Telomeric IGH losses detectable by fluorescence in situ hybridization in chronic lymphocytic leukemia reflect somatic VH recombination events.

Routine interphase fluorescence in situ hybridization (FISH) analysis of chronic lymphocytic leukemia (CLL) with LSI IGH/CCND1 assay, applied to differentiate CLL from leukemic mantle cell lymphoma, identified a subset of cases (42/174) with translocation-like IGH signal pattern. To unravel the underlying 14q32/IGH aberrations, 14 of these cases were subjected to cytogenetic, detailed FISH, and V(H) mutation analyses. FISH identified cryptic losses of various portions of the IGHV region in all 14 cases. Fine mapping of these V(H) deletions revealed a strict correlation between their distal border and localization of the used VH gene, suggesting that they are not oncogenic but reflect physiological events accompanying somatic V-D-J assembly. This hypothesis was further supported by FISH analysis of 20 CLL and hairy cell leukemia cases with the known V(H) usage showing a constant loss of sequences proximal to the used gene, identification of V(H) deletions in normal B cells, and their exclusive demonstration in B cell malignancies, but not of T cell and myeloid linage. Given that these cryptic physiological VH losses in B cells may seriously complicate analysis of B cell leukemia/lymphoma and lead to false conclusions, FISH users should take them into consideration when interpreting IGH aberrations in these malignancies.

V(H)3-48 and V(H)3-53, as well as V(H)3-21, gene rearrangements define unique subgroups in CLL and are associated with biased lambda light chain restriction, homologous LCDR3 sequences and poor prognosis.

This study determined IgV(H) gene usage in 228 chronic lymphocytic leukaemia patients to investigate associations between gene usage and other biological or clinical characteristics. V(H)3-48 [N=8] and V(H)3-53 [N=4] gene rearrangements showed biased lambda light chain restriction and were predominantly found in female patients with short lymphocyte doubling time but without adverse prognosis cytogenetics. Overuse of V(L)3-21(Vlambda2-14) gene and highly homologous LCDR3 sequences were found in V(H)3-48 patients. V(H)3-21 gene usage [N=18, 7.9%] was associated with poor prognosis, overuse of V(L)3-21(Vlambda2-14) gene and highly homologous heavy- and light-chain CDR3 sequences, but was not associated with poor prognosis chromosomal aberrations.

MDR-1, but not MDR-3 gene expression, is associated with unmutated IgVH genes and poor prognosis chromosomal aberrations in chronic lymphocytic leukemia.

Two P-glycoprotein (P-gp) genes, MDR-1 (ABCB1) and MDR-3 (ABCB4), have been identified in humans. This study was designed to investigate whether associations exist between expression of MDR-1 and MDR-3 P-gp and other markers of poor prognosis and/or prior exposure to therapeutic agents in chronic lymphocytic leukemia (CLL). IgVH mutational status, gene usage, CD38 positivity, FISH analysis and clinical information were available on all patients. Twenty-one of 101 patients tested showed MDR-3 P-gp positivity. Associations with markers of poor prognosis or prior chemotherapy did not reach statistical significance, but MDR-3 P-gp positive patients had significantly shorter survivals than MDR-3 P-gp negative patients. MDR-1 P-gp expression (18/25) showed a strong association with unmutated IgVH genes and adverse prognosis cytogenetics (p = 0.015, p = 0.014, respectively), but was independent of prior exposure to chemotherapeutic agents. These results suggest a role for MDR-1 and MDR-3 in chemoresistant disease. This study highlights the value of determining MDR phenotype in CLL patients prior to treatment, to allow the design of novel drug regimens containing agents that reverse MDR function.

Detection of tumor-derived antigen receptor DNA by polymerase chain reaction in the serum of patients with B-cell chronic lymphocytic leukemia.

Tumor-derived DNA is detectable in the serum of patients. In this study, tumor derived and mononuculear cell DNA from 50 patients with B-cell chronic lymphocytic leukemia (B-CLL) was amplified by polymerase chain reaction (PCR) and analyzed using polyacrylamide electrophoressis and Genescan analysis. DNA was demonstrated in 86% of serum samples. Genescan analysis has significantly enhanced the sensitivity and specificity of detection of tumor-derived DNA.

Routine analysis of IgVH mutational status in CLL patients using BIOMED-2 standardized primers and protocols.

Current methods for the detection of IgVH mutational status in chronic lymphocytic leukemia (CLL), which identifies 2 subgroups of patients with significantly different outcomes, are laborious, expensive and do not lend themselves to a routine diagnostic setting. With the introduction of BIOMED-2 primers, a rapid protocol is now available. This study evaluated the protocol by examining DNA from 100 CLL patients. Conventional methods using RNA, and fluorescence in-situ hybridization (FISH) analysis for recurring chromosomal abnormalities, were carried out on 30 and 60 of these patients, respectively. There was complete concordance between the BIOMED-2 protocol and the RNA based method, both in mutational status and gene usage, whilst unmutated IgVH genes showed association with 17p13 and 11q23 deletions, and trisomy 12, associated with poor and intermediate outcomes, respectively. This study demonstrates that it is feasible to use the BIOMED-2 protocol in the diagnostic profile of CLL patients, obviating the need for inclusion of surrogate markers such as ZAP-70.

Improved laboratory diagnosis of bacterial and fungal infections in patients with hematological malignancies using PCR and ribosomal RNA sequence analysis.

During October 1999 to November 2000, 98 blood culture specimens from the same number of febrile episodes originating from 49 patients with hematological malignancies were examined for the presence of eubacteria and fungi based on 16S rRNA gene and the 5.8, 18 and 28S rRNA combined with in vitro PCR amplification and sequencing, in addition to conventional blood culture laboratory techniques. Nineteen of the samples were associated with positive blood cultures. Eubacterial (16S rRNA) PCR detected bacterial DNA in 26 febrile episodes, i.e. in an additional 7 febrile episodes than blood-culture alone. The species identified by partial 16S rRNA gene sequencing were as follows Staphylococcus spp (n = 6), Staphylococcus epidermidis (n = 5), Acinetobacter spp (n = 5), Escherichia coli (n = 2), Enterobacter agglomerans (n = 2), Campylobacter spp (n = 1), Citrobacter spp (n = 1), Corynebacterium spp (n = 1), Enterobacter faecium (n = 1), Ralstonia spp (n = 1), Acidovorax spp. (n = 1) and Stenotrophomonas maltophilia (n = 1). Gram-positive bacteria were found in 12/27 (44.6%) and gram-negative bacteria were found in 15/27 (55.6%). After optimization of a PCR-based fungal detection method, none of the febrile episodes were shown to be attributable to fungi. The results of this study suggest that fungi are not common causal agents of febrile episodes in patients with a hematological malignancy at this centre and that molecular techniques can augment cultural methods in the diagnosis of causal agents of bacteremia in patients, so that appropriate antibiotic regimens may be commenced in patients with culture-negative episodes of infection.

Aggressive natural killer cell leukemia/lymphoma: case report, use of telesynergy and review of the literature.

Natural killer cell malignancies, although increasingly recognized, remain rare tumors within the USA and Europe. They are somewhat more common in Asia, and have been best characterized within this population. We present a case of a young Caucasian woman who presented acutely with an aggressive natural killer cell leukemia/lymphoma. Use of Telesynergy technology enabled a transatlantic telemedicine conference with colleagues in a center of expertise. Unfortunately the patient was ultimately refractory to both conventional chemotherapy and Campath-1H and her disease course was fulminant, as has been described previously in this condition. We review the possible therapeutic options for this extremely aggressive malignancy and briefly discuss our center's experience of telemedicine technology.

ABCB1 (MDR1) rs1045642 is associated with increased overall survival in plasma cell myeloma.

Multi-drug resistance (MDR) may compromise the successful management of haematological malignancies, impairing the effectiveness of chemotherapy. The P-glycoprotein (P-gp) drug efflux pump, encoded by the gene ABCB1 (MDR1), is the most widely studied component in MDR. A single nucleotide polymorphism (SNP) has been identified within ABCB1, rs1045642 (C3435T), which may alter P-gp substrate specificity and have an impact on the effectiveness of treatment, and hence overall survival (OS). We estimated the frequency of this SNP in the Northern Irish population and investigated its impact on the OS of patients with plasma cell myeloma (PCM). There was no significant difference in the frequency of rs1045642 between the PCM cohort and an age- and gender-matched control population. Findings within the PCM cohort suggest that rs1045642 genotype influences OS (p = 2 x 10(-2)). If confirmed in larger studies, these results suggest that genotyping rs1045642 may be a useful predictor of outcome in PCM and could indicate modified treatment modalities in certain individuals.

Mutated IgHV1-69 gene usage represents a distinct subgroup associated with indolent disease in chronic lymphocytic leukemia.

Biased IgHV gene usage in chronic lymphocytic leukemia (CLL) is well documented and suggests antigen involvement in leukemogenesis. IgHV1-69 is one of the most frequently rearranged IgHV genes in CLL and the majority of IgHV1-69 cases lack somatic hypermutation and display poor prognosis. However, its independent prognostic impact remains uncertain given reports showing a low proportion of mutated IgHV1-69 cases and stereotyped IgHV1-69 subsets with divergent clinical outcome. We assessed the frequency and clinical significance of IgHV1-69 gene usage in a cohort of 330 CLL patients. Functional IgHV1-69 gene rearrangements were detected in 32 cases (9.7%), 31 of which were characterised further. Seven (22.6%) were found to have undergone somatic hypermutation. This subgroup had shorter and more diverse complementarity determining region 3 (CDR3) sequences compared with unmutated IgHV1-69 cases. In addition, mutated IgHV1-69 gene status was associated with lower cell surface CD38 expression and less progressive disease as monitored by Binet staging, lymphocyte doubling time and requirement of chemotherapeutic intervention. To conclude, we present data confirming that IgHV1-69 gene rearrangements in CLL are not exclusively associated with unmutated IgHV status. In addition, we show that a somatically hypermutated subgroup may demonstrate more indolent characteristics despite the general association of IgHV1-69 gene usage with aggressive disease.