Formalin-fixed Specimens Research Articles

Utility of C3d and C4d immunohistochemical staining in formalin-fixed skin or mucosal biopsy specimens in diagnosis of bullous pemphigoid and mucous membrane pemphigoid

Bullous pemphigoid (BP) and mucous membrane pemphigoid (MMP) sometimes have overlapping clinical, histopathological, and direct immunofluorescence (DIF) features in the early stages. Complement deposition is an intrinsic component of the patho-mechanism of BP in contrast to MMP. Hence immunohistochemistry (IHC) for C3d and C4d may be helpful in differentiating the two disorders. Seventy-four patients of BP and 18 patients of MMP along with 10 negative controls were enrolled in this study. C3d and C4d IHC was performed in formalin-fixed skin biopsy specimens. C3d IHC staining in BP/MMP had a sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 59.2%/41.2%, 100%/100%, 100%/100%, 25.6%/50.0%, respectively. C4d IHC staining in BP/MMP had a sensitivity, specificity, PPV and NPV of 26.8%/17.6%, 100%/100%, 100%/100% and 16.1%/41.7%, respectively. Receiver operator analysis showed utility of C3d in diagnosing both BP [Area under curve (AUC) = 0.8, p = 0.0001] and MMP (AUC = 0.71; p = 0.001). C4d was useful in diagnosis of BP (AUC = 0.5; p = 0.0001), but not MMP (AUC = 0.6; p = 0.064). Hence, C3d is a better diagnostic modality for BP as compared to C4d, whereas C3d and C4d have lower diagnostic importance in MMP. C3d IHC can be employed in diagnosing BP when a second biopsy for direct immunofluorescence (DIF) is not possible or where a facility for IF microscopy does not exist.

In endometrial carcinoma, Ghrelin expression has a diagnostic role, and signal transductors and activators of transcription 5 can be a therapeutic target

Abstract Background and objective Endometrial carcinoma was ranked the sixth most common female cancer worldwide. Previous studies reported that Ghrelin–signal transductors and activators of transcription 5 (STAT5) axis could modulate many tumors’ behavior. Therefore, we investigated the expressions of Ghrelin and STAT5 in endometrioid carcinoma (EC). Moreover, we tried to find a diagnostic marker to differentiate atypical hyperplasia (AH) from well-differentiated EC. Patients and methods One hundred-eight formalin-fixed and paraffin-embedded specimens were cut, and each specimen was stained with STAT5 and Ghrelin separately using immunohistochemistry. Results STAT5 expression was detected in 78.6% of EC. This expression was significantly increased with increasing EC grade (P=0.04) and myometrial invasion depth (P=0.01). In contrast, there was a decrease in Ghrelin expression with lesion progression from cyclic endometrium, and endometrial hyperplasia to EC, which was statistically significant (P=0.002). Moreover, a negative association was noticed between Ghrelin expression and histological grades, depth of myometrial invasion, presence of lymph node metastasis, and tumor stage (P=0.01, 0.05, 0.003, and 0.002, respectively). Conclusion STAT5 is associated with differentiation and invasion in EC and can be targeted for therapeutic management. Additionally, Ghrelin can be a sensitive marker to distinguish AH and well-differentiated EC.

Chromogenic in situ hybridization technique for detecting porcine circovirus 3 in lung and lymphoid tissues.

Porcine circovirus 3 (PCV3) was recently reported in Malaysian commercial pig population in 2020 by conventional polymerase chain reaction (PCR), revealing a molecular prevalence of 17.02% in the sampled domestic pig population. This study aims to describe a chromogenic in situ hybridization (ISH) technique using digoxigenin (DIG)-labeled cloned PCV3 open reading frame 1 (ORF1) fragment DNA to detect and localize the PCV3 antigen in formalin-fixed, paraffin-embedded lung, and lymphoid tissue specimens. Since PCV3 was mainly detected in lung and lymphoid tissues, we obtained tissue specimens from these organs from the previous Malaysian PCV3 study. Digoxigenin-labeled ISH probes were designed to target a 69 bp region of PCV3 ORF1 spanning from the nucleotide positions (282-350). Light microscopy analysis revealed that chromogenic staining of PCV3 antigens was visualized within the cytoplasm of pneumocytes and lymphocytes, indicating positive ISH results. The results of molecular detection of PCV3 using PCR and ISH showed a high agreement of 90.91%, including for the negative PCV3 status for all samples. This study reports a chromogenic ISH technique using DIG-labeled probes targeting PCV3 ORF1 to detect PCV3 antigens in lung and lymphoid tissues. Despite the limited availability of PCV3 antibodies, ISH remains relevant for investigating PCV3 replication and pathogenesis and can be used complementarily with PCR for evaluating the localization of antigens in infected tissues.

Detection of human papillomavirus in archival bladder and ovarian cancer samples

ProblemHuman papillomavirus (HPV) is a well-established causative agent of cervical cancer. However, its role in the development of bladder and ovarian cancer is under debate. Current study evaluated the role of high-risk HPV genotypes 16 and 18 in triggering bladder and ovarian carcinomas. MethodsA total of 80 formalin-fixed and paraffin-embedded (FFPE) archival tissue specimens of both bladder (n = 40) and ovarian carcinoma (n = 40) were tested for the presence of HPV integrants and HPV 16 genotype and HPV 18 genotype. Results and conclusionThe PCR results showed that 5% (2/40) of bladder cancer samples were HPV positive with equal prevalence of HPV 16 in both sexes (1:1) while 22.5% (9/40) of ovarian cancer samples came positive for HPV with only one positive for HPV 16. These results indicate association of HPV with bladder and ovarian carcinomas, HPV 16 genotype being more prevalent than 18. The study emphasizes that HPV is a serious issue in low- and middle-income countries where existing methods of prevention are still inadequate or costly. Timely HPV screening can help lower the burden of HPV-induced carcinomas. Furthermore, systematic HPV vaccination program among the young population is paramount.

Gadolinium during human pregnancy following administration of gadolinium chelate before pregnancy.

Gadolinium (Gd), a toxic rare earth element, has been shown to dissociate from chelating agents and bioaccumulate within tissues, raising concerns about the possibility of their remobilization during pregnancy with subsequent free Gd exposures to developing fetuses. Gd chelates are among the most commonly used magnetic resonance imaging (MRI) contrast agents. This investigation was undertaken after the detection of elevated Gd (800-1000× higher than the usual rare earth element levels) in preliminary unpublished studies from the placentae of subjects in the NIH ECHO/UPSIDE Rochester Cohort Study and unpublished studies from placentae analyzed in formalin-fixed placental specimens from Surgical Pathology at the University of Rochester. Fifteen pregnancies with elevated Gd were studied (12 first pregnancies and 3 second pregnancies). Maternal bloods were collected from all three trimesters, maternal, and cord (fetal) bloods at delivery as well as placental tissue. Breastmilk was also collected from selected mothers. It was determined that Gd was present in maternal bloods from all three trimesters, and in cord bloods and breastmilk in both first and second pregnancies. These results emphasize the need to fully appreciate the implications of pre-pregnancy exposure to Gd chelates and its potential effects on maternal and fetal health.

Optimization of protein extraction for proteomic analysis of formalin-fixed and paraffin-embedding (FFPE) biopsy specimens from males with breast cancer.

e13119 Background: Male breast cancer corresponds to 1% of breast cancer diagnoses. Given this low incidence, the tumors detected have a worse prognosis and lethality. Formalin fixation and paraffin-embedding (FFPE) methods are the most common to maintain biopsied samples. It is a low-cost procedure and is a valuable resource for the study of pathological molecular mechanisms and research of potential biomarkers and therapeutic target molecules. However, in this technique cross-links are formed compromising protein extraction, resulting in low yield and quality for mass spectrometry analysis. Objective: To present an optimized method of protein extraction from male breast FFPE samples for proteomic analysis. Methods: Four protein extraction protocols were optimized and tested in female breast cancer FFPE samples from Haroldo Juaçaba Hospital/Cancer Institute of Ceará to preserve the male sample. Each protocol was performed using a total of eight 5uM tumor slides. Different deparaffinization processes were tested followed by distinct extraction buffers and variable incubation time. The characteristics of each of the protocols and optimizations are presented in the table. The quantification of the protein profile was carried out in nanodrop 2000. The protocol with the highest yield was used in the male breast cancer sample FFPE. Analysis of the protein profile in polyacrylamide gel (SDS PAGE) was performed. Results: Protocol IV presented the highest yield of protein (16.9 mg/mL) and protocols I, II, and III presented, 0.46 mg/mL and 0.28 mg/ml, and 2.3 mg/mL, respectively. Protocol IV was selected to be used in the male breast cancer sample and the yield was 14.26 mg/mL, similar to the female sample. Protein bands from male and female samples using protocol IV were detected on the SDS-PAGE gel. Conclusions: Protocol IV performed a satisfactory protein yield in breast cancer samples FFPE. [Table: see text]

Capturing age-related changes in the tumor microenvironment of luminal breast cancer using the multiplex immunohistochemistry technique, MILAN.

e15089 Background: The ‘immune landscape’ of luminal breast tumors is poorly described to date. We applied the ‘multiple iterative labeling by antibody ‘neodeposition’ (MILAN) technique for in-depth characterization of the tumor microenvironment of luminal-like breast cancer (BC) in relation to age. Methods: Newly diagnosed patients with early luminal BC (grade 2/3, ER+, HER2-, tumor size ≥1,5 cm) were prospectively included in UH Leuven between March 2014 and November 2015 and divided into 3 distinct age categories (young: 35-45 yrs.; middle: 55-65 yrs.; old: ≥70 yrs.). 55 patients (young: n = 12; middle: n = 16; old: n = 27) were included in the cohort. From each patient up to 2 cores from the tumor center (TC) and 2 cores from the invasive front (IF) were sampled from formalin-fixed and paraffin-embedded tumor resection specimens and embedded in tissue microarrays. The MILAN workflow included 20 rounds of three-color indirect immunofluorescence (FITC, TRITC, and Cy5 channel combined with 58 antibodies), image acquisition, and removal/stripping of the antibodies before any other stain. The MILAN data analysis pipeline was applied to study the expression of phenotypic and functional proteins at single-cell level including spatial resolution. These were compared between the different age groups using Wilcoxon's test (FDR correction). Results: We identified 1,5 million cells, of which 59% were epithelial cells (cancer and normal), 29% were immune cells, and the remaining 12% were endothelial or stromal cells. Within the immune cell fraction, T-helper (Th) cells, B-cells, T-cytotoxic (Tcy) cells, and NK-cells significantly decreased with aging while monocytes significantly increased in TC and IF (Table 1). The evaluation of the functional markers in the inflammatory cells revealed a decreased expression with aging of PD-L1 in Tcy cells (p < 0.001 for TC and IF), Th cells (p = 0.003 for IF), T-regulatory (Treg) cells (p = 0.038 for TC), monocytes (p < 0.001 for TC and IF), and B-cells (p = 0.023 for TC). Perforin expression significantly decreased with aging in Th cells (p < 0.05 for TC and IF) and monocytes (p < 0.001 for TC and IF). OX-40 expression significantly decreased with aging in Th cells (p = 0.001 for TC), Treg cells (p = 0.002 for TC), and B-cells (p = 0.012 for TC). Conclusions: Our results suggest important compositional and functional changes in the tumor inflammatory microenvironment during aging in patients with luminal BC. Further analysis on other cell types is warranted. [Table: see text]

Clinicopathological Value Of Epidermal Growth Factor Receptor (EGFR) And Ki-67 Expression In Colorectal Adenoma And Adenocarcinoma.

To evaluate epidermal growth factor receptor and Ki-67 immunohistochemical expression in colorectal adenoma and carcinoma cases, and to relate their expression with the available clinicopathological data. The retrospective study was conducted at the Faculty of Medicine, Kafrelsheikh University, Egypt, from September 2019 to October 2020, and comprised formalin-fixed and paraffin-embedded specimens related to cases of colorectal adenoma and those of colorectal carcinoma, who had no previous radiation or chemotherapeutic treatment. Immunohistochemical staining of all thecases was done using anti-epidermal growth factor receptor and anti-Ki-67 antibodies. Data was analysed using SPSS 20. Of the 70 cases, 20(28.5%) were of colorectal adenoma; mean age 52.95±13.47 years, and male-to-female ratio 1:1. The remaining 50(71.5%) cases had colorectal carcinoma; mean age 51.08±13.49 years, and male-to-female ratio 1.17:1. Epidermal growth factor receptor and Ki-67 overexpression related significantly to villous histopathological type and high-grade dysplasia in colorectal adenoma cases(p<0.05). In colorectal carcinoma cases, epidermal growth factor receptor overexpression related significantly to tumour grade (p<0.05). Ki-67 overexpression related significantly to increased pathological stage (p<0.05). Overexpression of epidermal growth factor receptor and Ki-67 was found to be an ominous sign of colorectal adenoma aggressiveness, and the risk of progression to colorectal carcinoma.

The Value Of PD-L1 Immunohistochemical Expression In Egyptian Urinary Bladder Carcinoma Patients.

To evaluate programmed death-ligand 1 immunohistochemical expression in the available variants of urinary bladder carcinoma, and to correlate its expression with the available clinicopathological features. The retrospective study was conducted at the Faculty of Medicine, Kafrelsheikh University, Egypt, from February 2020 to April 2021, and comprised formalin-fixed and paraffin-embedded specimens of urinary bladder carcinoma belonging to patients who had no history of radiotherapy or chemotherapy. Immunohistochemicalstaining of all cases was done using anti-programmed death-ligand 1 antibody. Data was analysed using SPSS 20. Of the 70 specimens, 58(82.86%) had been obtained through transurethral resection of bladder tumours and 12(17.14%) through radical cystectomy. Also, 53(75.7%) specimens belonged to males and 27(24.3%) to females. The age of the cases ranged 34-83 years, and 59(84.3%) were aged ≥45 years. There were 27(38.6%) noninvasive bladder tumours and 43(61.4%) were infiltrating bladder carcinomas. Positive programmed death-ligand 1 expression was detected in 42(60%) cases. Age, gender and histopathological type were not significantly associated with the expression of programmed death-ligand 1. Programmed death-ligand 1 could be considered a predictive marker for aggressive bladder carcinoma and its immunohistochemical expression may aid in identifying selective patients for targeted immunotherapy.

Anatomical Step-by-Step Dissection of Complex Skull Base Approaches for Trainees: Surgical Anatomy of the Frontotemporal and Orbitozygomatic Craniotomies.

Introduction Although many neuroanatomic atlases have been published, few have detailed complex cranial approaches and pertinent anatomic considerations in a stepwise fashion intended for rapid comprehension by neurosurgery students, residents, and fellows. Methods Five sides of formalin-fixed latex-injected specimens were dissected under microscopic magnification. The frontotemporal and orbitozygomatic approaches were performed by neurosurgical residents and fellows at different training levels with limited previous experience in anatomical dissection mentored by the senior authors (M.P.C. and M.J.L.). Meticulous surgical anatomic dissections were performed until sufficient visual and technical completion was attained, with parameters of effectively demonstrating key surgical steps for educational training purposes. Following the completion of dissection and three-dimensional photography, illustrative case examples were reviewed to demonstrate the relative benefits and optimal applications of each approach. Results The frontotemporal and orbitozygomatic approaches afford excellent access to anterior and middle skull base pathologies, as well as the exposure of the infratemporal fossa. Key considerations include head positioning, skin incision, scalp retraction, fat pad dissection and facial nerve protection, true or false MacCarty keyhole fashioning, sphenoid wing drilling and anterior clinoidectomy, completion of the craniotomy and accessory orbital osteotomy cuts, dural opening, and intradural neurovascular access. Conclusion The frontotemporal and orbitozygomatic approaches are core craniotomies that offer distinct advantages for complex cranial operations. Learning and internalizing their key steps and nuanced applications in a clinical context is critical for trainees of many levels. The orbitozygomatic craniotomy in particular is a versatile but challenging approach; operative-style laboratory dissection is an essential component of its mastery and one that will be powerfully enhanced by the current work.

MP17-14 IDENTIFYING CRIBRIFORM PATTERN IN MPMRI SCANS FROM 3D RECONSTRUCTED RALP SPECIMENS USING DEEP LEARNING

You have accessJournal of UrologyCME1 Apr 2023MP17-14 IDENTIFYING CRIBRIFORM PATTERN IN MPMRI SCANS FROM 3D RECONSTRUCTED RALP SPECIMENS USING DEEP LEARNING Ashwin Sachdeva, Dinisha Kadam, Avaneesh Meena, Nitin Singhal, Archie Parekh, Guillaume Antem, Yatin Jain, Pedro Oliveira, and Vijay Ramani Ashwin SachdevaAshwin Sachdeva More articles by this author , Dinisha KadamDinisha Kadam More articles by this author , Avaneesh MeenaAvaneesh Meena More articles by this author , Nitin SinghalNitin Singhal More articles by this author , Archie ParekhArchie Parekh More articles by this author , Guillaume AntemGuillaume Antem More articles by this author , Yatin JainYatin Jain More articles by this author , Pedro OliveiraPedro Oliveira More articles by this author , and Vijay RamaniVijay Ramani More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003237.14AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Amongst various Gleason 4 patterns, cribriform morphology (Gl4Cri) is unique, as it seems to correlate with an aggressive clinical course, distant metastases, and disease-specific mortality. However, the true value of Gl4Cri on PCa management has recently been challenged, given that cribriform morphology is frequently intermixed with other morphologic patterns (such as glomeruloid and intra-ductal carcinoma, IDC), and biopsy-based studies suffer from sampling bias. A methodology for precise identification and quantification of Gl4Cri would help inform the role of Gl4Cri in impacting PCa management (active surveillance vs surgery vs radiotherapy) and molecular germline testing. METHODS: Using deep learning based methods, we developed an algorithm for identification and quantification of cribriform and glomeruloid patterns in whole mount images of radical prostatectomy (RP) specimens. Whole-mount H&E-stained histopathological sections of formalin-fixed RP specimens showing a mixture of different GP4 subtypes were digitised. A neural network based on U-Net++ with EfficientNet backbone was trained to identify and separate cribriform and glomeruloid glands from benign, other tumour patterns, and stromal regions. Two pathologists annotated a training set of 13 RP samples and a validation set of 42 RP samples with perfect consensus. We then applied this new algorithm to label Gl4Cri on mpMRI images using reconstructed 3D histology, allowing the extent of Gl4Cri cancer to be mapped directly onto mpMRI for development of a radiomic signature. RESULTS: The model performed well on the validation set with AUCs (Area Under Curve) of 0.87 for cribriform and 0.81 for glomeruloid pattern identification. For the two patterns, the segmentation model achieved an F1-score of 0.83 and 0.80, respectively. Using deep learning, we developed a method for automated localisation of cribriform and glomeruloid growth patterns in H&E-stained RP whole slide images, which could precisely determine the proportion of each growth pattern on corresponding mpMRI scans. CONCLUSIONS: We developed a method for automated localisation of cribriform and glomeruloid growth patterns in radical prostatectomy images and demonstrated the ability to map these patterns within pre-biopsy mpMRI scans successfully. In the future, we intend to further improve the algorithm for IDC identification and conduct multicentre trials on larger data sets for validation of this approach. Source of Funding: NHS/Aira-Matrix Project PO01 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e218 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ashwin Sachdeva More articles by this author Dinisha Kadam More articles by this author Avaneesh Meena More articles by this author Nitin Singhal More articles by this author Archie Parekh More articles by this author Guillaume Antem More articles by this author Yatin Jain More articles by this author Pedro Oliveira More articles by this author Vijay Ramani More articles by this author Expand All Advertisement PDF downloadLoading ...

Maxillary Nerve as Landmark for Exposure of the Internal Maxillary Artery in Extracranial-Intracranial Bypass Surgery

To establish a new method for fast exposure of the internal maxillary artery (IMA) during extracranial-intracranial bypass surgery. To explore the positional relationship between the IMA and the maxillary nerve and pterygomaxillary fissure, 11 formalin-fixed cadaveric specimens were dissected. Three bone windows of the middle fossa were created for further analysis. Then the IMA length that could be pulled up above the middle fossa was measured after different degrees of removal of bony structure. The IMA branches under each bone window were also explored in detail. The top of the pterygomaxillary fissure was located 11.50 mm anterolateral to the foramen rotundum. The IMA could be identified just inferior to the infratemporal segment maxillary nerve in all specimens. After drilling of the first bone window, the IMA length that could be pulled above the middle fossa bone was 6.85 mm. After drilling of the second bone window and further mobilization, the IMA length that could be harvested was significantly longer (9.04 mm vs. 6.85 mm; P < 0.001). Removal of the third bone window did not significantly improve the IMA length that could be harvested. The maxillary nerve could be used as a reliable landmark for the exposure of the IMA in the pterygopalatine fossa. With our technique, the IMA could be easily exposed and sufficiently dissected without zygomatic osteotomy and extensive middle fossa floor removal.

PIK3CA Mutations as a Prognostic Factor in Patients With Residual Rectal Cancer After Neoadjuvant Chemoradiotherapy.

PIK3CA mediates various cellular processes, such as transformation, tumor initiation and proliferation, and resistance to apoptosis. This study was conducted to identify the clinical significance and prognostic effect of PIK3CA mutations in patients with residual rectal cancer who underwent surgery after neoadjuvant chemoradiotherapy (NACRT). Formalin-fixed and paraffin-embedded surgical specimens were collected from 128 patients between January 2006 and December 2011 and analyzed using real-time polymerase chain reaction for hotspot mutations in exons 9 and 20 of the PIK3CA gene. Of the 128 patients, 109 were analyzed and 19 were excluded because of poor DNA quality. Mutations in PIK3CA were identified in three patients (2.8%), all of which were detected in exon 20 of the PIK3CA gene. PIK3CA mutations significantly correlated with lymphatic invasion (p=0.016), lymph node metastasis (p=0.034), and higher pathological disease stage (p=0.040). By univariate analysis, patients with PIK3CA mutations were observed to have significantly shorter cancer-specific survival (CSS) (p=0.001) and disease-free survival (DFS) (p=0.006) than PIK3CA wild-type patients. However, PIK3CA mutations were not an independent prognostic factor for CSS (p=0.319) or DFS (p=0.219) in multivariate modeling. Our findings indicate that PIK3CA mutation plays a role in oncogenesis in rectal cancer and may be considered as a candidate therapeutic approach targeting the PIK3/Akt/mTOR pathway in patients with residual rectal cancer after NACRT.

Comparison Between the Supracerebellar Infratentorial and Precuneal Interhemispheric, Transtentorial Approaches to the Cerebellomesencephalic Fissure: An Anatomoradiological Study and Illustrative Cases.

The dorsal brainstem and cerebellomesencephalic fissure are challenging surgical targets. To afford a preferentially craniocaudal trajectory to this region, the precuneal interhemispheric transtentorial approach (PCIT) has been proposed. To didactically describe and compare exposures and anatomic indications of the supracerebellar infratentorial approach (SCIT) and PCIT to the cerebellomesencephalic fissure. Nine formalin-fixed, latex-injected cadaveric head specimens were used to perform a midline SCIT and bilateral PCITs and measure the distance of each approach. Twenty-four formalin-fixed specimens were used to measure the distance from the most posterior cortical bridging vein entering the superior sagittal sinus to the calcarine sulcus and the torcula. Fifty-one magnetic resonance images were reviewed to calculate the angle of each approach. Three illustrative surgical cases were described. Mean distances from the brain or cerebellar surface to the operative target of the PCIT and SCIT were 7.1 cm (range: 5-7.7 cm) and 5.5 cm (range: 3.8-6.2 cm), respectively. The SCIT provided direct access to structures of the quadrigeminal cistern bilaterally. The PCIT provided access from the ipsilateral inferior colliculus to the ipsilateral infratrochlear zone. The PCIT's benefit was the direct access it provided to the cerebellomesencephalic fissure because of its superior to inferior trajectory. The PCIT is indicated for unilateral lesions of the cerebellomesencephalic fissure and dorsal brainstem that harbor a craniocaudal long axis and do not have a superior extension beyond the superior colliculi. The SCIT is beneficial for lesions that extend bilaterally, have an anteroposterior long axis, or involve the Galenic complex.

Development of a Multiplex qPCR Assay for Fast Detection and Differentiation of Paracoccidioidomycosis Agents.

Classic paracoccidioidomycosis (PCM) is a potentially deadly neglected tropical systemic mycosis caused by members of the Paracoccidioides brasiliensis complex (P. brasiliensis s. str., P. americana, P. restrepiensis, and P. venezuelensis) and P. lutzii. The laboratorial diagnosis of PCM relies on observing pathognomonic structures such as the "steering wheel" or "Mickey Mouse" shape in the direct mycological examination, fresh biopsied tissue in 10% KOH, histopathological analysis, and/or the isolation of the fungus in culture. However, these procedures are time-consuming and do not allow for the speciation of Paracoccidioides due to overlapping morphologies. Here, we propose a new one-tube multiplex probe-based qPCR assay to detect and recognize agents of the P. brasiliensis complex and P. lutzii. Primers (Paracoco-F and Paracoco-R) and TaqMan probes (PbraCx-Fam, Plu-Ned, and Paracoco-Vic) were developed to target the rDNA (ITS2/28S) in the Paracoccidioides genome. A panel of 77 Paracoccidioides isolates revealed a 100% specificity (AUC = 1.0, 95% CI 0.964-1.000, p < 0.0001) without cross-reacting with other medically relevant fungi or human and murine DNA. The lower limit of detection was 10 fg of gDNA and three copies of the partial rDNA amplicon. Speciation using qPCR was in perfect agreement with AFLP and TUB1-RFLP markers (kappa = 1.0). As a proof of concept, we assessed a panel of 16 formalin-fixed and paraffin-embedded specimens from histopathologically confirmed PCM patients to reveal a significant sensitivity of 81.25% and specificity of 100% (AUC = 0.906 ± 0.05, 95% CI = 0.756-0.979, p < 0.0001, Youden index J = 0.8125). Our assay achieved maximum sensitivity (100%) and specificity (100%) using fresh clinical samples (n = 9) such as sputum, bronchoalveolar lavage, and tissue fragments from PCM patients (AUC = 1.0, 95% CI 0.872-1.000, p < 0.0001, Youden index J = 1.0). Overall, our qPCR assay simplifies the molecular diagnosis of PCM and can be easily implemented in any routine laboratory, decreasing a critical bottleneck for the early treatment of PCM patients across a vast area of the Americas.

A study of morphological variations of fissures and lobes of formalin fixed cadaveric lungs

Background: Variation of morphology of lung fissures and lobes affects the interpretation of results of radiological examination as well as line of action in cardiothoracic surgical procedures. A research was done to find out the incidence of variations of fissures and lobes in embalmed cadaveric lungs.&#x0D; Materials and methods: This is a cross sectional study. All the formalin fixed lung specimens present in dissection halls and museums of the Anatomy Departments were observed for any abnormal fissures that are adding accessory lobes to lungs. Data was entered and analyzed by using SPSS 22.0. Descriptive analysis was applied by using frequencies and percentages for qualitative variables.&#x0D; Results: Total 80 formalin-fixed specimens of lungs were examined and a total of 29 (36.25%) (%) were found to have incomplete fissures. It was observed that 14 (17.5 %) out of 80 lungs had incomplete oblique fissures, and out of these 6 (17.65%) were found in right lungs and 08 (19.05%) were present in left lungs, whereas 15 (39.47%) right lungs showed incomplete horizontal fissure. Among 80 lungs, 6 (7.5%) presented unusual accessory fissures and lobes, whereas 5 (14.7%) showed absent horizontal fissure reducing the number of lobes to two in right lung specimen.&#x0D; Conclusion: There is a prevalent incidence of incomplete horizontal fissure that must be kept in mind when investigating and treating any lung pathology.

Impact of CD34-dependent Micro Vessel Density on Periapical Odontogenic Cysts.

Odontogenic cysts belong to a type of lesions with endodontic origin that in some cases mimic even aggressive odontogenic tumors sharing with them similar radiographic features. Periapical cysts (PCs) belong to the inflammatory odontogenic cysts sub-category and rarely squamous cell carcinoma arises from their hyperplastic/ dysplastic epithelia. This study aimed to explore the impact of cluster differentiation 34 (CD34) protein expression combined with micro vessel density (MVD) on PCs. Forty-eight (n=48) archival, formalin-fixed, and paraffin-embedded PC tissue specimens were included in the study. Immunohistochemistry (IHC) was performed in the corresponding tissue sections using an anti- CD34 antibody. CD34 expression levels and also MVD in the examined cases were measured by implementing a digital image analysis protocol. CD34 over-expression (moderate to high staining intensity levels) were detected in 29/48 (60.4%) cases, whereas the rest of them (19/48-39.6%) were characterized by low levels of expression. Extended MVD was identified in 26/48 (50.1%) cases correlated with CD34 over-expression, epithelial hyperplasia (p-value=0.001), and marginally with inflammatory infiltration level in the examined lesions (p-value=0.056). CD34 over-expression combined with increased MVD is associated with a neoplastic-like (hyperplastic) phenotype in PCs as a result of increased neo-angiogenic activity. These histopathological characteristics rarely form an eligible substrate for squamous cell carcinoma onset in untended cases.

Invasive lobular triple negative carcinoma of breast: A clinico-pathological study done in south eastern part of India

Background: Triple Negative Breast Carcinomas (TNBC) are breast carcinomas which are negative for Estrogen receptor (ER) Progesterone receptor (PR) and no overexpression of Human Epidermal Growth Factor receptor-2 (HER-2). The five-year survival rate for breast carcinomas in general is good but the same is bad for TNBC. TNBC has got worse prognosis compared to hormone positive breast carcinomas. Aim: The aim of present study is to compare invasive lobular TNBC with invasive ductal TNBC with morphologic, immunohistochemical and prognostic parameters. Materials and Methods: One-year study was done at Tertiary Care Centre. Patients of all ages were included. Most of them were in 4th and 5th decade. Mammography, FNAC and Biopsy was done. Formalin-fixed mastectomy specimens received at MGM hospital are screened, diagnosed histopathologically for carcinoma of breast over a period of one year. Immunohistochemistry (IHC) was performed to diagnose TNBC. Results: Total no of cases – 72. No of Cases with positive ER, PR and or Her 2/neu are – 61. No of Cases with ER, PR and Her 2/neu negative (Triple negative) – 11 Percentage of Triple negative cases – 15 %. Most of the TNBCs show high grade and high mitotic count 74% and 82% respectively. Other hand, some cases 60% showed only ER and PR positivity without Her-2 and 25% showed only Her-2 positivity without ER and PR. Invasive lobular TNBC subtype is reported in 18%. Conclusion: Overall TNBCs are high grade aggressive tumors and are more common in younger age groups. Whereas TNBCs associated with invasive lobular carcinoma are more common in 5th decade and were more aggressive compared to other subtypes.

An effective rehydrating formula for reconditioning dried, irreplaceable heart specimens for teaching

Formalin-fixed whole heart specimens are essential for studying the anatomy of congenital heart defects. Because of the improvement in diagnostic imaging and treatment, and cultural transition, heart specimens with congenital heart defects have become more and more rare and irreplaceable but are at risk of accidental deterioration through dehydration during long-term storage. In this article, we share our experience in reconditioning our inadvertently dehydrated heart specimens using a simple Formol-Glycerol solution that allowed for rehydration and recovery of such specimens.

Cortactin expression in a Norwegian cohort of human papilloma virus negative oral squamous cell carcinomas of the mobile tongue.

Oral squamous cell carcinoma of the tongue (OTSCC) is the most common malignancy among oral squamous cell carcinomas and is frequently associated with an unfavorable prognosis. Local spread and distant metastasis are important causes of poor prognosis in OTSCC. Cortactin amplification and overexpression, a common molecular alteration in oral squamous cell carcinomas, have been linked to invasion and metastasis of tumor cells. However, the intra-tumor expression pattern and prognostic significance of cortactin in human papillomavirus (HPV) negative OTSCC is not fully investigated. Immunohistochemical analysis using tissue microarray consisting of formalin-fixed and paraffin-embedded HPV negative OTSCC (n=123) specimens showed overexpression of cortactin at tissue cores from invading fronts as compared to the corresponding center cores. High overall cortactin expression was found to be associated with advanced (larger) tumor size and the occurrence of distance metastasis. Kaplan-Meier survival analysis showed that patients with high overall cortactin expression were associated with reduced 5-year survival. Multivariate Cox regression analysis identified high cortactin expression to be an independent prognostic factor in OTSCC. Additionally, siRNA-mediated silencing of cortactin was found to suppress the proliferative and invasive abilities of OTSCC cells in an organotypic co-culture model. Overexpression of cortactin is a promising prognostic marker in HPV-negative OTSCC.