Stages Of Chronic Myeloid Leukemia Research Articles

Chronic Myeloid Leukemia in Pediatrics — First Results From Study CML-PAED II.

Abstract 342▪▪This icon denotes an abstract that is clinically relevant. Background:Chronic myeloid leukemia (CML) is a rare malignancy in pediatrics. In this decade -like in adults- imatinib meyslate (IMA) has been established also as first line treatment for children with CML while allogeneic stem cell transplantation (SCT) as treatment option is postponed for those cases becoming intolerant or refractory to tyrosine kinase inhibitor (TKI) treatment. However, results from controlled trials in children are lacking so far. We here report an analysis of pediatric data from patients (pts) with newly diagnosed Philadelphia-chromosome positive (Ph+) CML on up-front treatment with IMA. Pts and Methods:According to protocol CML-PAED II pediatric pts with confirmed diagnosis of Ph+ CML were treated in CP with IMA 300 mg/sqm once daily, while in accelerated phase (AP) or in blastic phase (BC) the dose was increased to 400 mg/sqm and 500 mg/sqm (bis daily), respectively. Initial and long-term clinical and laboratory data, treatment response and side effects were reported to the study center on standardized forms by the treating physician. Specimen from peripheral blood (pB) and bone marrow (BM) were assessed by cytogenetics and by quantitative RT-PCR for BCR-ABL transcript rates in central laboratories for standardized monitoring in three months intervals. Results:From 1. Jan 2004 until 31. Mrch 2009 a total of 51 pts (21 female, 30 male; median age: 10.6 yrs [range:1-20 yrs]) were registered: 10 pts with ongoing IMA treatment were recruited and analyzed retrospectively while 41 pts were enrolled prospectively from centers in Austria (n=1), Czech Rep. (n=6), Germany (n=40), Italy (n=1), Netherlands (n=1), Slovak Rep. (n=2). Stages of disease were: CP n=47; AP n=1; BC n=3 (two myeloid). Those four pts diagnosed in AP and BC underwent early SCT. Observed side effects in the whole group included: nausea (n=9), muscle pain (n=7), edema (n=3), rhabdomyolysis (n=1, short interruption of IMA), reduced blood cell count (n=2, short interruption of IMA in one pt), biochemical alterations in bone metabolism [for details see: N Engl J Med 2006;354:2006] (n = 8), impaired longitudinal growth (n=1, [Haematologica 2009;94:1177]). Two pts experienced intolerance (muscle pain) or toxicity (hepatic), respectively, therefore stopped IMA and were put on dasatinib after 4 and 10 months, respectively. Having achieved complete cytogenetic response (CyR) and 2 log-fold reduction of BCR-ABL transcript rate, one pt opted for SCT from her HLA-identical brother after 15 mo of treatment. Response rates in advanced stages of CML were as follows: in BC (n=3) two pts became hematological responders (HR), one pt exhibited partial HR. The only one pt diagnosed in AC exhibited partial CyR but complete HR. A landmark analysis in pts entering CML-paed II in CP exhibited that 2/42 pts (5%) had no complete HR at month 3; 2/28 (7%) had no complete CyR at month 12, and 2/19 (15%) pts achieved no major molecular response (MMR, defined as >0.1% BCR-ABL [Blood 2006;108:28–37]) at month 18 after start of IMA. Each two of those four patients with incomplete response (one pt with no CyR at month 12, one pt with no MMR at month 18) underwent SCT from a sibling donor and the other two pts stopped IMA and were put on dasatinib. With a median follow-up of 19 months (range: 0-63 months) all 47 pts diagnosed in CP are alive. Of note none of the six pts (median age at diagnosis: 5 yrs; range 1–13 years) treated by imatinib meanwhile for >36 months have opted for SCT. Conclusion:Keeping in mind that the number of pediatric pts is still small, IMA treatment for children and adolescents with CML in CP is associated -like in adults- with high treatment response rates. Refractoriness to IMA is uncommon and side effects seem tolerable, as only 10% of the total cohort stopped imatinib and were put on 2nd generation TKI. However, disturbances of bone metabolism and longitudinal growth impairment may be of special concern in this not yet outgrown cohort [N Engl J Med 2006;354:2006, Blood 2008;111:2538; Haematologica 2008;93:1101; Lancet 2008;372:111; Int J Hematol; 2009;89:251; Haematologica 2009;94:1177]. Only 3/47 pts not diagnosed in advanced phases of CML so far underwent SCT thus underlining that also in pediatrics SCT has been shifted to a second-line strategy for high-risk patients and those who failed therapy with IMA. Disclosures:Suttorp:Novartis : Research Funding. Thiede:Novartis: Research Funding.

A Phase 2 study of the Combination of Omacetaxine and Imatinib in the Treatment of Patients with Chronic Myeloid Leukemia (CML) in Advanced Stages or After Failure to Imatinib.

Abstract 2193Poster Board II-170 Background:Despite the success of imatinib mesylate in treating patients with CML, several pts develop resistance to therapy. In addition, response rate for pts in accelerated (AP) or blast (BP) phase is significantly lower than in chronic phase (cp) and responses are usually short-lived. Omacetaxine (homoharringtonine) is a plant alkaloid extracted from cephalotaxus fortunei, with in vitro and clinical activity in CML. In vitro experience has shown synergy of omacetaxine combined with imatinib (Ref). Aim:Evaluate the safety and efficacy of intravenous omacetaxine combined with imatinib in the treatment of patients with CML in CP, AP, or BP. Methods:Patients with CML were eligible if they had 1) CP resistant to single agent imatinib, or 2) AP or BP, regardless of prior exposure to imatinib. Pts received omacetaxine 2.5 mg/m2 by 24-hour continuous infusion daily for 5 days (days 1-5 of each cycle) every 4 weeks and imatinib, 400 mg daily for pts in CP and 600 mg daily for pts in AP or BP. Results:Fifteen pts were enrolled: 9 in BP, 3 in AP, and 3 in CP. The median age is 55 years (range, 21-77); median time from diagnosis to treatment 35 months (range, 1 to 139). Twelve pts (80%) were imatinib resistant. Three pts had not received imatinib (all in BP; one had received prior interferon and stem cell transplant). Eight pts had received treatment with at least one other tyrosine kinase inhibitor beside imatinib. Mutation analysis was performed in 14 out of 15 pts and 7 had mutations (T315I in 3, F317L in 2, F359V and Q252H one each). Patients received a median of 3 cycles 4 (1-34). Eight (53%) pts responded. Hematologic response was achieved in 7 of 14 evaluable pts (1 started in CHR): 3 CHR (all BP), and 4 BCP (3 BP, 1 AP). Cytogenetic responses were achieved in 4 (of 14)pts(28%): 2 complete , 1 partial and 1 minor (BP) and one of the pt in CR later achieved complete molecular response. Responses were usually transient although one pt (BP) remains in complete molecular remission 42 months from the start of therapy. One pt died of unrelated causes while in major cytogenetic response 12 months after start of therapy, and one pt changed therapy with an ongoing minor cytogenetic response after 13 months. Therapy was overall well tolerated and administered on outpatient basis. Grade 3-4 non hematologic toxicity (regardless of causality) included fatigue (20%), nausea & vomiting (13%), dizziness (7%) and weight loss (6%). Grade 3-4 hematologic toxicity included thrombocytopenia (93%), anemia (80%), and neutropenia (80%). Transient treatment interruptions were required in 6 patients because of myelosuppression. The median survival is 4.7 months. Three patients are alive, 35, 41 and 42 months from start of therapy: 1 still on therapy with complete molecular response; 1 with complete cytogenetic remission on bosutinib, and one (T315I) on hydroxyurea. Conclusion:Omacetaxine combined with imatinib is effective in patients with advanced stages of CML and has a favorable toxicity profile. Further studies with this combination are warranted, particularly in advanced phase CML. Disclosures:Cortes-Franco:Chemgenex: Research Funding; Novartis: Research Funding.

Beta-Catenin Mutations are not Observed in Chronic Myeloid Leukemia

Studies reporting activated Wnt signaling in all stages of chronic myeloid leukemia (CML) have demonstrated that deregulation of the pathway plays a role in the pathogenesis of this disease. Several reports have suggested mechanisms for the deregulated Wnt signaling and beta-catenin stabilization observed in CML. One possible mechanism for beta-catenin stabilization could be the acquisition of mutations at its N-terminal domain, especially in the third exon where it is marked via phosphorylation for degradation. We sought to determine whether mutations in the third exon of the beta-catenin gene are responsible for the observed Wnt activation in CML. We screened bone marrow specimens from 33 patients with CML in the chronic phase and also examined the K562 cell line for beta-catenin mutations. None of the patients nor the K562 cell line were found to carry mutations. Beta-catenin amino-terminal mutations are not observed or very rare and therefore are not the underlying mechanism of activated Wnt signaling in CML.

Genome-Wide Identification of Genes Required for Yeast Growth Under Imatinib Stress: Vacuolar H+-ATPase Function Is an Important Target of This Anticancer Drug

Imatinib is a highly selective tyrosine kinase inhibitor of the oncogenic kinase Bcr-Abl, the result of a chromosomal abnormality that is associated with chronic myeloid leukaemia (CML). Despite the success of this target-directed therapy, imatinib resistance is an emerging problem, especially in advanced stages of CML. In this study, we explored the yeast Saccharomyces cerevisiae as a model eukaryotic system to better understand the mode of action of imatinib, as well as potential mechanisms of resistance to this drug. Using a systematic approach, we screened a yeast haploid deletion collection with individual knockouts of most nonessential yeast genes, and identified 51 genes that are required for yeast resistance to imatinib. The genes identified are involved mainly in DNA repair and transcription control, cell cycle control and differentiation, vacuolar pH homeostasis, vesicular transport, and protein trafficking. Remarkably, approximately 80% of the genes identified in our screen have human orthologs. The vacuolar pH homeostasis function is associated to our dataset by 13 genes that encode subunits and assembly factors of the yeast vacuolar proton-translocating ATPase (V-ATPase). Further studies using fluorescence microscopy showed that physiological acidification of the vacuole is severely compromised following imatinib treatment of yeast cells, an effect that was found to be dose dependent. Results suggest that imatinib might act as an effective inhibitor of V-ATPase function in yeast, identifying V-ATPase activity and vacuolar function as novel imatinib targets.

The changing scene of allogeneic stem cell transplantation for chronic myeloid leukemia—a report from the German Registry covering the period from 1998 to 2004

Due to the recent changes in the indication to allogeneic stem cell transplantation (SCT) in chronic myeloid leukemia (CML), we retrospectively analyzed 1,716 patients with different CML stages who received an allograft from related (n = 767) or unrelated donors (n = 938) within the German Registry of Stem Cell Transplantation (DRST) from 1998 to 2004. Myeloablative conditioning was performed in 724/871 cases (83%), dose-reduced conditioning in 147/871 (17%). Annual transplantations were decreasing from 357 to 98 (28%) in the period of study, but the proportion of advanced cases was increasing from 32% (112/346) to 53% (50/94) of all SCTs. Stage of disease, intervals from diagnosis, and patients' age were independent prognostic parameters, while peripheral stem cells and unrelated transplantation seemed equal to bone marrow/related transplantation. This study demonstrates that allo-SCT still has an important role in advanced CML, which emphasizes the need for optimized transplantation strategies for these high-risk patients.

Expression of the CDH13 gene and BCR/ABL fusion gene in chronic myeloid leukemia patients and their relationship

To investigate the expression of the CDH13 gene and BCR/ABL fusion gene in chronic myeloid leukemia(CML) patients at different stages and explore their relationship. RNA was isolated from peripheral blood in 30 healthy adults, 22 primary CML patients and 25 blastic crisis of CML patients. We examined the expression of the CDH13 gene and BCR/ABL fusion gene using EVA Green real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The expression of the BCR/ABL fusion gene in the patients with blastic crisis CML was 4.72 folds as that of the patients with primary CML. The expression of the CDH13 gene in the primary and blastic crisis CML patients was significantly reduced to 36% and 25% of that in healthy controls, respectively. Moreover, negative correlation was found between the expressions of these two genes. The study showed that the reduction of the CDH13 expression at different clinical stage of CML may account for the defective cell adhesion in CML, and the expression of the CDH13 gene was probably down-regulated by the BCR/ABL fusion gene.

Aberrant methylation of the death‐associated protein kinase 1 (DAPK1) CpG island in chronic myeloid leukemia

The death-associated protein kinase 1 (DAPK1) gene is a candidate tumor suppressor (TSG) and the abnormal methylation of DAPK1 gene has been found in many carcinomas. The epigenetic changes of TSGs are now recognized as a mechanism contributing to the development of chronic myeloid leukemia (CML). To clarify the role of DAPK1 in CML, we examined the methylation status of DAPK1 in 49 patients with CML using methylation-specific polymerase chain reaction. The aberrant methylation of the DAPK1 gene was found in 25 of 49 (51.0%) CML cases, not in all controls. No correlation was found between DAPK1 gene methylation and the age, hematologic parameters, chromosomal abnormalities, the types and levels of bcr/abl transcripts of CML patients. However, correlation could be observed between the sex and the status of DAPK1 methylation in CML patients (R = 0.374, P = 0.008). Furthermore, there was a significant correlation between DAPK1 methylation and the stages of CML (R = 0.354, P = 0.013). The CML patients in accelerated phase (AP) and blast crisis (BC) had higher frequency of DAPK1 methylation than those in chronic phase (CP) (75.0% vs. 34.5%) (chi(2) = 7.776, P = 0.005). In one patient, the status of DAPK1 methylation became positive on the transition from CP to AP and BC. These results suggested that DAPK1 promoter methylation might play a significant role in the progression of CML.

Cadherin-13 in primary and blast crisis chronic myeloid leukaemia: declining expression and negative correlation with the BCR/ABL fusion gene

Expression of the CDH13 gene in chronic myeloid leukaemia (CML) patients at different clinical stages, and its relationship with the BCR/ABL fusion gene, is investigated. Expression of the CDH13 gene and the BCR/ABL fuse gene is investigated in peripheral blood from 30 healthy adults, 25 primary CML patients and 25 CML patients in blast crisis using the EvaGreen real-time reverse transcriptase polymerase chain reaction (RT-PCR). Results showed that BCR/ABL fusion gene expression in the blast crisis CML patients was 4.72-fold higher than that in patients with primary CML. Expression of CDH13 mRNA in primary and blast crisis CML patients was lower than in the healthy adults, reduced 64% and 75%, respectively. Expression of CDH13 showed a negative correlation with the BCR/ABL fusion gene. The data indicate that the decline of CDH13 expression accompanied the different clinical stages of CML and probably was involved in over-expression of the BCR/ABL fusion gene.

The Effects of BCR/ABL on DNA Damage and Repair Are Dependent on Apoptosis Competence

Chronic myeloid leukemia (CML) is a two-stage disease associated with the t(9;22) translocation. This translocation fuses the BCR gene with the ABL tyrosine kinase, forming the BCR/ABL oncogene. BCR/ABL is a constitutively activated tyrosine kinase, which causes the first stage of CML, chronic phase. However, it is still unknown why patients progress to the second phase, blast crisis, a phase marked by increased chromosomal abnormalities. Previous data from our lab suggests that cells expressing BCR/ABL have an increase in chromosomal translocations after DNA repair compared to control cells, as assessed by spectral karyotyping. We hypothesized that BCR/ABL alters the apoptotic threshold in response to DNA damage, and we sought to determine if cells lacking BCR/ABL that were unable to undergo apoptosis would show similar responses in DNA damage and repair assays to BCR/ABL expressing cells. In order to study this question we have used an interleukin 3 (IL3) dependent cell line that lacks expression of the pro-apoptotic proteins, Bax and Bak. These cells grow in the presence of IL3 but do not undergo apoptosis after cytokine withdrawal or genotoxic stress. We have generated a subclone of these cells that expresses BCR/ABL. This cell line, designated DBA, grows in the absence of IL3. As expected, growth of the cells in the absence of IL3 is suppressed by the ABL kinase inhibitor, dasatinib. BCR/ABL expressing and control cells proliferate at similar rates following DNA damage. After damage, both control cells and DBA cells have a delay in the G2/M phase of the cell cycle, which is modestly prolonged in BCR/ABL expressing cells. Both control and BCR/ABL expressing cells have a similar amount of DNA damage after irradiation as assessed by pulsed field gel electrophoresis. Additionally, the rate of repair of DNA double strand breaks is not significantly different in the two cell types. Spectral karyotype analysis is ongoing to determine whether error prone DNA repair in BCR/ABL cells is secondary to the effects of BCR/ABL on inhibition of apoptosis or reflects other proposed mechanisms of action of the oncogene.

BCR and BCR/ABL Regulation during Myeloid Differentiation in Healthy Donors and in Chronic Phase/Blast Crisis CML Patients

The fusion protein BCR/ABL leads to chronic myeloid leukaemia (CML). The corresponding fusion gene is under the transcriptional control of BCR promoter. It is known that in CML progenitors the ability to block myeloid differentiation is directly related to BCR/ABL levels. These observations open new questions about BCR/ABL and BCR expression control. However, up to date only few studies have been focused on the characterization of the BCR promoter, so little is known about the transcriptional regulation of this gene. We studied BCR expression in sorted myeloid precursors in healthy donors (HD) and in CML patients in Chronic Phase (CP) and Blast Crisis (BC). CML samples were also analyzed for BCR/ABL. The expression level was analyzed by Real Time PCR normalized against GUS as housekeeping gene, in haematopoietic stem cells (HSCs defined as CD34+/CD38−/Thy+/−), common myeloid and granulocyte-monocyte progenitors (CMPs and GMPs, respectively defined as CD34+/CD38+/IL−3Ralo/CD45RA− and CD34+/CD38+/IL−3Ralo/CD45RA+,) sorted by Fluorescence Activated Cell Sorting (FACS). Preliminarly, we analyzed the BCR and BCR/ABL mRNA halflife. We performed a RNA stability assay using Actinomycin D in the K562 cell line and in CP and BC samples: the halflife of both genes were comparable thus excluding major differences in RNA stability. Then, we analyzed BCR levels in healthy donors. A statistically significant BCR downregulation was noted during myeloid maturation in HD (HSCs [0.815±0.27SD] vs CMPs [0.176±0.1] and vs GMPs [0.167±0.17], p=0.0079 in both cases). The same analysis performed in CP patients showed that both BCR and BCR/ABL were downregulated upon committement to differentiation (BCR: HSCs [0.326±0.24] vs CMPs [0.0899±0.054], p=0.0078, and vs GMPs [0.0277±0.07], p=0.0008; BCR/ABL: HSCs [2.99±1.76] vs CMPs [0.781±0.41], p=0.0005 and vs GMPs [0.304±0.46], p=0.028). However, while BCR levels were lower in CML samples compared to HD (HSC samples [0.815±0.27 vs 0.326±0.24]: p=0.0120), probably due to haploinsufficiency, BCR/ABL values were higher than BCR ones, when compared to CML (HSCs: p=0.0020) and even to HD samples (HSCs: p=0.0074). These data indicate that in CML a potent upregulation of BCR/ABL compared to BCR is present, probably caused by a selective mechanism acting on BCR/ABL. In a limited set of BC samples (4 patients), the decrease of BCR and BCR/ABL was less evident and did not reach statistical significance (BCR: HSCs 0.339±0.235; CMPs: 0.135±0.12; GMPs: 0.126±0.06; BCR/ABL: HSCs: 8.58±5.78; CMPs: 5.55±7.39; GMPs: 3.36±2.61); thus as already indicated by Jamieson and colleagues (NEJM , 351:657, 2004), in BC the downregulation of BCR/ABL during maturation is impaired. Moreover, we detected a tendency for higher expression of BCR/ABL in BC than CP in all the subpopulations under study, which reached statistical significance only in GMPs (HSCs [8.58±5.78 BC vs 2.99±1.76 CP]: p=0.1483; CMPs [5.55±7.39 BC vs 0.781±0.41 CP]: p=0.1483; GMPs [3.36±2.61 BC vs 0.304±0.46 CP]: p=0.028); BCR was upregulated only in GMPs (HSCs [BC 0.339±0.235 vs 0.326±0.24 CP]: p=0.9399; CMPs [0.135±0.12 vs 0.0899±0.054]: p=0.4818; GMPs [0.126±0.06 vs 0.0277±0.07]: p=0.0755). In conclusion, BCR is physiologically downregulated during myeloid maturation, and this regulation is conserved in CP-CML, not only for BCR but also for BCR/ABL. In BC-CML, this mechanism of regulation seems to be impaired for BCR/ABL and possibly for BCR too. In addition, a potent upregulation of BCR/ABL compared to BCR is present in all stages of CML, probably caused by a selective mechanism acting on BCR/ABL.

Targeting of heat shock protein 32 (Hsp32)/heme oxygenase-1 (HO-1) in leukemic cells in chronic myeloid leukemia: a novel approach to overcome resistance against imatinib

Resistance toward imatinib and other BCR/ABL tyrosine kinase inhibitors remains an increasing clinical problem in the treatment of advanced stages of chronic myeloid leukemia (CML). We recently have identified the heat shock protein 32 (Hsp32)/heme oxygenase-1 (HO-1) as a BCR/ABL-dependent survival molecule in CML cells. We here show that silencing Hsp32/HO-1 in CML cells by an siRNA approach results in induction of apoptosis. Moreover, targeting Hsp32/HO-1 by either pegylated zinc protoporphyrine (PEG-ZnPP) or styrene maleic acid-micelle-encapsulated ZnPP (SMA-ZnPP) resulted in growth inhibition of BCR/ABL-transformed cells. The effects of PEG-ZnPP and SMA-ZnPP were demonstrable in Ba/F3 cells carrying various imatinib-resistant mutants of BCR/ABL, including the T315I mutant, which exhibits resistance against all clinically available BCR/ABL tyrosine kinase inhibitors. Growth-inhibitory effects of PEG-ZnPP and SMA-ZnPP also were observed in the CML-derived human cell lines K562 and KU812 as well as in primary leukemic cells obtained from patients with freshly diagnosed CML or imatinib-resistant CML. Finally, Hsp32/HO-1-targeting compounds were found to synergize with either imatinib or nilotinib in producing growth inhibition in imatinib-resistant K562 cells and in Ba/F3 cells harboring the T315I mutant of BCR/ABL. In summary, these data show that HO-1 is a promising novel target in imatinib-resistant CML.

Long-Term Significance of Achieving a Major Cytogenetic Response (MCyR) without a Complete Hematologic Response (CHR) among Patients (pts) with Chronic Myeloid Leukemia (CML) in Advanced Phase Treated with Second Generation Tyrosine Kinase Inhibitors (TKI).

The BCR-ABL TKI, dasatinib (D) and nilotinib (N), are effective in Philadelphia chromosome-positive (Ph+) leukemias, including all stages of imatinib-resistant CML and Ph+ ALL. For both dasatinib and nilotinib, the reported rates of MCyR are higher than the rates of CHR; that is, some pts achieve MCyR but may have cytopenias that make their hematologic response less than a CHR; this has been attributed to myelosuppression induced by TKI. At present it is not known whether pts with MCyR who do not have CHR have a similar outcome as those with a CHR. We studied 135 pts with accelerated (AP; n=84) or blast phase (BP; n=47) CML or Ph+ ALL (n=4) treated with D (59) or N (76) for imatinib resistance, to see whether the presence of cytopenias at the time of best response affected the prognosis. The median age was 55 yrs (range, 15 to 98) and 62 were female. 56 (41%) had bcr/abl mutations at the start of therapy. Overall 36 pts (27%) achieved MCyR. All the 36 pts who achieved a MCyR after treatment with N or D had received imatinib for an average time of 34 mos (range 4–78) and 24/36 pts had obtained a MCyR under imatinib. They started the new TKI 66 mos after first diagnosis (12–336). 2-yr survival rates for pts that achieved MCyR was 72% compared to 35% for those without MCyR (p<0.001). There was a trend for an inferior survival for those not having a concomitant CHR at the time of MCyR compared to those with CHR (2-yr survival 58% vs 82%; p=0.11). The rates of CHR and MCyR were 68% and 29%, respectively in AP; 32% and 21% in BP, and 50% and 50% in Ph+ ALL. Among pts in AP, 17/24 (70%) who achieved a MCyR, had a CHR at the time of best cytogenetic response; 6 of these 17 (35%) pts eventually failed (lost response or transformed), compared to 3/7 (43%) of those with no CHR at the time of MCyR (p=0.28). The results are similar when analyzing separately pts treated with N or D. 2-yr survival was 88% and 66% for those with and without concomitant CHR (p=0.29). Among the 10 pts in BP that achieved a MCyR, 5 had a CHR at the time of best cytogenetic response. Three (60%) of these pts eventually failed (lost response), compared to 4 failures among the 5 pts with no CHR at the time of MCyR (p=1.0). Survival rates at 2 yrs were 60% and 40% for those with and without concomitant CHR (p=0.29). Among the 2 patients with ALL that achieved MCyR, one had comcomitant CHR (treated with N) and the other did not have CHR (D); both of them failed. Of the 18 pts who failed after having achieved an MCyR, 7 had a mutation before starting treatment with a 2nd TKI (A433T, G250E, E355G, E459K, M244V, M315T, F317L), and 6 developed a new mutation after treatment (F317L, F311I, Y253H, G476C, E255K, H359V). Five of them occurred in pts with a concomitant CHR and 1 without a CHR. These results suggest that achievement of MCyR with residual cytopenias (ie, without a concomitant CHR) may not have the same favorable outcome as that of MCyR with concomitant CHR. Whether this is related to pt- or disease-related characteristics remains to be determined.

Identification of BCR-ABL point mutations conferring resistance to the Abl kinase inhibitor AMN107 (nilotinib) by a random mutagenesis study

AbstractPatients with advanced stages of chronic myeloid leukemia (CML) often manifest imatinib mesylate resistance associated with point mutations in BCR-ABL. AMN107 is a new higher-potency inhibitor of BCR-ABL. To identify mutations in BCR-ABL that could result in resistance to AMN107, a cDNA library of BCR-ABL mutants was introduced into Ba/F3 cells followed by selection in AMN107 (0.125-0.5 µM). A total of 86 individual, drug-resistant colonies were recovered, and the SH3, SH2, and kinase domains of BCR-ABL were sequenced. A total of 46 colonies had single point mutations in BCR-ABL, with a total of 17 different mutations, all within the kinase domain. The other 40 colonies had multiple point mutations and were not analyzed further. Each of the 17 single point mutants were reconstructed by site-directed mutagenesis of native BCR-ABL and found to be approximately 2.5- to 800-fold more resistant to AMN107 than native BCR-ABL. The mutations included 6 known imatinib mesylate–resistant mutations, including T315I, which showed complete resistance to AMN107. Interestingly, most AMN107-resistant mutants were also resistant to imatinib mesylate. These results may predict some of the resistance mutations that will be detected in clinical trials with this kinase inhibitor.

Monitoring of Chronic Myeloid Leukemia (CML) Imatinib Response by Fluorescence-In-Situ-Hybridization (FISH).

Abstract Glivec, a tyosine kinase inhibitor is the drug of choice for newly diagnosed chronic phase CML. Whilst Q-PCR is the most sensitive method to monitor response; cytogenetic analysis as well as FISH is routinely used in many laboratories. FISH has the advantage over marrow cytogenetic by allowing analysis of more cells, and cells in interphase, obviating necessity for an invasive marrow procedure. We performed 126 FISH studies using BCR-ABL dual color dual fusion translocation probe for 69 CML patients on imatinib. For each samples, 300 nucleated cells were counted. Clinical information was available from 57 patients (34 chronic-phase (CP), 7 accelerated phase (AP), 8 blastic phase (BT) and 8 post-BMT relapses). Most CP patients (94%) could achieve sustained complete hematological responses (CHR), including 14 of the 15 patients pre-treated with IFN. IFN pre-treated patients received imatinib generally late in the course of CP compared to those without (median 19M vs 0.8M, p&amp;lt;0.001). At 12–15 months, 61% CP patients were FISH-ve. Fewer IFN-pretreated patients achieved FISH-ve responses (41.7% vs 90%, p=0.03). With a mean follow-up of 25 months (4–55M), none of the FISH-ve patients had disease transformation while AP/BT developed in 2 FISH+ patients (0% vs 22%, p=0.14). In CML-AP, early institution of imatinib (1–10M) achieved more FISH-ve responses than late treatment (30–36M) and degree of FISH response after 6–12 months of imatinib in CML-AP appeared to predict prognosis. For patients treated in BT, 4 received imatinib upfront and 4 after remissions or reversion to CP after chemotherapy. FISH negativity in the first few months of imatinib treatment, however, did not predict better prognosis. Two patients remained FISH-ve and disease-free 52 months without transplant, suggesting imatinib may be useful as maintenance after chemotherapy induction for BT patients not eligible for transplant. The observation that imatinib after chemotherapy induction has better prognosis than imatinib upfront requires confirmation by larger studies. In post-BMT relapses (8 patients: 1 cytogenetic relapse, 4 CP relapses, 1 blastic relapse, 2 AP relapses), hematological response could be achieved in all. Half became FISH-ve at 6–9 months and remained so at latest follow-up (44–56M). Three patients (1 CP, all 2 AP) were FISH+ at 6–9 months (42–90% Ph+ cells) and remained FISH+ thereafter, with one progressed to blastic disease at 24 months. FISH response at 6–9 months, thus, appears to carry prognostic significance in post-transplant CML relapses. In summary, our initial experience suggests monitoring of imatinib response by FISH provides useful prognostic information in different stages of CML.

T315I-mutated Bcr-Abl in chronic myeloid leukemia and imatinib: insights from a computational study

The early stage of chronic myeloid leukemia is triggered by the tyrosine kinase Bcr-Abl. Imatinib mesylate, a selective inhibitor of Bcr-Abl, has been successful in chronic myeloid leukemia clinical trials, but short-lived remissions are usually observed in blast crisis patients. Sequencing of the BCR-ABL gene in relapsed patients revealed a set of mutants that mediate drug resistance. Previously reported work postulated that the missense T315I mutation both alters the three-dimensional structure of the protein binding site, thus decreasing the protein sensitivity for the drug, and does not feature a fundamental hydrogen bond that is critical for binding with imatinib. These speculations, however, were not supported by investigations at the molecular modeling level. Here, we present the results obtained from the application of molecular dynamics simulations to the study of the interactions between T315I Bcr-Abl and imatinib. For the first time, we show that, with respect to the wild-type system, the absence of the supposedly critical H-bond is not the only cause for the failure of receptor inhibition by imatinib, but also a plethora of other protein/drug interactions are drastically and unfavorably changed in the mutant protein.

Dose-dependent effects of Bcr-Abl in cell line models of different stages of chronic myeloid leukemia

Chronic myeloid leukemia (CML) is caused by Bcr-Abl, an activated tyrosine kinase. The amounts of Bcr-Abl mRNA and protein in cells from patients in blast crisis (BC) are higher than in those chronic phase (CP), indicating that their expression rises with disease progression. In order to study this phenomenon on cells with the same genetic background, we transfected the 32D cell line with the BCR-ABL transgene and selected clones with graded expression of Bcr-Abl within the range found in cells from CP to BC. In vitro, we found that Bcr-Abl exerted dose-dependent effects upon growth factor dependence, clonogenicity and migration. However, the relationship between Bcr-Abl expression and cellular adhesion to fibronectin was more complex: rather than a direct positive correlation, clones that expressed low, but not high, levels of Bcr-Abl were less adhesive than growth factor stimulated, parental BCR-ABL-negative 32D cells. This finding parallels the situation with normal and CML-CP progenitors where the latter exhibit defective adhesion to fibronectin. Treatment with the tyrosine kinase inhibitor imatinib mesylate reversed the adhesion deficient phenotype of clones expressing low levels of Bcr-Abl. When injected subcutaneously into syngeneic mice, cell lines expressing high levels of Bcr-Abl rapidly induced tumors, whereas low-expressing clones led to tumor formation only after a prolonged latency. These findings suggest that the level of Bcr-Abl may be essential in determining the phenotype of the leukemic clone at different stages of the disease.

Detection and quantification of BCR-ABL transcripts in patients with chronic myeloid leukemia by real-time quantitative reverse transcriptase polymerase chain reaction

To investigate the effect of real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) approach in chronic myeloid leukemia (CML) for detecting the minimal residual disease (MRD) or monitoring the treatment response and predicting the prognosis. Fifty-six CML patients, 39 males and 17 females, aged 39 (16 approximately 66), with disease history and frozen RNA specimens were studied, 31 of which were in the incipient chronic phase, 7 in the accelerated phase, and 17 in the rapidly progressing phase. Three or more frozen RNA specimens collected before and after treatment were preserved in 11 of the patients. Breakpoint cluster region-Abelson murine leukemia viral oncogene (BCR-ABL) of the patients in different CML stages was analyzed by RT-PCR approach. The BCR-ABL transcript of those patients remaining in chronic period after treatment decreased to 1/3 that of the baseline level six months after the initiation of treatment and then remained at that level. The BCR-ABL transcript of those in which progressing change occurred increased when such change occurred. After allogeneic transplantation of peripheral blood stem cells the BCR-ABL level decreased significantly. The median DoseN in the 17 progressing patients was 10 492, significantly higher than those of the 31 patients in chronic phase (5920) and in the 7 patients in accelerated phase (4444, both P < 0.05). The minimal residual disease and the treatment response were closely associated with the level and its variation of BCR-ABL transcripts, the transcripts level in blastic crisis was significantly higher than that in chronic phase or accelerated phase. Real-time quantitative RT-PCR is reliable and can be used to detect the minimal residual disease, monitor the treatment outcome, and predicting blastic crisis.

Immunohistochemical Characterisation of Vascular Endothelial Growth Factor (VEGF) and its Receptors Flt-1 and KDR in Chronic Myeloid Leukaemia (CML) Patients Treated with Imatinib Mesylate.

VEGF is a potent angiogenic regulator implicated in increased angiogenesis, which is characteristic of CML. VEGF interacts with its two tyrosine kinase (TK) receptors, Flt-1 (VEGFR-1) and KDR (Flk-1/VEGFR-2). CML therapy with imatinib mesylate, which targets BCR-ABL TK activity, induces haematological remission in 95% and complete cytogenetic remission (CCR)/ major response (MR) in 60% of cases. We describe the serial evaluation of patient bone marrow (BM) trephines and the analysis of the impact of imatinib on VEGF in all stages of CML during therapy. Consecutive CML (n=38) patients (25 males, 13 females, median age of 56 years (19 to 81) were sequentially analysed during the course of imatinib therapy. Chronic phase (CP) n=24, accelerated phase (AP) n=11 and blast crisis (BC) n=3 prior to imatinib therapy. BM examination was performed at diagnosis, at 1 month, then 3 monthly following imatinib therapy. Immunohistochemical analysis determined expression of VEGF, VEGFR1/Flt-1 or VEGFR2/KDR. Results were correlated with cytogenetic response. In normal BM cellular VEGF and its receptors were expressed in megakaryocytes and macrophages, but rarely in myeloid cells. VEGF was not expressed in erythroblasts, lymphocytes or plasma cells. A weak VEGFR1 signal was observed in monocytes/histiocytes and VEGFR2 in histiocytes only. VEGF expression in CML was detected as a diffuse cytoplasmic pattern in myeloid and monocyte precursors, classified as 4+ (very intense staining), 3+ (strong), 2+ (moderate), 1+ (weak), or 0 (completely negative) throughout. The intensity of staining varied and did not correlate with the stage of disease, but VEGF was strongly expressed in the megakaryocytes. Both VEGFR1/Flt-1 and VEGFR2/KDR were expressed in monocytic/myeloid population and in megakaryocytes. The staining intensity varied from borderline detectable to a very strong staining intensity. 14/38 (37%) of patients showed a reduction in VEGF expression, which was particularly marked with respect to VEGFR2 receptor staining. Of these, 10/14 patients (71%) achieved CCR, one MR (major response) and 3 minimal/NR (no response). All patients in this group had a reduction in BM cellularity, median 30% (15–70%). No change in VEGF receptor expression was detected in 10/38 (26%). Only 1 patient achieved CCR, 3 MR, with a minor/NR in the rest (60%), and a median BM cellularity of 30% (10–100%). Increasing VEGF expression was observed in 14/38 patients (37%). Here 6/14 (43%) patients showed NR (2 minor/minimal responses) with CCR in only 4 of the evaluable patients. The median BM cellularity was 25% (10–95%). 3/14 patients went on to develop progressive disease In summary, reduced expression of VEGF and its receptors, particularly KDR, can predict a favourable response to imatinib and correlates with a reduced BM cellularity and cytogenetic response. In patients with an increase or no change in VEGF expression there is a greater incidence of a poor cytogenetic response and a higher tendency to relapse despite a reduction in BM trephine cellularity, but a longer follow-up may be necessary.

HTERT, the Catalytic Component of Telomerase, Is Down-Regulated in the Hematopoietic Stem Cells of Patients with Chronic Myeloid Leukemia.

Telomere shortening and increased telomerase activity is associated with disease progression in chronic myeloid leukemia (CML). In order to gain insight into the biology of telomerase and its regulation in CML we have evaluated the expression levels of the components of telomerase, its regulators, and several telomeric associated proteins in the hematopoietic stem cells (HSC)/leukemic blast cells of 21 patients with this disorder, as compared to the HSC population of a group of healthy individuals, using quantitative real time PCR. In this study we have made the surprising observation that hTERT, the catalytic component of telomerase, is down-regulated in the CD34+/blast cells of the majority of CML patients in chronic phase (CP) (7 out of 9, p=0.0339) relative to CD34+ cells from normal controls. Furthermore hTERT was found to be significantly down-regulated in 2 out of 3 patients in accelerated phase (AP) and in 9 out of 9 patients in blast crisis (BC) relative to normal controls (p=0.0003). The expression levels of hTR, the telomerase RNA template, and the telomeric- associated proteins TEP1, TRF1, TRF2 and tankyrase were all up-regulated in the CD34+/blast cells of the majority of the CML patients in CP and AP, relative to normal control CD34+ cells. With the exception of TEP1, the expression levels of all these factors were highest in CP CML and, in the majority of patients, decreased during disease progression. We speculate that the up-regulation of tankyrase (positive regulator of telomere length) and TRF2 (protects critically shortened telomeres) may contribute to telomere maintenance in CML. The expression levels of hTERT and c-myc, a positive regulator of hTERT expression, correlate in all stages of CML (R2=0.623). The expression levels of c-myc decreased with disease progression, falling below the levels observed in normal CD34+ cells in the majority of patients in BC. The expression levels of hTERT were increased in the CD34+ cells of three additional CP CML patients in remission following successful treatment with imatinib, relative to both untreated CP CML patients (p=0.0126) and to normal individuals, reflecting a restoration of haematopoiesis towards normal. In CML the turnover of all types of Ph+ stem and progenitor cells is known to be increased. We suggest that the reduced (and presumably inadequate) expression levels of hTERT in CML CD34+/blast cells, and the consequent telomere shortening with each division, is the direct cause of the shortened telomeres frequently observed in CML patients. These data challenge the widely accepted view that hTERT expression is always increased in tumorigenesis. It is possible that the Bcr-Abl fusion product may play a role in the down-regulation of hTERT observed in the HSC of CML patients in this study.

Four Years of Follow-Up of 1027 Patients with Late Chronic Phase (L-CP), Accelerated Phase (AP), or Blast Crisis (BC) Chronic Myeloid Leukemia (CML) Treated with Imatinib in Three Large Phase II Trials.

BackgroundThis report updates the results of 3 large phase II studies of the orally available BCR-ABL tyrosine kinase inhibitor imatinib for patients (pts) in AP, BC and late chronic phase (L-CP) CML failing prior interferon therapy (Kantarjan et al, ASH 2003; Talpaz et al, ASH 2003).MethodsBetween August 1999 and June 2000, 1027 pts were enrolled in phase II trials for CML in L-CP (n=532), AP (n=235) or BC (n=260). Pts in L-CP were treated with 400 mg/day and pts in AP or BC with either 400 or 600 mg/day. Dose escalation up to 800 mg/d was allowed in the late-chronic phase study. Pts with a confirmed diagnosis of AP (n=181), BC (n=229) and late-chronic phase (n=454) were evaluated for efficacy. All pts were evaluated for safety. The median time from initial diagnosis to study entry was 32 months for L-CP pts.ResultsAs of 31-Jul-03, 5% patients with BC, 25% of CML-AP and 64% of L-CP patients still remain on treatment.At the recommended dose of 600 mg, an estimated 40% (AP) and 7% (BC) of patients remained progression-free at 36 months, and an estimated 55% (AP) and 14% (BC) patients were alive at 36 months after initiation of imatinib. The 3-year survival rates for pts with AP with a major cytogenetic response at 3 months were 85% vs. 52% for pts with no response (p<0.001). In L-CP patients with a median follow-up of 40 months, 65% of patients achieved a major cytogenetic response, which was complete in 52%. The cytogenetic responses were durable with an estimated 82% of the pts in continuos major cytogenetic response at 3 years. The estimated rates of progression-free survival and overall survival at 3 years were 80% and 88%. Pts with at least a minor cytogenetic response at 6 months ≤65% Ph+ cells) had an estimated 3-year survival rate of 96% vs. 86% for pts with a ( minimal response and 81% for pts with no cytogenetic response (p<0.001).ConclusionIn large phase II studies, continuous imatinib treatment is safe and has improved progression-free survival of patients at all stages of CML. Responses to imatinib are durable and are predictive of long-term outcomes. These results will be further updated at the meeting using a data base lock planned for 20-Sept-04 (using data collected up to 31-July-04, i.e. more than 4 years after the last pts enrollment).