Identification of molecular markers for detection of lymph node metastasis.

This work was funded by a Sponsored Research Grant from Cepheid, Sunnyvale CA.

Background: For reliable molecular detection of metastases it is necessary to have good markers. Ideally, these markers would be present in all tumor cells but completely absent lymph. One possibility is to use DNA mutations or methylation patterns that are specific to cancer cells. Unfortunately however, there are no known DNA changes that occur with high enough frequency to be practical for identification of metastatic disease. Therefore, our laboratory has focused on the use of mRNA markers. While it would be preferable if the mRNA markers were specific to cancer cells, this is not an absolute requirement and, in fact, most of the markers used in the literature are mRNA’s with restricted tissue distribution (tissue specific). In other words, the markers tend to be expressed not only in the tumor but also in the surrounding normal tissue from which the tumor is derived. For an mRNA species to be a useful metastasis marker, it needs to have high and frequent expression in primary tumors but consistently low expression in lymph nodes. The larger the difference in expression between tumors and lymph node, the better the marker may be for detection of small metastatic foci.

This project was designed to identify the best messenger RNA (mRNA) markers for detection of metastasis to lymph nodes. Six tumor types have been studied so far: Breast cancer, lung cancer (NSCLC), colon cancer, esophageal adenocarcinoma, squamous cell carcinoma of the head and neck (SCCHN) and melanoma.

Identification of Potential Markers: An extensive literature and public database survey was conducted to identify any potential markers relevant to the cancer types listed above. Resources for this survey included PubMed, OMIM, UniGene (http://www.ncbi.nlm.nih.gov), GeneCards (http://bioinfo.weizmann.ac.il/cards), and CGAP (http://cgap.nci.nih.gov). Our survey criteria were somewhat flexible but the goal was to identify genes with moderate to high expression in primary tumors and low expression in normal lymph nodes and/or blood. In addition, genes reported to be upregulated in tumors and genes with restricted tissue distribution were considered potentially useful. Finally, genes reported to be cancer-specific, such as the cancer testis antigens and hTERT, were evaluated.

Marker Screening: Screening of potential markers was conducted in two phases. All potential markers entered the primary screening phase and expression was analyzed in 6 primary tumors from each of the 6 cancer types and 10 benign lymph nodes obtained from patients without cancer (5 RNA pools with 2 lymph node RNA’s per pool). Markers that showed good characteristics for lymph node metastasis detection (i.e. high expression in primary tumors and low expression in benign lymph nodes) passed into a secondary screening phase. The secondary screen consisted of expression analysis on a larger set of primary tumors, histologically positive lymph nodes and benign lymph nodes from patients without cancer.

Results: Approximately 60 markers were analyzed in the primary screen and this data is available by clicking here: primary screen data. This data is organized alphabetically by marker name. Additional data and results of the secondary screening are available for specific tumor types using the following links: esophageal cancer (link2), SCCHN (link3), breast cancer (link4). Similar data will be made available for the other tumor types soon.