Aditya Bardia

Circulating Breast Tumor Cells Exhibit Dynamic Changes in Epithelial and Mesenchymal Composition

Cells in Transit(ion) Epithelial-mesenchymal transition (EMT) is a developmental program that converts adherent epithelial cells to a migratory mesenchymal state. This cell-fate change has been linked to tumor metastasis in preclinical models. To investigate whether EMT occurs in human cancer, Yu et al. (p. 580) isolated circulating tumor cells (CTCs) from breast cancer patients and analyzed their expression of epithelial and mesenchymal markers by RNA–in situ hybridization and RNA sequencing. Biphenotypic cells expressing both types of markers were rare in primary breast tumors but were enriched among CTCs, as were cells expressing only mesenchymal markers. Serial blood samples from one patient revealed that CTCs in the mesenchymal state declined in number when the patient responded to therapy but rebounded when the disease began to progress—a pattern repeated when a different therapy was administered. Thus, EMT may facilitate tumor cell dissemination in humans. Tumor cells circulating in the blood of cancer patients undergo a phenotypic change that may facilitate their spread. Epithelial-mesenchymal transition (EMT) of adherent epithelial cells to a migratory mesenchymal state has been implicated in tumor metastasis in preclinical models. To investigate its role in human cancer, we characterized EMT in circulating tumor cells (CTCs) from breast cancer patients. Rare primary tumor cells simultaneously expressed mesenchymal and epithelial markers, but mesenchymal cells were highly enriched in CTCs. Serial CTC monitoring in 11 patients suggested an association of mesenchymal CTCs with disease progression. In an index patient, reversible shifts between these cell fates accompanied each cycle of response to therapy and disease progression. Mesenchymal CTCs occurred as both single cells and multicellular clusters, expressing known EMT regulators, including transforming growth factor (TGF)–β pathway components and the FOXC1 transcription factor. These data support a role for EMT in the blood-borne dissemination of human breast cancer.

Circulating Tumor Cell Clusters Are Oligoclonal Precursors of Breast Cancer Metastasis

Circulating tumor cell clusters (CTC clusters) are present in the blood of patients with cancer but their contribution to metastasis is not well defined. Using mouse models with tagged mammary tumors, we demonstrate that CTC clusters arise from oligoclonal tumor cell groupings and not from intravascular aggregation events. Although rare in the circulation compared with single CTCs, CTC clusters have 23- to 50-fold increased metastatic potential. In patients with breast cancer, single-cell resolution RNA sequencing of CTC clusters and single CTCs, matched within individual blood samples, identifies the cell junction component plakoglobin as highly differentially expressed. In mouse models, knockdown of plakoglobin abrogates CTC cluster formation and suppresses lung metastases. In breast cancer patients, both abundance of CTC clusters and high tumor plakoglobin levels denote adverse outcomes. Thus, CTC clusters are derived from multicellular groupings of primary tumor cells held together through plakoglobin-dependent intercellular adhesion, and though rare, they greatly contribute to the metastatic spread of cancer.

Inertial Focusing for Tumor Antigen–Dependent and –Independent Sorting of Rare Circulating Tumor Cells

A multistage microfluidic chip is capable of sorting rare EpCAM+ and EpCAM− CTCs from cancer patients’ whole blood. Positive and Negative Outcomes Usually people want the good news first, to help cope with the bad news that inevitably follows. However, patients will soon desire both the positive and the negative outcomes together, according to the latest study by Ozkumur and colleagues. These authors have developed a multistage microfluidic device that is capable of sorting rare circulating tumor cells (CTCs) that are either positive or negative for the surface antigen epithelial cell adhesion molecule (EpCAM). EpCAM+ cells found in the bloodstream have long defined the typical CTC. Many sorting technologies have been developed to enumerate EpCAM+ CTCs in cancer patient’s blood; however, these cells are not always detectable in cancers with low EpCAM expression, like triple-negative breast cancer or melanoma. Ozkumur et al. engineered an automated platform, called the “CTC-iChip,” that captured both EpCAM+ and EpCAM− cancer cells in clinical samples using a series of debulking, inertial focusing, and magnetic separation steps. The sorted CTCs could then be interrogated using standard clinical protocols, such as immunocytochemistry. The authors tested the “positive mode” of their device using whole blood from patients with prostate, lung, breast, pancreatic, and colorectal cancers. After successfully separating out the EpCAM+ CTCs, they confirmed that the cells were viable and had high-quality RNA for molecular analysis, in one example, detecting the EML4-ALK gene fusion in lung cancer. Using the “negative mode” of their device, the authors were able to capture EpCAM− CTCs from patients with metastatic breast cancer, pancreatic cancer, and melanoma. The isolated CTCs showed similar morphology when compared with primary tumor tissue from these patients, suggesting that the microfluidic device can be used for clinical diagnoses—delivering both positive and negative news at once. Ozkumur et al. also demonstrated that CTCs isolated using the iChip could be analyzed on the single-cell level. One such demonstration with 15 CTCs from a prostate cancer patient reveals marked heterogeneity in the expression of mesenchymal and stem cell markers as well as typical prostate cancer–related antigens. The CTC-iChip can therefore process large volumes of patient blood to obtain not just EpCAM+ CTCs but also the EpCAM− ones, thus giving a broader picture of an individual’s cancer status and also allowing the device to be used for more cancer types. With the ability to further analyze the molecular characteristics of CTCs, this CTC-iChip could be a promising addition to current diagnostic tools used in the clinic. Circulating tumor cells (CTCs) are shed into the bloodstream from primary and metastatic tumor deposits. Their isolation and analysis hold great promise for the early detection of invasive cancer and the management of advanced disease, but technological hurdles have limited their broad clinical utility. We describe an inertial focusing–enhanced microfluidic CTC capture platform, termed “CTC-iChip,” that is capable of sorting rare CTCs from whole blood at 107 cells/s. Most importantly, the iChip is capable of isolating CTCs using strategies that are either dependent or independent of tumor membrane epitopes, and thus applicable to virtually all cancers. We specifically demonstrate the use of the iChip in an expanded set of both epithelial and nonepithelial cancers including lung, prostate, pancreas, breast, and melanoma. The sorting of CTCs as unfixed cells in solution allows for the application of high-quality clinically standardized morphological and immunohistochemical analyses, as well as RNA-based single-cell molecular characterization. The combination of an unbiased, broadly applicable, high-throughput, and automatable rare cell sorting technology with generally accepted molecular assays and cytology standards will enable the integration of CTC-based diagnostics into the clinical management of cancer.

Ex vivo culture of circulating breast tumor cells for individualized testing of drug susceptibility

Staying one step ahead of tumors Cancer treatments require continual adjustment. A drug that works initially will lose its potency as the tumor acquires new mutations that allow it to bypass the drugs lethal effects. To stay ahead of the tumor, oncologists need a noninvasive way to collect tumor cells from patients over the course of their treatment. Analyzing the mutations in these samples may help them choose the right drugs as the tumors change. In a small study of breast cancer patients, Yu et al. show that rare tumor cells circulating in the blood can be captured in viable form and used for this purpose. Science, this issue p. 216 Mutational analysis of tumor cells isolated from the blood of cancer patients may help optimize treatment selection. Circulating tumor cells (CTCs) are present at low concentrations in the peripheral blood of patients with solid tumors. It has been proposed that the isolation, ex vivo culture, and characterization of CTCs may provide an opportunity to noninvasively monitor the changing patterns of drug susceptibility in individual patients as their tumors acquire new mutations. In a proof-of-concept study, we established CTC cultures from six patients with estrogen receptor–positive breast cancer. Three of five CTC lines tested were tumorigenic in mice. Genome sequencing of the CTC lines revealed preexisting mutations in the PIK3CA gene and newly acquired mutations in the estrogen receptor gene (ESR1), PIK3CA gene, and fibroblast growth factor receptor gene (FGFR2), among others. Drug sensitivity testing of CTC lines with multiple mutations revealed potential new therapeutic targets. With optimization of CTC culture conditions, this strategy may help identify the best therapies for individual cancer patients over the course of their disease.

Malignant Melanoma in the 21st Century, Part 1: Epidemiology, Risk Factors, Screening, Prevention, and Diagnosis

Malignant melanoma is an aggressive, therapy-resistant malignancy of melanocytes. The incidence of melanoma has been steadily increasing worldwide, resulting in an increasing public health problem. Exposure to solar UV radiation, fair skin, dysplastic nevi syndrome, and a family history of melanoma are major risk factors for melanoma development. The interactions between genetic and environmental risk factors that promote melanomagenesis are currently the subject of ongoing research. Avoidance of UV radiation and surveillance of high-risk patients have the potential to reduce the population burden of melanoma. Biopsies of the primary tumor and sampling of draining lymph nodes are required for optimal diagnosis and staging. Several clinically relevant pathologic subtypes have been identified and need to be recognized. Therapy for early disease is predominantly surgical, with a minor benefit noted with the use of adjuvant therapy. Management of systemic melanoma is a challenge because of a paucity of active treatment modalities. In the first part of this 2-part review, we discuss epidemiology, risk factors, screening, prevention, and diagnosis of malignant melanoma. Part 2 (which will appear in the April 2007 issue) will review melanoma staging, prognosis, and treatment.

A microfluidic device for label-free, physical capture of circulating tumor cell clusters

Cancer cells metastasize through the bloodstream either as single migratory circulating tumor cells (CTCs) or as multicellular groupings (CTC clusters). Existing technologies for CTC enrichment are designed to isolate single CTCs, and although CTC clusters are detectable in some cases, their true prevalence and significance remain to be determined. Here we developed a microchip technology (the Cluster-Chip) to capture CTC clusters independently of tumor-specific markers from unprocessed blood. CTC clusters are isolated through specialized bifurcating traps under low–shear stress conditions that preserve their integrity, and even two-cell clusters are captured efficiently. Using the Cluster-Chip, we identified CTC clusters in 30–40% of patients with metastatic breast or prostate cancer or with melanoma. RNA sequencing of CTC clusters confirmed their tumor origin and identified tissue-derived macrophages within the clusters. Efficient capture of CTC clusters will enable the detailed characterization of their biological properties and role in metastasis.

Efficacy of Complementary and Alternative Medicine Therapies in Relieving Cancer Pain: A Systematic Review

PURPOSE Despite widespread popular use of complementary and alternative medicine (CAM) therapies, a rigorous evidence base about their efficacy for cancer-related pain is lacking. This is a systematic review of randomized controlled trials (RCTs) evaluating CAM therapies for cancer-related pain. METHODS RCTs using CAM interventions for cancer-related pain were abstracted using Medline, EMBASE, CINAHL, AMED, and Cochrane database. RESULTS Eighteen trials were identified (eight poor, three intermediate, and seven high quality based on Jadad score), with a total of 1,499 patients. Median sample size was 53 patients, and median intervention duration was 45 days. All studies were from single institutions, four had sample size justification, and none reported any adverse effects. Seven trials reported significant benefit for the following CAM therapies: acupuncture (n = 1), support groups (n = 2), hypnosis (n = 1), relaxation/imagery (n = 2), and herbal supplement/HESA-A (n = 1, but study was of low quality without control data). Seven studies reported immediate postintervention or short-term benefit of the following CAM interventions: acupuncture (n = 2), music (n = 1), herbal supplement/Ai-Tong-Ping (n = 1), massage (n = 1), and healing touch (n = 2). Four studies reported no benefit of CAM interventions (music, n = 2; massage, n = 2) in reducing cancer pain compared with a control arm. CONCLUSION There is paucity of multi-institutional RCTs evaluating CAM interventions for cancer pain with adequate power, duration, and sham control. Hypnosis, imagery, support groups, acupuncture, and healing touch seem promising, particularly in the short term, but none can be recommended because of a paucity of rigorous trials. Future research should focus on methodologically strong RCTs to determine potential efficacy of these CAM interventions.

Efficacy of Antioxidant Supplementation in Reducing Primary Cancer Incidence and Mortality : Systematic Review and Meta-analysis

OBJECTIVE To estimate the association between antioxidant use and primary cancer incidence and mortality and to evaluate these effects across specific antioxidant compounds, target organs, and participant subgroups. METHODS Multiple electronic databases (MEDLINE, Cochrane Controlled Clinical Trials Register, EMBASE, Science Citation Index) were searched from their dates of inception until August 2005 to identify eligible randomized clinical trials. Random effects meta-analyses estimated pooled relative risks (RRs) and 95% confidence intervals (CIs) that described the effect of antioxidants vs placebo on cancer incidence and cancer mortality. RESULTS Twelve eligible trials, 9 of high methodological quality, were identified (total subject population, 104,196). Antioxidant supplementation did not significantly reduce total cancer incidence (RR, 0.99; 95% CI, 0.94-1.04) or mortality (RR, 1.03; 95% CI, 0.92-1.15) or any site-specific cancer incidence. Beta carotene supplementation was associated with an increase in the incidence of cancer among smokers (RR, 1.10; 95% CI, 1.03-1.10) and with a trend toward increased cancer mortality (RR, 1.16; 95% CI, 0.98-1.37). Selenium supplementation was associated with reduced cancer incidence in men (RR, 0.77; 95% CI, 0.64-0.92) but not in women (RR, 1.00; 95% CI, 0.89-1.13, value for interaction, P< .001) and with reduced cancer mortality (RR, 0.78; 95% CI, 0.65-0.94). Vitamin E supplementation had no apparent effect on overall cancer incidence (RR, 0.99; 95% CI, 0.94-1.04) or cancer mortality (RR, 1.04; 95% CI, 0.97-1.12). CONCLUSION Beta carotene supplementation appeared to increase cancer incidence and cancer mortality among smokers, whereas vitamin E supplementation had no effect. Selenium supplementation might have anticarcinogenic effects in men and thus requires further research.

Diagnosis of Obesity by Primary Care Physicians and Impact on Obesity Management

OBJECTIVE To evaluate whether primary care physicians document obesity as a diagnosis and formulate a management plan. PATIENTS AND METHODS The Mayo Clinic primary care database was used to identify general medical examinations performed from November 1, 2004, to October 31, 2005, in a primary care clinic for obese patients (body mass index [BMI] equals 30). Data on demographic variables, BMI, comorbidities, documentation of obesity, and obesity management strategy were obtained through the database. Multivariate logistic regression analyses were conducted to estimate multivariate odds ratios (ORs) and 95 percent confidence intervals (CIs). RESULTS A total of 9827 patients were seen for a general medical examination. Of the 2543 obese patients, 505 (19.9 percent) had a diagnosis of obesity documented, and 574 (22.6 percent) had an obesity management plan documented. Older patients (OR, 0.97 per year; 95 percent CI, 0.96-0.98) and men (OR, 0.60; 95% CI, 0.47-0.76) were significantly less likely to be diagnosed as having obesity, whereas those with a BMI greater than 35 (OR, 2.54; 95 percent CI, 2.10-3.16), diabetes mellitus (OR, 1.40; 95 percent CI, 1.09-1.78), and obstructive sleep apnea (OR, 2.34; 95 percent CI, 1.79 to 3.07) were significantly more likely to have the diagnosis made. Staff physicians were less likely than residents to document obesity as a diagnosis (OR, 0.55; 95 percent CI, 0.44 to 0.69). Diagnosis of obesity was the strongest predictor of formulation of an obesity plan (OR, 2.39; 95 percent CI, 1.90 to 3.02). CONCLUSION Most obese patients did not have a diagnosis of obesity or an obesity management plan made by their primary care physician. Diagnosis of obesity results in a higher chance of formulation of an obesity plan.

Malignant Melanoma in the 21st Century, Part 2: Staging, Prognosis, and Treatment

Critical to the clinical management of a patient with malignant melanoma is an understanding of its natural history. As with most malignant disorders, prognosis is highly dependent on the clinical stage (extent of tumor burden) at the time of diagnosis. The patients clinical stage at diagnosis dictates selection of therapy. We review the state of the art in melanoma staging, prognosis, and therapy. Substantial progress has been made in this regard during the past 2 decades. This progress is primarily reflected in the development of sentinel lymph node biopsies as a means of reducing the morbidity associated with regional lymph node dissection, increased understanding of the role of neoangiogenesis in the natural history of melanoma and its potential as a treatment target, and emergence of innovative multimodal therapeutic strategies, resulting in significant objective response rates in a disease commonly believed to be drug resistant. Although much work remains to be done to improve the survival of patients with melanoma, clinically meaningful results seem within reach.