Improved Detection Rate of Cytogenetic Abnormalities in Chronic Lymphocytic Leukemia and Other Mature B-Cell Neoplasms With Use of CpG-Oligonucleotide DSP30 and Interleukin 2 Stimulation

Min Shi, MD, PhD; Matthew J. Cipollini, MA; Patricia A. Crowley-Bish; Anne W. Higgins, PhD; Hongbo Yu, MD, PhD; Patricia M. Miron, PhD


Am J Clin Pathol. 2013;139(5):662-669. 

In This Article

Abstract and Introduction


Detection of cytogenetic abnormalities requires successful culture of the clonal population to obtain metaphase chromosomes for study, and as such, has been hampered by low mitotic indices of mature B cells in culture. Our study presents data on the improved abnormality detection rate with the use of a CpG-oligonucleotide/interleukin 2 (OL/IL-2) culture protocol for mature B-cell neoplasms, including chronic lymphocytic leukemia (CLL) and non-CLL specimens. The increased detection rate of abnormalities, compared with unstimulated culture and traditional pokeweed mitogen culture, was statistically significant for both CLL and non-CLL neoplasms. For CLL specimens, our data also showed that for cytogenetically visible aberrations, OL/IL-2 was as, if not more, sensitive than detection with interphase fluorescence in situ hybridization (iFISH). Use of OL/IL-2 allowed a number of abnormalities to be detected, which were not covered by specific iFISH panels, especially balanced translocations. Therefore, OL/IL-2 stimulation improves diagnostic sensitivity and increases discovery rate of novel prognostic findings.


Mature B-cell neoplasms are common hematologic malignancies in the western hemisphere, with chronic lymphocytic leukemia (CLL) being the most prevalent.[1] Cytogenetic analysis is a useful adjunct to morphologic assessment for both diagnosis and prognosis, as has been particularly well-established in CLL.[2–5] However, conventional cytogenetic analysis has been hampered by a low mitotic index of mature B cells in culture.

For specific established recurring cytogenetic abnormalities, interphase fluorescence in situ hybridization (iFISH) assays are used to detect abnormalities in cells that do not divide readily in culture. FISH probes for a number of translocations diagnostic in B-cell neoplasms are commercially available [eg, t(11;14)(q13;q32) in mantle cell lymphoma, t(14;18)(q32;q21) in follicular lymphoma] as are CLL-specific FISH panels. In CLL, iFISH with a 4-assay panel (for detection of trisomy 12, and deletion of 13q, ATM, and TP53) has an approximately 80% abnormality detection rate and provides useful prognostic information because TP53 and ATM deletions correlate with poor outcome whereas isolated 13q deletion correlates with a favorable outcome.[2] However, iFISH is limited because of its targeted nature and thus cannot provide the comprehensive genomic assessment afforded by metaphase cytogenetic analysis of banded chromosomes. In addition, iFISH cannot contribute to discovery of new prognostic abnormalities that can be revealed by a complete cytogenetic analysis of metaphase chromosomes.

B-cell mitogens have been used to stimulate division of cells for chromosome analysis. Clinical cytogenetic laboratories have commonly used pokeweed mitogen (PWM), 12-O-tetradecanoyl-phorbol-13-acetate, and lipopolysaccharide with some success. For CLL, these mitogens typically yield an abnormality detection rate of approximately 40% to 50%.[6] Recently, clinical laboratories have started to assess protocols using synthetic CpG-oligodeoxynucleotides (CpG-ODN) to stimulate cell division in CLL specimens and enhance the detection rate of clonal aberrations[7–10] after research showed that these oligonucleotides induce proliferation, cytokine production, and high-affinity interleukin 2 (IL-2) receptor expression in CLL cells[11,12] through interaction with Toll-like receptor 9 on B cells.[13–15]

In the United States, studies largely from 1 group[8,9] showed improved abnormality detection with a protocol involving CpG-ODN in combination with PWM and phorbol myristate acetate. Because prior research studies showed that IL-2 prevents cell death of CLL cells activated by CpG-ODN, European groups introduced a protocol that combines IL-2 with CpG-ODN and have shown success with this culture method in CLL.[16]

Mature B-cell neoplasms are heterogeneous, and in addition to CLL, include follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, and hairy cell leukemia. Similar to CLL cells, the tumor cells in this group of disease generally do not respond well to the standard B-cell mitogens. CpG-oligonucleotide stimulation is not specific to CLL cells and has been shown to stimulate division of B cells more generally.[11,12,17–19] As such, we also investigated the impact of the combined use of CpG-oligonucleotide and IL-2 (OL/IL-2) protocol for detection of cytogenetic abnormalities in other mature B-cell neoplasms.

Herein we present data from our clinical cytogenetics laboratory that support improved abnormality detection for both CLL and other mature B-cell neoplasms with the use of OL/IL-2. Our abnormality detection rate for OL/IL-2 cultures is significantly higher than that for both PWM cultures and unstimulated (overnight Colcemid [ONC]) cultures. In addition to increasing the overall abnormality detection, our data also show that OL/IL-2 application detects more subclones with higher complexity than the other methods, with implications for possible additional prognostic stratification. Notably, many of the abnormalities detected with OL/IL-2 would not be detected with current standard FISH panels.