Isaksson 100930
EGFR status and downstream signaling in primary lung carcinoma:
Investigations on the DNA, RNA and protein levels
Isaksson S1, Salomonsson A1, Bendahl P-O1, Jönsson M1, Johansson L2, Jönsson P3,
Pettersson H4, Staaf J1, Jönsson G1, Planck M1*
Departments of Oncology 1, Pathology 2, and Thoracic Surgery 3, Lund University Hospital,
Sweden and Department of Molecular Medicine4, Malmö University Hospital, Sweden
*Correspondence to:
Dr. Maria Planck, Department of Oncology, Lund University Hospital, SE-221 85 Lund, Sweden.
Phone +46-46-177501, Fax +46-46-147327, E-mail maria.planck@med.lu.se
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Abstract
Background: Lung carcinomas frequently exhibit complex karyotypes and mutation patterns, involving a large
number of genes. The epidermal growth factor receptor (EGFR) pathway is targeted by recently developed
therapies for advanced disease. This study focus on how various parameters of EGFR status correlate within
the same primary lung tumors, differ between histological subtypes, and relate to involvement of other
receptor tyrosine kinases (e.g. HER2 and KIT) or to patterns of downstream signaling (through the RAS and
PI3K-AKT-PTEN pathways). Materials and Methods: Seventy-seven freshly frozen surgical specimens of
primary NSCLCs (n=61) and SCLC (n=16) were collected. Occurrence of mutations was examined with direct
DNA sequencing (EGFR, PIK3CA) or RT-PCR (KRAS). Gene amplifications / deletions were detected by
quantitative real-time PCR (EGFR) or microarray based comparative genomic hybridization (32k BAC arrays).
mRNA expression was investigated by RT-PCR (KIT) and microarray based gene expression analysis (Illumina).
Paraffin-embedded material was used for construction of tissue microarrays and immunohistochemical
analysis of expression of the EGFR, PTEN, P-AKT, KIT, HER2, ERK, and mTOR proteins. Results: In NSCLC,
whereas 2 tumors were positive and 19 were negative for all three variables of EGFR status, 30 tumors
exhibited positive protein expression alone, 5 were positive regarding gene amplification and protein
expression but harbored no mutation, and 5 tumors were mutation positive but had neither amplification nor
protein overexpression. In SCLC, positive EGFR protein expression was demonstrated in one and EGFR gene
amplification in 3 tumors, all mutation negative. KRAS mutations were observed in adenocarcinomas alone, 14
tumors. Summary: In NSCLC, EGFR protein expression correlated with amplifications but was negative in the
majority of tumors with mutation, thus further questioning the biological and clinical role for
immunohistochemical determination of EGFR status. EGFR, as well as its downstream signaling through KRAS,
were significantly less altered in SCLC compared to NSCLC (p=…and ….) whereas involvement of AKT and its
inhibitor, PTEN, were comparable between all subtypes of lung carcinoma.
KEYWORDS: LUNG CANCER ,
PIK3CA, KRAS
NSCLC, SCLC, AMPLIFICATION, MUTATION, PROTEIN EXPRESSION , EGFR, PTEN, P-AKT,
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INTRODUCTION
Lung cancer, with high incidence and poor prognosis, is the leading cause of cancer related death, accounting
for a third of all deaths from cancer worldwide1. Most lung cancer cases are classified as non-small cell lung
cancer (NSCLC), with adenocarcinoma and squamous cell carcinoma being the frequent histological subtypes.
Small cell lung cancer (SCLC) constitutes less than 20% of primary malignant lung tumors and, since very few
cases are available for surgical resection, the tumor biology is less understood in SCLC compared to NSCLC.
The development of targeted therapies directed against specific molecules involved in carcinogenesis
has recently increased the treatment possibilities. One main target is the epidermal growth factor receptor
(EGFR), inhibited by use of monoclonal antibodies or tyrosine kinase inhibitors2. The EGFR superfamily of
transmembrane receptors includes EGFR/erbB-1, HER2/erbB-2, HER3/erbB-3 and HER4/erbB-4. Binding of
ligand (epidermal growth factor (EGF), transforming growth factor-α (TGF-α), or neuregulins) to the
extracellular domain results in homo-or heterodimerization, consequently initiating tyrosine kinase activity of
the intracellular domains. Phosphorylated tyrosine residues then act as binding sites for signal transducers
which in turn initiate downstream intracellular signalling cascades, involving for example the RAS-RAF and
PI3K-AKT pathways, ultimately leading to carcinogenic events; cell proliferation, anti-apoptosis, metastasis and
angiogenesis3.
Lung tumorigenesis involves multiple genetic alterations and this complexity is challenging when
evaluating clinical and biological markers as prognostic or predictive of response to treatment with targeted
therapies. So far, non-smoking history, female sex, Asian ethnicity, adenocarcinoma, EGFR mutations,
increased EGFR gene copy number, and wild-type K-RAS, are factors that have been associated with sensitivity
to EGFR inhibitors4,5,6.
We characterized primary early stage SCLC and NSCLC regarding EGFR gene copy numbers, occurrence
of mutations in the tyrosine kinase domain of the EGFR gene, immunohistochemical expression of the EGFR
protein, and occurrence of mutations in the downstream K-RAS gene. …pAKT PTEN PIK3CA!!!!!!!!!!!!!!
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MATERIAL AND METHODS
Patients and tumors
Freshly frozen surgical specimens of primary lung carcinoma (62 NSCLCs and 16 SCLCs) were collected between
1989 and 2007 at the Lund University Hospital, Lund, Sweden. Clinical data were obtained from patient charts
(Supplementary table 1); Of the 78 patients, 40 (51%) were women and 38 (49%) men. Sixty patients had a
history of smoking, 11 patients (all NSCLC) were never-smokers, and data on smoking status was missing in 7
patients. Mean age at diagnosis was 66 years (range 37 -85 years). None of the patients had received
preoperative chemo/radiotherapy.
The tumors were reviewed by a pathologist and categorized as adenocarcinomas (48 tumors), squamous cell
carcinomas (14 tumors) or SCLC (16 tumors). In one case, radicality could not be judged but all other tumors
were evaluated as radically excised. Fifty-six tumors were staged as pT1-T2N0M0 according to the TNM
classification (1996). In addition, the series included one T3, one T4, one M1 (satellite tumor in another lobe
within the same lung), two N1 (bronchopulmonary ipsilateral) tumors, and one case of unknown N-status
(Supplementary table 1).
For 77 of the 78 cases the histopathological paraffin-embedded slides were available and the appropriate
blocks could be identified for construction of TMAs; Two 1 mm tissue cylinders were collected from each
tumor block and arranged in a recipient block using an electronic tissue arrayer (TMArrayer, Pathology
Devices, Inc.USA).
Microarray based CGH
DNA for array-based CGH analysis was extracted from the 62 freshly frozen tumor specimens using Proteinase
K (20mg/µl) digestion followed by phenol-chloroform purification according to published protocols.
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Microarrays used for the present aCGH investigations were produced at the SCIBLU Genomics-facilities, Lund
University, Sweden (http://www.lth.se/sciblu/services/dna_microarrays), using the 32k BAC Re-Array set Ver.
1.0 (BACPAC Resource Center, Children’s Hospital, Oakland Research Institute, US). This platform, including a
total of 32 433 BAC clones, provides 99 % coverage of the BAC fingerprint map (November 2001) and
comprises clones sampled from the RPCI-11, RPCI-13 (94%) and Caltech-D (6%) libraries. For all samples, 2µg of
tumor DNA and 1.5µg reference DNA (Promega Corporation, Madison, USA) was labeled with Cy3-dCTP and
Cy5-dCTP (Amersham Biosciences, Uppsala, Sweden), respectively, using BioPrime Array CGH Genomic
Labeling System (Invitrogen Life Technologies, Carlsbad, US). Purification of labeled DNA was done using the
Cyscribe GFX purification kit (Amershamn Biosciences) and the pooled tumor and reference DNA were mixed
with Human COT-1 DNA (1µg/µl, Invitrogen Life Technologies) and resuspended in a hybridization buffer
containing de-ionized formamide, Yeast-tRNA (Invitrogen Life Technologies) and dextrane sulphate. Array
slides were UV cross-linked (500 mJ/cm2) and pre-treated using “Pronto! Universal Microarray Hybridization
Kit” (Corning BV, Schiphol-Rijk, the Netherlands). Denatured and re-annealed DNA was applied to the arrays
and hybridization was performed in hybridization chambers (Corning BV) for 48-72 hours in a 37°C water bath.
Array slides were washed in post hybridization buffers with different contents of 20xSSC, 10% SDS and deionized formamide and scanned in an Agilent Microarray Scanner (Agilent Technologies, Palo Alto, CA).
Spots were identified using GenePix Pro. 4.1 (Axon Instruments) and the data was uploaded in BioArray
Software Environment (BASE) (Saal, et al. 2002). Background correction of the two channels was calculated
using the median-feature and median-local background intensities of the uploaded file. A signal to noise ratio
(SNR) was set to ≥ 5 for both channels and data was normalized using a pin-based lowess algorithm excluding
chromosome X. A moving average of 250 kbp was applied to the CGH-plotter tool and plots were created
excluding the X and Y chromosomes, and a noise constant was set to 25. Hierarchical cluster analysis was
performed in BASE using Pearson correlation coefficient distance measure. Cutoffs for gain and amplification
of the clones harboring the EGFR gene were set to log2 ratio 0.2 and 0.8, respectively.
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Direct DNA Sequencing
Mutations of exon 18 through 21 of the EGFR gene were analysed by direct DNA sequencing using the BigDye
Terminator Cycle Sequencing Kit v1.1 (Applied Biosystems®). The samples were initially purified and
subsequently amplified. PCR was performed in 10µL volumes using 80 ng (4µL 20ng/µL) template, 1, 75 µL
5xbuffer, 4 µL deionized water and 0, 5 µL Terminator reaction mix. The mutations in exons 18-21 were
determined using primers described in supplementary table 1. PCR was run under the following conditions; 25
cycles of denaturation at 96°C for 10 s, primer annealing at 50°C for 5 s and elongation at 60°C for 1 min.
Sequencing products were separated by capillary electrophoresis by ABI 3130xl Genetic Analyzer (Applied
Biosystems®). Analysis of the sequence curves was performed using the 3100 data collection software (Gene
Code Corporation®). To confirm the presence of sequence alterations, samples in which the studied sequences
were found to be nonsynonymous with the wild type EGFR sequence were resequenced after a repeated
extraction of DNA.
Quantitative real time-PCR
Quantitative real time-PCR was performed using Rotor Gene 3000 by Corbett Research and the binding dye
iTaqTM SYBR® Green Supermix (BIO-RAD). To determine the copy number of the EGFR gene we used the genes
for albumine and glucokinase as controls. The ratios were compared to similar ratios of control DNA. A
standard curve for each run was constructed from serial dilutions. The CT-threshold was set to 0.2.
Amplification mixes (20µL) contained 10 ng sample DNA, 10µL binding dye, 1µL primer and dH2O. Thermal
cycling conditions comprised 10 min at 95°C and 45 cycles at 95°C for 15 s, 55°C at 30s and 72°C at 30 s. All the
samples were analyzed in triplicate and the serial dilutions were performed in duplicates. Relative gene
expression was calculated using the Pfaffl method (supplementary table 2). The ratios presented are average
values of EGFR gene expression in relation to albumin and glucokinase. Ratio ≥ 1,5 signified amplification.
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KRAS mutation analysis
Mats – kan du komplettera?
Reverse transcriptase PCR
Annette – komplettera!
Immunohistochemical analysis
3 µm sections were cut out from the TMA blocks and EGFR protein expression was evaluated
immunohistochemically using the mouse monoclonal anti-human EGFR clone 2- antibody and the EGFR
pharmDxTM kit (Dako) according to the manufactor’s instruction manual. The membrane staining was scored
as 0 (negative) 1+ (weakly positive) 2+ (moderately positive) or 3+ (strongly positive) according to the EGFR
pharmDx interpretation guidelines. When there was lack of agreement in IHC scoring between the two TMA
cores from the same sample (Sofi – infoga detta! X/61 evaluable cases), the sample was scored according to
the most positive core. All samples were independently evaluated by three of the authors (SI, MJ, MP) with an
initial discrepancy in less than 2% of the judgments, and, after a consensus was achieved, these scorings were
confirmed by a pathologist (LJ).
Statistics
Pär-Ola ?
RESULTS
Seven of the NSCLC tumors (11%) harbored EGFR mutations, all summarized in table 2. Only two samples were
positive for both EGFR mutation and EGFR amplification (Tables 1 and 2) and there was thus no statistically
significant correlation between mutations and amplifications of the EGFR gene (p=0,18).
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61 of the NSCLC were available for analysis of EGFR protein expression (Table 4, fig 1). There was an
association between amplification and 3+ IHC staining of the tumors (p<0.001) although the two samples with
simultaneous EGFR amplification and mutation (table 3), were classified as 1+. Tumors with mutated but not
amplified EGFR (n=5) were classified as 0 (Table 4). Thus, in the IHC positive group (constituting 37/61 tumors)
the EGFR mutation frequency was 5% whereas 21% of the tumors in the IHC negative group (a total of 24 out
of 61 evaluable tumors) harboured EGFR mutations.
KRAS mutations were analyzed with RT-PCR. Of the 62 samples, 48 (77%) were wild type and 14 (23%) were
mutated (Table 3). KRAS and EGFR mutations were mutually exclusive. KRAS amplifications were identified
with aCGH in 5 (8%) of the tumors (Table 3).
Discussion
All major histological types of invasive lung carcinoma frequently exhibit molecular alterations that involve a
large number of genes, thus believed to have accumulated during a multistep carcinogenesis 7. To identify the
key molecules among multiple genetic alterations and to subsequently apply this knowledge on the
development of targeted therapies has therefore become a prioritized challenge in the fight against this
devastating disease. One validated target for lung cancer therapy is the EGFR pathway. EGFR is widely
expressed in lung cancer and has well-documented oncogenic activity through regulation of proliferation,
invasion, angiogenesis, and apoptosis8,9. We characterized early stage primary NSCLCs regarding alterations of
the EGFR pathway (EGFR copy numbers, mutations of the EGFR and KRAS genes, and immunohistochemical
staining of the EGFR protein).
We showed an absolute correlation between high amplification of EGFR as revealed by aCGH and increased
number of EGFR copies detected by qPCR (Table 1). Quantitative RT-PCR is thus reliable for validation of aCGHfindings.
Somatic EGFR mutations were found in 11% of the tumors. In line with previous data, all mutation positive
cases were adenocarcinomas and female patients were overrepresented (Table 2)10. We detected two
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insertions in exon 20 and one substitution in exon 21 although, as expected from previous studies, the most
common mutations were 15 /18 bp in-frame deletions in exon 19, proven as predictive markers for response
to EGFR inhibitors by several reports5,11,12.
Whereas strongly positive IHC correlated with EGFR amplifications, IHC turned out negative in the majority of
tumors with EGFR mutation in our study (Table 4). Our results thus further question the clinical use of IHC for
determination of EGFR status. Similar results can be found in previous reports demonstrating negative IHC
EGFR expression for about 40% of mutation positive tumors with unknown EGFR copy numbers13,14. The two
tumors with concomitant amplification and mutation of EGFR were classified as IHC 1+, pointing towards a
mechanism which results in lower IHC scoring when EGFR mutations are present in tumors with increased
EGFR gene copy numbers. Indeed, results from studies of mutant to wt allele ratios in lung tumors with
simultaneous EGFR copy gain and EGFR mutation, indicate that the mutant allele is selectively gained15. Of the
30 tumors in our series which were IHC positive without any revealed amplification or gain in the EGFR region,
only 3 were strongly stained, i.e. 3+. These tumors were all SqCC, perhaps suggesting that this histological
subtype might harbor mechanisms other than gene amplification that influence protein expression.
The high impact of alterations in EGFR and its downstream molecules on lung tumorigenesis is well described,
but how these different mechanisms of oncogenic activation interact within the very same tumors is less
frequently investigated. In total, we found KRAS mutations in 14 tumors (23%). Consistent with previous
reports, all patients were smokers or former smokers (Massarelli et al) and this was also true for patients
whose tumors demonstrated KRAS amplifications by aCGH in our study (5/62 tumors, table 3). In our material,
EGFR mutations and KRAS mutations were mutually exclusive as described in previous studies16 17. However,
EGFR amplifications and high level amplification of the 12p12.1 locus, containing KRAS (as revealed by aCGH)
were, in contrast, not mutually exclusive. Recurrent copy gains of the KRAS locus, demonstrated also in
genome wide studies by others, indicate functional significance of these copy number alterations18 (ref Weir et
al. Nature 2007). Furthermore, an association between activating KRAS mutations and KRAS copy number
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gain/amplification has been reported, suggesting that these gains undergo a positive selection, which is
stronger in mutant tumors, thus consistent with previous data on mutant to wt allele ratios of the EGFR gene
(ref Modrek et al. Molecular Cancer Research 2009 and Barmak Modrek). A relatively small overlap between
mutation and amplification was seen for KRAS (2 samples) and EGFR (2 samples) in our study. A possible
explanation for this could be that also wild-type alleles can act as tumor promoters if a selection pressure in
individual tumors modulates the copy numbers.
As mentioned above, EGFR mutations and EGFR amplifications in lung carcinoma are suggested as predictive
for positive outcome after treatment with EGFR inhibitors10,11. In our study, EGFR mutations and gene
amplifications affected the whole genome copy number profiles of the tumors in an unsupervised cluster
analysis of aCGH data. These correlations possibly reflect the importance of the EGFR pathway in NSCLC
tumorigenesis (Fig ). In light of this, one might postulate that the IHC patterns, with no correlation at all to
aCGH profiles, are less likely to mirror the true EGFR-related biology of the tumors (Fig ).
In conclusion, although high immunohistochemical EGFR expression was seen in tumors with amplification of
the EGFR gene, the consequent lack of expression in tumors with EGFR mutation again demonstrates the
limitations of EGFR immunohistochemistry as clinical predictor in lung cancer. Furthermore, and most
importantly, we postulate the existence of whole genome copy number profiles with respect to EGFR status
(amplification/mutation), thus again emphasizing the importance of this tumorigenic pathway in lung cancer
development. However, there is a need for confirming studies that correlate these and other biomarkers (e.g.
occurrence of extra EGFR gene copies, different EGFR mutation subtypes, KRAS mutations, MET amplification,
Akt overexpression, or LKB1 mutations) to distinct whole genome copy number profiles, and also to gene
expression and proteomic patterns.
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