Background

Relapsing multiple sclerosis (RMS) is characterized by recurrent episodes of inflammatory demyelination in the brain and spinal cord. Despite recommendations to consider disease activity and severity levels when determining the right treatment strategy, no consensus exists on how to classify patients with RMS into high- or low-risk groups to prioritize certain treatment options. Given that neurofilament light chain (NfL) may represent a prognostic and monitoring biomarker in MS that may be capable of assessing disease activity, a study was conducted to confirm the prognostic value of serum NfL (sNfL) for on-study disease activity and worsening in individuals with RMS.

This Journal Club discussion will focus on a pooled analysis of 2 phase 3 trials by Ziemssen and colleagues that evaluated and sought to confirm the prognostic capability of baseline sNfL for on-study disease activity and disease worsening in individuals with RMS who were randomly assigned to receive either oral teriflunomide 14 mg once daily or subcutaneous ofatumumab 20 mg every 4 weeks for up to 30 months.¹ A panel of neurology experts from the University of Texas Southwestern Medical Center in Dallas, led by Kyle Blackburn, MD, reviewed the results of the study and evaluated the implications on clinical practice.

Methods

The study cohort included 1882 patients with RMS from the phase 3 ASCLEPIOS 1/2 trials (ClinicalTrials.gov Identifiers: NCT02792218; NCT02792231), of whom 1746 had available baseline sNfL samples. The median baseline sNfL level was 9.3 pg/mL. The researchers stratified patients into ”high” (sNfL >9.3 pg/mL) and “low” (sNfL ≤9.3 pg/mL) classifications to prognosticate the annualized rate of new and enlarging T2 (neT2) lesions in years 1 and 2, the annualized relapse rate, the annual percentage change in whole brain and regional brain volume, and disability outcomes. The investigators performed similar analyses with patients who had been diagnosed within the last 3 years and were treatment naive at baseline.

Results

A total of 870 patients (49.8%) were classified into the high baseline sNfL group, while 876 patients (50.2%) were classified into the low baseline sNfL group. The baseline high sNfL vs low sNfL was deemed prognostic of increased on-study T2 lesion formation. At year 1, the relative increase was 158% in the ofatumumab treatment group (P <.001) and 69% in the teriflunomide treatment group (P <.001). This finding persisted into year 2, with a 65% increase in the ofatumumab treatment group (P =.124) and a 46% increase in the teriflunomide treatment group (P =.003).

Among patients with high sNfL who received ofatumumab, the rate of brain volume loss between years 1 and 2 was significantly lower than that observed in patients who received teriflunomide, yielding a relative reduction of 24.5% (-0.32 vs -0.43; P =.019). The effect of treatment with ofatumumab by baseline sNfL category was consistent for reduction in thalamic volume loss (relative reduction, 40.6%; -0.56 vs -0.94; P =.003). A single assessment of sNfL at baseline was not deemed prognostic for on-study relapses or worsening of disability. The researchers reported that these findings were similar in the subgroup of patients who were recently diagnosed and treatment-naive.

Discussion

---

Kyle Blackburn, MD, assistant professor; Natalia Gonzales Caldito, MD, fourth-year resident; and Victor Salinas, MD, PhD, fourth-year resident, Department of Neurology, University of Texas Southwestern Medical Center, Dallas participated in this journal club discussion.

---

Kyle Blackburn, MD: We selected this article for discussion because recent research has shown that NfL may represent a prognostic and monitoring biomarker in MS that could help assess disease activity.

Natalia, will you speak briefly about NfL?

Natalia Gonzales Caldito, MD: Yes, NfL is emerging as a key biomarker for MS. In this article, the researchers studied NfL as a biomarker for disease activity and also in estimating a patient’s response to treatment. High NfL levels have been found to correlate with brain volume loss so measuring sNfL levels may help identify patients at higher risk for brain damage.

KB: Right, NfL is a measure of neuro-axonal damage. Several studies in MS have found that high NfL levels seems to correlate with certain measures of disease activity. Typically, higher levels of NfL occur around the time of a relapse or development of new gadolinium-enhancing (Gd+) lesions. A decrease in NfL levels is observed in patients who are undergoing treatment for MS. I think people are really excited about this marker as a potential adjunct to the traditional ways in which we monitor disease activity in MS.

ASCLEPIOS 1/2 were 2 randomized trials assessing the efficacy of ofatumumab, which is one of the newer therapies for MS, vs teriflunomide. In these trials, they found that ofatumumab resulted in a significant reduction in the relapse rate and in the development of neT2 lesions and Gd+ lesions. This ultimately led the US Food and Drug Administration (FDA) to approve ofatumumab for the treatment of RMS. Dr Salinas, can you review the analysis of the trials?

Victor Salinas, MD, PhD: Yes, patients pooled from these 2 trials were stratified on the basis of their sNfL levels using a cutoff that was actually derived from the distribution of all sNfL levels across the patients, taking the median value at baseline to designate patients into a high sNfL group and low sNfL group. The study sought to determine what kind of prognostic information is contained in these baseline sNfL measurement groups. The researchers went on to compare aspects of these 2 cohorts, namely the appearance of neT2 lesions at various time points, relapse rates, disability progression, and other radiologic findings, including brain atrophy and localized brain atrophy.

KB: It is an interesting way to look at things. They were trying to assess the prognostic value of sNfL levels. They split the patients into 2 groups: approximately 50% above a certain threshold and 50% below it based upon that median sNfL level in the study. They then looked at the outcomes of the trial, eventually comparing the treatment outcomes between ofatumumab and teriflunomide. In clinical practice, we often will have a patient on an early disease course and we have to try to apply some logic or some measure like T2 lesion formation to decide whether the patient needs an aggressive treatment plan or just monitoring. It is really intriguing to think about having another data point like NfL to help us make the decision about how aggressive we should be in treating patients. Natalia, do you want to dive into the findings from the trial?

NGC: In addition to the 2 groups, there also was a subgroup of newly diagnosed patients who had no Gd+ T1 lesions at baseline. Based on the sNfL levels, the researchers investigated if there were any difference in disease progression outcomes. But, in the subgroup, the researchers also found that the higher the baseline sNfL, the greater the risk for neT2 lesions. I thought that was very interesting. I know with newly diagnosed patients, there is always a question as to which patient is going to do worse and have a more severe disease course down the line. With this new biomarker, we could potentially have more information to answer this question.

This trial also analyzed sNfL levels post treatment. It was expected that post treatment, when there is higher sNfL, there is a higher percentage of patients having more lesions within the next month. However, the reason behind the statistically significant difference in duration could not be explained. The investigators thought that perhaps it could be because of the shorter time and the sensitivity. It is also possible the Expanded Disability Status Scale (EDSS) is not sensitive enough for short-term changes and smaller changes that are harder to pick up with the EDSS.

Considering the change in brain volume post-treatment, we know that the most pronounced was thalamic atrophy. Atrophy rates in thalamic volumes were the most pronounced in the higher sNfL group, regardless of treatment assignment. There is a lot of information in this article but these are the findings that stood out to me the most. All of them align to the same idea: the higher the sNfL levels at baseline, the greater the lesions, atrophy, and perhaps tendency towards more significant disability outcomes.

KB: I think that is a really good way to summarize it. They found that if you had a high sNfL level, there was a higher rate of neT2 lesions and a higher rate of atrophy. Also, you specifically mentioned thalamic atrophy, which has always been an area of particular interest with MS. What were the clinical correlates to this, Victor?

VS: One detail that they could not find was a statistically significant difference between the 2 cohorts in the annualized relapse rate and that is something that I tried to delve into a little bit more myself. If you look at the methods, they looked at neT2 lesions and annualized relapse rate separately using these regression models. Curiously, for the neT2 lesions, their regression model did not take into account the presence of Gd+ T1 lesions, but the annualized relapse rate model did. You can see in Table 1 that the presence of Gd+ T1 lesions at baseline was higher for patients who were in the high baseline sNfL category.

It is possible that they may not have found a difference between the 2 cohorts, because it was already conveyed or controlled for by the presence of Gd+ T1 lesions at baseline. Of course, they later go on to show that when they looked at patients in the subgroup who lacked Gd+ T1 lesions at baseline, there was still a significantly higher rate of neT2 lesion formation in those with a high vs low baseline sNfL. But I thought that could be a potential reason why they did not see a difference in the annualized relapse rate.

I think that a key limitation in a lot of the clinical studies in MS, especially now with the availability of highly efficacious treatment, is that you have to accrue large patient samples and follow them for long periods of time in order to have relapses that can be compared between 2 cohorts. Of course, they also go on to talk about the confirmed disease worsening or progression, which is effectively a threshold change in the EDSS that is sustained after a certain period of time. They did not find a statistically significant difference, but you can see from the data that there is a trend towards increased disability progression between the 2 cohorts that changes with treatment.

KB: I think that you bring up really good points there. You are right, whenever study authors compared high vs low sNfL groups, they did not see a statistically significant difference in terms of relapse rates. Of course, they say that in both groups, the relapse rates were relatively low. It may be that we have not followed these patients for a long enough duration to be able to detect that difference. This is probably the same for understanding disability worsening. Certainly, the authors also mentioned that this trial was not powered to detect a difference in disability worsening with sNfL levels. In the results section, researchers looked at the sNfL levels and how the 2 different treatments in the high vs low sNfL groups affected the different outcomes that they were measuring. Natalia, will you comment on the effect of ofatumumab vs teriflunomide in these patients?

NGC: In patients treated with ofatumumab or teriflunomide, patients with a higher sNfL levels at baseline had higher atrophy rates. At 3 months and 6 months, they did not see the same results in patients in the low sNfL group but there was a significant difference in treatments outcomes. Meaning that, even when there was a high sNfL, ofatumumab was more efficacious in those markers than teriflunomide.

KB: Part of this goes back to the original ASCLEPIOS 1/2 trials. I think it is important to highlight that they did not just check sNfL levels at the beginning of the study. In the actual ASCLEPIOS 1/2 trials, they actually reported the sNfL levels. We did see a higher reduction in sNfL levels. The sNfL levels were lower in the ofatumumab-treated group vs the teriflunomide-treated group. In the results, they discuss the effect of ofatumumab on relapse rates, disability progression, and brain volume loss.

Another interesting analysis that I think is worth talking about, although not explicitly mentioned in the discussion, is that they looked at patients who did not have Gd+ lesions. I think that is really intriguing, and both of you have alluded to this. NfL is a marker that is often very elevated after a relapse or after a new Gd+ lesion. Dr Salinas, would you comment on the findings regarding patients who did not have Gd+ lesions?

VS: I think the results of those are in the supplementary figures, but they also discussed this in the text. It was the case that these patients had decreased neT2 lesion frequency in the cohort with high sNfL, which I thought was interesting. There is data showing that sNfL is a prognostic marker for progressive MS, which is characterized by the accrual of disability in the absence of T1 Gd+ lesions, or so-called active lesions. I see sNfL possibly integrating all of this. I think one of the next challenges would be to differentiate the value of sNfL as telling us something about active information and relapses vs the insidious accrual of disability or degeneration that happens gradually in patients. But going back to the original question, even in patients who were free of Gd+ lesions, there was indeed a higher frequency of T2 lesion formation in patients with elevated sNfL levels.

KB: Just to piggyback on that question, Victor, what do you think is the clinical implication for that? So again, we see patients in clinic, and we are having to advise them and make a decision on the appropriate disease-modifying therapy (DMT) based upon a lot of factors. But do you think this study makes an argument that sNfL should be part of that conversation?

VS: I think so. It can form part of our arsenal of data that we use to guide our patients to make the most informed decisions. Obviously, this paper and the ASCLEPIOS trials already provide evidence that ofatumumab is a more efficacious medication that has the ability to reduce disease activity in patients with high or low sNfL levels. I think in cases in which we have patients who might be initially more apprehensive about taking an infusion or injectable, this is data that we can use to convince them of the benefits.

We will still rely on the many clinical and demographic variables that we use to determine if a patient is at higher risk and if more efficacious treatments should be pursued. What I like about sNfL is that it seems to be able to integrate both clinical and radiographic information into a single measurement. Even with continued reliance on measures like EDSS for disability, we know that a lot of our patients suffer from other things that the EDSS doesn’t capture, including issues with sleep and mood. In this regard, the use of sNfL could help us make more informed, data-driven decisions for our patients.

KB: Natalia, what are your takeaways from this study?

NGC: I agree with everything that Victor said and I have 2 main takeaways. I think that sNfL can be used as a biomarker to assess for response to treatment in future trials. Also, I think in the clinic setting, it could be most helpful in cases in which the patient is not having obvious signs of aggressive MS but they are at that halfway point when it’s unclear if they will benefit from an infusion, injectable, or oral disease-modifying therapy. This also is true in those cases in which the patient is doing well, the EDSS is 0, but they have some lesions in the brain. The sNfL would give us that extra piece of information to help guide our decision making. However, given the novelty of this biomarker, I think that a question that has not been completely answered is the cutoff. How do we decide if the sNfL is high or low? I think that is something that still has to be further categorized.

KB: I think that is a very good point, Natalia. This study definitely divided patients into high and low groups based upon the median sNfL across the trial, but defining the meaningful cutoff is probably the biggest barrier to using sNfL in the clinical setting at the moment. Of course, there still are barriers in terms of age adjustments, because we know that NfL increases with age, so there needs to be some adjustment for age whenever an individual’s sNfL is calculated. So, yes, I agree, there definitely needs to be more standardization in terms of determining what is considered to be a high vs low sNfL level.

This article was edited for clarity and length.

Reference

Ziemssen T, Arnold DL, Alvarez E, et al. Prognostic value of serum neurofilament light chain for disease activity and worsening in patients with relapsing multiple sclerosis: results from the phase 3 ASCLEPIOS I and II trials. Front Immunol. 2022;13:852563. doi:10.3389/fimmu.2022.852563

Posted by Haymarket’s Clinical Content Hub. The editorial staff of Neurology Advisor had no role in this content’s preparation.

Reviewed July 2022

Background

Anti-CD20 therapies rapidly induce a significant depletion of peripheral B cells in patients with multiple sclerosis (MS). As individuals with MS have previously demonstrated impaired responses to COVID-19 vaccines, a study was undertaken to determine whether patients who receive a vaccine prior to initiation of anti-CD20 therapy will demonstrate better responses to a third dose of the COVID-19 vaccines.

This Journal Club discussion will focus on a pilot study by Moser and colleagues that evaluated the antibody response to a third dose of a COVID-19 vaccine in patients with MS who were either receiving long-term anti-CD20 therapy at the time they received the first 2 doses of the COVID-19 vaccine (CD20-vaccine cohort) or received the first 2 doses of the COVID-19 vaccine series prior to initiating anti-CD20 therapy (vaccine-CD20-vaccine cohort).¹ A panel of neurology experts from the University of Texas Southwestern Medical Center in Dallas led by Benjamin Greenberg, MD, MHS, reviewed the results of the study by Moser and colleagues and evaluated the implications on clinical practice.

Methods

A total of 15 participants with MS (women, 54%; mean age, 45.9 years) were enrolled into 2 cohorts. At baseline, the mean Expanded Disability Status Score of all participants was 4.5 years and the mean duration of MS was 9.7 years. The CD20-vaccine cohort included 4 participants while the vaccine-CD20-vaccine cohort included 11 participants. Both cohorts received a third dose, also referred to as a booster dose, of a COVID-19 vaccine prior to the initiation of the study while receiving anti-CD20 therapy. Antibody levels specific to the SARS-CoV-2 spike receptor-binding domain in peripheral blood samples were evaluated to compare the 2 cohorts.

Results

Among participants in the vaccine-CD20-vaccine cohort, 100% of participants experienced a measurable response to a third dose of a COVID-19 vaccine compared with 18% in the CD20-vaccine cohort. The mean antibody levels were significantly higher among the vaccine-CD20-vaccine cohort compared with the CD20-vaccine cohort (mean, 951.25±1137.96 binding antibody units (BAU)/mL vs 12.36±11.94 BAU/mL; mean difference, 938 BAU/ml [95% CI, 249-1629 BAU/mL]; P <.0001). Additionally, 75% of patients in the second cohort also demonstrated a 3.8- to 9.4-fold increase in SARS-CoV-2 spike antibody levels compared with the first 2 doses.

Discussion

---

Benjamin Greenberg, MD, MHS, professor and the Cain Denius Scholar in Mobility Disorders; Natalia Gonzales Caldito, MD, fourth-year resident; and Victor Salinas, MD, PhD, fourth-year resident, Department of Neurology, University of Texas Southwestern Medical Center, Dallas participated in this journal club discussion.

---

Benjamin Greenberg, MD, MHS (BG): Natalia, will you provide us with an overview of the study design?

Natalia Gonzales Caldito, MD (NGC): In this study, the researchers investigated the humoral response to recall antigen by COVID-19 vaccines in patients with MS who had received anti-CD20 therapies.

They separated the patients into 2 groups. The first group of patients was on anti-CD20 therapies before they received the 3 doses of vaccine. The second group received 2 doses of vaccine before starting anti-CD20 therapy and then received the third dose.

The investigators looked at the antibody responses in these 2 groups to identify if having received the COVID-19 vaccine prior to anti-CD20 therapy would make any difference in outcomes.

BG: If you had to give a 10-second, single-sentence summary to somebody, what would you say is the study’s big clinical question and how it might impact our patient treatment?

NGC: I think that the study’s most important question is whether it is important to have received the vaccine before initiating anti-CD20 therapy. That was my takeaway from this paper.

BG: I agree, but I might say it slightly differently. We have heard about concerns from the literature about the impact of anti-CD20 on preventing COVID-19 vaccine efficacy. We hear less about what anti-CD20 therapy does to an existing immunity after vaccination. So, this is about the patients who are already on therapy when they are vaccinated and it is also about patients who are vaccinated and about to start therapy. The question is whether anti-CD20 therapy will negate the vaccine.

Victor, will you walk us through the 2 cohorts? The study gives us some data about them and admittedly, this was a pretty small study.

Victor Salinas, MD, PhD (VS): The skewed demographics may in part reflect this, but certainly the study has 2 cohorts, as Natalia noted. One is a cohort that received 2 doses of the COVID-19 vaccine before the initiation of anti-CD20 therapy and the other cohort received 2 doses of the vaccine while on anti-CD20 therapy for a period of time. You can see that the mean age for the former was significantly younger than the latter. That is also reflected in the disease duration. Patients who received the booster after initiating anti-CD20 therapy had a disease duration of almost 6 years compared to more than 12 years for patients on long-term anti-CD20 therapy.

Those receiving the booster after initiating anti-CD20 therapy were less disabled, likely related to their shorter disease duration. Curiously, on average, this cohort used more immunomodulating therapies compared with patients on long-term anti-CD20 therapy (2.25 vs 1). I thought this was interesting, again reflecting the biases in the skewed small sample. But it made me think about age because we know that immune responses decline to some degree with age. Could age have been a factor in some of the results? Maybe, but I certainly do not think it explains all of differences. I also would be interested to see the effects of different types of medications on the outcomes. It would be interesting to review a larger study that looked at other types of cell therapy and how they affect the immune response to COVID-19 vaccination.

BG: Yes, and you know the duration of being on anti-CD20 therapy is the definition of the 2 cohorts. By definition, patients in the CD20-vaccine cohort had been on therapy for quite a while compared with the individuals in the vaccine-CD20-vaccine cohort.

So, we have these 2 groups. We have the CD20-vaccine cohort who are the patients on anti-CD20 therapy prior to being vaccinated. Then we have vaccine-CD20-vaccine cohort who are the patients who received both doses of the COVID-19 vaccine, started anti-CD20 therapy, and then received the booster dose. Patients in vaccine-CD20-vaccine cohort was immunologically naive to CD20-depleting therapy when vaccinated and patients in the CD20-vaccine cohort were already B-cell depleted when their first vaccine exposure occurred. The researchers analyzed the data in a couple of different ways. Natalia, I will start with you. Months after the booster dose, the researchers looked to see what percentage of patients had a measurable anti-spike protein antibodies. Will you walk us through the differences between the 2 cohorts on that data point?

NGC: In the CD20-vaccine cohort, only 18% (2/11) had measurable antibody response after booster dose. In the vaccine-CD20-vaccine cohort, 100% (4/4) developed antibody response. That tells us that the group who received the vaccine prior to anti-CD20 therapy had a much more robust response to the vaccine compared with the group who were vaccinated after anti-CD20 therapy.  

BG: Out of those 11 individuals who had been on long-term anti-CD20 therapy followed by the vaccine and then the booster, 9 of the 11 did not mount an antibody response in the absence of B cells, even with all 3 doses. This means that our patients who are on anti-CD20 therapies prior to being vaccinated have a limited ability to elicit an antibody response. 

The researchers give the absolute numbers in the paper and the absolute numbers of antibodies generated is significantly higher in patients in the vaccine-CD20-vaccine cohort. The researchers note that there is a dramatic increase in spike antibody levels in this group. Can you recall what percentage of that population had this dramatic increase in their antibody levels?

VS: Natalia alluded to this earlier and it was 75% of these patients.  You can see it in Figure 1A, where in 3 of 4 patients, the increase in the antibody levels was significantly increased after the booster.

BG: The researchers talk about the reasons for this in their discussion and I want to get your thoughts, too. But let us start with their reasoning. Victor, despite being B-cell depleted — and they show in the data that individuals in the vaccine-CD20-vaccine cohort also had a depletion in B cells — there was a significant increase in antibody levels in 75% of the individuals. How is that possible? What is their explanation mechanistically for how patients had a robust antibody response in that cohort despite the lack of B cells?

VS: Anti-CD20 therapy was initiated after patients received their first 2 doses of the COVID-19 vaccine. What they suggest is that before the existing B-cell population was depleted, it was educated based on the effects of the immunization. Then those B cells matured into different B-cell subsets, including plasmablasts that likely went on to different compartments within the body, including the bone marrow. Those were effectively resistant to depletion by the antibody such that when these patients received the booster, it stimulated antibody production from those cells.

Additionally, it is interesting that they note that the 3.8- to 9.4-fold increase in antibody levels after the booster was probably an underestimate based on the timing of when they measured the antibody levels. I think it is a compelling argument that warrants further investigation.

BG: Natalia, when the study authors are going through their explanation, they cite other studies. They talked about the OPERA I (ClinicalTrials.gov Identifier: NCT01247324) and OPERA II (ClinicalTrials.gov Identifier: NCT01412333) studies, which assessed the effect of ocrelizumab, an anti-CD20 agent, on humoral immunity markers in patients with MS² and was a much larger study than this one here. They referenced what happened to an individual’s preexisting immunity after being treated with an anti-CD20 therapy and found similar patterns to the study we are discussing.

NGC: The study authors did note that humoral immunity-based remains intact and does not wear off over time despite a patient receiving anti-CD20 therapy. I think that is a really important point to keep in mind when interpreting this study.

BG: Where I think we should end this discussion is in the clinical application of this. How should practitioners use this paper about antibody response for their patients with MS who are receiving anti-CD20 therapy and how should this study be applied to practice in the clinic? 

But first, it is worth mentioning that the authors acknowledge that there still can be T-cell responses even without an antibody response. This is an area with rich literature. In terms of antibody responses, how should this information affect the recommendations we provide our patients? I’ll give you an example and I am interested in your thoughts as well.

The authors of this paper talk about the notion of whether you should hold subsequent doses of anti-CD20 therapy to allow for B cells to replete somewhat before vaccinating patients against COVID-19 and then reinitiate anti-CD20 therapy after vaccination. Is there a role for this type of strategy or should clinicians try to administer all the appropriate vaccinations before initiating a patient on anti-CD20 therapy? Natalia, how are you going to incorporate this into your practice?

NGC: If I have a patient who comes to my practice and is in need of anti-CD20 therapy, I definitely would try to get them vaccinated before starting the anti-CD20 therapy. If I can get the patient even the first dose of the COVID-19 vaccine to develop some immunity, it would give the patient a better response to later doses of the vaccine. I am not sure whether studies have shown that patients with MS need extra doses to develop some type of significant immunity. Regardless, I would prefer to have patients vaccinated first and then begin anti-CD20 therapy if the patient is clinically stable.

BG: Victor, what are your thoughts in terms of the clinical application of the data?

VS: I agree with Natalia. It is pretty clear that when we encounter a patient who has not previously received anti-CD20 therapy, we should try to administer at least the first dose of a COVID-19 vaccine before initiating anti-CD20 treatment.

Concerning the other scenario that you described, it is worth looking at. In cases where you have an intuition about the patient’s individual clinical trajectory, you could potentially lengthen the duration between anti-CD20 therapy doses enough to where you get some degree of B-cell repopulation. The key is to know what the thresholds are. They are difficult to define, of course. Certainly, in other rheumatologic diseases in which the intervals are much longer, there may be some data that you can extrapolate. I don’t know, but I think it is worth looking at.

BG: Natalia, what would your next steps be in terms of researching these questions?

NGC: I think that that one of the future directions of this study is the need to look at timing, because even if we give a COVID-19 vaccine before anti-CD20 therapy, how long do we wait before initiating anti-CD20 therapy? Do we wait 4 weeks or 2 weeks? I think that needs to be clarified. One thing I noticed was that this study did not take into account the length of time between the administration of COVID-19 vaccines and anti-CD20 therapy. The study also did not specify how long a time had passed between anti-CD20 therapy and the booster, and I think that is also something that should be understood.

Interestingly, there is a study that showed no difference in antibody detectability according to vaccination timing since the last infusion of anti-CD20 therapy, specifically for ocrelizumab.³ However, I do not believe there is a great deal of data exploring the opposite situation (timing that has passed between vaccination and subsequent anti-CD20 therapy administration). Therefore, I think it is worth exploring this to understand if the timing makes any difference.

BG: Absolutely. In terms of time as a covariate, when you dose the COVID-19 vaccine, when you check for a titer relative to that dosing, and the timing of the anti-CD20 therapy are all variables that have to be taken into account. But I think we agree, and what I am hearing from both of you is that if you can get a patient vaccinated before you start an anti-CD20 therapy, it puts you in a great position clinically to respond to boosters and the challenges in the future. On the other hand, if you have been on anti-CD20 therapy when you get a COVID-19 vaccine for the first time, it is going to limit the efficacy of the vaccine even with subsequent doses and booster doses. It is something for our colleagues, practitioners, and patients to consider when choosing therapies and timing of therapies relative to vaccines.

VS: It is always interesting looking at data like this. We are looking at immunizations, which can be examined from the perspective of protecting people from infectious disease and public health and clearly there is a lot of benefit to vaccination. But we also can conceive of this as data on patients who are relatively immunocompromised. We are looking at the health of their immune system effectively by challenging them with some immunization and looking to see what kind of responses are able to mount. This is the kind of data that I think we are going to see more often as we try to transition away from relying on mouse models to looking at human data and allowing it to better inform our clinical decisions.

This article was edited for clarity and length.

References

1. Moser T, Otto F, O’Sullivan C, et al. Recall response to COVID-19 antigen is preserved in people with multiple sclerosis on anti-CD20 medications – a pilot study. Mult Scler Relat Disord. 2022;59:103560. doi:10.1016/j.msard.2022.103560 

2. Ziemssen T, Bar-Or A, Arnold D, et al. Effect of ocrelizumab on humoral immunity markers in the phase iii, double-blind, double-dummy, IFN β-1a–controlled OPERA I and OPERA II studies. 2016. Clin Neurophysiol. 128, e326–e327. CMSC Poster.

3. Novak S, Neilsen C, Holm DK, et al. Humoral response following SARS-CoV2 mRNA vaccination concomitant to ant-CD20 therapy in multiple sclerosis. Mult Scl Rel Dis. 2021;56:103521. doi:10.1016/j.msard.2021.103251

Posted by Haymarket’s Clinical Content Hub. The editorial staff of Neurology Advisor had no role in this content’s preparation.

Reviewed April 2022

Background

Anti-CD20 therapy has been shown to be highly effective at depleting the existing population of immune B cells in patients with multiple sclerosis (MS).¹ The goal of therapy is to remove impaired B cells from the immune system, setting the stage for repopulation with a healthier generation of new B cells. Little is known about the maturation and activation of the repopulated cells, or any other immune cells, once a course of anti-CD20 therapy has been completed in this setting.

This Journal Club discussion will focus on a study by Nissimov and colleagues that evaluated the character of the new population of B cells after anti-CD20 therapy with rituximab, as well as the effects of repopulation on the frequency, differentiation, and activity of T cells and myeloid cells.¹ The results, which showed differences in the B-cell populations before vs after anti-CD20 therapy, led our Journal Club reviewers to pose an intriguing question: Can anti-CD20 therapy be used for immune-system remodeling in the treatment of MS?

Methods

The study cohort included a heterogeneous population of 15 patients with relapsing-remitting MS who underwent B-cell depletion with rituximab 1000 mg.¹ The frequency and function of B cells, T cells, and myeloid cells were analyzed before administration of the anti-CD20 agent and at different time intervals over a period of 24 months following treatment.

The majority of B-cell types identified in the 15 study patients were either naive B cells (45.5% ± 3.1%; mean ± SEM) or memory B cells (36.8% ± 3.1%). The patients were then stratified into phenotypes by the ratio of naive B cells to memory B cells. A ratio of 1 or less was designated as memory/balanced type and a ratio greater than 1 was designated as naive type. For the balanced type, the difference between the frequencies of naive vs memory B cells could not exceed 5%.

Results

Anti-CD20 treatment was found to be effective for all study participants. When compared with the phenotypes recorded before the initiation of B-cell-depletion therapy, the repopulating B cells appeared both less mature and more activated.¹ The reappearing population had a much higher percentage of transitional B cells (58.8% ± 5.2%) compared with that before B-cell depletion (10.1% ± 1.9%), and the percentage of mature naive phenotypes was significantly altered (before treatment, 45.5% ± 3.1%;  after treatment, 25.1% ± 3.5%). The frequency of memory B cells decreased following treatment (before, 36.7% ± 3.1%; after, 8.9% ± 1.7%).

Enhanced expression of activation markers CD25 and CD69 was noted in the new population of B cells. Expression of CD25 rose from 2.1% (± 0.4%) prior to treatment to 9.3% (± 2.1%) after treatment; CD69 expression rose from 5.9% (± 1.0%) prior to treatment to 21.4% (± 3.0%) after treatment. The investigators also observed that the reappearing B cells expressed significantly higher levels of costimulatory CD40 and CD86.

Changes in T cells were also evident, including increases in naive CD4⁺ T cells (before, 11.8% ± 1.3%; after, 18.4% ± 3.4%) and CD8⁺ T cells (before, 12.5% ± 1.4%; after, 16.5% ± 2.3%), as well as decreases in terminally differentiated subsets of both CD4⁺ T cells (before, 47.3% ± 3.2%; after, 34.4% ± 3.7%) and CD8⁺ T cells (before, 53.7% ± 2.1%; after, 49.1% ± 2.7%).

Discussion

---

Benjamin Greenberg, MD, MHS, professor and the Cain Denius Scholar in Mobility Disorders; Natalia Gonzales Caldito, MD, fourth-year resident; and Victor Salinas, MD, PhD, fourth-year resident, Department of Neurology, University of Texas Southwestern Medical Center, Dallas participated in this journal club discussion.

---

Benjamin Greenberg, MD, MHS: We selected this article to review for a variety of reasons. One goal was to evaluate the findings in the context of the therapies that we currently use for MS. As you are both aware, we separate our therapies into broad categories. We have immunomodulatory therapies, immunosuppressive therapies, and then what some people call the “immune-remodeling” therapies, which involve administering a drug that depletes the immune system in order to regrow a new immune system. Classic examples of drugs used for immune remodeling are alemtuzumab and cladribine.

For the last decade at least, we have thought of anti-CD20 therapies — the ones currently approved for MS by the US Food and Drug Administration are ocrelizumab

and ofatumumab — as immune-suppressive therapies. You are used to using these drugs for the treatment of a variety of forms of MS. The idea is to start patients on the drug and keep them on it because as long as the immune system is suppressed, patients remain in remission. There is a growing question, however, about whether you can consider using an anti-CD20 drug as an immune-remodeling therapy.

For example, after suppressing the immune system for a period of time by inducing apoptosis of CD20-positive B cells, and as the drug wears off, is the patient’s immune system different from how it was prior to treatment?

I thought this paper by Nissimov and colleagues from Göttingen, Germany, would be interesting to explore because it looks from an immunologic perspective at what happens to the immune system over time after exposure to anti-CD20 therapy.

So Victor, can you provide an overview of the study design?

Victor Salinas, MD, PhD: I would say that the investigators profiled the B-cell populations in patients at various times before and after receiving medication by using cell surface markers to characterize different B cells.

BG: Yes, they did these samplings, as you mentioned, of the B cells both before and after treatment, and then they profiled several different compartments within the immune system, such as B cells, T cells, and even monocytes. Natalia, looking at this, how would you explain their categorization?

Natalia Gonzales Caldito, MD: The investigators categorized them into 5 different groups: transitional B cells, naive B cells, antigen-experienced B cells, memory B cells, and plasmablasts. Then they also divided the 15 patients into 2 groups according to immunophenotype.

BG: Along those lines, what are your thoughts about the phenotyping, using a paradigm of naive-type vs balanced-type?

NGC:  I thought that was an interesting way to do phenotyping. The investigators did it by the ratio of memory B cells to naive B cells. In the balanced type, the ratio was 1 or less, whereas in the naive group it favored the naive cells (ratio >1). In this paper, graph B in Figure 1 shows that while they make the distinction, the ratio of memory B cells to naive B cells is actually very similar in some patients.

BG: As they described it, there was a “heterogeneity” between patients. The authors assigned a cutoff point where, depending on whether the ratio is more than or less than 1, patients were split into 2 groups, but the patients in the middle are quite similar. What they are saying is that even though all of these patients have MS, they come to their B-cell-depleting therapy with different ratios of these B-cell types.

Also, before the investigators even go on to evaluate B-cell depletion, they looked at the association between B-cell populations and patients’ expanded disability status scale (EDSS) scores, as depicted by the interesting graphs in Figure 1. They definitely documented statistical significance in terms of positively correlating the frequency of naive B cells with the EDSS score. You look at that and see an r value of 0.9, which is a very significant r value. Victor, as someone who has spent a lot of time in the world of immunology and MS, what do you take away from that?

VS: Given that the EDSS data used to generate this graph were obviously from the present, the key question is: If we were to follow these patients, how would this relationship fare if we were to start with patients who are completely treatment naive? The study authors tell us that most of these patients had been treated with some agent prior to enrollment, so maybe there is some confounder — and clearly they have been treated with different agents, so I do not think that is necessarily a fair comparison — but it is pretty remarkable. This [naive B-cell status] has possible prognostic value that is worth looking into, and there’s also the question of whether it would be a useful biomarker for determining the efficacy of these therapies.

Although this is a very limited study with just 15 patients, it is the kind of relationship that you could build a research program around.

BG:  Yes, I agree with you. We think of MS as being a disease in which the immune system matures over time with antigen specificity against the cerebrospinal fluid (CSF) antigen, so you would expect more memory B cells than you have — meaning they have been primed and activated, they are antigen-specific, and they are on the hunt for CSF antigens — and that this would associate with higher disability scores. In fact, though, Figure 1F of this study shows that the reverse has happened: the more naive a population of B cells the patients had, the worse they fared.

I want to draw your attention to one of the things that the authors do, which is to remind us of the cytokine profiles associated with naive B cells vs memory B cells. They tell us that the naive B cells are the major producers of interleukin (IL)-10, whereas the memory B cells are much more likely to produce tumor necrosis factor-alpha (TNF-α), lymphotoxin, and other cytokines. So although we think of B cells as being disease-propagating in MS, you could consider a paradigm in which an overabundance of naive B cells is the thing driving disability and maybe there are protective memory B cells. It is just a different way of looking at the disease in terms of intrinsic B-cell populations, and I agree that it was counterintuitive to what you might expect.

What struck each of you the most about the data after B-cell depletion?

VS: One thing that the authors emphasize is the degree of heterogeneity in their repopulation dynamics, but clearly there is a robust result after B-cell treatment showing a change in the population subset that looks not as heterogeneous afterward since you end up with a larger population of naive B cells as opposed to memory B cells. I think that is a very robust result.

Obviously the next question is: what is the phenotype of naive B cells? The study authors do characterize it with a subset of cytokine and activation markers. We gleaned that they have a more activated phenotype, but this also gets us thinking about what else could be different, what the overall pattern might be, and in what other directions we can we go with these data.

BG: You’re right, I think it was a robust result. I do not think it was a surprise to see an augmentation of transitional B cells during repopulation after treatment with an anti-CD20 drug because you are going to repopulate from the bone marrow and the first cells we are going to see are transitional; so that did not shock me. Their IL-6 data surprised me.

You mentioned that they saw a more activated cell population. What do we do with those data?  We have treated a patient with a monoclonal antibody, we have depleted this population of naive and memory B cells, they start to grow back with transitional B cells, but then they are actually secreting more IL-6 than they would in their baseline state. Do you think we have enough data to say whether that would be a good, bad, or indifferent outcome in a patient with MS or are we not there yet?

VS: The intuition is that because the cell population is more activated, you get concerned about the phenomenon of repletion and making sure that the frequency of scheduling of this medication is such that we prevent B-cell population. The other issue is that this study is just looking at IL-6. These cells could be more complicated. There could be other cell types that have this, even the regulatory B cells. For all we know, these repopulated B cells may represent some sort of spectrum between regulatory B cells and naive B cells. Here, in this study, we have only looked at a subset of B-cell cytokines, so we have to explore this further. The main concern is that B cells are perhaps more activated, and we have to make sure that we prevent a rebound of clinical disease.

NGC: I was also surprised by why the repopulation led to a significant increase in IL-6. There were no clinical relapses in these 15 patients. so I do not know how to interpret this IL-6 increase. It is thought to be a proinflammatory cytokine related to MS disease, with some indication that IL-6 can be a marker in the CSF,² but we do not have enough information to see the significance in these patients. This needs to be studied further to ascertain whether IL-6 in the context of the repopulation of B cells is not as dangerous as it was before.

BG: Beyond the results related to activated B cells, both in terms of cell surface markers and cytokine production, there was a fascinating additional arm to this study, which was to phenotype the T-cell populations. I do not think this area has gotten enough attention over the years in terms of integrating the lymphocyte compartments between B cells and T cells.

Natalia, could you walk us through the first portion of T-cell data, outlining what they saw in terms of CD4⁺ and CD8⁺ T cells before and after B-cell depletion?

NGC: The main observation regarding the B-cell population was an increase in the CD4⁺ to CD8⁺ T-cell ratio, although the number of T cells did not change overall. For CD4⁺ T cells, the authors showed a proportional increase in naive, central memory, and effector CD4⁺ T cells, while levels of the terminally differentiated CD4⁺ T cells were decreased. Regarding CD8⁺ T cells, they saw similar changes. but the levels of memory CD8⁺ T cells were unchanged. This interesting observation made me think about how, although anti-CD20 therapies are supposed to target B cells, some studies show that T cells can also express CD20 on their surfaces.^3,4 The question is whether that is due to a lack of assimilation with the B cells or due to anti-CD20 treatments also affecting the B cells.

BG: Yes, I agree that the investigators were not able to differentiate between the T-cell population changes being an indirect consequence of depleted B cells vs a direct effect of the monoclonal antibody on those rare CD20-positive T cells. Sorting that out would be a fascinating immunologic exercise that would have repercussions for patients.

Within the discussion of the T-cell data, there was one paragraph, Victor, that — as someone who likes to talk about finding biomarkers for prognosis — I found fascinating. It was the paragraph on drawing correlations between the baseline B-cell phenotyping and what happened in terms of T-cell differentiation after therapy. I call it the “crystal ball paragraph,” where they looked at whether you had a memory-balance type vs naive-type B-cell population and what happened to T cells after depletion. Did that stand out to you and do you want to highlight the results of that analysis?

VS: I will admit that it does not [stand out to me]. I harbored other questions. One of my questions in general relates to the body of data itself. It struck me that, despite having known data for most of the patients (at least 10 of 15), the data were analyzed at the level of the population sample rather than the individual. I don’t understand why. When I see the error bars showing a statistical difference, it may just be due to the fact that the investigators were looking at medians or averages. What I would like to see is intra-individual trajectories because I would argue that there’s a significance to the trajectories between individuals. I actually did not have much confidence in the data as reported. 

I think you are talking about Figure 5, where they compare changes in the frequencies of the different T-cell subsets within the 2 populations that Natalia noted. I wanted to bring up a small point: I would have liked to see the structure of the data population. I guess there is a rationale for separating the population into naive vs balanced types, but is there actual structure in the data that supports that artificial separation? The authors did not provide this information. Those 2 things influenced the way that I looked at the study.

That said, the data do show, at least for naive cells (Figure 5A) before anti-CD20 therapy, that when the B cells re-emerged, there was a slight increase in the frequency of naive CD4⁺ T cells. It looks as if this is the case for CD8⁺ T cells too, albeit with some variability. The data show a similar increase in the frequency of central memory CD8⁺ and CD4⁺ T cells, and a decrease in the terminally differentiated cells. I think the authors emphasized that as well.

In the lower panels of Figure 5, CD62 is an activation marker and it is upregulated in cells that home to secondary lymphoid organs. This is an interesting result. It looks as if there is increased activation in the CD62 expression of CD4⁺, again suggesting that the T cells that emerge after repletion have this quasi-activated phenotype. This bears looking into, but I would like to see the data presented differently so I can see just how robust their conclusions are about the findings.

BG: Based on these data and what you have both discussed, particularly this repopulation of T cells after depletion, is it reasonable to explore anti-CD20 therapy as an immune remodeler? Is there a scientific basis to think about this as a strategy in the management of MS? After answering, can each of you briefly summarize what you consider to be a key takeaway from the paper?

NGC: I definitely think that, after reading this article and seeing the change in the immunophenotype of the B cells and T cells, anti-CD20 therapy could be considered for remodeling therapy. For me, this study brings home how anti-CD20 therapies affect not only B cells but also T cells, and it highlights the complexities behind these therapies.

VS: I also think the perspective that we may be resetting the immune system in some way is very interesting. For me, one of the big takeaways is that we can look at this study as an experiment in how we understand the immune system and its abnormal function in MS.  I think it is worth trying similar approaches to get at this question by using data from a larger number of patients.

BG: My conclusion is a combination of what both of you have said. I think it is worth looking at anti-CD20 therapy as a possible immune remodeler in some patients using their baseline immunologic phenotype. One thing I would take away from this study is that rather than treating all patients with MS in a clinical trial with the same therapy based on their initial diagnosis, a patient’s immunologic phenotype at baseline may determine the individual response or lack of response to a certain therapeutic paradigm. We may find that certain patients can use anti-CD20 monoclonal antibodies as an immune remodeler while others may just use this therapy as an immunosuppressant. I think this is definitely worthy of further study. This would be a nice way to tie together a patient’s specific biomarkers and immunologic phenotype to predict that person’s response to a given therapeutic intervention. 

NGC: Besides my concern about the small size of this study, I was also worried about the scheduling of the rituximab. Not every patient received rituximab every 6 months, which is standard practice in the clinic. Ideally, this study would be repeated in a more protocolized manner in the future.

This Q&A was edited for clarity and length.

References

1. Nissimov N, Hajiyeva Z, Torke S, et al. B cells reappear less mature and more activated after their anti-CD20-mediated depletion in multiple sclerosis.  Proc Natl Acad Sci USA. 2020;13;117(41):25690-25699. doi:10.1073/pnas.2012249117

2. Stampanoni Bassi M, Iezzi E, Drulovic J, et al. IL-6 in the cerebrospinal fluid signals disease activity in multiple sclerosis. Front Cell Neurosci. 2020;14:120. doi:10.3389/fncel.2020.00120

3. Vlaming M, Bilemjian V, Freile JÁ, et al. CD20 positive CD8 T cells are a unique and transcriptionally-distinct subset of T cells with distinct transmigration properties. Sci Rep. 2021;11(1):20499. doi:10.1038/s41598-021-00007-0

4. Palanichamy A, Jahn S, Nickles D, et al. Rituximab efficiently depletes increased CD20-expressing T cells in multiple sclerosis patients. J Immunol. 2014;193(2):580-586. doi:10.4049/jimmunol.1400118

Posted by Haymarket’s Clinical Content Hub. The editorial staff of Neurology Advisor had no role in this content’s preparation.

Reviewed February 2022