Aims Diffuse large B-cell lymphoma (DLBCL) is an aggressive and potentially fatal disease. Prediction of risk of relapse is based on clinical markers. There is a need for more accurate biomarkers to select patients for more aggressive first-line treatments. Peroxiredoxins (Prxs) are a family of potent antioxidant proteins. Their prognostic role in DLBCL is unknown.
Methods Altogether, 103 diagnostic biopsy samples from patients with DLBCL were immunohistochemically stained for Prxs I, II, III, V and VI.
Results Strong Prx VI expression was associated with the presence of B-symptoms. There were no other significant associations with traditional risk factors. Five-year disease-specific survival was 68.6% in patients with high cytoplasmic Prx VI intensity vs 97.0% in those with low intensity. In multivariate analysis, high Prx VI expression (HR 12.846, 95% CI 1.722 to 95.807, p=0.013) was an independent risk factor of lymphoma-associated death not related to International Prognostic Index score (HR 2.514, 95% CI 1.040 to 6.073, p=0.041).
Conclusions High intensity of cytoplasmic Prx VI expression in pretreatment DLBCL samples predicts worse outcome in patients with DLBCL. Whether Prx VI is associated with chemoresistance, and therefore a poorer outcome, needs to be evaluated. If Prx VI is a predictive marker and it proves causality, it would be crucial to study Prx VI ability to become a target enzyme for treatment.
- TUMOUR MARKERS
- MOLECULAR BIOLOGY
- MALIGNANT TUMOURS
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Diffuse large B-cell lymphoma (DLBCL) represents about 30% of all lymphomas. It is an aggressive disease with a potentially fatal outcome. Modern immunochemotherapy regimens have changed the prognosis of this lymphoma subtype, the median 5-year overall survival (OS) rate now being >75% for all patients with DLBCL. Unfortunately, in some patients the disease may become resistant and progress despite the therapy. Such patients should be selected in clinical trials when testing new forms of therapy.
Within cells, reactive oxygen species (ROS) are produced and removed as by-products of normal aerobic metabolism. ROS also modulate the intracellular redox environment both in physiological and pathological processes. When the production of ROS exceeds their removal, a state called oxidative stress develops. Oxidative stress has been suggested to play an important part in carcinogenesis.1 ,2 Peroxiredoxins (Prxs) are a group of redox state-regulating enzymes that play a part in the intracellular redox system. The major role of Prxs is to detoxify peroxides, such as in reduction of hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2). Expression of Prxs is induced under oxidative stress, and knockout studies have underlined their importance in cell survival.2–4 There are six members in the mammalian Prx family (I−VI). Prx isoenzymes are present in multiple locations in cells including nuclei, cytoplasm, mitochondria, lysosomes, endoplasmic reticulum and peroxisomes.2 The function of Prx VI differs from that of other Prxs because of the different structure of its catalytic site (so called 1-Cys Prx). It needs glutathione as an electron donor. Other Prxs receive electrons from thioredoxin.2 ,5 In a number of studies, the relationship between the amount of Prxs in tissue samples and the prognosis of several cancers has been explored, with controversial results.2 ,5–15 The biology of Prxs is largely unknown in lymphomas.1
In the present study, we retrospectively assessed the prognostic importance of Prxs in pretreatment DLBCL, that is, we wanted to see if the level of expression of Prx isoenzymes could predict prognosis.
Patients and sample collection
The samples were diagnostic biopsy samples from patients with de novo DLBCL. They were taken at Oulu University Hospital and Kuopio University Hospital in 2001−2010. Patients with primary central nervous system lymphoma were excluded.
Patient demographics are presented in table 1. Tissue samples were taken during normal lymphoma diagnostic procedures. The clinical data were collected from hospital records by researchers blinded to the immunohistochemistry results. Staging was based on medical history and clinical status, whole-body CT scanning, blood chemistry, and bone marrow aspirate and biopsy data. The patients were treated with modern rituximab-based chemotherapy including R-CHOP-21 (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone) or R-CHOEP-21 (etoposide added to R-CHOP).
For immunohistochemical analyses, 103 tissue samples were available. The samples consisted of lymph nodes (n=57) and extranodal biopsy samples (n=46) from several tissue origins (testis, n=7; tonsil, n=6; bone, n=5; bone marrow, n=4; stomach, n=4; bowel, n=3; breast, n=2; lung, n=2; nasal tissue, n=2; retroperitoneal tissue, n=2; appendix, n=1; laryngeal tissue, n=1; lip, n=1; mouth, n=1; pericardium, n=1; maxillary sinus, n=1; spinal canal, n=1; spleen, n=1; thyroid, n=1).
Immunostaining was performed as described previously,16 with a few modifications. The glass slides were incubated in 1.5% H2O2 solution instead of 3% H2O2, and they were washed with Tris-buffered saline instead of phosphate buffered saline between the different steps of staining. In the present study, immunostaining of nuclei was performed with Mayer’s haematoxylin (Reagena, Toivola, Finland). Prx antibody staining kits (table 2) were used according to the instructions of the manufacturer. Prx IV was excluded from staining because of the unavailability of suitable commercial antibody. Analysis of immunohistochemical staining was performed by an experienced haematopathologist (K-MH) together with principal investigator (MELK) blinded to the clinical data. Positivity was evaluated separately in the nuclei, cell membranes, adjacent T-lymphocytes and cytoplasm. The intensity of cytoplasmic staining was divided into three categories: low, moderate and strong. The strong intensity was seen in low-power field (×10 magnification), moderate in medium-power field (×20) and low intensity only with high-power field (×40 magnification). Only samples of 100% strong intensity in tumour cells were included in the group of strong intensity.
Statistical analysis was performed using IBM SPSS Statistics for Windows Software V.20 (IBM, released 2011; IBM SPSS Statistics for Windows, V.20.0, IBM, Armonk, New York, USA). Disease-specific survival (DSS) was calculated from the date of diagnosis to the date of lymphoma-specific death or to the last follow-up date. OS was calculated from date of diagnosis to the date of death from any cause or last follow-up date. Progression-free survival (PFS) was calculated from date of diagnosis to lymphoma progression or follow-up date. DSS, OS and PFS were analysed by using Kaplan–Meier analysis in which the cases lost to follow-up were censored on the date of the last follow-up. Significance was tested by log-rank comparison. All statistical immunohistochemical staining analyses were performed by analysing strong intensity versus others. In multivariate analysis, the Cox regression model was applied. Correlations with nominal variables were evaluated by using Fisher's test. Values of p<0.05 were considered to be statistically significant.
The cases represented a standard group of patients with lymphoma as regards age and gender (table 1). The median follow-up time was 53 months (range 0−112). Complete response was achieved with 90 of the patients (87.4%), partial response with 5 patients (4.9%) and progressive disease was detected in 8 patients (7.8%). There were 18 relapses in the total patient group of 103 cases. There were 22 lymphoma-specific deaths and 15 deaths due to other causes (eg, infections). Also, 20 of the patients represented germinal centre (GC) and 30 non-GC immunophenotype. From 53 of the patients, this immunophenotyping was missing. No significant difference was found between GC and non-GC groups in relation to prognosis (p=0.666).
Correlations with clinical disease presentation
Strong expression of cytoplasmic Prx VI was found to correlate with B-symptoms (p=0.002). No statistically significant correlation was found between any Prx versus other traditional risk factors, such as the International Prognostic Index (IPI), WHO performance status, Ann Arbor stage, age, elevated levels of lactate dehydrogenase or extranodal involvement (table 1). Positive adjacent T-cell Prx VI immunoreactivity correlated with non-GC immunophenotype (p=0.028). A similar trend was seen with Prx V expression in adjacent T-cells and non-GC phenotype, but the difference was not statistically significant (p=0.080). No other statistically significant differences were found between other Prx immunostaining and GC/non-GC immunophenotyping.
Correlations with outcome
Cytoplasmic staining intensity of Prx VI correlated with DSS (figure 2A), OS (figure 2B) and PFS (figure 2C). The 5-year DSS rate was 68.6% in the patient group with strong intensity vs 97.0% in the group showing low-staining and moderate-staining intensity (p<0.001). The 5-year OS rates were 58.0% vs 91.6% (p=0.001) and the PFS rates 60.9% vs 89.1% (p=0.001), respectively. There was a correlation between positive Prx VI staining in adjacent T-cells and better outcome, the 5-year DSS rate being 75.0% in the negative staining group vs 92.4% in the group showing positive staining of adjacent T-cells (p=0.045). OS rates were 68.0% vs 81.9% (p=0.068), and PFS rates were 64.2% vs 79.8% (p=0.061), respectively, although these differences were not statistically significant. Staining intensities of Prx I, II, III and V did not correlate with clinical outcome.
In Cox regression multivariate analysis, cytoplasmic Prx VI expression was an independent prognostic factor as regards lymphoma-related death (HR 12.846, 95% CI 1.722 to 95.807, p=0.013) compared with IPI as a single factor in the model (0–2 vs 3−5, HR 2.514, 95% CI 1.040 to 6.073, p=0.041).
This is the first study in which the expression of Prx proteins in DLBCL has been evaluated. The main finding was that strong cytoplasmic expression of Prx VI was associated with a poorer prognosis in patients with DLBCL. This was also seen in multivariate analysis: DSS, OS and PFS were all better in the patient group with low or moderate intensity of cytoplasmic Prx VI staining. In contrast, Prx VI positivity in adjacent but probably reactive benign T-lymphocytes correlated with better outcome when analysing DSS.
Prxs are a family of redox-regulating proteins that are able to reduce peroxynitrite alkylhydroxyperoxide or H2O2 and this also enables Prx participation in cellular signalling cascades.2 However, Prx isoenzymes not only have different subcellular locations, but they also have different structures at their catalytic sites. Prxs I−IV have 2-Cys subgroups, and Prx V is classified as atypical 2-Cys Prx. Prxs VI is the only 1-Cys Prx with a single-cysteine catalytic site and a need for glutathione as an electron donor.5 In addition, compared with 2-Cys Prxs, Prx VI seems only to regenerate via de novo protein synthesis after oxidative attack.17 In previous studies,5 ,9 ,11 ,12 ,14 Prx VI has been associated with the development of radiotherapy and chemotherapy resistance in leukaemia and in ovarian and breast carcinomas. Prx VI is also associated with the invasiveness and metastasis of breast, ovarian and lung carcinomas and squamous cell carcinoma of the tongue,6–12 ,15 which seem in part to be related to activation of the urokinase-type plasminogen activator and its receptor and also to increased glutathione peroxidase and calcium-independent phospholipase 2 activities.10
No previous studies have been carried out to evaluate the possible role of Prxs in lymphomas, although in these diseases chemotherapy has the most crucial role as regards achieving adequate treatment results. For example, the effects of doxorubicin and vinblastine are based on ROS-derived sublethal damage that drives the cell into apoptosis.18 Other redox-regulating enzymes have been little studied in lymphomas, although in one of our recent investigations strong thioredoxin expression was shown to be associated with poor PFS.19 Thioredoxin is also associated with the development of chemoresistance.20 Use of the redox signature score created by Tome et al1 suggested that decreased expression of catalase, manganese superoxide dismutase and glutathione peroxidase was associated with poor prognosis in DLBCL. However, the authors decided to omit Prxs from the redox signature score because at the time neither their regulation nor function was well characterised.1
The difference in patient survival in the current series was striking when classified according to cytoplasmic Prx VI expression: only one patient (3.0%) with a low-staining or moderate-staining result died from DLBCL during follow-up, even though 14 of these patients had high IPI scores. In multivariate analysis, Prx VI appeared to have more prognostic significance compared with the traditional IPI classification. Expression of Prx VI was not associated with any particular IPI score. In contrast, strong cytoplasmic Prx VI expression in reactive T-lymphocytes seemed to be associated with better survival, suggesting a protective effect. Our results may suggest that Prx VI expression in malignant B-cells could be associated with chemoresistance. Since different Prxs isoforms have distinct localisations and structures, it was not surprising that 2-Cys Prxs did not show any prognostic significance. In fact, 2-Cys Prxs may rather have tumour-protecting functions.21 We found a strong association between Prx VI immunoreactivity and prognosis. However, we do not know whether this adverse prognosis is caused by high Prx VI expression or whether this expression is a consequence of some other effect. On the basis of Prx VI biology and previous results concerning solid tumours,5 ,11 however, we suggest that Prx VI may primarily be related to chemoresistance and therefore to poorer prognosis.
We conclude that Prx VI is a promising prognostic biomarker in DLBCL, and we are currently evaluating its association with lymphoma chemoresistance in a lymphoma cell culture knockdown model. If this association can be verified, Prx VI silencing might in the future have therapeutic value when combined with chemotherapy.
Take home messages
There were no correlation between peroxiredoxin (Prx) I–III, V and clinical risk markers of diffuse large B-cell lymphoma (DLBCL).
Strong immunoreactivity of Prx VI was associated with B-symptoms (p=0.002), but with no other clinical risk markers.
Strong cytoplasmic expression of Prx VI was associated with poor prognosis in patients with DLBCL.
The causality of Prx VI should be evaluated in an in vitro model and later study the possible role as a treatment target.
We thank Mrs Anne Bisi and Mr Hamid Bur for their technical assistance in immunohistochemical staining.
Abstract in Finnish
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Handling editor Mary Frances McMullin
Contributors MELK collected the lymphoma patient data and samples, took part in the pathological analysis of immunohistochemical staining, carried out statistical analysis and wrote the manuscript. K-MH carried out pathological analysis of immunohistochemical staining and reviewed the manuscript. TT-H participated in planning the study and analysing the data; she reviewed the manuscript and funded the study. EJ, YS and PP collected the lymphoma patient data and samples and reviewed the manuscript. RB and PK took part in statistical analysis and reviewed the manuscript. OK planned the study, collected the patient samples and reviewed the manuscript.
Funding The Cancer Society of Northern Finland (to MELK), the Finnish Medical Association Duodecim (to PK and OK), the Finnish Cancer Society (to YS) and the Finnish Medical Foundation (to MELK).
Competing interests None.
Ethics approval The regional Ethics Committee of Oulu University Hospital (42/2010) and by the national Supervisory Authority for Welfare and Health (6622/05.01.00.06/2010). The principles of the Declaration of Helsinki were followed during data collection and management in this study.
Provenance and peer review Not commissioned; externally peer reviewed.
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