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Assessment of individual molecular response in chronic myeloid leukemia patients with atypical BCR-ABL1 fusion transcripts: recommendations by the EUTOS cooperative network.
Approximately 1-2% of chronic myeloid leukemia (CML) patients harbor atypical BCR-ABL1 transcripts that cannot be monitored by real-time quantitative PCR (RT-qPCR) using standard methodologies. Within the European Treatment and Outcome Study (EUTOS) for CML we established and validated robust RT-qPCR methods for these patients. BCR-ABL1 transcripts were amplified and sequenced to characterize the underlying fusion. Residual disease monitoring was carried out by RT-qPCR with specific primers and probes using serial dilutions of appropriate BCR-ABL1 and GUSB plasmid DNA calibrators. Results were expressed as log reduction of the BCR-ABL1/GUSB ratio relative to the patient-specific baseline value and evaluated as an individual molecular response (IMR). In total, 330 blood samples (2-34 per patient, median 8) from 33 CML patients (19 male, median age 62 years) were analyzed. Patients expressed seven different atypical BCR-ABL1 transcripts (e1a2, n = 6; e6a2, n = 1; e8a2, n = 2; e13a3, n = 4; e14a3, n = 6; e13a3/e14a3, n = 2; e19a2, n = 12). Most patients (61%) responded well to TKI therapy and achieved an IMR of at least one log reduction 3 months after diagnosis. Four patients relapsed with a significant increase of BCR-ABL1/GUSB ratios. Characterization of atypical BCR-ABL1 transcripts is essential for adequate patient monitoring and to avoid false-negative results. The results cannot be expressed on the International Scale (IS) and thus the common molecular milestones and guidelines for treatment are difficult to apply. We, therefore, suggest reporting IMR levels in these cases as a time-dependent log reduction of BCR-ABL1 transcript levels compared to baseline prior to therapy.
Publication Date: 2021-03-08
Journal: Journal of cancer research and clinical oncology

Dasatinib induces endothelial-to-mesenchymal transition in human vascular-endothelial cells: counteracted by cotreatment with bosutinib.
Adverse vascular events have become a serious clinical problem in chronic myeloid leukemia (CML) patients who receive certain BCR/ABL1 tyrosine kinase inhibitors (TKIs). Studies have shown that endothelial-to-mesenchymal transition (EndMT) can contribute to various vascular diseases. We investigated the effects of TKIs on the development of EndMT in human vascular-endothelial cells (VECs). Exposure of VECs to dasatinib, but not to other TKIs, produced a significant increase in the formation of spindle-shaped cells. This effect was accompanied by a significant increase in expression of the EndMT inducer transforming growth factor-β (TGF-β) and mesenchymal markers vimentin, smooth muscle alpha-actin, and fibronectin, as well as a significant decrease in expression of vascular-endothelial markers CD31 and VE-cadherin attributable at least in part to activation of ERK signaling. Inhibitors of TGF-β and ERK partially attenuated dasatinib-induced EndMT. Interestingly, bosutinib efficiently counteracted dasatinib-induced EndMT and attenuated dasatinib-induced phosphorylation of ERK. Taken together, these results show that dasatinib induces EndMT, which might contribute to the development of vascular toxicity, such as the pulmonary hypertension observed in CML patients receiving dasatinib. Bosutinib could play a distinct role in protecting VECs from EndMT.
Publication Date: 2021-01-05
Journal: International journal of hematology

RT-qPCR versus Digital PCR: How Do They Impact Differently on Clinical Management of Chronic Myeloid Leukemia Patients?
Real-time quantitative PCR (RT-qPCR) is the gold standard to quantify the BCR-ABL1 transcript for molecular response monitoring in chronic myeloid leukemia (CML) patients, and it plays a pivotal role in clinical decision-making process, even if it presents technical limits. Increasing data suggest that digital PCR (dPCR) is more accurate and reliable than RT-qPCR in CML minimal residual disease monitoring and in patients' selection for treatment discontinuation. But what about the identification of treatment discontinuation failures? We present the case of a CML patient enrolled both in a study aiming to comparatively assess molecular response by RT-qPCR and dPCR and in the progressive arm of the OPTkIMA trial. This is a phase III trial including CML patients randomized to receive a fixed versus a progressive intermittent tyrosine kinase inhibitor regimen. At 24 months, because of two consecutive detections of MR2.0 by RT-qPCR, the patient resumed daily treatment. Conversely, dPCR revealed a stability of molecular response and even a slight decreasing of transcript over time. An additional specimen was sampled one month after the first MR2.0 detection because of clinical decision: RT-qPCR resulted MR3.0 and dPCR confirmed the transcript's stability. Nowadays, the resumption of therapy is RT-qPCR-driven despite its limits in detection and robustness. In this case, according to dPCR, the patient could have continued intermittent treatment and the stability of response was then confirmed by RT-qPCR. So, dPCR could be able to better identify peculiar clinical response to therapy.
Publication Date: 2020-12-01
Journal: Case reports in oncology

Programme for Harmonization to the International Scale in Latin America for BCR-ABL1 quantification in CML patients: findings and recommendations.
Objectives The quantitation of BCR-ABL1 mRNA is mandatory for chronic myeloid leukemia (CML) patients, and RT-qPCR is the most extensively used method in testing laboratories worldwide. Nevertheless, substantial variation in RT-qPCR results makes inter-laboratory comparability hard. To facilitate inter-laboratory comparative assessment, an international scale (IS) for BCR-ABL1 was proposed. Methods The laboratory-specific conversion factor (CF) to the IS can be derived from the World Health Organization (WHO) genetic reference panel; however, this material is limited to the manufacturers to produce and calibrate secondary reference reagents. Therefore, we developed secondary reference calibrators, as lyophilized cellular material, aligned to the IS. Our purpose was both to re-evaluate the CF in 18 previously harmonized laboratories and to propagate the IS to new laboratories. Results Our field trial including 30 laboratories across Latin America showed that, after correction of raw BCR-ABL1/ABL1 ratios using CF, the relative mean bias was significantly reduced. We also performed a follow-up of participating laboratories by annually revalidating the process; our results support the need for continuous revalidation of CFs. All participating laboratories also received a calibrator to determine the limit of quantification (LOQ); 90% of them could reproducibly detect BCR-ABL1, indicating that these laboratories can report a consistent deep molecular response. In addition, aiming to investigate the variability of BCR-ABL1 measurements across different RNA inputs, we calculated PCR efficiency for each individual assay by using different amounts of RNA. Conclusions In conclusion, for the first time in Latin America, we have successfully organized a harmonization platform for BCR-ABL1 measurement that could be of immediate clinical benefit for monitoring the molecular response of patients in low-resource regions.
Publication Date: 2020-05-07
Journal: Clinical chemistry and laboratory medicine

Prospective assessment of NGS-detectable mutations in CML patients with nonoptimal response: the NEXT-in-CML study.
In chronic myeloid leukemia (CML) patients, tyrosine kinase inhibitors (TKIs) may select for drug-resistant BCR-ABL1 kinase domain (KD) mutants. Although Sanger sequencing (SS) is considered the gold standard for BCR-ABL1 KD mutation screening, next-generation sequencing (NGS) has recently been assessed in retrospective studies. We conducted a prospective, multicenter study (NEXT-in-CML) to assess the frequency and clinical relevance of low-level mutations and the feasibility, cost, and turnaround times of NGS-based BCR-ABL1 mutation screening in a routine setting. A series of 236 consecutive CML patients with failure (n = 124) or warning (n = 112) response to TKI therapy were analyzed in parallel by SS and NGS in 1 of 4 reference laboratories. Fifty-one patients (22 failure, 29 warning) who were negative for mutations by SS had low-level mutations detectable by NGS. Moreover, 29 (27 failure, 2 warning) of 60 patients who were positive for mutations by SS showed additional low-level mutations. Thus, mutations undetectable by SS were identified in 80 out of 236 patients (34%), of whom 42 (18% of the total) had low-level mutations somehow relevant for clinical decision making. Prospective monitoring of mutation kinetics demonstrated that TKI-resistant low-level mutations are invariably selected if the patients are not switched to another TKI or if they are switched to a inappropriate TKI or TKI dose. The NEXT-in-CML study provides for the first time robust demonstration of the clinical relevance of low-level mutations, supporting the incorporation of NGS-based BCR-ABL1 KD mutation screening results in the clinical decision algorithms.
Publication Date: 2019-12-27
Journal: Blood

Next-generation sequencing for BCR-ABL1 kinase domain mutation testing in patients with chronic myeloid leukemia: a position paper.
BCR-ABL1 kinase domain (KD) mutation status is considered to be an important element of clinical decision algorithms for chronic myeloid leukemia (CML) patients who do not achieve an optimal response to tyrosine kinase inhibitors (TKIs). Conventional Sanger sequencing is the method currently recommended to test BCR-ABL1 KD mutations. However, Sanger sequencing has limited sensitivity and cannot always discriminate between polyclonal and compound mutations. The use of next-generation sequencing (NGS) is increasingly widespread in diagnostic laboratories and represents an attractive alternative. Currently available data on the clinical impact of NGS-based mutational testing in CML patients do not allow recommendations with a high grade of evidence to be prepared. This article reports the results of a group discussion among an ad hoc expert panel with the objective of producing recommendations on the appropriateness of clinical decisions about the indication for NGS, the performance characteristics of NGS platforms, and the therapeutic changes that could be applied based on the use of NGS in CML. Overall, these recommendations might be employed to inform clinicians about the practical use of NGS in CML.
Publication Date: 2019-12-06
Journal: Journal of hematology & oncology

Molecular Mechanisms of Resistance to Tyrosine Kinase Inhibitors.
Chronic myeloid leukemia (CML) patients with constitutive activity of BCR-ABL1 oncoprotein frequently derive significant clinical benefits from tyrosine kinase inhibitors (TKIs). Point mutations in the ABL1 kinase domain (KD) are an important mechanism of TKI resistance in CML. In this review, we present molecular mechanisms of TKI resistance paying particular attention to drug resistance which allows for a survival advantage in CML. Sensitive disease monitoring is a required standard of care for management of CML. Screening of these mutations fail to explain 20-40% of resistant cases where activation of different survival pathways must be the main reason for resistance. Eliminating TKI resistance appears to be the most successful therapeutic way to decrease leukemic disease burden and potentiate cure. Advances on novel strategies for identifying and confronting drug resistance are rapidly altering management of CML that are resistant to TKI and expanding the landscape of available therapies.
Publication Date: 2019-08-30
Journal: Current hematologic malignancy reports

Clinical characteristics and prognostic significance of chronic myeloid leukemia with rare BCR-ABL1 transcripts.
The prognostic significance of rare BCR-ABL1 transcripts is uncertain in the tyrosine kinase inhibitor (TKI) era. In this retrospective study, 40 (1.7%) patients with rare BCR-ABL1 transcripts were identified from a cohort of 2331 chronic myeloid leukemia (CML) patients; 4 types of rare transcripts were identified, including e1a2 (0.9%), e19a2 (0.4%), e13a3 (0.1%), and e14a3 (0.3%). Compared to patients with the typical transcript, those with the e1a2 transcript had an inferior response to TKIs and a worse outcome. Patients with the e19a2 transcript had a high rate of early optimal response to TKIs, but most of them later lost the complete cytogenetic response (CCyR) due to BCR-ABL1 mutations, resulting in a poor prognosis. Patients with the e13a3/e14a3 transcript responded well to TKIs and had a good outcome. These findings indicate that the type of BCR-ABL1 transcript should be considered when determining the treatment for CML patients in the TKI era.
Publication Date: 2019-07-02
Journal: Leukemia & lymphoma

Prevalence and outcomes of uncommon BCR-ABL1 fusion transcripts in patients with chronic myeloid leukaemia: data from a single centre.
To explore the type, prevalence and outcomes in chronic myeloid leukaemia (CML) patients with uncommon BCR-ABL1 transcripts in the era of tyrosine kinase inhibitors (TKIs), uncommon BCR-ABL1 transcripts were screened in 4750 patients by multiplex polymerase chain reaction (PCR), and type-specific real-time quantitative PCR was regularly performed for molecular monitoring. A total of 19 uncommon transcripts, including e1a2, e1a3, e6a2, e8a2, e12a2, unusual e13a2, e13a3, unusual e14a2, e14a3 and e19a2 were identified in 83 (1·7%) patients. The three most frequent types were e19a2, e13a3/e14a3 and e1a2. Compared with the 571 newly diagnosed CML patients in chronic phase with common e13a2/e14a2 transcripts receiving frontline imatinib therapy, patients with the e19a2 (n = 16) and e1a2 (n = 11) transcripts had significantly reduced probabilities of 1-year complete cytogenetic response (CCyR, P = 0·0004 and 0·016) and major molecular response (MMR, P = 0·0018 and 0·0035), and patients with the e13a3/e14a3 transcript (n = 10) had significantly increased probabilities of 1-year CCyR (P = 0·0072) and MMR (P = 0·0073). Patients with the e19a2 transcript had low probabilities of 2-year event-free survival (EFS, P = 0·0004) and progression-free survival (P = 0·0067), and patients with the e1a2 transcript had low probability of 2-year EFS (P < 0·0001). Therefore, uncommon BCR-ABL1 fusion transcripts are rare and diverse in patients with CML and may be relevant for TKI therapy outcomes.
Publication Date: 2018-07-06
Journal: British journal of haematology

The BCR-ABL1 Inhibitors Imatinib and Ponatinib Decrease Plasma Cholesterol and Atherosclerosis, and Nilotinib and Ponatinib Activate Coagulation in a Translational Mouse Model.
Treatment with the second and third generation BCR-ABL1 tyrosine kinase inhibitors (TKIs) increases cardiovascular risk in chronic myeloid leukemia (CML) patients. We investigated the vascular adverse effects of three generations of TKIs in a translational model for atherosclerosis, the APOE*3Leiden.CETP mouse. Mice were treated for sixteen weeks with imatinib (150 mg/kg BID), nilotinib (10 and 30 mg/kg QD) or ponatinib (3 and 10 mg/kg QD), giving similar drug exposures as in CML-patients. Cardiovascular risk factors were analyzed longitudinally, and histopathological analysis of atherosclerosis and transcriptome analysis of the liver was performed. Imatinib and ponatinib decreased plasma cholesterol (imatinib, -69%,
Publication Date: 2018-06-28
Journal: Frontiers in cardiovascular medicine

Evaluation of cardiovascular ischemic event rates in dasatinib-treated patients using standardized incidence ratios.
With high survival rates for chronic myeloid leukemia (CML) patients treated with BCR-ABL1 tyrosine kinase inhibitors (TKIs), emerging consequences, such as arterial ischemic events, require consideration when evaluating treatment options. Cardiovascular ischemic event incidence in clinical trials was evaluated in 2712 dasatinib-treated patients with Philadelphia chromosome-positive (Ph+) leukemias from 11 first- and second-line trials (pooled), newly diagnosed CML patients treated with dasatinib or imatinib (DASISION), and prostate cancer patients treated with dasatinib or placebo plus docetaxel/prednisone (READY). Overall, 2-4% of dasatinib-treated patients had cardiovascular ischemic events. Most dasatinib-treated patients with an event had a history of and/or risk factor for atherosclerosis (pooled 77 with history/risk and event/96 with events; DASISION 8/10; READY 15/18). Most cardiovascular ischemic events occurred within 1 year of initiating dasatinib (pooled 69/96; DASISION 7/10; READY 16/18). Comparison of observed and expected event rates through standardized incidence ratios indicates that dasatinib does not increase risk for cardiovascular ischemic events compared with external reference populations.
Publication Date: 2017-05-24
Journal: Annals of hematology

A BCR-ABL1 cutoff of 1.5% at 3 months, determined by the GeneXpert system, predicts an optimal response in patients with chronic myeloid leukemia.
In chronic myeloid leukemia (CML) patients, 3-month BCR-ABL1 levels have consistently been correlated with further outcomes. Monitoring molecular responses in CML using the GeneXpert (Cepheid) platform has shown an optimal correlation with standardized RQ-PCR (IS) when measuring BCR-ABL1 levels lower than 10%, as it is not accurate for values over 10%. The aim of the present study was to determine the predictive molecular value at three months on different outcome variables using the Xpert BCR-ABL1 MonitorTM assay (Xpert BCR-ABL1). We monitored 125 newly diagnosed consecutive CML patients in the chronic phase (CML-CP) using an automated method: Xpert BCR-ABL1. Only 5% of patients did not achieve an optimal response at 3 months, and the 10% BCR-ABL1 cutoff defined by RQ-PCR (IS) methods was unable to identify significant differences in the probabilities of achieving a complete cytogenetic response (CCyR) (50% vs. 87%, p = 0.1) or a major molecular response (MMR) (60% vs. 80%, p = 0.29) by 12 months. In contrast, a cutoff of 1.5% more accurately identified differences in the probabilities of achieving CCyR (98% vs. 54%, p<0.001) and MMR (88% vs. 56%, p<0.001) by 12 months, as well as probabilities of treatment changes (p = 0.005). Therefore, when using the Xpert BCR-ABL1 assay, a cutoff of 1.5% at 3 months could with high probability identify patients able to achieve an optimal response at 12 months.
Publication Date: 2017-03-10
Journal: PloS one

Chronic myelogenous leukemia with acquired t(11;14)(q13;q32) CCND1-IGH: A case report and literature review.
Approximately 5-10% of chronic myeloid leukemia (CML) patients are found to have structural or numerical additional chromosomal abnormality (ACAs) in addition to the characteristic t(9;22)(q34;q11.2) BCR/ABL1 at the time of diagnosis. The prognostic significance of such additional chromosomal abnormalities has been controversial. Translocation t(11;14)(q13;q32) CCND1-IGH is typically associated with mantle cell lymphoma or a subset of plasma cell myeloma and is exceedingly rare in myeloid neoplasm. Here we report a unique case describing a patient found at diagnosis of chronic phase CML to have both the Philadelphia chromosome as well as t(11;14)-a rare cytogenetic combination. The patient was treated with imatinib with appropriate hematologic response but persistent disease by FISH and RT-PCR. She was switched to dasatinib and eventually achieved cytogenetic remission in both translocations, but still with persistent RT-PCR evidence of BCR-ABL1 fusion. As cyclin D1 is a regulatory subunit of cyclin-dependent kinases CDK4 and CDK6 and is required for the cells to progress through the G1 phase of the cell cycle, overexpression of cyclin D1 will likely promote cells into cell cycle. This may further augment proliferation in addition to upregulated ABL1 kinase activity in the index case. It may also contribute to the resistance to imatinib, as imatinib only targets on BCR-ABL fusion. Therefore, the addition of t(11;14)(q13;q32) may have significant implication in patient management.
Publication Date: 2016-11-05
Journal: Cancer genetics

In chronic myeloid leukemia patients on second-line tyrosine kinase inhibitor therapy, deep sequencing of BCR-ABL1 at the time of warning may allow sensitive detection of emerging drug-resistant mutants.
Imatinib-resistant chronic myeloid leukemia (CML) patients receiving second-line tyrosine kinase inhibitor (TKI) therapy with dasatinib or nilotinib have a higher risk of disease relapse and progression and not infrequently BCR-ABL1 kinase domain (KD) mutations are implicated in therapeutic failure. In this setting, earlier detection of emerging BCR-ABL1 KD mutations would offer greater chances of efficacy for subsequent salvage therapy and limit the biological consequences of full BCR-ABL1 kinase reactivation. Taking advantage of an already set up and validated next-generation deep amplicon sequencing (DS) assay, we aimed to assess whether DS may allow a larger window of detection of emerging BCR-ABL1 KD mutants predicting for an impending relapse. a total of 125 longitudinal samples from 51 CML patients who had acquired dasatinib- or nilotinib-resistant mutations during second-line therapy were analyzed by DS from the time of failure and mutation detection by conventional sequencing backwards. BCR-ABL1/ABL1%(IS) transcript levels were used to define whether the patient had 'optimal response', 'warning' or 'failure' at the time of first mutation detection by DS. DS was able to backtrack dasatinib- or nilotinib-resistant mutations to the previous sample(s) in 23/51 (45 %) pts. Median mutation burden at the time of first detection by DS was 5.5 % (range, 1.5-17.5 %); median interval between detection by DS and detection by conventional sequencing was 3 months (range, 1-9 months). In 5 cases, the mutations were detectable at baseline. In the remaining cases, response level at the time mutations were first detected by DS could be defined as 'Warning' (according to the 2013 ELN definitions of response to 2nd-line therapy) in 13 cases, as 'Optimal response' in one case, as 'Failure' in 4 cases. No dasatinib- or nilotinib-resistant mutations were detected by DS in 15 randomly selected patients with 'warning' at various timepoints, that later turned into optimal responders with no treatment changes. DS enables a larger window of detection of emerging BCR-ABL1 KD mutations predicting for an impending relapse. A 'Warning' response may represent a rational trigger, besides 'Failure', for DS-based mutation screening in CML patients undergoing second-line TKI therapy.
Publication Date: 2016-08-04
Journal: BMC cancer

Quantitative Analysis of Mutant Subclones in Chronic Myeloid Leukemia: Comparison of Different Methodological Approaches.
Identification and quantitative monitoring of mutant BCR-ABL1 subclones displaying resistance to tyrosine kinase inhibitors (TKIs) have become important tasks in patients with Ph-positive leukemias. Different technologies have been established for patient screening. Various next-generation sequencing (NGS) platforms facilitating sensitive detection and quantitative monitoring of mutations in the ABL1-kinase domain (KD) have been introduced recently, and are expected to become the preferred technology in the future. However, broad clinical implementation of NGS methods has been hampered by the limited accessibility at different centers and the current costs of analysis which may not be regarded as readily affordable for routine diagnostic monitoring. It is therefore of interest to determine whether NGS platforms can be adequately substituted by other methodological approaches. We have tested three different techniques including pyrosequencing, LD (ligation-dependent)-PCR and NGS in a series of peripheral blood specimens from chronic myeloid leukemia (CML) patients carrying single or multiple mutations in the BCR-ABL1 KD. The proliferation kinetics of mutant subclones in serial specimens obtained during the course of TKI-treatment revealed similar profiles via all technical approaches, but individual specimens showed statistically significant differences between NGS and the other methods tested. The observations indicate that different approaches to detection and quantification of mutant subclones may be applicable for the monitoring of clonal kinetics, but careful calibration of each method is required for accurate size assessment of mutant subclones at individual time points.
Publication Date: 2016-05-03
Journal: International journal of molecular sciences

A role for FOXO1 in BCR-ABL1-independent tyrosine kinase inhibitor resistance in chronic myeloid leukemia.
Chronic myeloid leukemia (CML) patients who relapse on imatinib due to acquired ABL1 kinase domain mutations are successfully treated with second-generation ABL1-tyrosine kinase inhibitors (ABL-TKIs) such as dasatinib, nilotinib or ponatinib. However, ~40% of relapsed patients have uncharacterized BCR-ABL1 kinase-independent mechanisms of resistance. To identify these mechanisms of resistance and potential treatment options, we generated ABL-TKI-resistant K562 cells through prolonged sequential exposure to imatinib and dasatinib. Dual-resistant K562 cells lacked BCR-ABL1 kinase domain mutations, but acquired other genomic aberrations that were characterized by next-generation sequencing and copy number analyses. Proteomics showed that dual-resistant cells had elevated levels of FOXO1, phospho-ERK and BCL-2, and that dasatinib no longer inhibited substrates of the PI3K/AKT pathway. In contrast to parental cells, resistant cells were sensitive to growth inhibition and apoptosis induced by the class I PI3K inhibitor, GDC-0941 (pictilisib), which also induced FOXO1 nuclear translocation. FOXO1 was elevated in a subset of primary specimens from relapsed CML patients lacking BCR-ABL1 kinase domain mutations, and these samples were responsive to GDC-0941 treatment ex vivo. We conclude that elevated FOXO1 contributes to BCR-ABL1 kinase-independent resistance experienced by these CML patients and that PI3K inhibition coupled with BCR-ABL1 inhibition may represent a novel therapeutic approach.
Publication Date: 2016-04-06
Journal: Leukemia

Co-occurrence of Myeloproliferative Neoplasms and Solid Tumors Is Attributed to a Synergism Between Cytoreductive Therapy and the Common TERT Polymorphism rs2736100.
The germline telomerase reverse transcriptase (TERT) rs2736100_C variant was identified as a susceptibility factor for a variety of solid tumors and recently for myeloproliferative neoplasms (MPN). LightCycler melting curve analysis was applied to detect risk alleles of TERT rs2736100_C and Janus kinase 2 (JAK2) rs12343867_C tagging 46/1 haplotype in 584 BCR-ABL1-negative MPN, 308 acute, and 86 chronic myeloid leukemia (AML and CML) patients and 400 healthy individuals. TERT rs2736100_C showed an increased allele frequency in BCR-ABL1-negative MPN patients compared with controls (62.7%±2.8% vs. 48.8%±3.5%, P < 0.0001) regardless of molecular background or disease type, but not in CML or AML. Combined TERT and JAK2 hetero- or homozygosity conferred even higher risk for classic MPN. Common complications (thrombosis, myelofibrosis, or leukemia) were not associated with the TERT variant; however, adverse survival was noted in TERT variant carrier polycythemia vera patients. MPN patients with the TERT CC genotype had a higher probability (44.4%) to die from solid tumors compared with TERT AC/AA individuals (5.3%; P = 0.004). TERT rs2736100_C carriers had increased risk of solid tumors independently from cytoreductive treatment [3.08 (1.03-9.26), P = 0.045]. TERT rs2736100_C polymorphism predisposes to the development of BCR-ABL1-negative MPN with the co-occurrence of solid tumors, especially with the usage of cytoreductive treatment. The high frequency of TERT variant in the classic MPN population highlights the importance of the avoidance of long-term cytoreductive treatment in MPN patients.
Publication Date: 2015-10-22
Journal: Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology

Molecular characterization and follow-up of five CML patients with new BCR-ABL1 fusion transcripts.
We report five chronic myeloid leukaemia (CML) patients in whom we identified and characterized undescribed BCR-ABL1 fusion transcripts. We investigated the precise features of the molecular rearrangements and the minimal residual disease follow-up for these five patients. Three resulted from new rearrangements between the BCR and ABL1 sequences (the breakpoints being located within BCR exon 13 in two cases and within BCR exon 18 in one case). The other two cases revealed a complex e8-[ins]-a2 fusion transcript involving a third partner gene, PRDM12 and SPECC1L, respectively. Moreover, single nucleotide polymorphism-array analysis performed in the latter two cases showed copy number alterations shared by the two patients, thus identifying genes that were deleted during rearrangement and suggesting their potential role in CML pathogenesis. Interestingly, we highlight that the prognosis of alterations, such as the presence of an e8a2 transcript or the deletion of various genes, which have been controversial, may be definitively erased by the introduction of tyrosine kinase inhibitors (TKIs).
Publication Date: 2015-08-08
Journal: Genes, chromosomes & cancer

Molecular response to imatinib in chronic myeloid leukaemia with a variant e13a3 BCR-ABL1 fusion.
The majority of chronic myeloid leukaemia (CML) patients express either e13a2 or e14a2 BCR-ABL1 transcripts. Variant fusion genes can arise, usually due to alternative splicing of either BCR or ABL1 exons, with molecular monitoring by quantitative PCR (qPCR) in response to tyrosine kinase inhibitor therapy rarely reported in such cases. A case of CML is described in which an e13a3 BCR-ABL1 fusion was characterised. A qPCR methodology was developed and applied prospectively to demonstrate a favourable molecular response to imatinib treatment. This case serves to highlight the requirement for molecular monitoring of those CML patients harbouring the e13a3 and other variant BCR-ABL1 transcripts.
Publication Date: 2015-01-13
Journal: Medical oncology (Northwood, London, England)

shRNA library screening identifies nucleocytoplasmic transport as a mediator of BCR-ABL1 kinase-independent resistance.
The mechanisms underlying tyrosine kinase inhibitor (TKI) resistance in chronic myeloid leukemia (CML) patients lacking explanatory BCR-ABL1 kinase domain mutations are incompletely understood. To identify mechanisms of TKI resistance that are independent of BCR-ABL1 kinase activity, we introduced a lentiviral short hairpin RNA (shRNA) library targeting ∼5000 cell signaling genes into K562(R), a CML cell line with BCR-ABL1 kinase-independent TKI resistance expressing exclusively native BCR-ABL1. A customized algorithm identified genes whose shRNA-mediated knockdown markedly impaired growth of K562(R) cells compared with TKI-sensitive controls. Among the top candidates were 2 components of the nucleocytoplasmic transport complex, RAN and XPO1 (CRM1). shRNA-mediated RAN inhibition or treatment of cells with the XPO1 inhibitor, KPT-330 (Selinexor), increased the imatinib sensitivity of CML cell lines with kinase-independent TKI resistance. Inhibition of either RAN or XPO1 impaired colony formation of CD34(+) cells from newly diagnosed and TKI-resistant CML patients in the presence of imatinib, without effects on CD34(+) cells from normal cord blood or from a patient harboring the BCR-ABL1(T315I) mutant. These data implicate RAN in BCR-ABL1 kinase-independent imatinib resistance and show that shRNA library screens are useful to identify alternative pathways critical to drug resistance in CML.
Publication Date: 2015-01-13
Journal: Blood