Breast Cancer Combined with Primary Lung Cancer: A Study of Clinicopathologic Features and Prognostic Factors

Summary

We present a single-center, pathology-validated workflow for breast cancer (BC) patients who develop a second primary lung cancer (pLC). Larger primary tumor size and higher Ki-67 define risk, while cohort-level OS and iDFS remain comparable. Findings support risk-stratified chest imaging and early pathology correlation in follow-up.

Abstract

We conducted a single-center retrospective study of patients with breast cancer (BC) who developed a second primary lung cancer (pLC) (BC-pLC; n = 54) versus BC only (n = 216) undergoing curative-intent surgery from 2012 to 2017, with follow-up through June 30, 2023. Clinicopathologic variables were compared; multivariable logistic regression estimated correlates of BC-pLC; survival was assessed with Kaplan-Meier estimates and Cox proportional-hazards models. Compared with BC, the BC-pLC cohort had higher frequencies of estrogen receptor-negative (ER-negative) and progesterone receptor-negative (PR-negative) tumors and more Ki-67-positive tumors; after multiplicity adjustment (Bonferroni), ER negativity and Ki-67 positivity remained significant, whereas human epidermal growth factor receptor 2 (HER2) did not. In multivariable analyses, larger tumor size (>2 cm) and Ki-67 positivity were independently associated with BC-pLC (odds ratio [OR] 3.02, 95% CI 1.29-7.38; OR 3.10, 95% CI 1.32-7.49). At the cohort level, differences in overall survival (OS) and invasive disease-free survival (iDFS) between BC-pLC and BC were not statistically significant after adjustment. Biomarker-stratified analyses showed worse OS for ER-negative tumors and nominally better iDFS for Ki-67-negative tumors. Tumor burden and proliferative activity can guide risk-stratified chest imaging surveillance and early pathology correlation to distinguish metastasis from new primaries during BC follow-up.

Introduction

Breast cancer (BC) is among the most common malignancies worldwide, and advances in screening and therapy have extended survival, increasing the likelihood of multiple primaries; notably, the risk of a second primary lung cancer (pLC) is higher in BC survivors than in the general population¹. Lung cancer (LC) remains a leading cause of cancer mortality, with persistently low 5-year survival despite therapeutic progress^2,3. Sex-specific patterns are increasingly recognized: the proportion of LC in women has risen, and tumors in women more often harbor actionable driver alterations such as EGFR mutations^4,5. The hormonal milieu (e.g., estrogen signaling) and age-both established carcinogenic factors-may contribute to the elevated LC risk observed among BC survivors, alongside shared exposures (tobacco, radiation, family history), treatment-related effects, heritable susceptibility, and overlapping oncogenic pathways (EGFR, KRAS, TP53, BRCA1)⁶.

BC with second primary LC (BC-pLC) may present synchronously (detected during the BC diagnostic work-up) or metachronously (arising months to years after BC)⁷. In practice, diagnosis and management are challenging. A substantial fraction of cases are first noted as pulmonary nodules on routine chest CT after BC treatment, yet radiologic appearances of pLC and pulmonary metastases from BC often overlap, leading to initial misclassification and delays in definitive tissue diagnosis^8,9. Approximately one-third of BC-pLC cases are synchronous (diagnostic interval <6 months)¹⁰. Treatment sequencing (breast-first vs lung-first) may influence perioperative risk and adjuvant options, but standardized guidance remains limited, and reported 5-year survival varies across series, partly reflecting differences in selection and management.

Multicenter registries offer breadth and statistical power, but single-center retrospective analyses provide unique depth: harmonized imaging pathways, uniform pathology workflows, and adjudication by a dedicated multidisciplinary team enable fine-grained clinicopathologic validation with internally consistent long-term follow-up¹¹. These features are particularly valuable when distinguishing intrathoracic metastasis from a second primary, where small classification errors can distort outcomes and treatment decisions. We acknowledge, however, that single-center design limits generalizability relative to registry-scale cohorts¹².

Practical applicability. By quantifying clinicopathologic correlates of BC-pLC (e.g., primary BC tumor size and Ki-67 proliferative index) and reporting survival with clear denominators and effect sizes, this study aims to support risk-stratified chest CT surveillance, earlier multidisciplinary review of indeterminate nodules, and tissue acquisition strategies tailored to distinguish metastasis from new primaries^13,14. In settings where imaging findings are equivocal, readily available clinicopathologic cues can prioritize patients for expedited pathology correlation.

Against this background, we sought to delineate the clinicopathologic profile of BC-pLC, identify factors associated with its occurrence, and assess prognostic implications using Kaplan-Meier and Cox models for overall survival (OS) and invasive disease-free survival (iDFS). To transparently position the contribution of a single-center design, we complement primary analyses with interval-stratified and treatment-adjusted models and clearly state methodological advantages and limitations.

Protocol

This retrospective study was approved by the institutional ethics committee 2024YX070 and conducted in accordance with the Declaration of Helsinki. Given the non-interventional, de-identified nature of the dataset and minimal risk, informed consent was waived per institutional policy. Data were stored on secure, access-controlled servers, and analyses used an anonymized, locked dataset with an auditable trail and a pre-specified data-freeze date.

Study design and setting
We performed a single-center retrospective cohort study including patients with breast cancer (BC) who developed a second primary lung cancer (pLC) (BC-pLC) and contemporaneous patients with BC only. All patients underwent curative-intent surgery between January 2012 and January 2017, with follow-up through June 30, 2023. The BC-pLC cohort required pathologic confirmation of both primary BC and primary lung cancer; the BC cohort included patients with primary BC only. Propensity score matching was not performed and is acknowledged as a limitation; to mitigate selection bias, covariates were pre-specified for multivariable adjustment and are reported transparently. Synchronous BC-pLC was defined as a diagnostic interval of ≤6 months between primaries and metachronous BC-pLC as >6 months. A flow diagram summarizing screening, exclusions, inclusion, and analyses is presented in Figure 1.

Eligibility criteria and case adjudication
Eligible participants were aged ≥18 years with pathologically confirmed early-stage primary BC, available core clinicopathologic variables, imaging for review, and blocks/slides for central pathology review when indicated. Patients were excluded if they had a concurrent malignancy at another site, radiologic or pathologic evidence of pulmonary metastasis from BC, metastatic or recurrent BC at the index diagnosis, or incomplete follow-up data. Classification of lung lesions as primary lung cancer rather than metastatic BC was based on histomorphology and, when available, an immunophenotypic profile consistent with lung origin (e.g., TTF-1/Napsin A for adenocarcinoma and p40 for squamous differentiation). Discordant or indeterminate cases were resolved by multidisciplinary consensus (breast surgery, thoracic surgery, radiology, pathology).

Imaging protocol and surveillance
During BC care, chest imaging followed institutional pathways that included baseline staging CT at diagnosis and surveillance CT typically every 6-12 months during the first 2-3 years, and annually thereafter, or earlier if symptoms or abnormal findings prompted imaging. Acquisition parameters were standardized per radiology protocols. Because surveillance intensity can affect detection and stage distribution, potential detection/lead-time bias was addressed in sensitivity analyses by considering imaging intensity and calendar period.

Pathology workflow and immunohistochemistry
Tumor specimens were fixed in neutral buffered formalin, 4 %, typically 6-24 min/mm of tissue thickness (conventionally totaling 6-24 h for routine specimens), dehydrated, and paraffin-embedded before sectioning. Sections of 3-4 µm were cut and mounted on charged slides. Heat-induced epitope retrieval was performed using either citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) at 95-100 °C for approximately 20 min, followed by cooling at room temperature for 10 min. Primary antibodies for ER, PR, HER2, and Ki-67 were applied according to validated laboratory protocols with typical incubation of 30-60 min at room temperature, polymer-based secondary detection, and DAB development for 5-10 min; slides were counterstained with hematoxylin for 30-60 s, dehydrated, cleared, and mounted. ER and PR positivity were defined as ≥1% tumor-cell nuclear staining. HER2 followed ASCO/CAP 2018 criteria (3+ by IHC or amplification by in situ hybridization when 2+ and equivocal). Ki-67 was dichotomized at a pre-specified cut-off of 14%. Two board-certified pathologists independently reviewed all slides, and discrepancies were resolved by consensus to standardize scoring.

Biospecimen storage conditions
Formalin-fixed, paraffin-embedded (FFPE) blocks were stored at controlled room temperature (18-25 °C), dry and dark, with ambient humidity maintained below 60%. Blocks were retained for ≥10 years per institutional policy, and re-cutting from the original block was performed whenever section age exceeded predefined stability windows. Unstained sections (3-4 µm) were stored in sealed slide boxes with desiccant, protected from light. For immunohistochemistry (ER, PR, Ki-67), unstained slides were refrigerated at 2-8 °C and stained within 8 weeks of sectioning; slides older than this threshold were discarded and re-cut. For in situ hybridization/FISH confirmation of equivocal HER2 results, unstained slides were used within 2-4 weeks of sectioning at 2-8 °C storage, or re-cut if older. Storage temperature and sectioning dates were logged in the laboratory information system, and any deviations triggered re-cutting prior to staining.

Data collection and management
Demographic and clinicopathologic variables were abstracted from the electronic medical record per a predefined data dictionary. Variables included age at diagnosis, menopausal status, hypertension, family history, primary tumor size, histologic subtype and grade, nodal status and number of examined nodes, lymphovascular invasion, and biomarker status for ER, PR, HER2, and Ki-67. Histologic subtypes and TNM staging were summarized descriptively in Table 1 and Table 2, with clarifying footnotes for abbreviations and subgroup definitions. Data quality procedures comprised range and logic checks and source verification by a second reviewer. Analyses were reproduced from a locked dataset with a documented codebook and manifest.

Outcomes and follow-up
Overall survival (OS) was measured from the date of cancer diagnosis to either death from any cause or the most recent follow-up. Invasive disease-free survival (iDFS) was calculated from the end of curative treatment to the occurrence of an invasive recurrence, the development of a new primary malignancy, or death from unrelated causes. Patients alive without events were censored at last contact. Follow-up was conducted via telephone contacts, clinic visits, and electronic medical record review through June 30, 2023. Cause-specific mortality was not adjudicated; competing-risks analyses were therefore not performed, and this limitation is noted in the Discussion.

Statistical analysis
Continuous variables were evaluated for normality using the Shapiro-Wilk test and summarized as median (IQR) or mean (SD) as appropriate, and categorical variables as n (%). Group comparisons used the t test or Wilcoxon rank-sum test and the chi-square test or Fisher's exact test where appropriate. For correlates of BC-pLC, univariable logistic regression was followed by multivariable logistic regression using a priori variables (tumor size category, ER, PR, Ki-67), with events-per-variable considerations, variance inflation factors for collinearity, and standard goodness-of-fit diagnostics. Survival was assessed with Kaplan-Meier estimates and log-rank tests and with Cox proportional-hazards models prespecifying age, tumor size, nodal status, ER, PR, HER2, Ki-67, and cohort (BC-pLC vs BC). Proportional-hazards assumptions were evaluated using Schoenfeld residuals. Missing data were handled with complete-case analysis, and the extent of missingness is reflected in denominators. Given multiple biomarker comparisons across ER, PR, HER2, and Ki-67, family-wise error control used Bonferroni adjustment with an adjusted ɑ of 0.0125, and nominal two-sided P values are also reported. All P values in the text and tables are paired with effect sizes and 95% confidence intervals.

Sensitivity and robustness analyses
Robustness was examined through interval-stratified survival analyses that contrasted synchronous cases with metachronous cases categorized as ≤12 months, 13-36 months, and >36 months, with adjusted hazard ratios reported alongside 95% confidence intervals and nominal P values. A 12 month landmark approach was used to mitigate guarantee-time bias where postoperative or adjuvant therapies might otherwise distort time-to-event estimates. Additional models adjusted for treatment classes where available, including lung surgery type, radiotherapy exposure, and systemic therapy categories (chemotherapy, targeted therapy, immunotherapy). Sensitivity analyses further considered imaging surveillance intensity and calendar period to address potential detection and secular-trend biases.

Reproducibility checkpoints
Key checkpoints influencing reproducibility included explicit adjudication criteria distinguishing primary lung cancer from metastatic BC, prespecified thresholds for ER, PR, HER2, and Ki-67, dual independent pathology reads with consensus resolution, transparent reporting of imaging surveillance intensity, and a priori covariate specification with multiplicity control. Any deviations from protocol were documented, including the rationale and an assessment of their potential impact on results.

Results

Baseline characteristics
The patient selection pathway is shown in Figure 1. Baseline clinicopathologic characteristics are summarized in Table 1. Median age was 51 years (IQR 45-59) in the BC cohort and 52 years (IQR 46-60) in the BC-pLC cohort. Compared with BC, the BC-pLC cohort had larger primary breast tumors and a higher proportion of T2-T4 disease; nodal metastasis was modestly more frequent in BC-pLC. Invasive ductal carcinoma pr...

Discussion

The rising recognition of multiple primary cancers reflects advances in imaging, therapeutics, and longitudinal surveillance, coupled with longer survivorship and the interplay of genetic and environmental factors^15,16. Using routinely collected clinicopathologic data, this study characterized breast cancer with a second primary lung cancer (BC-pLC) and examined correlates of occurrence and prognosis, with a focus on immunohistochemical markers-estrogen receptor ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Coverslips	Fisher Scientific	12?545?10	Used to cover stained tissue sections for microscopy
DAB Chromogen	Thermo Fisher Scientific	K346711	Used for the color development in immunohistochemistry
Estrogen Receptor (ER) Antibody	Thermo Fisher Scientific	MA1?10066	Monoclonal antibody for ER protein detection RRID: AB_2252608
Fluorescence In?Situ Hybridization (FISH) Kit	Abbott	32?10030	For confirmatory HER2 amplification studies
Formalin (4% Neutral Buffered)	Neomarkers, USA	–	For tissue fixation in histopathology studies
HER2 Antibody	Dako / Agilent	A0485	Monoclonal antibody for HER2 protein detection RRID: AB_2252608
Immunohistochemistry Staining Kit	Neomarkers, USA	–	For performing IHC assays on tumor samples
Ki67 Antibody	Dako / Agilent	M7240	Monoclonal antibody for Ki67 protein detection RRID: AB_2341197
Microtome	Leica	RM2255	For sectioning paraffin?embedded tissue samples
Mounting Media	Sigma?Aldrich	M2681	Used for mounting tissue sections onto slides
Paraffin	Sigma-Aldrich	P?6001	Used for tissue embedding in histology
Progesterone Receptor (PR) Antibody	Thermo Fisher Scientific	MA1?10176	Monoclonal antibody for PR protein detection
SPSS v23.0