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Keywords:

  • lung cancer;
  • WT1;
  • humoral immune response;
  • tumor marker;
  • prognostic marker

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

There are urgent needs to develop methods for early detection of nonsmall cell lung cancer (NSCLC) because of its increasing incidence and poor prognosis. Here, we analyzed the production of IgG antibody (WT1 Ab) against WT1 (Wilms' tumor gene) protein that was overexpressed in the majority of NSCLC. Enzyme-linked immuno-sorbent assay showed that WT1 Ab was produced in all of 91 NSCLC patients and 70 healthy individuals and that WT1 Ab titers were significantly higher in NSCLC patients compared with healthy individuals. When the cut-off level of WT1 Ab titers were fixed at mean + 3SD of those in healthy individuals, 26.4% of NSCLC patients had WT1 Ab titers over the cut-off level, and positive rates of WT1 Ab at each clinical stage were 25.0, 30.8 and 38.4% in stage I, II and III NSCLC, respectively. When WT1 Ab was combined with CEA or CYFRA for detection of NSCLC, positive detection rates increased from 25.0 to 34.1 and 31.8%, respectively, in stage I and from 38.4 to 69.2 and 46.1%, respectively, in stage III, but not changed in stage II. Western blot analysis showed that dominant subclass of WT1 Ab was Th1-type IgG2. Interestingly, elevation of WT1 Ab titers was significantly associated with longer disease-free survival in patients with stages I–III NSCLC. These results showed that WT1 Ab could be a useful marker for early detection of NSCLC and its prognostic prediction. These results also suggested that WT1-specific immune responses played an important role in anti-cancer immunity in NSCLC. © 2009 UICC

Lung cancer is the leading cause of cancer death in the world and nonsmall cell lung cancer (NSCLC) represents nearly 80% of the disease.1 Because 75% of lung cancer patients are diagnosed at stages of metastatic spread when therapies are rarely curative,2 early detection of localized lung cancer is the key to improve its clinical outcome.

Although chest X-ray is routinely used as a screening tool, its limitation to detect localized lung cancer has been evident because of its insufficient sensitivity.2 Recently, low-dose spiral computed tomography (CT) has been proposed as an early detection screening tool. However, despite its high sensitivity, specificity of CT in lung cancer detection is poor.3 Therefore, serum biomarkers with high sensitivity and specificity for diagnosis of lung cancer are needed. However, current serum biomarkers for NSCLC such as carcino embryonic antigens (CEA) and squamous cell carcinoma antigen do not have sufficient sensitivity and specificity required for early detection of NSCLC.4, 5

It is now clear that malignant transformation occurs by changes in cellular gene expression with subsequent clonal proliferation. Altered gene expression in malignant cells may lead to the expression of aberrantly expressed proteins recognized by host immune system. If autoantibodies against these antigens are produced at early stage of NSCLC when quantity of the tumor antigens in the circulation is very small, these antibodies should be useful markers for early detection of NSCLC. Moreover, because production of IgG antibodies needs helper T-cell functions and helper T cells play an important role in cellular immune responses, IgG antibodies against tumor antigens may be more useful markers for predicting prognosis in NSCLC patients compared with IgM antibodies.

The WT1 gene was originally isolated as a tumor suppressor gene responsible for a kidney neoplasm of the childhood, Wilms' tumor.6 However, we have demonstrated that the wild-type WT1 gene is overexpressed in leukemia7 and various types of solid cancers including lung,8, 9 gastric,10 esophageal,11 colorectal12 and pancreatic cancers.13 We proposed that the wild-type WT1 gene played oncogenic roles rather than tumor-suppressor functions in tumorigenesis of various types of cancers.14–16 We had previously demonstrated that IgG and IgM Ab against WT1 were produced at higher levels in patients with AML and MDS compared with normal individuals17 and that Th1 type subclasses of WT1 IgG Ab, IgG1, 2, and 3 were dominantly produced in the patients.18 On the other hand, production of WT1 Ab in patients with solid tumors remained undetermined.

In the present study, we demonstrate that high titers of Th1 type WT1 IgG antibody are detected in 26.4% of NSCLC patients and positive detection rate of WT1 Ab was higher than that of CEA or CYFRA in stages I and II NSCLC. Moreover, we describe that high titers of WT1 IgG Ab are associated with longer disease-free survival (DFS) in stages I–III NSCLC.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Serum samples

Serum samples were obtained from 91 patients with nonsmall cell lung cancer (54 adenocarcinoma, 29 squamous cell carcinoma, 4 large cell carcinoma, 2 pleomorphic carcinoma, 2 adeno-squamous cell carcinoma) before operation and from 70 healthy individuals under written informed consent at Dokkyo Medical University Hospital, Toneyama National Hospital, Osaka Rosai Hospital and Osaka University Hospital. Characteristics of the patients are shown in Table I. The samples were stocked at −80°C until use.

Table I. Characteristics of the Patients with NSCLC
 StageAge (median)Sex (M:F)Total
IIIIIIUnknown
Adenocarcinoma27591342–83 (70)32:2254
Squamous Cell Carcinoma1361948–85 (68)23:629
Large Cell Carcinoma400063–81 (72)4:04
Pleomorphic Carcinoma101060–822:02
Adeno-squamous Cell Carcinoma011074–751:12

Tissue samples

Tissue samples were obtained from 41 patients (40 adenocarcinoma and 1 squamous cell carcinoma) with stage I NSCLC at Osaka University Hospital under informed consent.

Construction of vectors for recombinant WT1 protein

To obtain recombinant full length WT1 protein and one each of three WT1 fragment proteins [WT-Fr1 (1–182aa), WT-Fr2 (180–324aa) and WT-Fr3 (318–449 aa)], corresponding region of the WT1 gene was amplified by PCR using Pfx polymerase (Invitrogen, Carlsbad, CA) and inserted into the pGEX-5X-3 vector (GE Healthcare Biosciences, Piscataway, NJ) that allowed the expression of the recombinant protein with an N-terminal GST tag. GST tagged, full length WT1 protein was purified by size fractionation using NA-1800 apparatus (Nippon Eido, Tokyo, Japan) according to the manufacturer's instructions. WT1 fragment proteins and GST protein were purified using Glutathione Sepharose 4 Fast Flow beads (GE Healthcare Biosciences) according to the manufacturer's instructions.

Enzyme-linked immunosorbent assay

Enzyme-linked immunosorbent assay (ELISA) was established to measure the titers of WT1 IgG Ab present in serum from patients and healthy individuals. ELISA 96 well plates (Multi Well Plate H Type Plate MS-8896F, Sumitomo Bakelite, Japan) were coated with 100 μl of recombinant GST tagged, full length WT1 protein (20 μg/ml) in immobilization Buffer (10 mM NaCO3, 30 mM NaHCO3, 0.02% NaN3, pH 9.6 overnight at 37°C. Plates were washed with Tris-buffered saline (TBS) and blocked with Blocking solution (TBS containing 0.05% Tween20 and 1% gelatin) at 37°C for 2 hr. Sera were diluted at 1:1,600 in Blocking solution and pre-absorbed by immobilized GST protein at 4°C overnight. Then, 100 μl of the diluted sera was added to each well for overnight incubation at 4°C. Plates were washed with TBST (TBS containing 0.05% Tween20) and incubated with ALP-conjugated goat anti-human IgG Ab (Santa Cruz Biotechnology, CA) diluted at 1:1,000 in TBST (TBS containing 1% Tween20) for 2 hr at room temperature. After washing, bound WT1 IgG Ab was visualized using 50 μl of BCIP/NBT kit (Nacalai Tesque, Kyoto, Japan) for each well. Then, absorbance at 550 nm was measured using a microplate reader MTP-32 (Corona Electric, Ibaraki, Japan). All sera were examined in duplicate. The titers of WT1 IgG Ab were calculated by interpolation from the standard line which was constructed for each assay from the results of simultaneous ELISA assay using serial diluted WT1 6F-H2 Ab using ALP-conjugated goat anti-mouse IgG Ab (diluted at 1:1,000; Santa Cruz Biotechnology) as the second Ab. WT1 Ab titers in the sera that produced the absorbance at 550 nm equal to that produced by 0.1 μg/ml of WT1 monoclonal 6F-H2 (Dako Cytomation, Golstrup, Denmark) Ab was defined as a 1.0 WT1-reacting-unit (WRU) in the ELISA system. Thus, WT1 IgG Ab titers of sera were measured to be [(concentrations of 6F-H2 mAb (μg/ml) corresponding to the absorbance at 550 nm produced by the diluted sera on the standard line) × (ratio of serum dilution) × 10] WRU. When titers of WT1 IgG Ab were high and out of measurable range in the ELISA system, the sera were diluted with Blocking solution to measurable levels and reanalyzed to determine the titers of WT1 IgG Ab.

Western blot analysis

For detection of WT1 IgG Ab, sera were diluted at 1:1,000, reacted with immobilized recombinant GST protein (200 ng) at 4°C overnight, and then reacted at room temperature overnight with GST tagged, full length WT1 protein or one each of three WT1 fragment proteins (200 ng) that was transferred onto PVDF membrane after SDS-PAGE. WT1 IgG Ab captured by the protein was visualized by ALP-conjugated anti-human IgG Ab or ALP-conjugated anti-human IgG subclass-specific Ab using a BCIP/NBT kit (Nacalai Tesque).

For analysis of relative amount of subclasses of WT1 IgG Ab and total IgG, 5 μl of diluted serum (diluted at 1:10 with 2 × Laemieli's SDS sample buffer) were loaded onto SDS-PAGE gel and the serum proteins were transferred onto PVDF membrane. Then, the membrane was reacted with ALP-conjugated anti-human IgG subclass-specific Ab at room temperature overnight and the bound Ab was visualized by using a BCIP/NBT kit.

ALP-conjugated anti-human IgG Ab was purchased from Santa Cruz Biotechnology and used at the dilution of 1:1,000. ALP-conjugated anti-human IgG1 (#05-3322), IgG2 (#05-3522), IgG3 (#05-3622) and IgG4 (#05-3822) Ab were purchased from Zymed Laboratories (San Francisco, CA) and used at the dilution of 1:500. WT1 monoclonal 6F-H2 antibody against 180aa residues in the amino terminal region of WT1 protein (Dako Cytomation), WT1 polyclonal C-19 antibody against 19aa residues near the carboxy terminus of WT1 protein (Santa Cruz Biotechnology) and GST antibody (Santa Cruz Biotechnology) were used as a first antibody. WT1 IgG Ab detected in Western blot analysis was scored into three categories; positive (WT1 IgG Ab was detected as a band with strong or intermediate density), weakly positive (WT1 IgG Ab was detected as a band with weak density), and negative (WT1 IgG Ab was not detected).

Immunohistochemistry

Formalin-fixed tissue sections of 3-μm thickness were cut from each paraffin-block. After dewaxing with xylene and rehydration through a graded series of ethanol, the sections were antigen retrieved using Pascal apparatus (Dako Cytomation) in 10 mM citrate buffer (pH 6.0). These sections were reacted with WT1 C-19 Ab (diluted at 1:100, Santa Cruz Biotechnology) at 4°C overnight and then reacted with Envision kit/HRP (Dako Cytomation) at room temperature for 30 min. After treatment with 0.7% H2O2 solution to reduce endogenous peroxidase activity, immunoreactive WT1 protein was visualized using DAB tablet (Wako, Osaka, Japan). The sections were then counterstained with hematoxylin.

Statistical analysis

The Welch's t test was used to calculate the difference between the titers of WT1 IgG Ab of NSCLC patients and those of healthy individuals. Statistical analysis for correlations between the titers of WT1 IgG Ab and the clinical parameters and was performed using Welch's t test or Kruskal–Wallis test. Statistical analysis for prognosis between NSCLC patients with or without elevated WT1 IgG Ab titers was performed by log-rank's test.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Establishment of ELISA system for measurement of WT1 IgG Ab

Enzyme-linked immunosorbent assay (ELISA) system was established to measure the titers of WT1 IgG Ab (WT1 Ab titers) in the sera from NSCLC patients and healthy individuals. In the ELISA system, WT1 IgG Ab was captured by GST tagged, full length WT1 protein and visualized using ALP-conjugated anti-human IgG Ab. WT1 Ab titers were calculated by interpolation from the standard line, which was constructed from the results of simultaneous ELISA assay using serial diluted WT1 monoclonal 6F-H2 Ab. The absorbance at 550 nm produced by serially diluted WT1 monoclonal 6F-H2 Ab showed a linear plot (R2 = 0.9612) against the concentrations of 6F-H2 Ab ranging from 0.008 to 1 μg/ml (Fig. 1a). WT1 Ab titers in the sera that produced the absorbance at 550 nm equal to that produced by 0.1 μg/ml of WT1 monoclonal 6F-H2 Ab was defined as a 1.0 WRU. Thus, WT1 IgG Ab titers of sera were measured to be [(concentrations of 6F-H2 mAb (μg/ml) corresponding to the absorbance at 550 nm produced by the diluted sera on the standard line) × (ratio of serum dilution) × 10] WRU. The standard line was reproducible with between-run coefficient of variation (CV) of 7.5% in 10 independent assays.

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Figure 1. Enhanced production of WT1 IgG Ab in NSCLC patients. (a) A representative standard line of the ELISA system for measurement of the titers of WT1 IgG Ab. The absorbance at 550 nm produced by serial dilution of WT1 monoclonal 6F-H2 Ab was measured by the ELISA system. The results are from four independent assays, and the mean ± SE is shown. WT1 Ab titer that produces the absorbance at 550 nm equal to that produced by 0.1 μg/ml of WT1 monoclonal 6F-H2 Ab in the ELISA system was defined as 1.0 WT1-reacting-unit (WRU). (b) Increased WT1 Ab titers in NSCLC patients. WT1 Ab titers were examined by the ELISA system in 91 NSCLC patients and 70 healthy individuals. Dotted line shows the cut-off level (2,910 WRU) of WT1 Ab titers, *p < 0.05. (c) Detection of WT1 IgG Ab by Western blot analysis in NSCLC patients. Western blot analysis was performed as described in “Material and Methods”. As representative results, those from three NSCLC patients (P16, P19 and P22) and healthy individual (H2) are shown. WT1 monoclonal 6F-H2 Ab and GST Ab were used to show the positions of GST-WT1 protein and GST protein, respectively.

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Elevation of WT1 IgG Ab in NSCLC patients

WT1 Ab titers (WRU) were examined in 91 NSCLC patients and 70 healthy individuals. WT1 IgG Ab was detected in all the samples examined (Fig. 1b). WT1 Ab titers ranged from 10 to 3,664 (median 392) and from 183 to 104,940 (median 1,135) WRU in healthy individuals and NSCLC patients, respectively. Therefore, WT1 Ab titers were significantly (p < 0.05) higher in NSCLC patients than in healthy individuals. When the cut-off level of WT1 Ab titers was fixed at 2,910 WRU, which was mean + 3SD of WT1 Ab titers in healthy individuals, 24 (26.4%) of 91 NSCLC patients had WT1 Ab over the cut-off level.

To confirm the presence of WT1 IgG Ab in sera from NSCLC patients and healthy individuals, sera of randomly selected 31 NSCLC patients and 27 healthy individuals were reacted with immobilized recombinant GST protein and then examined for WT1 IgG Ab by Western blot analysis using GST-WT1 protein as an antigen. WT1 IgG Ab was detected in all of the 31 NSCLC patients and 27 healthy individuals examined (Fig. 1c, upper panels). Furthermore, to confirm that the bands detected did not result from the reaction of GST portion of GST-WT1 protein with antibodies against GST in the patients' sera, Western blot analysis was performed using the sera which was pre-reacted with immobilized GST protein and purified GST protein as an antigen. As expected, sera of the patients and healthy individuals did not react with GST protein (Fig. 1c, lower panels). These results showed that Western blot analysis used here could specifically detect WT1 IgG Ab.

No correlation between WT1 Ab titers and clinical parameters in NSCLC patients

Whether or not WT1 Ab titers were correlated with clinical parameters was examined in NSCLC patients. As shown in Figure 2, no correlation was found between WT1 Ab titers and clinical parameters such as histology, clinical stages and age.

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Figure 2. No correlation between WT1 Ab titers and clinical parameters of NSCLC. Correlations between WT1 Ab titers and clinical parameters of NSCLC, histology (a), clinical stages (b) and age (c) are shown. n.s., Not significant.

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Epitope distribution of WT1 IgG Ab in NSCLC patients and healthy individuals

Epitope distribution of WT1 IgG Ab was examined in the 31 NSCLC patients and 27 healthy individuals by Western blot analysis using three purified, recombinant WT1 fragment proteins, WT-Fr1 (1–182aa), WT-Fr2 (180–324aa) and WT-Fr3 (318–449aa) as antigens (Fig. 3a). Sera from 30 (96.8%) of 31 NSCLC patients and 24 (88.9%) of 27 healthy individuals examined recognized two or three WT1 fragment proteins (Fig. 3b). These results indicated the presence of multiple antigenic epitopes within WT1 protein.

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Figure 3. Epitope distribution of WT1 IgG Ab. (a) Schema of WT1 fragment proteins. Recombinant WT1 fragment proteins were expressed in bacterial hosts. (b) Western blot analysis for epitope distribution of WT1 IgG Ab. Western blot analysis was performed. WT1 antibodies, 6F-H2 monoclonal Ab against 180 aa residues at the amino terminus of WT1 protein and C-19 polyclonal Ab against 19 aa residues near the carboxy terminus were used as positive control antibodies recognizing WT-Fr1 and WT-Fr3, respectively. Because WT1 antibody recognizing WT-Fr2 was not available, GST antibody was used to show the band responsible for WT-Fr2 with GST tag. Representative results of NSCLC patients (P5 and P44) and a healthy individual (H1) are shown.

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Dominant subclass of WT1 IgG Ab is IgG2

To examine whether humoral immune responses against WT1 protein were Th1- or Th2- type, subclasses of WT1 IgG Ab against WT-Fr3 protein, which was recognized by all of the sera examined, were examined by Western blot analysis in the 31 NSCLC patients and the 27 healthy individuals (Fig. 4). IgG2 was a dominant subclass of WT1 Ab in all of the patients and healthy individuals examined (Fig. 4a, upper panels). To confirm that this IgG2 dominance did not result from the reflection of the dominance of total IgG2 subclass, subclasses of total IgG were simultaneously determined (Fig. 4a, lower panels). Differently from the IgG2 dominance of WT1 IgG Ab, the dominant subclass of total IgG was IgG1. These results showed that dominant subclass of WT1 IgG Ab was IgG2. In NSCLC patients, positivity (see “Material and Methods”) of WT1 IgG1, IgG2, IgG3 and IgG4 subclass Ab were 22.6% (7 of 31), 64.5% (20 of 31), 19.4% (6 of 31) and 12.9% (4 of 31), respectively (Figs. 4a, 4b). In healthy individuals, positivity of WT1 IgG1, IgG2, IgG3 and IgG4 subclass Ab were 22.2% (6 of 27), 77.8% (21 of 27), 29.6% (8 of 27) and 29.6% (8 of 27), respectively.

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Figure 4. Subclass distribution of WT1 IgG Ab. Recombinant WT1 fragment protein (WT-Fr3, approximately 200 ng/lane) was subjected to Western blot analysis using sera from NSCLC patients and healthy individuals as first antibodies. Membrane bound WT1 IgG Ab was visualized using IgG subclass-specific second Ab. (a) Representative results of Western blot analysis for WT1 IgG Ab and IgG subclasses in NSCLC patients (P4 and P22) and healthy individuals (H11 and H18). Results of WT1 IgG Ab (upper panels) and total IgG (lower panels) subclasses were displayed in pairs for each patient and healthy individual. WT1 polyclonal C-19 Ab and IgG Ab were used to confirm the positions of the bands responsible for WT1 IgG Ab and IgG, respectively. (b) Positive rate of each subclass of WT1 IgG Ab produced in NSCLC patients and healthy individuals is graphically shown. (c) Semiquantitative nature of the Western blot analysis. Western blot analysis was performed for detecting IgG2 WT1 Ab, which was dominantly contained in the serum, by using WT1-Fr3 protein as an antigen and serially diluted serum from a NSCLC patient (P4) (upper). To confirm the band of WT1 IgG Ab, polyclonal WT1 Ab C-19 was used. The band density obtained from the diluted serum is graphically shown as the mean (columns) and SD (bars) of four independent experiments (lower).

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To confirm the semiquantitative nature of the Western blot analysis used here, it was performed by using WT-Fr3 protein as an antigen and serially diluted serum from a NSCLC patient (P4). As shown in Fig. 4c, the band density of IgG2 WT1 Ab, which was dominantly contained in the serum, decreased as the serum was diluted at the ratio of 1:200, 1:600 and 1:1,800, confirming that the Western blot analysis used here could semi-quantitatively detect WT1 IgG Ab.

Therefore, production of Th1-type, IgG2 WT1 Ab was enhanced in NSCLC patients, indicating that Th1-type humoral immune responses against WT1 protein were elicited in NSCLC patients.

WT1 Ab as a biomarker for detection of NSCLC

Positivity of WT1 Ab was compared with that of currently available tumor markers, CEA and CYFRA, in NSCLC patients. Clinical data of CEA and CYFRA were available for 70 (44 on stage I, 13 on stage II and 13 on stage III) and 59 patients (36 on stage I, 12 on stage II and 11 on stage III) with NSCLC, respectively. As shown in Figure 5, WT1 Ab titers did not correlate with serum levels of CEA (Fig. 5a) and CYFRA (Fig. 5b). Positive detection rate for WT1 Ab, CEA and CYFRA was 25.0 (11 of 44), 13.6 (6 of 44) and 11.1% (4 of 36) in stage I NSCLC patients; 30.8 (4 of 13), 23.1 (3 of 13) and 16.7% (2 of 12) in stage II patients and 38.5 (5 of 13), 46.2 (6 of 13) and 18.2% (2 of 11) in stage III patients, respectively (Fig. 5c). Because positive detection rate of WT1 Ab was 25.0%, which was higher than that of either CEA or CYFRA, in stage I NSCLC patients, it may be a useful marker for early detection of NSCLC.

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Figure 5. WT1 Ab level as a marker for detection of NSCLC. No correlation between WT1 Ab levels and levels of current NSCLC tumor marker, (a) CEA (n = 70) or (b) CYFRA (n = 59). Dotted line indicates the cut-off level for each marker. (c) Positive detection rate for NSCLC. (d) Overexpression of WT1 protein in stage I NSCLC. Expression of WT1 protein was examined by immunohistochemistry in stage I NSCLC. (a, b) Representative results of different cases are shown. WT1 protein is detected in brown.

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When WT1 Ab was combined with CEA or CYFRA for detection of NSCLC, positive detection rates increased from 25.0 to 34.1 and 31.8%, respectively, in stage I and from 38.4 to 69.2 and 46.1%, respectively, in stage III, but not changed in stage II (Fig. 5c). Thus, WT1 Ab is useful for sensitive detection of NSCLC in combination with CEA or CYFRA.

Overexpression of WT1 protein in early stage NSCLC

To confirm the expression of WT1 protein in early stage NSCLC, the expression of WT1 protein was examined by immunohistochemistry in 41 patients with stage I NSCLC. WT1 expression in cancer cells was scored into the following three categories: positive, weakly positive and negative staining meant strong, slightly stronger and similar one of lung cancer cells, respectively, compared with that of the alveolar epithelial cells of lung. In positive or weakly positive cases, WT1 protein was dominantly detected in the cytoplasm of lung cancer cells. WT1 protein expression in lung cancer cells was positive in 30 (73.2%), weakly positive in 7 (17.1%), and negative in the remaining 4 (9.8%), of 41 NSCLC patients (Fig. 5d). Thus, WT1 protein was overexpressed in 90.3% (37 of 41) of stage I NSCLC patients examined.

Elevation of WT1 Ab is associated with favorable prognosis in NSCLC patients

To examine whether or not enhanced humoral immune responses against WT1 were associated with lower relapse rates in patients with NSCLC, DFS rates were analyzed between two groups of NSCLC patients whose follow-up data after surgery were available: 20 patients with elevated WT1 Ab titers (52,110–3,514; median 8,777 WRU) and 59 patients without elevated WT1 Ab titers (2,648–183; median 655 WRU). In patients with elevated WT1 Ab titers, 1-, 2- and 3-year DFS rates of the patients were 100, 94.7 and 94.7%, respectively. In contrast, in patients without elevated WT1 Ab titers, 1-, 2- and 3-year DFS rates were 88.0, 82.8 and 77.4%, respectively. Kaplan–Meier curve and log rank's test showed that patients with elevated WT1 Ab titers had significantly (p = 0.002) longer DFS than those without them (Fig. 6). These results indicated that enhancement of humoral immune responses against WT1 was associated with favorable prognosis in patients with NSCLC.

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Figure 6. Kaplan–Meier curves for disease-free survival in 79 NSCLC patients. The results from NSCLC patients with elevated WT1 IgG Ab titers (elevated, n = 20) and without them (nonelevated, n = 59) are shown.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In the present study, we showed that WT1 IgG Ab was produced in all of the 91 NSCLC patients and 70 healthy individuals and that 26.4% of the NSCLC patients had WT1 IgG Ab titers over the cut-off level which was mean + 3SD of those of healthy individuals. These findings showed two aspects of WT1 specific immune responses in NSCLC patients. One was that, in consistence with our previous reports on patients with leukemia and MDS,17, 18 humoral immune responses were also elicited against cancer-derived WT1 protein in patients with NSCLC. The elevation of WT1 Ab titers were found even in stage IA NSCLC in which tumor size was less than 3 cm, indicating that the immune system could recognize cancer-derived WT1 protein and respond to it when its amount was still small. Another was the difference in the immune responses against WT1 protein in individual NSCLC patients. No differences were observed in clinical parameters such as histology, clinical stages and age among NSCLC patients with or without elevated WT1 Ab titers. Thus, the reason why a part of NSCLC patients highly produced WT1 Ab remains undetermined.

There is a great need to develop novel biomarkers for early detection of NSCLC. In the present study, we showed that positive detection rate of WT1 Ab was higher than that of CEA or CYFRA in stages I and II NSCLC. One reason for it should be the difference in the substances measured in these assays. Because the currently available cancer biomarkers such as CEA and CYFRA are based on the measurement of the substances released into blood from cancer cells, detection of such substances may be difficult until cancer becomes advanced and releases considerable amount of the substances. In contrast, measurement of WT1 Ab is an assay of antigen-specific humoral immune responses. Even if early cancer releases small amount of tumor antigens, host immune system is considered to be able to recognize small amount of antigens and respond to them. Through this process, a signal with antigenic tumor protein is biologically amplified to the detectable level.19 Furthermore, because WT1 Ab was an independent tumor marker that was not correlated with serum levels of CEA and CYFRA, combination of WT1 Ab with CEA or CYFRA increased the positive detection rates for NSCLC. Taken together, these results indicated that WT1 Ab was a useful serum marker for early detection of NSCLC.

Helper T cells are central regulators of both humoral and cellular immune responses.20 Th1 cells are primarily responsible for activating and regulating the development and persistence of cytotoxic T lymphocyte. In addition, Th1 cells activate antigen-presenting cells and induce the production of Th1 type antibodies (IgG1, 2 and 3), whereas Th2 cells promote that of Th2 type one (IgG4). In the present study, we analyzed the subclass of WT1 IgG Ab and showed that dominant subclass of WT1 IgG Ab was Th1-type IgG2 in NSCLC patients, indicating activation of Th1 type WT1-specific immune responses. These results might indicate that WT1-specific CD8+ cytotoxic T lymphocyte (CTL) responses were more activated in NSCLC patients with elevated WT1 Ab titers compared with those without them. Interestingly, high titers of WT1 IgG Ab were associated with longer DFS in NSCLC patients. This may be explained by the activated WT1-specific CD8+ CTL responses in NSCLC patients with elevated WT1 Ab titers. Another possible explanation is a direct cytotoxic effect of WT1-specific CD4+ T lymphocytes on NSCLC cells. Recently, direct recognition and lysis of leukemia cells by WT1-specific CD4+ T lymphocytes in an HLA class II-restricted manner were reported.21 Because elevated WT1 Ab titers were considered to be associated with activation of WT1-specific CD4+ T lymphocytes and because HLA class II molecules were expressed in NSCLC cells,22 it was possible that WT1-specific CD4+ T lymphocytes exert their cytotoxic activities against NSCLC. These findings and our results presented here indicated that WT1-specific immune responses played an important role in anti-tumor immunity against NSCLC and that WT1 IgG Ab was a good marker for prognostic prediction of NSCLC.

Although many reports show the production of antibodies against autoantigens in cancer patients,23–25 that of these antibodies in healthy individuals remains unclear. This study showed that WT1 IgG Ab was produced in all of the healthy individuals examined. The titers of WT1 IgG Ab in these individuals were low and dominant subclass of WT1 IgG Ab was Th1-type IgG2. Thus, Th1-type humoral immune responses were weakly elicited in healthy individuals. Moreover, our previous report showed that WT1-specific CTLs exist at a percentage of 0.04 ± 0.02% of CD8+ T-cells in peripheral blood mononuclear cells of five healthy volunteers.26 The frequency seems to be higher than that of CTLs against other antigens such as gp100 or Tyrosinase.27 These results indicated that WT1 protein elicited both humoral and cellular immune responses in healthy individuals, indicating its high immunogenecity. As for the source of WT1 antigen in healthy individuals, one possible source of WT1 antigen is normal WT1-expressing organs such as urogenital systems and mesothelium.28 Although these organs express WT1 protein at high levels, no injury was observed in the patients vaccinated with WT1 peptide in clinical trials,29 suggesting that the normal WT1-expressing organs are protected from WT1-specific immune responses by unknown mechanism. Another source may be undetectable tumors expressing WT1. It is considered that tumors develop in healthy individuals but are eliminated by host immune surveillance system before tumors grow to detectable levels.30 Because WT1 protein is highly immunogenic, WT1-specific immune responses may function as one of immune surveillance system against cancer in healthy individuals.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors thank Dr. Yoshinori Iwatani (Department of Biomedical Informatics, Osaka University Graduate School of Medicine) for his advice on ELISA.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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