HPV E6/E7 mRNA in situ hybridization in endocervical adenocarcinoma: implications for prognosis and diagnosis

Background Human papillomavirus (HPV) E6/E7 mRNA in situ hybridization (HPV E6/E7 RNAscope) appears to be a sensitive and specific technique in detecting transcriptionally active HPV. We aimed to examine the diagnostic utility of this technique in endocervical adenocarcinoma (ECA), to explore the prognostic factors for ECA patients and develop a clinically useful nomogram based on clinicopathological parameters to predict it. Methods We retrospectively analyzed 200 patients with ECA who had undergone surgery at Sun Yat-sen University Cancer Center from 2010 and 2014. The diagnostic performance of HPV E6/E7 RNAscope were evaluated by receiver operating characteristic (ROC) curve. A prognostic nomogram model including HPV E6/E7 RNAscope was generated based on multivariate Cox regression analysis, then we compared the predictive accuracy of the prognostic model with FIGO staging and treatment using concordance index (C-index), time-dependent ROC (tdROC), and decision curve analysis (DCA). Results The sensitivity and specificity of HPV E6/E7 RNAscope for distinguishing HPV-associated adenocarcinoma (HPVA) from non-HPV-associated adenocarcinoma (NHPVA) in the whole cohort were 75.8% and 80%, respectively. According the univariate analysis and multivariate logistic regression analysis, age, lymphovascular invasion (LVI), lymph node involvement (LNI), and HPV E6/E7 RNAscope were valuable predictive factors for OS. These parameters were incorporated into the nomogram model (nomogram A) compared with FIGO stage and treatment. The C-index of nomogram A for predicting OS was 0.825, which was significantly higher than FIGO stage (C-index = 0.653, p = 0.002) and treatment (C-index = 0.578, p < 0.001). Conclusions HPV E6/E7 RNAscope is highly specific for ECA, and the 4-variable nomogram showed more accurate prognostic outcomes in patients with ECA.

The Expression of HPV E6/E7 mRNA in Situ Hybridization in HPV Typing-negative Cervical Cancer

Background: High-risk human papilloma virus (HR-HPV) persistent infection is the major tumorigenesis factor for cervical cancer (CC) and cervical intraepithelial neoplasia (CIN). However, the incidence of HPV-negative CC is approximately 5%-30% with different HPV detection methods. HR-HPV E6/E7 mRNA in situ hybridization (RISH) can detect HPV-driven tumours. Our study aimed to explore whether HPV typing-negative CC was caused by HPV infection.Methods: The records of CC patients with HPV typing results who had undergone cervical biopsies, cervical conization or hysterectomies were collected from April 2018 to September 2021 at the Affiliated Hospital of Weifang Medical University. RISH was detected using RNAscope chromogenicin in the tissues of patients with CC. Immunohistochemistry (IHC) was performed to evaluate the expression of p16INK4a and Ki67. CC patients with positive HPV typing in the same period were collected as controls.Results: A total of 308 women with HPV typing results were enrolled, and 30 (9.74%) cases of HPV typing were negative, including 22/30 (73.3%) cases of squamous cell carcinoma (SCC) and 8/30 (26.7%) cases of adenocarcinoma. In the CC patients who were HPV typing-negative, 28/30 (93.3%) were positive for RISH and p16INK4a block+ staining, which contained 22/22 (100%) SCCs and 6/8 (75%) adenocarcinomas. RISH was positive in 278/278 (100%) HPV typing-positive CCs, which included 232/232 (100%) SCCs and 46/46 (100%) adenocarcinomas. While 273/278 (98.2%) showed p16INK4a block+ staining, the five negative cases were SCC. Positive RISH in HPV typing-negative CC was significantly lower than in the HPV typing-positive group (P=0.002, 95% CI: 0.848-1.027). However, this significant difference only existed in adenocarcinoma, and positive RISH in HPV typing-negative SCC was the same as in the HPV typing-positive group. No significant differences were seen in the expression of p16INK4a and Ki67 (P=0.291, 95% CI: 0.863-1.047 and P=0.174, 95% CI: 0.905-1.033).Conclusions: HPV typing may cause misdiagnosis in 9.74% of CC patients, and HPV E6/E7 mRNA can detect the majority of CC with HPV-related status even in HPV typing-negative patients. This approach could provide a novel option to accurately detect HR-HPVs in cervical tumours and help to eliminate the percentage of misdiagnosed HPV-related cases.

HPV E6/E7 mRNA In Situ Hybridization in Endocervical Adenocarcinoma: Implications for Prognosis and Diagnosis

Background: Human papillomavirus (HPV) E6/E7 mRNA in situ hybridization (HPV E6/E7 RNAscope) appears to be a sensitive and specific technique in detecting transcriptionally active HPV. We aimed to examine the diagnostic utility of this technique in endocervical adenocarcinoma (ECA), to explore the prognostic factors for ECA patients and develop a clinically useful nomogram based on clinicopathological parameters to predict it.Methods: We retrospectively analyzed 200 patients with ECA who had undergone surgery at Sun Yat-sen University Cancer Center from 2010 and 2014. The diagnostic performance of HPV E6/E7 RNAscope were evaluated by receiver operating characteristic (ROC) curve. A prognostic nomogram model including HPV E6/E7 RNAscope was generated based on multivariate Cox regression analysis, then we compared the predictive accuracy of the prognostic model with FIGO staging and treatment using concordance index (C-index), time-dependent ROC (tdROC), and decision curve analysis (DCA).Results: The sensitivity and specificity of HPV E6/E7 RNAscope for distinguishing HPV-associated adenocarcinoma (HPVA) from non-HPV-associated adenocarcinoma (NHPVA) in the whole cohort were 75.8% and 80%, respectively. According the univariate analysis and multivariate logistic regression analysis, age, lymphovascular invasion (LVI), lymph node involvement (LNI), and HPV E6/E7 RNAscope were valuable predictive factors for OS. These parameters were incorporated into the nomogram model (nomogram A) compared with FIGO stage and treatment. The C-index of nomogram A for predicting OS was 0.825, which was significantly higher than FIGO stage (C-index = 0.653, p=0.002) and treatment (C-index = 0.578, p < 0.001).Conclusions: HPV E6/E7 RNAscope is highly specific for ECA, and the 4-variable nomogram showed more accurate prognostic outcomes in patients with ECA.

Somatosensory processing deficits and altered cortico-hippocampal connectivity in Shank3b-/- mice

Abnormal tactile response is considered an integral feature of Autism Spectrum Disorders (ASDs), and hypo-responsiveness to tactile stimuli is often associated with the severity of ASDs core symptoms. Patients with Phelan-McDermid syndrome (PMS), caused by mutations in the SHANK3 gene, show ASD-like symptoms associated with aberrant tactile responses. However, the neural underpinnings of these somatosensory abnormalities are still poorly understood. Here we investigated, in Shank3b-/- adult mice, the neural substrates of whisker-guided behaviors, a key component of rodents' interaction with the surrounding environment. To this aim, we assessed whisker-dependent behaviors in Shank3b-/- adult mice and age-matched controls, using the textured novel object recognition (tNORT) and whisker nuisance (WN) test. Shank3b-/- mice showed deficits in whisker-dependent texture discrimination in tNORT and behavioral hypo-responsiveness to repetitive whisker stimulation in WN. Notably, sensory hypo-responsiveness was accompanied by a significantly reduced activation of the primary somatosensory cortex (S1) and hippocampus, as measured by c-fos mRNA in situ hybridization, a proxy of neuronal activity following whisker stimulation. Moreover, resting-state fMRI showed a significantly reduced S1-hippocampal connectivity in Shank3b mutant mice. Together, these findings suggest that impaired crosstalk between hippocampus and S1 might underlie Shank3b-/- hypo-reactivity to whisker-dependent cues, highlighting a potentially generalizable form of dysfunctional somatosensory processing in ASD.

Prognostic nomogram for patients with invasive endocervical adenocarcinoma incorporating HPV E6/E7 mRNA in situ hybridization and clinical characteristics

Background: Human papillomavirus (HPV) E6/E7 mRNA in situ hybridization (HPV E6/E7 RNAscope) appears to be a sensitive and specific method. We aimed to examine the diagnostic and prognostic utility of this technique in endocervical adenocarcinoma (ECA) and build a useful prognostic nomogram model using this approach. Methods: The model was constructed from a retrospective study of 200 patients with ECA who had undergone surgery at Sun Yat-sen University Cancer Center between 2010 and 2014. The model's predictive efficiency and discriminative capability were defined by a concordance index (C-index) and calibration curve. Results: The overall sensitivity and specificity of HPV E6/E7 RNAscope for distinguishing HPV-associated adenocarcinoma (HPVA) from non HPV-associated adenocarcinoma (NHPVA) in the whole cohort were 75.8% and 80%, respectively, compared to 60.2% and 90.0% for p16 immunohistochemistry, 80.5% and 80.0% for HPV DNA, and 63.3% and 50% for HPV genotype. The independent factors derived from multivariable analysis of the whole cohort to predict overall survival (OS) were age, lymphovascular invasion (LVI), lymph node involvement (LNI), and HPV E6/E7 RNAscope, which were all incorporated into the nomogram with (nomogram B) or without (nomogram A) FIGO stage and treatment. The C-index of nomogram A for predicting OS was 0.825 (p = 0.002 and p < 0.001), which was significantly higher than the C-index for FIGO stage (0.653) and treatment (0.578). No significant difference occurred between nomograms A (0.825) and B (0.836). Furthermore, a risk stratification system was established to accurately stratify patients with ECA into two subgroups with significantly different prognosis. Conclusions: HPV E6/E7 RNAscope is highly specific for ECA, and the proposed nomogram using HPV E6/E7 RNAscope showed more accurate outcome in terms of prognosis in patients with ECA.

Development of a Rat Model for Glioma-Related Epilepsy

Seizures are common in patients with high-grade gliomas (30–60%) and approximately 15–30% of glioblastoma (GB) patients develop drug-resistant epilepsy. Reliable animal models are needed to develop adequate treatments for glioma-related epilepsy. Therefore, fifteen rats were inoculated with F98 GB cells (GB group) and four rats with vehicle only (control group) in the right entorhinal cortex. MRI was performed to visualize tumor presence. A subset of seven GB and two control rats were implanted with recording electrodes to determine the occurrence of epileptic seizures with video-EEG recording over multiple days. In a subset of rats, tumor size and expression of tumor markers were investigated with histology or mRNA in situ hybridization. Tumors were visible on MRI six days post-inoculation. Time-dependent changes in tumor morphology and size were visible on MRI. Epileptic seizures were detected in all GB rats monitored with video-EEG. Twenty-one days after inoculation, rats were euthanized based on signs of discomfort and pain. This study describes, for the first time, reproducible tumor growth and spontaneous seizures upon inoculation of F98 cells in the rat entorhinal cortex. The development of this new model of GB-related epilepsy may be valuable to design new therapies against tumor growth and associated epileptic seizures.

Transcriptome-Wide Analyses Provide Insights into Development of the Hedychium Coronarium Flower, Revealing Potential Roles of PTL

Background:The flower of Hedychium coronarium possesses highly specialized floral organs: a synsepalous calyx, petaloid staminodes and a labellum. The formation of these organs is controlled by two gene categories: floral organ identity genes and organ boundary genes, which may function individually or jointly during flower development. Although the floral organogenesis of H. coronarium has been studied at the morphological level, the underlying molecular mechanisms involved in its floral development still remain poorly understood. In addition, previous works analyzing the role of MADS-box genes in controlling floral organ specification in some Zingiberaceae did not address the molecular mechanisms involved in the formation of particular organ morphologies that emerge later in flower development, such as the synsepalous calyx formed through intercalary growth of adjacent sepals. Results:Here, we used comparative transcriptomics combined with Real-time quantitative PCR and mRNA in situ hybridization to investigate gene expression patterns of ABC-class genes in H. coronarium flowers, as well as the homolog of the organ boundary gene PETAL LOSS (HcPTL). qRT-PCR detection showed that HcAP3 and HcAG were expressed in both the petaloid staminode and the fertile stamen. mRNA in situ hybridization showed that HcPTL was expressed in developing meristems, including cincinnus primordia, floral primordia, common primordia and almost all new initiating floral organ primordia.Conclusions:Our studies found that stamen/petal identity or stamen fertility in H. coronarium was not necessarily correlated with the differential expression of HcAP3 and HcAG. We also found a novel spatio-temporal expression pattern for HcPTL mRNA, suggesting it may have evolved a lineage-specific role in the morphogenesis of the Hedychium flower. Our study provides a new transcriptome reference and a functional hypothesis regarding the role of a boundary gene in organ fusion that should be further addressed through phylogenetic analyzes of this gene, as well as functional studies.

Irregular adaxial–abaxial polarity rearrangement contributes to the monosymmetric-to-asymmetric transformation of Canna indica stamen

In flowering plants, lateral organs including stamens develop according to the precise regulation of adaxial–abaxial polarity. However, the polarity establishment process is poorly understood in asymmetric stamens. Canna indica (Zingiberales: Cannaceae) is a common ornamental plant with an asymmetric stamen comprising a one-theca anther and a petaloid appendage. In this study, we depicted the monosymmetric-to-asymmetric morphogenesis of C. indica stamen, and the morphogenesis of the monosymmetric stamen of a sister species was used as a contrast. We chose a HD-ZIP III gene family member and a YABBY family member as the adaxial and abaxial polarity marker genes, respectively, and tested their expression using mRNA in situ hybridization. The expression patterns of the two genes changed dynamically and asymmetrically during the stamen development process. Compared with their homologues in Arabidopsis thaliana, these two genes exhibited some specific expression patterns. We hypothesize that the distinctive adaxial–abaxial polarity participates in the irregular morphogenesis of C. indica stamen, which mediates the putative stamen-to-petaloid staminode conversion in this species.