Glucocorticoid therapy and risk of bladder cancer

Background: Use of immunosuppressive drugs post organ transplantation, and prolonged use of glucorticoids for other conditions have been associated with subsequent risk of certain malignancies, that is, skin cancers and lymphoma. There is evidence that the incidence of bladder cancer is also elevated among organ transplant recipients, however, it is unknown whether other groups of patients, that is, those taking oral glucocorticoids, likewise are at an increased risk. Methods: In a population-based case–control study in New Hampshire, USA, we compared the use of glucocorticoids in 786 bladder cancer cases and in 1083 controls. We used unconditional logistic regression analysis to compute adjusted odds ratios (ORs) associated with oral glucocorticoid use. Results: In our analysis, the risk of bladder cancer was related to a history of prolonged oral glucocorticoid use (OR=1.85, 95% CI=1.24–2.76, adjusted for age, gender and smoking). Associations with oral glucocorticoid use were stronger for invasive tumours (OR=2.12, 95% CI=1.17–3.85) and tumours with high (3+) p53 staining intensity (OR=2.35, 95% CI=1.26–4.36). Conclusion: Our results raise the possibility of an increased risk of bladder cancer from systemic use of glucocorticoids, and a potential role of immune surveillance in bladder cancer aetiology.

With an estimated 68 810 new cases in 2008, bladder cancer is the fifth most commonly diagnosed cancer in the United States of America (Scosyrev et al, 2009). Several risk factors have been identified as potential causes of bladder cancer, including tobacco use, occupational exposure to aromatic amines and polycyclic aromatic hydrocarbons (Johansson and Cohen, 1997;Silverman et al, 2006), and treatment with cyclophosphamide (Kinlen, 1985;Baker et al, 1987;Radis et al, 1995;Volkmer et al, 2005). Glucocorticoids, often in combination with other immunosuppressive drugs, are a part of post-transplant therapy and alone are used to treat other acute or chronic inflammatory conditions, including rheumatoid arthritis, inflammatory bowel disease and asthma (Zoorob and Cender, 1998). There is evidence of an enhanced risk of certain types of cancer, including skin cancers and lymphomas, among organ transplant recipients (Birkeland et al, 1995(Birkeland et al, , 2000Kyllonen et al, 2000;Adami et al, 2003;Vajdic et al, 2006) and prolonged users of immunosuppressive drugs, such as glucorticoids, for other conditions (Karagas et al, 2001;Sorensen et al, 2004;Jensen et al, 2009). The incidence of bladder cancer is two-to four-fold higher among organ transplant recipients (Buzzeo et al, 1997); however, the risk among patients using oral glucocorticoids for other reasons is unknown. Therefore, we examined the potential risk of bladder cancer associated with glucocorticoid use in non-transplant recipients as part of an ongoing population-based case -control study of bladder cancer conducted in New Hampshire, USA.

Study group
Through the New Hampshire State Department of Health and Human Services' rapid reporting Cancer Registry, we identified newly diagnosed cases of bladder cancer among New Hampshire residents, aged 25 -74 during the 1 July 1994 to 31 December 2001 period. For efficiency, we shared controls with a study of nonmelanoma skin cancer covering a diagnostic period of 1 July 1993 to 30 March 2000, along with additional controls frequency matched to the bladder cancer cases by age and gender (Wallace et al, 2009). Controls o65 years of age were selected using population lists obtained from the New Hampshire Department of Transportation. Controls 65 years of age and older were chosen from data files provided by the Centers for Medicare & Medicaid Services (CMS) of New Hampshire, as described previously (Fortuny et al, 2007).

Interviews
We conducted standardized personal interviews with the study participants to obtain information on demographic traits, use of tobacco (including frequency, duration and intensity of cigarette smoking), alcohol and other exposures. We requested the original paraffin-embedded tumour specimen for histopathology re-review by the study pathologist who classified tumours according to WHO 1973 and WHO ISUP criteria . Owing to the high concordance rates for overall diagnosis (490%), we classified subjects based on the original pathologist's diagnosis; whereas tumour morphology, extent of disease and grade were based solely on the standardized histopathology re-review by the study pathologist. Immunohistochemical analysis of the tumours was carried out for TP53 using a monoclonal antibody (BioGenex, San Ramon, CA, USA), and scored for intensity and the percentage of tumour cells staining positively as markers of tumour severity, as the number of tumours having TP53 mutations increases with the degree of invasiveness of the tumour (Kelsey et al, 2004) and may represent an aetiologically distinct subgroup of tumours (Kelsey et al, 2005;Wallace et al, 2009). We obtained informed consent from each participant and all procedures and study materials were approved by the Committee for the Protection of Human Subjects at Dartmouth College.

Drug use assessment
Participants were asked if their doctor had ever prescribed glucocorticoids or steroids as pills, injections or inhalers for 1 month or longer before their reference date (defined as the diagnosis date of the cases and a comparable date randomly assigned to the controls). Those who responded positively were considered users and were asked the age they were first treated, the condition for which the glucocorticoids were prescribed, the name of the drug, dose and duration of the treatment. Those who responded that they did not use glucocorticoids for at least 1 month were considered non-users. To aid recall, we developed a list of glucocorticoids and other immunosuppressive drugs (trade name, generic name and description) and a pictorial guide showing the most commonly used drugs grouped by pill colour, size and shape. To minimize potential reporting bias, we did not reveal the specific hypotheses-of-interest to either the interviewer or participant, and we did not inform the interviewers of the case -control status of participants.

Statistical analysis
We computed odds ratios (ORs) and their 95% confidence intervals (CIs) for bladder cancer associated with the use of glucocorticoids before the reference date using unconditional logistic regression, taking into account multiple confounding factors (Breslow and Day, 1980). In addition to age and sex, we also adjusted for the potential confounding effects of smoking status (current, former and never). Further, we assessed the possibility that education, as a marker of socioeconomic status, could act as a potential confounder, but the inclusion of this variable did not significantly influence our results, and therefore was not included in the final models.
We conducted a combined analysis of all bladder cancers, as well as specifically on pathologically confirmed transitional cell carcinomas. In addition, we carried out a case-only analysis comparing glucocorticoid users to non-users according to subgroups defined by the extent of disease (low grade noninvasive, high grade non-invasive, invasive or carcinoma in situ) and TP53 staining intensity (o3 or 3 þ ) to examine whether glucocorticoid use was associated with tumour aggressiveness.

RESULTS
A total of 824 cases took part in the study (85% response rate of those eligible), and from 786 (95%) we obtained data on the use of oral glucocorticoids. A total of 1119 controls took part in the study (70% of those eligible), and from 1083 (97%) we obtained data on the use of oral glucocorticoids (Table 1). Cases were more likely male than were controls, but did not differ by age. A higher percentage of cases than controls were current smokers, who reported a family history of bladder cancer and did not have education beyond high school or technical college (Table 1). Overall, 5% of controls and 8% of cases reported a history of oral glucocorticoid use for 1 month or longer. Prednisone accounted for 87% of the reported oral glucocorticoid use. Of the histologically reviewed cancers, 98% were transitional cell (urothelial) carcinomas (TCC), 61% were non-invasive low grade, 8% were non-invasive high grade, 27% were invasive and 4% were in situ carcinomas (Table 1).

Associations with glucocorticoid use
Any glucocorticoid use was associated with an increased risk of bladder cancer, and the association was stronger for oral use (OR ¼ 1.85, 95% CI ¼ 1.24 -2.76) than inhaled use (Table 2). Although there was not a clear trend in risk by categories of duration of use, the OR was highest among those who used oral glucocorticoids for 5 years or longer (OR ¼ 3.39, 95% CI ¼ 0.98 -

Subgroups by tumour characteristics
Restriction to transitional cell carcinomas did not affect our risk estimate for oral glucocorticoid use (TCC OR ¼ 1.85, 95% CI ¼ 1.22 -2.81). Further, using a case -case approach, we found higher ORs associated with more advanced disease stage and grade (Table 3). Oral glucocorticoid users had somewhat higher odds of having TP53 high-intensity stained tumours than TP53 negative tumours.

DISCUSSION
Immunosuppressive therapy, usually a combination of cytotoxic drugs and glucocorticoids, is commonly prescribed to organ transplant recipients, in order to prevent allograft rejection. Glucocorticoids, alone or in combination with other immunosuppressive drugs, are used to help treat many chronic inflammatory conditions, such as rheumatoid arthritis and asthma. Long-term immunosuppressive therapy has been shown to increase the risk of many types of malignancies among organ transplant recipients (Birkeland et al, 1995(Birkeland et al, , 2000Kyllonen et al, 2000;Adami et al, 2003;Vajdic et al, 2006). Further, previous research has found increased risk of skin cancers and non-Hodgkin's lymphoma among longterm glucocorticoid users (Karagas et al, 2001;Sorensen et al, 2004;Jensen et al, 2009). Our findings suggest that long-term glucocorticoid users may have an increased risk of bladder cancer, although to a lesser extent than organ transplant recipients. Previous large cohort studies have found risk of bladder cancer in transplant recipients to be elevated two-to five-fold over the general population, a smaller effect size than seen with skin cancer or lymphoma (Kyllonen et al, 1994;Buzzeo et al, 1997;Adami et al, 2003). Bladder cancer is a far less common disease than skin cancer, and thus it is not surprising that cohort studies would observe few if any cases (Master et al, 2004;Kamal et al, 2007). An exception was a Taiwanese study that reported bladder cancer as the most common type of post-transplant malignancy with an incidence of 4.1% among kidney transplant recipients (Wu et al, 2004).
In non-transplant cohorts, cyclophosphamide and other drugs with immunosuppressive effects also have been linked to increased risk of bladder cancer. Several previous studies have found that cyclophosphamide therapy in patients with Wegener's granulomatosis is associated with an increased risk of bladder cancer (Travis et al, 1995;Talar-Williams et al, 1996;Knight et al, 2004). Patients with rheumatoid arthritis treated with long-term cyclophosphamide (Radis et al, 1995) and patients on cyclophosphamide or azathioprine for underlying autoimmune disorders (e.g., rheumatoid arthritis, Crohn's disease) had reported increased risks of bladder cancer (Kinlen, 1985). In another study, patients with rheumatic disease on multiple drugs, including azathioprine, methotrexate, cyclophosphamide and chlorambucil were found to have an enhanced risk of neoplasms, including in the bladder (Asten et al, 1999). In a more recent cohort study, rheumatoid arthritis patients on either methotrexate or tumour necrosis factor alpha (TNF-a) inhibitors, were twice as likely to develop bladder cancer as the general population (Setoguchi et al, 2006).
An enhanced risk of bladder cancer also has been observed in other immunosuppressed populations, including HIV-infected and AIDS patients. Manfredi et al (2006), published a case series  Glucocorticoid therapy and bladder cancer K Dietrich et al and literature review with thirteen reports of HIV-infected patients with bladder carcinoma. Although the prevalence of bladder cancer is less than Kaposi's sarcoma, lymphoma and cervical cancer, this report is consistent with our observations and suggests the necessity of further research addressing the association of bladder cancer with immunosuppression. Further in recent work, Roberts et al (2008) found evidences of polyoma virus in urothelial carcinomas of renal transplant patients, suggesting a potential aetiological role of polyoma virus in post-transplant bladder malignancies. However, they acknowledge that it is possible that tumour cells are more susceptible to BK virus infection than normal cells, and thus that the infection is a consequence rather than a cause. Still this hypothesis may warrant further investigation. It is conceivable that our findings were because of chance, unmeasured confounding or other biases. Differential misclassification due to recall bias is possible, although we attempted to minimize this through the use of a pictorial guide of common medications. Moreover, it seems unlikely that bladder cancer patients would recognize oral glucocorticoids as a possible aetiological factor. Another advantage of our study was that both cases and controls were drawn from the general population; among cases, over 95% reported having a valid driver's license (those o65 years) or being enrolled in Medicare (those 65 years and older), ensuring our control group was represented within our case group. Our study population was relatively large, although the statistical power diminished in certain categories (i.e., by duration of use). We assessed the potential confounding effects of multiple factors and confounding by indication was unlikely, as we did not find systematic differences in the ORs for oral glucocorticoid use by reason for use. A weaker association observed with inhaled steroids is plausible because of the minimal systemic effects of inhaled steroids (Hardman et al, 1996;Zoorob and Cender, 1998). Oral glucocorticoid users appeared to be at an enhanced risk of specifically developing invasive, TP53 positive (3 þ staining intensity) bladder cancer compared with non-users. This is supported by previous research that has observed more rapid progression of transitional cell carcinomas in immunosuppressed patients (Wang et al, 2002). This is perhaps because tumours are able to progress more quickly with diminished immunosurveillance. Nonetheless, our findings will need to be confirmed or refuted in future studies. If an enhanced risk of bladder cancer, particularly advanced disease is confirmed, it might indicate the need for closer monitoring of individuals who regularly take glucocorticoids.