REFLECTION ON THE DISCOVERY OF CARCINOEMBRYONIC ANTIGEN, PROSTATE-SPECIFIC ANTIGEN AND CARBOHYDRATE ANTIGENS CA125 and CA19.9

Introduction

One of the current hottest areas of research is discovery and validation of novel biomarkers for many diseases, including cancer. For the latter, 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 intensely utilized for discovery of novel cancer biomarkers, the yield is poor. In my previous analyses, I pinpointed to the possible reasons for such failures and I proposed remedies for avoiding common and repetitive mistakes (1–4). Others have done the same, including some articles in this issue (5, 6).

It is highly astonishing that some of the most useful cancer biomarkers have been discovered in the 60s (e.g. carcinoembryonic antigen, CEA and alpha-fetoprotein), the late 70s (PSA) and the early 80s (CA125, CA19.9 and some others). The discoverers of these biomarkers achieved their goals by using classic analytical techniques, especially more or less primitive (with today's standards) immunological and chromatographic assays, or by taking advantage of the monoclonal antibody technology which originated in 1975. If we compare the technological tools of the 60s to the 80s with those that we have today, and the volume of data that we can generate per unit of time with the new high-throughput technologies, we can conclude that at least up to now, the contemporary technologies have not delivered the promised goods in the arena of cancer biomarker discovery. We should admire the pioneers of cancer biomarkers, who used more ingenuity and inventiveness and less technology to reach their goals. Every major discovery, such as the four representative ones described below, has a story behing it, with a group of characters, and resembles a movie with a script, actors and a director. Obviously, we could not cite all those unsung heores (from technicians, to graduate students and post-docs) who participated in these discoveries, but we can certainly identify the “Directors”. We asked four of them here to comment on the team and the environment associated with their discoveries, the impact of these discoveries in clinical care and their projected future in cancer research. As others said before, we should use the past to derive important lessons that could shape the future. I hope that these essays on the discovery of four major and clinically useful biomarkers will teach us some lessons which we can use to overcome the current difficulties with cancer biomarker discovery, and hope that this field will become more fertile in the years to come.

CA125: A SERENDIPITOUS BIOMARKER

The discovery of CA125 was a serendipitous event, arising from attempts to provide more effective therapy for patients with ovarian cancer. As a medical student at Harvard, I had spent two years with Dr. Hal Dvorak studying maturation of the immune response in guinea pigs (7, 8). After completing medical internship at Johns Hopkins, I had spent 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 optimizing intratumoral immunotherapy in a guinea pig hepatoma model using Bacillus Calmette Guerin (BCG), a live attenuated strain of Mycobacterium bovis widely used in the early 20^th Century as a vaccine against tuberculosis. Herb and Bert had found that the intense inflammatory response to the 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 NCI with 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 had joined the faculty at the Dana Farber Cancer Institute. In establishing my own laboratory, I had wanted to apply the principles that I had learned in Bethesda to develop effective immunotherapy for a visceral cancer. Ovarian cancer seemed to be an ideal candidate, where clinically important metastases were found on the surface of the peritoneal cavity. Intraperitoneal administration of an immunostimulant might induce sufficient chronic inflammation on the peritoneal surface 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 and so we joined forces, combining our two laboratories. Bob and Ross had adapted a murine model for ovarian cancer developed by Dr. Stanley Order, where 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 could prolong survival of tumor bearing mice and the addition of a rabbit anti-murine ovarian cancer heteroantiserum further prolonged survival. In my initial studies, the synergistic anti-tumor activity of the two agents was shown to depend upon antibody dependent cell mediated cytotoxicity (ADCC) mediated by macrophages that were attracted into the peritoneal cavity and activated by the C. parvum (11).

In the days before Translational Research, we had translated this murine model directly to the clinic, treating ovarian cancer patients with residual peritoneal disease following conventional chemotherapy using repeated intraperitoneal administration of C. parvum through a peritoneal dialysis catheter. An objective response rate of 30% was observed with two complete responses lasting 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 specific antibody against human ovarian cancer. Using the then new monoclonal antibody technology developed by Kohler and Milstein (13), we had developed the first monoclonal antibodies against human ovarian cancer. The 125^th promising clone was designated OC (Ovarian Cancer) 125 and the cancer antigen recognized by this antibody became CA125. Expression of CA125 was found in normal amnion, Mullerian duct and peritoneum during fetal development and in normal adult endometrium, lung and cornea, but not normal ovary (14, 15). Approximately 80% of ovarian cancers expressed significant amounts of CA125, but it soon became apparent that CA125 was shed from the cancer cell surface and could be found in supernatants from cultured ovarian cancer cells, limiting its potential for serotherapy. A shed antigen might, however, prove valuable as a biomarker to monitor response to treatment, filling 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 - a homologous double determinant radioimmunoassay was developed using OC125 to capture and to detect CA125 taking advantage of the fact that the multiple identical peptide subunits of the high molecular weight mucin bound to OC125 (16). Elevated levels of CA125 were found in sera from 90% of patients with advanced ovarian cancer and in 50% from patients with stage I disease. False positive elevations were found with benign effusions and gynecologic conditions including endometriosis and uterine fibroids. Other malignancies could elevate CA125 including endometrial, fallopian tube, breast and lung cancer.

Over the last 3 decades, the CA125 assay has evolved into a heterologous double determinant assay (CA125II) that utilizes two epitopes: one recognized by OC125 (14) and the other by the M11 antibody developed by Tim O'Brian (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 equivalent to CA125 or CA125II. As these assays have different limits for normal values and different calibration curves, only one assay should be used consistently to monitor a particular patient. Over the years, the strengths and limitations of CA125 have been defined for a number of indications.

Monitoring response to treatment

The CA125 assay was originally developed to monitor response to chemotherapy. When the biomarker is elevated, CA125 tracks progression or regression of ovarian cancer with >90% accuracy. Persistent elevation of CA125 following primary chemotherapy was found to be a highly specific marker (>90%) for residual ovarian cancer, leading to approval by the FDA in 1987, four years after the initial publication of the assay. Despite high specificity, the biomarker is not optimally sensitive for detecting persistent disease. CA125 can return to normal levels and residual disease can be found in up to half of cases at second look operations.

Estimating prognosis

The rate at which CA125 declines following surgery and during chemotherapy correlates statistically with prognosis. A short apparent half-life for CA125 is associated with prolonged survival. This correlation has not, however, proven sufficiently precise to aid in the care of individual patients.

Detection of recurrent disease

Sequential monitoring of CA125 after surgery and chemotherapy for women in a complete clinical remission can detect disease recurrence with a lead time of 3 to 4.8 months in 70% of patients. Whether monitoring recurrence actually benefits patients has been debated. Only one study has evaluated this question directly (18) and this trial, though negative, has significant limitations in that increases of CA125 within the normal range were not considered, only 25% of women participating received optimal treatment for recurrent disease and secondary surgical cytoreduction was performed in only a small fraction of patients (19). While each patient must decide whether she wants to be monitored with CA125, earlier detection of disease does provide additional time for participation in clinical trials and for administration of the several drugs known to have activity against the disease.

Referral to gynecologic oncologists

Several studies have documented improved outcomes when patients are referred to specially-trained gynecologic oncologists for their primary operations. Despite this fact, less than half of patients receive their primary surgery from gynecologic oncologists. CA125 has aided in identifying patients with pelvic masses who are likely to have ovarian cancer. Preoperative diagnosis has depended upon age, physical examination and imaging with ultrasonography, MRI or computerized tomography. Elevation of serum biomarkers has also been utilized to increase the accuracy of differential diagnosis. Integrating biomarker, clinical and imaging data has required mathematical analysis. The Risk of Malignancy Index (RMI) has been developed in the United Kingdom and includes menopausal status, CA125 and imaging (20). The OVA1 algorithm developed by Drs. Zhen Zhang, Dan Chan and Eric Fung working with Vermillion, includes five serum biomarkers (CA125, β₂microglobulin, transferrin, apoplipoprotein A1, and transthyretin) that are used in combination with imaging data (21). A Risk of Malignancy Algorithm (ROMA), developed by Drs. Steven Skates and Richard Moore working with Fujirebio utilizes CA125 and HE4 to triage patients for operation with a specially trained surgeon (22). The ROMA has proven more sensitive than the RMI in a direct comparison (23). The OVA1 and ROMA have not been compared directly. In different trials, the two assays exhibit comparable sensitivity (90%), but the ROMA is somewhat more specific (24).

Early detection

Five year survival for ovarian cancer patients has increased significantly over the last three decades, but rates of long-term survival have not changed, related, in large part, to diagnosis at a late stage. Up to 90% of patients can be cured when disease is detected in stage I, whereas less than 30% of patients are cured in stage III–IV. Given the prevalence of ovarian cancer in the post-menopausal population (1 in 2,500) any screening strategy must have high sensitivity for early stage disease (>75%) and very high specificity (>99.6%) in order to achieve a positive predictive value (PPV) of 10%, i.e., 10 operations for each case of ovarian cancer detected.

The Prostate, Lung, Colon and Ovary screening trial in the United Sates had screened postmenopausal women with CA125 and transvaginal sonography (TVS) and found no improvement in survival (25). How these modalities are used, however, matters. While a single determination of CA125 lacks the requisite sensitivity and specificity, greater PPV can be attained by performing TVS in a fraction of women with rising CA125. CA125 rises progressively with ovarian cancer, but remains stable over time with benign disease. The UKCTOCS trial, conducted in the United Kingdom by Drs. Usha Menon and Ian Jacobs, has randomized more than 200,000 postmenopausal women at average risk to 3 groups: 1) controls who receive routine care (101,359); 2) annual TVS in all women (50,639); and 3) annual CA125 followed by TVS in <2% of women with rising values (50,640) judged by the Risk of Ovarian Cancer (ROCA) algorithm developed by Steven Skates (26). The entire trial is powered to detect an improvement in survival and will be completed in 2015. Only the prevalence phase of the trial has been reported and a shift in stage was observed with a near doubling in the fraction of early stage (I–II) cancers detected. In contrast to the 25% of cancers usually diagnosed in stage 1 or 2, 48% of cancers detected by screening were in early stage. CA125 followed by transvaginal ultrasound detected 89% of the ovarian cancers. CA125 followed by ultrasound prompted 2.8 operations per case (O/C) compared to 36.2 O/C with annual ultrasound alone. Moreover, ovarian cancers appeared to develop 2 years before they were detected by conventional means, suggesting that annual screening will be effective.

With Dr Karen Lu, the MDACC SPORE has conducted a smaller trial over the last 10 years in 4,543 postmenopausal women at average risk using the third arm of the UKCTOCS trial with annual CA125 followed by TVS (27). Less than 0.9% of participants have been referred for TVS after each annual screening and 2.6% have been referred over multiple years on study. Eleven operations have been prompted by the ROC algorithm and have detected 6 cases of ovarian cancer – two borderline IA and four invasive high grade in Stages IA, IC, IC, and IIB. With a PPV of 60% for all cancers and 40% for invasive cancers, no more than 3-operations will be required to detect each case of ovarian cancer using this strategy. All invasive cases were detected during screening and two of the four were still within the normal range for CA125.

Using serum samples from the Prostate, Lung, Colon and Ovary (PLCO) screening trial performed in the United States, CA125 levels were found elevated in only 40% of patients prior to diagnosis (28). Panels of biomarkers have been evaluated by proteomic and multiplex techniques to increase the sensitivity of CA125 for early stage and pre-clinical disease (29). The most promising panel developed to date includes CA125, HE4, CA72.4 and MMP-7. A new algorithm is being developed and a new trial planned to determine specificity and positive predictive value of the new algorithm during annual screening. Each assay is being developed on a nanobioochip that will permit rapid assay from a drop of blood obtained by fingerstick at point of service (30).

Biology

CA125 (MUC16) may contribute to ovarian cancer pathogenesis (31). The MUC16 molecule, cloned by Dr. Ken Lloyd (32) and Tim O'Brien (33), is a high molecular weight (1 MDa) mucin with 1) an N-terminal domain, 2) up to 60 repeating tandem subunits containing identical sequences of 156 amino acids, 3) a membrane spanning domain, and 4) a short cytoplasmic tail with a phosphorylation site. The extracellular domain is highly glycosylated and can bind to mesothelin, possibly facilitating attachment of metastatic cancer cells to mesothelial cells on the peritoneal surface. Signaling through the intracellular domain does not affect proliferation, but can regulate migration, invasion and xenograft growth. In ovarian cancer cells, overexpression of MUC16 appears to relate to transcriptional or postranslational regulation rather than to amplification in most cases. In normal mice, CA125 is not required for normal development or reproduction (34) but may regulate susceptibility to neoplasia in aging animals. Much remains to be discovered regarding the role of MUC16 in health and disease.

Carcinoembryonic Antigen (CEA): Past, Present and Future

By the early-1960s, when the work on the Carcinoembryonic Antigen (CEA) was begun, studies had revealed little regarding unique molecular structures in human cancer that might be useful in the diagnosis and/or treatment of these diseases (35). It had, however, been shown in studies of artificially-induced and transplantable tumors in inbred mice that the existence of Tumor-Specific Transplantation Antigens (TSTA) did, indeed, exist, but did not necessarily lead to tumor rejection (35). Hence, the possibility that human tumors contained comparable, unique molecular structures, that would not prove adequately immunogenic to induce cancer rejection, was certainly feasible.

The problem, in the case of human cancer, however, was that of obtaining appropriate control tissue for comparison with the tumor tissue under consideration (35). It was for this reason that colon cancer was initially chosen for study since this tumor does not extend intramurally for more than 6 or 7 cm either distal or proximal to the cancerous tissue, in the gross (36). Because appropriate surgical technique frequently requires fairly extensive colonic resection, this allowed us to compare the central tumor with areas of normal bowel mucosa taken more than 7 cm away from either side of the tumor.

Normal and corresponding cancer tissues from the same donors, were compared immunologically by the techniques of immunologic tolerance and antiserum absorption (36). After a variety of analyses, a single distinctive antigenic moiety was found which was initially believed to be colon tumor-specific, but was then found to be generalized to all endodermal-derived digestive system cancers (37).

Indeed, the same molecule was found to exist in embryonic and fetal digestive tissues, obtained by spontaneous abortion, in the first and second trimesters of gestation. It had apparently disappeared, with the technology available at that time, by the third trimester, and did not reappear until tumor transformation had occurred, a phenomenon that was termed “derepressive-dedifferentiation”. Hence, the name Carcinoembryonic Antigen (CEA) was applied to the material in question (37), and effectively ushered in the field of Oncodevelopmental Biology.

CEA, now also designated by the international CD coding, as CD66e, was subsequently purified in our laboratories, and a virtually complete structural analysis of this GPI-bound cell surface glycocalyceal glycoprotein followed (35). We then demonstrated that CEA was released into the circulation where it could be detected by the radioimmunoassay in bowel cancer patients (35). With this, and other comparably sensitive techniques, CEA has been detected in low concentrations in normal bowel, and in over 70% of all human cancers.

The serum assay for CEA was the first clinical marker to achieve widespread use, and after some 45 years of scrutiny, remains the most widely used, and most useful, tumor marker assay, worldwide. It has been the standard against which all other tumor markers of clinical significance have been measured, despite the fact that the CEA does not approach the perfection of complete tumor-specificity that one would desire of an ideal tumor marker. However, the clinical significance and utility of CEA is well established and is a routine test in assisting in the diagnosis and management of bowel cancer patients, and those with other forms of cancers as well (35).

As the only marker the organization recommends, ASCO guidelines recommend serum CEA testing as a useful guide to the effectiveness of systemic therapy and as preoperative guide for staging and surgical planning. Thus, quarterly CEA assays are recommended for three years in patients with stage II or III colon or rectal cancer who are candidates for further surgery or systemic therapy (38). In addition, the National Comprehensive Cancer Network recommends serial CEA testing for five years in patients with T2 or higher disease, if the patient is a candidate for resection of isolated metastases. Thus, other than for population screening, the CEA assay remains a standard in all stages of colorectal malignancy.

Further work resulted in the elucidation of the structure of the gene that codes for the protein core of CEA (35), and remains the central character of the new CEACAM (CEA Cell Adhesion Material) (35) nomenclature of the 29 CEA gene family members. This family, itself, forms a subgroup of the Ig gene superfamily (35) and studies of the CEA and its family members continue undiminished (39, 40).

Although tumor markers, for the diagnostic roles that they play, need not have biological roles when initially defined, ongoing studies of the CEA molecule, and it's family members, have been performed vis-à-vis their functions in embryological life, in cell differentiation, in intercellular adhesion, and in carcinogenesis (35). CEA demonstrates a relatively unique form of intercellular reciprocal 2-point adhesion between CEA molecules, and important relationships to one or more integrins, and fibronectin, in the intercellular matrix. Hence, the role of CEA in metastatic potential becomes increasingly interesting (36).

The CEA system, in addition to the radioimmunoassay role for which it is most frequently used, has also been shown to be of importance in tumor imaging (35) and immunopathology. Its role in the biological treatment of cancer patients continues to expand annually with numerous clinical trials, such as that of the naked CEA gene incorporated-DNA vaccines, and of drug and isotope “homing” in conjunction with partial hepatectomy in cancers that have metastasized to the liver (35).

The advent of cancer genomics and biopharmaceuticals will obviously have enormous impacts on the areas of cancer prediction and diagnosis and of cancer treatment. Tumor markers such as CEA will likely become a footnote in the field of cancer diagnosis and treatment. But, hopefully, CEA will have been of some significance in moving the field forward.

PSA Discovery to Application: A Historic Journey

Shortly after I joined Roswell Park Memorial Institute in 1970 as a new staff scientist, my department chair took me to see Dr. Gerald Murphy, institute director and urologist. Dr Murphy warmly accepted me into the Roswell Park family and said, “Ming, you may do your tumor marker research and any research you want, but make sure that you do prostate cancer research too”. I replied, “Yes, Sir”. Thus, began my journey to PSA!

As an active investigator of CEA at the dawn of the cancer biomarker era, I welcomed the PSA project as an addition to my research portfolio. This “marching order” from the institute prompted me to submit an NIH grant application, entitled “Antigen Markers in Diagnosis of Prostate Cancer”. I proposed “We will search for prostate cancer-specific or associated antigens…Usefulness of the prostate tumor antigen as a marker for the presence of early tumor and for the evaluation of treatment will be determined”. My goal was to discover a new prostate cancer marker and to develop a simple blood test for the early detection of prostate cancer.

At that time, three quarters of prostate cancer were detected when they already had developed metastasis. The commonly used blood test for diagnosis was PAP, which was developed in 1938. Unhappily, its elevation was always a gloomy finding.

In this article, I would like to share with you some of my reflections on the discovery of the PSA and the development of the PSA test. This was a team effort. Basic science and laboratory support was provided by my own group at Roswell Park. Clinical support was provided by NPCP.

In the beginning, I worked with my own technicians. A few years later, with the support from NIH and ACS, I greatly expanded my research project, including the addition of new staff scientists and postdoctoral fellows. By means of immunochemical techniques, we used the extracts of prostate tumor as the immunogens to prepare antiserum as a reagent to differentiate prostate cancer from normal prostate.

After many years of scientifically challenging and technically difficult research, we published our first paper on PSA regarding its identification and purification in Investigative Urology in 1979 (41). The senior author, Ming Wang, was a staff scientist in my department. Monospecific antiserum and purified PSA (Mr 34,000) were obtained. Prostate specific antigen was initially abbreviated as PA. As evidence of the importance of this new discovery, this paper was cited as one of the twelve most significant articles in prostate oncology in the AUA centennial issue of the Journal of Urology, February 2002. Additionally, the Journal of Urology republished this paper as a Milestone in Urology in March 2002.

With PSA and antiPSA antiserum, we were able to show circulating PSA in prostate cancer patients, which was published in July 1980 (42). The senior author, Larry Papsidero, was a postdoctoral fellow. Shortly thereafter we developed the PSA blood test and demonstrated its diagnostic potential, which was published in December 1980 (43). The senior author, Manabu Kuriyama, was a postdoctoral fellow. It is worthwhile to note that, using this paper as reference, our PSA work was cited by AACR Centennial in 2007 as a Landmark Scientific Discovery during the past century of cancer research.

Through the NPCP, the clinical application of PSA was evaluated without delay (44). Both prognostic and monitoring value of PSA were evident immediately. A significant finding was noticed early on in our clinical study: in patients with localized cancer who received curative therapy, the usefulness of PSA in detection of early disease recurrence was always demonstrated.

The study of the biological nature of PSA was undertaken simultaneously. We reported the protease activity of PSA in 1984 with Yoshihito Ban, a postdoctoral fellow, as the senior author. The 240 amino acid sequence, determined by staff scientist Rueming Loor and colleagues, led to the conclusion that PSA is a chymotrypsin-like protease, which forms the basis of today's antichymotrypsin “complexed” vs. “free” forms of PSA. This area of investigation was pursued productively a few years later by other researchers.

It should be noted that the clinical application of PSA was based upon the prostate specificity of the PSA molecule. Prostate epithelial cell specificity of PSA was established in 1981. This prostate specificity was further assured by a series of monoclonal anti-PSA antibodies generated shortly thereafter in 1983. The availability of anti-PSA monoclonal antibodies and a simplified purification procedure of PSA from seminal plasma published in 1982 allowed large scale and mass production of the essential reagents for the PSA test and their standardization.

Additionally, our PSA patent issued in 1984 greatly facilitated the transfer of our PSA technology to the biomedical industry. Our PSA patent was non-exclusively transferred to the biomedical industry, which in turn subsequently has made PSA reagent and test kits readily available since 1986, when the FDA approved its use. Consequently clinical applications of PSA were extensively investigated and led to the widespread use of PSA in patient care around the globe.

One of the most significant impacts of PSA is the dramatic shift of the profile of newly diagnosed prostate cancer. The proportion of men with advanced cancers at the time of diagnosis was 75% prior to the PSA era. Today 90% of prostate cancers are detected before the disease has spread to other organs. In essence, I have accomplished my research goal that was proposed in my NIH grant application submitted 40 years ago. PSA has helped achieve a 99% 5-year survival rate for prostate cancer. PSA also is an important factor in reducing the mortality rate of prostate cancer by almost 50% over the past 20 years.

Considering this progress, it is incomprehensible that the USPSTF recently has recommended the abandonment of the PSA test for prostate cancer screening. Their sole rationale is “screening may benefit a small number of men but will result in harm to many others”. Since then, the AMA has criticized the composition of USPSTF, as it includes neither oncologists nor urologists. Nationally recognized experts in the care of prostate cancer patients have disagreed with this recommendation by pointing out that the USPSTF report was based on flawed clinical trials and contained errors and misinterpretation (45). It is important that PSA-based screening should continue with an informed decision-making process. Men with average risk and with at least a 10-year life expectancy should begin conversation with their physicians at age 50. Men in higher risk groups should review the risk/benefit information at age 40.

Like any diagnostic test, this simple PSA has its strength and weakness, but it is the best that is currently available. The focus of discussion should be on how to use PSA in assisting patient care. We should not turn back the clock to the time when too many men experienced painful and unnecessary death from prostate cancer that was detected too late.

Reflections on CA19-9: from Discovery and Structural Analysis to Function and Drug Design

CA19-9 is a functional cell surface carbohydrate antigen that is the only FDA-approved marker for monitoring pancreatic cancer progression. As a functional marker, CA19-9 is being explored as a potential measure for a clinical endpoint as well as a target for the development of novel therapies. I have had the good fortune and privilege to be involved in its discovery, structural elucidation, function and drug design resulting in a molecular mechanism that promotes the understanding of a variety of disease states, while offering the potential to intervene with novel therapeutic compounds.

My interest in functional carbohydrates stems from formative years studying embryonic cell adhesion in Malcolm Steinberg's lab at Princeton University. Convinced of the importance of carbohydrates as recognition molecules, I started my career under the mentorship of one of the pioneers in Glycobiology, Victor Ginsburg at NIH. At that time new methods were needed to identify carbohydrate ligands recognized by protein receptors. I developed a simple technique of binding such receptors directly to thin layer silica gel plates treated to immobilize chromatograms of separated glycolipids extracted from tissues. Soon thereafter we were approached by Hilary Koprowski of the Wistar Institute who was using the new exciting technology of monoclonal antibodies to distinguish tumor from normal antigens on cell surfaces. This was the era in the early 1980's when Cesar Milstein, Niels Jerne and Georges Kohler received the Nobel Prize in Physiology or Medicine (1984) for the “discovery of the principle for the production of monoclonal antibodies.” Applying this technology to cell surfaces allowed the detection of specific novel antigens on tumor cells as seen through the eyes of the immune system. Many of these antibodies revealed aberrant forms of glycosylation in tumor cells which are completely missed in the current restricted use of genomics to study tumor markers due to the fact that carbohydrates are secondary gene products and are not simply detected by a genomics approach. Two antibodies (1116-NS-19-9 and 1116-NS-52a) were sent to us from the Wistar Institute that displayed the best specificity for colorectal cancer and appeared to be carbohydrates in nature due to their resistance to proteases but sensitivity to glycosidases. Upon analysis by the new method of immunostaining thin layer chromatograms of glycolipids extracted from colorectal cancer cells, we detected a novel monosialylganglioside as the antigen for both antibodies (46). Once identified, we scaled up purification of this monosialylganglioside and determined its structure through chemical techniques including GC/MS. The structure of the monosialoganglioside detected by both antibodies was a new carbohydrate structure identified as sialylated lacto-N-fucopentaose II (47) or more commonly known as sialyl Le^a. Excitement over this novel tumor-associated carbohydrate antigen promoted the founding of Centocor which developed a diagnostic assay with one of these antibodies (1116-NS-19-9) and thus the carbohydrate tumor marker sialyl Le^a received the immunologist's nomenclature of CA19-9. While the structural analysis was performed on simple gangliosides, we analyzed the major source of the CA19-9 antigen in patients' sera and to our surprise made the novel discovery that it was expressed mainly on mucins secreted from these adenocarcinomas into the bloodstream (48).

While elevated in both gastrointestinal and pancreatic cancer, serum levels of CA19-9 show the highest sensitivity and specificity for the detection of pancreatic cancer in symptomatic patients (approx. 80% and 90%, respectively). The core carbohydrate structure of CA19-9 contains a fucose linkage that is under control of the Lewis blood group system and those individuals who are Lewis negative (Le^a-b-) represent about 5 to 7% of the population, lack the fucosyltransferase needed to synthesize the CA19-9 structure and are negative for this assay.

Serum CA19-9 is the most extensively studied and clinically useful biomarker for pancreatic cancer and is the only FDA-approved validated assay for monitoring pancreatic cancer patients. Studies have shown significant decrease in survival in patients with high preoperative serum levels of CA19-9. Likewise, high postoperative CA19-9 levels also have a significant influence on lower survival rates of patients and can be considered a prognostic indicator of metastatic disease. More importantly, at least 8 different clinical studies have reported that pancreatic cancer patients showing a decrease in CA19-9 during chemotherapy (responders) have a significant increase in survival over treated patients with constant or rising CA19-9 levels during treatment (49) which has prompted discussions on the potential future use of CA19-9 as a clinical endpoint.

Almost 10 years after discovering the structure of sialyl Le^a (CA 19-9), we were approached by Eugene Butcher of Stanford University for assistance in discovering the structure of a carbohydrate receptor for an adhesion molecule expressed on endothelial cells of blood vessels that functions in the extravasation of immune cells during an inflammatory response. The adhesion molecule is now known as E-selectin and we quickly discovered that it bound sialyl Le^a (CA19-9). More specifically, we described a trisaccharide domain shared by both sialyl Le^a and its isomer sialyl Le^x (found on immune cells) as the true binding epitope for E-selectin (50). This was an exciting time in the lab as we now understood the function of CA19-9 and why it was a prognostic indicator of metastatic disease. We hypothesize that pancreatic cancer cells expressing high levels of sialyl Le^a (CA19-9) readily bind to E-selectin on the vascular walls thereby hijacking the inflammatory pathway for extravasation of cells from the bloodstream and promoting metastasis. Support for this theory also comes from studies on E-selectin. Colorectal cancer patients with a genetic polymorphism of E-selectin (S128R) resulting in greater E-selectin-mediated cell adhesion show significant decreased survival over an 8 year period. Other studies show that elevated levels of serum E-selectin are associated with higher prevalence of metastatic disease and combining measures of serum CA19-9 with serum E-selectin improves prediction of metastatic spread. One interesting study further shows significant increase in survival of colon cancer patients expressing high levels of CA19-9 on their tumors by inhibiting expression of E-selectin by treatment with cimetidine over a 10 year period (51).

Thus, CA19-9 is a prognostic marker of disease for pancreatic cancer as it functions in the process of metastatic spread of cancer cells that strongly express it on the cell surface. As we have identified a small trisaccharide domain within CA19-9 that binds E-selectin and is responsible for this function, we were presented with an opportunity to design a small molecule mimic of this domain as a novel glycomimetic drug to inhibit metastasis. Our first glycomimetic design (GMI-1070) included other domains required to inhibit all three selectins (E, P, and L), and showed efficacy in pre-clinical models of both inflammation and cancer (52, 53). GMI-1070 is now in Phase II clinical trials to treat sickle cell patients in vaso-occlusive crisis and was recently partnered with Pfizer in one of the largest licensing deals in the biotech industry for 2011. Interest in this novel glycomimetic antagonist validates this approach and we are now focusing our new design on a more restricted glycomimetic of CA19-9 to develop an orally available potent E-selectin-specific antagonist to be used in combination therapy with standard of care treatments for both solid and liquid tumors.

Non-Standard Abbreviations

CEA

carcinoembryonic antigen

CA125

carbohydrate antigen 125

CA19.9

carbohydrate antigen 19.9

ADCC

antibody dependent cell-mediated cytotoxicity

OC

ovarian cancer

RMI

risk of malignancy indes

ROMA

risk of malignancy algorithm

RVS

transvaginal synography

PPV

positive predictive value

ROCA

risk for ovarian cancer

TSTA

tumor-specific transplantation antigens

ASCO

American Society of Clinical Oncology

Ig

immunoglobulin

PSA

prostate-specific antigen

PAP

prostatic acid phosphatase

NIH

National Institutes of Health

NPCP

National Prostatic Cancer Project

AUA

American Urological Association

ACS

American Cancer Society

AACR

American Association for Cancer Research

USPSTF

U. S. Preventive Services Task Force

AMA

American Medical Association