Chromogenic In Situ Hybridization Research Articles

Exploring the prognostic value of circular RNAs in pancreatic ductal adenocarcinoma using genome-wide expression profiling

Background/ObjectivesMedian survival of pancreatic ductal adenocarcinoma (PDAC) is around eight months and new prognostic tools are needed. Circular RNAs (circRNAs) have gained interest in different types of cancer. However, only a few studies have evaluated their potential in PDAC. We aimed to identify the most differentially expressed circRNAs in PDAC compared to controls and to explore their potential as prognostic markers. MethodsUsing frozen specimens with PDAC and controls, we performed RNA sequencing and identified 20,440 unique circRNAs. A custom code set of capture- and reporter probes for NanoString nCounter analysis was designed to target 152 circRNAs, based on abundancy, differential expression and a literature study. Expression of these 152 circRNAs was examined in 108 formalin-fixed and paraffin-embedded surgical PDAC specimens and controls. The spatial expression of one of the most promising candidates, ciRS-7 (hsa_circ_0001946), was evaluated by chromogenic in situ hybridization (CISH) using multi-punch tissue microarrays (TMAs) and digital imaging analysis. ResultsBased on circRNA expression profiles, we identified different PDAC subclusters. The 30 most differentially expressed circRNAs showed log2 fold changes from −3.43 to 0.94, where circNRIP1 (hsa_circ_0004771), circMBOAT2 (hsa_circ_0007334) and circRUNX1 (hsa_circ_0002360) held significant prognostic value in multivariate analysis. CiRS-7 was absent in PDAC cells but highly expressed in the tumor microenvironment. ConclusionsWe identified several new circRNAs with biomarker potential in surgically treated PDAC, three of which showed an independent prognostic value. We also found that ciRS-7 is absent in cancer cells but abundant in tumor microenvironment and may hold potential as marker of activated stroma.

Histopathological Tracing of HPV Genotypes 6 and 11 in Pediatric Patients in Medical City with Chronic Palatine and Pharyngeal Tonsillitis.

HPVs are considered to have high-oncogenic risk. These genotypes have been proven to have a causal link to cancers, in pediatric and youth patients, with high rates of HPV presence in the tonsillar tissues. A prospective case-control research for determining HPV 6/11 genotypes in tonsillar specimens of children who underwent operations in the otolaryngology departments of the Medical City Complex, Baghdad, Iraq, for their non-oncologic palatine and pharyngeal tonsillar hypertrophies. This study enrolled 102 tonsillar tissues, 82 from pediatric patients aged from 4 to 12 years and who underwent tonsillectomies for non-oncologic palatine and pharyngeal tonsillar hypertrophies; 38 specimens were from single operations while 22 were multiple specimens from the same pediatric patients, represented as a total of 44 tissues). In addition, trimmed nasal tissues from 20 patients, with unremarkable pathological changes, were included as the control group. For HPV 6/11 DNA detection, specific DNA probes were used for the chromogenic in situ hybridization (CISH) technique. In the palatine tonsillar hypertrophied tissue group, 26.2% of the tissues revealed positive CISH signals for HPV 6/11 DNA. Regarding the pharyngeal tonsillar hypertrophied tissues, 22.5% of the specimens expressed positive CISH reactions. Among the 22 pediatric patients who had combined pharyngeal and palatine tonsillectomies, in 22.7% both sites expressed positive signals. No positive-CISH reactions were documented in the control nasal tissues. Statistically a significant difference was seen when compared to the control group. Significant rates of HPV were observed which pointed to the spread of HPV, among other STIs, and in mothers of at least this studied pediatric group. Also, this represented a critical mark as reservoir tissue sites, allowing transmission to other mucosal tissue localizations, playing part in their pathogenesis.

In Situ Hybridization of Brain Slices.

In situ hybridization (ISH) is an important technique for identifying gene expression at the cellular level in various organs, including brain slices. This approach hybridizes nucleic acid probes to cellular mRNA, allowing the detection of transcriptional products. Recent advances have enabled RNA preservation in formalin-fixed paraffin-embedded (FFPE) samples, making ISH applicable to brain tumor diagnosis and research. Here, we provide a concise overview of the standard application of chromogenic ISH in neuroscience research and neuropathology practice using FFPE blocks of brain slice sections.

CSF1 expression in xanthogranulomatous epithelial tumor/keratin-positive giant cell-rich tumor

“Xanthogranulomatous epithelial tumor” (XGET) and “keratin-positive giant cell-rich soft tissue tumor” (KPGCT), two recently described mesenchymal neoplasms, likely represent different aspects of a single entity. Both tumors are composed of only a small minority of tumor cells surrounded by large numbers of non-neoplastic inflammatory cells and histiocytes, suggesting production of a paracrine factor with resulting “landscape effect,” as seen in tenosynovial giant cell tumor. Recent evidence suggests that the paracrine factor in XGET/KPGCT may be CSF1, as in tenosynovial giant cell tumor. We hypothesized that CSF1 is overexpressed in XGET/KPGCT. To test our hypothesis, we performed quantitative real time PCR (qPCR) for CSF1 expression and CSF1 RNAscope chromogenic in situ hybridization (CISH) on 6 cases of XGET/KPGCT. All cases were positive with CSF1 CISH and showed increased expression of CSF1 by qPCR. Our findings provide additional evidence that the CSF1/CSF1R pathway is involved in the pathogenesis of XGET/KPGCT. These findings suggest a possible role for CSF1R inhibition in the treatment of unresectable or metastatic XGET/KPGCT.

Chromogenic in situ hybridization reveals specific expression pattern of long non-coding RNA DRAIC in formalin-fixed paraffin-embedded specimen

Long non-coding RNA (lncRNA) plays an important role in the regulation of gene expression in normal and cancer cells. We previously discovered a novel tumor-suppressive lncRNA, DRAIC, in prostate cancer cells. Subsequent studies have demonstrated that DRAIC is dysregulated in various malignancies and exhibits a tumor-suppressive or pro-oncogenic function. However, details regarding its expression pattern in normal and cancerous tissues remain largely unknown. In this study, we performed chromogenic in situ hybridization (CISH) using RNAscope technology to assess DRAIC expression in formalin-fixed paraffin-embedded (FFPE) specimens. In the neuroendocrine-differentiated cancer cell line VMRC-LCD, CISH revealed a diffuse localization of DRAIC in the cytoplasm as well as specific accumulation in the nuclear compartment. DRAIC expression was comprehensively analyzed using tissue microarrays containing 89 normal and 155 tumor tissue samples. DRAIC was weakly expressed in normal epithelial cells of the colon, bronchiole, kidney, prostate, and testis. Conversely, DRAIC was moderately to highly expressed in some cancer tissues, including prostate adenocarcinoma, invasive ductal carcinoma of the breast, neuroendocrine carcinoma of the esophagus, lung adenocarcinoma, and small cell lung carcinoma. While DRAIC knockdown did not affect VMRC-LCD cellular viability and invasive ability, gene expression related to the neuroendocrine and cancer-related pathways was altered. Our expression analysis revealed the specific expression pattern of DRAIC in normal and cancerous FFPE tissues. The results presented here may lead to the elucidation of additional novel functions of DRAIC.

Chromogenic detection of telomere lengths in situ aids the identification of precancerous lesions in the prostate.

Telomeres are terminal chromosomal elements that are essential for the maintenance of genomic integrity. The measurement of telomere content provides useful diagnostic and prognostic information, and fluorescent methods have been developed for this purpose. However, fluorescent-based tissue assays are cumbersome for investigators to undertake, both in research and clinical settings. A robust chromogenic in situ hybridization (CISH) approach was developed to visualize and quantify telomere content at single cell resolution in human prostate tissues, both frozen and formalin-fixed, paraffin-embedded (FFPE). This new assay (telomere chromogenic in situ hybridization ["Telo-CISH"]) produces permanently stained slides that are viewable with a standard light microscope, thus avoiding the need for specialized equipment and storage. The assay is compatible with standard immunohistochemistry, thereby allowing simultaneous assessment of histomorphology, identification of specific cell types, and assessment of telomere status. In addition, Telo-CISH eliminates the problem of autofluorescent interference that frequently occurs with fluorescent-based methods. Using this new assay, we demonstrate successful application of Telo-CISH to help identify precancerous lesions in the prostate by the presence of markedly short telomeres specifically in the luminal epithelial cells. In summary, with fewer restrictions on the types of tissues that can be tested, and increased histologic information provided, the advantages presented by this novel chromogenic assay should extend the applicability of tissue-based telomere length assessment in research and clinical settings.

Genome Stability Profiles of Titania Nanosurface Technology for Biomedical Applications

The emergence of nano-based products for advanced biomedical applications has underlined the need for toxicogenomic studies which govern genome stability endpoints. In the present study, the genome stability profiles on cell-TNA interaction were assessed via nuclear staining profile by a fluorescent microscope, DNA ploidy profile by flow-cytometry, metaphase chromosome analysis, DNA topoisomerase II alpha (TOP2A) profiling by chromogenic in situ hybridization (CISH), cytokinesis-blocked micronucleus assay (CBMN), reactive oxygen species (ROS) measurement and gene expression profile by real-time polymerase chain reaction (RT-PCR). Findings from this study demonstrated that no significant genomic instability risk was observed from cell-TNA interaction, especially at clastogenic and aneugenic levels. Findings also suggest that cell-TNA interaction could involve the regulation of ribonucleoprotein complex and cell metabolism associated with the cellular adaptation process towards the nanosurface. Further comprehensive studies involving in vivo models are needed to support this work.

Evaluation of HER2 Positivity Based on Clinicopathological Findings in HER2 Borderline Tumors in Iranian Patients with Breast Cancer.

Human epidermal growth receptor-2 (HER2) gene amplification is an important predictive and prognostic factor in breast cancer treatment. However, the expression of HER2 determined by immunohistochemistry (IHC) is considered as borderline in some cases, and confirmation of the HER2 status by either fluorescent in situ hybridization (FISH) or chromogenic in situ hybridization (CISH) is necessary for correct treatment decision-making. Considering the high cost of FISH and CISH, we aimed to investigate whether clinicopathological findings of the tumor could predict the HER2 status. A retrospective study was performed using the data from 584 patients with breast cancer with HER2-borderline disease, confirmed by IHC. Final HER2 status, pathologic tumor size and type, nodal involvement, Ki67 index, presence of estrogen and progesterone receptors (ER, PR), lymphovascular invasion (LVI), and stage were retrieved from the clinical records. One hundred twenty-one (20.7%) patients were HER2-positive according to the FISH or CISH results. Logistic regression analysis showed that the pathologic size was positively associated with HER2 positivity with an odds ratio (OR) of 1.02 (95% CI: 1.01-1.04). In addition, the adjusted OR illustrated a statistically significant association between HER2 positivity and PR negativity (OR= 2.22, 95% CI: 1.29-3.83). In HER2 borderline breast cancer, HER2 positivity significantly increases with tumor size and PR negativity. Further studies are recommended that may find an applicable model to predict the actual status of HER2 in borderline cases.

Exo-erythrocytic development of Leucocytozoon parasites (Haemosporida, Leucocytozoidae) in song thrushes Turdus philomelos

Leucocytozoon parasites (Haemosporida, Leucocytozoidae) are haemosporidians whose diversity, exo-erythrocytic development and potential vectors are the least studied. The knowledge about their exo-erythrocytic development and pathogenicity is fragmentary, resulting in an incomplete comprehension of the impact of these parasites on avian hosts. For a long time, Leucocytozoon infections were considered benign to wild birds, even though they were virulent in poultry and responsible for some wild bird population declines. This study aimed to investigate the presence of Leucocytozoon species exo-erythrocytic stages in song thrushes Turdus philomelos using conventional histological techniques (sections stained by H&E) and chromogenic in situ hybridization (CISH). Tissues from ten birds (seven naturally infected and three opportunistic samplings) were examined using both methods. Parasite lineages were identified from blood samples using PCR-based techniques. Leucocytozoon species meronts were found in five individuals (in four birds using H&E staining protocol, and in three in CISH-treated histological sections). Meronts were found mainly in the kidneys, but some meronts were also present in the lungs. It was possible to observe different maturation stages of meronts in the same bird individual, indicating an asynchronous development. Cytomeres were readily visible in developing meronts. One megalomeront-like structure was present close to a blood vessel in the heart. It was covered with a prominent capsular-like wall. No inflammatory reaction or necrosis was seen in the tissues surrounding the meronts or the megalomeront-like structure. We could confirm the transmission of three Leucocytozoon lineages (lTUPHI14, lSTUR1 and lTUPHI13) in Europe, and add evidence of the transmission of two Plasmodium lineages, including Plasmodium circumflexum (pTURDUS1), and Haemoproteus asymmetricus (hTUPHI01). We call for further research to better understand Leucocytozoon parasite exo-erythrocytic development.

HER2 Status in RAS and BRAF Wild-Type Metastatic Colorectal Cancer: A Portuguese Study.

Colorectal cancer (CRC) is the second-most deadly cancer worldwide. However, there remains a scarcity of precision treatmentsavailable for this type of cancer. Amplification or overexpression of human epidermal growth factor receptor 2 (HER2+) is a well-established therapeutic target in gastric and breast cancer. HER2is positive in approximately 5% of CRC cases and has been implicated in resistance to therapy with anti-epidermal growth factor receptor antibodies. The aim of this study was to evaluate HER2 status in RAS and BRAF wild-type metastatic CRC (mCRC) and its correlation with survival outcomes. A single-center retrospective analysis of RAS and BRAF wild-type mCRC patients undergoing systemic treatment was conducted from July 2014 to September 2020. Tissue HER2 status was determined by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH) and/or chromogenic in situ hybridization (CISH). HER2+ was defined as IHC3 (+) or IHC2 (+) through FISH or CISH (+). Fifty-nine patients were included. The median age of all the included patients was 64 years (33-82). Four patients had HER2+ tumors (7%). Four patients had HER2+ tumors (7%).The majority of HER2+ mCRC cases were males (n=3) and left-sided CRC (n=3). All patients received FOLFIRI plus cetuximab as first-line treatment. At the median follow-up of 24.0 months, patients with HER2-negative mCRC presented with a median overall survival (mOS) of 39.4 months (95% confidence interval (CI) 32.7-46.0) and the four patients with HER2+ mCRC had a mOS of 20.4 months(95% CI; 9.5-31.3; p=0.07). In HER2-negative patients, themedian PFS (mPFS) was 11.3 months (95% CI; 9.2-13.4) vsHER2-positive patients with a mPFS of 10.9 months (95% CI; 1.3-20.4;p=0.47). To our knowledge, this is the first study reporting HER2+ in mCRC patients in a Portuguese population and the HER2+ rate was consistent with previous studies. Our study suggests that HER2+ may potentially be a marker that is able to predict poor prognosis in RAS and BRAF wild-type mCRC.

Chromogenic in situ hybridization technique for detecting porcine circovirus 3 in lung and lymphoid tissues.

Porcine circovirus 3 (PCV3) was recently reported in Malaysian commercial pig population in 2020 by conventional polymerase chain reaction (PCR), revealing a molecular prevalence of 17.02% in the sampled domestic pig population. This study aims to describe a chromogenic in situ hybridization (ISH) technique using digoxigenin (DIG)-labeled cloned PCV3 open reading frame 1 (ORF1) fragment DNA to detect and localize the PCV3 antigen in formalin-fixed, paraffin-embedded lung, and lymphoid tissue specimens. Since PCV3 was mainly detected in lung and lymphoid tissues, we obtained tissue specimens from these organs from the previous Malaysian PCV3 study. Digoxigenin-labeled ISH probes were designed to target a 69 bp region of PCV3 ORF1 spanning from the nucleotide positions (282-350). Light microscopy analysis revealed that chromogenic staining of PCV3 antigens was visualized within the cytoplasm of pneumocytes and lymphocytes, indicating positive ISH results. The results of molecular detection of PCV3 using PCR and ISH showed a high agreement of 90.91%, including for the negative PCV3 status for all samples. This study reports a chromogenic ISH technique using DIG-labeled probes targeting PCV3 ORF1 to detect PCV3 antigens in lung and lymphoid tissues. Despite the limited availability of PCV3 antibodies, ISH remains relevant for investigating PCV3 replication and pathogenesis and can be used complementarily with PCR for evaluating the localization of antigens in infected tissues.

Utility of GLI1 RNA Chromogenic in Situ Hybridization in Distinguishing Basal Cell Carcinoma From Histopathologic Mimics

Basal cell carcinoma (BCC) is the most common human malignancy and is a leading cause of nonmelanoma skin cancer-related morbidity. BCC has several histologic mimics which may have treatment and prognostic implications. Furthermore, BCC may show alternative differentiation toward a variety of cutaneous structures. The vast majority of BCCs harbor mutations in the hedgehog signaling pathway, resulting in increased expression of the GLI family of transcription factors. GLI1 immunohistochemistry has been shown to discriminate between several tumor types but demonstrates high background signal and lack of specificity. In this study, we evaluated the utility of GLI1 RNA chromogenic in situ hybridization (CISH) as a novel method of distinguishing between BCC and other epithelial neoplasms. Expression of GLI1 by RNA CISH was retrospectively evaluated in a total of 220 cases, including 60 BCCs, 37 squamous cell carcinomas (SCCs) including conventional, basaloid, and human papillomavirus infection (HPV)-associated tumors, 16 sebaceous neoplasms, 10 Merkel cell carcinomas, 58 benign follicular tumors, and 39 ductal tumors. The threshold for positivity was determined to be greater than or equal to 3 GLI1 signals in at least 50% of tumor cells. Positive GLI1 expression was identified in 57/60 BCCs, including metastatic BCC, collision lesions with SCC, and BCCs with squamous, ductal, or clear cell differentiation or with other unusual features compared to 1/37 SCCs, 0/11 sebaceous carcinomas, 0/5 sebaceomas, 1/10 Merkel cell carcinomas, 0/39 ductal tumors, and 28/58 follicular tumors. With careful evaluation, GLI1 RNA CISH is highly sensitive (95%) and specific (98%) in distinguishing between BCC and nonfollicular epithelial neoplasms. However, GLI1 CISH is not specific for distinguishing BCC from most benign follicular tumors. Overall, detection of GLI1 RNA by CISH may be a useful tool for precise classification of histologically challenging basaloid tumors, particularly in the setting of small biopsy specimens, metaplastic differentiation, or metastatic disease.

Histopathologic and molecular characterization of Erethizon dorsatum papillomavirus 1 and Erethizon dorsatum papillomavirus 2 infection in North American porcupines (Erethizon dorsatum).

Erethizon dorsatum papillomavirus 1 (EdPV1) and Erethizon dorsatum papillomavirus 2 (EdPV2) are associated with cutaneous papillomas in North American porcupines (Erethizon dorsatum). This study defined gross, histopathologic, and molecular characteristics of viral papillomas in 10 North American porcupines submitted to the New York State Animal Health Diagnostic Center. Investigation for the presence of EdPV1 and EdPV2 DNA via polymerase chain reaction (PCR) was performed in 9 of the 10 (90.0%) porcupines, and all porcupines were investigated for the detection and localization of EdPV1 and EdPV2 E6 and E7 nucleic acid via chromogenic in situ hybridization (CISH). Next-generation sequencing (NGS) was performed in 2 porcupines. Papillomas were diagnosed on the muzzle (n = 4), caudal dorsum (n = 1), upper lip (n = 1), chin (n = 1), gingiva (n = 2), and nasal planum (n = 1). Histologically, the lesions consisted of hyperplastic epidermis or epithelium with orthokeratotic keratin, prominent keratohyalin granules, and intranuclear inclusion bodies. PCR identified EdPV1 in 6 of 9 samples and EdPV2 in the remaining 3 samples. NGS resulted in 100% genome coverage of EdPV1 and 76.20% genome coverage of EdPV2 compared with GenBank reference sequences, with 99.8% sequence identity to the complete EdPV2 L1 gene of a novel subtype recently identified in France. Hybridization patterns in 9 of the 10 (90.0%) porcupines were characterized by strong nuclear signals in the superficial epidermis, with strong nuclear and punctate cytoplasmic signals in the stratum spinosum and basale. In one animal, CISH suggested dual EdPV1 and EdPV2 infection.

Exo-erythrocytic development of two Haemoproteus species (Haemosporida, Haemoproteidae), with description of Haemoproteus dumbbellus, a new blood parasite of bunting birds (Emberizidae)

Avian haemosporidians are widespread parasites categorized into four families of the order Haemosporida (Apicomplexa). Species of the subgenus Parahaemoproteus (genus Haemoproteus) belong to the Haemoproteidae and are transmitted by Culicoides biting midges. Reports of death due to tissue damage during haemoproteosis in non-adapted birds have raised concerns about these pathogens, especially as their exo-erythrocytic development is known for only a few Haemoproteus spp. More research is needed to better understand the patterns of the parasites’ development in tissues and their impact on avian hosts. Yellowhammers Emberiza citrinella (Emberizidae) and common house martins Delichon urbicum (Hirundinidae) were screened for Haemoproteus parasites by microscopic examination of blood films and PCR-based testing. Individuals with single infection were selected for histological investigations. H & E-stained sections were screened for detection and characterization of the exo-erythrocytic stages, while chromogenic in situ hybridization (CISH) and phylogenetic analysis were performed to confirm the Haemoproteus origin and their phylogenetic relationships. Haemoproteus dumbbellus n. sp. was discovered in Emberiza citrinella single-infected with the lineage hEMCIR01. Meronts of H. dumbbellus n. sp. developed in various organs of five of six tested individuals, a pattern which was reported in other Haemoproteus species clustering in the same clade, suggesting this could be a phylogenetic trait. By contrast, in Delichon urbicum infected with the Haemoproteus lineage hDELURB2, which was linked to the more distantly related parasite Haemoproteus hirundinis, only megalomeronts were found in the pectoral muscles of two of six infected individuals. All exo-erythrocytic stages were confirmed to be Haemoproteus parasites by CISH using a Haemoproteus genus-specific probe. While the development of meronts seems to be typical for species of the clade containing H. dumbbellus, further investigations and data from more species are needed to explore whether a phylogenetic pattern occurs in meront or megalomeront formation.

Evaluation of HER2 Gene Amplification using CISH in Patients with HER2 2+ (equivocal) Breast Carcinoma based on Immunohistochemistry in Imam Khomeini Cancer Institute from 2016 to 2018

Background: Human epidermal growth factor 2 (HER2) is known to be an important prognostic factor in breast cancer. Numerous studies have shown HER2+ breast cancers have reduced overall survival and recovery time, as well as the efficacy of anti-HER2 therapies along with chemotherapy in improving disease outcomes. For this reason, it is recommended that all patients with breast cancer should be evaluated for HER2 status. This study aimed to assess the HER2 gene amplification by the CISH method in evaluating the HER2 status in patients with immunohistochemistry (IHC) 2+ (equivocal) results. Methods: This retrospective cross-sectional study examined HER2 status based on the Chromogenic in situ hybridization (CISH) method in 280 breast carcinoma samples with an initial 2+ (equivocal) score in IHC. The relationship between HER2 amplification and hormone receptors (estrogen and progesterone) and Ki67 level was also investigated. Results: In sixty samples (21.4%), the HER2 gene was amplified based on the CISH method. The majority (215, 76.8%) of the samples were negative and 5 (1.8%) samples were indeterminate. No significant relationship was observed between HER2 amplification, estrogen receptor (p=0.143), and ki.67 protein level (p=0.977). There was a significant inverse relationship between HER2 amplification and progesterone receptor positivity (p=0.007). Conclusion: These results demonstrate that CISH is a helpful method to assess HER2 status in equivocal breast cancer and is positive (amplified) in about 21.4% of them.

Diverse Expression Patterns of EBV Oncogenes (LMP2A, EBV-Encoded microRNA, and EBV-encoded dUTPase) in EBV Associated Gastric Carcinoma and their Association with Viral Loads.

The chromogenic in situ hybridization (CISH) test is the gold standard for detecting Epstein-Barr virus (EBV)-associated gastric carcinoma (GC). Real-time (RT) PCR method is also a sensitive test that can detect the viral load in samples. As such, three EBV oncogenes were investigated in this study. RNA extraction and cDNA synthesis were performed on GC tissues of nine patients, who were previously confirmed to have EBVGC subtype. In addition, 44 patients that had positive RT-PCR but negative CISH results were also included as the control group. TaqMan RT-PCR analysis was performed to determine the expression of EBV-encoded microRNAs, and the expression of EBV-encoded dUTPase, as well as LMP2A, was analyzed by SYBR Green RT-PCR. EBV-encoded microRNAs and LMP2A were identified in 2 out of 9 (22%) EBVGC subtypes. In addition, EBV-encoded dUTPase was detected in 4 out of 9 (44.5%) EBVGC subtypes. EBV-encoded dUTPase was also expressed in a sample of the control group. The expression of LMP2A, EBV-encoded microRNAs, and EBV-encoded dUTPase viral oncogenes in patients with high EBV viral loads indicates that these expressions correlate with viral loads. Our findings indicate that the EBV-encoded dUTPase gene may have a role in EBVGC patients' non-response to treatment and might be considered a Biomarker-targeted therapy.

False positivity in break apart fluorescence in-situ hybridization due to polyploidy.

In-situ hybridization (ISH) is a diagnostic tool in the detection of chromosomal anomalies, which has important implications for diagnosis, classification and prediction of cancer therapy in various diseases. Certain thresholds of number of cells showing an aberrant pattern are commonly used to declare a sample as positive for genomic rearrangements. The phenomenon of polyploidy can be misleading in the interpretation of break apart fluorescence in-situ hybridization (FISH). The aim of this study is to investigate the impact of cell size and ploidy on FISH results. In sections of varying thickness of control liver tissue and non-small cell lung cancer cases, nuclear size was measured and the number of MET chromogenic ISH and ALK FISH (liver) or ALK and ROS1 FISH (lung cancer) signals was manually counted and quantified. In liver cell nuclei the number of FISH/chromogenic ISH signals increases with nuclear size related to physiological polyploidy and is related to section thickness. In non-small cell lung cancer cases tumour cells with higher ploidy levels and nuclear size have an increased chance of single signals. Furthermore, additional lung cancer samples with borderline ALK FISH results were examined with a commercial kit for rearrangements. No rearrangements could be demonstrated, proving a false positive ALK FISH result. In case of polyploidy there is an increased likelihood of false positivity when using break apart FISH probes. Therefore, we state that prescribing one single cut-off in FISH is inappropriate. In polyploidy, the currently proposed cut-off should only be used with caution and the result should be confirmed by an additional technique.

New Presentation of CD27 Deficiency; Coronary Ectasia and COVID-19.

CD27 is a costimulatory receptor involved in the maturation of the innate and adaptive immunity. CD27, through interaction with CD70, plays a role in the control of Epstein-Barr virus (EBV) infection. CD27 deficiency leads to an immune dysregulation disease characterized by EBV susceptibility. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) might put patients with primary immunodeficiency at risk for adverse outcomes. Chromogenic in situ hybridization (CISH) study was performed to detect EBV in the lymphoma tissue. Genetic analysis of the patient was done with Whole Exome Sequencing and detected variant was confirmed with PCR-Sanger sequencing. Here we report a 20-month-old boy with CD27 deficiency who developed lymphoma and coronary artery ectasia and had been infected with SARS-CoV-2. Clinical and laboratory findings were incompatible with atypical Kawasaki syndrome or multisystem inflammatory syndrome in children (MIS-C). As CD27 deficiency is a rare immune defect, publishing clinical data about the identified patient(s) can shed light on our knowledge about the related phenotype and the spectrum of clinical manifestations associated with CD27 deficiency. Thus, our findings expanded the spectrum of manifestations beyond EBV infection, highlighting this unusual cardiac sequela that could be related to EBV infection, lymphoma, or an underlying disease.

Oesophageal squamous papilloma in paediatric population: a single-centre case series

PurposeOesophageal squamous papilloma (OSP) is a rare epithelial lesion with an unclear aetiology, found incidentally in upper gastrointestinal endoscopy (UGE). We evaluate the epidemiology, general features and endoscopic and histological...

Metaplastic Breast Carcinoma (MBC), Primary Squamous Cell Carcinoma Subtype in A 49-Year-Old Woman: A Case Report

Introduction: Metaplastic Breast Carcinoma (MBC) is quite a challenging case because metaplastic breast cancer is one of the rarest subtypes of invasive breast cancer. It is reported that MBC occurs only 0.2 to 1% throughout the world. The metaplastic changes can be squamous cells or other mesenchymal cell types. Clinically, MBC presents as a large palpable mass and may be associated with rapid growth. The size of MBC tends to be larger compared with other types of invasive breast cancer ranging from 1 to more than 10 cm. Although there are several main categories of MBC, some carcinoma can be difficult to classify due to their unusual histologic patterns. This case report study is to provide a clinicopathological overview and approach to MBC.Case Presentation: We reported a 49-year-old woman who suffered from a breast mass that rapidly grew for less than one year. The microscopic findings showed squamous cell carcinoma. While molecular studies revealed triple negative results for hormone receptors although Human Epidermal Growth Factor Receptor 2 (HER2) overexpression was unusual (< 5%). Then, we confirmed with chromogen in situ hybridization (CISH) and there was no gen amplification for HER2. Microscopically, we found ductal carcinoma in situ and this finding supported breast origin.Conclusions: Metaplastic carcinoma did not have any specific and distinctive signs clinically. Metaplastic carcinoma can be monophasic (with only a metaplastic component) or biphasic with two or more components. As treatment options, our patient received conventional chemotherapy. Metaplastic breast cancer is reported to have a lower response rate to conventional adjuvant chemotherapy and worse clinical outcome after chemotherapy than other forms of triple-negative breast cancer.