Rapid phenotypic and genomic change in response to therapeutic pressure in prostate cancer inferred by high content analysis of single circulating tumor cells

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

Modifying a sealed tube zinc reduction method for preparation of AMS graphite targets: Reducing background and attaining high precision

The sealed tube zinc reduction method for converting CO2 to graphite for AMS C-14 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 similar to 50,000 C-14 years when analyzing for C-14 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 delta C-13 of our zinc-reduced graphite is usually lighter by 2-37 parts per thousand than the CO2 from which it is made, but this is corrected for in our system by simultaneous measurement of C-13/C-12 along with C-14/C-12 by the spectrometer. This method is suitable for C-14 enriched samples, as well as natural abundance C-14 samples, especially those with modern C-14 contents. With improved precision and background, we believe that many disciplines can benefit from this technique because of its low cost and rapid production of graphite. (c) 2007 Elsevier B.V. All rights reserved

Sequential monitoring of single-cell copy number variation in metastatic prostate cancer

The high-definition circulating tumor cell (HD-CTC) assay provides for an enrichment-free approach to CTC identification. Here, we utilized the HD-CTC approach to study androgen receptor (AR) expression combined with single-nucleus sequencing for genome-wide analysis of copy number variation (CNV) in sequential CTCs samples obtained from a patient with metastatic prostate cancer treated with abiraterone acetate (an androgen synthesis inhibitor). At baseline, before initiation of abiraterone treatment, we observed a balanced proportion of AR-negative and AR-positive CTCs. During a brief period of clinical response (marked with a decreased serum PSA and decreased pain) the proportion of AR-positive CTCs declined followed by a rapid increase associated with clinical progression (increased PSA and pain). CNV analysis of single CTCs revealed multiple genomic rearrangements, such as AR amplification along with the chromosomal gains and losses typical of prostate cancer, in multiple cells at baseline. During treatment response, the frequency of CNV alterations significantly declined, followed by a reemergence to a pattern of multiple, complex alterations associated with clinical progression. Detailed analysis of the CNV profiles revealed that many abnormalities were commonly shared between the CTC populations, but a number were unique to the AR+ resistant/hormone refractory CTC population including increased MYC amplification alteration and the AR amplicon that include additional adjacent genes. Remarkably, the reconstruction of tumor lineage history based on the CTC genomic profiles enables us to trace and identify the precise treatment time point where the putative therapy-resistant CTC clone emerges, under therapeutic pressure, until it eventually expanded to become the AR+ resistant CTC population at the point of therapeutic relapse. Overall, our results demonstrated that the integration of the HD-CTC enumeration technology with protein expression and single cell genomic analyses could successfully be applied to real time monitoring of ADT therapy emergent change in a prostate cancer patient, and may provide a direct roadmap for personalized cancer medicine in the near future