Isolation and Structural Analysis of Genomic Variants of Herpes Simplex Virus Type 2

The original aim of the project was to study recombination in HSV-2 strain HG52 using restriction enzyme sites as unselected markers. As no relevant DNA sequence data was available for HSV-2 it was decided to isolate site deletion mutants by enrichment selection of spontaneously occurring variants. The restriction enzyme Xba I was chosen as it makes only four staggered cuts in the HSV-2 genome and a virus, HG52X163X3X53, lacking all four Xba I sites was isolated following three rounds of enrichment selection. However, during the screening procedures involved in the isolation of HG52X163X3X53, a large number of variants with genomic alterations was identified and the work described in this thesis has concentrated on the analysis and characterization of these variants. Of the variants isolated after the initial enrichment selection of HG52 DNA five have been studied in detail. Three (HG52X85/4, HG52X85/5 and HG52X86) have deletions from IRL ranging in size from 2. 5x10e6 daltons to 6x10e6 daltons. Under immediate early conditions VmwIE64 production by HG52X85/5 and HG52X86 in which only the 3' part of IE1 is removed is reduced despite their deletions ending approximately 1kb downstream from the 3' end of UL54 which encodes this polypeptide. HG52X85/4 which also has a deletion ending approximately 1kb downstream of UL54 but in which an entire copy of IE1 is removed makes VmwIE64 in normal amounts. Of the two remaining variants one, HG52X192, has a deletion of approximately 1x10 6 daltons in each copy of RL. The final variant, HG52X19, has a deletion of approximately 9x10e6 daltons which removes the entire internal copy of the long repeat and half of the short repeat. The long segment of the genome is fixed in the prototype orientation whilst the short region inverts inefficiently through the undeleted part of the repeats (the internal copy of the 'a' sequence being deleted). The genome has non-HSV DNA inserted across the deletion. The inserted DNA has been reiterated to give different copy numbers and hence a heterogeneous genome population. It has been demonstrated that the inserted DNA is of bovine origin -presumably the result of recombination with the calf thymus DNA used as a carrier during transfection. To investigate if the enrichment selection procedure was responsible for the production of the unexpectedly high proportion of variants, isolates from untreated HSV stocks were studied. Of the fifty plaques of HG52 analysed, twelve differed significantly from the wild type structure. However, the plaques, five of HG52/5 and seven of HG52/10, represented only two variants with the same genome structures as HG52X86 and HG52X192 respectively. No significant variation was found in the stocks of other HSV strains examined (ie. HSV-2 strains 333 and 186 and HSV-1 strains 17 and KOS or plaque purified HG52 isolates (eg. tsl)). Variation was found in HSV-1 strain McKrae but its relevance was impossible to assess as the history of the stock is unknown. The conclusion has been drawn that variation has arisen in HG52 in the absence of enrichment selection although the structure of HG52X19 must have resulted from the transfection procedure. Three variants, HG52X163X12, HG52X163X14 and HG52X163X21, were isolated following the second round of enrichment selection using as the parent HG52X163 which lacks the 0.7m.c. Xba I site. HG52X163X12 has a deletion removing sequences from 0.94m.c. to 0.99m.c. (ie. part of U S and almost all of TRS) . The isolation of this variant demonstrates that genes US 10, 11, 12 and one copy of IE3 and one copy of oriS are non essential, at least in vitro. The two remaining variants, HG52X163X14 and HG52X163X21, both have deletions removing the same region as from HG52X163X12. However, the deleted sequences are replaced by sequences between 0.83m.c. -0.91m. c. in the inverted orientation thereby effectively deleting US between 0.94-0.96m.c. while extending the short repeats by 6kb. The only differences found between HG52X163X14 and HG52X163X21 were the loss of the 0.91m.c. Xba I site and a small insert containing a Hind III and EcoR I site at the 0.94m.c. Xba I site in the latter. It is possible that the enrichment selection procedure was responsible for the generation of these three variants as the rearrangements appear to be associated with the Xba I sites at 0.91 and 0.94m.c

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

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