Supported Grants

2021 DDF YOUNG INVESTIGATOR RESEARCH GRANT
Immunologic Phenotyping of Patients with Neuromuscular Immune-related Adverse Events after
Checkpoint Inhibitor Therapy

LAY LANGUAGE ABSTRACT

Immune checkpoint inhibitors are a class of cancer therapies that activate the body’s immune system to fight tumor cells. These medications have improved survival for patients with many types of treatment-resistant cancers. However, activation of the immune system can also cause damage in non-cancer cells, including nerves and muscles. This causes neuromuscular toxicity that can cause severe illness and even death, which limits the potential benefit of these lifesaving medications. The immune system pathways that trigger and sustain neuromuscular toxicity are unclear. We plan to use a cutting-edge technique called single-cell RNA sequencing to characterize the types of cells present in muscle, nerve, and spinal fluid samples from patients with neuromuscular toxicities. In each sample, we will identify the different types of immune cells and non-immune cells present, the genes that these cells express, and the receptors that immune cells have on their membranes. This will allow us to map out the abnormalities in affected tissues and understand how neuromuscular damage occurs. This type of analysis has not been systematically performed in neuromuscular samples from patients with these toxicities. We hope to show that single-cell RNA sequencing and analysis can be successfully performed on these samples. We will analyze whether specific immune signatures are associated with neuromuscular toxicities, their severity, and their appearance on biopsy. We will also analyze whether the pathways involved in toxicities are different from those involved in the anti-tumor activity; if so, then we could target these pathways with medications to limit toxicities while maintaining the effectiveness of these medications.
Our overall goal is to understand what causes neuromuscular toxicities so that we can predict who is at risk and provide optimal treatment. If our study is successful in this patient population, we hope to extend this type of analysis to other autoimmune neuromuscular diseases.

TECHNICAL ABSTRACT

Background: Immune checkpoint inhibitors (ICIs) are potent anti-tumor drugs but are associated with treatment-limiting toxicities called immune-related adverse events (irAEs). irAEs affecting the peripheral nervous system have unique clinical features and can have fulminant and even fatal presentations. The pathomechanisms underlying neuromuscular irAEs are not fully elucidated. Single-cell RNA sequencing (scRNA-seq) analysis is a cutting-edge unbiased methodology that is well-suited to defining the cellular and transcriptional programs driving and sustaining neuromuscular irAEs.

Objective and Hypothesis: Prior studies have shown striking T-cell perturbations in patients with irAEs in both tumor tissue and tissues affected by toxicity. Distinct gene expression and T-cell receptor signatures are present in tissues with toxicity and can differ based on irAE severity. Further, immune signatures associated with toxicity are distinct from those associated with anti-tumor efficacy. Our main objective is to demonstrate feasibility of scRNA-seq in neuromuscular tissues to define the gene expression and T-cell receptor signatures in neuromuscular irAEs. We hypothesize that (1) a break in tolerance in the tissue microenvironment underlies neuromuscular irAE presentations and is mediated by complex interactions of immune and non-immune cell populations, which likely involves the expansion of cytotoxic T-cell lymphocytes that contribute to sustaining local tissue damage; (2) specific gene expression and T-cell receptor signatures are associated with severe/fulminant irAE-NMs but not associated with anti-tumor efficacy; and (3) distinct transcriptional programs expressed in muscle tissue are associated with different types of immune cell infiltrate (T-cells, B-cells and macrophages) and the presence of complement deposition on muscle biopsy.

Specific Aims: The specific aims are three-fold: (1) Demonstrate the feasibility of high-dimensional scRNA-seq data generation and analyses in neuromuscular samples to define the cellular and transcriptional programs characterizing neuromuscular irAEs; (2) Collect detailed clinical data via retrospective chart review for stratified analyses of whether specific immune signatures on scRNA-seq analysis are associated with irAE-NM severity and/or ICI anti-tumor response; and (3) Collect histologic data for stratified analyses of whether specific immune signatures on scRNA-seq analysis are associated with the presence and type of immune cell infiltrates and the presence of complement deposition on muscle biopsy.

Study Design: We will analyze 15 neuromuscular samples (including skeletal muscle, nerve tissue, and cerebrospinal fluid) during clinical biopsies and rapid autopsies from patients with clinically confirmed neuromuscular irAEs. These tissues will be processed to create single-cell suspensions for immune cells and single-nuclei suspensions for non-immune cells. These suspensions undergo reverse transcription to create gene expression libraries, which are then sequenced. We will use established computational frameworks to define all the cell types and states present in skeletal muscle, nerve tissue, and CSF using a marker-free approach and identify aberrant signaling pathways. We will perform further downstream analyses to define which immune cell types dominate the immune infiltrate in neuromuscular irAEs, characterize the transcriptional programs altered across immune and non-immune cells compared to control tissue (derived from published single-cell transcriptomic datasets), and assess whether similar cell types and transcriptional programs are commonly altered across all patients analyzed. We will also sequence all T-cell receptors in the samples to define the differences in T-cell types and states present in neuromuscular irAE, assess whether particular T-cells associated with neuromuscular irAE are characterized by dysregulated transcriptional regulatory programs, and assess the relevance of T-cell receptor repertoire diversity as a clinical predictor. We will also correlate specific gene expression and T- cell receptor signatures with clinical and pathologic features to determine whether specific patterns are associated with neuromuscular irAE severity, ICI anti-tumor response, the presence and type of immune cell infiltrate on muscle biopsy, and the presence of complement deposition on muscle biopsy.

Scientific Relevance: This project will provide insights into the cellular and molecular perturbations that occur in neuromuscular irAEs related to ICI therapy, a group of newly defined immune-mediated neuromuscular disorders. Understanding how neuromuscular irAEs are triggered and sustained is key to developing new treatment strategies to limit the toxicities of these lifesaving medications. Additionally, successful application of this methodology to neuromuscular irAEs will enable future transcriptome studies in idiopathic autoimmune neuromuscular diseases.

ABOUT DR. BURTON

Dr. Leeann Burton is a neuromuscular neurologist at the Massachusetts General Hospital and an Instructor of Neurology at Harvard Medical School. She graduated from Hamilton College with a degree in neuroscience and received her medical degree from Harvard Medical School. She completed her internal medicine internship at the Massachusetts General Hospital, followed by neurology residency and a clinical fellowship in neuromuscular medicine at the Massachusetts General Hospital and Brigham and Women’s Hospital.

Her clinical practice is dedicated to patients with neuromuscular diseases. She has a particular interest in immune-mediated disorders such as myasthenia gravis and inflammatory myopathies.

She is involved in clinical research programs around myasthenia gravis and immune-related adverse events of cancer immunotherapies. She also has an interest in drug development and emerging therapeutics for neuromuscular disease and is the current Anne B. Young fellow in translational neuroscience.

ABOUT DDF

Dysimmune Diseases Foundation (DDF) was created in 2005 as a non-profit health foundation. Our goals are: to support access to treatment for patients with autoimmune disorders, to foster support for patients, families, and physicians through advocacy for autoimmune disease research and treatments, and to fund research into the cause, treatment, and cure for autoimmune diseases with a focus on neuromuscular autoimmune diseases