Niki Stavroyianni

Molecular evidence for EBV and CMV persistence in a subset of patients with chronic lymphocytic leukemia expressing stereotyped IGHV4-34 B-cell receptors.

The chronic lymphocytic leukemia (CLL) immunoglobulin repertoire is uniquely characterized by the presence of stereotyped B-cell receptors (BCRs). A major BCR stereotype in CLL is shared by immunoglobulin G-switched cases utilizing the immunoglobulin heavy-chain variable 4-34 (IGHV4-34) gene. Increased titers of IGHV4-34 antibodies are detected in selective clinical conditions, including infection by B-cell lymphotropic viruses, particularly Epstein–Barr virus (EBV) and cytomegalovirus (CMV). In this context, we sought evidence for persistent activation by EBV and CMV in CLL cases expressing the IGHV4-34 gene. The study group included 93 CLL cases with an intentional bias for the IGHV4-34 gene. On the basis of real-time PCR results for CMV/EBV DNA, cases were assigned to three groups: (1) double-negative (59/93); (2) single-positive (CMV- or EBV-positive; 25/93); (3) double-positive (9/93). The double-negative group was characterized by heterogeneous IGHV gene repertoire. In contrast, a bias for the IGHV4-34 gene was observed in the single-positive group (9/25 cases; 36%). Remarkably, all nine double-positive cases utilized the IGHV4-34 gene; seven of nine cases expressed the major BCR stereotype as described above. In conclusion, our findings indicate that the interactions of CLL progenitor cells expressing distinctive IGHV4-34 BCRs with viral antigens/superantigens might facilitate clonal expansion and, eventually, leukemic transformation. The exact type, timing and location of these interactions remain to be determined.

Anti-CD20-based therapy of B cell lymphoma: state of the art

Over the last 5 years, studies applying the chimeric anti-CD20 MAb have renewed enthusiasm and triggered world-wide application of anti-CD20 MAb-based therapies in B cell non-Hodgkins lymphoma (NHL). Native chimeric anti-CD20 and isotope-labeled murine anti-CD20 MAbs are currently employed with encouraging results as monotherapy or in combination with conventional chemotherapy and in consolidation of remission after treatments with curative intent (ie after/ in combination with high-dose chemotherapy and hematopoietic stem cell rescue). On the available experience, anti-CD20 MAb-based therapeutic strategies will be increasingly integrated in the treatment of B cell NHL and related malignancies.

Whole-exome sequencing in relapsing chronic lymphocytic leukemia: clinical impact of recurrent RPS15 mutations

Fludarabine, cyclophosphamide, and rituximab (FCR) is first-line treatment of medically fit chronic lymphocytic leukemia (CLL) patients; however, despite good response rates, many patients eventually relapse. Although recent high-throughput studies have identified novel recurrent genetic lesions in adverse prognostic CLL, the mechanisms leading to relapse after FCR therapy are not completely understood. To gain insight into this issue, we performed whole-exome sequencing of sequential samples from 41 CLL patients who were uniformly treated with FCR but relapsed after a median of 2 years. In addition to mutations with known adverse-prognostic impact (TP53, NOTCH1, ATM, SF3B1, NFKBIE, and BIRC3), a large proportion of cases (19.5%) harbored mutations in RPS15, a gene encoding a component of the 40S ribosomal subunit. Extended screening, totaling 1119 patients, supported a role for RPS15 mutations in aggressive CLL, with one-third of RPS15-mutant cases also carrying TP53 aberrations. In most cases, selection of dominant, relapse-specific subclones was observed over time. However, RPS15 mutations were clonal before treatment and remained stable at relapse. Notably, all RPS15 mutations represented somatic missense variants and resided within a 7 amino-acid, evolutionarily conserved region. We confirmed the recently postulated direct interaction between RPS15 and MDM2/MDMX and transient expression of mutant RPS15 revealed defective regulation of endogenous p53 compared with wild-type RPS15. In summary, we provide novel insights into the heterogeneous genetic landscape of CLL relapsing after FCR treatment and highlight a novel mechanism underlying clinical aggressiveness involving a mutated ribosomal protein, potentially representing an early genetic lesion in CLL pathobiology.

Recurrent cytogenetic findings in subsets of patients with chronic lymphocytic leukemia expressing IgG-switched stereotyped immunoglobulins

We report a remarkable association of recurrent (stereotyped) cytogenetic aberrations with two subsets of chronic lymphocytic leukemia (CLL) cases expressing IgG-switched, stereotyped B-cell receptors (BCRs). Comparison with cases with heterogeneous BCRs showed that these recurrent cytogenetic aberrations were subset-biased. These findings further support a role for antigen in CLL development.

Lymphocyte subpopulation imbalances, bone marrow hematopoiesis and histopathology in rituximab-treated lymphoma patients with late-onset neutropenia

Lymphocyte subpopulation imbalances, bone marrow hematopoiesis and histopathology in rituximab-treated lymphoma patients with late-onset neutropenia

Origin and diversification of the clonogenic cell in multiple myeloma : lessons from the immunoglobulin repertoire

The study of immunoglobulin genes in multiple myeloma over the last decade has provided important information regarding biology, ontogenetic assignment, disease evolution, pathogenic consequences and tumor-specific therapeutic intervention. Detailed analysis of VH genes has revealed the clonal relationship between switch variants expressed by the bone marrow plasma cell and myeloma progenitors in the marrow and peripheral blood. Regarding VH usage, a bias was found against the V4-34 gene encoding antibodies with cold agglutinin specificity (anti-l/i), thus explaining in part the absence of autoimmune phenomena in myeloma compared to other B cell lymphoproliferative disorders. However, in some studies a substantial number of cases analyzed were carrying the rearranged Humκv325 Vκ gene, known to be over utilized by B cell chronic lymphocytic leukemia clones and possessing autoantibody binding activity. VH genes accumulate somatic hypermutations following a distribution compatible with antigen selection, but with no intraclonal heterogeneity. The analysis of Vκ genes indicates a bias in usage of VκI family members; somatic hypermutation, in line with antigen selection, of the expressed Vκ genes is higher than any other B cell lymphoid disorder. Similar conclusions were reached for Vλ genes; in this case, the analysis raises the controversial issue of N nucleotide insertion at Vλ–Jλ junctions, apparently as a result of TdT activity. A complementary imprint of antigen selection as evidenced by somatic hypermutation of either the VH or VL clonogenic genes has been observed. The absence of ongoing somatic mutations in either VH or VL genes gives rise to the notion that the cell of origin in myeloma is a post-germinal center memory B cell.

Ofatumumab in poor-prognosis chronic lymphocytic leukemia: a Phase IV, non-interventional, observational study from the European Research Initiative on Chronic Lymphocytic Leukemia

We report the largest retrospective, phase IV non-interventional, observational study of ofatumumab therapy in heavily pre-treated patients with poor-prognosis chronic lymphocytic leukemia. Total number of patients was 103; median age was 65 years (range 39–85). Median number of prior lines of therapy was 4 (range 1–13), including, in most cases, rituximab-, fludarabine- and alemtuzumab-based regimens; 13 patients had been allografted. Of 113 adverse events, 28 (29%) were considered to be directly related to ofatumumab. Grade 3–4 toxicities included neutropenia (10%), thrombocytopenia (5%), anemia (3%), pneumonia (17%), and fever (3%). Two heavily pre-treated patients developed progressive multifocal leukoencephalopathy. On an intention-to-treat analysis, the overall response rate was 22% (3 complete response, 1 incomplete complete response). Median progression-free and overall survival times were 5 and 11 months, respectively. This study confirms in a daily-life setting the feasibility and acceptable toxicity of ofatumumab treatment in advanced chronic lymphocytic leukemia. The complete response rate, however, was low. Therefore, treatment with ofatumumab should be moved to earlier phases of the disease. Ideally, this should be done in combination with other agents, as recently approved for ofatumumab plus chlorambucil as front-line treatment for patients unfit for fludarabine. This study is registered at clinicaltrials.gov identifier:01453062.

Molecular evidence for transferrin receptor 2 expression in all FAB subtypes of acute myeloid leukemia

We examined transferrin receptor (TfR) 1 and TfR2 mRNA expression in 50 acute myeloid leukemia (AML) patients by RT-PCR, with primers specific for exons 15-17 (TfR1), 3-5 (TfR2-alpha) and 4-5 (TfR2-beta) of the corresponding gene. There were 4/50 TfR1-negative (-), 3/50 TfR2-alpha mRNA (-) and 13/50 TfR2-beta mRNA (-) cases; only three cases were TfR1/2 mRNA (-). No significant correlations were identified between TfR2-beta mRNA negativity and specific FAB subtypes, karyotype or attainment of complete remission. These findings suggest that: (i) TfR2 expression is not restricted to erythroid cells, and (ii) iron import proteins might complement each other in AML cells.

Immunoglobulin kappa gene repertoire and somatic hypermutation patterns in follicular lymphoma.

Immunoglobulin kappa gene usage and somatic mutation patterns were studied in a series of 47 IGKV-J rearrangements amplified in 42 follicular lymphoma (FL) cases. The IGKV1-39/1D-39 gene predominated and was significantly over-represented compared to normal cells, autoreactive cells or other B cell lymphomas. The impact of somatic hypermutation varied significantly; nevertheless, mutation distribution patterns indicated pressure for preservation of the B cell receptor. In conclusion, the present series demonstrates biased usage of IGKV genes in FL and alludes to the important role of immunoglobulin kappa light chains in antigen selection of the clonogenic B cells in FL.

The histone methyltransferase EZH2 as a novel prosurvival factor in clinically aggressive chronic lymphocytic leukemia.

The histone methyltransferase EZH2 induces gene repression through trimethylation of histone H3 at lysine 27 (H3K27me3). EZH2 overexpression has been reported in many types of cancer and associated with poor prognosis. Here we investigated the expression and functionality of EZH2 in chronic lymphocytic leukemia (CLL). Aggressive cases with unmutated IGHV genes (U-CLL) displayed significantly higher EZH2 expression compared to indolent CLL cases with mutated IGHV genes (M-CLL); furthermore, in U-CLL EZH2 expression was upregulated with disease progression. Within U-CLL, EZH2high cases harbored significantly fewer (p = 0.033) TP53 gene abnormalities compared to EZH2low cases. EZH2high cases displayed high H3K27me3 levels and increased viability suggesting that EZH2 is functional and likely confers a survival advantage to CLL cells. This argument was further supported by siRNA-mediated downmodulation of EZH2 which resulted in increased apoptosis. Notably, at the intraclonal level, cell proliferation was significantly associated with EZH2 expression. Treatment of primary CLL cells with EZH2 inhibitors induced downregulation of H3K27me3 levels leading to increased cell apoptosis. In conclusion, EZH2 is overexpressed in adverse-prognosis CLL and associated with increased cell survival and proliferation. Pharmacologic inhibition of EZH2 catalytic activity promotes apoptosis, highlighting EZH2 as a novel potential therapeutic target for specific subgroups of patients with CLL.