Accurate differentiation between primary tumors and metastatic lesions is a cornerstone in the clinical management of malignancies. Establishing the tissue of origin is essential for guiding treatment strategies, determining prognosis, and assessing eligibility for targeted therapies or clinical trials. In the era of personalized oncology, distinguishing whether a tumor represents a primary neoplasm or a secondary metastasis is more critical than ever [1–3].

Traditional diagnostic modalities such as computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and histopathological examination provide foundational insights into tumor morphology and anatomical localization. However, significant histological overlap can occur among tumors originating from different organs, particularly in poorly differentiated or undifferentiated cancers. This challenge is especially pronounced in cases of cancers of unknown primary (CUP), where despite extensive diagnostic workup, the origin remains elusive. In such scenarios, immunohistochemistry (IHC) serves as a crucial ancillary technique by identifying lineage-specific antigen expression, thus aiding in the determination of tumor origin [1,2,4].

IHC utilizes monoclonal or polyclonal antibodies to detect antigens associated with specific tissue lineages. Commonly used cytokeratin markers such as CK7 and CK20 help characterize adenocarcinomas of the gastrointestinal tract, breast, lung, and urothelial system. Thyroid transcription factor-1 (TTF-1) is one of the most reliable markers for pulmonary adenocarcinoma and thyroid neoplasms [2,3]. However, studies have reported occasional TTF-1 expression in colorectal adenocarcinomas and other non-pulmonary tumors, suggesting potential diagnostic pitfalls if interpreted in isolation [4,5].

Similarly, CDX2, a nuclear transcription factor, is widely recognized as a highly sensitive and specific marker for colorectal origin [1]. When used alongside CK7/CK20, CDX2 enhances diagnostic accuracy in both primary and metastatic intestinal adenocarcinomas. The combined use of TTF-1 and Napsin A has been shown to improve diagnostic yield in lung adenocarcinomas, particularly in fine-needle aspiration and small biopsy samples [6].

Despite their widespread application, IHC markers can vary in their expression patterns depending on tumor differentiation, antibody clone specificity, and laboratory protocols. Therefore, interpretation often relies on comprehensive marker panels tailored to the clinical and radiological context, rather than on single-marker analysis. Continued validation of emerging markets and integration with molecular diagnostics will be essential to improve the accuracy and reliability of tumor origin determination in complex metastatic cases.

Given this context, a systematic review of the literature is essential to consolidate current evidence regarding the diagnostic performance of immunohistochemical markers in differentiating primary from metastatic tumors. This review aims to provide a comprehensive synthesis of data across various tumor types and organ systems to guide accurate pathological diagnosis and clinical decision-making.

Search Strategy

A focused and systematic literature search was conducted to identify relevant studies evaluating the diagnostic utility of immunohistochemical (IHC) markers in differentiating primary from metastatic tumors. Four major electronic databases—PubMed, Embase, Scopus, and Web of Science—were searched for articles published between January 2000 and April 2024. The search strategy utilized a combination of Medical Subject Headings (MeSH) and free-text keywords including “immunohistochemistry,” “TTF-1,” “CDX2,” “CK7,” “CK20,” “SATB2,” “Napsin A,” “p40,” “lung adenocarcinoma,” and “colorectal carcinoma.” Boolean operators such as AND/OR were applied to maximize the retrieval of relevant studies. The review process adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.

Eligibility Criteria

Studies were considered eligible for inclusion in this review if they met the following criteria:

Original human research articles evaluating the diagnostic accuracy of immunohistochemical markers in distinguishing primary tumors from metastases;

Reported diagnostic metrics such as sensitivity, specificity, or overall accuracy;

Utilized validated immunohistochemical markers, including TTF-1, CDX2, SATB2, Napsin A, CK7, CK20, or p40;

Published in English with full-text availability.

Exclusion criteria included:

Case reports, reviews, editorials, or conference abstracts;

Studies that did not report quantifiable diagnostic performance measures;

Experimental studies conducted on animals or cell lines.

Twelve eligible articles were ultimately included in the qualitative synthesis, all of which involved immunohistochemical analysis of human tumor samples relevant to lung and colorectal carcinomas.

Data Extraction

Two reviewers independently screened all retrieved articles by title and abstract. Full-text review was conducted for studies that met the preliminary inclusion criteria. Any disagreements during selection were resolved by consensus. A standardized data extraction form was used to gather information on the year of publication, study design, number of cases, tumor type (primary or metastatic), IHC markers assessed, and key diagnostic outcomes such as sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy.

Quality Assessment

The methodological quality and risk of bias of the included studies were assessed using the QUADAS-2 (Quality Assessment of Diagnostic Accuracy Studies-2) tool. This validated instrument evaluates four domains: patient selection, index test execution and interpretation, reference standard, and the flow and timing of the study. Two reviewers conducted the assessments independently. Inconsistencies were resolved through discussion and mutual agreement. Most studies demonstrated moderate to high methodological quality, with low risk of bias in the majority of domains.

Study Selection

The initial literature search and screening process resulted in the identification of 12 studies that met the inclusion criteria. These studies were published between 2003 and April 2024 and collectively involved over 3,800 tumor samples. All included studies were original human research evaluating the diagnostic accuracy of immunohistochemical (IHC) markers in distinguishing primary tumors from metastatic lesions, particularly in the context of lung and colorectal cancers. The study selection process is depicted in the PRISMA flow diagram.

Figure 1. PRISMA Flow Diagram of Study Selection Process

Overview of Included Studies

The included studies were conducted across multiple academic institutions and pathology laboratories. They primarily focused on immunohistochemical analysis of tumor tissues using markers such as thyroid transcription factor-1 (TTF-1), caudal-type homeobox 2 (CDX2), cytokeratins (CK7, CK20), SATB2, Napsin A, and p40. The tumor types examined included primary and metastatic lung adenocarcinomas, squamous cell carcinomas, colorectal carcinomas, and metastatic gastrointestinal adenocarcinomas. Sample sizes ranged from 60 to 600 specimens per study, with some articles focusing on specific diagnostic panels in cytology specimens, pleural metastases, or resected tissues.

Diagnostic Performance of Key Immunohistochemical Markers

The diagnostic performance metrics reported in the included studies are summarized below:

These values are consistent across multiple studies, although minor variations were noted due to differences in antibody clones, tissue preparation methods, and interpretation thresholds.

Utility of Marker Combinations and Panels

The included studies emphasized that marker panels provide higher diagnostic accuracy than individual markers. Key findings include:

TTF-1 and Napsin A: When used together, this combination showed excellent performance in diagnosing lung adenocarcinomas, particularly on fine-needle aspiration (FNA) specimens. Fatima et al. (2011) reported sensitivities above 85% and specificity greater than 95%.

CDX2 and SATB2: Strongly supported the diagnosis of colorectal carcinoma. Elnady et al. (2022) highlighted that SATB2, in conjunction with CDX2, provides complementary nuclear staining with high diagnostic yield, particularly in metastatic settings.

CK7/CK20 Expression Profile: Bayrak et al. (2012) reported that a CK7−/CK20+ phenotype was highly specific for colorectal origin, whereas CK7+/CK20− patterns were associated with upper GI, breast, or ovarian sources.

p40 and TTF-1 Co-expression: Walia et al. (2017) and Savari et al. (2023) demonstrated that co-expression of these markers in rare biphenotypic non-small cell lung carcinomas (NSCLCs) may assist in subclassification and guide therapy.

Overall Observations

Collectively, the studies confirmed that:

Organ-specific IHC panels provide superior diagnostic precision over single-marker strategies.

Nuclear transcription factors (e.g., TTF-1, CDX2, SATB2) offer greater specificity compared to cytoplasmic markers (e.g., CK7/CK20).

Combined use of markers minimizes diagnostic ambiguity, especially in cases of poorly differentiated tumors or metastases with unknown primary origin.

The consistent performance across multiple studies reinforces the clinical value of these markets and supports their continued use in routine pathology practice for tumor origin determination.

This systematic review consolidates evidence from 12 high-quality studies encompassing over 3,800 tumor samples, underscoring the vital role of immunohistochemical (IHC) markers in differentiating primary from metastatic tumors. Accurate identification of the tumor’s tissue of origin is central to effective cancer management, influencing therapeutic decisions, prognostication, and eligibility for targeted treatment. In diagnostically challenging cases such as poorly differentiated carcinomas or cancers of unknown primary (CUP), IHC provides essential diagnostic clarity by detecting tissue-specific antigen expression patterns.

Among the most validated markers, thyroid transcription factor-1 (TTF-1) and caudal-type homeobox 2 (CDX2) have shown consistently high specificity for pulmonary and colorectal adenocarcinomas, respectively. A meta-analysis by Shen et al. demonstrated the robust diagnostic utility of TTF-1 for identifying pulmonary adenocarcinoma in pleural and other serous metastases, highlighting its value in fluid-based cytological evaluations [7]. However, its occasional expression in thyroid and gynecologic malignancies necessitates careful interpretation. Similarly, CDX2 has been well validated in colorectal adenocarcinomas; studies by Werling et al. and Hansen et al. confirm its high diagnostic reliability, with Hansen et al. also emphasizing its prognostic value in stage II colon cancer [8].

The CK7/CK20 immunophenotypic profile remains a foundational tool for IHC-based screening. A CK7−/CK20+ pattern is highly suggestive of colorectal origin, while CK7+/CK20− is more consistent with breast, pulmonary, or gynecologic tumors. Nonetheless, Bayrak et al. reported that overlapping expression in upper GI and pancreatic tumors can reduce the specificity of this pattern [9]. As such, combining CK7/CK20 with other organ-specific markers such as CDX2, SATB2, PAX8, and GATA3 enhances diagnostic confidence and accuracy.

Multimarker panels have emerged as the standard of care in complex cases. For lung adenocarcinomas, the combination of TTF-1 and Napsin A significantly improves diagnostic accuracy, especially in cytology samples [6]. In the context of colorectal tumors, SATB2—a nuclear transcription factor—has demonstrated high specificity and sensitivity. Elnady et al. showed that SATB2 complements CDX2 in distinguishing both primary and metastatic colorectal carcinoma with excellent accuracy [10]. Additional support comes from Asgari-Karchekani et al., who noted CDX2’s correlation with survival and clinicopathologic features in colorectal cancer patients [11].

In rare biphenotypic non-small cell lung carcinomas, co-expression of TTF-1 and p40 has diagnostic implications. Savari et al. identified distinct clinicopathological and genomic features in such tumors, which require nuanced interpretation to avoid misclassification [12]. These findings highlight the need for a tailored, context-aware approach when interpreting co-expressed markers.

Despite the broad utility of IHC, limitations persist. Technical inconsistencies in antibody clones, staining protocols, and interobserver variability can influence diagnostic interpretation. Additionally, antigenic loss in heavily pretreated or poorly differentiated tumors may result in false negatives. Aberrant marker expression in tumors outside their canonical profiles further emphasizes the importance of comprehensive clinical and pathological correlation.

To mitigate these challenges, integrating molecular diagnostics such as next-generation sequencing (NGS), RNA expression profiling, and DNA methylation analysis with IHC is becoming increasingly important. These tools offer complementary genomic insights that enhance tumor classification and may identify actionable mutations, especially in CUP or diagnostically ambiguous cases.

In summary, this review affirms that organ-specific IHC panels, when applied in combination and interpreted within the clinical context, remain indispensable for distinguishing primary from metastatic tumors. The future of diagnostic oncology lies in a multimodal strategy that integrates traditional IHC with molecular technologies to improve diagnostic accuracy, minimize uncertainty, and facilitate personalized cancer therapy.

Immunohistochemical (IHC) markers remain a cornerstone in differentiating primary from metastatic tumors, particularly when conventional histopathology is limited by poor differentiation or ambiguous morphology. The application of organ-specific IHC panels such as TTF-1 for pulmonary tumors, CDX2 for colorectal origin, PAX8 for renal and Müllerian primaries, and GATA3 for breast and urothelial carcinomas significantly enhances diagnostic accuracy and facilitates informed clinical decision-making. Nevertheless, the reliability of IHC can be affected by technical variability, antigen preservation, and interpretive discrepancies, highlighting the need for standardized staining protocols and robust diagnostic algorithms. Future progress in this field will depend on the continued validation of emerging markers and the integration of IHC with advanced molecular technologies, including next-generation sequencing (NGS), transcriptomic profiling, and epigenetic classifiers. These multimodal strategies hold promise for improving diagnostic precision, particularly in cancers of unknown primary and poorly differentiated malignancies, ultimately enabling more targeted and personalized therapeutic interventions.

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