Scalpel Biopsy Research Articles

A cytomics-on-a-chip platform and diagnostic model stratifies risk for oral lichenoid conditions

ObjectivesA small fraction of oral lichenoid conditions (OLC) have potential for malignant transformation (MT). Distinguishing OLCs from other oral potentially malignant disorders (OPMD) can help prevent unnecessary concern or testing, but accurate identification by non-expert clinicians is challenging due to overlapping clinical features. In this study, the authors developed a cytomics-on-a-chip tool and integrated predictive model for aiding the identification of OLCs. Study DesignAll study subjects underwent both scalpel biopsy for histopathology and brush cytology. A predictive model and OLC Index comprising clinical, demographic, and cytologic features was generated to discriminate between subjects with lichenoid (OLC+) (N=94) and non-lichenoid (OLC−) (N=237) histologic features in a population with OPMDs. ResultsThe OLC Index discriminated OLC+ and OLC− subjects with area under the curve (AUC) of 0.76. Diagnostic accuracy of the OLC Index was not significantly different from expert clinician impressions, with AUC of 0.81 (p=0.0704). Percent agreement was comparable across all raters, with 83.4% between expert clinicians and histopathology, 78.3% between OLC Index and expert clinician, and 77.3% between OLC Index and histopathology. ConclusionThe cytomics-on-a-chip tool and integrated diagnostic model have the potential to facilitate both the triage and diagnosis and risk stratification of patients presenting with OPMDs and OLCs.

Adjunctive Techniques and Diagnostic Aids in the Early Detection of Oral Premalignant Disorders and Cancer: An Update for the General Dental Practitioners.

Oral cancer (OC), a potentially fatal disease, is a major health concern across the world. It is reported to be the sixth most common cancer in the world with a disappointingly low 5-year survival rate, despite major advances in onco-medicine in the past three decades. The low 5-year-survival rate is associated with late diagnosis of the disease, while OC diagnosed at early stages enjoy a much higher 5-year-survival rate, comparatively. Although the oral cavity is one of the most easily accessible parts of the body for cancer screening, OC is typically diagnosed at later stages. The delay in diagnosis is one of the factors for the poor 5-year survival rate and high mortality and morbidity among patients. Therefore, an early diagnosis is of utmost importance. Visual and tactile examination and scalpel biopsy are still considered the gold standard for definitive diagnosis of oral potentially malignant disorder (OPMD) and OC. Nevertheless, adjunctive techniques could be employed to increase the ability to distinguish benign abnormalities from dysplastic/malignant changes. These would also aid in identifying areas of dysplasia/early OC that are not visible to the naked eye and tackle the delay in diagnosing OPMD/OC. These adjunctive tools are not a replacement for visual and tactile examination but are supplementary aids. They could be used to screen healthy patients for the presence of any occult cancerous change and evaluate the biological potential of clinically abnormal mucosal lesions, thus enabling early recognition and diagnosis which might increase survival rate and reduce mortality and treatment-associated morbidity.

Comparison of Oral Mucosal Biopsies Done Using Scalpel and Diode Lasers

Background:As dental lasers are becoming more popular in the branch of oral medicine for its various advantages and applications, this study was carried out to evaluate better mode of obtaining oral biopsies which is a common and inevitable procedure for providing final diagnosis in majority of conditions.Methodology:In this study, a total of 60 patients who required biopsy for final diagnosis of oral mucosal lesions as part of diagnosis in department of Oral Medicine were selected, out of which 30 were subjected to scalpel biopsies and 30 patients were subjected to diode laser biopsies. A 980 nm Zolar plus diode laser was used for the study. Out of 30 patients who were subjected to laser biopsies, 3W continuous mode settings were used for 15 patients and 3W pulsed mode was used for 15 patients. The specimens were sent to Department of Oral Pathology for histopathological evaluation to provide the final diagnosis. The time taken for each patient, volume of local anesthesia, during operative, postoperative pain scale, and co-relation of provisional and final diagnosis was noted for comparison purpose and the pathologist comments, peripheral tissue damage and artifact's for each slide were noted.Results:The study results showed the postoperative pain was comparatively less in diodlaser than scalpel, the lasers were patient friendly as the heammorage was negligible when compared to scalpel and suturing was not required. The pulsed mode in diode laser was advantageous over continuous mode when amount of thermal damage and postoperative pain score was compared.Conclusion:The results showed that oral biopsies can be made better using diode lasers, by having thorough knowledge on the device.

Generalized actinomycosis of the pelvic organs. Clinical case

Here, a clinical case of generalized actinomycosis of the pelvic organs occuring very rarely usually observed in immunocompromised patients is presented. Patient T. referred to the women's clinic with complaints of prolonged genital discharge, itching, burning, and occasional sub-fever. The patient had a history of prolonged hormonal immunosuppressive therapy. After a thorough examination, the patient was consulted by two oncologists. Preliminary diagnosis: suspicio cancer coli; suspicio cancer vaginae. In order to clarify the diagnosis, an in-hospital multifocal scalpel biopsy was performed. The biological material obtained was sent out for intravital pathological and anatomical examination; no signs of malignancy were found. To prevent complications during surgery, the patient underwent separate diagnostic scraping of the uterine cavity and cervical canal after repeated sanitation of the vagina and additional patient examination; hysteroscopy; vulval biopsy; colpocentesis. The final diagnosis was made after obtaining the results of in-surgery microbial culture on nutrient media. Active growth of actinomycetes was detected in the material obtained. The final clinical diagnosis was pelvic actinomycosis, generalized form. Patient T was discharged in satisfactory condition after antibacterial treatment according to the diagnosis for further under gynecologist observation at the women's clinic.

Rose Bengal Staining, Microbiopsy and Scalpel Biopsy as a Cytology-cumHistopathology based Diagnostic Scheme for Oral Dysplasias- A Pilot Study

Introduction: Most Oral Squamous Cell Carcinoma (OSCC) is preceded by Oral Potentially Malignant Disorders (OPMDS). These disorders if diagnosed early can be prevented from converting into full blown malignancies. This points towards an ever increasing need for a more accurate, less invasive diagnostic tool to detect these lesions early. Aim: To analyse and compare the accuracy of 1% Rose Bengal (RB) dye and oral microbiopsies (using dermatologic ring curettes) with conventional scalpel biopsy in diagnosing oral epithelial dysplasias. Materials and Methods: This crosssectional pilot study, included a total number of 26 male patients, 40-60 years old, with oral white and red mucosal lesions attending the Outpatient Department (OPD) of Teerthanker Mahaveer Dental College, Moradabad, Uttar Pradesh, India. Thorough clinical examination, 1% RB staining, microbiopsy, and scalpel biopsy was performed on all included participants. Parameters assessed included clinical signs indicative of dysplasia in red and white lesions such as increase thickness, nodularity, atrophic mucosa, erosion,ulcers and change in colour of mucosa with positive history of tobacco smoking. Hyperchromatic areas owing to increase stain intake (due to increased nuclear cytoplasmic ratio) were obtained after staning with RB dye and histopathological indicators of epithelial dysplasia (cellular and architectural changes) were observed after both microbiopsy and scalpel biopsy. Chi-square test was done to compare results of 1% RB staining and microbiopsies with scalpel biopsies. Results: Total 26 male patients were included with a mean age of 50 years. There was no statistically significant difference in the accuracy of microbiopsies (p=0.913) and 1% RB dye (p=0.393) as compared to conventional scalpel biopsy in delineating the epithelial dysplastic changes associated with OPMDS. Conclusion: Oral microbiopsy, is a relatively novel, accurate, and less invasive diagnostic tool. It has proved to be as effective as scalpel biopsy in diagnosing and grading epithelial dysplasia. Through this article, it was proposed that 1% RB staining, microbiopsy, and scalpel biopsy can be used in conjugation as a part of cytology-cum-histopathology based diagnostic scheme for oral clinically suspicious lesions

Effectiveness of a New Technique for Oral Cancer Screening – A Pilot Study

Introduction: Screening programs with the use of specific diagnostic tools in asymptomatic patients are useful in identifying suspicious oral lesions and aid in the early diagnosis of oral cancer. The objective of the present study was to compare the oral rub and rinse technique with the conventional exfoliative cytology in the screening of oral malignant and potentially malignant diseases. Materials and Methods: An oral cancer screening program was conducted in the Dakshina Kannada district of Karnataka, India. The oral rub and rinse technique was performed on patients who had red/white lesions in the oral mucosa followed by the conventional exfoliative cytology. Scalpel biopsy was performed to confirm for presence or absence of malignancy in cases wherever indicated. Descriptive statistics (frequency and percentage) were used in the present study. Results: A total of 848 subjects were screened for oral cancer and precancer. About 112 participants had premalignant/malignant lesions and biopsy was performed on 30 subjects. Of these, 27.7% were Class I smears, 39.3% were Class II smears, 22.3% were Class III smears, 4.5% were Class IV smears, and 6.2% were unsatisfactory using the conventional technique, whereas the oral rub and rinse technique showed 26.8% Class I smears, 42.9% Class II smears, 19.6% Class III smears, 6.2% Class IV smears, 0.9% Class V smears, and 3.6% unsatisfactory. Conclusion: Although both the techniques could detect malignancy, the oral rub and rinse technique showed better cellular clarity and sample adequacy when compared to conventional exfoliative cytology, which makes it a practical tool in resource-challenged settings.

Point-of-care characterization and risk-based management of oral lesions in primary dental clinics: A simulation model.

ObjectivesOral potentially malignant disorders (OPMDs) encompass histologically benign, dysplastic, and cancerous lesions that are often indistinguishable by appearance and inconsistently managed. We assessed the potential impact of test-and-treat pathways enabled by a point-of-care test for OPMD characterization.Materials and methodsWe constructed a decision-analytic model to compare life expectancy of test-treat strategies for 60-year-old patients with OPMDs in the primary dental setting, based on a trial for a point-of-care cytopathology tool (POCOCT). Eight strategies of OPMD detection and evaluation were compared, involving deferred evaluation (no further characterization), prompt OPMD characterization using POCOCT measurements, or the commonly recommended usual care strategy of routine referral for scalpel biopsy. POCOCT pathways differed in threshold for additional intervention, including surgery for any dysplasia or malignancy, or for only moderate or severe dysplasia or cancer. Strategies with initial referral for biopsy also reflected varied treatment thresholds in current practice between surgery and surveillance of mild dysplasia. Sensitivity analysis was performed to assess the impact of variation in parameter values on model results.ResultsRequisite referral for scalpel biopsy offered the highest life expectancy of 20.92 life-years compared with deferred evaluation (+0.30 life-years), though this outcome was driven by baseline assumptions of limited patient adherence to surveillance using POCOCT. POCOCT characterization and surveillance offered only 0.02 life-years less than the most biopsy-intensive strategy, while resulting in 27% fewer biopsies. When the probability of adherence to surveillance and confirmatory biopsy was ≥ 0.88, or when metastasis rates were lower than reported, POCOCT characterization extended life-years (+0.04 life-years) than prompt specialist referral.ConclusionRisk-based OPMD management through point-of-care cytology may offer a reasonable alternative to routine referral for specialist evaluation and scalpel biopsy, with far fewer biopsies. In patients who adhere to surveillance protocols, POCOCT surveillance may extend life expectancy beyond biopsy and follow up visual-tactile inspection.

Clinico-pathological correlations of macular lesions in leprosy, using a new histological grading system

In macular forms of pauci-bacillary (PB) leprosy as the dermal infiltrate is less, greater precision in evaluation is needed both for diagnosis, and for assessment of the efficacy of treatment. With this in view, a new numerical grading system on a scale from 0-4, with intervening decimals, was recently published to describe each specific pathology of the dermal nerves and appendages. This retrospective study of smear negative macular lesions, has utilized this grading system for comparison and follow up. Fifteen new patients with PB macular leprosy (TT, BT) were divided into two groups – Group A without deformity (ten patients), and Group B with deformity (five patients). All the patients had pre-treatment scalpel biopsies. In the pre-treatment biopsies in Group B the average grade of dermal granuloma was 1.3 compared to 0.5 in Group A. Two patients in Group A who were given daily Rifampicin and Dapsone for five and six months respectively, showed marked reduction in the infiltrate. In many biopsies a linear serpiginous infiltrate (horizontal) was present in the superficial dermis. This new grading system is useful for quantifying the dermal pathology in PB macular leprosy, for comparison of duration of treatment, or evaluation of newer drugs in studies, and for assessing resolution of the lesion by the pathologist. Keywords: Macular leprosy (PB), Histological grading, Dermal granuloma, Neural infiltrate and destruction, Linear serpiginous infiltrate.

A quick and effective method of photomicrography in dermato – pathology

A quick and effective method of photomicrography in dematopathology has been devised using the idea of longitudes and latitudes as on a geographical map. In a horizontally oriented scalpel biopsy section, ten equi-distant vertical imaginary lines are drawn starting from the extreme left as V 0/10 and the extreme right as V 10/10. In addition, five equi-distant imaginary horizontal lines are drawn with H 0/5 on the stratum corneum and H 5/5 below the subcutis. The position of a particular field at higher magnification should be then noted at the scanner position (4x objective) for proper orientation.

Nuclear F-actin Cytology in Oral Epithelial Dysplasia and Oral Squamous Cell Carcinoma

Oral cavity cancer has a low 5-y survival rate, but outcomes improve when the disease is detected early. Cytology is a less invasive method to assess oral potentially malignant disorders relative to the gold-standard scalpel biopsy and histopathology. In this report, we aimed to determine the utility of cytological signatures, including nuclear F-actin cell phenotypes, for classifying the entire spectrum of oral epithelial dysplasia and oral squamous cell carcinoma. We enrolled subjects with oral potentially malignant disorders, subjects with previously diagnosed malignant lesions, and healthy volunteers without lesions and obtained brush cytology specimens and matched scalpel biopsies from 486 subjects. Histopathological assessment of the scalpel biopsy specimens classified lesions into 6 categories. Brush cytology specimens were analyzed by machine learning classifiers trained to identify relevant cytological features. Multimodal diagnostic models were developed using cytology results, lesion characteristics, and risk factors. Squamous cells with nuclear F-actin staining were associated with early disease (i.e., lower proportions in benign lesions than in more severe lesions), whereas small round parabasal-like cells and leukocytes were associated with late disease (i.e., higher proportions in severe dysplasia and carcinoma than in less severe lesions). Lesions with the impression of oral lichen planus were unlikely to be either dysplastic or malignant. Cytological features substantially improved upon lesion appearance and risk factors in predicting squamous cell carcinoma. Diagnostic models accurately discriminated early and late disease with AUCs (95% CI) of 0.82 (0.77 to 0.87) and 0.93 (0.88 to 0.97), respectively. The cytological features identified here have the potential to improve screening and surveillance of the entire spectrum of oral potentially malignant disorders in multiple care settings.

Methylene Blue as a Diagnosis and Screening Tool for Oral Cancer and Precancer.

To evaluate the accuracy of methylene blue (MB) for diagnosing oral cancer and precancer. PubMed, Cochrane Database, Embase, Web of Science, SCOPUS, and Google Scholar. Two authors working independently reviewed 6 databases from their dates of inception until April 2020. Studies exploring oral mucosal disorders as detected by MB were assessed. True-positive, true-negative, false-positive, and false-negative data were extracted for each study. Methodological quality was evaluated with the Quality Assessment of Diagnostic Accuracy Studies tool (v 2). Seven prospective and retrospective studies (N = 493) were included. The diagnostic odds ratio of MB was 20.017 (95% CI, 10.65-37.63, I2 = 23%). The area under the summary receiver operating characteristic curve was 0.699. Sensitivity was 0.903 (95% CI, 0.84-0.94, I2 = 54%), and specificity was 0.68 (95% CI, 0.60-0.75, I2 = 0%). The correlation between the sensitivity and the false-positive rate was -0.17, indicating an absence of heterogeneity. Regarding diagnostic accuracy, MB had high sensitivity but low specificity, suggesting that it cannot be recommended as a replacement for the currently used standard of a scalpel biopsy with histologic assessment. Instead, it should be used as an adjunct to conventional assessment because of its low toxicity and price.

Comparison of tissue artifacts in punch and scalpel biopsies of oral and maxillofacial lesions: A systematic review and meta-analysis

AimThe aim of this systematic review and meta-analysis was to compare the occurrence of tissue artifacts in punch and scalpel biopsies of oral and maxillofacial lesions. MethodsElectronic searches were conducted in four databases (PubMed, Scopus, Web of Science and Ovid). Study selection, data extraction, and quality assessment of the included articles were performed independently by two authors. An evaluation of the strength of the evidence (GRADE) and meta-analysis were conducted. Odds ratio and confidence intervals (CI) were provided. ResultsAfter the removal of duplicates, 466 references were identified. Four studies evaluating artifacts, such as crush, fragmentation, splits, hemorrhage, curling, and orientation artifacts and also those induced by improper surgical removal, were included. Specimens obtained using scalpel biopsy were 2.98 times more likely (CI=1.53–5.80) to present crushes and 12.36 times more likely (CI=2.64–57.83) to present splits than specimens procured using punch biopsy. Scalpel biopsies were also 2.40 times more likely to present fragmentation than punch biopsies (CI=1.01–5.72). No significant differences between scalpel and punch biopsies were found concerning the presence of hemorrhage, curling, or orientation artifacts and those induced by improper surgical removal. After sensitivity analysis, samples obtained from scalpel biopsy were 6.18 times more likely to present hemorrhage than those from a punch biopsy (CI=2.21–17.28). Based on the GRADE evaluation, the confidence in the effect estimate of the sub-group analysis of crush and fragmentation was moderate. For the other subgroup analysis, the confidence was low or very low. ConclusionPunch biopsies were less likely to produce artifacts, such as crush, fragmentation, splits, and hemorrhage, than biopsies obtained with a scalpel.

Comparison of the efficacy of cytodiagnostic instruments in exfoliative cytology

Aims and Objectives: The aims and objectives of the study were to determine the efficacy of wooden spatula, cytobrush, and toothbrush in obtaining cytology smears for early detection of oral premalignant lesions and also to compare the analysis of cytology smears with the histopathological diagnosis. The smears were observed and analyzed under a compound light microscope. Materials and Method: Fifty patients of clinically diagnosed oral leukoplakia patients attending the outpatient department of oral pathology and microbiology were considered for the study. The smear samples were obtained using wooden spatula, cytobrush, and toothbrush. The samples were manually analyzed in a double-blinded fashion. Histopathological diagnosis was confirmed using scalpel biopsy and a comparison was made between cytological and histopathological diagnosis. Statistical analysis was performed using Chi-square test. Results: In our study, toothbrush cytology was comparatively better (P

Evaluation of the Accuracy of Liquid-Based Oral Brush Cytology in Screening for Oral Squamous Cell Carcinoma.

This study evaluates the accuracy of the results of liquid-based oral brush cytology and compares it to the histology and/or the clinical follow-ups of the respective patients. A total of 1352 exfoliated specimens were collected with an Orcellex brush from an identical number of oral lesions, then cytological diagnoses were made using liquid-based cytology. The final diagnoses in the study were 105 histologically proven squamous cell carcinomas (SCCs), 744 potentially malignant lesions and 503 cases of traumatic, inflammatory or benign hyperplastic oral lesions. The sensitivity and specificity of the liquid-based brush biopsy were 95.6% (95% CI 94.5–96.7%) and 84.9% (95% CI 83.0–86.8%), respectively. This led to the conclusion that brush biopsy is potentially a highly sensitive and reliable method to make cytological diagnoses of oral neoplasia. The main advantage of a brush biopsy over a scalpel biopsy is that it is less invasive and is more tolerated by the patients. Therefore, more lesions can be screened and more cancers can be detected at an early stage.

DNA cytometry of exfoliated cells in the diagnosis of oral potential malignant disorders

To evaluate the diagnostic efficiency of oral mucosa disease, especially oral squamous cell carcinoma (OSCC) and oral potential malignant disorders (OPMDs) by DNA cytometry compared with histopathological diagnosis, so as to find a convenient, simple and low-invasive method for screening and follow-up. 203 subjects with OSCC, OPMDs and other oral mucosa disease without dysplasia according to the inclusion criteria and exclusion criteria were recruited from Peking University School and Hospital of Stomatology. The mean age was (52.44±13.55) years, 98 males and 105 females. Brush biopsy was taken before scalpel biopsy at the same site. The brush biopsy sample was screened by moticytometer system for DNA cytometry after Feulgen stain, and histopathological examination were taken for the scalpel tissue. Data from DNA cytometry were used to calculate the parameters, such as sensitivity, specificity, positive and negative predictive values, odds ratios, Youden index (YI), positive and negative likelihood ratios, compared with the golden standard, histopathological diagnosis. DNA cytometry and histopathological diagnosis were performed back to back. Totally, 42 OSCC and 4 tumor in situ (TIS), 39 oral leukoplakia (OLK) with dysplasia (17 mild dysplasia, 13 medium dysplasia and 9 severe dysplasia), 29 OLK with hyperplasia, 1 verrucous OLK, 83 oral lichen planus (OLP) and 5 inflammation were included in our research. We grouped the OSCC, TIS and dysplasia as the positive group and others without dysplasia as the negative group, the sensitivity of DNA cytometry was 79.07%, the specificity was 81.20%, and the diagnostic accuracy was 80.30%,We grouped the OSCC and TIS as the tumor group, OLP, OLK with hyperplasia and inflammation as the non-tumor group, The sensitivity of DNA cytometry in diagnosing OSCC and TIS was 95.65%, and the specificity was 81.2%, The diagnostic accuracy was 85.28%. positive predictive values 66.67%, negative predictive values 97.94%, ratio odds 95, positive likelihood ratio 5.09, negative likelihood ratio 0.05, and Youden index 0.77. For the dysplasia, we grouped the different dysplasia together as the dyaplasia group, OLP, OLK with hyperplasia and inflammation as the non-tumor group, the sensitivity of DNA cytometry in diagnosing dyaplasia is 60%, the specificity is 81.2%. The diagnostic accuracy is 75.8%, positive predictive values 52.17%, negative predictive values 85.59%, ratio odds 6.48, positive likelihood ratio 3.19, negative likelihood ratio 0.49, and Youden index 0.41. DNA cytometry is convenient and low-invasive, which can be used as an adjuvant method for screening the early OSCC and OPMDs, monitoring the prognosis of OSCC after surgery. Further large-scale and long period prospective studies are necessary to validate the better value of DNA cytometry.

Combination of Cytopathology and DNA Ploidy Increases the Performance of Oral Epithelial Dysplasia Prediction in Oral Potentially Malignant Disorders

Background: Grading of oral epithelial dysplasia (OED) by a pathologist is currently the key guide used for treatment planning of oral potentially malignant disorders (OPMDs). Conventional oral examination (COE) clinically detects OPMDs but may not predict their risk status to transform to cancer. Therefore, there is a need for a reliable test to predict OED in OPMDs. Aim: This study was conducted to evaluate COE, liquid based cytology (Cytopath) and DNA image cytometry (Ploidy) in predicting OED in OPMDs. Methods: A total of 179 patients from Malaysia, India and Indonesia underwent COE followed by brush biopsies and scalpel biopsies. Brush-biopsy samples were analyzed for cytopathology and DNA ploidy at Dental Faculty, University of Malaya. Histopathological findings of presence/absence of OED were used as the reference standard. Calculations for sensitivity (Sn), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV) and accuracy (A) were done for individual tools and in combinations. The Youden index (Sn+Sp-1) was used as a measure of overall performance. The relevant medical ethics committees of the different research locations approved the study. Results: For COE, the sensitivity (Sn) was high (100%) and the specificity (Sp) was low (5.9%), while both Cytopath and Ploidy showed a low sensitivity (Sn) (28.6% and 22.2%) and high specificity (Sp) (94.3% and 82.3%). All 3 tools individually have high negative predictive value (NPV) for predicting presence of OED (COE-100%, Cytopath-66.7%, Ploidy-78.5%). When combining outcomes from all 3 tools, the best performance indicated by Youden index (42.1) is which defines a positive case when both COE and Cytopath show abnormal. In general, using results from at least 2 tools had better Youden indices than using these tools individually. Conclusion: COE as a screening tool by virtue of its high Sn would be a suitable first level diagnostic test, while the Cytopath and the Ploidy individually with high Sp may be used as a second level test to predict presence of OED. Combining the COE with cytopathology would be the best combination for a high performance of the tools. Cytopathology (when performed by a trained cytologist) would allow for most of the false positives from the first level test to be correctly identified as true negative at the second level. Longitudinal data are needed to assess which of these may correctly identify the malignant potential of OPMDs. Acknowledgment: Grant: High Impact Research - Ministry of Higher Education (HIR-MOHE UM000025/C3)

Risk Stratification of Oral Potentially Malignant Disorders in Fanconi Anemia Patients Using Autofluorescence Imaging and Cytology-On-A Chip Assay

Fanconi anemia (FA) is a hereditary genomic instability disorder with a predisposition to leukemia and oral squamous cell carcinomas (OSCCs). Hematopoietic stem cell transplantation (HSCT) facilitates cure of bone marrow failure and leukemia and thus extends life expectancy in FA patients; however, survival of hematologic malignancies increases the risk of OSCC in these patients. We developed a “cytology-on-a-chip” (COC)–based brush biopsy assay for monitoring patients with oral potentially malignant disorders (OPMDs). Using this COC assay, we measured and correlated the cellular morphometry and Minichromosome Maintenance Complex Component 2 (MCM2) expression levels in brush biopsy samples of FA patients’ OPMD with clinical risk indicators such as loss of autofluorescence (LOF), HSCT status, and mutational profiles identified by next-generation sequencing. Statistically significant differences were found in several cytology measurements based on high-risk indicators such as LOF-positive and HSCT-positive status, including greater variation in cell area and chromatin distribution, higher MCM2 expression levels, and greater numbers of white blood cells and cells with enlarged nuclei. Higher OPMD risk scores were associated with differences in the frequency of nuclear aberrations and differed based on LOF and HSCT statuses. We identified mutation of FAT1 gene in five and NOTCH-2 and TP53 genes in two cases of FA patients’ OPMD. The high-risk OPMD of a non-FA patient harbored FAT1, CASP8, and TP63 mutations. Use of COC assay in combination with visualization of LOF holds promise for the early diagnosis of high-risk OPMD. These minimally invasive diagnostic tools are valuable for long-term surveillance of OSCC in FA patients and avoidance of unwarranted scalpel biopsies.

Cytological study of DNA content and nuclear morphometric analysis for aid in the diagnosis of high-grade dysplasia within oral leukoplakia

To quantitatively examine the DNA content and nuclear morphometric status of oral leukoplakia (OL) and investigate its association with the degree of dysplasia in a cytologic study. Oral cytobrush biopsy was carried out to obtain exfoliative epithelial cells from lesions before scalpel biopsy at the same location in a blinded series of 70 patients with OL. Analysis of nuclear morphometry and DNA content status using image cytometry was performed with oral smears stained with the Feulgen-thionin method. Nuclear morphometric analysis revealed significant differences in DNA content amount, DNA index, nuclear area, nuclear radius, nuclear intensity, sphericity, entropy, and fractal dimension (all P<.01) between low-grade and high-grade dysplasia. DNA content analysis identified 34 patients with OL (48.6%) with DNA content abnormality. Nonhomogeneous lesion (P=.018) and high-grade dysplasia (P=.008) were significantly associated with abnormal DNA content. Importantly, the positive correlation between the degree of oral dysplasia and DNA content status was significant (P=.004, correlation coefficient=0.342). Cytology analysis of DNA content and nuclear morphometric status using image cytometry may support their use as a screening and monitoring tool for OL progression.

MicroRNA from brush biopsy to characterize oral squamous cell carcinoma epithelium.

Few cancers are diagnosed based on RNA expression signatures. Oral squamous cell carcinoma (OSCC) is no exception; it is currently diagnosed by scalpel biopsy followed by histopathology. This study sought to identify oral tumor epithelial microRNA (miRNA) expression changes to determine if these changes could be used to diagnose the disease noninvasively. Analysis of miRNA profiles from surgically obtained OSCC tissue, collected under highly standardized conditions for The Cancer Genome Atlas, was done to determine the potential accuracy in differentiating tumor from normal mucosal tissue. Even when using small 20 subject datasets, classification based on miRNA was 90 to 100% accurate. To develop a noninvasive classifier for OSSC, analysis of brush biopsy miRNA was done and showed 87% accuracy in differentiating tumor from normal epithelium when using RT‐qPCR or miRNAseq to measure miRNAs. An extensive overlap was seen in differentially expressed miRNAs in oral squamous cell carcinoma epithelium obtained using brush biopsy and those reported in saliva and serum of oral squamous cell carcinoma patients in several studies. This suggested that nonselective release of these miRNAs into body fluids from tumor epithelium was largely responsible for the changes in levels in these fluids seen with this disease. Using a variation in mirRPath we identified the KEGG pathway of neurotrophin signaling as a target of these miRNAs disregulated in tumor epithelium. This highlights the utility of brush biopsy of oral mucosa to allow simple acquisition of cancer relevant miRNA information from tumor epithelium.

Abstract LB-248: Minimally-invasive “cytology-on-chip” assay combined with continuous risk index for screening and surveillance of oral cancer in patients with Fanconi anemia

Abstract Fanconi Anemia (FA) is an autosomal recessive disorder caused by mutations of DNA repair genes. The risk of oral cancer (OC) among FA patients is 800-times higher than in the general population, occurring at younger ages. Hematopoietic stem cell transplantation (HSCT), which greatly extends the life-expectancy in FA patients further increases the risk of OC in these patients. Patients with FA cannot tolerate chemo-and radiation therapy; hence, early detection of OC is critical to improve survival. Objective: To investigate the role of a numerical index for monitoring OC progression applied to FA patients in combination with a microfluidic “cytology-on-chip” assay and autofluorescence visualization (AFV) using VELscope (Visually Enhanced Lesion scope). Methods: Patients attending the Meeting for Adults with FA, held in Baltimore, Maryland in March 2014 underwent a conventional oral examination followed by AFV. Transepithelial brush samples of mucosal lesions suspected of being oral potentially malignant disorders (OPMD) were obtained using Orcellex® brush (Rovers Medical Devices, Oss, The Netherlands) and transported in ThinPrep® CytoLyt® for cytology-on-chip assays. A numerical OC risk index (0-100) developed and validated on 506 prospectively recruited non-FA patients with OPMD was applied to site-matched brush biopsy samples of FA patients. Results: A total of 28 FA patients (Age range: 18-61 yrs., M = 9, F = 19) participated in this study, of whom 13 have had HSCT. The majority of these lesions (89%) revealed loss of fluorescence (LOF+ve) with AFV. Compared to a cohort of prospectively recruited non-FA patients with OPMD (mean = 36.71, SD = 33.60), the mean OC risk index score for FA patients was 50.66 (SD = 32.83). Additionally, HSCT-recipient FA patients exhibited higher risk index scores (mean = 58.69, SD = 32.16) compared to non -HSCT-recipient FA patients (mean = 40.34, SD = 33.08). The frequency of nuclear aberrations such as micronuclei, bi- and poly-nucleated cells was significantly higher in FA patient cells than healthy controls and significantly higher in LOF+ve compared to LOV-negative OPMD. Two samples exhibited significantly higher WBC (white blood cell) counts, an indicator of inflammation; both were from LOF+ve OPMD found in HSCT-recipient FA patients. Conclusion: FA patients who are HSCT recipients have significantly higher OC risk index scores as well as increased prevalence of LOF+ve OPMD that demonstrate higher incidence of nuclear aberrations and inflammation. These findings are consistent with recent reports that HSCT in FA patients increases their risk for oral cancer. Therefore, use of the OC numerical index and cytology-on-chip assay in combination with AFV may improve the efficacy of conventional oral examination for long-term surveillance of OC in this high-risk patient population, while minimizing unwarranted scalpel biopsies. Citation Format: Timothy J. Abram, Pierre N. Floriano, Rameez Raja, Nancy Bass, Anne Gillenwater, Nadarajah Vigneswaran, John T. McDevitt. Minimally-invasive “cytology-on-chip” assay combined with continuous risk index for screening and surveillance of oral cancer in patients with Fanconi anemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-248.