The HLA ligandome of oropharyngeal squamous cell carcinomas reveals shared tumour-exclusive peptides for semi-personalised vaccination.

BACKGROUND: The immune peptidome of OPSCC has not previously been studied. Cancer-antigen specific vaccination may improve clinical outcome and efficacy of immune checkpoint inhibitors such as PD1/PD-L1 antibodies. METHODS: Mapping of the OPSCC HLA ligandome was performed by mass spectrometry (MS) based analysis of naturally presented HLA ligands isolated from tumour tissue samples (n = 40) using immunoaffinity purification. The cohort included 22 HPV-positive (primarily HPV-16) and 18 HPV-negative samples. A benign reference dataset comprised of the HLA ligandomes of benign haematological and tissue datasets was used to identify tumour-associated antigens. RESULTS: MS analysis led to the identification of naturally HLA-presented peptides in OPSCC tumour tissue. In total, 22,769 peptides from 9485 source proteins were detected on HLA class I. For HLA class II, 15,203 peptides from 4634 source proteins were discovered. By comparative profiling against the benign HLA ligandomic datasets, 29 OPSCC-associated HLA class I ligands covering 11 different HLA allotypes and nine HLA class II ligands were selected to create a peptide warehouse. CONCLUSION: Tumour-associated peptides are HLA-presented on the cell surfaces of OPSCCs. The established warehouse of OPSCC-associated peptides can be used for downstream immunogenicity testing and peptide-based immunotherapy in (semi)personalised strategies.

Polyfunctionality of CD4+ T lymphocytes is increased after chemoradiotherapy of head and neck squamous cell carcinoma.

BACKGROUND: For head and neck squamous cell cancer (HNSCC), standard therapy consists of surgery, radiation, and/or chemotherapy. Antineoplastic immunotherapy could be an option in an adjuvant setting and is already in palliation. A functional immune system is a prerequisite for successful immunotherapy. However, effects of the standard-of-care therapy on the patients' immune system are not fully understood. METHODS: Peripheral blood mononuclear cells (PBMC) were collected from patients with HNSCC (n = 37) and healthy controls (n = 10). PBMC were stimulated with staphylococcal enterotoxin B (SEB). Simultaneous expression of various cytokines was measured in CD4+ and CD8+ T cells by multicolor flow cytometry, and polyfunctional cytokine expression profiles were determined on a single-cell basis. RESULTS: Expression levels of all measured cytokines in CD4+ T cells were higher in patients after chemoradiotherapy (CRT) as compared to untreated HNSCC patients or normal controls. After CRT, the frequency of polyfunctional CD4+ T cells, which simultaneously expressed multiple cytokines, was significantly increased as compared to untreated patients (p < 0.01). CONCLUSION: CRT increases polyfunctionality of CD4+ T cells in HNSCC patients, suggesting that standard-of-care therapy can promote immune activity in immune cells. These polyfunctional CD4+ T cells in the blood of treated HNSCC patients are expected to be responsive to subsequent immunotherapeutic approaches.

[Immunotherapy Against Head and Neck Cancer Stem Cells]. / Immuntherapie gegen Tumorstammzellen bei Plattenepithelkarzinomen im Kopf-Hals-Bereich.

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.

[Immunoglobulin G4 (IgG4)-related disease. A review of head and neck manifestations]. / Immunglobulin-G4(IgG4)-assoziierte Erkrankung. Gesichertes und Kontroverses der häufigsten Kopf-Hals-Manifestationen.

Immunoglobulin G4 (IgG4)-related disease (also known as hyper-IgG4 disease) is a recently defined emerging condition with highly heterogeneous clinicopathological features and variable disease manifestations. This disorder is characterized by unifocal or multifocal (multiorgan) involvement by tumefactive plasma cell-rich inflammatory infiltrates associated with prominent fibrosclerosis. This not uncommonly interferes with organ function resulting in diverse clinical symptoms. The autoimmune pancreatitis represents the prototype of this disease; however, to date almost all organs have been reported to be involved in this disorder. In the head and neck area several presentations of this disease may be encountered in salivary glands, lacrimal glands, thyroid gland, lymph nodes, soft tissue of the neck, ear and sinonasal tract. However, IgG4 positive plasma cells are occasionally prominent in non-specific chronic inflammatory conditions of the head and neck and the oral cavity unrelated to autoimmune diseases or systemic disorders, thus representing diagnostic pitfalls. The diagnosis of IgG4-related disease should be based on a combination of typical histological, clinical and serological findings.

[MIF in head and neck cancer: a new therapeutic target ?]. / Le MIF dans les cancers des voies aéro-digestives supérieures: une nouvelle thérapie ciblée?

Macrophage migration inhibitory factor is a critical proinflammatory cytokine produced by cells of innate and adaptive immune system. MIF plays a key role in cell cycle regulation and in the pathogenesis of many cancers. Recently, MIF has been studied in the upper aerodigestive tract cancer for its involvement in tumor progression, invasion, proliferation and cell motility. In addition, MIF appears to be a mediator in angiogenesis and in the development of metastasis and locoregional lymph node, which are often associated with a poor prognosis. The mechanisms of action responsible for MIF involvement in tumor progression are not completely elucidated. However, the main effects of MIF are mediated by the CD74 receptor. MIF binding to its receptor is responsible for the activation of several signaling pathways (ERK1/2 - MAPK, JAB1 - CSN5, PI3K - Akt), the inhibition of p53 and the stimulation of angiogenic factors including VEGF and IL-8. The overexpression of MIF also causes a reduction of the anti-tumor activity of the immune system. Finally, MIF could be an interesting biomarker in the diagnosis and monitoring of upper aerodigestive tract cancers. In this paper, we assess the state of knowledge of MIF involvement in upper aero-digestive tract cancers and we analyze the therapeutic perspectives.

[Prophylactic and therapeutic vaccines against human papilloma virus]. / Prophylaktische und therapeutische Vakzinen gegen humane Papillomviren.

Infection with human papilloma virus (HPV) has been identified as the cause of recurrent papillomatosis and of a subgroup of squamous cell carcinomas of the head and neck. A change in prevalence of these lesions, especially for oropharyngeal carcinoma, can be expected as a consequence of the introduction of prophylactic HPV vaccines for young women, targeting the most frequent high- and low-risk HPV subtypes. Vaccination for the major low-risk HPV types has proven to be highly effective against genital warts and activity against papillomatosis can be expected. The possibilities of prophylactic HPV vaccination as well as new developments and the rationale for therapeutic vaccines are discussed on the basis of the current literature.

[The significance of perioperative immunonutrition]. / Stellenwert der perioperativen Immunonutrition.

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.

Mononuclear phagocytes in head and neck squamous cell carcinoma.

The head and neck squamous cell carcinoma microenvironments contain many immune cells and their secretory products. Many of these cells belong to the mononuclear phagocyte system. The aim of this review is to study the interactions between mononuclear phagocytes and head and neck squamous cell carcinoma tissue. The role of inflammation in tumours and the cytokine interleukin-6 will be highlighted. Future therapy strategies in the treatment of head and neck cancer might be directed towards mononuclear phagocytes and their cytokine production.

[T-cells activated with a trifunctional bi-specific antibody in head and neck cancer]. / Removab aktivierte Lymphozyten bei Plattenepithelkarzinomen des oberen Aerodigestivtraktes.

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.

[Long-term changes in peripheral blood leukocyte and lymphocyte populations in ENT-carcinoma patients. A flow cytometric study in 346 ENT-carcinoma patients and 31 healthy controls]. / Veränderungen von peripheren Blutzellpopulationen bei HNO-Karzinompatienten. Eine flowzytometrische Studie mit 346 akut erkrankten und therapierten Patienten bis 20 Jahre nach Abschluss der Therapie und 31 Kontrollpersonen.

BACKGROUND AND AIM: The aim of this investigation was the quantification of leukocyte/lymphocyte populations in the peripheral blood of ENT-carcinoma patients before and long after therapy. PROBANDS AND METHOD: PBL T-lymphocytes and NK-cells were examined in 346 patients and 31 controls using flow cytometry. In 248 participants additional populations of leukocytes, monocytes, granulocytes, lymphocytes in toto, CD4+- and CD8+-lymphocytes, CD4+8+-lymphocytes, B-lymphocytes, activated T-lymphocytes, NK-cells and CD3+56+-T-lymphocytes were studied. RESULTS: We demonstrated a long-term reduction in T-lymphocytes (CD4+-lymphocytes) in patients more than 10 years after receiving therapy, caused mainly, but not solely, by radio/chemotherapy. We also showed a long-term, significant increase in NK-cells after more than 10 years in patients following therapy without renewed carcinoma in comparison to controls, patients prior to therapy or patients with a recurrence of carcinoma. CONCLUSIONS: Due to long-term deficits in CD4+-lymphocytes, and considering the possible protective effect of NK-cells in treated patients, an immune-supportive therapy is recommended.

[The expression of zeta-chain of the T cell receptor as prognostic marker for patients with head and neck cancer]. / Die Expression der zeta-Kette des T-Zell-Rezeptors als Prognosemarker bei Kopf-Hals-Karzinomen.

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.

[IL-2 gene therapy in ENT carcinomas]. / IL-2 Gentherapie bei HNO-Karzinomen.

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 predictive value of serum interleukins in recurrent respiratory papillomatosis: a preliminary study.

OBJECTIVE: IL-2 is the primary interleukin responsible for activation of the cell-mediated (Th1) arm of the immune response. Our objective was to determine whether a correlation existed between circulating levels of interleukin-2 as well as its soluble receptor (sIL-2R) and the clinical course of recurrent respiratory papillomatosis. METHODS AND MATERIALS: Fifteen children with a histological diagnosis of RRP were recruited. Age at the time of study, time since first diagnosis, and number of surgical interventions were recorded. The number of surgically treated recurrences per year was then calculated. We obtained serum samples from each of these 15 children and from 10 normal control subjects. We then performed in vitro determination of serum IL-2 and soluble IL-2 receptor levels using enzyme-linked immunosorbent assay (ELISA) techniques. RESULTS: IL-2 was significantly lower (136.6 vs. 199.9 pg/mL, P =.035) in papilloma patients than in control subjects. IL-2R was also lower in papilloma patients (531.7 vs. 785.8 U/mL, P =.025). There was no statistical age difference between the papilloma and control groups. Among patients with papillomatosis, IL-2 and sIL-2R levels were significantly higher in those with aggressive disease (>4 surgically treated recurrences per year) versus non-aggressive disease (179.2 vs. 99.2 pg/mL, P =.024; and 697 vs. 387 U/mL, P =.022). Age was also significantly lower in the aggressive papilloma group (P =.002). CONCLUSIONS: Levels of interleukin-2 and IL-2 receptor were significantly lower in patients with recurrent respiratory papillomatosis compared with normal children. These data support the presence of an aberrant cell-mediated immune response in children with recurrent respiratory papillomatosis.

[Immunosuppression caused by head and neck surgery]. / Einfluss tumorchirurgischer Eingriffe im Kopf-Hals-Bereich auf das Immunsystem.

The immunomodulating effect of primary surgical intervention was investigated in 33 patients with squamous cell carcinomas of the oral cavity, pharynx and larynx. An operation time longer than 7 h was significantly associated with a decrease in lymphocytes, CD4(+) T-lymphocytes and CD8(+) T-lymphocytes. The CD4/CD8-ratio as a marker for the down-regulation of the cellular immune response was slightly decreased but was still in the normal range. CD4-lymphocyte counts increased 7 days after operation while CD8 lymphocytes were found elevated 4 weeks after surgery. The in vitro stimulation of the lymphocytes was impaired for 1 to 4 weeks. Interleukins, interferon-gamma and tumor necrosis factor did not show any changes after surgery. Decreases of lymphocytes, especially CD4(+) and CD8(+) lymphocytes, were significantly associated with the time of operation and the volume of blood loss. Extensive trauma, age, different kinds of anesthesia and intensive care interventions were not associated with specific immunomodulating effects, although these factors might be responsible for suppressing cytokine responses.

[Induction of antitumor immune response in the mouse model after vaccination with B7.1 expressing tumor cells]. / Induktion einer antitumoralen Immunantwort im Mausmodell nach Vakzinierung mit B7.1 experimierenden Tumorzellen.

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.

Impairment of T-cell activation in head and neck cancer in situ and in vitro: strategies for an immune restoration.

BACKGROUND: The rationale for the study was based on the hypothesis that decreased or absent expression on tumor cells of adhesion molecules, the class I or class II major histocompatibility complex (MHC) molecules, or costimulatory molecules might be responsible, in part, for the poor ability of squamous cell carcinoma of the head and neck (SCCHN) to induce generation of antitumor effector cells in vitro and in vivo. OBJECTIVE: To investigate expression of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function associated antigen-3 (LFA-3) and distribution of the costimulatory molecules, B7.1, B7.2, and CD40, and of class I and class II MHC molecules on SCCHN cells in situ and on SCCHN cell lines. SETTING: University medical centers. DESIGN: Expression of ICAM-1, LFA-3, MHC molecules, B7.1, B7.2, and CD40 was evaluated in human SCCHN biopsy specimens by immunohistochemistry and on SCCHN cell lines by flow cytometry. To confirm our hypothesis that impaired T-cell activation observed in patients with SCCHN is caused by the absence of costimulatory B7 molecules, a B7-negative SCCHN cell line was transduced with the B7.1 gene, using a retroviral vector, and tested in mixed lymphocyte tumor cocultures. RESULTS: In contrast to abundant expression of ICAM-1, LFA-3, class I MHC molecules, and CD40, the absence of B7.1, B7.2, and class II MHC molecules on tumor cells was observed in situ and in vitro. Lymphocytes and antigen-presenting cells in inflammatory infiltrates surrounding tumor cell clusters expressed both costimulatory and adhesion molecules. The SCCHN lines negative for B7.1 and class II MHC antigens failed to induce proliferation of T cells in mixed lymphocyte tumor cocultures. However, when these cell lines were transduced with the B7.1 gene, their ability to induce T-cell proliferation in mixed lymphocyte tumor cocultures was restored. CONCLUSIONS: The absence of B7 protein or class II MHC antigen expression on human SCCHN cells is responsible for the failure of these tumors to induce proliferation of T cells in vitro. Transduction of the B7.1 gene into SCCHN restores the ability of the tumor to induce T-cell proliferation in vitro.