Major Cytogenetic Remission Research Articles

Application of a Short Tandem Repeat Based PCR Assay for Chronological Monitoring of Myelodysplastic Syndrome (MDS) Patients with Deletion of Chromosome 5q Following Lenalidomide Treatment

IntroductionDeletion of chromosome 5q (del(5q)) defines a distinct clinical subtype of myelodysplastic syndromes (MDS) and qualifies patients to specific treatment with Lenalidomide (LEN). Therefore, detection and monitoring of this deletion is an important element in routine clinical diagnostics for determining molecular response. Current methodologies for performing these analyses consist of cytogenetics, fluorescence in situ hybridization (FISH) or microarrays. All of these methods have downsides due to the high demands to the input material, i.e. viable cells or necessity for large amounts of high quality genomic DNA (gDNA). To perform quantitative assessment of cytogenetic lesions in low quantity or residual material we here present the establishment of a PCR-based assay for interrogation of del(5q) in MDS, based on the allelic loss at heterozygous short tandem repeat (STR) loci within deleted regions.MethodsGenomic DNA was isolated from bone marrow (BM) and peripheral blood (PB) of n=86 MDS del(5q) patients. 49 non-del(5q) MDS patients were used as controls. Serial chronological BM samples (n=95) following treatment with LEN from n=40 del(5q) patients, who were enrolled in the LEMON-5 trial from the German MDS study group, were analysed. Using 10ng DNA, 12 fluorochrome-labelled PCR amplicons of STR loci located between chromosomal bands 5q21 and 5q31 were amplified in a single optimized multiplex-PCR reaction. Subsequently, amplicon fragment analysis was carried out via capillary electrophoresis and allele size quantification of heterozygous STR loci was performed. Finally, the degree of skewing in the allelic ratios of all informative STR markers was averaged and translated into an allelic burden of del(5q).ResultsPaired quantitative correlation of clone sizes using STR-PCR and interphase FISH was carried out in n=34 samples and revealed highly concordant results with r²=0.924. The diagnostic accuracy of the PCR assay was evaluated by receiver operating characteristic (ROC) analysis and revealed an area under the curve of 0.989 (sensitivity and specificity of 0.977 and 0.948, respectively). Prior to treatment with LEN, clone sizes as determined by STR-PCR were heterogeneous (mean: 57%, range: 11-91%). During follow-up analysis, while cytogenetic analyses failed (e.g. metaphase failure) in 7/40 (18%) cases, our STR-PCR assay successfully generated estimates of del(5q) cell burden in all available samples. Upon LEN treatment, n=12 patients achieved major cytogenetic remission (absence of del(5q)-positive metaphases). The mean clone size carrying del(5q) determined by STR-PCR in that group was 7% (range 3 - 10%) and significantly increased compared with n=15 patients who reached minor cytogenetic response (defined as 50% reduced aberrant metaphases, mean 13%, range 5 - 39%, p=0.025). Intriguingly none of n=6 patients without cytogenetic response achieved a del(5q) clone size of less than 35% as determined by STR-PCR (mean 46%, range 35 - 66%), highlighting the correlation of PCR based follow-up analysis with currently used cytogenetic methods for response evaluation. Finally in n=93 matched PB and BM samples a correlation of del(5q)-frequency in BM versus PB showed r²=0.81. Moreover, in 96% of samples in which the BM still showed clone sizes >10%, we reliably detected del(5q) in corresponding PB cells with a robust sensitivity of 5% deleted cells.DiscussionWe present a highly adaptable tool for precise measurement of large chromosomal deletions, requiring only minute amounts of genomic DNA. It shows a very good quantitative correlation with established methods and good diagnostic accuracy. Most importantly, this PCR based assay does not require dividing cells so it can be performed from PB, which shows a sufficient correlation with clone sizes in BM and rarely involves the risk of underrepresentation of del(5q)-clones in PB, possibly allowing the use of PB as a regular specimen for clone size monitoring. Thus, especially in the context of serially monitored patients this assay represents an alternative method for less invasive tracking of cytogenetically aberrant clones. DisclosuresPlatzbecker:Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Boehringer: Research Funding. Schlenk:Janssen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Research Funding; Boehringer-Ingelheim: Honoraria; Teva: Honoraria, Research Funding; Arog: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees. Bug:TEVA Oncology, Astellas: Other: Travel Grant; NordMedica, Boehringer Ingelheim, Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene, Novartis: Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Imatinib Mesylate Effectiveness in Chronic Myeloid Leukemia with Additional Cytogenetic Abnormalities at Diagnosis among Black Africans

Imatinib mesylate provides good results in the treatment of CML in general. But what about the results of this treatment in CML associated with additional cytogenetic abnormalities at diagnosis among black Africans? For this, we retrospectively studied 27 cases of CML associated with additional cytogenetic abnormalities, diagnosed in the department of clinical hematology of the University Hospital of Yopougon in Côte d'Ivoire, from May 2005 to October 2011. The age of patients ranged from 13 to 68 years, with a mean age of 38 years and a sex ratio of 2. Patients were severely symptomatic with a high Sokal score of 67%. CML in chronic phase accounted for 67%. The prevalence of additional cytogenetic abnormalities was 29.7%. There were variants of the Philadelphia chromosome (18.5%), trisomy 8 (14.8%), complex cytogenetic abnormalities (18.5%), second Philadelphia chromosome (14.8%), and minor cytogenetic abnormalities (44.4%). Complete hematologic remission was achieved in 59%, with 52% of major cytogenetic remission. The outcome was fatal in 37% of patients. Death was related in 40% to hematologic toxicity and in 30% to acutisation. The median survival was 40 months.

Chronic myeloid leukemia presenting with bilateral central retinal vein occlusion and massive retinal infiltrates

Visual impairment as the initial symptom of leukemia, especially chronic myeloid leukemia, is rare, occurring in approximately 3% of all leukemias in children. We describe the case of a 14-year-old boy with vision loss attributable to bilateral central retinal vein occlusion and massive retinal infiltrates. Bone marrow biopsy demonstrated the BCR-ABL translocation, and the patient was diagnosed with chronic myeloid leukemia. A major cytogenetic remission was achieved 6 months after initiation of therapy.

Clinical Significance of Complete Cytogenetic Response (CCyR) and Major Molecular Response (MMR) Achieved with Different Treatment Modalities Used As Frontline Therapy In Chronic Myeloid Leukemia (CML) Chronic Phase (CP)

Abstract 745▪FN2▪This icon denotes a clinically relevant abstract Background:Recent studies have shown that treatment of CML-CP with second generation tyrosine kinase inhibitors (2G-TKI) dasatinib and nilotinib result in a higher rate of CCyR and MMR and these responses are achieved earlier. Whether the same level of response at a given time-point achieved with different treatment modalities has the same implications for long-term outcome is unclear. Aim: To determine whether the early achievement of CCyR or MMR with different treatment modalities has similar long-term prognostic implications among patients with CML CP. Methods: The outcome of 485 patients treated in consecutive or parallel clinical trials of TKI’s as initial therapy for CML-CP were analyzed. Patients were treated with imatinib 400mg (IM400) (n=73), imatinib 800mg (IM800) (n=208), nilotinib (n=105), and dasatinib (n=99). Patients were followed uniformly with cytogenetics and PCR every 3 months for the first 12 months, then every 6 months. We reviewed the rates of CCyR and MMR at early time points, and analyzed the probability of long-term event-free survival (EFS), transformation-free survival (TFS) and overall survival (OS) according to the achievement of early responses. We then compared whether such outcome was similar for patients who achieved such response with different treatment modalities. For EFS an event was defined to include any of the following: loss of CCyR, in addition to standard IRIS definition of loss of complete hematologic remission (CHR), loss of major cytogenetic remission (MCyR), progression to accelerated phase (AC) or blast crisis (BC), or death due to any cause on treatment. Results: CCyR at any time was achieved by 94% of patients treated with nilotinib, 96% of those on dasatinib, 87% with IM400 and 91% with IM800. Rates of MMR (as defined by BCR-ABL/ABL ≤0.1% IS) of patients receiving nilotinib was 88%, dasatinib 86%, IM800 87% and IM400 73%. At six months, the rates of CCyR were 91%, 94%, 85%, 53% respectively. Corresponding rates at 12 months were 95%, 96%, 90% and 66%. The rate of MMR at 12 months was 86%, 74%, 81%, and 55%, respectively, and at 18 months 88%, 80%, 83%, and 67%. For patients taking nilotinib 9% had lost CCyR at 36mo, with dasatinib 6%, IM800 5%, and IM 400 13%. The median time to achieve MMR was 6 months for patients treated with nilotinib and dasatanib, compared to 6 months with IM800 and 9 months with IM400. For the total population, those achieving a CCyR at 6 months had a better EFS rate at 24 months (97%) compared to those not achieving this response (79%). Similarly, CCyR at 12 months correlated with a similar benefit in EFS (98% vs 72%). Achievement of CCyR and MMR at 18 months resulted in a 24 month probability of EFS of 98% compared to 87% for those with CCyR but no MMR and 71% for those with no CCyR. We then compared the results by treatment arm. Results are shown in Table 1. At two-year landmark, early achievement of CCyR by 6-mo was associated with higher EFS (but not OS) with all treatment modalities. Achieving MMR by 18 mo was a positive predictor of 24-mo EFS with IM and Dasatinib, but pts with Nilotinib so far had an excellent outcome regardless of 18-mo response. Conclusions: Time to achieving CCyR and MMR with frontline TKI’s in CML-CP, reflects similar prognostic implications regardless of the modality of frontline therapy used to achieve it. However, earlier achievement of CCyR with nilotinib or dasatinib results in a higher percentage of patients obtaining the long-term benefit associated with early responses. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The Effect of Imatinib Mesylate for Newly Diagnosed Philadelphia Chromosome-Positive, Chronic-Phase Myeloid Leukemia in Sub-Saharan African Patients: The Experience of Côte d'Ivoire

Imatinib mesylate, showed encouraging activity in chronic myelogenous leukemia. However, there are few data regarding his efficacy and response monitoring in Sub-Saharan African patients. Our objective was to assess response to imatinib mesylate (Glivec) in Côte d'Ivoire patients with newly diagnosed Chronic Myeloid Leukemia (CML). From May 2005 to September 2009, we treated 42 patients (40 years; range 16–69) with Philadelphia chromosome (Ph+) positive in chronic phase CML with oral imatinib mesylate at daily doses of 400 mg. Overall survival (OS) and frequency of complete or major cytogenetic remission (CCR/MCR) were evaluated. At a median follow up of 32 (range 7.6–113) months, the CHR rate in our study group was 76%. A major CR was found in 19 patients (45%) with 17% and 29% complete and partial CR respectively. There were no significant differences in the incidence of major cytogenetic response by known prognostics factors. Median time to CHR was 8 months (range 0.4–25), and 16 months (range: 0.1–36) for CR. Projected 5-year OS rate was 72% (95%CI 42–88). We conclude that imatinib therapy sub-Saharan African CML patients is very promising and has favorably changed the prognosis for black African patients with CML.

Impact of additional chromosomal aberrations and BCR-ABL kinase domain mutations on the response to nilotinib in Philadelphia chromosome-positive chronic myeloid leukemia

Additional chromosomal aberrations in Philadelphia chromosome-positive chronic myeloid leukemia are non-random and strongly associated with disease progression, but their prognostic impact and effect on treatment response is not clear. Point mutations in the BCR-ABL kinase domain are probably the most common mechanisms of imatinib resistance. We assessed the influence of additional chromosomal aberrations and BCR-ABL kinase domain mutations on the response to the second-generation tyrosine kinase inhibitor nilotinib after imatinib-failure. Standard cytogenetic analysis of metaphases was performed to detect additional chromosomal aberrations and the BCR-ABL kinase domain was sequenced to detect point mutations. Among 53 patients with a median follow-up of 16 months, of whom 38, 5 and 10 were in chronic phase, accelerated phase and blast crisis, respectively, 19 (36%) had additional chromosomal aberrations and 20 (38%) had BCR-ABL kinase domain mutations. The 2-year overall survival rate of all patients with-out additional chromosomal aberrations (89%) was higher than that of patients with such aberrations (54%) (P=0.0025). Among patients with chronic phase disease, overall survival at 2 years was 100% and 62% for patients without or with additional chromosomal aberrations, respectively (P=0.0024). BCR-ABL kinase domain mutations were associated with lower remission rates in response to nilotinib, with 9 of 20 (45%) of these patients achieving a major cytogenetic remission as compared to 26 of 33 (79%) patients without mutations (P<0.05). However, overall survival was not affected by BCR-ABL kinase domain mutations. Whereas BCR-ABL kinase domain mutations may confer more specific resistance to nilotinib, which will predominantly affect response rates, the presence of additional chromosomal aberrations may reflect genetic instability and, therefore, intrinsic aggressiveness of the disease which will be less amenable to subsequent alternative treatments and thus negatively affect overall survival. Conventional cytogenetic analyses remain mandatory during follow-up of patients with chronic myeloid leukemia under tyrosine kinase inhibitor therapy.

High rates of durable response are achieved with imatinib after treatment with interferon plus cytarabine: results from the International Randomized Study of Interferon and STI571 (IRIS) trial

Imatinib is the standard of care for newly diagnosed chronic-phase chronic myeloid leukemia. The largest randomized clinical trial of imatinib was the multinational IRIS trial in which 1106 patients were randomized to receive either imatinib 400 mg/day or a standard regimen of interferon-alpha plus cytarabine. Patients were allowed to cross over to the opposite treatment for intolerance, lack of response, disease progression, and, following release of the initial efficacy data, reluctance to remain on therapy with interferon-alpha plus cytarabine. The safety and efficacy of imatinib in patients who crossed over from interferon-alpha plus cytarabine to imatinib is reported here. Of 553 patients originally assigned to interferon-alpha plus cytarabine, 65% crossed over to imatinib, of whom 67% continue to receive treatment. After a median of 54 months of imatinib treatment on study, 93% achieved complete hematologic remission, 86% achieved major cytogenetic remission, and 81% achieved a complete cytogenetic remission as the best observed response. Estimated rates of freedom from progression to accelerated or blast phase and overall survival were 91% and 89%, respectively, at 48 months after starting imatinib. This is the largest analysis to date describing the efficacy of imatinib in patients who have received prior therapies for chronic myeloid leukemia and it demonstrates excellent responses to this treatment. (ClinicalTrials.gov identifier: NCT00006343).

Update of recent studies in chronic myeloid leukemia

Update of recent studies in chronic myeloid leukemia

Randomized Comparison of Imatinib 400 Mg Vs. Imatinib + IFN Vs. Imatinib + AraC Vs. Imatinib after IFN Vs. Imatinib 800 Mg: Optimized Treatment and Survival. Designed First Interim Analysis of the German CML Study IV

In spite of favorable response and survival results for the majority of CML patients on imatinib therapy, in a substantial minority imatinib fails or shows suboptimal responses. A treatment optimization study was therefore designed to compare in a randomized fashion standard imatinib vs. imatinib + interferon alpha (IFN) vs. imatinib + low dose araC vs. imatinib after IFN (for low- and intermediate-risk patients) or vs. imatinib 800 mg (for high-risk patients). Inclusion criteria were newly diagnosed BCR/ABL positive CML in chronic phase. In July 2005, randomization to the arms imatinib + araC and imatinib after IFN was discontinued and recruitment for imatinib 800 mg was expanded to low- and intermediate-risk patients. Primary goals are: rates of hematologic, cytogenetic and molecular remissions, duration of chronic phase, overall survival, adverse events and analysis of subsequent allografting. Since its activation in 7/2002, 1203 patients have been randomized. The current evaluation represents the first of three designed, statistically adjusted interim analyses of 710 patients randomized by the end of 2005 with a followup of at least 2 years. Analysis was according to intention to treat. 666 patients (545 with primary imatinib, 121 with primary IFN) were evaluable for hematologic, 621 for cytogenetic, and 631 for molecular responses. Median age was 53 years, 60% were male, median values were for Hb 12.5 g/dl, WBC 71.2/nl and platelets 384/nl, 35% had low, 53% intermediate and 12% high risk (Euro score). Median observation time was 3.5 years. Median duration of IFN pretreatment was <4 months. At 1 year, the cumulative incidence of complete hematologic remission (CHR) was 82.3% and 74.4%, of major cytogenetic remission (MCR) 65.6% and 40.6%, of complete cytogenetic remission (CCR) 52% and 19.7%, and of major molecular remission (MMR) 33.2% and 4.7% for primary imatinib and IFN therapies, respectively. At 3 years, the cumulative incidence of CHR was 96.4% and 93.8%, of MCR 89.5% and 89.1%, of CCR 85.2% and 78.5%, and of MMR 79% and 63% for primary imatinib and IFN therapies, respectively. 5-year-survival probability of all patients currently exceeds 90% (94% for imatinib-, 91% for IFN-based therapy, Figure 1). Event free survival after two years (no progression, no death, CCR within the first 18 months, no loss of CHR or MCR) was 80.3%. 36 patients died, 51 patients were transplanted in first chronic phase, and 80 patients progressed, 43 of which were switched to alternative treatments (16 to new drugs, 18 to transplantation, 9 received both). Type and severity of adverse events (AE) did not significantly differ from those reported previously. Hematologic AEs (leukopenia, thrombocytopenia) were most frequent in the imatinib 800 mg arm. Nonhematologic AEs (gastrointestinal) were most frequent in the combination arms and with imatinib 800 mg. In no case recruitment had to be changed due to superiority or inferiority of any arm. This applies also to the high dose imatinib arm where earlier response might translate into better survival. In conclusion, this first interim analysis shows favorable survival and long term response rates. Imatinib in combination with, or after, IFN or with low dose araC are feasible and equally safe treatment alternatives. More definite information will be provided by the next interim evaluation after recruitment has been terminated. [Display omitted]

A Retrospective Study of Introferon-Alpha Treatment on Chinese Patients with Chronic Myeloid Leukemia

Introduction: Chronic Myeloid Leukemia (CML) is one of the myeloproliferative malignancies characterized by the reciprocal t(9;22)(q34;q11) chromosome, which causes the generation of Bcr-Abl fusion protein. Interferon-alpha (IFN-alpha) was introduced into clinical application from 1980s' and showed its treatment benefit mainly in chronic phase CML (CML-CP) patients including achieving hematological remission, prolonging survival. Our retrospective study aimed to evaluate its clinical efficacy as well as toxicities in Chinese CML-CP patients.Methods: This is a retrospective, multicentric clinical trial. Chinese CML patients in chronic phase received IFN-alpha treatment from 1992 to 2006 were enrolled. Other agents such as hydroxyurea or cytarabine treatment were permitted during treatment excepted for tyrosine protein kinase inhibitors.Results: 1037 CML-CP patients were into analysis, the median age was 42 yrs (range 9–80) and male: female ratio of 1.6:1. The median time for interferon treatment was 12 months (range from 1 month to 136 months). 86.32% of patients received 300Mu of interferon, three times a week, 10.88% in 300Mu of daily injection, while another 2.8% in 500Mu of interferon treatment, three times a week. Main of additional treatment included hydroxyurea, which was used in 8.9% of patients, and cytarabine treatment in 3.3% of patients. We evaluate the best response reaction during IFN-alpha treatment, 496 out of 1037 patients obtained hematological remission (47.8%). 33 in complete cytogenetic remission (CCR, 3.2%) but unfortunately 60% of them were lost CCR after median of 6–11 months; 89 in partial cytogenetic remission (8.6%); 40 in major cytogenetic remission (3.9%); 301 in minor cytogenetic remission (29%); 78(7.5%) of CML-CP patients worsen into accelerate phase or blast crisis. Non-Hematological toxicities during IFN-alpha treatment mainly included flu-like symptom (24.9%) and fatigue (23.5%) which were transient and most of patients got recovery after second injection whereas 20% of them could not endurable. While hematological toxicities were neutropenia (22.1%, 33% of them were in grade III or IV), and thrombocytopemia (16.9%, 33% of them in grade III or IV).Conclusion: The IFNalpha is one candidate agent for CML-CP patients in China with moderate toxicities during treatment, but it has little influence on achieving or maintaining complete cytogenetic remission.

Allogeneic Stem Cell Transplantation (SCT) in Advanced Chronic Myeloid Leukemia (CML) Patients after Tyrosine Kinase Inhibitor (TKI) Therapy

In contrast to chronic phase CML patients (pts), in whom imatinib is the treatment of choice, in advanced CML pts allogeneic SCT remains the only therapeutic option with a curative potential. In order to adress the impact of pre-transplant parameters such as remission status, duration of disease, baseline therapies and cytogenetic and/or mutation status we analysed advanced CML pts with TKI pre-treatment (imatinib, nilotinib and/or dasatinib) who subsequently received an allogeneic stem cell transplant. A total of 24 pts (15 males, 9 females) was studied (median age 43,8 years, range: 21 – 58). Of these 7 pts were in 1. chronic phase (CP), 10 in 2nd or 3rd CP, 2 in accelerated phase (AP) and 5 in blast crisis (BC). Eleven of the 17 CP pts had either a CHR (complete hematologic remission) or HR at SCT and seven had a major cytogenetic remission (MCyR, including 4 CCyR). Cytogenetics and molecular analysis at SCT showed additional chromosomal abnormalities in 9/21 and point mutations in the BCR-ABL kinase domain in 2/17 pts (E255V & Y253H). All pts received imatinib prior to SCT, in 4 pts a 2nd line TKI therapy (dasatinib or nilotinib) and in one patient a 3rd line TKI therapy (dasatinib) was given. In 8/24 pts additional chemotherapy or irradiation after the last TKI were given prior to conditioning therapy. In 23/24 pts a myeloablative conditioning regimen was given. 15 pts were transplanted from an unrelated and 9 pts from an HLA identical family donor. The median follow-up of all patients is 19.8 months (range: 0 – 71). 12/24 pts died (SCT failure: n= 1, infection: n=5, GvHD: n=3, relapse: n=3). Risk stratification by basline parameters showed a statistically relevant survival advantage for patients with at least a MCyR and no non-TKI therapy prior to conditioning therapy (14 versus 10 pts; 14 versus 5 months; p=0.036; Chi-square test). Likewise, a survival benefit was identified for pts receiving SCT with at least a baseline HR (15 versus 9 pts; 11 versus 5 months; p=0.011; Chi-square test). Conversely, no statistically relevant differences were observed when pts were stratified upon additional chromosomal alterations, donor origin or stage of disease at baseline. In summary our data confirm the feasibility and benefit of allogeneic SCT in advanced CML patients following TKI treatment. Relevant pre SCT risk factors that could be identified as part of this analysis included baseline response and the absence of a non-TKI treatment prior to the conditioning therapy. Further studies are required to optimize the pretransplant treatment of advanced CML patients that are eligible for allo SCT.

Clonal Hematopoiesis in Philadelphia Chromosome-Negative Bone Marrow Cells of Chronic Myeloid Leukemia Patients Receiving Tyrosine Kinase Inhibitors

Patients with chronic myeloid leukemia (CML) are experiencing prolonged survival due to successful therapy with tyrosine kinase inhibitors. However, some CML patients who have achieved longstanding remissions with these agents harbor clonal cytogenetic abnormalities in their Philadelphia chromosome negative (Ph-) bone marrow cells. Because CML patients in remission often have peripheral blood count abnormalities, including cytopenias, we investigated whether these patients may have developed myelodysplastic syndrome (MDS) within the Ph- cell population. Bone marrow samples from 26 CML patients who had achieved a major cytogenetic remission (MCyR) with tyrosine kinase inhibitor therapy between 2 and 15 years after diagnosis were evaluated; 6 patients had advanced disease prior to their last therapy, 20 were in chronic phase. At the time of evaluation, 2 of the patients were receiving imatinib, 23 dasatinib, and 1 PHA739358. At least one peripheral blood lineage was abnormal in 21 patients, of whom 7 had pancytopenia. Routine metaphase cytogenetics (MC) revealed a persistent clonal chromosomal abnormality in 10% of the Ph- metaphases in 5 patients (+8 in 2, −7 in 2, and 20q- in 1). We hypothesized that clonal hematopoiesis might exist in additional patients and applied single nucleotide array (SNP-A) based karyotyping and X-linked human androgen receptor (HUMARA) clonality assay to further delineate the nature of the hematopoietic defect in these patients. HUMARA was performed on bone marrow samples and germ-line DNA from peripheral blood T lymphocytes of the female patients. Clonality, as assessed by skewing of X-chromosome inactivation in bone marrow cells compared to germline control cells, could not be demonstrated in the12 female patients. SNP-A karyptyping using 250K Affymetrix SNP array confirmed the known cytogenetic abnormalities. Several microdeletions were found, but comparison with purified T lymphocytes demonstrated that these “lesions” represented germ line-encoded copy number variants. However, SNP-A karyotyping revealed the presence of uniparental disomy (UPD) involving chromosome 17(p12-pter) in bone marrow, but not germ line cells, from one male patient with normal karyotype by routine MC. In the context of secondary AML, del17p or UPD17 have been observed always in the presence of del7/q and 5q and were associated with poor prognosis. However, in our patient UPD17 occurred as a sole defect. Because in our studies in AML, UPD of chromosome 17p was found in association with p53 mutations, genomic sequencing of this gene was performed. A 5 bp deletion destroying the splice acceptor region of exon 6 was identified in bone marrow cells from this patient. Alternative splicing leading to loss of exon 6 was predicted to result in a frame shift and premature introduction of a stop codon. These methods revealed clonal hematopoiesis in the Ph- bone marrow cells of 6/26 patients with longstanding CML in remission from tyrosine kinase inhibitors and persistent peripheral blood abnormalities. The approaches used here probably underestimate the frequency of this condition, as oligoclonal populations may be present in numbers below the limit of assay sensitivity. The Ph- clonal bone marrow populations have cytogenetic and molecular features in common with MDS. After a median follow up of two years, one patient with monosomy 7 developed acute myeloid leukemia, but longer follow up will be required to determine the natural history of the Ph- clonal disorders.

CELSG CML 11 “ISTAHIT” Phase III Study – Planned Interim Analysis: High Doses of Imatinib Mesylate (800mg/day) Significantly Improve Rates of Major and Complete Cytogenetic Remissions (MCR, CCR) in Pretreated Ph+/BCR-ABL+ CML Patients in Chronic Phase.

We have recently reported results from the first planned interim analysis of a multicenter, randomised, 2-arm - phase III study comparing imatinib standard dose (400 mg/day; arm A) with imatinib high dose (HD) induction (800 mg/day; HD arm B) followed by imatinib standard dose maintenance (400 mg/day) in pretreated Ph+/BCRABL+ CML patients in chronic phase (CP; Blood,110,1048a). The first planned interim analysis was performed after 50% of the patients had been treated for 12 months (mo) since randomisation. A total of 227 patients were randomized. Pretreatments included hydroxyurea (96%), interferon (72%), busulfan (17%) and “others” (26%; mainly AraC +/− combinations). Although rates of complete haematological responses did not differ significantly between the 2 arms at 3, 6 and 12 mo, significantly more patients achieved a major (MCR) and a complete cytogenetic response (CCR) at 3 mo (MCR: 21% arm A, 37% arm B, p=0.01; CCR: 6% arm A, 25% arm B, p<0.001) and 6 mo (MCR: 34% arm A, 54% arm B, p=0.009; CCR: 20% arm A, 44% arm B, p<0.001). At 12 mo, following dose reduction of imatinib to 400 mg/d for “maintenance” at mo 6 in the HD arm B, the rates of MCR (the primary endpoint of the study) were comparabel (59% arm A, 57% arm B). Nevertheless, there was still a clear trend to higher rates of CCR (37% arm A, 48% arm B) in the HD arm B, but at the time of the interim analysis these values did not reach statistical significance. In contrast to non-hematological toxicities, grade 3/4 hematological toxicities were significantly more common in the HD arm B (anemia: 2% arm A, 14% arm B; leukopenia: 24% arm A, 46% arm B; thrombocytopenia: 15% arm A, 39% arm B; p≤0.02). Updated results reveal, that the cumulative median doses of imatinib were 400 mg (arm A) and 767 mg (arm B), respectively. The median days of dose interruptions during the first 6 mo were not significantly different (12.5 days arm A, 13 days arm B; p=0.78). Nevertheless, significantly more patients (65.1%) remained on the initial 400mg dose of imatinib in arm A as compared to the initial 800mg dose in the HD arm B (45.6%; p=0.009). 35% of the patients that had to be dose reduced in arm B were reduced to 600mg/day, 63% to 400mg and 2% to 300mg. The cumulative rates of MCR in patients with no dose reduction/interruption were higher in the HD arm B (71%) compared to arm A (59%, p=0.232). Moreover, the cumulative rates of CCR were significantly better for patients with no dose reduction/interruption in the HD arm B (61%) compared to the patients with no dose reduction/interruption in arm A (36%, p=0.014). In addition, in the HD arm B the cumulative rates of CCR were significantly better for patients with no dose reduction/interruption (61%) compared to patients in the HD arm B with an interruption and/or dose reduction (34.9%; p=0.017).Conclusions: These updated phase III data not only support the concept of more rapid and higher rates of cytogenetic remissions (MCR, CCR) when higher doses of imatinib are applied, they also suggested that patients that are capable to tolerate continous high dose imatinib may have a better outcome.

Results of the AFR07 Prospective Study in De Novo Philadelphia Positive ALL Patients Aged over 55 Years: Efficacy and Safety of a Glivec® Based Induction Followed by Maintenance Therapy with Glivec® and Pegasys®.

Our previous report concluded that an imatinib (Glivec®) based induction may be proposed as front line therapy in elderly patients (pts) with Philadelphia positive acute lymphocytic leukemia (Ph+ ALL) (Rea et al. Leukemia , 2006; 20:400–3). We decided to test this hypothesis and to evaluate the value of maintenance therapy with Pegasys® and imatinib in a prospective study of pts aged over 55 years with de novo Ph+ ALL. The protocol consisted in IV injections of vincristine 2 mg combined with 2 days of dexamethasone 40 mg PO repeated weekly for 4 weeks as induction and then monthly for 4 months as consolidation. Imatinib was administered at 800 mg per day during the induction period and at 600 mg/d continuously with 6 mercaptopurine during consolidation. Pts in CR were allocated to maintenance with SC injections of Pegasys 45μg once a week and continuous administration of imatinib during 2 years. BCR-ABL analysis were performed in a centralized laboratory according to the ENL recommendations. 25 pts were included from May 2005 to July 2007. Median age was 68 years and sex ratio 0,92. After a median follow up of 13.2 months (1 to 27), median overall survival and DFS were not reached. The estimated proportion of pts alive and free of relapse at 18 months was 53.7%. The CR rate was 84%. Induction failures (n=4) were due to death in 3 cases and intolerance in 1 case. Extra haematological grade 2 to 4 adverse events during induction were mainly related to vincristine (ileus in 6 cases, neuropathy in 5 cases). Median duration of neutropenia (<1 G/l) was 18 days (0 to 32). Median duration of thrombocytopenia (<100 G/l) was 25 days (9 to 45). During consolidation 4 pts died (3 from infections and one from unrelated digestive haemorrhage) and 1 patient relapsed. Eleven pts were evaluable for maintenance. Three pts relapsed after 1, 4.5 and 8.4 months, one patient died from unrelated cause. Six pts are in continuing complete remission (63.6%). Two other pts undervent autologous HSCT. Cytogenetic analysis was performed at diagnosis in 18 patients. In 10 cases (55.5%) the Ph+ chromosome was associated with complex abnormalities or monosomy 7. All pts were in major cytogenetic remission after induction with a 69.2% CCR rate. 40% of pts in CR obtained a complete molecular remission (CMR) in blood and half of them were also in CMR in bone marrow (BM). None of the pts with BM CMR relapsed whereas 2 relapses occurred in pts with CMR in blood but not in BM suggesting that BM CMR is of prognostic value. BCR-ABL TK domain mutations were systematically detected at each time point by sequencing. No mutation was found at diagnosis. At relapse, only one patient out of 4 was mutated (G250E). The AFR07 protocol is highly effective in elderly patients with Ph+ ALL. Only 4 patients relapsed. Two third of the patients treated with imatinib and Pegasys® maintenance are in continuing CR. We observed 4 deaths in CR related to haemorrhage and sepsis. Second generation TKIs will be tested in futur joint European trials.

Sustained remissions and low rate of BCR‐ABL resistance mutations with imatinib treatment chronic myelogenous leukemia in patients treated in late chronic phase: A 5‐year follow up

The introduction of Imatinib (IM) has significantly altered the treatment for CML, although only limited follow-up results are available. As failure of Interferon-alpha had been associated with poor prognosis and results of IM-treatment in this patient group may allow earlier estimation of long-term benefits for early chronic phase patients. Therefore we prospectively analyzed the quality and duration of remissions and the rate of BCR-ABL resistance mutations occurring in patients treated with IM, if they were intolerant or refractory to interferon. Fifty-nine patients were included and median follow up is 4.75 years. Haematologic remission rate was 92% and 62% of patients achieved at least major cytogenetic remission. There were no major differences between patients failing or being refractory to IFN. Major molecular response was observed in 67% of patients evaluable. For the entire group, median PFS was 4.3 years and OS was 82% at 4.75 years. Haematologic and complete cytogenetic remissions, but not molecular responses were correlated with improved PFS and OS. In patients with progression or inadequate response, BCR-ABL kinase domain mutations were detected in 3/28 patients. IM resulted in prolonged remission rates in a substantial proportion after IFN-failure/intolerance, with no differences in these patient groups. Quality of hematologic and cytogenetic, but not molecular response was associated with duration of remissions and survival. A low rate of resistance-mutations was detected, suggesting that alternative mechanism may play an important role in disease progression.

Dasatinib induces significant hematologic and cytogenetic responses in patients with imatinib-resistant or -intolerant chronic myeloid leukemia in accelerated phase

AbstractTreatment options are limited for patients with imatinib-resistant or -intolerant accelerated phase chronic myeloid leukemia (CML-AP). Dasatinib is a novel, potent, oral, multitargeted kinase inhibitor of BCR-ABL and SRC-family kinases that showed marked efficacy in a phase 1 trial of patients with imatinib-resistant CML. Results are presented for 107 patients with CML-AP with imatinib-resistance or -intolerance from a phase 2, open-label study further evaluating dasatinib efficacy and safety. At 8 months' minimum follow-up, 81%, 64%, and 39% of patients achieved overall, major (MaHR), and complete hematologic responses, respectively, whereas 33% and 24% attained major and complete cytogenetic remission. Of 69 patients who achieved MaHR, 7 progressed. Seventy-six percent of patients are estimated to be alive and progression-free at 10 months. Response rates for the 60% of patients with baseline BCR-ABL mutations did not differ from the total population. Dasatinib was well tolerated: most nonhematologic adverse events (AEs) were mild to moderate; no imatinib-intolerant patients discontinued dasatinib because of AEs. Although common (76% of patients with severe neutropenia), cytopenias were manageable through dose modification. In summary, dasatinib induced significant hematologic and cytogenetic responses in patients with imatinib resistance or intolerance, was well tolerated, and may represent a potent new therapeutic option for CML-AP. Further follow-up is warranted. This trial was registered at www.clinicaltrials.gov as #CA180005.

Dynamics of Cytogenetic Aberrations in Philadelphia Chromosome Positive and Negative Hematopoiesis during Dasatinib Therapy of Chronic Myeloid Leukemia Patients after Imatinib Failure.

Clonal cytogenetic aberrations of the Philadelphia chromosome (Ph) positive hematopoiesis have been associated with the natural evolution of CML to advanced disease. Clonal aberrations of Ph negative metaphases have been described after treatment with interferon alpha or imatinib in a minority of patients (pts) with cytogenetic response. Conflicting data suggest selection of preexisting clones vs. induction of aneuploidy by tyrosine kinase inhibitors. The prognostic impact of aberrations in the Ph negative hematopoiesis for the individual pts remains to be determined. Dasatinib is a multitargeted agent inhibiting both ABL and SRC tyrosine kinases. The efficacy and safety of dasatinib has been demonstrated in phase I and II studies in pts with Ph positive CML after failure of imatinib therapy. We sought to evaluate the effect of dasatinib on Ph positive clones with additional cytogenetic aberrations and the frequency of novel aberrations in Ph positive and Ph negative metaphases during dasatinib therapy. 71 pts (40 m, 31 f) with Ph positive CML after failure of imatinib therapy have been evaluated. Median age was 58 years (range, 28–78), median time from diagnosis 73 months (range, 14–231). Pts were in chronic phase (CP, n=50), accelerated phase (AP, n=6), myeloid (n=8) or lymphoid (n=7) blast crisis (BC). Pretreatment consisted of imatinib (n=71), hydroxyurea (n=48), interferon alpha (n=49), cytosine arabinoside (n=11) and nilotinib (n=2). Dasatinib therapy was commenced at a dose of 100–140 mg/day, median duration of dasatinib therapy was 9 months, (range, 1–16). Prior to dasatinib therapy, 22 pts (31%) demonstrated additional chromosomal aberrations in the Ph positive clone indicating BCR-ABL independent mechanisms of resistance. Of these, 8 pts were in CP (16%), 5 in AP (83%), and 9 in BC (60%). In 35 pts (49%), BCR-ABL mutations associated indicating a BCR-ABL dependent mechanism of resistance were observed. 9 pts (13%) showed both clonal evolution and BCR-ABL mutations. Two pts (3%) had trisomy 8 as an aberration of the Ph negative clone at baseline. During dasatinib therapy, 33 pts (46%) achieved major cytogenetic remission, 26 (37%) being complete. From pts with clonal evolution, 3 (14%) achieved a major with 2 (9%) complete cytogenetic remissions. Novel aberrations of the Ph positive clone appeared during dasatinib therapy in 6 pts (8%), aberrations of the Ph negative clone in 3 pts (4%). In total, 5/71 pts (7%) showed clonal aberrations of Ph negative metaphases after consecutive imatinib/dasatinib therapies. None of these pts had morphological indications for secondary neoplastic changes, like myelodysplasia or acute leukemia. In conclusion, dasatinib was efficacious in pts with clonal cytogenetic aberrations as a marker of BCR-ABL independent imatinib resistance. However, cytogenetic response was delayed compared to pts without additional aberrations. Preexisting clonal aberrations of the Ph negative hematopoiesis were uncovered by a simultaneous gradual elimination of the Ph positive clone. In addition, a minority of pts demonstrated novel aberrations in Ph negative cells. The prognostic significance of aneuploidy of the Ph negative hematopoiesis remains to be evaluated by long term observation.

Pilot trial of a synthetic breakpoint peptide vaccine in patients with chronic myeloid leukemia (CML) and minimal disease

6514 Background: CML is characterized by a tumor-specific fusion oncoprotein, BCR-ABL. Peptides spanning the B3A2 breakpoint of BCR-ABL can elicit MHC restricted T cell responses and clinical responses. No such data have previously been reported for vaccines targeting B2A2. Methods: We initiated a clinical trial administering synthetic analog breakpoint specific peptide vaccines for either B3A2 or B2A2 breakpoints to CML patients with major or complete cytogenetic remission. Measurable minimal disease was determined by either quantitative polymerase chain reaction (RQ-PCR) or nested PCR for BCR-ABL. Vaccine was administered with GM-CSF and Montanide ISA 51 subcutaneously. Eleven vaccinations were planned over the course of 1 year with the first 5 doses administered bi-weekly. Results: Eleven of 20 planned patients have been accrued to the study. Eight had documented cytogenetic remission and 3 were BCR-ABL positive via FISH analysis. Seven of the 11 patients had measurable BCR-ABL transcript levels as determined by RQ-PCR and all 11 were positive using the nested technique. Of the 5 patients positive for BCR-ABL (by RQ-PCR) before vaccination, 3 converted from RQ-PCR positive to negative while the transcript levels in the other patients decreased by approximately 1 log after 5 doses. All 5 of these patients remained positive using the nested PCR technique. Vaccination was well tolerated with local skin reactions at the injection sites. Immunologic reactivity was assessed ex vivo by CD4 autologous proliferation assay and T cell interferon (IFN) secretion (ELISPOT) assay. Ten of 11 patients were immunologically unreactive to the peptides prior to the vaccinations. Following the 5th dose of vaccine, 8/8 patients (including 2 with B2A2) tested showed significant immunologic responses by one of the above assays. Conclusions: These preliminary results suggest vaccination with synthetic analog peptides derived from CML proteins results in immunologic responses and may be associated with molecular improvement. Clinical efficacy of these vaccines in reducing/ eliminating minimal disease has yet to be established. [Table: see text]

Gene expression signature of primary imatinib-resistant chronic myeloid leukemia patients

Although the selective tyrosine kinase inhibitor imatinib is successfully used in the treatment of chronic myeloid leukemia (CML), inherent mechanisms confer primary resistance to leukemic patients. In order to search for potentially useful genes in predicting cytogenetic response, a retrospective gene expression study was performed. Leukocyte RNA isolated before imatinib from interferon-alpha-pretreated chronic phase CML patients (n=34) with or without major cytogenetic remission (< or =35% Philadelphia (Ph)+ metaphases) during the first year of treatment was comparatively analyzed using Affymetrix U133A chips. Using support vector machines for gene classification, an outcome-specific gene expression signature consisting of 128 genes was identified. Comparative expression data of specific genes point to changes in apoptosis (e.g. casp9, tumor necrosis factor receptor-associated protein 1, hras), DNA repair (msh3, ddb2), oxidative stress protection (glutathione synthetase, paraoxonase 2, vanin 1) and centrosomes (inhibitor of differentiation-1) within primary resistant patients. Independent statistical approaches and quantitative real-time reverse transcriptase-polymerase chain reaction studies support the clinical relevance of gene profiling. In conclusion, this study establishes a candidate predictor of imatinib resistance in interferon-alpha-pretreated CML patients to be subjected to future investigation in a larger independent patient cohort. The resulting expression signature point to involvement of BCR-ABL-independent mechanisms of resistance.

Feasibility of Administering Oblimersen (G3139; Genasense) with Imatinib Mesylate in Patients with Imatinib Resistant Chronic Myeloid Leukemia (CML) - Cancer and Leukemia Group B Study 10107.

The CALGB studied the feasibility and effect of oblimersen (G3139; Genasense) with imatinib mesylate in patients with primary imatinib resistant CML. We hypothesized that CML cells that are resistant to imatinib mesylate (i.e., less than a complete hematologic remission after 8 weeks of treatment or less than a major cytogenetic remission after 6 months of treatment) are no longer being driven to proliferate by Bcr/Abl tyrosine kinase activity alone. Instead, the anti-apoptotic protein Bcl-2 would regulate one of the pathways controlling growth and/or viability. Thus, blocking both Bcr/Abl and Bcl-2 simultaneously with imatinib mesylate and G3139, respectively, would result in hematologic and cytogenetic improvement. Preclinical data suggested synergy. G3139 was administered via continuous intravenous infusion with an ambulatory pump over 10 days every 21 days, along with daily oral imatinib. Doses of both drugs were escalated in 3 cohorts; the initial dose of imatinib was 600 mg/day. The median age of the 21 enrolled patients (13 females and 8 males) who received any therapy was 54 years (range, 25–74), with 13 Caucasians and 8 African Americans. Twelve patients had primary imatinib resistance and 8 had secondary imatinib resistance (data are not yet available on 1 patient). Five patients had never achieved complete hematologic remission while only two patients had achieved major cytogenetic remission while on a prior imatinib therapy. Nine patients received 4 mg/kg/day G3139 and 600 mg of imatinib, 7 patients received 7 mg/kg/day G3139 and 600 mg of imatinib, and 5 patients received 7 mg/kg/day G3139 and 800 mg of imatinib. Progressive CML after the first (4 patients), second (1 patient), third (2 patients) or fourth course (1 patient) was noted in 8 patients whose imatinib dose had been reduced from 800 mg to 600 mg/day to comply with study entry requirements. Nine patients completed all 4 planned courses. Grade IV thrombocytopenia and fatigue (both at 7 mg/kg G3139 and 600 mg of imatinib) and deep vein thrombosis (at 7 mg/kg G3139 and 800 mg of imatinib) were noted in one patient each. One patient with primary imatinib resistance achieved complete hematologic remission following 2 courses of therapy at 4 mg/kg G3139 and 600 mg of imatinib. No patients had cytogenetic responses. Although the combination of G3139 and imatinib is safe and feasible, we did not detect clinical benefit in these patients with imatinib-resistant CML using these doses and schedule.