Abdominal aortic aneurysm (AAA) is a fatal clinical condition with high mortality. Computed tomography angiography (CTA) imaging is the preferred minimally invasive modality for the long-term postoperative observation of AAA. Accurate segmentation of the thrombus region of interest (ROI) in a postoperative CTA image volume is essential for quantitative assessment and rapid clinical decision making by clinicians. Few investigators have proposed the adoption of convolutional neural networks (CNN). Although these methods demonstrated the potential of CNN architectures by automating the thrombus ROI segmentation, the segmentation performance can be further improved. The existing methods performed the segmentation process independently per 2D image and were incapable of using adjacent images, which could be useful for the robust segmentation of thrombus ROIs. In this work, we propose a thrombus ROI segmentation method to utilize not only the spatial features of a target image, but also the volumetric coherence available from adjacent images. We newly adopted a recurrent neural network, bi-directional convolutional long short-term memory (Bi-CLSTM) architecture, which can learn coherence between a sequence of data. This coherence learning capability can be useful for challenging situations, for example, when the target image exhibits inherent postoperative artifacts and noises, the inclusion of adjacent images would facilitate learning more robust features for thrombus ROI segmentation. We demonstrate the segmentation capability of our Bi-CLSTM-based method with a comparison of the existing 2D-based thrombus ROI segmentation counterpart as well as other established 2D- and 3D-based alternatives. Our comparison is based on a large-scale clinical dataset of 60 patient studies . The results suggest the superior segmentation performance of our Bi–CLSTM-based method by achieving the highest scores of the evaluation metrics, e.g., our Bi-CLSTM results were 0.0331 higher on total overlap and 0.0331 lower on false negative when compared to 2D U-net++ as the second-best. Abdominal aortic aneurysm (AAA) is a life-threatening clinical condition that needs long-term follow-up care and management. AAA is characterized as the abnormal focal dilation of the aorta that exceeds its nomotopic diameter by more than 50% . If leftComputed tomography angiography (CTA) imaging has been the preferred minimally invasive data modality for the follow-up observation of AAA that typically does not cause any noticeable physical symptoms. The use of postoperative CTA imaging allows for the quantitative assessment in the evolution of AAA by imaging specialists. The quantitative assessment should involve the detection of the thrombus region of interest (ROI) and the precise measurement of its morphologic properties, including maximum diameter, volume, and shapes, which, when manually performed by humans, is time-consuming and implies inter- and intra-observer variability .The intensity values of thrombus ROIs tend to overlap with those of adjacent tissues and organs a;The geometric shape of thrombus ROIs tends to be irregular, and its position could appear across any part of the abdominal aortic passway b,c;Parts of ROIs tend to be occluded by metal artifacts introduced by stent grafts. c,d.To facilitate the quantitative evaluation of thrombus ROIs, computerized segmentation of thrombus ROIs in postoperative CTA imaging was actively investigated ,9,10. TrIn addition, the traditional segmentation methods often involve user interaction and rely on prior knowledge. The segmentation performance was highly reliant on the careful adjustment of multiple parameters, which affects the robustness for use in clinical settings.Recent advances in deep learning research using medical imaging continue to address some automation, parameter tuning, user interaction, clinical robustness, and applicability ,12. Few In this work, we propose an automated thrombus ROI segmentation method to utilize not only the spatial features of a target image, but also the volumetric coherence among adjacent images. We propose the adoption of a recurrent neural network, bi-directional convolutional long short-term memory (Bi-CLSTM) , which cThe remainder of this paper is described as follows. In The traditional methods ,9,10,22 Machine learning- and deep learning-based methods have been proposed for enhanced segmentation of thrombus ROIs. Maiora et al.  used a rRecurrent neural networks are a type of deep learning architecture that is capable of learning coherence between a sequence of data. The LSTM architecture improved the learning process of the recurrent neural networks by addressing the vanishing gradient problems. LSTM architectures have been demonstrated to show notable performance in handling a large variety of sequential problems, e.g., natural language processing , machineN) to five for the best segmentation performance but we demonstrated that the different values of the sequence length had no significant influence on the segmentation performance.The whole process of our Bi-CLSTM-based segmentation method for thrombus ROIs in a postoperative CTA image volume is shown in The architecture of Mask R-CNN  for the Mask R-CNN produced three output branches, including (i) classification, which lets us know whether each region proposal belonged to the foreground class (thrombus ROIs); (ii) regression that estimated bounding box coordinates; and (iii) spatial attention map which produced binary prediction for the class. We only used the spatial attention map for our Bi-CLSTM-based segmentation method. The classification and regression branches used a fully connected (FC) layer with 1024 neurons, and the spatial attention map branch used FCN . We usedOur Bi-CLSTM-based segmentation method consisted of two residual blocks and a CLSTM layer (Conv LSTM) as shown in We built an evaluation dataset of 60 patient studies who went through EVAR operation for the treatment of AAA . Each patient study had a thrombus ROI in the abdominal aorta. To the best of our knowledge, our evaluation dataset is the largest in the number of patient studies. We observed that the geometrical structure and position of thrombus ROIs exhibited considerable variability in the evaluation dataset, and the noises and artifacts caused by postoperative stent grafts were dispersed randomly see . We beliThe evaluation dataset was acquired by Gachon University Gill Hospital, Republic of Korea, and had been collected for 9 years from 2012 to 2020. The age range of the evaluation dataset is from 51 to 88; this age range includes the elderly population over 65 in which AAA usually occurs . A totalThe ground truth (GT) segmentation masks of thrombus ROIs were manually annotated by a senior imaging specialist with more than 10 years of clinical experience. The reliance on the sole annotator may imply that inter and intra-observer variability was present in the evaluation dataset. We generated the ground truth of 2D bounding boxes by fitting rectangles to segmentation masks. We excluded the images from each patient study, where thrombus ROIs did not exist; in total, the number of images was 1637 for use in our evaluation. The full contrast range was rescaled to 8 bits between 0 and 255 without window-level adjustment to minimize human intervention. The window-level contrast adjustment transforms images to focus on specific organs and tissues to obtain better segmentation results. However, it may require the image specialist to manually define the optimal window-level depending on patient studies and scanning devices. So, we decided to use the consistent full contrast range. We could leverage the augmented data to train classifiers to increase reliability. In our method, elastic deformation  and rotaWe compared our method with three 2D-based methods  in our Bi-CLSTM-based method on the thrombus ROI segmentation results. We experimented with the three different lengths of image sequences ranging from 3, 5, to 7. The results show that our Bi-CLSTM-based method with the sequence of 5 images had the highest for the Dice, Jaccard, and FP metrics; the TO and FN metrics were high with the longest sequence of 7 images. We, however, note that there were no significant differences among all three sequence lengths. We chose 5 as the default sequence length due to its advantage in lower memory consumption when compared to the longest length of 7.In The results demonstrate the capability of our Bi-CLSTM-based method to precisely segment thrombus ROIs in post-operative CTA image volumes, where different artifacts and noises commonly appear. The comparative validation with the large-scale patient studies suggests the robustness and utility of our Bi-CLSTM-based method over the four existing counterparts ,19,20,21We attribute our superior segmentation performance to the novel use of Bi-CLSTM which was designed to take into account the volumetric coherence of image sequences. The ROI segmentation of an image could be refined with additional features learned from adjacent images. The value of the volumetric coherence was experimentally validated from the comparison results with the 2D-based segmentation methods ,19,21, wWe demonstrated the enhanced segmentation capability of our Bi-CLSTM-based method over the 3D-based counterpart  by showiWe valued the over-segmentation tendency of our Bi-CLSTM-based method, instead of the under-segmentation tendency from the existing methods ,19,20,21The current results may imply that our Bi-CLSTM-based segmentation method is limited in clinical generalization and robustness because we validated it only with a single medical institution. Medical institutions vary in, for example, imaging scanner manufacturers, image acquisition protocols, and clinical routines. These variations have a major impact on the quality of postoperative CTA image volumes and the segmentation performance for thrombus ROIs. As future work, we plan to achieve a certain level of clinical generalization and robustness by validating our method with multi-site medical institutions.Our results see  demonstrWe proposed a noise-robust Bi-CLSTM-based segmentation method for thrombus ROIs in postoperative CTA image volumes. Our Bi-CLSTM-based segmentation method approximates the thrombus ROIs of a target image and then refines the challenging perturbation parts, e.g., the boundaries, using the volumetric coherence of the adjacent images, thereby improving overall segmentation performance. The thorough validations with a large-scale clinical dataset of 60 patient studies suggest that our method is superior to the existing 2D-based and 3D-based counterparts ,19,20,21


Rechallenge with EGFR inhibitors represents a promising strategy for patients with RAS wild type (WT) colorectal cancer (CRC) but definitive selection criteria are lacking. Recently, the RAS WT status on circulating tumor DNA (ct-DNA) emerged as a potential watershed for this strategy. Our study explored the liquid biopsy-driven cetuximab rechallenge in a RAS and BRAF WT selected population.CRC patients with RAS and BRAF WT both on tumor tissue and on ct-DNA at baseline receiving rechallenge with cetuximab were eligible for our analysis. Ct-DNA was analyzed for RAS-BRAF mutations with pyro-sequencing and nucleotide sequencing assays. Real-time PCR and droplet digital PCR were performed to confirm the RAS-BRAF mutational status.p = 0.048); anti-EGFR free interval > 14 months and anti-EGFR free interval > 16 months were associated with improved mPFS  and with improved mOS . Previous lines >2 were correlated with improved mPFS  and with improved mOS . In a multiple logistic regression model, only the anti-EGFR free interval was confirmed to be a significant predictor for mOS and mPFS.A total of 26 patients were included in our analysis. In the global population, RR was 25.0%, median overall survival (mOS) was 5.0 months, and median progression-free survival (mPFS) was 3.5 months. Previous response to anti-EGFR was associated with improved mPFS (5.0 vs. 2.0 months, HR: 0.26, Liquid biopsy-driven cetuximab rechallenge was confirmed to be effective. The clinical outcome was consistent with available results from phase II studies. In addition to the molecular selection through the analysis of ct-DNA for RAS, the long anti-EGFR free interval is confirmed as a prospective selection criterion for this therapeutic option. The anti-epidermal growth factor receptor (EGFR) antibodies  have a key role in the treatment of metastatic colorectal cancer. The resistance to anti-EGFR antibodies represents a challenging issue in this context. In particular the genetic heterogeneity of colorectal cancer along with the dynamic nature of tumor biology are at the basis of secondary resistance to EGFR blockade . The mecThe rechallenge strategy with anti-EGFR might then represent a promising therapeutic weapon aiming to overcome secondary resistance to anti-EGFR in the light of the knowledge about the pulsatile behavior of RAS clones under EGFR blockade pressure. The basic idea supporting the concept of rechallenge with anti-EGFR is the possibility to successfully treat patients previously exposed and resistant to such drugs after an anti-EGFR interval in which tumor cells may have gone back to prevalent RAS wild-type (WT) status after developing a prevalent RAS mutant status due to anti-EGFR pressure.Santini et al.  firstly p = 0.03).Subsequently, Cremolini et al.  prospectSimilarly, Masuishi et al.  conducteResults from CHRONOS, a multicenter phase II trial of liquid biopsy-driven anti-EGFR rechallenge with panitumumab , have beHowever, selection criteria allowing clinicians to reliably select patients most likely to benefit from such a treatment approach are yet to be defined. This is of particular clinical relevance when we consider that metastatic colorectal cancer patients who are potential candidates for a rechallenge with anti-EGFR may also be eligible, at the same time, for other therapeutic options with higher level of evidence , 11. In Further studies are urgently needed to better understand the prognostic and predictive factors along with the better timeline for rechallenge strategy. The present study explored the role of liquid biopsy-driven rechallenge strategy with EGFR blockade in molecularly and clinically selected metastatic colorectal cancer patients.http://www.medcalc.org; 2014). For study purposes, the optimal anti-EGFR free interval threshold was defined according to receiver operating characteristic (ROC) curve analysis. The association between categorical variables was estimated by Fisher exact test for categorical binomial variables. Survival probability over time was estimated by the Kaplan–Meier method. Significant differences in the probability of survival between the strata were evaluated by log-rank test. Multiple logistic regression was used to assess the role of significant variables in the univariate analysis. The study was performed in accordance with the protocol, which was approved by the AOU Cagliari Ethical committee  along with all experimental procedures. Written informed consent was obtained for all patients enrolled into the analysis, and methods were carried out in accordance with the Declaration of Helsinki.We conducted a multicenter retrospective analysis in patients with molecularly selected colorectal cancer who underwent rechallenge with anti-EGFR antibodies. The aim of the study was to evaluate the correlation of several putative predictive/prognostic factors for rechallenge strategy with clinical outcome. Patients were included in our analysis if they met the following criteria: histologically confirmed RAS and BRAF wild-type colorectal cancer, first-line anti-EGFR-based therapy with documentation of progression to first-line therapy within 4 weeks after the last administration of anti-EGFR, rechallenge with anti-EGFR antibody , and measurable disease according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. To be eligible, patients must also have circulating tumor DNA (ct-DNA) RAS-BRAF WT profile at the rechallenge baseline. The ct-DNA was analyzed at the rechallenge baseline for RAS-BRAF mutations using pyro-sequencing (PyroMark Q24 MDx Workstation) and nucleotide sequencing  assays. Real-time PCR (Idylla) and droplet digital PCR (QX200 System) were performed to confirm the RAS-BRAF mutation status. The limit of detection for RAS BRAF mutations was 5%. Tumor response was evaluated by clinicians’ assessment according to the Response Evaluation Criteria in Solid Tumors (RECIST v1.1). Several clinical variables including previous response to anti-EGFR containing therapy, anti-EGFR free interval, and previous lines for metastatic disease > 2 were evaluated in relation to outcome in terms of RR, mPFS, and mOS. Statistical analysis was performed with the MedCalc Statistical Software version 14.10.2 ; long anti-EGFR free interval > 14 months and long anti-EGFR free interval > 16 months were correlated with improved mPFS  and with mOS . Previous lines >2 for metastatic disease were correlated with improved mPFS  and mOS . The optimal anti-EGFR free interval threshold identified for predicting prognosis was 14 months . Likewise, the anti-EGFR free interval >16 months maintained the independent role for PFS (p = 0.020) among the significant variables in the univariate analysis . None of them were statistically correlated to RR at multivariate analysis.In a multiple logistic regression model, the anti-EGFR free interval >16 months as well as the anti-EGFR free interval >14, among the variables , maintained their independent role for OS  colorectal cancer patients. Notably, the response rate and the mPFS reported in the heavily pretreated population  Table 1 Previous response to anti-EGFR, long anti-EGFR free interval, and previous lines >2 were correlated to better clinical outcome in our clinical series. In particular, previous responders with longer anti-EGFR free interval  achieved the best outcome from rechallenge with anti-EGFR. PFS and OS were significantly longer in patients with long anti-EGFR free interval, consistent with results from phase II trials of clinical-driven rechallenge with anti-EGFR , 8. A nuAll this, in the light of the exponential decay kinetics of RAS mutant alleles after EGFR blockade suspension , suggestNevertheless, the results of the CHRONOS study  are partOur study has some limitations; in particular, the small sample size along with the retrospective nature of the study deeply influenced the interpretations of our findings. This analysis, therefore, must be considered exploratory and caution is needed in data interpretation.Taking into account the current data regarding secondary resistance to EGFR blockade and the biological rationale for rechallenge strategy, the results of the present study are impressive and promising for future development. This especially applies in the form in which the results underlying the hypothesis of delaying the introduction of rechallenge with anti-EGFR would maintain efficacy in later lines. The liquid biopsy-driven anti-EGFR rechallenge is confirmed to be viable in clinical practice, and it should be considered the main tool for selected patients. Previous response to anti-EGFR and EGFR free interval might just represent a surrogate factor substituting in fact for acquired gene alteration status. Nevertheless, given the difficulties in applying the molecular methods from interventional liquid biopsy-driven anti-EGFR rechallenge studies, these clinical predictive factors maintain their value and a combination of the two approaches is also possible in the interest of greater effectiveness.The up-to-date results from prospective liquid biopsy-driven rechallenge strategy trials , 14 are The raw data supporting the conclusions of this article will be made available by the authors upon request.The studies involving human participants were reviewed and approved by AOU Cagliari Ethical committee . Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.Author GiuP reports advisory role for BMS, MSD, Roche, Pierre-Fabre, Novartis, and Incyte.The remaining 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Is psychotropic medication use associated with differences in the risk of COVID-19 infection among adults with serious mental illness?In this cohort study of 1958 inpatients with serious mental illness in a statewide psychiatric hospital system, the use of second-generation antipsychotic medications was associated with a decreased risk of COVID-19 infection; the largest association was observed with the use of paliperidone. Valproic acid use was associated with an increased risk of infection.These results suggest that individual psychotropic medications are associated with differential risks of COVID-19 infection among patients with serious mental illness. Individuals with serious mental illness are at increased risk of severe COVID-19 infection. Several psychotropic medications have been identified as potential therapeutic agents to prevent or treat COVID-19 but have not been systematically examined in this population.To evaluate the associations between the use of psychotropic medications and the risk of COVID-19 infection among adults with serious mental illness receiving long-term inpatient psychiatric treatment.This retrospective cohort study assessed adults with serious mental illness hospitalized in a statewide psychiatric hospital system in New York between March 8 and July 1, 2020. The final date of follow-up was December 1, 2020. The study included 1958 consecutive adult inpatients with serious mental illness (affective or nonaffective psychoses) who received testing for SARS-CoV-2 by reverse transcriptase–polymerase chain reaction or antinucleocapsid antibodies and were continuously hospitalized from March 8 until medical discharge or July 1, 2020.Psychotropic medications prescribed prior to COVID-19 testing.COVID-19 infection was the primary outcome, defined by a positive SARS-CoV-2 reverse transcriptase–polymerase chain reaction or antibody test result. The secondary outcome was COVID-19–related death among patients with laboratory-confirmed infection.Of the 2087 adult inpatients with serious mental illness continuously hospitalized during the study period, 1958 (93.8%) underwent testing and were included in the study; 1442 (73.6%) were men, and the mean (SD) age was 51.4 (14.3) years. A total of 969 patients (49.5%) had laboratory-confirmed COVID-19 infection that occurred while they were hospitalized; of those, 38 (3.9%) died. The use of second-generation antipsychotic medications, as a class, was associated with decreased odds of infection , whereas the use of mood stabilizers was associated with increased odds of infection . In a multivariable model of individual medications, the use of paliperidone was associated with decreased odds of infection , and the use of valproic acid was associated with increased odds of infection . Clozapine use was associated with reduced odds of mortality in unadjusted analyses .In this cohort study of adults hospitalized with serious mental illness, the use of second-generation antipsychotic medications was associated with decreased risk of COVID-19 infection, whereas the use of valproic acid was associated with increased risk. Further research is needed to assess the mechanisms that underlie these findings. This cohort study evaluates the associations between the use of psychotropic medications and the risk of COVID-19 infection among adults with serious mental illness receiving long-term inpatient psychiatric treatment. Patients with psychiatric disorders are more likely to have medical comorbidities associated with worse outcomes and have a higher mortality rate from COVID-19 independent of these medical risk factors.9 suggesting that antipsychotic treatment is unlikely to account for increased mortality risk. However, individual medications within and across pharmacologic classes may differ in their associations with infection and adverse outcomes. Several psychotropic medications—including some first-generation antipsychotics  and antidepressants  were identified as potential therapeutic agents based on in vitro evidence of anti–SARS-CoV-2 activity. Clinical evidence to support these in vitro findings has been mixed. Although several studies have established an association between the use of antidepressants15 and a less severe course of infection in patients with COVID-19, small observational studies have found no association between the use of haloperidol16 or chlorpromazine17 and the severity of COVID-19 infection. These medications and several other psychotropic drugs, including second-generation antipsychotics, may affect the host response to COVID-19 by modifying the balance between proinflammatory and anti-inflammatory cytokines and through other mechanisms.19 Several of these medications are common treatments for adults with serious mental illness, but their associations with the risk of COVID-19 infection have not been systematically examined in this population, to our knowledge.Identifying factors associated with the risk of infection among inpatients with serious mental illness is of critical importance given their susceptibility to severe infection. A study of adults with serious mental illness infected with COVID-19 found equivalent mortality rates among those taking antipsychotic medications compared with those not taking antipsychotic medications,The primary aim of this study was to assess the risk of COVID-19 infection associated with psychopharmacologic treatments among adult long-term inpatients with serious mental illness in a statewide psychiatric hospital system in New York. We also aimed to assess the risk of COVID-19 mortality among patients with laboratory-confirmed infection.STROBE) reporting guideline for cohort studies.In this retrospective cohort study, we used data from the electronic health record (EHR) and a centralized COVID-19 registry used by all psychiatric hospitals operated by the New York State (NYS) Office of Mental Health (OMH). The study protocol was reviewed by the institutional review board of the Nathan S. Kline Institute for Psychiatric Research and deemed exempt from the requirement of informed consent based on use of deidentified data and designation of non–human participants research. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology  testing of suspected cases began March 1, 2020, using multiple commercial laboratories; as of March 24, 2020, RT-PCR testing was performed by a single laboratory (Bioreference Laboratories Inc). The NYS-OMH made universal testing available to all hospitalized patients beginning May 7, 2020; patients were offered screening by RT-PCR and antibody testing using the ARCHITECT SARS CoV-2 antinucleocapsid protein immunoglobulin G (IgG) assay (Abbott). This assay has a reported sensitivity of 100% and specificity of 99.6% for detecting SARS-CoV-2 within several days after infection,Included in this study were adult inpatients (≥18 years of age) with serious mental illness  who received testing for SARS-CoV-2 by RT-PCR or by serum IgG assay and were continuously hospitalized from March 8, 2020, until medical discharge for COVID-19 or July 1, 2020. Patients who were admitted within 30 days prior to testing were excluded because we assessed medication exposure during the 30 days prior to testing, and information about medications could not be confirmed for patients admitted during this assessment window  prescribed prior to their index test date, (2) used for at least 7 days, and (3) received via scheduled administration (as-needed medications were excluded). Medications were grouped by pharmacologic class: first-generation antipsychotic, second-generation antipsychotic, mood stabilizer, benzodiazepine, or antidepressant (eTable 1 in the The medication exposure period was defined based on the date of SARS-CoV-2 test and the type of test (RT-PCR or antibody). The date of the first positive test result was used as the index date for patients with any positive test result. A positive RT-PCR test result was considered a proxy of active infection, and the exposure period was limited to 30 days. Patients who were transferred to a medical facility for COVID-19 treatment prior to receiving confirmatory testing were also considered to have active infection, and the exposure period included 30 days prior to transfer. For patients without a positive RT-PCR test result or medical transfer for COVID-19 treatment, the date of the SARS-CoV-2 IgG test was used as the index date. Because this was considered a measure of prior infection, the exposure period included the full study period prior to testing .International Statistical Classification of Diseases and Related Health Problems, Tenth Revision medical billing category codes  or unknown. Missing data for race and ethnicity and smoking status were assigned to unknown categories. Medical comorbid conditions included chronic respiratory disease, diabetes, heart disease, and hypertension, using We first used unadjusted binary logistic regression to estimate associations between baseline characteristics and psychopharmacologic classes, with COVID-19 infection as the primary outcome  and COVIP < .10 significance level.22 Binary logistic regression was used to estimate the association between use of each medication and infection using odds ratios (ORs) and 95% CIs. Three types of ORs were estimated: (1) unadjusted, (2) adjusted for age and sex, and (3) fully adjusted for age and sex in addition to covariates that were significantly associated with infection in binary logistic regression (P < .05). Because patients could have been exposed to multiple medications within and across drug classes, medications were retained in each step to empirically adjust for polypharmacy. A Bonferroni-type correction was used to adjust for multiple comparisons within medication classes.Statistical tests were 2-sided. Multivariable statistical modeling retained individual medications with at least 5% exposure in the study population from psychopharmacologic classes identified as being associated with infection at the P < .10) and medications significantly associated with infection (P < .05) after adjusting for age and sex. All analyses were conducted using SAS, version 9.4 (SAS Institute Inc).A similar approach was used to assess the association between medication exposure and mortality among patients with a positive SARS-CoV-2 test result. Medications retained for statistical modeling included those from drug classes associated with mortality  were excluded owing to lack of testing information, and 4 were excluded owing to admission within 30 days prior to the test result . No difference in the rate of infection was observed in association with benzodiazepine or antidepressant exposure.Patients prescribed second-generation antipsychotics were less likely to test positive for COVID-19 infection than those not prescribed second-generation antipsychotics , whereas patients prescribed mood stabilizers were more likely to test positive than patients who were not exposed to medications in this class  . First-gEach of the second-generation antipsychotics , mood stabilizers , and first-generation antipsychotics  with at least 5% exposure in the study population were retained for regression modeling of infection . After aP = .10). Chlorpromazine, clozapine, paliperidone, risperidone, olanzapine, and valproic acid were retained based on their significant association with infection after adjusting for age and sex. Clozapine was the only medication associated with decreased odds of mortality in the unadjusted model ; this association did not remain statistically significant after adjusting for age and sex  or after additional adjustment for medical risk factors   . There w23 This finding is consistent with prior studies that have found increased rates of infection in congregate settings25 and increased mortality after infection among patients with serious mental illness.7 The potential harms and benefits associated with several psychotropic medications have been explored in preclinical and clinical studies since the start of the COVID-19 pandemic,19 but, to our knowledge, this is the largest study to systematically assess associations between the use of individual medications and the risk of COVID-19 infection among inpatients with serious mental illness.We investigated COVID-19 infection and mortality among long-term inpatients with serious mental illness in a NYS-operated psychiatric hospital system during the first wave of the pandemic, with a focus on the association between exposures to psychotropic medications and adverse outcomes. Nearly half of the patients in this cohort had laboratory-confirmed COVID-19 infection, and infection-related fatality was more than 4 times higher than estimates from the general population in New York during the same time period.10 However, there are other drug-protein interactions that may interfere with the viral life cycle. SARS-CoV-2 3c-like protease and viral RNA-dependent polymerase are critical enzymes for viral protein processing and genome replication, respectively. An in silico screening analysis identified paliperidone as one of the leading drugs to bind these enzymes with high affinity.26The use of second-generation antipsychotics, as a class, was associated with decreased infection. The largest effect size was observed in association with paliperidone. The use of second-generation antipsychotics was also found to have a protective association with mortality, although the association was not statistically significant, likely owing to insufficient sample size. These findings are contrary to what would be expected based on in vitro evidence showing that first-generation antipsychotics , but not second-generation antipsychotics, interact with sigma-1-receptors to reduce SARS-CoV-2 replication.27 clozapine use was not associated with an increased risk of COVID-19 infection or death in the present study. In fact, unadjusted estimates suggested a significant protective association. This finding stands in contrast to prior EHR studies that found an increased risk of COVID-19 infection associated with clozapine treatment29 but is consistent with a prior study limited to inpatients that found a lower risk of infection and a lower risk of symptomatic disease in association with clozapine use.30 One potential explanation for this discrepancy is surveillance bias because patients receiving clozapine in the general community may be more likely than other outpatients to undergo testing for COVID-19. Because 94% of patients in this cohort underwent testing, surveillance bias was less likely. Furthermore, this is the largest study to date of COVID-19–infected patients prescribed clozapine. Clozapine is unique among antipsychotics in its ability to enhance the T helper cell type 1 response that supports antiviral immune response and is blunted in schizophrenia.31 Further research is needed to determine whether clozapine may protect against severe COVID-19 infection.Although there have been concerns about clozapine use during the pandemic as a risk factor for pneumonia and potential toxic effects during acute infection,32 we found a significant increase in infection and a similar  increase in mortality. Valproic acid downregulates angiotensin-converting enzyme 2 in endothelial cells,33 and several studies have shown that the downregulation of membrane-bound angiotensin-converting enzyme 2 may impair immune function and contribute to poor outcomes in the setting of COVID-19 infection.34 Additional factors may confound the association between valproic acid use and COVID-19 risk. For example, patients are typically prescribed mood stabilizers, such as valproic acid, to manage mood lability and other manic symptoms; these symptoms may negatively affect adherence to infection prevention measures, such as mask wearing and social distancing.To our knowledge, this is the first study to assess the association between valproic acid use and COVID-19 risk. Although it has been hypothesized that the use of valproic acid could reduce the risk of COVID-19 infection or reduce viral load by its association with angiotensin-converting enzyme 2 and transmembrane serine protease 2,35 There were no COVID-19–related deaths among patients prescribed escitalopram (n = 88), venlafaxine (n = 53), bupropion (n = 43), or fluvoxamine (n = 25).Risk of mortality was lower among patients taking antidepressants. Although the association was not statistically significant in this cohort, this finding is consistent with larger studies that found reduced risk of adverse outcomes associated with antidepressant use.This study has several strengths. Restricting the sample to inpatients with chronic psychotic disorders minimized differences in risk associated with community exposures, underlying psychiatric diagnosis, medication adherence, and access to care, thus facilitating assessment of the association between psychotropic medications and outcomes. Implementation of universal screening and use of antibody testing to detect prior infection helped identify patients who may not have received confirmatory testing by RT-PCR when infected owing to lack of test availability or other factors.36 had not yet been implemented. The availability of tests and criteria for testing evolved during the study period and varied across facilities. Duration of medication use, medication dose, and polypharmacy may have been associated with differences in outcomes and were not explored in this analysis. In addition, medication exposure after infection could not be evaluated for patients who were transferred to outside facilities for medical treatment. Medication adherence, although likely high, was not directly assessed. Additionally, the study population represents a subset of adults with serious mental illness: those with persistent and severe illness, chronically exposed to psychotropic medications, who reside in long-term state psychiatric hospitals. The association between the use of psychotropic medications and the risk of COVID-19 infection may differ between patients with less severe psychiatric disease and those without prior medication exposure.This study also has several limitations. During the study period, the pandemic was at its first peak in New York. Interventions, including regular testing of staff and a statewide vaccination campaign, which successfully limited the spread of COVID-19 during the second surge of the virus,Exposures to several psychotropic medications were associated with risk of COVID-19 infection among inpatients with serious mental illness; decreased risk was observed with the use of second-generation antipsychotics, with paliperidone use associated with the largest effect size. Valproic acid use was associated with an increased risk of infection. Further research is needed to replicate our findings, evaluate their generalizability, and explore underlying mechanisms.


The results of 17 assays are summarized. This harmonization effort allowed to ensure that all Belgian laboratories would report positive PCR results in the same semi-quantitative manner to clinicians and to the national database which feeds contact tracing interventions.From early 2020, a high demand for SARS-CoV-2 tests was driven by several testing indications, including asymptomatic cases, resulting in the massive roll-out of PCR assays to combat the pandemic. Considering the dynamic of viral shedding during the course of infection, the demand to report cycle threshold (Ct) values rapidly emerged. As Ct values can be affected by a number of factors, we considered that harmonization of semi-quantitative PCR results across laboratories would avoid potential divergent interpretations, particularly in the absence of clinical or serological information. A proposal to harmonize reporting of test results was drafted by the National Reference Centre (NRC) UZ/KU Leuven, distinguishing four categories of positivity based on RNA copies/mL. Pre-quantified control material was shipped to 124 laboratories with instructions to setup a standard curve to define thresholds per assay. For each assay, the mean Ct value and corresponding standard deviation was calculated per target gene, for the three concentrations (10 The rapid scale-up of testing capacity in combination with a testing strategy linked to contact tracing, including intense screening of asymptomatic persons, resulted in the massive roll-out of polymerase chain reaction (PCR) assays to combat the pandemic of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). Most PCR assays report in cycle threshold (Ct) values that can highly vary across different methods and laboratories, complemented with supply chain issues that forced many laboratories to adopt multiple SARS-CoV-2 PCR methods , causing5 copies/mL [4 copies/mL [6 copies/mL [5 RNA copies/mL [3 RNA copies/mL. An arbitrary threshold of >107 RNA copies/mL was set to define a person as contagious with high certainty, since an overall peak viral load of 108 RNA copies/mL was reported to be reached prior to or within the first days of symptoms onset [In November 2020, a proposal to move towards a semi-quantitative way of reporting RT-PCR test results was drafted by the National Reference Centre (NRC) for Respiratory Pathogens at UZ/KU Leuven. Since no consensus on thresholds with respect to viral load and its link to contagiousness was available in literature at that time, data on which to formulate the proposal were mainly derived from in vitro studies that were characterized by an important heterogeneity of methods, study population and potential impact of pre-analytical factors on the quality of viral cell culture experiments using clinical material. The majority of these published studies report thresholds based on Ct values instead of actual viral loads in copies/mL, complicating the evaluation of cut-off values for cell culture experiments. A range of Ct values from 24 up to 35 was reported as threshold value for a so-called negative cell culture, implicating the absence of a cytopathogenic effect (CPE) when the viral material was cultivated, mainly including studies making use of clinical specimens ,12,13,14opies/mL ,17,18,19opies/mL . Among topies/mL . Based oms onset .3 RNA copies/mL for the nucleocapsid gene while not detected for the other two target genes, however this category is not further discussed in this manuscript. All four categories (very strongly positive to weakly positive) gave rise to contact tracing and quarantine measures, in cases where it was informed by additional clinical information. In order for an infection to be considered as old or cleared, the criteria as proposed by the RAG in June 2020 [After thorough discussion within the Molecular Microbiology expert group of the department Quality of Laboratories of the Belgian national public health institute Sciensano, and the risk assessment group (RAG) coordinated by Sciensano, advice to report in a semi-quantitative manner was formulated , dividinune 2020  still neune 2020 .To be able to report in a semi-quantitative manner, pre-quantified SARS-CoV-2 control material was prepared at the NRC for Respiratory Pathogens at UZ/KU Leuven. The control material, accompanied by a letter providing guidelines to set up a standard curve, was shipped to all laboratories in Belgium that were declared to perform SARS-CoV-2 PCR assays in a routine diagnostic setting. By doing so, the laboratories were able to define thresholds for the PCR assay(s) used in their respective laboratory, to allow the reporting of SARS-CoV-2 positive test results in the four described categories without the use of a quantitative PCR assay or the need to report Ct values. Through the detailed setup of a standard curve, the correlation between RNA copies/mL present in the control material to the Ct values or equivalent metric used for their respective assay, would be defined and could later be used to report in a semi-quantitative manner to clinicians as well as to the healthdata.be platform of Sciensano, improving infection control measures .−1 to 10−7) were prepared in triplicate to quantify the viral concentration by the use of the SARS-CoV-2 PCR assay on Alinity m (Abbott), used in routine diagnostics and set up as a quantitative assay at the NRC (for further details see next paragraph). Dilution series were prepared in Sigma MMTM medium  starting from 200 µL of the prepared stock. Each dilution of a total volume of 2 mL was distributed to three vials to allow triplicate testing. Whole-genome sequencing (WGS) according to a previous published protocol [The NRC for Respiratory Pathogens at UZ/KU Leuven cultured a SARS-CoV-2 PCR-positive clinical sample dated 27 January 2021, on Vero E6 cells ,29, follprotocol ,31 was pQuantitative determination of the viral load of the stock material was possible due to the earlier setup of a standard curve for the Alinity m SARS-CoV-2 assay, using the SARS-CoV-2 molecular control SCV2MQC01-B , a commercially available control with known genome equivalents. In April 2020, this Qnostics control was used at first to set up a standard curve for an early Belgian SARS-CoV-2 isolate, quantifying at that time the viral load of the cell culture using a lab-developed test (LDT) on QuantStudio Dx . The Apr8 to 10 copies/mL, complemented with a negative control, were proposed to evaluate the possibility to report test results in a semi-quantitative way. An important condition that needs to be met when reporting in this way is the possibility to be able to thoroughly evaluate the test result of the internal control (IC) to account for underestimation of the viral load category. Each dilution was recommended to be tested in triplicate to allow calculating the average of these measurements for each gene that is targeted in the respective PCR assay. The advice was given to implement the average thresholds for the gene that was found to be associated with the highest sensitivity complemented with the knowledge on circulating variants at the time.The pre-quantified SARS-CoV-2 material was shipped on dry ice to 124 recognized COVID-19 testing laboratories across Belgium ,33, usinThe laboratories that participated in this harmonization effort were asked to report results of the standard curve(s) they set up, to the NRC, in order to process the data and to prepare a summary comparing the different PCR assays in use in Belgium. Despite this summary, it was advised to implement the cut-off values as determined by the standard curve setup in the respective laboratory, following procedures as validated in the routine diagnostic flow, rather than implementing standard thresholds resulting from data of multiple laboratories. Solely, the PCR assay that was used to set up the standard curve was considered when processing the results, not the sample preparation specifics, extraction method nor the specific equipment used to perform the PCR.2 coefficient of ≥0.95 were considered. Finally, during data analysis, the results of a laboratory could still be excluded when inclusion of the respective results caused an increase in the standard deviation (SD) of 1 or more, as such a standard curve was considered to be an outlier compared to the results shared by other participating laboratories.Since a large number of laboratories participated to this harmonization effort, criteria were defined that needed to be fulfilled in order for the respective results of the standard curve to be included in the downstream data analysis. At first, the standard curve of a particular PCR assay needed to be set up by a minimum of two laboratories to make a comparison possible. This automatically entailed that LDT assays were not considered. Secondly, data were only used when each dilution series was measured in duplicate or triplicate. As a third criterion, only results derived from a standard curve with a R7, 105 and 103 RNA copies/mL) that determine the classification of the four categories. In case the SD exceeded 2, the summary of the respective RT-PCR assay was not included in the further data analysis since the dispersion of the results that different laboratories submitted, was simply too high to rely on the respective results. SD values exceeding 1 were included, however noted to carefully interpret the average values since results across the laboratories appeared to be scattered.For each individual PCR assay, the mean Ct value or equivalent metric, with the corresponding SD, was calculated per target gene of the respective assay, based on the results derived from the standard curve set up by each individual laboratory. The mean Ct value was calculated for the three concentrations  on Alinity m (Abbott) were used to calculate the viral load of the SARS-CoV-2 control material, as detailed in the 9 copies/mL or in fact 1.10 × 109 genome equivalents) was obtained for the stock called ‘SARS-CoV-2 strain BetaCoV/Belgium/GHB-0127/2021’. WGS classified the strain to the type 20A according to Nextclade [Triplicate testing of dilution series , TaqPath COVID-19 CE-IVD RT-PCR Kit , ARIES® SARS-CoV-2 Assay   and GeneFinderTM COVID-19 Plus RealAmp kit  , Xpertatories) . The 17 n = 127), envelope , RNA-dependent RNA polymerase , spike , open-reading frame 1ab , ORF8 (n = 4) and non-structural protein 2 .Since more than one SARS-CoV-2 gene is targeted for most PCR assays, results were analyzed for all different genes targeted in each respective PCR assay. Based on sensitivity, accuracy and reproducibility complemented with knowledge at the time of the analysis on circulating SARS-CoV-2 variants and their potential impact on the performance of RT-PCR assays, advice was provided on which gene to use in the context of semi-quantitative reporting. When considering only the assays that were used by more than one Belgian laboratory, but weighting the number of laboratories that have implemented each particular SARS-CoV-2 assay, the following order of SARS-CoV-2 genes that are most often targeted by PCR assays used in routine diagnostics in Belgium was obtained: nucleocapsid  and the laboratories that only measured the dilution series once, the range of R7, 105 and 103 RNA copies/mL) that determine the classification of the four categories. When more than one SARS-CoV-2 gene was targeted, a gene for semi-quantitative reporting was proposed as discussed earlier.The mean Ct value or equivalent metric, with the corresponding SD, was calculated per target gene for these 17 PCR assays, based on the results provided by the participating laboratories . The meaInitially all categories were interpreted as SARS-CoV-2 positive test results, followed by contact tracing and giving rise to equal quarantine and isolation measures, unless information was available to consider an infection as old or cleared . More reThe rapid scale-up of testing capacity, combined with a testing strategy linked to contact tracing, including asymptomatic screenings, resulted in the massive role of PCR assays to combat the SARS-CoV-2 pandemic. As Ct values can highly vary across methods and even between laboratories using the same PCR assay, a semi-quantitative approach based on RNA copies/mL was chosen to provide more details to clinicians with respect to the stage of infection, although additional clinical and/or serological evidence remains key for interpretation. Out of 124 laboratories performing daily routine diagnostic tests for SARS-CoV-2, 91 participated to this study, enabling a considerable impact of the study’s outcome on the national testing practice. Considering the number of PCR assays that the 91 participating laboratories conduct on a weekly basis, it was estimated that over 85% of the testing activity of Belgium is covered. Thanks to the harmonization effort initiated by the NRC UZ/KU Leuven distinguishing four categories of SARS-CoV-2 positivity (very strongly positive to weakly positive), many Belgian laboratories currently report in a similar semi-quantitative manner to clinicians and to healthdata.be, improving infection control measures. Providing more detailed information on the stage of infection by using the proposed semi-quantitative categories, moving beyond reporting of a purely qualitative test result, allows clinicians and the national contact tracing services to rapidly identify recent infections and subsequentially to embank superspreading transmission events. Moreover, it facilitates the follow-up of long-term SARS-CoV-2 positive patients and the potential impact of such repeated positive PCR results on the clinical management of these individuals as well as its impact on the overall hospital hygiene management. Nevertheless, as previously noted, a weakly positive test result certainly can also reflect the early stage of a new SARS-CoV-2 infection, next to an old, cleared infection, warranting the need to carefully interpret such PCR test results.7 RNA copies/mL a wide variety of Ct values was observed, ranging from 6.2 to 20.3, while for the categories determined by a viral load of respectively 105 RNA copies/mL and 103 RNA copies/mL, the following ranges were reported: Ct 12.7 to 27.4, and Ct 19.1 to 34.4. The lowest Ct values were observed for the PCR assay Abbott RealTime SARS-CoV-2, substantially lower compared to the Ct values obtained for the other 16 PCR assays included in the analysis. In at least two other studies, lower Ct values have also been reported for this assay [3 RNA copies/mL), a higher variability was observed and is to be expected compared to samples with a higher viral load (105 and 107 RNA copies/mL), increasing the uncertainty range for the categories of moderate positive and weakly positive compared to the categories very strongly and strongly positive, possibly impacting the interpretation of these test results. Of note, when interpreting the results of this study, correct interpretation is key since, in this study, no absolute reference material was used to calibrate the SARS-CoV-2 control material. The standard Qnostics, expressed in genome equivalents instead of RNA copies/mL, was used since, at the time of initial calibration of the standard curve performed at the NRC UZ/KU Leuven, no absolute reference material was available. While it is more correct to express the viral concentration of the control material in genome equivalents, this term is not widely used nor easily interpretable. Currently, a WHO International Standard (NIBSC code: 20/146) is available [Despite the attempt to harmonize the reporting of SARS-CoV-2 PCR positive test results at a national level, we need to recognize the remaining limitations when reporting laboratory test results in the form of SARS-CoV-2 positivity categories as measured in the large majority of participating laboratories based on Ct values . To summis assay ,40 relatvailable , charactThe sampling method, including the execution, type of sampling material, sampling site and virus transport medium, have all been reported to have an important impact on the outcome of PCR test results ,43. In tWhen weighting the number of laboratories that have implemented each particular SARS-CoV-2 assay, the nucleocapsid was identified to be the gene most often targeted by PCR assays used in routine diagnostics in Belgium. While the detection of viral RNA by PCR assays is the gold-standard method for COVID-19 diagnosis, dropouts or shifts in Ct values of the genes targeted in the PCR assay can serve as indicators for the presence of particular variants of concern (VOC). Evaluating mismatches between the set of primers and probes used during RT-PCR and the SARS-CoV-2 genomes of a variety of VOCs, is important to assess the impact of these VOCs on the performance of routine diagnostic tests . VariousOverall, the large variability in Ct values reported between PCR assays and even between laboratories using the same SARS-CoV-2 assay advocates to not simply report pure Ct values. By implementing semi-quantitative categories of SARS-CoV-2 positivity, a compromise was initiated between the request of clinicians to detail SARS-CoV-2 test results to a maximum and a correct laboratory estimate of viral load. Especially laboratories that have implemented several PCR assays in parallel, due to the shortage of reagents and the need to upscale their testing capacity, should be cautious when directly reporting Ct values to clinicians. We strongly encourage these laboratories to invest into harmonization of PCR test results through the use of standards to control the process or through the implementation of the proposed SARS-CoV-2 positivity categories to balance the estimation of viral load based on Ct values. Although through this harmonization effort a summary of mean Ct values and associated SD has become available for the 17 most used PCR assays in Belgium, it is of particular importance that laboratories should never implement these thresholds as such but should only rely on standard curve data obtained in their own laboratory setting, following the standard operating procedures in place. Furthermore, standard curves have been initiated for the period February to March 2021, using pre-quantified control material dating from January 2021, after which additional PCR assays have been implemented in some laboratories and followed by the emergence of numerous SARS-CoV-2 VOCs and variants of interest, of which some have been reported to impact PCR performance ,48,49,50The indirect link between the SARS-CoV-2 PCR positive test result and the infectivity needs to be interpreted with caution. A positive PCR test does not necessarily entail the presence of viable virus , as someDue to the massive roll-out of PCR assays, including in the context of intense screening of asymptomatic persons, the demand to report Ct values rapidly emerged. Since Ct values can be affected by a number of factors and can highly vary between assays and laboratories, it was decided to move towards a semi-quantitative approach to provide more details to clinicians with respect to the stage of infection, and indirectly the potential link with infectivity. Thanks to this harmonization effort, many Belgian laboratories currently report SARS-CoV-2 positive PCR results in the same semi-quantitative manner, distinguishing four categories of positivity based on RNA copies/mL, to clinicians and to healthdata.be, improving infection control measures.


ObjectiveTo assess the efficacy of oncologic healthcare during the COVID-19 pandemic on patients with head and neck squamous cell carcinoma (SCC) in a tertiary university hospital in Germany.MethodsThis retrospective, cross-sectional, observational study included 94 patients with newly diagnosed head and neck squamous cell carcinoma during a two-year period. Patients were assigned to two date-dependent groups; referrals before (group A) and during (group B) the COVID-19 pandemic. Time intervals from the symptom(s) onset to diagnosis, diagnosis to treatment, and treatment initiation to completion were recorded. Furthermore, TNM stages and the application of reconstructive surgery with free tissue transfer were determined. Patients’ outcomes and characteristics were compared between the two groups. Finally, a comprehensive literature review was carried out to identify similar epidemiological studies.ResultsThe symptom-to-diagnosis interval was longer during the COVID-19 pandemic . The intervals from diagnosis to treatment and treatment initiation to end of treatment were approximately the same in both groups . The T-and N-stages were not higher during the pandemic . Patients who presented with distant metastatic disease and those who underwent reconstructive surgery with free tissue transfer were observed more frequently in the lockdown phase .ConclusionThis study suggests the consistent diagnostic and therapeutical performance of the tertiary oncologic healthcare in Berlin, Germany, despite the challenges that patient care units faced during the COVID-19 pandemic. In March 2020, the World Health Organization (WHO) declared the global spread of the severe acute respiratory syndrome Coronavirus type 2 (SARS-CoV-2) as a pandemic . Since tThe primary aim of this study is to evaluate the management efficacy of HNC patients during the Coronavirus disease 2019 (COVID-19) pandemic and address two main questions: did we notice any delays in referrals, diagnoses, and treatment times? Did patients present with higher tumor stages or more advanced locoregional/distal metastases?The secondary aim is to provide the results from current epidemiological trials related to the same subjects in order to get insights into how patients with HNC were being cared for during the COVID-19 era.Study design and ethicsThis trial was conducted as a mono-centric, retrospective, cross-sectional observational study in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines Figure  that havPatients’ selection and stratificationThe study’s cohort was selected from 239 consecutive patients who presented with head and neck malignancies in the Otorhinolaryngology-Head and Neck Surgery Department of Charité University Hospital, Berlin, from March 22, 2019 to March 22, 2021. This research period was divided into two equal subperiods of one year each and comprised of patients who were diagnosed before (group A) and after (group B) the federal state’s imposition of lockdown on March 22, 2021 . In particular, demographic characteristics , site of cancer , symptom-diagnosis interval (SDI - time between the first symptom related to cancer manifestation and diagnosis through biopsy), diagnosis-treatment interval , treatment-end-of-treatment interval , clinical/pathological TNM-stage  and free tissue transfer reconstruction  were registered.A special focus was laid on recording meticulously the time and type of each diagnostic/therapeutical step. The therapeutical concepts consisted of surgical resection only, the surgical procedure followed by adjuvant radio(chemo)therapy [R(C)T] as well as primary R(C)T. Drop-outs after diagnostics or after receiving part of the therapeutical procedure were also recorded.Statistical analysesThe statistical analyses were performed at the Institute of Biometry and Clinical Epidemiology of the Charité-Universitätsmedizin Berlin (iBikE) using IBM SPSS Statistics V.24 . The Shapiro-Wilk test was used to analyze the distribution of all variables, while Pearson's chi-square test was used for comparisons of categorical and the Mann-Whitney U tests of numerical data. The global level of significance was set at α=0.05.Comprehensive literature reviewA comprehensive literature review was conducted according to PRISMA guidelines. The aim was to identify epidemiological observational studies that quantified treatment delays and upstage head and neck cancer during the COVID-19 pandemic. The PubMed/MEDLINE and Cochrane Library databases were used. The search terms "COVID-19," "coronavirus," "pandemic," "head and neck cancer," "oncologic patients," "treatment delay," "tumor size," and "upstage" were identified in 31 articles published from January 2020 to January 2022. The search strategy extended to the references of the identified articles, and 12 were chosen for full-text review. Seven articles reported quantitative measurements and, thus, met the inclusion criteria.Figure Table Table General aspectsOverall, patients’ accessibility to health services, along with their health-seeking behaviour, has been compromised massively during the COVID-19 pandemic for several reasons .The worldwide exponential surge of SARS-CoV-2 infections within a few months at the beginning of 2020 overwhelmed the majority of health systems and altered the clinical routine. Medical and nursing personnel shifted to strengthen the first line of care against the novel virus. Elective surgical procedures were postponed and surgical activity was limited to acute or subacute indications to release anesthesiology capacities for critical care units . FurtherThe surgical and interventional workforce adhered to guidelines conscientiously  to reducGovernments across the world imposed radical restrictions on social life and mobility freedom, leading to a high burden, anxiety, and uncertainty for patients, who adopted a more self-effacing approach to their health problems; many postponed their regular checkups, neglected follow-ups, and preferred self-treatment strategies for potentially serious symptoms in the face of Covid-19 contamination risk . InadequOncologic patients have been the focus of attention since the beginning of the pandemic due to the crucial time frame of their therapeutical management . SeveralCurrent epidemiological dataThe impact of the COVID-19 pandemic at the community level was also perceptible. According to a report from the UK, cancer referral cases by general practitioners dropped dramatically . In our We performed a proportionally similar number of surgical procedures or surgeries followed by adjuvant therapy in both cohorts Table . Whilst This seems paradoxical at first, but there is a possible explanation. At first glance, complex surgery seems to be more resource-demanding and envelope the risk of postoperative critical care . The altFive out of seven published studies reported more advanced T-and N-stages of HNC patients during the pandemic -20,22,24With the limitation of a small sample size, we observed a statistically significant increase in primary metastatic cases. A closer analysis of these cases revealed that three out of five patients initially presented with a T-stage of T4 (the rest T3 and T2), all of them were N-positive, and had a mean symptom to diagnosis interval of approximately ten weeks. Whilst time to diagnosis is associated with tumor progression and upstaging of disease via locoregional and distant metastases ,21, we cFuture considerationsThe ongoing COVID-19 pandemic has taught us many useful lessons for the future. It highlighted the necessity of establishing pandemic protocols and implementing them rapidly in patient care to minimize risk while maximizing safety and outcome . AdequatIt is obvious that we cannot draw final assumptions regarding the impact of the pandemic on the HNC population yet. We definitely need more multi-center, prospective studies to clarify the consequences of the pandemic on a global scale. International networks, such as CovidSurg, offer great platforms for collaboration and contribution in order to produce high-yield clinical data in the COVID-19 era . From noModern technologies and artificial intelligence can contribute to overcoming similar emergency situations. The establishment of telehealth services, such as call consultations or video sessions, is an optimal tool to decongest the overrun outpatient clinics . AutomatLimitations and biasIn this study, we tried to minimize bias through the inclusion of patients newly diagnosed with HNC, exclusively SCC, in the most common head and neck localizations. Head and neck cancer patients with recurrence or previous malignancy of another kind have been excluded because of their higher awareness and reflexes. The investigation period was comprised of a one-year pandemic and encompassed all possible variabilities: peaks and minimums, within and after the lockdown. The control period was the previous year to enhance comparability. The institutions’ direction, personnel, and organisation remained constant and provided the same level of health services throughout the two-year period. The personnel have been spared from infection by COVID-19.Nevertheless, the design was mandatory and retrospective. Despite careful examination of the patients’ files, we cannot exclude minor aberrations in subjective clinical data, such as the symptom-diagnosis interval. The monocentric character poses a geographical limitation, and thereafter, conclusions are not applicable nationwide. A subsite-or grading-related data analysis could not be conducted because of the modest number of cases.During the COVID-19 pandemic, our data showed consistent diagnostic and therapeutic performance in a tertiary university setting in Berlin, Germany. By highly prioritizing HNC patients' treatment to the detriment of elective cases, we did not notice any significant delays, more advanced primary tumor size, or locoregional metastases in our patients during this period. Nonetheless, these findings reflect a small geographical area, and the current epidemiological data is too sparse to draw conclusions on the real impact of the pandemic on healthcare.


ALK) mutation, also known as the diamond mutation in non–small-cell lung cancer (NSCLC), has been treated with tremendous success since it was first reported in 2007. Alectinib, a second generation ALK-Tyrosine kinase inhibitor (TKI), has been reported to have significantly longer progression- free survival (PFS) than first generation ALK inhibitors in untreated ALK positive NSCLC. However, the clinical efficacy of ALK-TKIs on rare ALK fusions remains unclear. In recent years, with the popularity of next-generation sequencing (NGS) technology, an increasing number of novel ALK fusion partners have been reported, but the responses are heterogeneous among different ALK fusions. Considering the inconsistent reactions, the clinical efficacy of ALK-TKIs in rare ALK gene fusions remains to be evaluated in more cases.The anaplastic lymphoma kinase . See Intergenic Region (IGR)-ALK rearrangements detected by DNA sequencing, which had limited clinical response to ALK-TKIs but showed sensitivity to chemotherapy combined with bevacizumab therapy in patient 2, with a PFS of over 1 year up till the last follow‐up assessment.We present two cases of patients with lung adenocarcinoma harboring two novel IGR-ALK fusions.In summary, our cases emphasize the need for comprehensive molecular analysis of different ALK fusion partners at the DNA level to formulate accurate treatment strategies and provide a certain therapeutic reference for these two types of novel  ALK gene rearrangement is a common driving oncogene in patients with NSCLC, approximately 3%-5% of non-squamous NSCLC harbor an ALK rearrangement, predominantly in younger, female patients who are light- or non-smokers (EML4) is the most common fusion partner gene -ALK exon20 (abundance: 9.7%) fusion was identified by a NGS assay based on 139-gene panel  (TP53 (abundance: 42.6%) and ERBB4 (abundance: 18.7%) were also identified in the same tumor tissue (see ), thus bronchus stents were placed to alleviate tracheal stenosis. To clarify the genetic alterations, a NGS analysis was recommended again after disease progression. However, the patient refused because of the high cost. We then re-evaluated the alterations of the coughed-up tissues by IHC, of note, IHC revealed positive expression of ALK-D5F3 and BRAF-V600E . Considering the resistance of this patient to alectinib, and the fact that lorlatinib was not accessible, alectinib was then replaced with ceritinib, 450 mg daily, combined with dabrafenib and trametinib. Despite switching to second-line treatment, tumor markers increased sharply  revealed a mass in the right middle lobe and enlarged lymph node in the mediastinum. The mediastinal lesion surrounded the right main bronchus. She underwent ultrasonography-guided percutaneous right cervical lymph node biopsy. The pathologic examination showed poorly differentiated adenocarcinoma  (IGR (downstream ZIC4)–ALK exon20 (abundance: 22.6%) fusion was found by a NGS assay based on 425-gene panel   chemotherapy plus bevacizumab anti-angiogenic therapy, and continued to receive pemetrexed and bevacizumab as maintenance therapy. Tumor markers dropped significantly. The patient’s best response was partial response (PR) and maintained SD during the treatment, with intracranial metastasis maintained stable throughout the course of treatment , achieving a PFS of over 1 year up to the last follow-up assessment.A 55-year-old Chinese male with no history of smoking or alcohol consumption visited our hospital on 24 October, 2020 with chief complaints of discovering lung occupation and aphasia for 4 days. PET-CT revealed a mass in the right lower lobe as well as pleural effusion, with bilateral pulmonary, multiple lymph node, bone, adrenal gland, and brain metastases. A pathologic diagnosis of lung adenocarcinoma was established by percutaneous needle biopsy of the left supraclavicular lymph node   can detect gene fusions at the DNA level and has been considered the gold standard for ALK gene rearrangements, but it can produce false-positive results owing to unproductive rearrangements , and we further emphasize the need for comprehensive molecular analysis of different ALK fusion partners at the DNA level to formulate accurate treatment strategies.Based on literature retrieval, this is the first report to demonstrate the presence of a novel The original contributions presented in the study are included in the article/Written informed consent was obtained from the participant for the publication of this case report.WL and YW contributed to the planning and supervision of the study. SL contributed to data curation, visualization and drafted the manuscript. HS and JW contributed to manuscript revision. HL contributed to the review of clinical data. YF contributed to the literature research and discussion. 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Pancreatic cancer (PC) is one of the most lethal cancers; caused by family history, obesity, diabetes, and smoking, it has a 2–9% five-year survival rate. However, patients diagnosed by endoscopic ultrasound (EUS) already have an advanced stage of PC, indicating the difficulty of surgical resection. Recently, laser ablative treatment with a diffusing applicator has been proven to be feasible for treating advanced PC. Despite the potential application for treating PC, further evaluation of acute and chronic tissue responses are essential to determine the efficacy and safety of diffusing applicator under EUS guidance. In this study, EUS-guided diffusing applicator-assisted laser ablation was evaluated to quantify the extent of the tissue response after the delivery of various energy levels.Endoscopic ultrasound (EUS)-guided cylindrical interstitial laser ablation (CILA) procedures can be used to treat unresectable pancreatic cancer (PC). The aim of this study was to investigate the acute responses of pancreatic tissue after EUS-guided CILA in vivo in porcine models. Eight pigs were tested to compare the effects of different energy levels on pancreatic tissue ablation. A 1064 nm laser system was used to deliver 5 W through a diffusing applicator. The EUS-guided CILA was performed under four different energies: 200, 400, 600, and 800 J. Three days after the experiments, histological analysis was performed. The CILA consistently generated circular coagulated necrosis (CN) in the cross-sectioned pancreatic tissue. The ablation diameter was linearly dependent on the total energy delivery. The area of the CN initially increased with total energy delivery but became saturated at 600 J. The width of the degenerative parenchyma (DP) in the native tissue beyond the CN region increased with the total energy up to 600 J, and then decreased afterward. EUS-guided CILA can be a feasible approach for treating PC. Further animal studies will investigate the chronic responses of the pancreatic tissue to examine the efficacy and safety of the proposed method for clinical translation. Pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer (PC), is a highly fatal disease. It is generally caused by family history, obesity, diabetes, and/or smoking, and it has a 2–9% five-year survival rate . AlthougPrevious studies have reported the development of EUS-guided cylindrical ILA (CILA), which uses a diffusing applicator to ablate pancreatic tissue in a predictable manner ,10. UnliThe current study aimed to investigate the acute responses of pancreatic tissue after laser treatment and to comparatively quantify the extent of tissue ablation after the delivery of various energy levels. We hypothesized that the EUS-guided CILA could control the dimensions of the treated pancreatic tissue by adjusting the total energy delivery. Four energy levels were applied to identify the optimal conditions for EUS-guided CILA in vivo in porcine models. Histological analysis was performed to evaluate the acute responses of the pancreatic tissue after CILA.A diffusing applicator  was prepared by micromachining a fiber tip surface. An active element of 5 mm was fabricated to irradiate the laser light circumferentially for tissue ablation . A 19-G A total of eight male pigs  were used to conduct the in vivo EUS-guided CILA tests (N = 2 per energy group). All animal experimental procedures followed the Korean National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committee of Knotus, Korea (permit number: IACUC 21-KE-442). Prior to the ablation tests, all the animals were injected with a 0.1 mL/kg mixture of zoletil and xylazine (1:1 ratio) for general anesthesia. Subsequently, a ventilator provided isoflurane (1–2%) and oxygen (2 L/min) for the animals to maintain general anesthesia during the experiments. A 19 G biopsy needle was initially inserted through the endoscopic channel, and the stomach was punctured under endoscopic visualization to reach the pancreatic tissue. After ultrasound imaging confirmed the location of the needle, the needle was moved back 10 mm, and the diffusing applicator was inserted through the inner lumen of the biopsy needle. All the treated samples of the pancreas were extracted three days after the experiments and were fixed in 10% formalin for a week. Then, the tissue samples were sectioned into 5 μm slices and stained with hematoxylin and eosin. The stained tissue slides were scanned using a digital slide scanner . The Motic “DSAssistant” software was used to observe and photograph the biological and structural changes in the treated tissue. The tissue slides were selected from three ablation points in the pancreatic tissue for each group. Then, the extent of laser ablation (LA) was estimated quantitatively by measuring the ablation diameter and necrotic area in the treated pancreatic tissue. The distance between the inflammatory band located at the edge of the central necrotic area and native tissue (NT) was measured to assess the width of the degenerative parenchyma (DP), as this indicates the ablative impact on the NT beyond the necrotic area.p < 0.05 represents statistical significance.All the data and graphs are presented as means ± standard deviation for quantitative evaluation. For the nonparametric tests, SPSS software  was used to perform Kruskal–Wallis and Mann–Whitney U tests to compare multiple groups and two groups, respectively. Blood tests were conducted pre- and post-operatively and before euthanasia for animal monitoring . Althoug2 for 600 J vs. 169.2 ± 52.5 mm2 for 800 J; p = 0.43).p < 0.05 vs. 200 and 400 J).TM EUS-RFA probe  and an EUS-RFA needle  [3). Crio et al. reported EUS-guided RFA in eight patients with locally advanced PDAC, and the mean volume of thermal necrosis only accounted for 30% of the tumor mass (range 5.8–73.5%) after one-day and one-month treatments [3), multiple punctures with the RFA needles were required to cover a large tumor, eventually increasing undesired complications, such as mild abdominal pain and peripancreatic effusion [3 in volume with a single application) [EUS-guided ablation therapies have been widely used as alternative treatments for PC, mainly owing to their advantages regarding real-time imaging and focal ablation. Multiple case reports on EUS-guided RFA have demonstrated results from clinical trials using both the Habidh Korea) ,12. EUS-eatments . Owing teffusion ,14. In oication) ,16. Afteication) .Instead of a flat fiber, the current study used a diffusing applicator with cylindrical light emission for circumferential LA of the pancreatic tissue, aiming to generate uniform ablation lesions  and a large volume of thermal capability  for in vivo, ex vivo, and numerical simulations ,10. DuriTM injection followed by a saline solution flush should be performed before and after LA to enhance the contract-EUS image for optimal treatment performance in terms of the ablation lesion and position of the diffusing applicator [EUS guidance is a beneficial technique for defining the position of both the biopsy needle and the diffusing tip in the pancreatic tissue so as to prevent adverse injury to the surrounding organs during laser irradiation. However, in this study, the position of the diffusing applicator was sometimes difficult to visualize during the EUS monitoring owing to the low reflection of the ultrasound signals. Therefore, to ensure the accurate positioning of the diffusing applicator, a small highly reflective material should be attached to the proximal end of the applicator tip to induce the reflected US signals for EUS detection during the procedure. Additionally, an intravenous SonoVueplicator ,19.The histological analysis demonstrated a well-defined demarcation between the native and necrotic tissue three days after the EUS-guided CILA . The curIn addition, the current study demonstrates the acute responses of the pancreatic tissue 3 days after EUS-guided CILA. However, further studies should be performed to confirm the long-term safety and efficacy of EUS-guided CILA for clinical translations. Thus, various assessments will be performed in 2-month survival studies to compare acute and chronic responses in terms of the extent of ablation and changes in digestive enzymes  ,22. FurtAlthough the current study showed the clinical feasibility of EUS-guided CILA for treating pancreatic tissue, limitations remain. For example, the number of mini pigs was relatively small (N = 8) compared with that of other investigations ,24. The The current in vivo studies confirmed that EUS-guided LA using a diffusing applicator generates a uniform, predictable ablation lesion and can regulate ablation areas by adjusting the total energy delivery. EUS-guided CILA can be a feasible way to effectively treat PC. Further long-term in vivo studies will investigate the complete removal of the pancreatic tumor and the healing response of the pancreatic tissue to ensure the enhanced efficacy and safety of the proposed EUS-guided CILA for clinical translation.


Drosophila, Insulin-Producing cells (IPCs) present in the fly brain, analogous to vertebrate pancreas, control growth, metabolism and lifespan. Regulation of gene expression by microRNAs is strongly believed to be crucial in determining adult lifespan, however, no systematic approach has been conducted so far to study the role of microRNAs in the IPCs in the aging process. Most of the current work has been focused on deciphering the roles of microRNAs in the IPCs during developmental stages. Here, we report the results of a mini-screen performed to identify microRNAs that function in the adult IPCs in regulating lifespan and neuronal integrity.Various genetic, molecular and environmental factors influence the lifespan of an organism, which includes the highly conserved insulin signaling pathway. In The lifespan of organisms is controlled by both genetic and environmental factors. Nutrition is one of the key environmental factors that regulates the lifespan of an organism . Among the molecular pathways, insulin signaling , TOR signaling  and JNK  signaling  have been reported to affect lifespan. Various other factors like oxidative stress (Liochev 2013), metabolic status, telomere length , and DNA damage  have previously been implicated to regulate lifespan in mammals.Drosophila, 8 insulin-like peptides (DILPs), similar to vertebrate insulin/IGFs have been discovered to date. Of which DILP2, 3 and 5 are produced by the insulin-producing cells (IPCs) located in thepars intercerebralisregion of theDrosophilabrain. In addition to the IPC DILPs, DILP6 is produced from the fatbody, DILP7 from the intestine and DILP8 from the imaginal discs and ovaries .The insulin/insulin-like growth factor (IGF) pathway links nutritional status to growth, metabolism and lifespan. Indilp2 mutants . Increased lifespan caused by dietary restriction is mediated by the neuronal DILPs . Overexpression ofdilp6 in the adult head and abdominal fatbody, has also been demonstrated to increase longevity, possibly through the regulation ofdilp2 . Recent data also suggests thatdilp1 could play a role in regulating lifespan . In a recent study, age-related decrease in dilp3 levels with age was implicated to be responsible for Intermediate-Term Memory Impairment inDrosophila .Earlier studies have demonstrated an increase in lifespan in flies that are defective for insulin signaling . Ablation of the IPCs was also found to increase the lifespan . A significant increase in longevity was observed inDrosophila, was found to promote insulin signaling and body growth, by regulating its target genes,u-shaped(ush), anddilp6, in the fatbody .miR-278 mutants were found to be insulin resistant and that miR-278 acts through regulation of theexpandedtranscript .microRNAs are short non-coding RNAs that post-transcriptionally regulate the levels of various target mRNAs that they bind to via sequence complementarity (Ambros 2001). microRNAs are known to either regulate insulin levels directly or regulate insulin signaling in the responsive tissues. miR-8 insugarbabe . miR-11 has been shown to regulate pupal size via its target RAS85D by regulating the gene expression ofilp2, 3and5from the IPCs . miR-7 regulates the production and secretion of insulin-like peptides from the IPCs via its target,DrosophilaF-actin capping protein alpha . A large genetic screen, identified that the conserved miR-9a regulates body growth by targeting sNPFR1 in the IPCs inDrosophila .Quite a few microRNAs have been discovered to regulate the levels of insulin-like peptides directly. miR-14 has previously been discovered to regulate the insulin-like peptides and thereby the metabolism via its targetEip74E . An age-dependent increase in all the three let-7-C miRNAs in both adult males and females flies, was observed . All of this work suggests the possibility that the level of various microRNAs in each cell type or cell could be dynamic and change in an age-dependent manner as well.Recently various studies have been carried out to estimate the levels of all microRNAs in the fly brain/head to discover any role that microRNAs could play in aging. The level of miR-34 was found to go up in the head of flies with age and thus regulate lifespan via its targetchinmoand thereby regulate lifespan . miR-969 , miR-305 , miR-277  and miR-282  independently were also found to play a role in the regulation of lifespan inDrosophila. Few microRNAs were discovered to circulate in the hemolymph ofDrosophilaand were found to accumulate in tissues in an age-dependent manner . Thus highlighting the importance of studying the role any microRNAs may play in regulating lifespan.Adult specific overexpression of miR-184 and let-7 led to significant changes to fat metabolism and life span in Drosophila (Gendron and Pletcher. 2017). Both let-7 and miR-125 were found to regulateDrosophila with the use of atsdilp2GAL4; tubulinGAL80line. This approach was taken to ensure that the effects seen are not caused by reducing the levels of these microRNAs during stages prior to adulthood. We also carried out a climbing assay on day 24, as a readout for neuronal integrity in response to aging in these flies.Here, we carried out a mini-genetic screen to identify microRNAs which when downregulated in the IPCs play a role in regulating lifespan. We analyzed the effects of downregulation of 7 conserved microRNAs which are known to be expressed in the fly head. The screen was carried out by expressing microRNA-sponges  specifically in the Insulin-producing cells in the adult maleA significant increase in lifespan was observed when miR-100 sponge was expressed in the IPCs of adult male flies . In a prior study, miR-100 levels were found to increase with age . Therefore it would be interesting to verify any role that the microRNA may play in regulating DILPs from the IPCs in adult flies. To check for any early or late changes in death, we also carried out a Fleming-Harrington weighted log rank-test Test wherein, flies started dying later when miR-312 sponge (p=0.0294) and miR-285 (p=0.0459) sponge was expressed in the IPCs of adult male flies, without affecting the overall lifespan . Expression of miR-184 sponge in the IPCs led to a similar but not statistically significant increase in survival during early lifespan (p=0.0567). None of the other microRNA sponges tested showed any effects on lifespan. The climbing ability of flies in response to expression of miR-375 (p=0.00653) and miR-312 sponges (p=0.00656) in the IPCs was found to be significantly higher than that of the control . Expression of miR-100 sponge in the adult IPCs also improved the climbing capacity of older flies  . In a screen carried out previously, overexpression of miR-312 and miR-285 in the IPCs led to an increase in wing size . Further experiments are needed to verify the role that miR-100, miR-375, miR-285 and miR-312 play in the IPCs in neuronal function and aging.Survival assayOC, flies were allowed to mate at 29OC for two days. Batches of 10-15 male flies per vial were provided fresh food every alternate day with dead flies being recorded every day. A Log rank test and a weighted-log rank test with a Fleming-Harrington test were performed using the OASIS 2 software.Larvae were raised on standard media at 18Climbing assayClimbing assay was carried out using long vials. Flies were transferred to assay vials and acclimatised for 10 minutes, the vial was tapped thrice in an arrhythmic fashion, and the number of flies that cross 5 cms in 3 seconds were noted. This assay was carried out thrice with a gap of about 3 minutes between each trial. A student's t-test was carried out to calculate if there were any significant changes if any.Statistical analysisPrism 8 was used to plot all the data. OASIS 2  was used for the statistical analysis for the lifespan experiments. A log-rank test was carried out to check for differences in lifespan while a Fleming-Harrington test was carried out to check for differences in early or late survival. The percentage of flies crossed 5 cms in 3 secs was calculated. The percentages were compared with the control using a student's t-test.Fly stocksdilp2Gal4 is described in Ikeya et al. 2002,tstubulin-Gal80(7017),and1118 w(5905) are originally from Bloomington Drosophila Stock Center.