Tumor Characteristics and Treatment Responsiveness in Pembrolizumab-Treated Non-Small Cell Lung Carcinoma

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors sought to identify factors underlying differences in response to pembrolizumab between patient groups. Although some results were obtained, the reader failed to gain valuable information. The authors can then carefully consider the purpose of the study and refine the research strategy, including increasing the number of cases.

 

Here are some suggestions based on existing content:

 

1. Analysis of the significance of differences in clinical characteristics between different groups.

 

2. Evaluation criteria for CD8+ lymphocyte levels.

 

3. The Kaplan-Meiser survival curves of the 4 groups are in Figure 2A.

 

4. Lines 194-196, which groups of patients are clinically responding? In Figure 2B, p 0.05 for rg&pd and p0.05 for SD&PD. What p values?

 

5. Sequencing data of gene expression and mutations should be provided as attachments, and raw sequencing data should be uploaded to public databases.

 

6. Are lines 255-267 misplaced or not deleted?

 

​7. Reference 22 Analysis of differences between tumor-stromal ratios.

Author Response

Reviewer 1

Comments and Suggestions for Authors

The authors sought to identify factors underlying differences in response to pembrolizumab between patient groups. Although some results were obtained, the reader failed to gain valuable information. The authors can then carefully consider the purpose of the study and refine the research strategy, including increasing the number of cases.

• We express our sincere gratitude to the reviewer for providing thorough and thoughtful comments. In the revised manuscript, we have made efforts to refine the purpose of the study. The data derived from this study emphasizes the significance of detailed pathological characteristics in predicting treatment responsiveness in pembrolizumab-treated NSCLC cases. However, we acknowledge that the relatively small number of cases is one of the limitations of our study. Future studies with a larger sample size will be necessary to validate our findings.

 

Here are some suggestions based on existing content:

1. Analysis of the significance of differences in clinical characteristics between different groups.

• We appreciate this suggestion. The clinical characteristics have been statistically analyzed and presented in Table 1. The revised table is shown below-

Table 1. Clinicopathologic characteristics

Characteristic	Overall, N = 841	Responsive, N = 421	Stable, N = 231	Progressive, N = 191	p-value2
age	65 (61, 74)	68 (62, 76)	65 (60, 71)	64 (57, 70)	0.2
gender					0.6
Female	48 (57%)	23 (55%)	15 (65%)	10 (53%)
Male	36 (43%)	19 (45%)	8 (35%)	9 (47%)
diagnosis					0.3
Adenocarcinoma	51 (61%)	26 (63%)	15 (65%)	10 (53%)
Squamous cell carcinoma	18 (22%)	7 (17%)	6 (26%)	5 (26%)
Adenosquamous carcinoma	1 (1.2%)	0 (0%)	1 (4.3%)	0 (0%)
NSCLC	13 (16%)	8 (20%)	1 (4.3%)	4 (21%)
smoking					0.7
Current	27 (33%)	12 (30%)	9 (41%)	6 (32%)
Former	46 (57%)	22 (55%)	12 (55%)	12 (63%)
Never	8 (9.9%)	6 (15%)	1 (4.5%)	1 (5.3%)
secondMalignancy	11 (13%)	6 (14%)	4 (17%)	1 (5.3%)	0.5
1 Median (IQR); n (%)
2 Kruskal-Wallis rank sum test; Pearson’s Chi-squared test; Fisher’s exact test

 

2. Evaluation criteria for CD8+ lymphocyte levels.

• This has been clarified as follows: The levels for CD8 TILs were recorded from molecular profiling data (Omniseq Inc) from patients’ medical record. Per report, CD8+ lymphocytes were analyzed using RNA sequencing. Interpretation of gene expression for CD8 TIL is done as follows: Each gene is compared to a reference population derived from 735 unique tumors, normalized to a value between 1 and 100 and scored as the percentile (relative) rank (%rank). CD8 TILs gene expression is expressed as high for genes ranked 75-100 (highly inflamed), moderate for genes ranked 25-74 (moderate inflamed), and low for genes ranked 0-24 (non-inflamed). In our analyses, the tumor was categorized as inflamed if the CD8 gene expression profile was high or moderate and non-inflamed if the expression profile was reported as low.

3. Lines 194-196, which groups of patients are clinically responding? In Figure 2B, p 0.05 for rg&pd and p0.05 for SD&PD. What p values?

• We apologize for this confusion. This statement has been revised as follows-

In the treatment-responsive group (complete and partial response), 29 of 42 patients (69.0%) showed tumor infiltrating CD8⁺ lymphocytes. In the non-responsive group (stable disease and progressive disease), only 17 of 42 patients (40.5%) showed intratumoral CD8⁺ lymphocytes (p <0.05). There was no significant difference between SD and PD groups.

 

4. Sequencing data of gene expression and mutations should be provided as attachments, and raw sequencing data should be uploaded to public databases.

• Summary of mutation profile is provided in the supplemental table. Unfortunately, we don’t have access to raw sequencing data currently.

•  

Group	Mutation_TP53	Mutation_KRAS	Mutation_Other
RG	C238F		NOTCH1
	P278R, H179Q		CDKN2A, RAF1, TERT PROMOTER SNV, STK11, ATM
	D148Y		NF1, APC, PIK3R1
			ATM
	C733G	G12V	BRAF, AR E710D
	E271Q	G12V	STK11
	e343x	g13d	atm, gata3, kit
			BRAF, PIK3R1
	Y205f		CDKN2A, ATM,  APC
		G12S	BRCA2,
	A159P, R175H		CDKN2A, NF2, APC, CDH1, PIK3R1, SMAD4, FBXW7
		G12C
	E286A, R306	Q61L	BRAF, IDH, MARP2K, PIK3CA,
	R337L	G12C	CDKN2A, VHL
		G12C	ABL1
		G12C	TSC2
	H193L		PIK3R1
			NRAS, BRAF
	g262v		CCND1, PIK3CA, CDKN2A, APC, CD274, PDCD1LG2, PIK3R1
		G12C	CTNNB1, BRCA1, PIK3CA
	T123M	Q61H
	K132N		KIF5Be15-RETe12, CDKN2A
	G266X,		NF1,TSC2, RET
		G12C	BRAF, ATM, SMAD4
	Y236C		NFE2L2, PTEN, CDKN2A
	C135F		TSC2, FGFR3e17-TACC3e10, RB1
			FBXW7
		G12A	NF1, IFITM3
	E171X		TSC2, STK11
	r337l		BRCA2, NRAS, APC, NF2, PIK3R1, TET2
		C12A
	Y	G12C
	Splice site SNV		BRAF(V600E), SMAD4
			CDKN2A,
		G12C	CDKN2A,
	R273L		MET, NRAS, ROS1
	V173G
	A159P		BRCA2, EGFR, NFE2LE, pik3r1, ATM, TERT2
			EGFR SLICE SITE SNV
		G12A	ATM, PIK3CA
SD	C445del
	R175H		CDKN2A, PIK3CA, ATM
			BRCA2
		G12C
		g12v
			EGFR, CDKN2A, NET, CDK6, BRAF EGFR
	R249S	G12C
		G13D	NRS, KEAP1, STK11
		G12F	BRCA2, ATM
	R273L	G12V	BAP1
			NF1
		G12C
		G12V
	G245V		STK11,FGFR2, APC, PDGFRA
			PIK3CA
			FGFR2, EZH2, TERT PROMOTER SNV, NOTCH1
		Q61R	APC
	E349fs
	N131Y, G154fs	G12C	TSC1, KIT , NTRK1. AR, ATM, KIT AMPLIFICATION
			BRCA2
PD		G13D
			EGFR
	S166X		EGFR, CTNNB1
		G12V	APC, NF1, PDCD1LG2
		g12c	CDKN2A, TET2
	L114FfsX35		GATA3
		G12V
		G12C	ATM, VHL
	c517g		PTEN, FGFR2
	G266R		DDR2, CDKN2A, GATA3, STK11
	H193L		ATM, MARPK1
		G12C
			EGFR, EML4-ALK, ARID1A
	V157F	G12C	APC
	E204X
	R273H, P142F		NRAS
		G12V

•  

5. Are lines 255-267 misplaced or not deleted?

• Our apology for this editorial error. This has been corrected.

​6. Reference 22 Analysis of differences between tumor-stromal ratios.

We agree.

Reviewer 2 Report

Comments and Suggestions for Authors

The study seems to be excellent. The only issue is that material gained from biopsy and surgery is not comparable. First of all You do not mention whether it was fine needle biopsy or core biopsy. Secondly material resected during surgical procedure differs in pathological microscopic view as a lot of different processes ( including inflammation and lymphocyte migration) is generated also by surgical procedure itself. It will be marvelous to continue this study and to make patients group more uniform, thanks to analysis of   tissue samples acquired exclusively surgically. 

Author Response

Reviewer 2

The study seems to be excellent. The only issue is that material gained from biopsy and surgery is not comparable. First of all, you do not mention whether it was fine needle biopsy or core biopsy. Secondly material resected during surgical procedure differs in pathological microscopic view as a lot of different processes (including inflammation and lymphocyte migration) is generated also by surgical procedure itself. It will be marvelous to continue this study and to make patients group more uniform, thanks to analysis of   tissue samples acquired exclusively surgically. 

• We greatly appreciate these comments. Regarding the biopsy samples, the tissue was collected through core needle biopsies. While the sample comprised both biopsies and resection specimens, the majority consisted of biopsy tissue, given that patients with advanced disease stage are rarely treated with surgical resection of the tumor. It would be meaningful to have a more uniform population of samples involving surgically resected tissue when a reasonable sample size becomes available in the future. We acknowledge the fact that surgical resection can initiate tissue inflammation, often involving neutrophils rather than lymphocytes. These limitations are discussed in the revised manuscript.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The author made modifications based on the review comments and has no further suggestions.