Autoimmune Myositis The Role of the Laboratory in Diagnosis and Phenotyping Sponsored by LabCorp Save

Hello. It's a pleasure to be here today and have the opportunity to speak to you about autoimmune myositis and the role of the laboratory in the diagnosis and phenotyping of SID. My name is Stanley Nades. I am a medical science liaison and discipline director in rheumatology, autoimmunity and immunology at LabCorp. And it's always fun to speak about rheumatologic topics as a fellow rheumatologist.

So I think for many of you, you understand the clinical presentation of myositis, but just to level set for those of our listeners who may be new to the area, patients with myositis present with weakness and pain of the proximal muscles of the pelvic and shoulder girdle. They're on histology, muscle biopsy, there's degeneration and regeneration of muscle fibers, and infiltration with inflammatory cells, largely mononuclear cells. Typically, there's elevated muscle enzymes, although there are instances where muscle enzymes such as creatinine kinase may be normal. So, one has to keep that in mind. When there's inflammatory skin lesions, now call our myositis dermatomyositis, and so we have clinically, over the years, thought of dermatomyositis and myositis as somewhat different, but at the same time, they really are part of the same spectrum.

Besides clinical features, muscle enzymes, and muscle biopsy, myopathic changes on electron myography are very important. And of course, one wants to avoid, doing the muscle biopsy before, the electromyography so as to not have, damage that you're looking at. Where you want to do a biopsy can be guided by electromyography. And what are the findings? One finds needle insertional instability, that as you insert the needle, you get a burst of electrical activity.

We see polyphasic action potentials, whereas the cumulative action potential is when given a signal to that area of the muscle, the action potentials are various amplitudes because of the variable function of the fibers due to injury. And then obviously there's action potential dropout in a multiphasic action potential because some of the fibers are gone. So back in the days, certainly before I had my gray hair and I was coming up as a rheumatology fellow in the early '80s, we relied on a classification system for myositis that was developed by Bowen and Peter in 1975, and it was a syndromic classification. And so we classified patients as idiopathic polymyositis or idiopathic dermatomyositis, again, based on clinical findings. We also separated out a group of polymyositis and dermatomyositis associated with malignancy.

There was childhood dermatomyositis, which often looked a bit different, and polymyositis, these being associated with vasculitis, which was more prominent in children than adults. And then we had a classification of overlap syndromes with myositis, and these are typical rheumatologic diseases such as lupus, Sjogren's syndrome, systemic sclerosis, and rheumatoid arthritis. And finally, inclusion body myositis, and we'll talk more about that as we go along. As we've evolved over time, we think of the idiopathic inflammatory myopathies, and we think of them in part. Our thinking has not only been shaped now by the syndromes, but also by what we see in terms of muscle, histology.

And so we see inflammatory myopathies, and we have classified these, in part, based on antibody profiles. We're going to spend a fair amount of time talking about the antibody profiles, but also some observations regarding histology. So we now tend to think of idiopathic inflammatory myopathies as being grouped into immune mediated necrotizing myositis, statin therapy induced myositis in that group, Adult polymyositis and dermatomyositis, these are predominantly seen in women more so than men, and typically they're seen in overlap syndromes with other rheumatic diseases. And juvenile dermatomyositis, of course, hence the name would suggest in children, and inclusion body myositis, which is predominantly seen in middle aged men. The idiopathic inflammatory myopathies often have extra muscular symptoms such as skin rashes, lung disease, joint pain, arthritis, Raynaud phenomenon, and mechanics hands.

And here are some examples of the kinds of extra articular, in this instance, cutaneous, this group, cutaneous manifestations that we see. So, there's Gotren's papules, and these are erythematous papules rough in skin over the extensor surfaces of the joints. And we see that here and here, We can see what has been called a shawl sign or a necklace sign, and here you see the erythematous changes of this rash around the neck and over the shoulders, much as a shawl would be worn. And then there's the heliotrope rash that's periorbital violaceous discoloration with some local tissue swelling. So the differential diagnosis before we jump to autoimmune myositis for muscle and pain, as patients will often present, can be broad, and one has to think about other diagnoses before rushing to assume an autoimmune myositis.

There are a number of genetic myopathies, many listed here, that are of late onset, so they may first appear in young adulthood, for example. There are metabolic myopathies, such as glycogen storage diseases, fatty acid oxidation defects, mitochondrial disorders they can also appear latent. One of the clues that we're dealing with a metabolic myopathy could come from patient history suggesting that after intense exercise, they have muscle weakness, or after a long period of food denial, they have muscle symptoms. And so keep metabolic myopathies in mind. There are a number of neurological diseases that can present as mimics of myositis in terms of muscle pain and weakness, such as amyotrophic lateral sclerosis and chronic inflammatory demyelinating polyneuropathy.

And of course, for rheumatologists, our old friend fibromyalgia, in which patients will often complain of muscle pain and weakness. There are a number of endocrinopathies that are in the differential diagnosis, such as thyroid disease, acromegaly, Addison's disease, and diabetic myonecrosis. There are nutritional deficiencies, such as vitamin D, that can present with muscle pain and weakness. There are a number of drugs that can be associated with muscle pain and weakness, such as immune checkpoint inhibitors. Certainly toxins, people who work on farms might be exposed to phosphate based insecticides, for example, can present with muscle weakness and pain.

Environmental agents such as radiation myositis, graft versus host disease. Sarcoidosis occasionally can mimic muscle myositis by presenting with muscle pain and weakness, and there, the diagnosis is made by finding non caseating granulomas on muscle biopsies. Infection, trichinosis, and a number of viruses can present with muscle pain and weakness. There are a number of viral infections that can lead to myositis, and so one has to be aware of those. Sometimes we'll see myositis with influenza A and B, enteroviruses such as Coxsackie B virus, which can affect muscles as well as cardiac muscle, as well as skeletal muscle.

HIV associated myositis. We have seen cases of HIV with inclusion body myositis, polymyositis, and necrotizing myositis phenotypes. So one always has to be aware. HTLV-one, particularly endemic in areas in Japan, thanks to the early Portuguese sailors who settled in a number of villages. And as they transmigrated around the horn of Africa, picked up HTLV and brought it to Africa.

So one needs to think about this, particularly if you're in places with large Japanese populations. Epstein Barr virus and SARS CoV-two has been associated with myositis at times. There are a number of other viruses that will rarely cause myositis: mumps virus, parainfluenza virus, one of my favorite viruses, parvovirus B19, varicella zoster, West Nile, adenovirus, cytomegalovirus, dengue, herpes simplex virus two, and hepatitis B and C viruses. So the role of the laboratory began to evolve with the discovery of JO-one, which is an antibody to JO-one. And JO-one is a histidyl transfer RNA synthetase.

This is the enzyme that loads codon RNA to allow now that amino acid to be placed into a protein. So antibody to JO-one was discovered in 1980 by Nishikai and Mars Reichlin using Octamony double immunodefusion. For many of you, you'll remember this is a method that's played a critical role in the discovery of many of our autoantibodies in the rheumatology space. It's no longer used. It's been supplanted by, in many instances, by other more efficient methodologies.

But actually, we still use, alkalone double immunodefusion, for example, in looking at hypersensitivity pneumonitis precipitants, in the allergy pulmonary space. And so for those of you who may not be familiar with octolony, one takes a petri dish, puts a small layer of agarose gel that's hardened, punches out a number of wells adjacent to each other, places in one well, the antigen. In this case, it would have been a calf thymus extract, and then the other well, the patient's serum, and the antigen and the antibody would diffuse through the gel. And where the antibody and gel meet, because it's a decreasing concentration gradient as you move away from the well, Where they meet at a zone where there's optimal concentrations to form a interlocking lattice of antigen and antibody, you form a precipitant line and you can see it as a white line. And so that's how JO-one was ultimately discovered.

Then, in 1992, confirming JO-one and also finding a number of other autoantibodies in patients with myositis, Here Akata and his colleagues used immunoprecipitation of HeLaCil extracts with patient serum. So basically, they would radio label HeLaCils with S35, either in the form of methionine or cysteine. And this is a method we still use in myositis, using either HeLa cells or K562 cells, as we'll talk about in a bit. But then taking the immunoprecipitate, washing it, putting it onto a polyacrylamide gel, allowing the proteins that were precipitated to band out, based on move through the gel and based on their molecular weight, and then, take a, x-ray film, put it over the gel blot, and make an autoradiograph and look where the bands that were radioactive lit up, and that would be the molecular weight of your protein. So that's how a number of these other proteins were initially discovered.

So over time, we've seen the addition of autoantibodies to the test menu and correlating them with the clinical pathologic picture, we have developed a system for phenotyping. Now there have been a number of autoantibodies that have been discovered over the years, and we're going to walk through some of them. These are antibodies associated with myositis, Newer antibodies like CCAR1 and SP4, which we'll talk about, are important in terms of helping us to interpret cancer risk, for example, with an antibody like TIF1 gamma, and we'll talk about that. So the accumulation of antibodies and finding them associated with particular disease has allowed this classification format to become the most, popular. What we see are immune mediated necrotizing myopathies associated with HMGCR, typically statin induced and not related to statins per se, is a signal recognition protein, antibody.

And then there's a group of patients with dermatomyositis who have a number of autoantibodies, MDA-five, TIF-one gamma, NXP-two, MI-two, SAE. And then there's a group that we have overlap typically with them. So classic group is the antisynthetase antibodies, because they have a peculiar syndrome associated with myositis and interstitial lung disease. These are a number of antibodies in this group that target specific transfer RNA synthetases, and so we'll go through these, JO-one being the first discovery. We also have PL-seven and PL-twelve, EJ, OJ, KS, ZO, and or TIER.

And so these are targeting specific transfer RNAs, and they present in a similar fashion with mild or even absent, clinically absent, myositis, but pulmonary involvement. There are a number of other autoantibodies that are associated with overlap myositis. There's Rho-fifty two, it's the 52 kilodalton version of SSA, Ku, PMSCL, and RNP. And lastly, we see in about forty, fifty percent of patients with inclusion body myositis, that myositis which is associated with inclusion bodies and muscle fibers, we see an antibody to CN1A. So we're going to walk through some of these.

Many of these antibodies we consider myositis specific autoantibodies because they are associated essentially with myositis in contrast to antibodies that we might see in myositis but are also associated with other rheumatic diseases. We call those myositis associated autoantibodies. So the myositis specific autoantibodies, they're highly selective and mutually exclusive. It's very uncommon to have more than one antibody in a patient. They tend to be associated with particular clinical phenotypes within the myositis spectrum, as I suggested to you in the previous slide.

And they target either nuclear or cytoplasmic components of the cell that those proteins, or nuclear proteins, are involved in very critical functions within the cells, such as gene transcription, protein translocation, or antiviral innate immune responses. And so those that target the cytoplasmic enzymes that catalyze the binding of specific amino acids to their transfer RNA define the antisynthetase antibody syndromes. So let's start looking at some of these individual autoantibodies. So in the immune mediated necrotizing myositis group, we have HMGCR, and this stands for a MATHFUL, which is three hydroxy-three methylglutaryl coenzyme A reductase. This is an enzyme that plays a critical rate limiting, role, in the cholesterol synthesis.

It was discovered in 2010 by Immunoprecipitation. And associated with the statin use in these patients is an upregulation of HMGCR in muscle tissue, and it's thought that the upregulation, the increased HMGCR leads to cryptic epitopes on that, in the cholesterol synthesis pathway that break tolerance to muscle. There's genetic susceptibility, includes HLA DRB1 eleven oh one allele. What's interesting is that we do see patients who have HMGCR who are not statin users, and that ostensibly they have never been treated with a statin. But for some of these patients, we find that they have encountered statins in food products.

So for example, so one has to ask very carefully about a diet history and supplementation. So for example, red yeast rice, which is a dietary supplement, it contains Monocolin K lovastatin, which is generated by fermenting the yeast in the presence of a fungus, Monascus purpureus strains. And there's an increased cancer association with those who have HMGCR antibody. The other antibody that we see associated with immune mediated necrotizing myositis is signal recognition protein, SRP antibody, and SRP is a cytosolic ribonucleoprotein, which is in the endoplasmic reticulum. And that protein is responsible for directing nascent polypeptides to post translational modification.

It's a complex molecule. It's got a 7s RNA and has six polypeptides of various molecular weights. So we believe that those antibodies to signal recognition protein impair myoblast regeneration, and so we find a significant proportion, depending on the study, eighteen to thirty nine percent of patients with immune mediated necrotizing myositis have this antibody. Patients with this antibody are characterized typically by acute onset proximal muscle weakness with prominent or scattered myonecrosis and a paucity of, or very few, lymphocyte infiltrates in their muscle biopsy. But despite this, they have marked damage of their muscle.

The mean age is 40 to 50 years. There's a male dominance, female to female ratio is 1.6 to 3.6, again, depending on your study. There is an association with HLA, DRB10803 and B5001 and DQA10194. And as a consequence of the marked muscle damage, there's high CPK encountered typically. About twenty percent of these individuals will have an associated interstitial lung disease, so if you have signal recognition protein, it's important to think about, interstitial lung disease.

These people have a good response to immunotherapy, at least in some studies, and so, it's typically a good, prognosis. Now we're going to move into the dermatomyositis group, TIF1 gamma antibody. TIF1 gamma is a 155 kilodalton nuclear protein. It's a transcription factor, transcription intermediary factor one gamma. There is a homologous TIF-one alpha, which has a molecular weight of 140, and sometimes we'll see antibodies to both TIF-one gamma and also TIF-one alpha.

Patients who have TIF-one gamma have severe cutaneous disease. They may have normal muscle strength and muscle enzymes, but they get, a certain percentage of them, interstitial lung disease, about eight point six percent. So you have severe cutaneous disease, you might be fooled and have normal muscle strength or even normal muscle enzymes, you have to look for ILD in a minority. What is critical to be aware of is that patients with TIF-one gamma have increased cancer risk. But there are two new antibodies, which unfortunately are not yet in the commercial space, that have been associated with amelioration of the cancer risk, which without those antibodies, the odds ratio of having a cancer, is nine point three seven beyond normal population, and typically lung, breast, gastric, hematologic, and colon.

People who have CCAR1, which is cell division cycle and apoptosis regulatory protein one antibody, have about half a reduction in the risks, so there's some moderation. And patients with SP4 antibody have a marked amelioration of the cancer risk. So patients who have without these two antibodies, you really need to look very hard for an occult malignancy. Those who have CCA1, I'd still look fairly hard, But for SP4, the data would suggest that you can do routine cancer screening and make sure the patient has had a breast exam if appropriate, colonoscopy, a chest x-ray, a good physical exam, and not necessarily as you might with CCR1 or the absence of other antibody, need to look for PET scan scanning to find an occult malignancy. Another antibody in the dermatomyositis group is the nuclear matrix protein two antibody, or NXP2.

This is a nine thirty nine amino acid protein. It's distributed in the nuclear matrix, and so it has domains for matrix binding as well as RNA binding. It's seen in juvenile dermatomyositis and is associated with subcutaneous edema and calcinosis, And as a consequence of this, we see severe muscle disease with contractures. In adults, we can see either polymyositis or dermatomyositis, which is associated in dysphagia with a significant majority of patients at sixty one percent and a significant number who have interstitial lung disease, mostly older individuals. Now, the critical thing is when you see the patient first, you know, the risk of finding evidence of interstitial lung disease is low, only about nine percent of the population, but over time, several more will develop interstitial lung disease.

So one needs to stay vigilant and periodically, think about, looking for interstitial lung disease, certainly careful about retaking a history of pulmonary symptoms and occasionally thinking about, you know, doing, intermittent spirometry. Cancer associated in adults, not in children. And so, again, another myositis antibody in the malignancy group. MI2, another antibody associated with dermatomyositis. MI2 autoantibodies target the chromo domain in helicase DNA binding protein four.

And what is a chromo domain? A chromo domain is a chromatin organizer modifier. A protein of 40 to 50 amino acids commonly found in proteins associated with remodeling and manipulation of chromatin. And so that suggests the potential role for MI2 in terms of remodeling chromatin and manipulating it, and so MI2 antibodies recognize this helicase DNA binding protein four, which is a transcriptional repressor. Adult and juvenile dermatomyositis is typically what's seen.

About ten percent of all autoimmune myositis are associated with MI2, so it's one that will be encountered. It's a classic dermatomyositis. Patients with MI2 antibody have hallmark cutaneous disease. If they have MI2 with a classic dermatomyositis, they're going to have higher CPK levels versus those who don't have MI2, and they have greater muscle damage. There's myofibr necrosis in the mesial involvement and macrophage infiltration.

But fortunately, these patients tend to have a good response to therapy. Another antibody in the dermatomyositis group is the small ubiquitin like modifier-one, or SUMA-one activating enzyme, better known as SAE. And so SAE activates a ubiquitin protein, UBA2, to promote somylation of proteins. And what is somylation? Somylation refers to the activity of SUMO, small ubiquitin like modifier one, and it's part of a family of proteins of which there are five, one through five, each are about 10 to 20 kilodaltons in size.

And so SUMA biases a becomes conjugated by lysine to its target protein substrate. And once SUMA conjugation occurs, the substrate's affinities for other proteins, its localization, and its ability to block ubiquitin mediated degradation, is affected, and so it impacts on cell function. So the SAE antibody is associated with severe cutaneous involvement, mild muscle involvement. It's associated with dysphagia, interstitial lung disease, fever and weight loss, and it's fairly responsive to immunotherapy, but yet it has significant mortality, and that's typically due to malignancy. MDA-five Myositis, MDA-five is myeloma differentiation associated protein five, and this protein is a RIG I like receptor double stranded RNA helicase enzyme.

And what these proteins do is they sit in the cytoplasm as part of our innate immune response, and they chew up long viral double stranded DNA. And so that's the role for MDA5 antibody to it is associated with adult and juvenile dermatomyositis. And what's interesting and what's unique about MDA-five is that it's associated with lesions, papules that ulcerate. And you can see them here in what we would think of as a more typical Gotren's papule distribution. But what's unique about MDA-five is that you see papules on the flexor surface in the palm, and you can see small papules at the creases on the radial and ulnar aspects of the fingers, and you'll see them at adjacent fingers, and hence the term kissing papules because they lie next to each other in two fingers, for example.

So, the other unique thing about MDA-five is that patients will also complain about oral mucosal pain. And I should mention that these ulcers and papules will often ulcerate, and there's marked calcinosis. MDA-five is associated with rapidly progressive interstitial lung disease, and that is the major cause of morbidity and mortality in these patients along with the cutaneous disease. And so it's important to aggressively treat the interstitial lung disease and this. And people have used clearly high dose steroids, calcinorin inhibitors, and cyclophosphamide.

And a recent study from a Japanese group have identified good response to control of the myositis, the cutaneous symptoms, and the interstitial lung disease long term with very early aggressive intervention with triple therapy. So, the overlap myosidities include the transfer RNA synthetase antibodies, as we said, JO1 is antihistatile, PL-seven is antithreonine, and so on. And for those of you who also do ANA by IFA in your, myositis patients, JO-one, PL-seven, and PL-twelve can be picked up on ANA IFA screening if your laboratory reports cytoplasmic staining because these will give you a speckled cytoplasmic staining. So, these are the ones that are typically tested now, JO1, PL7, PL12, EJ, and OJ antibodies, KS, and ZO are relatively new and not widely available commercially yet, but they're coming. Overlapped myositis anti tRNA synthetase antibodies, as we've mentioned, are associated with myositis, but again, the myositis may be mild asymptomatic, and patients may present more with pulmonary disease.

These patients will have interstitial lung disease, they'll also have Raynaud's phenomenon, arthritis, and what we call mechanic's hands, this roughened, thickened skin over the hands. So when you think about myositis, you have to be aware of the antibodies associated with interstitial lung disease, again, the antisynthetase antibodies, MDA-five, NXP-two, and SAE. When you think about, how often you're going to encounter these antibodies, clearly, if you look at JO-one, about eleven percent in the setting of, dermatomyositis, The other, antisynthetase antibodies are not very common, but collectively, the antisynthetase antibodies make about twenty percent of dermatomyositis, and a significant number of patients who would be called polymyositis. What I wanted to point out with this slide is the cancer associated myositis is twenty five percent EJ. I wouldn't be too misled because the studies are very small and because EJ is not very common.

But if you look at the more common, antibodies that are associated with, cancers, NXP-two and TIF-one gamma, you should definitely, keep cancer in mind in those patients. So now there are a number of other autoantibodies that we refer to as myositis associated antibodies, and these are seen in the setting of other rheumatic diseases, frequently without myositis, but they can be seen in overlap syndromes. And so anti PMSCL100 and systemic sclerosis overlap syndromes, These are associated with digital ulcers and ILD, interstitial lung disease. We've talked about SSA 52 kilodalton version, not the 60 kilodalton version seen in SLE, Sjogren's syndrome, rheumatic rheumatoid arthritis, systemic sclerosis, dermatomyositis, malignancies, fibromyalgia, and NA positive individuals. So if you're thinking about SSA positivity in someone who you suspect may have a myositis or an overlap syndrome, you have to be careful to look for the 52 kilodalton protein specifically.

Many of our tests that are done for SSA use an immunobead method, which is a multi immunobead method, which is good for detecting SSA, but there are two separate beads in that array. One is for 52 kilodaltons and one is for the 60 kilodaltons. But when the commercial testing was approved by the FDA, the individual analyte beads were not approved by the FDA, but rather they were bunched, they were batched together. And so one will get a report for SSA, and one doesn't know if it's the 52 kD version or the 60 kD version that made it positive or both. And so one has to think about doing an ELISA, which is an alternative method, which can isolate, RO52 and report on antibodies specifically to so that's good to think about.

And we see Rho52 antibody in a host of other diseases, lupus Sjogren's syndrome, rheumatoid arthritis, systemic sclerosis, again, dermatomyositis. We can see it in malignancies, you know, as a significant proportion of individuals with fibromyalgia complain of sickest symptoms, dry eyes, dry mouth, and who may have, a RO-fifty two antibody, and we can see it in a number of individuals who were just ANA positive. Anti Ku antibody is another myositis associated antibody, and that's associated with systemic sclerosis overlap syndromes. Typically, patients have skin changes, myositis, renounced phenomenon, sickest symptoms. There are a number of ribonucleoproteins that are considered myositis associated antibodies, certainly anybody to U1RNP, which is, seen in mixed connective tissue disease.

And when U1RNP is in, high titer and is isolated in the absence of other autoantibodies that we associate with rheumatic diseases, we think of mixed connective tissue disease, but it can also occur with other antibodies in the setting of lupus and idiopathic immune, myositis as part of an overlap. The U2RNP is commonly associated with scleroderma myositis overlap syndromes, and U3RNP, also known as vivolaren, is mostly seen in systemic sclerosis myositis overlaps, in which typically you see this in four to ten percent of patients who have systemic sclerosis, and less than two percent with limited sclerosis. Patients who have U3RNP have associated with it, a pulmonary arterial hypertension, as well as myositis, and they can have cardiac and renal involvement. And finally, inclusion body myositis. Inclusion body myositis in a significant minority of patients will have associated with it CN1A antibody.

CN1A stands for cytosolic five prime nucleotides 1A. This is a muscle enzyme that hydrolyzes adenosine nucleoside monophosphates, releasing inorganic phosphate and the corresponding nucleosides. So it has an impact on, metabolism of nucleic acids. It's predominantly seen in patients over 50 years of age. The testing for CN1A antibody will be positive in about forty six point nine percent of these patients with inclusion body myositis, but its specificity is high.

You don't see this test being positive in patients with diseases other than spontaneous inclusion body myositis. CPK levels tend to be mildly elevated, and histologically, if you look at the patients who have CN1A antibody, compared to those who don't, they tend to have fewer rim vacuoles, hence the inclusion bodies, in muscle tissue. So, it's suggesting that there may be a different process, in those who are negative for the antibody with, spontaneous, inclusion body myositis. Patients who have the antibody, however, are more frequently going to have dysphagia than those who don't have the antibody. So that always needs to be looked at.

And the importance of dysphagia was struck home, I'll tell you an old doc story, was struck home early in my residency, being asked to see a woman who had a respiratory arrest. And the story was that she had been admitted the night before and with suspected myositis, had mild muscle weakness, and given a regular diet. And so she had a bolus of food that blocked her airway. She had dysphagia and was unable to move it, had an arrest, and was resuscitated fortunately, but spent a very long period of time on a respirator because of complication of her muscle weakness, before she was able to recover. And probably writing for a liquid diet, or at least a soft diet, would have saved her journey through the ICU.

So it's very important to keep these other symptoms, other system system, not just the skeletal muscle and mind, and taking care of our patients. So how do we test for these antibodies? The gold standard test is immunoprecipitation using radiolabeled HeLa cells or K562 cells. K562 cells are a hematologic cell line, and HeLa cells made famous by Helen Lax, who donated HeLa cells to the world of medical research and patient care. So, one does a gel electrophoresis of the immunoprecipitate and makes an autoradiograph, puts an x-ray over.

This is considered the gold standard as it's available in a number of research based laboratories, often with prolonged turnaround times. And it's still a method that we use, and there may be other commercial labs using it, although there are a number of labs that have stepped away from this to look at other methods that allow the labs not to use radioactivity in the lab, which is complex in terms of managing. And as you can look at the gel, interpretation requires a very informed clinical laboratory scientist to do this because one has to look at a line on essentially a blot and know where the approximate molecular weight is and where the protein that precipitated, that's now radioactive, is seen on gel. And the other thing that is confounding is that you'll notice, for example, TIF1 gamma runs at near MI2, has some bands. And let's look at NXP2 and MDA5 since they're really single bands and not complexes of bands.

Here's an example. NXP2 is running at about 150 kilodaltons, MDA-five is running at about 150 kilodaltons. And so if you were looking at these in isolation without the labels for the positive controls that have been put on this blot, you would have a hard time knowing which protein you were looking at. And so, as backup, there have been a number of other methods that have been used to look for some of these autoantibodies. So, line blots have been used.

And a line blot avoids the uncertainty of assessing an autoradiograph block location, so you know what is the correct molecular weight. Basically, it's a Western blot. And a typical Western blot will use a cell lysate, run lysate out on a gel. The proteins separate based on the molecular weight and mobility through the gel. And then typically, one puts a filter paper over the gel.

It's hard to work with a gel because it's like Jell O. And so one puts a firmer filter paper over the gel, and the proteins, based on charge, will migrate onto the filter paper, sometimes helped by putting the gel and the filter paper in an electrical field. But now, once the proteins are on the gel, or excuse me, on the filter paper, you can cut out a strip and you can take the strip, and put it into a well, add patient serum, and if there's antibody to any of the components, it'll glom on. You rinse off what doesn't stick. You come back with typically a goat anti human IgG that is conjugated to alkaline phosphatase, and in the presence of a color developer, you see a band.

Again, on a Western blot, you have the challenge understanding what is the molecular weight. Or if you see where you think the molecular weight is, is it 150, 155, or if it's 150, is it your protein of interest, or is it another protein that happened to be labeled and was pulled down by an irrelevant antibody? And so to resolve this, manufacturers have taken purified proteins or recombinant proteins and put them onto a filter strip in a small chip and then take these chips and line them up on a plastic strip backing. And so now you have a whole array of targeted proteins that you can do a Western like blot and some mind blot, but you know if it's positive, you know what you put on that chip, and you know where it is. And so, you can identify it easily.

And you could also make this assay semi quantitative by doing densitometry on the line on the blood. For select antibodies, we have ELISAs, which are confirmatory and can be quantitative. And so at LabCorp, we have a number of panels for myositis specific antibodies. We have a full panel, if you will, MyoMarker III plus profile. It's performed at our Rheumatology Legacy Laboratory in Calabasas, California.

There are a number of markers that are used in the myositis specific antibodies, and we also include a number of myositis associated antibodies. And to help look at subsets of these antibodies. We have a number of other laboratory tests available. We have an antisynthetase profile, and we have serum recognition protein in MI2. We do these on gels by radioimmunoprecipitation and to help differentiate antibodies like NXP2 from MDA5, for example, we have ELISAs which are analyte specific, and we have one for TIF1 gamma, MDA-five, NXP-two, and SAE-one.

We also have ELISAs for HMGCR antibody and for CN1A antibody for the statin induced myositis for HMGCR and the CN1A for inclusion body myositis. And so with that, I say thank you. Thank you for your time and for listening, and I would be happy to entertain questions in the question and answer period. Thank you. Hello.

Let me start by, saying thank you for listening, and I do wanna make one comment. When I was back in medical school, I I asked what did HeLa stand for in as in HeLa cells, and I was told Helen Lang. That was misinformation. And so I occasionally, slip, since that was embedded so long ago. But the person who contributed the HeLaCils was a woman named Henrietta Lacks.

And to say she contributed was a misnomer, simply or misrepresentation because they were taken from her. This is long before the days of informed consent. And as a consequence, she created without any benefit to herself or without any knowledge to herself, an industry that has generated billions of of HeLa cells, that has helped medical research, along the way. For those of you who are interested to learn about her tragic story, Rebecca Sklute published a book, The Immortal Life of, Henrietta Lacks. So it's it's a good read.

I'm happy to answer some questions now. The first is how to interpret a negative HMG CR when a patient with necrotizing myopathy has been treated and their CPK level is back to normal. I think I think this question gets to the heart of a bigger question, is that are these autoantibodies, pathologic, pathogenic, or are they biomarkers? And we suspect given the role of HMGCR in cholesterol synthesis, that these antibodies are, playing a role in pathogenesis. And so finding a patient who has been treated, who had perhaps had a positive HMGCR, beforehand, with necrotizing myopathy and the CPK levels are back to normal and the HMGCR antibody is now negative, I think is a very positive thing.

If on the other hand, the HMGCR antibody was never positive, then, one has to think about signal recognition, protein and also about perhaps other causes. Perhaps there are other autoantibodies yet to be discovered in the autoimmune necrotizing myositis group that have yet to be described. So, assuming that the patient had HMGCR antibody at the onset of therapy, this is a positive, outcome. So the next question is, is there a preference in treatment for IgG versus Rituxan and corticotropin? The mainstay of therapy has been corticosteroids, particularly prednisone initially in high doses, and then for steroid sparing, immunosuppressive agents.

And typically methotrexate and azathioprine have been used. The there are a number of other therapies that have been tried with, variable success, some role in decreasing steroid dependence such as hydroxychloroquine. Hydroxychloroquine may be helpful in skin manifestations of dermatomyositis. In the question of IVIG, as a standalone treatment, it has not, been, completely effective, but it does play a role, in reducing steroid dependence. And and the same can be said for Rituxan.

There's limited studies with IVIG and Rituxan, however. The one, study, RIM study, Rituxan in myositis, did not meet, its primary endpoint, but was helpful in demonstrating some steroid sparing effect. And so these these drugs may play a role, but they, they're not mainstay of therapy. For corticotropin, I think most investigators, most physicians will use, prednisone rather than corticotropin, although as our gel, has been used and, does have some benefit. So let's go down.

We we still have some time. And let's see. Is myositis accepted as a clinical term? I think it is. It has traditionally been accepted, by physicians in terms of thinking of myositis, inflammation of muscles.

But I think the what we are talking about today is autoimmune myositis, you know, clearly inflammation of the muscle that is associated with an autoimmune response. Sarcoidosis, for example, has its own entity, if you will. We would not call that autoimmune myositis, although one wonders if there's an autoimmune process in place or perhaps an infectious process. But the, but autoimmune myositis is what we're talking about today. In terms of accepting this as a term, there is an ICD 10 code for, autoimmune myositis.

Myositis. There's also one for, myositis, comma, unspecified. And so these are diagnostic labels that are associated with ICD 10 codes for classification of of of diseases. Let's see what else we have. Okay.

Is the phenotype classification associated with autoantibodies a widely accepted classification scheme? I would say yes. This was rolled out and stressed, to a great deal as early as 2018 in the second, global conference on myositis, and I think this has continued to be broadly accepted as an appropriate, classification scheme. Okay. And let's see.

Are there good markers for muscle injury other than creatine phosphokinase? The answer is yes. Clearly, aldolase may be used as as a marker as well as the other, enzymes that we associate with muscle injuries such as AST, ALT, LDH, but just remember they're also found in liver and, other tissues. The other thing to be aware of is that, the CP CPK or CK, is, you know, has a muscle form and muscle CK. There are two isoforms, an m form and a b form.

And you can find elevated, CK, in, muscle injury. Obviously, it's predominantly, It comes as a dimer. And there's a small amount of MB, but the brain has a fair amount of BB. And so, if there is evidence of neurological injury, then it behooves you to do iso isoenzyme, characterization to make sure this is indeed coming from muscle. The other is following a myocardial infarction.

You can see, MB, elevated as well. So, again, you have to put your work in, context. Okay. Another question. Can, creatinine kinase be spuriously elevated and not due to muscle injury?

That is indeed true. You know, you can see elevated CK in, following muscle and intramuscular injections or following an EMG. So certainly, you want to measure your, CK, prior to doing your EMG or giving your patient an intramuscular injection. There are some drugs that can raise the CKs such as colchicine, chloroquine, or d penicillamine. These are drugs that we often encounter.

And there there are some drugs that can decrease excretion such as barbiturates, morphine, and diazepam. So one has to always put any laboratory finding in the clinical context. And so I think at that, I think our our time is up, unfortunately. If there are additional questions, feel free to, reach out to me. I'm always happy to speak to colleagues and, to help address questions or concerns.

So, again, thank you for attending, and, be well.