Daniel R. Zollinger

Emerging technologies focused on the detection and quantification of circulating tumor DNA (ctDNA) in blood show extensive potential for managing patient treatment decisions, informing risk of recurrence, and predicting response to therapy. Currently available tissue-informed approaches are often limited by the need for additional sequencing of normal tissue or peripheral mononuclear cells to identify non-tumor-derived alterations while tissue-naïve approaches are often limited in sensitivity. Here we present the analytical validation for a novel ctDNA monitoring assay, FoundationOne®Tracker. The assay utilizes somatic alterations from comprehensive genomic profiling (CGP) of tumor tissue. A novel algorithm identifies monitorable alterations with a high probability of being somatic and computationally filters non-tumor-derived alterations such as germline or clonal hematopoiesis variants without the need for sequencing of additional samples. Monitorable alterations identified from tissue CGP are then quantified in blood using a multiplex polymerase chain reaction assay based on the validated Signatera^TM assay. The analytical specificity of the plasma workflow is shown to be 99.6% at the sample level. Analytical sensitivity is shown to be >97.3% at ≥5 mean tumor molecules per mL of plasma (MTM/mL) when tested with the most conservative configuration using only two monitorable alterations. The assay also demonstrates high analytical accuracy when compared to liquid biopsy-based CGP as well as high qualitative (measured 100% PPA) and quantitative precision (<11.2% coefficient of variation).

A majority of patients with metastatic colorectal cancer (mCRC) experience recurrence post curative-intent surgery. The addition of adjuvant chemotherapy has shown to provide limited survival benefits when applied to all patients. Therefore, a biomarker to assess molecular residual disease (MRD) accurately and guide treatment selection is highly desirable for high-risk patients. This feasibility study evaluated the prognostic value of a tissue comprehensive genomic profiling (CGP)-informed, personalized circulating tumor DNA (ctDNA) assay (FoundationOne^®Tracker) (Foundation Medicine, Inc., Cambridge, MA, USA) by correlating MRD status with clinical outcomes. ctDNA analysis was performed retrospectively on plasma samples from 69 patients with resected mCRC obtained at the MRD and the follow-up time point. Tissue CGP identified potentially actionable alterations in 54% (37/69) of patients. MRD-positivity was significantly associated with lower disease-free survival (DFS) (HR: 4.97, 95% CI: 2.67–9.24, p < 0.0001) and overall survival (OS) (HR: 27.05, 95% CI: 3.60–203.46, p < 0.0001). Similarly, ctDNA positive status at the follow-up time point correlated with a marked reduction in DFS (HR: 8.78, 95% CI: 3.59–21.49, p < 0.0001) and OS (HR: 20.06, 95% CI: 2.51–160.25, p < 0.0001). The overall sensitivity and specificity at the follow-up time point were 69% and 100%, respectively. Our results indicate that MRD detection using the tissue CGP-informed ctDNA assay is prognostic of survival outcomes in patients with resected mCRC. The concurrent MRD detection and identification of actionable alterations has the potential to guide perioperative clinical decision-making.

Action potential conduction along myelinated axons depends on high densities of voltage-gated Na⁺ channels at the nodes of Ranvier. Flanking each node, paranodal junctions (paranodes) are formed between axons and Schwann cells in the peripheral nervous system (PNS) or oligodendrocytes in the CNS. Paranodal junctions contribute to both node assembly and maintenance. Despite their importance, the molecular mechanisms responsible for paranode assembly and maintenance remain poorly understood. βII spectrin is expressed in diverse cells and is an essential part of the submembranous cytoskeleton. Here, we show that Schwann cell βII spectrin is highly enriched at paranodes. To elucidate the roles of glial βII spectrin, we generated mutant mice lacking βII spectrin in myelinating glial cells by crossing mice with a floxed allele of Sptbn1 with Cnp-Cre mice, and analyzed both male and female mice. Juvenile (4 weeks) and middle-aged (60 weeks) mutant mice showed reduced grip strength and sciatic nerve conduction slowing, whereas no phenotype was observed between 8 and 24 weeks of age. Consistent with these findings, immunofluorescence microscopy revealed disorganized paranodes in the PNS and CNS of both postnatal day 13 and middle-aged mutant mice, but not in young adult mutant mice. Electron microscopy confirmed partial loss of transverse bands at the paranodal axoglial junction in the middle-aged mutant mice in both the PNS and CNS. These findings demonstrate that a spectrin-based cytoskeleton in myelinating glia contributes to formation and maintenance of paranodal junctions. SIGNIFICANCE STATEMENT Myelinating glia form paranodal axoglial junctions that flank both sides of the nodes of Ranvier. These junctions contribute to node formation and maintenance and are essential for proper nervous system function. We found that a submembranous spectrin cytoskeleton is highly enriched at paranodes in Schwann cells. Ablation of βII spectrin in myelinating glial cells disrupted the paranodal cell adhesion complex in both peripheral and CNSs, resulting in muscle weakness and sciatic nerve conduction slowing in juvenile and middle-aged mice. Our data show that a spectrin-based submembranous cytoskeleton in myelinating glia plays important roles in paranode formation and maintenance.

In myelinated axons, K⁺channels are clustered in distinct membrane domains to regulate action potentials (APs). At nodes of Ranvier, Kv7 channels are expressed with Na⁺channels, whereas Kv1 channels flank nodes at juxtaparanodes. Regulation of axonal APs by K⁺channels would be particularly important in fast-spiking projection neurons such as cerebellar Purkinje cells. Here, we show that BK/Slo1 channels are clustered at the paranodal junctions of myelinated Purkinje cell axons of rat and mouse. The paranodal junction is formed by a set of cell-adhesion molecules, including Caspr, between the node and juxtaparanodes in which it separates nodal from internodal membrane domains. Remarkably, only Purkinje cell axons have detectable paranodal BK channels, whose clustering requires the formation of the paranodal junction via Caspr. Thus, BK channels occupy this unique domain in Purkinje cell axons along with the other K⁺channel complexes at nodes and juxtaparanodes. To investigate the physiological role of novel paranodal BK channels, we examined the effect of BK channel blockers on antidromic AP conduction. We found that local application of blockers to the axon resulted in a significant increase in antidromic AP failure at frequencies above 100 Hz. We also found that Ni²⁺elicited a similar effect on APs, indicating the involvement of Ni²⁺-sensitive Ca²⁺channels. Furthermore, axonal application of BK channel blockers decreased the inhibitory synaptic response in the deep cerebellar nuclei. Thus, paranodal BK channels uniquely support high-fidelity firing of APs in myelinated Purkinje cell axons, thereby underpinning the output of the cerebellar cortex.