Karyotype Results From CpG Oligodeoxynucleotide Stimulated Chronic Lymphocytic Leukemia (CLL) Cultures Are Consistent Among Laboratories: a CLL Research Consortium (CRC) Study.
Authors: Heerema Nyla A. , Byrd John C. , Cin Paola Dal , Dell' Aquila Marie L , Koduru Prasad , Aviram Ayala , Smoley Stephanie , Rassenti Laura Z. , Greaves Andrew , Brown Jennifer R , Rai Kanti R , Kipps Thomas J. , Kay Neil E. , van Dyke Daniel 
Published: October 2019

Abstract
Abstract 1614
Poster Board I-640

Background
Cytogenetic abnormalities in B-cell CLL are important prognostic indicators for predicting disease progression and treatment response. Until recently, only interphase cytogenetics has been readily applicable to clinical use in CLL due to the inability of traditional mitogens to promote effective metaphase cytogenetic identification. Recently, cytokine or CpG oligonucleotide stimulation has been utilized to promote efficient metaphase analysis. One concern with this approach is the actual clonality and reproducibility of such clones when examined by different cytogenetic laboratories. The CLL Research Consortium (CRC) conducted a prospective trial to test (1) whether CpG would enhance detection of abnormal clones and (2) whether the CRC cytogenetic laboratories would achieve similar results when testing the same patient samples.

Methods
Initially, one laboratory compared stimulation of CLL cells using CpG versus pokeweed mitogen (PWM) + 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in the same samples. To test the reproducibility of CpG stimulation, fresh blood samples from 1 normal control and 12 CLL patients were collected at one site and distributed (blinded to phenotype and prior cytogenetic results) to 5 CRC cytogenetic laboratories where they were cultured for 3 days using CpG stimulation, harvested, banded and analyzed utilizing standard laboratory procedures. When possible, each laboratory analyzed 20 metaphases per case. Interphase FISH analysis for CLL (including probes for chromosomes 6q, 11q, 12, 13q, 17p, and IGH/CCND1) was also done on each sample.

Results
In the PWM+TPA versus CpG comparison, more cases were found to have clonal abnormalities with CpG stimulation (147 of 229, 64%) than with PWM+TPA (110 of 229, 48%; P=0.0005). All clonal abnormalities detected in the PWM+TPA cultures were also observed in the CpG cultures. In the 5-laboratory CpG comparison, the results were compiled and compared to the concurrent FISH results. The CpG karyotypes were concordant with the FISH results. The control case had a normal CpG-stimulated karyotype in all 5 laboratories. Three cases exhibited a complex karyotype; one of these also exhibited 11q-, one had 17p-, and one had neither. The cytogenetic descriptions varied modestly among labs, but all identified the complex clones. Every case including the control exhibited some nonclonal abnormal cells, but no pattern was detected among the 5 labs. When an abnormal clone was found by only 1 or 2 labs, it was present in only a very small number of cells. A small 13q- clone was observed in 2 cases in 1 and 2 labs (3 of 20 and 5 of 50 cells, respectively), and was confirmed by interphase FISH. A single lab found 2 of 30 cells with a t(12;15) in one case.

Conclusions
Abnormal clones in CLL are more readily detected with CpG stimulation than with traditional B-cell mitogens. The clonal abnormalities revealed by CpG stimulation are consistent with the “gold standard” interphase FISH results, and are reproducible among different cytogenetic laboratories. We have no evidence that CpG oligonucleotides create new clonal B-cell populations in vitro during 3 days of cell culture. CpG stimulation in vitro reveals clonal cytogenetic abnormalities and complexity in CLL that should be evaluated as potential independent prognostic parameters.

Disclosures
Kay: Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees; Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding.