Leveraging Molecular Techniques
A team of researchers from MolecularMD was focused on PTEN, a tumor suppressor that exerts its effects via dephosphorylation. Loss of PTEN function has been associated with multiple cancers, including endometrial cancer, glioblastoma, melanoma, and prostate cancer. Loss of PTEN activity can result from more than one independent aberration, including mutations in the coding regions (exons) of the PTEN gene, genomic deletions, or promoter methylation, in which the PTEN gene may be present and unadulterated but epigenetic changes to the regulatory DNA upstream of the gene block expression, resulting in loss of the PTEN protein.
The researchers from MolecularMD evaluated FFPE tissue samples using three different methods, allowing them to assess PTEN status at both the DNA and protein levels. Both “may be of important for clinical decision-making because loss of PTEN is associated with resistance to anti-EGFR therapies,” they explained.
Using a Sanger sequencing assay on genomic DNA extracted from FFPE tissue sections and amplified with PCR they could detect known hot-spot mutations in specific exons. The automated immunohistochemistry assay they developed was validated in cell lines and FFPE tissues. If >10% of the tumor cells in a sample stained, the sample was defined as positive for PTEN protein. The two-color dual chromogenic and silver in situ hybridization (ISH) assay was scored as follows: >20% loss of PTEN signal identifies tumors having heterozygous loss of the PTEN gene; >30% loss of PTEN signal indicates homozygous loss of the PTEN gene.
Assessment of 22 FFPE tissue specimens showed that of the nine samples identified as wild-type by Sanger sequencing, seven had normal PTEN protein expression on IHC. The fact that two samples were IHC-negative suggests that mechanisms other than mutations in the coding regions of the PTEN gene are responsible for the loss of PTEN protein. Of the 13 samples found to have hot-spot mutations on Sanger sequencing, seven showed loss of PTEN protein on IHC; the other six samples had normal PTEN protein expression.
This finding also supports the observation that mutations in gene coding region hot-spots are not sufficient on their own to predict PTEN protein expression, and vice versa. The authors concluded that complementary molecular diagnostic methods that assess the mechanisms underlying changes in gene and protein expression are needed for accurate clinical assessment of PTEN status in FFPE tissue samples.