Chromogenic In Situ Hybridization Research Articles

ANXA2 expression in African American triple-negative breast cancer patients

Our aim was to determine the role of Annexin A2 (AnxA2), which we have previously found to contribute to the aggressiveness of TNBC, with AA TNBC patients and clinical outcome. We analyzed TCGA breast cancer database (n = 1098) to observe AnxA2 expression within breast cancer subtypes and is correlation with overall survival. Further, we examined breast tissue specimens (n = 119) through chromogenic in situ hybridization (CISH) and specimen were scored independently by two pathologists in a blinded study. In our TCGA analysis, high expression of AnxA2 was correlated with poor survival in patients with TNBC. AnxA2 gene expression was not correlated with poor survival in other breast cancer subtypes. AnxA2 average CISH intensity score (CISH score = 0, null expression to 3, high expression) for TNBC was significantly higher in comparison to estrogen receptor and/or progesterone receptor positive, human epidermal growth factor positive, and non-malignant tissues. Furthermore, AnxA2 average score was significantly higher in AA TNBC patients (CISH average score = 2.45 ± 0.3266) in comparison to Caucasian TNBC patients (CISH average score = 1.1 ± 0.4069). AnxA2 is overexpressed in TNBC, implicating AnxA2 as a contributor to the aggressive biology of TNBC in AA women.

Evaluating HER2 Gene Amplification Using Chromogenic In Situ Hybridization (CISH) Method In Comparison To Immunohistochemistry Method in Breast Carcinoma.

BACKGROUND:In patients with breast cancer, HER2 gene expression is of a great importance in reacting to Herceptin treatment. To evaluate this event, immunohistochemistry (IHC) has been done routinely on the basis of scoring it and so the patients were divided into 4 groups. Lately, as there have been disagreements about how to treat score 2 patients, chromogenic in situ hybridization (CISH) and florescence in situ hybridization (FISH) are introduced. Since CISH method is more convenient than FISH for gene amplification study, FISH has been substituted by CISH.AIM:The current study is conducted in order to investigate whether using CISH is a better method comparison to IHC method for determines HER2 expression in patients with breast cancer in.METHODS:In this cross-sectional descriptive analytical study, information of 44 female patients with invasive ductal breast cancer were gathered from Imam Reza and Omid Hospital in Mashhad. IHC staining was done for all patients in order to determine the level of HER2 expression, and after scoring them into 4 groups of 0, +1, +2 and +3, CISH staining was carried out for all 4 groups. At the end, results from both methods were statistically evaluated using SPSS software V.22.0.RESULTS:The average age of patients was 50.2 with the standard deviation of 10.96. Using IHC method was observed that 2.6% (1 patient), 26.3% (10 patients), 65.8% (25 patients) and 5.3% (2 patients) percentage of patients had scores of 0, +1, +2 and +3. On the other hand, CISH method showed 36 patients (90%) with no amplifications and 4 (10%) with sever amplifications. In a comparative study using Fisher’s exact test (p = 0.000), we found a significant relation between IHC method and CISH method indicating that all patients showing severe amplifications in CISH method, owned scores of +2 and +3 in IHC method.CONCLUSION:According to the present study and comparing the results with similar previous studies, it can be concluded that CISH method works highly effective in determining HER2 expression level in patients with breast cancer. This method is also able to determine the status of patients with score +2 in IHC for their treatment with herceptin.

Phosphaturic mesenchymal tumor of the nasal cavity: Clinicopathologic correlation is essential for diagnosis

Phosphaturic mesenchymal tumor (PMT) is a rare neoplasm in which the tumor cells produce fibroblast growth factor 23 (FGF23), leading to oncogenic osteomalacia and thus a distinct clinical presentation. However, the pathologic findings of PMT are often non-specific and variable, especially in tumors occurring in the head and neck. We present a case of a 66-year-old female who presented with osteomalacia-related symptoms and was found to have a nasal cavity mass. Histopathologic examination was suggestive of PMT but certain characteristic features were lacking, requiring confirmation of the diagnosis by chromogenic in situ hybridization (CISH) assay for FGF23 mRNA. The patient's symptoms and laboratory abnormalities resolved upon resection of the tumor.

Haploid Germ Cells Generated in Organotypic Culture of Testicular Tissue From Prepubertal Boys.

While in mice various studies have described the completion of spermatogenesis in vitro using either organotypic culture of prepubertal testicular tissue or 3D culture of isolated cells, in humans it has not been possible to achieve germ cell differentiation from immature testicular tissue (ITT). In our study, we evaluated the ability of human ITT to differentiate via a long-term organotypic culture of frozen–thawed 1 mm3 testicular fragments from five prepubertal boys in two different culture media. Tissue and supernatants were analyzed at regular intervals up to day 139. Sertoli cell (SC) viability and maturation was evaluated using immunohistochemistry (IHC) for SOX9, GDNF, anti-Mullerian hormone (AMH) and androgen receptor (AR), and AMH concentration in supernatants. Spermatogonia (SG) and proliferating cells were identified by MAGE-A4 (for SG) and Ki67 (for proliferating cells) via immunohistochemistry (IHC). Apoptotic cells were studied by active caspase 3. To evaluate Leydig cell (LC) functionality testosterone was measured in the supernatants and steroidogenic acute regulatory protein (STAR) IHC was performed. Germ cell differentiation was evaluated on Hematoxylin-Eosin histological sections, via IHC for synaptonemal complex 3 (SYCP3) for spermatocytes, Protein boule-like (BOLL) for spermatocytes and round spermatids, angiotensin-converting enzyme (ACE), protamine 2 and transition protein 1 (for elongated spermatids) and via chromogenic in situ hybridization (CISH). We reported the generation of meiotic and postmeiotic cells after 16 days of culture, as shown by the histological analyses, the presence of differentiation markers and the increase of haploid germ cells. We showed SC viability and maturation by a decrease of AMH secretion in the supernatants (p ≤ 0.001) while the number of SOX9 positive cells did not show any variation. A decrease of spermatogonia (p ≤ 0.001) was observed. The number of apoptotic cells did not vary. LC functionality was shown by the increase in STAR expression (p ≤ 0.007) and a peak in testosterone secretion, followed by a reduction (p ≤ 0.001) with stabilization. According to our knowledge, this is the first report of generation of haploid cells in human ITT. Differentiating germ cells have to be further evaluated for their ability to complete differentiation, their fecundability and epigenetic characteristics.

Abstract 5136: Development of an RNA hybridization technique for the in situ visualization of bacteria in cancer tissues

Abstract There is currently a great interest in understanding direct interactions between human-associated microbial populations and the tumor microenvironment. In this study, we aimed to develop a chromogenic in situ hybridization (CISH) assay for universal detection of bacteria in formalin fixed paraffin embedded (FFPE) cancer tissues. This was accomplished using the RNAscope assay (Advanced Cell Diagnostics) with a CISH probe set designed to target the most highly conserved region in the bacterial 16S ribosomal RNA (rRNA) gene. Optimization of the 16S rRNA CISH assay was conducted using FFPE mouse colon, Mycobacterium tuberculosis infected rabbit lung, and Propionibacterium acnes infected mouse prostate tissues. Positive control (peptidylprolyl isomerase B- PPIB) and negative control (maize –Zm) stains were also conducted on the tissues. We additionally developed immunohistochemistry (IHC) assays with antibodies against lipopolysaccharide (LPS) and lipoteichoic acid (LTA) to visualize gram negative and gram positive bacteria, respectively, as a means to verify the results of the CISH assay. As our laboratory has a specific interest in the interactions between prostate infections and prostate cancer, we used this technique to interrogate a series of 10 radical prostatectomy specimens that contained a very high degree of acute and chronic inflammation, and were suspicious for the presence of an infectious organism. Both CISH and IHC were conducted in FFPE radical prostatectomy samples. The 16S rRNA CISH assay detected bacteria in all of the control tissues. Interestingly, 3 of the 10 human radical prostatectomy samples that we examined showed bacterial 16S rRNA signal concentrated in areas with acute inflammation where the glandular lumens were filled with neutrophils. Two of the radical prostatectomy samples that showed positive bacterial 16S rRNA CISH signal also showed positive LPS staining indicative of the presence of gram negative bacteria. The remaining positive sample was suspicious for a gram positive intracellular organism. This study shows preliminary data supporting robust detection of bacteria in a subset of human prostate samples. We expect the frequency of bacterial infections of this nature present in radical prostatectomy specimens would be low, and that we were only able to identify these cases because we screened for samples with very high levels of inflammation. Detection of both bacterial RNA and protein in the tissues corroborates the strength and reliability of the 16S rRNA CISH. The use of these two methods concurrently can provide valuable information on bacterial presence in a range of clinical samples. In the future, we plan to use these methods to probe for bacterial presence and in understanding the spatial dynamics of the urinary microbiome. We expect that this method may also be of broader use in the study of many infection-associated cancers. Citation Format: Eva Shrestha, Karen S. Sfanos. Development of an RNA hybridization technique for the in situ visualization of bacteria in cancer tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5136.

Abstract 5189: Evaluation of thymidylate synthase mRNA expression in circulating tumor cells from non-metastatic colon and rectal patients

Abstract Background: colorectal cancer is the third leading cause of cancer death in the United States. With different behaviors, rectal cancer represents worse prognosis, even in the locally advanced setting, with therapy following three steps, consisting of neoadjuvant chemoradiation, surgery and adjuvant chemotherapy. For non-metastatic colon cancer, the main treatment is surgery and then, patients can or not undergo adjuvant chemotherapy. Studies have shown that circulating tumor cells (CTCs) may be involved in metastatization process. Thymidylate synthase (TYMS) acts in the 5-Fluorouracil metabolism, and when overexpressed, can confer resistance to treatment, which is commonly used for neoadjuvant treatment (rectal cancer) and in combination with oxaliplatin for adjuvant treatment (colon cancer). Excision repair cross complementation Group 1 (ERCC1) is a protein involved in damage DNA repair process that is described to confer resistance to platinum-based chemotherapy. Objective: evaluate the TYMS messenger RNA (mRNA) expression in CTCs isolated from patients with locally advanced colon and rectal cancers; and its predictable value in treatment resistance. For patients with colon cancer, we also evaluated the ERCC1 protein expression in order to verify the adjuvant therapy resistance related to oxaliplatin. Methods: We collected 10 mL of blood before the beginning of treatment and processed it on ISET® (Rarecells) device. RNAscope® Technology is a novel chromogenic in situ hybridization (CISH) assay for detection of target RNA within intact cells. The expression of TYMS target gene was performed using the kit from ACDbio, as described on the protocol, but standardized to cytology. Results: there were included 12 patients of each type of tumor. We analyzed samples from 12 rectal cancer patients, and found 10 with overexpression of TYMS mRNA in CTCs. Curiously, of the two patients who had TYMSneg mRNA expression; one had complete pathological response and the other substantial down-staging. In the positive cases, 50% presented response (partial or complete) and 50% present no response and systemic progression. This result will better understood when we perform the evaluation of the protein TYMS. Among the samples from colon cancer patients, we found 8 out 12 as CTCs TYMSpos mRNA expression. Unfortunately, our recent data does not allow us to do analysis of recurrence. However, one of the positive cases had recurrence. Among the 4 TYMSneg, one had recurrence. This can be explained by the positive immunocytochemical expression of the ERCC1 protein, which confers resistance to oxaliplatin. Our next steps are to expand the CISH experiments in a larger group of patients as well as to evaluate the TYMS protein expression in the same cases. Conclusion: our preliminary results point molecular analysis of CTCs as predictor marker of colon/rectal cancer treatments. Citation Format: Bianca Troncarelli Flores, Emne Ali Abdallah, Virgílio Souza e Silva, Celso Abdon Mello, Samuel Aguiar, Renata Mayumi Takahashi, Vanessa Silva Alves, Bruna Elisa Kupper, Ludmilla T. Chinen. Evaluation of thymidylate synthase mRNA expression in circulating tumor cells from non-metastatic colon and rectal patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5189.

Kappa and Lambda Determination by Single Colored Immunohistochemistry and Chromogenic in Situ Hybridization Techniques in B-Cell Malignancies

Background: kappa and lambda light chains detection in bone marrow trephine sections help in the determination of B-cell clonality through evaluation of light chain restriction. Aim of the Work: was to compare the efficacy of single color detection-based immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) in evaluating kappa/lambda expression in tissues harboring B-lymphoid lesions. Patients and Methods: Forty patients were enrolled in this study. They were divided into three groups chronic lymphocytic leukemia (CLL/SLL) group I (n=13), non-Hodgkin lymphoma (NHL) group II (n=24) and hairy cell leukemia (HCL) group III (n=3). The 24 NHL cases comprised of (11 diffuse large B-cell lymphoma, 6 mantle cell lymphomas, 3 marginal zone lymphoma, 2 lymphoplasmacytic lymphoma, 1 follicular lymphomas and 1 Burkitt’s lymphoma). Kappa and lambda light chains were detected in their bone marrow trephine sections using single colored immunohistochemistry, chromogenic in situ hybridization and the results were compared to the flowcytometry as reference method. Results: Light chain restriction (LCR) was detected by FCM in 100% of the cases followed by CISH (52.1%; 12/23) of the cases and finally IHC (43%; 18/40). Background: kappa and lambda light chains detection in bone marrow trephine sections help in the determination of B-cell clonality through evaluation of light chain restriction. Aim of the Work: was to compare the efficacy of single color detection-based immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) in evaluating kappa/lambda expression in tissues harboring B-lymphoid lesions. Patients and Methods: Forty patients were enrolled in this study. They were divided into three groups chronic lymphocytic leukemia (CLL/SLL) group I (n=13), non-Hodgkin lymphoma (NHL) group II (n=24) and hairy cell leukemia (HCL) group III (n=3). The 24 NHL cases comprised of (11 diffuse large B-cell lymphoma, 6 mantle cell lymphomas, 3 marginal zone lymphoma, 2 lymphoplasmacytic lymphoma, 1 follicular lymphomas and 1 Burkitt’s lymphoma). Kappa and lambda light chains were detected in their bone marrow trephine sections using single colored immunohistochemistry, chromogenic in situ hybridization and the results were compared to the flowcytometry as reference method. Results: Light chain restriction (LCR) was detected by FCM in 100% of the cases followed by CISH (52.1%; 12/23) of the cases and finally IHC (43%; 18/40). Conclusion: Both conventional CISH and IHC are effective in determining monoclonality in cases of mature B- cell neoplasm that has plasmacytic differentiation and with high amount of cytoplasmic Ig light chains such as MZL and LP. However, they are not effective in determining monoclonality in cases with low amount of Ig light chain such as cases of pregerminal and germinal center lymphoma. Yet, CISH is more informative than IHC due to the lack of background staining which allowed for greater discrimination between absence and presence of monoclonality.

Abstract P5-07-04: Nuclear miR-133 expression is associated with breast cancer progression and survival

Abstract Background:MiRNAs (microRNAs) can function as the tumor suppressor gene or oncogene to affect carcinogenesis, therapy response and progression in human cancer. The prevailing view is that miRNAs function to regulate mRNA stability and translation in the cytoplasm. However, multiple studies have detected miRNAs in the nuclear compartment. Nuclear miRNAs have been proven to regulate the functions of nuclear protein and synthesis of nuclear miRNA. We have found circulating muscle-enriched miR-133a can serve as prognostic biomarkers for breast cancer. Herein, we aim to explore the subcellular distribution, prognostic significance and treatment response of miR-133a in Taiwanese breast cancer. Methods: Circulating miR-133a levels were conducted with the serum from 188 patients prior to surgery and from 82 healthy controls. The subcellular distribution patterns of miR-133a were analyzed by Chromogenic In Situ Hybridization (CISH) in paraffin embedded tissue sections from 224 cases. Cell viability and cell cycle were determined by XTT assay and flowcytometry. Protein and mRNA levels were examined by western blotting and RT-qPCR (TaqMan) assay. Results: Our results showed that circulating miR-133a levels of breast cancer patient are significantly higher than those of health subjects, suggesting that increased miR-133a secretion from unknown tissues or cells may promote carcinogens in Taiwanese breast cancer. Using the receiver operating characteristic (ROC) curve to examine the diagnostic value of circulating miR-133a in breast cancer (AUC=0.834), we found that increased miR-133a levels could serve as a diagnostic biomarker in accessing Taiwanese breast cancer risk. Nevertheless, CISH analysis revealed that nuclear miR-133a frequency was inversely correlated with tumor grade, stage and lymph node metastasis (p=0.029, 0.012, and 0.0008), and cytoplasmic miR-133a intensity was positively correlated with recurrence (p=0.010). Furthermore, high frequency of nuclear miR-133a was correlated with a better overall survival rate (p=0.039) and improved the survival rate of patients who received hormone therapy (p=0.009). To further investigate the role of nuclear miR-133a, an additional miR-133a, denoted as Nu-miR-133a, ending in nuclear localization element was constructed and found to be highly enriched in the nucleus. Ectopic expression of wild-type and Nu-miR-133 inhibited proliferation of breast cancer cells. Both wild type miR-133a and Nu-miR-133a increased S phase arrest as well as the levels and foci of γ-H2AX. Unexpectedly, we found that wild type miR-133a decreased ATM expression but activated p38 MAPK. Notably, H2AX can be also phosphorylated by p38 MAPK kinase in addition to ATM and ATR. The underlying mechanism between nuclear miR-133a and potentially p38-mediated DNA damage response (DDR, γ-H2AX and S phase arrest) will be needed further elucidation. Conclusions: In conclusion, we provide the first evidence that nuclear miR-133a may play a crucial role in the development and treatment response of breast cancer potentially through its mediated DDR. The interplay among nuclear miR-133a, p38 and DDR require further investigations. Citation Format: Hou M-F, Chen F-M, Yang S-F, Dai H-Y, Yeh Y-T. Nuclear miR-133 expression is associated with breast cancer progression and survival [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-07-04.

Assessment the of Amplification HER-2/neu Gene by Chromogenic In Situ Hybridization (CISH) Compared to Immunohistochemistry (IHC) Method in Gastric Cancer

Abstract Background: Gastric cancer has a highmortality rate and often has a poor treatment outcome. The HER2/neu gene target therapy has been known as a potential way for treatment. Objectives: The goal of our study was assessment the relation between chromogenic in situ hybridization (CISH) and immunohis- tochemistry (IHC)methods in determining the best diagnosticmethod for gastric cancer. Methods: In this historical cohort study, 50 gastric cancer samples were analyzed by CISH and IHC. The relation between clinical- pathological parameters of HER2/neu was also analyzed. Alive patients were followed from 2009 through 2012 for the main out- comes (mortality). The results of these twomethods, in terms of sex, age, tumor size, grading, staging, tumor location,metaplasia, presence of necrosis and ulceration, vascular invasion, the TNM system, mucin or signet producing adenocarcinoma cells and pa- tient survival rates were compared. Results: There was no significant difference between IHC and CISH regarding the sex, age, tumor size, grading, staging, tumor lo- cation,metaplasia, presence of necrosis and ulceration, vascular invasion, the TNMsystem,mucin or signet producing adenocarci- noma cells and patient survival rates. Comparison of TNMscores by these twomethods showed no significant relationship between IHC and staging, but a statistically significant difference between CISH and different N staging, (P < 0.05) was assessed. Conclusions: Comparison between IHC and CISH showed the only significant relationship between CISH and different N staging. Therefore, low amplified CISH was a better diagnosticmethod for gastric cancer, compared to low expression in IHC.

Detection Human Papilloma Virus genotype (16/18) in Iraqi Women Patients with endometrial carcinoma by using Chromogen - Insitu Hybridization (CISH) Technique

Aim: To investigate the possible association between human papillomavirus (HPV) and endometrial carcinoma. Does HPV play any role in the initiation or prognosis of endometrial adenocarcinomas among Iraqi women patients. Method: To determine the relationship between HPV and endometrial carcinoma , a retrospective study was done. The study was carried out on 30 patients with hisopathologically confirmed primary endometrial adenocarcinoma ,samples were collected from each patients , as well as twenty (20) endometrial tissues from control individuals with no cancer. Chromogen In situ hybridization (CISH) was used to detect HPV DNA genotype 16 /18 in endometrial tissues. Results: HPV 16/18 was detected in 19/30 (63.3%) tumor sample while negative cases were 11/30 (36.7%) of tumor sample and HPV 16/18 was detected in 5/20(25.0%) of endometrial control group while negative in 15/20 (75.0%) cases of endometrial control group. Conclusion: Our results revealed that high risk of HPV(16 / 18 ) being the most frequent type in Iraqi women patients with endometrial adenocarcinoma. Association between HPV infection and endometrial adenocarcinoma was observed and this research considered the first study which evaluated score ,intensity and pattern of replication system of HPV by using CISH technique which reflected role of HPV in endometrial carcinogensis.

Determination of HER-2/neu gene Status by Chromogenic in Situ Hybridisation Assay on Borderline (2+) Immunohistochemistry Cases in Patients with Invasive Breast Carcinoma: An Experimental Study on Preserved Tissue

HER-2 (also known as HER-2/neu) is a member of the epidermal growth factor (EGF) receptor family. The amplification of oncogene HER-2 is presented in 20 to 30% of breast cancers and results in an increase of the protein expression. HER-2 over expression has also been shown to correlate with poor prognosis. It is associated with poorly differentiated high grade tumors with lymph node involvement, greater risk of recurrence and relative resistance to some types of chemotherapy. The receptor is however a target for treatment with anti HER-2 antibody trastuzumab (Herceptin). Immunohistochemistry analysis for HER 2 scoring is subjective, requires trained personnel and expertise. One of the main concerns with IHC is that there is evidence of significant inter-observer variation in the assessment of staining, which can lead to misclassification of HER2 status. Chromogenic in situ hybridization (CISH) testing is sensitive and specific in detecting HER-2/neu gene amplification. Direct evaluation of gene amplification using CISH assay is a reliable method for routine diagnostic evaluation of HER2/neu status in breast cancer patients, especially in specimens showing 2+ IHC score. This observational experimental study was carried out in the Department of Pathology, Bangabandhu Sheikh Mujib Medical University, Dhaka, during the period of July 2014 to June 2015, The aim of this study was to assess HER-2 expression accurately in equivocal immunohistochemistry (2+) invasive breast cancer cases by Chromogenic in situ hybridization (CISH) and to associate the findings of CISH assay with the histologic prognostic and predictive factors (eg: tumor size, regional lymph node metastases, tumor grade and type. A total of 20 archival paraffin tissue blocks and IHC slides with IHC score 2+ for HER-2 were included in this study. All the slides were reviewed. CISH assay was done on section from archival paraffin block.CISH assay showed amplification of the HER-2 neu gene in 30% of cases. Majority (60%) was nonamplified. In two cases the results were unsatisfactory for interpretation. The differences were not statistically significant (p&gt;0.05) among three groups (amplified, nonamplified and unsatisfactory) regarding the baseline characteristics (age and sex) with CISH. All the results of CISH assay in association with tumor size, tumor grade and lymphnode metastasis were not statistically significant. HER-2 / neu status is the one of the most important prognostic and predictive factor. It is a target for treatment with anti HER-2 antibody trastuzumab (Herceptin). Detection of HER-2/ neu status by IHC may sometimes be difficult and inaccurate, specially in IHC score 2+ cases. Based on the findings of the study, CISH can be considered a useful, simple and reproducible method for detecting HER-2/ neu gene amplification in cases with borderline (2+) immunohistochemistry score , and patients may be benefited from Trastuzumab therapy.

PP06 HER2 Evaluation By CISH And SISH In Breast Cancer: A Meta-Analysis

Introduction:Molecular techniques play a critical role in identifying breast cancer patients with overexpressed human epidermal growth factor receptor-2 (HER2). New bright field techniques such as chromogenic in-situ hybridization (CISH) and silver in-situ hybridization (SISH) have emerged to overcome some of the challenges associated with the reference standard, fluorescence in-situ hybridization (FISH). We conducted a literature review and synthesis to characterize the accuracy of HER2 tests, and inform decisions about test selection.Methods:We searched MEDLINE and EMBASE databases using these eligibility criteria: studies evaluating invasive breast cancer samples which examined agreement between CISH or SISH, and FISH, and reported sensitivity, specificity, or concordance. We performed a bivariate meta-analysis of sensitivity and specificity using a generalized linear mixed model in Stata. We used likelihood ratio tests from meta-regression to compare accuracy between HER2 tests.Results:The search identified 4,475 articles, of which thirty-one were included. A total of thirteen studies (43%) evaluated dual-color SISH, twelve single-color CISH, and six dual-color CISH. The summary estimates for sensitivity and specificity were, respectively, 0.97 (95%CI 0.83–0.99) and 0.99 (95%CI 0.96–1.00) for single-color CISH, 0.98 (95%CI 0.92–0.99) and 0.98 (95%CI 0.91–0.99) for dual-color CISH; 0.92 (95%CI 0.86–0.95), and 0.96 (95%CI 0.91–0.98) for SISH. Significantly higher specificity was reported for single-color CISH than SISH (chi-square 4.12; p = 0.04), while dual-CISH had higher sensitivity than SISH (chi-square: 4.63; p = 0.03). These differences were not maintained when studies with cohorts enriched with equivocal samples were excluded.Conclusions:The agreement between new bright field tests (SISH and CISH) and FISH is high (&gt;92 percent). Indirect comparison of HER2 tests indicated that overall CISH performance exceeds that of SISH. However, low agreement between SISH and FISH in equivocal cases affects these comparative estimates. The pooled estimates from this meta-analysis can help inform future HER2 test selection decisions.

CD36 Neuronal Identity in the Olfactory Epithelium.

CD36 scavenger receptor is expressed in a subpopulation of olfactory sensory neurons (OSNs). These neurons express canonical olfactory signaling machinery; however, not all odorant receptors (ORs) are coexpressed with CD36. In situ hybridization (ISH) enables the detection of nucleic acids in tissues, cells, or isolated chromosomes. The development of nonradioactive and stable labeling probes almost 30years ago, allowed to routinely perform this technique employing different labeling strategies in one experiment. ISH is widely used in the field of neurobiology of the sense of smell, providing valuable neuroanatomical information regarding the molecular organization of the olfactory epithelium (OE). Here we show a method for studying CD36+-OSNs and provide a detailed protocol for chromogenic ISH, one- or two-color fluorescent ISH, which can be combined with immunofluorescence and are suitable for Cd36 mRNA probing simultaneous to other transcripts and/or proteins labeling.

Downregulation of N6-methyladenosine binding YTHDF2 protein mediated by miR-493-3p suppresses prostate cancer by elevating N6-methyladenosine levels.

Recent evidence suggests that m6A modifications regulate the progressions of several types of tumors. YTHDF2, an m6A reader, has been implicated in the regulation of hepatocellular carcinoma (HCC). miR-493-3p has been defined as tumor suppressor that inhibits the progressions of several types of cancers. However, the functions and mechanisms of YTHDF2 and the indirect m6A regulated role of miR-493-3p in prostate cancer (PCa) remains to be elusive. In this study, immuno-histochemical (IHC) staining and chromogenic in situ hybridization (CISH) were performed to find YTHDF2 was frequently upregulated but miR-493-3p was downregulated in both PCa tissues and cell lines (DU-145 and PC3) which was negatively correlated with each other. Knock down of YTHDF2 significantly elevated m6A levels, and inhibited the cell proliferation and migration of DU-145 and PC3 cell lines. The dual-luciferase reporter assay confirmed YTHDF2 as the direct target of miR-493-3p. In addition, forced expression of miR-493-3p consistently elevated the m6A levels and inhibited proliferation and migration with the knock down of YTHDF2. In contrast, overexpression of YTHDF2 and inhibition of miR-493-3p conversely reduced m6A levels. Additionally, the rescue experiments revealed that inhibition of miR-493-3p abrogated the suppression of proliferation and migration induced by si-YTHDF2. To conclude, YTHDF2 and miR-493-3p, as two crucial m6A regulators, are involved in the progression of PCa by indirectly modulating m6A levels. In view of these promising results, YTHDF2 and miR-493-3p may provide new insights into the carcinogenesis and new potential therapeutic targets for PCa.

Microfluidic-assisted chromogenic in situ hybridization (MA-CISH) for fast and accurate breast cancer diagnosis

Chromogenic in situ hybridization (CISH) is a common technique that is used for human epidermal growth factor receptor 2 (HER2) assessment in breast cancer diagnosis. CISH is based on the enzymatic amplification of DNA markers, generating a colorimetric image that can be visualized by a bright-field microscope and does not require a fluorescence detection setup. However, CISH is still characterized by a laborious and lengthy staining process, and is expensive due to the cost of the DNA probe solution. In this paper, we present a new technique named microfluidic-assisted chromogenic in situ hybridization (MA-CISH) in which hydrodynamic flow patterns in a microfluidic chip are used to improve hybridization efficiency between probe and target, thereby reducing the hybridization time and the consumption of probe solution. We optimized the MA-CISH protocol with a fractional factorial design of experiments using a SKBR3 breast cancer cell line. Optimized MA-CISH exploits discontinuous flow of DNA probe solution, enabling the reduction of the hybridization time from overnight down to 2h and requiring 70% less probe solution, while achieving a better signal quality than the standard CISH technique. Optimized MA-CISH was successfully tested for several breast cancer tissue samples, showing a good correlation with standard CISH results as evaluated on adjacent slides. Finally, an automatic scoring software was developed to facilitate CISH signal evaluation, and showed good agreement with the traditional counting protocol.

Digital image analysis of HER2 immunohistochemistry in gastric- and oesophageal adenocarcinoma: a validation study on biopsies and surgical specimens.

To test the validity of diagnostics incorporating digital image analysis (DIA) for human epidermal growth factor 2 (HER2) immunohistochemistry (IHC) in gastro-oesophageal adenocarcinomas, as an alternative to current standard diagnostics using manual scoring. We included 319 consecutive gastro-oesophageal adenocarcinomas (232 biopsies and 87 surgical specimens). DIA was applied to determine HER2 IHC classification, using both standard breast cancer (BC) and modified gastro-oesophageal cancer (GEC) cut-offs. Consensus manual scores were established by four independent observers. Chromogenic in-situ hybridization (CISH) was performed on all 2+ cases by manual scoring, DIA or both. HER2 status was considered positive in 3+ and CISH-positive 2+ cases. Overall agreement between DIA and consensus manual scores was 76.5% (weighted κ = 0.66, BC cut-offs) and 85.6% (weighted κ = 0.80, GEC cut-offs). Agreement was similar for biopsies and surgical specimens. All disagreement occurred in the manual IHC equivocal cases. DIA resulted in a reduction of 2+ cases: 75.8% with BC cut-offs and 46.5% with GEC cut-offs. HER2 status was positive in 48 cases (15%) with standard diagnostics and DIA using GEC cut-offs, and 46 cases (14.4%) using BC cut-offs (all with CISH in 2+ cases). Considering standard diagnostics as a reference, DIA showed 93.8% sensitivity and 99.6% specificity (BC cut-offs) or 97.9% sensitivity and 99.6% specificity (GEC cut-offs). DIA is a reliable and feasible alternative to manual HER2 IHC scoring in gastro-oesophageal adenocarcinoma, both in biopsies and surgical specimens, leading to a reduction of 2+ cases for which subsequent ISH testing is required.

Detection of Epstein–Barr virus and Cytomegalovirus in Iraqi Acute Lymphoblastic Leukemia patients

Objective : determination the percent of infection of EBV and CMV infections in Iraqi patient infected with ALL .Methods : the study have concentrated to determine the rate of EBV and CMV infections by detecting EBV and CMV antibodies in both the IgM and IgG classes of immunoglobulins by ELISA method and Indirect Immunofluorescence Test ( IIFT) as well as chromogenic in situ hybridization (CISH) for detecting Epstein-Barr virus (EBV) EBER RNA and CMV DNA . Results : from total 50 ALL patients were involved in this study , 50% were having viral infection (22% patients infected with EBV and 28% infected with CMV) this may be referred that in present study the viral causes may be equal to other unknown etiological factors of ALL collectively. anti-EBV Ig G and EBER RNA were detected in( 24%,12) of all ALL patients and anti EBV Ig M was found only in (4%,2) of ALL patients and patients with positive EBV Ig M also were positive for anti EBV Ig G and EBER RNA while anti-CMV IgG and CMV DNA were detected in (28%,14) of all ALL patients involved in the study and only (8%,4) of ALL patients showed positive anti CMV Ig M and patients with positive anti CMV Ig M also were positive for anti CMV Ig G and CMV DNA. Conclusion : The presence of elevated levels of EBV-infected cells or CMV-infected cells within the peripheral blood may be risk factors for developing ALL and the incidence of CMV infection (28%) was slightly greater than the rate of EBV infection (24%) in ALL patients and this may be referred that in present study the viral causes may be equal to other unknown etiological factors of ALL collectively.

Abstract 5684: NSABP FC-7 correlative study: HER2 amplification (amp) in circulating cell-free DNA (cfDNA) in metastatic colorectal cancer (mCRC) resistant to anti-EGFR therapy (tx)

Abstract Background: FC-7 is a phase Ib study hypothesizing that dual ERBB blockade (neratinib and cetuximab) could overcome an acquired resistance mechanism to anti-EGFR tx in KRAS/NRAS/BRAF/PI3KCA wild type (wt) mCRC patients (pts). Previously we demonstrated a 36% rate of HER2 amp in post- anti-EGFR biopsies (bxs). Assay of plasma cfDNA is minimally invasive and may provide comprehensive genomic profiling. We tested the accuracy of a cfDNA assay for HER2 amp, comparing results from matched bxs to evaluate molecular changes in HER2 copy number. Methods: 23 pts with mCRC resistant to anti-EGFR tx were enrolled and treated with cetuximab and neratinib. 17 paired bxs (6 post-tx bxs had insufficient tumor) were assessed for HER2 amp using chromogenic in situ hybridization (CISH) (Dako PharmDxTM). HER2 was considered amplified when HER2/CEP17 ratio was ≥2. cfDNA was extracted from plasma of 11 pts after 1st line anti-EGFR tx but before addition of cetuximab and neratinib. 5-30 ng of DNA was sequenced across 512 exons in 54 genes with HiSeq (Illumina). Copy number variants (CNV) were quantified using a customized pipeline (Guardant 360). One plasma sample failed to yield sufficient cfDNA. 10 pts had comparison of cfDNA with pre- or post- anti-EGFR bx. Results: 4 of 9 plasma samples (1 non evaluable) had HER2 amp, all concordant with pre- or post- anti-EGFR tx tissue. 3 pts converted to HER2 amp after anti-EGFR tx (pts 3, 5, 7), and for 2 of these conversions (pts 5, 7), the amplification was evident in cfDNA. PtPre- anti-EGFR CISH RatioPost- anti-EGFR CISH RatioPost- anti-EGFR cfDNA CNVConcordance of cfDNA with pre- or post-anti-EGFR Tissue Samples1Neg (1.0)Neg (1.3)Non evaluableNA2Pos (2.5)Neg (1.5)Pos (2.66)Pre-Tx3Neg (1.0)Pos (3.7)NegPre-Tx4Neg (1.0)Neg (1.8)NegBoth5Neg (1.2)Pos (2.7)Pos (2.48)Post-Tx6Pos (&amp;gt;10)QNSPos (21.86)Pre-Tx7Neg (1.1)Pos (2.0)Pos (2.20)Post-Tx8Neg (1.3)Neg (1.1)NegBoth9Neg (1.0)Neg (1.3)NegBoth10Neg (1.0)Neg (1.5)NegBoth Conclusion: In this sample set, HER2 amp detected in plasma-derived cfDNA from mCRC pts resistant to anti-EGFR tx is comparable to amp assessed in primary or metastatic tissue. Given advantages of liquid bx relative to repeated tissue bxs, measurement of HER2 amp in cfDNA may be of utility in monitoring pts for resistance to anti-EGFR tx. This will need to be confirmed in a trial incorporating both plasma and tissue samples. Support: Puma Biotechnology, Inc. Citation Format: S. Rim Kim, Ashok Srinivasan, Carmen J. Allegra, Samuel Jacobs, Rebecca J. Nagy, Richard B. Lanman, AmirAli Talasaz, Patrick Gavin, Rekha Pal, Peter C. Lucas, Kay Pogue-Geile. NSABP FC-7 correlative study: HER2 amplification (amp) in circulating cell-free DNA (cfDNA) in metastatic colorectal cancer (mCRC) resistant to anti-EGFR therapy (tx) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5684. doi:10.1158/1538-7445.AM2017-5684

P16 expression in cutaneous squamous cell carcinoma of the head and neck is not associated with integration of high risk HPV DNA or prognosis

Head and neck cutaneous squamous cell carcinoma (HNcSCC) can present with cervical metastases without an obvious primary. Immunohistochemistry for p16 is established as a surrogate marker of human papillomavirus (HPV) in oropharyngeal cancer. p16 expression in HNcSCC needs to be elucidated to determine its utility in predicting the primary site. The aim of this study was to evaluate the rate of p16 expression in HNcSCC and its association with prognostic factors and survival. p16 immunohistochemistry was performed on 166 patients with high risk HNcSCC (2000-2013) following histopathology review. Chromogenic in situ hybridisation (CISH) for HPV was performed. Fifty-three (31.9%) cases showed strong, diffuse nuclear and cytoplasmic p16 expression including 14 (41%) non-metastatic and 39 (29.5%) metastatic tumours (p=0.21). HPV CISH was negative in all cases. p16 expression significantly increased with poorer differentiation (p=0.033), but was not associated with size (p=0.30), depth of invasion (p=0.94), lymphovascular invasion (p=0.31), perineural invasion (p=0.69), keratinisation (p=0.99), number of involved nodes (p=0.64), extranodal extension (p=0.59) or survival. Nearly 32% of HNcSCCs, particularly poorly differentiated HNcSCCs, show p16 expression. A primary HNcSCC should be considered in p16 positive neck node metastases in regions with high prevalence of HNcSCC. p16 expression is not associated with improved survival in HNcSCC.

Chromosome 7 Multiplication in EGFR-positive Lung Carcinomas Based on Tissue Microarray Analysis.

Epidermal growth factor receptor (EGFR) over-activation is observed in significant proportions of non-small cell lung carcinomas (NSCLC). Our aim was to investigate the role of chromosome 7 multiplication with regard to its influence in EGFR expression, combined or not with gene amplification. Using tissue microarray technology, fifty (n=50) primary NSCLCs were cored and re-embedded into the final recipient block. Immunohistochemistry (IHC) and also chromogenic in situ hybridization (CISH) were performed. EGFR expression at any level was detected in 40/50 (80%) cores. Over-expression was observed in 23/40 (57.5%) cases. Gene amplification was identified in 11/50 (22%) cases whereas chromosome 7 polysomy in 8/50 (16%) cases. Pure chromosome 7 multiplication alone led to low or moderate levels of expression. Overall EGFR expression was correlated with gene (p=0.001) and interestingly with chromosome 7 centromere numerical imbalances (p=0.004). EGFR expression is associated not only with amplification, but also with chromosome 7 centromere multiple copies. Chromosome 7 multiplication -due to centromere region amplification or true polysomy- is critical for applying monoclonal antibody targeted therapeutic strategies excluding the pure non-amplified cases.