DNA methylation profiling of myelodysplastic syndromes and clinical response to azacitidine: A multicentre retrospective study.

Real-world data have revealed that a substantial portion of patients with myelodysplastic syndromes (MDS) does not respond to epigenetic therapy with hypomethylating agents (HMAs). The cellular and molecular reasons for this resistance to the demethylating agent and biomarkers that would be able to predict the treatment refractoriness are largely unknown. In this study, we shed light on this enigma by characterizing the epigenomic profiles of patients with MDS treated with azacitidine. Our approach provides a comprehensive view of the evolving DNA methylation architecture of the disease and holds great potential for advancing our understanding of MDS treatment responses to HMAs.

e14a2 Transcript Favors Treatment-Free Remission in Chronic Myeloid Leukemia When Associated with Longer Treatment with Tyrosine Kinase Inhibitors and Sustained Deep Molecular Response.

e13a2 and e14a2 are the most frequent transcript types of the BCR::ABL1 fusion gene in chronic myeloid leukemia (CML). The current goal with tyrosine kinase inhibitors (TKI) is to achieve sustained deep molecular response (DMR) in order to discontinue TKI treatment and remain in the so-called treatment-free remission (TFR) phase, but biological factors associated with these goals are not well established. This study aimed to determine the effect of transcript type on TFR in patients receiving frontline treatment with imatinib (IM) or second-generation TKI (2G-TKI). Patients treated at least 119 months with IM presented less post-discontinuation relapse than those that discontinued IM before 119 months (p = 0.005). In addition, cases with the e14a2 transcript type treated at least 119 months with IM presented a better TFR (p = 0.024). On the other hand, the type of transcript did not affect the cytogenetic or molecular response in 2G-TKI treated patients; however, the use of 2G-TKI may be associated with higher and earlier DMR in patients with the e14a2 transcript.

Optimization of monocyte gating to quantify monocyte subsets for the diagnosis of chronic myelomonocytic leukemia.

BACKGROUND: The presence of >94% classical monocytes (MO1, CD14++/CD16-) in peripheral blood (PB) has an excellent performance for the diagnosis of chronic myelomonocytic leukemia (CMML). However, the monocyte gating strategy is not well defined. The objective of the study was to compare monocyte gating strategies and propose an optimal one. METHODS: This is a prospective, single center study assessing monocyte subsets in PB. First, we compared monocyte subsets using 13 monocyte gating strategies in 10 samples. Then we developed our own 10 color tube and tested it on 124 patients (normal white blood cell counts, reactive monocytosis, CMML and a spectrum of other myeloid malignancies). Both conventional and computational (FlowSOM) analyses were used. RESULTS: Comparing different monocyte gating strategies, small but significant differences in %MO1 and percentually large differences in %MO3 (nonclassical monocytes) were found, suggesting that the monocyte gating strategy can impact monocyte subset quantification. Then, we designed a 10-color tube for this purpose (CD45/CD33/CD14/CD16/CD64/CD86/CD300/CD2/CD66c/CD56) and applied it to 124 patients. This tube allowed proper monocyte gating even in highly abnormal PB. Computational analysis found a higher %MO1 and lower %MO3 compared to conventional analysis. However, differences between conventional and computational analysis in both MO1 and MO3 were globally consistent and only minimal differences were observed when comparing the ranking of patients according to %MO1 or %MO3 obtained with the conventional versus the computational approach. CONCLUSIONS: The choice of monocyte gating strategy appears relevant for the monocyte subset distribution test. Our 10-color proposal allowed satisfactory monocyte gating even in highly abnormal PB. Computational analysis seems promising to increase reproducibility in monocyte subset quantification.

Association between Germline Single-Nucleotide Variants in ADME Genes and Major Molecular Response to Imatinib in Chronic Myeloid Leukemia Patients.

Imatinib is the most common first-line tyrosine kinase inhibitor (TKI) used to treat chronic-phase chronic myeloid leukemia (CP-CML). However, only a proportion of patients achieve major molecular response (MMR), so there is a need to find biological factors that aid the selection of the optimal therapeutic strategy (imatinib vs. more potent second-generation TKIs). The aim of this retrospective study was to understand the contribution of germline single-nucleotide variants (gSNVs) in the achievement of MMR with imatinib. In particular, a discovery cohort including 45 CP-CML patients was analyzed through the DMET array, which interrogates 1936 variants in 231 genes related to the absorption, distribution, metabolism and excretion (ADME) process. Variants statistically significant in the discovery cohort were then tested in an extended and independent cohort of 137 CP-CML patients. Finally, a total of 7 gSNVs (ABCG1-rs492338, ABCB11-rs496550, ABCB11-rs497692, CYP2D6-rs1135840, CYP11B1-rs7003319, MAT1A-rs4934027 and SLC22A1-rs628031) and one haplotype in the ABCB11 gene were significantly associated with the achievement of MMR with first-line imatinibtreatment. In conclusion, we identified a genetic signature of response to imatinib in CP-CML patients that could be useful in selecting those patients that may benefit from starting imatinib as first-line therapy, therefore avoiding the toxicity related to second-generation TKIs.

Flow cytometry to detect bone marrow involvement by follicular lymphoma.

BACKGROUND: High-quality data on bone marrow involvement (BMI) assessed by flow cytometry (FC) in follicular lymphoma (FL) is lacking. AIMS: We set up a prospective protocol with a 10-color tube and acquisition of 500.000 leukocytes on a Nav flow cytometer for evaluation of BMI in FL by FC. MATERIALS AND METHODS: FC was compared with a combination of histopathology and IGH gene rearrangement, which were considered the gold standard. We also compared BMI by FC with PET. RESULTS: Fifty-two patients were included (median 67 years, 54% female). BMI by FC was seen in 35 (67%), with a median involvement of 1.2% (interquartile range: 0.3%-7%) of leukocytes. Comparison with the gold standard revealed two false negatives and two false positives (potentially true involvement undetected by the gold standard). BMI by PET was seen in 14/46 (30%). Immunophenotype of FL in the bone marrow was highly heterogeneous. The most common phenotypic abnormality was dim expression of CD19 (>0.5 log loss in 30% of patients). CD10 was negative in 13 (37%) and incompletely positive (overlap with the negative population) in a further 8 (28%) while entirely positive only in 14 (48%). Other abnormalities (loss of CD20, gain or loss of CD79b, expression of CD43, and substantial loss of CD45) were rare. Computational analysis by means of FlowSOM confirmed the heterogeneous phenotype, with FL from different patients clustering in unrelated metaclusters. CONCLUSION: BMI by FL was frequent and immunophenotype was heterogeneous. However, this protocol enabled detection of FL in bone marrow in the vast majority of patients with bone marrow involvement by the gold standard.

Influence of Telomere Length on the Achievement of Deep Molecular Response With Imatinib in Chronic Myeloid Leukemia Patients.

Tyrosine kinase inhibitors have dramatically changed the outcome of chronic myeloid leukemia (CML), and nowadays, one of the main treatment goals is the achievement of deep molecular responses (DMRs), which can eventually lead to therapy discontinuation approaches. Few biological factors at diagnosis have been associated with this level of response. Telomere length (TL) in peripheral blood cells of patients with CML has been related to disease stage, response to therapy and disease progression, but little is known about its role on DMR. In this study, we analyzed if age-adjusted TL (referred as "delta-TL") at diagnosis of chronic phase (CP)-CML might correlate with the achievement of DMR under first-line imatinib treatment. TL from 96 CP-CML patients had been retrospectively analyzed at diagnosis by monochrome multiplex quantitative PCR. We observed that patients with longer age-adjusted telomeres at diagnosis had higher probabilities to achieve DMR with imatinib than those with shortened telomeres (P = 0.035 when delta-TL was studied as a continuous variable and P = 0.047 when categorized by the median). Moreover, patients carrying long telomeres also achieved major molecular response significantly earlier (P = 0.012). This study provides proof of concept that TL has a role in CML biology and when measured at diagnosis of CP-CML could help to identify patients likely to achieve DMR to first-line imatinib treatment.

Divergent leukaemia subclones as cellular models for testing vulnerabilities associated with gains in chromosomes 7, 8 or 18.

Haematopoietic malignancies are frequently characterized by karyotypic abnormalities. The development of targeted drugs has been pioneered with compounds against gene products of fusion genes caused by chromosomal translocations. While polysomies are equally frequent as translocations, for many of them we are lacking therapeutic approaches aimed at synthetic lethality. Here, we report two new cell lines, named MBU-7 and MBU-8, that differ in complete trisomy of chromosome18, a partial trisomy of chromosome 7 and a tetrasomy of the p-arm of chromosome 8, but otherwise share the same mutational pattern and complex karyotype. Both cell lines are divergent clones of U-937 cells and have the morphology and immunoprofile of monocytic cells. The distinct karyotypic differences between MBU-7 and MBU-8 are associated with a difference in the specific response to nucleoside analogues. Taken together, we propose the MBU-7 and MBU-8 cell lines described here as suitable in vitro models for screening and testing vulnerabilities that are associated with the disease-relevant polysomies of chromosome 7, 8 and 18.

Impact of BCR-ABL1 Transcript Type on Response, Treatment-Free Remission Rate and Survival in Chronic Myeloid Leukemia Patients Treated with Imatinib.

The most frequent BCR-ABL1-p210 transcripts in chronic myeloid leukemia (CML) are e14a2 and e13a2. Imatinib (IM) is the most common first-line tyrosine-kinase inhibitor (TKI) used to treat CML. Some studies suggest that BCR-ABL1 transcript types confer different responses to IM. The objective of this study was to correlate the expression of e14a2 or e13a2 to clinical characteristics, cumulative cytogenetic and molecular responses to IM, acquisition of deep molecular response (DMR) and its duration (sDMR), progression rate (CIP), overall survival (OS), and treatment-free remission (TFR) rate. We studied 202 CML patients, 76 expressing the e13a2 and 126 the e14a2, and correlated the differential transcript expression with the above-mentioned parameters. There were no differences in the cumulative incidence of cytogenetic responses nor in the acquisition of DMR and sDMR between the two groups, but the e14a2 transcript had a positive impact on molecular response during the first 6 months, whereas the e13a2 was associated with improved long-term OS. No correlation was observed between the transcript type and TFR rate.

FMOD expression in whole blood aids in distinguishing between chronic lymphocytic leukemia and other leukemic lymphoproliferative disorders. A pilot study.

BACKGROUND: Within the hematopoietic compartment, fibromodulin (FMOD) is almost exclusively expressed in chronic lymphocytic leukemia (CLL) lymphocytes. We set out to determine whether FMOD could be of help in diagnosing borderline lymphoproliferative disorders (LPD). METHODS: We established 3 flow cytometry-defined groups (CLL [n = 65], borderline LPD [n = 28], broadly defined as those with CLLflow score between 35 and -20 or discordant CD43 and CLLflow, and non-CLL LPD [n = 40]). FMOD expression levels were determined by standard RT-PCR in whole-blood samples. Patients were included regardless of lymphocyte count but with tumor burden ≥40%. RESULTS: FMOD expression levels distinguished between CLL (median 98.5, interquartile range [IQR] 37.8-195.1) and non-CLL LPD (median 0.012, IQR 0.003-0.033) with a sensitivity and specificity of 1. Most borderline LPDs were CD5/CD23/CD200-positive with no loss of B-cell antigens and negative or partial expression of CD43. 16/22 patients with available cytogenetic analysis showed trisomy 12. In 25/28 (89%) of these patients, FMOD expression levels fell between CLL and non-CLL (median 3.58, IQR 1.06-6.21). DISCUSSION: This study could suggest that borderline LPDs may constitute a distinct group laying in the biological spectrum of chronic leukemic LPDs. Future studies will have to confirm these results with other biological data. Quantification of FMOD can potentially be of help in the diagnosis of phenotypically complex LPDs.

Spanish Guidelines for the use of targeted deep sequencing in myelodysplastic syndromes and chronic myelomonocytic leukaemia.

The landscape of medical sequencing has rapidly changed with the evolution of next generation sequencing (NGS). These technologies have contributed to the molecular characterization of the myelodysplastic syndromes (MDS) and chronic myelomonocytic leukaemia (CMML), through the identification of recurrent gene mutations, which are present in >80% of patients. These mutations contribute to a better classification and risk stratification of the patients. Currently, clinical laboratories include NGS genomic analyses in their routine clinical practice, in an effort to personalize the diagnosis, prognosis and treatment of MDS and CMML. NGS technologies have reduced the cost of large-scale sequencing, but there are additional challenges involving the clinical validation of these technologies, as continuous advances are constantly being made. In this context, it is of major importance to standardize the generation, analysis, clinical interpretation and reporting of NGS data. To that end, the Spanish MDS Group (GESMD) has expanded the present set of guidelines, aiming to establish common quality standards for the adequate implementation of NGS and clinical interpretation of the results, hoping that this effort will ultimately contribute to the benefit of patients with myeloid malignancies.

m7FLIPI and targeted sequencing in high-risk follicular lymphoma.

Patients with follicular lymphoma (FL) refractory to front-line immunochemotherapy (ICT) have a poor overall survival (OS). Gene mutation analysis may be more accurate than classical risk factors to pick out these patients before treatment. This study aimed to describe the prevalence of selected genetic mutations in a cohort of patients with high-risk FL. Twenty-five patients with FL refractory to front-line ICT and 10 non-refractory patients matched for age, sex, and FLIPI score were included. We sequenced 18 genes (custom targeted sequencing panel) previously reported to potentially have prognostic impact, including the seven genes necessary to determine m7FLIPI risk. The 35 patients had a median age of 62. The FLIPI and FLIPI2 were high in 27 (84%) and 14 (48%), respectively. Three-year progression-free survival (PFS) and OS probabilities were 25% (95% CI, 13%-41%) and 53% (34%-69%), respectively. There were 73 variants in the 18 genes among the 35 patients. The median number of mutations per patient was 1 (interquartile range, 0-3). The most commonly mutated genes were CREBBP (11 of 35, 31%) and EP300 (10 of 35, 29%). EP300 mutations were associated with refractoriness to treatment (10 of 25 among refractory and 0 of 10 among non-refractory). In conclusion, in this study, patients with high-risk follicular lymphoma were genetically heterogeneous.

Impact of mutational studies on the diagnosis and the outcome of high-risk myelodysplastic syndromes and secondary acute myeloid leukemia patients treated with 5-azacytidine.

Myelodysplastic syndromes (MDS) are stem cell disorders caused by various gene abnormalities. We performed targeted deep sequencing in 39 patients with high-risk MDS and secondary acute myeloid leukemia (sAML) at diagnosis and follow-up (response and/or relapse), with the aim to define their mutational status, to establish if specific mutations are biomarkers of response to 5-azacytidine (AZA) and/or may have impact on survival. Overall, 95% of patients harbored at least one mutation. TP53, DNMT3A and SRSF2 were the most frequently altered genes. Mutations in TP53 correlated with higher risk features and shorter overall survival (OS) and progression free survival (PFS) in univariate analysis. Patients with SRSF2 mutations were associated with better OS and PFS. Response rate was 55%; but we could not correlate the presence of TET2 and TP53 mutations with AZA response. Patients with sAML presented more variations than patients with high-risk MDS, and usually at relapse the number of mutations increased, supporting the idea that in advanced stages of the disease there is a greater genomic complexity. These results confirm that mutation analysis can add prognostic value to high-risk MDS and sAML patients, not only at diagnosis but also at follow-up.

DNA methylation profile in chronic myelomonocytic leukemia associates with distinct clinical, biological and genetic features.

Chromosomal abnormalities are detected in 20-30% of patients with chronic myelomonocytic leukemia (CMML) and correlate with prognosis. On the mutation level, disruptive alterations are particularly frequent in chromatin regulatory genes. However, little is known about the consequential alterations in the epigenetic marking of the genome. Here, we report the analysis of genomic DNA methylation patterns of 64 CMML patients and 10 healthy controls, using a DNA methylation microarray focused on promoter regions. Differential methylation analysis between patients and controls allowed us to identify abnormalities in DNA methylation, including hypermethylation of specific genes and large genome regions with aberrant DNA methylation. Unsupervised hierarchical cluster analysis identified two main clusters that associated with the clinical, biological, and genetic features of patients. Group 1 was enriched in patients with adverse clinical and biological characteristics and poorer overall and progression-free survival. In addition, significant differences in DNA methylation were observed between patients with low risk and intermediate/high risk karyotypes and between TET2 mutant and wild type patients. Taken together, our results demonstrate that altered DNA methylation patterns reflect the CMML disease state and allow to identify patient groups with distinct clinical features.

CD34 expression and the outcome of nucleophosmin 1-mutated acute myeloid leukemia.

CD34 positivity has been considered as an adverse prognostic factor in acute myeloid leukemia (AML). Although nucleophosmin 1-mutated (NPM1m) AML is usually CD34 negative, this marker may be expressed at diagnosis or acquired at relapse in a variable number of cases. Our objective was to ascertain if CD34 expression has any influence on the general outcome of this form of acute leukemia. Analysis of clinical outcome (complete remissions, relapses, disease-free survival, and overall survival) was performed depending on the degree of expression of CD34 determined by flow cytometry, in 67 adult patients with NPM1m AML. CD34 expression did not have any influence on the variables analyzed whatever the percentage of blasts expressing this marker. In contrast to other forms of AML, CD34 expression is not an unfavorable prognostic factor in NPM1m AML, neither at diagnosis nor at relapse.

Targeted deep sequencing improves outcome stratification in chronic myelomonocytic leukemia with low risk cytogenetic features.

Clonal cytogenetic abnormalities are found in 20-30% of patients with chronic myelomonocytic leukemia (CMML), while gene mutations are present in >90% of cases. Patients with low risk cytogenetic features account for 80% of CMML cases and often fall into the low risk categories of CMML prognostic scoring systems, but the outcome differs considerably among them. We performed targeted deep sequencing of 83 myeloid-related genes in 56 CMML patients with low risk cytogenetic features or uninformative conventional cytogenetics (CC) at diagnosis, with the aim to identify the genetic characteristics of patients with a more aggressive disease. Targeted sequencing was also performed in a subset of these patients at time of acute myeloid leukemia (AML) transformation. Overall, 98% of patients harbored at least one mutation. Mutations in cell signaling genes were acquired at time of AML progression. Mutations in ASXL1, EZH2 and NRAS correlated with higher risk features and shorter overall survival (OS) and progression free survival (PFS). Patients with SRSF2 mutations associated with poorer OS, while absence of TET2 mutations (TET2wt) was predictive of shorter PFS. A decrease in OS and PFS was observed as the number of adverse risk gene mutations (ASXL1, EZH2, NRAS and SRSF2) increased. On multivariate analyses, CMML-specific scoring system (CPSS) and presence of adverse risk gene mutations remained significant for OS, while CPSS and TET2wt were predictive of PFS. These results confirm that mutation analysis can add prognostic value to patients with CMML and low risk cytogenetic features or uninformative CC.

Highly variable mutational profile of ASXL1 in myelofibrosis.

OBJECTIVE: Somatic mutations in ASXL1 seem to have a negative prognostic impact in patients with several myeloid neoplasms, including myelofibrosis (MF). The aim of this work was to determine the prevalence and profile of ASXL1 mutations in MF. METHODS: We analyzed mutations in ASXL1 in 70 consecutive MF patients from 8 Spanish hospitals by means of Sanger sequencing, as well as JAK2, CALR, and MPL mutations. RESULTS: ASXL1 mutations were found in 16/70 (23%) of cases, most commonly p.Gly646TrpfsX12 (5/16). Most mutations (13/16) were frameshift mutations. Of 54 ASXL1- wild-type patients, 32 (59%) had at least one single nucleotide polymorphism (SNP), 27 of them had g.78128C>T, g.79017A>C, and g.79085T>C [triple SNP (TSNP) patients]. The 5-yr overall survival probability of TSNP patients was 67% (95% CI, 43-91%) vs. 90% (95% CI, 77-100%) in ASXL1-WT patients (P = 0.152). CONCLUSION: ASXL1 mutations were found in 23% of cases, p.Gly646TrpfsX12 being the most frequent. About 85% of mutations were found only in individual cases and 46% had not previously been reported, a pattern also seen in other series. Fifty percent of ASXL1-WT patients had a combination of three specific SNPs that might have a prognostic correlation that needs to be determined in larger series.