Genetic mapping of novel modifiers for ApcMin induced intestinal polyps' development using the genetic architecture power of the collaborative cross mice.

BACKGROUND: Familial adenomatous polyposis is an inherited genetic disease, characterized by colorectal polyps. It is caused by inactivating mutations in the Adenomatous polyposis coli (Apc) gene. Mice carrying a nonsense mutation in the Apc gene at R850, which is designated ApcMin/+ (Multiple intestinal neoplasia), develop intestinal adenomas. Several genetic modifier loci of Min (Mom) were previously mapped, but so far, most of the underlying genes have not been identified. To identify novel modifier loci associated with ApcMin/+, we performed quantitative trait loci (QTL) analysis for polyp development using 49 F1 crosses between different Collaborative Cross (CC) lines and C57BL/6 J-ApcMin/+mice. The CC population is a genetic reference panel of recombinant inbred lines, each line independently descended from eight genetically diverse founder strains. C57BL/6 J-ApcMin/+ males were mated with females from 49 CC lines. F1 offspring were terminated at 23 weeks and polyp counts from three sub-regions (SB1-3) of small intestinal and colon were recorded. RESULTS: The number of polyps in all these sub-regions and colon varied significantly between the different CC lines. At 95% genome-wide significance, we mapped nine novel QTL for variation in polyp number, with distinct QTL associated with each intestinal sub-region. QTL confidence intervals varied in width between 2.63-17.79 Mb. We extracted all genes in the mapped QTL at 90 and 95% CI levels using the BioInfoMiner online platform to extract, significantly enriched pathways and key linker genes, that act as regulatory and orchestrators of the phenotypic landscape associated with the ApcMin/+ mutation. CONCLUSIONS: Genomic structure of the CC lines has allowed us to identify novel modifiers and confirmed some of the previously mapped modifiers. Key genes involved mainly in metabolic and immunological processes were identified. Future steps in this analysis will be to identify regulatory elements - and possible epistatic effects - located in the mapped QTL.

The role of parotidectomy for advanced cutaneous squamous cell carcinoma of the head and neck.

PURPOSE: Regionally metastatic cutaneous squamous cell carcinoma of the head and neck (CSCCHN) is usually managed surgically; however, the role of parotidectomy remains controversial. Herein we elucidate the controversy and present our experience. METHODS: We retrospectively analyzed disease variables, extent of parotidectomy, and pathologic characteristics in association to outcome measures of all advanced CSCCHN patients who underwent definitive surgical resection from 2008 to 2018. RESULTS: Sixty-seven patients were enrolled, of whom 47 (70%) underwent parotidectomy; 27 superficial and 20 that included deep lobe resection. Parotidectomy had improved 5-year overall survival (OS) and disease-free survival (DFS) when neck was clinically involved (67.6% vs. 22.2%, P = 0.003 and 75.8% vs. 33.3% P = 0.002, respectively). Elective parotidectomy did not confer survival benefit for patients with no clinical involvement of the parotid gland (41.7% vs. 35%, P = 0.977). Recurrent disease was predictive for parotid metastases (P = 0.034). Thirty-nine patients received adjuvant radiotherapy, which significantly improved OS and DFS versus surgery alone (70.7% vs. 38.1%, P = 0.004 and 77.8% vs. 57.9%, P = 0.014, respectively). CONCLUSION: Parotidectomy was associated with improved survival of cervically spread CSCCHN.

Genetic analysis of intestinal polyp development in Collaborative Cross mice carrying the Apc (Min/+) mutation.

BACKGROUND: Colorectal cancer is an abnormal tissue development in the colon or rectum. Most of CRCs develop due to somatic mutations, while only a small proportion is caused by inherited mutations. Familial adenomatous polyposis is an inherited genetic disease, which is characterized by colorectal polyps. It is caused by inactivating mutations in the Adenomatous polyposis coli gene. Mice carrying and non-sense mutation in Adenomatous polyposis coli gene at site R850, which designated Apc (R850X/+) (Min), develop intestinal adenomas, while the bulk of the disease is in the small intestine. A number of genetic modifier loci of Min have been mapped, but so far most of the underlying genes have not been identified. In our previous studies, we have shown that Collaborative Cross mice are a powerful tool for mapping loci responsible for phenotypic variation. As a first step towards identification of novel modifiers of Min, we assessed the phenotypic variation between 27 F1 crosses between different Collaborative cross mice and C57BL/6-Min lines. RESULTS: Here, C57BL/6-Min male mice were mated with females from 27 Collaborative cross lines. F1 offspring were terminated at 23 weeks old and multiple phenotypes were collected: polyp counts, intestine length, intestine weight, packed cell volume and spleen weight. Additionally, in eight selected F1 Collaborative cross-C57BL/6-Min lines, body weight was monitored and compared to control mice carry wildtype Adenomatous polyposis coli gene. We found significant (p < 0.05) phenotypic variation between the 27 F1 Collaborative cross-C57BL/6-Min lines for all the tested phenotypes, and sex differences with traits; Colon, body weight and intestine length phenotypes, only. Heritability calculation showed that these phenotypes are mainly controlled by genetic factors. CONCLUSIONS: Variation in polyp development is controlled, an appreciable extent, by genetic factors segregating in the Collaborative cross population and suggests that it is suited for identifying modifier genes associated with Apc (Min/+) mutation, after assessing sufficient number of lines for quantitative trait loci analysis.

Heritability and coefficient of genetic variation analyses of phenotypic traits provide strong basis for high-resolution QTL mapping in the Collaborative Cross mouse genetic reference population.

Most biological traits of human importance are complex in nature; their manifestation controlled by the cumulative effect of many genetic factors interacting with one another and with the individual's life history. Because of this, mouse genetic reference populations (GRPs) consisting of collections of inbred lines or recombinant inbred lines (RIL) derived from crosses between inbred lines are of particular value in analysis of complex traits, since massive amounts of data can be accumulated on the individual lines. However, existing mouse GRPs are derived from inbred lines that share a common history, resulting in limited genetic diversity, and reduced mapping precision due to long-range gametic disequilibrium. To overcome these limitations, the Collaborative Cross (CC) a genetically highly diverse collection of mouse RIL was established. The CC, now in advanced stages of development, will eventually consist of about 500 RIL derived from reciprocal crosses of eight divergent founder strains of mice, including three wild subspecies. Previous studies have shown that the CC indeed contains enormous diversity at the DNA level, that founder haplotypes are inherited in expected frequency, and that long-range gametic disequilibrium is not present. We here present data, primarily from our own laboratory, documenting extensive genetic variation among CC lines as expressed in broad-sense heritability (H(2)) and by the well-known "coefficient of genetic variation," demonstrating the ability of the CC resource to provide unprecedented mapping precision leading to identification of strong candidate genes.

Pre-resection Intraoperative Core Biopsies in Oral Tongue Cancer-A Pilot Study.

OBJECTIVE: To assess a novel intraoperative core biopsy technique to provide enhanced guidance in partial glossectomies. METHODS: All patients diagnosed with squamous cell carcinoma of the oral tongue were eligible for study participation. Following anesthesia, the planned resection and three points midway between the gross tumor and the intended ablation were marked. A core biopsy was performed with a needle spring on each point and sent for frozen sections. The initially planned resection was executed if the cores returned free of tumor. In case of a positive core biopsy, a new 1-1.5 cm margin was marked around that point. The main outcome measure was the closest final margin diameter, especially the deep ones. Other outcome measures were the core biopsies' sensitivity, specificity, and negative predictive value. Complications were recorded. RESULTS: The final margins of 10 patients undergoing intraoperative core biopsies and 20 matched controls were analyzed. One patient had two positive cores and final negative margins after modifying the resection accordingly. Another patient had a positive biopsy diagnosed only on final pathology, and one close final margin. Patients that were operated with the new technique had larger margins compared to the controls: median (interquartile range) closest margin 5.95 (3.97; 9.63) mm versus 4 (2.25; 5) mm (p = 0.074) and median deep margin 8.6 (6.16; 10) mm versus 5 (3;10) mm (p = 0.411), respectively. There were no complications. CONCLUSION: A novel pre-resection intraoperative biopsy technique is presented. Core biopsies taken during glossectomies have the potential to prevent inadequate margins. LEVEL OF EVIDENCE: 3 Laryngoscope, 133:2232-2236, 2023.

Surgically Treated Advanced Cutaneous Squamous Cell Carcinoma of the Head and Neck: Outcome Predictors and the Role of Adjuvant Radiation Therapy.

OBJECTIVES: Advanced cutaneous squamous cell carcinoma of the head and neck (CSCCHN) is associated with poor outcome despite multimodality therapy. Comprehensive risk stratification may pinpoint the most suitable adjuvant treatment. This study aimed to evaluate the outcomes of surgically treated locoregional CSCCHN and to identify prognostic indicators of treatment outcomes. METHODS: We retrospectively analyzed disease variables, pathologic characteristics, and management in association with treatment outcomes of all consecutive advanced CSCCHN patients who underwent surgical resection at Tel Aviv Sourasky Medical Center. RESULTS: From 2008 to 2018, 74 patients met the inclusion criteria. Only perineural invasion (PNI) was significantly associated with worse overall survival (OS) (P = .001). Location within the facial "mask areas" was significantly associated with pathologically negative cervical disease (P = .001). Forty-seven patients underwent adjuvant radiation therapy (RT) which significantly improved OS and disease-free survival versus surgery alone (P = .025 and P = 0.035, respectively). CONCLUSION: PNI was associated with worse OS in surgically treated advanced CSCCHN. Adjuvant RT conferred better outcomes despite high risk features.

Safety and immunogenicity of a synthetic carbohydrate conjugate vaccine against Shigella flexneri 2a in healthy adult volunteers: a phase 1, dose-escalating, single-blind, randomised, placebo-controlled study.

BACKGROUND: Shigella remains in the top four pathogens responsible for moderate to severe diarrhoea in children below 5 years of age. The shigella O-specific polysaccharide (O-SP) is a promising vaccine target. We developed a conjugate vaccine prototype incorporating a unique well defined synthetic oligosaccharide hapten, chemically designed for optimal antigenic, conformational, structural, and functional mimicry of the O-SP from Shigella flexneri 2a (SF2a). We aimed to assess the safety, tolerability, and immunogenicity of this original synthetic oligosaccharide-based vaccine candidate, SF2a-TT15, conceived to drive the antibody response towards the key protective determinants of the native lipopolysaccharide antigen, in a first-in-human phase 1 study. METHODS: We did a first-in-human, dose-escalating, single-blind, observer-masked, randomised, placebo-controlled study at the Clinical Research Center of Tel Aviv Sourasky Medical Center (Israel). Participants were healthy adults aged 18-45 years with low titres of serum SF2a-specific IgG antibodies. 64 eligible participants were assigned to one of two cohorts. 32 participants in each of the two cohorts were randomly assigned via computer-generated algorithm in a stepwise manner to receive the 2 µg (cohort 1) and 10 µg oligosaccharide dose (cohort 2) of the SF2a-TT15 vaccine candidate non-adjuvanted or adjuvanted with aluminium hydroxide (alum) or matching placebos. The vaccine was administered as three single intramuscular injections into the arm, 28 days apart. The primary outcome was the incidence and severity of adverse events, which were assessed in the intention-to-treat safety population analysis including all participants who were randomly assigned and received at least one vaccine or placebo injection. The immunogenicity endpoints were secondary outcomes and were analysed in all participants who were randomly assigned, received all of the assigned injections before the time of the immunogenicity assessment, and provided blood samples for immunological follow-up (per-protocol immunogenicity analysis). The study is registered with ClinicalStudies.gov, NCT02797236 and is completed. FINDINGS: Of 203 volunteers initially screened, 64 participants were enrolled between Sept 20, 2016, and Sept 26, 2017. In each of the two cohorts, 12 participants received the adjuvanted vaccine, 12 received the non-adjuvanted vaccine and eight received the matching placebo (four each). The SF2a-TT15 glycoconjugate was well tolerated at both doses. No serious or severe adverse events occurred. Overall, seven (88%) of eight to 12 (100%) of 12 in each group of volunteers had one adverse event or more after receiving the study agents with the majority of adverse events, 300 (98%) of 307, considered mild in intensity. Of the seven adverse events defined as moderate in severity, one (nausea) was suspected to be related to the vaccine candidate. At all post-immunisation days and for both oligosaccharide doses, whether adjuvanted or not, SF2a-TT15 induced significantly higher serum IgG anti-SF2a lipopolysaccharide geometric mean titres (GMTs) as compared with baseline or with the corresponding GMTs in placebo recipients (p<0·01). After one injection, the non-adjuvanted 10 µg oligosaccharide dose induced a 27-times increase in IgG GMT (5080 vs 189) and the non-adjuvanted 2 µg oligosaccharide dose induced a five-times increase (1411 vs 283), compared with baseline. Alum enhanced the specific IgG response at 2 µg oligosaccharide dose after the third injection (GMTs 3200 vs 1176, p=0.045). INTERPRETATION: SF2a-TT15 was safe and well tolerated and induced high titres of anti-SF2a LPS IgG antibodies. These results support further evaluation of this original synthetic oligosaccharide-protein conjugate vaccine candidate for safety, immunogenicity, and protective efficacy in target populations. FUNDING: The European Union Seventh Framework Programme.

Single-Day Simulation-Based Training Improves Communication and Psychiatric Skills of Medical Students.

BACKGROUND: Use of standardized (or simulated) patients (SP) is considered an effective teaching method for improving clinical and communication skills. This study assesses the effect of a single-day simulated patients (SP)-based training course on medical students' communication and basic skills in clinical psychiatry during their psychiatry rotation in a university-affiliated tertiary medical center. METHODS: Forty-two third-year medical students participated. Communication and basic skills in clinical psychiatry were evaluated by a modified Four Habits Coding Scale (4HCS) and the psychiatric interview coding scale before and after SP training. An actual patient interview by the students 1 week after the training was evaluated by an attending psychiatrist blinded to the student's score during the SP-based training. Self-report questionnaires on satisfaction from the training and its impact on their self-confidence were administered at the end of training. FINDINGS: The mean pre-training 4HCS score of 33.9 increased to 52.3 post-training (p < 0.001). The mean psychiatric interview coding scale score increased from 4.33 to 5.36 (p = 0.002). The self-report questionnaire yielded a mean score of 4.21 on a 1-5 Likert scale, implying high levels of satisfaction and self-confidence. CONCLUSIONS: A single SP-based training course of medical students sufficed to improve clinical and communication skills in psychiatric settings and enhance their subjective perception of those skills.

Reducing Stigma Toward Psychiatry Among Medical Students: A Multicenter Controlled Trial.

OBJECTIVE: To examine the effect of a novel antistigma intervention curriculum (ASIC) in reducing stigma toward psychiatry among medical students. METHODS: Medical students from 8 hospitals in central Israel were divided into intervention (n = 57) and control (n = 163) arms. The students completed the 30-item Attitudes Toward Psychiatry (ATP-30) and the Attitudes Toward Mental Illness (AMI) scales at psychiatry rotation onset and conclusion. The ASIC was designed to target prejudices and stigma through direct informal encounters with people with serious mental illness (SMI) during periods of remission and recovery. Supervised small-group discussions followed those encounters to facilitate processing of thoughts and emotions that ensued and to discuss salient topics in psychiatry. The study was conducted between November 2017 and July 2018. RESULTS: Significant between-group differences were found at endpoint for attitudes toward psychiatry and psychiatric patients (P < .001). Although changing attitudes toward psychiatry as a career choice was not part of the ASIC, a significant between-group difference emerged by endpoint (P < .001). CONCLUSIONS: Implementation of an ASIC that includes contact with individuals with lived SMI experience followed by supervised small-group discussions is effective in reducing stigma in medical students' perceptions of people with mental illness and psychiatry. Further evaluation is warranted with regard to the long-term destigmatizing effects of an ASIC. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT03907696.

Collaborative Cross Mice Yield Genetic Modifiers for Pseudomonas aeruginosa Infection in Human Lung Disease.

Human genetics influence a range of pathological and clinical phenotypes in respiratory infections; however, the contributions of disease modifiers remain underappreciated. We exploited the Collaborative Cross (CC) mouse genetic-reference population to map genetic modifiers that affect the severity of Pseudomonas aeruginosa lung infection. Screening for P. aeruginosa respiratory infection in a cohort of 39 CC lines exhibits distinct disease phenotypes ranging from complete resistance to lethal disease. Based on major changes in the survival times, a quantitative-trait locus (QTL) was mapped on murine chromosome 3 to the genomic interval of Mb 110.4 to 120.5. Within this locus, composed of 31 protein-coding genes, two candidate genes, namely, dihydropyrimidine dehydrogenase (Dpyd) and sphingosine-1-phosphate receptor 1 (S1pr1), were identified according to the level of genome-wide significance and disease gene prioritization. Functional validation of the S1pr1 gene by pharmacological targeting in C57BL/6NCrl mice confirmed its relevance in P. aeruginosa pathophysiology. However, in a cohort of Canadian patients with cystic fibrosis (CF) disease, regional genetic-association analysis of the syntenic human locus on chromosome 1 (Mb 97.0 to 105.0) identified two single-nucleotide polymorphisms (rs10875080 and rs11582736) annotated to the Dpyd gene that were significantly associated with age at first P. aeruginosa infection. Thus, there is evidence that both genes might be implicated in this disease. Our results demonstrate that the discovery of murine modifier loci may generate information that is relevant to human disease progression.IMPORTANCE Respiratory infection caused by P. aeruginosa is one of the most critical health burdens worldwide. People affected by P. aeruginosa infection include patients with a weakened immune system, such as those with cystic fibrosis (CF) genetic disease or non-CF bronchiectasis. Disease outcomes range from fatal pneumonia to chronic life-threatening infection and inflammation leading to the progressive deterioration of pulmonary function. The development of these respiratory infections is mediated by multiple causes. However, the genetic factors underlying infection susceptibility are poorly known and difficult to predict. Our study employed novel approaches and improved mouse disease models to identify genetic modifiers that affect the severity of P. aeruginosa lung infection. We identified candidate genes to enhance our understanding of P. aeruginosa infection in humans and provide a proof of concept that could be exploited for other human pathologies mediated by bacterial infection.

Dissecting the Effect of Genetic Variation on the Hepatic Expression of Drug Disposition Genes across the Collaborative Cross Mouse Strains.

A central challenge in pharmaceutical research is to investigate genetic variation in response to drugs. The Collaborative Cross (CC) mouse reference population is a promising model for pharmacogenomic studies because of its large amount of genetic variation, genetic reproducibility, and dense recombination sites. While the CC lines are phenotypically diverse, their genetic diversity in drug disposition processes, such as detoxification reactions, is still largely uncharacterized. Here we systematically measured RNA-sequencing expression profiles from livers of 29 CC lines under baseline conditions. We then leveraged a reference collection of metabolic biotransformation pathways to map potential relations between drugs and their underlying expression quantitative trait loci (eQTLs). By applying this approach on proximal eQTLs, including eQTLs acting on the overall expression of genes and on the expression of particular transcript isoforms, we were able to construct the organization of hepatic eQTL-drug connectivity across the CC population. The analysis revealed a substantial impact of genetic variation acting on drug biotransformation, allowed mapping of potential joint genetic effects in the context of individual drugs, and demonstrated crosstalk between drug metabolism and lipid metabolism. Our findings provide a resource for investigating drug disposition in the CC strains, and offer a new paradigm for integrating biotransformation reactions to corresponding variations in DNA sequences.