Bowel Disease Research Foundation
of The Association of Coloproctology of Great Britain and Ireland
Advancing the cure and treatment of bowel disease
Royal College of Surgeons of England
35-43 Lincoln's Inn Fields
London
WC2A 3PE
Tel: 020 7869 6945
E-mail: mhall@bdrf.org.uk
Web: www.bdrf.org.uk
Research Project Final Report
Principal investigator
Mr Graham
Branagan
Institution Salisbury NHS Foundation
Trust
Project Title
HomeR: Development of HPV genotyping as a predictive
biomarker of chemo-radiotherapy response in patients with Rectal
Cancer
Start date
1st Feb 2014
Finish
date
31st December 2014
Introduction: Large bowel cancer is a major cause of death worldwide.
The cause of non-genetic cancer is poorly understood with a number of
(max 500 words)
potential causative agents. Recent evidence suggests that high-risk
human papillomavirus (HPV) can be identified in >40% of large bowel
Please use the following cancer tissue compared with 6% of tissues from normal patients.
Further evidence suggests i) HPV is more likely to be found in tumours
format:
of the rectum rather than the colon ii) HPV is associated with less
a) Problem addressed,
advanced disease. HPV is also associated with other cancers especially
background and
of the throat and in these patients is associated with improved responses
to radiotherapy and improved survival.
strategic significance
Methods: HPV genotyping was performed on rectal cancer specimens
b) Method(s) used
from 2 centres (Manchester and Salisbury). Funding supported tissue
c) Hoped for results
collection, DNA extraction and HPV genotyping to identify 3 HPV
d) Actual results and
genotypes.
Hoped for results: Patients with HPV positive rectal cancer would
implications for
treatment/understanding have a greater likelihood of a complete response to chemoradiotherapy
(CRT)and that HPV would therefore act as a biomarker for response to
CRT
Actual results: There was no association between the presence of HPV
and complete response to CRT
Lay Summary
Background (purpose for Human papilloma virus (HPV) is an established aetiological factor for
the development of ano-genital and oro-pharyngeal cancers, but less
project)
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
chemoradiotherapy (CRT) [2]. A parallel hypothesis has not been tested
in rectal cancer.
Introduction
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 waitandsee 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 was to extend an established HPV genotyping
research programme, using highsensitivity 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 pretreatment 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).
Methods
Setting: Salisbury NHS Foundation Trust and the University of
Manchester (2006-2013)
Patients: Pre-treatment biopsy tissue in patients with rectal
adenocarcinoma undergoing CRT.
Cases: Patients with rectal cancer who had either a complete clinical
or pathological response to CRT were identified from both centre
databases. ‘Real-world’ definitions of complete responses i.e. as
determined by the MDT were used.
Tissue transfer: Whole paraffin-embedded tissue blocks were
transferred to Manchester.
DNA extraction: DNA was extracted from all the blocks in the NHS
BRC Biobank laboratory following GCLP QC standards. QIAamp
DNA FFPE tissue kit was used for purification of genomic DNA from
formalin-fixed, paraffin-embedded tissues. H&E staining was
performed on the slides of the first and last cuts for each sample for
pathological examination (to confirm presence of adenocarcinoma). The
quality of the DNA was checked using polymerase chain reaction
(PCR) to detect the presence of a house-keeping gene (beta-2microglobulin). If the quality of samples was adequate, all the DNA
samples underwent whole genome amplification (WGA). The quality of
the DNA was checked again using PCR (determination of the presence
of beta-2-microglobulin).
Bowel Disease Research Foundation
of The Association of Coloproctology of Great Britain and Ireland
Advancing the cure and treatment of bowel disease
Royal College of Surgeons of England
35-43 Lincoln's Inn Fields
London
WC2A 3PE
Tel: 020 7869 6945
E-mail: mhall@bdrf.org.uk
Web: www.bdrf.org.uk
Laboratory measurements: Multiplex PCR was performed for all HPVs
done in this study.
Using two sets of primers for each HPV genotype, higher sensitivity /
accuracy for HPVgenotyping was performed. Each PCR was
be performed with appropriate positive controls 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.
Planned statistical analysis:
Analyses for objective (i) were 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 used logistic regression to test differences
between complete responders versus others, allowing for adjustment for
potential confounders identified in the baseline analysis.
Results and discussion
Any HPV positivity was noted in 25% of tumours. Individual
genotype frequencies were: HPV16, 16%; HPV18, 6%; HPV33, 3%;
HPV6, 3%. In multivariate models that included age, gender, pretreatment T-size and N status, there was no association between
either any HPV positivity (OR = 1.095, 95% CIs: 0.394, 3.044) or
HPV16 positivity (OR = 1.072, 95% CIs: 0.332, 3.465), and complete
response to CRT.
Conclusion
In this stage 2 biomarker discovery study using high-sensitivity HPV
multi-valent assays, we found tumour HPV positivity rates lower than
those reported elsewhere in the literature. We found no clear ‘signal’
to take forward HPV genotyping for validation as a predictive
biomarker for chemo-radiotherapy response in patients with rectal
cancer.
Recommendations for
future work
Based on these reults there are no obvious future research avenues for
HPV genotyping in rectal cancer and response to radiotherapy
References
(author, title, date of
publication)