Purpose: Patients with mutations of epidermal growth factor receptor (EGFR) receive more benefit from EGFR-tyrosine kinase inhibitor treatment. However, usually such treatment is used to treat advanced lung cancer and only small biopsy samples are available for mutational analysis. We used immunohistochemistry to examine recently developed antibodies specific to major hotspot mutations of L858R and DEL E746-A750.

Experimental Design: We used five series of lung cancers: 47 non–small cell lung cancers (NSCLC) to evaluate various types of EGFR mutations, a consecutive series of 238 NSCLCs to study the sensitivity and specificity, 11 NSCLCs with both EGFR mutation and amplification to examine the spatial distribution, 32 patients treated with gefitinib to compare clinical responses, and 15 NSCLCs to explore changes associated with acquired T790M mutation.

Results: Each antibody specifically recognized the corresponding mutation but also recognized other types of mutations. Overall specificity and sensitivity were 96% and 47%, respectively. The positive reaction showed heterogeneous distribution that agreed with the expression of the total EGFR molecule, part of which was associated with gene amplification. A clinical response to gefitinib treatment correlated with the reaction, although one of the two patients with a positive reaction responded well despite having the wild-type EGFR. Acquired T790M mutation did not change the reaction to the antibodies.

Conclusions: On some characteristics, the positive reaction to mutation-specific antibodies differs from the molecular EGFR mutation. Therefore, this study revealed that not all patients with EGFR mutations can be selected using these mutation-specific antibodies. Clin Cancer Res; 16(13); 3349–55. ©2010 AACR.

Translational Relevance

Mutation analysis of epidermal growth factor receptor (EGFR) plays an important role in the initial treatment in many patients with advanced lung cancers, in which only small biopsy samples are usually available. Immunohistochemistry has an advantage in examining such small biopsy specimens. In this study, we evaluated recently developed antibodies, which were generated to recognize major hotspot mutations of L858R and DEL E746-A750. Each antibody was specific to the corresponding mutation. However, the positive reaction showed some characteristics that differed from those of the molecular mutation, including recognition of different mutation types, association with expression of the total EGFR molecule, and heterogeneous distribution of the positive reaction. A positive reaction correlated generally with a clinical response to EGFR-TKI treatment, but not all patients with EGFR mutations can be selected using these mutation-specific antibodies.

Epidermal growth factor receptor (EGFR) is a receptor with a tyrosine kinase domain and plays essential roles in physiologic and neoplastic conditions (1, 2). Mutation in the tyrosine kinase domain has been identified in a subset of lung cancer (35), and its mutational status is associated with the clinical response to EGFR-tyrosine kinase inhibitor (TKI) treatment (6, 7). This association solved the enigma of why female, nonsmoking, adenocarcinoma patients of East Asian origin with lung cancers have a higher response to EGFR-TKIs; it is because patients with these characteristics have a higher incidence of EGFR mutation (8, 9). Similarly, much has been elucidated about the clinical response to the EGFR-TKIs and the characteristics of EGFR tyrosine kinase mutation (10, 11). Patients with EGFR-mutated lung cancers who initially achieve a marked response to EGFR-TKI treatment eventually develop progression of the disease during the course of treatment, which is caused, at least partly, by a secondary mutation, such as T790M (12, 13). The results of several clinical trials indicate that EGFR gene copy number is also a response predictor (6), and that an exaggerated form of increased copy number, EGFR amplification, is associated with progression of adenocarcinoma (14, 15).

Increasing clinical evidence suggests that patients with EGFR mutation receive more benefits from EGFR-TKI treatment (2, 6). EGFR-TKI treatment has been shown recently to be superior to carboplatin-paclitaxel as an initial treatment (16). Therefore, mutation analysis plays an important role in the initial treatment in many patients with advanced lung cancers. Many techniques are available to examine EGFR mutation, including direct sequencing, Scopion amplified refractory mutation system (ARMS), polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP), peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR clamp, smart amplification process (SMAP), and cycleave PCR (17). Because EGFR-TKI treatment is used mainly to treat advanced stages of lung cancer, mutation analysis is done with a small biopsy sample, which is embedded in paraffin in most cases. However, DNA extracted from paraffin-embedded tissues is often degraded and mixed with a significant amount of normal tissue. Yu et al. (18) recently developed mutation-specific rabbit monoclonal antibodies against EGFR with the E746-A750 deletion in exon 19 or the L858R point mutation and showed that these antibodies can be applied to the immunohistochemical detection of the mutations using paraffin-embedded tissue. Because these antibodies are available commercially, we applied them in lung cancer samples with various EGFR mutation types. We also analyzed tissues from a cohort of patients with lung cancer in a region with a high incidence of EGFR mutation. Furthermore, we correlated these data with the clinical response to EGFR-TKI treatment and in lung cancers with EGFR amplification and secondary T790M mutation.

Patients and samples

We used five independent data sets in this study. To evaluate various types of EGFR mutations, 47 cases of non–small cell lung cancer (NSCLC) were selected from a database held at the Department of Pathology and Molecular Diagnostics, Aichi Cancer Center, Nagoya, Japan. To determine the sensitivity and specificity in a cohort, we used tissue microarray data from a consecutive series of 238 NSCLCs that had been surgically resected from 2002 to 2003 in the Aichi Cancer Center. In this cohort, invasive tumors <1 cm in diameter were excluded because of difficulties in generating the tissue microarray. A part of this cohort has been reported (19), and the mutational status of the NSCLCs has been examined. To compare the immunohistochemical reactions with the clinical response to gefitinib treatment, we used 32 patients, whose data were reported previously (7). Using 10 adenocarcinomas and an adenosquamous cell carcinoma, whose copy numbers have been reported, we compared the spatial distribution between a positive reaction to the mutation-specific antibodies and gene copy number. In each tumor, three independent portions were selected to reflect the morphologic characteristics, and the status of the EGFR mutation and amplification were determined (14). To analyze the T790M mutation, 15 NSCLCs from nine patients were selected from the database. Six were patients who were treated with gefitinib and subsequently developed resistance caused by a secondary T790M mutation (19, 20). In the other three patients, simultaneous mutation of L858R and T790M was detected in the surgically resected primary adenocarcinomas, but the patients were not treated with EGFR-TKI before surgery. All specimens examined in this study were obtained from primary lung tumors, except for the T790M analysis, in which recurrent tumors of skin and lymph nodes were used. This study was a part of a comprehensive lung cancer research program, which had been approved by an institutional review board. Written informed consent for participation in the program was obtained from each patient.

Immunohistochemistry

Immunohistochemical examination proceeded according to the standard avidin-biotin-peroxidase complex method using monoclonal rabbit antibodies against L858R and DEL E746-A750 (clone 43B2 and clone 6B6, respectively, Cell Signaling Technology). Antigens were retrieved by microwave for 15 minutes in EDTA buffer (pH 9.0). To access expression of total EGFR molecule, we used the EGFR pharmDX kit (DAKO). Briefly, 4-mm-thick formalin-fixed and paraffin-embedded tissue sections were deparaffinized in xylene, treated with 0.3% hydrogen peroxide in methanol for 20 minutes to block endogenous peroxidase activity, microwaved for antigen retrieval, and incubated with 10% normal goat serum for 30 minutes to block nonspecific binding. The rabbit monoclonal antibodies were then applied as the primary antibody at a dilution of 1:100 at 4°C overnight. The subsequent procedure followed the manufacturer's instructions (Vectastain ABC Elite kit, Vector Laboratories). The sections were incubated with biotinylated anti-rabbit antibody for 40 minutes at room temperature and then with the ABC complex for 30 minutes. Color was developed by the diaminobenzidine reaction. In between each incubation step, the sections were washed with cold PBS.

Tissue microarray slides stained with these antibodies were digitized using an Aperio ScanScope (Aperio Technologies, Inc.) and were then analyzed using the TMALab Microarray Analysis Tool. The tissue cores scoring 1+ or more based on membrane algorithms were reevaluated under a microscope, and a tissue core was considered positive when an appropriate positive reaction was confirmed. Other staining was done using normal-sized sections of the tumor. In these sections, the positive intensity was scored as 1+, 2+, and 3+, which were equivalent to the intensity based on the computer-quantified analysis of tissue microarrays. A tumor was recorded as positive when 10% or more of the tumor cells had an intensity score of 1+ or more. The immunohistochemical reactions were evaluated by the last author noted (Y.Y.) and at least one of the other authors.

EGFR mutation and amplification

Mutation data for the EGFR gene were obtained from the database, which has been analyzed using direct sequencing of reverse transcription-PCR products for fresh frozen tissues, and/or a cycleave PCR technique (L858R and T790M) and fragment analysis (deletion of exon 19) of paraffin-embedded tissues. These methods have been described elsewhere (7, 19, 20). To access the amplification status of EGFR, we used data previously reported (14). Briefly, we examined three portions of each individual tumor using two methods: quantitative PCR with microdissected fragments and fluorescent in situ hybridization.

Positive reaction among a spectrum of EGFR mutations

We first examined the specificity of the two antibodies specific to the L858R point mutation and in-frame deletion in exon 19 (DEL E746-A750) using lung cancers with the spectrum of EGFR mutations listed in Table 1. Anti-DEL antibody showed a positive reaction in 4 of 16 mutation types in exon 19 (Fig. 1). All of these involved E746-A750. DEL E746-A750insK was positive, whereas DEL E746-A750insRP and DEL L747-A750insP were negative. The other deletions in exon 19 were negative. In contrast, anti-L858R mutation–specific antibody reacted in all 12 lung cancers with the L858R mutation, which involves a different pattern of nucleotide substitution (CTG → CGT). Additional point mutations adjacent or near the L858R mutation (A859S and A861S) also showed a positive reaction. Of note, these antibodies were positive for other types of mutations, including G719C, DEL L747-T751insQ, and A769insASV with anti-L858R–specific antibody, and D770insSVD with anti-deletion–specific antibody (Fig. 1), although such positive reactions were not always seen in the tumors with these mutation types. In addition, anti-L858R–specific antibodies frequently showed a faint reaction in tumors with deletional mutation in exon 19.

Table 1.

Positive reactions among a spectrum of EGFR mutations

GenotypeImmunohistochemistry
nAnti-L858R AbAnti-DEL Ab
Exon 18 (n = 5) 
    G719A 
    G719C 
    G719S 
Exon 19 (n = 21) 
    DEL E746-A750 
    DEL E746-A750 
    DEL E746-A750 (2253 A → G T751T) 
    DEL E746-A750 ins K 
    DEL E746-A750 ins RP 
    DEL L747-A750 ins P 
    DEL L747-P753 insS 
    DEL L747-S752 
    DEL L747-S752 insV 
    DEL L747-T751 ins A 
    DEL L747-T751 ins Q 
    DEL S721-I759 
    DEL T751-A755 DEL E758-I759 ins A 
    DEL T751-I759 ins S 
    E746 ins VPVAIK 
    K744 ins KIPVAI 
Exon 20 (n = 7) 
    A769 ins ASV 
    D770 ins SVD 
    H773 ins NPH 
    P772 ins A 
Exon 21 (n = 14) 
    L858R(CTG → CGG) 11 11 
    L858R(CTG → CGT) 
    L858R & A859S 
    L858R & A861S 
Total 47 19 
GenotypeImmunohistochemistry
nAnti-L858R AbAnti-DEL Ab
Exon 18 (n = 5) 
    G719A 
    G719C 
    G719S 
Exon 19 (n = 21) 
    DEL E746-A750 
    DEL E746-A750 
    DEL E746-A750 (2253 A → G T751T) 
    DEL E746-A750 ins K 
    DEL E746-A750 ins RP 
    DEL L747-A750 ins P 
    DEL L747-P753 insS 
    DEL L747-S752 
    DEL L747-S752 insV 
    DEL L747-T751 ins A 
    DEL L747-T751 ins Q 
    DEL S721-I759 
    DEL T751-A755 DEL E758-I759 ins A 
    DEL T751-I759 ins S 
    E746 ins VPVAIK 
    K744 ins KIPVAI 
Exon 20 (n = 7) 
    A769 ins ASV 
    D770 ins SVD 
    H773 ins NPH 
    P772 ins A 
Exon 21 (n = 14) 
    L858R(CTG → CGG) 11 11 
    L858R(CTG → CGT) 
    L858R & A859S 
    L858R & A861S 
Total 47 19 
Fig. 1.

Representative positive reactions of lung adenocarcinomas with DEL E746-A750 mutation stained with anti-DEL antibody (A), with L858R point mutation stained with anti-L858R antibody (B), with A769 ins ASV mutation in exon 20 stained with anti-L858R antibody (C), and with G719A mutation stained with anti-L858R antibody (D).

Fig. 1.

Representative positive reactions of lung adenocarcinomas with DEL E746-A750 mutation stained with anti-DEL antibody (A), with L858R point mutation stained with anti-L858R antibody (B), with A769 ins ASV mutation in exon 20 stained with anti-L858R antibody (C), and with G719A mutation stained with anti-L858R antibody (D).

Close modal

Correlation of the positive reaction with EGFR genotype

We then examined a consecutive series of 238 lung cancers, and the results are summarized in Table 2. The overall sensitivity and specificity using the two specific antibodies were 92% and 37%, respectively. The anti-DEL–specific antibody had higher specificity and sensitivity than the L858R-specific antibody (99% and 40% versus 97% and 36%, respectively). Four tumors with a false-positive reaction were reexamined using normal-sized sections but produced the same results. Because 226 of the 238 tumors (95%) were evaluated using at least three tissue microarray cores, the distribution of the positive reaction was compared between the cores of individual tumors. Of 33 NSCLCs with a positive reaction, the reaction was heterogeneously distributed in 15 (45%).

Table 2.

Relationship between genotype and positive reaction to the mutation-specific antibodies

Histologic subtypeGenotypeTotal
Wild-type (n = 151)DEL Exon19 (n = 41)L858R (n = 37)Other type (n = 9)
Adenocarcinoma 96 39 37 181 
Other types* 55 57 
Immunohistochemical reaction 
Negative 147 24 25 205 
Positive 17 12 33 
    Anti-DEL antibody 16 17 
    Anti-L858R antibody 12 18 
    Heterogeneous 15 
Histologic subtypeGenotypeTotal
Wild-type (n = 151)DEL Exon19 (n = 41)L858R (n = 37)Other type (n = 9)
Adenocarcinoma 96 39 37 181 
Other types* 55 57 
Immunohistochemical reaction 
Negative 147 24 25 205 
Positive 17 12 33 
    Anti-DEL antibody 16 17 
    Anti-L858R antibody 12 18 
    Heterogeneous 15 

*Other histologic types include 11 large cell carcinoma, four small cell lung cancer, five adenosquamous carcinoma, and one carcinoid tumor.

Heterogeneous denotes different staining intensity in at least one of the tissue microarray cores in individual tumors.

Gene amplification and mutation-specific antibody reaction

We recently reported that EGFR is amplified heterogeneously in individual tumors and that this amplification was associated with invasive growth. To test whether this heterogeneous amplification is associated with a similar pattern of distribution of the positive reaction to the mutation-specific antibodies, we examined the correlation between the amplification and the positive reaction with mutation-specific and conventional EGFR antibodies. Using the amplification data of the individual tumors in the previous study (14), we compared the positive reaction with amplification status. The intensity of the positive reaction correlated significantly with the relative EGFR copy number EGFR (one-way ANOVA, P < 0.01; Fig. 2), whereas the amplification status did not correlate with the expression of total EGFR (one-way ANOVA, P = 0.355). Both expressions of mutation-specific and total EGFR was associated (one-way ANOVA, P < 0.05). These findings suggest that the positive reaction to mutation-specific antibodies is associated with EGFR amplification but that the amplification is not the sole determinant of a positive reaction.

Fig. 2.

Positive reaction of mutation-specific antibodies (Ab) and gene copy number. Top, the relationship of the gene copy number with the intensity of the positive reaction to mutation-specific antibodies (left) and total EGFR antibody (right). An intense positive reaction (score 3+) tended to show gene amplification with both antibodies. Similar to EGFR amplification, the positive reaction was also distributed heterogeneously. In general, a positive reaction correlated with the expression of the total EGFR molecule (bottom left), but this was not always the case (bottom right).

Fig. 2.

Positive reaction of mutation-specific antibodies (Ab) and gene copy number. Top, the relationship of the gene copy number with the intensity of the positive reaction to mutation-specific antibodies (left) and total EGFR antibody (right). An intense positive reaction (score 3+) tended to show gene amplification with both antibodies. Similar to EGFR amplification, the positive reaction was also distributed heterogeneously. In general, a positive reaction correlated with the expression of the total EGFR molecule (bottom left), but this was not always the case (bottom right).

Close modal

Prediction of the EGFR-TKI response using mutation-specific antibodies

We expected that the positive reaction would be associated with a clinical response to EGFR-TKI treatment because the positive reaction was seen nearly exclusively in lung cancer with the EGFR mutation. In a series of 32 patients treated with gefitinib, 12 (63%) of 19 lung cancers with EGFR mutation were positive for mutation-specific antibodies, and 10 (71%) of 14 lung cancers with a positive reaction responded clinically to gefitinib treatment (Table 3). Of note, one patient whose lung cancer was positive for the mutation-specific antibodies showed good response to the gefitinib treatment despite having the wild-type EGFR gene.

Table 3.

Response to gefitinib treatment and positive reaction to the mutation-specific antibodies

Response to gefitinibMutation-specific antibodies
PositiveNegative
Responded 10 
    EGFR, wild-type 
    EGFR, mutated 
Did not respond 11 
    EGFR, wild-type 10 
    EGFR, mutated 
Response to gefitinibMutation-specific antibodies
PositiveNegative
Responded 10 
    EGFR, wild-type 
    EGFR, mutated 
Did not respond 11 
    EGFR, wild-type 10 
    EGFR, mutated 

Reaction of tumors with secondary resistant mutations

Our data suggest that conformational changes of EGFR protein similar to L858R or E746-A750 in other mutation types might affect the positive reaction because both anti-L858R and anti-DEL antibodies reacted to the other types of mutations, such as G719C and D770insSVD (Table 1). Conversely, secondary mutations acquired in association with drug resistance might affect the reaction pattern. We examined lung cancers with T790M mutation in addition to the classic L858R and deletional mutations. Six lung adenocarcinomas with acquired resistance to gefitinib treatment had a positive reaction, and this finding is similar to that found in the primary lung adenocarcinomas examined in five patients (Table 4). The reaction in three primary lung adenocarcinomas with both L858R and T790M point mutations was similarly positive. These findings suggest that the secondary T790M mutation did not affect the positive reaction.

Table 4.

Secondary mutation and positive reaction to the mutation-specific antibodies

Case IDGenotypeImmunophenotype
PrimarySecondaryPrimarySecondary
With gefitinib treatment 
    1 DEL DEL + T790M Anti-DEL: 1+ Anti-DEL: 2+ 
    2 L858R+V834L L858R + V834L + T790M Anti-L858R: 2+ Anti-L858R: 3+ 
    3 E747-753 insS E747-753 ins S + T790M Anti-DEL: 3+ Anti-DEL: 2+ 
    4 E745-750 insK E745-750 insK + T790M Anti-DEL: 3+ Anti-DEL: 3+ 
    5 Not available L858R + H776R + T790M Not available Anti-L858R: 2+ 
    6 L858R L858R + T790M Not available Anti-L858R: 3+ 
Without gefitinib treatment 
    1 L858R + T790M   Anti-L858R: 2+ 
    2 L858R + T790M   Anti-L858R: 2+ 
    3 L858R + T790M   Anti-L858R: 1+ 
Case IDGenotypeImmunophenotype
PrimarySecondaryPrimarySecondary
With gefitinib treatment 
    1 DEL DEL + T790M Anti-DEL: 1+ Anti-DEL: 2+ 
    2 L858R+V834L L858R + V834L + T790M Anti-L858R: 2+ Anti-L858R: 3+ 
    3 E747-753 insS E747-753 ins S + T790M Anti-DEL: 3+ Anti-DEL: 2+ 
    4 E745-750 insK E745-750 insK + T790M Anti-DEL: 3+ Anti-DEL: 3+ 
    5 Not available L858R + H776R + T790M Not available Anti-L858R: 2+ 
    6 L858R L858R + T790M Not available Anti-L858R: 3+ 
Without gefitinib treatment 
    1 L858R + T790M   Anti-L858R: 2+ 
    2 L858R + T790M   Anti-L858R: 2+ 
    3 L858R + T790M   Anti-L858R: 1+ 

Similar to the original report by Yu et al. (18), our data confirm the specificity of the mutation-specific antibodies. In this study, data from tissue microarray and gefitinib-treated group revealed that the overall specificity of these antibodies to EGFR mutation was 96%. In contrast, the overall sensitivity of 47% based on both data sets was much lower than the 92% reported by Yu et al (18). A possible explanation for this discrepancy may be the relatively low detection rate for deletional mutation in exon 19. As shown in Table 1, anti-DEL–specific antibody detected only the mutation type of DEL E746-A750 and that with minor modification at the edge of the deleted region, including DEL E746-A750insK. In our cohort of 511 patients with NSCLCs from 2000 to 2006, DEL E746-A750 and its minor variants, which were expected to be positive for anti-DEL antibody, comprised 53% of the 105 deletions in exon 19 (data not shown). However, anti-L858R antibody also showed a lower sensitivity (40%), suggesting that other mechanisms may be involved.

Because these two antibodies reacted to the corresponding mutated form of EGFR protein, the positive reaction should be reflected in the expression status of EGFR. In general, EGFR expression is associated with increased EGFR copy number but not with mutational status (21, 22), and this finding was confirmed in our study. It is likely that the positive reaction to the mutation-specific antibodies is also affected by the EGFR gene copy number. A strongly positive reaction was found almost exclusively in tumors with EGFR amplification, as shown in Fig. 2. Similarly, a low-level expression of mutated protein is associated with the expression of total EGFR, and thus, tumors without detectable EGFR expression should be negative for the mutation-specific antibodies. However, a positive reaction was not detected in some tumors, even when total EGFR protein was expressed, as shown in Fig. 2. Some other mechanisms, such as differences in posttranscriptional modifications or in the detection thresholds, might be involved.

Interestingly, although these two antibodies were generated to be specific for the L858R point mutation or for the deletion in exon 19, these antibodies reacted with other mutations (Fig. 1C and D). Because the antibody recognizes a part of the conformational composition, the positive reaction suggested that other mutation types generate or form a conformational composition similar to that of L858R and the deletion in exon 19. We examined whether conformational changes caused by T790M secondary resistant mutation lose such conformational composition, but we found that T790M did not change the composition recognized by these mutation-specific antibodies. Another point of interest is that one of two lung cancers with a positive reaction, which did not harbor EGFR mutation, showed a clinical response to gefitinib treatment. About 10% of EGFR-TKI responders do not have EGFR mutation (7, 23), and analysis of lung cancer with this false-positive reaction may provide a clue to identifying another mechanism responsible for the response to EGFR-TKI treatment.

This study reveals some pitfalls in applying these antibodies in place of a mutation test. First, the immunohistochemical results have clinical significance only when the reaction to the antibodies was positive. The overall sensitivity was relatively low (47%), and one fourth of immunohistochemically negative lung cancer may have had the EGFR mutation. Second, the antibodies may react to insertion mutation in exon 20, which is resistant to EGFR-TKI treatment. In this study, two (29%) of seven lung cancers with an insertion in exon 20 showed a positive reaction. Third, even when the antibodies showed a positive reaction, ∼10% of such lung cancers may not have had an EGFR mutation. The clinical significance of these false-positive reactions should be examined in a larger cohort, as mentioned above. Fourth, tumor heterogeneity may cause a false-negative result. The heterogeneous distribution is crucial especially in the diagnosis of biopsy specimens. Table 2 is a summary of the results of the tissue microarray analysis, which used tissue cores to simulate real biopsy samples. The microarray analysis showed that 46% of lung cancer samples with a positive reaction showed a different reaction in at least one of the three or four cores. Taken together, these findings suggest that the clinical utility of these antibodies may be less than expected.

In summary, we examined EGFR mutation–specific antibodies. The antibodies showed specific reactions to EGFR mutations. However, the positive reaction showed some characteristics that differed from those of the molecular mutation, including recognition of different mutation types, association with expression of the total EGFR molecule, and heterogeneous distribution of the positive reaction. Despite a limited number of patients examined, a positive reaction correlated generally with a clinical response to EGFR-TKI treatment. Although these antibodies were relatively specific, not all patients with EGFR mutations can be selected using these mutation-specific antibodies.

No potential conflicts of interest were disclosed.

We thank Noriko Shibata, Motoko Nimura, and Akiko Yoshinari for the excellent technical assistance with the molecular genetic experiments.

Grant Support: Ministry of Education, Culture, Sports, Science and Technology of Japan and research grant for Princess Takamatsu Cancer Research Fund (08-24018).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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