BOWEL DISEASE RESEARCH FOUNDATION
of The Association of Coloproctology of Great Britain and Ireland
APPLICATION FORM FOR THE BDRFOBSERVATIONAL RESEARCH GRANTS
Applications should be a maximum of 4 pages, including any images, and also accompanied by a CV (1 side of A4) for each
applicant. Please send the form with CVs electronically as onesingle Word document using 12pt typeface.
1.Name of the investigator / Mr Graham Branagan2. Job title / Consultant colorectal surgeon
3. Email address /
4. Tel. No. / Work: 01722 336262 ext. 4483 mobile: 0775740280
5. Institution / Sponsor: Salisbury NHS Foundation Trust
Collaborator: The Christie NHS Foundation Trust &
University of Manchester (UoM)
6. Name of supervisor / Not applicable
7. Name of Co-investigators / Salisbury:
Mr Graham Branagan, Consultant Colorectal Surgeon
Dr Clare Fuller, Consultant Pathologist
Manchester:
Professor Andrew Renehan, Consultant Colorectal Surgeon
Dr Ian Hampson, Lead Scientist, Viral Oncology Group, UoM
Dr Ivona Baricevic-Jones, Post-doctoral scientist, Viral Oncology Group, UoM
Dr Matthew Sperrin, Lecturer in Biostatistics, UoM
8. Ordinary member of ACPGBI / Mr Graham Branagan
9. Title of Project / HomeR: Development of HPV genotyping as a predictive biomarker of chemo-radiotherapy response in patients with Rectal Cancer
10. Length of project / 12 months
Funding requested
11.a Total amount / £30,00011.b Breakdown of costs / Salisbury
Tissue retrieval: £2,600
Tissue transfer: £800 / Manchester
Post-doc scientist salary: £15,000
Laboratory consumables: £4,000
Tissue retrieval: £1,600
DNA extraction: £3,000
ABI Veriti PCR thermo-cycler: £3,000
11.c How many instalments / Not applicable
11.d In which months instalments need to be paid / Project start date: 1stFebruary 2014
11.e Justification of support requested / The requested funding is for the following:
- Retrieval of targeted tissue samples under existing biobank retrospective ethics arrangements; TISUE transfer to Manchester
- 6 month salary for a post-doctoral scientist (IB-J), already in post in Hampson laboratory, Manchester
- Laboratory consumables (e.g. primers) and updating PCR thermo-cycler equipment
- DNA extraction from the paraffin blocks using established SOPs
12. Has the institution administering
funding agreed exemption from overheads ? / Yes No
Project Details
13. Background to project
Human papilloma virus (HPV) is an established aetiological factor for the development of ano-genital and oro-pharyngeal cancers, but less appreciated, HPV is also associated with the development of rectal cancer [1]. Moreover, in ano-genital and oro-pharyngeal cancers, the presence of HPV16, the commonest oncogenic genotype, is associated with better prognosis and improved response to treatment, mainly chemo-radiotherapy (CRT) [2]. A parallel hypothesis has not been tested in rectal cancer.
In the UK, there are approximately 15,000 new cases of rectal cancer per year. Surgery is the mainstay of treatment. Locally advanced disease is treated initially with ‘downstaging’ pre-operative CRT, followed by surgery 8 to 15 weeks later, but this combination is associated with considerable peri-operative mortality and long-term morbidity. In 15% to 20% of cases, CRT may result in complete disappearance of tumour. In patients without residual tumour on imaging and endoscopy (clinical complete response), a wait-and-see policy (omission of surgery with follow-up) might be considered an alternative to major resection - and represents a new paradigm for treating rectal cancer - but to-date, there are no predictors for this response, and no recognised adjuvants to enhance this response.
The aim of this study is to extend an established HPV genotyping research programme, using high-sensitivity assays in anal cancer (Renehan & Hampson, Manchester), to rectal cancer, with the two-fold objectives to:
i)Describe the proportions of HPV 16, 18 and 33 in pre-treatment rectal cancer specimens, and relations with age, gender and stage (CRUK biomarker classification: BM Discovery Stage 1), and;
ii)Test the hypothesis that HPV16 positivity is greater in patients who subsequently develop a complete response compared with those without a complete response to CRT (CRUK biomarker classification: BM Discovery Stage 2).
We consider these objectives as ‘proof of concept’, forming a platform for future biomarker validation (see below).
Knowns and unknowns: (i) There is evidence of a causal association between HPV infection and rectal cancer based on Bradford-Hill criteria of analogy, biological plausibility, strength of association, and to a lesser extent the criteria of consistency, specificity and coherence [1]; (ii) a recent meta-analyses estimated that HPV is present in 42% of colorectal tumours versus 6% of non-diseased normal mucosa [3]; (iii) many of the earlier studies (reviewed in [3]) used crude methodologies (e.g. Southern blots and immunohistochemistry of the oncoproteins) and probably underestimated prevalence; (iv) most series are small - only four studies had greater than 100 patients [3]; (v) HPV16 appears to the dominant genotype; but HPV18 and 33 also appear dominant in some series [3]; (vi) there is yet no consistent 'correlation' between HPV positivity and tumour location or stage [3]; and (vii) there is no substantive study of HPV positivity and treatment outcome.
14. Methodology
Setting: Salisbury NHS Foundation Trust and the University of Manchester (2006-2013)
Patients: Pre-treatment biopsy tissue in patients with rectal adenocarcinoma undergoing CRT.
Study design: The design is shown in the flow diagram (Figure 1) – consists of two levels corresponding to the two objectives. Level I is a baseline characterisation (descriptive) study; level II is a case-control study.
Controls: From both centre databases, patients with rectal cancer who underwent CRT and subsequently proceeded to respective surgery with visible tumour present (non- or partial responders) – considered the ‘normal’ clinical pathway – will be identified and tissue blocks retrieved.
Cases: Patients with rectal cancer who had either a complete clinical or pathological response to CRT will be identified from both centre databases. For Manchester, we will additionally use the OnCoRe database (a BDRF funded project of clinical complete responders across the North West of England) to identify cases. We will use ‘real-world’ definitions of complete responses i.e. as determined by the MDT.
Tissue transfer: Whole paraffin-embedded tissue blocks will be transferred to Manchester.
DNA extraction:DNAwill be extracted from all the blocks in the NHS BRC Biobank laboratory following GCLP QC standards. QIAamp DNA FFPE tissue kit will be used for purification of genomic DNA from formalin-fixed, paraffin-embedded tissues. H&Estaining will be performedon the slides of the first and last cuts for each sample for pathological examination (to confirm presence of adenocarcinoma). The quality of the DNA will be checked using polymerase chain reaction (PCR) to detect the presence of a house-keeping gene (beta-2-microglobulin). If the quality of samples is adequate, all the DNA samples will undergo whole genome amplification (WGA). The quality of the DNA will be checked again using PCR (determination of the presence of beta-2-microglobulin).
Laboratory measurements: Multiplex PCR will be performed for all HPVs that will be done in this study. Using two sets of primers for each HPV genotype, higher sensitivity/accuracy for HPV genotyping will be performed. This approach is novel and increases sensitivity of each HPV genotype. Each PCR will be performed with an appropriate positive control as well as with negative control that will be run at the beginning of each PCR and at the end of each PCR to avoid any contamination(Figure 2).
Planned statistical analysis: Analyses for objective (i) will be standard chi-squared tests (e.g. proportions by gender); Mann-Whitney tests (e.g. median age by HPV positivity); and trends across ordinal categories (e.g. proportions by stage).
For objective (ii), we will use logistic regression to test differences between complete responders versus others, allowing for adjustment for potential confounders identified in the baseline analysis.
Power calculations: We stipulate from the outset that to demonstrate ‘proof of concept’ the minimal odds ratio is 3.0. Based on deliverable tissue retrieval in the timeframe and clinical practices of the two centres, the control sample size = 120. The estimated cases sample size = 40 i.e. a 1:3 case-control design. We estimate the HPV16 positivity among responders will be 69%; and among non-responders will be 36% (chosen to achieve an odds ratio of 4, and population prevalence of HPV16 positivity of 42%). The power at these sample sizes to show this difference is 95% (sampsi command in STATA).
In a worst-case scenario analyses, if we only retrieve and extract high-quality DNA in 30 cases (complete responders), the power to show difference is 88%. If the HPV positivity in the responders arm were only 63% (odds ratio = 3) the power to show difference is 80%.
Existing Infrastructure: Manchester – the Manchester Cancer Research Centre (MCRC) Biobank will retrieve the targeted archival tissues. An already funded biobank technician (through the Christie Surgical Oncology Research Group – lead: Renehan) will facilitate this. DNA will be extracted through the Manchester Central Biobank (site of Hampson laboratory) using existing protocols. The HPV detection work will be performed by a project-dedicated post-doctoral scientist experienced in these techniques, the Hampson laboratory, a leading Viral Oncology laboratory.
Salisbury – targeted archival tissue will be retrieved through the Pathology Department (Fuller). Clinical data will be taken from existing NBOCAP-compliant databases at both centres.
Data/tissue transfer agreements have been commonly used between Manchester and other UK centres.
Ethics: A strength of this study is the MCRC Biobank existing ethics – this allows tissue retrieval from clinical archives without need for individual patient retrospective consent (see supporting letter). The ‘Salisbury arm’ of the proposal will be suitable for proportionate ethics review, reducing delays.
Platform for future research: If our hypothesis is upheld, two research directions will emerge:
- Biomarker validation i.e. retrospective 2x2 interactive (predictive) analysis of HPV positivity versus HPV negativity in patients within a prospective trial of CRT versus no CRT;
- Development of adjuvants to enhance radiosensitivity – ultimately reducing treatment-related toxicity. Examples include: (i) engineering HPV oncoproteins into HPV-negative HNC cells; and (ii) AKT inhibition (e.g. nelfinavir) specifically enhances radiosensitvity in HPV positive tumours (based on oropharyngeal cancer studies) [4].
5. References (if any): Maximum of 4
1. Burnett-Hartman AN, Newcomb PA, Potter JD (2008) Infectious agents and colorectal cancer: a review of Helicobacter pylori, Streptococcus bovis, JC virus, and human papillomavirus. Cancer Epidemiol Biomarkers Prev 17: 2970-2979.
2. O'Rorke MA, Ellison MV, Murray LJ, Moran M, James J, et al. (2012) Human papillomavirus related head and neck cancer survival: a systematic review and meta-analysis. Oral Oncol 48: 1191-1201.
3. Lorenzon L, Ferri M, Pilozzi E, Torrisi MR, Ziparo V, et al. (2011) Human papillomavirus and colorectal cancer: evidences and pitfalls of published literature. Int J Colorectal Dis 26: 135-142.
4. Lui VW, Grandis JR (2012) Primary chemotherapy and radiation as a treatment strategy for HPV-positive oropharyngeal cancer. Head Neck Pathol 6 Suppl 1: S91-97.
16. Sample size and source of statistical advice (if appropriate) / Controls = 120; cases = 40. Total = 160 Power calculations in main text.17. Have you applied for or acquired funding from any other sources(s)? / Yes No
If Yes, where?
Lay summary (200-300 word summary)
Problem addressed, back ground and strategic significance: Large bowel cancer is a major cause of death worldwide. The cause of non-genetic cancer is poorly understood with a number of potential causative agents. Recent evidence suggests that high-risk human papillomavirus (HPV) can be identified in >40% of large bowel cancer tissue compared with 6% of tissues from normal patients. Further evidence suggests i) HPV is more likely to be found in tumours of the rectum rather than the colon ii) HPV is associated with less advanced disease. HPV is also associated with other cancers especially of the throat and in these patients is associated with improved responses to radiotherapy and improved survival.
Methods: HPV genotyping will be performed on rectal cancer specimens from 2 centres (Manchester and Salisbury). Funding will support tissue collection, DNA extraction and HPV genotyping to identify 3 HPV genotypes
Results of this research: We hope to confirm that high risk HPV can be identified in a proportion of patients with rectal cancer and compare whether patients with HPV positive rectal cancers respond better to radiotherapy and whetehr this impacts on recurrence and survival..
Changes to the current position: This study may add to the body of evidence that HPV is associated with rectal cancer and identify whether HPV positive tumours respond better to radiotherapy.
NAMEGraham William Branagan
TELEPHONE01725 518478(H)07775 740280 (M)
,
D.O.B.19.11.68
GMC No.4105260
HOSPITALSalisbury Foundation Trust (SFT)
POSTConsultant Colorectal Surgeon
RESEARCH
Master of Surgery, University of Portsmouth July 2002.
PIfor the Mercury II low rectal cancer study
PI for DREAMS
PI for LARS
PI for ISAAC
Past member of the Research and Audit Committee of the ACPGBI.
Past member of the NCRI anal cancer subgroup.
Reviewer for Digestive Diseases, BJS, Surgical Oncology, Colorectal Disease and the Annals of the Royal College of Surgeons, Annals of Surgery, Diseases of the Colon and Rectum and the international Journal of Urology .
Publications:One book chapter, two invited articles, twenty-three peer-reviewed articles.
Presentations:Five invited lectures, 13 oral presentations (international / national).
MOST RECENT PUBLICATIONS
Barker T, Branagan G, Wright E, Crick A, McGuiness C, Chave H. Vertical rectus abdominis myocutaneous flap reconstruction of the perineal defect after abdominoperineal excision is associated with low morbidity. Colorectal Dis. 2013 May 15. doi: 10.1111/codi.12286. [Epub ahead of print]
Byrne BE, Branagan G, Chave HS. Unselected rectal cancer patients undergoing low anterior resection with defunctioning ileostomy can be safely managed within an Enhanced Recovery Programme.Tech Coloproctol. 2013 Feb;17(1):73-8.
Foster JD, Pathak S, Smart NJ, Branagan G, Longman RJ, Thomas MG, Francis N. Reconstruction of the perineum following extralevator abdominoperineal excision for carcinoma of the lower rectum: a systematic review.Colorectal Dis. 2012 Sep;14(9):1052-9
Dabbas N, Adams K, Chave H, Branagan G. Current practice in abdominoperineal resection: an email survey of the membership of the Association of Coloproctology.Ann R Coll Surg Engl. 2012 Apr;94(3):173-6
How P, Stelzner S, Branagan G, Bundy K, Chandrakumaran K, Heald RJ, Moran B. Comparative quality of life in patients following abdominoperineal excision and low anterior resection for low rectal cancer.Dis Colon Rectum. 2012 Apr;55(4):400-6
CURRICULUM VITAE
Surname / Forename(s)Fuller Clare Elizabeth
Work address:
Salisbury District Hospital
Salisbury
Wiltshire
SP2 8BJ UK
Telephone No: 01722 429001
Fax No: 01722 341499
E-mail address:
GMC registration no: 3091894
Qualifications:
BM BCh MA FRCPath
Present Post
Consultant Histopathologist 1994-present
Previous appointments:
Lecturer Pathology Sheffield Medical School 1990-1994
Clinical research experience:
Recently Mecury II and BACCHUS, supplying blocks, slides and reports for multicentre national studies
Clinical research training (eg, ICH GCP/EU Directive etc) including date of course attended:
None
Signed:Clare FullerDated:30.07.2013
Andrew Renehan PhD FRCS FRCS(Gen Surg)
Current post: Professor in Cancer Studies and Surgery andHonorary Consultant Colorectal Surgeon, Peritoneal Tumour Service at the Department of Surgery, the Christie NHS Foundation Trust, Manchester.
Academic affiliation: Faculty Institute of Cancer Sciences, University of Manchester
I work a split 50: 50 clinical: academic biweekly job plan.
Tel (W): 0161 446 3157 e-mail:
Prestigious award: Hunterian Professor 2011/2012
Research / Editorial roles:
STROBE reporting revision group/COSMOS writing group
Diabetes & Research Cancer Consortium
NICE Colorectal Cancer Guidelines Development group
ACPGBI Research & Audit Committee
NCRN ano-rectal trials subgroup
COLOFOL steering committee– European trial in colorectal cancer follow-up
Dutch CEA follow-up in colorectal cancer trial advisory group
Member of editorial board: Cochrane Collaboration Colorectal Cancer Group
Member of editorial board: British Journal of Surgery
Lead cancer & obesity research group, Manchester CRC & MRC Health eResearch Centre
Grants awarded: last 2 years
2011: Novo Nordisk Independent research award £50,000
2011-2013: NAEDI Cancer screening in type 2 diabetes £122,000
2012-2014: NORD/Christie charitable funds £103,000
2012-2013: Sanofi-Pasteur MPS Independent research award £30,000
2013-2014: WCRF global attributable risk of BMI to cancer incidence - £500,000 (Co-investigator)
2013-2015: Pancreatic Cancer UK £74,000
2013-2015: Bowel Disease Research Foundation £94,000
2013-2018: MRC Health eResearch Centre - £5.5M (Co-investigator)
List of Key Publications (selected since 2012)
1. McDonald JR, O'Dwyer ST, Rout S, Chakrabarty B, Sikand K, Fulford PE, et al. Classification of and cytoreductive surgery for low-grade appendiceal mucinous neoplasms. Br J Surg 2012;99(7):987-92.
2. Renehan AG. Insulin analogues and cancer risk: the emergence of second-generation studies. Diabetologia 2012;55(1):7-9.
3. Renehan AG, Flood A, Adams KF, Olden M, Hollenbeck AR, Cross AJ, et al. Body Mass Index at Different Adult Ages, Weight Change, and Colorectal Cancer Risk in the National Institutes of Health-AARP Cohort. Am J Epidemiol 2012.
4. Renehan AG, M Z, Egger M. Obesity and cancer risk: seeking new mechanistic insights from epidemiology [commissioned]. Nature Reviews Cancer 2012.
5. Renehan AG, Solomon M, Zwahlen M, Morjaria R, Whatmore A, Audi L, et al. Growth hormone receptor polymorphism and growth hormone therapy response in children: a Bayesian meta-analysis. Am J Epidemiol 2012;175(9):867-77.
6. Renehan AG, Yeh HC, Johnson JA, Wild SH, Gale EA, Moller H. Diabetes and cancer (2): evaluating the impact of diabetes on mortality in patients with cancer. Diabetologia 2012.
7. Parkin E, O’Reilly DA, Adam R, Kaiser GM, Laurent C, Elias E, et al. The effect of hepatic steatosis on survival following resection of colorectal liver metastases in patients without pre-operative chemotherapy [in press] DOI:10.1111/hpb.12007. HPB 2013.
8.. Sperrin M, Marshall AD, Higgins V, Buchan IE, Renehan AG. Levelling off of adult body mass index trends in England: a latent class analysis of cross-sectional surveys (1992-2009). PLoS Medicine 2013.
Curriculum Vitae
Dr Ian N. Hampson
Date of birth: 26th February, 1954
Degrees: 1974-1977 BSc. Hon, Bio Sci, University of Lancaster
1978-1982 PhD. Faculty of Medicine, PICR, Christie Hospital, Manchester, UK.