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Abstract Background A predictive assay for late radiation toxicity would allow more personalized treatment planning, reducing the burden of toxicity for the more sensitive minority, and improving the therapeutic index for the majority. In a previous study in prostate cancer patients, the γ-H2AX foci decay ratio (γ-FDR) was the strongest predictor of late radiation toxicity. The current study aimed to validate this finding in a more varied group of patients with pelvic cancer. Additionally, the potential correlation between the γ-FDR and patient-reported outcomes was investigated. Methods Prostate and gynecological cancer patients with ≥ 24 months of follow-up were included in the current analysis. Toxicity was evaluated by physician (CTCAE version 4) and patient (EORTC questionnaires). γ-FDRs were determined in ex vivo irradiated lymphocytes. Correlation between γ-FDR and toxicity was assessed using both linear and logistic regression analyses. The highest toxicity grade recorded during follow-up was used. The association between global quality of life and γ-FDR was tested by comparing the change in quality of life over time in patients with γ-FDR

Background: Oxaliplatin-based hyperthermic intraperitoneal chemotherapy (HIPEC) using the original 30 min protocol has shown limited benefits in patients with peritoneal metastasis of colorectal cancer (PMCRC), likely due to the short duration, which limits drug penetration into tumor nodules. Bevacizumab, an antiangiogenic antibody that modifies the tumor microenvironment, may improve drug delivery during HIPEC. This in silico study evaluates the availability of oxaliplatin within tumor nodules when HIPEC is performed after bevacizumab treatment. Methods: Using a computational fluid dynamics (CFD) model of HIPEC, the temperature and oxaliplatin distribution within the rat abdomen were calculated, followed by a model of drug transport within tumor nodules located at various sites in the peritoneum. The vascular normalization effect of the bevacizumab treatment was incorporated by adjusting the biophysical parameters of the tumor nodules. The effective penetration depth values, including the thermal enhancement ratio of cytotoxicity, were then compared between HIPEC alone and HIPEC combined with the bevacizumab treatment. Results: After bevacizumab treatments at doses of 0.5 mg/kg and 5 mg/kg, the oxaliplatin availability increased by up to 20% and 45% when HIPEC was performed during the vascular normalization phase, with the penetration depth increasing by 1.5-fold and 2.3-fold, respectively. Tumors with lower collagen densities and larger vascular pore sizes showed higher oxaliplatin enhancement after the combined treatment. Bevacizumab also enabled a reduction in the oxaliplatin dose (up to half at 5 mg/kg bevacizumab) while maintaining effective drug levels in the tumor nodules, potentially reducing systemic toxicity. Conclusions: These findings suggest that administering oxaliplatin-based HIPEC during bevacizumab-induced vascular normalization could significantly improve drug penetration and enhance treatment efficacy.

Normal tissue reactions vary significantly among patients receiving the same radiation treatment regimen, reflecting the multifactorial etiology of late radiation toxicity. Predicting late radiation toxicity is crucial, as it aids in the initial decision-making process regarding the treatment modalities. For patients undergoing radiotherapy, anticipating late toxicity allows for planning adjustments to optimize individualized care. Various dosimetric parameters have been shown to influence the incidence of late toxicity, and the literature available on this topic is extensive. This narrative review examines patient-related determinants of late toxicity following external beam radiotherapy for pelvic tumors, with a focus on prostate and cervical cancer patients. In Part I, we address various methods for quantifying radiation toxicity, providing context for interpreting toxicity data. Part II examines the current insights into the clinical risk factors for late toxicity. While certain factors—such as previous abdominal surgery, smoking behavior, and severe acute toxicity—have consistently been reported, most of the others show inconsistent associations. In Part III, we explore the influence of genetic factors and discuss promising predictive assays. Single-nucleotide polymorphisms (SNPs) likely elevate the risk in specific combinations. Advances in artificial intelligence now allow for the identification of SNP patterns from large datasets, supporting the development of polygenic risk scores. These innovations hold promise for improving personalized treatment strategies and reducing the burden of late toxicity in cancer survivors.

Abstract Background Partial breast irradiation (PBI) is standard of care in low-risk breast cancer patients after breast-conserving surgery (BCS). Pre-operative PBI can result in tumor downstaging and more precise target definition possibly resulting in less treatment-related toxicity. This study aims to assess the pathologic complete response (pCR) rate one year after MR-guided single-dose pre-operative PBI in low-risk breast cancer patients. Methods The ABLATIVE-2 trial is a multicenter prospective single-arm trial using single-dose ablative PBI in low-risk breast cancer patients. Patients ≥ 50 years with non-lobular invasive breast cancer ≤ 2 cm, grade 1 or 2, estrogen receptor-positive, HER2-negative, and tumor-negative sentinel node procedure are eligible. A total of 100 patients will be enrolled. PBI treatment planning will be performed using a radiotherapy planning CT and -MRI in treatment position. The treatment delivery will take place on a conventional or MR-guided linear accelerator. The prescribed radiotherapy dose is a single dose of 20 Gy to the tumor, and 15 Gy to the 2 cm of breast tissue surrounding the tumor. Follow-up MRIs, scheduled at baseline, 2 weeks, 3, 6, 9, and 12 months after PBI, are combined with liquid biopsies to identify biomarkers for pCR prediction. BCS will be performed 12 months after radiotherapy or after 6 months, if MRI does not show a radiologic complete response. The primary endpoint is the pCR rate after PBI. Secondary endpoints are radiologic response, toxicity, quality of life, cosmetic outcome, patient distress, oncological outcomes, and the evaluation of biomarkers in liquid biopsies and tumor tissue. Patients will be followed up to 10 years after radiation therapy. Discussion This trial will investigate the pathological tumor response after pre-operative single-dose PBI after 12 months in patients with low-risk breast cancer. In comparison with previous trial outcomes, a longer interval between PBI and BCS of 12 months is expected to increase the pCR rate of 42% after 6–8 months. In addition, response monitoring using MRI and biomarkers will help to predict pCR. Accurate pCR prediction will allow omission of surgery in future patients. Trial registration The trial was registered prospectively on April 28th 2022 at clinicaltrials.gov (NCT05350722).

AbstractIntroduction Hyperthermic IntraPEritoneal Chemotherapy (HIPEC) aims to treat microscopic disease left after CytoReductive Surgery (CRS). Thermal enhancement depends on the temperatures achieved. Since the location of microscopic disease is unknown, a homogeneous treatment is required to completely eradicate the disease while limiting side effects. To ensure homogeneous delivery, treatment planning software has been developed. This study compares simulation results with clinical data and evaluates the impact of nine treatment strategies on thermal and drug distributions.Methods For comparison with clinical data, three treatment strategies were simulated with different flow rates (1600-1800mL/min) and inflow temperatures (41.6–43.6 °C). Six additional treatment strategies were simulated, varying the number of inflow catheters, flow direction, and using step-up and step-down heating strategies. Thermal homogeneity and the risk of thermal injury were evaluated.Results Simulated temperature distributions, core body temperatures, and systemic chemotherapeutic concentrations compared well with literature values. Treatment strategy was found to have a strong influence on the distributions. Additional inflow catheters could improve thermal distributions, provided flow rates are kept sufficiently high (>500 mL/min) for each catheter. High flow rates (1800 mL/min) combined with high inflow temperatures (43.6 °C) could lead to thermal damage, with [Formula: see text] values of up to 27 min. Step-up and step-down heating strategies allow for high temperatures with reduced risk of thermal damage.Conclusion The planning software provides valuable insight into the effects of different treatment strategies on peritoneal distributions. These strategies are designed to provide homogeneous treatment delivery while limiting thermal injury to normal tissue, thereby optimizing the effectiveness of HIPEC.

Summary: Severe combined immunodeficient mice are typically used for xenografting experiments and show reliable tumor engraftment; however, their Prkdscid mutation renders them highly sensitive to irradiation. Here, we describe a protocol that allows safe local irradiation of tumor xenografts in immunodeficient mice. We detail the steps for the establishment and handling of patient-derived cancer cultures, subcutaneous injection of cancer cells on the mouse hind limb, localized irradiation in mice, tumor monitoring, and tumor characterization via histological and immunohistochemical assessment.For complete details on the use and execution of this protocol, please refer to Dings et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

IntroductionIn patients with limited peritoneal metastasis (PM) originating from colorectal cancer, cytoreductive surgery (CRS) followed by hyperthermic intraperitoneal chemotherapy (HIPEC) is a potentially curative treatment option. This combined treatment modality using HIPEC with mitomycin C (MMC) for 90 minutes proved to be superior to systemic chemotherapy alone, but no benefit of adding HIPEC to CRS alone was shown using oxaliplatin-based HIPEC during 30 minutes. We investigated the impact of treatment temperature and duration as relevant HIPEC parameters for these two chemotherapeutic agents in representative preclinical models. The temperature- and duration- dependent efficacy for both oxaliplatin and MMC was evaluated in an in vitro setting and in a representative animal model.MethodsIn 130 WAG/Rij rats, PM were established through i.p. injections of rat CC-531 colon carcinoma cells with a signature similar to the dominant treatment-resistant CMS4 type human colorectal PM. Tumor growth was monitored twice per week using ultrasound, and HIPEC was applied when most tumors were 4-6 mm. A semi-open four-inflow HIPEC setup was used to circulate oxaliplatin or MMC through the peritoneum for 30, 60 or 90 minutes with inflow temperatures of 38°C or 42°C to achieve temperatures in the peritoneum of 37°C or 41°C. Tumors, healthy tissue and blood were collected directly or 48 hours after treatment to assess the platinum uptake, level of apoptosis and proliferation and to determine the healthy tissue toxicity.ResultsIn vitro results show a temperature- and duration- dependent efficacy for both oxaliplatin and MMC in both CC-531 cells and organoids. Temperature distribution throughout the peritoneum of the rats was stable with normothermic and hyperthermic average temperatures in the peritoneum ranging from 36.95-37.63°C and 40.51-41.37°C, respectively. Treatments resulted in minimal body weight decrease (

IntroductionCytoReductive Surgery (CRS) followed by Hyperthermic IntraPeritoneal Chemotherapy (HIPEC) is an often used strategy in treating patients diagnosed with peritoneal metastasis (PM) originating from various origins such as gastric, colorectal and ovarian. During HIPEC treatments, a heated chemotherapeutic solution is circulated through the abdomen using several inflow and outflow catheters. Due to the complex geometry and large peritoneal volume, thermal heterogeneities can occur resulting in an unequal treatment of the peritoneal surface. This can increase the risk of recurrent disease after treatment. The OpenFoam-based treatment planning software that we developed can help understand and map these heterogeneities.MethodsIn this study, we validated the thermal module of the treatment planning software with an anatomically correct 3D-printed phantom of a female peritoneum. This phantom is used in an experimental HIPEC setup in which we varied catheter positions, flow rate and inflow temperatures. In total, we considered 7 different cases. We measured the thermal distribution in 9 different regions with a total of 63 measurement points. The duration of the experiment was 30 minutes, with measurement intervals of 5 seconds.ResultsExperimental data were compared to simulated thermal distributions to determine the accuracy of the software. The thermal distribution per region compared well with the simulated temperature ranges. For all cases, the absolute error was well below 0.5°C near steady-state situations and around 0.5°C, for the entire duration of the experiment.DiscussionConsidering clinical data, an accuracy below 0.5°C is adequate to provide estimates of variations in local treatment temperatures and to help optimize HIPEC treatments.

Hyperthermic intraperitoneal chemotherapy (HIPEC) is administered to treat residual microscopic disease after debulking cytoreductive surgery. During HIPEC, a limited number of catheters are used to administer and drain fluid containing chemotherapy (41–43 °C), yielding heterogeneities in the peritoneum. Large heterogeneities may lead to undertreated areas, increasing the risk of recurrences. Aiming at intra-abdominal homogeneity is therefore essential to fully exploit the potential of HIPEC. More insight is needed into the extent of the heterogeneities during treatments and assess their effects on the efficacy of HIPEC. To that end we developed a computational model containing embedded tumor nodules in an environment mimicking peritoneal conditions. Tumor- and treatment-specific parameters affecting drug delivery like tumor size, tumor shape, velocity, temperature and dose were assessed using three-dimensional computational fluid dynamics (CFD) to demonstrate their effect on the drug distribution and accumulation in nodules. Clonogenic assays performed on RKO colorectal cell lines yielded the temperature-dependent IC50 values of cisplatin (19.5–6.8 micromolar for 37–43 °C), used to compare drug distributions in our computational models. Our models underlined that large nodules are more difficult to treat and that temperature and velocity are the most important factors to control the drug delivery. Moderate flow velocities, between 0.01 and 1 m/s, are optimal for the delivery of cisplatin. Furthermore, higher temperatures and higher doses increased the effective penetration depth with 69% and 54%, respectively. We plan to extend the software developed for this study toward patient-specific treatment planning software, capable of mapping and assist in reducing heterogeneous flow patterns.

Background Hyperthermic intraperitoneal chemotherapy (HIPEC) is administered to treat residual microscopic disease after cytoreductive surgery (CRS). During HIPEC, fluid (41–43 °C) is administered and drained through a limited number of catheters, risking thermal and drug heterogeneities within the abdominal cavity that might reduce effectiveness. Treatment planning software provides a unique tool for optimizing treatment delivery. This study aimed to investigate the influence of treatment-specific parameters on the thermal and drug homogeneity in the peritoneal cavity in a computed tomography based rat model. Method We developed computational fluid dynamics (CFD) software simulating the dynamic flow, temperature and drug distribution during oxaliplatin based HIPEC. The influence of location and number of catheters, flow alternations and flow rates on peritoneal temperature and drug distribution were determined. The software was validated using data from experimental rat HIPEC studies. Results The predicted core temperature and systemic oxaliplatin concentration were comparable to the values found in literature. Adequate placement of catheters, additional inflow catheters and higher flow rates reduced intraperitoneal temperature spatial variation by −1.4 °C, −2.3 °C and −1.2 °C, respectively. Flow alternations resulted in higher temperatures (up to +1.5 °C) over the peritoneal surface. Higher flow rates also reduced the spatial variation of chemotherapy concentration over the peritoneal surface resulting in a more homogeneous effective treatment dose. Conclusion The presented treatment planning software provides unique insights in the dynamics during HIPEC, which enables optimization of treatment-specific parameters and provides an excellent basis for HIPEC treatment planning in human applications.

Purpose: Enhancing immune responses in triple negative breast cancers (TNBCs) remains a challenge. Our study aimed to determine whether magnetic iron oxide nanoparticle (MION) hyperthermia (HT) can enhance abscopal effects with radiotherapy (RT) and immune checkpoint inhibitors (IT) in a metastatic TNBC model. Methods: One week after implanting 4T1-luc cells into the mammary glands of BALB/c mice, tumors were treated with RT (3 × 8 Gy)±local HT, mild (HTM, 43 °C/20 min) or partially ablative (HTAbl, 45 °C/5 min plus 43 °C/15 min),±IT with anti-PD-1 and anti-CTLA-4 antibodies (both 4 × 10 mg/kg, i.p.). Tumor growth was measured daily. Two weeks after treatment, lungs and livers were harvested for histopathology evaluation of metastases. Results: Compared to untreated controls, all treatment groups demonstrated a decreased tumor volume; however, when compared against surgical resection, only RT + HTM+IT, RT + HTAbl+IT and RT + HTAbl had similar or smaller tumors. These cohorts showed more infiltration of CD3+ T-lymphocytes into the primary tumor. Tumor growth effects were partially reversed with T-cell depletion. Combinations that proved most effective for primary tumors generated modest reductions in numbers of lung metastases. Conversely, numbers of lung metastases showed potential to increase following HT + IT treatment, particularly when compared to RT. Compared to untreated controls, there was no improvement in survival with any treatment. Conclusions: Single-fraction MION HT added to RT + IT improved local tumor control and recruitment of CD3+ T-lymphocytes, with only a modest effect to reduce lung metastases and no improvement in overall survival. HT + IT showed potential to increase metastatic dissemination to lungs.

Background: The peritoneum is a common site for the formation of metastases originating from several gastrointestinal and gynecological malignancies. A representative preclinical model to thoroughly explore the pathophysiological mechanisms and to study new treatment strategies is important. A major challenge for such models is defining and quantifying the (total) tumor burden in the peritoneal cavity prior to treatment, since it is preferable to use non-invasive methods. We evaluated ultrasound as a simple and easy-to-handle imaging method for this purpose. Methods: Peritoneal metastases were established in six WAG/Rij rats through i.p. injections of the colon carcinoma cell line CC-531. Using ultrasound, the location, number and size of intraperitoneal tumor nodules were determined by two independent observers. Tumor outgrowth was followed using ultrasound until the peritoneal cancer index (PCI) was ≥8. Interobserver variability and ex vivo correlation were assessed. Results: Visible peritoneal tumor nodules were formed in six WAG/Rij rats within 2–4 weeks after cell injection. In most animals, tumor nodules reached a size of 4–6 mm within 3–4 weeks, with total PCI scores ranging from 10–20. The predicted PCI scores using ultrasound ranged from 11–19 and from 8–18, for observer 1 and 2, respectively, which was quite similar to the ex vivo scores. Conclusions: Ultrasound is a reliable non-invasive method to detect intraperitoneal tumor nodules and quantify tumor outgrowth in a rat model.

Purpose: A proposed mechanism for the enhanced effectiveness of hyperthermia and doxorubicin (Dox) combinations is increased intracellular Dox concentrations resulting from heat-induced cell stress. The purpose of this study was to determine whether specific varied Dox and heat combinations produce measurable effects greater than the additive combination, and whether these effects can be attributed to heat-induced increases in intracellular Dox concentrations. Methods: HCT116, HT29 and CT26 cells were exposed to Dox and water bath heating independently. A clonogenic survival assay was used to determine cell killing and intracellular Dox concentrations were measured in HCT116 cells with mass spectrometry. Cells were exposed to heating at 42 °C (60 min) and 0.5 µg/ml of Dox at varying intervals. Synergy was determined by curve-fitting and isobologram analysis. Results: All cell lines displayed synergistic effects of combined heating and Dox. A maximum synergistic effect was achieved with simultaneous cell exposure to Dox and heat. For exposures at 42 °C, the synergistic effect was most pronounced at Dox concentrations

Abstract Background Combined radiotherapy and hyperthermia is a well-established alternative to chemoradiotherapy for advanced stage cervical cancer patients with a contraindication for chemotherapy. Pre-clinical evidence suggests that the radiosensitizing effect of hyperthermia decreases substantially for time intervals between radiotherapy and hyperthermia as short as 1–2 h, but clinical evidence is limited. The purpose of this study is to determine the effect of the time interval between external beam radiotherapy (EBRT) and same-day hyperthermia on in-field recurrence rate, overall survival and late toxicity in women with advanced stage cervical cancer. Methods Patients with advanced stage cervical cancer who underwent a full-course of curative daily EBRT and (4–5) weekly hyperthermia sessions between 1999 and 2014 were included for retrospective analysis. The mean time interval between EBRT fractions and same-day hyperthermia was calculated for each patient; the median thereof was used to divide the cohort in a ‘short’ and ‘long’ time-interval group. Kaplan-Meier analysis and stepwise Cox regression were used to compare the in-field recurrence and overall survival. Finally, high-grade (≥3) late toxicity was compared across time-interval groups. DNA repair suppression is an important hyperthermia mechanism, DNA damage repair kinetics were therefore studied in patient biopsies to support clinical findings. Results Included were 58 patients. The 3-year in field recurrence rate was 18% and 53% in the short (≤79.2 min) and long (>79.2 min) time-interval group, respectively (p = 0.021); the 5-year overall survival was 52% and 17% respectively (p = 0.015). Differences between time-interval groups remained significant for both in-field recurrence (HR = 7.7, p = 0.007) and overall survival (HR = 2.3, p = 0.012) in multivariable Cox regression. No difference in toxicity was observed (p = 1.00), with only 6 and 5 events in the short and long group, respectively. The majority of DNA damage was repaired within 2 h, potentially explaining a reduced effectiveness of hyperthermia for long time intervals. Conclusions A short time interval between EBRT and hyperthermia is associated with a lower risk of in-field recurrence and a better overall survival. There was no evidence for difference in late toxicity.

Electric permeabilization of cell membranes is the main mechanism of irreversible electroporation (IRE), an ablation technique for treatment of unresectable cancers, but the pulses also induce a significant temperature increase in the treated volume. To investigate the therapeutically thermal contribution, a preclinical setup is required to apply IRE at desired temperatures while maintaining stable temperatures. This study’s aim was to develop and test an electroporation device capable of maintaining a pre-specified stable and spatially homogeneous temperatures and electric field in a tumor cell suspension for several clinical-IRE-settings. A hydraulically controllable heat exchange electroporation device (HyCHEED) was developed and validated at 37 °C and 46 °C. Through plate electrodes, HyCHEED achieved both a homogeneous electric field and homogenous-stable temperatures; IRE heat was removed through hydraulic cooling. IRE was applied to 300 μL of pancreatic carcinoma cell suspension (Mia PaCa-2), after which cell viability and specific conductivity were determined. HyCHEED maintained stable temperatures within ±1.5 °C with respect to the target temperature for multiple IRE-settings at the selected temperature levels. An increase of cell death and specific conductivity, including post-treatment, was found to depend on electric-field strength and temperature. HyCHEED is capable of maintaining stable temperatures during IRE-experiments. This provides an excellent basis to assess the contribution of thermal effects to IRE and other bio-electromagnetic techniques.

Cytoreductive surgery (CRS) followed by hyperthermic intraperitoneal chemotherapy (HIPEC) is a treatment with curative intent for peritoneal metastasis of colorectal cancer (CRC). Currently, there is no standardized HIPEC protocol: choice of drug, perfusate temperature, and duration of treatment vary per institute. We investigated the temperature-dependent effectiveness of drugs often used in HIPEC. Methods: The effect of temperature on drug uptake, DNA damage, apoptosis, cell cycle distribution, and cell growth were assessed using the temperature-dependent IC50 and Thermal Enhancement Ratio (TER) values of the chemotherapeutic drugs cisplatin, oxaliplatin, carboplatin, mitomycin-C (MMC), and 5-fluorouracil (5-FU) on 2D and 3D CRC cell cultures at clinically relevant hyperthermic conditions (38–43 °C/60 min). Results: Hyperthermia alone decreased cell viability and clonogenicity of all cell lines. Treatment with platinum-based drugs and MMC resulted in G2-arrest. Platinum-based drugs display a temperature-dependent synergy with heat, with increased drug uptake, DNA damage, and apoptosis at elevated temperatures. Apoptotic levels increased after treatment with MMC or 5-FU, without a synergy with heat. Conclusion: Our in vitro results demonstrate that a 60-min exposure of platinum-based drugs and MMC are effective in treating 2D and 3D CRC cell cultures, where platinum-based drugs require hyperthermia (>41 °C) to augment effectivity, suggesting that they are, in principle, suitable for HIPEC.

Background: Efficacy of current treatment options for cervical cancer require improvement. Previous in vitro studies have shown the enhancing effects of the addition of PARP1-inhibitors to chemoradiotherapy and thermoradiotherapy. The aim of our present study was to test efficacy of different combinations of treatment modalities radiotherapy, cisplatin, hyperthermia and PARP1-inhibitors using in vitro tumor models, ex vivo treated patient samples and in vivo tumor models.Materials and Methods: In vitro clonogenic survival curves (0–6 Gy) show that PARP1-i (4–5 M Olaparib) enhances both chemoradiotherapy (0.3–0.5 µM cisplatin) and thermoradiotherapy (42 °C for 1 h) in SiHa, CaSki and HeLa cells. A cervical cancer mouse model and freshly obtained in-house developed patient-derived organoids were used to examine the effects of different treatment combinations. For the in vivo study, human cervical cancer (SiHa) cells were injected in the right hind leg of athymic nude mice. In vivo mouse experiments show that PARP1-i enhances thermoradiotherapy or chemoradiotherapy by reduction of tumor volumes. Five cycles of treatment were applied with the following doses per cycle: irradiation 3 Gy, hyperthermia 1 h at 42 °C, cisplatin at 2 mg/kg, and twice PARP1-i at 50 mg/kg.Results: Quadruple treatment, combining radiotherapy, hyperthermia, cisplatin and PARP1-i, was very effective but also lead to severe side effects causing severe weight loss and death. In contrast, thermoradiotherapy or chemoradiotherapy with addition of PARP1-i, were effective without serious side effects.Conclusion: The triple combinations are promising options for potentially more effective treatment of locally advanced cervical cancer without more toxicity.