Associations between patient-reported late effects and systemic cytokines in long-term survivors of head and neck cancer treated with radiotherapy.

PURPOSE: Head and neck cancer (HNC) treatment may lead to late effects and impaired health-related quality of life of survivors. Knowledge on long-term late effects after radiotherapy (RT) and potential underlying biological mechanisms is lacking. We assessed the prevalence of xerostomia, dysphagia, and chronic fatigue (CF) in HNC survivors ≥ 5 years post-RT, and examined associations between pro-inflammatory cytokines and late effects. METHODS: In a cross-sectional study, 263 HNC survivors treated between 2007 and 2013 were enrolled. They completed validated questionnaires assessing xerostomia and dysphagia (the EORTC QLQ-H&N35), and CF (the Fatigue Questionnaire), and underwent blood sampling and clinical examination. Pro-inflammatory cytokines were analyzed in 262 survivors and 100 healthy age- and gender-matched controls. RESULTS: Median time since treatment was 8.5 years. The proportions of survivors reporting xerostomia, dysphagia, and CF were 58%, 31%, and 33%, respectively, with a preponderance of females. We found no significant associations between IL-6, IL-8, IP-10, TARC, TNF, or ENA-78 and the three late effects. The odds of having elevated levels of IL-6 and IP-10 were significantly higher in the survivors compared to the controls. CONCLUSIONS: More than one-third of long-term HNC survivors experienced xerostomia, dysphagia, and CF. Persistent inflammation, with elevated systemic cytokines, was not associated with these late effects, although HNC survivors had higher levels of some cytokines than the controls. IMPLICATIONS FOR CANCER SURVIVORS: This study provides new knowledge on late effects that can serve as grounds for informing patients with HNC about risk of late effects more than 5 years after RT.

Proton-dynamic therapy following photosensitiser activation by accelerated protons demonstrated through fluorescence and singlet oxygen production.

We demonstrate excitation of photosensitisers (PSs) by accelerated protons to produce fluorescence and singlet oxygen. Their fluorescence follows a pattern similar to the proton energy loss in matter, while proton-derived fluorescence spectra match the photon-induced spectra. PSs excited in dry gelatin exhibit enhanced phosphorescence, suggesting an efficient PSs triplet state population. Singlet oxygen measurements, both optically at ~1270 nm and through the photoproduct of protoporphyrin IX (PpIX), demonstrate cytotoxic singlet oxygen generation by proton excitation. The singlet oxygen-specific scavenger 1,4-diazabicyclo[2.2.2]octane (DABCO) abrogates the photoproduct formation under proton excitation, but cannot countermand the overall loss of PpIX fluorescence. Furthermore, in two cell lines, M059K and T98G, we observe differential cell death upon the addition of the PS cercosporin, while in U87 cells we see no effect at any proton irradiation dose. Our results pave the way for a novel treatment combining proton therapy and "proton-dynamic therapy" for more efficient tumour eradication.

Consequences of leaf calibration errors on IMRT delivery.

IMRT treatments using multi-leaf collimators may involve a large number of segments in order to spare the organs at risk. When a large proportion of these segments are small, leaf positioning errors may become relevant and have therapeutic consequences. The performance of four head and neck IMRT treatments under eight different cases of leaf positioning errors has been studied. Systematic leaf pair offset errors in the range of +/-2.0 mm were introduced, thus modifying the segment sizes of the original IMRT plans. Thirty-six films were irradiated with the original and modified segments. The dose difference and the gamma index (with 2%/2 mm criteria) were used for evaluating the discrepancies between the irradiated films. The median dose differences were linearly related to the simulated leaf pair errors. In the worst case, a 2.0 mm error generated a median dose difference of 1.5%. Following the gamma analysis, two out of the 32 modified plans were not acceptable. In conclusion, small systematic leaf bank positioning errors have a measurable impact on the delivered dose and may have consequences for the therapeutic outcome of IMRT.

Formates and dithionates: sensitive EPR-dosimeter materials for radiation therapy.

Polycrystalline formates and dithionates are promising materials for EPR dosimetry, as large yields of radiation induced stable radicals are formed with a linear dose response. Rapid spin relaxation rates were detected in many of the substances, indicating that a high microwave power can be applied during EPR acquisition in order to improve sensitivity. Different techniques used to further improve the sensitivity, such as the replacement of 7Li with 6Li or exchange of protons with deuterons in the corresponding crystalline matrices and metal ion doping are discussed. It is concluded that formates and dithionates may be up to 10 times as sensitive as L-alpha-alanine.

Development of an extensive set of 16S rDNA-targeted primers for quantification of pathogenic and indigenous bacteria in faecal samples by real-time PCR.

AIMS: The microbiota of the human intestinal tract constitutes a complex ecosystem. We report the design and optimization of an extensive set of 16S rDNA-targeted species- and group-specific primers for more accurate quantification of bacteria from faecal samples with real-time PCR. METHODS AND RESULTS: A linear range of quantification between 0.1-10 pg and 10 ng of specific target genome was obtained, which corresponds to detection of ca 30-4500 to 1.9 x 10(6)-6.0 x 10(6) target bacterial genomes. Functionality of the assays was confirmed by quantification of target bacterial DNA from faecal DNA preparations of healthy volunteers and irritable bowel syndrome (IBS) patients. Additionally, spiking of faecal preparations with Helicobacter pylori, Clostridium difficile or Campylobacter jejuni was used to confirm the accurate and sensitive quantification. CONCLUSIONS: Real-time PCR is a very sensitive and precise technique for an extensive quantitative evaluation of gut microbiota and is feasible for detection of human pathogens from faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: To design and optimize an extensive set of real-time PCR assays targeting a large group of predominant and pathogenic GI microbial species for further use in updating the current knowledge of the putative role of gut microbiota in health and disease.

Genetic heterogeneity and functional properties of intestinal bifidobacteria.

AIMS: The aim of the present study was to compare several molecular methods for the identification and genotyping of bifidobacteria, and further to investigate genetic heterogeneity and functional properties of bifidobacterial isolates from intestinal samples of Finnish adult subjects. METHODS AND RESULTS: A total of 153 intestinal bifidobacterial isolates were included in initial screening and 34 isolates were further characterized. Identification results obtained with PCR-ELISA and ribotyping were well in accordance with each other, while randomly amplified polymorphic DNA (RAPD) gave tentative identification only to Bifidobacterium bifidum and to 65% of the B. longum isolates. The most commonly detected species were B. longum biotype longum followed by B. adolescentis and B. bifidum. In addition, B. animalis (lactis), B. angulatum and B. pseudocatenulatum were found. Ribotyping and pulsed-field gel electrophoresis (PFGE) proved to be discriminatory methods for bifidobacteria, but also RAPD revealed intraspecies heterogeneity. Besides two B. animalis (lactis) isolates with very close similarity to a commercially available probiotic strain, none of the intestinal isolates showed optimal survival in all tolerance (acid, bile and oxygen) or growth performance tests. CONCLUSIONS: Several species/strains of bifidobacteria simultaneously colonize the gastrointestinal tract of healthy Finnish adults and intestinal Bifidobacterium isolates were genetically heterogeneous. Functional properties of bifidobacteria were strain-dependent. SIGNIFICANCE AND IMPACT OF THE STUDY: Applicability of ribotyping with the automated RiboPrinter System for identification and genotyping of bifidobacteria was shown in the present study.

The influence of autologous tumor fibroblasts on the radiosensitivity of squamous cell carcinoma megacolonies.

PURPOSE: To study the influence of tumor fibroblasts on radiosensitivity and stem cell fraction of tumor cells in squamous cell carcinoma megacolonies by determining colony cure and clonogen survival. METHODS AND MATERIALS: Murine squamous cell carcinoma cells (AT478c) grown as flat but multilayered megacolonies were co-cultured with pre-irradiated tumor fibroblasts derived from the same carcinoma, and irradiated with 1, 2, 4, or 8 fractions. Recurrent clones and their growth pattern in situ were recorded. From megacolony cure data and clonogen survival data, the clonogen number and the parameters of cellular radiosensitivity were calculated. RESULTS: The curability of the co-cultured megacolonies, as determined by TCD50 values, was significantly increased compared to the megacolonies without fibroblasts (p < 0.01). Both the megacolony cure and clonogen survival data suggested a decrease of the clonogen fraction in the co-cultured megacolonies. CONCLUSIONS: The presence of tumor fibroblasts increases megacolony radiosensitivity. This is due to a decrease in the fraction of clonogens in the tumor megacolony, apparently caused by a downregulation of the stem cell fraction of the tumor cells.

Regrowth delay analysis of irradiated tumors in the curative dose region.

PURPOSE: To develop a method to analyze regrowth delay times, including cured tumors. METHODS AND MATERIALS: Regrowth delay measured in the in vitro squamous cell carcinoma megacolony system following fractionated irradiation was analyzed using two different approaches. A conventional regrowth delay analysis based on means or medians of dose groups was contrasted with a one-step procedure using the inverse of individual regrowth delay times, where cured tumors are represented by an infinite delay time. Two data sets with different proportions of cured tumors were compared in this way. In addition, for both approaches, confidence limits were also generated by the bootstrap technique. RESULTS: The parameter estimated was the alpha/beta ratio. The inverse analysis yielded similar results as the conventional analysis, but with much smaller confidence limits. When the dataset was stripped by stepwise removal of lower dose groups with no cures, the estimates of the alpha/beta ratio remained definitely more robust in inverse analysis. Results of the bootstrap procedure confirmed the accuracy of the calculated confidence intervals. CONCLUSION: Regrowth delay analysis using reciprocal regrowth delay times is a useful tool, especially when the data shows large variations, or a substantial fraction of the dataset falls into the curative dose region.