Cancer is a leading cause of death in China, and the number of affected individuals is increasing, although different methods of treatment for cancer, for example, surgery, radiotherapy, chemotherapy, and endocrine therapy have been improved tremendously in recent decades. These factors, along with the information that about 98% of health-screened people show diseases or illness, some of them linked to development of pre-malignancies and malignancies [1–3], indicate that health screening is an important part of improving the quality of life.

Cancer can be treated effectively, if discovered at an early stage. Non-invasive serological methods for early detection of tumours may reveal potential malignancies before assessment by imaging techniques is possible, and thus increase the possibility of curing patients. Although a number of tumour markers have been identified [4–6], a review of the literature shows that measurements of most tumour markers alone are often insufficient to diagnose malignancies in health screening [3]. Guidelines for the use of tumour markers in a range of malignancies have been set up and published by the American Society of Clinical Oncology (ASCO), the American Cancer Society (ACS), the (U.S.) National Cancer Institute (NCI), and the (U.S.) National Comprehensive Cancer Network (NCCN). Pre-malignancies are easier to cure than malignancies. Detecting pre-malignancies with routine screening could prevent the occurrence of most human cancers. In vivo imaging techniques have been developed to discover small-size tumours, but the techniques are expensive. Furthermore, such imaging techniques cannot distinguish pre-cancerous lesions from malignancies very well. Even serologic tumour markers have limited sensitivity and specificity, with un-excepted high false positivity. Therefore, development of new techniques for discovery of pre-cancerous diseases is still needed [6].

Thymidine kinase (TK) activity in serum is a tumour growth-related marker which has been used for prognosis and monitoring of treatment of lymphoma and leukaemia since 1980 [7–10], and also to some extent in patients with solid tumours [9–11]. No immunohistology based on TK1 was available due to lack of useful anti-TK1 antibodies. However, the development by us of new generations of chicken polyclonal (IgY) [12] and mouse monoclonal anti-TK1 antibodies [13] extended the clinical use of TK1 to almost all types of solid human tumours, for both serum [12,14–27] and immunohistology [12,13,28] analysis. With these anti-TK1 antibodies, TK1 protein concentration in serum (STK1) is now useful for prognosis [17,22,26,27], treatment monitoring [14,16,18,19,21–23], and discovery of recurrence [17–19,21,24] of various types of solid tumours, in addition to lymphoma and leukaemia, showing an increasingly important role of TK1 in the clinical setting. Serological TK1 seems also to be useful for early screening of hyperplasia/neoplasia [1–3,15,19,20] and thus could be used as a warning system for development of malignancies later in life.

In this study we investigated the use of a sensitive chemiluminescent dot blot assay for determination of STK1 as a biomarker for pre-malignancy/malignancy in health screening. We combined previously published health screening results [1–3] with new, extensive health screening data (n = 7,080), all obtained in China between 2005 to 2011, involving 35,365 people in total. We compared health conditions in relation to TK1 protein concentration in the serum of people living in large and moder with people living and working in the countryside, where industrial pollutions (land-based oil field) probably influence health conditions in different ways compared to those of city dwellers. The TK1 serum assay is a commercial kit based on human TK1 chicken (IgY) antibodies produced by SSTK Ltd. (Shenzhen, China). These TK1 antibodies are the only TK1 antibodies available today with a sensitivity and specificity high enough to d iscriminate between, one the one hand, healthy people and people of non-malignant diseases, and on the other, people with pre-malignancies and malignancies. The present extensive study confirmed the results of the earlier studies, showing the usefulness of serological TK1 for health screening.

TK1 is an enzyme linked to DNA synthesis, and thus expressed in proliferating cells, both non-malignant and malignant. The concentration and activity of TK1 in blood serum of cancer patients are elevated, while TK1 in serum of healthy people is low or undetectable [10,12,32]. However, in some healthy people showing acute illness (infection, inflammation) or exhibiting other physiological changes (menstruation, blood donor, surgery), TK1 could increase transiently. The reason(s) behind the differences in the STK1 levels of healthy people and people with malignancies are still not fully understood. One possible explanation is linked to cell death. It is known that normal, non-malignant cells die in G1/G0 of the cell cycle, where the intracellular concentration of TK1 is low, while tumour cells die also in S/G2 stage of the cell cycle, where TK1 concentration is high [10,32], generating more TK1 in serum. It is also possible that a factor in serum affects the stability of TK1 in serum, acting differently in healthy people compared to cancer patients [15]. However, it cannot be excluded that TK1 is excreted from living normal and tumour cells in different ways. Since TK1 in serum shows transient increases when people have infection/inflammation due to activation of the immune system, it cannot be excluded that at least part of the elevated TK1 concentration in the serum of cancer patients is a result of a stimulated immune system and is not only excreted from tumour cells. Thus, when judging the implication of an elevated STK1 value as a risk of development of malignancies, reasons for the transient elevated STK1 values other than potential malignancies should also be taken in account [10]. Therefore, access to medical histories of patients is of importance.

The present health screening study of more than 35,000 people shows that TK1 protein concentration in serum (STK1) could be a useful biomarker for detection of people at the risk to develop malignancies later in life. The STK1 assay used is able to discriminate between tumour patients and healthy people, and people with various illnesses/non-malignant proliferation diseases (ROC value 0.96, likelihood (+) 233.73, sensitivity 0.798, specificity 0.997). The number of false-positive cases is low (1/300) compared to other biomarkers in use today, for example, mammography and PSA. Thus, the high ROC and likelihood (+) value show that the STK1 assay meets the requirements of a health screening test. Furthermore, since most malignant patients (65–90%) express an STK1 concentration >2.0 pM [15], it is reasonable to suspect that people with STK1 values >2.0 pM, but still not showing visible tumours, have an increased risk to develop malignancies later in life. On the other hand, people with STK1 values <2.0 pM likely have a lower risk to develop malignancies. We suggest that people with STK1 values >2.0 pM should recheck their STK1 values and health conditions within 3–6 months, while people with STK1 values <2.0 pM have their annual health test.

We found that the frequency of STK1 values varied, depending on living conditions, that is, 0.8% of the city dwellers and 5.8% of the land-based oil-field workers. Both these values are higher than the cancer incidence rate of China today (0.2–0.3%) [31]. A cancer incidence rate is based on the whole population, but could differ in subgroups of people, depending on living conditions. It is possible that those people living and working in an oil-field area are exposed to pollut that lead to higher frequency of proliferation-related diseases, such as pre-malignancy/malignancy. The significantly different STK1 values found in this study between the oil drilling/oil transportation staff and the people working in the oil company service/office suggest that the working conditions for the people participating in this study affect the frequency of malignancy-related diseases, and as a consequence, the STK1 values. It should also be noted that about 85% of the people in the STK1 elevated group are pre-malignant cases. However, not all pre-malignant cases ended up in malignancies. For example, only about 10–20% of women with pre-malignant breast hyperplasia show a higher risk of developing breast malignancy [30]. In the present study, about 95% of the breast hyperplasia was of lobular type, in accordance with a Chinese study of women showing about 83% of breast adenoids of lobular hyperplasia type [33]. Lobular neoplasia broadly defines the spectrum of changes within the lobule, ranging from atypical lobular hyperplasia (ALH) to lobular carcinoma in situ (LCIS). These types of lesions are associated with an increased risk for developing subsequent invasive breast cancer [34–36]. Thus, if the STK1 value is correlated to pre-malignant diseases, the frequency of people with elevated STK1 values should be higher than the cancer incidence rate.

The number of people with malignancies in this study was about 30 times less than expected from the cancer incidence rate of China (0.01% versus expected 0.2–0.3%) [31]. However, those with malignancies were exclusively found in the group of people with elevated STK1 values, indicating that the cut-off value of STK1 2.0 pM is useful for identifying people with malignancies in health screening. The reason for the limited number of people with malignancies found is that people who participate in health screening regard themselves as healthy, with no obvious symptoms. In China today, those with obvious symptoms for malignancies (pain, reduced weight, blood in urine/faeces, fever, etc.) contact the hospital directly.

A more interesting subject is how many people with elevated STK1 will develop malignancies later in life. Since the development of malignancies from pre-malignancies takes rather a long time, such studies need 10–15 years follow-up. In this study we were able to follow some of the people (24%) of the elevated STK group for six years. We found that there was about 30 times higher risk to develop new malignancies in the elevated STK1 group compared to the normal STK1 group.

The elevated STK1 value also correlated to moderate/severe type of hyperplasia of breast and prostate, but not to low-risk hyperplasia. No such correlation was found in people with HBV and fatty liver of moderate/high risk, or to with obesity. This further supports the notion that STK1 specifically reflects changes in proliferation of pre-malignancies, but not of pre-malignancies that do not yet show uncontrolled proliferation, indicating the proliferation specificity of TK1 in serum. This is in accordance with the observation that women with breast hyperplasia show a high risk of developing breast malignancy [30].

The higher frequency of breast and prostate hyperplasia found among city-dwelling people compared to those living in the countryside is in accordance with previous reports. The frequency of breast and prostate hyperplasia is increasing in city residents of China. The frequency of breast hyperplasia is now 30–50% of women aged around 40 years [30], and 18% of men with prostate hyperplasia [37]. On the other hand, only 2.1% of women living in the countryside in China show breast hyperplasia [38]. Although the significance of prostate benign hyperplasia (PBH) and atypical adenoma hyperplasia (AAH) for the development of prostate carcinoma is still debated, there seems to be a link to progression of malignancy [39–43].

Obesity is now considered to be a multifunctional chronic disease, resulting from interactions between genetic and environmental factors [44]. Obesity is a significantly increased risk factor of mortality in cancer and accounts for about 20% of the more frequent type of malignancies [45]. In an epidemiological investigation of 401,215 participants [46], an increased risk of mortality in cancer between 1.50 and 4.21 times was found, depending on the type of malignancy.

This study indicates that the chemiluminescent dot blot STK1 assay can distinguish between people with low or high risk of developing malignancies, before any indication of visible tumours. Hence, TK1 in serum is useful in health screening as a reliable tumour-proliferating marker alone or in combination with routine inspections for an early risk warning assessment of malignant process. The STK1 assay can be applied to different occupations and groups, particularly in cancer frequency high-risk areas such as industries with chemical exposures, and among elderly people. STK1 is a reliable marker for assessment of tumour cell proliferation that can achieve the goal of “early detection and early treatment of tumours”.