Using biomarkers representing proteins with different biological functions may allow complementarity in cancer detection, resulting in increased sensitivity without compromising specificity.
Cancer is a significant health problem in Asia1 and around the world2, being the leading cause of mortality in Taiwan, Thailand, Singapore, Korea, Hong Kong, and Japan. The disease is becoming an increasingly important health concern in countries such as Malaysia and The Philippines as the impact of communicable diseases decreases with the widespread supply of clean water and sanitation facilities. Cancer currently is the third and fourth leading cause of death in The Philippines and Malaysia, respectively. For most cancers, detection of asymptomatic early-stage disease, when the tumors are still localized, is critical for effective treatment and possibly full recovery3. Unfortunately, most cancers are detected at later stages, after they have invaded the surrounding tissue or metastasized to distant sites. Current screening methods include imaging (e.g.mammography), endoscopy (e.g.colonoscopy and sigmoidoscopy for colon cancer), cytology ((e.g.Papanicolau or Pap test for cervical cancer), and blood test (e.g.PSA test for prostate cancer). Of all currently used screening methods, the Pap test developed for cervical cancer is the most effective, resulting in a dramatic decrease of mortality from this cancer. Other routine screening methods, mammography for breast cancer, serum prostate specific antigen (PSA) testing for prostate cancer, and fecal occult blood testing and colonoscopy for colon cancer, have also reduced mortality through early detection although to a lesser extent.
Unfortunately, none of these tests possesses high sensitivity (probability that someone with cancer will have a positive test) for early disease or specificity (probability that someone without cancer will have a negative test). As a result, only about 50% of breast cancers, 56% of prostate cancers, and 35% of colorectal cancers are diagnosed at the early stages, and these cancers remain the three leading causes of cancer death, respectively, after lung cancer. In another example, over 80% of the two deadliest cancers, ovarian and pancreatic, are diagnosed at late stages, where the chances of survival are dismal, due to asymptomatic nature of these cancers at early stages4,5.
Biomarkers - Improving cancer detection
Most detection methods in use to date identify fully developed cancer, not the pre-malignant or early lesions amenable to resection and cure. For example, imaging methods, such as mammography ortransvaginal ultrasound, visualize tumors at >1 cm diameter, fecal occult blood is the result of invasive tumour. On the other hand, Pap test and endoscopy (colonoscopy, sigmoidoscopy, and others) are sometimes able to detect pre-cancerous lesions. Another problem is that in many organs, for example, prostate or colon, pre-neoplastic lesions are much more common than aggressive cancers, and only 10% or less develop into a malignant tumour6. Therefore, the development of a screening modality that (i) will have high sensitivity for early disease, and (ii) would diagnose cancers at very early, possibly pre-clinical stages, and (iii) would be capable of discrimination of aggressive cancers vs. benign or non-aggressive tumors, is strongly desired.
Blood-based tests may be good candidates as early cancer screening tools, since: (i) blood collection is simple and minimally invasive; (ii) tumour biomarkers may come not only from the tumour but from other organs and tissues and may represent the systemic response to tumour growth. As such, these proteins may be secreted into the bloodstream at the very early stages of tumourigenesis, when tumour itself is undetectable by conventional imaging methods. Presently, very few blood biomarkers have proven useful for diagnosing primary cancer. Only three biomarkers, including serum PSA for prostate cancer, and bladder tumour antigen (BTA) and nuclear matrix protein-22 as diagnostic markers for bladder cancer7,8 are currently approved by FDA and are sensitive enough for screening and early detection in selected populations.
Despite the success of PSA in detecting early stage prostate cancer, its use to screen patients for prostate cancer remains controversial due to overdiagnosis. Three other serum markers have been approved by the FDA for cancer diagnosis: alpha-fetoprotein (AFP) for hepatocellular carcinoma and testicular cancer, catecholamines for neuroblastoma, and immunoglobulins for multiple myeloma9. Because the assays for these proteins are neither sensitive nor specific enough for use as the sole screening method for early cancer detection, all are used as an adjunct to other direct detection and diagnostic methods. The identification of a new class of cancer associated serum proteins, and the validation of sensitive and specific predictive assays, would expand the current clinical capabilities for early cancer detection and diagnosis and further reduce cancer mortality.
The intensive search for new biomarkers that would allow overcoming the hurdles of early cancer detection is currently under way. Recent advances in biomarker development using gene arrays10 in addition to proteomic technologies, including two-dimensional electrophoresis and mass spectrometry11,12 have facilitated the discovery of several new biomarkers. Recently, the FDA has approved a small number of new urine-based biomarkers, including bladder tumour antigen (BTA) and nuclear matrix protein-22 as diagnostic markers for bladder cancer7,8. Three serum biomarkers of ovarian cancer, apolipoprotein A1, truncated form of transthyretin, and a cleavage fragment of inter-alpha-trypsin inhibitor heavy chain H4, were identified using mass spectrometry approach13.
After new promising biomarkers are discovered, they must be validated for their ability to discriminate patients with cancer from healthy individuals. No single biomarker is likely to have 100% sensitivity and specificity for a specific cancer. Instead, combinations (panels) of several biomarkers seem to be a promising alternative for the use in clinical laboratories. There are several benefits of using combinations of multiple biomarkers. First, since each cancer is represented by several histologies, and even each histologic type is heterogeneous, several biomarkers may help to recognize all cancer subtypes. As mentioned earlier, multiple biomarkers should represent not only proteins secreted directly by tumour but also proteins representing systemic host response to tumour growth. These latter biomarkers will depend less on tumour size and may be measurable early in the tumour development. Finally, using biomarkers representing proteins with different biological functions may allow for complementarity in cancer detection, resulting in increased sensitivity without compromising specificity. Usefulness of combining three serum biomarkers along with CA125 for increased sensitivity and specificity in ovarian cancer was demonstrated in three publications. Two biomarker combinations, CA 125, CA 72-4, CA 15-3, and M-CSF (13), and CA 125, apolipoprotein A1, truncated form of transthyretin, and a cleavage fragment of inter-alpha-trypsin inhibitor heavy chain H4 (13) substantially improved test accuracy over CA 125 alone, with a sensitivity of 70-73% at a specificity of 97-98%. A panel of 4 biomarkers, leptin, prolactin, IGF-II, and osteopontin, reportedly exhibited a sensitivity of 95% at a specificity of 95% (14)14.
At the University of Pittsburgh Cancer Institute, we have developed highly sensitive and specific biomarker panels for early diagnosis of a variety of cancers, with preliminary results appearing very promising for ovarian (sensitivity 90%/specificity 98%), pancreatic (sensitivity 96%/specificity 98%), and endometrial (sensitivity 98%/specificity 98%) cancers. These assays are able, in addition to distinguishing cancer cases from healthy controls, to also discriminate malignant from benign tumors, thus increasing the specificity of assays. Importantly, each panel was able to identify a specific cancer but not other cancers. Furthermore, work is in progress for developing an assay for breast cancer in premenopausal women that substantially surpasses the results of mammographic screening. These assays will be further validated in retrospective trials in which large populations of healthy individuals were screened yearly and blood samples were obtained from them over the course of over 10 years. Among the individuals who were diagnosed with cancers, blood samples preceding the diagnosis are now available to determine the interval prior to diagnosis when biomarker panels indicate the presence of cancer. Subsequent prospective clinical trials will determine the effect of early detection on mortality from these cancers.
Conclusion
Serum-based panels of multiple biomarkers hold a great promise for better and more efficient early diagnosis of cancers. Such tests are not only more convenient and less expensive, but also may demonstrate superior sensitivity and specificity in comparison to conventional screening methods. Blood-based assays may detect cancer at very early, potentially pre-clinical stages when the probability of efficient therapy and complete cure is the highest. Development of such assays for early detection may shift cancer therapy towards the development of new strategies aimed at treatment of very early or pre-cancerous lesions.
References:
1. Smith, M. Overview of Cancer in Asia. Oncology Forum, 6: 3-7.
2. Greenlee, R. T., Hill-Harmon, M. B., Murray, T., and Thun, M. Cancer statistics, 2001. CA Cancer J Clin, 51: 15-36, 2001.
3. Smith, R. A., Mettlin, C. J., Davis, K. J., and Eyre, H. American Cancer Society guidelines for the early detection of cancer. CA Cancer J Clin, 50: 34-49, 2000.
4. Menon, U. and Jacobs, I. Screening for ovarian cancer. Best Pract Res Clin Obstet Gynaecol, 16: 469-482, 2002.
5. Brand, R. The diagnosis of pancreatic cancer. Cancer J, 7: 287-297, 2001.
6. Manne, U., Srivastava, R. G., and Srivastava, S. Recent advances in biomarkers for cancer diagnosis and treatment. Drug Discov Today, 10: 965-976, 2005.
7. Konety, B. R. and Getzenberg, R. H. Urine based markers of urological malignancy. J Urol, 165: 600-611, 2001.
8. Mungan, N. A., Vriesema, J. L., Thomas, C. M., Kiemeney, L. A., and Witjes, J. A. Urinary bladder cancer test: a new urinary tumor marker in the follow-up of superficial bladder cancer. Urology, 56: 787-792, 2000.
9. Wagner, P. D., Maruvada, P., and Srivastava, S. Molecular diagnostics: a new frontier in cancer prevention. Expert Rev Mol Diagn, 4: 503-511, 2004.
10. Weeraratna, A. T. Discovering causes and cures for cancer from gene expression analysis. Ageing Res Rev, 4: 548-563, 2005.
11. Ornstein, D. K. and Petricoin, E. F., 3rd Proteomics to diagnose human tumors and provide prognostic information. Oncology (Williston Park), 18: 521-529; discussion 529-532, 2004.
12. Wulfkuhle, J. D., McLean, K. C., Paweletz, C. P., Sgroi, D. C., Trock, B. J., Steeg, P. S., and Petricoin, E. F., 3rd New approaches to proteomic analysis of breast cancer. Proteomics, 1: 1205-1215, 2001.
13. Skates, S. J., Horick, N., Yu, Y., Xu, F. J., Berchuck, A., Havrilesky, L. J., de Bruijn, H. W., van der Zee, A. G., Woolas, R. P., Jacobs, I. J., Zhang, Z., and Bast, R. C., Jr. Preoperative sensitivity and specificity for early-stage ovarian cancer when combining cancer antigen CA-125II, CA 15-3, CA 72-4, and macrophage colony-stimulating factor using mixtures of multivariate normal distributions. J Clin Oncol, 22: 4059-4066, 2004.
14. Mor, G., Visintin, I., Lai, Y., Zhao, H., Schwartz, P., Rutherford, T., Yue, L., Bray-Ward, P., and Ward, D. C. Serum protein markers for early detection of ovarian cancer. Proc Natl Acad Sci U S A, 102: 7677-7682, 2005
