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OBJECTIVES: To examine the impact of positive surgical margin (PSM) laterality on failure after radical prostatectomy (RP). A PSM can influence local recurrence and outcomes after salvage radiation. Unlike intrinsic risk factors, a PSM is caused by intervention and thus iatrogenic failures may be elucidated by analyzing margin laterality as surgical approach is itself lateralized. PATIENTS AND METHODS: We reviewed 226 RP patients between 1991 and 2013 with PSM. Data includes operation type, pre/postoperative PSA, surgical pathology, and margin type (location, focality, laterality). The median follow-up was 47 months. Biochemical recurrence after RP was defined as PSA ≥ 0.1 ng/mL or 2 consecutive rises above nadir. Ninety-two patients received salvage radiation therapy (SRT). Failure after SRT was defined as any PSA ≥ 0.2 ng/mL or greater than presalvage. Kaplan-Meier and Cox multivariate analyses compared relapse rates. RESULTS: The majority of PSM were iatrogenic (58%). Laterality was associated with differences in median relapse: right 20 versus left 51 versus bilateral 14 months (P < 0.01). Preoperative PSA, T-stage, Gleason grade, and laterality were associated with biochemical progression on univariate and multivariate analyses. Right-sided margins were more likely to progress than left (hazard ratio, 1.67; P = 0.04). More right-sided margins were referred for SRT (55% right vs. 23% left vs. 22% bilateral), but were equally salvaged. Only T-stage and pre-SRT PSA independently influenced SRT success. CONCLUSIONS: Most PSM are iatrogenic, with right-sided more likely to progress (and sooner) than left sided. Margin laterality is a heretofore unrecognized independent predictor of biochemical relapse and hints at the need to modify the traditional unilateral surgical technique.

Pyrrole-imidazole (Py-Im) polyamides are synthetic non-genotoxic minor groove-binding small molecules. We hypothesized that Py-Im polyamides can modulate the cellular response to ionizing radiation. Pre-treatment of cells with a Py-Im polyamide prior to exposure to ionizing radiation resulted in a delay in resolution of phosphorylated γ-H2AX foci, increase in XRCC1 foci, and reduced cellular replication potential. RNA-sequencing of cell lines exposed to the polyamide showed induction of genes related to the ultraviolet radiation response. We observed that the polyamide is almost 10-fold more toxic to a cell line deficient in DNA ligase 3 as compared to the parental cell line. Alkaline single cell gel electrophoresis reveals that the polyamide induces genomic fragmentation in the ligase 3 deficient cell line but not the corresponding parental line. The polyamide interferes directly with DNA ligation in vitro. We conclude that Py-Im polyamides may be further explored as sensitizers to genotoxic therapies.

A blood-based, low cost alternative to radiation intensive CT and PET imaging is critically needed for cancer prognosis and management of its treatment. "Liquid biopsies" of circulating tumor cells (CTCs) from a relatively non-invasive blood draw are particularly ideal, as they can be repeated regularly to provide up to date molecular information about the cancer, which would also open up key opportunities for personalized therapies. Beyond solely diagnostic applications, CTCs are also a subject of interest for drug development and cancer research. In this paper, we adapt a technology previously introduced, combining the use of micro-scale vortices and inertial focusing, specifically for the high-purity extraction of CTCs from blood samples. First, we systematically varied parameters including channel dimensions and flow rates to arrive at an optimal device for maximum trapping efficiency and purity. Second, we validated the final device for capture of cancer cell lines in blood, considering several factors, including the effect of blood dilution, red blood cell lysis and cell deformability, while demonstrating cell viability and independence on EpCAM expression. Finally, as a proof-of-concept, CTCs were successfully extracted and enumerated from the blood of patients with breast (N = 4, 25-51 CTCs per 7.5 mL) and lung cancer (N = 8, 23-317 CTCs per 7.5 mL). Importantly, samples were highly pure with limited leukocyte contamination (purity 57-94%). This Vortex approach offers significant advantages over existing technologies, especially in terms of processing time (20 min for 7.5 mL of whole blood), sample concentration (collecting cells in a small volume down to 300 μL), applicability to various cancer types, cell integrity and purity. We anticipate that its simplicity will aid widespread adoption by clinicians and biologists who desire to not only enumerate CTCs, but also uncover new CTC biology, such as unique gene mutations, vesicle secretion and roles in metastatic processes.

Pyrrole (Py) - Imidazole (Im) polyamides are a class of non-genotoxic DNA-binding small molecules with modular sequence recognition and high affinity. A polyamide targeted to the sequence 5’-WGWWCW-3’, corresponding to the Androgen Receptor (AR) Response Element (ARE) half-site, suppresses AR positive prostate tumor xenografts. Evidence for inhibition of DNA dependent processes including allosteric inhibition of AR-DNA interactions and RNA Polymerase II transcription are observed in vitro. It is hypothesized that polyamides may displace DNA repair elements recruited after genotoxic insult and thereby sensitize cells to ionizing radiation.

Glioblastoma multiforme is a grade IV astrocytoma arising de novo or progressing from lower grade II and III gliomas. Currently the etiology of glioma and prediction of disease progression are unknown. Recent epidemiological studies have suggested that hormonal factors, including estrogen treatment, may impact glioma risk however these studies have been limited to case-control trials and retrospective cohort studies. Here, we evaluate results from the Women’s Health Initiative, which involved 161,808 women with robust data regarding hormone exposures. During a median of 12.7 years of follow-up, 167 cases of glioma (130 cases of GBM) were ascertained. The relationship between gliomas and hormone therapy (HT; estrogen-alone [E-alone] or estrogen plus progestin [E+P]) was evaluated using Cox proportional hazards models as well as Kaplan-Meier time-to-event analysis. There was no association with gliomas for the E-alone group (HR=0.76, 95% CI=0.43,1.36) but there was an inverse association for E+P (HR=0.48, 95% CI= 0.26, 0.88, p=0.02) after accounting for patient, hormone exposure and reproductive factors. Kaplan-Meier survival analysis demonstrated a significant reduction in time-to-incidence for the E+P group (p=0.0035). Findings from the matched case-control arm of the WHI trial did not demonstrate significant impact of HT on glioma incidence. The results of this study suggest a reduction in glioma risk after treatment with estrogen plus progesterone however a further large scale case-controlled study is warranted to evaluate the impact of HT on this disease.

Circulating tumor cells (CTCs) are cancer cells shed from a primary tumor, which enter the bloodstream and have the potential to metastasize. The detection and isolation of CTCs in bodily fluids can aid in cancer prognosis, characterizing genetic mutations for targeted therapies, and studying the biological mechanisms of metastasis. CTCs are very rare; there may only be tens of CTCs among millions of white blood cells and billions of red blood cells in one milliliter of blood. Current approaches for CTC isolation each have unique advantages and disadvantages: these techniques are often limited by speed, variable gene expression for immunocapture, long sample preparation steps, cost, and ability to deliver viable cells at high purity. Here, we use of arrays of microscale laminar fluid vortices to quickly isolate CTCs from large volumes of blood at high purity and without labels. Under high flow rates, large cells (> 15 μm) become stably trapped in laminar microvortices that form in simple rectangular reservoirs, while smaller red and white blood cells pass through. The large cancer cells are maintained in the microvortices, allowing for solution exchange, followed by release on lowering flow rate. The device is able to purify, enrich, and release a small volume (∼300 μL) of concentrated, viable cancer cells from blood at high throughput, concentration, and purity. The vortex chip successfully separated a variety of cancer cell lines from blood (∼20% capture efficiency), including those from melanoma, ovarian, breast, lung, and prostate cancer cell lines, with ∼10,000 fold enrichment. Captured cells remained viable and could be cultured off-chip. Finally, CTCs were successfully extracted and enumerated from the blood of patients with breast (N=4, 25-51 CTCs per 7.5 mL) and lung (N=8, 23-317 CTCs per 7.5 mL) cancers, and their sizes were measured. Importantly, samples were highly pure with limited leukocyte contamination (purity 57-94%). Preliminary comparisons with FDA-approved CellSearch system highlighted improved results for CTC enumeration from breast and lung samples. Cells isolated from the vortex chip can also be utilized for downstream molecular analysis. We successfully utilized a qPCR-based approach to detect specific KRAS point mutations from lung cancer cells spiked into pleural fluids and then isolated using the vortex chip and demonstrated improved detection sensitivity. The high purity of our approach should allow for improved molecular sequencing results, and additional molecular analyses of CTCs from patient blood samples are underway, utilizing next generation transcriptome sequencing technologies. This vortex approach offers significant advantages over existing technologies, especially in terms of processing time, low cost, simplicity, cell integrity and purity. The ability to obtain viable CTCs provides flexible opportunities for the clinicians and biologists who desire to not only enumerate CTCs, but also uncover new CTC biology, such as unique gene mutations, vesicle secretion and roles in metastatic processes. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A197. Citation Format: Elodie Sollier, James Che, Derek E. Go, Westbrook M. Weaver, Nicolas Kummer, Matthew Rettig, Jonathan Goldman, Nicholas Nickols, Dino Di Carlo, Rajan P. Kulkarni. Size-selective isolation of viable and pure CTCs for molecular analysis using vortex technology. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A197.