Prolonged allogeneic marrow engraftment following nonmyeloablative conditioning using 100 cGy total body irradiation and pentostatin before and pharmacological immunosuppression after transplantation.

In a canine model of dog leukocyte antigen (DLA)-identical nonmyeloablative marrow transplantation including postgrafting immunosuppression with cyclosporine (CSP) and mycophenolate mofetil (MMF), engraftment was only transient with 100 cGy total body irradiation (TBI) conditioning, indicating suboptimal pretransplant immunosuppression. In contrast, grafts after 200 cGy TBI were durable in 11/12 recipients. We hypothesized that addition of pentostatin before transplantation could, in part, substitute for 100 cGy TBI. Pharmacokinetic studies showed pentostatin significantly inhibited adenosine deaminase in canine lymphocytes. Eight dogs were conditioned with 6x4 mg/m pentostatin and 100 cGy TBI, whereas two dogs received 3x4 mg/m pentostatin plus 100 cGy TBI. All were given MMF/CSP posttransplant. All showed initial engraftment; four remained stable mixed chimeras for >32 weeks. The median duration of engraftment was 13 (range 9 to >39) weeks, which was significantly longer than in six historical controls conditioned with 100 cGy TBI alone (median 10, range 3-12 weeks) (P=0.01).

A minority of concurrent monoclonal lymphocytes and plasmacytic cells sharing light chains are genetically related in putative lymphoplasmacytic lymphoma.

Flow cytometric cell sorting combined with molecular gene rearrangement analysis was used to detect and to further characterize simultaneously occurring phenotypically distinct B cell monoclonal lymphoid and monoclonal plasma cell populations from 38 individual specimens. By sorting and subsequent gene rearrangement analysis, separate or identical monoclonality genotypes could be revealed and confirmed. In only 13 of 38 specimens, the B lymphoid cells and plasma cell populations showed an identical genotypic profile, while 25 had non-identical profiles (including 4 process control specimens). The majority of the genotypically identical group had a phenotype consistent with Waldenström's/lymphoplasmacytic lymphoma (WM/LPL), while WM/LPL phenotype was present in 16/25 of the non-identical cases. Proof of an identical monoclonal genotype for plasmacytic and B-lymphoid cell populations must be used to define WM/LPL as a distinct entity in the clinical setting of monoclonal lymphoid and plasma cells expressing the same light chains. Conversely, the confirmation of genotypically distinct populations can significantly improve confidence in diagnostic and prognostic decisions in specimens with B lymphoid lymphomas and a concurrent, possibly smoldering myeloma or multiple myeloma. These techniques are requisite in future clinical studies for diagnosis and prognosis in these diseases.

Minimal disease detection and confirmation in hematologic malignancies: combining cell sorting with clonality profiling.

BACKGROUND: In this study we demonstrate the technical application of flow cytometry and cell sorting combined with gene-rearrangement clonality profiling to detect and confirm minimal disease in 2 leukemia and 2 lymphoma cases. METHODS: Specimens with low percentages (0.05%-5%) of abnormal lymphoid populations were identified by flow cytometry. The abnormal lymphoid populations were sorted by flow cytometry, and the purified tumor populations along with unsorted fractions were subsequently analyzed for the presence of clonal gene rearrangements by PCR and fluorescence-based capillary electrophoresis fragment analysis. RESULTS: In 3 cases, distinct clonality profiles could be detected in the purified tumor cell fraction, and suspicious amplicons of identical sizes were detected among the polyclonal backgrounds in the unsorted specimens. For 1 patient, a monoclonal signal was detected in the sorted tumor cell fraction but not in the unseparated bone marrow specimen containing 0.05% abnormal lymphoblasts. A subsequent bone marrow specimen containing 4.8% recurring leukemia cells tested positive with a clonality profile that matched the previous profile in the sorted cell population. CONCLUSIONS: The described method integrating 2 technologies allows genotypic confirmation of an aberrant population detected by immunophenotype to increase diagnostic certainty. This strategy provides a sensitive tool for disease monitoring without the need for patient-specific primer design and assay optimization required for quantitative PCR analysis.