BM Blast Count Research Articles

Myelodysplastic Syndromes in Latin-America - Results from a Novel International Registry: Re-Glam

Background: It is very well known that information provided by clinical records and organized system platforms (registries) offer a holistic view of MDS patients' characteristics and therapeutic aspects in real-life. The primary objective of this study was to analyze data from a novel MDS Latin-American Registry. Methods: The registry started in April 2022. From April to July, patients with MDS or CMML diagnosed since January 2014 were reported from Argentina, Brazil, Colombia, Mexico, and Uruguay. The Research Electronic Data Capture (REDCap) platform was used, consisting of 298 fields to describe patient demographics; their biological characteristics; clinical manifestations; comorbidities; laboratory features; bone marrow studies; risk classification; treatments, and outcomes. According to the Ethics Committee, all subjects had to sign an inform consent. Descriptive statistics were used to summarize demographic, and clinical data, as well as treatment characteristics - overall and by risk groups. Results: A total of 231 patients were included in this analysis. The mean age was 62.8 ± 16.9 years and the male to female ratio was 1.1:1. Baseline characteristics are described in Table 1. According to WHO's 2016 classification, the following categories were reported: MDS-SLD 4.4%; MDS-RS 1.3%; MDS-MLD 34.5%; MDS-RS-MLD 4.4%; MDS-EB 24.9%; MDS with isolated del(5q) 2.2%; unclassifiable MDS 0.4%; therapy-related MDS 7.0%; CMML 10.9%; MDS-U 1.3%; MDS/MPN-RS-T 0.4%; aCML 0.4%; other 7.9%. The IPSS-R was: 3.4% Very Low; 62.3% Low; 17.9% Intermediate; 10.1% high-risk; and 6.3% very high-risk. The majority of patients had de novo MDS (95.2%), and 8.6% had hypoplastic MDS. The mean BM blast count was 3.5±4.3%. Iron staining was performed in 28.1%. BM biopsies were performed at the moment of diagnosis in 77.5% of cases, reported as hypercellular in 90.0% of them and with fibrosis (> grade 1) in 32%. Immunohistochemistry was performed in 53.7% and flow cytometry in 47.8% of patients. Cytogenetics was performed in 93.9% and FISH in 26.9%. Abnormal karyotype was detected in 38.2%. An NGS panel was performed in 35 (15.2%) patients. Low-risk MDS accounts for the majority of patients in our registry (58%). Erythropoietin was given in 73 (69.5%) and growth factor therapy in 13 (11%) patients. Red blood cell transfusion was given to 59 (50%) patients. 31 (26%) of them received second-line treatment: 29 with HMA, and 2 with lenalidomide. 5 (4.2%) underwent allogenic transplant. 16 (13.7%) progressed to AML. The median overall survival was 4.19 (2.7-6.40) years. High-risk MDS was reported in 42% of patients. Regarding treatment, 69 (80.2%) received HMA (AZA=65 and DEC=4), 4 (4.6%) chemotherapy, and 8 (9.3%) of them were administered a venetoclax-based therapy. Fifteen (17.4%) were transplanted and 46 (53.4%) progressed to AML. The median overall survival was 1.5 (1.2-2.0) years. Conclusions: This novel and international registry showed preliminary MDS data from Latin-America. Our results revealed younger age at diagnosis, high number of patients undergoing allogenic transplant and OS according to literature. The expansion of this registry certainly improves our clinical practice. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

T-MDS Is a Distinct Clinical and Pathological Entity Characterized By Better Survival Compared to t-AML

Introduction: Therapy-related myeloid neoplasm (t-MN) includes acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) that occur as a complication of DNA-damaging therapies. The World Health Organization recommends considering t-MN as a single entity. Whether t-MDS and t-AML have distinct characteristics and outcomes is not known. The aim of this study was to compare clinicopathological characteristics and outcomes of t-MDS and t-AML.Methods: All patients diagnosed with t-MN based on the World Health Organization criteria were identified. Bone marrow biopsies, cytogenetic, and next-generation sequencing (NGS) were obtained at the treating physician's discretion. Pathogenic/likely pathogenic variants (PV) were called based on the standard criteria. Overall survival (OS) was calculated from the time of t-MN diagnosis until the last follow up using Kaplan-Meier analysis using Wilcoxon test. For survival analysis comparing chemotherapy to best supportive care (BSC) only, patients were censored at the time of allogeneic stem cell transplant (SCT). Multivariate analysis was performed using Cox proportional hazard method and corrected using false discovery rate (FDR). Statistical analysis was performed using JMP (v14.1, SAS Institute) and significance was defined as P<0.05.Results: We identified 554 patients, of which 180 (32.4%), 365 (65.8%), and 9 (1.6%) presented as t-AML, t-MDS, and t-MDS/MPN respectively. t-MDS/MPN patients were excluded from further analysis due to a small number. Clinical and laboratory characteristics of the t-AML and t-MDS cohorts is shown in Table 1. t-AML patients were significantly more anemic and thrombocytopenic at presentation. As expected, t-AML had a higher peripheral blood and BM blast count. There was no difference in proportion of patients with chromosomal abnormalities, though a statistically significantly higher proportion of t-MDS patients had chromosome 5 abnormality, 5q deletion, monosomy 5, chromosome 7 abnormality, monosomy 7 compared to t-AML patients. On other hand, 11q23 (mixed lineage leukemia, MLL) rearrangement was more common in t-AML compared to t-MDS (9.3% vs. 2.8%, P=0.005).A higher proportion of t-MDS patients had PV detected by NGS compared to t-AML (92.9% vs. 85.6%, P=0.038). A higher proportion of t-MDS patients had PV in TP53 (37.6% vs. 21.4%, P=0.004) and ASXL1 (23% vs. 11.7%, P=0.016) genes; whereas a higher proportion of t-AML patients had PV in RAS (18.9% vs. 9.1%, P=0.013) and WT1 (8.1% vs. 2.9%, P=0.05) genes.One hundred twenty-eight (35%) of 365 t-MDS patients progressed to t-AML. The presence of any chromosomal abnormality at t-MDS diagnosis predicted a higher risk of transformation to t-AML (χ 2 3.9, P=0.03).t-AML patients had a significantly shorter OS compared to t-MDS (9.2 vs. 19.7 months, P<0.0001, Figure A). This difference persisted when stratified by no disease modifying therapies (BSC only) 2 vs. 17 months (P<0.0001, Figure B), as well as among those who received at least one line of chemotherapy (14 vs. 24.6 months, P<0.001). Finally, a higher proportion of patients with t-AML underwent SCT and there was a trend towards improved survival for t-AML patients (vs. t-MDS 52.9 vs. 20.7 months, P=0.07) from the time of transplant.Multivariate analysis for OS performed to control for all the variables that were different at presentation (except for blast count), showed that t-MDS (as opposed to t-AML) phenotype at diagnosis, and undergoing SCT were independent predictors of improved survival (Table 2).Conclusion: t-MDS and t-AML have distinct clinical, cytogenetic, and genetic features at presentation. In the absence of disease modifying therapies, t-AML is a more aggressive phenotype, consistently associated with a shorter survival. Even after controlling adverse risk features, t-AML phenotype had a shorter survival compared to t-MDS. [Display omitted] DisclosuresLitzow: AbbVie: Research Funding; Astellas: Research Funding; Amgen: Research Funding; Actinium: Research Funding; Pluristem: Research Funding; Jazz: Other: Advisory Board; Omeros: Other: Advisory Board; Biosight: Other: Data monitoring committee. Hiwase: Novartis: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees.

Prognostic Impact of Concurrent DNMT3A, FLT3 and NPM1 Gene Mutations in Acute Myeloid Leukemia Patients

BackgroundAcute myeloid leukemia (AML) is one of the rapidly progressing malignancies which is characterized by unregulated proliferation of hematopoietic precursors. Technological improvements enhanced the availability of genetic AML biomarkers. The clinical relevance of these molecular markers for AML diagnosis, planning of therapy and risk stratification are increasing evidently. MethodsIn current study, one hundred newly diagnosed AML patients before receiving induction chemotherapy were included, they were subjected to clinical examination, cytochemical and morphological analysis of blood cells, flow cytometric, cytogenetic and molecular genetic analysis for detection of NPM1, FLT3-ITD and DNMT3A mutations. Direct sequencing analysis for detection of NPM1 and DNMT3A genes mutations were done. FLT3 /ITD gene mutation was detected by gel electrophoresis after PCR amplification. ResultsAccording to genetic markers, our AML patients are classified in to further 8groups. AML patients with three DNMT3A/FLT3/NPM1 gene mutations (AML DNMT3A /FLT3/NPM1) this group of patients presented with a heavy disease burden, had an elevated WBC in comparison to other groups (70 vs. 41 × 103/μL; P = .019), and BM blast counts (71% vs. 55.6%, P < .02). When comparing eight groups for death event there were significant difference among groups; P = .005, group 1 (AML DNMT3A /FLT3/NPM1) showed rapid decline of the cumulative overall survival. There was a significant difference among 8 groups as regards response to treatment after 14 days (P = .02), group 7 AML with (DNMT3A +NMP1) gene mutations showed better response to treatment (100%), groups 1 and 3 AML with (NPM1+DNMT3A +NMP1) gene mutations and AML with isolated FLT3-ITD showed no response to treatment after 14 days. And as regards response to treatment after 28 days, the eight groups showed no significant difference (P = .14). ConclusionOur study supports adverse prognostic effect of presence of DNMT3A gene mutation either alone or in the presence of FLT3 and/or NPM1 gene mutations.

Expression of SATB2 in Acute Myeloid Leukemia and Its Prognostic Significance

Background: Human special AT-rich sequence-binding protein-2 (SATB2) is a novel DNA binding protein, which is involved in regulating gene expression. Recent studies conducted to evaluate the clinical significance of SATB2 in multiple malignant carcinomas, and have yielded controversial results. The purpose of this study is to investigate the expression status of SATB2 and its prognostic significance in AML patients.Methods: Total RNA was extracted from bone marrow or plasma of 87 patients with AML and 21 health volunteers. The expression of SATB2 was examined by Realtime quantitative PCR. The relationship of SATB2 expression and other clinicopathological charactors and its prognosis significance were statistically analyzed.Results: The relative expression of SATB2 was significantly up-regulated in AML patients (median value 0.061, range: 0.007-0.328) in comparison to those of healthy volunteers (median value 0.033, range: 0.008-0.077; P < 0.001) , and SATB2 expression level was much lower in patients who had achieved CR (median value 0.005, range: 0.001-0.006;P < 0.001) compared with newly diagnosed AML patients(median value 0.010, range: 0.002-0.027;P < 0.001).Furthermore, the expression of SATB2 was markedly associated with gender(P =0.043), WBC count(P =0.001), platelet count (P =0.025), BM blast count (P = 0.007) and Complete Remission (P < 0.001). Patients with higher SATB2 expression had a relatively poor overall survival(OS) (P = 0.009) and short disease-free survival(DFS) (P < 0.001). Moreover, SATB2 expression was an independent prognostic factors for both OS (P = 0.001) and DFS (P = 0.006).Conclusions: SATB2 was significantly up-regulated in AML patients in comparison to healthy donors. Patients who had achieved CR usually had a lower level of SATB 2 expression compared with newly diagnosed AML patients. The over-expression of SATB2 is associated with poor prognosis in AML patients, and was an independent prognostic factor for both OS and DFS. DisclosuresNo relevant conflicts of interest to declare.

Adoptive Immunotherapy with Cord Blood (CB) Grafts Targeting Shared Inherited Paternal Antigens (IPA) and/or Non-Inherited Maternal Antigens (NIMA) during Induction Treatment for High Risk Myeloid Malignancies: Safety and Preliminary Results

BackgroundSeveral groups have reported use of haploidentical peripheral blood donor cells as adjunct to chemotherapy in remission induction of myeloid leukemia (Colvin et al, BBMT 2009, Gue et al, Blood 2011). Moreover, CB grafts have been associated with greater disease control and enhanced graft-versus-leukemia responses, as a consequence of the fetal-maternal interactions during pregnancy (van Rood et al, PNAS 2009 and PNAS 2012).We evaluated prospectively the feasibility and tolerability of infusing partially HLA matched cord blood units (CBUs) with specific alloreactive properties after salvage chemotherapy to patients (pts) with refractory myeloid leukemia.Patients and MethodsPts with confirmed diagnosis of AML or MDS, according to WHO classification (excluding acute promyelocytic leukemia) who had received two or more previous induction/re-induction regimens were eligible. All pts were treated at NYPH and all CBUs were obtained from the NCBP. The study was performed under FDA IND and was approved by the IRBs of both institutions. Pts received induction chemotherapy followed by CBU infusion (1-3 days later) without any immunosuppression.CBUs eligible for the study contained a pre-cryopreservation Total Nucleated Cell (TNC) dose of 0.5-2.5x107/kg. They were HLA matched at 4-6/6, HLA-A, -B intermediate resolution, -DRB1 high resolution. CBUs had undergone HLA typing of the mother, thus allowing determination of the paternal and maternal antigens. CBUs with shared IPA targets and/or NIMA match were selected for cell therapy.ResultsA total of 34 patients underwent CBU search. Mean CBU number per search was 134 (range: 7-250) with 58% potentially eligible CBUs per pt (range: 5-159). A total of 143 CBUs underwent testing including HLA confirmatory typing. At least one eligible CBU was identified for each pt. Higher HLA allele level match was preferred in the final selection.Of the 34 pts, 23 did not receive treatment because of progressive disease and death (n=8); other clinical trials/treatments (n=6); hematopoietic stem cell transplants (HSCT) (n=9); while one is currently undergoing evaluation. Ten pts have been treated (Table); 8 had AML, 7 with primary induction failure (PIF); one had myeloid blast phase CML and one with high risk MDS. Nine pts had poor risk karyotype and 4 had prior allografts. Median age was 52 years (range: 34-67); median number of prior treatments was 11 (2-15) and median HSCT-CI was 3 (0-7).Of the CBUs infused, six were 5/6 and four were 4/6 HLA matched. The allele level compatibility and presence of shared IPA targets and/or NIMA matches are shown in the Table. CBU median TNC was 90x107 (range: 63-151) and they tended to be “older” with a median cryopreservation time of 10 years. CBUs provided a mean cell dose of 1.66 x 107 TNC/kg (range: 1.1-2.4). Mean post-thaw CD34+ and CD3+ cell doses were 52x103/kg (range: 20-90) and 0.34x107/kg (range: 0.24-0.52; SD: 0.08), respectively, with excellent post-thaw CD34+ viability (93.5%-98.3%).No CBU infusion reactions or cytokine release syndrome were observed. One pt, recipient of a prior allograft with no history of GVHD, experienced skin rash 1 week after CBU infusion. The skin biopsy was consistent with GVHD. No GVHD was observed in the other 9 pts. One pt died of disseminated adenovirus infection early after treatment; viremia was present at the initiation of chemotherapy.Transient CBU chimerism was detected in 5 of the 10 pts as early as one week and as late as 6 months after CBU infusion. Four pts with median pre-treatment BM blast counts of 56% (range 13-94) had objective hematologic responses (Cheson B JCO 2003) with median post-treatment blast counts of 0% (range 0-12), 3 with morphologic leukemia free state and one with a partial response. One patient had aplastic marrow but with flow cytometry evidence of persistent leukemia; while 4 pts recovered with morphologic persistent leukemia. Six pts went on to receive HSCT. Two subsequently relapsed and one died of disseminated adenovirus, while 3 pts remain in remission.ConclusionUse of CBUs as adoptive immunotherapy for pts with refractory myeloid leukemia represents a safe, well tolerated treatment, in which the potential for alloreactivity can be enhanced through the selection of CBUs by targeting shared IPA and NIMA present in the patients. Further, smaller CBUs, unlikely to be adequate grafts for adult transplantation, can be utilized for cell therapy. [Display omitted] DisclosuresRoboz:Cellectis: Research Funding; AbbVie, Agios, Amgen, Amphivena, Array Biopharma Inc., Astex, AstraZeneca, Celator, Celgene, Clovis Oncology, CTI BioPharma, Genoptix, Immune Pharmaceuticals, Janssen Pharmaceuticals, Juno, MedImmune, MEI Pharma, Novartis, Onconova, Pfizer, Roche Pharmace: Consultancy. Ritchie:Celgene: Consultancy, Other: Travel, Speakers Bureau; Pfizer: Consultancy, Other: Research funding to my institution; Incyte: Consultancy, Speakers Bureau; Astellas Pharma: Other: Research funding to my institution; NS Pharma: Other: Research funding to my institution; Bristol-Myers Squibb: Other: Research funding to my institution; Novartis: Consultancy, Other: Research funding to my institution, and travel, Speakers Bureau. Lee:Amgen: Consultancy; Baxalta: Consultancy; Clinipace: Consultancy; Alexion Pharmaceuticals: Consultancy.

Analysis of Clonal Evolution of AML Using Simultaneous Single-Cell DNA/RNA Analysis

Background Acute myeloid leukemia (AML) was defined by an increase of immature myeloid cells, or blasts that exceed ≥20% in bone marrow or peripheral blood. Many lines of evidence suggest that the development of AML is shaped by clonal evolution through multiple rounds of positive selection driven by newly acquired mutations, ultimately leading to an increased blast count. This process has been analyzed in detail in the case of progression from myelodysplastic syndromes (MDS) to secondary AML (sAML), which is invariably accompanied by expansion of cells that acquired new driver alterations, generating clonal substructures in many cases (Walter et al. NEJM. 2012, Makishima et al. Nat. Genet. 2015). However, it has not been fully elucidated how these newly acquired mutations contribute to increased blast cells that define AML. Results In order to understand how driver mutations contribute to the phenotype of blasts, we first focused on the driver mutations that have known to be enriched in sAML, including those in IDH1/2, NPM1, FLT3,NRAS, KRAS, PTPN11, CBL and WT1, and compared BM blast count (BC) and mutant cell fraction (MCF) of each driver mutation in 27 cases with sAML. Compared with BC, IDH1- or IDH2-mutated cells exhibited a larger MCF in most cases, suggesting that newly acquired IDH1/2 mutations contribute clonal expansion but only a part of the expanded cells undergo differentiation block and the remaining cells can differentiate into mature cells. Of interest, we observed lower MCFs than BC in approximately half of the cases with signaling pathway mutations, including FLT3 and RAS pathway (NRAS, KRAS, PTPN11 and CBL) mutations, in which MCFs for signaling pathway mutation accounted for less than 2/3 of BC, which was also observed in de novo AML cases. In fact, signaling pathway mutations in two representative cases were confirmed to account only for 30.4% and 3.4% of blast cells, using ddPCR of the blast cells collected as the CD45dim SSClow fraction, which were confirmed to show a blast morphology. These results suggest a possibility that the presence of mutant cells might affect the phenotype of the surrounding unmutated cells. Thus, to investigate the mechanism of such non-cell autonomous effects of mutations on blast cell morphology, we developed an advanced single-cell sequencing platform that enables simultaneous measurements of both mutations and gene expression profiles at a single-cell level and applied this to the analysis of immature (CD34+ Lin-) BM cells from 2 sAML cases with multiple RAS pathway mutations showing disproportionately small MCF compared to BC, in which gene expression of mutated and unmutated cells were evaluated separately. The same BM faction in 13 healthy donors was also analyzed as normal control. In single-cell mutation analysis, multiple RAS pathway mutations in both cases represented independent clones. As expected, cells carrying each RAS pathway mutation at sAML showed an immature myeloid phenotype. However, most of the cells, even carrying MDS mutations alone, also exhibited an immature myeloid phenotype similar to the RAS pathway mutated cells, although the latter cells showed upregulated RAS signaling compared with the former cells. Cells solely carrying MDS mutations in MDS phase showed multi-lineage differentiation, which was no longer observed in those cells in sAML phase. This was in contrast to another case who acquired MYC amplification on sAML progression, where nearly all cells having MYC-amplification showed an immature myeloid phenotype, whereas the remaining MDS clones lacking MYC-amplification retained multilineage differentiation even at the sAML phase. These results suggest that RAS mutants might have a non-cell autonomous effect on the surrounding cells including those hematopoietic cells lacking those mutations and other stromal cells, preventing their differentiation to mature cells, although we cannot exclude another possibility that altered BM microenvironment could influence the phenotype of both mutated and unmutated cells. Conclusions Although an acquisition of new mutations is essential for the progression of MDS to sAML, our results suggest that the blast cell phenotype may not solely be determined by cell-intrinsic effects of such mutations, but non-cell autonomous effects of mutated cells (and possibly also of an altered BM microenvironment) may have a role in increased blast count and therefore AML progression. Disclosures Inagaki: Sumitomo Dainippon Pharma Co., Ltd.: Current Employment. Nakagawa:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Ogawa:KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Factors Predicting Long-Term Survival Following CD19 CAR T-Cell Therapy in Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia

Introduction Chimeric antigen receptor (CAR) T-cell therapy targeting CD19 has demonstrated promising efficacy in patients with relapsed and refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). However, relapse after CART remains a major issue. Here, we analyzed the factors related to long-term efficacy, including overall survival (OS), leukemia-free survival (LFS) and cumulative relapse rate (CRR), following CAR-T therapy in 231 R/R B-ALL patients who achieved complete remission (CR) within one month after CAR T-cell therapy. Patients and Methods From April 2017 to March 2019, 254 patients with R/R B-ALL were enrolled onto one of five different clinical trials (NCT03173417; ChiCTR-ONC-17012829; NCT02546739; ChiCTR1800016541; and NCT03671460) at our center and received a second generation CD19+ CAR T-cell infusion. The median infused CAR T-cell dose was 3×105/kg (range: 0.2-10×105/kg). The CAR-T/T-cell ratio and the CD19+ B lymphocyte percentage in PBLC samples from 159 of the patients were analyzed using flow cytometry on day 0, 4, 7, 11, 14, 21, and 30 following CAR T-cell infusion. We performed single continuous variate factors analysis on the influence of the CAR-T/T-cell ratio and the percentage of CD19 + B-lymphocytes in day 30 post-infusion PBLC samples on the OS, LFS, and CRR. We also analyzed the impact of patient age, BM blast count, CAR-T-cell dose, and the interval time between CAR-T-cell therapy and consolidation allogeneic hematopoietic stem cell transplantation (allo-HSCT) on OS and LFS. Results Among 254 patients, 231 cases achieved CR within one month after CART therapy. A total of 211 CR patients had long-term follow-up of more than 30 days with a median follow-up of 12 months (1 to 29 months). On day 30 post CAR T-cell infusion, the median CAR-T/T-cell ratio in PBLC samples was 0.51% (range: 0%-44.8%), with 59 of 169 patients (34.9%) having a CAR-T/T-cell ratio of ≥1% and 85 of 169 patients (50.3%) with a CAR-T/T-cell ratio of ≥0.5%. The median percentage of CD19+ B lymphocytes in PBLC on day 30 was 0.0% (range: 0.0%-9.4%), of which 157 of 169 patients (92.9%) had &amp;lt;0.5% CD19+ B-cell lymphocytes, and 137 of 169 patients (81.1%) had &amp;lt;0.1% CD19+ B lymphocytes on day 30. Using a single continuous variate factors analysis, we found that increasing BM blasts and percentage of CD19+ B-lymphocytes in PBLC samples on day 30 correlated with a worse OS and LFS (Table 1). BM blasts of ≥70% were statistically significantly correlated with a worse OS and LFS when compared to BM blasts of &amp;lt;70% (2-year OS of 52.6% vs. 65.0%, p=0.041; 2-year LFS of 43.3% vs. 58.6%, p=0.023). Unlike the BM blast data, for the CD19+ B-lymphocytes percentage in PBLC samples on day 30, we not identify a cut-off threshold. The CAR-T/T-cell ratio in PBLC samples on day 30 had no influence on OS or LFS. Unfortunately, the CAR-T/T- cell ratio and CD19+ B-lymphocyte percentage data beyond day 30 following CAR T-cell therapy was lacking for most patients and further analysis could not be performed to understand the impact of these factors on long-term survival. In our analysis, CAR T-cell dose, the interval time between CAR T-cell infusion and allo-HSCT did not significantly correlate with OS, LFS, or relapse. The remaining 184 patients in CR received a consolidation allo-HSCT after a median interval time of 67 days post CAR T-cell therapy (range: 30-334 days). Thirty-two of these patients (17%) relapsed with a median time to relapse of 221 days (57-490 days). The remaining 27 patients received CAR T-cell therapy only and 11 (41%) relapsed with a median time to relapse of 100 days (53-398 days). None of the four factors above had an influence on the CRR in either the bridging into allo-HSCT group or the CAR-T only group (Table 2). Conclusions Using a single continuous variate factors analysis, we found that a high BM blast count and the percentage of CD19+ B-lymphocytes in PBLC samples from R/R ALL patients on day 30 predicted a worse OS and LFS while age, the CAR-T/T-cell ratio on day 30, CAR-T cell dose, and the interval time between CAR-T cell infusion and allo-HSCT had no clear impact on long-term outcomes. Disclosures No relevant conflicts of interest to declare.

The clinical significance of the alternative Wilms tumor gene overexpression-hypermethylation signature in acute myeloid leukemia.

Wilms tumor 1 (WT1) gene is overexpressed in numerous cancers, including acute myeloid leukemia (AML). The alternative WT1 gene (AWT1) is generated from alternative transcription start site in the WT1 first intron and encodes an N-terminal-truncated protein lacking the repressor domain. Although WT1 overexpression is a common feature in AML, the expression levels of the AWT1 and its underlying epigenetic alterations, as well as their clinical relevance in AML remain unknown. Quantitative assessment of AWT1 gene transcripts was performed using real-time polymerase chain reaction (PCR). Bisulfite PCR followed by pyrosequencing was done to determine the methylation status of the AWT1 promoter. The bone marrow samples were collected at diagnosis and after completion of induction chemotherapy from 80 newly diagnosed AML patients. Forty non-malignant BM samples were recruited as controls. The AWT1 was significantly overexpressed in AML patients. Robust hypermethylation of the AWT1 promoter was found to be a highly specific and sensitive marker for AML (p < 0.001). Significant positive correlations between the AWT1 expression and methylation levels with BM blast counts at both initial diagnosis and after induction therapy were observed (p < 0.001). AWT1 overexpression at the initial diagnosis of AML was found to be an independent negative factor for complete remission response after induction therapy (p = 0.014). The AWT1 gene overexpression-hypermethylation signature is a characteristic marker that positively correlates with the leukemic burden in AML. AWT1 overexpression at AML diagnosis is an independent negative predictor for CR after induction chemotherapy.

EZH2 Mutations and Impact on Clinical Outcome Analyzed in 1604 Patients with Acute Myeloid Leukemia

EZH2 Mutations and Impact on Clinical Outcome Analyzed in 1604 Patients with Acute Myeloid Leukemia

Clinical Characteristics and Outcome in IDH1/2 Mutant AML Patients - Analysis of 3898 Newly Diagnosed Patients with Acute Myeloid Leukemia

Clinical Characteristics and Outcome in IDH1/2 Mutant AML Patients - Analysis of 3898 Newly Diagnosed Patients with Acute Myeloid Leukemia

Relationship between Altered Gene Expression and DNA Methylation of the DLK1-DIO3 region in Azacitidine-Treated Patients with Myelodysplastic Syndromes and Acute Myeloid Leukemia with Myelodysplasia-Related Changes

INTRODUCTIONMyelodysplastic syndromes (MDS) represent a heterogeneous spectrum of hematopoietic stem cell disorders characterized by inefficient hematopoiesis, peripheral blood cytopenia, dysplasia in one or more myeloid cell lineages, and a tendency to evolve into acute myeloid leukemia (AML). Hypomethylating agent azacitidine (AZA) is a standard therapy for patients with higher-risk MDS and AML with myelodysplasia-related changes (AML-MRC).The DLK1-DIO3 region is located on chromosome 14q32 and contains one of the largest miRNA clusters in the genome. Functions of these miRNAs have been linked to apoptosis and proliferation. Their expression is under the control of a differentially methylated region (DMR) located in the promoter of MEG3 gene. Significant overexpression of these miRNAs has previously been found in MDS and acute promyelocytic leukemia (APL). In APL, this upregulation has been linked to hypermethylation of MEG3-DMR. AIMSWe examined (i) expression of the 14q32 miRNAs and mRNAs (DLK1, RTL1, DIO3, MEG3, and MEG8) and (ii) DNA methylation of MEG3-DMR in patients with higher-risk MDS and AML-MRC before and during AZA treatment with the aim to reveal whether the overexpression of these miRNAs is epigenetically regulated in myelodysplasia. METHODSThe study comprised 24 paired samples of CD34+ bone marrow cells obtained from 12 patients with higher-risk MDS and AML-MRC before and during AZA therapy. miRNA profiling was done using Agilent microarrays and mRNA levels of the genes located within the 14q32 region were assessed by Illumina microarrays. Measurement of MEG3 level by RT-qPCR confirmed the microarray results on an independent cohort (81 MDS and 14 AML-MRC patients, and 13 controls). Methylation status of MEG3 promoter was investigated by amplicon bisulfite sequencing. RESULTSWe studied levels of miRNAs and mRNAs encoded in the DLK1-DIO3 region and found that 50 % of untreated patients showed strong upregulation of expression activity within the locus. Following AZA therapy, we observed a significant reduction in the miRNA/mRNA expression to nearly normal levels, suggesting that expression activity might be affected by hypomethylation. Interestingly, expression levels of miRNAs and mRNAs positively correlated, indicating that transcriptional activity was regulated uniformly throughout the region.Evaluation of expression data with clinical features in the pretreatment samples revealed that expression activity was related to BM blast count, patient diagnosis, and outcome. High expression was significantly associated with the diagnosis of AML-MRC and poor outcome, whereas low expression with MDS and favorable outcome. No chromosomal abnormality or cytogenetic risk category was linked to the expression changes.In the patients before treatment, we revealed a significant hypermethylation of a part of the MEG3-DMR that closely preceded the MEG3 start codon. This hypermethylation was seen in 50 % of the patients and negatively correlated with expression activity. Interestingly, the hypermethylation was associated with longer progression-free survival after initiation of AZA therapy (hazard ratio = 0.220, P = 0.03).Following AZA therapy, we identified an apparent decrease of methylation in another part of MEG3-DMR. This hypomethylation might be attributed to the hypomethylating effect of AZA and could therefore influence the deregulated expression of the miRNA cluster.Finally, we investigated the potential application of the deregulation observed in this locus for prognostic purposes regarding the responsiveness to AZA treatment. Although, we identified changes in expression and methylation levels before AZA therapy, these changes were not associated with future response status. CONLUSIONSHere, we correlated changes in expression of 14q32 miRNAs and mRNAs with specific methylation changes in its regulatory sequences in higher-risk MDS and AML-MRC patients treated with AZA. We thoroughly investigated a complex regulatory mechanism within this region and based on our data, we assumed that epigenetic alterations affect expression activity in the locus. However, also other mechanisms such as influence of some transcription factors may play yet unrecognized roles in the resulting state.Supported by grants 17-31398A, 16-33617A, 16-33485A, and 00023736 (Ministry of Health of the Czech Republic). DisclosuresNo relevant conflicts of interest to declare.

Correlation between Bone Marrow Fibrous Proliferation and Prognosis of Acute Myeloid Leukemia

To investigate the correlation between the bone marrow fibrous proliferation and the prognosis of acute myeloid leukemia(AML). The quantitative method was used to analyze the reticulin fiber density (RFD) of AML patients. the bone marrow sections from 39 primary AML patients and 35 normal controls were collected to compare the RFD between these 2 groups. The prognosis value of RFD for AML were estimated by using appropriate statistical analysis. RFD in primary AML was significantly higher than that in normal controls(2.41%±0.23% vs 1.14%±0.06%)(P<0.05). Relapse-free survival(RFS) analysis showed that the patients with RFD more than 1.68% indicated poor RFS, and the overall survival(OS) analysis showed that patients with RFD more than 2.66% indicated poor overall survival (P<0.05). Besides, there were no relationship between RFD and the BM blast count (r=0.01) and WBC counts (r=0.04) at diagnosis(P>0.05). The RFD in bone marrow is a high risk factor in poor prognosis of AML patients.

Comparable Outcomes of Therapy-Related and De Novo Myelodysplastic Syndrome after Allogeneic Hematopoietic Stem Cell Transplantation

Therapy-related myelodysplastic syndrome (t-MDS) is a well-known complication of conventional chemoradiotherapy used to treat a variety of hematologic malignancies as well as solid tumors. Because of poor prognosis of t-MDS when treated with conventional therapy, allogeneic hematopoietic stem cell transplantation (HSCT) is usually recommended with encouraging results reported.Herein, we retrospectively analyzed outcomes in of 309 adult patients with t-MDS (n =101) and de novo MDS (n=208) receiving allogeneic HSCT from 2005 to 2015 at MD Anderson Cancer Center and compared their outcomes adjusted for disease characteristics.The study cohort had median age of 59 (range, 18-77) at HSCT. Histological subtype at diagnosis was RAEB-1 and -2 in 120 (39%) and CMML in 30 (10%) patients. When cytogenetics were classified by monosomal karyotype (MK) defined by Breems et al., 87 patients (27%) had MK-positive (MKpos), 99 patients (32%) MK-negative (MKneg) and 100 patients (32%) good risk cytogenetics (including normal karyotype (n=88) and del(5q), del(12p), del(20q) (n=12)). Donors were matched related donor (MRD, n=177), matched unrelated donor (MUD, n=114) or haploidentical donor (n=18). Most patients received a myeloablative conditioning regimen (n=181, 59%). Hematopoietic stem cell comorbidity index (HCT-CI) was ≥3 in 144 (47%) patients.Among t-MDS and de novo MDS; median age, donor type and conditioning intensity were not different (Table 1). Compared with de novo MDS, t-MDS patients had more MKpos (20% vs. 46%, p<0.001) less good risk cytogenetics (38% vs. 10%, p<0.001) and more HCT-CI ≥3 (31% vs. 79%, p<0.001). The median time from diagnosis to HSCT was shorter in t-MDS (6 mo. vs. 9 mo., p<0.001).The median follow up of 139 survivors was 37 (range, 3-125) months. Outcome analyses were stratified for t-MDS and de novo MDS patients due to different disease characteristics observed.Univariate analyses showed that MKpos was associated with a poor prognosis for the cumulative incidence of progression both in t-MDS and de novo MDS compared with good cytogenetics (HR=4.3, p=0.05, HR=6.9, p<0.001, respectively) (Figure 1). BM blast count ≥3% at HSCT was also associated with increased incidence of progression in both groups (HR=1.5, p=0.2, HR=1.9, p=0.02, respectively). This enabled us to analyze t-MDS and de novo MDS together for multivariate regressions which revealed that MKpos patients with BM blast count ≥3% had the highest risk for disease progression compared with patients without MKpos and with BM blast count <3% (HR=5.8) (Table 2). Patients with MKpos but with BM blast count <3% also had higher incidence of progression (HR=3.9). The outcomes by risk groups for progression was comparable for t-MDS and de novo MDS (Data not presented).For progression free survival (PFS), univariate analyses revealed that MKpos had poor prognosis for both in t-MDS and de novo MDS compared with good-risk cytogenetics (HR=2.9, p=0.025 and HR=8.4, p<0.001, respectively). MKneg were also associated with inferior PFS compared with good-risk cytogenetics in both t-MDS and de novo MDS (HR=2.1, p=0.1 and HR=2.3, p=0.001, respectively). BM blast count ≥3% also decreased PFS both in t-MDS and de novo MDS for PFS (HR=1.6, p=0.07 and HR=1.8, p=0.004, respectively). When t-MDS and de novo MDS were analyzed together for multivariate regressions, we found that MKpos patients with BM blast count ≥3% had lowest PFS while patients good risk cytogenetics had the best outcomes independent of their BM blast count (Table 2). There was no impact of t-MDS on PFS and similar to progression, the outcomes were not inferior in t-MDS (Data not presented).In conclusion, although t-MDS has been felt to have a relatively poor prognosis after HSCT, our results suggest that t-MDS has comparable outcomes with de novo MDS after allogeneic HSCT when adjusted for disease characteristics. Patients with MKpos in t-MDS and de novo MDS have very high incidence of disease progression and low PFS and represent a target population for innovative transplant strategies. [Display omitted] [Display omitted] [Display omitted] DisclosuresCiurea:Cyto-Sen Therapeutics: Equity Ownership; Spectrum Pharmaceuticals: Other: Advisory Board.

A 5-Day Cytoreductive Chemotherapy Followed By Haplo-Identical HSCT (FA5-Bucy) As a Tumor-Ablative Regimen Improved the Survival of Patients with Advanced Hematological Malignancies

Background: Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) has been used when HLA-matched siblings are not available. Conditioning regimens aim to reduce tumor burden prior to HSCT and provide sufficient immunoablation.Patients and Interventions: We report the outcome of haplo-HSCT in 63 consecutive patients (19 females/44 males) with high-risk or relapsed/refractory hematological malignancies (n=29-AML; 8-sAML; 19-ALL; 5-advanced-MDS; 2-CML-BC), followed at our Center from 2/2013 to 12/2015. Median age was 20 years (range: 1.1-49). Twenty-one patients achieved remission prior to transplant, while 42 did not (7%-98% BM blasts). No patient had severe infection or organ failure before HSCT. Patients received FA5-BUCY registered on http:// ClinicalTrials.gov (NCT02328950), i.e., 5-day salvage chemotherapy (Fludarabine/Ara-C) and conditioning (Busulfan/Cyclophosphamide). GvHD prophylaxis included ATG, CsA, MMF and short-term MTX.Results: All patients received stem cells from bone marrow and peripheral blood, and achieved successful engraftment, except two who died before. With a median follow-up of 269 days (120-1081), 42/63 patients are still alive and disease-free. Two-year OS and RFS were similar in patients not in remission and in those in complete remission (61.3% vs 56.3%, p=0.88; 58.3% vs 56.3%, p=0.991). Non-relapse mortality and relapse incidence were 22.2% and 11.1%, respectively. Severe acute-GvHD occurred in 4/63 patients. Transplant-related mortality was low at day+100 (17.5%) and for the entire study period (20.6%). Unexpectedly, few patients experienced mild-to-moderate toxicity, and main causes of death were infection and GvHD. BM blast counts, age,and donor-recipient gender-pairs did not affect the outcome. Less chemotherapy cycles prior to HSCT might result in more favorable outcome.Conclusions: Thus, haplo-HSCT with FA5-BUCY appears promising for advanced disease, especially when TBI and amsacrine, used for FLAMSA, are not available and in pediatric patients for whom TBI is not recommended. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Prognostic Impact of Chromosomal Translocations in 751 Korean Patients with Primary Myelodysplastic Syndrome

Introduction Chromosomal translocations are rare in myelodysplastic syndrome (MDS) and their impact on overall survival (OS) and response to hypomethylating agents (HMA) is unknown.Methods The prognostic impact of the revised International Prognostic Scoring System (IPSS-R) and for chromosomal translocations was assessed in 751 patients from the Korea MDS Registry. Patients with secondary MDS, chronic myelomonocytic leukaemia, and patients with AML and a BM blast count of 20-30% who were classified as having refractory anaemia with excess blast (RAEB)-t according to the French-American-British classification were excluded. Cytogenetic aberrations were counted following the International Working Group on MDS Cytogenetics (IWGMC) consensus guidelines. Complex karyotype (CK) was defined as having 3 or more chromosomal abnormalities. Monosomal karyotype (MK) was defined as the presence of 2 or more autosomal monosomies or 1 single autosomal monosomy in combination with structural abnormalities.Results The median age of the patients was 65 years, and 457 (61.9%) were male. The median follow-up time was 71.7 months for survivors (range, 0.1 - 230.5). The most common WHO subtype was refractory cytopaenia with multilineage dysplasia (29.7%), followed by RAEB-1 (19.8%), RAEB-2 (18.4%), refractory cytopaenia with unilineage dysplasia (15.4%), and MDS-unclassifiable (11.9%). More than half of patients received disease-modifying treatment; 381 (50.7%) received hypomethylating agents (HMAs), and 83 (11.1%) received haematopoietic stem cell transplantation (HSCT). IPSS-R effectively discriminated patients according to leukaemia evolution risk and OS.We identified 40 patients (5.3%) carrying translocations. Among these, 46 translocations involving 72 breakpoints were identified including balanced translocations in 13 (28.3%) and unbalanced translocation in 33 (71.7%). Translocations were found as a part of CK in 30/46 cases (65.2%) and as a part of MK in 21/46 (45.7%). Frequently involved chromosomes were chromosome 7 (n = 13); ch1 (n = 12); ch5 (n = 9); ch12 (n = 6); ch6, ch11, and ch14 (n = 5); ch10 and ch20 (n = 4); and ch16, ch17, and X (n = 3) in decreasing order. To the best of our knowledge, 43 (93.5%) of the 46 translocations have not been previously described in MDS, nor in AML or CMML. Patients with translocation also had a higher percentage of BM blasts and included a significantly larger proportion of RAEB-1 and RAEB-2. Translocation presence was associated with a shorter OS (median, 12.0 versus 79.7 months, P < 0.01), and multivariate analysis demonstrated that translocations (hazard ratio [HR] 1.64 [1.06-2.63]; P = 0.03) as well as age, sex, IPSS-R, and CK were independent predictors of OS. In the IPSS-R high and very high risk subgroup (n = 260), translocations remained independently associated with OS (HR 1.68 [1.06-2.69], P = 0.03) whereas HMA treatment was not associated with improved survival (median OS, 20.9 versus 21.2 months, P = 0.43). However, translocation carriers exhibited enhanced survival following HMA treatment (median 2.1 versus 12.4 months, P = 0.03).Conclusion Our data suggest that chromosomal translocation is an independent predictor of adverse outcome and has an additional prognostic value in discriminating patients with MDS having higher risk IPSS-R who could benefit from HMA treatment. Further studies in other populations are needed to validate the clinical relevance of translocation in patients with MDS. DisclosuresLee:Amgen: Membership on an entity's Board of Directors or advisory committees.

A 5-day cytoreductive chemotherapy followed by haplo-identical hsct (FA5-BUCY) as a tumor-ablative regimen improved the survival of patients with advanced hematological malignancies.

Haplo-HSCT has been used when HLA-matched siblings are not available. Conditioning regimens aim to reduce tumor burden prior to HSCT and provide sufficient immunoablation. We report the outcome of haplo-HSCT in 63 consecutive patients from 2/2013 to 12/2015 (19 females/44 males) with high-risk or relapsed/refractory hematological malignancies (n=29-AML; 8-sAML; 19-ALL; 5-advanced-MDS; 2-CML-BC). Median age was 20 years (range: 1.1-49). Twenty-one patients achieved remission prior to transplant, while 42 did not. Patients received FA5-BUCY, i.e., 5-day salvage chemotherapy (Fludarabine/Ara-C) and conditioning (Busulfan/Cyclophosphamide). GvHD prophylaxis included ATG, CsA, MMF and short-term MTX. All patients received stem cells from bone marrow and peripheral blood, and achieved successful engraftment, except two who died before. With a median follow-up of 269 days (120-1081), 42/63 patients are still alive and disease-free. Two-year OS and RFS were similar in patients not in remission and in those in complete remission (61.3% vs 56.3%, p=0.88; 58.3% vs 56.3%, p=0.991). Non-relapse mortality and relapse incidence were 22.2% and 11.1%, respectively. Severe acute-GvHD occurred in 4/63 patients. Transplant-related mortality was low at day+100 (17.5%) and for the entire study period (20.6%). Unexpectedly, few patients experienced mild-to-moderate toxicity, and main causes of death were infection and GvHD. BM blast counts, age, and donor-recipient gender-pairs did not affect the outcome. Less chemotherapy cycles prior to HSCT might result in more favorable outcome. Thus, haplo-HSCT with FA5-BUCY appears promising for advanced disease, especially when TBI and amsacrine, used for FLAMSA, are not available and in pediatric patients for whom TBI is not recommended.

Short telomere length and its correlation with gene mutations in myelodysplastic syndrome

BackgroundTelomere erosion can lead to genomic instability and cancer progression. It has been suggested that the shortest telomere, not the average telomere length (TL), is critical for cell viability. Some studies have shown shorter TL in myelodysplastic syndrome (MDS) patients but the critically short telomeres, the variability of TL within individual patient has not been evaluated. Thus, we aimed to investigate the TL of MDS patients and assessed the association of TL with recurrent genetic mutations in MDS.MethodsWe measured the TL of bone marrow nucleated cells for diagnostic samples at a single-cell level by quantitative fluorescence in situ hybridization (Q-FISH) for 58 MDS patients and analyzed the minimum, median, average, standard deviation, average of the 0th to 10th percentile TL within a patient, and the proportion of cells with TL that is shorter than the lowest 10th percentile of the normal control (NC). The correlations of TL to clinical parameters, cytogenetic results, and genetic mutations were assessed.ResultsMDS patients showed eroded telomeres and narrow distribution compared to the NC (P < 0.001, P = 0.018, respectively). Patients with mutation showed significantly lesser cells with short TL, below the lowest 10th percentile of the NC (P = 0.017), but no differences in TL were found according to mutations/cytogenetic abnormalities except for CSF3R mutation. However, those patients with a high percentage (≥80 %) of cells with short TL showed poorer overall survival (P = 0.021), and this was an independent prognostic factor, along with TP53, U2AF1 mutation, and high BM blast count (P = 0.044, 0.001, 0.004, 0.012, respectively).ConclusionsThe shortest TL, which determines the fate of the cell, was significantly shorter, and higher burden of cells with short TL were found in MDS, which correlated with poor survival, suggesting the need to measure TL in single cells by Q-FISH.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0287-9) contains supplementary material, which is available to authorized users.

Increased TACE (Tumor necrosis factor-alpha±-converting enzyme; ADAM17) Activity Associates with Decreased CD62L Expression, Increased Soluble CD62L Plasma Levels and Predicts Molecular Response to Nilotinib Therapy in Patients with Early Chronic Phase Chronic Myelogenous Leukemia (CML-CP): Results from an ENEST1st Substudy

▪Background and Aim: Tyrosine kinase inhibitors (TKI) imatinib and dasatinib modulate immune responses in vitro and in vivo. Immunological surveillance in the MRD-situation might be of particular relevance for long-term control or even elimination of CML-repopulating stem cells. Moreover, baseline immunological characteristics may be associated with response to TKI therapy. Little is known about potential immune-modulatory effects of nilotinib in vivo. The ENEST1st study (NCT01061177) evaluated the role of first-line nilotinib therapy in CML-CP. The primary endpoint was the MR4 rate at 18 months. A comprehensive immunological monitoring program within this ENEST1st substudy characterized baseline and therapy-induced immunological variables to correlate them with biological disease characteritics and clinical response parameters.Methods: Peripheral blood was taken prior to treatment initiation and after 6 and12 months (mo) from 52 patients. Samples were analyzed by nine colour flow cytometry employing six panels of optimized antibodies to determine various leukocyte populations (e.g. T cell subpopulations including Treg and NKT cells, NK cells, B cells, monocytes, MDSC, dendritic cell subsets). Plasma concentrations of soluble CD62L (sCD62L) and TACE (tumor necrosis factor-α-converting enzyme; ADAM17, CD156b), the metalloproteinase inducing proteolytic cleavage of CD62L from the cell surface, were either measured by ELISA or (in case of the enzymatic activity of TACE) using a fluorogenic assay. Changes in immune cell parameters were correlated to biological disease features and clinical endpoints.Results: The most striking finding of this study is the drastic loss of the lymph-node homing marker CD62L on immune cells (T cell subsets and granulocytes) at baseline (basCD62L), which increased back to normal levels during nilotinib therapy. The proportion of basCD62L+ cells among both CD4+ and CD8+ T cell subsets significantly correlated with Sokal score (both as continous and categorial variable, i.e. high vs. low/int). Low basCD62L expression levels on both T cell subsets correlate with increased spleen size, higher BM and PB blast and WBC counts as well as it correlates to higher BCR-ABL copy numbers at almost all time points during treatment. Similarly, lower basCD62L on either CD4+ or CD8+ T cells is linked to a longer duration to reach the respective molecular endpoint. Patients reaching MR4 at 18 months (primary study endpoint) had significantly higher levels of basCD62L on both CD4+ (p=0.02) and CD8+ (p=0.008) T cells. Consequently, MR4 at 18 months was attained in a significantly higher percentage of patients in the basCD62hi compared to the CD62lo patients (63% vs. 13.0%). Vice versa, patients who reached MR4 at 18 months had significantly higher proportions of basCD62L expressing cells among both CD4+ and CD8+ T cells. Moreover, as depicted by a cumulative response rate, patients with high proportions of basCD62Lhi T cells, achieved MMR and MR4 significantly earlier and in a higher proportion throughout the observation period. A detailed characterization of other T cell differentiation marker (CD45RA, CD45R0, CD28, CD27, and CD95) did not reveal significant baseline T cell subset alterations as explanation for altered CD62L expression. In contrast to low basCD62L surface expression levels, its shed form sCD62L is significantly increased at diagnosis but subsequently drops back during nilotinib therapy. Similar to surface CD62L expression, also sCD62L associates with biological disease features and molecular response to nilotinib. Finally, low CD62L surface expression was associated with elevated sCD62L levels and increased proteolytic activity but not total amount of TACE.Conclusion: At baseline, increased proteolytic activity of TACE sheds CD62L from the immune cell surface. During nilotinib therapy, TACE activity gets normalized leading to re-expression of CD62L on T cells and vice versa a drop of sCD62L. Low baseline T cell expression levels of CD62L and increased sCD62L levels correlate to a more aggressive CML phenotype and are linked to inferior molecular response to nilotinib in early CML-CP. Larger prospective studies including also other TKIs are needed to confirm the prognostic relevance of sCD62L/CD62L expression as response-prediction marker, as this marker is easy to measure by ELISA in plasma samples or flow-cytometry. DisclosuresMustjoki:Finnish Cancer Institute: Research Funding; Sigrid Juselius Foundation: Research Funding; Academy of Finland: Research Funding; the Finnish Cancer Societies: Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Signe and Ane Gyllenberg Foundation: Research Funding. Loskog:RePos Pharma AB: Membership on an entity's Board of Directors or advisory committees; Vivolux AB: Membership on an entity's Board of Directors or advisory committees; Lokon Pharma AB: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; NEXTTOBE AB: Membership on an entity's Board of Directors or advisory committees; Alligator Bioscience AB: Patents & Royalties. Gjertsen:Haukeland University Hospital: Research Funding. Giles:Novartis: Consultancy, Honoraria, Research Funding. Ossenkoppele:Pfizer: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Porkka:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria.

Low expression of circulating microRNA-328 is associated with poor prognosis in patients with acute myeloid leukemia.

BackgroundDysregulation of circulating miR-328 has been identified in several tumors and is associated with prognosis of patients. However, the expression pattern of miR-328 and the impact on prognosis has not yet been studied in acute myeloid leukemia (AML). The purpose of this study is to investigate the expression status of miR-328 and its clinical significance in AML patients.MethodsRNA was extracted from plasma of 176 patients with newly diagnosed AML and 70 healthy volunteers. The miR-328 expression was examined by Realtime quantitative PCR. The association of circulating miR-328 expression with clinicopathological factors and prognosis of AML patients was statistically analyzed.ResultsThe expression of miR-328 was significantly downregulated in AML patients (median value 22.99, range: 3.63-242.0) compared with those of healthy controls (median value 89.17, range: 12.05-397.7; P < 0.001), and miR-328 expression was markedly increased in patients after treatment than before (23.40 ± 1.76 vs. 46.61 ± 3.83, P < 0.001). Moreover, low levels of miR-328 were associated with a higher white blood cell count and BM blast count (P = 0.026 and P = 0.003, respectively), and lower hemoglobin and platelet count (P = 0.004 and P = 0.022, respectively). Patients with low miR-328 expression had a relatively poor overall survival (P = 0.022) and shorter relapse-free survival (P = 0.008) than those with high miR-328 expression. In addition, low miR-328 expression was an independent prognostic factors for both OS (P = 0.017) and RFS (P = 0.023).ConclusionsCirculating miR-328 downregulation is a common event and is associated with poor clinical outcome in AML patients.

Flow cytometry immunophenotypic analysis of Philadelphia-negative myeloproliferative neoplasms: Correlation with histopathologic features.

Compared with the proven utility of flow cytometry immunophenotyping (FCI) analysis in the workup of myelodysplastic syndromes (MDS), immunophenotypic alterations in myeloproliferative neoplasms (MPN) have been less studied and the potential utility of FCI is not defined. Bone marrow (BM) samples of 83 Philadelphia-negative MPN patients were assessed by multicolor FCI including 27 with essential thrombocythemia (ET); 17 polycythemia vera (PV); 33 primary myelofibrosis (PMF) and 6 MPN-unclassifiable (MPN-U). The time interval from initial diagnosis of MPN to FCI analysis was 18 months (0-370). Ninety-five age-matched MDS patients with a similar BM blast count were included for comparison. Immunophenotypic alterations, either in CD34(+) cells or myelomonocytic cells, were detected in 82 of 83 (99%) MPN cases. FCI abnormalities were more frequently observed in cases with substantial myelofibrosis but not different between PMF and fibrotic stage of ET/PV. Furthermore, FCI abnormalities were more frequent in cases with ≥5% BM blasts and/or circulating blasts (P = 0.006); as well as cases with an abnormal karyotype (P = 0.036); but not associated with morphologic dysplasia or JAK2 mutation status. Comparing with MDS, FCI abnormalities were overall less pronounced in MPN cases (P = 0.001). MPNs exhibit frequent immunophenotypic alterations, more pronounced in cases with adverse histopathologic features. These findings illustrate that immunophenotypic alterations are a part of constellational findings in MPN, and correlate progressively with disease stage. The study results also suggest a role of FCI in diagnosis of MPN and monitoring disease over time and after therapy.