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Keywords:

  • Meningioma;
  • grade;
  • invasiveness;
  • immunohistochemistry;
  • histopathology

Abstract

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

Meningiomas are slow-growing neoplasms that recur locally. Their morphologic grading does not always correlate with patient outcome. We evaluated the status of several immunohistochemical markers with histopathologic parameters in various grades of meningioma.Eighty-eight meningioma specimens were examined immunohistochemically to determine the status of Ki-67, cyclin D1, epidermal growth factor receptor (EGFR), cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF), and bcl-2. Several clinical and pathological parameters were investigated.Forty-nine Grade I, 33 Grade II, and 6 Grade III meningiomas were observed. VEGF and Ki-67 expression was correlated with higher tumor grade. The association between grade and other immunohistochemical markers expression was not significant. A correlation was observed between COX-2 expression and invasiveness to the brain or adjacent soft tissue. Tumor recurrence was correlated with brain or adjacent soft tissue invasion. We also observed a relationship between VEGF level and COX-2 expression, and they were both correlated with necrosis.Immunohistochemical evaluation of VEGF, COX-2, and Ki-67 expression can provide information regarding the behavior of meningiomas, particularly for cases in which histological grading is not straightforward.

Meningiomas are common among intracranial tumors and are estimated to account for 15–20% of all primary brain tumors [1], with an annual incidence of up to 13 per 100 000 persons [2]. Ninety percent of meningiomas are classified as histologically benign and are typically curable after complete removal [1]. A minority of these tumors present with clinical and pathologic features, suggesting aggressive clinical behavior, and are histologically classified as atypical (World Health Organization grade II) and malignant (WHO grade III) [2, 3]. However, even histologically benign meningiomas often recur, thus, their morphologic grading does not always correlate with patient outcome [4]. These limitations of the current grading system for predicting tumor behavior have prompted a search for other approaches to identify more aggressive meningiomas.

Brain invasion is defined as irregular, tongue-like protrusions of tumor cells that infiltrate the parenchyma, without intervening of the meningeal tissue layer. Brain invasion is a powerful predictor of shorter recurrence-free survival; according to the WHO classification, otherwise benign meningiomas with brain invasion should be considered as WHO grade II [2]. Invasion into adjacent tissues such as the skull, scalp, and even paranasal sinus does not alter the grade, but can make resection more difficult and shows a high recurrence rate and poor prognosis [5].

The prognostic significance of diverse proliferative indices in meningiomas has been assessed in various studies, and evaluation of the proliferative potential of tumors may be used as an ancillary technique in predicting the clinical course for these patients [6-9]. Cyclooxygenase-2 (COX-2), an enzyme that converts arachidonic acid into prostaglandins, plays a critical role in the development of various tumors including those of the colon, pancreatic, prostate, lung, and head and neck. This enzyme can stimulate gene transcription, tumoral growth, angiogenesis, and metastasis; inhibit apoptosis; and cause resistance to chemotherapy [10-12]. COX-2 overexpression is also frequently observed in brain tumors. Numerous studies have demonstrated the presence of COX-2 in gliomas, indicating the therapeutic effectiveness of COX inhibitors in gliomas [13, 14]. However, there are relatively few reports regarding COX-2 expression in meningiomas [10, 15, 16].

Increased vascular permeability and angiogenesis are essential for tumorigenesis. Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a prime regulator of vascular permeability and angiogenesis. The VEGF system has been shown to be upregulated in various types of tumors, and there is evidence for interactions between this system and COX-2-derived prostaglandins [17].

A wide variety of normal and neoplastic tissues express epidermal growth factor receptor (EGFR). Its overexpression has been detected in a number of human tumors including breast, lung, head and neck, and colorectal carcinomas. EGFR protein expression has been suggested to be of prognostic importance [18]. Immunoreactivity of EGFR protein has been also observed in central nervous system tumors such as meningiomas and gliomas, suggesting EGFR involvement in the proliferation and/or differentiation of meningothelial cells [18, 19].

In addition, other biological parameters such as apoptosis, cell cycle-related factors including bcl-2 and cyclin D1, and hormonal receptor status have been investigated in several studies. However, their predictive values have not led to the development of a comprehensive molecular prognostic model [20-22].

On the basis of the limitations of the current grading system for predicting tumor behavior, we assessed whether these markers can be used to predict and distinguish differences in prognosis among meningioma cases, even cases of the same-grade meningioma.

The goal of this study was to establish a relationship between the expression of Ki-67, cyclin D1, EGFR, COX-2, VEGF, and bcl-2 and histological grading with other histopathologic parameters, including invasion into adjacent tissues, to determine the value of these molecular markers for predicting disease prognosis.

Materials and methods

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

Patients and tissue samples

Histologic sections of meningiomas resected between 2000 and 2004 were retrieved from the archives of the Department of Hospital Pathology at Seoul St. Mary's Hospital. Eighty-eight cases (24 men and 64 women) were enrolled. This subset consisted of all meningioma patients within the given period for which sufficient tumor tissue was available for immunohistochemical studies. No patients had chemotherapy, radiotherapy, or embolization therapy preoperatively. Clinicopathological information was obtained for all cases, including that on sex, age, brain, or adjacent soft tissue invasion, mitotic index, cellularity, uninterrupted patternless or sheet-like growth, foci of spontaneous or geographic necrosis, prominent nucleoli, small-cell population with a high nucleus/cytoplasmic ratio, and site of tumor. We evaluated the presence of brain invasion only for cases in which we could identify brain tissue on hematoxylin and eosin (H&E) sections. In addition, adjacent soft tissue invasion was defined as invasion to adjacent connective tissues of the scalp or paranasal sinus beyond the dura. Only dural invasion was excluded as adjacent soft tissue invasion. The study was performed after receiving approval from the local institutional review board (IRB).

H&E-stained slides were re-evaluated by 2 pathologists, and the diagnosis was reconfirmed based on the 2007 WHO classification of nervous system tumors [2]. Cases included 24 meningothelial, 17 fibrous, 3 psammomatous, 2 transitional, 2 angiomatous, 1 secretory, 33 atypical, and 6 malignant (including 1 rhabdoid) meningiomas. Clinicopathological features of the 88 cases are shown in Table 1.

Table 1. Clinicopathologic characteristics of 88 patients with various grades
CharacteristicAllGrade IaGrade IIGrade IIIb
  1. a

    Grade I meningiomas consist of 2 angiomatous, 17 fibrous, 24 meningothelial, 3 psammomatous, 1 secretory, and 2 transitional subtypes.

  2. b

    Grade III meningiomas consist of 1 rhabdoid and 5 anaplastic subtypes.

No. of patients8849 (55.7%)33 (37.5%)6 (6.8%)
Age (years) 
Mean

55.9

(range: 24-83)

55.0

(range: 24-83)

57.4

(range: 25-77)

55.7

(range: 33-70)

Sex 
Male24 (27.3%)10104
Female64 (72.7%)39232
Location of tumor 
Intracranial80 (90.9%)42326
Intraspinal8 (9.1%)710

Immunohistochemistry and staining interpretation

Tissue sections (4 μm thickness) were prepared from formalin-fixed, paraffin-embedded tissues and mounted onto poly-l-lysine-coated slides. Immunohistochemical staining for EGFR (5B7; Ventana Medical Systems, Tucson, AZ, USA; prediluted), Ki-67 (MIB-1; Dako, Glostrup, Denmark; dilution 1:50), COX-2 (cx229; Cayman Chemical Company, Ann Arbor, MI, USA; dilution 1:800), VEGF (Santa Cruz Biotechnology, Santa Cruz, CA, USA; dilution 1:150), bcl-2 (124; Dako; dilution 1:50), and cyclin D1 (SP4; Thermo Scientific, Waltham, MA, USA; dilution 1:50) was conducted. Tissue sections were subjected to antigen retrieval by heating with a microwave processor at 95 °C. Sections were then incubated in a Coplin jar at room temperature for 30 min and subsequently washed with 0.05 mol/L TRIS-buffered saline. Next, the slides were incubated with antibodies described above overnight at 4 °C. Detection was performed using a standard biotin streptavidin detection system (Dako, Carpinteria, CA, USA). In addition, 3,3-diaminobenzidine was used as the chromogen, and sections were counter-stained with hematoxylin. All stains were performed using a Dako Autostainer.

Ki-67-positive cells showed nuclear staining and the Ki-67 labeling index was assessed by counting at least 1 000 adjacent cells in at least 10 high-power fields (HPFs) from the areas of greatest proliferation counts. Averages were expressed as percentages. Cells were considered Ki-67-positive if diffuse nuclear staining was observed; in principle, only tumor cells would be counted. To assess COX-2, VEGF, and bcl-2 protein expression, a cutoff value of 10% was used to define positivity for minimization of false-positive results. Cyclin D1 staining was approximated and scored from 0 to 3 + , with 0 being negative, 1 +  showing staining in <25%, 2 +  showing staining in 26% to 50%, and 3 +  showing staining in more than 50% of neoplastic cells. Staining was also scored as semiquantitatively negative (no staining); 1 +  (weakly positive); 2 +  (moderately positive); and 3 +  (strongly positive). Positivity for EGFR expression was defined as >10% of tumor cells with any membrane staining above background level. Cytoplasmic staining without associated membrane staining was reported as negative.

Positive controls included colonic adenocarcinoma and a normal colon specimen for determining COX-2 and VEGF expression, respectively. A case of gastric adenocarcinoma, with known immunoreactivity, was used as a positive control for EGFR, bcl-2, and Ki-67. Mantle cell lymphoma tissue was used as a positive control for cyclin D1. Negative controls were assessed without primary antibody. Non-neoplastic cells were excluded from counting.

Statistical analysis

Data were analyzed using SPSS for Windows version 13.0 (SPSS, Chicago, IL, USA). Statistical analysis was performed to compare tumor grades, recurrence, and various histopathologic parameters with Ki-67 index, bcl-2, cyclin D1, COX-2, EGFR, and VEGF expression. Correlation among immunohistochemical markers was also analyzed. Categorical variables were analyzed using χ2 or Fisher exact test and multivariate analysis was performed using backward stepwise regression analysis in which variables were removed at each step to define the independent contribution of each factor. A p-value of ≤0.05 was considered statistically significant.

Results

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

Clinicopathologic features

Characteristics of the patient population are summarized in Table 1. Of the tumors in the 88 patients who underwent operation, 49 (55.7%) were of grade I, 33 (37.5%) were of grade II, and 6 (6.8%) were of grade III. Meningioma was more common in female patients with a ratio of 2.67:1. Patient ages at the time of surgery ranged from 24 to 83 years (mean age, 55.9 years). The tumor site was determined based on radiographic studies and operative findings; in 80 cases (90.9%), the tumors were intracranial and 8 were intraspinal. Mitotic activity ranged from 0 to 24/10 HPFs (mean: 1.03), and the mitotic activity of atypical/malignant meningiomas was significantly higher than that of benign meningiomas. Brain or adjacent soft tissue invasion was present in 10 (11.4%) meningiomas including 8 cases of adjacent soft tissue invasion and 2 cases of brain invasion in only grade II meningioma. In addition, 11 (12.5%) meningiomas recurred (not shown in Table 1), which included 7 grade II or III cases and 4 grade I cases. All but 1 meningioma case showed the same histologic grade and subtype at initial operation and at reoperation due to tumor recurrence. One case of histopathologic transition from grade I to grade II was observed. We observed no correlation between aggravation of tumor grades and recurrence.

Mitotic activity and brain or adjacent soft tissue invasion were significantly correlated with tumor recurrence. There was no correlation between grade and brain or adjacent soft tissue invasion and tumor site. Higher tumor grade was strongly associated with increased cellularity, uninterrupted patternless or sheet-like growth, foci of spontaneous or geographic necrosis, prominent nucleoli, and small-cell population with a high nucleus/cytoplasmic ratio. To determine the significance of differences among tumor subtypes, we evaluated the correlation between tumor subtypes and various clinicopathological parameters. However, tumor subtypes alone failed to show statistically significant differences. Analyses of the correlation between tumor location, recurrence, and various histopathologic parameters are summarized in Table 2.

Table 2. Relationship between tumor location, recurrence, and various histopathologic parameters in 88 patients
p-valueGradeInvasionMitosisCellularitySheetNucleoliNecrosisSCb
  1. a

    Parameters considered statistically significant.

  2. b

    SC: small cell population with high nucleus/cytoplasmic ratio.

Location0.0570.9150.3480.2470.038a0.1680.029a0.915
Recurrence0.1680.000a0.001a0.6260.3460.8050.1520.446

Immunohistochemistry

Immunohistochemical characteristics of various molecular markers are shown in Tables 3. Twenty-nine cases with a Ki-67 labeling index ≥4% were observed (33%). Mean Ki-67 labeling indices for grades I, II, and III meningioma were 2.4%, 7.7%, and 16.0%, respectively. Positive correlations were found between the Ki-67 labeling index and higher histological grade of the meningiomas.

Table 3. Results of 88 cases with various immunohistochemical markers
 No of positive cases
AllGrade IGrade IIGrade III
  1. a

    LI, labeling index.

  2. b

    Grading system for Cyclin D1P (proportion) staining: 0, negative; 1 + , <25%; 2 + , 26−50%; 3 + , >50%.

  3. c

    Grading system for Cyclin D1I (intensity) staining: 0, negative; 1 + , weakly positive; 2 + , moderately positive; 3 + , strongly positive.

Bcl-246 (52.3%)23203
COX-211 (12.5%)641

Ki-67 LIa

(mean,%)

4.9(range, 0−40)2.4(range, 0−18)7.7(range, 0−30)16.0(range, 0−40)

Cyclin D1Pb&

Cyclin D1Ic

86 (97.7%)47336
EGFR29 (33.0%)15122
VEGF45 (51.1%)20205

In addition, a strong correlation between Ki-67 labeling index and mitotic figures, increased cellularity, prominent nucleoli, and necrosis were noted. Forty-five cases (51.1%) showed positive expression of VEGF protein. Positive VEGF reactivity occurred in the cytoplasm of tumor cells (Fig. 1A). There were statistically significant differences in VEGF expression among meningioma grades; thus, overexpression of VEGF protein is correlated with increased tumor grade. In addition, VEGF expression was positively correlated with necrosis and COX-2 expression. COX-2 immunoreactivity was noted in the cytoplasm (Fig. 1B). COX-2 expression was significantly associated with brain or adjacent soft tissue invasion and necrosis. We attempted to define the relative contribution of ancillary markers to tumor invasiveness using multivariate analyses with the backward stepwise method. Invasion was significantly correlated with sex (p = 0.039), tumor recurrence (p = 0.000), Ki-67 index (p = 0.035), and COX-2 expression (p = 0.003) (data not shown). In addition, bcl-2 expression was correlated with cellularity. However, cyclin D1 and EGFR expression showed no correlation with other immunohistochemical markers and histopathologic parameters including meningioma grade. In addition, there was no significant correlation between immunohistochemical markers and tumor recurrence.

image

Figure 1. (A) Most neoplastic cells within this section of meningioma exhibit positive cytoplasmatic immunohistochemical staining for VEGF (rhabdoid meningioma, grade III according to WHO criteria, × 400). (B) Immunohistochemical finding for COX-2 proteins shows cytoplasmatic reaction in neoplastic cells (atypical meningioma, grade II according to WHO criteria, × 400).

Download figure to PowerPoint

Relationships between histopathologic parameters and immunoreactivity of molecular markers are shown in Table 4, while Table 5 shows relationships between immunoexpression among molecular markers.

Table 4. Relationship between histopathologic parameters and immunoexpression of molecular markers in 88 patients
p-valueBcl-2COX-2Ki-67 LIVEGF
  1. a

    Parameters considered statistically significant.

Grade0.2610.9350.019a0.030a
Invasion0.8790.005a0.0530.939
Mitosis0.3801.0000.008a0.157
Cellularity0.036a0.6260.032a0.140
Sheet0.1970.2650.4860.325
Nucleoli0.5720.0840.036a0.408
Necrosis0.5900.035a0.015a0.001a
Small cell0.2330.4460.8330.205
Location0.3800.2620.6160.121
Recurrence0.7510.6230.7970.375
Table 5. Relationship between immunoexpression among molecular markers in 88 patients
p-valueBcl-2COX-2Ki-67 LIVEGF
  1. a

    Parameters considered statistically significant.

Bcl-2-0.4200.7030.516
COX-20.420-0.3460.005a
Ki-67 LI0.7030.346-0.595
VEGF0.5160.005a0.595-

Discussion

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

In our series of 88 meningiomas treated at a single institution and diagnosed according to the 2007 WHO classification of nervous system tumors, we confirmed that tumor grade was strongly associated with all histologic para-meters for meningioma grading criteria. Meanwhile, invasion into surrounding tissues such as bone and soft tissue makes tumor resection more difficult, but does not change the grade [23]. No correlation was observed between tumor grade and brain or adjacent soft tissue invasion in our study. This lack of correlation may be explained, in part, by the inclusion of adjacent soft tissue invasion as well as brain invasion in our statistical analysis. However, invasion into adjacent soft tissues, like brain invasion, results in a high recurrence rate and poor prognosis [5]. Therefore, we recommend that meningiomas with invasion into adjacent soft tissues should be monitored extremely closely with radiographic follow-up. We also observed a significantly higher rate of recurrence in brain- or soft tissue-invasive meningiomas. This result indicates that despite recurrence of brain-invasive meningiomas being well-recognized [24], adjacent soft tissue invasion may also be associated with tumor recurrence. In addition, our data confirm that most meningiomas (90.9%) recur with a similar histology, both at first operation and recurrence. This indicates that meningioma recurrence is not associated with an increased potential for tumor growth. We attempted to define the role of ancillary markers; however, there was no significant correlation between immunohistochemical markers and tumor recurrence.

Prognostic prediction based on tumor location has led to conflicting results. Among intracranial and intraspinal meningiomas, no obvious regional differences regarding proliferative activity have been observed [4]. However, genetic studies by Gezen et al. have demonstrated a loss of 1 homologue of chromosome 22 localized on the long arm in patients with spinal meningioma; a tumor suppressor gene may be present at this site [25]. These results suggest that the prognosis for intraspinal meningiomas is less favorable than that for intracranial meningiomas. Presently, most intraspinal meningioma cases can be categorized as WHO grade I (7/8, 87.5%), but the occurrence of solid-sheet formation and necrosis was statistically higher in intraspinal meningiomas, although we did not perform chromosomal studies. Additional studies are needed to confirm our data.

Immunohistochemistry of the Ki-67 antigen has been widely used in numerous studies of meningiomas as a powerful prognostic marker and supplement to histopathology for meningioma grading. A high Ki-67 index is associated with aggressiveness and poor prognosis for meningiomas [4, 26]. Although we observed no significant correlation between Ki-67 labeling index and tumor recurrence, a strong correlation was observed between Ki-67 labeling index and tumor grade as well as various histopathologic parameters. In addition, tumor invasiveness was significantly influenced by Ki-67 labeling index according to multivariate analysis. These results are consistent with those of previous reports [4, 7, 8, 27, 28]. Thus, Ki-67 labeling index may be helpful, particularly in borderline cases of meningiomas in which mitoses are difficult to identify or other histopathologic criteria for atypical meningioma are not clear.

VEGF has been implicated as a critical factor in the formation of peritumoral brain edema associated with meningiomas [15, 29]. Sakuma et al. revealed that the expression of VEGF-A is related to the development of peritumoral brain edema with meningiomas and histological grade; thus, VEGF-A expression may influence the prognosis of patients with meningiomas [30]. Furthermore, VEGF is thought to play an essential role in the positive regulation of tumor angiogenesis by promoting the migration, proliferation, and tube formation of endothelial cells; thus, up-regulation in meningiomas confirms its role as a pro-angiogenic factor [31]. Presently, VEGF was shown to be correlated with grading of meningiomas and necrosis. Our findings indicate that overexpression of VEGF is correlated with poor prognosis of meningiomas by the mechanisms mentioned above. Thus, in the clinical setting, VEGF can be used as a prognostic marker for meningiomas. However, the prognostic value of VEGF expression regarding recurrences of these tumors appears questionable [24, 32]. VEGF secretion from microscopic remaining tumor lesions after surgery may induce neovascularization, which promotes meningioma recurrence, suggesting that high VEGF expression is significantly related to recurrence and poor prognosis of meningioma [32]. However, in another study, despite more prominent vascularity in high-grade tumors, no difference in VEGF expression has been observed between tumor grades of meningiomas [33]. We did not observe a significant association between VEGF expression status and tumor recurrence. However, few studies examining VEGF as a prognostic factor in meningiomas have been conducted. As information regarding VEGF status in meningiomas increases, correlation of this marker with diagnostic potential and clinical outcomes will become possible.

COX-2 plays an important role in neoplasm development in various manners. For brain neoplasms, increased levels of COX-2 have been noted in human gliomas, demonstrating an association between COX-2 protein expression, clinical aggressiveness and poor survival [34]. Moreover, the utility of COX-2 inhibitors on proliferation and the invasion ability of cultured glioma cell lines have been reported [35]. In meningiomas, Lin et al. revealed that the association between tumor grade and COX-2 expression is highly significant based on the 1993 WHO classification. More aggressive tumors were associated with increasingly high levels of COX-2 [10]. In our study, COX-2 expression was significantly correlated with necrosis and brain or adjacent soft tissue invasion in meningioma patients. Moreover, we showed that COX-2 expression had a major impact on adjacent tissue invasion according to multivariate analysis. A hypothesis regarding increased COX-2 expression in more aggressive meningioma states that COX-2 levels may be an indicator of ischemia. COX-2 expression appears to be elevated near areas of necrosis, which may reflect increased angiogenesis [10]. According to Ragel et al., overexpression of COX-2 contributed to tumor growth in mouse xenograft models with meningioma cell lines by increasing angiogenesis and cellular proliferation, as well as by decreasing apoptosis [36]. In addition, the study revealed that celecoxib, a selective COX-2 inhibitor, decreases meningioma growth with evidence of decreased microvascular density, increased apoptosis, and decreased COX-2 and VEGF expression [36, 37]. Thus, the association between COX-2 and meningioma provides a new therapeutic strategy for treatment using COX-2 inhibitors, either as an adjunct or in combination with radiation therapy. Additional systemic studies are necessary before COX-2 inhibitors can be used in clinical practice.

We observed a correlation between COX-2 and VEGF expression. A previous study reported a trend of moderate to high COX-2 levels with VEGF expression, although no statistical association between COX-2 expression and VEGF was observed [15]. VEGF interacts with COX-derived prostaglandins in angiogenesis [17]. Thus, COX-2 as well as VEGF may be ancillary molecular markers used to indicate aggressive meningioma behavior.

The bcl-2 and cyclin D1 proteins have been implicated in prolonged cell survival by inhibiting apoptosis and influencing the cell cycle [6, 38]. Several studies demonstrated that bcl-2 and cyclin D1 are useful as proliferative markers in defining subgroups of meningiomas with more aggressive biological behavior [6, 20, 38]. However, another study did not observe an alteration of the cyclin D1 gene in non-astrocytic brain tumors, including meningiomas [39]. We were unable to demonstrate other correlations of bcl-2 and cyclin D1 with histopathologic parameters and other molecular markers, except for cellularity in various grades of meningiomas.

Several studies have demonstrated a significant association between the staining intensity/percentage of EGFR and tumor grade in meningiomas [19, 40]. However, Andersson et al. identified no relationship between EGFR expression level and clinical outcome using immunohistochemical and quantitative real-time PCR analyses [40]. We observed no correlation between EGFR and tumor grade.

In summary, our data revealed a statistically significant correlation between expression of Ki-67 and VEGF proteins and meningioma grade; COX-2 expression was correlated with several histopathologic parameters, including necrosis and brain or adjacent tissue invasion and with VEGF expression. These molecular factors may be helpful for determining meningioma grade in histologically controversial cases and identifying tumors with benign histological features, but unfavorable clinical outcomes.

The authors wish to acknowledge the financial support to the Songeui Scholarship Fund made in the program year of 2006.

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  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
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