Advanced HNCs often evade standard therapies. The lack of alternative treatment options limit patient prognosis. Here, we investigated the effect of alternative immunotherapies in a tumor mouse model.

Das fortgeschrittene HNC entgeht häufig den Standardtherapien. Der Mangel an alternativen Therapieoptionen limitiert deshalb häufig die Patientenprognose. In dieser Studie haben wir den Effekt von Therapiealternativen in einem Tumormausmodell untersucht.

Exosomes, virus-sized vesicles, carry protein cargos resembling those of their parent cells and modify immune cell functions. Here, we address the query whether exosomes derived from Human Papilloma Virus-positive (HPV+) or HPV-negative (HPV-) HNC cell lines differ in molecular and immunoregulatory profiles.

Exosomen, Virus-große Vesikel, tragen die Proteine ihrer Elternzellen und modifizieren Immunzellfunktionen. Hier untersuchen wir, ob sich Exosomen von Humanem Papillomvirus-positiven (HPV+) oder HPV-negativen (HPV-) HNC-Zelllinien durch ihr molekulares oder immunregulatorisches Profil unterscheiden.

Ca2+ signals are critical for T-cell function, and Ca2+ influx is an early event in T-cell activation. TEX, ubiquitously present in the tumor microenvironment and plasma, carry a cargo of suppressive and stimulatory molecules. TEX bind to the surface of immune cells, suppressing functions of some T cells while up-regulating functions of others. To determine whether binding of TEX or exosomes produced by normal cells (EXO) to primary T cells results in delivery of signals regulating activation, human PBMC or different freshly-isolated T-cell subsets were co-incubated with PKH-26 labeled exosomes isolated from normal or HNSCC plasma specimens and from supernatants of HNSCC cell lines as described by us (Muller et al., J. Immunol. Methods, 2014). CD4+ T cell, CD8+ T cell and CD4 + CD39+ Treg were isolated from normal PBMC using AutoMACS. Binding of PKH-26-labeled TEX or EXO by T cells, NK cells or monocytes was evaluated following co-incubation for 24–96 h using an AMNIS image cytometer. The strength of Ca2+ influx induced by TEX or EXO in various T cells was measured by confocal microscopy after staining the T cells with a Fura-2 or Fluo-4 dye. AMNIS flow data showed that binding of TEX and EXO to T cells was time and dose-dependent, but exosomes were not internalized even after prolonged [96 h] co-incubation. Ca2+ influx was time dependent and was observed only after co-incubation of T cells with TEX but not (or only minimally) with EXO. Among T cells, CD4 + CD39+ Treg were preferentially targeted by TEX, showing the strongest Ca2+ influx within 30 min. In AMNIS, TEX binding to Treg gave a significantly stronger signal than that measured with CD4+ or CD8+ T cells. This strong, sustained Ca2+ influx induced in Treg by TEX is consistent with our previous functional data demonstrating selective TEX-mediated Treg activation and proliferation. The data also emphasizes the fact that activation of Treg functions by TEX is regulated through signals delivered to the Treg surface and does not require exosome up-take or their internalization by the target cells.

PURPOSE:TG4010 is a recombinant MVA vector expressing the tumor-associated antigen MUC1 and IL2. We explored the effect two schedules of TG4010 on PSA in men with PSA progression.PATIENTS AND METHODS:A randomized phase II trial was conducted in 40 patients with PSA progression. Patients had PSA doubling times less than 10 months, with no overt evidence of disease. Patients received either weekly subcutaneous injection (sc) of TG4010 10(8) pfu for 6 weeks, then one injection every 3 weeks or sc injection of TG4010 10(8) pfu every 3 weeks.RESULTS:The primary endpoint of a 50% decrease in PSA values from baseline was not observed. Nevertheless, 13 of 40 patients had a more than two fold improvement in PSA doubling time. Ten patients had their PSA stabilized for over 8 months. Therapy was well tolerated.CONCLUSIONS:Although the primary endpoint was not achieved, there is evidence of biologic activity of TG4010 in patients with PSA progression, further investigation in prostate cancer is warranted.

BACKGROUND:Abnormalities of the major histocompatibility complex (MHC) antigens by tumour cells impair the cellular immune response and promote tumour evasion from immune surveillance. So far, studies analysing the MHC class II expression levels in head and neck cancer have been limited.OBJECTIVES:Therefore, we investigated the constitutive and interferon (IFN)-gamma-regulated expression profiles of MHC class II antigen processing machinery (APM) in various head and neck cancer cell lines and also analysed the MHC class II expression in head and neck cancer lesions.METHODS:Using immunohistochemistry, flow cytometry, and reverse transcriptase-polymerase chain reaction analyses we investigated the expression pattern of various components of the MHC class II APM in biopsies and cell lines from head and neck cancers. Furthermore, we analysed the class II transactivator (CIITA) and HLA-DR promoter activity in head and neck cancer cells by transient transfection of specific luciferase promoter constructs and finally studied the methylation pattern of the CIITA promoter using methylation sensitive restriction enzymes.RESULTS:Head and neck cancer cell lines analysed in vitro lacked constitutive MHC class II surface expression. Despite the IFN-gamma-mediated induction at the mRNA level, six out of 10 cell lines did not show any relevant MHC class II surface expression. This phenomenon might be attributed to a post-transcriptional dysregulation of specific MHC class II APM components. One cell line displayed a loss of IFN-gamma-induced CIITA-expression that corresponded to impaired MHC class II surface expression, which could be linked to hypermethylation of the IFN-gamma-responsive CIITA-promoter IV. In vivo, immunohistochemistry analyses of 35 patients revealed that about 86% of head and neck cancer tissues exhibit a negative or only marginally positive staining, whereas 14% displayed a heterogeneous or highly positive MHC class II surface expression. There was no statistical correlation between tumour differentiation and the MHC class II expression in head and neck cancer lesions.CONCLUSIONS:Taken together these results suggest a high frequency of MHC class II abnormalities in head and neck cancer in vitro and in vivo, which could occur at different steps of the antigen processing pathway. This information may have a significant impact on practical and clinical aspects of tumour immunotherapeutic strategies.

Head and neck carcinomas are histologically and clinically heterogeneous. While squamous cell carcinomas (SCC) are characterized by lymphogenous spread, adenoid cystic carcinomas (ACC) disseminate preferentially hematogenously. To study cellular and molecular mechanisms of organ‐specific metastasis, we used SCC and ACC cell lines and tumor tissues, obtained from patients with primary or metastatic disease. Comprehensive analysis at the mRNA and protein level of human chemokine receptors showed that SCC and ACC cells exhibited distinct and nonrandom expression profiles for these receptors. SCC predominantly expressed receptors for chemokines homeostatically expressed in lymph nodes, including CC chemokine receptor (CCR) 7 and CXC chemokine receptor (CXCR)5. No difference in expression of chemokine receptors was seen in primary SCC and corresponding lymph node metastases. In contrast to SCC, ACC cells primarily expressed CXCR4. In chemotaxis assays, ACC cells were responsive to CXCL12, the ligand for CXCR4. Exposure of ACC cells to cisplatin resulted in upregulation of CXCR4 on the cell surface, which was repressed by the transcriptional inhibitor, α‐amanitin. Treatment of ACC cells with CXCL12 resulted in the activation of Akt and ERK1/2 pathways. Furthermore, CXCL12 suppressed the rate of apoptosis induced by cisplatin in ACC cells, suggesting that signaling via CXCR4 may be part of a tumor cell survival program. Discrimination of the chemokine receptor profile in SCC and ACC in vitro and in tissues provided insights into their distinct biologic and clinical characteristics as well as indications that chemokine receptors might serve as future therapeutic targets in these malignancies. © 2005 Wiley‐Liss, Inc.

General practitioner Newtownabbey, County Antrim, Northern Ireland (b Belfast 1955; q Queen's University, Belfast, 1979; DCH, MRCOG, MRCGP), died from metastatic breast cancer on 9 October 2005. Lorraine joined Notting Hill Medical Practice over 20 years ago and soon became a valued partner. She had many attributes and skills in obstetrics, diabetic treatment, and forensic medical work, and was a great asset to the practice. She leaves a husband, Trevor, and two daughters.

4501 We have previously reported the initial results of a Phase II clinical trial evaluating the feasibility of a prime/boost vaccine strategy using vaccinia virus and fowlpox virus expressing human PSA in patients with hormone-dependent prostate cancer. 64 eligible patients with biochemical progression after local therapy were randomly assigned to three treatment arms: (A) fowlpox-PSA (rF-PSA) by intramuscular injection every six weeks for four doses, (B) rF-PSA for three doses followed by vaccinia-PSA (rV-PSA) given by intradermal injection, or (C) rV-PSA followed by three rF-PSA vaccines. Vaccination was well tolerated without evidence of autoimmunity. Our initial analysis was conducted at a median follow-up of 19.1 months and demonstrated that 45.3% of men were free of PSA progression and 78.1% were free of clinical progression with a trend favoring arm C. The patients enrolled on this trial have been followed for PSA response and clinical outcome and we now report data with a median follow-up of 50 months. The median time to PSA progression is 9.2 and 9.1 months for arms A and B respectively, compared to 18.2 months for arm C (rV-PSA prime and rF-PSA boost; p=0.15 by Log Rank Test). The median time to clinical progression has still not been reached for any treatment group with 80% of men in arms A and B free of disease progression compared to 90% of men in arm C free of clinical progression (p=0.73 by log rank test). These results suggest that men with hormone-dependent prostate cancer and a rising PSA may derive long-term clinical benefit from vaccinations with poxviruses expressing PSA. A trend in PSA and clinical response favoring patients receiving a prime vaccination with rV-PSA followed by booster immunizations with rF-PSA appears to persist at 50 months median follow-up. Collectively, these results establish the feasibility of a poxvirus vaccine strategy for men with early hormone-dependent prostate cancer recurrences, and support future phase III studies. No significant financial relationships to disclose.

Lymphozyten, die gleichzeitig CD4 und CD25 exprimieren sind essentiell für die Selbst-Toleranz und werden als regulatorische T-Zellen (Treg) bezeichnet. Treg verhindern Auto-Immunerkrankungen durch Hemmung von autoreaktiven Lymphozyten. Die körpereigene Immunantwort gegen Kopf-Hals-Karzinome (HNC) ist insuffizient und T-Zellen scheitern in der Eliminierung von malignen Zellen. Diese Dysfunktion könnte auf die Präsenz von Treg zurückzuführen sein, die die Immunreaktion gegen diese Tumoren suprimieren. Lymphozyten von 11 HNC-Patienten und 10 Kontrollen (NC) wurden mit diversen Differenzierungsmarker gefärbt und mittels Durchflusszytometrie analysiert. HNC-Patienten besaßen einen deutlich höheren Anteil an CD4+CD25+ Zellen (meanx=5.9% aller CD4+) als NCs (meanx=2.8%). In HNC-Patienten zeigten diese Treg in 47% einen CCR7- Phänotyp (NCs=36.1%). Die CD4+CD25-CCR7--Population (CCR7-CD4+-non-Treg) umfasste in HNC-Patienten 21.7% (NCs=7.4%). Dies ergab eine Ratio von CCR7--T reg zu CCR7--non-Treg in HNC Patienten von 2.2: 1 (NCs=5: 1). Der expandierte Pool an CD4+CD25+ Lymphozyten bei HNC-Patienten kann einen Aspekt der inadäquaten Immunantwort gegenüber diesen Tumoren darstellen. Der hohe Anteil an CCR7- Treg zeigt, dass diese Zellen bereits sekundäre lymphatische Organe durchwandert haben und rezirkulieren. Die gegenüber NCs verminderte Ratio von CCR7--T reg vs. -non-Treg kann ein Indiz dafür sein, dass Treg in Geweben wie Primärtumor oder tumorbefallenen Lymphknoten akkumulieren und dort die Immunantwort gegen HNC-Zellen hemmen.

s for the 19th Annual Scientific Meeting of the International Society for Biological Therapy of Cancer, San Francisco, California, November 4-7, 2004: Immune Monitoring

Präsenz, Funktion und Verteilung von tumorspezifischen T-Zellen bestimmen die Möglichkeit des Patienten, eine effektive Antitumor-Immunantwort mit T-Zellen aufzubauen. Bisher wurden T-Zellen, spezifisch für p53-Tumorantigene, nur in der Zirkulation von Patienten mit Kopf-Halstumoren nachgewiesen. Es ist unbekannt, ob tumorspezifische T-Zellen auch im Tumor lokalisiert sind und ob sich dort auch regulatorische T-Zellen (Treg) finden. Tumor infiltrierende Lymphozyten (TIL) und periphere mononucleäre Lymphozyten (PML) wurden von 9 HLA-A2.1+ Tumorpatienten isoliert und mit Tetrameren und Antikörpern markiert, um tumorspezifische T-Zellen und Treg. durchflusszytometrisch nachzuweisen. Die Häufigkeit von p53-Tetramer+CD3+CD8+ T-Zellen war in TIL gegenüber PML für beide getesteten Tetramere signifikant erhöht. Gleichzeitig war die Häufigkeit von Treg. im Tumor dreifach höher als in der Peripherie. Unsere Ergebnisse zeigen, dass p53-Tetramer+ T-Zellen nicht nur in der peripheren Zirkulation vorkommen, sondern bevorzugt auch im Tumor. Aus diesem Ergebnis, zusammen mit der Tatsache, das im Tumor auch eine erhöhte Zahl von Treg. lokalisiert sind, ergibt sich die Frage nach der Funktionalität dieser tumorspezifischen T-Zellen. Der erhöhte Anteil von Treg. zusammen mit früheren Ergebnissen unserer Arbeitsgruppe, die die Dysfunktion von T-Zellen in Tumorpatienten zeigen impliziert, dass die Tumorumgebung nicht optimal für immunologische Antitumorreaktionen ist. Dieser Umstand sollte bei zukünftigen immuntherapeutischen Impfstrategien besondere Berücksichtigung finden.