Annals of Oncology Advance Access originally published online on February 27, 2008
Annals of Oncology 2008 19(6):1053-1059; doi:10.1093/annonc/mdn006
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lung cancer |
EGFR regulation by microRNA in lung cancer: correlation with clinical response and survival to gefitinib and EGFR expression in cell lines
1 TGen Clinical Research Services at Scottsdale Healthcare, Scottsdale, AZ, USA
2 Department of Medical Oncology, University of Colorado Cancer Center, Aurora, CO, USA
3 Department of Surgery, Tokyo Medical University, Tokyo, Japan
4 Department of Pathology, University of Colorado at Denver and Health Sciences Center, Aurora, CO, USA
* Correspondence to: G. J. Weiss, MD, TGen Clinical Research Services at Scottsdale Healthcare, 10510 N 92nd Street, Suite 200, Scottsdale, AZ 85258, USA. Tel: +1-480-323-1350; Fax: +1-480-323-1359; E-mail: gweiss{at}tgen.org
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Abstract |
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Background: Allelic loss in chromosome 3p is one of the most frequent and earliest genetic events in lung carcinogenesis. We investigated if the loss of microRNA-128b, a microRNA located on chromosome 3p and a putative regulator of epidermal growth factor receptor (EGFR), correlated with response to targeted EGFR inhibition. Loss of microRNA-128b would be equivalent to losing a tumor suppressor gene because it would allow increased expression of EGFR.
Patients and Methods: We initially showed that microRNA-128b is a regulator of EGFR in non-small-cell lung cancer (NSCLC) cell lines. We tested microRNA-128b expression levels by quantitative RT–PCR, genomic copy number by quantitative PCR, and mutations in the mature microRNA-128b by sequencing. We determined whether microRNA-128b loss of heterozygosity (LOH) in 58 NSCLC patient samples correlated with response to gefitinib and evaluated EGFR expression and mutation status.
Results: We determined that microRNA-128b directly regulates EGFR. MicroRNA-128b LOH was frequent in tumor samples and correlated significantly with clinical response and survival following gefitinib. EGFR expression and mutation status did not correlate with survival outcome.
Conclusion: Identifying microRNA regulators of oncogenes could have far-reaching implications for lung cancer patients including improving patient selection for targeted agents, development of novel therapeutics, or development as early biomarkers of disease.
Key words: epidermal growth factor receptor, gefitinib, microRNA, non-small-cell lung cancer
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introduction |
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TopAbstractintroductionmethodsresultsdiscussionfundingAcknowledgementsReferences |
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Lung carcinoma remains the leading cause of cancer death worldwide for both men and women. Non-small-cell lung cancer (NSCLC), which accounts for
88% of lung cancer cases [1], presents in advanced inoperable stages 75% of the time [2]. Systemic chemotherapy remains palliative and modestly effective. Recent therapeutic advances include the use of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) including gefitinib and erlotinib. It is thought that these drugs would be most effective with patient selection on the basis of target expression. However, the presence of epidermal growth factor receptor (EGFR) by immunohistochemical (IHC) staining is a poor predictor of survival benefit to those patients receiving treatment with EGFR-TKIs [3]. Debate is ongoing as to whether EGFR mutation in the tyrosine kinase domain (EGFR exons 18–21) [4] or high EGFR gene copy number or gene amplification by FISH correlates better with response and survival to EGFR-TKIs [5]. These predictors are not mutually exclusive, with 24%–43% of patients having concurrent mutation and high copy number [6, 7]. In NSCLC patient populations, EGFR mutation prevalence is 10%–23% (in Caucasians) [5] and 30%–40% (in East Asian populations) [8], while high EGFR copy number and/or amplification by FISH has a prevalence of 22%–45% [5, 9]. Thus, the most adequate predictor for identifying patients who would benefit from TKIs is still under debate.
In our search for additional factors that might predict patient response to EGFR-TKIs, we considered the recently identified regulators, microRNAs. MicroRNAs are a new class of small noncoding RNAs of 21–25 nucleotides recently implicated in cancer biology [10]. These RNA fragments posttranscriptionally regulate gene expression by binding to complementary sequences in the 3' untranslated region (3'UTR) of the target messenger RNA (mRNA) [11]. This can ultimately lead to repression of protein translation and downregulation of protein expression [12]. Deregulation of microRNAs is emerging as an important area of study in carcinogenesis because their regulatory capabilities can drastically influence cell physiology [13].
We queried whether a microRNA could regulate EGFR and be a predictor of response to EGFR-TKIs. Genomic loss of a microRNA capable of downregulating EGFR would be expected to allow increased EGFR expression, thereby offering a more robust target for the EGFR-TKI. We searched TargetScan 3.1 (http://www.targetscan.org) for candidate microRNAs that regulate EGFR and inquired if the predicted microRNA regulators of EGFR were localized to chromosomal regions known to be frequently lost in lung cancer. We identified EGFR as a potential target of microRNA-128b, a microRNA located on chromosome 3p. Allelic loss in chromosome 3p is one of the most frequent and earliest genetic events in lung carcinogenesis, with up to 96% loss in lung cancer and 78% loss in preneoplastic or preinvasive lung epithelial samples [14]. We assessed microRNA-128b's ability to regulate EGFR in several lung cancer lines and determined whether loss of heterozygosity (LOH) in tumor samples from Japanese patients who received EGFR-TKI therapy was associated with response and survival.
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methods |
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microRNA computational predictions
TargetScan 3.1 (http://www.targetscan.org) was consulted to identify microRNAs predicted to target EGFR. MicroRNA-binding predictions were confirmed by manually analyzing the EGFR 3'UTR for binding sites of microRNA-128b. Only four microRNAs were predicted to regulate EGFR and microRNA-128b was chosen for follow-up because it is located on chromosome 3p.
NSCLC cell lines
Representative NSCLC cell lines H157, H520, and H3255 were selected for representative histology, range of sensitivity to gefitinib, and range of EGFR IHC staining. Lines were maintained in RPMI media supplemented with 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT) in a humidified incubator with 5% CO2.
cytogenetics
We analyzed the three NSCLC lines by classical and molecular cytogenetics. After harvesting, cells were submitted to giemsa/trypsin/leishman banding using a CytoVision workstation (Applied Imaging Corp-AI, Santa Clara, CA) [15]. Spectral karyotyping (SKY) assays and analysis were carried out with reagents and equipment from Applied Spectral Imaging (ASI, Vista, CA) also according to protocol published elsewhere [15]. For comparative genomic hybridization (CGH) analyses, DNA from the cell lines was labeled with Spectrum Red deoxyuridine triphosphate (dUTP) (SR) using nick translation (Vysis/Abbott Laboratories, Des Plaines, IL); DNA from normal subjects were labeled with Spectrum Green dUTP (SG) and used as reference. The SR-labeled DNA (tumor or normal) and the SG-labeled reference DNA were combined in a ratio of 1 : 1.5, respectively, and competitively hybridized to normal metaphase spreads [16]. Chromosomal abnormalities were interpreted [17].
PCR and sequencing methods for genomic DNA
PCR and direct sequencing were accomplished following methods previously described [18]. PCR primers were designed for the two predicted binding regions in the EGFR 3'UTR (chromosome 7p) and the region containing the mature microRNA-128b (chromosome 3p) (supplemental table S1, available online). Sequencing to detect the EGFR exon 21 L858R point mutation and EGFR exon 19 deletion was carried out as previously described [6]. Purified PCR products were directly sequenced by the University of Colorado Cancer Center DNA Sequencing Core.
To determine relative microRNA-128b expression, quantitative RT–PCR (qRT–PCR) was carried out using the Applied Biosystems Taqman system, followed by quantitation against RNU6B control according to manufacturer's instructions. Each sample was analyzed in triplicate on each quantitation run. Cell line results were normalized to the lowest ratio (H3255) to determine relative microRNA-128b expression [19].
DNA copy number of green fluorescent protein (GFP) was measured to determine transfection equivalence between controls and cell line treatment conditions (supplemental table S1, available online).
EGFR immunohistochemistry
Formalin-fixed NSCLC cell lines and patient tumor slides were collected and stained with anti-EGFR antibody (anti-EGFR clone 31G7, Zymed, San Francisco, CA), as previously described [18, 20]. EGFR IHC intensity was determined (range 0–400), where the percentage of positive tumor cells per slide (0%–100%) was multiplied by the staining intensity (1, negative or trace; 2, weak; 3, moderate; 4, intense) [20]. All grading was carried out by a board-certified pathologist (WAF).
GFP reporter constructs and expression
The 3'UTR of EGFR is encoded in exon 28. Using genomic DNA from H157, empty vector GFP and GFP-EGFR-3'UTR constructs were made (details in supplementary section, available online). H157 cells were then transfected using the lipophilic reagent Effectene Transfect Reagent (301427, Qiagen, Valencia, CA) with either the empty vector GFP or GFP-EGFR-3'UTR construct at a concentration of 1000 ng of transfection product. Following 48 h of incubation, GFP protein was quantified by western blot (described in "microRNA transfections and western blotting") and mRNA was quantified by qRT–PCR (described above) with beta-actin serving as an internal control (primers listed in supplemental table S1, available online). Equal transfection was confirmed by GFP message quantitation.
microRNA transfections and western blotting
Transient transfections of microRNA mimics and inhibitors were accomplished using HiPerfect (Qiagen). Mimic of microRNA-128b was purchased from Dharmacon (C-300139-01-0010, Boulder, CO) and inhibitor of microRNA-128b was purchased from Ambion (17000, Foster City, CA). NSCLC cells were seeded at 300–400 K per 60 mm plate and transfected with 4 nM microRNA-128b inhibitor or 4 nM microRNA-128b mimic. Immunoblot of protein lysates was probed for proteins of interest and imaged (see supplementary section, available online).
patients and clinical database
DNA copy number of microRNA-128b was assessed retrospectively in 58 samples of DNA extracted from microdissected primary surgically resected NSCLC tumors obtained with written informed consent from patients receiving care and follow-up at Tokyo Medical University. Patients were treated with gefitinib upon relapse with a majority receiving at least one other systemic therapy. Clinical and biologic information (sex, age at diagnosis, surgical date, histology, surgical stage, smoking history, line of therapy, gefitinib treatment start date, and outcome) was available for all patients (supplemental table S3, available online). Tumor measurements were assessed by response evaluation criteria in solid tumors criteria [21] at Tokyo Medical University.
DNA quantitative PCR
A standard curve was created by amplifying genomic DNA from the H157 line with primers for the microRNA-128 DNA locus (supplemental table S1, available online, forward and reverse microRNA-128b primers). Cystic fibrosis transmembrane conductance regulator (CFTR) was used as the standard reference gene (supplemental table S1, available online, forward and reverse CFTR primer) [22]. DNA copy number was determined (see supplementary section, available online).
statistical analysis
For all tests, a level of P <0.05 was considered statistically significant. The 
2 test or Fisher's exact test for count data was used to analyze proportions among the factors studied. To determine which factors had an influence on response to gefitinib, logistic regression was carried out, with response or stable disease considered a positive outcome and progressive disease negative. The Kaplan–Meier method was used to estimate the probability of survival as a function of time. Survival was calculated from the date of first gefitinib treatment to the date of death from any cause; all other patients were censored at the time of their last follow-up. Significant differences between survival curves were analyzed using the log-rank test. Multivariate analysis of the relative importance of the factors to survival was done using the Cox proportional hazards method. Pearson correlations were calculated using Microsoft Excel 2002 (Microsoft Corporation, Redmond, WA). All other calculations were carried out using R statistical software (http://cran.r-project.org/).
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We hypothesized that a microRNA regulates EGFR, and if that microRNA were lost in lung lesions, it would be consistent with losing a tumor suppressor gene thereby allowing increased expression of EGFR. We queried the TargetScan 3.1 database (http://www.targetscan.org) for microRNAs predicted to target EGFR. We examined the EGFR-3'UTR to look for potential microRNA-128b-binding sites (supplementary figure 1, available online). Two sites for microRNA-128b binding have been predicted within the EGFR-3'UTR. While there may be additional microRNAs that regulate EGFR, we choose to limit our analysis to microRNA-128b because of its genomic location on 3p22, a region of the genome commonly deleted in lung cancer. Therefore, we conducted further studies of the regulation of EGFR by microRNA-128b. In addition, we studied patient samples to determine whether LOH of microRNA-128b correlated with response to EGFR-TKI therapy.
cell line studies
To determine whether microRNA-128b could regulate EGFR, we evaluated three NSCLC cell lines (H157, H3255, and H520) for microRNA-128b and EGFR mRNA expression and EGFR protein expression in response to mimics and inhibitors of microRNA-128b. We initially confirmed that genomic alteration of microRNA-128b is a frequent event in lung cancer cell lines. SKY and CGH were used to determine chromosome loss or rearrangement. Relative expression of microRNA-128b was determined by qRT–PCR. Two of three lung cancer cell lines showed losses and/or rearrangements involving 3p22 (Table 1). In the H157 line, both microRNA-128b copy number and expression were consistently lower than in the H520 cell line, which has no chromosome 3p change (Table 1). The H3255 line showed a complete loss by both copy number and expression for microRNA-128b. Direct sequencing of the relevant genomic DNA regions did not find any mutations in either microRNA-128b locus (applicable to H157 and H520 only) or the two predicted microRNA-128b-binding sites in the EGFR-3'UTR of all three cell lines. Thus, two of three cell lines showed reduced or absent levels of microRNA-128b by qRT–PCR as a result of loss or rearrangement, and therefore would be expected to have increased levels of EGFR protein. RNA extracted from two benign lung epithelium lines (BLE) had greater microRNA-128b expression compared with that of the highest expressing lung cancer cell line, H520 (factor of at least 98) (Table 1).
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IHC was carried out and confirmed high EGFR protein expression in both H157 and H3255, both of the cell lines with an alteration of chromosome 3p22. On the contrary, H520, which did not show a loss or arrangement in chromosome 3p22, had no detectable EGFR and high expression levels of microRNA-128b (Table 1). On two BLE specimens, EGFR staining was limited to the basal layer, indicating that high levels of microRNA-128b can prevent EGFR dysregulation and overexpression in normal lung.
Sensitivity to gefitinib correlated with microRNA-128b copy number (r = 0.97) better than microRNA-128b expression (r = 0.55). H3255, which lacks both microRNA-128b copy number and expression, is exquisitely sensitive to EGFR-TKI. However, this cell line also harbors a mutation in EGFR [4] and the contribution of EGFR mutation versus that of microRNA-128b in vitro is beyond the scope of this paper.
Confirmation that microRNA-128b acts through binding of the 3'UTR of EGFR was confirmed by placing the EGFR-3'UTR in a GFP reporter plasmid. NSCLC line H157 expressing microRNA-128b was successfully transfected with GFP or GPF-EGFR-3'UTR reporters. GFP protein was lower in those cells receiving the GFP-EGFR-3'UTR reporter as compared with those transfected with GFP (Figure 1A). GFP DNA copy number and mRNA were quantified to assure equivalent transfection rates between cells receiving GFP or GFP-EGFR-3'UTR.
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Confirmation that microRNA-128b regulates EGFR was accomplished by treating the same NSCLC cell lines with mimics of microRNA-128b or inhibitors to microRNA-128b (as described in "methods"). Figure 1B shows the immunoblots and qRT–PCR results following microRNA-128b mimic and inhibitor transfections. In the EGFR-expressing cell line, H157, microRNA-128b inhibitor treatment resulted in upregulation of EGFR protein expression, while microRNA-128b mimic treatment resulted in downregulation of EGFR protein expression as expected. The EGFR-expressing cell line, H3255, which lacks microRNA-128b expression, did not have an alteration in EGFR protein levels with transfections. Similarly, H520, the cell line with no EGFR protein by both IHC and immunoblotting, lacked EGFR protein change following transfection with microRNA-128b mimic or inhibitor. However, EGFR mRNA expression levels were altered after transfections in both H3255 and H520. These results demonstrate that EGFR can be regulated through sequences in its 3'UTR and is specifically regulated by microRNA-128b, which can regulate both EGFR protein and mRNA expression.
clinical studies
To determine whether LOH of microRNA-128b correlates with clinical response or survival in 58 NSCLC patients treated with gefitinib, we carried out quantitative PCR on DNA extracted from microdissected primary NSCLC tumors samples. There were 49 lung adenocarcinomas and 9 squamous cell carcinomas. There were 34 males and 24 females, all of whom progressed to stage 4 lung cancer and received treatment with gefitinib. Forty-two patients had EGFR IHC staining carried out, and in those patients, high EGFR was associated with disease presentation at a later stage (P = 0.02). Gender was significantly associated with disease control (response + stable disease) to gefitinib treatment (P = 0.02), with women showing less disease progression than men. This did not translate into an overall survival benefit, however (P = 0.24). Improved survival after initiation of gefitinib therapy was observed with adenocarcinoma histology (P = 0.01) and 
3 lines of therapy (P = 0.002). EGFR IHC intensity had no correlation with survival (P = 0.56) and patients aged 70 and older had similar benefit compared with their younger counterparts (P = 0.20). The incidence of EGFR exon 19 deletion and exon 21 L858R point mutation was 31% of the 58 samples (Table 2). Individually, EGFR exon 19 and exon 21 status did not correlate with disease control (P = 0.15 and 0.18, respectively), while the combination did have a significant correlation (P = 0.01). However, neither EGFR exon 19 deletion, nor EGFR exon 21 L858R point mutation, nor the combination had a significant correlation with survival (P = 0.19, 0.48, and 0.49; respectively).
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MicroRNA-128b LOH was frequent (55%, n = 58) (Table 2). Loss of microRNA-128b was significantly associated with improved disease control to gefitinib treatment (P = 0.03). Improved survival was observed in those patients with a microRNA-128b LOH with an observed benefit of 23.4 versus 10.5 months (P = 0.02) (Figure 2). Multivariate analysis using a Cox proportional hazard model including all factors available for the 58 patients revealed that gender and histology contributed significantly to survival, while smoking status, EGFR mutation status, and stage at diagnosis did not (Table 3). Loss of microRNA-128b and
3 lines of therapy had a trend to significance (P = 0.11 and 0.08) in this model. When modeled individually, however, only histology, line of treatment, and microRNA-128b LOH were significant (P = 0.02, 0.006, and 0.02, respectively), whereas gender was no longer significant (P = 0.12). The best model appeared to consist of histology, line of treatment, and microRNA-128b, with all three factors contributing significantly (P = 0.02, 0.03, and 0.03, respectively) (Table 3). EGFR intensity, as measured by IHC, and EGFR mutation status were not found to be significant in any model (data not shown).
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discussion |
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LOH of chromosome 3p is a common and early event in lung carcinogenesis, as is the dysregulation of EGFR expression. With the recent report of microRNA involvement in cancer [10], we decided to investigate a possible role of microRNAs in lung cancer. Using available bioinformatics, we found that microRNA-128b could provide a functional link between a common genetic abnormality in lung cancer, loss of 3p22, and the frequently dysregulated and overexpressed gene, EGFR. Our findings are similar to those reported for the Ras gene implicated in lung cancer and regulated by microRNA let-7 [23]. Let-7g resides on chromosome 3p21.2 and expression levels of let-7g were on average 30% less than normal adjacent tissue in seven of eight NSCLC samples [23]. LOH of microRNA-128b, which resides on the 3p22 locus, would be equivalent to losing a tumor suppressor gene because microRNA-128b downregulates the expression of EGFR.
MicroRNA-128b appears to play a role in EGFR regulation in BLE. BLE microRNA-128b expression exceeded that of H520 by almost 100-fold, and in BLE specimens, EGFR IHC expression is limited to the basal layer. If a deletion in 3p22 occurred in BLE, we would suspect a decrease in microRNA-128b expression levels leading to EGFR dysregulation and overexpression, providing a link between LOH at 3p, microRNA-128b, and early lung carcinogenesis.
While we observed microRNA-128b regulation of mRNA levels in H520 and H3255, eliciting a protein change when EGFR protein is not expressed or highly overexpressed can be a challenge. There may be optimal conditions in which microRNA-128b mimic or inhibitor can alter EGFR protein in these two lines. Other factors may be regulating an increased turnover of the EGFR protein, for example, H3255 line is known to harbor the exon 21 L858R point mutation.
From our cell line and clinical specimen analyses, loss of 3p22.3, which harbors microRNA-128b, is a frequent event in lung cancer and loss of this locus would result in increased expression of EGFR and increased sensitivity to EGFR-TKI therapy. Women had improved disease control, a well-known clinical factor associated with response to EGFR-TKI therapy [4, 24, 25]. In addition, our findings correlate with other reports that adenocarcinoma histology [26] and line of treatment, but not EGFR IHC intensity [3, 6, 7] or age [18], are significantly associated with improved survival. As patients are subjected to increasing lines of therapy, the likelihood of response and benefit diminish; therefore, it is not surprising that patients who received gefitinib no later than third-line therapy had improved survival. In this study, 72% of cases had tumor available for EGFR IHC staining. The lack of correlation between microRNA-128b LOH and EGFR IHC intensity highlights the complexity of the ultimate mechanism of protein expression as observed by IHC in clinical specimens. Consistent with other work, EGFR exon 19 deletion and exon 21 L858R point mutation correlated with disease control [6, 7]; however, it was not significantly associated with improved survival [7]. There was overlap among microRNA-128b LOH and EGFR mutation status (56%), but only microRNA-128b LOH was significantly associated with survival benefit in patients treated with gefitinib.
This is a unique finding that has not been previously reported. MicroRNA-128b LOH and its significant correlation with positive patient response and survival with EGFR-TKI treatment may provide a biologic explanation for the impact of chromosomal loss in one area of the genome on gene expression from another part of the genome. We acknowledge that the sample set is retrospective, relatively small, and limited to East Asian samples; further analysis in additional patient cohorts is warranted. Furthermore, analysis of microRNA-128b in a cohort of advanced NSCLC patients who do not receive EGFR-TKI treatment is necessary to determine whether microRNA-128b LOH is a positive prognostic factor for patient survival or is a predictive factor of survival benefit restricted to patients receiving EGFR-TKI therapy. These investigations support the contention that microRNA regulation could have far-reaching implications in cancer research including screening for appropriate selection of patients to receive targeted agents, novel therapeutics, or development of early biomarkers of disease.
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International Association for the Study of Lung Cancer to G.J.W.; National Cancer Institute Specialized Program of Research Excellence in Lung Cancer (P50-58187) to P.A.B.; Early Detection Research Network (U01-CA85070) to W.A.F.
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Acknowledgements |
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We thank Carol Amato, Jennifer Caputo, and Natalie Thomas for technical assistance and Dan Chan, PhD, and Todd Pitts for providing reagents used in this experiment. Conflict of interest statement: PAB: <$10K consulting and honoraria from BMS/ImClone and <$10K consulting and honoraria from Astrazeneca; FRH: <$10K consulting and honoraria, research funds <$10K from Astrazeneca; WAF: <$10K consulting and honoraria, research funds <$10K from Astrazeneca. The other coauthors report no conflict of interest.
Received for publication November 7, 2007. Revision received December 19, 2007. Accepted for publication January 2, 2008.
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