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Keywords:

  • urine cytology;
  • ProEx C;
  • urothelial atypia;
  • sensitivity;
  • specificity

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. CONFLICT OF INTEREST DISCLOSURES
  8. REFERENCES

BACKGROUND

ProEx C is an antibody cocktail targeting the expression of topoisomerase IIα and minichromosome maintenance protein-2. ProEx C staining is being used to assist in diagnoses of the gynecological specimens. This study was designed to determine the utility of ProEx C in urine cytology samples for improving the detection of urothelial carcinomas where a significant number of urine cytology specimens are diagnosed as “atypia.”

METHODS

Sixty urinary specimens (12 negative, 13 positive, and 35 atypical cases) were stained with ProEx C, and ProEx C results were recorded as positive when nuclear staining was only seen in at least one morphologically atypical urothelial cell.

RESULTS

All 12 benign cytology samples showed negative staining with ProEx C. Twelve of 13 cases that had a malignant cytologic diagnosis showed a positive nuclear staining of the malignant cells. Eighteen of 35 cases with atypical cytologic diagnoses showed positive nuclear staining. Of the 35 cases with “atypia,” 17 had a malignant histopathologic follow-up. In this study, ProEx C stain had an overall sensitivity of 78.4%, specificity of 95.7%%, positive predictive value of 96.7%, and negative predictive value of 73.3% for the detection of urothelial carcinoma.

CONCLUSIONS

ProEx C stain is a useful adjunct test to urine cytologic analysis, even in specimens with limited cellularity. In urinary smears, this test is most useful in stratification of the “atypical” diagnoses into benign and malignant subsets. To the authors' knowledge, this is the first study of ProEx C application in urine cytology as an adjunct marker for detection of urothelial carcinoma. Cancer (Cancer Cytopathol) 2013;121:320–8. © 2013 American Cancer Society.

There are approximately 50,000 new cases of urothelial carcinoma with 10,000 deaths each year in the United States.[1] Cystoscopy and cytology have been the primary modalities used to detect and monitor urothelial carcinomas.[2, 3] It has been well demonstrated that cytology has a high specificity but low sensitivity for bladder cancer detection, particularly if the urothelial neoplasia is of a lower grade. Several potential adjunct markers have been used to improve the sensitivity and specificity of the urinary tract cytology diagnoses.[4-9] Here, we have explored usage of ProEx C as an aid in improving the sensitivity of urine cytology cancer diagnoses. ProEx C is a novel antibody cocktail targeting the expression of topoisomerase IIα and minichromosome maintenance protein 2 (MCM2).[10-12] Both proteins are overexpressed in the cell nucleus during aberrant S-phase induction of neoplastic and human papillomavirus–infected cells.[13-15] Several studies have demonstrated ProEx C as an adjunct marker for assessing dysplasia in gynecologic specimens, especially in cervical biopsies and smears.[15-23] In this study, we have attempted to use ProEx C on urine cytology specimens where a significant number of them are diagnosed as “atypical,” a designation that triggers unwarranted additional procedures that may result in patient discomfort and/or increase in cost. This study was designed to determine the utility of ProEx C in urine cytology samples for improving the precision of detection of urothelial carcinomas.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. CONFLICT OF INTEREST DISCLOSURES
  8. REFERENCES

Specimen Criteria and Collection

The Institutional Review Board of the David Geffen School of Medicine at UCLA has approved this study (IRB# 11-001781). The patients, who had urine cytology specimens with surgical pathology follow-up, were studied from July 2010 through June 2011. Residual fluids from the liquid-based ThinPrep urinary specimens were used to make a slide for the Papanicolaou (Pap) and subsequent immunohistochemistry (IHC) stains with ProEx C. After Pap staining, the smears were screened microscopically to confirm presence of the urothelial cells and the initial cytologic diagnoses.

ProEx C Immunostaining

ProEx C antibody cocktail (BD ProEx C IHC), BD SureDetect Detection Reagents kit, and BD SureDetect SiHa Cell Control slides were obtained from BD Diagnostics-TriPath, Burlington, NC. The staining procedures were performed manually by following the published procedure in the manufacturer product insert. The ThinPrep Pap-stained slides, after the cytological evaluation, were destained in 1% hydrochloric acid/70% ethanol for 5 minutes, then washed with phosphate-buffered saline solution. Subsequently, they were restained with ProEx C using the manual IHC technique along with positive and negative controls (SiHa cells). Minimal artifacts and/or loss of cellularity were encountered due to destaining and restaining.

ProEx C Staining Evaluation

Large atypical cells were scored for positivity or negativity of ProEx C staining by IHC. ProEx C was recorded as positive in urine cytology samples when nuclear staining was seen in at least one morphologically atypical urothelial cell (Figs. 1 and 2) and as negative if no staining was observed. If only the small parabasal cells were positive, the smear was scored as negative for ProEx C. The umbrella cells were excluded from scoring, although none were stained with ProEx C, similar to the inflammatory cells. The interpretations for the study, on Pap- and IHC-stained slides, were made by a cytotechnologist (M.C.) and a cytopathologist (N.A.M.) blindly without the knowledge of the official cytologic and/or histologic follow-up diagnoses. The number of large atypical cells with and without ProEx C reactivity was estimated and recorded for each slide. The same procedure was followed for the small parabasal cells. Positivity or negativity of each case was based only on the reactivity of the large atypical cell(s) regardless of the small parabasal cells (Tables 1 and 2).

image

Figure 1. Papanicolaou stain from a malignant cytology (case #21, Table 1) shows a single cluster of highly atypical urothelial cells with increased nuclear-to-cytoplasmic ratio and dark chromatin (A, 100× objective). ProEx C immunostain shows positive nuclear staining of the cellular cluster (B,100× objective). Hematoxylin and eosin stain from the follow-up surgical biopsy specimen shows a high-grade urothelial carcinoma (C, 20× objective).

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image

Figure 2. An example of “atypia” by cytology, case #54 in Table 2, is displayed. There were rare highly atypical cells present in this urine sample. Papanicolaou stain illustrates such a cell (A, 100× objective). ProEx C immunostain shows positive nuclear staining of a single large atypical cell (B,100× objective). Hematoxylin and eosin stain from the follow-up surgical biopsy specimen shows an invasive high-grade urothelial carcinoma (C, 20× objective).

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Table 1. Summary of the Patients With Definitive Cytology Diagnoses and Emphasis on ProEx C Test Results in Group I
No.SexAgeSpecimenProEx CNo. of ACsNo. of SMsCytology DiagnosisHistology Diagnosis
Total#PosTotal#Pos
  1. Abbreviations: AC, atypical cell; CA, urothelial cell carcinoma; CIS, carcinoma in situ; FN, false negative reaction on atypical cells; HG, high grade; IR, irrigation; LG, low grade; N, negative reaction on atypical cells; NCFU, negative cytology follow-up; P, positive reaction on atypical cells; #Pos, estimated number of cells positive for ProEx C; RPW, renal pelvic wash; SM, small parabasal cell; Total, estimated number of the cells on the slide; VU, voided urine.

1M62VUN0000NegativeNCFU
2M75VUN00102–3NegativeBenign
3M30IRN0000NegativeBenign
4M22IRN00102–3NegativeBenign
5M20IRN00<50NegativeBenign
6M80IRN00<51NegativeBenign
7F23IRN00<50NegativeBenign
8M70IRN00102–3NegativeBenign
9M62IRN00102–3NegativeBenign
10M53IRN00<100NegativeBenign
11M54IRN00102–3NegativeBenign
12M85IRN00102–3NegativeBenign
13M58IRFN10000HG CA/CISLG CA
14M69IRP>502000HG CA/CISHG CA
15M87IRP>10300HG CA/CISHG CA
16M57RPWP>502000HG CA/CISHG CA
17F67IRP>10>200HG CA/CISHG CA
18M85VUP>502000HG CA/CISHG CA
19M88IRP>10>200HG CA/CISHG CA
20M74IRP>10>200HG CA/CISHG CA
21F65IRP>502000HG CA/CISHG CA
22M79VUP502000HG CA/CISHG CA
23M88IRP>5020>201HG CA/CISHG CA
24M43VUP>10>2>10>2HG CA/CISHG CA
25M55VUP>502000HG CA/CISHG CA
Table 2. Summary of The Patients With Diagnosis of “Atypia” by Cytology and Emphasis on ProEx C Test Results in Group II
No.SexAgeSpecimenProEx CNo. of ACsNo. of SMsCytology DiagnosisHistology Diagnosis
Total#PosTotal#Pos
  1. Abbreviations: AC, atypical cell; CA, urothelial cell carcinoma; FN, false negative reaction on atypical cells; FP, false positive reaction on atypical cells; IR, irrigation; LG, low grade; N, negative reaction on atypical cells; #Pos, estimated number of cells positive for ProEx C; P, positive reaction on atypical cells; SM, small parabasal cell; Total, estimated number of the cells on the slide; RPW, renal pelvic wash; UB, ureteral brush; VU, voided urine.

26M63IRN20>102AtypiaBenign
27M56RPWN30>105AtypiaBenign
28M70VUN3000AtypiaBenign
29M76VUN3000AtypiaBenign
30M68VUN3000AtypiaBenign
31M70VUN3000AtypiaBenign
32M88IRN3000AtypiaBenign
33M68IRN3000AtypiaBenign
34M68IRN30>10>5AtypiaBenign
35M59IRN30>10>5AtypiaBenign
36M66IRFP31>50>10AtypiaBenign
37M57IRFN<10000AtypiaHG CA
38M57IRFN<10000AtypiaHG CA
39M60IRFN>10050AtypiaHG CA
40M67VUFN>10050AtypiaLG CA
41F59IRFN20>100AtypiaLG CA
42M82IRFN>10050AtypiaHG CA
43M69VUFN<10000AtypiaLG CA
44M66IRP>502000AtypiaHG CA
45F55VUP<10100AtypiaHG CA
46M68IRP<10100AtypiaLG CA
47F50VUP<102–300AtypiaLG CA
48M50VUP<102–300AtypiaHG CA
49M54UBP>502000AtypiaHG CA
50M50IRP>10100AtypiaLG CA
51M90VUP<102–300AtypiaHG CA
52M84VUP>102–300AtypiaHG CA
53F94IRP>502000AtypiaHG CA
54M90VUP10500AtypiaHG CA
55M80IRP>102–300AtypiaHG CA
56F94IRP10500AtypiaHG CA
57F70IRP>502000AtypiaHG CA
58M69IRP<102–3>102–3AtypiaHG CA
59M67IRP>10500AtypiaHG CA
60F31IRP<101–200AtypiaLG CA

Study Design

The patients were divided into 2 groups: group I, those with a definitive cytological diagnosis of either benign or malignant (Table 1), and group II, those with a cytological diagnosis of “atypia” (Table 2) where the pertinent data are tabulated. This study was intended to evaluate the usefulness of performing ProEx C immunostain on urine samples when a diagnosis of “urothelial atypia” was rendered. The overall ProEx C reactivity among all cases is shown in Table 3. The results were compared to the histopathology finding, which served as the gold standard. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were determined (Table 4).[24]

Table 3. Results of ProEx C Stain Reactions on the Urine Specimens in Relation to the Cytology Diagnoses
 Cytology Diagnoses (n = 60)
 Group IGroup II
Stain ReactionBenign (n = 12)Malignant (n = 13)Atypical (n = 35)
ProEx C +01218
ProEx C –12117
Table 4. Test Performance of ProEx C Reactivity in the Combined and Individual Groups
 All GroupsGroup IGroup II
  1. Abbreviations: NPV, negative predictive value; PPV, positive predictive value.

Total (n)602535
Positive (n)291217
Negative (n)221210
False positive (n)101
False negative (n)817
Sensitivity78.4%92.3%70.8%
Specificity95.7%100.0%90.9%
PPV96.7%100.0%94.4%
NPV73.3%92.3%58.8%

Statistical Analysis

The chi-square test, with 2 degrees of freedom, was used to assess the assay performance. This comparative statistical analysis was carried out to evaluate the ProEx C staining for each group. The applied nonparametric chi-square statistical analysis was to compare the values in the expected versus observed groups (χ2) as specified in Table 5. A P value of .05 or less was considered a significant difference between the 2 compared groups. Statistica, version 10, from StatSoft Inc. (Tulsa, Okla)[25] and Office Excel 2010 (Microsoft, Redmond, Wash) were used for the tabulations and the statistical analyses.

Table 5. Results of ProEx C Staining of the Cells in Cytology Slides With Histopathology Follow-Up Diagnoses (n = 60) and the 2 × 2 Chi-Square Analysis in the 2 Groups
 Group I Chi-Square P < 0.00001Group II Chi-Square P = 0.0007
 Benign Cytology (n = 12)Malignant Cytology (n = 13)Atypical Cytology (n = 35)
Stain ReactionsBenign HistologyMalignant HistologyBenign HistologyMalignant HistologyBenign HistologyMalignant Histology
  1. The data columns displayed in bold were used for the 2 × 2 chi-square analyses.

ProEx C +00012117
ProEx C –12001107

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. CONFLICT OF INTEREST DISCLOSURES
  8. REFERENCES

A total of 60 patients were identified who had histopathology follow-up diagnoses after the initial cytological interpretations, except for one case that was followed up by only urinary cytology (case #1, Table 1). On the basis of the cytological diagnoses, the 60 cases were divided into 2 groups and tabulated in Tables 1 and 2.

Group I: Patients With Definitive Cytology Diagnoses

There were 25 cases (41.7% of the total) in this group, consisting of 3 females and 22 males with ages ranging from 20 to 88 years (Table 1). Twelve cases had benign/negative cytology diagnoses, whereas 13 had positive cytology diagnoses of high-grade carcinoma/carcinoma in situ. All cytology diagnoses were concordant with histopathology. Among the patients with benign/negative cytology, 2 had voided urine and 10 had bladder irrigation specimens. The ProEx C results were negative, because there were no atypical cells in the samples of these cases. However, all the samples except 2 (cases #1 and #3, Table 1) had small parabasal cells, of which 7 cases had positive reactions for ProEx C, serving as positive internal controls. Among the patients with positive/malignant cytology, 4 had voided urine, 8 had bladder irrigation, and 1 had renal pelvic wash samples. The histology follow-up of 12 cases was high-grade urothelial carcinomas, whereas 1 case had a low-grade papillary urothelial carcinoma (case #13, Table 1). The ProEx C results were positive in 12 cases that had high-grade urothelial carcinomas and negative in the case with low-grade papillary carcinoma. An example of a positive ProEx C reaction is shown in Fig. 1. The case with the negative ProEx C reaction (case #13, Table 1) was subsequently classified false negative.

Group II: Patients With Nondefinitive (Atypia) Cytology Diagnoses

There were 35 cases (58.3% of the total) in this group, consisting of 7 females and 28 males ranging from 31 to 94 years in age (Table 2). Eleven patients had benign and 24 had malignant diagnoses on histology follow-up. Among the cases with the benign histopathology diagnoses, 4 had voided urines, 6 had bladder irrigations, and 1 had renal pelvic wash. The ProEx C results were negative in 10 cases and positive in 1, where the latter was classified as a false positive reaction (case #36, Table 2). In addition to a few positive parabasal cells, this case demonstrated a single atypical cell which was also positive with the IHC stain, in contrast to malignant urothelial cells (case #23, Table 1) where generally more positive cells were present. Among the cases with malignant diagnoses on histopathology follow-up, 8 had voided urines, 15 had bladder irrigations, and 1 had ureteral brush. The histology diagnoses were high-grade carcinoma in 17 patients and low-grade papillary carcinoma in 7 cases (Table 2). The ProEx C results were positive in 17 cases of which 13 had high-grade urothelial and 4 had low-grade papillary urothelial carcinomas. An example is shown in Fig. 2. The ProEx C result was negative in 7 cases that were subsequently classified as false negative (cases #37-#43, Table 2). Three of the false negative cases had low-grade and 4 had high-grade urothelial carcinomas. Among these false negative cases, neither the atypical cells nor the parabasal cells (4 cases, #39-#42, Table 2) were immunoreactive with the stain, suggestive of possible issues related to technical difficulties.

ProEx C Reactivity Performance

Table 3 provides a bird's-eye view of the ProEx C staining distribution between the 2 groups. In group I, all 12 cases with a benign cytology diagnosis were negative for the stain. In contrast, all 13 cases with a malignant cytology diagnosis were positive except for 1 that was classified as false negative. Consequently in group I, the staining test performances were 92% sensitivity, 100% specificity, 100% PPV, and 92% NPV (Table 4).

In group II, on one hand, 11 (31.4%) of the 35 samples with a cytology diagnosis of “atypia” were shown to be benign by histopathologic follow-up, of which all were negative for ProEx C, except for 1 case that was classified as false positive (case #36, Table 2). On the other hand, the remaining 24 (68.6% in this group) were determined to be malignant by histopathologic analysis (Table 2), of which 17 were positive for ProEx C and 7 were classified as false negative for the stain. The staining performance in this group yielded 71% (sensitivity), 91% (specificity), 94% (PPV), and 59% (NPV). All performance values including the overall parameters are displayed in Table 4.

Using the McNemar-Fisher exact chi-square analysis in a 2 × 2 table, a P value of < .00001 was obtained when the positive and negative ProEx C results were compared among the patients with benign and malignant diagnoses (group I). A similar statistical analysis was carried out in group II obtaining a P value of .0007, again among the patients who were determined to be benign and malignant by histopathology after the initial diagnosis of “atypia” by cytologic analysis (Table 5).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. CONFLICT OF INTEREST DISCLOSURES
  8. REFERENCES

Since the introduction of urine cytology 6 decades ago as a means of detecting urothelial neoplasms, it has remained a cancer screening test despite its well-known limitations.[26-31] When a definitive benign or malignant diagnosis is made by cytology on urinary samples, it is highly reliable. It is, however, limited when a neoplastic lesion, particularly a low-grade tumor, becomes indistinguishable from a reactive process. Therefore, whereas urine cytology has a critical role in the evaluation of urothelial cells, the cytological diagnosis of urothelial carcinoma can be very challenging.[32] In general, urine cytology has been used as a screening tool to detect urothelial cancers in high-risk patients, as an initial test for individuals with hematuria, and as a follow-up test in patients with a previous diagnosis of urothelial cancer to rule out the tumor recurrence.[33] When a diagnosis of “atypical” cytology is made on urinary specimens, invasive procedures may be triggered that result in patient's discomfort, anxiety, and/or increase in cost. To remedy this situation, several markers have been introduced to improve the accuracy of urinary cytology. Cytokeratin-20 has been evaluated as an adjunct marker in cases of atypical cytology. A malignant process is favored when this marker is positive.[34-36] Cheng et al has proposed that prostate stem cell antigen may be another useful adjunct marker to supplement urine cytology.[37] Multiple studies have evaluated the usefulness of NMP-22 (nuclear matrix protein 22).[38-41] Other ancillary tests such as ImmunoCyt[42, 43] and UroVysion[4-6] have also been used; however, they are expensive and require special expertise and certification for the test interpretation. Yet, none of these markers has been accepted as a simple and universal marker of the neoplastic cells in urine cytology. In addition, Burger et al have used MCM2 antibody in surgical specimens to evaluate the risk of recurrence in bladder cancers. They have shown that MCM2 predicts recurrence of the neoplasia in stage Ta/T1 more accurately than cytokeratin-20, ki-67, and histologic grade.[44]

In this study, we have introduced immunocytochemical staining of urothelial cells with ProEx C. This marker, heretofore, has been mainly used as a molecular marker for differentiating neoplastic/dysplastic cells in the gynecological specimens. We demonstrate that this antibody cocktail could also serve as an adjunct marker in urine cytology. As shown in Tables 1 and 4, ProEx C staining results highly correlate with the cytology and histopathology findings where definitive diagnoses have been made in group I. It appears that when a definite diagnosis is made by cytology, there is no need for performing the ProEx C stain on the specimen. The value of the stain becomes apparent in a significant number of the urinary cytology cases (group II, ∼60% of the cases in this study) when a diagnosis of “atypia” is made (Table 2). It appears that a large number of the cases, 27 of 35 (cases #26-#35 and #44-#60, Table 2), have benefited from staining with ProEx C by stratifying the cytology diagnoses. The remaining 8 cases in this group have resulted in the false reactions. In a relative sense, the number of false negative reactions (case #13, Table 1 and cases #37-#43, Table 2) is higher than the false positive reactions in this study (case #36, Table 2), yielding a high specificity and PPV but a lower sensitivity and NPV (Table 4). We presume that the false negative (if they are indeed false negative) reactions are mostly technical in nature due to the variability inherent in the manual methodologies. The basis of our presumption is the general stainability of the normal small parabasal cells with ProEx C. The parabasal cells in 4 of the cases (cases # 39-#42, Table 2) did not exhibit ProEx C staining, which we attribute to isolated technical malfunctions. Because the respective large atypical cells also did not stain with ProEx C, we assume that these 4 cases are outliers resulting from the same technical variability. Henceforth, on the basis of this assumption of technical malfunctions, we removed the 4 cases from Table 4 and retabulated in Table 6, resulting in significant improvements in the assay performance. To aid in circumventing a misinterpretation of a negative stain arising from such technical malfunctions in the future, one may also use a histologic section of normal urothelium with an intact parabasal cell layer as positive control on all cases simultaneously stained with this marker, particularly when using a well-controlled automated platform.

Table 6. Presumptive Test Performance of ProEx C Reactivity in the Combined and Individual Groups After Eliminating 4 of the False Negative Reactions in Group II on the Assumption of Being Outliers
 All GroupsGroup IGroup II
  1. Abbreviations: NPV, negative predictive value; PPV, positive predictive value.

Total (n)562531
Positive (n)291217
Negative (n)221210
False positive (n)101
False negative (n)413
Sensitivity87.9%92.3%85.0%
Specificity95.7%100.0%90.9%
PPV96.7%100.0%94.4%
NPV84.6%92.3%76.9%

In addition, in this study, 8 cases (1 patient in group I and 7 patients in group II) had a low-grade urothelial neoplasia on histopathology follow-up. Four of these patients were classified as false negative for ProEx C; 1 patient in group I (case #13, Table 1) and 3 patients in group II (cases #40, #41, and #43, Table 2). Although an argument can be made for a lower sensitivity of ProEx C in low-grade urothelial cancers, the low number of the low-grade papillary carcinomas in our series does not allow for such a definitive conclusion. Future large-scale studies are required to address this point.

We are confident that in a larger study with longer follow-up periods, all assay performance parameters would be 95% or better provided the staining with ProEx C is performed on a tightly controlled automated platform. The “false positive” reaction (case #36, Table 2) may have been an early indication of neoplastic development even though the immediate histopathology follow-up has been negative. This assumption may be clarified during a longer follow-up period.

In conclusion, ProEx C seems to be a promising and simple adjunct tool in urine cytology and further helps by avoiding expensive and time-consuming tests such as immunofluorescent and/or fluorescence in situ hybridization assays. This stain is particularly useful when the urine samples have scant cellularity with only a few atypical cells present, creating difficulty in cytologic interpretation.

FUNDING SOURCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. CONFLICT OF INTEREST DISCLOSURES
  8. REFERENCES

This study was supported by a Translational Research Fund grant from the UCLA Department of Pathology and Laboratory Medicine. This work was not supported, either financially or in any manner, by BD Company, which is the supplier of ProEx C antibody.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. CONFLICT OF INTEREST DISCLOSURES
  8. REFERENCES
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