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Clinical Cancer Research Vol. 12, 5978-5986, October 15, 2006
© 2006 American Association for Cancer Research

Human Cancer Biology

The Potential of BORIS Detected in the Leukocytes of Breast Cancer Patients as an Early Marker of Tumorigenesis

Vivien D'Arcy1,5, Ziedulla K. Abdullaev2, Naresh Pore1, France Docquier1, Verónica Torrano3, Igor Chernukhin1, Melissa Smart1, Dawn Farrar1, Metodi Metodiev1, Nelson Fernandez1, Carlos Richard4, M. Dolores Delgado3, Victor Lobanenkov2 and Elena Klenova1

Authors' Affiliations: 1 Department of Biological Sciences, University of Essex, Colchester, Essex, United Kingdom; 2 Molecular Pathology Section, Laboratory of Immunopathology, National Institute of Allergy and Infectious Diseases, NIH, Rockville, Maryland; 3 Grupo de Biología Molecular del Cáncer, Departamento de Biologia Molecular, Unidad de Biomedicina, Consejo Superior de Investigaciones Cientificas, Universidad de Cantabria; 4 Servicio de Hematología, Hospital Universitario Marqués de Valdecilla, Instituto de Formación e Investigación Marqués de Valdecilla, Santander, Spain; and 5 Helen Rollason Cancer Care Laboratory, Anglia Ruskin University, Chelmsford, Essex, United Kingdom

Requests for reprints: Elena Klenova, Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, United Kingdom. Phone: 44-0-1206-874868; Fax: 44-0-1206-872592; E-mail: klenovae{at}essex.ac.uk.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Brother of the regulator of imprinted sites (BORIS) is a novel member of the cancer-testis antigen gene family. These genes are normally expressed only in spermatocytes but abnormally activated in different malignancies, including breast cancer. The aim of this study was to investigate the expression of BORIS in the leukocytes of breast cancer patients and the correlation between BORIS levels and clinical/pathologic variables.

Experimental Design: Leukocytes were obtained from whole blood of 87 breast cancer patients and 52 donors not diagnosed with cancer. BORIS protein was detected in leukocytes by immunohistochemical staining; the immunoreactivity score (IRS) of each sample was determined. Additionally, BORIS expression was assessed by Western blot analysis and real-time reverse transcription-PCR.

Results: We describe significantly high levels of BORIS (IRS = 4.25 ± 0.034) in a subpopulation of leukocytes, the neutrophil polymorphonuclear granulocytes, in 88.5% of breast cancer patients. Increased IRS for BORIS in these patients correlated with increased tumor size. In comparison, 19.2% samples from the control group were BORIS positive with only very low levels of BORIS (IRS = 0.25 ± 0.009).

Conclusion: We report here the novel finding of BORIS expression in polymorphonuclear granulocytes of breast cancer patients. This tumor-related occurrence is a phenomenon not observed in donors with injuries and immune and inflammatory diseases. Detection of BORIS in a high proportion of patients with various types of breast tumors indicates that BORIS can be a valuable early blood marker of breast cancer. We conclude that BORIS represents a new class of cancer biomarkers different from those currently used in medical practice.

The identification of markers to distinguish between normal cells, tumorigenic cells, and different stages of cancer pathology is of critical importance for cancer diagnosis and prognosis. Cancer biomarkers can be classified into two types: cellular and humoral markers. Cellular markers are associated with cancer cells and are used in the pathologic assessment of biopsy material. Examples of breast cellular markers include estrogen receptor, HER2, c-erbB-2 (1–4), and those of prostate markers are cadherins, G-actin, tissue transglutaminase type II, {alpha}-methylacyl CoA racemase, pim-1, and hepsin (5–7). One disadvantage in the use of these markers is that their analysis requires invasive procedures to obtain tissues. Other cellular markers, such as mammaglobin and B305D-C, are associated with circulating cancer cells (8); the difficulties in detection of cancer-associated molecules in these cells lie in the very low numbers of the circulating cancer cells in blood. Humoral cancer markers are characterized by their appearance in body fluids (urine, blood, etc.) in amounts exceeding normal physiologic concentrations. These markers can be released on tumor disintegration or may be secreted by tumors. Such markers are very attractive as less invasive procedures are required to obtain the specimen fluid for analysis.

Significant efforts at the cutting edge of technology are currently directed to the identification of suitable new cancer markers in body fluids, blood in particular. Examples of such markers already identified include prostate-specific antigen (9), the mesothelioma markers, soluble mesothelin-related proteins (10), and others (11–13). Several breast cancer markers such as nectin-4 (14), matrix metalloproteinase-9 protein (15), monocyte chemoattractant protein-1 (16), relaxin (17), and CA15-3 and CA27-29 (products of the MUC-1 gene; ref. 18) have also been described. The recent findings that cell-free DNA can be shed into the bloodstream as a result of tumor cell death holds potential for molecular diagnosis and prognosis (19); circulating tumor DNA with abnormal methylation patterns has also been considered as a potential cancer-specific biomarker (20).

At present, there are no established circulating tumor markers available for clinical use in the determination of cancer susceptibility, screening, diagnosis, and prognosis. Tumor markers currently available lack sensitivity for early cancer and specificity for malignancy; therefore, there is a continuing quest to identify a more sensitive and accurate circulating marker specific or applicable for a range of human cancers. The identification of such a marker would be of great clinical value.

Brother of the regulator of imprinted sites (BORIS) is a paralogue of the transcription factor, CTCF, or CCCTC-binding factor, an evolutionarily conserved protein with features of a tumor suppressor (21–23). In contrast to the ubiquitous CCCTC-binding factor, BORIS expression pattern is restricted to testis and normally BORIS is not present in females. Our previous results showed that during male germ-line development, CCCTC-binding factor and BORIS are expressed in a mutually exclusive manner; this may be important for epigenetic reprogramming occurring in these cells during development (21, 22). Expression of BORIS in normally BORIS-negative cells may lead to cell transformation (21), and BORIS has been found to be aberrantly activated in various human cancers, including female cancer such as uterine (endometrial) and breast tumors.6,7 BORIS therefore represents a novel member of the cancer-testis antigen (CTAG) gene family that comprises genes normally detected only in testis but aberrantly activated in different malignancies (24, 25). Recent reports also show that BORIS expression in normal cells leads to derepression of several cancer-testis genes (MAGE-A1, NY-ESO-1, and others; refs. 26, 27).

In this study, we show that BORIS is present in the leukocyte fraction of patients with breast cancer and we also investigate the potential of BORIS as a blood cancer biomarker.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patients and controls
Blood samples were collected before breast surgery from patients treated at the Colchester General Hospital (Essex, United Kingdom). Samples were also collected from individuals not diagnosed with cancer to be used as a control. All blood samples were obtained with previous written consent. Clinical information on tumor stage, tumor grade, tumor size, estrogen receptor status, progesterone receptor status, HER-2 receptor status, lymph node metastasis, preoperative and postoperative chemotherapy, menopausal state, and age of patient was received from the Colchester General Hospital. The control group of donors provided information about their gender, age, family history of cancer, and present or recent disease/condition. We analyzed a total of 87 blood samples from patients diagnosed with breast cancer and 52 samples from the control group. The latter was subdivided into two groups: healthy donors (36 blood samples) and donors with injuries and immune and inflammatory diseases (16 samples).

Procedures
Preparation of the leukocyte fraction from the peripheral blood cells. Blood fractions were separated by a buoyancy density method using Histopaque 1119-1 (Sigma, St. Louis, MO) according to the instructions of the manufacturer. The leukocyte fraction was collected after centrifugation and cell smears were prepared from ~2.5 x 104 cells in Nunc eight-well chamber slides. The cells were then fixed with 4% formaldehyde in a PBS solution (0.01 mol/L phosphate buffer, 0.0027 mol/L KCl, 0.137 mol/L NaCl, pH 7.4) for 30 minutes at room temperature, washed with PBS, and stored at 4°C.

Immunohistochemical staining and assessment of BORIS levels in the leukocyte fraction. Immunohistochemical analysis was done by staining leukocytes with the Vectastain Elite ABC standard kit (Vector Laboratories, Burlingame, CA) as suggested by the manufacturer. Immunohistochemical staining was evaluated by using the immunoreactivity score (IRS) as previously described (28). In brief, the percentage of BORIS-positive cells was divided into four categories (<10%, 11-50%, 51-80%, and >80%, with corresponding scores of 1, 2, 3, and 4, respectively), whereas the staining intensity was given a value between 0 (no detectable immunostaining) and 3 (strong immunostaining). The IRS (0-12) was then calculated by multiplying the score values. Scoring was done in a blinded fashion by two independent scorers, with each slide read twice; the results in Tables 1 , 2 , and 3 represent the average score for each sample. Morphologic assessment of cells in the leukocyte fraction was done by examining fixed slide preparations stained with Giemsa (Sigma) using established cytologic criteria.


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  		Table 1. Assessment of the leukocytes obtained from 36 healthy donors for BORIS IRS

 

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  		Table 2. Assessment of the leukocytes obtained from 16 donors with injuries and immune and inflammatory diseases for BORIS IRS

 

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  		Table 3. Assessment of the leukocytes obtained from 87 patients diagnosed with breast cancer for BORIS IRS

 
RNA isolation, reverse transcription, and real-time PCR analysis. Total RNA was isolated from leukocytes with RNeasy Protect Kit (Qiagen, Valencia, CA) according to the instruction of the manufacturer. RNA was then converted to cDNA using random primers and Thermoscript reverse transcriptase (Invitrogen, Carlsbad, CA). Real-time reverse transcription-PCR (RT-PCR) analysis was done as previously described (26) using the Applied Biosystems 7900HT Fast Real-time PCR System. BORIS primers/probe sequences were as follows: 5'-CCCATTGTGCCACCATCA-3' (forward), 5'-AGCATGCAAGTTGCGCATAT-3' (reverse), and 6FAM-ACGGAAAAGCGACCTAC-MGB (BORIS probe). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers/probe mixture was purchased as Predeveloped Assay (Applied Biosystems, Foster City, CA).

Western blot analysis. For Western blot analysis, lysates from leukocytes were prepared according to Klenova et al. (29) with modifications. Western blot assay was conducted as previously described with anti-BORIS or anti-{alpha}-tubulin (Sigma) antibodies (22). Quantification of the bands was done with the Image J software8 and values were obtained from the BORIS/{alpha}-tubulin ratio.

Statistical analysis. Statistical analysis was carried out using unpaired Student's t test. Values were considered as significant when the probability was below the 5% confidence level (P ≤ 0.05).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Immunohistochemical analysis of BORIS expression in the leukocyte fractions of blood samples from healthy donors and donors with injuries and immune and inflammatory diseases. The level of BORIS in leukocyte fractions from 52 donors not diagnosed with cancer was assessed. The donors were initially subdivided into healthy donors, 36 samples, and those with injuries and immune and inflammatory diseases, 16 samples. Immunochemical analysis of the 36 healthy donor samples revealed that 6 (16.67%) were BORIS positive (Table 1). Staining with only the secondary antibody did not show any nonspecific background (an example is given in Fig. 1A ). Significantly, the IRS values observed in the BORIS-positive samples in this cohort were very low, with a mean IRS value of 0.250 ± 0.0153 (Fig. 2 ). The BORIS-positive specimens from this cohort comprised five females and one male, all with history of familial cancer.


Figure 1
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  		Fig. 1. A, immunohistochemical staining of the population of the leukocyte fraction from a healthy donor with the anti-BORIS antibody (right). Left, background staining with hematoxylin plus secondary antibodies only. Magnification, x100. B, immunohistochemical staining of the population of the leukocyte fraction from breast cancer patients with the anti-BORIS antibody; examples of leukocyte fractions with different IRS immunostained with the anti-BORIS antibody. The IRS values were calculated as described in Materials and Methods. Magnification, x100. C, real-time RT-PCR and Western blot analyses of BORIS in leukocytes with different IRS. Top, real-time RT-PCR: expression levels of BORIS mRNA were calculated using the comparative CT method ({Delta}{Delta}Ct) and normalized to GAPDH expression. Numbers above columns represent fold change relative to the lowest BORIS/GAPDH mRNA ratio (designated as 1.0). For each sample, measurements were done at least in triplicates. Bars, SD. Bottom, Western blot analysis: cellular extracts were prepared from ~5 x 106 cells; total protein concentration was determined for each sample and equal amount (20 µg) of total protein loaded onto SDS-PAGE. Samples were electrophoretically separated, blotted, and probed with the anti-BORIS antibody. The same membrane was stripped and reprobed with the {alpha}-tubulin antibody (loading control). The developed films were scanned and images quantified. The ratios of the intensity of the BORIS bands over the intensity of the corresponding {alpha}-tubulin bands were determined and expressed as fold change relative to the lowest BORIS/{alpha}-tubulin ratio (designated as 1.0). Numbers below each lane represent these results. D, immunostaining of the population of the leukocyte fractions from a healthy donor (left) and a breast cancer patient (middle) with the anti-BORIS antibody and Giemsa. BORIS can be detected in PMNs in the leukocyte fraction obtained from a breast cancer patient, but not in the control blood sample obtained from a healthy donor. Magnification, x100. Examples of BORIS-positive PMNs (colored arrows) are shown in the magnified boxes on the right.

 

Figure 2
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  		Fig. 2. Comparison between BORIS levels (expressed in IRS values) in the leukocytes of breast cancer patients and the phenotypes of matching tumors (grouped as shown in Table 3). DCIS, ductal carcinoma in situ; IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma; Inflammatory, injuries and immune and inflammatory diseases; Control, combined "Healthy donors" and "Inflammatory" groups; Miscellaneous, rare tumors and mixed phenotype tumors; All tumor types, the entire tumour group, all phenotypes are included in the analysis. An unpaired Student's t test was carried out between the leukocyte fraction of the control and tumor groups. Columns, mean IRS value; bars, SE. **, statistically significant results.

 
We also assessed BORIS levels in the group of 16 donors with injuries and immune and inflammatory diseases. Four (25%) samples in this group were BORIS positive (Table 2; Fig. 2); the mean IRS value was 0.250 ± 0.018. All four were females with a history of familial cancer. As the IRS value in this group of donors did not significantly differ from the first cohort ("healthy donors"), we combined both groups into one group (n = 52), which we will refer to as the "control group". The IRS value for BORIS in the control group has been calculated as 0.250 ± 0.009 (Fig. 2).

Analysis of BORIS expression in the leukocyte fractions of blood samples from breast cancer patients by immunohistochemical staining, Western blot, and real-time RT-PCR. Next, we investigated the levels of BORIS in blood samples obtained from breast cancer patients. For this purpose, leukocyte fractions from 87 individuals diagnosed with breast cancer were immunostained with the anti-BORIS antibodies and the IRS values were calculated (Table 3; Fig. 1B). We found that 88.5% (77 of 87 samples) were BORIS positive. The IRS values determined by immunohistochemical staining were in good agreement with the Western blot data. Correlation was also observed between the amount of BORIS protein and the levels of BORIS mRNA obtained by the real-time RT-PCR analysis of the same samples (Fig. 1C).

Morphologic analysis of the leukocyte fractions was done by examining fixed slide preparations stained with Giemsa. Analysis revealed that most of the BORIS-positive cells were neutrophil polymorphonuclear granulocytes (PMN), with characteristic lobulated nucleus, segmented into two to five lobes connected by thin chromatin strands, and cytoplasm with fine azurophilic granules (Fig. 1D).

The mean IRS for BORIS in the leukocyte fraction from all breast cancer patients, regardless of tumor phenotype, was calculated and compared with the mean IRS from the control group. The mean value from breast cancer patients was 4.25 ± 0.034 and that from the control group was 0.250 ± 0.009 (Fig. 2). The IRS values for BORIS in the leukocyte fraction of breast cancer patients were also categorized according to the diagnosed histologic type of the matching breast tumor (Fig. 2). The calculated mean IRS values were 4.25 ± 0.099 for ductal carcinoma in situ, 4.17 ± 0.094 for invasive ductal carcinoma, 3.29 ± 0.402 for invasive lobular carcinoma, and 4.78 ± 0.17 for miscellaneous mixed (rare tumors and mixed phenotype tumors, the last 18 specimens at the bottom of Table 3). Using an unpaired Student's t test, we confirmed that the difference was significant for all categories of blood specimens obtained from breast cancer patients with the following P values: invasive ductal carcinoma, P ≤ 0.0001; invasive lobular carcinoma, P ≤ 0.0001; ductal carcinoma in situ, P ≤ 0.0001; miscellaneous tumor, P ≤ 0.0001; and "all tumors," P ≤ 0.0001.

In summary, the data described in this section show elevation in BORIS levels in the leukocyte fractions obtained from breast cancer patients with different histologic types of breast cancer. This elevation was significant compared with BORIS levels in the leukocyte fractions obtained from the control group of donors.

BORIS levels in the leukocyte fractions from blood of breast cancer patients are increased with the size of the matching tumor. We then investigated a possible correlation between BORIS levels in the leukocyte fractions and the size of the matching breast tumor. For this analysis, tumors were divided into four groups according to their size: <10, 10 to 15, 16 to 24, and >25 mm (Fig. 3 ). The mean IRS for BORIS in the leukocyte fractions obtained from the patients with tumors <10 mm in size was 3.2 ± 0.296; in comparison with the control group (0.250 ± 0.009), the BORIS levels were significantly higher. The mean IRS values increased progressively with increasing tumor size, achieving 4.9 ± 0.15 in patients with tumor size >25 mm. Statistical evaluation of this data showed significant differences between BORIS levels in the leukocyte fractions obtained from patients with tumors of all sizes and BORIS levels in the leukocyte fractions from healthy donors (control; P ≤ 0.0001, each group versus control).


Figure 3
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  		Fig. 3. Comparison between BORIS levels (expressed in IRS values) in the leukocytes of breast cancer patients and size of matching tumors. Four groups of tumor sizes were analyzed: tumors <10, 10 to 15, 16 to 24, and >25 mm in diameter were analyzed. BORIS levels were assessed in the entire tumor group; all phenotypes were included in the analysis. Columns, mean IRS value BORIS; bars, SE. **, significant results (Student's t test).

 
A possible correlation between BORIS levels in PMNs and other clinical variables (tumor stage, tumor grade, estrogen receptor status, progesterone receptor status, HER-2 receptor status, lymph node metastasis, preoperative chemotherapy, menopausal state, and age of patients) was also assessed. No significant correlation has been found in these studies.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The high levels of BORIS protein in the leukocyte fraction of patients with breast cancer was an unexpected finding. We therefore aimed to investigate whether BORIS could serve as a biomarker in blood, with a potential use in the early diagnosis of patients with breast cancer. For this purpose, we analyzed BORIS content in the leukocytes obtained from both breast cancer patients and individuals not diagnosed with cancer. For quantitative assessment of BORIS levels, we used a standard technique commonly used in clinical practice, immunohistochemical staining of leukocyte smears, and scored the immunoreactivity. The IRS for BORIS determined by this method reflected fairly the BORIS protein content in leukocytes as confirmed by Western blot analysis. Using the patient clinical information, we also evaluated whether there was a relationship between BORIS levels and clinical/pathologic variables.

Our data described in this report suggest that the appearance of BORIS in PMNs is a specific tumor-related phenomenon. The presence of BORIS is not linked to general effects of growing tumors on metabolism or previously received treatment because (i) BORIS is detected at significantly higher levels in PMNs of 88.5% of breast cancer patients with all types of breast cancer, including ductal carcinoma in situ, a very early, noninvasive form of cancer (30, 31); (ii) higher levels of BORIS have been detected in tumors of various sizes, including very small tumors (<10 mm in size); (iii) only 3 of 87 patients received preoperative chemotherapy; the IRS values for BORIS in these three cases were comparable with the rest of the group (IRS 4-6; Table 3); (iv) such a phenomenon has not been observed in donors with injuries and immune and inflammatory diseases and (v) was not gender and age related. Furthermore, it proved to be very difficult to experimentally induce BORIS in BORIS-negative total leukocytes using genotoxic drugs, or DNA-demethylating agents (except using short-term treatments with a relatively high dose of 5-AzadC), and inhibitors of histone deacetylase (26, 27)9; such compounds are commonly used to activate expression of cancer-testis genes (32–34). These observations imply that much more specific mechanisms are to be involved in the induction of BORIS in PMNs in cancer patients.

Based on the findings described above, we envisage that detection of BORIS in leukocytes may become a useful tool in the early diagnosis of breast cancer. However, low levels of BORIS observed in some blood samples from the control group of donors indicate the complexities of BORIS detection in leukocytes. These levels are comparable with the low levels of BORIS in leukocytes of breast cancer patients as determined by immunohistochemical staining and Western blot analysis (Fig. 1B and C). It is possible that the presence of BORIS at low levels in the control group of donors reflects normal fluctuations of BORIS around the baseline. However, the fact that 8 of 10 BORIS-positive donors from the control group had strong family history of cancer can be very significant; positive staining for BORIS in these cases may be associated with an undiagnosed cancerous condition. Further research is required to explore and explain these observations. For diagnostic purposes, more informative quantitative method for BORIS detection in blood, such as ELISA, or methods based on cell sorting will require development; these investigations are currently being carried out.

A detailed morphologic analysis of the leukocytes revealed that cells positive for BORIS were PMNs. PMNs are members of the granulocyte family of leukocytes and are the most abundant circulating blood leukocytes (35). Mature PMNs are differentiated cells; however, they are metabolically active and synthesize RNA and proteins (35, 36). The importance of PMNs in innate immunity has been well established and recent findings place PMNs as an essential element of adaptive immunity (37–39). Several reports have shown a role for human or mouse PMNs in tumor rejection (40, 41); it is perhaps not surprising that these cells carry a marker (BORIS) that appears in patients diagnosed with breast cancer. The role of PMNs in antitumoral defense is, however, dual as they can inhibit or promote tumor growth depending on the microenvironment and functional state. It is therefore important to understand how the function of PMNs in antitumoral response may be regulated and whether BORIS can be involved in these processes. BORIS has features of a transcription factor and when up-regulated in PMNs may play an active role by changing expression patterns of subsets of genes important for PMN survival and inflammatory and antitumoral immune response. This can be achieved by direct regulation of the DNA targets by BORIS or by epigenetic alterations (e.g., changes in DNA methylation). Alternatively, BORIS can be simply deposited in granulocytes without playing an active role. Our current laboratory studies, which include profiling of proteins and identification of BORIS DNA targets in BORIS-positive granulocytes, are aimed at understanding the underlying molecular events associated with BORIS appearance in granulocytes.

What could be the origin of BORIS associated with PMNs? Our data show that BORIS can be expressed endogenously because there is a correlation between the levels of BORIS mRNA and protein; both are significantly increased in PMNs of breast cancer patients. In this situation, BORIS synthesis can be induced in response to factors released by the tumorigenic cells and/or by other cells of the immune system; these factors may have an effect on transcriptional regulation of the BORIS gene within the PMNs (the "signaling hypothesis"). However, the possibility that accumulation of BORIS protein in granulocytes may be a result of phagocytosis of BORIS-positive tumor tissues cannot be excluded (the "phagocytosis hypothesis"). In this case, PMNs could be attracted to the sites of inflammation by cytokines and chemokines secreted by the tumor itself or by tumor infiltrating leukocytes, such as T cells and macrophages (42, 43), or by antibodies produced by B-lymphocytes (44). Further detailed investigations will be required to clarify this issue.

Another intriguing aspect of this phenomenon is the nature of the PMNs expressing BORIS. Are they circulating mature cells or a population of expanded precursors? Our calculations of the percentage of PMNs in leukocyte fractions in breast cancer patients versus healthy controls revealed no significant difference between the two groups (84 ± 5.7% and 81 ± 7%, respectively; n = 10 randomly selected specimens).10 Thus, the presence of BORIS is most likely to be a characteristic feature of circulating PMNs.

We also investigated the possibility of BORIS accumulation in donor blood plasma. However, BORIS was not detected in blood plasma samples from either breast cancer patients or healthy donors. This was determined by immunoprecipitation of plasma proteins with the anti-BORIS antibody in combination with Western blot analysis and mass spectrometry and was further validated by analysis of tryptic plasma peptides using nano-liquid chromatography/tandem mass spectrometry following a previously described procedure (45).11 Therefore, it is not likely that BORIS could be taken up by PMNs from blood plasma.

In this study, we assessed the clinical significance of BORIS in the leukocyte fraction obtained from breast cancer patients and observed a positive correlation between IRS for BORIS in leukocytes from breast cancer patients and tumor size. A positive relationship between IRS for BORIS in the leukocyte fraction and the size of the matching breast tumor (Fig. 3) may be explained by more active signaling in the surrounding and adjacent tissues from larger tumors, thereby affecting BORIS production in leukocytes (the signaling hypothesis). On the other hand, more significant infiltration of the tumor by PMNs may occur with the increase of tumor size (the phagocytosis hypothesis).

In summary, in this study we have identified significantly higher levels of BORIS in the leukocyte fractions of patients diagnosed with breast tumors when compared to the control group of donors. To our knowledge, there are no previous reported observations of the presence of tumor-specific antigen in cell types other than tumor. Our novel findings therefore suggest the potential use of BORIS in blood leukocytes as a valuable marker for early detection of breast cancer, which may lead to the development of early intervention and prevention strategies.


   Acknowledgments
 
We thank F. MacNeill, P. Murray, C. Chandrasekharan, S. Marsh, K. Rooke, T. Dearson, M. Marshall, K. Reeve, I. Seddon, A. Colclough, R. Gooch, Z. Gatta, M. Hamblin, D. Rigg and colleagues, Copford Ward, Constable Wing Theatre and University of Essex staff for help with tissue and blood collection and patient information, S. Pack, D. Loukinov and H.C. Morse III, for helpful discussions, and the reviewers for helpful comments.


   Footnotes
 
Grant support: Breast Cancer Campaign (F. Docquier and E. Klenova), Breast Cancer Research Trust (M. Smart and E. Klenova), Association for International Cancer Research (I. Chernukhin and E. Klenova), Research Promotion Fund from the University of Essex (F. Docquier, I. Chernukhin, and E. Klenova), grant FISPI041083 from Spanish Ministerio de Sanidad y Consumo (M.D. Delgado), the Biomedical Research Collaboration Grant from the Wellcome Trust (E. Klenova and M.D. Delgado), Ph.D. studentship from the Biotechnology and Biological Sciences Research Council, United Kingdom (V. D'Arcy), predoctoral fellowship from Fondo Investigación Sanitaria and from University of Cantabria (V. Torrano), Ph.D. studentship from the University of Essex (D. Farrar), and Essex Rivers NHS Trust (N. Pore).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: V. D'Arcy, Z.K. Abdullaev, N. Pore, and F. Docquier contributed equally to this work. N. Pore is a Clinical Fellow.

6 J.I. Risinger, G.V.R. Chandramouli, G.L. Maxwell, et al. Global expression analysis of cancer testis genes in uterine cancers reveals a high incidence of BORIS expression (submitted for publication). Back

7 V. D'Arcy, F.M. Docquier, N. Pore, et al. BORIS, a cancer-testis gene that shares an 11 zinc finger DNA-binding domain with the tumor suppressor gene CTCF, is aberrantly expressed in breast tumors (manuscript in preparation). Back

8 http://rsb.info.nih.gov/ij/. Back

9 V. Lobanenkov, unpublished data. Back

10 C. Richard, M.D. Delgado, unpublished data. Back

11 E. Klenova, I. Chernukhin, M. Metodiev, unpublished data. Back

Received 12/16/05; revised 5/31/06; accepted 6/23/06.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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