Nicole R. Parker

Pancreatic stellate cells: partners in crime with pancreatic cancer cells.

Pancreatic stellate cells (PSC) produce the stromal reaction in pancreatic cancer, but their role in cancer progression is not fully elucidated. We examined the influence of PSCs on pancreatic cancer growth using (a) an orthotopic model of pancreatic cancer and (b) cultured human PSCs (hPSC) and human pancreatic cancer cell lines MiaPaCa-2 and Panc-1. Athymic mice received an intrapancreatic injection of saline, hPSCs, MiaPaCa-2 cells, or hPSCs + MiaPaCa-2. After 7 weeks, tumor size, metastases, and tumor histology were assessed. In vitro studies assessed the effect of cancer cell secretions on PSC migration and the effect of hPSC secretions on cancer cell proliferation, apoptosis, and migration. Possible mediators of the effects of hPSC secretions on cancer cell proliferation were examined using neutralizing antibodies. Compared with mice receiving MiaPaCa-2 cells alone, mice injected with hPSCs + MiaPaCa-2 exhibited (a) increased tumor size and regional and distant metastasis, (b) fibrotic bands (desmoplasia) containing activated PSCs within tumors, and (c) increased tumor cell numbers. In vitro studies showed that, in the presence of pancreatic cancer cells, PSC migration was significantly increased. Furthermore, hPSC secretions induced the proliferation and migration, but inhibited the apoptosis, of MiaPaCa-2 and Panc-1 cells. The proliferative effect of hPSC secretions on pancreatic cancer cells was inhibited in the presence of neutralizing antibody to platelet-derived growth factor. Our studies indicate a significant interaction between pancreatic cancer cells and stromal cells (PSCs) and imply that pancreatic cancer cells recruit stromal cells to establish an environment that promotes cancer progression.

Immunohistochemistry for SDHB divides gastrointestinal stromal tumors (GISTs) into 2 distinct types.

The Carney triad (CT) is gastrointestinal stromal tumor (GIST), paraganglioma, and pulmonary chondroma. The GISTs of CT show different clinical, molecular, and morphologic features to usual adult GISTs but are similar to the majority of pediatric GISTs. We postulated that these GISTs would show negative staining for succinate dehydrogenase B (SDHB). We performed SDHB immunohistochemistry on GISTs arising in 5 individuals with CT, 1 child, 7 individuals with GIST in young adulthood including 2 with germline KIT mutations, 3 individuals with neurofibromatosis 1, one 63-year-old female with multifocal gastric epithelioid GIST with lymph node metastases, and 104 consecutive unselected individuals with apparently sporadic GIST. The GISTs and paragangliomas arising in CT, the pediatric GIST, and the multifocal gastric GIST from the 63-year-old showed negative SDHB staining. GISTs from the 7 young adults and 3 with neurofibromatosis were SDHB positive. Of the unselected GISTs, 101 (97%) were positive. One of the negative GISTs arose in a 48-year-old female with previous recurrent multifocal gastric GISTs and the other 2 arose in females also in their 40s with gastric GISTs with epithelioid morphology. We conclude that negative staining for SDHB is characteristic of the GISTs of CT and the subgroup of pediatric GISTs which it resembles. Furthermore, when negative staining occurs in apparently sporadic GISTs in adults, the GISTs show morphologic and clinical features similar to pediatric and CT type GISTs. GISTs may therefore be divided into type 1 (SDHB positive) and type 2 (SDHB negative) subtypes.

Molecular heterogeneity in glioblastoma: potential clinical implications.

Glioblastomas, (grade 4 astrocytomas), are aggressive primary brain tumors characterized by histopathological heterogeneity. High-resolution sequencing technologies have shown that these tumors also feature significant inter-tumoral molecular heterogeneity. Molecular subtyping of these tumors has revealed several predictive and prognostic biomarkers. However, intra-tumoral heterogeneity may undermine the use of single biopsy analysis for determining tumor genotype and has implications for potential targeted therapies. The clinical relevance and theories of tumoral molecular heterogeneity in glioblastoma are discussed.

Intratumoral heterogeneity identified at the epigenetic, genetic and transcriptional level in glioblastoma.

Heterogeneity is a hallmark of glioblastoma with intratumoral heterogeneity contributing to variability in responses and resistance to standard treatments. Promoter methylation status of the DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT) is the most important clinical biomarker in glioblastoma, predicting for therapeutic response. However, it does not always correlate with response. This may be due to intratumoral heterogeneity, with a single biopsy unlikely to represent the entire lesion. Aberrations in other DNA repair mechanisms may also contribute. This study investigated intratumoral heterogeneity in multiple glioblastoma tumors with a particular focus on the DNA repair pathways. Transcriptional intratumoral heterogeneity was identified in 40% of cases with variability in MGMT methylation status found in 14% of cases. As well as identifying intratumoral heterogeneity at the transcriptional and epigenetic levels, targeted next generation sequencing identified between 1 and 37 unique sequence variants per specimen. In-silico tools were then able to identify deleterious variants in both the base excision repair and the mismatch repair pathways that may contribute to therapeutic response. As these pathways have roles in temozolomide response, these findings may confound patient management and highlight the importance of assessing multiple tumor biopsies.

Overexpression of miR-210 is associated with SDH-related pheochromocytomas, paragangliomas, and gastrointestinal stromal tumours

miR-210 is a key regulator of response to hypoxia. Pheochromocytomas (PCs) and paragangliomas (PGLs) with germline SDHx or VHL mutations have pseudohypoxic gene expression signatures. We hypothesised that PC/PGLs containing SDHx or VHL mutations, and succinate dehydrogenase (SDH)-deficient gastrointestinal stromal tumours (GISTs), would overexpress miR-210 relative to non-SDH or -VHL-mutated counterparts. miR-210 was analysed by quantitative PCR in i) 39 PC/PGLs, according to genotype (one SDHA, five SDHB, seven VHL, three NF1, seven RET, 15 sporadic, one unknown) and pathology (18 benign, eight atypical, 11 malignant, two unknown); ii) 18 GISTs, according to SDHB immunoreactivity (nine SDH-deficient and nine SDH-proficient) and iii) two novel SDHB-mutant neurosphere cell lines. miR-210 was higher in SDHx- or VHL-mutated PC/PGLs (7.6-fold) compared with tumours without SDHx or VHL mutations (P=0.0016). miR-210 was higher in malignant than in unequivocally benign PC/PGLs (P=0.05), but significance was lost when benign and atypical tumours were combined (P=0.08). In multivariate analysis, elevated miR-210 was significantly associated with SDHx or VHL mutation, but not with malignancy. In GISTs, miR-210 was higher in SDH-deficient (median 2.58) compared with SDH-proficient tumours (median 0.60; P=0.0078). miR-210 was higher in patient-derived neurosphere cell lines containing SDHB mutations (6.5-fold increase) compared with normal controls, in normoxic conditions (P<0.01). Furthermore, siRNA-knockdown of SDHB in HEK293 cells increased miR-210 by 2.7-fold (P=0.001) under normoxia. Overall, our results suggest that SDH deficiency in PC, PGL and GISTs induces miR-210 expression and substantiates the role of aberrant hypoxic-type cellular responses in the development of these tumours.

Factors that May Influence the Willingness of Cancer Patients to Consent for Biobanking

BACKGROUND Broad or general consent given by cancer patients for their tissue, blood, and clinical information to be stored in institutional biorepositories is fundamental to enable future ethical translational cancer research. The decision to consent for biobanking will contribute to the development of advanced diagnostic and prognostic tests, as well as new therapies to improve patient outcomes. While the rate of patient participation in biobanking programs is generally reported as high worldwide, few studies have investigated factors that may influence this decision. Biobanking at our medical research institute, an associated public (government-run) university hospital, and private hospital has been established for over 20 years, with collection of certain tumor types embedded in the research culture of these institutions. In this study, we investigated factors that may influence a cancer patients decision to give broad consent for biobanking of their specimens. METHODS Data on patient consent were collected over a 6-month period from both government and private hospitals associated with our medical research institute. Factors considered included gender, patient age at surgery, type of malignancy (breast, duodenal, cervical, endometrial, gastric, liver, esophageal, ovarian, pancreatic, pelvic, uterine, or vulval), type of institution where surgery was performed, and timing of consent. RESULTS Of 171 cancer patients, 159 (93%) gave broad consent for biobanking of their tissue and blood specimens for future cancer research projects receiving ethical and scientific approval. None of the factors analyzed was shown to influence a patients decision to contribute biological specimens and clinical data to a biorepository for future medical research. CONCLUSION Biobanking for future ethically and scientifically approved research projects in an established institution is an initiative that receives strong support from patients undergoing cancer surgery, independent of factors including gender, age, type of tumor, type of institution where surgery was performed, or timing of consent.

Glioblastoma Recurrence Correlates With Increased APE1 and Polarization Toward an Immuno-Suppressive Microenvironment

While treatment with surgery, radiotherapy and/or chemotherapy may prolong life for patients with glioblastoma, recurrence is inevitable. What is still being discovered is how much these treatments and recurrence of disease affect the molecular profiles of these tumors and how these tumors adapt to withstand these treatment pressures. Understanding such changes will uncover pathways used by the tumor to evade destruction and will elucidate new targets for treatment development. Nineteen matched pre-treatment and post-treatment glioblastoma tumors were subjected to gene expression profiling (Fluidigm, TaqMan assays), MGMT promoter methylation analysis (pyrosequencing) and protein expression analysis of the DNA repair pathways, known to be involved in temozolomide resistance (immunohistochemistry). Gene expression profiling to molecularly subtype tumors revealed that 26% of recurrent post-treatment specimens did not match their primary diagnostic specimen subtype. Post-treatment specimens had molecular changes which correlated with known resistance mechanisms including increased expression of APEX1 (p < 0.05) and altered MGMT methylation status. In addition, genes associated with immune suppression, invasion and aggression (GPNMB, CCL5, and KLRC1) and polarization toward an M2 phenotype (CD163 and MSR1) were up-regulated in post-treatment tumors, demonstrating an overall change in the tumor microenvironment favoring aggressive tumor growth and disease recurrence. This was confirmed by in vitro studies that determined that glioma cell migration was enhanced in the presence of M2 polarized macrophage conditioned media. Further, M2 macrophage-modulated migration was markedly enhanced in post-treatment (temozolomide resistant) glioma cells. These findings highlight the ability of glioblastomas to evade not only the toxic onslaught of therapy but also to evade the immune system suggesting that immune-altering therapies may be of value in treating this terrible disease.

21. Audit of primary brain tumours at royal north shore hospital from 2007 to 2011

Accurate pathological diagnosis and classification of brain tumours guides therapy and underpins translational research. We reviewed the pathological diagnosis of all primary brain tumours undergoing resection or biopsy at the Royal North Shore Hospital, an adult tertiary referral institution with a catchment population of 1.2 million during the period 2007 to 2011. Patients age ranged from 15 to 92. There were 779 cases, comprising 382 (49.04%) astrocytic, 11 (1.41%) oligoastrocytic, 35 (4.49%) oligodendroglial (0.13%) choroid plexus, 7 (0.90%) embryonal, 34 (4.36%) ependymal, 14 (1.80%) haemangioblastoma, 276 (35.43%) meningeal, 11 (1.41%) 15 (1.93%) neuronal and mixed neuronal-glial, 35 (4.49%) and 1 (0.13%) pineal. Of the astrocytic tumours 84% were glioblastoma, 10.5% anaplastic astrocytoma, 3% diffuse astrocytoma and 2% pilocytic astrocytoma. Meningeal tumours were subtyped into the following breakdowns: (72%) meningioma, (19%) atypical meningioma, (4.8%) haemangio-pericytoma, (2.8%) anaplastic meningioma, (0.7%) papillary, meningioma, (0.7%) anaplastic haemangiopericytoma and (0.34%) rhabdoid meningioma. Oligodendroglial tumours were subtyped into the following breakdowns: (74%) anaplastic oligodendroglioma, (26%) oligo-dendroglioma. Ependymal tumours were broken down into the following subcategories: (62%) ependymoma, (32%) myxopapillary ependymoma, (3%) anaplastic ependymoma and (3%) subependymoma. The incidence of most tumour subtypes in our audit was comparable to published rates in large-scale hospital-based studies. The most notable exception to this was the unusually high proportion of glioblastomas and haemangioblastomas which occurred at a rate that was double that seen in similar studies presumably because of a referral bias towards this quaternary centre.

Abstract B39: Intratumoral heterogeneity of DNA repair pathways in glioblastoma

Heterogeneity is a hallmark of glioblastoma with intratumoral heterogeneity contributing to variability in responses and resistance to standard treatments. DNA repair mechanisms are key elements involved in the response to temozolomide with epigenetic silencing of the O6-methylguanine methyltransferase (MGMT) promoter being a predictive biomarker for temozolomide response. However, response to temozolomide is highly variable and not always predicted by MGMT promoter methylation status. The mismatch repair (MMR) and base excision repair (BER) pathways have also been shown to be involved in treatment response with aberrations in these pathways leading to chemo-resistance and poor response to therapy. Thus changes in these pathways may confer resistance to temozolomide which is independent of MGMT methylation. Further, intratumoral heterogeneity in these pathways may also exacerbate resistance leading to worse outcomes. This study investigated intratumoral heterogeneity in glioblastoma with a particular focus on the DNA repair pathways. The cohort comprised 14 cases of glioblastoma with 2 - 6 tumor tissue biopsies (5-10mm3) per case resected from regions at least 1cm apart. Classification of transcriptional subtype was performed by gene expression profiling (Fluidigm, Taqman assays). Pyrosequencing was used to identify MGMT promoter methylation. Expression of MMR and BER genes was determined using qRT-PCR and Taqman assays and deep sequencing of these genes was performed using the MiSeq Illumina platform and Avadis NGS software. Gene expression profiling using two different limited gene-sets classified tumor specimens into the 3 major transcriptional subtypes, proneural, classical and mesenchymal, with strong concordance. These clustering techniques were then applied to tumor biopsies from the same individual. Transcriptional intratumoral heterogeneity defined as biopsies from the same individual being classified into different subtypes was identified in 40% of the patients. Intratumoral heterogeneity was also identified in the DNA repair pathways. Variability in MGMT methylation status was found in 14% of cases. In each case the percentage methylation varied up to 4-fold and the methylation status was independent of transcriptional classification. Intratumoral variation in the expression of the MMR genes MSH2 and PMS2 was identified in 15% and 20% of cases respectively and in 50% and 30% of cases for the BER genes PARP1 and APEX1. Significant heterogeneity within specimens was not identified for MMR genes MSH6 or MLH1. Targeted next generation sequencing of these 6 genes confirmed the presence of intratumoral heterogeneity at the mutation level. Up to 80 sequence variants were identified in each specimen, with 35 – 56 variants common across all specimens from a case, up to 20 shared between at least 2 specimens in a case and between 1 and 37 unique to each specimen within a case. This study identified intratumoral heterogeneity of DNA repair pathways in glioblastomas, at the genomic, transcriptional and mutational levels. These pathways have roles in the responsiveness of glioblastomas to temozolomide. As such, intratumoral heterogeneity may confound patient management. Therefore, these results highlight the importance of assessing results from multiple tumor biopsies in order to correctly manage glioblastoma patients and their treatment. Citation Format: Nicole R. Parker, Amanda L. Hudson, Peter Khong, Jonathon F. Parkinson, Rowan Ikin, Zhangkai Jason Cheng, Fatemeh Vafaee, Helen R. Wheeler, Viive M. Howell. Intratumoral heterogeneity of DNA repair pathways in glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B39.

Abstract 3881: A novel class of structure-based cyclin-dependent kinase inhibitors

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Deregulation of the Cdk/Rb/E2F axis is a common feature among cancers. Several promising ATP-competitive Cdk inhibitors are undergoing clinical testing as anticancer therapeutics. However a weakness of ATP-competitive Cdk inhibitors is that all kinases share a highly conserved ATP binding site, making it difficult to selectively inhibit cell cycle Cdks. Further, there is significant functional redundancy among cell cycle Cdks, so the ideal Cdk inhibitor would block the activity of multiple cell cycle Cdks while not affecting other kinases. Nature utilizes several modes of regulation that are specific to the Cdks that involve (1) the inhibitory proteins p21, p27, and p57, (2) inhibitory phosphorylation on a tyrosine residue near the ATP binding pocket, (3) activating phosphorylation on the “T-loop” site, and (4) binding of an activating Cyclin subunit. Usurping one of these natural Cdk-specific control mechanisms may be the best hope for developing potent and selective anticancer Cdk inhibitors. Comparison of the crystal structures of Cyclin A/Cdk2/p27 and Cyclin A/Cdk2 complexes indicated that p27 binding causes a conformational change, which opens a pocket on the surface of Cdk2. We hypothesized that a small molecule that binds to this pocket would force Cdks to adopt an inactive conformation. In silico molecular docking was performed to identify compounds predicted to bind to this pocket. Multiple compounds identified in this screen inhibited the proliferation of breast cancer cells. Inhibition of proliferation correlated with a decrease in the soluble levels of Cdk1, Cdk2, and Cdk4, and an increase in these proteins in the insoluble fraction. Video microscopy of cells stably expressing a GFP-Cdk4 fusion protein showed aggregate formation over time in the presence of the Cdk inhibitors and that the aggregates dissipated after the drug was removed. Proliferation studies performed in parallel also indicated that the effects of the Cdk inhibitors were largely reversible within the first 24 hours. One of the compounds, NSC117024, preferentially inhibited the proliferation of BT549 breast cancer cells over nontransformed mammary epithelial cells. Inhibition of BT549 proliferation was observed at NSC117024 concentrations as low as 780 nM. Interestingly, suppression of cancer cell division by the compounds occurred as well or better in cells lacking p21 and p27. The compounds directly inhibited Cdk2 activity in kinase assays. Together the results suggest that the novel family of Cdk inhibitors mimic the actions of p21 and p27 and may preferentially inhibit the proliferation of a subset of breast cancer cells. Current efforts are directed toward optimizing NSC117024 antiproliferative activity, developing a biotinylated NSC117024 affinity probe to verify compound specificity, and identifying the genetic features of cancer cells that render them sensitive to NSC117024. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3881.