A novel approach for extracting DNA from formalin-fixed paraffin-embedded tissue using microwave.

BACKGROUND: Formalin-fixed paraffin-embedded (FFPE) tissue is the gold standard procedure for tissue preservation. However, the extraction of DNA is a cumbersome procedure as the extracted DNA is fragmented because of the cross-linking effect of formalin. Hence, the aim of the present study is to extract DNA from FFPE tissues using different techniques with a specific objective of comparing the extracted DNA, both quantitatively and qualitatively. METHOD: Ten samples of FFPE tissues were retrieved from the archives of the Department of Oral Pathology. Total genomic DNA was extracted by different methods which included QIAamp DNA FFPE Tissue Kit, Norgen DNA FFPE Tissue Kit, phenol-chloroform method, mineral oil-based extraction, M/10 NaOH solution method, and microwave method. A 280-base pair sequence was selected for evaluation of downstream amplification. RESULTS: The statistical analysis was performed using unpaired student's t-test to compare the DNA yields and quality obtained by microwave methods with other methods using SPSS software. Total genomic DNA retrieved by the microwave method was superior to other methods both qualitatively and quantitatively. CONCLUSION: DNA extraction from FFPE tissues is an onerous task as irreversible bonds form between the nucleic acid during fixation which are difficult to break during DNA retrieval. Hence, the microwave method provides good total genomic DNA which gives better downstream results when compared with other methods.

Role of phosphatase and tensin homolog in pathogenesis of ameloblastoma: An immunohistochemical study.

BACKGROUND: Altered molecular signaling pathways in ameloblastoma have been identified to play a pivotal role in the mechanism of oncogenesis, differentiation, and tumor progression. Phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin pathway is one of the signaling pathways that are associated with the pathogenesis of ameloblastoma. Phosphatase and tensin homolog (PTEN) controls cell migration and proliferation. It monitors the level of the Akt and maintains cellular integrity. The present study was aimed to study the immunoexpression of PTEN in ameloblastoma to understand its role in the pathogenesis of ameloblastoma. MATERIALS AND METHODS: Twenty cases of ameloblastoma and ten cases of normal tooth germ were subjected to immunohistochemical staining against PTEN. RESULTS: Strong PTEN immunopositivity was seen in the tooth germs, while weak positivity was seen in the ameloblastoma. The immunoscore for PTEN was calculated by adding the percentage score and the intensity score. Seventeen cases showed the reduced PTEN expression in the epithelial component of ameloblastoma. The unpaired t-test showed a statistically significant difference in the mean PTEN immunoscore in tooth germ and ameloblastoma. CONCLUSION: The study showed reduced PTEN immunoreactivity, which plays a role in the pathogenesis of ameloblastoma, through Akt pathway.

Assessment of Interleukin-18 gene polymorphism and serum levels in oral lichen planus in an Indian population.

BACKGROUND: Oral lichen planus (OLP) is a chronic, inflammatory disease with uncertain etiology. The aim of this study was to assess Interleukin-18 (IL-18) gene polymorphism and serum levels in OLP cases of Indian origin and to compare them with a control population of similar background. METHODS: The assessment of single-nucleotide polymorphisms (SNPs) of IL-18 gene at promoter regions -137(G/C) and -607(C/A) was done in 70 OLP cases and 70 healthy controls using sequence-specific primer-polymerase chain reaction (SSP-PCR). In a subset of this cohort, comprising of 41 OLP cases and 41 controls, serum IL-18 levels were assessed using enzyme-linked immunosorbent assay (ELISA). RESULTS: Mean serum levels of IL-18 among OLP cases were significantly higher when compared to controls. Genotypic and allelic frequencies of IL-18 at position -137(G/C) showed that GG genotype and allele G was significantly higher in OLP cases, whereas, GC genotype and C allele was high in the control group. Polymorphism of IL-18 at position -607(C/A) showed no significant differences. CONCLUSIONS: Gene polymorphism at -137GG genotype and allele G seems to be associated with genetic susceptibility to OLP whereas -137GC and allele C may have a protective role against its development. However, our study lacks clear statistical correlation, the differences observed could be caused by sampling problems and the results could not be fully representative of Indian patients with OLP. Further studies are warranted to explore the role of IL-18 genetic polymorphisms in OLP development.

Genetic alteration of Exon 5 of the PTEN gene in Indian patients with ameloblastoma.

OBJECTIVE: The PI3K/Akt/mTOR pathway is one of the signaling pathways associated with the pathogenesis of ameloblastoma. The phosphatase and tensin (PTEN) homologue controls cell migration and proliferation. It monitors the level of Akt and maintains cellular integrity. The aim of the present study was to study the genetic alteration of Exon 5 of the PTEN gene in Indian patients with ameloblastoma. STUDY DESIGN: Total DNA was extracted from formalin-fixed paraffin-embedded tissue samples from 20 cases with solid multicystic ameloblastoma (SMA) and from 10 cases with normal tooth germ. Exon 5 of the PTEN gene, was assessed for its role in the pathogenesis of ameloblastoma. RESULTS: Five of 20 cases of SMA showed genetic alteration. Of these cases 3 (15%) showed silent mutation, 1 (5%) showed change in amino acid sequence from valine to glutamic acid, and 1 (5%) showed nonsense-mediated mRNA decay. CONCLUSIONS: The present study showed 25% somatic mutational frequency in exonic region 5 of the PTEN gene. This may indicate its role in the pathogenesis of ameloblastoma.

Fungal osteomyelitis of maxillofacial bones: Rare presentation.

Osteomyelitis is defined as inflammation of the medullary cavities, haversian system and adjacent cortex of bone. Osteomyelitis involving maxilla is quite rare when compared to that of mandible. Fungal osteomyelitis is very rarely seen and documented in the maxillofacial area. It is devastating to patients if it is invasive in nature. A prospective study was undertaken from December 2011 to December 2013. Biopsied hard tissue bits were decalcified and sections were stained with H&E, periodic acid Schiff and Grocott methenamine silver. Culture sensitivity was carried out in all cases. Surgical intervention followed by antifungal therapy was advocated to treat the patients. The current series showed five cases of fungal osteomyelitis which included candidal osteomyelitis, aspergillosis and mucormycosis with slight predominance of maxilla over mandible. Osteomyelitis of facial bones needs to be investigated thoroughly as there is no difference in clinical presentation between bacterial and fungal osteomyelitis. The diagnostic workup with biopsy and culture sensitivity helps to identify the pathogen at the earliest. Appropriate antifungal treatment needs to be advocated for successful treatment.

Primary malignant melanoma of oral cavity: A report of three rare cases.

Oral malignant melanoma (OMM) is a rare tumor of melanocytic origin, accounting for 20-30% of malignant melanomas at the mucosal surface and 16% intra-orally. Hard palate and maxillary gingiva are the most common involved sites. In this case series, we present varying patterns of presentation of three cases of OMM with one case of distant metastasis. All cases in the current series presented at an advanced stage and died within a year of diagnosis. In conclusion, due to the aggressive clinical course and poor prognosis of this deadly lesion, it is of paramount importance to maintain a high index of suspicion for early detection and diagnosis for any pigmented lesion in the oral cavity.

Cementoblastoma Solely Involving Maxillary Primary Teeth--A Rare Presentation.

Cementoblastoma is a rare benign neoplasm of odontogenic ectomesenchyme origin, involving the roots of any tooth, which occurs predominantly in second and third decade of life. Very few cases of cementoblastoma associated with a primary tooth or having a maxillary presentation have been reported in the past. Here, a rare case of a ten year old boy who presented to the department with a swelling in maxillary posterior region since one month is being discussed. The radiographic presentation was mimicking an odontoma. The final diagnosis was cementoblastoma. We have advocated the use of polarized microscopy to support the histopathological diagnosis with respect to its cemental origin. Cementoblastoma should be considered in the differential diagnosis of radio-opaque lesions in the transitional dentition.

Calcifying ghost cell odontogenic tumor (CGCOT) with predominance of clear cells: a case report with important diagnostic considerations.

An 18-year-old female reported painful swelling in the left maxillary region 4 months after the extraction of tooth #15. Because incisional biopsy suggested ameloblastoma with clear-cell changes, segmental resection was performed. The microscopic examination of the excised tissue revealed histomorphologic diversity, with features of typical calcifying ghost cell odontogenic tumor (CGCOT) and a significant clear-cell component constituting more than 50% of the tumor mass without any features of malignancy. Clear cells were periodic acid-Schiff (PAS) positive and mucicarmine negative. CK19 was strongly positive in the solid tumor islands and variably positive in clear cells. A definitive diagnosis of CGCOT with predominance of clear cells was confirmed. Because both squamous metaplasia and clear-cell components were present in the ameloblastic follicles independent of each other, the presence of abundant calcifications favored the diagnosis of CGCOT with a clear-cell component over that of an ameloblastoma with clear-cell changes. The importance of identifying the histomorphologic features of the 2 entities, ameloblastoma and CGCOT, is highlighted. This appears to be the second case reported in the literature, to the best of our knowledge.

Post endodontic Aspergillosis in an immunocompetent individual.

UNLABELLED: Non-invasive aspergillosis in immunocompetent individuals subsequent to post endodontic treatment can involve the maxillary antrum. An early and accurate diagnosis will aid in prompt and effective treatment. A 35 year old female patient reported with a painful nasomaxillary swelling. Previous records revealed the failure of the endodontic treatment of maxillary left second premolar which was subsequently extracted. Root piece was accidently left behind which resulted in a painful nasomaxillary swelling. The extraction socket was curetted and tissue was sent for histopathological examination, which revealed abundant septate fungal hyphae with numerous spores characteristic of Aspergillus. The patient showed marked improvement in the symptoms with systemic itraconazole at 3 months follow up and complete resolution occurred within 6 months. Inclusion of aspergilloma infections in the differential diagnosis is advocated when patients present with post-endodontic nasomaxillary swelling. KEY WORDS: Aspergillosis, fungal sinusitis, post root canal treatment.

Biochemical and Cytological Comparison of Keratocystic Odontogenic Tumours to Nonkeratinising Odontogenic Cysts Fluid.

AIM: To evaluate the levels of albumin, prealbumin, total protein, inorganic phosphate and presence of keratinocytes in the cystic fluid for the diagnosis and appropriate treatment planning of keratocystic odontogenic tumours and other non keratinizing odontogenic cysts. MATERIALS AND METHODS: Fifteen keratocystic odontogenic tumour and 15 controls were studied. The cystic fluid was aspirated and analysed to determine the levels of albumin, prealbumin, total protein, inorganic phosphate and the presence of keratinocytes. The data collected was statistically evaluated using Mann Whitney U-Test and Student's t-test. RESULTS: A highly significant difference (p<0.0001) was seen when a comparison of Prealbumin, total protein, inorganic phosphate and presence of keratinocytes was made between keratocystic odontogenic tumour and non keratinizing odontogenic cysts. The presence of albumin also showed a significant difference (p<0.01). CONCLUSION: A combined analysis of total protein, albumin, prealbumin, inorganic phosphorous and detection of epithelial squames may be used as a diagnostic adjunct in the preoperative diagnosis of keratocystic odontogenic tumour in a minimally invasive and highly accurate fashion.

Analysis of salivary antioxidant levels in different clinical staging and histological grading of oral squamous cell carcinoma: noninvasive technique in dentistry.

OBJECTIVE: To estimate and Compare of salivary antioxidant level {Uric acid (UA), Glutathione S Transferase (GST) and Superoxide dismutase (SOD)} between healthy control and study group (oral squamous cell carcinoma patients).Further comparison of sub division of study group on the basis of clinical staging and histological grading. MATERIALS AND METHODS: The study group consists of 50 cases of squamous cell carcinoma and 50 healthy patients. These parameters were estimated by spectrophotometer. The biochemical values of this study were subjected to statistical analysis i.e. Independent t-test, ANOVA and Tukey test. RESULT: UA suggested statistically significant changes in saliva of clinical staging and histological grading of oral squamous cell carcinoma (SCC) patients. Salivary SOD level between well to poorly differentiated SCC showed a progressive increase although it is not statistically significant. CONCLUSION: Salivary analysis of antioxidant is simple, non-invasive technique which may be useful as diagnostic, prognostic and therapeutic marker.

Intra-osseous jaw lesions in paediatric patients: a retrospective study.

OBJECTIVE: This study was undertaken to comprehensively analyze the distribution of intra-osseous paediatric jaw lesions (0-16 years) and to correlate the same with the data which has been published in the literature. STUDY DESIGN: A total of 171 hard tissue paediatric pathologies obtained from the archives of Department of Oral Pathology, Maulana Azad Institute of Dental Sciences, India were retrospectively reviewed over a period of 6 years. All the cases were analyzed for gender, site, radiographic findings, histopathological diagnosis, treatment modality and recurrence rate. RESULTS: The present study revealed 54/171 odontogenic cysts, 45/171 odontogenic tumours, 33/171 bone pathologies, 2 malignant tumours, 1 connective tissue pathology and 36/171 miscellaneous category cases. The highlights of this analysis showed a relatively higher incidence of odontogenic tumours (26.3%) as compared to those seen in other studies which have been published in literature. KCOT and ameloblastoma (solid and unicystic) were the most frequently diagnosed tumours. Also, one case of dentigerous cyst which was converted into calcifying ghost cell odontogenic tumour was a unique feature which was noted in the current study. CONCLUSION: In the current study, a preponderance of odontogenic pathosis was seen in the paediatric age group which was studied.