Application of a digital PCR method for WT1 to myeloid neoplasms in CR and deep ELN WT1 molecular response (< 10 copies).

Bone marrow WT1 mRNA levels assessed by the ELN method are useful to establish prognostic correlations in myeloid malignancies treated with chemotherapy or hematopoietic stem cell transplantation (HCT). Those patients with WT1 levels below ten copies have a good outcome. However, some of these patients relapse. To further characterize this group of cases, we applied a new and sensitive digital (ddPCR) WT1 method. A consecutive series of 49 patients with treated myeloid malignancies and with an ELN WT1 quantitation of < 10 copies were included in the study. All cases (47 AML and 2 MDS) have received intensive chemotherapy or HCT. One to four micrograms of total RNA were retrotranscribed to obtain ≥ 10,000 ABL1 copies using the ELN protocol. Only those cases with a good quality cDNA were used in the ddPCR WT1 test. The ddPCR Gene Expression WT1 Assay of Bio-Rad© was used to perform the PCR amplification, and the microdroplets were quantified in the Bio-Rad's QX200 droplet reader. Eighteen patients showed a negative WT1 ddPCR assay (0 copies/µl), whereas 31 cases were positive (results ranged from 1 to 15.2 copies/µl). Survival analysis showed statistically significant differences in terms of OS between both groups, 83 ± 8% vs. 46 ± 9% (p = 0.024). A statistically significant correlation was also found between ddPCRWT1 results and CD123+ cell number detected by flow cytometry (p = 0.024). Larger series of patients tested with the current ddPCRWT1 method will solve whether it could be used to stratify patients with myeloid malignancies achieving deep WT1 molecular response (< 10 copies).

Frequency and clinical impact of CDKN2A/ARF/CDKN2B gene deletions as assessed by in-depth genetic analyses in adult T cell acute lymphoblastic leukemia.

Recurrent deletions of the CDKN2A/ARF/CDKN2B genes encoded at chromosome 9p21 have been described in both pediatric and adult acute lymphoblastic leukemia (ALL), but their prognostic value remains controversial, with limited data on adult T-ALL. Here, we investigated the presence of homozygous and heterozygous deletions of the CDKN2A/ARF and CDKN2B genes in 64 adult T-ALL patients enrolled in two consecutive trials from the Spanish PETHEMA group. Alterations in CDKN2A/ARF/CDKN2B were detected in 35/64 patients (55%). Most of them consisted of 9p21 losses involving homozygous deletions of the CDKNA/ARF gene (26/64), as confirmed by single nucleotide polymorphism (SNP) arrays and interphase fluorescence in situ hybridization (iFISH). Deletions involving the CDKN2A/ARF/CDKN2B locus correlated with a higher frequency of cortical T cell phenotype and a better clearance of minimal residual disease (MRD) after induction therapy. Moreover, the combination of an altered copy-number-value (CNV) involving the CDKN2A/ARF/CDKN2B gene locus and undetectable MRD (≤ 0.01%) values allowed the identification of a subset of T-ALL with better overall survival in the absence of hematopoietic stem cell transplantation.

Inhibition of serotonin receptor type 1 in acute myeloid leukemia impairs leukemia stem cell functionality: a promising novel therapeutic target.

Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous neoplasia with poor outcome, organized as a hierarchy initiated and maintained by a sub-population with differentiation and self-renewal capacities called leukemia stem cells (LSCs). Although currently used chemotherapy is capable of initially reducing the tumor burden producing a complete remission, most patients will ultimately relapse and will succumb to their disease. As such, new therapeutic strategies are needed. AML cells differentially expressed serotonin receptor type 1 (HTR1) compared with healthy blood cells and the most primitive hematopoietic fraction; in fact, HTR1B expression on AML patient samples correlated with clinical outcome. Inhibition of HTR1s activated the apoptosis program, induced differentiation and reduced the clonogenic capacity, while minimal effect was observed on healthy blood cells. In vivo regeneration capacity of primary AML samples was disrupted upon inhibition of HTR1. The self-renewal capacity remaining in AML cells upon in vivo treatment was severely reduced as demonstrated by serial transplantation. Thus, treatment with HTR1 antagonists showed antileukemia effect, especially anti-LSC activity while sparing healthy blood cells. Our results highlight the importance of HTR1 in leukemogenesis and LSC survival and identify this receptor family as a new target for therapy in AML with prognostic value.

The expression level of BAALC-associated microRNA miR-3151 is an independent prognostic factor in younger patients with cytogenetic intermediate-risk acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease whose prognosis is mainly related to the biological risk conferred by cytogenetics and molecular profiling. In elderly patients (⩾60 years) with normal karyotype AML miR-3151 have been identified as a prognostic factor. However, miR-3151 prognostic value has not been examined in younger AML patients. In the present work, we have studied miR-3151 alone and in combination with BAALC, its host gene, in a cohort of 181 younger intermediate-risk AML (IR-AML) patients. Patients with higher expression of miR-3151 had shorter overall survival (P=0.0025), shorter leukemia-free survival (P=0.026) and higher cumulative incidence of relapse (P=0.082). Moreover, in the multivariate analysis miR-3151 emerged as independent prognostic marker in both the overall series and within the unfavorable molecular prognostic category. Interestingly, the combined determination of both miR-3151 and BAALC improved this prognostic stratification, with patients with low levels of both parameters showing a better outcome compared with those patients harboring increased levels of one or both markers (P=0.003). In addition, we studied the microRNA expression profile associated with miR-3151 identifying a six-microRNA signature. In conclusion, the analysis of miR-3151 and BAALC expression may well contribute to an improved prognostic stratification of younger patients with IR-AML.

MicroRNA expression at diagnosis adds relevant prognostic information to molecular categorization in patients with intermediate-risk cytogenetic acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease, and optimal treatment varies according to cytogenetic risk factors and molecular markers. Several studies have demonstrated the prognostic importance of microRNAs (miRNAs) in AML. Here we report a potential association between miRNA expression and clinical outcome in 238 intermediate-risk cytogenetic AML (IR-AML) patients from 16 institutions in the CETLAM cooperative group. We first profiled 670 miRNAs in a subset of 85 IR-AML patients from a single institution and identified 10 outcome-related miRNAs. We then validated these 10 miRNAs by individual assays in the total cohort and confirmed the prognostic impact of 4 miRNAs. High levels of miR-196b and miR-644 were independently associated with shorter overall survival, and low levels of miR-135a and miR-409-3p with a higher risk of relapse. Interestingly, miR-135a and miR-409-3p maintained their independent prognostic value within the unfavorable molecular subcategory (wild-type NPM1 and CEBPA and/or FLT3-ITD), and miR-644 retained its value within the favorable molecular subcategory. miR-409-3p, miR-135a, miR-196b and mir-644 arose as prognostic markers for IR-AML, both overall and within specific molecular subgroups.

Acute myeloid leukemia with translocation (8;16)(p11;p13) and MYST3-CREBBP rearrangement harbors a distinctive microRNA signature targeting RET proto-oncogene.

Acute myeloid leukemia (AML) with t(8;16)(p11;p13) (t(8;16) AML) has unique clinico-biological characteristics, but its microRNA pattern is unknown. We analyzed 670 microRNAs in seven patients with t(8;16) AML and 113 with other AML subtypes. Hierarchical cluster analysis showed that all t(8;16) AML patients grouped in an independent cluster. Supervised analysis revealed a distinctive signature of 94-microRNAs, most of which were downregulated, including miR-21 and cluster miR-17-92. The mRNA expression analysis of two known transcription factors of these microRNAs (STAT3 and c-Myc, respectively) showed significant downregulation of STAT3 (P=0.04). A bioinformatic analysis showed that 29 of the downregulated microRNAs might be regulated by methylation; we treated a t(8;16) AML sample with 5-aza-2'-deoxycytidine (5-AZA-dC) and trichostatin A and found that 27 microRNAs were re-expressed after treatment. However, there was no difference in methylation status between t(8;16) and other AML subtypes, either overall or in the microRNA promoter. Cross-correlation of mRNA and microRNA expression identified RET as a potential target of several microRNAs. A Renilla-luciferase assay and flow cytometry after transfection with pre-microRNAs confirmed that RET is regulated by miR-218, miR-128, miR-27b, miR-15a and miR-195. In conclusion, t(8;16) AML harbors a specific microRNA signature that is partially epigenetically regulated and targets RET proto-oncogene.