Why recent studies relating normal tissue response to individual radiosensitivity might have failed and how new studies should be performed

Purpose: New insights into the kinetics of late complications occurring after radiation therapy indicated that all patients have a constant risk of developing late tissue complications. These observations might have a great impact on studies relating normal tissue complications to individual radiosensitivity. Methods and Materials: Data previously published by Peacock et al. were used for analysis. In this study, 39 breast cancer patients with severe reactions (responders) were compared with 65 matched patients showing no reactions (nonresponders). Cellular radiosensitivity as measured in vitro in terms of D-0.01 did not show significant differences between the two groups, both for high-dose-rate (5.84 +/- 0.06 vs. 5.85 +/- 0.07 Gy) and low-dose-rate (7.44 +/- 0.10 vs. 7.56 +/- 0.09 Gy) irradiation. Results: A theoretical distribution was calculated for the individual radiosensitivity of patients with Grade less than or equal to 1, Grade 2, or Grade 3 reactions under the following assumptions: (1) The variation of the individual radiosensitivity is described by a normal distribution. (2) All patients and not only a subgroup have a risk of developing late complications. Based on the normal distribution of low-dose-rate data (mean value [MV] = 7.56 Gy, standard deviation [SDI = 0.5 Gy), a total of 200 hypothetical patients were divided into three groups: a resistant group with a sensitivity greater than or equal to(MV + SD), a normal group with a sensitivity between MV - SD and MV + SD, and a sensitive group :S(MV - SD), the relative fractions being 16%, 68%, and 16%, respectively. It was assumed that these groups differed in the risk of developing late complication; for Grade 3 the annual incidence rate was set at 1%, 2%, and 4% and for Grade 2 at 5%, 10%, and 20% per year, respectively. It was shown that the mean cellular sensitivity calculated for Grade 3 (7.39 +/- 0.10 Gy) or Grade 2 patients (7.46 +/- 0.06 Gy) was slightly but not significantly lower than that of Grade less than or equal to 1 patients (7.65 +/- 0.04 Gy). This result demonstrated that even if the risk was assumed to depend clearly on the individual radiosensitivity, significant differences in the mean cellular sensitivity between responders and nonresponders; were not expected, just as found by Peacock et al. It was shown that a significant correlation between these two parameters could be detected only when the risk was analyzed separately for each group of patients. Conclusion: Most data published so far aiming at establishing a relationship between cellular radiosensitivity and the risk of late complications might need to be reevaluated, because the questions asked up to now were inadequate to arrive at a meaningful answer. (C) 2003 Elsevier Inc

In HPV-Positive HNSCC Cells, Functional Restoration of the p53/p21 Pathway by Proteasome Inhibitor Bortezomib Does Not Affect Radio- or Chemosensitivity

Human papillomavirus (HPV) associated squamous cell carcinomas of the head and neck region (HPV+ HNSCCs) harbor diverging biological features as compared to classical noxa-induced (HPV−) HNSCC. One striking difference between subtypes is that the tumor suppressor gene TP53 is usually not mutated in HPV+ HNSCCs. However, p53 is inhibited by viral oncoprotein E6, leading to premature proteasomal degradation. We asked whether bortezomib (BZM), a clinically approved inhibitor of the proteasome, can functionally restore p53 and investigated in how far this will result in an enhanced radio- or chemosensitivity of HPV+ HNSCC cell lines. For all four HPV+ cell lines tested, BZM led to functional restoration of p53 and transactivation of downstream protein p21. In HPV+ cells, BZM also restored the radiation-induced p53/p21 transactivation. Consistently, in HPV+ cells, a restored G1 arrest as well as enhanced apoptosis were seen when BZM was given prior to irradiation (IR) or cisplatin (CDDP). BZM alone reduced the clonogenic survival of both HPV− and HPV+ cells. However, if BZM was combined with IR or CDDP, BZM did not significantly enhance radio- or chemosensitivity of HPV+ or HPV− HNSCC cell lines

In cancer cell lines inhibition of SCF/c-Kit pathway leads to radiosensitization only when SCF is strongly over-expressed

Background and purpose: The SCF/c-Kit pathway is often overexpressed in human tumors leading to an enhanced tumorigenesis, proliferation and migration. It was now tested for NSCLC and prostate cancer cells growing in 2D and 3D whether the inhibition of this pathway can be used to achieve a significant radiosensitization and whether a respective biomarker may be identified. Material and methods: Experiments were performed with different cancer cell lines (NSCLC: H23, H520, H226, H1975 and PrCa: DU145) growing either under 2D or 3D conditions. Expression of SCF and c-Kit was determined by RT-PCR and Western blot, SCF was knocked down by siRNA, c-Kit was inhibited by ISCK03 inhibitor and cell survival was determined by colony formation assay. Results: There is a profound variation in the expression of both c-Kit and SCF with no association between each other. Neither levels did correlate with the respective cellular radiosensitivity determined for 2D or 3D with only a trend seen for SCF. Knock-down of SCF was generally found to result in no or only minor reduction of plating efficiency or cellular radioresistance. A significant reduction was only obtained for H520 cells characterized by an extreme over-expression of SCF. The inhibition of c-Kit by a specific inhibitor was also found to result only in minor radiosensitization. Conclusion: Generally, the SCF/c-Kit pathway does not have a dominant effect on both, cell survival and radioresponse and, as a consequence, knockdown of this pathway does not result in a strong effect on radioresistance, except when SCF is strongly over-expressed

Dual PI3K/mTOR Inhibitor NVP-BEZ235 Leads to a Synergistic Enhancement of Cisplatin and Radiation in Both HPV-Negative and -Positive HNSCC Cell Lines

The standard of care for advanced head and neck cancers (HNSCCs) is radiochemotherapy, including cisplatin. This treatment results in a cure rate of approximately 85% for oropharyngeal HPV-positive HNSCCs, in contrast to only 50% for HPV-negative HNSCCs, and is accompanied by severe side effects for both entities. Therefore, innovative treatment modalities are required, resulting in a better outcome for HPV-negative HNSCCs, and lowering the adverse effects for both entities. The effect of the dual PI3K/mTOR inhibitor NVP-BEZ235 on a combined treatment with cisplatin and radiation was studied in six HPV-negative and six HPV-positive HNSCC cell lines. Cisplatin alone was slightly more effective in HPV-positive cells. This could be attributed to a defect in homologous recombination, as demonstrated by depleting RAD51. Solely for HPV-positive cells, pretreatment with BEZ235 resulted in enhanced cisplatin sensitivity. For the combination of cisplatin and radiation, additive effects were observed. However, when pretreated with BEZ235, this combination changed into a synergistic interaction, with a slightly stronger enhancement for HPV-positive cells. This increase could be attributed to a diminished degree of DSB repair in G1, as visualized via the detection of γH2AX/53BP1 foci. BEZ235 can be used to enhance the effect of combined treatment with cisplatin and radiation in both HPV-negative and -positive HNSCCs

Comparison of stem-loop sequences in H2B genes.

<p>Alignment of the stem-loop sequences of H2B genes performed with ClustalW2 multiple sequence alignments tools (<a href="http://www.ebi.ac.uk/Tools/msa/clustalw2/" target="_blank">http://www.ebi.ac.uk/Tools/msa/clustalw2/</a>). Highly expressed histones are marked in grey. Stem loop sequence is shown in orange, bases that are different from canonical are underlined.</p

Expression of the histone H2B gene complement in different cell lines.

<p>Expression of different H2B genes in the indicated cell lines was analyzed by qRT-PCR. Relative expression values between the individual genes were normalized using diploid genomic DNA (see materials and methods) and indicated as “Rel. gDNA units”. Mean±SD, n = 3.</p