Muscle Visualization and Evaluation Methods for Immune Checkpoint Inhibitor-Related Myositis

Summary

Immune checkpoint inhibitors (ICIs) revolutionize cancer therapy but risk myositis; early multimodal evaluation (clinical, biomarkers, EMG, imaging, biopsy) optimizes diagnosis, management, and protocol standardization amid diagnostic challenges.

Abstract

Immune checkpoint inhibitors (ICIs) have fundamentally transformed the landscape of cancer therapy, yet their administration frequently correlates with immune-related adverse events (irAEs), amongst which myositis, an inflammatory disorder affecting the muscles, stands out. The prompt identification and thorough assessment of myositis associated with ICIs are imperative for effective therapeutic management and the enhancement of patient prognoses. In this report, we present a detailed evaluation of muscle function aimed at accurately diagnosing and appraising ICI-related myositis. Essential diagnostic methodologies, such as clinical evaluations, assessments of muscle strength, the analysis of laboratory biomarkers, electromyography (EMG), imaging modalities, and muscle biopsies, are scrutinized. The complexities involved in differentiating ICI-induced myositis from other autoimmune disorders, as well as non-autoimmune conditions, are thoroughly explored. A focused evaluation of muscle-specific factors is essential for refining diagnostic precision, optimizing patient management strategies, and establishing standardized protocols for assessment. By addressing these critical aspects, we can facilitate a more nuanced understanding of ICI-related myositis, ultimately leading to improved outcomes for patients affected by this condition. This comprehensive approach not only aids in the timely recognition of myositis but also contributes to the broader aim of maximizing the efficacy of immunotherapy in oncological settings.

Introduction

Immune checkpoint inhibitors (ICIs) have become a cornerstone in the treatment of various malignancies, offering a new dimension in cancer therapy by modulating the immune system to recognize and attack cancer cells¹. However, this novel class of drugs is not without its challenges, as it can lead to immune-related adverse events (irAEs) that may affect any organ system, including the musculoskeletal system².

Myositis, a condition characterized by inflammation and damage to muscle tissue, is one such irAE that usually manifests as muscle weakness, pain, and elevated muscle enzymes³. The incidence of ICI-related myositis, while rare, carries significant morbidity and could severely impact patients' quality of life and cancer treatment outcomes⁴.

Previous research into the mechanisms underlying immune checkpoint inhibitor (ICI)-related adverse events has focused on the role of the immune system's hyperactivation. ICIs, such as anti-PD-1 and anti-CTLA-4 antibodies, enhance T-cell responses against tumors but may also lead to autoimmunity by disrupting the balance of immune tolerance. Studies have shown that the activation of autoreactive T cells and the resultant cytokine release can trigger inflammatory responses in various organs, contributing to the development of adverse effects, including arthritis, myositis, and colitis^5,6. Understanding these mechanisms is vital for developing strategies to predict, prevent, and manage these adverse events more effectively.

The clinical presentation of ICI-related myositis often includes non-specific symptoms such as muscle weakness, myalgia, and fatigue, which can complicate diagnosis⁷. The weakness could affect the eye muscles, face and throat muscles, neck muscles, as well as shoulder girdle and pelvic girdle muscles. Patients may present with difficulties opening their eyes, speaking, swallowing, lifting their head or limbs, or breathing. In addition, some patients have involvement of the myocardium, often manifesting as difficulty breathing, chest pain, and heart palpitations. Severely, it could progress to heart failure, cardiogenic shock, and malignant arrhythmias. ICI-related myositis can also present with myasthenia gravis (MG)-like syndrome and respiratory failure. Reported characteristics of patients with ICI-myositis are summarized in Table 1^7,8,9. The condition can occur within days to months after the initiation of ICI therapy and may persist for months to years, even after discontinuation of the ICI¹⁰. The affected muscles can limit mobility and may incur permanent damage, necessitating joint replacement surgery in severe cases¹¹. Therefore, understanding the evaluation methods for ICI-related myositis is essential for effective patient management.

The diagnosis of ICI-related myositis is based on clinical symptoms, elevated serum muscle enzymes (creatine kinase) and inflammatory markers, including T cell activation, electromyography (EMG), and muscle biopsy if necessary^12,13,14.These evaluations help distinguishidiopathic inflammatory myopathies (IIM) from ICI-related myositis.

The management of ICI-related myositis typically involves corticosteroids as the first line of treatment¹⁵. In cases unresponsive to steroids, additional immunosuppressive therapy, such as methotrexate, azathioprine, mycophenolate mofetil, rituximab, or abatacept may be considered^16,17,18. The decision to rechallenge with ICIs after myositis resolution requires careful consideration of the risks and benefits, with close monitoring for recurrence of irAEs¹⁹.

Here, we aim to provide a comprehensive overview of the evaluation methods for ICI-related myositis, including the clinical presentation, diagnostic approach, imaging techniques, and management strategies. We will also discuss the potential pitfalls and challenges in the diagnosis and management of this serious irAE, highlighting the need for a multidisciplinary approach to ensure optimal patient care.

CASE PRESENTATION:
A 70-year-old Asian male presented with progressive drooping eyelids and limb weakness in the past 20 days. He also experienced dizziness, heart palpitations, and difficulty in swallowing and breathing within the first week of symptom presentation. His muscle weakness exacerbated rapidly, and he was bedridden, unable to move, eat, or talk within 3 weeks of symptom onset, and mechanical ventilation was required due to CO₂ retention.

Diagnosis, Assessment, and Plan:
The patient underwent comprenhensive medical history review and systemic review. His muscle weakness and other symptoms appeared two weeks after the initiation of anti-PD-1 immunotherapy for squamous cell carcinoma. Physical examination revealed generalized muscle weakness, respiratory muscle failure, and complete bilateral ptosis. Laboratory tests, including creatine kinase level, cardiac troponin I levels, electrocardiograms, and MRI of bilateral thigh muscles helped identifying the muscle damage. A muscle biopsy confirmed inflammatory myopathy. Autoantibody panels for myositis and myasthenia gravis were negative. Flow cytometry staining of CD38 and HLA-DR on circulating CD8 T cells confirmed T cell activation in relation to anti-PD-1 therapy.

The patient was diagnosed with ICI-related myositis, likely induced by the anti-PD-1 therapy, after ruling out other conditions through negative neostigmine and repetitive nerve stimulation tests, along with the absence of relevant autoantibodies.

Symptoms such as generalized muscle weakness, respiratory muscle failure, bilateral ptosis, and difficulty moving the tongue, alongside abnormal MRI findings and histopathological evidence of muscle fiber damage, supported the diagnosis of ICI-related myositis.

Differential diagnoses included idiopathic inflammatory myopathies, myasthenia gravis, and neurological, infectious, or metabolic disorders, which were excluded through specific tests.

The patient was started on methylprednisolone (80 mg daily) and abatacept (125 mg weekly) to manage the inflammatory process. The rationale was to reduce inflammation and prevent further muscle damage. After 4 weeks, the glucocorticoid dose was tapered, and abatacept was discontinued. The patient showed improvement in muscle strength and functional recovery.

No significant complications were noted, though careful monitoring of immune responses and the gradual tapering of treatment was necessary to avoid flare-ups or further muscle damage. The patient responded well to the treatment regimen, regaining the ability to open eyes, eat, talk, and walk independently.

Protocol

All patients provided written consent, and the study was approved by the Ethics Committee of Renji Hospital (ID: 2013-126), Shanghai, China.
NOTE: When ICI-myositis is suspected, a thorough diagnostic evaluation is essential. ICI-related myositis requires a comprehensive diagnostic approach that includes various evaluation methods to confirm the diagnosis, assess disease severity, and monitor treatment response. A detailed evaluation protocol is described here.

1. Medical history and symptom assessment

1. Obtain full medical history of patients with a history of cancer who have received immune checkpoint inhibitors (ICIs) targeting PD-1/PD-L1 and/or CTLA-4. Identify patients presenting with new-onset muscle weakness, dyspnea, or chest pain following ICI therapy. Table 2 displays a standardized symptom checklist. Review medical history for pre-existing diagnosis of myositis.
2. Assess the timing of symptom onset in relation to ICI treatment. Record the initiation of symptoms after the first or second cycle of ICI therapy.
   NOTE: ICI-myositis is likely to develop shortly after the first 1 or 2 cycles of ICI. If symptoms occur after multiple cycles or after discontinuation of ICI, the following causes should be considered: 1) non-ICI-induced myositis: such as paraneoplastic syndrome or myopathy caused by tumor progression; 2) recurrence or coexistence of autoimmune diseases: pre-existing dermatomyositis may recur during ICI treatment, or there may be pathological processes driven by other autoantibodies (such as anti-TIF1γ antibodies or anti-striated muscle antibodies); 3) Infections or toxicities from other medications: Infections causing myositis or neuro-muscular toxicities from other chemotherapeutic agents (such as paclitaxel) should be excluded.

2. Physical examination

1. Assessment of skin manifestations
   1. Observe the patient's skin from head to toe, and assess whether the patient has the characteristic rashes of dermatomyositis (such as heliotrope rash, Gottron's papules, or shawl sign).
2. Joint involvement evaluation
   1. Assess the patient for complaints of joint pain, swelling, or stiffness, which may indicate coexisting inflammatory arthritis.
   2. Evaluate each joint by checking for elevated skin temperature over the affected joint, palpating for tenderness at the joint site, and assessing the range of motion of the joint.
   3. Differentiate between weakness caused by joint inflammation and weakness due to muscle involvement. The differences between muscle weakness and joint stiffness are listed in Table 3.
3. Cardiopulmonary examination
   1. Conduct a comprehensive assessment of respiratory function.
      1. Inquire about any symptoms such as dyspnea or the presence of lung crackles.
      2. Perform auscultation of the chest to detect abnormal heart sounds or murmurs.
   2. Examine for signs of heart failure by assessing for peripheral edema (swelling in extremities), jugular vein distention.
   3. Assess the severity of heart failure through the detection of B-type natriuretic peptide (BNP) and echocardiography.
4. Neurological examination
   1. Perform a basic neurological assessment to rule out other potential causes of weakness.
   2. Conduct a comprehensive examination.
      1. Evaluate the patient's level of consciousness.
      2. Assess cranial nerve function.
      3. Test motor function, including muscle strength, muscle tone, and coordination.
      4. Vigilantly examine sensory function, paying attention to superficial and deep sensations.
      5. Assess deep tendon reflexes and superficial reflexes.
   3. If symptoms such as sensory loss, areflexia, and positive pathological signs are present, a neurology consultation should be requested.

3. Muscle evaluation

1. Manual muscle testing (MMT)
   1. Position the patient comfortably, either seated or lying down, depending on the muscle group being evaluated.
   2. Instruct the patient to perform a specific movement against resistance, such as raising an arm or leg.
   3. Apply resistance at the specific joint or muscle group during the movement.
   4. Grade the muscle strength based on the following scale: 0: No muscle contraction; 1: Visible contraction without movement; 2: Movement with gravity eliminated; 3: Movement against gravity; 4: Movement against moderate resistance; 5: Normal strength against full resistance.
      NOTE: It is recommended that examiners undergo joint training to reduce the heterogeneity of the examination assessment.
2. Conduct imaging techniques.
   1. Perform muscle magnetic resonance imaging (MRI) to assess the presence and extent of muscle involvement. Document any observed muscle edema and inflammation, noting whether changes appear diffuse or focal. Prioritize T1-weighted, T2-weighted fat-suppressed (T2-FS), and STIR (short-tau inversion recovery) sequences.
      ​NOTE: T1-weighted imaging highlights structural abnormalities, such as fatty infiltration (appearing as bright hyperintense signals) and fibrosis, providing a baseline assessment of chronic muscle damage. T2-FS and STIR sequences are critical for detecting active inflammation. These sequences suppress fat signals, enhancing sensitivity to edema (free water content in inflamed tissues), which manifests as hyperintense areas. STIR is particularly useful for identifying subtle or diffuse edema. Alternatively, imaging methods such as muscle ultrasound or PET/CT scan can be used²⁰.
3. Perform electrophysiologic examination.
   NOTE: ICI-related myositis, often mimicking myasthenia gravis (MG) with symptoms such as ptosis and oculomotor dysfunction^8,21, leading to misdiagnosis, underscores the need for comprehensive electrophysiological evaluation, including needle electromyography (EMG), nerve conduction studies (NCS), and low-frequency and high-frequency repetitive nerve stimulation (RNS).
4. Evaluate respiratory muscle strength: Perform a pulmonary function test to evaluate respiratory muscle strength, particularly the diaphragm.
5. Exercise tolerance test: 6-min walk test (6MWT)
   1. Explain the purpose of the test and the importance of pacing to the patient.
   2. Measure and record baseline heart rate, blood pressure, and oxygen saturation.
   3. Have the patient walk back and forth in a flat, straight corridor for 6 min.
   4. Instruct the patient to walk as far as possible at their own pace, allowing them to stop if necessary.
   5. Encourage the patient at regular intervals, noting the distance walked and any signs of fatigue, dyspnea, or discomfort.
   6. Record the total distance walked and post-exercise heart rate and oxygen saturation.
   7. Interpret the results by comparing them to established normative values and the patient's clinical condition.
      NOTE: The 6MWD for healthy adults typically ranges from 400-700 m. For different age and gender groups, the normal values for 6MWD may vary. A walking distance of less than 150 m may indicate severe cardiopulmonary dysfunction; between 150 m and 425 m may suggest moderate cardiopulmonary dysfunction; and greater than 425 m may belong to mild cardiopulmonary dysfunction.

4. Laboratory testing

1. Muscle enzyme testing: Measure the levels of the following muscle enzymes in serum samples: creatine kinase (CK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine transaminase (ALT).
2. Inflammatory marker assessment: Assess the levels of the following non-specific inflammatory markers in serum: serum ferritin, C-reactive protein (CRP), serum amyloid A, and erythrocyte sedimentation rate (ESR).
3. Measure CD38 and HLA-DR expression on T cells using flow cytometry to provide a relatively specific indication of T cell activation¹⁴.
   1. Perform surface staining of cells with fluorochrome-conjugated CD38 and HLA-DR antibodies, followed by flow cytometry analysis with gating on specific cell subsets.
   2. Quantify positive cell percentages and mean fluorescence intensity (MFI) in biparametric scatter plots to evaluate activation or functional states.
4. Cardiac troponin I (cTnI) testing:
   1. Measure cTnI level to assess potential cardiac involvement in ICI-related myositis patients with heightened disease activity and poor prognosis.
   2. In patients with elevated cTnI levels continuously monitor troponin levels. Conduct electrocardiograms (ECG) to detect signs of life-threatening ICI-myocarditis.
   3. Consider coronary angiography for patients being evaluated for acute coronary syndrome.
   4. When hemodynamic instability or arrhythmias ocurrs, prioritize echocardiography for rapid assessment of cardiac function and acute complications (e.g., pericardial effusion).
5. Autoantibody testing
   1. Test for the presence of myositis-specific autoantibodies (MSA) and myositis-associated autoantibodies (MAA) in patient serum samples. The commonly used method is line blot assay.
   2. Review the autoantibody status in ICI-myositis patients with pre-existing autoantibodies. Record any development of new MSA or MAA.
   3. Refer to the Table 4 that summarizes different autoantibodies relevant to ICI-related myositis and IIM for detailed identification.

5. Pathology

1. Muscle biopsy preparation
   1. Conduct a thorough physical examination to identify appropriate biopsy sites based on the following: 1) Presence of decreased muscle strength; 2) Evidence of myoedema on MRI; 3) Irritable EMG findings indicating active inflammation.
   2. Prioritize muscles showing decreased strength for biopsy, avoiding sites that exhibit severe muscle atrophy or fatty infiltration.
   3. If a muscle exhibits a manual muscle test (MMT) score < 3/5, perform an MRI to select an optimal biopsy site.
   4. For ICI-related myositis, perform the biopsy on the contralateral side of the muscle with abnormal EMG findings to minimize needle-induced muscle damage.
2. Specimen handling
   1. Wrapping the muscle specimen
      1. Wrap the collected muscle specimen in gauze that has been moistened with physiological saline.
      2. Place the wrapped specimen into a culture container filled with dry ice to prevent drying and degradation of muscle structure.
   2. Fixing the muscle specimen
      1. Select a portion of the biopsied muscle, approximately 5 mm in diameter and 1 cm in length.
      2. Fix this portion vertically on a small piece of cork.
      3. Secure the muscle with gum tragacanth, mixed lightly with water.
      4. Ensure that the tissue is stable and does not fall over.
      5. Avoid excessive application of gum tragacanth that could bury the muscle.
   3. Freezing process
      1. Cooling isopentane
         1. Cool isopentane using liquid nitrogen in a well-ventilated area.
         2. Ensure that the isopentane is sufficiently cooled before proceeding.
      2. Immersing the tissue
         1. Grasp the cork holding the tissue with tweezers.
         2. Quickly place the cork and tissue into the cooled isopentane.
         3. Move the cork back and forth to ensure even freezing of the tissue.
         4. Keep the tissue in isopentane for approximately 20-30 s.
      3. Drying and storage
         1. Transfer the tissue from the isopentane to a box containing dry ice.
         2. Leave the tissue in the dry ice for about 1 h to allow the isopentane to evaporate.
         3. After evaporation, place the tissue in a sealed plastic bag or a Bayer bottle.
         4. Store the tissue in a freezer at temperatures below -70 °C.5.4.
   4. Histological processing
      1. Sectioning the frozen muscle specimens
         1. Remove the frozen muscle specimen from the freezer.
         2. Use a cryostat to section the specimen into 6 µm slices(range 5-10µm).
      2. Staining procedures
         1. Perform hematoxylin and eosin (HE) staining on the sections to visualize general tissue architecture using standard procedures.
         2. Conduct enzyme histochemistry to assess specific enzymatic activities (modified gomori trichrome [MGT], nicotinamide adenine dinucleotide tetrazolium reductase [NADH-TR], SDH, ATPase at pH 4.6/pH 10.6, acid phosphatase [ACP], alkaline phosphatase [ALP], periodic acid-schiff[PAS], oil red O [ORO]) within the muscle tissue²².
         3. Perform immunohistochemistry using the following markers: major histocompatibility complex I (MHC-I) and II (MHC-II), C5b-9, myxovirus resistance protein A (MxA), lymphocyte markers (CD3, CD4, CD8, CD20), and macrophage marker (CD68)²³.
   5. Interpretation of results
      1. Assessment of inflammatory infiltrates and pathways
         1. Examine the stained sections for patterns of muscle fiber necrosis.
         2. Look for endomysial macrophage infiltrates and myophagocytosis.
         3. Observe for strong positivity of MHC class I and II in areas of inflammation, presenting with a focal pattern on the sarcolemma.
         4. Note the presence of C5b-9 on necrotic myofibers in a non-specific pattern.
         5. Identify the abundance of lymphocytes, particularly CD8+ T cells, in the endomysium.
      2. Special considerations for DM-associated MSAs
         1. In cases with DM-associated MSAs, look for typical DM-like myopathology, such as perifascicular atrophy.
         2. Assess for severe vascular damage leading to muscle infarction.
         3. Evaluate the expression of type I interferon-inducible genes if possible²⁴.

Results

A flowchart is used to evaluate suspected ICI-myositis (Figure 1). The patient reported no pre-existing presentation or family history of IIM, nor known exposure to toxins. A review of medical history revealed that his symptoms presented 2 weeks after receiving the first round of anti-PD-1 immunotherapy for squamous cell carcinoma of the right upper lung.

Upon physical examination, the patient was conscious but unable to communicate due to generalized muscle weakn...

Discussion

The landscape of cancer treatment has been revolutionized by the advent of immune checkpoint inhibitors, which have demonstrated unprecedented benefits in terms of durable responses across a spectrum of malignancies²⁵. However, the accompanying irAEs have emerged as a significant challenge due to their multi-systemic nature and unpredictable severity²⁶. Most studies to date have concentrated on well-known irAEs affecting endoc...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ATPase Staining Kit (pH 10.6)	Solarbio	G2380	Reagent
ATPase Staining Kit (pH 4.6)	Solarbio	G2380	Reagent
C5b-9	Dako	M0777	Antibody
CD20	ZSGB-BIO	ZM-0039	Antibody
CD3	MXB	MAB-0740	Antibody
CD4	MXB	RMA-1086	Antibody
CD68	ZSGB-BIO	ZM-0060	Antibody
CD8	MXB	MAB-1031	Antibody
Cryostat	Leica Biosystems	CM1950	Instrument
Gum tragacanth powder	Ourchem	69013283	Chemical
Hematoxylin	BASO	BA4041	Chemical
Isopentane	Ourchem	XW7878406	Chemical
MHC-I	invitrogen	14-9983-82	Antibody
MHC-II	invitrogen	MA1-25914	Antibody
Modified Gomori Trichrome Stain	Solarbio	G3510	Reagent
MxA	Millipore	MABF938	Antibody
NADH-TR Staining Kit	Solarbio	N8120	Reagent
Oil Red O Staining Kit	Solarbio	G1261	Reagent
PAS Staining Kit	BASO	BA4080B	Reagent
SDH Staining Kit	Solarbio	G2000	Reagent