Potential Resistance Mechanisms Revealed by Targeted Sequencing from Lung Adenocarcinoma Patients with Primary Resistance to Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitors (TKIs)

Accepted Manuscript Potential resistance mechanisms revealed by targeted sequencing from lung adenocarcinoma patients with primary resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) Jia Zhong, Lei Li, Zhijie Wang, Hua Bai, Gai Fei, Jian Chunduan, Jun Zhao, Minglei Zhuo, Yuyang Wang, Shuhang Wang, Wanchun Zang, Meina Wu, Tongtong An, Guanhua Rao, Jie Wang PII:

S1556-0864(17)30663-9

DOI:

10.1016/j.jtho.2017.07.032

Reference:

JTHO 667

To appear in:

Journal of Thoracic Oncology

Received Date: 24 February 2017 Revised Date:

19 June 2017

Accepted Date: 20 July 2017

Please cite this article as: Zhong J, Li L, Wang Z, Bai H, Fei G, Chunduan J, Zhao J, Zhuo M, Wang Y, Wang S, Zang W, Wu M, An T, Rao G, Wang J, Potential resistance mechanisms revealed by targeted sequencing from lung adenocarcinoma patients with primary resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), Journal of Thoracic Oncology (2017), doi: 10.1016/ j.jtho.2017.07.032. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Potential resistance mechanisms revealed by targeted sequencing from lung adenocarcinoma patients with primary resistance to epidermal growth factor receptor

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(EGFR) tyrosine kinase inhibitors (TKIs)

Jia Zhong1#, Lei Li2#, Zhijie Wang1, Hua Bai3, Gai Fei2, Jian Chunduan3, Jun Zhao1, Minglei Zhuo1, Yuyang Wang1, Shuhang Wang1 , Wanchun Zang2, Meina Wu1, Tongtong An1,

1

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Guanhua Rao2, and Jie Wang3*

Key laboratory of Carcinogenesis and Translational Research (Ministry of Education),

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Department of Thoracic Medical Oncology-I, Peking University Cancer Hospital & Institute, Beijing, China； 2

Novogene Bioinformatics Institute, Beijing, China；

3

Department of Medical Oncology, Cancer hospital Chinese Academy of Medical Sciences

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*

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&Peking Union Medical College, Beijing, China；

Jia Zhong and Lei Li contribute equally to this work.

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Corresponding author: Jie Wang, [email protected];

ACCEPTED MANUSCRIPT Background:

EGFR-TKIs

lung adenocarcinoma. However,

have

greatly

approximately

improved

the

5%-10%

lung

prognosis

of

adenocarcinoma

patients with EGFR sensitive mutations have primary resistance to EGFR-TKIs treatment. The underlying mechanism is unknown.

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Methods: This study used next-generation sequencing (NGS) to explore the mechanisms of primary resistance by analyzing 11 patients with primary resistance and 11 patients sensitive to EGFR-TKIs. NGS targeted sequencing was performed on the Illumina X platform for 483 cancer-related genes. EGFR mutation was initially

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detected by amplification-refractory mutation system (ARMS).

Results: Potential primary resistance mechanisms were revealed by mutations unique

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to the EGFR-TKIs resistance group. Among the 11 resistant patients, 45% (5/11) harbored a known resistance mechanism, such as MET amplification de novo T790M mutation, or overlapping T790M and PTEN loss and Her2 amplification. In 6 of 11 resistant cases (54%), potential novel mutations that might lead to drug resistance were identified (including TGFBR1 mutation and/or EGFR structure rearrangement

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MTOR mutation; TMPRSS2 fusion gene and MYC amplification). By analyzing somatic mutation patterns, the frequency of C:G→T:A transitions in the patients with primary resistance was significantly higher than that in sensitive group and occurs

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more frequently in the non CpG region (Cp(A/C/T)→T). Discussion: The mechanisms of EGFR-TKIs primary resistance may be highly heterogeneous. Mutations in EGFR and its downstream pathway as well as mutations

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that affect tumor cell function are related to primary resistance. Somatic single-nucleotide mutation patterns might associated with EGFR-TKIs primary resistance.

Keywords: Lung adenocarcinoma, EGFR-TKIs, NGS, primary resistance

ACCEPTED MANUSCRIPT Introduction Lung

cancers

harboring

mutations

sensitive

to

epidermal growth factor receptor (EGFR) demonstrate good objective responses for treatment with EGFR-TKIs (tyrosine kinase inhibitors). However, according to

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previous clinical trials [1-3], approximately 5%-10% of lung adenocarcinoma patients with EGFR sensitive mutations have primary resistance to EGFR-TKIs treatment. Meanwhile, for EGFR-TKIs sensitive patients, secondary resistance due to drug resistance invariably emerges during treatments from 8 to 16 months [1-3].

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Currently, some mechanisms of acquired resistance to EGFR-TKIs have been revealed, including T790M mutation [4], MET amplification [5], or PIK3CA mutation

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[6]. New drugs targeting these mechanisms, such as osimertinib [7], have been developed to overcome secondary EGFR-TKIs resistance. However, little is known about the molecular background of primary resistance. Primary resistance to EGFR-TKIs is defined as patients with EGFR sensitive mutant adenocarcinoma who have disease progression in <90 days（3months） without any evidence of objective

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response while receiving EGFR-TKIs [8]. Thus, primary resistance has become the main problem of EGFR-TKIs treatment.

Several studies have shown that primary resistance to EGFR-TKIs is associated

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with KRAS mutations and ALK rearrangement. However, coexistence of EGFR and KRAS mutations occurs in less than 1% of lung cancer patients [9], and EGFR mutations coexist with ALK rearrangement in less than 2% of lung adenocarcinoma

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[10]. The acquired resistance related molecular mutations such as T790M and MET amplification could also exist in pre-treatment tissues, and contribute to primary resistance to EGFR-TKIs [5, 11, 12]. However, these mechanisms cannot explain all cases of primary resistance in clinical practice. Recently, several preclinical and retrospective studies demonstrated that BIM(BCL-2 like 11) deletion polymorphisms, a germline mutation, could predict poor response to EGFR-TKIs treatment [13, 14]. But whether BIM deletion can predict primary resistance to EGFR-TKIs is controversial: several studies did not find any difference between BIM deletion or wild type patients in EGFR-TKIs treatment [15, 16].

ACCEPTED MANUSCRIPT The underlying molecular mechanisms of primary resistance to treatment with EGFR-TKIs are not fully elucidated. Therefore, we conducted this exploratory study to identify novel potential mechanisms of primary resistance to EGFR-TKIs by next-generation sequencing.

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Materials and Methods Ethics statement

This study was approved by the Ethics Committee of Peking University Cancer

samples.

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Sample collection and DNA extraction

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Hospital. All patients signed informed consent for use of their tumor tissue and blood

For most patients, FFPE materials were used in this study, except one paitent (Tumor ID TSCR-T) can provide primary tumor tissues collected during surgery and frozen immediately in liquid nitrogen. A 5 µM section of a hematoxylin and eosin-stained slide was reviewed by a pathologist to evaluate the tumor purity and

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circle the tumor area. For some patients, white blood cells from blood samples were available for somatic variant calling.

Genomic DNA was extracted from tumor tissue and blood samples using

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TIANamp Genomic DNA Kit (Tiangen, Beijing, China). The quality of purified DNA samples was examined by 3 methods: agarose gel electrophoresis to analyze the degree of DNA degradation; Nanodrop (Thermo Fisher Scientific, Waltham, MA,

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USA)) analysis of OD260/280 for DNA purity; and Qubit 2.0 fluorometric quantitation (Life Technologies) to detect DNA concentration. Library construction, hybridization and sequencing Genome DNA was randomly sheared into 150-300 bp segments by Covaris S220

Focused-ultrasonicator(Covaris Inc, MA, USA). After end repair and dA-tailing, indexed adaptors were ligated to DNA fragments to perform pre-capture PCR. We adapted a customized 483 gene panel based on Agilent liquid-phase hybrid capturing system for target enrichment. This panel was designed to target a total of 483 cancer related human protein-coding genes; intron regions of 19 genes; and 3

ACCEPTED MANUSCRIPT SNP sites, including C8orf34, UGT1A9, and UGT1A1. The coverage of exon regions and intron regions is above 99.9%. Exon region and 3 SNP sites were detected, intron region of 19 genes were detected for gene fusion events. The total length of both exon and intro regions is about 2.3 Mbp. Manufacturers’ recommendations were followed

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during liquid-phase hybridization step. Following library enrichment, post-capture PCR was performed to yield final sequencing libraries. The fragment size of prepared library was qualified using 2100 Bioanalyzer (Agilent Technologies) and library concentration was quantified using

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qPCR NGS library quantification kit (Agilent Technologies). Multiplexed libraries were sequenced at the Novogene sequencing facility using HiSeq platform (Illumina,

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San Diego, CA). Quality control and sequence alignment

Quality Control (QC) was conducted to filter out adaptor sequences and low quality reads. Q20 and Q30 of all samples were above 90% and 85%, respectively. The sequencing reads were aligned to the hg19 human reference genome using the

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Burrows-Wheeler Transform in the program BWA (0.6.2-r126)[17] with default parameters. Duplications were removed using Picard (http://picard.sourceforge.net). In this study, all samples with coverage of the target region above 99% and the

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mapping rate were no less than 95%. The effective depth of most samples was above 200x, and the average effective depths were 491.6x. Variant calling

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Somatic single nucleotide variants (SNVs) were called using MuTect (v1.1.5) [18]

and somatic InDels were called using strelka [19]. Somatic mutations were filtered at several thresholds, including a read depth of greater than 50X, and no less than 5 reads containing the alternate allele. Structure variation events were identified with in-house software, and fusion events were only retained if they were supported by more than 4 split-reads on two genes. Function annotations were made with ANNOVAR [20], COSMIC [21] and the clinvar database. Enrichment using the Kyoto Encyclopedia of Genes & Genomes (KEGG) was performed with the DAVID online server [22]. To analyze mutation signatures, germline mutations were called by

ACCEPTED MANUSCRIPT MuTect. The copy number of genes was analyzed by ExomeCNV [23], with the circular binary segmentation (CBS) algorithm. A control set for somatic copy-number variation (CNV) was generated from white blood cell samples from some patients.

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For patients with tumor samples that lacked white blood cell samples, a pre-calculated control set from multiple white blood cell samples was used. In the final report, we only kept CNV events of 6 drug-related genes with copy numbers of at least 2.7: EGFR, ERBB2, MET, MYC, FGFR1, and FGFR2.

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Results Patient Characteristics

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A total of 415 patients with lung adenocarcinoma from Peking University Cancer Hospital were enrolled in the study. All patients harbored EGFR-activating mutations, and had a treatment history of EGFR-TKIs (including gefinib, erlotinib, or icotinib) between Jan 2006 to Jan 2015. All 415 patients harbored mutations known to associate with EGFR-TKI’s activity based on previous studies, including EGFR exon

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19 deletion, exon 21 L858R, exon 18 G719X, and exon 21 L861Q. The median follow-up time for these patients was 22.3 months (range 3.1 months-65.5 months). 41.2% of the patients (171/415) received EGFR-TKIs as first line treatment. At the

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last follow up, 22.4% of the patients (93/415) were still receiving EGFR-TKIs treatment. The median progression-free survival(PFS) was 8.5 months. 20.2% of the patients (84/415) exhibited primary resistance to EGFR-TKIs treatment. Among

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resistant patients, 11 cases had sufficient tumor tissue for NGS analysis and met the filtering criteria to be included in this study. For comparison, another 11 EGFR-TKIs sensitive cases (PFS>12months) meeting these standards were also included. The filtering criteria was as follows: 1) after DNA extraction, the DNA was tested by electrophoresis on a 1% agarose gel and the main band was at least 600bp; 2) effective coverage with a mean sequencing depth of over 200x; 3) the absolute difference of mean depth and median depth of all bins of 483 gene panel was less than 50; 4) all the reads align to hg19 and the mapping rate was at least 95%. The baseline clinical characteristics including age, gender, smoking status, EGFR mutation and

ACCEPTED MANUSCRIPT types of EGFR-TKIs was well balanced between the sensitive and primary resistant group(Supplementary Table1). Patients with primary resistance showed unique somatic variations To explore possible mechanisms of primary resistance to EGFR-TKIs treatment,

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11 lung adenocarcinoma patients with primary resistance were included in the study; all resistant patients had archival EGFR-TKIs naive tissues for NGS. For comparison, 11 EGFR-TKI sensitive samples were also sequenced. Eight of the 11 patients in each group had paired white blood cells available. Since several important mutations have

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low alteration allele frequency, higher sequencing depth is required for mutation calling. Thus, we focused on the genome region of 483 cancer-associated genes by

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targeted NGS technology. Under strict quality control, the extracted DNA was sequenced to high, uniform coverage with a mean sequencing depth of 491.6x. The mapping rate ranged from 98.41% to 99.77%, and percent coverage of the target region was greater than 99.75% (Supplementary Fig 1).

The number of single nucleotide variations (SNVs) and insertion-deletions

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(InDels) was different from patient to patient. The average numbers of somatic SNVs or InDels were 9.75 or 2.625 for the sensitive group, and 18.625 or 2 for the resistance group. The differences in the numbers of SNVs/InDels between the two groups were

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not statistically significant (P=0.43 and 0.91) (Supplementary Fig 2). We also analyzed the allelic frequency of EGFR mutations in the two cohorts. The allelic frequency of EGFR exon 19 deletion was significantly higher in sensitivity group

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(0.158 vs. 0.059, P=0.035). The median allelic frequency of EGFR exon 21 L858R was also higher in the sensitivity group, but the difference was not statistically significant (0.42 vs. 0.22, P=0.99). To eliminate detection bias, we further analyzed somatic SNVs/InDels that were

1) detected in at least two pre-treatment tissue specimens 2) and were unique in the resistant group. These SNVs/InDels were defined as high frequency somatic variations. Besides the common EGFR sensitive mutation and a high prevalence of mutations in TP53, patients in the resistance group contained three unique highly frequent SNVs/InDels including: 1) the negative regulator of Wnt signaling (RNF43)

ACCEPTED MANUSCRIPT [24]; 2) the components of a protein complex for cell-cycle arrest and DNA damage (MTOR and RICTOR) [25]; and 3) cell adhesion proteins in the immunoglobulin superfamily (DSCAM)[26] (Fig 1). Besides these three mutations, we also found that patient TG93348 had a mutation in TGFBR1 Glu223X and patient TG81531

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(post-treatment sample) harbored a TGFBR1 Val1222fs mutation. The two TGFBR1 mutations were frameshift deletion and a stop-gain mutation, respectively, which may relate to EGFR-TKIs resistance. NF1 is reported to have predictive value of EGFR-TKIs[27]. In this study, NF1 mutation uniquely exit in the primary resistant

exon48: c.7175G>A: p.Gly2392Glu).

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group, but occurred in only one patient’s pre-treatment tissue(NF1: NM_001042492:

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To reduce the bias may be caused by the small patient cohorts, we compared these six mutations identified in 8 paired resistance patients (RNF43, MTOR, RICTOR, DSCAM,

TGFBR1 and NF1) to 33 lung adenocarcinoma patients with

EGFR mutation in The Cancer Genome Atlas (TCGA) using two-side fisher exact test [28]. The occurrence of RNF43, MTOR and RICTOR are significantly higher in the

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Patients in the resistant group were likely to have other mutations coexisting with EGFR sensitive mutations, including EGFR L858M (exon 21), L11F (exon 1), G719A (exon 18), and T790M (exon 20). Intragenic mutations in EGFR were considered to

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be a possible mechanism for TKI resistance; such mutations included T790M in exon 20, L747S in exon 19, and an insertion in exon 20 [29, 30]. Four patients with uncommon mutations were found in this study including G719A, L858M, L11F and

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L861Q. The former three coexist with EGFR sensitive mutations, while the latter exists alone. Additionally, we also found EGFR chromosome structure rearrangement in patient TG74187 and patient TG77905; these two patients both belong to the EGFR-TKIs primary resistance group. No such chromosome structure rearrangement was found in the EGFR-TKIs sensitive group (Supplementary Table 2). We also tried to detect fusion genes in both the sensitive and resistant groups. TMPRSS2-ALPL rearrangement was found in one primary resistant patient. TMPRSS2-ERG fusion gene was reported with high prevalence in a prostate tumor,

ACCEPTED MANUSCRIPT and as a regulator of insulin-like growth factor-1 receptor (IGF1R) [31]. No fusion gene was detected in the EGFR-TKIs sensitive group. Copy number aberrations were also analyzed in the two cohorts. MET amplification and HER2 (ERBB2) copy number gain only exist in the primary

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resistant group; PTEN loss was found in one patient in the resistant group. MYC amplification was found in two sensitive patients and one primary resistant patient. MYC copy number gains in the primary resistant patient (13.6 folds) were notably higher than that of the sensitive patients (4.8 and 2.9 folds). EGFR amplification was

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both found in sensitive and resistant groups (Supplementary Table 3).

BIM deletion polymorphism detection was made for both groups. One patient in

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the sensitive group and one patient in the resistant group had the BIM deletion. We inferred potential primary resistance mechanism according to the high frequency mutations unique in EGFR-TKIs primary resistance group. Among the 11 EGFR-TKIs primary resistant patients, 45% (5/11) harbored a known resistance mechanism (2 patients had MET amplification; 1 patient had T790M mutation; 1

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patient had co-existing T790M mutation and PTEN loss; and 1 patient had Her-2 amplification), while 54%（6/11）harbored novel mutations that may lead to drug resistance (1 patients had TGFBR1 mutation; 1 patient had EGFR structure

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rearrangement; 1 patient had EGFR structure rearrangement and TGFBR1 mutation in post-treatment tissue; 1 patient had MTOR mutation; 1 patient had TMPRSS2 fusion gene; and 1 patient had EGFR L861Q, MYC amplification and BIM deletion). 3/11

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(27.3%) patients had multiple resistance mechanism (Table 2). Overall, these data suggested that the mechanism of EGFR-TKIs primary resistance is highly heterogeneous.

ACCEPTED MANUSCRIPT Signaling Pathways Possibly Related to EGFR-TKIs Primary Resistance To further explore genetic differences between the two groups, genes unique to each group were analyzed with the pathway database in the Kyoto Encyclopedia of Genes & Genomes (KEGG). Overall, we observed 20 and 5 pathways in the resistant

resistant samples were involved in more pathways (Figure 2).

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and sensitive cohorts, respectively. Compared to sensitive samples, mutations in

For the EGFR-TKIs primary resistant group, the pathways include: 1) EGFR and its downstream pathway, such as Jak-STAT signaling pathway, ErbB signaling

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pathway, and MAPK signaling pathway; 2) pathways that relate to tumor cell function, such as cell cycle and focal adhesion; and 3) immune related pathways, such as the T

(Supplementary Table 4a).

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cell receptor signaling pathway. These pathways were found in resistant group only

Pathways related to epithelial-mesenchymal transition (EMT) were found in both groups. These pathways include the TGF-beta signaling pathway in the sensitive group and the Wnt signaling pathway in both groups. However, the mutations in the

4b).

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Wnt pathway were not the same between the groups (Supplementary Tables, 4a and

Unique pathways in the resistant cohort included the ErbB pathway and its

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downstream MAPK and Jak-STAT signaling pathway; pathways related to cell functions such as cell cycle and focal adhesion; and pathways associated with

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immune response (Figure 2A).

Single-nucleotide Mutation signature for the Primary resistant group

For the somatic and germline mutation signature, patients in both groups showed

similar patterns, either single nucleotide changes, including A>T/T>A, A>C/T>G, A>G/T>C, C>A/G>T, C>T/G>A, C>G/G>C (Supplementary Fig 3A), or subdivided mutation patterns such as transversions, A->G, Cp(A/C/T)->T, (C/T)p*CpG->T and (A/G)p*CpG->T (Supplementary Fig. 3B and Supplementary Table 5). We examined the somatic mutation signature in more detail. For a limited number of somatic mutations in each sample, we pooled results of all samples in each group, and compared the sum of frequency. We found that the most prevalent changes

ACCEPTED MANUSCRIPT were C>T/G>A transitions, and the frequency was higher in the primary resistance group (51.3%) than in the sensitive group (43.6%; Fig. 3A). This contrast between resistant and sensitive group indicates a possible influence of cytosine spontaneous deamination (C>T/G>A) on EGFR-TKIs primary resistance. For the subdivided five

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mutation patterns, we found the frequency of transversions in the sensitive group was notably higher than that in the primary resistant group (46.2% vs. 32.0%). In addition, the C>T change in the non CpG region (Cp(A/C/T)->T) occurred more frequently in the primary resistant group when compared with the sensitive group (32.0% vs.

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15.4%; Fig. 3).

Considerting the possible association of smoking status and C>T/G>A nucleotide

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transitions, we analyze the smoking status of the two groups (Table 2). The occurrence of C>T/G>A transition was not different between the smoking and the non-smoking groups(P=0.55)

Dynamic change in samples before and after EGFR-TKIs treatment Among the 11 EGFR-TKIs primary resistant patients, one patient provided both

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pre- and post EGFR-TKI treatment samples. This patient is a 56 year-old woman, who carried an EGFR 19 deletion in the adenocarcinoma. She experienced a PFS of 1.17 months after treatment with first line gefitinib (first generation EGFR-TKIs). We

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made a target sequence of 483 cancer-associated genes from her paired samples. We found 9 somatic mutations present in the pre-treatment tissue (MTOR, ETV6, TP53, STAT1, IL7R, ETV1, EGFR, RICTOR, TGFBR1), and measured the frequency of

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these somatic mutations in the post-treatment sample from the same patient, who underwent primary resistant of gefitinib treatment. Eight of the 9 mutations (with the exception of IL7R) were also prevalent in the DNA of the post-treatment tumor. The WNK3 mutation emerged in the post-treatment sample (Fig 4). We further analyzed the non-synonymous coding mutations and found increased allele frequencies of TP53, TGFBR1, and WNK3, and decreased allele frequencies of IL7R and EGFR. This patient underwent EGFR-TKIs treatment, so it is not surprising to find decreased frequency of EGFR. Notably, this primary resistant patient also presented elevated frequencies of TGFBR1, a high frequency somatic variation in the primary resistant

ACCEPTED MANUSCRIPT group. Taken together, our data showed that EGFR-TKIs can also target EGFR sensitive mutations for EGFR-TKIs primary resistant patients, and resistant mutations accumulate under the selection pressure of EGFR-TKIs. Discussion

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Understanding of the mechanisms of primary resistance to EGFR-TKIs in the patients with sensitive mutations is still extremely limited. Here, we utilized samples from two groups of patients with Non-small cell lung cancer (NSCLC) harboring EGFR-sensitive mutations which were sensitive and primary resistant to EGFR-TKIs

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treatment to investigate the potential resistant mechanisms of resistance. Our data indicate that the mechanism of EGFR-TKI primary resistance is highly heterogeneous.

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Using a genome-wide analysis, we identified several genetic mechanisms related to primary resistance, including somatic mutations, signaling pathways alterations, and single-nucleotide mutation patterns. Our findings provide new insight into EGFR-TKI primary resistance in the era of genotype-directed personalized treatment. In the genomic analysis of 11 primary resistance patients, molecular changes in

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EGFR and its downstream pathways is the most common. In the present study, 2 patients had T790M mutation along with EGFR sensitive mutations before treatment. T790M accounts for approximately 50% of acquired resistance [6]. T790M mutation

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can also initially co-exist with EGFR-activating mutations, and patients with this mutation had shorter PFS in EGFR-TKIs treatment than patients without it [32]. Several studies have indicated that a germline T790M mutation induces drug-free

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tumor progression [33, 34]. We have reason to regard the presence of T790M as a cause of primary resistance. Four uncommon mutations were found in this study including G719A, L858M, L861Q, and L11F. The former three were sensitive mutations according to previous studies [35, 36]. The oncogenicity of L11F and its sensitivity to EGFR TKI have not been fully elucidated. Because L11F locates in the extracellular region of EGFR, it is probably a neutral mutation. Uncommon mutations have shorter PFS and lower objective response rates than classical mutations (such as exon 19 deletion and exon 21 L858R) [36, 37]. Prospective trials are needed to validate uncommon EGFR mutations as possible mechanisms of primary resistance.

ACCEPTED MANUSCRIPT EGFR chromosome structure rearrangements were found only in the primary resistance group. Although the function of this interesting “exon skipping” phenomenon is unknown, it provided a new version for study of primary resistance mechanisms.

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The allelic frequency of EGFR mutations in the sensitive group was higher than the primary resistance group. Wu et.al found the EGFR mutation abundance could predict benefit from EGFR-TKIs treatment for NSCLC[38]. The potential heterogeneity might explain the underlying mechanism of primary resistance to

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EGFR-TKIs.

In our study, mutations in EGFR-downstream genes including MET amplification,

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PTEN loss and Her-2 amplification were associated with primary resistance to EGFR-TKI. Two of the 11 resistant patients had MET amplification. According to previous studies, activation of MET by hepatocyte growth factor (HGF) was well recognized as a secondary resistance mechanism to EGFR-TKIs [5]. The prevalence of these genomic alternations in treatment-naïve specimens with EGFR mutations is

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only 1.5% [39], which is lower than the occurrence in primary resistant patients in our study (2/11, 18.2%). PTEN is a tumor suppressor gene that negatively regulates the PI3K/Akt/mTOR pathway [40], thus the loss of PTEN[41] was also regarded as a

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mechanism for EGFR-TKIs acquired resistance. Our study found a single patient with PTEN loss that coexisted with EGFR sensitive mutations in the pre-treatment tissue. A recent study identified 54.5% (12/22) of patients with primary resistance had loss of

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PTEN, suggesting the deletion of PTEN as a candidate for primary resistance [42]. One patient in this study had HER-2 amplification. HER-2 amplification [43] was also a potential mechanism of acquired resistance to EGFR inhibitors. Reduced expression of neurofibromin, the RAS GTPase-activating protein encoded by the NF1 gene, was related to Erlotinib treatment [27]. In our study, one patient have NF1 mutation in the resistant group. Previously study has shown that NF1 low expression level is reported to be associated with primary EGFR-TKIs resistance[27]. However, whether NF1 mutation will result in low expression of NF1 is unknown. NF1 mutation is only a possible primary resistance of EGFR-TKIs. In a word, mutations in

ACCEPTED MANUSCRIPT EGFR downstream genes enable the cancer cell to maintain its intracellular growth signaling pathways in the presence of EGFR-TKIs. These somatic changes found in pre-treatment tissues are possible mechanisms of primary resistance and suggest that the clonal selection of these genomic alternations during EGFR-TKIs treatment

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resulted in TKI resistance. TGFBR1 mutation was a high frequency mutation unique in the EGFR-TKIs primary resistant group. Previous research indicated that the activation of classical TGF-β signaling pathway was related to secondary resistance to EGFR-TKIs in lung

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adenocarcinoma [44]. TGFBR1 is one of the receptor in the TGF-β pathway. The activation of TGF-β pathway was related to stem cells and epithelial-mesenchymal

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transition (EMT) [45, 46], which might cause EGFR-TKIs resistance. Stem cells and EMT are activated by either Smad dependent pathways or Smad-independent pathways [47, 48]. Yamashita et al. reported that the TGFBR1 kinase activity is required in the activation of Smad-independent TGF-β pathways [49]. In our study, we also found a high frequency of mutations in mTOR and MAPK signaling

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pathways, which are also involved in Smad-independent pathways. This indicates the mechanism of primary resistance might be the activation of Smad-independent TGF-β pathways through TGFBR1.

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By analyzing somatic single-nucleotide mutation patterns, we found the frequency of C>T/G>A transitions in the primary resistance group was higher than that in sensitive group (43.6% vs. 51.3%). This phenomenon indicated cytosine

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spontaneous deamination C>T/G>A was positively associated with EGFR-TKIs primary resistance [50]. We found germline variations in the primary resistant group occurred more frequently in non CpG region (Cp(A/C/T)->T). According to the literature, a higher prevalence of C>T mutations are related to ultraviolet exposure [51] and C>T mutations were often observed in cancer caused by tobacco smoking [50]. In our study, the C>T/G>A transition enriched in the primary resistance group but lacks correlation with smoking status (P=0.55). Our study first time associated somatic single-nucleotide mutation patterns with EGFR-TKIs primary resistance, though validation by additional studies is needed.

ACCEPTED MANUSCRIPT We also found somatic mutations related to tumor cell function, such as cell cycle and cell adhesion. DSCAM is a high frequency mutation in the resistant group. It is the member of the cell adhesion immunoglobulin superfamily [26]. MTOR and RICTOR, which are the components of a protein complex for cell-cycle arrest and

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DNA damage, are also prevalent in the resistant group, [25]. MYC (c-Myc) is a regulator gene that plays a role in cell cycle progression, apoptosis and cellular transformation [52], and its amplification in lung cancer was first reported in 1983 [53]. Of note, MYC amplification in the primary resistant group (13.6 fold) was

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significantly higher than that of the sensitive patients (4.8 and 2.9 folds). According to the literature, MYC amplification was regarded as an independent poor-prognostic

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factor for Disease-free Survival (DFS) and Overall Survival (OS) in lung adenocarcinomas [54, 55], and MYC inhibitors have recently been employed in preclinical studies. Therefore, combination therapy with MYC inhibitors and EGFR-TKIs seems to be a promising strategy for overcoming primary resistance to EGFR-TKIs in lung cancer.

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Therapeutic impact was also considered in this study. One patient provided both pre- and post- EGFR-TKI treatment samples. The increased allele frequencies of resistant mutations, and decreased allele frequencies of sensitive mutations can be

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attributed to a preferential response of subclones of sensitive mutations in tumors with heterogeneous tumor cell populations[56]. Heterogeneity that derive from genetic differences and arise through clonal evolution may also be one of the explanations of

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EGFR-TKIs resistance. Despite this interesting finding, our study has some limitations. First, this is a

retrospective study in a single center and additional prospective studies will be necessary to verify the results. Second, only 11 primary resistant patients had enough available tissue and white blood cell samples for NGS; we have considered expanding the sample size for future studies. Finally, although possible primary resistant mechanisms were examined in this study, the definite relationship between these factors and primary resistance require molecular experiments to confirm. In summary, our study identified potential primary resistance mechanisms for

ACCEPTED MANUSCRIPT each patient in the primary resistant group. Our data suggest that molecular mechanisms of primary resistance are highly heterogeneous. Of note, we are the first to demonstrate an association between single-nucleotide mutation patterns and EGFR-TKI primary resistance.

Further investigations with larger cohorts and

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experimental research are warranted to further explore these potential mechanisms. We believe that our study contributes to the understanding of primary resistance and the ongoing efforts to develop

more personalized treatment.

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[1]. Rosell, R., et al., Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre,

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[51]. Pleasance, E.D., et al., A comprehensive catalogue of somatic mutations from a human cancer genome. Nature, 2010. 463(7278): p. 191-6. [52]. Hirsch, C.L., et al., Myc and SAGA rewire an alternative splicing network during early somatic cell reprogramming. Genes Dev, 2015. 29(8): p. 803-16. [53]. Little, C.D., et al., Amplification and expression of the c-myc oncogene in human lung cancer cell lines. Nature, 1983. 306(5939): p. 194-6. [54]. Iwakawa, R., et al., MYC amplification as a prognostic marker of early-stage lung adenocarcinoma identified by whole genome copy number analysis. Clin Cancer Res, 2011. 17(6): p. 1481-9.

ACCEPTED MANUSCRIPT [55]. Seo, A.N., et al., Clinicopathologic and prognostic significance of c-MYC copy number gain in lung adenocarcinomas. Br J Cancer, 2014. 110(11): p. 2688-99. [56]. Bai, H., et al., Influence of chemotherapy on EGFR mutation status among patients with

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non-small-cell lung cancer. J Clin Oncol, 2012. 30(25): p. 3077-83.

ACCEPTED MANUSCRIPT Figure legend Figure 1: High frequency somatic variations for the EGFR-TKIs sensitive group and primary resistant group. Each group has 8 patients that provided paired white blood cell samples as a control. In the EGFR-TKIs sensitive group, patient 3531-ca has two mutation sites in EGFR. One is an exon 19 deletion

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and the other is a synonymous mutation. A low frequency EGFR exon 19 deletion was detected by ARMS in patient 1363-ca, but failed to find it by NGS.

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Figure 2: Signaling pathway analysis for the EGFR-TKIs sensitive group and primary resistant group. The horizontal axis indicates gene count for each pathway. Each pathway showed over three gene differences between

A. B.

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the two cohorts. Signaling pathways for the EGFR-TKIs primary resistant group. Signaling pathways for the EGFR-TKIs sensitive group.

Figure 3: Somatic single nucleotide mutation signatures for the EGFR-TKIs sensitive group and primary resistant

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group. A limited number of somatic mutations for each group were integrated and analyzed by group as a whole. A.

Somatic single nucleotide changes for both groups.

B.

Five mutation patterns for somatic mutations for both groups.

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Figure 4: Dynamic changes in mutant allele frequencies for one EGFR-TKIs primary resistant patient. TG74187 is the biopsy tissue sample before icotinib treatment. TG81531 is the re-biopsy tissue after icotinib progression.

B.

Mutant allele frequency change for all the somatic mutations before and after treatment.

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A.

Dynamic changes of somatic mutations that might cause alterations in the protein level.

ACCEPTED MANUSCRIPT Table 1:Comparison of six mutations to TCGA.

size，

in，TCGA， Primary，resistance， group，in，our，study，

RNF43，

MTOR，

RICTOR，

DSCAM，

TGFBR1，

NF1，

33，

0，

1，

1，

2，

1，

1，

8，

2，

3，

3，

2，

1，

1，

，

0.034，

0.019，

0.019，

0.169，

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Lung，adenocarcinoma，

0.36，

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P，value，

Number，of，patients，with，mutation，

Sample，

Database，

0.36，

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Table 2: Detailed characteristecs for patients of both groups and potential primary resistance mechanisms Smoking，

Nature，of，

status，

specimens，

55，

60，pack-year，

7653-wbc，

49，

No，

7871-wbc，

65，

No，

1593-ca，

，

47，

No，

2935-ca，

6193-wbc，

60，

No，

sensitive，

2028-ca，

4840-wbc，

51，

sensitive，

1363-ca，

6719-wbc，

sensitive，

3315-ca，

sensitive，

Gender，

Histology，

line，

Stage，

EGFR，mutation，

TKI，PFS，

sensitive，

1936-ca，

6515-wbc，

Surgical，

male，

adenocarcinoma，

1，

IV，

L858R，

21.46，

sensitive，

3531-ca，

Surgical，

female，

adenocarcinoma，

2，

IV，

19del，

12，

sensitive，

2035-ca，

male，

adenocarcinoma，

2，

IV，

L858R，

16，

BIM，deletion;，EGFR，amplification(24.46);，

sensitive， sensitive，

Surgical，

male，

adenocarcinoma，

1，

IV，

Braf，mutation;，MAPK，mutation，

Surgical，

female，

adenocarcinoma，

1，

No，

Needle，biopsy，

female，

adenocarcinoma，

2，

70，

30，pack-year，

Surgical，

male，

adenocarcinoma，

1，

6364-wbc，

49，

No，

Surgical，

male，

adenocarcinoma，

1，

3114-ca，

，

54，

No，

Surgical，

female，

adenocarcinoma，

sensitive，

1791-ca，

6285-wbc，

49，

20，pack-year，

Needle，biopsy，

male，

sensitive，

1505-ca，

，

66，

No，

Needle，biopsy，

49，

No，

63，

resistant， resistant，

TCSR-T， TG95755，

wbcZTLBL15 03029， wbcZTLBL15 03037，

Needle，biopsy，

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Age，

19del，

21.1，

IV，

L858R，

20，

IV，

19del，

13，

L858R，

47.5，

IV，

19del，

20，

2，

IV，

L858R，

24，

adenocarcinoma，

2，

IV，

19del，

36.9，

female，

adenocarcinoma，

1，

IV，

L858R，

11，

Fresh，surgical，

female，

adenocarcinoma，

1，

IV，

No，

Surgical，

female，

adenocarcinoma，

1，

IV，

L861Q，

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WBC，ID，

I

IV，

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Tumor，ID，

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Group，

T790M

L858R，

0.67， 1，

Potential，mechanism，of，TKI，resistance，

， ，

， ， ， ， ， EGFR，R476K， ， PTEN，loss;T790M;，RICTOR，and，DSCAM，mutation， MYC，amplification(13.6);，BIM，deletion;，NF1，mutation;， MTOR,，RICTOR，and，DSCAM，mutation，

T4499，

，

58，

No，

Needle，biopsy，

female，

adenocarcinoma，

1，

IV，

L858R，

1，

MET，amplification(4.9)，

resistant，

3037-ca，

8142-wbc，

67，

No，

Needle，biopsy，

female，

adenocarcinoma，

1，

IV，

19del，

2.3，

MTOR，mutation，

63，

No，

Surgical，

female，

adenocarcinoma，

1，

IIIb，

L858R，

2.96，

Her2，amplification(17.66);，RNF43，mutation，

74，

No，

Needle，biopsy，

female，

adenocarcinoma，

3，

IV，

2.37，

EGFR，structure，rearrangement，

Needle，biopsy，

male，

resistant，

TG88205，

resistant，

TG77905，

wbcZTLBL15 03021， wbcZTLBL15 03025，

T2111，

，

75，

40，pack-year，

resistant，

4769-ca，

9168-wbc，

50，

No，

TG74187T

wbcZTLBL15

G81531，

03024，

56，

No，

resistant， resistant，

T1751，

resistant，

TG93348，

， wbcZTLBL15 03020，

L858M L858R,19del，

adenocarcinoma，

2，

IV，

L858R，

1，

MET，amplification(2.9)，

Surgical，

male，

1，

IV，

L858R，

1，

TMPRSS2-ALPL，fusion，gene，

Needle，biopsy，

male，

adenocarcinoma，

1，

IV，

L11F,19del，

1.17，

adenocarcinoma，

65，

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resistant，

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resistant，

5，pack-year，

Surgical，

male，

adenocarcinoma，

2，

58，

No，

Needle，biopsy，

female，

adenocarcinoma，

1，

I

IV， IV，

G719A T790M 19del， 19del，

EGFR，structure，rearrangement;，TGFBR1，mutation;，MTOR， and，RICTOR，mutation，

1.33，

T790M，

1，

TGFR1，mutation，and，RNF43，mutation，

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