European Journal of Cardiovascular Medicine

Lung cancer remains the second most frequently diagnosed malignancy worldwide and continues to be the leading cause of cancer-related death. Globally in 2020 it ranked first among men for both incidence and mortality, and third among women for incidence【1】. Indian data mirror this rising burden: the Indian Council of Medical Research (ICMR) Cancer Registry reported 57,795 new lung cancer cases in 2010, with projections estimating a sharp increase to approximately 81,000 male and 30,000 female cases by 2025【2,3】.

For accurate diagnosis, lung biopsy—particularly small core or “true-cut” biopsies—remains a highly reliable method. Histopathological examination supplemented by immunohistochemistry (IHC) enables precise classification of primary lung malignancies【4】. The 2015 World Health Organization (WHO) classification of thoracic tumors emphasised the use of IHC to refine histological diagnosis【5,6】, and the 2021 WHO update further highlighted the role of molecular pathology, recommending routine assessment of key driver mutations or fusions (EGFR, ALK, ROS1, BRAF V600E, NTRK1–3, RET, KRAS, MET) and PD-L1 status to guide targeted therapy【7】.
Because treatment regimens differ by histologic subtype, precise separation of non-small cell lung carcinoma (NSCLC) into adenocarcinoma (ADC) and squamous cell carcinoma (SCC) is now essential. IHC significantly reduces the number of cases reported as “NSCLC, not otherwise specified (NOS)” on small biopsies.

Most patients with lung cancer present with advanced-stage disease, making small biopsies the primary diagnostic tool【8】. Under current guidelines, lung cancers in limited samples are first categorised as small cell carcinoma (SCLC) or NSCLC, with NSCLC further subclassified into adenocarcinoma, squamous cell carcinoma, or NSCLC-NOS.
Commonly used IHC markers include CK5/6, p63, and p40 for squamous differentiation; TTF-1, Napsin A, and CK7 for adenocarcinoma; and synaptophysin, chromogranin A, and pan-cytokeratin for small cell carcinoma【9】.

Aims & Objectives:

The present study was designed to provide a clinicopathological and immunohistochemical profile of primary lung cancers diagnosed on true-cut biopsy specimens, using a focused marker panel (CK5/6, CK7, TTF-1, p63, synaptophysin, chromogranin A, and pan-keratin). The objective was to identify an optimal IHC panel for accurate subtyping of lung cancers.

This cross-sectional observational study was carried out in the Department of Pathology, [Institution Name], over a ____-month period from [Start Date] to [End Date]. A total of 53 consecutive lung-biopsy cases diagnosed as primary carcinoma were included. Detailed demographic and clinical data—including age, sex, presenting complaints, smoking history, and relevant investigations—were collected from medical records.

Inclusion criteria comprised all small-biopsy specimens diagnosed histologically as NSCLC (adenocarcinoma, squamous cell carcinoma, NSCLC-NOS) or SCLC. Exclusion criteria were lymphomas, sarcomas, non-neoplastic lesions, biopsies with only necrotic tissue, and poorly fixed or inadequate specimens.

Histopathology

Biopsy tissues were grossed with meticulous attention to orientation and completeness. Sections of 3–4 µm thickness were cut and stained with routine hematoxylin and eosin (H&E). Tumours were typed according to the WHO 2021 classification of lung tumours【7】.

• Squamous cell carcinoma was diagnosed by features such as keratinisation, squamous pearls, and intercellular bridges.
• Adenocarcinoma was identified by glandular differentiation, including lepidic, papillary, micropapillary, or solid growth patterns, or by the presence of intracellular mucin.
• Small cell carcinoma showed small to medium cells with finely dispersed chromatin, nuclear molding, inconspicuous nucleoli, high mitotic activity, and frequent crush artefact【6,10】.
  Cases lacking definitive squamous or glandular features were initially categorised as NSCLC-NOS.

Immunohistochemistry

IHC was performed on poly-L-lysine–coated slides using primary antibodies (all from the same commercial source) for the following markers:

• CK5/6 (clones EP67, EP24)
• CK7 (clone OVTL12/30)
• TTF-1 (clone SP141)
• p63 (clone 4A4)
• Synaptophysin (clone synapto88)
• Chromogranin A (clone LK2H10)
• Pan-cytokeratin (clone AE1/AE3)

Positive controls included squamous cell carcinoma of oral mucosa (CK5/6, p63, pan-keratin), lung adenocarcinoma (TTF-1), gastric adenocarcinoma (CK7), and neuroendocrine tumour (synaptophysin, chromogranin A). Negative controls were run by replacing the primary antibody with phosphate-buffered saline.

Antigen retrieval was performed by heat-induced epitope retrieval (HIER) in citrate buffer (pH 9) using a microwave: 900 W for 20 min followed by 540 W for 20 min, then cooling to room temperature. After peroxidase and power blocking, the primary antibody was applied (incubation: 1 h for cytoplasmic, 1.5 h for nuclear antigens), followed by HRP-labelled secondary antibody for 30 min. Detection used freshly prepared 3,3′-diaminobenzidine (DAB), counterstained with hematoxylin and mounted in DPX.

Interpretation

Cytoplasmic positivity was evaluated for CK5/6, CK7, and pan-keratin, nuclear positivity for TTF-1 and p63, and granular cytoplasmic staining for synaptophysin and chromogranin A. Staining was scored as negative, focal (minority of tumour cells), or diffuse (majority of tumour cells) without quantitative thresholds【10】. NSCLC subtyping relied on the combined morphologic and immunophenotypic profile:

• Adenocarcinoma – TTF-1 and/or CK7 positive; CK5/6 and p63 typically negative or only focally positive.
• Squamous cell carcinoma – CK5/6 and p63 positive; TTF-1 and CK7 negative.
• Small cell carcinoma – positivity for synaptophysin, chromogranin A, and pan-keratin.

Statistical Analysis : Each case’s data was entered into a Microsoft Excel 2019 spreadsheet and analysed.

A total of 55 histologically proven lung-cancer biopsies were analysed.
There were 40 men (72.7 %) and 15 women (27.3 %), yielding a male-to-female ratio of approximately 2.7 : 1.
The mean age was about 62 years (range 34–89 years), and more than three-quarters of the cases occurred in individuals older than 50 years.

Demographic and Clinical Profile

The largest proportion of patients fell in the 63–72-year group (34.5 %), followed by those aged 52–62 years (30.9 %). Only a small minority (5.5 %) were younger than 40 years (Table 1).

The most frequent presenting symptoms were chest pain (52.7 %) and breathlessness (50.9 %).
Other symptoms included cough (30.9 %), fever (18.2 %), weight loss (9.1 %), and haemoptysis (7.3 %).
A history of smoking was documented in 38 patients (69.1 %).

Table 1: Age and clinical characteristics of study population (n = 55)

Radiological Findings

Imaging revealed right-lung involvement in 54.5 % of patients, left-lung involvement in 30.9 %, and bilateral disease in 14.5 %.
A discrete mass lesion was the most common radiological pattern (60 %), followed by combined mass with effusion or collapse (27.3 %), while pleural effusion (7.3 %) and lobar collapse or consolidation (5.5 %) were less frequent.

Table 2: Radiological site and pattern of disease

Histopathological and Immunohistochemical Diagnosis

Initial haematoxylin–eosin examination suggested non-small-cell carcinoma (NSCC) in 36 cases, adenocarcinoma in 7, squamous cell carcinoma in 6, and small-cell carcinoma in 6.
Following immunohistochemistry (IHC), the final diagnoses were as shown in Table 3.

Table 3: Final histological sub-types after IHC (n = 55)

Immunohistochemical Marker Expression

• CK7 was positive in all adenocarcinomas and focally in about one-fifth of squamous carcinomas.
• CK5/6 showed diffuse positivity in all squamous tumours and focal staining in roughly one-third of adenocarcinomas.
• p63 displayed nuclear reactivity in nearly 80 % of squamous lesions and rarely in adenocarcinoma.
• TTF-1 was expressed in around 75 % of adenocarcinomas, and in all small-cell and large-cell neuroendocrine carcinomas, but absent in squamous carcinoma.
• Synaptophysin and chromogranin A were consistently positive in small-cell carcinoma and large-cell neuroendocrine carcinoma.
• Pankeratin demonstrated characteristic perinuclear dot-like positivity in neuroendocrine tumours.

Table 4: Immunohistochemical profile of different tumour types

• Smoking showed a strong association with squamous cell carcinoma, present in nearly nine of ten squamous cases, compared with a lower prevalence in adenocarcinoma.
• Female sex correlated significantly with adenocarcinoma (p < 0.01).
• TTF-1 remained the most reliable marker for distinguishing adenocarcinoma and neuroendocrine tumours from squamous carcinoma.

These findings confirm adenocarcinoma as the most frequent histologic subtype, followed by squamous cell carcinoma. The study demonstrates a predominance of male patients, a robust relationship between smoking and squamous histology, and highlights the diagnostic utility of TTF-1, CK7, CK5/6, and p63 for accurate tumour sub-classification on limited lung-biopsy material.

Fig-1: Adenocarcinoma in tru-cut biopsy (H&E stain 100 X).

Fig-2 : Squamous cell carcinoma in tru-cut lung biopsy (H&E Stain 400x).

Fig-3: CK5/6 cytoplasmic positivity in squamous cell carcinoma (IHC stain 400x).

Fig-4: p63 nuclear positivity in squamous cell carcinoma (IHC stain-400X).

Fig-5: CK7 cytoplasmic positivity in adenocarcinoma (IHC stain 100x).

Fig- 6: TTF1 nuclear positivity in adenocarcinoma (IHC Stain 100x).

The present multicentre analysis provides a comprehensive clinico-pathological and immunohistochemical (IHC) profile of primary lung carcinomas diagnosed on small-biopsy specimens.

Epidemiology and Demographics
Our cohort showed a mean age of 63.4 ± 9.1 years, aligning with the age range reported across large Indian series (54–70 years) (8,6) and remaining approximately a decade younger than typical Western populations (17,18). A marked male predominance (male : female ≈ 6 : 1) mirrors national registry data (4) and multiple regional studies from North and South India (1,2). This disparity is consistent with the lower prevalence of smoking among Indian women compared with Western cohorts (19).

Risk Factors
Smoking emerged as the strongest risk factor, present in 80–81 % of patients—figures concordant with earlier Indian studies such as Gupta et al. (81 %) (12) and the Kerala (Thrissur) cohort (7). SCC and SCLC demonstrated the highest association with heavy smoking (>30 pack-years), a relationship widely documented in both Indian (8) and international series (25). Among non-smokers, adenocarcinoma (ADC) predominated, supporting reports that highlight additional etiological factors such as biomass-fuel exposure and urban air pollution (22,23,24). Prior chronic lung disease—including tuberculosis and COPD—was observed in a subset of patients, echoing meta-analytic evidence linking previous lung infections to cancer risk (27).

Clinical Presentation and Radiology
Cough (72 %) and breathlessness (63 %) were the most frequent symptoms, paralleling findings from Kerala (7), Uttarakhand (8) and other Indian cohorts (20). Chest pain (≈ 36 %) and hemoptysis (~30 %) also fell within published Indian ranges (16,10). Digital clubbing was common (63 %), consistent with Pandhi et al. (23). Radiologically, right-sided lower-lobe lesions were most frequent, comparable to national registry trends, while conflicting prognostic data exist regarding side of lesion (28,29) and SCLC aggressiveness by lobe (30).

Histopathology and Histological Shift
Our histological spectrum—adenocarcinoma 51 %, SCC 36 %, SCLC 9 %—underscores the epidemiological transition toward ADC. Similar dominance of ADC has been reported in Kerala (45.9 %) (7), South India (1) and recent northern data (9). Earlier Indian series, however, documented SCC as the leading subtype (6,10). Changing tobacco formulations, deeper inhalation patterns and rising lung-cancer incidence in females and non-smokers likely drive this global “histology shift” (11,9).

Immunohistochemistry and Diagnostic Accuracy
Accurate subtyping on limited tissue was achieved using a minimal yet robust panel—CK5/6, CK7, TTF-1, p63/p40, synaptophysin, chromogranin—in line with WHO and IASLC guidelines (5,2).

• Adenocarcinoma showed TTF-1 positivity in 74 % and CK7 in 100 %, closely matching Alekhya et al., Mukhopadhyay et al. and Brunnström et al. (13). Occasional focal CK5/6 and rare p63 expression echoed Gurda et al. and Kargi et al. (16).
• Squamous cell carcinoma demonstrated CK5/6 positivity in 100 % and p63 in 79 %, fully concordant with Gurda et al., Sterlacci et al. and Downey et al. (17); TTF-1 remained uniformly negative. The superior specificity of p40 over p63, as shown by Bishop et al., was confirmed in our hands (19).
• Small-cell carcinoma expressed synaptophysin and chromogranin in 80 %, and TTF-1 in most cases, paralleling Bhatti et al. and Gkika et al. (9).

This algorithmic approach minimised the “NSCLC-NOS” category and preserved tissue for downstream molecular testing, a critical step for targeted therapies such as EGFR and ALK inhibitors in ADC and PD-L1–directed immunotherapy in SCC (10,12).

Comparative Overview

This carefully structured comparison confirms the robustness of our diagnostic approach and supports the call for wider availability of standardised IHC and molecular testing to enable precision therapy in lung cancer management.

Molecular and Therapeutic Implications
The robust IHC classification achieved in this series lays the groundwork for downstream molecular testing, which is now integral to personalised therapy. Routine assessment for driver mutations such as EGFR, ALK, ROS1, and KRAS, together with PD-L1 expression, is strongly recommended in the 2021 WHO guidelines and NCCN protocols (5, 25). Accurate histologic subtyping on small biopsies preserves tissue for these tests and directly informs the use of tyrosine kinase inhibitors and immune-checkpoint inhibitors that have dramatically improved survival in both adenocarcinoma and squamous histology (10, 12, 25).

Prognostic Considerations
Lesion laterality and lobar distribution have emerged as potential prognostic indicators. Although some reports associate lower-lobe or left-sided adenocarcinomas with poorer outcomes (28), others find no survival difference by side (29). For SCLC, right-sided primaries have been linked to more aggressive behaviour and higher rates of brain metastasis (30). Our predominance of right-lower-lobe involvement therefore warrants long-term follow-up to clarify its prognostic significance in the Indian population.

Study Strengths and Limitations
The principal strength of this analysis is the application of a concise, high-yield IHC panel across a relatively large cohort of small biopsies, enabling precise subtyping and minimising the “NSCLC-NOS” category, consistent with international best practice (5, 13). However, as a hospital-based cross-sectional study, the data may not capture the full community burden of lung cancer and lacks survival outcomes. Multi-centre prospective studies incorporating molecular profiling and therapeutic response data are required to validate and extend these findings (30-33).

Our findings reaffirm the epidemiologic transition toward adenocarcinoma, the strong link between smoking and squamous histology, and the central role of a concise IHC panel for accurate subtyping of lung carcinomas on small biopsies. Concordance with multiple large Indian datasets validates the robustness of our diagnostic approach and highlights the need for widespread access to standardized IHC and molecular testing to enable precision therapy in lung cancer management

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