Clinical, biological, and prognostic implications of SF3B1 co-occurrence mutations in very low/low- and intermediate-risk MDS patients.

SF3B1 is a highly mutated gene in myelodysplastic syndrome (MDS) patients, related to a specific subtype and parameters of good prognosis in MDS without excess blasts. More than 40% of MDS patients carry at least two myeloid-related gene mutations but little is known about the impact of concurrent mutations on the outcome of MDS patients. In applying next-generation sequencing (NGS) with a 117 myeloid gene custom panel, we analyzed the co-occurrence of SF3B1 with other mutations to reveal their clinical, biological, and prognostic implications in very low/low- and intermediate-risk MDS patients. Mutations in addition to those of SF3B1 were present in 80.4% of patients (median of 2 additional mutations/patient, range 0-5). The most frequently mutated genes were as follows: TET2 (39.2%), DNMT3A (25.5%), SRSF2 (10.8%), CDH23 (5.9%), and ASXL1, CUX1, and KMT2D (4.9% each). The presence of at least two mutations concomitant with that of SF3B1 had an adverse impact on survival compared with those with the SF3B1 mutation and fewer than two additional mutations (median of 54 vs. 87 months, respectively: p = 0.007). The co-occurrence of SF3B1 mutations with specific genes is also linked to a dismal prognosis: SRSF2 mutations were associated with shorter overall survival (OS) than SRSF2wt (median, 27 vs. 75 months, respectively; p = 0.001), concomitant IDH2 mutations (median OS, 11 [mut] vs. 75 [wt] months; p = 0.001), BCOR mutations (median OS, 11 [mut] vs. 71 [wt] months; p = 0.036), and NUP98 and STAG2 mutations (median OS, 27 and 11 vs. 71 months, respectively; p = 0.008 and p = 0.002). Mutations in CHIP genes (TET2, DNMT3A) did not significantly affect the clinical features or outcome. Our results suggest that a more comprehensive NGS study in low-risk MDS SF3B1mut patients is essential for a better prognostic evaluation.

Detection of erythropoiesis-stimulating agents in a single dried blood spot.

The use of dried blood spots (DBS) for anti-doping purposes would facilitate an increase in the number of blood samples because it eliminates the need for specialized personnel and involves minimal invasiveness, reduced costs, stability, and easy transportation and storage. Here, the electrophoretic methodology established by the World Anti-Doping Agency (WADA) to detect erythropoiesis-stimulating agents (ESAs) has been adapted to evaluate their applicability to DBS. A qualitative procedure to detect recombinant erythropoietin (rEPO), novel erythropoiesis-stimulating protein (NESP), and continuous erythropoietin receptor activator (CERA) in a single DBS was optimized and validated. For rEPO and NESP, confirmation was performed in finger-prick DBS from a pilot study and an administration patients study, respectively. For CERA, detection capabilities were evaluated in DBS prepared with modeled-blood spiked with known concentrations of the protein. Main validation parameters concerning DBS sampling such as stability, hematocrit influence, and blood type (capillary vs. venous) described minor variations. Onsite drying appeared not to be essential before transport. Intra- and inter-day variation range was 2.9%-23.5%. Linearity was maintained (r ≥ 0.9) and ESAs were robustly recovered (CV ≤ 20.2%). The validated method permitted the detection of treated subjects after 48 hours and 17 days of rEPO and NESP administration, respectively. The reproduction of a CERA pharmacokinetics showed good possibilities for the method with a detection window that could reach 16 days after its actual administration. Thus, results provided here reinforce the suitability of DBS blood sampling for the analysis of ESA misuse in sports drug testing.

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.

Bone marrow VEGFC expression is associated with multilineage dysplasia and several prognostic markers in adult acute myeloid leukemia, but not with survival.

Vascular endothelial growth factor C (VEGFC) stimulates leukemia cell proliferation and survival, and promotes angiogenesis. We studied VEGFC expression in bone marrow samples from 353 adult acute myeloid leukemia (AML) patients and its relationship with several clinical, cytogenetic, and molecular variables. We also studied the expression of 84 genes involved in VEGF signaling in 24 patients. We found that VEGFC expression was higher in AML patients with myelodysplasia-related changes (AML-MRC) than in patients with non-AML-MRC. We also found an association between VEGFC expression and the patient cytogenetic risk group, with those with a worse prognosis having higher VEGFC expression levels. No correlation was observed between VEGFC expression and survival or complete remission. VEGFC expression strongly correlated with expression of the VEGF receptors FLT1, KDR, and NRP1. Thus, in this series, VEGFC expression was increased in AML-MRC and in subgroups with a poorer prognosis, but has no impact on survival.

Clinical and biological significance of isolated Y chromosome loss in myelodysplastic syndromes and chronic myelomonocytic leukemia. A report from the Spanish MDS Group.

Isolate loss of chromosome Y (-Y) in myelodysplastic syndromes (MDS) is associated to a better outcome but it is also well described as an age-related phenomenon. In this study we aimed to analyze the prognostic impact of -Y in the context of the IPSS-R cytogenetic classification, evaluate the clinical significance of the percentage of metaphases with isolated -Y, and test whether finding -Y may predispose to over-diagnose MDS in patients with borderline morphological features. We evaluated 3581 male patients from the Spanish MDS Registry with a diagnosis of MDS or chronic myelomonocytic leukemia (CMML). -Y was identified in 177 patients (4.9%). Compared with the 2246 male patients with normal karyotype, -Y group showed a reduced risk of leukemic transformation that did not translate into a survival advantage. The overall survival and the risk of leukemic transformation were not influenced by the percentage of metaphases with -Y. The -Y group was not enriched in patients with minor morphologic traits of dysplasia, suggesting that the better outcome in the -Y group cannot be explained by enrichment in cases misdiagnosed as MDS. In conclusion, our results support the current recommendation of classifying patients with -Y within the very good risk category of the IPSS-R for MDS and rule out a selection bias as a possible explanation of this better outcome. An analysis of the molecular basis of MDS with isolated -Y would be of interest as it may provide a biological basis of protection against progression to acute leukemia.

Enumerating bone marrow blasts from nonerythroid cellularity improves outcome prediction in myelodysplastic syndromes and permits a better definition of the intermediate risk category of the Revised International Prognostic Scoring System (IPSS-R).

The Revised International Prognostic Scoring System (IPSS-R) has been recognized as the score with the best outcome prediction capability in MDS, but this brought new concerns about the accurate prognostication of patients classified into the intermediate risk category. The correct enumeration of blasts is essential in prognostication of MDS. Recent data evidenced that considering blasts from nonerythroid cellularity (NECs) improves outcome prediction in the context of IPSS and WHO classification. We assessed the percentage of blasts from total nucleated cells (TNCs) and NECs in 3924 MDS patients from the GESMD, 498 of whom were MDS with erythroid predominance (MDS-E). We assessed if calculating IPSS-R by enumerating blasts from NECs improves prognostication of MDS. Twenty-four percent of patients classified into the intermediate category were reclassified into higher-risk categories and showed shorter overall survival (OS) and time to AML evolution than those who remained into the intermediate one. Likewise, a better distribution of patients was observed, since lower-risk patients showed longer survivals than previously whereas higher-risk ones maintained the outcome expected in this poor prognostic group (median OS < 20 months). Furthermore, our approach was particularly useful for detecting patients at risk of dying with AML. Regarding MDS-E, 51% patients classified into the intermediate category were reclassified into higher-risk ones and showed shorter OS and time to AML. In this subgroup of MDS, IPSS-R was capable of splitting our series in five groups with significant differences in OS only when blasts were assessed from NECs. In conclusion, our easy-applicable approach improves prognostic assessment of MDS patients.

Excess mortality in the myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are the commonest hematologic malignancies in the elderly. Since many patients with MDS actually die from age-related ailments, the very disease burden of MDS remains largely unknown. This registry-based study was aimed at investigating the excess mortality attributable to MDS. We analyzed 7,408 adult patients diagnosed with primary MDS from 1980 to 2014. Excess mortality was estimated by comparing the patients' survival with that expected in the matched general population. Median age of patients was 74 years, 58% were males, and 65% belonged to the lower risk categories of the Revised International Prognostic Scoring System (IPSS-R). Excess mortality accounted for three-fourths of the all-cause mortality and was mainly driven by factors unrelated to leukemic transformation. Excess mortality increased with the IPSS-R risk category [Incidence rate ratio (IRR): 2.1, 95% CI: 1.9-2.3; P < .001]. Older age and male sex retained an independent association with higher excess mortality after discounting demographic effects. Excess mortality increased in the most recent periods just in the higher risk IPSS-R categories (IRR: 1.2; 95% CI: 1.1-1.3 when comparing periods 2007-14, 2000-06, and 1980-99). In conclusion, MDS carry a significant excess mortality, even in the lower risk categories, that is mainly driven by factors unrelated to leukemic transformation, and increases with older age, male sex, and poorer risk categories. Excess mortality has increased in recent years in the higher risk patients, which might be ascribed to a parallel increase in age-related comorbidities. Our results claim for more comprehensive treatment strategies for patients with MDS. Am. J. Hematol. 92:149-154, 2017. © 2016 Wiley Periodicals, Inc.

Erythroleukemia shares biological features and outcome with myelodysplastic syndromes with excess blasts: a rationale for its inclusion into future classifications of myelodysplastic syndromes.

Erythroleukemia was considered an acute myeloid leukemia in the 2008 World Health Organization (WHO) classification and is defined by the presence of ≥50% bone marrow erythroblasts, having <20% bone marrow blasts from total nucleated cells but ≥20% bone marrow myeloblasts from nonerythroid cells. Erythroleukemia shares clinicopathologic features with myelodysplastic syndromes, especially with erythroid-predominant myelodysplastic syndromes (≥50% bone marrow erythroblasts). The upcoming WHO revision proposes to eliminate the nonerythroid blast cell count rule and to move erythroleukemia patients into the appropriate myelodysplastic syndrome category on the basis of the absolute blast cell count. We conducted a retrospective study of patients with de novo erythroleukemia and compared their clinico-biological features and outcome with those of de novo myelodysplastic syndromes, focusing on erythroid-predominant myelodysplastic syndromes. Median overall survival of 405 erythroid-predominant myelodysplastic syndromes without excess blasts was significantly longer than that observed in 57 erythroid-predominant refractory anemias with excess blasts-1 and in 59 erythroleukemias, but no significant difference was observed between erythroid-predominant refractory anemias with excess blasts-1 and erythroleukemias. In this subset of patients with ≥50% bone marrow erythroblasts and excess blasts, the presence of a high-risk karyotype defined by the International Prognostic Scoring System or by the Revised International Prognostic Scoring System was the main prognostic factor. In the same way, the survival of 459 refractory anemias with excess blasts-2, independently of having ≥20% bone marrow blasts from nonerythroid cells or not, was almost identical to the observed in 59 erythroleukemias. Interestingly, 11 low-blast count erythroleukemias with 5 to <10% bone marrow blasts from total nucleated cells showed similar survival than the rest of erythroleukemias. Our data suggest that de novo erythroleukemia is in the spectrum of myelodysplastic syndromes with excess blasts and support its inclusion into future classifications of myelodysplastic syndromes.

Considering Bone Marrow Blasts From Nonerythroid Cellularity Improves the Prognostic Evaluation of Myelodysplastic Syndromes.

PURPOSE: WHO classification of myeloid malignancies is based mainly on the percentage of bone marrow (BM) blasts. This is considered from total nucleated cells (TNCs), unless there is erythroid-hyperplasia (erythroblasts ≥ 50%), calculated from nonerythroid cells (NECs). In these instances, when BM blasts are ≥ 20%, the disorder is classified as erythroleukemia, and when BM blasts are < 20%, as myelodysplastic syndrome (MDS). In the latter, the percentage of blasts is considered from TNCs. PATIENTS AND METHODS: We assessed the percentage of BM blasts from TNCs and NECs in 3,692 patients with MDS from the Grupo Español de Síndromes Mielodisplásicos, 465 patients with erythroid hyperplasia (MDS-E) and 3,227 patients without erythroid hyperplasia. We evaluated the relevance of both quantifications on classification and prognostication. RESULTS: By enumerating blasts systematically from NECs, 22% of patients with MDS-E and 12% with MDS from the whole series diagnosed within WHO categories with < 5% BM blasts, were reclassified into higher-risk categories and showed a poorer overall survival than did those who remained in initial categories (P = .006 and P = .001, respectively). Following WHO recommendations, refractory anemia with excess blasts (RAEB)-2 diagnosis is not possible in MDS-E, as patients with 10% to < 20% BM blasts from TNCs fulfill erythroleukemia criteria; however, by considering blasts from NECs, 72 patients were recoded as RAEB-2 and showed an inferior overall survival than did patients with RAEB-1 without erythroid hyperplasia. Recalculating the International Prognostic Scoring System by enumerating blasts from NECs in MDS-E and in the overall MDS population reclassified approximately 9% of lower-risk patients into higher-risk categories, which indicated the survival expected for higher-risk patients. CONCLUSION: Regardless of the presence of erythroid hyperplasia, calculating the percentage of BM blasts from NECs improves prognostic assessment of MDS. This fact should be considered in future WHO classification reviews.

Response to erythropoietic-stimulating agents in patients with chronic myelomonocytic leukemia.

BACKGROUND: The efficacy of erythropoietic-stimulating agents (ESA) in chronic myelomonocytic leukemia (CMML) is unknown. Our objective was to analyze erythroid response (ER) and overall survival (OS) in a series of 94 patients with CMML treated with ESA. METHODS: We analyzed a series of 94 patients with CMML treated with ESA included in the Spanish and Düsseldorf-MDS registries. FINDINGS: ER was observed in 64% of patients and red blood cell (RBC) transfusion independence in 31%. The median duration of ER was 7 months (range, 0-88). CPSS and EPO level were significantly associated with ER in multivariate analysis (P = 0.003). Considering only patients with CPSS low- or intermediate-1-risk group, the absence of RBC transfusion dependence and erythropoietin (EPO) level predicted ER (P = 0.003 and P = 0.008, respectively). In multivariate analysis, only the EPO level retained its prognostic value (P = 0.029). Achievement of ER correlated with a better survival since ER evaluation (P = 0.016). INTERPRETATION: The CPSS and EPO levels are adequate tools to select CMML patients with symptomatic anemia who may benefit from treatment with ESA. A significant ER to ESA is expected in anemic patients with low/intermediate-1 CMML risk by the CPSS and a low endogenous serum EPO level.

Trisomy 8, a Cytogenetic Abnormality in Myelodysplastic Syndromes, Is Constitutional or Not?

Isolated trisomy 8 is not considered presumptive evidence of myelodysplastic syndrome (MDS) in cases without minimal morphological criteria. One reason given is that trisomy 8 (+8) can be found as a constitutional mosaicism (cT8M). We tried to clarify the incidence of cT8M in myeloid neoplasms, specifically in MDS, and the diagnostic value of isolated +8 in MDS. Twenty-two MDS and 10 other myeloid neoplasms carrying +8 were studied. Trisomy 8 was determined in peripheral blood by conventional cytogenetics (CC) and on granulocytes, CD3+ lymphocytes and oral mucosa cells by fluorescence in situ hybridization (FISH). In peripheral blood CC, +8 was seen in 4/32 patients. By FISH, only one patient with chronic myelomonocytic leukemia showed +8 in all cell samples and was interpreted as a cT8M. In our series +8 was acquired in all MDS. Probably, once discarded cT8M by FISH from CD3+ lymphocytes and non-hematological cells, +8 should be considered with enough evidence to MDS.

Darbepoetin alfa administered once every three weeks for the treatment of anemia in elderly patients with non-myeloid tumors receiving chemotherapy.

AIMS AND BACKGROUND: The present study aims to describe the hematological response to darbepoetin alfa (DA) under daily clinical practice conditions in anemic elderly patients with non-myeloid tumors receiving chemotherapy. METHODS AND STUDY DESIGN: This was a prospective, observational, multicenter study in elderly (≥65 years) patients with non-myeloid cancer receiving DA (500 µg every 3 weeks) for chemotherapy-induced anemia (hemoglobin [Hb] level ≤11.0 g/dL). RESULTS: A total of 102 anemic patients with solid tumors and 51 with hematological malignancies were included in 28 centers in Spain. Mean age (±SD) was 73.4 (±5.8) years, and mean baseline Hb level was 10.0 (±0.8) g/dL. DA was administered for a median of 8 weeks. Of the 115 subjects with a post-baseline Hb value, the percentage of patients who achieved a hematopoietic response (Hb increase ≥2 g/dL or reaching ≥12 g/dL without transfusions in the previous 28 days) was 69.7% (95% CI 56.1% to 83.3%). Functional Assessment of Cancer Therapy-Fatigue subscale scores increased during the study (median change 1.0 [Q1 -5.0, Q3 9.0], P = 0.04). One patient (0.7%) experienced a non-serious adverse reaction (cutaneous rash). CONCLUSION: The study results suggest that DA is an effective and well-tolerated therapy for the treatment of chemotherapy-induced anemia in elderly patients.

Transfusion dependence development and disease evolution in patients with MDS and del(5q) and without transfusion needs at diagnosis.

Patients with isolated del(5q) and MDS are considered to have good prognosis as compared to other MDS subtypes. Most patients suffered of anemia and 50% of them required transfusions at diagnosis. It is known that for patients with MDS and del(5q) in transfusion dependence(TD), Lenalidomide is the first choice treatment. However, there are no data regarding natural evolution of anemia in patients diagnosed in MDS and del(5q) without TD, factors that may impact on the development of TD or disease outcome. In the present study we have performed a retrospective multicenter analysis on 83 patients with low-int 1 MDS and del(5q) without TD. During the study 61 patients became TD at a median of 1.7 years and only the Hb level 9 g/dL was associated with poorer TFS (p = 0.007) in the multivariate analysis. Among these 61 TD patients, 49 received treatment (19 Lenalidomide). Median follow up was 48 months, estimated OS at 2 and 5 year was 92% and 50% respectively. In the multivariate analysis for OS, platelets <100,000 mm(-3) and Lenalidomide treatment retained the statistical significant impact. LFS at 2 and 5 years was 86% and 73% respectively, and median time to sAML was 8.16 years (CI 95%: 6.05-10.27). In the multivariate analysis only thrombocytopenia retained statistical significance. In summary, this retrospective study show that level of Hb is an important parameter in order to determine the time until TD, it should be also stressed the importance of an early treatment in order to prevent TD development and shorter survival.

Results of treatment with azacitidine in patients aged ≥ 75 years included in the Spanish Registry of Myelodysplastic Syndromes.

The tolerability of azacitidine (AZA) allows its administration in elderly patients. The objective of this study was to analyze the clinical and biological characteristics, transfusion independence (TI), overall survival (OS) and toxicity in a series of 107 patients ≥ 75 years of age from the Spanish Registry of Myelodysplastic Syndromes (MDS) treated with AZA. The median age (range) was 78 (75-90) years. According to the World Health Organization (WHO) classification, 86/102 (84%) had MDS, 10/102 (10%) had mixed myeloproferative/myelodysplastic disorder and 6/102 (6%) had acute myeloblastic leukemia. Regarding MDS by the International Prognostic Scoring System on initiation of AZA, 38/84 (45%) were low-intermediate-1 risk and 46/84 (55%) were intermediate-2-high risk. Ninety-five patients (89%) were red blood cell or platelet transfusion dependent. The AZA schedule was 5-0-0 in 39/106 (37%) patients, 5-2-2 in 36/106 (34%) patients and 7 consecutive days in 31/106 (29%) patients. The median number of cycles administered was 8 (range, 1-30). Thirty-eight out of 94 (40%) patients achieved TI. Median OS (95% confidence interval [CI]) was significantly better in patients achieving TI (n = 38) compared to patients who did not (n = 56) (22 [20.1-23.9] months vs. 11.1 [4.8-17.5] months, p = 0.001). No significant differences were observed in TI rate and OS among the three different schedules. With a median follow-up of 14 (min-max, 1-50) months, the median OS (95% CI) of the 107 patients was 18 (12-23) months and the probability of OS (95% CI) at 2 years was 34% (22-46%). Cycles were delayed in 31/106 (29%) patients and 47/101 patients (47%) were hospitalized for infection. These results show that treatment with AZA was feasible and effective in this elderly population, with 40% achieving TI, having a better OS than patients not achieving it. The schedule of AZA administration did not affect efficacy and toxicity.

Single nucleotide polymorphism array karyotyping: a diagnostic and prognostic tool in myelodysplastic syndromes with unsuccessful conventional cytogenetic testing.

Cytogenetic aberrations identified by metaphase cytogenetics (MC) have diagnostic, prognostic, and therapeutic implications in myelodysplastic syndromes (MDS). However, in some MDS patients MC study is unsuccesful. Single nucleotide polymorphism array (SNP-A) based karyotyping could be helpful in these cases. We performed SNP-A in 62 samples from bone marrow or peripheral blood of primary MDS with an unsuccessful MC study. SNP-A analysis enabled the detection of aberrations in 31 (50%) patients. We used the copy number alteration information to apply the International Prognostic Scoring System (IPSS) and we observed differences in survival between the low/intermediate-1 and intermediate-2/high risk patients. We also saw differences in survival between very low/low/intermediate and the high/very high patients when we applied the revised IPSS (IPSS-R). In conclusion, SNP-A can be used successfully in PB samples and the identification of CNA by SNP-A improve the diagnostic and prognostic evaluation of this group of MDS patients.

Prognostic value of trisomy 8 as a single anomaly and the influence of additional cytogenetic aberrations in primary myelodysplastic syndromes.

Trisomy 8 is the most common chromosomal gain in myelodysplastic syndromes (MDS), however, little is known about the features of MDS with isolated trisomy 8 and the influence of additional cytogenetic aberrations. We determined the characteristics and prognostic factors of 72 patients with trisomy 8 as a single anomaly and analysed also the impact of other aberrations added to trisomy 8 in another 62 patients. According to our study, MDS with isolated trisomy 8 was more frequent in men, with more than one cytopenia in most patients (62%) and having about 4% bone marrow blasts. The multivariate analysis demonstrated that platelet count and percentage bone marrow blasts had the strongest impact on overall survival (OS). The median OS for isolated trisomy 8, trisomy 8 plus one aberration (tr8 + 1), plus two (tr8 + 2) and plus three or more aberrations (tr8 + ≥3) was 34·3, 40, 23·4 and 5·8 months, respectively (P < 0·001). Trisomy 8 confers a poorer prognosis than a normal karyotype in MDS patients with ≥5% bone marrow blasts. This study supports the view that MDS with isolated trisomy 8 should be included in the intermediate cytogenetic risk group.