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Keywords:

  • non-Hodgkin's lymphoma;
  • antinuclear autoantibodies;
  • autoimmune disease;
  • HEp2 cells;
  • indirect immunofluorescence technique

Abstract

  1. Top of page
  2. Abstract
  3. Patients, materials and methods
  4. Patients
  5. Indirect immunofluorescence technique (IIF)
  6. Farr analysis
  7. Enzyme-linked immunosorbent assay (ELISA) analysis
  8. Immunoblotting technique
  9. Clinical features of the patients
  10. Statistical analysis
  11. Results
  12. Detection of ANA
  13. ANA titre
  14. Detection of autoantibodies other than ANA
  15. Clinical features of patients with detectable aAbs or ANA
  16. Antibody detection and patient outcome
  17. Discussion
  18. Acknowledgments
  19. REFERENCES

Summary. The presence of antinuclear autoantibodies (ANA) was investigated in a large cohort of patients with non-Hodgkin's lymphoma (NHL) in order to assess their frequency, specificity and prognostic relevance. ANA were analysed in 347 patients with different histological subgroups of NHL and in 213 controls using an indirect immunofluorescence technique on HEp2 cells. As the appearance of autoantibodies may be found after treatment of NHL, samples were collected at the time of diagnosis of NHL before any therapy. Sixty-six (19%) NHL patients and 12 (5·6%) patients from the control group displayed ANA. The prevalence between the two groups was found to be significantly higher in NHL patients (P < 0·0001) with a marked increased prevalence in follicular and mantle cell lymphoma subgroups. Autoantibodies directed against mitotic proteins or mitotic-associated proteins were found in 6·9% of NHL patients versus 0·5% in the control group (P < 0·001), with a significantly increased incidence in follicular and mantle cell lymphoma subgroups (P < 0·0001). Some 28% of the patients with positive ANA displayed clinical symptoms that could correspond to classical autoimmune manifestations, this frequency appearing to be higher in the marginal zone/mucosa-associated lymphoid tissue lymphoma subgroup. These data demonstrate a significant incidence of ANA before any treatment in NHL occurrence, which seems to be higher in some histological subgroups with particular ANA, such as ANA directed against mitotic proteins or mitotic-associated proteins.

The concept of an association between autoimmunity and non-Hodgkin's lymphoma (NHL) is based on reports of patients with NHL who displayed clinical or biological signs of autoimmunity (Chandor, 1988; Swissa et al, 1992; Levine, 1994; Abu-Shajran et al, 2001). Conversely, patients suffering from autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus (SLE) or Sjogren's syndrome are prone to develop lymphoma. The biological hallmark of autoimmunity includes different autoantibodies (aAbs) and especially antinuclear aAbs (ANA) (Swissa et al, 1992; Timuragaoglu et al, 2000). Specific treatment of NHL patients is also known to induce cellular lysis, leading to an autoimmune process with the production of different aAbs such as ANA or autoimmune clinical manifestations. This phenomenon was demonstrated after therapy with fludarabine (Tiplady et al, 2000), other immunoregulatory drugs or radiation (Tamura et al, 1981; Fleischmann et al, 1996; Abu-Shajran et al, 2001). These observations of ANA detection in patients after treatment led us to question whether the production of aAbs, especially ANA, could occur before any treatment.

Consequently, we analysed the presence of ANA in the sera of NHL patients obtained at the time of the diagnosis of lymphoma, before any specific treatment. In addition, the occurrence of clinical or biological signs that could be ascribed to autoimmune manifestations was analysed for the patients showing ANA.

As different lymphoma entities may represent pathological equivalents to distinct B- and T-cell populations, this study was performed for the different histological subgroups of NHL.

Patients

  1. Top of page
  2. Abstract
  3. Patients, materials and methods
  4. Patients
  5. Indirect immunofluorescence technique (IIF)
  6. Farr analysis
  7. Enzyme-linked immunosorbent assay (ELISA) analysis
  8. Immunoblotting technique
  9. Clinical features of the patients
  10. Statistical analysis
  11. Results
  12. Detection of ANA
  13. ANA titre
  14. Detection of autoantibodies other than ANA
  15. Clinical features of patients with detectable aAbs or ANA
  16. Antibody detection and patient outcome
  17. Discussion
  18. Acknowledgments
  19. REFERENCES

Sera from 347 patients with NHL were examined for the presence of ANA. These sera were collected from the Department of Clinical Haematology (Centre Hospitalier Lyon-Sud, Hospices Civils de Lyon) in consecutive patients providing informed consent. They were selected at the time of diagnosis before any specific treatment of NHL. The 347 patients consisted of 202 males and 145 females, with a median age of 63 years (range 17–86 years). All patient cases were classified according to the criteria of the Revised European–American Classification of Lymphoid Neoplasm (Harris et al, 1994), and the study population included 43 small lymphocytic, 49 mantle cell, 70 marginal zone, 37 mucosa-associated lymphoid tissue (MALT), 54 follicular, 60 diffuse large B-cell lymphomas and 33 T-cell lymphomas. As a control population, 213 sera (115 males and 98 females, median age 60 years, range 42–76 years) were selected from individuals who were undergoing examination in different clinical departments of the same hospital and had no evidence of malignancy or known autoimmune disease. All sera were frozen at −80°C before use.

Detection of ANA.  This detection was performed using HEp2 cells. This cell line, routinely used for the detection of ANA, was originally thought to be derived from an epidermoid carcinoma of the larynx, but was subsequently found, based on isoenzyme analysis, HeLa marker chromosomes and DNA fingerprinting, to have been established via HeLa cell contamination (Chen, 1988). Sera diluted at 1:80 were incubated on HEp2 cells (Bio-Rad, Marnes la Coquette, France) for 30 min at room temperature. After three washes in phosphate-buffered saline (PBS), pH 7·4, the slides were incubated with a goat anti-human immunoglobulin (Ig)G (F(ab′)2) conjugated to fluorescein isothiocyanate (diluted at 1:100) (Bio-Rad) for 30 min at room temperature. The slides were examined using an Olympus fluorescence microscope. The different nuclear immunofluorescence patterns were noted: homogeneous, speckled, nucleolar, proteins of the mitotic apparatus or mitotic-associated proteins such as type A, B, C or F centromere proteins (CENPs), centrosome, centriole, mitotic spindle microtubules, mid-body or spindle-associated proteins, nuclear mitosis antigen (NuMA) (Hollingsworth et al, 1996; Rattner et al, 1998). The cytoplasmic patterns were also considered. A classical titration of each ANA that was positive at a titre of 1:80 was performed by serial 1:2 dilution until 1:5120 for the homogeneous, speckled or nucleolar patterns and until 1:320 for the proteins of the mitotic apparatus or mitotic-associated protein patterns. A titre = 1:160 was interpreted as a positive result. Depending on the immunofluorescence staining pattern, ANA were characterized further by detecting more specific antidouble-stranded(ds) DNA aAbs or antiextractible nuclear antigen (ENA) aAbs.

Detection of other autoantibodies.  Sera giving a cytoplasmic pattern on HEp2 cells were analysed for the presence of antismooth muscle (ASMA), antimitochondria or antiliver kidney microsomes (anti-LKM). The detection was performed with sera diluted at 1:30 on cryostat mouse stomach and kidney sections (Biomedical, BMD, Marne la Vallée, France).The detection of antiendomysium aAbs was performed with sera diluted at 1:5 on cryostat monkey oesophagus sections (Bio-Rad). The immunofluorescence technique was identical to the technique used for ANA detection.

Farr analysis

  1. Top of page
  2. Abstract
  3. Patients, materials and methods
  4. Patients
  5. Indirect immunofluorescence technique (IIF)
  6. Farr analysis
  7. Enzyme-linked immunosorbent assay (ELISA) analysis
  8. Immunoblotting technique
  9. Clinical features of the patients
  10. Statistical analysis
  11. Results
  12. Detection of ANA
  13. ANA titre
  14. Detection of autoantibodies other than ANA
  15. Clinical features of patients with detectable aAbs or ANA
  16. Antibody detection and patient outcome
  17. Discussion
  18. Acknowledgments
  19. REFERENCES

A radioimmunological test was used to detect the antidsDNA aAbs (Dade Behring, Paris, France) according to the manufacturer's instructions. This test was performed on sera giving a homogeneous pattern on HEp2 cells with fluorescent chromosomes; this immunofluorescence pattern is often associated with antidsDNA aAbs, which are used for their sensitivity and specificity in the diagnosis of SLE.

Enzyme-linked immunosorbent assay (ELISA) analysis

  1. Top of page
  2. Abstract
  3. Patients, materials and methods
  4. Patients
  5. Indirect immunofluorescence technique (IIF)
  6. Farr analysis
  7. Enzyme-linked immunosorbent assay (ELISA) analysis
  8. Immunoblotting technique
  9. Clinical features of the patients
  10. Statistical analysis
  11. Results
  12. Detection of ANA
  13. ANA titre
  14. Detection of autoantibodies other than ANA
  15. Clinical features of patients with detectable aAbs or ANA
  16. Antibody detection and patient outcome
  17. Discussion
  18. Acknowledgments
  19. REFERENCES

The major anti-ENA aAbs [Ss-A/Ro, Ss-B/La, Smith (Sm), U1Ribonucleoprotein (RNP), topoisomerase 1 (Scl70), histidyl-tRNA-synthetase (JO1)] or the aAbs directed against the M2 mitochondria (pyruvate dehydrogenase: PDH) and against the liver kidney microsomes type 1 (LKM1) were detected using ELISA techniques according to the manufacturer's instructions (BMD) as well as for the antithyroperoxidase and antithyroglobulin aAbs (Pharmacia, Saint-Quentin en Yvelines, France).

Immunoblotting technique

  1. Top of page
  2. Abstract
  3. Patients, materials and methods
  4. Patients
  5. Indirect immunofluorescence technique (IIF)
  6. Farr analysis
  7. Enzyme-linked immunosorbent assay (ELISA) analysis
  8. Immunoblotting technique
  9. Clinical features of the patients
  10. Statistical analysis
  11. Results
  12. Detection of ANA
  13. ANA titre
  14. Detection of autoantibodies other than ANA
  15. Clinical features of patients with detectable aAbs or ANA
  16. Antibody detection and patient outcome
  17. Discussion
  18. Acknowledgments
  19. REFERENCES

This technique was used for the identification of antimitochondria and anti-LKM aAbs that were not identified using the ELISA techniques. Mitochondrial proteins and reticulum proteins were extracted from pig heart and rat liver according to previously described methods (Beaufay et al, 1974; Penin et al, 1982). The immunoblotting technique has been described elsewhere (Fabien et al, 1999). Briefly, the nitrocellulose strips were incubated with human sera (diluted at 1:100) for 2 h. The complexes were revealed using a goat anti-human IgG (H + L) conjugated to peroxidase (diluted at 1:400, Bio-Rad) and the peroxidase substrate (hydrogen peroxide and 4-chloro 1-naphthol). Positive and negative sera controls for antimitochondria or anti-LKM aAbs were included in each experiment.

Clinical features of the patients

  1. Top of page
  2. Abstract
  3. Patients, materials and methods
  4. Patients
  5. Indirect immunofluorescence technique (IIF)
  6. Farr analysis
  7. Enzyme-linked immunosorbent assay (ELISA) analysis
  8. Immunoblotting technique
  9. Clinical features of the patients
  10. Statistical analysis
  11. Results
  12. Detection of ANA
  13. ANA titre
  14. Detection of autoantibodies other than ANA
  15. Clinical features of patients with detectable aAbs or ANA
  16. Antibody detection and patient outcome
  17. Discussion
  18. Acknowledgments
  19. REFERENCES

Clinical data were analysed for the patients showing positive ANA or identified cytoplasmic aAbs. Clinical symptoms and biological findings that could be ascribed to autoimmune manifestations were retrospectively examined from the patient's data file, especially arthralgia, skin lesions, myalgia, Raynaud's syndrome, xerostomia, pericarditis, autoimmune haemolytic anaemia (AIHA), positive direct Coombs' test, cryoglobulinaemia, monoclonal gammopathy along with aAbs for the biological signs.

Statistical analysis

  1. Top of page
  2. Abstract
  3. Patients, materials and methods
  4. Patients
  5. Indirect immunofluorescence technique (IIF)
  6. Farr analysis
  7. Enzyme-linked immunosorbent assay (ELISA) analysis
  8. Immunoblotting technique
  9. Clinical features of the patients
  10. Statistical analysis
  11. Results
  12. Detection of ANA
  13. ANA titre
  14. Detection of autoantibodies other than ANA
  15. Clinical features of patients with detectable aAbs or ANA
  16. Antibody detection and patient outcome
  17. Discussion
  18. Acknowledgments
  19. REFERENCES

The χ2 test was used to analyse the statistical significance of the presence of the aAbs in NHL compared with the control group and between the different histological NHL subgroups. A P-value < 0·05 was considered as statistically significant. The influence of the presence of ANA and aAbs on progression-free survival and overall survival was estimated using the log-rank test.

Detection of ANA

  1. Top of page
  2. Abstract
  3. Patients, materials and methods
  4. Patients
  5. Indirect immunofluorescence technique (IIF)
  6. Farr analysis
  7. Enzyme-linked immunosorbent assay (ELISA) analysis
  8. Immunoblotting technique
  9. Clinical features of the patients
  10. Statistical analysis
  11. Results
  12. Detection of ANA
  13. ANA titre
  14. Detection of autoantibodies other than ANA
  15. Clinical features of patients with detectable aAbs or ANA
  16. Antibody detection and patient outcome
  17. Discussion
  18. Acknowledgments
  19. REFERENCES

Sixty-six (30 women, 36 men) out of the 347 sera from patients with NHL (19%) and 12 (five women, seven men) out of the 213 sera from the control group (5·6%) displayed ANA with a titre  1:160 (Table I). This difference between the two groups was found to be significant (P < 0·0001), with a higher prevalence of aAbs in follicular lymphomas (25·9%), mantle cell lymphomas (24·5%) and T-cell lymphomas (21·2%). The homogeneous pattern (Fig 1) was found to be the most frequent immunofluorescent pattern with a significant difference for the follicular and mantle cell lymphoma and for T-cell lymphoma, marginal zone B and MALT lymphomas (P < 0·001, P < 0·05 respectively) in comparison with the control group.

Table I.  Prevalence and immunofluorescence patterns of ANA in NHL patients and in the control group.
 Control n = 213All NHL n = 347Histological subgroups of NHL
LL n = 43MCL n = 49MZL + MALT n = 108FL n = 54TCL n = 33DLCL n = 60
  • A serum can present several patterns of immunofluorescence such as a homogeneous and a nucleolar or a mitotic protein pattern. In this case, the serum is considered to be positive only once for the count of the total number of positive ANA.

  • MAP, mitotic-associated proteins; CMA, components of the mitotic apparatus.

  • Lymphocytic lymphoma (LL), mantle cell lymphoma (MCL), marginal zone B lymphoma (MZB), mucosa-associated lymphoid tissue lymphoma (MALT), follicular lymphoma (FL), T-cell lymphoma (TCL) and diffuse large B-cell lymphoma (DLCL).

  • Statistical tests between NHL and the control group: *P < 0·05, **P < 0·01, ***P < 0·001, ****P < 0·0001.

No. of ANA-positive patients1266  7121814  7  8
%  5·619****16·3*24·5****16·7**25·9****21·2***13·3*
Nuclear patterns
 Homogeneous  222  2  5  6  5  2  2
 %  0·9  6**  4·710***  5·5*  9***  3*  3
 Speckled  714  1  1  4  2  1  5
 %  3·3  3·7  2  2  3·7  4  3  8·3
 Nucleolar  311  1  0  4  1  3  2
 %  1·4  3·2  2  0  3·7  2  9  3
 MAP or CMA  124  2  7  7  6  1  1
 %  0·5  6·9***  4·6*14·3****  6·511·1****  3  1·7
 Others  0  2  1    1  
 %  0  0·01  2    2  
 Cytoplasmic patterns2065  6  627  8  513
 %  9·318·7**1412·225***14·815·221·7**
image

Figure 1. IIF pattern of positive ANA observed in NHL patients on HEp2 cells. (A) Homogeneous; (B) nucleolar; (C) speckled subtype SSA; (D) speckled subtype Sm/RNP; (E) speckled non-identified subtype; (F) centriole; (G) centromeres, protein types A, B, C; (H) centromeres, protein type F. Magnification level: ×40.

Download figure to PowerPoint

Autoantibodies directed against the components of the mitotic apparatus or the mitotic-associated proteins were found in 6·9% of NHL versus 0·5% in the control group (P < 0·001). Considering the different histological entities of NHL, these aAbs were significantly more frequent in the follicular and mantle cell lymphoma subgroups compared with the control population (P < 0·0001), and in the mantle cell lymphoma subgroup compared with the other NHL subgroups (P < 0·05). Various immunofluorescence patterns were observed with a characteristic labelling of the CENP-A, -B, -C (3/24) or a labelling of the CENP-F (12/24) giving a speckled pattern of staining on metaphase nuclei with no staining of interphase nuclei. Staining of the centriole (5/24) and staining of the mid-body (4/24) were also observed (Fig 1). No staining of NuMA was found.

Identification of ANA showed that one out of 22 patients with homogeneous ANA was positive for antidsDNA aAbs (15·6 IU/ml with a cut-off = 4·5 IU/ml). Two of 14 patients with speckled ANA were positive for anti-SS-A/Ro aAbs.

No control patient was positive for the antidsDNA aAbs, for the anti SS-A/Ro aAbs or for the components of the mitotic apparatus or the associated mitotic proteins.

ANA titre

  1. Top of page
  2. Abstract
  3. Patients, materials and methods
  4. Patients
  5. Indirect immunofluorescence technique (IIF)
  6. Farr analysis
  7. Enzyme-linked immunosorbent assay (ELISA) analysis
  8. Immunoblotting technique
  9. Clinical features of the patients
  10. Statistical analysis
  11. Results
  12. Detection of ANA
  13. ANA titre
  14. Detection of autoantibodies other than ANA
  15. Clinical features of patients with detectable aAbs or ANA
  16. Antibody detection and patient outcome
  17. Discussion
  18. Acknowledgments
  19. REFERENCES

Within the homogeneous, speckled and nucleolar patterns observed for 47 sera, the ANA titre varied from 1:160 to 1:320 for the control sera and from 1:160 to 1:5120 for the NHL patients (Table II). For the components of the mitotic apparatus or the associated mitotic protein patterns observed in 24 sera, the titre was always superior to 1:320.

Table II.  ANA titres in the positive NHL (47/347) and control (12/213) groups showing homogeneous, speckled and nucleolar patterns.
 nTitre
1:1601:3201:6401:12801:25601:5120
NHL4718174323
 LL4111  1
 MCL633    
 MZL+14541112
 MALT
 FL853    
 TCL632 1  
 DLCL914211 
Controls1275    

Detection of autoantibodies other than ANA

  1. Top of page
  2. Abstract
  3. Patients, materials and methods
  4. Patients
  5. Indirect immunofluorescence technique (IIF)
  6. Farr analysis
  7. Enzyme-linked immunosorbent assay (ELISA) analysis
  8. Immunoblotting technique
  9. Clinical features of the patients
  10. Statistical analysis
  11. Results
  12. Detection of ANA
  13. ANA titre
  14. Detection of autoantibodies other than ANA
  15. Clinical features of patients with detectable aAbs or ANA
  16. Antibody detection and patient outcome
  17. Discussion
  18. Acknowledgments
  19. REFERENCES

Beside the presence of ANA, aAbs directed against cytoplasmic components were found on HEp2 cells in 65 NHL sera (18·7%) with a predominance in marginal zone and MALT lymphomas (25%, P < 0·001) compared with other subtypes. Among these 65 positive sera, seven have been characterized further. Two patients were positive for aAbs directed against actin, one against M2 mitochondria (PDH, 78 kDa), one against another mitochondrial antigen of 65 kDa, one against LKM1 and one against endomysium. Two of these seven sera were also positive for ANA. The other 58 positive sera that gave a cytoplasmic pattern contained aAbs directed against proteins within or associated to the filaments of the cytoskeleton, to the lysosomes or peroxysomes. Among the control patients, 20 (9·3%) were positive for anticytoplasmic aAbs, but none of them was positive for antiactin, antimitochondria, anti-LKM or antiendomysium aAbs. The difference in aAbs detection between lymphoma patients and controls was statistically significant (P < 0·01).

Four of 39 positive ANA sera (10%) obtained from NHL patients were positive for antithyroperoxidase aAbs or for antithyroglobulin aAbs, whereas no positivity was observed for the 12 ANA-positive controls.

Clinical features of patients with detectable aAbs or ANA

  1. Top of page
  2. Abstract
  3. Patients, materials and methods
  4. Patients
  5. Indirect immunofluorescence technique (IIF)
  6. Farr analysis
  7. Enzyme-linked immunosorbent assay (ELISA) analysis
  8. Immunoblotting technique
  9. Clinical features of the patients
  10. Statistical analysis
  11. Results
  12. Detection of ANA
  13. ANA titre
  14. Detection of autoantibodies other than ANA
  15. Clinical features of patients with detectable aAbs or ANA
  16. Antibody detection and patient outcome
  17. Discussion
  18. Acknowledgments
  19. REFERENCES

Complete clinical and biological data were available before any specific treatment for 40 NHL patients (21 women, 19 men) who were positive for ANA or an identified anticytoplasmic aAb (Table III). The clinical and biological data of these patients were analysed to identify any association between ANA and signs of autoimmunity. Seven patients presented classical, well-defined autoimmune diseases such as rheumatoid arthritis (one case), coeliac disease (one case), Sjogren's syndrome (two cases, which presented MALT lymphoma), hypothyroidism (two cases) and Raynaud's syndrome (one case). In addition, three patients had a positive Coombs' test (with haemolytic anaemia in one patient), and two presented antiphospholipid antibodies (one of these cases had both anticardiolipid and positive Coombs' test and presented with skin ulcers). Therefore, 11 (28%) out of these 40 patients had unequivocal manifestations of autoimmunity. Eight other patients had symptoms that could eventually be considered as manifestations of autoimmune diseases such as inflammatory rheumatismal diseases (two cases), other arthralgia (four cases) and various skin manifestations such as purpura and urticaria (three cases; one of these cases also had arthralgia). Among the 11 patients with marginal zone or MALT lymphomas, seven had autoimmune manifestations. Monoclonal gammapathy (10 cases; IgM, 7 and IgG, 3) and hypogammaglobulinaemia (one case) were also encountered in this group of patients. Given the relatively low general frequency of clinical and biological symptoms of autoimmunity in the general population, these findings suggest that ANA detection was more likely to be associated with those clinical or biological features, although some patients with high antibody titres did not present any symptoms.

Table III.  Clinical and/or biological signs of autoimmunity.
PatientsStatus gender/ age (years)NHL subtypeClinical symptoms of autoimmunityANA and other autoantibodies
  1. See legend to Table I for abbreviations.

 1W/57LLANA 160 speckled
 2M/71LLANA 640 homogeneous
 3W/57MCLHypothyroidismANA 320 homogeneous, hypogammaglobulinaemia
 4W/72MCLANA 160 homogenous, M component (IgM)
 5W/66MCLANA 160 granular + mitotic-associated aAbs
 6M/74MCLANA 320 homogeneous
 7M/52MCLANA 320 homogeneous, positive Coombs' test
 8M/53MCLANA 160 homogeneous
 9W/68MZLChronic arthritisANA 160 homogeneous +  anticentriole aAbs
10W/70MZLANA 160 speckled + nucleolar > 160, M component (IgM)
11W/60MZLAutoimmune haemolytic anaemia, cutaneous ulcersANA 2560 speckled, antidsDNA aAbs (15·6 UI/l), anticardiolipid aAbs, M component (IgM), positive Coombs' test
12W/77MZLANA 2560 anti-dot aAbs, anti-M2 mitochondria aAbs (PDH)
13M/72MZLRaynaud's syndromeAnti-actin aAbs
14M/63MZLArthralgiaANA 160 homogeneous, M component (IgM)
15M/59MZLANA 160 homogeneous
16W/57MALTInflammatory rheumatism, Sjogren's syndromeANA 320 homogeneous
17W/47MALTSjogren's syndrome, fibromyalgiaANA 320 speckled anti SS-A and SS-B aAb, M component (IgM)
18M/69MALTANA 320 nucleolar
19M/51MALTCoeliac diseaseAnti-actin aAb
20W/83FLANA 320Speckled, anticardiolipids aAbs, M component (IgM)
21W/67FL ANA 160 homogeneous
22W/66FLHypothyroidism, arthralgiaANA 160 homogeneous
23W/60FLArthralgiaANA 160 homogeneous
24W/57FLGiant urticariaANA 320 homogeneous, M component (IgG)
25M/52FLANA 320 homogeneous, M component (IgM)
26M/55FLANA 160 speckled
27M/60FLArthritisANA 160 nucleolar
28W/79TCLChronic inflammatory purpuraANA 160 homogeneous
29M/16TCLANA 640 nucleolar
30M/35TCLANA 320 nucleolar
31M/32TCLANA 160 homogeneous
32M/68DLCLANA 640 nucleolar, M component (IgG)
33W/72DLCLANA 2560 antidots aAbs, anti-M2 mitochondria aAbs (PDH), positive Coombs' test
34W/58DLCLANA 640 speckled
35W/54DLCLRheumatoid arthritisANA 640 speckled
36W/42DLCL ANA 2560 homogeneous
37W/46DLCL ANA 640 speckled and nucleolar
38M/50DLCLArthralgia, myalgia, cutaneous nodositisANA 320 speckled
39M/53DLCL ANA 320 homogeneous, M component (IgG)
40M/49DLCLArthralgiaANA 320 speckled

Antibody detection and patient outcome

  1. Top of page
  2. Abstract
  3. Patients, materials and methods
  4. Patients
  5. Indirect immunofluorescence technique (IIF)
  6. Farr analysis
  7. Enzyme-linked immunosorbent assay (ELISA) analysis
  8. Immunoblotting technique
  9. Clinical features of the patients
  10. Statistical analysis
  11. Results
  12. Detection of ANA
  13. ANA titre
  14. Detection of autoantibodies other than ANA
  15. Clinical features of patients with detectable aAbs or ANA
  16. Antibody detection and patient outcome
  17. Discussion
  18. Acknowledgments
  19. REFERENCES

In order to evaluate whether patients with ANA or aAbs had a different disease outcome compared with those without these biological markers, time to progression and overall survival were assessed in these patients. The outcome was not found to be significantly different between these two populations.

Discussion

  1. Top of page
  2. Abstract
  3. Patients, materials and methods
  4. Patients
  5. Indirect immunofluorescence technique (IIF)
  6. Farr analysis
  7. Enzyme-linked immunosorbent assay (ELISA) analysis
  8. Immunoblotting technique
  9. Clinical features of the patients
  10. Statistical analysis
  11. Results
  12. Detection of ANA
  13. ANA titre
  14. Detection of autoantibodies other than ANA
  15. Clinical features of patients with detectable aAbs or ANA
  16. Antibody detection and patient outcome
  17. Discussion
  18. Acknowledgments
  19. REFERENCES

Many studies have reported that patients suffering from NHL could display clinical or biological features of autoimmunity, especially aAbs such as ANA (Swissa et al, 1992; Timuragaoglu et al, 2000). However, these initial findings were observed in some patients after treatment and may represent the effect of NHL-specific treatment, known to induce the production of different aAbs (Fleischmann et al, 1996; Tiplady et al, 2000). To clarify this point further, the presence of ANA was analysed in our study at the time of diagnosis of NHL and before any specific treatment. This analysis showed that 66 out the 347 sera (19%) from patients with NHL displayed ANA with a titre ≥ 160. A lower frequency was observed for the control group sera (5·6%), and the difference between the two groups was significant (P < 0·001). Previous studies have indicated the percentage of ANA in NHL patients to be as high as 32%, but these analyses were performed during or after lymphoma treatment (Swissa et al, 1992; Timuragaoglu et al, 2000). Another bias that can increase the frequency of positive ANA is the choice of the cut-off level for sera positivity. A previous study showed that healthy individuals generally have negative ANA but can be regarded as positive depending on the cut-off level (Adams & Mutasim, 2000). One-third of healthy persons had a positive ANA test at a titre of 1:40. This percentage decreased when the cut-off was higher i.e. 13%, 5% and 3% for titres of 1:80, 1:160 and 1:320 respectively (Adams & Mutasim, 2000). A cut-off of 1:160 was chosen in our study to avoid this bias; 5·6% of the control patients were positive, which is in agreement with Adams & Mutasim (2000). Moreover, a cross-matched age population was used as the control group in the present study as older patients are known to have a higher frequency of ANA. Given the higher prevalence of NHL in the elderly, the presence of ANA could then be just a reflection of age (Huminer et al, 1990; Xavier et al, 1995). We can thus conclude that the presence of ANA in our population resulted not only from the treatment of NHL, a low cut-off level or the old age of the lymphoma population, but really represented a specific biological abnormality. Therefore, ANA can frequently be detected before the diagnosis of NHL. Interestingly, other antibodies such as antioncoprotein and antitumour suppressor gene antigen aAbs have already been described in other cancer patients (Pasquinelli et al, 1992; Winter et al, 1992; Disis & Cheever, 1996; Montenarh et al, 1998; Stockert et al, 1998; Abu-Shajran et al, 2001; Zhang et al, 2001a).

In addition to the homogeneous pattern, speckled and nucleolar patterns of ANA were observed for 3·7% and 3·2% of the NHL patients respectively. Considering the identification of ANA, antidsDNA was found in 4·7% of the patients positive for the homogeneous ANA pattern. Only three of the 45 positive ANA cases had a homogeneous, speckled and nucleolar pattern. A precise identification of the target of the other ANA could be of interest as these antigens may eventually correspond to oncogenic proteins known to be implicated in the development of certain NHL, and would constitute a recognized set of nuclear autoantigens (Fernandez-Madrid & Tomkiel, 2000). In the present study, aAbs directed against the proteins of the mitotic apparatus or against the mitotic-associated proteins were detected. Indeed, the use of cycling cells i.e. HEp2 cells, some of which may be in mitosis, as substrate for ANA screening has made it possible to recognize aAbs directed to these proteins. This recognition is based on the staining of well-defined sites of the mitotic apparatus, i.e. mitotic poles, spindles, centrosomal area or intercellular bridge. The pattern of some aAbs in our study corresponds to the aspect given by the aAbs directed against the CENP-A, -B, -C or -F described by Rattner et al (1993); anti-CENP-F aAbs have been described previously in a high proportion of patients with neoplasia especially in breast and lung carcinoma (Casiano et al, 1995; Rattner et al, 1997). The other predominant mitotic protein found to be the target of these ANA corresponded to the centriole. These aAbs have not yet been described in NHL but only in Raynaud's phenomenon and scleroderma (Tuffanelli et al, 1983; Sato et al, 1994).

NHL is categorized into different entities based on histological, immunological, genetic and major clinical features (Rattner et al, 1998). The frequency and the specificity of ANA could therefore depend on the type of histological subgroup. We found that the frequency of ANA was especially high for the follicular and mantle cell lymphomas. It was difficult to compare this finding with other published data as only one study detailed the NHL subgroups, reporting that there was no increase in ANA in T-cell lymphoma (Peterson et al, 1998). We observed a higher frequency of aAbs directed against the proteins of the mitotic apparatus or against the mitotic-associated proteins in the follicular and mantle cell lymphomas. Mantle cell lymphoma is a lymphoproliferative lymphoma with a high rate of cycling cells and proliferation. The production of aAbs can result from the exacerbation of an apoptotic mechanism observed in this type of lymphoma. The germinal centre, from which follicular lymphomas appear to originate, is also characterized by a large number of apoptotic cells. Numerous antigens can be modified during the apoptotic process, with these modifications leading to an autoimmune process (Rodenburg et al, 2000; Rovere et al, 2000).

In parallel to ANA, the frequency of aAbs directed against identified cytoplasmic antigens was evaluated and showed a higher prevalence in the marginal zone/MALT and diffuse large B-cell lymphomas. Some aAbs have been identified as antiactin or antimitochondria aAbs that are known to be correlated with autoimmune hepatitis and primary biliary cirrhosis, respectively, or antiendomysial aAbs associated with coeliac disease. The other anticytoplasmic aAbs also described in a case of a centrocytic–centroblastic NHL could not be identified but are of unknown significance for autoimmune disease (Barrios et al, 2000).

The correlation between the presence of ANA and the autoimmune clinical manifestations was then evaluated. Well-defined and incomplete autoimmune syndromes have been described in NHL, such as thyroiditis, autoimmune haemolytic anaemia, rheumatoid arthritis and Sjögren's syndrome, but also urticaria phenomena, cold agglutinin disease, nephrotic syndrome, vasculitis, SLE and scleroderma (Duhrsen et al, 1987; Polliack & Lugassy, 1992; Gronbaek et al, 1995; Jonsson et al, 1999; Sallah et al, 2000; Granel et al, 2001). Considering the association between the diseases, autoimmune disease can develop in the course of the lymphoproliferative disorder but can also precede the diagnosis of lymphoma (Gronbaek et al, 1995). In our study, clinical data have been analysed for 40 patients with ANA and/or cytoplasmic aAbs. More than a quarter of these patients displayed some autoimmune clinical symptoms or well-defined autoimmune disease, i.e. Sjogren's syndrome, hypothyroiditis, coeliac disease, rheumatoid arthritis, with a range from 22% to 73%, depending on the histological subtype of NHL. The highest incidence was observed for the marginal zone B and MALT lymphomas. Duhrsen et al (1987) reported a lower frequency (3·5%), but it was an overall frequency and not just the prevalence within the ANA-positive patients (Jonsson et al, 1999). The risk of occurrence of B-cell NHL in Sjogren's syndrome has been estimated to be almost 50 times greater than in the age-matched control subjects (Rustin et al, 1988; McCurley et al, 1990) with a high incidence of marginal zone lymphoma (Mellemkjaer et al, 1997; Mariette, 1999). There is also an increased risk, of up to 75-fold, of malignant lymphoproliferative disease in Hashimoto thyroiditis; a majority of the patients diagnosed with a primary thyroid lymphoma had histological evidence of previous Hashimoto thyroiditis (Aozasa et al, 1986, 1987; Ansell et al, 1999). A frequent association was also observed between coeliac disease and T-cell lymphoma or MALT lymphoma (Ortiz-Hidalgo & Wright, 1992; Tursi & Gasbarrini, 1999). We did not observe any occurrence of SLE reported in NHL, although the overall cancer incidence was significantly increased by 30% in SLE patients compared with that of the general population (Asherson et al, 1991; Mellemkjaer et al, 1997). The frequency of AIHA was also low in our study (2·4%), but AIHA generally appears at diagnosis or during the course of the disease rather than before the diagnosis of NHL (Gronbaek et al, 1995). In our study, no significant over-representation of B-cell or T-cell phenotype was found in patients with pre-existing clinical autoimmune signs, as reported previously (Duhrsen et al, 1987; Gronbaek et al, 1995). Within the B-cell types, the marginal zone B/MALT lymphomas showed a higher frequency but with a moderate statistical difference (P < 0·05).

The majority of the patients with positive ANA or cytoplasmic aAbs did not display autoimmune clinical symptoms, demonstrating the lack of strict correlation between the presence of such aAbs and autoimmune symptoms or disease. The prognostic significance of the frequency of ANA in NHL has also been evaluated, as it was demonstrated that the presence of such aAbs in the serum of patients with breast cancer was associated with higher risk of recurrence or metastases in a follow-up of 2 years (Wasserman et al, 1975). Furthermore, some data suggest that the presence of ANA or dsDNA aAbs seemed to be correlated with a better clinical outcome in patients with small-cell lung (Blaes et al, 2000) and colorectal (Syrigos et al, 2000) cancers. Such prognostic association was not found in NHL patients in a previous study (Timuragaoglu et al, 2000) or in the present large study of 347 patients.

The association between autoimmune disease and NHL is still not fully understood. Some have speculated that ANA represent an important component of the natural aAbs repertoire and might participate in an antitumour immunosurveillance (Torchilin et al, 2001). A possible link is the CD5+ B-cell population, representing 20% of B cells in adult blood and spleen (Gadol & Ault, 1986). These CD5+ B cells constitute a functionally distinct subset of autoantibodies producing cells that can contribute to the association between B-lymphoproliferative disorders and specific autoimmune diseases (Youinou et al, 1993). Accumulated evidence indicates that the autoreactive B repertoire frequently undergoes malignant transformation (Borche et al, 1990), and it has been postulated that challenge of this autoreactive repertoire by self-antigens could create conditions for malignant transformation to occur (Dighiero, 1998). However, this CD5+ lineage usually contributes essentially to small lymphocytic and mantle cell lymphoma. Recently, the role of memory CD5-B cells was emphasized, and marginal zone lymphoma may develop from this immunologically distinct subpopulation. Interestingly, a higher percentage of homogeneous ANA and other aAbs was found in this subpopulation, and those patients were more likely to present with autoimmune symptoms. However, they did not represent the subgroup with the highest frequency of ANA.

Fas mutations can also be implicated in the association between lymphoma and autoimmunity. These mutations have been identified in 60% of MALT lymphomas, 21% of diffuse large B-cell lymphomas, 6% of follicle centre cell lymphomas and 50% of anaplastic large-cell lymphoma (Gronbaek et al, 1998).

In conclusion, these data demonstrate a significant incidence of ANA before any treatment in NHL patients. This occurrence seems to be higher in some histological subgroups with specific ANA, such as the ANA directed against proteins of the mitotic apparatus or mitotic-associated proteins found in mantle cell lymphoma. Further characterization of these ANA should be performed as they may reflect specific protein alterations in lymphoma cells. Indeed, different aAbs have already been detected before the diagnosis of cancer, such as the aAbs directed againstthe p62, Koc and the CENP-F described in patients before the diagnosis of hepatocarcinoma (Reeves, 2001; Zhang et al, 2001b), as well as for aAbs directed against some neuronal proteins (Hu, Ri, Yo) in the paraneoplastic neurological syndromes discovered before the diagnosis of small-cell lung, breast or ovary carcinomas (Antoine & Honnorat, 2000). As a consequence, it would be of great interest to identify the target antigen of ANA that may be used as markers for some NHL subgroups.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Patients, materials and methods
  4. Patients
  5. Indirect immunofluorescence technique (IIF)
  6. Farr analysis
  7. Enzyme-linked immunosorbent assay (ELISA) analysis
  8. Immunoblotting technique
  9. Clinical features of the patients
  10. Statistical analysis
  11. Results
  12. Detection of ANA
  13. ANA titre
  14. Detection of autoantibodies other than ANA
  15. Clinical features of patients with detectable aAbs or ANA
  16. Antibody detection and patient outcome
  17. Discussion
  18. Acknowledgments
  19. REFERENCES

The authors thank all the clinical departments of the CHLS that worked with the control group population, Dr Martine Ffrench (Laboratoire d'Hématologie, HEH, Lyon, France) for helpful discussion, and Mrs Paola Damaso for secretarial help. This work was supported in part by the Hospices Civils de Lyon.

REFERENCES

  1. Top of page
  2. Abstract
  3. Patients, materials and methods
  4. Patients
  5. Indirect immunofluorescence technique (IIF)
  6. Farr analysis
  7. Enzyme-linked immunosorbent assay (ELISA) analysis
  8. Immunoblotting technique
  9. Clinical features of the patients
  10. Statistical analysis
  11. Results
  12. Detection of ANA
  13. ANA titre
  14. Detection of autoantibodies other than ANA
  15. Clinical features of patients with detectable aAbs or ANA
  16. Antibody detection and patient outcome
  17. Discussion
  18. Acknowledgments
  19. REFERENCES
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