BRIEF COMMUNICATION Evaluation of Minimal Residual

Evaluation of minimal residual disease by interphase FISH in multiple myeloma: does complete remission exist?

F Genevieve1, M Zandecki1, J-L Laı¨2, B Hennache3, J-L Faucompre3, L Stalnikiewicz4, F Bauters4 and T Facon4

1Laboratoire d’He´matologie, Angers; 2Laboratoire de Ge´ne´tique, Lille; 3Laboratoire de Biochimie, Lille; and 4Service des Maladies du Sang, Hoˆpital Huriez, Lille, France

As in other hematological malignancies, the achievement of a apparent complete remission after high-dose therapy complete remission (CR) is important in multiple myeloma but (autologous stem cell transplantation, 11 patients; high-dose is still based on common cytological and electrophoretic cri- 2 teria. In this report, we studied 14 patients who achieved an melphalan 140 mg/m , three patients) and who had sufficient apparent CR following high-dose therapy using fluorescence material for FISH analysis. Tumor mass was assessed by the in situ hybridization (FISH) analysis. Although the results were Durie and Salmon classification.7 Six patients were male and difficult to interpret in two patients, 12 of 14 patients had median age was 54 years (range 38–65). The M-component unequivocal persistence of abnormal plasma cells in their bone was G kappa in four patients, G lambda in four patients, A marrow. Our results suggest that only a few patients, if any, lambda in four patients and lambda light chain in two are in true CR following one course of high-dose therapy and are in favor of post-transplantation treatments. patients. Staging was IA (symptomatic) in two patients, IIA in Keywords: multiple myeloma; complete remission; autologous one patient, IIIA in eight patients and IIIB in three patients. stem cell transplantation; fluorescence in situ hybridization; cytogen- After treatment (2–21 months, median 4.5 months), electro- etics; plasma cells phoresis and immunofixation of serum and urine were performed to assess biochemical remission at the time of bone marrow FISH analysis. Bone marrow plasmacytosis ranged from Introduction 0.1% to 3.5% (median 0.6%, 1000 cells enumerated). No patient had a detectable M-component on electropheresis. Complete remission (CR) in multiple myeloma (MM) is com- Nevertheless, only four patients had a negative immunofix- monly defined by achievement of bone marrow plasmacytosis ation (three patients with an A lambda isotype and one patient Ͻ5% and disappearance of the M-component in serum and with a lambda light chain isotype). All patients had cyto- urine. Although the achievement of a complete remission is genetic abnormality identified at diagnosis either by conven- considered as a strong prognostic factor in high-dose therapy tional cytogenetics (6 patients) or after fluorescence in situ protocols, the current parameters used to define remission are hybridization (FISH) (8 patients). Conventional cytogenetic probably insufficient.1 Eventually, nearly all patients will analyses were performed as previously reported,8 and relapse after the achievement of this so-called complete interphase FISH was performed at diagnosis and after remission, favoring the fact that the abnormal clone is quiesc- remission as follows. ent rather than is eliminated. Recent data demonstrate that nearly all patients display cytogenetic changes within their plasma cells.2 So, cytogenetic analysis of bone marrow Interphase FISH study plasma cells (BMPC) could help to define complete remission, but conventional cytogenetics is hampered by very low num- Mononuclear cell separation of EDTA anticoagulated bone bers of BMPC. By contrast, fluorescence in situ hybridization marrow (sternal bone or iliac posterior crest) was performed (FISH) might be an adequate technique, as previously shown by density gradient centrifugation (density = 1.077; MSL; Euro- in MM,3 monoclonal gammopathy of undetermined signifi- bio, Les Ulis, France), cells centrifuged and slides stored at cance,4 and circulating plasma cells.5 In this study, we evalu- −20°C until use. For all patients tested after treatment, bone ated interphase fluorescence in situ hybridization (FISH) for marrow samples were aspirated by the same day as for bio- the detection of minimal residual disease in 14 MM patients chemical studies. For the specific study of plasma cells we who achieved an apparent complete remission after high- used interphase FISH coupled to immunological detection of dose therapy. monotypic light chain. Biotinylated probes for chromosome Nos 3, 7, 8, 9, 11, 15, 13/21 and X were all purchased from Oncor (Gaithersburg, MD, USA). Cytocentrifuge slides were Patients and methods fixed in cold acetone (5 min), in 4% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.40), rinsed and allowed Patients to dry for 15 min. The step corresponding to immunostaining of BMPC was performed as follows. After rehydratation of The study concerns 14 patients with multiple myeloma diag- slides with PBS, diluted (1/100) rabbit polyclonal anti-human nosed in our center according to Durie6 between January light chains (either ␬ or ␭) antibody, directly labeled with FITC 1992 and February 1995. These were consecutive patients in (Dako, Glostrup, Denmark), was applied for a brief period (5 min); next, slides were rinsed with PBS, air dried, and dehy- drated in graded alcohols. The step corresponding to the Correspondence: T Facon, Service des Maladies du Sang, Hoˆpital hybridization of DNA probe was done as follows. Slides were Claude Huriez, CHRU, 59037, Lille, France; Fax: 33 3 20 44 47 08 denaturated for 5 min at 72°C in 70% formamide solution (in Received 17 July 1998; accepted 1 December 1998 2ϫ sodium saline citrate); 10 ␮l of hybridization mixture was Brief communication F Genevieve et al 642 + + + + + + 37 56 31 67 41 25 35 23 35 18 25 19 31 11

(months) + + + + + + − − + + − + − − 15(34%) − 15(12%) 13/21(14%) 13/21(43%) + − 11(39%) 11(57%) − + + 9(16%), 13/21(71%) 13/21(45%), 8(17%) 9(26%) 9(26%), 7(45%) 11(16%), 9(11%), 11(15%), 11(14%) 9(33%) + − − − + + + + + + + + 7(22%), 3(31%), X(18%), 7(12%), X(65%), X(57%), 8(18%), 3(21%), 7(14%), 9(33%), 9(22%), 3(19%), 13/21(54%) 3(33%), disomy for chromosome 11 + + − + − − − − + + + − − −

treatment analyzed cells) 14, + 2, × 11 9,add(16)(p11), + + 2, 8, × 16 − 9 − + 7, + 13,add(14)(q32), mar 5, − + + 9, 4, − 22, − 5, − − 21, 21 14 0.9 119 21 + − + 5,del(6)(q22), + 9, 13,add(14)(q32), + 18, − − 15 21 0.3 51 X, + 11 20 1.4 89 11 4 0.6 52 − 12, + + 17, − + 11 10 1.1 132 9, 11, 9, 11 20 0.1 53 8, + + + + + − 9, 7, 8 3 1.6 78 7, 7, 9, 9 4.5 0.5 50 11 3 0.3 77 3mar 13,t(11;14)(q13;q32),add(17)(p11) 6, + + − − + + + + + − − X,del(2)(p22), 3, 3, 7, 3, 3, 8, 3, 3, X, X,dic(1;2)(p11;q37), − Y, + + + − + − − − 22, − − − − X, − 16,add(19)(p or q13), 15, 49,XY,del(1)(q23),del(2)(p?), + 44,X, 43,X, − 50, add(17)(p11), 40,X, Results of cytological and cytogenetic analyses and clinical course of the 14 patients

BMPC % Cytogenetic changes at diagnosis Months after BMPC % No. PC Detailed interphase FISH study (% of Table 1 CHI 35 karyotype:All deaths are relatedBMPC, to bone MM marrow except plasma for cells; patient PC, LEC plasma who cells. had 2 toxic death during a second transplant. 0.6 92 ELI 24 FISH: FAV 23 karyotype: 46,X, GAR 36 FISH: OLZ 53 karyotype: 2 3.5 70 THICHE 10 27 FISH: karyotype: 3.5 0.6 52 EGO 10 FISH: VAN 37 FISH: LUC 34 karyotype: 13 0.4 54 DUP 10 FISH: PAR 22 FISH: LER 18 karyotype: 12 2.3 81 Patient Data at diagnosisLEC 10 FISH: Data after treatment Relapse Survival Brief communication F Genevieve et al 643 applied next (0.5 ␮l of biotin-labeled probe in 9.5 ␮l of Hybri- chromosomal abnormalities and could escape the condition- sol VI (Oncor), denaturation for 2 min at 72°C), coverslipped ing regimen would likely be involved in the relapse. Indeed, and sealed with rubber cement. After one night of hybridiz- numerous chromosomal changes are usually common ation at 37°C, amplification of the biotinylated probe was per- between diagnosis and relapse, although sequential clonal formed using successive layers of Texas red avidin (Vector, evolution at relapse can be observed.16 In the future, a Burlingame, CA, USA), biotinylated anti-avidin antibody sequential FISH study at different evolutive phases of the (Vector), and Texas red avidin once again, as previously disease would probably be of interest. described.4 Slides were next counterstained with 4,6-diamino- The patients reported in our series represent 35% (14/40) phenyl indole (DAPI) and analyzed under a fluorescence of myeloma patients receiving high-dose therapy in the study microscope. Trisomy or monosomy were ascertained when period in our center. Nine out of these 14 patients relapsed. Ͼ8% or Ͼ15% plasma cells exhibited three or one signal, Six patients are still alive, including two patients in relapse. respectively (except for 13/21 probe). All patients were tested Three patients (patients 6, 8 and 11) who still had a few with two or three different probes. In each patient at least 100 demonstrable plasma cells have long survival without pro- and 50 cells were enumerated at diagnosis and after treat- gression. Thus, the persistence of a few abnormal plasma cells ment, respectively. does not preclude long progression-free survival. In our opinion, these results show that most if not all of myeloma patients, selected using usual complete remission Results and discussion criteria, are not in complete remission after interphase FISH study, due to the persistence of cytogenetically abnormal Results of the cytological and cytogenetic analyses, as well as BMPC. clinical course of the patients are presented in Table 1. Twelve patients who had either trisomy of monosomy within their bone marrow plasma cells (BMPC) at diagnosis exhibited the References same anomaly within their BMPC after treatment and were thus not in complete remission. In these patients, trisomic and 1 Attal M, Harrouseau JL, Stoppa AM, Sotto JJ, Fuzibet JG, Rossi JF, monosomic cells were clearly over the thresholds, ranging Casassus P, Maisonneuve H, Facon T, Ifrah N, Payen C, Bataille R. A prospective randomized trial of autologous bone marrow from 12 to 57% and 17 to 71%, respectively. All these results transplantation and chemotherapy in multiple myeloma. New Engl were concordant using either two (six patients) or three (six J Med 1996; 335: 91–97. patients) different probes. In the last two patients, the 2 Zandecki M, Laı¨ JL, Facon T. Multiple myeloma: almost all patients remission status was as follows: one patient had −3, +11 at are cytogenetically abnormal. Br J Haematol 1996; 94: 217–227. diagnosis and −3 (33%) but disomy for chromosome 11 after 3 Flactif M, Zandecki M, Laı¨ JL, Bernardi F, Obein V, Bauters F, autograft, and one patient could only be tested with the 13/21 Facon T. Interphase fluorescence in situ hyridization (FISH) as a powerful tool for the detection of aneuploidy in multiple myel- probe (54% abnormal cells). Eventually, at least 12 of 14 oma. Leukemia 1995; 9: 2109–2114. patients had unequivocal persistence of an abnormal plasma 4 Zandecki M, Obein V, Bernardi F, Soenen V, Flactif M, Laı¨ JL, cells clone within their bone marrow. François M, Facon T. Monoclonal gammopathy of undetermined In this study we used a modified FISH technique and not a significance: chromosome changes are a common finding within polymerase chain reaction analysis. Although less sensitive, it plasma cells. Br J Haematol 1995; 90: 693–696. was sufficiently powerful, however, to clearly detect persistent 5 Zandecki M, Bernardi F, Genevieve F, Laı¨ JL, Preudhomme C, Flactif M, Cosson A, Bauters F, Facon T. Involvement of peripheral cytogenetic abnormalities within the BMPC from most (12 of blood cells in multiple myeloma: chromosome changes are the 14) patients tested after intensive treatment. Interphase and rule within circulating plasma cells but not within B lymphocytes. metaphase FISH have been demonstrated to be sensitive Leukemia 1997; 11: 1034–1039. methods for detecting minimal residual disease in leukaemias 6 Durie BGM. Staging and kinetics of multiple myeloma. In: Wiernik and lymphomas.9–13 In multiple myeloma, a recent report PH, Canellos GP, Kyle RA, Schiffer CA (eds). Neoplastic Diseases using interphase FISH with a chromosome 9 centromeric of the Blood. Churchill Livingstone: New York, 1991, pp 439–451. 7 Durie BGM, Salmon SE. A clinical staging system of multiple myel- probe detected a persistent trisomy 9 in one of four patients oma. Correlation of measured myeloma cells mass with presenting after autograft, two of three bone marrow collections and one clinical features, response to treatment and survival. Cancer 1975; of 10 peripheral blood stem cell transplants.14 Although the 36: 842–854. gain of chromosome 9 is one of the most frequent cytogenetic 8 Laı¨ JL, Zandecki M, Mary JY, Bernardi F, Izydorczyk V, Flactif M, abnormalities found in MM, a higher rate of positivity may be Morel P, Jouet JP, Bauters F, Facon T. Improved cytogenetics in expected using a patient-specific panel of probes. Molecular multiple myeloma: a study of 151 patients including 117 at diagnosis. Blood 1995; 85: 2490–2497. monitoring of minimal residual disease (MRD) in MM has also 9 Heerema NA, Argyropoulos G, Weetman R, Tricot G, Secker- 15 been reported. In a recent report of Corradini et al, a PCR Walker LM. 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Leukemia 1994; 8: 587–594. cation as a result of the intensive treatment; (2) premalignant 11 El-Rifai W, Ruutu T, Vettenranta K, Elonen F, Saarinen UM, Volin plasma cells that share a karyotype and clonal immunoglob- L, Knuutila S. Follow-up residual disease using metaphase-FISH ulin phenotype with the malignant tumor cell; (3) residual in patients with acute lymphoblastic leukemia in remission. Leu- tumor cells that retain the capability to replicate and eventu- kemia 1997; 11: 633–638. 12 Tanaka K, Arif M, Eguchi M, Kumaravel TS, Ueda R, Ohno R, ally regrow the malignant tumor. This last hypothesis is in Iwato K, Kyo T, Dohy H, Kamada N. Application of fluorescence agreement with the clinical course of virtually all patients who in situ hybridization to detect residual leukemic cells with (9;22) ultimately relapse. Residual BMPC which share the initial and (15;17) translocations. Leukemia 1997; 11: 436–440. Brief communication F Genevieve et al 644 13 Kasprzyk A, Secker-Walker LM. 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