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pmid:32292720
Gene Expression Profiles Identified Novel Urine Biomarkers for Diagnosis and Prognosis of High-Grade Bladder Urothelial Carcinoma.
Yuxuan Song · Donghui Jin · Ningjing Ou · Zhiwen Luo · Guangyuan Chen · Jingyi Chen · Yongjiao Yang · Xiaoqiang Liu
Frontiers in oncology · 2020 doi:10.3389/fonc.2020.00394
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Complete file
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High-grade bladder urothelial carcinoma (HGBC) is aggressive with a high risk of recurrence, progression, metastasis, and poor prognosis. Therefore, HGBC clinical management is still a challenge. We performed the present study to seek new urine biomarkers for HGBC and investigate how they promote HGBC progression and thus affect the prognosis based on large-scale sequencing data. We identified the overlapped differentially expressed genes (DEGs) by combining GSE68020 and The Cancer Genome Atlas (TCGA) datasets. Subsequent receiver operating characteristic (ROC) curves, Kaplan-Meier (KM) curves, and Cox regression were conducted to test the diagnostic and prognostic role of the hub genes. Chi-square test and logistic regression were carried out to analyze the associations between clinicopathologic characteristics and the hub genes. Ultimately, we performed gene set enrichment analysis (GSEA), protein-protein interaction (PPI) networks, and Bayesian networks (BNs) to explore the underlying mechanisms by which ECM1, CRYAB, CGNL1, and GPX3 are involved in tumor progression. Immunohistochemistry based on The Human Protein Atlas and quantitative real-time polymerase chain reaction based on urine samples confirmed the downregulation and diagnostic values of the hub genes in HGBC. In conclusion, our study indicated that CRYAB, CGNL1, ECM1, and GPX3 are potential urine biomarkers of HGBC. These four novel urine biomarkers will have attractive applications to provide new diagnostic methods, prognostic predictors and treatment targets for HGBC, which could improve the prognosis of HGBC patients, if validated by further experiments and larger prospective clinical trials.", "claims": [{"id": "claim-method", "statement": "To identify DEGs in GSE68020 and TCGA BLCA datasets between BC patients and non-tumor healthy controls, we utilized limma R package (12).", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:32292720", "locator": "sections[5] \"RNA Data Processing and Identification of Differentially Expressed Genes\" paragraph 2", "quote": "To identify DEGs in GSE68020 and TCGA BLCA datasets between BC patients and non-tumor healthy controls, we utilized limma R package (12)."}], "state": "complete"}, {"id": "claim-data-access", "statement": "Publicly available datasets were analyzed in this study. 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"access": "open", "state": "verified", "reader": "read_table", "reader_status": "available", "identifier": "section[17]/table-wrap[1]#T2", "version": null, "note": "Relationship between expression of the five DEGs and clinicopathological characteristics in BC patients from TCGA.", "structure": {"pmcid": "PMC7118735", "section_index": 17, "section_title": "Correlations Between Expression Levels of the 5 Hub Genes and Clinical Outcomes in TCGA BLCA Dataset", "row_count": 30, "column_count": 17}, "table": {"label": "Table 2", "caption": "Relationship between expression of the five DEGs and clinicopathological characteristics in BC patients from TCGA.", "rows": [["Clinical characteristics", "Total (N)", "CRYAB expression", "P-value", "ECM1 expression", "P-value", "GPX3 expression", "P-value", "CGNL1 expression", "P-value", "CRNN expression", "P-value"], ["(A)", "", "High", "Low", "", "High", "Low", "", "High", "Low", "", "High", "Low", "", "High", "Low", ""], ["Histological grade"], ["High grade", "380", "198 (52.1%)", "182 (47.9%)", "<0.001", "199 (52.4%)", "181 (47.6%)", "<0.001", "196 (51.6%)", "184 (48.4%)", "0.006", "190 (50.0%)", "190 (50.0%)", "0.383", "192 (50.5%)", "188 (49.5%)", "0.176"], ["Low grade", "20", "2 (10.0%)", "18 (90.0%)", "", "1 (00.3%)", "19 (05.0%)", "", "4 (20.0%)", "16 (80.0%)", "", "8 (40.0%)", "12 (60.0%)", "", "7 (35.0%)", "13 (65.0%)", ""], ["UICC stage"], ["Stage I+II", "130", "43 (33.1%)", "87 (66.9%)", "<0.001", "47 (12.4%)", "83 (21.8%)", "<0.001", "52 (40.0%)", "78 (60.0%)", "0.006", "57 (43.8%)", "73 (56.2%)", "0.094", "71 (54.6%)", "59 (45.4%)", "0.189"], ["Stage III+IV", "271", "158 (58.3%)", "113 (41.7%)", "", "153 (40.3%)", "118 (31.1%)", "", "148 (54.6%)", "123 (45.4%)", "", "143 (52.8%)", "128 (47.2%)", "", "129 (47.6%)", "142 (52.4%)", ""], ["pT stage"], ["T1", "4", "0 (0.0%)", "4 (100.0%)", "0.052", "1 (00.3%)", "3 (00.8%)", "0.356", "2 (50.0%)", "2 (50.0%)", "1.000", "2 (50.0%)", "2 (50.0%)", "1.000", "3 (75.0%)", "1 (25.0%)", "0.623"], ["T2+T3+T4", "367", "193 (52.6%)", "174 (47.4%)", "", "191 (50.3%)", "176 (46.3%)", "", "189 (51.5%)", "178 (48.5%)", "", "184 (50.1%)", "183 (49.9%)", "", "184 (50.1%)", "183 (49.9%)", ""], ["pM stage"], ["No (M0)", "193", "78 (40.4%)", "115 (59.6%)", "0.988", "72 (18.9%)", "121 (31.8%)", "0.409", "77 (39.9%)", "116 (60.1%)", "0.215", "84 (43.5%)", "109 (56.5%)", "0.686", "91 (47.2%)", "102 (52.8%)", "0.485"], ["Yes (M1)", "11", "5 (45.5%)", "6 (54.5%)", "", "6 (01.6%)", "5 (01.3%)", "", "7 (63.6%)", "4 (36.4%)", "", "6 (54.5%)", "5 (45.5%)", "", "4 (36.4%)", "7 (63.6%)", ""], ["pN stage"], ["No (N0)", "234", "108 (46.2%)", "126 (53.8%)", "<0.001", "109 (28.7%)", "125 (32.9%)", "0.029", "116 (49.6%)", "118 (50.4%)", "0.430", "103 (44.0%)", "131 (56.0%)", "0.002", "125 (53.4%)", "109 (46.6%)", "0.106"], ["Yes (N1+N2+N3)", "128", "84 (65.6%)", "44 (34.4%)", "", "75 (19.7%)", "53 (13.9%)", "", "69 (53.9%)", "59 (46.1%)", "", "78 (60.9%)", "50 (39.1%)", "", "57 (44.5%)", "71 (55.5%)", ""], ["Age (years)"], ["≤ 60", "106", "39 (36.8%)", "67 (63.2%)", "0.002", "48 (12.6%)", "58 (15.3%)", "0.271", "44 (41.5%)", "62 (58.5%)", "0.045", "50 (47.2%)", "56 (52.8%)", "0.516", "58 (54.7%)", "48 (45.3%)", "0.246"], [">60", "297", "162 (54.5%)", "135 (45.5%)", "", "153 (40.3%)", "144 (37.9%)", "", "157 (52.9%)", "140 (47.1%)", "", "151 (50.8%)", "146 (49.2%)", "", "143 (48.1%)", "154 (51.9%)", ""], ["gender"], ["Female", "105", "55 (52.4%)", "50 (47.6%)", "0.551", "55 (14.5%)", "50 (13.2%)", "0.551", "55 (52.4%)", "50 (47.6%)", "0.551", "51 (48.6%)", "54 (51.4%)", "0.756", "57 (54.3%)", "48 (45.7%)", "0.293"], ["Male", "298", "146 (49.0%)", "152 (51.0%)", "", "146 (38.4%)", "152 (40.0%)", "", "146 (49.0%)", "152 (51.0%)", "", "150 (50.3%)", "148 (49.7%)", "", "144 (48.3%)", "154 (51.7%)", ""], ["(B)", "", "OR (95%CI)", "", "OR (95%CI)", "", "OR (95%CI)", "", "OR (95%CI)", "", "OR (95%CI)", ""], ["Histological grade (high vs. low)", "400", "9.79 (2.77, 62.14)", "0.002", "20.89 (4.27, 377.21)", "0.003", "4.26 (1.53, 15.08)", "0.011", "1.50 (0.61, 3.91)", "0.386", "2.38 (0.90, 7.43)", "0.100"], ["UICC stage (III+IV vs. I+II)", "401", "2.83 (1.83, 4.41)", "0.000", "2.30(1.49.3.54)", "0.000", "1.80 (1.18, 2.77)", "0.006", "1.43 (0.94, 2.18)", "0.095", "0.85 (0.56, 1.29)", "0.440"], ["pN stage (N1+N2+N3 vs. N0)", "362", "2.23 (1.43, 3.50)", "0.000", "1.62 (1.05, 2.52)", "0.029", "1.19 (0.77, 1.84)", "0.431", "1.98 (1.28, 3.09)", "0.002", "0.76 (0.49, 1.18)", "0.222"], ["pM stage (M1 vs. M0)", "204", "1.23 (0.34, 4.22)", "0.741", "2.02 (0.59, 7.22)", "0.261", "2.64 (0.77, 10.35)", "0.132", "1.56(0.45, 5.57)", "0.477", "0.74 (0.19, 2.54)", "0.642"], ["Age (>60 vs. ≤ 60)", "403", "2.06(1.31, 3.27)", "0.002", "1.28 (0.82, 2.01)", "0.271", "1.58 (1.01, 2.48)", "0.046", "1.16 (0.74, 1.81)", "0.516", "0.86 (0.55, 1.34)", "0.498"], ["Gender (male vs. female)", "403", "0.87 (0.56, 1.36)", "0.551", "0.87(0.56, 1.36)", "0.551", "0.87 (0.56, 1.36)", "0.551", "1.07 (0.69, 1.68)", "0.756", "0.68 (0.43, 1.06)", "0.091"]], "row_count": 30, "column_count": 17, "numeric_cells": 263, "header_rows": 1, "locator": "section[17]/table-wrap[1]#T2"}}, {"id": "asset:table:pmid:32292720:4", "role": "table", "format": "jats-table", "media_type": "application/vnd.jats.table+json", "uri": null, "sha256": "sha256:b89daeee4f84af205ceae7f8b07ca2ba2942b47860f69b9f6d8deff980d41240", "bytes": 1075, "access": "open", "state": "verified", "reader": "read_table", "reader_status": "available", "identifier": "section[17]/table-wrap[2]#T3", "version": null, "note": "Univariate and multivariate Cox analyses between expression levels of the five DEGs and patient survival based on TCGA.", "structure": {"pmcid": "PMC7118735", "section_index": 17, "section_title": "Correlations Between Expression Levels of the 5 Hub Genes and Clinical Outcomes in TCGA BLCA Dataset", "row_count": 13, "column_count": 5}, "table": {"label": "Table 3", "caption": "Univariate and multivariate Cox analyses between expression levels of the five DEGs and patient survival based on TCGA.", "rows": [["Items", "Univariate Cox", "P-value", "Multivariate Cox", "P-value"], ["Histological grade", "-", "0.996", "-", "-"], ["UICC stage", "1.51 (1.06, 2.15)", "0.024", "1.27 (0.85, 1.9)", "0.245"], ["pT stage", "-", "0.997", "-", "-"], ["pM stage", "2.09 (0.75, 5.80)", "0.158", "1.29 (0.44, 3.8)", "0.641"], ["pN stage", "2.18 (1.30, 3.66)", "0.003", "1.97 (1.08, 3.58)", "0.026"], ["Age", "2.30 (1.09, 4.85)", "0.029", "2.58 (1.18, 5.64)", "0.018"], ["Gender", "0.59 (0.34, 1.03)", "0.062", "0.61 (0.34, 1.09)", "0.095"], ["CRYAB", "1.26 (1.03, 1.55)", "0.026", "1.12 (0.83, 1.52)", "0.443"], ["ECM1", "1.42 (1.18, 1.71)", "<0.001", "1.44 (1.17, 1.78)", "0.001"], ["CGNL1", "1.19 (0.71, 1.98)", "0.511", "0.98 (0.52, 1.85)", "0.957"], ["CRNN", "1.16 (0.86, 1.57)", "0.339", "0.94 (0.68, 1.3)", "0.700"], ["GPX3", "0.97 (0.82, 1.15)", "0.716", "0.81 (0.65, 0.99)", "0.043"]], "row_count": 13, "column_count": 5, "numeric_cells": 42, "header_rows": 1, "locator": "section[17]/table-wrap[2]#T3"}}, {"id": "asset:table:pmid:32292720:5", "role": "table", "format": "jats-table", "media_type": "application/vnd.jats.table+json", "uri": null, "sha256": "sha256:78b76d14f8b3dbed0a4750a7b6e0f00e139bfd31c40c452de46ef8334c1d3e69", "bytes": 4029, "access": "open", "state": "verified", "reader": "read_table", "reader_status": "available", "identifier": "section[19]/table-wrap[1]#T4", "version": null, "note": "GO functional and KEGG pathway enrichments of PPI networks based on the four gene modules.", "structure": {"pmcid": "PMC7118735", "section_index": 19, "section_title": "PPI Network Analyses and Module Functional Enrichment", "row_count": 26, "column_count": 12}, "table": {"label": "Table 4", "caption": "GO functional and KEGG pathway enrichments of PPI networks based on the four gene modules.", "rows": [["CGNL1", "GPX3", "ECM1", "CRYAB"], ["GOterm/pathway", "Description", "FDR", "GOterm/pathway", "Description", "FDR", "GOterm/pathway", "Description", "FDR", "GOterm/pathway", "Description", "FDR"], ["BP (GO)"], ["GO:0051694", "Pointed-end actin filament capping", "0.0067", "GO:1990748", "Cellular detoxification", "3.60E-14", "GO:0002576", "Platelet degranulation", "6.94E-22", "GO:0007601", "Visual perception", "5.15E-07"], ["GO:0031032", "Actomyosin structure organization", "0.0067", "GO:0098869", "Cellular oxidant detoxification", "3.53E-12", "GO:0010466", "Negative regulation of peptidase activity", "7.33E-06", "GO:0051260", "Protein homooligomerization", "0.00011"], ["GO:0030036", "Actin cytoskeleton organization", "0.0135", "GO:0006749", "Glutathione metabolic process", "9.98E-09", "GO:0030162", "Regulation of proteolysis", "4.12E-05", "GO:0003008", "System process", "0.00013"], ["GO:0016338", "Calcium-independent cell-cell adhesion via plasma membrane cell-adhesion molecules", "0.0135", "GO:0006979", "Response To Oxidative Stress", "3.25E-08", "GO:0010951", "Negative regulation of endopeptidase activity", "0.00021", "GO:0050877", "Nervous system process", "0.00018"], ["GO:0007015", "Actin filament organization", "0.0172", "GO:0034599", "Cellular response to oxidative stress", "9.65E-08", "GO:0051336", "Regulation of hydrolase activity", "0.00059", "GO:0032387", "Negative regulation of intracellular transport", "0.00051"], ["MF (GO)"], ["GO:0005523", "Tropomyosin binding", "0.0027", "GO:0016209", "Antioxidant activity", "7.12E-13", "GO:0004866", "Endopeptidase inhibitor activity", "8.89E-05", "GO:0005212", "Structural constituent of eye lens", "1.02E-19"], ["GO:0008092", "Cytoskeletal protein binding", "0.0426", "GO:0016491", "Oxidoreductase activity", "2.24E-08", "GO:0030234", "Enzyme regulator activity", "0.0011", "GO:0051082", "Unfolded protein binding", "0.00069"], ["GO:0005515", "Protein binding", "0.0426", "GO:0004784", "Superoxide dismutase activity", "2.19E-07", "GO:0004867", "Serine-type endopeptidase inhibitor activity", "0.0087", "GO:0042802", "Identical protein binding", "0.0031"], ["—", "—", "—", "GO:0003824", "Catalytic Activity", "1.87E-05", "GO:0002020", "Protease binding", "0.0137", "GO:0001540", "Amyloid-beta binding", "0.0074"], ["—", "—", "—", "GO:0043295", "Glutathione binding", "0.00033", "GO:0008201", "Heparin binding", "0.0173", "—", "—", "—"], ["CC (GO)"], ["GO:0043296", "Apical junction complex", "0.00015", "GO:0031970", "Organelle envelope lumen", "0.0016", "GO:0031089", "Platelet dense granule lumen", "6.28E-32", "—", "—", "—"], ["GO:0005911", "Cell-cell junction", "0.00015", "GO:1904813", "Ficolin-1-rich granule Lumen", "0.0472", "GO:0005576", "Extracellular region", "7.36E-10", "—", "—", "—"], ["GO:0005923", "Bicellular tight junction", "0.0023", "GO:0044444", "Cytoplasmic part", "0.0472", "GO:0044421", "Extracellular region part", "7.40E-06", "—", "—", "—"], ["GO:0044430", "Cytoskeletal part", "0.0024", "GO:0044429", "Mitochondrial part", "0.0472", "GO:0005615", "Extracellular space", "4.37E-05", "—", "—", "—"], ["GO:0019897", "Extrinsic component of plasma membrane", "0.0024", "GO:0044421", "Extracellular region part", "0.0472", "GO:0031012", "Extracellular matrix", "0.0012", "—", "—", "—"], ["KEGG"], ["hsa04530", "Tight junction", "0.0018", "hsa00480", "Glutathione metabolism", "1.92E-09", "—", "—", "—", "hsa04141", "Protein processing in endoplasmic reticulum", "0.0216"], ["hsa05152", "Tuberculosis", "0.0265", "hsa04213", "Longevity regulating pathway-multiple species", "7.82E-05", "—", "—", "—", "—", "—", "—"], ["hsa04145", "Phagosome", "0.0265", "hsa04146", "Peroxisome", "0.00012", "—", "—", "—", "—", "—", "—"], ["—", "—", "—", "hsa05418", "Fluid shear stress and atherosclerosis", "0.00038", "—", "—", "—", "—", "—", "—"], ["—", "—", "—", "hsa05014", "Amyotrophic lateral sclerosis", "0.0022", "—", "—", "—", "—", "—", "—"]], "row_count": 26, "column_count": 12, "numeric_cells": 123, "header_rows": 2, "locator": "section[19]/table-wrap[1]#T4"}}, {"id": "asset:table:pmid:32292720:6", "role": "table", "format": "jats-table", "media_type": "application/vnd.jats.table+json", "uri": null, "sha256": "sha256:66d9922e67f97994348e7bed128832476525112b36424043d337b4c8f2b5790b", "bytes": 969, "access": "open", "state": "verified", "reader": "read_table", "reader_status": "available", "identifier": "section[21]/table-wrap[1]#T5", "version": null, "note": "Comparison of immunohistochemistry expression level between normal bladder and HGBC by Fisher's Exact test from The Human Protein Atlas.", "structure": {"pmcid": "PMC7118735", "section_index": 21, "section_title": "Immunohistochemistry From the Human Protein Atlas", "row_count": 12, "column_count": 8}, "table": {"label": "Table 5", "caption": "Comparison of immunohistochemistry expression level between normal bladder and HGBC by Fisher's Exact test from The Human Protein Atlas.", "rows": [["Gene name", "", "N", "Immunohistochemistry expression level", "P-value"], ["", "", "", "Negative (–)", "Weakly positive (+)", "Positive (++)", "Strongly positive (+++)", ""], ["GPX3", "Normal bladder tissue", "2", "0", "2", "0", "0", "0.016"], ["", "HGBC tissue", "8", "7", "1", "0", "0", ""], ["ECM1", "Normal bladder tissue", "2", "0", "2", "0", "0", "0.001"], ["", "HGBC tissue", "9", "9", "0", "0", "0", ""], ["CRYAB", "Normal bladder tissue", "5", "5", "0", "0", "0", "0.439"], ["", "HGBC tissue", "9", "8", "0", "0", "1", ""], ["CGNL1", "Normal bladder tissue", "3", "3", "0", "0", "0", "0.569"], ["", "HGBC tissue", "10", "9", "1", "0", "0", ""], ["CRNN", "Normal bladder tissue", "4", "2", "2", "0", "0", "0.477"], ["", "HGBC tissue", "7", "5", "2", "0", "0", ""]], "row_count": 12, "column_count": 8, "numeric_cells": 55, "header_rows": 2, "locator": "section[21]/table-wrap[1]#T5"}}, {"id": "asset:table:pmid:32292720:7", "role": "table", "format": "jats-table", "media_type": "application/vnd.jats.table+json", "uri": null, "sha256": "sha256:e668cbfde5e5b30b786f3c1a12a13135e8a8245fd1d4951a13664228f40d3bda", "bytes": 766, "access": "open", "state": "verified", "reader": "read_table", "reader_status": "available", "identifier": "section[22]/table-wrap[1]#T6", "version": null, "note": "Characteristics of enrolled participants from Tianjin validation cohort.", "structure": {"pmcid": "PMC7118735", "section_index": 22, "section_title": "Expression of GPX3, ECM1, CRYAB, CGNL1, and CRNN in Tianjin Validation Cohort", "row_count": 15, "column_count": 5}, "table": {"label": "Table 6", "caption": "Characteristics of enrolled participants from Tianjin validation cohort.", "rows": [["", "BC patients (n = 30)", "Controls (n = 30)", "chi-square", "P-value"], ["Age (years)"], ["≤60", "14 (46.67%)", "12 (40.00%)", "0.271", "0.602"], [">60", "16 (53.33%)", "18 (60.00%)", "", ""], ["Gender"], ["Male", "13 (43.33%)", "16 (53.33%)", "0.601", "0.438"], ["Female", "17 (56.67%)", "14 (46.67%)", "", ""], ["UICC stage"], ["Stage I", "7 (23.33%)", "", "", ""], ["Stage II", "8 (26.67%)", "", "", ""], ["Stage III", "9 (30.00%)", "", "", ""], ["Stage IV", "6 (20.00%)", "", "", ""], ["Histological grade"], ["Low grade", "13 (43.33%)", "", "", ""], ["High grade", "17 (56.67%)", "", "", ""]], "row_count": 15, "column_count": 5, "numeric_cells": 22, "header_rows": 1, "locator": "section[22]/table-wrap[1]#T6"}}], "gaps": ["asset:data:geo:GSE68020 not downloaded", "asset:data:geo:GSE68020 licence not read"]}, "analysis": {"state": "partial", "methods": [{"id": "method-1", "family": "differential_expression", "specific": "limma_voom", "label": "Differential expression analysis", "dsl_operations": ["normalise_counts", "differential_expression_fit", "fdr_bh"], "missing_operations": [], "expressible": true, "parameters": {}, "data_shapes": ["count_matrix_with_condition"], "evidence": {"score": 1.0, "band": "verbatim", "quote": "To identify DEGs in GSE68020 and TCGA BLCA datasets between BC patients and non-tumor healthy controls, we utilized limma R package (12)."}}, {"id": "method-2", "family": "survival_analysis", "specific": "kaplan_meier", "label": "Survival analysis", "dsl_operations": ["survival_km"], "missing_operations": [], "expressible": true, "parameters": {}, "data_shapes": ["time_to_event_with_censoring"], "evidence": {"score": 0.7, "band": "medium", "quote": "Kaplan-Meier"}}, {"id": "method-3", "family": "survival_analysis", "specific": "cox_proportional_hazards", "label": "Survival analysis", "dsl_operations": ["cox_ph"], "missing_operations": [], "expressible": true, "parameters": {}, "data_shapes": ["time_to_event_with_censoring"], "evidence": {"score": 0.7, "band": "medium", "quote": "Cox regression"}}, {"id": "method-4", "family": "survival_analysis", "specific": "log_rank_test", "label": "Survival analysis", "dsl_operations": ["logrank_test"], "missing_operations": [], "expressible": true, "parameters": {}, "data_shapes": ["time_to_event_with_censoring"], "evidence": {"score": 0.8, "band": "high", "quote": "log-rank"}}, {"id": "method-5", "family": "regression_modelling", "specific": "logistic", "label": "Regression modelling", "dsl_operations": ["glm_logit_fit"], "missing_operations": [], "expressible": true, "parameters": {}, "data_shapes": ["predictors_and_outcome"], "evidence": {"score": 0.7, "band": "medium", "quote": "logistic regression"}}, {"id": "method-6", "family": "network_analysis", "specific": "ppi_network", "label": "Network / pathway analysis", "dsl_operations": ["build_graph", "centrality_scores", "pathway_enrich"], "missing_operations": [], "expressible": true, "parameters": {}, "data_shapes": ["edge_list"], "evidence": {"score": 0.7, "band": "medium", "quote": "protein-protein interaction"}}, {"id": "method-7", "family": "contingency_analysis", "specific": "chi_square", "label": "Categorical (contingency table) analysis", "dsl_operations": ["chi_square_test"], "missing_operations": [], "expressible": true, "parameters": {}, "data_shapes": ["categorical_cross_tabulation"], "evidence": {"score": 0.7, "band": "medium", "quote": "Chi-square"}}], "targets": [], "gaps": ["method.reader_differs_from_extractor"]}, "capability": {"state": "complete", "requirements": [{"capability_id": "capability:computational_analysis", "label": "Computational analysis platform", "assay_families": ["meta_analysis", "machine_learning", "survival_analysis", "statistical_regression", "pca", "molecular_docking", "network_analysis", "differential_expression", "gwas"], "availability_state": "owned", "tier_unlocked": "R1", "candidate": "Automated Assays Laravel application and simulated adapters"}], "gaps": []}, "readiness": {"state": "complete", "tier": "R1", "blocking_axis": "none", "blocked_on": "nothing:replicable_now", "axes": {"method": {"tier": "R1", "expressible": true, "required_operations": ["normalise_counts", "differential_expression_fit", "fdr_bh"], "missing_operations": [], "basis": "The paper names the method it used and the corpus's method extractor maps it onto the DSL catalogue: limma_voom."}, "capability": {"tier": "R1", "capability_id": "capability:computational_analysis", "availability_state": "owned", "basis": "The computational analysis is owned. The physical bench the paper used is not, so a physical validation cannot be run here."}, "data": {"tier": "R1", "reachable": true, "accessions": ["GSE68020"], "resolved_accessions": ["GSE68020"], "statements": ["Publicly available datasets were analyzed in this study. The data can be found in Gene Expression Omnibus (GEO: http://www.ncbi.nlm.nih.gov/geo/), The Cancer Genome Atlas (TCGA: http://tcga-data.nci.nih.gov/tcga/), and The Human Protein Atlas (THPA: http://www.proteinatlas.org/). GSE68020 dataset was from GEO; BLCA (Bladder Urothelial Carcinoma) dataset was from TCGA; and immunohistochemistry was from THPA. All the data are open access. Summarized detailed characteristics of immunohistochemistry data from THPA are in Supplementary Table 1."], "basis": "The paper names GSE68020 in Gene Expression Omnibus (GEO) and the accession resolves in the repository. No dataset was downloaded and no licence was read."}}, "can_run": {"dsl_operations": ["normalise_counts", "differential_expression_fit", "fdr_bh"], "assets": ["source:pmid:32292720", "data:geo:GSE68020", "asset:table:pmid:32292720:1", "asset:table:pmid:32292720:2", "asset:table:pmid:32292720:3", "asset:table:pmid:32292720:4", "asset:table:pmid:32292720:5", "asset:table:pmid:32292720:6", "asset:table:pmid:32292720:7"], "steps": ["run_analysis"]}, "must_build": {"dsl_operations": [], "converters": [], "readers": [], "capabilities": [], "data_gaps": ["data:geo:GSE68020"]}, "gaps": []}, "review": {"state": "partial", "machine_critique": {"kind": "extraction_review", "reader": {"model": "MiniMax-M3.1-Flash", "provider": "minimax", "provider_family": "minimax", "read_at": "2026-10-31T18:19:10Z", "independence": "single_reader"}, "evidence_check": {"method": "verbatim", "availability": "verbatim"}, "calls": 3, "usage": {"prompt_tokens": 41116, "completion_tokens": 1686, "cached_tokens": 13832}}, "human_critique": null, "stages": [{"id": "extraction", "state": "complete", "reader": "MiniMax-M3.1-Flash", "provider": "minimax"}], "gaps": ["review.independent_reader", "review.machine_critique", "review.human_critique"]}, "execution": {"state": "absent", "status": "not_run", "mode": null, "runs": [], "gaps": ["execution.not_run"]}, "provenance": {"entities": [{"id": "pmid:32292720", "type": "paper", "label": "pmid:32292720"}, {"id": "source:pmid:32292720", "type": "manuscript", "label": "PMID 32292720 full text"}, {"id": "data:geo:GSE68020", "type": "dataset", "label": "GEO GSE68020"}], "activities": [{"id": "activity-reading-sci-2026-11-01", "type": "classification", "used": [{"id": "pmid:32292720", "type": "paper", "label": "pmid:32292720"}, {"id": "source:pmid:32292720", "type": "manuscript", "label": "PMID 32292720 full text"}, {"id": "data:geo:GSE68020", "type": "dataset", "label": "GEO GSE68020"}], "agent": "agent:corpus-assay-engine"}, {"id": "activity-read-32292720", "type": "extraction", "used": ["source:pmid:32292720"], "agent": "agent:minimax-reader"}, {"id": "activity-resolve-GSE68020", "type": "accession_resolution", "used": ["data:geo:GSE68020"], "agent": "agent:corpus-assay-engine"}], "agents": [{"id": "agent:corpus-assay-engine", "type": "software", "label": "Corpus assay engine"}, {"id": "agent:minimax-reader", "type": "software", "label": "MiniMax literature reader"}], "source_run_id": "reading-sci-2026-11-01", "dsl_catalogue_fingerprint": "343fb30756ba6ec3", "capability_table_fingerprint": "2cb3e42f047d2ecf", "snapshot_id": "ba34b49490d9bfaf"}, "lineage": {"parent_id": "pmid:32292720", "parent_hash": "sha256:16d6cc1469a21251e617e66d9bfcce8bb7b6bd320ed4636f9282e865d52c6e38", "fork_of": null, "generation": 7}, "gaps": ["asset:data:geo:GSE68020 licence not read", "asset:data:geo:GSE68020 not downloaded", "execution.not_run", "method.reader_differs_from_extractor", "review.human_critique", "review.independent_reader", "review.machine_critique"], "assays": [{"role": "data_generation", "definition_id": "ihc", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.93, "evidence": ["and GPX3 are involved in tumor progression. 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