Fluid biopsy in patients with metastatic prostate, pancreatic and breast cancers

Abstract

Hematologic spread of carcinoma results in incurable metastasis; yet, the basic characteristics and travel mechanisms of cancer cells in the bloodstream are unknown. We have established a fluid phase biopsy approach that identifies circulating tumor cells (CTCs) without using surface protein-based enrichment and presents them in sufficiently high definition (HD) to satisfy diagnostic pathology image quality requirements. This 'HD-CTC' assay finds >5 HD-CTCs mL(-1) of blood in 80% of patients with metastatic prostate cancer (n = 20), in 70% of patients with metastatic breast cancer (n = 30), in 50% of patients with metastatic pancreatic cancer (n = 18), and in 0% of normal controls (n = 15). Additionally, it finds HD-CTC clusters ranging from 2 HD-CTCs to greater than 30 HD-CTCs in the majority of these cancer patients. This initial validation of an enrichment-free assay demonstrates our ability to identify significant numbers of HD-CTCs in a majority of patients with prostate, breast and pancreatic cancers.

Figure 1

Gallery of representative HD-CTCs found in cancer patients. Each HD-CTC is cytokeratin positive (red), CD45 negative (green), contains a DAPI nucleus (blue), and is morphologically distinct from surrounding white blood cells.

Figure 2

Mean observed SKBR3s plotted against expected SKBR3s. Four aliquots of normal control blood was spiked with varying numbers of SKBR2 cells to produce 4 slides with approximately 10, 30, 100, and 300 cancer cells per slide. The mean of each quadruplicate is displayed as well as error bars noting standard deviation.

Figure 3

Comparison of CTC counts between two separate processors on 9 different cancer patient samples. CTC/mL counts ranged from 0 to 203.

Figure 4

Gallery of representative clusters that are found in most patients with cancer. Clusters range from 2 to over 30 HD-CTCs. Each HD-CTC is cytokeratin positive (red), CD45 negative (green), contains a DAPI nucleus (blue), and is morphologically distinct from surrounding nucleated cells.

Figure 5

Gallery of candidate HD-CTCs that were excluded because they lacked various morphologic or morphometric inclusion criteria. A) Cytokeratin intensity too dim. B) Nuclear size too small. C) Cytokeratin insufficiently circumferential (surrounds less than 2/3 of nucleus) D) Cytokeratin too dim, although appears to be a cluster of two very large cells. E) Nucleus shows apoptotic disintegration changes. F) Nucleus too small and cytoplasm insufficiently circumferential; appears to be a cell in late apoptosis. G) Nucleus too small (same size as surrounding WBC nuclei). H. Cytokeratin present, but not circumferential. I) Cytoplasm insufficiently circumferential, nucleus too small.

Figure 6

Representative images of types of suspected CTCs found in a single prostate cancer patient. Top Row: Suspected CTC that is negative for Cytokeratin and CD45, but has a nucleus that is large and looks like other HD-CTCs found in this patient. Middle Row: Typical HD-CTC that is cytokeratin positive, CD45 negative, with a DAPI nucleus. Bottom Row: HD-CTC cluster of 4 cells.