Chromogenic In Situ Hybridization Research Articles

Can Epstein-Barr virus play a role in upper urinary tract urothelial carcinomas?

Upper urinary tract urothelial carcinomas are very rare tumours with different biological behaviours. The Epstein-Barr virus, which is the first known oncogenic virus, is being investigated for various malignant tumours. It is known that this virus is associated with nasopharyngeal carcinoma, as well as multiple haematological malignancies, head and neck and gastric cancers. We aimed to determine the presence of the Epstein-Barr virus in upper urinary tract urothelial carcinomas using chromogenic in situ hybridisation (CISH). A total of 44 upper urinary tract urothelial carcinomas from two different centres were included. Demographic data and survival rates were obtained from hospital records. One demonstrative paraffin block from each case was stained using Epstein-Barr encoded RNA (EBER) with an automated CISH procedure. The positivity of EBER was statistically analysed for prognostic factors. Among all patients, 38 were male and 6 were female. The mean age of the patients was 65.93 years. At the time of the study, 15 patients had died and 29 were alive. EBER-CISH positivity was found in 13 patients. Four showed strong EBER-CISH expression and nine showed weak expression. EBER-CISH positivity was not statistically related to any of the prognostic factors or to overall survival. Although EBER-CISH positivity showed no significant relation with prognostic factors, it was observed in one-third of all cases. Therefore, we think that the Epstein-Barr virus may have a role in the pathogenesis of upper urinary tract urothelial carcinomas. This finding needs to be supported by larger studies.

Immunohistochemical over-expression of HER2 does not always match with gene amplification in invasive bladder cancer

BackgroundHER2 is a potential target of therapy in urothelial cancer (UC). Pathological case stratification according to HER2 gene amplification or HER2 protein overexpression was critical for patients’ selection in previous unsuccessful clinical trial with HER2 targeting agents. Study designWe evaluated the HER2 overexpression by immunohistochemistry (IHC) together with the amplification of the HER2 gene with chromogenic(CISH) and fluorescent (FISH) in situ hybridization in a cohort of 61 patients covering the whole spectrum of bladder UC variants, using a tissue microarray (TMA) approach. ResultsIHC was available in all the 61 cases while ISH in 37 and FISH in 42. At IHC, 2/61 cases (3%) were scored 3+; 2 (3%) scored 2+; 2 (3%) scored 1+; the remaining 55 (91%) scored 0. At CISH analysis 10/37 cases (27%) were amplified, 6 cases with HER2 amplification showed positive HER2 IHC (3+, 2+, 1+). Seven cases with IHC score 0 were amplified at CISH. FISH analysis revealed an amplification in 5/42 cases (12%). The total number of HER2amplified cases was different between chromogenic and fluorescent ISH with 5 cases amplified using FISH compared to 10 with CISH. ConclusionsIn clinical trials with HER2 targeting agents the candidate patients should be investigated not only by IHC but also by ISH, independently of the IHC results. Since also usual type UC can overexpress HER2 we recommend to extend the patients’ selection to all the histotypes of bladder cancer other than the micropapillary type.

The Epstein-Barr virus and osteosarcoma in Iranian children.

e23543 Background: This study was conducted to describe the possible association between EBV infection and OS in Iranian children. Methods: This study carried out at Kermanshah university hospital on 48 OS tissue and ten non-cancerous bone tissue as a control group by three laboratory methods. The real-time polymerase chain reaction (RT-PCR) and Chromogenic in-situ hybridization (CISH) done to detect the Epstein Barr Encoded RNA (EBER1) expression as long as immunohistochemistry staining (IHC) to detect EBV latent membrane protein-1 (LMP-1) in tumor cells and controls. Overall-survival (O-S), prognosis free survival (PFS) and disease-free survival (DFS) provided based on the Kaplan-Meier method. Results: Specimen considered positive when EBER using PCR and CISH associated with LMP-1 using IHC stain were expressed separately in the same sample. Six out of eighty-four (12.5%) of OS cases were simultaneously EBV positive in all three methods within the tumor cells as compared to the control group. (P < 0.22) Association between EBV positivity and histologic characteristics of OS cases such as nodal involvement (P < 0.04), metastasis (P < 0.04), Ki67 reactivity (as proliferation index) (P < 0.03), and tumor stage (P < 0.05) were significant. EBV Positive patients show worse clinical symptoms in older age as well as the worse prognosis compared with negative patients. EBV positive patients presented shorter DFS, PFS, and OS. Conclusions: Our research could not find a statistical association between EBV infection and OS in Iranian children significantly. Meanwhile, we found, EBV infection could interfere in the invasive behavior of tumors. [Table: see text]

Pathological complete response rates in HER2 amplified breast cancers based on levels of HER2 amplification.

e12632 Background: Untreated HER2 (Human Epidermal Growth Factor) amplified breast cancer has poorer prognosis, compared to those with HER2 negative status with shorter time to relapse and increased incidence of metastases and higher mortality. Both Trastuzumab and Pertuzumab (humanized monoclonal antibodies against HER2) have shown to improve PCR (Pathological Complete Response) rates when used in the neoadjuvant setting. However, whether the quantitative level of HER2 amplification affects PCR rates or disease-free survival/overall survival has not been well studied. If the level of HER2 amplification correlates with PCR rates, this could be a tool for clinicians to use as a predictive marker for response to therapy. Methods: This is a retrospective chart review of community oncology clinic patients with HER2 amplified breast cancer Stage I-III to evaluate the rates of PCR after HER2 targeted neo-adjuvant therapy categorized by HER2 CISH (Chromogenic in situ hybridization) amplification and stratified based on levels of amplification of < 3, 3 to 5, or > 5. Pathology reports were reviewed for report of PCR. Inclusion criteria was women age 19 to 90, HER2 positive biopsy proven breast cancer, Stage I-III, patients treated with neoadjuvant chemotherapy, adequate renal function, and left ventricular ejection fraction within normal range. Exclusion criteria was pregnancy, metastatic disease, history of other malignancies, and impaired renal or cardiac function. Results: The data consisted of 36 unique patients, 25% of whom had a HER2 below 3, 22.2% had a HER2 between 3 and 5, and 52.8% had a HER2 value > 5. There was found to be a statistically significant association between the level of HER2 amplification and PCR rates with increased amplification of HER2 relates with increased PCR rates (p value < 0.0176). Both three-year disease-free survival and three-year overall response rates were not statistically significantly associated with HER2 category (p value -0.2691 and 0.4692 respectively). Conclusions: The information from this study may introduce a future systematic approach to further risk stratify patients based upon their quantitative HER2 amplification level to help predict response to therapy. We have noted that a level of HER2 amplification > 5 led to significant association with PCR compared to < 3 and 3-5 values.

MiR-18a and miR-18b are expressed in the stroma of oestrogen receptor alpha negative breast cancers

BackgroundPreviously, we have shown that miR-18a and miR-18b gene expression strongly correlates with high proliferation, oestrogen receptor -negativity (ER−), cytokeratin 5/6 positivity and basal-like features of breast cancer.MethodsWe investigated the expression and localization of miR-18a and -18b in formalin fixed paraffin embedded (FFPE) tissue from lymph node negative breast cancers (n = 40), by chromogenic in situ hybridization (CISH). The expression level and in situ localization of miR-18a and -18b was assessed with respect to the presence of tumour infiltrating lymphocytes (TILs) and immunohistochemical markers for ER, CD4, CD8, CD20, CD68, CD138, PAX5 and actin. Furthermore, in two independent breast cancer cohorts (94 and 377 patients) the correlation between miR-18a and -18b expression and the relative quantification of 22 immune cell types obtained from the CIBERSORT tool was assessed.ResultsCISH demonstrated distinct and specific cytoplasmic staining for both miR-18a and miR-18b, particularly in the intratumoural stroma and the stroma surrounding the tumour margin. Staining by immunohistochemistry revealed some degree of overlap of miR-18a and -18b with CD68 (monocytes/macrophages), CD138 (plasma cells) and the presence of high percentages of TILs. CIBERSORT analysis showed a strong correlation between M1-macrophages and CD4+ memory activated T-cells with mir-18a and -18b.ConclusionsOur study demonstrates that miR-18a and miR-18b expression is associated with ER- breast tumours that display a high degree of inflammation. This expression is potentially associated specifically with macrophages. These results suggest that miR-18a and miR-18b may play a role in the systemic immunological response in ER− tumours.

Predicting Effects of Clinicopathological Variables on Her2 Gene Amplification by Chromogenic in situ Hybridization (CISH) in IHC Her2 (2+) Breast Cancer Patients; A Study from Iran.

Background & Objective:The her2 amplification plays an important role in breast cancer management. Therefore, there is a need for using supplementary molecular methods in IHC equivocal cases. Present study has been conducted to determine the effects of clinicopathological variables on her2 gene amplification by chromogenic in situ hybridization (CISH) in IHC Her2 (2+) breast cancer individuals.Methods:A cross-sectional study was conducted in Zaferanyeh Laboratory collaborated with Shahid Beheshti University of Medical Sciences (Tehran-Iran; 2015-2018). All pathological data related invasive breast cancer patients with equivocal IHC results were included. CISH method was performed as a supplementary technique. The associations between histopathologic variables, status of Ki-67 index, progesterone and estrogen receptors (PR & ER) with her2 amplification by CISH were investigated and analyzed. The level of significance was considered as P-value < 0.05.Results:Totally, 239 patients with mean age of 53.2 years were studied. CISH identified her2 gene amplification in 51 subjects (21.3%). The type of tumor (invasive ductal carcinoma), the tumor grade, and the value of Ki-67 index were directly correlated with her2 amplification. Significant negative associations were also observed between CISH results and ER and PR expression.Conclusion:As her2 gene amplification was identified in 21.3% of invasive breast cancer patients with equivocal IHC results, it is supposed that applying CISH method may consider as a potentially valuable supplementary method. Results have also shown that higher grades of tumor, invasive ductal carcinoma, absences of hormone receptors and high Ki-67 index significantly correlated with the her2 amplification.

Utilization of RNA in situ Hybridization to Understand the Cellular Localization of Powassan Virus RNA at the Tick-Virus-Host Interface.

Skin is the interface between an attached, feeding tick and a host; consequently, it is the first line of defense against invading pathogenic microorganisms that are delivered to a vertebrate host together with tick saliva. Central to the successful transmission of a tick-borne pathogen are complex interactions between the host immune response and early tick-mediated immunomodulation, all of which initially occur at the skin interface. The focus of this work was to demonstrate the use of RNA in situ hybridization (RNA ISH) as a tool for understanding the cellular localization of viral RNA at the feeding site of Powassan virus (POWV)-infected Ixodes scapularis ticks. Intense positive staining for POWV RNA was frequently detected in dermal foci and occasionally detected in hypodermal foci after 24 h of POWV-infected tick feeding. Additionally, duplex chromogenic RNA ISH staining demonstrated co-localization of POWV RNA with Mus musculus F4/80 RNA, CD11c RNA, vimentin RNA, Krt14 RNA, and CD3ε RNA at the feeding site of POWV-infected ticks. In future studies, RNA ISH can be used to validate transcriptomic analyses conducted at the tick-virus-host cutaneous interface and will provide cellular resolution for specific gene signatures temporally expressed during infected tick feeding. Such a systems biology approach will help create a more refined understanding of the cellular and molecular interactions influencing virus transmission at the cutaneous interface.

In Situ Localization of Human Cytomegalovirus DNA and Translated Proteins of BRCA1 and BRCA2 Genes in Histological Specimens from Patients with Serous Epithelial Ovarian Carcinoma in Baghdad Province

HCMV has been detected in various types of tumors. Besides its well-describedoncomodulation, recentlyan oncogenic properties of HMCV per se as well asits direct transforming role in infected cells have beendescribed.HCMV in serous ovarian adenocarcinoma and its relation to BRCA1and BRCA2 was scarcelystudied.The study was designed to examine cellular dysregulation mediated by the concordant protein expressionsof BRCA1 and BRCA2 tumor suppressor genes with HCMV in tissues from ovarian cancers.Eighty ovarian tissues were examined for HCMV-DNA and BRCA1 & BRCA2 genes expression. Thosesamples belonged to (40) patients diagnosed with ovarian cancer and (40) from apparently normal ovariantissues. The detection of HCMV-DNA was done by chromogenic in situ hybridization (CISH) whereas thetranslated proteins of the expressed BRCA1 & BRCA2 genes by immunohistochemistry (IHC).Positive signals of HCMV-DNA -CISH reactions in malignant serous epithelial ovarian tumors, was detectedin 45% (18 out of 40)tissues and followed by the apparently healthy ovarian control tissues (12.5%,5 outof 40 tissues). The difference of the HCMV in ovarian cancers and control was highly significant. Thetranslated proteins of the expressed BRCA1 & BRCA2 genes was detected by IHC in 60% (24 out of 40tissues) of malignant serous epithelial ovarian tumors while no signal was reported in the control tissues.The difference between the percentages of BRCA1 as well as BRCA2proteins detection in ovarian cancer &control group was statistically significant (<0.05).The significant detection of either HCMV o rBRCA1 & BRCA2 genes expression in the studied ovariancancer tissues could support a role for that virus along with these genes in ovarian carcinogenesis.

Are Immunohistochemical Markers Useful in Phenotypic Gastric Cancer Classification?

To identify useful markers for prognostic and therapeutic purposes, The Cancer Genome Atlas (TCGA) provided a molecular classification of gastric cancers (GCs). Previous studies have used immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) to define immunophenotypic surrogate markers of the molecular alterations. Some critical issues concerning the correct definition of immunophenotypic groups have emerged in these studies that employed tissue microarrays (TMAs). We performed an immunophenotypic classification by evaluating MLH1, p53, HER2, E-cadherin, and Epstein-Barr virus (EBV) on the whole section of the surgical GC samples compared to most of the studies conducted on TMAs. We also investigated the immunohistochemical expression of PD-L1, a known therapeutic target. We identified the following immunophenotypic groups: EBV (2.9%); mismatch repair deficient (MMR-D) (7.2%); overexpressed p53 and/or HER2+ (61.4%); aberrant E-cadherin (11.4%); and normal pattern (17.1%). The use of surgical samples emphasized that some immunohistochemical markers were not useful for properly classifying the GC specimens. We can state that EBV (significantly correlated to PD-L1 expression) and MMR-D GCs are well-defined groups, mutually exclusive, and easily assessable with IHC and CISH, and could be candidates for immunotherapy with PD-1/PD-L1 inhibitors. As regards p53, our findings suggest that IHC assessment may be responsible for a misclassification of GC groups. Immunohistochemical evaluation of E-cadherin needs to be standardized, particularly in terms of the heterogeneous cytoplasmic/membranous staining pattern. Whether to consider the normal-pattern group as a separate category remains to be clarified. Because GC specimens with known therapeutic targets account for only 40%, we suggest reviewing the immunophenotypic classification to find new therapeutic targets, such as PD-L1, MLH1, and HER2.

Molecular profiling of gastric cancer in a population with high HIV prevalence reveals a shift to MLH1 loss but not the EBV subtype.

The human immunodeficiency virus (HIV) pandemic heavily affects sub‐Saharan Africa. It is associated with persistently active Epstein‐Barr virus (EBV) infection. To determine if this translates into increased frequency of EBV‐associated gastric cancer (EBVaGC), we evaluated molecular profiles of gastric cancer (GC) in Zambia. Patients with GC or premalignant gastric lesions were enrolled in Lusaka, Zambia. We used patients without any of these lesions as a control group. Chromogenic in situ hybridization (CISH) on tumor tissue was used to identify EBVaGC. To identify the microsatellite unstable subtype, immunofluorescence staining for MutL homolog 1 (MLH1) was used. Exposure to EBV and HIV was assessed serologically. We enrolled 369 patients (median age 52 years [IQR 41‐65]; 198 (54%) female). Of these, 72 (20%) had GC and 35 (9%) had gastric premalignant lesions (PL). CISH identified EBVaGC in 5/44 (11%) of those with adequate tissue, while MLH1 loss was identified in 29/45 (64%). Both GC and PL were associated with the highest titers of antibodies to Early antigen‐diffuse (OR 2.5, 95% CI 1.0‐6.1, P = .048 and OR 3.9, 95% CI 1.1‐12.9, P = .03, respectively) at high concentrations. Human immunodeficiency virus infection was associated with a range of antibodies to EBV, but not with any cancer subtype. Despite the association of HIV with persistent EBV, the proportion of EBVaGC in Zambia is similar to populations with a low prevalence of HIV infection. The proportion of microsatellite unstable tumors may be higher than other populations.

Haemosporidioses in wild Eurasian blackbirds (Turdus merula) and song thrushes (T. philomelos): an in situ hybridization study with emphasis on exo-erythrocytic parasite burden

BackgroundPasserine birds are frequently infected with diverse haemosporidian parasites. While infections are traditionally considered benign in wild birds, recent studies demonstrated mortalities of passerine species due to exo-erythrocytic development of the parasites, which can damage organs in affected hosts. However, exo-erythrocytic development remains insufficiently investigated for most haemosporidian species and thus little is known about the virulence of tissue stages in wild passerine birds. The aim of the present study was to investigate natural haemosporidian infections in deceased Eurasian blackbirds (Turdus merula) and song thrushes (Turdus philomelos) and to determine parasite burden and associated histological effects.MethodsFor molecular analysis, blood and tissue samples from 306 thrushes were screened for Plasmodium, Haemoproteus and Leucocytozoon parasites by nested PCR. For the detection of parasite stages in organ samples, tissue sections were subjected to chromogenic in situ hybridization (CISH) using genus- and species-specific probes targeting the rRNAs of parasites. Exo-erythrocytic parasite burden was semi-quantitatively assessed and histological lesions were evaluated in haematoxylin–eosin-stained sections.ResultsBy PCR, 179 of 277 Eurasian blackbirds and 15 of 29 song thrushes were positive for haemosporidians. Parasites of all three genera were detected, with Plasmodium matutinum LINN1 and Plasmodium vaughani SYAT05 showing the highest prevalence. CISH revealed significant differences in exo-erythrocytic parasite burden between lineages in Eurasian blackbirds, with P. matutinum LINN1 frequently causing high exo-erythrocytic parasite burdens in various organs that were associated with histological alterations. Song thrushes infected with P. matutinum LINN1 and birds infected with other haemosporidian lineages showed mostly low exo-erythrocytic parasite burdens. Two Eurasian blackbirds infected with Leucocytozoon sp. TUMER01 showed megalomeronts in various organs that were associated with inflammatory reactions and necroses.ConclusionThis study suggests that P. matutinum LINN1, a common lineage among native thrushes, regularly causes high exo-erythrocytic parasite burdens in Eurasian blackbirds, which may result in disease and mortalities, indicating its high pathogenic potential. The findings further illustrate that the same parasite lineage may show different levels of virulence in related bird species which should be considered when assessing the pathogenicity of haemosporidian parasite species. Finally, the study provides evidence of virulent Leucocytozoon sp. TUMER01 infections in two Eurasian blackbirds caused by megalomeront formation.

A Simple Method to Assess Estrogen Receptor Gene (ESR1) Amplification in Paired Biopsies from Primary Tumor and Recurrence in Breast Cancer Patients Receiving Endocrine Therapy

Globally, Breast Cancer (BC) is the leading cause of cancer death among women. About 75% of patients are diagnosed with hormone-dependent tumors and are set to receive Endocrine Therapy (ET) targeting the estrogen receptor. Unfortunately, a significant proportion of these patients develops ET resistance. Still controversial, studies have proposed that Estrogen Receptor-Alpha Gene (ESR1) alterations may underlie ET resistance. Here, we describe the use of a Chromogenic in Situ Hybridization (CISH) assay for the assessment of ESR1 amplification in primary tumors and recurrences. This assay could be a useful clinical tool with therapeutic implications for estrogen receptor positive BC patients.

Cyclin D1 Gene Numerical Imbalances in Laryngeal Squamous Cell Carcinoma: A Tissue Microarray Grid Based Analysis.

Background:Deregulation of critical proteins involved in cell cycle stability, such as cyclins, is a frequent genetic event in the development and progression of solid malignancies. Concerning laryngeal squamous cell carcinoma (LSCC), cyclin D1 oncogenic transformation leads to an aberrant protein expression and seems to affect the biological behaviour of the neoplasm. The aim of this study was to determine the correlation of cyclin D1 numerical imbalances with the corresponding protein expression levels in LSCCs. Material and Method:Using tissue microarray (TMA) technology, fifty (n=50) histologically confirmed primary LSSCs were cored at a diameter of 1.5 mm. Immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) analyses were applied. Concerning the screening process in CISH slides, a novel real-time reference and calibration grid platform was implemented. Results:Protein overexpression was observed in 22/50 (44%) cases; whereas, gene amplification was seen in 13/50 (26%) cases (p=0.02). Combined protein/ gene deregulation was associated with the stage of malignancy (p= 0.0014, p=0.001), whereas overall protein expression was strongly correlated with the grade of tumour (p= 0.001). Conclusion: Cyclin D1 gene amplification led to aberrant protein expression in LSCCs and it was also correlated with an aggressive biological behaviour. To best of our knowledge, this study was the first described grid based CISH analysis under conventional bright field microscopy for detecting gene numerical imbalances while providing a novel and accurate description for screening-mapping process in the entire slide area.

Agarose cell block and ancillary molecular tests enhance diagnostic efficacy of liquid-based cytology samples

Background The concomitant use of cytology smears and cell block (CB) preparations for ancillary techniques represents a useful diagnostic performance, especially in suspicious malignant cases or with equivocal cytological morphology. CBs prepared by the agarose-based technique have a high morphologic quality. Agarose prevents notable loss of diagnostic cells during CB preparation. This can be used as a maximal investment for the residual supernatant liquid-based cytology (LBC) material. Aim To study the use of agarose CBs from residual LBC material as a diagnostic tool added to cytology smears in fluid and fine-needle aspiration cytology. Patients and methods The current study included 65 cases utilizing LBC slides, and agarose CBs prepared from the residual LBC material. Ancillary studies were performed on CBs using immunomarkers: CK-7, CK-20, ER, PR, CEA, calretinin, pan-cytokeratin, Her2, CK5/6, P63, TTF-1, HMB-45, and S-100, as well as chromogenic in-situ hybridization (CISH) Her2. Results The majority of our studied agarose CBs (89.2%) had morphologic criteria of the diagnostically superior category. Immunohistochemistry on agarose CBs raised the sensitivity from 85.3 to 100% in effusion cytology and from 92.9 to 100% in aspiration cytology. In this study, 60 out of 65 studied cases needed further diagnostic verification with consequent use of ancillary techniques on agarose CBs including immunohistochemistry and CISH. Regarding effusion cytology samples, immunomarkers enabled subtyping of 21 cases of metastatic adenocarcinoma (14 cases of noncolorectal origin and seven cases of metastatic colorectal adenocarcinoma). Moreover, verification of the origin of malignant cells in effusion cytology samples could be reached (mesothelioma was proved in 4/6 cases and disproved in two cases that were reported as metastatic adenocarcinoma). Moreover, the inconclusive cases of effusion cytology could be solved; 9/14 cases were confirmed as benign and the remaining five cases were reported as metastatic adenocarcinoma. In our study, two cases of metastatic breast carcinoma in pleural effusion were requested for hormonal profile (one showed positivity to ER, PR, and Her2 by immunocytochemistry and the other was triple negative, and Her2 negativity was confirmed by CISH performed on the agarose CB). In pulmonary specimens, immunohistochemical subtyping of nonsmall cell carcinoma cases was possible (6/8 cases were confirmed as squamous cell carcinoma, and the remaining two cases were adenocarcinomas). In the studied five cases of lymph node aspirates, the provisional broad diagnosis of metastatic undifferentiated malignancy could be further specified by immunohistochemical application of certain tumor markers; their choice was suggested according to morphologic criteria of LBC material. Of these five cases, there was one case of metastatic malignant melanoma. Another two cases were highly suggestive of thyroid origin. Unfortunately, two of these metastatic undifferentiated cases had diagnostically unsuitable CBs, and no further studies could be done. Four lymph node aspirates were inconclusive for metastasis and were reclassified by immunohistochemistry as three cases of reactive lymphadenopathy and one case of metastasis. Accordingly, final cytologic diagnosis could be achieved in 63 (97%) of our total 65 studied cases. The complementary use of agarose CBs helped in diagnosis of 54 (96.4%) of 56 cases that needed ancillary tests. Conclusion Agarose CBs prepared from LBC residues, together with cytology smears, can achieve significant and accurate results when coupled with different ancillary studies.

Human Mammary Tumor Virus, Human Papilloma Virus, and Epstein-Barr Virus Infection Are Associated With Sporadic Breast Cancer Metastasis

Background:Viral cause of sporadic breast cancer (SBC) has been suggested based on the experimental murine model of mammary tumor caused by mouse mammary tumor virus (MMTV), Epstein-Barr virus (EBV), and human papillomavirus (HPV). While some studies have demonstrated the presence of viral sequences of MMTV, HPV, and EBV in breast cancer cells, others failed. These contradictions may be attributed to the geographical distribution of breast cancer incidence and/or technical variations. In the current study, we aimed to investigate the correlation of MMTV, HPV, and EBV infections with the development of breast cancer in Jordanian patients.Methods:One hundred SBC tissue samples were subjected to laser capture microdissection for the selection of tumor cells populations. Fluorescence polymerase chain reaction (PCR) was used to detect the presence of the MMTV env-like sequences. Real-time PCR was used for HPV and EBV detection, and EBV was further confirmed by chromogen in situ hybridization (CISH).Results:Mouse mammary tumor virus, HPV, and EBV were detected in SBC in 11%, 21%, and 23%, respectively. Only 3 of 52 (5.7%) positive cases demonstrated multiple virus infections. However, 49 of 52 (94%) of the positive cases revealed the presence of 1 type of viral sequences. Consequently, 52% of the studied breast cancer cases were infected with at least 1 type of the aforementioned viruses.Conclusions:The current cohort suggests that MMTV, HPV, and EBV have a potential role in the development of breast cancer and adding more reasons to proceed with the quest of a possible viral origin of breast cancer.

Detection of Individual RNA in Fixed Cells and Tissues by Chromogenic ISH.

Visualization of RNA molecules in situ helps to better understand the functions of expressed genes. Currently, most conventional in situ hybridization methods for visualization of individual RNAs are based on fluorescence detection. Herein we present a chromogenic in situ hybridization protocol for visualization of single RNA molecules in fixed cells and tissues. The protocol is based on padlock probing and rolling circle amplification to generate detectable chromogenic signal from single RNA molecules. Chromogenic signal can avoid background autofluorescence and can be preserved for a longer period than fluorescence signal.

ORAL HAIRY LEUKOPLAKIA IN A PEDIATRIC PATIENT UNDER CORTICOSTEROID THERAPY

Oral hairy leukoplakia (OHL) is a condition caused by Epstein-Barr virus (EBV) infection, usually associated with immunosuppressed patients. This study aims to report a case of atypical clinical presentation of OHL involving a 9-year-old girl, without previous history of immunosuppression. During anamnesis, the person in charge reported that the patient had recently started treatment for allergic dermatitis. Clinically, there were white corrugated lesions on both lateral tongue borders. Under the OHL diagnosis hypothesis, an incisional biopsy was performed. Microscopically, underlying the keratin layer, there were keratinocytes with pale cytoplasm, perinuclear haloes, and nuclei with margination of chromatin (nuclear beading). The final diagnosis was confirmed by chromogenic in situ hybridization (CISH) for EBV detection test. The occurrence of OHL in childhood is extremely uncommon, even in pediatric patients who are positive for human immunodeficiency virus (HIV). OHL should be included in the differential diagnosis when assessing white lesions in the oral mucosa. Oral hairy leukoplakia (OHL) is a condition caused by Epstein-Barr virus (EBV) infection, usually associated with immunosuppressed patients. This study aims to report a case of atypical clinical presentation of OHL involving a 9-year-old girl, without previous history of immunosuppression. During anamnesis, the person in charge reported that the patient had recently started treatment for allergic dermatitis. Clinically, there were white corrugated lesions on both lateral tongue borders. Under the OHL diagnosis hypothesis, an incisional biopsy was performed. Microscopically, underlying the keratin layer, there were keratinocytes with pale cytoplasm, perinuclear haloes, and nuclei with margination of chromatin (nuclear beading). The final diagnosis was confirmed by chromogenic in situ hybridization (CISH) for EBV detection test. The occurrence of OHL in childhood is extremely uncommon, even in pediatric patients who are positive for human immunodeficiency virus (HIV). OHL should be included in the differential diagnosis when assessing white lesions in the oral mucosa.

High-throughput RNA sequencing from paired lesional- and non-lesional skin reveals major alterations in the psoriasis circRNAome

BackgroundPsoriasis is a chronic inflammatory skin disease characterized by hyperproliferation and abnormal differentiation of keratinocytes. It is one of the most prevalent chronic inflammatory skin conditions in adults worldwide, with a considerable negative impact on quality of life. Circular RNAs (circRNAs) are a recently identified type of non-coding RNA with diverse cellular functions related to their exceptional stability. In particular, some circRNAs can bind and regulate microRNAs (miRNAs), a group of RNAs that play a role in the pathogenesis of psoriasis. The aim of this study was to characterize the circRNAome in psoriasis and to assess potential correlations to miRNA expression patterns.MethodsWe used high-throughput RNA-sequencing (RNA-seq), NanoString nCounter technology and RNA chromogenic in situ hybridization (CISH) to profile the circRNA expression in paired lesional and non-lesional psoriatic skin from patients with psoriasis vulgaris. In addition, 799 miRNAs were profiled using NanoString nCounter technology and laser capture microdissection was used to study the dermis and epidermis separately.ResultsWe found a substantial down-regulation of circRNA expression in lesional skin compared to non-lesional skin. We observed that this mainly applies to the epidermis by analyzing laser capture microdissected tissues. We also found that the majority of the circRNAs were downregulated independently of their corresponding linear host genes. The observed downregulation of circRNAs in psoriasis was neither due to altered expression levels of factors known to affect circRNA biogenesis, nor because lesional skin contained an increased number of inflammatory cells such as lymphocytes. Finally, we observed that the overall differences in available miRNA binding sites on the circRNAs between lesional and non-lesional skin did not correlate with differences in miRNA expression patterns.ConclusionsWe have performed the first genome-wide circRNA profiling of paired lesional and non-lesional skin from patients with psoriasis and revealed that circRNAs are much less abundant in the lesional samples. Whether this is a cause or a consequence of the disease remains to be revealed, however, we found no evidence that the loss of miRNA binding sites on the circRNAs could explain differences in miRNA expression between lesional and non-lesional skin.

Automatic quantification of HER2 gene amplification in invasive breast cancer from chromogenic in situ hybridization whole slide images.

Human epidermal growth factor receptor 2 (HER2), a transmembrane tyrosine kinase receptor encoded by the ERBB2 gene on chromosome 17q12, is a predictive and prognostic biomarker in invasive breast cancer (BC). Approximately 20% of BC are HER2-positive as a result of ERBB2 gene amplification and overexpression of the HER2 protein. Quantification of HER2 is performed routinely on all invasive BCs, to assist in clinical decision making for prognosis and treatment for HER2-positive BC patients by manually counting gene signals. We propose an automated system to quantify the HER2 gene status from chromogenic in situ hybridization (CISH) whole slide images (WSI) in invasive BC. The proposed method selects untruncated and nonoverlapped singular nuclei from the cancer regions using color unmixing and machine learning techniques. Then, HER2 and chromosome enumeration probe 17 (CEP17) signals are detected based on the RGB intensity and counted per nucleus. Finally, the HER2-to-CEP17 signal ratio is calculated to determine the HER2 amplification status following the ASCO/CAP 2018 guidelines. The proposed method reduced the labor and time for the quantification. In the experiment, the correlation coefficient between the proposed automatic CISH quantification method and pathologist manual enumeration was 0.98. The -values larger than 0.05 from the one-sided paired -test ensured that the proposed method yields statistically indifferent results to the reference method. The method was established on WSI scanned by two different scanners. Through the experiments, the capability of the proposed system has been demonstrated.

Optimization of trypsin treatment condition utilizing immunohistochemistry for chromogenic in situ hybridization.

Chromogenic in situ hybridization (CISH) is a molecular technique used to visualize specific genes. Both heat treatment and protease treatment play important roles for the success of CISH on formalin-fixed paraffin-embedded (FFPE) tissue sections. In contrast to heat treatment, the optimal condition of protease treatment may vary depending on each sample. Because trypsin has a substrate specificity to cleave lysine and arginine, we hypothesized that trypsin could effectively degrade histones rich in lysine and arginine and that the removal of histones from DNA following heat treatment could improve CISH results. We selected 21 patients with lung adenocarcinoma previously known to be positive or negative for anaplastic lymphoma kinase (ALK) gene rearrangement and used FFPE tissue sections collected from these patients. Then, we assessed histone degradation among the following protease treatments; trypsin, pepsin, and proteinase K, and compared the ALK CISH results with results obtained using commercially available kits and these protease treatments. The results showed that trypsin effectively degraded histones. Additionally, compared with the other treatments, ALK CISH with trypsin treatment showed the most evaluable cells and the smallest standard deviation. Our study suggests that the degradation of histones by trypsin subsequent to heat treatment might improve CISH results.