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Year : 2020  |  Volume : 31  |  Issue : 6  |  Page : 904-910
Pancytokeratin immunostained tumor buds and cytoplasmic pseudofragments are reliable early predictive variables for regional lymph node metastatic risk assessment of oral squamous cell carcinoma

1 Department of Oral Pathology and Microbiology, Faculty of Dental Sciences, M.S. Ramaiah University of Applied Sciences, Bangalore, Karnataka, India
2 Department of Oral and Maxillofacial Surgery, Faculty of Dental Sciences, M.S. Ramaiah University of Applied Sciences, Bangalore, Karnataka, India

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Date of Submission01-Feb-2019
Date of Decision16-Apr-2019
Date of Acceptance20-Oct-2020
Date of Web Publication22-Mar-2021


Context: The 5-year survival rate in patients with cervical lymph node metastasis is reported to be 20–36% post surgery as compared to 63–86% in patients without nodal involvement. This necessitates assessing the metastatic potential of OSCC patients. Objectives: To evaluate the role of Pancytokeratin immunostained tumour buds and cytoplasmic pseudofragments with other histopathological and immunohistochemical variables in predicting metastatic risk of Oral Squamous Cell Carcinoma (OSCC). Settings and Design: Retrospective study on archival tissues of OSCC available from the Department of Oral Pathology and Microbiology. Methodology: Totally, 40 samples of 117 histopathologically diagnosed OSCC samples were selected that displayed metastatic risk variables like invasive tumour front pattern, lymphovascular invasion, tumour buds and cytoplasmic pseudofragmentation and were grouped into 20 each of metastatic and non-metastatic OSCC. 5 normal oral mucosa samples were included in the control group. The 45 tissues were stained with congo red to assess tumour - associated tissue eosinophilia (TATE) and immunohistochemically evaluated for tumour budding and cytoplasmic pseudofragmentation using pancytokeratin, proliferation (Ki-67), microvessel density (MVD)(CD31) and lymphatic vessel density (LVD)( LYVE-1). Statistical Analysis: Pearson's Chi square test and Man Whitney U test were used and analysed by Statistical Package for the Social Sciences (SPSS) software version 20.0. Results: Metastatic OSCC showed significantly high number of tumour buds (p = 0.001), cytoplasmic pseudofragments (p = 0.008), higher tumour grade (p = 0.038), lymphovascular invasion (p = 0.008) and LVD (p = 0.013), aggressive invasive tumour front pattern (p = 0.001) compared to non-metastatic OSCC. Conclusion: Pancytokeratin immunostained tumour buds, cytoplasmic pseudofragments and higher LYVE-1 expression may be used as independent predictors for OSCC metastasis. This study highlights the importance of recognizing the early metastatic risk variables that navigates the surgeon in planning appropriate therapy for OSCC.

Keywords: Carcinoma, immunohistochemistry, lymphatic metastasis, mouth neoplasms, squamous cell

How to cite this article:
Sowmya S V, Rao RS, Prasad K. Pancytokeratin immunostained tumor buds and cytoplasmic pseudofragments are reliable early predictive variables for regional lymph node metastatic risk assessment of oral squamous cell carcinoma. Indian J Dent Res 2020;31:904-10

How to cite this URL:
Sowmya S V, Rao RS, Prasad K. Pancytokeratin immunostained tumor buds and cytoplasmic pseudofragments are reliable early predictive variables for regional lymph node metastatic risk assessment of oral squamous cell carcinoma. Indian J Dent Res [serial online] 2020 [cited 2023 Feb 6];31:904-10. Available from:

   Introduction Top

Oral cancer is third among varied cancer types in India. Early detection of cancer would help to improve the morbidity of the patient.[1]

Metastasis is a dreadful outcome of cancer progression, with 90% mortalities rather than primary tumours.[2] The existing predictive modalities for metastatic behaviour of Oral Squamous Cell Carcinoma (OSCC) by the tumour thickness, histopathology, gene or protein expression is challenging due to its heterogenous clinical presentation. There are instances wherein small primary oral tumours could metastasize to regional lymph nodes, without its clinical or radiographic evidence.[3] Prediction of metastasis in OSCC would navigate the surgeon to avoid inappropriate treatment. Currently, the choice of treatment is dependent on clinical T stage or tumour size, primary tumour site and grade, follow-up compliance or the possibility of occult metastasis.

Various biomarkers for molecular and pathway based mechanisms have been identified for metastasis. The expression of biomarkers may correlate with the histopathological features, clinical staging and the biological behaviour.[4] However, the independent predictive role of pancytokeratin stained tumour budding, cytoplasmic pseudofragmentation with other metastatic risk variables have been least explored in OSCC. Hence, an attempt has been made to assess the role of immunostained tumour buds and cytoplasmic pseudofragments with other variables in predicting metastatic risk of OSCC.

   Methodology Top

A retrospective study initially included 117 histopathologically diagnosed archival OSCC samples from the Department of Oral Pathology and Microbiology, from January 2014 to February 2017. Of these, 40 tissue samples that showed the presence of one or more of the metastatic risk variables like tumour budding, cytoplasmic pseudofragmentation, invasive tumour front pattern and lymphovascular invasion were subjected to histopathological and immunohistochemical evaluation.

The study group included primary OSCC with and without lymph node metastasis showing one or more of the above mentioned risk variables. The control group comprised of normal oral mucosa specimens. Patients with any prior treatment for OSCC and recurrent OSCC samples were excluded from the study.

Histopathological examination

The selected and histopathologically confirmed 40 OSCC tissue samples were further grouped into metastatic (20 samples) and non-metastatic (20 samples) based on positive or negative lymph node involvement confirmed by the case records. The control group included 5 archival tissue specimens of normal oral mucosa. Interpretation was done using a binocular compound light microscope (Olympus, model CX21FS1, Japan).

The 45 samples were evaluated for the variables, invasive tumour front pattern, lymphovascular invasion and tumour grade using Hematoxylin and eosin (H & E) stained slides; tissue eosinophilia using a special stain, Congo red (Puchtler's staining kit -AMD labs, Avistains amyloid staining kit, Bengaluru); and immunohistochemical analysis using pan CK for tumour buds and cytoplasmic pseudofragmentation; Ki-67 for proliferation index, CD31 for MVD and LYVE-1 for LVD.

Immunohistochemical analysis

Totally, 4μm sections of 45 selected tissues each were deparaffinized at 56-60°C for 15 min and transferred to two changes of xylene bath for 5 min. each. This was followed by rehydration with descending grades of ethanol for 5 min each and immersed in 0.3% hydrogen peroxide and methanol for 30 min to abolish endogenous peroxidase activity. The sections were heated in a microwave oven for 20 min to facilitate antigen retrieval. Subsequently, primary monoclonal antibodies, anti- pan-cytokeratin (pan-CK (AE1/AE3), rabbit/mouse, 1:50 dilution; Dako, Denmark), anti-human Ki-67 monoclonal antibody (MIB-1, 1:50; Dako Denmark), anti-CD31 (mouse, 1:100, Dako, Denmark) diluted in 1% bovine serum albumin and phosphate buffered saline (PBS), was added and overnight incubation was done at 4°C in a moist chamber. IHC staining was done with Envision system (HRP based two-step IHC staining method). Breast carcinoma tissues were used as positive controls for IHC staining and negative controls were performed by omitting the primary antibody.

Antirabbit IgG peroxidase-linked secondary antibodies at 1:100 dilutions were used for 60 min at room temperature after PBS wash. The sections were subjected to streptavidin conjugated HRP for 30 min at normal room temperature. The reaction was appreciated using diaminobenzidine tetrahydrochloride (DAB) for 4 min with Tris buffer, counterstained by Meyer's haematoxylin and dehydrated in graded alcohols; excess fluid was drained and mounted in DPX.[4]

Interpretation of Metastatic risk variables

All samples were evaluated using a compound light microscope independently by 3 investigators and the mean scores were considered for statistical analysis.

Invasive tumour front (ITF) was graded from Type 1 to Type 5 according to the criteria given by Brandwein-Gensler et al., 2005, where Type 1: broad pushing front, Type 2: finger-like pushing pattern, Type 3: Islands at tumour periphery greater than 15 cells per island, Type 4: individual tumour cells infiltrating at interface, strands of infiltrating tumour cells, tumour islands composed of 15 cells or less and Type 5: tumour satellites of varying size with ≥1 mm distance from normal tissue at the tumour-host interface.[5]

Lymphovascular invasion was graded as positive or negative based on the presence or absence of tumour cells or nests in the lymphatic or vascular channels.[6] However, tumour grade was assigned based on a descriptive modified Broder's system and graded as well, moderate and poorly differentiated according to the degree of differentiation and keratinization of tumour cells.[7] Each Congo red stained section for eosinophils/TATE was evaluated at the invasive tumour front region in the peri- and intratumoral inflammatory infiltrate. Areas of degenerated muscle and necrosis were excluded. Eosinophils per 10 high power fields (hpf) were noted and the mean score was considered for each case. Further, they were categorised into mild, moderate and marked having eosinophil counts of <25, 25-75 and >75 cells per hpf [Figure 1]d.[8]
Figure 1: Photomicrographs showing strands of infiltrating tumor cells in (a) (arrow) and tumour satellites in (b) (arrow) at the invasive tumour front (H&E, ×100). Lymphovascular invasion is shown in (c) (arrow) (H&E, ×400); Tissue eosinophils with red granules are depicted in (d) (arrow) (Congo red stain, ×400)

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Small clusters of five or more cells/isolated single cells scattered in the stroma beyond the ITF were considered as tumour buds. Ten areas of pancytokeratin immunostained slides were selected wherein budding/sprouting was extensive [Figure 2]a and the number of buds was counted using the 20X objective lens [Figure 2]b. The average score was taken and graded as low grade with <10 buds and high grade with ≥10 buds.[9],[10]
Figure 2: Photomicrographs showing numerous tumour buds at the invasive tumour front demonstrated using Pancytokeratin immunostaining, ×100- (a); ×200- (b); Cytoplasmic pseudofragments × 100- (c); ×200- (d); Ki-67 immunopositive nuclei of cancer cells at a magnification of × 100-(e); ×400- (f); CD31 immunopositive blood vessels (×100)-(g); LYVE-1 immunopositive lymph vessels (×200)- (h)

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Tumour budding areas at the invasive front with 2 μm in diameter, non-nucleated, without nuclear fragmentation, uniform positivity for cytokeratin, smoothly contoured with no inflammation were considered as cytoplasmic pseudofragments and evaluated by pancytokeratin immunostaining [Figure 2]c. Based on the criteria by S. El Gendi et al. (2011), the number of fragments/field under 20X objective were categorized into low (0–9 fragments) and high grades (≥10 fragments) [Figure 2]d.[11]

Tumour cells that showed brown nuclear Ki-67 immunostaining irrespective of their intensity were counted as positive [Figure 2]e. Cells with the absence of brown staining were assessed as negative. The control tissue for staining used was that of breast carcinoma. The intensity of staining was scored as mild for light brown colour, moderate for dark brown and intense for very dark brown colour.[12]

Ki-67 Labelling index (LI) was assessed semi-quantitatively by counting the number of positive cells per 100 cells in OSCC using 40X objective [Figure 2]f.[13]

All the slides were screened using a 4X objective lens to identify the areas that contained the highest number of positively stained vessels (hot spots). Lymphatic vessels and microvessels were counted at the ITF using 20X objective. Any brown-staining endothelial cell or endothelial cell cluster which clearly separates from adjacent microvessels, tumour cells and other connective tissue elements was counted as an individual microvessel. Vessel lumens, although usually present, were not necessary for a structure to be defined as a microvessel. LVD and MVD were defined as the number of lymphatic vessels or microvessels per optical field, respectively.[2],[14],[15] 5 such fields were evaluated and the mean was considered for statistical analysis.

Statistical analysis

Statistical analysis using Statistical Package for the Social Sciences (SPSS) software version 20.0 was done. The variables- tumour buds, cytoplasmic pseudofragmentation, lymphovascular invasion, tumour grade, invasive tumour front pattern and Ki-67 staining intensity were assessed using Pearson's Chi square test. Descriptive statistics using a non-parametric test, Mann Whitney U test, was applied for the variables- Ki-67 labeling index, CD31 immunoexpression for microvessel density and tumour-associated tissue eosinophilia (Congo red). A 'p value' of <0.05 was considered as statistically significant.

   Results Top

The following variables were assessed for the H and E and IHC stained tissue sections of the selected 45 cases.

Applying the criterion of Brandwein-Gensler et al. for assessing ITF[5] to the present study, it was found that 12 cases (60%) of metastatic OSCC showed Type 5 pattern [Figure 1]a & [Figure 1]b whereas absence of such pattern was noted in non- metastatic group. Statistical analysis revealed a significant difference in the invasive tumour front pattern between metastatic and non- metastatic groups of OSCC (p value = 0.001). 11 cases in metastatic OSCC showed presence of lymphovascular invasion in contrast to only 3 cases in the non-metastatic group with a significant P value of 0.008. 14 cases (70%) of non-metastatic OSCC were well differentiated whereas majority of the metastatic OSCC showed moderate [8 cases (40%)] and poor differentiation [6 cases (30%)] with a statistically significant difference (p value- 0.038). However, comparison of TATE between the study groups of OSCC did not reveal statistically significant difference (p = 0.115) [Table 1]. High grade tumour buds of 16 cases were observed in metastatic OSCC as compared to non metastatic (6 cases) with a statistically significant difference between the groups (p value = 0.001). Also, the metastatic OSCC group (20) showed high grade cytoplasmic pseudofragmentation in 11 cases (55%) in contrast to 3 cases (15%) of the non-metastatic group with a significant P value of 0.008 [Table 1].
Table 1: Comparison of histopathological risk variables between metastatic and non- metastatic OSCC groups

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Comparison of Ki-67 immunostaining intensity of OSCC did not reveal significant difference (p = 0.254) whereas the labelling index between metastatic and non-metastatic OSCC groups showed statistically significant difference (p = 0.303). Comparison of MVD using CD31 immunoexpression [Figure 2]g between the study groups of OSCC did not reveal statistically significant difference (p = 0.144) whereas a P value of 0.013 was obtained between metastatic and non-metastatic OSCC groups on immunostaining with LYVE-1 for the lymph vessels [Figure 2]h, [Table 2].
Table 2: Comparison of immunostaining using Ki-67, CD31 and LYVE-1 between metastatic and non- metastatic OSCC groups

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   Discussion Top

The present study assessed some of the histopathological and immunohistochemical risk variables for metastasis. Invasive tumour front (ITF) is the most advanced region of the tumour that presents as three to six cell layers or detached tumour cell groups of the OSCCs.[5] The ITF usually comprises of less differentiated and highly dissociated population of tumour cells. It provides important information about the tumour's invasive and metastatic potential as it is composed of the most aggressive cells.[16],[17] The greater presence of Type 4 and 5 patterns in metastatic OSCC in the current study are in accordance with the findings of M Akhter et al. who also correlated these patterns with regional metastasis.[18] Margaret Brandwein-Gensler et al. (2005) also observed Type 4 & 5 patterns to be associated with poor overall survival and local recurrence.[5]

Lymphovascular invasion is defined as the presence of tumour cells or nests in the lymphatic or vascular channels [Figure 1]c. It has been associated with the aggressive tumour behaviour and metastasis in malignancies. The number of OSCC cases showing lymphovascular invasion was greater in metastatic group. Similar results were observed by Adel et al. who evaluated the associations between lymphatic and vascular invasion, clinicopathological manifestations and their impact on OSCC patient outcomes after treatment. They found significant associations between lymphatic and vascular invasion, overall stage, nodal metastasis and pathologic differentiation in pre-surgical patients.[6]

There was greater association of moderate and poor grades of OSCC with the metastatic group as compared to non-metastatic in the present study which was similar to the findings of M. Akter et al. where significant association of Anneroth's (p = 0.002), Broder's grading (p = 0.012) and metastasis of OSCC was appreciated.[18]

Tumour-associated tissue eosinophilia (TATE) is described as 'eosinophilic stromal infiltration of a tumour not associated with tumour necrosis or ulceration'. Megha Jain et al. (2014) showed significantly raised eosinophil counts in non-metastatic in comparison to metastatic group of OSCC and concluded that eosinophilia is a favourable histopathological prognostic factor in OSCC.[8] However, the present study showed contrasting results for TATE. It has been suggested that the role of eosinophils in OSCC remains controversial with both immunoprotective and tumour promoting effects favouring metastasis thereby influencing the prognosis of a tumour.[19]

The presence of high grade tumour buds in significant numbers in the metastatic group is in concordance with the study of Almangush et al. 2014, with ≥5 clusters of tumour buds at the invasive front and ≥4 mm of invasion depth associated with poor prognosis in early OSCC of tongue.[20] Okado et al. assessed the prognostic significance of tumour budding and Ln5-γ2 expression immunohistochemically and correlated the findings with clinicopathological parameters in SCC of the external auditory canal (EAC). They found shorter survival times and poor prognosis of patients whose tumours had high budding grade and Ln5-γ2 expression and concluded that assessment of tumour buds served as prognostic predictors.[21]

Cytoplasmic pseudofragments showed significantly high grades in metastatic OSCC compared to the non-metastatic group. S. El-Gendi et al., studied the tumour margin and determined the degree of tumour budding (TB), cytoplasmic pseudofragmentation using β- catenin immunoexpression in colorectal carcinoma. They found a significant relation between TB and cytoplasmic pseudofragments. Also, they emphasised the use of pancytokeratin to demonstrate tumour buds and cytoplasmic pseudofragments that are caused by dysregulation of EMT through Wnt pathway, suggestive of tumour aggressiveness.[11]

Ki-67/MIB-1 is a marker for cellular proliferation, expressed in all phases of cell cycle except G0 and early G1 phase of cell cycle. It is a nuclear protein that correlates with survival, metastasis and prognosis of OSCC. The present study results are in favour of the research of Barberet al.[22] Malignant growth in tumour cells is caused by only a small subgroup of proliferative cells and Ki-67 reflects the total fraction of tumour cells in proliferation including those that undergo terminal differentiation. Therefore, they do not develop phenotypic characteristics that worsen the prognosis.[23],[24] However, contrasting results were observed by H. Myoung et al. (2006), wherein a high Ki-67 immunoreactivity was observed in patients with neck metastasis in comparison to non-metastatic group and concluded that Ki-67 could act as a promising prognostic marker in OSCC patients.[25]

A tumour growth beyond 1–2 mm3 requires not only proliferation but must be capable of inducing new capillary vasculature growth from the host. It is believed that increased angiogenesis facilitates greater propensity for invasion and metastases. Quantification of angiogenesis in tissues can be made by counting microvessels in a particular area of the IHC stained slides. CD31/PECAM-1 (Platelet endothelial cell adhesion molecule) is expressed by endothelial cells of blood vessels and is concentrated on the cell membrane and at the junctions between adjacent cells. The results of MVD in the present study was similar to that of Monteiro-Amado et al., 2013 and T. N. Suresh et al., 2015. The negative correlation between MVD and metastasis may be attributed to difference in vascularisation at different sites of the oral cavity.[26],[27] Studies with lesions from the same oral site need to be evaluated for ascertaining the results.

Lymphangiogenesis is the formation of new lymphatic vessels in OSCC that would enhance tumour growth by removing waste products of metabolism and promoting metastasis of tumour cells into lymph nodes. Lymphangiogenesis was assessed using a lymphatic specific marker; LYVE-1 (Lymphatic vessel endothelial hyaluronan receptor-1) which is a surface endocytic receptor for hyaluronan, that shares 41% homology with CD44 – a metastasis related molecule. Hyaluronan is an extracellular glycosaminoglycan involved in cell adhesion and migration. The metabolism of hyaluronan (>80%) is regulated by the lymphatic system. The uptake and transport of hyaluronan in the lymph is controlled by LYVE-1. The preferential tendency of tumours for regional lymph node metastasis may be attributed to lymphangiogenesis.[28] LVD of the present study is in accordance with the study of Shunichi Yoshida et al., 2006. LYVE-1 positivity of the lymph vessel endothelial cells is due to differentiation of lymph vessels. The capacity of cancer cells to invade lymph vessels and promote lymph node metastasis increases in lymphangiogenesis.[29]

To the best of my knowledge, the assessment of pancytokeratin immunostained tumour buds and cytoplasmic pseudofragments have been least employed for predicting the metastatic risk in OSCC. These variables could serve as simple and cost-effective early predictors of metastasis in OSCC. The observations of the present study may provide baseline data for further research in this field wherein histopathological examination facilitates the prediction potential of metastatic OSCC cases.

Subjective assessment employed to assess the risk variables is the limitation of the present study. Further studies may be carried out using larger sample size and computer- aided software to predict the metastatic potential of OSCC.

   Conclusion Top

Assessment of metastatic risk variables has been challenging in oral squamous cell carcinoma. Although various histopathological and immunohistochemical risk variables have been employed till date, their application as independent assessment tools remains controversial. In the present study, two of the recently identified histopathological variables namely tumour buds and cytoplasmic pseudofragments that were assessed using pan cytokeratin immunostaining have shown favourable results in OSCC for the distinction of metastatic and non- metastatic cases. The other histopathological variables like pattern of invasive tumour front, tumour grade and lymphovascular invasion have also shown significant differences between the study groups suggesting that these parameters may be used as individual variables for predicting the metastatic risk of OSCC. Also, the immunohistochemical evaluation of lymphatic vessel density using LYVE-1 could be a reliable marker for differentiating metastatic from non-metastatic OSCC.

Therefore, this study highlights the importance of recognising the risk variables to help navigate the surgeon in the early prediction of metastatic OSCC cases and plan appropriate therapy.

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Conflicts of interest

There are no conflicts of interest.

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Correspondence Address:
Dr. S V Sowmya
Department of Oral Pathology and Microbiology, Faculty of Dental Sciences, M.S. Ramaiah University of Applied Sciences, Gnanagangothri Campus, New BEL Road, M S R Nagar, Bangalore - 560 054, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijdr.IJDR_101_19

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