Kisspeptin Signalling in the Hypothalamic Arcuate Nucleus Regulates GnRH Pulse Generator Frequency in the Rat

Kisspeptin and its G protein-coupled receptor (GPR) 54 are essential for activation of the hypothalamo-pituitary-gonadal axis. In the rat, the kisspeptin neurons critical for gonadotropin secretion are located in the hypothalamic arcuate (ARC) and anteroventral periventricular (AVPV) nuclei. As the ARC is known to be the site of the gonadotropin-releasing hormone (GnRH) pulse generator we explored whether kisspeptin-GPR54 signalling in the ARC regulates GnRH pulses.We examined the effects of kisspeptin-10 or a selective kisspeptin antagonist administration intra-ARC or intra-medial preoptic area (mPOA), (which includes the AVPV), on pulsatile luteinizing hormone (LH) secretion in the rat. Ovariectomized rats with subcutaneous 17β-estradiol capsules were chronically implanted with bilateral intra-ARC or intra-mPOA cannulae, or intra-cerebroventricular (icv) cannulae and intravenous catheters. Blood samples were collected every 5 min for 5–8 h for LH measurement. After 2 h of control blood sampling, kisspeptin-10 or kisspeptin antagonist was administered via pre-implanted cannulae. Intranuclear administration of kisspeptin-10 resulted in a dose-dependent increase in circulating levels of LH lasting approximately 1 h, before recovering to a normal pulsatile pattern of circulating LH. Both icv and intra-ARC administration of kisspeptin antagonist suppressed LH pulse frequency profoundly. However, intra-mPOA administration of kisspeptin antagonist did not affect pulsatile LH secretion.These data are the first to identify the arcuate nucleus as a key site for kisspeptin modulation of LH pulse frequency, supporting the notion that kisspeptin-GPR54 signalling in this region of the mediobasal hypothalamus is a critical neural component of the hypothalamic GnRH pulse generator

Duration of androgen deprivation therapy with postoperative radiotherapy for prostate cancer: a comparison of long-course versus short-course androgen deprivation therapy in the RADICALS-HD randomised trial

Background Previous evidence supports androgen deprivation therapy (ADT) with primary radiotherapy as initial treatment for intermediate-risk and high-risk localised prostate cancer. However, the use and optimal duration of ADT with postoperative radiotherapy after radical prostatectomy remains uncertain. Methods RADICALS-HD was a randomised controlled trial of ADT duration within the RADICALS protocol. Here, we report on the comparison of short-course versus long-course ADT. Key eligibility criteria were indication for radiotherapy after previous radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to add 6 months of ADT (short-course ADT) or 24 months of ADT (long-course ADT) to radiotherapy, using subcutaneous gonadotrophin-releasing hormone analogue (monthly in the short-course ADT group and 3-monthly in the long-course ADT group), daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as metastasis arising from prostate cancer or death from any cause. The comparison had more than 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 75% to 81% (hazard ratio [HR] 0·72). Standard time-to-event analyses were used. Analyses followed intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov , NCT00541047 . Findings Between Jan 30, 2008, and July 7, 2015, 1523 patients (median age 65 years, IQR 60–69) were randomly assigned to receive short-course ADT (n=761) or long-course ADT (n=762) in addition to postoperative radiotherapy at 138 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 8·9 years (7·0–10·0), 313 metastasis-free survival events were reported overall (174 in the short-course ADT group and 139 in the long-course ADT group; HR 0·773 [95% CI 0·612–0·975]; p=0·029). 10-year metastasis-free survival was 71·9% (95% CI 67·6–75·7) in the short-course ADT group and 78·1% (74·2–81·5) in the long-course ADT group. Toxicity of grade 3 or higher was reported for 105 (14%) of 753 participants in the short-course ADT group and 142 (19%) of 757 participants in the long-course ADT group (p=0·025), with no treatment-related deaths. Interpretation Compared with adding 6 months of ADT, adding 24 months of ADT improved metastasis-free survival in people receiving postoperative radiotherapy. For individuals who can accept the additional duration of adverse effects, long-course ADT should be offered with postoperative radiotherapy. Funding Cancer Research UK, UK Research and Innovation (formerly Medical Research Council), and Canadian Cancer Society

Adding 6 months of androgen deprivation therapy to postoperative radiotherapy for prostate cancer: a comparison of short-course versus no androgen deprivation therapy in the RADICALS-HD randomised controlled trial

Background Previous evidence indicates that adjuvant, short-course androgen deprivation therapy (ADT) improves metastasis-free survival when given with primary radiotherapy for intermediate-risk and high-risk localised prostate cancer. However, the value of ADT with postoperative radiotherapy after radical prostatectomy is unclear. Methods RADICALS-HD was an international randomised controlled trial to test the efficacy of ADT used in combination with postoperative radiotherapy for prostate cancer. Key eligibility criteria were indication for radiotherapy after radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to radiotherapy alone (no ADT) or radiotherapy with 6 months of ADT (short-course ADT), using monthly subcutaneous gonadotropin-releasing hormone analogue injections, daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as distant metastasis arising from prostate cancer or death from any cause. Standard survival analysis methods were used, accounting for randomisation stratification factors. The trial had 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 80% to 86% (hazard ratio [HR] 0·67). Analyses followed the intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov, NCT00541047. Findings Between Nov 22, 2007, and June 29, 2015, 1480 patients (median age 66 years [IQR 61–69]) were randomly assigned to receive no ADT (n=737) or short-course ADT (n=743) in addition to postoperative radiotherapy at 121 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 9·0 years (IQR 7·1–10·1), metastasis-free survival events were reported for 268 participants (142 in the no ADT group and 126 in the short-course ADT group; HR 0·886 [95% CI 0·688–1·140], p=0·35). 10-year metastasis-free survival was 79·2% (95% CI 75·4–82·5) in the no ADT group and 80·4% (76·6–83·6) in the short-course ADT group. Toxicity of grade 3 or higher was reported for 121 (17%) of 737 participants in the no ADT group and 100 (14%) of 743 in the short-course ADT group (p=0·15), with no treatment-related deaths. Interpretation Metastatic disease is uncommon following postoperative bed radiotherapy after radical prostatectomy. Adding 6 months of ADT to this radiotherapy did not improve metastasis-free survival compared with no ADT. These findings do not support the use of short-course ADT with postoperative radiotherapy in this patient population

Effect of intra-arcuate nucleus (ARC) and intra-medial preoptic area (mPOA) administration of kisspeptin-10 (KP) on LH secretion.

<p>Representative examples illustrating the effects of intra-ARC infusion of (A) 400 nl aCSF or (B) 100 pmol KP in ovariectomized 17βestradiol-replaced rats. C, Summary showing the effect of KP on LH secretion, calculated by comparing the mean area of under LH profile 2 h before with 1 h after its administration. Representative examples illustrating the effects of intra-mPOA infusion of (D) 400 nl aCSF or (E) 100 pmol KP in ovariectomized 17βestradiol-replaced rats. F, Summary showing the effect of intra-mPOA KP on LH secretion. LH secretion was dramatically increased immediately after KP treatment in both nuclei, which lasted about 1 h in most experimental animals. *P<0.05 versus aCSF control group at the same time point. ^#P<0.05 versus 10 pmol KP treatment group at the same time point; N = 5–7 per group.</p

Schematic illustration of the intra-cerebral microinjection sites.

<p>Bilateral cannulae were positioned in the medial preoptic area (mPOA) at bregma −0.26 mm or the hypothalamic arcuate nucleus (ARC) at bregma −3.30 mm according to the rat brain atlas of Paxinos and Watson (19). Closed triangles represent the location of the cannulae tips. Ac, anterior commissure; 3v, third cerebral ventricle.</p

Effect of intra-arcuate nucleus (ARC) administration of kisspeptin antagonist (Kiss-antag) on pulsatile LH secretion.

<p>Representative examples illustrating the effects of intra-ARC injection of (A) of 500 nl aCSF (3 injections at 30 min intervals), (B) 10 pmol Kiss-antag (3 injections at 30 min intervals) or (C) 50 pmol Kiss-antag (3 injections at 30 min intervals) in ovariectomized 17βestradiol-replaced rats. D, Summary showing the inhibitory effect of Kiss-antag on LH pulse frequency. *P<0.05 versus aCSF control group at the same time point. ^#P<0.001 versus Kiss-antag (10 pmol ×3) at the same time point; N = 5–6 per group.</p

Effect of intracerebroventricular (icv), intra-medial preoptic area (mPOA) or intra-arcuate nucleus (ARC) administration of kisspeptin antagonist, peptide 234 (Kiss-antag), on LH pulse amplitude (ng/mg) in ovariectomized 17β-estradiol-replaced rats.

<p>*2 rats continued to show LH pulses during kisspeptin antagonist infusion<b>/</b>not significant from baseline value (P = 0.28).</p><p>LH pulse amplitude (Mean ± SEM) is indicated before, during and post administration of antagonist or artificial cerebrospinal fluid (aCSF).</p

Effect of intracerebroventricular (icv) administration of kisspeptin antagonist (Kiss-antag) on LH pulse frequency.

<p>Representative examples illustrating the effects of continuous icv infusion of (A) aCSF (4 µl/h for 3 h) or (B and C) Kiss-antag (2.5 nmol/h for 3 h) in ovariectomized 17βestradiol-replaced rats. Pulsatile LH secretion was either completely suppressed during the period of Kiss-antag infusion (B) or LH pulse interval was significantly prolonged by Kiss-antag (C). D, Summary showing the inhibitory effect of Kiss-antag on pulsatile LH secretion. ^†P<0.001 versus aCSF control group at the same time point. ^#P<0.001 versus Kiss-antag treated group during the time of infusion; N = 5–6 per group. *LH pulse.</p

Effect of intra-medial preoptic area (mPOA) administration of kisspeptin antagonist (Kiss-antag) on pulsatile LH secretion.

<p>Representative examples illustrating the effects of intra-mPOA injection of (A) of 500 nl aCSF (3 injections at 30 min intervals), (B) 10 pmol Kiss-antag (3 injections at 30 min intervals) or (C) 50 pmol Kiss-antag (3 injections at 30 min intervals) in ovariectomized 17βestradiol-replaced rats. D, Summary showing there was no inhibitory effect of Kiss-antag on LH pulse interval. N = 6–8 per group.</p