Deep sequencing of the T-cell receptor repertoire in CD8+ T-large granular lymphocyte leukemia identifies signature landscapes.

New massively parallel sequencing technology enables, through deep sequencing of rearranged T-cell receptor (TCR) Vß complementarity-determining region 3 (CDR3) regions, a previously inaccessible level of TCR repertoire analysis. The CDR3 repertoire diversity reflects clonal composition, the potential antigenic recognition spectrum, and the quantity of available T-cell responses. In this context, T-large granular lymphocyte (T-LGL) leukemia is a chronic clonal lymphoproliferation of cytotoxic T cells often associated with autoimmune diseases and various cytopenias. Using CD8(+) T-LGL leukemia as a model disease, we set out to evaluate and compare the TCR deep-sequencing spectra of both patients and healthy controls to better understand how TCR deep sequencing could be used in the diagnosis and monitoring of not only T-LGL leukemia but also reactive processes such as autoimmune disease and infection. Our data demonstrate, with high resolution, significantly decreased diversity of the T-cell repertoire in CD8(+) T-LGL leukemia and suggest that many T-LGL clonotypes may be private to the disease and may not be present in the general public, even at the basal level.

Fanconi Anemia germline variants as susceptibility factors in aplastic anemia, MDS and AML.

Using next generation sequencing we have systematically analyzed a large cohort of 489 patients with bone marrow failure (BMF), including myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), aplastic anemia (AA), and related conditions for the presence of germline (GL) alterations in Fanconi Anemia (FA) and telomerase genes. We have detected an increased frequency of heterozygous FA gene mutations in MDS and to lesser degree in AML suggesting that the presence of one normal allele may not be completely protective and indeed heterozygous FA lesions may have a long latency period before hematologic manifestation. In contrast, GL telomerase gene mutations were not associated with increased disease risk. When compared to large control cohorts, we have not detected an increased frequency of damaging variants among telomerase complex genes, including those previously believed to be involved in the pathogenesis of AA. Our results may suggest that while low penetrance and delayed disease onset can confound identification of genetic predisposition factors, GL FA alterations can be also associated with MDS.

Dynamics of clonal evolution in myelodysplastic syndromes.

To elucidate differential roles of mutations in myelodysplastic syndromes (MDS), we investigated clonal dynamics using whole-exome and/or targeted sequencing of 699 patients, of whom 122 were analyzed longitudinally. Including the results from previous reports, we assessed a total of 2,250 patients for mutational enrichment patterns. During progression, the number of mutations, their diversity and clone sizes increased, with alterations frequently present in dominant clones with or without their sweeping previous clones. Enriched in secondary acute myeloid leukemia (sAML; in comparison to high-risk MDS), FLT3, PTPN11, WT1, IDH1, NPM1, IDH2 and NRAS mutations (type 1) tended to be newly acquired, and were associated with faster sAML progression and a shorter overall survival time. Significantly enriched in high-risk MDS (in comparison to low-risk MDS), TP53, GATA2, KRAS, RUNX1, STAG2, ASXL1, ZRSR2 and TET2 mutations (type 2) had a weaker impact on sAML progression and overall survival than type-1 mutations. The distinct roles of type-1 and type-2 mutations suggest their potential utility in disease monitoring.

Inherited and Somatic Defects in DDX41 in Myeloid Neoplasms.

Most cases of adult myeloid neoplasms are routinely assumed to be sporadic. Here, we describe an adult familial acute myeloid leukemia (AML) syndrome caused by germline mutations in the DEAD/H-box helicase gene DDX41. DDX41 was also found to be affected by somatic mutations in sporadic cases of myeloid neoplasms as well as in a biallelic fashion in 50% of patients with germline DDX41 mutations. Moreover, corresponding deletions on 5q35.3 present in 6% of cases led to haploinsufficient DDX41 expression. DDX41 lesions caused altered pre-mRNA splicing and RNA processing. DDX41 is exemplary of other RNA helicase genes also affected by somatic mutations, suggesting that they constitute a family of tumor suppressor genes.