Consuming a Ketogenic Diet while Receiving Radiation and Chemotherapy for Locally Advanced Lung Cancer and Pancreatic Cancer: The University of Iowa Experience of Two Phase 1 Clinical Trials

Abstract

Ketogenic diets are low in carbohydrates and high in fat, which forces cells to rely more heavily upon mitochondrial oxidation of fatty acids for energy. Relative to normal cells, cancer cells are believed to exist under a condition of chronic mitochondrial oxidative stress that is compensated for by increases in glucose metabolism to generate reducing equivalents. In this study we tested the hypothesis that a ketogenic diet concurrent with radiation and chemotherapy would be clinically tolerable in locally advanced non-small cell lung cancer (NSCLC) and pancreatic cancer and could potentially exploit cancer cell oxidative metabolism to improve therapeutic outcomes. Mice bearing MIA PaCa-2 pancreatic cancer xenografts were fed either a ketogenic diet or standard rodent chow, treated with conventionally fractionated radiation (2 Gy/fraction), and tumor growth rates were assessed daily. Tumors were assessed for immunoreactive 4-hydroxy-2-nonenal-(4HNE)-modfied proteins as a marker of oxidative stress. Based on this and another previously published preclinical study, phase 1 clinical trials in locally advanced NSCLC and pancreatic cancer were initiated, combining standard radiation and chemotherapy with a ketogenic diet for six weeks (NSCLC) or five weeks (pancreatic cancer). The xenograft experiments demonstrated prolonged survival and increased 4HNE-modfied proteins in animals consuming a ketogenic diet combined with radiation compared to radiation alone. In the phase 1 clinical trial, over a period of three years, seven NSCLC patients enrolled in the study. Of these, four were unable to comply with the diet and withdrew, two completed the study and one was withdrawn due to a dose-limiting toxicity. Over the same time period, two pancreatic cancer patients enrolled in the trial. Of these, one completed the study and the other was withdrawn due to a dose-limiting toxicity. The preclinical experiments demonstrate that a ketogenic diet increases radiation sensitivity in a pancreatic cancer xenograft model. However, patients with locally advanced NSCLC and pancreatic cancer receiving concurrent radiotherapy and chemotherapy had suboptimal compliance to the oral ketogenic diet and thus, poor tolerance.

Figures

Figure 1. Schematic overview of the ketolung (1A) and ketopan (1B) clinical protocols

Subjects consumed a 4:1 ketogenic diet for a period of 5.5 – 7 weeks while receiving standard of care radiation and chemotherapy for their respective cancers. Subjects were followed for up to 1 year after completing the ketogenic diet assessing for subsequent adverse events.

Figure 2. Mice fed KDs had lower blood glucose and higher blood ketone levels than mice fed std chow

Mice (N=3 per group) were fed 4:1 KetoCal® or standard (std) chow for 25 days. A) β-hydroxybutyrate (βHB) levels were taken via tail vein stick at various times in the first two weeks of diet via Precision Xtra ketone monitoring system. B) Mouse blood taken via cardiac puncture at sacrifice was assayed for HbA1c using Crystal Chem mouse assay kit. Analysis using one way ANOVA with Newman Keul’s posttest yielded *p

Figure 3. Feeding a Ketogenic Diet Sensitizes Mia PaCa-2 Human Pancreatic Cancer Xenografts to Radiation

Nude mice (5–6 animals per group) were injected with Mia Paca-2 cells in their right flank and tumors were allowed to grow to approximately 4 mm in maximum diameter. Mice were irradiated (IR) with 6 fractions of 2 Gy over a period of 2 weeks. Control mice with Mia PaCa-2 xenografts were fed ad libitum standard rodent chow (Control) or 4:1 KetoCal® ketogenic diet (Keto) 2 days prior to first radiation dose and continued until two days following last radiation dose for a total of 16 days. A) Kaplan-Meier survival plots for Control, Keto, IR and IR + Keto treatment groups. Keto + IR resulted in significantly longer survival than all groups alone p

Figure 4. KD increases 4HNE-modified proteins in pancreas cancer xenografts

Equivalent protein quantities isolated from pancreas xenograft animals treated as in Figure 3 (N=3 from each treatment group) were blotted onto PVDF membrane and stained with a polyclonal antibody against 4HNE-modified proteins. (A) Negative (−) and positive (+) controls represent the immunoreactivity derived from Mia Paca-2 cell homogenates treated with and without 100 µM 4HNE for 1 hour. (B) shows the quantification of dot blots by Image J analysis with normalization to the background. Error bars represent ± 1 SEM. One way ANOVA with Newman-Keuls Multiple Comparison Test demonstrated that homogenates from KD fed mice were significantly greater than mice fed standard mouse chow (control and IR)(*p

Figure 5. Circulating levels of ketones in subjects consuming a 4:1 ketogenic diet while receiving concurrent radiation and chemotherapy in NSCLC and pancreas cancer as determined by clinical lab values (A, C, E) as well as by the fingerstick method (B, D, F)

Day 0 indicates the day subjects began the ketogenic diet. Subjects were typically in ketosis by day 3 of the diet. There was not a significant difference in weight between subjects who prematurely stopped the ketogenic diet as opposed to those who consumed the ketogenic throughout the course of radiation and chemotherapy (data not shown).

Figure 6. Serum glucose levels in subjects compliant with the 4:1 ketogenic diet

Figure 7. Lung and pancreas cancer subjects on a 4:1 ketogenic diet have significantly higher plasma protein carbonyl content when compared to their baseline pre-diet value

Blood from subjects with lung cancer (Lung-2, Lung-5, Lung-6) and pancreatic cancer (Panc-1) were drawn weekly and the carbonyl content was measured in plasma and normalized to pre-diet levels. Subjects were in confirmed ketosis and data points represent weekly values. *pt-test