Open Access

Impact of CD34-dependent Micro Vessel Density on Periapical Odontogenic Cysts

MATHIOU VASILIKI 1
TSIAMBAS EVANGELOS 2 3
MAIPAS SOTIRIOS 3
THYMARA IRENE 3
PESCHOS DIMITRIOS 4
LAZARIS ANDREAS C. 3
  &  
KAVANTZAS NIKOLAOS 3

1Mathiou Dental Clinic, Athens, Greece

2Department of Cytology, 417 Veterans Army Hospital (NIMTS), Athens, Greece

31st Department of Pathology, Medical School, National and Kapodistrian University, Athens, Greece

4Department of Physiology, Medical School, University of Ioannina, Ioannina, Greece

Cancer Diagnosis & Prognosis Mar-Apr; 3(2): 189-193 DOI: 10.21873/cdp.10200
Received 21 December 2022 | Revised 21 March 2023 | Accepted 23 January 2023
Corresponding author
Evangelos Tsiambas, MD, MSc, Ph.D., 17 Patriarchou Grigoriou E’ Street, Ag. Paraskevi, 153 41 Athens, Greece. E-mail: tsiambasecyto@yahoo.gr

Abstract

Background/Aim: Odontogenic cysts belong to a type of lesions with endodontic origin that in some cases mimic even aggressive odontogenic tumors sharing with them similar radiographic features. Periapical cysts (PCs) belong to the inflammatory odontogenic cysts sub-category and rarely squamous cell carcinoma arises from their hyperplastic/ dysplastic epithelia. This study aimed to explore the impact of cluster differentiation 34 (CD34) protein expression combined with micro vessel density (MVD) on PCs. Materials and Methods: Forty-eight (n=48) archival, formalin-fixed, and paraffin-embedded PC tissue specimens were included in the study. Immunohistochemistry (IHC) was performed in the corresponding tissue sections using an anti- CD34 antibody. CD34 expression levels and also MVD in the examined cases were measured by implementing a digital image analysis protocol. Results: CD34 over-expression (moderate to high staining intensity levels) were detected in 29/48 (60.4%) cases, whereas the rest of them (19/48-39.6%) were characterized by low levels of expression. Extended MVD was identified in 26/48 (50.1%) cases correlated with CD34 over-expression, epithelial hyperplasia (p-value=0.001), and marginally with inflammatory infiltration level in the examined lesions (p-value=0.056). Conclusion: CD34 over-expression combined with increased MVD is associated with a neoplastic-like (hyperplastic) phenotype in PCs as a result of increased neo-angiogenic activity. These histopathological characteristics rarely form an eligible substrate for squamous cell carcinoma onset in untended cases.
Keywords: Periapical, odontogenic, cyst, microvascular, density, CD34

Among lesions with endodontic origin, odontogenic cysts are frequently identified and clinically categorized as developmental and inflammatory (1). According to their radiographical features they are classified as multi- or mono-unicolar cystic lesions characterized by precise and distinct borders. Interestingly, in some cases they mimic odontogenic tumors due to similar and non-discriminating radiographic features. In these cases, differential diagnosis is only available by histology (2). More specifically, inflammatory odontogenic cysts include a spectrum of sub-types such as residual, paradental, and periapical cysts (PCs), whereas developmental cysts comprise mainly gingival, eruption, glandular, keratocyst, and dentigerus, respectively. Histologically, PCs demonstrate a non-keratinized stratified squamous epithelium as a formation of one or two thin cell layers combined with fibrous connective tissue substrate infiltrated by inflammatory cells. Although rare, primary intraosseous squamous cell carcinoma arises from their hyperplastic/dysplastic epithelia in untended, misdiagnosed cases (3,4).

Hyperplasia in a fragment of PCs is correlated with increased inflammation and angiogenesis. In order to evaluate different neo-angiogenesis levels inside malignant tissues derived from oral and odontogenic epithelia, many studies have focused on cluster differentiation 34 (CD34) protein expression combined with micro vessel density (MVD) in them (5-9). CD34 (gene band: 1q32.2) is a ~120-kDa cell surface-transmembrane phosphoglycoprotein acting as an adhesion molecule between cells (10). Naive stem hematopoietic, vascular-associated progenitor and also mature murine mast cells express the marker in high levels (11-13). Furthermore, CD34 enhances T-cell motivation leading to lymph node infiltration (14). Concerning MVD measurement in PCs, CD34 is a sensitive and specific marker based on immunohisto-chemistry (IHC) protocols implementation in a variety of tissue substrates including cysts and neoplastic lesions such as ameloblastomas and oral carcinomas (15,16). In the current experimental, research study, we explored the impact of CD34 protein expression combined with MVD on a PCs series using modern techniques.

Materials and Methods

Study group. A series of forty-eight (n=48) archival, formalin-fixed, and paraffin-embedded PC tissue specimens were analyzed. Among patients, 26 were males, whereas the rest of them (n=22) females. The National and Kapodistrian University Medical Ethics Committee consented to the use of these tissues in the 1ST Department of Pathology, Medical School, National and Kapodistrian University, Athens, Greece for research purposes, according to World Medical Association Declaration of Helsinki. The tissue samples were fixed in neutral-buffered formalin. Hematoxylin and eosin (H&E)-stained slides of the corresponding samples were reviewed for confirmation of histopathological diagnoses. All lesions were classified according to the histological typing criteria of World Health Organization (WHO) for oral and especially odontogenic lesions (17).

Antibodies and IHC assay. Ready-to-use mouse monoclonal anti-CD34 (clone QBEnd10, Dako, Glostrup, Denmark) antibody was selected at a dilution of 1:200. The IHC assay was implemented on 4 μm serial tissue sections obtained from the corresponding tissue blocks. Firstly, the slides were de-paraffinized followed by rehydration. According to the manufacturer’s guidelines, the hyperoxide/streptavidin/biotin protocol (Dako) combined with EnVision FLEX TBS (Tris-buffered saline: 0.05 MTris/HCl, 0.15 MNaCl, pH=7.6) wash Buffer (20×) was applied. Diaminobenzidine-tetrahydrochloride (DAB–Dako) containing 0.1% hydrogen peroxide was used as a chromogen. After its incubation, the tissue sections were counterstained using Hematoxylin, for 5 min, dehydrated, and finally cover-slipped. The primary antibody was omitted in the case of control slides. An automated staining system (Biogenex, Fremont, CA, USA) was used for the current IHC procedure. Endothelial cell continuous, membranous staining was considered suitable for the marker, according to manufacturers’ data sheets (Figure 1A). Normal endothelia expressing the protein were used as the control group.

Digital image analysis (DIA) assay. CD34 protein expression levels and MVD levels were estimated quantitatively by measuring the corresponding CD34 staining intensity levels (densitometry calculation) in the stained endothelial cells and their number, respectively. A DIA assay was implemented based on a semi-automated system (hardware: Microscope CX-31, Olympus, Melville, NY, USA; Digital camera, Sony, Tokyo, Japan; Windows XP/NIS-Elements Software AR v3.0, Nikon Corp., Tokyo, Japan). According to the digitized algorithm, CD34-related stained areas were detected (5 optical fields at ×400 magnification) and a digital database including the corresponding snapshots was constructed. A specific macro (membranous expression pattern) was assessed as a matrix for the measurements. Based on an algorithm, an extensive spectrum of continuous grey scale values (0-255) at the RedGreenBlue (RGB) color spectrum was eligible for calculating different protein expression levels (Figure 1B). Staining intensity values decreasing to 0 correspond to a progressive protein over-expression. In contrast, increased values to 255 lead a progressive loss of its staining intensity. MVD levels were measured as a number of CD34-stained endothelial ring-like structures (vessels) per high power optical field. Total results for CD34/MVD and DIA values are demonstrated in Table I.

Statistical analysis. In order to analyze statistically the extracted results, we applied the statistics software package Statisticav. 6.0 (StatSoft Power Solutions Dell, TIBCO Soft, Palo Alto, CA, USA). Quantitative variables were presented as mean±standard deviation, whereas qualitative variables were presented in frequency tables. To evaluate the relationship between qualitative and quantitative variables, because of the small number of subjects in each group, the nonparametric Mann–Whitney and Kruskal–Wallis tests were applied. To evaluate the relationship between independent qualitative variables, where appropriate, the chi-square test for linear trend and Fisher exact test were applied. Statistical significance (p) was evaluated in pairs and differences <0.05 were considered statistically significant. Total IHC results and differences (p-values) are described in Table I.

Results

All examined PC cases expressed the marker in different levels. CD34 over-expression (moderate to high staining intensity levels) was detected in 29/48 (60.4%) cases, whereas the rest of them (19/48-39.6%) were characterized by low levels of expression. Increased MVD was identified in 26/48 (50.1%) cases -as an absolute number of endothelial ring-like structures -and correlated with CD34 over-expression, epithelial hyperplasia (p-value=0.001), and marginally with inflammatory infiltration level in the examined lesions (p-value=0.001), whereas no correlation was established with sex (p-value=0.534).

Discussion

Approaching the non- or neoplastic dental lesions, periodontitis is a frequent and progressively severe inflammation mediated by bacteria accumulation combined or not with other factors such as low-level mouth hygiene conditions and tobacco chronic consumption (18). More specifically, its onset includes gingivitis presenting clinical signs such as swollen gums, bleeding, plaque, and teeth pain. Besides periodontitis, peri-implantitis is characterized by similar clinical signs Interestingly, there is a genetic predisposition in specific populations (19). Furthermore, chronic colon inflammation (colitis) leads indirectly to periodontitis by altering specific metabolic pathways (20). Additionally, Helicobacter pylori (H. pylori) stomach infection was suggested to be present in chronic periodontitis patients, but their exact relation is under investigation (21). In conjunction, apical periodontitis is the result of chronic endodontic inflammation mediated by bacteria of the Porphyromonas spp. including predominantly Porphyromonas endodontalis, and Porphyromonas gingivalis (22,23).

Odontogenic cysts - combined or not with periodontitis - represent frequent lesions in adults. Referring to PCs, they are characterized histologically by non-keratinized stratified squamous epithelium including thin (one or two) layers combined with excessive fibrous connective tissue and inflammatory infiltration. Interestingly, there is a variety of non-malignant non-endodontic periapical lesions (NMNPLs) that mimic pure PCs (24). Additionally, pathological entities that share similar histological characteristics with PCs include squamous odontogenic tumors and non-neoplastic lesions with epithelial hyperplasia inside a radicular cyst (25).

In this study, we explored the role of CD34 protein expression combined with MVD in a series of PC tissues using IHC and DIA methods. We observed that CD34-dependent MVD was increased significantly in cases with epithelial hyperplasia and marginally with inflammatory infiltration. It seems that PCs incorporating these specific histological features demonstrate a more aggressive biological behavior (increased angiogenesis). Another study co-analyzed receptor for advanced glycation end products (RAGE), S100, and CD34 molecules in a series of periapical granuloma tissues (26). They reported elevated CD34 protein expression leading to endothelial hyperplasia (increased angiogenesis) in these inflammatory cystic lesions. Similarly, interleukin (IL)-17 – a molecule that acts as a cytokine- has been found to be over-expressed in apical periodontitis lesions (periradicular cysts and granulomas) in conjunction with CD34 (27). Both of them promote inflammation and angiogenesis in them, respectively. CD34 over-expression is also observed in regenerative endodontic processes providing healing of periradicular lesions (28). Furthermore, quantitative digital analysis of MVD rates in PCs and other oral-dental lesions is superior compared to conventional eye-based evaluation. In the current study, we estimated MVD levels in a fast and accurate way using a digitized algorithm based on CD34 IHC expression levels. Our previous studies on oral malignancies are concordant with similar experimental studies (29-35). All of them suggest and enhance this practice because it provides a systematic screening and mapping of immunostained slides.

In conclusion, CD34 over-expression combined with increased MVD is correlated with a neoplastic-like (hyperplastic) phenotype in PCs as a result of increased neo-angiogenic and inflammatory activity. A subset of PCs characterized by elevated MVD rates demonstrate significant epithelial hyperplasia and increased inflammatory infiltration, mimicking neoplastic lesions. These histopathological characteristics rarely form an eligible substrate for squamous cell carcinoma onset in untended cases.

Conflicts of Interest

The Authors have no conflicts of interest to declare in relation to this study.

Authors’ Contributions

VM, ET: design of the study, ET, VM: manuscript writing, IT, DP, ACL, NK: academic advisors: SM: collection and management of references’ data. All Authors read and approved the final manuscript.

References

1 Wang LL & Olmo H Odontogenic cysts. StatPearls [Internet]. PMID: 34662043.
2 Rioux-Forker D Deziel AC Williams LS & Muzaffar AR Odontogenic cysts and tumors. Ann Plast Surg. 82(4) 469 - 477 2019. PMID: 30856625. DOI: 10.1097/SAP.0000000000001738
3 Lukandu OM & Micha CS Primary intraosseous squamous cell carcinoma arising from keratocystic odontogenic tumor. Oral Surg Oral Med Oral Pathol Oral Radiol. 120(5) e204 - e209 2015. PMID: 25953635. DOI: 10.1016/j.oooo.2015.03.006
4 Hegde U Sheshanna SH Jaishankar HP & Prasad RR Primary intraosseous squamous cell carcinoma ex-odontogenic cyst. J Cancer Res Ther. 16(3) 683 - 685 2020. PMID: 32719292. DOI: 10.4103/jcrt.JCRT_606_16
5 Seifi S Shafaie S & Ghadiri S Microvessel density in follicular cysts, keratocystic odontogenic tumours and ameloblastomas. Asian Pac J Cancer Prev. 12(2) 351 - 356 2011. PMID: 21545193.
6 Naumov GN Bender E Zurakowski D Kang SY Sampson D Flynn E Watnick RS Straume O Akslen LA Folkman J & Almog N A model of human tumor dormancy: an angiogenic switch from the nonangiogenic phenotype. J Natl Cancer Inst. 98(5) 316 - 325 2006. PMID: 16507828. DOI: 10.1093/jnci/djj068
7 Sales CB Buim ME de Souza RO de Faro Valverde L Mathias Machado MC Reis MG Soares FA Ramos EA & Gurgel Rocha CA Elevated VEGFA mRNA levels in oral squamous cell carcinomas and tumor margins: a preliminary study. J Oral Pathol Med. 45(7) 481 - 485 2016. PMID: 26861159. DOI: 10.1111/jop.12398
8 Kademani D Lewis JT Lamb DH Rallis DJ & Harrington JR Angiogenesis and CD34 expression as a predictor of recurrence in oral squamous cell carcinoma. J Oral Maxillofac Surg. 67(9) 1800 - 1805 2009. PMID: 19686913. DOI: 10.1016/j.joms.2008.06.081
9 Mohtasham N Babakoohi S Salehinejad J Montaser-Kouhsari L Shakeri MT Shojaee S Sistani NS & Firooz A Mast cell density and angiogenesis in oral dysplastic epithelium and low- and high-grade oral squamous cell carcinoma. Acta Odontol Scand. 68(5) 300 - 304 2010. PMID: 20586672. DOI: 10.3109/00016357.2010.494622
10 Lanza F Healy L & Sutherland DR Structural and functional features of the CD34 antigen: an update. J Biol Regul Homeost Agents. 15(1) 1 - 13 2001. PMID: 11388737.
11 Folkman J Fundamental concepts of the angiogenic process. Curr Mol Med. 3(7) 643 - 651 2003. PMID: 14601638. DOI: 10.2174/1566524033479465
12 López-Graniel CM Tamez de León D Meneses-García A Gómez-Ruiz C Frias-Mendivil M Granados-García M & Barrera-Franco JL Tumor angiogenesis as a prognostic factor in oral cavity carcinomas. J Exp Clin Cancer Res. 20(4) 463 - 468 2001. PMID: 11876537.
13 Pandiar D & Shameena P Immunohistochemical expression of CD34 and basic fibroblast growth factor (bFGF) in oral submucous fibrosis. J Oral Maxillofac Pathol. 18(2) 155 - 161 2014. PMID: 25328292. DOI: 10.4103/0973-029X.140718
14 Alaeddini M Salah S Dehghan F Eshghyar N & Etemad-Moghadam S Comparison of angiogenesis in keratocystic odontogenic tumours, dentigerous cysts and ameloblastomas. Oral Dis. 15(6) 422 - 427 2009. PMID: 19413675. DOI: 10.1111/j.1601-0825.2009.01566.x
15 Mărgăritescu C Pirici D Stîngă A Simionescu C Raica M Mogoantă L Stepan A & Ribatti D VEGF expression and angiogenesis in oral squamous cell carcinoma: an immunohistochemical and morphometric study. Clin Exp Med. 10(4) 209 - 214 2010. PMID: 20376688. DOI: 10.1007/s10238-010-0095-4
16 Guzmán-Medrano R Arreola-Rosales RL Shibayama M Silva-Olivares DA Bologna-Molina R & Rodríguez MA Tumor-associated macrophages and angiogenesis: a statistical correlation that could reflect a critical relationship in ameloblastoma. Pathol Res Pract. 208(11) 672 - 676 2012. PMID: 23041027. DOI: 10.1016/j.prp.2012.09.001
17 Barnes L Eveson JW Reichart P & Sidranksy D Classification of Tumors. Pathology and Genetics of Head and Neck Tumors. Lyon, IARC Press. pp. 283 2005.
18 Janaphan K Hashem I Smith C Holmes S & Chatzopoulou D Periodontal disease as a primary cause of surgical site infection in fractures of the mandible: is smoking a confounding variable. Br J Oral Maxillofac Surg. 60(10) 1424 - 1429 2022. PMID: 36400684. DOI: 10.1016/j.bjoms.2022.08.001
19 Turkmen M & Firatli E The study of genetic predisposition on periodontitis and peri-implantitis. Niger J Clin Pract. 25(11) 1799 - 1804 2022. PMID: 36412285. DOI: 10.4103/njcp.njcp_19_22
20 Yuan G Chen J Wang X Hu F Zhang X & Chen X Serum metabolomics provides clues in understanding colitis exacerbating experimental periodontitis in female mice. Arch Oral Biol. 145 105583 2023. PMID: 36395563. DOI: 10.1016/j.archoralbio.2022.105583
21 Tsimpiris A Tsolianos I Grigoriadis A Moschos I Goulis DG & Kouklakis G Association of Chronic Periodontitis with Helicobacter pylori Infection in Stomach or Mouth: A Systematic Review and Meta-Analysis. Eur J Dent. PMID: 36400109. DOI: 10.1055/s-0042-1756690
22 Astorga J Hernández M Bravo D & Hoare A Evaluation of PCR primers to identify Porphyromonas endodontalis in apical periodontitis clinical samples. Arch Microbiol. 204(10) 652 2022. PMID: 36173466. DOI: 10.1007/s00203-022-03260-7
23 Bordagaray MJ Fernández A Garrido M Astorga J Hoare A & Hernández M Systemic and extraradicular bacterial translocation in apical periodontitis. Front Cell Infect Microbiol. 11 649925 2021. PMID: 33816354. DOI: 10.3389/fcimb.2021.649925
24 Modi K Padmapriya R Elango S Khandelwal P Arul B & Natanasabapathy V Nonmalignant nonendodontic lesions mimicking periapical lesions of endodontic origin: A systematic review. J Conserv Dent. 25(3) 214 - 225 2022. PMID: 35836562. DOI: 10.4103/jcd.jcd_13_22
25 Sala-Pérez S Marco-Molina V & Gay-Escoda C Squamous odontogenic tumor-like proliferation in a radicular cyst: A case report. J Clin Exp Dent. 5(5) e298 - e301 2013. PMID: 24455099. DOI: 10.4317/jced.51056
26 Takeichi O Hatori K Kamimoto A Oka S Ogiso B & Saito I Receptor for advanced glycation end products (RAGE)-expressing endothelial cells co-express AGE and S100 in human periapical granulomas. J Dent. 39(10) 679 - 685 2011. PMID: 21864645. DOI: 10.1016/j.jdent.2011.07.010
27 Ajuz NC Antunes H Mendonça TA Pires FR Siqueira JF Jr & Armada L Immunoexpression of interleukin 17 in apical periodontitis lesions. J Endod. 40(9) 1400 - 1403 2014. PMID: 25043329. DOI: 10.1016/j.joen.2014.03.024
28 Meschi N Hilkens P Lambrichts I Van den Eynde K Mavridou A Strijbos O De Ketelaere M Van Gorp G & Lambrechts P Regenerative endodontic procedure of an infected immature permanent human tooth: an immunohistological study. Clin Oral Investig. 20(4) 807 - 814 2016. PMID: 26250796. DOI: 10.1007/s00784-015-1555-8
29 Costa Neto H Andrade ALDL Carmo AFD Freitas RA & Galvão HC Involvement of tryptase-positive mast cells and angiogenesis in the growth of inflammatory odontogenic cysts. Braz Oral Res. 35 e061 2021. PMID: 34076187. DOI: 10.1590/1807-3107bor-2021.vol35.0061
30 de-Freitas CT de-França GM Gordón-Núñez MA Santos PP de-Lima KC & Galvão HC Myofibroblasts and increased angiogenesis contribute to periapical cystic injury containment and repair. Med Oral Patol Oral Cir Bucal. 25(5) e584 - e591 2020. PMID: 32388520. DOI: 10.4317/medoral.23605
31 Lima SC Rizo VH Silva-Sousa YT Almeida LY Almeida OP & León JE Immunohistochemical evaluation of angiogenesis and tryptase-positive mast cell infiltration in periapical lesions. J Endod. 37(12) 1642 - 1646 2011. PMID: 22099897. DOI: 10.1016/j.joen.2011.08.024
32 Pereira T Dodal S Tamgadge A Bhalerao S & Tamgadge S Quantitative evaluation of microvessel density using CD34 in clinical variants of ameloblastoma: An immunohistochemical study. J Oral Maxillofac Pathol. 20(1) 51 - 58 2016. PMID: 27194862. DOI: 10.4103/0973-029X.180929
33 Segatelli V de Oliveira EC Boin IF Ataide EC & Escanhoela CA Evaluation and comparison of microvessel density using the markers CD34 and CD105 in regenerative nodules, dysplastic nodules and hepatocellular carcinoma. Hepatol Int. 8(2) 260 - 265 2014. PMID: 26202507. DOI: 10.1007/s12072-014-9525-9
34 Papanikolaou V Chrysovergis A Mastronikolis N Tsiambas E Ragos V Peschos D Stavraka C Roukas D & Kyrodimos E Topoisomerase IIa protein expression patterns in laryngeal squamous cell carcinoma. Anticancer Res. 40(2) 807 - 811 2020. PMID: 32014923. DOI: 10.21873/anticanres.14012
35 Chrysovergis A Papanikolaou VS Tsiambas E Ragos V Peschos D & Kyrodimos E Digital analysis of BCL2 expression in laryngeal squamous cell carcinoma. Anticancer Res. 39(3) 1253 - 1257 2019. PMID: 30842155. DOI: 10.21873/anticanres.13235