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Recently, we defined “CML-like” subtype of BCR::ABL1-positive acute lymphoblastic leukemia (ALL), resembling lymphoid blast crisis of chronic myeloid leukemia (CML). Here we retrospectively analyzed prognostic relevance of minimal residual disease (MRD) and other features in 147 children with BCR::ABL1-positive ALL (diagnosed I/2000–IV/2021, treated according to EsPhALL (n = 133) or other (n = 14) protocols), using DNA-based monitoring of BCR::ABL1 genomic breakpoint and clonal immunoglobulin/T-cell receptor gene rearrangements. Although overall prognosis of CML-like (n = 48) and typical ALL (n = 99) was similar (5-year-EFS 60% and 49%, respectively; 5-year-OS 75% and 73%, respectively), typical ALL presented more relapses while CML-like patients more often died in the first remission. Prognostic role of MRD was significant in the typical ALL (p = 0.0005 in multivariate analysis for EFS). In contrast, in CML-like patients MRD was not significant (p values > 0.2) and inapplicable for therapy adjustment. Moreover, in the typical ALL, risk-prediction could be further improved by considering initial hyperleukocytosis. Early distinguishing typical BCR::ABL1-positive ALL and CML-like patients is essential to enable optimal treatment approach in upcoming protocols. For the typical ALL, tyrosine-kinase inhibitors and concurrent chemotherapy with risk-directed intensity should be recommended; in the CML-like disease, no relevant prognostic feature applicable for therapy tailoring was found so far.

Several studies have reported that chronic myeloid leukaemia (CML) patients expressing e14a2 BCR::ABL1 have a faster molecular response to therapy compared to patients expressing e13a2. To explore the reason for this difference we undertook a detailed technical comparison of the commonly used Europe Against Cancer (EAC) BCR::ABL1 reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assay in European Treatment and Outcome Study (EUTOS) reference laboratories (n = 10). We found the amplification ratio of the e13a2 amplicon was 38% greater than e14a2 (p = 0.015), and the amplification efficiency was 2% greater (P = 0.17). This subtle difference led to measurable transcript-type dependent variation in estimates of residual disease which could be corrected by (i) taking the qPCR amplification efficiency into account, (ii) using alternative RT-qPCR approaches or (iii) droplet digital PCR (ddPCR), a technique which is relatively insensitive to differences in amplification kinetics. In CML patients, higher levels of BCR::ABL1/GUSB were identified at diagnosis for patients expressing e13a2 (n = 67) compared to e14a2 (n = 78) when analysed by RT-qPCR (P = 0.0005) but not ddPCR (P = 0.5). These data indicate that widely used RT-qPCR assays result in subtly different estimates of disease depending on BCR::ABL1 transcript type; these differences are small but may need to be considered for optimal patient management.

This work investigated patient-specific genomic BCR-ABL1 fusions as markers of measurable residual disease (MRD) in chronic myeloid leukaemia, with a focus on relevance to treatment-free remission (TFR) after achievement of deep molecular response (DMR) on tyrosine kinase inhibitor (TKI) therapy. DNA and mRNA BCR-ABL1 measurements by qPCR were compared in 2189 samples (129 patients) and by digital PCR in 1279 sample (62 patients). A high correlation was found at levels of disease above MR4, but there was a poor correlation for samples during DMR. A combination of DNA and RNA MRD measurements resulted in a better prediction of molecular relapse-free survival (MRFS) after TKI stop (n = 17) or scheduled interruption (n = 25). At 18 months after treatment cessation, patients with stopped or interrupted TKI therapy who were DNA negative/RNA negative during DMR maintenance (green group) had an MRFS of 80% and 100%, respectively, compared with those who were DNA positive/RNA negative (MRFS = 57% and 67%, respectively; yellow group) or DNA positive/RNA positive (MRFS = 20% for both cohorts; red group). Thus, we propose a “traffic light” stratification as a TFR predictor based on DNA and mRNA BCR-ABL1 measurements during DMR maintenance before TKI cessation.

Background Effectiveness of L-asparaginase administration in acute lymphoblastic leukemia treatment is mirrored in overall outcome of patients. Generally, leukemia patients differ in their sensitivity to L-asparaginase; however, the mechanism underlying their inter-individual differences is still not fully understood. We have previously shown that L-asparaginase rewires the biosynthetic and bioenergetic pathways of leukemia cells to activate both anti-leukemic and pro-survival processes. Herein, we investigated the relationship between the metabolic profile of leukemia cells and their sensitivity to currently used cytostatic drugs.Methods Altogether, 19 leukemia cell lines and primary leukemia cells from 11 patients were used. Glycolytic function and mitochondrial respiration were measured using Seahorse bioanalyzer. Sensitivity to cytostatics was measured using MTS assay and/or absolute count and flow cytometry. Mitochondrial membrane potential was determined as TMRE fluorescence.Results We characterized the basal metabolic state of the cells derived from different leukemia subtypes using cell lines and primary samples and assessed their sensitivity to cytostatic drugs. We found that leukemia cells cluster into distinct groups according to their metabolic profile, which is mainly driven by their hematopoietic lineage of origin from which they derived. However, majority of lymphoid leukemia cell lines and patients with lower sensitivity to L-asparaginase clustered regardless their hematopoietic phenotype together with myeloid leukemias. Furthermore, we observed a correlation of specific metabolic parameters with sensitivity to L-asparaginase. Greater ATP-linked respiration and lower basal mitochondrial membrane potential in cells significantly correlated with higher sensitivity to L-asparaginase. No such correlation was found in other tested cytostatic drugs.Conclusions These data support the prominent role of the cell metabolism in the treatment effect of L-asparaginase. Based on these findings metabolic profile could identify leukemia patients with lower sensitivity to L-asparaginase with no specific genetic characterization.

Acute lymphoblastic leukaemias (ALL) with 51-67 chromosomes are defined as high hyperdiploid (HHD) and are generally associated with good prognosis. However, several studies show heterogeneity in HHD ALL and suggest that the favourable prognosis is associated rather with higher ploidy defined by DNA index (DNAi) ≥ 1.16 or with a presence of specific single or combined trisomies. HHD ALL with DNAi < 1.16 are only rarely studied separately. Using single nucleotide polymorphism array, we analysed 89 childhood HHD ALL patients divided into groups with lower (

We used the genomic breakpoint between

Introduction: In chronic myeloid leukemia (CML) resistant to tyrosine kinase inhibitors (TKI), detection of mutations in the BCR-ABL1 kinase domain (KD) is routinely performed on transcript level. To determine the level of BCR-ABL1 KD mutation is important to follow kinetics of resistant CML cells and therapeutically prevent progression. However, the mutation types and levels are not always reliable predictors of subsequent dynamics of mutation-bearing clones and of corresponding clinical consequences (Willis, 2005; Khorashad, 2006; Preuner, 2012). DNA analysis enables more precise quantification of (sub)clonal levels and thus might be more reliable approach to monitor dynamics of BCR-ABL1 KD mutations. Aim: To study clonal evolution of resistant CML cells using genomic quantification of mutated BCR-ABL1 KD by droplet digital PCR (ddPCR). Methods: BCR-ABL1 mutation analysis on transcript level was performed using next generation sequencing (NGS) (Nextera XT; Illumina) and on DNA level using allele-specific ddPCR assays detecting T315I, E255K and Y253H (Bio-Rad). The level of genomic BCR-ABL1 mutation was determined as a copy number of mutation divided by a copy number of genomic BCR-ABL1 fusion. Quantification of genomic BCR-ABL1 was performed by ddPCR using patient-specific primers and probes designed to detect individual fusions. ALB (albumin) quantification was used as a control of DNA load/cell numbers. For analyses, mRNA and DNA extracted from KCL-22 cell line resistant to imatinib (IM) and from leukocytes of a patient who developed T315I during TKI therapy were used. Results: KCL-22 cell line is characterized by 2 Ph chromosomes and by ability to develop resistance by acquisition of BCR-ABL1 mutations early after the exposure to IM. We repeatedly found, that during early cultivation in the presence of IM, BCR-ABL1-T315I transcript increased up to maximum of 50%. Subsequently, after 2 months, BCR-ABL1-E255K transcripts became detectable and increased over time to 100%, while T315I decreased to un-detectable levels. To study the observed kinetics, we isolated 4 clones resistant to 4 µM IM that expressed 1) 50% of T315I, 2) 50% of E255K and 3) 30% of Y253H. In the fourth clone, no BCR-ABL1 mutation was detected, but mutation acquisition was found in KRAS, RUNX1 and ATRX. The levels of mutated BCR-ABL1 transcripts in mutation bearing clones remained stable over time. DNA analyses confirmed the same level of mutated BCR-ABL1 and revealed that in all resistant clones, only 1 Ph chromosome carried the BCR-ABL1 mutation (T315I, E255K or Y253H). Based on quantification of genomic BCR-ABL1 fusion and albumin we found, that the un-mutated BCR-ABL1 fusion was duplicated in Y253H clone, explaining the 30% level of Y253H. To follow a clonal evolution, we mixed the 4 KCL-22 resistant clones and analyzed BCR-ABL1 KD mutations at both mRNA and DNA levels during exposition to IM. We found that T315I clone overgrew other 3 clones in the mixture over time and 1 Ph chromosome remained mutated. These data confirm the T315I mutation being the most resistant; however, the data from the original cell culture, where the 100% E255K clone overgrew the 50% T315I cells, demonstrate, that a less resistant mutation might dominate the culture if present on both Ph chromosomes (as revealed by DNA analysis). We compared mRNA and DNA approach in 14 samples collected during individualized treatment management of a CML patient, who developed T315I during TKI therapy. The first mutation detection was during warning response preceded by eight samples negative by mRNA-NGS approach; DNA ddPCR analysis reliably detected T315I mutation in 7 of these 8 samples. Six mRNA positive samples were positive by DNA approach, which showed the same level of T315I. Conclusions: Allele-specific ddPCR together with quantification of BCR-ABL1 genomic fusion represents highly sensitive and reliable method providing fast and precise quantification of BCR-ABL1 mutations. A single DNA analysis is able to uncover clinically relevant events including BCR-ABL1 amplification or additional mutation acquisition, which presumably influence fitness of leukemic cells and clonal evolution during therapeutic interventions. The information provided by DNA mutational analysis may thus refine prediction of mutation kinetics and consequently improve management of progressed CML and Ph+ ALL. Support GACR 18-18407S, MZCR 00023736, AZV 15-31540, AZV 16-30186A Disclosures Klamova: Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Ernst:Novartis: Research Funding. Soverini:Incyte Biosciences: Consultancy; Novartis: Consultancy; Bristol Myers Squibb: Consultancy. Machova:Bristol-Myers Squibb: Consultancy, Other: Educational grant funding; Incyte: Consultancy; Novartis: Consultancy.

Acute lymphoblastic leukaemias (ALL) with 51-67 chromosomes are defined as high hyperdiploid (HHD) and are generally associated with good prognosis. However, several studies show heterogeneity in HHD ALL and suggest that the favourable prognosis is associated rather with higher ploidy defined by DNA index (DNAi) ≥ 1.16 or with a presence of specific single or combined trisomies. HHD ALL with DNAi 1.16 are only rarely studied separately. Using single nucleotide polymorphism array, we analysed 89 childhood HHD ALL patients divided into groups with lower (1.16; n = 34) and higher (≥1.16; n = 55) DNAi. We assessed treatment response, presence of secondary aberrations, mutations in RAS pathway genes and CREBBP and also gene expression profile (GEP) to reveal differences between the two subgroups. Cases with 51-54 chromosomes had DNAi 1.1-1.16 and cases with 55-67 chromosomes had DNAi ≥ 1.16. The groups with lower and higher DNAi had distinct response to early treatment and distinct GEP. The better response of the group with higher DNAi was associated with specific trisomies (trisomy of chromosome 10 or combined with trisomies 4 and/or 17). Our results suggest that cytogenetically defined HHD ALL can in fact be divided into two biologically distinguishable subgroups and that DNAi 1.16 is a relevant value to separate between the two. © 2016 Wiley Periodicals, Inc.

Background Both high hyperdiploidy (HeH) and the translocation t(9;22)(q34;q11) are recurrent abnormalities in childhood B-cell acute lymphoblastic leukemia (ALL) and both are used in current classification to define different genetic and prognostic subtypes of the disease. The coexistence of these two primary genetic aberrations within the same clone is very rare in children with ALL. Here we report a new case of a 17-year-old girl with newly diagnosed ALL and uncommon cytogenetic and clinical finding combining high hyperdiploidy and a cryptic BCR/ABL1 fusion and an inherited Charcot-Marie-Tooth neuropathy detected during the induction treatment. Results High hyperdiploid karyotype 51,XX,+X,+4,+14,+17,+21 without apparent structural aberrations was detected by conventional cytogenetic analysis and multicolor FISH. A cryptic BCR/ABL1 fusion, which was caused by the insertion of part of the ABL1 gene into the 22q11 region, was proved in HeH clone by FISH, RT-PCR and CGH-SNP array. In addition, an abnormal FISH pattern previously described as the deletion of the 3′BCR region in some BCR/ABL1 positive cases was not proved in our patient. Conclusion A novel case of extremely rare childhood ALL, characterized by HeH and a cryptic BCR/ABL1 fusion, is presented and to the best of our knowledge described for the first time. The insertion of ABL1 into the BCR region in malignant cells is supposed. Clearly, further studies are needed to determine the genetic consequences and prognostic implications of these unusual cases.

Background: Acute lymphoblastic leukemias (ALL) with 51-67 chromosomes in leukemic cells are defined as high-hyperdiploid (HHD). In childhood, these leukemias comprise 25-30% of all cases, typically arise from B lymphocyte precursors and are generally associated with good prognosis. Besides the number of chromosomes, the hyperdiploid ALLs can be determined also by DNA index (established by flow cytometry), representing ratio of DNA content in leukemic vs. normal diploid cell. Cases with DNA index >=1.16 and 50 chromosomes and DNA index = 1.16) and, according to some studies, particularly with cases characterized by "triple-trisomy", i.e. concurrent gain of chromosomes 4, 10 and 17. However, as the triple-trisomy significantly overlaps with HDI-HHD, it is not clear whether the favorable prognosis is driven rather by the triple-trisomy itself or by the higher ploidy of these patients (with most cases having DNA index >= 1.16). In this study we aimed to analyze biological and clinical features of HHD leukemias, compare LDI-HHD and HDI-HHD cases and verify prognostic role of triple-trisomy of chromosomes 4, 10 and 17. Patients and methods: We tested 75 patients with hyperdiploid childhood ALL defined by the presence of 51-67 chromosomes (46 HDI-HHD, 29 LDI-HHD). To determine the type of hyperdiploidy we used flow cytometry (DNA index) and whole genome single nucleotide polymorphism (SNP) array. SNP array also enabled precise determination of amplified chromosomes as well as partial gains and losses and calculation of a SNP array-based "theoretical" DNA index. From clinical features we analyzed final risk stratification of patients (based in this subgroup on early treatment response measured at day +8 by morphology ("prednisone response"), at day +15 by flow cytometry and at day +33 and week +12 by PCR quantification of immunoglobulin a T-cell receptor rearrangements) and their outcome. Results: Our data show that correlation of DNA content in leukemic cells determined by flow cytometry and by SNP-array is very high (Spearman correlation: rho = 0.96; p-value < 2,2e-16). Besides the HHD patients we analyzed DNA index and SNP array in 53 non-high hyperdiploid patients ( Conclusion: High hyperdiploidy can be determined by karyotype, SNP array and also by flow cytometry, where cases with DNA index > 1.1 are highly likely to carry > 50 chromosomes. Patients with high hyperdiploidy over 50 chromosomes form a subgroup of childhood ALL with a generally very good prognosis. However, some heterogeneity within this group is present. Our data suggest that patients with LDI-HHD are more often stratified into high risk treatment. On the other hand, patients with triple-trisomy of chromosomes 4, 10 and 17 are characterized by a rapid response to initial therapy, which is not just a result of coexisting HDI-HHD status. Supported by grant IGA MZ NT14350/3. Disclosures No relevant conflicts of interest to declare.

Background: The exact role of minimal residual disease (MRD) testing in childhood BCR-ABL1+ ALL is still unclear and MRD levels are used rather for stem cell transplantation (SCT) indication than for frontline treatment stratification. There are two standard targets used for MRD monitoring in these ALLs: Ig/TR rearrangements and the fusion transcript quantification. However, despite similar sensitivity of the methods, we previously showed a poor correlation between them in some patients. Overall, 20% of samples negative by Ig/TR MRD were BCR-ABL1 positive (even at high levels). By demonstrating presence of BCR-ABL1 within the non-lymphoid (Ig/TR-negative) population in the non-correlating samples we showed that multilineage involvement is at least partly responsible for the discrepancy (Zaliova, Leukemia 2009). Here we established MRD monitoring based on the quantification of the patient-specific genomic (intronic) BCR-ABL1 breakpoint sequence, compared MRD levels obtained from different approaches and analyzed their prognostic significance. Methods: Our cohort totals 35 children diagnosed with minor-BCR-ABL1+ ALL. The BCR-ABL1 genomic breakpoint was found using multiplex long distance DNA PCR. The patient-specific intronic fusion sequences were used for MRD quantification by qPCR. We quantified MRD levels in 386 bone marrow (BM) samples. The results of genomic BCR-ABL1 quantification were compared with Ig/TR and BCR-ABL1 transcript levels. Samples with >1 log difference in MRD levels were considered non-correlating. For correlation analysis of methods, double-negative (DN) samples were excluded. Overall survival (OS) rates were calculated according to Kaplan-Meier and compared by log-rank test. Results: Analysis of MRD in BM samples confirmed poor correlation between Ig/TR and BCR-ABL1 mRNA quantification (Spearman correlation coefficient R=0.70; n=166 non-DN samples). Moreover, we also saw poor correlation when comparing the two DNA methods (Ig/TR vs. BCR-ABL1 DNA, R=0.69; n=254 non-DN samples) with 21% of samples positive by BCR-ABL1 (at levels up to 10e-1) while negative by Ig/TR. While in some patients the correlation of the DNA-based methods was very good, others had several subsequent follow-up samples with poor correlation (> 1 log difference) suggesting distinct biology of the disease. MRD levels determined by BCR-ABL1 DNA quantification had the best predictive value for overall survival (the best separation of survival curves was at week 12 of treatment with 10e-3 cut-off: BCR-ABL1 DNA, p=0.0003; Ig/TR, p=0.017; BCR-ABL1 mRNA, not significant). The proportion of samples with BCR-ABL1 DNA levels higher than Ig/TR by more than 1 log increased with time on treatment (day 15: 10% (2/20); day 33: 26% (7/27); week 12: 39% (7/18); week 22: 50% (7/14)). Importantly, presence of samples with this poor correlation was prognostically relevant as patients with BCR-ABL1 levels higher than Ig/TR by more than 1 log at the end of induction treatment (day 33) had significantly worse outcome (2 years OS 84+/-9% (n=21) vs. 28+/-23% (n=6) for correlating and non-correlating patients, respectively; p=0.004). Conclusion: The BCR-ABL1 quantification at genomic level probably provides the most accurate measurement of leukaemic burden. Due to multi-lineage involvement in some Ph+ ALL as well as occasional presence of subclones with different Ig/TR markers, quantification of the BCR-ABL1 gene breakpoint is more reliable than Ig/TR monitoring. Moreover, in contrast to the BCR-ABL1 transcript, measurement of the gene is not influenced by possible variability in expression between cell subtypes or during concurrent treatment. Importantly, the MRD levels determined by BCR-ABL1 DNA-based testing have higher predictive value than Ig/TR DNA or BCR-ABL1 transcript quantification. The poorer outcome observed in the subset of patients in whom BCR-ABL1 DNA-based MRD levels were higher than Ig/TR-based MRD detection possibly reflects multilineage involvement, suggesting, that some cases with minor-BCR-ABL1+ ALL are in fact CML cases identified in a lymphoid blast crisis. In view of their poor outcome, these cases might be candidates for early SCT or alternative treatments. The most reliable method for MRD detection in BCR-ABL1+ ALL needs to be further investigated within a large therapeutic protocol. Support: GAUK 554214; MH CZ-DRO UH Motol 00064203; NHMRC Australia APP1057746. Disclosures Machova Polakova: Bristol Myers-Squibb: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Introduction - Targeted therapy of chronic myeloid leukemia (CML) using tyrosine kinase inhibitors (TKI) very effectively suppresses the growth of leukemic clone to a clinically safe level of the disease. Therefore sensitive monitoring of molecular response (MR) based on standardized measurements of BCR-ABL1 transcript levels plays an important role for prediction either of optimal response or progression. Thus, its sensitive and accurate detection is very important for the individualized therapy, especially for a TKI stopping treatment management in patients achieving long-term deep MR. While the transcript levels may not exactly correspond to the number of leukemic cells, BCR-ABL1 quantification at the DNA level using patient-specific assays may bring more accurate information about residual disease. Moreover, BCR-ABL1 DNA quantification may contribute to a stratification of patients for whom the cessation of TKI therapy may be safe. Objectives - This work is focused on the comparison of BCR-ABL1 quantity at DNA and mRNA level in CML patients after TKI initiation until the MR achievement, and after TKI discontinuation within the EURO-SKI clinical trial (Europe Stops TKI in CML), respectively, and on determination of differences on BCR-ABL1 positivity/negativity of samples analyzed. Additionally, the BCR-ABL1 DNA data obtained from the measurements by qPCR and droplet digital PCR (ddPCR) were compared. Methods - Our study included analyses of 232 samples of 13 patients with CML in chronic phase (CP), who achieved deep MR during TKI treatment. Four of 13 patients (total of 85 samples analyzed) discontinued the treatment within the EURO-SKI study. The levels of BCR-ABL1 mRNA were determined by standardized RT-qPCR method and DNA levels were detected by qPCR with developed patient-specific qPCR assays after BCR-ABL1 genomic fusions characterization. The ddPCR method was performed on 72 samples using QX-200 Droplet Digital PCR System (Bio-Rad). The determined ratios of BCR-ABL1/GUSB (mRNA) and BCR-ABL1/albumin (DNA level) of follow-up samples of every patient were related to the diagnostic sample (considered as 100% level) for the most appropriate comparison of mRNA and DNA levels. Results - Standardized RT-qPCR and patient-specific qPCRs were able to detect the mRNA and DNA BCR-ABL1 levels, respectively, reduced by more than 5 logs from the levels at the time of diagnosis. We observed a significant correlation between the mRNA and DNA levels by comparing 148 paired BCR-ABL1 positive results (correlation coefficient r2=0.9055; P˂0.0001). When comparing the frequency of the BCR-ABL1 negative/positive results at the DNA vs. mRNA levels, BCR-ABL1 positivity at DNA level was found in 21 samples which were BCR-ABL1-negative at mRNA level. BCR-ABL1 mRNA was found either negative or positive at MR5.0 in 3/4 EURO-SKI patients within the period between MR achievement until the TKI discontinuation (19 samples analyzed). The level of BCR-ABL1 DNA was continuously positive in this period before the TKI cessation. The molecular relapse (i.e. MMR loss) was observed in all those 3 patients several months after the therapy cessation. The levels of BCR-ABL1 DNA detected by ddPCR method correlated with DNA qPCR results (r2=0.9684; P˂0.0001) and the obtained values were not significantly different. Conclusion - The levels of the BCR-ABL1 mRNA and DNA in peripheral blood (PB) correlated significantly, thus both consistently reflecting the course of CP-CML at the molecular level. Although much more patients with TKI cessation need to be tested for the presence of BCR-ABL1 DNA, our data suggest that quantification at the DNA level appears to be (at least in some cases) more sensitive method for detection of residual leukemic cells in PB in comparison to the mRNA quantification. The BCR-ABL1 DNA measurement may have a potential prognostic significance in deciding for eventual withdrawal of TKI therapy, which is currently being investigated within the clinical trials. Digital PCR enables absolute quantification without a need of patient-specific calibration curves and with the potentially higher sensitivity and accuracy for BCR-ABL1 DNA quantification. This work was supported by the project 15-31540A of the Czech Health Research Council, GAUK 554214 and EURO-SKI Research Consortium. Disclosures Klamova: Novartis: Consultancy, Honoraria, Research Funding; Bristol Myers-Squibb: Consultancy, Honoraria, Research Funding. Saussele:ARIAD: Honoraria; Pfizer: Honoraria, Other: Travel grant; BMS: Honoraria, Other: Travel grant, Research Funding; Novartis Pharma: Honoraria, Other: Travel grant, Research Funding. Mahon:Pfizer: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Machova Polakova:Bristol Myers-Squibb: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.