| Abstract|| |
Background: The characterization of hepatic metastases as having neuroendocrine origins is essential and the main markers currently used are chromogranin A (CgA) and synaptophysin (Syn). However, these markers may exhibit certain limitations, and the use of CD56 and CD57 can also be considered, although, due to low specificity, their use is discouraged. Aim: This study sought to compare the immunohistochemical expression of these markers in hepatic metastases of neuroendocrine neoplasms (NEN). Materials and Methods: Eighteen samples, were used for immunohistochemical staining with CgA, Syn, CD56, and CD57 antibodies. The immunostaining reactions were compared according to its intensity (I), the percentage of labeled cells (P), and a final score (I × P). Statistical agreement between the markers was also evaluated. Results: CD57 was expressed in the highest number of cases and also showed the most intense expression. CgA showed the highest number of cases with more than 80% positively stained area (72.2%), followed by CD57 (61.1%). The highest average score (I × P) was obtained for CD57 (9.1 ± 4.1). The best indices of agreement were between CgA and CD57 with respect to positivity (P = 0.021) and score (P = 0.014). According to the primary site, stomach/duodenum, lungs, and undetermined subgroups showed the highest average scores for CD57, followed by CgA. For the small bowel subgroup, the highest average score was obtained for CgA, followed by CD57. Conclusion: Our results highlight the importance of CD57 in the evaluation of hepatic metastases of NEN and indicate that this marker should be included with CgA and Syn in routine diagnostic panels.
Keywords: CD57 antigen, immunohistochemistry, liver, neoplasm metastasis, neuroendocrine tumors
|How to cite this article:|
Caroli-Bottino A, Mauricio AS, N. Pannain VL. CD57 as a routine neuroendocrine marker for liver metastasis. Indian J Pathol Microbiol 2020;63:38-43
|How to cite this URL:|
Caroli-Bottino A, Mauricio AS, N. Pannain VL. CD57 as a routine neuroendocrine marker for liver metastasis. Indian J Pathol Microbiol [serial online] 2020 [cited 2021 Jun 14];63:38-43. Available from: https://www.ijpmonline.org/text.asp?2020/63/1/38/277365
| Introduction|| |
Neuroendocrine neoplasms (NEN), although rare, have recently exhibited increased incidence in Western countries. This phenomenon can be partially attributed to advances in diagnosis that are mainly linked to the widespread use of endoscopic methods. Primary tumors are found most frequently in the gastrointestinal tract and lungs, whereas liver metastases represent the most common form of hematogenous spread.,, Approximately 60–75% of patients with NEN of the gastrointestinal tract present with hepatic metastases at the time of diagnosis.
The characterization of hepatic metastases as having neuroendocrine origins is essential. In new cases, such metastases are often identified before the primary tumor has been detected. In cases involving an established primary site, hepatic metastasis of NEN is associated with a worse prognosis.
NEN may exhibit different histological patterns (insular, trabecular, glandular, and mixed, among others) and different degrees of differentiation.,, Given this heterogeneity, an immunohistochemical examination that demonstrates the expression of neuroendocrine markers is recommended in all cases.,
The main markers currently used in the diagnosis of NEN in both the gastroenteropancreatic and pulmonary tracts are chromogranin A (CgA) and synaptophysin (Syn).,, In association, these two markers are sufficient to characterize the neuroendocrine phenotypes of most NEN and satisfy the standards proposed by certain authors, who have recommended positivity for at least 2 neuroendocrine markers to establish a diagnosis of NEN., However, these markers exhibit certain limitations such as the poor expression of CgA in poorly differentiated pulmonary tumors and certain well-differentiated tumors, particularly colorectal tumors;, in addition, Syn generally has relatively low specificity with immunoreactivity for other neoplasms such as carcinomas and adenomas of the adrenal cortex and pseudo-papillary solid tumors of the pancreas., Therefore, it is necessary to have other options for neuroendocrine markers.
For this purpose, CD56 (neural cell adhesion molecule (NCAM)) and CD57 (Leu-7) can also be considered. CD56 is a membrane glycoprotein involved in cell adhesion, normally found in neuronal and neuroendocrine cells, and also expressed in other normal (renal tubules and thyroid follicles) and neoplastic tissues (small cell lung carcinoma, renal tumors, ovarian cancer, and endometrial carcinoma)., CD57, also a membrane glycoprotein found in neural cells, is expressed in an even greater amount of tissues. It was initially used to characterize “natural killer” lymphocytes. Subsequently, it was found to be expressed also in neural tumors including schwannomas, neurofibromas, neuromas, and granular cell tumors, as well as renal adenomas.,, It may also be positive in pheochromocytomas and extra-adrenal paragangliomas, in addition to other neoplasms such as prostatic carcinomas, thymomas, metanephric adenomas, and in some small blue cell tumors., Thus, although very sensitive, CD56 and CD57 are considered having low specificity and their use is discouraged, particularly for assessments of NEN of the gastrointestinal tract.,,,,,,,,,,
Therefore, this study sought to compare the immunohistochemical expressions of CgA, Syn, CD56, and CD57 in hepatic metastases of NEN.
| Materials and Methods|| |
We assessed 18 hepatic metastases of NEN from patients who underwent wedge biopsy or segmentectomy. All tumors were analyzed via hematoxylin-eosin staining, and two representative 1.0 mm cylinders were obtained from paraffin blocks for the construction of a new tissue microarray (TMA) block with Pathology Devices TMArrayer™ (USA).
The primary sites were characterized as stomach/duodenum (five cases, all of them with digestive endoscopy and histopathology), small bowel (five cases, all of them with histopathology), pancreas (two cases, all of them with computed tomography and histopathology), and lungs (two cases with computed tomography, one of them also with histopathology). Four cases remained with undetermined primary tumors.
Immunohistochemical staining with CgA, Syn, CD56, CD57, and Ki67 antibodies was performed in tissue sections with a thickness of 4 μm from the TMA. Antibodies were purchased from Cell Marque™: monoclonal anti-CgA (1:200 dilution, 238-96, clone LK2H10); polyclonal anti-Syn (1:100 dilution, clone 336A-74); monoclonal anti-CD56 (1:100 dilution, 156M-84, clone 123C3.D5); monoclonal anti-CD57 (1:300 dilution, 157M-94, clone NK-1); and DAKO: monoclonal anti-Ki67 (1:200 dilution, M7240, clone MIB-1). The Novolink Max Polymer Detection System (Leica Biosystems, product no. RE7280-K) was used.
Scoring of immunostaining
Immunostaining reactions with the CgA, Syn, CD56, and CD57 antibodies were assessed using a semiquantitative “quick score” system for both staining intensity and the proportion of cytoplasmic cell staining. Intensity scores ranged from 0 to 3 (0 = negative; 1 = light; 2 = moderate; 3 = strong) and proportion scores ranged from 1 to 4 (0 = negative; 1 = 1–10%; 2 = 11–50%; 3 = 51–80%; 4 = 81–100%). These two scores were then multiplied to obtain a final score that ranged from 0 to 12. Cases that showed positivity for at least one of the tested neuroendocrine markers were included.
In cases involving a tumor of gastroenteropancreatic origin, Ki67 evaluation was performed in accordance with the 2010 WHO guidelines, with the number of counted nuclei ranging from 860 to 2565 per case.
Statistical analysis was performed using SAS® System 6.11 software (SAS Institute, Inc., Cary, North Carolina). For categorical data, the results of the descriptive analysis were expressed using frequencies and percentages. For numerical data, the results of the descriptive analysis were expressed using means and standard deviations. Agreement between markers was analyzed with respect to both positivity (using kappa (κ)) and staining score (using the intraclass correlation coefficient (ICC)). For both types of agreement, values were interpreted as follows: poor (<0.00); slight (0.00–0.20); fair (0.21–0.40); moderate (0.41–0.60); substantial (0.61–0.80); and almost perfect (0.81–1.00). Differences with P < 0.05 were regarded as significant.
This study was approved by our institution's Institutional Ethical Committee (record 49922315.3.0000.5257).
| Results|| |
Across the entire sample, CD57 was expressed in the highest number of cases (17/18—94.5%), followed by CgA (15/18—83.4%), Syn (9/18—50%), and CD56 (8/17—47.1%). CD57 also showed the most intense expression, with moderate/strong intensity in all cases, followed by CgA (13/15—86.6%), Syn (5/9—55.5%), and CD56 (4/8—50.0%) [Table 1]. With respect to the positively stained area, CgA showed the highest number of cases with more than 80% staining (72.2%), followed by CD57 (61.1%), Syn (27.8%), and CD56 (17.6%) [Table 1]. In accordance with these results, the highest average scores were obtained for CD57 (9.1 ± 4.1) [Figure 1] and CgA (7.5 ± 4.6) [Figure 2]. The average scores for Syn and CD56 were 2.9 ± 3.8 and 2.8 ± 4.2, respectively.
|Table 1: The intensity of marker expression and percentage of positive staining areas for chromogranin A (CgA), synaptophysin (Syn), CD56, and CD57 in liver metastases from neuroendocrine neoplasms|
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|Figure 1: Expression of CD57 in a liver metastasis of intestinal neuroendocrine tumor (200x)|
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|Figure 2: Expression of chromogranin A in the same case of liver metastasis shown in Figure 1 (200x)|
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The best indices of agreement between markers with respect to positivity were between CgA and CD57 (κ = 0.455, moderate agreement) and between Syn and CD56 (κ = 0.414, moderate agreement); however, only the former result was statistically significant (P = 0.021) [Table 2]. Moderate agreement with respect to score was also observed between CgA and CD57 (ICC = 0.493) and between Syn and CD56 (ICC = 0.526); both of these results were statistically significant (P = 0.014 and P = 0.011, respectively) [Table 2].
|Table 2: Concordance between positive immunohistochemical staining (κ) and the scores (ICC) in the whole sample|
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The number of cases with positive staining for each of the examined markers according to the primary site had been determined. For metastases with a known primary site, positive staining was most common for CD57 (observed in all cases), followed by CgA (12/14—85.71%), Syn (8/14—57.14%), and CD56 (6/11—54.54%). Among metastases with an indeterminate primary site, CD57 and CgA exhibited positive staining in most cases (3/4—75%).
An evaluation of marker expression in liver metastases according to the primary site revealed that in the stomach/duodenum, lungs, and undetermined subgroups, the highest average scores were obtained for CD57 (8.6 ± 3.6, 8.0 ± 5.7, and 9.0 ± 6.0, respectively), followed by CgA (5.4 ± 3.7, 2.0 ± 2.8, and 6.0 ± 5.16, respectively). The opposite result was observed for the small bowel subgroup, for which the highest average score was obtained for CgA (12.0 ± 0.0), followed by CD57 (10.2 ± 4.0). Only for the pancreas subgroup CD56 exhibited the highest average score (10.0 ± 2.8) with the same score obtained for CgA [Table 3].
|Table 3: Scores from CgA, Syn, CD56, and CD57 concerning the primary sites of the metastatic neuroendocrine neoplasms|
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Among NEN of the gastroenteropancreatic tract, seven NEN were classified as G2 neuroendocrine tumors (NET) (four in the stomach/duodenum, two in the small bowel and one in the pancreas), four NEN were classified as G1 NET (one in the stomach/duodenum, two in the small bowel and one in the pancreas), and one NEN with a primary site in the small intestine was classified as G3 neuroendocrine carcinoma (NEC). The two tumors with a primary pulmonary site were classified according to 2015 WHO guidelines as typical carcinoids. The metastases from undetermined primary sites were not classified.
| Discussion|| |
Few studies have evaluated neuroendocrine markers in hepatic metastases. In 2002, Van Eeden et al. found expression of CgA, Syn, CD56, and CD57 in 90.0%, 83.67%, 54.7%, and 87.7% of cases, respectively. However, in that study, metastases were evaluated in conjunction with primary tumors.
Recently, in an evaluation of hepatic metastases of gastroenteropancreatic NEN, Shen et al. found that the most frequently expressed marker was CgA, followed by Syn and CD56. In our series, for metastases with a similar origin and for all examined metastases, we obtained similar immunohistochemical results.
We did not find any previous study that evaluated the expression of CD57 in metastases of NEN; in our series, this marker was superior to CgA with respect to the percentage of cases with positive staining.
In our knowledge, this study involved the first NEN-related used method for the semi-quantification of marker expression via the determination of a score that accounts for staining intensity and percentage of the overall area with positive staining. For this purpose, the use of a TMA to perform immunohistochemical reactions was fundamental to ensure that all samples were subjected to the same conditions, thereby minimizing technical variations and subjectivity in interpretations that could otherwise have influenced analyses of staining reactions, particularly with respect to intensity.
Overall, for the studied cases, we obtained the highest mean score for CD57. Although CgA exhibited more than 80% staining in a greater number of cases, CD57 demonstrated strong staining intensity in most cases, resulting in a higher score. In fact, there was a statistically significant agreement between CgA expression and CD57 expression with respect to both percentages of cases with positive staining and score.
In 2003, Jirásek et al. utilized laser confocal microscopy to assess NEN of the gastroenteropancreatic tract and demonstrated the coexistence of cytoplasmic granules positive for CgA and CD57 in most neoplastic cells of tumors that they referred to as typical, which likely corresponded to well-differentiated tumors. In our series, almost all tumors with a known primary site were well differentiated based on current WHO classifications.,, Therefore, both strong staining intensity and a high percentage of positive cells should reflect the large quantity of these granules in tumor cells. Conversely, consistent with the observations reported by Jirasek et al. in 2003, the presence of these granules should be associated with the coexpression of these markers in most cases.
For the examined cases, better differentiation could also be correlated with high expression of CgA, which is dependent on the number of neurosecretory granules present in neoplastic cells and is therefore reduced when tumors dedifferentiate.
In contrast, we found more cases with negative staining and lower scores for Syn and CD56, which exhibited moderate agreement with respect to both positivity and score; for staining score, the agreement between these markers was statistically significant.
Syn staining is recognized as being well-preserved in poorly differentiated NEN due to the persistent presence of small synaptic vesicles in any neuroendocrine cell, regardless of differentiation status., In a 2012 study of liver metastases of gastroenteropancreatic NEN, Shen et al. found positive staining for Syn in 78.9% of cases, a finding that contrasted with our results. However, this percentage was obtained in analyses of both NET and NEC. When NEC was excluded, only 42.1% of tumors expressed Syn; these results corroborated the importance of this marker for less differentiated neoplasms.
We can attribute the absence of Syn expression in half of the examined cases to the good differentiation of tumors in our series, which included 11 G1 or G2 NET of the gastroenteropancreatic tract, 2 typical carcinoids of pulmonary origin,,, and only 1 NEC G3, which had a primary site in the small bowel.
CD56 is regarded as a good marker for the NET of the lung., Previous studies have demonstrated varying CD56 expression in typical and atypical carcinoids, with positive staining percentages of up to 90%. However, the importance of CD56 in the characterization of small cell carcinomas is well established; in this context, CD56 shows marked cytoplasmic expression.,, In this study, the lack of expression of CD56 in metastases with a pulmonary primary site can likely be attributed to the small number of such cases that were examined, both of which involved typical carcinoids.
In contrast, CD56 expression was mainly observed in metastases of NEN of the small bowel and pancreas, particularly the latter. Insufficient data are available in the literature for the analysis of these results due to the limited number of cases involving such tumors and certain authors' recommendations to avoid using CD56 in the gastroenteropancreatic tract.
The stratification of our series according to the primary site resulted in subgroups with few cases. Whenever possible, these subgroups were combined; for instance, metastases with primary sites in the stomach and duodenum, which are part of the so-called “foregut,” were categorized into a single subgroup. It was not possible to obtain more specific information regarding the locations of primary sites in the small bowel. In certain cases, it was impossible to identify the primary site using the methodology applied in this study. Although immunohistochemistry could help indicate primary sites in these cases, we chose to utilize only one criterion for this definition favoring the imaging methods.
The aim of this stratification was to determine whether any markers exhibited staining patterns for metastatic NEN that were related to a specific primary site. CgA and CD57 were the only markers present in hepatic metastases from all primary sites, including metastases from indeterminate primary sites, and exhibited the highest mean scores for metastases from all sites.
Our results highlight the importance of CD57 in the evaluation of hepatic metastases of NEN and indicate that this marker should be included with CgA and Syn in routine diagnostic panels.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Williams GT. Endocrine tumours of the gastrointestinal tract-selected topics. Histopathology 2007;50:30-41.
Hamilton SR, Aaltonen LA, editors. WHO Classification of Tumours of the Digestive System. Lyon: International Agency for Research on Cancer (IARC); 2000.
Niederle MB, Hackl M, Kaserer K, Niederle B. Gastroenteropancreatic neuroendocrine tumours: The current incidence and staging based on the WHO and European Neuroendocrine Tumour Society classification: An analysis based on prospectively collected parameters. Endocr Relat Cancer 2010;17:909-18.
Riihimaki M, Hemminki A, Sundquist K, Sundquist J, Hemminki K. The epidemiology of metastases in neuroendocrine tumors. Int J Cancer 2016;139:2679-86.
Steinmuller T, Kianmanesh R, Falconi M, Scarpa A, Taal B, Kwekkeboom DJ, et al
. Consensus guidelines for the management of patients with liver metastases from digestive (neuro) endocrine tumors: Foregut, midgut, hindgut, and unknown primary. Neuroendocrinology 2008;87:47-62.
Kloppel G. Classification and pathology of gastroenteropancreatic neuroendocrine neoplasms. Endocr Relat Cancer 2011;18(Suppl 1):1-16.
Rindi G, Kloppel G, Couvelard A, Komminoth P, Korner M, Lopes JM, et al
. TNM staging of midgut and hindgut (neuro) endocrine tumors: A consensus proposal including a grading system. Virchows Arch 2007;451:757-62.
Chetty R. An overview of practical issues in the diagnosis of gastroenteropancreatic neuroendocrine pathology. Arch Pathol Lab Med 2008;132:1285-9.
Capelli P, Fassan M, Scarpa A. Pathology - grading and staging of GEP-NETs. Best Pract Res Clin Gastroenterol 2012;26:705-17.
Rindi G, Wiedenmann B. Neuroendocrine neoplasms of the gut and pancreas: New insights. Nat Rev Endocrinol 2011;8:54-64.
Rindi G, Petrone G, Inzani F. 25 Years of neuroendocrine neoplasms of the gastrointestinal tract. Endocr Pathol 2014;25:59-64.
Rekhtman N. Neuroendocrine tumors of the lung: An update. Arch Pathol Lab Med 2010;134:1628-38.
Plockinger U, Rindi G, Arnold R, Eriksson B, Krenning EP, de Herder WW, et al
. Guidelines for the diagnosis and treatment of neuroendocrine gastrointestinal tumours. A consensus statement on behalf of the European Neuroendocrine Tumour Society (ENETS). Neuroendocrinology 2004;80:394-424.
Rindi G, Bordi C, La Rosa S, Solcia E, Delle Fave G. Gastroenteropancreatic (neuro)endocrine neoplasms: The histology report. Dig Liver Dis 2011;43(Suppl 4):356-60.
Lloyd RV. Practical markers used in the diagnosis of neuroendocrine tumors. Endocr Pathol 2003;14:293-301.
Ohara Y, Oda T, Hashimoto S, Akashi Y, Miyamoto R, Enomoto T, et al
. Pancreatic neuroendocrine tumor and solid-pseudopapillary neoplasm: Key immunohistochemical profiles for differential diagnosis. World J Gastroenterol 2016;22:8596-604.
Kloppel G, Anlauf M. Epidemiology, tumour biology and histopathological classification of neuroendocrine tumours of the gastrointestinal tract. Best Pract Res Clin Gastroenterol 2005;19:507-17.
Kontogianni K, Nicholson AG, Butcher D, Sheppard MN. CD56: A useful tool for the diagnosis of small cell lung carcinomas on biopsies with extensive crush artefact. J Clin Pathol 2005;58:978-80.
Mechtersheimer G, Staudter M, Moller P. Expression of the natural killer cell-associated antigens CD56 and CD57 in human neural and striated muscle cells and in their tumors. Cancer Res 1991;51:1300-7.
Kamel OW, Gelb AB, Shibuya RB, Warnke RA. Leu 7 (CD57) reactivity distinguishes nodular lymphocyte predominance Hodgkin's disease from nodular sclerosing Hodgkin's disease, T-cell-rich B-cell lymphoma and follicular lymphoma. Am J Pathol 1993;142:541-6.
Shen SS, Truong LD, Scarpelli M, Lopez-Beltran A. Role of immunohistochemistry in diagnosing renal neoplasms: When is it really useful? Arch Pathol Lab Med 2012;136:410-7.
Kloppel G, Rindi G, Anlauf M, Perren A, Komminoth P. Site-specific biology and pathology of gastroenteropancreatic neuroendocrine tumors. Virchows Arch 2007;451(Suppl 1):9-27.
Kloppel G, Couvelard A, Perren A, Komminoth P, McNicol AM, Nilsson O, et al
. ENETS Consensus guidelines for the standards of care in neuroendocrine tumors: Towards a standardized approach to the diagnosis of gastroenteropancreatic neuroendocrine tumors and their prognostic stratification. Neuroendocrinology 2009;90:162-6.
Klimstra DS, Modlin IR, Adsay NV, Chetty R, Deshpande V, Gonen M, et al
. Pathology reporting of neuroendocrine tumors: Application of the Delphic consensus process to the development of a minimum pathology data set. Am J Surg Pathol 2010;34:300-13.
Lantuejoul S, Moro D, Michalides RJ, Brambilla C, Brambilla E. Neural cell adhesion molecules (NCAM) and NCAM-PSA expression in neuroendocrine lung tumors. Am J Surg Pathol 1998;22:1267-76.
Zahel T, Krysa S, Herpel E, Stenzinger A, Goeppert B, Schirmacher P, et al
. Phenotyping of pulmonary carcinoids and a Ki-67-based grading approach. Virchows Arch 2012;460:299-308.
DeLellis RA. The neuroendocrine system and its tumors: An overview. Am J Clin Pathol 2001;115 Suppl 1:5-16.
Han CP, Kok LF, Lee MY, Wu TS, Ruan A, Cheng YW, et al
. Five commonly used markers (p53, TTF1, CK7, CK20, and CK34betaE12) are of no use in distinguishing between primary endocervical and endometrial adenocarcinomas in a tissue microarray extension study. Arch Gynecol Obstet 2010;281:317-23.
Bosman F, Carneiro F, Hruban RH, Theise ND, editors. WHO Classification of Tumours of the Digestive System. Lyon: International Agency for Research on Cancer (IARC); 2010.
Landis JR, Koch GG. The measurement of observer agreement for categorical data. Biometrics 1977;33:159-74.
Travis WD, Brambilla E, Nicholson AG, Yatabe Y, Austin JHM, Beasley MB, et al
. The 2015 World Health Organization Classification of Lung Tumors: Impact of genetic, clinical and radiologic advances since the 2004 classification. J Thorac Oncol 2015;10:1243-60.
Van Eeden S, Quaedvlieg PF, Taal BG, Offerhaus GJ, Lamers CB, Van Velthuysen ML. Classification of low-grade neuroendocrine tumors of midgut and unknown origin. Hum Pathol 2002;33:1126-32.
Shen YH, Chen S, Zhang WT, Ji Y, Yu L, Sun HC, et al
. Clinical analysis of gastroenteropancreatic neuroendocrine tumor with liver metastasis, compared with primary hepatic neuroendocrine tumor. J Cancer Res Ther 2014;10(Suppl 1):276-80.
Jirasek T, Hozak P, Mandys V. Different patterns of chromogranin A and Leu-7 (CD57) expression in gastrointestinal carcinoids: Immunohistochemical and confocal laser scanning microscopy study. Neoplasma 2003;50:1-7.
Hirabayashi K, Zamboni G, Nishi T, Tanaka A, Kajiwara H, Nakamura N. Histopathology of gastrointestinal neuroendocrine neoplasms. Front Oncol 2013;3:2.
Williams ED, Sandler M. The classification of carcinoid tumours. Lancet 1963;1:238-9.
Department of Pathology, Clementino Fraga Filho Universitary Hospital, R. Prof. Rodolpho Paulo Rocco 255, Ilha do Fundão, Rio de Janeiro, RJ, Zip Code: 21941-590
Source of Support: None, Conflict of Interest: None
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[Table 1], [Table 2], [Table 3]