IGVH 1-69 Gene Usage in CLL - Significance of Mutational Status

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IGVH1-69 GENE USAGE IN B-CLL - THE SIGNIFICANCE OF SOMATIC

HYPERMUTATION AND CRD3 STRUCTURAL FEATURES

101

Author Galligan, Leeona , Belfast, Belfast City Hospital Trust

Northern Ireland

Co-author(s) Catherwood, Mark , Belfast, Belfast City Hospital Trust

, Curly , Belfast, Belfast City Hospital Trust

, Denis , Belfast, Belfast City Hospital Trust

Background

B-CLL is a heterogenous disorder with a highly variable clinical

course. Although certain acquired cytogenetic aberrations, un-

mutated IgVH gene status, ZAP-70 positivity and an atypical

phenotype have been linked with more aggressive disease and a poorer

outcome, the search for further prognostic markers is ongoing. IgVH

3-48, 3-53 and 3-21 gene usage have previously been associated with

more aggressive disease and shorter survival times. In addition,

IgVH 1-69 is expressed at a high frequency in B-CLL supporting the

concept of antigenic selection in disease pathogenesis. It has been

suggested that comparison to germline immunoglobulin sequence using

the NCBI/IgBLAST database only may result in overestimation of

mutated IgVH frequency associated with IgVH 1-69 rearrangements in B-

CLL. Thus the clinical significance of IgVH 1-69 gene usage remains

to be clarified.

Aims

The aims of this study were to determine the frequency of VH1-69

gene usage in B-CLL, to assess the structural features of the CDR3

region, mutational status of the VH1-69 gene using the up-dated

IgBLAST and V-QUEST databases and to determine if patients with VH1-

69 rearrangements differ in clinical course to those patients with

alternative IgVH gene usage.

Methods

Two hundred and seventy patients were recruited for this study. IgVH

gene usage was determined using multiplex BIOMED-2 primers

(InVivoScribe Technologies) and protocol. IgVH mutational status was

determined by sequence analysis using BigDye chemistry and homology

comparison with the up-dated NCBI/IgBLAST and IMGT/V-QUEST

databases. Interphase FISH analysis was performed to screen for

common cytogenetic aberrations and serum thymidine kinase (TK)

levels were measured using a radioenzyme assay.

Results

We identified IgVH1-69 gene usage in 31/270 CLL patients (11.5%), 30

of which were characterised further. Our results demonstrate that it

is associated with un-mutated IgVH status (80% of cases) and a

preponderance of males (73% of cases). Poor/intermediate prognosis

cytogenetic abnormalities were detected in 10 of 15 (75%) cases

analysed. Although 21 patients (70%) were found to have Binet stage

A disease upon presentation, the majority have since progressed to

stage B or C. The median TK level was 16.1 U/L (>8.5 U/L is

associated with progressive disease). IgVH somatic hypermutation was

found in 6 cases (20%) and was associated with shorter CDR3 regions,

IGJH 4 gene usage and lower TK levels (median of 6.8 U/L).

Summary/conclusions

We have found that IgVH1-69 gene usage is over-expressed in B-CLL,

is associated with advanced disease and may have potential as a

supplemental predictive marker of disease progression. However, it

remains to be determined if IgVH1-69 gene usage is an independent

poor prognosis marker, as its association with un-mutated IgVH

status in most patients may be the overriding factor. We present

evidence that IgVH1-69 B-CLL cases can be divided into mutated (n=6)

and un-mutated (n=24) sub-groups and that mutated cases are defined

by short CRD3 regions and biased IGJH 4 gene usage. We are currently

investigating the possible common antigenic stimuli which may be

involved in leukaemogenesis in this sub-group.