Sir Peter MacCallum Department of Oncology - Research Publications
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ItemA multisite blinded study for the detection of BRAF mutations in formalin-fixed, paraffin-embedded malignant melanoma(NATURE PUBLISHING GROUP, 2013-04-15)Melanoma patients with BRAF mutations respond to treatment with vemurafenib, thus creating a need for accurate testing of BRAF mutation status. We carried out a blinded study to evaluate various BRAF mutation testing methodologies in the clinical setting. Formalin-fixed, paraffin-embedded melanoma samples were macrodissected before screening for mutations using Sanger sequencing, single-strand conformation analysis (SSCA), high resolution melting analysis (HRM) and competitive allele-specific TaqMan® PCR (CAST-PCR). Concordance of 100% was observed between the Sanger sequencing, SSCA and HRM techniques. CAST-PCR gave rapid and accurate results for the common V600E and V600K mutations, however additional assays are required to detect rarer BRAF mutation types found in 3-4% of melanomas. HRM and SSCA followed by Sanger sequencing are effective two-step strategies for the detection of BRAF mutations in the clinical setting. CAST-PCR was useful for samples with low tumour purity and may also be a cost-effective and robust method for routine diagnostics.
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ItemTargeted-capture massively-parallel sequencing enables robust detection of clinically informative mutations from formalin-fixed tumours(NATURE PUBLISHING GROUP, 2013-12-13)Massively parallel sequencing offers the ability to interrogate a tumour biopsy for multiple mutational changes. For clinical samples, methodologies must enable maximal extraction of available sequence information from formalin-fixed and paraffin-embedded (FFPE) material. We assessed the use of targeted capture for mutation detection in FFPE DNA. The capture probes targeted the coding region of all known kinase genes and selected oncogenes and tumour suppressor genes. Seven melanoma cell lines and matching FFPE xenograft DNAs were sequenced. An informatics pipeline was developed to identify variants and contaminating mouse reads. Concordance of 100% was observed between unfixed and formalin-fixed for reported COSMIC variants including BRAF V600E. mutations in genes not conventionally screened including ERBB4, ATM, STK11 and CDKN2A were readily detected. All regions were adequately covered with independent reads regardless of GC content. This study indicates that hybridisation capture is a robust approach for massively parallel sequencing of FFPE samples.
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ItemA phase I trial of imatinib in combination with mFOLFOX6-bevacizumab in patients with advanced colorectal cancer(SPRINGER, 2013-02-01)PURPOSE: Platelet-derived growth factor receptor (PDGFR) inhibition by reducing tumoral interstitial fluid pressure might increase the efficacy of chemotherapy. Imatinib inhibits PDGFR kinase activity at therapeutically relevant doses. This phase I study aimed to assess the maximal tolerated dose (MTD) of imatinib in combination with mFOLFOX6-bevacizumab in patients with advanced colorectal cancer and to identify pharmacokinetic (PK) interactions and toxicities. METHODS: Eligible patients had measurable disease and adequate organ function. On day-14, patients commenced imatinib daily plus bevacizumab (5 mg/kg/2 weekly). Two weeks later (day 1), patients were also treated with full dose mFOLFOX6-bevacizumab for 12 cycles. Blood samples were taken for PK. DLTs defined in the first 6 weeks. Standard dose escalation of imatinib, with 3 patient cohorts: planned dose levels (DL): DL1; 400 mg, DL2; 600 mg, DL3; 800 mg daily. RESULTS: Ten patients enrolled. DL1 3 patients, DL2 7 patients. DLTs observed in 3 of 6 patients in DL2: febrile neutropenia (2); Grade 3 infection and Grade 4 neutropenia (1). Neutropenia was most frequent AEs: Grade 3/4 in >60 % of patients overall. In DL2 pts, imatinib clearance was reduced post-chemotherapy (P < 0.05). Oxaliplatin and 5FU PK unchanged by imatinib. CONCLUSIONS: MTD was imatinib 400 mg plus full dose mFOLFOX-bevacizumab. Dose escalation of imatinib limited by neutropenia. Further study is warranted as imatinib can be delivered at levels that inhibit PDGFR.
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ItemAKT signalling is required for ribosomal RNA synthesis and progression of E mu-Myc B-cell lymphoma in vivo(WILEY-BLACKWELL, 2013-11-01)The dysregulation of PI3K/AKT/mTORC1 signalling and/or hyperactivation of MYC are observed in a high proportion of human cancers, and together they form a 'super signalling' network mediating malignancy. A fundamental downstream action of this signalling network is up-regulation of ribosome biogenesis and subsequent alterations in the patterns of translation and increased protein synthesis, which are thought to be critical for AKT/MYC-driven oncogenesis. We have demonstrated that AKT and MYC cooperate to drive ribosomal DNA (rDNA) transcription and ribosome biogenesis, with AKT being essential for rDNA transcription and in vitro survival of lymphoma cells isolated from a MYC-driven model of B-cell lymphoma (Eμ-Myc) [Chan JC et al., (2011) Science Signalling 4, ra56]. Here we show that the allosteric AKT inhibitor MK-2206 rapidly and potently antagonizes rDNA transcription in Eμ-Myc B-cell lymphomas in vivo, and this is associated with a rapid reduction in indicators of disease burden, including spleen weight and the abundance of tumour cells in both the circulation and lymph nodes. Extended treatment of tumour-bearing mice with MK-2206 resulted in a significant delay in disease progression, associated with increased B-cell lymphoma apoptosis. Our findings suggest that malignant diseases characterized by unrestrained ribosome biogenesis may be vulnerable to therapeutic strategies that target the PI3K/AKT/mTORC1/MYC growth control network.
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ItemSynergistic inhibition of ovarian cancer cell growth by combining selective PI3K/mTOR and RAS/ERK pathway inhibitors(ELSEVIER SCI LTD, 2013-12-01)BACKGROUND: Ovarian cancer is the major cause of death from gynaecological malignancy with a 5year survival of only ∼30% due to resistance to platinum and paclitaxel-based first line therapy. Dysregulation of the phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) and RAS/extracellular signal-regulated kinase (ERK) pathways is common in ovarian cancer, providing potential new targets for 2nd line therapy. METHODS: We determined the inhibition of proliferation of an extensive panel of ovarian cancer cell lines, encompassing all the major histotypes, by the dual PI3K/mTOR inhibitor PF-04691502 and a MEK inhibitor, PD-0325901. In addition, we analysed global gene expression, mutation status of key PI3K/mTOR and RAS/ERK pathway members and pathway activation to identify predictors of drug response. RESULTS: PF-04691502 inhibits proliferation of the majority of cell lines with potencies that correlate with the extent of pathway inhibition. Resistant cell lines were characterised by activation of the RAS/ERK pathway as indicated by differential gene expression profiles and pathway activity analysis. PD-0325901 suppressed growth of a subset of cell lines that were characterised by high basal RAS/ERK signalling. Strikingly, using PF-04691502 and PD-0325901 in combination resulted in synergistic growth inhibition in 5/6 of PF-04691502 resistant cell lines and two cell lines resistant to both single agents showed robust synergistic growth arrest. Xenograft studies confirm the utility of combination therapy to synergistically inhibit tumour growth of PF-04691502-resistant tumours in vivo. CONCLUSIONS: These studies identify dual targeted inhibitors of PI3K/mTOR in combination with inhibitors of RAS/ERK signalling as a potentially effective new approach to treating ovarian cancer.
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ItemNo Preview AvailableThe Cell-Cycle Regulator CDK4: An Emerging Therapeutic Target in Melanoma(AMER ASSOC CANCER RESEARCH, 2013-10-01)The recent clinical success of targeted therapies in melanoma directed at the oncogene BRAF validates the concept of targeting oncogenes. The p16-cyclin D-CDK4/6-retinoblastoma protein pathway (CDK4 pathway) is dysregulated in 90% of melanomas, and is, therefore, an obvious therapeutic target for this disease. The main outcome of CDK4 activation is the phosphorylation and, thus, inhibition of the retinoblastoma protein leading to G1-S cell-cycle transition. In addition, CDK4 directly phosphorylates other proteins that promote cell-cycle progression and inhibit both cell senescence and apoptosis. In preclinical studies, the response to CDK4 inhibition correlates with genomic changes that increase CDK4 activity, most notably where the tumor suppressor CDKN2A (p16(INK4A)) is deleted. A central question is whether melanomas with activating events in the CDK4 pathway have become "addicted" to this signaling pathway, in which case inhibition of CDK4 would not simply induce cell-cycle arrest but induce cell death and tumor regression. Recently, a number of selective CDK4/6 inhibitors have entered clinical trials, and these compounds are showing great promise in that they are well tolerated and show clinical benefit. This review discusses the CDK4 pathway, its dysregulation in melanoma, the consequences of CDK4 pathway inhibition, and potential novel combinational strategies for the treatment of melanoma.
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ItemNo Preview AvailableTargeting Oncogenic Drivers and the Immune System in Melanoma(AMER SOC CLINICAL ONCOLOGY, 2013-02-01)Melanoma is one of the most common cancers in Western countries but has defied the trend of reductions in age-adjusted mortality observed in most other cancers in recent years. Biologically, melanoma is characterized by a high propensity to metastasize at low tumor volumes necessitating the need for effective drug therapies to support efforts in prevention and early detection for reducing mortality. Efforts to study the clinical biology of melanoma have led to a new understanding of the disease, with genomic studies identifying several targetable oncogenes, in particular the protein kinases BRAF and KIT. Biologic studies have also identified a variety of immunologic targets, including the programmed death 1 (PD-1) and cytotoxic T-cell lymphocyte-associated antigen 4 (CTLA-4) inhibitory molecules expressed on T lymphocytes. After several decades of clinical trials that failed to demonstrate improvement in overall survival in patients with advanced melanoma, small molecule inhibitors of BRAF or MEK and inhibition of CTLA-4 can improve survival in patients with advanced disease. These early clinical studies have provided a great opportunity to improve mortality in melanoma with the significant potential of combinations of signaling inhibitors or signaling inhibitors combined with immunologic agents, particularly when used in the adjuvant setting, and to transform the care of patients with this most challenging of cancers.
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ItemBRAF-targeted therapy and immune responses to melanoma(LANDES BIOSCIENCE, 2013-06-01)Type I BRAF inhibitors and immunotherapy constitute two new exciting approaches for the treatment of advanced malignant melanoma. We have recently elucidated a role for host C-C chemokine receptor type 2 (CCR2) in the antineoplastic effects of type I BRAF inhibitors in mice, supporting the therapeutic potential of combining BRAF inhibitors with immunotherapy.
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ItemNo Preview AvailableHost immunity contributes to the anti-melanoma activity of BRAF inhibitors(AMER SOC CLINICAL INVESTIGATION INC, 2013-03-01)The BRAF mutant, BRAF(V600E), is expressed in nearly half of melanomas, and oral BRAF inhibitors induce substantial tumor regression in patients with BRAF(V600E) metastatic melanoma. The inhibitors are believed to work primarily by inhibiting BRAF(V600E)-induced oncogenic MAPK signaling; however, some patients treated with BRAF inhibitors exhibit increased tumor immune infiltration, suggesting that a combination of BRAF inhibitors and immunotherapy may be beneficial. We used two relatively resistant variants of Braf(V600E)-driven mouse melanoma (SM1 and SM1WT1) and melanoma-prone mice to determine the role of host immunity in type I BRAF inhibitor PLX4720 antitumor activity. We found that PLX4720 treatment downregulated tumor Ccl2 gene expression and decreased tumor CCL2 expression in both Braf(V600E) mouse melanoma transplants and in de novo melanomas in a manner that was coincident with reduced tumor growth. While PLX4720 did not directly increase tumor immunogenicity, analysis of SM1 tumor-infiltrating leukocytes in PLX4720-treated mice demonstrated a robust increase in CD8(+) T/FoxP3(+)CD4(+) T cell ratio and NK cells. Combination therapy with PLX4720 and anti-CCL2 or agonistic anti-CD137 antibodies demonstrated significant antitumor activity in mouse transplant and de novo tumorigenesis models. These data elucidate a role for host CCR2 in the mechanism of action of type I BRAF inhibitors and support the therapeutic potential of combining BRAF inhibitors with immunotherapy.
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ItemCombined inhibition of PI3K-related DNA damage response kinases and mTORC1 induces apoptosis in MYC-driven B-cell lymphomas(AMER SOC HEMATOLOGY, 2013-04-11)Pharmacological strategies capable of directly targeting MYC are elusive. Previous studies have shown that MYC-driven lymphomagenesis is associated with mammalian target of rapamycin (mTOR) activation and a MYC-evoked DNA damage response (DDR) transduced by phosphatidylinositol-3-kinase (PI3K)-related kinases (DNA-PK, ATM, and ATR). Here we report that BEZ235, a multitargeted pan-PI3K/dual-mTOR inhibitor, potently killed primary Myc-driven B-cell lymphomas and human cell lines bearing IG-cMYC translocations. Using pharmacologic and genetic dissection of PI3K/mTOR signaling, dual DDR/mTORC1 inhibition was identified as a key mediator of apoptosis. Moreover, apoptosis was initiated at drug concentrations insufficient to antagonize PI3K/mTORC2-regulated AKT phosphorylation. p53-independent induction of the proapoptotic BH3-only protein BMF was identified as a mechanism by which dual DDR/mTORC1 inhibition caused lymphoma cell death. BEZ235 treatment induced apoptotic tumor regressions in vivo that correlated with suppression of mTORC1-regulated substrates and reduced H2AX phosphorylation and also with feedback phosphorylation of AKT. These mechanistic studies hold important implications for the use of multitargeted PI3K inhibitors in the treatment of hematologic malignancies. In particular, the newly elucidated role of PI3K-related DDR kinases in response to PI3K inhibitors offers a novel therapeutic opportunity for the treatment of hematologic malignancies with an MYC-driven DDR.