Neurofilament Light Chain as a Biomarker of Global Cognition in Individuals With Possible Vascular Mild Cognitive Impairment.

BACKGROUND: Neurofilament Light Chain (NfL) is a biomarker of axonal injury elevated in mild cognitive impairment (MCI) and Alzheimer's disease dementia. Blood NfL also inversely correlates with cognitive performance in those conditions. However, few studies have assessed NfL as a biomarker of global cognition in individuals demonstrating mild cognitive deficits who are at risk for vascular-related cognitive decline. OBJECTIVE: To assess the relationship between blood NfL and global cognition in individuals with possible vascular MCI (vMCI) throughout cardiac rehabilitation (CR). Additionally, NfL levels were compared to age/sex-matched cognitively unimpaired (CU) controls. METHOD: Participants with coronary artery disease (vMCI or CU) were recruited at entry to a 24-week CR program. Global cognition was measured using the Montreal Cognitive Assessment (MoCA) and plasma NfL level (pg/ml) was quantified using a highly sensitive enzyme-linked immunosorbent assay. RESULTS: Higher plasma NfL was correlated with worse MoCA scores at baseline (ß = -.352, P = .029) in 43 individuals with vMCI after adjusting for age, sex, and education. An increase in NfL was associated with worse global cognition (b[SE] = -4.81[2.06], P = .023) over time, however baseline NfL did not predict a decline in global cognition. NfL levels did not differ between the vMCI (n = 39) and CU (n = 39) groups (F(1, 76) = 1.37, P = .245). CONCLUSION: Plasma NfL correlates with global cognition at baseline in individuals with vMCI, and is associated with decline in global cognition during CR. Our findings increase understanding of NfL and neurobiological mechanisms associated with cognitive decline in vMCI.

Host Expression Profiling From Diagnostic Coronavirus Disease 2019 Swabs Associates Upper Respiratory Tract Immune Responses With Radiologic Lung Pathology and Clinical Severity.

Background: COVID-19 presents with a breadth of symptomatology including a spectrum of clinical severity requiring intensive care unit (ICU) admission. We investigated the mucosal host gene response at the time of gold standard COVID-19 diagnosis using clinical surplus RNA from upper respiratory tract swabs. Methods: Host response was evaluated by RNA-sequencing, and transcriptomic profiles of 44 unvaccinated patients including outpatients and in-patients with varying levels of oxygen supplementation were included. Additionally, chest X-rays were reviewed and scored for patients in each group. Results: Host transcriptomics revealed significant changes in the immune and inflammatory response. Patients destined for the ICU were distinguished by the significant upregulation of immune response pathways and inflammatory chemokines, including cxcl2 which has been linked to monocyte subsets associated with COVID-19 related lung damage. In order to temporally associate gene expression profiles in the upper respiratory tract at diagnosis of COVID-19 with lower respiratory tract sequalae, we correlated our findings with chest radiography scoring, showing nasopharygeal or mid-turbinate sampling can be a relevant surrogate for downstream COVID-19 pneumonia/ICU severity. Conclusions: This study demonstrates the potential and relevance for ongoing study of the mucosal site of infection of SARS-CoV-2 using a single sampling that remains standard of care in hospital settings. We highlight also the archival value of high quality clinical surplus specimens, especially with rapidly evolving COVID-19 variants and changing public health/vaccination measures.

Genomic profiling of dedifferentiated endometrial carcinomas arising in the background of high-grade carcinoma: a targeted next-generation sequencing study.

AIMS: Our understanding of dedifferentiated endometrial carcinoma (DEC), a rare and aggressive malignancy, mainly reflects undifferentiated carcinomas (UC) arising in the setting of low-grade endometrial cancer (DEC-LG). However, cases of UC arising in the setting of high-grade EC (DEC-HG) have been noted in the literature. Our knowledge of the genomics of DEC-HG is limited. To characterise the molecular landscape of DEC-HC, targeted genomic sequencing and immunohistochemical analysis was carried out on seven DEC-HG and four DEC-LG. METHODS AND RESULTS: DEC-HG and DEC-LG, including undifferentiated and differentiated components, both showed a similar frequency and spectrum of mutations. ARID1A mutations were identified in 6/7 (86%) DEC-HG and 4/4 (100%) DEC-LG, while SMARCA4 mutations were present in 4/7 (57%) DEC-HG and in 1/4 (25%) DEC-LG. Concurrent SMARCA4/BRG1 protein loss by immunohistochemistry was observed in 3/4 and 1/1 SMARCA4 mutated DEC-HG and DEC-LG, respectively. Neither genomic alterations nor protein loss in SMARCB1/INI1 were observed in any of our cases. TP53 mutations were detected in 4/7 (57%) DEC-HG and in 2/4 (50%) DEC-LG, while mutation-pattern p53 immunohistochemistry expression was observed in 2/7 (29%) DEC-HG and none of the DEC-LG. MLH1 mutations were observed in 1/7 (14%) DEC-HG and 1/4 (25%) DEC-LG. MSH2 and MSH6 mutations were each detected in 1/7 (14%) DEC-HG, but neither was associated with corresponding loss of protein expression. CONCLUSION: The findings support expanding the definition of DEC to include DEC-HG, a previously under-recognised phenomenon with genomic similarities to DEC-LG.

Mesonephric-like adenocarcinoma of the female genital tract: novel observations and detailed molecular characterisation of mixed tumours and mesonephric-like carcinosarcomas.

AIMS: To report novel observations in five mesonephric-like adenocarcinomas (MLAs) of the female genital tract. METHODS AND RESULTS: We report two endometrial MLAs in association with endometrioid carcinoma and atypical hyperplasia and three (one endometrial, two ovarian) cases with a sarcomatoid component (mesonephric-like carcinosarcoma). Pathogenic KRAS mutations, which are characteristic of MLA, were identified in all cases although interestingly, in one of the mixed carcinomas, this was confined to the endometrioid component. The concurrent MLA, endometrioid carcinoma and atypical hyperplasia components in one case harboured identical EGFR, PTEN and CCNE1 mutations, suggesting that the atypical hyperplasia gave rise to a Müllerian carcinoma with both endometrioid and mesonephric-like components. The carcinosarcomas all contained a component of MLA and a sarcomatous component with chondroid elements. In the ovarian carcinosarcomas, the coexisting epithelial and sarcomatous components shared some mutations including KRAS and CREBBP, suggesting that they are clonally related. Furthermore, in one case CREBBP and KRAS mutations detected in the MLA and sarcomatous components were also detected in an associated undifferentiated carcinoma component, suggesting that it was clonally related to the MLA and sarcomatous components. CONCLUSIONS: Our observations provide additional evidence that MLAs have a Müllerian origin and characterise mesonephric-like carcinosarcomas in which chondroid elements appear to be characteristic. In reporting these findings, we provide recommendations for distinction between a mesonephric-like carcinosarcoma and a MLA with a spindle cell component.

PAXgene Fixation for Pancreatic Cancer: Implications for Molecular and Surgical Pathology.

Genomic profiling of pancreatic cancer using small core biopsies has taken an increasingly prominent role in precision medicine. However, if not appropriately preserved, nucleic acids (NA) from pancreatic tissues are known to be susceptible to degradation due to high intrinsic levels of nucleases. PAXgene fixation (PreAnalytix, Switzerland) represents a novel formalin-free tissue preservation method. We sought to compare the NA and histomorphological preservation of pancreatic cancer tissues preserved with PAXgene-fixed paraffin-embedding (PFPE) and formalin-fixed paraffin-embedding (FFPE). Tissues from 19 patients were obtained prospectively from pancreaticoduodenectomy specimens and evaluated by four gastrointestinal pathologists. The extracted NA were quantified by Nanodrop and Qubit and assessed for quality by qPCR, targeted next-generation sequencing (NGS) assay, and RNA-sequencing. Our results demonstrated that, when assessed blindly for morphological quality, the four pathologists deemed the PFPE slides adequate for diagnostic purposes. PFPE tissues enable greater yields of less fragmented and more amplifiable DNA. PFPE tissues demonstrated significantly improved quality control (QC) metrics in a targeted NGS assay including Median Absolute Pair-wise Difference (MAPD) scores. Our results support the use of PAXgene fixative for the processing of specimens from pancreatic cancers with the potential benefits of improved yields for more amplifiable DNA in low-yield biopsy specimens and its ideal use for amplicon-based NGS assays.

Multisite verification of the accuracy of a multi-gene next generation sequencing panel for detection of mutations and copy number alterations in solid tumours.

Molecular variants including single nucleotide variants (SNVs), copy number variants (CNVs) and fusions can be detected in the clinical setting using deep targeted sequencing. These assays support low limits of detection using little genomic input material. They are gaining in popularity in clinical laboratories, where sample volumes are limited, and low variant allele fractions may be present. However, data on reproducibility between laboratories is limited. Using a ring study, we evaluated the performance of 7 Ontario laboratories using targeted sequencing panels. All laboratories analysed a series of control and clinical samples for SNVs/CNVs and gene fusions. High concordance was observed across laboratories for measured CNVs and SNVs. Over 97% of SNV calls in clinical samples were detected by all laboratories. Whilst only a single CNV was detected in the clinical samples tested, all laboratories were able to reproducibly report both the variant and copy number. Concordance for information derived from RNA was lower than observed for DNA, due largely to decreased quality metrics associated with the RNA components of the assay, suggesting that the RNA portions of comprehensive NGS assays may be more vulnerable to variations in approach and workflow. Overall the results of this study support the use of the OFA for targeted sequencing for testing of clinical samples and suggest specific internal quality metrics that can be reliable indicators of assay failure. While we believe this evidence can be interpreted to support deep targeted sequencing in general, additional studies should be performed to confirm this.

Delayed MRI Enhancement of Colorectal Cancer Liver Metastases Is Associated With Metastatic Mutational Profile.

BACKGROUND/AIM: Individual tumor genomics plays a key role in determining patient prognosis, response to chemotherapy and in guiding therapy. In prior studies, it was shown that the degree of late enhancement of colorectal liver metastases (CRCLM) target tumor enhancement (TTE) as seen on magnetic resonance imaging (MRI) was associated with overall survival. In order to better understand the relationship between MRI enhancement and survival, the aim of this study was to characterize genomic profiles of tumors clustered by MRI TTE, and investigate the association between TTE and genetic mutations. MATERIALS AND METHODS: Matched tumor and normal tissue samples from patients with weak TTE and strong TTE were analyzed by Next-generation sequencing (NGS) technology using a custom colorectal cancer panel. RESULTS: We discovered a total of 42 non-synonymous somatic mutations from 10 patients with weak TTE and 26 with 10 patients with strong TTE. Adenomatosis Polyposis Coli (APC) was the most commonly altered gene, 18 of those APC mutations were found in the weak TTE and 9 in the strong TTE group. CONCLUSION: An association exists between TTE and mutational status of CRCLM, which may offer some explanation as to why TTE is associated with overall survival in patients with CRCLM.

MiR-139 Induces an Interferon-ß Response in Prostate Cancer Cells by Binding to RIG-1.

BACKGROUND: We previously identified a panel of five miRNAs associated with prostate cancer recurrence and metastasis. Expression of one of the down-regulated miRNAs, miR-139-5p, was significantly associated with a lower incidence of biochemical recurrence and metastasis. Transcriptome profiling of miR-139-expressing prostate cancer cells revealed up-regulation of genes involved in interferon (IFN) stimulation. The association between miR-139 and IFN-ß was further explored in this study. MATERIALS AND METHODS: We examined miR-139 transfected PC3, Du145 and LNCaP cells and the associated IFN response by transcriptome sequencing, immunoblotting and pulldown assays. RESULTS: Treatment of prostate cancer cells by miR-139 resulted in the up-regulation of IFN-related genes. Specifically, miR-139 induced expression of the IFN-ß protein. The ability of miR-139 to induce IFN-ß was due to its binding to RIG-1 and the induction of IFN-related genes was found to be dependent on RIG-1 expression. CONCLUSION: miR-139 acts as an immune agonist of RIG-1 to enhance IFN-ß response in prostate cancer cells.

MR-guided focused ultrasound liquid biopsy enriches circulating biomarkers in patients with brain tumors.

BACKGROUND: Liquid biopsy is promising for early detection, monitoring of response, and recurrence of cancer. The blood-brain barrier (BBB) limits the shedding of biomarker, such as cell-free DNA (cfDNA), into the blood from brain tumors, and their detection by conventional assays. Transcranial MR-guided focused ultrasound (MRgFUS) can safely and transiently open the BBB, providing an opportunity for less-invasive access to brain pathology. We hypothesized that MRgFUS can enrich the signal of circulating brain-derived biomarkers to aid in liquid biopsy. METHODS: Nine patients were treated in a prospective single-arm, open-label trial to investigate serial MRgFUS and adjuvant temozolomide combination in patients with glioblastoma (NCT03616860). Blood samples were collected as an exploratory measure within the hours before and after sonication, with control samples from non-brain tumor patients undergoing BBB opening (BBBO) alone (NCT03739905). RESULTS: Brain regions averaging 7.8 ± 6.0 cm3 (range 0.8-23.1 cm3) were successfully treated within 111 ± 39 minutes without any serious adverse events. We found MRgFUS acutely enhanced plasma cfDNA (2.6 ± 1.2-fold, P < .01, Wilcoxon signed-rank test), neuron-derived extracellular vesicles (3.2 ± 1.9-fold, P < .01), and brain-specific protein S100b (1.4 ± 0.2-fold, P < .01). Further comparison of the cfDNA methylation profiles suggests a signature that is disease- and post-BBBO-specific, in keeping with our hypothesis. We also found cfDNA-mutant copies of isocitrate dehydrogenase 1 (IDH1) increased, although this was in only one patient known to harbor the tumor mutation. CONCLUSIONS: This first-in-human proof-of-concept study shows MRgFUS enriches the signal of circulating brain-derived biomarkers, demonstrating the potential of the technology to support liquid biopsy for the brain.

CTNNB1 Mutations and Aberrant ß-Catenin Expression in Ovarian Endometrioid Carcinoma: Correlation With Patient Outcome.

CTNNB1 mutations and aberrant ß-catenin expression have adverse prognosis in endometrial endometrioid carcinoma, and recent evidence suggests a prognostic role of ß-catenin in ovarian endometrioid carcinoma. Thus, we aimed to determine the prognostic value of the CTNNB1 mutational status, and its correlation with ß-catenin expression, in a well-annotated cohort of 51 ovarian endometrioid carcinomas. We performed immunohistochemistry for ß-catenin and developed an 11-gene next-generation sequencing panel that included whole exome sequencing of CTNNB1 and TP53. Results were correlated with clinicopathologic variables including disease-free and disease-specific survival. Tumor recurrence was documented in 14 patients (27%), and cancer-related death in 8 patients (16%). CTNNB1 mutations were found in 22 cases (43%), and nuclear ß-catenin in 26 cases (51%). CTNNB1 mutation highly correlated with nuclear ß-catenin (P<0.05). Mutated CTNNB1 status was statistically associated with better disease-free survival (P=0.04, log-rank test) and approached significance for better disease-specific survival (P=0.07). It also correlated with earlier International Federation of Gynecology and Obstetrics stage (P<0.05). Nuclear ß-catenin, TP53 mutations, age, ProMisE group, surface involvement, tumor grade and stage also correlated with disease-free survival. There was no association between membranous ß-catenin expression and disease-free or disease-specific survival. CTNNB1 mutations and nuclear ß-catenin expression are associated with better progression-free survival in patients with OEC. This relationship may be in part due to a trend of CTNNB1-mutated tumors to present at early stage. ß-catenin immunohistochemistry may serve as a prognostic biomarker and a surrogate for CTNN1B mutations in the evaluation of patients with ovarian endometrioid neoplasia, particularly those in reproductive-age or found incidentally without upfront staging surgery.