts type and CML patients' characteristics, including MDR1 gene expression. Ninety-eight untreated chronic phase CML patients were studied. The type of BCR-ABL fusion transcripts and MDR1 gene expression were determined by reverse transcriptase polymerase chain reaction. B3a2 and b2a2 transcripts were found in 53 [54%] and 44 [45%] patients, respectively. One patient co-expressed b3a2/b2a2 and was excluded from analysis. The only difference in the clinical characteristics between the two groups was the platelets count, that was higher in b3a2(+) patients [791.3±441.3×109/L vs. 440.4±283.4×109/L in b2a2(+); P=0.007]. MDR1 over-expression [MDR1(+)] was observed in 48 patients (49.5%), more frequently in older patients >60 years [71% (24/34) vs. 38% (24/63) in younger; P=0.008], and was associated with a lower white blood cells (WBC) count [105.5±79.8× 109/L vs. 143.6±96.5×109/L in MDR1(−) cases; P=0.047]. On performing the analysis only within the MDR1(+) group, the b3a2(+) cases were characterized with a significantly higher platelets count [908.7±470.1×109/L vs. 472.9±356.1×109/L; P=0.006] and a lower WBC count [85.4±61.2×109/L vs. 130±93.9×109/L; P=0.004) compared to b2a2(+) patients. No similar differences were found between b3a2(+) and b2a2(+) groups with normal MDR1 levels. These results indicate that the type of BCR-ABL transcripts correlates with the hematological parameters of CML, however only in the subgroup of patients characterized by MDR1 over-expression.

Imatinib plus Peginterferon Alfa-2a in Chronic Myeloid Leukemia

Imatinib (400 mg daily) is considered the best initial therapy for patients with newly diagnosed chronic myeloid leukemia (CML) in the chronic phase. However, only a minority of patients treated with imatinib have a complete molecular remission. We randomly assigned 636 patients with untreated chronic-phase CML to receive imatinib alone at a dose of 400 mg daily, imatinib (400 mg daily) plus cytarabine (20 mg per square meter of body-surface area per day on days 15 through 28 of each 28-day cycle) or pegylated interferon (peginterferon) alfa-2a (90 μg weekly), or imatinib alone at a dose of 600 mg daily. Molecular and cytogenetic responses, time to treatment failure, overall and event-free survival, and adverse events were assessed. An analysis of molecular response at 12 months was planned. A superior molecular response was defined as a decrease in the ratio of transcripts of the tyrosine kinase gene BCR-ABL to transcripts of ABL of 0.01% or less, corresponding to a reduction of 4 log(10) units or more from the baseline level, as assessed by means of a real-time quantitative polymerase-chain-reaction assay. At 12 months, the rates of cytogenetic response were similar among the four groups. The rate of a superior molecular response was significantly higher among patients receiving imatinib and peginterferon alfa-2a (30%) than among patients receiving 400 mg of imatinib alone (14%) (P=0.001). The rate was significantly higher among patients treated for more than 12 months than among those treated for 12 months or less. Gastrointestinal events were more frequent among patients receiving cytarabine, whereas rash and depression were more frequent among patients receiving peginterferon alfa-2a. As compared with other treatments, the addition of peginterferon alfa-2a to imatinib therapy resulted in significantly higher rates of molecular response in patients with chronic-phase CML. (Funded by the French Ministry of Health and others; ClinicalTrials.gov number, NCT00219739.).

Lymphocytosis Following First-Line Treatment for CML In Chronic Phase with Dasatinib Is Associated with Improved Responses: A Comparison with Imatinib

AbstractAbstract 358 Background:Persistent expansion of clonal cytotoxic T cells or NK cells has been described in patients with chronic myeloid leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL) receiving dasatinib. These small studies have suggested a relationship between the development of T/NK lymphocytosis with both toxicity and improved response. A recent analysis of CML patients who were resistant to or intolerant of imatinib indicated that lymphocytosis occurs in ∼30% of patients in all stages of CML after treatment with dasatinib (Schiffer 2010 ASCO abstract #6553). In addition, lymphocytosis was associated with improved cytogenetic response and an increased incidence of pleural effusions. The DASISION study (CA180-056) compared dasatinib with imatinib in patients with newly diagnosed, previously untreated chronic phase CML. This subanalysis of the DASISION data was undertaken to determine the safety profile, responses, and outcomes in those patients with sustained lymphocytosis. Method:Patients enrolled in the DASISION study were retrospectively evaluated for lymphocytosis (N=516). Lymphocytosis was defined as lymphocytes >3.6 × 109/L on ≥2 occasions after 28 days of treatment. Immunophenotyping was not done as part of these studies. Rates of major cytogenetic response (MCyR) and complete cytogenetic response (CCyR), overall survival (OS), progression-free survival (PFS), and adverse events (AEs) were measured in those with and without lymphocytosis. The median follow-up was 14.0 and 14.3 months for dasatinib and imatinib, respectively. Result:Lymphocytosis occurred more frequently and sooner in patients treated with dasatinib compared with imatinib (23.6% vs 5.4% and 3.0 months vs 4.7 months, respectively). Dasatinib-treated patients who developed lymphocytosis had lower baseline Hasford risk scores, whereas imatinib-treated patients who developed lymphocytosis had high Hasford risk scores. In the presence or absence of lymphocytosis, MCyR and CCyR occurred more frequently with dasatinib as compared with imatinib. In dasatinib treated patients, lymphocytosis was associated with a higher MCyR rate (91.8% with vs 83.3% without) and CCyR rate (83.6% with vs 75.1% without). In imatinib-treated patients, lymphocytosis was associated with a lower MCyR rate (50.0% with vs 82.8% without) and CCyR rate (50.0% with vs 69.7% without). Patients with lymphocytosis, compared with those without it, had higher rates of pleural effusion (any grade) on dasatinib (18.0% vs 7.6%, respectively). Only 1 patient treated with imatinib, who did not have lymphocytosis, experienced pleural effusion. Dasatinib-treated patients with lymphocytosis, compared with those without it, had higher rates of fatigue (16.4% vs 9.1%,); this trend was reversed for imatinib-treated patients (7.1% vs 11.9%). Patients with lymphocytosis, compared with those without it had lower rates of myalgias and arthralgias (all grades) on both the dasatinib arm (11.5% vs 18.8%) and the imatinib arm (7.1% vs 24.2%). Conclusion:Lymphocytosis occurred much more frequently and sooner in patients treated with dasatinib than with imatinib. Lymphocytosis was associated with improved responses (MCyR and CCyR) in dasatinib-treated patients. An apparent inverse relationship was observed in imatinib-treated patients, although the number of patients with lymphocytosis was small. It remains to be determined how lymphocytosis, baseline Hasford risk score, and response are correlated. It is possible that some of the antileukemic effects of dasatinib are produced by an immunomodulatory mechanism. Lymphocytosis in dasatinib-treated patients was associated with an increased rate of pleural effusion and a possible decrease in myalgias and arthralgias. Longer follow-up is needed to assess whether lymphocytosis has an effect on PFS.Lymphocytosis, n (%)Cumulative MCyR, n (%)Cumulative CCyR, n (%)Patients on treatment with CCyR at 12 months, n (%)dasatinib (100 mg)Yes61 (23.6)56 (91.8)51 (83.6)50 (82)(n=258)No197 (76.4)164 (83.2)148 (75.1)143 (72.6)P value0.140.220.17imatinib (400 mg)Yes14 (5.4)7 (50)7 (50)7 (50)(n=258)No244 (94.6)202 (82.8)170 (69.7)160 (65.6)P value0.0070.140.26 Disclosures:Schiffer:Bristol-Myers Squibb: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Cellgenix: Consultancy; Bristol-Myers Squibb: Research Funding; Novartis: Research Funding. Cortes:Bristol-Myers Squibb: Research Funding. Saglio:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria. le Coutre:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria. Guilhot:Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Bahceci:Bristol-Myers Squibb: Employment. Dejardin:Bristol-Myers Squibb: Employment. Shah:Bristol-Myers Squibb: Consultancy; Novartis: Consultancy; Ariad: Consultancy.

Seven-Year Follow-up Data on Sequential Prospective Trials of Imatinib 400mg Vs 800mg Daily Schedule for Front-Line Treatment of Chronic Myeloid Leukemia (CML).

AbstractAbstract 3438 Background:The standard of care for most patients (pts) with CML has been imatinib mesylate at a dose of 400mg by mouth daily. Earlier studies have suggested that there may be a benefit to pts to start treatment at a higher dose as this may result in faster and more durable responses to imatinib. It is not yet known whether long-term event-free survival (EFS), transformation-free survival (TFS), and overall survival (OS) will be impacted by the higher dosing schedule. Objectives:To determine the long term responses and clinical benefit of imatinib 800mg daily versus 400mg daily dosing when used as upfront treatment strategy in CML. Methods:We conducted sequential prospective trials using imatinib 400mg or 800mg daily as initial therapy for patients with previously untreated chronic phase CML. Results:A total of 281 pts were included in these trials: 208 treated with 800mg and 73 with 400mg. The median follow-up for each group was 79 months (range: 3–107) and 110 months (range: 2–116). The overall, cumulative rate of complete cytogenetic response (CCyR) was 91% and 87%, respectively (p=0.49) for those treated with high- and standard-dose, and the cumulative rate of major molecular response (MMR) was 87% and 78%, respectively (p=0.06). Rates of CCyR at 12 months were 90% and 66%, respectively (p < 0.001), and MMR at 18 months 82% and 68%, respectively (p=0.04). A significantly better EFS (definition per IRIS criteria) was observed for the 800 mg group compared to that in the 400mg group (log-rank test, p=0.049; estimated 7-year EFS 86% vs 76% by Kaplan-Meier method). No significant differences were seen for survival free from transformation to accelerated and blast phase (p = 0.46) and overall survival (p = 0.27). For OS, thus far 19 pts in the 800mg group have died (2 probable CML-related, 3 unknown causes, 14 non CML related) compared to 13 pts (10 probable CML-related, 3 non CML-related) in the 400mg group. The table below shows the annual rate of events and transformation for each dose group.Actualno. at riskno. eventevent yearly %no. transformationyearly %400 mg1-yr7357452-yr6246233-yr5412124-yr5112125-yr5000006-yr4824007-yr44240no event occurred after the 7yr800 mg1-yr20842312-yr18463213-yr16642324-yr15632215-yr1462120.76-yr1281110.87-yr972222no event occurred after the 7yrno transformation occurred after the 7yrTreatment discontinuation for toxicity occurred in 16 (8%) pts treated with 800mg and 6 (8%) pts treated with 400mg. Conclusions:At 7-year follow up, pts treated with 800mg demonstrated a significantly better EFS (by IRIS criteria) compared to those treated with 400mg. There is a trend for a lower annual rate of events and transformation with the higher dose, particualry in the earlier years, but no difference in OS. These results suggest a modest benefit for patients treated with higher dose imatinib. Disclosures:Off Label Use: imatinib at dose of 800mg po daily for CML. Kantarjian:BMS, Pfizer and Novartis: Research Funding; Novartis: Consultancy. Verstovsek:Incyte Corporation: Research Funding. Ravandi:Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Speakers Bureau. Cortes:Pfizer: Consultancy, Research Funding; BMS: Honoraria, Research Funding; Novarits: Honoraria, Research Funding.

Functional Activity of the OCT-1 Protein Is Predictive of Long-Term Outcome in Patients With Chronic-Phase Chronic Myeloid Leukemia Treated With Imatinib

Organic cation transporter-1 (OCT-1) activity (OA), a measure of the OCT-1-mediated influx of imatinib into CML mononuclear cells (MNCs), is predictive of major molecular response (MMR) at 12 and 24 months in patients with untreated CML. We now report the impact of OA on loss of response, disease transformation, and survival after 5 years of imatinib. OA is defined as the difference in intracellular concentration of carbon-14-imatinib with and without OCT-1 inhibition. OA was measured in blood from 56 patients with untreated chronic-phase CML. More patients who had high OA (ie, > median OA value) achieved MMR by 60 months compared with patients who had low OA (89% v 55%; P = .007). A low OA was associated with a significantly lower overall survival (87% v 96%; P = .028) and event-free survival (EFS; 48% v 74%; P = .03) as well as a higher kinase domain mutation rate (21% v 4%; P = .047). These differences were highly significant in patients who averaged less than 600 mg/d of imatinib in the first 12 months but were not significant in patients averaging >/= 600 mg/d. Patients with very low OA (ie, quartile 1) were the only group who developed leukemic transformation (21% in quartile 1 v 0% in all other quartiles; P = .002). Measurement of OA pretherapy is a predictor for the long-term risk of resistance and transformation in patients with imatinib-treated CML. Early dose-intensity may reduce the negative prognostic impact of low OA. We propose that OA could be used to individualize dosage strategies for patients with CML to maximize molecular response and optimize long-term outcome.

Rekombinantes Interferon α-2c bei Ph-positiver chronischer myeloischer Leukämie: Ergebnisse einer multizentrischen Phase-II-Studie

In a Phase II study, 82 patients with Philadelphia chromosome(Ph)-positive chronic myelogenous leukaemia were treated with 3.5 million I.U./d of recombinant interferon alpha-2c (rIFN). 73 patients have so far been evaluated (42 male, 31 female, mean age 50 [12-87] years). At the start of therapy, 10 were in the accelerated phase (group 1) and 63 in the chronic phase, of whom 19 had received previous treatment with cytotoxics (group 2), while the remainder (group 3, n = 44) had received primary treatment with rIFN. There was short-term stabilization in 7 of the 10 group 1 patients, but none had complete haematological or cytogenetic remission. In contrast, the remission rate (complete or partial haematological remission) was 63% in the previously treated (group 2) and 66% in the previously untreated chronic phase patients (group 3). There was a reduction in the proportion of Ph-positive metaphases in 7 group 2 patients (11%) and in 10 group 3 patients (23%). Complete cytogenetic remission has so far been seen in 2 patients. Cytogenetic improvement occurred after 3 months at the earliest, and in some patients only after 12 to 19 months treatment. Differences in response to treatment were related to stage (prognostic staging system of Kantarjian et al.) in group 3 patients: complete or partial haematological remission was seen in 22 out of 25 patients with stage 1 disease, in 4 out of 7 in stage 2, and in only 3 out of 12 in stages 3 and 4.

A Novel Assay for Development of Chromosomal Lesions in Human CD34+ Cells Following Exposure to Ionizing Radiation Reveals Increased Susceptibility To Acquire Unstable Chromosomal Lesions in CML CD34+ Cells.

Acquisition of chromosomal lesions likely plays an important role in pathogenesis of primary and therapy-related leukemia, as well as in disease evolution in myeloproliferative and myelodysplastic disorders. However the process of acquisition of chromosomal lesions in hematopoietic stem cells (HSC) is not well understood. Development of persistent chromosomal abnormalities may involve several factors including acquisition of DNA lesions, repair of DNA damage, sensing of nonrepaired or misrepaired lesions and activation of cell cycle checkpoint and apoptotic pathways, and clonal growth advantage conferred by the lesions. In the current study we assessed the frequency, nature and kinetics of chromosomal lesions following exposure to genotoxic agents in normal human HSC and determined whether these were altered in CML, a prototypic HSC malignancy associated with genetic instability and acquisition of new chromosomal abnormalities during disease progression. CD34+ cells were selected from 4 normal donors and 4 newly diagnosed, untreated chronic phase CML patients. Cells were exposed to increasing doses of γ-radiation, cultured with growth factors and metaphase spreads assessed for development of chromosomal lesions. Chromosome painting was performed using chromosomes 1, 3, 5, 7, 11 and 21 probes, representing 32% of genomic DNA, with >100 metaphases scored per dose per time. Radiation exposure resulted in induction of chromosomal lesions in normal CD34+ cells in a dose-dependent manner. Chromosomal lesions were not seen in cells cultured without radiation exposure. The frequency of aberrant metaphases after 72 h culture (shown by cell cycle analysis to represent the first cell division for normal CD34+ cells) was 2.7% with 0.5 Gy, 4.8% with 1.0 Gy, 9.9% with 2.0 Gy and 23.5% with 4.0 Gy exposures. Chromosome aberrations observed at first division included both stable (translocations, insertions) and unstable (excess fragments, dicentrics) lesions. Subsequent results are shown for exposure to 2.0 Gy. The frequency of aberrant metaphases dropped to 5.3% at 144 h (3–4 cell divisions); only stable lesions persisted at this time. In contrast to normal CD34+ cells, first cell division in CML CD34+ cells was seen at 24 h, at which time 15.3% of the metaphases showed aberrations, whereas 11.2% of metaphases showed aberrations after 72 h (3–4 divisions) and 11.2% after 144 h (6–7 divisions). A striking difference between normal and CML cells was persistence of unstable aberrations after several cell divisions in the latter (42.9% unstable lesions present even after 6–7 divisions). These observations suggest impaired ability to sense and eliminate cells with chromosomal lesions or continued generation of such lesions after initial radiation exposure in CML cells. In conclusion we have developed a novel chromosome painting based assay for evaluation of acquisition of chromosomal lesions in primitive hematopoietic cells. We have demonstrated an inherent chromosomal instability that may contribute to clonal evolution and disease progression in CML CD34+ cells. This assay will provide a useful platform for: i) assessment of mechanisms underlying development of chromosomal lesions in response to DNA damage; and ii) assessing susceptibility to genotoxic agents, and allow improved understanding of pathogenesis and disease evolution in myeloid malignancies.

Two Year Data from a Prospective Safety Study Analyzing the Consequences of Imatinib Mesylate Inhibition of Sensitive Kinases Other Than bcr-abl in Patients with Previously Untreated Chronic Phase CML.

Imatinib Mesylate (IM) is a potent inhibitor of bcr-abl which also inhibits a number of other tyrosine kinases; c-abl, c-arg, PDGF-R, c-kit, and c-fms. IM inhibits these targets in vivo at clinically achievable concentrations, but the consequences of these actions in patients (pts) with CML are unknown. Gene knockout mice lacking these kinases have distinct phenotypes suggesting potential consequences of long-term inhibition (infertility, lymphopenia, emphysema, glomerular dysfunction). To determine if long-term IM therapy may lead to such features, we prospectively evaluated these parameters in a subset of pts with untreated chronic-phase CML enrolled on the ALLG trial of dose intensified IM. Analyses were performed at baseline, 6-, 12-, and 24-months. Results are presented as within patient comparisons of baseline and 24-month data as paired samples, unless stated otherwise. 103 pts were entered in the main study and 45 have consented to aspects of this sub-study, with ages ranging from 37–75 yrs (median 51), and average daily IM dose of at least 600 mg. There were minor but statistically significant declines in respiratory function (n=24); median vital capacity (4.9 to 4.4L; p=0.019), FEV1.0 (3.6 to 3.2L; p=0.007) and PEFR (9.0 to 7.2 L/sec; p=0.06), with no change in DLCO. There was no change in urinary protein excretion (p=0.45), male (testosterone / FSH / LH) (n=14; each p>0.08) or female hormone levels (oestradiol / progesterone / FSH / LH) (n=6; each p>0.16), but one of 3 men with serial analyses developed oligospermia. There were consistent falls (n=26) in all Ig classes, most evident for IgG and IgM with the greatest decline seen in the first year and stable levels thereafter. In the 20 pts analysed there were sustained and ongoing declines in CD3+, CD4+, and CD8+ cells, but no changes in CD19+ or CD56+ cells. Results are tabulated, with the change (Δ) from baseline to 24-months shown. There have been no instances of severe or opportunistic infections in these pts. Based on these data there have been no adverse effects of IM evident on male or female reproductive hormonal status, glomerular function, B- or NK-cell numbers, but one case of oligospermia, consistent and sustained effects on pulmonary function, T-cell subsets and all classes of Ig levels without any clinical consequences yet manifest. These effects of IM on sensitive kinases other than bcr-abl in pts with CML are important for both our understanding of the physiologic role of these kinases in humans and the delineation of the spectrum of potential adverse effects associated with long-term use of this class of drugs.Median valuesIgG (g/L)IgA (g/L)IgM (g/L)CD3+ (109/L)CD4+ (109/L)CD8+ (109/L)CD19+ (109/L)Baseline10.451.631.361.431.030.510.196 month9.91.550.761.120.670.350.2012 month8.71.490.711.060.650.380.1624 month8.51.550.680.900.510.280.15Δ−2.23−0.2−0.7−0.46−0.25−0.17−0.04P-value<0.00010.018<0.00010.00070.00030.0040.33

High-dose imatinib mesylate treatment in patients (Pts) with untreated early chronic phase (CP) chronic myeloid leukemia (CML): 2.5-year follow-up

6535 Background: The standard dose (SD) of imatinib for CP CML is currently 400 mg daily, but higher doses (HD) may be more effective. We conducted 2 consecutive trials using HD imatinib (i.e., 400mg twice daily) in previously untreated early CP CML pts. This is an updated analysis of the longer follow-up. Methods: A total of 175 previously untreated pts received HD imatinib. We compared the results with a previous study using SD imatinib (400mg/day) in untreated pts with early CP CML (N=50). Results: Cytogenetic and molecular responses were evaluable in 222 pts (N=49 at SD, 173 at HD) and 217 pts (N=46 at SD, 171 at HD), respectively. In HD group, Sokal risk classification was good in 69%, intermediate in 29%, and poor in 11% of pts. There were no differences in pre-treatment characteristics between two groups. The median age was 48 years in both groups. Median follow-up is 53 months for SD and 30 months for HD group. Patients treated with HD had a higher rate of complete cytogenetic responses (90% vs 78% with SD, p=0.03) and these occurred earlier, with 69% achieving this response after 6 months of therapy vs 45% with SD (p=0.001). The cumulative incidence of major molecular response was significantly better in HD group (p=0.03), and this response was also observed earlier in HD group: at 12 months 54% in HD and 24% in SD group had achieved this response (p=0.001). At 24 months, 19/70 (27%) evaluable pts with HD versus 3/31 (10%) of pts in SD group achieved complete molecular remission. Four pts (2%) in HD group and 4 pts (8%) in SD group have progressed to advanced phases (p=0.05). There was a trend in favor of the HD group for transformation-free-survival but it was not statistically significant (p=0.07). Overall survival is excellent in both groups (24 month survival, 99% with HD vs 98% with SD; p=0.24). Grade 3 or 4 hematologic toxicity was more frequent in HD group whereas extramedullary toxicity was similar in two groups. The median actual dose in HD group was 800 mg at 12 months, with 39% patients requiring dose reduction at some point. Conclusions: High-dose imatinib provides higher rates of complete cytogenetic responses and earlier molecular responses with some increase myelosupression. The long-term benefit of earlier responses remains to be demonstrated. [Table: see text]

HTERT, the catalytic component of telomerase, is downregulated in the haematopoietic stem cells of patients with chronic myeloid leukaemia

Telomere shortening is associated with disease progression in chronic myeloid leukaemia (CML). To investigate the biology and regulation of telomerase in CML, we evaluated expression of the telomerase components, its regulators and several telomeric-associated proteins. Quantitative real-time-polymerase chain reaction (PCR) was used to compare gene expression in the CD34+/leukaemic blast cells of 22 CML patient samples to the CD34+ cell population of healthy individuals. hTERT, the catalytic component of telomerase, was downregulated in eight of 12 chronic phase (CP) patients (P = 0.0387). Furthermore, hTERT was significantly downregulated in two of three patients in accelerated phase (AP) and seven of seven patients in blast crisis (BC), P = 0.0017. Expression of hTR and telomeric-associated proteins TEP1, TRF1, TRF2, tankyrase and PinX1 was high in the majority of CP and AP patients. With the exceptions of TEP1 and hTR, expression of these factors was highest in CP and decreased during disease progression. Expression of c-Myc, a positive regulator of hTERT transcription, correlated with hTERT expression and decreased with disease progression, falling below control levels in BC. hTERT levels were increased in CP patients following successful treatment with imatinib, relative to untreated CP patients. We suggest that reduced hTERT expression directly causes the shortened telomeres observed in CML.

A Randomized Trial of High-Dose (HD) Imatinib Mesylate (IM) with or without Peg-Interferon (PEG-IFN) and GM-CSF as Frontline Therapy for Patients with Chronic Myeloid Leukemia (CML) in Early Chronic Phase (CP).

Some studies suggest that HD IM may be more effective than standard dose in CP CML. Still, only a minority of patients (pts) may reach undetectable levels of Bcr-Abl transcripts, and the use of single agent therapy may eventually result in development of resistance. IFN is thought to be effective in CML due to its immunomodulatory properties and may be valuable for management of minimal residual disease. Pre-clinical studies suggest synergy with imatinib. GM-CSF combined with IFN is a potent stimulator of dendritic cells. We designed this randomized phase II study to investigate if adding PEG-IFN to imatinib may improve the rate of complete molecular responses and prolong remission duration. Pts with previously untreated CP CML started treatment with imatinib 800mg daily and randomized after 6 months (mo) of therapy to either continue HD IM alone or add PEG-IFN 0.5mcg/kg/week and GM-CSF 125 mg/m2 three times weekly. Pts were monitored with real-time PCR and cytogenetics (CG) every 3 mo for the 1st year and every 6 mo thereafter. Ninety-four pts have been registered to date: 49 randomized to IM alone and 45 to IM + PEG-IFN+GM. 70 (75%) have been followed for at least 6 months and 49 (52%) for 12 months. (First interim analysis done when 30 pts evaluable at 12 mo). Ten pts randomized to PEG-IFN did not start therapy (2 refused, 2 physicians decision, 6 off study before 6 mo because of noncompliance n=3, melanoma n=1, financial n=1, progressive disease n=1). Patient characteristics and response (intention to treat) are as follows:No./No. Evaluable (%)p valueOverallIM aloneIM+PEGIFN+GMMedian age (range), y48 (19–73)46 (19–73)50 (19–73)0.12Sokal (% low/int/high)71/20/963/25/1279/16/50.17CG CR Overall73/84 (87)40/45 (89)33/39 (85)0.74@ 12 mo44/49 (90)27/31 (87)17/18 (94)0.6312 mo BCR-ABL/ABL<0.05%20/49 (41)10/31 (32)10/18 (55)0.13Undetectable5/49 (10)3/31 (10)2/18 (11)1.0Median (range)0.1 (0–79.3)0.12 (0–79.3)0.04 (0–21.9)0.51Toxicity with HD imatinib was similar to previous reports. The most common grade ≥3 toxicity associated with PEG-IFN in the 26 pts included fatigue (n=7, 27%), rash (n=5, 19%), depression (n=3, 11%), and headache (n=2, 7%). Twelve pts have required dose reductions of PEG-IFN and 3 (13%) permanently discontinued it. The actual median (range) dose of IM at 12 months was 800 mg (600–800 mg) for IM alone and 800 mg (400–800 mg) for combination arm. Among pts randomized to PEG-IFN arm, the dose of IM had to be reduced after 6 mo (i.e., after start of PEG-IM) in 4 (16%) pts. We conclude that the combination of IM and PEG-IM as done here is associated with acceptable toxicity profile. There is a trend for improved response after 12 months with the combination.

CD34+CD7+ Leukemic Progenitor Cells May Be Involved in Maintenance and Clonal Evolution of Chronic Myeloid Leukemia

We analyzed CD34+ cells coexpressing CD7 in chronic myeloid leukemia (CML) in chronic phase (CP) or accelerated phase (AP) to clarify their role in progression or regression of the disease during treatment. Enumeration of CD34+CD7+ cells was done on bone marrow nucleated cells from normal donors and CML patients. Fluorescence in situ hybridization analysis was done on sorted CD34+CD7+and CD34+CD7- cells to examine the occupancy rate of each fraction by BCR-ABL+ cells with or without additional cytogenetic abnormalities. The proportion of CD34+CD7+cells was significantly affected by the treatment outcome and/or the disease status as follows: 20.5 +/- 10.4% in normal donors (n = 22), 18.1 +/- 10.2% in CP with major cytogenetic response (n = 14), 53.0 +/- 12.9% in CP at diagnosis (n = 18), 55.0 +/- 15.8% in CP with minor or no cytogenetic response (n = 28), and 70.2 +/- 18.1% in AP (n = 6). The proportion of CD34+CD7+cells decreased in parallel with cytogenetic improvement in individual patients. In six untreated CP patients, the ratio of BCR-ABL+ cells was comparable between each fraction. In three patients with major cytogenetic response, the ratio of BCR-ABL+ cells was remarkably lower in CD34+CD7- cells than in CD34+CD7+cells. In three AP patients with additional cytogenetic abnormalities, extra signals were detected at a much higher rate in CD34+CD7+ cells than in CD34+CD7- cells. Our results suggest that CD34+CD7+ cells may be involved in maintenance and clonal evolution of BCR-ABL+ cells in CML.

Imatinib Mesylate Does Not Inhibit BCR-ABL Kinase Activity in CML Stem Cells In Vitro.

Imatinib mesylate (IM, Gleevec, Novartis), a specific inhibitor of the tyrosine kinase BCR-ABL, promotes an impressive complete cytogenetic remission rate in patients with chronic phase Chronic Myeloid Leukaemia (CML). In most cases however, molecular disease persists and may lead to eventual relapse. IM selectively interacts with BCR-ABL thereby inhibiting activation of downstream targets, in particular phosphorylation of CrkL. We have previously demonstrated the sensitivity of rare Ph+ CD34+ CML stem cells (<5% of total CD34+ cells) to the anti-proliferative but, not the pro-apoptotic effects of IM, hence their persistence in vitro. We hypothesised that these rare cells may also behave differently from total CD34+ cells in terms of their kinase response to IM. The effect of IM on the phosphorylation status of CrkL was therefore investigated, in total CD34+ and in primitive CD34+/38- cells from 6 untreated chronic phase CML patients. Ph+ CD34+ and CD34+/38- cells were cultured in serum free medium for up to 72h, in the presence and absence of IM (5mM). After 16 and 72h the phosphorylation status of CrkL was measured with an antibody specifically designed to recognise only the phosphorylated form of the protein. After 16h total CD34+ cells showed a significant dephosphorylation of CrkL in response to IM. However, after 72h the remaining viable BCR-ABL+ cells failed to show any de-phosphorylation of CrkL, consistent with an IM selected, resistant population of stem cells. Primitive CD34+/38- cells from the same patients exhibited no de-phosphorylation of CrkL after either 16 or 72h, confirming their innate/inherent resistance to the drug. These data show, for the first time, that very primitive CML cells exhibit BCR-ABL dependent resistance (i.e. BCR-ABL kinase remains active during drug exposure) and suggest that the drug is not reaching or binding its target at inhibitory concentrations. Novel approaches to eliminate these cells will be critical to the development of curative strategies for CML.