Chemoradiation therapy is the mainstay for treatment of locally advanced, borderline resectable pancreatic cancer. Pharmacologic ascorbate (P-AscH−, i.e., intravenous infusions of ascorbic acid, vitamin C), but not oral ascorbate, produces high plasma concentrations capable of selective cytotoxicity to tumor cells. In doses achievable in humans, P-AscH− decreases the viability and proliferative capacity of pancreatic cancer via a hydrogen peroxide (H2O2)-mediated mechanism. In this study, we demonstrate that P-AscH− radiosensitizes pancreatic cancer cells but inhibits radiation-induced damage to normal cells. Specifically, radiation-induced decreases in clonogenic survival and double-stranded DNA breaks in tumor cells, but not in normal cells, were enhanced by P-AscH−, while radiation-induced intestinal damage, collagen deposition, and oxidative stress were also reduced with P-AscH− in normal tissue. We also report on our first-in-human phase I trial that infused P-AscH− during the radiotherapy “beam on.” Specifically, treatment with P-AscH− increased median overall survival compared with our institutional average (21.7 vs. 12.7 months, P = 0.08) and the E4201 trial (21.7 vs. 11.1 months). Progression-free survival in P-AscH−–treated subjects was also greater than our institutional average (13.7 vs. 4.6 months, P < 0.05) and the E4201 trial (6.0 months). Results indicated that P-AscH− in combination with gemcitabine and radiotherapy for locally advanced pancreatic adenocarcinoma is safe and well tolerated with suggestions of efficacy. Because of the potential effect size and minimal toxicity, our findings suggest that investigation of P-AscH− efficacy is warranted in a phase II clinical trial.
These findings demonstrate that pharmacologic ascorbate enhances pancreatic tumor cell radiation cytotoxicity in addition to offering potential protection from radiation damage in normal surrounding tissue, making it an optimal agent for improving treatment of locally advanced pancreatic adenocarcinoma.
Over 55,000 patients are projected to be diagnosed with pancreatic adenocarcinoma in 2018, making it the 12th most common cancer in the United States (1). Despite this modest incidence, it is the fourth deadliest cancer among both men and women (12.6 per 100,000) in the United States, with 44,330 patients expected to succumb to the disease in 2018 (1). This lethality is likely due to a combination of late stage at diagnosis and poor survival at all stages. In fact, 29% of patients present with regional disease and 52% with metastatic disease, with respective 5-year survival rates of 11.5% and 2.7% (1, 2).
Although there is a significant effort devoted to identify novel systemic agents for advanced disease, there is a tremendous clinical need to improve therapies for patients diagnosed with localized disease (2). One autopsy series demonstrated that approximately 30% of all stages of patients with pancreatic cancer died as a result of local disease burden, irrespective of clinical stage at diagnosis, suggesting that patients with advanced disease may benefit from improved local disease treatment (3). Surgery is one option for local therapy; however, the majority of patients with advanced disease are not amenable to resection. An alternative to surgery is radiotherapy in combination with chemotherapy, but most patients will still experience local treatment failure and eventually succumb to their disease (1, 4). Thus, it is clear that a significant segment of patients with pancreatic cancer could benefit from improvements in the efficacy of radiotherapy. Chemoradiation therapy may also be initiated in this patient population as a first-line therapy or after induction chemotherapy if there has been no metastatic progression (4). The Eastern Cooperative Oncology Group (ECOG)-E4201 study established 50.4 Gy radiotherapy delivered over 28 fractions plus gemcitabine as a superior regimen to gemcitabine alone for locally advanced disease with an increase in overall survival from 9.2 months to 11.1 months (5).
The dose limitation of radiation in pancreatic cancer is dictated by damage to surrounding tissue, in particular the small intestine (6). The expected complication rate when the intestine receives a total dose of 50 Gy in 1.8 or 2 Gy fractions is 5% but rapidly increases to 50% when radiation doses reach a total dose of 60 Gy (7). The maximum recommended conventional radiation dose for the treatment of pancreatic adenocarcinoma is 54 Gy, which poses a 9.4% to 24.4% complication rate (6, 8). Unfortunately, efforts to find radioprotection agents that could potentially allow increased radiation dosing while avoiding concurrent tumor protection have been relatively unsuccessful (9).
Vitamin C (ascorbic acid, ascorbate) is traditionally considered a donor antioxidant, yet is capable of behaving as a pro-oxidant at higher pharmacologic doses (P-AscH−), producing H2O2 in the extravascular space (10–12). Intravenous dosing is required to bypass the intestinal mechanisms that tightly control oral uptake (only micromolar concentrations achievable) and safely allows plasma concentrations to achieve levels 100-fold higher than oral administration (mmol/L concentrations achievable; refs. 13, 14). The H2O2 produced in the extravascular space at these levels is selectively cytotoxic to cancer cells (15, 16). Although the exact mechanism is still under study, H2O2 is thought to diffuse across the cell membrane and subsequently react with the relatively high levels of catalytic metals sequestered in cancer cells to produce toxic radical species (15, 17). A recent phase I clinical trial combining P-AscH− with gemcitabine for advanced pancreatic cancer produced millimolar levels of plasma P-AscH−, demonstrated excellent tolerability, and suggested a synergistic effect with chemotherapy (13, 18).
More recently, the radiosensitizing effects of P-AscH− on cancer have been elucidated. Preclinical studies confirm that P-AscH− potentiates radiation-induced cytotoxicity via a H2O2-mediated mechanism, decreasing tumor growth and prolonging survival in an animal model (19). A selective radiosensitizing agent with a protecting effect on surrounding tissue is novel and could potentially liberate patients from the current radiation dose limitations used, allowing more aggressive treatment of local disease. The goal of the present study is to determine the differential toxicity of P-AscH− in pancreatic cancer cells versus normal cells and to evaluate the safety, tolerability, and potential efficacy of P-AscH− in a first-in-human phase I clinical trial combined with radiation and gemcitabine for locally advanced pancreatic cancer.
Materials and Methods
Human pancreatic cancer cell lines, MIA PaCa-2, PANC-1, and normal fetal human intestinal epithelial cell line, FHs 74 Int, were purchased from the ATCC and not further authenticated or tested. MIA PaCa-2 and PANC-1 were maintained as described (15, 16, 19). Patient-derived pancreatic cancer cell line, 403, from the Medical College of Wisconsin Surgical Oncology Tissue Bank (20, 21) was cultured in Dulbecco's Modified Eagle's Media Nutrient Mixture F-12 supplemented with 5% FBS, penicillin/streptomycin, human recombinant EGF, bovine pituitary extract, hydrocortisone, and human recombinant insulin. Human umbilical vein endothelial cells (HUVEC) were from Lonza and cultured in Endothelial Basal Medium-Plus supplemented with bovine brain extract, hydrocortisone, human epidermal growth factor (hEGF), 2% FBS and getamincin/amphotericin-B. The immortalized normal pancreatic ductal epithelial cells H6c7 were cultured in keratinocyte-serum-free medium with supplements (2.5 μg human recombinant EGF and 25 mg bovine pituitary extract; ref. 22). All cells were maintained in a humidified atmosphere of 95% air/5% CO2 at 37°C and passaged routinely for use until passage 20.
Stock solutions of ascorbate (pH 7.0) were made under argon and stored in screw-top sealed test tubes at 4°C. Ascorbate concentration was verified using, ϵ265 = 14,500/M/cm (23). The solution can be kept for several weeks without significant oxidation due to the lack of oxygen.
Clonogenic survival assay
Clonogenic survival assays were performed as previously described (19). Briefly, cells were plated and allowed to attach for 24 to 48 hours. Cells were treated with P-AscH− (1 and 2 mmol/L or 10 and 20 pmol/cell, respectively) for 1 hour in DMEM/10% FBS followed by irradiation at 0.5 to 2 Gy. Cells were immediately trypsinized, counted, and seeded into 6-well plates at varying densities. FHs 74 Int and H6c7 cells were seeded on top of feeder cells that had been irradiated at 30 Gy. The dishes were maintained in a 37°C, 4% O2, for 10 to 14 days to allow colony formation. The colonies were then fixed with 70% ethanol, stained with Coomassie Blue, and counted (colonies containing > 50 cells were scored). Plating efficiency was determined by the formula: (number of colonies formed/number of cells inoculated) × 100.
Protein (20–40 μg) was electrophoresed in a 4% to 20% Bio-Rad ready gel and then electrotransferred to an Immobilon PVDF membrane (EMD Millipore). Membranes were blocked in 5% bovine serum albumin for 2 hours and then treated with anti–γH2AX (Ser 139) antibody (1:1,000; EMD Millipore). Horseradish peroxidase (HRP)–conjugated goat anti-rabbit or goat anti-mouse (1:50,000; Chemicon International) was used as a secondary antibody. Anti-actin (1:4,000; Sigma-Aldrich) was used as a loading control. Blots were treated with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and exposed to autoradiography film. To determine the role of oxidative stress in P-AscH−-radiosensitization on both tumor and normal tissue, 4HNE was used a marker of lipid peroxidation and is a well-established marker of tissue oxidative stress (27) Mouse jejunal and tumor samples were excised and processed to determine 4-hydroxy-2-nonenal-(4HNE) modified proteins as previously described (19, 26). Briefly, jejunal and tumor tissues were homogenized in 1.3 mmol/L diethylenetriaminepentaacetic acid (DETAPAC), 10 mmol/L butylated hydroxytoluene (BHT) and a complete mini-protease inhibitor (Roche Diagnostics) and assayed for protein concentration utilizing the Bradford assay. Protein (50 μg), in PBS, was blotted onto PVDF membranes. Blots were incubated with the primary antibody recognizing the Michael addition product of 4HNE-modified cellular proteins diluted 1:2,000 overnight at 4°C (28). Secondary antibody incubation was performed for 1 hour with an HRP-conjugated goat anti-rabbit polyclonal antibody (1:25,000). Immunoreactive protein was detected using chemiluminescence (ECL Plus Western Blotting Detection System) on X-ray film. Analysis was performed by comparing 4HNE immunoreactive protein normalized to protein loading determined by staining with Ponceau S stain. Normalized average integrated densities were determined with ImageJ software.
Thirty-day-old athymic nude mice (Foxn1nu) were obtained from Envigo. The nude mice protocol was reviewed and approved by the Institutional Animal Care and Use Committee of The University of Iowa. The animals were housed four in a cage and were fed a sterile commercial stock diet and filtered tap water, ad libitum. Animals were allowed to acclimate in the unit for 1 week before any manipulations were performed. Mice were injected with with 3.0 × 106 MIA PaCa-2 pancreatic cancer cells, and the tumors were allowed to grow to a size of 5 to 6 mm before treatments were initiated. Prior to radiation treatments, animals were anesthetized with 80 to 100 mg/kg ketamine and 10 mg/kg xylazine i.p. and shielded in a lead block such that only the lower body was irradiated, sparing the head and chest. Animals were euthanized by CO2 asphyxiation.
Mice were randomized to receive P-AscH− (4 g kg−1, i.p., twice daily) or an equivalent volume of 1 M NaCl (4 g/kg, i.p., twice daily) for 2 days. On the third day, the animals received 10 Gy abdominal radiation (Pantak Therapax DXT 300, X-rays). The animals were shielded to protect the thoracic organs when the abdomen was irradiated. Dose rate for the abdominal radiation was 1.38 Gy/minute. In the in vivo experiments, the mouse was in a lead shield, resulting in just the tumor being irradiated. Dosimetry was used, assuming the mouse leg is 1 cm thick and dose rate is to mid-leg while the mouse is in a body/leg container, which is corrected for the depth of the container. The dose rate was 1.38 Gy/minute. In separate groups of animals with tumor xenografts, mice received the same dosing schedule of P-AscH− or NaCl, but then had 5 Gy radiation delivered to the tumors. Treatment continued with either P-AscH− or NaCl on the fourth and fifth days; then all mice were sacrificed on day 6. Jejunal tissue was obtained and evaluated with hematoxylin/eosin for tissue microstructure and trichrome blue for collagen deposition. A comparative pathologist blinded to the study assigned semiquantitative scores to each jejunal section according to the grading system outlined in Supplementary Table S1. Mitochondrial damage was investigated using electron microscopy. Jejunal tissue was fixed in 2.5% glutaraldehyde in PBS overnight at 4°C and then post fixed with 1% osmium tetroxide for 1 hour. Following serial alcohol dehydration (50%, 75%, 95%, and 100%), the samples were embedded in Epon 12 (Ted Pella). Ultramicrotomy was performed, and ultrathin sections (70 nm) were post stained with uranyl acetate and lead citrate. Samples were visualized with a JEOL 1230 transmission electron microscope at the Central Microscopy Research Facility of The University of Iowa. The number of total mitochondria visualized were counted, as well as the number of damaged mitochondria.
The glutathione assay was kindly performed by the Antioxidant Enzyme Core Facility at The University of Iowa. Following incubation, jejunum slices were homogenized in 5% 5-sulfosalicylic acid (Sigma). The samples were then centrifuged to remove precipitating proteins, and the supernatants were assayed for total glutathione (GSH) content by the method of Griffith (24). Glutathione disulfide (GSSG) was measured by adding 20 μL of a 1:1 mixture of 2-vinylpyridine and ethanol per 100 μL of sample and incubating for 2 hours prior to assaying as described previously (25). The SSA precipitated protein was resuspended in 0.1 N NaOH, and protein levels determined using the BCA Assay Kit (Thermo Scientific). Glutathione determinations were normalized to the protein content of whole homogenates.
Phase I Clinical Trial
This clinical trial was conducted under an established investigator-sponsored Investigational New Drug. Compliant with International Committee of Medical Journal Editors policy, the trial was listed on clinicaltrials.gov prior to enrollment (NCT01852890). Approval was obtained from The University of Iowa IRB-01 (Biomedical). The trial was conducted under Good Clinical Practice consistent with International Council on Harmonization guidance document [ICH E6(R2)]. Written or signed informed consent was obtained from all participating patients.
Study design and statistical analysis
This was a single-institution, open-label, phase I study of P-AscH− administered concurrently with gemcitabine and radiotherapy. The P-AscH− dose was escalated via Storer's Two-Stage Design BD for phase I trials (26). Design B enrolls only a single participant per dose cohort until a dose-limiting toxicity is identified. The cohort was then expanded (Design D) to a 3-subject evaluation and continues with 3 subjects per cohort for subsequent cohorts. A minimum of 6 subjects must be evaluated at the recommended phase II dose with ≤1 dose-limiting toxicity occurring in those 6.
Patients with histologically or cytologically diagnosed pancreatic adenocarcinoma and indication for concurrent gemcitabine and radiotherapy were invited to participate in this trial. Because P-AscH− infusions may cause red blood cell hemolysis in those deficient in the glucose-6-phosphate dehydrogenase (G6PD) enzyme, G6PD-deficient patients were excluded from the study (lower limit of normal 7 U/g hemoglobin, ARUP Laboratories). Minimum Karnofsky performance status was 60. Required initial laboratory values included an absolute neutrophil count of ≥1,500/mm3, platelet count of ≥100,000/mm3, leukocytes > 3,000/mm3, creatinine of ≤1.5 mg/dL or creatinine clearance ≥60 mL/minute, total bilirubin ≤2 × upper limit of normal (ULN), transaminases ≤2.5 × ULN, and a prothrombin time and international normalized ratio within normal institutional limits. Key exclusion criteria included active comorbidities such as end-stage congestive heart failure, unstable angina, or myocardial infarction within 6 months of enrollment. Consistent with Good Clinical Practice, subjects had to be capable of providing informed consent; legally authorized representatives and proxy consent were not allowed. The final eligibility criterion was tolerance of a test dose of 15 g pharmacologic ascorbate (Mylan Pharmaceuticals) in 250 mL sterile water for human use. Eligibility was independently confirmed by the Data and Safety Monitoring Committee of the Holden Comprehensive Cancer Center.
This study is the first to actively infuse P-AscH− during the radiation “beam on” time. P-AscH− infusion was initiated at least 20 minutes prior to radiotherapy delivery. Subjects were then transferred to the linear accelerator for therapy. After radiation, subjects were returned to the infusion area for the remainder of their infusion.
The schema is provided (Fig. 1). Subjects were assigned the P-AscH− dose as per open cohort slots (50 g in 750 mL, 75 g in 1,000 mL, or 100 g in 1,500 mL sterile water for human use). P-AscH− was infused daily. Gemcitabine was administered according to ECOG-E4201 with an intravenous infusion at a dose of 600 mg/m2 over 30 minutes, once weekly for 6 weeks (5). Intensity modulated radiotherapy (IMRT) was delivered with either Siemans Oncor or Elekta Versa HD treatment machines as either 50.4 Gy in 28 fractions or 50 Gy in 25 fractions as determined most appropriate by the treating radiation oncologist. Four-dimensional (4D) computed tomography–based simulation was performed for each subject. The 4D CT data sets included 10 respiratory phases: 20%, 40%, 60%, 80%, and 100% inspiration phases, and 80%, 60%, 40%, 20%, and 0% expiration phases. If target motion due to respiration was ≥1 cm, respiratory gating was used for treatment planning and delivery to limit target motion to ≤1 cm. In situations where the target motion was ≤1 cm, an internal target volume was generated using the 100% inspiration and 0% expiration phases. For subjects with residual disease at the time of simulation, the gross tumor volume (GTV) was defined as the primary tumor and/or any suspicious (≥1 cm) regional lymph nodes. The clinical target volume (CTV) was a 1.5 cm expansion on the GTV. The planning treatment volume (PTV) was defined as the CTV plus a 0.5 cm expansion in all directions. For subjects receiving adjuvant radiotherapy: a CTV volume was generated using the celiac axis, superior mesenteric artery, portal vein, preoperative tumor volume, hepaticojejunostomy or pancreaticojejunostomy, aorta and surgical clips. A PTV was generated by expending the CTV by 0.5 cm. Surrounding normal structures including the liver, each kidney, spinal cord, duodenum, and bowel were contoured for dose-volume histogram generation and analysis of normal tissue radiation dose constraints. Subject positioning was confirmed prior to each treatment using either kilovoltage (kV) or megavoltage (MV) conebeam CT.
Dose-limiting toxicities were defined as the following events, regardless of attribution to the P-AscH−: vomiting resulting in hypokalemia that did not respond to treatment; vomiting resulting in hypotension that did not respond to treatment; grade 4 febrile neutropenia, grade 4 intraabdominal hemorrhage; or grade 4 weight loss. Also, any serious adverse event (21 CFR§312.32) with a causality to the P-AscH− infusion was deemed a dose-limiting toxicity. Subjects continued P-AscH− treatment throughout combination therapy. Blood samples were drawn weekly, pre- and post-infusion (≤15 minutes). Blood samples were stored on ice in a cooler and transferred to a dark refrigerator until prepared for assay. No P-AscH− degradation was expected to occur during processing and storage conditions.
Response and toxicity criteria
Adverse events were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) 4.03. Consistent with recent FDA guidance on risk-based monitoring and safety assessments (27, 28), assessment to P-AscH− was required for all serious adverse events or adverse events ≥ grade 3. Lower-graded events were assumed to be related to tumor burden, side effects of antineoplastic therapy, and activities of daily life. Causality of P-AscH− to the adverse event was determined by the sponsor investigator (J.J. Cullen) as per Federal Code.
Sample preparation for plasma and red blood cells
Whole blood (NaHeparin 75 USP units, BD Vacutainer green top blood collection tube, 4 mL) was collected from clinical trial participants. Samples were centrifuged at 2,000 relative centrifugal force for 15 minutes, and then red blood cell–free platelet-depleted plasma was collected and stored frozen at −80°C in multiple aliquots for subsequent P-AscH− or F2-isoprostane measurement and other biochemical analyses. Ascorbate levels in subject plasma samples were determined with a published plate reader-based assay as described in Vislisel and colleagues with minor modifications to detect ascorbate concentrations at nutritional (μmol/L) and post-intravenous P-AscH− infusion levels (mmol/L; ref. 29). Minor modifications to this protocol have been described and implemented in this trial (13, 29).
Measurement of isoprostanes
Isoprostanes are arachidonic acid derivatives, formed from nonenzymatic, free radical peroxidation. F2-isoprostanes are chemically stable, and found in all biological fluids, making them the most ideal current measure of systemic oxidative stress (30). Plasma samples were collected prior to initiating chemoradiotherapy, 3 weeks into chemoradiotherapy, and at the completion of chemoradiotherapy for F2-isoprostane comparison. To quantify F2-isoprostane levels, gas chromatography-mass spectrometry was utilized (Eicosanoid Core Laboratory, Vanderbilt University, Nashville, TN). This assay was chosen because it has been shown to be capable of detecting changes indicative of in vivo oxidative stress in plasma samples and is verifiable (30–32).
There were 19 subjects enrolled in the comparator arm of the study. These subjects met the same inclusion criteria as subjects receiving P-AscH−. All subjects received standard regimens of concurrent gemcitabine and radiotherapy with curative intent. Additional clinical data for the comparator subjects are available in Supplementary Table S2. Blood was obtained in 4 subjects undergoing concurrent chemoradiation to determine F2-isoprostane levels. Approval was obtained from The University of Iowa IRB-01 (IRB 201508733, J.J. Cullen, PI).
Differential effects of P-AscH− on radiation-induced clonogenic cell death
MIA PaCa-2, 403, H6c7, and FHs 74 Int cells were treated with P-AscH− (1 mmol/L, 10 pmol/cell) and radiation (1 Gy). Individually, P-AscH− (1 mmol/L) and radiation (1 Gy) induced clonogenic cell death in the MIA PaCa-2 pancreatic cancer cell line but not in the 403 pancreatic cancer cell line. The combination treatment, however, did induce clonogenic cell death in both cell lines (Fig. 2A and B). These data are consistent with previous studies of P-AscH− combined with radiation on pancreatic cancer cell lines (19). To determine whether H2O2 was responsible for the cytotoxic effects of P-AscH− and radiation, the 403 cell lines was treated with bovine catalase (100 U/mL) and clonogenic survival was determined. In addition, P-AscH− and P-AscH− combined with radiation decreased clonogenic survival, which was reversed with catalase pretreatment (Supplementary Fig. S1). In contrast, the H6c7 nontumorigenic pancreatic ductal epithelial cell lines did not demonstrate significant cytotoxicity with a combination treatment of P-AscH− and radiation (Fig. 2C). Furthermore, the FHs 74 Int cell line experienced a recovery in clonogenic survival capability when radiated cells were treated with P-AscH−, suggesting a possible radioprotective effect in this normal cell line (Fig. 2D).
Differential effects of P-AscH− on radiation-induced DNA damage
All cell lines were treated with P-AscH− (5 mmol/L, 5 pmol/cell) for 1 hour and radiated (5 Gy). Protein was collected at 5and 60 minutes after radiation to determine γH2AX peak. In the pancreatic cancer cell lines, cotreatment with 1 hour of P-AscH− enhanced γH2AX immunoreactivity at 5 and 60 minutes (Fig. 2E). In contrast, γH2AX immunoreactivity was decreased at these time points when the nontumorigenic cell lines (HUVEC and FHs 74 Int) were treated with IR and P-AscH− (Fig. 2F and G). To quantify and further clarify the DNA-damage response, a quantitative PCR analysis of DNA damage was determined using a recently described and published method from our group (16). Using quantitative PCR method, the formation and removal of DNA damage were measured in cells. In MIA PaCa-2 cells, P-AscH− (5 mmol/L) plus radiation (5 Gy) increased the frequency of DNA lesions (0.31 ± 0.05 lesions/10 kb; kb = kilobase pairs; means ± SEM, n = 3) compared with cells irradiated with 5 Gy only (0.09 ± 0.01 lesions/10 kb). In contrast, FHs 74 Int cells had a decrease in the frequency of DNA lesions from (0.17 ± 0.05 lesions/10 kb, means ± SEM, n = 3, P < 0.05 vs. MIA PaCa-2) in cells irradiated alone, to undetectable levels in cells treated with P-AscH− (5 mmol/L) plus 5 Gy. These data are consistent with the Western blots for γH2AX immunoreactivity in nontumor and tumor cell lines treated with P-AscH− and radiation.
P-AscH− limits radiation-induced intestinal damage and collagen deposition
The clinical effect of radiation damage on the small intestine is demonstrated by villous blunting, loss of crypt cells, and collagen deposition. Semiquantitative analysis was conducted by a comparative pathologist (K. Gibson-Corley), blinded to the study, according to a standard scoring system (Supplementary Table S1). The hematoxylin/eosin staining demonstrated radiotoxic effects of mouse jejunum following 10 Gy radiation, including extensive villous blunting and loss of crypt architecture (Fig. 3A). This effect was partially inhibited when radiated mice were treated with P-AscH− (Fig. 3A and B). Similarly, collagen deposition and permeation into the submucosa and lamina propria was significantly increased in mice treated with radiation alone (Fig. 3C). This effect was also partially ameliorated in mice treated with P-AscH− (Fig. 3C and D). The mitochondrial structure of jejunal epithelial cells from mice radiated with or without P-AscH− was analyzed utilizing electron microscopy. Radiation alone resulted in 91% damaged mitochondria characterized by swelling, vacuolization, loss of cristae structure, and membrane rupture (Fig. 3E). P-AscH− once again partially inhibited the radiation-induced mitochondrial damage (Fig. 3E and F). To determine the role of oxidative stress as a mechanism whereby P-AscH− ameliorates radiation-induced damage to normal tissue, a glutathione assay was performed on irradiated jejunum (Fig. 3G). The ratios of reduced glutathione (GSH) to oxidized glutathione disulfide (GSSG) were compared in mice with abdominal radiation alone and abdominal radiation with P-AscH−. Jejunum from mice treated with radiation and P-AscH− displayed lower levels of GSSG, suggesting that P-AscH− may be acting as a systemic antioxidant to normal tissue during radiation. In addition, 4HNE expression demonstrated that irradiated jejunal tissue treated with P-AscH− expressed lower levels of 4HNE than irradiated tumor tissue treated with P-AscH− (Fig. 3H). Taken together, these data suggest that P-AscH− radiosensitizes pancreatic tumors and acts as a pro-oxidant in irradiated tumor tissue, but may act as an antioxidant in radiated normal tissue.
Phase I Clinical Trial
Table 1 provides a clinical summary of the subjects enrolled in the clinical trial and the comparator group. A total of 16 subjects were enrolled for P-AscH− treatment from June 2014 to November 2016. Of the 16 subjects treated with P-AscH−, one subject withdrew due to chronic back pain exacerbation resulting from sitting during the infusion. Another subject withdrew after a grade 3 transient blood pressure elevations due to hypervolemia. The remaining 14 subjects completed all protocol therapy, making them evaluable for statistical purposes. Adverse events were reviewed monthly at investigator meetings and reviewed against protocol requirements for dose limiting toxicities. Average treatment duration was 5.7 weeks. No subjects were lost to follow-up.
Safety and toxicity
Average Karnofsky performance score both prior to chemoradiotherapy and post-chemoradiotherapy was 86 ± 2 (SEM). One subject experienced a decrease in Karnofsky score, 2 subjects showed an increase, and 11 showed no change. No adverse events occurred during the concurrent P-AscH− infusion with combination therapy administration or immediately thereafter. In general, adverse events were consistent with those previously described (Table 2). Three adverse events were considered attributable to P-AscH−: dry mouth, thirst, and transient blood pressure elevation. For the purposes of this clinical trial, transient blood pressure elevation is described as a blood pressure elevation (systolic pressure, diastolic pressure, or both) causing the post-infusion blood pressure grade to be higher than preinfusion grade. Grading was harmonized to CTCAE hypertension grading. Transient blood pressure elevation was considered a separate event from hypertension because the blood pressure elevation was typically not sustained beyond 30 minutes. Five subjects experienced a grade 3 transient blood pressure elevation at least once. Dry mouth and thirst were transient symptoms alleviated the same day as infusion. Only one dose-limiting toxicity occurred (subject 3, 75 g dose). Within 2 hours after P-AscH− infusion, subject 3 reported to a routine medical oncology visit. During the exam, the subject described left-sided chest heaviness followed by sharp stabbing pain lasting for about 20 minutes. Subject 3 was admitted for cardiac workup and observation and ultimately diagnosed with esophageal spasm (CTCAE noncardiac chest pain, grade 3); the event was filed with FDA through the MedWatch 3500A mechanism due to the contemporaneousness of the event to the P-AscH− infusion. Subject 3 was rechallenged without incident and continued on the trial. The cohorts were then expanded from Storer's Design B to Design D as per protocol. Table 2 provides the summary of maximum grade for toxicities for all treatments combined. Grade 3 and 4 hematologic toxicities included anemia (n = 1), white blood cell count decreased (n = 8), lymphocyte count decreased (n = 13), absolute neutrophil count decreased (n = 7), and platelet count decreased (n = 2). Hematologic toxicities were consistent with gemcitabine and radiotherapy.
Maximum tolerated dose
All 3 dose cohorts were evaluated. Due to the single dose-limiting toxicity, 6 subjects were evaluated in the 75 g dose cohort prior to escalating to 100 g. Of the 5 subjects treated at the 100 g dose, one subject's blood pressure elevated to 201/102 mmHg but resolved to 127/88 mmHg within 30 minutes. A second subject, who did not have a history of baseline hypertension, developed post-infusion elevated blood pressures that did not resolve as quickly, remaining in a systolic range of 140 to 150 mmHg. The fluid volume for a 100 g dose is 1500 mL and is less than the typical hydration volume used for cisplatin. For this reason, these adverse events were attributed as possible to the P-AscH− and were considered dose limiting. Both subject's doses were reduced to 75 g for the remainder of therapy. Given the increased blood pressure events, the increased infusion time (120 minutes vs. 180 minutes), and the diminished return on the average plasma ascorbate level, 100 g was determined to be the maximum tolerated dose and 75 g was selected as a recommended phase II dose.
Plasma ascorbate levels
Mean plasma ascorbate concentrations immediately upon completion of the i.v. infusion were 15 mmol/L for 50 g (n = 17; 95% CI, 13–17 mmol/L), 20 mmol/L for 75 g (n = 37; 95% CI, 19–21 mmol/L), and 20 mmol/L for 100 g (n = 32; 95% CI, 19–22 mmol/L), respectively. Plasma ascorbate levels in the 50-g cohort were significantly lower than the 75-g and 100-g cohorts (one-way ANOVA with post hoc Tukey analysis, n = 86, P < 0.05; Fig. 4A). Comparison of plasma ascorbate levels with subject metrics revealed the most predictable dosing on a per kg basis (Fig. 4B). Body mass index, body surface area, and height all correlated significantly with plasma ascorbate levels (Supplementary Fig. S2).
Despite the potential of hemolysis by P-AscH−, as determined from in vitro experiments (33), which could negatively affect the tumoricidal effects, the incidence of anemia is consistent with the published literature (5). Hemoglobin levels were collected periodically over the 6-week chemoradiation cycle (Supplementary Fig. S3). These data were analyzed by one-way repeated measure ANOVA for both P-AscH−–treated patients and comparator patients (Fig. 4C). There was no change in the Hgb level over time when P-AscH− was added to combination therapy–treated patients (P = 0.44). However, a change in the Hgb level over the course of combination therapy was detected in comparator patients (P < 0.01). Paired t tests did not detect any change in pre- versus post-combination therapy Hgb values (mean Δ Hgb −0.34 g/dL; 95% CI, −1.27 to 0.59 mg/dL, P > 0.05; Fig. 4D). However, a statistically significant decrease in Hgb was found in the 13 of 19 comparator patients with Hgb data available for analysis (mean Δ Hgb −1.47 g/dL; 95% CI, −2.21 to 0.73 mg/dL, P < 0.01, Fig. 4E). One of 14 subjects (7%) treated with P-AscH− suffered grade 3 anemia as defined by hemoglobin concentration. This also compared favorably to historical controls, where 6 of 34 subjects (18%) receiving combination therapy alone suffered grade 3 or 4 anemia as defined by hemoglobin concentration (5).
Despite concerns that P-AscH− may have acute hypotensive effects when given in dosages over 30 g (34), hypotension was not a realized adverse event (Table 2). To the contrary, transient elevation in blood pressure was documented (grade 2: n = 7; grade 3, n = 5; grade 4, n = 1). These elevations typically resolved within 30 minutes upon completion of infusion. Two subjects had shifts in hypertension (as defined by CTCAE, from grade 1 to grade 2) during active therapy. One returned to baseline posttreatment blood pressure levels, the other did not.
Plasma F2-isprostanes are a reliable marker of systemic lipid peroxidation and are used as markers of oxidative injury (30). F2-isoprostane levels from P-AscH−–treated subjects drawn prior to beginning combination therapy, during combination therapy (after the third week), and after completion of all 6 weeks of combination therapy, showed a significant decrease over time from 0.17 ± 0.01 ng/mL prior to treatment initiation to 0.09 ± 0.01 ng/mL at the end of the study (P = 0.02, means ± SEM, n = 14; Fig. 4F). In contrast, F2-isoprostane levels did not change over the course of treatment for comparator subjects treated with combination therapy alone starting at a level of 0.16 ± 0.02 ng/mL prior to treatment initiation to 0.13 ± 0.02 ng/mL at the conclusion of the trial (P = 0.88, means ± SEM, n = 4; Fig. 4F).
Eight of 14 subjects (57%) received all 6 gemcitabine doses, 5 received 5 doses, and 1 received 4 doses. Two subjects had a dose delay of 1 week (week 6) as absolute neutrophil count decreased and platelet count decreased. Eight subjects had gemcitabine dose reductions. All subjects completed radiotherapy. The average dose was 50.4 Gy. One subject received 50 Gy in 25 doses, and the remaining 13 subjects received 50.4 Gy in 28 doses. Eight of 14 subjects (57%) received all doses of P-AscH−. In 6 subjects, at least one dose of P-AscH− was withheld. Subjects received an average of 97% ± 5% of the prescribed ascorbate. Of the 364 doses of ascorbate prescribed, 354 were infused.
Although the trial was not prospectively powered to determine differences in survival, treatment with P-AscH− appears to be no worse than combination therapy when compared with The University of Iowa's median OS of 12.7 months (range, 3.1–54.6 months; log-rank test P = 0.08) and the historical median OS of 11.1 from ECOG-E4201 (5). Similarly, median progression-free survival (PFS) was 13.7 months (range, 2.4–43.8 months) compared with The University of Iowa's 4.6 months median (range, 1.9–26.5 months; P = 0.02; Fig. 5). Two subjects with borderline resectable disease became eligible for pancreaticoduodenectomy following trial completion. Subject #1 was initially staged with cT2N1M0 disease and considered borderline resectable due to impingement on the first jejunal branch of the gastroduodenal artery. Following initial FOLFIRINOX treatment, there was no longer evidence of lymphadenopathy, but there was disease progression at the interface between the tumor and the superior mesenteric artery and superior mesenteric vein. The subject enrolled in the clinical trial with P-AscH− and chemoradiation. After completing the trial, restaging imaging demonstrated disease regression. The subject was determined suitable for surgery and a pancreaticoduodenectomy was performed. Pathology demonstrated marked treatment effect and 17 lymph nodes negative for disease. Subject #4 was initially staged with cT3N1M0 disease and determined to be borderline resectable based on suspected invasion of the portal vein, abutment of the common hepatic artery and regional lymph node adenopathy. The subject initially underwent 2 cycles of FOLFIRINOX and 3 cycles of gemcitabine and nab-paclitaxel. Restaging CT scan demonstrated stable disease. The subject was enrolled in the clinical trial and received P-AscH− in combination with chemoradiation. After completion of the trial, restaging imaging showed disease regression with resolution of regional lymphadenopathy making the subject eligible for surgery. A pancreaticoduodenectomy was performed, and surgical pathology revealed no residual viable invasive adenocarcinoma; 17 lymph nodes were negative for disease. Both subjects are currently alive and are 44 and 35 months from diagnosis, respectively, without evidence of disease recurrence. At the time of submission of this article, 5 of 14 subjects remain alive and 3 subjects are without evidence of disease recurrence.
Our study demonstrates that P-AscH− selectively radioprotects normal cells and radiosensitizes tumor cells. P-AscH− reverses radiation-induced clonogenic cell death in nontumorigenic cells but radiosensitized a variety of human pancreatic cancer cell lines. A potential mechanism is the differential oxidative effect of radiation-induced DNA damage in cancer versus normal cells, which the present study demonstrates in vitro and in vivo. Radiation damage to the intestine is demonstrated by villous blunting, loss of crypt cells, collagen deposition, mitochondrial damage, and increased GSSG, which were all partially ameliorated with P-AscH−. Results from our first-in-human phase I clinical trial suggest that adding daily P-AscH− to combination therapy is safe for locally advanced pancreatic cancer. Although not statistically powered to demonstrate efficacy, PFS and OS are increased compared with similar patients treated at our institution as well as ECOG-E4201.
There are limitations to the present study. The mechanism underlying the opposing effects of P-AscH− radioprotecting normal cells while radiosensitizing tumor cells is an active area of investigation. A possible explanation is that the H2O2 produced by P-AscH− has a much greater detrimental effect on cancer cells than normal cells. Cancer cells, which have elevated levels of reactive metals, are likely to have increased formation of hydroxyl radical, causing DNA damage (15, 17). Normal cells, which have relatively lower fluxes of O2•, have lower levels of labile metals, and more abundant catalase to detoxify H2O2, leaving P-AscH− to act as an antioxidant alone (12, 16). Others have demonstrated that the NF-κB transcription factor RelB can determine the differential effects of P-AscH− in normal versus cancer cells (35). In addition, this phase I trial was not powered to make any conclusive statements regarding the efficacy of P-AscH− for locally advanced pancreatic cancer. Additionally, the retrospective comparator patients are not ideal controls for a variety of reasons including differences in chemotherapy regimens prior to chemoradiation, differing starting CA19-9 levels, and the retrospective nature of their data collection. Due to the retrospective nature of the data in the comparator patients, our study can only evaluate the reduced normal tissues toxicity in the in vitro and in vivo studies and not in human subjects at this time. However, we do believe this comparator group adds clinical context for which to interpret the potential of P-AscH− treatments for locally advanced disease in both historical controls and our institutional comparator subjects. The clinical data and pathologic findings in the resected surgical specimens from P-AscH−–treated patients are also encouraging.
There is growing evidence that P-AscH− is safe with suggestion of efficacy in a variety of cancer types (17, 36, 37). The safety of P-AscH− in combination with gemcitabine in metastatic pancreatic adenocarcinoma has been previously established (13). We establish here the safety and tolerability of P-AscH− administered concomitantly with radiotherapy and gemcitabine for locally advanced pancreatic adenocarcinoma. Fourteen subjects received the combined therapy in this clinical trial. Of these subjects, 57% received gemcitabine as prescribed (6 cycles) and 100% received the prescribed radiation dose, a major improvement from the published data whereby 29% completed all planned cycles of chemotherapy and 24% of subjects received less than 45 Gy of radiotherapy (5). The improved ability to remain adherent to chemoradiotherapy regimens may be partially explained by decreased systemic oxidative stress. Plasma F2-isoprostanes significantly decreased over the course of therapy in subjects treated with P-AscH− but not in comparator subjects. This phenomenon has been observed before in previous studies of patients receiving P-AscH− and gemcitabine (13). However, this trial evaluated F2-isoprostane levels in subjects treated with P-AscH−, gemcitabine, and radiotherapy, and furthermore compared these plasma levels to control subjects receiving gemcitabine and radiotherapy alone. These results taken together with the current in vitro and in vivo data suggest a potential systemic antioxidant effect in spite of local pro-oxidative treatments. In translational studies of glioblastoma, P-AscH− has been shown to synergize with radiation in vitro but protect tumor cells from radiation killing in vivo (38). These differences from our present study could be due to different ascorbate metabolism in the central nervous system, single versus multiple doses of radiation, tumor microenvironment, and different tumor types.
The effect size suggested by this first-in-human trial is promising. This trial demonstrates an increase in median overall survival compared with our institutional median (21.7 vs. 12.7 months, P = 0.08) and the ECOG-E4201 trial (21.7 vs. 11.1 months; refs. 5, 39–42). PFS in P-AscH−–treated subjects was also greater than our institutional median (13.7 vs. 4.6 months, P = 0.02) and the ECOG-E4201 trial (6.0 months; ref. 5).
In conclusion, P-AscH− enhances pancreatic tumor cell radiation cytotoxicity, in addition to offering potential protection from radiation damage in normal surrounding tissue, making it an optimal agent for potentially improving locally advanced pancreatic adenocarcinoma. This is the first human study to not only infuse P-AscH− in combination with chemoradiation but to infuse while the radiation beam is on. These clinical trial results suggest adding P-AscH− daily to radiotherapy is safe with minimal additional toxicity and a promising therapeutic effect. A phase II trial powered to determine efficacy of P-AscH− at a dose of 75 g, when added to current standard-of-care combination therapy for pancreatic adenocarcinoma is indicated.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Conception and design: M.S. Alexander, S.R. Schroeder, G.R. Buettner, B.A. Wagner, K.L. Bodeker, S. Vollstedt, H.A. Brown, B.G. Allen, J.J. Cullen
Development of methodology: M.S. Alexander, S.R. Schroeder, G.R. Buettner, B.A. Wagner, K.L. Bodeker, S. Vollstedt, J.J. Cullen
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M.S. Alexander, B.A. Wagner, J. Du, B.R. O’Leary, D.J. Berg, K.L. Bodeker, S. Vollstedt, H.A. Brown, B.G. Allen, J.J. Cullen
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M.S. Alexander, J.G. Wilkes, G.R. Buettner, B.A. Wagner, K. Gibson-Corley, B.R. O’Leary, J.M. Buatti, K.L. Bodeker, S. Vollstedt, B.G. Allen, J.J. Cullen
Writing, review, and/or revision of the manuscript: M.S. Alexander, J.G. Wilkes, G.R. Buettner, B.A. Wagner, K. Gibson-Corley, B.R. O’Leary, D.R. Spitz, J.M. Buatti, K.L. Bodeker, S. Vollstedt, H.A. Brown, B.G. Allen, J.J. Cullen
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M.S. Alexander, B.R. O’Leary, J.M. Buatti, K.L. Bodeker, S. Vollstedt, H.A. Brown, J.J. Cullen
Study supervision: K.L. Bodeker, S. Vollstedt, H.A. Brown, J.J. Cullen
Other (regulatory compliance): K.L. Bodeker
We acknowledge the staff at The University of Iowa College of Medicine and Holden Comprehensive Cancer Center Radiation and Free Radical Research (RFRR) Core for radiation services. We acknowledge the invaluable support of the ESR Facility. This study was supported by NIH grants P01 CA217797 (J. Cullen and D. Spitz), P30CA086862, CA184051 (J. Cullen), CA148062 (M. Alexander and J. Wilkes), CA169046 (G. Buettner), CA086862 (D. Spitz), and a grant from Gateway for Cancer Research (J. Cullen).
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