|Year : 2018 | Volume
| Issue : 2 | Page : 361-367
Morphometric computer-assisted image analysis of epithelial cells in different grades of oral squamous cell carcinoma
Chatterjee Ananjan1, Mahadesh Jyothi2, BL Laxmidevi2, Pillai Arun Gopinathan3, Salroo Humaira Nazir2, L Pradeep2
1 Department of Oral and Maxillofacial Pathology, Buddha Institute of Dental Sciences, Patna, Bihar, India
2 Department of Oral and Maxillofacial Pathology, Sri Siddhartha Dental College and Hospital, Sri Siddhartha Academy of Higher Education, Tumkur, Karnataka, India
3 Department of Oral and Maxillofacial Pathology, Sri Sankara Dental College, Varkala, Kerala, India
|Date of Web Publication||8-Mar-2018|
Dr. Pillai Arun Gopinathan
Department of Oral and Maxillofacial Pathology, Sri Sankara Dental College, Akathumuri, Varkala, Kerala
Source of Support: None, Conflict of Interest: None
Introduction: Oral squamous cell carcinoma (OSCC) accounts 94% of all malignant lesions in the oral cavity. In the assessment of OSCC, nowadays the WHO grading system has been followed widely but due to its subjectivity, investigators applied the sophisticated technique of computer-assisted image analysis in the grading of carcinoma in larynx, lungs, esophagus, and cervix to make it more objective.
Aims and Objectives: Access, analyze, and compare the cellular area (CA); cytoplasmic area (Cyt A); nuclear area (NA); nuclear perimeter (NP); nuclear form factor (NF); and nuclear-cytoplasmic ratio (N/C) of the cells in different grades of OSCC.
Materials and Methods: Fifty OSCC cases were obtained and stained with hematoxylin and eosin which were graded according to the WHO classification. The sections were subjected to morphometric analysis to analyze all the morphometric parameters in different grades of OSCC and subjected to one-way ANOVA statistical analysis.
Results: CA and Cyt A decreased from normal mucosa with dedifferentiation of OSCC. The NA and NP increased in carcinoma group when compared to normal mucosa but decreased with dedifferentiation of OSCC (P < 0.05). NF had no significance with normal mucosa and different grades of OSCC (P > 0.05), while N/C ratio increased from normal mucosa through increasing grades of OSCC, reaching the highest value in poorly differentiated squamous cell carcinoma (P < 0.05).
Conclusion: Both cellular and nuclear variables provide a more accurate indication of tumor aggressiveness than any single parameter. Morphometric analysis can be a reliable tool to determine objectively the degree of malignancy at the invasive tumor front.
Keywords: Invasive tumor front, morphometric analysis, oral squamous cell carcinoma
|How to cite this article:|
Ananjan C, Jyothi M, Laxmidevi B L, Gopinathan PA, Nazir SH, Pradeep L. Morphometric computer-assisted image analysis of epithelial cells in different grades of oral squamous cell carcinoma. J Can Res Ther 2018;14:361-7
|How to cite this URL:|
Ananjan C, Jyothi M, Laxmidevi B L, Gopinathan PA, Nazir SH, Pradeep L. Morphometric computer-assisted image analysis of epithelial cells in different grades of oral squamous cell carcinoma. J Can Res Ther [serial online] 2018 [cited 2019 Nov 22];14:361-7. Available from: http://www.cancerjournal.net/text.asp?2018/14/2/361/189423
| > Introduction|| |
The oral cavity is the preferable place in the head and neck region for the primary malignant tumor to manifest and oral squamous cell carcinoma (OSCC) accounts approximately 94% of all malignant lesions in the oral cavity.,, In the assessment of OSCC, histological grading based on Broder's classification was followed earlier.,, Since this grading system was subjective in nature and lacks consensus regarding its prognostic value, many other grading systems had been put forth. Bryne suggested that molecular and morphological characteristics at the invasive front area of various SCC may reflect tumor prognosis better than other parts of the tumor., The WHO grades OSCC by the assessment of the degree of keratinization, cellular and nuclear pleomorphism, and mitotic activity, and is followed widely.
To create the grading system more objective, sophisticated technique of computer-assisted morphometry was applied to investigate the cellular and the nuclear changes in correlation with the histological behavior of the lesions., The results have been more reliable, objective, and reproducible. It also may help to give a rapid and reliable diagnosis which has been applied with considerable success in pulmonary neuroendocrine neoplasms as shown by Marchevsky et al.
Aims and objectives
- To assess and analyze the cellular area (CA), cytoplasmic area (Cyt A), nuclear area (NA), nuclear perimeter (NP), and nuclear form factor (NF) in different grades of OSCC
- To assess and analyze the nuclear-cytoplasmic ratio (N/C) of the cells in different grades of OSCC
- To compare the CA, Cyt A, NA, NP, NF, and N/C in different grades of OSCC.
| > Materials and Methods|| |
The study was conducted on tissue sections which were obtained from the biopsy tissue specimens retrieved from the archives of the department. The study group comprised fifty various grades of differentiation based on the WHO classification of OSCC.
Fifty cases of OSCC were grouped as:
- Group 1: Twenty cases of well-differentiated squamous cell carcinoma (WDSCC)
- Group 2: Twenty cases of moderately differentiated squamous cell carcinoma (MDSCC)
- Group 3: Ten cases of poorly differentiated squamous cell carcinoma (PDSCC).
The control group comprised ten cases of normal oral mucosa from the healthy adult individuals which were obtained during minor oral surgical procedures.
- Incisional biopsy specimen only were included in the study
- Cases with the deepest invasive area with its invasive front were considered as that area shows the maximum amount of dysplastic features.
- No premalignant lesions and conditions were included in the study to reduce the bias between the premalignant and malignant conditions
- Metastatic lesions were not included in the study.
Sections of 4 microns thickness were stained with freshly prepared Harris hematoxylin and eosin (H and E). The stained sections were observed under a research microscope [Figure 1]: WDSCC, [Figure 2]: MDSCC, and [Figure 3]: PDSCC] and graded using the WHO grading system only.,
|Figure 1: H and E stained sections of well-differentiated squamous cell carcinoma without morphometric analysis|
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|Figure 2: H and E stained sections of moderately differentiated squamous cell carcinoma without morphometric analysis|
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|Figure 3: H and E stained sections of poorly differentiated squamous cell carcinoma without morphometric analysis|
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The sections were subjected to the morphometric image analysis system. The system comprised a microcomputer, a digitizer tablet, a drawing pen (stylus), and a video camera (CCD) attached to a light microscope (OLYMPUS BX40).
The measurements were made by moving the stylus (drawing pen) around the outline of each dysplastic cells, appearing on the monitor. Once the stylus completes the outline of the cell, the Jenoptik Speed XT Core 3 with Capture Pro Software (Jenoptik, Germany) automatically measured the cellular perimeter and CA. The NP and NA were also measured in the similar way.
A magnification of × 400 was used for the measurements [Figure 4]: WDCC, [Figure 5]: MDSCC, and [Figure 6]: PDSCC]. After the measurements, the image was captured using CCD camera which is attached to the trinocular research microscope so that it could be documented with both premeasured and postmeasured photomicrographs in case if there were any confusion in the statistical analysis.
|Figure 4: H and E stained sections of well-differentiated squamous cell carcinoma with morphometric analysis|
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|Figure 5: H and E stained sections of moderately differentiated squamous cell carcinoma with morphometric analysis|
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|Figure 6: H and E stained sections of poorly differentiated squamous cell carcinoma with morphometric analysis|
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- Four fields of sections were selected from the invasive front areas of all the grades of OSCC randomly
- An attempt was made to divide the cell population into large and small cells and then take the average measurements to increase the appropriateness of the study
- For each section, two large cells and two small cells and nuclei with clear, identifiable outline in each compartment is selected avoiding all the overlapping cells
- Histologically identifiable nonkeratinocytes such as melanocytes showing clear cell change and inflammatory cells, as well as cells showing degenerative changes and those undergoing mitosis were not measured
- For carrying out morphometric analysis of the normal mucosa, basal and parabasal layers were considered as these layers have least differentiated oral epithelial cells
- The actual measurements of the morphometric parameters were done by Image Analyzer Software ProgRes SpeedXT Core 3 (JENOPTIK optical system GmbH, Germany) after accurate calibrations are done using the stage micrometer
- The size of the cell and its nucleus is measured with the area and the perimeter
- Two large and two small cells were selected with a clear outline and which were not overlapping.
The CA, NA, and NP and other morphometric parameters such as the N/C and NF were calculated by the following formula:
- N/C ratio = N area/(C area − N area)
- NF = 4πNA/NP2 where π =22/7
All measurements were in microns (calibrations 1630 pixels = 140 μm, 1 μm = 0.001 mm) and was saved in the Microsoft Excel for further statistical analysis.
| > Results|| |
Fifty cases of different grades of OSCC along with ten normal tissues were stained with H and E, and all the obtained values were subjected to one-way ANOVA statistical analysis.
The CA of normal mucosa was highest, and it significantly decreased (P< 0.05), with dedifferentiation of OSCC [Table 1]. The Cyt A decreased gradually from WDSCC to MDSCC but drastically in PDSCC with an overall significant P < 0.05 [Table 2]. The mean NA of various grades of OSCC is more than twice the mean NA of the normal cells which is in contrast to cellular and cytoplasmic parameters. It was also seen that in WDSCC had the largest mean NA followed by MDSCC and PDSCC, respectively, with an overall significant P < 0.05 [Table 3].
In OSCC, the mean NP was found to be about 30 μm with not much deviation of values seen within the different grades with a statistical significance of P < 0.05 [Table 4]. Hence, it is analyzed that the mean NP of all grades of OSCC was about 1.5 times the value obtained from the normal cells.
With the obtained N P value, NF was also calculated from the formula (4πNA/NP2) and this parameter had no significance (P > 0.05) with the various grades of OSCC, as well as when compared with the normal tissue [Table 5]. The mean N/C ratio, when compared with various grades of OSCC, was found to be increased in carcinoma groups when compared to normal mucosa, and these values also increased significantly with increasing grades of OSCC [Table 6]. The N/C ratio of PDSCC has the highest value, which is 3.3 times the N/C ratio of normal cells, whereas MDSCC and WDSCC are about 2.7 times the N/C ratio of normal cells. The variation of the value for N/C ratio of WDSCC and MDSCC was not much, but it raised considerably in cases of PDSCC.
| > Discussion|| |
OSCC is the most common malignant neoplasm arising from the mucosal epithelium of the oral cavity,, and grading is of great importance as it has a direct correlation with the prognostic value. All grading systems that have been put forth have many pitfalls as grading scores differ largely due to interobserver variability. To make it more objective, many investigators have applied a sophisticated technique such as computer-assisted image analysis in the grading of carcinoma in larynx, lungs, esophagus, bladder, cervix, and many more.,,,,,,,,,, Studies in oral cavity have also been done using image analysis starting in potentially malignant disorders such as leukoplakia, lichen planus, oral submucous fibrosis, and epithelial dysplasias.,,,,,,,
Very few studies have applied the image analysis on different grades of OSCC to analyze the cellular and nuclear features in the invasive tumor front as it is of major significance for the prognosis of oral cancer providing better understanding of the mechanisms involved in tissue structure organization and cellular interplay at this biological “hot zone” of the tumors.,,,,
It was observed that there was a considerable amount of variation in the NAs in previous researches; therefore, in this study, it was an effort to consider two large and two small cells in every field of all cases of OSCC to get the overall mean measurements of the epithelial cells to improve the reproducibility of the study.
H and E stain is the most popular routine stain used as the gold standard in the field of histopathology by pathologists for medical diagnosis; hence, we used the stain in all the cases as the focus was on the cellular, cytoplasmic, and nuclear morphology rather than only nuclear contents.
In the present study, the WHO classification, was used to grade SCC into well, moderate, and poorly differentiated. Later the cellular, cytoplasmic and nuclear changes of various grades of OSCC were analyzed morphometrically at the invasive tumor front.
The mean CA in normal mucosa was the highest, and it decreased considerably in carcinoma group. With the dedifferentiation of OSCC, the mean CA reduced from 161.07 ± 29.48 to 127.03 ± 31.40 μm2 and MDSCC was found to be within these two ranges. Similar findings were obtained by Hande and Chaudhary, where it was put forth that normal mucosa had the maximum cellular size, but as cells became actively proliferating to trigger carcinoma, the cellular size considerably decreased with increasing grades of OSCC. Cowpe suggested that tissues undergoing malignant transformation show a reduction in the CA before a reduction in the NA.
The Cyt A for normal mucosa was maximum, and it started decreasing significantly with dedifferentiation of SCC. The difference in Cyt A for WDSCC and MODSCC was not obvious, but there was a radical reduction of Cyt A in the cases of PDSCC. Similar findings were observed with Hegde's cytomorphometric study, wherein there was a significant reduction in the mean nuclear and cytoplasmic diameters in the cases of OSCC. Hegde et al. suggested that there was the greatest reduction in the mean Cyt A of squames in SCC with histologic evidence of dysplasia. Thus, the findings can be supported by the statement that the major changes involved in normal cell maturation in oral epithelium are the changes in cell size and shape which may be due to the synthesis of more structural protein in the form of tonofilaments, the appearance of new organelles, and production of additional intercellular material.
Therefore, we can hypothesize that as the cells of carcinoma get dedifferentiated, they also synthesize equally less tonofilaments, organelles, and intercellular material. This further has a direct bearing on the size of the cell, as well as their cohesive properties. Another possible reason for the reduction of CA in increasing grades of OSCC could be due to loss of ability of cells to adhere to each other due to downregulation of E-cadherin expression in carcinomas leading to loss of cellular size and shape., Therefore, in this histomorphological study, we can say that CA and Cyt A are found to be inversely proportional to the increasing grades of OSCC.
When the NA was analyzed in normal mucosa (34.39 ± 3.41 μm2), it was half of the NA in the carcinoma group which ranged from 62 to 88 μm2. Analyzing the NA in different grades of OSCC, NA decreased with the dedifferentiation of OSCC. It was observed that NA increased considerably in the carcinoma group when compared to normal mucosa, but decreased within the grades of OSCC. As the NA decreased, it was quite obvious that even the NP should decrease with increasing grades of OSCC and the exact result was achieved in our study but not much of numerical deviation was observed between the various grades, but whatever difference was obtained, it was found to be statistically significant.
Our observations were in agreement with findings of Laitakari et al., who observed that nuclear size decreased with decreasing degree of differentiation and Sunitha et al. in the area of invasive tumor front also observed that the mean NA increased in descending order of histological grades of OSCC. Kinoshita et al. mentioned that increase in nuclear size in carcinomatous tissue is because of the fact as the cells proliferate in carcinogenesis, they possess more biological activity comprising increase in chromosomes related to abnormal division, increase of nuclear DNA content which in turn increases the size of the nucleus.
In the present study, when considering the various grades of OSCC, NA decreased significantly with increasing grades, which was in contradiction to various authors. Therefore, we hypothesize that this finding may be due to the fact that high-grade tumors show more mitotic activity, abnormal mitotic figures, less potential for DNA repair thereby increased DNA damage which ultimately leads to irregular nuclear shape thus finally decrease in nuclear size.
NF was assessed as described by Nandini and Subramanyam, and it was suggested that a perfect circle has a form factor of 1.0 and elliptical structures deviate considerably from unity toward zero as their degree of circularity becomes less perfect. Based on this concept, it was found that NF was less in the carcinoma group than normal mucosa suggesting that irregularity or loss of roundness prevails in the carcinoma group. Although NF decreased, there was no statistically significant relationship between normal and carcinoma group or within different grades of OSCC which was in accordance with Nandini and Subramanyam. Tan et al. and Sunitha et al. suggested that the nuclear shape, nuclear roundness, and ellipticity were not an important parameter to differentiate grades of SCC which could be due to inherent problems with tracing irregular nuclear contours of malignant cells for image analysis.
The N/C ratio was a contrasting parameter with respect to CA, Cyt A, and NA as this parameter was directly proportional to the increasing grades of OSCC. The scrutiny regarding N/C ratio fell in with the observations by several investigators as all of them proposed that N/C ratio increases in the carcinoma group when compared to normal mucosa and when various grades of SCC was taken into consideration, N/C ratio increased with dedifferentiation.,,,,,,
It could be hypothesized that the reduction in mean CA and mean Cyt A of the epithelial cells of OSCC is much more than the reduction in the mean NA of the respective cells.
From the overall findings, we could infer that there was not much of numerical deviation observed between the WDSCC and MDSCC cases in all the parameters, but PDSCC showed marked variation within the grades. Therefore, it might be tricky for one to differentiate between WDSCC and MDSCC by morphometric technique, but to differentiate PDSCC from lower grades becomes unmistakable.
There are several investigators who have done extensive researches on nuclear morphometry to assess and analyze only the nuclear features in various tissues of different grades of SCC. However, in the present study, along with the nuclear features such as NA and NP, even the CA and Cyt A seem important parameters that have to be considered while grading the carcinoma to increase the objectivity for better prognostic value.
| > Conclusion|| |
The combination of several cellular and nuclear variables provides a more accurate indication of tumor aggressiveness and behavior rather than any single parameter. A truly prospective study on large series of carcinoma patients is required to determine the practical use and validity of this objective system. In the present study, it was found that the morphology of all the cellular and nuclear features reflect cell biologic behavior and general activity.
The results of the research show a significant reduction in CA, cytoplasmic area, NA, and NP with increasing grades of OSCC, while the N/C ratio was increasing with the increasing grades of OSCC. NF had no significant relationship in various grades of OSCC.
This indicates that a combination of several cellular and nuclear variables provide a more accurate indication of tumor aggressiveness and behavior rather than any single parameter. Hence, the computer-assisted histomorphometric techniques can be used as a reliable tool to determine objectively the degree of malignancy of oral tissues at the invasive tumor front.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]