SUMMARY:We have delineated regions of interest at chromosome 2q21.2, 2q36.3, and 2q37.1 by deletion mapping of 114 urothelial cancers (UC). Altogether, 17%, 18%, and 63% of the G1, G2, and G3 tumors displayed loss of heterozygosity at chromosome 2q, respectively, The region at 2q21.2 was narrowed down to the LRP1B gene (NT_005129.6). Hemi-and homozygous deletion at the LRP1B gene region was seen in 31 of 114 UCs. Only 8% of the UCs with G1 and none with G2 tumors showed loss of heterozygosity at the LRP1B gene, whereas 49% of the G3 UCs had allelic loss at this region. RT-PCR analysis of the LRP1B gene showed the lack of expression of several exons in 2 of 9 cases analyzed. Our analysis suggests that the LRP1B gene is a candidate tumor suppressor gene in UCs. (Lab Invest 2002, 82:639 -643).C ancer of the urinary bladder is one of the most common tumors in the Western world. The majority of urothelial cancers (UC) are diagnosed as noninvasive tumors (Ta), whereas 20% to 25% of the cases show an invasive growth (T1-4) at the time of first presentation. From the clinical point of view, the question arises whether these two major groups of tumors are distinct entities or correspond to different stages of progression of a single tumor entity. Pioneering cytogenetic analyses before the chromosome banding era have suggested that the number of gross karyotype alterations predicts the clinical course of UCs, for example, recurrency and progression (Falor and Ward, 1978;Lamb, 1967). Later, several studies showed that allelic changes at specific chromosomal regions and alterations of tumor suppressor genes, such as PTEN, RB, and TP53, correlate with stage and grade of bladder cancers (for review see Knowles, 1999). Comparative genomic hybridization (CGH) studies also suggested quantitative differences of genetic changes, including DNA losses at chromosome 2q22-33, 2q32-qter, and 2q34-qter regions, between the noninvasive and invasive bladder cancers (Richter et al, 1997Simon et al, 1998Simon et al, , 2000. Loss of heterozygosity (LOH) at chromosome 2q is also associated with aggressive growth of head and neck and non-small cell lung carcinomas (Ransom et al, 1998;Shiseki et al, 1994). Recently, Liu et al (2000) identified a putative tumor suppressor gene LRP1B from the chromosome 2q21.2 region that was found to be homozygously deleted in several cancer cell lines, including the bladder cancer cell line VM-CUB-2. To delineate putative tumor suppressor gene regions, we analyzed 114 UCs for 20 microsatellite loci at the chromosome 2q including those from the LRP1B region. We identified three distinct regions of LOH in 40% of tumors and found a correlation between LOH at chromosome 2q and tumor grade. Results Three Target Regions of Allelic Loss at Chromosome 2qCGH analysis of 18 Grade 3 (G3) UCs of this series revealed a gain at chromosome 2p in 7 cases and loss of DNA at chromosome 2q in 11 cases (one example is shown in Fig. 2). Therefore, we evaluated score 2 at chromosome 2p as a duplication of one allele, but at chromoso...

Identification of prognostic and predictive genomic markers requires long-term clinical follow-up of patients. Extraction of high-quality DNA from archived formalin-fixed, paraffin-embedded material is essential for such studies. Of particular importance is a robust reproducible method of whole genome amplification for small tissue samples. This is especially true for high-resolution analytical approaches because different genomic regions and sequences may amplify differentially. We have tested a number of protocols for DNA amplification for array-based comparative genomic hybridization (CGH), in which relative copy number of the entire genome is measured at 1 to 2 mb resolution. Both random-primed amplification and degenerate oligonucleotide-primed amplification approaches were tested using varying amounts of fresh and paraffin-extracted normal and breast tumor input DNAs. We found that randomprimed amplification was clearly superior to degenerate oligonucleotide-primed amplification for arraybased CGH. The best quality and reproducibility strongly depended on accurate determination of the amount of input DNA using a quantitative polymerase chain reaction-based method. Reproducible and highquality results were attained using 50 ng of input DNA, and some samples yielded quality results with as little as 5 ng input DNA. We conclude that randomprimed amplification of DNA isolated from paraffin sections is a robust and reproducible approach for array-based CGH analysis of archival tumor samples. (J Mol Diagn 2005, 7:65-71)Genomic analysis of tumor DNA allows identification of alterations in sequence and copy number for individualized diagnostic, prognostic, and therapeutic decision making. This is especially relevant as personalized treatments for cancer patients based on specific genomic alterations become clinically available. Although DNA extracted from freshly acquired samples is optimum for these analyses, it is not always feasible to freeze away such samples given the constraints of clinical practice. Thus, having optimized protocols for extraction of DNA from formalin blocks is a necessary adjunct to recently developed clinical testing for tumor DNA alterations.One of the most useful approaches for analysis of DNA copy number alterations over the entire genome uses an array-based comparative genomic hybridization (CGH) analysis of DNA clones at 1-mb resolution.1-3 Current protocols for array CGH commonly require g quantities of high-quality DNA.4 Such material is generally not available from paraffin sections of formalin-fixed material, especially when tumors are small or require microdissection to remove contaminating normal or necrotic elements.A number of protocols have been reported for extraction and genomic amplification of archival material for application to standard (chromosome-based) or even array-based CGH, with varying quality of the resultant analyses. Degenerate oligonucleotide-primed (DOP) polymerase chain reaction (PCR) 5,6 and genomic representation amplification 7,8 have been tested using s...

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We sequenced the entire mitochondrial genome in 8 chromophobe renal cell carcinomas (RCCs) and corresponding normal kidneys. Our study disclosed 68 known and 45 new sequence variations occurring 132 and 45 times, respectively. We found 6 somatic nucleotide changes in 5 out of the 8 chromophobe RCCs. One A --> T substitution occurred in the D-loop region and an insertion of a 9-bp sequence in the noncoding region of the MTNC7. One G --> A substitution and one C --> T substitution were seen in the MTRNR1 and MTRNR2 genes, respectively. One C deletion in MTND5 and one T insertion in the MTND3 gene resulted in frameshift mutations in two tumors. All somatic alterations, with the exception of the 9-bp insertion, were heteroplasmic changes. Although somatic mtDNA mutations are found in chromophobe RCCs, their role in the maintenance of tumor cell phenotype or in tumorigenesis remains to be elucidated.

To define the possible role of the VHL gene in the development of sporadic renal cell carcinomas, 91 different parenchymal tumours of the kidney have been investigated for mutation of the VHL gene by single strand conformation polymorphism (SSCP) and/or heteroduplex (HD) techniques. Chromosome 3p deletion was detected in 98 per cent of non‐papillary renal cell carcinomas and in 25 per cent of chromophobe renal cell carcinomas. In 22 of the 43 non‐papillary renal cell carcinomas, abnormally migrating DNA bands were detected by SSCP and/or HD analysis. No mobility shift was seen in any of the 23 chromophobe renal cell carcinomas. In addition, 15 papillary renal cell tumours and ten renal oncocytomas, which are characterized by genetic changes other than loss of chromosome 3p sequences, were analysed for mutation of the VHL gene. None of these tumours showed abnormal migration patterns. The results indicate that mutation of the VHL gene is associated exclusively with the development of non‐papillary renal cell carcinoma.