AJCP / Original Article Utility of Estrogen Receptor, Progesterone Receptor, and HER-/neu Analysis of Multiple Foci in Multifocal Ipsilateral Invasive Breast Carcinoma Ellen G. East, MD, Judy C. Pang, MD, Kelley M. Kidwell, PhD, and Julie M. Jorns, MD From the Departments of Pathology and Biostatistics, University of Michigan, Ann Arbor. Key Words: Multifocal; Breast; Cancer; Carcinoma; Estrogen receptor; ER; Progesterone receptor; PR; HER-/neu CME/SAM Am J Clin Pathol December 05;44:95-959 DOI: 0.09/AJCPFWXP54OLILMU ABSTRACT Objectives: To determine the frequency of estrogen receptor (ER), progesterone receptor (PR), and HER-/neu (HER) testing multiple foci of ipsilateral invasive breast carcinoma at our institution and to evaluate resulting change in treatment recommendation. Methods: We identified 65 consecutive cases of multifocal invasive breast cancer over a 0-year period (005-04). Clinicopathologic features and treatment recommendation were assessed by slide and chart review. Results: Seventy (4.4%) of 65 patients had two or more foci tested. In the first 6 years (005-00), frequency of testing two or more foci was.6% and increased to 70.6% in 04. Seven (0%) of 70 had a clinically significant difference in ER/PR and/or HER status, five (7.%) with a difference in HER, one (.4%) in ER/PR, and one (.4%) in both ER/PR and HER. All cases with difference in status had different histology and/or the largest focus was the most positive one. Conclusions: Our findings support current recommendations to evaluate smaller tumor foci in multifocal invasive breast cancer if the focus is of different grade or histology. Additional features, including specific histology, grade, and ER, PR, and HER status of the largest focus, should also be considered when selecting cases for which testing of foci may be of benefit. Upon completion of this activity you will be able to: describe the College of American Pathologists (CAP) guidelines for performing estrogen receptor (ER), progesterone receptor (PR), and HER-/neu (HER) analysis in smaller tumor foci in multifocal ipsilateral invasive breast carcinoma. predict the likelihood of difference in ER, PR, and HER status between largest and smaller tumor foci in multifocal ipsilateral invasive breast carcinoma based on similarities and differences in histology and grade between the tumor foci. discuss possible exceptions to CAP guidelines for performing ER, PR, and HER analysis in smaller tumor foci in multifocal ipsilateral invasive breast carcinoma. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of AMA PRA Category Credit per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Exam is located at www.ascp.org/ajcpcme. The American Joint Committee on Cancer staging manual defines multifocal ipsilateral invasive breast carcinomas as infiltrating carcinomas in the same breast, which are grossly and macroscopically distinct and measureable using available clinical and pathologic techniques. Multifocal invasive breast cancers appear to arise most frequently from a single cancer clone and less often as synchronous independent primary tumors. -8 In the largest series to date (n = 8,95), Wolters et al 9 report an incidence of 0.8%; however, multifocality appears to be increasing, at least in part due to greater sensitivity of preoperative imaging techniques. 0 Multifocality has been previously associated with increased risk of axillary lymph node metastases, locoregional recurrence, and potentially worse outcome. -7 95 Am J Clin Pathol 05;44:95-959 Downloaded from https://academic.oup.com/ajcp/article-abstract/44/6/95/7660 DOI: 0.09/AJCPFWXP54OLILMU on 07 April 08
AJCP / Original Article Estrogen receptor (ER), progesterone receptor (PR), and HER-/neu (HER) status provides crucial prognostic and predictive information for patients with breast cancer. In the case of multifocal invasive breast cancer, the American Society of Clinical Oncology (ASCO) and College of American Pathology (CAP) guidelines recommend ER/PR analysis on at least one of the tumors, preferably the largest, 8 whereas HER guidelines do not provide specific recommendations. 9 The CAP biomarker template further recommends testing of smaller foci if they have different histology or grade than the largest focus. 0 However, others advocate for the testing of or even all foci, regardless of histology or grade, due to possible tumor heterogeneity and the potential to provide life-saving therapies that may not have been recommended based on testing of a single (largest) tumor., In practice, testing foci is variable. We aimed to evaluate the frequency of ER, PR, and HER testing of tumor foci in patients with multifocal ipsilateral invasive breast cancer at our institution and the resulting clinical therapy decisions. Materials and Methods Case Selection In accordance with internal review board protocol, we conducted an electronic database search from January 005 through December 04 using keywords breast, invasive, and multifocal, which initially yielded 556 cases. Cases were assessed for multifocality, defined as two or more discrete foci of ipsilateral invasive breast cancer separated by intervening normal breast tissue, via chart and slide review (E.G.E., J.C.P., and J.M.J.). Exclusion criteria were neoadjuvant therapy; recurrent, microinvasive, or transected tumors; or artifactual multifocality due to large biopsy site/ hematoma or potentially contiguous satellite foci. Only cases with slides available for re-review and confirmation of multifocality were included. A total of 65 cases meeting these criteria were identified. ER, PR, and HER Evaluation The cases represented tumors tested from internal and referral material; however, all immunohistochemical (IHC) (and corresponding H&E) slides and fluorescence in situ hybridization (FISH) reports from referral cases were reviewed at our institution by board-certified pathologists with expertise in breast pathology. When performed at our institution, ER (clone SP), PR (clone IE), and HER (clone 4B5) (Ventana Medical Systems, Tucson, AZ) IHC was initially performed. Staining was performed on the Ventana BenchMark XT platform, and results were obtained via US Food and Drug Administration approved and validated Ventana Image Analysis System or iscan Coreo System (Ventana Medical Systems), which provided a percentage ER/PR or semiquantitative (0, +, +, or +) score for HER via colorimetric and morphometric analysis of at least four images. Reflex HER FISH testing was performed when IHC yielded a (+) equivocal result and used PathVysion probes (Abbott Molecular Laboratories, Des Plaines, IL). In referral cases, IHC stains were reported as a percentage score for ER and PR and semiquantitative (0, +, +, or +) score for HER; these were assessed via manual review. HER FISH reports were reviewed, and HER status (negative, equivocal, or positive) based on HER/CEP7 ratio and HER copy number was reported. Reporting was per most recent ASCO/CAP guidelines at the time of review. 8,9, ER and PR were categorically stratified as negative, weak, and positive if there was less than %, % to 0%, and more than 0% staining, respectively. Data Collection and Analysis Clinicopathologic features were assessed by chart and slide review (E.G.E., J.C.P., and J.M.J.). Descriptive statistics and frequencies were tabulated for the sample. Patient and clinical characteristics were compared between those with only one focus tested vs those with two or more foci tested using the Wilcoxon rank sum test for continuous variables and c or Fisher exact test for categorical variables. Logistic regression on the binomial proportion of cases testing multiple foci out of all cases on year assessed the trend over time. We did not control for multiple comparisons due to the small sample size and descriptive nature of the study. SAS 9. (SAS Institute, Cary, NC) was used for all statistical analyses (K.M.K.). Results Features of Multifocal Invasive Breast Carcinomas Of the 65 patients, 07 (64.8%) had two foci, (9.4%) had three foci, and 6 (5.8%) had four or more foci. Additional clinicopathologic features are shown in Table. Ninety-five (57.6%) of 65 patients had only a single focus and 70 (4.4%) had two or more foci tested for ER, PR, and HER. Of those with two or more foci tested, 4 (48.6%) had testing on diagnostic specimens (core or excisional biopsy), and 6 (5.4%) had testing on both diagnostic and resection specimens. two or more foci was significantly associated only with different tumor histology, with (5.7%) of 70 with multiple foci tested vs one (.%) of 95 with a single focus tested having different tumor histology (P =.000). Downloaded from https://academic.oup.com/ajcp/article-abstract/44/6/95/7660 on 07 April 08 Am J Clin Pathol 05;44:95-959 95 DOI: 0.09/AJCPFWXP54OLILMU
East et al / ER, PR, and HER in Multifocal Cancer Table Clinicopathologic Features of Patients With Multifocal Ipsilateral Invasive Breast Carcinoma (n = 65) a Characteristic Value Age, mean (range), y 54 (6-9) No. of foci, median (range) (-9) Largest tumor Histology 9 (55.) -L 8 (7.0) ILC 9 (.6) Other 7 (4.) mbr grade 9 (7.6) 9 (55.8) 44 (6.7) Size, mean (range), cm. (0.-7) ER (n = 6) 9 (.6) Weak 5 (.) 9 (85.) PR (n = 6) 0 (8.4) Weak 5 (9.) 8 (7.4) HER (n = 6) 44 (88.) (.8) 6 (9.8) Other foci Different histology (7.) Different grade 5 (9.) T stage Ta 6 (.6) Tb 6 (5.8) Tc 6 (8.) T 57 (4.5) T (7.9) N stage N0 8 (49.7) Nmi 4 (8.5) Na 5 (.) Na (.9) Na 9 (5.5) Unknown (.) Final surgery Lumpectomy 54 (.7) Mastectomy (67.) Lymph node evaluation SLNB 9 (56.4) ALND 70 (4.4) (.) Postsurgical therapy (n = 57; 8 LTF) Radiation 87 (55.4) Endocrine 8 (8.5) Chemotherapy 66 (4.0) Herceptin (4.0) ALND, axillary lymph node dissection; ER, estrogen receptor; HER, HER-/neu;, invasive ductal carcinoma; -L, invasive ductal carcinoma with lobular features; ILC, invasive lobular carcinoma; LTF, lost to follow-up; mbr, modified Bloom-Richardson; PR, progesterone receptor; SLNB, sentinel lymph node biopsy. a Values are presented as number (%) unless otherwise indicated. 70% 60% 50% 40% 0% 0% 0% 0% or more foci tested Clinically significant difference in ER/PR and/or HER 005 006 007 008 009 00 0 0 0 04 Figure Frequency of estrogen receptor (ER), progesterone receptor (PR), and HER-/neu (HER) testing of tumor foci and clinically significant difference in patients with multifocal ipsilateral invasive breast carcinoma (n = 70). Table Categorical Differences in ER, PR, and HER Status Between Foci Tested in Patients With Multifocal Ipsilateral Invasive Breast Carcinoma (n = 70) Characteristic Weak/ No. (%) Weak/ Of note, two patients with only a single focus tested had testing performed on diagnostic core biopsy specimens of the tumor that corresponded to the smaller of two foci identified at resection. In both cases, histology and grade were similar. The tumor foci in one case measured 0.7 cm and 0.6 cm and in the second measured.9 cm and 0.7 cm. Overall frequency of testing two or more foci was.6% during the first 6 years of this study (005-00) and increased to 70.6% in 04. There was a significant increase in rate of testing multiple foci by year of 6.5% (P =.00) Figure. Cases in Which Multiple Foci Yielded Differing ER, PR, and/or HER Status ER (.4) (.4) (.4) a PR 8 (.4) 5 (7.) (.4) HER 0 (0) (.4) a 5 (7.) a ER, estrogen receptor; HER, HER-/neu; PR, progesterone receptor. a Clinically significant difference resulting in change in treatment recommendation (see Table ). Categorical differences for ER, PR, and/or HER were seen in 8 (5.7%) of 70 cases in which two or more foci were tested Table. Most frequently (0 of 8; 55.6%), the differences were due to categorical changes in ER/PR that did not meaningfully affect therapy decision; in nine patients, there was already a positive ER and/or PR result that supported the use of hormonal therapy Image, and for one patient, there was weak ER expression in one of two foci that did not change the decision to not recommend hormonal therapy. 954 Am J Clin Pathol 05;44:95-959 Downloaded from https://academic.oup.com/ajcp/article-abstract/44/6/95/7660 DOI: 0.09/AJCPFWXP54OLILMU on 07 April 08
AJCP / Original Article A B Image Example case in which categorical difference in estrogen receptor (ER) and progesterone receptor (PR) status did not affect clinical management. The two tumor foci have similar morphology, with one focus (A) having strong positive ER and weak PR staining and the second focus (B) having strong positive ER and negative PR staining. (H&E, immunohistochemistry 0; insets reduced to approximately 0% original size) Categorical difference was significantly associated with high grade in the largest focus and weak PR expression in any focus. Specifically, the largest tumor was modified Bloom-Richardson grade in (6.%) of 8 patients with categorical difference vs nine (7.%) of 5 without, and weak PR expression was seen in at least one focus in (66.7%) of 8 with categorical difference vs three (5.8%) of 5 without (P <.000). Clinically significant differences resulting in change in treatment recommendation were seen in seven (0%) of 70 cases in which multiple foci were tested. Five (7.%) patients had a significant difference in HER, one (.4%) in ER/PR, and one (.4%) in both ER/PR and HER status. Frequency of testing multiple foci and clinically significant difference are shown in Figure. In the seven cases with clinically significant difference in ER, PR, and/or HER status, tumor foci had different histology in three (4.9%) of seven cases and different grade in three (4.9%) of seven cases. In cases with HER discordance, five (8.%) of six had grade histology in the positive focus, three (50.0%) of six were HER positive in the largest focus, and HER IHC was equivocal in one or more focus in three (50.0%) of six cases. The case with differing ER/PR had weak expression in only one (the largest) of two foci tested, while the other was negative; in this case, postsurgical endocrine therapy was recommended by the treating oncologist, which the patient subsequently received. Clinicopathologic features of cases with clinically significant difference in ER, PR, and/or HER status are shown in Table, and example cases are shown in Image. Downloaded from https://academic.oup.com/ajcp/article-abstract/44/6/95/7660 on 07 April 08 Discussion Detection of multifocal ipsilateral invasive breast carcinoma is increasing.0 While ASCO/CAP guidelines recommend ER, PR, and HER testing of the largest focus, others advocate for testing of smaller or all tumor foci. Over a 0-year period, we have seen an increase in testing two or more foci at our institution, reaching 70.6% of multifocal cases in 04. However, only seven (0%) of 70 cases with multiple foci tested had clinically significant difference in ER, PR, and/or HER status between foci, resulting in differences in therapy recommendation. In our cohort, a clinically significant difference was more often due to discordance in HER (six of 70; 8.6%) than in ER/PR status (two of 70;.9%), and no cases had an ER/PR-positive smaller focus when the largest focus was ER/PR negative. Similarly, when testing multiple tumor foci, Garimella et al4 noted no major difference in ER/PR expression in their pilot study of cases. However, Buggi et al noted ER positivity in a smaller focus while the larger focus was ER negative in two (.8%) of cases, and Choi et al noted.% (two of 65) ER discordance between foci. Choi et al further described the discordant foci; in both cases, tumor foci had the same T stage and (high) grade but had different histology and molecular phenotypes, with less aggressive luminal types in ER-positive foci. Thus, it appears unlikely that ER/ PR testing of, smaller foci is beneficial unless it is of lower grade, similar size, or different histology. Specifically, histologic subtypes that are morphologically Am J Clin Pathol 05;44:95-959 955 DOI: 0.09/AJCPFWXP54OLILMU
East et al / ER, PR, and HER in Multifocal Cancer A B Image Example cases (case and foci numbers corresponding to those in Table ) with clinically significant difference in estrogen receptor (ER) (case ) and HER-/neu (HER) testing (cases and 7). Case : focus (A), invasive ductal carcinoma () with (+) equivocal HER immunohistochemistry (IHC) and positive fluorescence in situ hybridization (FISH) (HER/D7Z ratio =.75) and focus (B), with (+) equivocal HER IHC and negative FISH (HER/D7Z ratio = 0.95). Table Clinicopathologic Features of Cases With Clinically Significant Difference in ER, PR, and/or HER Status Between Foci Tested in Patients With Multifocal Ipsilateral Invasive Breast Carcinoma (n = 7) Case Focus Histology mbr Grade Size, cm ER PR HER IHC HER FISH -L -L ILC ILC ILC -L 0.8 0.7..9..5.5.5..4.9.4..0 0.8 Weak Weak (HER/D7Z =.75; HER/cell = 6) (HER/D7Z = 0.95; HER/cell = ) (HER/D7Z =.0; HER/cell = ) (HER/D7Z =.8; HER/cell = ) (HER/D7Z =.8) (HER/D7Z =.4; HER/cell = ) 4 5 6 7 ER, estrogen receptor; FISH, fluorescence in situ hybridization; HER, HER-/neu;, invasive ductal carcinoma; -L, invasive ductal carcinoma with lobular features; IHC, immunohistochemistry; ILC, invasive lobular carcinoma; mbr, modified Bloom-Richardson; PR, progesterone receptor. consistent with molecular luminal A/B subtypes and/or are typically ER positive such as invasive lobular, papillary, and mucinous carcinoma especially warrant evaluation due to higher likelihood of discordance. ASCO/CAP guidelines define ER/PR as positive if as few as % of tumor nuclei are positive. However, they also support discussion of pros and cons of endocrine therapy between the oncologist and patient in cases in which ER/PR expression is weak (ie, %-0%).8 Thus, in cases with weak expression, staining of blocks or foci in the case of multifocal disease may be helpful in determining treatment and thus is warranted if requested by the clinical 956 Am J Clin Pathol 05;44:95-959 Downloaded from https://academic.oup.com/ajcp/article-abstract/44/6/95/7660 DOI: 0.09/AJCPFWXP54OLILMU on 07 April 08 team. This scenario is relatively infrequent and in our series comprised just.0% (five of 65) with weak staining when testing the main tumor focus. HER difference, although also infrequent, more often resulted in clinically significant difference in status (six of 70; 8.6%). However, in three cases, the larger focus was the positive one, and different histology was seen in the other three cases. Thus, no case would have failed to have been identified as HER positive by current CAP recommendations.0 However, Buggi et al and Choi et al noted the low frequency of HER positivity in a smaller focus in.5% (four of ) and 6.% (four of 65) of cases,
AJCP / Original Article C D E F Image (cont) Case : focus (C), invasive lobular carcinoma with positive ER and (+) equivocal HER IHC and focus (D), with negative ER and (+) positive HER IHC. Case 7: focus (E), with positive ER and (0) negative HER IHC and focus (F), with positive ER and (+) positive HER. (H&E, IHC 0; insets reduced to approximately 0% original size) respectively. In the study by Choi et al, all had different histology and were high grade, and three of four were of the same T stage. Bethune et al5 also studied HER difference in multifocal breast carcinoma; they observed that.6% (four of 46) of cases were positive in a smaller tumor focus, and these all had either higher grade or different histology. Overall, these findings support CAP recommendations to perform HER testing on smaller foci if there is different histology or grade. They also support the recommendation by Bethune et al5 for testing of foci of similar size. In our series, half of the cases with HER discordance also had (+) equivocal IHC in one or more focus. Although not an equal comparison since more foci were stained in these cases, this was far less than 0.6% (4 Downloaded from https://academic.oup.com/ajcp/article-abstract/44/6/95/7660 on 07 April 08 of 65) observed in the entire cohort. Also, in our series and those cited previously, the positive focus was almost always high grade. Thus, it could be argued that testing of smaller foci could be warranted for those with prior equivocal results or high-grade histology. Changes to the most recent ASCO/CAP HER guidelines include recommendations to repeat testing on resection material for unifocal tumors if the tumor is high grade and prior testing was negative.9 This is largely due to concerns for tumor heterogeneity. Although not specified, testing of grade foci in multifocal cancer could be inferred; doing so would also likely identify rare cases in which only a smaller focus is HER positive. Additional larger studies would be helpful to further investigate these observations. Am J Clin Pathol 05;44:95-959 957 DOI: 0.09/AJCPFWXP54OLILMU
East et al / ER, PR, and HER in Multifocal Cancer ER/PR (largest focus) HER (largest focus) Weak foci is generally not indicated foci is often indicated and reasonable if requested foci if different histology or grade, especially if lower grade or subtype more likely to be positive foci is generally not indicated foci is often indicated and reasonable if requested foci if different histology, higher grade, or similar size; consider testing if smaller focus is high grade Figure Proposed algorithm for estrogen receptor (ER), progesterone receptor (PR), and/or HER-/neu (HER) testing of tumor foci in multifocal ipsilateral invasive breast carcinoma. Conclusions Multifocal ipsilateral invasive breast carcinoma is increasingly being identified. There has been no change in recommendation for testing of smaller foci by accrediting agencies, but we have observed a significant increase in this practice at our institution. Despite increase in use, ours and other studies show high concordance in ER, PR, and HER status between foci. They also support current CAP guidelines recommending foci be tested if they are of different histology or grade; however, minor modifications previously discussed and outlined in Figure may further improve selection of cases. Investigation of other histopathologic features may also prove beneficial in selection of cases for testing of small tumor foci. Corresponding author: Julie M. Jorns, MD, Dept of Pathology, University of Michigan, 500 E Medical Center Dr, Rm G, Ann Arbor, MI 4809; jjorns@umich.edu. This article was presented in part at the United States and Canadian Academy of Pathology (USCAP) annual meeting; March -7, 05; Boston, MA. References. Edge S, Byrd D, Compton C, et al, eds. AJCC Cancer Staging Manual. 7th ed. New York, NY: Lippincott; 00.. Brommesson S, Jonsson G, Strand C, et al. Tiling array-cgh for the assessment of genomic similarities among synchronous unilateral and bilateral invasive breast cancer tumor pairs. BMC Clin Pathol. 008;8:6.. Choi Y, Kim EJ, Seol H, et al. The hormone receptor, human epidermal growth factor receptor, and molecular subtype status of individual tumor foci in multifocal/multicentric invasive ductal carcinoma of breast. Hum Pathol. 0;4:48-55. 4. Dawson PJ, Baekey PA, Clark RA. Mechanisms of multifocal breast cancer: an immunocytochemical study. Hum Pathol. 995;6:965-969. 5. Middleton LP, Vlastos G, Mirza NQ, et al. Multicentric mammary carcinoma: evidence of monoclonal proliferation. Cancer. 00;94:90-96. 6. Teixeira MR, Pandis N, Bardi G, et al. Discrimination between multicentric and multifocal breast carcinoma by cytogenetic investigation of macroscopically distinct ipsilateral lesions. Genes Chromosomes Cancer. 997;8:70-74. 7. Ghazani AA, Arneson N, Warren K, et al. Genomic alterations in sporadic synchronous primary breast cancer using array and metaphase comparative genomic hybridization. Neoplasia. 007;9:5-50. 8. Noguchi S, Aihara T, Koyama H, et al. Discrimination between multicentric and multifocal carcinomas of the breast through clonal analysis. Cancer. 994;74:87-877. 9. Wolters R, Wockel A, Janni W, et al. Comparing the outcome between multicentric and multifocal breast cancer: what is the impact on survival, and is there a role for guideline-adherent adjuvant therapy? a retrospective multicenter cohort study of 8,95 patients. Breast Cancer Res Treat. 0;4:579-590. 0. Houssami N, Ciatto S, Macaskill P, et al. Accuracy and surgical impact of magnetic resonance imaging in breast cancer staging: systematic review and meta-analysis in detection of multifocal and multicentric cancer. J Clin Oncol. 008;6:48-58.. Chua B, Ung O, Taylor R, et al. Frequency and predictors of axillary lymph node metastases in invasive breast cancer. ANZ J Surg. 00;7:7-78.. Chung AP, Huynh K, Kidner T, et al. Comparison of outcomes of breast conserving therapy in multifocal and unifocal invasive breast cancer. J Am Coll Surg. 0;5:7-47.. Cabioglu N, Ozmen V, Kaya H, et al. Increased lymph node positivity in multifocal and multicentric breast cancer. J Am Coll Surg. 009;08:67-74. 4. Katz A, Strom EA, Buchholz TA, et al. The influence of pathologic tumor characteristics on locoregional recurrence rates following mastectomy. Int J Radiat Oncol Biol Phys. 00;50:75-74. 5. Tot T, Pekar G. Multifocality in basal-like breast carcinomas and its influence on lymph node status. Ann Surg Oncol. 0;8:67-677. 6. Vlastos G, Rubio IT, Mirza NQ, et al. Impact of multicentricity on clinical outcome in patients with T-, N0-, M0 breast cancer. Ann Surg Oncol. 000;7:58-587. 7. Weissenbacher TM, Zschage M, Janni W, et al. Multicentric and multifocal versus unifocal breast cancer: is the tumornode-metastasis classification justified? Breast Cancer Res Treat. 00;:7-4. 958 Am J Clin Pathol 05;44:95-959 Downloaded from https://academic.oup.com/ajcp/article-abstract/44/6/95/7660 DOI: 0.09/AJCPFWXP54OLILMU on 07 April 08
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