Formalin-fixed Tissue Specimens Research Articles

A Diagnostic Algorithm for Detection of Leishmania spp. in Human Fresh and Fixed Tissue Samples.

Leishmaniasis is an important travel-related parasitic infection in the United States. Treatment regimens vary by Leishmania species and require an accurate diagnosis. The sensitivity and specificity of diagnostic methods depend on the type and condition of specimen analyzed. To identify the best algorithm for detection of parasites in fresh and fixed tissue samples, we evaluated parasite cultures, two PCR methods, and Leishmania immunohistochemistry (IHC) in samples received by the CDC from 2012 through 2019. The sensitivity and specificity of IHC assays were evaluated in fresh specimens tested. Diagnostic accuracy for formalin-fixed tissue was evaluated by using PCR-based methods and IHC. Of 100 suspected cases with fresh tissue available, Leishmania spp. infection was identified by PCR in 56% (56/100) of specimens; from these, 80% (45/56) were positive by parasite culture and 59% (33/56) by IHC. Of 420 possible cases where only fixed specimens were available, 58% (244/420) were positive by IHC and/or PCR. Of these, 96% (235/420) were positive by IHC and 84% (204/420) by PCR-based methods. Overall parasite detection using all methodologies was similar for fresh and formalin-fixed tissue specimens (56% versus 58%, respectively). Although PCR-based methods were superior for diagnosis of leishmaniasis and species identification in fresh samples, IHC in combination with PCR increased the accuracy for Leishmania spp. detection in fixed samples. In conclusion, PCR is the most effective method for detecting Leishmania infection in fresh tissue samples, whereas for formalin-fixed samples, IHC and PCR-based methods should be used in combination.

Age-dependent increase of perineuronal nets in the human hippocampus and precocious aging in epilepsy.

Perineuronal nets (PNN) are specialized extracellular matrix (ECM) components of the central nervous system, frequently accumulating at the surface of inhibitory GABAergic interneurons. While an altered distribution of PNN has been observed in neurological disorders including Alzheimer's disease, schizophrenia and epilepsy, their anatomical distribution also changes during physiological brain maturation and aging. Such an age-dependent shift was experimentally associated also with hippocampal engram formation during brain maturation. Our aim was to histopathologically assess PNN in the hippocampus of adult and pediatric patients with temporal lobe epilepsy (TLE) compared to age-matched post-mortem control subjects and to compare PNN-related changes with memory impairment observed in our patient cohort. Sixty-six formalin-fixed and paraffin-embedded tissue specimens of the human hippocampus were retrieved from the European Epilepsy Brain Bank. Twenty-nine patients had histopathologically confirmed hippocampal sclerosis (HS), and eleven patients suffered from TLE without HS. PNN were immunohistochemically visualized using an antibody directed against aggrecan and manually counted from hippocampus subfields and the subiculum. PNN density increased with age in both human controls and TLE patients. However, their density was significantly higher in all HS patients compared to age-matched controls. Intriguingly, TLE patients presented presurgically with better memory when their hippocampal PNN density was higher (p < 0.05). Our results were compatible with age-dependent ECM specialization in the human hippocampus and its precocious aging in the epileptic condition. These observations confirm recent experimental animal models and also support the notion that PNN play a role in memory formation in the human brain. "Perineuronal nets" (PNN) are a specialized compartment of the extracellular matrix (ECM), especially surrounding highly active neurons of the mammalian brain. There is evidence that PNN play a role in memory formation, brain maturation, and in some pathologies like Alzheimer's disease, schizophrenia or epilepsy. In this study, we investigated the role of PNN in patients suffering from drug-resistant focal epilepsy compared to controls. We found that with increasing age, more neurons are surrounded by PNN. Similarly, all epilepsy patients but especially patients with better memory performance also had more PNN. This study raises further interest in studying ECM molecules in the human brain under physiological and pathophysiological conditions.

Predominance of T-bet-positive Th1 cells in infiltrating T-lymphocytes in most of active arteritis lesions of giant cell arteritis.

Immunological mechanisms through the activation of CD4-positive T-cells have been assumed to be involved in the pathogenesis of giant cell arteritis (GCA). Many studies employing frozen tissues of temporal artery biopsy, peripheral blood lymphocytes, and plasma of GCA patients have revealed the contribution of interferon-γ and interleukin-17 in both protein and mRNA levels. However, the analyses using formalin-fixed and paraffin-embedded (FFPE) tissue specimens, in which the correlation between histopathologic pictures and immunological circumstances would be elucidated, have been limited. Here, we performed the immunohistochemical analyses of infiltrating small lymphocytes in GCA lesions using FFPE specimens, especially of the subsets of CD4-positive T-cells by immunohistochemistry with antibodies against T-bet, GATA-3, RORγT, and Foxp3, which is the differentiation-specific transcription factor for Th1, Th2, Th17, and Treg cells, respectively. In these slides, the nuclear-positive staining is much more clearly and easily identifiable than the cytoplasmic staining for cytokines. The results indicate the predominance of T-bet-positive Th1 cells in infiltrating T-cells in most of active arteritis lesions of GCA. Furthermore, our data suggest the possible immunosuppressive microenvironment induced by T-reg cells and M2-type macrophages in the arteritis lesions throughout the course of GCA inflammation.

A high-throughput 3D X-ray histology facility for biomedical research and preclinical applications

Background The University of Southampton, in collaboration with the University Hospital Southampton (UHS) NHS Foundation Trust and industrial partners, has been at the forefront of developing three-dimensional (3D) imaging workflows using X-ray microfocus computed tomography (μCT) -based technology. This article presents the outcomes of these endeavours and highlights the distinctive characteristics of a μCT facility tailored explicitly for 3D X-ray Histology, with a primary focus on applications in biomedical research and preclinical and clinical studies. Methods The UHS houses a unique 3D X-ray Histology (XRH) facility, offering a range of services to national and international clients. The facility employs specialised μCT equipment explicitly designed for histology applications, allowing whole-block XRH imaging of formalin-fixed and paraffin-embedded tissue specimens. It also enables correlative imaging by combining μCT imaging with other microscopy techniques, such as immunohistochemistry (IHC) and serial block-face scanning electron microscopy, as well as data visualisation, image quantification, and bespoke analysis. Results Over the past seven years, the XRH facility has successfully completed over 120 projects in collaboration with researchers from 60 affiliations, resulting in numerous published manuscripts and conference proceedings. The facility has streamlined the μCT imaging process, improving productivity and enabling efficient acquisition of 3D datasets. Discussion &amp; Conclusions The 3D X-ray Histology (XRH) facility at UHS is a pioneering platform in the field of histology and biomedical imaging. To the best of our knowledge, it stands out as the world's first dedicated XRH facility, encompassing every aspect of the imaging process, from user support to data generation, analysis, training, archiving, and metadata generation. This article serves as a comprehensive guide for establishing similar XRH facilities, covering key aspects of facility setup and operation. Researchers and institutions interested in developing state-of-the-art histology and imaging facilities can utilise this resource to explore new frontiers in their research and discoveries.

Prevalence of Bovine leukemia virus in Egyptian women’s breast cancer tissues

ABSTRACT Bovine leukemia virus (BLV) infects cattle around the world and presents in commercial bovine products. Several studies have suggested that BLV may play a role in breast oncogenesis. This study aimed to determine the risk factors for BLV infection and the presence of the BLV gene in breast tissue. A total of 100 formalin-fixed and paraffin-embedded breast tissue specimens were collected (50 confirmed cancer samples and 50 negative samples). DNA was extracted from breast tissues, and nested PCR was done to detect the tax gene of BLV. Immunohistochemical staining for ER, PR, Her2/neu, and Ki-67 was applied. Bovine leukemia virus DNA was detected in 16% of breast cancer tissue samples and in only 4% of tissue samples without cancer. No statistically significant association was reported between the presence of BLV and tumor grade, stage, or LN metastasis. A high percentage of positivity (100%, 88%, and 75%) was reported for ER, PR, and Ki-67 in BLV-positive cancer samples, respectively. There was a significant association between the BLV tax gene and breast cancer in Egyptian women. Thus, there is a need to control BLV transmission to humans from cattle, and further investigations about its risk related to public health are highly required.

A high-throughput 3D X-ray histology facility for biomedical research and preclinical applications.

The University of Southampton, in collaboration with the University Hospital Southampton (UHS) NHS Foundation Trust and industrial partners, has been at the forefront of developing three-dimensional (3D) imaging workflows using X-ray microfocus computed tomography (μCT) -based technology. This article presents the outcomes of these endeavours and highlights the distinctive characteristics of a μCT facility tailored explicitly for 3D X-ray Histology, with a primary focus on applications in biomedical research and preclinical and clinical studies. The UHS houses a unique 3D X-ray Histology (XRH) facility, offering a range of services to national and international clients. The facility employs specialised μCT equipment explicitly designed for histology applications, allowing whole-block XRH imaging of formalin-fixed and paraffin-embedded tissue specimens. It also enables correlative imaging by combining μCT imaging with other microscopy techniques, such as immunohistochemistry (IHC) and serial block-face scanning electron microscopy, as well as data visualisation, image quantification, and bespoke analysis. Over the past seven years, the XRH facility has successfully completed over 120 projects in collaboration with researchers from 60 affiliations, resulting in numerous published manuscripts and conference proceedings. The facility has streamlined the μCT imaging process, improving productivity and enabling efficient acquisition of 3D datasets. The 3D X-ray Histology (XRH) facility at UHS is a pioneering platform in the field of histology and biomedical imaging. To the best of our knowledge, it stands out as the world's first dedicated XRH facility, encompassing every aspect of the imaging process, from user support to data generation, analysis, training, archiving, and metadata generation. This article serves as a comprehensive guide for establishing similar XRH facilities, covering key aspects of facility setup and operation. Researchers and institutions interested in developing state-of-the-art histology and imaging facilities can utilise this resource to explore new frontiers in their research and discoveries.

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.

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.

Plastination - A method for preservation of oral hard and soft tissue biopsy specimen v/s the conventional method of preservation with formalin.

Plastination is one of the most advanced method for preserving perishable biological specimen as well as tissue samples as for a longer period of time using polymers. To evaluate the changes in dimension of hard and soft tissue specimens after plastination procedure and compare it with the conventional method of preservation in formalin. Institution-based retrospective study. The study included 20 formalin-fixed soft tissue and 10 formalin-fixed hard tissue specimens. All the specimens were plastinated which involved four basic steps of fixation, dehydration and defatting, impregnation with polymer and curing of polymer followed by finishing and storage. The specimens were analysed for shrinkage and dimensional changes and changes in colour and consistency between formalin-fixed specimen and plastinated soft tissue and hard tissue specimen. Descriptive statistics were used. After plastination, soft tissues showed average shrinkage of 3.49%with a range of 0.80-7.90% in comparison to the original size. In case of teeth and hard tissue specimen, there was no evidence of dimensional changes or shrinkage before and after plastination. Changes in colour and consistency of the soft tissue specimens were also noted before plastination and after plastination. Although the plastinated specimens in the current study showed minimal shrinkage rate, they have proved to be an excellent alternative to formalin-fixed specimens as they are easy to handle and maintain, non-infectious, non-toxic, user-friendly.

The ALDH2, IGSF9, and PRDM16 Proteins as Predictive Biomarkers for Prognosis in Breast Cancer

IntroductionALDH2, IGSF9, and PRDM16 play crucial roles in regulating diverse cellular pathophysiologic functions. The current study was to evaluate the effect of the 3 proteins on clinicopathologic features and prognosis of patients with breast cancer. Materials and MethodsThe formalin-fixed and paraffin-embedded tissue specimens were collected from breast cancer patients by immunohistochemistry (IHC) were analyzed. ResultsOf the 216 patients enrolled, ALDH2 high expression was significantly correlated with the age (p = .040), larger tumor size (p = .001), LVI (p < .001), LNM (p < .001), advanced TNM staging (p < .001), PR (p = .027), HER2 status (p = .002), and molecular subtype (p = .003). IGSF9 low expression was significantly correlated with the LV1 (p = .024), LNM (p = .024), advanced TNM staging (p = .001). The low expression of PRDM16 was significantly correlated with age (p = .023), and LNM (p = .014). The A+I−P− expression (13.4%) were markedly correlated with lymphatic vessel invasion (LVI) (p < .001), lymph node metastasis (LNM) (p < .001), advanced TNM staging (p < .001). Furthermore, patients with A+I−P− expression had significantly advanced-stage breast cancer [stage III (72.4%) vs. (23.0%)]. Univariate and multivariate analysis identified variables (ie, larger tumor size, lymph node involvement, and A+I−P− expression) as independent prognostic factors for survival. ConclusionOur results reveal ALDH2 high expression, IGSF9 and PRDM16 low expression, A+I−P− expression was associated with advanced clinicopathological characteristics, and shorter OS and DFS in breast cancer patients. The 3 proteins may be potential prognosis markers and therapeutic targets for breast cancer patients.

Drug Resistance and Molecular Characteristics of Mycobacterium tuberculosis: A Single Center Experience.

In recent years, the incidence of tuberculosis (TB) and mortality caused by the disease have been decreasing. However, the number of drug-resistant tuberculosis patients is increasing rapidly year by year. Here, a total of 380 Mycobacterium tuberculosis (MTB)-positive formalin-fixed and paraffin-embedded tissue (FFPE) specimens diagnosed in the Department of Pathology of the Eighth Medical Center, Chinese PLA General Hospital were collected. Among 380 cases of MTB, 85 (22.37%) were susceptible to four anti-TB drugs and the remaining 295 (77.63%) were resistant to one or more drugs. The rate of MDR-TB was higher in previously treated cases (52.53%) than in new cases [(36.65%), p < 0.05]. Of previously treated cases, the rate of drug resistance was higher in females than in males (p < 0.05). Among specimens obtained from males, the rate of drug resistance was higher in new cases than in previously treated cases (p < 0.05). Of mutation in drug resistance-related genes, the majority (53/380, 13.95%) of rpoB gene carried the D516V mutation, and 13.42% (51/380) featured mutations in both the katG and inhA genes. Among the total specimens, 18.68% (71/380) carried the 88 M mutation in the rpsL gene, and the embB gene focused on the 306 M2 mutation with a mutation rate of 19.74%. Among the resistant INH, the mutation rate of −15 M was higher in resistance to more than one drug than in monodrug-resistant (p < 0.05). In conclusion, the drug resistance of MTB is still very severe and the timely detection of drug resistance is conducive to the precise treatment of TB.

Comparative Benefits of Tissue Marking by Poster Ink in Histopathology

Background: Tissue margin marking with India ink is important in decision making for surgeons. The present study was conducted to examine the reliability of different shades of locally available poster colours in tissue marking and to evaluate the colour perceptibility microscopically in comparison with similar tissues marked by India ink.&#x0D; Methodology: This experimental study was conducted at Department of Pathology, HBS Medical &amp; Dental College &amp; Hospital, Islamabad from 27th February 2021 to 29th April 2021. Sample size was fifty, collected through convenient sampling technique. Five types of formalin fixed tissue specimens were selected for the study so as to evaluate the effectiveness of poster ink marking on different tissue surfaces. From each specimen, four sections were taken from the margins. Three shades of poster colour (black, blue and green) were used to ink three sections while one section from each tissue type was marked with India ink. After complete tissue processing and routine haematoxylin and eosin (H&amp;E) staining, slides were examined microscopically. Scoring was done on a scale 0 to 3 on the basis of visibility.&#x0D; Results: The present study showed that poster colours inking of the tissues was quite reliable as compared to India ink. Most consistent results were achieved with black and blue colours as compared to green colour.&#x0D; Conclusion: Poster colours are reliable tool for tissue marking when India ink is not available. Their availability in a variety of colours provides them an edge over India ink.&#x0D; Key words: Eosin, India ink, Resection Margins

Elucidation of HER2/neu and Wilms’ tumor 1 immunohistochemical expression in endometrial carcinomas regarding the diagnostic and prognostic significance

Objective To confirm HER2 neu and Wilms’ tumor 1 (WT1) immunohistochemical expression in endometrial cancer and illustrate its possible diagnostic and prognostic significance in various clinicopathological parameters that both physicians and pathologists may find therapeutically valuable. Materials and methods A total of 50 formalin-fixed and paraffin-embedded tissue specimens were acquired and prepared for this investigation from the archival pathology files of Al-Hussein and Al-Zahraa University Hospitals between 2017 and 2020, after obtaining the informed consent and approval of the local ethical committee. The patient’s medical records and histopathologic data such as tumor size, myometrial invasion depth, lymph node invasion, histologic type of tumor, clinical presentation, and International Federation of Gynecology and Obstetrics stage were assessed using hematoxylin-eosin for all sections that were re-evaluated by HER2/neu and WT1 antibodies immunohistochemically. Results Positive HER2/neu and WT1 expressions were frequently associated with a worsening condition in patients with endometrial carcinomas (ECs) with a higher grade, stage, and nonendometrioid carcinoma type, reflecting the prognostic relevance of HER2/neu and WT1 expressions, especially in aggressive EC forms. Follow-up and more genetic concordance between EC subtypes could improve the predictive value of HER2/neu and WT1 expression in EC. For advanced endometrial malignancies, tailored chemotherapy may be valuable. Conclusions HER2/neu-positive and WT1-positive immunohistochemical staining results are generally coupled to ECs in their advanced forms, which have a higher grade, stage, and nonendometrioid carcinoma type, reflecting their prognostic implications, especially in aggressive EC types. However, additional validation is required before enrolling this panel in clinical settings, which could improve the value of prediction of HER2/neu and WT1 immunohistochemical markers in ECs. Moreover, targeted chemotherapy for aggressive endometrial cancers can be beneficial.

Oncoviruses and melanomas: A retrospective study and literature review.

The role of human oncoviruses in melanoma has been poorly investigated. The aim of this study was to investigate the association between oncoviruses and melanomas searching for human papillomavirus (HPV), Epstein Barr virus (EBV), and human herpesvirus 8DNA in melanoma specimens. Formalin-fixed and paraffin-embedded tissue specimens of cutaneous, mucosal, and ocular melanomas (OM) were selected from the Pathology Departments of the Galliera Hospital (Genoa) and the University Hospitals of Turin and Cagliari. Cutaneous and mucosal nevi have been collected as controls. The oncoviruses search has been performed with different polymerase chain reaction reagent kits.Fifty-four melanomas (25 mucosal, 12 ocular, and 17 cutaneous) and 26 nevi (15 cutaneous and 11 mucosal) specimens were selected. The detection rate for one of the investigated oncoviruses was 17% in mucosal, 20% in ocular, and 0% in cutaneous melanomas (CMs). Despite the differences between groups seeming remarkable, there was no statistical significance (p > 0.5). Our data do not support a primary role of oncoviruses in melanoma carcinogenesis; however, the finding of HPV and EBV DNA in a considerable fraction of mucosal and OMs suggests that these viruses may act as cofactors in the development of extra-CMs.

A Non-Hazardous Deparaffinization Protocol Enables Quantitative Proteomics of Core Needle Biopsy-Sized Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue Specimens.

Most human tumor tissues that are obtained for pathology and diagnostic purposes are formalin-fixed and paraffin-embedded (FFPE). To perform quantitative proteomics of FFPE samples, paraffin has to be removed and formalin-induced crosslinks have to be reversed prior to proteolytic digestion. A central component of almost all deparaffinization protocols is xylene, a toxic and highly flammable solvent that has been reported to negatively affect protein extraction and quantitative proteome analysis. Here, we present a ‘green’ xylene-free protocol for accelerated sample preparation of FFPE tissues based on paraffin-removal with hot water. Combined with tissue homogenization using disposable micropestles and a modified protein aggregation capture (PAC) digestion protocol, our workflow enables streamlined and reproducible quantitative proteomic profiling of FFPE tissue. Label-free quantitation of FFPE cores from human ductal breast carcinoma in situ (DCIS) xenografts with a volume of only 0.79 mm3 showed a high correlation between replicates (r2 = 0.992) with a median %CV of 16.9%. Importantly, this small volume is already compatible with tissue micro array (TMA) cores and core needle biopsies, while our results and its ease-of-use indicate that further downsizing is feasible. Finally, our FFPE workflow does not require costly equipment and can be established in every standard clinical laboratory.

Program death ligand-1 immunocytochemistry in lung cancer cytological samples: A systematic review.

In this era of personalized medicine, targeted immunotherapies like immune checkpoint inhibitors (ICI) blocking the programmed death‐1 (PD‐1)/program death ligand‐1 (PD‐L1) axis have become an integral part of treating advanced stage non‐small cell lung carcinoma (NSCLC) and many other cancer types. Multiple monoclonal antibodies are available commercially to detect PD‐L1 expression in tumor cells by immunohistochemistry (IHC). As most clinical trials initially required tumor biopsy for PD‐L1 detection by IHC, many of the currently available PD‐1/PD‐L1 assays have been developed and validated on formalin fixed tissue specimens. The majority (>50%) of lung cancer cases do not have a surgical biopsy or resection specimen available for ancillary testing and instead must rely primarily on fine needle aspiration biopsy specimens for diagnosis, staging and ancillary tests. Review of the literature shows multiple studies exploring the feasibility of PD‐L1 IHC on cytological samples. In addition, there are studies addressing various aspects of IHC validation on cytology preparations including pre‐analytical (e.g., different fixatives), analytical (e.g., antibody clone, staining platforms, inter and intra‐observer agreement, cytology‐histology concordance) and post‐analytical (e.g., clinical outcome) issues. Although promising results in this field have emerged utilizing cytology samples, many important questions still need to be addressed. This review summarizes the literature of PD‐L1 IHC in lung cytology specimens and provides practical tips for optimizing analysis.

In Vivo Tumorigenicity Assays Using Subcutaneous and Orthotopic Xenografts in Immunodeficient Mice.

Evaluation of tumorigenic potential of medulloblastoma cell lines in vivo has been the obvious and major next step following cell line driven research for last many years. Effect of changes in expression of gene/s or efficacy of anticancer drugs on tumor initiation and/or growth can be easily assessed by injecting genetically modified cell lines in vivo or by treating in vivo xenografts of established cell lines with newer inhibitors or anticancer drugs. These studies are easy to perform and to reproduce in comparison to patient derived xenografts owing to ease in propagating, maintaining, and modifying genetic makeup of cell lines. Here we describe standardized protocols of obtaining either subcutaneous or orthotopic xenografts of medulloblastoma cell lines in immunodeficient mice. Once established, tumor growth of xenografts can be assessed during the course of experiment by either employing a simple method using Vernier caliper or technically demanding but sensitive method like in vivo bioluminescence imaging. In addition, xenograft tumors of euthanized animals can be preserved as formalin-fixed tissue specimens for further histopathological, immunohistochemical, or molecular analysis.

Accelerated loss of oogonia and impaired folliculogenesis in females with Turner syndrome start during early fetal development.

How are germ cell numbers and initiation of folliculogenesis affected in fetal Turner syndrome (TS) ovaries? Germ cell development was severely affected already in early second trimester pregnancies, including accelerated oogonia loss and impaired initiation of primordial follicle formation in TS ovaries, while the phenotype in TS mosaic ovaries was less severe. Females with TS are characterized by premature ovarian insufficiency (POI). This phenotype is proposed to be a consequence of germ cell loss during development, but the timing and mechanisms behind this are not characterized in detail. Only few studies have evaluated germ cell development in fetal TS and TS mosaic ovaries, and with a sparse number of specimens included per study. This study included a total of 102 formalin-fixed and paraffin-embedded fetal ovarian tissue specimens. Specimens included were from fetuses with 45,X (N = 42 aged gestational week (GW) 12-20, except one GW 40 sample), 45,X/46,XX (N = 7, aged GW 12-20), and from controls (N = 53, aged GW 12-42) from a biobank (ethics approval # H-2-2014-103). The number of OCT4 positive germ cells/mm2, follicles (primordial and primary)/mm2 and cPARP positive cells/mm2 were quantified in fetal ovarian tissue from TS, TS mosaic and controls following morphological and immunohistochemical analysis. After adjusting for gestational age, the number of OCT4+ oogonia was significantly higher in control ovaries (N = 53) versus 45,X ovaries (N = 40, P < 0.001), as well as in control ovaries versus 45,X/46,XX mosaic ovaries (N = 7, P < 0.043). Accordingly, the numbers of follicles were significantly higher in control ovaries versus 45,X and 45,X/46,XX ovaries from GW 16-20 with a median range of 154 (N = 11) versus 0 (N = 24) versus 3 (N = 5) (P < 0.001 and P < 0.015, respectively). The number of follicles was also significantly higher in 45,X/46,XX mosaic ovaries from GW 16-20 compared with 45,X ovaries (P < 0.005). Additionally, the numbers of apoptotic cells determined as cPARP+ cells/mm2 were significantly higher in ovaries 45,X (n = 39) versus controls (n = 15, P = 0.001) from GW 12-20 after adjusting for GW. The analysis of OCT4+ cells/mm2, cPARP+ cells/mm2 and follicles (primordial and primary)/mm2 should be considered semi-quantitative as it was not possible to use quantification by stereology. The heterogeneous distribution of follicles in the ovarian cortex warrants a cautious interpretation of the exact quantitative numbers reported. Moreover, only one 45,X specimen and no 45,X/46,XX specimens aged above GW 20 were available for this study, which unfortunately made it impossible to assess whether the ovarian folliculogenesis was delayed or absent in the TS and TS mosaic specimens. This human study provides insights about the timing of accelerated fetal germ cell loss in TS. Knowledge about the biological mechanism of POI in girls with TS is clinically useful when counseling patients about expected ovarian function and fertility preservation strategies. This work was supported by the International Center for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC). N/A.

SMG9 Serves as an Oncogene to Promote the Tumor Progression via EMT and Wnt/β-Catenin Signaling Pathway in Hepatocellular Carcinoma.

Background/Aims: SMG9 participates in the nonsense-mediated mRNA decay process that degrades mRNA harboring nonsense mutations introduced either at the level of transcription or RNA processing. However, little is known about the role of SMG9 in hepatocellular carcinoma (HCC). The objective of this research was to clarify the effects of SMG9 expression on HCC progression.Methods: Microarray data were acquired from NCBI Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) database to bioinformatically analyze the differential expression of SMG9 between HCC patients and normal controls. SMG9 mRNA level was measured in sixteen sets of fresh tumor tissues and adjacent non-cancerous liver tissues (ANLTs) via reverse transcription-quantitative polymerase chain reaction (RT-qPCR). SMG9 protein expression was analyzed in ninety-five sets of paired formalin-fixed and paraffin-embedded tissue specimens by immunohistochemistry (IHC). In addition, clinicopathological features of SMG9 in HCC were checked. For in vitro studies, small interfering RNA (siRNA) was used to silence SMG9 expression for exploring biological functions and underlying mechanisms of SMG9 in SMMC-7721 and HepG2.Results: We found that SMG9 was upregulated in HCC tissues and SMG9 levels were closely related to TNM stage, tumor number and tumor size. Cox regression and Kaplan–Meier proportional hazards analyses showed that high expression of SMG9 was associated with poor patient survival. Furthermore, proliferation, apoptosis resistance, migration and invasion of both SMMC-7721 and HepG2 cells were suppressed by SMG9 inhibition. In addition, EMT and the Wnt/β-catenin signaling pathway were involved in SMG9-mediated HCC progression.Conclusions: SMG9 may serve as a potential novel prognostic biomarker and therapeutic target in HCC patients.