Despite advances in diagnostic and therapeutic modalities against lung cancer, little improvement in the prognostic outcome has been accomplished. Therefore, new screening methods and therapeutic approaches, including immunotherapy, are needed for the management of patients with lung cancer. Cancer germline (CG) or cancer testis antigen (CTA) genes are genes that are normally expressed in germline cells but can be expressed in various tumors as a result of demethylation [1,2]. These proteins have been considered to be targets for immunotherapy and to be prognostic factors because their expression is associated with the characteristic features of the neoplastic phenotype, including invasiveness, immune evasion, and metastatic capacity [1]. Brother of the regulator of imprinted sites (BORIS) is a newly described autosomal CG or CTA that has been proposed to function as an oncogene in human cancer through the dysregulation of the cancer epigenome [1]. However, its role during oncogenesis and the clinical implications of its expression are not clear [2].

The melanoma antigen gene family A (MAGEA) consists of several subtypes, including MAGEA1 to MAGEA12, that are also categorized as CG antigens [3]. During the past several years, the expression of MAGEA genes has been studied for application in cancer diagnosis and targeted immunotherapy [3?5]. The aim of this study was to investigate the expression of BORIS in the sputum of patients with lung cancer and its clinical implications.

Transcripts of MAGEA1?6 were detected using the Cancer Hunter kit (iC&G Co., Daegu, Korea) [5]. Briefly, reverse transcription reactions were carried out in a 20-μL reaction mixture containing 7 μL of the RT master mixture, 0.5 μL of the RNase inhibitor, 1 μL of RNase, and 11.5 μL of eluted RNA solutions. The reaction mixture was incubated at 42°C for 60 minutes and 95°C for 5 minutes and stored at ?20°C until needed for PCR. The first round of PCR was carried out in a 20-μL reaction mixture containing 8 μL of the PCR master mixture, 0.5 μL of the outer primers, 2 μL of the RT reaction products, and 9.5 μL of distilled water. The cycling parameters were as follows: denaturation was initiated at 94°C for 2 minutes, followed by 30 cycles of 94°C for 30 seconds, 60°C for 45 seconds, and 65°C for 60 seconds, with a final extension by incubation at 65°C for 5 minutes. After the first PCR, 20 μL of the nested PCR mixture containing 8μL of the PCR master mixture, 0.5 μL of the inner primers, and 11.5 μL of distilled water was added to the first PCR tube. Nested PCR was carried out under the same conditions as the first PCR except for the annealing temperature, which was set at 62°C. The nested PCR products were separated on 1% agarose gels impregnated with ethidium bromide (0.5μg/mL). The DNA sequences of primers for the MAGE are listed in Table 2.

Statistical analysis was performed using SPSS ver. 12.0 (SPSS Inc., Chicago, IL, USA). Categorical variables were compared by Fisher’s exact test and chi-square analysis with the Yates correction. Differences were considered significant if the p-value was less than 0.05.

The overall expression rate of BORIS in our patients with lung cancer was 36.0% (36/100) (Table 3, Fig. 1). BORIS expression was detected in 19 of the 50 squamous cell carcinomas (38.0%), 13 of the 36 adenocarcinomas (36.1%), and 4 of the 14 (28.6%) small-cell carcinomas. BORIS mRNA expression was not significantly associated with age, gender, or histologic type; however, it was significantly related to the pathologic cancer stage (p=0.004) and lymph node metastasis (p=0.001).

BORIS was expressed in 36 of the 74 patients (48.6%) with lymph node metastasis and in none of the patients without, indicating a significant association with lymph node metastasis (p=0.001). BORIS expression was detected in 92.6% of the cases with pathologic stage IIIb and IV diseases but in none of the patients with pathologic stage Ib disease. Therefore, BORIS expression was positively associated with the cancer stage (p=0.004).

The expression rate of MAGEA1?6 was 76% (76/100) for all lung cancer patients (Fig. 2). MAGE was expressed in 90% (45/50) of the patients with squamous cell carcinoma, 57.1% (8/14) of the patients with small-cell carcinoma, and 36.1% (13/36) of the patients with adenocarcinoma. The expression rate in squamous cell carcinoma was significantly higher than that in the other two histologic types (p=0.001). There was no significant difference in the MAGEA1?6 expression according to the pathologic cancer stage and lymph node metastasis. There was no significant correlation between the expression of BORIS and the expression of MAGE.

Lung cancer remains one of the most deadly diseases worldwide; therefore, more effective therapeutic approaches are urgently needed. Although resection is a therapeutic option for patients with non-small-cell lung cancer (NSCLC), most patients show a locally advanced or metastatic status at diagnosis. Immunotherapy offers a novel approach for the treatment of patients with NSCLC in both the adjuvant setting as post-primary treatment and as the primary treatment for advanced cancer. A number of promising candidate vaccines based on different types of antigenic stimulus have now been evaluated in clinical studies [6,7], including target protein-specific vaccines designed to induce responses against CGs such as MAGEA [3?7].

BORIS, a novel member of the CG family, is suspected to be a new oncogene with unknown functions [2,8] and is considered a potential molecular target for cancer immunotherapy [8]. In our study, the expression rate of BORIS in lung cancer patients was 36%. The expression of BORIS was not significantly different according to age, gender, or histologic type but was significantly associated with the pathologic cancer stage (p=0.004) and lymph node metastasis (p=0.001). The expression of MAGEA1?6 was not associated with the expression of BORIS.

In the case of breast cancer, the expression rate of BORIS was reported to be 70.7% [9] and BORIS expression was detected in 73 of the 95 (77%) endometrial cancers and 24 of the 31 (77%) uterine-mixed mesodermal tumors [10]. BORIS was also detected in 27% (n=63) of the melanoma tissue samples [11]. BORIS expression was also reported in ovarian cancer patients with advanced stage disease [12]. These reports indicate that the expression of BORIS varies according to the origin or histologic type of tumors. At present, there are no other data on BORIS expression in the sputum or the tissue of patients with lung cancer; therefore, our results cannot be compared or verified. In addition, it is likely that the expression rate in the sputum is lower than that in the tumor tissue itself.

There have been reports that the functions of BORIS in vi-tro may involve the expression of other CTAs such as MAGE or other proto-oncogenes [13]. Moreover, the aberrant expression of BORIS is associated with the up-regulation of candidate proto-oncogenes in multiple human malignancies [13,14]. The expression of BORIS might explain how the expression of the human retinoblastoma-related Rb2/p130 gene accelerates the progression and recurrence of lung tumors after treatment [14]. However, findings on the regulatory role of BORIS in the expression of other genes are inconclusive. A previous study reported that CTA expression is dependent on BORIS and BORIS regulates the CTA gene expression through methylation-dependent and -independent mechanisms [15]. In addition, another study suggested that the expression of BORIS and MAGE was not correlated in tumors expressing low levels of BORIS such as melanoma [11]. Our results suggest a similar low expression rate of BORIS (36.0%) in lung cancer. Moreover, the expression of BORIS was not associated with the expression of MAGEA1?6 in our patients.

Our study revealed that lymph node metastasis and pathologic cancer stage were significantly related to the expression of BORIS. This is supported by a report that the BORIS expression is significantly associated with the lymph node metastasis and is an independent poor prognostic factor in esophageal cancer [16].

BORIS has been studied as a target of immunotherapy [17], and our data suggest that 36% of the patients with lung cancer could be considered candidates for immunotherapy using BORIS. However, further studies with larger populations and survival analyses are needed to reach a solid conclusion about the prognostic implications of the BORIS expression in lung cancer.

BORIS could be a molecular target for immunotherapy in the 36% of patients with lung cancers that express the BORIS gene. The expression of BORIS in sputum might be a prognostic factor associated with a higher pathologic cancer stage and lymph node metastasis.