Abstract
Background
Hematoxylin & Eosin (H & E) stains have been conventionally used to establish the status of safe margins following resection of primary Oral Squamous Cell Carcinoma. Due to non-specificity of this stain, there is a possibility of false negative results. In this study, we have assessed the role of Immunohistochemistry (IHC) in establishing the status of safe margins.
Aim
To compare Hematoxylin & Eosin (H & E) and Immunohistochemistry (IHC) staining in identification of tumor cells in establishing the status of safe margins.
Methodology
This study included 14 cases diagnosed with OSCC. Following resection, the primary lesion was subjected to Histopathological analysis. 2 sets of HP slides were prepared from serial sectioning of the wax block prepared for each of the four margins. Both sets of slides were stained with H &E stain. One set of these slides was further stained with Pan CK marker (IHC) which is a cytokeratin marker to identify tumour cells.
Results
All the slides with H & E staining reported negative for tumor infiltration and 4 slides (3 patients) out of 56 were reported positive with PanCK marker. There was a statistically significant difference in the number of patients with positive margins using IHC as compared to H & E stain.
Conclusion
Immunohistochemistry using PanCK marker proved to be more efficient in the determination of status of safe margins than routine H & E staining.
Keywords: Immunohistochemistry, Hematoxylin and eosin stain, Safe margins, Pancytokeratin marker
Introduction
Oral cancer is one of the most fatal health problems faced by the mankind today [1] as the 5-year survival rate ranges from 30 to 80% in different regions of the world, and the overall mortality rate for oral squamous cell carcinoma (OSCC) remains high, at approximately 50% [2]. Recurrence in head and neck cancer ranges between 40 and 68% and is found to be due to loco-regional recurrence [3–7] following wide excision of the primary lesion. Surgical resection with safe margin is the widely used treatment protocol for resection of the primary lesion. The width of this margin varies between 1–2 cm or more depending on the histopathological differentiation [8].
The status of safe margins is established by histopathological analysis using hematoxylin & eosin (H & E) staining and is a widely used method due to its low cost, ease of performance and usefulness in verification of safe margins. However, there is a possibility of false-negative margin result with H & E staining [9]. As the islands of the tumor cells can be very small, they can be easily missed even with H & E staining. This limitation can be overcome with the use of immunohistochemistry (IHC), which supports the detection of specific protein expression at different locations of a tissue. Pancytokeratin (PanCK) marker is a cytokeratin marker that can be used to identify cytokeratin in tumor cells at the safe margin periphery [10]. In this study, we aim to compare the efficacy of H & E staining & IHC staining in studying the status of surgical safe margins.
Methodology
The present study was conducted to assess status of safe margins in patients diagnosed with oral squamous cell carcinoma at Al-Badar dental college and Hospital, Kalaburagi, Karnataka. Patients histopathologically diagnosed with OSCC and indicated for surgery as primary mode of management for OSCC were included in the study. Patients with recurrent squamous cell carcinoma, patients who have undergone radiation therapy and chemotherapy, patients who had distant metastasis at presentation and patients with any systemic condition contraindicating general anesthesia were excluded from the study.
Post-operatively, the four sides of the resected specimen margins were studied to establish the status of surgical safe margins. Following the preparation of the wax block, 2 sets of slides were prepared for each side by serial sectioning. Both set of slides were stained with H&E. Of these two stained sets, one set was used to analyze for safe margin, i.e., the presence/absence of tumor cells at the margin and the other set were further subjected to IHC staining followed by histopathological analysis for safe margins. PanCK marker which is a cytokeratin marker was used to mark the cytokeratin in tumor cells at the surgical safe margins, if present.
Results
| Correlation of patients with positive margins using PanCK marker & H & E Stain using McNemar's Test | |||||
|---|---|---|---|---|---|
| PanCK | HPE findings | P-value | |||
| N | % | n | % | ||
| Negative | 11 | 78.5% | 14 | 100.0% | 0.03* |
| Positive | 3 | 21.4% | 0 | 0.0% | |
| Statistically significant for a P-value of < 0.05 | |||||
A total of 112 slides were studied, 56 with H & E staining and 56 with PanCK marker. All the slides with H & E staining reported negative for tumor infiltration and 4 out of 56 slides were reported positive with PanCK marker. Two margins were positive in a single patient and 1 margin each in 2 patients, with a total of 3 patients (21.4%) with positive margins out of 14 patients. The results obtained were subjected to statistical analysis. Statistical analysis using McNemar’s test was performed to compare the results of H & E staining and IHC staining in assessing status of safe margins in OSCC. There was a statistically significant difference in the number of patients with positive margin using IHC as compared to H & E stain with a P-value of 0.03.
Discussion
Oral cancer is one of the most common cancers in India [1] with high morbidity & mortality. The most widely used and successful line of treatment is surgical resection with safe margin and neck dissection. The width of this margin varies between 1–2 cm or more depending on the histopathological differentiation [8]. Also, the size of the primary tumor affects both the choice and outcome of treatment. Tumor size is an important factor in determining the ability to obtain tumor-free margins and the dose necessary to cure patients treated by radiotherapy. Larger size at presentation is associated with an increased risk of local recurrence, increased cervical lymph node metastasis and poor survival [9].
Ravi SB et al. in their review on surgical margin and its evaluation in OSCC concluded that status of surgical resection is an important predictor of outcome for both local recurrence and overall survival in OSCC [11]. 10–30% of OSCC pts with histological negative margins report local recurrence. The reasons suggested were: (1) residual cancer cells under-treated after routine histopathological analysis described as MRC (minimal residual cancer) and (2) macroscopically undetected fields of genetically altered cells which have a potential to develop into pre-malignant malignant lesion as well as invasive carcinoma. Majority of these can be identified using IHC or genetic analysis [12, 13]. Local behavior of OSCC can lead to an early recurrence or point to a possible metastasis helping the surgeon/medical oncologist in planning aggressive post-op management and long-term follow up. The 5-year overall survival rate was reported to be 70.9% and 89.2%, respectively [14].
Patients with negative surgical margins are subjected to prophylactic radiotherapy. Patients with positive margins are subjected to therapeutic radiotherapy with or without chemotherapy. Since radiation with or without chemotherapy leads to significant morbidity, with the use of immunohistochemistry, we can reduce the number of patients exposed to radiation and chemotherapy.
Histopathological analysis of safe margins is established by H & E staining and is a widely used method due to its low cost, ease of performance and usefulness in verification of safe margins. However, it is easy to miss small islands or clusters of tumor cells in H & E staining, thereby resulting in many false-negative results [6]. Due to high rate of local recurrence, the significance of safe margin as established by H & E stain is not reliable and a large group of patients are hence over treated with significant personal and socio-economical impact. Loco-regional recurrence which ranges between 40 and 68% [3–6] in patients with early stage oral cavity squamous cell carcinoma after surgery remains a problem and can affect survival of the patients. Immunohistochemistry (IHC) due to its elaborate procedure and high cost is not used widely in assessing safe margins for detection of tumor cell nests and its presence in vascular spaces. It is generally used in histology to detect the presence of specific protein marker that can assist in accurate diagnosis in histologically similar & in difficult to diagnose cases [15]. Pancytokeratin (PanCK) marker (AE1/AE3) consistently stains all the keratinocytes within OSCC and also the normal, uninvolved keratinocytes within the surrounding tissue with high staining intensity. Cytokeratins are usually found in heterodimeric pairs of acidic and basic subunits of similar size. Its antibody is AE1/AE3 which targets the cytokeratins CK1, CK2, CK9 and CK10 detecting cells from the multilayered, cornified epidermal epithelium but does not detect CK17 or CK18. It is an antibody cocktail mixture that can detect multiple cytokeratins and reacts to multiple epithelial tissues. AE1/AE3 is the most consistent marker, with intensity and sensitivity rates averaging 99.1% and 99.9%, respectively.
This marker detects micro-metastasis and isolated tumor cells efficiently in surgical safe margins.
We conducted this study to analyze role of H & E staining & IHC staining in identification of surgical safe margins. All the margins were found to be negative with H & E staining. However, PanCK staining revealed 4 positive margins in 3 patients. This difference in patients with positive margins on H & E and IHC examination was statistically significant. Thus, PanCK marker proved to be an important diagnostic marker in studying safe margins.
Following surgical management of OSCC, to prevent recurrence, prophylactic radiation therapy is given in patients having negative margin clearance of 5 mm. In cases which have negative margin ranging from 2.2 to 5 mm, adjuvant chemotherapy is given [16]. Prophylactic radiation dosage is 45–50 Gy administered for 5–8 weeks [17]. In case of positive margins, the radiation dose is increased to 60 Gy [18].
In spite of prophylactic radiation therapy in patients with negative margins, we see 14.44% [18] of loco-regional recurrence. This could be due to false-negative reports with H & E staining. To overcome this error and compromised line of treatment, IHC proves to be helpful. A definitive surgical safe margin outcome in these cases helps in deciding the type of radiation therapy, i.e., prophylactic or therapeutic that needs to be given to a patient. Patients with negative margins on IHC may be considered for following-up without radiotherapy, thereby reducing significant morbidity that may result from complications like radiation induced mucositis, fibrosis, xerostomia, loss of taste, cervical atherosclerosis and osteoradionecrosis associated with radiotherapy, etc. However, other factors such as the size of tumor and nodal status should be taken into consideration.
PanCK marker is readily available in the market, and all the centers can make use of it in identification of safe margins in oral squamous cell carcinoma. IHC stain technique does not require any special equipment other than special stains. A short-term training is enough for technicians and pathologists to gain basic knowledge about stains and its techniques, which is sufficient to perform the staining.
However, IHC is costlier in comparison with the H&E stains, but, as compared to the expenditure incurred following recurrence due to false-negative margins and the treatment that follows, including surgery, therapeutic radiation or chemoradiation, results in higher cost.
Limitations of our study is the small sample size. Further studies can be conducted with larger sample size to see the accuracy and effectiveness of this staining technique.
It can be concluded that IHC can give more definitive results and can be used to determine the status of safe margins and thereby the need for post-op radiotherapy, especially prophylactic, which is given in patients with negative safe margins.
Acknowledgements
The authors extend their acknowledgement to Dr. Hina, Dr. Deepa and Dr. Ameena of Department of Oral Pathology, Al-Badar Rural Dental College & Hospital, Kalaburagi.
Declarations
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be constructed as a potential conflict of interest.
Ethical approval
This study was approved by Institutional Ethical Committee, Al-Badar Rural Dental College & Hospital, Kalaburagi (No. IEC/2019-20/06).
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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