Identification of chromosome 9 alterations and p53 accumulation in isolated carcinoma in situ of the urinary bladder versus carcinoma in situ associated with carcinoma

Abstract

Carcinoma in situ (CIS) of the urinary bladder is a flat, aggressive lesion and may be the most common precursor of invasive bladder cancer. Although chromosome 9 alterations are among the earliest and most prevalent genetic alterations in bladder cancer, discrepancy exists about the frequency of chromosome 9 losses in CIS. We analyzed 22 patients with CIS of the bladder (15 patients with isolated CIS, 7 patients combined with synchronous pTa or pT1 carcinomas) for gains and losses of chromosome (peri)centromere loci 1q12, 7p11-q11, 9p11-q12, and 9p21 harboring the INK4A/ARF locus (p16(INK4A)/p14(ARF)) and INK4B (p15(INK4B)) by multiple-target fluorescence in situ hybridization, and for p53 protein accumulation by immunohistochemistry. In 15 of 20 (75%) CIS lesions analyzed p53 overexpression was detected, whereas aneusomy for chromosomes 1 and 7 was identified in 20 of 22 (91%) CIS. In 13 of 22 (60%) CIS cases analyzed, 12 of which were not associated with a synchronous pTa or pT1 carcinoma, no numerical losses for chromosome 9 (p11-q12 and 9p21) were detected as compared with chromosomes 1 and 7. Furthermore 6 of 12 (50%) patients showed a metachronous invasive carcinoma within 2 years. In the remaining nine biopsies CIS lesions (40%) were recognized that showed losses of chromosome 9p11-q12 and 9p21, six of these were associated with a synchronous pTa or pT1 carcinoma. Three of these carcinomas were pTa and exhibited loss of 9q12 as well as a homozygous deletion of 9p21. The others were invasive carcinomas in which CIS lesions were also recognized that showed no numerical loss of chromosome 9, but did show an accumulation of p53. In conclusion our data demonstrate that predominantly isolated CIS lesions contained cells with no specific loss of chromosome 9, as opposed to CIS lesions with synchronous carcinomas that showed evidence of chromosome 9 loss. Furthermore our data strengthen the proposition that p53 mutations (p53 overexpression) precede loss of chromosomes 9 and 9p21 in CIS as precursor for invasive bladder cancer, as opposed to noninvasive carcinomas where chromosome 9 (9p11-q12) losses are early and frequently combined with homozygous deletions of 9p21.

Figure 1.

Histopathology, clinical data, and p53 immunohistochemical genetic analyses of CIS from 22 patients. a: □, no p53 accumulation; ▪, p53 accumulation; ┌, heterogeneity with respect to accumulation (no diffuse pattern, but distinct separate histological areas). b: □, disomy for 1c and/or 7c; ▪, aneusomy for chromosomes 1 and/or 7 (copy number >2). c: □, balanced chromosome copy numbers for 9c and 9p21 as compared with 1c and/or 7c copy numbers; ▪, loss of 9c and/or 9p21 (lower copy numbers). Copy numbers are estimated on basis of maximum number of FISH signals. ┌; Heterogeneity with respect to loss. d: HZD, homozygous deletion of 9p21; □, no HZD; ▪, HZD. e: Extreme large nuclei were recognized indicating polyploidization of aneuploid cells. f: Trisomy for 9c and 9p21. n.e., not evaluated; cyst., cystectomy.

Figure 2.

FISH for chromosomes 7, 9, and 9p21, and p53 immunohistochemistry of tissue sections from patients diagnosed with CIS of the bladder. A–D: Case 4. A: Hematoxylin staining showing three histological different areas, ie, nest of Brunn (area 1), normal epithelium (area 2), and CIS (area 3), with corresponding hybridization images in B, C, and D, respectively. The CIS region was classified as tetrasomic for 7c, 9c, and 9p21, whereas the other areas were disomic. FISH with directly labeled probes for chromosomes 7, 9, and 9p21 are designated as green (7c), red (9c), and blue (9p21); numbers (eg, 2/2/2) indicate the classified copy numbers for the three probes, respectively. E–H: Case 16. E and G: p53-positive staining in two different CIS areas, with corresponding FISH images in F and H, respectively. In F an aneusomy for both chromosomes without loss of chromosome 9. Arrowhead indicates polyploid cell (high copy numbers for both chromosomes). In H loss of chromosome 9 is detected. FISH with probes for chromosomes 1 and 9 in a dual-color tyramide signal amplification detection system, designated as green (1c) and red (9c). Numbers (eg, 4/4) indicate the classified copy numbers for the two probes, respectively. I–K: Case 19. I: Hematoxylin staining of a synchronous lesion demonstrating detached cells. J: Cells with a disomy for the three loci (stromal site). K: Selective loss of chromosome 9 together with a homozygous deletion of 9p21. Probe set, colors, and numbers see A–D. L–N: Case 21. L: Hematoxylin staining of a synchronous papillary tumor showing different graded areas G2 (area 1), G3 (area 2), and corresponding hybridization images in M and N, respectively. The G2 area was classified as disomic for 1c and monosomic for 9c, the high grade as tetrasomic for 1c and a disomic for 9c. For probe set, colors, and numbers see E–H.

Figure 3.

Schematic drawing of histopathological classification of tissue biopsy containing a pTaG2, pTaG3, and CIS lesion (A). The numbers (eg, 2/2) indicate the estimated copy numbers for chromosomes 7 (7c) and 9 (9c), respectively. In B, C, and D the frequency distribution of FISH signals per nucleus detected for chromosomes 7 and 9 in the low-grade, high-grade, and CIS lesion.