Low Dosage and Long-Term Use of Cyclophosphamide Improve the Survival of Patients with Systemic Lupus Erythematosus

Background: Cyclophosphamide (CTX) is an alkylating agent used in the treatment of systemic lupus erythematosus (SLE) for its potent immunosuppression effect. Many aspects of the use of CTX in SLE have been previously studied. However, its relation to mortality in SLE had rarely been mentioned. Thus we investigate the effect of cyclophosphamide on organ involvements and the overall and cause-specic mortality of SLE patients. Methods: Information about CTX prescription were taken from medical records in the Jiangsu Lupus database that was set up to collect data from SLE patients since their rst admission during 1999-2009 in Jiangsu province, China. Follow- up studies were carried out in 2010 and 2015 to check the survival status of the patients. Cox regression models were used to estimate hazard ratio (HR) and 95% CI. Kaplan-Meier model was used to assess the effect of CTX on mortality between organ involvements and non-involvements. Results: There were 221 deaths observed out of 2446 SLE patients. CTX users showed a lower mortality of SLE (8.4%) with adjusted HR (95% CI) of 0.74 (0.56-0.97), as compared to non-users. A decreased in overall mortality of SLE in low daily dosage of CTX, with adjusted HR (95% CI) of 0.54 (0.36-0.81) and a signicant dose-response pattern (P = 0.004) between overall mortality of SLE and long-term use of CTX with adjusted HR (95% CI) 0.53 (0.35-0.80) were observed. In cause-specic mortality analyses, protective effective of CTX was found to be insignicant. However, CTX could eliminate the differences in mortality between organ involvement and non-involvement, including renal, neuropsychiatric, cardiopulmonary, gastrointestinal and hematological involvements. Conclusion: Low daily dosage and long-term use of CTX lowers the risk of overall mortality of SLE. CTX might improve the survival of patients with renal, neuropsychiatric, cardiopulmonary, gastrointestinal


Background
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that presents with a variety of manifestations, which can be mild to severe. It ranges from arthritis or the classic rash to internal organs involvement mostly the kidneys or central nervous system [1]. The treatment regime depends mostly on glucocorticoids, antimalarials and immunosuppressive agents [2]. This has vastly improved the prognosis of SLE with an increase in the global 10-year survival rates of patients from 63.2% in the 1950s to 91.4% in 2010 [3]. Nevertheless there is signi cant high risk of mortality in SLE patients [4][5][6]. About one fourth of patients with diffuse proliferative lupus nephritis develop end stage renal failure. It is a great concern globally as it accounts for the majority of patient mortality in SLE [7]. In China, the infections are the leading cause of death in SLE over complications from kidney and central nervous system (CNS) [8].
Cyclophosphamide (CTX) is a chemotherapeutic agent that can improve outcomes in lupus nephritis (LN) [9, 10] and it has also been reported to have the therapeutic effects in both CNS and hematological involvement in lupus [11][12][13]. Lupus patients with thrombocytopenia have experienced improvement in platelet counts after treatment with CTX [13]. Several randomized controlled trials (RCTs) have demonstrated that treatments including CTX were superior to corticosteroid therapy alone in the long term due to their protective effect of renal function in patients with severe LN [9, 10, 14,15]. Furthermore, it was reported that patients with manifestation of neuropsychiatric SLE had responded well with CTX treatment where they priory failed to other immunosuppressive therapy [11]. However, few studies have estimated the effect of CTX on survival of SLE. This study aims to investigate the association between treatment with CTX and overall as well as cause-speci c mortality of SLE. Also we will assess what kind of the effect treated with CTX dosage and duration could prolong the survival of patients with SLE.

Study design and participants
In 2010, Jiangsu Lupus Collaborative Group in Jiangsu Province, China carried out a large-scale, multicentre retrospective study. All patients admitted for the rst time across 26 centres with complete medical record between 1 st January 1999 and 31 st December 2009 were enlisted in the study. Patients who satis ed four or more classi cation criteria of SLE revised and updated by the American College of Rheumatology (ACR) were included [16]. Follow ups were conducted in 2010 and 2015 respectively to monitor the survival status of the patients. More details about the study design can be found in previous studies [17][18][19].

Data collection and de nition
Following the approval of this study by the Institutional Review Board of Nanjing Drum Tower Hospital, a website (http://sys.91sqs.net/sle/Index/index.html) was set up to gather and manage data from different hospitals. The data collected from all the medical records since the rst hospitalization of SLE patients were demographic data, diagnostic information, medical history, organ involvements, laboratory tests, and type and dosage of medication.
The disease activity was measured by the SLE disease activity index 2000 (SLEDAI-2K), and the SLICC/ACR Damage Index (SDI) was used to evaluate damage to the organ system [20]. The laboratory variables which were de ned as positive were: anti-dsDNA positive, antinuclear antibody positive, anti-Sm positive, complement C3 < 0.8 g/l and complement C4 < 0.2g/l. The immunosuppressant used included CTX, hydrochloroquine, le unomide, azathioprine, methotrexate, mycophenolate mofetil, tripterygium, ciclosporin, tacrolimus and glucocorticoids.
The patients were grouped into CTX users and non-users based on the medical records. Patients who received CTX treatment in rst and subsequent hospital admission and discharge were classi ed as longterm use. The other patients treated with CTX were de ned as short-term use. The daily dosage of CTX administered was collected and in case a different dosage formula was used during the same treatment period, the mean daily dosage was calculated.

Statistical analysis
We used SAS version 9.3 (SAS Institute, Inc., Cary, NC, USA) to carry out statistical analyses. The median [interquartile range (IQR)] of continuous variables in a non-normal distribution between CTX users and non-users was compared using the Mann-Whitney U test. We used the χ 2 test to contrast the distribution of categorical variables. We calculated the hazard ratios (HRs) and corresponding 95% con dence intervals (CIs) and used Cox proportional hazards regression models to investigate the association between treatment of CTX and overall and cause speci c mortality. Ordinal values of CTX usage for the different category of dosage and duration were used in the regression models to estimate P value for trend. Strati ed analyses were performed based on the following factors: gender, age at diagnosis, SLEDAI score at rst admission, comorbidities, glucocorticoids and immunosuppressive medications, organ involvements and serology test. In addition we used the Kaplan-Meier model to compare the survival rate between organ involvement and non-involvement by CTX. Adjustments were made to the multivariate analysis models for potential confounding factors comprising of gender (male = 1, female = 0), SLEDAI score at admission (continuous), comorbidities (yes = 1, no= 0), glucocorticoid treatment on admission (yes = 1, no = 0), and immunosuppressive treatment on admission (yes = 1, no = 0). P-values were two-sided, with P less than 0.05 considered to be statistically signi cant.

Characteristics of SLE patients in the study
Among the 2446 patients with SLE, 44.0% (1077) were treated with CTX. The median (IQR) daily dose of CTX was 600 (400 -800) mg. The demographic and clinical characteristics of patients with SLE among CTX users and non-users are shown in table 1. The age of the participants is shown as median (IQR) of 31.6 (23.5 -39.6) year-old for CTX users and 32.5 (23.6 -41.1) year-old for non-users (P < 0.144). Majority of patients in the study were female and the proportion of CTX users were lower (85.0%), compared with non-users (99.5%) (P = 0.023). The SLEDAI score at admission is represented as median (IQR) of 14.0 (10.0 -20.0) for CTX users and 13.0 (9.0 -18.0) for non-users (P < 0.001). There was a higher prevalence of comorbidities (P < 0.001) as well as cardiopulmonary (P < 0.001) and renal (P < 0.001) involvement in CTX users. CTX non-users were likely to be taking glucocorticoid (P < 0.001) and immunosuppressive medication (P = 0.031). There were no differences in CTX users and non-users in terms of positive autoantibodies of anti-dsDNA, anti-Sm and antinuclear antibodies, and abnormal complement of C3 and C4.
Overall mortality There were 221 deaths (9.0%) out of 2446 patients with SLE. The HR and 95% CI for overall mortality of SLE patients according to the usage of CTX including dosage and the length are shown in Table 2. The mortality of SLE was 8.4% and 9.5% for CTX non-users and users, respectively. After adjustment was made to confounding factors, CTX users showed a lower mortality of SLE with adjusted HR (95% CI) of 0.74 (0.56 -0.97), as compared to non-users. A decreased in overall mortality of SLE was found in lower daily dosage of CTX, with adjusted HR (95% CI) of 0.54 (0.36 -0.81), but with insigni cant doseresponse pattern, (P trend = 0.259). A signi cant dose-response pattern was observed between overall mortality of SLE and a long time of using CTX (P trend = 0.004). The adjusted HR (95% CI) for long-term CTX use compared with non-users was 0.53 (0.35 -0.80).
Further analyses were made to establish the association between CTX use and overall mortality of patients with SLE strati ed by gender, age, SLEDAI score, comorbidities, organ involvements, serology test results and other medications. After adjustments were made to potential factors, we observed the associations between CTX use and mortality of patients with SLE were negative but turned to be insigni cant in some subgroups. However, the protection of CTX on mortality of SLE were still observed for female, SLEDAI score ³ 15 group, with neuropsychiatric, renal and hematological involvement, low serum C3, but without comorbidities, mucocutaneous, gastrointestinal and immunosuppressive treatment ( Table 3).
Comparisons of patient's survival treated by CTX Figure 1 presents the comparisons of Kaplan-Meier survival curves by CTX use according to organ involvement. Increased mortality were signi cantly observed in SLE patients with neuropsychiatric (P < 0.001), cardiopulmonary (P < 0.001), gastrointestinal (P = 0.025), renal (P < 0.001), and hematological (P = 0.002) involvements among CTX non-users; however, those differences in mortality between organ involvement and non-involvement were eliminated among CTX users (all P > 0.10). Moreover, high SLEDAI score (≥15) patients had higher mortality compared with low SLEDAI score (0-14) in CTX nonusers (P < 0.001) and users (P = 0.019), whereas such difference was weakened by CTX, but there were no differences for comorbidity, mucocutaneous and musculoskeletal involvement (Supplementary Figure  S1).

Discussion
As far as we know, this is the rst study that analyzed the association between CTX usage and overall as well as cause-speci c mortality in patients with SLE. In this large multi-centre retrospective study, we found an inverse association between CTX therapy and overall mortality in SLE patients. Patients having renal, neuropsychiatric, cardiopulmonary, gastrointestinal and hematological involvement may improve survivability if they were CTX users.
Up to now, there is only one study reported the association between CTX use and mortality of SLE patients. In South Korea, 413 patients who were diagnosed with SLE across three tertiary rheumatology centers between 1992 and 2016 were retrospectively evaluated; it was noted that the use of CTX was not signi cantly associated with mortality of SLE patients in univariable Cox analyses [21]. However, this study has not performed multivariable analyses for CTX use, and further cause-speci c mortality could not be examined due to the limited sample size. Moreover, previous studies reported that CTX improved renal but not overall survival [22]. Petri et al noted that clinical symptoms and survival of SLE patients with major organ injuries were signi cantly improved with pulsed high-dose CTX therapy [23]. In addition, there have been clinical studies that demonstrated the long-term e cacy of CTX in decreasing morbidity with no fatality cases in SLE patients [24].
In this present study, decreased overall mortality can be shown in patients taking low dosage of CTX (< 600mg/d), and a strong dose-response relationship was observed between decreased mortality of SLE and long-term CTX use. Previous studies have indicated that remission could be achieved with slow cumulative effect of CTX over time. Houssiau et al. showed in their study known as the "EURO-Lupus regimen" that low dose CTX provides a good alternative to high dose CTX as it yields good results clinically [9]. In their 10 years follow up, they found that low dose of CTX is bene cial in the long term as 70% of patients that took this regimen did not require any pulse of CTX following their initial therapy. However, they reported that there was no signi cant difference in mortality between the two groups treated with low dose and high dose of CTX respectively [10]. It should be noted that the patients' ethnicity was white European and most of them presented with disease of moderate severity. A recent network meta-analysis of 11 RCTs found that low dose CTX was the most e cient as induction therapy in LN and had the most effect in lowering the risk of serious infections [25]. Those evidences could support our ndings that slow cumulative effect of CTX over time might improve survival of SLE patients.
Consistent with previous studies among Chinese population, infection, neuropsychiatric and renal insu ciency were among the most common cause of death in this study [8,26]. Although decreased mortality from speci c causes of death were found to be associated with CTX users, we still do not have a su cient sample size to observed signi cant associations. However, our ndings showed that CTX has better protective effects against overall mortality in organ involvements such as neuropsychiatric, renal, cardiopulmonary, gastrointestinal and hematological involvements. Our ndings were consistent with the 2019 update of the EULAR recommendations for management of SLE that CTX is used for organ-threatening disease, especially renal, cardiopulmonary or neuropsychiatric involvement, as well as 'rescue' therapy in patients with life-threatening or refractory disease such as thrombocytopenia [27].
The mechanism of action of CTX is not well understood in spite of its heavy usage as an immunomodulating agent in the treatment of SLE. CTX is a prodrug that requires bioactivation through multiple hepatic isoenzymes of cytochrome P450 (CYP2B and CYP3A), which metabolize it to 4hydroxycyclophosphamide [28]. The acylic tautomer of 4-hydroxycyclophosphamide is then cleaved nonenzymatically into phosphoramide mustard which is the therapeutic active compound and is supposed to cause alkylation and cross-linking of DNA, thereby inhibiting DNA replication which leads to cell death There are several limitations in our study. Firstly, as this was an observational study, we could not entirely get rid of unmeasured residual confounding, though adjustments were made in SLEDAI score, comorbidities and medications in the treatment. Secondly, due to the nature of our study being a retrospective design, information bias might be present. Since the use of CTX was estimated from hospital records, the duration and dosage of CTX might be overestimate because adherence was not taken into consideration. It would probably lead to an underestimation of the observed negative association toward null, making our observed association conservative. Thirdly, this study might not include patients with milder SLE. Nevertheless, this does not deter our goal of evaluating the protective effective of CTX use on mortality as the most severe end-stage of the disease spectrum generally has the greatest risk of complications as well as death. Fourthly, this study enrolled patients of Asian Chinese ethnicity and the results might not re ect in patients of African-American ethnicities as they are less responsive to CTX therapy [37]. Despite those limitations, this study has some bene ts. Given that it is a large multicenter cohort study, we were able to perform both multivariate and strati ed analysis to investigate the association between CTX and overall and speci c causes of mortality. Moreover, the doseresponse relationships between duration and dosage of CTX and mortality in SLE were rstly estimated.

Conclusion
In conclusion, based on this retrospective cohort study, we can observed SLE patients treated with CTX have a decreased overall mortality, and is especially bene cial in low daily dosage and long-term use.
CTX use may improve the survival of SLE patients with vital organ involvements, especially renal, neuropsychiatric, cardiopulmonary, gastrointestinal and hematological involvements.

Availability of data and materials
The datasets in the present study are available from the corresponding author on reasonable request.