Immune modulation associated with vascular endothelial growth factor (VEGF) blockade in patients with glioblastoma.

BACKGROUND: Vascular endothelial growth factor (VEGF), in addition to being pro-angiogenic, is an immunomodulatory cytokine systemically and in the tumor microenvironment. We previously reported the immunomodulatory effects of radiation and temozolomide (TMZ) in newly diagnosed glioblastoma. This study aimed to assess changes in peripheral blood mononuclear cell (PBMC) populations, plasma cytokines, and growth factor concentrations following treatment with radiation, TMZ, and bevacizumab (BEV). METHODS: Eleven patients with newly diagnosed glioblastoma were treated with radiation, TMZ, and BEV, following surgery. We measured immune-related PBMC subsets using multi-parameter flow cytometry and plasma cytokine and growth factor concentrations using electrochemiluminescence-based multiplex analysis at baseline and after 6 weeks of treatment. RESULTS: The absolute number of peripheral blood regulatory T cells (Tregs) decreased significantly following treatment. The lower number of peripheral Tregs was associated with a CD4+ lymphopenia, and thus, the ratio of Tregs to PBMCs was unchanged. The addition of bevacizumab to standard radiation and temozolomide led to the decrease in the number of circulating Tregs when compared with our prior study. There was a significant decrease in CD8+ cytotoxic and CD4+ recent thymic emigrant T cells, but no change in the number of myeloid-derived suppressor cells. Significant increases in plasma VEGF and placental growth factor (PlGF) concentrations were observed. CONCLUSIONS: Treatment with radiation, TMZ, and BEV decreased the number but not the proportion of peripheral Tregs and increased the concentration of circulating VEGF. This shift in the peripheral immune cell profile may modulate the tumor environment and have implications for combining immunotherapy with anti-angiogenic therapy.

Glypican-1 modulates the angiogenic and metastatic potential of human and mouse cancer cells.

Cells isolated from many types of human cancers express heparin-binding growth factors (HBGFs) that drive tumor growth, metastasis, and angiogenesis. The heparan sulfate proteoglycan glypican-1 (GPC1) is a coreceptor for HBGFs. Here we show that both cancer cell-derived and host-derived GPC1 are crucial for efficient growth, metastasis, and angiogenesis of human and mouse cancer cells. Thus downregulation of GPC1 in the human pancreatic cancer cell line PANC-1, using antisense approaches, resulted in prolonged doubling times and decreased anchorage-independent growth in vitro as well as attenuated tumor growth, angiogenesis, and metastasis when these cells were transplanted into athymic mice. Moreover, athymic mice that lacked GPC1 exhibited decreased tumor angiogenesis and metastasis following intrapancreatic implantation with either PANC-1 or T3M4 human pancreatic cancer cells and fewer pulmonary metastases following intravenous injection of murine B16-F10 melanoma cells. In addition, hepatic endothelial cells isolated from these mice exhibited an attenuated mitogenic response to VEGF-A. These data indicate that cancer cell- and host-derived GPC1 are crucial for full mitogenic, angiogenic, and metastatic potential of cancer cells. Thus targeting GPC1 might provide new avenues for cancer therapy and for the prevention of cancer metastasis.

Discovery of a novel molecule that regulates tumor growth and metastasis.

The heparan sulfate proteoglycan, Glypican-1 (GPC1), significantly impacts the growth of pancreatic cancer cells in vivo and markedly attenuates tumor angiogenesis and metastasis in athymic mice. Interestingly, both cancer cell-derived and host-derived GPC1 play an important role in tumor development and spread. These data suggest that GPC1 may be a valid therapeutic target for pancreatic cancer.

The mitogen-activated protein kinase pathway plays a critical role in regulating immunological properties of BRAF mutant cutaneous melanoma cells.

The advent of drugs targeting the mitogen-activated protein kinase (MAPK) pathway has markedly changed the treatment of advanced-stage melanoma harboring BRAF mutations. However, drug resistance, through mechanisms not well elucidated, often occurs. A better understanding of how melanoma-derived immunologically active molecules change in response to MAPK inhibition of BRAF mutated (BRAF) and BRAF wild type (BRAF) melanomas could help identify promising treatment combinations of small molecule inhibitors and immunotherapy. To this aim, we treated 13 BRAF and 13 BRAF mutated human melanoma cell lines with either a specific BRAF inhibitor or an MEK1/2 inhibitor and analyzed changes in the secretion of 42 selected cytokines, chemokines, and growth factors. We also measured changes in the expression levels of immunologically relevant melanoma cell surface markers. The BRAF melanomas showed minimal changes in response to the inhibitors, whereas the BRAF cell lines showed, on average, a significant decrease in IFNα2, interleukin-7, Fractalkine, GCSF, GRO, TGFα2, interleukin-8, and VEGF, as well as a reduction in pERK and pMEK protein levels, upon MAPK pathway blockade. BRAF inhibition in BRAF cell lines also resulted in significant changes in the expression of several surface markers including upregulation of ß2-microglobulin as well as a decrease in MIC A/B and TRAIL-R2. These results indicate that MAPK pathway inhibition leads to changes in the immunological properties of mutant BRAF melanoma cells and lends support for future studies aimed at designing effective treatment strategies that combine BRAF and MEK inhibition with immunotherapy.

Tumor talk: understanding the conversation between the tumor and its microenvironment.

It was once believed that tumor growth, progression, and metastasis were intrinsically driven by the tumor. Instead, recent research has demonstrated that a solid tumor is surrounded by a complex matrix of cells, particularly fibroblasts, which support and even promote tumor progression. This matrix of stromal cells, also known as the tumor microenvironment (TME), plays a critical role in cancer and may represent a novel therapeutic target. As such, understanding the complex nature of how the tumor initiates and maintains communication, or a "conversation", with the TME is the focus of current investigations. We have previously shown that the most prevalent mutation found in melanoma, BRAFV600E, results in increased expression and secretion of several growth factors, cytokines, and matrix metalloproteinases, including factors that are able to activate fibroblasts. Targeted inhibition of the BRAFV600E mutation resulted in a decrease of secreted proteins into the TME and suggests that targeting the tumor also modifies the TME. Overall, this work, in combination with several additional studies discussed herein, provides strong evidence for the potential therapeutic benefits of targeting the TME, particularly signaling pathways within the fibroblasts, in conjunction with the tumor. This approach may result in extended drug resistance free survival, reduction in metastasis, and improved cytotoxic drug delivery.

Regulatory T cells are not a strong predictor of survival for patients with glioblastoma.

BACKGROUND: Regulatory T cells (Tregs) are potentially prognostic indicators in patients with glioblastoma. If differences in frequency of Tregs in tumor or blood account for substantial variation in patient survival, then reliably measuring Tregs may enhance treatment selection and improve outcomes. METHODS: We measured Tregs and CD3+ T cells in tumors and blood from 25 patients with newly diagnosed glioblastoma. Tumor-infiltrating Tregs and CD3+ T cells, measured by quantitative DNA demethylation analysis (epigenetic qPCR) and by immunohistochemistry, and peripheral blood Treg proportions measured by flow cytometry were correlated with patient survival. Additionally, we analyzed data from The Cancer Genome Atlas (TCGA) to correlate the expression of Treg markers with patient survival and glioblastoma subtypes. RESULTS: Tregs, as measured in tumor tissue and peripheral blood, did not correlate with patient survival. Although there was a correlation between tumor-infiltrating Tregs expression by epigenetic qPCR and immunohistochemistry, epigenetic qPCR was more sensitive and specific. Using data from TCGA, mRNA expression of Forkhead box protein 3 (FoxP3) and Helios and FoxP3 methylation level did not predict survival. While the classical glioblastoma subtype corresponded to lower expression of Treg markers, these markers did not predict survival in any of the glioblastoma subtypes. CONCLUSIONS: Although immunosuppression is a hallmark of glioblastoma, Tregs as measured in tissue by gene expression, immunohistochemistry, or demethylation and Tregs in peripheral blood measured by flow cytometry do not predict survival of patients. Quantitative DNA demethylation analysis provides an objective, sensitive, and specific way of identifying Tregs and CD3+ T cells in glioblastoma.

Protein requirements of infants: a reexamination of concepts and approaches.

Estimates of protein requirements of infants aged 3-4 mo by FAO/WHO/UNU (1.47 +/- 0.26 g.kg-1.d-1 as crude protein, N X 6.25) are judged to be overestimates. From simulation analyses we suggest that 1.1 +/- 0.1 - 0.2 g.kg-1.d-1 is a more reasonable estimate. This is consistent with statements that 1) breast milk that provides an average of 16 g protein/1000 kcal or a fixed-composition formula that contains 17 g protein/1000 kcal is adequate for essentially all such infants and 2) average protein intakes from that milk or formula would be approximately 1.65 or 1.75 g.kg-1.d-1, close to current average requirements estimates. It appears that there has been a difference in the concepts of requirement usually applied to infants and to adults and a systematic misinterpretation of breast-milk data in estimating requirements. A plea is issued for the application of epidemiologic approaches as a part of requirement estimation.

Dialysis utilization in the Toronto region from 1981 to 1992. Toronto Region Dialysis Committee.

OBJECTIVE: To analyse trends in the demand for and supply of dialysis in the Toronto region and to determine whether planned dialysis expansion will be sufficient to provide for the projected growth of the dialysis population. DESIGN: Descriptive analysis of data reported to the Toronto Region Dialysis Registry between 1981 and 1992, compared with provincial and national equivalents. SETTING: All secondary and tertiary care dialysis programs in the Toronto region participating in the registry. PATIENTS: All 504 existing patients enrolled in dialysis programs in 1981 and all 3794 new patients entering programs from 1982 to 1992. Patients with acute renal failure were excluded. MAIN OUTCOME MEASURES: Demand for dialysis: dialysis population at year end, age distribution, crude mortality rate and transplant rate. Supply of resources: distribution of modality (hemodialysis or peritoneal dialysis), number of patients treated per hemodialysis station, number of hemodialysis stations per million population and hemodialysis utilization index (actual/budgeted treatments). RESULTS: During the study period the number of dialysis patients in the Toronto region went from 504 to 1422, for an increase of 182.1%. The average rate of growth was 9.8% per year. Of the total increment of 918 patients from 1981 to 1992, 390 (42.5%) were 65 years of age or more; none the less, the average annual crude mortality rate remained relatively constant, at 13.8% to 17.3%. The transplantation rate declined from a peak of 20.2% in 1982 to 7.8% in 1992. During the study period the Toronto region had much higher numbers of dialysis patients, and hemodialysis patients, per hemodialysis station than the rest of Ontario or Canada. The region's hemodialysis utilization index was 101% in 1991 and 102% in 1992; the index in individual hospitals varied from 98% to 124% (85% was considered optimal). CONCLUSIONS: The growth of the dialysis population in the Toronto region has caused a critical shortage of resources. This trend can be attributed mainly to a decrease in the transplantation rate and an increase in the number of elderly patients entering dialysis programs, combined with insufficient funding for expansion of facilities. Continuation of this trend would be expected to limit universal access to this expensive, but life-sustaining therapy.

Evaluation of methods used to assess dietary intake: simulation analyses.

The choice of dietary methodology can affect the ability to detect and describe the relationship between dietary sodium intake and blood pressure. This is illustrated in this paper through the use of simulation modelling of the effect of using different dietary methods (food recalls or records covering different numbers of days, food frequency questionnaire estimates of a single diet component) and using urinary excretion as a proxy for intake. Both epidemiologic studies and experimental interventions are simulated. Although the data base used was simulated rather than real, an attempt was made to keep it realistic in relation to what might be seen in actual populations. From these analyses it can be inferred that with appropriate choice of methodology and study design, even low order relationships between sodium intake and blood pressure should be detectable. At a more general level, it may be concluded that while there is no perfect dietary methodology, there are preferred methodologies for defined purposes.

Racial differences in survival of patients on dialysis.

BACKGROUND: Recent studies have documented racial differences in the crude mortality rates of patients on dialysis. However, proper interpretation of these findings requires adjustment for potential confounders and comorbid risk factors between the racial groups. METHODS: We examined the clinical data on 3752 Caucasian patients, 451 Southeast Asian patients, 322 South Asian patients, and 319 black patients who were treated with hemodialysis or peritoneal dialysis under a Universal Health Care system in Toronto and prospectively followed between 1981 and 1995. In all patients, a number of comorbid risk factors for survival was assessed at the start of dialysis and was reassessed with their outcome status (that is, continued dialysis, transplantation, death, or loss to follow-up) at least every six months. Cox proportional hazards analysis was used to fit multivariate models predicting patient survival. Pairwise comparisons of the relative hazards of death between the racial groups were performed after stratifying for cardiovascular disease, diabetes mellitus, and hypertension at the start of dialysis, and were adjusted for differences in other comorbid risk factors. RESULTS: The risk of death in Caucasian patients was significantly increased when compared with Southeast Asian patients, South Asian patients, and black patients [multivariate relative hazards (95% CI): 1.63 (1.36 to 1.97), 1.36 (1.07 to 1.73), 1.34 (1.07 to 1.67), respectively]. Additionally, we detected an interaction between race and cigarette smoking (P < 0. 004), suggesting that in the dialysis patients who smoked, whites had a higher mortality risk compared with non-whites. CONCLUSIONS: Differences in patient survival on dialysis exist between racial groups. However, the genetic and environmental determinants that underlie these differences are presently unknown.

Autosomal dominant polycystic kidney disease in Toronto.

This study describes the Toronto, Ontario experience with autosomal dominant polycystic kidney disease (ADPKD). Patients were divided into three groups: Group 1, 19 families studied with genetic markers; Group 2, 80 pre-dialysis ADPKD patients followed by Toronto nephrologists in whom the incidence of non-renal complications and the mean age of onset of symptomatology is documented; Group 3, 4,449 individuals who entered end-stage renal failure (ESRF) in the Toronto region between the years 1981 and 1992, 320 with ADPKD and 4129 with other diseases. In this third group age of onset of ESRF, frequency, age and cause of death is compared between ADPKD and non-ADPKD. ADPKD caused by a gene different from that linked to chromosome 16 short-arm probes occurred at a frequency of between 8 and 17%. Incidence of hepatic cysts in ADPKD was similar to that of previous series, other organ involvement was underdiagnosed without deliberate screening, and incidence of symptomatic intracranial aneurysm was 1.25%. A 5% excess of patients with ADPKD died of cerebro-vascular accident. Years of survival after ESRF measured by life table analysis was significantly greater for ADPKD patients than for non-ADPKD patients. A high frequency of death due to infection still exists in ADPKD despite the reduction of invasive procedures in diagnosis and treatment, and despite the presumably improved recent methods of managing infection. The average age of onset of ESRF has been delayed by over six years, and average age of death of ADPKD patients at 63.9 years-old by 12.4 years since 1960.

Computational and experimental analysis reveals a novel Src family kinase in the C. elegans genome.

MOTIVATION: The complete genomes of a number of organisms have already been sequenced. However, the vast majority of annotated genes are derived by gene prediction methods. It is important to not only validate the predicted coding regions but also to identify genes that may have been missed by these programs. METHODS: We searched the entire C.elegans genomic sequence database maintained by the Sanger Center using human c-Src sequence in a TBLASN search. We have confirmed one of the predicted regions by isolation of a cDNA and carried out a phylogenetic analysis of Src kinase family members in the worm, fly and several vertebrate species. RESULTS: Our analysis identified a novel tyrosine kinase in the C.elegans genome that contains functional features typical of the Src family kinases that we have designated as Src-1. The open reading frame contains a conserved N-terminal myristoylation site and a tyrosine residue within the C-terminus that is crucial for regulating the activity of Src kinases. Our phylogenetic analysis of Src family members from C. elegans, Drosophila and other higher organisms revealed a relationship among Src kinases from C. elegans and Drosophila.