Correspondence 2211 S Savas¸an1 Barbara Ann Karmanos Cancer Ginsberg C, Trujillo J, Stass S, Champlin RE. Preliminary results of E Abella1,2 Institute and 1Children’s Hospital treatment with filgrastim for relapse of leukemia and myelodyspla- AJ Akhtar2 of Michigan, Division of sia after allogeneic bone marrow transplantation. New Engl J Med 1 2 1993; 329: 757–761. Y Ravindranath Hematology/Oncology and Bone 4 Shinohara K, Oeda E, Nomiyama J, Inoue H, Kamei S, Tajiri M, Marrow Transplantation Unit, Ichikawa T, Kuwaki T, Tachibana K. The levels of granulocyte col- Harper Hospital, Wayne State ony-stimulating factor in the plasma of the bone marrow aspirate University, Detroit, MI, USA in various hematological disorders. Stem Cells 1995; 13: 421–427. 5 Gabrilove JL, Jakubowski A. Granulocyte colony-stimulating fac- tor: preclinical and clinical studies. Hematol Oncol Clin North Am 1989; 3: 427–440. References 6 Keil F, Kalhs P, Haas OA, Fritsch G, Lechner K, Mannhalter C, Greinix HT. G-CSF stimulation of donor myelopoiesis prolongs ¨ 1 Terpstra W, Lowenberg B. Application of myeloid growth factors survival of relapsed BCR-ABL-positive acute lymphoblastic leuke- in the treatment of acute myeloid leukemia. Leukemia 1997; 11: mia after allogeneic marrow transplantation. Bone Marrow Trans- 315–327. plant 1996; 18: 655–657. 2 Rowe JM, Liesveld JL. Hematopoietic growth factors in acute leu- 7 Savas¸an S, Abella E, Karanes C, Ravindranath Y. Recurrent breast kemia. Leukemia 1997; 11: 328–341. relapses in a patient with acute lymphoblastic leukaemia following 3 Giralt S, Escudier S, Kantarjian H, Deisseroth A, Freireich EJ, And- allogeneic bone marrow transplantation. Acta Haematol 1997; ersson BS, O’Brien S, Andreeff M, Fisher H, Cork A, Hirsch- 871.

Acute myeloid leukemia following 3M (mitoxantrone, mitomycin and methotrexate) chemotherapy for advanced breast cancer

TO THE EDITOR were admitted in 1996 to our Department for acute leukemia. Clinical characteristics of the patients are shown in Table 1. The risk of secondary acute myeloid leukemia (AML) among Patient No. 1, a 44-year-old woman, was well until 4 years subjects with breast cancer receiving standard-dose cyclopho- earlier, when breast cancer occurred. She was treated with sphamide and doxorubicin has been reported equivalent to radical surgery plus six courses of cyclophosphamide, metho- that of the general population.1 Recently an increased fre- trexate and 5-fluorouracil (CMF). One year after the initial quency of therapy-related leukemias has been documented, diagnosis, she had recurrence of the disease with bone metast- paralleling the more widespread use of dose-intensive ther- ases and was treated with six courses of 3M followed by med- apies and the longer survival of cancer patients.2 roxyprogesteron acetate (MAP) and radiation therapy (RT) on Alkylating agents3 or more recently, epidophyllotoxins- the bone lesions. About 6 months from the start of 3M a related4 secondary AML have been described, but also decrease of white blood cells and platelets count was noted. growth factors or different drug combinations seem to affect Eighteen months later she was admitted to our Department cellular proliferation, especially in subjects with genetic because of a hemorrhagic syndrome: the bone marrow exam- susceptibility.5 ination revealed AML M0 according to FAB criteria. The Because of the effectiveness of chemotherapeutic agents patient died 4 days later. such as topoisomerase II inhibitors (epidophyllotoxins, anthra- Patient No. 2, a 60-year-old woman, had a history of breast cyclines and their derivates) it is important to define their leu- cancer treated with radical surgery at 50 years. Six years later, kemogenic potential in patients with advanced diseases and bone metastases developed and she was treated with eight mainly in adjuvant regimens for early cancer. courses of CMF and RT to lesions of the skeleton. At the age We describe three cases of secondary AML observed in the of 58, the disease progressed and she was therefore treated last year in our Institution, which occurred in women treated with seven courses of 3M followed by MAP. One year after with 3M (methotrexate, mitoxantrone, mitomycin) regimen for the end of the therapy, piastrinopenia and hemorrhagic syn- advanced breast cancer. drome occurred: bone marrow examination disclosed acute Clinical and biologic features are described and the poss- promyelocytic leukemia. All-trans retinoic acid therapy was ible relationship between these drugs and the onset of acute started and at +10 months from diagnosis the patient is alive leukemia is discussed. and well. Three women with a history of advanced breast cancer Patient No. 3 was a 52-year-old woman. She was well until treated with mitoxantrone (8 mg/m2 intravenously every 3 10 years earlier when she underwent radical surgery and weeks), mitomycin (8 mg/m2 intravenously every 6 weeks) and breast RT for breast cancer. Thirty months before admission, methotrexate (30 mg/m2 intravenously every 3 weeks) (3M) the patient received five courses of 3M followed by tamoxifen for disease recurrence to skeleton and skin. After that a pro- gressive anemia and leucopenia developed: bone marrow examination revealed refractory anemia. Transfusional ther- Correspondence: LMA Melillo apy was prescribed. Two weeks before admission acute mon- Received 20 February 1997; accepted 5 September 1997 ocytic leukemia developed; the patient was treated with Correspondence 2212 Table 1 Clinical and laboratory features at diagnosis of breast cancer patients developing acute myeloid leukemia

Patient No. Age FAB Latent period from 3M Previous treatment WBC Organomegaly or (months) for breast cancer (× 109/l) adenopathy

1 44 M0 24 Radical mastectomy + 6CMF 0.5 No 2 60 M3 12 Radical mastectomy + Tamoxifen 21.3 No 8CMF + RT 3 52 M5 30 Radical mastectomy + RT 43.4 No

mitoxantrone and intermediate doses of cytosine arabinoside, genetic abnormalities, but molecular analysis was not but she died a few days later. reported. In our series, however, Southern blot analysis Chromosome investigations were carried out at diagnosis detected a germline configuration of MLL gene. on bone marrow samples using direct or short term (24–48 h) Since methotrexate seems to have little leukemogenic poten- culturing techniques. Twenty well-spread GTG banded meta- tial,12 the increased risk of AML appears to be probably related phases were analyzed for each patient. to mitoxantrone, alone or by an interaction with mitomycin or Patient 1 had normal karyotype; patient 2 was found to methotrexate and/or previous therapies, including RT. have 46,XX,t(15;17)(q22;q12). In patient 3, cytogenetic In conclusion, the clinical features of our cases resemble analysis revealed a complex karyotype interpreted as those of secondary AML occurring after treatment with topo- 45,XX,del(3)(q12.q25), −7,−19,+mar. The chromosome 3 isomerase II agents: nevertheless, the lack of MLL rearrange- interstitial deletion was confirmed by whole chromosome ments in our series suggests a different pathway by which painting with a digoxygenin-labelled probe specific for chro- cytotoxic drugs and radiations interfere with the genetic pro- mosome 3 (Oncor, Gaithersburg, MD, USA) that did not show gram of hemopoietic cells. any translocation. Molecular analysis by RT-PCR assay was able to detect fusion transcript PML/RAR alpha (bcr3) in case 2. Southern blot analysis with 0.74 kb MLL cDNA probe to Acknowledgements detect MLL gene rearrangements revealed a germline con- figuration in all three examined cases. We wish to thank Dr Daniela Diverio and Dr MC Rapanotti, AML occurs in up to 15% of patients treated with cytotoxic Cattedra di Ematologia, Dipartimento di Biopatologia Umana, 3 ` drugs, radiation or both. Two different forms of secondary Universita La Sapienza, Rome for performing the molecular AML are described. The classic one, in patients treated with analysis. alkylating agents and/or RT, is characterized by a long latency period (3–5 years), a high incidence of preleukemic phase, LMA Melillo Divisione di Ematologia, frequent cytogenetic abnormalities involving chromosomes 5 MR Sajeva Ospedale ‘Casa Sollievo della Sofferenza’ 5 and 7 and an extremely poor prognosis. The second form, P Musto IRCCS, that occurs in patients who have received agents targeting G Perla S Giovanni Rotondo, DNA topoisomerase II, such as epidophyllotoxin derivates N Cascavilla Italy 4 (etoposide, teniposide) or anthracyclines and their derivates MM Minervini 6 (such as mitoxantrone) develops after a short latency period G D’Arena (2–3 years), a brief myelodysplastic phase and is associated M Carotenuto with myelomonocytic or monocytic morphology. In this form, most cases show a balanced translocation involving chromo- some 11q23 leading to rearrangement of MLL gene. This gene References encodes a transcription factor with extensive homology to the Drosophila homeotic regulator trithorax and is probably 1 Tallman MS, Gray R, Bennet JM, Variakojis D, Robert N, Wood involved in the regulation of differentiation pathway through WC, Rowe JM, Wiernik PH. Leukemogenic potential of adjuvant DNA interaction.7 Many different mechanisms (aberrant V-D- chemotherapy for early breast cancer: the Eastern Cooperative J recombination, homologous recombination between Alu- Oncology Group experience. J Clin Oncol 1995; 13: 1557–1563. repeats, and topoisomerase II-mediated non-homologous 2 Shepherd L, Ottaway J, Miles J, Levine M. Therapy related leuke- 8 mia associated with high-dose 4-epi-doxorubicin and cyclophos- recombination) seem to be involved in MLL rearrangements, phamide used as adjuvant chemotherapy for breast cancer. J Clin but a genetic susceptibility predisposing this region to altered Oncol 1994; 12: 2514–2515. recombinations cannot be excluded. 3 Levine EG, Bloomfield CD. Leukemias and myelodysplastic syn- Nowadays, detection of molecular MLL rearrangements by dromes secondary to drug, radiation, and environmental exposure. Southern blot analysis rather than structural abnormalities of Semin Oncol 1992; 19: 47–84. 11q23 (that comprises a heterogeneous group of fusion 4 Pui CH, Ribeiro RC, Hancock ML, Rivera GK, Evans WE, Rai- 9 mondi SA, Head DR, Behm FG, Mamboud MH, Sandlund JT, Crist genes) seems to identify this distinct subtype of secondary WM. Acute myeloid leukemia in children treated with epidophyl- AML and to define clinical syndromes and outcome more lotoxins for acute lymphoblastic leukemia. New Engl J Med 1991; specifically.10 325: 1682–1687. Recently, Cremin et al11 reported a 5% incidence of AML 5 Pedersen-Bjergaard J, Philip P, Larsen SO, Andersson M, Daugaard and myelodysplasias after adjuvant chemotherapy for breast G, Ersboll J, Hansen SW, Nielsen D, Sigsgaard C, Specht L, Oster- cancer using 2M and 3M regimens. Such cases show close lind K. Therapy-related myelodysplasia and acute myeloid leuke- mia. Cytogenetic characteristics and risk in seven cohorts of similarities with those observed in our Department: a short patients treated intensively for malignant disease in the Copen- latency time from therapy, a brief preleukemic phase and a hagen series. Leukemia 1993; 7: 1975–1986. generally poor prognosis; they also showed non-specific cyto- 6 Mitchell PLR, Treleaven J, Swansbury GJ. Secondary acute Correspondence 2213 myeloid leukemia (AML) and myelodysplasia (MDS) following 10 Chen CS, Sorensen PHB, Domer PH, Reaman GH, Korsmeyer SJ, mitozantrone given as adjuvant therapy for breast cancer. Proc Heerema NA, Hammond G, Kersey JH. Molecular rearrangements Am Soc Clin Oncol 1996; 15: 127 (Abstr.). on chromosome 11q23 predominate in infant acute lymphoblastic 7 Hunger SP, Tkachuk DC, Amylon MD, Link MP, Carroll AJ, Wel- leukemia and are associated with specific biologic variables and ban JL, Willman CL, Cleary ML. HRX involvement in de novo and poor outcome. Blood 1993; 81: 2386–2393. secondary leukemias with diverse chromosome 11q23 abnormali- 11 Cremin P, Flattery M, McCann SR, Daly PA. Myelodysplasia and ties. Blood 1993; 81: 3197–3203. acute myeloid leukemia following adjuvant chemotherapy for 8 Chen CL, Fuscal JC, Liu Q, Relling MV. Etoposide causes illegit- breast cancer using mitoxantrone and methotrexate with or with- imate V(D)J recombination in human lymphoid leukemic cells. out mitomycin. Ann Oncol 1996; 7: 745–746. Blood 1996; 88: 2210–2218. 12 Moder KG, Tefferi A, Cohen MD, Menke DM, Luthra HS. Hemato- 9 Caligiuri MA, Shichman SA, Strout MP, Mrozek K, Baer MR, Fran- logic malignancies and the use of methotrexate in rheumatoid kel SR, Barcos M, Herzig GP, Croce CM, Bloomfield CD. Molecu- arthritis: a retrospective study. Am J Med 1995; 99: 276–281. lar rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translo- cations. Cancer Res 1994; 54: 370–373.

Bone marrow reconstitution with normal autologous cells demonstrated by VNTR analysis after allogeneic bone marrow transplantation for chronic myelogenous leukemia

TO THE EDITOR

Allogeneic stem cell transplantation is the only presently available procedure for a potentially successful cure of chronic myeloid leukemia (CML) by which the patient’s hem- atopoietic cells are replaced by those derived from donor stem cells.1,2 Following BMT, engraftment, remission or relapse must be monitored by identifying the origin of cell popu- lations3 and the presence of residual Ph+ clones or other cyto- genetic rearrangements.4,5 In July 1990, a 23-year-old male with Ph+ CML received a BMT from his HLA-identical brother at the British Hospital (Montevideo, Uruguay). At that time, the patient was in second chronic phase after alpha-interferon and hydroxyurea treatment, and he had an Hb of 8.06 mmol/l, WBC 6 × 109/l and platelet count of 400 × 109/l. His bone marrow aspirate showed a normal myeloid sector, while cytogenetic analysis Figure 1 Southern blot of the patient’s DNA digested with BamHI + (lane 1), BglII (lane 2) and HindIII (lane 3) after hybridization with the demonstrated 10/20 46,XY, Ph , t(3;21) and 10/20 46,XY Ј cells. Molecular analysis by Southern blotting and hybridiz- 3 bcr probe. Arrows point to rearranged bands. ation with a 3Ј bcr probe (1.2 kb HindIII/BglII fragment) con- firmed the presence of a rearrangement within the M-BCR (major break-point cluster region) (Figure 1).6 Pre-BMT con- ditioning regimen was high-dose cyclophosphamide (50 mg/kg/per day for 2 days) and busulphan (4 mg/kg/per day for 4 days). GVHD prophylaxis was cyclosporin A at a dose of 10 mg/kg from day +1 to day +100. The number of non-T- depleted nucleated cells infused was 2.5 × 108 cells/kg. CFU- GM and CD34+ cell quantification was not performed. Bone marrow repopulation was confirmed on day +14 with a WBC count of 0.5 × 109/l and platelet count of 10 × 109/l. Platelet count reached 20 × 109/l on day +16, and 50 × 109/l on day +19. Last platelet and red cell transfusions were done on day Figure 2 Southern blot of BamHI DNA digests after hybridization +17. No CSF stimulation was used. For a short period at that with the pYNZ22 probe: donor’s DNA (lane 1), recipient’s DNA before BMT (lane 2) and recipient’s DNA after BMT (lane 3). time the patient developed acute GVHD grade I associated ´ with fever. Correspondence: M del Rosario Uriarte, Laboratorio Genia-Genetica ´ ˜ Molecular, Asociacion Espanola Primera de Socorros Mutuos, Bvar. Two months after BMT, cytogenetic and Southern blot Artigas 1465, 11200, Montevideo, Uruguay; Fax: 0598 248 7588 analyses were performed and subsequently repeated every 6 Received 16 October 1996; accepted 3 July 1997 months. In all these instances bone marrow cells exhibited a