The Translational Research Laboratory at MGH utilizes an adaptation of Applied Biosystems’ SNaPshot® pyrosequencing kit to determine genetic alterations in regions that are known to cause abnormal activation. The genetic material is extracted from standard paraffin blocks using a completely automated robotic platform. Then the regions in question are amplified and analyzed in a multiplex format.
“We do not know all the oncogenes that exist in human cancer, but as cancer gene discovery efforts continue, we will continue incorporating relevant mutations into our panel,” says Dr. Ellisen. The MGH Cancer Center is part of a national collaborative effort called the Lung Cancer Mutation Consortium funded by the American Recovery and Reinvestment Act of 2009. Together with 12 other cancer centers across the country, Mass General collects linked clinical, pathologic, and genomic data on over 1,000 patients.
The MGH Cancer Center is also involved in several clinical trials with investigational drugs directed against abnormally activated proteins or pathways. Currently, only a few FDA-approved cancer therapies are based on a genetic profile. Nevertheless, information that Dr. Ellisen has collected has already teased out a few unexpectedly activated pathways, especially in tumors that became resistant to the first line of therapy. This information may serve as a basis for development of new genetically targeted drugs, and additional studies may identify molecular predictors of response to therapy.
Patient outcome could be improved by using targeted therapies based upon the molecular signature of the original tumor. However, establishing the tissue of origin is quite difficult for many metastatic malignancies. Even after extensive immunohistochemistry profiling, the primary site remains unidentified for 30–60% of cases.
“Metastatic undifferentiated tumors retain at least some of the molecular signatures of the parent tissues, but the search for such signatures using mRNA chips was not initially very successful,” comments Federico A. Monzon, M.D., medical director of molecular diagnostics at The Methodist Hospital in Houston.
“Using high-density microarray platforms for clinical diagnostics proved to be challenging due to high interlaboratory preanalytical variability and computational complexity arising from interpreting thousands of data points,” he says. “The Pathwork TOO test developed by Pathwork Diagnostics seems to address both issues. In our studies it demonstrated the ability to issue unambiguous calls for 15 tissue types. It also showed good reproducibility in blinded studies conducted by four independent labs.”
The test is based on interpretation of the expression patterns of 1,550 genes in comparison with the patterns established for selected tumor types such as bladder, breast, and colorectal. In each signature, a single gene is only predictive in combination with other genes, requiring pairwise comparisons between 1,550 genes on the chip.
For each specimen, the test reduces this highly complex expression data into 15 numerical similarity scores (0 to 100). In order to develop the scoring system based on this multiplex pattern, the whole chip was trained on a large set of 2,039 specimens. Dr. Monzon and collaborators further validated the test in a blinded multicenter study with 547 metastatic or poorly differentiated primary tumor specimens.
The validation study resulted in 87.8% accuracy of determination, with only 5% of specimens with indeterminate results. The Pathwork test has since been validated on paraffin-embedded tissues and approved by the FDA.
“The paradox of validation studies for molecular profiling of unknown primary tumors,” continues Dr. Monzon, “lies in the fact that we cannot use tumors of unknown origin. By definition, these tissues lack the clear origin that could be defined by other means and thus cannot be used as a standard to evaluate performance. Future studies, thus, are focusing on the demonstration that clinical management based on expression profiling can improve treatment outcomes for patients with cancer of unknown primary.”