Computer simulation of glioma growth and morphology.

Despite major advances in the study of glioma, the quantitative links between intra-tumor molecular/cellular properties, clinically observable properties such as morphology, and critical tumor behaviors such as growth and invasiveness remain unclear, hampering more effective coupling of tumor physical characteristics with implications for prognosis and therapy. Although molecular biology, histopathology, and radiological imaging are employed in this endeavor, studies are severely challenged by the multitude of different physical scales involved in tumor growth, i.e., from molecular nanoscale to cell microscale and finally to tissue centimeter scale. Consequently, it is often difficult to determine the underlying dynamics across dimensions. New techniques are needed to tackle these issues. Here, we address this multi-scalar problem by employing a novel predictive three-dimensional mathematical and computational model based on first-principle equations (conservation laws of physics) that describe mathematically the diffusion of cell substrates and other processes determining tumor mass growth and invasion. The model uses conserved variables to represent known determinants of glioma behavior, e.g., cell density and oxygen concentration, as well as biological functional relationships and parameters linking phenomena at different scales whose specific forms and values are hypothesized and calculated based on in vitro and in vivo experiments and from histopathology of tissue specimens from human gliomas. This model enables correlation of glioma morphology to tumor growth by quantifying interdependence of tumor mass on the microenvironment (e.g., hypoxia, tissue disruption) and on the cellular phenotypes (e.g., mitosis and apoptosis rates, cell adhesion strength). Once functional relationships between variables and associated parameter values have been informed, e.g., from histopathology or intra-operative analysis, this model can be used for disease diagnosis/prognosis, hypothesis testing, and to guide surgery and therapy. In particular, this tool identifies and quantifies the effects of vascularization and other cell-scale glioma morphological characteristics as predictors of tumor-scale growth and invasion.

Computer simulation of glioma growth and morphology.

Despite major advances in the study of glioma, the quantitative links between intra-tumor molecular/cellular properties, clinically observable properties such as morphology, and critical tumor behaviors such as growth and invasiveness remain unclear, hampering more effective coupling of tumor physical characteristics with implications for prognosis and therapy. Although molecular biology, histopathology, and radiological imaging are employed in this endeavor, studies are severely challenged by the multitude of different physical scales involved in tumor growth, i.e., from molecular nanoscale to cell microscale and finally to tissue centimeter scale. Consequently, it is often difficult to determine the underlying dynamics across dimensions. New techniques are needed to tackle these issues. Here, we address this multi-scalar problem by employing a novel predictive three-dimensional mathematical and computational model based on first-principle equations (conservation laws of physics) that describe mathematically the diffusion of cell substrates and other processes determining tumor mass growth and invasion. The model uses conserved variables to represent known determinants of glioma behavior, e.g., cell density and oxygen concentration, as well as biological functional relationships and parameters linking phenomena at different scales whose specific forms and values are hypothesized and calculated based on in vitro and in vivo experiments and from histopathology of tissue specimens from human gliomas. This model enables correlation of glioma morphology to tumor growth by quantifying interdependence of tumor mass on the microenvironment (e.g., hypoxia, tissue disruption) and on the cellular phenotypes (e.g., mitosis and apoptosis rates, cell adhesion strength). Once functional relationships between variables and associated parameter values have been informed, e.g., from histopathology or intra-operative analysis, this model can be used for disease diagnosis/prognosis, hypothesis testing, and to guide surgery and therapy. In particular, this tool identifies and quantifies the effects of vascularization and other cell-scale glioma morphological characteristics as predictors of tumor-scale growth and invasion.

The biology and mechanism of chemoresistance of brain metastases

Primary brain neoplasms and metastases to the brain are generally resistant to systemic chemotherapy. The purpose of theses studies was to determine the mechanism(s) for this resistance. We have developed a model to study the biology of brain metastasis by injecting metastatic K1735 melanoma cells into the carotid artery of syngeneic C3H/HeN or nude mice. The resulting brain lesions are produced in the parenchyma of the brain. Mice with subcutaneous or brain melanoma lesions were treated intravenously with doxorubicin (DXR) (7 mg/kg). The s.c. lesions regressed in most of the mice whereas no therapeutic benefits were produced in mice with brain metastases. The intravenous injection of sodium fluorescine revealed that the blood-brain barrier (BBB) is intact in and around brain metastases smaller than 0.2 mm$\sp2$ but not in larger lesions, implying that the BBB is not a major obstacle for chemotherapy of brain metastases.^ Western blot and FACS analyses revealed that K1735 melanoma brain metastases expressed high levels of P-glycoprotein (P-gp) as compared to s.c. tumors or in vitro cultures. Similarly, K1735 cells from brain metastases expressed higher levels of mdrl mRNA. This increased expression of mdrl was due to adaptation to the local brain environment. We base this conclusion on the results of two studies. First, K1735 cells from brain metastases cultured for 7 days lost the high mdrl expression. Second, in crossover experiments K1735 cells from s.c. tumors (low mdrl expression) implanted into the brain exhibited high levels of mdrl expression whereas cells from brain metastases implanted s.c. lost the high level mdrl expression.^ To investigate the mechanism by which the brain environment upregulates mdrl expression of the K1735 cells we first studied the regulation of P-gp in brain endothelial cells. Since astrocytes are closely linked with the BBB we cocultured brain endothelial cells for 3 days with astrocytes. These endothelial cells expressed high levels of mdrl mRNA and protein whereas endothelial cells cocultured with endothelial cells or fibroblasts did not. We next cocultured K1735 melanoma cells with astrocytes. Here again, astrocytes (but not fibroblasts or tumor cells) uprelated the mdrl expression in K1735 tumor cells. This upregulation inversely correlated with intracellular drug accumulation and sensitivity to DXR.^ The data conclude that the resistance of melanoma brain metastases to chemotherapy is not due to an intact BBB but to the upregulation of the mdrl gene by the organ microenvironment, i.e., the astrocytes. This epigenetic mediated resistance to chemotherapy has wide implications for the therapy of brain metastases. ^

Predictive oncology: a review of multidisciplinary, multiscale in silico modeling linking phenotype, morphology and growth.

Empirical evidence and theoretical studies suggest that the phenotype, i.e., cellular- and molecular-scale dynamics, including proliferation rate and adhesiveness due to microenvironmental factors and gene expression that govern tumor growth and invasiveness, also determine gross tumor-scale morphology. It has been difficult to quantify the relative effect of these links on disease progression and prognosis using conventional clinical and experimental methods and observables. As a result, successful individualized treatment of highly malignant and invasive cancers, such as glioblastoma, via surgical resection and chemotherapy cannot be offered and outcomes are generally poor. What is needed is a deterministic, quantifiable method to enable understanding of the connections between phenotype and tumor morphology. Here, we critically assess advantages and disadvantages of recent computational modeling efforts (e.g., continuum, discrete, and cellular automata models) that have pursued this understanding. Based on this assessment, we review a multiscale, i.e., from the molecular to the gross tumor scale, mathematical and computational "first-principle" approach based on mass conservation and other physical laws, such as employed in reaction-diffusion systems. Model variables describe known characteristics of tumor behavior, and parameters and functional relationships across scales are informed from in vitro, in vivo and ex vivo biology. We review the feasibility of this methodology that, once coupled to tumor imaging and tumor biopsy or cell culture data, should enable prediction of tumor growth and therapy outcome through quantification of the relation between the underlying dynamics and morphological characteristics. In particular, morphologic stability analysis of this mathematical model reveals that tumor cell patterning at the tumor-host interface is regulated by cell proliferation, adhesion and other phenotypic characteristics: histopathology information of tumor boundary can be inputted to the mathematical model and used as a phenotype-diagnostic tool to predict collective and individual tumor cell invasion of surrounding tissue. This approach further provides a means to deterministically test effects of novel and hypothetical therapy strategies on tumor behavior.

FoxM1B transcriptionally regulates vascular endothelial growth factor expression and promotes the angiogenesis and growth of glioma cells.

We previously found that FoxM1B is overexpressed in human glioblastomas and that forced FoxM1B expression in anaplastic astrocytoma cells leads to the formation of highly angiogenic glioblastoma in nude mice. However, the molecular mechanisms by which FoxM1B enhances glioma angiogenesis are currently unknown. In this study, we found that vascular endothelial growth factor (VEGF) is a direct transcriptional target of FoxM1B. FoxM1B overexpression increased VEGF expression, whereas blockade of FoxM1 expression suppressed VEGF expression in glioma cells. Transfection of FoxM1 into glioma cells directly activated the VEGF promoter, and inhibition of FoxM1 expression by FoxM1 siRNA suppressed VEGF promoter activation. We identified two FoxM1-binding sites in the VEGF promoter that specifically bound to the FoxM1 protein. Mutation of these FoxM1-binding sites significantly attenuated VEGF promoter activity. Furthermore, FoxM1 overexpression increased and inhibition of FoxM1 expression suppressed the angiogenic ability of glioma cells. Finally, an immunohistochemical analysis of 59 human glioblastoma specimens also showed a significant correlation between FoxM1 overexpression and elevated VEGF expression. Our findings provide both clinical and mechanistic evidence that FoxM1 contributes to glioma progression by enhancing VEGF gene transcription and thus tumor angiogenesis.

FoxM1B regulates NEDD4-1 expression, leading to cellular transformation and full malignant phenotype in immortalized human astrocytes.

Our recent studies have shown that the FoxM1B transcription factor is overexpressed in human glioma tissues and that the level of its expression correlates directly with glioma grade. However, whether FoxM1B plays a role in the early development of glioma (i.e., in transformation) is unknown. In this study, we found that the FoxM1B molecule causes cellular transformation and tumor formation in normal human astrocytes (NHA) immortalized by p53 and pRB inhibition. Moreover, brain tumors that arose from intracranial injection of FoxM1B-expressing immortalized NHAs displayed glioblastoma multiforme (GBM) phenotypes, suggesting that FoxM1B overexpression in immortalized NHAs not only transforms the cells but also leads to GBM formation. Mechanistically, our results showed that overexpression of FoxM1B upregulated NEDD4-1, an E3 ligase that mediates the degradation and downregulation of phosphatase and tensin homologue (PTEN) in multiple cell lines. Decreased PTEN in turn resulted in the hyperactivation of Akt, which led to phosphorylation and cytoplasmic retention of FoxO3a. Blocking Akt activation with phosphoinositide 3-kinase/Akt inhibitors inhibited the FoxM1B-induced transformation of immortalized NHAs. Furthermore, overexpression of FoxM1B in immortalized NHAs increased the expression of survivin, cyclin D1, and cyclin E, which are important molecules for tumor growth. Collectively, these results indicate that overexpression of FoxM1B, in cooperation with p53 and pRB inhibition in NHA cells, promotes astrocyte transformation and GBM formation through multiple mechanisms.

Relationship of hospital volume to patient mortality, length of stay and total costs in patients receiving brain surgery for cancer: A nationwide analysis

Better morbidity and mortality outcomes associated with increased hospital procedural volume have been demonstrated across a number of different medical procedures. Existence of such a volume-outcome relationship is posited to lead to increased specialization of care, such that patients requiring specific procedures are funneled to physicians and hospitals that achieve a minimum volume of such procedures each year. In this study, the 2009 Nationwide Inpatient Sample is used to examine the relationship between hospital volume and patient outcome among patients undergoing procedures related to malignant brain cancer. Multiple regression models were used to examine the impact of hospital volume on length of inpatient stay and cost of inpatient stay; logistic regression was used to examine the impact of hospital volume on morbidity. Hospital volume was found to be a significant predictor of both length of stay and cost of stay. Hospital volume was associated with a lower length of stay, but was also associated with increased costs. Hospital volume was not found to be a statistically significant predictor of morbidity, though less than three percent of this sample died while in the hospital. Volume is indeed a significant predictor of outcome for procedures related to brain malignancies, though further research regarding the cost of such procedures is recommended.^

The role of deregulated E2F1 expression in brain tumors

Mutations disabling the retinoblastoma (Rb) pathway are among the most common in human cancers, including brain cancer. These mutations promote tumor development through deregulated control of the E2F family of transcription factors. E2F1 belongs to a class of E2F's identified as transcriptional activators and involved in the G1/S phase transition of the cell. However, E2F-1 presents with a paradox as it is considered to have membership in two gene classes, functioning as both an oncogene and a tumor suppressor. This unusual trait generates a degree of uncertainty on the role that E2F1 plays in the development or maintenance of any given tumor. Here we show that E2F1 functions as an oncogene in brain tumors through the generation of mice engineered to overexpress E2F1 specifically within glial cells and neuronal progenitors as directed by the GFAP promoter. Mice carrying the transgene develop with high penetrance a phenotype characterized by neurological deficits including paresia, ataxia, head tilt and seizures. MRI imagining of the tgE2F1 mice reveals a low incidence of mild hydrocephalus, and most notably, histological analysis demonstrates that 25% of tgE2F1 mice present with the spontaneous formation of malignant brain tumors. Overall these neoplasms show histological features from a wide range of aggressive brain cancers including medulloblastoma, choroid plexus carcinoma, primary neuroectodermic tumor and malignant gliomas. Isolation and characterization of astrocytes from the tgE2F1 animal reveals a highly proliferative population of cells with 55% ± 2.5 of the tgE2F1astrocytes, 35% ± 3.4 normal mouse astrocytes in S-phase and the acquired capacity to grow in anchorage independent conditions. Additionally tgE2F1 astrocytes show an aberrant phenotype with random chromosomal fusions and nearly all cells demonstrating polyploidy. Taken together, this model forces a comparison to human brain tumor formation. Mouse age as related to tumoral mimics the human scenario with juvenile tgE2F1 mice presenting embryonal tumors typically identified in children, and older tgE2F1 mice demonstrating gliomas. In this regard, this study suggests a global role for E2F1 in the formation and maintenance of multilineage brain tumors, irrefutably establishing E2F1 as an oncogene in the brain. ^

Genetic epidemiology of childhood brain tumors

A rare familial cancer syndrome involving childhood brain tumors (CBT), breast cancer, sarcomas and an array of other tumors has been described (Li and Fraumeni 1969, 1975, 1982, 1987). A survey of CBT identified through the Connnecticut Tumor Registry in 1984 revealed a high frequency of CBT, leukemia and other childhood cancer in siblings of CBT patients (Farwell and Flannery, 1984). Other syndromes such as neurofibromatosis and nevoid basal cell carcinoma syndrome have also been associated with CBT; however, no systematic family studies have been conducted to determine the extent to which cancer aggregates in family members of CBT patients. This family study was designed to determine the frequency of cancer aggregation overall or at specific sites, to determine the frequency of known or potentially hereditary syndromes in families of CBT patients, and to determine a genetic model to characterize familial cancer syndromes and to identify specific kindreds to which such a model(s) might apply. This study includes 244 confirmed CBT patients referred to the University of Texas M. D. Anderson Cancer Center between the years 1944 and 1983, diagnosed under the age of 15 years and resident in the U.S. or Canada. Family histories were obtained on the proband's first (parents, siblings and offspring) and second degree (proband's aunts, uncles and grandparents) relatives following sequential sampling scheme rules. To determine if cancer aggregates in families, we compared the cancer experience in the population to that expected in the general population using Connecticut Tumor Registry calendar year, age, race and sex-specific rates. The standardized incidence ratio (SIR) for cancer overall was 0.91 (41 observed (O) and 44.94 expected (E); 95% Confidence Interval (CI) = 0.65-1.24). We observed a significant excess of colon cancer among the proband's first degree relatives (O/E = 5/1.64; 95% CI = 1.01-7.65), in particular those under age 45 year. Segregation analysis showed evidence for multifactorial inheritance in the small percentage (N = 5) of the families. ^

Identification of genetic risk factors for cerebellar mutism in pediatric brain tumor patients

Following posterior fossa surgery for resection of childhood medulloblastoma and primitive neuroectodermal tumor (M/PNET), cerebellar mutism (CM) may develop. This is a condition of absent or diminished speech in a conscious patient with no evidence of oral apraxia, which can be accompanied by other symptoms of the posterior fossa syndrome complex, which includes ataxia and hypotonia. Little is known about the etiology. Therefore, we conducted a SNP, gene, and pathway-level analysis to assess the role of host genetic variation on the risk of CM in M/PNET subjects following treatment. Cases (n= 20) and controls (n= 53) were recruited from the Childhood Cancer Epidemiology and Prevention Center, in Houston, TX. DNA samples were genotyped using the Illumina Human 1M Quad SNP chip. Ten pathways were identified from logistic regression used to identify the marginal effect of each SNP on CM risk. The minP test was conducted to identify associations between SNPs categorized to genes and CM risk. Pathways were assessed to determine if there was a significant enrichment of genes in the pathway compared to all other pathways. There were 78 genes that reached the threshold of min P ≤0.05 in 948 genes. The Neurotoxicity pathway was the most significant pathway after adjusting for multiple comparisons (q=0.040 and q=0.005, using Fisher's exact test and a test of proportions, respectively). Most genes within the Neurotoxicity pathway that reached a threshold of minP ≤0.05 were known to have an apoptosis function, possibly inducing neuronal apoptosis in the dentatothalamocortical pathway, and may be important in CM etiology in this population. This is the first study to assess the potential role of genetic risk factors on CM. As an exploratory study, these results should be replicated in a larger sample. ^

Expression of p53, {\it * ras\/} and PCNA in human colonic neoplasms: Possible biomarkers of malignant potential

Colorectal cancer is a leading cause of cancer mortality and early detection can significantly improve the clinical outcome. Most colorectal cancers arise from benign neoplastic lesions recognized as adenomas. Only a small percentage of all adenomas will become malignant. Thus, there is a need to identify specific markers of malignant potential. Studies at the molecular level have demonstrated an accumulation of genetic alterations, some hereditary but for the most occurring in somatic cells. The most common are the activation of ras, an oncogene involved in signal transduction, and the inactivation of p53, a tumor suppressor gene implicated in cell cycle regulation. In this study, 38 carcinomas, 95 adenomas and 20 benign polyps were analyzed by immunohistochemistry for the abnormal expression of p53 and ras proteins. An index of cellular proliferation was also measured by labeling with PCNA. A general overexpression of p53 was immunodetected in 66% of the carcinomas, while 26% of adenomas displayed scattered individual positive cells or a focal high concentration of positive cells. This later was more associated with severe dysplasia. Ras protein was detected in 37% of carcinomas and 32% of adenomas mostly throughout the tissue. p53 immunodetection was more frequent in adenomas originating in colons with synchronous carcinomas, particularly in patients with familial adenomatous polyposis and it may be a useful marker in these cases. Difference in the frequency of p53 and ras alterationbs was related to the location of the neoplasm. Immunodetection of p53 protein was correlated to the presence of a mutation in p53 gene at exon 7 and 5 in 4/6 carcinomas studied and 2 villous adenomas. Thus, we characterized in adenomas the abnormal expression of two proteins encoded by the most commonly altered genes in colorectal cancer. p53 alteration appears to be more specifically associated with transition to malignancy than ras. By using immunohistochemistry, a technique that keeps the architecture of the tissue intact, it was possible to correlate these alterations to histopathological characteristics that were associated with higher risks for transformation: villous content, dysplasia and size of adenoma. ^