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

10586 General Poster Session (Board #48C), Mon, 1:15 PM-5:15 PM
Multiplex TaqMan assays for a 7-gene prognostic immune response score to
differentiate risk among women with ER-negative breast cancer. Presenting
Author: John J. Sninsky, Celera Corporation, Alameda, CA
Background: Teschendorff et al. (Breast Cancer Res 2008) reported a
7-gene, immune response-enriched gene expression classifier using microarrays
that was prognostic in women with ER-negative breast cancer. To
facilitate further evaluation of the prognostic value of a classifier in archival
fixed tumor tissue, we developed two multiplex TaqMan assays to quantify
the expression levels of these genes, then trained and validated a 7-gene
immune response score (IRS). Methods: RNA was extracted from FFPE
sections of 185 ER-negative breast cancer patient samples from Mayo
Clinic (n�81), Guy’s Hospital (n�50) and California Pacific Medical
Center (CPMC) (n�27). Patients (N� and N-) did not receive adjuvant
therapy and were followed for a minimum of 5 years. Amplification of i)
LY9, TNFRSF17, HLA-F, and IGLC2 and ii) XCL2, SPP1, C1QA and two
reference genes NUP214 and PPIG were carried out in two multiplex
RT-PCR TaqMan reactions and relative expression levels were determined
using the DDCT method. The training set, comprised of the Mayo Clinic
samples, was used to develop an IRS by using a summation of expression
levels of the 7 genes. Association of the IRS with distant metastasis was
assessed using a Cox proportional hazards model. The score was subsequently
tested in a validation set (Guy’s � CPMC). Results: The amplification
efficiency of each gene within the multiplex reactions was comparable
to singleplex reactions. Duplicate amplifications showed excellent assay
reproducibility and sensitivity (R2�0.98). The hazard ratio for a single
standard deviation decrease in the IRS was 1.45 (95% CI 0.99-2.12,
p�0.056) in the training set and 2.18 (1.32-3.61, p�0.002) in the
validation set. ROC curves of continuous IRS to predict distant metastasis
within 5 years had AUC of 0.74, 95% CI � 0.60-0.88. Conclusions: Robust
multiplex TaqMan assays for FFPE tissue extracted RNA were developed for
a 7-gene IRS. Increases in risk are associated with decreases in the IRS.
Immune related gene expression clearly plays an important role in the
spectrum of risk of ER-negative tumors. Additional signatures are in
development that further support this conclusion.
10588 General Poster Session (Board #48E), Mon, 1:15 PM-5:15 PM
Multisite validation of a 92-gene molecular classifier: Diagnostic accuracy
and clinical utility in metastatic cancer. Presenting Author: Catherine A.
Schnabel, bioTheranostics, Inc., San Diego, CA
Background: Accurate tumor classification is increasingly critical to individualized
care as patient outcomes improve with use of targeted cancer
therapies and predictive biomarkers. In metastatic cancers of uncertain or
unknown origin, identification of a primary site remains equivocal in a
significant number of cases. Gene expression signatures may improve
accuracy and specificity of tumor classification, however, large-scale,
blinded validation studies that demonstrate stable performance in metastatic
and poorly differentiated cases are currently lacking. Methods: Seven
hundred and ninety cases (51% female, 44% metastatic, 63% grade 2 and
3, 14% limited tissue specimens) representing 28 tumor types and 50
subtypes were processed and adjudicated between Mayo Clinic, UCLA and
Massachusetts General Hospital. Blinded FFPE tumor sections were
submitted and tested using a 92-gene RT-PCR assay (CancerTYPE ID,
bioTheranostics Inc.). Molecular predictions were evaluated for concordance
with the reference diagnosis; diagnostic accuracy between clinical
subsets was compared. Results: The 92-gene assay demonstrated overall
sensitivities of 87% for tumor classification and 82% for subtyping.
Forty-seven (5.9%) cases were considered unclassifiable. Comparative
analysis of performance between metastatic (n�329) and primary (n�414)
tumors showed no statistically significant difference in accuracy (85% vs
88%, p�0.16). Similarly, no significant decreases in performance were
observed across histological grades (p�0.58) and when comparing limited
tissue to excisional biopsy specimens (p�0.16). Strong precision was
demonstrated for accurate identification of a primary tumor in tissues
biopsied from common metastatic sites, reported as positive predictive
values of 100% for lung, brain and peritoneum, 92% for ovary and 80% for
liver. Conclusions: Results from this multisite, blinded study validate the
diagnostic accuracy of the 92-gene assay for classifying a diverse set of
tumors, and support its clinical utility in the diagnosis of metastatic tumors
to determine tissue of origin, and in the differential diagnosis of metastatic
vs new primary disease.
Tumor Biology
677s
10587 General Poster Session (Board #48D), Mon, 1:15 PM-5:15 PM
Specific and sensitive detection of BRAF and KRAS mutations in clinical
samples. Presenting Author: Vanessa Kelchner, Swift Biosciences, Inc,
Ann Arbor, MI
Background: The development of highly sensitive and specific genotyping
assays that are suitable for clinical research and molecular diagnostics
opens new opportunities for the detection, assessment, and research of
cancer. Swift Biosciences has developed myT Primer qPCR technology
which has unique structural and thermodynamic properties that enables
highly sensitive mismatch discrimination. The myT Primer reagents can
detect 1% mutant BRAF or KRAS present in a background of 103 wild-type
genomic DNA copies with no background signal from wild-type. A separate
ultrasensitive BRAF format has been developed which enables the detection
of a single mutant BRAF copy in � 104 wild-type genomic DNA copies,
representing 0.01% detection. Methods: To evaluate the performance on
clinical samples, DNA was prepared from 26 melanoma, 40 colorectal, and
49 lung tumor fresh frozen and FFPE samples, and their corresponding
normal adjacent samples. qPCR was performed using standard cycling
conditions in single tube format with allele-specific myT primers to assess
two BRAF alleles or seven KRAS alleles. Performance of the assay was
evaluated on the ABI 7500, CFX96, and Roche LightCycler. A subset of
samples were selected for validation by Sanger sequencing. Results:
Mutations of BRAF in melanoma, colorectal, and lung tumors were
observed to be 46%, 5%, and 2%, in concordance with published data for
the frequency of BRAF mutations. Mutations of KRAS in colorectal cancer
were observed to be in 17/40, or 42% of samples, consistent with
published reports of KRAS in colorectal cancer. 34% of lung cancer
samples had a KRAS mutation, also consistent with available statistics.
Mutation status was confirmed with Sanger sequencing in the subset of
selected samples. Conclusions: The extreme selectivity of the myT BRAF
Primers makes them well suited for genotyping of conventional FFPE and
frozen tissue samples, whereas the ultrasensitive format is ideal for use
with difficult samples such as needle biopsies, CTCs, and serum. The
extreme selectivity of these primers results in a definitive Yes/No answer
and eliminates the need to use a delta Ct method.
10589 General Poster Session (Board #48F), Mon, 1:15 PM-5:15 PM
A pilot study on the clinical value of 18F-sodium fluoride PET/CT in
advanced prostate cancer. Presenting Author: Joseph W. Kim, Medical
Oncology Branch, National Cancer Institute, National Institutes of Health,
Bethesda, MD
Background: We evaluated the clinical utility of 18F-sodium fluoride PET/CT
bone scan ( 18F-NaF) in the detection of bone metastases in patients (pts)
with prostate cancer in comparison with Technetium-99m MDP bone scan
(TcBS). Methods: In a prospective study, from October 2010-December
2011, 30 prostate cancer pts (ages 51-79), 21 with known bone metastases
and 9 without known bone metastases, had18F-NaF and a TcBS
performed. Abnormal foci of uptake on both TcBS and 18F-NaFwere classified as benign, malignant or indeterminate. Benign lesions included
uptake in the joints and linear uptake at the endplates of the vertebral
bodies consistent with degenerative changes. Malignant uptake on 18F-NaF scans was confirmed by characteristic osteoblastic features on CT. All TcBS
and 18F-NaF were reviewed by an experienced nuclear medicine physician.
For the patient-based analysis, scan results were categorized as positive
(POS) � any malignant lesion; indeterminate (IND) � not distinctly
malignant or benign; negative (NEG) � benign lesions only. Results: In the
lesion-based analysis, 21 of 30 (70%) pts had more malignant lesions
identified on 18F-NaF than on TcBS. The mean number of additional
malignant lesions per patient on 18F-NaF vs TcBS was 4. Eight of the 30 pts
had same number of malignant lesions identified in both studies. One of 30
pts had one less malignant lesion identified on 18F-NaF than on TcBS. CT
correlation by 18F-NaF PET/CT of this particular lesion did not confirm
osteoblastic feature. Malignant lesion distribution on 18F-NaF included:
spine (28%), thorax (26%), pelvis (24%), long bones (13%) and skull
(10%). In the patient-based analysis, 24 pts (80%) were POS by 18F-NaF, of whom 14 pts were POS, 8 were IND, and 2 were NEG by corresponding
TcBS; in the 4 pts with NEG 18F-NaF, zero were POS, 2 were IND and 2
were NEG by corresponding TcBS. Conclusions: 18F-NaF identified more
malignant lesions than TcBS. 18F-NaF may also add useful information in
the management of advanced prostate cancer pts with and without known
bone metastases.
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