Human squamous cell carcinomas of the head and neck chemoattract immune suppressive CD34(+) progenitor cells

CD34(+) progenitor cells have previously been shown to be mobilized in patients with squamous cell carcinoma of the head and neck (HNSCC). The present study showed that these CD34(+) cells inhibit the capacity of intratumoral lymphoid cells to become activated in response to stimulation through the TCR/CD3 complex. The mechanisms that could lead to the accumulation of CD34(+) cells within the tumor tissue were assessed. This was accomplished through in vitro studies that determined if HNSCC produce soluble factors that chemoattract CD34(+) cells. The migration of cord blood CD34(+) cells, which were used as a readily available source of progenitor cells, was stimulated by products derived from HNSCC explants and primary HNSCC cultures. This stimulated migration was due to chemotaxis because it was dependent on an increasing gradient of HNSCC-derived products. CD34(+) cells that were isolated from the peripheral blood of HNSCC patients were similarly chemoattracted to the HNSCC-derived products. The majority of the chemotactic activity produced by HNSCC could be attributed to vascular endothelial cell growth factor (VEGF). These studies indicate that HNSCC can chemoattract immune inhibitory CD34(+) progenitor cells through their production of VEGF.     

Epidemiologic survey of head and neck cancers in Korea

Head and neck cancers have never been systematically studied for clinical purposes yet in Korea. This epidemiological survey on head and neck cancer patients was undertaken from January to December 2001 in 79 otorhinolaryngology resident-training hospitals nationwide. The number of head and neck cancer patients was 1,063 cases in the year. The largest proportion of cases arose in the larynx, as many as 488 cases, which accounted for 45.9%. It was followed by, in order of frequency, oral cavity (16.5%), oropharynx (10.0%), and hypopharynx (9.5%). The male:female ratio was 5:1, and the mean age was 60.3 yr. Surgery was the predominant treatment modality in head and neck cancers: 204 (21.5%) cases were treated with only surgery, 198 (20.8%) cases were treated with surgery and radiotherapy, 207 cases (21.8%) were treated with combined therapy of surgery, radiotherapy, and chemotherapy. Larynx and hypopharynx cancers had a stronger relationship with smoking and alcohol drinking than other primary site cancers. Of them, 21 cases were found to be metastasized at the time of diagnosis into the lung, gastrointestinal tract, bone, or brain. Coexisting second primary malignancies were found in 23 cases. At the time of diagnosis, a total of 354 cases had cervical lymph node metastasis accounting for 42.0%.     

A natural cytokine mixture (IRX-2) and interference with immune suppression induce immune mobilization and regression of head and neck cancer

Prior studies indicate that combination immunotherapy of squamous cell cancer (SCC) of head and neck (H&N) with cytokines is feasible (Hadden et al., 1994). To induce immune regression of H&N SCC 20 stage II–IV patients received 3 weeks prior to surgery low dose cyclophosphamide (300 mg/M²), then 10 daily perilymphatic injections of a natural cytokine mixture (IRX-2) (150 units of IL-2 equivalence) and daily oral indomethacin and zinc.Tumorresponses, T-lymphocyte and subset counts, and toxicity were monitored. Six patients had major clinical responses (both complete [CR] and partial [PR]) without major toxicity. Five of 20 patients were lymphocytopenic (1242±88 mm³) prior to treatment and the immunotherapy induced marked significant increases in total lymphocyte counts, CD3+ T-cells, and both CD4+ and CD8+ T-cells as well as a population of CD3+, CD4−, and CD8− lymphocytes.The post treatment specimen of 18/20 patients showed histologically tumor fragmentation, overall reduction and diffuse infiltration with lymphocytes and plasma cells. Histologic tumor reductions in these patients averaged 44% and the lymphoid infiltration increased 4.7 fold from 9–42%. The immune infiltration of the tumor reflects varying degrees of both T- and B-cells and indicates immunization to the tumor. The immunization achieved may improve clinical control of H&N SCC by improving the possibility that surgical resection of advanced loco-regional disease will leave no viable tumor.     

The relationship between occupations and head and neck cancers

OBJECTIVE: The objective of this study was to investigate the relationship between occupation and head and neck cancers. PATIENTS AND METHODS: In this case-control study, 206 Turkish patients with head and neck cancers comprised the case group. The control group consisted of 206 age- and sex-matched patients without malignant disease. All patients completed a questionnaire regarding occupation; tobacco and alcohol consumption; educational status; and history of any systemic disease, benign head and neck disease, and cancer among family members. High-risk jobs were considered those in the industries of construction, wood, mining, metal, chemistry and agriculture. RESULTS: Patients with head and neck cancers worked in high-risk occupations more frequently than did controls [odds ratio (OR): 3.42, p<0.05]. Cancer risk decreased with the increase in time interval between quitting the high-risk job and time of interview. Smokers were at higher risk than nonsmokers (OR: 3.33, p<0.05). The risk was also higher in patients who drank alcohol regularly (OR: 1.59, p<0.05). However, occupation was found to be an independent high-risk factor for head and neck cancers in regression analysis. Frequency of benign head and neck disease and family history of cancer were not significant risk factors (p>0.05). CONCLUSION: Our analysis showed that occupation and smoking were significant independent risk factors for the development of head and neck cancers among workers.     

Anergy testing in patients with head and neck cancer

Patients with head and neck cancer were found to be deficient in were not clear [correction] Possible explanations include a change in T-lymphocyte numbers, particularly the helper/suppressor T-cell ratio, with the cause of this change still unknown. Tumor immunosuppressing factors and cancer-induced immunosuppression are proposed to be such causes. The deficiency of T cells resulted in an impaired cell-mediated immune response (CMIR), which lowered the host resistance, such facilitating the tumor to spread. As the CMIR can be evaluated by delayed hypersensitivity skin testing (= anergy screen), the objective of this study was to compare the CMIR function of patients with head and neck cancer to a non-cancer control group using this anergy screen. The study group consisted of 20 patients (17 males, 3 females, age range 10-76 years) with head and neck cancer, which were anti-HIV negative and had not received any therapy yet. The control group consisted of another 20 persons (17 males, 3 females, age range 21-72 years) without any cancer and who were also anti-HIV negative. Exclusion criteria were (1) eczema or skin disease in the area to be tested, (2) having received oral prednisolone within the last week and (3) an anti-HIV positive immune status. The antigens used in this study consisted of PPD (5 IU), tetanus toxoid (TT) (0.8 LF/ml and 1.6 LF/ml, Candida albicans (20 PNU/ ml and 200 PNU/ml), mumps-measles-rubella (MMR) vaccine (1:10 v/v and 1:5 v/v). The test was done by intradermal injection of 0.1 ml of each antigen. The anergy screen was considered positive when the test resulted in an erythema or induration larger than 5 mm at 72 hours after the injection. Complete anergy was diagnosed when there was no skin reaction at all, partial anergy when only 1 antigen tested positive and no anergy when there were positive skin reactions to two or more antigens. In the study group, 9 (45%) patients were diagnosed with complete anergy, 11 (55%) with partial anergy and none with no anergy, while in the control group, none were complete anergic, 3 (15%) were partially anergic and 17 (85%) had no anergy. There was a statistically significant difference (p < 0.01) between these two groups. In conclusion, patients with head and neck cancer seemed to have an impaired CMIR, with at least the partial anergy being statistical significantly different compared to the non-cancer group.     

Hit and run oncogeneses in head and neck cancers requires greater investigation

Head and neck cancers are unique in so far that two major oncogenic viruses, Epstein Barr virus (EBV) and Human papillomavirus (HPV) infect adjacent anatomy and cause nasopharyngeal and oropharyngeal cancers, respectively. Dominant recognized carcinogens are alcohol and tobacco but some head and neck cancers have been found to have mixed carcinogens (including betel leaf, areca nuts, slaked lime, viruses, etc.) involved in their oncogenesis and conversely, groups of patients with unknown or less dominant carcinogens involved in their development. These cancers may have had viral involvement in the past but then lost most of their viral nucleic acids (be they DNA and/or RNA) below a detection threshold, thus rendering them virus-negative. Some of these virus-negative tumors appear to have mutagenic signatures associated with virus-positive cancers,  for example, from the APOBEC defense mechanism which is known to mutate viral nucleic acids as well as cause collateral damage to host DNA, with subsequent development of strongly viral prejudiced mutational signatures. These mechanisms are likely to be less efficient at oncogenesis than traditional EBV and HPV oncogenes directly driving mutagenesis, thus accounting for the smaller frequencies of these cancers found. More profound investigations of these unusual tumors are warranted to dissect out these mechanistic pathways.     

Immunomagnetic separation for enrichment and sensitive detection of disseminated tumour cells in patients with head and neck SCC.

Screening for malignant cells in the blood and bone marrow was introduced as a strategy for the improved detection of tumour spread and may predict the development of distant metastases. The sensitivity of these approaches depends on several factors, including the choice of antibody for immunocytochemistry (ICC) and the number of cells examined. In this study criteria have been defined for scoring cells reactive with a pan-cytokeratin antibody as tumour, by comparing immunostained cells in clinical samples obtained from head and neck cancer patients and a control group without epithelial malignancy. When leucocyte subfractions are prepared by density gradient separation (DGS) from central venous blood obtained from patients with advanced head and neck squamous cell carcinoma (SCC) and screened by ICC, epithelial tumour cells sediment preferentially with the mononuclear cells but may also be detected in the granulocyte (GC) fraction. Some cases were found to have more tumour cells in the GC fraction. Similar results were seen in model experiments. To increase the sensitivity of the ICC approach, the efficiency of positive immunomagnetic selection (IMS) using Dynabeads coated with an antibody recognizing the Ber-EP4 epitope has been compared with negative IMS using anti-CD45 Dynabeads. Tumour cells were recovered from bone marrow aspirates for 2/17 cases using the positive enrichment technique and for 11/17 patients following negative IMS. These findings justify prospective studies incorporating negative IMS to establish the prognostic significance of these disseminated tumour cells for this group of patients.     

The characteristics of human papillomavirus DNA in head and neck cancers and papillomas

AIM: To determine the prevalence, type, physical state, and viral load of human papillomavirus (HPV) DNA in cases of head and neck cancer and recurrent respiratory papillomatosis (RRP). METHODS: The prevalence and type of HPV DNA was determined in 27 fresh frozen tissue specimens from patients with head and neck cancers and 16 specimens from 10 patients with RRP by MY09/MY11 and GP5+/GP6+ nested polymerase chain reaction (PCR) and subsequent restriction enzyme cleavage. The physical state of HPV DNA was analysed by E1, E2, and E1E2 specific PCRs and Southern blot hybridisation (SBH). RESULTS: HPV DNA was detected in 13 of 27 cancers and 10 of 10 papillomas. Both low risk HPV-6 and HPV-11 and high risk HPV-16 were present in cancers in low copy numbers, whereas papillomas exclusively harboured low risk HPV-6 and HPV-11. E1E2 PCRs failed to determine the physical state of HPV in cancers except one case where HPV-6 DNA was integrated. In contrast to cancers, all papillomas showed the episomal state of HPV DNA and a relatively higher viral load. CONCLUSIONS: Based on the prevalence, type, physical state, and copy number of HPV DNA, cancers and papillomas tend to show a different HPV DNA profile. The 100% positivity rate of low risk HPV types confirms the role of HPV-6 and HPV-11 in the aetiology of RRP.     

Demographic and risk factors in patients with head and neck tumors

The association between human papillomavirus (HPV) infection and the development of head and neck cancer has been documented recently. In this study on 86 head and neck cancer patients and 124 controls, data regarding demographics, behavioral risk factors, and risks related to HPV exposure were collected. HPV detection was carried out using polymerase chain reaction in the tumors and in oral exfoliated cells, and HPV typing by a reverse line blot assay specific for 37 HPV types. Sera were tested by an enzyme‐linked immunosorbent assay specific for HPV proteins. Head and neck cancer cases report significantly more oral‐anal contact (P = 0.02) and tobacco and alcohol use than controls (P = 0.001; P = 0.02, respectively). High‐risk HPV DNA was detected in 43% of oral washings of cases and 4% of controls (P < 0.0001). The association between the presence of high‐risk HPV DNA in oral exfoliated cells and in tumor tissues was statistically significant (adjusted P < 0.0001). The prevalence of HPV‐specific antibodies was significantly higher in cases than in controls (adjusted P < 0.0001). These results provide epidemiological and immunological evidence for HR HPV as a strong risk factor (OR = 44.3, P < 0.0001) for head and neck cancer, even after controlling for age, tobacco and alcohol use. The detection of high‐risk HPV DNA in oral exfoliated cells and HPV‐specific antibodies in serum can be considered as clinically relevant surrogate markers for the presence of a HPV‐associated head and neck cancer, with a high sensitivity (83%) and specificity (88%). J. Med. Virol. 81:878–887, 2009. © 2009 Wiley‐Liss, Inc.     

Cultures derived from peripheral blood CD34+ progenitor cells of head and neck cancer patients and from cord blood are functionally different

Patients with head and neck squamous cell carcinoma (HNSCC) have profound immune defects mediated, in part, by an increased number of immune suppressive CD34⁺ progenitor cells in their peripheral blood and tumor. One means of overcoming this immune suppression is to stimulate the CD34⁺ cells to differentiate into more mature, nonsuppressive progeny such as dendritic cells or monocytes. This study determined that CD34⁺ cells from the peripheral blood of HNSCC patients have the same potential to differentiate into dendritic cells as do human umbilical cord blood CD34⁺ cells following 12–16 days of culture with a cytokine cocktail. When compared functionally, the cultures that developed from CD34⁺ cells of cord blood were able to induce an allostimulatory response in naive T-cells, while the cultures that developed from patient CD34⁺ cells lacked allostimulatory ability. Both cultures expressed class II MHC (HLA-DR), but the proportion of cells expressing the costimulatory molecules CD80 and CD86 was significantly less in cultures that developed from HNSCC-patient CD34⁺ cells. Therefore, although the CD34⁺ cells from the peripheral blood of HNSCC patients can differentiate into dendritic cells, their allostimulatory capabilities are impaired, raising the question of their potential effectiveness in stimulating antitumor immune responses.     

Interferon-gamma and interleukin-10 production by mononuclear cells from patients with advanced head and neck cancer

OBJECTIVE:

This study aims to evaluate the production of interferon-gamma and interleukin-10 by stimulated peripheral blood mononuclear cells isolated from patients with supraglottic laryngeal cancer before and after surgical treatment.

METHODS:

Fourteen patients with advanced supraglottic laryngeal cancer were studied. Cultures of peripheral blood mononuclear cells isolated during the preoperative and late postoperative periods were stimulated with concanavalin A and Bacille Calmette-Guérin, and the supernatant concentrations of interferon-gamma and interleukin-10 were measured.

RESULTS:

For non-stimulated cultures, the interferon-gamma levels produced by the preoperative period and the late postoperative period cultures were lower than the levels produced by the control group cultures. The interferon-gamma levels after stimulation with concanavalin A were higher in the late postoperative period cultures than in the preoperative evaluation cultures. Stimulation with Bacille Calmette-Guérin led to the production of similar levels of interferon-gamma and interleukin-10 by all cultures; thus, stimulation increased the levels of interferon-gamma produced by both the preoperative and postoperative cultures relative to the levels produced by the corresponding unstimulated cultures.

CONCLUSION:

Patients with advanced supraglottic laryngeal cancer exhibit an in vitro deficiency in interferon-gamma secretion by mononuclear cells. Stimulated cells seem to recover this function during the postoperative period.     

Simultaneous radiochemotherapy in the treatment of inoperable, locally advanced head and neck cancers

The results of radiation therapy alone in locally advanced head and neck cancers are dismal with 5 year locoregional control rates not exceeding 15%. The addition of concomitant chemotherapy with cisplatin and more recently carboplatin has shown promising results. Twenty patients of inoperable stage III and IV oral or oropharyngeal cancers were treated with concomitant chemoradiation with carboplatin 300 mg/m2 i.v. on days 1, 21 and 42 of radiation therapy. Twelve (60%) patients had a complete remission. Thirteen patients were alive at a median follow up of 11 months. The treatment was well tolerated with only 2 patients requiring treatment interruptions for mucositis. Longer follow up would reveal any improvement in overall survival. The relative ease with which carboplatin/RT was administered suggests that other agents might be added as well.     

Regulatory T cells: immune suppression and beyond

Foxp3-expressing regulatory T cells (Tregs) were originally identified ascritical in maintaining self-tolerance and immune homeostasis. Theimmunosuppressive functions of Tregs are widely acknowledged and have beenextensively studied. Recent studies have revealed many diverse roles of Tregs inshaping the immune system and the inflammatory response. This review willdiscuss our efforts as well as the efforts of others towards understanding themultifaceted function of Tregs in immune regulation.     

Hyaluronan-grafted particle clusters loaded with Mitomycin C as selective nanovectors for primary head and neck cancers

CD44, a well-documented cell surface receptor, is involved in cell proliferation, migration, signaling, adhesion, differentiation and angiogenesis, which are important properties for normal and cancerous cell function. We recently developed particle clusters coated with hyaluronan (termed gagomers; GAG), and showed that they can deliver the insoluble drug paclitaxel directly into CD44-over-expressing tumors in a mouse tumor model. Here, we tested primary head and neck cancers (HNC) and normal cells taken from the same patient, and found that although CD44 expression in both types of cells was high, GAGs bind only to the cancerous cells in a selective manner. We next formulated the anti cancer agent mitomycin C (MMC) in the GAGs. MMC-based chemoradiation is a potential treatment for HNC, however, due to patient's toxicity, MMC is not part of the standard treatment of HNC. MMC encapsulation efficiency was about 70% with a half-life drug efflux of 1.2 ± 0.3 days. The Ex vivo study of the targeted MMC-GAG showed significant increase in the therapeutic effect on HNC cells (compared to free MMC), while it had no effect on normal cells taken from the same patient. These results demonstrate the specificity of the nanovectors towards head and neck cancers, which might be applicable as future therapy to many CD44-expressing tumors.     

Genetic imbalances with impact on survival in head and neck cancer patients

Chromosomal imbalances in 113 primary head and neck squamous cell carcinomas (HNSCCs) determined by comparative genomic hybridization were correlated with patients survival using custom-made computer software which enabled the assessment of individual chromosomal loci. The Kaplan-Meier analysis revealed that overrepresentations of 2q12, 3q21-29, 6p21.1, 11q13, 14q23, 14q24, 14q31, 14q32, 15q24, 16q22, and deletions of 8p21-22 and 18q11.2 were significantly associated with both shorter disease-free interval and disease-specific survival in this tumor collective. Multivariate Cox proportional hazards regression models consistently identified the gains of 3q21-29, 11q13, and the loss of 8p21-22 as independent prognostic markers carrying a higher significance than the nodal status as the only clinicopathological parameter with statistical importance. In addition, these three markers allowed a molecular dissection of the patients with low clinical risk (pN0 and pT2 tumors). Thus, the genomic data being derived from the evaluation of primary HNSCC enabled a stratification of the patients into subgroups with different survival highlighting the necessity of a genetically based tumor classification for refining diagnosis and treatment of HNSCC patients.     

Beta-HPV types in patients with head and neck pathology and in healthy subjects

BackgroundHuman papillomaviruses (HPV) are a heterogeneous group of viruses classified into five genera. The beta-HPV type (beta-PV) infection is very common but mostly asymptomatic in immunocompetent individuals. However, beta-PVs play a role in Epidermodysplasia verruciformis and possibly in non-melanoma skin cancer. Head and neck cancer (HNC) is a common cancer type worldwide and high-risk alpha-PV involvement in HNC has been extensively studied but beta-PV types have rarely been the focus of such studies.ObjectivesTo evaluate the prevalence of beta-PV types in HNC, subjects with non-malignant or potentially pre-malignant oral lesions, and healthy controls.Study designThe frequency of different beta-PVs in samples from oral (n = 35) and oropharyngeal (n = 35) cancer patients, gender- and age-matched healthy controls (n = 70), and subjects with various non-malignant or potentially pre-malignant oral lesions (n = 102) was assessed by a highly sensitive, bead-based, multiplex genotyping assay.ResultsOverall, 54.8% of all tested samples contained at least one beta-PV type. Even though the correlation between types found in lavage and tissue specimens from cancer patients was low, there was a large statistically significant difference between oropharyngeal cancer patients and matched controls for HPV5 (P = 0.003; OR = 15.58) and between both oral (P = 0.026; OR = 5.7) and oropharyngeal cancer patients (P = 0.002; OR = 25.5) and controls for HPV122. In addition, there was no correlation between the prevalence of alpha and beta-PVs in the study patients.ConclusionThe study provides new data on the prevalence of beta-PVs in HNC. HPV5 was found significantly associated with HNC as already observed by other studies. Additionally, the significant association of HPV122 with HNC might warrant further study as this type has not been extensively studied so far.     

Assessment of sensitivity and specificity of immunohistochemical staining of p53 in lung and head and neck cancers.

Thirty-two primary carcinomas of the lung and 17 carcinomas of the head and neck (HN) were systematically analyzed for p53 mutations in the highly conserved regions of the gene (exons 5-8). Frozen sections of the same tumors were stained immunohistochemically to assess the sensitivity and specificity of p53 expression as determined by the presence or absence of the protein. On the basis of histology, the lung tumors studied were divided into adenocarcinomas (AC; n = 15), squamous-cell carcinomas (SCC; n = 12), and large-cell carcinomas (LCC; n = 5). All the HN cancers were SCC. Mutations in the p53 gene were detected by direct sequencing of amplified polymerase chain reaction products in six AC of the lungs (40%), three SCC of the lungs (25%), and one LCC (20%), with an overall mutation frequency of 31%. Nine AC (60%) of the lungs, five SCC (42%), and four LCC (80%) were p53-positive by immunohistochemistry. Among HN cancers, p53 mutations were detected in seven tumors (41%). Nine HN tumors (53%) were positive for p53. Negative staining, despite the presence of p53 mutations, was confined to nonsense mutations with truncated p53 and to single-base mutations not causing any change in the amino acid. Although immunohistochemical staining for mutated p53 is sensitive and simple to perform as a screening method, it is not as specific for evaluation of p53 mutations in lung and HN cancers.     

双阴性调节性T细胞在免疫抑制中的作用及其机制

αβTCR+CD3+CD4-CD8-双阴性调节性T细胞(DN Treg细胞）被证实具有通过对效应性T细胞的直接杀伤作用从而抑制免疫应答的能力。DN Treg细胞与特异性抗原接触后增加其调节活性,该过程部分通过其对抗原呈递细胞表面MHC抗原肽复合物的识别作用介导。DN Treg细胞与靶细胞接触后可通过Fas和Fas配体之间的交互作用介导其杀伤效应。对DN Treg细胞功能特性、分子表达方式及其激活机制的深入研究将有助于探索临床新的治疗手段。