J Oral Pathol Med (2014) 43: 454–458 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

doi: 10.1111/jop.12157

wileyonlinelibrary.com/journal/jop

BRIEF REPORT

Neutrophil-tumor cell cannibalism in oral squamous cell carcinoma Sachin C. Sarode1,2, Gargi S. Sarode1 1

Department of Oral Pathology and Microbiology, Dr. D.Y. Patil Dental College and Hospital, Dr. D.Y. Patil Vidyapeeth, Pune, Maharashtra, India; 2Fulbright Doctoral and Research Fellow, The Ohio State University, College of Dentistry, Oral Pathology, Columbus, OH, USA

BACKGROUND: Cannibalism was recognized as a phenomenon seen mainly with the tumor cells ingesting other tumor cells. Recent reports have shown tumor cell engulfing other cells (xeno-cannibalism) as well, such as neutrophils, lymphocytes and erythrocytes. But no such finding has been reported in oral squamous cell carcinoma (OSCC) in the literature till date. OBJECTIVE: Retrospective histopathological analysis of OSCC for identification of neutrophil-tumor cell cannibalism (NTCC) and its correlation with clinico-pathological parameters. METHODS: The hematoxylin and eosin stained tissue sections of 500 OSCC cases were thoroughly screened at high power magnification (400X) for NTCC. Cases showing only frank NTCC were selected. Cases were subjected to immunohistochemical analysis using CD68 and lysozyme. RESULTS: Seven (1.4%) cases of OSCC which showed classical features of extreme NTCC on histopathological examination. Seventeen Cases (3.4%) showing occasional isolated NTCC were excluded. All the cases were poorly differentiated and showed cervical lymph node metastasis. Immunohistochemical analysis showed mild (+) to moderate (++) positivity in tumor cells for CD68 and lysozyme markers. CONCLUSION: NTCC in OSCC can predict the biological behavior and could serve as a useful prognostic marker in future. Tumor cell displaying macrophage phenotype and cell digestion could be mediated through lysosomal enzyme activity. J Oral Pathol Med (2014) 43: 454–458 Keywords: cell cannibalism; neutrophil tumor cell cannibalism; oral cancer; oral squamous cell carcinoma; xeno-cannibalism

Correspondence: Sachin C. Sarode, Department of Oral Pathology and Microbiology, Dr. D.Y. Patil Dental College and Hospital, Dr. D.Y. Patil Vidyapeeth, Sant-Tukaram Nagar, Pimpri, Pune – 18, Maharashtra, India. Tel: +91 99 22491465, Fax: +91 20 27423427, E-mail: [email protected] Accepted for publication December 10, 2013

Introduction Cellular cannibalism, defined as a large cell enclosing a slightly smaller one within its cytoplasm is a characteristic morphologic feature exclusively seen in aggressive malignancies. The earliest cell-in-cell phenomenon was described in tumor cells by Steinhaus in 1891 (1). The engulfed cell still remains alive when internalized, but the process implies its death. In this sense, this differs from several other types of cell engulfment like emperipolesis, phagocytosis, autophagy and entosis. Cellular cannibalism has been described previously in breast carcinoma (2), giant cell carcinoma of lung (3), gall bladder carcinoma, endometrial stromal carcinoma (4), malignant thymoma (5), malignant melanoma (6) etc. and is correlated well with the aggressiveness, degree of anaplasia, invasiveness and metastatic potential of the malignancy. Demonstration of an increased number of cannibalistic cells may also be helpful in grading breast carcinoma (2). In a recent study, Sarode GS et al. (7) are the first to report this phenomenon along with the concept of ‘complex cannibalism’ in oral squamous cell carcinoma (OSCC). Although cannibalism is used to differentiate benign tumors from malignant, recently it has been demonstrated in benign tumors like giant cell tumor of tendon sheath of localized type (8) and central and peripheral giant cell granuloma of oral cavity (9). Until recently, cannibalism was recognized as a phenomenon seen mainly with tumor cells ingesting other tumor cells. Recent reports have shown tumor cell engulfing other cells (xeno-cannibalism) as well, such as neutrophils, lymphocytes and erythrocytes (6, 10–17). But no such finding was reported in OSCC in the literature till date. With this view in mind, 500 cases of OSCC were retrospectively analyzed for the presence of neutrophil-tumor cell cannibalism (NTCC).

Materials and methods From the files of the Department of Oral Pathology and Microbiolgy, Dr. D.Y. Patil Dental College and Hospital, Pune (India), 500 cases of OSCC received between the year

++ Moderate Present 4 Individual cells Poor IVa M0 N2b (Present) T2 Exophytic

30/M

45/M

38/M

5

6

7

Gingivo-buccal complex

Poor IVa M0 N2b (Present) T2 Endophytic

68/M 4

Floor of mouth

Poor IVa M0 N2a (Abesent) T2 Endophytic

42/F 3

Tongue

4

++

Regional recurrence (1 year) Dead (1 year) + 3

Present

Mild

3

+

Follow up lost + 3

Present

Severe

6

+

Follow up lost + Moderate Present 4

Individual cell Individual cells Individual cells Poor IVa M0 N2a (Present) T2 Exophytic

Poor IVa M0 N2a (Present) T2 Exophytic

37/F 2

Buccal mucosa & floor of mouth Buccal mucosa

Poor IVa M0 N2b (Absent) T3 Endophytic

5

+

Dead (6 month) + + 8

Present

Moderate

5

++ ++ 6

Present

Moderate

8

++ ++ 60/M 1

Gingivo-buccal complex Tongue

Endophytic

T2

N2a (Present)

M0

IVa

Poor

Individual cells Individual cells Individual cells

5

Present

Severe

6

Lysosyme CD68 NTCC/HPF Tissue neutrophilia Abnormal mitosis Mitoses/HPF Invasion Pattern Grade Stage M T Site

TNM staging

N (Extracapsular invasion) Clinical presentation Age/ Sex

Majority of the cases were moderately differentiated (280; 56%), followed by well differentiated (180; 36%) and poorly differentiated (40; 8%). We came across 7 (1.4%) cases of OSCC which showed classical features of extreme NTCC on histopathological examination. Seventeen cases (23.4%) showing occasional isolated NTCC were excluded. The clinico-pathological data of seven cases is shown in Table 1. Since all the patients were from a rural and low socio-economic background with lack of awareness about OSCC, they presented to the OPD late or at an advanced stage of OSCC (patient related diagnostic delay = appraisal delay + illness delay + utilization delay; 18). One patient was presented with a second primary tumor (buccal mucosa and floor of mouth) which was diagnosed on the basis of the established criteria (19). Histopathological assessment of all the cases showed features of poorly differentiated OSCC. Malignant cells infiltrated the underlying connective tissue stroma in the form of individual cells with extreme cellular and nuclear pleomorphism. Abnormal mitoses were present in all the cases. Neutrophils were abundant and could be identified both within the malignant epithelial cells as well as in the stroma adjacent to the tumor cells. We observed both partial and complete neutrophil engulfment by tumor cells in all the cases. At many places multiple neutrophil engulfments by tumor cells was observed (Fig. 1). The internalized neutrophils showed different stages of degeneration. The neutrophils showed striking tumoral-centric distribution, decreasing in number away from the tumor cells. The maximum 8 and minimum 3 NTCC per high power field were observed with a mean value of 5.2. All patients underwent complete surgical excision of the tumor with radical neck dissection followed by postoperative radiotherapy over 50 Gy. In all the cases, the specimens showed

455

Sr. No.

Results

Table 1 Clinico-pathological characteristics of oral squamous cell carcinoma showing neutrophil-tumor cell cannibalism (NTCC)

Immunohistochemistry For the immunohistochemical study, 3-lm sections were obtained from the formalin-fixed paraffin-embedded surgical specimens of the lesions. Immunohistochemical staining was carried out using the streptavidin-biotin-peroxidase complex method. The following antigens were evaluated: CD68 (macrophage-associated monoclonal antibody, KP-1 clone, 1:50; BioGenex, Fremont, CA, USA), incubated at room temperature for 1 h; lysozyme (Rabbit polyclonal antibody; BioGenex), incubated at room temperature for 30 min. For CD68 the tissue sections were previously treated with citric acid in three cycles for 5 min and no pretreatment was recommended for lysozyme. Diaminobenzidine was used as chromogen. Lymph node was used as positive control for CD68 and lysozyme.

Prognosis

2000 and July 2013 were reviewed. All the hematoxylin and eosin stained tissue sections were thoroughly screened at high power magnification (400X) for NTCC. Only cases showing frank NTCC were selected. Quantification of NTCC was done by selecting four hot spots showing maximum density of this phenomenon. Tumor cells showing both partial and complete NTCC were also considered for quantification.

Local recurrence (4 month) Follow up lost

Neutrophil-tumor cell cannibalism SC Sarode and GS Sarode

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Figure 1 Photomicrographs showing neutrophil-tumor cell cannibalism. Single as well as multiple neutrophil cannibalism by tumor cells can be appreciated. (White arrow; HE stain, Magnification 400x).

clear surgical margins but the mucosal margins displayed moderate epithelial dysplasia. Two patients died after 6 month and 1 year, each showing local and regional recurrence after 4 months and 1 year respectively. Immunohistochemistry Immunohistochemical analysis showed mild (4) to moderate (3) cytoplasmic positivity for CD68 and lysozyme markers (Figs 2 and 3). The positivity was present only at the focal areas and not all the cells displayed positivity for CD68 and lysozyme.

Discussion The largest published series till date reported 11 fine needle aspiration cases with tumor cells showing NTCC (12). This series included carcinomas arising from the gallbladder (anaplastic carcinomas), small intestine (adenocarcinoma), pancreas (adenocarcinoma), breast (infiltrating duct carcinoma), larynx (squamous cell carcinoma), lung (small cell carcinoma), and disseminated tumor of unknown origin (anaplastic carcinoma). Two cases in this series were nonHodgkin lymphomas. The phenomenon of NTCC has also been documented by the light microscopy in pleomorphic (giant) cell carcinomas of lung, gall bladder, pancreas, and intestine (13–17). Caruso et al. (11) reported NTCC in two out of nine cases of advanced gastric carcinoma. Electron microscopic features showed the characteristic equipment of discrete primary and secondary granules, glycogen and lipid bodies. Neutrophils were present within vacuoles of adenocarcinoma cells and showed various phases of apoptotic changes. In OSCC, there are several well-recognized histological prognosticators like grade of the tumor, histological subtyping/categorization, tumor border being either infiltrative or more pushing, perineural invasion, lymphovascular invasion, bone involvement, sialoadenotropism/ductal invasion etc (20). Recently, cannibalism in OSCC has been proposed as an important histopathological prognosticator (7). But, till date NTCC has never been reported in OSCC. J Oral Pathol Med

Figure 2 Photomicrograph showing moderate cytoplasmic expression of CD 68 by tumor cells. (Magnification 400x).

In the present case series, we excluded the cases showing occasional NTCC for better understanding of OSCC showing prominent NTCC. Out of 500 cases, 40 (8%) were poorly differentiated and 7 (17.5% of poorly differentiated) displayed prominent NTCC. In all the seven cases, tumor cells were separated from each other as well as from the surrounding connective tissue stroma. The stroma was characterized by fewer blood vessels and displayed degenerative changes at focal areas (three cases). These findings suggest low nutrient supply to the tumor cells. It is quite conceivable that tumor has taken advantage of neutrophil phagocytosis for its survival. On the other hand, the possibility of this phenomenon for immune evasion can not be ignored. It has been generally considered that DNA from dying cells is degraded after apoptosis and, thus, inactivated. However, recent studies have demonstrated that horizontal DNA transfer between mammalian cells can occur through

Neutrophil-tumor cell cannibalism SC Sarode and GS Sarode

OSCC. We also recommend that future studies should focus on mechanism of cannibalism (both in vivo and vitro) involving lysozyme protease activity, cathepsin B activity, caveolae formation, and ezrin cytoskeleton integrity and function. Moreover, the horizontal transfer of DNA material from apoptotic cells to neoplastic cells and chromosomal instability of neoplastic cells will require further exploration.

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References

Figure 3 Photomicrograph showing moderate cytoplasmic expression of lysosyme by tumor cells. (Magnification 400x).

the uptake of apoptotic bodies, where genes from the apoptotic cells were transferred to neighboring cells phagocytosing the apoptotic bodies (21–25). The regulation of this process is poorly understood. p53, via the activation of p21, blocks normal cells from replicating transferred DNA from engulfed apoptotic bodies (24, 25). This may be a protection level against the propagation of potentially pathological DNA (24). It was shown that the ability of cells as recipient of horizontally transferred DNA was enhanced by deficiency of p53 or p21. It has been suggested that horizontal transfer of DNA from apoptotic bodies could be one explanation to the chromosomal instability observed in cancer cells (24). Recently, Krajcovic et al. (26) found that cell cannibalism by entosis, a form of cell engulfment involving live cells, also leads to polyploidy, as internalized cells disrupt cytokinesis of their engulfing cell hosts. By this mechanism, cannibalistic cell behavior can promote tumor progression by inducing aneuploidy. Cannibalism mediated chromosomal instability and aneuploidy can be one of the reasons for aggressive behavior of any cancer. All the cases were poorly differentiated with two patients having recurrences (after 4 months and 1 year) and two died (after 6 months and 1 year). Lymph node metastasis was observed in all the cases. These findings suggest NTCC as marker of aggressive behavior of OSCC. At focal areas, tumor cells showed positivity for CD68 and lysozyme markers. The tumor cells might acquire the properties of macrophages with death of internalized cell by lysozyme mediated cell digestion. As all the cells do not show positivity for these markers, future studies are needed on larger sample size to come to a final conclusion.

Conclusion We conclude that extreme NTCC has never been reported in OSCC. The tumor cells showed focal positivity for CD68 and lysozyme. As it is scientifically incorrect to draw any firm conclusions from the present case series due to a small sample size, we strongly recommend the need for future elaborate studies to evaluate the reliability of NTCC in predicting the prognosis, biological behavior and grading of

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Acknowledgement We would like to thank Dr. Atul Deshmukh for his assistance in immunohistochemistry.

Funding None declared.

Conflict of interest None declared.

Neutrophil-tumor cell cannibalism in oral squamous cell carcinoma.

Cannibalism was recognized as a phenomenon seen mainly with the tumor cells ingesting other tumor cells. Recent reports have shown tumor cell engulfin...
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