Kathryn Simpson

Is Serum or Plasma More Appropriate for Intersubject Comparisons in Metabolomic Studies? An Assessment in Patients with Small-Cell Lung Cancer

In clinical analyses, the most appropriate biofluid should be analyzed for optimal assay performance. For biological fluids, the most readily accessible is blood, and metabolomic analyses can be performed either on plasma or serum. To determine the optimal agent for analysis, metabolic profiles of matched human serum and plasma were assessed by gas chromatography/time-of-flight mass spectrometry and ultrahigh-performance liquid chromatography mass spectrometry (in positive and negative electrospray ionization modes). Comparison of the two metabolomes, in terms of reproducibility, discriminative ability and coverage, indicated that they offered similar analytical opportunities. An analysis of the variation between 29 small-cell lung cancer (SCLC) patients revealed that the differences between individuals are markedly similar for the two biofluids. However, significant differences between the levels of some specific metabolites were identified, as were differences in the intersubject variability of some metabolite levels. Glycerophosphocholines, erythritol, creatinine, hexadecanoic acid, and glutamine in plasma, but not in serum, were shown to correlate with life expectancy for SCLC patients, indicating the utility of metabolomic analyses in clinical prognosis and the particular utility of plasma in relation to the clinical management of SCLC.

Optimisation of circulating biomarkers of cell death for routine clinical use

BACKGROUND M30 and M65 enzyme-linked immunosorbent assays detect circulating cytokeratin 18 fragments released during caspase-dependent or total cell death, respectively, and have potential as biomarkers in epithelial cancers. While these assays have been validated, their robustness for routine clinical use is unknown. PATIENTS AND METHODS M30 and M65 were measured in matched serum and plasma samples from 31 lung cancer patients and 18 controls. RESULTS Time allowable between sample acquisition and processing is critical for assays in clinical use. A 4-h delay in processing at room temperature increased M30 (P < 0.0001), an effect minimised by incubation on ice. M30 and M65 in serum were resistant to processing variations including delays. Serum and plasma measurements correlated well although M30 but not M65 was lower in serum (P < 0.0005). Less variation between duplicate assays was observed in serum. Prolonged storage (-80 degrees C) led to increased M30 (12%, 6 months; 34%, 1 year). Sample dilution in the supplied assay diluent proved non-linear, whereas dilution in donor serum or porcine plasma restored linearity up to a ratio of 1 : 6. CONCLUSION We present recommendations that improve the reliability of these assays for clinical use and recommend serum as the preferred matrix with data more resistant to variations in collection.

Quantitative mass spectrometry-based techniques for clinical use: biomarker identification and quantification.

Abstract The potential for development of personalised medicine through the characterisation of novel biomarkers is an exciting prospect for improved patient care. Recent advances in mass spectrometric (MS) techniques, liquid phase analyte separation and bioinformatic tools for high throughput now mean that this goal may soon become a reality. However, there are challenges to be overcome for the identification and validation of robust biomarkers. Bio-fluids such as plasma and serum are a rich source of protein, many of which may reflect disease status, and due to the ease of sampling and handling, novel blood borne biomarkers are very much sought after. MS-based methods for high throughput protein identification and quantification are now available such that the issues arising from the huge dynamic range of proteins present in plasma may be overcome, allowing deep mining of the blood proteome to reveal novel biomarker signatures for clinical use. In addition, the development of sensitive MS-based methods for biomarker validation may bypass the bottleneck created by the need for generation and usage of reliable antibodies prior to large scale screening. In this review, we discuss the MS-based methods that are available for clinical proteomic analysis and highlight the progress made and future challenges faced in this cutting edge area of research.

Statistical Considerations of Optimal Study Design for Human Plasma Proteomics and Biomarker Discovery.

A mass spectrometry-based plasma biomarker discovery workflow was developed to facilitate biomarker discovery. Plasma from either healthy volunteers or patients with pancreatic cancer was 8-plex iTRAQ labeled, fractionated by 2-dimensional reversed phase chromatography and subjected to MALDI ToF/ToF mass spectrometry. Data were processed using a q-value based statistical approach to maximize protein quantification and identification. Technical (between duplicate samples) and biological variance (between and within individuals) were calculated and power analysis was thereby enabled. An a priori power analysis was carried out using samples from healthy volunteers to define sample sizes required for robust biomarker identification. The result was subsequently validated with a post hoc power analysis using a real clinical setting involving pancreatic cancer patients. This demonstrated that six samples per group (e.g., pre- vs post-treatment) may provide sufficient statistical power for most proteins with changes >2 fold. A reference standard allowed direct comparison of protein expression changes between multiple experiments. Analysis of patient plasma prior to treatment identified 29 proteins with significant changes within individual patient. Changes in Peroxiredoxin II levels were confirmed by Western blot. This q-value based statistical approach in combination with reference standard samples can be applied with confidence in the design and execution of clinical studies for predictive, prognostic, and/or pharmacodynamic biomarker discovery. The power analysis provides information required prior to study initiation.

Hypoxic human cancer cells are sensitized to BH-3 mimetic–induced apoptosis via downregulation of the Bcl-2 protein Mcl-1

Solid tumors contain hypoxic regions in which cancer cells are often resistant to chemotherapy-induced apoptotic cell death. Therapeutic strategies that specifically target hypoxic cells and promote apoptosis are particularly appealing, as few normal tissues experience hypoxia. We have found that the compound ABT-737, a Bcl-2 homology domain 3 (BH-3) mimetic, promotes apoptotic cell death in human colorectal carcinoma and small cell lung cancer cell lines exposed to hypoxia. This hypoxic induction of apoptosis was mediated through downregulation of myeloid cell leukemia sequence 1 (Mcl-1), a Bcl-2 family protein that serves as a biomarker for ABT-737 resistance. Downregulation of Mcl-1 in hypoxia was independent of hypoxia-inducible factor 1 (HIF-1) activity and was consistent with decreased global protein translation. In addition, ABT-737 induced apoptosis deep within tumor spheroids, consistent with an optimal hypoxic oxygen tension being necessary to promote ABT-737–induced cell death. Tumor xenografts in ABT-737–treated mice also displayed significantly more apoptotic cells within hypoxic regions relative to normoxic regions. Synergies between ABT-737 and other cytotoxic drugs were maintained in hypoxia, suggesting that this drug may be useful in combination with chemotherapeutic agents. Taken together, these findings suggest that Mcl-1–sparing BH-3 mimetics may induce apoptosis in hypoxic tumor cells that are resistant to other chemotherapeutic agents and may have a role in combinatorial chemotherapeutic regimens for treatment of solid tumors.

Circulating Biomarkers of Cell Death After Treatment with the BH-3 Mimetic ABT-737 in a Preclinical Model of Small-Cell Lung Cancer

Purpose: This study evaluated epithelial cell death ELISAs that measure circulating cytokeratin 18 in mice bearing small-cell lung cancer xenografts treated with a proapoptotic dose of the BH-3 mimetic ABT-737. Experimental Design: H146 tumor–bearing and non–H146 tumor-bearing severe combined immunodeficient (SCID)/bg mice were treated with ABT-737 or vehicle control. Plasma collected before and 2 to 360 hours after treatment was analyzed by M30 (caspase-cleaved cytokeratin 18) and M65 (intact and cleaved cytokeratin 18) ELISA. In parallel, tumors were interrogated for cleaved caspase-3 and cleaved cytokeratin 18 as biomarkers of apoptosis. Results: ABT-737–treated tumors regressed by 48 hours (P < 0.01) compared with controls, correlating with increased cleaved cytokeratin 18 (P < 0.01; 6 and 24 hours) and increased intact cytokeratin 18 (P < 0.01; 24 hours). Cleaved cytokeratin 18 levels decreased below baseline between 72 and 360 hours for ABT-737–treated and control mice whereas intact cytokeratin 18 decreased below the level of detection at 8 and 15 days in ABT-737–treated mice only. Apoptosis in tumors reflected changes in circulating cytokeratin 18 (cleaved caspase-3, P < 0.05 at 2 hours and P < 0.001 at 6, 12, and 24 hours; caspase-cleaved cytokeratin 18, P < 0.05 at 15 days, for drug treated versus controls). Conclusions: ABT-737 caused tumor regression by apoptosis in H146 xenografts that mapped to a drug-specific, early increase in circulating cleaved cytokeratin 18 that subsequently declined. Circulating, intact cytokeratin 18 levels correlated with tumor burden. Cleaved caspase-3 and caspase-cleaved cytokeratin 18 in tumor correlated with treatment (P < 0.05, 2 hours; P < 0.001, 6, 12, and 24 hours; cleaved caspase-3, P < 0.05, 15 days; caspase-cleaved cytokeratin 18), indicating that events in plasma were tumor derived. These circulating biomarker data will be translated to clinical trials wherein serial tumor biopsies are rarely obtained.

A caspase-3 ?death-switch? in colorectal cancer cells for induced and synchronous tumor apoptosis in vitro and in vivo facilitates the development of minimally invasive cell death biomarkers

Novel anticancer drugs targeting key apoptosis regulators have been developed and are undergoing clinical trials. Pharmacodynamic biomarkers to define the optimum dose of drug that provokes tumor apoptosis are in demand; acquisition of longitudinal tumor biopsies is a significant challenge and minimally invasive biomarkers are required. Considering this, we have developed and validated a preclinical ‘death-switch’ model for the discovery of secreted biomarkers of tumour apoptosis using in vitro proteomics and in vivo evaluation of the novel imaging probe [18F]ML-10 for non-invasive detection of apoptosis using positron emission tomography (PET). The ‘death-switch’ is a constitutively active mutant caspase-3 that is robustly induced by doxycycline to drive synchronous apoptosis in human colorectal cancer cells in vitro or grown as tumor xenografts. Death-switch induction caused caspase-dependent apoptosis between 3 and 24 hours in vitro and regression of ‘death-switched’ xenografts occurred within 24 h correlating with the percentage of apoptotic cells in tumor and levels of an established cell death biomarker (cleaved cytokeratin-18) in the blood. We sought to define secreted biomarkers of tumor apoptosis from cultured cells using Discovery Isobaric Tag proteomics, which may provide candidates to validate in blood. Early after caspase-3 activation, levels of normally secreted proteins were decreased (e.g. Gelsolin and Midkine) and proteins including CD44 and High Mobility Group protein B1 (HMGB1) that were released into cell culture media in vitro were also identified in the bloodstream of mice bearing death-switched tumors. We also exemplify the utility of the death-switch model for the validation of apoptotic imaging probes using [18F]ML-10, a PET tracer currently in clinical trials. Results showed increased tracer uptake of [18F]ML-10 in tumours undergoing apoptosis, compared with matched tumour controls imaged in the same animal. Overall, the death-switch model represents a robust and versatile tool for the discovery and validation of apoptosis biomarkers.

Tumourigenic non-small-cell lung cancer mesenchymal circulating tumour cells: a clinical case study

An explant model derived from EpCam negative mesenchymal non-small-cell lung (NSCLC) cancer circulating tumour cells (a ‘liquid biopsy’) recapitulates the histology of the donor patients diagnostic specimen and chemoresistance to cisplatin and pemetrexed. This proof-of-principal landmark model opens a new avenue for study of advanced NSCLC biology when tissue biopsies unavailable.

Sustained tumour eradication after induced caspase-3 activation and synchronous tumour apoptosis requires an intact host immune response

Effective anticancer treatments often result in the induction of large amounts of tumour cell death. In vivo, such dying tumour cells are a potential source of antigens for T-cell stimulation. Although apoptosis is generally considered nonimmunogenic, recent evidence suggests that some anticancer therapies that induce apoptosis can elicit antitumour immune responses. Here, a doxycycline-inducible, constitutively active caspase-3 (‘death switch’) was constructed in a murine tumour model to explore the impact of the host immune response to rapid, synchronous and substantial tumour cell apoptosis. In vitro, up to 80% of tumour cells underwent apoptotic cell death within 24 h and death was accompanied by the release of potential ‘danger signal’ molecules HMGB1 and HSP90. In vivo, death switch induction provoked rapid, pronounced tumour regression in immune-competent and immune-deficient mice, but sustained tumour eradication was observed only in immune-competent mice. Moreover, the majority of mice that were tumour free after death switch induction were protected from further tumour rechallenge. In addition, long-term remission after induction of the death switch was completely abrogated following depletion of CD8 T cells. These data suggest that sustained tumour eradication after substantial tumour apoptosis requires an antitumour host immune response that prevents tumour relapse. In many patients, cancer therapies produce encouraging initial responses that are only short lived. These results provide new insights that may have important implications for further development of strategies that result in long-term tumour clearance after initially effective anticancer treatment.

[18F]-FLT Positron Emission Tomography can be used to image the response of sensitive tumors to PI3-Kinase inhibition with the novel agent GDC-0941.

The phosphoinositide 3-kinase (PI3K) pathway is deregulated in a range of cancers, and several targeted inhibitors are entering the clinic. This study aimed to investigate whether the positron emission tomography tracer 3′-deoxy-3′-[18F]fluorothymidine ([18F]-FLT) is suitable to mark the effect of the novel PI3K inhibitor GDC-0941, which has entered phase II clinical trial. CBA nude mice bearing U87 glioma and HCT116 colorectal xenografts were imaged at baseline with [18F]-FLT and at acute (18 hours) and chronic (186 hours) time points after twice-daily administration of GDC-0941 (50 mg/kg) or vehicle. Tumor uptake normalized to blood pool was calculated, and tissue was analyzed at sacrifice for PI3K pathway inhibition and thymidine kinase (TK1) expression. Uptake of [18F]-FLT was also assessed in tumors inducibly overexpressing a dominant-negative form of the PI3K p85 subunit p85α, as well as HCT116 liver metastases after GDC-0941 therapy. GDC-0941 treatment induced tumor stasis in U87 xenografts, whereas inhibition of HCT116 tumors was more variable. Tumor uptake of [18F]-FLT was significantly reduced following GDC-0941 dosing in responsive tumors at the acute time point and correlated with pharmacodynamic markers of PI3K signaling inhibition and significant reduction in TK1 expression in U87, but not HCT116, tumors. Reduction of PI3K signaling via expression of Δp85α significantly reduced tumor growth and [18F]-FLT uptake, as did treatment of HCT116 liver metastases with GDC-0941. These results indicate that [18F]-FLT is a strong candidate for the noninvasive measurement of GDC-0941 action. Mol Cancer Ther; 12(5); 819–28. ©2013 AACR.