Recent years have seen development and implementation of anticancer therapies targeted to particular gene mutations, but methods to assay clinical cancer specimens in a comprehensive way for the critical mutations remain underdeveloped. We have developed UW-OncoPlex, a clinical molecular diagnostic assay to provide simultaneous deep-sequencing information, based on >500× average coverage, for all classes of mutations in 194 clinically relevant genes. To validate UW-OncoPlex, we tested 98 previously characterized clinical tumor specimens from 10 different cancer types, including 41 formalin-fixed paraffin-embedded tissue samples. Mixing studies indicated reliable mutation detection in samples with ≥ 10% tumor cells. In clinical samples with ≥ 10% tumor cells, UW-OncoPlex correctly identified 129 of 130 known mutations [sensitivity 99.2%, (95% CI, 95.8%-99.9%)], including single nucleotide variants, small insertions and deletions, internal tandem duplications, gene copy number gains and amplifications, gene copy losses, chromosomal gains and losses, and actionable genomic rearrangements, including ALK-EML4, ROS1, PML-RARA, and BCR-ABL. In the same samples, the assay also identified actionable point mutations in genes not previously analyzed and novel gene rearrangements of MLL and GRIK4 in melanoma, and of ASXL1, PIK3R1, and SGCZ in acute myeloid leukemia. To best guide existing and emerging treatment regimens and facilitate integration of genomic testing with patient care, we developed a framework for data analysis, decision support, and reporting clinically actionable results.

This paper describes an approach called ensemble decision aliquot ranking (eDAR) for isolating rare cells from peripheral blood. eDAR has a recovery of over 93% (n=9) with a zero false positive rate (n=8), and provides direct easy access to individual isolated live cells for downstream single-cell manipulation and analysis. We anticipate eDAR will enable new studies of various types of rare cells that circulate in blood.

Enumeration of circulating tumor cells (CTCs) has proved valuable for early detection and prognosis in cancer treatment. This paper describes an automated high-throughput counting method for CTCs based on microfluidics and line-confocal microscopy. Peripheral blood was directly labeled with multiple antibodies, each conjugated with a different fluorophore, pneumatically pumped through a microfluidic channel and interrogated by a line-confocal microscope. Based on the fluorescence signals and labeling schemes, the count of CTCs was automatically reported. Due to the high flow rate, 1 mL of whole blood can be analyzed in less than 30 minutes. We applied this method in analyzing CTCs from 90 stage IV breast-cancer patient samples, and performed a side-by-side comparison with the results of the CellSearch assay, which is the only method approved by U.S. Food and Drug Administration (FDA) at present for enumeration of CTCs. This method has a recovery rate for cultured breast cancer cells of 94% (n=9), with an average of 1.2 counts/mL of background level of detected CTCs from healthy donors. It detected CTCs from breast-cancer patients, ranging from 15 to 3375 counts/7.5 mL. Using this method, we also demonstrate the ability to enumerate CTCs from breast-cancer patients that were positive for Her2 or CD44+/CD24−, which is a putative cancer stem cell marker. This automated method can enumerate CTCs from peripheral blood with high throughput and sensitivity. It could potentially benefit the clinical diagnosis and prognosis of cancer.

The function of the zeta-globin promoter was studied using a series of zeta-globin promoter deletion constructs to drive luciferase expression in transiently transfected human erythroleukemia cells. The promoters were used without enhancers, or with enhancers derived from the beta-globin locus control region and the alpha-globin HS-40 enhancer. When transfected into K562 cells, which express zeta-globin, comparable amounts of activity were obtained from the -557 and -417 zeta-luciferase constructs and the alpha-luciferase constructs when no enhancers or the alpha-globin locus enhancers were used. When the constructs were transfected into OCIM1 cells, which do not express zeta-globin, the zeta-globin promoters were at best 20% as active as the alpha-globin promoters. When sequences from -417 to -207 5′ to the zeta-globin mRNA cap site were deleted, up to 95% of the zeta-globin promoter activity was lost in K562 cells. Reinsertion of these sequences into zeta-luciferase constructs missing the -417 to -207 region showed that the sequences lack classical enhancer activity. Point mutation of a GATA-1 site at -230 reduced promoter activity by 37%. Point mutation of a CCACC site at -240 had no effect. Electrophoretic mobility shift assays indicated that the -230 GATA-1 site has a relatively low affinity for GATA-1. These experiments show the presence of a strong positive-acting element, located between -417 and -207 bp 5′ to the zeta-globin mRNA cap site, is necessary for high-level promoter activity in K562 cells. This element requires GATA-1 and additional unknown factors for maximal activity.

BACKGROUND Quantitation of circulating tumor cells (CTCs) has utility in managing breast, colon and prostate carcinomas. OBJECTIVE Determine whether a commercially available CTC assay provides prognostic information in MCC and/or insight into treatment responses. METHODS We analyzed CTCs in 52 specimens from 34 MCC patients. RESULTS The presence of CTCs correlated with extent of disease at blood draw (p=0.004). Among 15 patients with regional nodal disease, CTC-negative patients had 80% disease-specific survival at 2 years after the test, versus 29% for CTC-positive patients (p=0.015). Among the entire cohort, those without CTCs had 72% MCC-specific survival while CTC-positive patients had 25% survival (n=34, median follow-up 19 months, p=0.0003). 57% of MCC patients had a cytokeratin “dot” visible in ≥20% of CTCs, a feature that was absent among CTCs from other carcinomas (zero of 13 cases). LIMITATIONS CTC assay was performed at variable times after diagnosis and heterogeneity in extent of disease affects interpretability of the data. CONCLUSION CTC detection in MCC is feasible and appears to add prognostic information, particularly in patients with regional nodal disease. It may also assist clinical management in certain situations, including differentiating metastatic MCC cells from those of other carcinomas.