Konference: 2015 57th ASH Annual Meeting - účast ČR
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.
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.
Datum přednesení příspěvku: 7. 12. 2015