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Immunotherapy against head and neck cancer stem cells Immunologic therapies like antibodies in solid tumors like squamous cell cancer of the head and neck are administered either alone or in combination with radiation and chemotherapy. Despite some respectable successes, the effect of this therapy reaches its limits due the ability of the tumor to escape the immune system. Cancer stem cells seem to play an important role in this process due to their intrinsic resistance to conventional therapy and the ability to regenerate tumor heterogeneity. This way they substantially contribute to the formation of recurrences and metastases. Therefore, future immunotherapies should target specifically this subpopulation, possibly in combination with other therapeutic modalities. In this review the immunologic features of cancer stem cells and their potential as target for immunotherapies is summarized., (© Georg Thieme Verlag KG Stuttgart · New York.)

Perioperative immunonutrition is aiming at modulating altered immunological and metabolic functions in the context of major surgery. It is defined as the supplementation of constitutionally essential substrates such as glutamine, arginine, omega-3-fatty acids or nucleotides. The application of such formula is recommended for patients undergoing major abdominal-surgical procedures and tumour surgery in the head neck area. The substitution should be given 5-7 days before and after the intervention.

Background: The trifunctional bi-specific antibody Removab bridges and activates CD3 positive T cells to EpCAM on carcinoma cells and simultaneously binds to an accessory immune cell, thus inducing tumor cell lysis. Following intravenous application, Removab may induce cytokine-related side effects resulting in a sepsis like syndrom. It was questioned, if peripheral mononuclear blood cells (PBMN) already opsonized with Removab could retain antitumor activity and induce less cytokine release than the mere antibody., Methods: PBMN of patients with head and neck cancer were incubated with Removab and the released cytokines were washed out. Then the Removab-opsonized PBMN were coincubated with genuine tumor cells of the same patient on a chorioallantois membrane for 24 h (T 24) and 48 h (T 48). Tumor cells coincubated with Cisplatin or solely cell culture medium served as control., Results: Coincubation of tumor cells with opsonized PBMN resulted in a 32 % decrease of viable cells at T 24 and a 37 % decrease at T 48, whereas viable cells increased by 10 % at T 24 or 3 % at T 48 when incubated with medium alone (p < 0.05). This tumor cytotoxicity was similar to that of Cisplatin (35 % at T 24/37 % at T 48)., Conclusion: In an autologous human ex vivo tumor system, Removab-opsonized PBMN induce tumor cell lysis with significantly reduced cytokine release after i. v. application.

Introduction: The zeta-chain as an important component of the T cell receptor (TCR) is involved in the transduction of intracellular signals and is therefore critical for T cell activation and subsequent induction of an anti-tumor response. In patients with squamous cell carcinoma of the head and neck (SCCHN) several mechanisms of immune suppression have been described. The expression of the TCR zeta-chain of both peripheral blood lymphocytes (PBLs) and tumor infiltrating lymphocytes (TILs) has been analysed in patients with SCCHN and correlated to clinical data., Methods: TIL and PBL from 47 patients with SCCHN were obtained. To determine expression of the zeta-chain in both PBLs and TILs, double immunostaining with mAbs and flow cytometric analysis was performed. The technique combined an intracellular staining with a surface staining., Results: As compared to healthy controls (n = 23), zeta-chain expression was significantly reduced (p < 0.002) in patients with SCCHN (n = 23) with lowest expression in those with UICC VI disease. Our results show a tight correlation between the loss of zeta-chain expression and the clinical aggressiveness of the tumor. Higher tumor stages frequently show a higher loss of the zeta-chain. In 11 patients zeta-chain expression of the PBL could be compared with TIL. Independent of the tumor stage the loss of the zeta-chain expression is much higher in TIL than in PBL. The loss of the zeta-chain expression also correlates with the progression of the disease. Patients with a high loss of the zeta-chain expression develop a recurrence more frequently., Conclusion: The loss of the zeta-chain documents a systemic immune defect, which even occurs in early tumor stages. Additionally to locoregional approaches future therapeutic strategies should also focus on systemic immunomodulation.

Suppressed cellular immunity is common in patients with squamous cell carcinoma of the head and neck (HNSCC). It was demonstrated in previous studies that administration of interleukin 2 (IL-2) results in enhanced antitumoral immunity in vitro as well as in vivo. Since the serum half-life of IL-2 is relatively short, repeated applications are necessary to achieve therapeutically effective serum concentrations, but this strategy might cause severe side effects. Therefore, methods that provide high local cytokine levels over a prolonged period of time without the need for repeated injections are desirable. Gene therapy as an innovative treatment approach using tumor cells stably transduced to produce IL-2 might meet these criteria. In vitro manipulated tumor cells, if readministered in the vicinity of non-manipulated tumor cells, may enhance a specific anti-tumor response in vivo without systemic side effects. The present manuscript reviews the current literature dealing with IL-2-protein and -gene therapy with special emphasis on head and neck cancer. Our own in vitro results with IL-2 gene therapy in conjunction with published data from other authors argue in favour of an in vivo approach for this therapeutic strategy that is currently in progress in our department.

The aim of the study was the induction of an antitumor immune response by genetic modification of tumor cells. This was done by transfecting the costimulatory molecule B7.1 into a murine tumor cell line SCCVII/SF in order to increase T cell recognition and to install an immunologic memory. One cohort of immunocompetent mice C3 H/HeN were injected with B7.1 expressing tumor cells, while the control group received parental B7.1 negative tumor cells. In a second step those immunized mice were rechallenged by parental tumor cells and tumor growth was compared to a new control group. Transfection with B7.1 prohibits outgrowth of the SCCVII cell line. Animals that have been vaccinated in this way are partially immune towards a secondary exposition to B7.1 negative tumor cells. Outgrowth of this recurrent tumor is slowed down. Such an immunization builds up an immunologic memory. Vaccination with B7.1 expressing tumor cells lead to a partial protective tumor immunity in the SCCVII-C3 H/HeN mouse model.

Aim: Until now no serological markers were available towards the diagnosis of squamous-cell carcinomas of the head and neck (HNSCC) particularly in the detection of posttherapeutic recurrent diseases and metastases. Previous reports described patients with malignant diseases of various tissues exhibiting a characteristic and highly significant alteration in the subclass composition of serum IgG, consisting of a reduction in %IgG1 and an increase of %IgG2. In this study we present for the first time results of this IgG-shift in patients suffering from HNSCC., Patients and Methods: A total of 111 patients was investigated at our clinic, all suffering from primary, histopathologically verified squamous cell carcinomas of the head and neck. These patients were investigated as to %Ig G1/IgG2 prior to any treatment. A second group consisted of 35 patients with local recurrences, 15 patients with distant metastases and 27 patients without tumour at the time of investigation, who were in observation for 1 to 5 years after primary treatment (T0). Thirdly, a total of 33 patients was included who were afflicted with a variety of benign diseases of the head and neck, such as chronic rhinosinusitis, chronic tonsillitis and also lateral or median cysts of the neck. Data of the three groups were compared with those of 174 healthy volunteer controls. 5 ml blood were taken from a forearm vein and the quantitation of subclasses IgG1, IgG2 and total IgG was performed by affinity chromatography. The single values obtained with all experimental groups and healthy controls showed normal distribution for primary cancer patients versus healthy control. Accordingly, significant differences between mean values were calculated with the two-sided Students t-test, and cut-off values were calculated as the arithmetic means of mean values obtained from the groups to be compared. Diagnostic sensitivities and specificities were defined as percentages of patients with %IgG1 smaller, and of healthy controls with %IgG1 greater than the cut-off value., Results: We found a highly significant alteration in the subclass composition of serum IgG, consisting of a reduction in %IgG1 and an increase in %IgG2 in our HNSCC groups. The present data suggest the changes in %IgG1 and %IgG2 as a useful serological tumour marker to detect primary or recurrent and/or metastatic squamous-cell carcinomas of the head and neck., Conclusion: The shift in %IgG1/%IgG2 exhibited diagnostic sensitivities and specificities comparable to, or--particularly at early tumour stages--by far higher than conventional serological tumour markers. Whereas conventional serological markers directly correspond to tumourogenically derived products, the shift in %IgG1/IgG2 represents an indirect marker, consisting of a change of the host's immune system due to the presence of malignant tumours.

Background: Squamous cell carcinoma is the most common cancer of the head and neck. The application of monoclonal hybridoma technology to the identification and characterization of molecules preferentially expressed in carcinoma cells may provide potentially important diagnostic and therapeutic tools., Methods: After immunization of mice with viable squamous cell carcinoma cells derived from human tumors of the upper aerodigestive tract hybridomas were selectively grown for production of monoclonal antibodies. Monoclonal antibodies produced by 690 hybridoma clones were tested on immunoreactions with frozen sections of human squamous cell carcinoma and reactive tonsils., Results: Immunohistochemical trials showed that monoclonal antibodies produced by hybridoma clone K-640 are strongly accumulated at the plasma membrane whereas no immunoreaction in squamous epithelium of the tonsil could be detected. By means of ELISA it could be shown that these antibodies belong to the class of IgM. Immunoblotting revealed that the antibodies recognize antigenes with approximate molecular weights of 52 and 54 kD., Conclusions: The experiments suggest that the hybridoma clone K-640 produces antibodies which bind to the plasmamembrane of the tumor cells. Further recloning steps of this hybridoma clone could possibly enhance the specificity of these antibodies for squamous cell carcinoma.

Objective: Mutations of the p53 gene are the most prevalent genomic changes in human malignancies. The detection of these mutations is usually carried out by gene sequencing or with immunohistochemical methods. A new approach is to search for p53 antibodies in serum of cancer patients. The cause for the production of antibodies against p53 is unclear. The aim of this study was to show the correlation between p53 pointmutations and p53 antibodies in patients with squamous cell carcinoma of the head and neck (SCCHN)., Methods: A semiquantitative ELISA was employed to screen the serum of 117 patients with SCCHN for p53 antibodies. Sixty-one tumor samples were evaluated with SSCP-analysis for point mutations in exons 4 to 9., Results: Twenty-three (19.7%) of the cancer patients had antibodies to p53. There was no significant difference in the frequency of p53 point mutations in exon 4 to 9 between the whole cancer patient group and the p53 antibody positive group., Conclusions: The accumulation of p53 in the tumor cell is one hypothesis as to how p53 becomes immunogenic. This leads to a decrease in tolerance after a certain amount of time. In contrast to this it is assumed that after a p53 point mutation, important protein determinants are changed and p53 becomes antigenic. The results of this study indicate that p53 point mutations in SCCHN seem to have no influence on the development of p53 antibodies. Therefore this study supports the theory that the loss of tolerance and subsequently the antibody production is induced by an accumulation of p53. The type of p53 point mutations on exon 4-9 seems to have no influence that p53 gains immunogenic potential in SCCHN:

Background: The mutated form of the nuclear tumor suppressor protein p53 plays an important role as oncogene in the malignant transformation of cells. A new serologic parameter are antibodies against the p53 protein, that are found in many carcinoma entities and localizations and may have a prognostic relevance. Antibodies against p53 in the serum of patients with squamous cell carcinoma of the head and neck (SCCHN) have not yet been described., Methods: A set of 117 sera from patients with SCCHN were screened for serum antibodies against p53 with ELISA (Dianova, Hamburg). The serum of 41 healthy blood donors served as controls. Tumor tissue of 45 patients was examined with ELISA for p53 protein accumulation., Results: In 23 patients (19.7%) with SCCHN p53 antibodies were demonstrated. The control sera were all negative for p53 antibodies. No correlation was found between the presence of p53 antibodies and tumorstage, tumor grading, age or sex of the patient or tumorlocalization. Thirteen of the 15 tumor samples from p53 antibody positive patients showed detectable levels of p53 protein., Conclusions: The frequency of p53 antibodies in patients with SCCHN is comparable with the occurrence of these antibodies in other malignancies. An accumulation of p53 in the tumor tissue seems to be necessary to initiate the antibody production. p53 antibodies seem to be an indicator for malignancy in SCCHN and may be of potential diagnostic and prognostic value.

Background: Several investigations showed increased levels of the soluble interleukin-2 receptor (sIL-2 R) in chronic and malignant diseases. A correlation between elevated sIL-2 R levels and disease activity or tumor size was observed in some patients. At present, there are no studies of sIL-2 R serum levels in patients with squamous cell carcinomas of the upper aerodigestive tract., Methods: SIL-2 R levels were measured in sera of 86 patients with squamous cell carcinoma of the head and neck and of 25 healthy controls with a sandwich ELISA., Results: Significantly elevated serum levels of sIL-2 R were detected in 49 out of 86 patients (56.9%) with squamous cell carcinoma of the head and neck. The mean serum level in the healthy controls was 437 U/ml compared to 895 U/ml in carcinoma patients. A correlation was found between tumor size, number of metastases and the sIL-2 R level. There was no correlation with histological type or tumor localization., Discussion: The physiological role of sIL-2 R is still unknown. Elevated levels of sIL-2 could be a sign of activation of the immune system. But they also might be involved in causing the disease. The sIL-2 R serum levels in patients with squamous cell carcinoma of the head and neck may serve as an unspecific indicator for tumor size and dissemination., Conclusion: Posttherapeutic titer controls could be of prognostic value. Additionally, sIL-2 R may help to select appropriate patients for an immunotherapy with recombinant IL-2.

Monoclonal antibodies were used to examine the presence and nature of tumor associated macrophages and T-lymphocytes in 26 cases of squamous cell carcinoma of head and neck. The content of macrophages varied according to the differentiation of the carcinoma: high differentiation was evidenced by a labelling index by an index between 13.5% and 23.7%, poor differentiation from 27.4% to 35.8%. T-lymphocytes were found where tumors were inflamed, but there was no significant correlation between tumor differentiation and infiltrating T-lymphocytes. The present results that prognosis decreases with the increasing number of tumor associated macrophages in squamous cell carcinoma.