Paraffin-embedded Biopsy Specimens Research Articles

Expressions of NeuroD and GAP43 as diagnostic markers for olfactory neuroblastoma

ObjectiveOlfactory neuroblastoma (ONB) is often difficult to pathologically distinguish from other small round cell tumors (SRCTs) arising in the nasal cavities. Although there are several diagnostic markers used for differential diagnosis of ONB, these molecules are also expressed in various neuronal derived tumors. Here, we examined the expression of NeuroD, GAP43, and olfactory marker protein (OMP) in ONB and non-ONB SRCT to determine their utility in the differential diagnosis of ONB. MethodsTwenty-six patients diagnosed with and treated for ONB at Kobe University Hospital between 1997 and 2017 with formalin-fixed, paraffin-embedded biopsy or surgical resection specimens were included. The expressions of NeuroD, GAP43, and OMP were immunohistochemically examined in these 26 ONB specimens and specimens from 13 SRCTs arising in the nasal cavities for reference. ResultsAmong the 26 ONB samples, focal, patchy, and marked staining for NeuroD was observed in 4, 3, and 9 samples, respectively. Focal, patchy, and marked GAP43 staining was observed in 5, 3, and 11 samples, respectively. Consequently, marked positive staining for either NeuroD or GAP43 was observed in 54% (14/26) of ONBs. Among the 13 SRCTs, marked staining for NeuroD was observed in two small cell carcinomas, one undifferentiated carcinoma, and one neuroendocrine carcinoma, whereas marked positive staining for GAP43 was observed only in one undifferentiated carcinoma. No specimen in this study exhibited OMP staining. ConclusionsOur results suggest possible roles of GAP43 immunostaining in the differential diagnosis of ONB.

030 Immune checkpoint inhibitor-induced bullous pemphigoid skin has elevated interleukin-4 and interleukin-13 expression and responds to IL-4R inhibition

Immune check point inhibitors (ICIs) have revolutionized the treatment of advanced malignancies but can induce skin toxicities including ICI-induced bullous pemphigoid (BP). The pathogenesis of ICI-induced BP is unclear and selection of effective treatment options that do not inhibit the anti-tumor response is a challenge. If cytokine expression in ICI-induced BP and conventional BP skin is similar, emerging therapies for conventional BP may be effective for ICI-induced BP as well. RNA-in situ hybridization was utilized to examine expression of IL4Rα ligands, IL4 and IL13, in formalin-fixed paraffin-embedded skin biopsy specimens from patients with conventional BP, ICI-induced BP, atopic dermatitis and normal, control skin.

In Situ Identification of Unknown Crystals in Acute Kidney Injury Using Raman Spectroscopy.

Raman spectroscopy is a well-established and powerful tool for in situ biomolecular evaluation. Type 2 crystal nephropathies are characterized by the deposition of crystalline materials in the tubular lumen, resulting in rapid onset of acute kidney injury without specific symptoms. Timely crystal identification is essential for its diagnosis, mechanism exploration and therapy, but remains challenging. This study aims to develop a Raman spectroscopy-based method to assist pathological diagnosis of type 2 crystal nephropathies. Unknown crystals in renal tissue slides from a victim suffered extensive burn injury were detected by Raman spectroscopy, and the inclusion of crystals was determined by comparing Raman data with established database. Multiple crystals were scanned to verify the reproducibility of crystal in situ. Raman data of 20 random crystals were obtained, and the distribution and uniformity of substances in crystals were investigated by Raman imaging. A mouse model was established to mimic the crystal nephropathy to verify the availability of Raman spectroscopy in frozen biopsy. All crystals on the human slides were identified to be calcium oxalate dihydrate, and the distribution and content of calcium oxalate dihydrate on a single crystal were uneven. Raman spectroscopy was further validated to be available in identification of calcium oxalate dihydrate crystals in the biopsy specimens. Here, a Raman spectroscopy-based method for in situ identification of unknown crystals in both paraffin-embedded tissues and biopsy specimens was established, providing an effective and promising method to analyze unknown crystals in tissues and assist the precise pathological diagnosis in both clinical and forensic medicine.

Granulomatous Mastitis with Corynebacterium Infection

Background: Idiopathic granulomatous mastitis (IGM) is a rare relapsing benign, chronic inflammatory breast disease characterized by infiltration of inflammatory cells including multinucleated giant cells. Recently, it has been suggested that infection with Corynebacterium species (spp.) may be involved in the onset of the disease. Therefore, the aim of this article is to summarize the previous IGM reports related to Corynebacterium spp.Methods: I used the terms "granulomatous mastitis Corynebacterium" in PubMed and "granulomatous mastitis Corynebacterium" with Japanese in Google Scholar, which resulted in 63 English articles and 71 Japanese articles. I read all the abstracts and summarized the recent articles with Corynebacterium in the title.Results: In 16 English articles and 4 Japanese ones, the most common Corynebacterium spp. was Corynebacterium kroppenstedtii, followed by Corynebacterium tuberculostearium. Conclusion: Corynebacterium infection is widely detected with IGM. In addition to bacterial culture, real-time polymerase chain reaction and formalin-fixed, paraffin-embedded biopsy specimens’ analysis can be used to detect Corynebacterium spp.

Outcome of Patients With Head and Neck Squamous Cell Carcinoma Can be Predicted by Expression of eIF4E and Osteopontin in Free Surgical Margins.

Squamous cell carcinoma of the head and neck (HNSCC) is recognized as the third most common cause of death. Incomplete resection of the primary tumor is the main cause of local recurrence and poor prognosis in HNSCC. Histologic assessment in order to determine "tumor-free" margins could be inadequate because of malignant transformation occurs at the molecular level earlier than the morphologic level. The present study aimed to evaluate the prognostic significance of eukaryotic initiation factor 4E (eIF4E) and Osteopontin in the tumor cells and histologically tumor free surgical margins of HNSCC. This cohort study was performed on 60 cases of HNSCC diagnosed at the Department of Pathology and treated at the Clinical Oncology Department, Faculty of Medicine, Zagazig University. Our enrolled formalin fixed paraffin embedded biopsy specimens with their matched tumor free surgical margins from resected head and neck squamous cell carcinoma were immunostaind for eIF4E and Osteopontin markers. 65% of our HNSCC patients had eIF4 E positive cytoplasmic immunostaining and 70% of them exhibited Osteopontin staining. Two-thirds of the dead patients exhibited high Osteopontin positive staining, whereas the surviving group did not exhibit this high expression. Concerning eIF4E, 85% and 5% of the dead patients showed high and low eIF4E expression, respectively. Disease-free survival (DFS) and overall survival were significantly (P=0.000) different between high and negative expression of Osteopontin, high and negative expression of eIF4E. 84% of patients with eIF4E positive margins and 75% with Osteopontin positive margins had local recurrence. In addition, negative expression of eIF4E is associated with highly significant better DFS and overall survival (P=0.000 and 0.001), respectively, in the margin negative expression status, while negative expression of Osteopontin was significantly associated with better DFS but of no significance in overall survival outcome. Our findings suggest that tumor-free surgical margins in HNSCC may be redefined as histologically Osteopontin and eIF4E negative resection margins. However, multicenter prospective studies are required to further evaluate their clinical utility in the surgical management of primary HNSCC.

Molecular Diagnosis of Cat Scratch Disease: a 25-Year Retrospective Comparative Analysis of Various Clinical Specimens and Different PCR Assays.

ABSTRACTCat-scratch disease (CSD), caused primarily by Bartonella henselae, is a common etiology of infectious regional lymphadenopathy. Lymphadenopathy is preceded by a primary inoculation lesion and may progress to suppuration. Laboratory diagnosis of CSD is hampered by the limitations of available confirmatory tests. PCR, in general, is highly sensitive and specific; however, clinical sensitivity in CSD varies greatly between studies. We aimed to identify clinical specimens and PCR assays best suited for CSD diagnosis using a national CSD registry and a uniform case definition. Different clinical specimens and PCR assays, including conventional and real-time PCR, were evaluated. PCR was positive in 335/390 (86%) CSD patients and 425/482 (88%) PCR tests. The highest PCR sensitivity was achieved in lymph node pus aspirates (96%; n = 278 tests) followed by primary lesions (88%; n = 50), lymph node fine needle aspirations (85%; n = 46), lymph node biopsy specimens (73%; n = 91) and paraffin-embedded lymph nodes (59%; n = 17), (P < 0.001). Sensitivity was similar in all types of PCR assays studied. PCR negative predictive value of pus aspirate and lymph node biopsy specimen patient groups was 82% and 72%, respectively. Specificity was 100% based on 125 non-CSD patients with negative PCR. In conclusion, the specimen type rather than the PCR assay type has a major impact on CSD molecular diagnosis. We assume that the inadequate sensitivity of the biopsy specimens was due to sampling errors or the presence of inhibitory factors. Primary lesions should be sampled more frequently for CSD diagnosis. Physicians should be aware of the low PCR negative predictive value of lymph node biopsy specimens.IMPORTANCE Polymerase chain reaction (PCR) for the detection of Bartonella henselae is an important tool for the diagnosis of cat scratch disease (CSD); however, clinical sensitivity varies greatly between studies. The current study shows that the specimen type, with pus aspiration, fine needle aspiration, and primary inoculation lesion having significantly higher sensitivity than fresh or formalin-fixed paraffin-embedded lymph node biopsy specimen, rather than the type of the PCR assay, whether a conventional or a real-time assay, has a major impact on the performance of diagnostic PCR for CSD. The new data provide new tools for the clinical microbiologist when interpreting the results of the PCR assays. Primary inoculation lesions, although easily accessible, are often neglected and should be sampled more frequently for molecular diagnosis of CSD. Physicians should be aware that negative PCR, particularly if performed on fresh or paraffin-embedded lymph node biopsy specimens, does not exclude CSD.

FGFR1 Amplification and Response to Neoadjuvant Anti-HER2 Treatment in Early HER2-Positive Breast Cancer

HER2-positive breast cancer (BC) is an aggressive subtype that affects 20–25% of BC patients. For these patients, neoadjuvant therapy is a good option that targets a pathological complete response (pCR) and more breast-conserving surgery. In effect, the outcomes of patients with HER2-positive BC have dramatically improved since the introduction of anti-HER2 antibodies such as trastuzumab (TZ) and/or pertuzumab (PZ) added to chemotherapy. This study sought to examine whether correlation exists between copy number variations (CNVs) in several genes related to the PI3K/AKT pathway (HER2, FGFR1, PIK3CA, AKT3 and MDM2) and the efficacy of anti-HER2 neoadjuvant treatment in patients with early HER2-positive BC. Forty-nine patients received TZ or PZ/TZ and chemotherapy as neoadjuvant treatment. Gene CNVs were determined by quantitative polymerase chain reaction on paraffin-embedded biopsy specimens. The response to 6 months of therapy was assessed by Miller–Payne grading of the tumor on surgical resection; grades 4 and 5, indicating >90% tumor reduction, were defined as a good response. A good response was shown by 64.5% and a pCR by 31.2% of patients. When stratified by anti-HER2 antibody received and gene CNV, it was found that patients with FGFR1 gene amplification or those with FGFR1 amplification treated with TZ alone showed a poor response (p = 0.024 and p = 0.037, respectively). In the subset of patients treated with TZ/PZ combined, the pCR rate was significantly lower among those showing FGFR1 amplification (p = 0.021). Although based on a small sample size, our findings suggest that patients with FGFR1 amplification might benefit less from anti-HER2 antibody therapy.

Genetic Diversity of Helicobacter pylori Isolates from Patients with Gastric Diseases in Isfahan.

Background:Helicobacter pylori (H. pylori), a spiral-shaped bacterium colonizing the human stomach, is generally acquired in childhood. This pathogen is highly diverse and can be used as genetic markers for predict the history of human migrations. This study aimed to determine the genetic diversity of H. pylori isolates from patients with dyspepsia by the multi-locus sequence typing (MLST) and update data on the prevalence of H. pylori among Iranian dyspeptic patients.Materials and Methods:In this descriptive cross-sectional study, 165 gastric biopsy specimens were obtained from patients with dyspepsia referred to Dr. Shariati Hospital of Isfahan, Iran, from April to July 2018. The status of H. pylori infection was determined by FISH in paraffin-embedded biopsy specimens. MLST of seven housekeeping genes was performed for 20 H. pylori isolates. The phylogenetic tree was plotted using CLC v8 and iTol software.Results:The overall prevalence of H. pylori infection was 53.3%. In the results of the analysis of MLST, a total of 14 new STs were recorded. The results of the global analysis showed that all the isolates, with a wide diversity, have a genetic affinity with members of the European population, such as Italy and Russia, and are in the hpEurope haplotype.Conclusion:Given the high prevalence of H. pylori infection in this region, early and accurate identification of patients seems necessary. Sequence analysis and determination of the origin of the phylogeny of strains can be effective in clinical management and monitoring of risk factors for chronic and recurrence of infection.

Effect of progestin therapy on steroid receptor status and cellular proliferation in low-grade endometrial adenocarcinoma

<h3>Objectives:</h3> Low grade endometrioid endometrial cancer is a hormone sensitive disease however little is known about the role of steroid receptors in the development or treatment of disease. Our objective was to determine if steroid receptor status, or change in receptor status, predicts response to progesterone therapy in women with early stage, low grade endometrial cancer. <h3>Methods:</h3> A retrospective cohort of women was identified who underwent primary progesterone therapy for treatment of grade 1 endometrial adenocarcinoma. Steroid receptor (SR) status as well as Ki-67 expression was assessed with immunohistochemistry on pre- and post-treatment formalin fixed paraffin embedded biopsy specimens. Automated image analysis on digitized immunostained slides was performed. Estrogen Receptor (ER), Progesterone Receptor (PR), Androgen Receptor (AR) and Glucocorticoid Receptor (GR) were scored with an H-score and Ki-67 was reported as percent positive. Clinical and pathologic variables, including response to progesterone therapy were collected. SR levels and clinical characteristics were compared using paired t- and Fischer exact tests. <h3>Results:</h3> Fourteen women were included in this analysis, the majority of whom (13/14) had a progestin IUD for treatment of their carcinoma and the majority (9/14) were non-surgical candidates due to medical co-morbidities. Eight women had resolution of adenocarcinoma and six women did not have a response to therapy. Median treatment duration was 4.8 months (range 3.3 – 9.3). Responders were more likely to be younger and pre-menopausal. Two patients had mismatch repair deficient tumors, neither responded to progestin therapy. Both ER and PR declined significantly after progestin treatment (median H-score pre vs post for ER 183 vs 104, p=0.013; PR 110 vs 40, p<.001). GR levels increased (23 vs 47, p=0.003). AR demonstrated variable response. Neither pre-treatment, post-treatment, nor change in SR levels were associated with response to progestin therapy. Women with a response to therapy had lower Ki-67 levels prior to treatment compared to women who did not respond to therapy (26% vs 39%, p=0.033). <h3>Conclusions:</h3> Both ER and PR levels declined during progestin treatment in primary low grade endometrial adenocarcinoma suggesting a negative feedback loop from PR to ER signaling at the level of protein expression. Neither pre-treatment SR levels, nor change in SR receptor status predicted a response to progestin therapy which suggests more complex context-specific interactions of SRs and their ligands. Pre-treatment cellular proliferation as a biomarker for response to hormonal therapy warrants further study.

Low-cost transcriptional diagnostic to accurately categorize lymphomas in low- and middle-income countries

Inadequate diagnostics compromise cancer care across lower- and middle-income countries (LMICs). We hypothesized that an inexpensive gene expression assay using paraffin-embedded biopsy specimens from LMICs could distinguish lymphoma subtypes without pathologist input. We reviewed all biopsy specimens obtained at the Instituto de Cancerología y Hospital Dr. Bernardo Del Valle in Guatemala City between 2006 and 2018 for suspicion of lymphoma. Diagnoses were established based on the World Health Organization classification and then binned into 9 categories: nonmalignant, aggressive B-cell, diffuse large B-cell, follicular, Hodgkin, mantle cell, marginal zone, natural killer/T-cell, or mature T-cell lymphoma. We established a chemical ligation probe-based assay (CLPA) that quantifies expression of 37 genes by capillary electrophoresis with reagent/consumable cost of approximately $10/sample. To assign bins based on gene expression, 13 models were evaluated as candidate base learners, and class probabilities from each model were then used as predictors in an extreme gradient boosting super learner. Cases with call probabilities < 60% were classified as indeterminate. Four (2%) of 194 biopsy specimens in storage <3 years experienced assay failure. Diagnostic samples were divided into 70% (n = 397) training and 30% (n = 163) validation cohorts. Overall accuracy for the validation cohort was 86% (95% confidence interval [CI]: 80%-91%). After excluding 28 (17%) indeterminate calls, accuracy increased to 94% (95% CI: 89%-97%). Concordance was 97% for a set of high-probability calls (n = 37) assayed by CLPA in both the United States and Guatemala. Accuracy for a cohort of relapsed/refractory biopsy specimens (n = 39) was 79% and 88%, respectively, after excluding indeterminate cases. Machine-learning analysis of gene expression accurately classifies paraffin-embedded lymphoma biopsy specimens and could transform diagnosis in LMICs.

Detection of coxsackievirus A6 in formalin-fixed, paraffin-embedded skin biopsy specimens using immunohistochemistry and real-time reverse-transcriptase PCR

BackgroundHand, foot, and mouth disease (HFMD), classically a childhood viral infection, has an atypical and severe clinical presentation in adults. Coxsackievirus A6 is a leading cause of atypical HFMD, but current diagnostic methods utilizing formalin-fixed, paraffin-embedded skin biopsy specimens often lack sensitivity and specificity. MethodsFormalin-fixed, paraffin-embedded skin biopsies from seven case patients with clinical and histopathological suspicion of atypical HFMD were evaluated by coxsackievirus A6 (CVA6) immunohistochemistry, enterovirus-specific conventional reverse transcriptase-PCR with subsequent Sanger sequencing targeting the 5’UTR, and CVA6-specific real-time PCR targeting the VP1 gene. ResultsThe CVA6-specific antibody demonstrated appropriate antigen distribution and staining intensity in keratinocytes in all cases. Conventional RT-PCR and sequencing also detected the presence of enterovirus, and CVA6-specific real-time RT-PCR analysis identified CVA6. ConclusionApplying these immunohistochemistry and molecular techniques to formalin-fixed, paraffin-embedded tissues, CVA6 was determined to be the causative infectious agent in seven cases of atypical hand, foot, and mouth disease.

Immunohistochemical analysis of the expression of selected cell lineage markers (CD4, CD8, CD68, c-Kit, Foxp3, CD56, CD20) in cutaneous variant of lichen planus.

Lichen planus (LP) is considered to be an immune-mediated disease of a not fully understood etiology. There are scarce data on the immune cells forming the band-like infiltration in cutaneous LP (CLP). The objective of the current study was to investigate the immunohistochemical pattern of cells forming the infiltrate in CLP by assessing the immunoexpression of selected cell lineage markers. The immunohistochemical analysis of the expression of CD4, CD8, CD20, CD56, CD68, c-Kit, and Foxp3 was performed in formalin-fixed paraffin-embedded (FFPE) biopsy specimens from 14 cases of CLP and 11 healthy volunteers. The expression of CD4 (P<0.001), CD8 (P <0.001), CD68 (P<0.001), Foxp3 (P<0.001), CD56 (v=0.019), and CD20 (P<0.001) was significantly higher in lesional skin in CLP compared to healthy controls. The ratio of CD4+ to CD8+ cells in the infiltrate was 1.75:1. The expression of Foxp3, CD56, and CD20 was markedly lower than the expression of CD4 and CD8. There was no statistically significant difference in c-Kit expression between CLP lesions and healthy skin (P=0.57). We found a wide variety of immune cells in the inflammatory infiltrate in CLP. The expression of CD4, CD8, CD68, Foxp3, CD56, and CD20 was significantly increased in CLP, while the expression of c-Kit was comparable in lesional skin and controls. The presence of various cell populations, including T regulatory cells, NK cells, and B cells, may indicate a complex pathogenesis of CLP.

Comparison of high-resolution melting analysis with direct sequencing for detection of FLT3-TKD, FLT3-ITD and WT1 mutations in acute myeloid leukemia

BackgroundAcute Myeloid Leukemia (AML) is a group of hematologic diseases characterized by a variety of clinically important genetic alterations. Genetic mutations affecting the FMS-like receptor tyrosine kinase-3 (FLT3) and Wilm's tumor (WT-1) genes are associated with poor prognosis in AML. In this work, efficiency of HRM method for detection of FLT3-ITD, FLT3-TKD, and WT-1 mutations was assessed in comparison with direct sequencing. MethodA total of 58 formalin-fixed, paraffin-embedded BM biopsy specimens of AML patients were analyzed. Mutation detection was performed by HRM method and the results were consequently compared with direct sequencing ResultsFLT3 and WT-1 mutations were detected in 21 (36.2%) and 3 (5.17%) samples, respectively. Among all FLT3 mutations, 10 (17.2%) and 11 (18.2%) samples were harboring the FLT3-ITD and-TKD gene mutations, respectively. Frequency of the FLT3-ITD was not statistically different in females (51%) and males (49%). Also, FLT3-TKD was more common in males although the differences in gender distribution were not statistically significant (P = 0.721 and P = 0.626, respectively). ConclusionsRegarded as the desirable characteristic, the present study is generally distinguished by the similar previous ones due to assessing the FFPE BM tissue from the perspective of the type of assessed sample. This discrepancy between our results and those in prior studies may be due to the disparity of the studied population size, adopted methods as well as the sample type. In this survey, regarding to low amount of extracted DNA from the paraffinized samples, the HRM method was efficient in determining the mentioned mutations.

Abstract 1580: Distinct pathways of lytic and blastic bone metastases in prostate cancer patients

Abstract The most common metastatic lesions of prostate cancer are in the bone and can be classified into three distinct pathologic subtypes: lytic, blastic, and an indeterminate mixture of both. There is no immunotherapy guidance for treating patients based on lytic or blastic tumor induced bone disease. To identify distinct tumor and stroma in lytic and blastic prostate cancer bone metastases, we investigated a cohort of decalcified formalin-fixed and paraffin-embedded (FFPE) patient bone biopsy specimens that contained metastatic prostate cancer with lytic or blastic features. Tissue sections were utilized for immunohistochemistry (IHC) staining, isolation of RNA for gene expression, and Digital Spatial Profiling (DSP) of changes in both the tumor and microenvironment. A diverse set of unique immune cell populations and signaling pathways to both lytic and blastic types of prostate cancer metastases were present. High proportions of macrophages were present in lytic tumor induced bone disease, and T cells were present in lytic and blastic samples. In blastic lesions, immune cells were enriched for pSTAT3 and components of the JAK-STAT pathway. In lytic lesions, immune cells exhibited enhanced pAKT activity and components of the PI3K-AKT pathway. Expression of key immune checkpoint targets were identified in blastic prostate cancer, providing new therapeutic targets for patients with bone metastases. To further investigate which individual cells comprising the stroma and tumor have active JAK-STAT signaling, we performed fluorescent multiplex-IHC (mIHC). A tumor and myeloid panel of antibodies was used to identify on a single cell resolution which specific cell types are enriched for pSTAT3 in the tumor microenvironment. The identification of pAKT or pSTAT3 as promising therapeutic targets could be rationally employed to individual patients. The presence of macrophages and T cells in samples suggest that conventionally “cold” prostate cancer bone metastases warrant reappraisal as capable immunotherapy responders. Biopsies could guide selection of patients into appropriate therapeutic interventions based on protein levels and RNA expression of desired targets in metastatic disease. Citation Format: Claire L. Ihle, Meredith D. Provera, Desiree M. Straign, E. Erin Smith, Susan M. Edgerton, Adrie Van Bokhoven, M. Scott Lucia, Philip Owens. Distinct pathways of lytic and blastic bone metastases in prostate cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1580.

Increased Serum Level and High Tissue Immunoexpression of Interleukin 17 in Cutaneous Lichen Planus: A Novel Therapeutic Target for Recalcitrant Cases?

Background Interleukin-17 is supposed to play an important role in the pathogenesis of oral lichen planus (OLP). However, there is scarce data in the literature on its significance in the cutaneous variant of the disease. Objectives To determine the serum level and tissue immunoexpression of IL-17 in cutaneous lichen planus (CLP). Methods Fifty-two adult patients with CLP, without any significant autoimmune or inflammatory conditions, were included in the first part of the study. The control group consisted of 27 age- and sex-matched healthy volunteers. Serum concentration of IL-17 was quantified using enzyme-linked immunosorbent assay (ELISA) kit. In the second part of the study, the tissue expression of IL-17 was assessed in archival paraffin-embedded biopsy specimens from CLP (n = 14) against normal control tissues (n = 11) using immunohistochemical assays. The expression was evaluated using Zeiss Axio Imager A2 light microscope. Positively stained cells were counted in 10 fields of view for biopsy specimen at 200x magnification, and the mean value was calculated. Results The serum level of IL-17 was significantly elevated in patients with CLP, compared with healthy volunteers (0.218 ± 0.221 ng/ml versus 0.126 ± 0.058 ng/ml, respectively; p = 0.025). No correlation was found between the serum concentration of IL-17 and patient age, gender, disease duration, extent of skin involvement, the presence or intensity of pruritus, and coexistence of mucosal lesions. In tissue samples from CLP lesions, significantly higher numbers of cells expressing IL-17 were found when compared to a healthy skin (p < 0.001). Conclusion Elevated serum concentration of IL-17 and high expression in a lesional skin support the hypothesis that IL-17 is implicated in the immunopathogenesis of CLP. These findings may constitute a premise for the future use of anti-IL-17 monoclonal antibodies in the treatment of severe and recalcitrant forms of CLP.

Development and Validation of a Deep Learning Algorithm for Gleason Grading of Prostate Cancer From Biopsy Specimens

For prostate cancer, Gleason grading of the biopsy specimen plays a pivotal role in determining case management. However, Gleason grading is associated with substantial interobserver variability, resulting in a need for decision support tools to improve the reproducibility of Gleason grading in routine clinical practice. To evaluate the ability of a deep learning system (DLS) to grade diagnostic prostate biopsy specimens. The DLS was evaluated using 752 deidentified digitized images of formalin-fixed paraffin-embedded prostate needle core biopsy specimens obtained from 3 institutions in the United States, including 1 institution not used for DLS development. To obtain the Gleason grade group (GG), each specimen was first reviewed by 2 expert urologic subspecialists from a multi-institutional panel of 6 individuals (years of experience: mean, 25 years; range, 18-34 years). A third subspecialist reviewed discordant cases to arrive at a majority opinion. To reduce diagnostic uncertainty, all subspecialists had access to an immunohistochemical-stained section and 3 histologic sections for every biopsied specimen. Their review was conducted from December 2018 to June 2019. The frequency of the exact agreement of the DLS with the majority opinion of the subspecialists in categorizing each tumor-containing specimen as 1 of 5 categories: nontumor, GG1, GG2, GG3, or GG4-5. For comparison, the rate of agreement of 19 general pathologists' opinions with the subspecialists' majority opinions was also evaluated. For grading tumor-containing biopsy specimens in the validation set (n = 498), the rate of agreement with subspecialists was significantly higher for the DLS (71.7%; 95% CI, 67.9%-75.3%) than for general pathologists (58.0%; 95% CI, 54.5%-61.4%) (P < .001). In subanalyses of biopsy specimens from an external validation set (n = 322), the Gleason grading performance of the DLS remained similar. For distinguishing nontumor from tumor-containing biopsy specimens (n = 752), the rate of agreement with subspecialists was 94.3% (95% CI, 92.4%-95.9%) for the DLS and similar at 94.7% (95% CI, 92.8%-96.3%) for general pathologists (P = .58). In this study, the DLS showed higher proficiency than general pathologists at Gleason grading prostate needle core biopsy specimens and generalized to an independent institution. Future research is necessary to evaluate the potential utility of using the DLS as a decision support tool in clinical workflows and to improve the quality of prostate cancer grading for therapy decisions.

Significance of nuclear factor - kappa beta activation on prostate needle biopsy samples in the evaluation of Gleason score 6 prostatic carcinoma indolence.

BackgroundThe goal of our study was to find out whether the immunohistochemical expression of nuclear factor-kappa beta (NF-κB) p65 in biopsy samples with Gleason score 3 + 3 = 6 (GS 6) can be a negative predictive factor for Prostate cancer (PCa) indolence.Patients and methodsStudy was conducted on a retrospective cohort of 123 PCa patients with initial total PSA ≤ 10 ng/ml, number of needle biopsy specimens ≥ 8, GS 6 on biopsy and T1/T2 estimated clinical stage who underwent laparoscopic radical prostatectomy and whose archived formalin-fixed and paraffin-embedded (FFPE) prostate needle biopsy specimens were used for additional immunohistochemistry staining for detection of NF-κB p65. Both cytoplasmic and nuclear NF-κB p65 expression in biopsy cores with PCa were correlated with postoperative pathological stage, positive surgical margins, GS and biochemical progression of disease.ResultsAfter follow-up of 66 months, biochemical progression (PSA ≥ 0.2 ng/ml) occurred in 6 (5.1%) patients, 3 (50%) with GS 6 and 3 (50%) with GS 7 after radical prostatectomy. Both cytoplasmic and nuclear NF-κB p65 expressions were not significantly associated with pathological stage, positive surgical margin and postoperative GS. Patients with positive cytoplasmic NF-kB reaction had significantly more frequent biochemical progression than those with negative cytoplasmic NF-kB reaction with PSA 0.2 ng/ml as cutoff point (p = 0.015) and a trend towards more biochemical progression with PSA ≥ 0.05 ng/ml as cutoff point (p = 0.068).ConclusionsCytoplasmic expression of NF-κB is associated with more biochemical progression and might be an independent prognostic factor for recurrence-free survival (RFS), but further studies including larger patient cohorts are needed to confirm these initial results.

Correlation between IL-10 Expression with Histopathology Type on Nasopharyngeal Carcinoma Patients

Background: Nasopharyngeal carcinoma (NPC) is a malignancy derived from lymphoepithelial tissue andnasopharyngeal epithelial cells. The response therapy of NPC evaluated from histopathological type, but insome patients with the same type was illustrated a different response of therapy. IL-10 expression expects topredict a better response therapy of NPC. IL-10 expression by immunohistochemical examination associatedwith the differentiation of tumors.Method: The study was analytic observational with the cross-sectional approach. Samples were collectedby consecutive sampling and formalin-fixed, paraffin-embedded biopsy specimens were obtained.Result: The result of IL-10 expression in Nasopharyngeal carcinoma (NPC) patients with histopathologicalWorld health organization (WHO) type I was obtained; From all the samples, there were 36.36% of strongpositive expression and 63.64% of frail positive expression. There were increases in the number of cells thatgive a strong expression on WHO type III. Statistical analysis using Fisher’s exact test was obtained p =0.040 with a contingency coefficient of 0.384.Conclusion: There was a moderate correlation between IL-10 expression and histopathological type in NPCpatients.

RNA Expression Analysis of Antibody Mediated Rejection after Heart Transplant with or without Donor Specific Antibodies

Antibody mediated rejection (AMR) is an increasingly studied cause of graft failure after heart transplantation. AMR diagnostic criteria previously required serum donor specific antibodies (DSA). However, the most recent criteria only requires pathological findings independent of DSA. This classification defined a subset of patients with AMR without known antibodies. To investigate this complication, we performed RNA sequencing using endomyocardial biopsy samples taken at the time of pathology confirmed AMR in the presence and absence of DSA. We evaluated the transcriptome profile of the paraffin-embedded endomyocardial biopsy specimens of 30 representative heart transplant recipients. Patients were categorized as having no evidence of AMR (controls), immunohistochemical findings of AMR (pAMR1i) in the presence of DSA (1i+D), pAMR1i in the absence of DSA (1i-D), histopathological findings of AMR (pAMR1h) in the presence of DSA (1h+D), pAMR1h in the absence of DSA (1h-D) or presence of both immunohistochemical and histopathological findings of AMR (pAMR2) in the presence of DSA. A total of 5 patients were analyzed per group. All were matched for previous mechanical circulatory support and time from transplant to biopsy. There was no differential RNA expression between patients with no AMR and those in the 1i-D group. There was also no differential expression between patients in the 1h+D and pAMR2 groups. The largest difference was seen between the no AMR and 1i+D groups and the no AMR and pAMR2 groups. In addition, there was RNA expression differences between patients with immunohistochemical evidence of AMR compared to those with histopathological findings, suggesting distinct mechanisms. These studies demonstrate that RNA expression profiling can be performed on endomyocardial biopsy tissue preserved in paraffin from the time of a rejection episode and that different expression patterns are observed dependent on the criteria used to diagnose AMR. In particular, AMR in the presence of DSA showed a different RNA expression profile than AMR without DSAs. The RNA expression associated with histological markers of AMR also appeared different than that associated with immunological markers. Overall, our findings suggest the existence of different subtypes of AMR that may rely on distinct mechanisms of injury.

Evaluation of Mutations in 23S rRNA, rdxA and frxA Genes of Helicobacter pylori in Paraffin-Embedded Gastric Biopsy Specimens from Iranian Gastric Cancer and Gastritis Patients.

Helicobacter pylori (H. pylori) infection is considered as one of the main cause of gastric cancer. Treatment failure of the infection often occurs due to antibiotic resistance. Herein, we aimed to evaluate the mutations in 23S rRNA gene of H. pylori which are associated with clarithromycin resistance and in rdxA and frxA genes of the bacterium which may be associated with metronidazole resistance, in paraffin-embedded gastric biopsies from patients with gastric adenocarcinoma and gastritis in Tabriz, the northwest of Iran. In the study, 80 paraffin-embedded tissue sections from 40 gastric cancer and 40 gastritis patients in the Imam Reza hospital, Tabriz, Iran were collected. The existence of ureC gene was verified by PCR method. Genotypical clarithromycin resistance was investigated by real-time PCR method and determination of the melting temperature. PCR reaction and sequencing were used for the evaluation of mutations in rdxA and frxA genes. The results of ureC amplification showed that DNA of H. pylori was present in the 82.66% of the obtained DNA samples. About 45.16% of samples were resistant to the clarithromycin and 53.22% of them were resistant to the metronidazole. Based on the results from real-time PCR, the frequency of mutations was as follow A2143G 64.28%, A2142G 44.44% and A2142C 1.11%. The mutations of rdxA gene were 66.66% missense, 30.30% frameshift and 3.03% non-sense. The mutations of frxA gene were 36.36% missense, 54.54% frameshift and non-sense 9.09%. A2143G mutation is the most frequent mutation among clarithromycin resistant genes in Iran. Also, missense and frameshift mutations are frequent in rdxA and frxA genes. Screening for these mutations could help researchers to investigate the most effective anti-H. pylori antibiotics and to prevent antibiotic resistance.