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PHARYNGOLOGY

Histopathological and immunohistochemical aspects of the oropharyngeal neoplasm in the ENT Clinic, Craiova

 Aspecte histopatologice şi imunohistochimice ale neoplasmului orofaringian în Clinica ORL din Craiova

Ioana-Cristina Oprişcan, Elena Ioniță, Carmen-Aurelia Mogoanţă, Florin Anghelina, Mircea-Sorin Ciolofan, Mihaela Mitroi, Alina-Nicoleta Căpitănescu, Iulică Ioniță, Irina Enache, Carmen Sîrbuleţ, Eduard-Andrei Gheorghe

First published: 30 mai 2023

Editorial Group: MEDICHUB MEDIA

DOI: 10.26416/ORL.59.2.2023.8111

Abstract

Introduction. Cancers of the head and neck, which include neo­plasia of the ENT or upper respiratory and digestive tracts, represent a wide variety of malignant diseases ori­gi­na­ting in the cells of the upper aerodigestive tract. Oro­pha­ryn­geal carcinoma represents 10-15% of head and neck neo­plasms. Objective of the study. The purpose of this pa­per is to review recent data on several topics, including HPV-positive risk factors for oral squamous cell carcinoma (OSCC), guidelines in diagnostic evaluation, treatment, prog­no­sis and prevention strategies. The purpose of the paper. A comparison of proliferation factors, degradation of the basal lamina and modulation of the connective tissue with the survival time of patients diagnosed with oro­pha­ryn­geal neoplasm. Materials and method. The study analyzed a group of 272 patients hospitalized in the ENT Clinic of the Craiova County Emergency Clinical Hos­pi­tal over a period of five years, between 1 January 2017 and 31 December 2021, diagnosed with oropharyngeal neo­plasm. Results. The present study included a number of 272 ma­lig­nant oropharyngeal tumors, of which the ma­jo­ri­ty were represented by squamous cell oropharyngeal car­ci­no­mas; well differentiated (19 cases; 6.99%), moderately dif­fe­ren­tia­ted (130 cases; 47.79%), poorly differentiated (110 cases; 40.44%); LMNH (four cases; 1.47%), one leio­myo­sar­co­ma (0.36%), one malignant synovioma (0.36%) and one adenoid cystic carcinoma (0.36%). Two patients re­fused biopsy (0.73%) and four patients preferred that the his­to­path­olo­gi­cal examination be performed in the private system as well (1.47%). Conclusions. Oropharyngeal squa­mous cell carcinoma accounts for 95% of all head and neck can­cers, and in the last decade its incidence has increased by 50%. 

 

Keywords
oropharyngeal carcinoma, squamous cell carcinoma, head and neck cancer

Rezumat

Introducere. Cancerele capului şi gâtului ce includ neo­pla­zii­le ORL sau ale căilor aeriene şi digestive superioare re­­pre­­zin­tă o mare varietate de boli maligne cu originea în celulele tractului aerodigestiv superior. Carcinomul oro­fa­rin­gian reprezintă 10-15% din neoplaziile capului şi gâtului. Obiec­ti­vul lucrării. Sco­pul acestei lucrări este de a revizui da­te­le recente despre mai multe subiecte, inclusiv factorii de risc HPV-pozitivi ai car­ci­no­mu­lui orofaringian cu celule scua­moa­se (OSCC), alături de li­niile directoare în evaluarea di­ag­nos­ti­cului, tratamentului, prog­nos­ti­cu­lui şi a strategiilor de prevenire. Scopul lucrării. Com­pa­ra­rea factorilor de pro­li­fe­ra­re, alături de degradarea laminei ba­za­le şi modularea ţe­su­tu­lui conjunctiv cu timpul de su­pra­vie­ţuire al pacienţilor diag­nos­ti­caţi cu neoplasm orofaringian. Materiale şi me­to­dă. Studiul a analizat un grup de 272 de pacienţi internaţi în Clinica ORL a Spitalului Clinic Judeţean de Urgenţă din Cra­io­va, pe o perioadă de cinci ani, între 1 ia­nua­rie 2017 şi 31 decembrie 2021, diagnosticaţi cu neoplasm oro­fa­rin­gian. Rezultate. Acest studiu a cuprins un număr de 272 de tu­mori maligne orofaringiene, dintre acestea ma­jo­ri­ta­tea fiind reprezentate de carcinoame orofaringiene cu celule scua­­moa­se, bine diferenţiate (19 cazuri; 6,99%), mo­de­rat di­fe­ren­ţiate (130 de cazuri; 47,79%), slab diferenţiate (110 ca­zuri; 40,44%), LMNH (patru cazuri; 1,47%), un leio­mio­sar­com (0,36%), un sinoviom malign (0,36%) şi un car­ci­nom ade­no­id chis­tic (0,36%). Doi pacienţi au refuzat biop­sia (0,73%) şi pa­tru pacienţi au preferat ca examenul histo­pa­to­lo­gic să fie efec­tuat şi în sistemul privat (1,47%). Concluzii. Car­ci­no­mul oro­fa­rin­gian cu celule scuamoase reprezintă 95% din toa­te formele de cancer de cap şi gât, iar în ultimul deceniu in­ci­den­ţa acestuia a crescut cu 50%. 
 

Cuvinte cheie
carcinom orofaringian carcinom cu celule scuamoase cancer cap şi gât

Introduction

Head and neck cancers, which include ENT or upper airway and digestive tract neoplasia, represent a wide variety of malignant diseases originating in the cells of the upper aerodigestive tract. Oropharyngeal carcinoma represents 10-15% of head and neck neoplasms(1).

In general, cancers, including oral squamous cell carcinoma (OSCC), arise from the accumulation of genetic alterations and epigenetic abnormalities in signaling pathways that are associated with cancer, resulting in phenotypes that facilitate the development of OSCC. Oral squamous cell carcinoma is a malignant neoplasm derived from the stratified squamous epithelium of the oral mucosa. Its pathogenesis is multifactorial, associated with cigarette smoke, alcohol and snuff, as well as the human papillomavirus (HPV)(2).

Cancer of the oral cavity is the sixth leading cause of cancer worldwide. In the United States of America alone, there are over 21,500 diagnoses of oral carcinomas each year and 6000 Americans die from it annually(3). The incidence of oral carcinoma varies worldwide, with estimates exceeding 40 per 100,000 in parts of France, southern Asia and Hungary. Ninety percent of these malignancies are squamous cell carcinomas.

Oral squamous cell carcinoma is by far the most important and frequent malignant neoplasm of the mucosa affecting the head and neck, and represents over 90% of all malignant neoplasms that develop in this region. 80-90% of head and neck cancers are thought to be associated with known risk factors such as smoking and alcohol abuse(4). However, 15-20% of head and neck cancers occur in people with no history of tobacco or alcohol use(5). We therefore believe that there are various etiological factors that interact in a multifactorial process for the induction and proliferation of neoplastic cells.

Oral malignant lesions develop through a series of histopathological stages: from average dysplasia (low grade), moderate and severe (high grade) to carcinoma in situ and then to invasive disease. The early detection of these premalignant oral lesions that will develop into invasive tumors is necessary to improve the prognosis of oral cancer. Due to the fact that there is no tool for delimiting the risk of development in the case of low-grade oral lesions, we cannot determine which of these cases require an aggressive surgical or medicinal intervention. Within these morphological processes, angiogenesis and lymphangiogenesis are essential processes for tumor growth and metastasis, because without a blood supply, incipient neoplasms cannot grow larger than 2 mm(3).

Materials and method

The present study included a number of 272 malignant oropharyngeal tumors, of which the majority were represented by squamous cell oropharyngeal carcinomas; well differentiated (19 cases; 6.99%), moderately differentiated (130 cases; 47.79%), poorly differentiated (110 cases; 40.44%); LMNH (four cases; 1.47%), one leiomyosarcoma (0.36%), one malignant synovioma (0.36%) and one adenoid cystic carcinoma (0.36%). Two patients refused biopsy (0.73%) and four patients preferred that the histopathological examination be performed in the private system as well (1.47%).

After clinical diagnosis and preoperative preparation, the patients received surgical treatment. The biological material collected from the surgical intervention rooms, respectively the tumor fragments, were sent to the pathological anatomy laboratory for histopathological diagnosis.

Immediately after harvesting, the biological material, right from the operating room, went into a 10% neutral formalin fixative solution for 48 hours at laboratory temperature in glass or plastic containers resistant to the action of formalin.

In our study, to evaluate the proliferative activity of cells from oral tumors, we used three immunohistochemical markers: PCNA, Ki67 and p53. For the evaluation of the angiogenesis process by immunohistochemistry techniques, there are several types of specific antibodies for marking endothelial cells: CD31, CD34, von Willebrand Factor (vW), CD105 (Podoplanin). In our study, we used an immunohistochemical marker specific for young endothelial cells – namely, the anti-CD34 antibody. This allowed us to highlight mature and immature blood capillaries. In fact, CD34-positive cells are considered endothelial precursor cells capable of forming new vessels in areas of tissue ischemia, initially unrelated to the preexisting vascular network.

Regarding the process of lymphangiogenesis, it has a major contribution to the formation of new lymphatic vessels, through the proliferation and germination of endothelial cells from preexisting lymphatic vessels(6). Lymphangiogenesis is a dynamic process during embryogenesis, but is largely absent under normal, postnatal physiological conditions. In adult individuals, lymphangiogenesis occurs only under certain pathological conditions, such as inflammation, tissue repair and tumor growth(7-9).

In our study, we used a specific D2-40 antibody to identify the lymph nodes in the tumor stroma. D2-40 is a monoclonal antibody, a gm. 40,000 dalton sialoglycoprotein, that reacts with a fixation-resistant epitope from the lymphatic endothelium. This property of the epitope makes this immunomarker extremely useful for the study of lymphatic vessels on formalin-fixed and paraffin-embedded histological preparations.

Results

In the evaluation of cell proliferation factors, we started from the fact that human and animal tissues are made up of labile cells that have a lifespan limited to days, months or years, after which they enter a natural process of cell death, called apoptosis, process by which the tissue and the organism as a whole maintain a relatively constant number of cells for a certain stage of its development and existence, maintaining its tissue and organic architecture, which leads to the preservation of local and general homeostasis. In normal tissues, cell division and death are controlled by specific, highly complex, some incompletely known, genetically encoded mechanisms.

Unlike normal tissues, malignant tumors and some premalignant lesions are characterized by a significant change in the processes of cell division and maturation, by a profound disruption of the genetic program, which leads to the appearance of serious pathological proces­ses. They disrupt the local homeostasis, then they alter the entire tissue, organ and organism, resulting in the appearance of the disease state. If normal cells have a well-defined mitotic rhythm and lifespan, cancer cells divide and grow uncontrollably, and the process of apoptosis is mostly altered.

Well-differentiated squamous cell carcinomas were formed by well-differentiated squamous cells, arranged in islands of various shapes and sizes, with pearls of keratin inside them, resulting from a process of “neoplastic maturation”. Inside the keratotic pearls, the cells appeared acidophilic, with pyknotic nucleus, with karyolysis, while in the rest of the cells, the nucleus were of various shapes and sizes, much larger than the nucleus of the normal epithelium, with obvious nucleolus and unevenly arranged chromatin, sometimes in the form of grunts. The shape and sizes of the neoplastic islands, as well as the keratotic pearls were highly variable.

Moderately differentiated carcinomas were formed by cords or islands of atypical, neoplastic, oval, elongated, round epithelial cells infiltrating the fibrous tumor stroma. At their periphery, the carcinomatous islands were delimited by fibrous stromal elements or inflammatory type cells. The nucleolus of the neoplastic cells had various shapes and sizes, most of them being hypochromic with large nucleolus.

Tumor cells frequently appeared as atypical cells dispersed diffusely in the stroma of the oral, lingual or labial mucosa, with rare intercellular bridges. Often, the tumor cells showed large, deformed, hyperchromic or hypochromic nucleolus, with incisions and buds, with multiple atypical mitoses. The cytoplasm of these cells appeared acidophilic, inhomogeneous, and the cell membrane showed inhomogeneous digitiform extensions, characteristic of cellular pleiomorphism.

Poorly differentiated cell carcinomas appeared as cell cords, islands, or epithelioid-appearing cells of various shapes and sizes, bearing no resemblance to the epithelium from which they arose. These different aspects of cancer cells lead us to believe that malignant tumors are heterogeneous, multicellular entities containing multiple cell lineages whose interactions with each other and with the extracellular matrix through paracrine secreted soluble molecules are dynamic and favor cell proliferation, movement and differentiation malignancies.

The tumor stroma resulting from the transformation of the chorion of the covering epithelium into stroma with particular characters, as an adaptation reaction of the innate mesenchyme to the tumor invasion, was most often infiltrated with inflammatory type cells. In this stroma, numerous large-caliber, congested blood vessels were highlighted, mainly arranged around the islands of neoplastic cells. Due to the high vascular density, this stroma has the role of nutrition and support for tumor cells. It is well known that, immediately after the rupture of the basement membrane and the penetration of cancer cells into the underlying connective tissue, an inflammatory reaction is triggered which will lead to the formation of a particular granulation tissue that will mature and complete a tumor connective tissue, also known as tumoral stroma.

In our study, all squamous cell carcinomas, regardless of the degree of differentiation, were intensely PCNA-positive, except for areas of “keratotic pearls” in well-differentiated squamous cell carcinomas. More than 90% of the nucleolus of the neoplastic cells were intensely positive for anti-PCNA antibodies. In other words, almost all tumor cells have the ability to multiply. In areas with keratotic pearls, the number of positive cells gradually decreased as they involuted. That is why we consider that the reaction to PCNA is extremely valuable when the difference between a simple dysplasia and a carcinoma in situ has to be made, the reaction being intensely positive when neoplastic proliferation is triggered.

In our study, we tried to evaluate not only the presence of angiogenesis vessels in oral squamous carcinoma, but also their number and the area occupied by these vessels in the different forms of oral carcinomas. Where the tumor stroma was heavily infiltrated with inflammatory type cells, more angiogenesis vessels of a much larger caliber were identified than in the fibrous tumor stroma. In the stroma dominated by collagen fibers, the number and size of angiogenesis vessels were much lower than in areas of tumor proliferation where the increased number of angiogenesis vessels correlated with the intensity of the chronic inflammatory infiltrate. These aspects we observed confirm the idea that angiogenesis is stimulated by cells of the immune system that are able to synthesize and secrete numerous factors with angiogenic potential.

The number of angiogenesis vessels in poorly differentiated carcinomas was approximately 166 vessels/mm2, in moderately differentiated carcinoma – approximately 88 vessels/mm2, and in well-differentiated carcinoma – 72 vessels/mm2. The vascular areas were also variable according to the degree of differentiation of the carcinoma. Thus, in poorly differentiated carcinoma it was about two times higher than in moderately differentiated carcinoma and about four times higher than in well-differentiated carcinoma.

Discussion

Chronic exposure to carcinogens causes genetic abnormalities in the cells of the oral mucosa. When these genetic anomalies determine the activation of proto-oncogenes and the inactivation of tumor suppressor genes, cells acquire new properties of growth and multiplication. It is clearly accepted by everyone that oral squamous cell carcinoma develops as a result of the accumulation of genetic errors in the same tissue.

Changes affecting the TP53 gene cause the accumulation of an abnormal protein in the nucleus, which can be highlighted by immunohistochemistry. Some studies have indicated that, in the oral cavity, the presence of p53 positive cells in the upper layers of the dysplastic epithelium may imply a risk of evolution towards carcinoma(10).

Evidence that the p53 pathway is very important in the biology of oral cancer has led to the use of such immunohistochemical analysis as a simple, rapid and inexpensive method in the case of potentially malignant lesions in an attempt to find a useful marker for predicting progression to oral squamous cell carcinoma.

The presence of large-caliber lymphatic vessels denotes their ability to transport tumor cells to the regional lymph nodes. In recent years, a large number of authors have reported the existence of a correlation between VEGF-C expression, lymphangiogenesis and metastasis in regional lymph nodes(11). According to some authors, there is a strong correlation between the density of lymphatic vessels and the expression of VEGF-C. The same study shows that there is no direct correlation between the density of lymphatic vessels and the presence of lymph node metastases(12).

Cancer epidemiology claims that 20-40% of tumors are caused by smoking, 2-4% are caused by excessive alcohol consumption, 10-70% are caused by the diet and lifestyle of the patients, 0.5-3% are determined by medicinal substances, 1-10% are determined by viral infections and 1-5% are determined by atmospheric pollution, therefore a significant percentage of cancers are mainly determined by the patients’ lifestyle, cancer becoming, from this point of view, a condition that also benefits from “prophylactic treatment”(13).

Conclusions

1. The present study included a number of 272 oropharyngeal malignant tumors, of which the majority were represented by squamous cell oropharyngeal carcinomas; well differentiated (19 cases; 6.99%), moderately differentiated (130 cases; 47.79%), poorly differentiated (110 cases; 40.44%); LMNH (four cases; 1.47%), one leiomyosarcoma (0.36%), one malignant synovioma (0.36%) and one adenoid cystic carcinoma (0.36%). Two patients refused biopsy (0.73%) and four patients preferred that the histopathological examination be performed in the private system as well (1.47%).

2. Well-differentiated squamous cell carcinomas were formed by well-differentiated squamous cells, arranged in islands of various shapes and sizes, with keratin pearls inside them, resulting from a process of “neoplastic maturation”.

3. Moderately differentiated carcinomas were formed by cords or islands of atypical, neoplastic, oval, elongated, round epithelial cells infiltrating the fibrous tumor stroma. At their periphery, the carcinomatous islands were delimited by fibrous stromal elements or inflammatory type cells. The nucleus of the neoplastic cells had various shapes and sizes, most of them being hypochromic with large nucleolus.

4. Poorly differentiated cell carcinomas appeared as cell cords, islands, or epithelioid-appearing cells of various shapes and sizes, bearing no resemblance to the epithelium from which they arose.

5. The number of angiogenesis vessels in poorly differentiated carcinomas was approximately 166 vessels/mm2, in moderately differentiated carcinoma – approximately 88 vessels/mm2, and in well-differentiated carcinoma – 72 vessels/mm2. The vascular areas were also variable according to the degree of differentiation of the carcinoma. Thus, in poorly differentiated carcinoma it was about two times higher than in moderately differentiated carcinoma and about four times higher than in well-differentiated carcinoma.

6. All squamous carcinomas, regardless of the degree of differentiation, were intensely positive for PCNA, except for areas of “keratotic pearls” in well-differentiated squamous carcinomas. More than 90% of the nucleolus of the neoplastic cells were intensely positive for anti-PCNA antibodies. In other words, almost all tumor cells have the ability to multiply.

7. Numerous studies have shown that in solid tumors an abnormal vasculature appears, which is a histopathological hallmark, where blood vessels are arranged adjacent to the tumor itself(14). The higher number of tumor vessels increase the risk of tumor cells entering the systemic circulation and giving distant metastases.

8. In addition to increasing the density of lymphatic vessels, lymphangiogenic growth factors also act to enlarge and dilate lymphatic vessels(15). Interestingly, the activation of VEGFR-2 causes expansion of lymphatic vessels, in contrast to VEGFR-3 which activates lymphangiogenesis and vascular sprouting more(16). The enlarged lymphatic vessels that drain the tumor allow the migration and metastasis of tumor cells towards the lymph node, most likely through the increase of lymphatic flow(17), and facilitate the metastasis of some groups of tumor cells.

9. The anatomical and clinical types of oropharyngeal cancer are dictated by the anatomical and histological characteristics of the region(18).

 

Acknowledgment: This work was supported by the grant POCU/993/6/13/153178, “Performanţă în cercetare” (“Research performance”), co-financed by the European Social Fund within the Sectorial Operational Program Human Capital 2014-2020.

 

Figure 1. Image of oral carcinoma with well-dif­fe­ren­tia­ted squamous cells, in which it can be noted the presence of carcinomatous islands of various sizes and shapes, with a tendency to form “keratosic pearls”, delimited by a tumor stroma strongly infiltrated with inflammatory type cells. Col. hematoxylin – eosin X 100
Figure 1. Image of oral carcinoma with well-dif­fe­ren­tia­ted squamous cells, in which it can be noted the presence of carcinomatous islands of various sizes and shapes, with a tendency to form “keratosic pearls”, delimited by a tumor stroma strongly infiltrated with inflammatory type cells. Col. hematoxylin – eosin X 100


 

Figure 2. Well-differentiated squamous cell carcinoma of the oral cavity with strongly positive reaction to PCNA. Immunolabeling with anti-PCNA antibodies. Eosin X 100
Figure 2. Well-differentiated squamous cell carcinoma of the oral cavity with strongly positive reaction to PCNA. Immunolabeling with anti-PCNA antibodies. Eosin X 100

 

Figure 3. Microscopic image of moderately differen­tia­ted carcinoma, in which the presence of neoplastic cells of oval, elongated, round shape, arranged in the form of cords or islands, with numerous nuclear and cellular atypia, is noted. Col. hematoxylin – eosin X 100
Figure 3. Microscopic image of moderately differen­tia­ted carcinoma, in which the presence of neoplastic cells of oval, elongated, round shape, arranged in the form of cords or islands, with numerous nuclear and cellular atypia, is noted. Col. hematoxylin – eosin X 100

 

Figure 4. Island of neoplastic cells from a moderately dif­fe­ren­tiated squamous cell carcinoma strongly re­­active to the Ki67 marker. Immunolabeling with anti-Ki67 antibodies. Eosin X 100
Figure 4. Island of neoplastic cells from a moderately dif­fe­ren­tiated squamous cell carcinoma strongly re­­active to the Ki67 marker. Immunolabeling with anti-Ki67 antibodies. Eosin X 100

 

Figure 5. Poorly differentiated oral mucosal carcinoma, with apparently reduced vascularity. Col. hematoxylin – eosin X 100
Figure 5. Poorly differentiated oral mucosal carcinoma, with apparently reduced vascularity. Col. hematoxylin – eosin X 100


 

Figure 6. Image of tumor stroma from a poorly dif­fe­ren­tiated carcinoma heavily infiltrated with inflammatory cells and large lymphatic vessels. Immunolabeling with anti-D2-40 antibodies. Eosin X 200
Figure 6. Image of tumor stroma from a poorly dif­fe­ren­tiated carcinoma heavily infiltrated with inflammatory cells and large lymphatic vessels. Immunolabeling with anti-D2-40 antibodies. Eosin X 200

Conflict of interest: none declared  
Financial support: none declared
This work is permanently accessible online free of charge and published under the CC-BY. 

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