Integrative analyses reveal signaling pathways underlying familial breast cancer susceptibility.
The main results reproduced, with only marginal, non-material deviations.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
- ✓Reported values were directly comparable
- 🟡Could not use the authors’ exact input data
- 🟡A deviation arose in the data or preprocessing
- 🟡A deviation was attributed to the published material
- 🟡Reported values were not (fully) derivable from the shared data
- 🟡The deviation was non-trivial in magnitude
- 🟡The central claim did not (fully) hold under reproduction
- 🟡Overall, the reproduction showed a material discrepancy
A 0–100 reproducibility-quality score from the per-question grades, shown as a z-score: standard deviations above (+) or below (−) the mean of comparable assessments.
▸Reproduction agent’s raw note
DESCRIBED WELL ENOUGH: partly. The authors' repo (srp33/BCRiskPathways @ fa9cb60) ships the 932 pathway gene sets, the GSE17072 class labels, the ML-Flex2 experiment config, and the EXACT permutation-p R code - enough to reproduce the structure of the normal-breast (Lim/Visvader = GSE17072) validation. It does NOT ship matrices/Visvader.txt nor the SVM wrapper (Internals/ gitignored), so the exact ML-Flex2 Java/Weka/R SVM and the Illumina probe->gene matrix are under-specified. PER BRIEF (don't chase the 20%; third-party/own implementation on the paper's data is equally valid), we rebuilt the gene matrix from the GEO series matrix (GPL6884->Entrez) and ran the DESCRIBED classifier (SVM-RFE + radial SVM via LIBSVM/scikit-learn, LOOCV) on all 931 evaluable pathways, then scored them with the authors' verbatim permutation-p logic. RESULT = 1:1-in-method, near-1:1-in-value for the headline pathway: REACTOME Integrin Cell Surface Interactions reproduced P=0.030 vs reported 0.038 (within-tol); KEGG Small Cell Lung Cancer P=0.040 vs 0.007 (both significant, same direction, magnitude off = partial); KEGG Focal Adhesion strongly significant (P=0.008), consistent with the paper highlighting it. The GSE17072 cohort itself (20 samples, 15 familial/5 control) reproduced exactly. NOT ATTEMPTED: the '9 of 45 pathways replicate' joint claim (needs GSE47862 PBMC + GSE19383 Bellacosa + cross-dataset rankPvalue/metap), the PBMC 45-pathway result (needs a non-shipped clinical-variables file), and the exome 94.2% BRCA1/2 figure (dbGaP-restricted data, data_restricted). HONESTY FLAG: 316/931 pathways reach P<0.05 in this single small cohort, so single-cohort significance is liberal - the paper's rigor comes from cross-cohort replication we did not reproduce. No fabrication signal: the two named reported p-values are derivable from the shipped data+method.
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Assessment versions
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v1 current initial assessment Score 67assessed: 2026-06-16 ⛓ b44639637d9f
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Provenance — full disclosure
When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-16
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-16no human curator yet
- Last updated
- 2026-08-05
Provisional, curator- or AI-assessed, and independently checkable. A reproduction outcome states what one attempt could reproduce — not a judgement of the authors.
Deep full-text extraction
Model: opusThe authors hypothesized that comparing molecular profiles of women from high-risk families who developed familial breast cancer (FBC) against high-risk women who did not, using a pathway-based multi-omic strategy across multiple tissues, could identify biological signaling pathways underlying FBC susceptibility.
- ★ Cell adhesion (cell-cell and cell-ECM) pathways are significantly and consistently dysregulated in women who develop familial breast cancer across multiple omic data types and tissues. finding
- ★ A pathway-based integrative strategy combining gene expression and exome-sequencing data can elucidate the biological basis of FBC where single risk variants cannot. method
- ★ REACTOME Integrin Cell Surface Interactions and KEGG Small Cell Lung Cancer pathways are the top-ranked pathways discriminating FBC women from controls in both PBMC and normal breast tissue. finding
- ★ Normal mammary epithelial cells from FBC women show decreased cell adhesion, increased cell death with adhesion-modulating drugs, and aberrant morphology (larger spread cells, fewer cell-cell contacts). finding
- ★ Disrupted cell adhesion processes in non-malignant cells may play a role in FBC development and serve as an indicator of breast cancer risk. mechanism
- Gene and protein expression for a subset of adhesion-related genes (VTN up, F-actin down) are concordantly dysregulated in FBC women. finding
- Pathway-level germline DNA variation differs between FBC women and controls consistent with expression-based dysregulation. finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| Gene expression microarray (transcriptomic profiling) | Peripheral blood mononuclear cells (PBMCs) from Utah and Ontario cohorts of women with breast cancer family history | none (FBC-affected vs unaffected high-risk women) | Pathway-level multigene classifier accuracy discriminating FBC vs controls | — |
| Exome sequencing | PBMCs from 35 Utah patients | none (FBC vs control) | Per-pathway germline DNA variant counts (likely pathogenic variants) | — |
| Gene expression profiling (pathway analysis) | Normal breast tissue from two independent cohorts (Lim et al, Bellacosa et al) | none (family history/BRCA1/2 carriers vs controls) | Genomic model score discriminating high-risk vs control | — |
| Western blotting | Snap-frozen breast epithelial tissue / primary mammary epithelial cell lysates from prophylactic surgery (high-risk) and breast reduction (control) women (n=27) | none (FBC vs control) | Protein levels of VTN, F-actin, FAK/PTK2, PTEN, ITGA6, ICAM2, p53, integrins; β-actin loading control | — |
| Fluorescence microscopy / immunostaining | Normal primary mammary epithelial cells from 24 patient cultures (prophylactic mastectomy FBC vs breast reduction controls) | none | Cell size, F-actin staining, focal adhesion staining, cell morphology | — |
| Cell-based functional adhesion assays and drug-response assays | Normal primary mammary epithelial cells from high-risk (prophylactic mastectomy) and control (breast reduction) women | drugs modulating cell adhesion capacity | Cell adhesion ability and cell death | — |
- – 45 pathways consistently and significantly discriminated FBC women from controls across Utah and Ontario cohorts and both omic data types (rank P < 0.05). 45 pathways
- – REACTOME Integrin Cell Surface Interactions was a top discriminating pathway in PBMCs. P = 0.004
- – KEGG Small Cell Lung Cancer pathway discriminated FBC women from controls. P = 0.005
- – Of the 45 pathways, 9 showed significant differences in two normal breast tissue data sets and when combining all 5 data sets (P < 0.05). 9 of 45 pathways
- – Integrin Cell Surface Interactions and Small Cell Lung Cancer pathways remained top-ranked in normal breast tissue cohorts. Integrin P = 0.038 (Lim), 0.030 (Bellacosa); SCLC P = 0.007 (Lim), 0.003 (Bellacosa)
- – Vitronectin (VTN) protein increased and F-actin protein decreased in FBC women vs controls by Western blotting.
- – FBC normal mammary epithelial cells showed significant differences in cell size, F-actin staining, and focal adhesion staining vs controls.
- ▼ FAK/PTK2 and PTEN protein levels trended lower in FBC patient samples compared to controls.
- pvalue P = 0.004 (REACTOME Integrin Cell Surface Interactions pathway, PBMC analysis)
- pvalue P = 0.005 (KEGG Small Cell Lung Cancer pathway, PBMC analysis)
- pvalue P = 0.012 (KEGG Focal Adhesion pathway)
- pvalue P = 0.007 (REACTOME Cell Surface Interactions at the Vascular Wall pathway)
- pvalue P = 0.030–0.038 (Integrin Cell Surface Interactions in Lim (0.038) and Bellacosa (0.030) normal breast cohorts)
- count 45 pathways (Pathways consistently significant across Utah/Ontario cohorts and both omic types)
- count n = 27 (Breast epithelial tissue cohort for Western blotting)
- count 35 patients (Utah patients whose exomes were sequenced)
Statistical methods review
Model: sonnetA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
The study used a multi-cohort, multi-omic design integrating gene expression microarray data from PBMCs and normal breast tissue with exome-sequencing data to identify signaling pathways dysregulated in women who developed familial breast cancer (FBC) versus unaffected high-risk controls. Pathway-level classification was performed with Support Vector Machines ranked by cross-validated accuracy, and germline variant burden was compared with Barnard's exact test; P-values were combined across cohorts via a rank-based approach and corrected for multiple testing with Storey's q-value in sensitivity analyses. Genomic findings were validated in cell-based functional assays, Western blotting, and fluorescence microscopy with significance reported at P < 0.05.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Support Vector Machines (SVM) classifier, accuracy-ranked cross-validation | Pathway-level gene expression discrimination between FBC-affected and unaffected high-risk women across Utah (training), Ontario (test), Lim et al, and Bellacosa et al cohorts | — | not stated |
| Barnard's exact test | Pathway-level germline DNA variant burden comparison between FBC and control groups in the exome-sequencing subset | 35 Utah patients | not stated |
| Rank-based P-value combination (plus three additional unstated combination methods in sensitivity analyses) | Aggregating pathway significance across multiple cohorts and omic data types; rank P-value < 0.05 threshold used for primary filtering | — | not stated |
| Storey's q-value (FDR correction) | Multiple-testing correction across 932 pathways in robustness/sensitivity analyses using alternative P-value combination methods | — | not stated |
| Unspecified test, P < 0.05 threshold | Western blot protein level comparison (VTN, F-actin, FAK/PTK2, PTEN) between FBC and control primary mammary epithelial cells | 27 | not stated |
| Unspecified test, P < 0.05 threshold | Fluorescence microscopy quantification of estimated cell size, F-actin staining, and focal adhesion staining across microscopy fields from selected patients | ~10 fields per patient across 10 selected patients (from 24 total cultures) | not stated |
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Pathway classification relied on SVMs ranked by cross-validated accuracy; the SVM itself does not produce a classical null-hypothesis P-value for pathway enrichment↳ Could also: Gene Set Enrichment Analysis (GSEA), CAMERA, or fgsea could also be applied to test pathway-level enrichment in differential expression and return permutation- or rotation-based P-values with established FDR procedures — These methods are widely adopted for pathway analysis in transcriptomic studies and produce enrichment scores and FDR-adjusted statistics that align with standard reporting conventions, which may facilitate cross-study comparisons
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P-values were combined across cohorts using a rank-based aggregation approach as the primary strategy↳ Could also: Fisher's method for combining P-values (Fisher's combined probability test) or the Stouffer Z-score method are standard meta-analytic alternatives for aggregating evidence across independent cohorts — Fisher's and Stouffer's methods have well-characterized null distributions, are widely implemented, and are explicitly framed as meta-analytic procedures, which may offer clearer statistical properties and reproducibility than a rank-based threshold
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Formal multiple-testing correction (Storey's q-value) was applied only in sensitivity analyses; the primary filter used an unadjusted rank P < 0.05 across 932 pathways↳ Could also: Applying Benjamini-Hochberg FDR or Bonferroni correction as the primary multiplicity control across all 932 tested pathways would also explicitly control the false-discovery or family-wise error rate from the outset — Designating a formal FDR procedure as the primary analysis step makes the multiplicity strategy more transparent and consistent with common practice in large-scale genomic pathway studies
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Barnard's exact test was used to compare pathway-level variant burden (proportion of samples with at least one variant) between FBC and control groups↳ Could also: Fisher's exact test is another standard choice for 2×2 contingency tables comparing variant presence/absence between two groups; burden tests or sequence kernel association tests (SKAT) could also aggregate rare variant effects at the pathway level — Fisher's exact test is more universally implemented and familiar; SKAT-type approaches additionally account for variant frequency and functional annotation, which can increase power when multiple rare variants contribute to pathway burden
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The Western blot and fluorescence microscopy group comparisons were reported only as P < 0.05 with no test name or exact P-values stated↳ Could also: Explicitly stating the test used (e.g., Mann-Whitney U, Student's t-test, permutation test) and reporting exact P-values alongside effect sizes (e.g., fold-change or Cohen's d with 95% CI) would also fully characterize the inferential procedure — Naming the test and reporting exact P-values and effect sizes allows assessment of distributional assumptions, practical significance, and reproducibility — this is particularly informative at small n (n = 27 Western blot; 10 patients for microscopy quantification)
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Dispersion in box plots was conveyed as IQR with whiskers to extreme values; no confidence intervals or variance estimates were reported for the SVM model scores or cell-based measurements↳ Could also: Bootstrapped 95% confidence intervals around the cross-validated accuracy or around the median genomic model score per group could also be reported to convey estimation uncertainty — Confidence intervals communicate precision of the group-level estimates independently of sample size and would complement IQR summaries, especially given the small cohort sizes where interval width is informative
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Data lineage
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What was reproduced
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Scope — pmid-26969729
Paper: Piccolo SR et al. "Integrative analyses reveal signaling pathways
underlying familial breast cancer susceptibility." Mol Syst Biol 2016.
PMID 26969729 · PMCID PMC4812528 · DOI 10.15252/msb.20156506
Authors' code: https://github.com/srp33/BCRiskPathways @ fa9cb603373a196d1370bd8ffd4b5d7d883f41de
Assigned data accession: GEO GSE17072 = the Lim et al. (Visvader) normal
breast tissue validation cohort (GPL6884 Illumina, 20 QC-passed samples).
The paper's computational components
| # | Result | Pipeline / tool | In scope? | Why |
|---|---|---|---|---|
| 1 | PBMC pathway dysregulation → 45 pathways (rank P<0.05), Dataset EV1 | SCAN normalization + ComBat + ML-Flex2 (SVM-RFE + radial SVM) + WGCNA rankPvalue, on Utah+Ontario PBMC (GSE47862) | partial | needs GSE47862 + a non-shipped clinical/demographic variables file (secure/epidemiologic.txt, used to exclude 334 genes) → exact PBMC result not reproducible. Not the assigned accession. |
| 2 | Normal-breast validation: 9/45 pathways replicate; e.g. REACTOME Integrin Cell Surface Interactions P=0.038 (Lim/GSE17072), KEGG Small Cell Lung Cancer P=0.007 (Lim) — Fig 3, Datasets EV1/EV12 | ML-Flex2 per-pathway LOOCV SVM-RFE → AUC → permutation P (code/CalculatePredictionMetrics.R), on GSE17072 with shipped genesets + class labels |
YES (primary) | assigned accession; data public; class labels + 932 gene sets + the exact permutation-P R code are all shipped. Matrix must be rebuilt from GEO. |
| 3 | Exome variant counts (941,507 loci → 6,908; 94.2% BRCA1/2 detection) | BWA+GATK+snpEff pipeline | NO | data are dbGaP phs001044 + "require security approvals" → data_restricted. |
| 4 | Wet-lab assays (adhesion, FAK inhibitor EC50, microscopy, Western blot) | bench / ImageJ / GraphPad | NO | non_pipeline (wet-lab). |
What we attempt (80/20)
Component 2 on GSE17072 — the clearly-specified, low-hanging, fully-shipped validation pipeline keyed to the assigned accession. Two pinnable claims: the Lim permutation P-value for (a) REACTOME Integrin Cell Surface Interactions (paper 0.038) and (b) KEGG Small Cell Lung Cancer (paper 0.007), plus the structure "≈9 of 45 / how many of 932 pathways are significant."
Honest fidelity note (the hard ~20%, not chased)
The permutation P-value is a deterministic function of the per-pathway LOOCV
AUC (CalculatePredictionMetrics.R: shuffle probabilities vs fixed labels, 1000
fixed seeds, P=(#≥AUC)/1000 + 1/1000). We reproduce that R step verbatim
(authors' own logic). The only reimplemented piece is the LOOCV SVM probability
vector: the authors' ML-Flex2 SVM wrapper (Internals/R/Predict.R) is
.gitignored, and matrices/Visvader.txt is not shipped, so the exact SVM
hyper-parameters and the Illumina probe→gene matrix construction are
under-specified. We therefore apply the described method (SVM-RFE + radial
SVM via scikit-learn's LIBSVM — the same LIBSVM the paper cites — feature
scaling on, gamma=1/n_features, nested-CV C tuning) to the paper's own data
(P16: a faithful method re-run on the paper's data is equally valid). Exact
bit-for-bit P-values are NOT expected; we report ours beside the paper's and
let the human auditor judge.
Assessments & scoring basis
Each contributor’s verdict, the per-question basis, and the auditable, itemised worksheet behind it.
An automated assessment. It can flag an open question for review but can never, on its own, record a discrepancy verdict (C5) against a paper.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
In-method, near-1:1-in-value reproduction of the GSE17072 (Lim/Visvader) normal-breast validation: the cohort (20 samples, 15/5) and the authors' exact permutation-p R code were reused, and the headline REACTOME Integrin pathway reproduced P=0.030 vs 0.038 (within-tol). Deviations are on our side — the unshipped SVM wrapper and expression matrix forced a self-chosen probe->gene collapse and approximate hyperparameters, producing one ~6x gap (KEGG Small Cell Lung Cancer 0.040 vs 0.007) while keeping direction and significance. No fabrication signal: both named p-values are derivable from the shipped data+method. Confirmation is limited, not full, because the paper's actual stringency (9/45 pathways replicating across 5 datasets) was out of scope and single-cohort significance is liberal (34% of pathways reach P<0.05).
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Reproduction footprint
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