ASH Panel 2001 - Three Case Studies of CLL

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Several case studies of CLL patients as presented at

ASH, 2001.

This very interesting stuff; read the full text

article at:

http://www.asheducationbook.org/cgi/content/full/2001/1/140

Chronic Lymphocytic Leukemia: Case-Based Session

Kanti R. Rai, Hartmut Döhner, J. Keating and

Emili Montserrat

Abstract

Drs. Hartmut Döhner, J. Keating, Kanti R. Rai

and Emili Montserrat form the panel to review chronic

lymphocytic leukemia (CLL) while focusing on the

clinical features of a particular patient. The pace of

progress in CLL has accelerated in the past decade.

The pathophysiological nature of this disease, as had

been known in the past, was based largely on the

intuitive and empiric notions of two leaders in

hematology, Dameshek and Galton. Now the

works of a new generation of leaders are providing us

with the scientific explanations of why CLL is a

heterogeneous disease, perhaps consisting of at least

two separate entities. In one form of CLL, the

leukemic lymphocytes have a surface immunoglobulin

(Ig) variable region gene that has undergone somatic

mutations, with tell-tale markers suggesting that

these cells had previously traversed the germinal

centers. Such patients have a distinctly superior

prognosis than their counterparts whose leukemic

lymphocytes IgV genes have no mutations (these are

indeed immunologically naive cells), who have a worse

prognosis. The introduction of fluorescence in situ

hybridization (FISH) technique has provided us with

new insights into the diverse chromosomal

abnormalities that can occur in CLL, and which have

significant impact on the clinical behavior and

prognosis of patients with this disease.

Major advances in therapeutics of CLL also have

occurred during the past decade. Two monoclonal

antibodies, Campath-1H (anti-CD52) and rituximab

(anti-CD20), and one nucleoside analogue, fludarabine,

have emerged as three agents of most promise in the

front-line treatment of this disease. Studies

currently in progress reflect our attempts to find the

most effective manner of combining these agents to

improve the overall survival statistics for CLL

patients. As in many other hematological malignancies,

high dose chemotherapy followed by autologous or

HLA-compatible allogeneic stem cells rescue strategies

are under study as a salvage treatment for a

relatively younger age group of CLL patients with poor

prognosis characteristics.

I. Prognostic Implications of Findings from

Cytogenetics and Molecular Genetics

Hartmut Döhner, MD*In recent years, important aspects

of CLL biology have been elucidated which relate to

its stage of differentiation and to its transforming

events. It has been shown that there are two variants

of CLL arising at different stages of B-cell

differentiation as reflected by the mutational status

of the immunoglobulin variable region (IgV) genes.

Furthermore, by using modern molecular cytogenetic

techniques, genomic aberrations can now be diagnosed

in approximately 80% of CLL cases. The genomic regions

recurrently affected by chromosomal deletions,

trisomies, and, less frequently, translocations

contain mostly as yet unknown tumor suppressor genes

and oncogenes. Both the IgV mutation status and the

pattern of genomic aberrations have been shown to have

a high predictive value for disease progression and

survival in CLL patients. The prognostic information

from these new genetic markers is independent from

that obtained by the conventional clinical markers.

Methodological Aspects of Genetic Analysis in CLL

In our case, initial diagnostic work-up in March 1997

included conventional chromosome banding analysis

which showed a normal karyotype. The technique of

conventional chromosome banding has been hampered in

CLL by the low in vitro mitotic activity of the clonal

B-cells. With this method, clonal chromosome

abnormalities are detected in only 40% to 50% of

cases.1 In many cases only normal metaphase spreads

are obtained, mostly due to the fact that despite the

use of B-cell mitogens, the mitotic cells originate

from non-leukemic T lymphocytes contained in the

specimens. This was shown by the study of Autio et al.

using the technique of sequential immunophenotyping

and karyotype analysis.2

The development of fluorescence in situ hybridization

(FISH) using genomic DNA probes has greatly enhanced

our ability to detect chromosome aberrations in tumor

cells. One major advantage of this technique is that

aberrations can be detected in both metaphase and

interphase cells, an approach commonly referred to as

interphase cytogenetics. Given the methodological

problems associated with conventional chromosome

banding in CLL, it was not surprising that the

spectrum and frequency of aberrations reported in the

various FISH studies differed considerably from the

results obtained in banding studies.3 Using molecular

cytogenetics, genomic aberrations can now be

identified in approximately 80% of CLL cases.

In our case, molecular cytogenetic work-up at the time

of disease progression in December 1998 revealed the

presence of a 13q and an 11q deletion. It is very

likely that these two abnormalities were already

present at the time of diagnosis but were missed by

the conventional cytogenetic technique. Conventional

chromosome banding studies can no longer be

recommended in the routine diagnostic work-up of a CLL

patient. The novel molecular cytogenetic techniques

are now recognized to provide the most reliable data.

Prognostic Impact of Genomic Aberrations

Based on conventional chromosome banding analysis,

trisomy 12 was the first abnormality that in

univariate analysis was associated with both shorter

treatment-free interval and shorter survival.1 Other

abnormalities associated with inferior survival were

11q and 17p deletion.4,5 In contrast, patients with

structural aberrations of chromosome 13, mostly 13q

deletions, and patients with a normal karyotype seemed

to have a favorable outcome.1

Owing to the methodological problems of conventional

chromosome banding it became necessary to reassess the

prognostic value of the genetic markers based on the

novel techniques. We recently reported data from a

molecular cytogenetic study in CLL evaluating the

incidence and prognostic significance of the most

important disease-associated genomic aberrations.6

Samples from 325 CLL patients were analyzed by FISH

using a comprehensive set of diagnostic DNA probes for

deletions in chromosome bands 6q21, 11q22-q23, 13q14,

17p13, for trisomies of bands 3q26, 8q24, 12q13, and

for translocations involving the immunoglobulin heavy

chain locus in band 14q32. Genomic aberrations were

detected in 268 of 325 cases (82%). The most frequent

aberration was 13q deletion (55%), followed by 11q

deletion (18%), 12q trisomy (16%) and 17p deletion

(7%). On the basis of regression analysis we proposed

a hierarchical model of genomic aberrations, in which

each patient was allocated to a single category. This

model comprised five major categories, i.e. patients

with a 17p deletion; patients with an 11q deletion but

not a 17p deletion; patients with 12q trisomy but not

a 17p or 11q deletion, patients with a normal

karyotype, and patients with a 13q deletion as sole

aberration (Table 1). The estimated median survival

time of the entire study group was 108 months; and the

median survival times for patients of the five major

categories were 32, 79, 114, 111, and 133 months,

respectively. Furthermore, the cytogenetic categories

were associated with distinct presenting clinical

features. Patients with 17p or 11q deletion had more

advanced disease stage compared to the other 3

categories, they were more likely to have

splenomegaly, mediastinal and abdominal

lymphadenopathy, and they had more extensive

peripheral lymphadenopathy; furthermore, these

patients had B-symptoms more frequently. Finally,

there were statistically significant differences in

disease progression among the five categories as

measured by the treatment-free interval: the median

treatment-free intervals for the groups with 17p

deletion, 11q deletion, 12q trisomy, normal karyotype,

and 13q deletion as sole aberration were 9, 13, 33,

49, and 92 months, respectively. Multivariate analysis

identified six significant prognostic factors: 17p

deletion, 11q deletion, age, Binet stage, serum

lactate dehydrogenase level, and white cell count.

View this table:

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Table 1. Incidence of the major cytogenetic risk

groups* in various studies.

The data from this single center study indicate that

genomic aberrations in CLL are important independent

predictors of disease progression and survival. It is

now important to investigate the impact of these

genomic aberrations prospectively in clinical trials

of the large cooperative groups. Table 1 shows

preliminary results from the prospective genetic study

within the CLL1 (Binet A patients) and the CLL3

(high-dose therapy followed by autologous

transplantation for patients with stage Binet B and C

disease) treatment trials of the German CLL Study

Group (GCLLSG). These data are consistent with our

single center data with respect to the overall

incidence of genomic aberrations. In the CLL1 trial

for patients with stage Binet A disease there is a

higher incidence of the 13q deletion as sole

abnormality and a lower incidence of the high-risk

groups 17p deletion and 11q deletion, whereas in the

CLL3 trial there is a higher incidence of the 11q

deletion group likely reflecting the higher disease

burden and the younger median age of these patients.

As shown by the data from the CLL1 trial, high-risk

genomic aberrations are detected in approximately 15%

of stage Binet A patients (Table 1). The case under

discussion belongs to this subgroup of patients. Not

unexpectedly, he developed rapid disease progression

with generalized lymphadenopathy, hematopoietic

insufficiency and B-symptoms. I wonder whether the

disease in December 2001 had undergone clonal

evolution when the patient relapsed with marked

disease activity following treatment with fludarabine.

Few data so far address this question by using

molecular cytogenetic techniques. We applied FISH for

sequential interphase cytogenetic studies on 55

patients over a median observation time period of 42

months (range 24-81 months).7 Clonal evolution was

found in 9 of the 55 (16%) patients. The most frequent

acquired changes were 17p deletion (4 cases), 6q

deletion (3 cases), 11q deletion (1 case), and

evolution from mono- to bi-allelic 13q deletion in 3

cases. Two thirds of the patients exhibiting clonal

evolution have died, compared to only 20% in the group

of patients without genetic evolution.

Prognostic Impact of the IgV Genes Mutational Status

I also wonder whether the mutational status of the

immunoglobulin variable (IgV) genes was assessed in

our patient.

One important issue of biological risk classification

in CLL relates to the stage of differentiation of the

malignant B cells. The process of differentiation can

be divided into a pregerminal, germinal and

postgerminal center phase. Selection and recombination

of variable (V), diversity (D) and joining (J) genes

as well as the insertion of nontemplated nucleotides

at the V-D and D-J junction are early events in the

pregerminal phase. In the later stage of

differentiation, the germinal center phase, the B

cells undergo somatic hypermutation. In the

microenvironment of the germinal center, somatic

mutations are introduced in the V(D)J-rearrangement.

This process occurs in part with and without antigen

stimulation.8 Although the B cells involved in CLL

were originally considered to be naïve, pregerminal

lymphocytes, more recent data indicate that somatic

IgV gene mutations are present in approximately 50

percent of CLL cases.9,10 These data indicate that

there are two variants of the disease " CLL, " a

pregerminal variant which originates from naïve B

lymphocytes showing no IgV gene mutation, and a

postgerminal variant which originates from memory B

lymphocytes exhibiting IgV gene somatic hypermutation.

Correlation of the IgV mutational status with clinical

data revealed that the presence of unmutated IgV

predicts for inferior prognosis.11,12 Based on this

observation, other differentiation markers such as the

CD38 expression level have been studied in CLL. In one

study, CD38 expression was shown to correlate with the

presence of unmutated IgV genes and an unfavorable

clinical outcome.12 The correlation of CD38 with

unmutated IgV genes or survival probability is

currently a matter of discussion.13,14 Ibrahim et al

have recently confirmed previous results on the

prognostic significance of CD38 expression in

multivariate analysis.15 However, their study did not

include IgV mutation status and genomic aberrations.

In our single center study, we analyzed 211 CLL

samples for the IgV mutation status.16 Eighty-eight

patients had their IgV genes mutated. Analogous to the

pivotal studies of Hamblin et al11 and Damle et al12,

the patients with mutated IgV genes had significantly

higher survival probabilities compared to the patients

with unmutated IgV genes. This could be shown for the

entire group of patients as well as for the subgroup

of patients (n = 131) with stage Binet A disease

(Figures 1 and 2). In agreement with Damle et al we

also observed an inverse correlation between CD38

expression and IgV gene mutation status. However, in

about a third of cases CD38 expression failed to

predict the IgV gene mutation status, and CD38

expression level > 30% was not significantly

associated with lower survival probability.

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