Cancer stem cells in head and neck squamous cell carcinoma

Recent studies have demonstrated that cancer stem cells (CSC) play an important role in the pathobiology of head and neck squamous cell carcinomas (HNSCC). This subpopulation of undifferentiated, self-renewing cells is responsible for resistance to conventional anti-cancer therapy, cancer recurrence, metastasis and ability to form a heterogeneous tumor. CSC are identified on the basis of specific markers, including membrane proteins or cell enzymes, or by using their self-renewal properties. As their resistance to standard HNSCC treatment may eventually lead to the lack of treatment success, there is an urgent need to better understanding CSC biology and identify them as potential target new treatment modality.     

Konstitutive Expression des potentiellen Stammzellmarkers CD44 in permanenten HNSCC-Zelllinien

Background

Despite significant advances and the use of new diagnosic and therapy methods to treat head and neck squamous cell carcinoma (HNSCC), prognosis has improved only marginally in the last decades. Thus, there is an enormous need for novel immunotherapeutic approaches. It is becoming more and more obvious that stem cells play an important role in tumor development and progression. The identity of these cells and the underlying cellular and molecular mechanisms remain mostly unknown in HNSCC.

Material and methods

Solid tumors as well as cells from the peripheral blood of patients with HNSCC were analyzed by flow cytometry concerning the expression of different putative stem cell marker proteins.

Results

Distinct populations of CD44-positive (CD44+), lin-negative (lin-) potential stem cells could be identified in solid tumors of HNSCC patients with strong individual variations. Surprisingly, the potential stem cell marker CD44 was found to be constitutively expressed on the surface of all permanent HNSCC cell lines analyzed.

Conclusion

These data strongly suggest that CD44+ tumor stem cells may play a key role in establishment of permanent HNSCC cell lines, selecting especially robust cell entities that in real life might drive progression and metastasis of HNSCC. Individual analysis of tumor stem cell markers will be an important tool for innovative therapies and prognosis of patients with HNSCC. Additional stem cell markers will be investigated concerning their expression and cellular function.     

Zellen der Tumorfront

Background

With a frequency of about 90?%, head and neck squamous cell carcinomas (HNSCCs) are the most common malignancies of the upper aerodigestive tract. The cancer stem cell (CSC) hypothesis postulates that CSCs are the dangerous part of the tumor and are relevant to metastasis, invasiveness and resistance to chemotherapy.

Methods

Tissue samples taken from HNSCCs and normal mucosa were tested for the expression of several established CSC markers. The expression and activity of the matrix metalloproteinase MMP-9 was also investigated.

Results

Cells of the invasive tumor front expressed the basal stem cell markers CD44, ALDH1 and CK14. However, in contrast to the noninvasive basal cell layer of normal mucosa, HNSCC samples were also positive for active MMP-9, which lends the tumor its gelatinolytic activity.

Conclusion

These observations suggest a model in which cells of the invasive front are derived from the basal cell layer of normal mucosa and harbour the CSCs. Future studies should thus focus on the cells of the invasive front in particular, since the activity of these cells may form the basis for tumor recurrence and therapy resistance.     

Implication of stem cells in the biology and therapy of head and neck cancer

Stem cells play a central role in re- and generation of tissues. Special importance has been attributed to them in cancer biology. 2 entities can be discriminated: cancer infiltrating stem cells and cancer initiating stem cells. Infiltrating stem cells will be attracted to the tumor in order to be remodelled for tumor expansion, e. g. endothelial cells or other cancerous tissue components, yet these cells are per se benign. Malignant cancer stem cells are capable to generate a new tumor, histologically identical with the cancer they originate from. Many steps in cancer stem cell biology are not understood to date. It is still believed that CSC are only a minor cell fraction in tumor but capable to differentiate in hierarchical manner into any other tissue type. These stem cells are undergoing many steps of differentiation and dedifferentiation in a steady state. The factors of the micromilieu contributing to this are largely not understood. Still these steps are regarded as the key to tumor initiation, metastases and resistance to therapy. The biological functions and associated signal transduction pathways, e. g. self renewal pathways will be the key to future therapeutical strategies.     

Identification of Genes Overexpressed in Head and Neck Squamous Cell Carcinoma Using a Combination of Complementary DNA Subtraction and Microarray Analysis

Objectives/Hypothesis To discover unique genes specific for squamous cell carcinoma of the head and neck for eventual development as tumor markers and vaccine candidates. Study Design Molecular biological analysis of fresh‐frozen head and neck squamous cell cancer (HNSCC). Methods A subtractive library was made from two HNSCC and six normal tissues using a polymerase chain reaction (PCR)–based approach. Genes from this library were PCR amplified and placed on a microarray glass slide. RNA was prepared or obtained from 16 fresh‐frozen HNSCC and 22 normal tissue sources. Fluorescent probes were made from the polyA+ RNA derived from the tumor and normal tissues. The probes were hybridized to the glass slides and excited by a tuneable laser. One hundred seven of the genes showing the highest differential fluorescence value between tumor and normal tissue were identified by sequence analysis. Results Thirteen independent genes were found to be overexpressed in tumor tissues. Of these, nine were previously known: keratins K6 and K16, laminin‐5, plakophilin‐1, matrix metalloproteinase‐2 (MMP), vascular endothelial growth factor, connexin 26, 14–3‐3 sigma, and CaN19. The level of polyA+ RNA of these genes in the tumors was significantly different from the levels in normal tissue (P < .05). Four previously unidentified genes were also discovered to have increased expression in tumor tissue. Comparing the total tumor group (n = 16) to the normal group (n = 22), only one of these genes showed significant overexpression. Conclusion We report the identification of nine known genes that are significantly overexpressed in HNSCC as compared to normal tissue using subtractive and microarray technology. In addition, we present four previously unidentified genes that are overexpressed in a subset of tumors. These genes will be developed as tumor markers and vaccine candidates.     

The ARF-p16 gene locus in carcinogenesis and therapy of head and neck squamous cell carcinoma

OBJECTIVES/HYPOTHESIS: We have identified families with a high incidence of tumors including head and neck squamous cell carcinoma (HNSCC). The occurrence of melanoma in these kindreds suggested that the ARF-p16 gene may be involved in carcinogenesis. We wished to determine the gene defect associated with the familial predisposition to HNSCC and to determine whether restoration of the gene may have therapeutic benefit. STUDY DESIGN: Translational molecular research. METHODS: Molecular techniques were used to identify mutations of the ARF-p16 gene from the affected families and to test the activity of p16 and ARF mutants. In additional, HNSCC tumor tissue was analyzed to determine whether the wild-type p16 allele was lost or maintained. ARF-expressing adenoviruses were created, and their effect on HNSCC cell lines and normal head and neck epithelial cells was determined. RESULTS: Mutation of the ARF-p16 gene was found in two families with predisposition to develop HNSCC. Independent mutations detected in the germline DNA of both families inactivated p16, but not ARF, and the inactive mutant p16 allele segregated with disease within both families. The wild-type p16 allele was lost in HNSCC tumor tissue from both families. The efficacy of ARF in treatment of HNSCC was found to depend on retention of p53 activity within HNSCC tumor cells. Remarkably, ARF expression was found to kill cells, depending on loss of retinoblastoma activity. Because loss of retinoblastoma activity is nearly universal in tumors, ARF killed tumor cells that retained p53, but ARF spared normal cells. CONCLUSIONS: Our results support the recognition of a new clinical entity of familial head and neck cancer. We have shown that this syndrome is associated with inactivating mutations of the p16 gene that these mutations segregate with disease in two described families. Loss of the wild-type p16 allele in HNSCC tissue from both families strongly supports the role of the mutant p16 in carcinogenesis. We have also investigated the therapeutic utility of the alternate reading frame product of the p16 gene, ARF. The finding that ARF kills cells depending on loss of retinoblastoma activity and retention of p53 suggests that ARF may be effective in treatment of roughly 50% of head and neck cancers while sparing normal cells. Recognition of p16 mutations as an etiological factor in familial HNSCC provides an accessible tool for diagnosis of this syndrome. Clinical acceptance of familial head and neck cancer will ensure that patients are appropriately diagnosed and managed.     

Vascular endothelial growth factor receptor 2 (VEGFR2) expression in squamous cell carcinomas of the head and neck.

OBJECTIVES: Vascular endothelial growth factor receptor 2 (VEGFR2; Flk-1 [fetal liver kinase]/KDR [kinase insert domain containing receptor]) has been identified as a high affinity receptor for vascular endothelial growth factor (VEGF) on vascular endothelium. Head and neck squamous cell carcinomas (HNSCC) have already been shown to produce substantial amounts of VEGF. VEGFR2 is supposed to play a major role in tumor-neoangiogenesis. METHODS: We investigated 24 tumor specimens and 4 HNSCC cultured tumor cell lines for the incidence and distribution of VEGFR2 by immunohistochemistry using monoclonal antibodies (mAbs) and RT-PCR. RESULTS: Analysis of frozen sections by immunohistochemistry showed that in 90% of tumor specimens VEGFR2-positive cells were found which were associated with vascular endothelium. VEGFR2 was also expressed on tumor cells and vessels, which was confirmed by double immunolabeling of tumor cells with an a-cytokeratin mAb. Furthermore, 2 (JPPA, SCC9) of 4 HNSCC cultured tumor cell lines revealed positive VEGFR2 immunoreactivity. Synthesis of VEGFR2 mRNA on all 4 HNSCC cultured tumor cell lines (JPPA, SCC9, SCC25, and LFFR) and in 6 tumor specimens was confirmed by RT-PCR. In conclusion, our results showed that VEGFR2 is expressed in HNSCCs on tumor cells. VEGFR2 expression is associated with the beginning of vasculogenesis represented by accumulation of VEGFR2-positive cells budding into new vessels ("hot spots"). The focal expression pattern of VEGFR2 on tumor cells suggests an autocrine loop for VEGF in tumor cell growth.     

Molecular diagnostic alterations in squamous cell carcinoma of the head and neck and potential diagnostic applications

Head and neck squamous cell carcinoma (HNSCC) is a common malignancy that continues to be difficult to treat and cure. In many organ systems and tumor types, there have been significant advances in the understanding of the molecular basis for tumorigenesis, disease progression and genetic implications for therapeutics. Although tumorigenesis pathways and the molecular etiologies of HNSCC have been extensively studied, there are still very few diagnostic clinical applications used in practice today. This review discusses current clinically applicable molecular markers, including viral detection of Epstein–Barr virus and human papillomavirus, and molecular targets that are used in diagnosis and management of HNSCC. The common oncogenes EGFR, RAS, CCND1, BRAF, and PIK3CA and tumor suppressor genes p53, CDKN2A and NOTCH are discussed for their associations with HNSCC. Discussion of markers with potential future applications is also included, with a focus on molecular alterations associated with targeted therapy resistance.     

Expression der immun- supprimierenden Zytokine TGF-β2 und Il-10 in Kopf-Hals-Tumoren Vergleich von Serumwerten und Gewebeexpression

Transforming growth factor-beta (TGF-beta) and interleukin 10 (Il-10) are cytokines that have a strong immunosuppressive ability. Their secretion by tumor cells is able to suppress an immunological response against tumor. Both factors have been shown to enhance tumor growth in glioblastomas and carcinoma of the breast. We determined the expression pattern of TGF-beta and Il-10 in squamous cell carcinomas of the head and neck (HNSCC) and a possible association with tumor stage and their pre-treatment cytokine serum levels. Cytokine expression in primary tumors and metastases of 21 patients with HNSCC was investigated by immunohistochemistry. To assess the TGF-beta2 and Il-10 levels in tumor patients before therapy 49 serum specimens were analyzed by ELISA. TGF-beta2 was detected in 95% of all tumor tissues analyzed and Il-10 in 79% of all tumors. TGF-beta2 was localized in tumor cells and tumor borders, while Il-10 was preferentially found in peritumoral connective tissue. Metastasizing tumors showed elevated pretreatment serum levels for TGF-beta2 and Il-10. There was no correlation between TGF-beta2 and Il-10 expression in tumor tissue and pretreatment serum levels. Our data show that the majority of HNSCC analyzed express TGF-beta2 and Il-10. A correlation between pretherapy elevated cytokine serum levels and tumor grade was shown.     

Molecular pathogenesis of head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) represents 6% of all cancers. The overall 5-year survival rate for patients with this type of cancer is among the lowest of the major cancer types and has not improved dramatically during the last decade. The pathological staging, in particular the nodal stage, is the most important factor in HNSCC. The lack of progress in head and neck oncology emphasizes the importance of molecular genetic studies to define alterations that may correlate with tumor behavior. The molecular alterations observed in HNSCC are mainly due to oncogene activation and tumor suppressor gene inactivation, leading to deregulation of cell proliferation. These alterations include gene amplification and overexpression of oncogenes such as ras, myc, EGFR and cyclin D1, and mutations and deletions leading to p16 and TP53 tumor suppressor genes inactivation. This article reviews the molecular changes commonly observed in HNSCC. The biological function of these markers and the potential clinical application are discussed. Advances in the understanding of the molecular basis of HNSCC will help in the identification of new molecular markers that could be used for a more accurate diagnosis and assessment of prognosis and may open the way for novel approaches to treatment and prevention.     

Expression of vascular endothelial growth factor receptors on tumor cells in head and neck squamous cell carcinoma

BACKGROUND: Angiogenesis is essential for the growth of solid tumors, including head and neck squamous cell carcinoma (HNSCC). Angiogenesis is regulated by angiogenic factors such as vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) 1, 2, and 3 known to be located on vascular endothelial cells (VECs). We hypothesize that VEGFRs are also expressed on HNSCC tumor cells in vitro and in vivo and likely control tumor function in vivo. DESIGN: Immunohistochemical analysis for VEGFR-1 (n = 13), VEGFR-2 (n = 21), and VEGFR-3 (n = 16) was performed on human HNSCC tumor samples. Specimens were analyzed for receptor expression and staining intensity. A cultured oral SCC cell line (SCC-25) and a pharyngeal SCC cell line (FADU) were also studied for receptor expression. RESULTS: The HNSCC tumor cells expressed VEGFR-1, VEGFR-2, and VEGFR-3 in all specimens evaluated. Staining for all 3 receptors was also found on tumor-associated macrophages and fibroblasts, except that VEGFR-2 was not present on fibroblasts. Staining intensity for VEGFR-1 and VEGFR-2 was significantly higher in tumor cells and macrophages than in VECs stained for the same receptor. Both cultured HNSCC cell lines demonstrated expression of all 3 receptors. CONCLUSIONS: This represents the first report of all 3 VEGFRs being expressed by HNSCC cells. These findings indicate that VEGF may be an autocrine regulator of tumor cell activity in addition to its known angiogenic effects on VECs. The presence of VEGFRs on tumor-associated macrophages and fibroblasts contributes to the complexity of the VEGF/VEGFR system in human cancer.     

Selective Delivery of a Therapeutic Gene for Treatment of Head and Neck Squamous Cell Carcinoma Using Human Neural Stem Cells

Objectives

Based on studies of the extensive tropism of neural stem cells (NSCs) toward malignant brain tumor, we hypothesized that NSCs could also target head and neck squamous cell carcinoma (HNSCC) and could be used as a cellular therapeutic delivery system.

Methods

To apply this strategy to the treatment of HNSCC, we used a human NSC line expressing cytosine deaminase (HB1.F3-CD), an enzyme that converts 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU), an anticancer agent. HB1. F3-CD in combination with 5-FC were cocultured with the HNSCC (SNU-1041) to examine the cytotoxicity on target tumor cells in vitro. For in vivo studies, an HNSCC mouse model was created by subcutaneous implantation of human HNSCC cells into athymic nude mice. HB1.F3-CD cells were injected into mice using tumoral, peritumoral, or intravenous injections, followed by systemic 5-FC administration.

Results

In vitro, the HB1.F3-CD cells significantly inhibited the growth of an HNSCC cell line in the presence of the 5-FC. Independent of the method of injection, the HB1.F3-CD cells migrated to the HNSCC tumor, causing a significant reduction in tumor volume. In comparison to 5-FU administration, HB1.F3-CD cell injection followed by 5-FC administration reduced systemic toxicity, but achieved the same level of therapeutic efficacy.

Conclusion

Transplantation of human NSCs that express the suicide enzyme cytosine deaminase combined with systemic administration of the prodrug 5-FC may be an effective regimen for the treatment of HNSCC.     

The Stem Cell Network model: clinical implications in cancer

This review will discuss the aspects of stem cell biology that can contribute to explain tumor development and why standard oncology treatments sometimes fail. We also propose an integrated model of tumor progression based on the putative occurrence of Stem Cell Networks (SCNs). In a SCN, the somatic stem cells are derived from a common embryonic stem cell, share a specific molecular profile and maintain a high degree of cell-cycle synchronization. In the study of cancer, the SCN model introduces an additional conceptual frame to the interpretation of both the cancer stem cell (CSC) hypothesis and the field cancerization concept. The CSC model may explain how the cancer fields develop, justifies their sizes and shapes, contribute to explain the local recurrences in patients with free margins, the second primary tumors, the success of organ preserving surgical approaches or the trend of different tumors to metastasize to certain locations. We propose that the SCN model of cancer provides some clues for further understanding tumor progression and raises promising experimental and clinical implications.     

Funktionelle Expression des VEGF-Rezeptors Flt-1 auf Plattenepithel- karzinomzellen

Vascular endothelial growth factor (VEGF) is one of the most potent factors in tumor-induced neoangiogenesis. After binding to its specific receptors KDR and FLT-1 on the endothelial cell surface cell proliferation and migration are stimulated. Recently there has been some evidence for the expression of these receptors on tumor cells. We investigated the protein and mRNA expression of KDR and FLT-1 in native tissues and tumor cell cultures from squamous cell carcinomas of the head and neck (HNSCC) and analyzed their in vitro functional significance for tumor cell proliferation and migration. Apart from the expected expression of VEGF receptors on endothelial cells we observed a tumor cell-specific localization of FLT-1 in 29 tumors and KDR in 16 of 37 tumors analyzed. Functional studies in vitro revealed that the addition of VEGF to HNSCC cells inhibited the proliferation and migration of these cells in a dose-dependent manner. Our data suggest a potential negative regulatory loop for VEGF and FLT1 when tumor cells have an insufficient blood supply.     

Expression of EphB4 in head and neck squamous cell carcinoma

EphB4 is a receptor tyrosine kinase that is expressed on epithelial cells during fetal life. It is also expressed on some venous endothelial cells. We conducted a study of six men with primary squamous cell carcinoma of the head and neck (HNSCC) that had metastasized to the cervical lymph nodes. Our goal was to determine if EphB4 is aberrantly expressed in cases of HNSCC and to determine if there is a qualitative difference between the expression of EphB4 on primary and metastatic tumors and its expression on normal mucosa adjacent to primary tumors. From each patient, we obtained specimens of the primary tumor, the nodal metastasis, and the adjacent normal mucosa, and we performed immunocytochemistry on each. We observed EphB4 expression in all primary and metastatic tumors and no expression in the normal tissue. In each of the six patients, expression was greater in the metastatic tumor than in the primary tumor. We conclude that EphB4 is a novel target in the treatment of HNSCC.     

Centrosome hyperamplification in head and neck squamous cell carcinoma: a potential phenotypic marker of tumor aggressiveness

OBJECTIVES/HYPOTHESIS: There is currently no single histological or genotypic marker that reliably predicts the biological behavior of head and neck squamous cell carcinoma (HNSCC). While multiple genetic mutations have been investigated, no single genotypic alteration has consistently correlated with tumor aggressiveness. Phenotypic markers may prove more predictive, because they can represent many different genetic alterations. We investigated the frequency of centrosome hyperamplification in HNSCC and examined its usefulness as a marker for tumor recurrence. STUDY DESIGN: Analysis of archived paraffin blocks using immunohistochemistry. METHODS: Eighteen patients who underwent resection of oral cavity squamous cell carcinoma were reviewed. Ten patients had cancers that recurred locally within 1 year of resection, and 8 patients were tumor free at 5 years. The amount of centrosome hyperamplification in the cancer specimens and all surgical margins was graded as follows: 0, none; 1+, rare hyperamplification; 2+, greater than 10% of cells per high-powered field; and 3 +, greater than 20% of cells per high-powered field. RESULTS: Centrosome hyperamplification was found in 17 of 18 tumors (94%). Grade 2+ or 3+ hyperamplification was found more in cancers that recurred (9 of 10) than in those that did not (3 of 8) and was more prevalent in the histologically normal margins of patients with recurrence (8 of 10) than in those without recurrent cancer (3 of 8). CONCLUSIONS: Our results demonstrate the extremely frequent occurrence of centrosome hyperamplification in HNSCC. Centrosome hyperamplification is a phenotypic marker for HNSCC and can reflect multiple genotypic changes. Its presence in histologically normal margins suggests that it may be useful for analysis of primary tumors and tumor margins.     

Immuntherapie von Kopf-Hals-Karzinomen

In order to improve the prognosis for patients with head and neck squamous cell cancer (HNSCC) the introduction of new therapeutic strategies is necessary. The concept of immunotherapy has been applied and improved for several years and recent studies have used tumor-specific antigens which facilitates targeted oncologic therapy. However, immunotherapy is hampered by the fact that immunosuppressive mechanisms are pronounced and relevant effector cells are suppressed, especially in patients with HNSCC. Successful immunotherapy could induce an antitumor immune response by restitution of these cell populations. Current anti-tumor immunotherapy includes unspecific immune stimulation, genetic modification of tumor and immune cells, the use of monoclonal antibodies, e.g. cetuximab, adoptive cell transfer and tumor vaccination. In the future, these biologic therapies alone or in combination with conventional therapeutic regimens could present a valuable therapeutic option for HNSCC patients.     

Familial head and neck cancer: molecular analysis of a new clinical entity

OBJECTIVE: The tumor suppressor gene p16 encodes a cyclin-dependent kinase inhibitor that normally inhibits cell proliferation by causing a G1 cell cycle arrest. The p16 gene is frequently mutated in a variety of somatic tumors, as well as in familial melanoma and familial pancreatic carcinoma. We identified a family with a high incidence of head and neck squamous cell carcinoma (HNSCC) and melanoma. Molecular analyses of the p16 gene locus in blood and tumor DNA from this family was performed to determine whether an association between germline p16 gene mutation and HNSCC exists. STUDY DESIGN: Molecular pedigree analyses. METHODS: Exon 2 of p16 was polymerase chain reaction amplified from blood, tumor, or nontumor DNA isolated from affected and unaffected members, then directly sequenced and compared with consensus p16 sequence. Cell cycle position of cells expressing wild-type or mutant p16 was determined by flow cytometry. RESULTS: Molecular analyses revealed a nonfunctional germline point mutation within exon 2 of the p16 gene that encodes a mutant p16 protein substituting proline at amino acid position 87 for the wild-type arginine (p16R87P). Relative to wild-type p16, p16R87P lost ability to cause a growth arrest following ectopic expression. The mutant (p16R87P) allele segregated with cancer predisposition in tested family members, and analyses of HNSCC tumor tissues demonstrated universal loss of wild-type allele. CONCLUSIONS: Significance of the mutant p16 (p16R87P) in HNSCC tumorigenesis is strongly suggested by its loss of cell cycle arrest activity and its retention in tumor tissue with simultaneous loss of the wild-type allele. Further, the germline p16 mutation segregated with cancer predisposition within the family. In aggregate, these data suggest that there is a direct causal relationship between the germline p16 mutation in this family and HNSCC tumorigenesis. Based on our observations, the spectrum of familial cancers associated with p16 mutations should include a new clinical entity, familial HNSCC.     

头颈鳞状细胞癌乏氧微环境与放疗抵抗相关性的研究进展

头颈鳞状细胞癌(HNSCC)的治疗方式有手术和放化疗.治疗后HSNCC的高复发率及其显著的转移能力是影响HNSCC患者预后的主要因素,且放化疗存在各种并发症,包括放射性口腔黏膜炎、局部软组织损伤等,严重影响患者生活质量.而肿瘤局部组织内乏氧造成的乏氧微环境与肿瘤治疗抵抗和复发的关系密切相关,如果能采取措施改善肿瘤微环境...