Purpose/Objective(s)The PULSAR paradigm, treats patients using a few large dose "pulses," delivered at least a week apart. These split treatments represent a radical break from conventional radiation treatments lasting six to nine weeks. The expectation is that therapy will be less toxic and will allow treatments to personalized based upon noted changes in tumor morphology, location and radiation response. Early pre-clinical experiments identified an optimal timing between radiation pulses with the use of immune checkpoint inhibitors (ICI). Other therapeutic combinations are being explored. Tumor Treating Fields (TTFields) is a novel noninvasive physical modality of cancer therapy that disrupts mitosis, causes replication stress, inhibits DNA damage repair, and sets up a conditional vulnerability to DNA damaging agents. This study combined the conditional vulnerabilities caused by TTFields exposure with a preclinical model of PULSAR to determine treatment efficacy for this combination.Materials/MethodsThe Inovivo system was used to generate Tumor Treating Fields across subcutaneously implanted tumors. Three distinct syngeneic mouse tumor models, LLC (Murine Lewis Lung Carcinoma), KPC63 (Pancreatic Cancer) and MC38 (Colon Cancer). Tumor growth delay (TGD) was the endpoint determined.ResultsIn the three mice models tested in vitro, TTFields exposure alone modestly decreased tumor volume. A difference in tumor growth rate between the heat (sham) and TTFields group was not statistically significant. TTFields combined with 8 or 10 Gy radiation pulses, depending on tumor model radiosensitivity, produced a significant TGD effect on the MC38 mouse model after one round. The second round of TTFields and radiation treatment further delayed TGD, in a synergistic manner, confirming that TTFields induces a conditional vulnerability that is particularly suited to the PULSAR concept. The combination of TTFields, PULSAR and an ICI is being tested and will be reported.ConclusionThe addition of TTFields with a pre-clinical model of PULSAR resulted in synergistic delays in tumor growth compared to the PULSAR strategy alone when tested using three different subcutaneous tumor models. This combination is well suited for the addition of an ICI. The PULSAR paradigm, treats patients using a few large dose "pulses," delivered at least a week apart. These split treatments represent a radical break from conventional radiation treatments lasting six to nine weeks. The expectation is that therapy will be less toxic and will allow treatments to personalized based upon noted changes in tumor morphology, location and radiation response. Early pre-clinical experiments identified an optimal timing between radiation pulses with the use of immune checkpoint inhibitors (ICI). Other therapeutic combinations are being explored. Tumor Treating Fields (TTFields) is a novel noninvasive physical modality of cancer therapy that disrupts mitosis, causes replication stress, inhibits DNA damage repair, and sets up a conditional vulnerability to DNA damaging agents. This study combined the conditional vulnerabilities caused by TTFields exposure with a preclinical model of PULSAR to determine treatment efficacy for this combination. The Inovivo system was used to generate Tumor Treating Fields across subcutaneously implanted tumors. Three distinct syngeneic mouse tumor models, LLC (Murine Lewis Lung Carcinoma), KPC63 (Pancreatic Cancer) and MC38 (Colon Cancer). Tumor growth delay (TGD) was the endpoint determined. In the three mice models tested in vitro, TTFields exposure alone modestly decreased tumor volume. A difference in tumor growth rate between the heat (sham) and TTFields group was not statistically significant. TTFields combined with 8 or 10 Gy radiation pulses, depending on tumor model radiosensitivity, produced a significant TGD effect on the MC38 mouse model after one round. The second round of TTFields and radiation treatment further delayed TGD, in a synergistic manner, confirming that TTFields induces a conditional vulnerability that is particularly suited to the PULSAR concept. The combination of TTFields, PULSAR and an ICI is being tested and will be reported. The addition of TTFields with a pre-clinical model of PULSAR resulted in synergistic delays in tumor growth compared to the PULSAR strategy alone when tested using three different subcutaneous tumor models. This combination is well suited for the addition of an ICI.

Locoregional recurrence (LRR) is the major cause of morbidity and mortality in patients with squamous cell carcinoma of the head and neck (HNSCC). For those patients that experience surgically unresectable LRR, median overall survival (OS) is less than one year. Approaches utilizing stereotactic ablative radiotherapy (SabR) and immuno-oncology agents such as Nivolumab (α-PD-1 inhibitor) in the salvage setting have become alternatives to conventional chemoradiation therapy. Despite improvements in OS, rates of acute oral mucositis (OM) and late toxicities (skin fibrosis) remain concerns with SabR in the context of re-irradiation. Here we present pre-clinical evidence that avasopasem manganese (GC4419), a highly selective superoxide dismutase mimetic that has received FDA Breakthrough Therapy Designation and is currently in a Phase III trial for reducing severe OM in patients undergoing chemoradiotherapy for locally advanced HNSCC (NCT03689712) also enhances the anti-tumor radiation response of HNSCC tumors. We demonstrate in pre-clinical animal models that avasopasem protects the normal mucosa of the mouse tongue from primary irradiation toxicity. We will also report the results of ongoing studies examining whether avasopasem protects murine mucosa from reirradiation as with SabR as a salvage therapy. Utilizing clonogenic survival assays, we demonstrate that avasopasem at physiologically achievable concentrations exhibits single agent anti-cancer activity and also enhances the response of HNSCC lines to irradiation. In tumor growth delay (TGD) experiments utilizing the syngeneic HNSCC tumor model, AT-84, when avasopasem is delivered in combination with biologically equivalent fractionation schedules of 17 Gy x 1 fxn, 10.24 Gy x 2 fxn, or 5 Gy x 5 fxn, an enhancement of the radiation response is achieved. This potentiation of the anti-tumor response with GC4419 is more pronounced at fraction doses exceeding the threshold considered for intensity modulated radiation therapy. In addition, when avasopasem is combined with radiation and an α-PD-1 inhibitor, a further enhancement of the radiation response is observed, indicating that it compliments radiotherapy combined with immunotherapy. The pre-clinical data strongly suggest that avasopasem should be combined with radio-immune therapy to not only enhance local tumor control, but also to provide normal tissue protection with SabR which may allow dose escalation and further improve treatment outcomes. Whether radiation combined with avasopasem and checkpoint inhibitor has an effect on metastasis is under examination.

Hypoxia-induced radioresistance has been well-recognized. However, targeting hypoxic cells in tumors historically has had no impact on clinical practice although recent clinical trials with nimorazole holds promise. We aim to use targeted metabolomics, an integrated profiling of changes in cellular metabolites to identify metabolic predictors and targets for hypoxia-induced radioresistance. AT84 cells were acclimated to normoxia (21%) or hypoxia (0.1%) for 6 hours, irradiated with one fraction of 10 Gy and then returned to normoxia or hypoxia for another 2 to 24 hours. Cells were harvested, lysed, extracted, and subjected to LC-MS as previously described (Cell Reports. 2014, 7 (5): 1679-1690). Multivariate analyses and modelling of the normalized data were carried out using Metaboanalyst 4.0 (http://www.metaboanalyst.ca). Univariate statistical differences of the metabolites between two groups were analyzed using a two-tailed student's t-test. We observed significant disturbances of multiple metabolic pathways in response to ionizing radiation in a time- and oxygen-tension dependent fashion. As early as two hours following radiation, the metabolism of glutamate, glucose, alanine, glutathione, urea/ammonia cycle, glycine and serine, purine and one carbon metabolites were affected. By 24h after irradiation, the most prominent, persistent changes were increased abundance of metabolites related to purine and methionine pathways. As expected, radiation decreased the cellular content of glutathione, likely representing increased consumption in response to oxidative stress. Similarly, the increased folate and decreased nucleic acids levels after radiation may represent increased DNA synthesis and repair. Various metabolites responded to radiation differently based on oxygen status. For example, the cellular content of Riboflavin, whose deficiency is associated with oxidative DNA and protein damage, increased 1.5 fold with radiation under normoxia but failed to increase under hypoxia. Taurine, a radio-protective amino acid, was decreased by 74 % in irradiated cells under hypoxia but did not change under normoxia. Furthermore, multivariate analyses indicate the Serine and Glycine pathways, important contributors to glutathione synthesis and maintenance of redox status, were significantly enriched in irradiated cells as a function of hypoxia. The metabolome represents a promising tool for investigating radiation tolerance mechanisms. In response to cellular stress from ionizing radiation, we observed acute global metabolic disarrangements in nutrient utilization, energy production, re-dox homeostasis, DNA synthesis and repair pathways. The persistently changed metabolites by 24 hours after irradiation are predominantly involved in DNA synthesis and repair. Serine, Glycine and Glutathione pathway may represent potential markers for tumor hypoxia and targets for radiosensitization.

Locoregional recurrence (LRR) is the major cause of morbidity and mortality in patients with squamous cell carcinoma of the head and neck (HNSCC). For those patients that experience surgically unresectable LRR, median overall survival (OS) is less than one year. Approaches utilizing stereotactic ablative radiotherapy (SAbR) and immune oncology agents such as Nivolumab (α-PD-1 agonist) in the salvage setting have become alternatives to conventional chemoradiation therapy with one randomized, Phase II clinical trial combining these two modalities currently recruiting (clinicaltrials.gov identifier: NCT02684253). Despite improvements in OS, rates of acute oral mucositis (OM) and late toxicities (skin fibrosis) remain a concern with SAbR, particularly in the context of re-irradiation. Here we present pre-clinical evidence that GC4419 (Galera Therapeutics), a highly selective superoxide dismutase mimetic that recently received FDA Breakthrough therapy status following a Phase II, randomized, placebo controlled trial for reducing the duration and incidence of severe OM in patients undergoing chemoradiation therapy for locally advanced HNSCC (NCT02508389) also enhances the anti-tumor radiation response of HNSCC tumors to irradiation. GC4419 slows the growth of a panel of HNSCC cell lines in vitro, and utilizing clonogenic survival assays, demonstrates single agent anti-cancer activity at physiologically achievable concentrations, while also enhancing the response of HNSCC lines to irradiation. In tumor growth delay (TGD) experiments utilizing the syngeneic HNSCC tumor model, AT-84, when GC4419 is delivered starting 30-60 minutes prior to irradiation with biologically equivalent fractionation schedules of 17 Gy x 1 fxn, 10.24 Gy x 2 fxn, or 5 Gy x 5 fxn, a significant enhancement of the radiation response is achieved. Furthermore, when GC4419 is combined with radiation and α-PD-1 inhibitor, a further enhancement of the radiation response is observed, indicating that GC4419 compliments radiotherapy combined with immune oncology therapies. This potentiation of the anti-tumor response with GC4419 is more pronounced at doses that exceed the threshold to be considered for intensity modulated radiation therapy (IMRT). Results from clinical trials and the accompanying pre-clinical data strongly suggest that GC4419 should be combined with radio-immune therapy to not only enhance local tumor control, but that the potential for normal tissue protection with SAbR also creates the opportunity for dose escalation and may further improvement in treatment outcome. Citation Format: Brock J. Sishc, Elizabeth M. Polsdofer, Yuanyuan Zhang, Debabrata Saha, Michael D. Story. The radioprotector GC4419 enhances the response of squamous cell carcinoma of the head and neck tumors to ionizing radiation and enhances radioimmune therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2922.

Tumor Treating Fields (TTFields) delivers low-intensity, intermediate frequency, alternating electric fields non-invasively to a tumor. Optune, a TTFields delivery device, has been approved for glioblastoma and clinical trials are ongoing for other cancers. Other mechanisms of action besides interference with mitosis have been identified and suggest alternative clinical strategies to the use of TTFields. Human NSCLC and pancreatic cell lines were used to examine TTFields effects in vitro while an tumor explant system was used for ex vivo studies. The Inovitro system was used to generate TTFields. Quantification of DNA damage was done by using 53BP1 and γ-H2AX foci. The DNA fiber assay was used to quantify newly synthesized DNA to examine replication fork dynamics. R loop detection was done by dot-blot and immunofluorescence using the S9.6 antibody. Clonogenic cell survival was performed with TTFields combined with specific agents (ionizing radiation (IR), cisplatin, olaparib, IR plus cisplatin, IR plus olaparib). We have developed an ex vivo model for lung tumors that are exposed to to TTFields. TTFields exposure decreased the expression of FANC/BRCA1 pathway genes resulting in the downregulation of DNA DSB repair of IR-induced DSBs. Most importantly, TTFields alone increased the number of γH2AX foci and the incidence of chromatid aberrations as a function of TTFields exposure time. Furthermore, TTFields exposure decreased the length of newly replicated DNA and increase R-loop formation as a function of exposure time, suggesting that TTFields induce replication stress. We hypothesized that by applying TTFields, a systems level conditional vulnerability develops (inhibition of DNA repair, increased replication stress, production of DSBs, reduced mitophagy, and other stresses) rendering cells more susceptible to agents that cause DNA damage or replication stress or interfere with its repair. In agreement with our hypothesis, NSCLC cell susceptibility to radiation increased when cells were exposed to TTFields prior to IR treatment compared to IR treatment followed by TTFields. TTFields also enhanced cisplatin toxicity in a synergistic manner. Furthermore, TTFields exposure of NSCLC and pancreatic tumor cells concomitant with the PARP inhibitor Olaparib followed by radiation resulted in synergistic cell killing compared to radiation or Olaparib alone or in combination. Our initial results of ex vivo tumor explants exposed to TTFields supports our in vitro results in that we see the down-regulation of key DNA repair pathway proteins as was seen in our in vitro studies. Our data identifies systems level perturbations to key molecular and biochemical pathways that render tumor cells vulnerable to a number of different agents. We suggest that TTFields would serve well as neoadjuvant therapy prior to radiation treatment and either prior to or concomitant with chemotherapy agents.

Tumor treating fields (TTFields) is a new physical modality of cancer therapy composed of low-intensity, intermediate frequency, alternating electric fields non-invasively applied to the region of the tumor. TTFields have revolutionized the treatment of recurrent and newly diagnosed glioblastoma with clinical trials ongoing for other cancers. Initial experiments revealed that a major mechanism by which TTFields kill cancer cells is through the disruption of mitosis. However, other mechanisms have now been identified. Based upon these newly described mechanisms we have examined the combination of TTFields with either cisplatin or a PARP inhibitor (Olaparib), either alone or in combination with radiation. A panel of 5 non-small cell lung cancer cell lines were exposed to TTFields for 24, 48 or 72h. Gene expression analysis, western analysis, DNA repair, DNA replication and cell survival were assessed as a function of time using combinations of TTFields, radiation, cisplatin or a PARP inhibitor. We found that the expression of the BRCA1 DNA damage repair pathway genes were significantly downregulated (P < 0.05) upon TTFields treatment which was confirmed at the protein level by western blot. TTFields treatment also slowed the repair of ionizing radiation-induced DNA damage compared to radiation alone which was evident by an increased number of DNA double strand break repair foci at any given time. TTFields alone caused the number of γH2AX foci to increase with time of exposure. The length of newly replicated DNA was reduced as a function of TTFields exposure time while the number and intensity of R-loop formations (DNA:RNA hybrid structures) increased, suggesting increased replication stress. Based upon these newly identified mechanisms of TTFields action, we hypothesized that by applying TTFields first, a conditionally vulnerable environment would develop rendering cells more susceptible to radiation, cisplatin or other agents such as PARP inhibitors that caused DNA damage, interfered with DNA repair or inhibited the resolution of stalled replication forks. We found that the effect of TTFields exposure concomitant with the PARP inhibitor Olaparib followed by radiation was synergistic compared to radiation or olaparib alone or in combination, although the degree of sensitization and synergy varied across the cell lines. We suggest that new strategies for TTFields application be considered based upon these newly identified mechanisms of action for TTFields. For example, the use of TTFields prior to radiation treatment (TTFields loading), and either prior to or concomitant with, chemotherapy agents that cause DNA damage or interfere with DNA repair or DNA replication fork resolution may result in more favorable clinical responses.

MicroRNAs (miRNAs) are small single-stranded RNAs, measuring approximately 22 nucleotides in length and regulate gene expression at the post-transcriptional level through mRNA destabilization or repressing protein synthesis. Dysregulation of microRNAs can lead to tumorigenesis through changes in regulation of key cellular processes such as cell proliferation, cell survival, and apoptosis. miR-125a-5p has been implicated as a tumor suppressor miRNA in malignancies such as non-small cell lung cancer and colon cancer. However, the role of miR-125a-5p has not been fully investigated in head and neck squamous cell carcinoma (HNSCC). We utilized a panel of tumor samples from patients with high risk HNSCC to identify an association between miR-125a-5p expression and disease recurrence followed by utilizing The Cancer Genome Atlas to investigate the association between low expression of miR-125a-5p and survival. We then performed cell proliferation assays as well as flow cytometric analysis of cell cycle using the HN5 and UM-SCC-22B cell lines. An in vitro scratch assay was used to assay for cell migration. Clonogenic assays were utilized to interrogate sensitivity of miR-125a-5p to ionizing radiation. Finally, putative targets of miR-125a-5p will be predicted using a combination of in silico methods, combining gene expression analysis and miRNA binding site conservation. miR-125a-5p downregulation was associated with recurrent disease in a panel of high risk HNSCC and then confirmed poor survival associated with low expression in HNSCC via The Cancer Genome Atlas, suggesting that miR-125a-5p acts as a tumor suppressor miRNA. We then demonstrated that miR-125a-5p regulates cell proliferation through cell cycle regulation at the G1/S transition. We also show that miR-125a-5p can alter cell migration and modulate sensitivity to ionizing radiation. Finally, we identified putative mRNA targets of miR-125a-5p, including ERBB2 , EIF4EBP1 , and TXNRD1 , which support the tumor suppressive mechanism of miR-125a-5p. In sum, our data suggests that miR-125a-5p acts as a tumor suppressor miRNA, has potential as a diagnostic tool and may be a potential therapeutic target for the management and treatment of squamous cell carcinoma of the head and neck.

TTFields have demonstrated significant improvement in the treatment of glioblastoma (GBM) with trials in other tumor sites ongoing or being planned. In this study we examined the mechanisms of TTField-induced cell death in combination with radiation and chemotherapy and changes in gene expression in NSCLC cells to determine the underlying mechanisms of TTField response and cell death. A panel of non-small cell lung cancer (NSCLC) cell lines was used. TTFields were generated using the Inovitro system developed by Novocure (Haifa, Israel). Experiments assessing the effects of TTF on cell proliferation, cell viability, cell cycle distribution, and clonogenic survival when combined with radiation and chemotherapy (Cisplatin) were conducted to assess the potential effectiveness of TTFields in the treatment of NSCLC. Longitudinal gene expression analysis was performed on representative NSCLC cell lines, which vary in their TTF response, over 72 hours of TTField exposure. TTField frequency was optimized for each NSCLC cell line (H157, A549, H1299, H1650, H4006) by measuring the TTField effect on proliferation. This frequency was then used for further experimentation. The TTField effect was found to be p53 independent in this panel of NSCLC cell lines. Clonogenic experiments with TTField alone showed a variable response as with proliferation, and the effect with radiation was additive when combined with 2 or 4 Gy irradiation. Also observed was an enhanced effect of TTField in combination with cisplatin. We studied gene expression changes in these NSCLC cell lines upon TTField treatment in order to understand the molecular mechanism underlying the difference in their responsiveness to TTField. Supervised clustering suggested that 1083 genes differentiated the sensitive and non-sensitive cell lines to TTField with 628 genes differentially expressed in TTField sensitive cell lines. Analysis of these differentially expressed genes revealed that majority of differentially expressed genes fall into Cell Cycle and Proliferation pathways and interestingly, the DNA damage pathway was negatively correlated. Chromosomal aberration studies indicated that there increased chromatid type aberrations in TTField conditions compared to control. Our study results suggest that TTFields differentially decrease cell proliferation and clonogenic survival in a panel of NSCLC cells. Radiation and cisplatin combined with TTField treatment revealed an enhanced effect on cell survival. TTFields alter signal transduction pathways associated with cell cycle, proliferation and DNA damage repair pathways. TTField exposure increases chromatid type aberrations in NSCLC cells and ongoing experiments are examining the effects of down-regulated DNA repair, specifically the BRCA repair pathway, including the impact on replication stress.

Purpose:Two‐dimensional GRID therapy, traditionally planned and delivered using a dedicated GRID block or MLC modulation, has shown clinical efficacy in treating bulky tumors. However, the large dose to normal tissues outside target can be limiting. We hypothesize that modulation in the third dimension will improve dose sparing of normal tissues, maximize the bystander effect within the target, and ultimately improve the therapy effectiveness. This study aims to investigate the feasibility of a three‐dimensional GRID technique using conventional LINACs to achieve a 3D lattice of high dose volumes within a target.Methods:Datasets of patient's having large tumor sizes were used to investigate the planning and delivering of 3D GRID using a Varian TrueBeam linac. Original patient contours of PTV are exported from a TPS to DICOManTX where 3D GRID targets are generated in programmable configurations. A structure of avoidance (SOA), i.e., PTV minus GRID targets, is also generated to facilitate inverse planning to achieve the desired pattern. The artificial structures were sent back to the TPS where an IMRT or VMAT plan is designed to deliver a desired high dose to GRID targets while minimizing the dose to the SOA as much as possible.Results:The programmable GRID target generator enables us to modify the target geometry to maximize the peak‐to‐valley ratio. Preliminary results show that plans based on spherical GRID targets achieve a higher peak‐to‐valley dose ratio compared with cylindrical targets. High dose spillage outside the target was eliminated. IMRT planning requires the number of beams to be larger than 16, while for VMAT the number of arcs should be at least 4 in order to achieve dosimetric goals.Conclusion:Planning and delivering 3D GRID therapy using conventional LINACs was shown to be feasible. More research and development are required before this new modality can be implemented clinically.

Use of radiation therapy (RT) has been limited for renal cell carcinoma (RCC) due to concerns of RT-resistance at conventional doses. However, clinical studies suggest that RCC are quite responsive to RT if higher ablative doses are used. Molecular mechanism for such dichotomy of response is not understood. Recently, validated tumor graft (TG) model of RCC from > 94 patients have been established at our institution, for which genomic and patient outcome information are available. We hypothesized that these TGs have intrinsic RT response characteristics which should mirror response in patients. Our goal was to determine the RT response of the TGs to ablative RT, and to identify molecular profile correlates. Established stable TGs were implanted onto the hind legs of NOD-SCID mice. TGs were irradiated with 0 Gray (Gy), 8 Gy, 16 Gy, or 24 Gy (n = 4-6/ dose level) in 1 fraction, to reflect the typical single fraction palliative and ablative RT dose used in clinical practice. Tumor growth curves were generated and analyzed. The Table describes characteristics and RT response of TGs studied to date which were aggressive tumors (high grade, high T stage). 3 TGs had good response to 8 Gy (> 80% reduction), while TG334 had modest response (40% reduction). TG374 displayed resistance to 8 Gy. Interestingly, this TG had both VHL and p53 mutations. The response of TG to 8 Gy was not reflective of its response to 24 Gy. For example, TG374 which was extremely resistant to 8 Gy, had excellent response to 24 Gy (87% reduction). All TGs had excellent response to 16 (data not shown) and 24 Gy RT (Table). Interestingly, in all TGs studied, despite excellent response to 24 Gy, re-growth/recurrence was noted upon extended long term follow up (Table, last column). In a pilot experiment, the recurrent TG334 tumors were re-challenged with ablative 16 Gy dose. These tumors continued to grow despite re-treatment, suggesting development of RT resistance. Validated TGs for RCC can be used to study intrinsic tumor response to standard and ablative RT. While most TGs respond well to ablative RT, surprisingly late recurrences were seen on extended follow up, suggesting escape pathways, and development of resistant cells. Molecular profiling of recurrent TGs is in progress, and additional TGs are being analyzed for RT response. All available data will be presented at time of meeting.Scientific Abstract 3506; TableTumor Grafts Treated with Radiation TherapyTGSourceHistologyGradeStageSize(cm)Mutations% Tumor Volume Reduction 55 Days after 8 Gy% Tumor Volume Reduction 55 Day after 24 GyTime to 2X Tumor Volume Growth Over Baseline after 24 Gy (Days)TG426Kidneyclear cell with rhabdoid features4T4N114.6-83 (+/-2)93 (+/- 1)97TG26Adrenalclear cellN/AN/AN/AVHL,BAP175 (+/-7)91 (+/- 2)111TG164Kidneysarcomatoid with clear cell features4T4Nx11.3-80 (+/- 5)95 (+/-1)130TG334Kidneyunclassified3T3bN014.5-40 (+/- 23)70 (+/-12)66TG374Kidneyclear cell with sarcomatoic and rhabdoid features4T4Nx14.5VHL, P53No Response87 (+/- 4)Not reached till D100 Open table in a new tab

Decreased expression of DAB2IP tumor suppressor in prostate cancer (PCa) has been associated with worse prognosis and radiation resistance. EZH2, an upstream protein shown to down regulate DAB2IP, has also been associated with aggressive PCa. We investigated the role of DAB2IP and/or EZH2 as prognostic biomarkers following radiation therapy (RT) in high-risk PCa patients. Immunohistochemistry was performed and scored by an expert genitourinary pathologist. Freedom from biochemical failure (FFBF) was determined using the Phoenix definition. Castrate resistance-free survival (CRFS) was determined when ≥2 episodes of rising prostate-specific antigen (PSA) occurred while on standard hormone therapy. Distant metastasis-free survival (DMFS) was determined from clinical data review. Log-rank test and Cox regression were used to determine significance of DAB2IP and EZH2 levels with clinical outcome. Fifty-four patients with high-risk PCa (stage ≥ T3a, or Gleason score ≥ 8, or PSA ≥ 20) treated with RT from 2005-2012 at UT Southwestern were evaluated. 28.3% (13/46) of patients revealed DAB2IP-reduction while 71.7% (33/46) retained DAB2IP. Nearly all patients expressed EZH2 (97.9%), supporting prior reports that EZH2 expression precedes DAB2IP reduction. For EZH2, the intensity level was grade (G) 0 in 1 (2.1%), G1 in 9 (18.8%), G2 in 29 (60.4%), and G3 in 9 (18.8%) patients. Median follow-up was 34.0 months (mos) (range, 6.7-76.1 mos) for DAB2IP-reduced patients, 29.9 mos (range, 6.1-84.6 mos) for DAB2IP-retained patients, and 32.6 m (range, 2.8-84.6 mos) for the EZH2 study. Patients with reduced DAB2IP demonstrated worse outcome compared to patients retaining DAB2IP, including FFBF (2-year = 73% vs 93%; 4-year = 37% vs 89%; p = 0.04; HR = 3.65), DMFS (2-year = 90% vs 97%; 4-year = 36% vs 97%; p = 0.05), and CRFS (4-year = 50% vs 90%; p = 0.02). Intensity of EZH2 expression trended toward significance for worse FFBF and CRFS (p = 0.07). Patients with reduced DAB2IP or highest intensity (G3) EZH2 expression exhibited even worse FFBF (p = 0.03; HR = 5.65). Six of the 7 patients that developed CRPC had reduced DAB2IP or G3 EZH2. Even with modest follow-up and after controlling for duration of hormone therapy, DAB2IP remains significant. This study suggests that DAB2IP loss is a potent biomarker for identifying high-risk PCa patients that portends worse outcome despite definitive RT. EZH2 is expressed in most high-risk PCa tumors evaluated, and is a less potent discriminator of outcome in this group of patients. DAB2IP status in combination with degree of EZH2 expression may be useful for determining patients with worse outcome within the high-risk patient population.

Purpose: Radiation therapy (RT) is one of the primary modalities for treatment of non-small cell lung cancer (NSCLC). However, due to the intrinsic radiation resistance of these tumors, many patients experience RT failure, which leads to considerable tumor progression including regional lymph node and distant metastasis. This preclinical study evaluated the efficacy of a new-generation cyclin-dependent kinase (Cdk) inhibitor, AZD5438, as a radiosensitizer in several NSCLC models that are specifically resistant to conventional fractionated RT.Methods and Materials: The combined effect of ionizing radiation and AZD5438, a highly specific inhibitor of Cdk1, 2, and 9, was determined in vitro by surviving fraction, cell cycle distribution, apoptosis, DNA double-strand break (DSB) repair, and homologous recombination (HR) assays in 3 NSCLC cell lines (A549, H1299, and H460). For in vivo studies, human xenograft animal models in athymic nude mice were used.Results: Treatment of NSCLC cells with AZD5438 significantly augmented cellular radiosensitivity (dose enhancement ratio rangeing from 1.4 to 1.75). The degree of radiosensitization by AZD5438 was greater in radioresistant cell lines (A549 and H1299). Radiosensitivity was enhanced specifically through inhibition of Cdk1, prolonged G(2)-M arrest, inhibition of HR, delayed DNA DSB repair, and increased apoptosis. Combined treatment with AZD5438 and irradiation also enhanced tumor growth delay, with an enhancement factor ranging from 1.2-1.7.Conclusions: This study supports the evaluation of newer generation Cdk inhibitors, such as AZD5438, as potent radiosensitizers in NSCLC models, especially in tumors that demonstrate variable intrinsic radiation responses. (C) 2012 Elsevier Inc.

DAB2IP loss has been associated with aggressive prostate cancer behavior. Preclinical data suggests that loss of DAB2IP plays a significant role in prostate cancer cell survival following exposure to ionizing radiation due to enhanced double strand break (DSB) repair, and resistance to apoptosis. Therefore, we performed a pilot study to determine whether DAB2IP status could serve as a novel biomarker indicative of radiation therapy (RT) effectiveness in high risk prostate cancer patients. Patients with high-risk disease (stage ≥ T3a, or Gleason score (GS) ≥ 8, or Prostate Specific Antigen (PSA) ≥ 20) treated with definitive RT between 2005-2011 at UT Southwestern were identified. Immuno-histochemistry (IHC) analysis for DAB2IP protein was performed on their biopsy specimens. DAB2IP status was scored in the tumors by an expert GU pathologist. Biochemical recurrence-free survival (BRFS) of patient cohorts with and without DAB2IP loss was determined using the Phoenix definition. Log rank test was used to correlate BRFS with DAB2IP levels. Univariate analysis of BRFS to pretreatment PSA, GS, stage, age, DAP2IP status was performed. Twenty-four patients treated for high risk prostate cancer were evaluated. DAB2IP loss was seen in 8 patients (33.3%) whereas 16 patients (66.7%) expressed DAB2IP. Median f/u for DAB2IP patients was 19.4 months (m) (range 8.2 to 57.8 m) and 23.9 m (range 8.8 to 74.0 m) for the DAB2IP deficient group. Patients expressing DAB2IP exhibited markedly improved BRFS compared to patients with loss of DAB2IP (p = 0.027; log-rank test). The estimated 2 yr BRFS was 90% and 68.5% respectively for the DAB2IP present and deficient groups. At 4 yrs BRFS was 90% and 34.3% for the present and deficient groups respectively. Univariate analysis demonstrated DAP2IP status as the only variable with significant association to BRFS at this point of follow up. In this initial study, DAB2IP deficiency in patients portended a significantly worse BRFS after definitive treatment with RT. This is the first clinical evidence suggesting the importance of DAB2IP deficiency as a potential biomarker for predicting radiation response in patients with high risk prostate cancer. Limitations of this study include small sample size and short duration of follow up. We plan to extend this study to remaining patients identified (expected n = 137 patients). Additional studies planned include performing IHC staining of Ki-67, and markers of DNA-DSB repair proteins (H2AX, ATM, DNA-PKcs, 53BP1) which will be correlated with DAB2IP status. Implications for developing therapeutic intervention strategies to enhance radiation response for patients found to have DAB2IP deficient prostate tumors will be discussed.

Small animal irradiation provides an important tool used by preclinical studies to assess and optimize new treatment strategies such as stereotactic ablative radiotherapy. Characterization of radiation beams that are clinically and geometrically scaled for the small animal model is uniquely challenging for orthovoltage energies and minute field sizes. The irradiator employs a commercial x-ray device (XRAD 320, Precision x-ray, Inc.) with a custom collimation system to produce 1-10 mm diameter beams and a 50 mm reference beam. Absolute calibrations were performed using the AAPM TG-61 methodology. Beam's half-value layer (HVL) and timer error were measured with an ionization chamber. Percent depth dose (PDD), output factors (OFs) and off-axis ratios were measured using radiochromic film, a diode and a pinpoint ionization chamber at 19.76 and 24.76 cm source-to-surface distance (SSD). PDD measurements were also compared with Monte Carlo (MC) simulations. In-air and in-water absolute calibrations for the reference 50 mm diameter collimator at 19.76 cm SSD were measured as 20.96 and 20.79 Gy min(-1), respectively, agreeing within 0.8%. The HVL at 250 kVp and 15 mAs was measured to be 0.45 mm Cu. The reference field PDD MC simulation results agree with measured data within 3.5%. PDD data demonstrate typical increased penetration with increasing field size and SSD. For collimators larger than 5 mm in diameter, OFs measured using film, an ion chamber and a diode were within 3% agreement.

An x-ray image-guided small animal stereotactic irradiator was developed and characterized to enable tumor visualization and accurate target localization for small field, high dose irradiation. The system utilizes a custom collimation system, a motorized positioning system (x, y, θ), a digital imaging panel and operating software, and is integrated with a commercial x-ray unit. The essential characteristics of the irradiator include small radiation fields (1-10 mm), high dose rate (>10 Gy min(-1)) and submillimeter target localization. The software enables computer-controlled image acquisition, stage motion and target localization providing simple and precise automated target localization. The imaging panel was characterized in terms of signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR) and spatial resolution. Overall localization accuracy and precision were assessed. SNR, CNR and spatial resolution are 24 dB, 21 dB and 2.8 lp mm(-1), respectively, and localization accuracy is approximately 65 µm with 6 µm precision. With the aid of image guidance, system performance was subsequently used to evaluate radiation response in a rat orthotopic lung tumor effectively sparing normal tissues and in a mouse normal lung. The capabilities of 3D treatment and cone-beam computed tomography are presented for 3D localization and delivery as a work in progress.

Purpose: To develop a dual modality, optical and x‐ray, image guided stereotactic irradiator, and to improve tumor visualization and the accuracy of target localization in small animal irradiation. Method and Materials: A bioluminescence (BL) optical imaging system has been integrated into an existing x‐ray image‐guided stereotactic irradiator developed previously by our group. The new system provides precise target localization and accurate radiation delivery in three dimensions. All localization procedures are computer‐controlled. An air‐cooled CCD camera with sufficiently reduced dark current for better signal‐to‐noise ratio is employed for bioluminescence imaging. The CCD camera is positioned off‐axis, in the same transverse plane as the x‐ray radiation axis. To acquire BL images, the animal is rotated through a 30 degree angle towards the camera, and then back to the original (treatment) position for the x‐ray imaging. The BL images are registered to the x‐ray images for image guidance. Results: The dual modality image guided irradiator was evaluated using an orthotopic lung tumor in a rat. The anterior and lateral BL images clearly showed the tumor with high contrast, while the same tumor was visible only in the anterior view in the x‐ray images. This indicated that the x‐ray image guidance alone is not sufficient to provide the precise localization. On the other hand, the registered BL and x‐ray images overcome this limitation by combining information of tumor position in the BL image with the anatomy in the x‐ray image. Conclusion: The accuracy of tumor localization in small animal SBRT is dramatically improved by integrating optical imaging with x‐ray imaging. The developed system also enhances the sparing of dose limiting normal tissues and organs‐at‐risk (OARs) and has facilitated accurate assessment of experiments in small animal SBRT investigations.