Thymidine kinase 1: the key to predicting cancer recurrence
Dr. Kim L. O’Neill, Microbiology and Molecular Biology
Currently it is estimated that over 230,000 women will be diagnosed with breast cancer this year in the United States alone. Although treatments are improving, an alarming number of these cases will show cancer recurrence after treatment. A method to consistently predict cancer recurrence is crucial to better treat cancer and improve patient care. Thymidine Kinase 1 (TK1) has been found in abnormally high levels in serum and tissue of cancer patients compared to healthy patients. There is question to why TK1 is present in such high quantities when normally it aids in the DNA salvage pathway during S phase of the cell cycle and is otherwise virtually absent. It has also been shown that elevated TK1 levels in tissue is an early event in most cancers, and therefore has potential to be utilized in early diagnosis and could possibly be used to also predict cancer recurrence. We expect that TK1 is elevated in tissues of patients that later show cancer recurrence while patients that do not relapse have low levels of TK1. With further study of TK1, we may be able to differentiate between patients that later show recurrence and those that do not. Being able to predict patient recurrence for breast cancer patients would help provide better treatment to patients and possibly prevent relapse in the future.
To develop TK1 as a marker for cancer recurrence, we first obtained tissue slides from the Utah Valley Regional Medical Center. We were given breast cancer tissues as well as healthy breast tissue. The hospital is currently working to obtain pre-cancerous tissues that are then monitored for later cancer development to continue our study. In order to test our hypothesis that TK1 levels in breast tissue may predict cancer recurrence, we performed immunohistochemistry (IHC) staining to detect the presence of TK1 in the tissue samples that were obtained.
The tissue slides were prepared by removing the paraffin, rehydrating the tissues, and then performing antigen retrieval. These are necessary steps to prepare the tissues to be stained for TK1. The samples were blocked using Biomedical Care Sciences Serum Block to prevent background and stained using a rabbit anti-human TK1 antibody from Abcam. We then probed the slides with an anti-rabbit HRP secondary produced by Biomedical Care Sciences. Appropriate controls will also be prepared for each staining that included GAPDH as a positive control and the Biomedical Care Sciences Universal Block was used as a negative control. We used Vector Lab DAB enzymatic development kit which will form a brown precipitate when the anti-TK1 complex is present, ultimately determining if TK1 being overexpressed. The slides were then washed, counterstained with hematoxylin, and dehydrated. We mounted the slides with slide covers and Cytoseal for preservation. The tissues were imaged under a light microscope at a 20X increase.
As shown in Figure 1a-c, healthy breast tissue was stained for TK1. Figures 1a and 1b demonstrate the negative control and positive control, respectively. As expected there was little to no binding shown in Fig. 1a, dark brown precipitate in Fig. 1b, indicating the presence of the GAPDH complex and little to no binding in figure 1c. Fig. 1d-f shows the staining performed on breast cancer tissue. Fig. 1d pictures the staining with the negative control serum, Fig. 1e shows the GAPDH positive control, and Fig. 1f shows a distinct brown precipitate indicating the presences of TK1 complex binding to the tissue.
Since the negative controls are clean and the tissue remains blue (Fig. 1a & 1d), and both of the positive controls (Fig, 1b & 1e) are brown in both the healthy tissue and cancer tissue, we are confident that our IHC tissue staining method is consistent and accurate. Fig. 1c shows the healthy breast tissue which usually has very low levels of TK1 expression. As expected, the tissue has little to no staining when probed for TK1. Fig. 1f shows a distinct brown precipitate in the tissue indicating the overexpression of TK1 in the tumor tissue. This difference can be used to detect cancer and can be used as a definite marker to confirm breast cancer. Further studies must be performed in order to confirm TK1 as a marker for cancer recurrence when appropriate tissues are acquired.
In this study we were able to confirm that TK1 is upregulated in breast cancer tissue and not in healthy breast tissue. Although we were unable to acquire all of the necessary slides to finish this study, we were able to develop a staining method that was consistent and reliable. We are actively working with the hospital to obtain the recurrent tissue slides as quickly as possible to prepare for publication. The images that were obtained in this study are being used in a separate publication in the O’Neill lab to help confirm the upregulation of TK1 in cancer tissues and not normal tissues. This publication is currently under review and we expect will be publish shortly. Overall, this ORCA grant has aided in a very successful research project which will result in two publications in cancer research journals and has helped us to obtain the knowledge needed to better treat cancer and predict recurrence.