Abstract P5-04-30: Developing an immunohistochemistry protocol to detect neurofibromin as an effective, simple, and rapid method to identify NF1-negative breast cancer patients
Authors: Peng J , Zheng Z-y , Cakar B , Li J , Singh P , Szafrain AT , Stossi F , Dubrulle J , Mancini MA , Mao R , Miles G , Ellis MJ , Chang EC 
Published: May 2019

Background:Neurofibromin is a key tumor suppressor, well-known as a GTPase-Activating-Protein (GAP) to attenuate Ras signaling. It is encoded by the NF1gene, so named because its inactivation was first discovered to cause Neurofibromatosis type 1, an autosomal dominant genetic disorder. We have recently reported thatnonsense (NS) and frameshift (FS) mutations, but not missense mutations, in NF1are associated with a markedly higher risk of relapse and death in early stage ER+breast cancer after adjuvant tamoxifen monotherapy (Griffith et al. in press). Surprisingly, despite being best known as a GAP, no missense mutations inactivating NF1's GAP activity were found in our cohort. We have evidence that these NF1NS/FS mutations cause the resulting mRNAs to be degraded, leading to depletion of the entire NF1 protein. In a separate study that was presented here last year, we showed that NF1 is also an ER co-repressor, which partially explains why the loss of the single tumor suppressor NF1 is so detrimental — because this turns on two potent oncogenic pathways. Thus far there is no effective means to assess loss of NF1 protein in cancer. The objective of this project is to identify these aggressive NF1-negative breast cancers by establishing an immunohistochemistry (IHC) protocol with a valid NF1 antibody in order to properly find and treat them in the future.

Methods:A monoclonal antibody was raised against the C-terminus of NF1. Immunostaining as well as IHC was performed using a set of breast cancer cell lines with varying degrees of NF1 protein levels, including several NF1 null-like cell lines as negative controls. To assess whether the IHC protocol can be used on patients, NF1+and NF1–PDXs as well as patient biopsies were examined.

Results: We have purified a monoclonal antibody against NF1 (m376). By immunostaining, a strong NF1 signal can be seen in T47D cells, which have four copies of the NF1gene. In contrast, there was barely any signal in MDA-MB-175VII cells, which lack detectable NF1 due to NF1FS mutations. While NF1 appears mostly cytoplasmic, 10-15% can be seen in the nucleus. Nuclear NF1 levels can be further increased by the nuclear export blocker leptomycin-B, suggesting that NF1 is shuttled in and out of the nucleus. IHC staining confirmed these features of NF1. In addition, a weak nuclear signal can be seen in cancer cells carrying NF1FS mutations. Experiments are on-going to assess how to analyze tumor samples for NF1 loss and whether NF1FS mutations cause expression of truncated proteins that are nuclear.

Conclusion: Our results suggest that the m376 antibody has the potential to be used for IHC, provided that samples known to be NF1+or NF1–are included as controls. The success of this project will have particular clinical impact in telling us who should notbe treated by tamoxifen. Furthermore, we have an approved clinical trial protocol to assess the concept that these NF1–patients should instead be treated by combining a Ras pathway inhibitor and a SERD.

Citation Format: Peng J, Zheng Z-y, Cakar B, Li J, Singh P, Szafrain AT, Stossi F, Dubrulle J, Mancini MA, Mao R, Miles G, Ellis MJ, Chang EC. Developing an immunohistochemistry protocol to detect neurofibromin as an effective, simple, and rapid method to identify NF1-negative breast cancer patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-30.