Optimizing fresh specimen staining for rapid identification of tumor biomarkers during surgery

Connor W. Barth, Jasmin M. Schaefer, Vincent M. Rossi, Scott C. Davis, Summer Gibbs

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Rationale: Positive margin status due to incomplete removal of tumor tissue during breast conserving surgery (BCS) is a prevalent diagnosis usually requiring a second surgical procedure. These follow-up procedures increase the risk of morbidity and delay the use of adjuvant therapy; thus, significant efforts are underway to develop new intraoperative strategies for margin assessment to eliminate re-excision procedures. One strategy under development uses topical application of dual probe staining and a fluorescence imaging strategy termed dual probe difference specimen imaging (DDSI). DDSI uses a receptor-targeted fluorescent probe and an untargeted, spectrally-distinct fluorescent companion imaging agent topically applied to fresh resected specimens, where the fluorescence from each probe is imaged and a normalized difference image is computed to identify tumor-target distribution in the specimen margins. While previous reports suggested this approach is a promising new tool for surgical guidance, advancing the approach into the clinic requires methodical protocol optimization and further validation. Methods: In the present study, we used breast cancer xenografts and receiver operator characteristic (ROC) curve analysis to evaluate a wide range of staining and imaging parameters, and completed a prospective validation study on multiple tumor phenotypes with different target expression. Imaging fluorophore-probe pair, concentration, and incubation times were systematically optimized using n=6 tissue specimen replicates per staining condition. Resulting tumor vs. normal adipose tissue diagnostic performance were reported and staining patterns were validated via receptor specific immunohistochemistry colocalization. Optimal staining conditions were tested in receptor positive and receptor negative cohorts to confirm specificity. Results: The optimal staining conditions were found to be a one minute stain in a 200 nM probe solution (area under the curve (AUC) = 0.97), where the choice of fluorescent label combination did not significantly affect the diagnostic performance. Using an optimal threshold value determined from ROC curve analysis on a training data set, a prospective study on xenografts resulted in an AUC=0.95 for receptor positive tumors and an AUC = 0.50 for receptor negative (control) tumors, confirming the diagnostic performance of this novel imaging technique. Conclusions: DDSI provides a robust, molecularly specific imaging methodology for identifying tumor tissue over benign mammary adipose tissue. Using a dual probe imaging strategy, nonspecific accumulation of targeted probe was corrected for and tumor vs. normal tissue diagnostic potential was improved, circumventing difficulties with ex vivo tissue specimen staining and allowing for rapid clinical translation of this promising technology for tumor margin detection during BCS procedures.

Original languageEnglish (US)
Pages (from-to)4722-4734
Number of pages13
JournalTheranostics
Volume7
Issue number19
DOIs
StatePublished - 2017

Fingerprint

Tumor Biomarkers
Staining and Labeling
Neoplasms
Area Under Curve
Segmental Mastectomy
Heterografts
Adipose Tissue
Prospective Studies
Validation Studies
Optical Imaging
Fluorescent Dyes
Breast
Coloring Agents
Fluorescence
Immunohistochemistry
Breast Neoplasms
Technology
Morbidity
Phenotype

Keywords

  • Breast cancer
  • Breast conserving surgery
  • Dual probe difference specimen imaging
  • Dual probe imaging
  • Fluorescence
  • Image-guided surgery
  • Tumor margin assessment

ASJC Scopus subject areas

  • Medicine (miscellaneous)
  • Pharmacology, Toxicology and Pharmaceutics (miscellaneous)

Cite this

Optimizing fresh specimen staining for rapid identification of tumor biomarkers during surgery. / Barth, Connor W.; Schaefer, Jasmin M.; Rossi, Vincent M.; Davis, Scott C.; Gibbs, Summer.

In: Theranostics, Vol. 7, No. 19, 2017, p. 4722-4734.

Research output: Contribution to journalArticle

Barth, Connor W. ; Schaefer, Jasmin M. ; Rossi, Vincent M. ; Davis, Scott C. ; Gibbs, Summer. / Optimizing fresh specimen staining for rapid identification of tumor biomarkers during surgery. In: Theranostics. 2017 ; Vol. 7, No. 19. pp. 4722-4734.
@article{e5cf1da1dbdb4c37b6c380068634373f,
title = "Optimizing fresh specimen staining for rapid identification of tumor biomarkers during surgery",
abstract = "Rationale: Positive margin status due to incomplete removal of tumor tissue during breast conserving surgery (BCS) is a prevalent diagnosis usually requiring a second surgical procedure. These follow-up procedures increase the risk of morbidity and delay the use of adjuvant therapy; thus, significant efforts are underway to develop new intraoperative strategies for margin assessment to eliminate re-excision procedures. One strategy under development uses topical application of dual probe staining and a fluorescence imaging strategy termed dual probe difference specimen imaging (DDSI). DDSI uses a receptor-targeted fluorescent probe and an untargeted, spectrally-distinct fluorescent companion imaging agent topically applied to fresh resected specimens, where the fluorescence from each probe is imaged and a normalized difference image is computed to identify tumor-target distribution in the specimen margins. While previous reports suggested this approach is a promising new tool for surgical guidance, advancing the approach into the clinic requires methodical protocol optimization and further validation. Methods: In the present study, we used breast cancer xenografts and receiver operator characteristic (ROC) curve analysis to evaluate a wide range of staining and imaging parameters, and completed a prospective validation study on multiple tumor phenotypes with different target expression. Imaging fluorophore-probe pair, concentration, and incubation times were systematically optimized using n=6 tissue specimen replicates per staining condition. Resulting tumor vs. normal adipose tissue diagnostic performance were reported and staining patterns were validated via receptor specific immunohistochemistry colocalization. Optimal staining conditions were tested in receptor positive and receptor negative cohorts to confirm specificity. Results: The optimal staining conditions were found to be a one minute stain in a 200 nM probe solution (area under the curve (AUC) = 0.97), where the choice of fluorescent label combination did not significantly affect the diagnostic performance. Using an optimal threshold value determined from ROC curve analysis on a training data set, a prospective study on xenografts resulted in an AUC=0.95 for receptor positive tumors and an AUC = 0.50 for receptor negative (control) tumors, confirming the diagnostic performance of this novel imaging technique. Conclusions: DDSI provides a robust, molecularly specific imaging methodology for identifying tumor tissue over benign mammary adipose tissue. Using a dual probe imaging strategy, nonspecific accumulation of targeted probe was corrected for and tumor vs. normal tissue diagnostic potential was improved, circumventing difficulties with ex vivo tissue specimen staining and allowing for rapid clinical translation of this promising technology for tumor margin detection during BCS procedures.",
keywords = "Breast cancer, Breast conserving surgery, Dual probe difference specimen imaging, Dual probe imaging, Fluorescence, Image-guided surgery, Tumor margin assessment",
author = "Barth, {Connor W.} and Schaefer, {Jasmin M.} and Rossi, {Vincent M.} and Davis, {Scott C.} and Summer Gibbs",
year = "2017",
doi = "10.7150/thno.21527",
language = "English (US)",
volume = "7",
pages = "4722--4734",
journal = "Theranostics",
issn = "1838-7640",
publisher = "Ivyspring International Publisher",
number = "19",

}

TY - JOUR

T1 - Optimizing fresh specimen staining for rapid identification of tumor biomarkers during surgery

AU - Barth, Connor W.

AU - Schaefer, Jasmin M.

AU - Rossi, Vincent M.

AU - Davis, Scott C.

AU - Gibbs, Summer

PY - 2017

Y1 - 2017

N2 - Rationale: Positive margin status due to incomplete removal of tumor tissue during breast conserving surgery (BCS) is a prevalent diagnosis usually requiring a second surgical procedure. These follow-up procedures increase the risk of morbidity and delay the use of adjuvant therapy; thus, significant efforts are underway to develop new intraoperative strategies for margin assessment to eliminate re-excision procedures. One strategy under development uses topical application of dual probe staining and a fluorescence imaging strategy termed dual probe difference specimen imaging (DDSI). DDSI uses a receptor-targeted fluorescent probe and an untargeted, spectrally-distinct fluorescent companion imaging agent topically applied to fresh resected specimens, where the fluorescence from each probe is imaged and a normalized difference image is computed to identify tumor-target distribution in the specimen margins. While previous reports suggested this approach is a promising new tool for surgical guidance, advancing the approach into the clinic requires methodical protocol optimization and further validation. Methods: In the present study, we used breast cancer xenografts and receiver operator characteristic (ROC) curve analysis to evaluate a wide range of staining and imaging parameters, and completed a prospective validation study on multiple tumor phenotypes with different target expression. Imaging fluorophore-probe pair, concentration, and incubation times were systematically optimized using n=6 tissue specimen replicates per staining condition. Resulting tumor vs. normal adipose tissue diagnostic performance were reported and staining patterns were validated via receptor specific immunohistochemistry colocalization. Optimal staining conditions were tested in receptor positive and receptor negative cohorts to confirm specificity. Results: The optimal staining conditions were found to be a one minute stain in a 200 nM probe solution (area under the curve (AUC) = 0.97), where the choice of fluorescent label combination did not significantly affect the diagnostic performance. Using an optimal threshold value determined from ROC curve analysis on a training data set, a prospective study on xenografts resulted in an AUC=0.95 for receptor positive tumors and an AUC = 0.50 for receptor negative (control) tumors, confirming the diagnostic performance of this novel imaging technique. Conclusions: DDSI provides a robust, molecularly specific imaging methodology for identifying tumor tissue over benign mammary adipose tissue. Using a dual probe imaging strategy, nonspecific accumulation of targeted probe was corrected for and tumor vs. normal tissue diagnostic potential was improved, circumventing difficulties with ex vivo tissue specimen staining and allowing for rapid clinical translation of this promising technology for tumor margin detection during BCS procedures.

AB - Rationale: Positive margin status due to incomplete removal of tumor tissue during breast conserving surgery (BCS) is a prevalent diagnosis usually requiring a second surgical procedure. These follow-up procedures increase the risk of morbidity and delay the use of adjuvant therapy; thus, significant efforts are underway to develop new intraoperative strategies for margin assessment to eliminate re-excision procedures. One strategy under development uses topical application of dual probe staining and a fluorescence imaging strategy termed dual probe difference specimen imaging (DDSI). DDSI uses a receptor-targeted fluorescent probe and an untargeted, spectrally-distinct fluorescent companion imaging agent topically applied to fresh resected specimens, where the fluorescence from each probe is imaged and a normalized difference image is computed to identify tumor-target distribution in the specimen margins. While previous reports suggested this approach is a promising new tool for surgical guidance, advancing the approach into the clinic requires methodical protocol optimization and further validation. Methods: In the present study, we used breast cancer xenografts and receiver operator characteristic (ROC) curve analysis to evaluate a wide range of staining and imaging parameters, and completed a prospective validation study on multiple tumor phenotypes with different target expression. Imaging fluorophore-probe pair, concentration, and incubation times were systematically optimized using n=6 tissue specimen replicates per staining condition. Resulting tumor vs. normal adipose tissue diagnostic performance were reported and staining patterns were validated via receptor specific immunohistochemistry colocalization. Optimal staining conditions were tested in receptor positive and receptor negative cohorts to confirm specificity. Results: The optimal staining conditions were found to be a one minute stain in a 200 nM probe solution (area under the curve (AUC) = 0.97), where the choice of fluorescent label combination did not significantly affect the diagnostic performance. Using an optimal threshold value determined from ROC curve analysis on a training data set, a prospective study on xenografts resulted in an AUC=0.95 for receptor positive tumors and an AUC = 0.50 for receptor negative (control) tumors, confirming the diagnostic performance of this novel imaging technique. Conclusions: DDSI provides a robust, molecularly specific imaging methodology for identifying tumor tissue over benign mammary adipose tissue. Using a dual probe imaging strategy, nonspecific accumulation of targeted probe was corrected for and tumor vs. normal tissue diagnostic potential was improved, circumventing difficulties with ex vivo tissue specimen staining and allowing for rapid clinical translation of this promising technology for tumor margin detection during BCS procedures.

KW - Breast cancer

KW - Breast conserving surgery

KW - Dual probe difference specimen imaging

KW - Dual probe imaging

KW - Fluorescence

KW - Image-guided surgery

KW - Tumor margin assessment

UR - http://www.scopus.com/inward/record.url?scp=85032202913&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85032202913&partnerID=8YFLogxK

U2 - 10.7150/thno.21527

DO - 10.7150/thno.21527

M3 - Article

VL - 7

SP - 4722

EP - 4734

JO - Theranostics

JF - Theranostics

SN - 1838-7640

IS - 19

ER -