Cordula Petersen

HNSCC cell lines positive for HPV and p16 possess higher cellular radiosensitivity due to an impaired DSB repair capacity

BACKGROUND AND PURPOSE When treated by radiotherapy, patients with squamous cell carcinomas of the head and neck (HNSCC) positive for HPV and p16(INK4a) possess a clearly favorable prognosis as compared to those with HPV-negative HNSCC. The aim of this work was to study whether the better outcomes might be caused by an enhanced cellular radiosensitivity. MATERIALS AND METHODS The radiation response of five HPV/p16(INK4a)-positive and five HPV-negative cell lines was characterized with regard to cellular radiosensitivity by colony formation assay. Furthermore G1- and G2-arrest, apoptosis and residual DNA double-strand breaks (DSB) were analyzed by the colcemid-based G1-efflux assay, propidium iodide staining, the detection of PARP cleavage, the fluorescence-based detection of caspase activity and the immunofluorescence staining of γH2AX and 53BP1 foci. RESULTS On average, the cellular radiosensitivity of the HNSCC cell lines positive for HPV and p16(INK4a) was higher as compared to the sensitivity of a panel of five HPV-negative HNSCC cell lines (SF3=0.2827 vs. 0.4455). The higher sensitivity does not result from increased apoptosis or the execution of a permanent G1-arrest, but is rather associated with both, elevated levels of residual DSBs and extensive G2-arrest. CONCLUSIONS Increased cellular radiosensitivity due to compromised DNA repair capacity is likely to contribute to the improved outcome of patients with HPV/p16(INK4a)-positive tumors when treated by radiotherapy.

[18F]fluoroethylcholine-PET/CT imaging for radiation treatment planning of recurrent and primary prostate cancer with dose escalation to PET/CT-positive lymph nodes.

BackgroundAt present there is no consensus on irradiation treatment volumes for intermediate to high-risk primary cancers or recurrent disease. Conventional imaging modalities, such as CT, MRI and transrectal ultrasound, are considered suboptimal for treatment decisions. Choline-PET/CT might be considered as the imaging modality in radiooncology to select and delineate clinical target volumes extending the prostate gland or prostate fossa. In conjunction with intensity modulated radiotherapy (IMRT) and imaged guided radiotherapy (IGRT), it might offer the opportunity of dose escalation to selected sites while avoiding unnecessary irradiation of healthy tissues.MethodsTwenty-six patients with primary (n = 7) or recurrent (n = 19) prostate cancer received Choline-PET/CT planned 3D conformal or intensity modulated radiotherapy. The median age of the patients was 65 yrs (range 45 to 78 yrs). PET/CT-scans with F18-fluoroethylcholine (FEC) were performed on a combined PET/CT-scanner equipped for radiation therapy planning.The majority of patients had intermediate to high risk prostate cancer. All patients received 3D conformal or intensity modulated and imaged guided radiotherapy with megavoltage cone beam CT. The median dose to primary tumours was 75.6 Gy and to FEC-positive recurrent lymph nodal sites 66,6 Gy. The median follow-up time was 28.8 months.ResultsThe mean SUVmax in primary cancer was 5,97 in the prostate gland and 3,2 in pelvic lymph nodes. Patients with recurrent cancer had a mean SUVmax of 4,38. Two patients had negative PET/CT scans. At 28 months the overall survival rate is 94%. Biochemical relapse free survival is 83% for primary cancer and 49% for recurrent tumours. Distant disease free survival is 100% and 75% for primary and recurrent cancer, respectively. Acute normal tissue toxicity was mild in 85% and moderate (grade 2) in 15%. No or mild late side effects were observed in the majority of patients (84%). One patient had a severe bladder shrinkage (grade 4) after a previous treatment with TUR of the prostate and seed implantation.ConclusionsFEC-PET/CT planning could be helpful in dose escalation to lymph nodal sites of prostate cancer.

Individual Radiosensitivity Measured With Lymphocytes May Predict the Risk of Acute Reaction After Radiotherapy

PURPOSE We tested whether the chromosomal radiosensitivity of in vitro irradiated lymphocytes could be used to predict the risk of acute reactions after radiotherapy. METHODS AND MATERIALS Two prospective studies were performed: study A with 51 patients included different tumor sites and study B included 87 breast cancer patients. Acute reaction was assessed using the Radiation Therapy Oncology Group score. In both studies, patients were treated with curative radiotherapy, and the mean tumor dose applied was 55 Gy (40-65) +/- boost with 11 Gy (6-31) in study A and 50.4 Gy +/- boost with 10 Gy in study B. Individual radiosensitivity was determined with lymphocytes irradiated in vitro with X-ray doses of either 3 or 6 Gy and scoring the number of chromosomal deletions. RESULTS Acute reactions displayed a typical spectrum with 57% in study A and 53% in study B showing an acute reaction of Grade 2-3. Individual radiosensitivity in both studies was characterized by a substantial variation and the fraction of patients with Grade 2-3 reaction was found to increase with increasing individual radiosensitivity measured at 6 Gy (study A, p = 0.238; study B, p = 0.023). For study B, this fraction increased with breast volume, and the impact of individual radiosensitivity on acute reaction was especially pronounced (p = 0.00025) for lower breast volume. No such clear association with acute reaction was observed when individual radiosensitivity was assessed at 3 Gy. CONCLUSION Individual radiosensitivity determined at 6 Gy seems to be a good predictor for risk of acute effects after curative radiotherapy.

Proliferation and micromilieu during fractionated irradiation of human FaDu squamous cell carcinoma in nude mice

Purpose: Previous functional radiobiological experiments demonstrated a significant acceleration of repopulation after 3 weeks and reoxygenation after 12 days of radiotherapy of FaDu tumours. Owing to the temporal coincidence between repopulation and reoxygenation, it was hypothesized that the improved oxygenation status during fractionated irradiation might be the preceding stimulus for increased proliferation. The study investigated whether these changes in repopulation and re‐oxygenation are reflected by histological parameters of proliferation and the tumour micromilieu. Materials and methods: Human FaDu squamous cell carcinomas in nude mice were irradiated with three to 18 fractions of 3 Gy daily or every second day under normal blood flow and clamp hypoxia. At different time points, tumours were excised and stained for Ki67, BrdUrd, epidermal growth factor receptor (EGFR) and markers of the micromilieu (HOECHST 33342, pimonidazole, ER‐MP12). Results: On average, Ki67 and BrdUrd labelling indices decreased initially and increased again at later times during the course of fractionated radiotherapy. A similar kinetic pattern was found for the staining intensity of the EGFR. The vascular density in the viable tumour area remained constant during the whole course of irradiation, while the perfused fraction of vessels decreased within the first week of irradiation and returned to baseline values after 2 weeks. There was a corresponding increase in perfusion and a decrease in cellular hypoxia. Conclusions: The histological results were in surprisingly good agreement with the kinetics of clonogen repopulation and re‐oxygenation determined previously using functional assays. The results support that the kinetics of repopulation of FaDu squamous cell carcinoma in response to fractionated irradiation are determined not only by intracellular processes, but also by a complex interaction of proliferation parameters with a changing microenvironment.

Clinical management of primary vulvar cancer

AIMS Vulvar cancer is a rare disease with increasing incidence over the last decades. Treatment includes surgical, radio- and chemotherapeutical options; however, due to the low incidence of the disease and the lack of randomised trials many questions regarding indication of different treatment approaches remain unanswered. This article discusses the current literature to elaborate recommendations for the management of primary vulvar cancer in clinical routine. METHODS We reviewed the available literature on treatment of invasive vulvar cancer with emphasis on therapeutic strategies such as surgery and radio/chemotherapy. RESULTS Surgery of the primary tumour and the groins remain the cornerstone of treatment in vulvar cancer with a strong trend towards a less radical approach in early stage disease. Complete vulvectomy was replaced by radical local excision with plastic reconstruction and the sentinel node technique was implemented to avoid the morbidity of complete groin dissection in node negative patients. In patients with advanced primary disease, treatment decisions are still a challenge. Criteria for the indication and performance of chemo/radiotherapy of the vulva/groins/pelvis are still not fully established and vary between different countries and institutions due to the low level of evidence. Often an individualised therapeutic approach aside from guidelines is necessary to treat these patients adequately. CONCLUSIONS To enable reasonable treatment decisions and avoid unnecessary morbidity, treatment in specialised centres should be intended at any time. Clinical studies performed by several study groups on an international level are urgently needed to further improve therapy.

Selective inhibition of the epidermal growth factor receptor tyrosine kinase by BIBX1382BS and the improvement of growth delay, but not local control, after fractionated irradiation in human FaDu squamous cell carcinoma in the nude mouse.

Purpose: To investigate the effect of BIBX1382BS, an inhibitor of the epidermal growth factor receptor tyrosine kinase, on proliferation and clonogenic cell survival of FaDu human squamous cell carcinoma in vitro, and on tumour growth and local tumour control after fractionated irradiation over 6 weeks in nude mice. FaDu human squamous cell carcinoma is epidermal growth factor receptor positive and significant repopulation during fractionated irradiation was demonstrated in previous experiments. Materials and methods: Receptor status, receptor phosphorylation, cell cycle distribution, cell proliferation and clonogenic cell survival after irradiation were assayed with and without BIBX1382BS (5 µM) in vitro. Tumour volume doubling time, BrdUrd and Ki67 labelling indices and apoptosis were investigated in unirradiated tumours growing in NMRI nude mice treated daily with BIBX1382BS (50 mg kg−1 body weight orally) or carrier. Tumour growth delay and dose–response curves for local tumour control were determined after irradiation with 30 fractions within 6 weeks. Results: BIBX1382BS blocked radiation‐induced phosphorylation of the epidermal growth factor receptor and reduced the doubling time of FaDu cells growing in vitro by a factor of 4.9 (p=0.008). Radiosensitivity in vitro remained unchanged after incubation with BIBX1382BS for 3 days and decreased moderately after 6 days (p=0.001). BIBX1382BS significantly reduced the volume doubling time of established FaDu tumours in nude mice by factors of 2.6 when given over 15 days (p<0.001) and 3.7 when applied over 6 weeks (p<0.001). When given simultaneously to fractionated irradiation, growth delay was significantly prolonged by an average of 33 days (p=0.003). Local tumour control was not improved by BIBX1382BS. The radiation doses necessary to control 50% of the tumours locally were 63.6 Gy (95% confidence interval 55; 73) for irradiation alone and 67.8 Gy (60; 77) for the combined treatment (p=0.5). Conclusions: Despite clear antiproliferative activity in rapidly repopulating FaDu human squamous cell carcinoma and significantly increased tumour growth delay when combined with fractionated irradiation, local tumour control was not improved by BIBX1382BS. The results do not disprove that epidermal growth factor receptor inhibition might enhance the results of radiotherapy. However, the results imply that further preclinical investigations using relevant treatment schedules and appropriate endpoints are necessary to explore the mechanisms of action and efficacy of such combinations.

Local tumor control probability modeling of primary and secondary lung tumors in stereotactic body radiotherapy

BACKGROUND AND PURPOSE To evaluate whether local tumor control probability (TCP) in stereotactic body radiotherapy (SBRT) varies between lung metastases of different primary cancer sites and between primary non-small cell lung cancer (NSCLC) and secondary lung tumors. MATERIALS AND METHODS A retrospective multi-institutional (n=22) database of 399 patients with stage I NSCLC and 397 patients with 525 lung metastases was analyzed. Irradiation doses were converted to biologically effective doses (BED). Logistic regression was used for local tumor control probability (TCP) modeling and the second-order bias corrected Akaike Information Criterion was used for model comparison. RESULTS After median follow-up of 19 months and 16 months (n.s.), local tumor control was observed in 87.7% and 86.7% of the primary and secondary lung tumors (n.s.), respectively. A strong dose-response relationship was observed in the primary NSCLC and metastatic cohort but dose-response relationships were not significantly different: the TCD90 (dose to achieve 90% TCP; BED of maximum planning target volume dose) estimates were 176 Gy (151-223) and 160 Gy (123-237) (n.s.), respectively. The dose-response relationship was not influenced by the primary cancer site within the metastatic cohort. CONCLUSIONS Dose-response relationships for local tumor control in SBRT were not different between lung metastases of various primary cancer sites and between primary NSCLC and lung metastases.

RAD51 overexpression is a negative prognostic marker for colorectal adenocarcinoma

RAD51 is the central protein in the homologous recombination pathway and is therefore of great relevance in terms of both therapy resistance as well as genomic stability. By using a tissue microarray analysis of 1,213 biopsies taken from colorectal adenocarcinomas (CRCs), we investigated whether RAD51 expression can be used as a prognostic marker as well as potential associations between this and the expression of other proteins known to be related to CRC. Strong RAD51 expression was observed in 1% of CRC, moderate in 11%, weak in 34% and no expression in 44%. No correlation was found between RAD51 expression and clinicopathological parameters. RAD51 expression correlated significantly (p = 0.001) with overall survival, with a median survival of 11 months for patients with strong, 46 with moderate, 76 with weak and 68 with negative expression. Multivariate analyses revealed that in addition to tumor stage (p < 0.0001) and nodal status (p < 0.0001), RAD51 expression is also an independent prognostic parameter (p = 0.011). Strong RAD51 expression was found to be associated with the loss of the two DNA mismatch repair proteins MSH (p = 0.0003), MLH (p = 0.002) and β‐catenin (p = 0.012) as well as with elevated p21 (p = 0.003) and EGFR expression (p = 0.0001). However, a correlation with overall survival could only be found for EGFR expression (p = 0.008), although no added benefit in risk stratification could be determined when evaluated together with RAD51. Overexpression of RAD51 is a predictor of poor outcome in CRC. This finding indicated the promise of future studies using RAD51 as a prognostic marker and therapeutic target.

The extreme radiosensitivity of the squamous cell carcinoma SKX is due to a defect in double-strand break repair

PURPOSE Squamous cell carcinomas (SCCs) are characterized by moderate radiosensitivity. We have established the human head & neck SCC cell line SKX, which shows an exceptionally high radiosensitivity. It was the aim of this study to understand the underlying mechanisms. MATERIALS & METHODS Experiments were performed with SKX and FaDu, the latter taken as a control of moderate radiosensitivity. Cell lines were grown as xenografts as well as cell cultures. For xenografts, radiosensitivity was determined via local tumour control assay, and for cell cultures using colony assay. For cell cultures, apoptosis was determined by Annexin V staining and G1-arrest by BrdU labelling. Double-strand breaks (DSBs) were detected by both constant-field gel electrophoresis (CFGE) and gammaH2AX-foci technique; DSB rejoining was also assessed by in vitro rejoining assay; chromosomal damage was determined by G01-assay. RESULTS Compared to FaDu, SKX cells are extremely radiosensitive as found for both xenografts (TCD(50) for 10 fractions 46.0Gy [95% C.I.: 39; 54 Gy] vs. 18.9 Gy [95% C.I.: 13; 25Gy]) and cell cultures (D(0.01); 7.1 vs. 3.5Gy). Both cell lines showed neither radiation-induced apoptosis nor radiation-induced permanent G1-arrest. For DSBs, there was no difference in the induction but for repair with SKX cells showing a higher level of both, slowly repaired DSBs and residual DSBs. The in vitro DSB repair assay revealed that SKX cells are defective in nonhomologous endjoining (NHEJ), and that more than 40% of DSBs are rejoined by single-strand annealing (SSA). SKX cells also depicted a two-fold higher number of lethal chromosomal aberrations when compared to FaDu cells. CONCLUSIONS The extreme radiosensitivity of the SCC SKX seen both in vivo and in vitro can be ascribed to a reduced DNA double-strand break repair, resulting from a defect in NHEJ. This defect might be due to preferred usage of other pathways, such as SSA, which prevents efficient endjoining.

In tumor cells regulation of DNA double strand break repair through EGF receptor involves both NHEJ and HR and is independent of p53 and K-Ras status

PURPOSE The purpose of this study was to examine whether the epidermal growth factor receptor (EGFR) may be used as a general target to modulate DNA double strand break (DSB) repair in tumor cells. MATERIAL AND METHODS Experiments were performed with human tumor cell lines A549, H1299 and HeLa and primate cell line CV1. EGF, ARG and TGFα were used for EGFR activation, cetuximab or erlotinib for inhibition. Overall DSB repair was assessed by γH2AX/53BP1 co-immunostaining and non-homologous end-joining (NHEJ) and homologous recombination (HR) by using NHEJ and HR reporter cells; cell cycle distribution was determined by flow cytometry and protein expression by Western blot. RESULTS EGFR activation was found to stimulate overall DSB repair as well as NHEJ regardless of the ligand used. This stimulation was abolished when EGFR signaling was blocked. This regulation was found for all cell lines tested, irrespective of their p53 or K-Ras status. Stimulation and inhibition of EGFR were also found to affect HR. CONCLUSIONS Regulation of DSB repair by EGFR involves both the NHEJ and HR pathway, and appears to occur in most tumor cell lines regardless of p53 and K-Ras mutation status.