Patients In Complete Cytogenetic Remission Research Articles

Immature CML cells implement a BMP autocrine loop to escape TKI treatment

AbstractThe BCR-ABL specific tyrosine kinase inhibitors (TKI) changed the outcome of chronic myeloid leukemia (CML), turning a life-threatening disease into a chronic illness. However, TKI are not yet curative, because most patients retain leukemic stem cells (LSC) and their progenitors in bone marrow and relapse following treatment cessation. At diagnosis, deregulation of the bone morphogenetic protein (BMP) pathway is involved in LSC and progenitor expansion. Here, we report that BMP pathway alterations persist in TKI-resistant patients. In comparison with patients in complete cytogenetic remission, TKI-resistant LSC and progenitors display high levels of BMPR1b expression and alterations of its cellular localization. In vitro treatment of immature chronic phase CML cells with TKI alone, or in combination with interferon-α, results in the preferential survival of BMPR1b+ cells. We demonstrated persistent and increasing BMP4 production by patients' mesenchymal cells with resistance. Patient follow-up revealed an increase of BMPR1b expression and in BMP4 expression in LSC from TKI-resistant patients in comparison with diagnosis, while remaining unchanged in sensitive patients. Both leukemic and nonleukemic cells exhibit higher BMP4 levels in the bone marrow of TKI-resistant patients. Exposure to BMP2/BMP4 does not alter BCR-ABL transcript expression but is accompanied by the overexpression of TWIST-1, a transcription factor highly expressed in resistant LSC. By modulating BMP4 or BMPR1b expression, we show that these elements are involved in TKI resistance. In summary, we reveal that persistence of BMP alterations and existence of an autocrine loop promote CML-primitive cells' TKI resistance.

Characterization of Genomic Events Other than Ph and Evaluation of Prognostic Influence on Imatinib in Chronic Myeloid Leukemia (CML): A Study on 1449 Patients from India

Background: Analysis of Philadelphia (Ph) chromosome, a hallmark of chronic myeloid leukemia (CML) plays an important role in disease monitoring of the targeted drug Imatinib. Apart from Ph, genomic imbalances such as additional chromosomal abnormalities (ACAs) of major route occur during transformation of the disease and show negative impact on prognosis. Objective: The present study was carried out to investigate frequencies of ACAs, genomic deletions, complex Ph variants and their prognostic influences in a large cohort of newly diagnosed CML-CP (chronic phase) and CML-AP/BP (accelerated/blast phase). Material & Methods: Retrospective, single institutional study on 1367 cases of CML-CP and 82 cases of CML-AP/BP between 2009 and 2015, using conventional cytogenetics along with fluorescence in situ hybridization. Results: Of the 1367 patients in CML-CP, 1041 patients who completed 12 - 18 months of Imatinib therapy showed complete cytogenetic remission (CCyR) rates of 76% and 82% at 12 and 18 months respectively. Imatinib induced 81% and 33% CCyR in CML-AP and CML-BP respectively. Frequencies of ACAs in CML-CP, AP and BP were 2%, 27% and 67% respectively. Patients in chronic and AP/BP phase with ACAs showed resistance to Imatinib (p p p < 0.210 respectively). In a cohort of 112 patients in CCyR, development of new clonal abnormalities, more frequently trisomy 8 was detected in Ph negative clone. Conclusion: Our data demonstrated that Imatinib as a frontline therapy had significantly improved management of CML. However, ACAs play an important role in resistance to Imatinib, both in chronic and acute phase, which may limit sole ABL targeted therapy.

Detection of Dormant Chronic Myeloid Leukemia Clones in the Bone Marrow of Patients in Complete Molecular Remission

Several methods are available to detect MRD in patients with CML in complete molecular remission (CMR) and taking tyrosine kinase inhibitor (TKI) therapy. We performed clonogenic assays on mononuclear bone marrow cells from 14 patients. Of the 10 assessable samples, 6 were from patients in CMR and 4 from patients in complete cytogenetic remission but had detectable MRD using polymerase chain reaction (PCR) analysis (positive controls). At least 10 colonies per sample were microaspirated and individual colonies were subjected to PCR analysis. Of the 6 patients in CMR, 5 harbored breakpoint cluster region abelson (BCR-ABL1) negative colonies but in 1 sample, 1 of the 10 colonies analyzed was positive for BCR-ABL1. Of the 4 patients with evidence of MRD in peripheral blood, 2 had negative and 2 had positive BCR-ABL1 colonies. MRD is still detectable using clonogenic assays in some patients with CML after achieving CMR using TKI therapy, which is likely responsible for relapse on TKI discontinuation. Because of the large number of single colonies that need to be analyzed, the use of clonogenic assays in clinical practice to determine the feasibility of TKI discontinuation is not recommended.

The Significance of BCR-ABL Transcripts after Allogeneic Stem Cell Transplantation for Chronic Myeloid Leukemia

The discovery of the BCR-ABL fusion gene was a landmark in our understanding of the molecular basis of chronic myeloid leukemia (CML) [1]. This led rapidly to the demonstration that BCR-ABL transcripts could be identified using the PCR [2]. Next, it was shown that some patients in complete cytogenetic remission after an allogeneic transplantation had no detectable BCR-ABL transcripts in their blood or bone marrow [3]. A technically rather demanding technique for quantitating BCR-ABL transcripts was then developed [4], which showed that, at least for CML patients, blood and marrow gave comparable results. The advent of Taqman methodology greatly simplified measurement of BCR-ABL transcripts in the blood of transplant recipients and today results are routinely expressed as a ratio of BCR-ABL transcript numbers related to a control gene. This ratio is usually reported as a percentage on a log scale, where 100% reflects a hypothetical untreated patient with CML. A patient with a 3-log reduction (0.1%) in BCR-ABL transcript numbers is said to have achieved a major molecular response and a patient with no detectable transcripts is said to be in complete molecular response (CMR). Another landmark was the report by Kolb and colleagues in 1990 that patients in relapse after an allotransplantation could be treated successfully by infusion of lymphocytes collected from the original donor togetherwith interferon [5]. Later, it was shown that this effect resulted from the donor lymphocytes and not interferon [6]. The capacity for these donor lymphocyte infusions (DLI) to restore CMR is presumed to be due to a “graft-versus-leukemia” effect and this observation remains one of the most convincing examples of the potential beneficial effect of immune therapy in man. Kaeda et al. reported in 2006 that patients in seeming complete remission posttransplantation sometimes had BCR-ABL transcripts detected at low levels, yet relapse was not inevitable and subsequent testing may show no evidence of transcripts without any further therapy [7]. Because of the potential to treat the patients in relapse with DLI, it became important to agree on a definition of relapse and it was proposed that a patient with a transcript level of >.02% on 3

Is Major Molecular Response a Safe Haven Against Blast Crisis in Cml Patients Treated with Imatinib?

Abstract 3760The introduction of tyrosine kinase inhibitors (TKIs) has proved to be a major advance in the management of patients with chronic myeloid leukemia in chronic phase (CML-CP) although the clinical benefit seems to be limited to those patients who achieve complete cytogenetic remission (CCyR). Patients who achieve CCyR are believed to have an excellent prognosis, but a very small proportion of those patients may lose the response and progress to blastic phase. In this work we studied a cohort of 210 patients who achieved CCyR (142 of them were in MMR) on imatinib as first line therapy in order to identify the incidence of blastic transformation in this population.Between June 2000 and December 2010, 282 consecutive adult patients with CML-CP were seen at our institution, of whom, 210 achieved CCyR on imatinib 400 mg daily as first-line therapy. The median follow-up was 69 months. Of the 210 patients in CCyR, 5 (2.3%) progressed to blastic transformation (BT) (3 myeloid and 2 lymphoid). The Sokal risk was low in 3 patients and intermediate in two. The median time to achievement of CCyR in these 5 patients was 18 months (range 9–20 months). Two of these patients also achieved a major molecular response (MR3), one achieved a 4 log reduction on the international scale and two patients had a CCyR with no MR3. The median time to achievement of MR3 (or better) in the three patients was 24 months (range 18–28 months). After achieving CCyR all 5 patients had progressive increases in their BCR-ABL1 transcript levels over a period of a few months leading to BT without an intervening accelerated phase. None of the patients had a ABL1 kinase domain mutation prior to transformation to advanced phase, but two patients subsequently developed a new mutation at the time of BT (M244V and T315I, respectively). The 8-year cumulative incidence (CI) of BT after reaching CCyR was 2.3% and was 2.8% when calculated from diagnosis (Figure 1). The median time from achieving CCyR to BT, was 18 months (range 12–40 months). The median time from the onset of an increase in BCR-ABL1 transcripts to BT was 6 months (range 4–7 months). All 5 patients had good compliance with TKIs.All 5 patients were treated by allogeneic stem cell transplantation after BT in their second chronic phase. One patient proceeded to a transplant after combination chemotherapy and TKI, but 4 others only received a TKI prior to transplant (3 dasatinib and 1 nilotinib). All 5 patients subsequently died due to transplant related causes. The 8-year CI of BT after attaining CCyR was 2.3%. MR3 does not appear to completely protect against BT as 3 of the 5 patients with MR3 lost the response and progressed to BT. In our series we could not identify a time point beyond which patients may be safe from blastic transformation, which continues to be a rare but catastrophic event in responding patients. [Display omitted] Disclosures:Goldman:Novartis, Bristol Myers-Squibb, and Amgen: Honoraria. Marin:Novartis: Research Funding; BMS: Research Funding.

Equivalence of BCR-ABL Transcript Levels

AbstractAbstract 2495 Introduction:Response to therapy in patients with chronic myeloid leukemia (CML) is monitored by both cytogenetic assessment of bone marrow metaphases and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) of peripheral blood samples. Although standardization of BCR-ABL quantification according to the international scale (IS) provides results comparable between different laboratories, current treatment recommendations are still predominantly based on cytogenetic analyses. The aim of this work was to determine a BCR-ABL expression level according to the IS (BCR-ABLIS) which corresponds to complete cytogenetic remission (CCyR). Patients:1,329 paired cytogenetic and molecular assessments of 557 chronic phase CML patients on imatinib-based therapies from the German CML study IV, all during follow-up, have been considered. Median number of samples per individual was 3 (range 1–24). Only molecular evaluations derived from peripheral blood and from patients expressing b2a2 and/or b3a2 BCR-ABL transcripts were considered. Participating laboratories provided results standardized according to the international scale. For cytogenetic analyses at least 20 metaphases, evaluated by conventional chromosome banding analysis, were required. Paired molecular and cytogenetic analyses were performed within an interval of <3 days. Methods:Data was split randomly at the rate of 2:1 into learning and validation sample. 10,000 subsamples that contained no more than one observation per patient were repeatedly drawn randomly from the original learning sample, according to the bootstrap method Using these subsamples, the BCR-ABLIS expression level providing the best discrimination between CCyR and no CCyR was determined choosing the smallest p-value of the exact Fisher test. Only cut-off levels with p-values which were significant after adjustment for multiple testing were considered.To confirm these results, again 10,000 subsamples were randomly drawn from the original validation sample. For the cut-off points discovered in the learning sample, sensitivity and specificity for predicting a CCyR from the molecular data and the p-values from the Fisher test were calculated within each of these subsamples. Results:1329 cytogenetic analyses using a median of 25 metaphases (range, 20–30) of which 75% were in CCyR were compared with 1329 molecular data with a median BCR-ABLIS expression level of 0.055% (range, 0–237%). In the learning sample the most frequently found BCR-ABLIS value was 0.35%, but all values between 0.2% and 1.1% were similarly well-suited for separating between patients in CCyR and not in CCyR. Based on this range, two potential cut-off levels were chosen for validation, 0.35% and 1%. While the first one represents a value, where one would rather expect to underestimate the number of CCyRs, the latter represents a value, where one would rather expect to overestimate the number of CCyRs. For the level of 0.35% the p-value was below 0.05 for 67.2% of the 10,000 subsamples, the median p-value was 0.017. With a median value of 88.4%, CCyR cases were correctly identified while cases that were not in CCyR were correctly assessed with a median rate of 71.4%. For the 1% BCR-ABLIS level, the p-value was below 0.05 for 72.0% of the 10,000 subsamples with a median p-value of 0.011. At a median value of 96.0%, CCyR cases were correctly identified while cases that were not in CCyR were correctly assessed with a median rate of 60.0%. When restricting on BCR-ABLIS values between 0.1 and 10.0%, the median concordance rate was 76%. Conclusions:Compared to molecular analyses, cytogenetic assessment is imprecise and limited with regard to sensitivity. Thus, it is not surprising to find a range of molecular data equivalent to CCyR but not a one-to-one cut-off point. However, for practical reasons it seems desirable to have such a cut-off point. Since lack of CCyR is considered a failure criterion according to the ELN treatment recommendations, it is suggested to keep the proportion of patients small that are considered erroneously not to be in CCyR. Additionally, a considerable amount of patients in major molecular remission or even better is present in the sample, so there might be a slight bias towards lower cut-off values. Thus, BCR-ABLIS expression of <1% is considered to be equivalent to CCyR in chronic phase CML patients treated with imatinib with a median concordance rate of 89%. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Hochhaus:Novartis, BMS, MSD, Ariad, Pfizer: Consultancy Other, Honoraria, Research Funding. Müller:Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

Telomere loss in Philadelphia-negative hematopoiesis after successful treatment of chronic myeloid leukemia: Evidence for premature aging of the myeloid compartment

Telomere shortening, a well-known marker of aging and cellular stress, occurs under several conditions in the hematopoietic compartment, including aplastic anemia and following iatrogenic noxae. We decided to verify whether pathological telomere erosion also arises in restored Philadelphia-negative (Ph-negative) hematopoiesis following successful treatment of chronic myeloid leukemia (CML). Eighty-one CML patients in complete cytogenetic remission were compared to 76 age-matched healthy subjects. Myeloid cells of CML patients had shorter telomeres than controls (6521bp vs 7233bp, p<0.001). This difference was specific for the myeloid compartment, since it was not observed in lymphoid cells (6774bp vs 6909bp, p=0.620). Acquired Ph-negative cytogenetic abnormalities (p=0.010), lack of complete molecular remission (p=0.016) and age (p=0.013) were independent predictors of telomere shortening. Telomere dynamics were assessed over a median follow-up period of 22 months. We documented accelerated non-physiological ongoing telomere shortening in 17/59 CML patients (28%). Patients experiencing grade 2–4 hematological toxicity, during CML remission possessed significantly shorter telomeres compared to those lacking toxicity (p=0.005 for any toxicity, p=0.007 for anemia). CML patients suffer from significant and often ongoing telomere stress resulting in premature and selective aging of the myeloid compartment which might have long-term consequences on function and integrity of Ph-negative hematopoiesis.

Molecular Monitoring of Imatinib in Chronic Myeloid Leukemia Patients in Complete Cytogenetic Remission: Does Achievement of a Stable Major Molecular Response at any Time Point Identify a Privileged Group of Patients? A Multicenter Experience in Argentina and Uruguay

Monitoring minimal residual disease (MRD) by real-time quantitative polymerase chain reaction (RT-PCR) in chronic myeloid leukemia (CML) patients is mandatory in the era of tyrosine kinase inhibitors. Achieving a major molecular response (MMR) at 12 and 18 months predicts a better progression and event-free survival. The objective of this prospective, multicentric study was to evaluate MRD by standardized RT-PCR in 178 patients with chronic-phase CML who were treated with imatinib at different institutions in Argentina and Uruguay and to determine if achievement of a stable MMR (BCR-ABL transcript levels < 0.1%) identifies a low-risk cytogenetic relapse group. The median age of the patients was 50 years, and 55% of them had received imatinib as first-line therapy. BCR-ABL transcript levels were measured after achievement of complete cytogenetic remission (CCyR) and at 6-month intervals. MMR was detected in 44% patients at the start of the study. This value increased to 79% at month 36 of evaluation. Complete molecular response (CMR) also increased from 24% to 52% of patients. Not achieving a stable MMR determined a higher risk of cytogenetic relapse (9% of MMR patients not achieving an MMR vs. 1% of patients who achieved MMR). Patients with sustained MMR had a significantly better cytogenetic relapse-free survival at 48 months (97% vs. 87%; P = .008) but showed no differences in overall survival. Patients who did not remain in CCyR changed treatment. A stable MMR is a strong predictor for a durable CCyR. Standardized molecular monitoring could replace cytogenetic analysis once CCyR is obtained. These results emphasize the validity and feasibility of molecular monitoring in all standardized medical centers of the world.

Cytogenetic and SNP Array Study of CML Patients In Complete Cytogenetic Remission.

AbstractAbstract 3433The development of Ph− clones with new cytogenetic abnormalities has been reported among CML patients with complete cytogenetic remission (CCR) following diverse therapies such as Interferon and Imatinib (Gleevec). The etiologies for these abnormalities have not been characterized and have been attributed to drug effect, or underlying genetic instability in the bone marrow cells preceding or concomitant to the development of CML.Here we report on 31 patients in CCR who, in addition to standard karyotyping, were also analyzed by high-resolution SNP 6.0 array-based genomic copy number analysis.Of the 31 patients studied, 18 were male and 13 female. Their median age was 51 (range 19–71 years). Patients were in CCR for durations ranging from 6+ months to 156+ months (median duration of 24+ months). The duration of major molecular response (MMR) was similar to the duration of the CCR, apart from one patient where it was shorter (36 months versus 84 months).Past medical therapies included Imatinib only in 17 patients, Interferon-α followed by Imatinib in 11 patients, and Interferon only in 3 patients. Two of the patients also received low-dose Cytarabine in addition to Interferon and Imatinib, four received Dasatinib in addition to Interferon and Imatinib, and one received Nilotinib in addition to Imatinib.At the time of the sample collection four of the patients received Dasatinib, one received Nilotinib, three patients received no treatment, and 23 received continuous treatment with Imatinib.Cytogenetic testing disclosed normal karyotype in 24 of the patients. In two of the patients cytogenetic abnormalities were seen in only 1/20 analyzed cells – a finding not considered significant. Of the remaining patients, one had del20q in 11/20 analyzed cells. One had inversion of chromosome 12q (q15, q24.1 in 30/30 cells). One patient had an additional Y chromosome in 2/20 cells, one had deletion of the Y chromosome on 6/20 cells, and one had an extra chromosome 9 in 2/20 analyzed cells.SNP 6.0 array profiling was done on paired DNA samples from purified CML patient-derived CD34+/CD38- cells as well as buccal DNA. Data were analyzed using the previously validated software tool dChipSNP. Acquired genomic copy number aberrations (aCNA) were identified through visual inspection of paired heatmap displays and polymorphic copy number variants (CNVs) excluded. In total, one aCNA was identified in this cohort that was located on chromosome 20 (case #1 with corresponding cytogenetic finding of deletion of 20q) at physical position 30.856–48.002 Mb). Therefore, small or micro-aCNA do not exist in CML patient CCR marrows at appreciable frequencies or at sufficient (>25%) clonal representation.Based on this limited study sample, SNP arrays did not identify any additional genomic changes to the standard karyotype analysis. However, the recurrent presence of low frequency genomically abnormal clones in the bone marrow of these remission patients cannot be ruled out. Disclosures:No relevant conflicts of interest to declare.

BCR-ABL1 transcript levels increase in peripheral blood but not in granulocytes after physical exercise in patients with chronic myeloid leukemia

In chronic myeloid leukemia (CML) treatment response is determined by measurements of BCR-AB1L transcripts in peripheral blood by quantitative real-time PCR (qRT-PCR) and a 2–5 fold increase is considered a warning sign. The BCR-ABL1 gene is mainly expressed in myeloid cells whereas quantification of BCR-ABL1 is performed on the nucleated cell fraction of peripheral blood. Hence, leukocyte composition of the nucleated cell fraction may affect the result of BCR-ABL1 quantification. The aim of this study was to investigate if changes in leukocyte composition of peripheral blood had any effect on BCR-ABL1 transcript levels in CML patients. Six CML patients in complete cytogenetic remission (CCgR) performed a maximal physical exercise test. Blood samples were collected before exercise, at maximal exhaustion and after exercise. A biphasic increase in leukocyte count was observed and the relative proportion of granulocytes in peripheral blood changed significantly after exercise compared with baseline (p < 0.001). The BCR-ABL1 transcript level increased significantly following exercise, in nucleated cell fraction of peripheral blood (p < 0.05) but not in isolated granulocytes. In the nucleated cell fraction, the mean BCR-ABL1 transcript level was 3.3-fold (range 0.7–6.8) higher 180 min after exercise compared with baseline (p < 0.01). In conclusion, physical exercise induced significant increases in BCR-ABL1 transcript levels concomitant with changes in leukocyte content of peripheral blood. We therefore suggest that variations in leukocyte composition of peripheral blood, causing pre-analytic variations that affect BCR-ABL1 quantification, have to be accounted for. Consequently, small variations in BCR-ABL1 transcript levels should be interpreted cautiously in CML patients in CCgR.

Naturally occurring CD4 + CD25 + FOXP3 + T-regulatory cells are increased in chronic myeloid leukemia patients not in complete cytogenetic remission and can be immunosuppressive

Clinical presentation of chronic myeloid leukemia (CML) requires not only the deregulated tyrosine kinase BCR-ABL, but also the failure of an immune response against BCR-ABL-expressing cells. T-cell responses against BCR-ABL and other antigens are well-described, but their relevance to the in vivo control of CML is unclear. The suppressive role of naturally occurring T regulatory (T-reg) cells in antitumor immunity is well-established, although little is known about their role in modulating the T-cell response to BCR-ABL. Naturally occurring T-reg cells were characterized and quantified by flow cytometry in 39 CML patients and 10 healthy donors. Their function was studied by observing their effect on responses to purified protein derivative, a recall antigen, and on the response of an autologous T-cell line recognizing BCR-ABL. T-reg cells were CD4(+), CD25(+), FOXP3(+), CD127(low), and CD62L(high). T-reg numbers in patients in complete cytogenetic remission were significantly lower than in patients not in complete cytogenetic remission (p < 0.01). T-reg cell depletion using anti-CD25 selection enhanced proliferative responses to purified protein derivative. Furthermore, the interferon-γ and/or granzyme-B production of effector cells specific for viral peptides or a BCR-ABL HLA-A3-restricted peptide was inhibited when autologous T-reg cells were present. Taken together, these data suggest a role for T-reg cells in limiting immune responses in CML patients and this may include immune responses to BCR-ABL. The increased frequency of T-reg cells in patients with high levels of BCR-ABL transcripts indicates that an immune mechanism may be important in the control of CML.

Effective Targeting of Quiescent Chronic Myelogenous Leukemia Stem Cells by Histone Deacetylase Inhibitors in Combination with Imatinib Mesylate

Imatinib mesylate (IM) induces remission in chronic myelogenous leukemia (CML) patients but does not eliminate leukemia stem cells (LSCs), which remain a potential source of relapse. Here we investigated the ability of HDAC inhibitors (HDACis) to target CML stem cells. Treatment with HDACis combined with IM effectively induced apoptosis in quiescent CML progenitors resistant to elimination by IM alone, and eliminated CML stem cells capable of engrafting immunodeficient mice. In vivo administration of HDACis with IM markedly diminished LSCs in a transgenic mouse model of CML. The interaction of IM and HDACis inhibited genes regulating hematopoietic stem cell maintenance and survival. HDACi treatment represents an effective strategy to target LSCs in CML patients receiving tyrosine kinase inhibitors.

The Plateau of BCR-ABL Transcript Level ≥0.1% May Select CML Patients in Complete Cytogenetic Remission for Mutation Analysis.

Abstract 2216Poster Board II-193 Introduction.The major cause of the imatinib resistance is the presence of point mutations within the ABL kinase domain of BCR-ABL that are occurred in approximately 35-70% of patients displaying resistance to imatinib. Identification of mutation also in imatinib responders is associated with disease progression (Khorashad et al. 2008, J Clin Oncol 29:4806). According to ELN (European LeukemiaNet) patients should be screened for mutations in the case of: 1) hematological resistance / relapse, 2) cytogenetic resistance / relapse, 3) 5 – 10-fold increase of BCR-ABL transcript, 4) prior to therapy with 2nd generation TKIs, and 5) at 3-month intervals under therapy with 2nd generation TKIs. The BCR-ABL rise fold threshold for mutation analysis is highly discussed nowadays. Recently Press et al. (Blood 2009, prepublished online) identified a 2.6-fold increase in BCR-ABL RNA as the optimal cutoff for predicting a concomitant KD mutation, with a sensitivity of 77% (94% if including subsequent samples). Aims.In this study, we aimed to detect BCR-ABL mutation in patients responding to imatinib with CCgR expressing the plateau of BCR-ABL transcript level ≥0.1% (IS; International Scale) in a minimum of 3 subsequent samples (minimal duration 6-9 months) and to evaluate the risk of disease progression in this group of patients. Materials and methods.From 140 CML patients in CCgR after imatinib therapy, 32 showed constant BCR-ABL transcript levels ≥0.1% after the initial reduction. Altogether 134 samples of peripheral blood of these 32 patients were tested for mutation in BCR-ABL kinase domain by direct sequencing. Patients with constant BCR-ABL values <0.1% were not included because of the limitation of mutation detection by sequencing as shown our tests of reproducibility and repeatability. Results.Median follow-up of BCR-ABL transcript level plateau within the range of evaluated variability of RQ-PCR analysis was in 32 patients 12 months (range 6-64); 22/32 patients were treated with imatinib in the first line. Mutation was detected by direct sequencing with the measured sensitivity of 20-100% of mutant BCR-ABL present in the sample with the minimum copy number of total BCR-ABL 100 (corresponding to 0.1% of BCR-ABL measured by our RQ-PCR) in 9/32 patients (28%); 5/9 were in the first line imatinib treatment. Loss of CCgR or 1 log rise of BCR-ABL was observed in 5/9 patients median 5 months (range 4-17) since first detection of mutation. One patient with no mutation relapsed 12 months after the start of the BCR-ABL plateau. In 5/32 patients without mutation the BCR-ABL level significantly decreased after the 1st plateau to the levels that stayed unchanged for a median of 11 months (range 7-28); three of them achieved major molecular response (MMR; <0.1% BCR-ABL). Conclusion.In our study, patients expressing plateau of BCR-ABL transcript level ≥0.1% did not achieve MMR and were at more risk to lose their responses. We suppose that the fluctuation of BCR-ABL values ∼0.1% within the consecutive samples should not be classified as MMR achievement. Only levels constantly below 0.1% should be defined as MMR. The BCR-ABL constant levels ≥0.1% clearly select patients in CCgR for mutation analysis. This approach highly reduces the number of examinations for mutation in CML responders presenting a cost-effective alternative applicable in clinical practice.Supported by MZOUHKT2005 and research grant from Bristol-Myers Squibb. Disclosures:Machova Polakova:Bristol-Myers Squibb: Research Funding.

Low or undetectable numbers of Philadelphia chromosome-positive leukemic stem cells (Ph+CD34+CD38neg) in chronic myeloid leukemia patients in complete cytogenetic remission after tyrosine kinase inhibitor therapy

Low or undetectable numbers of Philadelphia chromosome-positive leukemic stem cells (Ph + CD34 + CD38 neg ) in chronic myeloid leukemia patients in complete cytogenetic remission after tyrosine kinase inhibitor therapy

Low or Undetectable Numbers of Philadelphia Chromosome Positive (Ph+) Leukemia Cells in the Primitive (CD34posCD38neg) Stem Cell Fraction in Chronic Myeloid Leukemia (CML) Patients during Tyrosine Kinase Inhibitor Therapy.

Targeted tyrosine kinase inhibitors (TKIs) efficiently induce rapid hematologic and cytogenetic remission in most chronic myeloid leukemia (CML) patients. However, in vitro experiments have suggested that the most primitive CML stem cells residing in the CD34posCD38neg fraction are relatively resistant to TKIs. The prevalence of these stem cells in vivo in patients under TKI therapy is unclear. The aim of this project was to analyze the effect of TKI therapy on Ph+ leukemia stem cell pool in patients and to analyze the proportion of Ph+ cells in different stem cell fractions. A total of 26 chronic phase CML patients were included in the study. 18 patients were treated with imatinib, 5 with dasatinib, and 3 with bosutinib. The median time of TKI treatment was 20 months (range 3–72 months). Large volume (median 30 ml, range 5–55 ml) of bone marrow (BM) aspirate was collected and mononuclear cells (MNC) were isolated. CD34pos cells were separated with paramagnetic beads and further sorted into CD34posCD38pos and CD34posCD38neg cell populations with multicolor flow cytometry in order to analyze progenitor cell fractions of different maturation stage. Proportion of Ph+ cells was determined with interphase FISH by counting 1000 cells in each fraction. The median yield of MNCs from 30 ml of BM aspirate was 280x106 cells resulting in a median of 32 000 CD34posCD38neg cells (range 1000–91000). High-sensitivity counting of the proportion of Ph+ cells was feasible with a median number of counted interphase nuclei of 1005. During TKI therapy the CD34pos cells expressing highest CD38 antigen level were already mostly differentiated into B-cell lineage (CD19 positive). The CD34pos cells expressing low CD38 antigen levels expressed markers of more primitive cells such as C-kit (CD117) and CD133. Of 26 patients with CML, 19 were in complete cytogenetic remission (CCyR) when assessed by metaphase FISH of non-fractionated BM cells (1000 cells analyzed). Only 3 patients had single Ph+ cells in CD34pos cell fractions (less than 1%). In remainder of patients, all progenitor cell fractions, including the most primitive CD34posCD38neg cells, were negative for Ph+ cells. 3 patients had 0–1% of Ph+ cells in non-fractionated BM sample. One of them had 0.2% of Ph+ cells in CD34posCD38neg fraction, but the other 2 patients had 0/1000 Ph+ stem cells. 4 patients had a partial cytogenetic response (5–20% of Ph+ cells in non-fractionated BM sample). Again, the proportion of Ph+ cells was not increased in the most primitive CD34posCD38neg cell fraction. Interestingly, patients who had discontinued imatinib treatment had lower level of Ph+ cells in different CD34pos fractions (median 0.1%) when compared to non-fractionated BM (median 9.3%). Based on our data, in chronic phase CML patients, TKI therapy eradicates most Ph+ CD34pos progenitor cells. Unexpectedly, leukemic stem cells were not enriched in the most primitive CD34posCD38neg cell fraction in vivo. These results differ from the in vitro studies, where CD34posCD38neg cells have been shown to be resistant to TKIs. This could be due to non-physiological conditions (growth factor sensitivity, other cytokines) in cell culture assays. In addition, leukemic stem cells in vivo may be located in the subcortical hypoxic stem cell niche in the BM and are less likely to be aspirated. Our data underline the tremendous proliferative potential of very rare stem cells in CML patients in CCyR, as is evident after discontinuation of TKI therapy. Future studies evaluating the kinetics of disappearance of Ph+ cells from stem cell fractions during TKI therapy and the location of residual Ph+ stem cells in the BM are warranted and may give important information on the depth of the therapy response. Furthermore, this knowledge may aid in targeting therapy to these cells and finding curative treatment strategies in CML.

Implication of STAT-5 Recruitment in the Expression of IRF-8 and in the Impairment of Human Plasmacytoid Dendritic Cells Differentiation Induced by BCR-ABL and GM-CSF

It is currently established that the immune system plays a critical role in the control of chronic myelogenous leukemia (CML). Several cell populations including T cells, dendritic cells (Dc) and NK cells have been shown to exhibit potential anti-leukemic activities. We have previously shown that plasmacytoid dendritic cells (pDc) differentiation and IFN-a production was impaired along the chronic phase (CP) of CML and that the Tyrosine- Kinase (TK) activity of BCR-ABL was responsible for this impairment. Moreover, patients in complete cytogenetic remission (CCyR) after imatinib mesylate (IM) or IFN-a therapy present a partial restoration of the pDc compartment and IFN-a production. Some recent results (Esashi et al., Immunity, 2008) indicate that, in mice, STAT-5 but not STAT-3, block the expression of IRF-8, a transcription factor required for the differentiation of pDc and that the GM-CSF-induced STAT5 activation interferes with pDc differentiation. As STAT3 and STAT5 proteins are important targets of BCR-ABL TK activity, we postulate that their recruitment could be responsible for the CML pDc impairment and could be involved in the persistent pDc dysfunction in CR patients.We decided to investigate the effect of BCR-ABL or GM-CSF on STAT-3, STAT-5 and IRF8 status ex vivo, on CMN of patients who have achieved sustained CCyR and major molecular response (MMR) after IFN-a therapy and in whom treatment has been discontinued (n=8) compared to CMN of healthy subjects (HS) (n=5). The same investigations were also performed in vitro with the model of pDc differentiation of CD34+ haematopoietic progenitors from CP patients and HSs, using the combination of Stem Cell Factor, Flt3-Ligand and Thrombopoietin,.Flow cytometry analysis showed that the homeostasis of the IFN-a production was altered in CCyR patients : pDc (determined by the coexpression of CD303, CD123 and CD4), represent 34.6 ± 14.9 % of the IFN-a producing cells after stimulation by Influenza virus and intracellular detection of IFN-a, whereas in healthy donors (HD) IFN-a producing cells were principally pDc (72.1 ± 20.5 %of IFN-a+ cells, p<0.05 in comparison with the CCyR patients group). The IRF-8 expression analysed by flow cytometry was also deregulated as low levels of IRF8 were detected in these cells when compared with pDc of HD (based on mean fluorescence analysis of the IRF-8 staining). Moreover, higher levels of P-STAT5 were observed in CMN and pDc from CCyR patients (65.1 ± 18 % of pDc was P-STAT5 +) than from HD (0.75 ± 0.66 % of pDc are P-STAT5 +, p<0.01 in comparison with the CR patients group). The involvement of Human STAT5 in the repression of pDc differentiation was confirmed in our in vitro model. We showed that GM-CSF blocked the pDc differentiation of normal CD34+ cells and enhanced the P-STAT5 levels. We showed higher levels of P-STAT5 along differentiation of CD34+ cells of CML patients in CP, in correlation with the lack of differentiation and maturation of pDc.Altogether, our results suggest that in Human STAT5 is a target for BCR-ABL and GM-CSF and is responsible for the defect of pDc differentiation and IFN-a production. After CCyR, the pDc defect is only partially restored, principally because a STAT5 recruitment persists, possibly induced by soluble factors produced by the residual disease.

Monitoring Minimal Residual Disease by Quantitative PCR in Chronic Myeloid Leukemia Patients in Complete Cytogenetic Remission

The landscape of chronic myeloid leukemia (CML) has radically changed since the introduction of tyrosine kinase inhibitor (TKI), imatinib (IM), now considered as standard therapy. Although excellent cytogenetic responses are obtained, minimal residual disease still persists in a proportion of patients (pts) when measured by serial molecular monitoring by quantitative real-time polymerase chain reaction (RQ-PCR) to measure BCR-ABL transcript levels (Baccarani M et al. Blood 2006; 108:1809–20). We monitored BCR-ABL transcript levels by RQ-PCR in 176 chronic phase (CP) –complete cytogenetic response (CCyR) CML pts treated with IM. Median follow-up from start of therapy with IM was 35 (6–80) months. Pts were recruited from 33 centers in Argentina and 2 in Uruguay. Median follow up from the first assessment at our Institution was 18 (6–32) months. Seventy nine patients (45%) had received interferon as 1st line prior to IM and 97/176 (55%) pts received imatinib as 1st line. Eighty eight percent (155/176) pts had received IM 400mg/d and 12% (21/176) 600–800mg at study initiation. Fifty four percent presented with low Sokal score at diagnosis. Peripheral blood samples were tested by RQPCR every 6 months. Major molecular response (MMR) was defined as BCR-ABL/ABL ratio of <0,1% on the Internationale Scale. Rise in transcript levels was immediately reconfirmed. Cytogenetic and mutational analyses were performed if rise in transcripts was confirmed. Overall, 48% had MMR at the initial evaluation (baseline), and this increased to 57% at last follow-up (month 18). No patient with MMR achievement lost CCyR. Only 5 pts lost CCyR, never having achieved MMR (p=0.01). All patients could be divided in 3 groups according to transcript level outcome: 61% decline (at least 1 log reduction of BCR-ABL/ABL ratio), 27% stable (no log variation), 13% rise (increasing 1 log of BCR-ABL/ABL ratio). Among 136 pts with follow up at month 18, we observed (Table 1):Molecular Response At baselineDecline in transcript levels %(pts)Stable transcript levels %(pts)Rise in transcript levels %(pts)Total %(pts)CMR ≥ 4 log red <0,01%/U MMR ≥ 3 log red < 0,1% (N:60)20(12)63(38)17(10)44(60)No MMR > 0,1% (N:76)47(36)47(36)5(4)56(76)Total35 (48)55(74)10(14)100(136)CMR: complete molecular response, U: undetectableFrom the group of pts with rise in transcript levels, 5 pts lost CCyR, none lost complete hematologic response. Overall, 5%(9/176) pts eventually changed therapy to a 2nd generation TKI: 5 pts with cytogenetic relapse and 4 pts with increase in transcript levels.Our results confirm that molecular responses continue improving over time and a significant number of pts achieve undetectable transcript levels with continued imatinib therapy. Achievement of MMR is associated with sustained cytogenetic response. These results emphasize the validity and feasibility of molecular monitoring in all areas of the world.

Monitoring response to imatinib by quantitative real-time polymerase chain reaction (RQ-PCR) in chronic phase, chronic myeloid leukemia patients in complete cytogenetic remission

18013 Background: Chronic myeloid leukemia (CML) landscape has radically changed since treatment with imatinib. As the majority of CML patients (pts) treated with imatinib achieve a complete cytogenetic remission (CCyR), molecular studies that measure the level of bcr/abl transcripts to determine minimal residual disease are mandatory. The degree of reduction of total leukemia cell mass by imatinib as measured by RQ-PCR correlates with progression-free survival. Methods: We monitored bcr-abl transcript levels by RQ-PCR in 169 CML pts in chronic phase (CP) that achieved CCyR with a median follow up of 15 months (range 6–26). Median age was 51 (range 18–82), 83 pts (49%) were newly diagnosed and 86 (51%) had been previously treated with interferon-alfa; 54% had low sokal score, 29% intermediate and 17% were high risk. At baseline, pts were evaluated by FISH and RQ-PCR, transcript quantification by RQ-PCR was measured every 6 months from baseline at 6, 12 and 18 months. Results: At present 78 pts completed 4 evaluations at month 0, 6, 12 and 18. Mayor molecular response (MaMR: > 3 log red in bcr-abl transcript levels) was obtained in 62% at month 18 compared to 45% obtained at study initiation. Minor (Mi)MR at month 18 was of 38% vs 50% at initial evaluation. The pts could be divided into three groups according to molecular responses: 1- in 34 pts transcript levels continued to decline during the observation period, 2- in 38 pts transcript level reached a plateau and 3- in 6 pts transcript levels increased. From pts who obtained MiMR at baseline, 54% showed decrease in transcript levels obtaining MaMR in 44% of pts. Nine pts changed treatment to a second line tyrosine kinase inhibitor, causes were: lost CCyR: 3, lost hematologic response: 1, molecular relapse: 4 and intolerance to treatment: 1. Conclusion: The pattern of residual disease after achieving CCyR on imatinib is variable: some patients show progressive reduction of transcript levels, others reach a plateau showing stable transcript levels and other show rising levels of bcr-abl defining a group of patients were therapeutic decisions are mandatory. No significant financial relationships to disclose.

Chromosomal Abnormalities in the Early Stem Cell Population of CML Patients in Complete Cytogenetic Remission.

Clonal cytogenetic abnormalities, most commonly involving chromosomes 7 and 8, are detectable by conventional karyotyping in Ph-negative metaphases of some chronic myeloid leukemia (CML) patients with a major cytogenetic response (MCyR) to imatinib. It is unknown whether these abnormalities involve the primitive progenitor cell compartment, and whether their frequency in this compartment may exceed the frequency detected by karyotyping. To answer these questions we analyzed lineage-negative CD34+/CD38− and CD34+/CD38+ cells from CML patients in complete cytogenetic response (CCyR), using by fluorescent in situ hybridization (FISH) for chromosome 7 and 8 abnormalities and BCR-ABL.Methods: Mononuclear cells (MNC) were selected from the bone marrow of patients with CCR by Ficoll-Hypaque density gradient centrifugation and enriched for lineage-negative cells using an immunomagnetic column. Lineage-negative cells were further sorted into CD34+/38− and CD34+/38+ cells by multicolor FACS. Interphase FISH analysis was performed using 7 LSI D7S522 Spectrum Orange / CEP7 Spectrum Green (chromosome 7), CEP8 Spectrum Aqua (chromosome 8) and LSI BCR/ABL +9q34 TriColor Dual Fusion Probe. Thus far, 5 CML patients with CCR (3 with a normal karyotype and 2 with trisomy 8) and 1 normal control have been analyzed.Results: Of the three CML patients in CCR with normal karyotype, one had 9% deletion 7q (internal cut off of 0.5%) and the second 1.2% trisomy 8 (just over the internal cut off of 1%) cells in the CD-34+/38− population, while the CD-34+/38+ cell population did not show abnormalities. The third CCR patient had no abnormalities in the CD-34+/38− and CD-34+/38+ cell populations. Two CML patients in CCR with 45% trisomy 8 abnormal cells by conventional cytogenetics had 44% and 60% trisomy 8 positive cells in the CD34+/38+ population (two few CD34+/CD38− cells were available for analysis). No BCR-ABL signal was detected in any cell. In the healthy control, the CD34+/38+ cells were normal, but 4.1% of CD34+/38− showed a deletion of 7q.Conclusion: Clonal chromosomal of chromosomes 7 and 8 in Ph-negative primitive hematopoietic progenitor cells may be more common than suggested by conventional karyotyping. A larger cohort of CML patients in CCR and healthy individuals is under study to determine if this phenomenon is indeed related to CML or occurs also in normals. Results will have implications for the interpretation of karyotypes in patients with hematologic malignancies.

Abnormally Small BCR-ABL Transcripts in CML Patients before and during Imatinib Treatment.

Neoplastic cells bearing fusion genes that express an activated tyrosine kinase may set the scene for accumulation of genetic lesions by dysregulating DNA damage repair mechanisms and causing genetic instability. The observation that the BCR-ABL fusion gene alters pre-mRNA splicing in a variety of other genes including Ikaros and PYK2 supports this hypothesis. However, the only current evidence for acquired genetic change in the BCR-ABL gene itself is limited to finding mutations in the BCR-ABL kinase domain in patients treated with imatinib mesylate (IM). Here we report the observation that some patients with CML have abnormally small BCR-ABL transcripts both before and during treatment. Patients with sub-optimal response to IM are screened for mutations specifically within the BCR-ABL kinase domain by performing nested PCR, thereby excluding amplification of the non-translocated ABL allele. In the first round PCR amplification is performed across the fusion and the amplicons generated are subjected to a second round to yield an expected 863 bp (containing ABL exons 4 through 9 and thus the entire BCR-ABL kinase domain) PCR fragment. Smaller amplicons were observed in 49 (9.9%) of the 494 CML patients investigated. There was marked variation in the mRNA species when the abnormally small amplicons were subjected to direct sequencing; we found exon skipping, intra-exon splicing and insertion of intronic sequences. Similarly, in some cases the open reading frame was maintained whilst in others there were frame shifts leading to premature stop codons. The commonest finding, (22 of the 49 patients) was skipping of ABL exon 7 from codons 362 to 424, which includes the activation loop of the kinase domain. The smaller amplicons persisted even after the first round products had been diluted to 1:160. We also noted that the normal 863 bp fragment was present in some cases but was not detectable in others; its absence could reflect preferential amplification of the smaller transcripts. In a number of cases the same abnormally short amplicons were identified in the same patient studied serially on three or more separate occasions. We subsequently performed a second round of nested PCR with primers designed to amplify across the BCR-ABL junction such that the product included sequences from BCR 13 to ABL exon 9. With these new primers the detection frequency of abnormally small transcripts was increased. Furthermore, we observed the smaller transcripts in all of the 12 patients tested prior to beginning IM therapy. We then sought to determine if the normal ABL allele was involved; in order to avoid amplifying the BCR-ABL kinase domain, we performed a single round of PCR and restricted the analysis to patients in complete cytogenetic remission (CCyR). Only the expected 863 bp amplicon was observed in cDNA samples from 19 CML patients in CCyR whose BCR-ABL/ABL ratios ranged from 0.01 to 0.98. Furthermore, the smaller amplicons were not observed in cDNA samples from 20 normal individuals. We conclude that these abnormalities may result either from aberrant alternative splicing or from spontaneous deletions, or from a combination of both mechanisms. They may be a manifestation of the genetic instability believed to be an integral feature of CML.