Detecting gene copy number alterations by Oncomine Comprehensive genomic profiling in a comparative study on FFPE tumor samples

Abstract

Copy number alterations (CNAs) play a fundamental role in cancer development and constitute a potential tool for tailored treatments. The CNAs recognition in formalin fixed paraffin embedded (FFPE) material for diagnostic purposes has relied for years mainly on fluorescence in situ hybridization. The introduction of other procedures, such as Next-Generation Sequencing has dramatically improved CNAs discovery at genome-wide level. The detection of CNAs by NGS in FFPE material is, nonetheless, a complex issue, which still requires validation studies. Herein, the CNAs detection by a widely used NGS assay (Oncomine Comprehensive Assay plus^®, OCA+) were evaluated in 14 FFPE samples mirroring diagnostic daily practice and compared to a whole-genome assay. OCA+, a targeted DNA panel, showed lower CNAs detection sensitivity and equal specificity for gains and losses. According to proprietary software pipeline, OCA+ accurately identified gains characterized by CN ≥ 5,2. No significant threshold maximizing the difference between true and false positive losses was found. Orthogonal FISH tests validated seven CNAs characterized by CN gain ≥ 6 or complete loss. Considering the CNAs growing significance in precision medicine, our findings further prompt towards a robust validation of NGS detection in FFPE materials.

Fig. 1

Example of OCA + output in A061 sample. a: Log2 ratios (LogR) of genomics segments along the chromosomes (CHRs). Horizontal brown bars show LogR clusters after genomic segmentation. b: Log odds ratios for each heterozygous single nucleotide variants (SNPs) in the assay. Horizontal brown bar shows inferred segment with similar Log odds ratio. c: total (black horizontal lines) and minor allele (red horizontal lines) copy number estimation for each of the identified genomic segments.

Fig. 2

OCA+ CNAs standard analysis metrics results. (a) boxplot showing the overall OCA+ sensitivity (median = 0,16 ) and specificity (median = 0,99 ) for gains (red) and losses (blue) assuming Oncoscan (OCS) as reference method; (b) The distributions of the copy number annotated either as TP or FP in the whole cohorts were proved to be significantly different through Mann–Whitney test; (c) gain and loss (d) receiving operating curves (ROC) results.

Fig. 3

OCA+ - OCS comparison extended analysis results. (a) panels showing the overall OCA+ sensitivity (red bars) and specificity (blue bars) of extended analysis. (b) As obtained in standard analysis (Fig. 2), the distributions of the copy number annotated either as TP or FP were proved to be significantly different through Mann–Whitney test.

Fig. 4

Figure showing a comparison between OCA + small CNAs and FISH results obtained in A063 sample. a: tumor copy number plot reported by OCA+. FGFR1 amplification and CDKN2A loss are very small and indicated by black arrows. In line with OCA + prediction, FISH patterns were suggestive of FGFR1 amplification in tight clusters (b1, white arrows) and CDKN2A FISH homozygous loss (c1, white arrows). Internal controls are present (red insets in b1 and c1), indicated by red arrows and magnified in b2 and c2.

Fig. 5

Figure showing a comparison between OCA + large genomic region CNAs (a) and FISH results (b and c) obtained in A061 sample. a): tumor copy number plot reported by OCA+. The CDKN2a loss is small while most of the 11q arm (including CCND1) was gained. b): CDKN2a FISH pattern (white arrows) indicated homozygous loss as predicted by OCA+. CCND1 FISH showed copy gains (up to six copies/cells) of intact CCND1. Interestingly and in contrast to what shown for FGFR1 (Fig. 4), the CCND1 gene copies are not arranged in clusters (indicated by with arrows). Internal control patterns are indicated by red arrows for both probes.