Mature dendritic cells enriched in regulatory molecules may control regulatory T cells and the prognosis of head and neck cancer

We previously reported that regulatory T (Treg) cells expressing CTLA-4 on the cell surface are abundant in head and neck squamous cell carcinoma (HNSCC). The role of expanded Treg cells in the tumor microenvironment of HNSCC remains unclear. In this study, we reveal that the tumor microenvironment of HNSCC is characterized by the high expression of genes related to Treg cells, dendritic cells (DCs), and interleukin (IL)-17-related molecules. Increased expression of IL17A, IL17F, or IL23A contributes to a favorable prognosis of HNSCC. In the tumor microenvironment of HNSCC, IL23A and IL12B are expressed in mature dendritic cells enriched in regulatory molecules (mregDCs). The mregDCs in HNSCC are a migratory and mature phenotype; their signature genes strongly correlate with Treg signature genes in HNSCC. We also observed that IL17A was highly expressed in Th17 cells and exhausted CD8⁺ T cells in HNSCC. These data suggest that mregDCs in HNSCC may contribute to the prognosis by balancing Treg cells and effector T cells that produce IL-17. Targeting mregDCs may be a novel strategy for developing new immune therapies against HNSCC.     

Tumour-infiltrating lymphocytes predict response to definitive chemoradiotherapy in head and neck cancer

Background:

We aimed to investigate the prognostic value of tumour-infiltrating lymphocytes'' (TILs) expression in pretreatment specimens from patients with head and neck squamous cell carcinoma (HNSCC) treated with definitive chemoradiotherapy (CRT).

Methods:

The prevalence of CD3+, CD8+, CD4+ and FOXP3+ TILs was assessed using immunohistochemistry in tumour tissue obtained from 101 patients before CRT and was correlated with clinicopathological characteristics as well as local failure-free- (LFFS), distant metastases free- (DMFS), progression-free (PFS) and overall survival (OS). Survival curves were measured using the Kaplan–Meier method, and differences in survival between the groups were estimated using the log-rank test. Prognostic effects of TIL subset density were determined using the Cox regression analysis.

Results:

With a mean follow-up of 25 months (range, 2.3–63 months), OS at 2 years was 57.4% for the entire cohort. Patients with high immunohistochemical CD3 and CD8 expression had significantly increased OS (P=0.024 and P=0.028), PFS (P=0.044 and P=0.047) and DMFS (P=0.021 and P=0.026) but not LFFS (P=0.90 and P=0.104) in multivariate analysis that included predictive clinicopathologic factors, such as age, sex, T-stage, N-stage, tumour grading and localisation. Neither CD4 nor FOXP3 expression showed significance for the clinical outcome. The lower N-stage was associated with improved OS in the multivariate analysis (P=0.049).

Conclusion:

The positive correlation between a high number of infiltrating CD3+ and CD8+ cells and clinical outcome indicates that TILs may have a beneficial role in HNSCC patients and may serve as a biomarker to identify patients likely to benefit from definitive CRT.     

Increased expression of immunosuppressive molecules on intratumoral and circulating regulatory T cells in non-small-cell lung cancer patients

The expression patterns of immunosuppressive molecules on regulatory T (Treg) cells have not been elucidated in non-small-cell lung cancer (NSCLC) patients. In this study, a total of 88 patients including 53 patients with NSCLC, 17 patients with lung non-malignant diseases, and 18 healthy volunteers were enrolled. Increased number of total CD4⁺CD25⁺FoxP3⁺ Treg cells and elevated expressions on the surface of several inhibitory molecules including CTLA-4, LAG-3 and PD-1 have been observed in the peripheral blood of NSCLC patients. We found that intratumoral Treg cells from NSCLC patients express the highest levels of co-inhibitory molecules compared to Treg cells isolated from tumor adjacent tissues or from peripheral blood of cancer patients, which is in consistent with the enhanced immunosuppressive function of these co-inhibitory molecules. Moreover, the number of Treg cells and their functional surface molecules increased during the progression of lung cancer. Elevated plasma levels of TGF-β and IL-10 in NSCLC patients were also observed in NSCLC patients compared to that in healthy volunteers. Our findings further support the role of Treg cells in the tumor microenvironments in NSCLC patients.     

Immune‐checkpoint molecules on regulatory T‐cells as a potential therapeutic target in head and neck squamous cell cancers

Immune‐checkpoint inhibitors improve the survival of head and neck squamous cell carcinoma (HNSCC) patients. Although recent studies have demonstrated that the tumor immune microenvironment (TIME) has critical roles in immunotherapy, the precise mechanisms involved are unclear. Therefore, further investigations of TIME are required for the improvement of immunotherapy. The frequency of effector regulatory T‐cells (eTregs) and the expression of immune‐checkpoint molecules (ICM) on eTregs and conventional T‐cells (Tconvs) both in peripheral blood lymphocytes (PBL) and tumor‐infiltrating lymphocytes (TIL) from HNSCC patients were analyzed by flow cytometry and their distributions were evaluated by multi‐color immunofluorescence microscopy. High frequency eTreg infiltration into HNSCC tissues was observed and high expressions of CD25, FOXP3, stimulatory‐ICM (4‐1BB, ICOS, OX40 and GITR) and inhibitory‐ICM (programmed cell death‐1 [PD‐1] and cytotoxic T‐lymphocyte‐associated protein‐4 [CTLA‐4]) were found on invasive eTregs. In contrast, the expression of stimulatory‐ICM on Tconvs was low and the expression of inhibitory‐ICM was high. In addition, ICM‐ligands (programmed cell death‐1 [PD‐L1], galectin‐9 and CEACAM‐1) were frequently expressed on cancer cells. PD‐L1 and galectin‐9 were also expressed on macrophages. PD‐1⁺ T‐cells interacted with PD‐L1⁺ cancer cells or PD‐L1⁺ macrophages. This suggested that in TIL, eTregs are highly activated, but Tconvs are exhausted or inactivated by eTregs and immune‐checkpoint systems, and ICM and eTregs are strongly involved in the creation of an immunosuppressive environment in HNSCC tissues. These suggested eTreg targeting drugs are expected to be a combination partner with immune‐checkpoint inhibitors that will improve immunotherapy of HNSCC.     

Characteristics of CD4+CD25+ regulatory T cells in the peripheral circulation of patients with head and neck cancer

Patients with squamous cell carcinoma of the head and neck (SCCHN) have depressed antitumour immunity. The presence of CD4+CD25+ (Treg) cells in these patients might be, in part, responsible for downregulation of antitumour immune responses. To evaluate the frequency and characteristics of Treg in the peripheral circulation of patients with SCCHN, we used multicolour flow cytometry. Expression of CCR7, CD62L, zeta chain and Annexin V binding to Treg and non-Treg CD4+ lymphocyte populations were evaluated. Treg were confirmed to be Foxp3+ and GITR+. The Treg frequency was significantly elevated in patients with active disease and those with no evidence of disease (NED) following curative therapies. Both Treg and non-Treg CD4+ T cells in patients were significantly enriched in CCR7- and CD62L- cell subsets. Although Treg in patients contained a higher proportion of double negative (CCR7-CD62L-) cells, the majority of Tregs were CCR7-CD62L+. The proportion of Annexin V+CD4+ T cells was higher in patients (P<0.00005) than normal controls (NC), and Treg were significantly more sensitive to apoptosis than non-Treg in patients and NC. Expression of zeta was reduced in all subsets of CD4+ T cells obtained from patients vs NC. The data suggest that Treg in patients with SCCHN largely contain T cells with the 'effector' phenotype, which bind Annexin V and have low zeta expression, consistent with their activation state and a rapid turnover in the peripheral circulation.     

Regulatory T cells expressing abundant CTLA-4 on the cell surface with a proliferative gene profile are key features of human head and neck cancer

FOXP3⁺ regulatory T (Treg) cells suppress anti-tumor immunity. The suppression of Treg cells is regulated by cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), whose expression on the cell surface is tightly regulated. Here we found that Treg cells expressing abundant CTLA-4 on the cell surface (surface-CTLA-4⁺ Treg) were expanded in human head and neck cancer tissues. RNA sequencing of surface-CTLA-4⁺ and surface-CTLA-4⁻ Treg cells infiltrating human head and neck cancer tissues revealed that surface-CTLA-4⁺ Treg cells have a previously undescribed gene expression profile correlating to cell cycle, cell proliferation, and DNA replication. Moreover, surface-CTLA-4⁺ Treg cells were PD-1⁺, actively proliferated and associated with CD45RA⁻ FOXP3^high Treg cells with strong suppressive function. Thus, surface-CTLA-4⁺ Treg cells with a proliferative gene expression signature and phenotype are key features of head and neck cancer. Targeting surface-CTLA-4⁺ Treg cells might be new strategies to evoke effective immune responses to head and neck cancer.     

Rehabilitation problems of head and neck cancer patients.

Head and neck cancer and its treatment result in varying degrees of disability affecting various organ systems. Ideal treatment of such patients requires a unit capable of managing problems in the areas of: Reconstructive surgery, maxillofacial prosthodontia, dentistry, deglutition disorders, and psychological, social, and vocational rehabilitation. Provision of such facilities in an integrated manner will give the patient the optimal chance for rehabilitation from the complex disabilities occurring in head and neck cancer.     

CD19+CD24hiCD38hiBregs involved in downregulate helper T cells and upregulate regulatory T cells in gastric cancer

Regulatory B cells (Bregs) play a critical role in inflammation and autoimmune disease. We characterized the role of Bregs in the progression of gastric cancer. We detected an increase in Bregs producing IL-10 both in peripheral blood mononuclear cells (PBMCs) and in gastric tumors. Multicolor flow cytometry analysis revealed that a subset of CD19⁺CD24^hiCD38^hi B cells produces IL-10. Functional studies indicated that increased Bregs do not inhibit the proliferation of CD3⁺T cells or CD4⁺ helper T cells (Th cells). However, Bregs do suppress the secretion of IFN-γ and TNF-α by CD4⁺Th cells. CD19⁺CD24^hiCD38^hiBregs were also found to correlate positively with CD4⁺FoxP3⁺ regulatory T cells (Tregs). Neutralization experiments showed that Bregs convert CD4⁺CD25⁻ effector T cells to CD4⁺FoxP3⁺Tregs via TGF-β1. Collectively, these findings demonstrate that increased Bregs play a immunosuppressive role in gastric cancer by inhibiting T cells cytokines as well as conversion to Tregs. These results may provide new clues about the underlying mechanisms of immune escape in gastric cancer.     

Cell-mediated immune response in head and neck cancer patients

The immune reactivity of 100 head and neck cancer patients was studied by means of DNCB, candidine, blastic transformation with PHA, and lymphocyte counts. DNCB reactivity was strong in only 21% of the population and was found impaired in patients with advanced primitive tumors, with more than one primitive tumor, and in patients after radiation. Candidine reactivity was impaired in bigger primitive tumors, positive nodes, and advanced stages, as well as in postradiation patients. Blastic transformation was significantly worse in patients over 70 years and tended to be lower in patients with more than one primitive tumor. Good blastic transformation was also lowered in postradiation patients.     

Long-term intraarterial chemotherapy infusion of ambulatory head and neck cancer patients

The complications of percutaneous intraarterial infusion chemotherapy of the head-and-neck-cancer patient has dampened enthusiasm for this approach. A totally implantable infusion pump system circumvents many of these complications and will in the long term enhance the opportunity to expand upon the advantages of infusion chemotherapy for the benefit of all cancer patients.     

Parenteral hyperalimentation in surgical patients with head and neck cancer: A randomized study

Sixty-nine patients were entered in a randomized study to determine the usefulness and practicality of parenteral hyperalimentation (TPN) in preparing and supporting patients with head and neck cancer undergoing radical resections. The patients were stratified by nutritional status and prognosis and randomization were done within each strata to TPN or control. Minimum full TPN was given at 35 calories/kgm/day for at least 14 days postoperatively. Eight patients received preoperative TPN also. Control patients received customary enteral alimentation by feeding tubes. Under the conditions of this particular study, the administration schedules, and type of solutions used, we were unable to demonstrate any superiority of TPN over conventional enteral nutrition in terms of immune parameters, wound healing, complications, and survival.     

Epigenetic editing and tumor-dependent immunosuppressive signaling in head and neck malignancies

Head and neck cancer (HNC) comprises a heterogeneous variety of malignant tumors, characterized by a relatively high tumor mutation burden. Previous data have revealed that immune system dysfunction appears to serve a key role in the development and progression of HNC and established immunosuppression is vital for evading the host immune response. Despite progress in chemotherapy and radiotherapy, the survival rate of patients with HNC is still low. Therefore, the present review discusses the development of novel immunotherapy approaches based on the various immune cell signaling routes that trigger drug resistance and immunosuppression. Additionally, the present review discusses the epigenetic alterations, including DNA methylation, histone modifications, chromatin remodeling and non-coding RNAs that drive and support HNC progression. Furthermore, the role of cancer-associated fibroblasts, tumor macrophages and myeloid cells in tumor-related immunosuppression are considered. Specifically, the molecular immune-related mechanisms in the tumor microenvironment, which lead to decreased drug sensitivity and tumor relapse, and strategies for reversing drug resistance and targeting immunosuppressive tumor networks are discussed. Deciphering these molecular mechanisms is essential for preclinical and clinical investigations in order to enhance therapeutic efficacy. Furthermore, an improved understanding of these immune cell signaling pathways that drive immune surveillance, immune-driven inflammation and tumor-related immunosuppression is necessary for future personalized HNC-based therapeutic approaches.     

Local control in advanced head and neck cancer by combined modalities of treatment

From July 1979 to January 1983, 20 patients with locally advanced head and neck cancer were treated with a combination of chemotherapy and irradiation with or without surgery. A majority of the patients were in the age range of 45 to 54 years. Eighty-five percent of the patients were male. Seventy-five percent of the patients had oral cavity lesions, the tongue being the most common site. Eighty percent of the patients had T₄ lesion and 35% had N₃ disease in the neck. A majority of the patients had combination chemotherapy, including bleomycin, methotrexate, and cis-platinum (BMP). All patients received irradiation with megavoltage equipment and 55% of patients received a dose of 5,000 to 6,000 rads in 5–6 weeks time. The tumor was converted to be resectable in ten patients. Nine patients (45%) had the neck and primary tumor completely controlled, while six patients (30%) had partial control. Six of the ten patients who had resection had the tumor controlled at the primary site and neck. The median duration of follow-up is 12 months (range, 4–32 months). The median survival of the whole group of patients is 12.5 months. A brief review of the current literature is also done in this paper.     

Impact of multidisciplinary team management in head and neck cancer patients

Background:

We analysed the outcomes of 726 cases of primary head and neck cancer patients managed between 1996 and 2008, including those managed in the multidisciplinary clinic or team setting (MDT) and those managed outside of an MDT by individual disciplines (non-MDT) in the same institution.

Methods:

Data were collected from the Hospital Based Cancer Registry and a database within the Head and Neck Cancer Clinic. Univariable comparisons and multivariable analyses were performed using a logistic regression model. Survival by staging was analysed. Comparisons of management and outcomes were made between MDT and non-MDT patients.

Results:

395 patients (54%) had been managed in the MDT vs 331 patients (46%) non-MDT. MDT patients were more likely to have advanced disease (likelihood ratio χ²=44.7, P<0.001). Stage IV MDT patients had significantly improved 5-year survival compared with non-MDT patients (hazard ratio=0.69, 95% CI=0.51–0.88, P=0.004) and more synchronous chemotherapy and radiotherapy (P=0.004), and the non-MDT group had more radiotherapy as a single modality (P=0.002).

Conclusions:

The improved survival of MDT-managed stage IV patients probably represents both the selection of multimodality treatment and chemotherapeutic advances that these patients received in a multidisciplinary team setting by head and neck cancer specialists as opposed to cancer generalists in a non-MDT setting.     

Hyperthermia and irradiation of head and neck squamous cancer cells causes migratory profile changes of tumour infiltrating lymphocytes

Purpose: CD4⁺CD25⁺FoxP3⁺ regulatory T-cells (Treg) are responsible for immunoevasion mechanisms induced by cancer. Specific chemokines such as CCL22 are presumed to mediate active Treg trafficking into the tumour site. In this context, the effects of irradiation and hyperthermia of tumour cells on Treg migration and the CCL22 concentration in the tumour cell supernatants after treatment were studied. Moreover, the relationship between CCL22 concentration and Treg cell migration was also examined.

Materials and methods: Treg and CD4⁺CD25^? T-cells were isolated from human peripheral blood. Supernatants were obtained from primary cell cultures derived from head and neck carcinoma patients. Tumour cell cultures were treated with a dose of 2 Gy and hyperthermia (41.5°C) or with hyperthermia or irradiation alone. Cancer cell culture supernatants were then used for a transmigration assay.

Results: Treg and CD4⁺CD25^? T-cells showed an increased transmigration towards supernatants of hyperthermia-treated tumour cells. After combined application of hyperthermia and irradiation, Treg migration was similar to control levels, but CD4⁺CD25^? migration was still enhanced. Irradiation caused a significantly decreased Treg influx, whereas the CD4⁺CD25^? T-cell migration was not altered after the same treatment. Changes of Treg chemotaxis could be attributed to a treatment-associated escalation of the CCL22 in the tumour cell supernatants.

Conclusion: The combination of irradiation and hyperthermia is able to modify transmigration of tumour infiltrating lymphocytes beneficially and individually. In this in vitro system hyperthermia alone negatively impacts the immune response by selectively recruiting Treg, whereas hyperthermia with the addition of irradiation negates this effect.     

Molecular biology in head and neck cancer

Major changes in the treatment of head and neck cancer are possible today because of the knowledge that we have on the molecular biology of these tumors. Different pathways are active in the development of this cancer and field cancerization is a major problem for the cure in early stage disease. Epidermal growth factor signal transduction pathway is now the principal target for this disease. New therapeutic strategies such as monoclonal antibodies and small molecules have appeared, however no more than 20% of the patients have objective responses with these therapies. Consequently, new alternatives of treatment in the basis of the understanding of molecular biology are necessary to increase the number of patients that can be cured in the future. Supported by an unrestricted educational grant by Bristol-Myers Squibb.     

Synchronous cancers in patients with head and neck cancer

BACKGROUND:

Second primary malignancies (SPMs) are the leading cause of death in survivors of head and neck squamous cell carcinoma (HNSCC). Synchronous SPMs are of significant clinical interest because they potentially can be identified by screening procedures at the time of diagnosis of the index cancer. Recently, human papillomavirus (HPV) has emerged as a distinct risk factor for oropharyngeal head and neck squamous cell carcinoma (HNSCC), differing from classic tobacco/alcohol‐associated HNSCC, suggesting that there also may be distinct patterns of synchronous SPMs.

METHODS:

The authors performed a population‐based cohort study in 64,673 patients in the National Cancer Institute Surveillance, Epidemiology, and End Results registry (1979‐2008), defining risks of synchronous SPM in patients with HNSCC who were diagnosed before and after the emergence of prevalent HPV‐associated oropharyngeal HNSCC. Excess risk was calculated using standardized incidence ratios (SIR) and excess absolute risk per 100 patients.

RESULTS:

Among patients with HNSCC, the SIR of synchronous SPM was 5.0, corresponding to 2.62 excess cases per 100 patients. The site with the highest excess risk of a second cancer was the head and neck (SIR, 41.4), followed by the esophagus (SIR, 21.8), and lung (SIR, 7.4). The risk of synchronous SPM changed markedly over time for patients with oropharyngeal HNSCC. In the 1970s and 1980s, oropharyngeal cancers carried the highest risk of SPM. Risk began to dramatically decline in the 1990s; and currently, oropharyngeal cancers carry the lowest risk of synchronous SPM.

CONCLUSIONS:

The current data are consistent with the etiologic shift of oropharyngeal HNSCC, from a primarily tobacco‐associated malignancy associated with significant field cancerization of the upper aerodigestive mucosa, to a malignancy primarily caused by oncogenic human papillomavirus. Cancer 2013. © 2013 American Cancer Society.