Single-Cell Analysis and Next-Generation Immuno-Sequencing Show That Multiple Clones Persist in Patients with Chronic Lymphocytic Leukemia.
The immunoglobulin heavy chain (IGH) gene rearrangement in chronic lymphocytic leukemia (CLL) provides a unique molecular signature; however, we demonstrate that 26/198 CLL patients (13%) had more than one IGH rearrangement, indicating the power of molecular technology over phenotypic analysis. Single-cell PCR analysis and next-generation immuno-sequencing identified IGH-defined clones. In 23% (18/79) of cases whose clones carried unmutated immunoglobulin heavy chain variable (IGHV) genes (U-CLL), IGH rearrangements were bialleic with one productive (P) and one non-productive (NP) allele. Two U-CLL were biclonal, each clone being monoallelic (P). In 119 IGHV-mutated (M-CLL) cases, one had biallelic rearrangements in their CLL (P/NP) and five had 2-4 distinct clones. Allelic exclusion was maintained in all B-clones analyzed. Based on single-cell PCR analysis, 5/11 partner clones (45%) reached levels of >5x10(9) cells/L, suggesting second CLL clones. Partner clones persisted over years. Conventional IGH characterization and next-generation sequencing of 13 CLL, 3 multiple myeloma, 2 Waldenstrom's macroglobulinemia and 3 age-matched healthy donors consistently identified the same rearranged IGH sequences. Most multiple clones occurred in M-CLL, perhaps indicative of weak clonal dominance, thereby associating with a good prognosis. In contrast, biallelic CLL occurred primarily in U-CLL thus being associated with poor prognosis. Extending beyond intra-clonal diversity, molecular analysis of clonal evolution and apparent subclones in CLL may also reflect inter-clonal diversity.
Department of Oncology, University of Alberta and Cross Cancer Institute, Edmonton, Canada.
Jitra
J
Kriangkum
Department of Oncology, University of Alberta and Cross Cancer Institute, Edmonton, Canada.
Sarah N
SN
Motz
Department of Oncology, University of Alberta and Cross Cancer Institute, Edmonton, Canada.
Tanner
T
Mack
Manitoba Institute of Cell Biology, Winnipeg, Canada.
Sara
S
Beiggi
Department of Oncology, University of Alberta and Cross Cancer Institute, Edmonton, Canada.
Eva
E
Baigorri
Department of Oncology, University of Alberta and Cross Cancer Institute, Edmonton, Canada.
Hemalatha
H
Kuppusamy
Department of Oncology, University of Alberta and Cross Cancer Institute, Edmonton, Canada.
Andrew R
AR
Belch
Manitoba Institute of Cell Biology, Winnipeg, Canada.
James B
JB
Johnston
Department of Oncology, University of Alberta and Cross Cancer Institute, Edmonton, Canada.
Linda M
LM
Pilarski
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001402:B-Lymphocytes / Q000276:immunology*; D002999:Clone Cells / Q000276:immunology; D005260:Female; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics / Q000276:immunology* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D011379:Prognosis; D059010:Single-Cell Analysis
D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region