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Targeted Mutation Detection in Advanced Breast Cancer Using MammaSeq Identifies RET as a Potential Contributor to Breast Cancer Metastasis

Smith, Nicholas and Gyanchandani, Rekha and Priedigkeit, Nolan and Hartmaier, RJ and Chen, Yijing and Gurda, Gregorz and Lucas, PC and Brufsky, Adam and Puhalla, Shannon and Bahreini, Amir and Kota, Karthik and Wald, Abigail and Nikiforov, YE and Nikiforova, MN and Oesterreich, S and Lee, AV (2018) Targeted Mutation Detection in Advanced Breast Cancer Using MammaSeq Identifies RET as a Potential Contributor to Breast Cancer Metastasis. Master's Thesis, University of Pittsburgh. (Unpublished)

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The lack of any reported breast cancer specific diagnostic NGS tests inspired the development of MammaSeq, an amplicon based NGS panel built specifically for use in advanced breast cancer. In a pilot study to define the clinical utility of the panel, 46 solid tumor samples, plus an additional 14 samples of circulating-free DNA (cfDNA) from patients with advanced breast cancer were sequenced and analyzed using the OncoKB precision oncology database. We identified 26 clinically actionable variants (levels 1-3) annotated by the OncoKB precision oncology database, distributed across 20 out of 46 solid tumor cases (40%), and 4 clinically actionable mutations distributed across 4 samples in the 14 cfDNA sample cohort (29%). The mutation allele (MAF) frequencies of ESR1-D538G and FOXA1-Y175C mutations correlated with CA.27.29 levels in patient-matched blood, indicating that MAF may be a reliable marker for disease burden. Interestingly, 4 of the mutations found in metastatic samples occurred in the gene RET, an oncogenic receptor tyrosine kinase. In an orthogonal study, the lab has recently identified RET as one of the most recurrently upregulated genes in breast cancer brain metastases. Interestingly, the ligand for RET is the family of glial-cell derived neurotrophic factors (GDNF), a growth factor secreted by glial cells of the central nervous system. This lead to the hypothesis that RET overexpression facilitates breast cancer brain metastasis in response to the high levels of GDNF, while RET activating point mutations increase metastatic capacity without specific organ tropism. While the effect of GDNF treatment on proliferation in 2D was limited, in ultra-low attachment (ULA) plates we saw a significant increase in anchorage independent growth of MCF-7 cells. To determine if GDNF acts as a chemoattractant for RET positive BrCa cells, we utilized a transwell migration assay, with GDNF as the sole chemoattractant. When RET was overexpressed, there was a visual increase in cell migration. Together, these studies demonstrate the clinical feasibility of using MammaSeq to detect clinically actionable mutations in breast cancer patients, and provides provisional data supporting the investigation of RET signaling as a potentially targetable mediator of breast cancer brain metastasis.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Smith, Nicholasnis84@pitt.edunis84
Gyanchandani, Rekhareg31@pitt.eduREG31
Priedigkeit, Nolannmp50@pitt.edunmp50
Hartmaier, RJrjh70@pitt.eduRJH70
Chen, Yijing
Gurda, Gregorz
Lucas, PCpcl8@pitt.eduPCL8
Brufsky, Adamadb5@pitt.eduADB5
Puhalla, Shannonslp55@pitt.eduSLP55
Bahreini, Amir
Kota, Karthik
Wald, Abigailaib7@pitt.eduAIB7
Nikiforov, YEyen1@pitt.eduYEN1
Nikiforova, MNmnn4@pitt.eduMNN4
Oesterreich, Ssto16@pitt.eduSTO16
Lee, AVavl10@pitt.eduAVL10
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairWang, Janeqjw1@pitt.eduqjw1
Thesis AdvisorLee, Adrianavl10@pitt.eduavl10
Committee MemberOesterreich, Steffi
Committee MemberKohanbash, Gary
Date: 2 August 2018
Date Type: Publication
Defense Date: 27 June 2018
Approval Date: 2 August 2018
Submission Date: 29 July 2018
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Number of Pages: 80
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Molecular Pharmacology
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: NGS, rearranged during transfection
Date Deposited: 02 Aug 2018 13:01
Last Modified: 02 Aug 2018 13:01


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