Insights into the role of JAK2-I724T variant in myeloproliferative neoplasms from a unique cohort of New Zealand patients.

OBJECTIVES: This study aimed to compile bioinformatic and experimental information for JAK2 missense variants previously reported in myeloproliferative neoplasms (MPN) and determine if germline JAK2-I724T, recently found to be common in New Zealand Polynesians, associates with MPN. METHODS: For all JAK2 variants found in the literature, gnomAD_exome allele frequencies were extracted and REVEL scores were calculated using the dbNSFP database. We investigated the prevalence of JAK2-I724T in a cohort of 111 New Zealand MPN patients using a TaqMan assay, examined its allelic co-occurrence with JAK2-V617F using Oxford Nanopore sequencing, and modelled the impact of I724T on JAK2 using I-Mutant and ChimeraX software. RESULTS: Several non-V617F JAK2 variants previously reported in MPN had REVEL scores greater than 0.5, suggesting pathogenicity. JAK2-I724T (REVEL score 0.753) was more common in New Zealand Polynesian MPN patients (n = 2/27; 7.4%) than in other New Zealand patients (n = 0/84; 0%) but less common than expected for healthy Polynesians (n = 56/377; 14.9%). Patients carrying I724T (n = 2), one with polycythaemia vera and one with essential thrombocythaemia, had high-risk MPN. Both patients with JAK2-I724T were also positive for JAK2-V617F, found on the same allele as I724T, as well as separately. In silico modelling did not identify noticeable structural changes that would give JAK2-I724T a gain-of-function. CONCLUSION: Several non-canonical JAK2 variants with high REVEL scores have been reported in MPN, highlighting the need to further understand their relationship with disease. The JAK2-I724T variant does not drive MPN, but additional investigations are required to exclude any potential modulatory effect on the MPN phenotype.

Impact of More Detailed Measures of Disease Severity on Racial Disparities in Cardiac Surgery Mortality among Native Hawaiians and Pacific Islanders.

Studies that examine racial disparities in health outcomes often include analyses that account or adjust for baseline differences in co-morbid conditions. Often, these conditions are defined as dichotomous (Yes/No) variables, and few analyses include clinical and/or laboratory data that could allow for more nuanced estimates of disease severity. However, disease severity - not just prevalence - can differ substantially by race and is an underappreciated mechanism for health disparities. Thus, relying on dichotomous disease indicators may not fully describe health disparities. This study explores the effect of substituting continuous clinical and/or laboratory data for dichotomous disease indicators on racial disparities, using data from the Queen's Medical Center's (QMC) cardiac surgery database (a subset of the national Society of Thoracic Surgeon's cardiothoracic surgery database) as an example case. Two logistic regression models predicting in-hospital mortality were constructed: (I) a baseline model including race and dichotomous (Yes/No) indicators of disease (diabetes, heart failure, liver disease, kidney disease), and (II) a more detailed model with continuous laboratory values in place of the dichotomous indicators (eg, including Hemoglobin A1c level rather than just diabetes yes/no). When only dichotomous disease indicators were used in the model, Native Hawaiian and other Pacific Islander (NHPI) race was significantly associated with in-hospital mortality (OR: 1.57[1.29,2.47], P=.04). Yet when the more specific laboratory values were included, NHPI race was no longer associated with in-hospital mortality (OR: 1.67[0.92,2.28], P=.28). Thus, researchers should be thoughtful in their choice of independent variables and understand the potential impact of how clinical measures are operationalized in their research.

Clonal dynamics limits detection of selection in tumour xenograft CRISPR/Cas9 screens.

Transplantable in vivo CRISPR/Cas9 knockout screens, in which cells are edited in vitro and inoculated into mice to form tumours, allow evaluation of gene function in a cancer model that incorporates the multicellular interactions of the tumour microenvironment. To improve our understanding of the key parameters for success with this method, we investigated the choice of cell line, mouse host, tumour harvesting timepoint and guide RNA (gRNA) library size. We found that high gRNA (80-95%) representation was maintained in a HCT116 subline transduced with the GeCKOv2 whole-genome gRNA library and transplanted into NSG mice when tumours were harvested at early (14 d) but not late time points (38-43 d). The decreased representation in older tumours was accompanied by large increases in variance in gRNA read counts, with notable expansion of a small number of random clones in each sample. The variable clonal dynamics resulted in a high level of 'noise' that limited the detection of gRNA-based selection. Using simulated datasets derived from our experimental data, we show that considerable reductions in count variance would be achieved with smaller library sizes. Based on our findings, we suggest a pathway to rationally design adequately powered in vivo CRISPR screens for successful evaluation of gene function.

Valve-sparing operations after Ross procedure: a single-center experience.

Background: The Ross procedure has demonstrated excellent long-term results, with restoration of life-expectancy in patients with severe aortic valve dysfunction. However, reintervention after Ross can occur, and herein we describe our center's experience with redo surgery after previous Ross procedures. Methods: We searched our prospective database for aortic valve-repair and recruited all adult (≥18 years) patients who have undergone valve-sparing root replacements (VSRRs) and/or aortic valve-repair after Ross procedure between July 2001 and July 2022. Univariable logistic regression analysis was performed to identify variables affecting early mortality. Survival, freedom-from-valve-reintervention and freedom-from-aortic regurgitation (AR) grade ≥3 were analyzed with the Kaplan-Meier method. Results: A total of 63 patients were recruited for this study. Indication for reoperation after Ross was aortic aneurysm without AR in 17 (27%), aortic aneurysm with AR in 27 (43%), and isolated AR in 19 (30%) patients. Median follow-up time was 7.82 years. The majority of patients (76%) had undergone the free root technique during their index Ross operation. Cumulative survival, after redo surgery following Ross, was 98.4% [95% confidence interval (CI): 89.3-99.8%] at 1 year, 96.3% (95% CI: 88.2-98.3%) at 5 years, and 92.4% (95% CI: 87.1-98.0%) at 10 years. Freedom-from-reoperation on the aortic valve at 1 year was 98.4% (95% CI: 97.0-99.8%), at 5 years was 96.7% (95% CI: 87.6-99.0%), and 79.7% (95% CI: 71.1-88.3%) at 10 years. Conclusions: Long-term survival after redo surgery following the Ross operation is excellent. The data support our aggressive valve-sparing approach after Ross.

Functional and pathomorphological anatomy of the aortic valve and root for aortic valve sparing surgery in tricuspid and bicuspid aortic valves.

The aortic valve (AV) is a three-dimensional structure, with leaflets that are suspended within the functional aortic annulus (FAA). These structures (AV and FAA) are therefore intrinsically connected and disease of just one component can independently lead to AV dysfunction. Hence, AV dysfunction can occur in the setting of entirely normal valve leaflets. However, as these structures are functionally inter-connected, disease of one component can lead to abnormalities of the other over time. Thus, AV dysfunction is often multifactorial. Valve-sparing root procedures require an in-depth understanding of these inter-relationships, and herein we are providing a detailed account of some of the most pertinent anatomical relationships.

Three decades of reimplantation of the aortic valve-the Brussels experience.

Background: Over the last three decades, the importance of native valve preservation has increasingly become evident. Valve-sparing root replacement procedures, such as the reimplantation or remodeling technique, are therefore being progressively used for aortic root replacement and/or aortic valve repair. Herein, we are summarizing our single-center experience with the reimplantation technique. Methods: We queried our prospective database for aortic valve repair and recruited all adult (≥18 years) patients who have undergone valve-sparing root replacement with the reimplantation technique between March 1998 and January 2022. We subcategorized the patients into three distinct groups: root aneurysm without aortic regurgitation (AR) (grade ≤1+), root aneurysm with AR (grade >1+) and isolated chronic AR (root <45 mm). Univariable logistic regression analysis was performed to identify variables of interest, which were further analyzed by multivariable Cox-regression analysis. Survival, freedom from valve reintervention, and freedom from recurrent regurgitation, were analyzed with the Kaplan-Meier method. Results: A total of 652 patients were recruited for this study; 213 patients underwent reimplantation for aortic aneurysm without AR, 289 patients for aortic aneurysm with AR, and 150 patients with isolated AR. Cumulative survival was 95.4% (95% CI: 92.9-97.0%) after 5 years, 84.8% (80.0-88.5%) after 10 years, and 79.5% (73.3-84.5%) after 12 years, which was comparable to the age-matched Belgian population. Older age (HR 1.06, P≤0.001) and male gender (HR 2.1, P=0.02) were associated with late mortality. Freedom from reoperation on the aortic valve at 5 years was 96.2% (95% CI: 93.8-97.7%), and 90.4% (95% CI: 87.4-94.2%) at 12 years. Age (P=0.001) and preoperative left ventricular end-diastolic dimension (LVEDD) (P=0.03) were associated with late reoperation. Conclusions: Our long-term data supports our reimplantation approach as a viable option for aortic root aneurysms and/or aortic regurgitation, with long-term survival that mirrors that of the general population.

Complex Patterns of Genomic Heterogeneity Identified in 42 Tumor Samples and ctDNA of a Pulmonary Atypical Carcinoid Patient.

Tumor evolution underlies many challenges facing precision oncology, and improving our understanding has the potential to improve clinical care. This study represents a rare opportunity to study tumor heterogeneity and evolution in a patient with an understudied cancer type. A patient with pulmonary atypical carcinoid, a neuroendocrine tumor, metastatic to 90 sites, requested and consented to donate tissues for research. 42 tumor samples collected at rapid autopsy from 14 anatomically distinct sites were analyzed through DNA whole-exome sequencing and RNA sequencing, and five analyzed through linked-read sequencing. Targeted DNA sequencing was completed on two clinical tissue biopsies and one blood plasma sample. Chromosomal alterations and gene variants accumulated over time, and specific chromosomal alterations preceded the single predicted gene driver variant (ARID1A). At the time of autopsy, all sites shared the gain of one copy of Chr 5, loss of one copy of Chr 6 and 21, chromothripsis of one copy of Chr 11, and 39 small variants. Two tumor clones (carrying additional variants) were detected at metastatic sites, and occasionally in different regions of the same organ (e.g., within the pancreas). Circulating tumor DNA (ctDNA) sequencing detected shared tumor variants in the blood plasma and captured marked genomic heterogeneity, including all metastatic clones but few private tumor variants. This study describes genomic tumor evolution and dissemination of a pulmonary atypical carcinoid donated by a single generous patient. It highlights the critical role of chromosomal alterations in tumor initiation and explores the potential of ctDNA analysis to represent genomically heterogeneous disease. Significance: DNA sequencing data from tumor samples and blood plasma from a single patient highlighted the critical early role of chromosomal alterations in atypical carcinoid tumor development. Common tumor variants were readily detected in the blood plasma, unlike emerging tumor variants, which has implications for using ctDNA to capture cancer evolution.

Chromosomal Aberrations Accumulate during Metastasis of Virus-Negative Merkel Cell Carcinoma.

Merkel cell carcinoma is a rare, aggressive skin tumor initiated by polyomavirus integration or UV light DNA damage. In New Zealand, there is a propensity toward the UV-driven form (31 of 107, 29% virus positive). Using archival formalin-fixed, paraffin-embedded tissues, we report targeted DNA sequencing covering 246 cancer genes on 71 tumor tissues and 38 nonmalignant tissues from 37 individuals, with 33 of 37 being negative for the virus. Somatic variants of New Zealand virus-negative Merkel cell carcinomas partially overlapped with those reported overseas, including TP53 variants in all tumors and RB1, LRP1B, NOTCH1, and EPHA3/7 variants each found in over half of the cohort. Variants in genes not analyzed or reported in previous studies were also found. Cataloging variants in TP53 and RB1 from published datasets revealed a broad distribution across these genes. Chr 1p gain and Chr 3p loss were identified in around 50% of New Zealand virus-negative Merkel cell carcinomas, and RB1 loss of heterozygosity was found in 90% of cases. Copy number variants accumulate in most metastases. Virus-negative Merkel cell carcinomas have complex combinations of somatic DNA-sequence variants and copy number variants. They likely carry the small genomic changes permissive for metastasis from early tumor development; however, chromosomal alterations may contribute to driving metastatic progression.

A Case of Multivessel Coronary Artery Disease and Anomalous Origin of the Right Coronary Artery With a Malignant Course Presenting With Non-exertional Chest Discomfort and Brugada Phenocopy.

Coronary artery anomalies (CAAs) are an uncommon cause of chest pain in the younger population. Misdiagnosis can be detrimental and lead to sudden cardiac deaths. We present a 62-year-old male with a past medical history significant for chest pain history with a workup in 2001 presumed to be non-cardiac in origin from bronchial asthma. He presented from a Micronesian Island for the evaluation of non-exertional chest discomfort. Further workup showed a Brugada type I pattern on ECG and ST wave depressions on anterolateral and inferior leads with associated AVR elevation on exercise stress testing. Further ischemic workup with coronary angiography revealed right dominant circulation with three-vessel coronary artery disease (CAD), including mid-left anterior descending (LAD) artery chronic total occlusion (CTO) with the right to left collaterals, left circumflex, and right coronary artery (RCA) with the accompanied anomalous origin of RCA. The patient underwent surgical correction of the anomalous RCA and coronary artery bypass grafting for the multi-vessel CAD. CAAs are usually found incidentally during ischemic workups similar to this case. Patients with CAAs can be managed conservatively with caution regarding physical activity. However, high-risk patients will warrant surgical treatment to avoid sudden cardiac death. The diagnosis of CAAs can be challenging and prone to misdiagnosis and maltreatment. It may be beneficial to pursue this in younger patients with ischemia-like symptoms. Further studies should be performed to identify the true incidence and guide medical practitioners regarding the risks, costs, and benefits of diagnosing and surgically treating CAAs in the general population.

Innate immune checkpoint inhibitor resistance is associated with melanoma sub-types exhibiting invasive and de-differentiated gene expression signatures.

Melanoma is a highly aggressive skin cancer, which, although highly immunogenic, frequently escapes the body's immune defences. Immune checkpoint inhibitors (ICI), such as anti-PD1, anti-PDL1, and anti-CTLA4 antibodies lead to reactivation of immune pathways, promoting rejection of melanoma. However, the benefits of ICI therapy remain limited to a relatively small proportion of patients who do not exhibit ICI resistance. Moreover, the precise mechanisms underlying innate and acquired ICI resistance remain unclear. Here, we have investigated differences in melanoma tissues in responder and non-responder patients to anti-PD1 therapy in terms of tumour and immune cell gene-associated signatures. We performed multi-omics investigations on melanoma tumour tissues, which were collected from patients before starting treatment with anti-PD1 immune checkpoint inhibitors. Patients were subsequently categorized into responders and non-responders to anti-PD1 therapy based on RECIST criteria. Multi-omics analyses included RNA-Seq and NanoString analysis. From RNA-Seq data we carried out HLA phenotyping as well as gene enrichment analysis, pathway enrichment analysis and immune cell deconvolution studies. Consistent with previous studies, our data showed that responders to anti-PD1 therapy had higher immune scores (median immune score for responders = 0.1335, median immune score for non-responders = 0.05426, p-value = 0.01, Mann-Whitney U two-tailed exact test) compared to the non-responders. Responder melanomas were more highly enriched with a combination of CD8+ T cells, dendritic cells (p-value = 0.03) and an M1 subtype of macrophages (p-value = 0.001). In addition, melanomas from responder patients exhibited a more differentiated gene expression pattern, with high proliferative- and low invasive-associated gene expression signatures, whereas tumours from non-responders exhibited high invasive- and frequently neural crest-like cell type gene expression signatures. Our findings suggest that non-responder melanomas to anti-PD1 therapy exhibit a de-differentiated gene expression signature, associated with poorer immune cell infiltration, which establishes a gene expression pattern characteristic of innate resistance to anti-PD1 therapy. Improved understanding of tumour-intrinsic gene expression patterns associated with response to anti-PD1 therapy will help to identify predictive biomarkers of ICI response and may help to identify new targets for anticancer treatment, especially with a capacity to function as adjuvants to improve ICI outcomes.

The Utility of Acid-Fast Bacillus (AFB) and Fungal Cultures in Orthopaedic Infections.

Introduction When diagnosing suspected orthopaedic-related infections, fungal and acid-fast bacilli (AFB) cultures are often obtained intraoperatively. These cultures are difficult and time-consuming to grow and increase healthcare costs. This study aimed to quantify the rate of positive AFB and fungal cultures in orthopaedic infections and to compare potential risk factors for a positive result.  Methods Orthopaedic surgical cases for suspected infection at one institution from March 2013 through December 2019 were included. Data were collected on patient demographics and procedure characteristics for patients with surgical AFB or fungal lab tests ordered on the day of surgery. Results Of the 813 patients for whom intraoperative AFB or fungal cultures were ordered, 3.8% (N=31) had a positive result. Of the 31 positive results, 30 were from fungal cultures and one was from AFB cultures. Patients with a positive versus negative culture result did not differ significantly by age, sex, American Society of Anesthesiologists (ASA) score, diabetes, obesity, or HIV/AIDS. In both unadjusted and adjusted analyses, peripheral vascular disease (PVD) was associated with higher odds of a positive fungal culture result (adjusted OR (aOR)=3.5, 95%CI=1.3-8.4). Likewise, in both unadjusted and adjusted models, a hand/foot operating region was associated with higher odds of a positive fungal culture result compared with all other regions (aOR=4.2, 95%CI=1.9-9.8). Conclusion Intraoperative fungal and AFB cultures may not need to be obtained except in orthopaedic surgical cases for hand or foot infections or in patients with PVD.

A Critical Period for Development of Cerebellar-Mediated Autism-Relevant Social Behavior.

The cerebellum has been increasingly implicated in autism spectrum disorder (ASD) with many ASD-linked genes impacting both cerebellar function and development. However, the precise timing and critical periods of when abnormal cerebellar neurodevelopment contributes to ASD-relevant behaviors remains poorly understood. In this study, we identify a critical period for the development of ASD-relevant behaviors in a cerebellar male mouse model of tuberous sclerosis complex (TSC), by using the mechanistic target of rapamycin (mTOR) inhibitor, rapamycin, to pharmacologically inhibit dysregulated downstream signaling. We find independent critical periods during which abnormal ASD-relevant behaviors develop for the two core ASD diagnostic criteria, social impairments and behavioral flexibility, and delineate an anatomic, physiological, and behavioral framework. These findings not only further our understanding of the genetic mechanisms underlying the timing of ASD-relevant behaviors but also have the capacity to inform potential therapies to optimize treatment interventions.SIGNIFICANCE STATEMENT No targeted treatments currently exist for autism spectrum disorder (ASD). This complex developmental disorder has established links to genetic and circuit aberrations, yet the precise timing and coordination of these underlying mechanisms that contribute to the spectrum of physiological and behavioral abnormalities remains unclear. Cerebellar pathology is consistently seen in ASD individuals; therefore, we sought to identify the specific windows for cerebellar involvement in the development of ASD-relevant behaviors. Using pharmacologic treatment paradigms, we outline distinct critical periods of developmental vulnerability for ASD-relevant social and inflexible behaviors. From this study, we posit a refined window of time during which ASD symptoms develop that will inform therapeutic timing.

Outcomes of Anatomic Lung Resection for Cancer Are Better When Performed by Cardiothoracic Surgeons.

BACKGROUND: Anatomic lung resection (ALR) outcomes are superior for cardiothoracic surgeons (CTSs) by analysis of Medicare; National Inpatient Sample; South Carolina Office of Research and Statistics; and Surveillance, Epidemiology, and End Results databases. Similar findings have been reported for all noncardiac thoracic procedures using the American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) database. Our aim was to further delineate outcome differences between CTSs and general surgeons (GSs) specifically for ALR. METHODS: A retrospective analysis of 15,574 nonemergent, nonpediatric ALR for lung cancer was conducted using the ACS-NSQIP 2013 to 2017 database. Included procedures were all ALR for lung cancer. Surgeons were classified as CTSs or GSs. Other specialties were excluded. Preoperative characteristics and 30-day outcomes were compared by bivariate (chi-square test) and multivariate analysis. Multivariate analysis was conducted by multiple logistic regression. RESULTS: CTSs performed 14,172 (91.0%) of included procedures, and GSs performed 1402 (9.0%). A thoracoscopic approach was utilized at a similar rate (49.08% for CTSs vs 49.71% for GSs; P = .747). The extent of resection differed in a statistically, but not clinically, significant fashion. CTS patients had a higher rate of preoperative dyspnea (22.66% for CTSs vs 17.62% for GSs; P < .001). Procedures performed by CTSs had a lower risk-adjusted odds ratio of overall morbidity, pulmonary morbidity, sepsis or septic shock, bleeding requiring transfusion, and length of stay greater than the median (5 days). CONCLUSIONS: ALR outcomes are superior for CTSs when compared with GSs. This is consistent with prior studies looking at this specific subset of patients and studies looking at a different subset of patients using the ACS-NSQIP database.

Genomic and signalling pathway characterization of the NZM panel of melanoma cell lines: A valuable model for studying the impact of genetic diversity in melanoma.

Melanoma is a disease associated with a very high mutation burden and thus the possibility of a diverse range of oncogenic mechanisms that allow it to evade therapeutic interventions and the immune system. Here, we describe the characterization of a panel of 102 cell lines from metastatic melanomas (the NZM lines), including using whole-exome and RNA sequencing to analyse genetic variants and gene expression changes in a subset of this panel. Lines possessing all major melanoma genotypes were identified, and hierarchical clustering of gene expression profiles revealed four broad subgroups of cell lines. Immunogenotyping identified a range of HLA haplotypes as well as expression of neoantigens and cancer-testis antigens in the lines. Together, these characteristics make the NZM panel a valuable resource for cell-based, immunological and xenograft studies to better understand the diversity of melanoma biology and the responses of melanoma to therapeutic interventions.

Accessing a New Dimension in TP53 Biology: Multiplex Long Amplicon Digital PCR to Specifically Detect and Quantitate Individual TP53 Transcripts.

TP53, the most commonly-mutated gene in cancer, undergoes complex alternative splicing. Different TP53 transcripts play different biological roles, both in normal function and in the progression of diseases such as cancer. The study of TP53's alternative RNA splice forms and their use as clinical biomarkers has been hampered by limited specificity and quantitative accuracy of current methods. TP53 RNA splice variants differ at both 5' and 3' ends, but because they have a common central region of 618 bp, the individual TP53 transcripts are impossible to specifically detect and precisely quantitate using standard PCR-based methods or short-read RNA sequencing. Therefore, we devised multiplex probe-based long amplicon droplet digital PCR (ddPCR) assays, which for the first time allow precise end-to-end quantitation of the seven major TP53 transcripts, with amplicons ranging from 0.85 to 1.85 kb. Multiple modifications to standard ddPCR assay procedures were required to enable specific co-amplification of these long transcripts and to overcome issues with secondary structure. Using these assays, we show that several TP53 transcripts are co-expressed in breast cancers, and illustrate the potential for this method to identify novel TP53 transcripts in tumour cells. This capability will facilitate a new level of biological and clinical understanding of the alternatively-spliced TP53 isoforms.