Small sizes and indolent evolutionary dynamics challenge the potential role of P2RY8-CRLF2-harboring clones as main relapse-driving force in childhood ALL.

The P2RY8-CRLF2 fusion defines a particular relapse-prone subset of childhood acute lymphoblastic leukemia (ALL) in Italian Association of Pediatric Hematology and Oncology Berlin-Frankfurt-Münster (AIEOP-BFM) 2000 protocols. To investigate whether and to what extent different clone sizes influence disease and relapse development, we quantified the genomic P2RY8-CRLF2 fusion product and correlated it with the corresponding CRLF2 expression levels in patients enrolled in the BFM-ALL 2000 protocol in Austria. Of 268 cases without recurrent chromosomal translocations and high hyperdiploidy, representing approximately 50% of all cases, 67 (25%) were P2RY8-CRLF2 positive. The respective clone sizes were ≥ 20% in 27% and < 20% in 73% of them. The cumulative incidence of relapse of the entire fusion-positive group was clone size independent and significantly higher than that of the fusion-negative group (35% ± 8% vs 13% ± 3%, P = .008) and primarily confined to the non-high-risk group. Of 22 P2RY8-CRLF2-positive diagnosis/relapse pairs, only 4/8 had the fusion-positive dominant clone conserved at relapse, whereas none of the original 14 fusion-positive small clones reappeared as the dominant relapse clone. We conclude that the majority of P2RY8-CRLF2-positive clones are small at diagnosis and virtually never generate a dominant relapse clone. Our findings therefore suggest that P2RY8-CRLF2-positive clones do not have the necessary proliferative or selective advantage to evolve into a disease-relevant relapse clone.

Immunogenotype changes prevail in relapses of young children with TEL-AML1-positive acute lymphoblastic leukemia and derive mainly from clonal selection.

PURPOSE: Variations of the immunogenotype and TEL deletions in children with TEL-AML1+ acute lymphoblastic leukemia support the hypothesis that relapses derive from a persistent TEL-AML1+ preleukemic/leukemic clone rather than a resistant leukemia. We aimed at elucidating the relationship between the immunogenotype patterns at diagnosis and relapse as well as their clinical and biological relevance. PATIENTS AND METHODS: Immunoglobulin and T-cell receptor gene rearrangements were analyzed in 41 children with a TEL-AML1+ acute lymphoblastic leukemia and an early (up to 30 months after diagnosis; n = 12) or late (at 30 months or later; n = 29) disease recurrence by a standardized PCR approach. RESULTS: In 68% of the patients (group I), we identified differences in the immunogenotype patterns, whereas no changes were observed in the remaining 32% (group II). The divergence resulted more often from clonal selection than clonal evolution and consisted predominantly of losses (0-6, median 5) and/or gains (0-4, median 1) of rearrangements. The frequency and number of clonal immunoglobulin/T-cell receptor rearrangements in group I was higher at diagnosis (2-13, median 5) than at relapse (2-7, median 4), whereas it was the lowest in group II (1-5, median 3). Although group I children were younger at diagnosis, there was no correlation between particular immunogenotype patterns and remission duration. CONCLUSION: These findings imply that the clonal heterogeneity in younger children most likely reflects an ongoing high recombinatorial activity in the preleukemic/leukemic cells, whereas the more uniform repertoire observed in older children mirrors end-stage rearrangement patterns of selected cell clones that evolved during the prolonged latency period.

Identification and characterisation of clonal incomplete T-cell-receptor Vdelta2-Ddelta3/Ddelta2-Ddelta3 rearrangements by denaturing high-performance liquid chromatography and subsequent fragment collection: implications for minimal residual disease monitoring in childhood acute lymphoblastic leukemia.

Incomplete T-cell-receptor delta (TCR-delta) rearrangements are widely used for detection of minimal residual disease in childhood acute lymphoblastic leukemia. In a substantial number of cases both alleles are rearranged and polymerase chain reaction (PCR) products have either to be cloned or excised and reamplified from acrylamide gels. Here we describe a novel HPLC-based method for identification and characterization of clonal TCR-delta targets. Clonality of PCR amplified TCR-delta products was examined on a high-resolution micropellicular DNASep matrix (WAVE Nucleic Acid Fragment Analysis System, Transgenomic) and subsequently classified into clonal, biclonal or negative samples. Vdelta2-Ddelta3 and Ddelta2-Ddelta3 rearrangements were analyzed by denaturing high-performance liquid chromatography (DHPLC), using triethylammonium acetate as an ion-pairing reagent, with a linear acetonitrile gradient at 50 degrees C. Biclonal elution profiles consisted of two individual homoduplex peaks and one heteroduplex peak unique for each patient sample. For characterization of biclonal rearrangements DHPLC separated samples were subjected to a second run and individual clonal peaks were collected. A software-controlled fragment collector was arranged in tandem with the HPLC system for this purpose and purified PCR products were collected in a time-dependent manner. Fractions were air dried and subsequently sequenced directly. The specificity of the observed patient specific sequences was tested via real time quantitative PCR on a LightCycler system.