The main objective of this study was to estimate the prevalence of human papillomavirus-DNA (HPV-DNA) in the saliva of sexually active women with HPV-related cervical intraepithelial neoplasias (CIN) and compare the findings with a healthy control group. The secondary objectives were: 1) to determine the concordance between genital and oral HPV types in sexually active women with HPV-related CIN; 2) to analyze whether sexual habits influence the presence of HPV-related CIN; 3) to determine whether sexual habits influence the presence of oral HPV. Saliva samples were collected from 100 sexually active women, 50 with HPV-related CIN and 50 healthy subjects presenting normal cytology. PCR assay was used to detect HPV-DNA.p =0.766). As for the concordance between genital and oral HPV types in women with HPV-related CIN, concordance was only observed for HPV-16, whereby among 22 women with genital HPV-16, only one (4.54%) also presented oral HPV-16. Regarding the possible influence of sexual habits on the presence of cervical pathology and presence of oral HPV, it was found that marital status, age at first intercourse, number of lifetime sexual partners, and condom use are related with the presence of cervical pathology ; condom use was also found to be related to the presence of oral HPV (p<0.001). The prevalence of oral HPV infection in saliva samples was 14% in women with HPV-related CIN, while in the healthy group it was 12%, without statistically significant difference ( The prevalence of HPV-DNA in the saliva of sexually active women with HPV-related CIN is similar to healthy women. The concordance between genital and oral HPV types is low. Both the presence of cervical pathology and the presence of oral HPV are related to sexual habits. Wider sample size is required to confirm this results. Key words:Cervical intraepithelial neoplasias, HPV, saliva, cervix, cancer. Cervical cancer is the second most common cancer among women around the world, and is more frequent (>85% of cases) in developing countries  . It is tHPV infection of the cervix, is usually related to the appearance of different degrees of cervical intraepithelial neoplasias (CIN), which range from CIN I lesions  that retain the capacity to maintain a complete life cycle with virion release; CIN II ; and CIN III . The two latter classifications produce an increase in the expression of two proteins with oncogenic capacity: the E6 and E7 ; these facilitate the accumulation of mutations in the cell genome, which allows the cancer to progress .In general, infection by HPV among young sexually active women is frequent, but clears up in 80% of cases due to effective immune responses, mediated by CD4+ Th1 response. HPV has various immune evasion systems, as its life cycle is exclusively epithelial and there are no viraemic phases, no cell destruction, and viral replication and virion release is not accompanied by inflammatory processes. Moreover, virion-producing cells that express large amounts of viral antigens are located on the surface of the epithelium away from the immune cells, which are also scarce in the cervical area . The keyRegardless of whether or not HPV clears up by cervical auto-immune response, sexual behavior has changed in recent decades, the age of sexual initiation has decreased, and lifetime numbers of sexual partners have increased. There has also been a rise in the frequency of oral-to-genital sex, which, in the U.S.A. is more common among Whites (75%), followed by Hispanics (63%), and Afro-Americans (62%) . These cOver 650.000 patients are diagnosed with head and neck squamous cell carcinoma (HNSCC) worldwide every year and over 350,000 die from it . IncreasThe main aim of this study was to estimate the prevalence of HPV-DNA in the saliva of sexually active women with HPV-related CIN compared with a healthy group. The secondary objectives were: 1) to determine the concordance between genital and oral HPV types in sexually active women with HPV-related CIN; 2) to analyze whether sexual habits influence the presence of HPV-related CIN; and 3) to determine whether sexual habits influence the presence of oral HPV.- Recruitment and patient characteristicsThe study protocol was approved by the University of Murcia Ethics Committee and was carried out between May 2015 and February 2018 at the Obstetrics and Gynecology Unit, Santa Lucía University Hospital, Cartagena (Spain). Subjects were treated according to guidelines established by the Declaration of Helsinki for medical research involving human subjects. All subjects provided their informed consent to participate. Subjects were recruited applying inclusion criteria recorded in the hospital database: women with and without HPV-related CIN. The entire protocol was carried out by a single clinician.Inclusion criteria in the study group were as follows: patients aged over 18 years, sexually active women with HPV-related CIN, patients willing to provide informed consent to take part in the study.For the control group the inclusion criteria were: patients aged over 18 years, sexually active women without HPV-related CIN, patients willing to provide informed consent to take part in the study.None of the patients who fulfilled the inclusion criteria and were invited to take part in the trial refused to do so. To calculate a representative sample size, a power of 80% was required . A total of 50 sexually active women with HPV-related CIN and 50 sexually active women without HPV-related CIN were included in this transversal clinical study.Having received their informed consent in writing, a detailed anonymous questionnaire was administered in order to collect information about age, smoking, drinking, and sexual habits.- Sample collection and HPV-DNA detectionAll saliva samples (n=100) were collected under equal conditions . Patients were placed in a relaxed position and looking downwards. Patients were asked to rinse the mouth vigorously and gargle with 10 ml normal saline for a total of 30 seconds . Specimens were collected in sterile tubes and refrigerated to minimize degradation of salivary proteins until further processing. The saliva samples were centrifuged at 2000 rpm at 4ºC for 10 min, 2 ml supernatant saved, followed by re-suspension of the cell pellet in 10 ml PBS, which was centrifuged at 2000 rpm at 4ºC for 10 min, then re-suspended in 3 ml PBS, and split into two aliquots. All samples were stored locally at -80ºC until DNA purification, amplification, and HPV-DNA detection.All samples were analyzed by the same geneticist in a blinded fashion. DNA was purified from all samples by use of a Puregene DNA Purification Kit  . The pre- Statistical analysisData were analyzed using the SPSS 20.0 statistics program . A descriptive study was made of each variable. The Kolmogorov-Smirnov normality test and Levene's homogeneity of variance test were applied; the data showed normal distribution and so were analyzed using parametric tests. Associations between the different qualitative variables were studied using Pearson’s chi-squared test. Associations between different quantitative variables were studied using Student’s t-test for two related samples. Adjusted odds ratios and confidence intervals were calculated using multiple logistic regression models considering the binaries “presence of HPV-related CIN” and “presence of oral HPV” as the outcome variables, and using marital status, age at first intercourse, number of lifetime sexual partners, oral-to-genital sex, and condom use as covariates. Statistical significance was accepted for p≤0.05.p=0.101), educational level (p=0.505), smoking (p=0.683), and alcohol consumption (p=0.517). Nevertheless, a higher percentage of women with HPV-related CIN were single or not in a sTable couple relationship (88% compared with 34% of control subjects), had had >4 children (16% compared with 4%), had had their first sexual intercourse aged ≤16 years (30% compared with 10%), had had a number of lifetime sexual partners ≥2 (98% compared with 60%), practiced oral-to-genital sex (92% compared with 70%), had had some sexually transmitted infection (16% compared with 4%), and a lower percentage of women in this group used condoms (34% compared with 74%); with statistically significant differences   and 50 healthy control subjects without HPV-related CIN were included in this transversal clinical study. Mean patient age was 36.92 ± 6.69. Both groups were homogenous in terms of age (ctively) .p=0.766). Only two (4%) women in the group with HPV-related CIN presented more than one type of oral HPV (two types) and only one women (2%) in the healthy control group, without statistically significant difference (p=0.842). When oral HPV types were compared between groups, nine different types were diagnosed in seven women with HPV-related CIN and HPV-DNA in saliva, the most common type being 45 but no significant difference was found in comparison with the control group . In the same way, it was observed that of the seven types diagnosed in six of the women without HPV-related CIN and HPV-DNA in saliva, the most frequently occurring type was 39 but without significant difference in comparison with its presence in women with HPV-related CIN   .Regarding the characteristics of genital HPV in women with HPV-related CIN, 50% presented multiple types, making a total of 93 types, the most common being type 16 (23.65%) . When thp<0.001; p=0.017; p=0.002; and p<0.001 respectively).When sexual habits were analyzed to determine whether these exert any influence on the presence of HPV-related CIN, it was found that marital status, age at first intercourse, number of lifetime sexual partners, and condom use were all related to presence of HPV-related CIN   (p<0.001).Lastly, when sexual practices  were analyzed to determine whether they had any influence on the presence of oral HPV, only condom use was found to be related to the presence of oral HPV . Similar results have been obtained by other authors such as Adamopoulou et al., .The main aim was to estimate the prevalence of HPV-DNA in the saliva of sexually active women with HPV-related CIN compared with a healthy control group. Prevalence was found to be similar in both groups, 14% in women with HPV-related CIN compared with 12% in women without HPV-related CIN, without statistically significant difference , among 22 women with genital HPV-16, only one (4.54%) also presented oral HPV-16; while for the other 92 HPV genital types, no concordance was found with oral types of HPV. Similar findings have been reported by authors such as Sayyah-Melli et al.,  who geno. Similar results were obtained by authors such as Saini et al., , it was found that marital status, age at first intercourse, number of lifetime sexual partners, and condom use were related to the presence of HPV-related CIN . Similar results were obtained by Schlecht et al., . The results of the present study analyzed by means of a logistic regression model found that the practice of oral-to-genital sex showed no relation to the presence of oral HPV (p=0.288), but the use or non-use of condoms did (p<0.001) indicating that oral-to-genital sex does not increase the risk of oral HPV infection providing a condom is used.Regarding the present study’s third secondary objective , the use or not of condoms was strongly related to the presence of oral HPV ( et al., , who in (et al.,  in theirIn conclusion, this study shows that the prevalence of HPV-DNA in the saliva of sexually active women with HPV-related CIN is similar to that of healthy control subjects and that the concordance between genital and oral HPV types is low, which may indicate that the natural history of oral and cervical infection differs; both the presence of cervical pathology and the presence of oral HPV are related to sexual behaviors. Nevertheless, wider sample size is required to confirm this results.


Data regarding coronavirus disease 2019 (COVID-19) description are still limited in pediatric oncology. The French society of pediatric oncology (SFCE) initiated a study to better describe COVID-19 presentation and evolution in patients followed in French pediatric oncology and hematology wards. By describing COVID-19 in this specific population, we aimed to identify the patients who may be the most at risk of severe COVID-19 and establish specific recommendations.Data regarding coronavirus disease 2019 (COVID-19) description are still limited in pediatric oncology. The French society of pediatric oncology (SFCE) initiated a study to better describe COVID-19 in patients followed in French pediatric oncology and hematology wards. All patients diagnosed with COVID-19 and followed in a SFCE center were enrolled. Data from medical records were analyzed for all patients enrolled up to the end of May 2020. Data were available for 37 patients. Thirty-one were children under 18 years of age. Nineteen patients were female. Seventeen patients had a solid tumor, 16 had a hematological malignancy and four recently underwent hematopoietic stem cell transplantation (HSCT) for non-oncological conditions. Twenty-eight patients presented symptoms, most often with fever, cough, rhinorrhea and asthenia. Ground-glass opacities were the most frequent radiological finding with abnormalities mostly bilateral and peripherally distributed. Twenty-four patients received chemotherapy a month prior to COVID-19 diagnosis. Most patients did not require hospitalization. Three patients required oxygen at the time of diagnosis. In total, five patients were admitted in an intensive care unit because of COVID-19 and one died from the disease. Children and young adults treated for a cancer and/or with a HSCT may be at risk for severe COVID-19 and should be closely monitored. Coronavirus disease 2019 (COVID-19), resulting from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) appears to affect children less severely than adults, with majority of benign forms and asymptomatic cases . Data arTo characterize clinical presentation and outcomes of children, adolescent and young adults with cancer and infected by SARS-CoV-2, the French society of pediatric oncology (Société Française de lutte contre les Cancers et leucémies de l’Enfant et de l’adolescent—SFCE) initiated a study (PEDONCOVID) to retrospectively and prospectively collect data regarding COVID-19 in this specific population. France has been so far one of the most affected countries with currently a total of 188,450 cases and 28,943 deaths . We descAs of the 28th of May 2020, 41 patients with COVID-19 have been declared in SFCE centers. Most of patients were from Eastern France and the region around Paris, consistent with the distribution of the epidemic in whole population . Thirty-Of the 37 patients, 31 were children under the age of 18 (mean: 11.2 years (1–25)). Nineteen patients (51%) were female. Seventeen patients had a solid tumor, 16 had a hematological malignancy and four recently underwent HSCT for non-oncological conditions. Nine patients were treated for a cancer relapse. Contact with an infected person was reported for 19 patients, with a median estimated incubation period of 9.5 days (data available for 10 patients). Twenty-eight patients presented symptoms .All patients were tested for SARS CoV-2 infection by PCR on a nasopharyngeal swab, which was positive in 34 patients (92%). Two patients were diagnosed with positive specific IgM serology and one patient was diagnosed upon typical clinical and radiological findings. Thoracic computed tomography-scan was performed in fifteen patients and was abnormal in all but one case. Ground-glass opacities were the most frequent abnormality (9 cases). Abnormalities were mostly bilateral and peripherally distributed. Blood count results were available for 31 patients. Lymphopenia below 0.5 G/L was found in thirteen patients. Neutropenia below 0.5 G/L was found in thirteen patients. Six patients presented concomitant neutropenia and lymphopenia. C-reactive protein (CRP) dosage results were available for 24 patients. Fifteen patients had an elevated CRP, with a CRP mean level of 41.6 mg/L (15–280). Four patients had a CRP above 50 mg/L. Liver enzymes were measured in seventeen patients, nine of whom had elevations greater than twice the upper limit of normal. A co-pathogen was found in blood samples of two patients . Twenty-four patients received chemotherapy, on average 12.6 days (0–35) prior to COVID-19 diagnosis, with a mean number of drugs of 2.1 (1–5). Nine patients were currently (or recently) treated with less than 1 mg/kg/day of corticosteroids, five with cyclosporin A and one with tacrolimus. Overall, 20 patients were hospitalized at diagnosis but most of them did not require hospitalization specifically to manage COVID-19. There were significant discrepancies among pediatric oncology and hematology wards regarding schedule triage, rules of admission to hospital and SARS-CoV-2 testing. Seventeen patients received antibiotics, with ten patients presenting febrile neutropenia. Only one patient received remdesivir and two received hydroxychloroquine outside of a clinical trial.In total, five patients were admitted into an ICU because of COVID-19 and one died from the disease. Their main characteristics are described in Notably, four patients had been heavily treated for their cancer and/or recently underwent HSCT. Except for these patients, there were only asymptomatic to moderate forms of infection, with a median follow-up of 21 days (0–58). For the 13 patients for whom PCR had been repeated and these data were available, the mean time to a negative PCR was 16.5 days (7–28). COVID-19 delayed oncology treatment in 16 patients for an average of 14 days.The objective of this study was to describe the clinical presentation of COVID-19 in the pediatric oncology and hematology population, not to determine the incidence of COVID-19. Indeed, each center may have different screening strategies and the data collected in this study do not allow for the calculation of the incidence of COVID-19. Our data show that clinical and radiological descriptions of COVID-19 are quite similar to those reported in adults, and that incubation period or virus clearance appear to be the same in this population as what has been described before. Most of patients developed a mild or even an asymptomatic form of the disease. However, as we previously stated,  and in cOncologic treatment was delayed in almost half of cases, which indicates that COVID-19 impacted the care of patients even if most cases were mild. Notably, some patients had their treatment as planned and did not develop any complications. However, every situation is unique and clinicians who followed these patients made their decision regarding the patients’ own history and potent risk factors, so that no general recommendation to pursue oncologic treatment may be stated from this study. On the contrary, delaying a non-urgent oncologic treatment appears to be wise in the context of suspected or proven infection, as suggested by most clinicians . ConversA striking difference between our data and the first published reports is the sex ratio ,5. ThougBiological findings are difficult to interpret since a high proportion of patients recently received drugs that could alter blood tests. However, profound neutropenia and profound lymphopenia were found in almost one third of patients. Even if that can be explained by the oncologic treatment received, the infection likely participated and increased the cytopenias. Lymphopenia is common with SARS-CoV-2 infection, and it appears that the deeper the lymphopenia, the more severe the disease ,20. Our Clinicians should pay attention to lung lesions caused by COVID-19 for patients who may receive treatments that could themselves damage lung tissue. Indeed, delaying lung irradiation or administration of drugs with known lung toxicity may be considered for infected patients. However, this delay should not be too long to avoid the risk of progression. In our cohort, the mean delay in treatment administration was 14 days, which appears reasonable regarding both this risk of disease progression and the risk of complications related to COVID-19. Of course, making the decision of delaying treatment and the length of this delay should be considered carefully and should depend on the situation.Until we get more data, we advocate not reassuring parents regarding the mild forms of COVID-19 in patients treated for malignancies. We recommend that patients with severe hematological malignancies and/or receiving heavily immunosuppressive treatments should be carefully watched and protected from the COVID-19 risk. This shall be of interest for patients in South Asia and Latin America who are now facing the COVID-19 pandemic and countries facing the second wave.All patients followed in a SFCE center and diagnosed with a SARS-CoV-2 infection who had undergone anti-cancer treatment in the past 6 months were enrolled in the study. We also included all patients from SFCE centers who underwent a hematopoietic stem-cell transplantation (HSCT) for any reason and with immunosuppressive therapy either ongoing or interrupted less than 6 months prior. Since SFCE centers may provide care to children and adolescents as well as in young adults, there was no limit of age to enroll patients in the study.Diagnosis criteria for SARS-CoV-2 infection were as follows: (1) biologically proven infection with positive polymerase chain reaction (PCR) or positive specific positive IgM SARS-CoV-2 serology, (2) clinical and radiological diagnosis with at least two (if any contact with a suspected or confirmed COVID-19 case) or three (if no notion of contact) clinical signs  and computed-tomography (CT)-scan signs  compatible with SARS-CoV-2 infection. All patients and their families were informed of this study. The study was approved by the ethics committee of the coordinating center (N04/2020/ROU). This study is registered in clinicaltrial (NCT04433871).We deliver here the first description of the French pediatric oncology and hematology cohort of patients diagnosed with SARS-CoV-2 infection, which to our knowledge is the largest ever published to date. Consistent with previous reports ,5, relat


Nasopharyngeal carcinoma (NPC) is a malignant tumour of the head and neck affecting localised regions of the world, with the highest rates described in Southeast Asia, Northern Africa, and Greenland. Its high morbidity rate is linked to both late-stage diagnosis and unresponsiveness to conventional anti-cancer treatments. Multiple aetiological factors have been described including environmental factors, genetics, and viral factors , making NPC treatment that much more complex. The most common forms of NPCs are those that originate from the epithelial tissue lining the nasopharynx and are often linked to EBV infection. Indeed, they represent 75–95% of NPCs in the low-risk populations and almost 100% of NPCs in high-risk populations. Although conventional surgery has been improved with nasopharyngectomy’s being carried out using more sophisticated surgical equipment for better tumour resection, recent findings in the tumour microenvironment have led to novel treatment options including immunotherapies and photodynamic therapy, able to target the tumour and improve the immune system. This review provides an update on the disease’s aetiology and the future of NPC treatments with a focus on therapies activating T cell immunity. Nasopharyngeal carcinoma (NPC) is a malignant tumour of the head and neck that originates from the epithelial tissue lining the nasopharynx. Every year, over 80,000 new cases of NPC are diagnosed and 50,000 NPC-related deaths are recorded worldwide . NPC typBased on the current World Health Organisation (WHO) pathologic classification, NPCs are grouped into keratinising squamous cell carcinoma (KSCC) and non-keratinising carcinoma. The latter group is further subdivided into non-keratinising differentiated carcinoma (NKDC) and non-keratinising undifferentiated carcinoma (NKUC) ,4. Patient’s five-year survival rate remains low as it is often diagnosed at late stages (30–40%) ,5. AlthoIn most of the world, NPC is a rare disease affecting fewer than 1 out of 100,000 people per year. Nevertheless, some regions or populations show a higher incidence of NPC. For instance, NPC has a rare incidence of <1/100,000 among Caucasians and in Europe. Some parts of South China, notably Guangdong and Hong Kong, have the highest levels of incidence in the world  . The resInterestingly, it has been described that men are three times more likely to develop NPC than women . It was Similar to all cancers, NPC is multifactorial and is caused by a number of etiologic factors. Other than its clear association to the Epstein-Barr virus (EBV), some environmental and genetic factors have been linked to higher risks of developing NPC. D4S405 and D4S3002 markers on chromosome 4 [As mentioned above, although Chinese people who migrate outside of China show a lower NPC-incidence, they still have an increased risk in comparison to the average population. This key information confirms that environment and genetic factors both contribute to the emergence of NPC. Indeed, families with many members developing NPC have been studied, yet there are no clear genetic markers that fully elucidate the seemingly strong predisposition for NPC in these cases . Studiesmosome 4 . TERT/CLPTM1L-encoding region. A 2016 meta-analysis study carried out on over 8000 people of Chinese descent identified that a variation in the tert/clptm1l locus (Chr 5p15.33) was linked to an increased NPC risk [Ras-dependent oncogenic transformation in lung cancer [Another reported susceptibility locus for NPC is the NPC risk . Indeed,us Chr 5p.33 was lg cancer . Thus, fgstm1) is linked to increased NPC susceptibility in all high-risk populations [Other loci already identified in carcinogenesis are studied for their potential involvement in NPC. When deleted, the glutathione S-transferase M1 . As with all member of this family, it is a double-stranded DNA virus containing 85 genes found in the nucleoid. The nucleus-like region is bound by an icosahedral nucleocapsid that measures 100–120 nm in diameter and is made up of 162 capsomeres. The space between the nucleocapsid and the outer envelope is lined with the tegument, a protein-rich matrix. This envelope contains proteins and surface glycoprotein projections that originate from the cell’s nuclear membrane. The projections help the virus bind to the target cell.NPC is widely recognised as an EBV-associated malignancy. EBV is part of the locus 155391: G>A) can be linked to increased NPC susceptibility in Southern China [Nonetheless, as EBV infects most of the world’s population, it was hypothesized that certain strains of EBV were responsible for specific NPC endemic regions. Indeed, some EBV variants can be significantly correlated with high- incidence of NPC in endemic regions ,46. Fengrn China . Furtherrn China . Evidencrn China . This varn China . This brThe Epstein-Barr virus latently infects more than 90% of the world’s adult human population and its association with NPC is thought to be mediated by an interplay of environmental  factors and genetic predisposition . In NPC, the EBV virus expresses a type II latency program and is present in virtually all poorly differentiated and undifferentiated non-keratinising (WHO type II and III) NPCs.EBV-associated NPC expresses a type II latency program, and tumour cells typically express the latent membrane proteins 1, 2A, and 2B  and EBNA1, all of which have limited immunogenicity. In addition, several EBV non-coding RNAs primarily EBER1 and EBER2, and BamHI-A rightward transcripts (BARTs) and BamHI-A rightward frame 1 (BARF1) of EBV are expressed abundantly and are detected consistently in NPC ,54,55,56EBNA1 is frequently expressed in NPC and is a dominant target for CD4 T cells. LMP1 and LMP2 are expressed in approximately 50% of all NPC tumours. Although LMP1 is poorly immunogenic, LMP2 proteins are sufficiently more immunogenic hence they are now putative targets for EBV-directed immunotherapy, such as cytotoxic T cells ,57,58,59NPC-related EBV antigens LMP1, LMP2A/B, EBNA1, EBER and EBV-encoded RNA each have distinct effects on growth, differentiation and the host’s immune response. Collectively, they likely contribute to the development of NPC by promoting cell transformation and angiogenesis, inhibition of apoptosis, induction of stem-cell-like phenotype and enhancement of cell motility. Pressure-driven selective evolution constantly fosters the emergence of new EBV variants ,63. ThesIt is important to note that NPC, while associated with EBV and the expression EBV proteins, is an entity that encompasses a broad range of other distinct molecular aberrations that may also be targets for immunotherapies .Before treating the patient, the stage of the cancer must be determined using the AJCC Cancer Staging Method. This classification system takes into consideration three main factors: (T) the characteristics of the main tumour mass; (N) the status of cancer spread in the lymph nodes (LNs); and (M) the status of metastasis outside the head and neck. The patient is assessed using the chart in As a lymphoepithelioma, NPC is highly linked to the host’s immune system. It has been shown that EBV favours carcinogenesis by evading the immune response . AlthougDue to the deep-seated localisation of NPC, surgery to remove the tumour is generally not applicable. However, nasopharyngectomy is a possible treatment for locally recurrent NPC. Different surgical methods exist including the maxillary swing, midface degloving, transpalatal, transmaxillary and trans-infratemporal fossa approaches, but all of these surgeries remains invasive . In receENPG techniques have also been used in later recurrences including rT3 and rT4 diseases . In one ® system  in preclinical studies allowed a transoral palate-sparing approach to the nasopharynx [Another minimally invasive approach is the use of robotics in nasopharyngectomy. Tsang et al. used this technique with the da Vinci S system  on a cohort of twelve patients . At the opharynx . On the other hand, a craniofacial resection has been proposed for the removal of advanced rT3 and rT4 recurrent tumours . A monocWhen diagnosed at early stages, NPC is classically treated with either radiotherapy and/or chemotherapy with over 90% five-year survival rate . In a PhTargeted therapies were being tested for all types of cancers at the turn of the century. These molecular agents target the increased cell growth and resistance to cell death attributed to tumour cells. Monoclonal antibodies are used to target the EGFR pathway and block multiple key effector Tyrosine-kinases. In lung cancer, they are now the gold standard treatment for patients. However, clinical trials have shown only modest advantages for NPC patients. Major toxic side-effects were recorded including some cases of Grade 5 tumour haemorrhaging with pazopanib  and suniOne of the main characteristics of NPC is the presence of a massive leukocyte infiltrate within the primary tumour. The latter consists of tumour-infiltrating T cells (TILs), B cells, dendritic cells, macrophages, monocytes, and eosinophils whose recruitment is facilitated by the production of inflammatory cytokines by tumour cells ,85. CytoThe majority of these components participate in the establishment of NPC immunosuppressive microenvironment, protecting it from the host’s immune response. Given the strong immunosuppressive TME and the EBV-linked nature of NPC, when the first immunotherapies were tested in clinical trials, NPC posed as a good candidate for this novel treatment. Immunotherapy engulfs many types of approaches, e.g. EBV vaccines, adoptive immune cell therapy and immune checkpoint inhibitors .We discussed above how viral proteins expressed in NPC contribute to its progression. The strategy is to target essential viral proteins or RNAs to weaken the viral drivers of NPC ,90. It is well established that NPC expresses viral proteins, mainly EBNA1-3 and LMP1-2 that are involved in carcinogenesis. However, evidence shows that the best strategies target multiple EBV proteins simultaneously. Thus, an EBV vaccine would increase the availability of viral antigens and ultimately enhance the EBV-specific immune response. Phase I studies using highly immunogenic fusion protein expressed by a recombinant virus showed an increase in the EBV-T cell population , whereasA peptide-based vaccine strategy was proposed by our team for EBV-associated malignancies including NPC . Six pepViral RNAs are also interesting targets as they are expressed in all EBV-latencies. However, their exact functions remain elusive and their diversity makes them difficult targets. Nevertheless, RNA-targeting drugs such as Ribavirin, a nucleoside inhibitor, is already being used to treat other viral diseases such as Hepatitis C and viral haemorrhagi fever, although these are RNA viruses. To our knowledge, no major developments of EBV-RNA-based treatments are currently underway. in vitro and then reinjected into the patients [Given the strong T-cell response mounted towards EBV, it seems logical to try to enhance such an immune response. The two main leads use autologous dendritic cells or T cell that are enhanced patients . A Phasepatients . Out of patients . The sampatients . Nevertheless, it is worth mentioning that, although cell therapy has shown promising results, it remains very costly and technically difficult. Thus, other, cheaper and more accessible therapies are also being developed. Given that most key immune regulatory checkpoints are expressed in NPC cells, targeting them using checkpoint inhibitors seems logical. Programmed death-1 (PD-1) is found on the surface of activated B and T cells and is an inhibitory molecule that favours immune tolerance. PD-1 interacts with members of the B7 family: PD-Ligand 1 (PD-L1) and PD-Ligand 2 (PD-L2) . HoweverPhotodynamic therapy (PDT) is a new promising treatment for cancers. It involves the administration of a photosensitiser, followed by a local illumination of tumour tissue with light of a wavelength complementary to the absorption spectrum of the photosensitiser. The photosensitiser produces reactive oxygen species (ROS) when activated by light. ROS’ in turn induce direct cytotoxicity of tumour cells as well as indirect destruction of tumour cells through vascular damage . It has 2 light. All 12 patients responded to treatment, among who three remained disease-free after 12 months and three others achieved useful palliation. The only complication observed was skin hypersensitivity. An interesting study conducted by Li et al. was designed to evaluate the clinical response of Photofrin PDT in patients with relapsed NPC. Thirty patients were divided into two groups of either Photofrin PDT or chemotherapy. Six months after treatments, PDT showed better local response and nasal cavity obstruction remission than chemotherapy [An initial study conducted by Lofgren et al. on recurrent or persistent forms of NPC was designed to determine the efficacy of PDT in five patients who were unresponsive to radiotherapy . Four paotherapy .® PDT in one patient with residual NPC (T4N0M0) after failing to respond to chemotherapy and radiotherapy [® before illumination. At the end of the therapy, a remarkable long-term response was described with no tumour progression and no lymph node metastasis. The same team conducted a similar study on 21 patients which showed that Foscan® PDT is effective in treating local failures of NPC with a depth of less than 10 mm [® PDT and once again this treatment was well tolerated by patients and showed efficacy against residual or recurrent NPC [® PDT [® before illumination. After the first PDT, two patients are disease-free at 38 and 71 months, one patient remains disease-free after a second PDT treatment, another one is currently living with the disease and two patients died. Temporary pain during swallowing was observed in all patients.Following these promising studies, other trials were carried out involving other photosensitisers. For instance, Indrasari et al. reported the efficiency of Foscanotherapy . He was an 10 mm . Nyst etrent NPC . SimilarC [® PDT . All patin vivo before they can be used as a therapy.Moreover, the effects of other promising photosensitisers such as 5-aminolevulinic acid (ALA) and its hexyl ester (ALA-H) were assessed on NPC cells . Wu founThus, all these studies tend to indicate that PDT has the potential to be an effective local treatment for recurrent/persistent NPC with only side effects linked to photosensitivity.Interestingly, it has been shown that PDT shuts down the tumour microvasculature and stimulates the host’s immune system. In contrast to surgery, radiotherapy and chemotherapy that are mostly immunosuppressive, PDT causes acute inflammation, expression of heat-shock proteins, invasion and infiltration of the tumour by leukocytes and might increase the presentation of tumour-derived antigens to T cells . Indeed, PDT produces tumour-cell destruction in the context of an acute inflammation that acts as a “danger signal” to the innate immune system. Activation of the innate immune system increases the priming of tumour-specific T lymphocytes that have the ability to recognise and destroy distant tumour cells and, in addition, lead to the development of an immune memory that can prevent recurrence of the cancer at a later time. PDT may also be successfully combined with immunomodulating strategies that are capable of overcoming or bypassing the escape mechanisms employed by the progressing tumour to evade immune attack .in vivo. Thus, their results identified PS and PD as novel ICD inducers that could be effectively combined with PDT in cancer therapy [As is well described, anti-cancer therapy is more successful when it can also induce an immunogenic form of cancer cell death (ICD). Therefore, when developing new treatment strategies, it is extremely important to choose methods that induce ICD and thereby activate an anti-tumour immune response leading to the most effective destruction of tumour cells. A very interesting work analysed whether the widely clinically used photosensitisers, photosens (PS) and photodithazine (PD), can induce ICD when used PDT. Using dying cancer cells induced by PS-PDT or PD-PDT, the authors notably demonstrated the efficient vaccination potential of ICD  therapy .Otherwise, a very recent investigation from our team has suggested that PDT, which is an effective therapy in the treatment of pancreatic ductal adenocarcinoma (PDAC), also activates the immune system and could be considered as a real adjuvant for anti-cancer vaccination. The authors developed a new photosensitiser (PS-FOL/PS2) that is associated with an addressing molecule (folic acid) targeting the folate receptor 1 (FOLR1) with a high affinity . Folate specifically binds to FOLR1 which is expressed in 100% of PDACs or over-expressed in 30% of cases. Interestingly, they observed a significant increase in the proliferation of activated human PBMCs and T cells when cultured with PDAC cell-conditioned media subjected to PS-FOL/PDT .Moreover, in the context of squamous cell carcinoma, a very interesting study has shown that, besides causing direct cytotoxic effects on illuminated tumour cells, PDT causes damage to the tumour vasculature and induces the release of proinflammatory molecules. Indeed, pre-clinical and clinical studies on squamous cell carcinoma have demonstrated that PDT can affect both the innate and adaptive arms of the immune system. Besides stimulating tumour-specific cytotoxic T-cells capable of destroying distant untreated tumour cells, PDT leads to the development of anti-tumour immune memory. The immunological effects of PDT make the therapy more effective also when used for treatment of bacterial infections, due to an enhanced infiltration of neutrophils into the infected regions that seems to potentiate the outcome of the treatment .in vitro study suggested that PDT using Zn-BC-AM photosensitiser might indirectly reduce tumour growth through the modulation of cytokine production. Indeed, by examinating the effects of EBV infection on proinflammatory cytokines secretion by NPC cells after Zn-BC-AM PDT, they showed that a light dose of 0.25–0.5 J/cm2 on Zn-BC-AM PDT-treated HK-1-EBV cells induce a higher level of IL-1α and IL-1β secretion than the non-treated HK-1 cells. The production of IL-1β appeared to be mediated via the IL-1β-converting enzyme (ICE)-independent pathway. In contrast, the production of angiogenic IL-8 was downregulated in both HK-1 and HK-1-EBV cells after Zn-BC-AM PDT [In the field of NPC, an C-AM PDT .The impact of PDT on tumour reduction through cytokines has also been confirmed by studying patients with residual or recurrent NPC who had received PDTs between 2005 and 2011. Pre- and post-test examination were used to test the hypothesis of the cytokines level difference before and after PDTs. The authors described that PDT in NPC either residual or recurrence patients after receiving radiation or in chemo-radiation therapy will yield a well-responding therapy, compared to those who did not receive PDT, and inducing systemic anti-tumour response which was marked by the increased level of immune-response cytokines . in vivo work characterised immune responses triggered by redaporfin-PDT and demonstrated that vascular-PDT leads to a strong neutrophilia, systemic increase of IL-6, increased percentage of CD4+ and CD8+ T cells producing IFN-γ or CD69+ and increased CD4+/CD8+ T cell ratio. At the tumour bed, T cell tumour infiltration disappeared after PDT but reappeared with a much higher incidence a day later. In addition, they showed that the therapeutic effect of redaporfin-PDT is highly dependent on neutrophils and CD8+ T cells but not on CD4+ T cells [Finally, a recent study has demonstrated in mice bearing CT26 tumours that vascular PDT with redaporfin PS, using a low light dose delivered at a high fluence rate not only destroys the primary tumour but also reduces the formation of metastasis, thus suggesting anti-tumour immunity. Indeed, this  T cells .Arguably, major benefits might be achieved with immunostimulating approaches that induce appropriate tissue-based inflammation. PDT and vascular-PDT in a proinflammatory regimen achieved a successful transition from innate to adaptive anti-tumour immunity and transformed the immunosuppressive tumour microenvironment into a more favourable homing for anti-tumour immunity. This therapy may offer new opportunities to improve systemic NPC treatments.When diagnosed at early stages, NPC is classically treated with radiotherapy and/or chemotherapy (CRT) with over 90% five-year survival rate . UnfortuConsidering the surgery option for NPC, endoscopic nasopharyngectomy was the first technique used for the resection of early stage recurrences and has shown encouraging results . Indeed,Regarding targeted therapies, clinical trials have shown only modest advantages for NPC patients with also major toxic side-effects  leading Furthermore, when the first immunotherapies were tested in clinical trials, NPC posed as a good candidate given the strong immunosuppressive tumour microenvironment and its EBV-linked nature. Indeed, it is well established that NPC expresses viral proteins, mainly EBNA1-3 and LMP1-2, that are involved in carcinogenesis. However, evidence shows that the best strategies are the ones that simultaneously target multiple EBV proteins. Thus, an EBV vaccine would increase the availability of viral antigens and ultimately enhance the EBV-specific immune response. Viral RNAs are also interesting but elusive targets that have not yet pursued in upcoming NPC therapies. in vitro and then reinjected into the patients [Moreover, given the strong T-cell response mounted towards EBV, cell therapy protocols are of great intertest. The two main leads either use autologous dendritic cells or T cell that are enhanced patients . Like alMost key immune regulatory checkpoints are expressed in NPC cells which in part explains local immune tolerance towards the tumour. Thus, testing immune checkpoint inhibitors in NPC was met with high expectations. As the complex interplay of EBV and NPC continues to be unraveled, it is likely that immunotherapeutic strategies will merge into mainstream clinical practice and offer durable remissions in patients with advanced NPC who are this day incurable.Finally, photodynamic therapy (PDT) is a new promising treatment for several cancers, including NPC. Arguably, major benefits might be achieved with immunostimulating approaches that induce appropriate tissue-based inflammation. PDT and vascular-PDT in a proinflammatory regimen achieved a successful transition from innate to adaptive anti-tumour immunity and transformed the immunosuppressive TME into a more favourable homing for anti-tumour immunity. This therapy may offer new opportunities to improve systemic NPC treatments.In conclusion, there has recently been substantial progress in the understanding of NPC biology. Thus, the search for compelling new diagnostic tools and treatments is gaining momentum. The repertoire of treatment options is widening, resulting in lower morbidity rates for locally recurrent NPC patients. Advances in therapeutics, namely immunotherapy and photodynamic therapy, have shown promising results. With all these exciting recent advances, we are looking forward to future studies, which will further improve our understanding of NPC and significantly improve the current management of NPC patients.


The advantages of photodynamic diagnostic technology using 5-aminolevulinic acid (ALA-PDD) have been established. The aim of this prospective cohort study was to evaluate the usefulness of ALA-PDD to diagnose upper tract urothelial carcinoma (UT-UC) using the Olympus VISERA ELITE video system.We carried out a prospective, interventional, non-randomized, non-contrast and open label cohort pilot study that involved patients who underwent ureterorenoscopy (URS) to detect UT-UC. 5-aminolevulinic acid hydrochloride was orally administered before URS. The observational results and pathological diagnosis with ALA-PDD and traditional white light methods were compared, and the proportion of positive subjects and specimens were calculated.A total of 20 patients were enrolled and one patient who had multiple bladder tumors did not undergo URS. Fifteen of 19 patients were pathologically diagnosed with UT-UC and of these 11 (73.3%) were ALA-PDD positive. Fourteen of 19 patients were ALA-PDD positive and of these 11 were pathologically diagnosed with UC. For the 92 biopsy specimens that were malignant or benign, the sensitivity for both traditional white light observation and ALA-PDD was the same at 62.5%, whereas the specificities were 73.1% and 67.3%, respectively. Of the 38 specimens that were randomly biopsied without any abnormality under examination by both white light and ALA-PDD, 11 specimens (28.9%) from 5 patients were diagnosed with high grade UC. In contrast, four specimens from 4 patients, which were negative in traditional white light observation but positive in ALA-PDD, were diagnosed with carcinoma in situ (CIS).Our results suggest that ALA-PDD using VISERA ELITE is not sufficiently applicable for UT-UC. Nevertheless, it might be better particularly for CIS than white light and superior results would be obtained using VISERA ELITE II video system.Trial registration: The present clinical study was approved by the Okayama University Institutional Review Board prior to study initiation (Application no.: RIN 1803–002) and was registered with the UMIN Clinical Trials Registry (UMIN-CTR), Japan (Accession no.: UMIN000031205). Photodynamic diagnostic (PDD) technology using 5-aminolevulinic acid (ALA-PDD) is currently being used for the diagnosis of brain tumors in Japan. For patients with bladder cancer, the sensitivity and specificity of ALA-PDD using scopes and video systems produced by Karl Storz SE & Co. KG  can be as high as 75.8% and 68.2%, respectively . Thus, iUpper tract urothelial carcinoma (UT-UC) includes renal pelvic and ureteral cancer, which are the same as bladder cancer from a pathological perspective. Although standard treatment for UT-UC is total nephroureterectomy and partial cystectomy, techniques involving endoscopic diagnosis and resection with URS have been developed , 4. HoweThe aim of this prospective cohort study was to evaluate the usefulness of ALA-PDD for diagnosis of UT-UC using the VISERA ELITE video system produced by Olympus Co., Ltd.All methods were carried out in accordance with relevant guidelines and regulations.We carried out a pilot, prospective interventional, non-randomized, non-contrast and open label cohort study. Enrollment began following approval by the review board. Patients who were suspected of having UT-UC and were undergoing ureterorenoscopy (URS) were enrolled. Since this study was a pilot study, 20 cases were considered based on the annual number of cases treated at our institution. The primary endpoint was the proportion of positive subjects and secondary endpoints were sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and adverse events for ALA-PDD.Patients that were enrolled in the study were adults who were suspected of having UT-UC and were undergoing URS. All patients were aware of the conditions associated with the disease, and provided informed consent to participate. All enrolled patients agreed to use contraception until 1 month after the administration of 5-ALA. Exclusion criteria were patients with porphyria, allergy to porphyrin or 5-ALA, hepatic dysfunction, use of drugs that induced photosensitivity, pregnancy, or severe underlying diseases such as cardiovascular diseases or infection.On the day of the URS, 20 mg/kg of 5-aminolevulinic acid hydrochloride (5-ALA HCl) was orally administered over 180 min (range: 120–240 min) before the scheduled insertion time for the ureteroscope. After oral administration of 5-ALA HCl, patients drank 250 mL of an L-arginine-intensive drink and 500 mL of fruit juice to maintain blood pressure. At the time of examination, normal observations using white light and ALA-PDD using visible blue light were carried out using a two-color LED light source . All endoscopic images were recorded.After the procedures, patients had proper shading to prevent photosensitivity for up to 48 h after ingesting 5-ALA HCl and light exposure of less than 500 lx was maintained. Hematologic and biochemical examinations were performed on postoperative days (POD) 1 and 3 and the occurrence of adverse events was noted.5-ALA HCl was supplied as a white powder similar to that for ALAGLIO by SBI Pharmaceuticals . The chemical compound was stored according to strict quality control guidelines and detailed usage records were maintained by the Division of Clinical Research of New Drugs and Therapeutics in the Center for Innovative Clinical Medicine, Okayama University Hospital.The endourological techniques and URS procedures performed at our institution for observation of the upper urinary tract and ablation of tumors were previously described , 12. AllThe observational results and pathological diagnoses, the proportion of positive subjects, positive specimens, sensitivity, and specificity were calculated. Adverse events were also investigated and severity was categorized using the Clavien-Dindo classification.Characteristics of the 20 patients enrolled in this study are shown in Table The median (IQR) time from oral administration of 5-ALA HCl to biopsy was 246 (227–269) minutes. One patient had multiple tumors in the bladder and URS was aborted. Among the 19 remaining patients, biopsy was not performed because of absence of any abnormality in one patient, for whom URS was performed as follow-up. Fifteen patients were pathologically diagnosed with UT-UC and of these, 11 (73.3%) were ALA-PDD positive. Fourteen of 19 patients were ALA-PDD positive and of these, 11 were pathologically diagnosed with UC. Among the 50 specimens that are ALA-PDD positive, 25 (50%) were pathologically diagnosed with UC, 17 (34%) were not malignant, and the status for 8 (16%) could not be determined because the specimens were too small. Among the 7 patients that were ALA-PDD positive and white light negative  in 6 patients and hypotension requiring vasopressors (Grade II) in one patient. No allergic episodes, cardiovascular complications or photosensitivity were observed in our study.In this prospective cohort study to evaluate the effectiveness of ALA-PDD for UT-UC diagnosis using the VISERA ELITE video system, the sensitivity and specificity were 62.5% and 67.3%, respectively. Of the 38 specimens that were randomly biopsied without any abnormality under examination by both white light and ALA-PDD, 11 specimens (28.9%) from 5 patients were diagnosed with high grade UC. On the other hand, four biopsy specimens that had abnormal findings with ALA-PDD but not with normal white light observation were diagnosed with CIS.ALA-PDD was approved only for bladder cancer by Japanese national health insurance in 2017. This approach is expected to have value not only for bladder cancer diagnosis but also for treatment, namely complete resection at TURBT –16. On tInterestingly, 11 of the 38 (28.9%) specimens that exhibited no abnormalities in either white light or ALA-PDD examinations were pathologically diagnosed with high grade UC. The reasons for this result are unclear, but some abovementioned factors may have contributed to this outcome. To our knowledge there are no studies that examined random biopsy samples taken from the upper urinary tract, and additional investigations are needed.According to European guidelines , diagnosAdverse events of ALA-PDD have been reported as liver toxicity, allergic episode, cardiovascular complications, photosensitivity, hypotension, nausea and vomiting , 25. In The present study has several limitations. Although this pilot study was prospective, it was performed at a single center, was non-randomized and included small number of patients (n = 20). Second, there were 2 patients who underwent protocol URS after endoscopic laser ablation and cognitive bias could be associated with the previous procedure. Third, we evaluated the usefulness of ALA-PDD only in the diagnostic process and not the treatment process. Fourth, we did not compare endoscopic devices including semirigid/flexible ureteroscopes, light cables, and video systems, and did not evaluate their compatibility with ALA-PDD. Despite these limitation, the ureteroscopy with ALA-PDD might be more useful for diagnosis in patients with UT-UC, particularly with CIS.Our results of this pilot study suggest that ALA-PDD using VISERA ELITE is not sufficiently applicable for UT-UC. Nevertheless, it might be better particularly for CIS than white light and superior results would be obtained using VISERA ELITE II video system. Further studies on patient selection, endoscopic equipment and image processing methods are needed for the broad application of ALA-PDD for UT-UC diagnosis.


Autism spectrum disorder (ASD) describes a collection of neurodevelopmental disorders characterized by core symptoms that include social communication deficits and repetitive, stereotyped behaviors often coupled with restricted interests. Primary challenges to understanding and treating ASD are the genetic and phenotypic heterogeneity of cases that complicates all omics analyses as well as a lack of information on relationships among genes, pathways, and autistic traits. In this study, we re-analyze existing transcriptomic data from simplex families by subtyping individuals with ASD according to multivariate cluster analyses of clinical ADI-R scores that encompass a broad range of behavioral symptoms. We also correlate multiple ASD traits, such as deficits in verbal and non-verbal communication, play and social skills, ritualistic behaviors, and savant skills, with expression profiles using Weighted Gene Correlation Network Analyses (WGCNA). Our results show that subtyping greatly enhances the ability to identify differentially expressed genes involved in specific canonical pathways and biological functions associated with ASD within each phenotypic subgroup. Moreover, using WGCNA, we identify gene modules that correlate significantly with specific ASD traits. Network prediction analyses of the genes in these modules reveal canonical pathways as well as neurological functions and disorders relevant to the pathobiology of ASD. Finally, we compare the WGCNA-derived data on autistic traits in simplex families with analogous data from multiplex families using transcriptomic data from our previous studies. The comparison reveals overlapping trait-associated pathways as well as upstream regulators of the module-associated genes that may serve as useful targets for a precision medicine approach to ASD. Autism spectrum disorder (ASD) is a neurodevelopmental disorder in which symptoms typically appear within the first 3 years of life. According to the Diagnostic and Statistical Manual of Mental Disorders-5th Edition (DSM-5), a diagnostic guide created by the American Psychiatric Association for mental disorders , individRecently, researchers have explored reducing clinical heterogeneity by selecting individuals with specific ASD traits based on severity scores from the Autism Diagnosis Interview-Revised (ADI-R) diagnostic instrument –5. For rFor genetic and quantitative trait association analyses, we further divided the three core domains probed by the ADI-R  into subcategories  which led to the identification of both subtype- and trait-associated genetic variants , 10. SavDespite the demonstrated association of genetic variants and autistic traits, there is still relatively little understanding of the relationship between gene expression and ASD traits. In this regard, Weighted Gene Co-expression Network Analysis (WGCNA) is a software tool designed to explore the relationships between gene expression profiles and external information, such as case-control status or a specific condition . It has The primary goals of this study are to: (1) determine whether subtyping of the ASD probands from simplex families (having only one affected child) by cluster analyses of scores from the ADI-R improves the ability to determine gene expression  differences between subtyped cases and controls over unsegregated cases and controls, (2) use WGCNA to investigate the association of gene networks with various traits of ASD, and (3) compare the gene expression modules and pathways associated with selected autistic traits in simplex families vs. those in multiplex families to identify both differences and similarities.The main goal of this study was to re-analyze existing transcriptomic data on a group of autistic probands and their respective siblings from the Simons Simplex Collection (SSC) in order to determine whether subtyping of ASD probands by multivariate cluster analysis of ADI-R item severity scores can facilitate detection of gene expression differences between cases from phenotypic subgroups and their non-autistic siblings. In addition, we sought to interrogate and compare relationships between gene networks and autistic traits in cases from both simplex and multiplex families. K-means cluster (KMC) analysis. This analysis showed an optimum separation of cases with K = 3, indicating three phenotypic subgroups. This was in contrast to the four phenotypic subgroups that optimally distinguished ASD cases in multiplex families  were obtained from the SSC . As described previously for multiplex families , 123 scofamilies . Unsuperfamilies .GSE37772). The expression data had been obtained using the Illumina Whole Human Genome Array Human REF-8 version 3.0 following the manufacturer's standard protocol . These data were obtained using custom-printed TIGR 40K human arrays containing 39,936 human cDNA probes, as previously described. ADI-R data for the individuals in this study were obtained from the Autism Genetics Research Exchange, with subtyping performed on adjusted ADI-R scores as previously described  was used to visualize the separation of cases and controls based on the subgroup-associated DEGs. Based on the PCA results, we used a more stringent p-value of ≤ 0.03 to optimize the separation of Mild cases from their respective sibling controls.Differential gene expression analysis was performed using Multi-experiment Viewer (MeV) software for microarray analyses  which emAnother goal of this study was to investigate the correlation between gene expression profiles and autistic traits. The three core domains considered in the ADI-R diagnostic tool  with external information . For WGCNA analyses of autistic traits in simplex families, normalized gene expression data for 24,526 probes  from 63 male probands were used to identify gene networks that associated with the six autistic traits. Similarly, WGCNA analysis of autistic traits in multiplex families utilized normalized gene expression data for 28,592 probes  from 81 male individuals with ASD. This transcriptomic data was derived from the study of 7).WGCNA is an R-package primarily designed for co-expression analysis of transcriptomic data . In thisIngenuity Pathway Analysis (IPA) software  was used for network prediction analyses to identify canonical pathways, biological functions, and diseases enriched among DEGs from our subtype-dependent transcriptomic analyses as well as those over-represented among genes in significant trait-associated gene modules from WGCNA analyses. The Fisher Exact Test, as implemented by IPA software, was used to determine the significance of enrichment with respect to a given pathway, function, or disease using IPA Knowledgebase as a reference gene set. IPA was also used for comparison analysis of pathways and upstream regulators associated with DEGs from simplex and multiplex samples.https://bioinfogp.cnb.csic.es/tools/venny/. The CASIO Keisan Online Calculator http://keisan.casio.com/exec/system/1180573201 was then used to determine hypergeometric distribution probabilities for over-representation of module genes within the SFARI dataset with significance determined by an upper cumulative q ≤ 0.05.Trait-associated module genes were compared to a list of 910 known autism risk genes in the SFARI Gene database . Venny 2Differential gene expression analysis of each subtype shown in IPA was used to identify over-represented canonical pathways, diseases, and neurological functions among the DEGs from each of the three ASD subgroups. WGCNA was used to identify gene clusters (modules) correlated with clinical autistic traits in 63 male probands using their respective cumulative ADI-R trait scores and normalized expression data for all detectable genes. Inasmuch as ASD is a neurodevelopmental disorder predominantly affecting males , we decip = 9.25E-13; 189 genes) and cognitive impairment , which are comorbidities frequently seen in ASD , suggesting that deficits in non-verbal communication are major contributors to the overall phenotype of ASD in the simplex population.IPA was used to identify canonical pathways that were enriched among the module genes collectively contributing to verbal, non-verbal, and social deficits in individuals with ASD from the simplex population. The complete sets of significantly over-represented pathways for each trait are presented in n in ASD , 33. In WGCNA was also used to identify gene modules that correlated with selected autistic traits in males from multiplex families that were included in our previous gene expression study of ASD subtypes  for the p-values) for pathway enrichment ranging from 7.9 to 12.5 and from 8.6 to 11.3, respectively. Mitochondrial dysfunction and oxidative phosphorylation are also significantly over-represented among genes associated with the non-verbal and savant traits. As for the simplex samples, there are both unique and overlapping ASD-associated pathways among the four traits represented in IPA was used to identify pathways and functions over-represented among module genes significantly associated with five ASD traits in the multiplex population. The gray module was not included in the pathway analysis because it represents the default module comprised of genes not correlated in any other module. The complete lists of significant trait-associated pathways are provided in p = 5.7E-22), non-verbal (p = 3.69E-09), and savant (p = 8.36E-10) traits, while a related disorder, ataxic gait, is associated with play skills (p = 4.09E-04) and insistence on sameness or ritualistic behaviors (p = 5.79E-06). Genes for cognitive impairment are enriched for both verbal (p = 1.9E-15) and savant (p = 1.26E-08) traits, although the shared modules that are significant for these two traits show inverse correlation with respect to gene expression and cumulative trait score.With respect to neurological diseases, motor dysfunction and movement disorders are significantly associated with verbal , the savant skills are also associated with genes linked to severe mental retardation (p = 2.00E-03) and cognitive impairment (p = 4.89E-03), thus highlighting their relevance to intellectual ability and cognition.To better distinguish pathways differentiating verbal and savant skills, IPA was used to analyze genes in modules unique to each trait . For the verbal trait, these 8 modules included the black, blue, brown, cyan, darkgreen, greenyellow, plum1, and skyblue. For the savant trait, these modules included grey60, lightyellow, royalblue, and tan. q-values of 0.012 (simplex), 7.62E-06 (multiplex), and 0.044 (overlapping), thus confirming relevance of these genes to ASD.While the WGCNA analyses of autistic traits clearly show differences in the gene networks associated with ASD in simplex and multiplex families, we also sought to find similarities as well as differences among all the trait-associated genes and biological networks. A direct comparison of all trait-associated genes from the simplex  and multiplex  individuals with ASD shows an overlap of 1,754 genes. Hypergeometric distribution analyses were then conducted to determine the over-representation of 910 autism risk genes  from the SFARI Gene database within each set of trait-associated genes as well as the overlapping set of genes. These analyses show that trait genes from both simplex and multiplex populations as well as the overlapping set are significantly enriched in SFARI genes, with hypergeometric distribution upper cumulative p-values of overlap: 2.48E-25, 7.8E-23, and 1.06E-20, respectively).IPA was then used to identify canonical pathways over-represented among the overlapping genes. q-value of 0.001 for enrichment in SFARI genes), pathways, and functions relevant to ASD. Among the significantly enriched ASD-relevant pathways associated with the Language phenotype are those involved in axonal guidance, actin cytoskeleton signaling, synaptogenesis, GABA receptor, and neuregulin signaling , in this study, we use WGCNA to identify gene modules that may relate to specific traits of ASD. These traits included deficits in verbal ability (spoken language), non-verbal communication, play skills, social interaction, insistence on sameness/rituals, and manifestation of savant skills. As shown in Although we have previously reported on differential gene expression in three subtypes of ASD from multiplex families , we wereTP53, and the proto-oncogene MYC are both regulators of cell cycle progression, growth, and apoptosis, processes that are notably deregulated in ASD  are considerably greater for the multiplex population 18 vs. 7 modules). These results suggest that there may be a greater burden with respect to gene dysregulation and network disruption in individuals with ASD from multiplex families. Despite these differences, there are shared canonical pathways that appear to underlie ASD traits in both populations albeit to different extents, as shown in d in ASD , 41. Thed in ASD , 43, andd in ASD , 45. Hepd in ASD . While Hd in ASD  and majod in ASD  as well d in ASD . Notably modules.d in ASD , 50–52. d in ASD , 53–55. An obvious limitation of this study is that our results are based on transcriptomic analyses of lymphoblastoid cell lines. It has been argued that peripheral tissues are not the ideal experimental model to understand brain development despite the fact that many neurological functions and signaling pathways relevant to autism have been identified in LCL and other peripheral tissues, such as whole blood, lymphocytes, and natural killer cells , 56–62. This study shows that heterogeneity reduction by phenotypic subtyping of individuals with ASD enhances the ability to identify more differences in autism-relevant gene expression, pathways, and functions between probands and siblings from simplex families in comparison to those obtained with a combined case group. These findings thus replicate those of our earlier study that applied this subtyping approach to transcriptomic analyses of the multiplex ASD population. While we used multivariate cluster analyses of ADI-R severity scores for the subtyping, we anticipate that other approaches to reduce heterogeneity among individuals with ASD , or functional MRI profiles) may also improve the ability to detect biological differences at the genome-wide level.To our knowledge, this is the first study to use WGCNA to analyze gene networks in the context of a continuous severity spectrum of autistic traits rather than with a dichotomous diagnosis of ASD. Perhaps the most important findings from this study are the associations of specific ASD traits with expressed gene networks and embedded pathways that may serve as useful targets for precision medicine in autism spectrum disorders.GSE37772 and GSE15402.Publicly available datasets were analyzed in this study. This data can be found here: Gene Expression Omnibus (GEO); Accession numbers The studies involving human participants were reviewed and approved by GWU Institutional Review Board, Office for Human Research. Written informed consent from the participants' legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.VH conceived of and designed the study, performed subgroup phenotyping, pathway, functional and hypergeometric analyses, and wrote the manuscript. CB was responsible for gene expression and WGCNA analyses as part of her Master's thesis research and contributed to manuscript preparation. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The coronavirus disease 2019 (COVID-19) has created unprecedented challenges on the healthcare system. The aim of this multi-centre study was to measure the impact of COVID-19 on IR services in the UK.Retrospective cross-sectional study of IR practice in six UK centres during the COVID-19 pandemic was carried out. All therapeutic IR procedures were identified using the respective hospital radiology information systems and COVID-19 status found on the hospital patient record systems. The total number of therapeutic IR procedures was recorded over two time periods, 25/03/2019–21/04/2019 (control group) and 30/03/2020–26/04/2020 (COVID-19 group). The data points collected were: procedure type, aerosol-generating nature, acute or elective case, modality used, in- or out-of-hours case and whether the procedure was done at the bedside (portable).n = 93), and 15% of total cases (n = 141) were classed as aerosol-generating procedures. A 66% rise in cholecystostomy was noted during COVID-19. Image-guided ablation, IVC filters, aortic stent grafting and visceral vascular stenting had the greatest % decreases in practice during COVID-19, with 91.7%, 83.3%, 80.8% and 80.2% decreases, respectively.A 31% decrease in overall number of IR procedures was observed during COVID-19 compared to the control group (1363 cases vs 942 cases); however, the acute work decreased by only 0.5%. An increase in out-of-hours work by 10% was observed. COVID-19 was suspected or laboratory proved in 9.9% of cases  contains supplementary material, which is available to authorized users. The novel coronavirus disease 2019 (COVID-19) was officially declared a pandemic by the World Health Organization on 11 March 2020. During the subsequent weeks, the rising number of new COVID-19 cases and hospital admissions in the UK resulted in increased pressures on the national health service (NHS). In an attempt to tackle these issues, the UK government issued a national lockdown on 23 March 2020. Despite these strict measures, cases continued to rise and COVID-19 death rates peaked in mid-April [Like other healthcare systems around the world that have quickly adapted their acute services, NHS hospitals swiftly responded to the pandemic, by halting elective services, to increase capacity for the expected surge in patients requiring urgent respiratory support. COVID-19 has brought new challenges for interventional radiology (IR), both in terms of workflow and IR preparedness; however, IR has continued to provide acute and emergency treatments due to the unique value that IR has to offer in terms of adaptability and offering minimally invasive procures (MIPs), the majority of which are done under local anaesthetic which are not aerosol-generating procedures (AGPs) , 3.The aim of this multi-centre study was to review the impact of COVID-19 on IR services in terms of workload and case mix.This study was a retrospective multi-centre cross-sectional study of IR practice during the COVID-19 pandemic. No formal ethical approval was required as these were audit data. The study was registered and approved within each Trust’s audit department. No identifiable patient data were kept or reported. This study required no internal or external funding. The authors have no conflicts of interest to declare.All therapeutic IR procedures were identified using the respective hospital radiology information systems and COVID-19 status found on the hospital patient record systems.The number of IR procedures was recorded over two time periods, 25/03/2019–21/04/2019 (control group) and 30/03/2020–26/04/2020 (COVID-19 group).The study period for 2020 was selected to encompass the peak in COVID-19 cases throughout the UK and capture the subsequent effect on the provision of IR services. The same 4-week interval in 2019 was captured which formed a control group for comparing the impact of COVID-19.This was replicated in six NHS trusts and health boards  to measure the variation in baseline workload and subsequent impact from COVID-19.For all recorded IR cases, we included type of procedure , acute/emergency or time-critical elective case, modality used, out of hours or in hours and whether the procedure was done at the bedside (portable) guidance for what constitutes as AGPs was from Sect. Time-critical elective procedures were defined as an intervention that was planned or booked in advance of the routine admission to hospital . These rAcute emergency procedures were defined as inpatient cases done for urgent, possibly life- or limb-threatening conditions, for example, Neuro-IR/stroke intervention, aortic stent grafting for ruptured aneurysm, embolisation for intra-abdominal bleeding, peripheral intervention for acute limb ischaemia, image-guided drainage for infective collections, pneumothoraces or tense ascites.For the 2020 (COVID-19) group, we included COVID-19 status at the time of procedure which was split into three categories: confirmed COVID-19 case if polymerase chain reaction (PCR) test positive, suspected COVID-19 based on clinical or imaging evidence (even if PCR test negative) or not suspected with or without COVID-19 negative PCR test.The absolute number and types of cases during the pre-COVID-19 period and COVID-19 period are shown in Table The impact of COVID-19 on acute IR work is site variable. Three out of the six sites report a decrease in workload, whereas the other 50% have performed more cases in the acute setting when compared to the pre-COVID-19 time period. The resulting effect on all site average is a 0.5% decrease in acute work. On the other hand, the trend for impact on elective work was a uniform decrease across all sites with an overall decrease of 58% (range 25%–80%). CT-guided procedures had the greatest decrease in workload (53.3%) compared to fluoroscopy-guided (33.3%) and ultrasound-guided (14.6%) procedures.The IR case mix during the COVID-19 pandemic across the six centres is highlighted in Fig. The most common procedures performed during the COVID-19 period were vascular access, abdominal drainages, peripheral vascular intervention and nephrostomies ± ureteric stenting. The areas of IR that had the greatest decrease in activity during the pandemic were image-guided ablation (91.7% reduction), placement of IVC filters (83.3% reduction), aortic stent grafting (80.8% reduction) and visceral vascular stenting (80.2% reduction).The total number of gallbladder drains performed increased by 67% during the COVID-19 pandemic. The out-of-hours work also increased by 10.4% during the pandemic.During the 2020 study period, there were 22 IR cases (2.3%) of confirmed COVID-19. An additional 71 cases were treated as suspected COVID-19 (7.5%). No IR physicians or allied healthcare professionals were directly infected from the cases of confirmed or suspected COVID-19.The COVID-19 pandemic had a profound immediate impact on IR workload. We report a 31% decrease in the number of cases performed in the first 4 weeks of the COVID-19 lockdown when compared to an equivalent time period from 2019. The six sites reported a range of decreasing workload from 10 to 40%. Existing published data have measured the impact of COVID-19 on other hospital specialities, with a focus on acute and elective subgroups –12. We hn = 93) were either suspected or laboratory proven COVID-19, and 15% of total cases (n = 141) were classified as AGPs. As radiology is often perceived as a non-patient facing speciality, the staff can be miscategorised as relatively low risk from COVID-19 when compared to other hospital environments. However, this study clearly highlights the significant risk to staff members who work within the IR sub-speciality and challenges this notion, however thankfully there were no recorded cases of infection amongst IR team members from the confirmed or suspected cases. Furthermore, this represents a significant impact on day-to-day practice as these cases required extra planning, resources and safety. Complete personal protective equipment (PPE) was worn by all the staff involved in the cases which placed a greater emphasis on clear communication and team-work during the procedure.A major aim of this study was to quantify, and attempt to qualify, the impact of the national COVID-19 safety measures within the hospitals delivering the care. The procedure-related risk of the COVID-19 infection depends on many factors, which can be broadly considered as operator, patient and environment. We highlighted the COVID-19 status of the patient and AGPs as two of the most important surrogate markers for procedure-related exposure. It is important to note that infection control remains a continuously evolving topic. We have used the risk standards during the period of observation to maintain the context of perceived and actual risk. For example, a major NHS Foundation Trust at the time instructed staff to only perform cardiopulmonary resuscitation on cardiac arrest patients if they were in the emergency department and all staff members were wearing PPE . In our National figures reported by Public Health England state 2.11 million Accident and Emergency (A&E) attendances in April 2019 compared to only 916,000 attendances in 2020, which represents some of the lowest figures in recent times . A decreOur results show a site-specific variability in the impact on acute work. We believe the variation in reported acute work is due to several reasons despite the same national guidance given to all the sites. The sites within our study vary in size, and therefore, the IR departments will have a different volume of baseline work, available staff and capacity to run a ‘COVID-19 appropriate’ rota. It is also important to appreciate that the impact of COVID-19 in the UK is geography dependent. These factors generate the need to develop locally specific policies to ensure ongoing safety. Furthermore, acute work in IR is heavily influenced by workload in other clinical specialities, and we often provide complementary patient care. Due to the safety concerns of viral transmission secondary to AGPs, MIPs were preferred in managing acutely unwell patients. This may explain why acute IR services have remained busy and not seen the same dramatic decreases seen elsewhere, i.e. a negligible 0.5% decrease in acute work between the two time periods. In the context of an overall decrease in emergency cases across the NHS, one assumption would be that the proportional work done by IR has increased with the finding of a 10% rise in out-of-hours work unsurprising. Uncertain working conditions require clinicians to be flexible in their approach, and IR services in this study have been able to adapt and successfully deliver front-line patient care, likely attributable to efficient methods of workflow reconfiguration and availability to treat patients. Similar trends have been observed in the US, where IR procedural volumes have decreased by a significantly smaller amount compared to other surgical, cardiac and endoscopy services .Upper gastrointestinal endoscopy procedures are classified as AGPs with increased risk of virus transmission. This led to consensus guidance from the British Society of Gastroenterology, American College of Gastroenterology, European Society of Gastrointestinal Endoscopy and Asian Pacific Society for Digestive Endoscopy that elective endoscopies should be suspended . This reSurgical practice has also been greatly affected during the pandemic with the British Intercollegiate General Surgery Guidance on COVID-19 stating that ‘whenever non-operative management is possible (such as for early appendicitis and acute cholecystitis), this should be implemented’ . The theIn contrast to the acute setting, there was a uniform decrease in elective cases performed as per the national guidance given. Remarkably, however, all sites continued providing elective services, used to help patients requiring time-critical treatment, or those waiting longer than expected due to treatment delays, including renal dialysis and fistula work. The proposed aim was to reduce the immediate backlog of patients as the NHS headed into uncertain times. The ability to deliver routine services amidst a respiratory viral pandemic is essential and intrinsic to IR, particularly important as a second COVID-19 wave is looming, with our data highlighting areas where IR can step in to support other acute services . This fuThe limitations of this study are the retrospective design. The study includes relatively small numbers of certain IR procedures; however, the multi-centre design allowed for a wider geographic area to be sampled to give a more accurate representation of the true impact of COVID-19 on IR services. Only the peak period of the COVID-19 pandemic was sampled (4 weeks) rather than the entire UK lockdown period.IR has continued to provide emergency treatment whilst maintaining some time-critical elective services throughout the pandemic highlighting the adaptability of IR in supporting many specialties. The unique skill set of IRs allows them to play a key role during COVID-19 and future system-wide crises.Supplementary file1 (DOCX 103 kb)Below is the link to the electronic supplementary material.


Severe acute respiratory illness caused by 2019 novel coronavirus (2019-nCoV), officially named severe acute respiratory syndrome coronavirus (SARS-CoV-2) in late December 2019 is an extremely communicable disease. World Health Organization (WHO) declared coronavirus disease 2019 (COVID-19) as a pandemic as it has spread to at least 200 countries in a short span of time. Being a new disease there is lack of information about pathogenesis and proliferation pathways of this new coronavirus. Currently there is no effective treatment for coronavirus infection; major effort is to develop vaccine against the virus and development of therapeutic drugs for the disease. The development of genome-based vaccine and therapeutic antibodies require thorough testing for safety and will be available after some time. In the meanwhile, the available practical approach is to repurpose existing therapeutic agents, with proven safety record as a rapid response measure for the current pandemic. Here we discuss the presently used repurposed drugs for COVID-19 and the potential for ivermectin (IVM) to be used as a therapeutic option in COVID-19. The severe acute respiratory syndrome caused by coronavirus 2 (SARS-CoV-2) has been detected as the pathogenic virus in the current outbreak which started in the city of Wuhan, Hubei Province of China in early December 2019. The Centre of Disease Control and Prevention in China, detected a new type of corona virus on 7th January 2020 causing coronavirus disease of 2019 (COVID-19), provisionally named as 2019 novel coronavirus (2019-nCoV) by World Health Organization (WHO) and later renamed as SARS-CoV-2 by International Committee on Taxonomy of Virus  that makes this agent a broad antiviral agent . Though n = 99) has concluded that the combination was not more effective than standard care treatment and had similar recovery process in severe COVID-19  and EC90  values of 0.77 μM and 1.76 μM, respectively , including SARS-CoV-2 with ECectively .50 of favipiravir against SARS-CoV-2 was 61.88 μM/L in Vero E6 cells  polymerase, inhibiting viral replication. In vitro, the ECE6 cells .Streptomyces avermitilis and purified by Campbell, has greater potency and lower toxicity . IVM, an anthelminthic drug is used to treat various parasitic infestations. This includes river blindness (onchocerchiasis), head lice, scabies, lymphatic filariasis, ascariasis, entrobiasis, strongyloidiasis and trichuriasis . IVM wastoxicity . They actoxicity . The peatoxicity .Independent of its antiparasitic action, IVM has demonstrated broad spectrum antiviral property in vitro. IVM is shown to be effective in vitro against RNA and deoxyribonucleic acid (DNA) viruses, including human immunodeficiency virus-1 (HIV-1), dengue virus (DENV), influenza, Venezuelan equine encephalitis virus (VEEV) and Zika virus . NuclearPseudorabies virus (PRV) is the causative agent for pseudorabies (PR), which is an important swine disease. Virus can cause lifelong infection in pigs by residing in the trigeminal ganglia. Vaccines are used widely but are unable to provide absolute protection. Use of IVM, blocked the nuclear/nucleolar translocation of the accessory subunit of the DNA polymerase UL42 and reduced the lesions in mice caused by PRV infection in vitro and in vivo . This im50 for inhibition of YFV replication was between 3.1 nM and 6.3 nM. IVM proved less active against DENV in the CPE reduction assay (EC50 0.1 mM), although inhibition in virus yield reduction assays was observed (EC50 0.7 mM). IVM has proved to be most potent against YFV and inhibit the in vitro replication of DENV, JEV and WNV, though less efficiently  and DENV nonstructural (NS1) protein 5. In DENV, a single daily oral dose of IVM has resulted in a significant reduction in serum level of viral NS1 protein, but no change in viral load has been observed . Infectiiciently . Liposomiciently . Recentliciently .IVM is used in a dose of 0.15 mg/kg–0.2 mg/kg body weight for most of the parasitic infestations as oral tablet and is well tolerated. It will be reasonable to use similar dose empirically in COVID-19 patients with mild to moderate symptoms, PCR positive for SARS-CoV-2 virus, and without comorbidities on first encounter, till there is conclusive evidence from randomised controlled trials. Rarely adverse effects such as seizure, hypotension and worsening of asthma are observed with IVM and should be avoided in patients with history of allergy, liver disease and asthma. A combination of IVM (200 mcg/day once stat) with doxycycline (200 mg/day for 5 days) can also be attempted empirically as it has been administered previously for parasitic infections too.COVID-19 pandemic has affected over 200 countries in a span of less than 4 months burdening the entire health care system with no viable drug which can inhibit the viral transmission or infectivity. Most of the drugs which are being used to treat this novel disease are not so effective and the development of vaccines is distant in near future, thus there is enormous requirement to repurpose the available drugs using concrete evidences. Present circumstances demand a therapeutically effective option against SARS-CoV-2 without delay. IVM which is a widely used as antiparasitic drug has shown to have antiviral activity in in vitro studies against HIV, dengue, influenza, VEEV and Zika virus. The broad-spectrum antiviral activity of IVM against both RNA and DNA viruses offers an added advantage to its potential use over other antiviral drugs. Studies are available for its use against RNA virus and have also been tested for its effectiveness against SARS-CoV-2 in vitro. Based on evidences it seems prudent to use IVM empirically and conduct well designed controlled randomised trials to prove its efficacy in vivo.