Volume 90 � Number 5S � Supplement 2014 Poster Presentations S71

263Noninvasive, Highly Specific Detection of T790M-MediatedAcquired Resistance in EGFR Mutant NSCLC Using a Novel DropletDigital PCR (ddPCR)eBased Cell-free Plasma DNA (cfDNA) AssayTranslational Research

A.G. Sacher,1,2 S. Mach,1 M. Messineo,3 D.M. Jackman,1 P.A. Janne,1,3

C.P. Paweletz,3 and G.R. Oxnard1; 1Dana-Farber Cancer Institute, Boston,

MA, 2University of Toronto, Toronto, ON, Canada, 3Belfer Institute for

Applied Cancer Science, Boston, MA

Purpose/Objective(s): The T790M mutation is the most common cause

of acquired resistance to EGFR kinase inhibitors, occurring in more than

50% of patients. Promising mutant-specific third generation EGFR kinase

inhibitors are being developed. However, selection of candidates for these

therapies requires invasive re-biopsy for tissue genotyping. We recently

reported on a new assay for plasma genotyping using ddPCR of cfDNA.

This plasma genotyping assay has the potential to noninvasively detect

T790M and accelerate delivery of appropriate therapies e potentially

obviating the need for biopsy entirely. We hypothesize that this assay can

be used to accurately detect the presence of the EGFR T790M resistance

mutation compared to standard tissue genotyping.

Materials/Methods: We identified patients (pts) with advanced EGFR

mutant NSCLC and acquired resistance to erlotinib that underwent

rebiopsy and plasma collection on 4 IRB-approved protocols. Rebiopsy

specimens underwent clinical EGFR genotyping. Plasma was collected in

EDTA tubes, cfDNA extracted, and EGFR genotype quantified using

ddPCR assays for L858R, exon 19 del, and T790M. Serial plasma geno-

typing on treatment was performed for a subset of pts with acquired

resistance initiating new treatment.

Results: A total of 41 pts undergoing rebiopsy for acquired resistance to

erlotinib had plasma available for analysis. Standard tissue genotyping on

these specimens detected the T790M resistance mutation in 26 pts (63%).

Plasma genotyping for T790M was concordant with rebiopsy T790M

status in 32/41 pts (78%). Plasma genotyping for T790M exhibited a

specificity of 93% (14/15 pts - false positive rate 7%), sensitivity of 70%

(18/26 pts) and a positive predictive value of 95% (18/19 pts) in this cohort

of patients. Posttreatment plasma specimens were available for 11 pts with

detectable T790M; the 6 pts with a partial response on imaging had a

significant decrease in plasma concentration compared to the 5 pts without

a response (mean 373 copies/mL decrease vs 22 copies/mL increase,

PZ.05). Treatment included platinum-doublet (1 pt), second generation

EGFR TKI (2 pts), and third-generation EGFR TKI (7 pts).

Conclusions: Plasma genotyping of cfDNA with ddPCR can detect

potentially actionable resistance mutations in EGFR mutant NSCLC with

acquired resistance to kinase inhibitors. The high specificity and positive

predictive value of this assay support its further development as a tool for

therapy selection despite its modest sensitivity. The quantitative nature of

this assay may also allow for early prediction of treatment failure. Clinical

development of this assay is ongoing.

Author Disclosure: A.G. Sacher: None. S. Mach: None. M. Messineo:

None. D.M. Jackman: G. Consultant; Genentech, Foundation Medicine.

P.A. Janne: G. Consultant; Pfizer (uncompensated). K. Advisory Board;

BI, Roche, Genentech, Astra-Zeneca, Sanofi, Chugai, Merrimack, Forma,

Clovis. C.P. Paweletz: None. G.R. Oxnard: K. Advisory Board; Clovis

Oncology, Genentech, BI, Novartis, Astellas, Chugai, Astra-Zeneca. Q.

Patent/License Fee/Copyright; Patent pending plasma genotyping assay.

264Tumor Biomarker Evaluation of 6785 Patients for CombinationTreatment Strategies in NSCLCTranslational Research

R. Feldman,1 J. Xiu,1 and R. Salgia2; 1Caris Life Sciences, Phoenix, AZ,2University of Chicago, Chicago, IL

Purpose/Objective(s): Non-small cell lung cancer (NSCLC) exhibits

activation of multiple tumor pathways. Presence of multiple aberrations

may account for drug resistance as well as strategies for combination

therapies. We examined concurrent aberrations of biomarkers in NSCLC to

present an overview of potential patient cohorts who may benefit from

such combinations.

Materials/Methods: 6785 NSCLC cases were evaluated. Specific testing

was performed and included a multiplatform approach: sequencing

(Sanger, NGS), protein expression (IHC), and gene amplification (CISH/

FISH).

Results: The EGFR mutation (MT) rate in this population was 12.7%

(135/1059), of which 57% (55/96) overexpressed EGFR (IHC) and 61%

(169/277) had EGFR gene amplification (FISH). This demonstrates the

dependence on the EGFR pathway and potential importance of dual in-

hibition with cetuximab and EGFR TKIs. Sixty-six percent (63/96) and 7%

(6/90) of EGFR MT patients were MET high (IHC) and amplified (CISH),

respectively, suggesting potential benefit from dual targeting of EGFR and

MET. Interestingly, TP53 mutations were observed in 54% (70/129) of

EGFR MT which has important implications for resistance to EGFR TKIs

and possible cross-resistance to radiation therapy. ALK translocations were

observed in 101 of 3611 (2.8%) patients, among which 19% (7/36), 3% (1/

29) and 2% (1/48) carried concurrent EGFR, METand HER2 amplification

(ISH), respectively, suggesting the potential for combining crizotinib with

agents such as cetuximab, onartuzumab, or trastuzumab. A BRAF muta-

tion was observed in 3.3% (34/1061), among which EGFR and MET

expression was high by IHC (both have been implicated in resistance to

BRAFi in other tumor types) in 58% (15/26) and 48% (12/25), respec-

tively, indicating benefit from combination of newly approved dabrafenib

with cetuximab or onartuzumab.

Conclusions: This is one of the largest analyses in the United States of

biomarkers in NSCLC. Our study shows the nonexclusive activation of

multiple pathways. These results also show the importance of multiplat-

form testing as both protein, gene copy number, and genetic alterations

were detected.

Author Disclosure: R. Feldman: A. Employee; Caris Life Sciences. J.

Xiu: A. Employee; Caris Life Sciences. R. Salgia: None.

265Prediction of NSCLC Survival With the PAM50 Breast CancerSignatureTranslational Research

J.M. Siegfried,1,2 Y. Lin,2 S. Dacic,2 L.P. Stabile,2 B. Diergaarde,2 H. Lin,2

T. Nukui,2 and M. Romkes2; 1University of Minnesota, Minneapolis, MN,2University of Pittsburgh, Pittsburgh, PA

Purpose/Objective(s): Lung tumors often express the estrogen receptor b(ERb) and progesterone receptor (PR). We hypothesized that breast cancer

survival signatures may also predict lung cancer survival.

Materials/Methods: We examined the relationship between disease-free

survival (DFS) and gene expression using 2 breast cancer survival signatures

(Oncotype DX and PAM50) in 104 early-stage (IA and IB) non-small cell

lung cancers. Criteria for cases were: fresh-frozen tumor tissue fromprimary

T1N0 or T2N0 adenocarcinoma or squamous cell lung cancer with no

neoadjuvant therapy; smoking history, age, sex, tumor size, and outcome

were known.mRNAexpressionwas examinedwith the IlluminaHumanHT-

12 v4BeadChip.Datawere subjected to background subtraction and quantile

normalization. Supervised Principal Component Analysis was used to

evaluate prediction of DFS for each gene panel as a group and by using the

top genes showing differential expression in the lung cancer cohort. All re-

sults were subjected to 10-fold cross-validation. A validation cohort of 64

lung cancer cases of all stages was also examined.

Results: Genes from the Oncotype DX panel showed no differential

expression and had no ability to separate cases based on DFS. For the

PAM50 gene panel, 3 probes (CXXC5, FGFR4, and FOXC1) showed

significant differential expression (P<.05) and 23 probes showed dif-

ferential expression at P<.1. The PAM50 gene panel was able to

separate lung cancer cases based on DFS (HR 1.9, PZ.03); PAM50

genes with differential expression at P<.1 also separated the cohort into

4 prognosis groups (PZ.008 for trend). The worst prognosis group had a

HR of 4.76 (PZ.002). These genes were next analyzed in a subset of 44

International Journal of Radiation Oncology � Biology � PhysicsS72

stage I cases for whom ERb immunohistochemistry (IHC) scores were

known. ERb was detectable in all cases. Comparing ERb high (Allred

score of 4 or more, NZ 20) and ERb low (Allred score of 4 or less, N Z24) cases, the top PAM50 genes were more informative in ERb high

cases (HR 11.7, PZ.0007) compared to ERb low cases (HR 3.38,

PZ.045). Similar analyses in the Oncotype DX gene panel did not

improve DFS prediction. We next validated the top PAM50 genes in a

different cohort of 64 NSCLC cases, which included all stages and

histologies and had ERb IHC scores. The top PAM50 genes were also

able to predict DFS in this cohort: HR 2.19, PZ.034 in all cases and HR

3.24, PZ.042 in ERb high cases. Pathway analysis indicated that the

informative PAM50 genes describe a network that contains the ER, PR,

HER2/HER3, neuregulins, FOXC1, FGFR4, and cyclin E.

Conclusions: These results suggest that genes involved in interactions

between ER, PR and HER signaling may be predictive of NSCLC survival,

especially in early-stage lung cancer that is ERb positive, and provide

further evidence for the importance of hormonal pathways in the biology

of lung cancer.

Author Disclosure: J.M. Siegfried: E. Research Grant; NIH, V Founda-

tion, Somalogic, Inc. K. Advisory Board; Free to Breathe. Y. Lin: E.

Research Grant; NIH. S. Dacic: E. Research Grant; NIH. L.P. Stabile: E.

Research Grant; NIH. B. Diergaarde: E. Research Grant; NIH. H. Lin:

None. T. Nukui: None. M. Romkes: E. Research Grant; NIH.

266Next-Generation Sequencing (NGS) in Advanced Lung Cancer in theCommunity to Prospectively Guide Clinical Trial SelectionTranslational Research

Z. Ma,1 T.M. Bauer,1,2 P.K. Chandra,3 D.J. Haynes,4 J.L. Prescott,3

D.M. Stults,1 S.F. Jones,1 J.D. Hainsworth,1,2 J.R. Infante,1,2

H.A. Burris,1,2 and D.R. Spigel1,2; 1Sarah Cannon Research Institute,

Nashville, TN, 2Tennessee Oncology, Nashville, TN, 3PathGroup,

Nashville, TN, 4Sarah Cannon Research UK, London, United Kingdom

Purpose/Objective(s): We launched a molecular profiling program in 10/

2012 at a single community practice in middle Tennessee. This program

was designed to discover molecular alterations with proven/potential

therapeutic significance to match pts to mutation-selective clinical trials.

Here we report the lung cancer cohort findings.

Materials/Methods: Tissue specimens from advanced lung cancer pts

were tested by NGS with 1000X coverage in a CLIA/CAP-certified lab to

detect hotspot mutations in 35 genes. Results were reported to the treating

physician <12 days of tissue procurement. Results were stored in a

database to enable correlation with clinical outcomes.

Results: From 10/2012-12/2013, 1039 pts were profiled across tumor

types; 238 (23%) were from lung cancer pts. 18% (43/238) of the lung

samples were inadequate for testing. Of the 195 pts with sufficient tissue,

80 (41%) had >1 mutation. The mutation frequency by histology was:

adenocarcinoma 59% (54/91), squamous 22% (10/45), large cell 67% (4/

6), and small cell 18% (5/28). The most frequent mutations were KRAS

and EGFR (16% and 12%, respectively). Fifty-two of 195 (27%) pts

have been prospectively enrolled to clinical trials. Twenty-three of 52

(44%) pts have “actionable” mutations. Mutations were considered

“actionable” if there is a known treatment that specifically targeted the

mutation or a target along the same molecular pathway. The other 29 pts

have either no mutation detected or nonactionable mutations. Among 23

enrolled pts with actionable mutations, 8 pts were enrolled to at least 1

treatment trial of agent(s) matched to the mutations, including 2 of 4

(50%) pts with FGFR mutations, 2 of 7 (29%) pts with MET mutations, 1

of 4 (25%) pts with SMO mutations and 1 of 24 (4%) pts with EGFR

mutations. Fifteen pts were enrolled to trials not matched to their mu-

tations including 7 pts in immunotherapy trials, 3 pts in antibody-drug

conjugate trials, and 4 pts with cytotoxic agents. Response rates and

durations will be presented.

Conclusions: Prospectively identifying lung cancer mutations andmatching

pts to clinical trials by NGS is feasible in the community. Forty-one percent

of ptswith lung cancer had at least onemutation. Twenty-sevenpercent of the

(52/195) pts were prospectively enrolled to clinical trials. Forty-four percent

(23/52) pts have actionable mutations and 8 of them were enrolled to

matched trials. As NGS panels expand and tissue procurement improves,

genomic profiling in the community could potentially broaden treatment and

clinical trial options for patients with lung cancer.

Author Disclosure: Z. Ma: None. T.M. Bauer: None. P.K. Chandra: A.

Employee; PathGroup. F. Honoraria; Pfizer. G. Consultant; Pfizer. M.

Stock; PathGroup. D.J. Haynes: None. J.L. Prescott: A. Employee;

PathGroup. D.M. Stults: None. S.F. Jones: None. J.D. Hainsworth:

None. J.R. Infante: None. H.A. Burris: None. D.R. Spigel: None.

267Genomic Mechanisms Underlying the Pathogenesis of Non-SmallCell Lung Cancer (NSCLC) in Young PatientsTranslational Research

Y. Feng,1 V. Varadan,2 S. Fink,2 W. zhang,3 L. Yin,3 A. Liu,4 K. Guda,2

and P.C. Ma3; 1University Hospital, Case Medical Center, Cleveland, OH,2Case Western Reserve University Comprehensive Cancer Center,

Cleveland, OH, 3Cleveland Clinic Taussig Cancer Institute, Cleveland,

OH, 4Cleveland Clinic Pathology and Laboratory Medicine Institute,

Cleveland, OH

Purpose/Objective(s): The incidence of NSCLC in patients (pts) under 45

years of age is w2% of total cases, with annual newly diagnosed cases

reaching 4500 in United States alone. Majority of these pts are diagnosed

at an advanced stage with poor outcomes. The underlying causative

mechanisms in the pathogenesis and progression in these pts remain

largely unknown, and could be different from elderly pts. The objective of

this study is to examine the genomic features of NSCLC in young pts

which therefore offer new opportunities for the development of novel

prognostic markers and therapeutic avenues.

Materials/Methods: We identified a total of 20 pts (60% female) diag-

nosed with NSCLC at an age of � 45, who underwent surgical resection

for the primary tumors or metastatic lesions from 2000 to 2012. Genomic

DNA from FFPE tumor samples and paired normal lung tissue/peripheral

blood was subjected to whole exome sequencing, and variant calling

performed using GATK and/or SOAPsnp algorithms to identify somatic

mutations in individual tumors. Pathway and protein-protein interaction

(PPI) network analysis on mutant genes was performed using KEGG/NCI-

PID databases and HotNet suite, respectively.

Results: Majority of the tumors had adenocarcinoma (nZ12) or squamous

cell (nZ4) histology. Six pts never smoked, while the others had a median

30 pack-year history. As expected, we found a significantly higher muta-

tion rate in smokers (Median, 3.47/Mb) compared to never-smokers

(Median, 0.76/Mb), with G:C/T:A transversions being more common in

smokers, and C:G/T:A transitions among never-smokers. Key driver

genes such as TP53 (50%) and KRAS (17%) showed mutations exclusively

in smokers, whereas EGFR mutations (14%) were observed specifically in

never-smokers. More important, global pathway/PPI analysis of mutant

genes revealed distinct sub-networks associated with cell adhesion/EMT

processes with a 7-fold enrichment in mutation frequency in these young

pts when compared to their overall frequencies in the COSMIC/TCGA

lung cancer dataset.

Conclusions: Our study identified novel candidate genes/pathways that

may play a key role in young age-at-onset NSCLC. Successful validation

of our findings would eventually lead to tailored therapies benefiting

younger pts.

Author Disclosure: Y. Feng: None. V. Varadan: None. S. Fink: None. W.

Zhang: None. L. Yin: None. A. Liu: None. K. Guda: None. P.C. Ma:

None.

268A Plasma MicroRNA Panel to Diagnose Esophageal Squamous CellCarcinoma and Predict the Effect of Radiation TherapyTranslational Research

Q. Yu,1 B. Li,1 and S. Fu2; 1Department of Radiation Oncology, Sixth

People’s Hospital of Jiao Tong University, Shanghai, China, 2Department