Paraffin-embedded Tissues Of Patients Research Articles

The role of miRNA in colorectal cancer diagnosis: A pilot study.

Despite recent advances in diagnosis and treatment, colorectal cancer (CRC) remains the third most common cancer worldwide, and has both a poor prognosis and a high recurrence rate, thus indicating the need for new, sensitive and specific biomarkers. MicroRNAs (miRNAs/miRs) are important regulators of gene expression, which are involved in numerous biological processes implicated in tumorigenesis. The objective of the present study was to investigate the expression of miRNAs in plasma and tissue samples from patients with CRC, and to examine their potential as CRC biomarkers. Using reverse transcription-quantitative PCR, it was revealed that miR-29a, miR-101, miR-125b, miR-146a and miR-155 were dysregulated in the formalin-fixed paraffin-embedded tissues of patients with CRC, compared with the surrounding healthy tissue, and these miRNAs were associated with several pathological features of the tumor. Bioinformatics analysis of overlapping target genes identified AGE-RAGE signaling as a putative joint regulatory pathway. miR-146a was also upregulated in the plasma of patients with CRC, compared with the healthy control group, and had a fair discriminatory power (area under the curve, 0.7006), with 66.7% sensitivity and 77.8% specificity. To the best of our knowledge, this distinct five-miRNA deregulation pattern in tumor tissue, and upregulation of plasma miR-146a, were shown for the first time in patients with CRC; however, studies on larger patient cohorts are warranted to confirm their potential to be used as CRC diagnostic biomarkers.

Clinicopathological analysis of primary thyroid non-Hodgkin lymphoma: a single-center study.

Primary thyroid lymphoma (PTL) is very rare. The aim of this study was to describe the clinical characteristics, pathological features, and survival outcomes of primary thyroid non-Hodgkin lymphoma, including the diagnostic value of clonal immunoglobulin (Ig) gene rearrangements in diagnosing mucosa-associated lymphoid tissue (MALT) lymphoma of the thyroid gland with co-existing Hashimoto thyroiditis (HT). Paraffin-embedded tissues of patients diagnosed in our institute with PTL between 2007 and 2021 were collected. Clinicopathological features and follow-up data were retrospectively analyzed. Analysis of clonal Ig gene rearrangements were performed in patients with suspected PTL. Of the 22 patients included in our study, 17 were female. The median age at diagnosis was 65.5 years (range, 44-80 years). We detected 11 cases of diffuse large B-cell lymphoma (DLBCL), 10 cases of MALT lymphoma, and 1 case of T-cell lymphoma. A total of 18 patients (81.8%) had early stage (I/II) disease. B-cell lymphoma showed diffuse positivity for CD20 but negative markers for thyroid-origin cells. Double-hit and triple-hit lymphomas were not found in the 8 DLBCL cases, and 2 cases of HT with lymphoid tissue dysplasia were diagnosed with MALT lymphoma with gene rearrangement testing. The median follow-up time was 47 months (range, 4-160 months), and the overall survival was 80% for MALT lymphoma (8 of 10), 72.7% for DLBCL lymphoma (8 of 11), and 100% for T-cell lymphoma (1 of 1). There was no significant difference between the groups. PTL mostly affects middle-aged and older adult females with HT, and its main histological type is non-Hodgkin B-cell lymphoma. For patients with HT along with a histologically benign lymphoepithelial lesion, the identification of clonal Ig gene rearrangements is important for differential diagnosis. The prognosis between DLBCL and MALT is not statistically significant.

Correlation between C-MYC and BAX expression with various Ann Arbor stages in B-cell large cell type of Non-Hodgkin lymphoma

Diffuse large B-cell lymphoma (DLBCL) is one of the B-cell large cell types of non-Hodgkin lymphoma (NHL) that has poor prognosis with highly variable clinical course. Various prognostic factors have been proposed to predict this, but the results were variable. C-MYC is a proto-oncogen that can cause overexpression leading to the increased of tumor cells proliferation. BAX is a main proapoptotic member of the BCL-2 family proteins that regulates apoptotic function. The study aimed to analyze correlation of c-MYC and BAX protein with various Ann Arbor stages in B-cell large cell type of NHL. This cross-sectional study was performed on 39 formalin fixed paraffin-embedded tissue of patients diagnosed as B-cell large cell type of NHL during January 2017 - December 2019 in Anatomical Pathology Laboratory at Dr. Soetomo General Hospital, Surabaya. To assess the expression of c-MYC and BAX, the immunohistochemistry examination was performed. Immunoexpression of C-MYC and BAX were evaluated according to the number of positive tumor cells divided by the total number of tumor cells and calculated in percentage. There was no difference in C-MYC (p = 0.877) and BAX (p = 0.093) expression with various Ann Arbor stages in B-cell large cell type of NHL. There was no correlation between c-MYC with BAX expression in various Ann Arbor stages in B-cell large cell type of NHL (rs = 0.206, p = 0.209). This indicated that C-MYC and BAX expression alone could not to be used as parameters to predict the outcome of the B-cell large cell type of NHL via Ann Arbor stages.

Correlation between CXCR4 and MMP-2 Expression with T Stage in Clear Cell Renal Cell Carcinoma

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer with high mortality. Escalation of T stage associates with worse survival. Proper and significant biomarker examination necessary to determine the predictive factor and the opportunity of targeted therapy as well. Upregulated CXCR4 expression on cancer cell promotes its aggressive growth, dissemination, metastasis, and conventional therapy resistance. Overexpression of CXCR4 may induce MMP-2 activity on tumor cell, an enzyme that degrade extracellular matrix and basal membrane particularly, which associated with tumor invasion and metastasis. Those biomarkers activity might be advantaged under hypoxic microenvironment in ccRCC. This study aimed to analyze the role and correlation of CXCR4 and MMP-2 expression with various T stages in ccRCC. Analytic observational study with cross-sectional approach was conducted on 43 formalin-fixed paraffin-embedded tissue of patients diagnosed as ccRCC in Anatomical Pathology Laboratory of Dr. Soetomo Hospital throughout January 2015 until December 2020. CXCR4 and MMP-2 expression were evaluated by immunohistochemistry. Statistical analysis with Kruskal-Wallis and Spearman test were utilized to analyze the expression difference and correlation. Difference of both CXCR4 (p=0.016) and MMP-2 (p=0.029) expression were obtained on various T stages. There was a significant positive correlation between CXCR4 expression (p=0.001) and MMP-2 expression (p=0.002) with T stage, also between CXCR4 with MMP-2 expression in various T stage of ccRCC (p=0.000). In conclusion, this study exhibited that CXCR4 and MMP-2 expression showed synergistic and positive correlation with T stage and might be considered as the basis of targeted therapy in ccRCC.

The significance of immunoexpression of Polo Like Kinase 1 (PLK1) in retinoblastoma

Background: Retinoblastoma is an intraocular malignant neoplasm of retinal neurosensory origin and often occurs in childhood. The average global survival rate is less than 50%. An increase in the pT stage is associated with worse survival. Through its distinct protein-binding and phosphorylation activities, Polo Like Kinase 1 (PLK1) is essential in mitotic activity. PLK1 protein expression has been proposed as a novel prognostic marker for many cancers. This study aims to analyze the correlation between PLK1 with pT stages of retinoblastoma. Methods: An observational analytic study with a cross-sectional approach was conducted on 56 formalin-fixed paraffin-embedded tissue of patients diagnosed with retinoblastoma from January 2016 - December 2020 in Dr. Soetomo General Academic Hospital, Surabaya. The samples were divided into four groups based on pT-stage retinoblastoma. Immunohistochemistry for PLK1 was applied to all models and evaluated semi-quantitatively using an immunoreactivity score. Results: There were significant differences in PLK1 expression in each pT stage of retinoblastoma (p=0.000014). There was a significant correlation between PLK1 and the pT stage of retinoblastoma (p=0.0001). Conclusion: The increase of PLK1 expression in retinoblastoma is associated with an increased pT stage. PLK1 plays a role in the proliferation of retinoblastoma tumor cells, hence increasing the pT stage.

Comparison of 3 PCR-based assays for microsatellite instability detection in formalin-fixed paraffin-embedded tissues of patients with colorectal cancer.

e15042 Background: Routine assessment of microsatellite instability (MSI) status in colorectal cancer (CRC) patients can help with initial screening for Lynch syndrome, prediction of tumor prognosis and selection of patients for immunotherapy. Immunohistochemistry (IHC) and PCR-based approaches represent the current clinical laboratory standard for the evaluation of MSI status. Both Bethesda and Promega marker panels have been used widely in the world and the Bethesda panel and the six mononucleotides panel (NR21/BAT25/NR24/NR27/MONO27/BAT26) have been approved by the National Medical Products Administration (NMPA) in China. The Idylla MSI Test, a fully automated system based on real-time PCR, which incorporates 7 novel MSI loci (ACVR2A/BTBD7/DIDO1/MRE11/RYR3/SEC31A/SULF2) can perform MSI analysis within 150min using 1-3 tissue slides. The aim of this study was to evaluate the performance of the three 3 PCR-based assays for MSI detection including Idylla MSI Test, Bethesda and that six mononucleotides panel. Methods: One hundred and fifty-three formalin-fixed paraffin-embedded (FFPE) tumor tissues from patients with CRC were collected and their MSI status were analyzed using 3 PCR-based assays, respectively, i.e. the Idylla MSI, “2B3D” NCI panel and the six mononucleotides panel. All samples were previously evaluated by IHC method. Results: In the all 153 patients, IHC identified 27 cases to be MMR-deficient (dMMR), and 126 to be MMR-proficient (pMMR). Using IHC as a reference, the Idylla MSI had a higher consistency that showed overall agreement, sensitivity and specificity as 93.46%, 92.89% and 93.65%, respectively, while the corresponding values of the six mononucleotides panel were 84.31%, 92.59% and 82.54% respectively, and the “2B3D” NCI panel showed a sensitivity of 92.16%, a specificity of 88.89% and an overall concordance of 92.86%. The PCR-based approaches identified 11 more MSI-H cases than IHC while 22 dMMR cases identified by IHC were detected by all the 3 assays, 2 dMMR by 2 assays, 1 dMMR by one assay only and 1 dMMR by neither assays above. Moreover, the Idylla MSI showed advantages in ease of use, turnaround time (TAT) and small dose of tissue input. Conclusions: Our findings support that the Idylla MSI assay provides a rapid, reliable, and ease-to-use solution to MSI detection in Chinese CRC patients with high sensitivity and specificity. The inconsistent cases between IHC and PCR-based approaches need a further analysis.

Evaluation of gene expression levels in the diagnosis of lung adenocarcinoma and malignant pleural mesothelioma.

This study aims to evaluate gene expression levels in the diagnosis of lung adenocarcinoma and malignant pleural mesothelioma both which have a distinct treatment and prognosis. Between January 2012 and January 2014, 12 newly diagnosed patients with a lung adenocarcinoma, 12 patients with malignant pleural mesothelioma, and eight healthy individuals as the control group were included. After treatment of the fresh samples of lung adenocarcinoma stored at -80°C for ribonucleic acid isolation, and paraffin-embedded tissues of patients with malignant pleural mesothelioma were deparaffinized, complementary deoxyribonucleic acid synthesis and expression of 84 genes associated with deoxyribonucleic acid repair were analyzed via real-time polymerase chain reaction assay. According to the expression of tumor cells, expression of each fold change was calculated. The BRCA1, BRCA2, CDK7, MLH3, MSH4, NEIL3, SMUG1, UNG, XRCC2, and XRCC4 genes showed more than five-fold higher expression in the patients with lung adenocarcinomas, compared to the control group. The patients with malignant pleural mesothelioma showed a five-fold higher expression in the APEX2, BRCA1, BRCA2, CDK7, MLH1, MLH3, MSH3, MSH4, NEIL3, PARP2, PARP3, PMS1, RAD50, RAD51, RAD51B, RAD51D, RAD52, RPA3, SMUG1, UNG, XPA, XRCC2, and XRCC4 genes, compared to the control group. Comparing malignant pleural mesothelioma with lung adenocarcinoma cases, we found that CDK7, MLH1, TREX1, PRKDC, XPA, PMS1, UNG, and RPA3 genes were overexpressed. Our study results showed differences between expression profiles of deoxyribonucleic acid repair genes in lung adenocarcinoma and malignant pleural mesothelioma cells. Based on our study results, we suggest that TREX1, PRKDC, and PMS1 genes may play a key role in the differential diagnosis of these two entities.

NOX1 Regulates Collective and Planktonic Cell Migration: Insights From Patients With Pediatric-Onset IBD and NOX1 Deficiency.

Genetic defects of pediatric-onset inflammatory bowel disease (IBD) provide critical insights into molecular factors controlling intestinal homeostasis. NOX1 has been recently recognized as a major source of reactive oxygen species (ROS) in human colonic epithelial cells. Here we assessed the functional consequences of human NOX1 deficiency with respect to wound healing and epithelial migration by studying pediatric IBD patients presenting with a stop-gain mutation in NOX1. Functional characterization of the NOX1 variant included ROS generation, wound healing, 2-dimensional collective chemotactic migration, single-cell planktonic migration in heterologous cell lines, and RNA scope and immunohistochemistry of paraffin-embedded patient tissue samples. Using exome sequencing, we identified a stop-gain mutation in NOX1 (c.160C>T, p.54R>*) in patients with pediatric-onset IBD. Our studies confirmed that loss-of-function of NOX1 causes abrogated ROS activity, but they also provided novel mechanistic insights into human NOX1 deficiency. Cells that were NOX1-mutant showed impaired wound healing and attenuated 2-dimensional collective chemotactic migration. High-resolution microscopy of the migrating cell edge revealed a reduced density of filopodial protrusions with altered focal adhesions in NOX1-deficient cells, accompanied by reduced phosphorylation of p190A. Assessment of single-cell planktonic migration toward an epidermal growth factor gradient showed that NOX1 deficiency is associated with altered migration dynamics with loss of directionality and altered cell-cell interactions. Our studies on pediatric-onset IBD patients with a rare sequence variant in NOX1 highlight that human NOX1 is involved in regulating wound healing by altering epithelial cytoskeletal dynamics at the leading edge and directing cell migration.

Comparison detection methods for EGFR in formalin-fixed paraffin-embedded tissues of patients with NSCLC

Epidermal growth factor receptor (EGFR) is an important gene in the development of lung cancer. Non-small cell lung cancer (NSCLC) is the most common lung cancer. In the present study, the expression of EGFR in 717 patients with NSCLC was detected by Ventana automatic immunohistochemical technique, and the samples was verified by Real-time PCR, and then the results were compared with the data acquired by next-generation sequencing technology (NGS), which is the high throughput, multiple sites for EGFR gene mutation testing. The expression of Ventana EGFR in 717 cases of NSCLC was detected by immunohistochemistry, and the positive rate was 60.70 % (435 / 717). The mutation rate of EGFR was 57.60 % (413/717). The coincidence rate of Ventana EGFR immunohistochemical assay and Real-time PCR assay reached 94.94 %, and the two had high consistency. The coincidence rate of Ventana EGFR immunohistochemical assay and NGS were high correlation. Based on these results, Ventana EGFR automatic immunohistochemical detection has high accuracy, simple operation process, low price and easy interpretation. It can be used as the preferred method for EGFR detection.

NRP-1 and KDR polymorphisms are associated with survival time in patients with advanced gastric cancer.

Neuropilin-1 (NRP-1), a member of the NRP-family, has been reported to be vital for tumor angiogenesis, growth and metastasis. As a co-receptor of vascular endothelial growth factor (VEGF), NRP-1 can bind to VEGF and meditate vascular development through the VEGF-VEGF receptor 2 (VEGFR2) signaling pathway. Furthermore, NRP-1 is capable of binding with platelet-derived growth factor (PDGF) to regulate the PDGF-PDGF receptor (PDGR) signaling pathway in tumor angiogenesis. In the present study, The DNA was obtained from the paraffin-embedded tissues of patients with advanced gastric cancer (AGC), amplified using PCR and subsequently sequenced to determine the polymorphisms within NRP-1, VEGFR2 [kinase insert domain receptor (KDR)] and PDGF. The effect of the functional polymorphism of the aforementioned genes on the overall survival (OS) and progression-free survival (PFS) of 81 patients with advanced gastric cancer was examined. Three single nucleotide polymorphisms (SNPs) of KDR were significantly associated with clinical outcomes. The rs1870377 TT genotype was positively associated with longer OS and PFS times compared with the AA+AT genotype (PFS, P=0.012; OS, P=0.038), the rs7692791 wild-type TT genotype was positively associated with longer PFS time and the rs2034965 AA+GA genotype was associated with shorter OS time (P=0.034). With regards to the SNPs of NRP-1, the rs2065364 AA genotype was significantly associated with improved OS and PFS times (PFS, P=0.023; OS, P=0.045). Following multivariate analysis using Cox proportional hazards regression models, patients with the KDR rs7692791 TT genotype experienced a longer PFS time compared with those with the CT genotype (P=0.016), and patients with the NRP-1 rs2065364 variant-type AA genotype still experienced a longer PFS time compared with those patients with the AG+GG genotypes (P=0.006). Regarding OS, the results demonstrated that the KDR rs2034965 AG+GG genotypes presented with a significant reduction in OS time (P=0.029), and that the KDR rs1870377 AT+AA genotypes had worse OS times compared with the wild-type TT genotype (P=0.021). In addition, increased mortality risk and AGC progression were significantly associated with the number of adverse alleles for combinations of NRP-1 rs2065364 and KDR rs1870377. In conclusion, the data from the present study demonstrated that the selected KDR and NRP-1 gene polymorphisms may be potential prognostic biomarkers in AGC.

EGFR T790M detection in formalin-fixed paraffin-embedded tissues of patients with lung cancer using RNA-based in situ hybridization: A preliminary feasibility study.

BackgroundFollowing drug resistance in patients with lung cancer treated by EGFR TKIs, a biopsy is required to obtain sufficient cancer tissue for T790M detection in order to select potential beneficiaries suitable for third‐generation EGFR TKIs, such as osimertinib. The purpose of this study was to explore the feasibility of using a new in situ analysis technique based on RNA target sequences to detect EGFR T790M in lung cancer.MethodsA total of 28 formalin‐fixed paraffin‐embedded (FFPE) samples from 24 lung adenocarcinoma patients archived in Peking Union Medical College Hospital from 2015 to 2016 were collected. The BaseScope T790M detection technique by in situ hybridization on FFPE slides was used to analyze the mutation of EGFR T790M, and then the results were compared with the data acquired by Scorpions ARMS assay, which is the so‐called gold standard for EGFR gene mutation testing. The sensitivity and specificity of the BaseScope T790M detection technique were preliminarily evaluated.ResultsOf the 28 FFPE specimens, the average proportion of T790M‐positive cells was 35.78% ± 17.68% in 18 samples with EGFR T790M, confirmed by Scorpions ARMS assay, Compared with real‐time PCR assay, the sensitivity and specificity of BaseScope T790M were all 100% in our cohort.ConclusionBaseScope T790M assay could be completed on only one FFPE slide and the visualized molecular result overplayed with histomorphological information perfectly, so it may be the alternative method for EGFR T790M evaluation. BaseScope assay has potential clinical utility, and it will be necessary to carry out validation with a large number of cases.

Increasing Heme Oxygenase-1-Expressing Macrophages Indicates a Tendency of Poor Prognosis in Advanced Colorectal Cancer

Background: Many cancers express heme oxygenase-1 ­(HO-1) at a higher frequency than healthy tissues, and this elevated expression is associated with cancer prognosis. Here, we aim to clarify the correlation between HO-1-expressing macrophage numbers and clinicopathological parameters of advanced colorectal cancer. Materials and Methods: Formalin-fixed and paraffin-embedded tissues of patients with advanced colorectal cancer were used. To detect HO-1 expression in macrophages, immunohistochemistry was performed. The number of positive cells was measured. Furthermore, HO-1 mRNA in colorectal cancer was examined by reverse transcription polymerase chain reaction. Results: Among the HO-1-negative and HO-1-positive groups, 58.02 and 85.00% of cases, respectively, were positive for lymph node metastasis. The disease-free survival (DFS) time was significantly shorter (p < 0.05) in the ­HO-1-positive group (2.44 years) than in the HO-1-negative group (4.09 years). However, according to the Cox proportional-hazards regression model, the HO-1-positive group could not be a risk factor of poor prognosis. HO-1 mRNA expression was confirmed in colorectal normal and cancer tissues. Conclusion: In this study, the correlation between HO-1-expressing macrophages and clinicopathological parameters in the tumor microenvironment of colorectal cancer was studied for the first time, and the expression was associated with lymph node metastasis and shortening of DFS.

Predictive Value of Transforming Growth Factor-α and Ki-67 for the Prognosis of Skull Base Chordoma

We aimed to characterize the expression of transforming growth factor-α (TGF-α) and Ki-67 and to assess the relationship between TGF-α and Ki-67 expression and prognostic factors in skull base chordoma. We retrospectively analyzed the data from 46 patients with skull base chordoma. The follow-up duration ranged from 1 to 168 months (mean, 74.1). The survival data were statistically analyzed using the Kaplan-Meier method and multivariate Cox regression analysis. The expression of TGF-α and Ki-67 were detected by immunohistochemical staining of paraffin-embedded patient tissue specimens. The total resection (TR) group had longer overall survival compared with the non-TR group (P=0.042). The TR group also had longer progression-free survival (PFS) than did the non-TR group (P= 0.046). The group with a high Ki-67 labeling index (Ki-67LI) had shorter overall survival than did the group with a low Ki-67LI (P=0.039). Also, the group with a high Ki-67LI had significantly shorter PFS than did the group with a low Ki-67LI (P= 0.016). Moreover, the group with high TGF-α expression had significantly shorter PFS compared with the group with low TGF-α expression (P= 0.005). Our results have shown that high levels of TGF-α and Ki-67 are associated with shorter PFS in patients with chordoma. We have confirmed the role of Ki-67 as a functional molecular marker of poor prognosis. We also identified TGF-α as a potential novel biomarker for predicting prognosis for patients with skull base chordoma.

Identifying Mcl-1 protein dependencies using dimerization-specific antibody biomarker for predicting response to targeted apoptosis inducing therapies.

7038 Background: The anti-apoptotic Bcl-2 family proteins facilitate pro-survival and resistance to anti-cancer therapies. Measuring the function of these proteins has shown utility in predicting response to therapy. A method that expands the principle of BH3 profiling to solid tumors is presented. Measuring the occurrence of heterodimers of Myeloid Leukemia Cell Differentiation Protein (Mcl-1), and pro-apoptotic binding protein Bim is an indicator of cancer cell apoptotic priming state. The readout of Mcl-1 containing complex-specific biomarkers can identify survival dependencies in cancer cells potentially providing clinical utility in guiding cancer treatments. Methods: Engineered immunogens that recapitulate conformation-specific epitopes induced during binding of the Mcl-1/Bim protein complex were used to generate. monoclonal antibodies. One Heterodimer Specific Mcl-1 Bim (HsMcB) was chosen. The selective binding was confirmed using ELISA, fluorescence polarization, immunofluorescence microscopy and flow cytometry in Bim or Mcl-1 knockdown cells, and by immunohistochemistry in formalin-fixed paraffin-embedded patient tissue. Correlation of HsMcB measurements to BH3 profiling readouts from the Mcl-1 restricted Noxa peptide was explored. Results: HsMcB signal depends on both Mcl-1 and Bim protein levels. The ratio of the HsMcB to unbound protein (Mcl-1) signal ([HsMcB]/[Mcl-1]) was measured. Disruption of the complexes by BH3 mimetics targeted to Mcl-1 and depletion of Mcl-1 level using CDK9 inhibitors diminished the [HsMcB]/[Mcl-1] readout. We see correlations between these readouts and BH3 mimetic peptides readouts from the Mcl-1 specific Noxa and MS1 BH3 mimetic peptides in AML patient samples that have been treated with Mcl-1 inhibitors. Conclusions: Mcl-1 dependence is a predictive biomarker for venetoclax resistance and for response to Mcl-1 targeted therapies. Flow cytometric and IHC based measurements of a heterodimer complex offer a direct and simpler approach that harbors potential for use in clinical settings. Additional antibodies targeting Mcl-1/Bak and Mcl-1 Noxa complexes are being tested.

Establishment of a biobank for human lung tissues of congenital diaphragmatic hernia and congenital pulmonary airway malformation

BackgroundHuman tissue samples are an invaluable and little available source of information for translational studies of congenital lung diseases such as Congenital Diaphragmatic Hernia (CDH) or Congenital Pulmonary Airway Malformation (CPAM). PurposeWe aimed to establish a human lung tissue biobank of CDH and CPAM patients together with age-matched controls, coupled with a clinical database. MethodsPathology records from autopsies or surgical specimens for CDH and CPAM cases between 1980 and 2017 were reviewed. For surviving individuals, clinical patient data was obtained from corresponding pediatric surgery reports. Formalin-fixed, paraffin-embedded tissues of patients and age-matched controls were systematically stored for further translational studies. RNA integrity was determined on selected CDH blocks. ResultsA total of 16 CDH and 18 CPAM and age-matched control lung tissue blocks were included in our biobank. Ages ranged from 22 to 41 weeks of gestation (GA) in CDH (33.9 ± 6.35 weeks) and 26 weeks (GA) and 12 years in CPAM (2.3 ± 3.7 y). RNA isolation from CDH and control blocks yielded good RNA quality (OD 260/280 ratio: 2.01–2.09, OD 260/230 ratio: 2.04–2.09). ConclusionWe established a unique human biobank for CDH and CPAM tissues. The combination with clinical patient data will allow us to design future translational studies to improve our understanding of the disease pathogenesis of these congenital malformations.

Frequency of Epstein–Barr Virus DNA in Formalin-Fixed Paraffin-Embedded Tissue of Patients with Ductal Breast Carcinoma

Background:Ductal carcinoma is one of the most common breast cancer (BrC) among the women in the world. Several factors may involve in establishment of breast cancer. The role of viral infections have been investigated in BrC, Among them the association of Epstein Barr virus have been reported in the patients with breast cancer type ductal carcinoma. Thus this study was conducted to evaluate the rate of Epstein Barr virus in women with breast cancer type ductal carcinoma.Material and methods:A total of 72 formalin-fixed paraffin-embedded tissue blocks samples were collected from 37 (51.38%) women with breast cancer type ductal carcinoma and 35 (48.61%) samples of breast with fibro adenoma as control group. The DNA was extracted for all the samples. The detection of EBNA 3C EBV DNA was done by nested PCR. The results of positive were sequenced to confirm PCR product and determine EBV genotypes.Results:About 10/37 (27.02%) samples of ductal breast carcinoma were showed positive for EBNA 3C EBV DNA while 4/35 (11.42%) of fibro adenoma were positive for EBNA 3C EBV DNA (p= 0.095). Randomly 7 PCR products were sequenced and the results of sequencing EBNA 3C shows, the detected EBVDNA were type 1 EBV type.Conclusion:This study shows high prevalence of 27.02% EBV DNA type 1 was found in formalin-fixed paraffin-embedded tissue of Patients with ductal breast carcinoma. The outcomes of this study suggesting that EBV might have a significant role in breast cancer in Ahvaz city, south west region of Iran. However the expression of EBV oncoproteins, EBNA1, LMP1, and LMP2 require to be determined with ductal carcinoma cells. About 72.97% breast samples showed negative for EBVDNA. The role other viruses including Human cytomegalovirus, papilloma viruses and Merkel viruses are required to be investigated in further studies.

Relationship between expressions of OCT-4, CD117 and clinicopathological features and prognosis of patients with ovarian cancer

Objective To investigate the relationship between expressions of OCT-4, CD117 and clinicopathological features and prognosis of patients with ovarian cancer. Methods A total of 70 paraffin-embedded tissues of patients with ovarian cancer from January 2010 to February 2016 in Shanxi Provincial Cancer Hospital were collected. The expressions of OCT-4 and CD117 were detected by immunohistochemistry. Results OCT-4 was mainly expressed in cytoplasm, while CD117 was expressed in cell membrane and cytoplasm. The positive expression rate of OCT-4 was 74.3% (52/70), and the positive expression rate of CD117 was 68.6% (48/70). The positive expression rates of OCT-4 in ovarian cancer tissues with poorly differentiation and high CA125 levels (≥500 U/ml), no peritoneal effusion and sensitive to chemotherapy drugs were 92.1% (35/38), 87.5% (28/32), 88.9% (24/27), and 78.7% (48/61), respectively, which were higher than those in ovarian cancer tissues with well and moderately differentiation, low CA125 levels (<500 U/ml), peritoneal effusion and resistance to chemotherapy drugs, the differences were statistically significant (P values were 0.000, 0.020, 0.047, and 0.043). The positive expression rates of CD117 in ovarian cancer tissues with poorly differentiation and peritoneal effusion were 84.2% (32/38) and 79.1% (34/43), respectively, which were higher than those in ovarian cancer tissues with well and moderately differentiation and no peritoneal effusion, the differences were statistically significant (P values were 0.006 and 0.017). Patients with OCT-4-positive expression, peritoneal effusion, and poorly differentiation had a shorter overall survival time (all P < 0.05). The peritoneal effusion and differentiation were independent prognostic factors in patients with ovarian cancer (both P < 0.05). Conclusion OCT-4 can be used as an important reference for predicting drug sensitivity and evaluating prognosis in patients with ovarian cancer. Key words: Ovarian neoplasms; OCT-4; CD117; Immunohistochemistry; Prognosis

Clinical utility of immunohistochemistry using the novel anti-BRAF V600E antibody (clone RM8) for detection of the BRAF V600E mutant protein in papillary thyroid cancers

&lt;p&gt;&lt;strong&gt;Background:&lt;/strong&gt;&lt;strong&gt; &lt;/strong&gt;Molecular markers are gaining increasing importance as diagnostic and prognostic tools in patients with well differentiated thyroid cancers and BRAF V600E mutation has received wide attention in this regard &lt;strong&gt;Aim: &lt;/strong&gt;To evaluate the clinical value of immunohistochemistry (IHC) using anti-BRAF V600E antibody (clone RM8) for detection of the BRAF V600E mutant protein in formalin-fixed and paraffin-embedded tissues of patients with papillary thyroid carcinomas. &lt;strong&gt;Materials and Methods: &lt;/strong&gt;Patients who were managed for well differentiated thyroid cancers (&lt;em&gt;n &lt;/em&gt;= 79) during the years 2005 and 2006 were included in the study. We evaluated the fidelity of the RM8 antibody specific for the BRAF V600E and compared its detection accuracy to real time polymerase chain reaction (PCR), which was taken as the gold standard. &lt;strong&gt;Results: &lt;/strong&gt;Mutant BRAF V600E antibody was studied in 79 tissue sections, out of which 21 (26.5%) had staining for BRAF V600E in &amp;gt;20% of the tumour cells and these were considered positive. The BRAF staining was moderate in 10 (47.6%), strong in 9 (42.5%) and very strong in 2 (9.5%) of sections stained. There was a statistically significant concordance (&lt;em&gt;P &lt;/em&gt;= 0.000) with quantitative PCR (qPCR) for BRAF mutant taken as standard. (Kappa agreement: 0.881) Further, the receiver operating characteristics (ROC) curve showed that IHC can be used as a comparable standard to the qPCR. The highest possible sensitivity of 92% and specificity of 92.6% could be achieved by considering the cytoplasmic positivity of &amp;gt;20% of cells with moderate to strong intensity (AUC = 0.923) &lt;strong&gt;Conclusion: &lt;/strong&gt;Our study has shown that BRAF V600E IHC can be done in a conventional manner using rabbit monoclonal antibody RM8 on formalin-fixed and paraffin-embedded tissues of patients with papillary thyroid carcinomas. With a comparable diagnostic accuracy to the gold standard qPCR testing and with an added advantage of being cost effective, this technology can be considered for use as a first-line method for detection of BRAF V600E mutations, especially in resource constrained settings.&lt;/p&gt;

CYP2C19*2 status in patients with Stevens-Johnson syndrome and toxic epidermal necrolysis.

PurposeGenetic polymorphisms have been linked to an increased predisposition to developing certain diseases. For example, patients of Han-Chinese descent carrying the HLA-B*1502 allele are at an increased risk of developing Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) if given carbamazepine. Given the complexity of in vivo drug metabolism, it is plausible that the activity of enzyme systems unrelated to specific drug metabolism may be important. Although multiple biomarkers have been identified in unique ethnic groups, there has yet to be a study investigating the presence of the slow metabolizing allele of CYP2C19, denoted CYP2C19*2, in diverse groups and the risk of developing SJS/TEN.Patients and methodsThis study looked into the carrier status of CYP2C19*2, a poor metabolizing variant of CYP2C19, in patients diagnosed with SJS/TEN. We looked at its status in our series as a whole and when patients were divided by ethnicity. Genomic DNA was extracted from formalin-fixed paraffin-embedded tissue of patients with biopsy-proven SJS/TEN and real-time polymerase chain reaction was used to assess for the presence of CYP2C19*2.ResultsCYP2C19*2 status was determined in 47 patients. Twenty-nine of these 47 patients had a single medication implicated as causing their disease, and eight of these patients were heterozygous or homozygous for CYP2C19*2. There was insufficient evidence to conclude that the presence of CYP2C19*2 is an independent predictor of risk for developing SJS/TEN in our series as a whole. This analysis also confirmed that the frequency of the CYP2C19*2 polymorphism within the different ethnicities in our series did not vary statistically from reported ethnic rates.ConclusionOur study was unable to show a relationship between CYP2C19*2 status and predisposition toward SJS/TEN. We had a heterogeneous population, making it difficult to control for possible confounding factors.

Proof of the quantitative potential of immunofluorescence by mass spectrometry

Protein expression in formalin-fixed, paraffin-embedded patient tissue is routinely measured by Immunohistochemistry (IHC). However, IHC has been shown to be subject to variability in sensitivity, specificity and reproducibility, and is generally, at best, considered semi-quantitative. Mass spectrometry (MS) is considered by many to be the criterion standard for protein measurement, offering high sensitivity, specificity, and objective molecular quantification. Here, we seek to show that quantitative immunofluorescence (QIF) with standardization can achieve quantitative results comparable to MS. Epidermal growth factor receptor (EGFR) was measured by quantitative immunofluorescence in 15 cell lines with a wide range of EGFR expression, using different primary antibody concentrations, including the optimal signal-to-noise concentration after quantitative titration. QIF target measurement was then compared to the absolute EGFR concentration measured by Liquid Tissue-selected reaction monitoring mass spectrometry. The best agreement between the two assays was found when the EGFR primary antibody was used at the optimal signal-to-noise concentration, revealing a strong linear regression (R2=0.88). This demonstrates that quantitative optimization of titration by calculation of signal-to-noise ratio allows QIF to be standardized to MS and can therefore be used to assess absolute protein concentration in a linear and reproducible manner.