Copy number alterations in urothelial carcinomas: their clinicopathological significance and correlation with DNA methylation alterations

Abstract

The aim of this study was to clarify the genetic backgrounds underlying the clinicopathological characteristics of urothelial carcinomas (UCs). Array comparative genomic hybridization analysis using a 244K oligonucleotide array was performed on 49 samples of UC tissue. Losses of 2q33.3-q37.3, 4p15.2-q13.1 and 5q13.3-q35.3 and gains of 7p11.2-q11.23 and 20q13.12-q13.2 were correlated with higher histological grade, and gain of 7p21.2-p21.12 was correlated with deeper invasion. Losses of 6q14.1-q27 and 17p13.3-q11.1 and gains of 19q13.12-q13.2 and 20q13.12-q13.33 were correlated with lymph vessel involvement. Loss of 16p12.2-p12.1 and gain of 3q26.32-q29 were correlated with vascular involvement. Losses of 5q14.1-q23.1, 6q14.1-q27, 8p22-p21.3, 11q13.5-q14.1 and 15q11.2-q22.2 and gains of 7p11.2-q11.22 and 19q13.12-q13.2 were correlated with the development of aggressive non-papillary UCs. Losses of 1p32.2-p31.3, 10q11.23-q21.1 and 15q21.3 were correlated with tumor recurrence. Unsupervised hierarchical clustering analysis based on copy number alterations clustered UCs into three subclasses: copy number alterations associated with genome-wide DNA hypomethylation, regional DNA hypermethylation on C-type CpG islands and genome-wide DNA hypo- and hypermethylation were accumulated in clusters A, B(1) and B(2), respectively. Tumor-related genes that may encode therapeutic targets and/or indicators useful for the diagnosis and prognostication of UCs should be explored in the above regions. Both genetic and epigenetic events appear to accumulate during urothelial carcinogenesis, reflecting the clinicopathological diversity of UCs.

Fig. 1.

Validation of array CGH analysis by FISH. (A) Array CGH profiles of representative tissue specimens (T1 to T4). The signal ratios of the CDKN2 locus in T1, T2 and T3 corresponded to copy numbers of 0, 1 and 2, respectively, whereas the signal ratio in T4 did not correspond to any whole number. (B) Although the LSI p16 (9p21) SpectrumOrange/CEP 9 SpectrumGreen Probe corresponding to the CDKN2A gene revealed two signals in stromal cells and adjacent non-cancerous urothelial cells, it revealed no signal in cancer cells in T1. (C) FISH analysis using the same probe revealed one signal in cancer cells in T2. (D) FISH analysis using the same probe revealed two signals in cancer cells in T3. (E) FISH analysis using the same probe revealed copy number heterogeneity in T4: cancer cells in areas 1 and 2 showed two signals and one signal within a tumor, respectively. These findings can explain the array CGH profile of T4 in panel (A).

Fig. 2.

Copy number alterations and their clinicopathological impacts in UCs. The incidence of copy number alterations on chromosomes 1–22 in UCs (T1 to T49) is shown. Gains (copy number: ≥3) and losses (copy number: 1 or 0) are shown in the upper and lower halves, respectively. Copy numbers of 0, 1, 3 and more are shown in dark red, light red, light blue and dark blue, respectively. The homozygously deleted regions on 3q26.1 and 4q13.2 are indicated by arrows. Locations of the array CGH probe on which copy number alterations were significantly correlated (unpaired T-test with Bonferroni correction, P < 0.00714) with histological grade (a), depth of invasion (b), lymph vessel involvement (c), vascular involvement (d), tumor configuration (papillary versus non-papillary, (e) lymph node metastasis (f) and recurrence (g) of UCs are indicated by ‘X’ under each of the histograms for chromosomes 1–22.

Fig. 3.

Correlations between copy number alterations on representative chromosomes and clinicopathological parameters of UCs. The 49 UCs (T1 to T49) were divided into recurrence-negative (n = 42) and -positive (n = 7) cases (A, C and J), histologically low-grade (n = 19) and high-grade (n = 30) tumors (B, E, F, H and O), lymph node metastasis (pN)-negative (n = 44) and -positive (n = 5) tumors (D), lymph vessel involvement (Ly)-negative (n = 33) and -positive (n = 16) tumors (G, L and M), and papillary (n = 28) and non-papillary (n = 21) tumors (I, K and N). −, negative; +, positive. The incidence of copy number alterations on chromosomes 1 (A), 2 (B and C), 3 (D), 4 (E), 5 (F), 6 (G), 7 (H), 8 (I), 10 (J), 15 (K), 17 (L), 19 (M and N) and 20 (O) in each of the UC groups is shown. Gains (copy number: ≥3) and losses (copy number: 1 or 0) are indicated in the upper and lower halves, respectively. Copy numbers of 0, 1, 3 and more are shown in dark red, light red, light blue and dark blue, respectively.

Fig. 4.

Unsupervised two-dimensional hierarchical clustering analysis based on array CGH analysis of UCs (T1 to T49). Forty-nine patients with UCs were hierarchically clustered into three subclasses, clusters A (n = 4), B₁ (n = 12) and B₂ (n = 33), based on copy numbers. Copy numbers of 0 or 1 (loss), 2 (no change) and ≥3 (gain) on each probe are shown in blue, yellow and red, respectively. The cluster trees for tumors and probes are shown at the top and to the left of the panel, respectively.