Detection of Minimal Residual Disease by Polymerase Chain Reaction in Patients with Different Hematologic Diseases Treated by Bone Marrow Transplantation Liborio Stuppia, Giuseppe Calabrese, Paolo Guanciali Franchi, Paolo Di Bartolomeo, Adriano Antonucci, Rita Peila, Glauco Torlontano, and Giandomenico Palka ABSTRACT: Thirteen male patients affected by different hematologic diseases who underwent bone mar- row transplantation (BMT) with female donors were investigated by cytogenetic analysis and polymerase chain reaction (PCR) amplification of a DNA sequence specific for the Y chromosome. In six of these pa- tients, PCR showed the presence of the Y chromosome-related sequence; in only three of these did cytogenetic analysis confirm the presence of mixed chimerism. In the remaining three patients, the results of the PCR were confirmed by in situ hybridization on cell nuclei with a probe for the a-satellite of the Y chromosome. We compare results obtained with the two methods and discuss the meaning of the minimal residual disease detected by PCR in patients submitted to BMT. INTRODUCTION In recent years, allogeneic bone marrow transplantation (BMT) has proven the preferable treatment for patients af- fected by different hematologic diseases when an HLA- matched donor is available [1]. To verify stable BM engraft- ment or disease relapse, the transplant must be monitored by techniques allowing early assessment of mixed chime- rism after BMT. Cytogenetic analysis, which permits one to show karyotypic differences between donor and host cells [2-4] and use of recombinant DNA technology [5, 6] are the most widely used techniques [7]. Chromosome analysis, how- ever, although very useful in follow-up of transplanted pa- tients as a routine investigation, shows a low sensitivity in detecting minimal residual disease because it is capable of detecting only a few cycling cells; on the other hand, the recombinant DNA technologies have limited application be- cause they required use of radioactivity and are time- consuming and costly. Recently, the polymerase chain reaction (PCR) has proven an excellent tool in the study of minimal residual disease as well, including investigation of patients submitted to BMT [8-11]. This technique, which allows in vitro amplification From the Istituto di Citomorfologia Umana Normale e Patolog- ica C. N. R., Chieti (L. S.), Istituto di Biologia e Genetica (P. G. F., G. C., R. P., G. P.), Istituto di Morfologia (A.A.) and Cattedra di Ematologia (P D. B., G. T.), Universit& di Chieti, Divisione di Ematalogia, Ospedale di Pescara (P. D. B., G. T.), Pescara, Italy. Address reprint requests to: Professor Giandomenico Palka, via B. Buozzi 93, 65121, Pescara, Italy. Received May 22, 1992; accepted September 14, 1992. 88 Cancer Genet Cytogenet 65:88-92 (1993) 0165-4608/93/$06.00 of specific DNA sequences starting from a few template mol- ecules [12] shows high sensitivity in detecting minimal re- sidual clones, requires small DNA sample size, and is rapid and highly efficient. We report 13 patients who underwent BMT for different hematologic diseases investigated by PCR, cytogenetic analyses, and in situ hybridization. MATERIALS AND METHODS Patients Thirteen male patients who underwent BMT with female donors were included in the study. Three of these patients were transplanted for chronic myelogenous leukemia (CML) (patients 2, 4 and 6), three for acute nonlymphoblastic leuke- mia (ANLL) (patients 8, 9 and 13), two for Fanconi anemia (FA) (patients 7 and 10), one for refractory anemia (RA) (pa- tient 1), one for multiple myeloma (MM) (patient 3), one for chronic granulomatosis disease (CGD) (patient 5), one for myelodysplastic syndrome (MDS) (patient 11), and one for paroxysmal nocturnal hemoglobinuria (PNH) (patient 12). Two patients, affected by CML and MDS, respectively (pa- tients 2 and 11), received a second BMT after clinical, hemato- logic, and cytogenetic relapse of the disease. All patients were alive and without clinical-hematologic relapse in Decem- ber 1991, with a mean follow-up of 28 months (range 4-67 months). Cytogenetic Analysis Metaphase plates were obtained from BM samples cultured 24 hours in RPMI 1640, karyotypes were investigated by GTG © 1993 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010