The LPL/ADAM29 expression ratio is a novel prognosis indicator in chroniclymphocytic leukemia Pablo Oppezzo, Yuri Vasconcelos, Catherine Settegrana, Dominique Jeannel, Franc¸oise Vuillier, Magali Legarff-Tavernier,Eliza Yuriko Kimura, Ste´phane Bechet, Ge´rard Dumas, Martine Brissard, He´le`ne Merle-Be´ral, Mihoko Yamamoto,Guillaume Dighiero, and Fre´de´ric Davi, for the French Cooperative Group on CLL Although the zeta-associated protein of
clinical outcome, IGVH mutational status,
concordant results (L/A؉, ZAP-70؉ or
70 kDa (ZAP-70) is overexpressed in pa-
and ZAP-70 protein expression. IGVH mu-
L/A؊, ZAP-70؊). LPL and ADAM29 gene
tients with chronic lymphocytic leukemia
tational status, ZAP-70, and the LPL and
expression could also be determined by a
(CLL) displaying unmutated IGVH genes
ADAM29 mRNA ratios (L/A ratio) were
simple competitive multiplex reverse tran-
and poor prognosis, a previous microar-
predictive of event-free survival for the
scription PCR assay. Overall, quantifica-
ray study from our group identified over-
whole cohort and for patients with stage
tion of LPL and ADAM29 gene expression
expression of LPL and ADAM29 genes
A disease. In patients in stage B and C,
is a strong prognostic indicator in CLL,
among unmutated and mutated CLL, re-
the L/A ratio was an independent prognos-
providing better prognostic assessment
spectively. To assess the prognostic value
tic factor, whereas ZAP-70 did not predict
than ZAP-70 in advanced stages of the
of these genes, we quantified their expres-
survival. Simultaneous usage of the L/A
disease. (Blood. 2005;106:650-657)
sion by real-time quantitative polymerase
ratio and ZAP-70 expression allowed an
chain reaction (PCR) in a cohort of 127
almost perfect (99%) assessment of the
patients with CLL and correlated this with
IGVH status in the 80% of patients with
2005 by The American Society of Hematology
Chronic lymphocytic leukemia (CLL) displays a variable outcome.
debate.9-11 Genomic aberrations correlate well with either good The classical Rai1 or Binet2 staging systems provided a basis for (isolated 13qϪ) or poor (17pϪ; 11qϪ) prognosis in CLL,11,12 therapeutic stratification by allocating CLL cases into 3 major risk though their occurrence as a second malignant hit cannot be groups (low, intermediate, and high), according to tumor burden definitely excluded. The mutational status of immunoglobulin and the presence of anemia and thrombocytopenia. Asymptomatic heavy chain variable (IGVH) genes has been considered as the best patients with a low tumor burden (Binet stage A) do not benefit prognostic marker in CLL.13 In an initial study, we observed that at from treatment with chlorambucil.3 However, the disease in half of least half of CLL cases carried mutations using a cut-off of 98% these patients will progress and both staging systems fail to initially germline homology.14 This was further confirmed by others,15 some identify such patients. The advent of new treatments such as purine of whom also correlated the IGVH mutational status to clinical analogues and monoclonal antibodies directed against CD20 and behavior.8,16,17 Mutated (MT) cases usually demonstrate a favor- CD52 are able to induce complete remissions and may allow early able evolution when compared to unmutated (UM) cases (Ͼ 98% treatment for asymptomatic patients whose disease is likely to germline homology), which are characterized by progressive progress.3 Accurate identification of these patients is therefore disease, continuing treatment needs, and a high proportion of Serologic markers such as lactic dehydrogenase, ␤2-microglobu- Unexpectedly, recent reports indicated that ZAP70, normally ex- lin,4 soluble CD23,5 and thymidine kinase4,6 are essentially indica- pressed in T and natural killer (NK) lymphocytes, is also transcribed in tors of disease activity or tumor load or both, although some can CLL B cells lacking IGVH mutations and is associated with poor anticipate disease progression.7 Phenotypic expression of CD38 prognosis.18,19 Further series have confirmed these findings at the has been associated with aggressive disease,8 but the threshold protein level,20-23 suggesting a pivotal role for the zeta-associated protein level for positive cases, if it exists at all, remains a matter of of 70 kDa (ZAP-70) in prognosis prediction in CLL.
From the Unite´ d’Immuno-He´matologie et d’Immunopathologie, Institut Fundac˛ao de Amparo a` Pasquisa do Estado de Sa˜o Paulo (FAPESP). P.O. was supported by the French Academy of Medicine.
Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, Brazil; Service d’He´matologie P.O. and Y.V. contributed equally to this work.
Biologique, Hoˆpital Pitie´-Salpeˆtrie`re, Paris, France; De´partement desEcosyste`mes et Epide´miologie des Maladies Infectieuses, Institut Pasteur, A complete list of the members of the French Cooperative Group on CLL Paris, France; Service Immunologie, Hoˆpital Pitie´-Salpeˆtrie`re, Paris, France; and Centre de Recherche Vaccinale et Biome´dicale, Institut Pasteur, Paris,France.
Pitie´-Salpeˆtrie`re, 47 Boulevard de l’Hoˆpital, 75013, Paris, France; e-mail: Submitted August 30, 2004; accepted February 25, 2005. Prepublished online as Blood First Edition Paper, March 31, 2005; DOI 10.1182/blood-2004-08-3344.
The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is hereby Supported by grants from the French Cooperative Group on CLL and the marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Ministe`re de la Recherche et de la Technologie. Y.V. was supported byCoordenac˛a˜o de Aperfeic˛oamento de Passaol de Nı´vel Superior (CAPES) and 2005 by The American Society of Hematology BLOOD, 15 JULY 2005 ⅐ VOLUME 106, NUMBER 2 LPL AND ADAM29 EXPRESSION PREDICT SURVIVAL IN CLL In a study of gene expression profiling performed on 18 CLL IGVH sequences were considered as mutated if their homology with the cases, we identified a limited set of genes (n ϭ 85), which were closest germline counterpart was less than 98%.
expressed differentially between progressive UM and stable MTCLLs.24 We validated these results by real-time quantitative RNA isolation and cDNA synthesis
polymerase chain reaction (RQ-PCR) for 18 genes on the same Total cellular RNA was extracted using the RNeasy kit (Qiagen, Courta- cDNAs that were hybridized on the DNA chips. From these boeuf, France) following the supplier’s instructions. The integrity of RNA RQ-PCR experiments, 4 genes in addition to ZAP70 appeared to was assessed by visualization of the 18S and 28S RNA species on provide a better segregation of the 2 groups of CLL. These included electrophoresis in agarose gel after ethidium bromide staining. First-strand the lipoprotein lipase (LPL) and spartin (SPG20) genes, whose cDNA was synthesized from 2 ␮g total RNA, using Superscript II reverse expression was higher in UM cases, whereas disintegrin and transcriptase (RT; Invitrogen, Cergy-Pontoise, France) and oligodT orrandom hexamer primers.
metalloproteinase 29 (ADAM29) and nuclear receptor-interactingprotein 1 (NRIP1) genes were found at higher levels in MT cases.
Quantitative RT-PCR
These findings led us to investigate, in an independent and largerCLL series, whether these 4 genes (isolated or in combination) For gene expression analyses of LPL, SPG20, ADAM29, and NRIP1, we could provide significant prognostic information and compared performed RQ-PCR using the Light Cycler System (Roche Molecular their value with that of the IGVH mutational status and ZAP-70 Biochemicals, Mannheim, Germany) and the SYBR Green I dye. Primers used in this study (Table 1) were designed with the GeneRunner software(Hastings Software, Hastings, NY). RQ-PCRs were carried out on 100 ngreverse transcribed total RNA (cDNA) with the following parameters: 10minutes at 95°C for initial denaturation, then 40 cycles of 10 seconds at95°C, 5 seconds at 62°C, and 17 seconds at 72°C. The specificity of the Patients, materials, and methods
amplified products was verified by analysis of their respective meltingcurves as provided by the Light Cycler software. All reactions were done in Patients and samples
duplicate and each PCR run also included the 5 points of the calibration This multicenter retrospective study was undertaken on samples from 127 curve and a no template control. Estimation of the quality of cDNA for each patients whose diagnosis was made between October 1979 and February sample was done by quantification of an endogenous reference, the 2003, and identified from the registries of the Pasteur Institute and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The gene copy Pitie´-Salpeˆtrie`re hospitals, Paris, France (n ϭ 92), and the University number was calculated with a standard curve generated from serially Hospital of Sa˜o Paulo, Brazil (n ϭ 35). Inclusion criteria consisted of: (1) diluted (10-fold dilutions from 106 to 102 copies) plasmids containing the diagnosis of typical CLL based on morphologic and phenotypic analyses25; respective sequence-verified insert. Results were expressed as the ratio of (2) availability of frozen samples; (3) previous determination of IgVH mean of gene copy number/mean GAPDH copy number ϫ 100.
mutational status; and (4) patient’s informed consent according to Frenchand Brazilian regulations. Approval was obtained from the Pitie´-Salpeˆtrie`re Multiplex RT-PCR
Hospital, the Pasteur Institute, and the University Hospital of Sa˜o Paulo for We also evaluated the relative expression of LPL and ADAM29 by these studies. This series included 87 patients with stage A, 29 with stage B, multiplex RT-PCR, using the same primers than those for RQ-PCR (Table and 11 with stage C disease. Median follow-up time was 73 months for the 1). Different concentrations of primers, MgCl whole series (range, 1-291 months), 87 months for patients in stage A and triphosphate (dNTP) were first evaluated. Optimized PCR conditions were 50 months for patients in stages B and C. Progression was defined as change obtained with primers at the final concentrations of 0.5 ␮M for LPL and of clinical stage or need for treatment or both.
All analyses were carried out on frozen peripheral blood samples. For were performed on 100 ng cDNA and included an initial denaturation step 113 patients, samples had been taken at the time of diagnosis. In 14 stage A at 94°C for 5 minutes, followed by 29 cycles of 30 seconds at 95°C, 20 cases with a very stable lymphocytosis over time, they were obtained at seconds at 62°C, and 30 seconds at 72°C. Finally, the reaction was distance from diagnosis. Mononuclear cells had been separated by Ficoll- completed with a final elongation step at 72°C for 5 minutes. PCR products Hypaque gradient centrifugation and stored in liquid nitrogen. On thawing, were analyzed on 2% agarose gel electrophoresis stained with ethidium cell viability was first assessed by trypan blue exclusion and only samples bromide, where they appeared as a 445–base pair (bp) band for ADAM29 displaying more than 80% viability were further analyzed. In 5 cases, and a 410-bp band for LPL. Amplification of GAPDH was performed in leukemic B-cell populations were purified by negative magnetic selection using anti-CD3, anti-CD14, anti-CD16, and anti-CD56 monoclonal antibod-ies (Dynal, Oslo, Norway). Final purity was evaluated by flow cytometry to Multiparametric flow cytometry
be over 98%. Control samples from 20 healthy adult volunteers wereobtained using the same procedures and included peripheral mononuclear Flow cytometric analysis of ZAP-70 intracellular expression was per- blood cells (PBMCs, n ϭ 14) and purified peripheral blood B cells (n ϭ 6).
formed using the method described by Crespo et al20 with some minor In addition the T-cell line Jurkat was cultured in RPMI 1640 mediumcontaining 10% fetal calf serum, 2 mM glutamine, 1% sodium pyruvate,and penicillin-streptomycin.
Table 1. Sequences of primers used in RQ-PCR and multiplex PCR
Sequence (5؅ 3 3؅)
IGVH mutational status
The IGVH gene sequences were determined as previously described.26 Briefly, amplification of immunoglobulin heavy chain variable regions by PCR was performed on DNA from leukemic cells with consensus primers for the VH framework region 1 and JH genes as previously described26 or following the BIOMED-2 protocols.27 Purified PCR products were se- quenced either directly or after a cloning procedure using an automated DNA sequencer. Sequence data were analyzed using IgBLAST (http:// www.ncbi.nlm.nih.gov/igblast), the VBASE (http://vbase.mrc-cpe.cam.
ac.uk/), and the ImMunoGeneTics database (IMGT; http://imgt.cines.fr).
BLOOD, 15 JULY 2005 ⅐ VOLUME 106, NUMBER 2 modifications. Thawed mononuclear cells were fixed in 2% paraformalde- associations between individual variables and survival was calculated by hyde and were then permeabilized by incubation with phosphate-buffered the log-rank test. Univariate and multivariate regression analyses were done saline containing 0.1% saponin (Sigma, Saint-Quentin Favallier, France) according to the Cox proportional hazards regression model. Because the and 0.5% bovine serum albumin. One million cells were first incubated with biologic factors studied as prognostic indicators were strongly correlated 2.5 ␮g anti–ZAP-70 antibody (clone 2F3.2; Upstate Biotechnology, Lake with IGVH mutational status, it was inappropriate to test them simulta- Placid, NY) or irrelevant isotype-matched anti-CD14 monoclonal antibod- neously by Cox regression. Consequently, 2 Cox regression analyses were ies (Dako Cytomation, Trappes, France). After washing, they were incu- performed, with the first one testing IGVH mutational status, and the second bated with 1.5 ␮g F(abЈ)2 fluorescein isothiocyanate (FITC)–conjugated one testing the other selected factors, in both circumstances with adjustment goat antimouse antibody (Immunotech, Marseille, France). Cells were then for age, sex, and when appropriate the Binet staging. Variables with 2-tailed washed and incubated with phycoerythrin-cyanin 5 (PC5)–conjugated P values of less than .05 were considered to be significant. All analyses mouse anti-CD19 (Immunotech), allophycocyanin (APC)–conjugated mouse were done using SPSS Statistical Software, version 11.5 (SPSS, Chicago, IL).
anti-CD3, and APC-conjugated mouse anti-CD56 (BD Biosciences, SanJose, CA). Samples including at least 104 cells were further analyzed with aflow cytometer (FACSCalibur; BD Biosciences) and the use of Cell Quest Pro software (BD Biosciences). Lymphocyte cells were first selected onsize structure characteristics and then gated on B cells (CD19ϩ) and T and Patient characteristics
NK cells (CD3ϩCD56ϩ). Bi-parametric dot-plot graphs were obtainedseparately for cells that were stained for, respectively, CD3, CD56, and Table 2 summarizes the characteristics of the 127 patients. Eighty- ZAP-70, or CD19 and ZAP-70, or CD3, CD56, and CD14 (negative seven patients were in stage A, 29 in stage B, and 11 in stage C.
control). In those plots as well as in mono-parametric histograms, ZAP-70 Twenty disease-related deaths were recorded and 4 additional expression on CD3ϩCD56ϩ cells served to determine the percentage of deaths, unrelated to CLL, occurred among patients in stage A.
CLL cells that were positive for ZAP-70.
Fifty-three patients (42%) were allocated to the UM IGVH group, Statistical analyses
whereas 74 displayed a MT IGVH profile. The MT and UM caseswere heterogeneously distributed within Binet stages because 69% Expression levels of the 4 tested genes and of ZAP-70 protein were of patients in stage A displayed a MT IGVH profile, whereas 65% compared with the IGVH mutational status as a reference. Threshold values were UM among B and C cases (P Ͻ .001). Sex distribution was that could best discriminate MT from UM cases were first determined by also heterogeneous with a high proportion of male patients among plotting expression values against IGVH percentage of germline homology, UM cases (70%) and a slight female predominance (53%) in the and then further refined by calculating the Youden index28 and validityindex (the percentage of correctly classified cases). Thereafter, the perfor- MT group (P ϭ .05). Based on French Cooperative Group guide- mance indexes including sensitivity, specificity, and positive and negative lines, almost all patients with stage B and C disease received early predictive values were determined. Distributions of patients, according to treatment, whereas treatment was deferred for those with stage A Binet staging, sex, and IgVH mutational status were compared using ␹2 test.
disease until disease progression. This was the case for 22 patients Median follow-up periods were calculated for each Binet stage group.
Because CLL-related deaths were mainly observed in stages B and C (only4 cases in stage A), whereas disease progression was the most frequent Selection of LPL and ADAM29 as potential prognostic
event for patients in stage A, we evaluated event-free survival (EFS), from markers in CLL
diagnosis to date of disease progression or CLL-related death or lastfollow-up visit, for the whole cohort and patients in stage A. Overall Because our microarray study showed that LPL and SPG20 were survival was calculated only for patients in stage B and C. Survival analyses overexpressed among UM CLLs, whereas ADAM29 and NRIP1 were performed using the Kaplan-Meier method. Statistical significance of predominated in MT CLLs, we first evaluated expression of these 4 Table 2. Clinical and biologic characteristics of patients
Stages B؉C
Stages A؉B؉C
Lymphocyte doubling time, no (%)
IGVH genes, no (%)
ZAP-70, no (%)
L/A ratio, no (%)
NA indicates not applicable.
*Median values LPL AND ADAM29 EXPRESSION PREDICT SURVIVAL IN CLL Table 3. Correlation of gene expression with IGVH mutational status
Less than 1
Less than 3
3.5 or more
Less than 3.5
Less than 4
Less than 1
genes in an initial series of 71 patients. Quantification was be identical to those of the first cohort (Figure 1). ZAP-70 performed on total lymphocytes because preliminary experiments expression was measured by flow cytometry in leukemic B cells in in 5 patients showed similar results when compared to purified comparison with that of the patients’ T and NK cells. A cutoff value leukemic cells (data not shown). For each gene, we determined at 20% of cell positivity was found to provide the best correlation which expression levels could best segregate UM from MT cases using the Youden and validity indexes. Results showed that overall Median EFS for the entire cohort was 87 months in CLLs LPL and ADAM29 performed better than SPG20 and NRIP1 to displaying UM IGVH genes as compared to 149 months in MT predict UM and MT IGVH profiles, respectively (Tables 3 and 4).
patients (P Ͻ .001). It was 84 months for patients with an L/A ratio The concordance rate was 80% for ADAM29, 77% for LPL, 65% above 1 and 88 months for patients expressing ZAP-70, whereas for SPG20, and 55% for NRIP1. Next, we investigated whether a median EFS was not achieved for those with an L/A ratio below 1 combination of the most discriminating parameters by a simple 1:1 (P Ͻ .001) or ZAP-70Ϫ (P Ͻ .001; Figure 2A). Multivariate Cox LPL/ADAM29 (L/A) ratio could improve their individual perfor- regression showed that, with adjustment for age and sex, UM IGVH mance. With a calculated threshold of 1, the L/A ratio displayed and stage B or C were independently associated with disease better sensitivity and specificity than each marker taken individu- progression or CLL-related death with hazard ratios of, respec- ally. Positive predictive value (PPV) was 91% for UM cases andnegative predictive value (NPV) was 86% for MT patients, tively, 5.0 (P Ͻ .001) and 2.6 (P ϭ .01; Table 6). Similarly, an L/A providing a better performance than each individual marker (Tables ratio above 1 and stage B or C were independently and significantly 3 and 4). Thus the L/A ratio constituted the best marker reflecting associated with shorter EFS. ZAP-70, however, was not found to be the mutational status of IGVH genes in this cohort of 71 patients with CLL, with a concordance rate of 89%.
In patients with stage A disease, an identical median EFS of 87 months was observed for patients with UM IGVH genes, an L/A Reproducibility of LPL and ADAM29 quantification
ratio above 1, and expressing ZAP-70, whereas it was not achieved Next, we tested the reproducibility of LPL and ADAM29 quantifica- for cases with MT IGVH genes, an L/A ratio below 1, and ZAP-70Ϫ tion by RQ-PCR. This was done by comparing results obtained (all P Ͻ .001; Figure 2B). In multivariate Cox analyses, after from replicate samples for 4 patients, 2 overexpressing LPL and 2 adjustment for age and sex, both ZAP-70 and L/A ratio were overexpressing ADAM29. For each patient, 4 replicates were independent significant prognostic factors. This was also true for analyzed during the same reaction run (intrarun variability), and the IGVH mutational status (Table 6).
this was repeated on 3 different days (interrun variability). The The 40 patients in stage B and C were analyzed together for overall variability of these 12 replicates is shown in Table 5. Of evaluation of overall survival (OS; Figure 2C). There was a trend note, intrarun variability was always smaller than overall variabil- for longer OS in patients with MT than in those with UM IGVH ity, with the coefficient of variation (CV) being less than 0.5% for genes (128 versus 79 months; P ϭ .067). The L/A ratio was LPL and less than 1.1% for ADAM29 (data not shown).
predictive of survival because patients with a ratio above 1 had amedian OS of 79 months, whereas it was not yet reached at time of The L/A ratio is a strong predictor of prognosis in CLL
analysis for those with a ratio below 1 (P ϭ .03). In contrast, We evaluated the prognostic value of the L/A ratio in an extended ZAP-70 did not correlate with survival in this group (100 versus 80 cohort of 127 patients. This included the 71 initially studied months; P ϭ .28). By multivariate Cox analysis, an L/A ratio patients and 56 additional cases selected because of their prolonged below 1 was found as a significant prognostic factor, whereas follow-up and the availability of frozen samples. In addition we ZAP-70 was not independently associated with survival. In a compared its value to that of the mutational profile of IGVH genes separate Cox analysis, IGVH mutational status became a significant and ZAP-70 protein expression. LPL and ADAM29 were available prognosis factor, after adjustment for age and sex (Table 6).
for 119 patients, whereas ZAP-70 could be determined for 101patients and all 3 parameters for 93 patients. On this larger series,the L/A ratio once again provided a better concordance (90%) with Table 5. Reproducibility of RQ-PCR
IGVH mutational status than LPL (76%) or ADAM29 (82%) taken LPL
individually (data not shown), and threshold values were found to Table 4. Parameters calculated in relation to unmutated IGVH genes
for LPL, SPG20, and LPL/ADAM29 and to mutated IGVH genes for
ADAM29 and NRIP1
Overall variability of RQ-PCR quantification of LPL and ADAM29 gene expres- sion was evaluated on 12 replicates performed on 4 different CLL patients. This was done by testing each sample in 4 replicates during the same reaction (intrarun variability), this being repeated on 3 different days (interrun variability).
Ct indicates threshold cycle; SD, standard deviation; CV, coefficient of variation; BLOOD, 15 JULY 2005 ⅐ VOLUME 106, NUMBER 2 stage A and 3 stage B), whereas 9 had MT IGVH genes (7 stageA and 2 stage B). Four of the 6 patients with a ZAP-70ϪL/Aϩprofile displayed UM IGVH genes (3 stage A and 1 stage C),whereas they were MT in the remaining 2 cases (1 stage A and 1stage B). Of note, the mutational status of these 19 discordantcases would have been predicted correctly more often by theL/A ratio alone (13 cases) than by ZAP-70 expression alone (6cases). The small number of patients with discordant markers Figure 1. Correlations between LPL/ADAM29 ratio or ZAP-70 expression and
did not allow prognostic evaluation.
the IgVH gene mutational status. The threshold values calculated for the L/A ratio
( ϭ 1) and ZAP-70 ( ϭ 20%) showing the best concordance rate with the IGVH
Determination of the LPL and ADAM29 gene expression by a
mutational status, as determined by using the Youden index, are indicated by a simple qualitative multiplex RT-PCR assay
To simplify the assessment of LPL and ADAM29 gene expression, Correlation between L/A ratio, ZAP-70 expression, and IGVH
we developed a multiplex RT-PCR assay, where both genes were mutational status
simultaneously amplified generating PCR products of different Given the strong prognostic value of the 3 markers, we next size, respectively, 410 bp and 445 bp (Figure 3). This assay was assessed the correlation between them in the 93 cases for whom all then evaluated on 95 patients of our series. Experiments were done 3 parameters had been determined. Cases were scored as “positive” in duplicate on separate PCRs for 25 cases and showed a perfect or “negative” for a given marker based on expression values above reproducibility. Using our defined PCR conditions, the results were or below the thresholds. Concordance rate with IGVH mutational unambiguous in 89 cases with production of a single band or 2 status was 85% for ZAP-70, thus slightly lower than that obtained bands but with one clearly more intense than the other. They with the L/A ratio (92%; Table 7). As depicted in Table 7, all correlated with the RQ-PCR results because 34 of the 36 patients double-positive patients (ZAP-70ϩL/Aϩ; n ϭ 30) except one ex- with an L/A ratio above 1.0 expressed predominantly LPL in the pressed UM IGVH genes, and all double-negative patients (ZAP- multiplex assay, whereas 2 displayed products of both size with 70ϪL/AϪ; n ϭ 44) had MT IGVH genes. Although an almost roughly similar intensities (doublets). Alternatively, 55 of the 59 perfect correlation for all 3 markers was found for these cases (73 patients exhibiting a L/A ratio less than 1.0 expressed predomi- of 74, 99% accuracy), discordant profiles still accounted for nantly ADAM29, whereas 4 displayed doublets (Table 8). Thus, 20% of CLL patients. They included 13 patients expressing a inconclusive results (doublets) were observed for 6 patients. In 5 of ZAP-70ϩL/AϪ profile, whereas 6 patients were ZAP-70ϪL/Aϩ.
these 6 cases, the L/A ratio values were in the range of 0.7 to 1.5.
Among the 13 ZAP-70ϩL/AϪ cases, 4 had UM IGVH genes (1 There was, therefore, a very good concordance between the results Figure 2. Kaplan-Meier survival curves in CLL according to IGVH
mutational status, L/A ratio, or ZAP-70 expression. (A) EFS
probabilities for the total population. (B) EFS probabilities for patients
in stage A. (C) OS probabilities for patients with stages B and C
LPL AND ADAM29 EXPRESSION PREDICT SURVIVAL IN CLL Table 6. Prognostic factors for disease progression and
CLL-related death in multivariate Cox regression

Hazard ratio (95% CI)
All stages, EFS for all stages
Figure 3. Multiplex PCR determination of LPL and ADAM29 expression. LPL and
ADAM29 transcripts were amplified simultaneously; the PCR products were then separated by electrophoresis on agarose gel and visualized under UV illumination after ethidium bromide staining. Amplification of the GAPDH gene from the sametranscripts served as control of cDNA integrity. MWM indicates molecular weight marker; MT, mutated; UM, unmutated; B, purified B cells from a healthy individual; T, detected in any of the PBMCs and at a very low level in one of the purified B-cell samples. In addition the T-cell line Jurkat failed to EFS for Binet stage A
express either of these 2 genes. Thus, all or most of the LPL and ADAM29 transcripts that we measured in patient samples origi- nated from leukemic cells and not from background normal Discussion
The recent development of microarray technology has allowed the discovery of genes, which may have prognostic significance in OS for stages B and C
human tumors. A previous gene expression profiling study in CLL led us to identify a set of genes that appeared to segregate stable MT from progressive UM forms.24 In the present work, we show that the combined expression of 2 of them, LPL and ADAM29, constitutes a new prognostic marker in CLL.
LPL is a heparin-releasable enzyme bound to glycosaminogly- can components of the capillary endothelium and is particularly abundant in muscle, adipose tissue, and macrophages. Withapolipoprotein CII, LPL mediates the hydrolysis of triacylglycerol Multivariate analyses were done separately for IGVH or ZAP-70 and L/A ratio due component of circulating chylomicrons and very-low-density li- to the high concordance between the latter 2 parameters and the former.
poproteins. It plays a central role in lipid metabolism and trans- †ZAP-70 was not independently associated with disease progression or CLL- port.29,30 Mutations in the LPL gene are frequently associated with related death and was then eliminated in the final Cox model.
dyslipidemia and atherosclerosis.29,31 In line with our findings onnormal cells, other investigators have failed to detect LPL expres- obtained by RQ-PCR and qualitative multiplex PCR. Prognostic sion in normal purified B and T lymphocytes.32 However, they evaluation performed with LPL and ADAM29 gene expression found that it was expressed and secreted by NK cells, where it was determined by multiplex PCR produced identical results to those shown to modulate their cytotoxic activity. Reasons for its high obtained by RQ-PCR (data not shown).
expression in UM CLL B cells are unknown. Because UM CLL Bcells have been shown to be more responsive to stimulation through Expression of LPL and ADAM29 genes in normal
their antigen receptor,33,34 we speculate that LPL could play a role circulating cells
in lipid raft formation or stabilization, a biologic process known tobe important in B-cell activation.35 The ADAM29 gene encodes a Although experiments on purified and unpurified CLL cells showed member of the disintegrin and metalloproteinase family of trans- similar results, we wanted to evaluate a possible expression of LPL membrane proteins, which have been shown to mediate cell-to-cell and ADAM29 genes in normal cells, which might contaminate or cell-to-matrix interactions (or both) as well as the proteolytic patient samples. For this, we assessed their expression by multiplex shedding of cell surface molecules.36,37 In contrast to other ADAMs PCR in the peripheral blood cells of 20 healthy individuals. Very that are expressed in various tissues, ADAM29 transcripts are low level of expression of LPL was found in 3 of the 14 PBMCs highly restricted to the testis.38,39 Because its precise functions are tested and in 1 of the 6 purified B-cell samples. ADAM29 was not Table 8. Comparison of multiplex RT-PCR with RQ-PCR
Table 7. Groups of CLL patients according to L/A ratio and
L/A ratio
L/A ratio
ZAP-70 expression
less than 1
greater than 1
Positivity or negativity for ZAP-70 refers to expression values Ն20% or Ͻ20%, Cases were scored as ADAM29 or LPL according to the type of the predominant respectively. Positivity or negativity for L/A ratio refers to expression values Ն 1 PCR product obtained by multiplex RT-PCR. When 2 bands of roughly similar intensity were observed, they were qualified as doublet.
BLOOD, 15 JULY 2005 ⅐ VOLUME 106, NUMBER 2 so far unknown, its role in the biology of CLL remains unex- higher than that observed by Crespo et al20 and Orchard et al22 plained. The fact that for these 2 genes: (1) similar results were (respectively, 5.4% and 7.8%) but lower than that of Rassenti et al obtained on total CLL lymphocyte populations and from purified (23%). In this latter work, the investigators used another anti- leukemic cells, and (2) their absence of detection or very low level ZAP-70 antibody and a different gating strategy.23 These discor- in normal peripheral B cells, indicates that their overexpression dant results might be due to technologic reasons, in addition to the could be tumor specific. Alternatively, it might reflect a restricted type of patients studied. Further studies evaluating the different expression in a minor B-lymphoid subpopulation, which is ex- technologic aspects of its detection are clearly needed before panded in CLL. Further experiments on purified B-cell subsets are ZAP-70 expression can be used in a clinical perspective. Interest- warranted to prove whether these hypotheses are correct.
ingly, concordant results for ZAP-70, L/A ratio, and IGVH genes The discovery that ZAP70 gene expression could differentiate were found in about 80% of cases. Provided that these results are MT from UM CLLs has also emerged from microarray experi- confirmed in larger series of patients, combining ZAP-70 and L/A ments.18,19 Further studies confirmed its presence at the protein ratio quantification may represent an alternative to sequencing the level by flow cytometry and showed that it had prognostic IGVH genes in about 80% of patients.
impact.20-23 We evaluated the ability of the L/A ratio as well as We also describe an alternative method to assess simultaneous ZAP-70 expression and the IGVH mutational status to predict expression of LPL and ADAM29 by a multiplex competitive clinical outcome in our cohort of patients with CLL. In univariate RT-PCR technique. Although it was not informative in a minority analysis, all 3 parameters correlated with EFS in the whole of cases, in our hands this assay proved to be simple and population. In multivariate analysis, however, ZAP-70 was no reproducible. Its extension to a routine test, however, requires longer selected. For stage A patients, these 3 biologic parameters further evaluation by independent laboratories. The determination were predictive of EFS. When considering the patients with stage B of the IGVH mutational status is an expensive labor-intensive and C disease, the L/A ratio was the only parameter that correlated technique not well suited as a routine test in most clinical significantly with survival in univariate analysis. The IGVH UM laboratories. Simpler techniques such as flow cytometric quantifica- status became a significant risk factor only after adjustment for sex tion of ZAP-70 or PCR-based expression analysis of LPL and and age in multivariate analysis. Rassenti et al reported recently ADAM29 genes represent promising alternatives. Standardization that ZAP-70 was a better predictor of time from diagnosis to initial of these techniques, as well as evaluation of their cost, including therapy than the IGVH mutational status.23 The lack of information comparison of RQ-PCR and multiplex RT-PCR for LPL and concerning the patients’ clinical stage in this cohort makes ADAM29, will be important for future therapeutic protocols. A cost comparison with our data difficult. In our study, most of patients in comparison evaluation of the different techniques to assess progno- stages B and C received early treatment, whereas it was delayed in sis in stage A CLL is currently being performed in France.
those with stage A disease until progression was observed.
In summary, our study shows that LPL and ADAM29 constitute Therefore, only patients in stage A can be compared. As noted, new and strong prognosis markers in CLL. Importantly, they ZAP-70, the L/A ratio, and the IGVH mutational status were all provide better prognostic information than ZAP-70 for advanced similarly capable of predicting treatment-free survival for this CLL cases. In addition, combination of the L/A ratio with ZAP-70 group of patients. In contrast, ZAP-70 expression had no prognos- expression predicts accurately the IGVH mutational status in 80% tic value in patients in stages B and C. Similar findings have also of CLL cases, thus rendering sequencing unnecessary in these been reported by Crespo et al.20 It remains to be elucidated why ZAP-70, which is a strong prognostic parameter for patients instage A, does not predict outcome for patients in stages B and C.
The availability of biologic prognostic indicators such as the L/A Acknowledgments
ratio for stage B and C CLL cases may therefore be of greatimportance for future risk-adapted treatments. For the cases with We thank Dr Rodrigo Proto-Siquera and Dr Marco Antoˆnio Zago discordant results for ZAP-70, L/A ratio, and IGVH genes, it was for providing patient samples, Ariane Michel for expert technical unclear which of these 3 biologic parameters was the most reliable assistance, and Elizabeth Macintyre for critical reading of the prognostic indicator. Larger series will be necessary to determine which most accurately predict clinical evolution.
In the present study, we also evaluated the correlations between these 3 biologic prognostic markers. The concordance rate with Appendix
IGVH mutational status was slightly higher for the L/A ratio (92%) Members of the advisory board and scientific committee of the French than for ZAP-70 (85%). For this latter parameter, the threshold Cooperative Group on CLL are as follows, in alphabetic order: J. L. Binet, value that best correlated with the IGVH mutational status was B. Cazin, S. Chevret, F. Cymbalista, F. Davi, A. Delmer, G. Dighiero, M.
found to be 20%, similar to that previously described by Crespo et Divine, B. Dreyfus, C. Dumontet, J. P. Fermand, R. Garand, V. Leblond, M.
al20 and Rassenti et al,23 but higher than that used by Orchard et al22 Leporrier, V. Levy, K. Maloum, H. Merle-Be´ral, M. Michallet, L. Sutton, P.
(10%). The discordance rate with the IGVH status was slightly References
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