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Year : 2011  |  Volume : 54  |  Issue : 3  |  Page : 532-538
Assessment of HER2/Neu status by fluorescence in situ hybridization in immunohistochemistry-equivocal cases of invasive ductal carcinoma and aberrant signal patterns: A study at a tertiary cancer center

1 Department of Laboratory Medicine (Pathology and Molecular Diagnostics), Basavatarakam Indo-American Cancer Hospital and Research Institute, Hyderabad, India
2 Department of Medical Oncology, Basavatarakam Indo-American Cancer Hospital and Research Institute, Hyderabad, India

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Date of Web Publication20-Sep-2011


Introduction: HER-2/neu status determines the eligibility for targeted therapy with trastuzumab in breast carcinoma. Evaluation for HER-2/neu protein expression by immunohistochemistry (IHC) and gene amplification by fluorescence in situ hybridization (FISH) has become the gold standard. Aims: Since data on HER-2/neu assessment by IHC and FISH and studies regarding concordance between the results of the two techniques are limited, especially from India, we sought to study HER-2 gene amplification status by FISH in equivocal (2+) cases by IHC and also study aberrant signal patterns. Settings and Design: Mastectomies and breast core biopsies, equivocal for HER-2/neu protein expression, were analyzed for HER-2 amplification by FISH. Materials and Methods: IHC (DAKO) and FISH (PathVysion dual-probe system) tests were performed on 68 of 112 (after exclusion) 10% neutral buffered formalin (NBF)-fixed paraffin-embedded tissues and evaluated according to American Society of Clinical Oncology ASCO guidelines. Statistical Analysis Used: Chi-square (χ2 ) test and the two-tailed P value were applied using Graphpad Quickcels software, version 2006. Results: It was found that 73.5% of the IHC 2+ patients were negative for HER-2/neu amplification, 25% were positive (ratios ranging from 2.3 to 5.6) and 1 patient was equivocal (2.2). Retesting FISH HER-2 equivocal case on another tumor block by IHC demonstrated HER-2 overexpression of protein 3+, thus resolving the equivocal status. Polysomy and HER-2 genetic heterogeneity were seen frequently. Conclusions: The findings reiterate that IHC HER-2 equivocal cases are a heterogenous group and need FISH for further categorization. Low concurrence (25%) rate between both IHC and FISH results in the equivocal scenario can be attributed to tumors with polysomy 17 and HER-2/neu genetic heterogeneity.

Keywords: Breast cancer, FISH, HER-2/neu, invasive ductal carcinomas, unusual signal patterns

How to cite this article:
Murthy SS, Sandhya D G, Ahmed F, Goud K I, Dayal M, Suseela K, Rajappa SJ. Assessment of HER2/Neu status by fluorescence in situ hybridization in immunohistochemistry-equivocal cases of invasive ductal carcinoma and aberrant signal patterns: A study at a tertiary cancer center. Indian J Pathol Microbiol 2011;54:532-8

How to cite this URL:
Murthy SS, Sandhya D G, Ahmed F, Goud K I, Dayal M, Suseela K, Rajappa SJ. Assessment of HER2/Neu status by fluorescence in situ hybridization in immunohistochemistry-equivocal cases of invasive ductal carcinoma and aberrant signal patterns: A study at a tertiary cancer center. Indian J Pathol Microbiol [serial online] 2011 [cited 2020 Nov 30];54:532-8. Available from: https://www.ijpmonline.org/text.asp?2011/54/3/532/85087

   Introduction Top

HER-2/neu amplification is the key to eligibility for trastuzumab therapy, prognosis and predictive response in breast carcinomas. [1],[2],[3],[4],[5],[6],[7],[8],[9] American Society of Clinical Oncology/College of American pathologists (ASCO/CAP) guidelines recommend HER-2/neu testing in invasive component of breast tumor primarily by immunohistochemistry (IHC) and when equivocal (2+) or borderline, the same needs to be assessed by fluorescence in situ hybridization (FISH). [10],[11],[12],[13],[14],[15] A high rate of discordance between both assays has been reported, with 40% of the patients positive for protein expression by IHC would be FISH negative, while 16% FISH-positive patients would be IHC negative. [16] The role of HER-2 genetic heterogeneity and polysomy 17, copy number anomaly, in contributing to the discordance is controversial. To the best of our knowledge, data on HER-2 status by FISH from India is limited. Hence HER-2 gene status in all IHC-equivocal cases by FISH, as well as concordance rates between the results of the two assays, was determined, and interpretation of copy number anomalies is discussed herein.

   Materials and Methods Top

A total of 112 specimens of mastectomies and breast core biopsies, equivocal for HER-2/neu protein expression, referred during the period August 2008 to December 2009, were assessed for HER-2 amplification by FISH. Excluding all duct carcinoma in situ (DCIS) cases, review of slides and blocks from other referral centers (with debatable pre-analytic issues like fixation of tissues), post-chemotherapy cases and the cases with incomplete information about the patient, only 60.71% (68/112) of the patients could be included in the study. Further, a patient rendered equivocal for protein expression and gene amplification was reassessed by IHC on another paraffin block with different area of tumor. Although as a routine, IHC HER-2, 3+/1+ patients are not evaluated for gene amplification by FISH owing to the test being expensive, 7 IHC HER-2/neu 3+ cases and one 1+ case were evaluated for comparison. Approval of the institutional ethical committee was obtained, and patients were included in the study after obtaining their informed consent. Clinical and pathological information was collected. The tumors were graded according to the modified Scarff-Bloom-Richardson histological grading. [17] IHC and FISH assays were conducted blinded from clinical information.

Immunohistochemical Staining for HER-2/neu Protein Expression

Sections with thickness of 3-4 m each were obtained from 10% neutral buffer formalin-fixed, paraffin-embedded tumor tissues. The tissue sections were deparaffinized in xylene and dehydrated using alcohol. After rehydration with water, retrieval of antigen was achieved by micro-waving the sections in citrate buffer (pH, 6.0) at 100°C for 3 cycles (each cycle, 5 minutes) for HER-2/neu immunostaining. After blocking the endogenous peroxidase activity with 0.3% hydrogen peroxide for 5 to 10 minutes, the sections were stained for HER-2/neu using polyclonal anti-rabbit cerb-B2 antibody according to instructions of the manufacturer and using reagents included in cerb-B2 detection kit (DAKO). The excess antibody was washed away with TRIS wash buffer (pH, 7.5-7.6). The tissue was incubated with EnVision TM Peroxidase/DAB, Rabbit/Mouse detection system (DAKO) for half an hour. Excess antibody was washed away with TRIS wash buffer again. Peroxidase was demonstrated with 0.5% diamino-benzidine for 5 minutes, and the sections were counterstained with Gills hematoxylin. Positive and negative controls were performed at the same time for each section. Positive HER-2/neu staining was identified in the form of membrane staining. Scoring for HER-2 was based on the percentage of cells stained, as well as intensity of membrane staining. HER-2/neu membrane staining in tumor cells was scored from 0 to 3 according to ASCO guidelines. [18] The biopsy sections were scored 0, 1+, 2+ and 3+. Score of 0 and 1+ was considered negative for HER-2 protein expression. while 2+ score, (equivocal/borderline) was defined as complete membrane staining that is nonuniform or weak in less than 30% of tumor cells. A 3+ score indicated protein expression.

FISH Assay

FISH allows rapid visual and accurate cell-by-cell assessment of the level of gene amplification with information about distribution of gene copies in each cell of the histologic section. Briefly 4-micron paraffin-embedded sections were deparaffinized using xylene and dehydrated in absolute ethanol for 45 minutes. The slides were pretreated chemically and enzymatically to remove proteins that blocked DNA access. Chaotropic salt treatment was given with 1 N sodium thio-cyanate for 30 minutes at 80°C. The two probes - LSI HER-2 probe directly labeled in spectrum orange and Centromere enumerator probe (CEP) 17 labeled in spectrum green - provided in the PathVysion HER-2/neu DNA probe kit from Vysis, Inc., Downers Grove, IL, were denatured and then cohybridized with the tissue DNA. Post-hybridization washes were performed with 2XSSC/0.3% NP40 at 37°C, and slides were counterstained with DAPI. The number of hybridized orange and green signals per nucleus were scored in 50 morphologically intact and non-overlapping nuclei using a fluorescence microscope (Olympus BX41) equipped with appropriate filter set. Criteria for successful FISH analysis included identification of single-copy HER-2/neu gene status in normal cells within the specimen, uniform hybridization throughout the section, and limited DNA loss. The cutoffs for the variables were defined by objective biologically relevant thresholds. Discrimination of true HER-2/neu amplification required correction for chromosome 17 polysomy. The FISH-defined genetic variables include HER-2/neu gene copy number (signals per cell), average chromosome 17 copy number (signals per cell) and the average HER-2/neu gene-to-chromosome 17 signal ratio. The ratio was derived by the calculation of the mean of HER-2/neu amplifications relative to chromosome 17 signals. Interpretation of HER-2/neu gene amplification was based on the guidelines set by American Society of Clinical Oncology/CAP. [19] A HER-2-CEP17 ratio less than 1.8 indicated a non-amplified HER-2 gene status; a ratio in the range of 1.8-2.2 was "equivocal"; and if the ratio was greater than 2.2, then HER-2 gene status was "amplified". HER-2 genetic heterogeneity (GH) is also known to occur and influence the FISH result. If there are more than 5% or less than 50% of infiltrating tumor cells with a ratio higher than 2.2, HER-2 GH exists. If more than 50% of infiltrating tumor cells have a ratio more than 2.2, then the specimen is considered to be amplified. [20] Copy number anomalies such as polysomy 17, co-amplification of HER-2 and CEP17 signals influence the interpretation of the result. Polysomy is defined as >3 copies of chromosome 17 signals per cell. Disomy 17 is considered if copy number is in the range of 1.76-2.25 signals per cell. The range took into account that even a given tumor specimen predominantly disomy 17 could still show some deviation from average of 2 signals per cell, because of tumor's genetic instability, high mitotic index, and potential nuclear truncation through tissue sectioning. Based on the range of mean CEP17 copy number/cell, the following were defined: Hypodisomy, ≤1.75 signals per cell; polysomy, ≥2.26 signals per cell; low polysomy, 2.26-3.75 signals per cell; and high polysomy, ≥3.76 signals per cell. [21]

Statistical Analysis

To determine the association between two variables, χ2 test was applied. The two-tailed P value to test for significance between variables was calculated using Graphpad Quickcels software, version 2006.

   Results Top

Analysis of the 68 cases of invasive ductal carcinomas indicated that patients were in the age group of 22-80 years with a median of 52 years, with 70.5% of women over the age of 45 years. Grade II tumors were seen predominantly in 64.7% of the patients; and grade III, in 23.5%. Stage IIA disease was more frequent (57.3%), while stage IIB disease was less common (30.8%). Overall, hormone positivity was seen in about 61% of the patients. "ER+ PR+" status was seen in 52.9% of the patients, while "ER- PR-" status was seen in 39.7%. Lymph nodes were not involved in 61.7% of the patients. Evaluation of IHC-equivocal cases reflexed for HER-2 FISH analysis indicated that 50 out of 68 (i.e., 73.5%) 2+ patients were non-amplified for HER-2/neu gene with HER-2/CEP17 ratios ranging from 0.68 to 1.68 [Figure 1],[Figure 2],[Figure 3],[Figure 4]. FISH could not resolve the dilemma in 1 (1.5%) patient as the results were equivocal by FISH too. Retesting the same IHC and FISH HER-2/neu equivocal case on another tumor block by IHC demonstrated overexpression of HER-2 protein with a score of 3+ [Table 1].
Table 1: Data on clinical, histological, pathological and hormonal receptor status in infiltrating ductal carcinomas (n = 68) - number (%)

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Figure 1: HER-2/neu 1+ (negative for HER-2 protein expression by immunohistochemistry), (x400 DAB)

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Figure 2: HER-2/neu 3+ (positive for HER-2 protein expression by immunohistochemistry), (x400 DAB)

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Figure 3: HER-2/neu gene non-amplification and polysomy 17 (FISH)

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Figure 4: Positive for HER-2/neu amplification by FISH

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Correlation of the frequency of pathological features such as grade, stage of the disease, tumor size, nodal status, hormone receptor status with HER-2/neu status was performed to see the possible association of these features in the IHC 2+/equivocal scenario. An increase in frequency of cases with HER-2/neu gene amplification was found to be associated only with increase in tumor size and nodal involvement, though not statistically significant. Chi-square analysis indicated significant difference between stage I and IIA diseases ( P = 0.045). Correlation between hormone receptor positivity and negativity showed the association bordering on significance ( P = 0.090) [Table 2].
Table 2: Correlation of HER-2/neu status with grade/size/lymph node status of tumors in IHC HER-2/neu equivocal cases

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Studies on concordance between the results of the IHC and FISH assays in 3+ and 1+ categories of protein expression showed strong concordance, with 6 out of 7 cases having HER-2 gene amplification. However, 1 case of the 3+ overexpressors was non-amplified, and 73.5% of the weak overexpressors (2+) were negative for HER-2 gene amplification. Similarly a single case with IHC 1+ negative for HER-2 protein expression was also negative for gene amplification [Table 3].
Table 3: Concordance studies on HER-2 status between IHC and FISH assays (n = 76)

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Data was also evaluated for the presence of polysomy 17 and HER-2 genetic heterogeneity, and the results are presented in [Table 4]. It was found that 78.3% of the specimens were positive for polysomy 17, and 63.8% of the specimens were non-amplified. Polysomy-negative cases were non-amplified, with low polysomy in 69.2% of cases.
Table 4: Data on polysomy 17, HER-2 genetic heterogeneity and HER-2 gene amplification from 60 IDC cases

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Two cases presented with an aberrant signal pattern consisting of cells with clusters of CEP17 green signals overlaying the red HER-2 signals representing co-amplification of both the genes [Figure 5]. The HER-2 to CEP17 ratio, HER-2 copy number and CEP17 copy number for the first specimens were 1.07, 6.96 and 6.46, respectively; while for the second specimen, the corresponding observations were 1.12, 10.36 and 9.2.
Figure 5: Co-amplification of HER-2/neu gene and CEP17 signals

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   Discussion Top

The biological mechanisms of breast carcinogenesis are not fully understood.

Healthy breast cells show low protein expression and contain just 2 copies of the HER-2 gene per cell. Evidences indicate that HER-2/neu gene is overexpressed and/or amplified in 25% to 30% of invasive carcinomas. [1],[13] However an Indian study revealed HER2 neu overexpression in 46.37%; [7] In approximately 90% to 95% of these cases, overexpression is the direct result of gene amplification. [13] The occurrence of FISH equivocal cases with a ratio between 1.8 and 2.2 is extremely rare. In the current study, the observations of amplification of HER-2/neu in 25% of IHC 2+ patients is in stark contrast to the findings of 66.6% of HER-2/neu amplification in IHC 2+ cases by another group in India. [22] It could possibly be due to the different antibodies available in the market for IHC HER-2 evaluation. Therefore, nationwide multicentric studies with a standard protocol should be performed for analysis to arrive at a plausible conclusion. The 2+ IHC cases may be a heterogeneous group. Further, retesting a FISH-equivocal case by IHC on another tumor block as per the recommendations of ASCO guidelines helped in re-categorizing to 3+ status with the patient being eligible for trastuzumab treatment. These findings parallel the previous reports of existence of intratumoral and intercellular HER-2 heterogeneity in breast carcinomas. [23],[24],[25]

Current management of patients of carcinoma of the breast depends on the pathology and status of prognostic markers ER, PR, HER-2/neu, etc. Tumors with HER-2/neu amplification or overexpression are known to be aggressive, often higher grade, with a high proliferation/progression rate and a propensity for lymph node involvement. Between the pathological subgroups, the percentages of equivocal or 2+ tumors turning out to be FISH positive should not be different. Correlation studies on association of grade, stage, tumor size, nodal and hormone receptor status with HER-2/neu amplification status confirmed the same. Another study showed a statistically inverse relationship between hormone receptor status and HER-2 gene status.[22]

Several studies described concordance of as high as 79% to 100% in 3+ group [14],[15] ; and 12% to 36% for 2+ cases [10] between IHC and FISH results. An Indian group described that IHC 3+ (93.9%) and IHC-negative (85.9%) cases showed good concordance with corresponding FISH results. [22] In the current study, there was good concordance in 6 out of 7 IHC 3+ cases having HER-2 gene amplification. One IHC 1+ case was negative by FISH. HER-2/neu overexpression without concurrent HER-2/neu gene amplification was described in 1% to 20% of the breast tumors, and a small undetermined percentage of breast tumors demonstrated amplification of the gene without overexpression. [10],[26] The observations of low concurrence of HER-2 amplification (25%) with protein expression indicate weak association in equivocal cases. This emphasizes the fact that dual-colored FISH is the gold standard for the accurate measurement of HER-2/neu gene amplification in IHC-equivocal breast carcinomas. [19] In practice, discordant results between the two methodologies have been reported. Protein expression for HER-2/neu both in 3+ and 2+ cases has been attributed to variations in HER-2 testing due to the use of different antibodies for immunohistochemical studies; different scoring systems; adverse effects of variable fixation, which might have a detrimental effect on IHC; polysomy of chromosome 17; [26],[27] and post-transcriptional regulation leading to increased surface receptor expression. [11],[15] However, larger number of cases need to be assessed to arrive at a plausible conclusion.

Typically, HER-2 amplification occurs in the form of homogeneously staining regions (HSRs) visible in metaphase chromosomes of tumor cells and presenting as clusters of signals in interphase nuclei after in situ hybridization. These clusters may be fragmented through further rearrangements. A difficulty in the assessment of the status of the HER-2 gene is that its copy number increase may be related not to an amplification but to one of the copy number anomalies, polysomy of chromosome 17, which presents with unusual signal pattern. [5],[16],[28] Interpretation of such cases requires a deviation from considering HER-2-CEP17 ratio (the standard rule) in order not to report an inadequate HER-2 amplification status. Evidences from literature indicate that the interpretation of unusual signal patterns should include assessment of raw data on mean copy number of signals per nuclei of both HER-2/neu and CEP17 for determining polysomy and genetic heterogeneity. [29]

Polysomy 17 refers to a cell with ≥3 copies of the chromosome 17 centromere, with parallel increase of HER-2 and CEP17 copies with random distribution of both signals within the nucleus of a tumor cell. The HER-2-CEP17 ratio may be equal to or slightly greater than 1 and leads to the wrong interpretation that the tumor is negative for amplification. If the copy number of HER 2 is greater than 6 copies per cell, then interpretation is that it is a true amplification. This allows the distinction between a polysomy 17 and true HER-2 amplification. IHC 2+ expression has been shown to be relatively common in tumors with polysomy 17 but no HER-2 amplification. Further, polysomy 17 tumors do not overexpress the HER-2 mRNA or the protein at the high level required for efficacy of the trastuzumab therapy, and consequently polysomy tumors without HER-2 amplification are considered as functionally HER-2-negative tumors. [27] Hence it is important to differentiate tumors with true HER-2 amplification from those having a copy number increase of the gene simply related to polysomy 17. Recent studies have described polysomy 17 incidence ranging from 10% to 50%, depending on which tumors are assessed and which criteria are applied. [6],[21] Polysomy 17 was commonly seen in 78% of the cases. Thirty (63.8%) out of 47 specimens were negative for HER-2 gene amplification with low polysomy mean copy number of CEP17, with <3.75 signals being more common (80%). High polysomy with mean copy number of CEP17 signals being >3.75 signals per cell was seen in 20% of specimens with mean HER-2 copy number per cell ranging from 2 to 5.28 signals per cell. HER-2 genetic heterogeneity was evident in 23.3% of specimens. Tumors with polysomy 17 and negative for HER-2 amplification represent HER-2- negative tumors.

In contrast, 17 (36%) out of 47 specimens were positive for HER- 2 amplification, with majority presenting with low polysomy. Mean HER-2 copy number ranged from 6.0 to 16.32 signals per cell, with all specimens having more than 6 copies of HER-2 per cell. HER-2 genetic heterogeneity was very common. These cases represent tumors with true HER-2 amplification.

Evidences from the literature [21] and our study indicate that in the absence of HER-2 gene amplification, high or low polysomy is inadequate to cause significant increase in gene dosage and subsequent HER-2/neu protein overexpression. Cases with high polysomy with concurrent modest gene amplification (ratio, 2-5.28) may have an additive effect on gene dosage and protein expression.

Among the polysomy-negative cases, 9 out of 13 were HER-2 non-amplified, with majority of the specimens showing disomy and mean copy number of HER-2 ranging from 1.64 to 3.46. However, HER-2 copy numbers ranged from 3.38 to 9.22 in polysomy-negative but HER-2-amplified cases.

Other copy number anomalies that presented with unusual signal patterns include co-amplification of HER-2 and CEP17 signals and deletion of CEP17 signals. [16],[30] Co-amplification of the HER-2 and CEP17 genes is rare and reported in 1% of the cases. However, the same was observed in 2 cases. When interpretation is based on copy number of HER-2 signals(ASCO guidelines) the presence of greater than 6 copies per nucleus, equates to a true HER-2 amplification. Since the copy number of HER-2 signals was 10.36 and there were greater than 6 copies of HER-2, the specimens had HER-2 gene amplification despite the HER-2 to CEP17 ratio being equal to or a little more than 1. Centromeric CEP17 and HER-2 locus are far away from each other, and normally CEP17 is not included in the HER-2 amplicon. A complex translocation between HER-2 and CEP17 locus brings the two amplicons next to each other on the same chromosome, followed by amplification of both the genes resulting in co-amplification of both loci.

HER-2 genetic heterogeneity of breast carcinomas sometimes makes it difficult to accurately analyze the biologic properties of individual cancers, especially in antigens with heterogeneous staining patterns. The data has shown that heterogeneous expression of HER-2-amplified tumors was seen frequently in contrast to the phenomenon being a rare event (3.4%). It may be associated with poor prognosis/outcome depending on the HER-2 overexpressing subclone and represents a true biological phenomenon. [31]

To the best of our knowledge, this is the first report from Andhra Pradesh validating the use of IHC and FISH judiciously for HER-2/neu evaluation and in re-categorizing HER-2/neu status for determining eligibility for treatment with trastuzumab. In clinical practice, awareness of HER-2 and CEP17 copy number anomalies is necessary for correct interpretation to avoid misdiagnosis.

To summarize, HER-2 interpretation algorithm - first ascertain if signal patterns of the probes are usual or aberrant, involving copy number anomalies. If it is the former type, interpretation should be based on HER-2-CEP17 ratio and HER-2 genetic heterogeneity. If copy number anomalies are involved, evaluate the mean copy number of CEP17 signals and HER-2 signals; and if HER-2 copy number is >6 copies or/and HER-2 GH is seen in >50% invasive carcinoma cells, interpret the result as HER-2 amplified.

   Acknowledgments Top

We profusely thank Dr. Ch. Mohana Vamsy for providing breast carcinoma specimens for diagnosis and further evaluation. We thank Mrs. C Ajitha, Mrs. M. Padma, Mr. U. Sudhakar Reddy, Mr. U. Ravinder, Miss B. Manju and Miss D. Radhika for their excellent technical assistance for embedding and cutting of blocks, IHC and FISH.

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Correspondence Address:
Sudha S Murthy
Department of Laboratory Medicine (Pathology and Molecular Diagnostics), Basavatarakam Indo-American Cancer Hospital and Research Institute, Road No. 14, Banjara Hills, Hyderabad - 500 034
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.85087

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  [Table 1], [Table 2], [Table 3], [Table 4]

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