Tony Godfrey

PIK3CA is implicated as an oncogene in ovarian cancer

Ovarian cancer is the leading cause of death from gynecological malignancy and the fourth leading cause of cancer death among American women, yet little is known about its molecular aetiology. Studies using comparative genomic hybridization (CGH) have revealed several regions of recurrent, abnormal, DNA sequence copy number that may encode genes involved in the genesis or progression of the disease. One region at 3q26 found to be increased in copy number in approximately 40% of ovarian and other cancers contains PIK3CA, which encodes the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3-kinase). The association between PIK3CA copy number and PI3-kinase activity makes PIK3CA a candidate oncogene because a broad range of cancer-related functions have been associated with PI3-kinase mediated signalling. These include proliferation, glucose transport and catabolism, cell adhesion, apoptosis, RAS signalling and oncogenic transformation. In addition, downstream effectors of PI3-kinase, AKT1 and AKT2, have been found to be amplified or activated in human tumours, including ovarian cancer. We show here that PIK3CA is frequently increased in copy number in ovarian cancers, that the increased copy number is associated with increased PIK3CA transcription, p110α protein expression and PI3-kinase activity and that treatment with the PI3-kinase inhibitor LY294002 decreases proliferation and increases apoptosis. Our observations suggest PIK3CA is an oncogene that has an important role in ovarian cancer.

Elevated Levels of FMR1 mRNA in Carrier Males: A New Mechanism of Involvement in the Fragile-X Syndrome

Fragile-X syndrome is a trinucleotide-repeat-expansion disorder in which the clinical phenotype is believed to result from transcriptional silencing of the fragile-X mental retardation 1 (FMR1) gene as the number of CGG repeats exceeds approximately 200. For premutation alleles ( approximately 55-200 repeats), no abnormalities in FMR1-gene expression have been described, despite growing evidence of clinical involvement in premutation carriers. To address this (apparent) paradox, we have determined, for 16 carrier males (55-192 repeats), the relative levels of leukocyte FMR1 mRNA, by use of automated fluorescence-detection reverse transcriptase-PCR, and the percent of lymphocytes that are immunoreactive for FMR1 protein (FMRP). For some alleles with>100 repeats, there was a reduction in the number of FMRP-positive cells. Unexpectedly, FMR1 mRNA levels were elevated at least fivefold within this same range. No significant increase in FMR1 mRNA stability was observed in a lymphoblastoid cell line (160 repeats) derived from one of the carrier males, suggesting that the increased message levels are due to an increased rate of transcription. Current results support a mechanism of involvement in premutation carriers, in which reduced translational efficiency is at least partially compensated through increased transcriptional activity. Thus, diminished translational efficiency may be important throughout much of the premutation range, with a mechanistic switch occurring in the full-mutation range as the FMR1 gene is silenced.

Quantitative mRNA Expression Analysis from Formalin-Fixed, Paraffin-Embedded Tissues Using 5′ Nuclease Quantitative Reverse Transcription-Polymerase Chain Reaction

Analysis of gene expression and correlation with clinical parameters has the potential to become an important factor in therapeutic decision making. The ability to analyze gene expression in archived tissues, for which clinical followup is already available, will greatly facilitate research in this area. A major obstacle to this approach, however, has been the uncertainty about whether gene expression analyses from routinely archived tissues accurately reflect expression before fixation. In the present study we have optimized the RNA isolation and reverse transcription steps for quantitative reverse transcription-polymerase chain reaction (RT-PCR) on archival material. Using tissue taken directly from the operating room, mRNAs with half-lives from 10 minutes to >8 hours were isolated and reverse transcribed. Subsequent real-time quantitative PCR methodology (TaqMan) on these cDNAs gives a measurement of gene expression in the fixed tissues comparable to that in the fresh tissue. In addition, we simulated routine pathology handling and demonstrate that this method of mRNA quantitation is insensitive to pre-fixation times (time from excision to fixation) of up to 12 hours. Therefore, it should be feasible to analyze gene expression in archived tissues where tissue collection procedures are largely unknown.

Molecular cytogenetic analysis of consistent abnormalities at 8q12-q22 in breast cancer

Studies using comparative genomic hybridization (CGH) indicate that portions of chromosome arm 8q from 8q12 to 8qter are present at an increased relative copy number in a broad range of solid tumors. In this study we define an ∼ 1 Mb wide region that appears to be frequently abnormal in copy number or structure in breast cancer cell lines and primary tumors. This was accomplished by fluorescence in situ hybridization (FISH) with yeast artificial chromosomes (YACs) mapped to 8q12‐q22. Eleven breast cancer cell lines and ten primary tumors were analyzed. A minimal region of rearrangement was localized to the CEPH‐YAC 928F9 in three breast cancer cell lines with unbalanced translocation breakpoints mapping in this region. Unbalanced translocations also were detected in two primary tumors mapping between CEPH‐YAC clones 890C4 and 936B3, flanking 928F9. An increased copy number in the minimal region was detected in nine cell lines and in multiple primary tumors. This suggests the possibility that a single gene mapping to 928F9 is involved in breast cancer development or progression and may be deregulated by copy number increases in some tumors and by translocation in others. Four expressed sequence tags were mapped to YAC 928F9 and analyzed for rearrangements by Southern analysis and for abnormal expression by Northern analysis. Genes Chromosomes Cancer 22:105–113, 1998.

WHOLE GENOME AMPLIFICATION AND MOLECULAR GENETIC ANALYSIS OF DNA FROM PARAFFIN-EMBEDDED PROSTATE ADENOCARCINOMA TUMOR TISSUE

PURPOSEnOften tissues obtained from prostate adenocarcinoma tumors embedded in paraffin are heterogeneous in cell type and must be carefully microdissected to acquire tissue fragments that provide homogeneous aliquots of tumor clones. Such tissue fragments rarely contain sufficient DNA to perform genomic characterization needed as an early step in localizing relevant oncogenes or tumor suppressor genes. We report that PCR using a degenerate oligonucleotide primer (DOP-PCR) can be applied to DNA samples from microdissected paraffin-embedded prostate adenocarcinomas, and this provides sufficient product for fluorescent allelic imbalance measurements or comparative genomic hybridization (CGH).nnnMATERIALS AND METHODSnSamples were selected to be representative of those routinely obtained during prostatectomies, based on typical tumor stages (T2 and T3) and Gleason grades (range 3 +3 to 4 +5). For DNA analysis without prior DOP-PCR, only large tumors were selected to be sectioned. More than 50 specimens were analyzed. Close comparison of data obtained from analysis of DOP-PCR with those from non-DOP DNA was obtained on a subset 8 samples. To compare the allelic balance of DOP-PCR amplified DNA with that measured for non-DOP DNA, we analyzed allelic ratios on DNA from 5 different tissue samples processed by both microdissection and conventional sectioning.nnnRESULTSnSystematic comparison of allelic imbalance results shows close similarity between DOP-PCR amplified product and non-DOP DNA, indicating that PCR product is a valid representation of the tumor genome. In addition, the difference between allelic balance and imbalance is more distinctive when microdissection followed by DOP-PCR is performed. Performing CGH on products of DOP-PCR also shows distinctive regional copy number alterations in DNA from microdissected tumor tissue.nnnCONCLUSIONnEither of these procedures allows distinction between benign and malignant genomes, and also allows independent analysis of genomic alterations in different portions of tumors. They also may be applied clinically for genomic characterization of small foci that frequently appear in prostates of elderly men who are showing no obvious pathological symptoms of adenocarcinoma.

Allelic imbalance mapping of chromosome 16 shows two regions of common deletion in prostate adenocarcinoma

Allelic imbalance (AI) has now been reported on the long arm of chromosome 16 in several cancers including breast, prostate, hepatocellular carcinoma, and Wilms tumor. Such nonrandom AI is commonly associated with the presence of a tumor suppressor gene (TSG) at or near the tested locus. Previous studies in our laboratory indicated that prostate cancer genomes frequently exhibit a region of allelic loss near the q terminus of chromosome 16. Here we report a detailed, PCR based, allelic imbalance study at ten polymorphic loci on 16q. The data indicate that there are two common regions of 16q AI in prostate cancer, one at 16q21-22 (50% of informative cases) and another at 16q24.2-qter (56% of informative cases). These are similar to regions of 16q previously shown to exhibit AI in breast cancer. Neither of these regions shows correlation of AI with the clinical parameters; Gleason grade, tumor stage, or metastases.