Prevalence of targeted therapy-related genetic variations in NSCLC and their relationship with clinicopathological characteristics.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the most common cancer type in China. Targeted therapies have been used to treat NSCLC for two decades, which is only suitable for a subgroup of patients with specific genetic variations. The aim of this study was to investigate the prevalence of genetic variations leading to sensitivity or resistance to targeted therapies in NSCLC, and their relationship with clinicopathological characteristics of the patients.
METHODS: Tumor samples were collected from 404 patients who were diagnosed to have NSCLC and underwent surgery, transthoracic biopsy, bronchoscopy biopsy, or pleural aspiration in Sichuan Provincial People's Hospital from January 2019 to March 2020. Commercial amplification-refractory mutation system kits were used to detect targeted therapy-related genetic variations in those tumor samples. The prevalence of genetic variations and their relationship with patient clinicopathological characteristics were analyzed using statistical software, followed by subgroup analysis.
RESULTS: In all, 50.7% of the NSCLC patients had sensitive genetic variations to anti-EGFR therapies, and 4.9% of those patients had co-existing resistant genetic variations. Fusions in ALK, ROS1, or RET were found in 7.7% of the patients, including 2 patients with co-existing EGFR exon 19 deletion or L858R. EGFR exon 19 deletion and L858R were more common in female patients and adenocarcinoma. Further subgroup analysis confirmed the observation in female patients in adenocarcinoma subgroup, and in adenocarcinoma in male patients. In addition, smokers were more likely to have squamous cell carcinoma and KRAS mutation and less likely to have EGFR L858R, which were also confirmed after standardization of gender except KRAS mutations.
CONCLUSION: Nearly half of the NSCLC patients were eligible for anti-EGFR treatments. In NSCLC, female gender and adenocarcinoma may indicate higher chance of EGFR exon 19 deletion or L858R, and smoking history may indicate squamous cell carcinoma and EGFR L858R.

Introduction

Lung cancer is a leading cause of cancer-related death worldwide [1]. There were more than 2 million new lung cancer cases and 1.76 million lung cancer-related deaths globally in 2018 [2]. As reported by National Cancer Center of China, lung cancer is also the most common cancer type and cause of cancer-related death in China, with crude incidence of 572.6 and mortality of 458.7 in 1 million population [3]. The high mortality of lung cancer is partially related to the large proportion (more than 60%) of late-stage patients when they were firstly diagnosed [4]. Since surgery is no longer applicable for those late-stage lung cancer patients, non-surgical therapies are the mainstay of treatments, including chemotherapy, radiotherapy, targeted therapy, and immunotherapy [4].

Targeted therapeutics has emerged during the past two decades. Different from traditional chemotherapy and radiotherapy, targeted therapy is only suitable for a subgroup of patients with specific genetic variations in tumor. For example, gefitinib was approved to treat metastatic non-small cell lung cancer (NSCLC) carrying exon 19 deletion or L858R mutation in epidermal growth factor receptor (EGFR) [5, 6]. Crizotinib was approved to treat advanced NSCLC with rearranged anaplastic lymphoma kinase (ALK) or ROS1 gene [7, 8], and dabrafenib which targets a rare mutation in lung cancer (BRAF V600E) was approved for the treatment of BRAF-positive advanced NSCLC [9]. Recently, the first KRAS-targeted therapy (Sotorasib) has been approved to treat locally advanced or metastatic NSCLC carrying KRAS G12C mutations [10]. At the same time, companion diagnostic tests for genetic variations in EGFR, ALK, ROS1, BRAF, or KRAS were also approved by the administrations, and are required before those targeted therapies are given. In addition, targeted treatments for other genetic variations in NSCLC are currently under development, including RET fusion [11], human epidermal growth factor receptor 2 (HER2) insertion (NCT04382300), or MET exon 14 skipping mutations (NCT02897479). Besides the drug targets mentioned above, some genetic variations in NSCLC indicate resistance to targeted therapies. For example, NSCLC patients carrying mutations in KRAS are less responsive to gefitinib [12]. Similar genetic variations which indicate resistance to anti-EGFR therapies include EGFR exon 20 insertion, NRAS exon 12 or 13 mutations, and PIK3CA mutations [13-16].

Considering the associations between the status of those genetic variations and sensitivity/resistance of targeted therapies, it would be important to understand their prevalence in diseases. Previous studies have investigated the prevalence of individual genetic variation in NSCLC [17-20]. However, not many studies investigated the prevalence and concurrence of those genetic variations in the same patient cohort. In addition, although previous studies showed linkage between smoking and tumor mutation profile in NSCLC [21], few studies investigated their association with other clinicopathological characteristics, e.g. gender, alcohol consumption. We hypothesized that there could be significant association between patient clinicopathological characteristics and genetic variations of tumor in NSCLC, which could help further optimize the usage of targeted therapies in patients with different clinicopathological background.

The aim of this study was to investigate the prevalence of genetic variations which indicate sensitivity or resistance to targeted therapies in NSCLC, as well as the relationships between those genetic variations and clinicopathological characteristics of patients. Our study results showed significant associations between genetic variations and age (EGFR L858R), gender (EGFR exon 19 deletion, L858R), smoking history (EGFR L858R, exon 20 insertion, and KRAS mutation), histological type (EGFR exon 19 deletion, L858R), and tumor grade (ALK fusion). In addition, further analysis also revealed significant associations between gender and EGFR L858R, KRAS mutation, histological type in non-smokers, and associations between smoking history and histological type in both male and female patients, or EGFR L858R in male patients.

Material and methods

Patients

Tumor samples were retrospectively collected from patients diagnosed with NSCLC who underwent surgery, transthoracic biopsy, bronchoscopy biopsy, or pleural aspiration in Sichuan Provincial People’s Hospital from January 2019 to March 2020. The inclusion criteria included: 1) pathologically-proven NSCLC, e.g. lung adenocarcinoma, lung squamous cell carcinoma, or large cell carcinoma; 2) age ≥ 18 years; 3) with sufficient clinicopathological information. The exclusion criteria included: 1) samples with pathologically-proven small cell lung cancer; 2) sample collected in lung not being the primary lesion; 3) samples from NSCLC patients who received chemotherapy, radiotherapy, or targeted therapies; 4) samples from patients with unclear smoking history or clinical staging information. All those tumor samples were formalin-fixed and paraffin-embedded. After sectioning and H&E staining, pathological diagnosis for each sample was confirmed by two independent pathologists, and slides with tumor content < 30% were excluded from further analysis. This study complied with the Declaration of Helsinki and was approved by Institutional Review Board of Sichuan Provincial People’s Hospital. Since this was a retrospective non-interventional study, informed consent was waived by the Institutional Review Board, and all the data were collected and analyzed anonymously.

Amplification-refractory mutation system (ARMS)

Three to eight sections from each tumor sample were firstly de-paraffinized in xylene in a 1.5-ml Eppendorf tube. DNA and RNA were extracted using a commercial DNA/RNA co-extraction kit (FFPE DNA/RNA Extraction Kit, AmoyDx, China) following the manufacturer’s instruction. After measurement of A260/A280, specific gene variations in tumor samples were determined using cFDA-approved commercial kit (Human Ten-Genetic-Variation Co-Detection Kit, AmoyDx, China) which target genetic variations in EGFR, ALK, ROS1, RET, KRAS, NRAS, BRAF, HER2, or PIK3CA on ABI7500 Real Time PCR system (ThermoFisher, USA) following the manufacturer’s instructions.

Statistical analysis

Statistical analysis was performed using IBM SPSS 24.0 software (IBM, USA). The genetic variation results and clinicopathological characteristics from each patient were summarized and the prevalence of positivity, negativity, or a specific subgroup was calculated by dividing the number of positive/negative/subgroup cases by the total number of patients. Relationship between different genetic variations and patient clinicopathological characteristics (age, gender, smoking history, alcohol consumption, histological type, and tumor grade) were then analyzed using χ2 test and Fisher’s exact test. Difference was considered statistically significant if P < 0.05. When statistical difference was observed among three or more subgroups (e.g. stage I, II, III, and IV), post hoc analysis was performed between each of the subgroups using χ2 test and Fisher’s exact test. When significant association was observed between different clinicopathological characteristics, relationship between genetic variations and clinicopathological characteristics were further analyzed in subgroups.

Results

Patient characteristics

In all, there were 405 patients enrolled in this study, in which 1 patient was excluded due to treatment history of targeted therapies. Samples from the rest 404 treatment-naïve patients were finally included in our analysis. As shown in Table 1, the patients were almost evenly distributed in males and females in this cohort. Majority of the patients were non-smokers (68.6%), and did not consume alcohol regularly (87.4%). Adenocarcinoma is the dominant subtype of NSCLC (94.3%) in this cohort. The percentage of tumor grades were Grade 1, 30.7%; Grade 2, 13.9%; Grade 3, 17.1%; Grade 4, 38.4%. Pathological diagnosis of NSCLC was confirmed in all samples.

Table 1

Demographics of patients and tumor characteristics.

Factors	Number	% (out of 404 patients)
Age
≤ median (63)	214	53.0
> median (63)	190	47.0
Gender
Male	209	51.7
Female	195	48.3
Smoking
Smoker	125	30.9
Non-smoker	277	68.6
Unknown	2	0.5
Alcohol consumption
Yes	51	12.6
No	353	87.4
Histological type
Adenocarcinoma	381	94.3
Squamous cell carcinoma	23	5.7
Tumor grade
1	124	30.7
2	56	13.9
3	69	17.1
4	155	38.4
Primary (grade 1 or 2)	180	44.6
Advanced (grade 3 or 4)	224	55.4

Biomarker evaluation

As shown in Table 2, EGFR exon 19 deletion (21%) and L858R mutation (26.2%) were the most common genetic variations found in this patient cohort. The overall positive rate for those two genetic variations was 47.0% (190/404), with 1 patient carrying both EGFR exon 19 deletion and L858R mutation. Similarly, with 4 patients carrying dual mutations (G719X/S768I, G719X/S768I, L858R/L768I, and L858R/L861Q), the overall positive rate for those rare EGFR mutations were 3.7% (15/404). Two patients carrying T790M also had L858R mutation. In the 9 patients carrying EGFR exon 20 insertion, 1 patient also had exon 19 deletion.

Table 2

Prevalence of genetic variations.

Biomarkers	Number	% (out of 404 patients)
EGFR
exon 19 deletion	85	21.0
L858R	106	26.2
L861Q	5	1.2
G719X	8	2.0
S768I	4	1.0
exon 20 insertion	9	2.2
T790M	2	0.5
Fusion	1	0.2
dual mutations	8	2.0
ALK
fusion	18	4.5
ROS1
fusion	8	2.0
RET
fusion	5	1.2
KRAS
G12X, G13X	36	8.9
NRAS
Q61H	1	0.2
BRAF
V600X	5	1.2
HER2
exon 20 insertion	14	3.5
PIK3CA
H1047R/E545K	4	1.0
MET
exon 14 skipping	6	1.5

EGFR, epidermal growth factor receptor; ALK, anaplastic lymphoma kinase; HER2, human epidermal growth factor receptor 2.

In other gene variations tested, the overall positive rate for ALK, ROS1, or RET fusion were 7.7% (31/404), with two samples holding both RET fusion and sensitive mutations in EGFR (exon 19 deletion, or L858R). Three patients carried both KRAS mutations and EGFR exon 19 deletion, and one patient had both BRAF mutation and EGFR exon 19 deletion. In addition, three patients carried both PIK3CA mutation and EGFR mutations (2 patients with exon 19 deletions, and 1 patient with both L858R and T790M).

Relationship between genetic variations in tumor and patient clinicopathological characteristics

As shown in Tables 3 and 4, older patients (age > median) were associated with significantly less prevalence of EGFR L858R mutation, compared to younger patients (age ≤ median). In addition, male patients showed significantly lower prevalence of EGFR exon 19 deletion and L858R mutation but significantly higher prevalence of KRAS mutations, compared to female patients. Smoking was associated with significantly lower prevalence of EGFR L858R mutation and KRAS mutation. In the two subtypes of NSCLC, adenocarcinoma was significantly associated with higher prevalence of EGFR exon 19 deletion and L858R mutation, compared to squamous cell carcinoma. Significant association was also observed between tumor grade and ALK fusion, and post hoc analysis showed that ALK fusion was significantly more prevalent in grade II tumors compared to grade IV tumors (S1 Table). No significant difference was found between alcohol consumption and histological type of NSCLC, or gene variations in tumor.

Table 3

Association between genetic variations and demographics of patient.

		Age			Gender			Smoking			Alcohol consumption
		≤ 63	> 63	P	Male	Female	P	Smoker	Non-smoker	P	Yes	No	P
EGFR
exon 19 deletion	Positive	41	44	0.325	34	51	0.015	21	63	0.175	11	74	0.921
	Negative	173	146		175	144		104	214		40	279
L858R	Positive	65	41	0.045	35	71	<0.001	17	89	<0.001	9	97	0.136
	Negative	149	149		174	124		108	188		42	256
L861Q	Positive	2	3	0.669	3	2	1.000	2	3	0.648	1	4	0.493
	Negative	212	187		206	193		123	274		50	349
exon 20 insertion	Positive	7	2	0.182	3	6	0.324	3	6	1.000	2	7	0.317
	Negative	207	188		206	189		122	271		49	346
G719X	Positive	6	2	0.291	5	3	0.725	2	6	1.000	0	8	0.603
	Negative	208	188		204	192		123	271		51	345
S768I	Positive	1	3	0.346	2	2	1.000	1	3	1.000	1	3	0.418
	Negative	213	187		207	193		124	274		50	350
T790M	Positive	1	1	1.000	0	2	0.232	0	2	1.000	0	2	1.000
	Negative	213	189		209	193		125	275		51	351
fusion	Positive	0	1	0.470	0	1	0.483	0	1	1.000	0	1	1.000
	Negative	214	189		209	194		125	276		51	352
ALK
fusion	Positive	9	9	0.796	8	10	0.527	4	14	0.405	0	18	0.146
	Negative	205	181		201	185		121	263		51	335
ROS1
fusion	Positive	4	4	1.000	3	5	0.490	2	6	1.000	1	7	1.000
	Negative	210	186		206	190		123	271		50	346
RET
fusion	Positive	4	1	0.376	3	2	1.000	2	3	0.648	0	5	1.000
	Negative	210	189		206	193		123	274		51	348
KRAS
mutation	Positive	21	15	0.499	28	8	0.001	17	19	0.028	7	29	0.193
	Negative	193	175		181	187		108	258		44	324
NRAS
Q61H	Positive	0	1	0.470	1	0	1.000	1	0	0.311	1	0	0.126
	Negative	214	189		208	195		124	277		50	353
BRAF
V600X	Positive	3	2	1.000	2	3	0.676	1	4	1.000	0	5	1.000
	Negative	211	188		207	192		124	273		51	348
HER2
exon 20 insertion	Positive	7	7	0.821	8	6	0.680	3	11	0.563	2	12	0.693
	Negative	207	183		201	189		122	266		49	341
PIK3CA
H1047R/E545K	Positive	1	3	0.346	2	2	1.000	2	2	0.591	2	2	0.079
	Negative	213	187		207	193		123	275		49	351
MET
exon 14 skipping	Positive	3	3	1.000	5	1	0.217	4	2	0.079	1	5	0.558
	Negative	210	187		203	194		121	274		50	347

EGFR, epidermal growth factor receptor; ALK, anaplastic lymphoma kinase; HER2, human epidermal growth factor receptor 2.

Table 4

Association between genetic variations and tumor characteristics.

		Histological type			Tumor grade
		Adenocarcinoma	Squamous cell carcinoma	P	I	II	III	IV	P	Primary	Advanced	P
EGFR
exon 19 deletion	Positive	85	0	0.007	30	6	17	32	0.180	36	49	0.646
	Negative	296	23		94	50	52	123		144	175
L858R	Positive	106	0	0.003	34	17	20	35	0.589	51	55	0.391
	Negative	275	23		90	39	49	120		129	169
L861Q	Positive	5	0	1.000	3	0	0	2	0.394	3	2	0.660
	Negative	376	23		121	56	69	153		177	222
exon 20 insertion	Positive	9	0	1.000	3	1	3	2	0.548	4	5	1.000
	Negative	372	23		121	55	66	153		176	219
G719X	Positive	8	0	1.000	3	0	2	3	0.669	3	5	0.737
	Negative	373	23		121	56	67	152		177	219
S768I	Positive	4	0	1.000	2	0	0	2	0.597	2	2	1.000
	Negative	377	23		122	56	69	153		178	222
T790M	Positive	2	0	1.000	0	0	0	2	0.358	0	2	0.505
	Negative	379	23		124	56	69	153		180	222
fusion	Positive	1	0	1.000	0	0	0	1	0.657	0	1	1.000
	Negative	380	23		124	56	69	154		180	223
ALK
fusion	Positive	18	0	0.613	4	6	5	3	0.025	10	8	0.337
	Negative	363	23		120	50	64	152		170	216
ROS1
fusion	Positive	8	0	1.000	1	2	1	4	0.573	3	5	0.737
	Negative	373	23		123	54	68	151		177	219
RET
fusion	Positive	5	0	1.000	0	1	1	3	0.506	1	4	0.387
	Negative	376	23		124	55	68	152		179	220
KRAS
mutation	Positive	35	1	0.709	12	5	5	14	0.955	17	19	0.736
	Negative	346	22		112	51	64	141		163	205
NRAS
Q61H	Positive	1	0	1.000	0	0	0	1	0.657	0	1	1.000
	Negative	380	23		124	56	69	154		180	223
BRAF
V600X	Positive	5	0	1.000	0	1	1	3	0.506	1	4	0.387
	Negative	376	23		124	55	68	152		179	220
HER2
exon 20 insertion	Positive	14	0	1.000	7	2	1	4	0.397	9	5	0.131
	Negative	367	23		117	54	68	151		171	219
PIK3CA
H1047R/E545K	Positive	3	1	0.210	1	0	1	2	0.827	1	3	0.632
	Negative	378	22		123	56	68	153		179	221
MET
exon 14 skipping	Positive	6	0	1.000	1	1	1	3	0.885	2	4	0.696
	Negative	374	23		123	55	68	151		178	219

EGFR, epidermal growth factor receptor; ALK, anaplastic lymphoma kinase; HER2, human epidermal growth factor receptor 2.

Relationship between histological type, tumor grade, and demographics of patients

As shown in Table 5, male patients were significantly more likely to be smokers and alcohol consumers. Adenocarcinoma was significantly more prevalent in female patient and non-smokers, compared to male patients and smokers, respectively. Significant association was observed between tumor grade and age, gender, smoking. Furthermore, advanced tumor was significantly more prevalent in older patients, male patients, smokers, and alcohol consumers. Post hoc analysis showed that stage III and stage IV tumors were significantly more prevalent in older patients. Compared to stage I tumors, male patients and smoking patients were significantly more likely to have stage III and stage IV tumors (S1 Table).

Table 5

Association between demographics of patients and tumor characteristics.

	Age			Gender			Smoking			Alcohol consumption
	≤ 63	> 63	P	Male	Female	P	Smoker	Non-smoker	P	Yes	No	P
Gender
Male	101	108	0.053	-	-	-	117	90	<0.001	48	161	<0.001
Female	113	82		-	-		8	187		3	192
Histological type
Adenocarcinoma	205	176	0.171	189	192	<0.001	107	272	<0.001	47	334	0.513
Squamous cell carcinoma	9	14		20	3		18	5		4	19
Tumor grade
I	82	42	<0.001	51	73	0.019	24	100	0.007	9	115	0.159
II	34	22		28	28		18	37		7	49
III	27	42		37	32		24	44		10	59
IV	71	84		93	62		59	96		25	130
Primary (grade 1 or 2)	116	64	<0.001	79	101	0.005	42	137	0.003	16	164	0.043
Advanced (grade 3 or 4)	98	126		130	94		83	140		35	189

Association between genetic variations, histological type, tumor grade, and gender or smoking history of patients

Since the percentage of smokers was much higher in male patients than in females, we further studied the relationship among histological subtype, tumor grade, genetic variations, and gender and smoking history of patients. As shown in Table 6, in both male and female patients, smokers are significantly more likely to have squamous cell carcinoma than adenocarcinoma. In male patients, tumors from smokers were significantly less likely to carry EGFR L858R mutation. Interestingly, in non-smokers, male patients were more likely to have squamous cell carcinoma than females. In addition, in non-smokers, more male patients were positive in KRAS mutations than female patients. However, in non-smokers, more female patients were positive in EGFR L858R mutation than male patients.

Table 6

Comparison of EGFR L858R, EGFR exon 20 insertion, KRAS mutation, histological type, and tumor grade between smoker and non-smoker in different gender, and between male and female patients in smokers or non-smokers.

	Male			Female			Smoker			Non-smoker
	Smoker	Non-smoker	P	Smoker	Non-smoker	P	Male	Female	P	Male	Female	P
EGFR L858R
Positive	14	21	0.031	3	68	1.000	14	3	0.076	21	68	0.039
Negative	103	69		5	119		103	5		69	119
EGFR 20ins
Positive	2	1	1.000	1	5	0.225	2	1	0.181	1	5	0.667
Negative	115	89		7	182		115	7		89	182
KRAS mutation
Positive	16	12	0.943	1	7	0.289	16	1	1.000	12	7	0.003
Negative	101	78		7	180		101	7		78	180
Histological type
Adenocarcinoma	101	86	0.026	6	186	0.004	101	6	0.324	86	186	0.040
Squamous cell carcinoma	16	4		2	1		16	2		4	1
Tumor stage
Primary	38	40	0.078	4	97	1.000	38	4	0.440	40	97	0.247
Advanced	79	50		4	90		79	4		50	90

EGFR, epidermal growth factor receptor; EGFR 20ins, EGFR exon 20 insertion.

Similarly, since histological type was significantly associated with gender of patients, we further investigated the relationship among histological type, gender, and genetic variations. As shown in Table 7, in adenocarcinoma, female patients had significantly higher prevalence of EGFR exon 19 deletion and L858R compared to male patients. On the other hand, in male patients, adenocarcinoma was associated with higher prevalence of EGFR exon 19 deletion and L858R, compared to squamous cell carcinoma.

Table 7

Comparison of EGFR exon 19 deletion and L858R between adenocarcinoma and squamous cell carcinoma in different gender, and between male and female patients in adenocarcinoma or squamous cell carcinoma.

	EGFR exon 19 deletion			EGFR L858R
	Positive	Negative	P	Positive	Negative	P
Male
Adenocarcinoma	34	155	0.050	35	154	0.029
Squamous cell carcinoma	0	20		0	20
Female
Adenocarcinoma	51	141	0.568	71	121	0.555
Squamous cell carcinoma	0	3		0	3
Adenocarcinoma
Male	34	155	0.044	35	154	<0.001
Female	51	141		71	121
Squamous cell carcinoma
Male	0	20	-a	0	20	-a
Female	0	3		0	3

EGFR, epidermal growth factor receptor.

aChi-square test was not applicable due to lack of positive cases of EGFR exon 19 deletion or EGFR L858R.

Discussion

Several targeted therapies have been approved for the treatment of NSCLC harboring certain genetic variations in EGFR, ALK, ROS1, or BRAF [5-9] and it is required to determine the existence of those genetic variations in tumor before targeted therapies are given to patients. Better understanding of the prevalence of those drug targets in NSCLC and their relationships may provide more guidance to clinical practices.

Our study investigated common genetic variations in EGFR, fusions in ALK or ROS1, and BRAF V600X mutations in a cohort of NSCLC patients. For EGFR, there are overall 50.7% of NSCLC patients harboring sensitive genetic variations to anti-EGFR therapies (including exon 19 deletion, L858R, L861Q, G719X, and S768I). This is higher than the percentage (32.3%) reported by a previous systemic review by Zhang et al. [19], which could be due to the different ethnicity of the patient cohorts in the two studies (Chinese patients versus mixed ethnicity of patients), or different sensitivity of measurement methods used in the two studies (ARMS versus a mixture of measurement methods). In all, there were 4.9% (10/205) of those patients holding genetic variations resistant to anti-EGFR therapy, including 2 patients with EGFR T790M mutation, 1 patient with EGFR exon 20 insertion, 3 patients with KRAS mutations, 1 patient with BRAF V600X mutation (BRAF V600E was also indicated as resistant mechanism for Osimertinib in a previous study [22]), and 3 patients with PIK3CA mutations.

Prevalence of ALK, ROS1, or RET fusions was relatively lower (7.7%), and only 1.2% of the patients had BRAF mutations. HER2 exon 20 insertion was found in 3.5% of the patients, which is higher than the prevalence (2.2%) as reported in previous study by Arcila et al. [17], possibly due to different screening strategy. Similar to the previous study, HER2 exon 20 insertion did not co-exist with other genetic variations in EGFR, ALK, ROS1, RET, or BRAF, indicating a need for targeted treatments for this specific subgroup of patients. Different from the previous study by Arcila et al. [17] which find two cases with concurrent PIK3CA mutations and HER2 exon insertion, we did not find any co-existing PIK3CA mutations in our patient cohort. MET exon 14 skipping mutation was found in 1.5% (6/404) of the patients in our cohort, which is similar to previous reports (1.6 ~ 2.6% of NSCLC) [18]. In our cohort, MET exon 14 skipping mutation did not co-exist with other genetic variations. Similarly, previous studies also showed no co-existing MET exon 14 skipping mutation with other driver gene variations, except amplifications of EGFR in 4 cases [18].

In our results, EGFR L858R mutation was more commonly seen in younger patients, which is conflicting with previous reports by Zhang et al. [20], possibly due to different cut-off value for younger and older patients (age of 50 in Zhang et al. versus median age used for cut-off value in our study). EGFR exon 19 deletion and L858R mutation were more commonly observed in female patients and adenocarcinoma. Similar observation has been reported between L858R mutation and gender, smoking, and histological type in a Japanese cohort [23] and a Polish cohort [24]. Further subgroup analysis confirmed the increased prevalence of those genetic variations in female patients in adenocarcinoma subgroup, and also in adenocarcinoma in male patients, which was not performed in previous studies. Similar to previous findings [23, 25, 26], smokers were more commonly found to have squamous cell carcinoma and KRAS mutation, and less likely to have EGFR L858R mutation in our cohort, and those associations between smoking and histological type, EGFR L858R mutation (but not KRAS mutation) were also observed after standardization of gender. Similarly, squamous cell carcinoma, KRAS mutations, and EGFR L858R mutation were more commonly found in males than females. Interestingly, after further standardization of smoking status, we found that in non-smokers, male patients were more likely to have squamous cell carcinoma and KRAS mutations and less likely to have EGFR L858R mutation than female patients. Previous study by Xue et al. [26] and Ramlau et al. [24] found that KRAS mutation was more common in male patients in a Chinese cohort, and EGFR L858R mutation was more common in female patients in the Polish cohort, but further standardization of smoking status was not performed in the study. Study by Stapelfeld et al. [27] reported conflicting results that KRAS mutations were more frequently found in women than in men, possibly due to the different race of patients (Caucasian) investigated in that study.

In summary, our study investigated the prevalence of targeted therapy-related genetic variations in NSCLC, as well as the relationship between those genetic variations and clinicopathological characteristics of patients. Compared to previous studies, our results showed overall similar prevalence of the investigated genetic variations in NSCLC. In addition, we also observed higher prevalence of EGFR exon 19 deletion and L858R both in female patients and in adenocarcinoma. Different from previous studies, we further looked into subgroups and confirmed those observations in female patients in adenocarcinoma subgroup and in adenocarcinoma in male patients, which indicate that the observations above were not due to the cross-linkage between gender and histological type but independent association between gender/histological type and the genetic variations. Furthermore, we also observed that smokers were more frequently to have squamous cell carcinoma and KRAS mutations, and less frequently to have EGFR L858R. Similarly, different from previous studies, we also performed subgroup analysis and confirmed those observations (except KRAS mutations) after standardization of gender, further confirming the association between histological type, EGFR L858R mutation and smoking. Our findings further confirmed the association between gender, histological type, smoking, and sensitive genetic variations of anti-EGFR therapies, which may help clinicians and clinical investigator in the patient selection of currently-available or potential targeted therapies. Limitations of this study could be the small patient cohort which may lead to potential statistical bias. Investigations using large patient cohort are required to further confirm the findings.

Post hoc analysis results of the association between ALK fusion, age, gender, and smoking history of patients and tumor stage.

(DOCX)

Click here for additional data file.

List of genetic variations and clinicopathological characteristics of patients.

(XLSX)

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16 Nov 2021

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We look forward to receiving your revised manuscript.

Kind regards,

Rama Krishna Kancha

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

The reviewers opined that the study is interesting but the importance limited by the smaller cohort. In addition, several limitations regarding the presentation of data and statistical analyses were raised which need to be addressed.

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2. Please ensure that you have specified (1) whether consent was informed, (2) what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee and (3) If you are reporting a retrospective study of medical records or archived samples, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data from their medical records used in research, please include this information.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript:

“This work was supported by 2018 Research Grant of The Education Department of Sichuan Province (No. 18CZ0044) to XS, and Co-Research Grant of Science & Technology Department of Sichuan Province and Luzhou Government and Luzhou Medical College (No. 14JC0084) to XS.”

We note that you have provided funding information within the Acknowledgements. Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“This work was supported by 2018 Research Grant of The Education Department of Sichuan Province (No. 18CZ0044) to XS, and Co-Research Grant of Science & Technology Department of Sichuan Province and Luzhou Government and Luzhou Medical College (No. 14JC0084) to XS.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

4. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript is by and large repeating previous NSCLC studies and is focused on clinically actionable coding aberrations and patient characteristics in this cohort of patients. The rank order of frequency for specific aberrations recapitulates previous studies. However, the total frequencies identified in this study are remarkably higher than results from other studies. The authors address this, pointing out that it may come down to methodology and differences in the size of the cohorts (this one being smaller), which is a reasonable explanation.

The interesting finding is that among non-smokers, squamous cell carcinoma patients were more often male than female. Though as pointed out by the authors, this is a small cohort and it needs to be validated.

The technology is appropriate and sound. Statistical approach is adequate.

Critique:

The study is not necessarily novel nor addressing a research question/hypothesis and it is uncertain that any new knowledge is gained from it.

The description of results does not refer to data tables. In addition, currently there are only 2 tables, and they present limited information/data. Tables are lacking data analysis results described in the results section, columns for p-values should also be included.

Supplemental data files should list all testing results so that analyses can be repeated by others.

The product number provided does not appear to be useful to find the kit online. Please provide another way to identify the kit, such as name of the AmoyDx assay.

The background should be updated to include availability of KRAS targeted therapy.

Reviewer #2: Detailed comments have been included in the attachment.

**********

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Reviewer #1: No

Reviewer #2: No

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Submitted filename: PONE-D-21-30951_reviewer_comments.docx

Click here for additional data file.

24 Dec 2021

Comments from Editor:

The reviewers opined that the study is interesting but the importance limited by the smaller cohort. In addition, several limitations regarding the presentation of data and statistical analyses were raised which need to be addressed.

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response: Many thanks for your comments. We have checked up the style of our manuscript and made necessary revisions to meet the journal’s requirements. Please find the revisions in the revised manuscript.

2. Please ensure that you have specified (1) whether consent was informed, (2) what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee and (3) If you are reporting a retrospective study of medical records or archived samples, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data from their medical records used in research, please include this information.

Response: Since this is a retrospective non-interventional study, the informed consent was waived by the Institutional Review Board, and all the data were collected and analyzed anonymously. Those statements were also included in the “Patients” section of Material and Methods. Please find them in the revised manuscript.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript:

“This work was supported by 2018 Research Grant of The Education Department of Sichuan Province (No. 18CZ0044) to XS, and Co-Research Grant of Science & Technology Department of Sichuan Province and Luzhou Government and Luzhou Medical College (No. 14JC0084) to XS.”

We note that you have provided funding information within the Acknowledgements. Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“This work was supported by 2018 Research Grant of The Education Department of Sichuan Province (No. 18CZ0044) to XS, and Co-Research Grant of Science & Technology Department of Sichuan Province and Luzhou Government and Luzhou Medical College (No. 14JC0084) to XS.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Response: Very sorry for our mistakes. We have removed the funding information from the acknowledgement section. Please find the amended funding statements as follows:

This work was supported by 2018 Research Grant of The Education Department of Sichuan Province (No. 18CZ0044) to XS, and Co-Research Grant of Science & Technology Department of Sichuan Province and Luzhou Government and Luzhou Medical College (No. 14JC0084) to XS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

4. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

Response: Sorry for the missing information. We have inserted all data regarding the testing results (genetic variations results) and clinicopathological characteristics from each of the patients as supplementary information of the revised manuscript. Please find the details in S1 File.

Comments from Review #1:

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

________________________________________

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript is by and large repeating previous NSCLC studies and is focused on clinically actionable coding aberrations and patient characteristics in this cohort of patients. The rank order of frequency for specific aberrations recapitulates previous studies. However, the total frequencies identified in this study are remarkably higher than results from other studies. The authors address this, pointing out that it may come down to methodology and differences in the size of the cohorts (this one being smaller), which is a reasonable explanation.

The interesting finding is that among non-smokers, squamous cell carcinoma patients were more often male than female. Though as pointed out by the authors, this is a small cohort and it needs to be validated.

The technology is appropriate and sound. Statistical approach is adequate.

Response: Many thanks for your comments and kind suggestions. Please find our point-to-point response below.

Critique:

The study is not necessarily novel nor addressing a research question/hypothesis and it is uncertain that any new knowledge is gained from it.

Response: Thanks very much for your comments. We have added more words in the Introduction section to describe the rationale, knowledge gap, hypothesis, and findings of our study. Please find the revisions in the 3rd and 4th paragraphs of Introduction section of the revised manuscript.

The description of results does not refer to data tables. In addition, currently there are only 2 tables, and they present limited information/data. Tables are lacking data analysis results described in the results section, columns for p-values should also be included.

Response: Thanks very much for your suggestion. We have added five more tables (Table 3 - 7) to provide more details of the results. Relevant parts of the Results (section 3-5) and Discussion (paragraph 4) were also revised accordingly. Please find the revisions in the revised manuscript.

Supplemental data files should list all testing results so that analyses can be repeated by others.

Response: Thanks very much for your suggestion. We have listed all the testing results and clinicopathological characteristics of patients in supporting information. Please find the detailed information in S1 File.

The product number provided does not appear to be useful to find the kit online. Please provide another way to identify the kit, such as name of the AmoyDx assay.

Response: Very sorry for the misleading information provided. We have replaced the product number with the name of the AmoyDx assy kit. Please find the revisions in the 2nd section in Methods (Amplification-refractory mutation system (ARMS) section).

The background should be updated to include availability of KRAS targeted therapy.

Response: Very sorry for the missing information. Following your suggestions, we have revised the Introduction section to reflect the approval of KRAS-targeted therapy for the treatment of KRAS G12C-mutated NSCLC. Please find the revisions in the 2nd paragraph of the Introduction in the revised manuscript.

Reviewer #2: Detailed comments have been included in the attachment.

________________________________________

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Comments from Review #2:

The study is aimed at assessing the prevalence of sanitizing and resistant mutations among patients with cancer in China. The authors also aimed to understand if there were any associations between genetic alterations and clinicopathological characteristics of patients with cancer. They found that nearly half of the patients (N=404) were eligible for receiving anti-EGFR therapy. Gender could influence the histological type and KRAS mutation frequency among non-smokers.

Response: Many thanks for your comments and kind suggestions. Please find our point-to-point response below.

Title:

The title is indicative of what the study entails.

Response: Many thanks for your comments.

Abstract:

This should be a standalone section of the manuscript, and therefore, should adequately summarize all sections of the main text. Currently the abstract does not provide enough background or context for the study. Similarly, the methods section of the abstract could be more detailed.

Response: Many thanks for your suggestions. We have expanded the Background, Methods, Results, and Conclusion of the Abstract to provide more detailed information. Please find the revisions in the Abstract of the revised manuscript.

Introduction:

-Although this section provides sufficient background about the incidence of lung cancer globally and in China and also describes the current literature with respect to available therapeutic targets in NSCLC, the rationale for the current study and the current knowledge gap in existing literature have not been clearly highlighted.

Response: Thanks very much for your comments and sorry for the missing information. Following your suggestions, we have added the rationale for this study and the knowledge gap in Introduction. Please find the revisions in the 3rd and 4th paragraphs in the Introduction section of the revised manuscript.

-Also, this section does not adequately bring out the originality of the current work and the need for it.

Response: Thanks very much for your comments. We have inserted the background (originality) of this study and the gap of knowledge in the Introduction section, and also discussed the need for the conduction of this study in the same section. Please find the revisions in the 3rd paragraph of the Introduction section in the revised manuscript.

Materials and methods:

-The study design and setting have not been described in this section. The authors have also not mentioned if informed consent was obtained from the participants of this study and whether the study was performed in accordance with ethical standards laid out for studies involving human subjects.

Response: Sorry for the missing information. This was a retrospective non-interventional study and all the data were collected and analyzed anonymously. Therefore, the informed consent from the patients was waived by the Institutional Review Board. The study design complied with the Declaration of Helsinki and was approved by the Institutional Review Board. Those descriptions have been included in the “Patients” section of Materials and Methods. Please find them in the revised manuscript.

-The inclusion and exclusion criteria used for patient recruitment in the current study have not been described.

Response: Sorry for the missing description. We have inserted the inclusion criteria and exclusion criteria in the 1st part (Patients) of Material and Methods section. Please find it in the revised manuscript.

-Since, the aim of this study was to investigate the prevalence of genetic variations which indicate either sensitivity or resistance to targeted therapies in NSCLC, it is imperative to provide details about the line of treatment received by the patients. Currently, it is not clear from this section if the patients were treatment-naive or had developed resistance to a previous line of therapy.

Response: Sorry for the missing information. All the patients were treatment-naïve. In the exclusion criteria newly-added into the manuscript, patients who received chemotherapy, radiotherapy, or targeted therapies were excluded. We have further clarified this issue at the beginning of Results. Please find the revisions in the 1st section (Patients) of Material and Methods, and 1st section (Patient characteristics) of Results of the revised manuscript.

-This section currently does not describe the study endpoints.

Response: Sorry for the missing description. We have expanded the “Statistical analysis” section to describe the study endpoints, including prevalence of genetic variations and patient clinicopathological characteristics, and the detailed analysis on their relationships. Please find the detailed revisions in “Statistical analysis” section in Material and Methods in the revised manuscript.

Results:

This section does not provide information about how many patients were enrolled, how many were excluded, and how many were finally included in the analysis.

Response: Sorry for the missing information. We have inserted the numbers of patients enrolled, excluded, and finally included in our study at the beginning of Results. Please find the description in the 1st section (Patient characteristics) of Results in the revised manuscript.

Discussion:

The authors have mentioned that 50.7% of NSCLC patients harbored genetic variations sensitive to anti-EGFR therapies (including exon 19 deletion, L858R, L861Q, G719X, and S768I), and that this is higher than the percentage (32.3%) reported by a previous systemic review by Zhang et al.[16]. The authors could try to provide a reason for this considerably higher proportion observed in the current study. Could this be because of the differences in the ethnicity of the two cohorts?

Response: Many thanks for your suggestion. We have further discussed the reason for the higher percentage of EGFR mutations found in our study. Please find the revisions in the 2nd paragraph of the Discussion in the revised manuscript.

The discussion section does not highlight the novelty of the current research and its possible implications on future research and clinical and/or diagnostic practices.

Response: Many thanks for your comments and sorry for the missing information. We have summarized the novelty of our research in the last paragraph of Discussion. Please find the revisions in the revised manuscript.

The conclusions drawn from the results obtained have not been mentioned at the end of the manuscript text.

Response: Sorry for the missing description. We have further summarized our findings in the last paragraph of Discussion section. Please find the revisions in the revised manuscript.

Tables:

The total cohort size for which the percentages have been denoted needs to be mentioned in the table headers. Moreover, there is considerable duplication of information between table 2 and “biomarker evaluation” section of the results.

Response: Sorry for the missing information. We have added the total cohort size (“out of 404 patients”) in the headers of Table 1 and 2. Please find them in the revised manuscript. In addition, we also revised the “biomarker evaluation” section and removed the duplicated information with Table 2. Please find the revisions in the revised manuscript.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

6 Jan 2022

Prevalence of targeted therapy-related genetic variations in NSCLC and their relationship with clinicopathological characteristics

PONE-D-21-30951R1

Dear Dr. Sun,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Rama Krishna Kancha

Academic Editor

PLOS ONE

12 Jan 2022

PONE-D-21-30951R1

Prevalence of targeted therapy-related genetic variations in NSCLC and their relationship with clinicopathological characteristics

Dear Dr. Sun:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

Dr. Rama Krishna Kancha

Academic Editor

PLOS ONE