The Impact Of Novel Splicing Abnormalities Of BCR-ABL On The Pathogenesis Of CML

Abstract Background Chronic myeloid leukemia (CML) is effectively treated with tyrosine kinase inhibitors (TKIs), but reactivation of BCR-ABL frequently occurs through acquisition of kinase domain point mutations. The mechanism of resistance in patients without BCR-ABL kinase domain point mutation is still elusive. Previous studies have revealed the abnormal splicing of BCR-ABL kinase domain, including Exon8/9 junction 35bp insertion and exon-skipping of Exon 7 (T O'Hare et al, Blood 2011. Gaillard JB, et al. Mol Cancer Ther 2010). The insertion of 35 intronic nucleotides at the exon 8/9 splice junction introduces a stop codon after 10 intron-encoded residues and inactive tyrosine kinase activity. The effect of these splicing abnormalities on susceptibility of cells against TKIs is still controversial. Furthermore, the conventional direct sequence techniques could not evaluate splicing abnormalities in major molecular response (MMR)- complete molecular response (CMR) CML patients, who achieved clinically leukemia-free state with a small number residual CML stem cells. Aims The aim of this study is to evaluate the frequency and the patterns of splicing abnormalities of BCR-ABL in CML patients, especially who achieved MMR-CMR by TKI treatment. Methods We analyzed peripheral blood samples from healthy individuals and CML chronic phase patients. We extracted total RNA from these samples and synthesized cDNA, and then performed PCR-amplification of BCR-ABL kinase domain in CML patients and ABL kinase domain in healthy individuals, respectively. PCR products were subjected to the amplicon sequence: We deeply sequenced BCR-ABL fusion gene transcripts, and evaluated splicing forms of BCR-ABL by using HiSeq 2000 (illumina). Results We successfully established a novel analysis method, which can detect the pattern of splicing abnormalities even in MMR-CMR patients. Using the amplicon sequence technique, we detected abnormal splicing patterns of BCR-ABL in 5 out of 15 CML patients. We also found that the splicing abnormalities were not restricted to 35bp insertion at the exon8/9 junction, thus intronic retention of intron 8 and intron 9 could be frequently detected with or without the 35bp insertion in CML patients (Table 1). Of note, these abnormal splicing patterns always co-existed with wild type BCR-ABL transcripts in all 5 cases analyzed. In addition to the novel splicing abnormalities in CML, we unexpectedly found in healthy individuals that splicing abnormalities such as 35bp insertion at the exon8/9 junction and intronic retention could be detected in ABL1 transcripts (Table 1). This result suggests that this sort of splicing abnormalities could occur at a certain frequency in steady state human hematpoiesis, and is not specific to BCR-ABL. Summary / Conclusion We have newly established an analysis system to efficiently detect splicing abnormalities of BCR-ABL even in MMR-CMR CML patients. Using this highly efficient amplicon sequence technique, we identified novel splicing abnormalities both in healthy individuals and CML patients, and found that the wild type BCR-ABL transcripts always co-exist with abnormally spliced BCR-ABL transcripts. These results collectively suggest that splicing abnormalities within the ABL1 kinase domain are not specific to CML patients treated with TKIs, and that the detection of such kinase domain splicing abnormalities do not reflect insusceptibility of the remaining cells during TKI treatment. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Co-Crystal Structures of 7-Azaindole Inhibitors of Wild-Type and T315I Imatinib-Resistant Mutant Forms of the BCR-ABL Tyrosine Kinase.

Blood ◽

10.1182/blood.v110.11.1018.1018 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1018-1018

Author(s):

Hal A. Lewis ◽

Fred Zhang ◽

Richard Romero ◽

Pierre-Yves Bounaud ◽

Mark E. Wilson ◽

...

Keyword(s):

Crystal Structure ◽

Tyrosine Kinase ◽

Crystal Structures ◽

Kinase Domain ◽

Hinge Region ◽

Wild Type ◽

X Ray ◽

Abl Kinase ◽

Imatinib Resistant ◽

Mutant Forms

Abstract Chronic myelogenous leukemia (CML) arises from uncontrolled cell growth driven by a constitutively active BCR-ABL fusion protein tyrosine kinase, which is the product of the pathognomonic Philadelphia chromosomal translocation. Imatinib mesylate (Gleevec) is a BCR-ABL inhibitor used as a first line treatment of CML. Although imatinib is highly effective in chronic phase CML, in advanced disease patients frequently relapse due to the emergence of drug resistance. Approximately two-thirds of resistance is caused by point mutations in the BCR-ABL kinase domain, which give rise to active mutant forms of the enzyme that are insensitive to Gleevec. The T315I mutation represents one of the most common causes of resistance, is resistant to the second generation BCR-ABL inhibitors dasatinib and nilotinib, and represents an important and challenging target for discovery of next generation targeted CML treatments. We have applied X-ray crystallographic screening of our FAST™ fragment library and structure-guided hit-to-lead optimization to identify potent inhibitors of both wild-type and T315I mutant BCR-ABL. These efforts yielded a 7-azaindole compound series that exhibits binding to and inhibition of both wild-type and T315I BCR-ABL. Methods: Wild-type (with Y393F) and T315I Abl kinase domain protein were expressed in E. coli and purified to homogeneity. These proteins were crystallized in the presence of a reference inhibitor followed by addition of the 7-azaindole series compounds soaked into the preformed crystals to displace the reference compound, giving the desired co-crystal. X-ray diffraction data were recorded at the company’s proprietary synchrotron beamline SGX-CAT at the Advanced Photon Source. Three-dimensional enzyme-inhibitor co-crystal structures were determined by molecular replacement and refined to permit modeling of bound ligand. Results: Both wild-type and T315I Abl structures revealed enzyme in the active conformation with inhibitors bound to the kinase hinge region. The crystal structure of 2-amino-5-[3-(1-ethyl-1H-pyrazol-5-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl]-N,N-dimethylbenzamide in complex with T315I, illustrates the typical binding mode which is independent of the 315 residue, and therefore accounts for the compound inhibiting the T315I mutant form of BCR-ABL (see figure). The inhibitor binds to the hinge region of ABL utilizing hydrogen bonding to backbone carbonyl of Glu316 and NH of Met318, with the pyrazole ring stacking in a lipophilic pocket between Phe382 and Tyr253. In addition, the benzamide carbonyl participates in a hydrogen bond interactioin with the backbone-NH of Glu249 of the p-loop. Conclusions: X-ray crystallographic fragment screening and co-crystal structure studies have been successfully employed in discovery/optimization of 7-azaindole series compounds, yielding potent, selective inhibitors of both wild-type and imatinib-resistant forms of BCR-ABL. Figure Figure

Download Full-text

Loss of the Wild-Type Allele Contributes to Myeloid Expansion and Disease Aggressiveness in FLT3/ITD Knock-in Mice

Blood ◽

10.1182/blood.v118.21.866.866 ◽

2011 ◽

Vol 118 (21) ◽

pp. 866-866

Author(s):

Li Li ◽

Emily Bailey ◽

Sarah M Greenblatt ◽

David Huso ◽

Donald Small

Keyword(s):

Median Survival ◽

Conflicts Of Interest ◽

Lentiviral Vector ◽

Point Mutations ◽

Kinase Domain ◽

Wild Type Allele ◽

Wild Type ◽

Type Allele ◽

Lower Ratio ◽

The Impact

Abstract Abstract 866 Activating mutations of FLT3, either in the form of internal tandem duplication (ITD) mutations in the juxtamembrane domain or point mutations in the kinase domain, are one of the most frequent mutations in acute myeloid leukemia (AML). AML patients with FLT3/ITD mutations have poor prognosis. Loss of the wild-type FLT3 allele is associated with even worse prognosis when compared to those FLT3/ITD AML patients with the wild-type FLT3 allele still present. Also, FLT3/ITD patients with a high mutant-to-wild-type ratio have a significantly worse outcome than FLT3/ITD patients with a lower ratio. We have previously reported that heterozygous FLT3wt/ITD “knock-in” mice develop a slowly fatal MPN. In order to study the roles wild-type FLT3 play in the development of leukemia associated with FLT3/ITD mutations, we crossed FLT3wt/ITD mice with themselves or with FLT3 “knockout” (FLT3−/−) mice to obtain hemizygous (FLT3−/ITD) or homozygous (FLT3ITD/ITD) FLT3/ITD mice. Investigating phenotypic differences among them reveals the impact of wild-type FLT3 on the development of MPN resulting from FLT3/ITD mutations, and by extension, the effect on acute leukemia. FLT3−/ITD mice, with the loss of the wild-type allele, displayed a more severe MPN, as evidenced by even larger spleen, higher white blood counts and shorter survival, compared to FLT3wt/ITD mice. FLT3ITD/ITD mice had an even severe MPN compared to the FLT3−/ITD and FLT3wt/ITD mice. Fully transformed leukemia developed in some of the FLT3ITD/ITD (7%, 9/129), but not FLT3wt/ITD or FLT3−/ITD mice, with latency ranging from 139 to 304 days. Compared to FLT3wt/ITD mice, FLT3−/ITD and FLT3ITD/ITD mice displayed a further increase in the fraction of primitive hematopoietic cells, with notable increases in ST-HSCs and MPPs. Phosphorylation of STAT5, one of the key downstream targets for constitutively activated FLT3, was increased in FLT3wt/ITD, FLT3−/ITD and FLT3ITD/ITD mice compared to the wild-type control. FLT3wt/ITD, FLT3−/ITD and FLT3ITD/ITD BM also showed increased PU.1 expression and decreased GATA-1 expression, resulting in the subsequent expansion of granulocytic/monocytic/lymphocytic progenitors and a decrease in megakaryocytic/erythrocytic progenitors. It appears that the extent of myeloproliferation in FLT3/ITD mice correlates with loss of the wild-type allele (FLT3wt/ITD vs. FLT3−/ITD) and with the dose of mutant allele (FLT3−/ITD vs. FLT3ITD/ITD). In order to further explore the potential moderating effect of wild-type FLT3 expression on FLT3/ITD-associated MPN, we transduced wild-type FLT3 (wtFLT3, with the lentiviral vector co-expressing GFP) into lineage-depleted FLT3−/ITD CD45.2 BM cells and injected them into lethally irradiated CD45.1 recipients. When injected with sorted (GFP+) BM, vector alone-transduced GFP+FLT3−/ITD BM recipients died of MPN, with a median survival of 62 days. 100% of the recipients in the other three groups, i.e., those injected with vector alone-transduced GFP+ wild-type BM, wtFLT3-transduced GFP+ wild-type BM or wtFLT3-transduced GFP+ FLT3−/ITD BM, remained viable even after the point in time at which all of the recipients in the vector alone-transduced GFP+FLT3−/ITD group died. Similarly, recipients transplanted with unselected (including GFP+ and GFP− populations) vector alone-transduced FLT3−/ITD BM also died early, with a median survival of 73 days and overt signs of MPN. The percentages of GFP+ and GFP− cells in the BM of the dying recipients were comparable to those shortly after transplantation, indicative of the similar expansion ability of the GFP+ and GFP− populations in the BM. In contrast to the wtFLT3-transduced GFP+FLT3−/ITD BM recipients, which have a very prolonged survival, recipients injected with unselected wtFLT3-transduced FLT3−/ITD BM died of MPN, with a median survival of 91 days. Interestingly, 99% of the BM cells in the BM of the dying recipients were GFP−, demonstrating a proliferative/survival advantage for the FLT3−/ITD cells that had not been successfully transduced with wild-type FLT3. These results suggest that the presence of wild-type FLT3 delays and moderates the development of MPN caused by FLT3/ITD mutations. These results suggest that loss of the wild-type allele contributes to the development of a more severe phenotype. Thus, the wild-type FLT3 allele seemingly functions as a “tumor suppressor” in leukemia harboring FLT3/ITD mutations. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Deep Molecular Response Achieved By Second-Generation Tyrosine Kinase Inhibitors (TKIs) Can Lead to Stopping TKIs

Blood ◽

10.1182/blood.v126.23.1594.1594 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1594-1594

Author(s):

Tetsuro Ochi ◽

Taiichi Kyo ◽

Kayo Toishigawa ◽

Takeshi Okatani ◽

Ryota Imanaka ◽

...

Keyword(s):

Tyrosine Kinase ◽

Relapse Rate ◽

Median Duration ◽

Second Generation ◽

Conflicts Of Interest ◽

Univariate Analysis ◽

Chronic Phase ◽

Molecular Response ◽

Molecular Monitoring ◽

Tki Treatment

Abstract Background: Imatinib (IMA), a first-generation tyrosine kinase inhibitor (TKI), has enabled safer, more successful treatment of chronic myeloid leukemia (CML). Moreover, second-generation TKIs such as nilotinib (NIL) and dasatinib (DAS) have enabled achievement of deeper molecular responses than IM. TKIs have improved prognosis for CML patients, but lifelong continuation of TKIs lowers quality of life and places an economic burden on patients. Whether administration of TKIs can be stopped is thus an important question. Trials including the STIM trial have suggested that IMA can be stopped in CML patients who maintain complete molecular response (CMR) for >24 months, but little data is available regarding second-generation TKIs. Methods: Among adult CML patients in the chronic phase diagnosed at Hiroshima Red Cross Hospital & Atomic-bomb Survivors Hospital from May 1995 to September 2010, we analyzed patients who achieved and maintained CMR for >1 year on TKIs, and then stopped TKIs. We started TKI treatment with IMA in all patients and changed to NIL or DAS after March 2009, when second-generation TKIs became available in Japan. We continued each TKI for ³6 months, and for >12 months in most cases. Molecular monitoring was performed with BCR-ABL1 real-time quantitative PCR (RQ-PCR) using bone marrow or peripheral blood samples. Sensitivity of this RQ-PCR was 0.004%, corresponding to MR4.4. Relapse was defined as a loss of CMR. We provided TKI therapy for relapsed patients. RQ-PCR was performed every three months after relapse. Results: Stopping TKI was possible in 51 patients (32 males, 19 females). Observations were continued until June 2015, and the median duration of observation was 147 months (range, 59-257 months). Interferon (IFN)-α was administered to 18 patients. Median age at diagnosis was 44 years (range, 22-83 years). Two deaths were observed, with neither due to CML. Median duration of TKI treatment was 91 months (range, 29-160 months). Median interval from starting TKIs until achieving CMR was 41 months (range, 6-144 months), and that from achieving CMR to stopping TKIs was 20 months (range, 10-91 months). Median duration of observation from stopping TKIs was 42 months (range, 4-135 months). TKI treatment comprised IMA alone in 10 patients, IMA → NIL in 8, and IMA →NIL → DAS in 33. Relapse after stopping TKIs was observed in 14 cases. The period from stopping TKIs to relapse was 3 months in 12 patients, and 6 months and 18 months in 1 patient each. We treated all relapse patients with TKIs as patients chose, and all achieved 2nd CMR. Median period from relapse to 2nd CMR was 20.5 months (range, 6-40 months). In univariate analysis by Fisher's exact test, no correlation was seen between relapse rate and sex (male, n=32 vs. female, n=19; p=0.106), history of IFN-α therapy (yes, n=18 vs. no, n=33; p=0.525), duration from achieving CMR to stopping TKI (³24 months, n=34 vs, <24 months, n=17; p=0.183), and use of second-generation TKI (yes, n=34 vs. no, n=10; p=0.25). However, relapse rate was significantly lower in patients who received second-generation TKIs for ³24 months (n=23 vs. <24 months, n=10; p=0.0425). Conclusions: In our cohort, the rate of relapse after stopping TKIs was lower among patients who received second-generation TKIs for a longer period. This suggests that achieving deeper molecular response may be more important than maintaining CMR for a long time when trying to stop TKIs. The fact that most relapses after stopping TKIs occurred 3 months after stopping TKIs implies a need for careful molecular monitoring, particularly just after stopping TKIs. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Dynamics of BCR-ABL1 Transcripts during Nilotinib Therapy in Patients with Chronic Myeloid Leukemia (CML).

Blood ◽

10.1182/blood.v110.11.2961.2961 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2961-2961

Author(s):

Alfonso Quintás-Cardama ◽

Hagop Kantarjian ◽

Dan Jones ◽

Susan O’Brien ◽

Mary Beth Rios ◽

...

Keyword(s):

Transcript Level ◽

Kinase Domain ◽

Pcr Analysis ◽

Common Mechanism ◽

Molecular Response ◽

Wild Type ◽

Imatinib Resistance ◽

Abl Kinase ◽

Frontline Therapy

Abstract Background: Mutations in the BCR-ABL kinase domain are a common mechanism (40%) of imatinib-resistance. Nilotinib is approximately 30-fold more potent than imatinib against ABL kinase and has activity against most BCR-ABL kinase mutants, except T315I. We investigated the molecular response to nilotinib in patients (pts) receiving this agent both as frontline therapy as well as after intolerance or resistance to imatinib. Methods: 144 pts in chronic (CP) (n=63), accelerated (AP) (n=44) and blast (BP) (n=37) phase CML received nilotinib therapy. Twenty-seven pts in CP received nilotinib as frontline therapy. Quantitative reverse transcription PCR in peripheral blood samples was performed prior to nilotinib and every 3 mo thereafter. Results: The median BCR-ABL1/ABL1 ratio (%) at nilotinib start was 64.50 (range, 0.67-100), including 67.09 (range, 0.01–100) for pts in CP (100 [range, 6.87–100] for pts receiving frontline nilotinib therapy), 46.44 (range, 0.01–100) for AP, and 73.31 (range, 0.16–100) for BP. Fifty-two (45%) of 116 assessed pts (19/43 in CP, 23/40 in AP, and 10/33 in BP) harbored 26 different BCR-ABL1 mutants. The most common mutations were G250E (n=8), E355G (n=5), M351T (n=4), and T315I (n=4). The lowest PCR values for pts in CP, AP, and BP were achieved after 18 (0.20), 18 (0.88), and 18 (0.18) mos, respectively (0.1 at 12 mos for pts receiving frontline nilotinib therapy). After 24 mos of therapy, the median BCR-ABL1/ABL1 ratios for pts in CP, AP, and BP were 0.21, 6.99, and 0.05, respectively. BCR-ABL1/ABL1 ratio reductions occurred in 80 (72%) of 112 pts who had at least 2 PCR analyses during nilotinib therapy: <1-log in 22 (20%) pts (6 CP, 8 AP, 8 BP) after a median of 18 wks (range, 12 to 96); >1-log in 19 (17%) pts (10 CP, 6 AP, 3 BP) after a median of 36 wks (range, 11 to 96); >2-logs in 20 (18%) pts (12 CP, 6 AP, 2 BP) after a median of 48 wks (range, 12 to 96) and >3-logs in 19 (17%) pts (13 CP, 4 AP, 2 BP) after a median of 36 wks (range, 12 to 116). A major molecular response (MMR) was seen in 21 (19%) pts (12 CP, 7 AP, 2 BP). Nine (8%) pts achieved a complete molecular response (CMR; undetectable BCR-ABL1 transcripts), including 5 CP, 2 AP, and 2 BP. Among pts treated in CP followed for at least 6 months, MMR occurred in 7 (22%) of 32 pts treated after imatinib failure (median follow-up 23 mos) and 6 (55%) of 11 pts treated as frontline therapy (median follow-up 9 mos). CMR occurred in 2 (6%) and 3 (27%) pts, respectively. Fifty-three (47%) pts (21 CP, 22 AP, 10 BP) had at least 1 follow-up PCR analysis after their lowest transcript level, and in 7 pts the BCR-ABL1/ABL1 ratio increased >1 log (baseline mutations: M244V, G250E, wild-type, wild-type, A433T, E355G, Q252H), in 6 pts >2 logs (baseline mutation: F359V, E453K, wild-type, E355G, G250E, wild-type), and in 1 AP >3 logs (wild-type). Conclusion: Nilotinib therapy induces molecular responses in a significant number of pts both as frontline therapy and after imatinib failure. These responses can be observed across a wide variety of BCR-ABL kinase mutations. Longer follow-up is needed to define the stability and durability of MMR and CMR in these pts.

Download Full-text

Detection of BCR-ABL Mutations in Patients with Chronic Myeloid Leukemia Treated with Imatinib. Experience of One Centre.

Blood ◽

10.1182/blood.v114.22.4280.4280 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4280-4280

Author(s):

J. Valentin Garcia Gutierrez ◽

Jesùs Odriozola ◽

Javier Lopez ◽

Pilar Herrera ◽

Maria Calbacho ◽

...

Keyword(s):

Dna Sequencing ◽

Clinical Significance ◽

Conflicts Of Interest ◽

Acquired Resistance ◽

Chronic Phase ◽

Complete Response ◽

Kinase Domain ◽

Molecular Response ◽

Direct Dna Sequencing ◽

Abl Kinase

Abstract Abstract 4280 Introduction Imatinib (Imatinib) offers outstanding results in the treatment of chronic phase of chronic myeloid leukaemia (CML). However, there are still some patients who do not achieve an optimal response. There are several possible mechanisms both for primary refractoriness and for acquired resistance, but very often the blame falls in the appearance of ABL-kinase-domain (KD) mutations. Different mutations are related to variable degrees of resistance, ranging from the extreme refractoriness of the mutation T315I down to some which appear to have no clinical significance. Aims To define the clinical characteristics and outcome of patients treated in our centre, in which ABL-kinase-domain mutations through direct DNA sequencing have been analysed. Results In 24 of a total 86 patients, KD mutations were looked for. Screening for mutations was undertaken for one of three reasons: at diagnosis in 6 patients, suboptimal response (Leukemia-Net criteria) in 10 cases and Treatment-Failure (LN criteria) in 8 cases. Median time from diagnosis to ABL mutation detection was 38 months. No risk factors at diagnosis for the appearance of KD mutations have been found. Of the 7 mutations, 5 cases corresponded to treatment failures and 2 to patients in so-called suboptimal responses. One patient, after achieving a complete response to Imatinib, developed a T315I KD mutation and died due to blastic crisis despite having received second generation TKI. Another patient in failure (mutation G250E) achieved a molecular response with dasatinib. No mutations were found in 6 patients checked before treatment, but one of these developed a mutation G250E after 18 months, along with the criteria of a suboptimal response and achieved a mayor molecular response with dasatinib. Conclusions KD mutations are found in a proportion of patients in the situation of suboptimal response or failure to IM, more frequently in the latter in our experience, but the clinical significance of some of them is still unclear. On the contrary, DNA sequencing for screening at the moment of diagnosis offers little relevance in chronic phase CML. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Management of Philadelphia chromosome–positive acute lymphoblastic leukemia (Ph+ ALL)

Hematology ◽

10.1182/asheducation-2009.1.371 ◽

2009 ◽

Vol 2009 (1) ◽

pp. 371-381 ◽

Cited By ~ 52

Author(s):

Oliver G. Ottmann ◽

Heike Pfeifer

Keyword(s):

Tyrosine Kinase ◽

Second Generation ◽

Kinase Domain ◽

Tyrosine Kinase Domain ◽

Molecular Response ◽

Front Line ◽

Imatinib Resistance ◽

Term Survival ◽

Abl Tyrosine Kinase ◽

Tki Treatment

AbstractThe tyrosine kinase inhibitor (TKI) imatinib has become an integral part of front-line therapy for Ph+ ALL, with remission rates exceeding 90% irrespective of whether imatinib is given alone or combined with chemotherapy. Treatment outcome with imatinib-based regimens has improved compared with historic controls, but most patients who do not undergo allogeneic stem cell transplantation (SCT) eventually relapse. Acquired resistance on TKI treatment is associated with mutations in the bcr-abl tyrosine kinase domain in the majority of patients, and may be detected at low frequency prior to TKI treatment in a subset of patients. Second generation TKIs, eg, dasatinib and nilotinib, show activity against most of the bcr-abl tyrosine kinase domain (TKD) mutations involved in acquired imatinib resistance, but clinical benefit is generally short-lived. Accordingly, SCT in first complete remission (CR) is considered to be the best curative option. Molecular monitoring of minimal residual disease levels appears to have prognostic relevance and should be used to guide treatment. International standardization and quality control efforts are ongoing to ensure comparability of results. Mutation analysis during treatment relies increasingly on highly sensitive PCR techniques or denaturing HPLC and may assist in treatment decisions, eg, in case of molecular relapse. Results from current studies of second-generation TKI as front-line treatment for Ph+ ALL are promising and show high molecular response rates, but follow-up is still too short to determine their impact on remission duration and long-term survival. Strategies to improve outcome after SCT include the pre-emptive use of imatinib, which appears to reduce the relapse rate. In patients ineligible for transplantation, novel concepts for maintenance therapy are needed. These could involve novel immunotherapeutic interventions and combinations of TKI.

Download Full-text

Persistence of Abnormally-Spliced, Functionally-Dead BCR-ABL Variants Is a Critical Obstacle to Achieve Sustained Complete Molecular Response in CML Patients: Results of a Quantitative, Highly-Sensitive, Deep Sequencing Study

Blood ◽

10.1182/blood.v124.21.4525.4525 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4525-4525

Author(s):

Junichiro Yuda ◽

Toshihiro Miyamoto ◽

Jun Odawara ◽

Yoshikane Kikushige ◽

Yasuyuki Ohkawa ◽

...

Keyword(s):

Long Range ◽

Nested Pcr ◽

Clonal Selection ◽

Kinase Domain ◽

Transcript Variant ◽

Molecular Response ◽

Abl Kinase ◽

Transcript Variants ◽

Highly Sensitive ◽

Tki Treatment

Abstract Background: Tyrosine kinase inhibitors (TKI) have dramatically changed the treatment algorithm of chronic myeloid leukemia (CML). Despite improved outcomes in the TKI era, mutations in the BCR-ABL kinase domain are one of the major mechanisms of resistance to TKIs. In addition, recent studies have suggested that abnormally-spliced (AS) BCR-ABL transcript variants, such as retention of 35bp intronic nucleotides at Exon8/9 splice junction (BCR-ABL Ins 35bp) is one of the main obstacles for patients to exhibit "optimal response" to TKI (Gaillard JB, et al. Mol Cancer Ther 2010; T O'Hare, et al. Blood 2011). These BCR-ABL variants have a stop codon in kinase domain residues, resulting in generation of "function dead" BCR-ABL. We hypothesized that emergence of such functionally-dead BCR-ABL variants should render such CML clones insensitive to TKI, contributing to persistence of CML clone, even at molecular remission phases. Here, by combining a long-range nested PCR-amplification method and an analysis by high-throughput next generation sequencer (NGS), we have established a highly-sensitive assay system to track minimal amounts of AS BCR-ABL transcript variants. Methods: We analyzed 294 samples from 62 CML patients. Twenty-six patients were treated with Imatinib and Dasatinib, 13 with Imatinib alone, 9 with Dasatinib alone, 6 with Nilotinib alone, 6 with Imatinib and Nilotinib, and 2 with Dasatinib and Nilotinib. cDNA was synthesized from extracted RNA samples, and BCR-ABL was amplified by long-range nested PCR method. Splicing forms of BCR-ABL were then deep-sequenced by using HiSeq 1500 (illumina). Samples capable of achieving>10,000 read counts on ABL-1 Exon 8 were regarded as reliable to enroll into further statistical analysis. Results: In all 7 patients analyzed at diagnosis, CML cells have wild-type (WT) BCR-ABL but also have extremely small amount of AS BCR-ABL transcript variants. After TKI treatment, mainly two type of splicing variants became evident; the BCR-ABL Ins 35bp and Intron8-retained BCR-ABL that possesses inappropriately spliced Intron8 of ABL-1. In both cases, such transcripts fail to form functional BCR-ABL. As shown in Figure 1, along with TKIs treatment for 6 months, total amount of BCR-ABL quickly decreased, whereas percentages of AS BCR-ABL variants (BCR-ABL Ins 35bp and Intron8-retained BCR-ABL) gradually increased, suggesting that clones possessing AS BCR-ABL variants might become dominant by clonal selection through TKI treatment. In 55 patients with deep molecular response under TKI treatment for >1 year, 29 out of 55 (52%) patients achieved MR4.5, whereas the remaining 26 (48%) patients retained MR3.0, failing to achieve MR4.5. In 29 patients who have reached MR4.5, BCR-ABL variants were detected in 11 (38%) patients in any points during treatment. In contrast, within 26 patients who retained only MR3.0, 22 (85%) patients possessed BCR-ABL variants. Thus, emergence of AS BCR-ABL variants are significantly more frequent in patients retaining only MR3.0 (p<0.05). In these patients, BCR-ABL transcript variants were continually detected at several points of analysis. A representative patient who could not achieved MR4.5 is shown in Figure 2. After switching from Imatinib to Dasatinib, total amount of BCR-ABL gradually decreased, while AS BCR-ABL transcript variants did not, resulting in the poor response to Dasatinib. Discussion: Our highly-sensitive quantitation system for AS BCR-ABL variants revealed that the majority of major molecular responders (≥MR3.0) possess BCR-ABL variants, but these are more frequently found in patients failed to achieve MR4.5 (P<0.05). These data strongly suggest that selective persistence of AS BCR-ABL variants is one of the key mechanisms of TKI resistance in major molecular responders. CML clones with kinase-dead BCR-ABL should not be sensitive to TKI, should not be addicted to ABL kinase activity, and should therefore be difficult to be killed by TKI alone. Thus, quantitation of AS BCR-ABL variants might be critical to predict clinical outcomes of CML patients, for example, whether or not they relapse after TKI cessation. Figure 1. Molecular kinetics in early reduction of BCR-ABL after TKIs. Figure 1. Molecular kinetics in early reduction of BCR-ABL after TKIs. Figure 2. Transition of AS BCR-ABL transcript variant in case who achieved MR3.0 but not MR4.5 under TKIs treatment for more than 1-year. Figure 2. Transition of AS BCR-ABL transcript variant in case who achieved MR3.0 but not MR4.5 under TKIs treatment for more than 1-year. Disclosures No relevant conflicts of interest to declare.

Download Full-text

ABL Kinase Domain Mutations in Iranian Chronic Myeloid Leukemia Patients with Resistance to Tyrosine Kinase Inhibitors

Laboratory Medicine ◽

10.1093/labmed/lmaa052 ◽

2020 ◽

Author(s):

Mahboobeh Shojaei ◽

Hamid Rezvani ◽

Azita Azarkeivan ◽

Behzad Poopak

Keyword(s):

Chronic Myeloid Leukemia ◽

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitors ◽

Myeloid Leukemia ◽

Kinase Inhibitors ◽

Kinase Domain ◽

Response To Therapy ◽

Molecular Response ◽

Abl Kinase ◽

Kinase Domain Mutations

Abstract Objective Tyrosine kinase inhibitors (TKIs) are considered standard first-line treatment in patients with chronic myeloid leukemia. Because ABL kinase domain mutations are the most common causes of treatment resistance, their prevalence and assessment during treatment may predict subsequent response to therapy. Methods The molecular response in Bcr-Abl1IS was tested via quantitative real-time polymerase chain reaction. We used the direct sequencing technique to discover the mutations in the ABL kinase domain. The IRIS trial established a standard baseline for measurement – (100% BCR-ABL1 on the ‘international scale’) and a major molecular response (good response to therapy) was defined as a 3-log reduction in the amount of BCR-ABL1 – 0.1% BCR-ABL1 on the international scale. Results We observed 11 different mutations in 13 patients, including E255K, which had the highest mutation rate. A lack of hematologic response was found in 22 patients, who showed a significantly higher incidence of mutations. Conclusion Detection of kinase domain mutations is a reliable method for choosing the best treatment strategy based on patients’ conditions, avoiding ineffective treatments, and running high-cost protocols in patients with acquired resistance to TKIs.

Download Full-text

Long-Term Mutation Follow-up of Philadelphia-Chromosome Positive Leukemia Patients Treated with Second-Generation Tyrosine Kinase Inhibitors after Imatinib Failure Shows That Newly Acquired Bcr-Abl Kinase Domain Mutations Leading to Relapse Are Mainly Detected during the First Year.

Blood ◽

10.1182/blood.v112.11.2118.2118 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2118-2118 ◽

Cited By ~ 1

Author(s):

Simona Soverini ◽

Alessandra Gnani ◽

Sabrina Colarossi ◽

Fausto Castagnetti ◽

Francesca Palandri ◽

...

Keyword(s):

Tyrosine Kinase ◽

Median Time ◽

Kinase Inhibitors ◽

Initial Response ◽

Kinase Domain ◽

Abl Kinase ◽

Imatinib Resistant ◽

Tki Treatment ◽

Selection Of

Abstract Resistance to imatinib in Philadelphia-positive (Ph+) leukemia patients is often associated with selection of point mutations in the Bcr-Abl kinase domain (KD). Dasatinib and nilotinib are second-generation tyrosine kinase inhibitors (TKIs) with different binding modes with respect to imatinib, that have been shown to confer in vitro and in vivo activity against many Bcr-Abl mutated forms. However, both dasatinib and nilotinib have been shown to retain some ‘Achilles heels’, and they include both imatinib-resistant mutations (e.g., T315I) and some novel, inhibitor-specific ones. Selection of either type of KD mutations has frequently been observed in patients (pts) who relapse after an initial response to dasatinib or nilotinib and represents one of the major hurdles on the road to successful treatment of imatinib-resistant pts. We have monitored Abl KD mutation status in a total of 121 pts who received dasatinib (n= 78) or nilotinib (n=43) as 2nd TKI after imatinib failure since February 2005. Fifty-eight (48%) pts had chronic phase (CP) chronic myelogenous leukemia (CML), 63 pts (52%) had accelerated phase (AP) or blast crisis (BC) CML or Ph+ acute lymphoblastic leukemia (ALL). Median age was 55 years (range, 18–76); median time from diagnosis was 49 months (range, 4–181); median time on imatinib was 32 months (range, 4–66). Median follow-up of all pts who received a 2nd TKI is 7 months (range, 1–38). Median follow-up of pts who are still on 2nd TKI treatment is 32 months (range, 28–38). Relapses after an initial response have so far been observed in 46/121 pts. Thirty-eight out of these 46 pts had AP/BC CML or Ph+ ALL at the time 2nd TKI was started. Forty-one out of 121 (34%) pts have experienced relapse after an initial response during the first 12 months of 2nd TKI treatment (median time to relapse, 6,5 months; range 4–12 months), while only five of the 45 (11%) pts who were still on 2nd TKI treatment after >12 months have relapsed (at 13, 15, 18, 20 and 33 months, respectively). Interestingly, none of these 5 pts had never achieved more than a minor cytogenetic response (CgR), and 4/5 pts were receiving a reduced TKI dose because of toxicity. In 36/46 (78%) cases, relapse was associated with newly acquired Abl KD mutations. In particular 26/30 (87%) pts who relapsed on dasatinib and 10/16 (63%) pts who relapsed on nilotinib had evidence of a newly acquired KD mutation presumably responsible for treatment failure. Newly acquired mutations in pts who relapsed on dasatinib as 2nd TKI were T315I (n= 12 pts) F317L (n= 8 pts) T315A (n=3 pts); V299L (n=3 pts); F317I (n=2 pts); 2 pts had multiple mutations. Newly acquired mutations in pts who relapsed on nilotinib as 2nd TKI were E255K (n=3); E255V (n=2); Y253H (n=2); T315I (n=1); F359V (n=1); F359C (n=1). Sixteen pts (but none of those harboring the T315I) switched to dasatinib or nilotinib or high-dose imatinib as 3rd TKI and this rescued hematologic or even cytogenetic responses in a proportion of cases. Our observations suggest that: newly acquired mutations leading to relapse in Ph+ leukemia pts receiving dasatinib or nilotinib as 2nd TKI usually arise rapidly; the likelihood of mutation selection consistently decreases over time, and seems mainly confined to advanced phase pts and to pts with no or minor CgR; almost all (87%) cases who developed resistance to dasatinib had newly acquired KD mutations - suggesting that the higher potency with respect to imatinib can overcome Bcr-Abl gene amplification and that Src kinase inhibition may turn off Bcr- Abl-independent resistance mechanisms; a lower incidence (63%) of newly acquired KD mutations was observed in pts who developed resistance to nilotinib; with the exception of T315I, there is little if no overlap between dasatinib and nilotinib-resistant mutants, which may allow to regain responses by switching TKIs.

Download Full-text

Low-Level Bcr-Abl Kinase Domain Mutations Are Very Rare In Chronic Myeloid Leukemia Patients Who Are In Major Molecular Response After 12 Months of First-Line Nilotinib Therapy.

Blood ◽

10.1182/blood.v116.21.1666.1666 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1666-1666

Author(s):

Simona Soverini ◽

Angela Poerio ◽

Caterina Debenedittis ◽

Ilaria Iacobucci ◽

Sabrina Colarossi ◽

...

Keyword(s):

Screening Method ◽

Working Party ◽

Kinase Domain ◽

Molecular Response ◽

Healthy Individuals ◽

Major Molecular Response ◽

Low Level ◽

Log Reduction ◽

Abl Kinase

Abstract Abstract 1666 Bcr-Abl kinase domain (KD) mutations have been documented in a variable proportion of residual Ph+ cells in some patients (pts) with stable response to imatinib. In general, the higher the sensitivity of the screening method, the greater the number of pts found to harbour mutations – although the clinical significance of low-level mutations is still controversial. Nilotinib is an imatinib derivative more selective and more potent in Bcr-Abl inhibition – with reported major molecular response (MMR) rates at 12 months ranging between 44 and 85%. We wondered whether low-level Bcr-Abl KD mutations are detectable in pts in MMR on nilotinib. To address this issue, we retrospectively analyzed the samples collected after 12 months of therapy from 12 pts enrolled on the GIMEMA CML working party study of nilotinib 400 mg BID as frontline treatment of CML. These pts had all achieved MMR (>3-log reduction in Bcr-Abl transcript according to the IS) between 3 and 6 months from nilotinib start and had a Bcr-Abl/Abl ratio ranging from 0.009%IS and 0.02%IS at the time of analysis. In all 12 pts, MMR was maintained at last follow up (24 months after nilotinib start), with four pts achieving complete molecular response (CMR; >4.5-log reduction) at 18 months and one at 24 months. Pts were equally distributed across Sokal risk categories (low Sokal risk, n=4; intermediate Sokal risk, n=4; high Sokal risk, n=4). Screening for low level mutations was performed by cloning the Bcr-Abl KD (a.a.240-502) in a bacterial vector and sequencing 150 independent clones for each patient. To rule out false positive results, a mutation was considered to be present in a sample if it was detected on both strands of two or more independent clones. The KD of Abl in 3 healthy individuals was analyzed in parallel. Our approach showed evidence of Bcr-Abl KD mutations in only 1 out of 12 pts analyzed. In this high Sokal risk patient, a Q346L mutation was detected in 3/150 independent clones, and an additional T315I mutation was present in 2 out of these 3 clones. The Q346L has never been reported in imatinib-resistant pts, neither is it among the mutants emerged in the in vitro random mutagenesis screenings for nilotinib-resistant mutations – hence it should be devoid of any clinical relevance. The T315I, in contrast, is known to be highly insensitive to nilotinib both in vitro and in vivo. Nevertheless, in this patient the Bcr-Abl transcript level continued to decline down to CMR (achieved after 24 months from nilotinib start). It may also be hypothesized that the Q346L-T315I double mutant may have decreased degree of resistance with respect to the T315I mutant. The remaining 11 pts scored negative for mutations – showing only evidence of some single, mutated clones as also the three healthy individuals did. To increase the number of pts analyzed and assess whether high Sokal risk pts are more prone to develop low level mutations, we have now set up a massively parallel amplicon sequencing approach of the Abl KD on the Roche 454 GS FLX instrument, allowing to increase both throughput and sensitivity (a 100.000x coverage will allow to detect mutations with a lower detection limit of 0.01%). Our current results suggest that a) low level Bcr-Abl KD mutations seem to be very rare in pts in MMR after 12 months of nilotinib therapy, a milestone achieved by the majority of pts. This may be due to the higher efficacy of nilotinib resulting in a more rapid clearance of the reservoir of Ph+ cells where mutations arise; b) as hypothesized by some authors, tyrosine kinase inhibitor-resistant mutations at low levels do not always predict for subsequent relapse and should not trigger changes in therapy. Supported by: European LeukemiaNet, AIL, AIRC, PRIN 2008, Fondazione del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integrated program (PIO), Programma di Ricerca Regione – Università 2007 – 2009. Disclosures: Rosti: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Baccarani:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Honoraria; Pfizer: Consultancy.

Download Full-text