Research ArticleClinical Studies
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GNRH2 Polymorphism in Men With Prostate Cancer Treated With Androgen Deprivation Therapy

TRISTAN M. SISSUNG, SARAH LOCHRIN, TYLER LIU, KEITH SCHMIDT, JONATHAN STROPE, EMILY RISDON, HYOYOUNG CHOO-WOSOBA, DAVID J. VENZON, WIEM LASSOUED, HOUSSEIN A. SATER, BEATRIZ WALTER-RODRIGUEZ, DOUGLAS K. PRICE and WILLIAM D. FIGG
Anticancer Research September 2023, 43 (9) 4023-4030; DOI: https://doi.org/10.21873/anticanres.16590

Abstract

Background/Aim: Gonadotropin-releasing hormone 2 (GNRH2) is a poorly-studied peptide hormone that is widely distributed in the central nervous system and expressed in peripheral tissues of mammals. The non-synonymous rs6051545 variant in GNRH2 (A16V) has been linked to higher serum testosterone concentrations. This study investigated whether the A16V variant is associated with altered androgen-deprivation therapy (ADT) progression-free survival (PFS) and overall survival (OS). Patients and Methods: We examined the expression of GNRH2 in prostate tissue microarrays comprising normal tissue, prostatic hyperplasia, and prostate cancer using immunofluorescence. We also evaluated the GNRH2 genotype in 131 patients with prostate cancer who received ADT and compared PFS and OS between the variant and wild-type genotypes. Results: GNRH2 was detected in all prostate tissues, although expression did not vary with Gleason grade or disease stage (p=0.71). The GNRH2 A16V genotype was not associated with PFS or OS; however, univariate and multivariate analyses revealed Gleason score and definitive local therapy were each associated with PFS (p≤0.0074), whereas age and Gleason score were associated with OS (p≤0.0046). Conclusion: GNRH2 is expressed in normal, hyperplastic, and neoplastic prostate tissues; the A16V variant is not related to treatment outcome or survival.

Key Words:

In addition to their role in the hypothalamus-pituitary-gonadal (HPG) axis, gonadotropin releasing hormone receptors (GNRH-R1 and GNRH-R2) are expressed in the prostate (1-3). Intraprostatic GNRH-R1 is linked to antiproliferative, antiangiogenic, and antimetastatic signaling cascades (4). GNRH-R2, due to inclusion of a premature stop codon, may not signal following GNRH binding (5).

GNRH analogues and inhibitors exhibit different pharmacology in the pituitary than in tumors. Leuprolide and goserelin do not appear to affect prostate cell proliferation (2), suggesting that these agents do not act on GNRH receptors expressed in the prostate. Degarelix and cetrorelix appear to act as agonists (4) activating G-protein signaling that inhibits the growth of normal prostate stroma, normal prostate epithelia, benign prostatic hyperplasia (BPH), and hormone responsive prostate cancer cells grown in culture (2, 3). Human tissues and cancers utilize different intracellular GNRH signaling cascades (4), and the tumor environment expresses several splice variants of the GNRH receptor that are distinct from the pituitary (6). In particular, prostate cancer cells express splice variants 1 and 2 (SV1 and SV2) that each lack exons 1-3, while SV2 also lacks exon 7. Normal prostate expresses splice variant 3 (SV3) for which the open reading frame begins within exon 6. Thus, differential response to classical GNRH “agonists and antagonists” is plausible in prostate cells.

While GNRH2 has been poorly characterized in humans, it is widely distributed in the central nervous system and is expressed in peripheral tissues of mammals (4). In humans, it is expressed at low levels in the hypothalamus and at higher levels in hormone-responsive tissues, binding to GNRH-R1 instead of its cognate receptor (GNRH-R2), which harbors a premature stop codon (2-4, 7). GNRH2 expression is significantly reduced following androgen withdrawal or androgen receptor blockade in vitro or castration in vivo, whereas treatment with a synthetic androgen (R1881) restores GNRH2 expression. GNRH2 positivity trended toward an inverse relationship with Gleason score in a small study of low-Gleason-grade biopsy samples, which is consistent with its role in inhibition of cancer aggressiveness (1). Therefore, variability in GNRH2 expression and function may underlie inter-individual variation in androgen deprivation therapy (ADT) outcomes.

A single study has evaluated whether any genetic variants in GNRH2 are associated with the risk of prostate cancer progression during ADT (8). In this study, a nonsynonymous variant (A16V; rs6051545) was associated with a slight increase in the risk of progression after adjustment for age and an approximate 33% increase in serum testosterone in Japanese individuals. We hypothesized that GNRH2 variants may be associated with clinical outcome in Caucasian and African American men with prostate cancer who were treated with ADT.

Patients and Methods

Patients and treatment. Data were obtained from medical record review of a cohort of 131 individuals who were enrolled in clinical trials within the intramural program of the National Cancer Institute taking place between 1990 and 2014 (NCT00001266, NCT00001295, NCT00027326, NCT00030095, NCT00032825, NCT0004527, NCT00060528, NCT00089609, NCT00090545, NCT00096551, NCT00113984, NCT00436956, NCT00514072, NCT00634647, NCT00942578, NCT01090765, NCT01240551, NCT01441089, NCT01553188, NCT01683994, and NCT01875250). Informed consent was obtained from all subjects before trial participation, and the present study was approved by the NCI Institutional Review Board.

Demographic and treatment parameters of the cohort are listed in Table I. Patients were treated with a GNRH agonist (goserelin acetate, leuprorelin acetate, triptorelin pamoate), a GNRH antagonist (degarelix acetate) and/or an antiandrogen (bicalutamide or flutamide). Some also received radical prostatectomy, radiation therapy, and/or brachytherapy. ADT failure was defined as an increase in prostate-specific antigen (PSA) after the initiation of primary ADT therapy. Data pertaining to demographic parameters (age, race, and BMI) and disease-related parameters (PSA and Gleason) were also collected from patients’ medical records.

View this table:
Table I.

Genotype vs. demographics, disease parameters, and prior therapy.

Genotyping. DNA was extracted from the plasma using a QiaBlood DNA extraction kit according to the manufacturer’s instructions (Qiagen, Valencia, CA, USA) and stored at 4°C. Genotyping was conducted using the following PCR primers: (rs6051545) F1 5′-CCCTGTCCATTAGAGCAGCC-3′, and R1 5′-GCTCGCTTTCCT CC AGGG-3′. All 50-μl PCR reactions contained 1 × PCR buffer, 2 mM each of four deoxynucleotidetriphosphates (dNTPs), 1.5 mM magnesium chloride, and 1 unit of Platinum Taq DNA polymerase (ThermoFisher Scientific, Waltham, MA, USA). PCR conditions for the thermocycler are described as follows: 94°C for 5 min, 40 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s, with a final seven-minute cycle at 72°C. After amplification, samples were purified using shrimp alkaline phosphatase (SAP) and Exonuclease I (ExoI). The 20-μl reaction consisted of SAP, ExoI, 10X dilute buffer, water, and the PCR product. The conditions for the reaction are as follows: 37°C for 90 min and then 70°C for 20 min to deactivate the enzymes in the reaction.

Direct nucleotide sequencing PCR was conducted using the Big Dye Terminator Cycle Sequencing Ready Reaction kit V1.1 (ThermoFisher Scientific). The 20-μl reaction consisted of water, BigDye Terminator, diluted F1 or R1 primer solution, and the purified DNA. The samples were run on a thermocycler for 25 cycles at 94°C for 5 min, 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min. Samples were purified using the Optima DTR™ Ultra 96-Well Plate Kit (Edge Biosystems, San Jose, CA, USA) to remove the BigDye Terminator from the DNA samples, and the samples were run on an ABI Prism 3130 xl Genetic Analyzer (Applied BioSystems, Foster City, CA, USA). Sequence analysis was performed using Sequence Analysis v5.2 to determine the genotype of each of the samples.

Immunofluorescence experiments. 5 μm-thick formalin-fixed paraffin-embedded sections from tissue microarray (PR484a: 24 cases/48 cores from US BIOMAX) were stained using Opal multiplex kits, according to the manufacturer’s protocol (PerkinElmer, Waltham, MA, USA), for a panel of DAPI, and GNRH2. Multiplex immunofluorescence scans were captured by a PerkinElmer Vectra Polaris. Mean fluorescence intensity was measured in prostate epithelium (benign or malignant) using open source QuPath v 0.2.0 (stable version).

Availability of data. Datasets generated and analyzed during the current study are not publicly available due to National Institutes of Health regulations but are available from the corresponding author on reasonable request.

Statistical considerations. Tests of genotype frequencies for Hardy–Weinberg equilibrium were performed using the exact conditional distribution of the chi-squared statistic. Probabilities of progression-free and overall survival were estimated using the Kaplan–Meier method. Associations of survival outcomes with categorical factors (race, +/− AR-inhibitor, local therapy, Gleason score and genotype) were analyzed using the log-rank test. For continuous factors (BMI, age and log transformed PSA), the likelihood ratio test of the Cox proportional hazards model, with the exact method for handling ties, was applied. Genotypes were compared with age, BMI, and PSA using the Kruskal–Wallis test, while genotypes were compared to race, Gleason score, and localized treatment using the exact chi-squared test. For the multivariate analyses, Cox proportional hazards regression analyses with backward selection were performed, and hazard ratios were assessed using the Wald test. The numbers of patients included in the regression analyses were limited due primarily to missing values in the combinations of potential risk factors tested in the models at the start of backward selection. p-Values from the large number of exploratory analyses were not corrected for multiple comparisons, but a slightly conservative level of p<0.01 was chosen for noteworthy results.

Results

Genotype vs. patient characteristics. Progression-free survival (PFS) was assessed in 131 patients who were successfully genotyped for GNRH2 (rs6051545). To determine whether genotype was associated with demographics, disease parameters, and prior therapy, the 16V variant was compared to age, BMI, race, PSA, Gleason score, use of AR inhibitors, and localized therapy (Table I). None of these characteristics were different between genotype groups at the nominal statistical criterion in the present analysis (i.e., p<0.01). Notably, however, patients carrying the 16V variant were potentially more likely to receive radiotherapy only (p=0.019), and African Americans were possibly more likely to inherit the 16V variant than Caucasians (p=0.026). Genotype frequencies were in Hardy-Weinberg equilibrium among Caucasians and African Americans (p≥0.71).

Expression of GNRH2 in prostate tissue. A previous report suggests GNRH2 expression is regulated by androgens, but GNRH2 positivity appeared to only be marginally non-significantly related to lower Gleason score (1). To clarify whether GNRH2 expression is related to Gleason score, we tested tissue microarrays containing normal prostate (n=3), prostatic hyperplasia (n=13), Gleason 2 (n=2), Gleason 3 (n=1), Gleason 4 (n=17), Gleason 5 (n=8), and metastatic disease (n=1). Unfortunately, we were not able to obtain genotype information for these samples. This analysis revealed that GNRH2 is expressed in healthy prostate and prostate cancer tissues (Figure 1A). GNRH2 was mostly expressed on the cell membrane and cytoplasm of the prostatic epithelium (benign and malignant). GNRH immunofluorescence intensity was not associated with the stage or grade of prostate cancer (p=0.71; Figure 1B).

Figure 1.
Figure 1.

Immunofluorescence was conducted on prostate tissue microarrays using a GNRH2-specific antibody. (A) Representative images of GNRH2 expression are presented for normal prostate, prostatic hyperplasia, prostatectomy tissue (Gleason 5), and metastatic prostate cancer. (B) Mean fluorescence intensity was compared between different tissues. *All images were reviewed by a pathologist.

Associations of genotype, demographics, and disease parameters with survival outcomes. Since a previous report determined that the 16V variant was associated with higher risk of prostate cancer progression during ADT in Japanese patients (8), we examined whether this variant was associated with ADT clinical outcomes in the present cohort of patients.

To account for other factors potentially affecting PFS, time from ADT start to PSA nadir, time from PSA nadir to progression, and overall survival (OS), the models included age, BMI, race, PSA, Gleason score, AR inhibitor use, and local therapy along with genotype (Table II).

View this table:
Table II.

Associations of genotype, demographics, and disease parameters with survival outcomes (univariate analysis).

BMI, race, PSA, AR inhibitor therapy, and GNRH2 rs6051545 genotype were unrelated to any of the survival characteristics we studied. Age was related to OS (p=0.0046), but only trended toward an association with PFS (p=0.0176); older patients generally had poorer survival than younger ones. Those with Gleason score below 8 had prolonged PFS and OS (p=0.0098 and p=0.0023, respectively; Figure 2A and F), and those who received a radical prostatectomy had estimated median PFS more than 14 months shorter than other patients (p=0.0017; Figure 2B) but had a much smaller difference in OS (p=0.0757). Additional Kaplan–Meier analyses of PFS, PFS after PSA nadir, and OS are included in Figure 2. Analysis of time from ADT initiation to PSA nadir and time from PSA nadir to progression was hampered by lower numbers of individuals who had PSA nadir data.

Figure 2.
Figure 2.

Kaplan–Meier analyses were conducted for PFS after ADT vs. (A) Gleason score, and (B) local therapy. Kaplan–Meier analysis was also compared with (C) race for patients who had not reached PSA nadir. Similar analyses were conducted for PFS after PSA nadir vs. (D) Gleason score, (E) local therapy. Overall survival of patients with low vs. high Gleason score is also included (F).

For multivariate analyses, Cox proportional hazards models for each survival outcome were tested by initially including all the potential risk factors above; the models with the strongest selected associations are reported (Table III). Gleason score and local therapy (RP vs. none) were associated with PFS (HR=2.04, p=0.0074 and HR=2.28, p=0.0024, respectively). Age (HR=1.04, p=0.0036) and Gleason (HR=2.04, p=0.0027) were associated with OS. Further, additional analysis with the Cox model was performed to investigate any interaction effects between those factors with small p-values from the multivariate analyses. There was only one result of interest, showing a potential interaction effect between the local therapy and Gleason score at the time from PSA nadir to progression outcome (Table III). Specifically, the hazard of the local RP therapy seems to decrease more than 50% from that of no therapy at Gleason scores of 5-7 (HR=0.42) but to be about 2.6 times the hazard of no therapy at Gleason scores of 8-10 (HR=2.63), with the 95% confidence interval of the HR estimate excluding 1. Tests for differences between the GNRH2 genotypes 16A/A vs. the others in these models indicated no important associations with the outcomes (0.8<HR<0.95 with 16A/A as the reference, p>0.38 for all four outcomes).

View this table:
Table III.

Results of multivariate analysis.

Discussion

The A16V variant in GNRH2 was not associated with any demographic, disease characteristic, or clinical parameter. GNRH2 expression was also not associated with prostate tumor grade. Multivariate analysis revealed that Gleason score was related to ADT PFS and OS.

The present results are inconsistent with a prior finding that A16V variant carriers had poor PFS on ADT after adjusting for age and another study that documented a marginally non-significant trend between GNRH2 expression and Gleason score (1, 8). In light of the wide confidence intervals in this study and others, the present results may be consistent with a weak positive association between genotype and PFS. We confirmed that GNRH2 was expressed at detectable levels in all prostate tissues examined (1). These results are also consistent with the Protein Atlas data in which a high percentage of prostate cancer and prostate tissue express GNRH2 at high levels compared to other tissues (9). The current findings validate several studies demonstrating that Gleason score is associated with ADT long-term outcomes, particularly when adjusting for age (10-14).

Unfortunately, this retrospective analysis could not ascertain the degree to which individual patients expressed GNRH2, and further studies should examine whether GNRH2 expression can be exploited for predictive or prognostic value. ADT therapeutic outcomes may also be influenced by GNRH2 expression in prostate tumors (2, 3) given that ADT disposition is influenced by GNRH2 in cellular assays (1). Last, A16V may influence ADT outcomes in Japanese men but not Caucasians (8). Despite its high level of expression in prostate and prostate cancer, very few studies have been published examining GNRH2 in prostate cancer and further research is warranted.

Conclusion

GNRH2 is expressed in the normal prostate and prostate cancer. While such expression may affect prostate biology and the treatment of prostate cancer, polymorphic variants in GNRH2 are not associated with clinical grade of prostate cancer or treatment outcome.

Footnotes

  • Authors’ Contributions

    TMS: Conception, experimental design, analysis and interpretation, writing; SL: data collection, analysis and interpretation; TL: data collection, analysis and interpretation; KS: data collection, analysis and interpretation; JS: data collection, analysis and interpretation; ER: data collection, analysis and interpretation; HCW: analysis and interpretation, writing; DJV: analysis and interpretation, writing; WL: data analysis and interpretation; HAS: data collection, analysis and interpretation, writing; BWR: data collection, analysis, and interpretation; DKP: analysis and interpretation; WDF: conception, experimental design, analysis and interpretation, writing, final approval of manuscript, agreement to be accountable.

  • Conflicts of Interest

    The Authors declare no conflicts of interest in relation to this study.

  • Funding

    NIH Intramural Funds, ZIA BC 010627.

  • Received June 23, 2023.
  • Revision received July 17, 2023.
  • Accepted July 19, 2023.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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GNRH2 Polymorphism in Men With Prostate Cancer Treated With Androgen Deprivation Therapy
TRISTAN M. SISSUNG, SARAH LOCHRIN, TYLER LIU, KEITH SCHMIDT, JONATHAN STROPE, EMILY RISDON, HYOYOUNG CHOO-WOSOBA, DAVID J. VENZON, WIEM LASSOUED, HOUSSEIN A. SATER, BEATRIZ WALTER-RODRIGUEZ, DOUGLAS K. PRICE, WILLIAM D. FIGG
Anticancer Research Sep 2023, 43 (9) 4023-4030; DOI: 10.21873/anticanres.16590
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