An artistic depiction of metastatic cancer cells (http:\www.
eliax.com)
When it comes to treating a cancer patient, time is of the essence for accurately identifying the type and malignancy of a tumor. A misdiagnosis of malignant tumors can be the difference between life and death for a any advanced stage cancer patient. There are many cancer types that are currently resistant to conventional DNA damaging chemotherapy agents such as cisplatin and nitrosoureas , a DNA alkylating agent, which are highly cytotoxic to tumors and to normal cells to a certain extent by inducing DNA mutations. However, only a subset of patients respond to cisplatin and nitrosoureas since these tumors over-express certain DNA repair enzymes such as ERCC1-XPF that renders them resistant to chemotherapy. This enzyme consists of two subunits with ERCC1 having a DNA repair function while XPF binds to region of DNA that is damaged. In normal mammalian cells, this enzyme is needed to protect cells from DNA damage induced by oxidative stress and delays aging. On the other hand, tumors expressing high amounts of this enzyme confers them resistance to chemotherapy or UV induced damage making cancer treatment more cumbersome and complicated. In tumor cells, ERCC1-XPF has proof-reading activity and catalyzes the repair of both DNA base pair mutations induced by UV irradiation and DNA strand breaks promoted by chemotherapeutic agents.
Current clinical protocols used to detect the presence of this biomarker by immunohistochemistry or by Western blot assays (a 2 dimensional assay used to measure the levels of proteins in cells or tissue samples) have not been well established and is controversial as different hospitals and clinics use different antibodies with varying affinities and specificities to detect the presence of this enzyme immunohistologically in tumor cells.
Researchers at the University of Pittsburgh Cancer Institute, a state of the art research institute located in Shadyside hospital, have devised and optimized a protocol to efficiently screen ERCC1-XPF in tissue samples. More importantly, this novel clinical protocol may predict how efficient chemotherapy will work in cancer patients. Furthermore, the authors of the study published in the journal of Cancer Research this month found that the levels of ERCC1-XPF differ from tumor to tumor; tumors that contained low levels of this enzyme are efficiently killed by UV induced irradiation or by chemotherapy while tumors bearing high levels of this protein are resistant to conventional chemotherapy. In their study, the team exhaustively evaluated the specificity of a large panel of antibodies that are currently used to measure ERCC1-XPF in tumors. In fact, their study showed that the most popular antibody (clone 8F-1) used in many research and pathology labs to diagnose the presence of this biomarker in tumors is not very specific for recognizing ERCC-XPF as it recognizes additional non-specific nuclear proteins in tumors as shown by histological analysis of biopsies or by Western blot assays. This is a very concerning and grim report since the ramifications of using a flawed antibody for analyzing cancer biomarkers can lead to poor diagnosis and a poor prediction of patient prognosis. Their results showed that this antibody (clone 8F-1) is highly non-specific by immunocytochemistry and Western blot assays for measuring the levels of ERCC1-XPF in tissue biopsies compared to other antibodies that work much better. Overall, this study provides a reliable protocol for clinicians to measure the enzyme biomarker in clinical specimens and begs the question whether multiple antibodies may be required to determine the malignancy of tumors before providing a prescriptive recommendation for a personalized chemotherapy.
Final thoughts- Given the variability of many different biomarkers seen in tumors (ie., Her2, anaplastic lymphoma kinase, EGF receptors in breast cancer and lung cancer respectively), it is important to always tailor each chemotherapy for each patient. Unfortunately, many hospitals around Western Pennsylvania do not administer individualized therapies but use a generalized chemotherapy protocol to cancer patients as a first line of defense. It is worth noting that immunocytochemical and RT-PCR assays performed on tissue biopsies can take upto two weeks from the time the biopsy is collected to the time that the assays are performed and interpreted by a certified pathologist. Thus, by the time a more in-depth molecular analysis of the tumor is required on a cancer patient in order to devise a tailored cancer therapy, the patient has sustained the normal tissue has sustained significant damage during the initial round of chemotherapy and the cancer has progressed significantly to the point that may render the patient untreatable. To this end, better strategies for rapidly diagnosing the cancer in non-responsive patients in the future will depend on the use of highly reliable protocols and the use of automated high throughput assays such as RNA and protein microarrays, used to validate and confirm the grade of tumor malignancy by molecular biology means, and confirmed immunohistologically by a Cell Array Scan, a machine that has the capacity to analyze dozens of tissue samples immunohistochemically in a high throughput manner at a time. In the end, every clinical diagnosis generated by automated machines will have to be validated and confirmed by a pathologist since no diagnostic assay can match the human eye reliably to this date.
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Original press report: http://www.upmc.com/MediaRelations/NewsReleases/2009/Pages/Researchers-Develop-Biomarker-Measurement.aspx
References
Nikhil R. Bhagwat, Vera Y. Roginskaya, Marie B. Acquafondata, and Rajiv Dhir, M.D. Immunodetection of DNA Repair Endonuclease ERCC1-XPF in Human Tissue. Cancer Research 69(17): 6821-6839
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