Smoking and non-Hodgkin lymphoma: Case-control study in Italy
Tobacco smoking is a well-documented risk factor for several cancers, but the role of cigarette smoking in the etiology of non-Hodgkin lymphoma (NHL) is inadequately understood. Hepatitis C virus (HCV) has been associated with NHL, but the interaction between HCV and smoking habits has not yet been studied. Between 1999 and 2002, we conducted a case-control study on the association of HCV, smoking habits and NHL in 2 areas of northern and southern Italy. Cases were 225 consecutive patients (median age, 59 years) with a new diagnosis of NHL that were admitted to reference and general hospitals. Controls were 504 patients (median age, 63 years) admitted to the same hospitals as cases, for a wide spectrum of acute, nonneoplastic, nonimmune-, nor tobacco-related conditions. Current, heavy smokers (≥20 cigarettes/day) had an odds ratio (OR) of NHL of 2.10 (95% confidence interval, CI: 1.07–4.12) compared to never smokers. The association between smoking and NHL was consistent across strata of sex and age. Compared to never smokers, current smokers of ≥20 cigarettes/day had ORs of 1.14 (95% CI: 0.37–3.56) for B-cell-low-grade, 2.10 (95% CI: 0.94–4.67) for B-cell-intermediate and high-grade, and 25.84 (95% CI: 1.95–342.17) for T-cell NHL. The effect of tobacco smoking and HCV were independent on the relative risk, leading a 4-fold elevated risk in current smokers HCV positive subjects. Tobacco smoking and hepatitis C virus (HCV) have been associated to non-Hodgkin lymphoma (NHL), but the interaction between HCV and smoking habits has not yet been studied. Our study confirms that tobacco is related to NHL, and reports on the combined effect of tobacco smoking and HCV. Infection acted together according to a multiplicative model, leading to a 4-fold elevated risk in current smokers HCV positive subjects. © 2005 Wiley-Liss, Inc.
Tobacco smoking is a well-documented risk factor for several cancers, but an international working group of experts had recently concluded that no clear evidence of an association with smoking is present for haemolymphopoietic tumors other than myeloid leukemia.1 However, some prospective2, 3, 4 and case-control studies5, 6, 7, 8, 9, 10, 11, 12 reported some direct association between NHL and tobacco smoking, though these were often restricted to selected histological subtypes. Among the studies examining such relationship by NHL subtype, follicular lymphoma emerged as the one with the most consistent association.3, 4, 9, 10, 11, 12 Increased risks were observed also for other NHL subtypes, including low-grade6 or high-grade NHL.5, 6 However, other prospective4, 13 and case-controls studies14, 15, 16, 17, 18, 19, 20, 21, 22 found no consistent association between tobacco and NHL risk.
A high prevalence of hepatitis C virus (HCV) infection in patients with B-cell NHL has been reported in several epidemiological studies conducted in many countries, including Italy.23, 24, 25, 26, 27 However, the interaction between tobacco smoking and HCV-infection remains an open issue.
The aim of our study is provide additional information on the role of tobacco smoking, to potential differences in risk by NHL subtype, and the interaction between tobacco and HCV in the etiology of NHL, analyzing the data from a case-control study conducted in Italy.
Materials and methods
Between January 1999 and July 2002, we conducted a case-control study on NHL in the province of Pordenone, North-eastern Italy, and in Naples, southern Italy.26 Cases were patients between 18 and 84 years with incident, histologically confirmed NHL. They were admitted to the National Cancer Institute, Aviano, the “Santa Maria degli Angeli” General Hospital, Pordenone, the “Pascale” National Cancer Institute and 2 General Hospitals, Naples. Cases were classified according to the International Classification of Disease for Oncology,28 and NHL subtypes were classified using the Revised European-American Lymphoma/World Heath Organization classification.29, 30 A total of 240 NHL cases were identified, of whom, 7 were excluded because of previous or concomitant cancer. Among the remaining 233 patients, 2 refused participation and 6 patients did not provide blood samples, thus leaving 225 cases (participation rate: 97%). Human immunodeficiency virus (HIV)-test was negative for all cases, and the median age was 59 years.
Controls were patients between 18 and 84 years, admitted for a wide spectrum of acute conditions to the same hospitals as NHL cases. Specifically excluded from the control group were patients admitted for malignant diseases, conditions related to alcohol and tobacco consumption, hepatitis, any chronic diseases that might have changed lifestyle habits and hematological, allergic and autoimmune diseases. A total of 554 controls were contacted, of whom 550 accepted to participate. Blood samples were available for 504 controls (median age: 63 years), with a participation rate of 92%. Of these, 27% were admitted for trauma; 23% for nontraumatic orthopaedic diseases, 22% for acute surgical conditions, 14% for eye diseases and 14% for a variety of other illnesses.
All study participants signed an informed consent form, according to the recommendations of the Ethical Committee of the Aviano National Cancer Institute. Trained interviewers administered a structured questionnaire to cases and controls during their hospital stay. The questionnaire included information on sociodemographic indicators, tobacco smoking, alcohol drinking, dietary habits, behaviors and exposures that entailed risk of HCV transmission. Information on smoking included smoking status (never, ex-smoker and current smoker), history of number of cigarettes and/or cigars smoked per day, grams of tobacco for pipe smoking, age at starting and duration of the habit. Smokers were defined as individuals who had smoked at least 1 cigarette per day for a continuous period of 1 year or more. Ex-smokers were individuals who had abstained from smoking for at least 1 year before NHL diagnosis or interviews (for controls). Information on tobacco was satisfactorily reproducible.31
Each case and control provided a 15 ml sample of blood the day the interview took place. Sera were screened for antibodies against HCV using a 3rd-generation MEIA (AxSYM HCV, version 3.0; Abbott, Wiesbaden, Germany). Positive samples were tested for HCV antibodies, using a 3rd-generation LIA (Innogenetics, Ghent, Belgium), and for serum HCV RNA, using Amplicor version 2.0 (Roche, Pleasanton, CA). Finally, HCV genotyping of HCV RNA-positive samples was performed using a 2nd-generation LiPA (Innogenetics). Samples were considered HCV-positive when HCV antibodies or HCV RNA were detected.
Odds ratios (OR), and the corresponding 95% confidence intervals (CI), were computed by unconditional multiple logistic regression including terms for age, sex, center, years of education, place of birth and seropositivity for HCV.32 We also evaluated departure from multiplicativity of the ORs by fitting logistic regression models containing HCV seropositivity and cigarette smoking, as well as their cross-products.
Table I shows the distribution of NHL cases and controls according to sex, age, education, place of birth, occupation, HVC test and tobacco smoking. Education and occupation were unrelated to NHL risk. Subjects born in the South of Italy had an OR of 2.07 (95% CI: 1.21–3.55) compared to those born in the North. A positive HCV test was associated with NHL risk, with an OR of 2.64 (95% CI: 1.62–4.30). Compared to never smokers, the multivariate OR was 0.87 (95% CI: 0.55–1.39) for ex-smokers and 1.33 (95% CI: 0.87–2.03) for current smokers. Among current smokers, the ORs were 1.13 (95% CI: 0.71–1.81) for <20 cigarettes/day, and 2.10 (95% CI: 1.07–4.12) for ≥20 cigarettes/day.
Table I. Distribution of 225 Cases of Non-Hodgkin Lymphoma and 504 Controls with Corresponding Odds Ratios (OR) and 95% Confidence Intervals (CI)1 for Selected Characteristics: Italy, 1999–2002
|Sex|| || || || || || |
| Male||120||(53.3)||341||(67.7)|| || |
| Female||105||(46.7)||163||(32.3)|| || |
|Age (years)|| || || || || || |
| <45||47||(20.8)||104||(20.6)|| || |
| 45-64||107||(47.6)||177||(35.1)|| || |
| ≥65||71||(31.6)||223||(44.3)|| || |
|Education (years)|| || || || || || |
| <7||97||(43.1)||251||(49.8)||14|| |
| χtrend|| || || || || ||0.61; p = 0.43|
|Place of birth|| || || || || || |
| North-Center||98||(43.6)||248||(49.2)||14|| |
|Occupation2|| || || || || || |
| White collar||75||(34.6)||177||(35.8)||14|| |
| Blue collar||87||(40.1)||226||(45.7)||1.02||(0.66–1.59)|
|HCV test|| || || || || || |
| Negative||181||(80.4)||459||(91.1)||14|| |
|Tobacco Smoking2|| || || || || || |
| Never-smoker||90||(40.2)||182||(36.1)||14|| |
| Cigarettes/day|| || || || || || |
| χ trend3|| || || || || ||3.58; p = 0.06|
Table II shows the relation of tobacco smoking with NHL in separate strata of sex, age and histological subtypes (B-cell low-grade; B-cell intermediate/high-grade; and T-cell NHL). The ORs in male smokers were 1.30 (95% CI: 0.66–2.56) for <20 cigarettes/day and 2.37 (95% CI: 1.08–5.22) for ≥20 cigarettes/day. Corresponding values for females were 1.00 (95% CI: 0.49–2.03) and 2.46 (95% CI: 0.31–19.75). With reference to age, the OR for smokers <60 years old was 1.22 (95% CI: 0.66–2.28) for <20 cigarettes/day and 2.77 (95% CI: 1.13–6.79) for ≥20 cigarettes/day. Corresponding values for smokers ≥60 years old were 1.00 (95% CI: 0.46–2.15) and 1.69 (95% CI: 0.57–5.03). Table II also considers histological subtype. Cases of NHL were classified according to the REAL/WHO classification (B-cell-low-grade, B-cell-intermediate and high-grade and T-cell). Compared to never smokers, current smokers of ≥20 cigarettes/day had ORs of 1.14 (95% CI: 0.37–3.56) for B-cell-low-grade, 2.10 (95% CI: 0.94–4.67) for B-cell-intermediate and high-grade, and 25.84 (95% CI: 1.95–342.17) for T-cell NHL.
Table II. Distribution of 225 Cases of Non-Hodgkin Lymphoma and 504 Controls with Corresponding Odds Ratio (OR) and 95% Confidence Intervals (CI)1 for Smoking Habits in Separate Strata of Sex, Age, and Histological Subtypes: Italy, 1999–2002
|Sex|| || || || || |
| Male|| || || || || |
| OR (95% CI)||14||0.92 (0.49–1.72)||1.30 (0.66–2.56)||2.37 (1.08–5.22)||p = 0.03|
| Female|| || || || || |
| OR (95% CI)||14||1.15 (0.53–2.48)||1.00 (0.49–2.03)||2.46 (0.31–19.75)||p = 0.67|
|Age (years)|| || || || || |
| <60|| || || || || |
| OR (95% CI)||14||1.29 (0.66–2.49)||1.22 (0.66–2.28)||2.77 (1.13–6.79)||p = 0.05|
| ≥60|| || || || || |
| OR (95% CI)||14||0.69 (0.36–1.33)||1.00 (0.46–2.15)||1.69 (0.57–5.03)||p = 0.46|
|Histological subtypes|| || || || || |
| B-cell:low-grade|| || || || || |
| OR (95% CI)||14||1.38 (0.70–2.73)||1.56 (0.75–3.22)||1.14 (0.37–3.56)||p = 0.49|
| B-cell:intermediate- and high-grade|| || || || || |
| OR (95% CI)||14||0.57 (0.31–1.02)||0.81 (0.45–1.45)||2.10 (0.94–4.67)||p = 0.26|
| T-cell|| || || || || |
| OR (95% CI)||14||3.32 (0.66–16.77)||7.84 (1.31–47.09)||25.84 (1.95–342.17)||p<0.01|
When we considered a more detailed histological subtype classification, the ORs for current versus never smokers were 1.91 for CLL (chronic lymphocytic leukemia)/SLL (small lymphocitic lymphoma), 1.44 for plasmocytoid, 1.82 for follicular, 1.16 for mucosa-associated lymphoid tissue (MALT), 0.91 for diffuse large B-cell lymphoma (DLBCL), 1.18 for immunoblastic diffuse large B-cell lymphoma and 5.52 for Burkitt/Burkitt- like lymphomas.
Table III considers the combined effect of smoking habits and HCV virus. In comparison with subjects that were never/ex-smokers HCV negative, the ORs were 1.41 (95% CI: 0.96–2.09) for current smokers, HCV negative, 2.70 (95% CI: 1.54–4.76) for never/ex-smokers, HCV positive and 3.97 (95% CI: 1.65–9.54) for current smokers, HCV positive. The OR was 11.48 (95% CI: 1.11–118.96) for HCV positive subjects and heavy smokers.
Table III. Odds Ratio (OR) and 95% Confidence Interval (CI)1 for Non-Hodgkin Lymphoma by Hepatitis C Virus seropositivity and Smoking Habits. 225 Cases and 504 Controls: Italy, 1999–2002
|Negative|| || || || || |
| cases:controls||115:324||65:135||42:100||23:35|| |
| OR (95% CI)||15||1.41 (0.96–2.09)||1.20 (0.77–1.87)||2.13 (1.15–3.95)||16|
|Positive|| || || || || |
| cases:controls||30:35||14:10||11:9||3:1|| |
| OR (95% CI)||2.70 (1.54–4.76)||3.97 (1.65–9.54)||3.37 (1.31–8.67)||11.48 (1.11–118.96)||2.73 (1.69–4.42)|
|HCV+ or HCV−|| || || || || |
| OR (95% CI)4||16||1.42 (0.98–2.05)||1.19 (0.79–1.79)||2.18 (1.20–3.96)|| |
The present findings are consistent with several epidemiological studies reporting a direct association between tobacco and NHL.2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 21 Some of those studies found increased NHL risks in smokers of 1 gender and/or in certain age groups. Given the low number of cases, these apparent differences are however likely due to chance alone.
Follicular NHL was the subtype with the most consistent association across studies with odds ratios ranging between 1.8 and 3.2.3, 4, 9, 10, 11, 12 However, an increased risk was also observed in other NHL subtypes, including B-cell high grade NHL (ORs between 1.7 and 2.3).5, 6 We also found a significant association between heavy smoking and T-cell NHL subtype (OR = 25.8). This finding, however, should be taken with caution since it is based on only 2 cases and 36 controls. Small sample sizes, which result from stratification by histological subtypes of NHL, are a common problem affecting various studies.
In our study, the combined effect of smoking and HCV infection on the risk of NHL was compatible with a multiplicative model, suggesting an independent effect of the 2 factors. HCV is a RNA virus belonging to the family of flaviviruses, is hepatotropic and causes hepatitis, cirrhosis33 and induces autoimmune manifestations, most notably essentially mixed cryoglobulinemia (EMC). EMC is characterized by cutaneous vasculitis, nephritis, peripheral neuropathy and clonal B-cell lymphoproliferations, which can evolve into NHL.27 Tobacco smoke contains substances shown to be leukemogens,1, 34 to have immunomodulatory effects35 and to decrease the serum levels of some immunoglobulins.36, 37 Tobacco and HCV seem therefore to act at different stages of the process of NHL carcinogenesis. The effect of tobacco smoking on white blood count, T-cell subsets or natural killer cells, and chronic antigenic stimulation, which leads to the clone of B lymphocyte growth, is not completely clear. However, chronic exposure to tobacco smoking or nicotine is thought to be related to lymphomagenesis through induction of T-cell anergy.35
Among the limitations of our study, there is its small sample size, which does not allow to adequately address subgroups or interactions. An additional potential limitation is the use of hospital controls, which may differ from the general population in relation to their smoking habits.38 However, we included in the comparison group only subjects admitted for a wide spectrum of acute, nonneoplastic, nonimmunological diseases, unrelated to tobacco smoking and hepatitis.39 Still, smokers may have been hospitalized more frequently and longer than nonsmokers.40 On the other hand, any such bias should lead to an underestimation of the real association in our study. Nonetheless, the exclusion from the analyses of each major sources of controls (namely, acute surgery, nontraumatic conditions and traumatic conditions) did not modify any of our results. The practically complete participation (much greater than that of most population-based studies),41 the availability of serum samples from all cases and controls, in addition to the comparable catchment's areas for cases and controls, and the satisfactory reproducibility of information on tobacco smoking31 contribute to the strength of our study.
The combined exposure to current smoking and HCV led to a 4-fold excess risk. However, this estimate should be taken with caution due to the small number of subjects exposed to both factors (14 cases and 10 controls) and to the potential bias introduced in case of diseases related to the exposure under study in the control group.39
In conclusion our findings confirm that tobacco is a nonnegligible risk factor for NHL, with an approximately 2-fold elevated risk for heavy smokers compared to never smokers. The association with tobacco was consistent across strata of sex and age. Tobacco smoking and HCV infection acted independently on NHL risk, leading to a grossly elevated risk for heavy smokers, HCV positive subjects.
The authors thank Dr. A. Pinto and Dr. A. Carbone for the collaboration to the study, Mrs. O. Volpato for study coordination, Dr. G. Laconca, Dr. M. Grimaldi and Dr. O. Manganelli for their help in data collection, and Mrs L. Mei for editorial assistance. We also acknowledge the technical personnel of the Microbiology and Immunology Division, General Hospital of Pordenone for the HCV antibody and virological testing.