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 Previous Article | Table of Contents
Blood, Vol. 96 No. 1 (July 1), 2000:
pp. 377-379
CORRESPONDENCE
 |
To the Editor: |
Expression of unmutated VH genes is a
detrimental prognostic factor in chronic lymphocytic
leukemia
Chronic lymphocytic leukemia (CLL) is recognized as a
heterogeneous disease.1 Although the clinical staging
systems (Rai et al2 and Binet et al3) provide
useful methods of assessing patient prognosis, they do not provide an
understanding of the basis of the heterogeneity. CLL cells are
monoclonal B cells expressing low levels of surface immunoglobulin and,
based on the expression of CD5, appear to phenotypically correspond to
mantle-zone naive B cells. Because such cells normally do not pass
through the lymphoid follicle during maturation, it would be expected
that the V region genes that make up the immunoglobulin they express
would not be mutated. But Schroeder and Dighiero4 showed
that the immunoglobulin VH genes expressed in about half of
a series of 75 CLL patients contained mutations as if they had matured
in a lymphoid follicle.
Recent work from Hamblin et al5 and Damle et
al6 has shown that the presence or absence of
immunoglobulin V gene mutations predicts the natural history of the
disease. They found that the presence of unmutated V genes was
associated with a much poorer prognosis, even within the group of
patients in Binet stage A. Importantly, Damle et al showed a strong
correlation between expression of CD38 and the presence of unmutated V
genes, suggesting that surface phenotyping might be able to identify
patients with a poorer prognosis without the requirement for
immunoglobulin gene sequencing. We have analyzed the immunoglobulin V
genes in 39 of our patients (25 with familial CLL) to investigate this
question further.
DNA was extracted from peripheral blood lymphocytes and subjected to
polymerase chain reaction (PCR) amplification using primers for the
VH and VL genes, as previously
reported.7,8 The amplified fragment was ligated into pMOS
Blue T-vector and transformed into MOS Blue competent cells (Amersham
Life Science, Buckinghamshire, England). Recombinant plasmids were
purified from transformed bacteria and selected by restriction
analysis. Nucleotide sequencing was performed as previously
reported.8 Inserts were sequenced in 2 directions and from
multiple independent clones. Sequences were compared with GenBank-EMBL
and V base databases.
The mean age of the patients was 62 years (range, 35-95), and the
male-female ratio was 0.95; 29 patients were in stage A, 7 in stage B,
and 3 in stage C. Twelve (31%) patients expressed unmutated
immunoglobulin; details of the the gene rearrangements are shown in
Table 1. Greater than 98% homology
with the germline sequence was required to be designated as unmutated.
Polymorphisms in the V region genes can account for this degree of
disparity. Twenty-seven patients (69%) expressed mutated
immunoglobulin; details of the gene rearrangements and mutations are
shown in Table 2. Twenty-two of the
27 patients with immunoglobulin gene mutations displayed greater than
5% difference compared to their germline counterpart. Further analysis
was performed to assess whether the high mutation rate was due to
antigen-driven selection.
In the absence of antigen-driven selection, replacement (mutations that
change the protein sequence) and silent mutations (those that do not
change protein sequence) are typically distributed randomly throughout
the protein sequence. A lower than expected frequency of replacement
mutations is circumstantial evidence of pressure to maintain the
protein sequence due to essential function. By contrast, if the number
of replacement mutations exceeds that expected by chance alone, a
positive selection process can be inferred. In immunoglobulin genes,
clustering of replacement mutations within the complementarity
determining regions (CDRs), which form the antigen contact points, is
the product of positive selection in the lymphoid follicle. Given the
lengths of the CDRs and the framework regions of the molecule, randomly
distributed mutations would result in a CDR-framework mutation ratio
of less than 0.3. In this series, 20 of 22 cases with greater than 5% mutation rate had a CDR-framework mutation ratio of at least
0.3.
A second test of directed versus random mutation is the ratio of
replacement mutations to silent mutations. Random mutation processes
produce a ratio of 2.9 of replacement (R) to silent (S) mutations
(mutations in the third nucleotide of a codon are often silent). In
this series, 14 of the 22 cases with a mutation rate greater than 5%
exhibited an R-S ratio of 3 or greater in their CDRs. Nine of these had
R-S ratios of less than 2.9 in their framework regions, strong evidence
for selective pressure on the CDRs.
Thus, our results confirm previous reports suggesting that at least 2 morphologically indistinguishable forms of CLL exist, one expressing
unmutated V genes, as expected based on phenotype, and one expressing
mutated V genes in spite of a cell-surface phenotype associated with
antigen-naive B cells.
The median followup time was 7 years overall (range, 2-21); 5 years
(range, 2-11) for the unmutated group, and 8 years (range, 3-21) for
the mutated group. The male-female ratio was not significantly different (P = .2) between the 2 groups: 1.4 in the unmutated group and 0.8 in the mutated group. Most stage A patients (24 of 29)
expressed mutated VH genes; most stage B and C patients expressed unmutated VH genes (7 of 10)
(P = .036). Among the 12 patients expressing unmutated
VH genes, 8 died of CLL-related causes, compared to 6 of 27 patients with mutated VH genes dying, 2 from CLL-related
causes and 4 from unrelated causes. Overall (P = .0007) and
CLL-specific survival (no more than 10 4) was shorter
in patients with unmutated V genes. Among stage A patients, all 5 with
unmutated V genes experienced disease progression within 5 years,
compared to 7 of 22 evaluable patients in the group with mutated V
genes (P = .013). CLL-specific survival was also shorter in
stage A patients with unmutated V genes (3 of 5 dead) than in stage A
patients with mutated V genes (2 of 24 dead) (P = .0098). We
did not examine CD38 expression on the tumor cells.
Thus, as previously reported, CLL exists in at least 2 forms, those
with mutated V genes and those with unmutated V genes. Patients with
unmutated V genes are more likely to present with advanced disease,
more likely to progress clinically, and more likely to die of CLL than
those with mutated V genes.
Karim Maloum
Frédéric Davi
Hélène Merle-Béral
Département d'Hématologie
Hôpital
Pitié-Salpêtrière
Paris, France
Otto Pritsch
Christian Magnac
Françoise Vuillier
Guillaume Dighiero
Unité d-Immuno-Hématologie et d'Immunopathologie
Institut Pasteur
Paris, France
Xavier Troussard
Service d'Hématologie Clinique
Caen, France
Francesca F. Mauro
Clinica de la Sapienza
Rome, Italy
Jacques Bénichou
Département de Biostatistique
CHU Rouen
Rouen,
France
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