The Role of 5-Hydroxymethylcytosine as a Potential Epigenetic Biomarker in a Large Series of Thyroid Neoplasms.

Cytosine modifications at the 5-carbon position play a critical role in gene expression regulation and have been implicated in cancer development. 5-Hydroxymethylcytosine (5hmC), arising from 5-methylcytosine (5-mC) oxidation, has shown promise as a potential malignancy marker due to its depletion in various human cancers. However, its significance in thyroid tumors remains underexplored, primarily due to limited data. In our study, we evaluated 5hmC expression levels by immunohistochemistry in a cohort of 318 thyroid tumors. Our analysis revealed significant correlations between 5hmC staining extension scores and nodule size, vascular invasion, and oncocytic morphology. Nuclear 5hmC staining intensity demonstrated associations with focality, capsule status, extrathyroidal extension, vascular invasion, and oncocytic morphology. Follicular/oncocytic adenomas exhibited higher 5hmC expression than uncertain malignant potential (UMP) or noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP), as well as malignant neoplasms, including papillary thyroid carcinomas (PTCs), oncocytic carcinomas (OCAs), follicular thyroid carcinomas (FTCs), and invasive encapsulated follicular variants of PTC (IEFV-PTC). TERT promoter mutation cases showed notably lower values for the 5hmC expression, while RAS (H, N, or K) mutations, particularly HRAS mutations, were associated with higher 5hmC expression. Additionally, we identified, for the first time, a significant link between 5hmC expression and oncocytic morphology. However, despite the merits of these discoveries, we acknowledge that 5hmC currently cannot segregate minimally invasive from widely invasive tumors, although 5hmC levels were lower in wi-FPTCs. Further research is needed to explore the potential clinical implications of 5hmC in thyroid tumors.

Rapid Remote Online Evaluation in Endoscopic Diagnostics: An Analysis of Biopsy-Proven Respiratory Cytopathology.

BACKGROUND: This prospective study assesses the use of rapid remote online cytological evaluation 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 analyses of 314 Rapid Remote Online Cytological Evaluations in total (154 imprint cytologies, 143 fine needle aspirations and 17 brush cytologies) performed on 239 patients at the LungenClinic Grosshansdorf. During on-site evaluation via telecytology, the time requirement was recorded and the findings were compared with the cyto-/histological and final diagnoses. RESULTS: By means of rapid remote online evaluation, findings of 86 cytological benign, 190 malignant and 38 unclear diagnoses were recorded (Ø assessment time, 100 s; range, 11-370 s). In 27 of the 37 specimens with unclear diagnoses, the final findings were malignant tumours and only 6 were benign changes. The diagnosis of another 4 of these 37 findings remained unclear. Excluding these 37 specimens, rapid remote online evaluation achieved a sensitivity of 90.5% with a specificity of 98.5% and a correct classification rate of 92.4% 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 the 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.

Melanocytic tumors with spitzoid morphology: correlation of clinicopathologic features and FISH analysis.

BACKGROUND: A subset of melanocytic tumors with spitzoid morphology may lead to potential inaccurate diagnosis and lack of assessment of malignancy potential. In this study, we aimed to evaluate melanocytic tumors with spitzoid morphology using conventional melanoma FISH (RREB-1, CCND1, MYB and CEP6) and 9p21 FISH (CDKN2A) probes and compare the probe results with clinical and histopathological features. METHODS: This study is a multicentric retrospective study including three centers, Istanbul University-Cerrahpasa, Cerrahpasa School of Medicine, Department of Pathology, Acibadem University, School of Medicine, Department of Pathology and ETA Pathology Laboratory. The pathology reports in archives of these three centers between 2015 and 2017 have been reviewed for cases diagnosed as atypical Spitz tumor or melanoma with Spitzoid features. These cases were selected for the study. We analyzed 39 cases of atypical Spitz tumor (AST), 10 cases of melanomas with spitzoid features for clinicopathological data and chromosomal alterations, targeting RREB-1 (6p25), CCND1 (11q13), MYB (6q23), together with 9p21 (CDKN2A), using FISH methodology. RESULTS: Thirty out of total 49 cases showed chromosomal alterations by 4-probe melanoma FISH assay, 22 (56.4%) cases were ASTs, and 8 (80%) cases were melanomas. Eighteen out of 49 cases showed homozygote deletion by 9p21 FISH assay, 12 (30.8%) cases were ASTs, and 6 (60%) cases were melanomas. When histopathological data were compared with FISH results, a statistically significant correlation was found between 9p21 FISH positivity (homozygous deletion) and presence of deep mitosis (p < 0.05). In addition, epidermal consumption (p = 0.07) and increased mitotic activity (p = 0.05) were more frequent in cases with homozygous 9p21 deletion, but these differences did not reach statistical significance. When the clinical features were considered, there was a statistically significant correlation between 9p21 FISH positivity and the diameter (p < 0.05). There was no statistically significant correlation between melanoma FISH assay and any of the histopathological or clinical data. DISCUSSION: These data suggest that 9p21 FISH positivity correlated with more worrisome histopathologic and clinical features, such as deep mitosis, increased mitotic activity, epidermal consumption, and larger lesion size, so these features are precious, pointing out spitzoid lesions with higher risk. However, there is a need for further studies using FISH or similar techniques in order to provide more accurate prognostic information in lesions Blank morphology.

Endometrial Staining of CD56 (Uterine Natural Killer), BCL-6, and CD138 (Plasma Cells) Improve Diagnosis and Clinical Pregnancy Outcomes in Unexplained Infertility and Recurrent IVF Failures: Standardization of Diagnosis with Digital Pathology.

In women with unexplained infertility (UI) and recurrent in vitro fertilization (IVF) failures, the etiology is often unclear. Endometrial immune perturbations and the use of immune markers associated with these dysregulations are of great interest in the diagnosis and treatment of UI. However, reliable biomarkers and standardized quantification methods are lacking. Here, to address endometrial immune dysregulation in UI patients with recurrent IVF failures, we performed endometrial tissue sampling and immunostaining of CD56 (uNK), CD138, and BCL-6. Of these cases, 57.9% had positive CD56 in the endometrial stroma, while 46.1% had positive BCL-6 in the glandular epithelium, and 14.5% of the cases were found to be positive for CD138. Combined staining rates were 60.5%, 68.4%, and 71.05% for (CD56 or BCL-6), (CD56 or CD138), and (CD56, BCL-6, or CD138), respectively. There was a significant correlation between CD56 and BCL-6 positivity, while CD138 positivity was an independent parameter. After the recommended targeted therapy, pregnancy rates were found to increase from 58.5% to 61.6% and 73.8% in CD56-positive, (CD56- or BCL-6-positive), and (CD56-, BCL-6-, or CD138-positive) cases, respectively. Notably, a retrospective evaluation of digital pathology and light microscopy results showed a significant correlation. This study suggests that the examination of CD56, BCL-6, and CD138 in the same endometrial sample may be an effective method in determining the etiology of UI and reaching an early diagnosis and treatment options. Moreover, digital pathology can be used in the evaluation of CD56 and BCL-6 to provide objective, rapid, and reliable results.

Tubule-U-Net: a novel dataset and deep learning-based tubule segmentation framework in whole slide images of breast cancer.

The tubule index is a vital prognostic measure in breast cancer tumor grading and is visually evaluated by pathologists. In this paper, a computer-aided patch-based deep learning tubule segmentation framework, named Tubule-U-Net, is developed and proposed to segment tubules in Whole Slide Images (WSI) of breast cancer. Moreover, this paper presents a new tubule segmentation dataset consisting of 30820 polygonal annotated tubules in 8225 patches. The Tubule-U-Net framework first uses a patch enhancement technique such as reflection or mirror padding and then employs an asymmetric encoder-decoder semantic segmentation model. The encoder is developed in the model by various deep learning architectures such as EfficientNetB3, ResNet34, and DenseNet161, whereas the decoder is similar to U-Net. Thus, three different models are obtained, which are EfficientNetB3-U-Net, ResNet34-U-Net, and DenseNet161-U-Net. The proposed framework with three different models, U-Net, U-Net++, and Trans-U-Net segmentation methods are trained on the created dataset and tested on five different WSIs. The experimental results demonstrate that the proposed framework with the EfficientNetB3 model trained on patches obtained using the reflection padding and tested on patches with overlapping provides the best segmentation results on the test data and achieves 95.33%, 93.74%, and 90.02%, dice, recall, and specificity scores, respectively.

Favorable locoregional control in clinically node-negative hormone-receptor positive breast cancer with low 21-gene recurrence scores: a single-institution study with 10-year follow-up.

BACKGROUND: Recent studies have shown a lower likelihood of locoregional recurrences in patients with a low 21-gene recurrence score (RS). In this single-institution study, we investigated whether there are any associations between different cutoff values of 21-gene RS, histopathological factors, and outcome in patients with long-term follow-up. METHODS: The study included 61 patients who had early-stage (I-II) clinically node-negative hormone receptor-positive and HER2-negative breast cancer and were tested with the 21-gene RS assay between February 2010 and February 2013. Demographic, clinicopathological, treatment, and outcome characteristics were analyzed. RESULTS: The median age was 48 years (range, 29-72 years). Patients with high histologic grade (HG), Ki-67 ≥ 25%, or Ki-67 ≥ 30% were more likely to have intermediate/high RS (≥ 18). Based on the 21-gene RS assay, only 19 patients (31%) received adjuvant chemotherapy. At a median follow-up of 112 months, 3 patients developed locoregional recurrences (4.9%), which were treated with endocrine therapy alone. Among patients treated with endocrine treatment alone (n = 42), the following clinicopathological characteristics were not found to be significantly associated with 10-year locoregional recurrence free survival (LRRFS): age < 40 years, age < 50 years, high histological or nuclear grade, high Ki-67-scores (≥ 15%, ≥ 20%, ≥ 25%, ≥ 30%), presence of lymphovascular invasion, luminal-A type, multifocality, lymph node positivity, tumor size more than 2 cm, RS ≥ 18, and RS > 11. However, patients with RS ≥ 16 had significantly poorer 10-year LRRFS compared to those with RS < 16 (75% vs. 100%, respectively; p = 0.039). CONCLUSIONS: The results suggest that patients with clinically node-negative disease and RS ≥ 16 are more likely to benefit from adjuvant chemotherapies. However, those with RS < 16 have an excellent outcome and local control in long-term follow-up with endocrine treatment alone.

Intramural Component of Venous, Lymphatic, and Perineural Invasion in Colon Cancer: A Threat or an Illusion?

Background: Extramural venous invasion is an independent predictor of poor outcome in colorectal cancer, whereas the significance of the intramural component of venous and lymphatic and perineural invasion is unclear. Aims: To evaluate the prognostic impact of intramural components for venous, lymphatic, and perineural invasions and the relation of these invasion patterns with clinicopathological features in patients with colon cancer. Study Design: A retrospective cross-sectional study. Methods: The analysis included 626 patients with colon cancer in stages II and III. All patients were divided into four categories (no invasion, intramural invasion only, extramural invasion only, or both intramural and extramural invasions) for vascular invasion, lymphatic invasion and perineural invasion. The primary outcomes were 5-year disease-free and overall survival. Results: Right-sided (for vascular invasion, 24.7% vs. 33.9%, p = 0.007; for perineural invasion, 34.5% vs. 41.5%, p = 0.034) and dMMR tumors (for vascular invasion, 13.5% vs. 33.5, p < 0.001; for perineural invasion, 25% vs. 41.4%, p = 0.004) exhibited less venous and perineural invasion. Compared with no invasion, presence of intramural invasion only, did not exert any effect on disease-free or overall survival for vascular invasion, lymphatic invasion, and perineural invasion. Multivariate analyses revealed that the presence of both intramural and extramural invasion was independently associated with poor disease-free and overall survival for venous (hazard ratios: 2.39, p = 0.001; hazard ratios: 2.46, p = 0.001), lymphatic (hazard ratios: 2.456, p < 0.001; hazard ratios: 2.13, p = 0.02) and perineural invasion (hazard ratios: 2.99, p < 0.001; hazard ratios: 2.68, p < 0.001), respectively. Conclusion: Our data strongly advocates the importance of reporting intramural and extramural components of invasion since the presence of intramural invasion alone may not be considered as a high-risk factor for systemic recurrence.

One step nucleic acid amplification (OSNA) for detection of lymph node metastasis during robotic radical prostatectomy for prostate cancer: A pilot study.

INTRODUCTION: The OSNA technique is based on reverse transcription loop-mediated DNA amplification for the detection of cytokeratin 19 (CK19) messen-ger RNA (mRNA). The purpose of our paper, which represents the first study in the literature, is to test the accuracy of this method in the detection of lymph node metastases in patients undergoing robotic radical prostatectomy with lymph node dis-section. METHODS: Our cohort consisted of patients that have undergone robotic radical prostatectomy with extended lymph node dissec-tion. Lymph nodes were evaluated with imprint technique and then with frozen section examination. The remaining tissue was evaluated by OSNA method. Lymph nodes were defined as 'neg-ative' or 'positive' according to mRNA copy number. RESULTS: 7 patients and 25 lymph nodes were included in our cohort. Two patients were found negative with all pathology methods. In one patient the standard stains revealed a suspi-cious outcome but it was positive for micrometastasis with OSNA. In another patient the outcome was positive for standard stains and negative for OSNA. Finally, 2 patients were found positive for OSNA and negative for imprint methods. CONCLUSIONS: One Step Nucleic Acid Amplification (OSNA) method using CK19 seems to fail in detection of lymph node metastases in prostate cancer patients undergoing radical prostatectomy and lymph node dissection.

Expression of vascular endothelial growth factor in follicular cell-derived lesions of the thyroid: Is NIFTP benign or precancerous?

Objective: Vascular endothelial growth factor (VEGF) is an angiogenic factor that plays an important role in physiological and pathological angiogenesis of the thyroid. The aim of the current study was to determine the expression characteristics of VEGF in follicular cell-derived lesions of the thyroid and to assess whether a new entity noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) is precancerous. Material and Methods: Patients diagnosed with 33 follicular adenomas (FA), 41 invasive follicular variant papillary thyroid cancer (IN-FVPTC), and 40 NIFTP in surgical resection materials were evaluated retrospectively. Immunostaining was performed on 5-µm paraffin tissue sections. The percentages of immunostaing for VEGF were evaluated on pathological materials. We used a percentage of labeled thyrocytes score (0, no labeling; 1, <30%; 2, 31-60%; 3, >60%) and an intensity score (0, no staining; 1, weak; 2, intermediate; 3, strong). The sum of two scores were accepted as the total score. Results: Mean ages of the FA, IN-FVPTC, and NIFTP groups were 44.7 ± 11.7 years, 46.9 ± 13.6 years, 43.2 ± 15.4 years, respectively and the mean VEGF immunostaining scores were 44.7 ± 29.3, 50.2 ± 32.54, 4 ± 26.3 respectively. Although there was no statistically significant difference (p= 0.347), the total score of the NIFTPs was higher than the scores of the FA (mean= 3.9 ± 1.8) and IN-FVPTC(mean= 4.3 ± 1.9) groups with a mean value of 4.6 ± 1.7. This result was remarkable. There was no statistically significant difference between tumor diameters and staining percentages (p= 0.750). Conclusion: Even if there were no statistical differences for VEGF immunostaining, it was high in NIFTPs. Since we know the role of VEGF in tumorigenesis, we can hypothesize that NIPTP can be precancerous. Our argue should be corroborated by a large prospective study.

Autologous omentum transposition for regeneration of a renal injury model in rats.

BACKGROUND: After renal trauma, surgical treatment is vital, but sometimes there may be loss of function due to fibrosis. This study aimed to evaluate the effect of autologous omentum flaps on injured renal tissues in a rat model. METHODS: A total of 30 Wistar albino rats were included and randomly divided equally into a control group and four intervention groups. Iatrogenic renal injuries were repaired using a surgical technique (primary repair 1 group and primary repair 2 group) or transposition of the autologous omentum (omentum repair 1 group and omentum repair 2 group). Blood samples were taken preoperatively and on the 1st and 7th postoperative days in all groups and on the 18th postoperative day in the control and two intervention groups. All rats were sacrificed on the 7th or 18th day postoperatively, and their right kidneys were taken for histopathological evaluation. RESULTS: The mean urea level significantly decreased from day 1 to day 7 and from day 1 to day 18 in the omentum repair 2 group (P = 0.005 and P = 0.004, respectively). There were no other significant changes in urea or creatinine levels within the intervention groups (P > 0.05). There was no significant correlation between the urea and creatinine levels and the histological scores (P > 0.05). The primary repair 1 and 2 groups had significantly higher median granulation and inflammation scores in the kidney specimen than the control and omentum repair groups (P < 0.05). The omentum repair 2 group had significantly lower median granulation and inflammation scores in the surrounding tissues than the primary repair 2 group (P < 0.05). The completion score for the healing process in the kidney specimen was significantly higher in the omentum repair groups than in the primary repair groups (P < 0.05). The omentum repair 2 group had significantly lower median granulation and inflammation scores in the surrounding tissues than the primary repair 2 group (P < 0.05). Granulation degree in the kidney specimen was strongly and positively correlated with the inflammation degree (r = 0.824, P < 0.001) and foreign body reaction in the kidney specimen (r = 0.872, P < 0.001) and a strong and negative correlation with the healing process completion score in the kidney (r = - 0.627, P = 0.001). Inflammation degree in the kidney specimen was strongly and positively correlated with the foreign body reaction in the kidney specimen (r = 0.731, P = 0.001) and strongly and negatively correlated with the healing process completion score in the kidney specimen (r = - 0.608, P = 0.002). CONCLUSION: Autologous omentum tissue for kidney injury repair attenuated inflammation and granulation. Additionally, the use of omental tissue to facilitate healing of kidney injury may theoretically lead to a more effective healing process and reduced fibrosis and tissue and function loss.

Histologically benign PI-RADS 4 and 5 lesions contain cancer-associated epigenetic alterations.

BACKGROUND: The detection rate of clinically significant prostate cancer has improved with the use of multiparametric magnetic resonance imaging (mpMRI). Yet, even with MRI-guided biopsy 15%-35% of high-risk lesions (Prostate Imaging-Reporting and Data System [PI-RADS] 4 and 5) are histologically benign. It is unclear if these false positives are due to diagnostic/sampling errors or pathophysiological alterations. To better understand this, we tested histologically benign PI-RAD 4 and 5 lesions for common malignant epigenetic alterations. MATERIALS AND METHODS: MRI-guided in-bore biopsy samples were collected from 45 patients with PI-RADS 4 (n = 31) or 5 (n = 14) lesions. Patients had a median clinical follow-up of 3.8 years. High-risk mpMRI patients were grouped based on their histology into biopsy positive for tumor (BPT; n = 28) or biopsy negative for tumor (BNT; n = 17). From these biopsy samples, DNA methylation of well-known tumor suppressor genes (APC, GSTP1, and RARß2) was quantified. RESULTS: Similar to previous work we observed high rates of promoter methylation at GSTP1 (92.7%), RARß2 (57.3%), and APC (37.8%) in malignant BPT samples but no methylation in benign TURP chips. Interestingly, similar to the malignant samples the BNT biopsies also had increased methylation at the promoter of GSTP1 (78.8%) and RARß2 (34.6%). However, despite these epigenetic alterations none of these BNT patients developed prostate cancer, and those who underwent repeat mpMRI (n = 8) demonstrated either radiological regression or stability. CONCLUSIONS: Histologically benign PI-RADS 4 and 5 lesions harbor prostate cancer-associated epigenetic alterations.

Gamma-irradiated SARS-CoV-2 vaccine candidate, OZG-38.61.3, confers protection from SARS-CoV-2 challenge in human ACEII-transgenic mice.

The SARS-CoV-2 virus caused the most severe pandemic around the world, and vaccine development for urgent use became a crucial issue. Inactivated virus formulated vaccines such as Hepatitis A and smallpox proved to be reliable approaches for immunization for prolonged periods. In this study, a gamma-irradiated inactivated virus vaccine does not require an extra purification process, unlike the chemically inactivated vaccines. Hence, the novelty of our vaccine candidate (OZG-38.61.3) is that it is a non-adjuvant added, gamma-irradiated, and intradermally applied inactive viral vaccine. Efficiency and safety dose (either 1013 or 1014 viral RNA copy per dose) of OZG-38.61.3 was initially determined in BALB/c mice. This was followed by testing the immunogenicity and protective efficacy of the vaccine. Human ACE2-encoding transgenic mice were immunized and then infected with the SARS-CoV-2 virus for the challenge test. This study shows that vaccinated mice have lowered SARS-CoV-2 viral RNA copy numbers both in oropharyngeal specimens and in the histological analysis of the lung tissues along with humoral and cellular immune responses, including the neutralizing antibodies similar to those shown in BALB/c mice without substantial toxicity. Subsequently, plans are being made for the commencement of Phase 1 clinical trial of the OZG-38.61.3 vaccine for the COVID-19 pandemic.

The crosstalk between H. pylori virulence factors and the PD1:PD-L1 immune checkpoint inhibitors in progression to gastric cancer.

BACKGROUND: The progression to gastric cancer has been linked to chronic infection with Helicobacter pylori (H. pylori). Immune checkpoint inhibitors (programmed cell death -1, PD-1; programmed cell death -ligand 1, PD-L1) have a role in cancer immune escape. The relationship between H. pylori virulence factors with PD-1, PD-L1 T helper 1 (Th1), T helper 17 (Th17), and regulatory T cell (Treg) response genes, has not been thoroughly investigated in the development of gastric cancer. Therefore, we evaluated how H. pylori virulence factors influence the expression levels of immune-related genes in the development of gastric immunopathology. METHODS: A total of 92 gastric tissues of normal controls and patients with gastritis, gastric ulcer, and gastric cancer were examined for the expression of immune-checkpoint inhibitor genes (PD-1 PD-L1), Th1 (interferon- γ, IFN-γ), Th17 (interleukin- 17, IL-17, Retinoic-acid-receptor- related orphan nuclear receptor gamma t, RORγ-t), and Treg (Forkhead box P3, FOXP3) response genes with quantitative real-time PCR (qRT-PCR). Furthermore, correlation of H. pylori virulence factors' (cytotoxin-associated gene A, cagA; vacuolating cytotoxin gene A, vacA (s1,s2,m1,m2); blood group antigen-binding adhesin gene A, babA, duodenal ulcer promoting gene A, dupA; the putative neuraminyllactose-binding hemagglutinin homolog, hpaA; neutrophil-activating protein A napA; outer inflammatory protein A, oipA; urease A, ureA; and urease B, ureB) genotypes with a degree of inflammation and density of H. pylori were investigated. Next, the relationship between H. pylori virulence factors and immune-checkpoint inhibitor genes, and T-cell response genes was evaluated. Eventually, a decision tree model was developed to determine the clinical outcome of patients using expression data. RESULTS: The intensity of PD-1 and PD-L1 mRNA expression was increased significantly in gastric tissue of patients with gastric ulcer (PD-1: 2.3 fold, p=0.01; PD-L1: 2.1 fold, p=0.004), and gastric cancer (PD-1: 2 fold, p= 0.04; PD-L1: 1.8 fold, p=0.05) compared with control subjects. Also, PD-1: PD-L1 expression was significantly higher in patients with gastritis, who were infected with a marked density of H. pylori compared with its mildly infected counterparts. Furthermore, a novel negative correlation was found between PD-1 (r= -0.43) and PD-L1 (r= -0.42) with FOXP3 in patients with gastritis. CagA-positive H. pylori strain's negative association with PD-L1 expression (r=-0.34) was detected in patients with gastritis. Interestingly, PD-1 mRNA expression correlated positively with vacA s2/m2, in gastritis (r=0.43) and ulcer (r=0.43) patients. Furthermore, PD-1: PDL1 expression negatively correlated with vacA m1/m2 (r=-0.43 for PD-1; r=-0.38 for PD-L1) in gastritis patients. Moreover, an inverse correlation of PDL1 was present with vacA m1 (r=0.52) and vacA s1/m1 (r=0.46) versus vacA m2 (r=-0.44) and vacA m1 (r=0.52) and vacA s1/m2 (r=-0.14) in ulcer patients, respectively. Also, a correlation of vacA m2 (r=-0.47) and vacA s1/s2 (r= 0.45) with PD-1 was detected in ulcer patients. In addition, a novel negative correlation between FOXP3 mRNA levels and napA was shown in patients with gastritis and ulcer (r=-0.59). Finally, a computer-based model that was developed showed that knowing the expression levels of PD-L1, RORγ-t, and vacA s1/m2 would be useful to detect the clinical outcome of a patient. CONCLUSION: Our results suggested that PD-1:PD-L1 immune checkpoint inhibitors were increased in gastric pre-cancerous lesions that progress to gastric cancer. Herein, we report the relationship between H. pylori virulence factors and expression of host immune checkpoint inhibitors for diagnostic prediction of gastric malignancies using computer-based models.

Characterisation of the Leishmania donovani/L. infantum Hybrid Isolated from an Autochothonous Kala-Azar Patient: Preliminary Results of an In Vivo Model

Objective: In the present study, preliminary outcomes of the in vivo assessment of a Leishmania donovani/L. infantum hybrid isolated from a hospitalised patient with visceral leishmaniasis in Manisa and identified through analysis of the Leishmania-specific ITS-1, hsp70 and cpb gene regions are presented in comparison with reference strains of L. donovani and L. infantum. Methods: Three different study groups [(SG); n=16 mice each] and a control group (n=8 mice) were established with female Balb/C mice weighing 25-30 g. Reference L. donovani (MHOM/IN/1980/DD8), reference L. infantum (MHOM/TN/1980/IP1) and a L. donovani/L. infantum hybrid (MHOM/TR/2014/CBVL-LI/ LD), stored in liquid nitrogen, were thawed, cultured and incubated at 25 °C. A 15-µL dose of 1x108/mL promastigotes of three strains was applied to the tail veins of mice in the SG. After the mice were sacrificed, the liver and spleen tissues were removed and stored for immunological, immunohistochemical and pathological analyses. Results: The presence of infection in the liver and spleen tissues of mice was detected both by a specific enzyme-linked immunosorbent assay test and from the recovery of Leishmania promastigotes from liver and spleen tissues in NNN medium. However, Leishmania amastigotes were not observed in the touch biopsy smears of livers or spleens in either of the SGs. In addition, no evidence of tissue damage was identified in the SGs after immunohistochemical staining (with antibodies against IL-9, CD-117, MBP, CD163, CD4, CD8 and CD31). Conclusion: The obtained results show that hybrid Leishmania and reference L. donovani and L. infantum strains reached the liver and spleens of Balb/C mice in SGs but were of no pathological consequence. Yet, these three Leishmania isolates caused skin lesions when applied subcutaneously in Balb/C mice in another study. The findings presented in this study will be reassessed upon completion of the project, once the final results are obtained.

Histopathological characteristics and CD163 immunostaining pattern in fibrous papule of the face.

BACKGROUND: Signs of inflammation including epidermal interface changes, spongiosis, and dermal inflammation as well as pagetoid dyskeratosis are rarely described in fibrous papule (FP). We aimed to describe the inflammatory parameters, the rate of pagetoid dyskeratosis, along with CD163 immunohistochemical staining as an adjunctive diagnostic tool in FP. METHODS: Histopathology samples of all biopsy-proven FP cases were retrieved from archives and investigated for inflammatory parameters, presence of pagetoid dyskeratosis, as well as CD163, CD10, and CD34 immunostaining pattern of dermal spindle/stellate or multinucleate cells (graded from 0 to 4). RESULTS: Thirty-two cases of FP were identified. A high rate of inflammatory parameters including interface changes (20/32), spongiosis (31/32), and dermal lymphocytic inflammation (31/32) were detected. Pagetoid dyskeratosis was identified in eight out of 32 cases (25%). A grade 4 staining revealing a strong dendritic pattern was confirmed in all FP cases with CD163 immunohistochemistry including atypical variants such as granular FP, compared with CD10 (11/32) and CD34 (3/32). CONCLUSION: The dendritic cellular proliferation in FP may represent an inflammatory response to various stimuli; pagetoid dyskeratosis is a relatively common and underrecognized epidermal feature and CD163 immunostaining may be used as an adjunctive diagnostic tool in unusual histopathological subtypes.

Non-muscle invasive bladder cancer tissues have increased base excision repair capacity.

The molecular mechanisms underlying the development and progression of bladder cancer (BC) are complex and have not been fully elucidated. Alterations in base excision repair (BER) capacity, one of several DNA repair mechanisms assigned to preserving genome integrity, have been reported to influence cancer susceptibility, recurrence, and progression, as well as responses to chemotherapy and radiotherapy. We report herein that non-muscle invasive BC (NMIBC) tissues exhibit increased uracil incision, abasic endonuclease and gap-filling activities, as well as total BER capacity in comparison to normal bladder tissue from the same patient (p < 0.05). No significant difference was detected in 8-oxoG incision activity between cancer and normal tissues. NMIBC tissues have elevated protein levels of uracil DNA glycosylase, 8-oxoguanine DNA glycosylase, AP endonuclease 1 and DNA polymerase ß protein. Moreover, the fold increase in total BER and the individual BER enzyme activities were greater in high-grade tissues than in low-grade NMIBC tissues. These findings suggest that enhanced BER activity may play a role in the etiology of NMIBC and that BER proteins could serve as biomarkers in disease prognosis, progression or response to genotoxic therapeutics, such as Bacillus Calmette-Guérin.

Preclinical Assessment of Efficacy and Safety Analysis of CAR-T Cells (ISIKOK-19) Targeting CD19-Expressing B-Cells for the First Turkish Academic Clinical Trial with Relapsed/Refractory ALL and NHL Patients

Objective: Relapsed and refractory CD19-positive B-cell acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL) are the focus of studies on hematological cancers. Treatment of these malignancies has undergone recent transformation with the development of new gene therapy and molecular biology techniques, which are safer and well-tolerated therapeutic approaches. The CD19 antigen is the most studied therapeutic target in these hematological cancers. This study reports the results of clinical-grade production, quality control, and in vivo efficacy processes of ISIKOK-19 cells as the first academic clinical trial of CAR-T cells targeting CD19-expressing B cells in relapsed/refractory ALL and NHL patients in Turkey. Materials and Methods: We used a lentiviral vector encoding the CD19 antigen-specific antibody head (FMC63) conjugated with the CD8-CD28-CD3ζ sequence as a chimeric antigen receptor (CAR) along with a truncated form of EGFR (EGFRt) on human T-lymphocytes (CAR-T). We preclinically assessed the efficacy and safety of the manufactured CAR-T cells, namely ISIKOK-19, from both healthy donors' and ALL/NHL patients' peripheral blood mononuclear cells. Results: We showed significant enhancement of CAR lentivirus transduction efficacy in T-cells using BX-795, an inhibitor of the signaling molecule TBK1/IKKƐ, in order to cut the cost of CAR-T cell production. In addition, ISIKOK-19 cells demonstrated a significantly high level of cytotoxicity specifically against a CD19+ B-lymphocyte cancer model, RAJI cells, in NOD/SCID mice. Conclusion: This is the first report of preclinical assessment of efficacy and safety analysis of CAR-T cells (ISIKOK-19) targeting CD19-expressing B cells in relapsed/refractory ALL and NHL patients in Turkey.

Variants of Papillary Thyroid Carcinoma: An Algorithmic Cytomorphology-Based Approach to Cytology Specimens.

BACKGROUND: Thyroid cancer accounts for 1% of cancer cases in developed countries, in which papillary thyroid carcinoma (PTC) is the most common type. There are multiple variants of PTC described to date, some of them with aggressive behavior and poor clinical outcome. These variants are well described and accepted in recent guidelines of many international societies, and the prognostic and management implications are well laid out. Due to their established clinical importance and to guide appropriate surgical management, it is now imperative in clinical practice, including cytopathology, to differentiate aggressive variants from nonaggressive ones. This review aims to describe the variants of PTC and to provide a practical algorithmic approach to facilitate the cytological diagnosis of these variants. SUMMARY: Subtyping PTC variants on fine needle aspiration cytology (FNAC) is challenging even for the most experienced cytopathologist. To facilitate a correct subtyping on FNAC, we propose a stepwise approach that is mainly designed for conventional smear methodology. This approach requires first to stratify the lesions into oncocytic and nononcocytic features before analyzing further details in cell morphology and pattern. Key Messages: (1) Subtyping in PTC is possible on cytopathology. (2) The main aim of the cytopathologist is to differentiate aggressive from nonaggressive variants. (3) The subtyping of PTC can help in the surgical management of the patients.

The Role of Telecytology in the Primary Diagnosis of Thyroid Fine-Needle Aspiration Specimens.

INTRODUCTION: The Acibadem Health Group (AHG) has been using telepathology/digital pathology stations since 2006. In 2013, the system was changed from videoconferencing to digital pathology (whole-slide imaging) utilizing 3DHISTECH scanners and software. In 2017, digital cytology started to be used for routine cytopathologic diagnosis for thyroid fine-needle aspiration (FNA) cases. MATERIAL AND METHODS: Two hundred and twenty-seven thyroid cases were received for analysis using telecytology (TC) during the period from November 2017 to May 2018. Rapid on-site evaluation was performed at the Atakent Hospital of the AHG by a cytotechnologist and scanned on the same day. For every case, there were Diff-Quik- and Papanicolaou-stained FNA smears. Each glass slide was digitized with a 3DHISTECH whole-slide scanner in 1 focal Z-plane at ×40 magnification. RESULTS: Two hundred and twenty-seven thyroid FNA specimens were retrieved, of which 25 had histologic follow-up. Samples were classified as nondiagnostic in 3%, benign in 74%, atypia of undetermined significance/follicular lesion of undetermined significance in 13%, suspicious for follicular neoplasia/follicular neoplasia in 3%, suspicious for malignancy in 4%, and malignant in 3%. When only the "suspicious for malignancy" and "malignancy" categories were considered positive tests, cytology sensitivity and specificity using TC for diagnosis was 100%. CONCLUSIONS: Our data demonstrate that TC is suitable to provide a primary diagnosis in daily routine cytology practice. Despite the promising results, there were some challenges stemming from the novelty of using TC for the primary diagnosis. The study also addresses both advantages and disadvantages of TC in daily practice to increase the efficiency of the technique in primary diagnosis.

Peptide Profile Differences of Noninvasive Follicular Thyroid Neoplasm with Papillary-Like Nuclear Features, Encapsulated Follicular Variant, and Classical Papillary Thyroid Carcinoma: An Application of Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

Introduction: The lack of papillary structures and faint and/or unclear core features of follicular variant of papillary thyroid carcinoma (FV-PTC) may hamper the definitive fine needle aspiration biopsy -based diagnosis. Recently, the nomenclature of noninvasive encapsulated FV-PTC was revised to "noninvasive follicular thyroid neoplasms with papillary-like nuclear features" (NIFTP). However, it remains inconclusive whether or not the peptide patterns differ between NIFTP and encapsulated FV-PTC. The main objectives of this study were to investigate the viability of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) in the pathological assessment of NIFTP and to evaluate the discriminatory power of MALDI MSI for the classification of classical variant of PTC (CV-PTC), NIFTP, and encapsulated FV-PTC. Methods: MALDI MSI was employed to investigate the changes in peptide profiles from 21 formalin-fixed paraffin-embedded (FFPE) tissue samples (n = 7 from each group of CV-PTC, NIFTP, and FV-PTC). Six out of seven FV-PTC FFPE tissue samples were encapsulated FV-PTC; only one was infiltrative FV-PTC. Liquid chromatography-tandem mass spectrometry was used for the identification of the peptide signals detected in MALDI MSI. Results: Using receiver operating characteristics analysis, 10 peptide signals distinguished NIFTP from normal thyroid parenchyma (area under the curve [AUC] >0.80). To evaluate the discriminatory power of MALDI MSI, statistically significant peptide signals (n = 88) within three groups were used for hierarchical clustering. The method had high discriminatory power for distinguishing CV-PTC from NIFTP and FV-PTC (encapsulated and infiltrative). The majority of the NIFTP and encapsulated FV-PTC were clustered together, indicating that NIFTP could not be distinguished from encapsulated FV-PTC. However, infiltrative FV-PTC FFPE tissue samples had the furthest distance from all the NIFTP cases. High signal intensities of S100-A6, vimentin, and cytoplasmic actin 1 were detected in FV-PTC, prelamin A/C in CV-PTC, and 60S ribosomal protein L6 and L8 in NIFTP tissues. Conclusions: MALDI MSI, a powerful tool combining histological and mass spectrometric data, enabled the differentiation of NIFTP from normal thyroid parenchyma. Although NIFTP is a recent definition that replaces noninvasive encapsulated FV-PTC, the peptide profiles of NIFTP and encapsulated FV-PTC were found to be similar.