Novel markers to detect HER2 amplification in Breast cancer

Overexpression of HER2 in breast cancer is an important prognostic and predictive biomarker, assessed using immunohistochemistry (IHC) and in situ hybridization (ISH). More than 20% of tumours are graded equivocal on IHC and is send for reflex testing via ISH. In situ hybridization (ISH) is an expensive assay and is not available widely in resource limiting areas. Therefore, we propose that genes found significantly co-expressed with HER2 in breast cancer can be used as surrogate markers for HER2 in breast cancer which can detect HER2 positivity on IHC itself. This hypothesis is based on analysis of publicly available datasets from the Gene Expression Omnibus (GEO) database. The genes found most significantly correlated with HER2 expression were PGAP3 (r = 0.85), GRB7 (r = 0.82), STARD3 (r = 0.78), CDK12 (r= 0.68), PSMD3 (r= 0.67) and GSDMB (r = 0.63). We hypothesize that these identified surrogate markers for HER2 amplification which can be detected on IHC can detect HER2 amplification status in HER2 equivocal tumors based on IHC staining alone and will reduce the number of HER2 2+ (equivocal) category tumours.

Autoimmune Peripheral Nervous System Hyperexcitability

A 25-year-old man was seen for assessment of progressive pain. He had a distant history of Guillain-Barré syndrome at age 8 years, at which time he had symmetrical proximal and distal weakness of the upper and lower extremities with loss of ambulation. No facial weakness, dysarthria, dysphagia, ptosis, diplopia, or respiratory weakness occurred. At his initial evaluation there was touch hypersensitivity of the muscles and skin. He had no weakness or cognitive involvement, although the pain made it difficult for him to concentrate. His creatine kinase value improved with hydration, but pain and muscle twitching persisted. On examination, he had diffuse extremity and truncal fasciculations and myokymia and reported pain in not only the areas of twitching but also other areas of his extremities and trunk. On neurophysiologic testing, fibular and tibial motor compound muscle action potentials were decreased in amplitude, with normal ulnar and median motor responses. Needle electromyography of muscles proximally and distally showed diffuse spontaneous firing of muscles ranging in frequency with waxing and waning characteristics. These findings were thought to be consistent with a primary hyperexcitable disorder of muscles with a superimposed old polyradiculoneuropathy and possibly a myopathy. Expanded autoimmune neuroimmunologic testing of serum identified immunoglobulin G-directed cerebellar molecular staining consistent with voltage-gated potassium channel autoantibodies. Radioimmunoprecipitation assay identified voltage-gated potassium channel-immunoglobulin Gs and led to reflex testing for contactin-associated protein 2-immunoglobulin G; autoantibodies were positive. Computed tomography of the chest with contrast was performed, and lymphadenopathy was identified. The patient was clinically diagnosed with contactin-associated protein 2 - immunoglobulin G–positive Isaacs syndrome. A trial of high-dose gabapentin was attempted, with only mild benefits. Next, intravenous immunoglobulin was initiated. Diabetes developed, and he was hospitalized requiring initiation of insulin. His condition is now managed variably with intravenous immunoglobulin and scheduled daily gabapentin. The immune system has long been recognized to help regulate pain via non- immunoglobulin G–mediated mechanisms. Specifically, cytokines decrease the nociceptive nerve fiber thresholds and are released after diverse tissue insults. This allows for speeded healing by increased blood flow and protection of the region by pain guarding mechanisms. It is now recognized that, in rare cases, immunoglobulin G-mediated autoimmunity can lead to otherwise idiopathic pain disorders.

Molecular characterisation of pancreatic ductal adenocarcinoma with NTRK fusions and review of the literature

AimsThe majority of pancreatic ductal adenocarcinomas (PDACs) harbour oncogenic mutations in KRAS with variants in TP53, CDKN2A and SMAD4 also prevalent. The presence of oncogenic fusions including NTRK fusions are rare but important to identify. Here we ascertain the prevalence of NTRK fusions and document their genomic characteristics in a large series of PDAC.MethodsWhole genome sequencing and RNAseq were performed on a series of patients with resected or locally advanced/metastatic PDAC collected between 2008 and 2020 at a single institution. A subset of specimens underwent immunohistochemistry (IHC) analysis. Clinical and molecular characterisation and IHC sensitivity and specificity were evaluated.Results400 patients were included (resected n=167; locally advanced/metastatic n=233). Three patients were identified as harbouring an NTRK fusion, two EML4-NTRK3 (KRAS-WT) and a single novel KANK1-NTRK3 fusion. The latter occurring in the presence of a subclonal KRAS mutation. Typical PDAC drivers were present including mutations in TP53 and CDKN2A. Substitution base signatures and tumour mutational burden were similar to typical PDAC. The prevalence of NTRK fusions was 0.8% (3/400), while in KRAS wild-type tumours, it was 6.25% (2/32). DNA prediction alone documented six false-positive cases. RNA analysis correctly identified the in-frame fusion transcripts. IHC analysis was negative in the KANK1-NTRK3 fusion but positive in a EML4-NTRK3 case, highlighting lower sensitivity of IHC.ConclusionNTRK fusions are rare; however, with emerging therapeutic options targeting these fusions, detection is vital. Reflex testing for KRAS mutations and subsequent RNA-based screening could help identify these cases in PDAC.

Reflex ROS1 IHC Screening with FISH Confirmation for Advanced Non-Small Cell Lung Cancer—A Cost-Efficient Strategy in a Public Healthcare System

ROS1 rearrangements are identified in 1–2% of lung adenocarcinoma cases, and reflex testing is guideline-recommended. We developed a decision model for population-based ROS1 testing from a Canadian public healthcare perspective to determine the strategy that optimized detection of true-positive (TP) cases while minimizing costs and turnaround time (TAT). Eight diagnostic strategies were compared, including reflex single gene testing via immunohistochemistry (IHC) screening, fluorescence in-situ hybridization (FISH), next-generation sequencing (NGS), and biomarker-informed (EGFR/ALK/KRAS wildtype) testing initiated by pathologists and clinician-initiated strategies. Reflex IHC screening with FISH confirmation of positive cases yielded the best results for TAT, TP detection rate, and cost. IHC screening saved CAD 1,000,000 versus reflex FISH testing. NGS was the costliest reflex strategy. Biomarker-informed testing was cost-efficient but delayed TAT. Clinician-initiated testing was the least costly but resulted in long TAT and missed TP cases, highlighting the importance of reflex testing. Thus, reflex IHC screening for ROS1 with FISH confirmation provides a cost-efficient strategy with short TAT and maximizes the number of TP cases detected.

Hepatitis C Diagnosis: Simplified Solutions, Predictive Barriers, and Future Promises

The simplification of current hepatitis C diagnostic algorithms and the emergence of digital diagnostic devices will be very crucial to achieving the WHO’s set goals of hepatitis C diagnosis (i.e., 90%) by 2030. From the last decade, hepatitis C diagnosis has been revolutionized by the advent and approval of state-of-the-art HCV diagnostic platforms which have been efficiently implemented in high-risk HCV populations in developed nations as well as in some low-to-middle income countries (LMICs) to identify millions of undiagnosed hepatitis C-infected individuals. Point-of-care (POC) rapid diagnostic tests (RDTs; POC-RDTs), RNA reflex testing, hepatitis C self-test assays, and dried blood spot (DBS) sample analysis have been proven their diagnostic worth in real-world clinical experiences both at centralized and decentralized diagnostic settings, in mass hepatitis C screening campaigns, and hard-to-reach aboriginal hepatitis C populations in remote areas. The present review article overviews the significance of current and emerging hepatitis C diagnostic packages to subvert the public health care burden of this ‘silent epidemic’ worldwide. We also highlight the challenges that remain to be met about the affordability, accessibility, and health system-related barriers to overcome while modulating the hepatitis C care cascade to adopt a ‘test and treat’ strategy for every hepatitis C-affected individual. We also elaborate some key measures and strategies in terms of policy and progress to be part of hepatitis C care plans to effectively link diagnosis to care cascade for rapid treatment uptake and, consequently, hepatitis C cure.

Implementation of SARS-CoV2 Screening in K-12 Schools using In-School Pooled Molecular Testing and Deconvolution by Rapid Antigen Test

SARS-CoV2 testing is one component of a multi-layered mitigation strategy to enable safe in-person school attendance for the K-12 school population. However, costs, logistics, and uncertainty about effectiveness are potential barriers to implementation. We assessed early data from the Massachusetts K-12 public school pooled SARS-CoV2 testing program, which incorporates two novel design elements: in-school “pod pooling” for assembling pools of dry anterior nasal swabs from 5-10 individuals, and positive pool deconvolution using the BinaxNOW antigen rapid diagnostic test (Ag RDT), to assess the operational and analytical feasibility of this approach. Over three months, 187,597 individual swabs were tested across 39,297 pools from 738 schools. The pool positivity rate was 0.8%; 98.2% of pools tested negative and 0.2% inconclusive, and 0.8% of pools submitted could not be tested. Of 310 positive pools, 70.6% had an N1 or N2 Ct value ≤ 30. In reflex testing (performed on specimens newly collected from members of the positive pool), 92.5% of fully deconvoluted pools with N1 or N2 target Ct ≤30 yielded a positive individual using the BinaxNOW test performed 1-3 days later. However, of 124 positive pools with full reflex testing data available for analysis, 32 (25.8%) of BinaxNOW pool deconvolution testing attempts did not detect a positive individual, requiring additional reflex testing. With sufficient staffing support and low pool positivity rates, pooled sample collection and reflex testing were feasible for schools. These early program findings confirm that screening testing for K-12 students and staff is achievable at scale with a scheme that incorporates in-school pooling, RT-PCR primary testing, and Ag RDT reflex/deconvolution testing.