Changes in circulating exosome molecular profiles following surgery/(chemo)radiotherapy: early detection of response in head and neck cancer patients.

BACKGROUND: Head and neck cancers (HNSCC) are highly immunosuppressive. Plasma-derived exosomes of HNSCC patients carry immunomodulatory molecules, and their cargo correlates with clinical parameters. Here, we evaluated the exosomal molecular profile for early detection of treatment failure in locally advanced HNSCC patients treated with conventional therapy. METHODS: Plasma from 17 HNSCC patients was collected before, during, and after treatment by surgery with adjuvant (chemo)radiation and at recurrence. Exosomes were isolated by size-exclusion chromatography. Total exosomal protein (TEP) was used to estimate exosome load and on-bead flow cytometry to evaluate relative fluorescence intensity (RFI) of tumour-associated and immunoregulatory proteins on exosomes. Exosomal effects on the activity of and adenosine production by T cells was assessed by flow cytometry and mass spectrometry. RESULTS: TEP and the ratio of tumour-/immune-cell-derived exosomes varied during and after therapy with an overall decrease in the tumour-free follow-up but an increase at recurrence. RFI values of immunoregulatory proteins on exosomes, their ability for T cell inhibition and adenosine production changed during and after therapy. PD-L1 was the earliest discriminator for treatment failure and disease-free survival. CONCLUSIONS: Monitoring of plasma exosomes during therapy represents a promising opportunity for early detection of treatment failure and risk stratification to delay/avoid recurrence.

[Tumor biology of oropharyngeal carcinoma]. / Tumorbiologie des Oropharynxkarzinoms.

BACKGROUND: Etiologically, oropharyngeal squamous cell carcinoma (OPSCC) can be divided into OPSCC caused by noxious agents and human papillomavirus (HPV)-driven carcinoma. These types differ with regard to clinical features and prognosis-differences which are rooted in the underlying molecular biology of the tumor. OBJECTIVE: The aim of this work is to provide an overview of the molecular biological characteristics of the genetics, epigenetics, and immunology of OPSCC. MATERIALS AND METHODS: A literature review was performed on a selection of genetic, epigenetic, and immunological factors characterizing OPSCC. RESULTS: The understanding of genetic aberrations and their consequences for cancerogenesis and tumor biology is increasing. Epigenetic phenomena are complementing functional relationships. However, epigenetic mechanisms of gene regulation are complex and much research is still required in this field. Immunological aspects of cancer molecular biology have moved into the focus in light of recent advances in the field of immunotherapy. CONCLUSION: The tumor biology of OPSCC is primarily defined by its HPV status. Additionally, HPV-independent genetic, epigenetic, and immunological signatures are being defined. From these advances, rationales for new treatment concepts may evolve.

Age-related changes in T lymphocytes of patients with head and neck squamous cell carcinoma.

INTRODUCTION: The number of aging cancer patients has increased continuously and will do so further in the future. The immune system of elderly people experiences critical changes over the time. Therefore, tumor-induced changes in the immune system are believed to differ in young and elderly cancer patients as well. METHODS: The effect of aging on the immune system was measured in peripheral blood lymphocytes (PBL) of healthy volunteers (n = 48, 21-84 yrs.) divided into three different age groups. Seventy years was set as a cut-off for defining subjects as elderly. Results were compared to two groups of adult cancer patients, which donated PBL and tumor infiltrating lymphocytes (TIL): young cancer patients (40-69 yrs.; blood: n = 13; TIL: n = 17) and elderly cancer patients (70-90 yrs.; blood: n = 20; TIL: n = 15) with head and neck squamous cell carcinoma (HNSCC). Frequencies and phenotypes of CD4+ and CD8+ T cells as well as regulatory T cells (Treg) were assessed by flow cytometry. RESULTS: We observed lower frequencies of CD8+ cytotoxic T cells during aging in both groups. Frequencies of tumor infiltrating regulatory T cells were significantly higher than in the peripheral blood but showed a significant decline in older tumor patients. With increasing age, expression of immunosuppressive CD73 and CCR7 was lower and expression of PD1 elevated on peripheral T cells in healthy volunteers and tumor patients. CONCLUSION: Immunosenescence takes place in healthy donors and cancer patients. Our results suggest that in elderly tumor patients, the immune system is impaired and the tumor-induced immune escape is less pronounced. The increased expression of PD1 implies the potential for effective immunotherapies in elderly, as treatment with checkpoint inhibitors could be more beneficial for elderly HNSCC patients.

Adenosine production in mesenchymal stromal cells in relation to their developmental status.

BACKGROUND: Mesenchymal stromal cells (MSC) are multipotent progenitor cells found in the tumor microenvironment. They have an innate and regulatory immune activity, and they are able to produce immunosuppressive adenosine (ADO) via their ectonucleotidases CD39 and CD73. The present study explores ADO metabolism of MSC in relation to their developmental status. METHODS: We analyzed MSC (n = 6), chondrogenic progenitor cells (CPC, n = 8), and chondrocytes (n = 8) for surface markers by flow cytometry. The ability to hydrolyze ATP and to produce ADO was tested by luminescence assays and mass spectrometry. RESULTS: Significant differences in the surface marker expression of MSC, CPC, and chondrocytes were seen. While the expression of CD73 was observed to be the same on all cell types, the expression of the ectonucleotidase CD39 was significantly increased on MSC. Consequently, production of ADO was most abundant in MSC as compared with chondrocytes and CPC. CONCLUSION: Mesenchymal stromal cells are potent producers of ADO and are, therefore, able to increase immunosuppression. As MSC differentiate into chondrocytes, they lose this ability and may take on other functions.

Ungual warts: comparison of treatment with intralesional bleomycin and electroporation in terms of efficacy and safety.

BACKGROUND: Ungual warts are considered the most common benign nail tumour, and they are caused by the human papillomavirus. Despite the numerous treatments reported in the medical literature, ungual warts are considered frustrating, with high relapse rates and a potential risk of nail dystrophy. Bleomycin is a therapeutic option showing a good safety profile and high cure rates. OBJECTIVE: To evaluate the efficacy of electrochemotherapy using intralesional bleomycin for the treatment of ungual warts in comparison with intralesional bleomycin alone and describe the side-effects related to the use of both techniques. METHODS: This was a prospective, randomized, double-blind, controlled clinical trial. Forty-four 18- to 60-year-old female and male patients with ungual warts of only one finger were included. The patients were divided into two treatment groups: GA - intralesional bleomycin; and GB - electroporation and intralesional bleomycin. Following a single application, the patients were followed up for 180 days. RESULTS: The patients' mean age was 36 years for GA and 37 years for GB. Most patients were female (68%). Of 22 patients in GA completing the study, 11 (50%) achieved the cure, while 18 (85.7%) of 21 patients completing the study in GB showed cure. A significant association of patients with or without cure after the GA and GB treatments (P = 0.022) was observed. None of the patients in either group had systemic side-effects. Independent of the technique used, all the participants considered the adverse effects tolerable. CONCLUSION: The intralesional use of bleomycin associated with electroporation for the treatment of ungual warts (both periungual and subungual) showed a statistically superior cure when compared with intralesional bleomycin alone. Side-effects were more frequently observed in the electrochemotherapy with bleomycin group than in the bleomycin monotherapy group.

Treatment-induced changes of lymphocyte subsets in patients with adenoid cystic carcinoma of the head and neck.

PURPOSE: Adenoid cystic carcinoma (ACC) of the head and neck is a rare and highly malignant tumor, characterized by perineural growth and early distant metastases. The composition of immune cells in the peripheral blood and the tumor microenvironment is critical to tumor growth and control. However, little is known about the frequency and function of the relevant immune cell subsets in this entity. METHODS: In ACC patients (n = 11) and matched healthy donors (n = 11), the frequency of peripheral blood T and B cells was measured by flow cytometry at different treatment stages of disease (24 samples). Cells were further characterized by their expression of CCR7, PD-1, CD39 and CD73. Tumor-infiltrating lymphocytes (TIL) were analyzed by immunohistochemistry for ten patients and for three patients by flow cytometry. RESULTS: CD4+ T cells had significantly lower frequency after radiotherapy (RT). All other cell frequencies, including Treg, were stable through course of the disease. In B cells, CD73 was reduced after RT. CCR7 expression on T and B cells in patients with relapse/metastases (R/M) differed significantly from patients with active disease. PD-1 remained stable. Treg were more present in TIL compared to peripheral blood. CONCLUSION: Composition of lymphocyte subgroups behaves similar to squamous cell carcinoma in the head and neck, except for Treg, which remained stable. Nevertheless, the CD4+/Treg ratio was lower after RT, which could stand for an immunosuppressive effect in these patients. Therefore, it could be beneficial treating ACC with combined RT and immunomodulatory drugs.

[Subcutaneous foreign body granuloma after neck dissection and adjuvant radiation]. / Subkutane "Perlschnur" nach Neck-Dissection unter adjuvanter Radiatio.

This article presents an oncologic patient with oropharyngeal cancer. After surgery with bilateral neck dissection and adjuvant radiation, the patient developed foreign body granuloma in the area of neck dissection in addition to cervical and mediastinal granuloma. Possible differential diagnoses in this situation are sarcoidosis or tumor-derived sarcoid-like lesions, but also metastases. Therefore, intensified follow-up is particularly important for oncologic patients developing granulomas.

Early Supported Discharge for patients with febrile neutropenia - Experience at a large district hospital in the UK.

Neutropenic sepsis can be life threatening, with mortality 2-21%. The heterogeneity of patients referred with "suspected neutropenic sepsis" has led to strategies being developed to risk-stratify patients and identify those with a low risk of septic complications that could be managed in the outpatient setting, such as The Multinational Association for Supportive Care in Cancer score (MASCC). Outcomes for patients referred with suspected neutropenic sepsis were assessed before and after use of MASCC guided early-supported discharge. 50/123 (41%) patients over 24 months were eligible for early-supported discharge. 26/50 patients had same-day discharge, 14 had overnight admission, 8 stayed 2 nights and 2 stayed 3 nights. Patients received on average 2 follow-up telephone consultations. There were 5 readmissions (10%) and no adverse events. In comparison group; 8 patients over 3-months would have been suitable, potentially saving 40 bed-days. This shows MASCC guided early-supported discharge is safe and cost-effective.

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.

[B cells in head and neck oncology]. / B-Zellen in der Kopf-Hals-Onkologie.

As immunotherapy is becoming increasingly important in the treatment of head and neck cancer, a fundamental understanding of the immunological relationships in the tumor microenvironment is required. The importance of tumor-infiltrating B cells (TIL-B) has been largely neglected so far. In the current literature, however, a significant influence of B cells on tumor growth is described, so that this cell population is now also perceived as a therapeutic target structure. Regulatory B cells (Breg) represent a subset of B cells with immunosuppressive properties. In addition to the secretion of IL-10, Breg can be defined by their ability to produce adenosine. Adenosine is known as an immunosuppressive messenger in the tumor microenvironment whose effect can be prevented by immunotherapeutic approaches. Understanding the tumor immunological relationships, including the different B­cell functions, can help to effectively combine standard approaches including surgery or radiochemotherapy with immunotherapy. In the present article, recent findings on B cells and adenosine in head and neck cancer are described.

Marked improvement in the success rate of medical management of early pregnancy failure following the implementation of a novel institutional protocol and treatment guidelines: a follow-up study.

PURPOSE: To analyze the success rate, time to passage of tissue and subjective patient experience of a newly implemented protocol for medical management of early pregnancy failure (EPF) over a 2-year period. METHODS: A retrospective chart review of all patients with early pregnancy failure primarily opting for medical management was performed. 200 mg mifepristone were administered orally, followed by a single vaginal dose of 800 mcg misoprostol after 36-48 h. We followed-up with our patients using a written questionnaire. RESULTS: 167 women were included in the present study. We observed an overall success rate of 92 %, defined as no need for surgical management after medication administration. We could not identify predictive values for success in a multivariate regression analysis. Most patients (84 %) passed tissue within 6 h after misoprostol administration. The protocol was well tolerated with a low incidence of side effects. Pain was managed well with sufficient analgesics. Responders to the questionnaire felt adequately informed prior to treatment and rated their overall experience as positive. CONCLUSION: The adaption of the institutional medical protocol resulted in a marked improvement of success rate when compared to the previously used protocol (92 vs. 61 %). We credit this increase to the adjusted medication schema as well as to targeted physician education on the expected course and interpretation of outcome measures. Our results underscore that the medical management of EPF is a safe and effective alternative to surgical evacuation in the clinical setting.

Novel calibration tools and validation concepts for microarray-based platforms used in molecular diagnostics and food safety control.

Commercial platforms consisting of ready-to-use microarrays printed with target-specific DNA probes, a microarray scanner, and software for data analysis are available for different applications in medical diagnostics and food analysis, detecting, e.g., viral and bacteriological DNA sequences. The transfer of these tools from basic research to routine analysis, their broad acceptance in regulated areas, and their use in medical practice requires suitable calibration tools for regular control of instrument performance in addition to internal assay controls. Here, we present the development of a novel assay-adapted calibration slide for a commercialized DNA-based assay platform, consisting of precisely arranged fluorescent areas of various intensities obtained by incorporating different concentrations of a "green" dye and a "red" dye in a polymer matrix. These dyes present "Cy3" and "Cy5" analogues with improved photostability, chosen based upon their spectroscopic properties closely matching those of common labels for the green and red channel of microarray scanners. This simple tool allows to efficiently and regularly assess and control the performance of the microarray scanner provided with the biochip platform and to compare different scanners. It will be eventually used as fluorescence intensity scale for referencing of assays results and to enhance the overall comparability of diagnostic tests.

[Immunotherapy of head and neck tumors: Highlights of the ASCO Meeting 2015]. / Immuntherapie von Kopf-Hals-Karzinomen: Highlights des ASCO-Kongresses 2015.

BACKGROUND: In recent years, new immunotherapeutic drugs have become available: the so-called immune checkpoint modulators. With these drugs, unprecedented treatment results have been achieved in different malignant diseases; primarily malignant melanoma, but also in various other malignomas. These achievements have revolutionized the oncologic treatment landscape. This quickly expanding research field, driven by revolutionary treatment results, has put immunotherapy in the focus of attention. OBJECTIVE: Due to rapid developments in the field of immunotherapy, this article aims at introducing, illustrating, and summarizing the field of modern immunotherapy, based on recently presented clinical data from the Annual Meeting of the American Society of Clinical Oncology (ASCO) 2015. MATERIALS AND METHODS: The most important ASCO Meeting 2015 immunotherapy trials for head and neck squamous cell carcinoma (HNSCC) were identified, summarized, and discussed with respect to the current state of research. RESULTS: The oncologic landscape of clinical trials is currently dominated by the new immune checkpoint modulating drugs. Also for HNSCC, a variety of clinical trials and substances are under way. The current primary focus of these trials is targeting and inhibiting the programmed death 1 (PD-1) axis. Cancer immunotherapy with immune checkpoint modulating drugs seems to be independent of human papilloma virus (HPV) status. Robust predictive markers for patient selection are not yet available. CONCLUSION: Current data from clinical trials with immune checkpoint modulators are promising. In the coming years, integration of these drugs into clinical routine can be expected. With regard to the public health economic burden and potential adverse events, the identification of predictive markers for patient selection is a major task for future trials.

[Botulinum toxin and rejuvenation of the eye]. / Toxine botulique et rajeunissement du regard.

AIM OF THE STUDY: Treatments with botulinum toxin in the forehead and periorbital areas may induce disappointing or even paradoxical results. Our study, focused on this area aimed at refining injection techniques by analyzing muscular balances and comparing the effect according to injection doses and topography. METHODS: This experimental study has been carried out in the form of 2 session workshops, with volunteers duly informed of the study contents and giving their informed consent. It was conducted by physicians and surgeons members of SAMCEP* (Société Avancée de Médecine et Chirurgie Esthétique et Plastique). The botulinum toxin was onabotulinumtoxin A. Results were evaluated 15 days after treatment, in regard to global eyebrow position, eyebrow head and tail position; muscle interactions; lines above the eyebrow. Eleven case reports and their results are shown and discussed. CONCLUSION: Our study underlines two important insights: muscle balances and "border areas", between orbicularis oculi and corrugator, key features for eyebrow head, and between frontalis and orbicularis oculifor eyebrow tail.

Headspace measurements of irradiated in vitro cultured cells using PTR-MS.

A pilot study was performed to evaluate a new concept for a radiation biodosimetry method. Proton transfer reaction-mass spectrometry (PTR-MS) was used to find out whether radiation induces changes in the composition of volatile organic compounds (VOCs) in the headspace of in vitro cultured cells. Two different cell lines, retinal pigment epithelium cells hTERT-RPE1 and lung epithelium cells A-549, were irradiated with gamma radiation at doses of 4 Gy and 8 Gy. For measuring the cell-specific effects, the VOC concentrations in the headspace of flasks containing cells plus medium, as well as of flasks containing pure medium were analyzed for changes before and after irradiation. No significant radiation-induced alterations in VOC concentrations in the headspace could be observed after irradiation.

Cross-reactive cytotoxic responses. H-2 restricted are more specific than anti-H-2 responses.

Cross-reactive T-cell cytotoxicity is seen when cytotoxic responses are generated in mixed lymphocyte cultures either between mouse strans which differ at the major histocompatibility complex, H-2, or between H-2b mutant strains and the strain from which they were derived. This cross-reactivity can be measured with [51Cr] labeled target cells from a number of different H-2 haplotypes, and the pattern of cross-reaction indicates that the target antigens are unlikely to be any of the serologically defined public specificities. In contrast, the specificity of H-2 restricted cytotoxic responses, such as that to the male-specific antigen, H-Y, is exquisite, and male cells from strains of mice carrying H-2 haplotypes other than the responder have never been found to act as appropriate targets. The contrast between the specificity of anti-H-2 and H-2 restricted responses may argue for a greater idiotypic homogeneity of the cells makiing H-2 restricted responses, and the greater specificity of these responses may be necessary for their biological function.

Discrimination of cancerous and non-cancerous cell lines by headspace-analysis with PTR-MS.

Proton transfer reaction mass spectrometry (PTR-MS) has been used to analyze the volatile organic compounds (VOCs) emitted by in-vitro cultured human cells. For this purpose, two pairs of cancerous and non-cancerous human cell lines were selected:1. lung epithelium cells A-549 and retinal pigment epithelium cells hTERT-RPE1, cultured in different growth media; and 2. squamous lung carcinoma cells EPLC and immortalized human bronchial epithelial cells BEAS2B, cultured in identical growth medium. The VOCs in the headspace of the cell cultures were sampled: 1. online by drawing off the gas directly from the culture flask; and 2. by accumulation of the VOCs in PTFE bags connected to the flask for at least 12 h. The pure media were analyzed in the same way as the corresponding cells in order to provide a reference. Direct comparison of headspace VOCs from flasks with cells plus medium and from flasks with pure medium enabled the characterization of cell-line-specific production or consumption of VOCs. Among all identified VOCs in this respect, the most outstanding compound was m/z = 45 (acetaldehyde) revealing significant consumption by the cancerous cell lines but not by the non-cancerous cells. By applying multivariate statistical analysis using 42 selected marker VOCs, it was possible to clearly separate the cancerous and non-cancerous cell lines from each other.

Preferential suppression of CXCR4-specific strains of HIV-1 by antiviral therapy.

To initiate infection, HIV-1 requires a primary receptor, CD4, and a secondary receptor, principally the chemokine receptor CCR5 or CXCR4. Coreceptor usage plays a critical role in HIV-1 disease progression. HIV-1 transmitted in vivo generally uses CCR5 (R5), but later CXCR4 (X4) strains may emerge; this shift heralds CD4+ cell depletion and clinical deterioration. We asked whether antiretroviral therapy can shift HIV-1 populations back to R5 viruses after X4 strains have emerged, in part because treatment has been successful in slowing disease progression without uniformly suppressing plasma viremia. We analyzed the coreceptor usage of serial primary isolates from 15 women with advanced disease who demonstrated X4 viruses. Coreceptor usage was determined by using a HOS-CD4+ cell system, biological and molecular cloning, and sequencing the envelope gene V3 region. By constructing a mathematical model to measure the proportion of virus in a specimen using each coreceptor, we demonstrated that the predominant viral population shifted from X4 at baseline to R5 strains after treatment. Multivariate analyses showed that the shift was independent of changes in plasma HIV-1 RNA level and CD4+ cell count. Hence, combination therapy may lead to a change in phenotypic character as well as in the quantity of HIV-1. Shifts in coreceptor usage may thereby contribute to the clinical efficacy of anti-HIV drugs.

Influence of photodynamic therapy on peripheral immune cell populations and cytokine concentrations in head and neck cancer.

BACKGROUND: Photodynamic therapy (PDT) represents a palliative treatment option for a selected group of patients with head and neck squamous cell carcinoma (HNSCC). PDT induces a local inflammatory reaction with the potential to initiate antitumor immune responses. However, the systemic impact on peripheral immune cells has not been described so far. METHODS: HNSCC patients (n=9) were treated with PDT in a palliative setting. All patients had previously undergone several oncologic treatment regimens. Blood samples were taken before, during and after PDT. Age-matched healthy donors served as control group (NC, n=15). The frequency and absolute number of T- and B-lymphocytes, CD4+CD39+ regulatory T-cells (Treg) and NK-cells were measured by 10-color flow cytometry. Serum concentrations of T cell related cytokine panel, including HMGB1, IL-6, IL-10 and perforin were measured by bead array and ELISA. RESULTS: In heavily pretreated HNSCC patients, the number and frequency of Treg and NK-cells were increased as compared to NC. PDT induced a further increase of the frequency of Treg and NK-cells in the peripheral blood. Additionally, the serum concentrations of HMGB1, IL-6 and IL-10 showed a significant elevation after treatment with simultaneously decreased perforin levels. CONCLUSION: Although PDT is a local treatment regimen, a systemic inflammatory response with altered peripheral immune cell populations and cytokine concentrations is visible. The increased Treg and NK cell numbers after PDT support the hypothesis that PDT may successfully be combined with NK cell or T cell activating immune checkpoint modulators in HNSCC patients to improve HNSCC specific immunity.