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HomemistoryLatest Research: Tear Biomarkers

Latest Research: Tear Biomarkers

A team of researchers led by Professor Mark Willcox, Chief Scientific Officer at Sydney’s Brien Holden Vision Institute, are searching for biomarkers in tears that could indicate life-threatening diseases such as breast cancer. Further research into the bio-activity of the biomarkers could have a major impact on the prognosis of breast cancer for the 1.5 million women around the world who succumb to the disease each year.

According to the National Institute of Health, U.S.A, the official definition of a biomarker is “a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.”

Our group has been interested in examining the use of tears as potential biomarkers for disease. We have received funding from the Australian Research Council to study whether there are changes in the proteins in the tear film during diabetes and prostate cancer, and are now turning our research efforts towards breast cancer. We believe that the proteins expressed in tears may change during disease processes, and these changes could be useful in monitoring those at risk of getting disease, the pro



gression of the disease, and the effectiveness of therapies to treat the disease.

Screening Misses Tumours

Breast cancer continues to be the leading cause of cancer-specific death in women in western countries including Australia. Screening using mammograms is currently the best available approach for detection of breast cancer in people over 50 years. Small lesions are frequently missed and may not be visible with mammograms, particularly in young women and women with dense breast tissues.1 Therefore, a significant proportion of tumours detected in women undergoing regular screening have already disseminated, and patients present with invaded lymph nodes or general metastases at the time of diagnosis.

These findings suggest that it is useful to develop tear film analysis to provide a simple non-invasive test for the diagnosis and/or management of breast cancer…

The one to two year regular mammogram screening program does not allow for the detection of tumours with a high growth rate at an early stage. Although patient survival has improved over the past few decades due to screening programs and postoperative adjuvant systemic therapies (hormone therapy and chemotherapy), many patients die from metastatic relapse. The marker currently used for breast cancer monitoring is an increase in the serum cancer antigen CA 15-3 (the product of the MUC-1 gene). However, CA 15-3 measurements from plasma are not helpful in diagnosis, especially in patients with early stage cancers, and are not useful in the therapeutic decisionmaking of patients with breast cancer.2

Traditional prognostic markers (age at diagnosis, tumour size, hormonal receptor status, tumour grade) are not sufficient for precise risk-group discrimination in breast cancer.3 Molecular markers that can potentially be used to identify small lesions that are not visible with imaging techniques could provide an opportunity to treat breast cancer early, before it invades surrounding tissue. Because breast cancer is heterogeneous, there is likely to be no single marker that can be used for early diagnosis and progression monitoring.

Unique Advantages In Tears

Tears are a unique source of body fluid and contain mucins, glycoproteins, unglycosylated proteins, peptides and lipids. The major tear proteins are lysozyme, lactoferrin, secretory immunoglobulin A (sIgA), lipocalin and lipophilin. The protein components of tears can change in local eye disease,4 systemic diseases such as diabetes5 and cancers.6,7,8,9

The tear proteome has been proposed to contain around 500 proteins.10 Doughty reported that components of the tear film include proteins, inflammatory mediators (including various interleukins) from serum/plasma leakage from the conjunctival vasculature.11 Cancer biomarkers could be members of the immune system, fragments of tumourassociated antigens, oncofetal proteins, adhesion proteins, acute phase proteins and others, which could be leaked from blood to tears through conjunctival vasculature and cause aberrant levels of proteins present in tears.

Tears have a number of advantages as a fluid for biomarker investigation compared with the most commonly used fluid, which is serum. Firstly, its collection is a non-invasive procedure. This ease of collection also enables multiple samples to be collected from a single subject over a period of time to monitor cancer progression or regression.

Secondly, tear proteins/peptides tend to be relatively small in size with a higher thermodynamic stability and are less complex and interactive than in serum. The lower range of proteins and proportion of serum albumin and immunoglobulins in tears compared to serum, make proteomic analysis more straightforward, as demonstrated by studies in our laboratories.6,7

Positive Study Results

These results suggest good potential for cancer diagnosis by analysing tear fluid proteins. Several studies have identified certain antigens in serum or tissue as prospective markers for breast cancer.12,13 It is already known that tears contain many of these potential markers. For example, tears contain MUC1 (aka CA 15-3),14 and it is often differentially glycosylated to other areas of the body, and MUC1614 which when bound with mesothelin (which is also found in tears) may facilitate the implantation and spread of tumors.15

Tears also contain the mucin-like protein Deleted in Malignant Brain Tumor (DMBT)-1. Reduced amounts of DMBT-1 in other areas of the body have been associated with increased risk of developing breast cancer.16 Tears can contain several of the cytokines and hormones that change in serum during breast cancer (IL-1, IL-6, IL-Ra, IL-8, MCP-1, Prolactin).17,18

Tears also contain acute phase proteins such as α-2-macroglobulin. However, none of these potential markers in tears have been validated for their ability to screen for breast cancer and may not be useful for early cancer detection required for population screening. Quantitative proteomic analysis of tears in prostate cancer and breast cancer has been carried out recently in our laboratory.19 Our results support the potential use of tear fluid for cancer detection diagnosis. We intend to search for novel human tear biomarkers using modern proteomic approaches.

Our research team found that lacryglobin (mammaglobin, a low molecular weight protein) could be detected in tears from breast cancer patients with bone metastasis but not in healthy controls, suggesting that lacryglobin in human tears may be a potential marker for breast cancer.6 Later on, mammaglobin was further confirmed to be a potential marker for metastasis of breast cancer cells to lymph nodes.20

Furthermore, we also demonstrated that a number of differences in peptide mass fingerprint were found between the tears of healthy dogs and the dogs with cancer.21Differences were found in levels of actin and albumin and in an unidentified protein, which may be analogous to human lacryglobulin.21

These findings suggest that it is useful to develop tear film analysis to provide a simple non-invasive test for the diagnosis and/or management of breast cancer. Lebrecht et al recently demonstrated a biomarker panel from tear fluids that allows breast cancer patients to be differentiated from healthy women.8 The same group further confirmed that the diagnostic pattern in tear fluid differentiated cancer patients from controls with a specificity and sensitivity of approximately 90 per cent using the same technique.9 These results further confirm that tears may be a potential source of biomarkers for breast cancer. A disadvantage of the technique used by Lebrecht et al is the difficulty in identifying biomarkers.

More Studies Planned

We plan to examine in more detail, with more refined proteomic techniques than have been used previously, the tear film differences between women with breast cancer and controls. The significance of these studies is the likely discovery of markers that can be used for early diagnosis and staging of breast cancer, and monitoring breast cancer progression and/or the effects of treatment.

These studies may also point the way for future research in understanding whether the bioactivity of the proteins found is related to disease progression and remission, so that new treatments and prevention methods can be developed. This may well have a major impact on the prognosis of breast cancer for the tens of thousands of older women who succumb to the disease each year. The use of tear samples may also provide a less invasive method for detecting breast cancer.

Professor Mark Willcox is a world leading contact lens researcher and the Chief Scientific Officer at the Brien Holden Vision Institute in Sydney. He is also Professor at the School of Optometry and Vision Science, UNSW.

Dr. Zhenjun Zhao is the tear biomarker project leader at Brien Holden Vision Institute, Sydney, Australia.Dr. Yong Li is a researcher with the Cancer Care Centre, St. George Hospital and St George Clinical School, University of New South Wales. He was awarded the Best Medical Research Award-NHMRC Achievement Award in 2010.

Dr. Bradley Walsh is the founding director and Chief Scientific Officer at Minomic International Pty Ltd, Sydney, Australia.

References:

1. Antman K, Shea S (1999) Screening mammography under age 50. JAMA 281: 1470-1472 2. Kumachi F, Basso SM, Brandes AA, Pagano D, Ermani M (2004) “Relationship between tumor markers CEA and CA 15-3, TNM staging, estrogen receptor rate and MIB-1 index in patients with pT1-

2 breast cancer. Anticancer Res 24: 3221-3224

3. Fitzgibbons PL, Page DL, Weaver D, Thor AD, Allred DC, Clark GM, Ruby SG, O’Malley F, Simpson JF, Connolly JL, Hayes DF, Edge SB, Lichter A, Schnitt SJ (2000) Prognostic factors in breast cancer. College of American Pathologists Consensus Statement 1999. Arch Pathol Lab Med 124: 966-978

4. Tiffany JM (2003) Tears in health and disease. Eye 17: 923-926

5. Herber S, Grus FH, Sabuncuo P, Augustin AJ (2002) Changes in the tear protein patterns of diabetic patients using two-dimensional electrophoresis. Adv exp Med Biol 506: 623-626

6. Evans V, Vockler C, Friedlander M, Walsh B, Willcox MD (2001) Lacryglobin in human tears, a potential marker for cancer Cli Exp Ophthalmol 29: 161-163

7. Li Y, Cozzi PJ, Zhao J, Russell PJ, Walsh BJ, Willcox M (2007) Identification of novel biomarkers in tears for prostate cancer diagnosis and prognosis. Advances in Proteomics in Cancer Research, AACR special conference. 27 Feb- 2 Mar, Amelia Island, Florida, USA 2007

8. Lebrecht A, Boehm D, Schmidt M, Koelbl H, Schwirz RL, Grus FH (2009A) Diagnosis of breast cancer by tear proteomic pattern. Cancer Genomics Proteomics 6: 177-182.

9. Lebrecht A, Boehm D, Schmidt M, Koelbl H, Grus FH (2009B) Desorption/Ionisation Time-of-flight Mass Spectrometry to Detect Breast Cancer Markers in Tears and Serum. Cancer Genomics Proteomics 6: 75-83.

10. de Souza GA, Godoy LMF, Mann M (2006) Identification of 491 proteins in the tear fluid proteome reveals a large number of proteases and protease inhibitors. Genome Biol 7:R72

11. Doughty M (2002) The cornea and conjunctival surfaces in relation to the tear film. In: Korb DR, Craig J, Doughty MJ, Smith G, omlinson, A (eds.) The tear film: structure, function and clinical examination. London: Butterworth-Heinemann.

12. Kim BK, Lee JW, Park RL, Shin YS, Lee WY, Lee KA, Ye S, Hyun H, Kang KN, Yeo D, Kim Y, Ohn SY, Noh DY, Kim CW (2009) The multiplex bead array approach to identifying serum biomarkers associated with breast cancer. Breast Cancer Res 11: R22

13. Jesneck JL, Mukherjee S, Yurkovetsky Z, Clyde M, Marks JR, Lokshin AE, Lo JY (2009) Do serum biomarkers really measure breast cancer? BMC Cancer 9: 164

14. Mantelli F, Argueso P (2008) Functions of ocular surface mucins in health and disease Current Opinion Allergy Clin Immunol 8: 477-83

15. Kaneko O, Gong L, Zhang J, Hansen JK, Hassan R, Lee B, Ho M (2009) A binding domain on mesothelin for CA125/MUC16. J Biol Chem 284: 3739-3749

16. Tchatchou S, Riedel A, Lyer S, Schmutzhard J, Strobel-Freidekind O, Gronert-Sum S, Mietag C, D’Amato M, Schlehe B, Hemminki K, Sutter C, Ditsch N, Blackburn A, Hill LZ, Jerry DJ, Bugert P, Weber BH, Niederacher D, Arnold N, Varon-Mateeva R, Wappenschmidt B, Schmutzler RK, Engel C, Meindl A, Bartram CR, Mollenhauer J, Burwinkel B. Identification of a DMBT1 polymorphism associated with increased breast cancer risk and decreased promoter activity. Hum Mutat. 2010 Jan;31(1):60-6.

17. Thakur A, Willcox MDP, Stapleton F (1998) The proinflammatory cytokines and arachidonic acid metabolites in human overnight tears: homeostatic mechanisms. J Clin Immunol18: 61-70

18. Mathers WD, Stovall D, Lane JA, Zimmerman MB, Johnson S (1998) Menopause and tear function: the influence of prolactin and sex hormones on human tear production. Cornea 17: 353-358

19. You J, Fitzgerald A, Cozzi PJ, Zhao Z, Graham P, Russell PJ, Walsh BJ, Willcox M, Zhong L, Wasinger V. Li Y. et al (2010) Post-translation modification of proteins in the tear. Electrophoresis 31: 1853-1861

20. Ouellette RJ, Richard D, Maicas E (2004) RT-PCR for mammaglobin genes, MGB1 and MGB2, identifies breast cancer micrometastases in sentinel lymph nodes. Am J Clin Pathol 121: 637-643

21. de Freitas Campos C, Cole N, Van Dyk D, Van Dyk D, Walsh BJ, Diakos P, Almeida D, Torrecilhas A, Laus JL, Willcox MD (2008) Proteomic analysis of dog tears for potential cancer markers. Res Vet Sci 85: 349-352

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