Copy Number Monitoring from Tumor-Only Targeted Gene Sequencing in Hematological Malignancies

The occurrence of copy number variation (CNV) observed in hematological malignancies could be up 20%~70%, variable in specific subtypes, but the available molecular diagnosis directed to the clinic are always limited among SNVs and Indels. In contrast, the copy number variation which cover broader genome region has not yet come to be implemented in clinical examination. Recently, the CNV in hematological malignancies have been extensively investigated from gene level aberration such as TP53, to arm-level or chromosomal aberrations such as iAMP21, -7q and trisomys. The detection methods also experience a lot, from conventional PCR, aCGH and snpArray to current FISH and MLPA. Despite the various amendments of those methods, their ability in CNV calling is restricted. Now with the great progresses in sequencing technology and more accumulated knowledge on CNV in diseases, it is quite necessary to evaluate the value in clinical translation.

We adopted read depth-based CNV caller ONCOCNV to review the copy number aberrations (CNAs) in 2,049 tumor-only samples from targeted gene sequencing, with certain number of unrelated samples as a reference pool. We retrospectively inspected the CNAs in the hematological malignancies patients, including ALL (n=851), AML (n=1008) and MDS (n=190) patients. By combining mutation and karyotype results, the comprehensive analysis of these results were made.

The mutations profile revealed that 663 (32.3%) cases had mutations and ten genes FLT3, NRAS, ASXL1, TET2, TP53, RUNX1, CEBPA, WT1, KRAS and NPM1 were among the top list in our patients group with incidence rate above 5%. In contrast, 432 (21.3%) cases have CNAs and the RUNX1, EZH2 and TP53 are the most common genes with copy number aberration with incidence rate above 5%. Among them, TP53 and RUNX1 were both recurrently involved in mutation and copy number aberration. In the 145 samples with TP53 mutations, 42 (28.9%) of them also occurred copy number deletion. As reported by Stegel A et al., 67% of case with TP53 mutation were accompanying with deletion and the co-occurrence had negative impact on in AML, MDS, ALL and CLL (Leukemia. 2017 Mar;31(3):705-711). While RUNX1, a transcription factor located in chromosome 21q22, plays an important role hematological malignancies, mutations (137 cases) and deletions (6 cases) were both detected. Furthermore, 166 cases detected copy number gain of RUNX1, 44 (26.5%) of them were sole RUNX1 copy number gain and the others accompany with CNAs in genes on different chromosomes. Unexpectedly, copy number aberrations were also found in 50 (2.4%) patients without mutations and karyotype abnormalities.

The copy number aberration constitutes a major part of aberrations in hematological malignancies, but they were always underestimated. If we want to achieve precision medical in hematological cancer patients, the molecular diagnosis should be comprehensive, then the copy number aberration must be provided. Especially, when come to genes with great significance in classification and prognosis, such as RUNX1, the copy number gain of RUNX1 may prompt to further screen out the iAMP21-ALL and trisomy 21 by FISH and Karyotype. Among the rare 3 iAMP21-ALL cases previously confirm by FISH signal of RUNX1, all indicated copy number gain of RUNX1 ranging from 3 to 5 by deep sequencing, and the only case with low copy number (cn = 3) was detected under 28% leukemia cells. In summary, we recommend monitoring the copy number aberration of related genes from targeted sequencing as an essential part of molecular diagnosis in hematological malignancies.