Effect of PROtons versus photons on IMMUNOlogical function in head and neck cancer (PRO-IMMUNO): a pilot study.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer
worldwide (1,2). Traditional factors associated with these cancers include
tobacco use and alcohol consumption (1,3). Prognosis of locally advanced HNSCC
is in general poor, with a 5-year overall survival of only 40% (53). Another
risk factor associated with HNSCC is human papillomavirus (HPV) leading to
oropharyngeal cancer (OPC) (2,3). These HPV-positive (HPV+) OPCs behave
clinically distinct from HPV-negative (HPV-) OPCs, and have a substantially
better prognosis. .

Standard treatment for locally advanced HNSCC of the oropharynx, hypopharynx
and larynx, and irresectable squamous cell carcinoma of the oral cavity is
radiotherapy with or without chemotherapy. For patients with
recurrent/metastatic (R/M) HNSCC who have progressive disease after
(chemo)radiation, immunotherapy with a programmed death 1 (PD-1) antibody
improves overall survival and has become the standard of care in this
palliative setting (6,7). Because of the poor prognosis of locally advanced
head and neck cancer, strategies to escalate the treatment are examined in the
curative setting, for example by immunotherapy. The phase 3 JAVELIN Head & Neck
100 study (8) compared concurrent chemoradiation (photons) plus placebo with
concurrent chemoradiation (photons) plus avelumab (programmed death ligand 1
(PD-L1) inhibitor) followed by avelumab maintenance in both HPV+ and HPV-
locally advanced HNSCC. The interim analysis did not show an improvement in
progression-free survival and overall survival. However, high PD-L1 status at
baseline (i.e. >=25%) showed a trend towards a better progression-free survival
upon treatment with avelumab and chemoradiation. In addition, pembrolizumab
(PD-1 inhibitor) concurrent with radiotherapy (photons) did not improve
progression-free and overall survival in locally advanced HNSCC compared to
cetuximab concurrent with radiotherapy (photons). Lack of selection of patients
based on PD-L1 status could be a reason for the negative result. Other
potential reasons for the disappointing result could be the ddestruction of
activated T-cells during photon radiotherapy or changes in the tumour immune
microenvironment caused by photon radiotherapy. Recent advances in radiotherapy
for the treatment of HNSCC include the use of proton therapy instead of photon
radiation, aimed at sparing healthy tissues surrounding the target volume
including lymphocytes, while providing equal tumour control.

Currently, there is some preclinical and clinical evidence that protons are
more effective than photons to maintain immunological function (9-11). The
consensus view is that fractionated photon radiation can hinder immunological
function, in part due to exposure of lymphocytes in the *off-target* low dose
radiation field, resulting in lymphopenia (9,12). A randomized phase II study
in esophageal cancer showed reduced treatment-related lymphopenia after
concurrent chemoradiotherapy with protons compared to photons, which was
related to much lower *off target* dose obtained with protons (11). In
addition, the immunogenicity of radiation increases with high-linear energy
transfer (LET) radiation like protons compared to low LET X-rays, like photons
(9,10).

If proton therapy is less detrimental for immunological function or if proton
therapy is able to maintain immunological functioning during (chemo)radiation,
proton therapy might be beneficial in the response to immunotherapy. Then,
proton therapy could be an attractive field for further research examining
proton (chemo)radio-immunotherapy next to other treatments. So, clinical data
on systemic immunological function and dynamic changes in the tumour immune
microenvironment during photon versus proton (chemo)radiation for locally
advanced HNSCC are needed to get more insight in the radiation-induced
immunogenicity of proton versus photon radiation. Better understanding of
systemic immunological function and dynamic changes in the tumour immune
microenvironment could guide the development and design of future studies. An
essential question that first needs to be addressed is whether proton therapy
is able to maintain immunological function in locally advanced HNSCC patients
compared to photons.

We aim to establish the difference in immunological function of HNSCC patients
undergoing (chemo)radiation with protons versus photons. These preliminary data
are required to follow-up with larger studies to compare the effect of proton
(chemo)radio-immunotherapy next to other approaches.

This is a pilot prospective observational study, comprising patients with stage
III-IV HNSCC treated with standard of care chemoradiation either with photons
(n=10) or protons (n=10), or with standard of care radiation alone with photons
(n=10) or protons (n=10). Patients will be assigned for protons or photons
based on the guidelines of the National Indication Protocol for Proton therapy.
There is only a preference for proton therapy in case the risk of long-term
toxicity (i.e. xerostomia and/or dysphagia) can be reduced with proton therapy.
The risk of toxicity can be examined in a radiation treatment plan comparison
(photons versus protons) before the start of (chemo)radiation. The probability
of being selected for protons mainly depends on the location of the primary
tumour site in relation to the most important organs at risk and not by tumour
extension (TN-stage), which is the main driver of outcome in terms of local
control and survival.

Immunological function will be evaluated by peripheral blood samples. Blood
samples will be collected at baseline, during (chemo)radiation (end of week 3
and/or before delivery of cycle 4 of chemotherapy) and after completion of
(chemo)radiation (week 9, week 12, week 20, week 34 and week 60, respectively 1
week, 5 weeks, 3 months, 6 months and 12 months after completion of
(chemo)radiation). To quantify immunological function, PBMCs collected during
(chemo)radiation and after (chemo)radiation will be compared with that before
(chemo)radiation (week 0), using IFN-γ-ELISPOT to screen for the presence of
T-cell responses to viral peptides, such as sars-cov-2, CEF (CMV, EBV,
Influenza) and E6 and E7 of HPV-16. Furthermore, flow cytometry panels will be
used to determine global changes in immune cell proficiency.

Histological evaluation will take place at baseline and week 3 to examine
changes in immune infiltration within tumour tissue during proton versus photon
(chemo)radiation. This biopsy part of the study is optional for the patient.
Archival tissue from the biopsy that was taken at diagnosis will be used for
the baseline assessments. An extra biopsy at baseline will only be taken in
case of insufficient or unavailable tumour tissue of the diagnostic biopsy.
Biopsy at week 3 week will be taken for all patients who agree to participate
in this optional part of the study.

Study related visits will be (as much as possible) combined with regular
hospital visits.

(Chemo)radiation either with protons or photons is standard-of-care in HNSCC.
The study procedures require seven visits to the hospital, which will be mostly
combined with the standard-of-care visits. During these visits blood will be
drawn (100 mL) for the collection of PBMC. We will try to combine these
venapunctures with routine blood tests as part of the standard of care and
standard of care follow up. We will minimize the number of extra vena
punctures.
In case the patient has given additional written informed consent, maximum two
additional tumor biopsies will be obtained.

We expect that the burden and risks associated with participation will be
minimal. Minimal risks are associated with a biopsy, like minimal risk of
(radiation-)ulcer, bleeding and/or infection.