Application of HIPEC simulations for optimizing treatment delivery strategies.

INTRODUCTION: 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 CEM4310 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.

Demonstration of treatment planning software for hyperthermic intraperitoneal chemotherapy in a rat model.

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.

HyCHEED System for Maintaining Stable Temperature Control during Preclinical Irreversible Electroporation Experiments at Clinically Relevant Temperature and Pulse Settings.

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.

Increased uptake of doxorubicin by cells undergoing heat stress does not explain its synergistic cytotoxicity with hyperthermia.

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 <0.5 µg/ml. Increased intracellular concentrations of Dox in HCT116 cells caused by heat-stress did not generate a concomitant thermal enhancement. Conclusions: Simultaneous exposure of HCT116 cells to heating and Dox is more effective than sequential exposure. Heat-induced cell responses are accompanied by increased intracellular Dox concentrations; however, clonogenic survival data do not support this as the cause for synergistic cytotoxicity.

Enhancing the abscopal effect of radiation and immune checkpoint inhibitor therapies with magnetic nanoparticle hyperthermia in a model of metastatic breast cancer.

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.

Enhancing radiosensitisation of BRCA2-proficient and BRCA2-deficient cell lines with hyperthermia and PARP1-i.

Poly(ADP-ribose)polymerase1 (PARP1) is an important enzyme in regulating DNA replication. Inhibition of PARP1 can lead to collapsed DNA forks which subsequently causes genomic instability, making DNA more susceptible in developing fatal DNA double strand breaks. PARP1-induced DNA damage is generally repaired by homologous recombination (HR), in which BRCA2 proteins are essential. Therefore, BRCA2-deficient tumour cells are susceptible to treatment with PARP1-inhibitors (PARP1-i). Recently, BRCA2 was shown to be down-regulated by hyperthermia (HT) temporarily, and this consequently inactivated HR for several hours. In this study, we investigated whether HT exclusively interferes with HR by analysing thermal radiosensitisation of BRCA2-proficient and deficient cells. After elucidating the equitoxicity of PARP1-i on BRCA2-proficient and deficient cells, we studied the cell survival, apoptosis, DNA damage (γ-H2AX foci and comet assay) and cell cycle distribution after different treatments. PARP1-i sensitivity strongly depends on the BRCA2 status. BRCA2-proficient and deficient cells are radiosensitised by HT, indicating that HT does not exclusively act by inhibition of HR. In all cell lines, the addition of HT to radiotherapy and PARP1-i resulted in the lowest cell survival, the highest levels of DNA damage and apoptotic levels compared to duo-modality treatments. Concluding, HT not only inhibits HR, but also has the capability of radiosensitising BRCA2-deficient cells. Thus, in case of BRCA2-mutation carriers, combining HT with PARP1-i may boost the treatment efficacy. This combination therapy would be effective for all patients with PARP1-i regardless of their BRCA status.

The clinical benefit of hyperthermia in pancreatic cancer: a systematic review.

OBJECTIVE: In pancreatic cancer, which is therapy resistant due to its hypoxic microenvironment, hyperthermia may enhance the effect of radio(chemo)therapy. The aim of this systematic review is to investigate the validity of the hypothesis that hyperthermia added to radiotherapy and/or chemotherapy improves treatment outcome for pancreatic cancer patients. METHODS AND MATERIALS: We searched MEDLINE and Embase, supplemented by handsearching, for clinical studies involving hyperthermia in pancreatic cancer patients. The quality of studies was evaluated using the Oxford Centre for Evidence-Based Medicine levels of evidence. Primary outcome was treatment efficacy; we calculated overall response rate and the weighted estimate of the population median overall survival (mp) and compared these between hyperthermia and control cohorts. RESULTS: Overall, 14 studies were included, with 395 patients with locally advanced and/or metastatic pancreatic cancer of whom 248 received hyperthermia. Patients were treated with regional (n = 189), intraoperative (n = 39) or whole-body hyperthermia (n = 20), combined with chemotherapy, radiotherapy or both. Quality of the studies was low, with level of evidence 3 (five studies) and 4. The six studies including a control group showed a longer mp in the hyperthermia groups than in the control groups (11.7 vs. 5.6 months). Overall response rate, reported in three studies with a control group, was also better for the hyperthermia groups (43.9% vs. 35.3%). CONCLUSIONS: Hyperthermia, when added to chemotherapy and/or radiotherapy, may positively affect treatment outcome for patients with pancreatic cancer. However, the quality of the reviewed studies was limited and future randomised controlled trials are needed to establish efficacy.

Enhancement of Radiation Effectiveness in Cervical Cancer Cells by Combining Ionizing Radiation with Hyperthermia and Molecular Targeting Agents.

Hyperthermia (HT) and molecular targeting agents can be used to enhance the effect of radiotherapy (RT). The purpose of this paper is to evaluate radiation sensitization by HT and different molecular targeting agents (Poly [ADP-ribose] polymerase 1 inhibitor, PARP1-i; DNA-dependent protein kinase catalytic subunit inhibitor, DNA-PKcs-i and Heat Shock Protein 90 inhibitor, HSP90-i) in cervical cancer cell lines. Survival curves of SiHa and HeLa cells, concerning the combined effects of radiation with hyperthermia and PARP1-i, DNA-PKcs-i or HSP90-i, were analyzed using the linear-quadratic model: S(D)/S(0) = exp - (αD + ßD²). The values of the linear-quadratic (LQ) parameters α and ß, determine the effectiveness at low and high doses, respectively. The effects of these sensitizing agents on the LQ parameters are compared to evaluate dose-dependent differences in radio enhancement. Combination of radiation with hyperthermia, PARP1-i and DNA-PKcs-i significantly increased the value of the linear parameter α. Both α and ß were significantly increased for HSP90-i combined with hyperthermia in HeLa cells, though not in SiHa cells. The Homologous Recombination pathway is inhibited by hyperthermia. When hyperthermia is combined with DNA-PKcs-i and PARP1-i, the Non-Homologous End Joining or Alternative Non-Homologous End Joining pathway is also inhibited, leading to a more potent radio enhancement. The observed increments of the α value imply that significant radio enhancement is obtained at clinically-used radiotherapy doses. Furthermore, the sensitizing effects of hyperthermia can be even further enhanced when combined with other molecular targeting agents.

Decay of γ-H2AX foci correlates with potentially lethal damage repair and P53 status in human colorectal carcinoma cells.

The influence of p53 status on potentially lethal damage repair (PLDR) and DNA double-strand break (DSB) repair was studied in two isogenic human colorectal carcinoma cell lines: RKO (p53 wild-type) and RC10.1 (p53 null). They were treated with different doses of ionizing radiation, and survival and the induction of DNA-DSB were studied. PLDR was determined by using clonogenic assays and then comparing the survival of cells plated immediately with the survival of cells plated 24 h after irradiation. Doses varied from 0 to 8 Gy. Survival curves were analyzed using the linear-quadratic formula: S(D)/S(0) = exp-(αD+ßD(2)). The γ-H2AX foci assay was used to study DNA DSB kinetics. Cells were irradiated with single doses of 0, 0.5, 1 and 2 Gy. Foci levels were studied in non-irradiated control cells and 30 min and 24 h after irradiation. Irradiation was performed with gamma rays from a (137)Cs source, with a dose rate of 0.5 Gy/min. The RKO cells show higher survival rates after delayed plating than after immediate plating, while no such difference was found for the RC10.1 cells. Functional p53 seems to be a relevant characteristic regarding PLDR for cell survival. Decay of γ-H2AX foci after exposure to ionizing radiation is associated with DSB repair. More residual foci are observed in RC10.1 than in RKO, indicating that decay of γ-H2AX foci correlates with p53 functionality and PLDR in RKO cells.

Mild hyperthermia inhibits homologous recombination, induces BRCA2 degradation, and sensitizes cancer cells to poly (ADP-ribose) polymerase-1 inhibition.

Defective homologous recombination (HR) DNA repair imposed by BRCA1 or BRCA2 deficiency sensitizes cells to poly (ADP-ribose) polymerase (PARP)-1 inhibition and is currently exploited in clinical treatment of HR-deficient tumors. Here we show that mild hyperthermia (41-42.5 °C) induces degradation of BRCA2 and inhibits HR. We demonstrate that hyperthermia can be used to sensitize innately HR-proficient tumor cells to PARP-1 inhibitors and that this effect can be enhanced by heat shock protein inhibition. Our results, obtained from cell lines and in vivo tumor models, enable the design of unique therapeutic strategies involving localized on-demand induction of HR deficiency, an approach that we term induced synthetic lethality.

Genetic markers of late radiation toxicity in the era of image-guided radiotherapy: lower toxicity rates reduce the predictive value of γ-H2AX foci decay ratio in patients undergoing pelvic radiotherapy.

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 < or ≥ 3.41, a previously established threshold. RESULTS: Eighty-eight patients were included. Physician-assessed and patient-reported cumulative grade ≥ 2 toxicity was 25% and 29%, respectively; which is much lower than in the previous cohort (i.e., 51% CTCAE grade ≥ 2). Patients with toxicity exhibited less favorable dose-volume parameters. In men, these parameters showed significant improvement compared to the previous cohort. The proportion of patients with a low γ-FDR increased with severity of toxicity, but this trend was not statistically significant. In addition, a γ-FDR < 3.41 was not correlated with the development of moderate to severe toxicity. Post-treatment decline in global quality of life was minimal, and similar for patients with γ-FDR < or ≥ 3.41. CONCLUSIONS: In the present study, the γ-H2AX foci decay ratio could not be validated as a predictor of late radiation toxicity in patients with pelvic cancer. Improved radiotherapy techniques with smaller irradiated bladder and bowel volumes have probably resulted in less toxicities. Future studies on genetic markers of toxicity should be powered on these lower incidences. We further recommend taking persistency, next to severity, into consideration.

Radiosensitization by Hyperthermia Critically Depends on the Time Interval.

PURPOSE: Hyperthermia is a potent sensitizer of radiation therapy that improves both tumor control and survival in women with locally advanced cervical cancer (LACC). The optimal sequence and interval between hyperthermia and radiation therapy are still under debate. METHODS AND MATERIALS: We investigated the interval and sequence in vitro in cervical cancer cell lines, patient-derived organoids, and SiHa cervical cancer hind leg xenografts in athymic nude mice and compared the results with retrospective results from 58 women with LACC treated with thermoradiotherapy. RESULTS: All 3 approaches confirmed that shortening the interval between hyperthermia and radiation therapy enhanced hyperthermic radiosensitization by 2 to 8 times more DNA double-strand breaks and apoptosis and 10 to 100 times lower cell survival, delayed tumor growth in mice, and increased the 5-year survival rate of women with LACC from 22% (interval ≥80 minutes) to 54% (interval <80 minutes). In vitro and in vivo results showed that the sequence of hyperthermia and radiation therapy did not affect the outcome. CONCLUSIONS: Shortening the interval between hyperthermia and radiation therapy significantly improves treatment outcomes. The sequence of hyperthermia and radiation therapy (before or after) does not seem to matter.

Validation of thermal dynamics during Hyperthermic IntraPEritoneal Chemotherapy simulations using a 3D-printed phantom.

Introduction: CytoReductive 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. Methods: In 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. Results: Experimental 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. Discussion: Considering 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.

Elevated temperatures and longer durations improve the efficacy of oxaliplatin- and mitomycin C-based hyperthermic intraperitoneal chemotherapy in a confirmed rat model for peritoneal metastasis of colorectal cancer origin.

Introduction: In 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. Methods: In 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. Results: In 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 (<10%) and only 7/130 rats did not reach the endpoint of 48 hours after treatment. Conclusions: Both elevated temperatures and longer treatment duration resulted in a higher platinum uptake, significantly increased apoptosis and lower proliferation in PM tumor lesions, without enhanced normal tissue toxicity. Our results demonstrated that oxaliplatin- and MMC-based HIPEC procedures are both temperature- and duration-dependent in an in vivo tumor model.

A Comparison between Patient- and Physician-Reported Late Radiation Toxicity in Long-Term Prostate Cancer Survivors.

Patient-reported outcome measures (PROMs) are advocated for the monitoring of toxicity after radiotherapy. However, studies comparing physician- and patient-reported toxicity show low concordance. In this study, we compared physician- and patient-reported toxicity in long-term prostate cancer survivors after radiotherapy, and we determined the correlation with a presumable risk factor for late toxicity: γ-H2AX foci decay ratio (FDR). Patients formerly included in a prospective study were invited to participate in this new study, comprising one questionnaire and one call with a trial physician assistant. Concordance was calculated for seven symptoms. Gamma-H2AX FDRs were determined in ex vivo irradiated lymphocytes in a previous analysis. Associations between FDR and long-term prevalence of toxicity were assessed using univariable logistic regression analyses. The 101 participants had a median follow-up period of 9 years. Outcomes were discordant in 71% of symptomatic patients; in 21%, the physician-assessed toxicity (using CTCAE) was higher, and, in 50%, the patients reported higher toxicity. We did not find a correlation between presence of toxicity at long-term follow-up and FDR. In conclusion, patients assigned greater severity to symptoms than the trial physician assistant did. Consideration of both perspectives may be warranted to provide the best care.

Gamma-H2AX Foci Decay Ratio as a Stronger Predictive Factor of Late Radiation Toxicity Than Dose-Volume Parameters in a Prospective Cohort of Prostate Cancer Patients.

PURPOSE: Late radiation toxicity is a major dose-limiting factor in curative cancer radiation therapy. Previous studies identified several risk factors for late radiation toxicity, including both dose-volume factors and genetic predisposition. Herein, we investigated the contribution of genetic predisposition, particularly compared with dose-volume factors, to the risk of late radiation toxicity in patients treated with highly conformal radiation therapy. METHODS AND MATERIALS: We included 179 patients with prostate cancer who underwent treatment with curative external beam radiation therapy between 2009 and 2013. Toxicity was graded according to the Common Terminology Criteria for Adverse Events version 4.0. Transcriptional responsiveness of homologous recombination repair genes and γ-H2AX foci decay ratios (FDRs) were determined in ex vivo irradiated lymphocytes in a previous analysis. Dose-volume parameters were retrieved by delineating the organs at risk (OARs) on CT planning images. Associations between risk factors and grade ≥2 urinary and bowel late radiation toxicities were assessed using univariable and multivariable logistic regression analyses. The analyses were performed using the highest toxicity grade recorded during the follow-up per patient. RESULTS: The median follow-up period was 31 months. One hundred and one patients (56%) developed grade ≥2 late radiation toxicity. Cumulative rates for urinary and bowel grade ≥2 late toxicities were 46% and 17%, respectively. In the multivariable analysis, factors significantly associated with grade ≥2 late toxicity were transurethral resection of the prostate (P = .013), γ-H2AX FDR <3.41 (P = .008), and rectum V70 >11.52% (P = .017). CONCLUSIONS: Our results suggest that impaired DNA double-strand break repair in lymphocytes, as quantified by γ-H2AX FDR, is the most critical determining factor of late radiation toxicity. The limited influence of dose-volume parameters could be due to the use of increasingly conformal techniques, leading to improved dose-volume parameters of the organs at risk.

Non-Invasive Imaging and Scoring of Peritoneal Metastases in Small Preclinical Animal Models Using Ultrasound: A Preliminary Trial.

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.

Hyperthermia-Based Anti-Cancer Treatments.

Hyperthermia is an adjuvant local anti-cancer treatment using temperatures exceeding the physiologically optimal level, typically 40-43 °C for approximately one hour [...].

PARP1-Inhibition Sensitizes Cervical Cancer Cell Lines for Chemoradiation and Thermoradiation.

Radiotherapy plus cisplatin (chemoradiation) is standard treatment for women with locoregionally advanced cervical cancer. Both radiotherapy and cisplatin induce DNA single and double-strand breaks (SSBs and DSBs). These double-strand breaks can be repaired via two major DNA repair pathways: Classical Non-Homologous End-Joining (cNHEJ) and Homologous Recombination. Besides inducing DNA breaks, cisplatin also disrupts the cNHEJ pathway. Patients contra-indicated for cisplatin are treated with radiotherapy plus hyperthermia (thermoradiation). Hyperthermia inhibits the HR pathway. The aim of our study is to enhance chemoradiation or thermoradiation by adding PARP1-inhibition, which disrupts both the SSB repair and the Alternative NHEJ DSB repair pathway. This was studied in cervical cancer cell lines (SiHa, HeLa, C33A and CaSki) treated with hyperthermia (42 °C) ± ionizing radiation (2-6 Gy) ± cisplatin (0.3-0.5 µM) ± PARP1-inhibitor (olaparib, 4.0-5.0 µM). Clonogenic assays were performed to measure cell reproductive death. DSBs were analyzed by γ-H2AX staining and cell death by live cell imaging. Both chemoradiation and thermoradiation resulted in lower survival fractions and increased unrepaired DSBs when combined with a PARP1-inhibitor. A quadruple modality, including ionizing radiation, hyperthermia, cisplatin and PARP1-i, was not more effective than either triple modality. However, both chemoradiation and thermoradiation benefit significantly from additional treatment with PARP1-i.

Simulating drug penetration during hyperthermic intraperitoneal chemotherapy.

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.