Detection of Clonal CD34+19' Progenitors in Bone Marrow
From www.bloodjournal.org by guest on December 22, 2014. For personal use only. Detection of Clonal CD34+19' Progenitors in Bone Marrow of BCL2-IgH-Positive Follicular Lymphoma Patients By E.A. Macintyre, C. Belanger, C. Debert, D. Canioni, A.G. Turhan, M. Azagury, 0. Hermine, B. Varet, G. Flandrin, and C. Schmitt The frequent occurrence of BCU-lgH rearrangements in follicular lymphoma (FL) makes detection of low numbers of tumor cells possible by polymerase chain reaction (PCR). The presence of BCU-lgH in the bone marrow (BM) and peripheral blood of many FL patients at the time of autografting has led to the suggestion that selection of the CD34enriched fraction may lead t o reinfusion of lower numbers of tumorcells. To address this issue, we PCR-amplifiedBCUIgH fromfluorescence-activated cell sorting (FACSI-purified BM CD34+ and CD34- fractions in seven FL patients showing a PCR-detectable translocation in the major breakpoint regionof BCLZ, five of whichshowed morphological BM involvement. The total CD34' fraction showed diminished but residual positivity in the first two cases tested. Therefore, BM cells from theremaining five patients weresorted for theCD34+19- immature population, the CD34'19' B-cell precursors, and the CD34-19+ mature B-cell fraction. The CD34+19-subpopulation was negative in four offive, despite evident BMinfiltration in three cases. In contrast, the CD34'19+ fraction was positive in all three cases tested. These cells represented 0% t o 50% (mean, 18%) of the total CD34' population, suggesting that, if reinfusion of BCUIgH-positive cells plays a role in postautograft relapse in FL, therapeutic CD34 selection procedures should include additional purging of the CD34+19+B-cell precursors or, at least, assessment of the proportion of CD19+ cells in the CD34' fraction and its correlation with clinicaloutcome postreinfusion. 0 1995 by The American Society of Hematology. F The latter include the CD34+19+ B-cell precursors (BCPs), representing about 10% to20% of total CD34+ cells.10"2 Although CD34 is not observed on mature lymphoma cells," involvement of the IgH locus in the t( 14; 18) suggests that the early oncogeneic events may take place at the CD34' BCP stage at the time of IgH rearrangement. The CD34 BM fraction has been shown, to restore hemopoiesis in patients receiving myeloablative therapy for breast cancer and myeloma, with a tumor cell depletion of 2 to 3 log^.'^.'^ More recently, successful hemopoietic reconstitution has been shown after reinfusion of the CD34-enriched BM fraction in non-Hodgkin's lymphoma, including FL, with an apparent reduction in tumor load.16 The punty of the CD34+ fraction depends on the method of cell separation; large scale techniques suitable for clinical use are most commonly based on immunoabsorption and result in a highly variable punty ranging from 10% to90%.16." Higher purity can be obtained by fluorescence-activated cell sorting (FACS), either on the initial mononuclear sample or on the semipure CD34+ fraction, thus permitting virtually 100% purity.'* Before evaluating the potential benefit on DFS of tumor cell purging by CD34+ cell selection on patients autografted OLLICULAR LYMPHOMA (FL) is a mature B-cell non-Hodgkin's lymphoma with a characteristic chromosomal abnormality involving the BCL2 gene on chromosome 18q21 and the Ig heavy chain (IgH) locus on chromosome14q32, with theirjuxtapositioningleading to deregulated expression of BCL2.' Because the majority of breakpoints inboth genes are clustered, it is possible to detect approximately 70% of t(14; 18)-positive FL by polymerase chain reaction (PCR) from DNA using primers specific for the BCL2 major breakpoint region (MBR) or, more rarely, the minor cluster region and a consensus primer for the JH segments of the IgH gene. The length of BCL2-IgH PCR product generated varies between patients as a function of the precise breakpoint position in BCL2, the presence of IgH D segments, and modification of the junctional sequence. The sensitivity of this technique has allowed the demonstration that the vast majority of patients show BCLZ IgH rearranged DNA in the bone marrow (BM) at diagnosis, including in apparently uninvolved samples, and that many also show PCR positivity in the peripheral blood(PB).*" High-dose therapy with autologous BM or PB stem cell (PBSC) support can improve disease-free survival (DFS) in low-grade FL in chemosensitive relapse? Contamination by tumor cells has been shown in both BM and PBSCs at the time of autologous tran~plantation.~.~ In view of the potential implication of these cells in relapse after autologous BM transplantation (ABMT), certain investigators have attempted in vitro immunologic purging of the graft using B-cell-specific antibodies.""On analysis of these purged fractions, Gribben et a17 found an association between the ability to purge BM to PCR negativity and DFS post-autologous BM transplantation> although these results have not been confirmed by others. To minimize reinfusion of BCL2-IgH-positive cells, indirect purging by selection of the CD34' fraction has been proposed. CD34, a marker of hematopoietic progenitors is expressed on approximately 1% to 4% of normal BM nucleated cells, including multipotential stem cells, often identified by their CD34+/CD38- lineage marker (1in)- phenotype, and the more mature, lineage-committed, precursor ~ e l l s . Blood, Vol 86, No 12 (December 15), 1995: pp 4691-4698 From the Departments of Laboratory and Clinical Hematology, Pathology, and CNRS URA 1461, H6pital Necker Enfants-Malades and Universite' Paris V, Paris, France; and CNRS URA 625 H6pital de la PitiP-Salpe'tritre, Paris, France. Submitted June 21, 1995; accepted August 8, 1995. Supported by the Ligue Nationale Contre le Cancer Comite' de Paris, the Fondation Contrela Leucemie, the Fondation pour la Recherche Medicale, and the Assistance Publique des H6pitaux de Paris. Address reprint requests to E. Macintyre, MD, PhD, hboratoire d'He'matologie, Tour Pasteur, H6pital Necker Enfants-Malades, 149 rue de Stvres, 75743 Paris Cedex, France. The publication costsof this article were defrayedin part by page chargepayment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1995 by The American Society of Hematology. ~ ~ 0006-4971/95/8612-0030$3.00/0 ~ 4691 From www.bloodjournal.org by guest on December 22, 2014. For personal use only. MACINTYRE ET AL 4692 for FL, we have evaluated the tumor cell content of highly purified FACS-sorted CD34'BM cells from FL patients with a high tumor mass and a BCLZIgH rearrangement. We show that the CD34'/CD19' BCP fraction is BCL2-IgHpositive, whereas the CD34+/CD19- population containing more primitive cells is negative in the majority of patients studied. MATERIALS AND METHODS Patient Material and Cell Fractionation Patients with FL characterized by a BCL2-IgH rearrangement detectable by PCR were selected for study. BM aspirate samples were obtained with informed consent. Low-density BM cells were isolated by Ficoll gradient separation (Pharmacia Biotech (FR), St Quentin Yvelines, France). After removal of an aliquot for confirmation of PCR positivity, cells were labeled with anti-CD34-fluorescein isothiocyanate (HPCA-2; Becton Dickinson, San Jose, CA) and, in certain cases, anti-CD19-phycoerythrin (B4, Coulter clone; Coulter Immunology, Hialeah, FL) monoclonal antibodies. After washing, the CD34' or CD34' 19+,CD34- 19+,and CD34+19- fractions were isolated on a FACStar-Plus (Beckton Dickinson) equipped with an argon laser tuned at 488 nm. Purity of all cases was controlled by reanalysis of an aliquot of the sorted cells. DNA Preparation and BCLZ-IgH PCR Amplifcation DNA was isolated by classical techniques (proteinase-K digestion and phenol chloroform extraction) on diagnostic samples and by a rapid lysis technique on sorted subpopulations. In the latter case, cells were resuspended at 0.5 to 1.5 X lo4 cells/pL in 10 mmol/L Tris HC1 (pH 7.5), IO mmol/L KCI, 2.5 mmol/L MgCI2,0.1 mg/mL gelatine, 0.45% NP40, 0.45% Tween 20, and 0.45% Triton X-l00 and were incubated for 1 hour at 60°C in the presence of 0.2 mg/ mL proteinase K, followed by boiling for 10 minutes. PCR amplification of the t(14; 18) BCL2-IgH rearrangement was undertaken in the presence of 10 pL of lysed cells (0.5 to 1 .S X 10' cells whenever possible), 10 mmoVL Tris HCI (pH 8.3), 50 mmol/L KCI, 2 mmoll L MgCI2, 0.2 mmol/L dNTP, 0.2.5 pmol/L oligonucleotide primers specific for the MBR region of the BCL2 gene (5"TATGGTGGTTTGACCTTTAG-3') and the JH segments of the IgH gene (5"ACCTGAGGAGACGGTGACCAGGGT-3'),'y and 2 U Taq polymerase (Perkin Elmer/Cetus) in a total volume of 50 pL. Amplification was performed for 35 cycles on a Biometra Trio-Thennoblock apparatus (Biometra, Gottingen, Germany) with the following protocol: first denaturation step 3 minutes at 9 T C , 35 cycles at 92°C for 30 seconds, hybridization at 56°C for I minute, and polymerization at 72°C for I minute, increased to 10 minutes inthe last cycle. Nested amplification was performed for 35 cycles on 1 pL of PCR product using the same reaction conditions, but with internal MBR (5'-TTAGAGAGTTGCTTTACGTG-3') and JH (S'GACGGTGACCAGI TGGTClTICCCCKT'GCCC-3') primers and an annealing temperature of 50°C. Analysis of 13 pL of PCR product was undertaken after single and nested PCR by electropheresis on 8% polyacrylamide and visualization after ethidium-bromide staining. The sensitivity of the reaction was estimated by concurrent amplification of log dilutions of DNA from RL cells (a BCL2-IgH-rearranged cell line kindly provided byDr J. Gribben, Dana Farber Institute, Boston, MA) diluted in the HL60 myeloid cell line. In view of the variability in thenumber of cells analyzed in certain fractions andto exclude false-negative results caused by PCR inhibitors, the BCL2-IgH PCR results were compared with those obtained by PCR amplification of a single exon of the RAGI gene for 35 cycles, using oligonucleotide primers aspreviously described.20Inthe presence of amplifiable DNA, these primers generate a fragment of 364 bp. Confirmation of thespecificity of the amplified bands obtained was performed by transferring thePCR products from polyacrylamide to nylon membranes (Hybond-N; Amersham, Buckinghamshire, UK) by electrotransfer and hybridization with an internal "P BCL2 probe ( S ' CACAGACCCACCCAGAGCCC-3') labeled with polynucleotide kinase. RESULTS Patient Characterisitics Seven FL patients were analyzed, including one follicular small cell and five follicular mixed. Lymph node biopsy was not performed in one case (unique patient number [UPN] 469) because the patient presented in leukemic phase with 12 X 109L circulating lymphocytes. BM subpopulations were analyzed at diagnosis in five patients and at progressive disease in two (UPN 96 and 173). To maximize the probability of detecting BCL2-IgH-positive cells in the CD34 fraction, the majority of patients analyzed (5 of 7) were chosen for their high tumor mass and/or evident BM involvement (Table l). Morphological involvement was not observed in the BM aspirate analyzed in two patients, although both were PCR-positive (Table 2). In one of these patients (UPN 636), iliac crest BM biopsy performed 1 month earlier had shown infiltration by FL. All patients were shown to have a BCL2IgH rearrangement involving the MBR by PCR, confirmed onat least two samples from blood, BM, orlymph node biopsy specimens in all cases. As expected, the sizes of amplified products varied from approximately 170 to 280 bp after single-stage amplification and from 150 to 260 after internal amplification (Fig l). Detection of BCL2-&H in the CD34' BM Fraction In the first two cases, analyzed with progressive disease, we undertook to determine whether the bulk CD34' fraction contained the BCLZIgH rearrangement previously identified in the BM. Both patients showed BCL2-IgH by PCR in the CD34' fraction, albeit at a lower level than thatin the CD34fraction containing the mature B cells. Single-stage, external amplification, which repeatedly showed a sensitivity of detection of IO-* to or approximately 1 tumor cell in 100 to 1,000, was sufficient for detection of BCLZIgH in the CD34- fraction in both cases, whereas internal PCR (sensitivity to 10-') was required for detection of BCL2-IgH in the CD34' DNA. UPN 173 was consistently positive on repeat testing, whereas UPN 96 was positive after internal PCR in two of four reactions, thus suggesting positivity at the limit of detection corresponding to a Poisson distribution of BCLZIgH fragments in the cell lysate. Alternatively, the relative weakness of the BCLZIgH in this sample may be related to the lower levelRAG signal (Table 2). Inview of these findings, we undertook to determine whether the contamination of the BM fraction expressing CD34 corresponded to the subpopulation of cells that express both CD34 and CD19, ie, the BCPs. Quantification of BM Subpopulations The BM cells from the remaining five patients were aspirated within 1 month of diagnosis and sorted for the CD34'/ From www.bloodjournal.org by guest on December 22, 2014. For personal use only. BCL2- M143 F147 ND CD34'19' PROGENITORS IGH MARROW4693 IN FL Table 1. Clinical Details and Distribution of BM SubwDulations in FL Patients UPN 173 96 469 454 23 636 635 Sed Age fyr) F149 M148 MI64 MI62 MI53 BM Involvement* Yes* ND (30%) Yes Yes (45%) 100Yes (20%) 50 Yes (11%) No§ 95No CD34+ Total f%) 0.8 0.84 0.28 1.9 1.2 1.12 0.65 CD34'19+ CD34- Total (%kt (%H (%J ND ND ND 14 0 50 20 5 CD34'19- 40 86 80 36 CD34-19f%) CD34-19' (96) ND 32 ND 69 ND ND 26 7.7 4.7 1.3 0.7 14 35 43 42 35 35 37 33 All percentages, apart from the CD34' subfractions, refer to the proportion of the mononuclear cell population expressing a given marker. Sorted cells were initially selected on the basis of size and scatter. Abbreviations: M, male; F, female; ND, not done. Morphological infiltration of the sorted BM aspirate. t Expressed as the percentage of CD34' cells. Percentage infiltration not specified. 0 BM biopsy performed 1 month earlier showed infiltration. * CD19--immature fraction, the CD34+19+BCP fraction, and the CD34-19+ mature B-cell-containing fraction (Fig 2). Purity of the CD34+ fraction was always greater than 95%on reanalysis. The proportion of mononuclear cells expressing CD34 in all seven patients varied from 0.28% to 1.9% (mean, 1.0%), with the lowest figure being observed in the patient (UPN 469) with the highest level of infiltration by morphologically identified malignant cells. These figures are at the lower limit of those previously published (1% to 4%),8*9 presumably because of the infiltration by mature malignant B cells. However, the two patients without evident morphological involvement of the BM aspirate (UPN 635 and 636) did not show a higher proportion of CD34-positive cells. The proportion of CD34-19' cells, including the mature malignant cells, correlated, as expected, with the extent of BM infiltration, being highest in the case showing 45% FL cells and lowest in the two cases without evident infiltration (Table 1). The CD34+19+ BCPfraction showed lower level CD34 expression than did the CD34'19- fraction (Fig 2), as previously reported." The percentage ofCD19' cells within the CD34+ fraction was highly variable, ranging from 0% to 50% (mean, 18%; see Table l), and did not correlate with BM involvement. Preferential BCL2-IgH Detection in the CD34+19+ Subpopulation PCR detection of BCL2-IgH rearranged DNA was undertaken in the CD34'19- hemopoietic progenitor fraction, in the CD34+19+ BCP fraction, and in the CD34-19+ mature B-cell fraction. The latter subset was positive in four of five cases, including the two without evident infiltration, showing that this fraction contained mature tumor cells in the majority of cases (Table 2 and Fig 1). One case (UPN 23) was negative. Control amplification of the RAG gene, present in single Table 2. PCR Detection of BCU-lgH in BMCD34 Cells in FL PresorVDiagnosis' UPN 173 96 Ext Int ++ ++ CD34- CD34+ Ext Int - +C - +/kt Control Ext Int ++ + + ++ ++ CD34+19- 469 454 23 636 635 ++ ++ ++ + - Int Control Ext Int ++ +l- ++ ND + +I-* - - ND ND ND - - + - - - - ++ CD34-19+ CD34+19' Ext ++* +* Control + ++ ++ ++ +++I~ ++ ++ + Control Ext Int Control ++ ++ ++ - - +l-* ++ ++ ++ ++ ++ ++ +I - ++ ++ ++ A total of 0.5 to 1.5 x lo6 cells/PCR were analyzed unless otherwise stated. Int, nested PCR (sensitivity, to Control, RAG amplification; ND, not Abbreviations: Ext. single stage PCR (sensitivity, lo-' to done. In certain patients analyzed within 1 month of diagnosis, the BM sample positivity was not reconfirmed presort, and the results given are those obtained in the diagnostic sample. In the remaining patients, the diagnostic and presort samples gave identical results. t Positive in two of four independent PCR reactions. * A total of 2 x lo3 cells analyzed. § A total of 350 cells analyzed. From www.bloodjournal.org by guest on December 22, 2014. For personal use only. MACINTYRE ET AL 4694 INT. RAG tested, including the two cases without BM involvement (UPN 635 and 636; see Fig I). Therefore, these three cases tested were positive in the CD34'19' BCP fraction but negative in the CD34'19- immature fraction. The PCR positivity was detected after single-stage amplification in one patient and clearly and reproducibly detected after internal PCR in the two others. PCR analysis of sorted cells cannot exclude the possibility that the positivity observed in the BCP fraction is due to contamination by a small number of mature malignant B cells. The fact that the positivity was only observed in the BCP compartment and not in the CD34'/ CD19- population argues against this possibility, although the CD19 expression and the weaker positivity of CD34 in the BCP double-positive population (Fig 2) means that contamination is more likely in this compartment. Contamination of the CD34' 19- fraction would occur preferentially fromthe CD34-19- residual population. This population was shown to be positive in two of three patients tested (data I I Fig 1. PCR detection ofBCL2-lgH in the sorted BM subpopulations. Detection of BCL2-IgHrearrangementsafter single-stage (EXTI and nested (INTI amplificationwas undertaken in the sorted BM subpopulations and compared with thediagnostic sample(Dg) and with serial 10-fold dilutions 110" to of the BCL2-lgH-positive cell line, RL. PBL, PB lymphocyte-negativecontrol; H20, no DNA-negative control; mwm, molecular weight markers. Integrity of the cell lysates was assessed by amplification of sequences from the RAG1 gene. W UPN 469 o l2 0 0 10' copy and, therefore, indicative of the amount of amplifiable DNA present, showed minimal positivity in this case (Table 2), thus suggesting the presence of an inhibitor. CD34'19-. The CD34'19- fraction was BCL2-IgHnegative in four of five patients tested. Despite evident BM contamination in UPN 469, 454, and 23, this subpopulation was negative by both external and internal PCR in two patients, including UPN 23 who showed the highest CD34'19' content. Adequate amplification of the RAG control DNA from this fraction was observed in both cases. UPN 469 was weakly positive by external PCR and strongly so by nested PCR. This case contained veryfewCD34' cells (total, 0.28%) and a massive tumor load (45% morphological infiltration and 26% CD34-19' cells: see Fig 2); therefore, it is possible that the positivity observed in this fraction represents cellular contamination by mature tumor cells. Both CD34' 19- subpopulations from cases without evident infiltration were PCR-negative for BCL2-IgH by both external and internal PCR (Table 2 and Fig l). Therefore, these data show that, in the majority of patients, in a qualitative analysis in which the PCR efficacy reproducibly detects one tumor cell in 10.000 to 100,000, BCL2-IgH rearranged DNA cannot be shown in the CD34' fraction from which the CD19' BCPs have been excluded. CD34'19'. The number ofCD34'19' cells was insufficient for analysis in the two patients with the lowest number of cells in this subpopulation (UPN 469 and 454).The CD34'19' BCP fraction was positive in all three cases l00 100 10' 102 o l3 104 CD34-FITC UPN 23 l00 100 101 ld o l3 0 l4 CD34-FITC Fig 2. FACS profiles of sorted BM cells. BM mononuclearcells from two representative cases were sorted on the basisofCD34 ICD36fluorescein isothiocyanate)andlor CD19 (CD19-phycoerythrin) expression. The proportions of cells within thedifferent sorted fractions are detailed in Table 2. From www.bloodjournal.org by guest on December 22, 2014. For personal use only. CD34'19+ BCLZ- IGH MARROW PROGENITORS IN FL not shown). The BCP subpopulation represented 5% to 50% of the total CD34+ fraction in these three patients (Table 1). Therefore, it is likely that the positivity shown here would also have been detected if the total CD34-positive fraction containing both CD19- and CD19+ cells had been analyzed. In contrast, given the very low number of CD34+19+ cells in UPN 454, it is probable that the total CD34+ fraction would have been BCL2-IgH-negative, even if the rare CD34+19+ BCP cells were BCL2-IgH-positive. Thus, it is possible to estimate that, of the seven patients analyzed in this study, at least five (UPN 173, 96, 469, 23, and 636), and potentially also UPN 635, showed BCL2-IgH in the CD34-positive fraction. DISCUSSION This study shows that, in highly purifed FACS-sorted BM fractions from patients with BCL2-IgH-positive FL, the total CD34-positive fraction is positive in at least five of seven cases and that this positivity is found predominantly, or uniquely, in the CD34+19+ BCP fraction in the majority of cases. These results are in discordance with those recently described by Gorin et a l , 1 6 in which the majority (8 of 9) of informative FL samples studied were BCL2-IgH-positive in the CD34- and were negative in the CD34+ BM fractions, although the relative intensity of the signals observed was not discussed. These samples were all shown to have morphologically uninvolved BM on aspirate and biopsy at the time of analysis, although the majority had histological BM involvement at diagnosis, thus potentially explaining the different results. Therefore, in the present series, they correspond most closely to UPN 635 and, to a lesser extent, UPN 636. An alternative possible explanation for the negativity in the column-selected CD34+ fraction is that this technique selects only those cells expressing high levels of CD34 (CD34hi).Because we and others have shown that the BCP CD34+19+fraction expresses lower level CD34, column selection could select against CD34+19+BCPs." The demonstration that 11% (range, 1% to 69%) of CD34+ cells selected in the Gorin et all6 study expressed CD19, which is similar to the incidence observed here, argues against this explanation. On the other hand, the median purity of the CD34+ fractions analyzed by Gorin et all6 was only 49% (range, 12% to 80%), which is in keeping withthat reported by others."Inview of the data presented here, itwould be interesting to correlate the PCR results obtained with the proportions of CD34'19+ BCP and of CD34-19+ mature B cells in the individual CD34+ fractions analyzed. The aim of this study was twofold, (1) to assess a highly purified CD34-positive fraction in patients at high risk of tumor contamination in order to adequately address the appropriate issues in subsequent larger scale clinical studies and (2) to attempt to identify at which stage of B-lymphoid development the oncogenic BCL2-IgH rearrangement occurs. AS discussed above, it is unlikely that the positivity we have observed in the CD34+19+BCP population is purely because of contamination by mature B cells, although only in situ analysis will allow definitive demonstration that the BCL2-IgH rearrangement occurs in a CD34+ cell. However, for practical, therapeutic purposes, the purity of the fractions 4695 obtained here is markedly superior to that obtained from the majority of immunoadsorption columns, suggesting that these observations can be extrapolated to analysis of the populations obtained from the cell separators currently under evaluation. From a clinical point of view, the observation that the CD34'19-BM fraction was PCR-negative for BCL2-IgH in four of five patients tested, including in patients with massive tumor infiltration, whereas the CD34+19' BCP fraction was positive in all cases tested, suggests that, if reinfusion of tumor cells contibutes to relapse in FL, therapeutic strategies should be based not only on postive selection of CD34+ cells but also on additional negative purging of CD19+ cells. The recent demonstration of marked reduction in tumorigenicity in BCP acute lymphoblastic leukemia after CD 19-mediated immunotherapy provides encouraging data in support of such ~ t r a t e g i e sThe . ~ ~data presented here obviously do not address the issue of whether these BM subpopulations have tumorigenic potential. This would require analysis of the in vitro or in vivo growth characteristics of the sorted cells. Alternatively, because the CD34+19+ BCL2IgH-positive fraction reproducibly showed lower level CD34 expression than did the CD34+19- fraction, therapeutic selection strategies could include selection of only CD34hi cells. It is well established that the human hemopoietic cells able to initiate long-term cultures in human are exclusively found in the CD34hiBM fraction."." Although the number of patients included in this study is limited and merits confirmation in a larger series, the data presented here show that, at a minimum, all CD34-based therapeutic strategies should include evaluation of the percentage of CD34'19+ (and CD34-19') cells in the CD34' fraction and correlation of these parameters with the risk of relapse and the DFS. The marked variability in the proportion of CD34+ BM cells expressing CD19 (1% to 69%) observed by Gorin et all6 emphasizes the importance of monitoring these parameters. Furthermore, it will be important to determine whether the CD19+ BCP cells can be removed from the CD34+ fraction using available techniques without affecting engraftment. Several groups are currently evaluating the use of the PBSC CD34 fraction after mobilization with chemotherapy with or without growth factor. Unselected PBSCs from patients with FL have been shown to be BCL2-IgH-positive PCR by in the majority of patients The CD34 antigen is expressed on approximately 0.1% of circulating cells, with an increase to approximately 1% after mobilization. Several studies suggest that the proportion of CD34positive cells expressing CD19 is lower in PBSCs than in BM, both in healthy individuals and in patients, including a limited number of NHL Furthermore, mobilization with chemotherapy and growth factors increases the percentage of myeloid precursors, with a diminution in the proportion of CD34+/CD19+ cells to approximately Based on these findings and on the observations presented here, it is possible that the PBSC CD34-positive fraction may be less heavily contaminated by BCL2-IgH than its BM counterpart. However, the extreme variability in the proportion of BM CD34 cells expressing CD19 in lymphoma,16 as discussed above, suggests that incidence of CD34+19+BCPs should be assessed in a larger number of PBSC collections in FL. From www.bloodjournal.org by guest on December 22, 2014. For personal use only. 4696 From amore fundamental point of view,normal Blymphoid development starts in the BM CD34’ population with expression of CD19 and CD10 (pro-B cells), followed by cytoplasmic p heavy-chainexpression and surface expression of the pre-B-cell receptor, consisting of a p heavy chain and a surrogate light chain (I,!JLC).’~.” Antigen-independent selection, potentially mediated by the pre-B-cell receptor, may occur this at If appropriately selected, the BMcellsacquire surfaceIg (sIgM’) p heavy- and K or A light-chain expression(mature Bcells). During this developmentalprocessthe IgH locus undergoes first D-J rearrangement followed by complete V-DJ rearrangment during thepro-B stage, and, if sucessful,subsequently by light-chain rearrangement. Migration of mature B cells from the BM to the lymph nodes, where the processes of somatic mutation and antigen-drivenselection take place, occurs after V-D-J rearrangement. Positive and negative selection of BCPs remains incompletely understood, but it is probable that at least partof these processes occur in the BM independently from antigen-driven selection. The BCL2-IgH rearrangement represents an error of IgH rearrangement thatplaces BCL2 under the controlofthe IgHregulatory sequences, thusleading to itsderegulated expression andimmortalization of the clone viaits antiapoptotic effect34 but not directly to enhanced tumorgenicity. This may merely serve to preserve the clone until a further oncogenic eventoccurs.Theobservation that 50% ofhealthy individuals have low levels of BCL2-IgH rearrangement in the blood3’.’‘ is in keeping with this and indicates that the detection of a low level BCL2-IgH rearrangement, per se, maynotindicate contamination by malignant lymphoma cells. It is unlikely that the BCL2-IgH rearrangements we observed in the CD34’ 19’ fraction correspond to these low level “physiological” rearrangements; however, becausethe PCR products comigrated in all cases with those observed in pathological material analyzed at diagnosis. Alternatively, deregulated BCL2 expression may play a more activerole in disruption of theearly stages of Blymphoid development. Analysis of BCL2-IgH breakpoints has suggested that the BCL2-IgH rearrangement results from an initial recombination involving homologous chi-like sequences in MBR and the IgH D segments and subsequent modification of the rearranged BCL2-DHsequences by classical heptamer-nonamer-mediated recombinase activity, includingD-Jrearrangment.” Therefore,the initial translocationmaypreceed orconcurwith D-J and V-DJ rearrangement.The demonstration of BCL2-IgH rearranged DNA in the BM CD34’19’ fraction is in keeping with this possibility. If this is the case, it suggeststhattheinitial oncogenic event in the multistep development of FL occurs in theBMbefore migration to thelymph nodeandthat deregulated BCL2 expression could disrupt the early stages of pro- and pre-B-cell development. The effect of deregulated BCL2 expression on theearly stages of B-cell development is not clear, although, based on analysis of B lymphoid development in BCL2 transgenic SCID mice, it could potentially play a role in rescuing BCPs which do not show an appropriate pre-B-cell receptor from negative selection.3x The known association of FL and autoantibody formation may represent the clinical outcome of this. MACINTYRE ET AL Vescioet al have recentlyusedPCRamplificationof clone-specific IgH rearrangements to analyze theexpression of CD34onBMmalignantcells in multiple myeloma (MM).’’ These investigators showed that, in patients with significant BM infiltration, simple selection of CD34’ cells onanimmunoadsorption column led to a 1 3 - to 2.5-log reduction in tumor load, but residual positivity in all patients tested. However, further purification of the total CD34 fraction by FACS sorting led to loss of the tumor-specific IgH rearrangement in all cases, with an assay sensitivity comparable with that obtained with BCL2-IgH in the present study. Althoughanalysis of the CD34’CD19’ fractionwasnot performed in the Vescio study,comparison of these data suggests that the V-D-J tumormarker used in MM is restricted to a more mature BM population than BCL2-IgH in FL. This may reflect the possibility that the primary oncogeniceventsoccur in a more maturepopulation in MM than in FL. In keeping with this, MM tumor cells represent accumulation of a mature B-lymphoid clonethat has already In contrast, the malignant undergone somatic mutati~n.’”~” cells in FL represent expansion of a B-cell clone that is in the process of undergoing somatic mutation and, therefore, shows several subclones, each with different somatic mutations in the IgH V-D-J rearrangments. Alternatively, V-D-J rearrangements may merely represent a more mature tumor marker than BCL2-IgH rearrangement, as suggested above. To address this issue, it would be interesting to compare the the clonal V-D-J recapacity to detect BCL2-IgHand arrangementin the same CD34’ 19+and CD34’ 19- BM fractions in FL.However, detection of IgH V-D-J rearrangement in FL by PCR is hampered by the processes of continuing somatic mutation and subclone formation.4i In conclusion,the demonstration thathighlypurified CD34BMfractions in FLcontaintheBCL2-IgH tumor marker has important implications for the choice of autografting strategies in FL. Thepreferential positivity observed in the CD34’19’ BCP fraction suggests that additional negative purging of CD19’ cells should be considered if reduction of BCL2-IgH-positivecells is considered necessary andor desirable. These datashould aid the design of clinical protocols aiming to evaluate the impact of CD34 selection in the treatment of this disorder. ACKNOWLEDGMENT WethankPatriciaLetutour,DanielleOrsolani,andYvette Leboeuf for technical assistance; Narette Atkhen for providing patient material; Frederic Davi, Laurent Ferradini (Paris, France) and Gilles Salles(Lyon,France)forcriticalcomments;andMoniqueLillier for photographic assistance REFERENCES 1. Korsmeyer S: BcI-2 initiatesanewcategory of oncogenes: Regulators of cell death. Blood 80:879, 1992 2. Gribben JG, Freedman AS, Woo SD, Blake K, Shu RS, FreeGS, Nadler LM: All advanced stage man G, Longtine JA. Pinkus non-Hodgkin’s lymphomas with a polymerase chain reaction amplifiable breakpoint of bcl-2 have residual cells containing the bcl-2 rearrangementatevaluationandaftertreatment.Blood78:3275, 1991 3 . Gribben J, Neuberg D, Barber M, Moore J, Pesek K, Freedman From www.bloodjournal.org by guest on December 22, 2014. For personal use only. 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For personal use only. 1995 86: 4691-4698 Detection of clonal CD34+19+ progenitors in bone marrow of BCL2-IgH- positive follicular lymphoma patients EA Macintyre, C Belanger, C Debert, D Canioni, AG Turhan, M Azagury, O Hermine, B Varet, G Flandrin and C Schmitt Updated information and services can be found at: http://www.bloodjournal.org/content/86/12/4691.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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