Introduction

Cervical lymphadenopathy is a common manifestation of underlying pathologies related to the head and neck, with a wide range of differential diagnoses. Non-neoplastic conditions more frequently affect lymph nodes compared to neoplastic processes.[1,2] Among neoplastic lesions, lymph node metastasis is most prevalent, with the primary site often being of squamous cell origin, as noted in few studies. Recent studies highlight the role of Human Papilloma Virus(HPV) in causation of head and neck squamous cell carcinoma.[3,4]

Fine-needle aspiration cytology (FNAC) is generally the initial assessment tool for cervical lymphadenopathy. However, cervical lymph node metastasis of squamous cell carcinoma (SCC) poses diagnostic challenge on FNAC when the tumour is well differentiated and lack nuclear atypia.[5] Many times, secondary changes like necrosis, abscess formation or cystic change may occur in the metastatic lymph node making the diagnosis on cytology a difficult task. [6, 7] Also many benign conditions like infected keratinous cyst or squamous metaplasia mimic well differentiated SCC, adding to the diagnostic dilemma. Repeat aspirate or guided FNAC may demand time, cost and expertise.

Recently, Hara H et al introduced the Five Parameter System (FPS) which is useful in differentiating oral SCC from in situ carcinoma on cytology. [8] This system focuses solely on assessing keratinized cellular findings for cytological examination of oral well-differentiated squamous cell carcinoma (SCC) and carcinoma in situ (CIS) specimens. These specimens can sometimes lack nuclear atypia, making them difficult to categorize accurately within The Bethesda System (TBS). [8]

Furthermore, same authors tested the utility of this system in differentiating oral SCC from in situ carcinoma when nuclear atypia is present and concluded that recognition of the following five parameters which are concentric arrangement of orangeophilic cells, large number of cells, bizarre-shaped cells without nuclear atypia, keratoglobules and uneven filamentous cytoplasm are useful for the cytological diagnosis of oral SCC. [9]

Hence, the present study was planned to determine the usefulness of five parameter system in diagnosing metastatic deposits of SCC to lymph node with secondary changes.

Objectives:

To determine the utility of five parameter system in diagnosing metastatic SCC showing secondary changes like cystic degeneration or abscess formation

Material and Methods

The present study was conducted at a tertiary care rural hospital. Institutional Ethics Committee clearance (IEC) was obtained from IEC-AIMS approval number being 42/2018 dated 10^th January 2018. This is a cross sectional study, conducted for a period of 04 years, from 2018 to 2022. The study population composed of 70 cases and 45 controls. Sampling technique used was purposive sampling. The study population composed of all the patients who had cervical lymphadenopathy who underwent the procedure of fine needle aspiration in the department of pathology in the institute with a cytological diagnosis of metastatic squamous cell carcinoma (SCC) with cystic change or abscess formation, with biopsy confirmation wherever available. Forty five cases of inflamed keratinous cyst diagnosed on FNAC were taken as controls in the study. Written informed consent was obtained before the study from all the study participants after providing adequate information about the study. The demographics of study population were recorded from case records for all the patients.

The FNAC slides from all 634 patients presenting with cervical lymphadenopathy were reviewed by two pathologists, each with over ten years of cytology experience. For this study, all the cases in whom FNAC diagnosis of metastatic SCC to cervical lymph nodes with inflammation/cystic change/abscess formation and had histological confirmation wherever available were selected. In deep seated lymph nodes where biopsy was not feasible, however which had a clinically confirmed primary, radiology was used as a supportive tool for delineating metastatic lymph nodes deposits which were then confirmed by FNAC. The procedures followed in the present study adhered to the ethical guidelines of the Declaration of Helsinki.

Inclusion criteria:

Cases: FNAC cases of metastatic SCC to cervical lymph nodes with cystic change or abscess formation, with biopsy confirmation wherever available. Amongst cases, those smears which showed presence of >30 diagnosable epithelial cells were included.

Controls: 45 cases of inflamed keratinous cyst diagnosed on cytology were taken as controls. Among controls those smears which has >200 squamous epithelial cells smeared on slide were included.

All smears from both cases and controls were evaluated for the Five Parameter System(FPS) focusing on cytoplasmic features which included concentric arrangement of orangeophilic cells, large number of orangeophilic cells, bizarre shaped orangeophilic cells, keratoglobules and dyskeratosis. (Figure 1 to 4)

Figure 1: A and B: FNAC smears showing concentric arrangement of squamous cells in cases i.e. metastatic deposits of squamous cell carcinoma. (Hematoxylin and Eosin, x 200)

Figure 2: FNAC smears showing orangeophilic cells in cases i.e., metastatic deposits of squamous cell carcinoma. (Hematoxylin and Eosin, x 200)

Figure 3: A and B: FNAC smears showing bizarre shared orangeophilic cells in cases i.e., metastatic deposits of squamous cell carcinoma. (Hematoxylin and Eosin, x 200)

Figure 4: FNAC smears showing keratoglobules in cases i.e., metastatic deposits of squamous cell carcinoma. (Hematoxylin and Eosin, x 200)

Statistical analysis:

Descriptive statistics was used like mean and standard deviation, percentages and proportions. Difference between two groups was analysed using unpaired t test with p<0.05 set as statistical significance.

Results

The total number of FNACs done on patients with cervical lymphadenopathy at the study institute, during the study period was 634 which constituted 8.05% of the total FNACs done. A total of 70 cases and 45 controls were included in this study. Of the total 70 cases, 56 cases of SCC were well-differentiated SCC and 14 cases were poorly differentiated SCC. Demographic characteristics of study population is depicted in Table 1.

The FNAC smears of all the cases and controls were evaluated using the five parameter system, Among the five parameters assessed, in the cases; the most frequently observed feature was large number of orangeophilic cells, seen in 58 cases (82.8%). This was followed by uneven filamentous cytoplasm in 50 cases (71.4%), bizarre-shaped orangeophilic cells in 46 cases (65.7%), a concentric arrangement of cells in 6 cases (8.57%), and keratoglobules in 2 cases (2.85%). In contrast, none of these characteristics were present in the control group (Table 2).

Out of the 70 cases of metastatic SCC, 56 were well-differentiated and 14 were poorly differentiated. (Table 3, Figure 5) Concentric cell arrangements were observed in 5 cases (8.92%) of well-differentiated SCC and in 1 case (7.8%) of poorly differentiated SCC. A large number of orangeophilic cells appeared in 50 cases (89.2%) of well-differentiated SCC and 8 cases (57.14%) of poorly differentiated SCC. Bizarre-shaped orangeophilic cells were found in 35 cases (62.5%) of well-differentiated SCC and in 11 cases (78.57%) of poorly differentiated SCC. Keratoglobules were present in 2 cases (3.57%) of well-differentiated SCC, while none were seen in poorly differentiated SCC. Uneven filamentous cytoplasm was noted in 47 cases (83.92%) of well-differentiated SCC and 3 cases (21.42%) of poorly differentiated SCC (Table 3 and Figure 5). Over all there was statistically significant difference between the frequency of all the 5 parameters among cases and control (unpaired t test, p<0.05)

Figure 5: Graphical representation of comparison of 5 parameter system between well differentiated SCC and poorly differentiated SCC

Discussion

Finding of keratinized squamous cells in a lymph node is a strong pointer to metastasis of SCC, however, in the presence of cystic degeneration or abscess formation, which is commonly seen in cervical metastasis of SCC, FNAC smears demonstrate necrosis and acute suppuration, whereas atypical cells are very scanty. Moreover, the degree of atypia is either less or may be attributed to the reactive changes, leading to false negative interpretation. Though lymph nodes and keratinous cyst can be differentiated clinically, this differentiation is not straightforward and ambiguities do occur. For example, the cystic lesions, when inflamed or rupture, develop pain, erythema and adhesions which may mimic inflamed lymph nodes. Similarly, the lymph nodes when they undergo suppuration or cystic changes may appear as soft fluctuant mass, mimicking cystic lesions. Moreover, at cytology, the inflammatory atypia and metaplastic changes in an inflamed keratinous cyst is a common pitfall for a false positive diagnosis of SCC, especially if the diagnosis is based solely on keratin presence. The five-parameter system is an indispensable adjunct tool in these diagnostically ambiguous cases which takes into consideration the more reproducible cytoplasmic keratinization-based features rather than nuclear characteristics alone. Hence, the present study emphasizes the usefulness of FPS in differentiating metastatic SCC from its benign mimickers, especially in cytologically equivocal or paucicellular aspirates.

Lymph node metastasis in cervical region have primary tumours more commonly in the oropharynx. tonsil and base of tongue. HPV is a well-established risk factor for the squamous cell carcinoma in these region with significant prognostic and therapeutic implications. Imaging modalities like Computed Tomography, Magnetic Resonance Imaging along with FNAC plays a crucial role in the diagnostic workup. [5, 6]

False negative rate in metastatic squamous cell carcinoma is a cause of concern since most are well differentiated and lack nuclear atypia. Hara et al (2017) observed in their study, that the five parameter system was successful in discriminating oral SCC.[9] These five parameter system (FPS) is based on cytoplasmic features and hence was proposed to be useful in cases which lacked nuclear atypia. Recently, Hara et al (2022), applied this FPS in oral SCC with apparent nuclear atypia. [8] (Table 4)

In the present study, we attempted to apply the FPS in metastatic SCC, especially in cases which showed inflammation and cystic change.

The current study investigated cytomorphological characteristics of metastatic squamous cell carcinoma (SCC) in patients with cervical lymphadenopathy, focusing on differences between well-differentiated and poorly differentiated cases. A comparative analysis of these cases with controls revealed distinct cytological features that can aid in the identification and differentiation of metastatic SCC.

A previous study by Hara et al (2017) has showed high frequency of orangeophilic cells (100%) in SCC cases. [9] Similar observations were made in the present study. The high frequency of orangeophilic cells, observed in 89.2% of well-differentiated and 57.14% of poorly differentiated cases in our study, underscores their diagnostic significance in SCC. This characteristic was the most frequently observed feature among the five parameters assessed, seen in 82.8% of all cases, yet absent in controls. This finding aligns with previous studies that highlight the presence of orangeophilic cells as a hallmark of SCC due to the presence of keratinized cytoplasm, a key feature in well-differentiated tumors. [8] The higher incidence of orangeophilic cells in well-differentiated cases may reflect the greater degree of keratinization compared to poorly differentiated SCC.

The presence of bizarre-shaped orangeophilic cells, observed in 62.5% of well-differentiated and 78.57% of poorly differentiated SCC, further supports the cytomorphological variations within SCC subtypes. This parameter was seen in 65.7% of all SCC cases, indicating its diagnostic utility in identifying malignant features within SCC, particularly in distinguishing between SCC subtypes based on cellular morphology. The higher frequency of these cells in poorly differentiated cases may be attributed to increased cellular atypia, a hallmark of lower differentiation grades in SCC. Similar observations of increased bizarre-shaped orangeophilic cells (83.3%) were documented by Hara et al. [8]

The concentric arrangement of cells, although less frequent, was observed in 8.92% of well-differentiated and 7.8% of poorly differentiated SCC cases. While this feature is not as prominent as orangeophilic or bizarre-shaped cells, its presence still contributes to the identification of SCC, as it reflects the organized, albeit disrupted, cellular architecture commonly seen in neoplastic lesions. Hara et al has documented the concentric arrangement of cells in 75% of the cases of SCC, which is high compared to our study. [8]

Keratoglobules were documented in all the cases of SCC (100%) by Hara et al. [8] in the present study, Keratoglobules, noted in only 3.57% of well-differentiated cases and absent in poorly differentiated cases, were the least common feature identified. Their limited occurrence suggests that keratoglobules may not be a reliable diagnostic marker in SCC, particularly for poorly differentiated cases. In contrast to Hara et al. who included FNAC of primary SCC in their study, the present study included FNAC from metastatic SCC deposits in lymph nodes. Most of these cases harbour necrosis, inflammation or demonstrate cystic degeneration, wherein keratinization may be incomplete or disrupted. Formation of concentric cell arrangements and the presence of keratoglobules reflect mature keratinization; and the same is reduced in metastasis, especially in the presence of secondary changes. Likewise, the variation in cellularity and tumour differentiation in primary and metastatic process may have contributed to the decreased prominence of keratinized structures.The absence of keratoglobules in poorly differentiated cases may correlate with reduced keratinization, a characteristic of lower differentiation. While their lower frequency may limit their utility as primary diagnostic criteria, these features, when present, could serve as ancillary markers, further substantiating the diagnosis of SCC.

Uneven filamentous cytoplasm, observed in 83.92% of well-differentiated and 21.42% of poorly differentiated cases, ranked second in frequency among the parameters. This characteristic, present in 71.4% of all SCC cases, highlights its relevance as a diagnostic feature, especially for well-differentiated tumors. This result is in concordance with the study by Hara et al, in which the uneven filamentous nature of the cytoplasm has been documented in 100% the cases of SCC. [8] The uneven filamentous nature of the cytoplasm may indicate the presence of keratinized strands, a prominent feature of well-differentiated SCC, which is typically reduced or absent in poorly differentiated variants.

The complete absence of these cytological features in the control group underscores their potential specificity. This specificity enhances the diagnostic accuracy of FNAC in these cases, offering a rapid, minimally invasive, and cost-effective approach to the diagnosis of metastatic SCC.

Limitations

Limited sample size and single institution study is the major limitation of the present study. In the study conducted by Hara et al, authors included primary SCC cases, however the present study had a strict inclusion criteria and specifically focused on the metastatic deposits of SCC with cystic degeneration which is less common and is a more problematic area, especially in the context of the overlap with benign cystic keratinous lesions. Hence, the present study cohort reflects real incidence patterns over 4 years. Moreover, the present study is one among the few studies evaluating FPS in metastatic SCC. Further studies with large sample size will be imperative to test the diagnostic value of FPS

Conclusion

Five parameter system can serve as a powerful tool in FNAC diagnosis of metastatic deposits of SCC in challenging situations such as presence of cystic change and abscess formation. The distinct cytomorphological features, such as the presence of orangeophilic cells, bizarre-shaped cells, and uneven filamentous cytoplasm, provide valuable diagnostic markers for metastatic SCC, and helps in distinguishing between well-differentiated and poorly differentiated forms

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