A mid-treatment break and reassessment maintains tumor control and reduces toxicity in patients with hepatocellular carcinoma treated with stereotactic body radiation therapy.

BACKGROUND AND PURPOSE: Patients with hepatocellular carcinoma (HCC) commonly have underlying liver dysfunction with variable tolerance to liver stereotactic body radiation therapy (SBRT). We hypothesized that insertion of a 1-month mid-treatment break would allow us to adapt treatment to the individual patient response, thereby reducing toxicity without compromising local control (LC). MATERIALS AND METHODS: We analyzed HCC patients receiving 3-5 fraction SBRT at our institution from 2005 to 2017. Over this time, patients were offered enrollment on prospective trials assessing individualized adaptive SBRT. Based on normal tissue complication probability and modeling of changes in liver function following a 1-month treatment break between fractions 3 and 4, patients could receive a total of 3 or 5 fractions. Patients not on trial received 3 or 5 fractions without a break. Toxicity was defined as a ≥2 point rise in Child-Pugh (CP) score within 6 months of SBRT. RESULTS: 178 patients were treated with SBRT to 263 HCCs. Median follow-up was 23 months. 86 treatments had a 1-month break. 1-Year LC was 95.4%; this was not different between patients treated with or without a break (p = 0.14). Controlling for tumor size and dose a break was not associated with inferior LC (HR: 0.58, 95%CI: 0.1-3.34, p = 0.54). 54 patients experienced a ≥2 point rise in CP score. Controlling for the number of prior liver directed therapies and mean liver dose, a treatment break reduced the odds of toxicity (OR: 0.42, 95% CI: 0.17-1.03, p = 0.06). CONCLUSION: A one-month mid-treatment break and reassessment may reduce the odds of treatment related toxicity without compromising LC.

Treatment data and technical process challenges for practical big data efforts in radiation oncology.

The term Big Data has come to encompass a number of concepts and uses within medicine. This paper lays out the relevance and application of large collections of data in the radiation oncology community. We describe the potential importance and uses in clinical practice. The important concepts are then described and how they have been or could be implemented are discussed. Impediments to progress in the collection and use of sufficient quantities of data are also described. Finally, recommendations for how the community can move forward to achieve the potential of big data in radiation oncology are provided.

Statistical controversies in clinical research: building the bridge to phase II-efficacy estimation in dose-expansion cohorts.

BACKGROUND: Regulatory agencies and others have expressed concern about the uncritical use of dose expansion cohorts (DECs) in phase I oncology trials. Nonetheless, by several metrics-prevalence, size, and number-their popularity is increasing. Although early efficacy estimation in defined populations is a common primary endpoint of DECs, the types of designs best equipped to identify efficacy signals have not been established. METHODS: We conducted a simulation study of six phase I design templates with multiple DECs: three dose-assignment/adjustment mechanisms multiplied by two analytic approaches for estimating efficacy after the trial is complete. We also investigated the effect of sample size and interim futility analysis on trial performance. Identifying populations in which the treatment is efficacious (true positives) and weeding out inefficacious treatment/populations (true negatives) are competing goals in these trials. Thus, we estimated true and false positive rates for each design. RESULTS: Adaptively updating the MTD during the DEC improved true positive rates by 8-43% compared with fixing the dose during the DEC phase while maintaining false positive rates. Inclusion of an interim futility analysis decreased the number of patients treated under inefficacious DECs without hurting performance. CONCLUSION: A substantial gain in efficiency is obtainable using a design template that statistically models toxicity and efficacy against dose level during expansion. Design choices for dose expansion should be motivated by and based upon expected performance. Similar to the common practice in single-arm phase II trials, cohort sample sizes should be justified with respect to their primary aim and include interim analyses to allow for early stopping.

A natural BH3 mimetic induces autophagy in apoptosis-resistant prostate cancer via modulating Bcl-2-Beclin1 interaction at endoplasmic reticulum.

A natural BH3-mimetic, small-molecule inhibitor of Bcl-2, (-)-gossypol, shows promise in ongoing phase II and III clinical trials for human prostate cancer. In this study we show that (-)-gossypol preferentially induces autophagy in androgen-independent (AI) prostate cancer cells that have high levels of Bcl-2 and are resistant to apoptosis, both in vitro and in vivo, but not in androgen-dependent (AD) cells with low Bcl-2 and sensitive to apoptosis. The Bcl-2 inhibitor induces autophagy through blocking Bcl-2-Beclin1 interaction, together with downregulating Bcl-2, upregulating Beclin1, and activating the autophagic pathway. The (-)-gossypol-induced autophagy is dependent on Beclin1 and Atg5. Our results show for the first time that (-)-gossypol can also interrupt the interactions between Beclin1 and Bcl-2/Bcl-xL at endoplasmic reticulum, thus releasing the BH3-only pro-autophagic protein Beclin1, which in turn triggers the autophagic cascade. Oral administration of (-)-gossypol significantly inhibited the growth of AI prostate cancer xenografts, representing a promising new regimen for the treatment of human hormone-refractory prostate cancer with Bcl-2 overexpression. Our data provide new insights into the mode of cell death induced by Bcl-2 inhibitors, which will facilitate the rational design of clinical trials by selecting patients who are most likely to benefit from the Bcl-2-targeted molecular therapy.

Metabolic alterations: a biomarker for radiation-induced normal brain injury-an MR spectroscopy study.

PURPOSE: To assess if interval changes in metabolic status in normal cerebral tissue after radiation therapy (RT) can be detected by 2D CSI (chemical shift imaging) proton spectroscopy. MATERIALS AND METHODS: Eleven patients with primary brain tumors undergoing cranial radiation therapy (RT) were included. 2D-CSI MRS was performed before, during, and after the course of RT with the following parameters: TE/TR 144/1500 ms, field of view (FOV) 24, thickness 10 mm, matrix 16 x 16. The metabolic ratios choline/creatine (Cho/Cr), N-acetylaspartate (NAA)/Cr, and NAA/Cho in normal brain tissue were calculated. RESULTS: NAA/Cr and Cho/Cr were significantly decreased at week 3 during RT and at 1 month and 6 months after RT compared to values prior to RT (P < 0.01). The NAA/Cr ratio decreased by -0.19 +/- 0.05 (mean +/- standard error [SE]) at week 3 of RT, -0.14 +/- 0.06 at the last week of RT, -0.14 +/- 0.05 at 1 month after RT, and -0.30 +/- 0.08 at 6 months after RT compared to the pre-RT value of 1.43 +/- 0.04. The Cho/Cr ratio decreased by -0.27 +/- 0.05 at week 3 of RT, -0.11 +/- 0.05 at the last week of RT, -0.26 +/- 0.05 at 1 month after RT and -0.25 +/- 0.07 at 6 months after RT from the pre-RT value of 1.29 +/- 0.03. Changes in Cho/Cr were correlated with the interaction of the radiation dose and dose-volume at week 3 of RT, during the last week of RT (P < 0.005), and at 1 month after RT (P = 0.017). CONCLUSION: The results of this study suggest that MRS can detect early metabolic changes in normal irradiated brain tissue.

Radiation improves gene delivery by a novel transferrin-lipoplex nanoparticle selectively in cancer cells.

Selective gene transfer to tumor is critical in cancer gene therapy. We previously used ionizing radiation to improve adenovirus uptake in intrahepatic tumors but liver cytotoxicity associated with the viral administration still occurred. Here, we explore the potential of radiation for improving gene delivery by a virus-mimicking nanoparticle, transferrin (Tf)-cationic liposome-DNA complex (Tf-lipoplex). Transduction by Tf-lipoplex was highly efficient in various cell lines and further increased by radiation in a dose- and time-dependent manner. This radiation induction, which was associated with an increase in Tf-lipoplex uptake (3- to 4-folds in hepatocytes WB and lung cancer cells, LLC1), was absent when a Tf-deficient complex was used or abolished by the presence of free Tf, suggesting that Tf receptor (TfR) interaction is required for radiation induction. Radiation (10-20 Gy) markedly induced transgene (LacZ) expression in LLC1 xenografts (3.5- to 7.4-folds), correlating with increased plasmid content and TfR expression in irradiated tumors. Moreover, Tf-lipoplex-mediated gene expression was not observed in the liver or other normal tissues regardless of radiation treatment. We conclude that radiation improves Tf-lipoplex gene delivery selectively to tumor cells both in vitro and in vivo. Our findings may provide insight in developing ligand-specific lipoplex for molecularly targeted cancer gene therapy.

Role of epidermal growth factor receptor degradation in gemcitabine-mediated cytotoxicity.

We have recently reported that treatment with gemcitabine, a potent chemotherapeutic agent and radiation sensitizer, stimulates phosphorylation of the epidermal growth factor receptor (EGFR). Because phosphorylation of EGFR is known to precede receptor degradation, we hypothesized that gemcitabine treatment may also result in EGFR degradation. In two human head and neck cancer cell lines, UMSCC-1 and UMSCC-6, we demonstrated an approximately 80% decrease in total EGFR levels at 72 h after a 2-h treatment with 1 muM gemcitabine. Neither cisplatin nor 5-fluorouracil, which are used to treat head and neck cancer, caused EGFR degradation. EGFR downregulation did not occur at the level of transcription, as assessed by reverse transcription-polymerase chain reaction (RT-PCR), but instead occurred via phosphorylation and ubiquitination of the receptor along a proteosome/lysosome-mediated pathway. Inhibition of EGFR degradation, by either pretreatment with the EGFR tyrosine kinase inhibitor gefitinib or by exposure to the proteosome/lysosome inhibitor MG132, significantly reduced gemcitabine-induced cell death. These results suggest that EGFR degradation may be a novel mechanism for gemcitabine-mediated cell death. These findings also indicate that caution should be exercised when combining gemcitabine with agents that may prevent EGFR degradation, such as EGFR tyrosine kinase inhibitors administered in a suboptimal sequence or proteosome inhibitors.

The role of Bcl-X(S) in radiation sensitivity.

Bcl-X(S) is a pro-apoptosis member of the Bcl2 family that has been shown to induce cell death and enhance chemosensitivity. We have investigated the effect of Bcl-X(S) overexpression on radiation sensitivity. Using a tetracycline-repressible system, we found that removal of tetracycline for 16 h induced Bcl-X(S) and reduced the surviving fraction of NIH 3T3 cells to 25%. However, radiation sensitivity was not significantly affected by Bcl-X(S) expression; the mean inactivation doses for Bcl-X(S) repressed and Bcl-X(S) induced cells were 2.7 +/- 0.3 and 2.3 +/- 0.1 Gy, respectively. We conclude that Bcl-X(S) induces cell death without affecting radiation sensitivity. These results suggest that mitochondrial pathways to apoptosis may not have a significant role in survival after irradiation.

The potential of 5-fluorocytosine/cytosine deaminase enzyme prodrug gene therapy in an intrahepatic colon cancer model.

Colorectal cancer can metastasize to the liver, but remain liver confined for years. A critical step in developing treatments for intrahepatic cancer involves assessment in an orthotopic intrahepatic model. The purpose of this study was to develop a noninvasive intrahepatic tumor model to study the efficacy of 5-flucytosine/yeast cytosine deaminase (5FC/yCD)-based gene therapy for liver tumors. Luciferase expressing human colorectal carcinoma (HT-29luc) cells were generated by retroviral infection and implanted in the left liver lobe of nude mice. The bioluminescence was measured every week for a period of 1 month, then animals were killed and tumors were measured by calipers. After we found a correlation between photon counts and tumor size, animals were implanted with tumors composed of either 0%, 10%, or 100% yCD/HT-29luc cells, and treated with 5FC. Tumor bioluminescence was measured during treatment and tumor histology examined at the time of death. We found that 5FC caused significant regression of yCD expressing tumors. Furthermore, visible tumors at the time of death, which emitted little bioluminescence, contained little or no viable tumor. We then developed an adenoviral vector for yCD. Intraperitoneal administration of adenovirus containing yCD led to the production of yCD enzyme within intrahepatic tumors. These results suggest that (1) intrahepatic cancer responds to 5FC when cells express yCD; (2) the luciferin-luciferase system permits non-invasive real time imaging of viable intrahepatic cancer; and (3) this system can be used to carry out gene therapy experiments using yCD adenovirus.

The reproducibility of organ position using active breathing control (ABC) during liver radiotherapy.

PURPOSE: To evaluate the intrafraction and interfraction reproducibility of liver immobilization using active breathing control (ABC). METHODS AND MATERIALS: Patients with unresectable intrahepatic tumors who could comfortably hold their breath for at least 20 s were treated with focal liver radiation using ABC for liver immobilization. Fluoroscopy was used to measure any potential motion during ABC breath holds. Preceding each radiotherapy fraction, with the patient setup in the nominal treatment position using ABC, orthogonal radiographs were taken using room-mounted diagnostic X-ray tubes and a digital imager. The radiographs were compared to reference images using a 2D alignment tool. The treatment table was moved to produce acceptable setup, and repeat orthogonal verification images were obtained. The positions of the diaphragm and the liver (assessed by localization of implanted radiopaque intra-arterial microcoils) relative to the skeleton were subsequently analyzed. The intrafraction reproducibility (from repeat radiographs obtained within the time period of one fraction before treatment) and interfraction reproducibility (from comparisons of the first radiograph for each treatment with a reference radiograph) of the diaphragm and the hepatic microcoil positions relative to the skeleton with repeat breath holds using ABC were then measured. Caudal-cranial (CC), anterior-posterior (AP), and medial-lateral (ML) reproducibility of the hepatic microcoils relative to the skeleton were also determined from three-dimensional alignment of repeat CT scans obtained in the treatment position. RESULTS: A total of 262 fractions of radiation were delivered using ABC breath holds in 8 patients. No motion of the diaphragm or hepatic microcoils was observed on fluoroscopy during ABC breath holds. From analyses of 158 sets of positioning radiographs, the average intrafraction CC reproducibility (sigma) of the diaphragm and hepatic microcoil position relative to the skeleton using ABC repeat breath holds was 2.5 mm (range 1.8-3.7 mm) and 2.3 mm (range 1.2-3.7 mm) respectively. However, based on 262 sets of positioning radiographs, the average interfraction CC reproducibility (sigma) of the diaphragm and hepatic microcoils was 4.4 mm (range 3.0-6.1 mm) and 4.3 mm (range 3.1-5.7 mm), indicating a change of diaphragm and microcoil position relative to the skeleton over the course of treatment with repeat breath holds at the same phase of the respiratory cycle. The average population absolute intrafraction CC offset in diaphragm and microcoil position relative to skeleton was 2.4 mm and 2.1 mm respectively; the average absolute interfraction CC offset was 5.2 mm. Analyses of repeat CT scans demonstrated that the average intrafraction excursion of the hepatic microcoils relative to the skeleton in the CC, AP, and ML directions was 1.9 mm, 0.6 mm, and 0.6 mm respectively and the average interfraction CC, AP, and ML excursion of the hepatic microcoils was 6.6 mm, 3.2 mm, and 3.3 mm respectively. CONCLUSION: Radiotherapy using ABC for patients with intrahepatic cancer is feasible, with good intrafraction reproducibility of liver position using ABC. However, the interfraction reproducibility of organ position with ABC suggests the need for daily on-line imaging and repositioning if treatment margins smaller than those required for free breathing are a goal.

Phase I trial of radiation dose escalation with concurrent weekly full-dose gemcitabine in patients with advanced pancreatic cancer.

PURPOSE: The primary objective of this phase I trial was to determine the maximum-tolerated dose of radiation that could be delivered to the primary tumor concurrent with full-dose gemcitabine in patients with advanced pancreatic cancer. PATIENTS AND METHODS: Thirty seven patients with unresectable (n = 34) or incompletely resected pancreatic cancer (n = 3) were treated. Gemcitabine was administered as a 30-minute intravenous infusion at a dose of 1,000 mg/m(2) on days 1, 8, and 15 of a 28-day cycle. Radiation therapy was initiated on day 1 and directed at the primary tumor alone, without prophylactic nodal coverage. The starting radiation dose was 24 Gy in 1.6-Gy fractions. Escalation was achieved by increasing the fraction size in increments of 0.2 Gy, keeping the duration of radiation constant at 3 weeks. A second cycle of gemcitabine alone was intended after a 1-week rest. RESULTS: Two of six assessable patients experienced dose-limiting toxicity at the final planned dose level of the trial (42 Gy in 2.8-Gy fractions), one with grade 4 vomiting and one with gastric/duodenal ulceration. Two additional patients at this dose level experienced late gastrointestinal toxicity that required surgical management. CONCLUSION: The final dose investigated (42 Gy) is not recommended for further study considering the occurrence of both acute and late toxicity. However, a phase II trial of this novel gemcitabine-based chemoradiotherapy approach, at a radiation dose of 36 Gy in 2.4-Gy fractions, is recommended on the basis of tolerance, patterns of failure, and survival data.

The investigational new drug XK469 induces G(2)-M cell cycle arrest by p53-dependent and -independent pathways.

PURPOSE: XK469 (2-[4-(7-chloro-2-quinoxalinyloxy) phenoxy]propionic acid), a synthetic quinoxaline phenoxypropionic acid derivative, has broad activity against murine tumors and is entering Phase I clinical development as a topoisomerase IIbeta inhibitor. This study investigated the underlying molecular mechanism of XK469's effects on the cell cycle. EXPERIMENTAL DESIGN: Growth inhibition, cell cycle arrest, induction of p53 and p21 mRNA and protein, and cdc2 phosphorylation and kinase activity were studied in treated cells from the H460 lung cancer line and p21 and p53 knockout cells of the HCT 116 colon cancer line. RESULTS: XK469 arrested H460 cells at G(2)-M, which was associated with cdc2 phosphorylation and decreased cdc2 kinase activity. Moreover, XK469 stabilized p53 and subsequently increased p21(WAF1/CIP1). Furthermore, HCT116 p21(-/-) cells were less sensitive than wild-type cells to XK469-induced growth inhibition, but p53(+/+) and p53(-/-) cells were equally sensitive despite the absence of p21 induction in the p53(-/-) cells. CONCLUSIONS: When considered with published data, our study suggests a complex mechanism of XK469-mediated anticancer activity involving multiple pathways, including p53-dependent and -independent G(2)-M arrest via inactivation of cdc2-cyclin B1 kinase activity.

Profiling of cancer cells using protein microarrays: discovery of novel radiation-regulated proteins.

The advent of DNA microarray technology will likely have a major impact on the molecular classification and understanding of human cancer. Obtaining a global perspective of proteins expressed in cancer cells is considerably more challenging. Here we describe a microarray-based platform that can be used to measure protein levels and activities in a complex biological milieu such as a cellular lysate. Using a protein microarray made up of 1920 elements (146 distinct antibodies) we were able to monitor alterations of protein levels in LoVo colon carcinoma cells treated with ionizing radiation. The protein microarray approach revealed radiation-induced up-regulation of apoptotic regulators including p53, DNA fragmentation factor 40/caspase activated DNase, DNA fragmentation factor 45/inhibitor of caspase activated DNase, tumor necrosis factor-related apoptosis-inducing ligand, death receptor 5, decoy receptor 2, FLICE-like inhibitory protein, signal transducers and activators of transcription 1alpha, and uncoupling protein 2, among others. Consistent with this observation, an increased percentage of apoptosis was observed in irradiated LoVo cells. Interestingly, we also observed radiation-induced down-regulation of carcinoembryonic antigen, a prototypic cancer biomarker. Selected proteins assessed by microarray were validated by traditional immunoblotting. Taken together, our work suggests that protein/antibody microarrays will facilitate high-throughput proteomic studies of human cancer and carcinogenesis.

Recent advances in the use of radiosensitizing nucleosides.

Chemotherapeutic drugs that perturb nucleotide metabolism have the potential to produce substantial sensitization of tumor cells to radiation treatment. The clinical effectiveness of fluoropyrimidines as radiosensitizers has been proven in multiple randomized trials. The development of oral fluoropyrimidine formulations may allow protracted exposure without the need for indwelling intravenous lines and infusion pumps. These agents may also provide more selective radiosensitization and are likely to be widely incorporated into chemoradiotherapy regimens for patients with gastrointestinal malignancies. Gemcitabine has been well studied in the laboratory, with respect to mechanisms of radiosensitization and strategies that may increase the therapeutic index. Clinical trials based on these studies are now defining the role of this radiosensitizing nucleoside. Issues regarding the use oral fluoropyrimidines and gemcitabine need to be viewed in the context of both local and distant disease control, given the potential systemic activity of these agents.

Partial irradiation of the liver.

The use of three-dimensional radiotherapy (RT) and the prospective follow-up of patients for radiation-induced liver disease (RILD) have led to a more quantitative understanding of the partial organ tolerance of the liver compared with previous estimates based on clinical judgment alone. Parameters of both the Lyman normal tissue complication probability (NTCP) model and a local damage-organ injury (D-I) NTCP model have been fit to clinical data from patients who have received hepatic radiation. Based on analyses of over 180 patients, the liver exhibits a large volume effect and a low threshold volume for RILD. Mean liver dose is associated with RILD, and no cases of RILD have been reported in patients with a mean liver dose of less than 31 Gy. Most recent estimates of the partial liver tolerance to RT suggest that if less than 25% of the normal liver is treated with RT, then there may be no upper limit on dose associated with RILD. Estimates of the liver doses associated with a 5% risk of RILD for uniform irradiation of one third, two thirds, and the whole liver are 90 Gy, 47 Gy, and 31 Gy, respectively.