Correlation of the treatment sensitivity of patient-derived organoids with treatment outcomes in patients with head and neck cancer (SOTO): protocol for a prospective observational study

Abstract

Introduction

Organoids have been successfully used in several areas of cancer research and large living biobanks of patient-derived organoids (PDOs) have been developed from various malignancies. The characteristics of the original tumour tissue such as mutation signatures, phenotype and genetic diversity are well preserved in organoids, thus showing promising results for the use of this model in translational research. In this study, we aim to assess whether we can generate PDOs from head and neck squamous cell carcinoma (HNSCC) samples and whether PDOs can be used to predict treatment sensitivity in HNSCC patients as well as to explore potential biomarkers.

Methods and analysis

This is a prospective observational study at a single centre (Guy’s and St Thomas’ NHS Foundation Trust) to generate PDOs from patients’ samples to assess treatment response and to correlate with patients’ treatment outcomes. Patients will be included if they are diagnosed with HNSCC undergoing curative treatment (primary surgery or radiotherapy) or presenting with recurrent or metastatic cancers and they will be categorised into three groups (cohort 1: primary surgery, cohort 2: primary radiotherapy and cohort 3: recurrent/metastatic disease). Research tumour samples will be collected and processed into PDOs and chemosensitivity/radiosensitivity will be assessed using established methods. Moreover, blood and other biological samples (eg, saliva) will be collected at different time intervals during treatment and will be processed in the laboratory for plasma and peripheral blood mononuclear cell (PBMC) isolation. Plasma and saliva will be used for circulating tumour DNA analysis and PBMC will be stored for assessment of the peripheral immune characteristics of the patients as well as to perform co-culture experiments with PDOs. SOTO study (correlation of the treatment Sensitivity of patient-derived Organoids with Treatment Outcomes in patients with head and neck cancer) uses the collaboration of several specialties in head and neck cancer and has the potential to explore multiple areas of research with the aim of offering a valid and effective approach to personalised medicine for cancer patients.

Ethics and dissemination

This study was approved by North West-Greater Manchester South Research Ethics Committee (REC Ref: 22/NW/0023) on 21 March 2022. An informed consent will be obtained from all participants prior to inclusion in the study. Results will be disseminated via peer-reviewed publications and presentations at international conferences.

Trial registration number

NCT05400239.

Correspondence to Dr Anthony Kong; [email protected]

STRENGTHS AND LIMITATIONS OF THIS STUDY

• A prospective study to generate a living biobank of head and neck squamous cell carcinoma patient-derived organoids and treatment sensitivity of organoids can be correlated with clinical outcome of patients.

• Allows collection of patients’ fresh tissue and other biological samples at different time points in a prospective manner as well as access to archival samples for translational research.

• Allows collaboration with different disciplines with potential of multiple subprojects across several areas of research.

• Difficulty in collecting blood and other biological samples at all time points due to logistic issues in scheduling this with patients’ routine clinical visits.

• Heterogeneity of patients included in the study and large cohort is required to fully assess the potential of this approach.

Introduction

Head and neck cancer

Head and neck cancer is a heterogeneous group of diseases and squamous cell carcinoma is the most common histology, accounting for more than 90% of cases.¹ Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer worldwide with around 350 000 deaths reported due to the disease in 2018.^{2 3} Moreover, the incidence of HNSCC is anticipated to increase by 30% by 2030 and estimated to be 1.08 million new cases annually.⁴ Several risk factors have been associated with HNSCC including exposure to environmental pollutants and human papillomavirus (HPV), excess alcohol intake and tobacco smoking.⁵

The treatment approach varies for each individual patient and is mostly guided by the anatomical site, disease characteristics, stage, functional outcome and patients’ wishes. The management of most locally advanced HNSCC (combination for International Cancer Control stages III and IV) requires a multimodality treatment approach as well as multidisciplinary care.⁶ Surgery and/or radiotherapy with or without concurrent platinum chemotherapy are the primary modalities of curative treatment for patients presenting with local or locoregional HNSCC. The treatment delivered is frequently associated with significant morbidity and a deterioration in quality of life, which can be life-long in many patients.⁷ Despite the delivery of aggressive multimodality treatment, less than 60% of patients will be disease-free in 3 years.³

HPV positive oropharyngeal patients respond better to chemoradiation (CRT) compared with HPV negative, tobacco-induced oropharyngeal cancer patients.⁸ These patients also have better 3-year rates of overall survival (82.4% vs 57.1%) compared with patients with HPV-negative tumours.⁹ Apart from HPV status, there is no accurate way of predicting which patients will respond to curative treatment and there is no easy way to test radiosensitivity and chemosensitivity of these tumours. There is a need, therefore, to develop an accurate test to evaluate the sensitivity ex vivo so that we can predict which patients are likely to be resistant to radiotherapy or platinum chemotherapy and therefore more likely to relapse.

Patient-derived organoids

Preclinical models based on immortalised cancer cell lines and xenograft studies have been important in elucidating the mechanisms of late-stage tumour development as well as studying drug resistance. However, large-scale drug screens based on these models have resulted in a high failure rate of preclinical compounds in clinical trials, which demonstrates their limitations.^{10 11}

Organoid is defined as ‘a 3D structure grown from stem cells and consisting of organ-specific cell types that self-organises through cell sorting and spatially restricted lineage commitment’.¹² Organoids are derived from two main types of stem cells: pluripotent embryonic stem cells and their synthetic-induced pluripotent stem cell counterparts and organ-restricted adult stem cells.^12–14 Sato et al was the first to establish 3D epithelial organoids using a single leucine-rich repeat containing G protein-coupled receptor 5 (LGR5) and intestinal stem cell¹⁵ and this protocol formed the basis of various adult stem cell-derived organoid culture protocols. Nowadays, long-term tumour organoid cultures have been established from a wide range of human epithelial tissues, including colon,¹⁶ liver,¹⁷ oesophagus,^{16 18} stomach,^{19 20} pancreas,^{21 22} lung,²³ breast,²⁴ prostate,^{25 26} bladder,^{27 28} ovary^{29 30} and endometrium.³¹

Large living biobanks of patient-derived organoids (PDOs) have been developed from a number of malignancies, including tissue derived from both primary and metastatic sites.^{16 17 21 23 24 27 29–42} The tissue to develop an organoid culture line is usually a specimen taken from the surgical resection site;^{16 43} however, in recent years scientists have succeeded in establishing organoid culture lines from other tissue sources such as the needle biopsy for hepatocellular carcinoma,⁴⁴ endoscopic biopsy from Barrett’s oesophagus,¹⁶ ascitic fluid for both pancreatic and ovarian cancers^{45 46} and endoscopic ultrasound-scan-guided fine-needle biopsy for pancreatic ductal adenocarcinoma.⁴⁷ Initially, the viability of fresh samples was a determining factor for the successful establishment of the organoid culture line, thus the specimen should have been processed within hours from collection. However, Tsai et al published robust methods of fresh human tumour sample cryopreservation which was later thawed to generate successful gastrointestinal organoid cultures.⁴⁸

One of the most important advantages of the organoid cultures is that the characteristics of the original tumour tissue such as mutation signatures, phenotype and genetic diversity are well preserved in organoids, as evidenced by the analysis of multiple long-term organoid cultures.^{20 24 28 34 38 44 49–51} Moreover, it has been shown that organoids derived from tumour samples preserve the histopathological characteristics of the original tumour both in vitro and following xenotransplantation.³⁵ Therefore, xenotransplantation can be used to validate drug responses in a more representative in vivo environment. In breast cancer, a living biobank was generated from over a hundred patients and most of the breast organoids retained expression of their original receptor status: progesterone receptor, oestrogen receptor and human epidermal growth factor 2.²⁴

Organoids have also been successfully used in other areas of cancer research such as interrogating mutational processes, creating a genetic cancer model and exploring possible causative agents in cancer development, such as infections.⁵² For example, organoid technology has been used to investigate the role of microbiome in tumourigenesis.⁵³ Moreover, two reports used healthy human intestinal organoids and exploited CRISPR–Cas9 genome editing to introduce combinations of common colorectal cancer (CRC) driver mutations to generate CRC progression models.^{54 55} Micro-injection of gastric organoids with Helicobacter pylori (H. pylori) creates a strong primary inflammatory response⁵⁶ and this model has been used to show how H. Pylori colonises the gastric epithelium.⁵⁷

In head and neck cancer, Tanaka et al were the first to report 3D cancer organoid cultures derived from patient’s cancer tissue with a success rate of 30.2%.⁵⁸ All organoid lines demonstrated similar histopathological features to the original tumour and their corresponding 2D cell lines were used for in vitro and in vivo drug screening with promising results.⁵⁸ Moreover, in a more recent study by Prof Clevers’ group, the establishment of 65 tumour organoid lines is described, which closely recapitulate molecular and genetic characteristics of the original tumour.^{38 59} Moreover, organoid lines were used to assess radiosensitivity in vitro, for extensive drug screening as well as for xenotransplantation in vivo, further supporting a personalised treatment approach.^{38 59}

Promising results have been published following drug screening using organoid biobanks.^{23 25 34} Profiling of PDOs can be used to identify genetic and/or epigenetic changes that cause drug resistance, thus allocating patients to specific treatments. In metastatic gastrointestinal cancer (oesophageal and colorectal cancer), Vlachogiannis et al explored organoid culture as a means to predict treatment response by using a compound library of drugs either investigated in clinical trials or used in clinic and they reported a positive predictive value of 88% and a negative predictive value of 100%.⁶⁰ These findings further highlight the utility of PDOs to proceed for personalised cancer treatment for each patient.⁶⁰ Importantly, a large biobank of matching normal tissue organoids is also generated, which offers a unique opportunity to screen drugs that exclusively target cancer cells and not healthy tissues. Using this approach, we can reduce treatment-derived toxicities for patients.⁶¹

Biomarkers

The term biomarker describes a measurement variable that is associated with the disease outcome.⁶² Circulating tumour DNA (ctDNA) has been identified as a promising and reliable biomarker for noninvasive molecular profiling, monitoring and predicting response to systemic treatment^{63 64} as well as detecting minimal residual disease (MRD) in gastric cancer,⁶⁵ lung cancer,⁶⁶ colon cancer⁶⁷ and breast cancer.⁶⁸

In head and neck cancer, a more recent publication explored the use of a 26-gene next-generation sequencing panel to detect MRD in locally advanced HNSCC and has shown promising results in predicting disease progression and survival.⁶⁹ However, the assay was assessed in a limited number of patients and the sampling time was anytime between 1 and 12 weeks following treatment. Thus, larger cohorts are needed to fully assess the potential of this approach, and it is also very interesting to explore the changes in the clearance of ctDNA on more frequent time intervals to identify the optimal sampling time. Moreover, in a cohort of 93 patients with HNSCC, saliva and plasma samples were screened for HPV (HPV 16 and 18) and somatic mutations (TP53, PIK3CA, NOTCH1, FBXW7, CDKN2A, NRAS and HRAS). Plasma ctDNA was shown to be a more sensitive biomarker than salivary ctDNA for oropharynx, larynx and hypopharynx cancer; plasma ctDNA: 86%–100% versus salivary ctDNA: 47%–70%. However, in oral cancer, salivary ctDNA had a better sensitivity than plasma (100% vs 80%). This could be explained by the fact the ctDNA is more readily available in the saliva due to the location of the tumour.⁷⁰

The immune system plays a key role in the initiation and tumour progression, and several studies have shown a critical link between the pathogenesis of HNSCC and immune cells. Some suggested markers for treatment response include the HPV status, programme death-ligand 1 (PD-L1) and HSP70 expression of the tumour, tumour-infiltrating lymphocyte count such as CD3-natural killer cells and CD8+T cells.^{71 72} However, these biomarkers can mainly be assessed on tumour samples. Thus, it is crucial to use peripheral blood for the identification of prognostic biomarkers. Chemotherapy and radiotherapy are established treatment options for HNSCC and can result in noticeable immune-related changes, such as the function and phenotype of immunocompetent effector cells. Several studies have shown a possible predictive role of the immune system composition and response to treatment including HNSCC.^73–77

Study rationale

The use of organoids in conjunction with genomic analysis from patient-derived tumour samples has the potential to stratify and identify effective cancer therapies for individual patients by (a) comparing the response of individual tumours to specific drugs in order to provide personalised recommendations to manage patient care; (b) assessing how tumours respond and develop resistance to in order to further understand the mechanisms; (c) determining the next drug treatment option when standard clinical options are not available or not considered effective and (d) creating a database of drug sensitivity to tumour genetics to recommend potential therapeutic strategies.

Aims and objectives

This study aims to generate PDOs from patients’ tumour samples and to collect preliminary data on the ability of PDOs to predict patients’ treatment response and whether their radiosensitivity and chemosensitivity can be correlated with their survival outcome.

The primary objective is to assess the percentage of successful generated organoids from tissues in head and neck cancer patients.

Secondary objectives are:

• To assess the sensitivity of radiotherapy, platinum (cisplatin and/or carboplatin) chemotherapy or cetuximab or immunotherapy or their combination in PDOs.

• To correlate the treatment sensitivities of PDOs above with the treatment outcome of patients undergoing primary surgery and adjuvant radiotherapy+/−concurrent platinum chemotherapy (cohort 1) (figure 1).

• To correlate the treatment sensitivities of PDOs above with the treatment outcome of patients undergoing primary radiotherapy+/−platinum chemotherapy (cohort 2) (figure 1).

• To correlate the sensitivities of platinum chemotherapy and/or cetuximab or immunotherapy of PDOs with the treatment outcome of recurrent or metastatic HNSCC patients (cohort 3) (figure 1).

Figure 1. Sample collection summary. The patient population will be split into three different cohorts and samples will be collected at different time points. mls, millilitres.

Exploratory objectives are:

• To correlate the treatment sensitivities of PDOs with the detection rates of plasma ctDNA (all cohorts).

• To assess and correlate the histopathological, genomic and transcriptomic features of patients’ samples with PDOs.

• To assess the sensitivities of PDOs to various targeted therapies based on their genomic profiles.

• Collection of archival tissues, blood samples (whole or processed for ctDNA and peripheral blood mononuclear cell (PBMC)), saliva (for ctDNA), urine or stool samples for other translational research.

The study objectives, endpoints and outcomes are summarised in table 1.

Table 1

Summary of study objectives, endpoints and outcomes

Objectives	Endpoints/measures	Outcomes
Primary
Ability to generate PDOs	Percentage of individuals with successfully generated PDOs	Rates of successful organoid generation
Secondary
PDO sensitivity to treatment	IC50 doses and dose-response curves	Effect on the viability and size of PDOs
Correlate PDO treatment sensitivity with outcomes of patients in:
Cohort 1	IC50 doses and dose-response curves correlated with the recurrence rates and disease-free survival of patients	The ability of PDOs to predict recurrences and survival outcomes of these patients
Cohort 2	IC50 doses and dose-response curves correlated with the complete metabolic response (PET-CT) and residual disease or salvage neck dissection	The ability of PDOs to predict complete metabolic response and treatment outcome of these patients
Cohort 3	IC50 and dose-response curves correlated with the objective response rates of patients undergoing the same treatment	The ability of PDOs to predict response to the same systemic treatments that the patients are receiving
Exploratory
Correlate PDO sensitivity with plasma ctDNA	IC50 doses/response of PDOs correlated with ctDNA and biomarkers in plasma before and after treatment	The ability of the PDOs to predict minimal residual disease and persistent disease
Correlate histological and genetic profiles of PDOs with tissue samples	IHC staining, exome sequencing and RNA sequencing of PDOs and patients’ samples	The ability of the PDOs to recapitulate the histopathological and genomic features of human samples
PDO sensitivity to various targeted therapies based on genomic profiles	IC50 doses of targeted therapies based on actionable genetic mutations	The ability of the PDOs to predict responses to various targeted therapies based on genomic profiles
Collection of blood and other biological samples for translational research	Conduct translational research including TMB, PD-L1 testing and co-culturing PDOs with PBMCs to assess immunotherapy response	The ability of the PDOs to facilitate high-quality translational research that will help to predict treatment response

ctDNA, circulating tumour DNA; IHC, immunohistochemistry; PBMC, peripheral blood mononuclear cell; PD-L1, programme death-ligand 1; PDO, patient-derived organoid; PET-CT, positron emission tomography computed tomography; TMB, tumour mutational burden.

Methods and analysis

Study design

This is a prospective observational study to generate PDOs from patients’ samples to assess treatment response and correlate with patients’ treatment outcomes. We would do a pilot study during year 1 to establish the pathways required and to assess the success rate in generating PDOs. During this time, we will also assess the willingness of patients to participate in the study as well as that of clinicians to help with recruitment. We aim to recruit 20 patients for the study in year 1 with specific criteria for progression beyond year 1 (see below). This will allow us to assess the method used to generate PDOs and determine the success rate of growing PDOs using our methods. Additionally, we will be able to evaluate the clinical relevance of PDOs including their role in drug screening and predicting therapeutic responses.

Patient population, screening and consent

Inclusion and exclusion criteria

Patient population will be chosen if they fit the following eligibility criteria:

• Patients with HNSCC undergoing curative treatment (primary surgery or radiotherapy) or presenting with recurrent or metastatic cancers.

• Age >18 years old.

Patients will be excluded if they are unable to give informed consent, for example, mental disability or vulnerable adults.

Patient groups

For the year 1 pilot study, the number of patients chosen for each cohort is the expected number of patients that we anticipate recruiting within 12 months based on the number of HNSCC patients that we treat at our centre. The patient population will be split into three different cohorts (figure 1). The sample sizes for each group will be determined based on the practicality of tissue collection and patient availability. The surgical group comprises 10 patients, as it is more feasible to obtain tissue samples from patients undergoing surgery. The RT group consists of five patients, as these patients often do not require a biopsy beforehand, making tissue collection more challenging. Similarly, the recurrent disease group also includes five patients, as these patients may not have had a primary tissue biopsy or surgery, making sample collection more difficult. The study aims to capture the heterogeneity in the HNSCC population and the outcomes from different treatment modalities.

For year 1 pilot study, the expected number of patients to be recruited in each cohort is as follows:

Cohort 1 (~10 patients)

• Cohort 1a (surgically resectable disease); only surgery: patients with surgically resectable locally advanced disease who will have surgery but will not undergo adjuvant radiotherapy+/−concurrent chemotherapy.

• Cohort 1b (surgically resectable disease); postoperative radiotherapy group: patients with surgically resectable locally advanced disease who will undergo adjuvant radiotherapy+/−concurrent chemotherapy following surgical resection.

Cohort 2 (~5 patients)

Patients suitable for primary radical radiotherapy+/−concurrent chemotherapy who agree to have additional research biopsy.

Cohort 3 (~5 patients)

Patients with recurrent or metastatic disease who agree to have additional research biopsy or a sample taken during surgery.

Following year 1 pilot study, additional 20–40 patients will be recruited and the number of patients in each cohort may vary.

Recruitment and consent

This will initially be a single centre study during year 1 pilot study which will be carried out at King’s College London and Guy’s and St Thomas’ NHS foundation trust. The study can be opened to other sites for participation after year 1 pilot study if appropriate local ethical approval is obtained. The potential participants will be identified from multidisciplinary team (MDT), surgical and oncology clinics. HNSCC patients suspected to have primary or recurrent HNSCC will be asked to consent to an additional research biopsy. This additional research biopsy will be used to generate PDOs in the laboratory (figure 2). For those surgically resectable patients, we may ask for additional resected sample once adequate samples have been obtained for routine diagnostic purposes. For these patients, the resectable samples will be used to obtain more PDOs regardless of whether the biopsy samples were successful in generating PDOs. Moreover, for the surgical patients, we will request samples from the normal surrounding resected tissue to create normal tissue organoids to be used as the control. Once the PDOs are successfully generated in the laboratory, they will be tested for sensitivities to various treatments including radiotherapy, systemic treatments such as cisplatin, carboplatin, cetuximab and immunotherapy as well as the combination. Successfully generated tumour organoids will be stored in the lab and can be used for future research.

Figure 2. Sample processing. Samples collected include tumour and relevant normal tissue samples at baseline (+-recurrence) for generation of PDOs and genetic analysis, blood and saliva samples (baseline and at different time points during treatment) for PBMC isolation/analysis and ctDNA analysis. ctDNA, circulating tumour DNA; PBMC, peripheral blood mononuclear cell.

If the patients are subsequently found to be ineligible due to no availability of fresh tissues for organoid generation, they will be given a new ID as a screen failure patient. However, the blood or biological samples collected can be used for translational research if this was consented to in their informed consent form (ICF). In addition, if any of these patients subsequently re-consent to SOTO study due to availability of fresh tissues for organoid generation, we will link the new SOTO ID with the screen failure ID so that we can process any previously collected samples and link the research results with the new SOTO ID.

Withdrawal of consent

The right of a participant to refuse participation without giving reasons will be respected. The participant will remain free to withdraw at any time from the study without giving reasons and without prejudicing his/her further treatment and will be provided with a contact point where he/she may obtain further information about the study. If a patient withdraws from the study, the research team can retain any tissue samples, DNA/RNA samples and organoids/cell lines that have been created up until the time of the patient’s withdrawal if the patient agrees. Any unused samples taken for research purpose can be destroyed if patients request to do so. Any demographic and medical information already provided or results from tests already performed on their samples will continue to be used in the study; however, no further data or sample collection will be performed. We will monitor the percentage of participants who withdraw consent from the study after taking part. Since the primary outcome is the rate of successful generated organoids and will not be affected by the dropouts of subjects, we will not need to recruit more participants to back fill those who may have withdrawn or dropped out once the fresh tissues are obtained from patients following consent.

Treatment visits

This is an observational study, and the patients will continue their clinical visits and follow-up for 5 years as per normal standard of care. We will obtain the treatment outcome from the medical records of patients.

Sample collection

For each patient recruited into the study, the following samples will be collected at the time points listed in online supplemental table S1a–1d.

Tissue sample and organoids processing

Research tumour samples will be taken while the patient is undergoing an examination under anaesthesia, or surgical resection or biopsy as standard of care. Up to four tumour samples will be requested each time (online supplemental table S2) if possible, to be obtained. For surgical resection specimens, normal tissue samples (up to four specimens) will be provided if available (ideally 5 cm or more away from the edge of the lesion). Tissue samples will be collected in a tube containing an appropriate medium for the generation of PDOs or snap-frozen for future translational research analysis (eg, genetic analysis). Moreover, we will be able to request formalin-fixed paraffin-embedded (FFPE) archival tissue from the head and neck pathology team.

Tumour and/or normal samples will be processed into PDOs within 24 hours of collection using an established methodology.³⁹ If immediate processing is not possible, samples will be stored at 4°C and processed within 24 hours. Briefly, samples will be washed with antibiotics, minced into small fragments and digested using a tissue-specific digestion solution (eg, 0.13% trypsin). After digestion, the cell clusters will be resuspended in the appropriate medium and filtered. The cell pellet will then be mixed with a basement membrane matrix and plated in small domes, covered with a tissue-specific medium composed of growth factors and nutrients. Organoids will be passaged on reaching confluency.

Once an established line is generated, sensitivity to systemic treatment and radiotherapy will be assessed using markers of viability at different time points following treatment. We will then correlate the treatment sensitivity of PDOs with the treatment outcomes of the patients from whom the tissues were obtained.

Cohort 1: surgically resectable locally advanced disease

The tumours of these patients would have been surgically resected and so the responses of these organoids to radiotherapy and chemotherapy cannot be directly assessed. Most of these patients may then undergo adjuvant radiotherapy+/–concurrent chemotherapy. It is anticipated that the radiosensitivity and platinum chemosensitivity may correlate with the outcome of these patients. We will therefore assess whether those PDOs with lower IC50 would have longer disease-free survivals. If PDOs could be derived from both biopsy and resected samples from these patients, we will compare the differences in responses of these PDOs generated from either biopsy or resected samples. We will use the normal tissue organoids from the periphery of the surgical specimen to confirm that response to the treatments is tumour specific.

Cohort 2: primary radiotherapy +/– platinum chemotherapy

We will culture HNSCC-PDOs and treat with irradiation and the same platinum chemotherapy drugs (cisplatin or carboplatin) as the corresponding patient. A few of these patients may undergo neoadjuvant chemotherapy before definitive chemoradiation. In this case, we will assess the responses and sensitivities of these organoids to the same chemotherapy drugs, usually docetaxel, cisplatin and 5FU (TPF). For patients undergoing primary radiotherapy with or without concurrent chemoradiation, positron emission tomography computed tomography (PET-CT) scan is done at 3 months after the completion of treatment to assess metabolic response. We will correlate the treatment sensitivity of PDOs as determined by the IC50 and dose-response curves with the complete metabolic responses by PET-CT scan and the rates of residual disease as well as salvage neck dissection.

Cohort 3: recurrent or metastatic HNSCC

The first-line treatment for the recurrent or metastatic HNSCC patients had been platinum-based chemotherapy+/−5FU chemotherapy with or without cetuximab (oral cavity squamous cell carcinoma only). Pembrolizumab monotherapy is now approved as a first-line treatment for HNSCC patients with PD-L1 positive (combined pathology score ≥ 1) tumours in the UK. Patients with PD-L1 negative tumours or those with rapidly progressing disease will continue to receive platinum-based chemotherapy as first-line treatment and may receive nivolumab as second-line treatment after prior platinum therapy. We will correlate the treatment sensitivities of PDOs to these drugs with the radiological responses from the HNSCC patients.

Blood sample and other biological samples for translational research

We aim to collect up to 40ml (which was changed to 80ml in the subsequent amendment) of blood in EDTA bottles at the different time points as shown in online supplemental table S1a–1d. Blood will be processed immediately in the laboratory for plasma and PBMC isolation (figure 2). PBMC will be stored for assessment of the peripheral immune characteristics of the patients at different time points of their treatment journey as well as to perform experiments with PDOs. Plasma will be cryopreserved for ctDNA analysis. Saliva samples will be pre-processed to remove debris before cryopreservation. We will then process plasma and saliva samples to extract ctDNA using commercial kits. A next-generation sequencing panel, focusing on the most commonly mutated genes in HNSCC, will be used. Bioinformatics analysis will be conducted to identify and validate biomarkers relevant to treatment sensitivity and patient outcomes. Participants will be asked to give generic consent for their samples, derived organoids and/or cell lines and linked data to be transferred and used in future research. Based on this consent blood, saliva, oral swab, urine and stool samples will be stored for future translational research. All material will be handled in accordance with the Human Tissue Act 2004 and other relevant legislation relating to the use of cell lines.

End of study definition

The patient recruitment for the initial pilot study will be 1 year from the commencement of the study at Guy’s Hospital. We will assess whether the endpoints and outcomes are met at the end of the pilot study but the patients will be followed up for their long-term treatment outcome and survival as per normal standard of care. We will continue to obtain relevant information on patients’ treatment and survival outcome from patients’ medical records. We will submit the protocol for a major amendment towards the end of year 1 so that SOTO study will continue as a prospective observation study for up to additional 2 years of recruitment if further funding is obtained.

The criteria for progression beyond year 1 pilot study are set as below:

• More than 50% of the approached participants will consent to taking part in SOTO study.

• At least 30% of the tissue samples will be successfully used to generate organoids.

• Further funding is obtained to continue beyond year 1.

Study organisation

The study is sponsored by Guy’s Cancer Centre via Wilson Olegario Philanthropy and conducted under the support of King’s Clinical Trial Unit according to their local procedures. The laboratory work will be done at the facilities of King’s College London under the supervision of Dr Anthony Kong.

Study timeline

Participant recruitment began on 10 June 2022. We completed the year 1 pilot study with the recruitment of 20 patients and received an extension (R&D number 305689) to recruit an additional 20–40 patients over the next 2 years. The current expected date to complete recruitment is June 2025 with the study completion date to be 12 months after the last patient.

Statistical considerations

The first year of recruitment will serve as a pilot study for this observation study and no formal power calculation is performed for year 1. We expect more than 50% of the approached participants will consent to taking part in SOTO study and at least 30% of the tissue samples will be successfully used to generate organoids. We will define successfully generated organoids as those that form organoid structures and can be passaged successfully beyond 4 weeks. However, we will also collect information on the rate of those organoids that form structure and are successfully passaged but could not be propagated beyond 4 weeks. A successful pilot study in year 1 will be defined as one that meets the predefined expectation. The data from year 1 pilot study may be used to calculate the sample size required to extend the observation study beyond year 1 if appropriate, which will be submitted as a major amendment.

Patient and public involvement

We previously involved patient and public involvement (PPI) members to discuss the use of organoids to correlate the treatment outcome during the conception of SOTO study. PPI members were also asked to read and advise on the changes required for the patient information sheet of SOTO study. We will involve PPI members for reporting or dissemination plans of the study results in the future.

Ethics and dissemination

This study was approved by North West-Greater Manchester South Research Ethics Committee (REC Ref: 22/NW/0023) on 21 March 2022. Any further amendments to the protocol or study documents will be reviewed by the sponsor, sent to the REC for review and approval. No changes will be implemented until approval has been received from the REC and approved by the Trust R&D department if required. The chief investigator will notify the REC of the end of the study within 90 days of the end of the study. If the study is ended prematurely, the chief investigator will notify the REC, including the reasons for the premature termination. Within 1 year after the end of the study, the chief investigator will submit a final report with the results, including any publications/abstracts, to the REC. An informed consent will be obtained from all participants prior to inclusion in the study. Personal data recorded on all documents will be regarded as strictly confidential and will be handled and stored in accordance with the General Data Protection Regulation 2016/679 and the Data Protection Act (2018). No serious adverse events are expected to occur following the tissue sampling involved in this study, as they will be done during standard care procedures. Participants will be followed up by their treating clinicians as per normal practice. The study findings will be published in a peer-reviewed journal and disseminated at appropriate conferences, departmental and scientific meetings.

Ethics statements

Patient consent for publication

Not applicable.

Footnote

Contributors AK (principal investigator) is the guarantor. AK was responsible for the conceptualisation, methodology, resources and overall supervision of the study. IV, AK, JD, AP and RM developed the methodology. Patient selection was carried out by AK, IV and LC. Sample collection was conducted by IV, CC, PYKC, RH-S, HG-W and LC. The original protocol was prepared by AK and IV. The manuscript was reviewed and edited by LC, GW, TG-U, J-PJ, SC, TN and AK. All authors reviewed the manuscript and approved the final version of the manuscript.

Funding This study has received funding from Guy’s Cancer Charity via Wilson Olegario Philanthropy (grant number: C210302 and C210302A).

Competing interests AK received fees for consulting, advisory, speaker’s roles and/or research funding from PUMA BioTechnology, AstraZeneca, Merck, MSD, Bristol-Myers Squibb, and Avvinity Therapeutics.

Patient and public involvement Patients and/or the public were involved in the design, or conduct, or reporting or dissemination plans of this research. Refer to the Methods section for further details.

Provenance and peer review Not commissioned; externally peer reviewed.

Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.

References

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