Prevalence of Variants of Uncertain Significance in Patients Undergoing Genetic Testing for Hereditary Breast and Ovarian Cancer and Lynch Syndrome.

Hereditary Breast and Ovarian Cancer (HBOC) and Lynch Syndrome (LS) are the most common inherited cancer syndromes identified with genetic testing. Testing, though, commonly reveals variants of uncertain significance (VUSs). This is a retrospective observational study designed to determine the prevalence of pathogenic mutations and VUSs in patients tested for HBOC and/or LS and to explore the characteristics of the VUS population. Patients 18-80 years old that met NCCN criteria for HBOC and/or LS genetic screening were tested between 2006 and 2020 at Mount Auburn Hospital in Cambridge, Massachusetts. A total of 663 patients were included in the study, with a mean age of 50 years old and 90% being females. Pathogenic mutations were identified in 12.5% and VUSs in 28.3%. VUS prevalence was associated with race (p-value = 0.019), being particularly higher in Asian populations. Patients with a personal history of breast cancer or family history of breast or ovarian cancer were more likely to have a VUS (personal breast: OR: 1.55; CI: 1.08-2.25; family breast: OR: 1.68; CI: 1.08-2.60, family ovarian OR: 2.29; CI: 1.04-5.45). In conclusion, VUSs appear to be detected in almost one third patients tested for cancer genetic syndromes, and thus future work is warranted to determine their significance in cancer development.

Evaluation of the Design, Development, and Performance of a Mass-Flow Based, Open-Source Buffer Manufacturing System.

Buffer solutions are a critical component of the manufacturing process for therapeutic proteins and other biomolecules. The traditional way to make and use buffers is space and resource intensive, creating operational bottlenecks that impact efficiencies and costs. Here we describe a full-scale, current Good Manufacturing Practices (cGMP) capable buffer stock blending system that has an open-source, configurable design and that overcomes the challenges of traditional buffer preparation. The system comprises simplified control and operation using mass flow to provide on-demand supply of buffer solutions. The system also has self-cleaning capability and is amenable to be operated as a closed system. The data will demonstrate the excellent performance and capabilities of the system as well as illustrate its potential transformative impact on biomanufacturing.

The Function of extravascular coagulation factor IX in haemostasis.

INTRODUCTION: The majority of clotting factor IX (FIX) resides extravascularly, in the subendothelial basement membrane, where it is important for haemostasis. AIM: We summarize preclinical studies demonstrating extravascular FIX and its role in haemostasis and discuss clinical observations supporting this. We compare the in vivo binding of BeneFIX® and the extended half-life FIX, Alprolix® , to extravascular type IV collagen (Col4). METHODS: Three mouse models of haemophilia were used: the FIX knockout as the CRM- model and two knock-in mice, representing a CRM+ model of a commonly occurring patient mutation (FIXR333Q ) or a mutation that binds poorly to Col4 (FIXK5A ). The murine saphenous vein bleeding model was used to assess haemostatic competency. Clinical publications were reviewed for relevance to extravascular FIX. RESULTS: CRM status affects recovery and prophylactic efficacy. Prophylactic protection decreases ~5X faster in CRM+ animals. Extravascular haemostasis can explain unexpected breakthrough bleeding in patients treated with some EHL-FIX therapeutics. In mice, both Alprolix® and BeneFIX® bind Col4 with similar affinities (Kd~20-40 nM) and show dose-dependent recoveries. As expected, the concentration of binding sites in the mouse calculated for Alprolix® (574 nM) was greater than for BeneFIX® (405 nM), due to Alprolix® binding to both Col4 and the endothelial cell neonatal Fc receptor. CONCLUSION: Preclinical and clinical results support the interpretation that FIX plays a role in haemostasis from its extravascular location. We believe that knowing the CRM status of haemophilia B patients is important for optimizing prophylactic dosing with less trial and error, thereby decreasing clinical morbidity.

N_LyST: a simple and rapid screening test for Lynch syndrome.

AIMS: We sought to use PCR followed by high-resolution melting analysis to develop a single closed-tube screening panel to screen for Lynch syndrome. This comprises tests for microsatellite instability (MSI), MLH1 methylation promoter and BRAF mutation. METHODS: For MSI testing, five mononucleotide markers (BAT25, BAT26, BCAT25, MYB, EWSR1) were developed. In addition, primers were designed to interrogate Region C of the MLH1 promoter for methylation (using bisulphite-modified DNA) and to test for mutations in codon 600 of BRAF. Two separate cohorts from Nottingham (n=99, 46 with MSI, 53 being microsatellite stable (MSS)) and Edinburgh (n=88, 45 MSI, 43 MSS) were tested. RESULTS: All the cases (n=187) were blind tested for MSI and all were correctly characterised by our panel. The MLH1 promoter and BRAF were tested only in the Nottingham cohort. Successful blinded analysis was performed on the MLH1 promoter in 97 cases. All MSS cases showed a pattern of non-methylation while 41/44 cases with MSI showed full methylation. The three cases with MSI and a non-methylated pattern had aberrations in MSH2 and MSH6 expression. BRAF mutation was detected in 61% of MSI cases and 11% of MSS cases.Finally, 12 cases were blind screened by using the whole panel as a single test. Of these, five were identified as MSS, four as MSI/non-LS and three as MSI/possible LS. These results were concordant with the previous data. CONCLUSION: We describe the Nottingham Lynch Syndrome Test (N_LyST). This is a quick, simple and cheap method for screening for Lynch syndrome.