Need for an accurate molecular diagnosis to assess the donor origin of leukemia relapse after allogeneic stem cell transplantation.

BACKGROUND AND OBJECTIVES: Leukemia relapse occurring in donor cells after allogeneic hematopoietic stem cell transplantation has been reported in rare cases. Cytogenetic analysis and molecular probing of variable number of tandem repeats (VNTRs) have been used to confirm this unusual event in the few cases so far reported in the literature. The aim of this study was to demonstrate that extensive molecular characterization of leukemic cells at diagnosis and relapse may be necessary to avoid many technical pitfalls possibly leading to an erroneous diagnosis of leukemia relapse in donor cells after allogeneic transplantation. DESIGN AND METHODS: We report the case of a 49- year old man who received an allogeneic transplantation from his HLA-identical sister because of BCR-ABL+ acute lymphoblastic leukemia (ALL). After having achieved complete hematologic and molecular remission, two years later an overt leukemia relapse occurred with cytogenetic findings suggesting a leukemia relapse in donor cells. The donor or patient origin of leukemic cells at relapse was further investigated by fluorescence in situ hybridization (FISH) karyotyping, reverse transcription (RT) polymerase chain reaction (PCR) analysis of BCR-ABL chimeric transcripts, PCR amplification of several VNTRs and the Y chromosome-specific DYS14 sequence and finally by amplification, cloning and sequencing of the CDRIII region of the immunoglobulin heavy chain (IgH) gene. RESULTS: At the time of relapse, conventional and FISH karyotyping revealed the presence of a Phl+ chromosome and a female karyotype in all the 25 metaphases analyzed and PCR amplification of the Y chromosome-specific DYS14 sequence was negative. Moreover, the molecular evaluation of hematopoietic chimerism performed by the NZ-22 VNTR allowed us to demonstrate that at the time of relapse, a consistent proportion of hematopoietic cells was of donor origin. However, the molecular cloning and sequencing of the CDRIII region of the immunoglobuin heavy chain (IgH) gene rearrangement in leukemic blasts at diagnosis and relapse demonstrated their identity thus formally proving the patient origin of both leukemic clones. INTERPRETATION AND CONCLUSIONS: While the simplest interpretation of the apparent female karyotype at relapse is the consequence of a loss of the Y chromosome which in leukemic blasts took place along with duplication of an X-chromosome, this case strongly emphasizes the need for accurate and extensive molecular characterization to prove the donor origin of a leukemia relapse after allogeneic transplantation.

Seven novel additional small mutations and a new alternative splicing in the human dystrophin gene detected by heteroduplex analysis and restricted RT-PCR heteroduplex analysis of illegitimate transcripts.

Around 35% of Duchenne and Becker muscular dystrophy (DMD/BMD) patients cannot be identified by techniques which identify major DMD rearrangements in the dystrophin gene. In order to characterize the gene defect in these patients, we screened 40 exons of the dystrophin gene by heteroduplex analysis on genomic DNA in 50 affected Italian males. Using conventional heteroduplex analysis and a modified heteroduplex analysis on restricted RT-PCR products of illegitimate transcripts, restricted RT-PCR heteroduplex analysis, we were able to identify 7 novel small mutations and a new alternative splicing involving exon 25 of the dystrophin gene in peripheral blood lymphocytes and skeletal muscle transcripts.