REVIEW Clinical Relevance of Minimal Residual Disease Monitoring in Non

Clinical relevance of minimal residual disease monitoring in non-Hodgkin’s lymphomas: a critical reappraisal of molecular strategies P Corradini1, M Ladetto2, A Pileri2 and C Tarella2

1Bone Marrow Transplantation Unit, Istituto Scientiﬁco HS Raffaele, Milan; and 2Divisione Universitaria di Ematologia–Azienda Ospedaliera S Giovanni Battista, Turin, Italy

Although current treatments can induce clinical complete neoplasms. In the NHL setting, several studies have been pub- remissions in the vast majority of patients with indolent lym- lished on the prognostic signiﬁcance of minimal residual dis- phoma, most of them actually relapse, because of the persistence of residual tumor cells which are undetectable using con- ease (MRD) detection. Most of these studies focus on indolent ventional diagnostic procedures. Polymerase chain reaction lymphomas: small lymphocytic lymphoma/chronic lympho- (PCR)-based methods are increasingly used for minimal cytic leukemia (SLL/CLL),17,18 follicular (FCL)19–21 and mantle residual disease detection (MRD), and provide useful prognos- cell lymphomas (MCL).20,22 This is mostly because these tic information. In this review, current approaches for MRD tumors, unlike more aggressive NHL histotypes, are dissemi- detection in indolent lymphomas are summarized. In addition, nated disorders with frequent microscopic or submicroscopic the prognostic aspects of molecular monitoring after transplantation procedures are discussed. The experience accumulated bone marrow (BM) and peripheral blood (PB) involvement, over the past decade shows that PCR analysis has a prognostic and thus they represent ideal targets for MRD evaluation with impact in several therapeutic programs including conventional PCR-based assays.23,24 and high-dose regimens. Major advantages coming from the In this review, we summarize the characteristics of the most introduction of molecular monitoring in clinical programs have frequently used strategies for PCR detection of residual disease been: (1) a rapid evaluation of the anti-tumor activity of innov- in lymphoma patients. The clinical relevance of molecular ative treatments; and (2) an early identiﬁcation of patients with a high-risk of disease recurrence. monitoring after autologous or allogeneic transplantation will Keywords: minimal residual disease in lymphomas also be discussed.

Introduction PCR strategies for monitoring residual disease in indolent lymphomas During the last 10 years, the treatment of non-Hodgkin’s lymphoma (NHL) has dramatically changed.1,2 Major advances in The essential requirement for a PCR assay is the presence of the field have been the introduction of high-dose chemo- DNA sequences that are constantly detectable in the neoplas- therapy regimens followed by autologous or allogeneic trans- tic population and constantly undetectable in normal hemo- plantation of hematopoietic cells,3–8 the development of novel poietic cells. From these sequences primers and probes can drugs such as purine analogues9 and monoclonal anti- be designed and used for MRD detection. There are two bodies,10 and the use of innovative methods for ex vivo categories of tumor molecular markers: tumor-specific manipulation of autologous stem cells.11–19 Treatment evol- translocations, and antigen-receptor rearrangements. Tumor ution has been particularly remarkable in low grade subtypes, translocations have two major advantages: first, MRD detec- where palliative approaches have been progressively substi- tion is relatively simple since it involves only a PCR amplifi- tuted by treatments aimed at improving disease-free survival cation possibly followed by probe hybridization; second, and possibly curing at least patients younger than 60–65 these chromosonal aberrations are pathogenetically related to years. the neoplastic process, they represent a stable molecular Despite the introduction of these novel approaches, a size- marker, and they are usually not detected in normal cells. The able fraction of patients achieving clinical complete remission main disadvantage of translocation-based markers is that they actually relapse and die of their disease. The relapse is pre- provide a molecular marker only for a subset of patients. Such sumably caused by the persistence of small numbers of lym- a fraction is highly variable depending on the lymphoma sub- phoma cells that are below the limit of detection of standard type: FCLs harbor the t(14;18) translocation in approximately 60–80% of cases,19 whereas t(11;14) translocation is detect- diagnostic procedures. Therefore, considerable effort has been 22 made over the past decade to develop new techniques that able in 70–100% of MCL patients. Moreover, in several NHL allow the detection of extremely small numbers of tumor cells. subtypes no chromosomal translocation is presently available Among these techniques, the most sensitive and widely used for MRD evaluation. is the polymerase chain reaction (PCR). Its use has been exten- The rearrangement of immunoglobulin heavy-chain (IgH) sive in several hematological neoplasms, and the persistence and T cell receptor genes (TCR), has a wider applicability and of PCR detectable tumor cells has been correlated with can be used as molecular markers in the vast majority of increased likelihood of relapse in both myeloid and lymphoid lymphoid tumors. However, their use for PCR detection of MRD is somehow more complex than chromosomal translocations since it requires the sequencing analysis of rearranged variable region in order to design tumor-specific primers and Correspondence: P Corradini, Bone Marrow Transplantation Unit, Isti- 25,26 tuto Scientifico HS Raffaele, Via Olgettina 60, 20132 Milano, Italy; probes. In addition, clonal evolution is a potential prob- 27 Fax: 0039 02 26434760 lem associated with the use of IgH or TCR rearrangements. Received 5 May 1999; accepted 9 July 1999 Clonal evolution, however, is a phenomenon peculiar to more Review P Corradini et al 1692 immature disorders such as acute lymphoblastic leukemia, reproducible methods for quantitative analysis of MRD is and it does not take place in mature lymphoid tumors such required. This is of particular interest in evaluating the kinetic as indolent lymphomas and myelomas. of the tumor clone in those diseases in which the persistence So far, the three most widely used clonal markers in NHL of PCR detectable tumor cells is frequently observed with stan- patients are the t(14;18), the t(11;14) and the IgH gene dard qualitative methods. Traditional quantitative and semi- rearrangement. The t(14;18) was the first lesion routinely quantitative PCR approaches of end-product DNA analysis, employed for MRD analysis in NHL and it is detectable in limiting dilution strategies and competitive PCR are labour approximately 60–80% of FCL patients and in 20% of diffuse intensive, show a limited dynamic range and often give non- large cell lymphomas.28,29 In this translocation, the Bcl-2 reproducible results, and thus are of limited applicability in proto-oncogene on chromosome 18 is juxtaposed to the IgH the clinical setting.34 Recently, the introduction of the TaqMan locus on chromosome 14. Breakpoints are located at two technology and the development of analytical thermal cyclers main clusters 3Ј to the Bcl-2 coding region, named major have allowed the development of extremely powerful systems breakpoint region (MBR) and minor cluster region (mcr).30,31 for quantitative analysis. These methods are highly accurate, The t(14;18) translocation upregulates expression of the Bcl-2 mostly because they allow direct measurement of quantitative gene product that induces prolonged cell survival by blocking data at the beginning of the exponential phase of PCR ampli- programed cell death. The two breakpoint regions require dif- fication and thus eliminate biases taking place during late ferent sets of primers for PCR amplification. MRD evaluation post-exponential phases of the reaction and during post-PCR is performed by PCR amplification of the junctional region manipulation of samples. Several different tumor translo- using a 5Ј primer derived from the Bcl-2 locus and a 3Ј primer cations including the t(14;18) as well as the IgH rearrange- derived from the JH region of IgH genes. PCR products can ment35–37 have been used as targets for MRD detection using be hybridized to a Bcl-2 specific probe or, alternatively, TaqMan-based approaches. However, large studies, reamplified using a second set of internal primers: the so- investigating the prognostic relevance of quantitative called nested-PCR. Both hybridization and nested-PCR are evaluation of MRD in lymphoid tumours, are still awaited. extremely sensitive and specific techniques and can routinely detect one lymphoma cell in 105–106 normal cells.20,21 The t(11;14) fuses the Bcl-1 locus with the IgH locus and Molecular analysis as a tool to assess the efficacy of in is detected by cytogenetic or Southern blot analysis in the vast vitro purging procedures majority of MCL patients (70–100%). This translocation acti- vates the cyclin D1 (PRAD-1/CCND1) gene. Cyclin D1 is a High-dose chemotherapy regimens followed by autologous G1 cyclin that participates in the control of cell cycle pro- transplantation with BM or peripheral blood progenitor cells gression at the G1- to S-phase transition. Overexpression of (PBPC) are increasingly used for the treatment of indolent lym- this cyclin induces a shortened G1 phase and may function phomas.3,4,6–8 However, since these neoplasms have a high as an oncogene, cooperating with other oncogenic factors in degree of PB and BM involvement, stem cell grafts are often cellular transformation. Breakpoints on chromosome 11 have contaminated by occult tumor cells that can contribute to dis- been found dispersed over more than 100 kb of genomic ease relapse.38 In vitro purging of stem cell grafts has been DNA. Three clusters have been identified so far; the most regarded as a potentially useful method to obtain a significant important is named the major translocation cluster and con- reduction and possibly the eradication of contaminating tumor tains approximately 30–50% of these translocations. This cells. PCR analysis has been extensively used in this setting: DNA region represents a suitable target for PCR amplification. the efficacy of purging procedures in different diseases has Because of the large breakpoint areas, less than 30% of MCL been evaluated giving the percentage of patients in which patients will have a rearrangement that can be detected by grafts were successfully cleared from residual lymphoma cells. PCR.22,32 PCR detection of the t(11;14) is usually performed In addition, correlations, between infusion of PCR-negative employing semi-nested PCR: two consecutive amplifications grafts and disease-free survival, have been investigated.19,20,24 are performed, using the same 3Ј primer derived from the JH Investigators from Dana Farber Cancer Institute, Boston, region and two 5Ј primers derived from the major translo- have pioneered large-scale in vitro purging techniques: anti- cation cluster of the Bcl-1 locus. This approach can usually B cell monoclonal antibodies together with complement- detect one lymphoma cell out of 105 normal cells.22 mediated lysis have been used to purge marrow grafts from The IgH gene rearrangement has been used in different sub- patients with FCL, SLL/CLL and MCL.18,23,24 Significant differ- types of NHL, including SLL/CLL, FCL and MCL.17,18,22,25,26,33 ences between lymphoma subtypes were observed: in FCL This method relies on the amplification and sequencing of the residual tumor cells became undetectable in approximately IgH rearranged variable region (VDJ). VDJ amplification is per- 50% of patients, and the reinfusion of a PCR-negative graft formed using consensus primers derived from the leader or was correlated with a better disease-free survival.24 A similar framework regions of IgH genes.26 Amplified VDJs are then finding has been observed in a panel of 21 SLL/CLL patients: sequenced to identify the three complementarity-determining a correlation between the reinfusion of PCR-positive grafts and regions (CDRs). These regions code for the antigen-binding increased likelihood of relapse has been documented.18 How- site and are unique to each B cell clone. From the CDR2 and ever, very recent data on a larger panel of SLL/CLL patients CDR3, oligonucleotide primers and probes are usually seem to provide less optimistic results with more than 75% designed. This method can provide a tumor marker in of patients still harboring residual tumor cells after in vitro approximately 80% of lymphoma and myeloma patients. PCR purging.39 The same purging procedure has been applied to analysis using the IgH rearrangement has specificity and sensi- MCL patients, giving unsatisfactory results. Marrow grafts tivity similar to translocation-based assays.26 resulted virtually always PCR-positive after the in vitro treat- A potential limitation of PCR detection of residual disease ment with monoclonal antibodies.22 is the qualitative and not quantitative nature of the analysis. It should be pointed out, however, that the use of in vitro Although qualitative analysis has been extremely useful in purging for FCL patients is still a matter of controversy. Pappa several clinical settings, the development of accurate and et al21 have used in vitro purging for FCL patients, and they Review P Corradini et al 1693 have shown a clearance of PCR-detectable lymphoma cells in ston reported a high rate of molecular remissions after conven- less than 15% of cases. There is not a clear explanation for tional-dose chemotherapy (66%). They could demonstrate a these conflicting findings. Both differences between purging correlation between molecular response during the first year procedures, and sensitivity/specificity of PCR assays have of therapy and failure-free survival.43 It is somehow difficult been postulated. to compare these results with those obtained after purged or Although most purging studies in NHL were performed unpurged autografting. In the MD Anderson study, the using negative selection, other purging techniques have been achievement of molecular response was to a certain extent recently developed. Positive selection of CD34+ cells has independent of the clinical response: one-third of patients in recently gained considerable interest. Some studies have CR became PCR-negative, and one-third of patients in partial shown that CD34+ selection was successful in eradicating remission became PCR-negative as well. In contrast, the data residual lymphoma cells from both PBPC and BM grafts in from autografting studies derive from CR patients only. A FCL patients.14,40 In contrast, CD34+ selection was largely recent report from Czuczman et al44 has opened a novel field unsuccessful in SLL/CLL patients.17 Thus, both negative and of investigation: they have shown that conventional chemo- positive selection methods proved to be successful in eradicat- therapy in combination with anti-CD20 chimeric antibody is ing PCR-detectable disease in a significant proportion of FCL able to produce durable clinical and molecular remissions. patients. Comparative studies are probably required to define The interesting point is that conventional chemotherapy with which approach should be considered more appropriate and or without monoclonal antibodies may be able to produce cost-effective. molecular remissions. A careful long-term monitoring is required to ascertain the clinical relevance of molecular data in this setting. Randomized trials between conventional and Role of molecular monitoring in the prediction of relapse high-dose chemotherapy are probably required. following conventional and high-dose chemotherapy Molecular monitoring has been also performed in MCL and treatments SLL/CLL patients. These studies, however, have involved a smaller panel of patients. In SLL/CLL Provan et al showed a An important application of MRD assays is the molecular pattern similar to that observed in FCL, but with less satisfac- monitoring of patients achieving complete remission. The tory results.18 Molecular follow-up data from SLL/CLL patients term ‘complete remission’ is used to define a category of undergoing high-dose chemotherapy followed by autografting patients in whom there is no evidence of disease using stan- of unmanipulated PBPC were quite disappointing. Although dard laboratory and clinical procedures. This condition clinical complete remissions were frequently achieved, the encompasses different clinical situations including complete persistence of PCR positivity after transplantation represented eradication of the tumor clone, long-term persistence of tumor a nearly constant finding.45 In the MCL subtype, the vast cells with no tendency to relapse, and presence of residual majority of patients showed persistent PCR positivity after tumor cells giving rise to overt relapse during the subsequent transplantation using both purged or unpurged autograft- follow-up period. While conventional approaches are unable ing.20,22 These data are consistent with the clinical finding that to distinguish between these situations, sequential analysis of these patients are not cured by autologous transplantation and MRD has provided novel insights on the behavior of the actually relapse. Preliminary results, in a novel strategy com- residual neoplastic population and allowed the identification bining high-dose chemotherapy and in vivo administration of of different prognostic subgroups. The studies with the longest chimeric anti-CD20 have been recently reported. PCR-nega- observation period have been performed in FCL patients tive PBPC grafts have been harvested, but the small number undergoing autologous transplantation with purged BM of patients and the short follow-up do not allow any definitive cells.19 PCR analysis was performed serially on BM and PB conclusions to be drawn as yet.46 samples obtained at different time-points after transplantation. Allogeneic transplantation has recently gained considerable Three major patterns of disease evolution have been observed: interest as salvage treatment for patients with relapsed or (1) some patients displayed persistent PCR positivity and sub- refractory indolent lymphomas. It has recently been published sequently relapsed; (2) others resulted PCR-negative immedi- that a significant proportion of these patients experiences a ately after autografting and then regained a PCR-positive prolonged disease-free survival.47,48 Unfortunately, few mol- status, and they are at high-risk for relapse; and (3) other ecular data are available for this group of patients. Several patients had a persistent PCR-negative status or achieved mol- long-term survivors experience prolonged clinical and molecular remission shortly after; these patients are characterized ecular remissions even among MCL patients.18,22,49 In the near by a low relapse rate.19 future, we expect a wider application of allogeneic transplan- A similar correlation between molecular monitoring and tation for the treatment of indolent lymphomas possibly disease-free survival has also been observed in patients associated with treatments aimed at modulating the graft-ver- undergoing high-dose chemotherapy followed by autografting sus-tumor immune response. There is little doubt that PCR- of unmanipulated PBPC. We have shown that patients, har- based monitoring of MRD, using both qualitative and quanti- vesting PCR-negative BM and/or PBPC grafts, displayed a per- tative methods, will be extremely helpful to evaluate the effec- sistent PCR negativity after transplantation. Among these tiveness of such treatments and to tailor specific therapeutic patients none have relapsed, while disease recurrences were modalities for patients at increased risk of relapse (ie frequently observed among patients with persistent PCR posi- programmed reinfusions of donor lymphocytes).50 tivity.20 Similar results in terms of correlation between PCR positivity and high incidence of relapse were also observed by Hardingham et al.41 In addition, Moos et al42 have shown Conclusion and future perspectives that the PCR status before autografting and the PCR status assessed at follow-up examinations are significant prognostic The treatment of indolent lymphomas is a rapidly evolving parameters for relapse-free survival in patients with FCL. field and novel treatments are constantly under evaluation. A recent report from the MD Anderson Cancer Center, Hou- However, the prolonged natural history of these neoplasms is Review P Corradini et al 1694 a potential obstacle to the rapid development of innovative References therapeutic strategies since several years are usually required 1 Armitage JO. Bone marrow transplantation for indolent lym- in order to draw definitive clinical conclusions on a given phomas. Semin Oncol 1993; 20: 136–142. treatment. In recent years, considerable experience has been 2 Coiffier B. Towards a cure in indolent lymphoproliferative dis- accumulated on PCR-based evaluation of MRD, and the cor- eases? Eur J Cancer 1995; 31A: 2135–2137. relation between PCR status and disease-free survival is now 3 Coiffier B, Philip T, Burnett AK, Symann ML. Consensus Confer- consolidated. Since PCR analysis can rapidly provide indi- ence on Intensive Chemotherapy Plus Hematopoietic Stem-Cell cations, about the effectiveness of innovative treatments, the Transplantation in Malignancies: Lyon, France, June 4–6, 1993. J Clin Oncol 1994; 12: 226–231. introduction of ‘molecular end-points’ in clinical studies 4 Freedman AS, Gribben JG, Neuberg D, Mauch P, Soiffer RJ, Ander- should be strongly encouraged in order to warrant a rapid son KC, Pandite L, Robertson MJ, Kroon N, Ritz J, Nadler LM. evaluation of novel therapeutic strategies. In addition, it is High-dose therapy and autologous bone marrow transplantation now clear that molecular remission represents a prognostic in patients with follicular lymphoma during first remission. Blood factor because it is a sort of measurement of the reduction of 1996; 88: 2780–2786. tumor burden. A similar consideration can be done for the 5 Gianni AM, Bregni M, Siena S, Brambilla C, Di Nicola M, Lom- bardi F, Gandola L, Tarella C, Pileri A, Ravagnani F, Valagussa evaluation of in vitro purging procedure: molecular analysis P, Bonadonna G. High-dose chemotherapy and autologous bone must be included in any protocol of in vitro manipulation of marrow transplantation compared with MACOP-B in aggressive stem cells. Another potentially important application of mol- B-cell lymphoma. New Engl J Med 1997; 336: 1290–1297. ecular analysis is the early treatment of patients who show 6 Philip T, Guglielmi C, Hagenbeek A, Somers R, Van der Lelie H, persistence (or increase, if quantitative approaches are used) Bron D, Sonneveld P, Gisselbrecht C, Cahn JY, Harousseau JL. of molecularly detectable disease, and thus are at high-risk of Autologous bone marrow transplantation as compared with salvage chemotherapy in relapses of chemotherapy-sensitive non- relapse. Several treatments including adoptive immunother- Hodgkin’s lymphoma. New Engl J Med 1995; 333: 1540–1545. apy, vaccination strategies and modulation of graft-versus-host 7 Bastion Y, Brice P, Haioun C, Sonet A, Salles G, Marolleau JP, disease will probably show greater effectiveness if used to Espinouse D, Reyes F, Gisselbrecht C, Coiffier B. Intensive therapy treat small residual tumor burdens instead of overt relapses. with peripheral blood progenitor cell transplantation in 60 patients This approach is already under evaluation in several hematol- with poor-prognosis follicular lymphoma. Blood 1995; 86: ogical tumors including chronic myelogenous leukemia and 3257–3262. 51,52 8 Bierman PJ, Vose JM, Anderson JR, Bishop MR, Kessinger A, Armit- acute promyelocytic leukemia. age JO. High-dose therapy with autologous hematopoietic rescue A potential problem associated with a more widespread for follicular low-grade non-Hodgkin’s lymphoma. J Clin Oncol use of PCR-based techniques is the lack of international 1997; 15: 445–450. standardization. Since small differences in assay 9 Wright SJ, Robertson LE, O’Brien S, Plunkett W, Keating MJ. The sensitivity/specificity can easily introduce significant biases in role of fludarabine in hematological malignancies. Blood Rev the interpretation of results, great caution should be used in 1994; 8: 125–134. 10 Leget G, Czuczman MS. Use of rituximab, the new FDA-approved comparing data obtained from different institutions. Centraliz- antibody. Curr Opin Oncol 1998; 10: 548–551. ation of MRD analysis should also be considered mandatory 11 Bensinger WI. Should we purge? Bone Marrow Transplant 1998; in multicentric studies, in order to obtain fully evaluable 21: 113–115. results. In future years, efforts should be undertaken to assure 12 Gee A. Purging of peripheral blood stem cell grafts. Stem Cells greater methodological homogeneity among different centers 1995; 13: 52–62. involved in molecular studies. In this view, a ‘consensus con- 13 Gorin NC, Lopez M, Laporte JP, Quittet P, Lesage S, Lemoine F, Berenson RJ, Isnard F, Grande M, Stachowiak J. Preparation and ference’ to define common guidelines for PCR assays could successful engraftment of purified CD34+ bone marrow progenitor represent a major progress in the field. cells in patients with non-Hodgkin’s lymphoma. Blood 1995; 85: The impact of PCR analysis in the management of indolent 1647–1654. lymphomas has been strong and therapeutic evolution in this 14 Lopez M, Lemoine F, Firat H, Fouillard L, Laporte JP, Lesage S, field has been greatly supported by data obtained from mol- Isnard F, Stachowiak J, Ferrer-Le Coeur F, Morel P, Najman A, ecular studies. However, it should be stressed that the value Douay L, Gorin NC. Bone marrow versus peripheral blood progenitor cells CD34 selection in patients with non-Hodgkin’s lym- of PCR negativity will change after the introduction of anti- phomas: different levels of tumor cell reduction. Implications for CD20 monoclonal antibody. The absence of circulating PCR autografting. Blood 1997; 90: 2830–2838. detectable cells may have a different clinical relevance when 15 Lowenberg B, Voogt P. Autologous stem-cell transplantation and obtained after months of high-dose chemotherapy or after a purging (editorial). J Clin Oncol 1996; 14: 2194–2196. single infusion of anti-CD20. New studies are awaited to vali- 16 Gribben JG. Attainment of molecular remission: a worthwhile date the prognostic value of PCR analysis in these novel thera- goal? J Clin Oncol 1994; 12: 1532–1534. 17 Scime` R, Indovina A, Santoro A, Musso M, Olivieri A, Tringali S, peutic settings. It cannot be excluded that PCR analysis will Crescimanno A, Montanari M, Felice R, Catania P, Mariani G, need to be partially readapted in order to address these Leoni P, Majolino I. PBSC mobilization, collection and positive future issues. selection in patients with chronic lymphocytic leukemia. Bone Marrow Transplant 1998; 22: 1159–1165. 18 Provan D, Bartlett-Pandite L, Zwicky C, Neuberg D, Maddocks A, Corradini P, Soiffer R, Ritz J, Nadler LM, Gribben JG. Eradication of polymerase chain reaction-detectable chronic lymphocytic leukemia cells is associated with improved outcome after bone mar- Acknowledgements row transplantation. Blood 1996; 88: 2228–2235. 19 Gribben JG, Neuberg D, Freedman AS, Gimmi CD, Pesek KW, Barber M, Saporito L, Woo SD, Coral F, Spector N, Rabinowe SN, We are grateful to the staff of the Molecular Hematology Lab- Grossbard ML, Ritz J, Nadler LM. Detection by polymerase chain oratory and in particular to Monica Astolfi, PhD and Claudia reaction of residual cells with the bcl-2 translocation is associated with increased risk of relapse after autologous bone marrow trans- Voena, PhD. This work was in part supported by AIRC plantation for B-cell lymphoma. Blood 1993; 81: 3449–3457. (Associazione Italiana Ricerca sul Cancro), and CNR special 20 Corradini P, Astolfi M, Cherasco C, Ladetto M, Voena C, Caracci- project ACRO grant No. 96.00.742.PF39 to CT. olo D, Pileri A, Tarella C. Molecular monitoring of minimal Review P Corradini et al 1695 residual disease in follicular and mantle cell non-Hodgkin’s lym- 38 Brenner MK, Rill DR, Holladay MS, Heslop HE, Moen RC, Buschle phomas treated with high-dose chemotherapy and peripheral M, Krance RA, Santana VM, Anderson WF, Ihle JN. Gene marking blood progenitor cell autografting. Blood 1997; 89: 724–731. to trace origin of relapse after autologous bone marrow transplan- 21 Pappa V, Wilkes S, Salam A, Young B, Lister T, Rohatiner A. Use tation. Lancet 1993; 341: 85–90. of the polymerase chain reaction and direct sequencing analysis 39 Donovan JW, Andersen NS, Poor CM, Bowers D, Gribben JG. to detect cells with the t(14;18) in autologous bone marrow from Prospective analysis of minimal residual disease detection in patients with follicular lymphoma, before and after in vitro treat- patients with CLL undergoing autologous and allogeneic BMT. ment. Bone Marrow Transplant 1998; 22: 553–558. Blood 1998; 92: 652a. 22 Andersen NS, Donovan JW, Borus JS, Poor CM, Neuberg D, Aster 40 Di Nicola M, Siena S, Corradini P, Bregni M, Ruffini PA, Milanesi JC, Nadler LM, Freedman AS, Gribben JG. Failure of immunologic M, Tarella C, Gianni AM. Elimination of bcl-2-IgH positive follicu- purging in mantle cell lymphoma assessed by polymerase chain lar lymphoma cells from blood transplants with high recovery of reaction detection of minimal residual disease. Blood 1997; 90: hematopoietic progenitors by the Miltenyi CD34+ cell sorting sys- 4212–4221. tem. Bone Marrow Transplant 1996; 18: 1117. 23 Berinstein NL, Reis MD, Ngan BY, Sawka C, Jamal HH, Kuzniar B. 41 Hardingham JE, Kotasek D, Sage RE, Gooley LT, Mi JX, Dobrovic Detection of occult lymphoma in the peripheral blood and bone A, Norman JE, Bolton AE. Significance of molecular marker-posi- marrow of patients with untreated early-stage and advanced-stage tive cells after autologous peripheral-blood stem-cell transplan- follicular lymphoma. J Clin Oncol 1993; 11: 1344–1352. tation for non-Hodgkin’s lymphoma. J Clin Oncol 1995; 13: 24 Gribben JG, Freedman AS, Neuberg D, Roy DC, Blake KW, Woo 1073–1079. SD, Grossbard ML, Rabinowe SN, Coral F, Freeman GJ. Immuno- 42 Moos M, Schulz R, Martin S, Haas R. The remission status before logic purging of marrow assessed by PCR before autologous bone and the PCR status after high-dose therapy with peripheral blood marrow transplantation for B-cell lymphoma. New Engl J Med stem cell support are prognostic factors for relapse-free survival in 1991; 325: 1525–1533. patients with follicular non-Hodgkin’s lymphoma. Leukemia 25 Corradini P, Voena C, Astolfi M, Ladetto M, Tarella C, Boccadoro 1998; 12: 1971–1976. M, Pileri A. High-dose sequential chemoradiotherapy in multiple 43 Lopez-Guillermo A, Cabanillas F, McLaughlin P, Smith T, Hage- myeloma: residual tumor cells are detectable in bone marrow and meister FB, Rodriguez MA, Romaguera MA, Sarris A, Pugh WC, peripheral blood cell harvests and after autografting. Blood 1995; Lee MS. The clinical significance of molecular response in indol- 85: 1596–1602. ent follicular lymphomas. Blood 1998; 91: 2955–2960. 26 Voena C, Ladetto M, Astolfi M, Provan D, Gribben JG, Boccadoro 44 Czuczman MS, Grillo-Lopez AJ, White CA, Saleh M, Gordon L, M, Pileri A, Corradini P. A novel nested-PCR strategy for the detec- LoBuglio AF, Jonas C, Klippenstein D, Dallaire B, Varns C. Treat- tion of rearranged immunoglobulin heavy-chain genes in B cell ment of patients with low-grade B-cell lymphoma with the combi- tumors. Leukemia 1997; 11: 1793–1798. nation of chimeric anti-CD20 monoclonal antibody and CHOP 27 Choi Y, Greenberg SJ, Du TL, Ward PM, Overturf PM, Brecher chemotherapy. J Clin Oncol 1999; 17: 268–276. ML, Ballow M. Clonal evolution in B-lineage acute lymphoblastic 45 Corradini P, Astolfi M, Voena C, Caracciolo D, Vitolo U, Benedetti leukemia by contemporaneous VH–VH gene replacements and G, Pileri A, Tarella C. High-dose chemotherapy and PBPC auto- VH–DJH gene rearrangements. Blood 1996; 87: 2506–2512. grafting inducedurable molecular remissions in follicular lym- 28 Lee MS, Chang KS, Cabanillas F, Freireich E, Trujillo J, Stass SA. phomas but not in small lymphocytic and mantle cell lymphomas. Detection of minimal residual cells carrying the t(14;18) by DNA Blood 1999; 92: 463a. sequence amplification. Science 1999; 237: 175–178. 46 Gianni AM, Magni M, Di Nicola M, Gandola L, Lombardi F, Das- 29 Bakhshi A, Jensen JP, Goldman P, Wright JJ, McBride OW, Epstein toli G, Matteucci P, Devizzi L, Bregni M, Campana S, Corradini AL, Korsmeyer SJ. Cloning the chromosomal breakpoint of t(14;18) P, Pileri A, Tarella C. In vivo purging of circulating CD34+ pro- human lymphomas: clustering around JH on chromosome 14 and genitor cells in low-grade lymphoma with Rituximab and high- near a transcriptional unit on 18. Cell 1985; 41: 899–906. dose chemotherapy. Blood 1999; 92: 119a. 30 Cleary ML, Galili N, Sklar J. Detection of a second t(14;18) break- 47 van Besien K, Sobocinski KA, Rowlings PA, Murphy SC, Armitage point cluster region in human follicular lymphomas. J Exp Med JO, Bishop MR, Chaekal OK, Gale RP, Klein JP, Lazarus HM, 1986; 164: 315–320. McCarthy PL, Raemaekers JM, Reiffers J, Phillips GL, Schattenberg 31 Cleary ML, Sklar J. Nucleotide sequence of a t(14;18) chromo- AV, Verdonk LF, Vose JM, Horowitz MM. Allogeneic bone mar- somal breakpoint in follicular lymphoma and demonstration of a row transplantation for low-grade lymphoma. Blood 1998; 92: breakpoint-cluster region near a transcriptionally active locus on 1832–1836. chromosome 18. Proc Natl Acad Sci USA 1985; 82: 7439–7443. 48 Verdonk LF, Dekker AW, Lokhorst HM, Petersen EJ, Nieuwenhuis 32 Tsujimoto Y, Yunis J, Onorato-Showe L, Erikson J, Nowell PC, HK. Allogeneic versus autologous bone marrow transplantation Croce CM. Molecular cloning of the chromosomal breakpoint of for refractory and recurrent low-grade non-Hodgkin’s lymphoma. B-cell lymphomas and leukemias with the t(11;14) chromosome Blood 1997; 10: 4201–4205. translocation. Science 1984; 224: 1403–1406. 49 Corradini P, Ladetto M, Astolfi M, Voena C, Tarella C, Bacigalupo 33 Zwicky C, Maddocks A, Andersen NS, Gribben JG. Eradication of A, Pileri A. Clinical and molecular remission after allogeneic polymerase chain reaction detectable immunoglobulin gene blood cell transplantation in a patient with mantle-cell lymphoma. rearrangement in non-Hodgkin’s lymphoma is associated with Br J Haematol 1996; 94: 376–378. decreased relapse after autologous bone marrow transplantation. 50 Mandingers CMPW, Meijerink JPP, Raemaekers JMM, Schatten- Blood 1996; 88: 3314–3322. berg AVMB, Mensink EJBM. Graft-versus-lymphoma effect of 34 Orlando C, Pinzani P, Pazzagli M. Developments in quantitative donor leucocyte infusion shown by real-time quantitative PCR PCR. Clin Chem Lab Med 1998; 36: 255–269. analysis of t(14;18). Lancet 1998; 352: 1522–1523. 35 Luthra R, McBride JA, Cabanillas F, Sarris A. Novel 5Ј exonucle- 51 Cross NC. Minimal residual disease in chronic myeloid leu- ase-based real-time PCR assay for the detection of kaemia. Hematol Cell Ther 1998; 40: 224–228. t(14;18)(q32;q21) in patients with follicular lymphoma. Am J 52 Diverio D, Rossi V, Avvisati G, DeSantis S, Pistilli A, Pane F, Saglio Pathol 1998; 153: 63–68. G, Martinelli G, Petti MC, Santoro A, Pelicci PG, Mandelli F, 36 Gerard CJ, Olsson K, Ramanatham R, Reading C, Hanania EG. Biondi A, Lo Coco FL. Early detection of relapse by prospective Improved quantitation of minimal residual disease in multiple reverse transcriptase-polymerase chain reaction analysis of the myeloma using real-time polymerase chain reaction and plasmid- PML/RARalpha fusion gene in patients with acute promyelocytic DNA complementarity determining region III standards. Cancer leukemia enrolled in the GIMEMA-AIEOP multicenter ‘AIDA’ trial. Res 1998; 58: 3957–3964. GIMEMA-AIEOP Multicenter ‘AIDA’ Trial. Blood 1998; 92: 37 Ladetto M, Donovan JW, Schlossman R, Anderson KC, Gribben 784–789. JG. Limitations of quantitative detection of immunoglobulin heavy chain (IgH) gene rearrangements in multiple myeloma (MM) patients using real-time PCR. Blood 1998; 92: 286a.