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.

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 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.

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 After Neoadjuvant Chemotherapy in Stage I-III Triple-Negative Breast Cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is characterized by a lack of estrogen and progesterone receptor expression and HER-2 gene amplification. Circulating tumor cells (CTCs) can be identified in 25 % of nonmetastatic breast cancer patients, and the identification of ≥1 CTC predicts outcome. This study was designed to determine whether CTCs present after neoadjuvant chemotherapy (NACT) predicted worse outcome in nonmetastatic TNBC patients. METHODS: CTCs were assessed in 57 TNBC patients with nonmetastatic TNBC after the completion of NACT. CTCs (per 7.5 ml blood) were identified using the Cell Search(®) System (Janssen). Log-rank test and Cox regression analysis were applied to establish the association of CTCs with relapse-free (RFS) and overall survival (OS). RESULTS: Median follow-up was 30 months, and mean age was 53 years. Fifty-four patients (95 %) had >2-cm tumors, 42 (84 %) were nuclear grade 3, and 42 (74 %) had positive axillary lymph nodes. One or more CTC was identified in 30 % of patients. CTC presence was not associated with primary tumor size, high grade, or lymph node positivity. Multivariate analysis demonstrated that detection of ≥1 CTC predicted decreased RFS (log-rank P = 0.03, HR 5.25, 95 % CI 1.34-20.56) and OS (log-rank P = 0.03, HR 7.04, 95 % CI 1.26-39.35). CONCLUSIONS: One or more CTCs present after NACT predicted relapse and survival in nonmetastatic TNBC patients. This information would be helpful in future clinical trial design of adjuvant treatments for TNBC patients who are at risk for relapse after completing NACT.

Small-molecule inhibition of a depalmitoylase enhances Toxoplasma host-cell invasion.

Although there have been numerous advances in our understanding of how apicomplexan parasites such as Toxoplasma gondii enter host cells, many of the signaling pathways and enzymes involved in the organization of invasion mediators remain poorly defined. We recently performed a forward chemical-genetic screen in T. gondii and identified compounds that markedly enhanced infectivity. Although molecular dissection of invasion has benefited from the use of small-molecule inhibitors, the mechanisms underlying induction of invasion by small-molecule enhancers have never been described. Here we identify the Toxoplasma ortholog of human APT1, palmitoyl protein thioesterase-1 (TgPPT1), as the target of one class of small-molecule enhancers. Inhibition of this uncharacterized thioesterase triggered secretion of invasion-associated organelles, increased motility and enhanced the invasive capacity of tachyzoites. We demonstrate that TgPPT1 is a bona fide depalmitoylase, thereby establishing an important role for dynamic and reversible palmitoylation in host-cell invasion by T. gondii.

Circulating tumor cells in non-metastatic triple-negative breast cancer.

Circulating tumor cells (CTCs) can be identified in approximately 25 % of stage I-III breast cancer patients; CTCs presence is a predictor of poor outcome in metastatic breast cancer, but little is known regarding the prognostic significance of CTCs in non-metastatic triple-negative breast cancer (TNBC) patients. The aim of this study was to determine whether CTCs predict worse outcome in non-metastatic TNBC patients. We evaluated CTCs in 113 patients with stages I-III TNBC at the time of definitive surgery. CTCs were assessed using the CellSearch System®. Progression-free and overall survival were defined as time elapsed between date of diagnosis and either date of clinical disease progression, death, or last follow-up. Log-rank test and Cox regression analysis were used to determine associations of CTCs with progression-free and overall survival. The median follow-up was 40 months. CTCs were identified in 23/113 (20 %) of patients. No primary tumor characteristic or lymph node status predicted the presence of CTCs. The identification of ≥2 CTCs predicted shorter progression-free (log rank P ≤ 0.001; hazard ratio 8.30, 95 % CI 2.61-26.37) and overall survival (log rank P = 0.0004; hazard ratio 7.19, 95 % CI 1.98-26.06) versus survival for patients with <2 CTCs. Two or more CTCs predict shorter progression-free and overall survival in TNBC patients. Larger studies are needed to determine whether CTC assessment provides beneficial information that could be used in stratifying TNBC patients at increased risk for disease progression. Finally, CTCs characterization could facilitate the development of novel treatment approaches for TNBC.

Chemical genetic screen identifies Toxoplasma DJ-1 as a regulator of parasite secretion, attachment, and invasion.

Toxoplasma gondii is a member of the phylum Apicomplexa that includes several important human pathogens, such as Cryptosporidium and Plasmodium falciparum, the causative agent of human malaria. It is an obligate intracellular parasite that can cause severe disease in congenitally infected neonates and immunocompromised individuals. Despite the importance of attachment and invasion to the success of the parasite, little is known about the underlying mechanisms that drive these processes. Here we describe a screen to identify small molecules that block the process of host cell invasion by the T. gondii parasite. We identified a small molecule that specifically and irreversibly blocks parasite attachment and subsequent invasion of host cells. Using tandem orthogonal proteolysis-activity-based protein profiling, we determined that this compound covalently modifies a single cysteine residue in a poorly characterized protein homologous to the human protein DJ-1. Mutation of this key cysteine residue in the native gene sequence resulted in parasites that were resistant to inhibition of host cell attachment and invasion by the compound. Further analysis of the invasion phenotype confirmed that modification of Cys127 on TgDJ-1 resulted in a block of microneme secretion and motility, even in the presence of direct stimulators of calcium release. Together, our results suggest that TgDJ-1 plays an important role that is likely downstream of the calcium flux required for microneme secretion, parasite motility, and subsequent invasion of host cells.

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.

Circulating tumour cells in non-metastatic breast cancer: a prospective study.

BACKGROUND: The identification of circulating tumour cells correlate with poor prognosis in metastatic breast cancer, but there are few data describing the importance of circulating tumour cells in patients with non-metastatic disease. Our aim was to establish if circulating tumour cells predicted worse outcome in patients with non-metastatic breast cancer. METHODS: We prospectively collected data on circulating tumour cells at the time of definitive surgery from chemonaive patients with stage 1-3 breast cancer from February, 2005, to December, 2010. We deemed eligible all patients with operable breast cancer presenting at The University of Texas MD Anderson Cancer Center (Houston, TX, USA). Patients were ineligible if they had bilateral breast cancer or any other malignancy within 5 years of the diagnosis of the present cancer. We measured circulating tumour cells with the CellSearch System (Veridex, Raritan, NJ). We correlated findings of circulating tumour cells with standard tumour characteristics, including tumour size and grade; oestrogen and progesterone receptor and human epidural growth factor receptor 2 (HER2) status; and axillary lymph node status with χ(2) or Fisher exact tests. We assessed outcomes at a median follow-up of 35 months. Log-rank test and Cox regression analysis was applied to establish the association of circulating tumour cells with progression-free and overall survival. FINDINGS: No patients reported adverse events or complications from blood collections. We identified one or more circulating tumour cells in 73 (24%) of 302 patients. Detection of one or more circulating tumour cells predicted both decreased progression-free survival (log-rank p=0·005; hazard ratio [HR] 4·62, 95% CI 1·79-11·9) and overall survival (log-rank p=0·01; HR 4·04, 1·28-12·8). INTERPRETATION: The presence of one or more circulating tumour cells predicted early recurrence and decreased overall survival in chemonaive patients with non-metastatic breast cancer. These results suggest that assessment of circulating tumour cells might provide important prognostic information in these patients. FUNDING: Society of Surgical Oncology, Morgan Welch Inflammatory Breast Cancer Program, The University of Texas MD Anderson Cancer Center, and the State of Texas Rare and Aggressive Breast Cancer Research Program.

Disseminated tumor cells predict survival after neoadjuvant therapy in primary breast cancer.

BACKGROUND: Tumor cells that disseminate to the bone marrow (disseminated tumor cells [DTCs]) have been identified in 30% of patients with stage I through II breast cancer (BC) and predict outcome. Neoadjuvant chemotherapy (NACT) is effective in reducing the size of primary tumors or eradicating lymph node metastases before surgery, but little is known regarding the presence or significance of DTCs after NACT. METHODS: The authors evaluated DTCs in 95 patients with clinical stage I through III BC. Bone marrow samples were collected after completion of NACT at the time they underwent surgery for primary BC. DTCs were assessed using an anticytokeratin antibody cocktail. Primary tumor markers, the extent of lymph node (LN) involvement, they type of NACT administered, and response to NACT were compared with presence of DTCs. Chi-square and Fisher exact tests were used for statistical analyses. RESULTS: The median patient age at diagnosis was 51 years, and the median follow-up was 24 months. Forty-six percent of patients had tumors classified as T1/T2, 20% had T3 tumors, 34.5% had T4 tumors, and 81% had lymph node metastasis before NACT. DTCs were identified in 26% of patients after NACT. No associations were observed between DTCs and primary tumor characteristics or LN involvement. A pathologic complete response was observed in 25 patients (26%) but was not predictive of DTCs after NACT (P = .83). DTCs after NACT predicted worse BC-specific survival (P < .02). CONCLUSIONS: The presence of DTCs was an independent predictor of outcome after NACT. The current results indicated that monitoring hematogenous micrometastatic disease after NACT may be useful in selecting patients who might benefit from additional systemic adjuvant therapies.

Role of COX-2 in tumorospheres derived from a breast cancer cell line.

BACKGROUND: Cyclooxygenase-2 (COX-2) expression in primary breast cancer predicts tumor cell dissemination to bone marrow, which is a risk factor for recurrence and distant metastasis. "Stem-like" phenotype may be important in cancer metastasis. METHODS: To investigate the role of COX-2 protein in breast cancer stem-like cells, we analyzed it by co-immunofluorescence in tumorospheres derived from the MCF7 estrogen receptor-positive breast cancer cell line. To evaluate COX-2 function we utilized a COX-2 inhibitor in a clonogenicity assay performed with tumorospheres-derived cells. RESULTS: We detected rare cells in tumorospheres (one cell per tumorosphere) with very high COX-2 expression (COX-2(high)). COX-2 transfected MCF7 cells were able to generate long-term tumorospheres culture, even though transfection efficiency was only one in a million cells. We detected expression of OCT4 in some COX-2(high) cells, supporting the hypothesis that these cells could be cancer stem-like cells. It is important that COX-2(high) cells showed less expression of Ki-67 than did neighboring cells, indicating that COX-2(high) cells may be progenitors of tumorospheres. Celecoxib inhibited the growth of tumorosphere cultures and the ability of tumorosphere-derived cells to form colonies in vitro, indicating an active role of COX-2 in these processes. However, 2 µM celecoxib failed to eradicate tumorosphere-initiating cells. Finally, we detected rare COX-2(high) cells among SUM149 inflammatory breast cancer cells growing on plastic in serum-containing medium; the SUM149 cell line produces a very high level of COX-2 protein. CONCLUSION: Our results support a role for COX-2 in stem-like breast cancer cells and suggest a mechanism behind a role for COX-2 in disseminated tumor cells, which are known to exhibit characteristic biomarkers and functional properties of stem-like cells.

Disseminated tumor cells in biologic subtypes of stage I-III breast cancer patients.

BACKGROUND: Triple receptor-negative breast cancers (TNBC) are higher grade and more likely to metastasize. Recurrences after 5 years are rare in TNBCs. Conversely, late recurrences are seen in estrogen receptor (ER)-positive (luminal) cancers. Disseminated tumor cells (DTCs) may be responsible for late recurrences. We compared rates of DTCs in basal and luminal subtypes. METHODS: We evaluated 205 stage I-III patients. DTCs were assessed from bone marrow aspirates using anti-cytokeratin (CK) antibody following cytospin, and the presence of ≥ 1 CK-positive cells was considered positive. Pathologic complete response (pCR) was defined as lack of invasive disease in primary tumor and regional lymph nodes after neoadjuvant chemotherapy (NAC). Statistical analyses used chi-square and Fischer's exact test. RESULTS: Median follow-up (f/u) was 27 months, and 40% of patients had NAC. Forty patients had TNBC, and 148 had luminal cancers. Seventeen percent of TNBC patients, and 27% of those with luminal subtype, had DTCs after NAC (P = NS). Following NAC, pCR occurred in 28% of TNBC and 23% of luminal patients. Luminal A subtypes were less likely to achieve pCR when compared with non-luminal A subtypes (16 versus 41%; P = 0.01). All TNBC patients who achieved pCR had complete eradication of DTCs, whereas 36% of luminal (A and B) subtypes had DTCs. CONCLUSIONS: DTCs were found in 29% of stage I-III patients. TNBCs were more likely to have complete eradication of DTCs after pCR. Further study is needed to determine whether DTCs are responsible for late recurrences in patients with luminal cancers.

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.

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.

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.

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.