Copy number variations in primary tumor, serum and lymph node metastasis of bladder cancer patients treated with radical cystectomy

Abstract

The aim of the present study was to analyze copy number variations (CNV) of multiple oncogenes and tumor suppressor genes in genomic DNA from primary tumor tissue, lymph node metastasis and cell-free DNA (cfDNA) from serum of 72 urothelial carcinoma of bladder (UCB) patients treated with radical cystectomy (RC), using multiplex ligation-dependent probe amplification (MLPA). We hypothesized that primary tumor and lymph node metastasis show similar CNV profiles, and CNV are more present in lymph node metastasis compared to primary tumor tissue. Samples from 43 (59.7%) patients could be analyzed. In total, 35 (83%), 26 (68%) and 8 (42%) patients had CNV in primary tumor, serum and lymph node metastasis, respectively. MYC, CCND1, ERBB2 and CCNE1 displayed the most frequent amplifications. In particular, CNV in ERBB2 was associated with aggressive tumor characteristics. CNV in both ERBB2 and TOP2A were risk factors for disease recurrence. The current findings show that CNV are present in various oncogenes and tumor suppressor genes in genomic DNA from primary tumor, lymph node metastasis and cfDNA from serum. CNV were more present in genomic DNA from primary tumor tissue compared to cfDNA from serum and genomic DNA from lymph node metastasis. Patients with CNV in ERBB2 and TOP2A are at increased risk for disease recurrence following RC. Further studies are necessary to validate, whether these genes may represent promising candidates for targeted-therapy.

Figure 1

Example of CNV in primary tumor and lymph node metastasis of one patient. The box plot shows data of leukocytes (reference), primary tumor and lymph node metastasis (as calculated by Coffalyser.Net software). The DNA probes are arranged by chromosomal locations. The target-specific probes have a blue and orange background in different hues (left), whereas the reference probes have a grey background (right). Only the dark blue points indicate significant CNV gains, whereas light blue and yellow points are ambiguous and not considered. As expected the leukocyte DNA does not show any CNV. The data were calculated by intra- and inter-sample comparisons. Intra-sample normalization was performed by dividing the fluorescence signal of each target-specific probe by the signal of every single reference probe in this probe. The median of all these ratios of this probe is the normalization constant. Subsequently, inter-sample comparison was performed by dividing the normalization constant of each probe of this sample by the average normalization constant of all reference (leukocyte) samples.

Figure 2

Kaplan–Meier plots of recurrence-free survival stratified by CNV in ERBB2 (A–D) and TOP2A (E) in 43 UCB patients treated with RC. Top curves (in blue) show UCB patients with no CNV (no genomic aberrations), and bottom curves (in red) show patients with CNV comprising DNA gains in the 13th exon (ERBB2_13. A), 23th exon (ERBB2_23, B), 30th exon (ERBB2_30, C) and all 3 exons patients of ERBB2 (D), as well as in both exons of TOP2A (E).