Cell-to-cell distances between tumor-infiltrating inflammatory cells have the potential to distinguish functionally active from suppressed inflammatory cells.

Beyond their mere presence, the distribution pattern of inflammatory cells is of special interest. Our hypothesis was that random distribution may be a clear indicator of being non-functional as a consequence of lack of interaction. Here, we have assessed the implication of cell-to-cell distances among inflammatory cells in anal squamous cell carcinoma and a possible association with survival data. Thirty-eight patients suffering from anal carcinoma were studied using tissue microarrays, double staining immunohistochemistry, whole slide scanning and image analysis software. Therapy consisted of concurrent radiochemotherapy. Numbers of stromal and intraepithelial tumor-infiltrating inflammatory cells (TIC) and the distances between cells were quantified. Double-staining of FoxP3(+) cells with either CD8(+), CD1a(+) or CD20(+) cells was performed. Measured cell-to-cell distances were compared to computer simulated cell-to-cell distances leading to the assumption of non-randomly distributed and therefore functional immune cells. Intraepithelial CD1a(+) and CD20(+) cells were randomly distributed and therefore regarded as non-functional. In contrary, stromal CD20(+) cells had a non-random distribution pattern. A non-random distance between CD20(+) and FoxP3(+) cells was associated with a clearly unfavorable outcome. Measured distances between FoxP3(+) cells were distinctly shorter than expected and indicate a functional active state of the regulatory T cells (Treg). Analysis of cell-to-cell distances between TIC has the potential to distinguish between suppressed non-functional and functionally active inflammatory cells. We conclude that in this tumor model most of the CD1a(+) cells are non-functional as are the intraepithelial CD20(+) cells, while stromal CD20(+) cells and FoxP3(+) cells are functional cells.

Radiosensitization by BRAF inhibitor therapy-mechanism and frequency of toxicity in melanoma patients.

BACKGROUND: Recent evidence suggests that ionizing radiation may be associated with unexpected side-effects in melanoma patients treated with concomitant BRAF inhibitors. A large multicenter analysis was carried out to generate reliable safety data and elucidate the mechanism. METHODS: A total of 161 melanoma patients from 11 European skin cancer centers were evaluated for acute and late toxicity, of whom 70 consecutive patients received 86 series of radiotherapy with concomitant BRAF inhibitor therapy. To further characterize and quantify a possible radiosensitization by BRAF inhibitors, blood samples of 35 melanoma patients were used for individual radiosensitivity testing by fluorescence in situ hybridization of chromosomal breaks after ex vivo irradiation. RESULTS: With radiotherapy and concomitant BRAF inhibitor therapy the rate of acute radiodermatitis ≥2° was 36% and follicular cystic proliferation was seen in 13% of all radiotherapies. Non-skin toxicities included hearing disorders (4%) and dysphagia (2%). Following whole-brain radiotherapy, rates of radiodermatitis ≥2° were 44% and 8% (P < 0.001) for patients with and without BRAF inhibitor therapy, respectively. Concomitant treatment with vemurafenib induced acute radiodermatitis ≥2° more frequently than treatment with dabrafenib (40% versus 26%, P = 0.07). In line with these findings, analysis of chromosomal breaks ex vivo indicated significantly increased radiosensitivity for patients under vemurafenib (P = 0.004) and for patients switched from vemurafenib to dabrafenib (P = 0.002), but not for patients on dabrafenib only. No toxicities were reported after stereotactic treatment. CONCLUSION: Radiotherapy with concomitant BRAF inhibitor therapy is feasible with an acceptable increase in toxicity. Vemurafenib is a more potent radiosensitizer than dabrafenib.

Radiochemotherapy induces a favourable tumour infiltrating inflammatory cell profile in head and neck cancer.

Head and neck squamous cell cancers (HNSSC) generate an immune-suppressive micro-environment by a specific pattern of tumour infiltrating inflammatory cells. The aim of our study was to evaluate the impact of radiochemotherapy on the numbers and composition of inflammatory cells and its influence on outcome. Fifty-eight patients suffering from oral cavity cancer were studied, whose therapy consisted of concurrent radiochemotherapy followed by surgery. Numbers and ratios of tumour infiltrating inflammatory cells were compared prior to and after radiochemotherapy. Intraepithelial and stromal location of tumour infiltrating inflammatory cells was analysed separately. Infiltration of CD3(+), CD4(+), CD25(+), FoxP3(+), CD8(+), Granzyme B(+), CD20(+) and CD68(+) cells predominated in the peritumoural stromal compartment, whereas CD1a(+) dendritic cells were found more frequently in the intraepithelial compartment. Neoadjuvant treatment was associated with a general decrease of tumour infiltrating inflammatory cells in both compartments. The CD8(+) and Granzyme B(+) cytotoxic cells decreased only slightly after RCT. In contrast, the decrease of FoxP3(+) regulatory T cells was more pronounced and the cytotoxic T-cell/FoxP3(+) ratio increased 2- to 3-fold in both compartments, respectively. Patients with high cytotoxic cell numbers, high dendritic cell numbers and a high ratio of cytotoxic cells to regulatory T cells had a better disease free survival. Concurrent radiochemotherapy of oral squamous cell carcinoma was shown to drive the composition of inflammatory cells in a direction which is supposed to be prognostically favourable.

Hyperthermia and irradiation of head and neck squamous cancer cells causes migratory profile changes of tumour infiltrating lymphocytes.

PURPOSE: CD4(+)CD25(+)FoxP3(+) regulatory T-cells (Treg) are responsible for immunoevasion mechanisms induced by cancer. Specific chemokines such as CCL22 are presumed to mediate active Treg trafficking into the tumour site. In this context, the effects of irradiation and hyperthermia of tumour cells on Treg migration and the CCL22 concentration in the tumour cell supernatants after treatment were studied. Moreover, the relationship between CCL22 concentration and Treg cell migration was also examined. MATERIALS AND METHODS: Treg and CD4(+)CD25(-) T-cells were isolated from human peripheral blood. Supernatants were obtained from primary cell cultures derived from head and neck carcinoma patients. Tumour cell cultures were treated with a dose of 2 Gy and hyperthermia (41.5 degrees C) or with hyperthermia or irradiation alone. Cancer cell culture supernatants were then used for a transmigration assay. RESULTS: Treg and CD4(+)CD25(-) T-cells showed an increased transmigration towards supernatants of hyperthermia-treated tumour cells. After combined application of hyperthermia and irradiation, Treg migration was similar to control levels, but CD4(+)CD25(-) migration was still enhanced. Irradiation caused a significantly decreased Treg influx, whereas the CD4(+)CD25(-) T-cell migration was not altered after the same treatment. Changes of Treg chemotaxis could be attributed to a treatment-associated escalation of the CCL22 in the tumour cell supernatants. CONCLUSION: The combination of irradiation and hyperthermia is able to modify transmigration of tumour infiltrating lymphocytes beneficially and individually. In this in vitro system hyperthermia alone negatively impacts the immune response by selectively recruiting Treg, whereas hyperthermia with the addition of irradiation negates this effect.

Analysis of radiation- and 5-FU-induced inhibition of cell proliferation by an automatic colony analyser.

Proliferation of Chinese hamster cells B-14 was inhibited by irradiation, by incubation with 5-fluorouracil (5-FU) and by a combination of both treatments. The reduction in proliferation was assayed by the colony formation test, which was evaluated by an automatic colony analyser according to the number and volume of the colonies. It was demonstrated that the number of colonies multiplied by the volume was equivalent to the number of cells in a Petri dish and is called total colony volume. Since this quantity reflects the entire proliferation of cells, it is a more sensitive parameter for measuring cell viability than the clonogenicity of cells. The drug-radiation interaction showed a supra-additive effect, if total colony volume is taken into account, while the traditional scoring of colonies yielded only an additive effect.

The effect of serum albumin on the radiolysis of DNA studied by constant field electrophoresis and compared to alterations caused by low molecular weight OH. scavengers.

After radiolysis of calf thymus DNA in 10(-2) mol dm-3 phosphate buffer at pH7 under N2, N2O and air the yields of double-strand breaks (dsb) have been determined by constant field electrophoresis. Double-strand (dsb) breaks were formed according to a linear-quadratic relationship with dose showing a lower G-value under aerobic than under anaerobic conditions (G (air) = 1.4 nmolJ-1; G (N2) = 2.1 nmolJ-1; G (N2O) = 4.9 nmolJ-1). To test the reliability of this system the effect of low molecular weight OH. scavengers which were already used in comparable work with plasmid DNA were studied. The results with plasmid DNA and calf thymus DNA obtained by different techniques of electrophoresis agreed quite well. Under N2 more protection was obtained with ethanol than with DMSO or with t-butanol. Under air, double-strand breakage was further decreased and reached the same level with all of these scavengers. Furthermore the constant field electrophoresis gives similar results as the low-angle light scattering technique for radiation induced double strand breakage of calf thymus DNA. When BSA was used at the same scavenger capacity as the low molecular weight scavengers, the protection against double strand breakage was less if radiolysis was carried out in the presence of proteins. Under anaerobic conditions the protection factor was 13 in the presence of BSA, while with DMSO or t-butanol this factor was about 100 and with ethanol 300. In contrast to the low molecular weight OH. scavengers oxygen enhanced radiation-induced double-strand breakage with BSA. It is assumed that protein peroxyl radicals may cause strand breakage.