The Second Department of Internal Medicine, Mie University School of Medicine, Tsu, Mie, Japan

Correspondence to: Dr K Nishii, The Second Department of Internal Medicine, Mie University School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan

We report a case of a patient with IgA multiple myeloma (MM) mobilized with etoposide and subsequently receiving high-dose melphalan (HDM) with stem cell support. She relapsed rapidly post transplantation. Southern blot and fluorescent in situ hybridization analysis showed MLL gene rearrangement in the myeloma cells, which was not detected in the sample at diagnosis or in the PBSC harvested with etoposide plus G-CSF. These observations suggest that clonal rearrangement of the MLL gene is caused by etoposide. Patients with MM undergoing HDM with stem cell rescue may be at an increased risk of not only secondary leukemia, but also secondary genetic abnormalities in myeloma cells, especially those receiving priming with etoposide for peripheral blood stem cell collection. Bone Marrow Transplantation (2001) 27, 555-558.

MLL gene; multiple myeloma; etoposide; stem cell priming; secondary leukemia

Multiple myeloma (MM) is a lymphoproliferative disorder characterized by clonal expansion of neoplastic plasma cells in the bone marrow (BM), and secretion of high amounts of monoclonal immunoglobulins. Characteristically, the neoplastic BM plasma cells in MM have undergone a switch recombination, and most patients secrete IgG or IgA. Intermittent melphalan and prednisone has for many years been the recommended treatment for patients with MM. Trials with other drug combinations have not led to any major improvements in clinical outcome. With conventional therapy, only a minority of patients achieves a complete response, and virtually all patients eventually succumb to progressive disease with a median survival of approximately 3 years. Recently, high-dose therapy (HDT) with autologous stem cell support has resulted in prolonged survival for MM patients.¹ The use of etoposide has been shown to be an effective and safe method for peripheral blood stem cell harvests (PBSCH), and an effective agent in tumor reduction in patients with malignancies.² Therefore, HDT and PBSC transfusion (PBSCT) is a common therapy for hematological malignancy, including MM.³ However, with improvements in survival following intensive chemotherapy, post-transplant therapy-related hematological malignancies are emerging as serious long-term complications.^4,5,6

We described here a MM patient (IgA : stage IIIa) who received PBSCH using etoposide and G-CSF who was then given PBSCT after HDT with melphalan. At 40 days post-PBSCT, aggressive neoplastic myeloma cells were increased in her BM, and moreover MLL gene rearrangement was detected in these cells. This is the first report that etoposide for stem cell priming in a MM patient may cause secondary MLL gene abnormalities in neoplastic plasma cells.

Case report

In March 1999, a 54-year-old female was referred to hospital for hypergammaglobulinemia. She had a punched-out lesion in the skull. Her IgA level was 4800 mg/dl with values of 343 mg/dl IgG and 9.9 mg/dl IgM. Immunoelectrophoresis showed M- but not Bence-Jones proteins. BM examination showed hypercellularity with 95% neoplastic plasma cells (Figure 1a). These neoplastic plasma cells expressed CD20, CD38, and IgA but not CD19 on their surface. The patient was diagnosed with IgA MM (stage IIIB according to the Durie-Salmon classification). Abnormal karyotypes were not detected in BM cells (Figure 2a). She received first-line treatment of three courses of VAD therapy (vincristine, doxorubicin, and dexamethasone). Following these treatments, plasma cells in her BM were significantly decreased and her serum IgA level dropped from 4800 to 125 mg/dl. In October 1999 she underwent PBSCH with etoposide (500 mg/m² once daily i.v. for 3 days) and G-CSF. However, in November 1999 her serum IgA increased again to 1120 mg/dl. Although a fourth course of VAD therapy was given, it did not improve the number of neoplastic plasma cells in BM or the IgA level. Therefore, she was treated with EDAP therapy (etoposide, cisplatin, dexamethasone and cytarabine). In January 2000, high-dose melphalan (HDM) (100 mg/m² once daily i.v. for 2 days) was given. After 48 h, PBSCT was performed. On day 19 after PBCST, the number of WBC had recovered and BM aspiration showed 1.5% neoplastic plasma cells. At 40 days post PBSCT, her serum IgA level was slightly increased to 222 mg/dl. BM aspiration showed hypercellularity with 98% blastic cells (Figure 1b). These cells expressed CD38, but not CD20 or CD19. Expression of cytoplasmic IgA was also detected in all blastic cells (data not shown). The rearrangement band of the IgL gene was the same as that before chemotherapy and the germline band was not detected in cells from BM by Southern blot analysis (Figure 3). Karyotype was hyperdiploid, but details were not analyzed (Figure 2b). These findings suggest that blastic cells were neoplastic plasma cells and these cells were of the same clone as at the onset. The probability of secondary leukemia or MDS was excluded because the germline band was not detected in the cells at relapse. EDAP therapy was re-started. However, it did not decrease the neoplastic plasma cells in her BM (Figure 4). Interestingly, Southern blot analysis showed a population of BM cells having the MLL gene rearrangement (Figure 3). The rearrangement of the MLL gene was also found in 28% of the blastic cells from BM by fluorescent in situ hybridization (FISH) analysis. This abnormality had not been detected in the cells at diagnosis or in those of PBSCH.

Discussion

In this report, we describe an MM patient who received HDM and PBSCT. However, 40 days after PBSCT, aggressive neoplastic plasma cell growth was found and these cells were more refractory to chemotherapy. Interestingly, rearrangement of the MLL gene was detected in these cells, but not in those from before chemotherapy. The rearrangement band of the IgL gene was the same as that before chemotherapy, and there were no germline band. These results suggest that aggressive neoplastic cells must be relapse of MM and not secondary leukemia or myelodysplastic syndrome (MDS). This MLL gene rearrangement in neoplastic plasma cells may have been caused by anticancer drugs. Leukemias with the MLL gene abnormality are clinically unique in that they are common in infants under 1 year of age and demonstrate biphenotypic or mixed-lineage features, and they are also secondary acute leukemias.⁴ In several previous studies on leukemia and MDS following conventional chemotherapy, the risk has been shown to increase with patient age and with exposure to alkylating agents,⁷ total body irradiation,⁸ and DNA topoisomerase II inhibitors such as etoposide.⁴ Of the cytogenetic characteristics of these secondary leukemias, deletions or loss of chromosomes 5 or 7 were the most common, and balanced translocations to chromosome bands 11q23 or 21q22 were often found.⁴ Most secondary leukemia cells with an 11q23 translocation induced by etoposide have a breakpoint in the MLL gene.⁹ In particular, patients treated with etoposide have been shown to have the shortest periods of 6 month to 3 years,¹⁰ compared with 5 to 10 years following treatment with alkylating agents and 6 to 8 years following radiation treatment.¹¹ In this patient, to determine whether the clone with the MLL gene abnormality was present prior to HDM, mononuclear cells taken at diagnosis and from the cells of PBSCH were studied for the MLL gene rearrangement with Southern blot and FISH analysis. There was no abnormality of the MLL gene detected in these samples. These findings suggest that the abnormality of the MLL gene in this MM patient may have been caused by etoposide, which was used for PBSCH. Krishnan et al⁶ reported that an increased risk was observed in patients primed with etoposide for PBSCH, which likewise was significantly associated with the development of secondary leukemia with 11q23/21q22 chromosomal abnormalities. Although a MLL gene rearrangement was found, the chromosomal abnormality with 11q23 was not detected in the same BM cells from this patient. Karyotypic information on MM is limited because of the low mitotic activity of plasma cells. Recently, many cytogenetic abnormalities have been found in MM cells by the FISH method.¹² These findings suggest that investigation of MLL gene abnormalities by Southern blot or FISH methods is very important in understanding the effects of etoposide, even if karyotypic aberrations are not detected in MM patients who received PBSCT.

To our knowledge, this is the first case in which a clonal abnormality of the MLL gene was detected in a refractory MM patient who received PBSCH with etoposide and PBSCT. It is not clear whether the new mutation in the MLL gene contributed to the relapse following therapy, because only 28% of the neoplastic plasma cells had this gene rearrangement. Other factors may also be important. Therefore, we conclude that further studies on a larger number of patients who received transplants for MM is necessary to examine the effects of etoposide in the stem cell priming regimen.

Acknowledgements

This work was supported by a Grant-in-Aid (1998) from the Mie Medical Research Foundation.

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Figure 1 Bone marrow smear (´ 1000). (a) At the onset of multiple myeloma (May 1999), dysplastic plasma cells are apparent. (b) At the plasma cell re-growth after PBSCT (March 2000), most of the cells are of blast-like morphology.

Figure 2 Karyotype analysis at the onset and at the relapse. (a) Karyotype of metaphase cells, 46,XX, derived from BM of the patient at diagnosis (May 1999). (b) Karyotype of a metaphase cells derived from BM of the patient at the relapse (March 2000). Several abnormal karyotypes such as 82, XX, 1q^-, 2q^-, 3q⁺, 5q^-, 6p⁺, 8p⁺, 15p⁺, 21p⁺ were found.

Figure 3 Southern blot analysis of MLL and IgL genes. Genomic DNA (3 g) was digested with BamHI overnight and hybridized with a probe for exons 5 to 10 of the MLL gene that was amplified with the primer used for RT-PCR-based analysis, or with a probe for IgL C gene. The rearranged band of the MLL gene was only detected in re-growth cells after PBSCT. The rearranged band of the IgL gene in the BM cells from relapse was the same size as that of before chemotherapy. Arrow shows the rearranged band. Lane 1, negative control (normal human BM); lane 2, BM samples at the diagnosis (May 1999); lane 3, sample from PBSCH (August 1999); lane 4, BM sample at the relapse after PBSCT (March 2000).

Figure 4 Clinical course.