Alexandre Theocharides

Clonal heterogeneity in polycythemia vera patients with JAK2 exon12 and JAK2-V617F mutations

We studied the lineage distribution of JAK2 mutations in peripheral blood of 8 polycythemia vera (PV) patients with exon 12 mutations and in 21 PV patients with JAK2-V617F. Using a quantitative allele discrimination assay, we detected exon 12 mutations in purified granulocytes, monocytes, and platelets of 8 patients studied, but lymphoid cells showed variable involvement and the mutation was absent in T cells. Endogenous erythroid colonies grew in all patients analyzed. One patient displayed erythroid colonies homozygous for the exon 12 mutation with evidence for mitotic recombination on chromosome 9p. In some patients with exon 12 mutations or JAK2-V617F, a proportion of endogenous erythroid colonies were negative for both JAK2 mutations. One patient carried 2 independent clones: one with an exon 12 mutation and a second with JAK2-V617F. The finding of clonal heterogeneity is compatible with the hypothesis that additional clonal events are involved in the pathogenesis of PV.

Deliberate removal of T cell help improves virus-neutralizing antibody production

The B cell response to lymphocytic choriomeningitis virus is characterized by a CD4+ T cell–dependent polyclonal hypergammaglobulinemia and delayed formation of virus-specific neutralizing antibodies. Here we provide evidence that, paradoxically, because of polyclonal B cell activation, virus-specific T cell help impairs the induction of neutralizing antibody responses. Experimental reduction in CD4+ T cell help in vivo resulted in potent neutralizing antibody responses without impairment of CD8+ T cell activity. These unexpected consequences of polyclonal B cell activation may affect vaccine strategies and the treatment of clinically relevant chronic bacterial, parasitic and viral infections in which hypergammaglobulinemia is regularly found.

Angiogenesis and vascular endothelial growth factor-/receptor expression in myeloproliferative neoplasms: correlation with clinical parameters and JAK2-V617F mutational status

Data on angiogenesis in the bone marrow of BCR‐ABL1‐negative myeloproliferative neoplasm (MPN) patients suggest an increase of the microvessel density (MVD) and vascular endothelial growth factor (VEGF) expression, but relations to the JAK2‐V617F status remain controversial. We performed immunohistochemical studies of MVD and VEGF‐expression in 100 MPN, including 24 essential thrombocythemia‐ (ET), 46 polycythemia vera‐ (PV), 26 primary myelofibrosis‐ (PMF), four myelodysplastic (MDS)/MPN‐ and 20 control reactive bone marrow cases, and correlated these findings with biological and clinical key data and the JAK2‐V617F status. We found significantly increased MVD, particularly that assessed by CD105, and VEGF expression in MPN compared to controls (PMF > PV > MDS/MPN > ET). We observed stronger association between CD105‐MVD and VEGF expression, fibrosis, and JAK2‐V617F mutant allele burden, compared to CD34‐MVD. MVD was strongly increased in MPN with high JAK2‐V617F mutant allele burden. Our study highlights the importance of newly formed CD105+ vessels in the bone marrow of MPN patients, and indicates that assessment of CD105‐MVD better reflects angiogenic activity in MPN. In addition, it provides evidence that despite the fact that angiogenesis is generally independent of the JAK2‐V617F status in MPN, new vessel formation might be linked to Jak2 effects in some cases with high JAK2‐V617F mutant allele burden.

Extralymphatic virus sanctuaries as a consequence of potent T-cell activation

T helper cells can support the functions of CD8+ T cells against persistently infecting viruses such as murine lymphocytic choriomeningitis virus (LCMV), cytomegalovirus, hepatitis C virus and HIV. These viruses often resist complete elimination and remain detectable at sanctuary sites, such as the kidneys and other extralymphatic organs. The mechanisms underlying this persistence are not well understood. Here we show that mice with potent virus-specific T-cell responses have reduced levels and delayed formation of neutralizing antibodies, and these mice fail to clear LCMV from extralymphatic epithelia. Transfer of virus-specific B cells but not virus-specific T cells augmented virus clearance from persistent sites. Virus elimination from the kidneys was associated with the formation of IgG deposits in the interstitial space, presumably from kidney-infiltrating B cells. CD8+ T cells in the kidneys of mice that did not clear virus from this site were activated but showed evidence of exhaustion. Thus, we conclude that in this model of infection, site-specific virus persistence develops as a consequence of potent immune activation coupled with reductions in virus-specific neutralizing antibodies. Our results suggest that sanctuary-site formation depends both on organ anatomy and on the induction of different adaptive immune effector mechanisms. Boosting T-cell responses alone may not reduce virus persistence.

The allele burden of JAK2 mutations remains stable over several years in patients with myeloproliferative disorders

This study shows that mutant allele burden remains stable overall several years in patients with JAK2 (V617F)-positive myeloproliferative disorders. In a retrospective single center study we determined the time course of the JAK2-V617F or JAK2 exon 12 allele burden in DNA from purified granulocytes from 48 patients with myeloproliferative disorders. The percentage of change between the first and last sample in JAK2-V617F positive patients without cytoreductive therapy (n=16) was only +9% during a follow-up of 36±13 months, reflecting a remarkably stable mutant allele burden. When treatment with hydroxyurea was initiated during the course of the study, we observed a significant decrease of the JAK2-V617F allele burden (n=6). However, in JAK2-V617F positive patients who were already on hydroxyurea treatment before the first blood sampling (n=14), we observed stable allelic ratios with a variance of only +3% during a follow-up of 34±16 months. Our data suggest that in untreated myeloproliferative disorders patients, from whom samples at diagnosis are not available, the JAK2 allele burden determined at later stages could be equally informative.

Systemic mastocytosis with associated myeloproliferative disease and precursor B lymphoblastic leukaemia with t(13;13)(q12;q22) involving FLT3

Systemic mastocytoses represent neoplastic proliferations of mast cells. In about 20% of cases systemic mastocytoses are accompanied by clonal haematopoietic non-mast cell-lineage disorders, most commonly myeloid neoplasms. A case of systemic mastocytosis carrying the characteristic mutation at codon 816 (D816V) in the KIT gene of mast cells, with two concurrent accompanying clonal haematopoietic non-mast cell-lineage disorders, chronic myeloproliferative disease, unclassifiable and precursor B lymphoblastic leukaemia is documented. Both accompanying clonal haematopoietic non-mast cell-lineage disorders carried the wild-type KIT gene, but had a novel t(13;13)(q12;q22) involving the FLT3 locus at 13q12. The chronic myeloproliferative disease, unclassifiable and the precursor B lymphoblastic leukaemia were cured by syngenous stem cell transplantation, but the systemic mastocytosis persisted for more than 10 years. The additional impact of molecular techniques on the correct diagnosis in haematological malignancies is highlighted, and evidence is provided that, apart from internal tandem duplications and mutations, FLT3 can be activated by translocations.