Abstract P3-02-03: Detection of mutations in single tumor cells collected by fine needle aspiration in a mouse xenograft breast cancer model using MALDI-TOF
Authors: Stilwell JL , Hobeida A , Birse RT , Ericson N , Ramirez AB , Hummel S , Irwin D , Kaldjian EP , Lyerly HK 
Published: February 2018

Abstract
Background: Breast fine needle aspiration (FNA) is less invasive than a core needle biopsy and reduces the risk of infection or injury to the patient. However, less tissue is available for analysis than with biopsy. The ability to detect and analyze single atypical cells for molecular abnormalities would allow the consideration of more widely adopting FNA for diagnosis. Here we demonstrate the feasibility of this approach in a mouse xenograft model of breast cancer.
Methods: Human-into-mouse MBA-MD-231 breast cancer xenograft tumors were aspirated using a technique that approximates the clinical procedure in patients. Cells from the FNA were prepared by two methods: 1) mixing the aspirate with transfer fluid and spreading onto a Superfrost® Plus slide using RareCyte's AccuCyte® process, and 2) spraying the aspirate directly onto a Superfrost Plus slide, then drying and fixing in ethanol. A single tumor was also disaggregated into suspension, filtered, mixed with transfer fluid and spread on to slides as in method 1 above as a control. Slides were fixed in formalin, stained on an automated immunostainer and imaged using the CyteFinder® digital fluorescence scanning microscope. Tumor cells were identified by positive nuclear, EpCAM, and cytokeratin staining, and negative CD45 staining. Tumor cells were picked from the slides and put into PCR tubes using the CytePicker® module. DNA from individual cells was amplified using the PicoPLEX® (Rubicon) whole genome amplification (WGA) kit. Quality control (QC) of the WGA reactions was performed by PCR of amplicons on eight different chromosomes. Specific gene regions surrounding 5 mutations present in MBA-MD-231 cells were amplified from the WGA products and scored for the mutations using a single PCR reaction iPLEX® Pro panel using the MassARRAY® platform (Agena Bioscience). A lung tumor panel was also run as a negative control.
Results: FNA tumor cells stained with epithelial markers similarly to cells from the disaggregated tumor control and were easily identified. Slides prepared by method 1 above spread into a uniform monolayer making it easier to pick individual cells. Cells from method 2 tended to clump making it more difficult to pick individual cells. Cells were thus picked only from method 1 slides. QC measurements of WGA products from individual cells demonstrated broad genome coverage of amplification; 10 of 14 cells exhibited 7 or more QC products out of 8. Point mutations in four genes (BRAF, KRAS, NF2, and TP53) and a deletion in one gene (CDKN2A) were measured in these cells by iPLEX® Pro chemistry on the MassARRAY® system and found in all cells picked, with all mutations identified in most cells. These mutations and the deletion were not detected in control WBCs.
Conclusions: Individual breast cancer cells were identified in FNA samples from xenograft tumors and molecularly characterized, verifying that the cells identified by positive staining were tumor cells. These results demonstrate the feasibility of detecting and verifying tumor cells in FNA samples in breast and other cancers.
Citation Format: Stilwell JL, Hobeida A, Birse RT, Ericson N, Ramirez AB, Hummel S, Irwin D, Kaldjian EP, Lyerly HK. Detection of mutations in single tumor cells collected by fine needle aspiration in a mouse xenograft breast cancer model using MALDI-TOF [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-02-03.