ANTICANCER RESEARCH 28 : 3897-3908 (2008)

Serological 1 (STK1) Indicates an Elevated Risk for the Development of Malignant Tumours ZHIHENG CHEN 1, HUI ZHOU 1, SHENGLAN LI 2, ELLEN HE 3,4 , JIYONG HU 5, JI ZHOU 5 and SVEN SKOG 3,4

1Healthy Centre and 2Medical Experimental Centre of the Third XiangYa Hospital, ZhongNan University, ChangSha, P.R. China; 3Department of Oncology, Karolinska Institute, Stockholm, Sweden; 4Sino-Swed Tongkang Molecular Bio-Med. Institute and 5Sino-Swed Tongkang Bio-Tech. Inc., Shenzhen, China

Abstract. Background: The role of serum tumour markers is between 5-8 years higher, as compared to the STK1 negative to reveal tumours not yet visible by imaging techniques. Here group, due to higher mean ages of persons with proliferative we examine the use of serum thymidine kinase 1 protein breast and prostate tissues. Thus, 83% of the STK1-positive (STK1) in health screening. Patients and Methods: Persons persons had diseases (benign, proliferation tissues, fatty (n=11,880) participating in health screening programs in liver, helicobacter pylori-positive and hepatitis B virus- China, during 2005-2007, were tested for STK1. STK1 was positive) related to malignancies. Conclusion: STK1-values measured by a sensitive chemiluminescence dot-blot assay. >2 pM may indicate an early risk for development of Medical examination of participants was carried out in malignancies years later. parallel. Results: The proportion of STK1-positive (>2 pM) individuals was 0.5%, corresponding to the cancer inciden ce The role of serum tumor markers is to detect small tumours rate of China. No malignant cases were found in the STK1- while they are still invisible by imaging or im palpable. A negative group, but two pre-malignant and one malignant number of serum tumour markers for malignancy have been case were found in the STK1-positive group. The low tested, but few of them are reliable for health screening of frequency of malignancies found was probably due to the cancer so far, because of lack of tumour-type specificity, relatively young population (mean age 40.4±13.4 years). In except for prostate-specific antigen ( PSA) for prostate (1). the STK1-positive group, there were 24% of persons with PSA gives elevated false-positive values, but could predict benign diseases (breast, liver, kidney), 37% with proliferati ve subsequent advanced cancer 25 years later if tested before an tissues (breast, prostate), 13% with fatty liver, 9% with age of 50 years (1). inflammatory reactions/virus infections (three hepatitis B We have shown in a number of clinical studies during the virus-positive persons) and 17% showed other types of last 10 years that thymidine kinase 1 (TK1) protein physiological changes not directly related to proliferation. In concentration in serum is a useful proliferation marker for the STK1-positive group, a significantly (p<0.001) higher prognosis of cancer and for monitoring the outcome of proportion of persons with proliferation of breast and tumour therapy (2-10). Recently, we started investigations to prostate tissues were found (37%), as compared to the determine if STK1 also could be used for screening of STK1-negative group (18%). Furthermore, the mean ages apparently healthy persons for malignancies. In this study, among the groups of persons with STK1-positive values were we report the results from three health-screening investigations, performed on 11,880 persons in China, during 2005 to 2007. Correspondence to: Sven Skog, Ph.D., Eningbölevägen 5B, 74961 Örsundsbro, Sweden. Tel: +46 734407152, e-mail: svenisak@ Patients and Methods hotmail.com / ZhiHeng Chen, MD, Healthy Centre of the Third XiangYa Hospital, ZhongNan University, China. Tel: +86 731 Health screening. The health screening was performed during 2005 8618588, Fax: +86 731 8618573, +86 13637403553, e-mail: to 2007, at the Third XiangYa Hospital Health Centre, Hogan [email protected] University, Hunan province, China, and corresponding to 11,278 persons. Another two health screenings were also carried out, but Key Words: Thymidine kinase 1, TK1, health screening, benign, with fewer people (Shenzhen, n=535, Dongguang, n=61). These cancer, malignancy, tumour, fatty liver, inflammation, infection, studies were not trials for STK1. STK1 was tested in order to see if virus. it could give any further medical information. The persons

0250-7005/2008 $2.00+.40 3897 ANTICANCER RESEARCH 28 : 3897-3908 (2008) participat ing in these health screenings did not represent a random Statistical analysis. The statistical analysis was performed using population of people, since they were mainly working in offices of Student’s unpaired t- test and chi-square. P-values less than 0.05 different types. All individuals from the Third XiangYa Hospital were regarded as statistically significant. Health Centre were tested for STK1 and examined for health status at the same time point. Standard diagnos tic methods for health Results examination were used (X-ray, ultrasound [for benign/malignancy], electrocardiograph y, urine, blood [sugar, glutamic pyruvic transaminase, hepatitis B virus positiv ity ], stones in gall bladder and The STK1 distributions of the STK1-negative and -positive kidney, tuberculosis, physical examination) (Table I). Regarding the persons from the Third XiangYa Hospital Health Centre are individuals from Shenzhen and Dongguang, only the STK1-positive shown in Figures 1 and 2. The mean STK1-value of the STK1- persons were examined (Table III). negative group was 0.29±0.25 pM, while the mean STK1-value of the STK1-positive group was 3.3±2.4 pM. Ninety-eight Characteri zation of STK1-positive individuals. The STK1-positive percent of the STK1-negative persons had STK1-values less than persons were subdivided into five groups depending on their diagnosis: benign, proliferation tissue, fatty liver, inflammation/virus infection 1.0 pM, and thus the STK1-negative group was well separated and others with diagnos es not directly related to proliferation (elevated from the STK1-positive group. No significant differences in glutamic pyruvic transaminase, gallbladderectoma, tonsillectoma, STK1-values were found according to the age of health persons appendectoma, slight angioten, enteritis, overweight, cholelithiasis, (data not shown). Similar results were found in the health cardiovascular disease, ST-wavelength abnormal , traumatic brain screening in Shenzhen (mean negative STK1 0.25±0.49, mean injury, anaemia). If more than one disease was present, the main positive STK1 3.3±2.4 pM) and Dongguang (mean negative disease was chosen as characterizing the individual. Only the STK1- STK1 0.70±0.22, positive STK1 2.7 and 3.7 pM). positive persons from the Third XiangYa Hospital Health Centre were follow ed up for 3 to 24 months. The STK1-positive persons from the Third XiangYa Hospital Health Centre were divided into five major groups Characterization of STK1-negative individuals. A majority of the depending on their different diagnos es (see Patients and STK1-negative persons had one or more different type s of health Methods) (Figure 3). The one high STK1-value (7.4 pM) in problems (breast benign/proliferati ve tissue, prostate proliferati ve the benign group was a person with cysts in the liver. One tissue, cysts, hepatitis B virus-positive, fatty liver, heart diseases person with proliferati ve breast tissue also had a pre- [cardiac enlargement, coronary disease, arteriosclerosis, myocardial malignancy in the gallbladder. The high STK1-value (17.6 infarction, irregular heart beat], blood pressure, stone (kidney, gall- pM, patient number 40) in the fatty liver group was from a bladder), tuberculosis, glutamic pyruvic transaminase and blood glycolipid) (Table II). Only few STK1-negative persons had no person who, as well as having a fatty liver and being health problems (n=201). In addition, there were 10 persons that had Helicobacter pylori -positive, was also overweight, malignancies of different types (breast n=5, cervical n=2, rectum hypertensive and had a low white blood cell count. Three n=1, noose papillary n=1, teratoma n=1) and 49 persons with months later, th eir STK1-value was 9.3 pM, but still high. The suspicious malignancies (cervical n=29, gastric n=5, prostate n=7, level of the Helicobacter pylori -positive value was also much breast n=3, others n=8) month s or years before participating in the elevated. The high STK1-value in the inflammation/virus health screening, but now regarded as cured. These persons were not infection group represents a person with duodenal ulcer (6.7 included in the estimation of the number of benign/proliferation tissue/pre-malignancies/malignancies. Instead, we used them as a pM). In the last group of more non-specific diagnos es, two reference of age distribution of persons with malignancies . rather high STK1-values were found: one person was reported to have kidney stone and traumatic brain injury (5.5 pM), the Serum TK1 concentration assay. STK1 protein concentration was other had cardiovascular disease and an abnormal ST- measured by an enhanced chemiluminescent (ECL) blot assay as wavelength (7.3 pM). In the Shenzhen health screening group, described by the manufacturer (commercial kit, SSTK Inc., two STK1-positive persons were found (0.4% positive), one Shenzhen, China) (8). Samples comprising 3 μl of serum were was hepatitis B virus-positive (3.7 pM) and one had anaemia directly applied to nitrocellulose membrane in duplicates. The serum (2.7 pM). In the Dongguan health screening group, eight samples were probed with anti-TK1 chicken IgY antibody raised against a peptide (residue 195-225, GQPAG PDNKE NCPVP STK1-positive persons were found (13.1% positive), GKPGE AVAAR KLFAPQ). TK1 peptide was dotted at different representing one with benign neck tumour, one with breast concentrations (20, 6.6 and 2.2 pM) as an extrapolation standard. mass, one with esophageal malignancy, two with stomach The intensities of the spots on the membrane were determined by ulce r, one with stomach pain, one with tong ue ulcer, and one CIS-1 imaging (SSTK Inc., Shenzhen, China). From the intensity with chronic pharyngitis ( Table III). of the TK1 standard of known concentrations, the concentration of The different disease groups of the persons from the Third the serum TK1 was calculated and expressed as pM (2, 5). XiangYa Hospital Health Centre were also expressed as Individuals with STK1-value with 2.0 pM and higher w ere regarded as STK1-positive. The ability of the STK1 dot blot assay to percentages (Figure 4). In Figure 4, benign and proliferati ve discriminate between health persons and persons with malignancies tissue groups were combined. Sixty-one percent of the STK1- was high, shown by its high receiver operating characteristic (ROC) positive persons were found to have benign or proliferative value (>0.90) (10). tissues, 13% had fatty liver, 9% inflammation/virus infections

3898 Chen et al : STK1 and Risk for Development of Malignant Tumors

Table I. Characteristics of the STK1-positive persons of the Third XiangYa Hospital Health Centre.

Case Gender Age STK1 Type of disease Follow-up no . (years) (pM)

1F52 2.1 Multiple liver cyst, double breast fibroma Unchanged condition 2M51 2.8 Atrophy cholecystitis, renal cyst (left) Two years after operation of gallbladder, suspicion of malignancy 3M30 2.4 Liver-angioma (benign) Unchanged condition 4M35 2.1 Fatty liver Unchanged condition 5F50 2.4 Rhinitis New: 18 Months after proliferation in double breast tissue, chronic gastritis 6F67 2.3 Liver cyst (benign) Unchanged condition 7M52 2.2 Proliferation in prostate gland Unchanged condition 8M51 2.2 Subcutaneous tumor, proliferation in prostate gland Unchanged condition 9F70 2.6 Liver-angioma (benign) Unchanged condition 10 M 43 6.4 Slight fatty liver Unchanged condition 11 M 40 2.1 Fatty liver Unchanged condition 12 F 44 2.2 Gland cystic hyperplasia in double breast, Unchanged condition squamous epithelium proliferation 13 F 34 2.1 Bearing premature, high leve l of glutamic pyruvic transaminase Unchanged condition 14 M 44 2.2 Soft tumour after operation, fatty liver Unchanged condition 15 F 70 2.0 Rheumatic heart disease, rhinitis, pharyngitis No more information 16 M 77 2.4 Hypertrophy of prostate, hepatitis B Two years after prostate operat ion , suspicion of virus-positive, gallstone malignancy, gallstone, renal cysts, hemangiomas 17 M 77 2.7 Proliferation in prostate Unchanged condition 18 M 42 2.9 Fatty liver Unchanged condition 19 M 72 2.6 Fatty liver Unchanged condition 20 M 38 2.2 Pharyngitis Liver angioma 21 F 50 2.1 After gallbladderectoma, tonsillectoma Two years after proliferation in breast tissue 22 F 48 2.1 Mild cerebral vascular tension increased, enteritis Unchanged condition 23 F 39 2.1 Left kidney hamartoma (multiple), Unchanged condition proliferation in breast 24 M 45 3.6 Proliferation in prostate Unchanged condition 25 F 54 2.6 Liver cyst 10 Months after TK1 in negative 26 F 31 2.6 Liver cyst Unchanged condition 27 F 56 3.7 Overweight Blood pressure increased 28 M 66 4.6 Fatty liver Prostate increased in size in 9 months 29 F 27 2.1 Proliferation in breast Unchanged condition 30 F 37 2.1 Proliferation in breast 6 Months after Nessler's cervical cyst (8 x 7mm) 31 F 49 2.1 Proliferation in double breasts, pre-malignant gallbladder Unchanged condition 32 M 52 3.7 Left renal tumor 4 Months after TK1 in negative 33 M 54 4.1 Hypertrophy of prostate Unchanged condition 34 M 55 5.5 Kidney stones (multiple), traumatic brain injury Unchanged condition 35 M 52 3.5 Hypertrophy of prostate, Helicobacter pylori -positive 7 Months after duoden al ulcer was found, hypertrophy of prostate 36 M 60 4.0 Hypertrophy of prostate Unchanged condition 37 M 41 7.4 Cyst in liver, gallbladder polyps (multiple) 4 Months after TK1 increased 13.9 pM 38 M 31 7.3 Cardiovascular disease, T-value abnormal Unchanged condition 39 M 57 2.2 Hypertrophy of prostate Unchanged condition 40 F 59 17.6 Fatty liver, Helicobacter pylori -positive Helicobacter pylori -positive, TK1 9.3 pM 41 F 41 2.0 Gallstone, renal cysts Unchanged condition 42 M 36 2.7 After appendectoma Unchanged condition 43 F 43 3.6 Proliferation in breast, cervical hypertrophy Nessler's cervical cyst, uterine fibroids 44 F 43 2.1 Proliferation in breast, cervical hypertrophy Unchanged condition 45 F 38 2.5 Hepatitis B virus-positive 4 Month s after STK1 in negative 46 F 32 3.8 Proliferation in breast Unchanged condition 47 M 33 4.2 Fatty liver Unchanged condition 48 F 40 2.1 Proliferation in breast New: 4 Months after revealing uterine fibroids 49 M 60 4.0 Proliferation in prostate gland Unchanged condition 50 M 44 3.8 Gallbladder polyps (multiple), hepatitis B virus-positive Unchanged condition 51 F 55 2.6 Multiple liver cyst Unchanged condition 52 F 44 2.2 Breast cystic hyperplasia Unchanged condition 53 F 30 2.8 Pharyngitis Unchanged condition 54 M 45 6.7 Duodenal ulcer, hepatitis B virus-positive Unchanged condition, C12 marker normal

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Table II. Number of STK1-negative persons of the Third XiangYa Table III. Characterization of the STK1-positive persons of the health Hospital Health Centre with different types of diseases. The total screenings of Shenzhen and Dongguang. percentage is more than 100% since some individuals had more than one type of disease. Screening Age Gender STK1 Type of disease (pM) Type of illness % Shenzhen, 2007 Breast, benign 2.8 Case n o. Breast, prolif erative tissue 9.2 65 33 M 3.7 Hepatitis B virus-positive Prostate, prolif erative tissue 8.5 139 26 F 2.7 Anaemia Cyst* 23.3 Hepatitis B virus-positive 8.5 Dongguang, 2007 Fatty liver 17.3 Case n o. Glutamic pyruvic transaminase 7.0 4 51 M 5.4 Neck, benign, under treatment Heart problem 20.9 6 55 M 2.0 Esophag eal malignancy Blood hypertension 7.2 9 44 M 2.0 Chronic pharyngitis Stone** 5.6 16 26 F 2.0 Long-term repeated tongue ulcer Tuberculosis 2.9 23 39 M 2.0 Gastric ulcer Blood glycolipid 9.2 30 52 F 2.4 Mass in breast about 4x3mm 35 30 F 5.6 Helicobacter pylori -positive, *Liver=40%, gall bladder =25%, kidney= 20%, cervical, skin=5%, gastric ulcer nose/t ong ue =3%, lung/rectum/prostate <1%; **gallbladder=70%, 45 40 F 5.0 Stomach pain kidne y=30%.

Figure 1. Individual STK1-values of 11,278 persons of STK1-negative and positive groups. The dotted line shows the cut-off value of 2.0 pM.

and another 17% had physiological changes not directly the STK1-positive persons from the Third XiangYa Hospital related to proliferation. There was no difference in the number Health Centre were followed up within 3 to 24 months after of STK1-positive woman and men among the various types of the first STK1 test (Table I). Twenty-two percent (12/54) of diseases, except for fatty liver where the number of men (n=6) them showed differences as compared to the first STK1 test: was significantly higher than the number of woman (n=1). two new breast proliferation cases, one new benign cervical When comparing the STK1-positive group with the STK1- case and one new cervical cyst case were found; in one person, negative one, the number of persons with proliferating breast the size of the prostate increased; in four persons, the STK1 or prostate tissue was significantly higher ( Table IV). All of values decreased, of which three fell to zero and in one person

3900 Chen et al : STK1 and Risk for Development of Malignant Tumors

Figure 2. Distributions of STK1-negative and STK1-positive persons.

Figure 3. Individual STK1-values of STK1-positive persons in relation to different types of diseases.

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Figure 4. Percentage of STK1-positive persons with different types of diseases.

Table IV. Number of persons with different type of diseases in the STK1-negative and STK1-positive groups of the Third XiangYa Hospital Health Centre. The number of STK1-positive persons with different types of diseases was also related to the total number of persons included in the study (11,278 persons).

STK1 Number of STK1-positive persons of total 11,278 persons (%) Type of illness Negative % Positive % P-value ( Chi-square)

Benign/cyst 25.3 24.1 0.650 0.12 Proliferation in breast 9.2 18.5 0.049 0.09 Proliferation in prostate 8.5 18.5 0.023 0.09 Hepatitis B virus-positive/inflammation 6.8 9.3 0.522 0.04 Fatty liver 17.3 13.0 0.47 0.06

STK1 increased (a liver cyst case); operation was performed in one of the person s with proliferation of prostate tissue and in one person with gallbladder problems , due to suspicious pre-malignancy/malignancy. The appearance of benign and malignant tumours among people are related to age. The benign tumours are more frequent between 30 to 50 years of age, while most malignant tumours start to appear after the age of 50 years and increase with age. Therefore, we determined the age distributions of the STK1- negative and STK1-positive groups and also of the persons with proliferating breast and prostate tissue to determine if persons with high STK1 values represent older ca ses, and thus are at higher risk for development of malignancies. The results are Figure 5. Age distributions of STK1-negative persons with no illness or I G G shown in Figure s 5-7 and Table V. diseases ( ); STK1-negative ( ) and STK1-positive persons ( ), and The STK1-negative and the STK1-positive persons from STK1-negative persons with malignancies years ago, but who were Third XiangYa Hospital Health Centre were divided into six symptom free ( xR ).

3902 Chen et al : STK1 and Risk for Development of Malignant Tumors

G G G G Figure 6. A, Age distributions of STK1-negative ( ) and -positive ( ) Figure 7. A, Age distributions of STK1-negative ( ) and -positive ( ) L females and STK1-negative females with STK1-values of 1.5-1.99 pM males and STK1-negative males with STK1-values of 1.5-1.99 pM ( ). L G G G G ( ). B, Age distributions of STK1-negative ( ) and -positive ( ) B, Age distributions of STK1-negative ( ) and -positive ( ) males with females with proliferating breast tissue. proliferating prostate tissue.

groups of each: i) all persons, ii) male and female separated, (n=49) whose relevant organ was partly or totally removed iii) persons with proliferating breast tissue, iv) persons with by surgery. Interestingly, the mean age of this group was 40 proliferating prostate tissue, v) STK1-negative persons who years, corresponding to the STK1-negative group with had malignancies before the health test, but were now benign and proliferating tissues (data not shown). The mean symptom free (post-operation), and vi) persons with no age of the health STK1-negative persons w as 30.9 years and illness or disease. The mean and median ages of all group s statistically significantly lower than the STK1-negative were higher among the STK1-positive persons by 3.3 to 7.5 persons showing different types of diseases, and also than years, depending on the group (see Table V), as compared the STK1-positive groups, as well as the STK1-negative to the STK1-negative group ( p=0.010). The appearance of malignant group ( p<0.001). malignancies in relation to age was further demonstrated in The cut-off value of 2.0 pM was set from the highest the group of persons with proliferating prostate tissue. The STK1-value found among health y persons (without any mean age was 58.9 years. More interestingly, there were no known diseases) in earlier investigations (2-8). Since the STK1-positive persons below 50 years of age, while in the STK1 distribution of the STK1-negative group was well STK1-negative prostate group, persons with proliferating separated from the cut-off value of 2.0 pM (mean value of tissue were found down to 20-30 years of age . The persons 0.29±0.25 pM), it is may be possibly to lower the cut-off that had malignancies (n=10) before the health test had a value and thus increase the number of STK1-positive persons. mean age of 54.7 years and this was statistically higher than However, the age distribution of the persons with STK1- in the STK1-negative group of healthy persons and STK1- values between 1.50-1.99 pM (mean age 41.5 years old) was negative persons with different types of disease s(p< 0.023). significantly different ( p=0.015) from that of the persons There was a group of persons with suspicious malignancies above the cut-off value of 2.0 pM (mean age 47.9 years)

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Table V. Mean and median ages, and maxi mum/minimum ages of STK1-negative (<2.0 pM) and STK1-positive (>2.0 pM) persons. The persons with malignancies participated in the health screening more than six months after the surgery, and reported no symptoms of their malignancies at the time of the health screening.

Type STK1 status Mean STD Median Max Min n

Hunan, 2005-2007 11278 Health, no illness Negative 30.9 8.0 29.0 66 21 All Negative 40.4 13.4 37.0 105 13 Positive 47.9 12.4 45.0 77 27 Male Negative 45.4 15.0 42.0 100 21 Positive 49.4 13.2 48.0 77 30 Female Negative 40.4 13.4 38.0 84 17 Positive 46.3 11.7 44.0 70 27 Breast Negative 36.7 9.5 36.5 71 17 Positive 40.9 7.1 43.0 52 27 Prostate Negative 58.0 15.6 60.0 100 22 Positive 58.9 10.7 55.6 77 45 All 1.5-1.99 pM Negative 41.5 14.4 38.0 88 23 Male 1.5-1.99 pM Negative 45.2 16.5 39.0 88 24 Female 1.5-1.99 pM Negative 36.5 9.2 37.0 58 23 Malignant all Negative 57.6 13.8 57.0 85 26

Shenzhen, 2007 535 All Negative 39.7 13.3 36 80 14 All Positive 36.8 15.4 30 60 23

Dongguang, 2007 61 All Negative 38.3 8.9 39 55 20 All Positive 41.1 11.2 39.5 55 23

(Table V). This indicates that persons with STK1-values (30). Therefore, proliferation-related diseases other than above 2.0 pM represent a group of persons with physiological malignancies must be interpreted by checking the patient’s conditions different from persons with STK1-values below medical history (9). 2.0 pM. In this study, we examined if STK1 could give any additional information of a person’s health condition, Discussion including discovery of benign, pre-malignancies and/or malignancies. We found that 61% of the STK1-positive Thymidine kinase (TK, ATP: thymidine 5-phosphotransferase, persons had benign and/or proliferative tissues and 22% had EC.2.7.1.21), an enzyme of the pyrimidine salvage pathway, virus/inflammatory-related diseases and fatty liver. All these catalyzes the phosphorylation of thymidine to thymidine diseases are related to malignancy in some way. The monophosphate (11, 12). TK enzyme in human cells appears remaining STK1-positive persons had other types of illness in two isozyme forms, a cytosolic (thymidine kinase 1, TK1) not directly related to proliferation (elevated glutamic and a mitochondrial (TK2) form (13). The level of TK1 rises pyruvic transaminase, gallbladderectoma, tonsillectoma, at the G 1/S boundary and increases dramatically to late appendectoma, slight angioten, enteritis, overweight, S-phase/ early G 2-phase during the , but is absent cholelithiasis, cardiovascular disease, abnormal ST-value, from quiescent cells (14-21). The TK1 enzyme is of high traumatic brain injury, anaemia). The number of STK1- considerable interest because its level is highly dependent on positive persons with proliferati ve breast or prostate tissues the growth stage of the cell. Therefore, TK1 is a useful marker was two times higher, as compared to STK1-negative for cell proliferation and hence for malignancy (10, 22-24). individual s, which was statistically significant. We also STK1 is almost undetectable in normal healthy persons, while found that the mean age of the STK1-positive group was the STK1 level increases significantly in persons with significantly higher than that of the STK1-negative group. malignant diseases (2-10, 24-26). Elevated levels of STK1 This is an interesting observation, since the risk of may also be found in hepatitis A (27), B, herpes group of developing malignancies increases with age. We believe that infections (28), and also in patients with anaemia (29), the higher STK1-values found among persons of higher ages cirrhotic liver (5) and sub-clinical reaction of cytomegalovirus indicate the presence of abnormal proliferati ve tissues, and

3904 Chen et al : STK1 and Risk for Development of Malignant Tumors developing malignancies. This was further demonstrated give unreasonable high false-positive values. In fact, the among the persons with proliferating prostate tissues who STK1-positive rate is in the range of the cancer incidence had a mean age of 58.9 years and coinciding with the age rate found among people in China today. The death rate of distribution of men with prostate malignancy. Thus, persons cancer in China is about 0.13% (female 0.09 and male above 45-50 years with STK1-values above 2.0 pM my have 0.16%) (Cancer Facts & Figures, Statistics for 2007, an increased risk for the presence of abnormal proliferati ve American Cancer Society, http://www.cancer.org). Using this tissues with a potential to develop malignancies, although not death rate value and supposing that about 40-50% of these yet discovered. The follow-up study of the STK1-positive Chinese persons with malignancies are cured, then the cancer persons also shows that STKl-values above 2.0 pM found at incidence rate (number of malignant persons/100,000 the first health test indicated abnormal events appearing persons) in China should be about 0.25-0.30%. The cancer several months later, for example appearance of proliferati ve incidence rate has also been estimated from cancer registries tissues and enlargement of the prostate. in China, and was 0.2% during 2005 (33). These estimated Although we only found two persons with pre-malignancy cancer incidence values are close to the number of STK1- and one person with malignancy, but a number of persons with positive persons with proliferation tissues found in this study benign and proliferative tissues, it should be remembered that (0.18%, 20 cases). Are the STK1-positive persons with benign tumours and proliferati ve tissues are not free of risks proliferative tissues those who years later develop for development of malignancies. The presence of benign malignancies? tumours increases the risk of developing malignancies 1.5- to STK1 was elevated in persons with inflammations and 2- fold within 14 years, while the same risk factor for virus infections. This is to be expected since inflammation proliferating tissues is 4- to 5- fold within 8 years (31). The and virus infections activate the immune system, leading to risks with benign tumours were further demonstrated in a enhanced cell proliferation of the immunological competent recent prostate study on 21,277 men during 30 years (1). It cells. It is known that the serum level of different types of was found that serological total PSA before 50 years o f age , growth factors (epidermal growth factor, transforming when no malignant prostate cancer was yet found, was a growth factor α, transforming growth factor ß, platelet- strong and statistically significant predicting factor for derived growth factor, fibroblast growth factor, insulin-like subsequent advanced cancer 25 years later. Furthermore, a growth factor-1, tumor necrosis factor) increases in persons fatty liver also increases the risk of development of cirrhosis, with inflammation (32). Hepatitis B virus-positiv ity also which is one major factor behind the development of liver increases the risk of development of liver cancer (32). cancer (32). It is also a fact that hepatitis B virus-positive is Malignancies associated with H. pylori infection has been one of the reasons for the development of liver cancer (32). reported (32). In some individuals, H. pylori infects the A possible reason for the few malignant persons found in corpus region of the stomach. This results in a more the present study could be the lack of a complete pathological widespread inflammation that predisposes not only to ulcer examination. None of the STK1-positive persons with in the corpus region, but also to gastric cancer (32, 34, 35). diagnosis of proliferative tissues were biopsy examined. Forty Fatty liver is caused by high alcohol intake and diabetes. percent (8/20) of the persons in this study with proliferative The fat metabolism of the liver cells is affected, leading to tissues had STK1-values higher than 3 pM and should have an accumulation of fat in the cytoplasm of liver cell s (fatty been regarded as indicating pre or even early malignancies liver). The formation of fatty liver increases the risk of rather than proliferati ve tissues. STK1-values above 3 pM in development of cirrhosis in the liver, a condition that is malignant patients were found to correlate to tumour classified as one of three major risks for development of liver pathology stages 2 and 3 (4, 6). However, a more likely malignancy (32). There is also a formation of intermediate explanation for the lack of persons with malignancies is filaments in liver cells due to high alcohol intake, called found in the age distribution. It is known that cancer Mallory’s hyaline, which is associated with acute frequency starts to increase significantly after 60 years of age. inflammation in the liver. Thus, although the reason for the The age distribution in this study shows that more than 95% elevated STK1 of fatty liver patients is primarily due to an of the participants were less than 60 years old, with a mean inflammation in the liver, the higher STK1 value may also age of 40 years. Thus, the frequency of malignancy among indicate a risk for development of liver cancer (32). the persons tested in this study should be low because it was In summary, we found that among the STK1-positive a relatively young population. persons, 83% were linked to diseases (pre-malignancies, The number of STK1-positive persons found in this study benign and proliferating tissues, H. pylori -positive, hepatitis is supported by another two independent health studies B virus-positive infection and fatty liver inflammation) that running at two different places in China (Fuji, 2007, 1,800 increase the risk of developing malignancies years later. persons, 1.7% positive (data not show); Hangzhou, 2007, Therefore, we conclude that STK1 could be a useful marker 761 ca ses, 0.9% positive (10)) showing that STK1 did not for an early risk of development of any type of malignanc y.

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30 Gronowitz JS, Larsson A, Källander CF, Claesson K, Sjöberg O, 34 Forbes GM, Warren JR, Glaser ME, Cullen DJ, Marshall BJ and Lernestedt JO, Frödin L and Tufveson G: Serum thymidine Collins BJ: Long-term follow-up of gastric histology after kinase in transplant patients: its relation to cytomegalovirus Helicobacter pylori eradication. J Gastroenterol Hepatol 11 : activity, renal transplant rejection and its use for monitoring of 670-673, 1996. antiviral therapy. Ann Clin Res 18 : 71-75, 1986. 35 Zhao D, Sun T, Zhang X, Guo Y, Yu D, Yang M, Tan W, Wang G 31 Tavassoli FA and Devilee P: World Health Organization: and Lin D: Role of CD14 promoter polymorphisms in Tumours of the Breast and Female Genital Organs. IARC Press Helicobacter pylori infection – related gastric carcinoma. Clin Lyon , p. 16, pp. 63-73, 2003. Cancer Res 13 : 2362-2368, 2007. 32 Underwood JCE: General and systematic pathology. London, Churchill Livingstone, p. 46, pp. 201-222, pp. 223-262, pp. 370- 375, pp. 410-432, 2004. 33 Yang L, Parkin DM, Ferlay J, Li L and Chen Y: Estimates of Received May 2, 2008 Cancer Incidence in China for 2000 and Projections for 2005. Revised July 15, 2008 Cancer Epidemiol Biomark Prev 14 : 243-250, 2005. Accepte d September 8, 2008

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