Serum Survivin in Patients with Rheumatoid Arthritis

Transcription

1 Med. J. Cairo Univ., Vol. 78, No. 1, September: , Serum Survivin in Patients with Rheumatoid Arthritis MAHMOUD M. MAHFOUZ, M.D.*; HODA ABD EL-SATAR, M.D.* and LAILA A. RASHED, M.D. * * The Departments of Internal Medicine* and Biochemistry**, Faculty of Medicine, Cairo University. Abstract Survivin is a member of the apoptosis inhibiting proteins. Suppression of apoptosis has been suggested as a key mechanism supporting selection and accumulation of distinct lymphocytes subsets in chronically inflamed joint tissues. The study was based on measuring the level of survivin in the sera of 35 RA and 15 matched control subjects. Results: Serum survivin is statistically higher in patients than control subjects with a mean value of 335 ± 119.3pg and 161±55.2pg respectively. Moreover, patients showing radiological evidence of osteopenia, cysts and erosions showed higher survivin values yet the differences were not significant. Patients were further subdivided into lower and higher survivin groups with cut off value of 325pg/ml. In patients with higher values, it was found that the mean grip strength and the length of morning stiffness, erythrocyte sedimentation rate (ESR), serum creatinine, platelet count and reduction of Hb were higher but the difference was insignificant. Patients with lower survivin values were younger and had shorter disease duration but the only unexpected results were the presence of statistically higher proportion of positive chest manifestations on chest X-ray and the presence of more swollen joints in those patients. Moreover, the mean serum survivin was positivity correlated to the length of morning stiffness and negatively correlated to the intensity of drug intake which showed only statistical difference with methotrexate. Regression analysis showed higher slope with disease modifying drugs than NSAIDs. Conclusions: The usefulness of survivin as a prognostic tool for the erosive outcome in RA is partly supported by the findings in this study, however the effect of drugs should be considered, it may be true in the cases taking no drugs, in our case the effect of DMARD seem to influence the relation pattern to a great extent. Key Words: Serum survivin Rheumatoid arthritis. Introduction SURVIVIN is a 142-amino-acid protein that belongs to the apoptosis inhibiting proteins (AIPs), which inhibits the activity caspase 3,7 & 9 and not Correspondence to: Dr. Mahmoud M. Mahfouz, The Department of Internal Medicine, Faculty of Medicine, Cairo University. the upstream initiator protease caspase 8. Survivin can thereby down regulates directly or indirectly, both death receptors mediated and mitochondrialmediated pathways of apoptosis [1]. A number of disturbances in the apoptosis machinery have been pointed out in RA patients. Inhibited apoptosis has been shown to contribute to the pathogenesis of experimental arthritis [2]. Fibroblasts from RA synovia are relatively resistant to apoptosis induced by extracellular Fas stimulation. Increased levels of soluble Fas in RA synovial fluid have been suggested as one possible explanation for this fact [3]. Bokarewa et al. [4]. Reported high levels of the anti-apoptosis survivin extracellularly in the plasma and synovial fluids of RA patients compared to a matched control subjects. Plasma survivin levels correlated strongly with their levels in synovial fluids. High levels of survivin were associated with erosive type of joint disease. They demonstrated that auto-antibody responses to survivin led to a more benign (non-erosive) course of RA. Patients with erosive RA (ERA) had a significantly higher level of survivin compared to non-erosive (NRA) patients. Comparison between the ERA patients with survivin higher or lower than 300pg/ml revealed beside erosivity an association between high level survivin and increased circulating C reactive protein as well as elevated WBC counts, indicating active inflammation. RA patients with high survivin were 16 times more likely to develop erosion in the joints compared with those with low levels of survivin after adjusting for the presence of RF, gender and the duration of RA. Survivin levels found in the supernatants and in the lysates of synovial fluids of the synovial fluid cells obtained from the same sample revealed a high correlation suggesting that survivin is produced and secreted locally in the joints of RA patients [4]. The level of survivin in the plasma 301

2 302 Serum Survivin in Patients with Rheumatoid Arthritis and synovial fluid, was found to be affected by the drugs taken by the patients, higher levels were seen in patients not receiving disease-modifying drugs (DMARD) at the time of the sampling. This effect was demonstrated regardless of the disease duration, age, WBC counts in the blood and synovial fluid and level of C reactive protein. Subjects and Methods The study included 35 RA patients recruited randomly from the patients attending the Rheumatology Department, Kasr El Aini Hospital and 15 matched controls. All patients were females, most patients were coming for follow-up and few were new cases. Active arthritis was defined as swelling, limitation of movement with either pain upon movement or tenderness. The morning stiffness if any was found out, and the presenting joint was noted. The presence of a family history, chest manifestations, secondary Sjogren s syndrome or subcutaneous nodules were found out. The type of disease onset was reported, blood pressure was measured for all the patients. Tenderness and swelling of all the joints were reported. The Ritchie index was calculated and the number of swollen joints was stated. X-ray for the hands was done and the presence of cystic changes, narrowing of the joint space, erosions, osteopenia and/or amalgamation was noted. The laboratory variables elicited included white blood cell count (WBC), hemoglobin (HGB), platelet count (PLT), erythrocyte sedimentation rate (ESR), Aspartate aminotransferase (AST), alanine aminotransferase (ALT), serum creatinine and blood urea. The presence or absence of urinary protein and rheumatoid factor (RF) were reported. The level of Survivin was measured in the blood for the patients and the controls. Blood was drawn from all participants after overnight fasting. The blood was left to clot for 30 minutes at room temperature then centrifuged at 3000 rpm for 20 minutes. The separated serum was kept frozen at 80c until analysis. Serum Survivin was measured by survivin titerzyme enzyme immuno-metric assay (EIA) Kit supplied by Assay Design, Inc. 800 Technology Drive USA. This kit used a monoclonal antibody to survivin immobilized on a micro titer plate to bind the survivin in both the standard and the samples, after a short incubation the excess sample or the standards were washed and a rabbit polyclonal antibody to survivin was added. This antibody binds to the survivin captured on the plate, after a short incubation the excess antibody was washed out then goat anti-rabbit IgG conju- gated to horseradish peroxidase was added. The excess conjugate was washed out and the substrate was added. After a short incubation the colour generated was read at 450nm and the concentration of total human survivin was calculated from the standard curve. The drugs received by the patients at the time of analysis were reported. The dose and duration was reported, in case the drug was discontinued, dose, duration and time since it was discontinued was recorded. The intensity of the drug intake was measured by the following: The dose of the drug was first divided by the lowest dose for the specific drug taken by any patient and the outcome multiplied by the corresponding duration of intake to come up with a uniform value. Statistical analysis was done by SPSS pc+. Descriptive variables were noted and the relation between the clinical, laboratory and disease activity was evaluated by Pearson correlation coefficients. Informed consent was obtained from all the patients. Results The study was designed to find out the relation of survivin with the different personal, clinical and laboratory findings in 35 Ra Egyptian patients and a matched control of 15 women having the same age and socioeconomic class as the study group. Comparison of the 2 groups is summarised in Table (1). The mean age for the RA group was 42.5 ± 10.8 and that for the control was 40.9 ±9.8 years. All were females. The mean serum survivin for the RA group was 335.0± 119.3pg/ml and that for the control 161.0±55.2pg/ml, the mean value was significantly higher than control subjects (p<0.0000). No other variables were compared between patients and control subjects. Patients were divided into 2 groups according to the serum survivin level into a low (<325) and high (325pg/ ml) or higher, the cut point was selected at the mean plus 3 SD (161+3*55) for the control group as done by Bokarewa et al. [4]. The number of patients in the first group amounted to 15 cases that for the second were 20 patients. Table (1): Mean age and serum survivin level for the RA and control. Ra Mean SD Mean Control Age Survivin (pg/ml) SD 55.2 t Sign N. Sig H. Sig

3 Mahmoud M. Mahfouz, et al. 303 The personal, clinical and laboratory finding in the 2 groups is summarised in (Table 2); the group with lower survivin was somewhat younger (41.8± 12.5 years) compared to the other group (43.1 ±9.6) though the difference was not significant. The disease duration (1.6 ±0.5 and 1.8±0.5) for the lower and higher groups was also not significantly different. The mean Ritche index, the number of swollen joints, the proportion of patients with chest manifestations and those with SRJN were surprisingly higher in the group with serum survivin less than 325pg/ml. The difference was only significant (p.048*) in case of the chest manifestations. The mean grip strength, and the length of morning stiffness indicating worse conditions, was higher in the group with serum survivin (more than 325pg/ml). The mean systolic blood pressure was similar in the 2 groups and the mean diastolic pressure was higher in the group having lower serum survivin. Though the differences were not significant. Regarding laboratory findings, the mean WBC, HGB, AST, ALT and urea were slightly higher with the group having lower serum survivin. The differences however did not reach the level of statistical significance. The means for the platelet count and the ESR mostly denoting disease activity were higher in the group with 325pg/ml of serum survivin or higher. The mean creatinine was also higher with that group. The mean values of RBCs and blood urea were almost the same in the 2 groups. The differences between the means were not different from what would be expected from a random fluctuation. The value of t and the level of significance is shown in the Table (2). Table (2): Mean and SD for the different variables according to the survivin level (pg/ml). Parameters < Stat Mean SD Mean SD t Sign Personal: Age Duration(year) Clinical: Ritchie Mean Grip X-Ray Swollen joints Morning stiffness Systolic blood pressure Diastolic bloodpressure Laboratory: WBC cc/ml RBC cc/ml PLT cc/ml HGB g/% AST mg/% ALT mg/% UREA mg/% Creatinine mg/% ESR Drugs: NSAID MTX * Hydroquine Steroid The mean drug intake was higher with the group having lower survivin, and this was seen with the drugs that were taken by a large proportion of the patients, some of the drugs namely Immuran, (taken by 3 patients) and Arthodos (taken by 4 patients) were not included in the analysis. The mean NSAID, MTX, and Hydroquine were appreciably higher in the group with lower survivin, the dose of steroids was also higher in this group however the difference was not as great as that for the other drugs. The difference was only significant in case of MTX. None of the other means was significantly different from the corresponding value for the other group.

4 304 Serum Survivin in Patients with Rheumatoid Arthritis Regarding radiological findings, the mean serum survivin level of the hands is summarised in (Table 3); the mean value in the group having cystic changes (6 cases) were equally distributed between the less and more than 325pg/ml, amounted to 346.8± 128.9, this mean was higher than the other group (332.6± 119.5), this was also seen with the 8 cases showing narrowing in the hand X-rays, where the mean was 366.4± and 325.7± for the positive (8 cases) and negative groups (17 cases) respectively. The difference between the means in the cases showing erosions (338.3 ± 115.0) compared to the other group (331.9 ± 126.5) was not as large as that for those showing cystic changes or narrowing. Patients with erosions was considered the worst prognostically and the one expected to show higher serum survivin level. Cases with osteopenia (11 cases) were accompanied by appreciably higher mean (383.1 ± 101.5) compared to any other group. The group with no osteopenia was also having the lowest mean (313.0 ± 122.3) the difference however was not significant (p.089). The cases having amalgamation of the hand joints had significantly lower means compared to the other group (p.006), however this group was limited to 2 cases. The correlation between the level of survivin and some of the relevant personal and clinical findings is shown in (Table 4), correlation with age, morning stiffness, disease duration, mean grip strength, Ritche index and the sum of the positive X-ray findings were not significant. It was negative with age, disease duration, Ritche index and the X-ray findings and positive with the other 2 variables. Some of the variables elicited showed certain cross correlations, age was negatively correlated with morning stiffness, mean grip and Ritche, the correlation was positive with disease duration and the X-ray findings. Morning stiffness was positively correlated with Ritche and the X-ray findings. The mean grip as expected was negatively correlated with the Ritche and the X-ray findings, and the Ritchie was positively correlated with the X-ray findings. It should be noted that the correlation was computed for the cases having positive reading for the 2 variables so the correlation was based on different number of pairs in each case so the level of significance would vary between the different groups. Since a significant correlation was found between the survivin level and the intake of MTX, it was interesting to find out if this would be extended to the different drugs and how the intake is cross correlated. The results are shown in (Table 5); survivin was negatively correlated with all drugs taken and was significantly correlated with MTX (r ). The relation was however quite complicated, the NSAID were significantly correlated with MTX, and Hydrokins. MTX was significantly correlated with the Hydroqine. Some of the correlations could not be computed because of the small number of the combined variables. It is important to note that combination therapy was usual in most of our cases and it was rare to find a patient taking a single drug. It is also relevant to note that the number of combinations were different in different cases and some of them were quite few indeed. The drug intake and the combination therapy was decided by the treating physician according to his judgment and the response of the patient. To examine the effect of the individual drugs on the survivin level the regression of survivin on the weighted drug intake (dose x duration) were computed, the results are shown in Table (6), the coeffient of correlation (r), the coeffient of determination (R squared), the regression equation and number of pairs in which survivin and the individual drugs was taken are shown in the table. The correlations have already been presented in Table (5), the coeffient of determination explaining the variability of survivin in blood by the intensity of drug intake; was highest with ARTH (0.543), so almost 50% of the variability of survivin could be explained by the variability of ARTH though the numbers were very small (4 pairs). It is interesting to note that the coeffients for correlation and determination were generally lower for NSTAD and STR compared to the DMARD. The 2 significant coefficients were those for MTXT and HYDR. The equations and the regression intercepts could be used to find out the survivin level by the corresponding value for the drug weight. With no MTXT the survivin level would be 410pg/ml. The value using HYDR would be very close (424pg/ml). It should be noted that in most of the cases more than one drug was taken simultaneously. The analysis according to the combination was not possible due to the small numbers of pairs however the survivin levels were quite close the minimum was 346 and the maximum was 425pg/ml. Table (3): Mean ± SD of serum survivin according to hand X-ray. Positive X-ray Mean SD Mean SD Negative X-ray t Sign X-ray: Cysts Narrowing Erosions Ostpenia Amalgamation

5 Mahmoud M. Mahfouz, et al. 305 Table (4): Correlation between survivin and clinical findings. Correlation Survivin Age Ms Durations Mean grip Ritchie AGE Morning stiffness Duration Mean grip Ritchie X-ray Table (5): Regression of survivin on the different drug weights. Drug r R squared Intercept N p Mtx Hydroquine Azathioprine Leflunomide Nsaids Discussion Suppression of apoptosis has been suggested as a key mechanism supporting selection and accumulation of distinct lymphocytes subsets in chronically inflamed joint tissues [5],synovial T- cells in RA are highly differentiated and are not expected to survive for prolonged time within inflamed joints unless their death is actively inhibited [6]. Bokarewa et al. [4] reported a higher mean survivin level in RA patients compared to a matched control, they also found that with RA patients. The level was significantly higher in patient having an erosive course of the disease. The serum survivin was closely correlated with that in the synovial fluid in the same patients. The patients recruited by the Swedish group were those having acute joint effusion to get synovial fluids. The serum survivin values in the present study were very close to those reported by Bokarewa et al. [4], they reported a survivin level of 121 ±2pg/ml for the healthy individuals and a mean of 330 ± 123pg/ml in the RA group. The corresponding figures in this study were 161 ±55 and 335± 119pg. The levels for the RA patients were the same, while that for the control was a bit higher than that in Sweden subjects. The RA patients were divided into 2 groups, a group < than 325pg/ml and another 325 or more, and the different personal, clinical and laboratory variables compared. This procedure was the same used by Bokarewa et al. [4], they found a significant association with the tendency of erosion of the joints. This was not clear in the present study. The level for the cases that showed erosions though slightly higher the differences were very small and insignificant. It may need major erosions to show since only cases with amalgamation showed significant differences. The effects of drugs on the level of survivin in the blood seem to be profound, and though the correlation was only significant with the intake of MTX it was negatively associated with all the other drugs. It is likely to be the same with the synovial fluids though, it was not determined in our patients. The magnitude of the coefficients of correlation was appreciably higher with the disease modifying drugs, including IMM ( 0.238), hydroquinine ( r ) and Arthos (r 0.737) compared to either the NSAID (0.199) or the steroids (0.190). A coefficients of correlations of this magnitude would be very highly significant if the number of the patients taking the drugs were higher. A lot of economic implications determine the availability of the drug intake in the type of patients showing up at Kasr El-Aini Hospital. Moreover the dose and combination of the drugs are determined by the attending physician according to the course of the disease and the response of the patient. No sample were taken from the synovial fluids, but the results published by Bokarewa et al. [4] point out that the levels are almost the same. Recent studies indicated that the induction of apoptosis in active RA synovial tissues is inhibited despite stimulation of the intracellular pathway(s) that lead to apoptosis. This inhibition of apoptosis was observed downstream of caspase-3 and may involve the caspase-3 inhibitors survivin [7]. The statement of Bokarewa et al. [4] about the usefulness of survivin as a prognostic tool for the erosive outcome in RA is partly supported by the findings in this study, however the effect of drugs should be considered, it may be true in the cases taking no drugs, in our case the effect of DMARD seem to influence the relation pattern to a great

6 306 Serum Survivin in Patients with Rheumatoid Arthritis extent. The regression line between MTX and survivin shown in the figure, is an example similar plots that could be constructed for other drugs if an ample size of patients could be included, a longitudinal design with patients not receiving any DMARD at the entry would be very useful [8,9]. Conclusion: Serum survivin may be used as a marker of erosive RA. Recent trials using antisense oligonuclotide targeting survivin mrna in treatment of cancers may be similarly tried in erosive RA to be added to the already exisisting list of new therapies (cytokines, angiogenesis inhibitors, and gene therapy). References 1- CHARD T. (2003): "An introduction to Radio immunoassay and Related Techniques 4 th ed. ", GORDAN J.D., et al.: J. Clin. Oncol., 21 (2): , LI F.: Survivin study: what is the next wave? J. Cell Physiol., 197: 8-29, BOKAREWA M., LINDBLAD S., BOKAREW D. and TARKOWSKI A.: Balance between survivin, a key member of apoptosis inhibitor family, and its specific antibodies determines erosivity in rheumatic arthritis. Arthritis Res. Ther., 7: R349-R HASUNUMA T., KAYAGAKI N., ASAHARA H., MO- TOKAWA S., KOBATA T., YAGITA H., SUMIDA T., OKUMURA K. and NISHIOKA K.: Accumulation of soluble Fas inflamed joints of patients with rheumatoid arthritis. Arthritis Rheum., 40: 80-86, PERLMAN H., LIU H., GEORGANAS C., KOCH A.E., SHAMIYEH E. and HAINES G.K.: 3 rd, RM Differential expression pattern of the antiapoptotic protein 2 and FLIP, in experimental arthritis. Arthritis Rheu. M, 44: , ANAK A.S.S.K., MALCOLM D.S., DAVID M.F., CHRIS- TOPHER A.H., et al.: Elevated expression of caspase-3 inhibitors, surviving and xiap correlates with low levels of apoptosis in active rheumatoid synovium, Arthritis Research and Therapy, 11: R13, PENNATI M., FOLINI M. and ZAFFARONI N.: Targeting survivin in cancer therapy: Fulfilled promises and open questions. Carcinogensis, 28 (6): 113-9, MITA A. C., MITA M.M., NAWROCKI S. T. and GILES F.J.: Survivin: Key regulator of mitosis and apoptosis and novel target for cancer therapeutics. Clin. Cancer Res., 14 (16): , 2008.