Correlation Between Anti-Topoisomerase I and C-Reactive Protein Antibody Level with Modified Rodnan Skin Score On Systemic Sceloris Patients

314 Indonesian Journal of Rheumatology Vol 12 Issue 2 2020

Correlation Between Anti-Topoisomerase I and C-Reactive Protein Antibody Level with Modified Rodnan Skin Score On Systemic Sceloris Patients

W isynu Tresnadi A.B

¹

, Sumartini Dewi

¹

, Rachmat Gunadi W achjudi

¹

1Rheumatology Division, Department of Internal Medicine, Faculty of Medicine Universitas Padjadjaran, RSUP Dr. Hasan Sadikin Bandung

A R T I C L E I N F O Keywords:

Anti-Topoisomerase I C-Reactive Protein Fibrosis

Systemic mRSS Systemic Sclerosis

Corresponding author:

Sumartini Dewi

E-mail addre ss:

Sumartini.dewi@unpad.ac.id All authors have reviewed and approve d the final version of the manuscri pt.

https://doi.org/10.37275/IJR.v12i2.164

A B S T R A C T

Systemic sclerosis is characterize d by extensive and progressive organ fibrosis processes leading to organ failure and death. Modifie d Rodnan Skin Score (mRSS) had been used as a clinical parameter of skin fibrosis. Anti - topoisomerase I and C-Reactive Protein (CRP) are potential biomarkers for assessing disease activity. The study was performe d to determine the association of anti-topoisomerase I and CRP antibodies with mRSS values.

We performe d an observational analytic study based on primary and secondary data. Systemic sclerosis patient sera data was obtaine d from Dewi S et al's study, taken from May 2015 to June 2017. Serum Anti - topoisomerase I antibody and CRP level analysis were performe d in December 2017.

Fifty six samples analyzed. Fifty four subjects (96.4%) out of 56 subjects are women with an average age of 37 ± 11 years, 41 subjects (73.3%) has disease duration over 2 years, 34 subjects (60.7%) has difuse system i c sclerosis, 41 subjects (73.3%) in steroid therapy and 50 subje cts (89.3%) in methotrexate therapy. The statistical analysis showed no correlati on between anti-topoisomerase I antibody and CRP levels with mRSS value s (r = 0.205, p = 0.064; r = -0.134, p = 0.167), but there was a positi ve correlation of anti-topoisomerase I antibody level with mRSS (r = 0,422 p = 0,007) and negative correlation between CRP level and mRSS (r = -0,511 p

= 0,001) in diffuse sclerosis systemic.

From this study we conclude d that anti -topoisomerase I antibody and CRP level were not correlated with mRSS, but in patient with diffuse system i c sclerosis there was a positive correlation of anti -topoisomerase I anti bo d y level with mRSS and negative correlation between CRP level and mRSS.

Introduction

Systemic sclerosis is a connective tissue autoimmune disease involving multisystem, chronic and progressive with an etiology that is not yet known.¹ Systemic sclerosis mostly affects women, aged between 40 to 55 years.^1,2 Systemic sclerosis has a high rate of morbidity, increased mortality in the last 20 years, and large funding.3,4 Treatment of systemic sclerosis is adjusted to the clinical situation of the patient. Measurement of disease activity in systemic sclerosis can use clinical parameters such as level of skin fibrosis, changes in organ involvement and laboratory parameters that describe

inflammatory activity, immune activity, fibrosis, and vascular.5 Modified Rodnan Skin Score (mRSS) is a measuring tool for assessing the thickness of the skin that has been proven in various clinical trials and correlates well with skin biopsy results as the gold standard to assess the degree of skin fibrosis.6 Modified Rodnan Skin Score better reflects skin damage that occurs compared to dynamic changes in the inflammatory process and fibrosis that are happening.

B cells play the process of fibrosis through three main mechanisms including the formation of

Indonesian Journal of Rheumatology

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autoantibodies, direct stimulation of fibroblasts by B cells with contact-dependent mechanisms, and the production of profibrotic cytokines such as Interleukin 6 (IL-6) and Transforming Growth Factor Beta (TGF-β) which increase collagen deposition and inhibit collagen degradation. Anti-topoisomerase 1 serum antibody levels are biologically potential markers in objectively measuring the activity of systemic sclerosis.2 B cells in systemic sclerosis produce interleukin-6 (IL-6) which stimulates fibroblasts to produce collagen.7 Serum IL-6 levels correlate with skin fibrosis.8 Increased serum IL-6 levels are seen in elevated levels of CRP in patients with systemic sclerosis.8,9 C-Reactive Protein (CRP) is an inflammatory marker produced and released by the liver under stimulation of cytokines such as Interleukin 6 (IL-6), Interleukin 1 (IL-1) and Tumor Necroting Factor α (TNF-α).10 This research is expected to provide information on anti- topoisomerase 1 antibodies and C-Reactive Protein in the pathophysiology of fibrosis in systemic sclerosis and potentially as a biological markers to measure disease activity in systemic sclerosis.

Research Methods

This research used an analytic observational approach by taking primary and secondary data. The primary data were taken from examination of anti- topoisomerase I and CRP antibody levels. Secondary data consisted of mRSS scores, age, sex, duration of illness, and treatment history. Anti-topoisomerase I and CRP levels were tested on serum samples from all systemic sclerosis patients who participated in the study “Uji Klinis Tersamar Ganda Ekstrak Herba

Ciplukan Terhadap Perbaikan Klinis Kelainan Kulit, Proses Inflamasi, Imunologi dan Fibrosis pada Pasien Skleroderma” by Dewi S. et al. who had been stored at -80oC at the Prodia laboratory in December 2017.

Bivariate analysis was performed with the Spearman Rank correlation test. Multivariate correlation analysis was performed when there is a significant correlation in bivariate analysis. Sub- analysis is done if there is no relationship between anti-topoisomerase I and CRP antibody levels with mRSS in bivariate analysis based on cutaneous type systemic sclerosis consisted of limited type and diffused type systemic sclerosis.

Results

There are 56 serum samples of systemic sclerosis patients that can be analyzed. Characteristics of research subjects are shown in Table 4.1. Most subjects were female, 54 subjects (96.4%), with an average age of 37 ± 11 years. The duration of illness suffered by patients more than 2 years was found in 41 subjects (73.3%). Diffused type systemic sclerosis was found in 34 subjects (60.7%). Patients with more than 2 years of illness were found in 41 subjects (73.3%). Diffused type systemic sclerosis was found in 34 subjects (60.7%). Based on the Kolmogorov Smirnov normality test with Lilliefors Significance Correction the results obtained are age variables, body mass index, and the mRSS scale 100 values were normally distributed, whereas CRP levels and anti-topoisomerase I antibodies were not normally distributed.

Table 4.1 Research Data Characteristics

Characteristics (unit)

N=56

n (%) Average ± SB

Median (min-max) or

Age (years) 37 ± 11

Sex

Male 2 (3,6)

Female 54 (96,4)

316 Systemic sclerosis types

Limited 22 (39,3)

Diffused 34 (60,7)

Duration of disease

≤ 2 years 15 (26,8)

> 2 years 41 (73,2)

BMI (Kg/m

²

) 20,35 ± 3,21

Medication history

Methotrexate 50 (89,3)

Steroids 41 (73,2)

Cyclophosphamide 1 (1,8)

Aspilets 26 (46,4)

mRSS 17 (4 – 36)

mRSS scale 100 28,2 ± 16,2

Anti-Topoisomerase I

(unit/mL) 185,8 (0,0 – 263,3)

CRP (pg/mL) 0,256 (0,016 – 7, 689)

Bivariate analysis was performed to test the significance of the relationship of each variable.

Correlation test uses the Spearman Rank.

Transforming the mRSS variable from ordinal to numeric (interval) with a scale method of 100 before correlation analysis.

Table 4.2 Correlation of Anti-Topoisomerase I antibodies and C-Reactive Protein (CRP) with mRSS

Variables mRSS scale 100

Correlation

coefficient (r) p-value Anti-Topoisomerase I

(unit/mL) 0,205 0,064

CRP (pg/mL) -0,134 0,162

Notes: analysis uses Spearman Rank correlation Table 4.2 shows the results of the correlation

coefficient of anti-topoisomerase I and CRP antibodies to the value of mRSS, respectively r

= 0.205, p = 0.064; r = -0.134, p = 0.167 (p>

0.05) which is statistically insignificant or in other words shows there is no correlation between levels of anti-topoisomerase I and CRP antibodies to the value of mRSS. Su-banalysis was done because there was no correlation between the levels of anti-topoisomerase I and CRP antibodies to the mRSS value.

The results of bivariate analysis based on systemic sclerosis types in Table 4.3 shows that in the populations of limited type systemic

sclerosis with a correlation coefficient between the levels of anti-topoisomerase I antibodies to the value of mRSS r = 0.142, p = 0.265 with (p>

0.05) there is no correlation between the levels

of anti-topoisomerase I antibodies to the value

of mRSS. In the diffused type systemic sclerosis

population there is a positive correlation

between the levels of anti-topoisomerase I

antibodies to the mRSS value with a moderate

correlation (r = 0.422 p = 0.007), meaning that

an increase in anti-topoisomerase I antibody

levels will be followed by an increase in the value

of mRSS.

317 Table 4.3 Correlation of Anti-Topoisomerase I antibodies with mRSS based on Systemic Sclerosis

Types Systemic

Sclerosis

Types Variables

mRSS Scale 100 Correlation

coefficient

(r) p-value Limited Anti-Topoisomerase I (unit/mL) 0,142 0,265 Diffused Anti-Topoisomerase I (unit/ml) 0,422 0,007 Note: analysis used Spearman Rank correlation

The results of the bivariate analysis based on the type of systemic sclerosis in Table 4.4 show that in populations of limited type systemic sclerosis with a correlation coefficient between CRP levels and mRSS values with r = 0.060, p = 0.0.395, with (p> 0.05) there is no correlation between CRP antibody levels to the mRSS value.

In diffused type systemic sclerosis populations there is a proven negative correlation of CRP levels to mRSS values with moderate correlations (r = -0.511 p = 0.001), meaning that a decrease in CRP levels in the blood will coincide with an increase in mRSS values.

Table 4.4 Correlation of C-Reactive Protein (CRP) Levels with mRSS based on Systemic Sclerosis Types

Systemic Sclerosis

Types Variables

mRSS Scale 100 Correlation

coefficient

(r) p-value

Limited CRP (pg/mL) 0,060 0,395

Diffused CRP (pg/mL) -0,511 0,001*

Note: analysis used Spearman Rank correlation

Discussion

The results showed that the mean age of study subjects was 37 ± 11 years, this corresponds to the highest onset of systemic scellosis in the third and fourth decade.¹ The mean age differs from previous studies by Muangchan et al.¹¹ On systemic sclerosis population in Canada, the mean age of patients was 55.39 ± 12.13 years, the age difference in the two study populations was suspected because of racial differences in the two populations, there were differences in autoantibody expression and genetic factors that play a role in the pathophysiology of systemic sclerosis in the Caucasian population compared to the Asian population. The most of the study subjects were female, 54 subjects (96.4%).¹

This is consistent with the incidence of systemic sclerosis in general that occurs in women. Similar to the results of research conducted by Muangchan. et al.11, Ohtsuka et al.^9,10 and Hasegawa et al.¹² where the majority of systemic sclerosis patients in this study were women.

Most of the patients in this study had diffused type systemic sclerosis in 41 subjects (60.7%), different to the previous studies of anti- topoisomerase I and CRP antibodies by Muangchan et al.¹¹ and Hasegawa et al.¹² were dominated by patients with limited type of systemic sclerosis. This is incompatible with the epidemiology of systemic sclerosis, where systemic sclerosis is more limited type than diffused type.¹ This situation is probably

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caused by the course of limited type of systemic sclerosis that runs slowly so the act of seeking treatment is carried out when the disease starts severe.¹ The effect of disease duration is also shown by the greater number of patients suffering from systemic sclerosis for more than 2 years in the study, in contrast to patients in the studies of Muangchan et al.¹¹ and Hasegawa et al.¹² who suffer more early systemic sclerosis (≤ 2 years). This situation can affect the results of the study because the level of anti-topoisomerase I antibodies is inversely proportional to the duration of the disease.¹²

Therapy in this study subject could influence the results because the study subjects were not new patients but old patients who were stable in treatment. The use of steroids and methotrexate in this study was recorded at 73.2% and 89.3%. Very different from previous studies by Muangchan et al.¹¹ with steroid use of only 14.3% and methotrexate of 9.3%. The use of steroids and methotrexate in most patients in the study can have an influence on the results of the study.^13-16 Walker et al.¹⁷ studies to examine the clinical risk and organ manifestations in patients with systemic sclerosis shows that anti- topoisomerase I antibodies are more common in patients with diffused type systemic sclerosis (60% in diffused type systemic sclerosis; 36.1% in limited type systemic sclerosis). Anti-topoisomerase I antibodies are found about 20% of systemic sclerosis patients and mainly associated with diffused type systemic sclerosis.² Previous studies by Horimoto et al.¹⁸ showed no correlation between limited type systemic sclerosis and anti-topoisomerase I antibodies. Horimoto et al.¹⁸ studies also showed no correlation between mRSS values and levels of anti- topoisomerase antibodies in patients with limited type systemic sclerosis, as shown in this study.

Research by Gourh et al.¹⁹ explains that positive results on the examination of anti-topoisomerase 1 antibodies in patients with systemic sclerosis depend on PTPN22 gene genotype CT / TT gene polymorphisms, this is seen in Caucasian, Hispanic

and black patients. Research on PTPN22 gene polymorphisms has never been done in Indonesia.

The positive correlation between levels of anti- topoisomerase I antibodies in diffused type systemic sclerosis patients with mRSS rates in this study is in accordance with previous studies by Hasegawa et al.¹² The results of this study support that anti- topoisomerase I antibodies play a role in the pathogenesis of fibrosis in patients with diffused type systemic sclerosis.

One possible lack of correlation between anti- topoisomerase I antibody levels and mRSS is the presence of systemic sclerosis has antibody and serological expression that is compatible with systemic sclerosis but there are no manifestations of fibrosis in the skin. This type of sclerosis is called sine scleroderma systemic sclerosis. This type of systemic sclerosis only occurs in less than 5% of patients with systemic sclerosis. The difference in the pathogenesis of systemic sclerosis of sine scleroderma with other types of systemic sclerosis is not yet known, but currently experts believe that sine scleroderma systemic sclerosis is part of a limited type of systemic sclerosis.

The results of this study differ from previous studies by Muangchan et al.¹¹ who concluded that there was a positive correlation with a small to moderate degree between CRP levels and mRSS values. A meta-analysis by Muangchan et al.¹¹ also explains the role and correlation between CRP levels and mRSS values.

CRP levels in this study were (median (min-max)) 0.256 (0.016-7, 689) pg/ml, this is different from large studies of CRP levels in patients with systemic sclerosis by Muangchan et al.¹¹ with average CRP levels 9.54 ± 19.98 (pg/mL), CRP values considered high if more than 8 pg/ml. Kelley et al.²⁰ explained that CRP levels in Asian populations were lower compared to African-American and Caucasian populations. Low levels of CRP conventionally measured in this study allow the explanation by Ohtsuka et al.⁹, that conventionally examined CRP

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does not provide useful information for monitoring inflammatory changes in SSc.

C-Reactive Protein is a protein consisting of 2 types, namely pCRP (pentameric CRP) and mCRP (monomeric CRP), this division is based on the conformational form of CRP.²¹ Examination of conventional CRP levels detects pCRP more, while mCRP levels can be detected with Hs-CRP.²² CRP levels in serum samples stored below -80 °C, such as in this study, will remain stable even up to 11 years.²³ The process of thawing and freezing repetitive serum samples can change the conformation of pCRP to mCRP in vitro so that if conventional CRP tests are obtained the results of CRP tend to be low but if examined with Hs-CRP will get higher CRP levels.²³

CRP levels that are negatively correlated with the degree of fibrosis as measured by mRSS have never been found in previous studies. Different conclusions obtained in this study compared with previous large studies, may be due to several reasons including;

CRP levels tend to persist in advanced systemic sclerosis, use of systemic glucocorticoids, the speed of CRP decreases faster than the decrease in mRSS values, and CRP levels that depend on clinical and serological phenotypes of systemic sclerosis and are associated with CRP RS1205 gene polymorphism.

73.2% of patients in this study had systemic sclerosis for more than 2 years, so it was classified as advanced systemic sclerosis. Research by Ohtsuka et al.⁹ shows that CRP levels tend to persist with longer duration of disease, CRP is more elevated in the early stages of the disease when cytokines cause more inflammation. A smaller increase in CRP levels in the later stages of the disease occurs when the fibrosis process is greater than inflammation.² CRP levels are one of the biomarkers used in the European Disease Activity Index, but because the study population is mostly done on Caucasian races, the results may not be the same if used for the systemic sclerosis population in Asia. C-Reactive Protein reflects the inflammatory process more than fibrosis, therefore CRP levels for monitoring fibrosis directly in patients

with systemic sclerosis cannot be applied yet.²⁴ The use of CRP levels as a marker of disease activity is limited by its low specificity for systemic sclerosis.²⁴

73.2% of patients in this study were on glucocorticoid treatment. Research by Liu et al.¹³ shows that administration of systemic steroids can reduce the production of cytokines such as IL-6 and IL-1, while CRP is mainly produced by hepatocytes via IL-6 stimulation as an innate nonspecific immune response to systemic inflammation.² Monitoring of mRSS values based on the course of the disease from a longitudinal study by Czirzak et al.²⁵ shows that mRSS will change 3-6 months after treatment, while CRP levels circulate in the blood for 6-10 hours after the acute inflammatory process and tissue destruction. CRP levels peak within 48-72 hours and then decrease, but in chronic inflammatory processes such as systemic sclerosis the levels can persist.²⁶ In diffused type systemic sclerosis the process of skin thickening increases in the early stages of the disease, peaking within 12-18 weeks, then experiencing periodic decreases as the duration of the disease.

Interleukin-6 in this study was not represented by CRP levels. C-Reactive Protein is only one of the markers of inflammation produced when IL-6 increases. Interleukin-6 as a profibrotic cytokine according to research by Stuart et al.²⁷, the increase is not always in line with an increase in CRP, this is caused by systemic connective tissue diseases such as systemic lupus erythematosus and systemic sclerosis, the normal acute phase response by the liver is often lacking. The mechanism underlying this liver disorder requires further investigation, but not because due to failure to produce an appropriate cytokine signal.²⁷ Research might be better by including the IL-6 level variable.

.28.29.28. Krylov et al.²⁸ in their study found that CRP levels were higher in patients with diffused type systemic sclerosis compared with limited types.

According to Wypasek et al.²⁹ the CRP RS1205 gene has 3 genotypes namely RS1205 CC, RS 1205 CT and

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RS1205 TT and based on the Krylov et al.²⁸ study in patients with systemic sclerosis CRP levels were higher in patients with CRP genes RS1205 CC. Krylov et al.²⁸ study was conducted in a population of Caucasian patients in the Russian region. Research on the CRP RS1205 gene subtype in Asian populations, especially Indonesia, has never been done before.

This study has several limitations that can affect the research results. Limitations in this study include: The study subjects were not new patients but were old patients with a duration of disease more than 2 years and had received therapy that could be a confounding factor in the results of the study, low levels of C-reactive protein were measured by conventional CRP examination, serum samples that were used in this study although stored at -80^oC, but there have been several thawing and freezing cycles that will affect the results of this study, this study did not predict differences in CRP results in Caucasian and Asian races which turned out to be different, since the initial study sample size was not calculated based on differences in types of systemic cutaneous sclerosis so that conclusions cannot be drawn precisely.

Conclusion

This study can be concluded that there is no relationship between anti-topoisomerase I and CRP antibody levels with mRSS values, but in diffused type systemic sclerosis there is a positive correlation between anti-topoisomerase I antibody levels with mRSS, and a negative correlation between CRP levels with mRSS values.

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