Immunohistochemical expression of CD57 in different histopathological grades of oral squamous cell carcinoma.

INTRODUCTION

Oral cancer ranks 15 th in global average mortality for the year 2022, according to the Global Cancer Statistics Database (GLOBOCAN) from the World Health Organization’s International Agency for Research (WHO/IARC). World Health Organization 2024 reports 188,438 deaths, with 130,808 deaths in males and 57,630 in females with oral cancer.[ 1 ] Torre et al .[ 2 ] have reported that the 5-year survival rate for patients with oral cancer is 40%–50%, and there is a reduction in quality of life in many of the survivors. South and South East Asia observe the highest incidence of oral cancer as well as mortality, and countries like India, Sri Lanka, Pakistan, and Bangladesh have more cases of oral cancer.[ 3 ] There are a variety of risk factors, including betel quid chewing, smoking, and alcohol consumption.[ 4 ] Human Papillomavirus infection is also reported as an important risk factor by Marur et al .[ 5 ] In recent years, the incidence of oral cancer has shown a significant upward trend, highlighting its urgency as a global public health issue.[ 6 ]

Exposure of the genes/proteins/RNA molecules of the mucosa lining to harmful endogenous and exogenous agents is a frequent implication of carcinogenesis.[ 7 ] Malignant transformation from a benign tumour shows an escalation of immune cells in tumour stroma, during the initiation, promotion, and progression of genetic and epigenetic disturbances of malignant epithelial cells. The microenvironment, commonly referred to as the tumour stroma, is needed for nutritional support and for the removal of waste products.[ 8 ]

The tumour microenvironment (TME) consists of, endothelial cells, pericytes, mesenchymal cells, adipocytes, and innate and adaptive immune cells.[ 9 ] The tumour cells in large quantities are targeted through natural killer (NK) cells by a number of inherent immune cells, including granulocytes, dendritic cells, macrophages, NK cells, and mast cells. In the absence of any priming or prior activation, the NK cells notice and control the progress of the tumour.[ 10 , 11 ]

By detecting the lack of expression of the major histocompatibility complex I, natural killer cells can detect cancerous cells and release cytokines and Human Natural Kill1 (HNK1) can be used to identify CD57 on natural killer cells.[ 12 ]

In the IV International Workshop, 1989, Leukocyte Antigens were designated as a cluster of differentiation, CD57 (cluster of differentiation 57), and it is a 110-kd glycoprotein that is stereotypically expressed by the NK cells. This attacks the cancer cells and impedes the tumour development.

The accumulation of CD57 + and CD8 + lymphocytes has been documented in several human malignancies, including oral squamous cell carcinoma (OSCC), renal cell carcinoma, gastric carcinoma, melanoma, multiple myeloma, Hodgkin’s lymphoma, acute and chronic myeloid leukaemia, and chronic lymphocytic leukaemia. Increased infiltration of these lymphocyte subsets has been correlated with reduced disease severity and improved clinical outcomes. CD-57 expressing tumour-infiltrating lymphocytes and NK cells express enhanced immune surveillance and cytotoxic activity.[ 13 ]

Stimulation with interleukin-12 ( IL-12 ) and interleukin-2 ( IL-2 ) promotes prolonged survival of NK cells and induces the secretion of interferon-gamma (IFN-γ), a critical effector cytokine. These findings suggest that heterogeneous NK cell populations, particularly those comprising CD57 + subsets, play a beneficial role in the eradication of neoplastic cells. Furthermore, the expression of CD57 has been correlated with disease progression and prognosis in OSCC, highlighting its potential utility in diagnostic pathology.

The study aims to analyse and compare the immunohistochemical expression of CD 57 in different histopathological grades of OSCC. The objectives included evaluating the overall staining intensity and area of stained cells in well, moderately and poorly differentiated oral squamous cell carcinoma sections using CD57 .

MATERIALS AND METHODS

The Institutional Research Committee and Institutional Ethics Committee of Vinayaka Mission’s Sankarachariyar Dental College, Salem, approved the study. Archival blocks were selected from the Department of Oral and Maxillofacial Pathology and Oral Microbiology, VMSDC, and comprised 10 blocks of histopathological confirmed cases of well differentiated squamous cell carcinoma, moderately differentiated and poorly differentiated, and poorly differentiated squamous cell carcinoma cases, each from different parts of the oral cavity. The lymphoma section was used as the positive control. Variants of squamous cell carcinoma and sections of patients on adjuvant therapy, like chemotherapy and radiotherapy, were excluded.

Serial sections of about 4 mm thickness using a standard rotary microtome were obtained from the archival wax blocks. Two sets of sections were prepared and used for both routine H&E and Immunohistochemical Study. For the IHC study, the sections were fixed on the positively charged hydrophobic poly-L-Lysine-coated microscopic slides. Mouse monoclonal - CD57 antibody was the primary antibody, and Endogenous block (Hydrogen peroxide block), Protein block, Horse radish peroxidase, and Chromogen DAB (Diaminobenzidine tetrahydrochloride) detection system are secondary antibodies, and the slides were mounted. Positive control for CD57 Staining was NK subset cells in Lymphoma [ Figure 1 ]. The sections were examined by one observer three times to reduce intra-observer variability and later seen by one more observer, and the results were compared and taken to avoid inter-observer variability. Results were tabulated and statistical analysis was conducted by using the Chi-square test and evaluate the statistical significance.

RESULTS

CD57 expressions in well, moderately, and poorly differentiated oral squamous cell carcinoma were calculated using the Klein et al . scoring system. The Klien et al . scoring system is:

The area of labelled cells was graded as

Grade 0: When none of the areas expressed CD57 ,

Grade 1: When <30% of the area expressed CD57 ,

Grade 2: When 30 to 60% of the area expressed CD57 ,

Grade 3: When >60% of the area expressed CD57 .

The intensity of expressed cells was graded

Numbers 0, 1, 2, and 3 are given as grades for No reaction, Weak intensity, Mild intensity, and Strong intensity respectively.

Final Score = A + B (Overall Expression) was denoted as 0–1 (Negative), 1–2 (Mild), >2–4 (Moderate), and >4–6 (Severe).

Among the 10 well-differentiated squamous cell carcinoma (SCC) cases, 7 (70%) demonstrated moderate CD57 expression, while 3 (30%) exhibited severe expression. Of the 10 moderately differentiated SCC cases, 3 (30%) showed mild, 6 (60%) moderate, and 1 (10%) severe CD57 expression. In contrast, among the 10 poorly differentiated SCC cases, 2 (20%) were negative for CD57 , whereas 8 (80%) displayed mild expression [ Table 1 and Figures 2 – 4 ].

Statistical analysis of overall positive and negative CD57 staining across the different histological grades of SCC was performed using the Chi-square test. The Chi-square value was 4.29 with a corresponding P value of 0.117, indicating no statistically significant association [ Table 2 ].

Out of 30 Oral squamous cell carcinoma cases of our study, the CD57 immuno expression was positive in 28 (93.33%) cases, the overall staining expression of CD57 was moderate to severe in 17 (57%) cases, mild immune expression was seen in 11 (37%) cases, and the negative result was seen in 2 (6%) cases. A Chi-square test was carried out for statistical analysis; the Chi-square value was 23.01 and the P value was found to be 0.001, which was highly significant [ Table 3 ].

In our study, when well-differentiated squamous cell carcinoma was compared with moderately-differentiated squamous cell carcinoma for staining intensity, Chi-square analysis showed a Chi-square value of 4.077 and an insignificant P value of 0.13 [ Table 4 ].

On comparing the immune expression of CD57 between moderately differentiated and poorly differentiated squamous cell carcinoma, the Chi-square analysis showed a Chi-square value of 2.222 and an insignificant P value of 0.136 [ Table 5 ].

On comparing the positive and negative staining of well and poorly-differentiated squamous cell carcinoma, by Chi square test, the Chi-square value was 20.0 and a highly significant P value of 0.001 [ Table 6 ].

DISCUSSION

Individuals with immunosuppressive disorders exhibit multiple predisposing factors that render them highly susceptible to the development and progression of oral cancers.[ 14 ] Immunity against tumours are executed by the mechanisms of humoral and cell-mediated mechanisms. Several researchers have validated that immune reactions thereby have exhibited that the innate immune system develops resistance against tumours. However, an innate immune reaction perhaps works against malignant transformation and explosion of the cells. Cell-mediated immunity includes natural killer cells and macrophages. In case of deficiency of these immune responses, the tumour develops.[ 14 ]

Mechanisms such as perforin/granzyme-mediated cytolysis and TRAIL- or FAS-mediated apoptosis are carried by CD57 killer tumour cells. Accumulation of CD57 + cells accrual is seen habitually in entities with numerous forms of cancer.[ 15 ] Lesser groups of leucocytes are infiltrated into the tumour stroma, instituted by NK cells. Conflicting T lymphocytes, which can only be stimulated by the antigens via major histocompatibility complex (MHC) class antigens, the cytotoxic effects of NK cells do not need foreign antigens. Perhaps, foreign antigen is triggered by the transformed or lessened MHC class I antigens. Hence, NK cytotoxicity recognizes and organizes the shield against tumours firsthand. Significant performance of CD57 can be appreciated by demonstrating the immune expression in well-differentiated squamous cell carcinoma and gradual decline in staining intensity in moderate and a further diminished expression and staining intensity in poorly differentiated Carcinomas, in this study.

In IHC staining, CD57 was perceived within the tumour stroma, and CD57-active cells were scarce and dispersed among mononuclear inflammatory infiltrate. Fairly contiguous to anaplastic tumour cells, minimal CD57 + cells are perceived. Sections that showed dysplastic epithelium adjacent to tumour epithelium have a better number of sub-epithelial CD57 + cells.[ 16 ]

Studies on head and neck tumour expression of CD57 by Karpathiou et al .[ 17 ] reported that there is a lesser penetration of cytotoxic T lymphocytes, NK cells, and dendritic cells. Fang et al .[ 18 ] in their study (2017) claimed a high CD57 expression in the early stage of the disease and found that strong CD57 manifestation in oral squamous cell carcinoma could be a self-regulating marker for longer existence.

Agarwal et al .[ 12 ] found that the CD57 mean labelling index in patients, who are alive was more than in dead persons. This study ended with the conclusion that a greater CD57 labelling index had a noteworthy association with the progression of disease and the status of life.

Taghavi et al .[ 11 ] pointed out that high CD57 expression was associated with an extended survival rate. The present study also showed a high CD57 accumulation in well-differentiated squamous cell carcinoma and a gradual decrease in staining intensity for moderate and further decreased or absence of staining expression for poorly differentiated squamous cell carcinomas which commonly show poor prognosis.

Other related studies including Turkseven and Oygür[ 19 ] confirmed that the lower intra-tumoral density of CD57 + cells in OSCCs showed decreased prognosis. Oral squamous cell carcinomas microenvironment is highly infiltrated by CD57 + immune cells, regardless of affected person’s age, tumour stage, or morphological grading as stated by de Sousa Lopes et al .,[ 20 ] Iida et al .[ 21 ] presented that amplified CD57 + T-cell penetration in the tumour microenvironment was a potent predictive indicator for oral squamous cell carcinoma. A favourable cytotoxic immune retort in contradiction of malignant cells was shown by the penetration of NK cells ( CD57 ) and CD8 + cells in the tumour microenvironment as stated by Zancope et al .[ 22 ]

Higher CD57 expression, particularly in well-differentiated OSCC, can reflect stronger NK cell or cytotoxic T-lymphocyte activity, suggesting better host immune response. They fall into the lower-risk group, and they may need less intensive treatment strategies and follow-ups. Reduced or absent CD57 expression in poorly differentiated OSCC can reflect impaired immune surveillance, which correlates with aggressive tumour biology, higher recurrence chances, and poorer prognosis. These patients may require aggressive multimodal therapy and shorter follow-ups.

CONCLUSION

Inflammation is a major factor affecting the biological behaviour of oral squamous cell carcinomas. Activated T lymphocytes and/or natural killer cells, which are key types of cells in cancer immunity, can express the surface molecule CD57 .

The primary aim of the current study was to evaluate CD57 in different histopathological grades of oral squamous cell carcinoma. Expression of CD57 in all the cases of well-differentiated squamous cell carcinoma, with staining intensity ranging from moderate to severe, in moderately differentiated squamous cell carcinoma, with staining intensity ranging from mild to severe, and in case of poorly differentiated squamous cell carcinoma, it was mild to negative. CD57 expression was identified to be diminishing as the grade of squamous cell carcinoma were increasing indicating that the prognosis of the patient will be poorer and the progression of the tumour will be faster. When clinical pathological parameters are compared, CD57 expression will surely help in prognosis prediction and individualized patient care.

But the present study was a single institutional cross-sectional study where the sample size was small and we were not able to do the clinical correlation, treatment modalities used for each patient and follow-up of survival status of the patient and these are the drawbacks of the study that can be improved in future studies.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.

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