Interlaboratory comparison of HER-2 oncogene amplification as detected by chromogenic and fluorescence in situ hybridization.

PURPOSE: Chromogenic in situ hybridization (CISH) is a new modification of the fluorescence in situ hybridization (FISH) technique for detection of oncogene amplification in archival tumor samples. In CISH, the oncogene probe is detected using a peroxidase reaction, allowing use of transmitted light microscopy. We compared detection of HER-2/neu amplification by CISH with a Food and Drug Administration-approved two-color FISH test in an interlaboratory setting. EXPERIMENTAL DESIGN: Formalin-fixed paraffin-embedded tumor samples from 197 breast cancers were analyzed for HER-2 amplification by CISH. Two-color FISH (PathVysion) CISH of 17 centromere was done if the observer considered it necessary to ascertain amplification status in tumors with borderline HER-2 CISH copy numbers. RESULTS: Paired CISH/FISH results were available from 192 (97%) of 197 cases, no clear difference in success rates of either method was observed. Centromere 17 CISH was considered necessary in seven tumors. CISH and two-color FISH results were concordant in 180 cases (93.8%). There were 92 and 88 tumors found HER-2 amplified and nonamplified, respectively, by both methods. Eight tumors were amplified by CISH but not by FISH, and four tumors exhibited the opposite condition (kappa coefficient 0.875). In 7 of 12 cases differences between the two methods could have related to a lack of CISH chromosome 17 information. The remaining cases were explained by difficult histology (ductal carcinoma in situ, poor representativity, dense lymphocytic infiltration, or intratumoral heterogeneity). CONCLUSIONS: These results indicate that CISH could provide an accurate and practical alternative to FISH for clinical diagnosis of HER-2/neu oncogene amplification in archival formalin-fixed breast cancer samples.

Progression to detrusor-muscle invasion in bladder carcinoma is associated with polysomy of chromosomes 1 and 8 in recurrent pTa/pT1 tumours.

Transitional cell carcinoma (TCC) provides a unique model of cancer recurrence and progression. Sequential tumours (n=100) from 57 patients with an index pTa or pT1 TCC were studied using fluorescence in situ hybridisation (FISH), to determine aberrations of chromosomes 1 and 8. Thirty-seven patients experienced recurrences; eleven developed muscle invasive tumours (pT2+). Polysomy of chromosomes 1 or 8 was associated with pT1 TCC (P=0.0017 and P=0.0037, respectively), but not with recurrence. Progression was associated with polysomy of chromosomes 1 (P=0.003) and 8 (P=0.011) in pTa/pT1 recurrences, but not with stage. In conclusion, patients who subsequently developed invasive TCC (pT2+) had significantly higher rates of aneusomy (90%) in their superficial cancers than those patients who did not progress (P=0.009). Investigation of sequential tumours in patients with recurrent and progressive TCC showed that polysomy of chromosomes 1 and 8 were linked to subsequent detrusor muscle invasion, but not recurrence per se.

A modified nick translation method used with FISH that produces reliable results with archival tissue sections.

Nick translation is used to label DNA and RNA to produce probes for in situ hybridization and Northern and Southern blotting. Fluorescence in situ hybridization (FISH) is a widely applied technique used to determine chromosomal and genetic anomalies in many biological samples. Initially the technique was applied to metaphase preparations, but the usefulness of detecting genetic anomalies in solid tumors in situ has resulted in the development of modified protocols. Formalin fixed paraffin processed tissue sections present novel challenges when applying FISH; the probes must be small (between 200 and 600 base pairs) and pretreatment is necessary before the probes can be applied to tissue sections, to promote probe access to target DNA. Here we report on a modification of a nick translation method to produce a probe that can reliably be used with FISH in paraffin processed tissue sections.

Identification of loci associated with putative recurrence genes in transitional cell carcinoma of the urinary bladder.

Following an earlier study linking monosomy 9 with recurrence of transitional cell carcinomas (TCCs) of the urinary bladder, 109 primary and recurrent TCCs (from 47 patients) were examined to explore genetic alterations at chromosome 9 associated with recurrence. Patient DNA was microdissected and extracted from archival tissue sections and analysed for loss of heterozygosity (LOH) at three regions on chromosome 9 where tumour suppressor genes (TSGs) are known to reside (INK 4A, DBC1, and TSC1). Patients were categorized into two groups, non-recurrent TCC (NR, n=18) and recurrent TCC (REC, n=29). It was noted that 12% of NR tumours, compared with 54% of REC primary tumours (p=0.01), had LOH at all informative markers spanning the TSC1 region. The risk of recurrence was significantly higher in patients with deleted TSC1 than in those who retained the TSC1 region (p=0.035). Levels of LOH at DBC1 or INK 4A were not significantly different in NR tumours than in REC primary tumours and recurrence-free survival was not affected by loss of either of these genes. Loss of all informative markers spanning chromosome 9 was observed in 0% of NR tumours compared with 25% of REC primary tumours (p=0.04). The probability of recurrence was also significantly increased in patients who had LOH at all informative markers spanning chromosome 9 (p=0.016), confirming earlier fluorescence in situ hybridization results. This study provides further evidence that recurrence in bladder cancer is a distinct event, with underlying molecular causes. It also identifies the TSC1 locus as a candidate for a TSG, which drives recurrence in a proportion of TCC patients. Loss of all informative markers, including those residing in the TSC1 region, spanning chromosome 9 was also linked to recurrence.