Ki-67 with Neutrophil-to-lymphocyte and CD4-positive/CD8-positive Ratios Predicts Early Response in Non-small Cell Lung Cancer

Summary

This protocol standardizes early response assessment in NSCLC patients receiving PD-1/PD-L1 inhibitors by integrating baseline NLR, flow-cytometric CD4/CD8 ratio, tumor Ki-67 immunohistochemistry scoring, and RECIST 1.1 evaluation after two cycles, with reproducibility-focused quality-control checkpoints throughout.

Abstract

This study evaluated the combined predictive value of tumor Ki-67 expression status, peripheral blood neutrophil-to-lymphocyte ratio (NLR), and the CD4-positive/CD8-positive T-cell ratio for short-term response assessment in patients with non-small cell lung cancer receiving PD-1/PD-L1 inhibitor therapy. We retrospectively analyzed 102 consecutive patients treated at Binhaiwan Central Hospital between January 2021 and October 2023 who completed two treatment cycles. Responses were assessed using Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1: 13 patients achieved complete response, 26 partial response, 54 stable disease, and 9 progressive disease. For analysis, we defined response as complete response plus partial response and non-response as stable disease plus progressive disease. Univariable screening identified candidate predictors associated with response status, which were entered into multivariable logistic regression adjusted for age, Eastern Cooperative Oncology Group performance status, and smoking history, identifying Ki-67 status, NLR, and the CD4-positive/CD8-positive ratio as independent predictors of short-term response classification. Receiver operating characteristic analysis supported improved discrimination for the combined biomarker model compared with single markers, highlighting a practical, minimally invasive approach for early response stratification in this clinical setting.

Introduction

Accounting for nearly 85% of lung malignancies, non-small cell lung cancer (NSCLC) is the most prevalent histological subtype and predominantly affects middle-aged and older adults in China¹. Its etiology is multifactorial and includes tobacco exposure, long-term contact with industrial dust or pollutants, hereditary susceptibility, and chronic exposure to environmental carcinogens²˒³. Early-stage NSCLC often presents with non-specific respiratory symptoms and can be misattributed to common airway conditions in primary care, contributing to delayed recognition and a high proportion of patient....

Protocol

1. Ethics and regulatory compliance

1. Obtain approval from the Ethics Committee of Binhaiwan Central Hospital of Dongguan (Approval No.: 2021045).
2. Ensure all procedures comply with the Declaration of Helsinki.
3. Confirm that written informed consent was obtained prior to treatment and document consent status in the study file.

2. Patient identification and eligibility screening

1. Identify consecutive patients diagnosed with non-small cell lung cancer who initiated programmed cell death protein 1/programmed death-ligand 1 inhibitor therapy between January 2021 and....

Results

Baseline characteristics
Baseline clinical characteristics of the 102 patients with non-small cell lung cancer are summarized in Table 1. The response group (complete response + partial response) included 30 males and 9 females, whereas the non-response group (stable disease + progressive disease) included 50 males and 13 females (p = 0.771). In the response group, 79.49% of patients were younger than 65 years, compared with 66.67% in the non-response group. No statistically signific.......

Discussion

Immune checkpoint inhibition has become an essential component of systemic therapy for advanced non-small cell lung cancer (NSCLC), yet clinically meaningful benefit remains concentrated in a subset of patients¹⁵˒¹⁶. This response heterogeneity, together with the cost and potential toxicity of prolonged treatment, makes early response stratification clinically relevant for follow-up planning and timely reassessment¹⁷. In this JoVE protocol, we d.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Automated hematology analyzer	Sysmex Corporation	XN-1000	Complete blood count and NLR calculation
Brightfield microscope	Olympus	BX53	Histological evaluation
Citrate antigen retrieval buffer (pH 6.0)	Abcam	ab93678	Heat-induced antigen retrieval
DAB substrate kit	Vector Laboratories	SK-4100	Chromogenic development
EDTA anticoagulant blood collection tube	BD Biosciences	367525	Peripheral blood collection
Flow cytometer	BD Biosciences	FACSCanto II	T-cell subset analysis
Flow cytometry tubes (12 75 mm)	BD Falcon	352058	Sample acquisition tubes
Formalin-fixed paraffin-embedded tumor tissue blocks	Clinical Pathology Archive	N/A	NSCLC tumor samples
Four-color T-cell subset reagent (CD3/CD4/CD8/CD45)	BD Biosciences	340499	For lymphocyte subset detection
Hematoxylin stain	Sigma-Aldrich	H3136	Nuclear counterstaining
HRP detection kit	Dako (Agilent)	K5007	Secondary antibody detection system
Ki-67 primary antibody (clone MIB-1)	Abcam	ab16667	Immunohistochemical detection
Microtome	Leica Microsystems	RM2235	Cutting 4 m FFPE sections
Phosphate-buffered saline (PBS)	Thermo Fisher Scientific	10010023	Cell washing and resuspension
Positively charged microscope slides	Thermo Fisher Scientific	J1800AMNZ	Tissue section mounting
Red blood cell lysing solution	BD Biosciences	349202	Erythrocyte lysis before flow cytometry
Statistical analysis software	IBM	SPSS v26.0	Data analysis