RNA degradation in human breast tissue after surgical removal: a time-course study.

There is much interest in the study of human malignancy using gene expression profiling techniques. Expression profiles obtained from microarrays utilize RNA extracted from the tissue in question. Currently, cell cultures or fresh tissue processed "quickly" are used in these studies. To our knowledge, there are no published reports of a time-course of RNA degradation in surgically removed breast tissue. Such a time-course study is critically needed. We obtained normal breast tissue from breast reduction surgery. Portions of breast tissue kept at room temperature were sampled and placed into RNAlater to preserve RNA at different time-points from 10 min to 3 h after the surgical removal. We evaluated total RNA integrity from each specimen using agarose gel electrophoresis and real-time quantitative RT-PCR analysis of four genes. Electrophoresis showed good-quality, intact RNA at all time points up to 3 h. Quantitative RT-PCR showed no difference in amplified products among all samples. Our study showed that there was no loss of RNA integrity in normal breast tissue for up to 3 h after surgical removal.

Ubiquinol cytochrome c reductase (UQCRFS1) gene amplification in primary breast cancer core biopsy samples.

OBJECTIVES: The ubiquinol cytochrome c reductase UQCRFS1 is a key subunit of the cytochrome bc1 complex (complex III) of the mitochondrial respiratory chain. The purpose of this study is to evaluate the significance of the ubiquinol cytochrome c reductase UQCRFS1 gene amplification in primary breast cancers. METHODS: Samples were obtained from image-guided core needle biopsies (CNB) in 40 patients with nontreated breast cancers. To examine UQCRFS1 gene amplification, we employed fluorescent in situ hybridization using BACs RP11-46I12 that harbors UQCRFS1 gene, RP11-110J19 that overlaps to RP11-46I12, and CA125 as a control gene. The amplification data were evaluated blindly of histopathological factors. RESULTS: Amplification of UQCRFS1 gene was found in 5 of 39 specimens (12.8%). The specimens with amplified UQCRFS1 gene were associated with high grade of cancer cells (P = 0.005). CONCLUSIONS: These results suggest that the UQCRFS1 gene appears to be involved in development of more aggressive phenotype of breast cancer.

FISH analysis of BRCA1 copy number in paraffin-embedded ovarian cancer tissue samples.

We investigated the BRCA1 gene copy number in unselected ovarian malignancies. Both additional genes (amplification) as well as deletion (loss of heterozygosity, LOH) are often thought to have a role in the initiation or progression of cancer. In addition, if there were little change, deletion studies might help identify BRCA1 mutation carriers. Forty-seven paraffin-embedded ovarian tissue blocks obtained between 1984 and 1997 were used for this study. A sample was "deletion-positive" when BRCA1-deleted cells in the tumor area were significantly different from the benign area. Twenty-five (53%) cases were found to be "deletion-positive". The average age of onset of "deletion-positive" patients was 50.8 years and of "deletion-negative" 57.8 years (P < 0.05). There was no statistical difference between groups in the staging, histology, or prognosis. A Kaplan-Meier study did show a trend towards poorer survival for "deletion-positive" patients. FISH permits unique molecular characterization of malignancies at a cellular level. Double amplification of HER-2 and c-myc predicts poor ovarian cancer survival. There appears to be a definite role for BRCA1 deletion in reducing the age of ovarian cancer onset and possibly in overall survival. Further FISH studies of this and other patient sets using additional molecular markers are needed.

CA125 and UQCRFS1 FISH studies of ovarian carcinoma.

OBJECTIVE: With the gene CA125 having recently been cloned, we chose to investigate the gene copy number of various ovarian cancer samples by FISH. As a control we chose BACs close to the chromosome 19 centromere. One of these BACs carries the gene UQCRFS1. METHODS: We developed FISH probes for CA125 and the UQCRFS1 region. We studied 22 touch preparations and 14 paraffin-embedded samples of ovarian carcinomas with known CA125 serum levels, two ovarian cancer cell lines, and one ascites sample from an ovarian cancer patient. The average copy number per cell of both probes was calculated. Metaphase analyses were done on cell lines and ascites cells to localize the signals. RESULTS: The CA125 gene mapped to 19p13.2. Three of 22 (13.6%) touch preparations and 1 of 14 (7.1%) paraffin samples had amplified levels of CA125. The cell lines and ascites sample did not have amplified CA125. Unexpectedly, 3 of 22 (13.6%) touch preparations, 1 of 14 (7.1%) paraffin samples, one cell line, and the ascites sample had amplification of the UQCRFS1 region. The amplification of the UQCRFS1 region occurred in the form of homogeneously staining regions (HSRs). Only one sample had coamplification of CA125 and UQCRFS1. CONCLUSIONS: CA125 was only sometimes modestly amplified in ovarian carcinoma, even when the serum CA125 level was highly elevated. Unexpectedly, the UQCRFS1 region was also sometimes amplified as HSRs. The UQCRFS1 protein is also known as complex III of the mitochondrial respiratory chain. This product may have an important role in malignant cells.

The use of FISH on breast core needle samples for the presurgical assessment of HER-2 oncogene status.

HER-2 status has been used in breast carcinoma as a prognostic marker to predict drug response and to select patients for trastuzumab treatment. Since immunohistochemistry (IHC) is thought to be less reliable, HER-2 testing with FISH is preferred. The analysis of HER-2 is usually performed on formalin-fixed paraffin tissue sections obtained from surgery. The use of paraffin sections is very time consuming and labor intensive. The objectives of this study were to (1) develop a simple and quick FISH protocol using touch imprints of breast core needle biopsies, eliminating the deparaffinization and pretreatment; and (2) make the HER-2 status available at the presurgical multidisciplinary treatment planning conference. A total of 50 core samples of breast carcinoma were obtained from image-guided core needle biopsy. Both FISH and IHC data were available for 46 cases. Forty-four of 46 cases (95.7%) were consistent. Two IHC 2+ cases were nonamplified (ratios of 0.99 and 1.09). It is expected that, in the near future, additional molecular markers will be used before surgery when the overall treatment plan is being developed. We conclude that HER-2 gene analysis by FISH on breast touch imprints is easily done and is a useful and reliable technique.