This study aims to determine which of 3 drug combinations best reduces the size of tumour prior to surgery for advanced melanoma and prevents the recurrence of melanoma after surgery.

The new drug options for advanced melanoma include oncogene-targeted therapy (such as dabrafenib, trametinib and vemurafenib) and immune checkpoint blockade (such as pembrolizumab, nivolumab and ipilimumab). These drugs have shown remarkable efficacy and have regulatory approval for metastatic disease. However, most patients with advanced disease eventually progress. It is unknown if earlier treatment with systemic therapy after surgery improves long term survival or what is the optimal sequencing or combination of therapy. An efficient method of assessing drugs and combinations in humans is critical, particularly as combinations of molecularly targeted and/or immune therapies may have similar signals for efficacy in pre-clinical models, and recapitulation of the human immune system in animal models is limited.

Neoadjuvant clinical trials in patients with resectable but bulky stage III/IV melanoma allows for the rapid evaluation of drug activity in humans utilising multiple clinical endpoints (metabolic response with Positron Emission Tomography [PET], clinical response with Computed Tomography [CT] imaging, pathological response, relapse-free survival and overall survival) and translational endpoints (morphological, genetic and immunophenotyping of tumour and blood).

Surgery remains the standard of care for resectable Stage III or IV melanoma, despite the recent drug therapy advances described above. The Food and Drug Administration (FDA) has recently expanded the approved use of ipilimumab to include a new use as adjuvant therapy for patients with resectable stage III / IV melanoma, to lower the risk of relapse following surgery. Neoadjuvant therapy in this group of patients may also result in improved survival rates and in the duration of local and distant disease control, with reduced surgical morbidity and the potential for early elimination of microscopic metastatic disease.

There is an emerging and rapidly growing evidence base of the value of combining targeted and immunotherapies in a number of histological subtypes of cancers. The support for a potential synergy between the two treatment modalities has been established, as has the increased toxicity profile. Both single agent BRAF inhibitors and combined BRAF and MEK inhibitors induce a marked clonal T cell infiltrate in responding melanoma metastases early during treatment (day 7-15), which is transient, and is not present at progression. Concurrently, melanoma tumour antigen and the programmed death-ligand 1 (PDL1) expression increase early during treatment.

It is unknown whether there is potential for converting a subset of patients who fail either immunotherapy or targeted therapy alone into long-term responders by treating with programmed cell death protein 1 (PD-1) inhibitors in conjunction with mitogen-activated protein kinases (MAPK) targeted therapies. Furthermore, it is unclear whether the PD-1 inhibitor would be best combined sequentially or concurrently with MAPK inhibitors. Mouse models have provided a clear rational for combining these treatments upfront, however there is no human tissue evidence to guide best combination strategies.

The question of how best to maximize clinical outcome via concurrent versus sequential targeted and immune therapy may be explored efficiently in the human neoadjuvant setting, with detailed interrogation of multiple biopsies early during treatment. Immunological, proteomic and genetic features in tissue and blood provide an in vivo assessment of tumour responsiveness to therapy. This may enable more selective application of therapeutic agents to patients who are more likely to benefit. Such findings would improve the therapeutic index and cost effectiveness of these agents. Earlier systemic therapy prior to surgery also means earlier targeting of distant micrometastases that could become the source of future disease relapse.

The rationale for this study design is therefore based on the hypothesis that one week of targeted therapy may be sufficient to induce an enhanced tumoral immunity to result in a higher pathological and clinical response using the 'Response Evaluation Criteria In Solid Tumors' (RECIST) guidelines when followed sequentially with pembrolizumab, than either pembrolizumab alone or the combination of targeted therapy and pembrolizumab upfront.

The potential for toxicities that could affect adherence to the combined study treatments are recognised, as additive, overlapping or unforeseen adverse events may occur with the triple combination. The adverse event profiles and safety-related interruption to treatment will therefore be assessed in conjunction with the objective responses.

The clinical and translational findings from this study have the potential to inform rational decisions regarding combinations of treatment both in the metastatic and the adjuvant settings. This is a critical study to inform future practice and future phase 3 clinical trials. The translational research performed on tissue biopsies and blood will provide mechanistic information to guide the selection of optimal combinations of therapies for phase 3 studies in the advanced and the adjuvant setting.

This is a phase II, randomised, open label, three arm, parallel group, clinical trial of neoadjuvant combined targeted and immune therapy for patients with BRAF V600 mutant resectable stage III (bulky regional stage IIIB-D, but excluding in transit disease) melanoma.

This translational study explores pathological and RECIST response rates for a 6-week duration of neoadjuvant therapy across 3 treatment arms. The key secondary outcomes to be measured include a detailed analysis of immunologic, proteomic and genetic biomarkers in tumour tissue and peripheral blood at weeks 1, 2 and 6 compared to baseline and correlated with clinical, metabolic and pathological response to neoadjuvant treatment, and relapse and overall survival to adjuvant treatment. In patients who relapse within 40 weeks of adjuvant treatment, further analysis of tumour tissue (if possible) will be undertaken. Relapse free and overall survival, surgical outcomes and adverse event profile will also be determined.

Sixty patients will be randomised to one of three treatment groups in a 1:1:1 ratio, with 20 patients in each treatment arm:

• "Sequential immunotherapy": Dabrafenib 150mg orally twice a day + Trametinib 2mg orally once a day for 1 week, then followed by treatment with Pembrolizumab 2mg/kg delivered intravenously at weeks 1, 3, and 6, then once every 3 weeks from week 6 for 50 weeks.
• "Concurrent immunotherapy": Dabrafenib 150mg orally twice a day + Trametinib 2mg orally once a day + Pembrolizumab 200mg intravenously once every 3 weeks for 6 weeks then Pembrlizuamb alone for a further 46 weeks after surgery.
• "Immunotherapy alone": Pembrolizumab 200mg intravenously once every 3 weeks alone for 52 weeks.

Allocation of treatment will be concealed prior to randomisation which will be performed via a web based system in permuted blocks and stratified by BRAF V600E mutation versus non BRAF V600E mutation (i.e. V600D, V600K, V600R, V600M).

Neoadjuvant treatment for all three arms will be administered for 6 weeks, followed by complete resection of tumour to no evidence of disease. Surgery is followed by 46 weeks of pembrolizumab adjuvant therapy or until disease relapse, death, intolerable adverse drug reactions or by withdrawal of patient consent. After 52 weeks of the study treatment phase, patients will be followed 3 monthly for relapse (and progression, following relapse) and survival for 5 years.

The biomarker component of this study will require blood samples and core biopsies of tumour tissue at the following time points:

• Baseline (PRE)
• Week 1 (EDT 1)
• Week 2 (EDT 2)
• Week 6 - complete lymph node dissection specimen (POST)
• At Relapse (RELAPSE) if applicable and available

Surveillance of disease during the 6 week neoadjuvant period will be undertaken with surgical assessments and with ultrasounds of the affected lymph node basin.

Inclusion Criteria:

• ≥18 years of age

• Written informed consent.

• Histologically confirmed, resectable American Joint Committee on Cancer (AJCC, 8th edition) stage IIIB, IIIC (Tx, T0, T1-4, N1b, N2b, N3b, M0) cutaneous melanoma or unknown primary melanoma with sufficient cutaneous and/or nodal disease to enable multiple excisional or core biopsies (at baseline, week 1 and week 2). 'Resectable' tumours are defined as having no significant vascular, central nervous system or bony involvement. Only cases where a complete surgical resection with tumour-free margins can safely be achieved are defined as resectable. Patients who may not have sufficient disease to enable multiple biopsies at weeks 1 and 2 will not be excluded, however the intention of the study is that at least one biopsy at these time points is required.

• Measurable disease according to RECIST version 1.1 criteria (≥ 10mm longest diameter for non-nodal lesions and / or ≥ 15mm in shortest diameter for lymph nodes) within 4 weeks of randomisation. 'Measurable' disease may be ascertained by CT or for cutaneous and superficial lesions, by caliper measurement with digital photography. CT preferred for all lesions where possible. PET imaging will be performed, but not used for the primary purpose of measuring response.

• BRAF V600 mutation positive on immunohistochemistry or a local molecular test (e.g. Oncofocus): a. A positive V600E immunohistochemistry stain at study entry should be formally quantified with a local molecular test following study entry (e.g. Oncofocus); b. Molecular BRAF mutation status should preferentially be confirmed using tissue taken from the presenting stage III / IV disease. Alternatively, archival primary tissue is also acceptable to confirm BRAF mutation status.

• Able to swallow and retain oral medication

• Eastern Cooperative Oncology Group (ECOG) performance status of 0-1

• Demonstrated adequate organ function as defined:

  1. Absolute neutrophil count (ANC) ≥1.5 109/L
  2. Platelets ≥100 109/L
  3. Haemoglobin ≥90g/L
  4. Serum creatinine OR measured or calculated creatinine clearance (CrCl) (Glomerular filtration rate [GFR] can also be used in place of creatinine or CrCl) ≤1.5 X upper limit of normal (ULN) OR ≥60 mL/min for patient with creatinine levels > 1.5 X institutional ULN.
  5. Serum total bilirubin ≤ 1.5 X ULN OR Direct bilirubin ≤ ULN for patients with total bilirubin levels > 1.5 ULN.
  6. Aspartate transaminase (AST) and Alanine transaminase (ALT) ≤ 2.5 X ULN OR ≤ 5 X ULN for patients with liver metastases.
  7. Albumin >25 g/L
  8. International Normalized Ratio (INR) or Prothrombin Time (PT)
  9. Activated Partial Thromboplastin Time (aPTT) ≤1.5 X ULN unless patient is receiving anticoagulant therapy as long as PT or PTT is within therapeutic range of intended use of anticoagulants. ≤1.5 X ULN unless patient is receiving anticoagulant therapy as long as PT or PTT is within therapeutic range of intended use of anticoagulants

• Anticipated life expectancy of > 12 months.

• Women of childbearing potential: a negative serum pregnancy test within 72 hours of first dose of study treatment and effective contraception from 14 days prior to study treatment until 4 months after the last dose.

• Men with a female partner of childbearing potential to use effective contraception from 14 days prior to study treatment until 4 months after the last dose.

Exclusion Criteria:

• In transit disease

• Uveal or mucosal melanoma.

• Prior anti-cancer treatment for melanoma, except for the following:

  1. surgery for a primary melanoma or previous stage III melanoma,
  2. adjuvant radiotherapy to the primary melanoma resected site or to lymph nodes for previous Stage III disease,
  3. previous adjuvant interferon or ipilimumab for resected stage II or III melanoma, Previous adjuvant treatment with PD-1 inhibitors or BRAF/MEK inhibitors is not permitted.

• Received any investigational drug within 28 days or 5 half-lives of the planned first dose of this study treatment.

• Known immediate or delayed hypersensitivity reaction or idiosyncrasy to drugs chemically related to the study treatments, their excipients and / or dimethyl sulfoxide (DMSO).

• Active infection requiring systemic therapy.

• Current use of any prohibited medication as described in protocol.

• Active autoimmune disease or a documented history of autoimmune disease or a syndrome requiring systemic steroids or immunosuppressive agents. Patients with the following are permitted to enrol:

  1. vitiligo,
  2. type I diabetes mellitus,
  3. residual hypothyroidism due to an autoimmune condition only requiring, and stable on hormone replacement,
  4. psoriasis not requiring systemic treatment,
  5. resolved childhood asthma or atopy,
  6. or conditions not expected to recur in the absence of an external trigger.

• A requirement for chronic systemic steroid therapy (> 10mg/kg per day of prednisone or equivalent) within two weeks before the planned first dose of study treatment or any on any other form of immunosuppressive treatment. Patients who require inhaled or intranasal corticosteroids (with minimal systemic absorption) may be continued if the patient is on a stable dose. Non-absorbed intra-articular steroid injections will also be permitted.

• A known history of another malignancy or concurrent malignancy unless the patient is disease-free for a minimum of 1 year, is completely treated and at low-risk of recurrence. The time requirement does not apply for patients with successful definitive resection or curative treatment of:

  1. Non-melanoma skin cancer (e.g. basal cell or squamous cell carcinoma of the skin),
  2. superficial bladder cancer,
  3. in situ carcinoma of the cervix,
  4. in situ breast cancer,
  5. atypical melanocytic hyperplasia or melanoma in situ
  6. other in situ carcinomas,
  7. multiple primary melanomas, or other treated low risk tumours.

• Known HIV, hepatitis B or C virus positive status or history of active tuberculosis (testing prior to randomisation is not required).

• Administration of a live vaccine with 30 days of planned first dose of study treatment. Seasonal influenza vaccines for injection are generally inactivated flu vaccines and are allowed, however intranasal influenza vaccines (e.g., Fluad®) are live attenuated vaccines, and are not allowed. Any vaccine is cautionary within 30 days.

• Patients with a history or evidence of cardiovascular risk including any of the following:

  1. QT interval corrected for heart rate using the Bazett formula ≥480 msec, a diagnosis of long QT syndrome (Roman-Ward or Jervell Lange-Nielsen syndromes)
  2. Taking medications known to prolong the QT interval.
  3. Uncorrectable electrolyte abnormal abnormality (e.g. hypo- or hyperkalaemia, hypomagnesaemia, hypocalcaemia)
  4. Uncontrolled arrhythmias, with the exception of atrial fibrillation which is controlled for > 30 days prior to randomisation.
  5. Patients with implanted cardioverter/defibrillators.
  6. Acute coronary syndromes (including myocardial infarction or unstable angina), coronary angioplasty or stenting within 6 months prior to randomisation.
  7. A history or current evidence of New York Heart Association (NYHA) ≥Grade 2 congestive heart failure
  8. A current left ventricular ejection fraction (LVEF) below than the lower limit of normal (LLN).
  9. Any abnormal cardiac valve morphology documented by echocardiogram which in the opinion of the investigator could interfere with the patient's safety.
  10. Treatment-refractory hypertension defined as a systolic blood pressure of >140 mm Hg and/or a diastolic pressure of >90 mm Hg, which cannot be controlled by anti-hypertensive treatment.

• Evidence or a risk of retinal vein occlusion (RVO) or central serous retinopathy (CSR), including:

  1. Presence of predisposing factors to RVO or CSR (e.g., uncontrolled glaucoma or ocular hypertension, uncontrolled hypertension, uncontrolled diabetes mellitus, or a history of hyperviscosity or hypercoagulability syndromes).
  2. Visible retinal pathology as assessed by ophthalmic examination that is considered a risk factor for RVO or CSR, such as evidence of new optic disc cupping.
  3. Intraocular pressure > 21 mm Hg as measured by tonography.
  4. Evidence of new visual field defects on automated perimetry.

• History or evidence of interstitial lung disease or active non-infectious pneumonitis.

• Serious or unstable pre-existing medical conditions or other conditions that could interfere with the patient's safety, consent, or compliance.

• Has known psychiatric or substance abuse disorders that would interfere with cooperation with the requirements of the trial.

• Has received prior therapy with an anti-PD-1, anti-PD-L1, or anti-PD-L2 agent or an agent directed to another co-inhibitory T-cell receptor (i.e. OX-40, CTLA-4).