The immune system and head and neck squamous cell carcinoma: from carcinogenesis to new therapeutic opportunities.

Immunol Res (2013) 57:52–69 DOI 10.1007/s12026-013-8462-3

IMMUNOLOGY & MICROBIOLOGY IN MIAMI

The immune system and head and neck squamous cell carcinoma: from carcinogenesis to new therapeutic opportunities Monika E. Freiser • Paolo Serafini • Donald T. Weed

Monika E. Freiser Paolo Serafini Donald T. Weed

Published online: 12 November 2013 Ó Springer Science+Business Media New York 2013

Abstract Head and neck squamous cell carcinomas (HNSCCs) exhibit complex interactions with the host immune system that may simultaneously explain resistance to various therapeutic modalities and that may also provide opportunities for therapeutic intervention. Discoveries in immunologic research over the last decade have led to an increased understanding of these interactions as well as the development of a multitude of investigational immunotherapies. Here, we describe the interaction between HNSCC and the immune system, including a discussion of immune cells involved with tumor carcinogenesis and the role of immune-modulating factors derived from tumors. We also describe the current immunotherapeutic approaches being investigated for HNSCC, including a discussion of the successes and limitations. With this review, we hope to present HNSCC as a model to guide future research in cancer immunology. Keywords Head and neck squamous cell carcinoma Immunosuppression Immunotherapy Tumor vaccines Antibody therapies Human papilloma virus

Introduction Head and neck squamous cell carcinomas (HNSCCs) represent about 95 % of tumors originating from the epithelium of the upper aerodigestive tract and globally account for the sixth most common malignancy [1]. Over 634,000 new cases were reported worldwide in 2008, and the case P. Serafini (&) Department of Microbiology and Immunology, University of Miami Miller School of Medicine, RMSB 3075, 1600 NW 10th Avenue, Miami, FL 33136, USA e-mail: [email protected] D. T. Weed (&) Division of Head and Neck Surgery, Department of Otolaryngology, University of Miami Miller School of Medicine, 1475 NW 12th Avenue, 3rd Flr, Suite 3550, Miami, FL 33136, USA e-mail: [email protected] M. E. Freiser University of Miami Miller School of Medicine, 1475 NW 12th Avenue, 3rd Flr, Suite 3550, Miami, FL 33136, USA e-mail: [email protected]

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fatality rate remains high, with about 56 % of patients dying from their disease [2]. This speaks to the limitations in treatment efficacy of the currently available standard of care treatment options employing combinations of surgery, radiotherapy, and chemotherapy. These limitations highlight a need for new therapeutic modalities and approaches in HNSCC, a need that is being met by an expanding role for immunotherapy as a fourth treatment option for this disease. Much has been discovered about the interaction of the immune system and HNSCC tumors, both in the development of the disease and in the mechanisms of tumor resistance. Understanding this interaction is essential to developing successful immunotherapeutic interventions.

Interaction between HNSCC and the immune system The immune system is intimately involved in the process of HNSCC carcinogenesis (Fig. 1a). Ideally, when an epithelial cell acquires a deleterious mutation that cannot be repaired, it should undergo apoptosis [3]. When this fails, the immune system should recognize the cell as abnormal

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Fig. 1 The interaction between immune system and HNSCC. a Anatomical location of HNSCC. The micro- and macro-environment of HNSCC is characterized by different immunological components that include lymph nodes, tonsils, resident immune cells of the mucosa (i.e., Langerhans cells), and immune-stimulatory elements (i.e., oral bacteria). b Schematic diagram of the interactions between HNSCC and the immune system. Langerhans cells and resident myeloid DCs or DCs infiltrating the mucosa uptake the tumor antigens (1) and migrate (2) to the draining lymph nodes to crosspresent them to the effector T cells (3). Once activated, T cells move

back to the tumor where they exert their tumoricidal action (4). To counteract the immune surveillance, neoplastic cells are edited to be ignored, to directly inhibit T-cell activity, or to secrete tumor-derived factors (TDF) that inhibit DC migration to the lymph nodes (5a) and alter the myelopoiesis (5b) inducing the generation of MDSCs and Tregs. These cells inhibit the immune surveillance (6) by inhibiting DC maturation and migration, by preventing T-cell expansion, by directly inhibiting the effector function of T cells or by promoting T-cell anergy

and attack it accordingly; this concept of immune system recognition and elimination of transformed cells is known as immunosurveillance, first introduced in 1970 by Burnet and Thomas [4, 5]. However, there is a failure of this paradigm with all clinically evident cancers, and growing evidence demonstrates that certain immune cells may in fact promote the development and survival of neoplastic cells. Perhaps the most basic evidence of a relationship between HNSCC and the immune system is that immunodeficiency increases HNSCC risk [6]. For example, premalignant leukoplakia develops in 13 % of renal transplant patients as compared to 0.6 % of age- and sexmatched individuals, and about 10 % will become frank HNSCC [7, 8]. Increased incidence of HNSCC has been observed in bone marrow transplant patients [9–11] and HIV-positive patients [12]. While HNSCC is not an AIDSdefining illness, HIV-positive patients develop HNSCC earlier in life with more advanced disease and poorer prognosis [13]. As significant as immunodeficiency may be in HNSCC risk elevation, most patients have normal immune systems when the cancer develops. In the immunocompetent patient, the process to blame is immunoediting [14].

Immunoediting selects for tumor cells Immunoediting is a concept first introduced by Dunn and Schreiber in 2002 [5, 15] that describes how tumor cells develop in the setting of a healthy immune system. Cancer immunoediting is composed of three processes: elimination, equilibrium, and escape. Immunosurveillance occurs during the elimination process, whereby the immune system recognizes transitioned cells and induces cell death through various mechanisms. Most preclinical lesions are likely cleared this way [15]. Of note, aerodigestive epithelial cells encounter foreign particles and bacteria more often than many tissues, and yet most patients do not suffer from perpetual mucosal inflammation. This natural tendency for tolerance may predispose to tolerance toward transitioned cells [16]. Those transitioned cells that are capable of surviving initially enter into the equilibrium phase, whereby Darwinian pressures continue to select for the tumor cells that develop mutations allowing continued survival. The escape process occurs once a tumor cell has the means to efficiently overcome the immune system. These three processes may co-exist in a collection of tumor cells. By the time a tumor becomes clinically evident, immunoediting has already selected for the cells that are

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best suited to survive in an immunologically intact environment. Mechanisms of immune system escape There are four main ways that selective pressures during the equilibrium process are thought to lead to HNSCC escape in immunocompetent patients: tumor cell evasion of detection, tumor resistance to attack, direct tumor cell inhibition of local immune function, and indirect inhibition of immune function by way of recruitment of immunosuppressive players [16]. While evidence exists supporting all four, it is now clear that selection of malignant cells with direct or indirect intrinsic immunosuppressive activity is particularly common. HNSCC cells evade immune system detection Head and neck squamous cell carcinoma cells may trick the immune system into disregarding them as normal cells. This can be achieved by decreasing the expression of surface MHC class I molecules [17] or by impairing their antigen processing machinery [16]. HNSCC may also lose immune-dominant epitopes during immunoediting [6]. However, immune system evasion is not thought to play a large role as most HNSCC tumors have abnormal antigens that are recognized by the immune system even though the immune system does not effectively react. Lists detailing many of these tumor-associated antigens (TAAs) have been published recently [16, 18–21]. In many cases, the specific CD8? T-cell targeting the TAA is actually present in the patient. Furthermore, patients’ TAA-specific CD8? T cells that are expanded in vitro and exposed to autologous tumor cells can lyse the tumor cells when incubated with stimulatory factors like IFN-c [6]. HNSCC cells resist immune attack Head and neck squamous cell carcinoma tumors may express receptors typically found on immune cells that induce anti-apoptotic pathways [6]. One such receptor is toll-like receptor 4 (TLR-4), which normally binds to lipopolysaccharide, found on bacteria. TLR-4 typically helps protect immune cells during natural killer (NK) cell attack, and on tumor cells may allow them to resist NKinduced apoptosis [22]. HNSCC cells express or secrete factors that directly inhibit the immune system As is the case with many cancers, most HNSCC tumors express or secrete factors that directly inhibit the immune system in the microenvironment. A number of apoptosis-

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promoting factors have been identified in HNSCC, including Galectin-1 [23], FASL [24, 25], TRAIL, and PDL-1 [26]. These factors expressed by the malignant cells seem to induce T-cell apoptosis when binding to the cognate receptor. A factor commonly present in human papilloma virus positive (HPV?) HNSCC cells is PDL-1, which binds with PD-1 on the T cells and promotes anergy, exhaustion, or apoptosis [27, 28]. Direct inhibition of local T cells by tumor cells is not the only immunosuppressive strategy present. Indeed, it is becoming clear that HNSCC cells can promote an aberrant hematopoiesis that affects the entire immune system. HNSCC cells indirectly inhibit the immune system by recruiting and directing an army of pro-tumoral cells It is now evident that HNSCC tumors secrete factors that alter normal hematopoiesis and induce the production and recruitment of cells from the innate and adaptive immune system to effectively counter and silence their anti-tumoral counterparts. These cells are part of a network of cells modulated by HNSCC tumors. It is important to understand this network in order to best design immunotherapeutic strategies.

Cellular network of immune modulation in HNSCC The HNSCC microenvironment usually contains a significant amount of non-cancerous stromal cells in addition to neoplastic cells; these include tumor-associated macrophages (TAMs), myeloid derived suppressor cells (MDSCs), immature dendritic cells (iDCs), and various types of T cells (Fig. 1b). These immune cells are not innocent bystanders; indeed, they play an important role in disease outcome. Depending on the composition and activation status of these cells and the surrounding signaling factors, the net effect can be either favorable or detrimental to tumor progression and metastasis. Table 1 summarizes the involvement of specific stromal cells and the net immunosuppressive effect. Effector T cells Cell-mediated immunity is considered to be the most important process for antitumor immunity; intact CD8?– CD4? T helper 1 (Th1) function is essential for effective immunosurveillance and tumor containment [4, 14]. As mentioned above, tumor antigen-specific CD8? T cells have been identified in patients, yet these cells are unable to fully perform their tumoricidal action because of a suppressive network orchestrated by the tumor [6]. HNSCC patients have fewer lymphocytes in their blood [29] but a

Immunology & Microbiology in Miami (2013) 57:52–69 Table 1 Cellular network of immune modulation Cell

Involvement

Net effect

Cytotoxic T cells (CD8?)

Recognize tumor antigens but are suppressed

CD8? T cells system impairment [36]

Helper T cells (CD4?)

T helper 1 suppressed, T helper 2 favored; T helper 2 cells express IL-4, IL-10, IL-13

Natural killer cells

NKG2D internalized, inhibitory receptors activated, reduced expression of cytotoxic substances, decreased CD1drestricted NK T cells

Humoral response favored, which is ineffective against tumor; cytotoxic activity inhibited [16] Failure to destroy tumor cells [36, 38]

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specimen correlates with better survival in HNSCC patients [31–33]. Nevertheless, defects are commonly found in the effector cells of patients with HNSCC. Even after curative resection of HNSCC cancers, patients have reduced T-cell counts for several years [34]. Circulating CD8? T cells in the blood of most HNSCC patients have an increased ratio of pro-apoptotic protein BAX to antiapoptotic factor Bcl-2 [25]. Intrinsic molecular defects are detectable in the T cells, including downregulation of the n-chain of the CD3 complex, a low responsiveness to IL-2 [35], and a reduced proliferative capability to mitogenic stimulation [36]. Natural killer cells

Dendritic cells

Impaired maturation; immature DCs lack sufficient CD80 and CD86 to co-stimulate; promote T regulatory cell proliferation

Tolerogenic environment; insufficient T-cell stimulation [48, 80]

Tumorassociated macrophages (TAMs)

M2 phenotype; produce IL-10 TGF-b, VEGF; secrete remodeling proteases

Impairs T helper 1/CD8? pathway and instead promotes production of Treg cells; contributes to tumor stabilization and angiogenesis [49]

Myeloid derived suppressor cells (MDSCs)

Produce TGF-b, peroxynitrite; induce local L-arginine starvation; Secrete remodeling proteases

Severe cytotoxic T-cell dysfunction [58]

Regulatory T cells (Tregs)

Secrete TGF-b, IL-10; induce local tryptophan starvation

T-cell anergy or death and APC cell dysfunction [36]

Cytokine profile

Imbalanced toward T helper 2/humoral phenotype

Impaired cell-mediated immunity [80]

B cells

Poor generation of highaffinity tumor antigen antibodies or antibodies produced do not inherently harm tumor cells

Impairment of phagocytic effector cells renders antibody recognition useless [80]

Complement cascade

Tumor cells express anti-complement factors

No complement damage to tumor cells [80]; complement proteins may promote mutagenesis [81]

Mast cells

Secrete proteolytic enzymes

Tumor migration and invasion [52]

relatively high presence of effector memory T cells [30], further suggesting T-cell recognition of tumor. Albeit stunted, effector T cells may still achieve some antitumoral effect: effector T-cell infiltration in a tumor

Natural killer cells of the innate immune system typically partake in immunosurveillance and antitumor immunity [37]. While NK cells should be able to clear tumor cells with decreased MHC class I expression and perform antibody-dependent cellular cytotoxicity, successful HNSCC cells inhibit NK function [36]. NK cell cytotoxicity depends on the relative stimulation of activating versus inhibitory receptors: tumor cells ensure that inhibitory signals dominate [38]. In addition, through secretion of TGF-b1 and a soluble form of MHC-I-related chain, tumor cells cause reduced expression of the activating receptors NKG2D and CD16 [36, 39]. Tumor cells resist NK attack by the production of anti-apoptotic molecules [22]. HNSCC patients have severe reductions in their circulating CD1d-restricted NK T (iNKT) cells [40], which are important for activating effector cells [36]. Dendritic cells Dendritic cells (DCs) have a critical function as antigenpresenting cells [41] but in HNSCC this function is also impaired. The environment in which HNSCC develops, aerodigestive tract mucosa, is rich in immature myeloid DCs and Langerhans cells [42]. Upon encountering inflammatory signals (IL-1, TNF-a, etc.) or microbial products (i.e., TLR ligands) under physiological conditions, DCs migrate to secondary lymphoid organs and mature into antigen-presenting cells capable of cross stimulating, priming, and promoting the expansion of effector T cells. Increased presence of DCs in tumor specimens is associated with longer disease-free survival and decreased recurrence [43, 44], and fewer DCs are found in high-grade tumors [45] and lymph nodes with metastatic disease [46]. To avoid immune system recognition, tumors frequently prevent DC maturation and instead attract abnormal, immature DCs (iDCs) that actually promote T-cell dysfunction [5]. Immature DCs express low levels of the necessary co-stimulatory CD80 and CD86

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molecules as well as MHC-II, which are necessary for antigen presentation to CD4? T cells. These tolerogenic DCs also induce production and proliferation of T regulatory cells, which are immunosuppressive immune cells discussed below. Defects in DC maturation are also visible systemically: HNSCC patients with advanced disease have fourfold reduced levels of circulating myeloid-derived DCs [47] as these cells accumulate in the lymph node sinuses and tumor site in an immature form [48]. In addition to iDCs, HNSCC tumors also simultaneously attract other immature myeloid cells, including macrophages, granulocytes, and myeloid-derived suppressor cells, which all contribute to a pro-tumoral environment.

residues of important signaling proteins in the IL-2 receptor pathway including JAK1, JAK3, STAT5, ERK, and AKT, effectively rendering T cells unresponsive to IL-2 [58]. In addition, MDSCs can produce high quantities of peroxynitrite which induces apoptosis [63] or anergy [64] in activated T cells. NOS and peroxynitrate metabolites have been found in HNSCC tumor beds [65, 66], and tumor infiltration with CD34? MDSCs [59, 67–69] is a negative prognostic factor. MDSCs, like TAMs, also secrete matrix remodeling proteases and series proteases, contributing to tumor dissemination [50–52].

Tumor-associated macrophages

CD4?CD25?Foxp3? regulatory T cells (Tregs) are particularly important for the maintenance of peripheral tolerance and have been implicated in tumor-induced immunosuppression by many studies. Tregs inhibit T-cell activity via a number of mechanisms [70], including via the production of TGF-b and IL-10 or the hydrolysis of extracellular ATP to ADP or AMP by the ectoenzyme CD39 [71]. Tregs also promote the generation of indoleamine-2,3-dioxygenase (IDO) positive tolerogenic DCs by expressing the cytotoxic T-lymphocyte antigen-4 (CTLA-4) and engaging CD80 and CD86 on DCs [5, 70]. An increase in CD4?CD25highFOXP3? has been associated with a poor prognosis in many cancers [72]. Controversy does exist, however, as FOXP3? cells infiltrating the tumor have been proposed either as a positive [73, 74] or as a negative prognostic factor in HNSCC [75, 76]. This controversy was recently resolved by using computer-assisted image quantification and by taking into consideration the subcellular localization of FOXP3 [77]. Indeed, FOXP3? can be expressed also by non-regulatory, activated effector T cells [78]. While in regulatory T cells FOXP3 is found mainly in the nucleus, in activated T cells FOXP3 seems to be localized mostly in the cytoplasm [79]. By taking advantage of these two conditions, a retrospective study in patients with oral squamous cell carcinoma found that while FOXP3 in the cytoplasm of tumor infiltrating CD4? cells correlated with favorable prognosis, its nuclear localization strongly predicted recurrence [77]. The ratio between nuclear and cytoplasmic FOXP3 expression within the tumor infiltrating CD4? T cells is proposed as a powerful prognostic marker in HNSCC [77].

Tumor-associated macrophages (TAMs) are macrophages known to support tumor progression via promotion of humoral immunity and hindrance of cell-mediated immunity [49]. TAMs are similar to M2 macrophages in that they produce IL-10 and transforming growth factor b (TGF-b) and secrete matrix remodeling proteases and serine proteases that are associated with more advanced tumor grade and metastasis [50–52], higher microvessel density in the tumor, and increased bioavailability of VEGF [36]. TAMs can also directly secrete VEGF [36, 53, 54]. A paracrine loop has been suggested between TAMs and tumor cells: tumor cells attract TAMs by secreting monocyte chemotactic protein-1 (MCP-1) and TGF-b1, and meanwhile, TAMs secrete VEGF, IL-8, TNF-a, and IL-1, which stimulate tumors to secrete more VEGF and IL-8 [1, 36, 55]. TAMs may contribute directly to extracapsular extension and lymph node metastasis as increased TAM infiltration correlates with tumors with these features [20, 56]. TAMs accumulate in the areas of fibrin deposition in the tumor and peritumoral tissue, which suggests that they aid in tumor matrix stabilization as well as angiogenesis [36, 57]. Myeloid derived suppressor cells MDSCs have emerged as a powerful mediator of the immunosuppressive state. Recruited directly by tumors via tumor-derived factors, MDSCs are CD34?CD33? immature myeloid cells that suppress CD8? T-cell proliferation and promote tolerance through a variety of mechanisms, including by producing TGF-b, removing L-arginine (L-Arg) from the microenvironment, and nitrosylating important T-cell motifs [58, 59]. L-Arg is essential for correct CD3 complex expression in T lymphocytes [60]. MDSCs upregulate the enzyme L-arginase or the enzyme nitric oxide synthase (NOS), both of which partake in L-Arg metabolism [61, 62]. MDSCs S-nitrosylate cysteine

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Regulatory T cells

Tumor-derived factors in HNSCC Head and neck squamous cell carcinoma cells effectively hijack the immune system and control distribution of supportive resources such as nutrients both locally and distantly via release of tumor-derived factors (TDFs) [16].


These soluble factors are also able to condition distant sites for eventual metastasis, leading to a tumor-driven macroenvironment beyond the local microenvironment. Numerous TDFs have been identified for HNSCC, including granulocyte–macrophage colony-stimulating factor (GMCSF), prostaglandin E2 (PGE2), vascular endothelial growth factor (VEGF), and various cytokines and chemokines. A number of these factors are not completely protumor or anti-tumor, but rather play a dual role depending on elements such as dose, concentration, and duration of exposure. Granulocyte macrophage-colony stimulating factor Granulocyte macrophage-colony stimulating factor appears to assume both anti-tumoral and pro-tumoral roles depending on variations in amount, systemic concentration, and duration of exposure. About 31 % of tested cancer lines including HNSCC secrete GM-CSF [82, 83], and it is associated with a negative prognosis [83]. At higher doses, GM-CSF recruits MDSCs and promotes their differentiation into effective pro-tumoral cells. Administration of tumortransduced GM-CSF, recombinant GM-CSF protein, or high doses of GM-CSF vaccines leads to emergence of MDSC cells [82, 84, 85], whereas administration of GMCSF neutralizing antibody in tumor conditioned media inhibits MDSC differentiation [86]. On the other hand, GMCSF has been shown to elicit powerful immune responses when combined with c-irradiated tumor cell vaccines in the clinical setting and as a result has been used as an immune adjuvant to augment antitumor immunity [87, 88]. GM-CSF is also given to decrease the incidence of mucositis during chemotherapy [89] as well as prevent potentially lifethreatening neutropenia. A bystander vaccine strategy was used in which the antigen dose and steric hindrance were maintained constant to test the dose–effect of GM-CSF, and a threshold was identified above which significant immunosuppression mediated by MDSC recruitment was induced [85]. This may also explain why clinical trials demonstrated a negative effect at doses between 100 and 500 lg, but not at repeated low doses of 40–80 lg for 1–5 days [90]. Prostaglandins Head and neck squamous cell carcinoma tumors frequently overexpress cyclooxygenase-2 (COX-2) [91, 92] as do MDSCs, leading to the production of the eicosanoid prostaglandin E2 (PGE2), which correlates with arginase overexpression, STAT3 and STAT1 phosphorylation, IL-10 and MIP-2 production, and resulting immunosuppression [93]. PGE2 is able to activate the suppressive phenotype in MDSCs and facilitate the differentiation of MDSCs from their hematopoietic precursors [94]. PGE2 can also induce

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Treg production [36]. Non-steroidal anti-inflammatory drugs, which target COX-2, have been shown to exert chemoprotective effects on HNSCC cancer development [95]. Vascular endothelial growth factor Released by most tumors, VEGF is well known for its role in tumor angiogenesis [96–98]. VEGF is also intimately involved with MDSCs, with evidence suggesting that VEGF administration leads to inhibition of DC development and an increased number of MDSCs [99]. Meanwhile, MDSCs secrete matrix metalloproteinase 9 (MMP-9), which breaks down matrix proteins and allows for VEGF permeation through the extracellular matrix [100]. In HNSCC, VEGF-A expression correlated with microvessel density, disease progression, a reduced number of mature DCs, and an increased number of immature DCs and MDSCs [101]. Elevated VEGF levels is a poor prognostic factor and correlates with tumor stage and lymph node status in HNSCC [102]. Cytokines Cytokines are released not only by recruited immunosuppressive immune cells, but also by tumor cells that release them to orchestrate a pro-tumoral effect [16]. HNSCC was shown to produce IL-13 and IL-4, which promote the T helper 2 response and an anti-inflammatory state [103, 104]. Many HNSCC tumors have IL-4 and IL-13 receptors (IL4Ra and IL13Ra), which are constitutively overexpressed, leading to excess arginase production and promotion of neoplastic proliferation [105]. IL4Ra expression on MDSCs and monocytes is required for their suppressive phenotype [106, 107] and survival [107]. Most HNSCC patients also have increased blood levels of IL-6 [108], which is associated with a poor prognosis. IL-6 affects the differentiation of myeloid lineages [109] by the activation of STAT3 and may inhibit the maturation of DCs in particular [110]. Introducing IL-6 antibodies leads to restoration of DC differentiation [111]. Indeed, bone marrow cells treated with GM-CSF and either IL-6 or G-CSF were shown to differentiate into suppressive MDSCs [112]. IL-6 also contributes to systemic symptoms seen in some HNSCC patients including weight loss, night sweats, and fevers [113]. Chemokines Chemokines are important chemotactic molecules that can not only bind to each other to form various signaling heterodimers [114, 115] but that can also bind to multiple receptors. These receptors, in turn, can be activated by

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different chemokines, and this allows for chemokine redundancy [116], robustness, integration [117], and synergy [118]. Consequently, individual chemokines have been shown both to promote tumor immunity, such as by recruiting T cells to the tumor site, and to promote tumor survival, such as by recruiting MDSCs and TAMs [119]. CCL5 can complex with CXCL4 to influence monocyte arrest; the same CCL5 can increase the antitumor effect of adoptively transplanted T cells, but only if intratumoral CD11c? cells are depleted [120]. CCL21 induces Th1 polarization and boosts DNA vaccine effects, and yet the same chemokine is secreted by many human tumors [121] and the expression of its receptor CCR7 correlates with metastasis in HNSCC [122]. There are many more examples of the contradictory roles of chemokines, but research is revealing more about the mechanisms behind each beneficial or deleterious role. For example, CCL2 is known to attract both cytotoxic T cells [123], MDSCs, and TAMs [124–127], yet T cells remain trapped in the stroma surrounding cancer cells, whereas TAMs and MDSC can freely travel within [128]. This occurs because reactive nitrogen species produced by TAMs and neoplastic cells induce nitration/nitrosylation of CCL2 that, once nitrosylated, can no longer recruit cytotoxic T cells, but that can still recruit myeloid cells to the tumor [129]. Understanding chemokine interactions may be imperative for designing immunotherapies capable of reaching the tumor and exerting the desired tumoricidal or immunostimulatory effect.


prevention strategies. Indeed, with the distribution of the HPV vaccine in children and young adults, evidence of a decrease in HNSCC in the vaccinated cohorts is emerging [132].

Immunotherapy for HNSCC For HNSCC patients, a number of immunotherapeutic strategies are being explored, and many treatments are currently being tested in clinical trials. There are two main categories of immunotherapeutic strategies: antigen nonspecific and antigen-specific. Antigen non-specific therapies are designed to broadly enhance the immune response, either by stimulating anti-tumoral mediators or by reversing the immunosuppression put in place by the tumor. Antigen-specific therapies are designed to cause a focused immune response toward the tumor cells directly. Both therapy types have yielded promising results, yet in most cases there are limitations that need to be addressed. Antigen non-specific therapies are frequently limited by systemic toxicity, and antigen-specific therapies are often limited by the powerful immunosuppressive environment put forth by the tumor. Both therapy types may exhibit variable efficacy depending on the specific mutations present in a given tumor. Combination immunotherapies are being explored in order to overcome these limitations.

Antigen non-specific therapies HPV1 HNSCC and the immune system

Immune stimulation: cytokine treatment

Whether a HNSCC tumor is HPV?or HPV- may have important implications for its interaction with the immune system. Notably, patients with HPV? tumors have a better survival than those with HPV- disease: three-year survival is 84 versus 57 %, and the median overall survival is fourfold higher at 131 versus 20 months [130]. What accounts for this difference? The answer may be that the immune system is better able to assume a cytotoxic role. In a preclinical study on immune-deficient mice, no difference in tumor growth was observed between transplanted HPV? and HPV- tumors, while in immune-competent mice, a significant delay in tumor progression was observed and 20–30 % of them cleared the tumor completely [131]. Tumor rejection was possible only when both HPV-specific CD4? and CD8? T cells were present. Indeed, HPV-infected cells are positive for the non-self, virus-associated E7 and E6 antigens that can evoke a powerful, generic, antiviral response. The differences between HPV? and HPV- tumors may become more important as we develop immunotherapies and immune

As detailed above, tumors utilize cytokines to orchestrate immunosuppression and create a pro-tumoral milieu. Cytokine treatments attempt to shift the balance away from the pro-tumoral, T helper-2 cytokine predominance and toward anti-tumoral T helper-1/cytotoxic predominance [80].

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Interleukin-2 Interleukin-2 (IL-2) is one of the earliest cytokines employed to treat HNSCC. Recombinant IL-2 given locally by peritumoral, [133] intranodal, or intraarterial infusion [134] to HNSCC patients leads to increases in intratumoral NK cells and activity of tumor infiltrating lymphocytes (TILs). These observation might be clinically significant since the injection of IL-2 preoperatively around the cervical lymph node chains prior to oral cavity or oropharynx squamous cell carcinoma resection and radiotherapy led to significant increases in disease-free and overall survival [135]. These local treatments are generally well tolerated,


whereas systemic IL-2 is not. Systemic toxicities are significant and include hypotension, capillary leak syndrome, and oliguria [134] with only partial response rates near 18 % [136]. Following the principle of local delivery, the fusion drug ALT-801, composed of IL-2 and a T-cell receptor specific for the p53/HLA-A*0201 complex, has been designed to target IL-2 to p53 positive tumors [133]. This treatment leads to higher efficacy and lower toxicity. Interleukin-12 Interleukin-12 (IL-12) has been used with a similar goal of immunostimulation in HNSCC patients. Intratumoral IL-12 injection led to increased B-cell tumor and lymph node infiltration, B-cell activation, and a highly significant IgG subclass switch measured in the plasma, indicating a switch to Th1 phenotype [93]. IL-12 increased NK cells in the tumor as well as led to a 128-fold increase in IFN-c mRNA in lymph nodes [137, 138]. IL-12 is currently being investigated in clinical trials [93] for efficacy. Interferon-gamma Systemic IFN-c therapy has been attempted in HNSCC patients with 3 of 8 responding with minimal toxicity in one study and 4 of 9 responding in a second study [139]. However, another trial failed to show any objective response [140]. Interferon-alpha IFN-a has been used in combination with cisplatin and 5fluorouracil in the treatment of advanced esophageal squamous cell carcinoma with an overall response rate of 55 % but was associated with significant toxicity. When combined with IL-12, IFN-a produced significant tumor regression in 2 of 11 advanced HNSCC patients [141]. A recent phase II trial investigated IFN-a combined with isotretinoin and vitamin E in locally advanced HNSCC, showing 5-year progression-free survival rate and overall survival rate of 80 and 81.3 %, respectively [93].

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IL-6, or other tumor-derived factors, while others target specific immunosuppressive populations such as MDSCs and T regulatory cells. Tyrosine kinase inhibitors Tyrosine kinases are commonly overexpressed and activated in many tumors including HNSCC [142]. Tumorderived factors promote also the aberrant activation of these kinases in the hematopoietic system, leading to the accumulation of, for example, MDSCs. Suntinib is a molecule that inhibits multiple tyrosine kinases (VEGFR-1, VEGFR-2, VEGFR-3, PDGFR, c-kit, ret, STAT3, etc.), and, as expected, shows potent effects against MDSCs [143]. It has been shown to reverse MDSC accumulation and induce tumor cell apoptosis in renal cell carcinoma. [144]. Clinical trials using suntinib in HNSCC patients demonstrated significant toxicities including lymphopenia, neutropenia, and thrombocytopenia [145] and poor therapeutic efficacy [146, 147]. A number of other selective kinase inhibitors are currently in development [142]. Some tyrosine kinase inhibitor drugs are monoclonal antibodies and will be discussed below. 1a,25-Dihydroxyvitamin D3 Vitamin D has been shown to induce the maturation of MDSCs into immune-stimulatory DCs [148, 149] as well as inhibit tumor production of VEGF and hypoxia factor 1a (HIF-1a) [150], which are involved with the induction of MDSCs by the tumor. Although it is not clear whether vitamin D acts on MDSCs directly or indirectly, it has been tested in HNSCC. Patients treated with vitamin D preoperatively had reduced amounts of MDSCs in the tumor and increased amounts of mature DCs and activated T cells (CD4? and CD8?) [151]. More importantly, the time to recurrence was doubled in the patients who received vitamin D. Nevertheless, more studies are needed to further characterize the impact of vitamin D on HNSCC survival. Cox-2 inhibitors

Reversal of immunosuppression Reversal of tumor-induced immunosuppression is the second class of antigen non-specific strategies employed in HNSCC. These strategies are designed to (1) reestablish a micro- and macro-environment favorable for immunosurveillance; (2) deplete the suppressive populations that are recruited by the tumor; and (3) block the molecular mechanism of negative regulators of the adequate immune response. Several of these therapies aim to inhibit aberrant intracellular pathways involving VEGF, PGE2, GM-CSF,

COX-2 inhibitors reverse the PGE2 overproduction by tumors and thus provide an opportunity to block MDSC differentiation and activation [152]. Celecoxib, a specific COX-2 inhibitor, when used in combination with radiotherapy and Erlotinib, an EGFR receptor blocker, led to 37 % progression-free survival and 60 % locoregional control at 1 year [153]. When combined with the EGFR receptor blocker gefitinib, 22 % of unresectable or metastatic HNSCC demonstrated a partial response [154]. While tolerated well during the study periods, celecoxib is

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associated with dose-dependent cardiovascular morbidity so its use in long-term treatment may be limited [155]. Phosphodiesterase inhibitors Phosphodiesterase-5 (PDE5) can be targeted to reverse MDSC suppression. Its inhibition, by controlling the intracellular concentration of cGMP, is sufficient to inhibit arginase 1, NOS2, and IL4a on MDSCs, thus reducing their suppressive ability [156]. Moreover, it has been shown that administration of the PDE5 inhibitors sildenafil or tadalafil was sufficient to significantly reduce tumor progression in murine models of breast and colon cancer [156], while in a lymphoma model was able to restrain MDSC-mediated expansion of tumor-specific Tregs [157]. More importantly, sildenafil was able to restore T-cell proliferation in anti-CD3/anti-CD28-stimulated peripheral blood mononuclear cells (PBMCs) from HNSCC patients [157]. Two clinical trials (NCT00843635, NCT 00894413) have recently closed to accrual utilizing daily tadalafil administration prior to standard of care surgery or chemoradiation therapy in patients with HNSCC. While results of these trials are not yet published, interim analysis of NCT00843635 suggests that PDE5 blockade lowers MDSC and Treg concentrations in the blood and tumor tissue and expands the pool of tumor-specific CD8? cells (unpublished data, updated September 2013). Alkylating agents Tregs are particularly sensitive to alkylating agents, and thus, alkylating agents may play an immunotherapeutic role. Indeed, cyclophosphamide or ifosphamide at low doses can decrease Treg concentration in the blood [158, 159]. Interestingly, when cyclophosphamide was combined with cisplatin and 13-cis retinoic acid, a response rate of 72 % for metastatic HNSCC was observed [160]. IRX-2 Combination therapies may be more effective than single agents. A novel drug named IRX-2 was developed that contains a mixture of the antitumoral cytokines IFN-c, IL-2, IL-6, IL-8, IL-1b, G-CSF, GM-CSF, and TNF-alpha, as well as agents that reverse immunosuppression, namely indomethacin and cyclophosphamide [161]. Zinc is also included as it is a necessary mineral for the development of cellular immunity. Interestingly, zinc deficiency is observed in 50 % of patients with HNSCC and correlates with higher cancer stage [162]. Recently tested in a phase II clinical trial, IRX-2 was given to Stage II-IVa HNSCC patients for 21 days preoperatively; 83 % had stable

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disease during treatment according to radiographic review, and overall survival was 69 % at 36 months [162]. Additional antigen non-specific therapies are currently being tested in other cancers; a recently published review discusses anticipated HNSCC clinical trials based on these additional therapies [36].

Antigen-specific therapies Antigen-specific therapies are designed to enhance tumorspecific responses generated by the natural immune system and include antibody therapies, adoptive cell transfer, and tumor vaccines. Antibody therapies A number of monoclonal antibodies have been developed against a variety of tumor cell surface molecules. When the antibody binds to the target molecule, it may inhibit the tumor cell by directly blocking the signaling pathway initiated by the molecule, and/or by initiating cell-mediated cytotoxicity resulting in tumor cell death. In cell culture, tumor cell apoptosis only occurs after antibody administration when lymphocytes are present [163, 164]. The precise mechanism of tumor lysis is not well understood: complement-dependent cytotoxicity can be seen with some antibody therapies, but this mechanism of cell death should occur rapidly, as should NK cell-mediated cell death, and yet tumor shrinkage is noted over weeks, not hours; this is more consistent with a T-lymphocyte-mediated lytic effect [164]. Fc gamma R polymorphisms on antibodies should variably influence any NK antibody-dependent cellular cytotoxicity, yet this is not well elucidated [164, 165]. The IgG subtype of the antibody appears to influence the efficacy of the therapy; IgG1 and IgG3 subclasses are more efficient than IgG2 and IgG4 at mediating lysis of target cells [164, 166, 167]. Lysis of normal cells is also a risk as normal cells also contain many of the surface antigen molecules, yet antibody therapy does not typically result in severe systemic toxicity [164]. The most widely studied antibodies for HNSCC are those targeting the epidermal growth factor receptor (EGFR), which is typically overexpressed on the majority of HNSCC cells [16]. Cetuximab, an immunotherapy currently FDA approved for HNSCC, targets EGFR, which is a receptor tyrosine kinase. A phase III trial in which cetuximab was added to radiotherapy demonstrated an increase in locoregional control from 14.9 to 24.4 months (p = 0.005) with limited toxicity [168]. Overall 5-year survival was also improved from 36.4 to 45.6 %, primarily in patients that experienced at least a grade 2 acneiform rash during treatment [169]. Efficacy of cetuximab and


other EGFR antibody therapies is limited by HNSCC resistance through upregulation of other receptor tyrosine kinases such as HER2, HER3, and MET, altered EGFR function after nuclear translocation, and altered expression of downstream effectors of EGFR signaling [142]. Combination therapy may be necessary to increase efficacy. A number of additional antibody therapies for HNSCC are also being studied, including ones that target HER2/neu, VEGF, CD45, CTLA-4, PDL-1, or carcinoembryonic antigen (CEA) [93]. Various monoclonal antibody therapies for HNSCC have been recently reviewed [36, 164, 170, 171]. Overall, antibody-based immunotherapy for HNSCC has the greatest clinical efficacy and the most widespread use in clinical practice today as compared to other immunotherapies. However, further investigation is needed in order to increase antibody effectiveness, perhaps by combining with agents that reverse local immunosuppression. Conjugated antibodies, in which the antibody is conjugated with a radioactive substance, drug, or toxin, are an alternative strategy for antibody therapy designed to take advantage of antibody specificity as a means of targeted delivery of a non-immunotherapeutic agent. One conjugated antibody tested in a HNSCC phase I clinical trial was 186 Re-labeled humanized bivatuzumab, which demonstrated that the drug was well tolerated and led to delivery of a median of 12.4 Gy to the tumor cells [172]. Studies evaluating the use of other labeled or loaded antibodies to treat various cancer cell types are ongoing [173]. Adoptive cell transfer Adoptive cell transfer involves harvesting and priming a patient’s own tumor antigen-specific effector cells before re-introducing them to the patient. In one such trial, 17 patients with HNSCC were injected in the thigh with irradiated autologous tumor cells admixed with GM-CSF followed by 3 additional injections of GM-CSF at the site [174]. The following week, the inguinal lymph nodes draining the injection site were resected, and the lymphocytes harvested from these nodes were activated with staphylococcal enterotoxin A and expanded in IL-2 in vitro. The CD3-enriched cells were then infused back into the patients peripherally. Three patients had disease stabilization where progression had been evident prior to the treatment. Another trial involved 4 patients in which harvested peripheral blood mononuclear cells (PBMCs) were collected by leukophoresis and then incubated ex vivo with catumaxomab, a monoclonal antibody that binds CD3? T cells with one arm and epithelial cell adhesion molecule (EpCAM) with the other [175]. Catumaxomabloaded PBMCs were shown to release IFN gamma and gramzyme B when coincubated with EpCAM? cells. After

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opsonization and cytokine washout, the cells were reintroduced into the patients. One patient showed stable disease for 6 months and another went into complete remission during the follow-up period of 27 months. A recent study made use of bimodal ex vivo expansion to expand harvested tumor infiltrating lymphocytes using anti-CD3 antibody, feeder cells, and high-dose IL-2. Just 17 days were required to create up to 3,500-fold expansion of the T cells, most of which were T-effector memory cells [31]. This method may be used in the future for clinical trials in HNSCC patients. Adoptive cell transfer techniques have also been studied for NK T cells, and one study demonstrated tumor regression in 5 out of 10 locally recurrent, operable HNSCC patients [176]. As with other immunotherapies, adoptive cell transfer has its limitations that still need to be overcome. Not only is the technique cumbersome and technically challenging, but also many tumor-specific lymphocytes that pre-exist in the patient are anergic to in vitro re-stimulation [177]. A new strategy involving gene modification of T lymphocytes may be a solution [135, 178]; PBMCs from patients are retrovirally transduced with a T-cell receptor (TCR) specific for the tumor, and additional genes that protect the lymphocytes from the tumor suppressive mechanisms can also be added [179]. In a study with melanoma patients, PBMCs were transfected with high-affinity T-cell receptors specific for p53–HLA-A2 complex and were shown to be able to recognize different human tumor cells lines expressing p53 [180]. As many HNSCC tumors express p53 [181, 182], this may become a potential therapy for HNSCC patients.

Anti-tumor vaccines Dendritic cell-based vaccines Dendritic cell counts are generally reduced in HNSCC patients, but patient monocytes can be used to generate autologous DCs in vitro, or allogeneic DC lines can be manipulated. These DCs loaded with TAAs such as p53 [183], tumor RNA [184, 185], or tumor DNA [36] are being used as vaccines in patients. These TAA-specific DCs are able to produce IFN-c in vitro [184, 186]. Stage I-IVa HNSCC patients with no active disease were vaccinated with autologous DC pulsed with p53 and a tetanusderived helper peptide. A phase I clinical trial [187] demonstrated the safety of this vaccine and an increase in p53-specific T cells in 11 of 16 patients. Frequencies and absolute numbers of Tregs were significantly decreased after vaccination. More importantly, disease-free survival at 24 months appeared to be favorable as compared to historical unvaccinated HNSCC patients.

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Peptide-based vaccines Peptide-based vaccines, also known as ‘‘Trojan’’ vaccines, utilize a penetrin peptide sequence derived from HIV-TAT, which allows the entire peptide to translocate through the cell membrane and penetrate directly into the endoplasmic reticulum and the Golgi apparatus [188]. Once there, they can form peptide–HLA complexes without the need of proteasome processing and TAP transportation. This therapeutic modality has been attempted in a pilot study with HNSCC patients. MAGE 3 and HPV-16 peptides fused to the penetrin peptide were used in 4 consecutive immunizations with GM-CSF and Montanide ISA 51 as adjuvant. Most patients demonstrated systemic immune response against the Trojan and HLA-II-restricted peptides [189]. Analysis of the tumor specimen from one patient after treatment revealed significant infiltration of MAGE-specific CD4? and CD8? T cells as well as large areas of apoptotic tumor cells. Phase II clinical trials using MAGE-A and HPV-16 antigens are currently ongoing [36]. Whole tumor vaccines Whole tumor vaccines attempt to target the whole repertoire of tumor antigens so as to minimize the chance that further immunoediting and subsequent tumor resistance. One technique involves re-injecting autologous irradiated tumor cells into patients along with GM-CSF and then harvesting the sentinel node lymphocytes that are theoretically sensitized to multiple TAAs for subsequent adoptive cell transfer [174], as already described above. Another strategy involves using oncolytic viruses with genes encoding immune-stimulatory cytokines such as GM-CSF [190]. The rationale for use of these viruses is that they selectively replicate in a tumor leading to lytic tumor cell death and also lead to release of tumor antigens along with stimulating cytokines, inducing a tumor-specific immune response that protects the host from local and distant recurrence. A phase I trial of OncoVEXGM-CSF, which consists of a herpes simplex virus expressing GM-CSF, was conducted on stage III/IV HNSCC patients. Preliminary data demonstrated that 6 of 8 patients had a complete pathologic response with only mild toxicity [191]. Another study was conducted in which the virus was injected intratumorally in stage III/IVA/IVB HNSCC patients in conjunction with neoadjuvant chemoradiotherapy [192]. All patients underwent neck dissection 6– 10 weeks later. 82.3 % of the patients showed tumor response by Response Evaluation Criteria in Solid Tumors, and pathologic complete remission was confirmed in 93 % of the patients at neck dissection. HSV was detected in injected and adjacent uninjected tumors at levels higher than the input dose, indicating viral replication. No patient

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developed locoregional recurrence, and disease-specific survival was 82.4 % at median follow-up of 29 months. Additional clinical trials with OncoVEX are ongoing.

Therapeutic HPV vaccines in HNSCC Therapeutic HPV vaccines are being developed that target E6 and E7, which are constitutively expressed in HPVinfected cells [193]. These vaccines include the use of HPV-E6 peptides, live attenuated listeria monocytogenes bacteria carrying E7 fusion protein, Trojan peptides, vaccinia based vaccines, naked DNA vaccines, and DC-based vaccines [194]. The vaccine utilizing listeria is interesting in that it takes advantage of the capacity of listeria to infect antigen-presenting cells and promote DC antigen crosspresentation; subsequently, effector T cells are activated while the innate immune system is simultaneously stimulated by the bacteria [195]. Preclinical data demonstrate that this technique induces the regression of established tumors by reducing the suppressive activity of Tregs and MDSCs at the tumor site, by promoting the chemotaxis and maturation of DCs, and by generating memory effector T cells [196]. In a phase I trial of refractory cervical cancer patients receiving the listeria-based vaccine, 1-year survival increased to 53 % from the historical 5 %, with a significant reduction in tumor size in 33 % [197]. Various clinical trials with HPV vaccines are ongoing [198].

Conclusion Recent advances in our understanding of the complex interactions between HNSCC and the immune system have led to a number of innovative immunotherapeutic strategies. While most of these strategies are still investigational, their existence represents both a hope for improved HNSCC survival in the near future and an opportunity to shape the future of immunologic cancer research. It is increasingly apparent that efforts to generate an effective anti-tumor immune response must be coupled to strategies that work to abrogate the strong immunosuppressive network orchestrated by the tumor. As more is discovered about the role of immune system in carcinogenesis, the opportunity for earlier intervention may also become possible. HNSCC remains a debilitating and deadly disease, yet through the enthusiastic study of its interaction with the immune system, the promise of immunotherapy is stronger than ever. Acknowledgments This work was supported by the Flight Attendant Medical Research Institute (FAMRI)’s young investigator award, by the Bankhead Coley Cancer Research Program Grant 2BF0650904.

Immunology & Microbiology in Miami (2013) 57:52–69 Conflict of interest of interest.

The authors declare that they have no conflict

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Breakthrough targeted therapeutic approaches to squamous cell carcinoma of the head and neck.

Pulmonary lymphangitic carcinomatosis from head and neck squamous cell carcinoma.

Genetic susceptibility to head and neck squamous cell carcinoma.

NLRP3 inflammasome activation promotes inflammation-induced carcinogenesis in head and neck squamous cell carcinoma.

Immune cell infiltration patterns and survival in head and neck squamous cell carcinoma.

MicroRNAs in Head and Neck Squamous Cell Carcinoma (HNSCC) and Oral Squamous Cell Carcinoma (OSCC).

Telomeres and telomerase in head and neck squamous cell carcinoma: from pathogenesis to clinical implications.

Expression of cdk6 in head and neck squamous cell carcinoma.

Immunotherapy for head and neck squamous cell carcinoma.

Human papillomavirus detection in head and neck squamous cell carcinoma.

Immunotherapy for head and neck squamous cell carcinoma.

X-linked FHL1 as a novel therapeutic target for head and neck squamous cell carcinoma.

New concepts in radiotherapy for advanced squamous cell carcinoma of the head and neck.

MicroRNA-138: a potential therapeutic target for head and neck squamous cell carcinoma (HNSCC).

Regulation of glycolysis in head and neck squamous cell carcinoma.

Cancer stem cells in head and neck squamous cell carcinoma.

Tumor microenvironment in head and neck squamous cell carcinoma.

Salvage surgery for head and neck squamous cell carcinoma.

Immunotherapy for Head and Neck Squamous Cell Carcinoma.

Novel Immunotherapeutic Approaches for Head and Neck Squamous Cell Carcinoma.

Panitumumab for locally advanced head and neck squamous-cell carcinoma.

Genetic events in head and neck squamous cell carcinoma revealed.

Chemotherapy in locally advanced head and neck squamous cell carcinoma.

Human papillomavirus-related head and neck squamous cell carcinoma variants.