Somatic hypermutation introduces bystander mutations that prepare SARS-CoV-2 antibodies for emerging variants.

Somatic hypermutation (SHM) drives affinity maturation and continues over months in SARS-CoV-2-neutralizing antibodies (nAbs). However, several potent SARS-CoV-2 antibodies carry no or only a few mutations, leaving the question of how ongoing SHM affects neutralization unclear. Here, we reverted variable region mutations of 92 antibodies and tested their impact on SARS-CoV-2 binding and neutralization. Reverting higher numbers of mutations correlated with decreasing antibody functionality. However, for some antibodies, including antibodies of the public clonotype VH1-58, neutralization of Wu01 remained unaffected. Although mutations were dispensable for Wu01-induced VH1-58 antibodies to neutralize Alpha, Beta, and Delta variants, they were critical for Omicron BA.1/BA.2 neutralization. We exploited this knowledge to convert the clinical antibody tixagevimab into a BA.1/BA.2 neutralizer. These findings broaden our understanding of SHM as a mechanism that not only improves antibody responses during affinity maturation but also contributes to antibody diversification, thus increasing the chances of neutralizing viral escape variants.

SARS-CoV-2 Omicron sublineages exhibit distinct antibody escape patterns.

SARS-CoV-2 neutralizing antibodies play a critical role in COVID-19 prevention and treatment but are challenged by viral evolution and the emergence of novel escape variants. Importantly, the recently identified Omicron sublineages BA.2.12.1 and BA.4/5 are rapidly becoming predominant in various countries. By determining polyclonal serum activity of 50 convalescent or vaccinated individuals against BA.1, BA.1.1, BA.2, BA.2.12.1, and BA.4/5, we reveal a further reduction in BA.4/5 susceptibility to vaccinee sera. Most notably, delineation of sensitivity to an extended 163-antibody panel demonstrates pronounced antigenic differences with distinct escape patterns among Omicron sublineages. Antigenic distance and/or higher resistance may therefore favor immune-escape-mediated BA.4/5 expansion after the first Omicron wave. Finally, while most clinical-stage monoclonal antibodies are inactive against Omicron sublineages, we identify promising antibodies with high pan-SARS-CoV-2 neutralizing potency. Our study provides a detailed understanding of Omicron-sublineage antibody escape that can inform on effective strategies against COVID-19.

Determining the reliability of rapid SARS-CoV-2 antigen detection in fully vaccinated individuals.

BACKGROUND: Rapid antigen detection tests (RADT) are commonly used as SARS-CoV-2 diagnostic tests both by medical professionals and laypeople. However, the performance of RADT in vaccinated individuals has not been fully investigated. OBJECTIVES: RT-qPCR and rapid antigen detection testing were performed to evaluate the performance of the Standard Q COVID-19 Ag Test in detecting SARS-CoV-2 breakthrough infections in vaccinated individuals. STUDY DESIGN: Two swab specimens, one for RT-qPCR and one for RADT, were collected from vaccinated individuals in an outpatient clinic. For comparison of RADT performance in vaccinated and unvaccinated individuals, a dataset already published by this group was used as reference. RESULTS: During the delta wave, a total of 696 samples were tested with both RT-qPCR and RADT that included 692 (99.4%) samples from vaccinated individuals. Of these, 76 (11.0%) samples were detected SARS-CoV-2 positive by RT-qPCR and 45 (6.5%) samples by the Standard Q COVID-19 Ag test. Stratified by Ct values, sensitivity of the RADT was 100.0%, 94.4% and 81.1% for Ct ≤ 20 (n=18), Ct ≤ 25 (n=36) and Ct ≤ 30 (n=53), respectively. Samples with Ct values ≥ 30 (n=23) were not detected. Overall RADT specificity was 99.7% and symptom status did not affect RADT performance. Notably, RADT detected 4 out of 4 samples of probable Omicron variant infection based on single nucleotide polymorphism analysis. CONCLUSION: Our results show that RADT testing remains a valuable tool in detecting breakthrough infections with high viral RNA loads.

No substantial preexisting B cell immunity against SARS-CoV-2 in healthy adults.

Preexisting immunity against SARS-CoV-2 may have critical implications for our understanding of COVID-19 susceptibility and severity. The presence and clinical relevance of a preexisting B cell immunity remain to be fully elucidated. Here, we provide a detailed analysis of the B cell immunity to SARS-CoV-2 in unexposed individuals. To this end, we extensively investigated SARS-CoV-2 humoral immunity in 150 adults sampled pre-pandemically. Comprehensive screening of donor plasma and purified IgG samples for binding and neutralization in various functional assays revealed no substantial activity against SARS-CoV-2 but broad reactivity to endemic betacoronaviruses. Moreover, we analyzed antibody sequences of 8,174 putatively SARS-CoV-2-reactive B cells at a single cell level and generated and tested 158 monoclonal antibodies. None of these antibodies displayed relevant binding or neutralizing activity against SARS-CoV-2. Taken together, our results show no evidence of competent preexisting antibody and B cell immunity against SARS-CoV-2 in unexposed adults.

Comprehensive analysis of Twitter usage during a major medical conference held virtually versus in-person.

BACKGROUND: Twitter has become one of the most important social media platforms in science communication. During scientific conferences, Twitter can facilitate the communication between audience and speakers present at the venue and can extend the reach of a conference to participants following along from home. To examine whether Twitter activity can serve as a surrogate parameter for attendance at the RSNA conferences in 2019 and in 2020, and to characterize changes in topics discussed due to the virtual character of the 2020 RSNA conference. METHODS: The Twitter API and R Studio were used to analyze the absolute number and frequency of tweets, retweets, and conference-related hashtags during the 2019 and 2020 RSNA conference. Topics of discussion were compared across years by visualizing networks of co-occurring hashtags. RESULTS: There was a 46% decrease in total tweets and a 39% decrease in individual Twitter users in 2020, mirroring a 43% decrease in registered attendees during the virtual conference. Hashtags related to social initiatives in radiology (e.g., "#radxx" and "#womeninradiology" for promoting women's empowerment in radiology or "#pinksocks," "#weareradiology" and "#diversityisgenius" for diversity in general) were less frequently used in 2020 than in 2019. CONCLUSION: Twitter and congress attendance were highly related and interpersonal topics underwent less discussion during the virtual meeting. Overall engagement during the virtual conference in 2020 was lower compared to the in-person conference in 2019.

Evaluation of a Rapid Antigen Test To Detect SARS-CoV-2 Infection and Identify Potentially Infectious Individuals.

The identification and isolation of highly infectious SARS-CoV-2-infected individuals is an important public health strategy. Rapid antigen detection tests (RADT) are promising tools for large-scale screenings due to timely results and feasibility for on-site testing. Nonetheless, the diagnostic performance of RADT in detecting infectious individuals is not yet fully determined. In this study, RT-qPCR and virus culture of RT-qPCR-positive samples were used to evaluate and compare the performance of the Standard Q COVID-19 Ag test in detecting SARS-CoV-2-infected and possibly infectious individuals. To this end, two combined oro- and nasopharyngeal swabs were collected at a routine SARS-CoV-2 diagnostic center. A total of 2,028 samples were tested, and 118 virus cultures were inoculated. SARS-CoV-2 infection was detected in 210 samples by RT-qPCR, representing a positive rate of 10.36%. The Standard Q COVID-19 Ag test yielded a positive result in 92 (4.54%) samples resulting in an overall sensitivity and specificity of 42.86 and 99.89%, respectively. For adjusted CT values of <20 (n = 14), <25 (n = 57), and <30 (n = 88), the RADT reached sensitivities of 100, 98.25, and 88.64%, respectively. All 29 culture-positive samples were detected by the RADT. Although the overall sensitivity was low, the Standard Q COVID-19 Ag test reliably detected patients with high RNA loads. In addition, negative RADT results fully corresponded with the lack of viral cultivability in Vero E6 cells. These results indicate that RADT can be a valuable tool for the detection of individuals with high RNA loads that are likely to transmit SARS-CoV-2.

Practical applications of deep learning: classifying the most common categories of plain radiographs in a PACS using a neural network.

OBJECTIVES: The goal of the present study was to classify the most common types of plain radiographs using a neural network and to validate the network's performance on internal and external data. Such a network could help improve various radiological workflows. METHODS: All radiographs from the year 2017 (n = 71,274) acquired at our institution were retrieved from the PACS. The 30 largest categories (n = 58,219, 81.7% of all radiographs performed in 2017) were used to develop and validate a neural network (MobileNet v1.0) using transfer learning. Image categories were extracted from DICOM metadata (study and image description) and mapped to the WHO manual of diagnostic imaging. As an independent, external validation set, we used images from other institutions that had been stored in our PACS (n = 5324). RESULTS: In the internal validation, the overall accuracy of the model was 90.3% (95%CI: 89.2-91.3%), whereas, for the external validation set, the overall accuracy was 94.0% (95%CI: 93.3-94.6%). CONCLUSIONS: Using data from one single institution, we were able to classify the most common categories of radiographs with a neural network. The network showed good generalizability on the external validation set and could be used to automatically organize a PACS, preselect radiographs so that they can be routed to more specialized networks for abnormality detection or help with other parts of the radiological workflow (e.g., automated hanging protocols; check if ordered image and performed image are the same). The final AI algorithm is publicly available for evaluation and extension. KEY POINTS: • Data from one single institution can be used to train a neural network for the correct detection of the 30 most common categories of plain radiographs. • The trained model achieved a high accuracy for the majority of categories and showed good generalizability to images from other institutions. • The neural network is made publicly available and can be used to automatically organize a PACS or to preselect radiographs so that they can be routed to more specialized neural networks for abnormality detection.

Longitudinal Isolation of Potent Near-Germline SARS-CoV-2-Neutralizing Antibodies from COVID-19 Patients.

The SARS-CoV-2 pandemic has unprecedented implications for public health, social life, and the world economy. Because approved drugs and vaccines are limited or not available, new options for COVID-19 treatment and prevention are in high demand. To identify SARS-CoV-2-neutralizing antibodies, we analyzed the antibody response of 12 COVID-19 patients from 8 to 69 days after diagnosis. By screening 4,313 SARS-CoV-2-reactive B cells, we isolated 255 antibodies from different time points as early as 8 days after diagnosis. Of these, 28 potently neutralized authentic SARS-CoV-2 with IC100 as low as 0.04 µg/mL, showing a broad spectrum of variable (V) genes and low levels of somatic mutations. Interestingly, potential precursor sequences were identified in naive B cell repertoires from 48 healthy individuals who were sampled before the COVID-19 pandemic. Our results demonstrate that SARS-CoV-2-neutralizing antibodies are readily generated from a diverse pool of precursors, fostering hope for rapid induction of a protective immune response upon vaccination.

Amplification of KRAS and its heterogeneity in non-Asian gastric adenocarcinomas.

BACKGROUND: Gastric cancer is one of the deadliest cancer entities worldwide. While surgery is the only curative treatment option in early tumors, for locally advanced and metastatic patients further therapeutic targets are needed. Several studies not only reported mutations but also amplifications of the KRAS locus in different cancer entities. More recently, KRAS amplification was discussed as a new therapeutic target. Little is known about the (prognostic) relevance and (heterogenic) distribution of KRAS amplification in gastric adenocarcinomas, especially in Non-Asian patients. METHODS: Amplification of the KRAS locus and corresponding protein expression was analyzed in 582 gastric adenocarcinomas employing fluorescence in-situ hybridization (FISH) and immunohistochemistry. Amplification status was correlated with clinico-pathological features, clinical outcome and molecular tumor data including a correlation to the TCGA subtypes of gastric carcinoma. RESULTS: KRAS amplification was detected in 27 out of 470 analysable tumors (5.7%) and correlated with protein expression of KRAS in all amplified tumors. Within the KRAS amplified gastric tumors 14/27 (51.9%) showed a heterogeneous distribution with also KRAS non-amplified tumor parts. According to TCGA 24 tumors (88.8%) were related to chromosomal instable tumors (CIN). The survival analysis of the entire patient cohort did not show any difference in overall survival in dependence on the KRAS status. However, a significant survival difference with a worse outcome for patients with KRAS amplified tumors was identified when analysing patients without neoadjuvant pre-treatment. CONCLUSIONS: We confirm the unfavorable prognosis of KRAS amplified tumors reported by other studies in (Asian) patient groups, at least in patients without neoadjuvant pre-treatment. Within KRAS amplified tumors we revealed intratumoral heterogeneity that may define a (more aggressive) tumor cell population which is more frequently observed in patients with lymph node metastases. Despite the heterogeneous distribution of KRAS amplified tumor clones, KRAS amplified locally advanced or metastasized gastric adenocarcinomas represent a therapeutically highly relevant tumor subgroup.

Identification of targeted therapy options for gastric adenocarcinoma by comprehensive analysis of genomic data.

BACKGROUND: So far only trastuzumab, pembrolizumab and ramucirumab have been approved by the FDA for targeted therapy in gastric cancer (GC). Here we report on potential targeted therapy options for gastric adenocarcinoma based on a novel analysis of "The Cancer Genome Atlas (TCGA)" database. METHODS: One hundred two FDA-approved targeted cancer drugs were compiled and molecular targets defined. Drugs were considered as potentially effective if targeted genes showed (1) an increase in copy number, (2) gain of function with oncogene activation, (3) specific alterations responsive to approved drugs. Additionally, genetic changes that confer drug resistance and/or sensitivity were evaluated. RESULTS: Fifty percentage of patients with GC may be treatable with non-GC but FDA-approved targeted cancer therapies. The major drug identified in our in silico study for GC is copanlisib, a PI3K inhibitor. In the TCGA patient database, our genetically based drug response prediction identified more patients with alterations sensitive to copanlisib compared to the already-GC-approved drug trastuzumab (20%, 78 out of 393 patients, vs. trastuzumab: 13%, 52 of 393 patients), which is mainly due to the high incidence of PIK3CA gain of function mutations within mutation hot spots. CONCLUSION: Our results demonstrate that various currently FDA-approved drugs might be candidates for targeted therapy of GC. For clinical trials, cancer patients should be selected based on the genomic profile of their tumor.

Technical Steps and Tips for Linear-Stapled Gastric Bypass Based on Personal Experience and the Classification of Intraoperative Complexity.

The most commonly performed Roux-en-Y gastric bypass (RYGB) procedure is difficult and has many technical variations. The individual patient's anatomic characteristics, like an exceptionally large left liver lobe, fatty mesentery with limited mobile Roux limb, difficulty in positioning a stapler, etc., can greatly increase the technical difficulty of this procedure. Challenging situations in laparoscopic gastric bypass surgery can be classified according to the intraoperative complexity. According to this classification scheme, all patients in laparoscopic linear-stapled gastric bypass can be classified into one of four types: Type I - ideal. Surgery is straightforward, and every operative technique is relatively routine. Type II - less-than-ideal. Some minor technical difficulties may occur; some operative techniques can be more difficult than others. Type III - problematic. Difficult, with some operative techniques considerably more difficult than others. Type IV - very difficult. Every operative step is very difficult. The goal of this article is to analyze the steps of laparoscopic linear-stapled RYGB with regard to personal experience and the classification of intraoperative complexity.

Video-Assisted Subcutaneous Destruction of the Sinus Tract with Vessel-Loop Drainage as Minimally-Invasive Surgical Treatment for Pilonidal Sinus Disease.

The video-assisted subcutaneous destruction of the sinus tract (VADST) is a novel, minimally-invasive technique for the treatment of pilonidal sinus disease (PSD). This is an advancement of the previously described subcutaneous destruction of the sinus tract and the removal of hairs as well as the long-term vessel-loop drainage of the wound channel (DST). Although the first results of this operation seemed to be promising, some of my colleagues felt that the blind approach to this procedure could be its potential "weak point". As a result of this critique, the procedure was enhanced with the subcutaneous video-assisted inspection of the natal cleft. Throughout most steps of VADST, like the widening of pilonidal pits with mosquito and/or Pean clamps, the subcutaneous destroying of the sinus tract, lifting the skin in a natal cleft with a curette, removing the hair with a Pean clamp and a subcutaneous vessel-loop drainage, were found to be similar to DST. The new steps involve the possibility of the video-assisted control of hair vestiges and bleedings as well as the removal of hair and debris under visual control. A rigid choledochoscope from Berci (Firma Richard Wolf GmbH, Knittlingen, Germany) was used for the subcutaneous endoscopic examination of the natal cleft area. Neither gas application nor water perfusion were necessary for this step. Three patients with simple forms of PSD, and one patient with an acute abscess formation, underwent the VADST procedure. The patients with simple forms of PSD had no adverse events during the first eight weeks postoperatively. The patient with an acute abscess formation developed a purulent inflammation that required a wide local excision with an open-wound healing. Due to the very small number of patients in this report, we were not able to asses this technique properly. Contrary to DST, we intend to use VADST, not only for the treatment of patients with simple forms (primary pilonidal sinus without abscessed inflammation with maximal three orifices, all of them inside the navicular area), but also for the treatment of complex PSD forms.

[Register of Difficult Surgical Situations]. / Register der schwierigen chirurgischen Situationen.

Every surgical problem that increases the likelihood of intraoperative and postoperative complications is considered to be a difficult surgical situation. Based on this definition, Korenkov et al. proposed to classify patients according to the following intraoperative difficulty levels (I to IV): (I) ideal situation (easy to operate, no problems), (II) fairly easy/manageable/simple (some minor difficulties may occur), (III) difficult/problematic (difficult to operate; some operative techniques are considerably more difficult than others), and (IV) very difficult (every operative step is difficult/challenging). Kaafrani et al. proposed a severity classification for intraoperative adverse events. Depending on the severity level, classes range from I (injury requiring no repair) to VI (intraoperative death). Clavien and colleagues published a globally established classification system for postoperative complications. In this classification, the severity of postoperative complications ranges from severity grade I (minimal deviation from the normal postoperative course) to severity grade V (death of patient). Based on the proposed classifications and the problems of individual surgical decision-making, we had the idea to create a Register of Difficult Intraoperative Situations (DIS register). The basic principle of such a register is the collection of an individual expert's experiences. The scientific analysis should focus on patients with apparent modifications in treatment due to difficult intraoperative situations. Registration and processing of enrolled cases will be performed anonymously based on an appropriate IT platform. The main goal of this register is to develop an accessible database for practising surgeons. This will provide an opportunity for every surgeon to find out what other surgeons did in similar situations.

Subcutaneous Destruction of Sinus Tract with Long-term Vessel Loop Drainage as a Minimally Invasive Surgical Treatment for Primary Pilonidal Sinus.

The surgical management of pilonidal disease ranges from minimally invasive techniques to wide excision with secondary wound healing. Hypothetical counterarguments for using the minimally invasive techniques are that these techniques only incompletely remove the sinus tract and that they allow the formation of subcutaneous cavities as a result of rapid closure of small skin incisions. In order to avoid these problems we used a vessel loop drainage after subcutaneous destruction of the sinus tract. In this technique we combined two procedures: a narrow excision of pilonidal orifices with the subcutaneous destruction of the sinus tract and the removal of hair as well as the long-term vessel loop drainage of the wound channel with repeated revisions of the wound channel for removing hair remnants. In order to assess the efficacy and morbidity of this method 5 patients with primary symptomatic pilonidal sinus underwent an operation as a pilot study. Up to the removal of the vessel loop the wound channel had been regularly inspected using a mosquito clamp. If during two successive inspections no hair from the wound was found the vessel loop was removed. No adverse events occurred such as abscess or recurrent pilonidal sinus. One female patient with a history of long-term use of morphine derivatives and psychotropic substances experienced persistent coccygeal pain that persisted longer than 6 weeks. The complete wound healing with the formation of a qualitative scar took place on the 21st postoperative day (for 3 patients) and on the 28th postoperative day (for 2 patients). Initial results suggest that this operation is an effective procedure for the treatment of primary simple pilonidal sinus disease and we will continue to systematically test it.

Individual surgery for gastric gastrointestinal stromal tumors.

The management of gastric gastrointestinal stromal tumors (GIST) is complex. Local tumor resection with disease-free resection margins without lymphadenectomy is the treatment of choice for primary non-metastatic tumors. This can be achieved with several techniques including wedge resection, transgastric resection, partial gastrectomy, total gastrectomy, and multivisceral resection. Open and minimally invasive surgical approaches can be considered. We describe the technical steps of the aforementioned procedures in relation to tumor size, tumor location (especially at or near the gastro-esophageal junction or pylorus), pattern of exophytic or intraluminal growth, adherence to surrounding structures, and other tumor-associated factors. Challenging situations in gastric surgery for GISTs are also discussed and categorized according to a classification of intraoperative complexity (1 [easy] to 4 [very difficult]). On the basis of this classification, we divided all patients with an indication for gastric GIST surgery into appropriate groups.

Metabolic programming of MEST DNA methylation by intrauterine exposure to gestational diabetes mellitus.

Epigenetic processes are primary candidates when searching for mechanisms that can stably modulate gene expression and metabolic pathways according to early life conditions. To test the effects of gestational diabetes mellitus (GDM) on the epigenome of the next generation, cord blood and placenta tissue were obtained from 88 newborns of mothers with dietetically treated GDM, 98 with insulin-dependent GDM, and 65 without GDM. Bisulfite pyrosequencing was used to compare the methylation levels of seven imprinted genes involved in prenatal and postnatal growth, four genes involved in energy metabolism, one anti-inflammatory gene, one tumor suppressor gene, one pluripotency gene, and two repetitive DNA families. The maternally imprinted MEST gene, the nonimprinted glucocorticoid receptor NR3C1 gene, and interspersed ALU repeats showed significantly decreased methylation levels (4-7 percentage points for MEST, 1-2 for NR3C1, and one for ALUs) in both GDM groups, compared with controls, in both analyzed tissues. Significantly decreased blood MEST methylation (3 percentage points) also was observed in adults with morbid obesity compared with normal-weight controls. Our results support the idea that intrauterine exposure to GDM has long-lasting effects on the epigenome of the offspring. Specifically, epigenetic malprogramming of MEST may contribute to obesity predisposition throughout life.

Unicentric castleman's disease located in the lower extremity: a case report.

BACKGROUND: Castleman's disease is a rare form of localized lymph node hyperplasia of uncertain etiology. Although the mediastinum is the most common site of involvement, rare cases occurring in lymph node bearing tissue of other localization have been reported, including only a few intramuscular cases. Unicentric and multicentric Castleman's disease are being distinguished, the latter harboring an unfavorable prognosis. CASE PRESENTATION: Here, we present a case of unicentric Castleman's disease in a 37-year-old woman without associated neoplastic, autoimmune or infectious diseases. The lesion was located in the femoral region of the right lower extremity and surgically resected after radiographic workup and excisional biopsy examinations. The tumor comprised lymphoid tissue with numerous germinal centers with central fibrosis, onion-skinning and rich interfollicular vascularization. CD23-positive follicular dendritic cells were detected in the germinal centers and numerous CD138-positive plasma cells in interfollicular areas. The diagnosis of mixed cellularity type Castleman's disease was established and the patient recovered well. CONCLUSIONS: In conclusion, the differential diagnosis of Castleman's disease should be considered when evaluating a sharply demarcated, hypervascularized lymphatic tumor located in the extremities. However, the developmental etiology of Castleman's disease remains to be further examined.

Laparoscopic gastric banding and individual bariatric surgery.

Different factors (strategic, anatomical, instrumental, etc.) can cause difficult surgical situations, especially in bariatric surgery. In a difficult surgical situation, the surgeon faces a dilemma as to whether to continue the intended operation "at all costs" or to deviate from the initially planned surgical procedure to some alternative technique or procedure. The dilemmatic nature of the difficult surgical situation in bariatric surgery has motivated us to discuss the following aspects because they all contribute to the problem: standards in bariatric surgery, deviation from standards, the role of experts, and the focus of research. We also propose to classify bariatric patients according to intraoperative difficulty (I to IV) as: (I) ideal cases (i.e., easy to operate, no problems), (II) not quite ideal cases (some minor difficulties may occur), (III) problematic cases (difficult to operate, some operative techniques are considerably more difficult than others), and (IV) very difficult cases (every operative step is difficult). We discuss the establishment of a registry of difficult surgical situations including the possibilities of deviating from the standard. Scientific analyses of such registries should focus on patients with apparent modifications in treatment (process deviations), but might also look at those with surprisingly good or bad results (outcome deviations). The technical steps of laparoscopic adjustable gastric banding (LAGB) have been chosen to illustrate this concept.