Hematological Alterations Related to Treatment with Teriflunomide and Dimethyl Fumarate in Multiple Sclerosis

Round 1

Reviewer 1 Report

Overall, this is a good peace of research work. Abstract: under conclusion it is mentioned that is "significant decrease" in leucocytes; could you pl mention significant decrease compared to what? baseline?

Materials and methods : any exclusion criteria? these may be patients on certain antibiotics, antimalarials, antiarrhythmics etc that could result in lymphocyte count changes?

Discussion: I dont know if one can, from this study conclude (As mentioned in line 160 of the discussion section) that DMF should be used as first option. There are numerous factors that have to considered and it is a matter of debate. I would rather rephrase this as we "recommend". 

Author Response

Thank you for all your comments.

I will try to respond all your questions.

1. The objective was to measure the effect of DMF or TERI on leukocyte count (time course), then 12-month data is compared to baseline data.
2. In effect, we have considered potential effects of concomitant medications, and MS patients did not take any drugs that could cause leukopenia. In case of current infection or treatment with corticosteroids the date was rejected.
3. I agree with you. But this is an author's opinion. I base this comment not only in the data of present series: a) It is well known that DMF cause lymphopenia and there is an immunosuppression with aging. In this sense, TERI is a safer treatment because it does not cause lymphopenia. b) It is well known that relapses and inflammatory activity in MRi are decreasing with the time course of MS -natural history of MS-. In this respect, MS patients do not need treatment with highly effective anti-inflammatory drugs, and concomitantly with more adverse effects. c) However, we have to treat our MS patients early with the most efficient drugs, in that way, most studies have shown that DMF is better than TERI in reducing relapses and new-T2 lesions in MRI.

Reviewer 2 Report

This manuscript examines the effect of dimethyl fumarate on lymphocyte and neutrophil numbers over a time course. Whilst the manuscript is clear and easy to read the subject matter lacks much originality and the analysis is cursory. There is some analysis of the major T cell subsets but surprisingingly there is no mention of the influences on B cell subsets or even B cells and it is therefore not surprising that the manuscript offers little over that already published (e.g. Mehta et al. 2019 Neurol 1919;92:e1724. Gandoglia et al. Neurol Neuroimmunol neuroinflamm 2017: 4:e403). As such it can offer little insight. It is known that lymphopenia does not provide suffieint insight to predict disease course but it does can have infectious consequences. The effects on absolute numbers of lymphocytes have been reported previously notably with dimethyl fumarate and therefore the study is largely confirmatory

Change Pg 1 Line ....remains unknown...to unproven, as several mechanisms have been described for each of these agents.

Table 1. Use a decimel point and not a comma 49,4 verses (49.4), 38,1 (38.1).

Having confidence intervals on the data on the figures may give some indication of variability

Author Response

Thank you for your comments.

I will try to respond all your comments.

1. This present work is based in the daily clinical practice. In the introduction item we say "The objective of this current study was to verify the influence of treatment with DMF and TERI on different elements of the white blood cells, especially on lymphocytes, in a series of patients with MS, and to compare our data with those published in the scientific literature from both clinical trials and studies performed in routine clinical practice.", then, by definition, is a confimatory study. The present study is not based in data from clinical trials and also we do not use methods that we cannot perform in our daily practice as flow cytometry (basic investigation study). However, real-life observational studies are very important to confirm data from clinical trials and to avoid any kind of bias. Routinely, we can measure T-cell subsets (CD8+, CD4+) but not B-cell subsets, and they do not have any major influency in our clinical practice. However, a great decrease in CD8+ T-cells could be related with the possibilty of develop PML like it occurs with natalizumab treatment (CD8+ T-cells are decreasing in brain of NTZ-treated MS patients).
2. If a mechanism is "unproven" is similar to "unknown". A drug can have several possible mechanisms of action, but it is also possible that these proposed mechanisms are not the underlying mechanisms.
3. Adjustments are done in the table.
4. A new figure is attached (SPSS bloxplot).

Author Response File: Author Response.docx

Reviewer 3 Report

The current study describes a comparison of teriflunomide (TERI) and dimethyl fumarate (DMF) on white blood cell populations in a cohort of 99 MS patients. Both TERI and DMF have been previously reported to alter and reduce immune cell populations, and the authors aim to compare effects of TERI and DMF. The authors observed enhanced decreases in leukocytes, lymphocytes and neutrophils with DMF compared to TERI, however there is some confusion in regards to the % decrease as described in the abstract and data presented (as mentioned below). Overall, these results are interesting and of note. However, it may be important to include a control or placebo group comparison in the study. The manuscript is generally well-written and organized, with only a few points to address prior to publication. Further points for consideration are included below.

The descriptions with respect to the immunce cell nos. in the abstract are not accurate; I believe the decrease in the immune cell nos. are at 6 months, whereas the authors tracked cell numbers for 30 months. In fact, after 30 months, neutrophil counts seem to be increased after 30 months.

Would it be possible to plot individual data points (for each patient) in Figure 1? This may better represent and depict data complexity and distribution.

Also Fig. 1, the X and Y axes need to be defined. If the y-axis is leukocyte count, this needs to be specified on the axis rather than title. Is the x-axis treatment duration?

Of importance, the y-axes on the graphs in Fig. 1, the origin should be at “0”.

There is no placebo or untreated patient group. Would there be any control/placebo group information available?

It would be helpful if the authors could describe the MS patient cohort further, whether patients comprised primary, secondary or RRMS forms. Should the cohort consist of a mixture, it may be interesting to see whether trends would vary according to MS type.

Some discussion with respect to MS and immunological susceptibility to infection may be helpful.

Author Response

Thank you for your comments.

I will try to respond all your comments.

1. The reported data in the abstract (12-months after treatment) are right and they are in agreement with data reported in the text (12 and 24-months after treatment). This is a real-world study from daily clinical practice and "we included patients with MS who had received treatment with TERI or DMF for at least 6 months". In the figure, data are shown at 6, 12, 18, 24 and 30 months, but the number of patients is different in each period (TERI group: baseline= 55, 6-month= 55, 12-month= 54, 18-month= 38, 24-month= 34, and 30-month= 18; and DMF group: baseline=44, 6-month=44, 12-month=43, 18-month= 29, 24-month= 23, and 30-month= 18). Then, the most exactly value is at 12-month (see abstract).
2. We do not compare DMF vs TERI, we have only measured the time course of leukocyte counts in every group, and we have compared the values in each period of time with baseline data. Leukocyte baseline data are significantly different in DMF and TERi groups. We analyze the trend of the data.
3. It is a real-world study, an observational study, there is not a placebo group.
4. All patients have a relapse-remiting MS form.
5. A new figure is attached (SPSS boxplot).

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

They authors have made two of the three changes I requested. They could easily have adopted them and yet they argue over the meaning of a word. They say unproven is unknown.

unknown means not known.

However, it is known that teriflunomide works by inhibitng pyrimidine synthesis by blocking dihydroorotate as a working mechanism, it is known that dimethyl fumarate blocks Nrf2 and importantly hydroxycarboxylic acid receptor 2.We know this from animal studies but it is difficult to definately prove in humans

Author Response:

I agree with what reviewer 2 comments and accordingly "unknown" should be changed to "unproven" as the reviewer has suggested. Thank you.