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�������������	���������������������������	P��FKSKS�K&M� �������!!�*+8!��"��$�,�55��5D:6$^6�*$e(h!(D�$=+�A challenge to use epidermal growth factor receptor (EGFR) mutations in serum as predictive and prognostic markers in patients with advanced non-small cell lung cancer (NSCLC)

Running title: Detection of EGFR mutations in serum DNA
Zhijun Li a, Wanru Cai b, Wenlong Bao c, Chuming Jiang c, Yongjun Zhang c,d,*
a Respiratory Department of Zhejiang Hospital (21 Ling Yin Road, Xihu District, Hangzhou, Zhejiang Province, 310013)
b Respiratory Department of Xinhua Hospital in Zhejiang Province (318 CaoWang Road , Gongsu District, Hangzhou, Zhejiang Province, 310005)
c Department of Integration of Traditional Chinese and Western Medicine
d Zhejiang Key Laboratory Diagnosis and Treatment Technology on Thoracic Oncology, Zhejiang Province, Zhejiang Cancer Hospital, Hangzhou, 310022, China
* Corresponding author at: Department of Integration of Traditional Chinese and Western Medicine, Zhejiang Cancer Hospital, 38 Banshan Road, Hangzhou, China, 310022
Tel: +86-571-8646 5336; Fax: +86-571-8646 1861
E-mail: zhangyongjun770323@163.com

Funded by: Zhejiang Provincial Traditional Chinese Medicine Foundation for Outstanding Young Talent (No. 2012ZQ005).
ABSTRACT
Objectives: Analysis of standard epidermal growth factor receptor (EGFR) mutations requires a minimum amount of tumor tissue, which may not be available in certain situations. In this study, we used peripheral blood samples from patients with advanced stage non-small cell lung cancer (NSCLC) to detect EGFR mutations with the purpose of guiding clinical use of EGFR-targeted therapy in lung cancer.
Materials and Methods: Peripheral blood sequencing was carried out in an automated 3730 sequencer folds: Peripheral blood samples were collected from 300 advanced NSCLC patients, including 155 adenocarcinoma and 145 squamous cell carcinoma. DNA was extracted from plasma of these patients for nested PCR amplification and plowed by analysis from nested PCR products of EGFR exons 18, 19, and 21 mutations. 
Results: No EGFR mutation was found from any of the blood samples of the 300 NSCLC patients.
Conclusion: These data indicate that it is difficult to identify EGFR mutations in circulating DNA of advanced stage NSCLC patients. According to our findings, detection of EGFR mutations in serum as a clinical method for deciding tyrosine kinase inhibitor therapy is not viable.
Keywords: 
Serum, Epidermal growth factor receptor (EGFR), Mutation, Non-small-cell lung cancer (NSCLC), Tyrosine kinase inhibitor
1. Introduction

Lung cancer is the most common fatal cancer in men and women in the United States, as well as worldwide (1,2). Non-small cell lung cancer (NSCLC) constitutes 80% of lung cancer cases, and at diagnosis about 70% of patients with lung cancer have advanced-stage disease. Only 15.9% of lung cancer patients live 5 years or longer after diagnosis (3). If NSCLC patients are not treated, the prognosis is a median survival of less than five months. Even so, NSCLC patients  survival is usually prolonged less than 6 months by chemotherapy, which is also often associated with significant toxicity (4).  
Epidermal growth factor receptor (EGFR) is a 170-kDa transmembrane glycoprotein that consists of an extracellular domain that recognizes and binds to specific ligands, a hydrophobic transmembrane domain, which is involved in interactions between two receptors within the cell membrane, and an intracellular domain that contains the tyrosine kinase enzymatic activity. EGFR is detectable in primary tumors in approximately 80% to 85% of NSCLC patients, although the levels of expression vary widely. Activation of the EGFR pathway results in the initiation of cancer proliferation, increased metastasis potential, and neoangiogenesis. EGFR tyrosine kinase has been implicated in the initiation and progression of NSCLC (5). Inhibition of EGFR kinase activities by EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib results in effective treatment for patients with NSCLC (6-8). Indeed, the Iressa Pan-Asia Study (IPASS) clinical trial in Asia and randomized controlled studies on advanced NSCLC have confirmed that EGFR- activating mutations are the main predictor of clinical outcome with TKI therapy (9-11). The most common EGFR-activating mutations were in exon 19 (Del19) and exon 21 (L858R), which are found in approximately 10% of Caucasian patients with NSCLC and up to 50% of Asian patients (12). So, the detection of EGFR mutations in tumor tissue can be used to as a sensitivity molecule target for tyrosine kinase inhibitor therapy (13).
Although testing of biopsied tumor tissue remains the recommended gold standard for detection of EGFR mutations, because these are limited by small biopsies with insufficient DNA, various methods have been used to detect somatic mutations of EGFR from DNA in the bloodstreams of NSCLC patients (13-15). Several groups have detected EGFR mutations in DNA isolated from plasma or serum samples and demonstrated correlations between the mutation status of serum and tumor tissue. There is 59%-92.9% agreement in EGFR mutation status between circulated DNA and tumor tissue samples (Table 1) (13, 15-21). 
Moreover, blood samples from patients with lung cancer contain much higher levels of DNA than blood from cancer-free patients. Most of this excess circulating DNA is believed to be released from the dying lung cancer cells at primary or metastatic sites (22). Thus, detection of EGFR mutations in serum DNA may provide a noninvasive and replicable source of genotypic information that might facilitate clinical decision making, including guidance for treatment, monitoring disease progress or effects due to EGFR-TKI, at the time of diagnosis and in the later course of the disease, especially in patients with NSCLC treated with TKIs (13, 14, 23).
Therefore, DNA from a patient s serum or plasma may be used as an alternative source of tumor DNA. Consequently, the number of studies evaluating the potential use of serum or plasma DNA in cancer diagnosis and prognosis has increased steadily in the past decade (22). To assess the clinical reliability of EGFR mutations detected from blood specimens, we analyzed EGFR exons 18, 19, and 21 mutations from nested PCR products using serum or plasma samples from 300 advanced NSCLC patients.

2. Methods

2.1. Patient characteristics

Three hundred patients with advanced NSCLC who were examined and treated at the Zhejiang Cancer Hospital in China from May 2012 to December 2012were enrolled in this study. Clinical information, including patients  gender, age, tumor histopathology, and smoking history were available for all patients. Histopathologic diagnosis for these advanced NSCLC cases included adenocarcinoma (ADC, n = 155) and squamous cell carcinoma (SCC, n = 145). All patients accepted from 1 to 4 cycles of platinum-based combined chemotherapy. Enrolled patients had no history of previous primary cancer other than lung cancer. All subjects provided informed consent, and the Ethics Committee of Zhejiang Cancer Hospital approved the study. 

2.2. Blood collection and DNA extraction

Peripheral blood samples were obtained from each of the 300 patients in the course of treatment and the separated blood samples were stored at  70 �C until used. Blood DNA was isolated from whole blood of these patients using the Axyprep Blood Genomic DNA Miniprep Kit (Axygen Biosciences, Union City, CA). The extraction was performed in accordance with the manufacturer s instructions. The extracted DNA was eluted in 50 �L buffer (provided by the kit) and stored at  20 �C until used.


2.3. Nested PCR amplification and purification

Amplification of exons 18, 19, and 21 were done in duplicate for each sample obtained from blood specimens. Primer sequences for EGFR exons 18, 19, and 21 nested polymerase chain reactions and extensions were designed by the Assay Designers software version 3.0 (Sequenom) and were processed in accordance with the standard protocols for iPLEX chemistry. Primers were synthesized by Sangon Biotech (Shanghai, China; Table 2). The first PCR assays were carried out in a 50-�L volume that contained 1 �L of extracted DNA, 1 �L of primer R1 and F1, and 0.5 �L of Taq DNA polymerase. DNA was amplified for 35 cycles at 95 �C for 3 min, 94 �C for 30 s, 55 to 60 �C for 35 s, 72 �C for 40 to 50 s; and then 5 to 8 minutes repaired extension at 72 �C. The second set of PCR reactions were carried out in a 25-�L volume that included 2 �L DNA from the first PCR products, 0.3 �L of primer R1 and F1, and 0.3 �L of Taq DNA polymerase. DNA was amplified at 95 �C for 5 min; the next 35 cycles at 95 �C for 30 s, 55 �C for 35 s, and 72 �C for 30 s; and then a 10-min extension at 72 �C. The PCR amplified fragment of exons 18, 19, and 21 are 395 bp, 590 bp and 365 bp, respectively. Nested PCR products were electrophoresed in 1% agarose gel, and only those PCR products that produced a positive band were purified using a PCR product purification kit (Sanprep, SK1141).

2.4. EGFR nested PCR amplification sequencing analysis

Before sequencing, PCR sequencing reaction for PCR products of EGFR exon 18, 19, and 21 was used by BigDye Terminator v1.1 kit (Applied Biosystems, USA) and performed in accordance with the Kit s instructions, and all PCR assays were carried out in a 20 �L volume, including 1 �L of purified PCR products, 8 �L of BigDye (2.5�), and 1 �� of primers (3.2 pmol/�L). The PCR sequencing reaction was performed at 96 �C for 1 min; and then 25 cycles at 96 �C for 10 s, 50 �C for 5 s, and 60 �C for 4 min. Sequencing was carried out in an ABI 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). All sequence variants were confirmed by sequencing the products of independent PCR amplifications.

2.5. Statistical analysis

In this study, no EGFR mutation was found in any samples from advanced NSCLC. Therefore, no statistical analysis was conducted.

3. Results

3.1. Patient characteristics

The subjects of the study consisted of 300 advanced-stage NSCLC patients, 217 men and 83 women (Table 2). Among them, 155 patients had ADC (82 men and 73 women), and the remaining 145 patients had SCC (135 men and 10 women). The mean age of the patients in the ADC and SCC groups was 56.30 years (range, 30 to 79 years) and 60.24 years (range, 42 to 85 years), respectively. Of the 300 subjects, 168 were former or current-smokers, including 3 women (all of whom had ADC); the others (132) had never smoked. At the time of blood collection, all patients had received 1 to 4 cycles of platinum-based combined chemotherapy.
To investigate the status of EGFR exon (18, 19, and 21) mutations from peripheral blood in advanced NSCLC patients, we collected a whole blood sample from each subject. After nested PCR amplification and purification, all PCR products underwent bidirectional sequencing on ABI 3730 sequencers via ABI Big Dye Terminator 3.1 chemistry. Unfortunately, no mutation of EGFR exons 18, 19, or 21 was detected in the serum DNA of the 300 patients, regardless of gender, age, ADC or SCC, or smoking history (Table 3). The results of DNA sequencing indicated the presence of EGFR wild-type sequences in all patients (Table 4).

4. Discussion

In the present study, we analyzed the serum samples of 300 advanced stage NSCLC patients for EGFR exons 18, 19, and 21 mutations through direct sequencing of nested PCR products. We found no mutations in EGFR exons 18, 19, or 21. Our results suggest that basing decisions regarding TKI therapy on the presence of EGFR mutations in serum has questionable value.
EGFR mutations activate EGFR pathways, and the most common EGFR-activating mutations have been found in exon 19 (Del19) and exon 21 (L858R) [12]. Although DNA from tumor tissue for detection of EGFR mutations is still considered the most direct and reliable source for sequence analysis, and is therefore the most widely used, DNA is often insufficient from small tumor tissue. Various methods have been used as alternative sources of tumor DNA to detect somatic mutations of EGFR, and one of these is DNA in the bloodstream of NSCLC patients (13-15). Several groups have detected EGFR mutations in DNA isolated from plasma or serum samples and showed some correlation between mutations in plasma and tumor tissue. The concordance has ranged from 59% to 92.9% for EGFR mutations between circulated DNA and tumor tissue samples (Table1) (13, 15-21). However, Song et al. (24) reported that no mutated DNA from tumor tissues resembled matched serum circulating DNA from 50 resectable patients, based on PCR amplification and direct sequencing. As in Song et al. s study, we failed to detect mutations in EGFR exons 18, 19, or 21 in advanced stage NSCLC patients using serum samples. 
EGFR mutations mainly occur in Asian, ADC, and women patients who have never smoked (25). In particular, exon 19 deletions and exon 21 (L858R) mutations account for up to 90% of all EGFR mutations, and are thereby predictors of response to the TKIs gefitinib and erlotinib. In the present study, we enrolled 300 advanced stage NSCLC patients, comprising 155 ADC and 145 SCC cases. Only 70 NSCLC patients had histopathologically diagnosed ADC who did not have a smoking history. It is also possible that the samples were too small to detect EGFR mutations from serum DNA in our study. Further study with larger populations may improve the detection of EGFR mutations. 
It is known that large amounts of tumor-derived DNA may be released from the primary or metastatic tumor mass in which cell necrosis or lysis of tumor cells occurs, resulting in elevated serum DNA levels (26). Therefore, freely circulating DNA is due not only to the necrosis or lysis of tumor cells, but also the renewal of normal cells. Although considerably degraded, DNA can be collected from a patient s serum or plasma and used as a surrogate source of tumor DNA. However, after platinum-based combined chemotherapy, many lung carcinoma cells from primary or metastatic sources are killed. Therefore, less tumor DNA is released into the bloodstream. Han et al. (27) found that EGFR mutations were detected in 39.4% (13/33) and 54.5% (18/33) of pre- and post- chemotherapy serum samples, respectively. Furthermore, because the DNA from tumor cells includes both wild and mutant types, the mixture of both may prevent the detection of mutations in serum or plasma. In this study, all of our enrolled patients had received from 1 to 4 cycles of platinum-based combination chemotherapy, and the small amount of circulating DNA in our samples may not have been enough to detect EGFR mutations.
In conclusion, no EGFR mutation was successfully detected using nested PCR and direct sequencing from blood specimens in 300 advanced stage NSCLC patients who had received platinum-based chemotherapy. Our present study indicates that it is difficult to identify EGFR mutations in DNA extracted from the whole blood of advanced stage NSCLC patients. Thus, we believe that the failure to detect EGFR mutations in whole blood precludes the use of EGFR mutations in serum as a clinical method for guiding treatment and monitoring disease progress. 
Conflict of interest statement
None declared
Acknowledgments
We thank Hailong Liu for his excellent technical support.
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Table 1 Different methods used to detect EGFR mutations in plasma and tissue samples of lung cancer patients.
Assay designnPre-or post-treatmentHistologic typeConcordanceHYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Kimura%20H%5BAuthor%5D&cauthor=true&cauthor_uid=17848912" Kimura et al., 2007 [16]Scorpion amplification refractory mutation system (ARMS) technology42Post-treatment31 ADC, 7 SCC, 4 LCC92.9%Bai et al., 2009 [17]Denaturing high-performance liquid chromatography230Post-treatment171 ADC, 55 SCC, 4 LCC79.7%Yung, et al., 2009 [15]Micro fluidics digital PCR35Pre-treatment35 ADC92%Rosell, et al., 2009 [18]Real-time fluorescence quantitative PCR164UnknownUnknown59%Brevet et al., 2011 [13]Mass spectrometry genotyping assay with a mutant-enriched PCR318 Pre-treatment30 ADC, 1 SCC61%23 Post-treatmentHuang et al., 2012 [19]Denaturing high-performance liquid chromatography822Unknown641 ADC, 104 SCC, 77 others77.0%Hu et al., 2012 [20]High-resolution melting assay4791.67%Kim et al., [21](Serum) the peptide nucleic acid locked5718 BSC40 ADC, 17 SCC87.7%Nucleic acid PCR clamp39 post-treatment(Tissue) genomic PCR and direct sequenceAbbreviation: ADC: adenocarcinoma; SCC: squamous cell carcinoma; LCC: Large cell carcinoma; others: Other types of NSCLC, BSC: Best supportive care

Table 2  Primer sequences listed by assay.
SequencesPrimerForwardReverseProduct length (bp)Sequencing endEGFR-E185 -ATGTCTGGCACTGCTTTC-3 5 -CTCACAGGACCACTGATTAC-3 395R endEGFR-E195 -CCCTCACCTTCGGGGTGCAT-3 5 -CTCCAGGCTCACCAAGAGCA-3 5905F endEGFR-E215 -TCAAGCCCAGGTCTCAACT-3 5 -CATTCACTGTCCCAGCAAG-3 365F endTable 3 Patients characteristics (n = 300).
ADCSCCMaleFemaleMaleFemale82 (27.3%)73 (24.3)135 (45%)10 (3.4%)Age (y)Mean56.355.060.255.1Range36-7930-7745-8542-75<6562 (20.7%)60 (20.0%)96 (32.0%)8 (2.66%)e"6520 (6.7%)13 (4.3%)39 (13.0%)2 (0.7%)Smokers62 (20.7%)3 (1.0%)103 (34.3%)0 (0%)Smoking history (y)0 (Never smoked)20 (6.7%)70 (23.3%)32 (10.6%)10 (3.4%)~104 (1.4%)1 (�a�6666666666666666666666666666666666666666666666666664@�4ck�emH	sH	nH	tH	_H`@`h�� 1��XD$$@&*CJOJ	PJQJ	^JaJ5KHnH	tH	\`@`h�� 2��XD$$@&*CJOJPJQJ^JaJ5KHnH	tH	\`@`h�� 3��XD$$@&*CJOJPJQJ^JaJ5KHnH	tH	\b@bh�� 4��XD$$@&,CJOJ	PJQJ	^J56KHnH	tH	\]V@Vh�� 5��XD$$@& CJOJ	PJQJ	^JKHnH	tH	\@\h�� 6��XD$$@&&CJOJ	PJQJ	^J6KHnH	tH	]\@\h�� 7��XD$$@&&CJOJ	PJQJ	^J6KHnH	tH	]Z@Zh�� 8��XD$$@&$CJOJ	PJQJ	^JaJKHnH	tH	`	@`h�� 9	��XD$$@&*CJOJ	PJQJ	^JaJ6KHnH	tH	]$A@�$؞���k=�W[SO:U@��:�����cB*`Jph�OJQJ^J>*.X@�.:_�OJQJ^J6]('@�(yb�l_(uCJaJ.W@�!.���pOJQJ^J5\6V@�16�]��v�����cB*`Jph��>*\�O�A\Balloon Text Char2 CJOJQJ^JaJKHnHtHb�O�QbIntense Quote Char%B*`Jph�OJQJ^J56\]b�O�abSubtitle Char1.CJOJ	PJQJ	^JaJ6@�KHnH	tH	]V�O�qVBody Text Char1 OJPJQJ^JaJKHnH	tH	`�O�`Heading 1 Char1*CJOJ	PJQJ	^JaJ5KHnH	tH	\T�O�TBody Text Char CJOJ
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Table 4  Exons18, 19, and 21 sequences.
EGFR SizeWild-type sequenceExon 18123 bpCTT GTG GAG CCT CTT ACA CCC AGT GGA GAA GCT CCC AAC CAA GCT CTC TTG AGG ATC TTG AAG GAA ACT GAA TTC AAA AAG ATC AAA GTG CTG GGC TCC GGT GCG TTC GGC ACG GTG TAT AAGExon 1999 bpGGA CTC TGG ATC CCA GAA GGT GAG AAA GTT AAA ATT CCC GTC GCT ATC AAG GAA TTA AGA GAA GCA ACA TCT CCG AAA GCC AAC AAG GAA ATC CTC GATExon 21156 bpGGC ATG AAC TAC TTG GAG GAC CGT CGC TTG GTG CAC CGC GAC CTG GCA GCC AGG AAC GTA CTG GTG AAA ACA CCG CAG CAT GTC AAG ATC ACA GAT TTT GGG CTG GCC AAA CTG CTG GGT GCG GAA GAG AAA GAA TAC CAT GCA GAA GGA GGC AAA
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