Oral Oncology xxx (2015) xxx–xxx

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Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology

Immunotherapy for head and neck squamous cell carcinoma Qiao Li, Mark E.P. Prince, Jeffrey S. Moyer ⇑ University of Michigan Comprehensive Cancer Center, 1500 E Medical Center Dr., Ann Arbor, MI 48109, United States

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Article history: Received 2 November 2014 Received in revised form 24 November 2014 Accepted 10 December 2014 Available online xxxx Keywords: Monoclonal antibody therapy Cytokine therapy Cancer vaccines T cell-based therapy Cancer stem cells

s u m m a r y Objectives: To review the current state of immunotherapy of head and neck squamous cell carcinoma. Materials and Methods: Review of the literature with emphasis on clinical trial data. Results: Patients with head and neck squamous cell carcinoma (HNSCC) have long been known to be immunosuppressed. This impairment of the immune system is believed, at least in part, to underlie the poor outcomes in this patient population. Modulating the immune system to improve cancer outcomes is an attractive concept in this difficult to treat population. Conclusion: New studies have started to unravel the mechanisms of immunosuppression and new therapies are being developed to exploit this new information. Ó 2015 Elsevier Ltd. All rights reserved.

Immune defects in HNSCC and immunotherapy Many human malignancies, including head and neck squamous cell carcinoma (HNSCC) are associated with quantitative and qualitative deficiencies in the immune system. Patients with HNSCC have lower absolute lymphocyte counts [1], higher immunosuppressive regulatory T (Treg) cells [2,3], and higher tumorassociated macrophages [4] when compared to healthy controls. These immune defects also include down-regulation of tumor leukocyte antigen expression [5] and spontaneous apoptosis of cytotoxic T-lymphocytes (CTLs) [6] along with the direct inhibition of immune defenses by the tumor secretion of vascular endothelial growth factor (VEGF) [7], prostaglandin E2 [8,9], transforming growth factor(TGF)-ß [10], and interleukin (IL)-10 [10]. Qualitative abnormalities also include impaired natural killer (NK) activity [11,12], impaired maturation of plasmacytoid dendritic cells (DCs) [13], and poor antigen-presenting function [14–17]. With an enhanced awareness of the immune defects present in HNSCC as well as much greater understanding of the basic mechanisms of the immune system in carcinogenesis, rationale therapeutic strategies are being developed to take advantage of this new knowledge. Five broad approaches are gaining popularity in the immunotherapy of HNSCC: (1) monoclonal antibody therapy; (2) cytokine therapy; (3) cancer vaccines; (4) T cell-based therapy, and (5) immunological targeting of HNSCC cancer stem cells.

⇑ Corresponding author at: University of Michigan Medical Center, 1500 E Medical Center Drive, TC 1904, Ann Arbor, MI 48109, United States. E-mail address: [email protected] (J.S. Moyer).

Conventional chemotherapy and radiation-conferred antitumor immune effects There is a greater appreciation for the role of the immune system in the mechanisms of tumor eradication with conventional treatment modalities. Conventional thinking is that cytotoxic chemotherapy and/or therapeutic external beam radiation act directly on the tumor. While certainly true in many instances, a growing body of preclinical and clinical data would suggest that the immune system plays a significant and meaningful role in tumor elimination with these standard treatment approaches. Initially introduced in 1953, the abscopal bystander effect is the regression of tumor outside of the radiation field [18]. Typically seen in melanoma, lymphoma, and renal-cell carcinoma, this effect is believed to be dependent on an intact immune system [19]. Several reports in humans have demonstrated distant tumor responses outside the radiation fields that were correlated with elevated tumor-specific antibodies and T-cell activation [20,21]. Taken together, these observations lend indirect support to the concept that the immune system is involved in chemoradiation. More direct support for the immune system’s involvement in chemoradiation comes from the danger signals released by dying cells. Apetoh et al. [22] describe a pathway in both mice and humans where dying tumor cells release a protein [high mobility-group box 1 (HMGB10)] that can activate the innate and adaptive immune system through Toll-like receptor 4 (TLR4) expressed on dendritic cells (DCs). In this study [22], patients with breast cancer who possess a TLR4 loss-of-function allele recur more quickly after chemoradiotherapy than those carrying the wild-type TLR4 allele.

http://dx.doi.org/10.1016/j.oraloncology.2014.12.005 1368-8375/Ó 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Li Q et al. Immunotherapy for head and neck squamous cell carcinoma. Oral Oncol (2015), http://dx.doi.org/10.1016/ j.oraloncology.2014.12.005

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High dose, or maximally tolerated doses (MTD), chemotherapy is well known to be immunosuppressive through direct effects on the immune system. However, low dose or metronomic chemotherapy may be more immunostimulating than immunosuppressive [23]. The immunostimulatory effects of metronomic chemotherapy include immunogenic cell death by increased antigen presentation [24–26], depletion of immunosuppressive Treg [27–30] and myeloid-derived suppressor cells (MDSC) [31], and enhancement of cytotoxic effector cells [32,33]. The effects of cytotoxic chemotherapy and radiation alone on the immune system make this an attractive approach to use in conjunction with immunotherapeutic strategies. Modulating the tumor microenvironment or the host immune system to be more receptive to systemic immunotherapy has strong support in preclinical models [34–37]. The use of chemotherapy and/or radiation along with immunotherapy in humans will be discussed below. Monoclonal antibody therapies Monoclonal antibody therapy is currently the most widely used form of immunotherapy in cancer patients [38]. Monoclonal antibodies directed against CD20 and HER-2 are standard of care in hematopoietic malignancies and breast cancer, respectively [38]. Epidermal growth factor receptor Monocloncal antibodies targeting the epidermal growth factor receptor (EGFR) are the most common immunotherapy in HNSCC [39]. EGFR leads to proliferation, survival, and metastasis in HNSCC and is mediated by oncogenic signaling along the RAS/MAPK and PI3K/Akt pathways [40]. While 90% of HNSCCs overexpress EGFR [41], high levels of EGFR do not predict response to cetuximab [42,43] and EGFR pathway inhibitors (intracellular tyrosine kinase) are not associated with meaningful clinical responses [44,45]. These observations argue for additional mechanisms of antitumor activity rather than just the inhibition of the signaling cascade with EGFR activation. Innate and adaptive immunity including antibody-dependent cellular cytotoxicity (ADCC) appear to have a significant role in the mechanism of action of cextuximab [46]. Cetuximab is the first and best studied monoclonal antibody against EGFR and is a chimeric IgG1 isotype containing a human constant region and a mouse variable region. Bonner et al. [47] studied 424 patients with HNSCC that were randomized to either radiation alone or radiation and cextuximab and found a significant increase in overall survival from 29 months to 49 months with the addition of cextuximab. Much of this survival advantage was seen in oropharyngeal patients where HPV status may have played a role in treatment efficacy. Nimotuzumab is also a human/mouse chimeric monoclonal antibody targeting EGFR and has been studied in several Phase II clinical trials of patients receiving definitive chemoradiation [48,49]. In both studies of advanced-stage HNSCC, the addition of nimotuzumab conferred a significant survival advantage. Panitumumab, a fully humanized monoclonal antibody specific to EGFR, has also been studied in HNSCC with less demonstrated efficacy. The SPECTRUM trial was a Phase 3 trial in 657 patients with recurrent or metastatic HNSCC comparing cisplatin and fluorouracil with and without panitumumab. The trial did not show improved overall survival but did demonstrate improved progression-free survival [50]. These finding are in contrast to the EXTREME study where overall survival was improved in patients who received cetuximab in addition to cisplatin or carboplatin and fluorouracil [51]. While one explanation for the difference between the studies may be study methodology, the fact that panitumumab is a fully humanized IgG2 isotype may be relevant.

Unlike cetuximab, panitumumab does not induce NK-DC cross priming [52]. It is unclear whether the different isotype of panitumumab, the lack of murine variable region, or other factors are responsible for the effect difference between panitumumab and cetuximab despite both binding to EGFR. However, one likely explanation is the difference in the immunogenicity (mouse versus human variable regions) between these different monoclonal antibodies. Vascularized endothelial growth factor Elevated pretreatment serum vascularized endothelial growth factor (VEGF) levels tend to indicate a more aggressive disease state and a poorer overall survival in advanced laryngeal carcinoma [53]. In a meta-analysis of 12 studies that examined VEGF levels in over 1000 HNSCC patients, tumors that overexpressed VEGF measured by immunohistochemistry were associated with 1.88 greater risk of death at 2 years [54]. Several studies have examined the use of bevacizumab in HNSCC. Bevacizumab is a recombinant humanized IgG1 monoclonal antibody that binds VEGF-A and was the first agent against this protein approved by the Food and Drug Administration. Fury et al. [55] undertook a phase 2 trial of bevacizumab with cisplatin and intensity-modulated radiation in advanced HNSCC that demonstrated a 2-year overall survival (OS) rate of 88% and progression-free survival (PFS) rate of 76%. Bevacizumab was also studied with standard radiation therapy and docetaxol with encouraging results. In 30 patients with over 3 years median follow-up, OS and PFS were 62% and 68%, respectively [56]. The combination of cetuximab and bevacizumab has also been investigated in recurrent or metastatic HNSCC. Forty-six patients were enrolled in this Phase 2 study that demonstrated a median PFS interval of 2.8 months and OS of 7.5 months in a patient population that was highly resistant to standard therapy (73% of patients recurred within 6 months of prior curative therapy) [57]. Despite the decrease in VEGF levels in patients treated with bevacizumab, the actual VEGF levels post-treatment have not been shown to be consistently associated with efficacy. Similar to cetuximab, bevacizumab has been shown, however, to have significant impacts on immunological parameters that are distinct from the VEGF pathways. In metastatic melanoma and ovarian cancer, treatment with bevacizumab is associated with significant increases in CD8+ effector cells [58,59] and a reduction in circulating T reg cells [58]. It would be reasonable to conclude that similar mechanisms may be present in HNSCC. Immune checkpoint blockade The immune response is a carefully orchestrated balance between potentiation and inhibition. While the lack of potentiation may be responsible for anergy and tumor progression, the lack of immune inhibition can result in autoimmunity and an unchecked immune response. In cancer immunotherapy, however, where immune suppressive mechanisms are elevated, pathways that limit the breaks on the immune system can be exploited. One pathway where this interaction is important in tumor formation is the stimulatory B7/CD28 pathway of antigen presenting cells and T cells and the inhibitory effects of CTLA4 and PD-1 on activated T cells that act as a break on this response. Monoclonal antibodies that block CTLA4 and PD-1 and release the breaks on the immune response have been used with success in several clinical trials. In 2010, ipilimumab became the first FDA-approved monoclonal antibody that blocks the inhibitory CTLA4 pathway. This approval was based on a randomized trial where ipilimumab alone was found to offer a survival benefit for patients with metastatic melanoma [60]. Long-term survival beyond 2 years was seen in

Please cite this article in press as: Li Q et al. Immunotherapy for head and neck squamous cell carcinoma. Oral Oncol (2015), http://dx.doi.org/10.1016/ j.oraloncology.2014.12.005

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18% of treated patients suggesting that this therapeutic approach could potentially have long-lasting immune effects after a relatively short course of treatment [61]. This approach would seem rationale in HNSCC given the fact that Tregs in the tumor microenvironment express high levels of CTLA4 [62,63]. A Phase 1b study of locally advanced HNSCC at the University of Pittsburgh School of Medicine examining ipilimumab with standard therapy (IMRT and cetuximab) is ongoing and has enrolled 3 patients as of January 2014 (NCT01935921) [40,46,64]. Whereas CTLA4 primarily limits effector T cell activation, the major role of PD1 is to limit T cell activity in the inflammatory immune response and to regulate autoimmunity [61]. PD1 is more broadly expressed than CTLA4 and is present on activated T cells, NK cells, B lymphocytes, monocytes, and DCs [61]. The ligand for PD1, PDL1, is found on the tumor surface of many solid tumor malignancies including HNSCC [65–69]. PDL1 is also found on myeloid cells in the tumor microenvironment [70–72]. Monoclonal antibodies to PD1 and PDL1 are currently being developed by various companies. In the first Phase 1 clinical trial of nivolumab, a fully humanized PD1 antibody (colon, renal, lung cancer, and melanoma), there were noted to be partial responses and a complete response that continued more than a year from the cessation of therapy [61,73]. Importantly, positive responses were correlated with increasing PDL1 expression on tumors [61]. Based on this early work, a multicenter, open-label, randomized study with pembrolizumab in patients with unresectable or metastatic melanoma showed significant benefit leading to accelerated approval by the FDA in September of 2014. Similarly, in a large Phase 3 study in metastatic melanoma of nivolumab compared to dacarbazine, nivolumab was associated with significant improvements in OS and PFS [74]. Seiwert et al. reported on toxicity data from pembrolizumab in patients with HNSCC and found that the medication is well tolerated in this patient population with few serious adverse events (NCT01848834) [75]. There are several trials with either pembrolizumab or nivolumab in HNSCC currently recruiting study participants. A Phase 3 study investigating nivolumab versus standard therapy in recurrent or metastatic HNSCC is ongoing (NCT02105636). Primary endpoints are PFS and OS with an estimated final data collection date in June 2016. A Phase 2 study examining pembrolizumab in recurrent or metastatic HNSCC after treatment with platinum-based and cetuximab therapy recently opened in October 2014 (NCT02255097). Primary outcomes to be evaluated are objective response rate (ORR) by RECIST. There are no definitive data yet available in HNSCC for CTLA4 and PD1 monoclonal antibody therapies. However the preclinical data and preliminary clinical data in other solid tumor malignancies are encouraging. CTLA4 or PD1 blockade will likely be more effective if combined with other immunologic modulating therapies, including standard treatment modalities, rather than used alone. These studies are an active area of interest for many investigators.

Cytokine therapy Cytokine-based immunotherapy has been used both locoregionally and systemically with limited success in HNSCC, albeit with significant toxicities in some patients. IL-2 is perhaps the most studied and has met with the most successes. The main function of the proinflammatory cytokine IL-2 is to promote antigen-specific clonal expansion of T cells as well as potentiate non-specific NK and LAK cell activity. Complete or partial responses have been reported with IL-2 therapy in HNSCC patients [76]. Peritumoral injections of IL-2 have been shown to increase T-cell cytotoxicity within the tumor microenvironment as well as increase CTL and NK cells in

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the peripheral blood of unresectable HNSCC patients [77]. Consistent with this observation, a Phase 3 randomized study of oral cavity and oropharynx SCC patients treated with perilymphatic IL-2 and surgery followed by radiation had a greater than 25% improvement in overall survival at 5 years [78]. In contrast, a Phase II trial of systemic IL-2 in refractory nasopharyngeal carcinoma was ineffective [79]. The difference in outcomes between these two studies could potentially be related to the mode of delivery, with systemic delivery resulting in more toxicity and/or less efficacy than perilymphatic delivery of IL-2. Low-dose perilymphatic IL-2 has also been used with success in combination with other cytokines. IRX-2 is a cell-derived, cytokine mixture of physiologic levels of IL-1, IL-2, IL-6, IL-8, tumor necrosis factor-alpha, interferon-gamma, G-CSF, and GM-CSF that is produced by stimulating human PBMCs with phytohemagglutinin under GMP conditions [80]. In combination with the immuneadjuvants cyclophosphamide and indomethacin, a Phase II study of IRX-2 in 27 previously untreated, resectable patients with Stage II-IV HNSCC showed promising results when given preoperatively. Tumor responses were seen in 16% of patients, and 74% of patients had either decreased or stable tumor size prior to definitive surgery [81]. Importantly, significant increases in tumor and regional lymphatic lymphocytic infiltration were observed in the IRX-treated cohort. Improved survival at 5 years was correlated with high overall lymphocyte infiltration in the tumor specimens [82]. Currently, a large multi-site international Phase 3 study is underway to validate these findings. Other single agent cytokines have been used in HNSCC patients, but on a limited basis with little clinical trial data available. A small Phase1/2 study of interferon-gamma was undertaken in 8 patients. Immunologic responses were evident in almost 50% of the patients with clinically measurable responses in 3 patients and stable disease in 4 patients [83]. Interferon-alpha has also been used in several small series in combination with cisplastin/5-fluorouracil [84], isotretinoin/vitamin E [85], and IL-2 [86] with mixed results. Interleukin-12 was studied in 10 patients with untreated HNSCC [87]. NK and B cell infiltration was increased in tumors and higher NK levels (CD56+ cells) of HNSCC patients, irrespective of IL-12 treatment, had a better overall survival than those with a low number.

Therapeutic cancer vaccines The goal of cancer vaccines is to generate clinically meaningful tumor regression through the activation of the adaptive immune system with the presentation of tumor–associated/specific antigens (TAA/TSA) in the context of a peptide–major histocompatibility complex (MHC) for T-cell recognition or on the surface of the tumor cell for B-cell recognition [88]. TAAs are also present on normal tissues, in contrast to TSA which are unique to the tumor, and therefore tend to be weekly immunogenic. Cancer vaccines seek to break this tolerant phenotype through several different vaccine strategies that have been tried in Phase 2 and 3 clinical trials: presentation of peptides or proteins with adjuvant, recombinant viruses, bacteria or yeast, whole tumor cells, or delivery of proteinor peptide-activated dendritic cells (DCs) [88]. The majority of clinical studies have been performed in melanoma, glioma, as well as prostate, colorectal, and pancreatic carcinoma as well as in HNSCC. DC vaccines take advantage of the potent antigen presenting capacity of DCs to stimulate primary and secondary T-cell and Bcell immune responses. In HNSCC, targeting mutated proteins in the p53 pathway has been an area of active investigation given the prevalence of these mutations in this patient population. A Phase 1b clinical trial of 16 patients with mostly advanced HNSCC (14/16) were treated with 3 interval injections of dendritic cells pulsed with wildtype (wt) p53 peptides [89]. There was no

Please cite this article in press as: Li Q et al. Immunotherapy for head and neck squamous cell carcinoma. Oral Oncol (2015), http://dx.doi.org/10.1016/ j.oraloncology.2014.12.005

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observed Grade 2–4 toxicity and 2-year disease free survival (DFS) was 88%. Importantly, only 4/16 patients had increased IFNgamma when presented with p53 peptide in Elispot assays, but p53-specific T-cell frequencies were observed in 11/16 patients. Interestingly, immune-inhibitory regulatory T (Treg) cells were decreased in vaccinated patients. Human papilloma virus (HPV) has been another target of cancer vaccine strategies in HNSCC. HPV is thought to be important in the pathogenesis of oropharyngeal SCC and is found in the majority of tumors of this subtype [90]. The oncoproteins E6 and/or E7 have been targets in both cervical malignancies as well as HPV+ HNSCC. A feasibility study of a peptide vaccine against HPV16 peptide was performed in 5 patients using a Trojan vaccine [91]. Four patients had a measurable immune response to relevant HLA-II peptides but not to HLA-I restricted peptides. Larger studies targeting E6/ E7 have been performed in cervical [92] and vulvar [93] intraepithelial neoplasia with encouraging clinical results. Adoptive T cell immunotherapy of HNSCC Adoptive T cell immunotherapy is a technique that involves the administration of externally manipulated T cells that have been derived from tumor infiltrating lymphocytes (TIL), ex-vivo modified T cells with engineered T cell receptors, or chimeric antigen receptor that links the T cell response with an external antibody to relevant antigen [38]. This approach was studied in six patients with HNSCC at the University of Michigan. Activated vaccineprimed lymph node (VPLN) cells were administered in conjunction with IL-2. Anti-CD3-activated VPLN cells secreted IFN-gamma and GM-CSF in response to autologous tumor cells but no significant tumor responses were noted. One of the main limitations of this approach, along with other vaccine approaches, has been the low frequency of tumor-reactive T cells generated in treated patients. Rosenberg and his colleagues have potentially found a technique to bypass this problem [94]. Using whole-exomic sequencing, they identified a point mutation that can be recognized by a Th1 population within the TIL and this population is expanded ex-vivo and adoptively transferred back to the patient. Tumor regression was observed for over 6 months along with the identification of polyfunctional Th1 cells in the peripheral blood. Immunological targeting of HNSCC cancer stem cells One promising area is targeting tumor-initiating or cancer stem cells (CSCs). CSC population in HNSCC is linked to treatment failure, recurrence and metastasis [95]. Work originally performed by Clarke et al. revealed the existence of a small subpopulation of highly tumorigenic cells in breast cancer that were given the label cancer stem cells [96]. Following this report, our group demonstrated that the cell surface marker CD44 could be used to identify the highly tumorigenic population of cells from HNSCC [97]. Subsequently, we showed that high levels of aldehyde dehydrogenase (ALDHhigh) activity is an even more specific marker for the CSC population in HNSCC [98]. Importantly, in vitro CSC-primed T cells have been shown to specifically target CSCs in vivo. CSC-specific CD8+ T cells were generated from human acute myeloid leukemia (AML) stem cells in 1999 by Bonnet et al., and have been proven to mediate tumor regression after injection into NOD/SCID mice [99]. In addition, Brown and colleagues isolated CD133+ brain tumor stem cells (BTSCs), and demonstrated that these BTSCs are susceptible to perforin-dependent CTL-mediated cytolysis [100]. In a more relevant study, Visus and colleagues generated aldehyde dehydrogenase 1 family member A1 (ALDHA1)bright CSC-specific CD8+ T cells by using antigenic peptide from ALDH1A1. They demonstrated that transfer of ALDH1A1-specific CD8+ T cells

eliminated ALDHA1bright cancer stem cells from head and neck squamous cell carcinoma, inhibited tumor growth and metastases, and prolonged survival of xenograft-bearing immunodeficient mice [101,102]. Our group recently generated an anti-CD3/antiCD133 bispecific antibody (BsAb) and bound it to the cytokineinduced killer (CIK) cells as effector cells (BsAb-CIK) to target CD133high CSCs. The killing of CD133high cancer stem cells by the BsAb-CIK cells was significantly higher than the killing by the parental CIK or by CIK cells bound with anti-CD3 (CD3-CIK) without CD133 targeting both in vitro and in vivo [103]. Together, these studies suggest that potential CSC-specific T cells can be generated in vitro for subsequent adoptive transfer into tumor-bearing hosts to target CSCs in vivo and eradicate tumors including HNSCC. Specifically targeting cancer stem cells with a DC vaccine is also an innovative and rationale approach that could specifically address the most replicative and therapy-resistant subpopulation of cancer cells. Utilizing a murine squamous cell carcinoma tumor model (SCC7), we recently reported that the administration of dendritic cells pulsed with ALDHhigh tumor cells (the cancer stem cells) is significantly more effective as an antigen source in inducing protective anti-tumor immunity than whole tumor lysate or the nonCSC cell population [104]. We have also demonstrated similar findings in vitro when human HNSCC tumors are treated with autologous DC pulsed with cancer stem cells (manuscript under review). It is becoming increasingly clear that cancer vaccines for the majority of solid tumor malignancies, including HNSCC, will be the most beneficial in limited disease settings as either a neoadjuvant or adjuvant treatment rather than a stand alone therapy. Adding more T cells or APCs to an immunosuppressed systemic host or tumor microenvironment will unlikely show success as a single modality therapy. Combinatorial approaches will be needed along with cancer vaccines including reversing immunosuppression, chemoradiotherapy, or other synergistic therapies. Conclusion The role of the immune system in the initiation and progression of carcinogenesis in HNSCC is only beginning to be understood. Immunosuppression in HNSCC has been known for many years, but the exact pathways are only now being elucidated. Taking advantage of this new information will undoubtedly allow clinicians to better care for this population by exploiting these pathways. Future clinical trials in HNSCC will need to consider immunotherapeutic strategies in conjunction with either standard approaches and/or other innovative therapies to achieve the best outcomes for this difficult to treat population. Conflict of interest statement The authors have no conflicts of interest as it relates to the information and/or data in this review manuscript. References [1] Kuss I, Hathaway B, Ferris RL, Gooding W, Whiteside TL. Imbalance in absolute counts of T lymphocyte subsets in patients with head and neck cancer and its relation to disease. Adv Otorhinolaryngol 2005;62:161–72. [2] Schaefer C, Kim GG, Albers A, Hoermann K, Myers EN, Whiteside TL. Characteristics of CD4+CD25+ regulatory T cells in the peripheral circulation of patients with head and neck cancer. Br J Cancer 2005;92:913–20 [see comment]. [3] Strauss L, Bergmann C, Szczepanski M, Gooding W, Johnson JT, Whiteside TL. A unique subset of CD4+CD25highFoxp3+ T cells secreting interleukin-10 and transforming growth factor-beta1 mediates suppression in the tumor microenvironment. Clin Cancer Res 2007;13:4345–54. [4] Li C, Shintani S, Terakado N, Nakashiro K, Hamakawa H. Infiltration of tumorassociated macrophages in human oral squamous cell carcinoma. Oncol Rep 2002;9:1219–23.

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