Bovine Meat and Milk Factor-like Sequences Are Frequently Detected in Renal Cell Carcinoma Tissues.

Previous studies have indicated a potential role of diet in the pathogenesis of renal cell carcinoma (RCC). Recently, circular bovine meat and milk factor (BMMF) DNAs have been identified in peritumoral tissues of human colon and breast cancers. Here, we investigated the prevalence of the DNA of these novel human pathogenic infectious agents in RCC and adjacent peritumoral renal tissues. DNA was extracted from formalin-fixed and paraffin-embedded (FFPE) RCC and peritumoral kidney tissues, including a test (n = 11) and a validation (n = 152) collection. BMMF1 and BMMF2 consensus primers were designed to screen for the presence of BMMF1- and BMMF2-like DNA. In addition, BMMF-specific PCR was performed on selected cases to test for the presence of additional regions of BMMF1 and BMMF2 genomes. A reference collection of hepatocellular carcinomas (HCCs; n = 60) and adjacent peritumoral liver tissues (n = 50) was also included. Our results demonstrated that BMMF1 and BMMF2 DNAs are frequently found in human RCC tissues and are particularly more prevalent in peritumoral kidney tissues. Of note, BMMF1 and BMMF2 genotype heterogeneity was higher in peritumoral kidney tissues compared to RCC tissues. This is the first study to directly test human FFPE tissues for BMMF1- and BMMF2-like DNA using consensus PCR and demonstrate BMMF DNA in neoplastic and peritumoral kidney tissues. The findings are in line with the recently proposed indirect etiopathogenetic role of BMMFs in, e.g., colorectal carcinogenesis. Follow-up studies are needed to explore the potential role of BMMFs in the etiopathogenesis of RCC.

Association of Coagulopathy and Inflammatory Biomarkers with Severity in SARS-CoV-2-Infected Individuals of the Al-Qunfudhah Region of Saudi Arabia.

BACKGROUND: Identifying prognosticators/predictors of COVID-19 severity is the principal focus for early prediction and effective management of the disease in a time-bound and cost-effective manner. We aimed to evaluate COVID-19 severity-dependent alteration in inflammatory and coagulopathy biomarkers. METHODS: A hospital-dependent retrospective observational study (total: n = 377; male, n = 213; and female, n = 164 participants) was undertaken. COVID-19 exposure was assessed by performing real-time PCR on nasopharyngeal (NP) swabs. Descriptive and inferential statistics were applied for both continuous and categorical variables using Rstudio-version-4.0.2. Pearson correlation and regression were executed with a cut-off of p < 0.05 for evaluating significance. Data representation by R-packages and ggplot2. RESULTS: A significant variation in the mean ± SD (highly-sever (HS)/moderately severe (MS)) of CRP (HS/MS: 102.4 ± 22.9/21.3 ± 6.9, p-value < 0.001), D-dimer (HS/MS: 661.1 ± 80.6/348.7 ± 42.9, p-value < 0.001), and ferritin (HS/MS: 875.8 ± 126.8/593.4 ± 67.3, p-value < 0.001) were observed. Thrombocytopenia, high PT, and PTT exhibited an association with the HS individuals (p < 0.001). CRP was correlated with neutrophil (r = 0.77), ferritin (r = 0.74), and WBC (r = 0.8). D-dimer correlated with platelets (r = -0.82), PT (r = 0.22), and PTT (r = 0.37). The adjusted odds ratios (Ad-OR) of CRP, ferritin, D-dimer, platelet, PT, and PTT for HS compared to MS were 1.30 (95% CI -1.137, 1.50; p < 0.001), 1.048 (95% CI -1.03, 1.066; p < 0.001), 1.3 (95% CI -1.24, 1.49, p > 0.05), -0.813 (95% CI -0.734, 0.899, p < 0.001), 1.347 (95% CI -1.15, 1.57, p < 0.001), and 1.234 (95% CI -1.16, 1.314, p < 0.001), respectively. CONCLUSION: SARS-CoV-2 caused alterations in vital laboratory parameters and raised ferritin, CRP, and D-dimer presented an association with disease severity at a significant level.

Mutational Analysis of Circulating Omicron SARS-CoV-2 Lineages in the Al-Baha Region of Saudi Arabia.

Purpose: Omicron (B.1.1.529) is one of the highly mutated variants of concern of SARS-CoV-2. Lineages of Omicron bear a remarkable degree of mutations leading to enhanced pathogenicity and upward transmission trajectory. Mutating Omicron lineages may trigger a fresh COVID-19 wave at any time in any region. We aimed at the whole-genome sequencing of SARS-CoV-2 to determine variants/subvariants and significant mutations which can foster virus evolution, monitoring of disease spread, and outbreak management. Methods: We used Illumina-NovaSeq 6000 for SARS-CoV-2 genome sequencing, MEGA 10.2 and nextstrain tools for phylogeny; CD-HIT program (version 4.8.1) and MUSCLE program for clustering and alignment. At the same time, UCSF Chimera was employed for protein visualization. Results: Predominant Omicron pango lineages in Al-Baha were BA.5.2/B22 (n=4, 57%), and other lineages were BA.2.12/21L (n=1, 14.28%), BV.1/22B (n=1, 14.28%) and BA.5.2.18/22B (n=1, 14.28%). 22B nextstrain clade was predominant, while only one lineage showed 21L. BA.5.2/22B, BA.5.2/22B harbored a maximum of n=24 mutations in the spike region. Twelve crucial RBD mutations: D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, and Y505H were identified except the lineage BA.5.2/22B in which F486V mutation was not observed. Critical deletions S106 in membrane protein NSP6, E31in nucleocapsid, and L24 in spike region were observed in all the lineages. Furthermore, we identified common mutations of Omicron variants of SARS-CoV-2 in therapeutic hot spot spike region: T19I, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, A653V, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, D1146D, L452R, F486V, N679K and D796Y. The effect of RBD-targeted mutations on neutralizing (NAbs) binding was considerable. Conclusion: The outcome of this first report on SARS-CoV-2 variants identification and mutation in the Al-Baha region could be used to lay down the policies to manage and impede the regional outbreak of COVID-19 effectively.

Association of mutation and expression of the brother of the regulator of imprinted sites (BORIS) gene with breast cancer progression.

INTRODUCTION: The BORIS, 11 zinc-finger transcription factors, is a member of the cancer-testis antigen (CTA) family. It is mapped to chromosome number 20q13.2 and this region is genetically linked to the early onset of breast cancer. The current study analyzed the correlation between BORIS mutations and the expression of the protein in breast cancer cases. MATERIALS AND METHODS: A population-based study including a total of 155 breast cancer tissue samples and an equal number of normal adjacent tissues from Indian female breast cancer patients was carried out. Mutations of the BORIS gene were detected by polymerase chain reaction-single standard confirmation polymorphisms (PCR-SSCP) and automated DNA sequencing and by immunohistochemistry for BORIS protein expression were performed. The observed findings were correlated with several clinicopathological parameters to find out the clinical relevance of associations. RESULTS: Of all the cases 16.12% (25/155) showed mutations in the BORIS gene. The observed mutations present on codon 329 are missense, leading to Val> Ile (G>A) change on exon 5 of the BORIS gene. A significant association was observed between mutations of the BORIS gene and some clinicopathological features like nodal status (p = 0.013), estrogen receptor (ER) expression (p = 0.008), progesterone receptor (PR) expression (p = 0.039), clinical stage (p = 0.010) and menopausal status (p = 0.023). The protein expression analysis showed 20.64% (32/155) samples showing low or no expression (+), 34.19% (53/155) with moderate expression (++), and 45.17% (70/155) showing high expression (+++) of BORIS protein. A significant association was observed between the expression of BORIS protein and clinicopathological features like clinical stage (p = 0.013), nodal status (p = 0.049), ER expression (p = 0.039), and PR expression (p = 0.027). When mutation and protein expression were correlated in combination with clinicopathological parameters a significant association was observed in the category of high (+++) level of BORIS protein expression (p = 0.017). CONCLUSION: The BORIS mutations and high protein expression occur frequently in carcinoma of the breast suggesting their association with the onset and progression of breast carcinoma. Further, the BORIS has the potential to be used as a biomarker.

Human polyomaviruses JCPyV and MCPyV in urothelial cell carcinoma: a single institution experience.

Objective: Urothelial cell carcinoma (UCC) is the most common type of urinary bladder. JCPyV and BKPyV have been detected in the urine and tissue of urothelial cell carcinomas (UCC) in immunocompetent patients. Here, we investigated the presence of several HPyVs in UCC samples using diverse molecular techniques to study the prevalence of HPyVs in UCC. Methods: A large single-institution database of urine cytology specimens (UCS; n = 22.867 UCS) has previously been searched for decoy cells (n = 30), suggesting polyomavirus infection. The available urine sediments and formalin-fixed paraffin-embedded (FFPE) tissue samples of UCC patients were tested for the presence of JCPyV-LTAg expression by immunohistochemistry (IHC) labeled with SV40-LTAg antibody (clone: PAb416) and subsequent PCR followed by sequencing. In addition, the presence of the oncogenic Merkel cell polyomavirus (MCPyV) and the presence of human polyomavirus 6 (HPyV6) and 7 (HPyV7) DNA were tested with DNA PCR or IHC. Results: Of the 30 patients harboring decoy cells, 14 were diagnosed with UCC of the urinary bladder (14/30; 46.6%) before presenting with decoy cells in the urine. The SV40-LTAg IHC was positive in all 14 UCC urine sediments and negative in the FFPE tissues. JCPyV-DNA was identified in all five available UCS and in three FFPE samples of UCC (three of 14; 21.4%). Two UCC cases were positive for MCPyV-DNA (two of 14; 14.3%), and one of them showed protein expression by IHC (one of 14; 7.1%). All specimens were HPyV6 and HPyV7 negative. Conclusion: Our findings show the presence of JCPyV in the urine and UCC of immunocompetent patients. Moreover, MCPyV was detected in two UCC cases. In total, five UCC cases showed the presence of either JCPyV or MCPyV. The evidence here supports the hypothesis that these viruses might sporadically be associated with UCC. Further studies are needed to confirm the relevance of JCPyV or MCPyV as a possible risk factor for UCC development.

Emerging role of human polyomaviruses 6 and 7 in human cancers.

BACKGROUND: Currently 12 human polyomaviruses (HPyVs) have been identified, 6 of which have been associated with human diseases, including cancer. The discovery of the Merkel cell polyomavirus and its role in the etiopathogenesis in the majority of Merkel cell carcinomas has drawn significant attention, also to other novel HPyVs. In 2010, HPyV6 and HPyV7 were identified in healthy skin swabs. Ever since it has been speculated that they might contribute to the etiopathogenesis of skin and non-cutaneous human cancers. MAIN BODY: Here we comprehensively reviewed and summarized the current evidence potentially indicating an involvement of HPyV6 and HPyV7 in the etiopathogenesis of neoplastic human diseases. The seroprevalence of both HPyV6 and 7 is high in a normal population and increases with age. In skin cancer tissues, HPyV6- DNA was far more often prevalent than HPyV7 in contrast to cancers of other anatomic sites, in which HPyV7 DNA was more frequently detected. CONCLUSION: It is remarkable to find that the detection rate of HPyV6-DNA in tissues of skin malignancies is higher than HPyV7-DNA and may indicate a role of HPyV6 in the etiopathogenesis of the respected skin cancers. However, the sheer presence of viral DNA is not enough to prove a role in the etiopathogenesis of these cancers.

Reactivation of BK Polyomavirus in Urine Cytology is Not Associated with Urothelial Cell Carcinoma.

BK polyomavirus (BKPyV) has been associated with some high-grade and special urothelial cell carcinoma (UCC) subtypes in immunosuppressed patients. Here, we evaluated the relationship of BKPyV-positive urine cytology specimens (UCS) with UCC. A large single-institution database was retrospectively searched for UCS positive for decoy cells, suggesting BKPyV infection. These were tested for the presence of BKPyV by PCR and immunohistochemistry (IHC) in urine sediments and formalin-fixed paraffin-embedded (FFPE) tissue samples of UCC. Decoy cells were reported in 30 patients out of the database with 22.867 UCS. Of these 30 patients, 16 (53.3%) had no history of UCC. Six patients out of these 16 had a history of transplantation, 4 had a history of severe chronic medical conditions, and 6 had no chronic disease. The other fourteen patients were diagnosed with either in situ or invasive UCC of the urinary bladder (14/30; 46.6%) prior to the detection of decoy cells in the urine. Nine of these UCC patients received intravesical treatment (BCG or mitomycin) after the first presentation with UCC. However, the clinical data on the treatment of the other five UCC patients was lacking. IHC identified BKPyV-positivity in the urine samples of non-UCC and UCC patients, while no BKPyV positivity was found in FFPE tissues of primary UCCs and metastases. In addition, BKPyV-PCR results revealed the presence of BKPyV DNA in the urine of the UCC cases, yet none in the UCC tissues itself. These data strongly indicate that BKPyV reactivation is not restricted to immunosuppression. It can be found in UCS of the immunocompetent patients and may be related to the intravesical BCG or mitomycin treatment of the UCC patients.

High Prevalence of Human Polyomavirus 7 in Cholangiocarcinomas and Adjacent Peritumoral Hepatocytes: Preliminary Findings.

Cholangiocarcinoma (CCA) is a rare biliary-duct malignancy with poor prognosis. Recently, the presence of the human polyomavirus 6 (HPyV6) has been reported in the bile of diverse hepatobiliary diseases, particularly in the bile of CCA patients. Here, we investigated the presence of novel HPyVs in CCA tissues using diverse molecular techniques to assess a possible role of HPyVs in CCA. Formalin-Fixed Paraffin-Embedded (FFPE) tissues of 42 CCA patients were included in this study. PCR-based screening for HPyVs was conducted using degenerated and HPyV-specific primers. Following that, we performed FISH, RNA in situ hybridization (RNA-ISH), and immunohistochemistry (IHC) to assess the presence of HPyVs in selected tissues. Of all 42 CCAs, 25 (59%) were positive for one HPyV, while 10 (24%) CCAs were positive for 2 HPyVs simultaneously, and 7 (17%) were negative for HPyVs. Of the total 35 positive CCAs, 19 (45%) were positive for HPyV7, 4 (9%) for HPyV6, 2 (5%) for Merkel cell polyomavirus (MCPyV), 8 (19%) for both HPyV7/MCPyV, and 2 (5%) for both HPyV6/HPyV7 as confirmed by sequencing. The presence of viral nucleic acids was confirmed by specific FISH, while the RNA-ISH confirmed the presence of HPyV6 on the single-cell level. In addition, expression of HPyV7, HPyV6, and MCPyV proteins were confirmed by IHC. Our results strongly indicate that HPyV7, HPyV6, and MCPyV infect bile duct epithelium, hepatocytes, and CCA cells, which possibly suggest an indirect role of these viruses in the etiopathogenesis of CCA. Furthermore, the observed hepatotropism of these novel HPyV, in particular HPyV7, might implicate a role of these viruses in other hepatobiliary diseases.

Presence of Human Papillomavirus and Epstein-Barr Virus, but Absence of Merkel Cell Polyomavirus, in Head and Neck Cancer of Non-Smokers and Non-Drinkers.

OBJECTIVE: Determine the presence and prognostic value of human papillomavirus (HPV), Epstein-Barr virus (EBV), Merkel cell polyomavirus (MCPyV), and cell cycle proteins in head and neck squamous cell carcinoma (HNSCC) of non-smokers and non-drinkers (NSND). METHODS: Clinical characteristics and tumors of 119 NSND with HNSCC were retrospectively collected and analyzed on tissue microarrays. RNAscope in situ hybridization (ISH) was used to screen for the presence of HPV and MCPyV mRNA. Immunohistochemistry was performed for expression of p16 as surrogate marker for HPV, Large T-antigen for MCPyV, and cell cycle proteins p53 and pRb. Positive virus results were confirmed with polymerase chain reaction. For EBV, EBV encoded RNA ISH was performed. Differences in 5-year survival between virus positive and negative tumors were determined by log rank analysis. RESULTS: All oropharyngeal tumors (OPSCC) (n = 10) were HPV-positive, in addition to one oral (OSCC) and one nasopharyngeal tumor (NPSCC). The other three NPSCC were EBV-positive. MCPyV was not detected. Patients with HPV or EBV positive tumors did not have a significantly better 5-year disease free or overall survival. Over 70% of virus negative OSCC showed mutant-type p53 expression. CONCLUSION: In this cohort, all OPSCC and NPSCC showed HPV or EBV presence. Besides one OSCC, all other oral (n = 94), hypopharyngeal (n = 1), and laryngeal (n = 9) tumors were HPV, EBV, and MCPyV negative. This argues against a central role of these viruses in the ethiopathogenesis of tumors outside the oro- and nasopharynx in NSND. So, for the majority of NSND with virus negative OSCC, more research is needed to understand the carcinogenic mechanisms in order to consider targeted therapeutic options.

Low prevalence of Merkel cell polyomavirus in human epithelial thymic tumors.

BACKGROUND: The etiology of thymic epithelial tumors is unknown. Murine polyomavirus strain PTA has been shown to induce thymomas in mice. Recently, using diverse molecular techniques, we reported the presence of human polyomavirus 7 (HPyV7) in thymic epithelial tumors. In the present study, we investigated the prevalence of Merkel cell polyomavirus (MCPyV) in thymic epithelial tumors. METHODS: Thirty-six thymomas were screened for MCPyV by PCR and subsequently tested by DNA and RNA in situ hybridization and immunohistochemistry. Twenty-six thymomas were diagnosed with myasthenia gravis (MG). RESULTS: MCPyV DNA was detected by PCR in 7 (19.4%) of the 36 thymic epithelial tumors and in six of these, the presence of MCPyV was confirmed by fluorescence situ hybridization. Of these, 3 (28.6%) revealed weak MCPyV LT-antigen protein expression. In addition, one of the MCPyV positive thymomas tested positive for MCPyV LT RNA with RNAscope. Of interest, two out of the three thymomas that previously tested positive for MCPyV by immunohistochemistry also tested positive for HPyV7. One of the 11 MG-negative and 2 of the 25 MG-positive were positive for MCPyV. CONCLUSIONS: MCPyV DNA and MCPyV protein expression can be detected in human epithelial thymoma; however, to a far lesser extent than HPyV7. Our data strongly indicate that because of its infrequent detection and weak expression, MCPyV is unlikely to play an important role in the etiopathogenesis of human thymomas.