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Original Article
KRAS status and clinical outcome in metastatic colorectal cancer patients treated with first-line FOLFOX chemotherapy
1Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY; 2Department of Biostatistics, University at Buffalo, Buffalo, NY
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Abstract
Background: Two previous first-line studies showed an improved trend in response rate (RR) and progression free survival (PFS) in metastatic colorectal cancer (CRC) patients with KRAS mutation. Others have reported a worsened outlook for metastatic CRC patients with KRAS mutation and a higher likelihood of metastatic disease to the lungs. In this study, we aimed to address the impact of KRAS on the pattern of metastatic disease at presentation and on RR and PFS with first-line 5-fluorouracil, leucovorin, and oxaliplatin (FOLFOX) chemotherapy.
Methods: Patients with CRC who underwent KRAS testing using DxS assay at Roswell Park Cancer Institute (RPCI) were identified. Patients with metastatic CRC treated with first-line FOLFOX +/- bevacizumab were assessed for response and survival using RECIST 1.1 guidelines. A two-sided Fisher's exact test was used to determine the statistical significance.
Results: 181 patients with metastatic CRC and KRAS testing were identified. 83/181 patients were treated with FOLFOX (+/- bevacizumab) in the first-line setting at RPCI and were evaluable as per study guidelines. KRAS mutation (MT) occurred in 40.31% cases. There was no difference in organ-metastases distribution, RR (56.60% in KRAS wild-type (WT) and 50% in KRAS mutant) or PFS (9.3 months KRAS WT and 8.7 months in KRAS MT) based on KRAS status.
Conclusion: In this single institute study, our findings do not support any predictive role for KRAS-MT in terms of response to FOLFOX first-line chemotherapy, or in terms of sites of metastatic disease at mCRC presentation.
Key words
metastatic colorectal cancer; KRAS; FOLFOX; cetuximab
J Gastrointest Oncol 2010; 1: 90-96. DOI: 10.3978/j.issn.2078-6891.2010.022
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Introduction
The treatment of metastatic colorectal cancer (mCRC) has
made significant progress in the past decade, including the
introduction of agents targeting epidermal growth factor
receptor (EGFR). The therapeutic success of monoclonal
antibodies against EGFR (cetuximab and panitumumab)
in treating patients with mCRC highlights the importance
of counteracting the EGFR pathway to control advanced
disease (1). In unselected patient populations, response to
anti-EGFR treatment has been modest, which prompted investigators to identify biomarkers that predict increased
likelihood of response in a subpopulation. Among a number
of potential biomarkers studied, mutational activation of
RAS oncogenes has emerged as the most important factor
for determining non-responsiveness to EGFR inhibitors.
KRAS is a protein which in humans is encoded by
the KRAS gene and functions as an essential component
of the EGFR signaling cascade. Activating mutations in
KRAS gene cause constitutively active Ras GTPase, which
leads to over-activation of downstream Raf/Erk/Map
kinase and other signaling pathways, resulting in cell
transformation and tumorigenesis (Fig 1) (2,3). KRAS
mutations are present in approximately 30% to 50% of colon
cancer specimens (4). Fearon and Vogelstein established a
stepwise hypothesis for colorectal cancer tumorigenesis and
delineated the importance of mutation in RAS gene as an
initiating event in the formation of malignant tumor (5).
Preclinical studies have suggested that constitutively
activated mutant KRAS can promote tumor invasion and
metastasis by stimulating matrix metalloproteases, cysteine
proteases, serine proteases, and urokinase plasminogen activator that facilitate migration through the basement
membrane (6,7,8). Despite such findings the role of KRAS
mutation in prognosis of mCRC patients is not clear. The
RASCAL study, which was the largest study designed to
analyze the prognostic value of KRAS status showed that
a glycine-to-valine mutation in codon 12 increased the
likelihood of disease relapse and a lower overall survival
(OS) (9). Multiple other studies with smaller sample size
did not demonstrate any impact of KRAS mutations on
survival (10,11,12). Even in the updated RASCAL II study,
the evidence of a statistically significant worse clinical
outcome was limited to stage III disease and was not
confirmed for other stages (13). These results are limited
by their retrospective nature and lack of adequate power to
detect significant differences.
The relationship between KRAS status of primary tumor
and stage at diagnosis as well as pattern of spread is also not
clear. Samowitz et al. reported that codon 12 mutations
in KRAS gene were found to be much more common in proximal tumors and were associated with advance stage
at presentation (14). Bazan and colleagues showed that
codon 12 mutation in tumor was associated with mucinous
histology and mutation in codon 13 was associated with
advanced Duke stage (15). In a retrospective study KRAS
mutation of the primary tumor was also associated with
higher incidence of metastatic disease to lungs (16).
Analysis of KRAS and BRAF mutation status in PETACC-3,
an adjuvant trial with 3,278 patients with stage II to III
colon cancer revealed that incidence of either mutation was
not significantly different according to tumor stage. KRAS
mutation was associated with grade of the tumor, while
BRAF mutation was associated with right-sided tumors,
older age, female gender, high grade, and MSI-high tumors.
KRAS mutations were not prognostically related to relapsefree
survival (RFS) or OS whereas BRAF mutation was not
prognostic for RFS, but was for OS, particularly in patients
with microsatellite instability-low (MSI-L) and stable
(MSI-S) tumors (17).
Multiple studies have demonstrated an association
between KRAS mutational status in the primary tumor
and resistance to EGFR inhibitors (cetux imab and
panitumumab) in patients with mCRC (18,19). Recently
based on convincing data, National Comprehensive Cancer
Network (NCCN) has also made recommendation that
patients with known KRAS mutations should not be treated
with EGFR inhibitors (20).
Although there is robust data regarding the association
of WT KRAS status and response to EGFR inhibitors,
the relationship between KRAS MT and response to first
line oxaliplatin based chemotherapy without anti-EGFR
antibodies is conf licting. Two previous first-line studies
showed an improved trend in response rate (RR) and
progression free survival (PFS) in mCRC patients with
KRAS MT, who were treated with first line chemotherapy
regimen including oxaliplatin without cetux imab or
panitumumab while others have reported a worsened
outlook for patients with KRAS MT who were treated
similarly (Table 1) (21,22).
In this study, we aimed to address the impact of KRAS
on the pattern of metastatic disease at presentation and on
clinical outcome with first line FOLFOX chemotherapy.
 
Figure 1 Epidermal growth factor receptor signal transduction pathway. * Common sites of mutation in colorectal cancer.
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Patients and methods
Study endpoints
The primary endpoint of this study was to compare the
progression free survival of KRAS WT and KRAS MT
CRC patients treated with first-line FOLFOX (with or
without bevacizumab) chemotherapy. Secondary endpoints
included overall survival, response rate, and pattern of metastatic disease in the KRAS WT and MT populations.
Patient population
All patients with metastatic colorectal cancer with a known
KRAS status and who were treated at Roswell Park Cancer
Institute (RPCI) with first-line FOLFOX or FOLFOX plus
bevacizumab were eligible for this study (Fig 2). Most of
these patients were treated at our institute. Patients who
received first line chemotherapy at a community hospital
were included in the study only if their imaging studies were
available for response evaluation.
Treatment plan
First line chemotherapy consisted of oxaliplatin 85mg/m2
infused over 2 hours; bevacizumab 5mg/kg intravenous
(I.V) over 10 minutes; leucovorin (LV) 400mg/m2 infused
during 2 hours, followed by fluorouracil (FU) as a 400mg/
m2 I.V. bolus on day 1 then a 2.4 grams/m2 continuous
infusion over 46 hours on a 14-day treatment cycle. Patients
receiving bevacizumab were dosed at 5mg/Kg every 2
weeks on day 1 of FOLFOX.
Efficacy assessment
CT images for all the patients were reviewed by the
investigators for evaluation of response. Response was
assessed according to revised RECIST (version 1.1) criteria
for response evaluation in solid tumors (23). Change in
tumor burden was classified as complete response (CR),
partial response (PR), stable disease (SD) or progressive
disease (PD). Patients with CR or PR were included in
overall response rate (ORR). Patients had imaging studies
every 4 cycles of chemotherapy. Best response after the start of chemotherapy was considered for ORR.
Statistical analysis
Response to treatment according to the mutational status
was evaluated using the Fisher’s exact test. Progression
free survival (PFS) was defined as the time from initiation
of FOLFOX (with or without bevacizumab) until first
evidence of radiographic progression or death, whichever
occurred earlier. Overall survival (OS) was calculated as the
period from the beginning of treatment to death or the last
follow-up at which point data were censored. OS and PFS
were estimated with the Kaplan-Meier algorithm. A P-value
P≤0.05 was considered to indicate statistical significance.
Test for differences in survival distributions was done using
the log-rank test. Statistical analysis and plots were done
using SAS, version 9.1, statistical software (SAS Institute
Inc., Cary, NC).
KRAS mutation detection
Tumor DNA was isolated from formaldehyde-fixed paraffinembedded
tissues and screened for the presence of KRAS
codon 12 and 13 mutations using a DxS K-RAS mutation
test kit (DxS Ltd). This assay detects 7 KRAS mutations
in codons 12 and 13 using qualitative real-time PCR assay
combining Scorpions® and ARMS® (allele-specific PCR)
technologies. Detectable mutations are; 1. Gly12Asp
(GGT>GAT) 2. Gly12Ala (GGT>GCT) 3. Gly12Val
(GGT>GTT) 4. Gly12Ser (GGT>AGT) 5. Gly12Arg
(GGT>CGT) 6. Gly12Cys (GGT>TGT) 7. Gly13Asp
(GGC>GAC). The method used in this kit is highly sensitive
and depending on the total amount of DNA present, can
detect approximately 1% of mutant in a background of wildtype genomic DNA.
Figure 2 Study scheme for assessment of outcome based on KRAS status in patients treated with first-line FOLFOX with or without Bevacizumab. CRC=colorectal cancer, mCRC= metastatic colorectal cancer, FOLFOX= Folinic acid, Fluorouracil, Oxaliplatin.
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Results
Patient demographics
We identified 191 patients with CRC who underwent
KRAS gene mutation testing using DxS assay at Roswell
Park Cancer Institute (RPCI). KRAS DsX assays were
performed between June 2008 and May 2009. 181/191
patients had conf irmed metastatic disease and were
included to assess the impact of KRAS status on the
pattern of metastatic disease. The sites of metastastic
disease at presentation were classif ied into 3 main
categories: liver, lung, and peritoneum. Only 83 of the
181 patients received first-line FOLFOX or FOLFOX plus
bevacizumab chemotherapy at RPCI and were subject
to efficacy analysis. Baseline patient characteristics are
summarized in Table 2.
KRAS mutation status and pattern of metastasis
Mutations in the KRAS were detected in 77 (40.31%)
tumors with the fol lowing distr ibutions: codon 13
mutat ions (Aspa r tate) 9.09%, codon 12 mutat ions
(Aspartate) 46.75%, (Valine) 19.48%, and (Alanine) 7.79%.
The most common site of metastasis was the liver, followed
by lung, and peritoneum. The pattern of metastases was not
significantly different between KRAS WT and MT patients
(Table 3). Liver metastases occurred in 63/104 (60.6%)
of KRAS WT and 41/77 (53.3%) of KRAS MT (P=0.36).
Lung metastases occurred in 34/104 (32.7%) of KRAS
WT and 24/77 (31.2%) of KRAS MT (P=0.87). Peritoneal
metastases occurred in 27/104 (26%) of KRAS WT and 13/77 (16.9%) of KRAS MT (P=0.15).
KRAS mutations and outcome with first-line FOLFOX
+/- bevacizumab
Out of 181 patients with metastatic disease, 83 received
first line FOLFOX (+/- bevacizumab) chemotherapy
at RPCI and were evaluable for response. Among
the response-evaluable patients, 44/53 (83.02%) and
24/30 (80%) received bevacizumab in combination
with FOLFOX in the KRAS WT and MT populations,
respectively (P= 0.771).
The best overall response rate was 56.60% (27/53
PR and 3/53 CR) in KRAS WT and 50% (15/30 PR) in
KRAS mutant patients (P=0.64). None of the patient
with KRAS mutation had CR. Twenty one patients
(39.6%) had stable disease in KRAS WT and 15 (50%)
in KRAS mutant patients (Table 3).
The median PFS was 9.3 months (95% CI, 7.85 to
10.78) in KRAS WT and 8.7 months (95% CI, 5.42
to 15.18) in KRAS MT populations (P=0.395, logrank
test) (Fig 3). Patients with resection of metastatic
disease after first-line FOLFOX (+/- bevacizumab) were
not included for estimation of PFS. Seven patients in
KRAS MT population and four patients in KRAS WT
population had resection of metastatic disease after first
line chemotherapy. Median OS was 34.8 months (95% CI, 23.5-42.5) in KRAS WT and not achieved in MT patients
(Fig 4).
  
Figure 3 Kaplan-Meier survival analysis for progression-free survival time according to KRAS status (P=0.3954).
Figure 4 Kaplan-Meier survival analysis for overall survival (OS) time according to KRAS status (P=0.7407). Median OS was not achieved.
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Discussion
Several studies have reported that WT KRAS status of tumor
is predictive of response to addition of EGFR inhibitors
(cetuximab or panitumumab) in chemotherapy regimens
involving oxaliplatin (FOLFOX or XELOX) (21,24). Although the combination of EGFR inhibitors with first-line FOLFOX
or XELOX significantly enhanced the clinical outcome
in patients with WT KRAS tumors in several studies,
the effect of KRAS status on patients receiving FOLFOX
alone or FOLFOX plus bevacizumab remains uncertain.
Table 1 summarizes effect of KRAS mutation on clinical
outcome of patients treated with FOLFOX or XELOX in
various studies. In the phase II OPUS (Oxaliplatin and
Cetuximab in First-Line Treatment of metastatic CRC)
study, patients with KRAS mutation had a trend to a better
response rate and PFS when treated with FOLFOX-4 alone
when compared to patients with WT KRAS. Similarly,
the phase III CAIRO2 (capecitabine, irinotecan, and
oxaliplatin in advanced colorectal cancer) study showed a
trend towards an improvement in response rate and PFS in
patients with KRAS mutation treated with capecitabine/
oxaliplatin plus bevacizumab compared to patients with
WT KRAS. In contrast, no trends in improved outcome
were noted in the PACCE (Panitumumab Advanced
Colorectal Cancer Evaluation), PRIME (Panitumumab
Randomized Trial In Combination With Chemotherapy
for Metastatic Colorectal Cancer to Determine Efficacy)
or COIN (Continuous Chemotherapy Plus Cetuximab or
Intermittent Chemotherapy with Standard Continuous
Palliative Combination Chemotherapy with Oxaliplatin
and a Fluoropy rimidine in First Line Treatment of
Metastatic Colorectal Cancer) studies when comparing
KRAS MT or WT patients receiving non-EGFR inhibitorcontaining
oxaliplatin-based therapy.
Interestingly in OPUS and CAIRO2 studies patients
with KRAS mutation who received cetuximab in
combination with FOLFOX or XELOX had significantly worse response rate and survival compared to similar group
who received only FOLFOX or XELOX. These findings
raised the concern that the addition of EGFR inhibitors to
FOLFOX or XELOX could impair the efficacy of oxaliplatin
component of the combined regimen in patients with KRAS
mutation.
In this study, we did not find any advantages to tumors with KRAS MT in terms of response or progression free survival with FOLFOX-based chemotherapy. In our
study, patients with KRAS mutation had response rate
of 50% with FOLFOX ± bevacizumab which was not
significantly different than that of patients with KRAS
WT (56.6%). These response rates are comparable to other
studies utilizing FOLFOX and bevacizumab as first line
chemotherapy in metastatic CRC patients. Both treatment
groups were well balanced in terms of bevacizumab use
(83.02% in KRAS WT type and 80% in KRAS MT) making
bevacizumab an unlikely confounder on the impact of
KRAS on outcome. William et al. have shown that benefit
derived from addition of bevacizumab to chemotherapy in
patients with mCRC is not affected by their KRAS status
(25).
In this study we also examined if KRAS status of tumor
was predictive of certain pattern of metastasis in patients
with metastatic CRC. Incidence of KRAS mutation in
our study was similar to other large studies (13). Cejas et
al. reported that tumors with KRAS mutation had higher
propensity to metastasize to lungs (16). We did not confirm
this finding in our study as tumors with KRAS wild type or
mutant status had similar propensity to metastasize to liver,
lung or peritoneum. In the RASCAL study it was suggested
that individual mutations may have different impact on tumor biology as glycine to valine mutation on codon 12
of the KRAS gene had significant association with more
aggressive biological behavior and worse outcome. The
incidence of predominant mutations (Glycine to Aspartate
and Glycine to valine on codon 12) in our study was similar
to the study by Cejas et al. making it an unlikely explanation
for different results.
In summary, our single institution experience does
not support any predictive role for KRAS-MT in terms
of response to FOLFOX first-line chemotherapy (in the
absence of anti-EGFR inhibitors), or in terms of sites of
metastatic disease at presentation.
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References
Cite this article as:
Sharma N, Saifo M, Tamaskar I, Bhuvaneswari R, Mashtare T, Fakih M. KRAS status and clinical outcome in metastatic colorectal cancer patients treated with first-line FOLFOX chemotherapy. J Gastrointest Oncol. 2010;1(2):90-96. DOI:10.3978/j.issn.2078-6891.2010.022
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