EXPERIENCE OF USING IMMUNOCYTOCHEMISTRY FOR IDENTIFYING AN UNKNOWN PRIMARY TUMOR

ABSTRACT

The article presents the experience of applying immunocytochemistry (ICC) in the diagnostic evaluation of cancer of unknown primary (CUP) within routine cytological practice. The diagnostic significance of ICC is substantiated as a method for refining the phenotypic characterization of tumor cells using minimally invasive material, including serous effusions. The principles of constructing diagnostic panels of immunomarkers are analyzed, emphasizing a sequential transition from broad lineage markers to organ-specific markers. Algorithmic approaches to ICC application (SCIP model) are described, along with issues related to standardization and quality control. It is demonstrated that systematic implementation of ICC improves diagnostic accuracy in CUP cases, shortens the time required to establish the probable primary site, and contributes to the optimization of therapeutic strategy.

АННОТАЦИЯ

В статье рассматривается опыт применения иммуноцитохимического исследования (ИЦХ) при синдроме невыявленного первичного очага (CUP) в условиях рутинной цитологической практики. Обоснована диагностическая значимость ИЦХ как метода уточняющей фенотипической характеристики опухолевых клеток на минимально инвазивном материале, в том числе при исследовании серозных выпотов. Проанализированы принципы построения диагностических панелей иммуномаркеров с последовательным переходом от маркеров широкой линии дифференцировки к органоспецифическим. Представлены алгоритмические подходы к применению ИЦХ (SCIP-модель), а также рассмотрены вопросы стандартизации и контроля качества исследования. Показано, что системное использование ИЦХ позволяет повысить диагностическую определенность при CUP, сократить сроки установления вероятного первичного очага и оптимизировать лечебную тактику.

Keywords: immunocytochemistry, cancer of unknown primary, CUP syndrome, cytological diagnosis, serous effusions, immunophenotype, diagnostic panels.

Ключевые слова: иммуноцитохимия, синдром невыявленного первичного очага, CUP, цитологическая диагностика, серозные выпоты, иммунный фенотип, диагностические панели.

Introduction. Cancer of unknown primary (CUP) remains one of the most challenging clinical situations in oncology, as metastatic involvement is detected before the primary source can be visualized and morphologically confirmed. In clinical practice, this scenario leads to a range of consequences, including prolongation of the diagnostic process (in which time is a critical component of treatment), expansion of instrumental examinations, and the risk of initiating therapy without sufficient confirmation of the tumor’s nature and without consideration of organ specificity.

In this context, a special place within the structure of CUP is occupied by metastatic involvement of serous cavities (pleural, peritoneal, and pericardial) and lymph nodes, in which cytological material is often the first and sometimes the only available substrate used for further diagnostic clarification.

The limitations of clinical and instrumental methods (including, for example, CT and/or MRI, endoscopy, PET and/or CT) are associated with the possible microscopic size of the primary lesion, its regression, or localization patterns that significantly hinder visualization. Morphological limitations of these approaches are also reflected in the fact that cytomorphology and even histology of a metastatic lesion do not always allow reliable assumption of the organ of origin, particularly in cases of poor differentiation, phenotypic atypia, and altered marker expression in metastases. In this context, cytological examination as a method of early and minimally invasive diagnostics requires reinforcement through the use of immunomarker panels specifically adapted to cytological specimens.

It should be separately noted that existing approaches to the diagnosis of unknown primary tumors in malignant effusions demonstrate the clinical effectiveness of identifying the organ of origin. The application of these approaches makes it possible to determine the subsequent treatment strategy for the patient (ranging from organ-specific systemic therapy to predominantly palliative interventions) [3].

The aim of the study is to describe the experience and prospects of using immunocytochemistry (ICC) for identifying an unknown primary tumor.

Research methodology. The methodological framework of the study is based on an analytical review and critical synthesis of contemporary publications devoted to immunocytochemistry (hereinafter referred to as ICC) in diagnostic cytology and its application in the search for the primary site in patients with CUP.

The literature selection was focused on studies and reviews describing:

1) the diagnostic effectiveness of combined cytomorphological and ICC approaches;

2) algorithmic structuring of marker panels for serous effusions and metastases of unknown origin;

3) comparative aspects of ICC and immunohistochemistry (hereinafter referred to as IHC);

4) requirements for validation, standardization, and quality control of ICC in laboratory practice.

An important methodological principle is the consideration of ICC as a test sensitive to pre-analytical and analytical variations, including the type of specimen carrier (smear, cytospin, liquid-based cytology, cell block), the method of fixation and/or preservation, storage conditions, specific features of antigen detection, and criteria for interpretation.

The contemporary context of quality management is considered separately; it is noted that ICC is widely applied in practice, yet it is characterized by high variability at all stages and insufficient standardization, and it imposes increased requirements for validation and control, including the use of control slides and participation in external quality assessment programs [10]. The practice-oriented component of the methodology is supplemented by an analysis of measures aimed at improving ICC reproducibility, which may enhance the reliability of interpretation when working with limited material and prove promising in specialist practice [6].

Results and discussion. Immunocytochemical examination is a laboratory method for detecting intracellular and membrane antigens in tumor and non-tumor cells of cytological specimens using specific monoclonal or polyclonal antibodies and visualization systems for the antigen–antibody complex.

The method is applied to various types of cytological material (smears, cytospins, liquid-based cytology preparations, and cell blocks) and is based on the specific binding of an antibody to a target antigen expressed by the cell.

Visualization of the reaction is performed using enzymatic (peroxidase, alkaline phosphatase) or fluorescent detection systems, which makes it possible to determine the localization of expression (cytoplasmic, membranous, nuclear) and to assess staining intensity. The diagnostic value of ICC lies in confirming the neoplastic nature of the cells, determining their line of differentiation, and establishing an immunoprofile that allows narrowing the range of possible primary sites in metastatic disease (Fig. 1).

Figure 1. Features of ICC, compiled by the author

Based on Fig. 1, it should be noted that conceptually ICC addresses two interrelated diagnostic tasks.

The first task consists in determining the line of tumor differentiation, that is, establishing its tissue origin based on the immunophenotype. This involves distinguishing epithelial, mesenchymal, lymphoid, melanocytic, neuroendocrine, and other variants of tumor differentiation through the use of broad tissue-specific markers. This stage makes it possible to confirm the malignant nature of the cells and assign the tumor to a specific morphogenetic class, thereby significantly narrowing the diagnostic search.

The second task of ICC is aimed at clarifying the organ specificity of the tumor, that is, at forming a probabilistic model of the primary site. At this stage, the expression profile of narrower, organ- or tissue-specific markers is analyzed, allowing the most probable anatomical localization of the metastatic source to be suggested. In the context of cancer of unknown primary (CUP), this stage is fundamentally important, as it determines the subsequent strategy of instrumental diagnostic work-up and the formation of the treatment plan.

A fundamentally important aspect of ICC application is the approach based on constructing diagnostic panels of immunomarkers (antibodies), with a sequential transition from broad, so-called lineage markers that determine the direction of tumor differentiation to narrower organ-specific markers, taking into account the clinical context, morphological features, and the quality of the cytological material.

For example, in the case of serous effusions and other fluid specimens, issues related to sample preparation are critical, since proper fixation and the preferred use of formalin-fixed cell blocks increase comparability with results obtained from FFPE tissues and reduce the risk of false-negative reactions due to altered antigenicity. Technical aspects of effusion preparation for ICC and the set of commonly used markers are considered from the standpoint of reproducibility as a key factor, as work with smear preparations increases the impact of background staining, cell loss, and variability in immunoreactivity [9].

Immunohistochemistry (IHC) is based on a tissue substrate with preserved architecture (FFPE block), which enables contextual evaluation of expression and allows for more standardized pre-analytical processing.

In contrast, ICC operates with cellular material (smears, cytospins, liquid-based cytology, cell blocks), in which architecture is limited or absent; however, the accessibility of the method increases in the setting of minimally invasive sampling and in situations where biopsy is difficult or contraindicated (Fig. 2).

Figure 2. Comparative analysis of ICC and IHC, compiled by the author

The pragmatic advantage of ICC in the clinical setting of CUP lies in the fact that cytological material is often the first confirmation of a tumor process; therefore, ICC makes it possible to avoid loss of time and proceed to further diagnostic clarification without waiting for a repeated invasive procedure.

Comparative reviews of applied ICC approaches in cytology indicate that the reliability of results increases significantly when cell blocks are used, since this technological approach brings ICC closer to IHC, thereby facilitating the transfer of protocols and interpretation criteria. In contrast, the application of markers on conventional smears requires more rigorous local validation and caution in the interpretation of borderline reactions [2]. At the same time, it is precisely ICC that expands the diagnostic window in real-world settings, when material is limited and clinical decisions must be made promptly.

It should also be noted that in CUP, ICC implements the principle of differential diagnostic narrowing, as the neoplastic nature and line of differentiation are first confirmed, followed by phenotyping that allows identification of the most probable primary site localizations.

The existing evidence base demonstrates that cytomorphology in isolation rarely allows sufficiently accurate identification of the primary site, whereas its combination with ICC panels provides a statistically significant increase in diagnostic certainty. In a prospective study focused on metastatic lesions without an identified primary source, the combined cytomorphological and ICC approach, for example, ensured high diagnostic accuracy of specific subtypes and enabled the clinician to guide the search for the primary localization based on the marker profile (in particular, using panels selected according to morphology and clinical data) [1].

Additional value of ICC is demonstrated in undifferentiated tumors, for which morphological classification is fundamentally limited. Studies devoted to FNAC substrates have shown that the inclusion of ICC helps categorize undifferentiated cases into clinically significant subgroups (carcinoma, lymphoma, sarcoma, neuroendocrine tumor, etc.), thereby enabling early decision-making regarding patient routing, selection of therapeutic strategy, and reduction in the need for immediate surgical biopsy, provided that antibodies are selected in a rational and cost-effective manner [4].

The practical aspect of identifying the primary site is particularly evident in situations where only previously stained cytology slides are available and repeat sampling is impossible. An approach based on obtaining cell blocks through scraping serves as evidence that ICC can be performed on material obtained by processing (destaining) informative smears, thereby allowing the probable source of metastasis to be established and additional invasiveness to be avoided. A series of clinical observations confirms the applicability of ICC in such cases for clarifying the primary site in metastatic lesions of the liver and lymph nodes when the standard method of cell block preparation is unavailable [5].

For the practical implementation of ICC in cancer of unknown primary (CUP) within routine cytological practice, two complementary components are of fundamental importance: the algorithmization of immunomarker application and systematic quality management at all stages of the examination.

With regard to algorithmization, in the examination of serous effusions and other fluid substrates, a stepwise diagnostic approach (Fig. 3) proves effective, based on the identification of a “second,” immunologically distinct cell population against the background of mesothelial and inflammatory elements. Such an approach involves the initial mapping of the cellular composition of the effusion using a basic panel that allows differentiation between mesothelium, lymphoid cells, and an epithelial tumor population. Within the SCIP approach (subtractive coordinate immunoreactivity pattern), the principle of “subtraction” of background cells is implemented through the analysis of coordinate immunoreactivity, after which additional organ-specific markers are applied to identify the most probable source of metastasis [8].

Figure 3. ICC algorithm in the examination of serous effusions in the context of CUP

In the context of CUP, it is assumed that ICC should be initiated immediately after the initial cytological conclusion confirming the presence of malignant cells or suspicion of metastatic involvement. It is fundamentally important to reserve a sufficient amount of material in advance for cell block preparation or for a standardized type of smears suitable for validated immunostaining, which helps avoid repeated invasive procedures and reduces the diagnostic interval.

The second component of implementation (quality control) essentially determines the clinical reliability of ICC results, especially in the context of CUP, where diagnostic decisions directly influence treatment strategy. Cytological material is characterized by high pre-analytical variability: differences in fixatives (alcohol fixation, formalin), preparation methods (smears, cytospins, liquid-based cytology), storage conditions, and cellularity significantly affect antigen preservation and the intensity of the immune reaction. Unlike IHC performed on FFPE blocks, ICC requires laboratory-specific validation of each antibody and each type of specimen carrier.

As noted in studies devoted to the application of ICC for predictive biomarkers in lung cancer, the transfer of validated IHC assays to cytological specimens is not possible without additional internal validation, precisely because of the multitude of pre-analytical variables [7]. The most standardizable form of cytological substrate remains the cell block, whereas non–cell block preparations are associated with the greatest interpretative difficulties. Therefore, the development of local protocols is proposed, regulating the type of fixation, minimum cellularity, selection of panels, criteria for positivity, and mandatory indication of study limitations in the final report.

Particular attention should be paid to the instrumental implementation of quality control. A promising direction is the use of on-slide controls placed on the same slide and subjected to an identical staining cycle, which increases the reliability of interpreting weak or equivocal reactions and reduces the risk of false-negative and false-positive results when cellularity is limited. The method of preparing on-slide controls from long-term preserved cell suspensions is considered a technologically feasible tool for enhancing ICC reproducibility under routine laboratory workload conditions [6].

Thus, the combination of a clearly structured algorithm (SCIP and stepwise immunomarker panels) and systematic quality control (validation, standardization, internal controls) makes it possible to significantly increase diagnostic certainty in CUP, shorten the time required to establish the probable primary site, reduce the need for repeated invasive procedures, and ensure an earlier transition to organ-oriented therapy.

Conclusion. ICC in cytological practice is an important method of diagnostic clarification in CUP, as it enables work with minimally invasive and often the only available material, thereby increasing the likelihood of determining the line of differentiation and identifying the primary site. The effectiveness of ICC is determined not only by the selection of markers but also by the manner of their application, namely the sequential transition from basic panels to organ-specific markers, as well as the use of algorithms (in particular, in serous effusions) and early reservation of material for ICC. A critical condition for the clinical reliability of ICC is quality management, as local validation of protocols with consideration of pre-analytical variables, the use of control slides, and the implementation of solutions such as on-slide controls are required. Thus, the experience of applying ICC in CUP should be considered from the perspective of opportunities and prospects for optimizing diagnostic timelines and increasing the validity of therapeutic decision-making in tumors of unclear origin.

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