Evaluation of Inactivation Methods for Rift Valley Fever Virus in Mouse Microglia.

Rift Valley fever phlebovirus (RVFV) is a highly pathogenic mosquito-borne virus with bioweapon potential due to its ability to be spread by aerosol transmission. Neurological symptoms are among the worst outcomes of infection, and understanding of pathogenesis mechanisms within the brain is limited. RVFV is classified as an overlap select agent by the CDC and USDA; therefore, experiments involving fully virulent strains of virus are tightly regulated. Here, we present two methods for inactivation of live virus within samples derived from mouse microglia cells using commercially available kits for the preparation of cells for flow cytometry and RNA extraction. Using the flow cytometry protocol, we demonstrate key differences in the response of primary murine microglia to infection with fully virulent versus attenuated RVFV.

17ß-estradiol promotes extracellular vesicle release and selective miRNA loading in ERα-positive breast cancer.

The causes and consequences of abnormal biogenesis of extracellular vesicles (EVs) are not yet well understood in malignancies, including in breast cancers (BCs). Given the hormonal signaling dependence of estrogen receptor-positive (ER+) BC, we hypothesized that 17ß-estradiol (estrogen) might influence EV production and microRNA (miRNA) loading. We report that physiological doses of 17ß-estradiol promote EV secretion specifically from ER+ BC cells via inhibition of miR-149-5p, hindering its regulatory activity on SP1, a transcription factor that regulates the EV biogenesis factor nSMase2. Additionally, miR-149-5p downregulation promotes hnRNPA1 expression, responsible for the loading of let-7's miRNAs into EVs. In multiple patient cohorts, we observed increased levels of let-7a-5p and let-7d-5p in EVs derived from the blood of premenopausal ER+ BC patients, and elevated EV levels in patients with high BMI, both conditions associated with higher levels of 17ß-estradiol. In brief, we identified a unique estrogen-driven mechanism by which ER+ BC cells eliminate tumor suppressor miRNAs in EVs, with effects on modulating tumor-associated macrophages in the microenvironment.

Measurement of Portal Vein Blood Circulating Tumor Cells is Safe and May Correlate With Outcomes in Resected Pancreatic Ductal Adenocarcinoma.

BACKGROUND: This study investigated the safety and feasibility of intraoperative portal vein blood (PVB) collection at the time of pancreatic ductal adenocarcinoma (PDAC) resection. Relationships of circulating tumor cells (CTCs) in PVB and peripheral blood (PB) with overall survival (OS) and recurrence-free survival were studied. METHODS: Patients undergoing PDAC resection were offered enrollment in a prospective liquid biopsy protocol. The patients had PB drawn before incision and PVB drawn before tumor mobilization, then again immediately after resection. Using standard CellSearch protocols, CTCs were identified and compared with OS. RESULTS: Of the 34 patients enrolled in this study, 23 (68%) underwent pancreaticoduodenectomy, 8 (23%) underwent distal pancreatectomy, and 3 (9%) underwent total pancreatectomy. Peripheral blood was available for 22 (65%) and PVB for 31 (91%) of the patients. No bleeding or thrombotic complications occurred with the PVB draws. The CTC counts per 7.5 mL of PVB collected before and after resection were highly correlated (R2 = 0.89). The study found CTCs in 11 (50%) of 22 PB samples and 22 (71%) of 31 PVB samples. The OS rate at 18 months was 92% for the patients with < 3 CTCs, 71% for the patients with ≥ 3 CTCs per 7.5 mL of PB (p = 0.30), 100% for the patients without PVB CTCs, and 70% for the patients with PVB CTCs (p < 0.01). CONCLUSIONS: Collection of PVB during PDAC resection is safe. In this pilot study, PVB CTC counts but not PB CTC counts were significantly correlated with OS. This opens the door for future studies on selective omission of adjuvant chemotherapy for patients treated preoperatively and tailored surveillance intensity for patients without PVB CTCs at PDAC resection.

The Role of Mastectomy in De Novo Stage IV Inflammatory Breast Cancer.

INTRODUCTION: The role of modified radical mastectomy (MRM) in patients with de novo stage IV inflammatory breast cancer (IBC) remains controversial. We evaluated the impact of MRM on outcomes in this population. METHODS: Ninety-seven women presenting with stage IV IBC were identified in an institutional database (2007-2016) and were stratified by receipt of MRM or no surgery (non-MRM). Demographic, clinicopathologic, and treatment factors were compared. Local-regional recurrence patterns were described and survival analyses were conducted. RESULTS: All patients initially received chemotherapy. Fifty-two patients (53.6%) underwent MRM; 47 received post-mastectomy radiation. Differences between the non-MRM and MRM groups included tumor receptor subtypes (hormone receptor-positive [HR+]/human epidermal growth factor receptor 2-positive [HER2+]: 4.4% vs. 19.2%; HR+/HER2-negative [HER2-]: 31.1% vs. 44.2%; HR-negative [HR-]/HER2+: 24.4% vs. 15.4%; and HR-/HER2-: 40.0% vs. 21.2%; p = 0.03), number of metastatic sites (3 vs. 2; p = 0.01), and clinical partial/complete response to chemotherapy (13.3% vs. 75.0%; p < 0.001). Of the 47 patients who completed trimodality therapy, 6 (12.8%) had a local-regional recurrence. Median overall survival (OS) was 19 months in the non-MRM group and 58 months in the MRM group (p < 0.001). On multivariable analysis, clinical N3 disease (hazard ratio 2.16, 95% confidence interval [CI] 1.07-4.37; p = 0.03) as well as tumor subtypes HR+/HER2- (hazard ratio 4.98, 95% CI 1.15-21.47; p = 0.03) and HR-/HER2- (hazard ratio 7.18, 95% CI 1.66-31.07; p = 0.008) were associated with decreased OS. Partial/complete response of distant disease to chemotherapy (hazard ratio 0.43, 95% CI 0.24-0.77; p = 0.005) and receipt of MRM (hazard ratio 0.52, 95% CI 0.29-0.93; p = 0.03) were independently associated with improved OS. CONCLUSIONS: In our retrospective study, MRM in de novo stage IV IBC patients is an independent factor associated with improved OS. Our findings strongly support the need for prospective randomized trials evaluating possible survival benefits of MRM in de novo stage IV IBC patients.

Mammalian Expression and In Situ Biotinylation of Extracellular Protein Targets for Directed Evolution.

Directed evolution is a powerful tool for the selection of functional ligands from molecular libraries. Extracellular domains (ECDs) of cell surface receptors are common selection targets for therapeutic and imaging agent development. Unfortunately, these proteins are often post-translationally modified and are therefore unsuitable for expression in bacterial systems. Directional immobilization of these targets is further hampered by the absence of biorthogonal groups for site-specific chemical conjugation. We have developed a nonadherent mammalian expression system for rapid, high-yield expression of biotinylated ECDs. ECDs from EGFR, HER2, and HER3 were site-specifically biotinylated in situ and recovered from the cell culture supernatant with yields of up to 10 mg/L at >90% purity. Biotinylated ECDs also contained a protease cleavage site for rapid and selective release of the ECD after immobilization on avidin/streptavidin resins and library binding. A model mRNA display selection round was carried out against the HER2 ECD with the HER2 affibody expressed as an mRNA-protein fusion. HER2 affibody-mRNA fusions were selectively released by thrombin and quantitative PCR revealed substantial improvements in the enrichment of functional affibody-mRNA fusions relative to direct PCR amplification of the resin-bound target. This methodology allows rapid purification of high-quality targets for directed evolution and selective elution of functional sequences at the conclusion of each selection round.

Correlation of circulating or disseminated tumor cells with the Oncotype DX Recurrence Score.

PURPOSE: New biomarkers are emerging to predict recurrence risk in women with early-stage breast cancer. High Oncotype DX Recurrence Score® (RS) is associated with worse disease-free and overall survival. Similarly, circulating tumor cells (CTCs, blood) and disseminated tumor cells (DTCs, bone marrow) have prognostic value in breast cancer. We investigated the association between high RS and CTCs or DTCs. METHODS: Using a prospective database, we evaluated patients with hormone receptor-positive/HER2-negative, node-negative invasive breast cancer from 1/2005 to 1/2017. RS was classified using TAILORx study cutoff points: low (< 11), intermediate (11-25), and high (> 25). CTCs were assessed using CellSearch® and DTCs using cytospin specimens of bone marrow aspirates. Positive result was defined as one or more CTCs or DTCs identified. Chi-square analyses were utilized to evaluate the relationship between RS and CTCs or DTCs. RESULTS: 233 patients were identified from a prospective database, of which 96 had RS results. Of these patients, 88 had CTC results and 58 had DTC results. CTCs were detected in 17/88 (19%) patients, while DTCs were detected in 20/58 (34%). Patients with high RS were not more likely to have CTCs (18%) compared to patients with low/intermediate RS (20%; p = 0.919). Similarly, high RS was not associated with DTC detection, with DTCs present in 40% of patients with high RS versus 33% with low/intermediate RS (p = 0.687). In the subgroup of patients ≤ 50 years, no associations were found between high RS and CTCs (p = 0.383) or DTCs (p = 0.234). CONCLUSIONS: High Oncotype DX RS did not correlate with CTCs in blood or DTCs in bone marrow in our study.

Circulating Tumor Cells and Early Relapse in Node-positive Melanoma.

PURPOSE: There is a need for sensitive, reproducible biomarkers for patients with stage III melanoma to guide clinical decision making. Circulating tumor cells (CTCs) can be detected in patients with melanoma; however, there are limited data regarding their significance in stage III disease. The aim of this study was to determine whether CTCs are associated with early relapse in stage III melanoma. EXPERIMENTAL DESIGN: We prospectively assessed CTCs at first presentation in clinic (baseline) for 243 patients with stage III melanoma. CTCs were measured using the CellSearch System. Relapse-free survival (RFS) was compared between patients with one or more baseline CTC versus those with no CTCs. Log-rank test and Cox regression analysis were applied to establish associations of CTCs with RFS. RESULTS: At least one baseline CTC was identified in 90 of 243 (37%) patients. Forty-five (19%), 67 (28%), 118 (49%), and 13 (5%) patients were stage IIIA, IIIB, IIIC, or IIID, respectively. CTC detection was not associated with substage, or primary tumor characteristics. Multivariable analysis demonstrated that the detection of ≥1 baseline CTC was significantly associated with decreased 6-month RFS [log-rank, P < 0.0001; HR, 3.62, 95% confidence interval (CI), 1.78-7.36; P < 0.0001] and 54-month RFS (log-rank, P = 0.01; HR, 1.69; 95% CI, 1.13-2.54; P = 0.01). CONCLUSIONS: ≥1 CTC was independently associated with melanoma relapse, suggesting that CTC assessment may be useful to identify patients at risk for relapse who could derive benefit from adjuvant therapy.

Circulating Tumor Cells in Breast Cancer.

With active screening for early detection and advancements in treatment, there has been a significant decrease in mortality from breast cancer. However, a significant proportion of patients with non-metastatic breast cancer at time of diagnosis will relapse. Therefore, it is suggested that the dissemination of bloodstream tumor cells (circulating tumor cells, CTCs) undetectable by currently available diagnostic tools occurs during the early stages of breast cancer progression, and may be the potential source of micrometastases responsible for treatment failures. Here, we review the clinical significance of CTCs, as detected by the FDA-approved CellSearch® System, in both metastatic and non-metastatic breast cancer patients. Studies so far suggest that CTCs are prognostic of poorer outcomes in breast cancer patients; however, there is currently insufficient data to support use of CTC data to guide treatment. Therefore, there are ongoing studies to evaluate the utility of assessing CTC phenotypes to develop personalized breast cancer treatment, which will be reviewed in this chapter.

Circulating Tumor Cells and Transforming Growth Factor Beta in Resected Pancreatic Adenocarcinoma.

BACKGROUND: Portal vein (PV) circulating tumor cells (CTCs) and elevated peripheral blood (PB) levels of biomarkers have been associated with poor outcomes in pancreatic ductal adenocarcinoma (PDAC). Although transforming growth factor-beta (TGFß) is associated with CTCs in breast cancer, there are limited data evaluating a comprehensive biomarker panel and CTCs in PDAC patients. The authors hypothesized that tumor progression biomarkers would be associated with PV CTCs. METHODS: PDAC patients at one institution were enrolled January to August 2018 and underwent preincision PB draws (T0) and on postoperative day 1 (T3), plus intraoperative PV draws before tumor manipulation (T1) and after resection (T2). CTCs were detected using CellSearch. Plasma biomarker levels (pg/mL) were measured with a multiplex bead assay. Patients were divided into two groups: high (≥3 CTCs/7.5 mL blood) versus low (<3). Clinicopathologic variables and biomarkers were compared in the two groups. RESULTS: Fourteen had complete blood draws with PDAC resection, with five demonstrating high CTCs. Fewer patients in the high-CTC group received preoperative radiation (78 versus 20%), whereas more of the high-CTC had pT3 tumors (80 versus 11%) (all P < 0.037). High-CTC patients demonstrated higher TGFß-2 levels (T0 [906 versus 586], T1 [1337 versus 627], T2 [1149 versus 445]), as well as higher TGFß-3 (T0 [320 versus 173], T2 [605 versus 120]) (all P < 0.021). CONCLUSIONS: PDAC patients with high CTCs demonstrated a distinct biomarker profile with elevated PB and PV levels of immunosuppressive cytokines (TGFß-2 and TGFß-3). These exploratory results prompt further study into interrupting TGFß signaling.

Pilot Study of Circulating Tumor Cells in Early-Stage and Metastatic Uveal Melanoma.

Nearly 50% of uveal melanoma (UM) patients develop metastatic disease, and there remains no current standard assay for detection of minimal residual disease. We conducted a pilot study to check the feasibility of circulating tumor cell (CTC) detection in UM. We enrolled 40 patients with early or metastatic UM of which 20 patients had early-stage disease, 19 had metastatic disease, and one was not evaluable. At initial blood draw, 36% of patients had detectable CTCs (30% in early-stage vs. 42% in metastatic), which increased to 54% at data cutoff (40% in early-stage vs. 68% in metastatic). Five early-stage patients developed distant metastases, 60% (3/5) had detectable CTCs before radiographic detection of the metastasis. Landmark overall survival (from study enrollment) at 24 months was statistically lower in CTC-positive vs. negative early-stage UM (p < 0.05). Within this small dataset, the presence of CTCs in early-stage UM predicted an increased risk of metastatic disease and was associated with worse outcomes.

Predicting treatment Response based on Dual assessment of magnetic resonance Imaging kinetics and Circulating Tumor cells in patients with Head and Neck cancer (PREDICT-HN): matching 'liquid biopsy' and quantitative tumor modeling.

BACKGROUND: Magnetic resonance imaging (MRI) has improved capacity to visualize tumor and soft tissue involvement in head and neck cancers. Using advanced MRI, we can interrogate cell density using diffusion weighted imaging, a quantitative imaging that can be used during radiotherapy, when diffuse inflammatory reaction precludes PET imaging, and can assist with target delineation as well. Correlation of circulating tumor cells (CTCs) measurements with 3D quantitative tumor characterization could potentially allow selective, patient-specific response-adapted escalation or de-escalation of local therapy, and improve the therapeutic ratio, curing the greatest number of patients with the least toxicity. METHODS: The proposed study is designed as a prospective observational study and will collect pretreatment CT, MRI and PET/CT images, weekly serial MR imaging during RT and post treatment CT, MRI and PET/CT images. In addition, blood sample will be collected for biomarker analysis at those time intervals. CTC assessments will be performed on the CellSave tube using the FDA-approved CellSearch® Circulating Tumor Cell Kit (Janssen Diagnostics), and plasma from the EDTA blood samples will be collected, labeled with a de-identifying number, and stored at - 80 °C for future analyses. DISCUSSION: The primary objective of the study is to evaluate the prognostic value and correlation of weekly tumor response kinetics (gross tumor volume and MR signal changes) and circulating tumor cells of mucosal head and neck cancers during radiation therapy using MRI in predicting treatment response and clinical outcomes. This study will provide landmark information as to the utility of CTCs ('liquid biopsy) and tumor-specific functional quantitative imaging changes during treatment to guide personalization of treatment for future patients. Combining the biological information from CTCs and the structural information from MRI may provide more information than either modality alone. In addition, this study could potentially allow us to determine the optimal time to obtain MR imaging and/ or CTCs during radiotherapy to assess tumor response and provide guidance for patient selection and stratification for future dose escalation or de-escalation strategies. TRIAL REGISTRATION: Clinicaltrials.gov ( NCT03491176 ). Date of registration: 9th April 2018. (retrospectively registered). Date of enrolment of the first participant: 30th May 2017.

Circulating Tumor Cells in Stage IV Melanoma Patients.

BACKGROUND: Management of stage IV melanoma patients remains a challenge. In spite of promising new therapies, many patients develop resistance and progression. The aim of this pilot study was to determine if circulating tumor cells (CTCs) are associated with shortened (180-day) progression-free survival (PFS) after a baseline CTC assessment in stage IV melanoma patients. STUDY DESIGN: A baseline CTC assessment was performed in 93 stage IV melanoma patients using a commercially available immunomagnetic system. The presence of 1 or more CTC was considered a positive result. A Cox multivariable regression model was used to evaluate the association between presence of CTCs at baseline and PFS, after adjusting for covariables. Kaplan-Meier curves and a log-rank test were used to summarize and compare unadjusted PFS for patients stratified by CTC positivity. RESULTS: Median follow-up was 17 months; mean age was 55 years. Thirteen of 93 (14%) patients had no evidence of disease (NED) at baseline CTC assessment. One or more CTC was detected in 39 of 93 (42%) of patients at baseline; CTCs were not associated with primary melanoma features or NED status. Twenty-eight of 93 (30%) patients progressed within 180 days of baseline draw, with 20 of 39 (51%) of the CTC-positive patients relapsing compared with 8 of 54 (15%) of the CTC-negative patients. In adjusted Cox models, a significant association was found suggesting worse PFS within 180 days for CTC-positive patients at baseline (vs CTC-negative) (hazard ratio 4.69, 95% CI 1.59 to 13.77, p = 0.005). CONCLUSIONS: One or more CTCs at baseline were associated with progression within 180 days in stage IV melanoma patients. This information warrants further study of CTCs as a means of identifying patients at high-risk for disease progression.

Circulating Tumor Cells in Breast Cancer Patients Treated by Neoadjuvant Chemotherapy: A Meta-analysis.

Background: We conducted a meta-analysis in nonmetastatic breast cancer patients treated by neoadjuvant chemotherapy (NCT) to assess the clinical validity of circulating tumor cell (CTC) detection as a prognostic marker. Methods: We collected individual patient data from 21 studies in which CTC detection by CellSearch was performed in early breast cancer patients treated with NCT. The primary end point was overall survival, analyzed according to CTC detection, using Cox regression models stratified by study. Secondary end points included distant disease-free survival, locoregional relapse-free interval, and pathological complete response. All statistical tests were two-sided. Results: Data from patients were collected before NCT (n = 1574) and before surgery (n = 1200). CTC detection revealed one or more CTCs in 25.2% of patients before NCT; this was associated with tumor size (P < .001). The number of CTCs detected had a detrimental and decremental impact on overall survival (P < .001), distant disease-free survival (P < .001), and locoregional relapse-free interval (P < .001), but not on pathological complete response. Patients with one, two, three to four, and five or more CTCs before NCT displayed hazard ratios of death of 1.09 (95% confidence interval [CI] = 0.65 to 1.69), 2.63 (95% CI = 1.42 to 4.54), 3.83 (95% CI = 2.08 to 6.66), and 6.25 (95% CI = 4.34 to 9.09), respectively. In 861 patients with full data available, adding CTC detection before NCT increased the prognostic ability of multivariable prognostic models for overall survival (P < .001), distant disease-free survival (P < .001), and locoregional relapse-free interval (P = .008). Conclusions: CTC count is an independent and quantitative prognostic factor in early breast cancer patients treated by NCT. It complements current prognostic models based on tumor characteristics and response to therapy.

Body mass index mediates the prognostic significance of circulating tumor cells in inflammatory breast cancer.

BACKGROUND: Obesity (BMI≥30) may be an etiologic and prognostic factor in inflammatory breast cancer (IBC). We examined the relationship between BMI, pathologic complete response (pCR), and circulating-tumor-cell (CTC) levels in IBC. METHODS: Cohort included IBC patients diagnosed 2005-2015 who had neoadjuvant chemotherapy during a prospective trial on CTCs and pathologic review describing pCR. Chi-square, logistic regression, and Cox proportional hazards models were used to identify clinicopathologic associations with event-free survival (EFS). RESULTS: Of 73 patients, 61 (84%) had CTC values, 22 (30%) achieved a pCR, and 39 (53%) were obese. There was no difference between obese and non-obese patients for pCR rates (31% vs. 29%, p = 0.90) or presence of CTCs (23% vs. 26%, p = 0.80). Among non-obese patients, CTCs were associated with worse EFS (HR 11.69, p < 0.01), but among obese patients, there was no difference in EFS between those with and without CTCs. CONCLUSIONS: BMI mediates CTCs' prognostic significance in IBC.

Tambora and the mackerel year: Phenology and fisheries during an extreme climate event.

Global warming has increased the frequency of extreme climate events, yet responses of biological and human communities are poorly understood, particularly for aquatic ecosystems and fisheries. Retrospective analysis of known outcomes may provide insights into the nature of adaptations and trajectory of subsequent conditions. We consider the 1815 eruption of the Indonesian volcano Tambora and its impact on Gulf of Maine (GoM) coastal and riparian fisheries in 1816. Applying complex adaptive systems theory with historical methods, we analyzed fish export data and contemporary climate records to disclose human and piscine responses to Tambora's extreme weather at different spatial and temporal scales while also considering sociopolitical influences. Results identified a tipping point in GoM fisheries induced by concatenating social and biological responses to extreme weather. Abnormal daily temperatures selectively affected targeted fish species-alewives, shad, herring, and mackerel-according to their migration and spawning phenologies and temperature tolerances. First to arrive, alewives suffered the worst. Crop failure and incipient famine intensified fishing pressure, especially in heavily settled regions where dams already compromised watersheds. Insufficient alewife runs led fishers to target mackerel, the next species appearing in abundance along the coast; thus, 1816 became the "mackerel year." Critically, the shift from riparian to marine fisheries persisted and expanded after temperatures moderated and alewives recovered. We conclude that contingent human adaptations to extraordinary weather permanently altered this complex system. Understanding how adaptive responses to extreme events can trigger unintended consequences may advance long-term planning for resilience in an uncertain future.

Circulating Tumor Cells in Breast Cancer Patients.

Breast cancer is the most commonly diagnosed cancer among women, resulting in an estimated 40,000 deaths in 2014.1 Metastasis, a complex, multi-step process, remains the primary cause of death for these patients. Although the mechanisms involved in metastasis have not been fully elucidated, considerable evidence suggests that metastatic spread is mediated by rare cells within the heterogeneous primary tumor that acquire the ability to invade into the bloodstream. In the bloodstream, they can travel to distant sites, sometimes remaining undetected and in a quiescent state for an extended period of time before they establish distant metastases in the bone, lung, liver, or brain. These occult micrometastatic cells (circulating tumor cells, CTCs) are rare, yet their prognostic significance has been demonstrated in both metastatic and non-metastatic breast cancer patients. Because repeated tumor tissue collection is typically not feasible and peripheral blood draws are minimally invasive, serial CTC enumeration might provide "real-time liquid biopsy" snapshots that could be used to identify early-stage breast cancer patients with micrometastatic disease who are at risk for disease progression and monitor treatment response in patients with advanced disease. In addition, characterizing CTCs might aid in the development of novel, personalized therapies aimed at eliminating micrometastases. This review describes current CTC isolation, detection, and characterization strategies in operable breast cancer.

Prognostic Value of Circulating Tumor Cells Identified Before Surgical Resection in Nonmetastatic Breast Cancer Patients.

BACKGROUND: Circulating tumor cells (CTCs) can be identified in approximately 25% of nonmetastatic breast cancer patients, and data are emerging regarding their prognostic significance. We hypothesized that CTCs identified before resection of the primary tumor would predict worse outcomes in nonmetastatic breast cancer patients. STUDY DESIGN: We performed CTC enumerations on 509 patients with nonmetastatic breast cancer as part of an IRB-approved study. The CTCs (per 7.5 mL blood) were identified using the CellSearch System (Janssen). The presence of ≥1 CTC meeting morphologic criteria for malignancy was considered a positive result. Log-rank test and Cox regression analysis were applied to establish the association of CTCs with relapse-free and overall survival. RESULTS: Median follow-up was 48 months and mean age was 53 years. Fifty-nine percent of patients (299 of 509) had tumors larger than 2 cm, and 46% (234 of 509) had positive lymph nodes. One hundred sixty-six patients received neoadjuvant chemotherapy (NACT) before CTC assessment, and 343 patients were chemonaïve. One or more CTC was identified in 43 of 166 (26%) NACT treated patients, and in 81 of 343 (24%) chemonaïve patients. Circulating tumor cells were not associated with tumor size, grade, or lymph node status (p = NS). Detection of 1 or more CTCs predicted decreased relapse-free (log-rank p < 0.001, hazard ratio [HR] 2.72, 95% CI 1.57 to 4.72; p < 0.001) and overall survival (log-rank p = 0.02, HR 2.29, 95% CI 1.12 to 4.67; p = 0.03) at 48 months of follow-up. CONCLUSIONS: One or more CTCs identified before resection of the primary breast tumor predicted worse relapse-free and overall survival, irrespective of primary tumor size, grade, or lymph node positivity.

Pooled Analysis of the Prognostic Relevance of Circulating Tumor Cells in Primary Breast Cancer.

PURPOSE: Although unequivocal evidence has shown the prognostic relevance of circulating tumor cells (CTC) in the peripheral blood of patients with metastatic breast cancer, less evidence is available for the prognostic relevance of CTCs at the time of primary diagnosis. EXPERIMENTAL DESIGN: We conducted a pooled analysis of individual data from 3,173 patients with nonmetastatic (stage I-III) breast cancer from five breast cancer institutions. The prevalence and numbers of CTCs were assessed at the time of primary diagnosis with the FDA-cleared CellSearch System (Janssen Diagnostics, LLC). Patient outcomes were analyzed using meta-analytic procedures, univariate log-rank tests, and multivariate Cox proportional hazard regression analyses. The median follow-up duration was 62.8 months. RESULTS: One or more CTCs were detected in 20.2% of the patients. CTC-positive patients had larger tumors, increased lymph node involvement, and a higher histologic tumor grade than did CTC-negative patients (all P < 0.002). Multivariate Cox regressions, which included tumor size, nodal status, histologic tumor grade, and hormone receptor and HER2 status, confirmed that the presence of CTCs was an independent prognostic factor for disease-free survival [HR, 1.82; 95% confidence interval (CI), 1.47-2.26], distant disease-free survival (HR, 1.89; 95% CI, 1.49-2.40), breast cancer-specific survival (HR, 2.04; 95% CI, 1.52-2.75), and overall survival (HR, 1.97; 95% CI, 1.51-2.59). CONCLUSIONS: In patients with primary breast cancer, the presence of CTCs was an independent predictor of poor disease-free, overall, breast cancer-specific, and distant disease-free survival. Clin Cancer Res; 22(10); 2583-93. ©2016 AACR.

Circulating Tumor Cells and Recurrence After Primary Systemic Therapy in Stage III Inflammatory Breast Cancer.

Inflammatory breast cancer (IBC) is rare and aggressive, with poor survival. While circulating tumor cells (CTCs) predict outcome in non-IBC patients, little data exists regarding their prognostic significance in IBC. This prospective study analyzed blood samples for CTCs from 63 stage III IBC patients to determine if CTCs present after primary systemic chemotherapy predicted relapse. CTC identification was not associated with tumor characteristics, lymph node positivity, or complete pathologic response to systemic therapy. At mean follow-up of 38 months, multivariable analysis demonstrated that detection of one or more CTCs predicted shortened relapse-free (log-rank P = 0.005, hazard ratio [HR] = 4.22, 95% confidence interval [CI] = 1.67 to 10.67, Cox P = 0.002) but not overall survival (log-rank P = 0.54, HR = 1.53, 95% CI = 0.41 to 5.79, Cox P = 0.53). All statistical tests were two-sided. In this study, CTCs after primary chemotherapy identified IBC patients at high risk for relapse.

Detection of circulating melanoma cells in the blood of melanoma patients: a preliminary study.

Significant prognostic heterogeneity exists within the substages of melanoma; therefore, novel prognostic biomarkers are needed to provide information on the risk of recurrence. Limited available data suggest prognostic significance for circulating melanoma cells (CMCs); there is a need for a sensitive, reproducible, and standardized identification technique. Using a semiautomated technology, we sought to determine whether CMCs could be identified reliably in stage I-IV melanoma patients and whether the presence of CMC correlated with known prognostic factors. CMCs were detected in the peripheral blood (7.5 ml) of patients with stage I-IV melanoma (n=89) using the CellSearch system. CD146 cells were immunomagnetically enriched; nucleated HMW-MAA/CD45/CD34 cells were considered CMCs. One or more CMCs was detected in 45% of all patients, varying with stage of disease (stages I/II, III, and IV: 35, 44, and 86%, respectively; P=0.03, for stage I/II vs. stage IV); 55% had one CMC, 32% had two CMCs, and 13% had three or more CMCs identified. The presence of CMCs in the blood was associated with histologic subtype, particularly in patients with stage I/II disease (superficial spreading 18% vs. acral lentiginous 75%). Using a semiautomated technique, CMCs can be identified in a significant number of melanoma patients. These data support further study with longer follow-up and longitudinal/serial time points to better determine the identification rates and prognostic significance of CMCs in stage I-IV melanoma patients.