Before the onset of treatment, sera were obtained from 1,833 patients at Chiba Cancer Center with different cancer types involving the esophagus (n=172), stomach (n=317), large intestine (n=262), liver (n=91), prostate (n=358), breast (n=364), lung (n=269), and 72 healthy donors. Age and stages were shown in Table I. Each serum sample was centrifuged at 3,000 x g for 5 min, and the resulting supernatant was stored at -80˚C until analyzed at Toho University. We avoided repeated thawing and freezing of the samples. This study was approved by the institutional ethics committee of the Chiba Cancer Center (#21-26) and the Toho University School of Medicine (#22-112, #22-047). Additionally, written informed consent was obtained from all patients. Patient recruitment was conducted from July 2008 to March 2010.

Serum samples were analyzed using an enzyme-linked immunosorbent assay, as previously described (16). Purified recombinant GAL-1 protein was coated onto 96-well microtiter plates (Maxisorp; Nunc). The absorbance was measured at 450 nm using a SUNRISE Microplate Reader (Tecan Japan Co., Ltd.). Gal-1 signals were evaluated by calculating the difference in absorbance between the wells containing galectin-1 and phosphate-buffered saline. Since the antibody titers are displayed in numerical value of absorbance, there is no unit of the protein amount.

Fisher's exact (two-sided) probability test was used to determine the differences between the two groups. All statistical analyses were performed using EZR (Saitama Medical Centre, Jichi Medical University; Saitama, Japan) (17), which is a graphical user interface for R (The R Foundation for Statistical Computing; version 2.13.0). A P-value <0.05 was considered to indicate a statistically significant difference.

s-GAL-1-Abs levels were divided into two groups; normal optical density (OD) values that were below the border level of 0.368 (calculated as the mean (0.053) + 3 standard deviations (0.105) of the values in healthy donors) and abnormal, or positive, values that were higher than 0.368 (Fig. 1). Mean OD values for each cancer type were as follows: 0.362 in HCC, 0.213 in lung cancer, 0.227 in esophageal cancer, 0.208 in colorectal cancer, 0.141 in prostate cancer, 0.136 in gastric cancer, and 0.179 in breast cancer.

The positive rate of serum anti-GAL-1 antibodies were greater than 10% in patients with HCC (16.7%), lung cancer (13.8%), esophageal squamous cell carcinoma (11.6%), and colorectal cancer (11.5%) (Fig. 2). Other cancer types demonstrated similar positive rates of around 6 to 7%. Although the overall positive rate of s-GAL-1-Abs was not higher than that of conventional tumor markers (13,18-21), it was found to be positive in various cancer types.

The differences of the positive rates of s-GAL-1-Abs among various cancer types might be associated with the positive rates of galectin-1 protein expression in the tumor tissues. Actually, the positive rates of galectin-1 protein expression were 57% in HCC (22), 51.5% in lung cancer (23), 30% in colorectal cancer (24), and 60.6% in gastric cancer (25). There were no reports on esophageal cancer, prostate cancer, and breast cancer. Although the information of protein expression was limited, there were no association with the positive rate of serum autoantibody.

Unlike conventional secretory tumor markers, autoantibody markers have been reported to be positive even in early stage cancer. However, the positive rates of advanced stages were not always higher than the positive rates of early stages. The exact reason why such paradoxical positive rates was unknow but potential immunosuppression in advanced stages might affect antibody reaction (26-28).

For the comparison of s-GLA-Abs according to the stages, each tumor was divided into Stage 0/I/II and III/VI (Fig. 3). HCC, colorectal cancer, and breast cancer showed higher positive rates in stage 0/I/II than in stage III/IV. Lung cancer, esophageal cancer, prostate cancer, and gastric cancer showed higher positive rates in stage III/IV than in stage 0/I/II.

Recent reports showed that galectin-1 targeting therapy may have a potential role to modify anti-cancer immune response (6,29). Galectin-1 vaccination or knockdown therapy could be one of the options for s-GAL-1-Abs positive cancer patients.

Figs. 2 and 3 showed just percentage of positive rates in each cancer types. These percentages were calculated by dividing the positive cases by all cases in Fig. 2. In Fig. 3, the percentages were calculated by dividing the positive cases by total cases at each stage.

In summary, the present study, of the 1,833 patients with seven different cancer types, showed that HCC and lung cancer patients showed relatively higher positive rates of s-GAL-1-Abs than the other cancer types. Based on its low sensitivity, the s-GAL-1-Abs may not be the first choice of a serum marker in other cancers. Such fundamental information of s-GAL-1-Abs in a wide range of tumor types could be a little help for further clinical studies of immune-therapy or immune-diagnosis.

Our present study did not include any survival data. Further studies are required in prospective manner to evaluate the prognostic significance and effects on treatment response of serum Galectin-1autoantibodies.