Reflection on the Discovery of Carcinoembryonic Antigen, Prostate-Specific Antigen, and Cancer Antigens CA125 and CA19-9

One of the hottest current areas of research is the discovery and validation of novel biomarkers for many diseases, including cancer. For cancers, the reality is that no new major cancer biomarkers have entered the clinic in the last 30 years. Despite the emergence of highly powerful genomic, proteomic, epigenomic, metabolomic, microarray, and other omic technologies, which have been used intensively for the discovery of novel cancer biomarkers, the yield has been poor. In previous analyses, I pinpointed the possible reasons for such failures, and I proposed remedies for avoiding common and repetitive mistakes (1–4). Other accounts have done the same, including some articles in this issue (5, 6). It is astonishing that some of the most useful cancer biomarkers were discovered in the 1960s [e.g., carcinoembryonic antigen (CEA)8 and α-fetoprotein], the late 1970s [prostate-specific antigen (PSA)], and the early 1980s [cancer antigen 125 (CA125), CA19-9, and some others]. The discoverers of these biomarkers achieved their goals by means of classic analytical techniques, especially the more or less primitive (by today's standards) immunologic and chromatographic assays, or by taking advantage of the then new monoclonal antibody technology, which originated in 1975. If we compare the technological tools of the 1960s to the 1980s with those we have today and the volumes of data that we can generate in the same time with the new high-throughput technologies, we can conclude that contemporary technologies have not delivered the goods promised in the arena of cancer biomarker discovery, at least to date. We should admire the pioneers of cancer biomarker research, who used more ingenuity and inventiveness and less technology to reach their goals. Every major discovery, such as the 4 representative examples described below, has a story behind it, a group of characters, and, similar to a movie, a script, actors, and a director. Obviously, we could not cite all the unsung heroes (technicians, graduate students, and postdocs) who participated in these discoveries, but we can certainly identify the “directors.” Four of these directors comment on their teams and the environment associated with their discoveries, the impact of these discoveries in clinical care, and their projected future in cancer research. As others have said, we should look to the past for the important lessons we can use to shape the future. I hope these essays on the discovery of 4 major and clinically useful biomarkers will teach us some lessons that can guide us in overcoming the current difficulties with cancer biomarker discovery, and I hope this field will become more fertile in the years to come. The discovery of CA125 was a serendipitous event that arose from attempts to provide more effective therapy for patients with ovarian cancer. As a medical student at Harvard, I had spent 2 years with Dr. Hal Dvorak studying maturation of the immune response in guinea pigs (7, 8). After completing a medical internship at Johns Hopkins, I spent the 3 years between 1972 and 1975 at the Biology Branch of the National Cancer Institute in Bethesda working with Drs. Herbert Rapp and Berton Zbar to optimize intratumoral immunotherapy in a guinea pig hepatoma model that used bacille Calmette–Guérin (BCG), a live attenuated strain of Mycobacterium bovis widely used in the early 20th century as a vaccine against tuberculosis. Herb and Bert had found that the intense inflammatory response to local injection of BCG could produce regression of syngeneic hepatoma transplants growing on the flanks of guinea pigs, eliminate regional lymph node metastases, and induce tumor-specific immunity (9). Their work paralleled ongoing clinical studies at the National Cancer Institute on intratumoral injection of cutaneous melanoma metastases and anticipated the intravesical administration of BCG to control superficial papillary carcinomas of the bladder, a treatment that is still widely used (10). After returning to Boston to complete a medical residency and fellowship in medical oncology, I joined the faculty at the Dana Farber Cancer Institute. In establishing my own laboratory, I wanted to apply the principles that I had learned in Bethesda to develop an effective immunotherapy for a visceral cancer. An ideal candidate seemed to be ovarian cancer, in which clinically important metastases developed on surfaces of the peritoneal cavity. We thought that intraperitoneal administration of an immunostimulant might induce a chronic inflammation on the peritoneal surface that would be sufficient to control metastatic disease. Dr. Robert Knapp, the head of Gynecologic Oncology at the Brigham and Women's Hospital, and his fellow, Dr. Ross Berkowitz, who now holds that position at the same institution, were pursuing a similar strategy, so we joined forces and combined our 2 laboratories. Bob and Ross had adapted a murine model for ovarian cancer developed by Dr. Stanley Order, in which murine ovarian cancer cells grew within the abdominal cavity, blocked diaphragmatic lymphatics, and induced ascites. Intraperitoneal injection of a heat-killed preparation of Corynebacterium (Propionobacter) parvum was able to prolong the survival of tumor-bearing mice, and adding a rabbit antimurine ovarian cancer heteroantiserum further prolonged survival. My initial studies showed that the synergistic antitumor activity of the 2 agents depended on antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by macrophages attracted into the peritoneal cavity and activated by C. parvum (11). In the days before translational research, we had translated this murine model directly to the clinic to treat ovarian cancer patients with residual peritoneal disease following conventional chemotherapy. We repeatedly administered C. parvum intraperitoneally through a peritoneal-dialysis catheter. An objective response rate of 30% was observed, and 2 complete responses lasted more than a year (12). Macrophages washed from the peritoneum demonstrated enhanced activity for ADCC, suggesting that therapy might be improved by the addition of a specific antibody against human ovarian cancer. We used the then new monoclonal antibody technology developed by Kohler and Milstein (13) to develop the first monoclonal antibodies against human ovarian cancer. The 125th promising clone was designated OC125 (for ovarian cancer 125), and the cancer antigen recognized by this antibody became CA125. We found that CA125 was produced in normal amnion, Müllerian duct, and peritoneum during fetal development and in normal adult endometrium, lung, and cornea, but not in normal ovary (14, 15). Approximately 80% of ovarian cancers produced substantial amounts of CA125, but it soon became apparent that CA125 was shed from the cancer cell surface. It was found in supernatants from cultured ovarian cancer cells, limiting its potential for serotherapy. A shed antigen, however, might prove valuable as a biomarker to monitor response to treatment and thus fill an unmet clinical need. Working with Dr. Vince Zurowski at Centocor—then a start-up company housed in a commercial incubator facility at the University of Pennsylvania—we developed a homologous double-determinant RIA with OC125 to capture and detect CA125 by taking advantage of the fact that multiple identical peptide subunits of the high molecular weight mucin bound to OC125 (16). Increased CA125 concentrations were found in sera from 90% of patients with advanced ovarian cancer and in 50% of patients with stage I disease. False-positive increases occurred with benign effusions and in gynecologic conditions such as endometriosis and uterine fibroids. Increased CA125 concentrations were found in other malignancies, including endometrial, fallopian tube, breast, and lung cancers. Over the last 3 decades, the CA125 assay has evolved into a heterologous double-determinant assay (CA125II) that uses 2 epitopes: one recognized by OC125 (14) and the other by the M11 antibody developed by Tim O'Brien (17). The CA125II assay has less day-to-day variation than the original CA125 assay. A number of other antibody-based assays have been shown to be equivalent to CA125 or CA125II. Because these assays have different limits for normal values and different calibration curves, consistent use of only 1 assay should be used to monitor a particular patient. Over the years, the strengths and limitations of CA125 have been defined for a number of indications. The CA125 assay was originally developed to monitor the response to chemotherapy. When the biomarker is increased, CA125 tracks the progression or regression of ovarian cancer with >90% accuracy. A persistent increase in CA125 after primary chemotherapy was found to be a highly specific marker (>90%) for residual ovarian cancer, which led to its approval by the US Food and Drug Administration (FDA) in 1987, four years after the initial publication of the assay. Despite its high specificity, the biomarker is not optimally sensitive for detecting persistent disease. CA125 can return to normal concentrations, and residual disease can be found at second-look operations in up to half of cases. The rate at which CA125 decreases after surgery and during chemotherapy correlates statistically with prognosis. A short apparent half-life for CA125 is associated with prolonged survival; however, this correlation has not proved sufficiently precise to aid in the care of individual patients. Sequential monitoring of CA125 after surgery and chemotherapy for women in a complete clinical remission can detect disease recurrence in 70% of patients with a lead time of 3 to 4.8 months. Whether monitoring recurrence actually benefits patients has been debated. Only 1 study has evaluated this question directly (18), and this trial, although it produced negative results, had important limitations: CA125 increases within the reference interval were not considered, only 25% of the participating women received optimal treatment for recurrent disease, and secondary surgical cytoreduction was performed in only a small fraction of the patients (19). 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The of Cancer by Drs. and has women at into 3 who care in all women and CA125 by in the of women with values as by the of Cancer developed by The is to detect an in survival and will be in The only to thus was for the of the trial, which a in with a in the fraction of and cancers In to the 25% of cancers in stage I or of the cancers by were in an early CA125 by of the ovarian cancers. CA125 by operations with operations with ovarian cancers to develop 2 years before were by conventional suggesting that would be Dr. the Cancer of has a the last years with women from the of the CA125 by who were at than of the have been for after each and have been multiple years on the The operations by the have of ovarian stage and 4 in and a of for all cancers and for cancers, no more than 3 operations would be with this to detect each of ovarian cancer. were during and 2 of the 4 were still within the reference interval for CA125. from the performed in the found CA125 concentrations in only of the patients before of biomarkers have been evaluated with and to increase the of CA125 for and disease The most promising developed to CA125, and A new is and a new is to the and the of this during assay is developed to be performed on a which will assay at the of with a of by CA125 as cell surface to ovarian cancer The which has been by Dr. and Tim O'Brien is a high molecular weight 1 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