C35 (2003)

Targeted immunotherapies have already shown their value in cancer treatment, including trastuzumab and rituximab. Encouraged by the success of these existing therapies, numerous research groups are hunting for other tumor-specific proteins that might make good targets for antibody therapy. Now, one research group at the University of Rochester thinks they might have identified one such antigen that is expressed almost exclusively in breast cancer cells. The leader of the team, Deepak m. Sahasrabudhe, MD, Professor of Medicine and Oncology at the University of Rochester (NY) School of Medicine and Dentistry and co-founder of Vaccinex, Inc., a privately owned company, also in Rochester, discussed the data in a teleconference last month arranged by the American Association for Cancer Research soon after the annual meeting was postponed. Dr. Sahasrabudhe's study was one of four selected by AACR to highlight studies that the organization deemed to be of particular scientific importance. 3 Criteria Dr. Sahasrabudhe noted that when the team first set out to identify a new breast cancer marker that would be valuable for immunotherapy, they laid out three criteria that any successful candidate had to meet. First, the protein had to be abundant in cancer cells and expressed in tumors derived from multiple individuals.Figure: Deepak m. Sahasrabudhe, MD: “We found that in high-grade tumors, about 34% were positive for both Her2/neu and C35, and an additional 31% were positive for C35 only and not for Her2/neu. None were positive for Her2/neu and negative for C35. We think this suggests that C35 is likely to be a prognostic factor.”Second, the target should not be expressed in normal tissue because such expression could lead to damage of healthy tissue by a therapeutic antibody. Third, the target had to be antigenic in humans—that is, induce human lymphocytes to make antibodies. To identify such a candidate gene, the researchers screened four breast cancer tumor cell lines against a cell line derived from normal ductal tissue. They came up with one gene, C35, that appears to meet all three of their criteria. The gene encodes a small 115 amino acid protein, but at this point the researchers can't say what the protein does or how it functions. Using both antibody and nucleic acid detection methods, Dr. Sahasrabudhe's team has found that the protein is frequently overexpressed in breast cancer cells but not in normal tissue. For example, when they examined primary breast carcinomas by Northern blot analysis, which tests whether the messenger RNA is present in the cells, they found that 22 of 38 samples (58%) expressed C35, but that normal tissues did not, except testes. When they used an antibody raised against C35 to detect the proteins via immunohistochemistry in breast cancer lumpectomy samples, they found that 65% of the tumors contained C35 protein, while the surrounding normal tissue did not. Interestingly, the C35 gene is located on chromosome 17, very close to the gene that encodes Her2/neu. With this information in mind, Dr. Sahasrabudhe looked to see if the two proteins were simultaneously expressed in high-grade tumors. “We found that in high-grade tumors—Grades II or III—about 34% were positive for both Her2/neu and C35, and an additional 31% were positive for C35 only and not for Her2/neu. None were positive for Her2/neu and negative for C35,” said Dr. Sahasrabudhe. “We think this suggests that C35 is likely to be a prognostic factor.” Caveats However, Mark D. Pegram, MD, Associate Professor of Medicine and Director of the Women's Cancer Program at the Jonsson Comprehensive Cancer Center at UCLA School of Medicine, said he is not convinced quite yet that C35 will be the next best cancer marker. “I hope it is,” Dr. Pegram said in an interview. “Unfortunately, in the history of breast cancer, most of these things that come up as potential markers fizzle out after a while, but this looks exciting at this point.” He noted that in order for the Rochester team to validate C35 as a marker and potential therapeutic target, they need to find out what the protein does and prove that it is directly related to pathogenesis.Figure: Mark D. Pegram, MD, noted that in order for C35 to be validated as a marker and potential therapeutic target, it would be necessary to find out what the protein does and prove that it is directly related to pathogenesis—for example, what happens in animal models where the gene is either overexpressed or knocked out and whether such alterations change the pattern of tumorigenesis.For example, he said he'd like to see what happens in animal models where the gene is either overexpressed or knocked out: Do such alterations change the pattern of tumorigenesis?, he asked. “A lot of markers look great at first, but until you prove they are involved in pathogenesis it's hard to know what to make of them. It is really unfortunate that the AACR meeting was canceled. I would have liked to see all of their data,” said Dr. Pegram, who is familiar with the team's work only through their abstracts from last year's ASCO annual meeting and the online abstracts from this year's AACR meeting, now rescheduled for July. Dr. Pegram also cautioned about putting too much weight on the differential expression of C35 and Her2/neu in the high-grade tumor samples. Detecting Her2/neu expression has been “notoriously difficult,” he noted, and that difficulty potentially leads to a disproportionate number of false negatives for that protein. Because of that complication, he would like to see the team check whether the C35 gene is co-amplified with the gene that encodes Her2/neu as detected by fluorescence in situ hybridization (FISH) methods, which has been the most reliable means of detecting the troublesome Her2/neu. Immunogenic For Dr. Sahasrabudhe's part, he too said that work needs to be done to understand the function of C35, but in the meantime the team is excited that the protein is immunogenic, and therefore meets all three of their criteria. When human lymphocytes are exposed to C35 in vitro, the cells make antibodies against the protein. Additionally, the researchers have found that C35 is expressed in the core of a tumor and seems to be released as these cells die. Together, these data “raise the possibility of being able to use both a vaccine and an antibody therapy targeted against C35 in breast cancer,” Dr. Sahasrabudhe said. The work on C35 was funded by Vaccinex.