Effect of janus kinase inhibitors and methotrexate combination on malignancy in patients with rheumatoid arthritis: a systematic review and meta-analysis of randomized controlled trials

Background Rheumatoid arthritis (RA) is a systemic autoimmune disease. The combination therapy of methotrexate (MTX) and Janus kinase inhibitor (JAKi) is commonly used. Patients with RA are at increased risk of malignancy, however, it remains unclear whether the combination therapy is associated with a higher risk. Objective To assess the malignancy risk among patients with RA receiving combination therapy of JAKi and MTX compared to MTX alone. Methods PubMed, Cochrane and Embase were thoroughly searched for randomized controlled trials (RCTs) in patients with RA receiving JAKi and MTX, from inception to July 2020. Primary endpoints were malignancy events, Non melanomatous skin cancer (NMSC) and malignancy excluding NMSC and secondary endpoints were serious adverse events (SAE), deaths. Risk ratio (RR) and 95% CI were calculated using the Mantel–Haenszel random-effect method. Results 659 publications were screened and 13 RCTs with a total of 6911 patients were included in the analysis. There was no statistically significant difference in malignancy [RR = 1.42; 95% CI (0.59, 3.41)], neither NMSC [RR = 1.44 (0.36, 5.76)] nor malignancies excluding NMSC [RR = 1.12 (0.40, 3.13)]. No statistically significant difference between the two groups for SAE [RR = 1.15 (0.90, 1.47)] and deaths [RR = 1.99 (0.75, 5.27)] was found. Conclusion The adjunction of JAKi to MTX is not associated with an increased risk of malignancy when compared to MTX alone. There is no increased risk of SAE and deaths when compared to MTX alone in patients with RA. Supplementary Information The online version contains supplementary material available at 10.1186/s13317-021-00153-5.


Introduction
Rheumatoid arthritis (RA) is a systemic autoimmune disease, estimated to affect approximately 0.5% to 1% of population [1]. Patients with RA are predisposed to an increased risk for malignancy, especially malignant lymphomas [2][3][4][5][6][7], lung cancers [5,6] and non-melanoma skin cancer [7]. Higher mortality was associated with the presence of cancer, varied by stage of malignancy [8]. Persistent inflammation triggers the development and progression of cancer [9,10]. It has been well-established that severity of inflammation in RA is positively correlated with the risk of lymphoma [11]. Taking the inflammation into control may reduce the risk of developing malignancy.
Janus kinase (JAK) can initiate lymphocyte activation, function and proliferation via tyrosine phosphorylation of the receptors and downstream signal transducer and activator of transcription (STAT) signaling [12]. JAK-STAT signaling pathways mediate a double-edged sword effect on both antitumor defense and tumor progression [13]. Inhibiting JAK-STAT pathways raises the concern of losing immune cell function to malignancy, as well as proposes the possibility of suppressing tumor formation.
JAK can be divided into 4 types: JAK1, JAK2, JAK3 and Tyrosine kinase 2 (Tyk2), responsive to myriad cytokines. Various Janus kinase inhibitors (JAKi) are being widely investigated in randomized controlled trials (RCTs) and proved efficacy in patients with RA [14]. JAKi consist of tofacitinib, selectively inhibiting JAK1 and JAK3, baricitinib, blocking JAK1 and JAK2, peficitinib, acting on all types of JAK, decernotinib, highly selective for JAK3, upadacitinib and filgotinib, selectively targeting JAK1. Limited evidence demonstrated no statistical difference in malignancy incidences in patients receiving tofacitinib compared to the general population [15]. Since most patients with RA are treated with combinations of traditional disease-modifying antirheumatic drugs (DMARDs), especially methotrexate, it is important to explore the safety profile of these therapies. The role of different JAKi in the risk of malignancy remains undetermined. Even scarce data exists, to see the malignancy outcomes of JAKi when used in combination with methotrexate.
In consideration of the current unclear malignancy risk of JAKi and MTX combination, we sought to explore a potential association between JAKi and MTX combination and malignancies in patients with RA.

Eligibility criteria
Two independent authors (VS and AM) screened all titles and abstracts for potential inclusion. Any discrepancy among the selected studies were resolved by a third author (RN). Double-blind RCTs that reported malignancy events in adult patients with RA receiving the combination of methotrexate with any JAKi, with methotrexate in the control arm. Studies that did not report malignancy outcomes were excluded. Exclusion criteria included reviews, editorials, letters, observational studies, non human studies. Long term extension studies with single arms and trials involving other biologic DMARDs were excluded as well.

Data extraction and quality assessment
Two independent investigators (VS and AM) performed the search and relevant studies were selected based on the inclusion and exclusion criteria. VS and AM extracted the data using a predefined data abstraction form: age, sex, duration of rheumatoid arthritis, mean number of tender and swollen joints, length of follow-up. Patient-years were calculated based on the extracted data. Any discordance between these two authors was resolved by the third author (RN). The Cochrane quality assessment tool for RCTs was used to assess risk of bias [16].

Outcomes of interest
The primary endpoints of interest were the incidence of malignancy, non melanomatous skin cancers (NMSC) and malignancies excluding NMSC. The secondary endpoints were incidence of serious adverse events (SAE) and deaths.

Statistical analysis
Extracted data were combined using Review Manager (RevMan) software (Cochrane collaboration) Version 5.4. Risk ratio (RR) of malignancies, NMSC, malignancies excluding NMSC, SAE, death was calculated with 95% confidence intervals (CIs) based on Mantel-Haenszel random-effect method. I 2 was used to evaluate heterogeneity among the studies (< 25% considered low heterogeneity and > 50% considered significant heterogeneity). Publication bias was assessed via the funnel plot for the primary endpoint. Sensitivity analysis was performed by leave-one-out method.
Results were reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocol (PRISMA-P) 2015 statement [17]. The protocol for this systematic review and meta-analysis was registered with PROSPERO (CRD42020201473).

Study characteristics
The baseline characteristics of the patients with RA included in the studies were comparable among the two groups, as summarised in Table 2. The length of followup of the trials ranged from 12 weeks to 24 months. The mean age for the JAKi and MTX combination group was 53 ± 11.7 years and 53 ± 11.8 years in the MTX alone group. The average duration of RA was 7.6 years in the MTX alone group and 8.2 years in the JAKi and MTX combination group. The crude incidence rates of malignancies (NMSC/non NMSC) in JAKi and MTX combination group and MTX group were, respectively, 1.086 (0.497/0.651) and 0.709 (0.202/0.409) per 100 patient years.

Primary outcomes
A total of forty patients suffering from malignancies were reported in the JAKi and MTX group among 3684 patient-years and seven malignancy events in the MTX group among 988 patient-years. There was no statistically significant difference in malignancy events (RR = 1.42; 95% CI 0.59 to 3.41, p = 0.44) between the combination group and the control group, seen in Fig. 2. We found a relatively low level of heterogeneity across all included RCTs (χ 2 = 5.35, df = 7, p = 0.62, I 2 = 0%). For the Mantel-Haenszel random methods, funnel plot showed no evidence of publication bias in all comparisons in Fig. 3. Considered separately, statistical differences remained undetectable for non melanomatous skin cancers (NMSC) (RR = 1.44; 95% CI 0.36 to 5.76, p = 0.61) (Fig. 4) and malignancies excluding NMSC (RR = 1.12; 95% CI 0.40 to 3.13, p = 0.82) (Fig. 5). Among 40 malignancy events in the JAKi and MTX combination group, 15 (37.5%) were NMSC and 24 (60%) were malignancies excluding NMSC. Among 7 malignancy events reported in MTX along group, 2 (28.5%) were NMSC and 4 (57%) were malignancies excluding NMSC. One malignancy in each group was not reported in detail. Among solid tumors for the MTX and JAKi combination group, the most common types were cervical cancer in 6 patients (25%), lung cancer in 5 patients (21%), breast cancer in 4 patients (17%) and ovarian cancer in 1 patient. Two cases of non-Hodgkin's lymphoma and two cases of melanoma were reported.

Secondary outcomes
Although more serious adverse events and deaths were reported in the combination therapy group, there was no statistically significant difference between the two groups with RR = 1.15 (95% CI 0.90 to 1.47, p = 0.26)    in SAE (Fig. 6) and RR = 1.99 (95% CI 0.75 to 5.27, p = 0.17) in deaths (Fig. 7).

Sensitivity analysis
The sensitivity analysis was performed for malignancy outcomes with leave one out method and did not show evidence of bias, as summarised in Table 3.

Risk of bias assessment
All RCTs (100%) adequately reported the generation of random sequence and 7 RCTs (53.8%) had adequate descriptions of concealed allocation. Blinding of participants, personnel, and outcome assessor was performed in all RCTs. Only 1 RCTs was unable to report the complete outcome. No selective reporting was discovered, seen in Fig. 8.

Discussion
Patients with RA are predisposed to a higher risk for malignancy [2][3][4][5][6][7]. It remains not entirely clear if the increased risk of malignancy is primarily related to the pathogenesis of the disease or due to the immunosuppressive therapy. Currently, the combination of JAKi and MTX is widely used in patients with RA. However, whether biologic therapy increases the risk of malignancy has not been well addressed. According to our results, combination therapy of JAKi and MTX did not show any statistically significant increase in the number of malignancies as compared to MTX alone, neither in NMSC nor in malignancies excluding NMSC. Moreover, there are no statistical significant differences that were discovered in the incidence of SAE and mortality among these two groups. The majority of SAE are associated with infections including herpes zoster virus (HZV), urinary tract infection (UTI) and upper respiratory tract infections (URTI). To be noticed, the exposure time of patients with RA to JAKi and MTX is positively correlated to SAE as evidenced by the trials with longer follow up [19,25,26]. Among all the trials included, the mortality incidences were similar between patients under JAKi and MTX combination therapy and MTX monotherapy except ORAL SCAN trial [25], in which the combination group had a higher proportion of deaths compared to MTX alone. The deaths were predominantly contributed to multiorgan dysfunction, acute respiratory distress syndrome (ARDS) and major cardiovascular events (MACEs). To our knowledge, this is the first systematic review exclusively to explore the safety profile, especially from the malignancy perspective, of JAKi and MTX combination therapy.
According to observational studies, patients with RA have an increased risk of malignancy [3,4,31]. This is well studied in non-Hodgkin's lymphoma (NHL), with diffuse large B-cell lymphoma (DLBCL) as the most common type of NHL among patients with RA. These patients are also at increased risk of non-hematologic malignancy such as lungs, kidney, nasopharyngeal carcinoma [32]. The pathophysiology is associated with organ damage from chronic inflammation, genetic mutations, and autoimmune B lymphocyte activation [3,32]. Viral infection, such as Epstein-Barr virus (EBV) infection may also play a role in the development of B cell lymphoma [33]. It was estimated that patients with RA have approximately 12-fold risk of developing lymphoma and twofold for lung cancer [34]. The risk of developing malignancy is closely related to disease activity [11]. Overtime, the stimulated immune cells from chronic inflammation may undergo malignant transformation, eventually leading to lymphoma. Interestingly, the chronic use of non-steroid anti-inflammatory drugs (NSAIDs) in these patients was associated with a decreased risk of developing colorectal cancer [34].
In terms of treatment, safety profiles, especially the effects on malignancy, should be carefully addressed. Previous studies have shown association between specific therapies and their corresponding risk of malignancy. The role of MTX on the incidence of malignancies remains controversial. Several population based studies did not show any increased risk of malignancies with MTX use compared to baseline risk among patients with RA [35,36]. MTX may even decrease the risk of lymphoma by suppressing immunologic activation in RA activity but at the same time can increase the risk of other lymphomas [37]. The effects of JAKi on the risk of malignancies still remain unclear. Although genetic variation of JAK-STAT pathway is a known risk factor for malignancy [38], it is unclear whether the use of JAKi would lead to a decreased incidence of malignancies. US Corrona RA registry, a 5-year prospective observational study, did not detect any statistically significant difference in developing malignancy between tofacitinib and other biologic DMARDs [39]. There was no increased risk of malignancy for tofacitinib in patients with RA, when compared to conventional synthetic DMARDs or tumor necrosis factor-α inhibitors per a meta-analysis of observational studies [40]. With the widespread use of JAKi, not only tofacitinib, and MTX among patients with RA, there is accumulating data from multiple RCTs and a definite necessity to explore the safety profile of the combination therapy.
It is important that the effect of dose and exposure time of JAKi and MTX combination on malignancy should be interpreted with caution. Among all the trials included in our study, ORAL SCAN [25] study using tofacitinib, reported the largest number of malignancies. This trial is also distinguished for the longest follow up period of 24 months. However, a definitive conclusion should not be drawn from this single study and more data from long term extension studies is warranted to establish the association between exposure length and incidence of malignancy. Moreover, there is no significant difference in risk of malignancy between high dose and low dose JAKi and MTX.
The results of our secondary analysis are consistent with previous studies, while still with few differences [41]. A previously published meta-analysis also showed no significant increase in the number of malignancies with JAKi treatment but it did not specifically account for the effect of MTX [41]. Safety profile of JAKi and MTX combination is comparable to MTX alone as reported in previous studies. Incidence of SAE and deaths in our analysis were similar to prior published studies. It was reported that higher doses of JAKi were associated with increased SAE, but studies included in our analysis had comparable SAE across different dose ranges of JAKi without large differences [41]. Although not included in our analysis, cardiovascular mortality was also not significantly high in patients receiving JAKi when compared to placebo [42]. Overall JAKi has an acceptable safety profile and combination with MTX did not change it.
There are several limitations in our study and the results should be carefully interpreted. First, the patientyear exposure in the control group was much less compared to the combination group. The increased number of malignancies in the combination group, may be partly related to longer inflammation exposure in the setting of RA. Secondly, long term extension studies were excluded as they were mostly single arm studies without a control group, which could result in potential bias. Last, the majority of malignancies have a latency period and develop over the course of months to years and some trials included in our analysis had relatively limited follow up duration which may underestimate the actual malignancy rate. The strengths of our study is that it included large patient-years to detect any differences among the groups. There was no heterogeneity among the studies included in our analysis and the results of sensitivity analyses are consistent with overall results, proving the robustness of the study.

Conclusion
Our meta-analysis showed combination therapy of JAKi and MTX did not increase the malignancies in rheumatoid arthritis patients when compared to MTX alone. SAE and deaths are also not significantly different among the two groups. These results have been consistent among all the studies included in the analysis suggesting overall acceptable safety profile of JAKi and MTX combination.