Sunil Kadam

Method Validation and Measurement of Biomarkers in Nonclinical and Clinical Samples in Drug Development: A Conference Report

No HeadingBiomarkers are increasingly used in drug development to aid scientific and clinical decisions regarding the progress of candidate and marketed therapeutics. Biomarkers can improve the understanding of diseases as well as therapeutic and off-target effects of drugs. Early implementation of biomarker strategies thus promises to reduce costs and time-to-market as drugs proceed through increasingly costly and complex clinical development programs. The 2003 American Association of Pharmaceutical Sciences/Clinical Ligand Assay Society Biomarkers Workshop (Salt Lake City, UT, USA, October 24–25, 2003) addressed key issues in biomarker research, with an emphasis on the validation and implementation of biochemical biomarker assays, covering from preclinical discovery of efficacy and toxicity biomarkers through clinical and postmarketing implementation. This summary report of the workshop focuses on the major issues discussed during presentations and open forums and noted consensus achieved among the participants on topics from nomenclature to best practices. For example, it was agreed that because reliable and accurate data provide the basis for sound decision making, biomarker assays must be validated in a manner that enables the creation of such data. The nature of biomarker measurements often precludes direct application of regulatory guidelines established for clinical diagnostics or drug bioanalysis, and future guidance on biomarker assay validation should therefore be adaptable enough that validation criteria do not stifle creative biomarker solutions.

A Phase 1 Dose Escalation, Pharmacokinetic, and Pharmacodynamic Evaluation of eIF-4E Antisense Oligonucleotide LY2275796 in Patients with Advanced Cancer

Purpose: The antisense oligonucleotide LY2275796 blocks expression of cap-binding protein eukaryotic initiation factor 4E (eIF-4E), an mRNA translation regulator upregulated in tumors. This phase I study sought an appropriate LY2275796 dose in patients with advanced tumors. Experimental Design: A 3-day loading dose, then weekly maintenance doses, were given to 1 to 3 patient cohorts, beginning with 100 mg and escalating. Plasma samples were collected to determine LY2275796 concentrations and tumor biopsies to quantify eIF-4E mRNA/protein. Results: Thirty patients with stage 4 disease received 1 or more LY2275796 dose. A dose-limiting toxicity was observed at 1,200 mg, with 1,000 mg the maximum-tolerated dose. Across all dose levels, most patients (87%) had only grade 1 to 2 toxicities. LY2275796 pharmacokinetics supported the dosing regimen. Comparison of pre- and postdose biopsies showed eIF-4E decreased in most patients. Fifteen patients had progressive disease, and 7 patients achieved stable disease (minimum of 6 weeks) as best response, with 2 patients on therapy for more than 3 months (one with melanoma, one with cystadenocarcinoma of the head/neck). Conclusions: LY2275796 was well tolerated up to 1,000 mg. Because tumor eIF-4E expression was decreased, but no tumor response observed, LY2275796 should be studied combined with other treatment modalities. Clin Cancer Res; 17(20); 6582–91. ©2011 AACR.

Tumor Survivin Is Downregulated by the Antisense Oligonucleotide LY2181308: A Proof-of-Concept, First-in-Human Dose Study

Purpose: Enhanced tumor cell survival through expression of inhibitors of apoptosis (IAP) is a hallmark of cancer. Survivin, an IAP absent from most normal tissues, is overexpressed in many malignancies and associated with a poorer prognosis. We report the first-in-human dose study of LY2181308, a second-generation antisense oligonucleotide (ASO) directed against survivin mRNA. Patients and Methods: A dose-escalation study evaluating the safety, pharmacokinetics, and pharmacodynamics of LY2181308 administered intravenously for 3 hours as a loading dose on 3 consecutive days and followed by weekly maintenance doses. Patients were eligible after signing informed consent, had exhausted approved anticancer therapies and agreed to undergo pre- and posttreatment tumor biopsies to evaluate reduction of survivin protein and gene expression. Results: A total of 40 patients were treated with LY2181308 at doses of 100 to 1,000 mg. Twenty-six patients were evaluated at the recommended phase 2 dose of 750 mg, at which level serial tumor sampling and [11C]LY2183108 PET (positron emission tomography) imaging demonstrated that ASO accumulated within tumor tissue, reduced survivin gene and protein expression by 20% and restored apoptotic signaling in tumor cells in vivo. Pharmacokinetics were consistent with preclinical modeling, exhibiting rapid tissue distribution, and terminal half-life of 31 days. Conclusions: The tumor-specific, molecularly targeted effects demonstrated by this ASO in man underpin confirmatory studies evaluating its therapeutic efficacy in cancer.

Dorrigocins: novel antifungal antibiotics that change the morphology of ras-transformed NIH/3T3 cells to that of normal cells. I. Taxonomy of the producing organism, fermentation and biological activity.

The dorrigocins are new secondary metabolites produced by submerged fermentation of a streptomycete which was isolated from a soil sample collected in Australia. The dorrigocins show moderate antifungal activity and reverse the morphology of ras-transformed NIH/3T3 cells from a transformed phenotype to a normal one. The producing culture was identified as Streptomyces platensis subsp. rosaceus strain AB1981F-75.

Quantitative Measurement of Cell-Free Plasma DNA and Applications for Detecting Tumor Genetic Variation and Promoter Methylation in a Clinical Setting

An elevated cell-free DNA (cfDNA) level is often reported in patients with advanced cancer and is thought to represent nuclear material from a distant inaccessible tumor. cfDNA can become a valuable source to monitor tumor dynamics and evaluate genetic markers for predictive, prognostic, and diagnostic testing. DNA extraction and quantification were optimized with plasma collected from 20 patients with advanced cancer and 16 healthy controls. Plasma cfDNA from patients with advanced cancer was evaluated for TP53 genetic variation and methylation status of CpG islands in several promoters of known disease-related genes. Tumor biopsy and corresponding plasma specimens were collected from study participants to determine whether the same genetic variations were present in both samples. The cfDNA isolation method provided a lower DNA detection limit of 144 pg, equivalent to DNA from approximately 24 cells. Normal pooled human plasma cfDNA averaged 110 copies/mL of the ACTB gene. Extracted cfDNA was suitable for gene-specific variant detection, sequencing, and promoter methylation analysis. DNA extracted from tumor biopsy and corresponding plasma specimens from two patients with advanced cancer revealed an identical, nonsynonymous variant present in both samples. Immunohistochemical analysis confirmed the TP53 mutant phenotype in the tumor specimens. Quantitative measurement of cfDNA represents a useful biomarker to follow treatment outcome and is a valuable tool with which to characterize specific genetic alterations for both patient selection and personalized treatment.

Precision profiling and components of variability analysis for Affymetrix microarray assays run in a clinical context.

Although gene expression profiling using microarray technology is widely used in research environments, adoption of microarray testing in clinical laboratories is currently limited. In an attempt to determine how such assays would perform in a clinical laboratory, we evaluated the analytical variability of Affymetrix microarray probesets using two generations of human Affymetrix chips (U95Av2 and U133A). The study was designed to mimic potential clinical applications by using multiple operators, machines, and reagent lots, and by performing analyses throughout a period of several months. A mixed model analysis was used to evaluate the relative contributions of multiple factors to overall variability, including operator, instrument, run, cRNA/cDNA synthesis, and changes in reagent lots. Under these conditions, the average probeset coefficient of variation (CV) was relatively low for present probesets on both generations of chips (mean coefficient of variation, 21.9% and 27.2% for U95Av2 and U133A chips, respectively). The largest contribution to overall variation was chip-to-chip (residual) variability, which was responsible for between 40 to 60% of the total variability observed. Changes in individual reagent lots and instrumentation contributed very little to the overall variability. We conclude that the approach demonstrated here could be applied to clinical validation of Affymetrix-based assays and that the analytical precision of this technique is sufficient to answer many biological questions.

A phase I dose-escalation study to a predefined dose of a transforming growth factor-β1 monoclonal antibody (TβM1) in patients with metastatic cancer

Transforming growth factor β (TGF-β) plays an important role in cancer. Monoclonal antibodies (mAb) designed to specifically block the TGF-β ligands, are expected to inhibit tumor progression in patients with metastatic cancer. TβM1 is a humanized mAb optimized for neutralizing activity against TGF-β1. The objective of this clinical trial was to assess the safety and tolerability of TβM1 in patients with metastatic cancer. In this phase I, uncontrolled, non-randomized, dose-escalation study, 18 eligible adult patients who had measurable disease per RECIST and a performance status of ≤2 on the ECOG scale were administered TβM1 intravenously over 10 min at doses of 20, 60, 120 and 240 mg on day 1 of each 28-day cycle. Safety was assessed by adverse events (as defined by CTCAE version 3.0) and possible relationship to study drug, dose-limiting toxicities and laboratory changes. Systemic drug exposure and pharmacodynamic (PD) parameters were assessed. TβM1 was safe when administered once monthly. The pharmacokinetic (PK) profile was consistent with a mAb with a mean elimination half-life approximately 9 days. Although anticipated changes in PD markers such as serum VEGF, bFGF and mRNA expression of SMAD7 were observed in whole-blood, suggesting activity of TβM1 on the targeted pathway, these changes were not consistent to represent a PD effect. Additionally, despite the presence of an activated TGF-β1 expression signature in patients’ whole blood, the short dosing duration did not translate into significant antitumor effect in the small number of patients investigated in this study

A Canonical Transforming Growth Factor Beta-Dependent Signaling Pathway is Present in Peripheral Blood Cells of Cancer Patients with Skeletal Metastasis

Background: Patients with advanced metastatic cancer often have elevated levels of circulating transforming growth factor beta (TGF-β) that is thought to stimulate receptor mediated signaling through phosphorylated SMAD transcription factors in peripheral blood mononuclear cells. Methods: To identify this TGF-β-dependent gene expression profile in cancer patients, we first evaluated a multi-gene expression profile from ex-vivo treated PBMCs with TGF-β1 stimulation. Change in expression, when challenged with a specific TGF-β receptor type kinase inhibitor was then derived to be ligand and inhibitor specific. Once this profile was established, we examined its role in identifying the activation of a canonical TGF-β1 signaling pathway in patient samples. Results: We discovered a 37 gene sub-set where the expression profile from ex-vivo treated PBMCs was significantly associated with SMAD phosphorylation in cancer patients. We found significant correlation between the ex-vivo derived expression signature and circulating levels of TGF-β1 in patient samples. Additionally, we report association between the expression profile and the presence of several plasma proteins in disease samples that are known to be concomitantly present with TGF-β- dependent pathway activation. Conclusion: An expression profile for TGF-β1 cytokine mediated-signaling in cancer patients is identified, which may serve as a biomarker to measure the pharmacodynamic effect of TGF-β inhibitors during clinical drug development and as marker of disease diagnosis.

Abstract 1738: Assay development for detecting cMET expression in circulating tumor cells (CTC), a potential patient tailoring marker for evaluation of cMET inhibitors

Hepatocyte growth factor and its receptor c-MET have been implicated in tumor formation and progression as well as drug treatment resistance to targeted agents such as EGFR inhibitors. cMET over-expression or gene amplification has been reported in a wide variety of human malignancies correlating with poor patient prognosis. A reasonable hypothesis for experimental agents targeting the cMET receptor is that patients with tumors expressing high levels of cMET may respond better. CTC counts are prognostic of survival in metastatic breast, colorectal, and prostate cancers (NEJM 2004;351:781-91; J Clin Oncol 2008;26:3213-21; Clin Can Res 2008;14:6302-9). CTCs are shed from tumors and are believed to be representative of cells migrating to form distal metastasis. The presence of these cells in blood provides an attractive and easy source for serial monitoring of tumors without the limitations of traditional solid tumor biopsy. We describe here the development of an assay that measures cMET expression in CTCs. The CTC assay was developed using the Veridex CellSearch® System, and RUO reagents for enumeration of CTCs and a Lilly proprietary cMET antibody (Ab) optD11. For assay development, cultured tumor cell lines with differing cMET expression levels representing solid epithelial tumor types were spiked into whole blood drawn into a CellSave tube. For initial assay development, mouse whole blood was used and results reproduced subsequently with human whole blood from healthy subjects. The spiked tumor cells in blood samples were recovered using the CellCapture™ Mouse/Rat CTC kit (for mouse blood) or CellSearch® CXC kit (for human blood), supplemented with the fluorescent dye (R- phycoerythrin) conjugated anti-cMET Ab. cMET in the recovered tumor cells was detected in the open/fourth channel on the CellTrack® Analyzer II®. Several proprietary and commercial cMET antibodies were screened for specificity and sensitivity. A commonly used commercially available cMET Ab (Santa Cruz Biotechnology C28) was not able to discriminate between cMET negative and positive cell lines. Results were confirmed by flow cytometry and by western blot analyses. optD11 was selected for CTC cMET expression assay development for the following reasons: a) ability to detect different cMET levels among positive cell lines (H441, MKN45, SNU5, SKOV3) while identifying SKBR3 as cMET negative; b) high affinity for cMET to allow for an image acquisition time of 0.02-0.04 second in the open channel; c) tolerance to the presence of LY2875358 (LA480), an experimental therapeutic anti-cMET Ab, allowing evaluation of cMET levels in CTCs post-treatment. The CTC assay for cMET expression, as well as a CTC assay measuring cMET gene amplification are currently being implemented in the early phase clinical studies for Lilly cMET inhibitors (JSBA, NCT01285037 & JTBA, NCT01287546). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1738. doi:1538-7445.AM2012-1738