KRAS Mutation in the Cell-Free DNA Predicts Survival in Advanced
Pancreatic Carcinoma Patients Treated with Gemcitabine and Erlotinib
Irene M. Hutchins, MD, William Holland, MS, Rebekah Tsai, MS, Leslie Snyder-Solis, MS, I-Yeh Gong, MD, Michael Tanaka, MD
Edward Kim, MD, PhD,, Philip Mack, PhD, Primo Lara, MD, Thomas Semrad, MD, MAS
Department of Internal Medicine, Division of Hematology & Oncology, University of California at Davis Medical Center, Sacramento, CA
INTRODUCTION
 Kirsten rat sarcoma (KRAS) gene
mutations are present in 80-95% of
pancreatic cancer tissue specimens,
however the prognostic value of KRAS
mutational status remains unclear.
RESULTS
SUMMARY OF RESULTS
 KRAS mutations were detected in the tumor tissue from 4 (80%) of the
5 patients with available tumor blocks.
 Plasma KRAS mutations were detected in the cell-free DNA from 10
(37%) of 27 patients, including 2 (50%) of those with known KRASmutated tumors.
EGFR
Biomarker groups (N=27)
 Erlotinib, an epidermal growth factor
receptor (EGFR) inhibitor, marginally
improves survival in advanced pancreatic
cancer treated with gemcitabine (median
survival 6.2 vs 5.9 months). [1]
 Patients with mutant plasma KRAS had significantly lower median PFS
(1.8 vs 4.6 months, p=0.014) and OS (2.8 vs 10.5 months, p=0.003)
compared to those without KRAS mutations detected in the plasma.
Progression-Free Survival
by Plasma KRAS
 KRAS mutations may constitutively
activate the molecular signaling cascade
downstream from EGFR, and are
therefore proposed to confer resistance
to EGFR inhibitors.
Overall Survival by Plasma KRAS
 AREG and EREG did not correlate with PFS or OS.
 After adjusting for age, gender, race, ethnicity, primary tumor site,
performance status, stage, and plasma DNA concentration, plasma
KRAS mutational status remained a strong predictor of overall survival
(HR 14.5, p=0.006).
 Limited availability of tumor tissue prohibits universal assessment of
KRAS mutational status in pancreatic cancer biopsy specimens.
DISCUSSION
 Attempts at detecting KRAS mutations non-invasively in the cell-free
DNA (i.e. circulating in the plasma) have yielded mixed results, with
detection rates ranging from 0% to 71%. [2-7]
 Plasma KRAS assays have not yet been utilized in clinical practice.
 A 1999 study found that the detection of mutant KRAS in cell-free DNA
was associated with shorter survival in pancreatic cancer [7], however
confirmatory studies are lacking.
 The epidermal growth factors amphiregulin (AREG) and epiregulin
(EREG) are involved in stimulating cell proliferation, and are
hypothesized to have a prognostic role in pancreatic cancer.
PURPOSE
 To determine the detection rate of KRAS mutations in the plasma of
advanced pancreatic cancer patients using a novel PCR assay.
 To evaluate the prognostic significance of KRAS mutations in the cellfree DNA, as well as AREG and EREG expression in patients with
advanced pancreatic cancer treated with gemcitabine and erlotinib.
METHODS
 Pre-treatment plasma samples were stored for 27 of 30 patients with
locally-advanced or metastatic pancreatic cancer enrolled in a phase II
clinical trial using gemcitabine and pharmacodynamically separated
erlotinib (UCDCC#211). Tumor blocks were stored for 5 patients.
 KRAS mutations can be detected non-invasively in the plasma of some,
but not all patients with KRAS-mutated pancreatic tumors.
Kaplan-Meier analysis demonstrates shorter progression free survival
in patients with KRAS mutations detected in the cell-free DNA.
Kaplan-Meier analysis demonstrates shorter progression-free survival
in patients with KRAS mutations detected in the cell-free DNA.
Kaplan-Meier analysis demonstrates shorter overall survival in
patients with KRAS mutations detected in the cell-free DNA.
Patient Distribution by Biomarker Category (N=27)
 Expression of the epidermal growth factors AREG and EREG do not
correlate with survival, possibly due to constitutive activation of tumor
cell proliferation pathways downstream from EGFR.
Low / Undetected
(n)
High / Detected
(n)
KRAS mutation
17
10
Mutation detected
DNA concentration
13
14
≥ Median (85.49 ng/mcL)
AREG
14
13
≥ Lower Limit Assay (15.6 pg/mL)
EREG
13
14
≥ Median (325.51 pg/mL)
Plasma Biomarker
)
Cut-point
 Further research is needed to determine if circulating mutant KRAS is
primarily a prognostic indicator or if it can be used as a predictive
biomarker for the efficacy of gemcitabine/erlotinib.
REFERENCES
Outcomes by Biomarker Category (Log-Rank Analysis)
Median Progression-Free Survival (months)
Progression Free Survival by Plasma KRAS
Plasma Biomarker
Low /
High /
HR
p-value
Detected
Undetected
KRAS mutation
4.6
1.8
2.89
0.014
Median Overall Survival (months)
Low /
High /
Undetected Detected
10.5
2.8
HR
p-value
4.69
0.003
 Progression-free survival (PFS) and overall survival (OS) were analyzed
using the Kaplan-Meier method, and log-rank tests were used to
compare survival distributions between groups. A Cox Regression
Model for OS was developed to adjust for clinical variables
1. Moore MJ, Goldstein D, Hamm J, et al. Erlotinib plus gemcitabine compared with
gemcitabine alone in patients with advanced pancreatic cancer: a phase III trial of
the National Cancer Institute of Canada Clinical Trials Group. Journal of clinical
oncology: official journal of the American Society of Clinical Oncology
2007;25:1960-6.
2. Sorenson GD. Detection of mutated KRAS2 sequences as tumor markers in
plasma/serum of patients with gastrointestinal cancer. Clinical cancer research: an
official journal of the American Association for Cancer Research 2000;6:2129-37.
3. Dianxu F, Shengdao Z, Tianquan H, et al. A prospective study of detection of
pancreatic carcinoma by combined plasma K-ras mutations and serum CA19-9
analysis. Pancreas 2002;25:336-41.
DNA concentration
2.6
2.1
0.69
0.368
5.0
9.0
0.74
0.525
AREG
2.6
2.1
0.79
0.582
5.0
7.1
0.92
0.867
4. Uemura T, Hibi K, Kaneko T, et al. Detection of K-ras mutations in the plasma DNA
of pancreatic cancer patients. Journal of gastroenterology 2004;39:56-60.
EREG
5.0
2.1
1.27
0.561
9.0
3.8
1.57
0.345
5. Marchese R, Muleti A, Pasqualetti P, et al. Low correspondence between K-ras
mutations in pancreatic cancer tissue and detection of K-ras mutations in
circulating DNA. Pancreas 2006;32:171-7.
SCORPION ARMS PCR
 Scorpion ARMS PCR was
used to detect KRAS codon
12 and 13 mutations.
 The presence of mutant KRAS in the circulating cell-free DNA
independently predicts shorter survival in patients with advanced
pancreatic carcinoma treated with gemcitabine and intermittent erlotinib.
 Surprisingly, detection of mutant KRAS in the plasma is not a surrogate
for plasma DNA concentration or cancer stage.
 Plasma AREG and EREG concentrations were measured using ELISAs.
 Cell-free DNA was extracted
from the plasma using the
Chemagen system (Perkin
Elermer), and quantified using
NanoDrop (Thermo Scientific).
 DNA levels ≥ median did not predict shorter survival compared to low
plasma DNA levels (9.0 vs 5.0 months, p=0.525).
Cumulative
2.6
5.9
Multivariate Analysis of Overall Survival A Cox Regression Model was developed to
adjust for clinical variables including age, gender, race, ethnicity, primary tumor site,
performance status, stage, and plasma DNA concentration. Plasma KRAS status remained
a strong independent predictor of overall survival (HR 14.5, p=0.006).
6. Dabritz J, Preston R, Hanfler J, Oettle H. K-ras mutations in the plasma
correspond to computed tomographic findings in patients with pancreatic cancer.
Pancreas 2012;41:323-5.
7. Castells A, Puig P, Mora J, et al. K-ras mutations in DNA extracted from the
plasma of patients with pancreatic carcinoma: diagnostic utility and prognostic
significance. Journal of clinical oncology : official journal of the American Society
of Clinical Oncology 1999;17:578-84.