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Acute myeloid leukemias (AMLs) are currently genomically characterized by karyotype, fluorescence in situ hybridization (FISH), real-time quantitative PCR, and DNA sequencing. Next-generation sequencing offers the promise of detecting all genomic lesions in a single run. However, technical limitations have hampered the detection of chromosomal rearrangements, so most studies are limited to somatic mutation assessment or require the use of RNA-based strategies. To overcome these limitations, we designed a targeted-DNA capture next-generation sequencing approach associated with easy-to-perform public bioinformatic tools for one-step identification of translocations, inversions, and somatic mutations in AML. Thirty well-characterized newly diagnosed myeloid leukemia patients (27 AML and 3 chronic myeloid leukemia) were tested with the panel. Twenty-three of 24 known rearrangements, as well as one novel fusion gene that could not be detected by karyotype/fluorescence in situ hybridization/real-time quantitative PCR, were detected. This strategy also identified all chromosomal breakpoints as potential targets for future high-sensitive minimal residual disease studies. In addition, mutation analysis revealed the presence of missense protein-coding alterations in at least 1 of the 32 genes evaluated in 21 of 30 patients (70%). This strategy may represent a time- and cost-effective diagnostic method for molecular characterization in AML., Supported by the Fundación Española de Hematología y Hemoterapia (FEHH) (M.I.P.-C. and M.G.-A.); the Spanish Association Against Cancer Scientific Foundation (AECC) (M.C.C.); Miguel Servet program CP13/00080 from the Instituto de Salud Carlos III (ISCIII), Ministerio de Ciencia, Innovación y Universidades, Spain (M.E.S.); and the “Beca de investigación FEHH-CRIS 2018” (C.J.). Partially supported by ISCIII research grants PI15/01706, CIBERONCCB16/12/00233, and “Una manera de hacer Europa” Innocampus, CEI-2010-1-0010.

To provide multi-centennial, annually-resolved records of climate for arid and semi-arid areas of Australia it is necessary to investigate the potential climate signals in tree species in this large region. Using a stable isotope and x-ray fluorescence approach to dendrochronology in Acacia cambagei, this study demonstrates short (10 years) proxies of temperature and precipitation are possible. Because rings in A. cambagei are difficult to see, precluding traditional dendrochronology, we used elemental abundances of Ca and Sr as an annual chronometer back to 1962. Radiocarbon analysis confirmed that our dating of wood from two trees. We compared δ13C and δ18O from the α-cellulose of the dated wood over the most recent 10 years (n = 10) to local climate records demonstrating significant relationships between δ18O and precipitation (r = −0.85, p < 0.002); mean monthly maximum temperature (r = 0.69, p < 0.03); and drought indexes (CRU scPDSI 0.5°, r = −0.89, p < 0.001) for February and March. Acacia cambagei may be useful in developing regional networks of climate proxies for drought. Using modern trees, in combination with architectural timbers, it may be possible to construct a multi-century, annually-resolved proxy-record of rainfall and temperature for semi-arid north-eastern Australia.

INTRODUCTION: Infection complications are important cause of morbidity and non-relapse mortality after allogeneic hematopoietic stem cell transplantation (allo-SCT), even in HLA-identical sibling donors. Although there are many studies associating single nucleotide polymorphisms (SNPs) and allo-SCT evolution, these are mainly related with graft versus host disease (GVHD) and/or transplant related mortality (TRM), with few focused on post-allo-SCT infections. This multicentric study analyzes the potential implication of SNPs with cytomegalovirus (CMV) reactivation and fungal infection (FI) development in allo-SCT with reduced intensity conditioning (RIC). MATERIALS AND METHODS: We included paired recipient-donor samples from 247 RIC allo-SCT for patients diagnosed of AML (35%), NHL (21%), MDS (16%), MM (10%), CLL (7%), HL (6%), or others (5%) in four centers in Spain. Flu-Bu conditioning was used in 55% of the transplants while Flu-Mel in 42%. A total of 52 SNP were analyzed from 44 different gene loci and genotyping was carried out by Sequenom Mass ARRAY platform. Allele frequencies were estimated by direct counting and compared between groups using Fisher's test. P-value RESULTS: Patient's characteristics are shown in Table 1. The median follow up for patients was 28 months (1-175), overall survival at 5 years was 45%, while the event-free survival at 5 years was 57%. Cumulative incidence of CMV reactivation at 3 years was 34% and of FI 11%. Transplant related mortality was 17%, and infection related mortality 14%. In univariate analysis, patients with genotype A/A in rs3093023 (CCR6) showed a lower incidence of CMV reactivation (table 2), along with acute GVHD were the variables associated in multivariate analysis. Regarding to FI, recipient genotype A/A in rs1494555 (IL7R), donor C/C in rs1800795 (FAS), and donor G/G in rs2302217 (MAdCAM-1) were associated with higher FI incidence (table 2), being the only variables related in multivariate analysis. CONCLUSION: This data suggest an association between rs3093023 (CCR6), rs1494555 (IL7R), rs1800795 (FAS) and rs2302217 (MAdCAM-1) polymorphisms with higher incidence of post-allo-SCT opportunist infection. Confirmation in larger series is required. FUNDING: RD12/0036/0069, CB16/12/00233 and 00480, PI12/02361 and ERDF, Innocampus; CEI-2010- 1-0010 Download : Download high-res image (236KB) Download : Download full-size image Disclosures No relevant conflicts of interest to declare.

The pathogenesis of diffuse large B-cell lymphoma (DLBCL) remains partially unknown. The analysis of the B-cell receptor of the malignant cells could contribute to a better understanding of the DLBCL biology. We studied the molecular features of the immunoglobulin heavy chain (IGH) rearrangements in 165 patients diagnosed with DLBCL not otherwise specified. Clonal IGH rearrangements were amplified according to the BIOMED-2 protocol and PCR products were sequenced directly. We also analyzed the criteria for stereotyped patterns in all complete IGHV-IGHD-IGHJ (V-D-J) sequences. Complete V-D-J rearrangements were identified in 130 of 165 patients. Most cases (89%) were highly mutated, but 12 sequences were truly unmutated or minimally mutated. Three genes, IGHV4-34 , IGHV3-23 , and IGHV4-39 , accounted for one third of the whole cohort, including an overrepresentation of IGHV4-34 (15.5% overall). Interestingly, all IGHV4-34 rearrangements and all unmutated sequences belonged to the nongerminal center B-cell–like (non-GCB) subtype. Overall, we found three cases following the current criteria for stereotyped heavy chain VH CDR3 sequences, two of them belonging to subsets previously described in CLL. IGHV gene repertoire is remarkably biased, implying an antigen-driven origin in DLBCL. The particular features in the sequence of the immunoglobulins suggest the existence of particular subgroups within the non-GCB subtype.

The PI3/AKT pathway is up-regulated in acute myeloid leukemia (AML), but its prognostic relevance in cytogenetically normal AML (CN-AML) is unclear. We evaluated RNA levels of AKT and two downstream substrates (FOXO3a-p27) in 110 de novo CN-AML, included in the Spanish PETHEMA therapeutic protocols. Patients with high FOXO3a gene expression displayed shorter OS (p = 0.015) and RFS (p = 0.048) than low FOXO3a expressers. Features selected in the multivariate analysis as having an independent prognostic value for a shorter survival were WBC > 50 × 109/L, age >65 years and high FOXO3a expression. We concluded that FOXO3a assessment could contribute to improve the molecular-based risk stratification in CN-AML. © 2009 Elsevier Ltd. All rights reserved., This work has been partially supported with the grants PI061351 and 00/0023-00 from the Spanish “Fondo de Investigaciones Sanitarias de la Seguridad Social”, 89/A/06 from “Gerencia Regional de Salud, Junta Castilla y León”, CR-USA Foundation-Spanish National Research Council (CSIC) Cooperative Agreement and CIC, IBMCC (USAL-CSIC), Spain.

We analyzed patients with hairy cell leukemia (HCL) to achieve a better understanding of the differentiation stage reached by HCL cells and to define the key role of the diversification of cell surface makers, especially CD25 expression.We analyzed 38 previously untreated patients with HCL to characterize their complete (VDJ(H)) and incomplete (DJ(H)) immunoglobulin (Ig) heavy chain (IgH) rearrangements, including somatic hypermutation pattern and gene segment use.A correlation between immunophenotypic profile and molecular data was seen. All 38 cases showed monoclonal amplifications: VDJ(H) in 97%, DJ(H) in 42%, and both in 39%. Segments from the D(H)3 family were used more in complete compared with incomplete rearrangements (45% vs. 12%; P.005). Furthermore, comparison between molecular and immunophenotypic characteristics disclosed differences in the expression of CD25 antigen; CD25(-) cases, a phenotype associated with HCL variant, showed complete homology to the germline in 3 of 5 cases (60%), whereas this characteristic was never observed in CD25(+) cases (P.005). Moreover, V(H)4-34, V(H)1-08, and J(H)3 segments appeared in 2, 1, and 2 CD25(-) cases, respectively, whereas they were absent in all CD25(+) cases.These results support that HCL is a heterogeneous entity including subgroups with different molecular characteristics, which reinforces the need for additional studies with a larger number of patients to clarify the real role of gene rearrangements in HCL.

At present, a major challenge in the initial diagnosis of leukemia of large granular lymphocytes (LGLs) is to establish the clonal nature of the expanded population. In the present study we have analyzed by flow cytometry immunophenotyping the TCR-Vβ repertoire of 98 consecutive cases of persistent expansions of CD4 + or CD8 +bright CD3 + /TCR-αβ + LGLs and compared the results with those obtained in molecular studies of TCR-β gene rearrangements. Fifty-eight cases were considered to be monoclonal in molecular studies whereas in the remaining 40 cases there was no evidence for monoclonality (11 cases were considered oligoclonal and 29 polyclonal). The TCR-Vβ repertoire was biased to the preferential use of one or more TCR-Vβ families in 96% of cases, a total of 124 TCR-Vβ expansions being diagnosed: one TCR-Vβ expansion in 71 cases and two or more TCR-Vβ expansions in 23 cases. The highest TCR-Vβ expansion observed in each case was higher among monoclonal (74 ± 19%) as compared to nonmonoclonal cases (24 ± 14%) ( P = 0.001), as did the fraction of LGLs that exhibited a TCR-Vβ-restricted pattern (86 ± 16% and 42 ± 23%, respectively; P = 0.0001); by contrast, the proportion of cases displaying more than one TCR-Vβ expansion was higher in the latter group: 7% versus 48%, respectively ( P = 0.001). Results obtained in oligoclonal cases were intermediate between those obtained in polyclonal and monoclonal cases and similar results were observed for CD4 + as for CD8 +bright T-cell expansions. TCR-Vβ familiesexpressed in CD8 +bright T-cell-LGL proliferations showed a pattern of distribution that mimics the frequency at which the individual TCR-Vβ families are represented in normal peripheral blood T cells. Assuming that a given proliferation of LGLs is monoclonal whenever there is an expansion of a given TCR-Vβ family of at least 40% of the total CD4 + or CD8 +bright T-cell compartment, we were able to predict clonality with a sensitivity of 93% and a specificity of 80%. By increasing the cut-off value to 60%, sensitivity and specificity were of 81% and 100%. In summary, our results suggest that flow cytometry immunophenotypic analysis of the TCR-Vβ repertoire is a powerful screening tool for the assessment of T-cell clonality in persistent expansions of TCR-αβ + LGLs.