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Hu and Voltage-Gated Calcium Channel (VGCC) Antibodies Related to the Prognosis of Small-Cell Lung Cancer

From the Department of Neurology, Haukeland University Hospital, University of Bergen, Bergen, Norway; Department of Oncology and Hematology, Næstved Hospital, Næstved, Denmark; and Neurosciences Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom.

Address reprint requests to S.E. Monstad, MSc, Department of Neurology, 200301028, Haukeland University Hospital, Jonas Liesvei 65, Bergen 5021, Norway; e-mail: sissel.monstad{at}helse-vest.no

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
PURPOSE: Hu antibodies previously have been associated with longer survival of patients with small-cell lung cancer (SCLC). Voltage-gated calcium channel (VGCC) antibodies play a pathogenic role in Lambert Eaton myasthenic syndrome, which is also associated with SCLC. These antibodies may reduce tumor growth in patients with the neurologic disease, but it is not clear whether they provide prognostic information in those without neurologic symptoms.

PATIENTS AND METHODS: Two hundred patients with SCLC (age 39 to 79 years; mean, 62.3 years; 129 males and 71 females) receiving chemotherapy were studied for the presence of Hu and VGCC antibodies. Sera were examined for Hu antibodies by an in vitro transcription-translation–based immunoprecipitation technique and by immunohistochemistry/dot blot. VGCC (P/Q subtype) antibodies were detected by radioimmunoassay. Survival analysis was used to analyze the data.

RESULTS: Hu antibodies were detected in 51 of 200 patients (25.5%) by in vitro transcription-translation–based immunoprecipitation and in 37 of 200 patients (18.5%) by immunohistochemistry or dot blot, whereas VGCC antibodies were detected in only 10 of 200 patients (5%). The presence of Hu antibodies did not correlate with VGCC antibodies, and there was no association between Hu or VGCC antibodies and the extent of disease or survival.

CONCLUSION: Hu and VGCC antibodies are found in a proportion of SCLC patients, irrespective of neurologic symptoms, but their presence does not correlate with the prognosis of the SCLC.

	INTRODUCTION

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Paraneoplastic neurologic syndromes arise as nonmetastatic manifestations in 1% of all cancers [1]. Many are associated with specific antibodies that can be used as diagnostic markers. The best example is the neuronal nuclear antibody type 1 (ANNA-1; also called anti-Hu), which is associated with small-cell lung cancer (SCLC) and paraneoplastic encephalomyelitis or sensory neuronopathy. Antibodies to voltage-gated calcium channels (VGCC) are also found in a proportion of patients with SCLC, usually with Lambert Eaton myasthenic syndrome, which is a peripheral antibody-mediated defect in neuromuscular transmission.

In patients with the neurologic disorders, Hu and VGCC antibodies appear to be associated with improved survival [2,3], suggesting that these antibodies reflect an effective immune response against the tumor. The presence of Hu antibodies could also influence the prognosis of SCLC in patients without neurologic disease. In a previous study, patients with SCLC and low levels of Hu antibodies were found to have limited cancer, whereas those with no antibodies more often had extensive disease [4]. Furthermore, the presence of Hu antibodies was correlated with a better treatment response and longer median survival than in those without such antibodies [5].

In this study we used highly sensitive immunologic assays to search for Hu and VGCC antibodies in 200 SCLC patients, to determine whether we could confirm a correlation with survival rates and disease markers that might be useful clinically.

	PATIENTS AND METHODS

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Patients
Two hundred unselected patients with newly diagnosed SCLC admitted to four hospitals in the Copenhagen Lung Cancer Study Group (Denmark) in the period June 1990 to January 1993 were included in the study. All patients had no prior cancer and either a histologic or a cytologic diagnosis of SCLC. Disease stage was defined as limited when the tumor was confined to one hemithorax and ipsilateral supraclavicular lymph nodes, excluding proven malignant pleural effusion and chest wall metastases. Lactate dehydrogenase (LDH) was measured in 194 of the patients. Performance status was scored according to the WHO system. It is not known whether some of the patients developed paraneoplastic neurologic syndromes. All patients received conventional chemotherapy including combinations of platin analogues, podophyllotoxin derivatives, alkylating agents, and vinca alkaloids. No patients received surgery or radiotherapy. Survival was determined from start of treatment to death or last visit. Sera were obtained before start of treatment and kept frozen at -80°C. Follow-up time for long-term survivors was at least 9 years. All patients signed informed consent for participation in the study.

Hu Antibody Detection
For in vitro transcription-translation (ITT) and immunoprecipitation, a Bluescript plasmid containing the HuD cDNA (No. 40986; the American Tissue Culture Collection, Manassas, VA) was electroporated into Escherichia coli XL1-Blue MRF' using a Bio-Rad gene pulser (Bio-Rad, Hercules, CA) at 12.5 kV/cm and 25 µF. Bacteria containing the plasmid were grown in Luria Bertani medium and plasmid DNA was purified using the Qiagen plasmid maxi kit (Qiagen, Hilden, Germany).

ITT was performed using the TNT coupled reticulocyte lysate system (Promega, Madison, WI) and the protein was 35S labeled 35S throughout its synthesis. The ITT product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by photostimulated luminescence imaging (Bio-image analyzer Bas 2000; Fuji Photo Film, Tokyo, Japan). The radiolabeled product had the expected molecular weight of 38 to 40 kd [6].

MultiScreen 96-well filtration plates (MABV N12; Millipore, Bedford, MA) were used for immunoprecipitation experiments. Each well was washed and blocked as previously described [7]. Sulfur-35-labeled HuD protein (5 x 10⁵ to 1 x 10⁶ cpm/well) and patient sera diluted 1:10 in incubation buffer (20 mmol/L Tris HCl, 150 mmol/L NaCl, 0.001% azide, 0.1% bovine serum albumin, and 0.15% Tween-20, pH 8.0) were incubated at 4°C overnight. The following day, 50 µL of a 50% (vol/vol) slurry of resuspended protein A-Sepharose (Pharmacia, Stockholm, Sweden) in incubation buffer was added to each well of the MultiScreen plates followed by the addition of the immune complexes. The plates were then incubated on a shaking platform for 45 minutes at 4°C, washed, and left to dry overnight. Finally, 20 µL of scintillation fluid (Microscint; Packard Sciences, Groningen, the Netherlands) was added to each well, and the amount of radiolabeled immunoprecipitate was measured in a beta counter (Topcount NXT microplate scintillation and luminescence counter; Packard).

Each patient serum sample was run in triplicate and the mean value of these three results was used. The results were expressed as a Hu index: (cpm sample - cpm negative control)/(cpm positive control - cpm negative control) x 1,000. A Hu index greater than 100 was considered as positive, based on the mean ± 2 standard deviations of 100 blood donors [7]. In other experiments using the ITT immunoprecipitation assay, we have detected only low levels of Hu antibodies in one of 150 controls. The sensitivity of the assay is indicated by the high levels of such antibodies in 10 patients with paraneoplastic encephalomyelitis or sensory neuronopathy.

Indirect Immunofluorescence and Dot Blot
Cryostat sections of snap-frozen rat cerebellar tissue were incubated with serum at various dilutions in phosphate-buffered saline (PBS) in a moist chamber at 4°C overnight. The slides were subsequently washed in PBS and incubated with fluorescein isothiocyanate-labeled rabbit antimouse immunoglobulin antibodies (F0200; DAKO A/S, Denmark) diluted 1:50 in PBS for 1 hour at 20°C. After the samples were washed and mounted on slides, the slides were examined by fluorescence microscopy (model DM IL; Leica Microsystems Ltd, Heerbrugg, Switzerland). Staining of neuronal nuclei with sparing of the nucleolus was considered suggestive of Hu antibody reactivity. Titers 500 have been defined as positive in previous experiments in line with other laboratories [8]. Hu reactivity was confirmed by dot blot. This was performed using a commercial kit (Anti-Onkoneurale Antigene Immuno Dot Blot Milenia; DPC Biermann GmbH, Germany) containing the recombinant proteins HuD, Yo (CDR62), Ri (Nova-1), and amphiphysin, according to the instructions of the manufacturer. Sera were used at the recommended dilution of 1:2,000.

VGCC Antibody Detection by Radioimmunoassay
The VGCC radioimmunoassay was performed using VGCCs extracted from human cerebellum and labeled with iodine-125-omega-conotoxin, as previously described [9,10]. The VGCC antibody concentration was expressed in picomoles per liter, and concentrations 50 pmol/L were considered as positive. The radioimmunoassay is used for routine diagnostics, and has a high specificity and sensitivity for anti-VGCC [9].

Statistical Analysis
Investigation of the dependence between Hu and the studied covariates (Table 1) was performed by Fisher's exact test for categoric variables. For age, a t test based on equal variance, as suggested by Levens test for homogeneity of variance, was used. The Kaplan-Meier survival curves were used to estimate survival for the positive and negative Hu groups (Fig 1). The log-rank test was used for univariate comparison of survival curves (Table 2). The Cox proportional hazards model was used to study the effect of several factors simultaneously on survival and to estimate hazard ratios (Table 3). To improve the fit of the survival models, the age variable was categorized into four groups. In addition to Hu, significant variables from the univariate analysis were used as candidates for the final Cox model. We attempted to enter medical sensible interactions into the model. The assessment of the proportional hazards assumption was done by comparing the Cox model with a time-dependent model [11]. Spearman correlation was used to compare the results of the two assays (ITT immunoprecipitation and immunofluorescence/dot blot), and Fisher's exact test was used to test independence between Hu and VGCC antibodies. StatXact (version 5; CYTEL, Cambridge, MA) was used to calculate the Fisher's exact tests. The survival analysis was performed with SPSS version 11 (SPSS Inc, Chicago, IL) and S-plus (version 6.0 professional release 1, Insightful Corp, Seattle, WA).

View larger version (12K): [in this window] [in a new window]   Fig 1. Kaplan-Meier survival curves for Hu-positive patients compared with Hu-negative patients.

	RESULTS

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Detection of VGCC Antibodies
VGCC antibodies were found in 10 (5%) of 200 SCLC patients. The concentrations varied from 50 to 945 pmol/L. There was no correlation between the presence of VGCC antibodies and sex (P = 1.0), but the presence of these antibodies was correlated with age (P = .02). Four of the patients with VGCC antibodies also had Hu antibodies, but there was no correlation between the presence of both paraneoplastic antibodies (P = .29).

Association of Hu Antibodies With Clinical Features of SCLC
Table 1 summarizes the variables studied and their association with Hu antibodies determined by ITT immunoprecipitation assay. There were no significant differences between performance status (P = .76), extent of disease (P = .34), LDH (P = .48), or survival (P = .18) among patients positive or negative for Hu antibodies. The survival data are illustrated by Kaplan-Meier curves in Figure 1. Similar correlations were also performed with Hu antibodies determined with immunofluorescence/dot blot, but no statistical significance was found (P = .29 for performance status; P = .82 for extent of disease; P = .90 for LDH; P = .65 for survival).

A univariate analysis was conducted to assess prognostic variables for survival (Table 2). Hu index (P = .18), VGCC (P = .60), or sex (P = .92) were not correlated, whereas performance status (P = .0007), extent of disease (P < .0006), LDH (P = .002), and age (P = .02) were all significantly associated with survival.

A multivariate Cox proportional hazards model was fitted to assess the relation between Hu index and survival (Table 3). The adjusted hazard ratio for Hu-positive patients compared with the Hu-negative patients was estimated to be 0.77, but this decreased risk for the patients with Hu antibodies was not significant (P = .13). However, survival was strongly correlated with extent of disease (P = .008), with an increased hazard ratio of 1.51 in the group with extensive disease compared with the group with limited disease. In addition, LDH (P = .01) and age (P = .012) gave significant results in the multivariate model.

To further investigate the association between the Hu index and survival, we divided the patients into four groups: index 100, index 101 to 249, index 250 to 500, and index more than 500. The univariate analysis gave no statistical association between the Hu index and survival (P = .09; Table 2). The statistical power of the multivariate analysis was low because of the limited number of observations, and therefore no data are given.

Association of VGCC Antibodies With Clinical Features of SCLC
VGCC antibodies were not correlated with performance status (P = .18), extent of disease (P = .52), LDH (P = 1.0), or survival (P = .6). Kaplan-Meier survival curves for those positive or negative for VGCC antibodies are illustrated in Figure 2.

View larger version (12K): [in this window] [in a new window]   Fig 2. Kaplan-Meier survival curves for voltage-gated calcium channel antibody (VGCC)-positive patients compared with VGCC-negative patients.

	DISCUSSION

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Previous reports have suggested a relation between Hu antibodies and the prognosis of SCLC. Dalmau et al [4] reported that seven nonparaneoplastic SCLC patients with anti-Hu had limited disease when diagnosed. Darnell and Deangelis [12] found radiologic regression of SCLC in two patients with paraneoplastic neurologic manifestations and Hu antibodies. Keime-Guilbert et al [2] reported similar median survival in 27 SCLC patients with paraneoplastic encephalomyelitis and anti-Hu, compared with matched SCLC controls without encephalomyelitis and Hu antibodies; however, they found a higher probability of survival at 30 months in the anti-Hu–positive patients. In a multicenter study of 170 SCLC patients without paraneoplastic neurologic disorders, patients with anti-Hu had a significantly better treatment response and longer median survival than those without detectable antibodies [5]. However, in line with our results, Graus et al [5], using Cox multivariate analysis, did not find Hu antibodies as an independent predictor for long-term survival.

Studies of Hu antibodies in unselected SCLC patients or patients without neurological disease have been infrequent. Graus et al [5] reported Hu antibodies in 16% of 170 patients with SCLC by Western blot, and a similar number using an enzyme-linked immunosorbent assay; these results compare well with the presence of Hu antibodies in 19% of our patients by dot blot. Recombinant HuD was used as antigen in these assays. However, we found that the prevalence of Hu antibodies on the basis of the dot blot technique was not correlated with performance status, extent of disease, or survival. In addition, we used an ITT immunoprecipitation assay, which was more sensitive than the dot blot, given that it detected Hu-antibodies in 25.5% of the patients. Even with this more sensitive assay, we did not find that the prevalence of Hu antibodies correlated with clinical measures.

Previously, it has been reported that only low levels of Hu antibodies were found in SCLC without paraneoplastic syndrome [4,7]. In contrast, we found that high levels, as found in patients with neurologic disorders, could be present in up to 13%, suggesting that high levels of Hu antibodies can be associated with SCLC without neurologic manifestations. Furthermore, we found no correlation between patients with low, medium, or high levels of Hu antibodies and survival.

The present group of patients matched those reported by Graus et al [5] regarding age, performance status, and extent of disease. However, only nine women were included in their study, and this could be of importance because female sex may be a good prognostic factor for SCLC [13]. We included 66 women, but did not find that female sex was an independent prognostic factor for SCLC. Furthermore, there was no predominance of women in the anti-Hu–positive group, contrary to previous findings [4]. We found performance status and disease extent to be the most important clinical factors for survival. Age and LDH were also significantly correlated with survival, and these data are all in line with other reports [14].

VGCC antibodies are usually believed to be highly specific for the presence of Lambert Eaton myasthenic syndrome. However, VGCC antibodies were recently reported in 41% of patients with paraneoplastic cerebellar degeneration and SCLC, of whom fewer than half had clinical evidence of Lambert Eaton myasthenic syndrome [10]. Similar to our results, this study did not show any significant difference in the median survival of the patients with or without detectable VGCC antibodies, although the number of patients was small. The fact that SCLC patients with Lambert Eaton myasthenic syndrome showed improved survival when compared with SCLC patients without Lambert Eaton myasthenic syndrome [3] may be because of a lead-time effect as a result of earlier tumor diagnosis in patients with the neurologic syndrome. Alternatively, it is possible that in SCLC patients with paraneoplastic neurologic syndromes and Hu and/or VGCC antibodies, the improved tumor prognosis may reflect parallel cellular immune responses that cause both the neurologic syndrome and improve tumor prognosis.

	Authors' Disclosures of Potential Conflicts of Interest

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The authors indicated no potential conflicts of interest.

	Acknowledgment

 
We thank Haukeland University Hospital and Innovest in Norway; and Dagmar Marshalls Fund and Fritz, Georg, and Marie Cecilie Glud's legacy in Denmark for support.

	NOTES

 
Authors' disclosures of potential conflicts of interest are found at the end of this article.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
1. Posner JB: Paraneoplastic syndromes. Neurol Clin 9:919–936, 1991[Medline]

2. Keime-Guilbert F, Graus F, Broet P, et al: Clinical outcome of patients with anti-Hu associated encephalomyelitis after treatment of the tumor. Neurology 53:1719–1723, 1999[Abstract/Free Full Text]

3. Maddison P, Newsom-Davis J, Mills KR, et al: Favourable prognosis in Lambert-Eaton myasthenic syndrome and small-cell lung carcinoma. Lancet 353:117–118, 1999[Medline]

4. Dalmau J, Furneaux HM, Gralla RJ, et al: Detection of the anti-Hu antibody in the serum of patients with small cell lung cancer: A quantitative Western blot analysis. Ann Neurol 27:544–552, 1990[CrossRef][Medline]

5. Graus F, Dalmau J, Rene R, et al: Anti-Hu antibodies in patients with small-cell lung cancer: Association with complete response to therapy and improved survival. J Clin Oncol 15:2866–2872, 1997[Abstract]

6. Tora M, Graus F, de Bolos C, et al: Cell surface expression of paraneoplastic encephalomyelitis/sensory neuronopathy-associated Hu antigens in small-cell lung cancer and neuroblastomas. Neurology 48:735–741, 1997[Abstract/Free Full Text]

7. Storstein A, Monstad SE, Nakkestad HL, et al: Paraneoplastic antibodies detected by a sensitive radiobinding assay. J Neurol (in press)

8. Moll JW, Vecht CJ: Immune diagnosis of paraneoplastic neurological disease. Clin Neurol Neurosurg 97:71–81, 1995[CrossRef][Medline]

9. Motomura M, Johnston I, Lang B, et al: An improved diagnostic assay for Lambert-Eaton myasthenic syndrome. J Neurol Neurosurg Psychiatry 58:85–87, 1995[Abstract/Free Full Text]

10. Graus F, Lang B, Pozo-Rosich P, et al: P/Q type calcium channel antibodies in paraneoplastic cerebellar degeneration with lung cancer. Neurology 59:764–766, 2002[Abstract/Free Full Text]

11. Grambsch P, Therneau TM: Proportional hazards tests and diagnostics based on weighted residuals. Biometrika 81:515–526, 1994[Abstract/Free Full Text]

12. Darnell RB, DeAngelis LM: Regression of small-cell lung carcinoma in patients with paraneoplastic neuronal antibodies. Lancet 341:21–22, 1993[CrossRef][Medline]

13. Wolf M, Holle R, Hans K, et al: Analysis of prognostic factors in 766 patients with small cell lung cancer (SCLC): The role of sex as a predictor for survival. Br J Cancer 63:986–992, 1991[Medline]

14. Yip D, Harper PG: Predictive and prognostic factors in small cell lung cancer: Current status. Lung Cancer 28:173–185, 2000[CrossRef][Medline]

Submitted January 3, 2003; accepted October 30, 2003.

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