The sealed tube zinc reduction method for converting CO 2 to graphite for AMS 14 C measurements was originally developed for rapid production of graphite in biomedical tracer experiments. The method was usually thought to have low precision and a high background. We have modified the zinc reduction method originally outlined in Vogel [J.S. Vogel, Radiocarbon 34 (3) (1992) 344] by carefully controlling the amounts of reagents (zinc, titanium hydride and Co or Fe catalyst) and now routinely obtain a precision of 2-3& and a relatively low background of $50,000 14 C years when analyzing for 14 C at the Keck Carbon Cycle AMS facility at UC Irvine. Fractionation of carbon isotopes does occur during graphitization and depends on the graphitization yield, which can be affected by the amounts of reagents used and other conditions. The d 13 C of our zinc-reduced graphite is usually lighter by 2-3& than the CO 2 from which it is made, but this is corrected for in our system by simultaneous measurement of 13 C/ 12

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 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 HDCTCs 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.

Circulating tumor cells (CTCs) have been implicated as a population of cells that may seed metastasis and venous thromboembolism (VTE), two major causes of mortality in cancer patients. Thus far, existing CTC detection technologies have been unable to reproducibly detect CTC aggregates in order to address what contribution CTC aggregates may have on metastasis or VTE. We report here an enrichment-free immunofluorescence detection method that can reproducibly detect and enumerate homotypic CTC aggregates in patient samples. We identified CTC aggregates in 43% of 86 patient samples. The fraction of CTC aggregation was investigated in blood draws from 24 breast, 14 non-small cell lung (NSCLC), 18 pancreatic, 15 prostate stage IV cancer patients, and 15 normal blood donors (NBD). Both single CTCs and CTC aggregates were measured to determine whether differences exist in the physical characteristics of these two populations. Cells contained in CTC aggregates had less area and length, on average, than single CTCs. Nuclear to cytoplasmic (N/C) ratio between single CTCs and CTC aggregates were similar. This detection method may assist future studies in determining which population of cells is more physically likely to contribute to metastasis and VTE.

Timely characterization of a cancer's evolution is required to predict treatment efficacy and to detect resistance early. High content analysis of single Circulating Tumor Cells (CTCs) enables sequential characterization of genotypic, morphometric and protein expression alterations in real time over the course of cancer treatment. This concept was investigated in a patient with castrate-resistant prostate cancer progressing through both chemotherapy and targeted therapy. In this case study, we integrate across four timepoints 41 genome-wide copy number variation (CNV) profiles plus morphometric parameters and androgen receptor (AR) protein levels. Remarkably, little change was observed in response to standard chemotherapy, evidenced by the fact that a unique clone (A), exhibiting highly rearranged CNV profiles and AR+ phenotype was found circulating before and after treatment. However, clinical response and subsequent progression after targeted therapy was associated with the drastic depletion of clone A, followed by the sequential emergence of two distinct CTC sub-populations that differed in both AR genotype and expression phenotype. While AR- cells with flat or pseudo-diploid CNV profiles (clone B) were identified at the time of response, a new tumor lineage of AR+ cells (clone C) with CNV altered profiles was detected during relapse. We showed that clone C, despite phylogenetically related to clone A, possessed a unique set of somatic CNV alterations, including MYC amplification, an event linked to hormone escape. Interesting, we showed that both clones acquired AR gene amplification by deploying different evolutionary paths. Overall, these data demonstrate the timeframe of tumor evolution in response to therapy and provide a framework for the multi-scale analysis of fluid biopsies to quantify and monitor disease evolution in individual patients.

Sampling circulating tumor cells from peripheral blood is ideally accomplished using assays that detect high numbers of cells and preserve them for downstream characterization. We sought to evaluate a method using enrichment free fluorescent labeling of CTCs followed by automated digital microscopy in patients with non-small cell lung cancer. Twenty-eight patients with non-small cell lung cancer and hematogenously seeded metastasis were analyzed with multiple blood draws. We detected CTCs in 68% of analyzed samples and found a propensity for increased CTC detection as the disease progressed in individual patients. CTCs were present at a median concentration of 1.6 CTCs per milliliter of analyzed blood in the patient population. Higher numbers of detected CTCs were associated with an unfavorable prognosis.