Kerstin Brüchner

Prognostic Value of Radiobiological Hypoxia during Fractionated Irradiation for Local Tumor Control

Background and Purpose: Previous experiments showed that the fraction of radiobiologically hypoxic tumor cells (rHF) in un-treated tumors did not accurately predict local tumor control after fractionated irradiation. Thus, the prognostic value of rHF determined during fractionated irradiation was investigated.Materials and Methods:Six human squamous cell carcinoma lines were transplanted into nude mice and then irradiated with 15 fractions over 3 weeks. Thereafter, single dose irradiation under normal and clamped blood flow was given. Local tumor control rates were used to calculate the rHF and the TCD50, i.e., the radiation dose necessary to control 50% of the tumors, after single dose irradiation. These values were compared with the in parallel determined TCD50 after 30 fractions in 6 weeks.Results:The rHF after 15 fractions varied between 28% and 100%. No correlation was found with the TCD50 after 30 fractions in 6 weeks. Single dose top-up TCD50 under ambient and clamp conditions after 15 fractions significantly correlated with TCD50 after 30 fractions in 6 weeks.Conclusion:rHF after 15 fractions is not a prognostic parameter for the outcome after fractionated irradiation. In contrast, the radiobiological parameters number of tumor stem cells, intrinsic radiosensitivity, and number of radiobiologically hypoxic tumor cells appear promising to predict outcome after fractionated irradiation.ZusammenfassungHintergrund und Fragestellung:Bisherige Experimente haben gezeigt, dass die radiobiologische Fraktion (rHF) in unbestrahlten Tumoren die Strahlenempfindlichkeit nach fraktionierter Bestrahlung nicht exakt vorhersagt. Hier wurde die rHF in fraktioniert bestrahlten Tumoren bestimmt, um den prognostischen Wert für die fraktionierte Bestrahlung zu ermitteln.Material und Methoden:Sechs menschliche Plattenepithelkarzinomlinien wurden in Nacktmäuse transplantiert und zunächst mit 15 Fraktionen in 3 Wochen bestrahlt. Anschließend wurden graduierte Einzeldosen unter normalen und abgeklemmten Blutflussbedingen appliziert. Aus den Tumorkontrolldaten wurden dieTCD50 (Strahlendosis zur Kontrolle von 50% der Tumoren) und die rHF berechnet und mit der TCD50 nach 30 Fraktionen in 6 Wochen verglichen.Ergebnisse:Die rHF nach 15 Fraktionen variierte zwischen 28 und 100% (Tabelle 1) und korrelierte nicht mit der TCD50 nach 6-wöchiger fraktionierter Bestrahlung (Abbildung 1). Dagegen korrelierten die TCD50-Werte nach Einzeldosisbestrahlung signifikant mit der TCD50 nach 6-wöchiger Bestrahlung.Schlussfolgerung:Die Fraktion strahlenbiologisch hypoxischer Tumorzellen nach 3 Wochen fraktionierter Bestrahlung scheint kein geeigneter prognostischer Parameter zu sein. Vielversprechend dagegen erscheinen die radiobiologischen Parameter Anzahl, intrinsische Strahlenempfindlichkeit und Anzahl radiobiologisch hypoxischer Tumorstammzellen, um das Ergebnis der fraktionierten Bestrahlung vorherzusagen.

Effect of [18F]FMISO stratified dose-escalation on local control in FaDu hSCC in nude mice

OBJECTIVE To investigate the effect of radiation dose-escalation on local control in hypoxic versus non-hypoxic hypoxic tumours defined using [(18)F]fluoromisonidazole ([(18)F]FMISO) PET. MATERIALS AND METHODS FaDu human squamous cell carcinomas (hSCCs) growing subcutaneously in nude mice were subjected to [(18)F]FMISO PET before irradiation with single doses of 25 or 35Gy under normal blood flow conditions. [(18)F]FMISO hypoxic volume (HV) and maximum standardised uptake value (SUVmax) were used to quantify tracer uptake. The animals were followed up for at least 120days after irradiation. The endpoints were permanent local tumour control and time to local recurrence. RESULTS HV varied between 38 and 291mm(3) (median 105mm(3)). Non-hypoxic tumours (HV below median) showed significantly better local control after single dose irradiation than hypoxic tumours (HV above median) (p=0.046). The effect of dose was significant and not different in non-hypoxic and in hypoxic tumours (HR=0.82 [95% CI 0.71; 0.93], p=0.002 and HR=0.86 [0.78; 0.95], p=0.001, respectively). Dose escalation resulted in an incremental increase of local tumour control from low-dose hypoxic, over low-dose non-hypoxic and high-dose hypoxic to high-dose non-hypoxic tumours. SUVmax did not reveal significant association with local control at any dose level. CONCLUSIONS The negative effect of [(18)F]FMISO HV on permanent local tumour control supports the prognostic value of the pre-treatment [(18)F]FMISO HV. Making the assumption that variable [(18)F]FMISO uptake in different FaDu tumours which all have the same genetic background may serve as an experimental model of intratumoural heterogeneity, the data support the concept of dose-escalation with inhomogeneous dose distribution based on pre-treatment [(18)F]FMISO uptake. This result needs to be confirmed in other tumour models and using fractionated radiotherapy schedules.

Impact of pre- and early per-treatment FDG-PET based dose-escalation on local tumour control in fractionated irradiated FaDu xenograft tumours

OBJECTIVE To investigate local tumour control after dose-escalation based on [18F]2-fluoro-2-deoxy-d-glucose (FDG) positron emission tomography (PET) obtained before and early during fractionated irradiation. MATERIALS AND METHODS 85 mice bearing FaDu xenografts underwent FDG-PET twice: first immediately prior to the first 2-Gy fraction of irradiation (PET1_0) and second after 18°Gy (PET2_18). After these 9 fractions, animals were randomly allocated to: (1) continuation of 2-Gy fractions (cumulative dose of 60°Gy; n=31), (2) dose-escalation with 3-Gy fractions (cumulative EQD2-dose 86.25°Gy [α/β-value: 10]; n=25), or (3) with 4-Gy fractions (cumulative EQD2-dose 116°Gy; n=29). The effects of SUVmax0°Gy, SUVmax18°Gy, and dose on local tumour control were analysed in two ways. First, the Cox proportional hazards model was used with two covariates: continuous SUVmax values and dose. Second, the Kaplan-Meier method was used, with tumours classified according to SUVmax greater than or less than (1) median maximum standardized uptake value (SUVmax) at PET1_0 and PET2_18, or (2) the cut-off value 2.5. RESULTS The multivariate Cox analysis revealed a significant negative association between higher SUVmax determined before start of treatment and local control (HR=1.59, [95% CI 1.04, 2.42], p=0.031), whereas higher dose had a significant positive effect (HR=0.95, [0.93, 0.98], p<0.001). In contrast, FDG uptake at 18Gy did not correlate with local control (HR=1.14, [0.53, 2.45], p=0.73). Neither FDG uptake prior to irradiation nor at 18Gy correlated with local control irrespective of the delivered dose (log-rank test) when using the median SUVmax values for stratification (SUVmax0Gy: 60Gy: p=0.25, 86.25Gy: p=0.47, 116Gy: p=0.88 and SUVmax18Gy: 60Gy: p=0.42, 86.25Gy: p=0.34, 116Gy: p=0.99). By contrast, stratifying the animals by the cut-off 2.5 at PET1_0 reveals a significant difference in local control for the 60Gy group (p=0.034), but not for the other dose groups. At PET2_18, no significant effect for any dose group was detected. CONCLUSIONS The multivariate Cox analysis revealed a significantly higher hazard of recurrence for mice with higher SUVmax determined before start of treatment. These results support the hypothesis that patients with high pre-therapeutic FDG uptake should be considered at increased risk of local failure and therefore as possible candidates for dose escalation strategies.

Selection of Genetically Distinct, Rapidly Proliferating Clones does not Contribute to Repopulation during Fractionated Irradiation in FaDu Squamous Cell Carcinoma

Abstract Zips, D., Petersen, C., Junghanns, S., Eicheler, W., Brüchner, K. and Baumann, M. Selection of Genetically Distinct, Rapidly Proliferating Clones does not Contribute to Repopulation during Fractionated Irradiation in FaDu Squamous Cell Carcinoma. Radiat. Res. 160, 257–262 (2003). Acceleration of clonogen repopulation during fractionated irradiation after about 3 weeks has been demonstrated previously in FaDu human squamous cell carcinoma in nude mice (Petersen et al., Int. J. Radiat. Oncol. Biol. Phys. 51, 483–493, 2001). Selection of genetically distinct, rapidly proliferating clones might contribute to this phenomenon. To address this question, three sublines (R1–R3) were established from FaDu tumors that recurred locally after fractionated irradiation. The tumors were retransplanted and irradiated under clamp hypoxia with single doses or with 18 × 3 Gy within 18 days or 36 days, followed by graded top-up doses. The results were compared with data obtained after the same treatment schedules in the parental tumor line. Histologies, tumor volume doubling times, and potential doubling times of FaDu sublines R1–R3 were not different from those of the parental line. The radiation dose required to control 50% of the tumors (TCD50) after single-dose irradiation of 37–38 Gy was the same for the FaDu sublines R1–R3 and the parental tumor. The top-up TCD50 values for the FaDu sublines R1–R3 after 18 fractions within 36 days were 14–17 Gy higher than those after 18 fractions within 18 days, indicating significant repopulation. The magnitude of this effect was not significantly different between the sublines R1–R3 or between these sublines and the parental FaDu tumors. The results indicate that selection of genetically distinct, rapidly proliferating clones does not contribute to the acceleration of repopulation during fractionated irradiation in poorly differentiated FaDu tumors.

An optimized small animal tumour model for experimentation with low energy protons

Background The long-term aim of developing laser based particle acceleration towards clinical application requires not only substantial technological progress, but also the radiobiological characterization of the resulting ultra-short and ultra-intensive particle beam pulses. After comprehensive cell studies a mouse ear tumour model was established allowing for the penetration of low energy protons (~20 MeV) currently available at laser driven accelerators. The model was successfully applied for a first tumour growth delay study with laser driven electrons, whereby the need of improvements crop out. Methods To optimise the mouse ear tumour model with respect to a stable, high take rate and a lower number of secondary tumours, Matrigel was introduced for tumour cell injection. Different concentrations of two human tumour cell lines (FaDu, LN229) and Matrigel were evaluated for stable tumour growth and fulfilling the allocation criteria for irradiation experiments. The originally applied cell injection with PBS was performed for comparison and to assess the long-term stability of the model. Finally, the optimum suspension of cells and Matrigel was applied to determine applicable dose ranges for tumour growth delay studies by 200 kV X-ray irradiation. Results Both human tumour models showed a high take rate and exponential tumour growth starting at a volume of ~10 mm3. As disclosed by immunofluorescence analysis these small tumours already interact with the surrounding tissue and activate endothelial cells to form vessels. The formation of delimited, solid tumours at irradiation size was shown by standard H&E staining and a realistic dose range for inducing tumour growth delay without permanent tumour control was obtained for both tumour entities. Conclusion The already established mouse ear tumour model was successfully upgraded now providing stable tumour growth with high take rate for two tumour entities (HNSCC, glioblastoma) that are of interest for future irradiation experiments at experimental accelerators.