Composite low grade lymphoma with two subpopulations in a same site is uncommon.

We herewith report
the case of an 80-year-old woman who presented with isolated bilateral dacryoadenomegaly. Pathological
examination of an incisional biopsy of her right lacrimal gland was consistent with a marginal zone
lymphoma. Flow cytometry immunophenotyping showed two distinct clonal B-cell populations expressing sIg
D lambda or sIg M kappa restriction in the lacrimal gland, blood, and bone marrow. Both B-cells populations
were sorted from peripheral blood for molecular biology investigations and comparison with molecular data
performed on tumor and bone marrow cells. IgH PCR performed on purified blood populations disclosed two
monoclonal peaks: 98 bp-sized peak in the sIg M kappa and a 107 bp in the sIg D lambda clones,
respectively. The lacrimal gland tumor expressed mainly sIg M kappa population, and showed a major 98
bp-sized peak coexisting with a very minor 107 bp peak. Cytogenetic studies showed a 46, XX,del (7)
(q22q32) karyotype. Bone marrow examination at diagnosis revealed the same B-cell clones distribution
than the one observed in blood with a dominant sIg D lambda population, a Genescan profile showing a
major peak of 107 bp and a minor peak of 98 bp. Chromosomal analysis disclosed a 46,XX,del (10) (?p14)
karyotype without detectable 7q deletion. To our knowledge, this observation represents the first reported
case of biclonal low grade lymphoma hidden behind a normal classical kappa/lambda Ig light chain ratio in
blood, but clearly demonstrated by the combination of three ancillary techniques (flow cytometry both
analytical and cell sorting, molecular biology, and cytogenetics) and analysis of different tissues (i.e., in this
case, lacrimal gland biopsy, blood, and bone marrow. Am. J. Hematol., 2007. © 2007 Wiley-Liss, Inc.

Abstract
Monoallelic deletion of 13q14.3 (13q14x1) is the most common abnormality in chronic
lymphocytic leukemia (CLL). The Recently described NZB miRNA15a/16-1 complex
are deleted or down-regulated as a part of this deletion , and Since bcl-2 mRNA is a
validated mir15a/16 target, increased bcl-2 expression maybe a direct result of and a
surrogate marker for mir15a/16 levels and/or the 13q14 status. We present a case of
Chronic Lymphocytic Leukemia (CLL) submitted to us twice with 3 month interval for
immunophenotyping evaluation by flow cytometry. The patient is a ------ year old
female with an ---- year history of CLL .

Experimental Design: Direct staining immunophenotyping method of intracellular

BCL-2 protein using the monoclonal anti-BCL-2 oncoprotein/ FITC clone 124 ,
together with mAb directed against CD5 CD19,CD20, CD23,.CD27,CD38,
kappa,lambda,CD45 . We Identify the BCL-2 Mean fluorescence intensity (MFI) and
examined the relationship between such levels and the proportion of leukemia cells within
the CLL population bearing 13q14 deletion deletions either (13q14x1,or 13q14x2),
sorting cell based on FS vs SC , and BCL-2 MFI were done. Interphase FISH was
performed on sorted cells using Vysis D13S319 spectrum orange / LSI 13q34 spectrum
green probe as a control .

Results: A seven-color flow cytometric evaluation was

performed. A population of 73% lymphoid cells was identified by
CD45 vs. SS gating. Using FS vs. SS gating 90.6% are small lymphoid cells
(SL1) shows (1% diploid cell ,78% 13q14x1, 21% 13q14x2, , with BCL-2
index=1.97) and and 6.38 % are large lymphoid cell (LL1) with ( 2% diploid
cell, 59% 13q14x1, 39% 13q14x2, with BCL-2 index = 3.54). We identified two
distinct malignant BCL-2 Sub-populations within CD19+CD5+ CLL cells (SL) ,
Each subpopulation had different BCL-2 Mean Fluorescence intensity (MFI),and
different percentage of (13q14x1,13q14x2).The First sub-population (34.4%)
brightly expressed BCL-2 (SLB) ,and shows 59%13x14x1, 41%13q14x2, the
second one (64.1%) dimly expressed BCL-2 (SLD) ,and shows 89% 13q14x1,
10.5 %13q14x2, 0.5 % diploid cell . Both sub-population show no difference in
expressing the following markers: kappa+
CD5+,CD19+,CD20+,CD27+,CD38-,CD23+,CD45+,

incontrast to the second sample coming to our lab after 3 month interval shows
the following 93.5 % are small lymphoid cells (SL2) shows ( 0.5 % diploid
cell , 61.5% 13q14x1, 38 % 13q14x2, , with BCL-2 index=2.2 ) and and 2.9 %
are large lymphoid cell (LL2) with ( 1.5% diploid cell, 52.5% 13q14x1, 46%
13q14x2, with BCL-2 index = 4.5) .

Conclusion:.

This case clearly demonstrates the importance of identification of more than one
BCL-2 sub-population within the B cell lymphoid clone. In this particular case, if
the populations had not been separately evaluated for BCL-2 and, the more
aggressive nature of 13q14x2 positive clone might have been missed. by the
combination of three ancillary techniques (flow cytometry both analytical and cell sorting, molecular biology,
and cytogenetics)

our observations point to the possibility of monitoring an aspect of the

evolution of the disease that might have profound clinical significance
simply by using immunophenotyping BCL-2 MFI . If sub-clones within a
patient harbor different BCL-2 MFI at any given time, we expect to
observe a differences in 13q14 status by comparing BCL-2 bright and
BCL-2 dim fractions. This type of analysis enabled us to time the
occurrence of events if we have the BCL-2 MFI base line for the clone
at the time of diagnosis, in summary within an overall apparently
constant leukemic burden, the outgrowth of a subclone with additional
genomic lesions might signal the start of a new phase of the disease.
Additional studies, combining data on fractionated BCL-2
subpopulations with clinical outcomes, In this particular case ,if the
populations had not been separately evaluated for BCl-2 ,the more
aggressive nature of 13q14x2 positive clone might have been
missed,this was .

In
This clinical trial confirms that Patients with higher BCL-2 MFI may be more likely to
experience such clonal evolution. These findings have important implications for both
clinical management and trials of early treatment for patients with high-risk, early-stage
CLL.

More generally,
 In one case, this involved a loss of
the p53 locus in the CD38_ fraction, a marker that was not
observed in the parallel CD38_ fraction (Table 2). More
generally,
our observations point to the possibility of monitoring an
aspect of
the evolution of the disease that might have profound
clinical
significance. Within an overall apparently constant leukemic
burden, the outgrowth of a subclone with additional genomic
lesions might signal the start of a new phase of the disease.
In summary, we have demonstrated that ROMA is a highly
sensitive CGH method to examine genomic changes in CLL.
We
have detected novel lesions, ascertained the frequencies of
gains
and losses with greater accuracy, and pinpointed candidate
genes.
The apparent continuing evolution of clones of CLL within a
patient may lead to improved understanding of the disease
and the
ability to identify patients at risk. Overall, the capabilities we
have
demonstrated here offer opportunities for selective patient
treatment
and the identification of new therapeutic targets. However,
over the course of the disease, clonal evolution can occur.
Among the acquired genetic aberrations, loss of the second
copy of 13q14.3 is not uncommon.

This case clearly

demonstrates the importance of correct identification of more than
one abnormal B lymphoid clone. In this particular case, if the
populations had not been separately evaluated for Zap-70 and
CD38 expression, the more aggressive nature of the lambda
positive clone might have been missed. In some CLL cases,
especially those with more than one clone, reagent combinations
that evaluate CD38 and Zap-70 expression for the entire CLL
population may even yield results that are prognostically
misleading.
This study revealed that there is a strong relationship between the levels of BCL-2 MFI
in CLL leukemia-cell , and the relative proportions of leukemia cells having either
(13q14x1 ,3q14x2 ). Studies are in progress to define whether these relationships can be
explained by altered expression of microRNA that map to 13q14.

This study reveals a relationship between the levels of TCL1 expression in CLL
leukemia-cell expression of ZAP-70, and the relative proportions of leukemia cells having
deletions at 11q. Studies are in progress to define whether these relationships can be
explained by altered expression of microRNA that map to 11q, which might also account
for the noted adverse prognosis of CLL that has deletions at 11q.

This case clearly

demonstrates the importance of correct identification of more than
one abnormal B lymphoid clone. In this particular case, if the
populations had not been separately evaluated for Zap-70 and
CD38 expression, the more aggressive nature of the lambda
positive clone might have been missed. In some CLL cases,
especially those with more than one clone, reagent combinations
that evaluate CD38 and Zap-70 expression for the entire CLL
population may even yield results that are prognostically
misleading.

Purpose:. Monoallelic deletion of 13q14.3 (13q14x1) is the most common abnormality

in chronic lymphocytic leukemia (CLL). The Recently described NZB miRNA15a/16-1
complex are deleted or down-regulated as a part of this deletion , and are inversely
correlated to Bcl2 expression in CLL patients. We present a case of Chronic
Lymphocytic Leukemia (CLL) submitted to us for immunophenotyping evaluation by
flow cytometry. The patient is a ------- year old female with an ---- year history of CLL .
A eight –color flow cytometry evaluation was performed .

Results:.
We identified two distinct malignant B-cell BCL-2 Sub-populations. Each
subpopulation had different BCL-2 Mean Flourecence intensity and 13q14
deletion status. The predominant B-lymphoid population
lymphoid cells) dimly expressed monoclonal kappa light chain and
also the following markers: HLA-DR, CD5, CD11c, CD19, CD22,
CD23 and CD45. They were clearly CD38 and Zap-70 negative.
The second population (33% of the lymphoid cells) dimly
expressed monoclonal lambda light chain and also the following
markers: HLA-DR, CD5, CD11c, CD19, CD22, CD23 and CD45.
Most significantly, this secondary minority clone was clearly CD38

and Zap-70 positive with two Distinct malignant B cell sub-populations. Each
population had different BCL-2 Main Fluorescence intensity ,and different cytogeneti
Isolated mono-allelic deletion of chromosome region 13q14 is seen in 40–60% of patients
with typical B-CLL and is associated with good risk disease. Bi-allelic deletion of 13q14
region has an overall incidence of 4–5% in typical B-CLL. It is not known if loss of both
allelic regions impacts on disease progression and survival. FISH analysis was performed
on 995 patients with CLL. 451 cases were assessed using 13q14 PAC probes 933-e9 and
273–2 (Dr Stilgenbauer, Ulm University, Germany) and 540 cases were assessed with the
B-CLL probe set ( 12/13q14/13q34 and p53/ATM probe sets, Abbott/Vysis).

Normal results for 13q14 were obtained in 501 (50.4%) cases, 450 (45.2%) had a mono-
allelic deletion of 13q14 and 44 (4.4%) cases had a bi-allelic deletion of 13q14. Control
probes (13q34 region) had normal patterns in all cases tested. Detailed analysis was
carried out to test the hypothesis that loss of both allelic regions conferred a poorer
prognosis by comparing the 44 cases with bi-allelic deletion of to 162 cases with mono-
allelic deletion good risk CLL. Cases with bi-allelic deletion have similar clinical
characteristics compared to cases with a mono-allelic deletion. There was no significant
difference in Binet stage between patients with a mono-allelic (84% A, 7% B, 9% C) or a
bi-allelic deletion of 13q14 (73% A, 13% B, 13% C). However, Binet stage was
significantly predicative of outcome independent of the cytogenetic abnormality.
Immunophenotypes were compared using the dChip program. Unsupervised hierarchical
cluster analysis showed that mono-allelic and bi-allelic deletion 13q14 CLL cases have
different protein expression profiles. CD5 and CD38 were more strongly expressed (1.23
fold increase, p = .06 and 2.36 fold increase, p = .02, repsectively), whereas CD22 and
CD23 had lower levels of expression (1.61 fold decrease, p = <.001 and 1.75 fold
decrease, p = <.001 respectively) in cases with bi-allelic deletion compared to mono-
allelic deletion. 12% of cases with bi-allelic deletion also have sub-populations of cells
with mono-allelic 13q14 deletions. 29% of cases with bi-allelic deletion (5.8% p53;
11.7% ATM; 13.5% +12) had additional cytogenetic abnormalities compared to just 6.5%
of cases with mono-allelic deletion (4.5% p53; 1.1% ATM; 0.6% +12)(p = <.001). The
overall survival between patients with a bi-allelic deletion compared to a mono-allelic
deletion was not significant but median follow-up is relatively short (25 months). Patients
with a bi-allelic deletion were significantly more likely to have disease progression and
require treatment (median progression free survival (PFS) 12 months) (p = <.001)
compared to patients with a mono-allelic deletion (median PFS 26 months) (progression
was defined as time to first theraputic intervention; 58% of cases with mono-allelic
deletion have not required treatment and 40% of cases with bi-allelic deletion have not
required treatment to date). PFS remained significant when cases with deletions of ATM
&p53 and +12 were excluded from analysis suggesting that the poor PFS is due to the bi-
allelic deletion of 13q14.

BACKGROUND AND OBJECTIVE: Monoallelic deletion of 13q14.3 (13q14x1) is the

most common abnormality in chronic lymphocytic leukemia (CLL). As a sole alteration,
it predicts a favorable outcome. Biallelic 13q14.3 (13q14x2) deletion or concomitant
13q14x1/13q14x2 has been scarcely evaluated in the literature. We present the clinical,
cytogenetic and fluorescence in situ hybridization (FISH) analysis of six CLL patients
with normal karyotypes and 13q14x2 and their comparison to cases with 13q14x1 as a
single abnormality. PATIENTS AND METHODS: A total of 103 CLL patients were
studied. Cytogenetic and FISH analysis were performed on stimulated peripheral blood
lymphocytes. Specific fluorescence DNA probes for CLL were used. RESULTS: Six out
of 103 (5.8%) patients showed normal karyotypes and 13q14x2. It was observed as a
single alteration in one patient and combined with 13q14x1 in five cases. Biallelic clones
were larger than monoallelic ones in 3/5 patients (60%). The comparison of clinical and
hematological data between 13q14x1 and 13q14x2 groups showed progression of the
disease in all 13q14x2 patients respect to 12/32 (37.5%) cases with 13q14x1 (P = 0.008),
significant differences in the distribution by Rai stage (P = 0.042) and a tendency of a
higher lactate dehydrogenase level in 13q14x2 patients (P = 0.054). Treatment free
survival for 13q14x2 group was 28.5 months, shorter than those observed in patients with
13q14x1 alone (49 months). CONCLUSIONS: Our data would suggest that 13q14x2
could represent a more aggressive FISH anomaly than 13q14x1 alone, probably as a
consequence of clonal evolution and/or due to the complete inactivation of this critical
region by mean of more complex mechanisms.

Deletion of a 13q14.3 region is by far the most common genomic alteration in CLL. In a
large cohort of CLL patients, the presence of deletion 13q as sole anomaly detected by
FISH was predominantly found in Binet stage A CLL and associated with a favorable
outcome (Dohner et al., N Engl J Med 2000). Further studies have evidenced some
heterogeneity among CLL cases with 13q deletion, such as the size of the clone carrying
the deletion, the existence of mono versus biallelic deletions, and the presence of other
concomitant genetic aberrations. Therefore, we aimed at analysing the impact of this
heterogeneity on the prognostic value of 13q14 deletion (del13q) in CLL.
Patients and methods In a cohort of 329 previously untreated newly diagnosed stage A
CLL, we detected del13q by FISH in 172 patients (52%) using the D13S319 probe.
Conventional cytogenetics was performed in the 105 cases with del13q followed in our
institution. The other important prognostic markers ( ZAP70, IgVH, CD38, proliferation
markers) and clinical progression were also available for all patients.
Results
We first studied the large cohort of stage A patients and found that deletion 13q had no
prognostic impact on PFS. When considering more specifically the presence of deletion
13q as sole anomaly (n=143), PFS was not significantly different from that of patients
with no aberration detected by FISH analysis (del 13q, del11q, del17p and trisomy 12)
(n=98). Moreover, the distribution of prognostic factors (ZAP70, sTK, mutational status,
CD38 expression, lymphocytosis) was not statistically different among these two groups.
We aimed at deciphering further these del13q cases through analysis of the percentage of
deleted cells, the presence of mono versus biallelic deletions, and the presence of
additional aberrations as detected by FISH and conventional cytogenetics in the 105
del13q cases followed in our institution.
The size of the del13q clone, as reflected by the percentage of del13q cells by FISH, was
highly variable, ranged from 7 to 90 %, and had no prognostic significance on PFS.
Monoallelic deletions were present in 77 cases, fully biallelic deletions in 9 cases , and
concomitant bi and monoallelic deletions in 19 cases. The 9 cases with biallelic deletions
had a significantly shorter PFS and were associated with other unfavorable prognostic
markers. As biallelic deletions are most likely to represent progression from monoallelic
cases, it is understandable that no clear prognostic impact was evidenced between cases
with monoallelic deletions and with concomitant variable amount of bi and monoallelic
deleted cells. Twenty cases (20 monoallelic and 6 biallelic) were further studied by array-
CGH. Minimal deleted region (MDR) was included in all deletions but the size of the
deletion was variable and in most cases much larger than MDR. Among the 6 bi allelic
cases, one of the deletions was restricted to the MDR in all cases, pointing out to the
importance of the level of miRNA expression.
Additional aberrations were found in 44/105 del13q cases. In 17 patients, one or more
alterations were detected by FISH techniques : del11q (n=8), trisomy 12 (n=8) or del17p
(n=5). By conventional cytogenetic all these aberrations were also detected, as well as
other rare ones in 16 additional cases, either isolated or associated in a complex
karyotype in 5 cases. Presence of additional aberrations had a significant unfavourable
impact on PFS, even when excluding del11q, del17p and tri12, and considering the non
recurrent aberrations that were detected by conventional cytogenetics only.
Conclusion
Presence of 13q deletion should not be considered as a good prognostic marker by itself
among stage A patients. Moreover, del13q cases are highly heterogeneous, and the
presence of deletion 13q should not be interpreted without considering both alleles or the
presence of concomitant genetic alterations.

Chronic lymphocytic leukemia (CLL) is the most common human leukemia and is
characterized by predominantly nondividing malignant B cells overexpressing the
antiapoptotic B cell lymphoma 2 (Bcl2) protein. miR-15a and miR-16-1 are deleted or
down-regulated in the majority of CLLs. Here, we demonstrate that miR-15a and miR-
16-1 expression is inversely correlated to Bcl2 expression in CLL and that both
microRNAs negatively regulate Bcl2 at a posttranscriptional level. BCL2 repression by
these microRNAs induces apoptopsis in a leukemic cell line model. Therefore, miR-15
and miR-16 are natural antisense Bcl2 interactors that could be used for therapy of Bcl2-
overexpressing tumors.

B cell lymphoma 2 (BCL2) is a central player in the genetic program of eukaryotic cells
favoring survival by inhibiting cell death (1). Overexpression of Bcl2 protein has been
reported in many types of human cancers, including leukemias, lymphomas, and
carcinomas (2). In follicular lymphomas and in a fraction of diffuse BCLs, the
mechanism of BCL2 activation was found to be the translocation t(14,18)(q32;q21),
which places the BCL2 gene under the control of Ig heavy chain enhancers, resulting in
deregulated expression of the gene (3, 4). B cell chronic lymphocytic leukemia (CLL) is
the most frequent adult leukemia in the Western world (5), and the malignant, mostly
nondividing, B cells of CLL overexpress Bcl2 (6). However, with the exception of <5%
of cases in which the BCL2 gene is juxtaposed to Ig loci (7), no mechanism has been
discovered to explain BCL2 deregulation in CLL.

We and others have previously reported that microRNAs (miRNAs) are a class of genes
involved in human tumorigenesis (refs. 8–15 and reviewed in refs. 16 and 17). In
animals, single-stranded miRNA binds specific mRNA through sequences that are
imperfectly complementary to the target mRNA, mainly to the 3′ UTR. The bound
mRNA remains untranslated, resulting in reduced levels of the corresponding protein or
can be degraded, resulting in reduced levels of the corresponding mRNA (18, 19).
Deletions and translocations involving two miRNAs, miR-15a and miR-16-1, located in a
cluster at 13q14.3, and their down-regulation was found in ≈65% of B cell CLL patients
(8). A germ-line mutation in miR-16-1/miR-15a primary precursor located 7 bp after the
3′ end of miR-16-1 caused low levels of miRNA expression in vitro and in vivo and was
associated with deletion of the normal allele (20). The presence of pathogenic mutations
in miR-15a/miR-16-1 indicate that these genes are involved in CLL, and that at least
some miRNAs can function as tumor suppressor genes. To decipher the miR-15a and
miR-16-1 involvement in CLL we pursued the identification of main targets with
importance for human tumorigenesis.

Here, we report the identification of a mechanism of regulation of BCL2 expression in

hematopoietic cancer cells consisting of posttranscriptional down-regulation by miR-15
and miR-16. This interaction has an important functional consequence: th

Bcl-2 and Bcl-xL are inhibitors of apoptosis frequently overexpressed in solid tumors.
The bcl-2 and bcl-xL mRNAs share a region of homology comprising nucleotides 605–
624 and 687–706, respectively, which differs by only three nucleotides. This sequence
does not occur in the proapoptotic splice variant bcl-xS. To test the possibility that
oligonucleotides targeting this region have the potential to down-regulate bcl-2 and bcl-
xL expression simultaneously, three 2′-O-methoxy-ethoxy-modified phosphorothioate
oligonucleotides were designed. These oligonucleotides differed in the number of
mismatches to bcl-2 and bcl-xL and in the number of nucleotides to which the
modifications were made. The effects of these oligonucleotides on bcl-2 and bcl-xL
expression, as well as their abilities to induce apoptosis, were assessed in small cell and
non-small cell lung cancer cell lines expressing different basal levels of bcl-2 and bcl-xL.
Although all oligonucleotides down-regulated bcl-2 and bcl-xL expression,
oligonucleotide 4625, which has no mismatching nucleotides to bcl-2 but three to bcl-xL,
two of which were modified by 2′-O-methoxy-ethoxy residues, showed the strongest
bispecific activity on the transcript and protein level. In all cell lines this bispecific
activity induced apoptotic cell death, as demonstrated by increased uptake of propidium
iodide, a 10–100-fold increase in caspase-3-like protease activity, and nuclear
condensation and fragmentation. This is the first report of a bcl-2/bcl-xL bispecific
antisense oligonucleotide that deserves attention as a therapeutic compound in lung
cancer and other malignancies in which bcl-2 and/or bcl-xL are overexpressed.

We identified two distinct malignant B-cell lymphoid populations. Each

population had different
phenotypic characteristics. The predominant B-lymphoid population (45% of
lymphoid cells) dimly
expressed monoclonal kappa light chain and also the following markers: HLA-
DR, CD5, CD11c, CD19,
CD22, CD23 and CD45. They were clearly CD38 and Zap-70 negative. The
second population (33%
of the lymphoid cells) dimly expressed monoclonal lambda light chain and also
the following markers:
HLA-DR, CD5, CD11c, CD19, CD22, CD23 and CD45. Most significantly, this
secondary minority clone
was clearly CD38 and Zap-70 positive

This case clearly demonstrates the importance of correct identification of more

than one abnormal B lymphoid clone. In this
particular case, if the populations had not been separately evaluated for Zap-70
and CD38 expression, the more aggressive
nature of the lambda positive clone might have been missed. In some CLL
cases, especially those with more than one clone,
reagent combinations that evaluate CD38 and Zap-70 expression for the entire
CLL population may even yield results that
are prognostically misleading.

The lesion lacks the typical zoning pattern of myositis ossificans, shows no direct
communication with native bone, and is extraarticular in location as opposed to synovial
osteochondromatosis. Soft tissue osteomas most commonly occur around the knee, the
foot, and the ankle. Soft tissue osteomas are rare tumors and this case is unusual in that it
occurs around the hip.
The TCL1 (T-cell leukemia/lymphoma1) oncogene is a coactivator of the AKT
oncoprotein, an essential molecule in the transduction of antiapoptotic signals in T and B
cells. Eµ-TCL transgenic mice with B cells with high TCL1 expression develop the
aggressive phenotype of chronic lymphocytic leukemia (CLL). Studies in human CLL
have found that expression of TCL1 correlates with high expression of ZAP-70 and use of
unmutated IgVH genes. The expression of TCL1 may be regulated in part by microRNA,
miR-29 and miR-181, which map to chromosome 11(11q). Because aberrations at 11q
have been associated with poor prognosis in CLL, we interrogated the relationship
between deletions at 11q and expression of TCL1 and ZAP-70. We used a direct
immunophenotyping method to investigate the relative co-expression levels of TCL1
and ZAP-70 within CLL cells and examined the relationship between such levels and the
proportion of leukemia cells within the CLL population bearing 11q deletions, as detected
by FISH analysis. Direct staining of intracellular TCL1 protein was performed by using
the monoclonal anti-TCL1 antibody (clone 1–21) labeled with Alexa647 in combination
with the established ZAP-70 protocol (NEJM 2004; 351:893[Abstract/Free Full Text])
together with mAb directed against CD5 and B cell surface antigens. Negative staining
levels were set using isotype control antibodies. FISH was performed on interphase nuclei
by using uniform and cross-validated procedures at all CRC sites using the CLL-panel
from Vysis. Chromosomal abnormalities were detected in 76% (520) of the 680 CLL
samples analyzed. Sixteen percent of the patients had leukemia cells with monoalleleic
deletions at 11q. We performed flow cytometry for intracellular co-expression TCL1 and
ZAP-70 on cryopreserved samples obtained from 25 CLL patient samples with varying
proportions of cells with the 11q deletion (10% to 98% abnormal cells with 11q deletion,
mean 70%) and 30 CLL samples lacking any chromosomal abnormalities. We detected
significantly higher levels of TCL1 in CLL cells that expressed ZAP-70 and/or unmutated
IgVH genes. The ZAP-70pos cases (39/55) had a median percent of TCL1pos cells of
34% compared to 15% for the ZAP-70neg cases. The cases using unmutated IgVH genes
(43/55) had a median percent of TCL1pos cells of 31%, which was greater than the 19%
median observed for cases that used mutated IgVH genes. Multiparameter analyses
revealed that the ZAP-70 positive fraction of each CLL clone had significantly higher
levels of TCL1 than did the ZAP-70 negative cells (mean=45% versus 29%, respectively,
p=0.002). We observed a significant difference between the expression levels of TCL1
for CLL cells that had deletions in 11q relative to that of CLL cells lacking any
chromosomal abnormalities (mean=41% versus 18%, respectively p=0.0002). In addition,
we observed a relationship between the levels of TCL1 expressed in leukemia cell
populations and the relative proportion of leukemia cells with deletions at 11q. This study
reveals a relationship between the levels of TCL1 expression in CLL leukemia-cell
expression of ZAP-70, and the relative proportions of leukemia cells having deletions at
11q. Studies are in progress to define whether these relationships can be explained by
altered expression of microRNA that map to 11q, which might also account for the noted
adverse prognosis of CLL that has deletions at 11q.
The Recently described NZB miRNA15a/16-1 mutation is syntenic to 13q14 deletion in
CLL. The mir15a/16-1 complex is thought to regulate bcl-2 expression. Therefore the
level of bcl-2 expression was examined more closely as it might be used to differentiate
mono-allelic deletion from the diploid CLL clone. Patients and Methods: Multicolor
flow-cytometry using a panel of anti-bcl-2 FITC (clone 124, DAKO Cytomation,
Glostrup, Denmark) and CD19, CD20, CD5, CD23, CD38, kappa, lambda, and CD45
was used. Bcl-2 expression was evaluated using the conventional bcl-2 index (Mean
fluorescence intensity (MFI) B cell/ MFI T cells) in 25 untreated CLL patients, and 10
normal donor controls. A modified ratio based on normal remaining (NR) polyclonal B
cells was also used (MFI clone/MFI NR) with a new gating strategy for bcl-2 expression
analysis. Results: The mean of the conventional bcl-2 index is significantly higher in
CLL patients [1.52 ± 0.53(SD)] compared to the control [0.5 ± 0.19(SD)], p<0.001
Student‘s t test. When comparing the mean of conventional bcl-2 index (clone/T cell)
versus the mean of the modified bcl-2 index (clone/NR) [1.78 ±0.61 (SD)] in only CLL
patients there was no significant difference. On the other hand, the rank order analysis of
the conventional and modified bcl-2 index showed an r=0.7, p< 0.001 indicating that both
methods give similar results. In addition to the high correlation between the conventional
and the modified ratio, the modified ratio was multimodal in distribution. Conclusions:
Bcl-2 expression varies largely among the lymphocyte sub-sets. The choice of the
polyclonal B cells allows better segregation of multimodal bcl-2 expression. Since bcl-2
mRNA is a validated mir15a/16 target, increased bcl-2 expression maybe a direct result
of and a surrogate marker for mir15a/16 levels and/or the 13q14 status. There are three
possibilities: no deletion (diploid), heterozygous deletion, or homozygous deletion. This
needs to be verified by fluorescence in situ hybridization (FISH) analysis of sorted CLL
B cells based on bcl-2 expression.
 We present a case of a peripheral blood submitted to us for
Leukemia/Lymphoma Evaluation by flow cytometry. The patient is
a 66 year old male with an 8 year history of CLL with both kappa
and lambda positive CLL clones identified in 1996 (kappa: lambda
equaled 3:1). A four-color flow cytometric evaluation was
performed. A population of 88% lymphoid cells was identified by
CD45 vs. SS gating. A complete phenotypic evaluation of the
lymphoid population was performed with both and an open-gate
strategy and a CD45 vs. SS gating strategy. All populations
present were evaluated for phenotypic expression as a quality
control. We identified two distinct malignant B-cell lymphoid
populations. Each population had different phenotypic
characteristics. The predominant B-lymphoid population (45% of
lymphoid cells) dimly expressed monoclonal kappa light chain and
also the following markers: HLA-DR, CD5, CD11c, CD19, CD22,
CD23 and CD45. They were clearly CD38 and Zap-70 negative.
The second population (33% of the lymphoid cells) dimly
expressed monoclonal lambda light chain and also the following
markers: HLA-DR, CD5, CD11c, CD19, CD22, CD23 and CD45.
Most significantly, this secondary minority clone was clearly CD38
and Zap-70 positive. Overall, if the two populations were not
separately analyzed, the kappa: lambda ratio of the entire B-cell
population would fall within the normal range of (equaled 1.36;
normal in our laboratory is approximately 1-2, with the ratio
equaling 1.5 in most clearly benign cases). This case clearly
demonstrates the importance of correct identification of more than
one abnormal B lymphoid clone. In this particular case, if the
populations had not been separately evaluated for Zap-70 and
CD38 expression, the more aggressive nature of the lambda
positive clone might have been missed. In some CLL cases,
especially those with more than one clone, reagent combinations
that evaluate CD38 and Zap-70 expression for the entire CLL
population may even yield results that are prognostically
misleading.