Liquid Biopsy for Early DiagNosis of Squamous Cell Carcinoma of the HeAd and NeCk rEgion (ENHANCE Study)
Conditions: Head and Neck Squamous Cell Carcinoma
Sponsor: Royal Marsden NHS Foundation Trust

 
 
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Title: Liquid Biopsy for Early DiagNosis of Squamous cell Carcinoma of the 
HeAd and NeCk rEgion (ENHANCE Study) 
 
 
Short Title: ENHANCE Study 
 
 
 
Protocol Version:  
 
v1.1 
Dated:  
 
 
18.07.2022 
 
 
 
 
 
RESEARCH REFERENCE NUMBERS 
 
 
IRAS Number: 
 
292104 
 
EudraCT Number: 
 
N/A 
 
ISRCT Number/Clinical 
 
N/A 
trials.gov Number: 
 
Sponsor Number: 
 
CCR5610 
 
Funders Number: 
 
829/R1554 – RM Partners 
 
 
 
 
Sponsor:  
 
 
The Royal Marsden NHS Foundation Trust 
 
 
  
Address:   
The Royal Marsden NHS Foundation Trust, Fulham Road, 
London SW3 6JJ 
 
Management:  
The Royal Marsden Clinical Trials Unit, Head & Neck 
Clinical Trials Unit  
 
Site Address: 
The Royal Marsden NHS Foundation Trust, Downs Road, 
Sutton, Surrey SM2 5PT 
 
 
 
 
 
 
 

 
 
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SIGNATURE PAGE 
 
The undersigned confirm that the following protocol has been agreed and accepted and that the 
Chief Investigator agrees to conduct the trial in compliance with the approved protocol and will 
adhere to the principles outlined in the Medicines for Human Use (Clinical Trials) Regulations 
2004 (SI 2004/1031), amended regulations (SI 2006/1928) and any subsequent amendments of 
the clinical trial regulations, GCP guidelines, the Sponsor’s SOPs, and other regulatory 
requirements as amended. 
I agree to ensure that the confidential information contained in this document will not be used for 
any other purpose other than the evaluation or conduct of the clinical investigation without the 
prior written consent of the Sponsor 
 
I also confirm that I will make the findings of the study publically available through publication or 
other dissemination tools without any unnecessary delay and that an honest accurate and 
transparent account of the study will be given; and that any discrepancies from the study as 
planned in this protocol will be explained. 
 
 
Chief Investigator: 
Signature:  
 
...................................................................................................... 
 
Date: 
  
....../....../...... 
Name: (please print): Dr Shree Bhide 
 
 
 
 
Statistician: 
Signature:  
 
...................................................................................................... 
Name: (please print): Maria Aresu 
 
 
 
 
Principle Investigator: 
Signature 
 
...................................................................................................... 
Name: (please print): 
 
 
 
 
Date: 
 
 
....../....../...... 
 
 
 
 
Date: 
 
....../....../...... 
 
 
 
 
 
 
 
 
 
 

 
 
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PARTICIPATING INVESTIGATORS AND CENTRES: 
 
 
Participating Centre: 
Kingston Hospital Foundation Trust 
 
Participating address: 
Galsworthy Rd, Kingston upon Thames 
KT2 7QB 
 
Principle Investigator: 
Mr Enyi Ofo 
Department: 
 
Head & Neck Clinic 
 
 
 
Galsworthy Rd, Kingston upon Thames 
 
 
 
KT2 7QB 
 
 
 
Email: enyi.ofo@nhs.net 
 
 
 
 
Participating Centre: 
London North West University Healthcare NHS Trust 
Participating address: 
Northwick Park Hospital, Watford Rd, Harrow 
 
 
 
HA1 3UJ 
 
Principle Investigator: 
Miss Zi-Wei Liu 
Department 
 
Lumps & Bumps clinic 
Northwick Park Hospital 
Watford Rd, Harrow 
 
 
 
HA1 3UJ 
 
 
 
Email: liu.ziwei@nhs.net 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
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KEY TRIAL CONTACTS AND PROTOCOL CONTRIBUTORS 
 
Chief Investigator 
Name:          Dr Shreerang Bhide 
Email:          Shreerang.Bhide@rmh.nhs.uk 
 
Trial Manager/ 
Trial email 
Name:          Jeane Guevara 
Email:           jeane.guevara@rmh.nhs.uk  
Trial Email : enhance@rmh.nhs.uk       
Sponsor 
The Royal Marsden NHS Foundation Trust 
For any enquiries, please contact the trial coordinator listed above or email 
your enquiry to: gcpcomplianceteam@rmh.nhs.uk  
Funder(s) 
RM Partners  
Statistician 
Name:         Maria Aresu 
Position:      Senior Statistician, The Royal Marsden Hospital, RDSU 
Email:          maria.aresu@rmh.nhs.uk 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
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1. BACKGROUND AND RATIONALE 
 
The 5-year survival for Head and Neck squamous cell carcinoma (HNSCC) across all TNM stage 
groups is approximately 50% [1]. Patients who are present with stage I & II disease have 
significantly better survival. When a patient  presents to their general practitioner (GP) with 
symptoms suggestive of HNSCC, they may be referred for urgent specialist input through the 
suspected cancer referral (SCR) pathway, which include dedicated neck lump clinics. Majority of 
the patients diagnosed via the SCR pathway present with Stage III &IV disease, which has a 
direct impact on outcomes.  
 
HNSCC is classified as an uncommon cancer and as such diagnosis and treatment is undertaken 
in specialist tertiary referral centres. The care of patients initially diagnosed with HNSCC in 
smaller secondary care hospitals is transferred to these specialist tertiary referral centres via the 
Inter Trust Transfer (ITT). ITT can introduce delays in treatment pathways resulting in a failure to 
achieve cancer treatment targets. Southwest London HNSCC diagnostic pathway review which 
was undertaken in February 2021 demonstrated that 40% of ITTs received were greater than 38 
days after referral and that  68% of the 62 day target breaches were in patients with ITT.  
 
HNSCC is known to shed fragments of DNA, called circulating tumor DNA (ctDNA) into the 
bloodstream [2]. We have developed novel ultra-sensitive (>90% sensitivity) next generation 
sequencing (NGS) assay for circulating HPV DNA [3] in patients with non-metastatic locally 
advanced head and neck cancer. Our current work involves ctDNA detection to cover the 
spectrum of genetic alterations in HNC using a single sequencing workflow to detect copy 
number aberrations (CNAs), HPV DNA (to cover 99.9% of HNC related HPV) and somatic 
mutations.  
 
The use of ultra-sensitive NGS assay for detection of ctDNA using a simple blood test (liquid 
biopsy) holds a great promise for cancer screening and early diagnosis and can lead to better 
survival results and less disease burden. This has been proven in proof of principle studies in 
nasopharyngeal cancer [4] (Chan N Engl J Med 2017; 377:513-522). Furthermore, this test can 
be administered in smaller secondary care hospitals in parallel to the ITT. With a quicker 
turnaround (1-2 weeks), the liquid biopsy can help expedite the patient journey through the 
cancer pathways reducing the incidence of cancer target breaches. In order to design studies to 
test this hypothesis we require preliminary data quantifying sensitivity and specificity of our assay 
in this setting.  
 
 
 

 
 
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2. STUDY OBJECTIVES 
 
2.1 Hypothesis 
 
The NGS assay has a high sensitivity and specificity for detection of ctDNA in early HNSCC 
 
2.2 Aims 
 
To test the feasibility of the NGS assay for detection of ctDNA in patients with early HNSCC 
 
 
3. STUDY SETTING 
 
3.1 Study Design 
 
This is a prospective observational sample collection study in patients referred to neck lump 
clinics via the SCR.  
 
3.2 End Points 
 
• 
Primary endpoint – Measure the sensitivity of the NGS assay for detection of ctDNA at 
baseline in patients with early HNSCC 
 
• 
Secondary end-point - Measure the specificity of the NGS assay for detection of ctDNA at 
baseline in patients with early HNSCC 
 
 
3.3 Inclusion/ Exclusion Criteria 
 
Inclusion Criteria 
 
• 
Patients referred for an Ultrasound guided fine needle aspiration (USFNA) in the neck 
lump clinic.  
 
Exclusion Criteria 
 
• 
Patient found to have a lump in the thyroid gland at the time of USFNA 
 
• 
Unable to give informed consent for biological sample collection. 
• 
Unable to safely participate in clinic sample collection 
 
4. TRIAL PROCEDURES 
 
4.1 Patient Identification and Consent 
 
The proposed observational sample collection study will recruit patients referred to the neck lump 
clinics via the SCR pathway.  Patients are classified as low and high risk in the neck lump clinic 
following clinical history and examination by the clinician. Patients classified as high risk are 

 
 
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referred for an USFNA. These patients will be approached for screening by a designated study 
research nurse and be provided with a patient information sheet (PIS). They will be made aware 
that definite eligibility will be determined following USFNA. Patients will only be eligible if they do 
not have a lump in their thyroid gland after undergoing USFNA. Following USFNA, eligible 
patients will be approached by the research nurse to address any concerns and to sign an 
informed consent.   
 
Blood sample will be collected from patients who sign the informed consent. As per the study 
procedure, the patient will be consented on the same day i.e. the day of the neck lump clinic, that 
they receive the consent form. They will be given adequate time to consider consenting to the 
study and have the opportunity to ask questions.  
 
The blood sample will be transferred to the Biobank at RMH via first class post for processing and 
storage. These samples will then be transferred in batches to ICR or CMP and analysed for the 
presence of and to quantify the levels of ctDNA. With advances in technologies for analysis we 
will also perform a complete genomic analysis.   
 
The study will be run at head and neck lump clinics at Kingston Hospital NHS Foundation Trust 
and London North West University Healthcare NHS Trust.  
 
4.2 Withdrawal criteria 
 
Patients may withdraw from the study at any time at their own request, or they may be 
discontinued at the discretion of the Principal Investigator. This can occur for the following 
reasons: 
• 
Patient decision  
• 
Ineligibility  
• 
PI decision  
If the patient is withdrawn from the study, the primary reason as well as the date of withdrawal will 
be recorded in the in the Excel Data Tool (combined eCRF and database). Should a patient 
withdraw consent for their samples to be used in the study, the samples will be destroyed. Data 
collected at the point of withdrawal will continue to be used by the study team.  
 
4.3 Study completion and end of trial definition 
 
The end of study accrual will be when the requisite numbers of patients have been recruited and 
the last blood sample is taken as per trial protocol. 
 
 

 
 
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5. DATA AQUISTITION AND HANDLING 
5.1 Clinical data 
 
We will be collecting identifiable data as part of the study . This information will be held by the 
study sponsor on an excel spread sheet stored on a Trust computer in line with local data 
governance policies and GDPR. Each patient enrolled in the study will have a unique study ID 
generated. 
The full dataset is available in an associated Excel Data Tool (combined eCRF and database). 
The dataset is divided into the following areas: 
 
1. General patient details: Demographics, smoking/alcohol  
2. Diagnosis – Histolgy/cytology from the USFNA 
5.2 Samples and Molecular data 
 
Biological specimens will be identified only using the study ID (link-anonymised). As such, no 
patient identifiable data will be processed outside of RMH.  
 
6. STORAGE AND ANALYSIS OF SAMPLES 
6.1 Specimens 
 
A 20ml blood sample (2x 10ml blood collection tube, Streck or other appropriate) for plasma 
extraction and collection of buffy coat or PBMCs will be taken.The blood will be centrifuged to 
separate the plasma and buffy coat and then labelled and stored at -80oC.  
 
6.2 Storage, labelling and postage of biological samples  
 
All the samples will be labelled in accordance with good clinical practice and local protocols. If the 
patient consents to participate in the study, the name of the study, the study ID and the date of 
acquisition should also be added to the label. Sample storage will be in compliance with the 
Human Tissue Act (HTA). 
 
The blood sample will be collected by the research nurse that attends the neck lump clinic. They 
will consent the patient, collect the sample and will arrange for the sample to be transferred to the 
Biobank. The research nurse will be adequately trained in the correct procedures for sample 
collection and transfer, ensuring procedures are compliant with the HTA. The blood sample will 
be transferred to Biobank at RMH via a courier or royal mail special delivery in an approved 
packaging for processing and storage. They will be link-anonymised and  transferred to ICR for 

 
 
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analysis, where samples will be stored in secure facilities. Samples labelled in an anonymised 
fashion with no identifiable features may be shared with external institutions and collaborators. 
Biological samples retained for the study will be encoded with a unique identifier and other patient 
identifiers will be removed prior to storage in order to maintain patient confidentiality. 
Laboratory researchers will not have access to any details that identify the patient in almost all 
circumstances. Certain key individuals within the study team will be able to link the unique 
identifier with the patient’s identification details. This will allow the collection of clinicopathological 
data to complement the molecular data results from the study. 
Extracted nucleic acids will be stored for future analysis in secure facilities at the Institute of 
Cancer Research. All samples will be held in compliance with the Human Tissue Act (HTA). 
 
The chief investigator will have overall responsibility for the samples and will delegate 
responsibilities to appropriately trained staff. While the samples are stored at the RMH Biobank , 
the biobank manager will have overall responsibility for the samples. The scientific officer based 
at ICR will request the samples to be transferred for analyses. The samples will be tracked at all 
times. Excess samples which are left over will be transferred back to the biobank and will be 
stored as per their SOPs, which include transfer to off-site facilites for long term storage.  
6.3 Molecular analyses  
Nucleic acid extraction 
Nucleic acids will be extracted from plasma with buffy coat used for germline nucleic acid 
sequencing. DNA samples will be subjected to sequencing analysis for identification of ctDNA. 
 
 
 
7. STATISTICS AND DATA ANALYSIS 
 
7.1 Statistical Considerations  
 
This is a pilot study that aims to investigate the feasibility of using HPV DNA as a biomarker of 
early diagnosis of HNSCC. To this effect, we will focus on identifying the sensitivity of the NGS 
assay in patients who been diagnosed with invasive cancer. A sensitivity of greater than 70% 
would be necessary for further investigation in a larger study. We hope to recruit 49 patients who 
undergo USFNA and are diagnosed with HNSCC and a further 121 patients who undergo USFNA 
and do not have invasive HNSCC. The standard ctDNA test will be compared against the tissue 
diagnosis of invasive cancer as per the tumour biopsy.  
 
 
 
 
 
7.2 Sample Size 

 
 
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Assuming disease prevalence at baseline of 99%, if ctDNA is detectable in at least 39 out of 49 
patients, this will enable us to demonstrate a sensitivity of at least 70% (i.e. rule out the maximum 
futility proportion), and assume a true sensitivity of 85% (the minimum efficacy proportion), with 
80% power and a one-sided type I error of 0.05.” 
If ctDNA is undetectable in at least 112 out of 121 patients, this will enable us to demonstrate a 
specificity of at least 85% (i.e. rule out the maximum futility proportion), and assume a true 
specificity of 95% (the minimum efficacy proportion), with 90% power and a one-sided type I error 
of 0.01 (Single stage phase II A’Hern design, software used nQuery v8.7.2). 
 
 
7.3 Statistical analysis  
 
The statistical analyses will be performed by Dr Ben O’Leary, who is a co-investigator on the 
project using the R statistical software package. Given, that the study will involve simple statistical 
analyses, an expert statistician will not be required to perform the analyses. However, we will ask 
the study statistician to check the analyses for accuracy.  
All quantitative data will be analysed for normality and presented accordingly as number of 
observations, measure of central tendency for HPV DNA copy number and variance. Qualitative 
data will be presented as a number of observations and percentages  
A Chi square test will also be performed to check for the association between plasma ctDNA and 
the diagnosis of viable invasive HNSCC.  
Sensitivity at baseline is defined as the proportion of participants with invasive HNSCC who have 
ctDNA detected. 
 
7.4 Study duration 
 
The study will continue until up to  49 patients with invasive HNSCC and 121 patients without 
invasive SCC,  have been recruited. The study will be completed once all 170 patients have been 
recruited and samples analysed. 
The study will be run at the head and neck lump clinics at Kingston Hospital NHS Foundation 
Trust and London North West University Healthcare NHS Trust. Based on previous year’s figures, 
approximately 200 patients are referred each year for USFNA following assessment in the clinic. 
Of these, 30-40 patients get diagnosed with HNSCC and 10-15 with Thyroid cancer. Therefore, 
we anticipate that this study will complete recruitment in 9-12 months.  
 
The primary analysis  will be performed once all 49 patients with invasasive HNSCC are recruited 
and samples analysed, whereas the secondary endpoint analysis will be performed once 121 
patients without invasive HNSCC  have their  samples analysed.  
 

 
 
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8. DATA HANDLING 
 Data collection tools and source document identification 
 
8.1 Source Data 
 
Clinical data will be collected directly onto the Excel Data Tool (combined eCRF and database). 
This will be stored in the electronic site file on the shared drive for clinical studies conducted by 
the H&N research team. In accordance with local data governance regulations for patient 
identifiable data. REDCAP might be used for data collection if it becomes available by the time 
the study starts to register patients. 
 
Data collection arrangement for sites outside RMH: A research nurse employed by RMH will be 
present at the neck lump clinics where recruitment will take place. They will enter the study data 
directly onto the Excel Data Tool (combined eCRF and database) on a secure trust laptop that 
they will carry with them to clinic.  
 
8.2 Data handling and record keeping 
 
The Excel Data Tool (combined eCRF and database) will be held in a folder on computers of the 
Royal Marsden Hospital NHS Foundation Trust and held for 5 years. This folder will have a 
security system to protect against unauthorized access. Each update of the data will be recorded 
and saved as a separate version in the folder so there will be no deletion of the entered data.  
Each participant will have their own trust medical record number (MRN) and a study ID to allow 
for identification of all the data reported for each participant and for pseudonymisation for the 
molecular analysis. 
The chief investigator (CI), Dr Shreerang Bhide, will be the custodian of the data submitted to the 
Royal Marsden Hospital. The CI and the Royal Marsden Hospital NHS Foundation Trust will keep 
records of all participating patients and all original signed informed consent forms. 
Data storage and management for the molecular analysis will be at the Institute of Cancer 
Research, London. Data will be pseudonymised with the study ID, encrypted and held in line with 
local data governance protocols.  
8.3 Access to data 
 
Direct access will be granted to authorised representatives from the Sponsor and the regulatory 
authorities to permit study-related monitoring, audits and inspections, in line with participant 
consent.  

 
 
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8.4 Archiving 
 
All study documents will be archived by the Royal Marsden Hospital NHS Foundation Trust 
following submission of the end of study report.  The site file and essential documents will be 
archived for 12 months after completion of the study.  These documents will be stored in a 
location determined by the Sponsor in line with their standard operating procedures.  Any 
destruction of essential documents will require authorisation from the Royal Marsden Hospital 
NHS Foundation Trust. 
 
 
 
9. TRIAL OVERSIGHT, MONITORING, INSPECTION  AND AUDIT 
 
9.1 Regulatory & Ethics Committee Approval 
 
The protocol will be submitted for ethical review to the Health Research Authority’s Integrated 
Research Application System’ (IRAS).  
The Study is sponsored by The Royal Marsden NHS Foundation Trust and will be approved by 
the Sponsor’s Committee for Clinical Research (CCR). The Royal Marsden NHS Foundation 
Trust will ensure that the study has received ethics approval from a research ethics committee 
(REC) and has received Health Research Authority (HRA) approval.  
 
9.2 Trial Management Group  
 
A Trial Management Group (TMG) will be set up and membership will include the Translational 
Lead Investigator, Clinical Lead Investigator, Co-Investigators and Senior Trial Manager. Principal 
Investigators at Kingston Hospital NHS Foundation Trust and London North West University 
Healthcare NHS Trust will be invited to join the TMG as appropriate. The TMG has operational 
responsibility for the conduct of the trial. The TMG is responsible for monitoring recruitment, 
safety and governance of the trial as well as collaborating with subsequent translational sub-
studies. The TMG will also review any safety concerns and can convene a meeting if significant 
concerns exist. 
 
 
10.  FINANCING, INDEMNITY & INSURANCE 
 
   
Where the Royal Marsden NHS Foundation Trust is either sponsoring or collaborating with 
externally sponsored research the NHS Litigation Authority will cover standard clinical negligence 
by employees, staff and health professionals employed by the Royal Marsden NHS Foundation 
Trust. For more information visit the following website:  
http://www.nhsla.com/Claims/Pages/Clinical.aspx.  
  

 
 
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There is unlimited liability and no excess. Insurance is provided under the Clinical Negligence 
Scheme for Trusts and there is no cover for non-negligence claims. 
  
For all notification of claims please contact the Board Secretariat. 
 
Where the Institute of Cancer Research is sponsoring the study there are no special 
compensation arrangements for this study. The NHS Litigation Authority covers standard clinical 
negligence of NHS employees, staff and health professionals under its Clinical Negligence 
Scheme for Trusts.  
 
For multicentre studies each participating site is responsible for ensuring insurance and indemnity 
arrangements are in place to cover the liability of the Principal Investigator. 
 
 
 
11. PUBLICATION POLICY  
 
Data arising from the study are owned by the Sponsor.  Findings will be submitted for publication 
in relevant H&N or Cancer peer reviewed journals.  Funders will be acknowledged in any 
subsequent reports.  Authorship will be granted in line with criteria defined by The International 
Committee of Medical Journal Editors.  
 
 
12. REFERENCES 
 
1. 
https://www.cancerresearchuk.org/health-professional/cancer-statistics/statistics-by-
cancer-type/head-and-neck-cancers. 
2. 
Bhide S, L.J., IGarcia-Murillas I, Cutts R,  Hurley T, Grove L, Nutting CM, Newbold K, 
Turner, Harrington KJ, Predicting response to radical  (chemo)radiotherapy (R-CRT) with 
circulating HPV DNA and tumor DNA (ctDNA) analysis in locally-advanced head and 
neck squamous cell carcinoma (LAHNC). J Clin Oncol 2017. 35 suppl; : p. abstr 6043). 
3. 
Lee, J.Y., et al., Predicting response to radical (chemo)radiotherapy with circulating HPV 
DNA in locally advanced head and neck squamous carcinoma. Br J Cancer, 2017. 
117(6): p. 876-883. 
4. 
Lam, W.K.J., et al., Sequencing-based counting and size profiling of plasma Epstein-Barr 
virus DNA enhance population screening of nasopharyngeal carcinoma. Proc Natl Acad 
Sci U S A, 2018. 115(22): p. E5115-E5124.