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Research Article
Timothy Su
Vanderbilt University Medical Center
Corresponding Author
ORCiD: https://orcid.org/0000-0002-7831-1947
Qiuyin Cai
Vanderbilt University Medical Center
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Purpose Tumor-infiltrating lymphocytes (TILs) have emerged as predictive biomarkers for cancer prognosis. The clinicopathological significance of spatial TIL subpopulations is not well studied due to lack of high-throughput scalable methodology for large population studies.
Methods We established a fluorescent multiplex immunohistochemistry (mIHC) method coupled with computer-assisted high-throughput quantitative analysis. We then evaluated associations of six TILs markers (CD3, CD8, CD20, CD56, FOXP3, and PD-L1) with breast cancer prognosis among 188 tumor samples from the Nashville Breast Health Study.
Results Our 5-plex fluorescent mIHC workflow was reliable, highly sensitive, non-interfering, and balanced labeling for three biomarkers per tissue section, which is applicable for high-throughput imaging quantification of spatial TILs in regular laboratory settings. In this study we found that the increased intratumoral CD56+ cells were associated with worse overall survival (OS) (HR, 4.89; 95% CI: 1.26-18.95, highest vs lowest tertiles; Ptrend = 0.022), suggesting the subset of immunosuppressive NK cell phenotypes within tumor bed. Increased stromal PD-L1+ cells (HR, 0.01; 95% CI, 0.00-0.89; Ptrend = 0.042) and a high stromal CD8+/FOXP3+ ratio (HR, 0.00; 95% CI, 0.00-0.12; Ptrend = 0.036) were associated with more favorable OS in stage III-IV breast cancer patients.
Conclusion We established a reliable 5-plex fluorescent mIHC and showed that CD56+, PD-L1, and CD8+/FOXP3+ ratio may be important biomarkers for breast cancer prognosis. Further studies with a larger sample size are warrant to further elucidate the association between TILs and breast cancer outcomes.
Keywords
Breast cancer, Tumor-infiltrating lymphocytes, Multiplex immunohistochemistry, Quantitative imaging analysis, Prognosis
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This preprint is under consideration at Breast Cancer Research and Treatment. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research Article
Timothy Su
Vanderbilt University Medical Center
Corresponding Author
ORCiD: https://orcid.org/0000-0002-7831-1947
Qiuyin Cai
Vanderbilt University Medical Center
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 1
Posted 15 Mar, 2021
No community comments so far
Review #? received
Received 21 Mar, 2021
Reviewers invited
Invitations sent on 09 Mar, 2021
Editor assigned
On 08 Mar, 2021
First submitted
On 08 Mar, 2021
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Purpose Tumor-infiltrating lymphocytes (TILs) have emerged as predictive biomarkers for cancer prognosis. The clinicopathological significance of spatial TIL subpopulations is not well studied due to lack of high-throughput scalable methodology for large population studies.
Methods We established a fluorescent multiplex immunohistochemistry (mIHC) method coupled with computer-assisted high-throughput quantitative analysis. We then evaluated associations of six TILs markers (CD3, CD8, CD20, CD56, FOXP3, and PD-L1) with breast cancer prognosis among 188 tumor samples from the Nashville Breast Health Study.
Results Our 5-plex fluorescent mIHC workflow was reliable, highly sensitive, non-interfering, and balanced labeling for three biomarkers per tissue section, which is applicable for high-throughput imaging quantification of spatial TILs in regular laboratory settings. In this study we found that the increased intratumoral CD56+ cells were associated with worse overall survival (OS) (HR, 4.89; 95% CI: 1.26-18.95, highest vs lowest tertiles; Ptrend = 0.022), suggesting the subset of immunosuppressive NK cell phenotypes within tumor bed. Increased stromal PD-L1+ cells (HR, 0.01; 95% CI, 0.00-0.89; Ptrend = 0.042) and a high stromal CD8+/FOXP3+ ratio (HR, 0.00; 95% CI, 0.00-0.12; Ptrend = 0.036) were associated with more favorable OS in stage III-IV breast cancer patients.
Conclusion We established a reliable 5-plex fluorescent mIHC and showed that CD56+, PD-L1, and CD8+/FOXP3+ ratio may be important biomarkers for breast cancer prognosis. Further studies with a larger sample size are warrant to further elucidate the association between TILs and breast cancer outcomes.
Figure 1
Figure 2
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