HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Hanno Huwer Gerhard Kalweit Hans-Joachim Schäfers Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Blaes, F. Articles by Schäfers, H.-J. Search for Related Content PubMed PubMed Citation Articles by Blaes, F. Articles by Schäfers, H.-J.

Ann Thorac Surg 2000;69:254-258
© 2000 The Society of Thoracic Surgeons

---

Original Articles

Antineural and antinuclear autoantibodies are of prognostic relevance in non-small cell lung cancer

^a Department of Neurology, University of the Saarland, Homburg, Germany
^b Department of Thoracic and Cardiovascular Surgery, University of the Saarland, Homburg, Germany

Address reprint requests to Dr Blaes, Department of Neurology, University of the Saarland, D-66421 Homburg, Germany;
e-mail: nefbla{at}krzsun.med-rz.uni-sb.de

	Abstract

Top Abstract Introduction Patients and methods Results Comment References

 
Background. Autoantibodies against nervous system structures have been proven to be a prognostic factor in small cell lung cancer. However, little is known about humoral autoimmunity in non-small cell lung cancer (NSCLC) and its prognostic significance.

Methods. We examined antineural antibodies (AnAb) and antinuclear antibodies (ANA) in the sera of 61 patients with NSCLC (histologically: 29 adenocarcinoma, 32 squamous cell carcinoma). Twenty-one patients had stage I NSCLC, 11 stage II, and 29 patients stage III. Autoantibody detection was done by immunofluorescence test; Western blotting was used as a confirmation test.

Results. Of the NSCLC patients, 27.8% were antineural antibody positive, and 32.7% had ANA. No differences were found between the histological groups. AnAb-positive patients showed a better survival in all patients (p = 0.005). There was also a higher survival of ANA-positive patients, but this was only significant in stage III (p = 0.0025). Cox regression analysis showed that antineural and antinuclear antibodies are a stage-independent prognostic factor in NSCLC.

Conclusions. Antineural and antinuclear autoantibodies are a stage-independent prognostic factor in patients with NSCLC and may represent an effective immune response to the tumor.

	Introduction

Top Abstract Introduction Patients and methods Results Comment References

 
Non-small cell lung cancer (NSCLC) represents a mixed group of tumors with differences in histology, clinical course, and response to treatment. Surgical resection is the preferred treatment in localized stages. The international tumor, lymph node, metastases (TNM) staging system has shown to be a predictor of survival of the patients after resection treatment [1, 2]. A number of studies have been carried out to evaluate the impact of variables on long-term prognosis. These investigations include the effect of aberrant gene expression, tumor-associated antigens, enzymes, hormones, and biological factors on survival [3]. However, little is known about the prognostic significance of tumor-associated autoimmune reactions. In small cell lung cancer (SCLC) patients, autoantibodies against tumor proteins cross-reacting with central nervous system structures have been described [4, 5]. Some of these autoantibodies were shown to have prognostic relevance for the sensitivity to chemotherapy in SCLC [6].

We therefore have examined the prognostic significance of antineural and antinuclear antibodies in surgically treated patients with NSCLC in a prospective study. Moreover, the occurence of antinuclear antibodies (ANA) was investigated to evaluate autoimmune phenomena in these patients.

	Patients and methods

Top Abstract Introduction Patients and methods Results Comment References

 
Patients
Serum samples of 61 consecutive NSCLC patients (mean age 66.9 years) with potentially curative resection treatment were obtained before receiving surgical therapy after informed consent. Histological examination revealed adenocarcinoma in 29 and squamous cell carcinoma in 32 patients. There were 21 patients with stage I disease (T1N0: 5, T2N0: 16), 11 with stage II (T1N1: 2, T2N1: 9), 20 patients had stage IIIA (T3N0: 3, T3N1: 4, T1N2: 2, T2N2: 7, T3N2: 4), and 9 patients stage IIIB (1 patient with T4N2 and 8 patients with satellite[s] in the primary tumor bearing lobe). The T4 tumor invaded the left atrium and could be completely resected. The minimum follow-up was 24 months; maximum follow-up was 41 months.

All patients underwent the following pretreatment staging investigations. After the diagnosis "NSCLC" by histological or cytological examination of specimens obtained by bronchoscopy or percutaneous needle biopsy, exercise electrocardiogram and body plethysmography were undertaken to confirm functional operability. Resectability of the tumors was proven by computed tomography as well as endoscopic methods, including rigid bronchoscopy with endobronchial biopsies. Metastatic spread was ruled out by abdominal ultrasound, isotope bone scan, computed tomography (CT) scanning of the brain, and CT scanning of the adrenal glands.

Mediastinoscopy was performed if mediastinal lymph node enlargement in the N2 and N3 regions were seen. Patients with mediastinoscopically proven N3 disease were treated nonsurgically, patients with N2 disease were treated by a combined modality that included systemic chemotherapy and subsequent surgery. Both groups were excluded from the present study. Extensive mediastinal lymph node dissection was routinely included in the resection treatment of NSCLC. Postoperatively patients were staged according to the international TNM staging system [1]. Satellite nodules within the primary tumor-bearing lobe were classified as T4 according to the recommendations of the American Thoracic Society and the European Respiratory Society in 1997. In patients with N2 disease and in patients with T3 or T4 tumors, postoperative irradiation was carried out. Patients who were considered as having T4 disease because of satellite nodes in the primary tumor-bearing lobe did not receive postoperative radio- or chemotherapy owing to T4.

Antibody detection
Indirect immunofluorescence test (IFT)
We used a commercial available kit ( Euroimmun, Lübeck, Germany) for the detection of antinuclear and anticerebellar antibodies according to the manufacturer’s instruction. In brief, unfixed frozen sections of a human epithelium cell line (Hep-2 cells) and human cerebellar cortex were incubated with the serum sample for 3 hours at room temperature. After washing in phosphate-buffered saline/Tween 2% (PBS-Tween), the sections were exposed to FITC-labeled goat anti-human IgG and IgM (Euroimmun) for 30 minutes. Immunofluorescence were viewed on a Zeiss immunofluorescence microscope (Zeiss, Oberkochen, Germany) by two independent investigators. The initial serum dilution was 1:32; all samples were diluted to end-point titers in immunofluorescence test (IFT). As negative controls, we used slides incubated with buffer only and buffer with the FITC-labeled antibody, respectively. Four sera known as positive for high-titer antinuclear antibody and two antineuronal (anti-Hu) antibody-positive sera were used as positive controls.

Western blotting
Western blotting using human cerebellar tissue was performed as described previously [7] with slight modifications. In brief, human cerebellum was homogenized in PBS, solubilized, and heated at 100°C. The proteins were seperated by electrophoresis on a preparative 4% to 12% gradient polyacrylamid gel (NUPAGE gel; Novex, San Diego, CA). Proteins were transferred to nitrocellulose in a tank blot unit at 500 mA for 1 hour. The nitrocellulose was cut into strips. Every strip was incubated with the patient’s serum (initial 1:200 diluted in PBS containing 2% dry milk) for 1 hour and washed three times in PBS. To visualize specific reactions, we used an alkaline phosphatase-marked goat anti-human IgG antibody with BCIP-NBT (Calbiochem, San Diego, CA) as a substrate. Sera were considered as positive for antineural antibodies if the titer in the IFT was greater than or equal to 1:100 and specific reactions in the Western blot were detectable. Antinuclear antibodies were detected only by IFT on Hep-2 cells; a serum titer of greater than or equal to 1:100 was considered as positive.

Statistics
Survivals were calculated from the date of operation, and described using the Kaplan-Meier method. Statistical evaluation was done by the log-rank test. In order to investigate the autoantibody findings being a stage-independent prognostic factor, Cox regression analysis has been used. A p value of less than 0.05 was being considered significant.

	Results

Top Abstract Introduction Patients and methods Results Comment References

 
A profile of the lung cancer patients studied is shown in Table 1. Autoantibodies were detectable in 33 out of 61 patients (54.1%). Of the adenocarcinoma patients, 51.7% were autoantibody positive (Ab-pos), and 56.2% of the squamous cell carcinoma patients were Ab-pos. There were no differences in the autoantibody frequency in the stages (stage I 52.3%, stage II 63.6%, stage III 51.7%). Autoantibody-positive patients had a significant better survival than autoantibody-negative patients (p = 0.0004; Fig 1).

View larger version (13K): [in this window] [in a new window]   Fig 1. Survival of autoantibody-positive and -negative (AnAb and/or ANA) NSCLC patients shown as Kaplan-Meier plot (p = 0.0004).

View larger version (218K): [in this window] [in a new window]   Fig 2. Immunofluorescence staining of a NSCLC serum on cerebellar tissue. Axonal staining around the Purkinje cells (serum dilution 1:500, magnification x500).

View larger version (57K): [in this window] [in a new window]   Fig 3. Western blot using cerebellar soluble protein fraction as antigens. (Lane S) Standard (Novex Mark 12); (lane 1) anti-Hu-positive NSCLC patient with a typical reactivity at 38 kDa; (lane 2) NSCLC patient with a 45-kDa reactivity and axonal staining, as shown in Figure 1; (lane 3) healthy control patient.

View larger version (14K): [in this window] [in a new window]   Fig 4. Survival functions of antineural antibody-positive and -negative NSCLC patients presented as Kaplan-Meier plots. (A) All patients (p = 0.005); (B) stage I and II (p = 0.056); (C) stage III (p = 0.026).

Antinuclear antibodies (ANA)
ANAs could be detected in the sera of 32.7% (20 of 61) of the patients. The most frequent binding pattern was homogenous (11 of 20, 55%), followed by speckled (5/20, 25%), nucleolar (2 of 20, 10%), and perinuclear (2/20, 10%) pattern on Hep-2 cells. The mean titer was 1:600 (range 1:100 to 1:2,000). There were no signs of a connective tissue disease in any patient.

Calculated for all patients, ANA-positive patients had a better survival than ANA-negative patients, but the difference did not reach statistical significance (p = 0.11). Although there was no difference in survival in stages I and II patients, the survival of ANA-positive patients in stage III was better than the ANA negatives (p = 0.0025; Fig 5). In a Cox regression analysis, ANA were a stage-independent prognostic factor (p = 0.002).

View larger version (13K): [in this window] [in a new window]   Fig 5. Survival functions of stage III patients grouped by ANA finding presented as Kaplan-Meier plots. Log-rank test showed a significantly better prognosis of the ANA-positive stage III patients (p = 0.0025).

	Comment

Top Abstract Introduction Patients and methods Results Comment References

The International Staging System for Lung Cancer provides a classification for the anatomic extent of NSCLC and has proven to be a predictor of prognosis [1, 2, 8]. Although survival curves do confirm the relationship between the anatomy represented by the TNM formula and the cancer biology, one may observe failure in patients with all favorable prognostic signs. Even in T1N0 disease, the recurrence rate is 11% to 25% [9, 10]. Thus, the malignant potential of the individual tumor is not determined completely by the TNM staging system. Clinical factors like performance status, weight loss, age, and gender present only a rough xposition of prognosis in patients with NSCLC.

Advances in the understanding of the biology of lung cancer have led to the identification of new classes of prognostic indicators, namely aberrant gene expression, tumor-associated antigens, enzymes, hormones, and biological factors like cell proliferation kinetics [3]. Mutation of the oncogenes of the rasfamily and the myc family were found to be associated with a poor prognosis in non-small cell lung carcinoma [11, 12]. Contrasting results were found concerning the significance of mutations of the tumor suppressor gene p53 [13, 14]. Epidermal growth factor receptor expression by the tumor cells, especially of squamous cell carcinomas, was used to develop new staging and therapeutic measures [15]. An overexpression was found to result in a worsening of the prognosis. Serum markers, in particular lactic dehydrogenase and neuron-specific enolase (NSE), were reported to have a prognostic significance for the response to chemotherapy in NSCLC [16]. Possibly, the elevation of these markers is based on a neuroendocrine differentiation of the tumor.

Although there is an increasing number of immunohistochemical and other biomarkers in the prognosis and diagnosis of NSCLC, little is known about humoral immunological factors in tumor patients and their relationship to survival. Recently, we reported that lung cancer patients have disturbances in the IgG subclass distribution [17]. Autoantibodies in lung cancer patients were first described by Hodson and associates in 1975 [18]. They found ANA in 12% of lung cancer patients, whereas 32.7% of our patients were ANA positive. Recently, another group described a frequency of 40% ANA positives in lung cancer patients [19]. The difference in detection rate between the results of Hodson and our series may derive from the different antigenic tissues used in the IFT. They used rat liver, while we used HEp-2 cells, a cell line established from a human laryngeal carcinoma. Possibly, a part of our detected ANA may represent a kind of antitumor reactivity that was not detectable with liver antigens. Another author reported autoantibodies against p53 (11% to 30% positive) [20] or against L-myc gene product (10% positive) [19] in lung cancer patients but not in healthy controls. The prognostic or pathogenic meaning of these antibodies, which were shown to be highly specific for tumor disease, is yet unclear.

There are many reports of autoantibodies against central nervous system structures in lung cancer patients [4–6]. Initially, these antibodies were detected in patients with paraneoplastic neurological diseases, mostly associated with SCLC. One of these autoantibodies, called anti-Hu or ANNA-1 (antineuronal nuclear antibody), reacts with a group of 35- to 40-kDa RNA-binding proteins, expressed in the tumor tissue as well as in neurons [4]. Because there are not only antineuronal reactions, but also autoantibodies, against glial cells, we used the whole cerebellar tissue in our tests and we used the term "antineural" for the reactivities described in our study. These autoantibodies are a heterogenous group, which shares the common quality of reacting with cerebellar tissue. In our investigation, there is a good correlation between these antineural reactivities and the survival in NSCLC. In lower stages, there was only a tendency to a better survival of antineural antibody-positive patients, whereas in stage III, this difference was significant. Graus and associates [6] reported that anti-Hu antibodies in patients with SCLC are associated with a complete response to chemotherapy and an improved survival. Another report described the frequent detection of autoantibodies against autologous tumor cell proteins in SCLC patients [21]. However, this group did not exclude the occurrence of ANA in their patients. Nevertheless, their patients with antitumor reactivity showed an improved survival compared with antibody-negative patients. These reports and the results of our study indicate that an immune response against tumor tissue may contribute to a better prognosis in lung cancer. Whether the autoantibodies are pathogenic for the tumor tissue or only a hallmark of a more T-cell-driven immune response remains to be determined. In a recent study, there was no evidence that autoantibodies against nervous system and tumor tissue have a cytotoxic effect on SCLC cell lines in vitro [22]. Moreover, another study showed evidence of cytotoxic T-cells against similar antigens in autoantibody-positive patients [23].

Because we have not performed histopathological investigation on the corresponding tumor samples, it is unclear whether these antineural autoimmunities also reflect a neuroendocrine differentiation of these tumors. In a few tumor samples, we found an association between antineural autoimmunity and neuroendocrine differentiation of the tumor, but the low number of samples does not allow statistical analysis (Blaes F, unpublished observations). In conclusion, antinuclear and antineural autoantibodies are a stage-independent prognostic factor for survival in NSCLC. Whether this represents a direct cytotoxicity of the autoantibodies or is associated with a cytotoxic T-cell response in these patients remains to be determined.

	Acknowledgments

 
This study was supported by the "Heinrich-Dietz-Stiftung for cancer research at the Saarland University," project-Nr. TS 117/2/97.

	References

Top Abstract Introduction Patients and methods Results Comment References

 

1. Mountain C.F. A new international staging system for lung cancer. Chest 1986;89(Suppl):225-233.
2. Naruke T., Goya T., Tsuchiya R., Suemasu K. Prognosis and survival in resected lung carcinoma based on the new international staging system. J Thorac Cardiovasc Surg 1988;96:440-447.[Abstract]
3. Fielding L.P. Molecular markers for the diagnosis and prognosis of lung cancer. Cancer 1992;69:1592-1599.[Medline]
4. Dalmau J., Furneaux H.M., Cordon-Cardo C., Posner J.B. The expression of the Hu (paraneoplastic encephalomyelitis/sensory neuronopathy) antigen in human normal and tumor tissues. Am J Pathol 1992;141:881-886.[Abstract]
5. Dalmau J., Furneaux H.M., Gralla R.J., Kris M.G., Posner J.B. Detection of the anti-Hu antibody in the serum of patients with small cell lung cancer. Ann Neurol 1990;27:544-552.[Medline]
6. Graus F., Dalmau J., Reñé R., et al. Anti-Hu antibodies in patients with small cell lung cancer. J Clin Oncol 1997;15:2866-2872.[Abstract]
7. Tanaka K., Yamazaki M., Sato S., Toyoshima I., Yamamoto A., Miyatake T. Antibodies to brain proteins in paraneoplastic cerebellar degeneration. Neurology 1986;36:169-172.
8. Mountain C.F. Surgical treatment of lung cancer. Crit Rev Oncol Hematol 1991;11:179-207.[Medline]
9. Thomas P., Rubinstein L., Lung Cancer Study Group. Cancer recurrence after resection. Ann Thorac Surg 1990;49:242-247.[Abstract]
10. Martini N., Bains M.S., Burt M.E., et al. Incidence of local recurrence and second primary tumors in resected stage I lung cancer. J Thorac Cardiovasc Surg 1995;109:120-129.[Abstract/Free Full Text]
11. Sugio K., Ishida T., Yokoyama H., et al. Ras gene mutations as a prognostic marker in adenocarcinoma of the lung without lymph node metastasis. Cancer Res 1992;52:2903-2906.[Abstract/Free Full Text]
12. Kawashima K., Nomura S., Hirai H., et al. Correlation of L-myc RFLP with metastasis, prognosis and multiple cancer in lung-cancer patients. Int J Cancer 1992;50:557-561.[Medline]
13. Kandioler D., Foedinger M., Mueller M.R., Eckersberger F., Mannhalter C., Wolner E. Carcinogen-specific mutations in the p53 tumor suppressor gene in lung cancer. J Thorac Cardiovasc Surg 1994;107:1095-1098.[Abstract/Free Full Text]
14. Wiman K.G. p53. Exp Cell Res 1997;237:14-18.[Medline]
15. Rusch V., Baselga J., Cordon-Cardo C., et al. Differential expression of the epidermal growth factor receptor and its ligands in primary non-small cell lung cancers and adjacent benign lung. Cancer Res 1993;53:2379-2385.[Abstract/Free Full Text]
16. Van Zandwijk M., Jassem E., Bonfrèr J.M.G., et al. Serum neuron-specific enolase and lactate dehydrogenase as predictors of response to chemotherapy and survival in non-small cell lung cancer. Semin Oncol 1992;19:37-43.[Medline]
17. Klotz M., Blaes F., Funke D., Kalweit G., Schimrigk K., Huwer H. Shift in the IgG subclass distribution in patients with lung cancer. Lung Cancer 1999;24:25-30.[Medline]
18. Hodson M.E., Turner-Warwick M. Autoantibodies in patients with bronchial carcinoma. Thorax 1975;30:367-370.[Abstract/Free Full Text]
19. Yamamoto A., Shimizu E., Ogura T., Sone S. Detection of autoantibodies against L-myc oncogene products in sera from lung cancer patients. Int J Cancer 1996;69:283-289.[Medline]
20. Lubin R., Zalcman G., Bouchet L., et al. Serum p53 antibodies as early markers of lung cancer. Nature Med 1995;1:701-702.[Medline]
21. Winter S., Sekido Y., Minna J., et al. Antibodies against autologous tumor cell proteins in patients with small cell lung cancer. J Natl Cancer Inst 1993;85:2012-2018.[Abstract/Free Full Text]
22. Verschuuren J.J., Dalmau J., Hoard R., Posner J.B. Paraneoplastic anti-Hu serum. J Neuroimmunol 1997;79:202-210.[Medline]
23. Albert M.L., Darnell J.C., Bender A., Francisco L.M., Bhardwaj N., Darnell R.B. Tumor-specific killer cells in paraneoplastic cerebellar degeneration. Nature Med 1998;4:1321-1324.[Medline]

This article has been cited by other articles:

				M Tschernatsch, E Stolz, M Strittmatter, M Kaps, and F Blaes Antinuclear antibodies define a subgroup of paraneoplastic neuropathies: clinical and immunological data J. Neurol. Neurosurg. Psychiatry, December 1, 2005; 76(12): 1702 - 1706. [Abstract] [Full Text] [PDF]

				F Blaes, M Jauss, J Kraus, P Oschmann, I Krasenbrink, M Kaps, and B Teegen Adult paraneoplastic opsoclonus-myoclonus syndrome associated with antimitochondrial autoantibodies J. Neurol. Neurosurg. Psychiatry, November 1, 2003; 74(11): 1595 - 1596. [Full Text] [PDF]

				M Ramos-Casals, M Garcia-Carrasco, M P Brito, A Lopez-Soto, and J Font Autoimmunity and geriatrics: clinical significance of autoimmune manifestations in the elderly Lupus, May 1, 2003; 12(5): 341 - 355. [Abstract] [PDF]

				M. D. Brundage, D. Davies, and W. J. Mackillop Prognostic Factors in Non-small Cell Lung Cancer* : A Decade of Progress Chest, September 1, 2002; 122(3): 1037 - 1057. [Abstract] [Full Text] [PDF]

				F. Brichory, D. Beer, F. LeNaour, T. Giordano, and S. Hanash Proteomics-based Identification of Protein Gene Product 9.5 as a Tumor Antigen That Induces a Humoral Immune Response in Lung Cancer Cancer Res., November 1, 2001; 61(21): 7908 - 7912. [Abstract] [Full Text] [PDF]

				F. M. Brichory, D. E. Misek, A.-M. Yim, M. C. Krause, T. J. Giordano, D. G. Beer, and S. M. Hanash An immune response manifested by the common occurrence of annexins I and II autoantibodies and high circulating levels of IL-6 in lung cancer PNAS, August 14, 2001; 98(17): 9824 - 9829. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Hanno Huwer Gerhard Kalweit Hans-Joachim Schäfers Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Blaes, F. Articles by Schäfers, H.-J. Search for Related Content PubMed PubMed Citation Articles by Blaes, F. Articles by Schäfers, H.-J.