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Here, we investigated for the first time the frequency and number of circulating tumor plasma cells (CTPC) in peripheral blood (PB) of newly diagnosed patients with localized and systemic plasma cell neoplasms (PCN) using next-generation flow cytometry (NGF) and correlated our findings with the distinct diagnostic and prognostic categories of the disease. Overall, 508 samples from 264 newly diagnosed PCN patients, were studied. CTPC were detected in PB of all active multiple myeloma (MM; 100%), and smoldering MM (SMM) patients (100%), and in more than half (59%) monoclonal gammopathy of undetermined significance (MGUS) cases (p <0.0001); in contrast, CTPC were present in a small fraction of solitary plasmacytoma patients (18%). Higher numbers of CTPC in PB were associated with higher levels of BM infiltration and more adverse prognostic features, together with shorter time to progression from MGUS to MM (p <0.0001) and a shorter survival in MM patients with active disease requiring treatment (p ≤ 0.03). In summary, the presence of CTPC in PB as assessed by NGF at diagnosis, emerges as a hallmark of disseminated PCN, higher numbers of PB CTPC being strongly associated with a malignant disease behavior and a poorer outcome of both MGUS and MM.

Immunosuppressive therapy (IST), consisting of antithymocyte globulin and cyclosporine A, is effective in refractory cytopenia of childhood (RCC), suggesting that, similar to low-grade myelodysplastic syndromes in adult patients, T lymphocytes are involved in suppressing hematopoiesis in a subset of RCC patients. However, the potential role of a T-cell-mediated pathophysiology in RCC remains poorly explored. In a cohort of 92 RCC patients, we prospectively assessed the frequency of T-cell receptor (TCR) β-chain variable (Vβ) domain skewing in bone marrow and peripheral blood by heteroduplex PCR, and analyzed T-cell subsets in peripheral blood by flow cytometry. TCRVβ skewing was present in 40% of RCC patients. TCRVβ skewing did not correlate with bone marrow cellularity, karyotype, transfusion history, HLA-DR15 or the presence of a PNH clone. In 28 patients treated with IST, TCRVβ skewing was not clearly related with treatment response. However, TCRVβ skewing did correlate with a disturbed CD4(+)/CD8(+) T-cell ratio, a reduction in naive CD8(+) T cells, an expansion of effector CD8(+) T cells and an increase in activated CD8(+) T cells (defined as HLA-DR(+), CD57(+) or CD56(+)). These data suggest that T lymphocytes contribute to RCC pathogenesis in a proportion of patients, and provide a rationale for treatment with IST in selected patients with RCC.

WNT signaling has been implicated in the regulation of hematopoietic stem cells and plays an important role during T-cell development in thymus. Here we investigated WNT pathway activation in childhood T-cell acute lymphoblastic leukemia (T-ALL) patients. To evaluate the potential role of WNT signaling in T-cell leukomogenesis, we performed expression analysis of key components of WNT pathway. More than 85% of the childhood T-ALL patients showed upregulated β-catenin expression at the protein level compared with normal human thymocytes. The impact of this upregulation was reflected in high expression of known target genes (AXIN2, c-MYC, TCF1 and LEF). Especially AXIN2, the universal target gene of WNT pathway, was upregulated at both mRNA and protein levels in ∼40% of the patients. When β-CATENIN gene was silenced by small interfering RNA, the cancer cells showed higher rates of apoptosis. These results demonstrate that abnormal WNT signaling activation occurs in a significant fraction of human T-ALL cases independent of known T-ALL risk factors. We conclude that deregulated WNT signaling is a novel oncogenic event in childhood T-ALL.

T-cell receptor (TCR) translocations are a genetic hallmark of T-cell acute lymphoblastic leukemia and lead to juxtaposition of oncogene and TCR loci. Oncogene loci become involved in translocations because they are accessible to the V(D)J recombination machinery. Such accessibility is predicted at cryptic recombination signal sequence (cRSS) sites ('Type 1') as well as other sites that are subject to DNA double-strand breaks (DSBs) ('Type 2') during early stages of thymocyte development. As chromatin accessibility markers have not been analyzed in the context of TCR-associated translocations, various genetic and epigenetic determinants of LMO2, TAL1 and TLX1 translocation breakpoint (BP) sites and BP cluster regions (BCRs) were examined in human thymocytes to establish DSB proneness and heterogeneity of BP site involvement in TCR translocations. Our data show that DSBs in BCRs are primarily induced in the presence of a genetic element of sequence vulnerability (cRSSs, transposable elements), whereas breaks at single BP sites lacking such elements are more likely induced by chance or perhaps because of patient-specific genetic vulnerability. Vulnerability to obtain DSBs is increased by features that determine chromatin organization, such as methylation status and nucleosome occupancy, although at different levels at different BP sites.

Clonal CD8(+)/T-cell receptor (TCR)αβ(+) T-cell large granular lymphocyte (T-LGL) proliferations constitute the most common subtype of T-LGL leukemia. Although the etiology of T-LGL leukemia is largely unknown, it has been hypothesized that chronic antigenic stimulation contributes to the pathogenesis of this disorder. In the present study, we explored the association between expanded TCR-Vβ and TCR-Vα clonotypes in a cohort of 26 CD8(+)/TCRαβ(+) T-LGL leukemia patients, in conjunction with the HLA-ABC genotype, to find indications for common antigenic stimuli. In addition, we applied purpose-built sophisticated computational tools for an in-depth evaluation of clustering of TCRβ (TCRB) complementarity determining region 3 (CDR3) amino-acid LGL clonotypes. We observed a lack of clear TCRA and TCRB CDR3 homology in CD8(+)/TCRαβ(+) T-LGL, with only low level similarity between small numbers of cases. This is in strong contrast to the homology that is seen in CD4(+)/TCRαβ(+) T-LGL and TCRγδ(+) T-LGL and thus underlines the idea that the LGL types have different etiopathogenesis. The heterogeneity of clonal CD8(+)/TCRαβ(+) T-LGL proliferations might in fact suggest that multiple pathogens or autoantigens are involved.

Artemis deficiency is known to result in classical T-B- severe combined immunodeficiency (SCID) in case of Artemis null mutations, or Omenn's syndrome in case of hypomorphic mutations in the Artemis gene. We describe two unrelated patients with a relatively mild clinical T-B- SCID phenotype, caused by different homozygous Artemis splice-site mutations. The splice-site mutations concern either dysfunction of a 5' splice-site or an intronic point mutation creating a novel 3' splice-site, resulting in mutated Artemis protein with residual activity or low levels of wild type (WT) Artemis transcripts. During the first 10 years of life, the patients suffered from recurrent infections necessitating antibiotic prophylaxis and intravenous immunoglobulins. Both mutations resulted in increased ionizing radiation sensitivity and insufficient variable, diversity and joining (V(D)J) recombination, causing B-lymphopenia and exhaustion of the naive T-cell compartment. The patient with the novel 3' splice-site had progressive granulomatous skin lesions, which disappeared after stem cell transplantation (SCT). We showed that an alternative approach to SCT can, in principle, be used in this case; an antisense oligonucleotide (AON) covering the intronic mutation restored WT Artemis transcript levels and non-homologous end-joining pathway activity in the patient fibroblasts.

Homozygous CD19 mutations lead to an antibody deficiency due to disruption of the CD19 complex and consequent impaired signaling by the B-cell antigen receptor. We studied the effects of heterozygous CD19 mutations on peripheral B-cell development and antibody responses in a large family with multiple consanguineous marriages. Sequence analysis of 96 family members revealed 30 carriers of the CD19 mutation. Lymphocyte subset counts were not significantly different between carriers and noncarriers in three different age groups (0-10 years; 11-18 years; adults). B cells of carriers had reduced CD19 and CD21 median expression levels, and had reduced proportions of transitional (0-10 years) and CD5(+) B cells (adults). CD19 carriers did not show clinical signs of immunodeficiency; they were well capable to produce normal serum Ig levels and had normal responses to primary and booster vaccinations. The frequency of mutated Vκ alleles was not affected. Heterozygous loss of CD19 causes some changes in the naive B-cell compartment, but overall in vivo B-cell maturation or humoral immunity is not affected. Many antibody deficiencies are not monogenetic, but likely caused by a combination of multiple genetic variations. Therefore, functional analyses of immune cell function should be carried out to show whether heterozygous mutations contribute to disease.