Single Cell Analysis of IG Genes in CLL: Cases with Multiple IGH Rearrangements Are Constituted of Several Independent Clones Even When Indistinguishable By Flow Cytometry

Konference: 2015 57th ASH Annual Meeting - účast ČR

Kategorie: Nádorová biologie/imunologie/genetika a buněčná terapie

Téma: 641. CLL: Biology and Pathophysiology, excluding Therapy: Poster

Číslo abstraktu: 4139

Autoři: Bc. Kamila Brázdilová; Mgr. Karla Plevová, Ph.D.; Mgr. Hana Skuhrová Francová; Mgr. Helena Kočková; MUDr. Magdalena Chmelíková; Mgr. Marek Borský; Mgr. Kateřina Burčková; Mgr. Barbara Kantorová; MvDr. Boris Tichý, Ph.D.; Dis. Hana Škabrahová; MUDr. Yvona Brychtová, Ph.D.; prof. MUDr. Jiří Mayer, CSc.; prof. MUDr. Michael Doubek, Ph.D.; prof. RNDr. Šárka Pospíšilová, Ph.D.

Current evidence suggests that in 2–5% of chronic lymphocytic leukemia (CLL) cases, multiple B cell clones – each expressing unique productive immunoglobulin gene rearrangements (P-IGH) – can be detected. However, only in 35% of them, coexistence of at least two leukemic clones was revealed by flow-cytometry (Plevova et al, Haematologica 2014). In the remaining 65% of cases, presence of multiple clones was presumed based on the number of immunoglobulin gene rearrangements detected, but underlying biological cause, also possibly including lack of allelic exclusion, was not satisfactorily proven at the level of single cells. Therefore, we developed a technique for simultaneous analysis of IGH, IGK and IGL rearrangements at the single cell level and performed further investigation of cases with multiple P-IGH.

In years 2006–2015, patients with multiple clonal P-IGHs were searched among 1670 CLL patients examined for IGHV mutation status. Identified cases were submitted to flow-cytometric measurement of surface markers CD5, CD19, CD20, CD23, CD43, CD45, sIgK, sIgL and FMC7 on sorted cells where possible. Based on this examination, patients with clonal P-IGHs exceeding the number of CLL populations distinguished by flow-cytometry were tested using single cell analysis. Patients with corresponding number of P-IGHs and CLL populations (i.e. confirmed as biclonal CLL according to the flow-cytometry and PCR-based IGH detection) served as a control of the technique accuracy. Single cell analysis technique was developed at our department to detect transcribed IGH, IGK and IGL simultaneously in individual cells. B cells from peripheral blood of CLL patients were separated by gradient centrifugation with depletion of non-B cells. Single CD19+cells were then sorted using FACS Aria III into 96-well plates containing lysis buffer, followed by 2 rounds of multiplex nested RT-PCR, capillary electrophoresis and Sanger sequencing. For each patient, 1–3 plates were analyzed. To consider an IG rearrangement clonal, it had to be detected at least in 3 wells.

We detected multiple clonal P-IGHs in 76/1670 (4,6%) CLL patients analyzed. Expression of surface markers was assessed by flow cytometry in 37/76 patients: In 24/37 (65%), the number of P-IGHs exceeded number of distinguished populations. Single cell analysis was performed in 16/24 cases, 15/16 patients displayed only one homogeneous CLL population by flow cytometry and 1/16 patient displayed two distinguished populations but three P-IGHs. Two patients with corresponding number of P-IGHs and CLL populations were used as a control. The median of wells tested per patient was 92 and P-IGH detection efficacy 83%. In 12/16 tested patients, as well as in two controls, no cell with more than one P-IGH was detected, confirming the expansion of multiple B-cell clones in all of them. Also, based on the structure of detected clonal IGH rearrangements in each case, a possibility of VH replacement was excluded. In 2 cases, we observed intraclonal diversification within one of the present clones (expressing either IGHV1-69, or IGHV3-72), a rare phenomenon described in CLL. In the remaining 4/16 cases we failed to detect one of the expected P-IGHs likely due to its underrepresentation in a sample, which is supported by results obtained from quantitative PCR with allele-specific primers in a bulk sample. Light chains were successfully detected in 10/12 analyzed cases and two controls; in 4 cases, single cell analysis revealed transcribed clonal IG rearrangements previously undetected in bulk samples. Importantly, each identified clonal IGK/IGL was repeatedly detected exclusively with only one distinct clonal P-IGH, thus constituting independent B cell receptors in independent leukemic clones.

We confirm and substantially extend the notion that oligoclonality is the major cause of multiple P-IGH detection in CLL. Obtained information on P-IGH and P-IGK/L pairing will help in further investigation of IG receptors in oligoclonal CLL, as the biological background of oligoclonality in CLL still remains to be elucidated. Supported by grants IGA NT13493-4/2012, MUNI/A/1180/2014 and AZV 15-30015A.

Disclosures: No relevant conflicts of interest to declare.

Datum přednesení příspěvku: 7. 12. 2015