AJTR Copyright © 2009-All rights reserved. Published by e-Century Publishing Corporation, Madison, WI 53711
Am J Transl Res 2012;4(2):229-239

Original Article
Biomarkers of the Hedgehog/Smoothened pathway in healthy

Sunil K Kadam, Bharvin K R Patel, Emma Jones, Tuan S Nguyen, Lalit K Verma, Katherine T Landschulz, Sergey
Stepaniants, Bin Li, John T Brandt, Leslie H Brail

Translational Medicine, Eli Lilly and Company, Indianapolis, IN 46285, USA; Oncology Discovery, Eli Lilly and
Company, Indianapolis, IN 46285, USA; European Early Phase Statistics, Eli Lilly and Company, Indianapolis, IN
46285, USA; Oncology-Statistics, Eli Lilly and Company, Indianapolis, IN 46285, USA; Covance Biomarker Center
of Excellence, Greenfield, IN 46214, USA; Covance Genomic Laboratory, Seattle, WA 98109, USA; Oncology
Business Unit, Eli Lilly and Company, Indianapolis, IN 46285

Received March 29, 2012; accepted April 15, 2012; Epub April 18, 2012; Published April 30, 2012

Abstract: The Hedgehog (Hh) pathway is involved in oncogenic transformation and tumor maintenance. The
primary objective of this study was to select surrogate tissue to measure messenger ribonucleic acid (mRNA)
levels of Hh pathway genes for measurement of pharmacodynamic effect. Expression of Hh pathway specific
genes was measured by quantitative real time polymerase chain reaction (qRT-PCR) and global gene
expression using Affymetrix U133 microarrays. Correlations were made between the expression of specific
genes determined by qRT-PCR and normalized microarray data. Gene ontology analysis using microarray data
for a broader set of Hh pathway genes was performed to identify additional Hh pathway-related markers in the
surrogate tissue. RNA extracted from blood, hair follicle, and skin obtained from healthy subjects was analyzed by
qRT-PCR for 31 genes, whereas 8 samples were analyzed for a 7-gene subset. Twelve sample sets, each with
≤500 ng total RNA derived from hair, skin, and blood, were analyzed using Affymetrix U133 microarrays.
Transcripts for several Hh pathway genes were undetectable in blood using qRT-PCR. Skin was the most
desirable matrix, followed by hair follicle. Whether processed by robust multiarray average or microarray suite 5
(MAS5), expression patterns of individual samples showed co-clustered signals; both normalization methods
were equally effective for unsupervised analysis. The MAS5- normalized probe sets appeared better suited for
supervised analysis. This work provides the basis for selection of a surrogate tissue and an expression analysis-
based approach to evaluate pathway-related genes as markers of pharmacodynamic effect with novel inhibitors
of the Hh pathway. (AJTR1203005).

Keywords: Hedgehog, smoothened, biomarkers, cancer, skin

Address all correspondence to:
Dr. Sunil K Kadam
Translational Medicine
Eli Lilly and Company
Indianapolis, IN 46285, USA.
Tel: 317 966 2930; Fax: 317 433-1071
E-mail: s.kadam@lilly.com