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.
Zehentner
Barbara K
BK
HematoLogics, Inc., Seattle, WA 98109, USA. Barbara@hematologics.com
Fritschle
Wayne
W
Stelzer
Tess
T
Ghirardelli
Keely M
KM
Hunter
Kimberly
K
Wentzel
Collette
C
Bennington
Richard
R
Hansen
Christian L
CL
Myerson
David
D
Kalnoski
Michael
M
Wells
Denise A
DA
Loken
Michael R
MR
D000328:Adult; D001402:B-Lymphocytes / Q000473:pathology; D002051:Burkitt Lymphoma / Q000175:diagnosis / Q000473:pathology; D005240:Feasibility Studies; D005260:Female; D005434:Flow Cytometry; D015321:Gene Rearrangement; D005838:Genotype; D019337:Hematologic Neoplasms / Q000175:diagnosis* / Q000473:pathology; D006801:Humans; D008224:Lymphoma, Follicular / Q000175:diagnosis / Q000473:pathology; D008297:Male; D008875:Middle Aged; D018365:Neoplasm, Residual; D014411:Neoplastic Stem Cells / Q000473:pathology; D016133:Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000473:pathology