Bone Marrow Transplantation, (1998) 22, 823–825  1998 Stockton Press All rights reserved 0268–3369/98 $12.00 http://www.stockton-press.co.uk/bmt Case report Long-lasting complete remission in leukemia after aggressive chemotherapy and CD34-selected autologous peripheral blood progenitor cell transplant: molecular follow-up of minimal residual disease

S Sica1, P Chiusolo1, P Salutari1, N Piccirillo1, L Laurenti1, E Ortu La Barbera1, FG Serra2 and G Leone1

1Istituto di Semeiotica Medica, Divisione di Ematologia; and 2Istituto di Anatomia Patologica, Universita` Cattolica Sacro Cuore, Roma, Italy

Summary: Case report

Plasma cell leukemia is a rare disease associated with In October 1995, a 58-year-old female, otherwise asympto- very poor survival with standard treatment. We report matic, came to our attention because a routine serum pro- a patient affected by plasma cell leukemia treated with tein electrophoresis disclosed the presence of paraproteins aggressive chemotherapy and autologous CD34-selected subsequently confirmed to be monoclonal IgG ␭ type at PBPC who achieved a complete remission now lasting immunofixation. At admission the physical examination more than 2 years. Molecular studies confirmed the was unremarkable. Hemoglobin was 8.0 g/dl, platelet count presence of minimal residual disease (MRD) despite the 146 × 109/l, WBC 8.3 × 109/l. On differential count, periph- absence of disease activity. High-dose chemotherapy eral blood neutrophils were 20%, lymphocytes 35%, mono- with stem cell rescue may be applied to selected patients cytes 10% and immature plasma cells 35%. Immunopheno- considering the impact of the treatment on survival. The type was available only on the peripheral blood: CD3 62%, meaning of molecular MRD in this setting is unclear. CD3/DR 17%; CD4 33%; CD8 39%; CD19 5%; CD57 Keywords: plasma cell leukemia; autologous stem cell 25%; CD16 13%. Bone marrow aspiration revealed a mass- transplantation; minimal residual disease ive plasma cell infiltration (65% immature plasma cells). Serum IgG was 5.9 g/l and free ␭ chains were detected in the urine. Serum LDH was 556 IU/l, total proteins were 11.4 g/dl, creatinine was 1.1 mg/dl, ␤2 microglobulin was Plasma cell leukemia (PCL) is a rare form of malignant not available at diagnosis. Skeletal bone survey did not plasma cell disease accounting for 1–4% of myelomas. PCL detect lytic lesions. is predominantly a disease of late adulthood and its course Cytogenetic analysis on bone marrow mononuclear cells is highly unfavorable with an estimated 5 year relative sur- disclosed the following karyotype: 17 normal mitoses vival rate of 13%. Current therapy of PCL is disappointing (46 XX), five pseudodiploid mitoses characterized by the and is based on the standard combination of and presence of complex rearrangement: isochromosome (21q), although more intensive therapeutic pro- 3q+, der(13). grammes including cyclophosphamide–etoposide or vincri- Three courses of 0.4 mg, stine, adriamycin and (VAD) are giving 9 mg/m2 and prednisone 40 mg from days 1 to 4 (VAD) promising results. We describe a patient with de novo PCL were planned. Laboratory evaluations before the third treated with intensive chemotherapy and autologous CD34- course showed the persistence of peripheral plasmacytosis selected peripheral blood progenitor cell tranplantation who with massive bone marrow infiltration. The patient was then achieved complete and sustained remission with a follow- switched to intermediate-dose cyclophosphamide and a up of 24 months. The course of PCL can be modified by complete remission was obtained at the end of two courses. the use of high-dose chemotherapy and autologous stem High-dose cyclophosphamide (7 g/m2) plus G-CSF 5 ␮g/kg cell transplantation in selected patients. was then administered. Peripheral blood stem cells were harvested and the product of two consecutive leukaphereses was submitted to positive immunoselection with Ceprate SC (Cellpro, Bothell, WA, USA). A total of 10 × 106/kg CD34+ cells was reinfused after conditioning with busulfan Correspondence: Dr S Sica, Istituto di Semeiotica Medica, Universita` Cat- 4 mg/kg p.o. in divided doses for 4 consecutive days and 2 tolica Sacro Cuore, Policlinico A Gemelli, 00168 Roma, Italy melphalan 90 mg/m day 5. Hemopoietic recovery after Received 7 April 1998; accepted 7 June 1998 CD34-selected PBPCT was complete, PMN Ͼ0.5 × 109/l Autologous stem cell transplant for plasma cell leukemia S Sica et al 824 were obtained at day +12 and a platelet count Ͼ50 × 109/l 20% of the differential white blood cell (WBC) or as an was obtained at day +40. absolute number greater than 2 × 109/l.3,4 PCL arises as a Immune reconstitution was characterized by a slow late manifestation in patients with or it recovery of the CD4+ lymphocyte subset with a persistent can occur de novo (primary PCL). Cytogenetic findings in reduction of CD4/CD8 (0.5 at 1 and 2 years of follow- PCL are most often characterized by complex karyotypes, up) and by the presence of activated T cells coexpressing with nonrandom abnormalities primarily involving chromo- CD3/DR for at least 2 years after PBPCT. Maintenance somes 1, 7, 8, 11, 14 and 13.5 The outcome for patients therapy with ␣-interferon (3 × 106 IU s.c. on alternate days) with PCL is disappointing with a 5 year relative survival was started 2 months after PBPCT. The patient is alive and rate of 13% in a recent report by Hernandez and co-work- in complete hematological and cytogenetic remission 24 ers.6 These results are primarily obtained with standard months after PBPCT still on maintenance therapy with ␣- treatment including a combination of one or more alkylat- interferon. ing agents with prednisone. Encouraging data have been obtained by the use of more intensive chemotherapeutic regimen (VAD or high-dose cyclophosphamide or VP-16). Molecular studies These regimens, although more toxic, were able to induce The evaluation of minimal residual disease (MRD) was per- complete remission and improve overall survival in selected formed through the molecular rearrangement of the heavy patients.4 Moreover, the use of myeloablative therapy with chain of the immunoglobulin by PCR technique using the stem cell support has been recently introduced in the man- primers FR3A (ACACGGCC/TC/GTGTATTACTGT) and agement of PCL responsive to chemotherapy and prelimi- JH (GACCCCGGTTCCTAGGGACCAGTGGC).1 Gen- nary results are encouraging.2–9 Our patient was unrespon- omic DNA from about 3 × 106 cells for each sample was sive to VAD but achieved a complete response after extracted according to standard protocols.2 The 100 ␮l PCR cyclophosphamide. After mobilization with high-dose reactions were performed as follow: 1 ␮g genomic DNA, cyclophosphamide + G-CSF a large number of PBPC was 30 pmol of each primer, 2.5 U of Taq polymerase collected and CD34+ cell selection was carried out. Hemo- (Amplitaq; Perkin Elmer-Cetus, Norwalk, CT, USA), poietic recovery after PBPCT was prompt and complete. 200 ␮mol/l of each dNTP, 5 to 10 ␮Ci of 32P-deoxycytidine Immune reconstitution was characterized by a slow recov- triphosphate (32P-dCTP; ICN Pharmaceuticals, The ery of CD4+ lymphocytes and by the appearance of acti- Netherlands), 20 mmol/l Tris-HCl (pH 8.3), 50 mmol/l KCl vated T cells persisting for at least 2 years after PBPCT. and 1.5 mmol/l MgCl2. After a denaturation step (7 min at At follow-up the patient is in complete remission at 2 94°C) amplification was performed by 30 cycles at 94°C years. Despite the absence of hematological, laboratory and for 1 min, at 56°C for 1 min, at 72°C for 1.5 min, with a clinical findings of the original disease, minimal residual final single extension step at 72°C for 7 min. All PCR reac- disease is still detectable at the molecular level. Currently, tions were carried out using DNA Thermal Cycler 480 the significance of disease persistence at the molecular level (Perkin Elmer-Cetus). Four microliters of PCR product in the purged graft and during follow-up is unknown and were separated on 6% polyacrylamide gel that was dried adequate follow-up of patients is required to validate its and exposed to X-ray film at room temperature for 12 h. role, if any, in the management of myeloma patients sub- The film was analyzed by scanner (Hewlett-Packard, mitted to high-dose chemotherapy and stem cell transplan- Geneva, Switzerland). The leukapheretic products showed tation. PCL, although a rare disease, should be treated with a persistent positivity of the monoclonal band. Purified intensive chemotherapy and autologous stem cell transplan- CD34+ cell fraction showed a positivity for the original tation at least in selected patients, considering the major rearrangement but to a lesser extent. At follow-up at 1 and impact of a correct strategy on survival. The role of purging 2 years, the monoclonal band still persists despite clinical in multiple myeloma and PCL is debatable10 and recent and laboratory remission (Figure 1). results showed no difference in a large series of myeloma patients receiving immunoselected CD34+ or unfractionated PBPC.11 These observations deserve serious consideration Discussion for economical, practical and ethical reasons in the design of new protocols. Plasma cell leukemia (PCL) is a rare disease occurring in 2–4% of newly diagnosed patients and it is characterized by a malignant proliferation of plasma cells massively Acknowledgements involving the peripheral blood, accounting for more than This work was supported in part by a grant from Associazione 12 34 56 7 Italiana Ricerca sul Cancro (AIRC).

References

1 Billeadeu D, Blackstadt M, Greipp P et al. Analysis of B- Figure 1 Molecular follow-up: (1) diagnosis; (2) leukapheretic product; lymphoid malignancies using allele-specific polymerase chain (3) CD34+ selected fraction; (4) bone marrow day +30; (5) bone marrow reaction: a technique for sequential quantification of residual day +60; (6) bone marrow month +12; (7) bone marrow month +24. disease. Blood 1991; 78: 3021–3029. Autologous stem cell transplant for plasma cell leukemia S Sica et al 825 2 Maniatis T, Fritsch EF, Sambrook J. Molecular Cloning: A 8 Sayeva MR, Greco MM, Cascavilla N et al. Effective autolog- Laboratory Manual. Cold Spring Harbor Press: Cold Spring ous peripheral blood stem cell transplantation in plasma cell Harbor, NY, 1982. leukemia followed by T-large granular lymphocyte expansion: 3 Kosmo MA, Gale RP. Plasma cell leukemia. Semin Hematol a case report. Bone Marrow Transplant 1996; 18: 225–227. 1987; 24: 202–208. 9 Barlogie B, Alexanian R, Dicke R et al. High-dose chemoradi- 4 Dimopoulos MA, Palumbo A, Delasalle KB et al. Plasma cell otherapy and autologous bone marrow transplantation for leukemia. Br J Haematol 1994; 88: 754–759. resistant multiple myeloma. Blood 1987; 70: 869–872. 5 Smadja N, Krulik M, Louvet C et al. Similar cytogenetic 10 Bensinger WI. Should we purge? Bone Marrow Transplant abnormalities in two cases of plasma cell leukemia. Cancer 1998; 21: 113–115. Genet Cytogenet 1991; 52: 123–129. 11 Schiller G, Vescio R, Freytes C et al. Autologous CD34-selec- 6 Hernandez JA, Land KJ, McKenna RW. Leukemias, mye- ted blood progenitor cell transplants for patients with loma, and other lymphoreticular neoplasms. Cancer 1995; 75: advanced multiple myeloma. Bone Marrow Transplant 1998; 381–394. 21: 141–145. 7 Yang CH, Lin MT, Tsay W et al. Autologous bone marrow transplantation for plasma cell leukemia. Transplant Proc 1992; 24: 1531–1532.