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CDH2/N-cadherin and early diagnosis of invasion in patients with ductal carcinoma in situ

  • Preclinical study
  • Published:
Breast Cancer Research and Treatment Aims and scope Submit manuscript

Abstract

Purpose

This proof-of-concept study investigates gene expression in core needle biopsies (CNB) to predict whether individuals diagnosed with ductal carcinoma in situ (DCIS) on CNB were affected by invasion at the time of diagnosis.

Methods

Using a QuantiGene Plex 2.0 assay, 14 gene expression profiling was performed in 303 breast tissue samples. Preoperative diagnostic performance of a gene was measured by area under receiver-operating characteristic curve (AUC) with 95% confidence interval (CI). The gene mRNA positivity cutoff was computed using Gaussian mixture model (GMM); protein expression was measured by immunohistochemistry; DNA methylation was evaluated by targeted bisulfite sequencing.

Results

mRNA from 69% (34/49) mammoplasties, 72% (75/104) CNB DCIS, and 89% (133/150) invasive breast cancers (IBC) were analyzed. Based on pre-and post-surgery DCIS chart reviews, 21 cases were categorized as DCIS synchronous with invasion and 54 DCIS were pure DCIS without pathologic evidence of invasive disease. The ectopic expression of neuronal cadherin CDH2 was probable in 0% mammoplasties, 6% pure DCIS, 29% synchronous DCIS, and 26% IBC. The CDH2 mRNA positivity in preoperative biopsies showing pure DCIS was predictive of a final diagnosis of invasion (AUC = 0.67; 95% CI 0.53–0.80; P = 0.029). Site-specific methylation of the CDH2 promoter (AUC = 0.76; 95% CI 0.54–0.97; P = 0.04) and measurements of N-cadherin, a pro-invasive cell–cell adhesion receptor encoded by CDH2 (AUC = 0.8; 95% CI 0.66–0.99; P < 0.005) had a discriminating power allowing for discernment of CDH2-positive biopsy.

Conclusions

Evidence of CDH2/N-cadherin expression, predictive of invasion synchronous with DCIS, may help to clarify a diagnosis and direct the course of therapy earlier in a patient’s care.

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Data Availability

All data needed to evaluate the conclusions in the paper are present in the paper or the supplemental information. COMPAi is available as a standalone application to qualified users.

Abbreviations

DCIS:

Ductal carcinoma in situ

CNB:

Core needle biopsy

IBC:

Invasive breast carcinoma

AUC:

Area under receiver-operating characteristic curve

CI:

Confidence interval

GMM:

Gaussian mixture model

FFPE:

Formalin-fixed paraffin-embedded

QG2:

QuantiGene Plex 2.0 Assay

ER:

Estrogen receptor alpha

PR:

Progesterone receptor

HER2:

Human epidermal growth factor receptor 2

IGF-IR:

Insulin-like growth factor I receptor

PPIB:

Peptidylprolyl isomerase B

GUSB:

Glucuronidase beta

gDNA:

Genomic DNA

FDA:

Food and Drug Administration

COMPAi:

Computer-assisted image analysis software

ROC:

Receiver-operating characteristic curve

CpG:

Cytosine-phosphate-guanine dinucleotides

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Acknowledgements

Biospecimens were provided by the National Cancer Institute-funded Cooperative Human Tissue Network, Tumor Tissue and Biospecimen Bank. and the Department of Pathology and Laboratory Medicine at the University of Pennsylvania. We are grateful to Caitlin Feltcher, Federico Valdivieso, Natalie Shih, Dee McGarvey, Deneen Mack, and Nicole Bollinger, and Gina Piermatteo for assistance with human tissue procurement and Li Ping for help with automated IHC. We thank Samantha Steeman for excellent assistance with editing and proofreading of this manuscript.

Funding

The authors received no external funding in support of this research.

Author information

Author notes

  1. Indira Prabakaran and Zhengdong Wu have contributed equally to this work.

Authors and Affiliations

  1. Department of Surgery, Division of Endocrine & Oncologic Surgery, Harrison Department of Surgical Research, Perelman School of Medicine, University of Pennsylvania, 416 Hill Pavilion, 380S University Avenue, Philadelphia, PA, 19104, USA

    Marina A. Guvakova, Indira Prabakaran, Daniel I. Hoffman, Ye Huang & Julia Tchou

  2. Department of Materials Science and Engineering, School of Engineering and Applied Science, 220 S 33rd St, Philadelphia, PA, 19104, USA

    Zhengdong Wu

  3. Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, 6 Founders, 3400 Spruce St, Philadelphia, PA, 19104, USA

    Paul J. Zhang

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  1. Marina A. Guvakova
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Contributions

M.A.G. designed and supervised all aspects of this study. I.P. acquired samples and performed QG2 assay and gDNA extraction. Z.W. performed statistical modeling. D.I.F., Y.H., J.T. reviewed biopsy charts. P.J.Z. re-reviewed archival tissue slides and pathology reports. I.P. constructed databases. M.A.G. and Z.W. analyzed the data, M.A.G. and I.P. wrote the paper. All authors approved the final manuscript.

Corresponding author

Correspondence to Marina A. Guvakova.

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Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This article does not contain any studies with animals performed by any of the authors. An approval from the University of Pennsylvania Institutional Review Board committee with a waiver of written informed consent was used for analysis of patients’ tissue and records.

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Electronic supplementary material

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Supplementary file1 Characteristics of the study population (PDF 30 kb)

Supplementary file2 The sample group comparison using Mann-Whitney U test with two-tailed exact p-values. (PDF 62 kb)

10549_2020_5797_MOESM3_ESM.pdf

Supplementary file3 Negative and positive tissue controls IHC –stained with an N –cadherin monoclonal antibody. Arrows, examples of microvasculature stained positive in IBC. Scale bar, 20µm. (PDF 31113 kb)

10549_2020_5797_MOESM4_ESM.pdf

Supplementary file4 Flow chart of inclusion and exclusion criteria of the study samples. *, preserved for the future patient care use; **, 5 years after treatment. HG, high grade, nonHG, non-high grade. (PDF 54 kb)

10549_2020_5797_MOESM5_ESM.pdf

Supplementary file5 Figure S3. Classification of CDH2-positive and CDH2-negative biopsy using GMM-based cutoff with confidence. A. GMM for overall (orange), similar to normal (green), and upregulated (red) gene expression in cancer are superimposed on histograms of frequency of CDH2 expression data in patients diagnosed with invasive breast cancer. B. Box plot depicts control CDH2 measurements in mammoplasties. Vertical lines in boxes, medians. Boxes. 25th and 75th percentile. Whiskers, 75th +1.5xIQR and 25th-1.5xIQR. X axis in A-C, mRNA levels in relative units. C. Graph for confidence calculated for invasive breast groups with changed gene expression as defined in Materials and Methods (blue). Step function plotted to illustrate minimal misclassification cutoffs as intersections of confidence and step function (green). The derivative of confidence with a half-height width plotted to define a range where the probability for sample to belong to either normal or changed group is similar (orange). The cutoff range was computed through the differentiation of confidence function followed by the calculation of the half-height width. D. Scatter plot for CDH2mRNA in the groups of mammoplasties (N, n=34), pure DCIS (DCIS, n=54), synchronous DCIS (DCIS w. INV, n=21), and IBC (n=133). E. Sample classification in normal (N), DCIS (DCIS) and DCIS with invasion (DCIS w. INV) groups based on three cutoffs. CDH2 positive and negative samples are depicted in red and green, respectively. The histogram, a summary of the proportion of CDH2 positive samples in each group classified with three cutoffs. (PDF 329 kb)

10549_2020_5797_MOESM6_ESM.pdf

Supplementary file6 The analysis of fourteen ROC models predicting high-grade DCIS. Forest plots; Y axis, 14 genes; X axis, AUC, bar, 95% CI (PDF 34 kb)

10549_2020_5797_MOESM7_ESM.pdf

Supplementary file7 A. The methylation ratios (Y axis = 0-1) were compared in 157 individual CpG site between reference group (CDH2-, CDH2mRNA levels ≤2.2; n=12) and test group (CDH2+, CDH2mRNA levels >2.2; n=12) of DCIS. Samples were coded and examined in a blind fashion. Arrow, CpG_25757619 site affected by hypermethylation. B. Comparison summary of the methylation differences at each of 157 examined CpG site between the test and reference groups of DCIS. A site with statistically significant difference in the methylation is in red. P-value, Student’s test. (PDF 156 kb)

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Guvakova, M.A., Prabakaran, I., Wu, Z. et al. CDH2/N-cadherin and early diagnosis of invasion in patients with ductal carcinoma in situ. Breast Cancer Res Treat 183, 333–346 (2020). https://doi.org/10.1007/s10549-020-05797-x

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