PURPOSE: Communication breakdowns in pediatric oncology can have negative consequences for patients and families. A detailed analysis of these negative encounters will support clinicians in anticipating and responding to communication breakdowns. METHODS: Semistructured interviews with 80 parents of children with cancer across three academic medical centers during treatment, survivorship, or bereavement. We analyzed transcripts using semantic content analysis. RESULTS: Nearly all parents identified negative communication experiences (n = 76). We identified four categories of contributors to negative experiences: individual (n = 68), team (n = 26), organization (n = 46), and greater health care system (n = 8). These experiences involved a variety of health care professionals across multiple specialties. Parents reported 12 personal consequences of communication breakdowns: emotional distress (n = 65), insufficient understanding (n = 48), decreased trust or confidence (n = 37), inconvenience (n = 36), medical harm (n = 23), decreased self-confidence (n = 17), decreased emotional support (n = 13), decreased engagement (n = 9), false hope (n = 9), decreased hope (n = 7), financial insult (n = 7), and decreased access to resources (n = 3). We identified five categories of supportive responses from clinicians: exploring (n = 8), acknowledging (n = 17), informing (n = 27), adapting (n = 27), and advocating (n = 18). Parents often increased their own advocacy on behalf of their child (n = 47). Parents also identified the need for parental engagement in finding solutions (n = 12). Finally, one parent suggested that clinicians should assume that communication will fail and develop contingency plans in advance. CONCLUSION: Communication breakdowns in pediatric oncology negatively affect parents and children. Clinicians should plan for communication breakdowns and respond by exploring, acknowledging, informing, adapting, advocating, and engaging parents in finding solutions.

Hepatocellular carcinoma (HCC) remains a deadly cancer, underscoring the need for relevant preclinical models. Male C3HeB/FeJ mice model spontaneous HCC with some hepatocarcinogenesis susceptibility loci corresponding to syntenic regions of human chromosomes altered in HCC. We tested other properties of C3HeB/FeJ tumors for similarity to human HCC. C3HeB/FeJ tumors were grossly visible at 4 months of age, with prevalence and size increasing until about 11 months of age. Histologic features shared with human HCC include hepatosteatosis, tumor progression from dysplasia to poorly differentiated, vascular invasion, and trabecular, oncocytic, vacuolar, and clear cell variants. More tumor cells displayed cytoplasmic APE1 staining versus normal liver. Ultrasound effectively detected and monitored tumors, with 85.7% sensitivity. Over 5000 genes were differentially expressed based on the GSE62232 and GSE63898 human HCC datasets. Of these, 158 and 198 genes, respectively, were also differentially expressed in C3HeB/FeJ. Common cancer pathways, cell cycle, p53 signaling and other molecular aspects, were shared between human and mouse differentially expressed genes. We established eigengenes that distinguish HCC from normal liver in the C3HeB/FeJ model and a subset of human HCC. These features extend the relevance and improve the utility of the C3HeB/FeJ line for HCC studies.

Hepatocellular carcinoma (HCC) is increasingly important in the United States as the incidence rate rose over the last 30 years. C3HeB/FeJ mice serve as a unique model to study HCC tumorigenesis because they mimic human HCC with delayed onset, male gender bias, a ~50% incidence, and susceptibility to tumorigenesis is mediated through multiple genetic loci. Because a human O6-methylguanine-DNA methyltransferase (hMGMT) transgene reduces spontaneous tumorigenesis in this model, we hypothesized that hMGMT would also protect from methylation-induced hepatocarcinogenesis. To test this hypothesis, wild type and hMGMT transgenic C3HeB/FeJ male mice were treated with two monofunctional alkylating agents: diethylnitrosamine (DEN) (0.025 µmol/g body weight) on day 12 of life with evaluation for glucose-6-phosphatase deficient (G6PD) foci at 16, 24, and 32 weeks or N-methyl-N-nitrosurea (MNU) (25 mg MNU/kg body weight) once monthly for 7 months starting at 3 months of age with evaluation for liver tumors at 12–15 months of age. No difference in abundance or size of G6PD foci was measured with DEN treatment. In contrast, it was unexpectedly found that MNU reduces liver tumor prevalence in wild type and hMGMT transgenic mice despite increased tumor prevalence in other tissues. hMGMT and MNU protections were additive, suggesting that MNU protects through a different mechanism, perhaps through the cytotoxic N7-alkylguanine and N3-alkyladenine lesions which have low mutagenic potential compared to O6-alkylguanine lesions. Together these results suggest that targeting the repair of cytotoxic lesions may be a good preventative for patients at high risk of developing HCC.

Bioluminescence imaging (BLI) of gene expression in live animals is a powerful method for monitoring development, tumor growth, infections, healing, and other progressive, long-term biological processes. BLI remains an effective approach for reducing the number of animals needed to monitor dynamic changes in gene activity because images can be captured repeatedly from the same animals. When examining these ongoing changes, it is sometimes necessary to remove rhythmic effects on the bioluminescence signal caused by the circadian clock’s daily modulation of gene expression. Furthermore, BLI using freely moving animals remains limited because the standard procedures can alter normal behaviors. Another obstacle with conventional BLI of animals is that luciferin, the firefly luciferase substrate, is usually injected into mice that are then imaged while anesthetized. Unfortunately, the luciferase signal declines rapidly during imaging as luciferin is cleared from the body. Alternatively, mice are imaged after they are surgically implanted with a pump or connected to a tether to deliver luciferin, but stressors such as this surgery and anesthesia can alter physiology, behavior, and the actual gene expression being imaged. Consequently, we developed a strategy that minimizes animal exposure to stressors before and during sustained BLI of freely moving unanesthetized mice. This technique was effective when monitoring expression of the Per1 gene that serves in the circadian clock timing mechanism and was previously shown to produce circadian bioluminescence rhythms in live mice. We used hairless albino mice expressing luciferase that were allowed to drink luciferin and engage in normal behaviors during imaging with cooled electron-multiplying-CCD cameras. Computer-aided image selection was developed to measure signal intensity of individual mice each time they were in the same posture, thereby providing comparable measurements over long intervals. This imaging procedure, performed primarily during the animal’s night, is compatible with entrainment of the mouse circadian timing system to the light cycle while allowing sampling at multi-day intervals to monitor long-term changes. When the circadian expression of a gene is known, this approach provides an effective alternative to imaging immobile anesthetized animals and can removing noise caused by circadian oscillations and body movements that can degrade data collected during long-term imaging studies.

Eighty percent of hepatocellular carcinoma (HCC) within the USA is diagnosed late when chemotherapy provides only 2 months increased survival. We fortuitously found that treatment with an alkylating agent, methylnitrosurea (MNU), prior to tumorigenesis reduced tumor prevalence later in life by ∼50% in C3HeB/FeJ male mice, a model of spontaneous HCC. A similar effect was observed with temozolomide (TMZ), a glioblastoma therapy that induces the same DNA damage at similar proportions to MNU. O6-methylguanine-DNA methyltransferase (MGMT) repairs the highly mutagenic and cytotoxic O6-methylguanine lesions induced by TMZ, and MGMT abundance influences therapy response in glioblastoma. MGMT is reduced in 1/3 of human HCC, leading us to hypothesize that TMZ could be effective in treating HCC. We proposed to combine TMZ with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, to maximize efficacy, reasoning that SAHA would allow TMZ greater access to DNA to induce greater levels of damage. We tested this drug combination in two human HCC cell lines, Huh7, with reduced MGMT expression, and SNU398, with higher expression. TMZ and SAHA synergistically reduced cell viability in both cell lines with multiple dose combinations. Based on this outcome, we tested the drugs in tumor-bearing C3HeB/FeJ mice. Surprisingly, DMSO treated solvent control mice showed reduced tumor burden. The DMSO treatment was repeated adding a sham control group and confirmed that DMSO significantly reduces tumor burden. The TMZ cohort also displayed a significantly lower tumor burden compared to the sham cohort, but no significant difference was found with SAHA alone or SAHA+TMZ. We previously showed that a hMGMT transgene protects from hepatocarcinogenesis in C3HeB/FeJ mice, but surprisingly, there is not additional protection when transgenic mice are treated with MNU. These results suggest that the more prevalent cytotoxic lesions induced by MNU and TMZ, e.g. N7-methylguanine, may be more important in HCC than O6-methylguanine. These lesions are recognized by methylpurine-DNA glycosylase (MPG) and repaired through base excision repair. Therefore, we directly tested the role of MPG in TMZ therapy through altered expression of MPG in Huh7 and SNU398 cell lines and showed that it does affect sensitivity to TMZ. Citation Format: Jessica A. Zavadil, Christi A. Walter. Improved therapeutics for hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3595.