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Table of Contents
ORIGINAL ARTICLE
Year : 2015  |  Volume : 2  |  Issue : 2  |  Page : 54-60

Histopathological and immunohistochemistry observations based bukhari grading for squamous cell carcinoma


1 Professor, Department of Pathology, American University of Barbados, Bridgetown, Barbados
2 Demonstrator, Department of Pathology, King Edward Medical University, Lahore, Pakistan
3 Assistant Professor, Department of Pathology, King Edward Medical University, Lahore, Pakistan
4 Professor, Department of Pathology, Gujraan Wala Medical College, Gujraan Wala, Pakistan
5 Professor of Medicine, Fatima Jinnah Medical College, Lahore, Pakistan

Date of Web Publication5-Jul-2017

Correspondence Address:
Mulazim Hussain Bukhari
Professor, Department of Pathology, American University of Barbados
Barbados
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Source of Support: None, Conflict of Interest: None


DOI: 10.5530/ami.2015.3.2

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  Abstract 


Background: Increasing incidence of Squamous Cell Carcinoma (SCC) has emphasized the challenges of managing this condition. Traditional microscopic information often fails, especially when based on H & E methods. Immunohistochemistry (IHC) and molecular studies in combination with traditional histopathology may fill this gulf. Aims: The study was conducted to introduce new a grading system based on both histopathological and biological correlation of SCC. Settings and Design: A descriptive study included 180 cases of SCC of the skin (all regions of skin and oral mucosa). Cellular proliferation index (Ki-67 and p53 expression) was studied in SCC by immunohistochemistry (IHC). This study was carried out in the Department of Pathology from January 2006 to December 2008. Methods and Material: The clinicopathological information regarding age, sex, primary tumor site, tumor size, local recurrence, distance metastasis and follow-up status was collected for each case. Patient outcome was verified and updated through the medical records. Five micron thick (5μm) sections were cut from archival formalin fixed, paraffin embedded specimens. The first section was stained with haematoxylin and eosin (H&E) for histopathological analysis. Other sections were stained immunohistochemically with p53 and Ki-67 and then independently scored for the expression of p53 proteins and Ki-67 index. Results: SCC was designated low, intermediate, and high tirade grades based on the sum of point values assigned to each 4 scores of histological differentiation, staging, expression of p53 protein and Ki-67 index. Expression of p53 was found to be related to the Ki-67 and the scores of histology and stages of SCC. A significant correlation was found among the newly assigned grades, stages (Spearman correlation = 0.721, P value = 0.000). The grades were also significantly correlated with other prognostic factors like local invasion, lymph node and distance metastasis (Kendall's Tau-b = 0.394;p-value = 0.00). Tumor recurrence was also significantly based on grades of SCC (Kendall's Tau–b = 0.966, P value = 0.025). Conclusion: It was concluded that a new grading system is an important prognostic indicator of squamous cell carcinoma. This practical approach has potential to improve clinical evaluation of SCC in understanding the pathological as well as clinical behavior of SCC.

Keywords: Malignancy grading system, Squamous cell carcinoma, Immunohistochemistry, Histopathology, P53, Ki-67, Surgical staging, Lymph node metastasis, Local invasion


How to cite this article:
Bukhari MH, Saba K, Qamar S, Khalil E, Niazi S, Imam SF. Histopathological and immunohistochemistry observations based bukhari grading for squamous cell carcinoma. Acta Med Int 2015;2:54-60

How to cite this URL:
Bukhari MH, Saba K, Qamar S, Khalil E, Niazi S, Imam SF. Histopathological and immunohistochemistry observations based bukhari grading for squamous cell carcinoma. Acta Med Int [serial online] 2015 [cited 2019 May 20];2:54-60. Available from: http://www.actamedicainternational.com/text.asp?2015/2/2/54/209653




  Introduction Top


Histological grading is a well-established pathological prognostic factor for most of the malignant neoplasms and the evaluation of genes, which affect these grades, are the common characteristic tools to assess the prognosis of patients. The Squamous Cell Carcinoma (SCC) grading system designed by Borders in 1921 is widely used in clinical laboratories. According to this system, there are four grades of SCC: I, II, III and IV. Each grade is determined by microscopic assessment of the proportion of mature cells to immature cells on Hematoxyllin and Eosin stained biopsy slides. Mature cells are typical cells which are differentiated towards normal keratinocytes. Immature cells are atypical and anaplastic that do not show differentiation toward normal keratinocytes.[1]

Jakobsson introduced another histological grading system for grading squamous cell carcinoma based on the microscopic characteristics of tumor cells and relationship of tumor with the host factors.[2],[3],[4],[5],[6] This system was successfully applied by pathologists and oncologists, but could not get popularity because it was time consuming.[7],[8]

This histological grading system of Jakobsson was further developed and updated by Anneroth et al. and by Bryne et al., in which a small number of variables were applied only to the interface between the tumor and the host. It has been shown to be prognostically more useful, more time effective, and to have good inter- and intra-observer reliability.[5],[7],[8],[9],[10],[11],[12],[13]

Evaluation of the quantitative distribution of p53 and Ki-67 has been applied in tumor pathology both for diagnostic and prognostic purposes.[14],[15],[16],[17],[18],[19]

Immunohistochemical detection of p53 and Ki-67 expression allows a quantitative measure of proliferation potential of a particular neoplasm. Hence, less proliferative tumors exhibit Ki-67 expression in a low percentage of its cells. In contrast, more aggressive cancers show Ki-67 expression in a high percentage of cells.[20],[21],[22],[23],[24],[25] P53 scoring is also correlated with the grades of tumor and is up regulated in high grade and aggressive carcinomas.[15],[16],[17],[18]

Cell proliferation is tightly coordinated with cell growth. The tumor-suppressor proteins, pRb and p53 exert a key role in coupling growth and proliferation by controlling both ribosome biogenesis and cell cycle progression. In tumors with altered pRb and p53 function the up-regulation of rRNA synthesis assures an adequate growth of cancer cells with uncontrolled cell cycle.[21]

Quantitative assessment of cell proliferation of any tumor is used to predict the biological behavior of a particular neoplasm therefore cell proliferation is of interest since abnormal cell proliferation appears to be a precursor of tumor genesis. Ki-67 is a nuclear protein proliferative marker and is expressed during active phases of cell cycle (G1, S, G2 and M) and is absent in the G0 “resting” phase.[18],[26],[27],[28],[29]

Detection of Ki-67 index by immunohistochemistry is a semi-quantitative measure of proliferation potential of a particular neoplasm. Well differentiated carcinomas with low proliferative potential usually exhibit Ki-67 expression in a percentage lower than 30% of its cells in contract to poorly differentiated aggressive carcinomas which have Ki-67 expression in more than 50% of cells.[18],[30]

The new grading system is based on histological values, surgical staging and proliferative index of the SCC that may be better in predicting prognosis, recurrence and response to treatment. The study was conducted to introduce new grading system based on both histopathological and prognostic behavior of the SCC.


  Materials and Methods Top


A descriptive study included 180 cases of SCC of the skin (all regions of skin and oral mucosa). Cellular proliferation index (Ki-67 and p53 expression) was studied in SCC by immunohistochemistry (IHC). This study was carried out from January 2006 to December 2008.

The clinicopathological information regarding age, sex, primary tumor site, tumor size, local recurrence, distance metastasis and follow-up status was collected for each case. Patient outcome was verified and updated through the medical records. Five micron thick (5μm) sections were cut from archival formalin fixed, paraffin embedded specimens. The first section was stained with haematoxylin and eosin (H&E) for histopathological analysis. Other sections were stained immunohistochemically with p53 and Ki-67 and then independently scored for the expression of p53 proteins and Ki-67 index.

Four scores were suggested and each indicates the proportion of mature cells to immature cells that are found when viewing the biopsy specimen under a microscope. Mature cells are classified as typical cells differentiating toward a normal phenotype. Immature cells are classified as atypical and undifferentiated compared with normal cells. The TNM staging (tumor, node, and metastasis) system was used to scores all tumors. The main histopathological risk factors were categorized against specific score and the grades were calculated by the sum of point values assigned to each of 4 scores of histological differentiation with other parameters. [Table 1]
Table 1: Scoring of histological features of tumor cells by Haematoxylin and Eosin

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To determine the grade of P53 and Ki-67 expression, 200 epithelial cells located across the whole epithelial layer were examined in a high-power field (40x). P53 and Ki-67 index was defined as the percentage of positive cells. Scoring was semi-quantitative. Immunoreactivity was considered positive when nuclear staining in cells of tumor was found. The intensity was graded as score 1 when positive in less than 5-10% of tumor cells, score 2 when positive between 10-30% and Score 3 when positive 30-50% of tumor cells, and score 4 when positive in more than 50% of tumor cells. All slides were blindly graded by two histopathologists with good correlation scores between them.[31]

For each biopsy, 4μm-thick sections were placed on glass slides and deparaffinized with Histoclear. Sections were then rehydrated through decreasing concentrations of alcohol (100%, 95%). They were then immersed in 0.01Mcitric acid (pH 6.0) in a microwave oven for 20 min to enhance antigen retrieval (positive and negative controls were also used). In some cases, microwaving was repeated for two additional cycles of 5 min each. The avidin-biotin peroxidase method was used employing an Immunotech 500 (Leica, Deerfield, IL) automated immunostainer according to the manufacturer's instructions.

Briefly the automated steps included blockage of endogenous peroxidase with 3% hydrogen peroxide and reaction with monoclonal antibodies against human p53 (clone D07; DAKO, Lahore, Pakistan) diluted 1:200 and Ki-67 (clone MIB-1) 1:50 diluted Monoclonal Mouse Antibody (DAKOC Lahore, Pakistan) for overnight respectively. After extensive washing, sections were incubated at room temperature for 30 min for p53 and 60 min for Ki-67 with biotinylated goat anti-mouse IgG antibody and then for 45 min with ABC (avidin-biotin peroxidase complexes) in a 1:25 final dilution. Diaminobenzidine (0.06%) (DAB) was used for 4 minutes as the final chromogen and were counterstained with Mayer's Hematoxylene for 4 minutes (Sigma Chemical Company, USA); All staining procedures were performed at 37°C. Stained slides were dried and were covered with glass cover slips.

Positive Control: Specimens consisting of invasive ductal carcinoma breast was used as positive controls for p53 and a lymph node with lymphoid hyperplasia for Ki-67 staining.

Negative Control: Parallel running additional sections in PBS not treated with antibody was used as negative control.

IHC reactivity for p53 was read by our newly adopted scoring system, based on the quantity and quality of brown pigmentation in the nuclei of cells. Cells having a positive nuclear reaction among each 500–1000 cells revealing a positive nuclear reaction in 5-10 high power fields were counted and the percentages were calculated for scoring the p53 protein expression. We also assessed the quality of staining of IHC in the nuclei of the keratinocytes on the basis of the granularity (color, shape, and size) of IHC stains in the nuclei of epithelial cells. To evaluate p53 staining two expert surgical pathologists performed the p53 counts. The observers independently evaluated the stained slides without knowledge of the clinicopathological profiles of these lesions. The presence of cells with clear and unequivocal nuclear staining was identified as positive cases. The count was based on a HPF of 0.159 mm2, which is defined as a field resulting from use of an ocular with an 18 mm2 field of view at 10 × magnification and 40 χ objective (with appropriate correction factors to compensate if different field oculars are used). The p53 protein-expression values were then compared with those in the controls, [15],[16],[17],[31] it was done according to nuclear immunoreactivity of p53. The grades were calculated by the sum of point values assigned to each of 4 scores of p53 expression with other parameter [Table 2].[15]
Table 2: Scoring of TNM staging of dermal squamous cell carcinoma

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To determine the grade of Ki-67 expression, 200 epithelial cells located across the whole epithelial layer were examined in a high-power field (40x). Ki-67 index was defined as the percentage of Ki-67 positive cells. Scoring was semi-quantitative. Immunoreactivity was considered positive when nuclear staining in cells of tumor was found. The intensity was graded as score 1 when positive in less than 5-10% of tumor cells, score 2 when positive between 10-30% and Score 3 when positive 30- 50% of tumor cells, and score 4 when positive in more than 50% of tumor cells. All slides were blindly graded by two histopathologists with good correlation scores between them.[31]

Data was analyzed by using SPSS version 16. The two-tailed P value was calculated. Spearman correlation coefficient was also used.[32] Kendall's Tau-b test was used to assess association of molecular grades with pathologic prognostic factors. Correlation between molecular grading and TNM staging was determined through Spearman Correlation Coefficient. The level of statistical significance was set to P < 0.05.


  Results Top


These scores were given on the basis of cytological feature (cell maturation, cytological morphology, nuclear features and appearance of nucleoli), presence of necrosis and mitosis. Well-differentiated SCC characterized by more normal appearing nuclei with abundant cytoplasm and extracellular keratin pearls was scored 1; moderately differentiated SCC exhibiting features of both well-differentiated and poorly differentiated lesions was scored 2. The poorly differentiated SCC showing a high degree of atypical nuclei with frequent mitoses, a greater nuclear-cytoplasmic ratio, and less keratinization was give score 3 while undifferentiated SCC with all the features of pleomorphism, solid pattern of growth, prominent nucleoli, profuse necrosis and marked mitotic activity was labeled under score 4 [Figure 1].
Figure 1: Photomicrograph showing SCC grade I, A (H&E 20x), D (IHC of p53 40x) and G (IHC of Ki-67 40x); SCC Grade II. B (H&E 20x), E (IHC of p53 40x) and H (IHC of Ki-67 40x); SCC Grade III. C (H&E20x), F (IHC of p53 40x) and I (IHC of Ki-67 40x);SCC Grade III

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The other important parameter was expression of p53 immunostaining reactivity in the malignant cells and these were also scored on the basis of percentage of reactive cells, and quality of reactivity. The other modality used in calculation of proliferative index is by Ki-67 staining. [Table 2]

The scores of three modalities were summated and named as are in present use. These grades are directly related to pathological prognostic factors of SCC like local invasion, lymph node and distance metastasis [Table 3],[Table 4].
Table 3: Scoring of Immunohistochemistry scores for Ki-67 index and p53 protein in tumor cells

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Table 4: Summation of all scores for molecular grades of squamous cell carcinoma

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The new grades of SCC were also important in staging the disease; clinical stages were compared and were found directly related to the grades of malignancy [Table 5]. New molecular grading was correlated with other important prognostic factors like in situ component, local invasion, lymph node involvement and distance metastasis and is also found to be significantly related to these grades. [Table 4]
Table 5: Correlation of molecular SCC with surgical staging of the (according to TNM system)

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Patients' follow-up was not possible for all cases; however few cases were kept under observation. No recurrence was seen in patients bearing tumor of grade I and II, 6/15 (40%) recurrence was seen in patients with high grade squamous cell carcinoma. [Table 5]

It has been seen that scores of proliferative markers, p53 immunohistochemistry and Ki-67 index were significantly related with histological score of SCC Five years survival was also related with scoring as it was better in grade I and gradually reduced to grade II and III, from 90% to 70% and 26% respectively.


  Discussion Top


The present grading system is an attempt to apply immunohistochemical and molecular genetics features in identifying clinicopathologic outcome of SCC. Recent years have witnessed an increasing emphasis on the role of nuclear DNA and its application in pathological diagnosis to predict prognosis and management of neoplasm The grading system and DNA content provides more objective and accurate criteria which relate the morphologic finding to biologic activity and growth patterns of oral cancer as compared to histologic differentiation alone.[33]

To make histological grading system more productive we made modifications in it. Our modified grading system is based on semi-quantitative assessment of morphological feature of tumor like the number of mature and immature cells, presence or absence of keratinization, evidence of cytological index, nuclear size and nuclear cytoplasmic ratio, count of mitosis and presence of necrosis for each grades of SCC. Apart from histologic grade, two molecular markers have been used to describe genetic mutation and proliferation, such as IHC expression of p53 and microscopic assessment of Ki-67 index.

In conventional methods oncologists were provided only histological information (Cytological features) while biological behavior was not considered in these methods. Now is the need to visualize the tumor at the molecular level. Because the SCC shows variable morphologic features and distinct molecular changes, understanding of molecular behavior will better predict clinical outcome than the traditional clinical and pathological standards.

Our grading system is more related with prognostic factors because it comprises biological markers, for example changes in proliferative markers and tumor suppressor genes that lead to cellular transformation and tumor progression. Loss of p53 tumor suppressor function is the most common genetic change in many solid tumors. Accumulation of the abnormal p53 protein usually the result of p53 gene mutation causes a prolonged half life of the protein and can be detected by immunohistochemical staining (IHC) in SCC. The current grading system of SCC has concentrated on the relationship of expression of the tumor suppressor gene p53 and the proliferation marker Ki-67. Pathologic prognostic factors (stage of tumor, local invasion, lymph node metastasis, distance metastasis, and recurrence of the disease) [Table 6].
Table 6: Correlation of pathological prognostic factors (lymph node, local and distance metastasis) of squamous cell carcinoma

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Mutation of p53 gene causes production of the abnormal protein with prolonged half-life resulting in its accumulation in the cell. This abnormal p53 protein can be detected by immunohistochemical staining (IHC) in oral squamous cell carcinomas. The expression is directly related to grading of SCC.[34],[36]

Increase in Ki-67 index shows the genetic instability of tumor cell population and is associated with prognosis of malignant tumors. These proliferation markers are easily detected by silver stain and have better reproducibility.[37],[38]

The study observed that grade I SCCs are low grade malignancies with good prognosis as most of the tumors were at stage 0, I and II. Grades of the tumors were directly related to the stages and up gradation of histological features from differentiation to anaplasia. We also think that these low grade tumors may progress to high grade malignancy if left unchecked and not treated with suitable modalities.

This molecular grading of SCC which is derived from the proliferative marker (Ki-67 index) and cell-cycle suppressive gene p53, divide the SCC into three separate grades because in past it was difficult for making decisions regarding treatment into subgroups of low genomic grade and high genomic grade with outcomes similar to those of low (well differentiated) and high histologic grade (Poorly differentiated), respectively. Now it is believed that this molecular information may change treatment recommendations usually prompting less use of chemotherapy.

Molecular Grade I tumors are low grade SCCs usually without distant metastasis and will need less aggressive therapy as compared to others and will show good response to postoperative radiation due to less genetic mutations and proliferation indies. Five years survival in our patients was 90%.

Molecular Grade II SCCs are intermediate group of diseases. No doubt it is histologically aggressive but has less potential of spread as compared to grade III due to less genetic mutations and proliferation indies. Five years survival in these patients was 70%.

These SCCs are high-grade tumors with more aggressive behavior and up regulation of genetic mutation and proliferation Indies. Majority of these tumors are at higher stages i.e., stage III and IV. We found that as the molecular grading advanced there was advancement in the stages of SCCs. Five years survival in these patients was 26%.This grading system had enough prognostic impact in our study because when it was correlated to pathological prognostic factors like in situ component, size, local invasion, lymph node invasion and distance metastasis, it was found to be significantly related with them. It was found that local invasion, lymph node metastasis and distance metastasis of the SCCs was up regulated with the upstaging of tumors grades. New molecular grading is as good for detecting local invasion, lymph node and distance metastasis. It is worth bearing in mind that p53 immunostaining and Ki-67 staining are less time consuming and less expensive than the other methods in use, like PCR or Microarray etc., but more reliable than histologically prepared H and E stained slides.


  Conclusion Top


SCC is a specific entity of diseases with a special spectrum of clinical, pathologic and molecular features. This new grading of SCC based on histological and immunohistochemical expression modalities shows the impact of diagnosis and prognostic classification of tumors. This technique will help in the prediction of response of individual patients to specific therapeutic regimens, and identification of novel tumor targets that have higher potential of recurrence. This system may help the oncologists to make decisions according to schedule.

The evidence behind this recommendation is that there was no recurrence among 20 of grade I and II patients followed after surgery and postsurgical radiation while 6/10 patients developed recurrence in grade III even receiving radiation and chemotherapy. The interesting feature among three of the patients was delayed wound healing due to neoadjuvant radiation compared with those who did not receive radiation.


  Acknowledgment Top


We are thankful for Dr Byron Elizabeth (MD), Victoria Eldon for editing our manuscript. We are also thankful for Prof Shahid Pervez for their help in doing immunomarkers.


  Authors Contribution Top


Bukhari MH conceived, designed and did statistical analysis & editing of manuscript. KS, SQ, EK, SN & helped manuscript writing. SFI did review and final approval of manuscript and Bukhari MH takes the responsibility and is accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved



 
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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