Frequent genetic alterations in simple urothelial hyperplasias of the bladder in patients with papillary urothelial carcinoma

Abstract

In order to understand the origin of bladder cancer, very early urothelial lesions must be investigated in addition to more advanced tumors. Tissue from 31 biopsies of 12 patients with urothelial hyperplasias and simultaneous or consecutive superficial papillary tumors were used to microdissect urothelium from 15- microm sections of biopsies. The biopsies were obtained with the recently developed highly sensitive diagnostic method of 5-aminolevulinic acid-induced fluorescence endoscopy (AFE). Besides flat and papillary urothelial neoplasms, the method of photodynamic diagnostics also detects simple urothelial hyperplasias as fluorescent positive lesions. In addition, 12 fluorescence-positive biopsies showing histologically normal urothelium were investigated. Fluorescence in situ hybridization was done using a dual color staining technique of biotinylated centromeric probes of chromosomes 9 and 17 and digoxigenin-labeled gene-specific P1 probes for chromosomes 9q22 (FACC), 9p21(p16/CDKI2), and 17p13(p53). Ten of 14 hyperplasias (70%) showed deletions of chromosome 9. In 7 out of 8 patients with genetic alterations in the hyperplasias the genetic change was also present in the papillary tumor. Six out of 12 samples of microdissected normal urothelium also showed genetic alterations on chromosome 9. Microdissection of urothelial lesions, obtained during AFE, has led to the first unequivocal documentation of genetic changes in urothelial lesions diagnosed as normal in histopathology. Thus, this technical approach is important to provide insight into the earliest molecular alterations in bladder carcinogenesis.

Figure 1.

A: ALA-induced fluorescence endoscopy of the bladder with a protoporphyrin IX red fluorescing flat lesion surrounded by blue autofluorescence (case 9). Histology of the red area revealed simple hyperplasia. B: Simple urothelial hyperplasia in the biopsy taken from the red fluorescing area in A. Thickened urothelium without papillary formations and disturbance of urothelial layering and inconspicuous nuclei are visible in a 5-μm frozen section stained with hematoxylin and eosin. Magnification, ×160. C: Methylene-blue stained 20-μm frozen section of a papillary tumor after microdissection. The papillary contours are clearly visible without significant amounts of residual stromal cells. The papillary tumor has been used as an example, since hyperplasias can be stripped more easily from the stromal compartment. Magnification, ×40. D: Fluorescence in situ hybridization using a centromeric probe for chromosome 9 and a gene-specific probe for chromosome 9p21 (p16) in metaphase spreads. Note the green signal on the short arm of chromosome 9. Magnification, ×1000. E: Fluorescence in situ hybridization using the probe for chromosome 9p21 (p16) in isolated nuclei of microdissected cells of the hyperplasia shown in A and B (case 8). The red signals are the centromeres of chromosome 9, whereas the green signals represent the gene-specific loci. There is a deletion of the 9p21 gene locus with two centromere signals and one gene-specific signal in the majority of the cells. Magnification, ×1000. F: Fluorescence in situ hybridization using the probe for chromosome 9p21 (p16) in isolated nuclei of microdissected cells of a hyperplasia (case 12). There is a homozygous deletion with one centromere signal and loss of the gene-specific signal in the majority of cells. Note rare nuclei with intact green gene-specific signal, which serves as an internal control for successful amplification. Magnification, ×1000.