Predictive Biomarkers For Response To Nivolumab In Head and Neck Squamous Cell Carcinoma
Conditions: HNSCC, DNA Damage, DNA Double Strand Break
Sponsor: Attikon Hospital

 
Study Title: Predictive Biomarkers for Response to 
Nivolumab in Head and Neck Squamous Cell Carcinoma 
(HNSCC) 
 
Principal Investigator: Amanda Psyrri, MD, PhD 
                                       Associate Professor 
                                       Attikon Hospital 
                                       Athens, Greece 12461 
                                       Email: dpsyrri@med.uoa.gr 
 
 
Date: August 3, 2018 
 
 
 

 
 
 
Study rationale 
Nivolumab is FDA-approved for the treatment of patients with 
recurrent/metastatic HNSCC whose disease has progressed 
within 6 months after platinum-based chemotherapy. The 
development of predictive biomarkers is needed to optimize 
patient benefit, minimize risk of toxicities and guide combination   
strategies. The greatest focus has been on tumor-cell 
Programmed Death Ligand 1 (PD-L1) expression. Although PD-
L1 positivity enriches for populations with clinical benefit, PD-L1 
testing alone is insufficient for patient selection in most 
malignancies. 
PD-L1 
expression 
can 
be 
transient, 
and 
intrapatient and even intratumor heterogeneity in PD- L1 tumor 
expression can exist. Therefore, tumor sampling at one time 
point might not accurately reflect the state of PD1 axis in a 
patient. 
Another 
important 
aspect 
is 
that 
PD-L1 
immunohistochemistry (IHC) alone does not take into account 
factors that could impede the anti-PD1 therapy response such as 
whether or not active immune cell engagement of the PD1 axis 
occurs in the tumor microenvironment or other concurrent 
immune suppressive pathways are present. Assessment of 
biomarkers 
at 
baseline 
may 
not 
predict 
benefit 
from 
immunotherapy. In a phase II study of ipilimumab in patients with 
metastatic melanoma baseline tumor- infiltrating lymphocyte 

status was not associated with clinical activity. However, 
increases in tumor infiltrating lymphocyte density in tumor biopsy 
samples collected after the second dose of ipilimumab was 
associated with significantly greater clinical activity with 
ipilimumab 
compared 
to 
samples 
without 
increases 
in 
lymphocyte density. For a better understanding of the 
mechanisms of resistance to nivolumab in HNSCC, The 
investigators propose to study a cohort of longitudinal HNSCC 
samples from recurrent/metastatic HNSCC patients (pts) treated 
with nivolumab and identify biomarkers of response and 
resistance. The investigators will specifically focus on modulation 
of immune phenotype (ImmR) following one cycle of nivolumab 
as surrogate biomarker for response to nivolumab. Mutational 
landscape and gene expression data will also be correlated with 
response to shed light in potential molecular pathways that 
should be co-targeted to reverse resistance to nivolumab. The 
investigators hypothesize that DNA Damage repair (DDR/R) 
pathways are strongly correlated with immune response. 
Primary Objective 
The primary objective of the current study is to prospectively 
explore candidate biomarkers from the tumor and tumor 
microenvironment for associations with clinical response to 
nivolumab in patients with recurrent/metastatic HNSCC treated 
with nivolumab. 

 
Primary Endpoint 
The primary endpoint will be the change in the percentage of 
immune cells that is caused by nivolumab treatment. Investigator 
assessment of best overall response (BOR), determined 
between the date of first dose and the last tumor assessment 
(TA), will be image-based and scored using RECIST 1.1. BOR 
will be defined as categorical variable with 3 levels { Benefit (CR, 
PR, SD lasting 6 months from the first nivolumab dose), non-
benefit (PD or SD lasting less than 6 months from the first 
nivolumab dose),  and unkown} 
 
Hypothesis 
The investigators hypothesize that modulation of immune 
phenotype following one cycle of nivolumab can be used as 
surrogate biomarker for response to nivolumab. In addition, 
alterations in the DDR/R network and the ImmR system 
contribute to the outcome of immunotherapy thus providing 
unique tools for discovery of biomarkers, therapeutic targets and 
their validation. 
Study Assessments 

Materials and Methods: Longitudinal tissue biopsies will be 
collected from HNSCC patients treated with nivolumab . Biopsies 
will be taken at baseline, 24-72 hours after the second cycle of 
nivolumab and at progression. The investigators will generate 
multidimensional profiling data (WES, nanostring, multiplex 
tissue biomarker imaging data).  Multidimensional profiling data 
will be analyzed to identify genomic and immune correlates of 
treatment response and resistance mechanisms of immune 
checkpoint blockade. 
 Patient cohort and tumor samples: The investigators will include 
recurrent/metastatic HNSCC patients who progressed after 
cisplatin-based chemotherapy and are to be treated with 
nivolumab. Tumor samples will be collected with appropriate 
written informed consent and analyzed. All tumor measurements 
will be performed by a physician formally trained in RECIST 1.1. 
Sample processing 
Diagnostic tumor FFPE block(s) will be retrieved for all 
consenting patients. 
 
1. 
Archival tumor tissue from primary debulking surgery or 
baseline biopsies will be used for Whole Exome Sequencing, 
Nanostring gene expression profiling and immunohistochemistry 

for the analysis of the immune landscape and tumor 
microenvironment. 
2. 
Paired core needle biopsies from post-cycle 1 and / or 
progression will be used for Nanostring gene expression profiling 
and immunohistochemistry for the analysis of the immune 
landscape and tumor microenvironment. 
Whole exome sequencing and targeted gene sequencing 
WES to identify gene expression associated with DNA repair and 
immune response. Targeted gene sequencing will identify the 
burden of somatic non-synonymous mutations. 
Nanostring gene expression profiling 
Multiple tumour, inflammatory- and immune related genes will be 
analyzed by multiplex Nanostring technology by using the 
nCounter 
PanCancer 
Immune 
Profiling 
770-plex 
gene 
expression Panel. This platform will be used to assess 
expression of genes related to immune signaling and attempt to 
define a gene set critical for clinical benefit from nivolumab. Data 
will be correlated with the immune landscape analysis (next 
section). 
Immune landscape 
 The 
assessment 
of 
the 
tumor-associated 
immune 
microenvironment will be performed in formalin-fixed, paraffin-

embedded tumor samples, at baseline and in cases in which 
post-therapy needle biopsy material is available. In these cases, 
the spatiotemporal dynamics of immune cell subsets will be 
quantified in both tumor nests and stroma, in order to highlight 
any increased homing, functional efficacy and clonal expansion 
in post-therapy tissues. The investigators hypothesize that the 
outcome would be determined by the spatial co-localization and 
close proximity of PD-1+ and PD-L1+ cells. Furthermore, other 
immune cell populations will be quantified by using lineage 
specific markers combined with functional markers, to assess the 
activation and differentiation status, by using multiplexed 
immunohistochemistry (IHC). 
The immune landscape in the tumor microenvironment will be 
correlated with the expression of PD-L1, PD-L2 and with the 
expression of molecules of the antigen presenting machinery in 
neoplastic 
epithelial 
cells, 
the 
gene 
expression 
profile 
(Nanostring) and the type and intensity of the peripheral blood 
immune responses. 
More specifically: 
1. 
Neoplastic epithelial cells will be examined for: 
a. 
The expression of PD-L1 and PD-L2 at the protein (IHC) 
level. 
 

b. 
The expression of HLA class I and HLA class II molecules, 
to evaluate the integrity of the antigen presenting machinery 
 
2. 
The immune microenvironment will be examined for: 
a. 
The presence of adaptive immunity cell populations e.g. 
CD3, CD8, CD20, Foxp3. 
 
b. 
The expression of PD-1 on different T cell subpopulations 
(multiplexed IHC for co-localization assessment). 
 
c. 
The expression of PD-L1 on stromal cells e.g. M1 and M2 
macrophages, Dendritic cells. 
 
d. 
Markers of tumor vasculature e.g. CD34 (endothelial cells) 
and D2-40 (lymphatics). 
  
Blood analyses 
1. 
PBMC (baseline, post-cycle 1 and at progression) will 
be used for: 
a. 
Quantification of T cell subsets by flow cytometry 
T cell subpopulations will be enumerated using a volumetric flow 
cytometric method and monoclonal antibody combinations for 

characterization of CD4, CD8, CD25, CD45RA, CD45RO, 
CD122, CD127, CD62L, CCR7, CTLA-4 and expression levels of 
PD-1. The main focus will be identification of shifts in effector 
and memory cells and finally populations of T cells that have 
been exhausted. 
b. 
Quantification of tumor-specific T cell responses. 
 
Assessment of peripheral blood T cell responses against known 
tumor-associated antigens (TAAs) expressed in most Head and 
Neck cancers, including HER-2, p53, HPV-related or Cancer 
testis antigens (or non TAA-specific, e.g. flu and tetanus, T cell 
responses as control) at baseline and after treatment. 
2. 
Serum 
(from 
baseline, 
post-cycle 
1 
and 
at 
progression) will be used for: 
In addition to T-cell responses, B-cell responses will be 
characterized. Serum obtained from patients at pre- and post-
treatment will be screened for the presence of antibodies 
directed against a panel of cancer testis antigens, and a panel of 
>9000 purified human proteins coated onto microarray slides 
(using the 'seromics' ProtoArray protein microarray assay from 
Invitrogen/Life Technologies). This protein microarray technology 
offers an unprecedented platform to simultaneously assay the 

serological response of patients to multiple antigens in a 
comprehensive fashion. 
3. 
PD-L1 expression on circulating tumor cells: The 
investigators will capture CTCs using the Parsotrix system and 
The investigators will then analyze PD-L1 expression using RT-
PCR. The investigators expect decrease in number of PD-L1 
expressing 
 CTCs following PD1 blockade (Strati…Psyrri: Prognostic 
significance of PD-L1 expressing CTCs in HNSCC Ann Onc Sept 
2017)). 
Secondary Objective 
The secondary objective of the current study is to assess 
candidate biomarkers for acquired resistance to nivolumab 
comparing baseline and post-progression specimens in patients 
with recurrent/metastatic HNSCC treated with nivolumab. 
 
Secondary Endpoints 
. Secondary Endpoints: 
1. Safety of performing a biopsy after second nivolumab dose 
2. Best overall response rate (BOR) according to RECIST 1.1 
criteria 

3. Number of participants with tolerability to the treatment 
4. The burden of somatic non-synonymous mutations in 
association with BOR and survival. 
5. The interferon-gamma gene signature in association with 
BOR and survival 
6. The expression of PD-L1 in association with BOR and 
survival 
7. The expression of human leukocyte antigens, HLA class I 
and HLA class II molecules in association with BOR and 
survival 
8. The presence of adaptive immunity cell populations 
9. The expression of PD-L2 in association with BOR and 
survival 
10. The expression of PD-L1 will be assessed in circulating tumor 
cells (CTCs) 
 
 
 
 
 

 
Statistical analysis 
Sample size calculation: Primary endpoint: Using a t-test at 
one-sided significance level 2.5% for a paired design, assuming 
the mean ratio of post- number of immune cells ETC will 
increase by 50% compared to the pre- number (ratio post/pre 
equal to 1.5; assumed standard deviation equal to 1), with the 
planned sample size of 50 cases, a power of 80% is achieved. 
References 
Hamid O, Schmidt H, Nissan A, Ridolfi L, Aamdal S, Hansson J, 
Guida M, Hyams DM, Gómez H, Bastholt L, Chasalow SD, Berman 
D. A prospective phase II trial exploring the association between 
tumor microenvironment biomarkers and clinical activity of ipilimumab 
in advanced melanoma. J Transl Med. 2011 Nov 28;9:204. doi: 
10.1186/1479-5876-9-204.   PubMed ID: 22123319