Abstract 3473: Sub-classification of prostate cancer circulating tumor cells (CTCs) by nuclear size reveals very-small nuclear CTCs in patients with visceral metastases
Authors: Chen Jie-Fu , Ho Hao , Lichterman Jake , Lu Yi-Tsung , Zhang Yang , Garcia Mitch A. , Chen Shang-Fu , Liang An-Jou , Zhau Haiyen E. , Hou Shuang , Ahmed Rafi S. , Luthringer Daniel J. , Huang Jiaoti , Li Ker-Chau , Chung Leland WK , Ke Zunfu , Tseng Hsian-Rong , Posadas Edwin M. 
Published: August 2015

Abstract
Background
Circulating tumor cells (CTCs) are an emerging biomarker in prostate cancer (PC). Most efforts have focused on enumeration, we and others have identified subsets within this pool of cells which may be clinically informative. Using the highly sensitive NanoVelcro technology for CTC isolation and fluorescence microscopy, our group sub-classified CTCs by nuclear size and focused on the correlation with sites of metastatic disease in advanced PC patients.
Methods
Blood samples were obtained from PC patients across the spectrum of metastatic states, i.e. no metastasis, non-visceral (osseous/lymph node) metastasis, and visceral metastasis. The patients were divided into training and validation cohorts. CTCs were captured from the blood and enumerated on NanoVelcro Chips. These were then subjected to pathologic review for cellular morphology and nuclear size. The distribution of nuclear sizes was analyzed using a Gaussian Mixture Model to sub-classify the CTCs. Correlations were made between CTC subpopulations and metastatic status. Individual CTCs were isolated by laser-capture microdissection for subsequent characterization focusing on cellular markers and transcriptomic signatures in comparison between vsnCTC and other CTC subpopulations.
Results
Statistical analysis and modeling of nuclear size distribution revealed 3 distinct subpopulations in the training cohort: large-nuclear CTCs (lnCTC), small-nuclear CTCs (snCTC), and very-small-nuclear CTCs (vsnCTCs). While the lnCTC subpopulation alone failed to distinguish metastatic disease from non-metastatic disease, snCTC + vsnCTC counts could make that distinction. Furthermore, vsnCTC counts were elevated in PC patients with visceral metastases when compared to those without visceral metastases (0.5 ± 0.5 versus 3.1 ± 5.2 cells/mL of blood, p = 0.005). This difference remained statistically significant in the validation cohort (0.6 ± 1.0 versus 3.5 ± 4.0 cells/mL of blood, p = 0.007). Serial enumerations for individual patients showed the emergence of vsnCTCs prior to detection of newly developed visceral lesions. The recurrence of vsnCTC in patients under treatment is also associated with radiographic progression of existing visceral lesions through therapy. Molecular characterization of vsnCTCs reveals differential expression of genes related to neuroendocrine, stem cell, and androgen biology.
Conclusions
Subsets of CTCs contain information on PC disease status when analyzed with pathologic approaches, such as nuclear size measurement. In particular, the detection of vsnCTCs correlated with the presence of visceral metastatic lesions and should be explored as a potential biomarker to identify patients at risk for developing this more aggressive form of PC.
Citation Format: Jie-Fu Chen, Hao Ho, Jake Lichterman, Yi-Tsung Lu, Yang Zhang, Mitch A. Garcia, Shang-Fu Chen, An-Jou Liang, Haiyen E. Zhau, Shuang Hou, Rafi S. Ahmed, Daniel J. Luthringer, Jiaoti Huang, Ker-Chau Li, Leland WK Chung, Zunfu Ke, Hsian-Rong Tseng, Edwin M. Posadas. Sub-classification of prostate cancer circulating tumor cells (CTCs) by nuclear size reveals very-small nuclear CTCs in patients with visceral metastases. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3473. doi:10.1158/1538-7445.AM2015-3473