Diagnostic and Prognostic value of Immunohistochemistry in Destructive Paediatric Maxillary Pathologies

Introduction: Establishing an accurate diagnosis and probable prognosis of ambiguous, extensive and destructive maxillary pathologies, is imperative for an appropriate, timely and effective treatment modality to be instituted. This is particularly true in the paediatric population, in order to ensure complete elimination of the lesion, with the least possible morbidity, debility, or interference with normal jaw growth.


Introduction
There often exists ambiguity in the diagnosis and management of large, destructive pathologies involving the maxilla in children 1,2 . Most of these destructive maxillary pathologies in the paediatric population, are either inflammatory, odontogenic or developmental in origin, arising from either the tooth forming apparatus or from epithelial remnants trapped within closing sutures of the craniomaxillofacial complex. They range from cysts and hamartomas to benign and even malignant neoplasms. Overlapping clinical, radiographic and histopathological features between the various lytic lesions such as Odontogenic cysts and tumours, compound the challenges and difficulties faced in distinguishing them from one another. Different pathologies vary widely in their clinical behaviour, aggressive and invasive potential, and tendency for recurrence and malignant transformation 3,4,5 , hence requiring vastly different management protocols.
HPE has remained to this day, the Gold Standard for making a confirmatory diagnosis of oral lesions, including destructive maxillary pathologies. On occasions, deceptively innocuous appearing unilocular maxillary lesions have unexpectedly been found to be ominous pathologies such as, benign or even malignant tumors 2 . Relative similarities in their clinical and radiographic presentation, as well as in their histological appearance, makes it both challenging as well as important to distinguish between the two, for an appropriate and timely management to be initiated 3 . It is at such times that IHC steps in as a valuable adjunct to HPE, aiding in an accurate identification of the pathology for an ideal treatment planning, thereon.
Too radical a treatment approach for an indolent and benign pathology can result in undesirable and unnecessary esthetic and functional morbidity in the child, while under-treatment of an aggressive pathology can lead to its persistence, recurrence or even malignant transformation. A precise and correct diagnosis is thus imperative for the institution of an ideal management protocol, in order to completely and effectively eliminate the pathology, while at the same time, ensuring the least possible morbidity, debility and risk of recurrence 3 .
IHC is the science of detection and localization of specific antigens (such as endocrine hormones, oncogene products or cell cycle participants) in biological tissue samples, by using labeled antibodies as reagents 4 . The ensuing Antigen-Antibody (Ag-Ab) interactions, resulting in the formation of immunological complexes, can be visualized using markers such as fluorescent dyes, chromogens, enzymes or colloidal gold.
IHC can be employed in two possible ways in the diagnosis and treatment planning of ambiguous maxillary pathologies in children 4 . One is, in their confirmatory identification by Tumor Immunodiagnosis, using 'Tumor Markers' (such as Calretinin, which is specific for odontogenic tumors like Ameloblastoma) that help to distinguish maxillary odontogenic cysts from odontogenic tumors 5 . The other is, evaluation of the proliferative activity of constituent cells of the pathology, by assessing their expression of various 'Cell Proliferation Markers' (Cell cycle associated antigens such as Ki-67 and PCNA) 6 , that give valuable information on the likely prognosis of the maxillary lesions.
Calretinin, is an intracellular antiapoptotic protein and a 'Tumor Marker', which is consistently expressed in all variants of Ameloblastoma and some cases of Keratocystic odontogenic tumors (KCOTs), whereas in none of the other cysts or tumors 5,7,8,9 , thus aiding in their differential diagnosis. In this Study of 25 cases of destructive maxillary pathologies in children, expression of Calretinin helped in distinguishing cystic lesions from odontogenic tumors, and in establishing a confirmatory diagnosis of ambiguous pathologies.
The biological behavior and probable prognosis of destructive maxillary pathologies in children, is rather difficult to predict on the basis of their clinical, radiographic and histological assessment alone 10,11 . Immunohistochemical expression of 'Cell Proliferative Markers / Cell cycle associated antigens' like Ki-67, PCNA (Proliferating Cell Nuclear Antigen), AgNORs (Silver staining Nucleolar Organizer Regions) 4 , can give valuable information in this aspect, as presumably, aggressiveness of any lesion is directly proportional to the proliferative activity of its constituent cells 6 . In this Study, 'Ki-67' and 'PCNA' were employed to assess the aggressive potential of each of the paediatric maxillary pathologies.
An extensive study has been carried out in 25 cases of large, destructive unilocular radiolucent lesions involving the maxilla in a paediatric population (age range of 5 to 16 years), over a period of two years. Details of the Immunodiagnostic procedures employed, management protocols implemented, results achieved and follow up findings have been presented in this study.

Aim And Objectives
Aim: To establish the value of IHC as a Diagnostic Marker to clinch the diagnosis and as a Prognostic Indicator to predict the aggressive potential of 25 cases of extensive maxillary lesions in children in the age range of 5 to 16 years, and thereby to formulate and institute the most ideal and appropriate treatment modality for each case.
Objectives a) To test the correlation between the histopathological typing of the paediatric destructive, unilocular maxillary lesions with their IHC analysis by using a 'Tumor Marker', namely Calretinin, in order to distinguish cysts from the tumors, and to arrive at a precise and conclusive confirmatory diagnosis. b) To select the most appropriate and effective treatment option for each case, based on the information on probable prognosis provided by the IHC expression of two 'Cell Proliferation Markers', namely Ki-67 and PCNA, by the constituent cells of the pediatric maxillary pathologies.

Materials And Methods
Sample and Sample Size: Twenty-five cases of large, unilocular, destructive, unilocular, radiolucent lesions of the maxilla, in a paediatric population in the age range of 5 to 16 years, were managed over a period of two years.

Ethical Clearance and Informed Consent:
All procedures performed on the patients (human participants) involved in this Study, were in accordance with the ethical standards of the institution and national research committee, as well as with the 1964 Helsinki declaration and its later amendments and comparable ethical standards. Institutional Ethical committee clearance was obtained prior to commencing the Study. This Study does not contain any new studies with human participants or animals performed by the author.
Informed written consent was obtained from all the individual participants in this study, including parents of the minors.
In all the 25 cases of destructive maxillary pathologies, HPE and IHC analysis were carried out of the biopsy specimens, in order to establish a confirmatory diagnosis and also to assess the clinical nature of the lesions, in order to formulate an ideal treatment plan for each individual case. Calretinin, an IHC Tumor marker, was employed to distinguish between Odontogenic cysts and tumours. Ki-67 and PCNA, both IHC Cell Proliferation markers, provided information on the biologic behaviour and aggressive potential of these lesions.
On the basis of the above information, an accurate diagnosis was arrived at and the most appropriate definitive management protocol was established for each of these different lesions on a case to case basis, in order to achieve the most ideal functional and esthetic outcome (Figures 1-12).

HPE and IHC Analysis:
Average size of the tissue samples tested were 1cm X 0.5 X 0.5cm. All samples were formalinfixed and paraffin embedded. The blocks were sectioned using a cryotome, into 4 to 7µm thick sections, that were placed on Super Frost slides for the Haematoxylin-Eosin and IHC staining. HPE was performed using Haematoxylin-Eosin stained sections observed under magnifications of 10X, 50X and 100X. IHC studies were carried out, by staining for Calretinin, Ki-67 and Proliferating Cell Nuclear antigen (PCNA).

IHC Technique:
Labelled secondary antibodies were used to detect the three antigens of interest. The Avidin-          Biotin Immunoperoxidase (ABI) staining technique was employed using Diaminobenzidine (DAB) as the chromogen for visualization of the Immunological complexes formed. Positivity was expressed as brown nuclear staining of the cells.
Steps in IHC Staining: Antigen retrieval of the Paraffin embedded tissue sections mounted on the slides, was carried out by the method of HIER (Heat Induced Epitope Retrieval), using a microwave. Specimen were blocked to avoid unspecific cross reaction and background staining. Presence of endogenous enzymes, such as peroxidase, which are preserved in paraffin were blocked by incubating the sections with a mixture of H 2 O 2 and methanol or incubating sections with 0.075% HCl in ethanol at room temp for 15 min before immunostaining.
Thereafter, the slides with the tissue specimen were  incubated overnight (12 hours) at 4 0 C with the Primary Monoclonal Antibody raised against the three Human Antigens of interest that were to be detected, namely, the Calretinin, Ki -67 Ag and Proliferating Cell Nuclear Antigen (PCNA). 25 to 50 µL of diluted Ab, a volume sufficient to completely cover the tissue was employed, so as to ensure that the tissue specimen would not dry out during incubation. Properly optimized working dilutions of the three Antibodies, in this case, 1:100 dilutions, were used.
The Primary Ab was unlabeled, while the secondary Ab was the labelled Ab. An egg white protein called Avidin has a very strong affinity for Biotin, so an Avidin-Horseradish Phosphate (HRP) Complex was used as the reporter molecule which forms the ABI complex which can be visualized or developed using DAB as the chromogen which is turned brown by the HRP. Thus, the nuclei expressing Ki 67 Ag and PCNA Ag turn brown.
As both Ki-67 and PCNA are Cell cycle associated antigens located within the nuclei, positivity was expressed as brown IHC Nuclear staining. Positivity for Calretinin, however, was expressed as both nuclear as well as cytoplasmic staining.

Calculation of LI / PI Values:
The Ki-67 and PCNA Labelling Index (LI) or the Index of Positivity / Proliferation Index (PI) was calculated by counting the number of positive cells in five histopathological fields, dividing by the total number of cells in that field and multiplying by 100.

Results
Confirmatory diagnosis for each destructive maxillary lesion was made by correlating the HPE and IHC analysis of the biopsy specimens (Table 1). Ten cases were diagnosed as Radicular / Periapical / Palatal cysts of inflammatory origin, associated with infected / non-vital teeth. These lesions demonstrated negativity for all three IHC markers. Eight cases were diagnosed as Dentigerous cysts associated with one or more impacted / unerupted teeth ( Table 1). Two of these Dentigerous cysts showed histological as well as Immunohistochemical evidence of ameloblastomatous transformation, by demonstrating positivity for Calretinin as well as Ki-67 and PCNA, while the remaining six exhibited nil immunoreactivity. Two cases were diagnosed as Adenomatoid odontogenic tumors (AOT) / Adenoameloblastomas, demonstrating positivity for Calretinin but not for Ki-67 or PCNA. Two cases were diagnosed as Unicystic Ameloblastomas of the luminal variety and two cases as Odontogenic keratocysts (OKC) / Keratocystic odontogenic tumors (KCOT), all of them demonstrating positivity for all three IHC markers. One case was found to be a large Globulomaxillary cyst with negative IHC expression (Table 1).
Treatment plan was formulated on a case to case basis, based on the diagnosis established by correlating the histopathological presentation with the expression of the IHC Tumor Marker, namely, Calretinin. The treatment plan was also guided by the likely prognosis of each case, as indicated by the expression IHC Cell Proliferation Markers, namely Ki-67 and PCNA, by the constituent cells of the lesions.
The Inflammatory pathologies and Odontogenic cysts which exhibited negativity for all three IHC markers, were managed conservatively. Those cystic lesions undergoing ameloblastomatous transformation, the Unicystic Ameloblastomas, Keratocystic odontogenic tumors and Adenoameloblastomas, all of which exhibited positivity for the IHC markers, were managed more aggressively, in order to ensure their complete elimination, leaving behind no residual cells or satellite cysts, so as to prevent future  recurrence of the lesion. The Ki-67 and PCNA Proliferation Index (PI) / Labelling index (LI) values were relatively low, that is <= 1.5, hence no case necessitated jaw resection / maxillectomy.
In the cases of unicystic ameloblastomas, the positivity for Calretinin, PCNA and KI-67 was exhibited mainly by the peripheral ameloblast-like cells lining the cystic cavity, followed by the areas of the epithelial islands within the cyst wall.

Discussion
Owing to the wide diversity in origin, etiopathogenesis and biological behaviour of different paediatric destructive maxillary pathologies, a precise and accurate diagnosis, followed by the most appropriate and effective management modality, that completely eliminates the lesion with least patient morbidity, is imperative for a successful esthetic and functional outcome. In this study IHC in conjunction with HPE, provided valuable information as to both, the diagnosis as well as the prognosis of these pathologies, contributing greatly to formulation of an ideal and efficacious treatment plan for each case.
In this Study, expression of Calretinin helped in distinguishing cystic lesions from odontogenic tumors, and also in identifying ameloblastomatous changes in Dentigerous cysts. This enabled implementation of the most appropriate surgical approach in each case.
Expression of 'Ki-67' and 'PCNA' was employed to assess the invasiveness, aggressiveness and recurrence potential of each of the paediatric maxillary pathologies in this Study. Negative expression of the IHC markers were suggestive of indolent and non-aggressive, slow growing pathologies, which were amenable to conservative treatment approaches, while positive IHC expression was suggestive of aggressive pathologies, meriting a more radial management approach to avoid possible recurrence.
In this Study of 25 cases of paediatric destructive, unilocular maxillary pathologies, at one end of the spectrum were those lesions which were found negative for all three IHC markers, thus ruling out a tumor, and indicative of a cystic lesion of low growth potential. These pathologies corroborated with the HPE findings of Periapical / Radicular cysts, Dentigerous cysts, Primordial and Globulomaxillary cysts. On the basis of the prognosis indicated by the HPE and IHC assessment, these pathologies were treated effectively using a conservative surgical approach of enucleation and curettage, accompanied by extirpation of the maxillary antral lining in those cases where the cyst lining was found contiguous with the lesion. The conservative approach ensured a successful elimination of the pathology, while at the same time producing the least functional morbidity and esthetic deformity.
At the other end of the spectrum of this Study, were those paediatric maxillary pathologies, which were found positive for all three IHC markers, and corroborated with the histological typing of Unicystic ameloblastomas or Dentigerous cysts undergoing ameloblastomatous transformation or OKCs. These lesions exhibited positivity for the Tumor marker, Calretinin, as well as the Cell proliferation markers, namely, Ki-67 and PCNA, albeit exhibiting low Labelling Index values (<1.5). These lesions were managed more aggressively, by enucleation, curettage, extirpation of the antral lining, peripheral ostectomy of the bony walls of the cavitation and chemical cauterisation of the antral walls with Carnoy's solution. The relatively aggressive treatment approach enabled complete elimination of the lesion, and also ensured minimising all possible chances of recurrence of the pathology, which would otherwise have necessitated repeat surgeries and possible future cosmetic deformity and functional debility.
All the cases of Periapical / Radicular and Palatal cysts (IHC Labelling Index Value = 0) were managed conservatively, by Root Canal Therapy of the involved tooth, followed by enucleation and curettage of the cystic lesion (Cases 5, 6 & 7; Figures 10, 11 & 12). This was followed by placement of fresh autologous Platelet Rich Fibrin within the bony cavity to enhance soft tissue healing as well as hasten subsequent reossification and bone fill. As, there was no case of malignant tumors or benign, aggressive pathologies with medium (LI >1.5) or high IH Labelling Index (LI >3) values, encountered in this study, no case was indicated for a more aggressive management protocol such as partial or complete maxillectomy.
There was no incidence of recurrence of the pathology in any of the 25 cases, during the entire follow up period, which ranged from one to two years. Excellent functional and esthetic outcomes were achieved in all the cases, with nil incidence of morbidity or functional debility. There were no neurological deficits observed in any case. Minor post-operative complications such as pain, swelling and edema resolved within three to five days following surgery. A superior bone fill and reossification of the lytic areas, was observed in those cases where fresh, autologous PRF had been placed intraoperatively, within the operative site prior to closure.
In this study, the IHC analysis of the biopsy specimen was corroborated with that of the final excised / enucleated specimen in all the cases, with no discrepancy in results noted in any case. Taking care to obtain a representative sample of the pathology at the time of biopsy, is essential for an accurate diagnosis to be made, and for the most ideal and effective treatment procedure to be implemented. A possible risk during biopsy of such destructive maxillary pathologies, could be to miss out the section of a Dentigerous cyst that might be undergoing ameloblastomatous transformation, and hence underreporting of the specimen with the subsequent treatment rendered, being more conservative than desired. Hence, a thorough histopathological and IHC examination of the entire pathology following excision is important for confirmation of the diagnosis.
A possible limitation of this Study could be the limited number of Cases observed and treated. Another drawback is that Immunohistochemistry is not always infallible as it is an intricate and technique sensitive lab procedure, where mistakes can occur during tissue processing and errors in antigen retrieval, yielding false positive or negative results. Hence, IHC must be viewed as a valuable adjunct to, rather than a substitute for Light microscopy.

Conclusion
Immunohistochemical analysis served as a valuable adjunct to histopathogical examination, in the management of 25 cases of destructive maxillary pathologies in the paediatric population. Calretinin served as a reliable IHC diagnostic marker, which helped in reaching a precise confirmatory diagnosis. Ki-67 and PCNA served as useful predictive and prognostic indicators, by providing valuable information about the nature of the lesions, in terms of their biological behavior, aggressiveness and tendency for recurrence. Armed with this information, the surgeon was able to make an informed and sound clinical decision, and employ the most efficacious management protocol for each case. This helped in achieving the best functional and esthetic outcomes in all the 25 cases, with the least possible morbidity, deformity or debility, and nil incidence of recurrence in any of the affected children.

Disclosure of potential Conflicts of Interest
The author of this article has not received any research grant, remuneration, or speaker honorarium from any company or committee whatsoever, and neither owns any stock in any company. The author declares that he / she does not have any conflict of interest.

Research involving human participants and /or animals
All procedures performed on the patients (human participants) involved were in accordance with the ethical standards of the institution and/or national research committee, as well as with the 1964 Helsinki declaration and its later amendments and comparable ethical standards.
Ethical approval: This article does not contain any new studies with human participants or animals performed by the author.
Informed Consent: Informed consent was obtained from all the individual participants in this study, including parents of the minors.

Funding
This study was not funded by any organization / society.