Despite continuous decline in incidence rates in both sexes, gastric cancer is still the fourth most common cancer worldwide, with 934,000 newly diagnosed cases per year ¹ and a vast annual death toll of more than 800,000, according to WHO 2004 statistics. With only little improvement over the past decades, the long-term survival from gastric cancer is poor, since patients are often diagnosed with advanced disease. In the USA, for example, five-year survival is 24% ² . In a hope for an overall improvement in the forlorn statistics, worldwide efforts are invested to shorten the time-to-diagnosis. Stomach cancer screening has been first introduced in Japan in 1963, followed by The Republic of Korea in 1996, and has recently commenced in less developed countries, with Venezuela, Chile, and Costa Rica adapting the Japanese model to implement pilot screening routines. Notably, the fact that to date no randomized trial of stomach cancer screening has ever been conducted sets hurdles for reliable assessment of efficacy of such policy. Nevertheless, data from recent prospective studies shows reductions in mortality from gastric cancer among participants in screening programs in Japan and Costa Rica ^{3– 5} . Importantly, in this regard, endoscopic procedures, although of undisputable great diagnostic value, face a lack of patient compliance, for being widely regarded as uncomfortable. Henceforth, to circumvent this obstacle, reliable biological markers, suitable for detection and monitoring of tumor growth in bodily fluids, have long been searched after, in both blood and gastric fluid. Understandably, being easily accessible, blood has long been the substance of choice for marker evaluation. However, as appears from the peer-reviewed sources, the vast majority of the proposed gastric cancer marker candidates share the lack of diagnostic potential. This flaw, expressed in low sensitivity and specificity parameters, obviously negates the idea of utilization of such markers in gastric cancer screening practice.

As new potential screening methodologies, such as capsule endoscopy ⁶ , arise, gastric fluid is becoming an attractive milieu for cancer diagnosis as it contains both secreted soluble and exfoliated cellular proteins from the entire gastric mucosa. Unfortunately, early reports on the diagnostic and prognostic utility of the preponderant indicators of neoplasia, such as carcinoembryonic antigen (CEA) and CA 19-9, yielded unsatisfactory results ^{7– 9} , thus diverting researchers’ attention to other biomarkers, including CA72-4. Historically, following its identification [a sialosyl-2–6-α-N-acetylgalactosaminyl epitope of TAG-72 mucin; ^{10– 15} ], and characterization, the applicability of the marker to cancer detection was examined in several neoplastic conditions. As second generation high-affinity monoclonal antibody (recognizing a different epitope; ¹⁶ ) has enabled establishment of a double-determinant immunoradiometric assay ^{17, 18} capable of detection of CA72-4 in bodily fluids of carcinoma patients, CA72-4 presence was assessed in the serum of patients diagnosed with gastrointestinal malignancies ^{19– 21} . These studies disclosed a complementarity between CA72-4 and CEA, as the former was often elevated in samples from cancer patients in which the levels of the latter remained low. Moreover, serum CA72-4 in patients during post surgical follow-up was predictive of recurrent disease ²⁰ . Several recently demonstrated lines of evidence also described CA72-4 as a potential serum and peritoneal wash-fluid marker of gastric malignant neoplasia, supreme to CEA ( ²² ; and references therein). In the present report we show that CA72-4, whose levels had to date been mainly assessed in serum samples ^{23– 26} , has the potential to become a major biomarker in the gastric fluid-based gastric cancer diagnosis.

The combination of a relatively high incidence, especially in northeast Asia, and a frequently late stage diagnosis of gastric cancer, due to the patient- or physician-side misinterpretation of symptoms, has proven deadly over the years, making the disease the second most frequent cause of cancer death worldwide ^{27, 28} . Generally, the cancer progression is aggressive upon late stage diagnosis with 5-year survival rates usually less than 30%. The existing means of screening are very accurate in providing a diagnosis, but face low patient-side compliance due to known discomfort associated with endoscopic procedures, thus imposing indirect constraints on the diagnostic value of fibreoptic endoscopy. The emerging capsule endoscopy, although holding a promise to circumvent the issue of compliance, still lacks diagnostic capabilities beyond visual identification of lesions. Thus, it will inevitably, require a reliable substitute for histological analysis, readily available through biopsy collection using a “conventional” endoscope, to outcompete the latter in screening efficiency, simplicity, and time-to-diagnosis. One such alternative is “lab-on-capsule” cancer marker-based molecular recognition of gastric malignancies. This option can be made possible through utilization of one or more marker(s) featuring high sensitivity and specificity in detection of gastric malignant tumors. In this work, we embarked upon identification of such a marker through assessment of the above mentioned parameters for a selected group of cancer markers, levels of which were measured in the gastric juice collected from patients with various disorders of the upper GIT, as well as in the gastric juice of patients in whom no disease was detected by either or both EGD and histopathology. During this study we have established several ELISA assays for markers previously implicated in relation with gastric cancer, such as: CEA ^{7– 9, 23} , pepsinogen II (PGII; ^{29– 33} ), regenerating islet-derived family, member 4 (RegIV; ^{34, 35} ), and cytokeratin 8 (CK8; ^{36– 38} ), and have also made use of commercially available assays for CA 19-9 ^{7– 9, 23} , Gastrin17 ³⁰ , and pepsinogen I (PGI; ^{31– 33} ). However, in our preliminary large-scale sampling analysis we failed to find any difference between the levels of these markers (including PGI/PGII ratio) in the gastric juice of cancer and non-cancer patients (data not shown). Quite the reverse, data generated in the course of this study points to a great diagnostic potential of CA72-4 direct measurements in gastric fluid. Notably in this regard, the CA72-4 concentrations in the gastric fluid of the majority of cancer patients were prominently elevated, compared to those measured in the gastric fluid obtained from patients with a completely normal stomach, patients with gastric inflammation, intestinal metaplasia or miscellaneous diseases of the upper gastrointestinal tract, including other malignant tumors.

One of the most obdurate hurdles to detection of cancer markers in gastric juice is the hostility of the gastric milieu imposed, among other factors, by high proteolytic activity and high acidity. Univariate linear regression analysis did not reveal a significant association between pH of gastric juice and CA72-4 concentrations. In multivariate linear regression analysis the “protective” effect of higher pH on CA72-4 concentrations was small, and failed to show statistical significance. In this regard, the apparent superiority of CA72-4 over other potential biomarkers in gastric juice may be speculated to stem from its relative stability in a wide range of pH, possibly due to the nature of the detected epitopes.

At a cut-off level of 100 U/ml CA72-4 had a sensitivity of 75% and a specificity of 89% for detection of gastric cancer. These data are also encouraging, although, with respect to the sensitivity parameter, the rather low number of cancer patients included in the study limits reliability of these findings and, generally, represents the most important drawback of our study. Gastric cancer was newly diagnosed in 8 out of 176 patients (5%), a proportion somewhat higher than that of cancer diagnoses expected in unselected patients undergoing EGD because of dyspeptic symptoms [1–2%; ³⁹ ). Notably, the IH is a tertiary referral center for patients with gastrointestinal diseases where gastric cancer patients are observed much more frequently. However, in most patients satisfactory endoscopic and histologic investigations have been performed prior to referral to IH. The experimental design did not allow performance of an EGD for reasons other than clinical, hence, certain patients could not be included in the study. Moreover, for final analysis of data only patients with newly diagnosed disease were selected in order to limit potential confounders. Interestingly, an ostensible decrease in CA72-4, measured in a single patient concomitantly with cancer detection (150 U/ml), was observed following chemotherapy (87 U/ml). Thus, in the future, effort should be invested in further exploration of this phenomenon and the possibility that CA72-4 may also be used to monitor treatment efficiency.

Contrary to sensitivity, the specificity data are based on analysis of a large number of gastric juice samples from patients with completely normal gastric findings or various gastric or other diseases, advocating for data reliability. We observed a trend towards lower intragastric CA72-4 concentrations in patients with pathologies other than gastric cancer and an accordingly high specificity of nearly 90% for CA72-4-based detection of gastric cancer at the cut-off level chosen. This is particularly encouraging for a putative screening marker, as low specificity would be associated with a large number of futile invasive diagnostic tests in patients that are, in fact, free from gastric cancer.

In conclusion, work will be needed to accurately establish the precise sensitivity and the cut-off level for cancer detection. However, more than a fourfold, and thus a highly biologically significant, increase in the mean value of CA72-4 in the cancer group patients in this study, compared to all other participants, along with the apparent high specificity of the marker-based cancer detection, underscore the validity of our findings, and suggest that CA72-4 may contribute to real-time detection of gastric cancer in the future.

Study participants gave written informed consent before any study related procedures were performed.