BCR-ABL Transcript Levels Research Articles

Evaluation of Molecular Response to Imatinib Mesylate Treatment in Iranian Patients With Chronic Myeloid Leukemia

BackgroundImatinib mesylate has revolutionized the treatment of patients with chronic myeloid leukaemia (CML); however, some patients fail to respond and have a poor prognosis. Evaluation of molecular response to imatinib is a sensitive method can help physicians make better and quicker therapeutic decisions in the course of this disease. This study aims to evaluate the molecular response to generic imatinib in Iranian patients with CML. Patients and MethodsThis prospective study consisted of 255 newly diagnosed patients with CML who received imatinib. Molecular response was analyzed at 3 and 6 months from the start of the treatment and then every 6 months, and long-term outcomes, including overall survival (OS) and progression-free survival (PFS), were evaluated. ResultsAt a median follow-up of 34.8 months (range, 3-84 months, (the OS and PFS at 7 years were 94.3% and 92.9%, respectively. Eighty–four-month PFS rates in patients with a BCR-ABLIS ≤ 10% at 3 months and BCR-ABLIS ≤ 1% at 6 months were significantly higher than patients who did not obtain these levels of BCR-ABL transcripts (P = .004 and P < .0001, respectively). The proportion of patients who achieved major molecular response (MMR) was 44.1%, 52.97%, and 60.75% at 12, 18, and 24 months, respectively. At 12, 18, and 84 months, the PFS rates in patients who achieved MMR were significantly higher than in patients who did not achieve MMR (P = .002, P < .0001, and P = .003, respectively). ConclusionsThe data of this prospective study are highly comparable with that from clinical trials and prospective international studies.

Laying the foundation for genomically-based risk assessment in chronic myeloid leukemia.

Outcomes for patients with chronic myeloid leukemia (CML) have substantially improved due to advances in drug development and rational treatment intervention strategies. Despite these significant advances there are still unanswered questions on patient management regarding how to more reliably predict treatment failure at the time of diagnosis and how to select frontline tyrosine kinase inhibitor (TKI) therapy for optimal outcome. The BCR-ABL1 transcript level at diagnosis has no established prognostic impact and cannot guide frontline TKI selection. BCR-ABL1 mutations are detected in ~50% of TKI resistant patients but are rarely responsible for primary resistance. Other resistance mechanisms are largely uncharacterized and there are no other routine molecular testing strategies to facilitate the evaluation and further stratification of TKI resistance. Advances in next-generation sequencing technology has aided the management of a growing number of other malignancies, enabling the incorporation of somatic mutation profiles in diagnosis, classification, and prognostication. A largely unexplored area in CML research is whether expanded genomic analysis at diagnosis, resistance, and disease transformation can enhance patient management decisions, as has occurred for other cancers. The aim of this article is to review publications that reported mutated cancer-associated genes in CML patients at various disease phases. We discuss the frequency and type of such variants at initial diagnosis and at the time of treatment failure and transformation. Current limitations in the evaluation of mutants and recommendations for future reporting are outlined. The collective evaluation of mutational studies over more than a decade suggests a limited set of cancer-associated genes are indeed recurrently mutated in CML and some at a relatively high frequency. Genomic studies have the potential to lay the foundation for improved diagnostic risk classification according to clinical and genomic risk, and to enable more precise early identification of TKI resistance.

SOX30 methylation correlates with disease progression in patients with chronic myeloid leukemia.

BackgroundOur previous study has reported that aberrant SOX30 methylation was associated with poor prognosis in AML, and it correlated with disease progression in MDS. Herein, we further determined SOX30 methylation and its clinical significance in the other myeloid malignance – chronic myeloid leukemia (CML).MethodsSOX30 methylation was examined by real-time quantitative methylation-specific PCR and bisulfite sequencing PCR, whereas SOX30 expression was detected by real-time quantitative PCR.ResultsSOX30 methylation was identified in 11% (10/95) CML patients. SOX30 methylation was associated with lower hemoglobin and platelets (P=0.006 and 0.032, respectively). Importantly, significant differences were observed in the distributions of clinical stages and cytogenetics (P=0.006 and 0.002, respectively). The frequency of SOX30 methylation in chronic phase (CP) stage occurred with lowest frequency (4/74, 5%), higher in accelerated phase (AP) stage (1/7, 14%), and the highest in blast crisis (BC) stage (12/31, 39%). In addition, SOX30 methylated patients tended to have a higher level of BCR-ABL transcript than SOX30 non-methylated patients (P=0.063). In two paired CML patients, SOX30 methylation showed lower density in CP stage (19% and 17%, respectively), and was significantly increased in BC stage (89% and 69%, respectively) during disease progression. Additionally, SOX30 methylated CML patients presented a lower SOX30 transcript level than SOX30 non-methylated CML patients (P=0.046).ConclusionOur study revealed that SOX30 methylation correlated with disease progression in chronic myeloid leukemia.

Responses to Dasatinib as a Second- and Third-Line Tyrosine Kinase Inhibitor in Chronic Phase Chronic Myeloid Leukaemia Patients

We retrospectively evaluated the efficacy and safety of dasatinib among 48 Chinese patients with chronic phase chronic myeloid leukaemia. The proportions of patients achieving the optimal molecular responses at 3, 6, and 12 months, a major molecular response (MMR) rate and a complete cytogenetic response (CCyR) rate were 87.0, 87.0, 72.2, 45.8, and 72.7% for patients with dasatinib as second-line therapy, and 34.8, 34.8, 33.3, 20.8, and 46.2% as third-line therapy, respectively. A BCR-ABL1 transcript level on the International Scale (BCR-ABL1^IS) of ≤10% at the initiation of ­dasatinib treatment was found to be associated with a higher probability of achieving MMR. Among patients with a ­BCR-ABL1^IS higher than 10% at initiation of dasatinib treatment, dasatinib showed better performance as a second-line therapy than as a third-line therapy. The patients who achieved an optimal molecular response at 3 months had a superior cumulative incidence of MMR and CCyR compared with patients who failed to achieve such a response. Dasatinib induced considerable responses as a second-line treatment, especially in patients with a BCR-ABL1^IS ≤10% at initiation of treatment, whereas the efficacy was limited in patients receiving third-line therapy with a BCR-ABL1^IS >10% at the initiation of treatment.

Somatic variants in epigenetic modifiers can predict failure of response to imatinib but not to second-generation tyrosine kinase inhibitors.

There are no validated molecular biomarkers to identify newly-diagnosed individuals with chronic-phase chronic myeloid leukemia likely to respond poorly to imatinib and who might benefit from first-line treatment with a more potent second-generation tyrosine kinase inhibitor. Our inability to predict these ‘high-risk’ individuals reflects the poorly understood heterogeneity of the disease. To investigate the potential of genetic variants in epigenetic modifiers as biomarkers at diagnosis, we used Ion Torrent next-generation sequencing of 71 candidate genes for predicting response to tyrosine kinase inhibitors and probability of disease progression. A total of 124 subjects with newly-diagnosed chronic-phase chronic myeloid leukemia began with imatinib (n=62) or second-generation tyrosine kinase inhibitors (n=62) and were classified as responders or non-responders based on the BCRABL1 transcript levels within the first year and the European LeukemiaNet criteria for failure. Somatic variants affecting 21 genes (e.g. ASXL1, IKZF1, DNMT3A, CREBBP) were detected in 30% of subjects, most of whom were non-responders (41% non-responders, 18% responders to imatinib, 38% non-responders, 25% responders to second-generation tyrosine kinase inhibitors). The presence of variants predicted the rate of achieving a major molecular response, event-free survival, progression-free survival and chronic myeloid leukemia-related survival in the imatinib but not the second-generation tyrosine kinase inhibitors cohort. Rare germline variants had no prognostic significance irrespective of treatment while some pre-leukemia variants suggest a multi-step development of chronic myeloid leukemia. Our data suggest that identification of somatic variants at diagnosis facilitates stratification into imatinib responders/non-responders, thereby allowing earlier use of second-generation tyrosine kinase inhibitors, which, in turn, may overcome the negative impact of such variants on disease progression.

Results of the European survey on the assessment of deep molecular response in chronic phase CML patients during tyrosine kinase inhibitor therapy (EUREKA registry).

The advent of tyrosine kinase inhibitor (TKI) therapies has revolutionized the treatment of chronic myeloid leukemia (CML). The European LeukemiaNet (ELN) recommends quantification of BCR-ABL1 transcripts by real-time quantitative PCR every 3months during TKI treatment. Since a proportion of patients in deep molecular response (DMR: MR4, MR4.5, MR5) maintain remission after treatment stop, assessment of DMR is crucial. However, systematically collected molecular data, monitored with sensitive standardized assays, are not available outside clinical trials. Data were collected on the standardized assessment of molecular response in the context of real-life practice. BCR-ABL1 transcript levels after > 2years of TKI therapy were evaluated for DMRby local laboratories as well as standardized EUTOS laboratories. Since standardized molecular monitoring is a prerequisite for treatment discontinuation, central surveillance of the performance of the participating laboratories was carried out. Between 2014 and 2017, 3377 peripheral blood samples from 1117 CML patients were shipped to 11 standardized reference laboratories in six European countries. BCR-ABL1 transcript types were b3a2 (41.63%), b2a2 (29.99%), b2a2/b3a2 (3.58%) and atypical (0.54%). For 23.72% of the patients, the initial transcript type had not been reported. Response levels (EUTOS laboratory) were: no MMR, n = 197 (6.51%); MMR, n = 496 (16.40%); MR4, n = 685 (22.64%); MR4.5, n = 937 (30.98%); MR5, n = 710 (23.47%). With a Cohen's kappa coefficient of 0.708, a substantial agreement between EUTOS-certified and local laboratories was shown. Multicenter DMR assessment is feasible in the context of real-life clinical practice in Europe. Information on the BCR-ABL1 transcript type at diagnosis is crucial to accurately monitor patients' molecular response during or after TKI therapy.

Predictive value of early molecular response for deep molecular response in chronic phase of chronic myeloid leukemia.

To investigate the association of 3- and 6-month BCR-ABL transcript levels on the international scale (BCR-ABLIS) and other factors with deep molecular response (DMR) achievement in chronic myeloid leukemia (CML)-chronic phase (CP) patients receiving tyrosine kinase inhibitor (TKI) therapy.We retrospectively analyzed the clinical data of 206 patients enrolled in our hospital between January 2010 and July 2018. These patients were initially diagnosed with CML-CP and received imatinib or nilotinib therapy. Early molecular response (EMR) was assessed based on BCR-ABLIS (IS: on the international scale) transcript level at 3 and 6 months. Potential factors impacting DMR achievement were identified using Cox proportional hazard regression models. The effects of EMR achievement on the cumulative incidence of MR4.0 were investigated via Kaplan–Meier analysis.Multivariate Cox regression analysis showed that a BCR-ABLIS transcript level at 3 and 6 months of TKI therapy was an independent factor for the achievement of MR4.0, which was nevertheless not related to age, gender, Sokal score, hemoglobin level, or white blood cell (WBC) count at the initial time of diagnosis. Patients achieving an EMR (EMR: 3-month BCR-ABLIS ≤10%, 6-month BCR-ABLIS <1%) were more likely to reach MR4.0 than patients failing to achieve EMR (P1 <.001, P2 <.001). Patients who had 3-month BCR-ABLIS ≤1% were more likely to reach MR4.0 than those who had 3-month BCR-ABLIS of 1% to 10% or >10% (P1 = .001, P2 <.001). Similarly, patients who had 6-month BCR-ABLIS ≤0.1% were more likely to achieve MR4.0 than those in the 0.1% to 1% and ≥1% groups (P1 <.001, P2 <.001). Also, a higher percentage of patients on nilotinib therapy achieved EMR compared with patients on imatinib therapy (93.3% vs 63.6% on 3-month nilotinib therapy, P = .001; 88.9% vs 59.9% on 6-month nilotinib therapy, P = .004).This study demonstrates that EMR, especially a 3-month BCR-ABLIS ≤1% and 6-month BCR-ABLIS ≤0.1%, have predictive value for DMR achievement. In addition, there is a higher percentage of patients receiving nilotinib therapy achieved EMR than that of those receiving imatinib therapy.

Rates of deep molecular response by digital and conventional PCR with frontline nilotinib in newly diagnosed chronic myeloid leukemia: a landmark analysis

ENESTnext (NCT01227577) was a single-arm, multicenter trial evaluating the rate of deep molecular response by 2 years in patients with newly diagnosed (within 6 months) chronic myeloid leukemia in chronic phase (CML-CP) treated with nilotinib 300 mg twice daily. Among 128 enrolled patients, 94 (73%) achieved major molecular response (MMR; BCR-ABL1 ≤ 0.1% on the International Scale [BCR-ABL1IS]) and 34 (27%) achieved confirmed MR4.5 (BCR-ABL1IS ≤0.0032% detectable or undetectable; primary endpoint) by 2 years. Three-month BCR-ABL1 levels were predictive of later responses. In exploratory analyses, digital polymerase chain reaction (PCR) detected BCR-ABL1 in 39.4% of samples from patients with confirmed MR4.5 and identified further decreases in BCR-ABL1 with continued nilotinib. Safety results, including cardiovascular events, were consistent with those in other nilotinib trials. These results further substantiate the molecular response rates associated with frontline nilotinib therapy and demonstrate the feasibility of monitoring very low BCR-ABL1 transcript levels using digital PCR.

Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph + ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1

Minimal residual disease (MRD) is a powerful prognostic factor in acute lymphoblastic leukemia (ALL) and is used for patient stratification and treatment decisions, but its precise role in Philadelphia chromosome positive ALL is less clear. This uncertainty results largely from methodological differences relating to the use of real-time quantitative PCR (qRT-PCR) to measure BCR-ABL1 transcript levels for MRD analysis. We here describe the first results by the EURO-MRD consortium on standardization of qRT-PCR for the e1a2 BCR-ABL1 transcript in Ph + ALL, designed to overcome the lack of standardisation of laboratory procedures and data interpretation. Standardised use of EAC primer/probe sets and of centrally prepared plasmid standards had the greatest impact on reducing interlaboratory variability. In QC1 the proportion of analyses with BCR-ABL1/ABL1 ratios within half a log difference were 40/67 (60%) and 52/67 (78%) at 10-3 and 36/67 (53%) and 53/67 (79%) at 10-4BCR-ABL1/ABL1. Standardized RNA extraction, cDNA synthesis and cycler platforms did not improve results further, whereas stringent application of technical criteria for assay quality and uniform criteria for data interpretation and reporting were essential. We provide detailed laboratory recommendations for the standardized MRD analysis in routine diagnostic settings and in multicenter clinical trials for Ph + ALL.

A new highly sensitive real-time quantitative-PCR method for detection of BCR-ABL1 to monitor minimal residual disease in chronic myeloid leukemia after discontinuation of imatinib.

Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL1 fusion protein, encoded by the Philadelphia chromosome, have drastically improved the outcomes for patients with chronic myeloid leukemia (CML). Although several real-time quantitative polymerase chain reaction (RQ-PCR) kits for the detection of BCR-ABL1 transcripts are commercially available, their accuracy and efficiency in laboratory practice require reevaluation. We have developed a new in-house RQ-PCR method to detect minimal residual disease (MRD) in CML cases. MRD was analyzed in 102 patients with CML from the DOMEST study, a clinical trial to study the rationale for imatinib mesylate discontinuation in Japan. The BCR-ABL1/ABL1 ratio was evaluated using the international standard (IS) ratio, where IS < 0.1% was defined as a major molecular response. At enrollment, BCR-ABL1 transcripts were undetectable in all samples using a widely-applied RQ-PCR method performed in the commercial laboratory, BML (BML Inc., Tokyo, Japan); however, the in-house method detected the BCR-ABL1 transcripts in five samples (5%) (mean IS ratio: 0.0062 ± 0.0010%). After discontinuation of imatinib, BCR-ABL1 transcripts were detected using the in-house RQ-PCR in 21 patients (21%) that were not positive using the BML method. Nineteen samples were also tested using a commercially available RQ-PCR assay kit with a detection limit of IS ratio, 0.0032 (ODK-1201, Otsuka Pharmaceutical Co., Tokyo, Japan). This method detected low levels of BCR-ABL1 transcripts in 14 samples (74%), but scored negative for five samples (26%) that were positive using the in-house method. From the perspective of the in-house RQ-PCR method, number of patients confirmed loss of MMR was 4. These data suggest that our new in-house RQ-PCR method is effective for monitoring MRD in CML.

Treatment progress of chronic myeloid leukemia

A great progress has been made in the treatment of chronic myeloid leukemia (CML) owing to the first generation tyrosine kinase inhibitor (TKI). Nowadays, more and more hematologists are eager to understand how to rapidly reduce the BCR-ABL transcripts level, and to get the standard therapeutic reactions, which emerges the second generation TKI. However, the first and second generation TKI have the potential of drug resistance, and thus the third and fourth generation TKI may resolve this problem. Imatinib drug discontinuation trial emphasizes the importance of digital polymerase chain reaction (PCR) and the probability of the second treatment-free remission. Key words: Leukemia, myelogenous, chronic, BCR-ABL positive; Tyrosine kinase inhibitor; Drug discontinuation trial

Effect of Imatinib on Bone Marrow Morphology and Angiogenesis in Chronic Myeloid Leukemia.

Background and Objectives Chronic myeloid leukemia (CML) is characterized by hyperproliferation of myeloid precursors, increased fibrosis, and neoangiogenesis in the bone marrow. Imatinib inhibits BCR-ABL tyrosine kinase produced due to reciprocal translocation t(9;22) in neoplastic CML cells. It reduces hyperproliferation of myeloid precursors and has been found to affect bone marrow fibrosis and angiogenesis. This study was done to assess the effect of imatinib on bone marrow morphology and angiogenesis in CML. Methods 31 newly diagnosed CML patients were evaluated before and after 3 months of imatinib therapy. A marrow morphological response (MMR) score was used to assess marrow cytological and histological features including grade of fibrosis. Mean microvessel density (MVD) was also assessed. Hematological parameters and BCR-ABL transcript levels were assessed in the peripheral blood. Results 86.21% of patients showed decrease in marrow cellularity with normalization of M:E ratio. 72.42% of patients had decrease in grade of fibrosis and 17.24% showed no change while 10.34% of patients showed progression of fibrosis grade. Patients with MMR score ≥ 2 (n=4) and those with progression of fibrosis grade (n=3) showed suboptimal molecular response (BCR-ABL transcripts > 10%). Pretherapy mean MVD of patients (14.69 ± 5.28) was higher than that of controls (6.32 ± 1.64). A significant reduction of 66.51% was observed in posttherapy mean MVD (4.98 ± 2.77) of CML patients (p<0.001). Conclusion Imatinib therapy in CML not only decreases marrow cellularity, but also helps towards normalization of bone marrow microenvironment by reducing fibrosis and angiogenesis.

Comparative Monitoring of Minimal Residual Disease (MRD) By RT-Quantitative (RT-qPCR) and Digital PCR (dPCR) in Ph+ Chronic Myeloid Leukemia (CML) Patients Treated with TKIs for Recognition of Stable Deep Molecular Response (DMR) and Identification of Best Candidates to TKIs Treatment Discontinuation

Current therapy of CML with tyrosine kinase inhibitors (TKIs) aims to achieve a Major Molecular Response to avoid the disease progression to blastic phase and, possibly, a Deep Molecular Response (DMR), to gain the opportunity for TKIs discontinuation and Treatment Free Remission (TFR). However, TFR strategies are not yet optimized, as well as the criteria for patients (pts) selection to TKI discontinuation are not well defined. The patient9s CML prognostic score, the duration of TKI treatment and "stable" DMR before discontinuation and the levels of DMR are still matters of debate. Due to its intrinsic limitations, RT-qPCR cannot be considered as an optimal tool for a precise detection of minimal BCR-ABL1 transcript levels and DMR monitoring. The digital PCR (dPCR) has emerged as a more sensitive and accurate method to detect and monitor the BCR-ABL1 minimal residual disease (MRD). This study focused on the BCR-ABL1 RT-q/dPCR comparative monitoring in CML pts treated with TKIs and with durable DMR (≥ 2 years), as conventionally assessed by RT-qPCR, before the enrollment. The aim was to evaluate the reliability and the efficiency of dPCR, as compared to RT-qPCR, for a better identification of "stable" DMR and for a better selection of the candidates for treatment discontinuation. 118 CML pts with durable DMR were enrolled and 493 peripheral blood samples were comparatively analyzed by both RT-qPCR, according to the last International Guidelines, and dPCR (QS3D Digital PCR System). RT-qPCR results were assessed as BCR-ABL1 %IS. dPCR results were assessed as number of BCR-ABL1 copies/□l of reaction and the molecular response was categorized as □ 0.468 or Starting from the first RTq/dPCR comparative assessment, the 118 pts were monitored every 3/4 months for at least 3 time points. Grouping the 118 pts according to the RT-qPCR molecular results at the enrolment, 50 cases (42%) resulted in the MR4.0group and 68 (58%) in the MR 4.5-MR5.0 group. No significant differences were observed between these 2 groups in terms of clinical and hematological characteristics. At the same time, the 118 pts were divided in 2 groups based on the comparative dPCR molecular results, according to the above mentioned cut-off value: 29 (25%) and 89 (75%) cases had dPCR values □ and □0.468 BCR-ABL1 copies/□l, respectively. No significant clinical differences were observed between these 2 groups. Out of these 118 pts, 87 (74%) discontinued the TKI treatment and 24 (28%) of them lost the DMR after a median time of 3 months (range 1-30). Based on dPCR, 23/87 (26%) and 64/87 (74%) had dPCR □ 0.468 and In order to reach a better recognition of stable DMR, which is strictly related with the probability to achieve and maintain TFR after TKIs discontinuation, we comparatively analyzed, by RTq/dPCR, the course of MRD during the follow up period. In the case of monitoring by RT-qPCR, a wide oscillation of the molecular responses was observed and they tended to be superimposable both in the group of pts with MR4.0 and in those with MR4.5 and 5.0. In the case of monitoring by dPCR, the values assessed at the enrolment and during the follow up revealed the capability of the dPCR to measure different and heterogeneous numbers of BCR-ABL1 copies in each pts. Moreover, the pts with a dPCR below the cut-off value of 0.468, at enrolment, were maintaining values (median 0,175; range 0-1,863), significantly lower (p This retrospective study show that the dPCR is more accurate and sensitive than conventional RT-qPCR for detecting and monitoring the BCR-ABL1 molecular levels and potentially able to improve the recognition of the stable DMR and selection of the candidates to TKI treatment discontinuation. Disclosures Bonifacio:Incyte: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Novartis: Research Funding; Bristol Myers Squibb: Consultancy. Gaidano:Morphosys: Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Roche: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. Rossi:Teva: Membership on an entity9s Board of Directors or advisory committees; Mundipharma: Honoraria; Sanofi: Membership on an entity9s Board of Directors or advisory committees; Janssen: Membership on an entity9s Board of Directors or advisory committees; Celgene: Membership on an entity9s Board of Directors or advisory committees; Abbvie: Membership on an entity9s Board of Directors or advisory committees; Pfizer: Membership on an entity9s Board of Directors or advisory committees; Novartis: Honoraria; Gilead: Membership on an entity9s Board of Directors or advisory committees; Sandoz: Honoraria; Jazz: Membership on an entity9s Board of Directors or advisory committees; Roche: Membership on an entity9s Board of Directors or advisory committees; BMS: Honoraria; Amgen: Membership on an entity9s Board of Directors or advisory committees. Gobbi:Novartis: Consultancy; Ariad: Membership on an entity9s Board of Directors or advisory committees; Pfister: Membership on an entity9s Board of Directors or advisory committees; Amgen: Consultancy; Janssen: Consultancy; Celgene: Membership on an entity9s Board of Directors or advisory committees. Voso:Celgene: Research Funding, Speakers Bureau. Abruzzese:Ariad: Consultancy; BMS: Consultancy; Novartis: Consultancy; Pfizer: Consultancy.

Dynamic Personalized Assessment of Outcome in Patients with Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia

Dynamic Personalized Assessment of Outcome in Patients with Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia

Second Attempt of TKI Discontinuation with Dasatinib for Treatment-Free Remission after Failing First Attempt with Imatinib: Treatment-Free Remission Accomplished By Dasatinib (TRAD) Trial

Introduction: A Canadian tyrosine kinase inhibitor (TKI) discontinuation trial is ongoing to determine if using dasatinib (DA) can lead to a successful treatment-free remission (TFR) after failing a first attempt of TKI discontinuation after imatinib (IM) treatment. The preliminary result indicate : 1) The 6-month molecular relapse-free survival (mRFS) rate is estimated as 58.0%; 2) DA re-treatment is feasible and safe, with achievement of excellent rates of MMR and MR4. We report here the preliminary analysis of the TFR rate at 6 months after DA discontinuation for the second TFR attempt. Methods and materials: This prospective clinical trial (BMS CA180-543, Clinicaltrial.gov NCT#02268370) has 3 phases: 1) IM discontinuation phase, 2) DA rechallenge phase, 3) DA discontinuation phase. Molecular relapse is defined as an increase in BCR-ABL transcript level MR4 for a 2nd TFR attempt. The null hypothesis was a TFR2 rate of 17.5% while the alternative hypothesis was a TFR2 rate of 35.0% and the study was designed to reject our null hypothesis if > 28% of patients remain in TFR after DA discontinuation. Results: As of Jun 15, 2018, 53 (40.4%) of 131 enrolled patients experienced molecular relapse after IM discontinuation with a mRFS rate of 58.0% at 12 months (95% CI, 42.1-71.0%). Of the 53 patients who lost response, 51 patients received DA. The incidence of MMR, MR4 and MR4.5 at 3 months was 97.7%, 89.9%, and 84.6%, respectively. 25/ 51 patients receiving DA attained MR4.5 for 12 months or longer and discontinued it for a 2nd TFR attempt (TFR2). 21/25 (84.0%) of these patients lost molecular response at a median of 3.7 months after DA discontinuation. The estimated TFR2 rate after DA discontinuation was 21.5±8.5% at 6 months (95% CI [7.9-39.5%], Fig 1A). Thus we cannot reject our null hypothesis based on this result. For risk factor analysis for maintaining TFR2, the variables analysed included Sokal risk score, IM duration, MR4/MR4.5 duration, monthly doubling time after IM discontinuation, time to molecular relapse after IM discontinuation, molecular relapse pattern after IM discontinuation (MMR loss vs MR4 loss), and BCR-ABL1 qPCR value prior to DA discontinuation. 1) Time to molecular relapse after IM discontinuation correlates with TFR2 (p 2) Molecular relapse pattern after IM discontinuation correlates with TFR2. The group who had loss of MMR after IM discontinuation lost molecular response faster after DA discontinuation (n=19; median 3.0 months) compared to those with two consecutive losses of MR4(n=6; 6.43 months; p=0.0435, HR 2.991; Fig 3B). 3) The group with 5.5 log reduction or deeper in BCR-ABL1 qPCR transcripts prior to DA discontinuation (n=19) showed a TFR2 rate of 28.7% at 6 months (median TFR2 duration of 4.04 months) versus 0% in the group with qPCR transcript level between 4.5 and 5.4 log reduction (n=6, median TFR2 duration of 2.89 months; p=0.017; Fig 3C). We did not identify any other risk factor for molecular relapse after DA discontinuation . The expansion kinetics of the leukemic clone after DA discontinuation is similar to that after IM discontinuation. Conclusion: These preliminary results suggest that rechallenge with DA after failing a first IM discontinuation attempt for TFR is well tolerated and effective as most cases rapidly regained at least MR4. However, more strict criteria should be considered for TFR2 attempt, including achievement of a 5.5 log reduction or deeper in BCR-ABL1 qPCR levels prior to the 2nd TKI discontinuation attempt, and a MR4 duration of more than 12 months. Disclosures Kim:Pfizer: Consultancy; Paladin: Consultancy; Novartis: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding. Busque:BMS: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Paladin: Consultancy. Savoie:Pfizer: Consultancy; Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Bence-Bruckler:Lundbeck: Membership on an entity9s Board of Directors or advisory committees. Delage:BMS: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity9s Board of Directors or advisory committees, Research Funding; AbbVie: Research Funding; Pfizer: Research Funding; Roche: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity9s Board of Directors or advisory committees. Liew:Novartis: Honoraria, Membership on an entity9s Board of Directors or advisory committees; BMS: Membership on an entity9s Board of Directors or advisory committees. Laneuville:BMS: Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding; Paladin: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding. Lipton:Bristol-Myers Squibb: Consultancy, Research Funding; ARIAD: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. Leber:Novartis Canada: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Novartis Canada: Honoraria, Membership on an entity9s Board of Directors or advisory committees.

Prognostic Implication of Early Molecular Response at 3 Months in Chronic Myeloid Leukemia Patients: A Comparison of Imatinib and Second Generation Tyrosine Kinase Inhibitors

Background: Although several studies have demonstrated that BCR-ABL1 transcript levels at 3 months were prognostically significant in patients treated with frontline imatinib (IM) and second-generation tyrosine kinase inhibitors (2G-TKIs), the decision to change treatment based on early molecular response (EMR) status remain under investigaiton. Because CML patients currently have multiple treatment options, the impact of early molecular milestones on long-term outcomes needs to be determined in each agent treated patients.Aims: The aim of this study is to evaluate the impact of 3-month early molecular response (EMR) on long-term outcomes of CML patients treated with IM and different 2G-TKIs, as additional information to guide clinical decisions on switching to a different TKI.Methods: 734 new CP CML patients who were treated with frontline IM (n = 366) or 2G TKIs (n = 368) were analyzed. Molecular responses were monitored using qRT-PCR assay in 3 month intervals by achieving major molecular response (MMR), and then 6 month intervals after achieving MMR. Main study objectives were to evaluate the long-term outcomes, including failure-free survival (FFS), progression-free survival (PFS), and overall survival (OS), according to 3-month EMR. FFS was measured from the date of treatment start until death, progression to AP or BP, or ELN failure on treatment, whichever came first. PFS and OS collected survivals on patients who were treated with other TKIs after frontline therapy discontinuation.Results: A total of 734 patients were treated with imatinib (IM; n = 366), dasatinib (DAS; n = 141), nilotinib (NIL; n = 89), and radotinib (RAD; n = 138). The median age was 43 years (range, 11-87). The percentages of patients with low, intermediate, and high Sokal risk scores were 37%, 38% and 23%, respectively, with 2% unknown risk. With a median follow-up of 85.6 (IM; range, 4.4-207.5) and 38.9 (2G-TKIs; range, 11.6-130.2) months, 460 (62.7%; 182 IM and 218 2G-TKIs) patients continue on the frontline therapy and 274 (37.3%) patients were permanently discontinued or changed to other TKIs due to intolerance (73 IM, 38 DAS, 2 NIL, 28 RAD), ELN failure (32 IM, 3 DAS, 2 NIL, 3 RAD), progression (3 IM, 1 DAS, 1 RAD), and others (60 warning, 3 pregnancy, 14 treatment-free remission study, and 9 follow-up loss). In 366 patients treated with IM, patients achieving BCR-ABL1 ≤ 10% at 3 months (n = 275, 75.1%) had a better outcomes in terms of 8-y OS (97.4% vs 89.7%, P <0.001), 8-y PFS (96.3% vs 88.6, P=0.002), and 8-y FFS (91.5% vs 74.6%, P<0.001) compared with those of the patients with BCR-ABL1 >10%. In 368 patients treated with 2G-TKIs, achievement of 3-month EMR (n = 331, 90.0%) was associated with a higher 8-y OS (98.6% vs 91.9%, P <0.001), 8-y PFS (98.2% vs 91.7, P=0.001), and 8-y FFS (96.5% vs 82.7%, P<0.001). The prognostic impact of 3-month EMR was observed in both IM and 2G-TKIs treated groups.Conclusions: Our data showed that EMR failure at 3 months could translate into poor survival outcomes in patients treated with frontline IM and 2G-TKIs.However, to confirm the benefit of an early switch of therapy, further investigations in a larger patient population with longer follow-up are needed. DisclosuresKim:Pfizer: Research Funding; BMS: Research Funding; Novartis: Research Funding; Ilyang: Research Funding.

Inactivation of Stage-Specific B-Cell Commitment Genes Generates Distinct Molecular Subtypes of BCR-ABL1 Lymphoblastic Leukemia

Background Philadelphia-chromosome positive acute lymphoblastic leukemia (Ph+ ALL), the most common form of ALL in adults, is a highly aggressive blood malignancy defined by the BCR-ABL1 fusion. Although inhibitors targeting the BCR-ABL1 oncoprotein, such as imatinib, have significantly improved clinical response rates for this disease, a subset of patients are refractory to therapy or respond initially but relapse soon after. ABL1 kinase domain mutations partly explain differential responses in patients; however, for the majority of cases, a molecular basis that can reconcile this clinical observation is lacking.Methods Flow-sorted blasts from 53 primary samples, 49 de novo Ph+ ALL and 4 lymphoid blast crisis CML, were subjected to RNA sequencing (RNA-seq) and whole genome sequencing (WGS). Response rates were tracked using BCR-ABL1 transcript levels from patient blood.Results Hierarchical clustering of transcriptome data produced two molecular subgroups of Ph+ ALL. One subgroup, which we termed ‘Core-B’, upregulated key regulators of B-lymphoid differentiation including IL7R and MS4A1 (CD20). By contrast, the second subgroup upregulated an expression program related to hematopoietic stem cell (HSC) and myeloid differentiation, with upregulation of KIT, CD34, MPO, CSF3R, and GATA3. We termed this subgroup ‘Aberrant-Stem-Myeloid’ (ASM). These subgroups displayed a striking disparity in response rates to intensive chemotherapy with imatinib. Whereas ‘Core-B’ patients showed highly durable responses often lasting many years, ‘ASM’ patients frequently relapsed (4% vs 43% relapse; p=0.007). We used WGS analysis to investigate the genetic basis of these molecular subtypes. ‘Core-B’ Ph+ ALLs were enriched for deletions in PAX5, a B-cell specification gene, and CDKN2A/B, tumor suppressors. The ‘ASM’ subtype lacked these genetic alterations; instead, these leukemias were enriched for deletions in EBF1, an early B-cell lineage factor that represses T-lymphoid and myeloid lineages and is expressed before PAX5 in B-cell lineage differentiation. Accordingly, blasts from ‘ASM’ leukemias with EBF1 deletions showed decreased CD19 antigen expression and upregulation of myeloid antigens by clinical flow cytometry. Rare cases with concurrent EBF1 and PAX5 deletions showed expression features of both ‘ASM’ and ‘Core-B’ leukemias. Mutations observed in myeloid leukemias (TET2, RUNX1) were only present in the ‘ASM’ subtype. Loss of IKZF1, found in 77% of cases, also displayed distinct patterns between the two subgroups; deletions leading to the dominant negative isoform (Ik6) were enriched in the ‘Core-B’ subgroup (45% vs. 14%; p=0.019) while monosomy 7 and large deletions encompassing IKZF1 were enriched in the ‘ASM’ leukemias (41% vs. 10%; p=0.017). In 1 of 4 diagnosis/relapse patients analyzed, a molecular switch from ‘Core-B’ at diagnosis to ‘ASM’ at relapse was observed. The diagnostic ‘Core-B’ clone from this patient harbored a PAX5 mutation that was lost at relapse, whereas the relapsed ‘ASM’ clone harbored trisomy 21 and a RUNX1 mutation. Altogether, our data suggest that the ‘ASM’ leukemias emerge through dysregulation of genes earlier in lympho-myeloid specification compared to ‘Core-B’ leukemias.These findings led us to investigate if the ‘ASM’ subtype originates from an HSC and the ‘Core-B’ subtype originates from a B-cell progenitor. We first looked at the distribution of the long (p210) and short (p190) isoforms of BCR-ABL1 in the two subtypes. The p210 isoform, also the hallmark of CML, is speculated to arise in an HSC, and the p190 is thought to arise in a B-cell progenitor. Neither the p190 or p210 BCR-ABL1 isoform was enriched in either subgroup. We resolved highly purified HSC and progenitor subsets from CD34+CD19- cells, functionally evaluated by methylcellulose assays, and subjected them to a sensitive nested-PCR strategy. Cases from both the ‘ASM’ and ‘Core-B’ subtypes showed HSC/myeloid progenitor involvement regardless of the BCR-ABL1 isoform. This data suggest that the cell-of-origin does not play a role in establishing the molecular subtype of leukemia blasts.Conclusion There are two distinct molecular subtypes of Ph+ ALL that demonstrate differential responses to treatment and emerge from independent mutational routes. Moreover, the key genetic determinants that form the molecular subtype are secondary driver alterations that lie downstream of BCR-ABL1. DisclosuresNo relevant conflicts of interest to declare.

Ruxolitinib in Combination with Dasatinib and Dexamethasone Is an Active and Well-Tolerated Chemotherapy-Sparing Oral Induction Regimen for Adults with Philadelphia Chromosome-Positive ALL: Results of a Phase I Trial

Introduction: Induction therapy consisting of dasatinib (DAS) and corticosteroids (CS) yields high rates of morphologic complete response (CR) in adults w/ newly diagnosed Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). However, only ~20% of patients (pts) achieve BCR-ABL1 response depth of Methods: We conducted a phase I trial (NCT02494882) w/ accelerated dose escalation of RUX. Pts w/ newly diagnosed Ph+ ALL received CS pre-phase (d -6 to 0) followed by DAS 140 mg daily (starting d1) in combination w/ dexamethasone (DEX) 10 mg/m2 PO daily (d1-24, then taper) and RUX (d1-84). RUX doses were 10 mg PO BID (n=1), 15 mg PO BID (n=1), 20 mg PO BID (n=1), and 25 mg PO BID (n=7). IT methotrexate was given as CNS prophylaxis (d22, 43, 64, 85). Post-induction therapy was left to the discretion of the treating physician. The primary objective was to determine the recommended phase 2 dose (RP2D) of RUX in this combination. Secondary objectives included assessment of response kinetics, including minimal residual disease (MRD) negativity by flow cytometry (FACS) and BCR-ABL1 transcript monitoring, and ABL kinase mutation development. Results: Ten pts enrolled (6M, 4F). Median age at time of screening bone marrow (BM) studies was 63.8 yrs (range, 25.7-77.4; 7/10 age ≥60). BCR-ABL1 transcripts were p190 (n=7), p210 (n=2), atypical (n=1). No dose-limiting toxicity (DLT) has been observed. Enrollment was expanded at the highest RUX dose level (25 mg PO BID). Grade (Gr) 3-4 non-hematologic toxicities possibly/probably attributable to DAS/RUX/CS were limited to Gr 3 hyperglycemia in the setting of CS (n=5), Gr 3 hypofibrinogenemia (n=1), and Gr 3 peripheral edema (n=1). Two pts discontinued DAS after completing the 84d induction due to symptomatic pleural effusions. All pts achieved morphologic CR or CR w/ incomplete hematologic recovery (CRi). Median log reduction in BCR-ABL1 transcript levels (Figure 1A) was 2.7 at d22 (range, 1.3-4.2, n=9), 3.4 at d43 (range, 1.0-CMR, n=8), and CMR at d85 (range, 3.2-CMR, n=5). By FACS, 7/10 achieved MRD Among 6 pts completing the 84d induction, next post-remission therapy included tyrosine kinase inhibitor (TKI) + concurrent blinatumomab (n=3), TKI + hyperCVAD (n=1), and allogeneic hematopoietic cell transplantation (alloHCT, n=1). Of the 4 pts not completing the 84d induction, next therapy included ponatinib + blinatumomab (n=2), ponatinib + hyperCVAD (n=1), or DAS alone (n=1). Overall, 3 pts experienced morphologic relapse, which was observed in the setting of T315I (n=2) or F317L (n=1) ABL kinase mutations. Median EFS and OS (Figure 1B&C) are not yet reached in this small group of pts at median follow-up of 11.2 months among survivors (range, 2.8-20.7). Conclusions: Chemotherapy sparing-induction consisting of DAS, RUX, and initial DEX was well tolerated among adults w/ newly-diagnosed Ph+ ALL and led to CR/CRi in all pts. The RP2D of RUX is 25 mg PO BID. Kinetics of BCR-ABL1 transcript reduction compare favorably w/ other series of TKI + steroid-based induction, w/ rapid achievement of median >3 log reduction in BCR-ABL1 transcripts. Correlation of response kinetics w/ JAK/STAT pathway inhibition may suggest mechanisms of resistance apart from ABL kinase mutations. In future chemotherapy-sparing induction approaches, incorporating ponatinib, asciminib, or other novel therapies (vs. DAS) during induction may suppress leukemic clones bearing T315I mutations and reduce early relapse rates. Disclosures Geyer:Dava Oncology: Honoraria. King:Genentech: Other: Advisory Board . Mauro:Bristol-Myers Squibb: Consultancy; Novartis: Consultancy, Research Funding; Pfizer: Consultancy; Takeda: Consultancy. Tallman:BioSight: Other: Advisory board; AROG: Research Funding; AbbVie: Research Funding; Cellerant: Research Funding; ADC Therapeutics: Research Funding; Daiichi-Sankyo: Other: Advisory board; Orsenix: Other: Advisory board. Levine:Gilead: Honoraria; Isoplexis: Equity Ownership; Epizyme: Patents & Royalties; Roche: Consultancy, Research Funding; Loxo: Consultancy, Equity Ownership; Novartis: Consultancy; C4 Therapeutics: Equity Ownership; Qiagen: Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; Prelude: Research Funding; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Research Funding; Imago: Equity Ownership. Douer:Jazz Pharmaceuticals: Consultancy; Gilead Sciences: Consultancy; Shire: Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Pfizer: Honoraria; Spectrum: Consultancy. Park:Novartis: Consultancy; Kite Pharma: Consultancy; Adaptive Biotechnologies: Consultancy; AstraZeneca: Consultancy; Juno Therapeutics: Consultancy, Research Funding; Amgen: Consultancy, Membership on an entity9s Board of Directors or advisory committees; Pfizer: Consultancy; Shire: Consultancy.

Multiparameter Single Cell Analysis to Identify Immune Cell States That May Contribute to Successful Treatment Discontinuation in Patients with Chronic Myeloid Leukemia

Background: Recently, numerous clinical trials have demonstrated that approximately half of CML patients who have achieved deep and durable remissions on TKI therapy can enjoy prolonged treatment-free remissions. A major question in the field concerns why treatment interruption is not universally successful. Two non-mutually exclusive theories have been proposed to explain successful discontinuation: CML stem cell exhaustion, and immune surveillance. Identification of mediators of CML stem cell exhaustion or successful immune surveillance could lead to adjunctive therapeutic approaches. We set out to determine potential immune mediators of disease control through interrogation of clinical samples using emerging technologies.Results: Consenting patients who discontinued TKI at our institution were grouped into three categories based upon BCR-ABL1 transcript kinetics following treatment discontinuation. Group 1 is characterized by stable undetectable or minimally detectable BCR-ABL1 transcripts. Group 2 exhibited molecular relapse, defined as loss of major molecular response (MMR) requiring treatment re-initiation, typically within the first 3-9 months of treatment discontinuation. Group 3 displays kinetics that are particularly intriguing, whereby the transcript level rises substantially upon treatment cessation, but remains below the MMR threshold despite remaining off TKI therapy. We hypothesize that disease control in group 3, and a subset of patients in Group 1, is mediated by the immune system.To investigate the immune system difference between these groups, we performed single cell immune profiling in the pre- (~ 1 week) and post- (~ 3 months) discontinuation PBMC samples obtained from 15 patients through CyTOF and 10x single cell RNA sequencing. Group 1 consist of 5 patients, group 2 consist of 8 patients, and group 3 consist of 2 patients, respectively. There were no meaningful differences in the immune cell composition prior to TKI discontinuation. Assessment of the immune phenotype 3 months after discontinuation in each group by CyTOF revealed an increase in the CD4+ T cell population in group 3 following TKI discontinuation. Specifically, memory CD4+ T cells and CD38+/HLA-DR+ activated CD4+ T cells were increased in group 3. While patients in groups 1 and 2 showed only decrease or moderate change in memory CD4+ T cells and activated CD4+ T cells, group 3 showed 53% increase of memory CD4+ T cells (21% to 32%) and 640% increase of activated CD4+ T cells (0.4% to 3.3%) following TKI discontinuation. While CD45RA-/CD4+ T cells were increased in abundance, the CD45RA- population was unchanged in CD8+ T cells. 10x single cell RNA sequencing of these samples is ongoing and will be presented.Conclusions: We have conducted multiparameter single cell immune profiling in CML patients with sustained deep molecular remission, loss of MMR, or maintenance of MMR despite a sustained rise in BCR-ABL1 transcript level. Our results suggest that an increase in memory and activated CD4+ T cells following TKI discontinuation may correlate with successful immune surveillance of CML. Assessment of a larger cohort of samples is required to confirm our findings. DisclosuresShah:ARIAD: Research Funding; Bristol-Myers Squibb: Research Funding.

BCR-ABL Kinase Domain Mutations Exist in Few Imatinib Treated Chronic Myeloid Leukemia (CML) Patients Exhibiting Stable Cytogenetic and Hematologic Responses

Introduction: Complete hematologic response (CHR) and complete cytogenetic response (CCyR) is observed in almost 80-90% freshly diagnosed chronic phase chronic myeloid leukemia patients treated with Imatinib, a tyrosine kinase inhibitor (TKI). The responses are usually persistent; however the residual disease may be detected molecularly by amplifying BCR-ABL transcript. Furthermore, the leukemogenic potential of the residual BCR-ABL positive cells is reflected when disease reoccurs after discontinuation of therapy. There are evidences of occurrence of mutations in patients who have achieved CCyR, which indicates their role in disease persistence and drug resistance.Patients and Methods: We performed BCR-ABL kinase domain (KD) mutation screening in 150 Imatinib treated CML patients (either resistant or sensitive to therapy) including 84 (56%) individuals manifesting CHR and and 67 (44.7%) with CCyR. For this purpose, nested reverse transcription PCR followed by direct sequence analysis was implemented as previously described. Statistical analysis was performed using SPSS version 19 (IBM, Chicago, IL, USA).Results and Conclusion: Thirty five of 150 patients exhibited mutations in BCR-ABL KD, out of which 11 (31.4%) had achieved CHR and 08 (22.9%) attained CCyR. Single rather than multiple mutated sites in ABL-KD was observed in most of mutant patients achieving CHR (27.3%) or CCyR (27.3%) (Figure 1). Eleven nucleotide substitutions were observed in more than one patient, including Imatinib sensitive individuals with CHR and CCyR (Table 1). Those include C-Helix, SH2 contact, Imatinib binding site (IBS), and A-loop mutations within ABL kinase domain. Overall survival among chronic myeloid leukemia patients achieving different levels of hematologic and cytogenetic response irrespective of mutation status show a favorable survival outcome in those attaining CHR and CCyR (Figure 2). Presence of mutations in cytogenetically stable patients of CML with good hematologic outcomes indicates a likely suboptimal response or TKI resistance during the course of treatment. It is believed by some researchers that mutations do not exist previously, but emerge after Imatinib exposure. Whereas, some believe that TKI's do not induce mutations, rather they select them. Many studies reported a rise in BCR-ABL transcript levels during follow up studies in patients who co-exhibited mutated ABL-KD and CCyR. This suggests that emergence of mutations is a disease evolution process independent of TKI therapy. Studies report that monitoring of nucleotide substitutions in optimally responding patients plays an important role in well managed therapeutic approach. Our studies reveal occurrence of ABL-KD mutations in patints with stable hematologic and cytogenetic response, therefore we suggest for regular mutation screening in CML paients irrespective of their response towards TKIs. DisclosuresNo relevant conflicts of interest to declare.