Integrative Analysis of Targeted Genomic Profiling and Immune Cell Infiltration in the Prognosis of Lung Adenocarcinoma

Summary

This protocol enables the integration of next-generation sequencing and multicolor immunofluorescence to profile lung adenocarcinoma. It aims to analyze the relationship between genomic heterogeneity and tumor-infiltrating immune cell density. This multidimensional approach facilitates a comprehensive evaluation of the tumor microenvironment for postoperative prognostic assessment.

Abstract

The prognosis of lung adenocarcinoma (LUAD) is traditionally evaluated via tumor-node-metastasis (TNM) staging, which does not account for the patient's immune status. This protocol integrates genomic profiling and immune microenvironment analysis to facilitate a more comprehensive postoperative prognostic evaluation. The method involves a retrospective analysis of paraffin-embedded tumor tissues using two primary techniques. First, next-generation sequencing (NGS) is performed with a customized 37-gene panel to identify mutations in driver genes and variants of uncertain significance. Second, multiplex immunofluorescence (mIF) is utilized to target markers including HLA-DR, CD68, CD163, CD206, PD-L1, and PanCK. This enables the quantification of spatial distribution and density for specific immune cell subpopulations across various tumor regions. This integrated approach enables the simultaneous assessment of genomic heterogeneity and tumor-infiltrating immune cell characteristics. The resulting data identifies specific combinations of mutational profiles—such as EGFR status—and immune cell densities. These integrated combinations enable the study of their collective impact on patient survival, offering a promising approach for the development of future lung cancer prognostic models. This protocol demonstrates a robust method for characterizing the complex biological features of the LUAD tumor microenvironment.

Introduction

LUAD is the most prevalent subtype of lung cancer, yet the average 5-year survival rate remains low at approximately 15%¹. Current prognostic evaluation primarily relies on the TNM staging system; however, this method often overlooks the patient's overall immune status, which may limit its predictive accuracy. Evidence suggests that both genomic heterogeneity and the tumor microenvironment (TME) play critical roles in LUAD tumorigenesis and disease progression^2,3.

In recent years, the transition from traditional Sanger sequencing to targeted NGS has enabl....

Protocol

This retrospective study was approved by the Medical Ethics Committee of Kunming Yan'an Hospital (Approval No. 2021-140-01). Informed consent was obtained from all participants.

Study design and sample collection
A cohort of 63 patients with pathologically or histologically confirmed LUAD who underwent surgical resection between 2015 and 2021 was enrolled. Exclusion criteria included the receipt of neoadjuvant chemotherapy, radiotherapy, or immunotherapy prior to surgery. Additionally, patients with concurrent malignancies, severe comorbidities, poor tissue quality, or incomplete clinical records were excluded fro....

Results

Clinical characteristics of patients with lung adenocarcinoma
A total of 63 patients who were diagnosed with LUAD and underwent surgical treatment in our hospital from 2015 to 2021 and met the inclusion criteria were collected in this study. The clinical characteristics of the included patients are shown in Table 1. Among the enrolled patients, the median age was 60 years (47-73 years), including 30/63 male patients (46.2%) and 33/63 female patients (53.8%). There were 23/63.......

Discussion

Integrated genomic and spatially resolved immunological profiling of LUAD
This study demonstrates an integrated workflow combining targeted NGS and mIF to characterize the multidimensional landscape of LUAD. Our genomic findings align with previous literature, particularly regarding ethnic variations in mutation frequencies. The observed EGFR mutation rate (approximately 40%-60%) is consistent with reported frequencies in Asian populations, which are significantly higher than those in West.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AlphaTSA Multiplex IHC Kit	alphaxbio Technology Co., Ltd.	AXT36025011	Multiplex immunofluorescence staining
Antifade Mounting Medium	Leagene Technology Co., Ltd.	IH0252, Lot0525A17	Multiplex immunofluorescence staining
CD163	Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd	ZM0428	RRID:AB_3714707;Multiplex immunofluorescence staining
CD206	Abcam, Inc	Cat# 5307-1	RRID:AB_10896526;Multiplex immunofluorescence staining
CD68	Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd	ZM0060	RRID:AB_2904190;Multiplex immunofluorescence staining
customlised cancer gene panel	TWIST Co., Ltd.		NGS Library Preparation
Deparaffinization Buffe	QIAGEN Co., Ltd.	19093	DNA Extraction HE staining; Multiplex immunofluorescence staining from FFPE Tissue Sections
Dynabead M-270 streptavidin	Thermo Co., Ltd.	65602	NGS Library Preparation
Fluorometer	ThermoFisher Scientific Co., Ltd.	QUBIT 4.0	NGS Library Preparation
HALO digital pathology image analysis platform	Indica Labs, Inc., Albuquerque	version 3.6.4134	HE staining and Multiplex immunofluorescence staining analysis
Hematoxylin-Eosin Staining Kit	Wuhan elabscience Technology Co., Ltd.	E-IR-R117	HE staining
HiPure FFPE DNA Kit	Guangzhou Magen Biotechnology Co., Ltd.	D6323	DNA Extraction from FFPE Tissue Sections
HLA-DR	HUABIO Co., Ltd.	ET1610-66	RRID:AB_3069950; Multiplex immunofluorescence staining
Illumina DNA sequencer	Illumina,  Inc	550DX	Sequencing
Neutral balsam	Beijing Solarbio Science & Technology Co., Ltd.	G8590	HE staining
Panck	Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd	ZM0069	RRID:AB_2941997;Multiplex immunofluorescence staining
PD-L1	R and D Systems	Catalog # AF1019	RRID:AB_354540;Multiplex immunofluorescence staining
TBST buffer	abclonla Technology Co., Ltd.	RM00013	HE staining and Multiplex immunofluorescence staining
UN-Blocker	homgen Co., Ltd.		NGS Library Preparation
VAHTS DNA Clean Beads	Vazyme Co., Ltd.	N411	NGS Library Preparation
VAHTS uniersal DNA library Prep kit for illumina	Vazyme Co., Ltd.	ND610	NGS Library Preparation
whole-slide imaging system	ZEISS Co., Ltd.	ZEISS AXIOSCAN 7 version3.3	HE staining and Multiplex immunofluorescence staining analysis
xGen Hybridization and Wash Kit	IDT Co., Ltd.	1080577	NGS Library Preparation