Oral Biopsy Research Articles

Accuracy, Sensitivity and Specificity of Oral Brush Cytology in the Diagnosis of Oral Epithelial Lesions

Background: Oral Squamous cell carcinoma (SCC) is highly prevalent in Pakistan than the rest of the world. Five-year survival rate is 80% if SCC is diagnosed at an early stage. The survival rate declines to 20% if diagnosed at a later stage. The objective of the present study was to find out the usefulness of oral brush cytology in detecting oral lesions.
 Material and Methods: In the present prospective observational study, 88 samples were collected from Maxillofacial Surgery Out-Patient Department (OPD), Liaquat University Hospital and ISRA University Hospital from July 2015 to December 2015. After taking written informed consent, oral brush cytology was performed and stained with standard Papanicolaou (PAP) staining protocol. The biopsy of the patients was performed by standard protocols of oral biopsy and specimen preserved in 10% buffered formalin. Cross tabulation between diagnosis of brush cytology and biopsy of same patients was done and accuracy, sensitivity and specificity were calculated.
 Results: According to the distribution of patients on brush cytology, 59.1% patients were diagnosed with malignant tumors, whereas 17.1% had benign tumors. On biopsy of the same patients, squamous cell carcinoma was confirmed in 72.7% and benign tumors were found in 17% of the patients. Comparison of brush cytology with biopsy of same patients revealed no significant difference. Accuracy, sensitivity and specificity of brush cytology of the oral cavity for detection of malignant tumor were calculated as 86.36%, 81.25% and 100% respectively. Whereas accuracy, sensitivity and specificity of brush cytology of the oral cavity for detection of benign and inflammatory conditions were 100%.
 Conclusion: Oral brush cytology has good accuracy, sensitivity and specificity for detection of oral epithelial lesions and can be useful in early detection of oral cancer as well as other lesions. Moreover, as an easy-to-do, painless and non-invasive procedure, it can be a good screening method for detection of oral lesions.

Dental Hygienists and Dentists as Providers of Oral Mucosa Screening and Brush Biopsies

Dental Hygienists and Dentists as Providers of Oral Mucosa Screening and Brush Biopsies

Clinical Outcome of Autologous Cultivated Oral Mucosal Epithelial Transplantation in Ocular Surface Reconstruction.

To evaluate the outcomes of autologous cultivated oral mucosal epithelial transplantation (COMET) in ocular surface reconstructive procedures. Twenty-five eyes of 24 patients who underwent COMET for ocular surface reconstruction were studied retrospectively from April 2011 to March 2014. Oral mucosal biopsy of 2 mm was harvested from all patients and cultured on amniotic membrane for 14 days. COMET was performed as a means to achieve corneal epithelization (group 1) and for fornix reconstruction (group 2). The extent of epithelization achieved (group 1) and the depth of the fornix reconstructed (group 2) were the primary outcome measures. Six eyes of 5 patients [2 chemical injury (CI), 3 Stevens-Johnson Syndrome (SJS)] underwent COMET for nonhealing of an epithelial defect within a month after insult or had a nonhealing defect since insult despite maximal medical/surgical therapy. Group 2 included 19 eyes of 19 patients (10 CI, 8 SJS, 1 ocular cicatricial pemphigoid) which underwent COMET for fornix reconstruction. Postsurgery, the cornea was fully epithelized in 66.67% of the eyes (n = 4) and partially epithelized in 33.33% of the eyes (n = 2). The overall improvement in surface epithelization was statistically significant (P = 0.046). Reconstruction of an anatomically deep fornix was achieved in 57% of the eyes (4 CI, 6 SJS and 1 ocular cicatricial pemphigoid). The fornix was partially formed in 26% of the eyes (4 CI and 1 SJS). The overall improvement in fornix reconstruction was considered statistically significant (P = 0.024). The follow-up ranged from 1 to 5 to 27 months with a mean follow-up of 18 months SD ±8.9. COMET, by providing an alternate source of epithelium, aids in faster epithelization and thus can be considered as an option in management of severe grade CI or SJS in the acute stage as well as in fornix reconstructive procedures in chronic stage of ocular surface disorders.

The optimal oral biopsy site for diagnosis of mucous membrane pemphigoid and pemphigus vulgaris.

Accepted 'standard practice' for the diagnosis of immunobullous disease is a perilesional sample for direct immunofluorescence (DIF). To compare diagnostic outcomes of a normal buccal punch biopsy (NBPB) with a perilesional biopsy (PLB) for mucous membrane pemphigoid (MMP) and pemphigus vulgaris (PV). A retrospective analysis of 251 DIF-positive patients with MMP and 77 DIF-positive patients with PV was undertaken. Parameters analysed included the intraoral sites of involvement and histopathological, DIF and indirect immunofluorescence (IIF) findings. For MMP, PLB was positive in 134 of 143 (93·7%) samples, compared with 129 of 144 (89·6%) by NBPB. The diagnostic sensitivities for PLB (81%, 39 of 48) and NBPB (77%, 37 of 48) among 48 patients who underwent both techniques were not significantly different (P = 0·62). In gingival-only MMP, PLB was positive in 63 of 69 (91%) and NBPB was positive in 63 of 75 (84%). For multisite MMP, PLB was positive in 71 of 74 (96%) and NBPB was positive in 66 of 69 (96%). In gingival-only MMP, biopsies from reflected alveolar mucosa in 17 consecutive patients were positive in 17 of 17 cases (100%). For PV, PLB was positive in 42 of 43 (98%), compared with 42 of 42 (100%) by NBPB. Histopathology was diagnostic in 93 of 134 (69·4%) cases of MMP and 38 of 41 (93%) cases of PV. IIF was positive in 126 of 197 (64·0%) MMP and 68 of 74 (92%) PV patient sera. In the largest series of combined oral DIF results in patients with MMP and PV, we have shown that NBPB is equivalent to PLB for the diagnosis of PV and multisite MMP, and is more sensitive than both histology and IIF. What's already known about this topic? The variation in sensitivity of oral biopsy sites for direct immunofluorescence (DIF) in the diagnosis of oral MMP and PV has not been studied in detail in large series of patients. Biopsy can be challenging due to difficult access and fragility of the oral mucosa. The diagnostic biopsy technique is therefore critical. What does this study add? We have shown that a normal buccal punch biopsy (NBPB) from uninvolved oral mucosa is as sensitive as a perilesional biopsy (PLB) for diagnosis of oral PV, and superior to serology and histology. For multisite MMP, NBPB is equivalent to PLB and is more sensitive than serology and histology. The oral punch biopsy technique on uninvolved buccal mucosa tissue is a simple and safe practical method for diagnosing oral PV and MMP.

Lichenoid reaction with granulomatous stomatitis: A retrospective histologic study of 47 patients

Lichenoid granulomatous reaction can be subcategorized into lichenoid granulomatous dermatitis or lichenoid granulomatous stomatitis. It is described in the literature as demonstrating a band-like lichenoid infiltration with clusters of histiocytic or granuloma formation. In this study, we presented a large case series of lichenoid granulomatous stomatitis including demographics, histological features, and subcategories and compared findings with current literature. We retrieved all cases diagnosed with lichenoid reaction with granulomatous inflammation between January 1, 2000, and August 1, 2016, from the University of Florida Oral Pathology Biopsy Service Archives. A total of 47 patients with tissue from 50 biopsy sites were included. The majority of the cases were noted in females (64%) with a mean age of 59years (range 30-88). Most patients were Caucasian, and the most common sites in descending order were: gingiva, buccal mucosa, vestibule, tongue, lip, palate, and occasional multifocal involvement. Clinical impressions by the treating clinicians included leukoplakia, dysplasia, carcinoma in situ, squamous cell carcinoma, lichen planus, vesiculobullous disease, trauma, and allergy. The histologic features of these cases ranged from lichenoid mucositis with numerous aggregates of histiocytes to well-formed granulomas, in accordance with previous classifications of similar dermatologic lesions. All cases revealed positive CD 68 in the histiocytic infiltrates and were negative for microorganisms utilizing acid-fast bacilli, Grocott methenamine-silver, and periodic acid-Schiff stains. Lichenoid granulomatous disease may be more common than previously reported; however, its etiology remains unknown and patients should be kept under long-term clinical follow-up.

SOFAT as a Putative Marker of Osteoclasts in Bone Lesions.

Secreted osteoclastogenic factor of activated T cells (SOFAT) is a novel activated human T-cell-secreted cytokine that induce osteoclastogenesis in a RANKL-independent manner. The aim of this study was to evaluate the immunohistochemical expression of SOFAT in intraosseous and extraosseous lesions. Thirty-two oral biopsies were divided into 2 groups: (1) intraosseous lesions-4 cases of cherubism, 5 central giant cell lesions, 3 osteoblastomas, 3 cementoblastomas, 2 periapical lesions and (2) extraosseous lesions-5 peripheral giant cell lesions, 5 cases of oral paracoccidioidomycosis, and 5 foreign body reactions. Immunohistochemistry was performed for SOFAT and tartrate-resistant acid phosphatase. Image analysis consisted of a descriptive evaluation of the immunohistochemical staining pattern observed. Tartrate-resistant acid phosphatase-positive lesions included those containing multinucleated giant cells (MGC) from both groups. SOFAT was positive in MGC of the intraosseous lesions group, except in periapical foreign body reactions as well as extraosseous lesions. SOFAT was shown to be a putative marker of osteoclasts, which proved useful to differentiate them from multinucleated macrophages. Osteoclast induction may be both dependent and independent from the RANK/RANKL/OPG pathway and independent from the bone microenvironment.

CDK4, CDK6, cyclin D1 and Notch1 immunocytochemical expression of oral brush liquid-based cytology for the diagnosis of oral leukoplakia and oral cancer.

This study aimed to investigate the feasibility of using oral liquid-based brush cytology (OLBC) coupled with immunocytochemistry as a minimally invasive approach to stratify the cancer risk in patients with oral leukoplakia. Fifty-five patients diagnosed with either oral leukoplakia (OLK) or oral squamous cell carcinoma (OSCC) were recruited. All patients underwent oral brush biopsy followed by surgical biopsy. 275 liquid-based cytology preparations were made. Pap-stained OLBC slides were assessed using the modified 2014 Bethesda Cytology system. The expression of CDK4, CDK6, cyclin D1, and Notch 1 was immunocytochemically analysed and compared against the histopathological diagnosis. A combined index score of OLBC grading and protein expression was calculated. A significant association was found between the definitive histopathological diagnosis and the cytological interpretation (P=0.0005). The index scores of CDK4, CDK6, and cyclin D1 were significantly associated with the development of disease from non-dysplastic epithelium to OSCC. No significant association was observed between the Notch 1 index score and disease stage. The diagnostic accuracy of OLBC showed the highest values of sensitivity, specificity, positive predictive value, negative predictive value, and accuracy: 84.6%, 70.4%, 73.3%, 82.6%, and 78.8%, respectively, compared with the cumulative protein index, CDK4/6 index, and the combined OLBC grading and CDK4/6 index. This study has also demonstrated the efficacy of the use of OLBC in the detection of OED and OSCC, and showed that the use of CDK4, CDK6, cyclin D1, and Notch 1 immunocytochemistry failed to improve the diagnostic accuracy of OLBC suggesting they are not useful in the early detection of OSCC.

DIFFERENTIAL EXPRESSION OF PD1 AND PDL1 IN ORAL POTENTIALLY MALIGNANT LESIONS AND ORAL SQUAMOUS CELL CARCINOMA:A PILOT STUDY

Background Programmed cell death protein 1 (PD-1, CD279) is a 50-55 kDa type I transmembrane receptor expressed by activated T and B cells, as well as subset of monocytes and dendritic cells (DCs). PD-1 and its ligands (PDL1, PDL2) are part of “checkpoint” immune recognition and peripheral tolerance system that emerged as a critical signaling pathway in cancer. PDL1 is expressed in various types of cancers and activation of PD1-PDL1 inhibits T-cell mediated cancer surveillance. Here we describe a quantitative, reproducible 2-color fluorescence-based protocol to determine the differential expression of PD1/PDL1 in oral biopsy specimens. Methods Histopathological samples with a diagnosis of hyperkeratosis (HK), OMPL (mild, moderate, severe dysplasia) and squamous cell carcinoma (OSCC) were selected from the archives of the Toronto Oral Pathology service, University of Toronto. FFPE sections were stained with monoclonal antibodies for PD1 and PDL1 (Abcam) and Alexa Fluor-labelled secondary antibodies allowing visualization of both proteins in the same section using a spinning disk confocal microscope (Quorum). PDL1 staining was assessed in basal/spinous layers of the epithelium while PD1 staining was assessed in inflammatory cells in tumor stroma/lamina propria. The mean fluorescent intensity (MFI) was quantified and normalized against background signal. Results Our results show a significant increase in PD1 expression in inflammatory cells in dysplasia and OSCC compared to hyperkeratosis. PDL1 expression in epithelial cells was significantly increased in OSCC but not in dysplasia or HK. The results suggest that PD1 increase in inflammatory cells precedes malignant transformation while PDL1 overexpression in epithelial cells only occurs after malignant transformation. Conclusion We developed a new quantitative method to study PD1/PDL1 expression in FFPE oral biopsy samples. The expression of PD1 and PDL1 may be used as predictive markers of transformation and the data may be used to develop early intervention in OPML using PD1 inhibitors.

CHARACTERIZATION OF THE INflAMMATORY INfiLTRATES IN ORAL EPITHELIAL DYSPLASIA AND ORAL SQUAMOUS CELL CARCINOMA USING A NEW MFCA METHOD

Oral cancer is a devastating disease and tumor associated inflammation is a key component of the tumor microenvironment. Current techniques to evaluate inflammatory infiltrate are based on a visual, operator-based quantification and may not accurately quantify specific inflammatory signatures. Objective To develop a method for characterizing the inflammatory infiltrate associated with oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC) using confocal microscopy and multichannel fluorescent colocalization analysis (MFCA). Methods We performed a retrospective analysis of 49 biopsy samples of lateral tongue lesions with a diagnosis of hyperkeratosis, ED and OSCC. The inflammatory infiltrate was identified using a combination of 2 primary antibodies for each cell type followed by staining with Alexa 488 or 555 tagged secondary antibodies for FIHC. Identification of the inflammatory cells was performed by 2-channel colocalization using a custom-made, semi-automated algorithm in Volocity 6.3. Results Using our novel analysis technique we identified and quantified neutrophils, TCD8, TCD4, eosinophils, plasma cells, B cells, Macrophages and NK cells in biopsy specimens. T-lymphocytes represented the main component of the inflammatory infiltrate in all specimens and there was a marked increase in inflammatory cell density from benign to OSCC lesions. Our results also showed that the CD4/CD8 ratio and neutrophils/lymphocytes ratio (NLR) had a progressive increase when moving from benign lesions to OSCC. Conclusions We described a new, method to quantify inflammatory infiltrates in oral biopsies. This semi-automated approach decreases operator bias and provides robust and reproducible data to study inflammation in tissue samples. Using this technique, we provide evidence that cancer progression is mirrored by progressive changes in the inflammatory infiltrate. Significance Understanding specific changes in cancer associated inflammation is essential to develop immune-targeted therapies. This technique and our current results will be further explored as a potential prognostic maker of oral cancer.

ODONTOGENIC MYXOMA: A 23-YEAR RETROSPECTIVE SERIES OF 38 CASES

Introduction Odontogenic myxoma (OMX) is an uncommon benign tumor arising in the jaw. Though it has some histologic overlap with other entities, correct diagnosis is imperative considering the aggressive nature, high recurrence rate, and necessity of radical surgical intervention in large sized lesions. Materials and Methods With IRB approval, a retrospective search of the University of Florida College of Dentistry Oral Pathology Biopsy Service archives from 1994-2017 for diagnosis of OMX of the mandible or maxilla was performed. Biopsy reports and original slides for each case were assessed and reviewed along with any accompanying radiographs to confirm the diagnosis. Immunohistochemical (IHC) staining was utilized to exclude entities with histologic overlap such as intraosseous myxoid neurofibroma. Results A total of 38 cases were included. The patients’ ages ranged between 13 to 82 years, with a mean age of 38.5 years. Females comprised two-thirds of the cases (n=25) versus males (n=13). The mandible was the most affected at 56% (n=21), followed by maxilla 34% (n=13) with 10% (n=4) not specified. Posterior jaw involvement was higher than anterior in both the mandible (n=17 versus n=1) and the maxilla (n=8 versus n=4). The right side was more commonly affected than the left side in both arches. Most lesions presented clinically as expansile masses with variable radiographic appearance, and the submitting providers’ clinical impressions included gelatinous masses, reactive gingival lesions, abscess, odontogenic lesions, fibro-osseous lesions, and soft tissue or bone neoplasms. In 30 cases (79%) the histologic diagnosis was compatible with OMX, while in 8 cases (21%) a more fibrous stroma was identified with diagnoses of fibromyxoma. Conclusion OMX may exhibit varied demographic and clinical profile and wide spectrum of histologic presentation. Oral and maxillofacial pathologists and surgical pathologists should be sentient of this variability of presentation for accurate diagnosis and management.

AMELOBLASTIC fiBRO-ODONTOMA: A DISTINCT ENTITY

Background Ameloblastic fibro-odontoma (AFO) is a benign odontogenic tumor first described by Hooker in 1967. Its etiology and behavior have long been debated, as some investigators have proposed that AFO may represent a stage in development of an odontoma. For this reason, AFO was eliminated from the most recent Odontogenic and Maxillofacial Bone Tumor section of the World Health Organization (WHO) Head and Neck classification system. Occasional AFOs, however, have been found in patients older than the proposed age for odontoma completion (22 years) or present as large radiolucent lesions consisting mainly of the ameloblastic fibroma (AF) pattern with only foci of mineralized product formation. Herein, we present seven cases of AFO, all of which demonstrate particularly aggressive radiographic and/or histopathologic features and do not support the contention that all AFOs represent maturing odontomas. Materials and Methods An IRB-approved retrospective search of the oral pathology biopsy services at the Universities of Kentucky and Florida between January 1, 1975 and January 1, 2018 was completed. Cases with appropriate histopathological and radiographic documentation were selected. Results Seven patient cases were identified with ages 8, 8, 12, 16, 17, 27, and 29 years. Six cases were from the posterior mandible, and one was located in the posterior maxilla extending into the maxillary sinus close to the floor of the orbit. Only two of the cases have follow-up information, both of which demonstrate no evidence of tumor following conservative treatment. Conclusion Although the majority of cases diagnosed as AFO likely represent developing odontomas, we present seven cases in which the clinical, histopathologic, and/or radiographic features suggest that AFO should exist as a distinct entity and be treated similarly to an AF.

CHRONIC ULCERATIVE STOMATITIS: A LICHENOID OR VESICULOBULLOUS DISEASE?

Objectives Chronic ulcerative stomatitis (CUS) is a rare disease of unknown etiology. The histopathologic features are similar to lichen planus, but direct immunofluorescence (DIF) studies show characteristic presence of IgG in basal and parabasal epithelial nuclei.This study will review a case series of CUS and assess if the entity is more similar to lichen planus or vesiculobullous diseases. Methods An IRB-approved retrospective search of CUS was performed within the archives of the UF Oral Pathology Biopsy Service between 2007 and 2017. Findings Seventeen cases, all female, were included. The median age was 64 years (range 47-83 years). Eleven patients were Caucasian, one was Asian, and one was African-American. Race was not specified in four cases. Buccal mucosa (8/17) was the most common location, followed by gingiva (7/17), buccal vestibule (1/17), and gingiva/buccal mucosa (1/17). The most common clinical presentations were pain/burning (13/17), erythema (13/17), whiteness (11/17), ulcerations/erosions (5/17), blisters/positive Nikolsky's sign (5/17), sloughing (2/17), striae (2/17), and recession (1/17). The clinical impression was lichen planus in 12 cases. Of these twelve cases, 4 included vesiculobullous disease as a differential. Four cases did not include a clinical impression and one listed erythema multiforme as the clinical impression. All cases were confirmed with DIF testing that showed a characteristic speckled pattern of IgG in basal and parabasal cells. Eleven of these cases were also positive for fibrinogen and two cases were faintly positive for C3. None of the cases were positive for IgA or IgM. Conclusion Since CUS has overlapping clinical, histological, and immunofluorescence features with lichen planus and vesiculobullous diseases, clinicians and pathologists should consider this unusual, but significant, entity whenever oral ulcerative diseases with mixed features are encountered.

DIAGNOSTIC CONCORDANCE AMONG PATHOLOGISTS INTERPRETING ORAL MUCOSAL BIOPSIES FROM INDIVIDUALS AFFECTED BY GVHD

Objective The objective of this study was to compare the final diagnosis agreement between 4 pathologists when they follow the Horn criteria and the NIH criteria for analysis of oral mucosa and minor salivary glands biopsies possible affected by GVHD. Findings 41 slides of oral mucosa and minor salivary glands obtained from individuals with GVHD were analyzed by 4 oral pathologists. Following the Horn criteria based on minor salivary gland and oral mucosa independently, all pathologists gave the same final diagnosis in 11 out of 41 cases, 3 of them agreed in 12 cases and 2 in 18 cases. Following NIH Consensus based on specific criteria in both epithelium and salivary glands, 4 pathologists gave the same final diagnosis in 9 out of 41 cases, 3 agreed in 24 cases and 2 in 7 cases. One case received 4 different diagnoses. To verify the agreement inter-observer in both oral cGVHD classifications, Kappa test showed to Horn classification k=0.22 and to NIH consensus k=0.35. Conclusions The concordance of the final diagnosis of oral mucosa biopsies of hematopoietic stem cell transplantation patients suspected of GVHD was very low when the pathologists followed both the Horn criteria and NIH consensus.

EXTRANODAL NK/T-CELL LYMPHOMA, NASAL-TYPE IN GUATEMALA: A CLINICOPATHOLOGIC ANALYSIS OF 76 CASES

Objective To describe the clinical, pathological and immunohistochemical features of extranodal natural killer/T-cell lymphoma, nasal type (ENKTL-NT) affecting Guatemalan patients. Study design Cases diagnosed as ENKTL-NT from 1985 to 2016 were retrieved from the files of the pathology laboratory at Centro Clinico de Cabeza y Cuello (Guatemala). Clinical data provided by clinicians or gathered directly from medical charts when available, microscopic features, and results of immunohistochemistry (IHC) and ISH-EBV were reviewed and recorded. Results Seventy-six cases were identified. Males were more commonly affected (65.7%) than females (34.3%), with a mean age of 34.3 ranging from 7 to 71 years. Most of the patients were of Mayan descent and low socioeconomic status. ENKTL-NT presented as an aggressive necrotizing midfacial process with rapid progression, affecting sinonasal, palatal and nasopharyngeal structures. Other features observed were: initial signs of edema and inflammation, rapidly progressing to ulceration or perforation of the hard palate, necrosis of nasal skin and mucosa, midface deformity, and in advanced stages, palpebral edema. Three patients presented lethal hemagophagocytic syndrome. Oral mucosa biopsies were more representative and adequate for IHC and ISH than the ones from nasal skin. Microscopically, lesions showed a diffuse atypical lymphoid infiltrate with angiocentric and angiodestructive pattern, with extensive necrosis and superimposed subacute inflammation. The neoplastic cells varied in size and sometimes were anaplastic. The ISH and IHC profile of these cases was: EBV+, LCA+, CD20-, CD3+, CD45RO+, CD30 variable, CD4+, CD8+, Granzyme-A+, Perforin+, CD56 + (except for 2 cases). The Ki-67 index was ≥ 80%. When clinical follow-up was obtained, only 30% survived and two patients presented a recurrence at 2 and 10 years respectively. Conclusion ENKTL-NT is an aggressive malignant EBV related lymphoma, with highly distinctive clinical, histopathological and immunohistochemical features.

IMMUNOHISTOCHEMICAL EXPRESSION OF EZH2 IN ATYPICAL PAPILLARY EPITHELIAL PROLIFERATIONS OF THE ORAL CAVITY: A POTENTIAL MARKER FOR MALIGNANT TRANSFORMATION

Background Enhancer of zeste homolog-2 (EZH2) is a member of the polycomb group PcG of proteins; the genes that are involved in transcriptional repression. Cell cycle regulation and cell proliferation is associated with EZH2 expression and EZH2 overexpression stimulates cell proliferation and invasiveness. Conversely, inhibition of EZH2 precludes cancer cell invasiveness through inhibition of cell proliferation. Atypical papillary epithelial proliferation (AEP) is a histologic diagnosis rendered for oral lesions with confounding microscopic features, neither overtly benign nor unequivocally malignant. Aim To evaluate EZH2 antibody expression through immunohistochemical testing to delineate the potential of malignant transformation in AEP by comparing and contrasting with unequivocally benign papillary lesions represented by inflammatory papillary hyperplasia (IPH) and malignant papillary lesions represented by papillary well differentiated squamous cell carcinoma (PSCC). Materials and Methods 10 cases each of AEP, IPH and PSCC were retrieved from the University of Florida, Oral Pathology Biopsy Service archive and stained with Anti-KMT6/EZH2 antibody. The cases were reviewed and the extent and pattern of EZH2 expression were assessed. The results were analyzed for statistical significance using Fischer's exact test. Results The pattern and intensity of EZH2 expression in AEP and PSCC demonstrated statistically significant differences when compared to IPH (p=0.002). In addition, the basal cell layer showed EZH2 expression in all the cases of AEP (100%) and PSCC (100%) but only 3 out of 10 (30%) in IPH (p=0.000), comparable to normal oral epithelial control tissue. Conclusion EZH2 expression in AEP is more similar to malignant processes than benign lesions. The pattern of basal cell layer expression of EZH2 could be a potential prognostic indicator of malignant transformation risk in oral AEP lesions. A subsequent study by our group to assess EZH2 expression with respect to clinical outcome in AEP lesions is ongoing.

THE EXPRESSION OF MAML2 GENE REARRANGMENT IN CASES OF GLANDULAR ODONTOGENIC CYSTS AND MUCOEPIDERMOID CARCINOMAS WITH OVERLAPPING HISTOLOGIC FEATURES

Objectives MAML2 expression has been demonstrated in the majority of mucoepidermoid carcinomas (MEC) arising in the salivary glands. MEC may also arise intraosseously in the jawbone (IMEC). Glandular odontogenic cyst (GOC) is an odontogenic cyst with some histologic overlap with IMEC. MAML2 expression has not been extensively studied in IMEC or in GOC. This study will test the reliability of MAML2 in distinguishing cases of IMEC from GOC that share similar histologic features. Methods An IRB-approved retrospective search of IMEC, GOC, and IMEC with prior history of GOC was performed within the archives of the UF Oral Pathology Biopsy Service from 1994-2017. Eight cases from four patients were selected with diagnoses of either IMEC with earlier GOC, GOC with IMEC features, or IMEC with GOC features. Tissue was available for six out of the eight cases, on which break apart fluorescent in situ hybridization (FISH) analysis was performed for the presence of MAML2 rearrangement. Findings Lesions from two of the patients were negative for the MAML2 gene rearrangement while lesions from the other two patients were positive for the MAML2 gene rearrangement. Conclusion Although it can be concluded that the two patients with positive translocation for MAML2 had a diagnosis of IMEC, the same conclusion could not be drawn for the two patients with negative translocation. Whether the cases that were negative for the translocation are GOCs with MEC-like islands or MAML2 negative IMEC could not be ascertained. Therefore, MAML2 gene rearrangement is not always dependable in differentiating IMECs and GOCs that share similar histologic overlap. The limited nature of the study due to small sample size precludes a more definitive conclusion. Collaboration vis-a-vis cases and data exchanges between oral and maxillofacial pathology centers may help achieve a better understanding of two uncommon, but clinically impactful, entities.

IMMUNOHISTOCHEMICAL ANALYSIS OF INFLAMMATORY RESPONSE IN VERRUCOUS CARCINOMA COMPARED TO CONVENTIONAL ORAL SQUAMOUS CELL CARCINOMA

Introduction Studies on inflammatory response to oral squamous cell carcinoma (OSCC) generally do not include verrucous carcinoma (VC), which typically carries a far better prognosis. While high CD8 expression is associated with favorable outcome in head and neck cancers, the role of CD4+ lymphocytes remains controversial. B cell involvement has been suggested to enhance T cell response. The aim of this study is to evaluate differences in inflammatory infiltrate immunohistochemistry (IHC) between OSCC and VC. Materials and Methods The archives of the UF College of Dentistry oral pathology biopsy service were retrospectively searched for OSCC and VC. Slides were reviewed and 10 cases of VC, 10 cases of well differentiated SCC (SCC- WD), and 10 cases of poorly differentiated SCC (SCC-PD) were selected for testing. IHC staining for CD4, CD8, and CD20 was performed for 30 selected cases. The results were assessed via Aperio Image Scope positive pixel count assessment and analyzed statistically using ANOVA comparison of means with significance measured atp Results A total of 90 scanned slides were evaluated. Analysis of the results showed no significant difference in mean scores of CD8 or CD20 across groups; however, there was significant difference in CD4 mean scores, with increasing scores noted from VC to SCC-WD to SCC-PD (p=0.002). The CD8:CD4 ratio was highest in VC followed by SCC-WD then SCC-PD, but the difference was not statistically significant. No significant difference in B: T cell ratio was observed between diagnostic groups. Conclusions This study demonstrated a lower level of CD4 positive lymphocytes and a slightly increased CD8:CD4 ratio within the VC infiltrate as compared to SCC. B lymphocyte involvement did not appear to differ between VC and SCC in this sample. Further studies may help elucidate the clinical implications of these differences.

A NEW HIGH-RESOLUTION INVASION TEST (HIT) CAN PREDICT MALIGNANT TRANSFORMATION IN ORAL EPITHELIAL DYSPLASIAS

Rationale Detecting the earliest signs of invasion and predicting transformation in oral potentially malignant lesions (OPMLs) can facilitate earlier treatment of oral squamous cell carcinoma (OSCC) and decrease morbidity and mortality. Here we described a new test to diagnose early invasion and predict malignant transformation in OPML. Methods Fluorescent immunohistochemist and multichannel colocalization were used to identify invadopodia markers FISH, cortactin and MMP14 in OSCC and OPML. The presence of invadopodia markers was calculated using 3-channel colocalization analysis based on a custom algorithm developed using Volocity Software. The threshold for colocalization was determined by linear least-square fit of the channel intensities and the product of the difference of the means was (PDM) was used to compare the area of colocalization (HIT score). This algorithm was applied to 80 cases (10 cases of non-dysplastic hyperkeratosis, 22 cases of epithelial dysplasias (ED), 20 cases of OSCC and 28 cases from patients who progressed from ED to OSCC) to determine the overall validity of the approach and establish cut off values. Results There was a significant and progressive increase in the colocalization of invadopodia markers (HIT score) in dysplasias and OSCC compared to control. The results showed that the HIT score could detect lesions that transformed to OSCC independently of the histopathological diagnosis with a sensitivity of 84% and specificity of 61.76%, PPV= 0.61 and NPV=0.84, AUC= 0.7653, likelihood ratio of 2.1, p Conclusion The HIT scores can predict malignant transformation in oral biopsies independent of the histopathological diagnosis. Larger prospective studies are needed to validate and assess the applicability of this test in combination with conventional histopathology.

Diverse Activity of IL-17+ Cells in Chronic Skin and Mucosa Graft-Versus-Host Disease

Excessive inflammatory environment in a course of chronic graft-versus-host disease (cGvHD) is associated with T-cell trafficking into inflamed tissues. This study focused on the identification of IL-17-producing cells in the tissue biopsies of cGvHD patients. Forty-one biopsy specimens of cGvHD lesions of the skin (n = 27), gastrointestinal tract (n = 9) and oral mucosa (n = 5), examined in 24 patients, were morphologically defined according to the NIH criteria and analyzed for the presence of cellular infiltrations including: IL-17+, FOXP3+ and CCR6+ cells. IL-17+ cells were identified in 26/27 skin and in all gut and oral mucosa biopsies, being more frequent in mucosa lesions than in the skin (11/14 vs 14/26, respectively; NS: not significant). Double staining documented that CD138+/IL-17+ cells were commonly seen in the gut than in the skin (5/8 vs 3/11, respectively; NS). In the skin, cells expressing trafficking receptor CCR6+ were more frequent than IL-17+ cells compared to the mucosa (23/26 vs 2/13, respectively; p < 0.0001). CCR6 was present on a majority of IL-17+ cells in all examined skin biopsies but only in 6 out of 11 digestive tract biopsies (p = 0.0112). FOXP3+ cells were identified only in five patients (with mild lesions) at least in one biopsy. In this study group, results documented that local expansion of IL-17-producing cells in the digestive tract correlate with moderate and severe clinical symptoms of cGvHD, in contrast to the skin, where IL-17+ cells are rather scarcely present (p = 0.0301) and the course of cGvHD is slowly progressing with final organ deterioration.

The Optimal Sequencing Depth of Tumor Biopsies for Identifying Clonal Cell Populations

The tumor content of a biopsy and the average depth of coverage are two essential aspects when performing DNA sequencing using next-generation sequencing technologies. The heterogeneous nature of cancer necessitates the identification of distinct clonal cell populations to better understand and treat cancer. Deep sequencing enables researchers to identify these populations, but no consensus on an optimal depth exists for identifying clonal populations. Data from eight deep-sequenced oral squamous cell carcinoma biopsies obtained from three stage IV patients, with various degrees of tumor content, were used to randomly down sample the depth before being subjected to cluster analysis. An increase in coverage resulted in an increase in resolution for clusters of mutations, enabling the identification of distinct clonal cell populations and clonal events. From a depth of 800×, limited gain in resolution can be achieved; and from a depth of 1200×, the resolution stabilizes. Overall, researchers should aim for an average depth of 1000× to 1200× when performing deep sequencing. The tumor content will, however, dictate the resolution and fidelity of the analysis, as an increase in tumor complexity increases the need for higher tumor content.