Purpose: The purpose of this study was to test the hypothesis that circulating tumor cells (CTCs) are present in patients many years after mastectomy without evidence of disease and that these CTCs are shed from persisting tumor in patients with breast cancer dormancy.Experimental Design: We searched for CTCs in 36 dormancy candidate patients and 26 age-matched controls using stringent criteria for cytomorphology, immunophenotype, and aneusomy.Results: Thirteen of 36 dormancy candidates, 7 to 22 years after mastectomy and without evidence of clinical disease, had CTCs, usually on more than one occasion. Only 1 of 26 controls had a possible CTC (no aneusomy). The statistical difference of these two distributions was significant (exact P ‫؍‬ 0.0043). The CTCs in patients whose primary breast cancer was just removed had a half-life measured in 1 to 2.4 hours. Conclusions:The CTCs that are dying must be replenished every few hours by replicating tumor cells somewhere in the tissues. Hence, there appears to be a balance between tumor replication and cell death for as long as 22 years in dormancy candidates. We conclude that this is one mechanism underlying tumor dormancy.

Amplification and overexpression of the HER-2 oncogene in breast cancer is felt to be stable over the course of disease and concordant between primary tumor and metastases. Therefore, patients with HER-2-negative primary tumors rarely will receive anti-Her-2 antibody (trastuzumab, Herceptin) therapy. A very sensitive blood test was used to capture circulating tumor cells (CTCs) and evaluate their HER-2 gene status by fluorescence in situ hybridization. The HER-2 status of the primary tumor and corresponding CTCs in 31 patients showed 97% agreement, with no false positives. In 10 patients with HER-2-positive tumors, the HER-2͞chromosome enumerator probe 17 ratio in each tumor was about twice that of the corresponding CTCs (mean 6.64 ؎ 2.72 vs. 2.8 ؎ 0.6). Hence, the ratio of the CTCs is a reliable surrogate marker for the expected high ratio in the primary tumor. Her-2 protein expression of 10 CTCs was sufficient to make a definitive diagnosis of the HER-2 gene status of the whole population of CTCs in 19 patients with recurrent breast cancer. Nine of 24 breast cancer patients whose primary tumor was HER-2-negative each acquired HER-2 gene amplification in their CTCs during cancer progression, i.e., 37.5% (95% confidence interval of 18.8 -59.4%). Four of the 9 patients were treated with Herceptin-containing therapy. One had a complete response and 2 had a partial response.

Screening for circulating tumor cells (CTCs) in blood has been an object of interest for evidence of progressive disease, status of disease activity, recognition of clonal evolution of molecular changes and for possible early diagnosis of cancer. We describe a new method of microchip-based immunomagnetic CTC detection, in which the benefits of both immunomagnetic assay and the microfluidic device are combined. As the blood sample flows through the microchannel closely above arrayed magnets, cancer cells labeled with magnetic nanoparticles are separated from blood flow and deposited at the bottom wall of the glass coverslip, which allows direct observation of captured cells with a fluorescence microscope. A polydimethylsiloxane (PDMS)-based microchannel fixed on a glass coverslip was used to screen blood samples. The thin, flat dimensions of the microchannel, combined with the sharp magnetic field gradient in the vicinity of arrayed magnets with alternate polarities, lead to an effective capture of labeled cells. Comparing to the commercially available CellSearch™ system, less (25%) magnetic particles are required to achieve a comparable capture rate, while the screening speed (at optimal blood flow rate of 10 mL/hour) is more than five times faster than those reported previously with a microchannel-based assay. For the screening experiment, blood drawn from healthy subjects into CellSave™ tubes was spiked with cultured cancer cell lines of COLO205 and SKBR3. The blood was then kept at room temperature for 48 hours before the screening, emulating the actual clinical cases of blood screening. Customized Fe3O4 magnetic nanoparticles (Veridex Ferrofluid™) conjugated to anti-Epithelial cell adhesion molecule (EpCAM) antibodies were introduced into the blood samples to label cancer cells, and the blood was then run through the microchip device to capture the labelled cells. After capture, the cells were stained with fluorescently labelled anti-cytokeratin, DAPI and anti-CD45. Subsequent immunofluorescence images were taken for the captured cells, followed by comprehensive computer aided analysis based on fluorescence intensities and cell morphology. Rare cancer cells (from ~1000 cells down to ~5 cells per mL) with very low tumor cell to blood cell ratios (about 1: 107~109, including red blood cells) were successfully detected. Cancer cell capture rates of 90% and 86% were demonstrated for COLO205 and SKBR3cells, respectively.

BackgroundFor community-dwelling older persons with dementia, the presence of multimorbidity can create complex clinical challenges for both individuals and their physicians, and can contribute to poor outcomes. We quantified the associations between level of multimorbidity (chronic disease burden) and risk of hospitalization and risk of emergency department (ED) visit in a home care cohort with dementia and explored the role of continuity of physician care (COC) in modifying these relationships.Methods and findingsA retrospective cohort study using linked administrative and clinical data from Ontario, Canada, was conducted among 30,112 long-stay home care clients (mean age 83.0 ± 7.7 y) with dementia in 2012. Multivariable Fine–Gray regression models were used to determine associations between level of multimorbidity and 1-y risk of hospitalization and 1-y risk of ED visit, accounting for multiple competing risks (death and long-term care placement). Interaction terms were used to assess potential effect modification by COC.Multimorbidity was highly prevalent, with 35% (n = 10,568) of the cohort having five or more chronic conditions. In multivariable analyses, risk of hospitalization and risk of ED visit increased monotonically with level of multimorbidity: sub-hazards were 88% greater (sub-hazard ratio [sHR] = 1.88, 95% CI: 1.72–2.05, p < 0.001) and 63% greater (sHR = 1.63; 95% CI: 1.51–1.77, p < 0.001), respectively, among those with five or more conditions, relative to those with dementia alone or with dementia and one other condition. Low (versus high) COC was associated with an increased risk of both hospitalization and ED visit in age- and sex-adjusted analyses only (sHR = 1.11, 95% CI: 1.07–1.16, p < 0.001, for hospitalization; sHR = 1.07, 95% CI: 1.03–1.11, p = 0.001, for ED visit) but did not modify associations between multimorbidity and outcomes (Wald test for interaction, p = 0.566 for hospitalization and p = 0.637 for ED visit). The main limitations of this study include use of fixed (versus time-varying) covariates and focus on all-cause rather than cause-specific hospitalizations and ED visits, which could potentially inform interventions.ConclusionsOlder adults with dementia and multimorbidity pose a particular challenge for health systems. Findings from this study highlight the need to reshape models of care for this complex population, and to further investigate health system and other factors that may modify patients’ risk of health outcomes.

Introduction At the time when metastatic disease is identified, assessment of human epidermal growth factor receptor (HER)2 status might help to optimize treatment decisions if HER2 status was not determined at first diagnosis and if HER2 positivity has been acquired during disease progression. Within this context, determination of serum HER2 or evaluation of HER2 status in circulating tumor cells (CTCs) may be of clinical relevance because metastatic tissue may be difficult to obtain for analysis as a result of its localization. The aim of this study was therefore to determine the HER2 status in serum and corresponding CTCs in patients with metastatic breast cancer whose primary tumors were HER2 negative or of unknown HER2 status.

Overexpression of urokinase plasminogen activator system or HER-2 (erbB-2) in breast cancer is associated with a poor prognosis. HER-2 overexpression is caused by HER-2 gene amplification. The anti-HER-2 antibody trastuzumab significantly improves clinical outcome for HER2-positive breast cancer. Drugs that target the uPA system are in early clinical trials. The aims of this study were to determine whether urokinase plasminogen activator receptor (uPAR) gene amplification occurs and whether analysis of individual tumor cells (TCs) in the blood or tissue can add information to conventional pathological analysis that could help in diagnosis and treatment. Analysis of individual TCs indicates that uPAR amplification occurs in a significant portion of primary breast cancers and also circulating tumor cells (CTCs) from patients with advanced disease. There was complete concordance between touch preps (TPs) and conventional pathological examination of HER-2 and uPAR gene status in primary tumors. There was also excellent concordance of HER-2 gene status between primary tumors and CTCs provided that acquisition of HER-2 gene amplification in CTCs was taken into account. Unexpectedly, gene amplification of HER-2 and uPAR occurred most frequently in the same TC and patient, suggesting a biological bias and potential advantage for coamplification. Expression of HER-2 and uPAR in primary tumors predicted gene status in 100 and 92% of patients, respectively.

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Combining the power of immunomagnetic assay and microfluidic microchip operations, we successfully detected rare CTCs from clinical blood samples. The microfluidic system is operated in a flip-flop mode, where a computer-controlled rotational holder with an array of microfluidic chips inverts the microchannels. We have demonstrated both theoretically and experimentally that the direction of red blood cell (RBC) sedimentation with regards to the magnetic force required for cell separation is important for capture efficiency, throughput, and purity. The flip-flop operation reduces the stagnation of RBCs and non-specific binding on the capture surface by alternating the direction of the magnetic field with respect to gravity. The developed immunomagnetic microchip-based screening system exhibits high capture rates (more than 90%) for SkBr3, PC3, and Colo205 cell lines in spiked screening experiments and successfully isolates CTCs from patient blood samples. The proposed motion controlled microchip-based immunomagnetic system shows great promise as a clinical tool for cancer diagnosis and prognosis.