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Single Nucleotide Polymorphism of the Human Kallikrein-2 Gene Highly Correlates With Serum Human Kallikrein-2 Levels and in Combination Enhances Prostate Cancer Detection

Robert K. Nam, William W. Zhang, John Trachtenberg, Eleftherios Diamandis, Ants Toi, Marjan Emami, Minnie Ho, Joan Sweet, Andrew Evans, Michael A.S. Jewett, Steven A. Narod

From the Division of Urology, Department of Medical Imaging, Department of Pathology, University Health Network; Department of Pathology and Laboratory Medicine, Mount Sinai Hospital; and the Department of Public Health Sciences, University of Toronto, Toronto, Canada.

Address reprint requests to Robert K. Nam, MD, Division of Urology, 2075 Bayview Ave, MG-406, Toronto, Ontario, Canada, M4N 3M5; email: robert.nam{at}utoronto.ca.

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Purpose: We examined the relationship between a mutant (T) for wild-type (C) allele substitution of the human kallikrein-2 gene (KLK2), circulating human kallikrein-2 (hK2) levels and prostate cancer risk.

Patients and Methods: We studied 1,287 consecutive men who underwent prostate biopsies because of an abnormal prostate-specific antigen level. Serum and DNA were obtained before biopsy. Cases were patients with cancer, and controls were patients with no cancer. The mutant and wild-type alleles of the KLK2 gene were designated as the T and C alleles, respectively.

Results: Of the 1,287 men, 616 had cancer, and 671 had no cancer. The overall distribution of the CC, CT, and TT KLK2 genotypes was 55.1%, 38.2%, and 6.8%, respectively. The median hK2 levels for men with the CC, CT, and TT genotypes were 0.24, 0.18, and 0.062 ng/mL and correlated with the genotypes, respectively (P = .0001). The adjusted odds ratios for prostate cancer for patients with the TT and CT genotypes compared with patients with the CC genotype, were 2.13 (95% confidence interval [CI], 1.3 to 3.5; P = .004) and 1.51 (95% CI, 1.2 to 2.0; P = .002), respectively. The adjusted odds ratio for prostate cancer for patients in the fourth quartile of hK2 compared with the first quartile was 4.33 (95% CI, 2.9 to 6.4; P = .0001). When combined, the adjusted odds ratio for having prostate cancer was 13.92 (95% CI, 6.6 to 29.2; P = .0001) for patients with high hK2 levels and at least one T allele.

Conclusion: The C/T polymorphism of the KLK2 gene and circulating levels of hK2 are correlated and, in combination, are highly predictive for prostate cancer.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PROSTATE CANCER is the most commonly diagnosed malignancy in males and is the second leading cause of cancer death.^1,2 Screening for prostate cancer has been recommended by the American Cancer Society for men who are older than 50 years with at least a 10-year life expectancy.³ Current screening tests include the measurement of serum levels of prostate-specific antigen (PSA) and digital rectal examination (DRE).⁴ Two large clinical trials are underway to confirm whether screening reduces prostate cancer mortality.^5,6

The sensitivity of PSA as a screening test is high, but the positive predictive value is relatively low for use in the general population.^7,8 Several benign conditions of the prostate gland can lead to elevated PSA levels.⁹ Variants of the PSA test, including PSA density, and the free to total PSA ratio have been developed, but they do not significantly enhance the predictive value of the PSA test.^10,11

A second problem related to PC screening is that the needle-core prostate biopsy may miss foci of cancer. With the aid of transrectal ultrasonography, a minimum of six needle-core samples are usually obtained from the prostate gland during the first biopsy session.¹² Because most tumors diagnosed through screening are microscopic and cannot be visualized radiographically, random samples are obtained in each anatomic zone of the prostate gland. Thus, malignant foci can be missed because of sampling. Patients who have no cancer detected on their first biopsy have a 15% to 30% of having cancer detected if a second biopsy is performed.^12,13 At present, there are no available tests to select patients who are at high risk for cancer on repeat prostatic biopsies.

Many new markers have been proposed to improve the detection of prostate cancer. Association studies have examined the significance of a number of candidate genes associated with prostate cancer. In particular, polymorphisms of the androgen receptor, vitamin D receptor, 5-alpha reductase enzyme, CYP17, and CYP3A4 genes have been associated with the presence of prostate cancer in one or more case-control studies.^14–18 Other candidate genes include the PSA and glutathione transferase genes, which have been linked to prostate cancer.^19,20 To date, no genetic test has been proven to be of clinical importance in diagnosing prostate cancer or in establishing high-risk groups.

We recently described the diagnostic value of the serum protein level of human kallikrein-2 (hK2) in screen-detected prostate cancer.²¹ A single nucleotide polymorphism for the human kallikrein-2 gene (KLK2) encoding for the hK2 protein has been described, consisting of a nucleotide change from cytosine to thymine on exon 5.²² It is not yet known whether this polymorphism is associated with a functional change in hK2 activity, with circulating levels of hK2, or with prostate cancer risk. In vitro, the two alleles code for an active and an inactive form of the protein product.²² The common allele codes for Arg²²⁶-hK2, which has trypsin-like activity; whereas the variant allele codes for Trp²²⁶-hK2, which has no detectable activity.²²

To determine whether the wild-type (C) for mutant (T) polymorphism predicts circulating levels of hK2 and prostate cancer risk, we examined serologic levels of hK2 and genotyped the C/T polymorphism of the KLK2 gene in 1,287 men who are at risk for having prostate cancer.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Study Subjects
Patients were drawn from a consecutive sample of 1,437 men who were referred to the Prostate Center of the University Health Network, between June 1998 and June 2000, because of either a PSA value of 4.0 ng/mL or greater or because of an abnormal DRE. No patient had a prior history of prostate cancer.

Of the 1,437 men, 23 patients were not capable of giving consent to participate in a research study. Of the remaining 1,414 men, 1,287 (91.0%) consented to participate. Blood samples were collected before clinical prostate examination. Plasma was separated from blood samples and was stored at -70°C. A urologic history was obtained, which was used to calculate the American Urological Association Symptom Score, which describes the severity of lower urinary tract voiding symptoms.²³ The results of DRE were recorded. A minimum of six ultrasound-guided needle biopsies was performed, with additional directed biopsies as needed, using an 18-gauge spring-loaded biopsy device (Bard Magnum, Murray Hill, NJ). The primary end point was the histologic presence of adenocarcinoma of the prostate. All research was conducted with informed consent and with the approval of the hospital research ethics board.

Repeat biopsies were offered to patients who did not have evidence of cancer on the initial prostate biopsy, but the decision to undergo repeat biopsy was made by the referring physician and patient. Of the 1,287 patients, 454 had cancer detected on the first biopsy session (35.3%). Of the remaining 833 men, 473 (56.8%) agreed to undergo one or more follow-up biopsies. An additional 153 cancers (32.4%) were detected on the second biopsy, and nine cancers (13.0%) were detected among 69 patients who had additional biopsies after a second negative biopsy.

Genetic Analysis
DNA samples from each patient were extracted from peripheral-blood leukocytes using standard protocols. We modified the technique described by Herrala et al²² to detect the polymorphism on exon 5 of the KLK2 gene, based on the complete sequence of the KLK2 gene.²⁴ We used the forward primer 5'GAG CTG GGA ATT GCT CTC AGT-3' and reverse primer 5'-TGC CAG AAC GTG AGG TGG AC-3'.

Genomic DNA (50 ng) was added to a 12.5-µL polymerase chain reaction (PCR) mix including 2 mmol/L of MgCl₂, 200 µmol/L of each deoxynucleotide triphosphate, 4% of dimethyl sulfoxide, 0.625 units of Taq DNA polymerase (Life Technologies, Rockville, MD), and 0.05 ng of forward and reverse primers. The PCR conditions included 32 cycles of 94°C for 30 seconds, 60°C for 30 seconds, 72°C 45 seconds, and 72°C for 10 minutes. Two units of Msp1 restriction endonuclease (New England Biolabs Ltd, Beverly, MA) were added to 6 µL of the PCR product to digest at 37°C overnight. The digested product was then separated on 2% agarose gel. To ensure for quality control of the restriction digests, we randomly duplicated 25% of samples for comparison. All gel readers were blinded to the primary end point and covariates.

Serologic Analysis
Three plasma proteins were examined, including free PSA, total PSA, and hK2. Both free and total PSA levels were measured using commercially available kits, performed on the Immulite chemiluminescence immunoassay system (Diagnostic Products Corporation, San Diego, CA). Total hK2 levels were measured using a new, time-resolved immunofluorometric assay.^21,25 Approximately 90% of hK2 is in the free form, but the assay measures both free and total forms of hK2.²⁵ The hK2 assay has a detection limit of 0.006 ng/mL for both plasma and serum samples and has less than 0.2% cross-reactivity to PSA.²⁵ hK2 levels were analyzed in five batches. Because the concentration of reagents and the calibration of the equipment were not consistently the same between the five batches, the hK2 levels were adjusted according to their calibration settings and to the overall distribution of all the hK2 levels.²⁶ All adjustments were performed in a blinded fashion to the outcome and to the hK2 genotype and were independently performed by Hybritech, Inc, a subsidiary of Beckman Coulter (San Diego, CA).

Data Analysis
We compared the frequencies of the KLK2 polymorphism between prostate cancer cases and controls and correlated the levels of hK2 with each genotype. Cases were patients who had adenocarcinoma of the prostate from any biopsy, and controls were patients who had no evidence of cancer in any of the biopsy samples. The distribution of the hK2 levels was compared between cases and controls. The odds ratios of the polymorphic alleles in predicting prostate cancer were estimated using multivariate, unconditional logistic regression modeling, controlling for age, serum PSA level, DRE, ethnic background, and the presence of lower urinary tract voiding symptoms. The mutant and wild-type alleles of the KLK2 gene were designated as the T allele and C allele, respectively.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Of the 1,287 men, the mean age at first biopsy was 65.5 years (range, 41.4 to 93.8 years). The mean PSA level was 12.3 ng/mL (range, 0.4 to 498.8 ng/mL). The majority of the patients were white (84.0%). Of the other patients, 8.2% were black, 5.4% were Asian, and 2.3% were from other ethnic backgrounds. Eleven percent of patients had at least one relative with prostate cancer.

Of the 1,287 men, 616 (48.9%) were found to have adenocarcinoma of the prostate on the initial or subsequent biopsy. Of the 671 men with no evidence of invasive cancer, 360 (53.7%) had a single biopsy, 251 (37.4%) had one repeat biopsy, and 60 (8.9%) had two or more repeat biopsies. Of the men with no cancer, 52 had normal prostate tissue, 465 had inflammation/benign prostatic hyperplasia, 31 had atypical small acinar–cell proliferation, and 123 had prostatic intraepithelial neoplasia.

Based on all biopsies, cases were defined as patients with cancer, and controls were men without any cancer. The mean age at biopsy of the cases (66.7 years) was higher than controls (64.4 years, P = .0001). Cases were more likely to have had an abnormal DRE and, on average, had a higher PSA level (Table 1). Asians had the lowest probability for prostate cancer (Table 1).

To determine whether the combination of the KLK2 genotype and circulating hK2 levels provided important information in the clinical setting, we examined how the KLK2 genotypes and circulating levels affect the likelihood of detecting prostate cancer for patients who present for their first and second biopsies. We did not examine how it affected the likelihood for patients who received two or more repeat biopsies because of limited sample size (n = 69).

Cancer Risk by hK2 Based on First Biopsy
The probability of having cancer detected at the first prostate biopsy was 35.3% (454 of 1,287 patients). Patients with the CC, CT, and TT genotype had a 31.5%, 38.7%, and 47.1% probability for having prostate cancer detected, respectively (² = 12.41, P = .002, Table 3). The median hK2 level was significantly higher for cases (0.24 ng/mL) than for controls (0.18 ng/mL, P = .0001). The adjusted odds ratio for prostate cancer detection by each KLK2 genotype and quartile ranged from 2.6 to 10.3 (Table 5).

Cancer Risk by hK2 Based on Second Biopsy (After an Initial Negative Biopsy)
A total of 833 men had a negative first biopsy, and of them, 473 (56.8%) had a second biopsy. The probability for having cancer at repeat biopsy was 32.4% (153 of 473 patients). Patients with the CC, CT, and TT genotype had a 30.3%, 34.7%, and 42.1% probability for having prostate cancer detected, respectively (P = .40) (Table 3). The median hK2 level was significantly higher for cases (0.25 ng/mL) than for controls (0.20 ng/mL, P = .0006). When combining the KLK2 genotypes and levels, the adjusted odds ratios for prostate cancer detection were also important predictors (Table 5).

Positive Predictive Value of hK2 and KLK2 Gene
To determine whether serum hK2 levels and the KLK2 genotype enhance prostate cancer detection, we calculated the changes in positive predictive values for subgroups of patients based on the combination of age, PSA level, and DRE results. To establish the ideal cutoff value for serum hK2, we used receiver operating characteristic curves for optimal sensitivity and specificity. Among the 1,287 patients, the subgroup of patients who had the lowest positive predictive value for prostate cancer detection (38.5%) were patients whose age was less than 70 years old, who had a PSA of less than 10 ng/mL, and who had a nonpalpable nodule on DRE (n = 494). For patients with an hK2 level of more than 0.175 ng/mL and who have at least one T allele of the KLK2 genotype compared with patients with an hK2 of 0.175 ng/mL or less or the CC KLK2 genotype, the positive predictive value increased from 36.2% to 53.0%, respectively (P = .008, Table 7). Further, even for patients at higher risk for prostate cancer (age ≥ 70 years old, PSA ≥ 10 ng/mL, or a palpable nodule on DRE), the positive predictive value significantly increased by the hK2 level and KLK2 genotype (Table 7).

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

Previous studies that have examined the significance of polymorphisms of other candidate genes have been case-case studies or case-control studies and have demonstrated little clinical utility. ^{14,15,17–20,27} The two prostate cancer susceptibility genes identified to date by linkage analysis have not been shown to provide predictive value for prostate cancer in the general population. Further studies will be required to evaluate the significance of the newly identified tumor suppressor gene (HPC1 or 2'-5'-oligoadenylate (2–5A)-dependent Rnase L).²⁸ However, the clinical impact of HPC1 is uncertain given that the observed frequency of a common mutation was found to be higher in noncancer controls than prostate cancer cases.²⁸ We and others examined missense variant alleles of the second prostate cancer gene, HPC2, in a smaller subset of patients in the present study and failed to find an association with prostate cancer risk.^29–31

Production of both PSA and hK2 is stimulated by androgens.^32–34 The genes encoding these proteins are located on chromosome 19, and the promoter regions of these genes contain androgen-response elements.^34,35 PSA has predominantly chymotrypsin-like protease activity, whereas hK2 has predominantly trypsin-like protease activity.^24,36 The primary function of the protease activity of PSA is to cleave semen proteins,³⁷ but the target for the protease activity of hK2 is unknown.³⁶

Several studies have now shown that patients with prostate cancer have significantly higher serum hK2 levels than patients without prostate cancer.^21,38–41 At present, no standardized cutoff value for hK2 has been established, and the distributions of the hK2 levels in cases and controls vary widely between each study. For example, Becker et al³⁹ reported mean hK2 levels of 0.079 ng/mL for patients with prostate cancer (n = 144), whereas the mean value for our cancer patients was 0.54 ng/mL (n = 616). The reason for this wide disparity has been attributed to differences in the reagent concentrations, the monoclonal antibodies used, calibration of the methods, and storage length.²⁶ Magklara et al⁴¹ reported a six-fold higher signal in the assay developed by Hybritech, Inc (a subsidiary of Beckman Coulter) compared with the assay we used. These potential factors remained consistent within the current study for cases and controls.

The observation that the inactive T allele of the KLK2 gene is associated with lower hK2 levels but with a higher prostate cancer risk seems to be paradoxical. However, men with the T allele might have an inherent predisposition to produce less hK2 in normal and malignant cells. Thus, increased hK2 production by prostate cancer cells would have a more pronounced association with prostate cancer risk for patients who have low baseline production of serum hK2 levels (ie, patients with the TT genotype) compared with patients who have high baseline hK2 levels (ie, patients with the CC genotype).

However, the variant allele itself may be related to prostate cancer development. Patients with the variant allele had a two-fold increase in risk for having prostate cancer, independent of serum hK2 levels. Herrala et al²² showed that in vitro, Arg²²⁶-hK2 (CC genotype) had only trypsin-like activity, whereas Trp²²⁶-hK2 (TT genotype) had no detectable enzymatic activity. The in vivo effects of a nucleotide change from C to T on exon 5 of the KLK2 gene are unknown. It is possible that the inactive protein may not be sensitive to the current monoclonal antibodies used by our hK2 assay, which would be consistent with our current findings.

In addition to the trypsin-like activity of hK2, other functions of hK2 have been linked to possible pathways of carcinogenesis. Several proteases are implicated in the invasion of tissues by tumor cells.⁴² Frenette et al⁴² showed that hK2 has plasmin-like activity and hypothesized that this could be the initiator of a proteolytic cascade leading to prostate cancer invasion. Also, hK2 may have indirect antiangiogenic properties similar to PSA.⁴³ Because hK2 has been shown to convert proPSA to an enzymatically active form of PSA,³⁶ hK2 may have effects in these pathways. Further study will be required to define the function of the Trp²²⁶-hK2 and Arg²²⁶-hK2 proteins.

Because patients have approximately a 30% chance of having cancer after an initial negative prostate biopsy,^12,13 patients with an initial negative biopsy were offered a repeat biopsy. This was done to reduce misclassification. At the time of analysis, 360 patients who had an initial negative biopsy did not undergo a repeat examination. The reasons for this included patient refusal, referring physician’s refusal, and loss to follow-up. Nevertheless, the effect of misclassification seemed to be minimal because the established risk factors of age, PSA level, and DRE were found to be strong predictors for prostate cancer in our model. To further determine whether misclassification of the controls adversely affected our results, we used a second control group in our study. This group consisted of patients with no evidence of cancer from two or more biopsies. Using this second control group, the adjusted odds ratio for prostate cancer for patients with the CT and TT genotype compared with patients with the CC genotype was 1.67 for the CT genotype (95% CI, 1.2 to 2.3; P = .002) and 3.93 for the TT genotype (95% CI, 1.8 to 8.5; P = .0005). Similar findings were present for the hK2 level. Thus, using a control group who had the least likelihood of having prostate cancer, the odds ratios for having prostate cancer increased, which makes it unlikely that potential misclassification of the cases and controls were significant.

We did not find a family history of prostate cancer to be a significant factor for our patients because our cohort was already prescreened with PSA and DRE. Family history of prostate cancer is an important risk factor for the general population but may not be as important among this group.⁴⁴ There were significant relationships between the KLK2 genotypes and ethnic background. No Asians had the TT genotype, whereas there seemed to be a higher proportion of blacks with the TT genotype. It is possible that differences in ethnic makeup of the cases and controls will lead to spurious effects. However, when the analysis was restricted to whites, the association between the KLK2 genotypes and prostate cancer risk was still significantly present.

The combination of the KLK2 C/T polymorphism and serum hK2 levels may help in the selection of patients for prostatic biopsy and further distinguish which patients are candidates for a second biopsy if the first biopsy is negative. The adjusted odds ratios for cancer after a repeat biopsy ranged from 2.2 to 14.5. These are among the highest odds ratios reported to date for any prostate cancer risk factor (Table 5).

Further, the combination of the KLK2 gene and serum hK2 levels enhanced the positive predictive value in addition to the established current risk factors for prostate cancer, including age, PSA level, and DRE results (Table 7). In particular, among patients who had a low positive predictive value for prostate cancer based on these factors (age < 70 years old, PSA < 10 ng/mL, and nonpalpable nodule on DRE), the effect of the KLK2 gene and serum hK2 level almost doubled the positive predictive value. This could be used to identify subgroups of men who are at high and low risk for having prostate cancer, which, in turn, could be used to aid in further management of these patients. Further confirmatory data will be required to validate these findings.

	ACKNOWLEDGMENTS

 
We thank Judith Finlay (Hybritech, Inc, a subsidiary of Beckman Coulter, San Diego, CA) for her assistance in adjusting the plasma hK2 levels according to current measurement standards.

	NOTES

 
Supported in part by the National Cancer Institute of Canada (Toronto) and the Canadian Urological Association Young Investigators Award.

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Submitted November 1, 2002; accepted April 2, 2003.

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