Conventional Hematoxylin Research Articles

Soft-tissue keratocyst: report of 3 new cases from Brazil.

To describe the clinicopathological features of 3 new cases of soft-tissue keratocyst (STK) of the buccal mucosa from Brazil. Clinical data from 3 patients diagnosed with STK were obtained from the archives of the Laboratory of Oral Pathology at the Federal University of Rio de Janeiro- Brazil from 2020 to 2023. Two oral pathologists reviewed conventional hematoxylin and eosin-stained slides of each case. Immunohistochemical stainings for CK19, Bcl-2, CD138, D2-40, EMA, and Ki-67 were performed in all cases. Case 1 was a 58-year-old man with a 2-year history of painless swelling of the right buccal mucosa, measuring approximately 5cm. Case 2 was of a 44-year-old man with a painful swelling in the left buccal mucosa lasting 3 years with drainage to the oral cavity. Case 3 was of a 74-year-old woman with a painful swelling in the left buccal mucosa of unknown duration. Microscopic evaluation of all 3 cases revealed a cyst lined with a few cell layers composed of columnar palisading basal cells and a corrugated parakeratin surface. The capsule comprised fibrous connective tissue with variable amounts of blood vessels with hemorrhage, nerve bundles, fat tissue, striated muscle fibers, and the absence of pilosebaceous units. No recurrence was observed after complete surgical removal. All epithelial layers of the 3 cases expressed positivity for CK19 and CD138, the basal cells were positive for Bcl-2 and D2-40, and the superficial epithelial layer was positive for EMA. All cases demonstrated a low proliferation index by Ki-67. This study represents a series of 3 cases of STK that affected the posterior buccal mucosa of middle-aged patients from Brazil, sharing histopathological and immunohistochemical features with odontogenic keratocyst.

Fumarate hydratase–deficient renal cell carcinoma: A single institution–based study of 29 patients by clinicopathological, immunohistochemical and genetic approaches

Fumarate hydratase (FH)-deficient renal cell carcinoma (RCC) is a renal neoplasm associated with FH loss, aggressive behaviour, and poor survival. We present a histopathological and immunohistochemical overview of FH-deficient RCC to infer significant features for its differential diagnosis. In this study, FH-deficient RCC tissue samples from patients who underwent surgical resection or biopsy at a single institution between July 1995 and August 2022 were reviewed by conventional haematoxylin and eosin staining, immunohistochemistry, and whole-genome analyses. Twenty-nine FH-deficient RCC specimens were examined ​based on immunohistochemistry findings regarding FH and S-(2-succino)cysteine (2SC). The histopathological findings included conspicuous nucleoli with a perinucleolar halo, resembling viral inclusion, eosinophilic cytoplasm, papillary and tubular growth patterns, and lack of stromal foam cell collection. Some tumours showed desmoplastic stroma, tumour-infiltrating lymphocytes, and solid growth pattern. One tumour presented low-grade oncocyte-like histomorphology. Widespread negativity for CD10, keratin 7, keratin 20, anaplastic lymphoma kinase, and GATA3 were observed in 24 specimens. All samples were positive for paired box gene 8, and the level of alpha-methylacyl-CoA racemase expression was variable. Whole-exome sequencing of 19 tumours revealed nonsynonymous mutations, including missense mutations, splice donors, splice acceptors, frameshifts, and deletions in 15 tumours. Eleven tumours showed novel mutations. In conclusion, results revealed generally unfavourable clinical presentations and outcomes with diverse variants of FH mutation. These findings, along with the histopathological and immunohistochemical features, can be used to guide diagnosis and treatment.

Significance of CD70, VEGF, and CD90 Immunohistochemical Expression in Colorectal Cancer.

Colorectal cancer is the 4th most reported reason for cancer death worldwide. It is a complex and multifaceted disease with diverse histopathological manifestations. CD70 is present on activated immune cells and is upregulated in patients who have finished adjuvant therapy. VEGF controls angiogenesis and demonstrates immuno-regulatory characteristics that inhibit the anticancer activity of immune cells. CD90 is an extracellular cancer stem cell marker and regulates apoptosis, cell migration, and T cell activation. to elucidate the prognostic potential of a combined immunohistochemical assessment of CD70, VEGF, and CD90 biomarkers in colorectal cancer tissues. Our study is a retrospective study done on 70 paraffin blocks (45 colorectal carcinoma cases and 11 adenomas, along with 14 cases of colitis serving as controls) were subjected to conventional Hematoxylin and Eosin stain for routine histopathological assessment and immunohistochemical staining for CD70, VEGF and CD90. Our investigation focused on evaluating the expression of CD70, VEGF, and CD90 in various colorectal tissues, including colitis, adenomas, and adenocarcinomas, as well as different grades of adenocarcinoma tissues, different stages of tumor invasiveness, and lymph node status. Our research revealed distinctive expression patterns of CD70, VEGF, and CD90 across different stages and grades of colorectal cancer. our research signifies the potential clinical utility of the CD70, VEGF, and CD90 triad as prognostic markers in colorectal cancer. The combined analysis of these markers emerged as a potent prognostic tool, offering valuable insights into disease progression. Our research showed that epithelial CD90 is the most sensitive marker for both tumor stage and lymph node status. CD70 and VEGF showed a statistically significant relation with lymph node status only and not with tumor stage.

Histological assessment of the effect of 50mW red laser on retina at variable time intervals: animal model.

To assess the histopathological effect of 50milliWatt red laser pointer on mice retinal tissue at variable exposure times. The experimental study was conducted in the Postgraduate Laboratory of the Unit of Medical Physics, Department of Physiology, College of Medicine, Mustansiriyah University, Baghdad, Iraq, from November 2020 to April 2021, and comprised albino mice. Histopathological effects on the retinal tissues were evaluated microscopically using conventional haematoxylin and eosin stain just after being exposed to commercially available 50 milliWatt red laser pointers with 630nm wavelength at 1, 2 and 3 minutes, grouped as R1, R2 and R3, respectively. The laser beam was focussed on the right eye of the mice sedated with sodium pentobarbital, reserving the left eye for use as a control group. Data was analysed using SPSS 24. Of the 12 mice, 4(33.3%) were in each of the 3 groups. There was significantly destructive effect of red laser on retinal outer cellular layers in all the groups compared to the controls (p<0.05). The effect was significant between R1 and R2 (p=0.0001), and between R2 and R3 (p=0.02). The handheld commercial red laser pointers could harmfully affect a wide range of mice retina and choroid.

Axially swept open-top light-sheet microscopy for densely labeled clinical specimens.

Open-top light-sheet (OTLS) microscopy offers rapid 3D imaging of large optically cleared specimens. This enables nondestructive 3D pathology, which provides key advantages over conventional slide-based histology including comprehensive sampling without tissue sectioning/destruction and visualization of diagnostically important 3D structures. With 3D pathology, clinical specimens are often labeled with small-molecule stains that broadly target nucleic acids and proteins, mimicking conventional hematoxylin and eosin (H&E) dyes. Tight optical sectioning helps to minimize out-of-focus fluorescence for high-contrast imaging in these densely labeled tissues but has been challenging to achieve in OTLS systems due to trade-offs between optical sectioning and field of view. Here we present an OTLS microscope with voice-coil-based axial sweeping to circumvent this trade-off, achieving 2 µm axial resolution over a 750 × 375 µm field of view. We implement our design in a non-orthogonal dual-objective (NODO) architecture, which enables a 10-mm working distance with minimal sensitivity to refractive index mismatches, for high-contrast 3D imaging of clinical specimens.

Spectrophotometry and Histological Staining Potential of Ethanol Extract of brassica Oleracea (Red Cabbage) on Selected Organs of Sprague Dawley rat

Objective: Plants with coloring and dyeing effects have widely been used in histological staining. Natural dyes are less expensive, nontoxic, renewable and sustainable resources, with minimal environmental impact compared to synthetic stains. Red cabbage is one of such natural dyes. The red color of the red cabbage derived from anthocyanin pigments confers a great advantage to red cabbage. In this study, the physiochemical and spectrophotometric characteristics of red cabbage as well as its histological staining potentials on various tissues were carried out to determine its tissue specificity. Methods:The ethanol extract of red cabbage was prepared using 100% ethanol, then pH, spectrophotometry, and concentration of the red cabbage ethanol extract were measured before the solution was divided into two equal volumes, and one part was added with 2ml glacial acetic acid while the other part was added with 10g of aluminum sulfate. The two solutions were used to stain 10% neutral-buffered formalin-fixed, paraffin-wax-embedded tissue sections separately. Stained sections were viewed with a photomicroscope. Results: The ethanol extract was acidic at a pH of 4.89 and the spectrophotometry graph showed 3 peak absorbances at the wavelengths of 395nm, 545nm and 670nm. The maximum absorbance was 0.795 at the wavelength of 395nm and this is indicative of the pigment cyanidin which is the anthocyanin present in red cabbage. The red cabbage ethanol extract with both glacial acetic acid and aluminum sulfate showed good histological details in tissues of kidney, stomach, lungs, liver, spleen, small intestine and skin. There was no difference between the red cabbage ethanol extract and the conventional hematoxylin and eosin stain (P&gt;0.05). Our study showed that red cabbage ethanol extract with acetic acid and aluminum sulfate is stable at 4oC, with color amber to light gold with a pH of 4.89 and a concentration of 1000 which gave good histological details. In general, our result indicates that red cabbage ethanol extract is an efficient stain for histological tissue structures and can be used as an eco-friendly alternative to eosin. Conclusion: Red cabbage ethanol extract stained basic structures of the cell such as the cytoplasm, muscle fibers and mucins. Therefore, red cabbage ethanol extract can be used in histological staining when appropriate modifications are done.

Improving the diagnosis of ductal carcinoma in situ with microinvasion without immunohistochemistry: An innovative method with H&E-stained and multiphoton microscopy images.

Ductal carcinoma in situ with microinvasion (DCISM) is a challenging subtype of breast cancer with controversial invasiveness and prognosis. Accurate diagnosis of DCISM from ductal carcinoma in situ (DCIS) is crucial for optimal treatment and improved clinical outcomes. However, there are often some suspicious small cancer nests in DCIS, and it is difficult to diagnose the presence of intact myoepithelium by conventional hematoxylin and eosin (H&E) stained images. Although a variety of biomarkers are available for immunohistochemical (IHC) staining of myoepithelial cells, no single biomarker is consistently sensitive to all tumor lesions. Here, we introduced a new diagnostic method that provides rapid and accurate diagnosis of DCISM using multiphoton microscopy (MPM). Suspicious foci in H&E-stained images were labeled as regions of interest (ROIs), and the nuclei within these ROIs were segmented using a deep learning model. MPM was used to capture images of the ROIs in H&E-stained sections. The intensity of two-photon excitation fluorescence (TPEF) in the myoepithelium was significantly different from that in tumor parenchyma and tumor stroma. Through the use of MPM, the myoepithelium and basement membrane can be easily observed via TPEF and second-harmonic generation (SHG), respectively. By fusing the nuclei in H&E-stained images with MPM images, DCISM can be differentiated from suspicious small cancer clusters in DCIS. The proposed method demonstrated good consistency with the cytokeratin 5/6 (CK5/6) myoepithelial staining method (kappa coefficient = 0.818).

Lymph Node Molecular Analysis with OSNA Enables the Identification of pT1 CRC Patients at Risk of Recurrence: A Multicentre Study.

Early-stage colorectal carcinoma (CRC)-pT1-is a therapeutic challenge and presents some histological features related to lymph node metastasis (LNM). A significant proportion of pT1 CRCs are treated surgically, resulting in a non-negligible surgical-associated mortality rate of 1.5-2%. Among these cases, approximately 6-16% exhibit LNM, but the impact on survival is unclear. Therefore, there is an unmet need to establish an objective and reliable lymph node (LN) staging method to optimise the therapeutic management of pT1 CRC patients and to avoid overtreating or undertreating them. In this multicentre study, 89 patients with pT1 CRC were included. All histological features associated with LNM were evaluated. LNs were assessed using two methods, One-Step Nucleic Acid Amplification (OSNA) and the conventional FFPE plus haematoxylin and eosin (H&E) staining. OSNA is an RT-PCR-based method for amplifying CK19 mRNA. Our aim was to assess the performance of OSNA and H&E in evaluating LNs to identify patients at risk of recurrence and to optimise their clinical management. We observed an 80.9% concordance in LN assessment using the two methods. In 9% of cases, LNs were found to be positive using H&E, and in 24.7% of cases, LNs were found to be positive using OSNA. The OSNA results are provided as the total tumour load (TTL), defined as the total tumour burden present in all the LNs of a surgical specimen. In CRC, a TTL ≥ 6000 CK19 m-RNA copies/µL is associated with poor prognosis. Three patients had TTL > 6000 copies/μL, which was associated with higher tumour budding. The discrepancies observed between the OSNA and H&E results were mostly attributed to tumour allocation bias. We concluded that LN assessment with OSNA enables the identification of pT1 CRC patients at some risk of recurrence and helps to optimise their clinical management.

Indocyanine green guided sentinel lymph node biopsy may have a high sensitivity for early (T1/T2) colon cancer: A prospective study in Indian patients.

Indocyanine green (ICG) dye guided near infrared fluorescence (NIR) imaging is a promising tool for mapping lymphatics. The aim of this study was to evaluate the role of ICG guided SLN biopsy in Indian colon cancer patients. Forty-eight patients of clinically staged T1-T3 node negative colon cancer underwent laparoscopic/open resection. Patients received colonoscopic peritumoral submucosal ICG injections for laparoscopic (n= 32) and subserosal injections for open resections (n= 16) followed by the detection of SLN using NIR camera. SLNs underwent conventional hematoxylin and eosin (H & E) staging with additional serial sectioning and immunohistochemistry for pancytokeratin antibody (ultra-staging). Detection rate and upstaging rate were the primary end points. Forty-eight patients were recruited. An average of 2.08 ± 1.27 SLNs were identified in 45 patients at a mean time of 8.2 ± 3.68 minutes with a detection rate of 93.75%. Mean age and mean BMI were 59.7 ± 12.54 years and 24.8 ± 4.09 kg/m2 , respectively. Eighteen patients had node positive disease, and SLN was false negative in four of these patients resulting in a sensitivity of 77.77% with a trend towards higher sensitivity for T1-T2 tumours (90% vs. 62.5%, p= 0.068). Upstaging rate was 10%. Negative predictive value (NPV) and accuracy of the procedure were 87.09% and 91.11%, respectively. ICG guided SLN biopsy can identify metastatic lymph nodes in colon cancer patients that can be missed on H & E staging with relatively higher sensitivity for early (T1/T2) tumours.

Multimodal recurrence scoring system for prediction of clear cell renal cell carcinoma outcome: a discovery and validation study

Improved markers for predicting recurrence are needed to stratify patients with localised (stage I-III) renal cell carcinoma after surgery for selection of adjuvant therapy. We developed a novel assay integrating three modalities-clinical, genomic, and histopathological-to improve the predictive accuracy for localised renal cell carcinoma recurrence. In this retrospective analysis and validation study, we developed a histopathological whole-slide image (WSI)-based score using deep learning allied to digital scanning of conventional haematoxylin and eosin-stained tumour tissue sections, to predict tumour recurrence in a development dataset of 651 patients with distinctly good or poor disease outcome. The six single nucleotide polymorphism-based score, which was detected in paraffin-embedded tumour tissue samples, and the Leibovich score, which was established using clinicopathological risk factors, were combined with the WSI-based score to construct a multimodal recurrence score in the training dataset of 1125 patients. The multimodal recurrence score was validated in 1625 patients from the independent validation dataset and 418 patients from The Cancer Genome Atlas set. The primary outcome measured was the recurrence-free interval (RFI). The multimodal recurrence score had significantly higher predictive accuracy than the three single-modal scores and clinicopathological risk factors, and it precisely predicted the RFI of patients in the training and two validation datasets (areas under the curve at 5 years: 0·825-0·876 vs 0·608-0·793; p<0·05). The RFI of patients with low stage or grade is usually better than that of patients with high stage or grade; however, the RFI in the multimodal recurrence score-defined high-risk stage I and II group was shorter than in the low-risk stage III group (hazard ratio [HR] 4·57, 95% CI 2·49-8·40; p<0·0001), and the RFI of the high-risk grade 1 and 2 group was shorter than in the low-risk grade 3 and 4 group (HR 4·58, 3·19-6·59; p<0·0001). Our multimodal recurrence score is a practical and reliable predictor that can add value to the current staging system for predicting localised renal cell carcinoma recurrence after surgery, and this combined approach more precisely informs treatment decisions about adjuvant therapy. National Natural Science Foundation of China, and National Key Research and Development Program of China.

DS24 Prospective study comparing ex vivo confocal laser scanning microscopy and frozen section histopathology of 23 basal cell carcinomas treated with Mohs micrographic surgery

Abstract Ex vivo confocal laser scanning microscopy (CLSM) is an emerging technique that is used for the rapid analysis of freshly excised tissue during Mohs micrographic surgery (MMS). Previous studies on fluorescence CLSM have shown 88% and 99% sensitivity and specificity, respectively, of diagnosing basal cell carcinomas (BCCs) vs. conventional histology. The fourth-generation Vivascope 2500 allows fresh tissue to be scanned after minimal preparation with acetic acid and acridine orange, creating digitally coloured mosaic images, mimicking conventional histological haematoxylin and eosin slides. The scanning speed of &amp;lt; 3 min creates the potential to improve patient experiences, allowing multiple stages and repair in a single session, reducing overall operating times and improving efficiency. After a 2018 pilot study on a limited case series, we have begun the process of testing the real-world use of this technology. We present an initial prospective case series to assess BCC excision of stage 1 Mohs specimens using the Vivascope 2500 vs. conventional frozen section histology. Caldicot approval was sought. Over a 3-month period, 23 cases of stage 1 facial BCCs on one Mohs list were processed. Samples were obtained as standard bevelled Mohs excisions, and Mohs maps were created for all cases. There were three false-negative BCCs, of which two were infiltrative BCCs with small foci missed on the initial review on CLSM images and one superficial BCC not visible due to lack of epidermal capture. Of the four false-positive cases, the foci appearing to be BCC included inflammation in one, follicular structures in two and conjunctival glands misinterpreted as BCC in one eyelid case. Time taken to process specimens and read the digital images by CLSM was compared with the time taken to obtain and read frozen section slides. The shortest time recorded for CLSM and frozen sections was 6 min and 31 min, respectively. The longest time recorded for CLSM and frozen sections was 20 min and 57 min, respectively. In summary, CLSM shows promise in MMS for selected BCCs. Improved techniques to flatten the epidermis for better visualization and further experience with image reading and interpretation have the potential to minimize errors.

Clinical Validation of Stimulated Raman Histology for Rapid Intraoperative Diagnosis of Central Nervous System Tumors

Stimulated Raman histology (SRH) is an ex vivo optical imaging method that enables microscopic examination of fresh tissue intraoperatively. The conventional intraoperative method uses frozen section analysis, which is labor and time intensive, introduces artifacts that limit diagnostic accuracy, and consumes tissue. SRH imaging allows rapid microscopic imaging of fresh tissue, avoids tissue loss, and enables remote telepathology review. This improves access to expert neuropathology consultation in both low- and high-resource practices. We clinically validated SRH by performing a blinded, retrospective two-arm telepathology study to clinically validate SRH for telepathology at our institution. Using surgical specimens from 47 subjects, we generated a data set composed of 47 SRH images and 47 matched whole slide images (WSIs) of formalin-fixed, paraffin-embedded tissue stained with hematoxylin and eosin, with associated intraoperative clinicoradiologic information and structured diagnostic questions. We compared diagnostic concordance between WSI and SRH-rendered diagnoses. Also, we compared the 1-year median turnaround time (TAT) of intraoperative conventional neuropathology frozen sections with prospectively rendered SRH-telepathology TAT. All SRH images were of sufficient quality for diagnostic review. A review of SRH images showed high accuracy in distinguishing glial from nonglial tumors (96.5% SRH vs 98% WSIs) and predicting final diagnosis (85.9% SRH vs 93.1% WSIs). SRH-based diagnosis and WSI-permanent section diagnosis had high concordance (κ = 0.76). The median TAT for prospectively SRH-rendered diagnosis was 3.7 minutes, approximately 10-fold shorter than the median frozen section TAT (31 minutes). The SRH-imaging procedure did not affect ancillary studies. SRH generates diagnostic virtual histologic images with accuracy comparable to conventional hematoxylin and eosin-based methods in a rapid manner. Our study represents the largest and most rigorous clinical validation of SRH to date. It supports the feasibility of implementing SRH as a rapid method for intraoperative diagnosis complementary to conventional pathology laboratory methods.

Verhoeff van Gieson staining in laser-irradiated gingival tissues

ABSTRACT There have been several studies on the use of the Verhoeff van Gieson staining method to demonstrate thermal effects on tissues. However, this method has rarely been used for the analysis of periodontal tissues. This study was undertaken to compare the quality and effectiveness of the Verhoeff van Gieson (VVG) staining method with conventional hematoxylin & eosin (H&E) in measuring the thermal effects in gingival tissues. Periodontal tissues around bovine mandibular teeth were treated using different surgical lasers (wavelengths of 10,600 nm, 970 nm, and 445 nm) at 2 W power setting. Measurements of the depth of the coagulation zone were recorded for all treatment groups in sample tissues stained with H&E as well as the VVG-staining method. Measures were interpreted by a trained pathologist. A statistical analysis was performed using the Wilcoxon signed-rank test to determine if there was a statistically significant difference between values recorded for the light penetration depth on tissues stained with each of the two staining methods. It was determined that there was no significant difference in the recorded values (P = 0.23). We have concluded that the VVG-stained tissues were better able to visualize the depth of thermal damage and thus may make it easier for someone not well trained to interpret the depth of light penetration in these tissues.

Cascaded Graph Convolution Approach for Nuclei Detection in Histopathology Images

Nuclei detection in histopathology images of cancerous tissue stained with conventional hematoxylin and eosin stain is a challenging task due to the complexity and diversity of cell data. Deep learning techniques have produced encouraging results in the field of nuclei detection, where the main emphasis is on classification and regressionbased methods. Recent research has demonstrated that regression-based techniques outperform classification. In this paper, we propose a classification model based on graph convolutions to classify nuclei, and similar models to detect nuclei using cascaded architecture. With nearly 29,000 annotated nuclei in a large dataset of cancer histology images, we evaluated the Convolutional Neural Network (CNN) and Graph Convolutional Networks (GCN) based approaches. Our findings demonstrate that graph convolutions perform better with a cascaded GCN architecture and are more stable than centre-of-pixel approach. We have compared our twofold evaluation quantitative results with CNN-based models such as Spatial Constrained Convolutional Neural Network (SC-CNN) and Centre-of-Pixel Convolutional Neural Network (CP-CNN). We used two different loss functions, binary cross-entropy and focal loss function, and also investigated the behaviour of CP-CNN and GCN models to observe the effectiveness of CNN and GCN operators. The compared quantitative F1 score of cascaded-GCN shows an improvement of 6% compared to state-of-the-art methods.

Frequent Intraluminal Growth of Large Muscular Veins in Surgically Resected Colorectal Cancer Tissues: A 3-Dimensional Pathologic Reconstruction Study

Although venous invasion (VI) is common in colorectal cancers (CRCs) and is associated with distant metastasis, the 3-dimensional (3D) microscopic features and associated mechanisms of VI are not well elucidated. To characterize the patterns of VI, 103 tissue slabs were harvested from surgically resected CRCs with ≥pT2. They were cleared using the modified immunolabeling-enabled 3D imaging of solvent-cleared organs method, labeled with multicolor fluorescent antibodies, including antibodies against cytokeratin 19, desmin, CD31, and E-cadherin, and visualized by confocal laser scanning microscopy. VI was classified as intravasation, intraluminal growth, and/or extravasation, and 2-dimensional and 3D microscopic features were compared. VI was detected more frequently in 3D (56/103 [54.4%]) than in conventional 2-dimensional hematoxylin and eosin-stained slides (33/103 [32%]; P < .001). When VI was present, it was most commonly in the form of intraluminal growth (51/56), followed by extravasation (13/56) and intravasation (5/56). The mean length of intraluminal growth was 334.0 ± 212.4 μm. Neoplastic cell projections extended from cancer cell clusters in the connective tissue surrounding veins, penetrated the smooth muscle layer, and then grew into and filled the venous lumen. E-cadherin expression changed at each invasion phase; intact E-cadherin expression was observed in the cancer cells in the venous walls, but its expression was lost in small clusters of intraluminal neoplastic cells. In addition, reexpression of E-cadherin was observed when cancer cells formed well-oriented tubular structures and accumulated and grew along the luminal side of the venous wall. In contrast, singly scattered cancer cells and cancer cells with poorly defined tubular structures showed loss of E-cadherin expression. E-cadherin expression was intact in the large cohesive clusters of extravasated cancer cells. However, singly scattered cells and smaller projections of neoplastic cells in the stroma outward of venous wall showed a loss of E-cadherin expression. In conclusion, VI was observed in more than half of the CRCs analyzed by 3D histopathologic image reconstruction. Once inside a vein, neoplastic cells can grow intraluminally. The epithelial-mesenchymal transition is not maintained during VI of CRCs.

Cytology Smears: An Enhanced Alternative Method for Colorectal Cancer pN Stage-A Multicentre Study.

Stage II colorectal cancer (CRC) recurrence remains a clinical problem. Some of these patients are true stage III CRC with a pN0 pathology stage. This large prospective multicentre cohort study aimed at evaluating the diagnostic ability of lymph node (LN) cytology smears to perform the pN stage and compare it with the conventional haematoxylin and eosin (H&E) pathology pN stage. Additionally, we used the One-Step Nucleic Acid Amplification (OSNA), a high-sensitive molecular method of LN staging. A total of 3936 fresh LNs from 217 CRC surgical specimens were examined by three methods, H&E, LN cytology smears, and OSNA. H&E detected 29% of patients with positive LNs, cytology smears 35%, and OSNA 33.2% (p &lt; 0.0001). H&E and cytology concordantly classified 92.2% of tumours, and 88.5% between OSNA and H&E. Cytology had 96.8% sensitivity and 90.3% specificity to discriminate positive/negative patients compared to H&E (p = 0.004), and 87.3% sensitivity and 89% specificity when compared to OSNA (p = 0.56). Patients with positive LNs detected by any of the three methods had significantly worse disease-free and overall survival. We conclude that pN stage accuracy for detecting positive LNs is superior with LN cytological smears than with conventional H&E, which would enable a better pN stage and management of early-stage CRC patients.

Staged Excision of Lentigo Maligna of the Head and Neck: Assessing Surgical Excision Margins With Melan A, SOX10, and PRAME Immunohistochemistry.

Staged excision has emerged as a superior treatment option for lentigo maligna (LM) of the head and neck when compared with conventional wide local excision. Assessing surgical excision margins for remaining LM poses a diagnostic challenge. To determine whether immunohistochemical (IHC) staining with SOX10 and preferentially expressed antigen in melanoma (PRAME) aids in diagnosing LM on excision margins compared with conventional hematoxylin and eosin and Melan A IHC staining. This study included cases of LM of the head and neck treated with staged excision. Histological findings were reviewed according to standard criteria for the diagnosis of LM and compared with the results after IHC staining for Melan A, SOX10, and PRAME. The cohort consisted of 35 sections. Based on hematoxylin and eosin and Melan A IHC staining, 23 sections were diagnosed as LM by the initial pathologist. Further staining with SOX10 IHC showed only 8 to be consistent with a diagnosis of LM and 9 revealing features of actinic melanocyte hyperplasia. PRAME was positive in 5 of the 8 cases of LM and negative in all 9 cases of actinic melanocyte hyperplasia (P = 0.009). The presence of melanocyte nests (P = 0.29) and pagetoid spread (P = 0.003) was the most reliable histological findings distinguishing LM from its mimics. SOX10 is a more specific and sensitive marker for melanocytes when assessing for LM on excision margins compared with Melan A. The addition of PRAME can be useful to confirm or exclude the diagnosis in challenging cases.

Diagnostic utility of Bcl-2 immunohistochemical expression in pediatric functional bowel obstruction cases with ganglionated specimens

BackgroundFunctional disturbances of the gastrointestinal tract are caused by a number of neurodysplastic conditions, including diseases that are rarer than Hirschsprung’s disease (HSCR), such as ganglion cell immaturity and intestinal neuronal dysplasia (IND). Bcl-2 shows positive immunoreactivity in degenerative and immature ganglion cells (IGCs). This work evaluates the implication of the immunohistochemical expression of Bcl-2 in cases of pediatric intestinal pseudo-obstruction (IPO) with ganglionated specimens.This 2-year prospective observational study was conducted from January 2017 to December 2019 on all intestinal specimens for cases referred from a pediatric surgery department with clinical data suggesting IPO (abdominal distension > 3 months, intermittent bilious vomiting, no radiological evidence of the transitional zone of HSCR or evidence of mechanical obstruction). The exclusion criteria were patients with IPO related to myopathic disorders, aganglionic biopsies, and specimens with inadequate tissue blocks. The same number of intestinal specimens of cases without IPO was used as a control group. All specimens underwent a histologic examination of ganglion cells and nerve bundles and were also evaluated for Bcl-2 immunohistochemical expression.ResultsTwenty-one specimens were analyzed, including six colonic resection specimens and 15 intestinal biopsies taken by full-thickness transanal biopsy (n = 12) or incisional biopsy from inadequately functioning stomas (n = 3). The mean ± standard deviation age of the patient cohort was 22 ± 7.4 months (range, 19–153 months), and there were 13 (61.9%) male patients. Bcl-2 protein was strongly positive in 57.1% of the cases, weakly positive in 19%, and negative in 23.8% as well as in all 21 control specimens. A highly significant correlation was observed between Bcl-2 expression and ganglion cell number (P < 0.001), where all hyperganglionic specimens showed strong positivity compared with the hypoganglionic and adequately ganglionic specimens. A positive association was also found between Bcl-2 expression and IND compared with other disorders of dysganglionosis (P = 0.04) and post-HSCR cases (P = 0.002).ConclusionBcl-2 immunohistochemistry is a valuable tool to diagnose allied disorders of HSCR through its expression in IGCs, which are difficult to identify by conventional hematoxylin and eosin staining.

Programmed cell death protein 1 (PD1) deficiency induces cardiac inflammation during baseline conditions and ischemia/reperfusion injury

Abstract Background The Programmed cell death protein 1 (PD1) immune checkpoint is densely expressed on cardiac endothelial cells. The increasing clinical application of immune checkpoint inhibitor therapy targeting PD1 for the treatment of advanced malignancies has revealed profound cardiovascular side effects that have recently paralleled in a preclinical model. However, the effects of PD1 deficiency during baseline conditions and myocardial injury are so far unknown. Purpose This study aims to assess the impact of PD1 deficiency on myocardia immunity, and to evaluate the relevance of PD1 signalling in cardiac disease. Methods C57BL/6J wild-type mice and C57BL/6J Pdcd1−/− mice were purchased and bred at the animal facility. In-vivo ischaemia/reperfusion (I/R) injury was applied to assess the response in cardiac injury. In brief, mice were anesthetised followed by lateral thoracotomy and ligation of the left coronary artery for 45 min. For flow cytometry, hearts were removed and subjected to an enzymatic digestion. Single cell solutions were stained with different antibody panels to assess cardiac immune cells and the expression of programmed cell death protein 1 ligand 1 (PDL1). Western blot was conducted after homogenization of snap-frozen heart tissue using specific primary and corresponding secondary antibodies. Immunofluorescence and conventional haematoxylin and eosin stain from 4 μm sections were used to visualize the distribution of PDL1 in cardiac tissue. Results While no overt cardiac phenotype was observed in Pdcd1−/− mice, a profound upregulation of pro-inflammatory cytokines was determined during baseline conditions, including interleukin (IL) 1α IL4, and extracellular-signal regulated kinase (ERK) 1/2. NADPH oxidase 1 (NOX1) which is involved in production of reactive oxygen species and endothelial injury response was downregulated in Pdcd1−/− mice. Following I/R injury, a significant decline in endothelial PDL1 expression was observed, which was attributed to changes in the area at risk, as shown by immunofluorescence staining. The infarct size following in-vivo I/R injury was not altered upon PD1-deficiency as determined by TTC staining. However, flow cytometry determined increased cell numbers of distinct leukocyte subsets during reperfusion-related inflammation. Conclusion Deficiency of PD1/PDL1 signalling shows distinct inflammatory changes in cardiac tissue at baseline and I/R injury. The results indicate that relevant PD1-related detrimental effects are not limited to complications from cancer therapy but can be expected in various forms of cardiovascular disease, hence requiring further investigations. Funding Acknowledgement Type of funding sources: Public hospital(s). Main funding source(s): Department of Cardiology and Vascular Medicine, West German Heart and Vascular Center, University Hospital Essen, Germany

A clinical decision support system optimising adjuvant chemotherapy for colorectal cancers by integrating deep learning and pathological staging markers: a development and validation study

The DoMore-v1-CRC marker was recently developed using deep learning and conventional haematoxylin and eosin-stained tissue sections, and was observed to outperform established molecular and morphological markers of patient outcome after primary colorectal cancer resection. The aim of the present study was to develop a clinical decision support system based on DoMore-v1-CRC and pathological staging markers to facilitate individualised selection of adjuvant treatment. We estimated cancer-specific survival in subgroups formed by pathological tumour stage (pT<4 or pT4), pathological nodal stage (pN0, pN1, or pN2), number of lymph nodes sampled (≤12 or >12) if not pN2, and DoMore-v1-CRC classification (good, uncertain, or poor prognosis) in 997 patients with stage II or III colorectal cancer considered to have no residual tumour (R0) from two community-based cohorts in Norway and the UK, and used these data to define three risk groups. An external cohort of 1075 patients with stage II or III R0 colorectal cancer from the QUASAR 2 trial was used for validation; these patients were treated with single-agent capecitabine. The proposed risk stratification system was evaluated using Cox regression analysis. We similarly evaluated a risk stratification system intended to reflect current guidelines and clinical practice. The primary outcome was cancer-specific survival. The new risk stratification system provided a hazard ratio of 10·71 (95% CI 6·39-17·93; p<0·0001) for high-risk versus low-risk patients and 3·06 (1·73-5·42; p=0·0001) for intermediate versus low risk in the primary analysis of the validation cohort. Estimated 3-year cancer-specific survival was 97·2% (95% CI 95·1-98·4; n=445 [41%]) for the low-risk group, 94·8% (91·7-96·7; n=339 [32%]) for the intermediate-risk group, and 77·6% (72·1-82·1; n=291 [27%]) for the high-risk group. The guideline-based risk grouping was observed to be less prognostic and informative (the low-risk group comprised only 142 [13%] of the 1075 patients). Integrating DoMore-v1-CRC and pathological staging markers provided a clinical decision support system that risk stratifies more accurately than its constituent elements, and identifies substantially more patients with stage II and III colorectal cancer with similarly good prognosis as the low-risk group in current guidelines. Avoiding adjuvant chemotherapy in these patients might be safe, and could reduce morbidity, mortality, and treatment costs. The Research Council of Norway.