Authors: Vassiliki Saloura , Aiman Fatima , Makda Zewde ... · various histologies of head and neck...

Title: Characterization of the T-cell receptor repertoire and immune microenvironment in patients with locoregionally advanced squamous cell carcinoma of the head and neck.

Authors: Vassiliki Saloura¥1, Aiman Fatima¥1, Makda Zewde1, Kazuma Kiyotani1, Ryan

Brisson1, Jae-Hyun Park1, Yuji Ikeda1, Theodore Vougiouklakis1, Riyue Bao2, Tanguy Seiwert1,

Nicole Cipriani3, Mark Lingen3, Everett Vokes1, Yusuke Nakamura1, 4

¥ These authors contributed equally to this work.

Affiliations: Department of Medicine1, Center for Research Bioinformatics2, Department of

Pathology3, Department of Surgery4, University of Chicago, Chicago, USA, Japan

Corresponding author: Vassiliki Saloura MD, 5841 S. Maryland Avenue, MC2115, Chicago,

IL, 60637, email address: [email protected]

Keywords: T-cell receptor repertoire, squamous cell carcinoma of head and neck, immune

markers, T-cell receptor sequencing, immune microenviroment

Figures: 5

Tables: 2

Word count: 3,344

Conflicts of Interest: The authors declare no potential conflicts of interest.

on May 1, 2020. © 2017 American Association for Cancer Research.clincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on April 25, 2017; DOI: 10.1158/1078-0432.CCR-17-0103

http://clincancerres.aacrjournals.org/

Statement of translational relevance

While immunotherapy was recently approved as a promising treatment option for patients with

recurrent/metastatic squamous cell carcinoma of the head and neck (SCCHN), its application in

the curative-intent setting is still under investigation. We used pretreatment tumor tissues from

44 patients with locoregionally advanced SCCHN and showed that tumor infiltrating T-cells

clonally expand and that patients with higher GRZB levels have significantly longer recurrence-

free survival independent of tumor, nodal stage or HPV status. Furthermore, HPV-positive

patients had lower degree of T-cell expansion compared to HPV-negative patients. Our data

suggest that this could be secondary to a lower percentage of HPV-positive patients expressing

HLA-A, thus making antigen presentation less efficient in these patients. These data underscore

the significance of the immune system in SCCHN biology and provide rationale for the

introduction of immunotherapy in the curative-intent setting.




Abstract

Purpose: Squamous cell carcinoma of the head and neck (SCCHN) is a lethal cancer with a

suboptimal 5-year overall survival of approximately 50% with surgery and/or definitive

chemoradiotherapy. Novel treatments are thus urgently awaited. Immunotherapy with

checkpoint blockade has emerged as a promising option for patients with recurrent/metastatic

SCCHN, however it has not been investigated in the curative-intent setting yet. The purpose of

this study was to investigate the T-cell receptor repertoire and the tumor microenvironment in

tumor tissues of SCCHN patients with locoregionally advanced disease.

Experimental design: We performed T-cell receptor sequencing of tumor tissues from 44

patients with locoregionally advanced SCCHN prior to treatment with definitive

chemoradiotherapy and correlated the T-cell clonality and the mRNA expression levels of

immune-related genes with clinicopathological parameters.

Results: Clonal expansion of T-cells was significantly higher in HPV-negative compared to

HPV-positive tumors, signifying more robust antigen presentation in HPV-negative tumors. The

latter was supported by the higher percentage of HPV-negative tumors expressing HLA-A

protein compared to HPV-positive tumors (p=0.049). Higher GRZB levels correlated significantly

with longer recurrence-free survival (log-rank, p=0.003) independent of tumor size, nodal stage

and HPV status.

Conclusion: Our findings support clonal expansion of T-cells in SCCHN patients with

locoregionally advanced disease and imply differences in the antigen presentation capacity

between HPV-negative and HPV-positive tumors. Elevated GRZB mRNA levels may also serve

as a favorable and independent predictor of outcome in SCCHN patients treated with

chemoradiotherapy. These data provide rationale for the introduction of immunotherapeutic

approaches in the curative-intent setting.




Introduction

Squamous cell carcinoma of the head and neck (SCCHN) is a lethal cancer which affects

approximately 500,000 people/year worldwide and is the sixth most common cancer in global

incidence1. The 5-year overall survival is approximately 50%2. In the locoregionally advanced

stage, chemoradiotherapy and surgery are the mainstay of treatment, but metastatic and

locoregional relapses are quite common, underlining the need for more effective treatment

strategies to achieve higher cure rates. More recently, immunotherapy has emerged as a

promising treatment option with pembrolizumab being expeditiously approved by the FDA for

recurrent/metastatic SCCHN in August 2016,3 its fourth indication after melanoma, non-small

cell lung cancer and renal cell carcinoma. Nivolumab was also shown to increase 1-year overall

survival to 36% compared to 16.6% with standard chemotherapy in a phase III trial.4 This

efficacy highlights the immunogenic nature of SCCHN and supports that immune-based

therapies are a rational and promising therapeutic platform for SCCHN.

CD8+ T-cell infiltration has been associated with better overall survival and response to

chemoradiotherapy in HPV-positive and HPV-negative patients with locoregionally advanced

SCCHN, though this still remains controversial and further studies are needed to confirm these

findings.5-9 Previous work has shown that CD8+ T-cell infiltration is essential for the response of

tumors to radiotherapy.10-11 Additionally, while radiotherapy alone may induce antitumor immune

responses, preclinical work has demonstrated that it also augments an immunosuppressive

tumor microenvironment through upregulation of PD-L1, and that combination of radiotherapy

with anti-PD-L1 blockade enhances the efficacy of radiotherapy through activation of CD8+ T-

cell-mediated cytotoxicity and a decrease in tumor infiltration by myeloid-derived suppressor

cells (MDSCs).12-14 These data support the notion that stimulation of the tumor immune

microenvironment may optimize tumor responses to radiotherapy. In this context, a recent

study15 evaluated the effects of definitive chemoradiotherapy on immune cell subsets,

chemokines and cytokines in pre- and post-treatment blood samples of 10-15 patients with




various histologies of head and neck cancer. The authors reported increases in blood circulating

CD8+ T-cells and also in the T-cell receptor (TCR) clonality in 2 of 3 patients examined.

Concurrently, an increase in MDSCs, regulatory T-cells (Tregs) and PD-1 positive T-cells was

detected, indicating that chemoradiotherapy induced both immune stimulatory as well as

immune suppressive effects. Although this study did not examine the microenvironment in tumor

tissues from patients with SCCHN, these results suggested that chemoradiotherapy may alter

the tumor microenvironment, and thus integration of immunotherapy with chemoradiotherapy in

the definitive setting would merit further investigation in SCCHN.

In this study, we pursued a comprehensive characterization of the tumor microenvironment in

locoregionally advanced SCCHN by evaluating factors that reflect the inflammatory status of the

tumor microenvironment, such as the TCR repertoire and various immune-related markers.

More specifically, we sought to investigate the pattern of TCR clonality and the expression of

immune-related genes in 44 patients with locoregionally advanced SCCHN prior to treatment

with definitive chemoradiotherapy, and to correlate these with clinicopathological characteristics,

such as HPV status, nodal and tumor stage, recurrence-free and overall survival. Overall, our

data give insight into the clonality of the T-cells infiltrating HPV-positive and HPV-negative

tumors, as well as the association of immune-related genes with survival, and provide rationale

for the integration of immunotherapy strategies in the treatment of locoregionally advanced

SCCHN.

Materials and Methods

Patient characteristics

The examined patient cohort consisted of 58 patients with locoregionally advanced SCCHN who

were previously treated with an organ-preservation approach with or without platinum-based

induction chemotherapy followed by one of the 5-fluorouracil and hydroxyurea (FHX)-based

chemoradiotherapy protocols at the University of Chicago, as previously described16. Of these




patients, 14 were excluded from the analysis due to low CD4 and CD8 mRNA levels compared

to the rest of the cohort. Of the 44 remaining patients, 39 (89%) had participated in (FHX)-based

chemoradiotherapy clinical protocols at the University of Chicago, ensuring the homogeneity of

the treatment received. The remaining 11% of patients received off protocol platinum-based

induction chemotherapy followed by (FHX)-based chemoradiotherapy and did not require

significant dose reductions or treatment interruptions. 36 out of 44 (82%) of these patients

received platinum-based induction chemotherapy followed by (FHX)-based chemoradiotherapy,

while 8 out of 44 (18%) received (FHX)-based chemoradiotherapy only. Table 1 shows the

clinicopathological characteristics of this patient cohort.

Patient samples

RNA was isolated from fresh frozen biopsies of 58 patients with locoregionally advanced

disease previous to treatment with or without platinum-based induction chemotherapy followed

by definitive chemoradiation with one of six 5-fluorouracil and hydroxyurea (FHX)-based

chemoradiotherapy protocols at the University of Chicago. Briefly, fresh biopsies were frozen in

OCT, a section was cut and stained with hematoxylin and eosin (H&E), and then reviewed by a

head and neck pathologist to confirm histology and tumor content. Guided by the H&E slides,

the OCT region with the highest tumor burden (≥60%) was cut from the OCT block for further

processing. RNA was isolated using the AllPrepRNA/DNA/Protein Mini kit (Qiagen, #80204) or

the RNA/DNA/Protein Purification Kit (Norgen Biotek, #47700) using an Ultrasonicator

(Covaris). Samples were obtained from the University of Chicago SCCHN tissue bank (Human

Tissue Resource Center). Use of tissues was approved by the University of Chicago Institutional

Review Board (IRB#8980).

Quantitative RT-PCR




To examine the expression levels of immune-related genes, quantitative RT-PCR from the

tumor and tumor bed cDNAs was conducted using TaqMan Gene Expression assays (Thermo

Fisher Scientific) and ABI ViiA 7 system (Applied Biosystems), according to the manufacturer's

instructions. The TaqMan probes for PD-1, PD-L1, PD-L2, OX40L, OX40, CTLA4, IDO1, B7-H3,

GRZB, PRF1, HLA-A, CD4, CD8, CD206, FoxP3 genes were purchased from Life

Technologies. Samples were run in triplicate and a probe for GAPDH was used for data

normalization.

TCR-β sequencing

The libraries for TCR-β sequencing were prepared using previously described methods.17-21

Briefly, cDNA with 5'-RACE adapter was synthesized. The TCR-β chains were amplified using a

reverse primer specific to the constant region and a forward primer for the SMART adapter.

Illumina sequence adapters with barcode sequence were then added using the Nextera XT

Index kit (Illumina, FC-131-2004). Sequencing was performed by 300-bp paired-end reads on

the Illumina MiSeq platform, using MiSeq Reagent v3 600-cycle kit (Illumina, MS-102-3003).

TCR-β repertoire analysis TCR-β repertoire analysis was performed using Tcrip software. Briefly, sequencing reads were

mapped to the human TCR-β reference sequences using Bowtie2 aligner, and then

decomposed to the V-D-J components of the CDR3s.17 The inverse Simpson’s DI value was

calculated to quantify the clonality of the TCR-β repertoires as previously reported.20,21 To

present the TCR-β repertoire of each sample, we used the Excel program (Microsoft) to

generate bar graphs and pie charts.

Immunohistochemistry in head and neck cancer tissue microarrays




The expression levels of HLA-A in 57 HPV-negative and 25 HPV-positive SCCHN sections were

examined by immunohistochemistry. SCCHN sections were derived from biopsies of patients

with local or locoregionally advanced disease previous to treatment with either surgery with or

without adjuvant chemoradiation, or definitive chemoradiation. Slides of paraffin-embedded

squamous cell carcinoma tumor specimens were deparaffinized, rehydrated and sections were

treated with antigen retrieval buffer (Bond TM Epitope Retrieval 2, AR9640, Leica

Microsystems) in a steamer for 20 min at 96°C. Anti-HLA-A antibody (Abcam ab52922) was

applied on tissue sections for 1h incubation at room temperature. Following TBS wash, the

antigen-antibody binding was detected with the Bond Refine polymer detection system

(DS9800, Leica Biosystems, Wetzlar, Germany). Tissue sections were briefly immersed in

hematoxylin for counterstaining of the nucleus and were covered with cover glasses. An expert

head and neck cancer pathologist and an additional reviewer blinded to clinical outcomes

performed semi-quantitative analysis of HLA-A staining using the following semiquantitative

scoring: tissues with moderate or strong staining in >50% of cancer cells were defined as

positive, and tissues with no staining, weak staining or moderate/strong staining in ≤ 50% of

cancer cells were defined as negative. The tissue samples were acquired with written informed

consent from all the participating patients following the relevant protocol approval by the

University of Chicago Institutional Review Board (IRB 12-2125 and IRB 12-2117). Accordingly,

the above experimental procedure involving patient samples was conducted in accordance with

the approved guidelines by the University of Chicago Institutional Review Board.

Statistical analysis

Gene expression level and TCR-β DI were compared between patient groups using the log-rank

test (Kaplan-Meier curves) and between different clinicopathological characteristics using the

Mann-Whitney U-test (two-tailed). Univariate and multivariate Cox-regression analysis was also




performed. Statistical analysis was carried out using Prism 6 (GraphPad) and R biostatistical

software. P < 0.05 was considered statistically significant.

Results

T-cells can clonally expand in SCCHN tumors.

To assess the TCR repertoire in the tumors of patients with locoregionally advanced SCCHN,

we obtained RNA from the tumors of 58 patients and we first examined CD8 and CD4 mRNA

expression levels. Of these samples, 14 were excluded due to low CD8 and CD4 mRNA

expression levels compared to the rest of the cohort, with 44 patients remaining for TCR-β and

immune-related gene analysis. Subsequently, we performed TCR-β sequencing by amplifying

the TCR-β cDNA using a MiSeq Illumina sequencing protocol. An average of 715,539 ± 61,940

(standard error of mean, SEM) sequence reads mapped to V, D, J and C segments were

obtained for TCR-β for all tumor samples. From these reads, 69,844 ± 7184 (SEM) were found

to be unique TCR-β complementarity determining region 3 (CDR3) clonotypes which are

important for the recognition of a unique antigen on an HLA molecule. The frequencies of the

CDR3 sequences were then calculated and clonal expansion for specific TCR-β CDR3

clonotypes was observed in both HPV-positive and HPV-negative tumors. Representative

examples of clonal expansion are shown in Figure 1A.

To assess the distribution of the TCR-β diversity among SCCHN patients, the TCR-β diversity

index (DI) was calculated as the inverse Simpson’s diversity index (1/Ds), whereby lower TCR-β

DI signifies more clonal expansion. The TCR-β DI among the 44 tumors ranged from 10 to

2,981 with a median value of 220 (Figure 1B).

HPV-negative SCCHN tumors exhibit more clonal expansion and express HLA-A more

frequently compared to HPV-positive SCCHN tumors.




Given their viral causality, we hypothesized that HPV-positive tumors would be more

immunogenic and would thus have more clonal expansion of T-cells directed to HPV-related

epitopes, compared to HPV-negative tumors. Contrary to our expectation, the TCR-β DI was

significantly lower in HPV-negative tumors compared to HPV-positive tumors (Mann-Whitney U-

test, p=0.002) (Figure 2A). This observation remained statistically significant after removal of

heavy smokers (≥10pack-years) from the HPV-positive group and light smokers (<10pack-

years) from the HPV-negative group (Supplementary Figure 1, Mann-Whitney U-test,

p=0.004). The sum of the percentages of the top 10 TCR-β CDR3 clonotypes for each tumor

was also calculated and ranged from 7% to 52% (average=22% ± 9.5%) in HPV-negative

tumors compared to 3% to 37% (average=14.6% ± 10.6%) in HPV-positive tumors and this

difference was statistically significant (Supplementary Figure 2, Mann-Whitney U-test, p=0.01).

The fact that the TCR-β DI was found to be lower in HPV-negative tumors implies that the T-

cell infiltration in these tumors is characterized by greater expansion of T-cell clones compared

to HPV-positive tumors. One explanation for this finding could be that the tumor

microenvironment of HPV-positive tumors and the chronic nature of the HPV-infection may

promote T-cell anergy or exhaustion. However, comparison of the mRNA levels of immune-

related genes associated with T-cell anergy and/or exhaustion, such as CTLA4 and PD-1, did

not reveal any significant differences between HPV-positive and HPV-negative tumors

(Supplementary Figure 3). Additionally, IDO1, PD-L1, PD-L2, CD206, a marker of myeloid

derived suppressor cells, and FOXP3/CD4, a ratio reflecting the presence of T-regulatory cells,

were not statistically different between the two groups (Supplementary Figure 3).

Another possibility that could explain the higher TCR-β DI in HPV-positive SCCHN tumors is

that, in these tumors, recognition of neoepitopes by T-cells may be less efficient due to defects

in HLA expression and/or antigen presentation. Given that the majority of antigens presented by

cancer cells that have been reported are mostly associated with HLA-A alleles22, we chose to

concentrate on HLA-A expression defects. To further assess whether HLA-A expression is




defective in HPV-positive compared to HPV-negative tumors, the protein levels of HLA-A were

compared between HPV-positive (N=25) and HPV-negative (N=57) patients using

immunohistochemistry in a separate cohort of SCCHN patients with locoregionally advanced

disease and available tissue microarrays. Results showed that 44% (11/25) of HPV-positive

tumors were HLA-A positive, compared to 68% (39/57) of HPV-negative tumors, a difference

which was statistically significant (Fisher’s exact test, p=0.049), indicating more frequent HLA-A

defects in HPV-positive SCCHN tumors (Figure 2B). Figure 2C shows representative examples

of HLA-A staining in HPV-positive (Figure 2C (i)) and HPV-negative patients (Figure 2C (ii)).

Correlation of the HLA-A IHC staining with the TCR-β DI was not feasible due to lack of

available RNA in this patient cohort.

Association of TCR-β DI with survival outcomes and other clinicopathological

characteristics.

Potential correlations of the TCR-β DI with recurrence-free and overall survival were examined

in HPV-positive and HPV-negative patients, but no associations were found (Supplementary

Figure 4). Associations of the TCR-β DI with T stage, N stage and tumor localization were also

examined, but no statistically significant differences were observed (Supplementary Figure 5).

High pretreatment levels of GRZB are associated with improved recurrence-free survival

in patients with SCCHN treated with definitive chemoradiotherapy.

Previous work has shown that CD8+ T-cell infiltration is associated with improved survival and

response to chemoradiotherapy of patients with HPV-positive and HPV-negative locoregionally

advanced SCCHN,6-9 however no studies have systematically evaluated the role of the tumor

microenvironment in the clinical outcome of these patients. Given several lines of preclinical

evidence supporting that an active immune response is critical for a successful radiotherapy

response,10 we hypothesized that SCCHN patients with higher levels of intratumoral immune-




related markers would exhibit longer disease-specific recurrence-free survival (RFS) and/or

overall survival (OS) after treatment with chemoradiotherapy.

To investigate this hypothesis, we analyzed the expression levels of CD8, CD4, FOXP3,

CD206, PD-1, CTLA4, OX40, IDO1, PD-L1, PD-L2, OX40L, B7-H3, GRZB (granzyme B), PRF1

(perforin) and HLA-A genes with RT-PCR in tumor samples from the previously described 44

patients with locoregionally advanced SCCHN and examined their association with disease-

specific RFS and OS of these patients. Univariate and multivariate Cox-regression analysis of

each of the immune markers with HPV status, tumor and nodal stage, which are known to affect

RFS and OS in SCCHN, was conducted. Patients with higher GRZB intratumoral levels had

significantly longer RFS compared to patients with lower GRZB levels (log-rank test, p=0.003),

and this relationship remained significant after multivariate analysis for HPV status, tumor and

nodal stage (p=0.032) (Figure 3A). Significantly longer RFS was also observed in patients with

higher levels of OX40 (log-rank test, p=0.008) and PD-L1 (log-rank test, p=0.027), however after

multivariate analysis the significance was lost, though a trend was still observed (p=0.052 for

OX40, p=0.09 for PD-L1) (Figure 3B, 3C). Higher levels of PD-L2 and HLA-A were also

associated with increased RFS with significance or trends to significance observed after

multivariate analysis for HPV status, tumor and nodal stage (p=0.042 for PD-L2, p=0.051 for

HLA-A) (Figure 3D, 3E). Interestingly, PD-L1, PD-L2 and HLA-A were also significantly and

positively associated with levels of GRZB and PRF1, indicating the presence of an active anti-

tumor immune infiltrate and the generation of adaptive immune resistance (Figure 4A, B). A

positive association with GRZB or PRF1 was also observed with B7-H3, CD206, FOXP3 and

CTLA4, further supporting the presence of adaptive immune resistance within SCCHN tumors

(Supplementary Figure 6). Longer RFS was also observed in patients with higher CD8 levels

(p=0.046), PRF1 (p=0.048), CD4 (p=0.052), OX40L (p=0.037) and CTLA4 (p=0.055), but these

associations were lost with multivariate analysis for HPV status, nodal and tumor stage. Results

of multivariate analysis are shown in Table 2. No associations were found between PD-1




(p=0.16), FOXP3 (p=0.20), B7-H3 (p=0.95), IDO1 (p=0.15) and CD206 (p=0.53) andRFS. OS

analysis did not reveal significant associations with any of the examined immune markers after

multivariate analysis (Supplementary Table 1).

These results indicate that increased levels of GRZB portend improved response to

chemoradiotherapy and maintenance of disease remission in SCCHN patients with

locoregionally advanced disease, independent of HPV status, nodal or tumor stage.

Furthermore, expression of PD-L1, PD-L2 and B7-H3, as well as immune suppressive cell

subtypes, such as MDSCs and T-regs, may develop as mechanisms of adaptive immune

resistance to an active immune infiltrate.

Association of intratumoral immune markers with other clinicopathological parameters.

We sought to further assess whether any of the aforementioned immune-related genes are

associated with other clinicopathological parameters, such as tumor size, nodal stage, primary

tumor location and HPV status. Interestingly, we found that higher B7-H3 levels correlated

significantly with high risk T-stage (Mann-Whitney U-test, p=0.009) (Figure 5A), implicating B7-

H3 as a mechanism of immune evasion in SCCHN. Additionally, HPV-positive tumors had

significantly higher levels of OX40 (Mann-Whitney U-test, p=0.037) and a trend towards

increased OX40L levels (Mann-Whitney U-test, p=0.07) (Figure 5B), implying that the

OX40/OX40L pathway may be important in enabling antitumor immune activity in HPV-positive

SCCHN. No other markers showed association with T-stage and HPV status and no other

significant associations were observed between immune-related markers, nodal stage and

primary location.

Association of intratumoral immune markers with TCR-β DI.

We performed additional correlations between the mRNA expression levels of the examined

immune markers and the TCR-β DI in our cohort of 44 patients. Results showed a significantly




positive association between the expression of CD4, CTLA4 and FOXP3 mRNA and the TCR-β

DI (CD4: r=0.44, p=0.0035, FOXP3: r=0.49, p=0.0009, CTLA4: r=0.43, p=0.0038)

(Supplementary Figure 7), signifying that higher numbers of T-regulatory cells may “dampen”

the function of cytotoxic T-cells and reduce clonal expansion of neoantigen-specific T-cells. This

finding is in accordance with multiple studies supporting the importance of T-regulatory cells in

SCCHN biology, with clinical trials actively investigating CTLA4 inhibition not only in

recurrent/metastatic SCCHN, but also in the locoregionally advanced, curative-intent setting23-25.

No significant associations were found between GRZB, PRF1, as well as the remaining immune

markers, with the TCR-β DI.

Discussion

This study is the first to investigate the TCR repertoire and its associations with various

clinicopathological parameters of prognostic significance in patients with locoregionally

advanced SCCHN prior to treatment with chemoradiotherapy. Our data support the pre-

existence of clonal expansion of T-cells in SCCHN tumors prior to treatment with

chemoradiotherapy, signifying the possible presence and recognition of specific neoepitopes by

T-cells. This finding provides a proof-of-concept for the application of immunotherapeutic

strategies that could potentiate and/or unleash the antitumor function of tumor infiltrating effector

T-cells that pre-exist and already recognize neoepitopes in patients with locoregionally

advanced SCCHN.

Another interesting finding of this study is the significantly higher clonal expansion of T-cells in

HPV-negative, smoking-related SCCHN tumors compared to HPV-positive SCCHN tumors. A

possible reason that could explain this finding is that HPV-positive SCCHN tumor cells may

have a defect in their antigen presenting machinery induced by HPV viral proteins, thus evading

recognition by effector T-cells. Indeed, our findings support that a significantly lower percentage




of HPV-positive SCCHN tumors express HLA-A protein compared to HPV-negative SCCHN

tumors, indicating an inherent defect in the expression of HLA-A in HPV-infected SCCHN

cancer cells. In accordance to this finding, previous work has also demonstrated that the HPV16

E7 viral oncoprotein interacts with histone deacetylases (HDACs) 1, 2 and 8 at the promoter

region of MHC class I genes and transcriptionally downregulates their expression in HPV-

infected cervical cancer cells.26 Furthermore, the HPV16 E5 protein has been shown to

decrease the expression of MHC class I proteins through their sequestration in the endoplasmic

reticulum of W12 cervical intraepithelial cells.27 Additionally, Albers et al has demonstrated

decreased protein expression of HLA class I antigens in HPV-positive SCCHN compared to

normal squamous epithelium28, while Tertipis et al has shown that the expression of the antigen

presenting machinery components, TAP2, LMP2 and LMP7, is decreased in HPV-positive

SCCHN cells.29 Another reason that could have explained the higher T-cell diversity in HPV-

positive SCCHN tumors is a more immunosuppressed tumor microenvironment in HPV-positive

tumors that could inhibit antigen-specific T-cell expansion, our data though did not reveal any

statistically significant differences in the expression of immune-related genes between HPV-

positive and HPV-negative SCCHN tumors. Finally, a possibility that could also explain the

lower TCR-β DI in patients with HPV-negative SCCHN is that the neoantigens present in

smoking-related SCCHN may be more immunogenic by binding more strongly to the HLA class

I and/or the T-cell receptor molecules, compared to HPV-related viral antigens. Interestingly,

while the mutation load in HPV-positive SCCHN is similar to that of HPV-negative SCCHN, 16, 30

it could be speculated that the abundance of HPV-related viral antigens may compete with

neoantigens present at potentially lower concentrations within the HPV-infected cancer cells,

thus leading to predominance of presentation of viral epitopes. Furthermore, it cannot be

excluded that smoking-induced mutations, characterized by transversions of purine nucleotides

to other purines and pyrimidine nucleotides to other pyrimidines, may lead to neoepitopes that

have different affinity to HLA class I molecules compared to the ones induced by the recently




described cytosine deaminase activity of the apolipoprotein B editing enzyme catalytic family of

enzymes (APOBEC) which is increased in HPV-positive tumors and is characterized by a

predominance of cytosine to thymidine mutations.30 In addition, it can be hypothesized that

HPV-infected patients may tend to express specific HLA class I molecules that have inherently

lower affinity to HPV-viral epitopes, which could be a mechanism of persistence or immune

evasion of the HPV virus. These hypotheses would merit further investigation as potential

causes of the difference in the TCR-β repertoire diversity between HPV-positive and HPV-

negative SCCHN tumors.

The lack of correlation of the TCR-β DI with survival outcomes could be explained by a

number of reasons. First, only 9 out of the 44 patients had recurrent disease, making the

survival analysis difficult to derive definite conclusions. Second, it cannot be excluded that the

clonally expanded tumor infiltrating T-cell subtypes include both CD8+ and CD4+ T-cells,

potentially also T-regs which may “dampen” an anti-tumor immune response and thus contribute

to worse survival outcomes. Third, even in the presence of clonal expansion of CD8+ T-cells

recognizing robust neoepitopes, the TCR-β DI alone does not take into consideration the

functional effect of the tumor microenvironment on effector T-cells. This is further supported by

the lack of association between GRZB and PRF1 with the TCR-β DI in our patient cohort,

indicating that expansion of effector T-cells does not necessarily imply effective antitumor

cytotoxicity.

Additionally, patients with locoregionally advanced SCCHN with longer RFS expressed higher

pretreatment levels of immune markers, compared to patients with shorter RFS. Specifically,

higher levels of GRZB and PD-L2 were significantly associated with longer RFS in patients

treated with chemoradiotherapy regardless of HPV status, tumor and nodal stage, suggesting

that the pre-existence of an intrinsically inflamed microenvironment enhances

chemoradiotherapy effects. Trends to significance were also found for OX-40, PD-L1 and HLA-

A. This is in accordance with preclinical data showing that the presence of CD8+ T-cells and the




induction of an active immune response is necessary for the antitumor effects of radiotherapy,10

as well as with recent data showing significant prolongation of survival by boosting an immune

response with PD-1 blockade in patients with metastatic SCCHN.4 The trend of a positive

association of HLA-A with longer RFS may also signify more robust antigen presentation,

leading to more effective antitumor inflammation. With the above in mind, it would be a rational

next step to evaluate the pretreatment levels of immune markers, such as GRZB, PD-L1, PD-

L2, OX-40 and HLA-A, as predictors of recurrence in patients with locoregionally advanced

SCCHN after chemoradiotherapy in a larger validation cohort and subsequently in a prospective

setting.

Expression levels of PD-L1, PD-L2 and B7-H3, which are expressed either by cancer or

stromal cells, as well as markers of immune suppressive cell subtypes, such as MDSCs

(CD206) and T-regs (CTLA4, FOXP3), were also found to be significantly and positively

associated with higher GRZB and PRF1 levels, indicating these pathways as potential

mechanisms of adaptive immune resistance in patients with locoregionally advanced SCCHN.

These findings suggest that targeting these mechanisms may be a promising approach to

potentiate the therapeutic effect of chemoradiotherapy in SCCHN.

Furthermore, our data show that higher levels of B7-H3 are significantly associated with high

risk tumor stage (T4). This implies that B7-H3 may be important in the progression of SCCHN to

more aggressive tumor stages by promoting immune evasion and thus warrants further

investigation as a modulator of the immunobiology of head and neck cancer. B7-H3, a member

of the B7/CD28 superfamily, is a potent inhibitor of CD8+ T-cell activation31 and has been

shown to be overexpressed in multiple cancer types, including SCCHN, while its expression in

normal tissues is limited. MGA271 is a humanized monoclonal antibody that is already being

investigated in phase I clinical trials, including patients with SCCHN.32 Furthermore, our findings

also suggest that HPV-positive SCCHN tumors have significantly higher OX40 levels compared

to HPV-negative SCCHN tumors. It is thus possible that HPV-positive patients may benefit more




from OX40 agonists which is currently under investigation in clinical trials for patients with

SCCHN.33-34

Limitations of this study are its hypothesis generating nature and its small sample size,

making larger cohorts necessary to validate these results and expand analysis in HPV-positive

versus HPV-negative patients. Furthermore, our TCR sequencing methodology did not

differentiate between CD4+ and CD8+ tumor infiltrating T-cells, which would require flow

cytometry analysis of fresh tumor samples. Additionally, due to the limited availability of cancer

tissues, we could not correlate the TCR-β DI with HLA-A protein expression in our main cohort

of 44 patients. Also, although HLA-A mRNA levels in our 44 patient cohort showed a trend for

lower expression in HPV-positive patients compared to HPV-negative patients, this was not

statistically significant (p=0.06), making any definite conclusion on the association of HLA-A with

the TCR-β DI precarious (Supplementary Figure 8). Finally, it would be important for future

studies to confirm the aforementioned immune marker correlations at the protein level.

In conclusion, to our knowledge this is the largest study exploring the TCR repertoire and the

immune tumor microenvironment of SCCHN tumors in the locoregionally advanced stage. Our

data suggest clonal expansion of T-cells infiltrating the tumors of patients with locoregionally

advanced SCCHN and provide rationale to introduce immunotherapeutic approaches in the

curative-intent setting, and to pursue the identification of relevant neoantigens. Our group is

currently pursuing the discovery of neoantigens in SCCHN, which could truly alter the treatment

paradigm in the curative-intent setting, either by the introduction of neoadjuvant or adjuvant

approaches through neoantigen-based personalized vaccines or personalized adoptive T-cell

transfer that could potentially enhance chemoradiotherapy effects or reduce recurrence rates. In

HPV-positive tumors, the T-cell infiltrate seems to be more diverse, and this could be secondary

to virally-induced defective expression of HLA class I molecules. Approaches that could restore

the HLA class I expression would thus merit further investigation as a method to increase

response to immunotherapy in HPV-positive SCCHN. Furthermore, locoregionally advanced




SCCHN patients with intrinsically inflamed tumors have longer recurrence-free survival after

chemoradiotherapy treatment, compared to patients who have lower levels of intratumoral

inflammatory markers, with GRZB and PD-L2 reaching statistical significance. These immune

markers could be further explored as predictive biomarkers to select patients with locoregionally

advanced SCCHN who could benefit from immune-boosting treatments prior to

chemoradiotherapy or from de-escalation of curative-intent treatment.

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Figure legends

Figure 1. Clonality of TCR-β in patients with locoregionally advanced SCCHN. (A)

Examples of the distribution of the TCR-β CDR3 unique clonotypes in HPV-negative and HPV-

positive patients. Pie charts depict clonotypes with more than 0.1% frequency in each patient.

Common colors among different pie charts do not represent identical clonotypes. The orange

portion of each pie chart contains clonotypes with less than 0.1% frequency. (B) TCR-β diversity

index (DI) distribution among 44 SCCHN tumors calculated as the inverse Simpson’s index

(1/D). Bars in orange and grey represent HPV-positive versus HPV-negative patients

respectively.

Figure 2. HPV-negative SCCHN tumors exhibit more clonal expansion and express higher

levels of HLA-A compared to HPV-positive tumors. (A) TCR-β DI in HPV-negative versus

HPV-positive patients (N=44, Mann-Whitney U-test, * p=0.002). Y axis is presented in

logarithmic scale. (B) Histogram representing the cumulative results of immunohistochemical

(IHC) analysis for HLA-A in a separate cohort of patients with locoregionally advanced SCCHN

(N=82). The percentage of HPV-negative (N=57) and HPV-positive (N=25) patients that are




HLA-A positive or HLA-A negative by IHC are presented. 68% of HPV-negative tumors were

HLA-A positive compared to 44% of HPV-positive tumors (Fisher’s exact test, p=0.049). (C)

Representative examples of IHC for negative HLA-A staining in HPV-positive (i) versus positive

HLA-A staining in HPV-negative patients (ii).

Figure 3. GRZB predicts recurrence in SCCHN patients treated with definitive

chemoradiotherapy. Recurrence-free survival Kaplan-Meier curves analyzed by log-rank test

for (A) GRZB (p=0.003), (B) OX40 (p=0.008), (C) PD-L1 (p=0.027), (D) PD-L2 (p=0.175), (E)

HLA-A (p=0.15). The median of mRNA values was used to dichotomize patients in high

versus low expressing groups. mRNA levels of immune markers were obtained by RT-PCR

from pretreatment samples of patients with SCCHN who received definitive chemoradiotherapy.

Normalization was performed by GAPDH.

Figure 4. Correlation of PD-L1, PD-L2 and HLA-A levels with GRZB and PRF1 in

pretreatment tumor samples of patients with locoregionally advanced SCCHN. (A) PD-L1,

PD-L2 and HLA-A correlate positively with GRZB (A) and (B) PRF1. Pearson’s correlation

coefficient and p-value was calculated for each association and shown in each graph separately

for each immune marker. mRNA levels were normalized by GAPDH.

Figure 5. Associations of clinicopathological characteristics with immune markers in

SCCHN. (A) B7-H3 is associated with high risk tumor stage (Mann-Whitney U-test, p=0.009).

RT-PCR for B7-H3 in patients low (T1-T3) versus high risk (T4) SCCHN tumor stage. mRNA

levels normalized by GAPDH. (B) HPV positive tumors are associated with higher levels of

OX40 (Mann-Whitney U-test, p=0.037). RT-PCR for OX40 in patients with HPV-positive versus




HPV-negative SCCHN tumors. OX40L also showed a trend for higher expression in HPV-

positive tumors (Mann-Whitney U-test, p=0.07). mRNA levels normalized by GAPDH.




Table 1. Clinicopathological characteristics of 44 patients with locoregionally advanced SCCHN. Clinicopathological Parameters Smoking status1

0-10py >10py

15 (34%) 29 (66%)

Stage1

I II III IVA IVB

0 (0%) 0 (0%) 2 (5%)

37 (84%) 5(11%)

T1

T1 T2 T3 T4

2 (5%)

11 (25%) 13 (30%) 18 (40%)

N1

N0 N1 N2a N2b-c N3 Nx

1 (2%) 5 (12%) 1 (2%)

31 (71%) 5 (11%) 1 (2%)

HPV status1

p16+ p16-

27 (61%) 17 (39%)

Anatomic site Tonsil/Base of tongue Oral cavity Larynx Hypopharynx Pharynx Other

26 (59%) 5 (11%) 5 (11%) 4 (9%) 2 (5%) 1 (2)%

1 Patients with missing data excluded




Table 2. Univariate and multivariate Cox-regression analysis of recurrence-free survival for immune markers and HPV status, tumor and nodal stage.

HPV: Human papilloma virus, T: tumor stage, N: nodal stage.

Log-rank Univariate Multivariate HPV + T + N P-value P-value P-value Hazard Ratio GZRB 0.003 0.019 0.032 0.087 [0.009-0.81] PD-L2 0.175 0.2 0.042 0.185 [0.03-0.94] HLA-A 0.15 0.17 0.051 0.249 [0.06-1] OX40 0.008 0.032 0.052 0.123 [0.01-1] PD-L1 0.027 0.047 0.09 0.255 [0.05-1.23] CTLA4 0.056 0.08 0.126 0.284 [0.05-1.42] OX40L 0.037 0.059 0.13 0.293 [0.05-1.44] PRF1 0.048 0.069 0.155 0.32 [0.06-1.53] CD4 0.052 0.076 0.21 0.358 [0.07-1.8] CD8 0.046 0.07 0.28 0.396 [0.07-2.13]




Published OnlineFirst April 25, 2017.Clin Cancer Res Vassiliki Saloura, Aiman Fatima, Makda Zewde, et al. advanced squamous cell carcinoma of the head and neckimmune microenvironment in patients with locoregionally Characterization of the tumor T-cell receptor repertoire and

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