Most oral squamous cell carcinomas (OSCCs) arise from oral epithelial dysplasia; however, there is no useful marker for early OSCC detection, likely owing to the inability to continuously observe the carcinoma sequence. We aimed to establish an experimental model to observe changes in the sequential expression pattern of mRNA and protein in the same rat using liquid-based cytology techniques. Cytology specimens were collected from a 4-nitroquinoline 1-oxide-induced rat tongue cancer model at 2, 5, 8, 11, 14, 17, and 21 weeks. We examined candidate biomarker expression using immunocytochemistry and quantitative real-time PCR. The percentage of positively stained nuclei was calculated as the labeling index (LI). All rats had OSCC of the tongue at 21 weeks. Brd4 (Brd4), Myc (c-Myc), and Tp53 (p53) mRNA levels were upregulated during progression from negative for intraepithelial lesion or malignancy to SCC. Brd4- and c-Myc-LI were increased in low- and high-grade squamous intraepithelial lesions and SCC specimens. p53-LI was significantly increased in SCC specimens. Our experimental model allowed the observation of sequential morphological changes and mRNA and protein expression patterns in the same rat during carcinogenesis. By reducing the false negative rate, BRD4 and c-Myc can be useful markers for the early detection of OSCC.

Human papillomavirus testing is increasingly central to screening programs for cervical cancer. Although HPV testing has excellent clinical sensitivity it suffers from modest clinical specificity. Cytology has taken an important role as a triage test, while more recently HPV16/18 genotyping has provided modest incremental benefits. Despite such progress issues remain to be addressed, some of which are how to: 1) improve access to the best methods, 2) decrease costs so more women can be screened, 3) simplify management algorithms, 4) Recognize the evolving diagnostic landscape created by widespread HPV vaccination. Part of the solution lies in improving the positive predictive value of the initial screen and reflex triage. An aspirational goal should be that most women attending colposcopy have true precancer; DNA methylation testing has a role to play here. Cervical cancers and CIN2/3 generally have high levels of methylation in target human and HPV genes. In contrast normal and healthy women with no intraepithelial lesions or malignancy usually have low methylation. Furthermore, there is an interesting and useful secular gradient of increasing cervical DNA methylation as women progress toward precancer and cancer. Recent results from clinical studies using commercially available routine qPCR-based DNA methylation tests have been encouraging, demonstrating the potential for higher sensitivity and specificity than current triage methods.

Equine Asthma Syndrome (EAS) is a common respiratory problem that affects horses of any age. In the severe EAS form (historically referred to as recurrent airway obstruction or RAO) the diagnosis, based mainly on history and clinical signs, it is definitively confirmed by the cytological examination of the bronchoalveolar lavage (BAL), also very useful in monitoring lower airway inflammation in response to environmental management and medication. Cytocentrifugated preparation is usually the staging method for the BAL cytological interpretation. To evaluate whether the BAL cytology in horses with severe EAS under different environmental conditions and before and after treatment can undergo significant changes, and at the same time to investigate whether in the cytological evaluation of the BAL there are interpretative differences between two methods of smear preparation of the collected fluid, a study was carried out on a series of BAL samples (n=48) collected in 8 withdrawals from 6 EAS-affected horses subjected to process exacerbation through environmental stimuli and then to pharmacological treatment. Each BAL fluid collected was equally divided into duplicate portions: one set up by cytocentrifugation and one by sediment from simple centrifugation. The cytological examination revealed any significant difference between the EAS-affected horses in all experimental phases (asymptomatic, early exacerbation phase, late exacerbation phase, and remission phase). Diagnostic interpretive comparison between the two BAL preparations then showed no significant differences in results, suggesting how they can be used indifferently in the evaluation of BAL under conditions of airway inflammation in the horse. Thus, the concentrated smear preparation appears to be an equally diagnostically useful method in conditions where there is no possibility of using an appropriate cytocentrifuge.

OBJECTIVE: Cervical cancer is a major cause of cancer-related mortality, necessitating effective screening and diagnostic methods. This study aimed to assess the performance of cervicovaginal smear (CVS) and Human papilloma virus (HPV)-DNA cotest. STUDY DESIGN: The pathology results of 225 female patients who underwent HPV-DNA testing with CVS between 2014 and 2022 and were subsequently diagnosed by colposcopic cervical biopsy or second CVS were retrospectively analyzed. RESULTS: CVS samples showed Atypical Squamous Cells of Undetermined Significance (ASCUS), Low-grade squamous intraepithelial lesion (LSIL), High-grade squamous intraepithelial lesion (HSIL) and cervical cancer. Concordance between the first and second diagnoses demonstrated moderate agreement for LSIL. ASCUS cases exhibited a significant correlation with HPV-DNA positivity and higher-grade cervical lesions. In biopsy, sensitivity and specificity for CIN 2 were 67.8% and 67.9%, respectively, while for CIN 3, they were 81.8% and 58.5%. HPV testing showed significant correlation with histopathologic results. In women over 40 years, more intraepithelial lesions were diagnosed compared to younger women (p<0.005). The conventional smear technique proved reliable in detecting high-grade lesions. CONCLUSION: Despite the limitations of our study, our results emphasize the value of HPV-DNA testing to avoid unnecessary interventions and to establish appropriate follow-up strategies.

Objective: Surgical eradication of malignant glioma cells is theoretically impossible. Therefore, reducing the number of remaining tumor cells around the brain-tumor interface (BTI) is crucial for achieving satisfactory clinical results. The usefulness of fluorescence-guided resection for the treatment of malignant glioma was recently reported, but the detection of infiltrating tumor cells in the BTI using a surgical microscope is not realistic. Therefore, we developed an intraoperative rapid fluorescence cytology system, and evaluated its clinical feasibility for the management of malignant glioma. Materials and methods: Twenty-five selected patients with malignant glioma (newly diagnosed: 17; recurrent: 8) underwent surgical resection under photodiagnosis using photosensitizer Talaporfin sodium and a semiconductor laser. Intraoperatively, a crush smear preparation was made from a tiny amount of tumor tissue, and the fluorescence emitted upon 620/660 nm excitation was evaluated rapidly using a compact fluorescence microscope in the operating theater. Results: Fluorescence intensities of tumor tissues measured using a surgical microscope correlated with the tumor cell densities of tissues evaluated by measuring the red fluorescence emitted from the cytoplasm of tumor cells using a fluorescence microscope. A “weak fluorescence” indicated a reduction in the tumor cell density, whereas “no fluorescence” did not indicate the complete eradication of the tumor cells, but indicated that few tumor cells were emitting fluorescence.Conclusion: The rapid intraoperative detection of fluorescence from glioma cells using a compact fluorescence microscope was a useful to evaluate the presence of tumor cells in the resection cavity walls, and provides surgical implications for the more complete resection of malignant gliomas.

The high prevalence of thyroid nodules and increased availability of neck ultrasound have led to an increased incidence of diagnostic thyroid fine needle aspirations, with approximately 20% yielding indeterminate results. The recent availability of molecular tests has helped guide the clinical management of these cases. This paper aims to review and compare three main commercially available molecular cytology platforms in the U.S. – Afirma GSC, Thyroseq GC, and ThyGeNEXT+ThyraMIR. Sequential improvements of the Afirma GSC and Thyroseq GC tests have led to higher positive and negative predictive values, sensitivity, and specificity. Comparative studies revealed similar diagnostic performance between these tests, with considerations for factors such as cost and processing time. Thyroseq GC provides detailed genomic information and specific management recommendations. ThyGeNEXT+ThyraMIR, though less studied, presents promising results, particularly in miRNA analysis for weak driver mutations. Challenges in interpreting results include variations in reporting and the evolving nature of testing platforms. Questions persist regarding cost-effectiveness and the utility of ultrasound characteristics in selecting candidates for molecular testing. While molecular testing has primarily served diagnostic purposes, advancements in understanding genetic alterations now offer therapeutic implications. FDA-approved options target specific genetic alterations, signaling a promising future for tailored treatments.

(1) Background: The prediction of cervical lesions evolution is a challenge for clinicians. The prospective study aimed to determine and compare the predictive accuracy of cytology, HPV genotyping, and p16/Ki67 dual staining alone or in combination with personal risk factors for the prediction of progression, regression or persistence of cervical lesions in human papillomavirus (HPV)-infected patients; (2) Methods: This prospective study included HPV-positive patients with or without cervical lesions that underwent follow-up. Descriptive statistics and sensitivity analysis for individual or combined index tests were used for data analysis; (3) Results: The highest predictive performance for the progression of cervical lesions was achieved by a model that comprised a Pap smear suggestive of high-grade squamous intraepithelial lesion (HSIL), the presence of 16/18 HPV strains, a positive p16/Ki67 dual staining result along with the presence of at least 3 clinical risk factors, which had a sensitivity (Se) of 74.42%, a specificity of 97.92%, an area under the receiver operating curve (AUC) of 0.961, and an accuracy of 90.65%. The prediction of cervical lesions regression or persistence was modest; (4) Conclusions: Multiple testing or new biomarkers should be used to improve HPV-positive patient surveillance, especially for cervical lesion regression or persistence prediction.

Purpose: To assess the tolerability, safety, and efficacy of an ophthalmic topical formulation containing helenalin from Arnica montana and hyaluronic acid 0.4% (HA) in patients with mild to moderate dry eye disease (DED) exhibiting positive Matrix Metalloproteinase 9 (MMP-9) test results. Methods: Tolerability and safety were evaluated in 24 healthy subjects. Participants were in-structed to routinely apply one drop of the formulation three times a day in the study eye, for a period of two weeks, followed by a clinical follow-up of 21 days. Efficacy was studied in 48 DED patients randomized into Study (Group 1/ receiving the study formulation) or Control (Group 2/ Receiving HA 0.4% eye lubricant) groups, for 4 weeks. Assessments included MMP-9 positivity, conjunctival impression cytology (CIC), Ocular Surface Disease Index (OSDI), non-invasive tear film breakup time (NIF-BUT), non-invasive average breakup time (NIAvg-BUT), ocular surface staining, Schirmer's test, and meibomiography. A crossover design with a four-week follow-up was applied to the control group. Results: Healthy subjects receiving the study formulation exhibited good tolerability and no ad-verse events. Group 1 showed significant MMP-9 reduction (25% positivity rate) unlike Group 2 (87.5% positivity rate). Group 1 displayed improved CIC (33.3% vs. 0% SSD) compared to Group 2 (29.1%). OSDI and NIF-BUT scores improved in both groups (p < 0.001). Only Group 1 showed improved NIAvg-BUT and Schirmer’s test scores (p < 0.001), while Group 2 did not (p > 0.05). MMP-9-positive subjects shifted from 25% to 91.6% in Group 1 and 87.5% to 20.8% in Group 2 after the follow-up. Conclusion: The topical formulation containing helenalin from Arnica montana and hyaluronic acid is well tolerated and has a good safety profile. Our formulation reduces DED sympto-matology and modulates the ocular surface inflammatory process; this is evidenced by the en-hancement of CIC, the improvement of DED-related tear film status, and the reduction of MMP-9 positivity rate.

Background: A plethora of data supports HPV-based screening to be the preferred strategy for cervical cancer prevention. The shift to a more sensitive firstline test brings the need of effective triage up for discussion. Currently, most algorithms apply cytology as triage of HPV-DNA positive women. This study compared the performance of a 7-type HPV-mRNA test to cytology. Methods: From 2019-01-01, until 2021-12-31, cervical samples from 58,029 women were examined at the University Hospital of North Norway. 30.5% (17,684/58,029) fulfilled the criteria for HPV-DNA primary screening. All positive samples were triaged by cytology and followed-up according to national guidelines through 2022. Additionally, a 7-type HPV-mRNA test was applied. Study endpoint was histologically confirmed high grade lesion (CIN2+). Results: 5.6% (990/17,684) had positive HPV-DNA test, 97.2% (962/990) with valid HPV-mRNA results. 55.5% (534/962) had abnormal cytology (ASC-US+) and 35.1% (338/962) had positive HPV-mRNA test. 13.9% (134/962) had CIN2+. Sensitivity (CIN2+) of cytology versus the HPV-mRNA test was 76.1% (102/134)) versus 73.1% (98/134)), p=0.67. The specificity was 47.8% (396/828) versus 71.0% (588/624), p<0.001. PPV was 19.1% (102/534) and 29.0% (98/338), p<0.001 respectively. The number of colposcopies per CIN2+ detected by cytology and HPV-mRNA test was 5.2 and 3.1. Conclusion: The 7-type HPV mRNA test was significant more specific than cervical cytology in triage of HPV-DNA positive women. Using this biomarker as threshold for colposcopy may better balance the benefits and harms of screening.

Urinary cytology is a useful, essential diagnostic method in routine urological clinical practice. Liquid-based cytology (LBC) for urothelial carcinoma screening is commonly used in the routine clinical cytodiagnosis because of its high cell collection rate. Since conventional screening processes by cytoscreeners and cytopathologists using microscopes is limited in terms of human resources, it is important to integrate new deep learning methods that can automatically and rapidly diagnose a large amount of specimens without delay. The goal of this study was to investigate the use of deep learning models for the classification of urine LBC whole-slide images (WSIs) into neoplastic and non-neoplastic (negative). We trained deep learning models using 786 WSIs by transfer learning, fully supervised, and weakly supervised learning approaches. We evaluated the trained models on two test sets (equal and clinical balance) with a combined total of 750 WSIs, achieving ROC-AUCs for WSI diagnosis in the range of 0.984-0.990 by the best model, demonstrating the promising potential use of our model for aiding urine cytodiagnostic processes.

Background: This prospective study assesses the use of Rapid Remote Online Cytological Evaluation analysis for diagnosing endoscopical achieved biopsies. It focuses on its effectiveness in identifying benign and malignant conditions using digital image processing. Methods: The study was conducted between April 2021 and September 2022 and involved a total of 314 Rapid Remote Online Cytological Evaluations (17 brush, 143 fine needle aspirations and 154 imprint cytologies) analyses performed on 239 patients at the LungenClinic Grosshansdorf. During on-site evaluation via telecytology, the time requirement was determined and the findings were compared with the cyto-/histological and final diagnoses. Results: By means of Rapid Remote Online Evaluation, 86 cytological benign and 190 malignant and 38 findings of unclear diagnosis were recorded (Ø assessment time 100 sec., range 11 - 370sec.). In 27 of the 38 cases with unclear diagnosis, the final findings were malignant tumours and only 6 were benign changes. The diagnosis of another five of these 38 cases remained unclear. Excluding these 38 findings, the Rapid Remote online cytology achieved a sensitivity of 78.6% with a specificity of 99.5% and a correct classification rate of 93.1% with regard to the final diagnosis of all cases. As expected, an increase in the sensitivity rate for the cytological detection of malignant tumours (76.1% vs. 92.5%) was found especially in fine-needle aspirations. Conclusions: Rapid remote online analysis allows fast quantitative and qualitative evaluation of clinically obtained cytological specimens. With a correct classification rate of more than 93%, sampling deficiencies can be corrected promptly and diagnostic and therapeutic approaches can be derived.

We performed urine cytology analysis of a pharmacologically induced diuresis for diagnostics of upper tract urothelial carcinoma. To evaluate the diagnostic value of cytology of pharmacologically forced diuresis, a first cohort of 77 consecutive patients with primary upper tract urothelial carcinoma treated with radical surgery was enrolled. To evaluate pharmacologically forced diuresis cytology as follow-up procedure, a second cohort of 1250 patients who underwent radical cystectomy for bladder cancer was selected. In the first cohort, sensitivity of cytology of pharmacologically forced diuresis in patients with invasive, high-grade, low-grade, and concomitant carcinoma in situ was 8%, 9%, 0% and 14%, respectively. In the second cohort, cytology of pharmacologically forced diuresis was positive in 30/689 (4.3%) of patients, in whom upper urinary tract recurrence was present in 21/30 (70%) and urethral recurrence in 8/30 (26%) of cases. As a follow-up tool, cytology of pharmacologically forced diuresis showed a sensitivity, specificity, and positive and negative predictive value of 60%, 99%, 70% and 98%, respectively. Summarized, as diagnostic tool, sensitivity of cytology of pharmacologically forced diuresis is slightly better in patients with invasive upper tract urothelial carcinoma and concomitant carcinoma in situ. As follow-up method, positive cytology of pharmacologically forced diuresis is strongly related to cancer recurrence and can reveal urethral recurrence. cytology of pharmacologically forced diuresis might be useful in patients with contraindications for imaging or with difficult endoscopic access to the upper urinary tract.

Marine invertebrates are model organisms in several areas of biological sciences, being a source of massive biological information. Although, the scientific relevance of marine invertebrates, the research with them can be limited for their tissue characteristics and troubles for the replication of physical and chemical properties of seawater. Thence, the main goal of this laboratory workflow is to provide a useful methodological approach to reduce the experimental limitations during the study of marine invertebrates. The present study describes experimental methodologies for the collection, transport, and maintenance of sessile tunicates. Also, an approach to observe and characterize, a diverse population of blood cells in marine invertebrates, by several cytological stains and electron microscopy. Lastly, suggestions and protocols to extract quality RNA from samples with high concentrations of salts, pigments, secondary metabolites, and polysaccharides. This methodological approach can be easily adapted to other marine invertebrates, moreover uses low-cost reagents and widely available laboratory equipment. Making possible the study of different types of marine animals in diverse locations.

Cervical cancer, primarily caused by high-risk human papillomavirus (HR-HPV) types 16 and 18, is a major global health concern. Persistent HR-HPV infection can progress from reversible pre-cancerous lesions to invasive cervical cancer, which is driven by the oncogenic activity of human papillomavirus (HPV) genes, particularly E6 and E7. Traditional screening methods, including cytology and HPV testing, have limited sensitivity and specificity. This review explores the ap-plication of p16/Ki-67 dual-staining cytology for cervical cancer screening. This advanced im-munocytochemical method allows for simultaneously detecting p16 and Ki-67 proteins within cervical epithelial cells, offering a more specific approach for triaging HPV-positive women. Dual staining and traditional methods are compared, demonstrating their high sensitivity and negative predictive value but low specificity. The increased sensitivity of dual staining results in higher detection rates of CIN2+ lesions, which is crucial for preventing cervical cancer progression. However, its low specificity may lead to increased false-positive results and unnecessary biopsies. The implications of integrating dual staining into contemporary screening strategies, particularly considering the evolving landscape of HPV vaccination and changes in HPV genotype prevalence are also discussed. New guidelines and further research are necessary to elucidate the long-term effects of integrating dual staining into screening protocols.

Introduction – Molecular tests can contribute to improve the preoperative diagnosis of thyroid nodules. Tests available are expensive and not adapted to different population. Aim – This study aimed to compare the cyto-histological genetic profile, and to evaluate the reliability of molecular tests on Ultrasound-guided Fine Needle Aspiration Cytology (US-FNAC) in accurately diagnosing differentiated thyroid carcinomas (DTCs) and predicting biologic behavior of papillary thyroid carcinomas (PTCs). Material and methods – The series included 259 patients with paired cyto-histological samples totalizing 518 samples. The genetic alterations were analyzed by PCR/Sanger sequencing. The association with clinicopathologic features was evaluated in PTCs. Results/Discussion – From the 259 patients included, histologies were 50 (19.3%) benign controls and 209 (80.7%) DTCs cases, from which 182 were PTCs; cytologies were 5.8% non-diagnostic, 18.2% benign, 39% indeterminate and 37.1% malignant. In histology, indeterminate nodules (n=101) were 22.8% benign and 77.2% malignant. Mutation frequencies in cytology and histology specimens were, respectively, TERTp: 3.7% vs. 7.9%; BRAF: 19.5% vs. 25.1%; RAS: 11% vs. 17.5%. The overall cyto-histological agreement of the genetic mutations was 94.9%, Cohen’s k=0.67, and in indeterminate nodules 95.7%, k=0.64. The identified mutations exhibited a discriminative ability in diagnosing DTC with a specificity of 100% for TERTp and BRAF, and of 94% for RAS, albeit with low sensitivity. TERTp and BRAF mutations were associated with aggressive clinicopathological features and tumor progression in PTCs (p<0.001). The obtained good cyto-histological agreement suggests that molecular analysis in US-FNAC may anticipate the genetic profile and the behavior of thyroid tumors, confirming malignancy and contributing to refer patients towards surgery.