Oncotarget

Research Papers:

PDCD1 (PD-1) promoter methylation predicts outcome in head and neck squamous cell carcinoma patients

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Oncotarget. 2017; 8:41011-41020. https://doi.org/10.18632/oncotarget.17354

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Diane Goltz, Heidrun Gevensleben, Joern Dietrich, Friederike Schroeck, Luka de Vos, Freya Droege, Glen Kristiansen, Andreas Schroeck, Jennifer Landsberg, Friedrich Bootz, Dimo Dietrich _

Abstract

Diane Goltz1,*, Heidrun Gevensleben2,*, Joern Dietrich3, Friederike Schroeck3, Luka de Vos3, Freya Droege4, Glen Kristiansen2, Andreas Schroeck3, Jennifer Landsberg5, Friedrich Bootz3 and Dimo Dietrich3

1Institute of Pathology, University Hospital Cologne, Cologne, Germany

2Institute of Pathology, University Hospital Bonn, Bonn, Germany

3Department of Otolaryngology, Head and Neck Surgery, University Hospital Bonn, Bonn, Germany

4Ear, Nose and Throat Clinic, University Hospital Essen, Essen, Germany

5Department of Dermatology and Allergy, University of Bonn, Bonn, Germany

*These authors contributed equally to this work

Correspondence to:

Dimo Dietrich, email: dimo.dietrich@gmail.com

Keywords: PD-1, PDCD1, HPV, head and neck squamous cell carcinoma, DNA methylation

Received: March 02, 2017     Accepted: April 11, 2017     Published: April 21, 2017

ABSTRACT

Background: Biomarkers that facilitate the prediction of disease recurrence in head and neck squamous cell carcinoma (HNSCC) may enable physicians to personalize treatment. In the current study, DNA promoter methylation of programmed cell death 1 (PDCD1, PD-1) was evaluated as a prognostic biomarker in HNSCC patients.

Results: High PDCD1 methylation (mPDCD1) was associated with a significantly shorter overall survival after surgical resection in both the discovery (HR = 2.24 [95%CI: 1.08–4.64], p = 0.029) and the validation cohort (HR = 1.54 [95%CI: 1.08–2.21], p = 0.017). In multivariate Cox proportional hazards analysis, PDCD1 methylation remained a significant prognostic factor for HNSCC (HR = 2.14 [95%CI: 1.19–3.84], p = 0.011). Further, mPDCD1 was strongly associated with the human papilloma virus (HPV) status.

Materials and Methods: mPDCD1 was assessed retrospectively in a discovery cohort of 120 HNSCC patients treated at the University Hospital of Bonn and a validation cohort of 527 HNSCC cases analyzed by The Cancer Genome Atlas Research Network.

Conclusions: PDCD1 methylation might aid the identification of HNSCC patients potentially benefitting from a radical or alternative treatment, particularly in the context of immunotherapies targeting PD-1/PD-L1.


<i>PDCD1</i> (<i>PD-1</i>) promoter methylation predicts outcome in head and neck squamous cell carcinoma patients | Goltz | Oncotarget

INTRODUCTION

Head and neck squamous cell carcinoma (HNSCC) is a major cause of death in Western countries accounting for an estimated incidence of 62,000 and 13,000 related deaths in the US in 2016 [1]. Major risk factors comprise exposure to chemical carcinogens such as tobacco and alcohol [2]. In addition, high-risk types of the human papilloma virus (HPV) are estimated to cause one fourth of head and neck cancer cases [37]. In over 90% of HPV-associated HNSCCs, HPV type 16 is identified as the causative agent [8]. Several studies have demonstrated that HPV-positive (HPV+) and HPV-negative (HPV) HNSCCs are separate entities associated with distinct etiology, clinical behaviour, treatment outcomes, imaging, pathological appearance, and molecular profiles [5, 9, 10].

Despite intensive local treatment, HNSCCs generally have an unsatisfactory prognosis due to the high percentage of locoregional tumor recurrence and distant metastasis [11]. As a consequence, these tumors do not only require the standard surgical and radiation treatments but additional effective systemic treatment. During the last couple of years, immunomodulatory therapies have increasingly emerged as a promising new treatment option for advanced malignancies. New insights on the interaction between tumor and host immune response have been particularly focusing on the programmed death-1 receptor (PD-1)/programmed death-1 ligand (PD-L1) pathway as potential therapy target in various tumor entities. Although HNSCC has traditionally been considered to be a very immunosuppressive or at least non-immunogenic tumor type, recent results from clinical studies of immune checkpoint modulating drugs have led to a resurgence of enthusiasm for immunotherapeutic approaches [12]. Currently, a variety of clinical trials and substances for the treatment of HNSCC are underway, primarily focussing on targeting and inhibiting the PD-1/PD-L1 axis [10]. Recently, the PD-1 checkpoint inhibitor pembrolizumab has gained regulatory approval for the treatment of recurrent/metastasized HNSCC [12]. So far, cancer immunotherapy with immune checkpoint modulating drugs seems to be independent of HPV status and may be successful even in PD-L1 low level expressing tumors [12, 13]. Robust predictive markers for patient selection, however, are not yet available [13].

Of note, Lyford-Pike et al. demonstrated that the PD-1 receptor ligand PD-L1 is differentially expressed among HPV+ and HPV- HNSCC [14], corroborating the role of the PD-1/PD-L1 pathway in HPV-related HNSCC immune resistance. While PD-1 receptor ligand PD-L1 has been shown to be expressed in various types of cancers [15], the immune inhibitory receptor PD-1 (also known as CD279 or PDCD1), a member of the extended CD28/CTLA-4 family, is known to be stably expressed only on T cells exposed to a chronic antigen [16, 17]. PD-1 expression has further been shown to be regulated by promoter methylation [18] and to be associated with biochemical recurrence-free survival in prostate cancer patients. Encouraged by these recent findings, we aimed to further complete the insight of the PD-1/PD-L1 pathway’s activity in HNSCC.

RESULTS

PDCD1 promoter methylation in HNSCC patients (discovery cohort)

For the analysis of PDCD1 methylation (mPDCD1) in the discovery cohort, a quantitative methylation-specific qPCR targeting the PDCD1 promoter region (Supplementary Figure 1) was used as recently described [19]. Median mPDCD1 of all HNSCC tumor tissues was 28.6%. The distribution of mPDCD1 levels did not differ across all categories of tumor location (mouth, oropharynx, hypopharynx, and larynx, Table 1A). Women presented with significantly higher mPDCD1 levels compared to men. Analysis of prognostic clinicopathological variables showed no correlation with age at initial diagnosis, tumor grade, pathologic T (pT) and N (pN) categories, or metastasis (Table 2). P16 expression as a surrogate marker for HPV-related HNSCC, however, showed a strong negative association with mPDCD1. Inversely, a history of smoking significantly correlated with mPDCD1 (Table 2).

Table 1A: Association of PDCD1 promoter methylation with clinicopathological data: Discovery cohort

Variable

All Patients

[%]

Median mPDCD1

p-value

mPDCD1low

[%]

mPDCD1high

[%]

p-value

Patient number

120

100

28.6

40

33.3

80

66.7

Sex

 Female

26

21.7

42.2

5

19.2

21

80.8

 Male

94

78.2

27.4

0.019*

35

37.2

59

62.8

0.085Χ

Patients with follow-up

115

95.8

 Mean [Months]

32 [29–34]

 Median [Months]

25

 Range [Months]

0-115

Localization

 Oral cavity

23

19.2

29.0

6

26.1

17

73.9

 Oropharynx

53

44.2

22.8

25

47.2

28

52.8

 Hypopharynx

9

7.5

27.3

3

33.3

6

66.7

 Larynx

35

29.2

32.4

0.15ε

6

17.1

29

82.9

0.026Χ

 Lip

0

0

Age [years]

 Mean

61.9 [59.1–62.8]

 Median

61

 n < Median

61

50.8

27.8

22

36.1

39

63.9

 n > Median

59

49.2

29.7

0.24*

18

30.5

41

69.5

0.52Χ

 Unknown

pT-category

 pTis.pT1/2

56

46.7

26.6

19

33.9

37

66.1

 pT3/4

43

35.8

28.8

0.50*

15

34.9

28

65.1

0.92Χ

 Unknown

21

17.5

Nodal status

 pN0

43

35.8

29.7

11

25.6

32

74.4

 pN1

17

14.2

29.5

4

23.5

13

76.5

 ≥ pN2

60

50

26.2

0.36ε

25

41.7

35

58.3

0.15Χ

 Unknown

0

Tumor grade

 G1

2

1.7

35.6

1

50.0

1

50.0

 G2

64

53.3

28.9

19

29.7

45

70.3

 G3

29

79.2

36.0

0.75ε

9

31.0

20

69.0

0.83Χ

 G4

0

0

 Unknown

25

20.8

Distant metastasis

 cM0

117

97.5

28.8

40

34.2

77

65.8

 cM1

2

1.7

33.1

0.85*

0

0

2

100

0.55ζ

 Unknown

1

0.8

Surgical margin

 R0

86

71.7

28.4

28

32.6

58

67.4

 R1

13

10.8

38.9

0.51*

4

30.8

9

69.2

0.90Χ

 Unknown

21

17.5

HPV (p16)

 p16-negative

64

53.3

37.3

15

23.4

49

76.6

 p16-positive

15

12.5

15.9

0.002*

10

66.7

5

33.3

0.001Χ

 Unknown

41

34.2

Smoking history

 Negative

13

10.8

15.9

8

61.5

5

38.5

 Positive

82

68.3

29.0

0.044*

27

32.9

55

67.1

0.047Χ

 Unknown

25

20.8

8.5

* - Mann-Whitney-Test; e - Kruskal Wallis; c - χ2 –test; ζ – Fisher’s exact test

Table 2: Correlation of PDCD1 promoter methylation with clinicopathological parameters

Discovery cohort

Validation cohort

Variable

Spearman’s ρ

p-value

Spearman’s ρ

p-value

Age

0.053

0.56

0.110

0.012¥

Pathological T category (pT)

0.055

0.59

0.148

0.001¥

Pathological nodal status (pN)

–0.118

0.12

–0.026

0.60

Distant metastasis (cM)

0.019

0.84

0.078

0.081

Tumor grade (WHO 2006)

0.070

0.50

–0.107

0.016¥

HPV association

–0.352

0.001¥

–0.404

< 0.001¥

History of smoking

0.208

0.043¥

0.068

0.13

B lymphocytes

NA

NA

–0.175

< 0.001¥

CD4pos T lymphocytes

NA

NA

–0.118

0.007¥

CD8pos T lymphocytes

NA

NA

–0.095

0.031¥

Macrophages

NA

NA

0.005

0.90

Dendritic cells

NA

NA

–0.073

0.10

P-values and correlation coefficients (Spearman’s r) are shown. PDCD1 methylation is analyzed as a continuous variable

¥ significant feature; NA: not available.

Since differential mPDCD1 was related to PD-1 expression in lymphocytes [18] and epithelial tumors are generally devoid of PD-1 expression [19], we speculated that differential mPDCD1 might reflect PD-1 regulation in the immune compartment. However, the number of cases did not guarantee sufficient power to support this assumption.

After dichotomization, the frequency of mPDCD1low was significantly higher in oropharyngeal SCC compared to all other categories of tumor location (mouth, hypopharynx, and larynx, Table 1A). In the discovery cohort, dichotomized mPDCD1high was significantly associated with shorter overall survival in the univariate Cox proportional hazards model (HR = 2.24 [95%CI: 1.08–4.64], p = 0.029) and Kaplan-Meier analysis (p = 0.025, Figure 1A).

Figure 1:

Figure 1: Kaplan-Meier analysis of overall survival in HNSCC patients in the discovery cohort stratified by PDCD1 methylation status (A). Kaplan-Meier analysis of overall survival in HNSCC patients in the validation cohort stratified by PDCD1 methylation status (B) as wells as stratified by B cell infiltration (C), CD4pos T cell infiltration (D), and CD8pos T cell infiltration (E) in the validation cohort. Patient classification into mPDCD1high and mPDCD1low as well as into cases with low and high immune cell content were based on the lower (mPDCD1) and upper tertile (immune cell infiltrates), respectively.

PDCD1 promoter methylation in HNSCC patients (validation cohort)

Since PD-1 expression has been mainly observed in immune cells [15], differential mPDCD1 seems to reflect changes in the lymphocyte compartment. In the validation cohort, mPDCD1 was related to the content of inflammatory cells in the tumor samples. According to histological data provided by The Cancer Genome Atlas (TCGA) Research Network, tumor samples were on average (mean) composed of 76.3% tumor cells (95%CI: 74.4–78.2%), 3.5% normal cells (95%CI: 2.4–4.5%), and 7.1% tumor infiltrating lymphocytes (95%CI: 6.0–8.3%). Subtypes of tumor infiltrating lymphocytes in the TCGA cohort as assessed by Li et al. [20] were correlated with mPDCD1. Tumor infiltrating B lymphocytes as well as CD4pos and CD8pos T lymphocytes inversely correlated with mPDCD1 (r = –0.175; p < 0.001 for B lymphocytes, r = –0.118; p = 0.007 for CD4pos T lymphocytes, and r = –0.095; p = 0.031 for CD8pos T lymphocytes, n = 514 for all, Table 2).

The expression of the major PD-1 ligand PD-L1, encoded by CD274, is vital for anti-tumor immune tolerance, enabling the tumor cells to escape from T cell attacks. In the series under investigation, promoter methylation of CD274 (mCD274) significantly correlated with mPDCD1 in tumor samples (r = 0.123; p = 0.005; n = 527). Further, mCD274 significantly correlated inversely with infiltrating CD8pos T lymphocytes (r = –0.136; p = 0.002; n = 514).

Clinicopathological correlation (validation cohort)

The analysis of clinicopathological parameters revealed a significant positive correlation of mPDCD1 with age at initial diagnosis and the pT category, while a significant negative correlation was seen with tumor grade (Table 2). P16 expression, as a surrogate marker for HPV association of the tumor, combined with HPV in situ hybridization data obtained by the TCGA network showed a strong association of mPDCD1 with HPV status (Table 2). Regarding immune cell infiltration, localization and tumor grade were associated with varying immune cell densities of all components (Table 1B), whereas the pT category was associated with alterations in B and CD4pos T lymphocyte densities. A positive HPV status was associated with increased B and CD8pos T lymphocytic infiltrates.

Table 1B: Association of PDCD1 promoter methylation with clinicopathological data: Validation cohort

PDCD1 promoter methylation and survival analyses (validation cohort)

Subsequently, we analyzed whether mPDCD1 and the analysis of immune cells allow for a risk stratification of patients. In the univariate Cox proportional hazard model, both continuous values of immune cell content (HR = 0.06 [95%CI: 0.01–0.38], p = 0.003 for B lymphocytes, HR = 0.04 [95%CI: 0.01–0.33], p = 0.002 for CD4pos T lymphocytes, and HR = 0.30 [95%CI: 0.09–0.97], p = 0.045 for CD8pos T lymphocytes) and mPDCD1 (HR = 2.52 [95%CI: 1.58–4.04], p < 0.001) served as strong prognostic factors (Table 3). Accordingly, patients classified as suffering from hypermethylated (mPDCD1high) tumors showed a significantly worse overall survival (HR = 1.54 [95%CI: 1.08–2.21], p = 0.017) compared to patients with hypomethylated (mPDCD1low) tumors. The prognostic value of dichotomized mPDCD1 was further confirmed by Kaplan-Meier analyses (Χ2 = 5.76, p = 0.016 for mPDCD1low and mPDCD1high, respectively; Figure 1B). For Kaplan-Meier analyses of immune cell infiltrates see Figure 1C1E).

Table 3: Univariate and multivariate Cox proportional hazards analyses on overall survival in HNSCC patients treated by radical surgical resection (validation cohort)

Univariate Cox
proportional hazards

Multivariate Cox
proportional hazards*

Variable

n

p-value

Hazard ratio
[95% CI]

p-value

Hazard ratio
[95% CI]

pT (pT3-4vs. pT1-2)

459

< 0.001¥

1.95 [1.35–2.82]

0.010

1.91 [1.17–3.13]

pN (pN2 vs. pN1 vs. pN0)

414

0.001¥

1.45 [1.18–1.79]

0.002

1.41 [1.13–1.76]

Tumor grade (WHO 2006)

499

0.82

1.03 [0.81–1.32]

NA

NA

Distant Metastases (clinical staging, cM1vs. cM0)

521

0.19

1.00 [0.97–1.01]

NA

NA

Surgical margin (R1 vs. R0)

460

0.067

1.48 [0.97–2.26]

NA

NA

HVP status (positive vs. negative)

107

0.53

0.66 [0.18–2.45]

NA

NA

History of smoking (smokers vs. non-smokers)

508

0.28

1.25 [0.84–1.87]

NA

NA

PDCD1 methylation (dichotomized, cut-off 23.1%)

521

0.017¥

1.54 [1.08–2.21]

NA

NA

PDCD1 methylation (logarithmic continuous variable)

521

< 0.001¥

2.52 [1.58–4.04]

0.011

2.14 [1.19–3.84]

B lymphocytes

514

0.003¥

0.06 [0.01–0.38]

0.66

0.48 [0.02–12.11]

CD4pos T lymphocytes

514

0.040¥

0.04 [ 0.01–0.33]

0.040

0.04 [0.00–0.86]

CD8pos T lymphocytes

514

0.045¥

0.30 [0.09–0.97]

0.28

2.68 [0.45–15.86]

Multivariate Cox proportional hazard analysis was conducted including only variables that showed significance in univariate analysis (pT, pN, PDCD1 methylation [logarithmic continuous variable], B lymphocytes, CD4pos T lymphocytes, and CD8pos T lymphocytes). Data for multivariate analysis was available for n = 402 patients.

¥ significant feature; NA: not available.

Since mPDCD1 did not correlate with major clinicopathological parameters, age and pT category excluded, we hypothesized that it might serve as an independent prognostic factor in HNSCC. In multivariate Cox Proportional Hazards analysis, continuous mPDCD1 (HR = 2.14 [95%CI: 1.19–3.84], p = 0.011) and continuous CD4pos T lymphocytic infiltrates (HR = 0.04 [95%CI: 0.00–0.86], p = 0.040) remained independent significant prognostic factors for overall survival when tested together with parameters with significant prognostic value in univariate Cox proportional Hazard analyses (pT,pN categories, B cell, and CD8pos T lymphocytic infiltrates, Table 3).

DISCUSSION

In the present study, we perceived mPDCD1 as a surrogate marker for immune cell infiltration. It was shown to have a considerable impact on the course of HNSCC patients. High mPDCD1 levels, applied as single or combined values, were linked to a significantly shorter overall survival after surgical resection. In univariate and multivariate Cox proportional hazards analysis, both immune cell infiltration and mPDCD1 methylation as continuous variables further served as highly significant prognostic factors in HNSCC, thereof mPDCD1 and CD4pos T lymphocytic infiltrates being independent and as powerful as pT and pN categories.

Aberrant promoter methylation of established or candidate tumor suppressor genes, in addition, has been shown to be essential for HPV-induced carcinogenesis in HNSCC [21], indicating the potential value of methylation as prognostic biomarker in this tumor entity [22]. Accordingly, mPDCD1 was significantly lower in HPV+ HNSCC and in tumors occurring in non-smokers, suggesting a major role in the PD-1 driven adaptive immune resistance in the subgroup of HPV+ HNSCC. While high B and CD8pos T lymphocytic immune cell infiltrates were associated with HPV persistence, neither CD4pos T lymphocytes nor tumor-associated macrophages and dendritic cell infiltrates were related to HPV status. In this context, it may be of importance that HPV+ HNSCCs contain a distinct population of PD-1 high expressing CD8 pos T cells [14], while the frequency of myeloid derived suppressor cells and tumor activated macrophages seems to be independent of HPV infection status in HNSCC [23]; an observation that could be reproduced in the present dataset.

Epigenetic alterations are involved in the regulation of gene expression in key biological processes, i.e. development, differentiation, alternative splicing, and genetic imprinting of various cell types [2426]. It seems reasonable, however, that the differential variation of mPDCD1 was secondary to alterations in the immune cell content in the present study; all the more so since epigenetic PDCD1 promoter control of PD-1 expression has been described for human T lymphocytes [17, 18]. These data are in line with the observation that mPDCD1 is roughly 100% in non-immunogenic cancer epithelium like the prostatic adenocarcinoma [19]. Nevertheless, it may be premature to rule out a tumor-intrinsic role of mPDCD1 in HNSCC. In fact, it must be noted that such associative analysis should be interpreted with caution and used to neither assume nor reject a direct regulation without additional experimental support.

Prolonged viral infections and cancer lead to chronic antigen exposure and can induce high expression levels of PD-1. PD-1 regulates the exhaustion of antigen-specific T cells, and T cells with high PD-1 expression consequently lose the ability to eliminate cancer. (see [15] for rev). In this context, Youngblood et al. reported that the PDCD1 promoter was fully demethylated in antigen-experienced PD-1highCD8pos T cells, whereas methylation levels were significantly lower in antigen-experienced PD-1lowCD8pos T cells [18]. We therefore speculated that the degree of T cell exhaustion may be very well reflected by their methylation levels of the PDCD1 promoter. Since PDCD1 promoter methylation was higher in tumors containing minor amounts of infiltrating lymphocytes and cancers with adverse prognosis, our findings are in line with the observation that tumors with dense lymphocytic infiltrates, like HPV-associated HNSCCs [27], altogether have a favourable course of disease. In addition, CD274 promoter methylation significantly correlated with PDCD1 methylation, suggesting that epigenetic regulation of the PD-1 receptor may be paralleled by PD-L1 induction in tumor tissue.

Not only is the PD1/PD-L1 axis involved in the reduction of immune effector responses in tumors, but it also affects T cell responses in secondary lymphoid tissues, moving the balance from T cell activation towards antigen tolerance. This modulation of the immune system is mediated via regulatory T cells (TRegs), a subpopulation of T cells which maintain self-tolerance and are also found in the immediate vicinity of tumors. Paradoxically, although inhibiting effector T cells, these receptors seem to enhance TReg cell activity or proliferation [15]. In this context, it would be crucial to define thresholds for PDCD1 methylation and consecutive PD-1 expression on T cells that establish anti-tumor immunity. With regards to the limitations of the present study, the application of our approach is generally not sufficient to precisely determine the levels of PDCD1 methylation and PD-1 expression in infiltrating immune cells, i.e. elucidation of PDCD1 in certain T lymphocytic strains, among others CD8pos and TRegs. To constrain the issue more profoundly, a different strategy needs to be adopted, whereby additional observations are collected by tissue digestion and detailed reworking on the microenvironment of the tumor. However, these studies need to be planned prospectively and are subject of our ongoing studies.

Although further mechanistic studies are clearly warranted in order to fully characterize the role of PD-1 expression in HNSCC, our results might imply that the densities of B and CD4pos/CD8pos T lymphocytic infiltrates may be easily estimated measuring mPDCD1 in HNSCC. This finding may be of significance for the future application of immunotherapies in HNSCC patients. Moreover, mPDCD1 might potentially serve as a predictive biomarker for the response to immunotherapies targeting the PD-1/PD-L1 axis.

Recent data obtained in the KEYNOTE-012 expansion cohort have shown that, similar to the initial HNSCC cohort of the trial, a higher response to pembrolizumab was observed in patients with HPV+ versus HPV- HNSCC [12]. However, emerging evidence supports the use of PD-1–targeted therapies to treat both groups of patients [14, 23]. Besides PD-L1 expression on the surface of tumor cells, mutational load, and the intensity of intratumoral CD8pos T cell infiltrates have each been proposed as distinct biomarkers of response to PD1 targeted therapies [13]. For HNSCC, mPDCD1 as a surrogate marker for immune cell infiltration may be of special value, since differential PD-L1 expression has failed to discriminate between patients prone to therapy failure and those with a reasonable response to [12, 28]. The activation of the co-inhibitory checkpoint molecule PD-1 in T lymphocytes and expansion of myeloid derived suppressor cells are considered the major mechanisms for tumors to escape from immune surveillance [29]. However, the latter factors are functionally interrelated and may often be found simultaneously in individual tumors. Therapies targeting the PD-1/PD-L1 pathway have shown excellent results in HNSCC. Reliable biomarkers predicting the response to treatment, however, are still lacking. So far, no data are available on the frequency or significance of promoter methylation regarding this immunomodulatory pathway. Our study is the first to show that PDCD1 methylation predicts the outcome in HNSCC patients, accordingly, potentially aiding the identification of HNSCC patients who might benefit from adjuvant treatment after radical surgical resection, particularly in the context of immunomodulatory therapies. Furthermore, PDCD1 methylation was shown to be associated with a HPV+ status, suggesting a major role of the PD-1 driven adaptive immune resistance in this tumor subgroup.

MATERIALS AND METHODS

Patients, ethics and clinical endpoint

Discovery cohort

Formalin-fixed and paraffin-embedded (FFPE) specimens from 120 patients diagnosed with HNSCC and having undergone surgical resection at the University Hospital Bonn between 1999 and 2013 were retrospectively included in the discovery cohort. Overall survival was considered as the endpoint of the study.

Validation cohort

The data from the validation cohort are based upon data generated by TCGA Research Network: http://cancergenome.nih.gov/. The TCGA cohort comprised fresh-frozen tissues from 527 patients with histologically confirmed HNSCC from several international centers involved in the TCGA project. From 50 patients matched normal adjacent tissues were available.

Clinical Endpoint

Overall survival (OS) was considered as the primary endpoint of the study. OS was censored after 5 years (1825 days). Clinical follow-up was available for 527 individuals.

Ethics

The study was approved by the Institutional Review Board at the University Hospital of Bonn. The TCGA Research Network obtained informed consent (written) from all patients included in the validation cohort. All experiments were performed in accordance with the relevant guidelines and regulations.

Sample preparation (discovery cohort)

For the analysis of mPDCD1, patient samples were processed according to the InnuCONVERT Bisulfite All-In-One Kit (Analytik Jena, Germany) as previously published [30].

PDCD1 quantitative methylation real-time PCR (discovery cohort)

mPDCD1 was determined by means of a methylation-specific real-time PCR assay targeting the PDCD1 promoter region [19]. The methylation-specific real-time PCR assay was duplexed with a second assay targeting a CpG-free region within the ACTB gene locus and allowing for the quantification of the total DNA, irrespective of its methylation [19]. PCR conditions (buffers, temperature cycling program, real-time PCR instrument) were applied as previously described [31]. The following primers and probes were used: PDCD1 forward primer, 5′-tcgaagcgaggttagaaatcgtt-3′; PDCD1 reverse primer, 5′-ccttcaaaaccgaaccgaatat-3′; PDCD1 probe, 5′-6-FAM-ttggcgcggttgtttggtttcgaga-BHQ-1-3′; ACTB forward primer, 5′-cccttaaaaattacaaaaaccacaa-3′; ACTB reverse primer, 5′-ggaggaggtttagtaagttttttg-3′; ACTB probe, 5′-Atto-647N-accaccacccaacacacaataacaaacaca-BHQ-2-3′. Each sample was measured in triplicate with an input of 25 ng of bisulfite-converted FFPE tissue DNA as quantified via UV. As a calibrator 3 ng of bisulfite-converted artificially methylated DNA (CpGenome Universal Methylated DNA, Merck Millipore, Billerica, MA, USA) was used. mPDCD1 was calculated with the ΔΔCT method as described earlier [31]).

Data processing (validation cohort)

TCGA methylation data were created by the TCGA Research Network (http://cancergenome.nih.gov/) using the Infinium HumanMethylation450 BeadChip (Illumina, Inc., San Diego, CA, USA). Methylation values for each bead pair comprised of a variant specific for the methylated and the unmethylated status, respectively, and were calculated by the formula 100*bead_intensity_methylated/(bead_intensity_methylated+ bead_intensity_ unmethylated) as previously described [32]. Data of level 2 from the TCGA Head and Neck Squamous Cell Carcinoma (TCGA-HNSCC) cohort were downloaded from the TCGA webpage. The five beads (cg20805133, cg00795812, cg27051683, cg17322655, cg03889044) located within the upstream CpG-island located in the PDCD1 promoter (Supplementary Figure 1) were selected and the mean methylation value of all five bead pairs from one patient sample was computed. For the quantification of CD274 promoter methylation, bead cg19724470 was chosen [33]. Data on immune cell infiltrates were adopted from [20].

Dichotomization of continuous methylation values and immune-cell infiltration

For detailed clinicopathological correlation and survival analyses, mPDCD1 values as well as quantitative data on immune cell infiltrates were considered as continuous variables and as dichotomized variables to obtain qualitative results. For the dichotomization of DNA methylation values, patients were stratified according to mPDCD1 tertiles (T1–3), in analogy to other three-level graduation systems commonly used in pathologic classifications (e.g. immunohistochemical staining intensity in immunoreactive scores). The cut-off was set between T1 (mPDCD1low) and T2/3 (mPDCD1high) and was 21.4% in the discovery cohort and 23.06% in the validation cohort, respectively. For immune cell infiltrates (B lymphocytes and T lymphocytes), the cut-off was set in an analogous manner between between T1/2 (B celllow, CD4poslow, CD8poslow) and T3 (B cellhigh, CD4poshigh, CD8poshigh).

Statistical analyses

Statistical analyses were performed using SPSS, version 22.0 (SPSS Inc., Chicago, IL). Statements regarding potential correlations of specific histological findings were made using the Spearman correlation coefficient. Comparisons were performed using the Wilcoxon-Mann-Whitney U test, the Kruskal-Wallis test, and the χ2 – test/Fisher Exact test. Survival analyses were performed using the Kaplan-Meier method, and differences between the patient groups were testes by the log rank test. Hazard ratios (HR) were calculated using univariate and multivariate Cox proportional hazards models. Continuous mPDCD1 values were logarithmized to base 2. P-values less than 0.05 were considered statistically significant.

Authors’ contributions

DG, HG, JL, and DD drafted the manuscript, conceived and coordinated the study. JD extracted and processed data from TCGA. FS, LdV, FD, GK, AS and FB provided patient material and data. All authors reviewed the manuscript.

ACKNOWLEDGMENTS

We would like to thank Carsten Golletz, Anna Pehlke, and Susanne Steiner for excellent technical assistance.

CONFLICTS OF INTEREST

A patent on PDCD1 methylation as prognostic and predictive biomarker is pending (inventor: Dimo Dietrich).

REFERENCES

1. Cohen EE, LaMonte SJ, Erb NL, Beckman KL, Sadeghi N, Hutcheson KA, Stubblefield MD, Abbott DM, Fisher PS, Stein KD, Lyman GH, Pratt-Chapman ML. American Cancer Society Head and Neck Cancer Survivorship Care Guideline. CA Cancer J Clin. 2016; 66:203–239.

2. Pelucchi C, Gallus S, Garavello W, Bosetti C, La Vecchia C. Cancer risk associated with alcohol and tobacco use: focus on upper aero-digestive tract and liver. Alcohol Res Health. 2006; 29:193–198.

3. Gillison ML, Koch WM, Capone RB, Spafford M, Westra WH, Wu L, Zahurak ML, Daniel RW, Viglione M, Symer DE, Shah KV, Sidransky D. Evidence for a causal association between human papillomavirus and a subset of head and neck cancers. J Natl Cancer Inst. 2000; 92:709–720.

4. Ang KK, Harris J, Wheeler R, Weber R, Rosenthal DI, Nguyen-Tan PF, Westra WH, Chung CH, Jordan RC, Lu C, Kim H, Axelrod R, Silverman CC, et al. Human papillomavirus and survival of patients with oropharyngeal cancer. N Engl J Med. 2010; 363:24–35.

5. Leemans CR, Braakhuis BJ, Brakenhoff RH. The molecular biology of head and neck cancer. Nat Rev Cancer. 2011; 11:9–22.

6. Zur Hausen H. The search for infectious causes of human cancers: where and why. Virology. 2009; 392:1–10.

7. zur Hausen H. Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer. 2002; 2:342–350.

8. Gillison ML, Castellsague X, Chaturvedi A, Goodman MT, Snijders P, Tommasino M, Arbyn M, Franceschi S. Eurogin Roadmap: comparative epidemiology of HPV infection and associated cancers of the head and neck and cervix. Int J Cancer. 2014; 134:497–507.

9. Slebos RJ, Yi Y, Ely K, Carter J, Evjen A, Zhang X, Shyr Y, Murphy BM, Cmelak AJ, Burkey BB, Netterville JL, Levy S, Yarbrough WG, et al. Gene expression differences associated with human papillomavirus status in head and neck squamous cell carcinoma. Clin Cancer Res. 2006; 12:701–709.

10. Schoppy DW, Sunwoo JB. Immunotherapy for Head and Neck Squamous Cell Carcinoma. Hematol Oncol Clin North Am. 2015; 29:1033–1043.

11. Ang KK, Chen A, Curran WJ Jr, Garden AS, Harari PM, Murphy BA, Wong SJ, Bellm LA, Schwartz M, Newman J, Adkins D, Hayes DN, Parvathaneni U, et al. Head and neck carcinoma in the United States: first comprehensive report of the Longitudinal Oncology Registry of Head and Neck Carcinoma (LORHAN). Cancer. 2012; 118:5783–5792.

12. Chow LQ, Haddad R, Gupta S, Mahipal A, Mehra R, Tahara M, Berger R, Eder JP, Burtness B, Lee SH, Keam B, Kang H, Muro K, et al. Antitumor Activity of Pembrolizumab in Biomarker-Unselected Patients With Recurrent and/or Metastatic Head and Neck Squamous Cell Carcinoma: Results From the Phase Ib KEYNOTE-012 Expansion Cohort. J Clin Oncol. 2016.

13. Topalian SL, Taube JM, Anders RA, Pardoll DM. Mechanism-driven biomarkers to guide immune checkpoint blockade in cancer therapy. Nat Rev Cancer. 2016; 16:275–287.

14. Lyford-Pike S, Peng S, Young GD, Taube JM, Westra WH, Akpeng B, Bruno TC, Richmon JD, Wang H, Bishop JA, Chen L, Drake CG, Topalian SL, et al. Evidence for a role of the PD-1:PD-L1 pathway in immune resistance of HPV-associated head and neck squamous cell carcinoma. Cancer Res. 2013; 73:1733–1741.

15. Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer. 2012; 12:252–264.

16. Francisco LM, Sage PT, Sharpe AH. The PD-1 pathway in tolerance and autoimmunity. Immunol Rev. 2010; 236:219–242.

17. McPherson RC, Konkel JE, Prendergast CT, Thomson JP, Ottaviano R, Leech MD, Kay O, Zandee SE, Sweenie CH, Wraith DC, Meehan RR, Drake AJ, Anderton SM. Epigenetic modification of the PD-1 (Pdcd1) promoter in effector CD4(+) T cells tolerized by peptide immunotherapy. Elife. 2014; 3.

18. Youngblood B, Oestreich KJ, Ha SJ, Duraiswamy J, Akondy RS, West EE, Wei Z, Lu P, Austin JW, Riley JL, Boss JM, Ahmed R. Chronic virus infection enforces demethylation of the locus that encodes PD-1 in antigen-specific CD8(+) T cells. Immunity. 2011; 35:400–412.

19. Goltz D, Gevensleben H, Dietrich J, Ellinger J, Landsberg J, Kristiansen G, Dietrich D. Promoter methylation of the immune checkpoint receptor PD-1 (PDCD1) is an independent prognostic biomarker for biochemical recurrence-free survival in prostate cancer patients following radical prostatectomy. OncoImmunology. 2016; 5:e1221555.

20. Li B, Severson E, Pignon JC, Zhao H, Li T, Novak J, Jiang P, Shen H, Aster JC, Rodig S, Signoretti S, Liu JS, Liu XS. Comprehensive analyses of tumor immunity: implications for cancer immunotherapy. Genome Biol. 2016; 17:174.

21. Lechner M, Fenton TR. The Genomics, Epigenomics, and Transcriptomics of HPV-Associated Oropharyngeal Cancer--Understanding the Basis of a Rapidly Evolving Disease. Adv Genet. 2016; 93:1–56.

22. Kostareli E, Hielscher T, Zucknick M, Baboci L, Wichmann G, Holzinger D, Mucke O, Pawlita M, Del Mistro A, Boscolo-Rizzo P, Da Mosto MC, Tirelli G, Plinkert P, et al. Gene promoter methylation signature predicts survival of head and neck squamous cell carcinoma patients. Epigenetics. 2016; 11:61–73.

23. Yu GT, Bu LL, Huang CF, Zhang WF, Chen WJ, Gutkind JS, Kulkarni AB, Sun ZJ. PD-1 blockade attenuates immunosuppressive myeloid cells due to inhibition of CD47/SIRPalpha axis in HPV negative head and neck squamous cell carcinoma. Oncotarget. 2015; 6:42067–42080. doi: 10.18632/oncotarget.5955.

24. Bestor TH, Edwards JR, Boulard M. Notes on the role of dynamic DNA methylation in mammalian development. Proc Natl Acad Sci U S A. 2015; 112:6796–6799.

25. Jones PA. Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat Rev Genet. 2012; 13:484–492.

26. Lev Maor G, Yearim A, Ast G. The alternative role of DNA methylation in splicing regulation. Trends Genet. 2015; 31:274–280.

27. Badoual C, Hans S, Merillon N, Van Ryswick C, Ravel P, Benhamouda N, Levionnois E, Nizard M, Si-Mohamed A, Besnier N, Gey A, Rotem-Yehudar R, Pere H, et al. PD-1-expressing tumor-infiltrating T cells are a favorable prognostic biomarker in HPV-associated head and neck cancer. Cancer Res. 2013; 73:128–138.

28. Mehra R, Seiwert TY, Mahipal A, Weiss J, Berger R, Eder JP, Burtness B, Tahara M, Keam B, Le DT, Muro K, Geva R, Chung HC, et al. Efficacy and safety of pembrolizumab in recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC): Pooled analyses after long-term follow-up in KEYNOTE-012. J Clin Oncol. 2016; 34:6012.

29. Kim HJ, Cantor H. The path to reactivation of antitumor immunity and checkpoint immunotherapy. Cancer Immunol Res. 2014; 2:926–936.

30. Holmes EE, Jung M, Meller S, Leisse A, Sailer V, Zech J, Mengdehl M, Garbe LA, Uhl B, Kristiansen G, Dietrich D. Performance evaluation of kits for bisulfite-conversion of DNA from tissues, cell lines, FFPE tissues, aspirates, lavages, effusions, plasma, serum, and urine. PLoS One. 2014; 9:e93933.

31. Dietrich D, Hasinger O, Banez LL, Sun L, van Leenders GJ, Wheeler TM, Bangma CH, Wernert N, Perner S, Freedland SJ, Corman JM, Ittmann MM, Lark AL, et al. Development and clinical validation of a real-time PCR assay for PITX2 DNA methylation to predict prostate-specific antigen recurrence in prostate cancer patients following radical prostatectomy. J Mol Diagn. 2013; 15:270–279.

32. Goltz D, Gevensleben H, Grunen S, Dietrich J, Kristiansen G, Landsberg J, Dietrich D. PD-L1 (CD274) promoter methylation predicts survival in patients with acute myeloid leukemia. Leukemia. 2017; 31:738–743.

33. Gevensleben H, Holmes EE, Goltz D, Dietrich J, Sailer V, Ellinger J, Dietrich D, Kristiansen G. PD-L1 promoter methylation is a prognostic biomarker for biochemical recurrence-free survival in prostate cancer patients following radical prostatectomy. Oncotarget. 2016; 7:79943–79955. doi: 10.18632/oncotarget.13161.


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