Annual Meeting Proceedings Part 1 - American Society of Clinical ...

292s Genitourinary Cancer
4560 Poster Discussion Session (Board #14), Mon, 8:00 AM-12:00 PM and
11:30 AM-12:30 PM
Development of a needle biopsy-based genomic test to improve discrimination
of clinically aggressive from indolent prostate cancer. Presenting
Author: Eric A. Klein, Glickman Urological and Kidney Institute, Cleveland
Clinic, Cleveland, OH
Background: Standardized, quantitative tools are needed to improve discrimination
of clinically aggressive from indolent prostate cancer at diagnosis.
We previously identified multiple genes and biological pathways in radical
prostatectomy (RP) specimens associated with clinical recurrence and
adverse pathology. We evaluated whether measurement of these genes in
prostate needle biopsy (Bx) specimens predicts adverse pathology - high
grade (GS � 4�3) and/or non-organ-confined disease (pT3) - in a separate
study of AUA low-intermed risk pts treated w/ RP. Methods: All AUA
low-intermed risk (cT1-T2a, GS 3�3/3�4) pts treated w/ RP �6 mos from
Bx at Cleveland Clinic (1999-2007) w/ available Bx tissue and �3 pos
cores were evaluated. Expression of 81 prognostic genes identified in
previous RP studies and 5 reference genes was quantitated by RT-PCR in
60 �m manually microdissected FPE prostate needle Bx tissue. Association
of gene expression w/ adverse pathology was analyzed by logistic
regression controlling false discovery rate (FDR) using Storey’s method.
Results: RT-PCR was successful for 167 of 171 (98%) pts (92 AUA low, 75
intermed). At RP, 58 pts (35%) had high-grade and/or non-organ confined
disease. 58 of 81(72%) genes predicted high grade and/or non-organconfined
disease (FDR�10%), including all stromal response and androgen
genes and the majority of cellular organization genes (82%).
Proportionately fewer proliferation (40%), stress response (29%), and
basal epithelial (25%) genes were associated w/ adverse path. Androgen
and cellular organization genes consistently predicted lower risk; stromal
response and proliferation genes predicted higher risk; (majority of std odds
ratios �1.5, comparable to the prognostic strength of ER in breast cancer).
Conclusions: Genes and biological pathways associated w/ clinically aggressive
prostate cancer in RP specimens can be reliably measured by RT-PCR
from fixed prostate needle biopsies and predict high-grade, non-organconfined
disease. These results support the potential value of a Bx-based
assay to inform selection of immediate treatment or active surveillance for
pts diagnosed with prostate cancer on needle Bx.
4562 Poster Discussion Session (Board #16), Mon, 8:00 AM-12:00 PM and
11:30 AM-12:30 PM
Predictive biomarkers in circulating tumor cells (CTC) from patients with
castration-resistant prostate cancer (CRPC) through genomic analysis.
Presenting Author: Daniel Costin Danila, Sidney Kimmel Center for
Prostate and Urologic Cancers, Memorial Sloan-Kettering Cancer Center,
New York, NY
Background: Mutations in the ligand binding domain of androgen receptor
(AR) in prostate cancer cells may alter their sensitivity to treatment with
specific antiandrogens. To predict for tumor sensitivity to treatment with
novel AR targeted therapies, we explored the frequency of mutation
detection and copy number alteration in CTC isolated from patients with
CRPC enrolled on trials with these targeted therapies. Methods: We used
fluorescence-activated cell sorting (FACS) methodology to enrich EpCAM� ,
CD45- , DAPI- cells. For mutation detection and genomic copy number
alteration in CTC in low number of cancer cells found in clinical samples,
we optimized next-gen deep sequencing by Illumina. Results: In patients
with progressive CRPC , �10 or �50 EpCAM� events (EPE) were isolated
by FACS in 88% or 58% of patients, in whom 32% and 10% had
unfavorable (�5 cells/7.5 ml) CTC counts using CellSearch. EPE, expressing
prostate-specific mRNAs, provide sufficient high quality DNA for
genomic sequencing and copy number analysis. Adequate coverage was
obtained from as few 50 EPE, with a recovery rate of 89% from FACS sorted
samples. The detection threshold of a mutation was established at 1:4
alleles. To further expand genomic profiling in CTC, we optimized Nimblegen
exome mutation detection by deep sequencing on HiSeq PE101. Our
initial analysis established the polymorphism frequency detection thresholds
in heterogeneous cell populations, and confirmed the sequencing
coverage. Somatic missense mutations in AR, APC and TP53 found in CTC
but not in paired WBC were confirmed by Sanger sequencing. In parallel,
copy number alterations in CTC are studied. Conclusions: Somatic mutations
detected in CTC isolated from patients with CRPC can serve as
predictive markers of tumor sensitivity to targeted therapies. We established
standard operating procedures for specimen processing, and confirmed
the sequencing coverage and polymorphism detection thresholds in
heterogeneous cell population. Currently we are proceeding to clinical
samples to study the associations between specific molecular alterations in
CTC as predictive markers of sensitivity and clinical outcomes.
4561 Poster Discussion Session (Board #15), Mon, 8:00 AM-12:00 PM and
11:30 AM-12:30 PM
Gene expression biomarkers to predict overall survival of prostate cancer
patients. Presenting Author: Chunde Li, Department of Clinical Oncology,
Karolinska University Hospital, Stockholm, Sweden
Background: Current clinical parameters are not accurate in the prediction
of overall survival of prostate cancer patients. Methods: Driven by cancer
stem cell hypothesis and by using a simple bioinformatics analysis, we
identified 641 genes with either consistently high or low level of expression
in human embryonic stem cells. We showed that these 641 genes had
strong power to classify cancers into different biological subtypes and
named them as embryonic stem cell gene predictors (ESCGPs). Nineteen
selected ESCGPs and five control genes were analyzed by multiplex
quantitative PCR in prostate fine needle aspiration (FNA) samples taken at
diagnosis. The cohort included 189 patients diagnosed during 1986-
2001. Of these patients, 97.9% had overall and cancer specific survival
data and 77.9% were treated by medical or surgical castration as the
primary treatment. The cohort was divided into a discovery and two
validation subsets. The univariate and multivariate Cox proportional hazards
and Kaplan-Meier plots were used in the survival analysis. A published
dataset was used for an external validation. Results: Ten gene markers F3,
WNT5B, VGLL3, CTGF, IGFBP3, c-MAF-a, c-MAF-b, AMACR, MUC1 and
EZH2, showed a significant correlation to overall or cancer specific survival.
Four of these genes, F3, IGFBP3, CTGF and AMACR, were independent of
all clinical parameters. An expression signature of F3, VGLL3 and IGFBP3
characterized patients into three subtypes. The median overall survival was
2.60 years in the high risk, 3.85 years in the intermediate risk and 7.98
years in the low risk subtype, corresponding to a HR of 5.86 (95% CI
2.91-11.78, p�0.001) for the high risk and 3.45 (95% CI 1.79-6.66,
p�0.001) for the intermediate risk over the low risk subtype. Two gene
markers, F3 and EZH2, were further verified by the external validation.
Conclusions: The new ESCGP gene markers and the expression signatures
can be used to predict the overall and cancer specific survival of prostate
cancer patients.
4563 Poster Discussion Session (Board #17), Mon, 8:00 AM-12:00 PM and
11:30 AM-12:30 PM
Generation of a prognostic cancer stem-like gene expression signature in
men undergoing radical prostatectomy for localized prostate cancer.
Presenting Author: Adrian Stuart Fairey, University of Southern California,
Los Angeles, CA
Background: Novel biomarkers are needed to predict recurrence after
radical prostatectomy for localized prostate cancer. Recent data suggest
that cancer stem-like cells (CSC) are present in prostate tumors, and the
CSC phenotype has been associated with an aggressive cancer phenotype.
We investigated whether tumor mRNA levels of genes typically expressed
by CSC are associated with disease recurrence. Methods: Clinically annotated
prostatectomy specimens (FFPE) were used for this nested casecontrol
study. Samples represented men who underwent radical
prostatectomy and either experienced a recurrence (PSA or clinical;
n�152) or no recurrence (controls; n�124) with minimum 5-year followup.
Men were excluded if they received neoadjuvant hormone therapy.
Cases and controls were frequency matched based on D’Amico risk group.
Laser capture microdissection with mRNA extraction and quantitative
RT-PCR were used to quantify the expression of 12 candidate CSCassociated
genes (ALDH1A1, Axin2, Bmi1, CD133, CD44a, CTNNB1/�catenin,
ITGA2/integrin �2, NANOG, Nkx3-1, Notch1, OCT 4, TACSTD2/
Trop2). Univariable and multivariable analyses were conducted to measure
associations with recurrence. Results: mRNA data were evaluable for 241 of
276 patients. Univariate Wilcoxon analysis showed that 4 genes (Axin2,
p�0.001; CD44a, p�0.036; OCT 4, p�0.017; TACSTD2, p�0.008) were
expressed at low levels in tumors of patients whose disease recurred.
Recursive partitioning analysis identified 3 genes significantly predictive of
disease recurrence (Axin2, NANOG, CTNNB1), with cutpoints yielding
odds ratios (OR) of 8.5 (Axin2low /NANOGhigh ;n�94, 95%CI 3.7-19.2) and
5.1 (Axin2low /NANOGvery high /CTNNB1low ; n�26, 95%CI 1.7-14.8).
Conclusions: We identified a novel CSC-associated 3 gene expression
signature with the potential to predict recurrence after radical prostatectomy.
Axin2 is known to interact with CTNNB1/�-catenin in the Wnt
signaling pathway, which in turn has been shown to regulate expression of
NANOG, a key protein that mediates pluripotency. In vitro experiments and
an independent cohort validation study are planned based on these novel
findings.
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