1152 - Optical genome mapping and fluorescence in situ hybridization is the best combination to identify genomic complexity and TP53 deletions in CLL patients

INTRODUCTION. Fluorescence in situ hybridization (FISH) is the gold-standard cytogenetic technique for the detection of the most frequent chromosomal alterations (CA) in chronic lymphocytic leukemia (CLL). In 2000, Döhner et al. established a hierarchical model for risk stratification including the following groups: isolated del13q, no abnormalities (normal), trisomy 12, del11q and del17p. Furthermore, complex karyotype (CK, ≥3 CA) identified by chromosome banding analysis (CBA) is also a poor prognostic and potentially predictive biomarker. Optical genome mapping (OGM) is a novel method that relies on the imaging of long DNA molecules (>250 Kb) labeled at specific sites. The unique pattern of labels throughout the genome allows their genomic location to be precisely mapped permitting the detection of both balanced and unbalanced abnormalities with high resolution. OGM has shown promising results for CK assessment in CLL (Puiggros et al, 2022). This study aims to determine the ability of OGM to detect poor prognostic biomarkers in CLL by evaluating the concordance with FISH and CBA results to investigate the potential of OGM as a routine diagnostic test.
METHODS. Fifty-six CLL patients from two European centers with FISH (13q, CEP12, ATM and TP53) and CBA data available were included. FISH was performed on fixed peripheral blood (PB) cells (n=39) or after IL2+DSP30 cultures (n=17). CBA was performed on IL2+DSP30 PB cultures (n=56) and in parallel TPA cultures (n=39). For OGM analysis, DNA was extracted from whole PB (n=22) or PBMC (n=34). Clinically relevant CA (2p, 8q, 12 gains; 6q, 8p, 11q, 13q, 15q, 17p deletions; IGH, IGK, IGL, BCL2 rearrangements), other translocations and CA ≥5Mb detected with OGM were compared with those obtained by conventional techniques.
RESULTS. Sixty-eight abnormalities were detected by FISH in the 56 patients. Of these, OGM detected 57 (84%). No additional variants were detected by OGM in the loci investigated by FISH. All FISH abnormalities present in >24% cells were identified by OGM. For the 11 abnormalities detected by FISH in ≤24% cells, OGM confirmed the aberration in four cases (4/11, range of FISH clonality percentage: 12-18%) but did not detect the abnormality in seven (7/11, range: 6-24%, median 16%). Importantly, this latter group included 27% (3/8) of cases with 17p deletions (range: 15-17%) (Table 1). If OGM was implemented as a stand-alone technique this would result in reclassification of 70% (7/10) of patients with discordant OGM/FISH results. More specifically, 4/7 patients would change from high-risk to low-risk (Table 1). Nevertheless, two of these discordant cases show high complexity by both CBA and OGM, which is a predictor of worse prognosis.Comparing CBA and OGM results, OGM detected a greater proportion of patients with CA (88% vs. 77%, p=0.193). The median number of aberrations was also higher with OGM (3 (1-65) vs 2 (1-13), p=0.469). Among the 13 patients with a normal karyotype, 6/13 showed 1 to 4 aberrations and 1/13 showed high complexity (23 CA) by OGM. Regarding patients with abnormal karyotype, OGM detected abnormalities in 42/43 patients but failed to detect an isolated 6q deletion present in 14% nuclei in one case. OGM also identified chromothripsis and chromoplexy, in 3 cases each with a further case demonstrating both.
Patients with CK (n=12; 8/12 with ≥5 CA) showed a significantly higher median number of aberrations by OGM compared with those with an abnormal non-CK (n=31) (10 (5-65) vs 3 (0-13), p < 0.001). Of note, 11 of the latter (35%) showed ≥5 aberrations by OGM (Figure 1), which was the minimum number of CA in the CK group. Remarkably, in cases with ≥5 CA by CBA (High-CK), the minimum number of aberrations by OGM was 6.
CONCLUSIONS. 1. OGM is a robust method to assess genome wide aberrations in CLL genome-wide; 2. Its independence of cell cultures and a higher genomic resolution increases the detection rate of genomic complexity. To define clear genomic complexity criteria by OGM, additional studies are needed and its clinical impact should be validated; 3. OGM sensitivity is limited and small cell clones ( < 15-25%) could be missed. Thus, FISH for del17p (TP53) detection should be maintained in the routine management of CLL patients.
ACKNOWLEDGEMENTS. 2021SGR00025, PI21/00022, FEHH-AstraZeneca; R.G-S supported by ACIF/2021/169 and CIBEFP/2022/35 from Generalitat Valenciana.