雌激素解救晚期内分泌治疗耐药乳腺癌的临床观察

目的 在晚期三线内分泌治疗耐药乳腺癌患者中,明确循环肿瘤细胞(CTCs)中FAS表达与雌激素疗效的关系,并研究雌激素在逆转内分泌治疗耐药时所起的作用。方法 20例检测到外周血中CTCs且其上FAS表达呈阳性的晚期三线内分泌治疗耐药乳腺癌患者应用雌二醇片剂2 mg口服,每日3次,4周为一周期。当雌二醇治疗临床获益的患者再次出现疾病进展时,给予阿那曲唑1 mg口服,每日1次,4周为一周期。每周期重复影像学检查和CTCs检测,并评价疗效。结果 20 例患者中失访1例,可供评价19例。应用雌二醇后,由实体瘤RECIST1.0标准判断疗效:CR 0例,PR 2例(10.5%),SD 5例(26.3%),PD 12例(63.2%),客观缓解率10.5%,临床获益率36.8%。中位无进展生存期6.0个月。由CTCs标准判断疗效:CR 0例,PR 2例(10.5%),SD 4例(21.1%),PD 13例(68.4%),客观缓解率10.5%,临床获益率31.6%。中位无进展生存期5.0个月。获益患者再次出现疾病进展并给予阿那曲唑治疗后,由实体瘤RECIST1.0标准判断疗效:CR 0例,PR 1例(14.3%),SD 2例(28.6%),PD 4例(57.1%),客观缓解率14.3%,临床获益率42.9%。中位无进展生存期4.0个月。由CTCs标准判断疗效:CR 0例,PR 1例(14.3%),SD 1例(14.3%),PD 5例(71.4%),客观缓解率14.3%,临床获益率28.6%。中位无进展生存期3.0个月。两种评价方法比较,差异无统计学意义。但在判定时间上,CTCs标准较影像学标准早1个月。结论 监测晚期三线内分泌治疗耐药乳腺癌患者CTCs中FAS的表达情况,给予雌激素治疗可取得一定疗效,并有可能恢复部分患者对芳香化酶抑制剂的敏感性,且无明显副作用。     

Prognostic significance of PD-L1 expression on circulating tumor cells in patients with head and neck squamous cell carcinoma

BackgroundSuccessful application of programmed death 1 (PD1) checkpoint inhibitors in the clinic may ultimately benefit from appropriate patient selection based upon predictive biomarkers. Molecular characterization of circulating tumor cells (CTC) is crucial for the investigation of molecular-targeted therapies while predictive biomarkers for response to PD1 checkpoint inhibitors are lacking. We sought to assess whether overexpression of PD-L1 in CTCs could be detected at baseline and at different timepoints during treatment in a prospective cohort of head and neck squamous cell carcinoma (HNSCC) patients and used to predict clinical outcome after treatment with curative intent.Patients and methodsWe developed a highly sensitive, specific and robust RT-qPCR assay for PD-L1 mRNA expression in EpCAM⁽⁺⁾ CTCs. In a prospective cohort of 113 locally advanced HNSCC patients treated with curative intent we evaluated PD-L1 expression in the EpCAM⁽⁺⁾ CTC fraction at baseline, after 2 cycles of induction chemotherapy (week 6) and at the end of concurrent chemoradiotherapy (week 15).ResultsPD-L1 overexpression was found in 24/94 (25.5%) patients at baseline, 8/34 (23.5%) after induction chemotherapy and 12/54 (22.2%) patients at the end of treatment. Patients with CTCs overexpressing PD-L1 at end of treatment had shorter progression-free survival (P = 0.001) and overall survival (P < 0.001). Multivariate analysis revealed that PD-L1 overexpression at end of treatment was independent prognostic factor for progression-free survival and overall survival. The absence of PD-L1 overexpression at the end of treatment was strongly associated with complete response with an odds ratio = 16.00 (95% CI = 2.76–92.72, P = 0.002).ConclusionsWe demonstrate that detection of CTCs overexpressing PD-L1 is feasible and may provide important prognostic information in HNSCC. Our results suggest that adjuvant PD1 inhibitors deserve evaluation in HNSCC patients in whom PD-L1⁽⁺⁾ CTCs are detected at the end of curative treatment.     

Circulating tumour cells in locally advanced breast cancer

Material and methods

A prospective study was conducted to determine the value of changes in circulating tumour cell (CTC) levels prior to and after the first cycle of neoadjuvant treatment in early prediction of pathologic response in locally advanced breast cancer (LABC). Two blood samples were obtained from 72 eligible LABC patients to isolate and enumerate CTCs before neoadjuvant chemotherapy started on day 1, and on day 21, immediately before second cycle administration.

Results

Sixty patients (83.3%) had <1 CTC in the first sample and response rates in this cohort were pathologic complete response (PCR) in 2 patients (5%), partial response (PR) in 35 (87.5%), stable disease (SD) in 2 (5%) and progressive disease (PD) in 1 (2.5%). Twelve patients (16.7%) had >2 CTCs in the first sample; these patients were more likely to have triple negative tumours. All 12 had fewer CTCs in the second sample. Response rates in this second cohort of 12 patients were PCR in 4 (34%), PR in 6 (50%), SD in 1 (8%) and PD in 1 (8%). PCR rate was markedly better in this second cohort (p<0.0042; OR 14.5, 95% CI 2.3–92).

Discussion

This study suggests that the presence of CTCs prior to neoadjuvant therapy might be a predictor of response to this therapy.     

Circulating tumor cell profiling for precision oncology

Analysis of circulating tumor cells (CTCs) collected from patient''s blood offers a broad range of opportunities in the field of precision oncology. With new advances in profiling technology, it is now possible to demonstrate an association between the molecular profiles of CTCs and tumor response to therapy. In this Review, we discuss mechanisms of tumor resistance to therapy and their link to phenotypic and genotypic properties of CTCs. We summarize key technologies used to isolate and analyze CTCs and discuss recent clinical studies that examined CTCs for genomic and proteomic predictors of responsiveness to therapy. We also point out current limitations that still hamper the implementation of CTCs into clinical practice. We finally reflect on how these shortcomings can be addressed with the likely contribution of multiparametric approaches and advanced data analytics.     

Circulating tumour cells: their utility in cancer management and predicting outcomes

Recent advances in technology now permit robust and reproducible detection of circulating tumour cells (CTCs) from a simple blood test. Standardization in methodology has been instrumental in facilitating multicentre trials with the purpose of evaluating the clinical utility of CTCs. We review the current body of evidence supporting the prognostic value of CTC enumeration in breast, prostate and colorectal cancer, using standardized approaches, and studies evaluating the correlation of CTC number with radiological outcome. The exploitation of CTCs in cancer management, however, is now extending beyond prognostication. As technologies emerge to characterize CTCs at the molecular level, biological information can be obtained in real time, with the promise of serving as a 'surrogate tumour biopsy'. Current studies illuminate the potential of CTCs as pharmacodynamic and predictive biomarkers and potentially their use in revealing drug resistance in real time. Approaches for CTC characterization are summarized and the potential of CTCs in cancer patient management exemplified via the detection of epidermal growth factor receptor mutations from CTCs in patients with non-small cell lung cancer. The opportunity to learn more about the biology of metastasis through CTC analysis is now being realized with the horizon of CTC-guided development of novel anticancer therapies.     

Expected clinical applications of circulating tumor cells in breast cancer

Tumor cell invasion and intravascular filtration lead to the presence of circulating tumor cells (CTCs) in peripheral blood. CTCs have, thus, been counted in patients with cancer to analyze metastatic mechanisms or in the hope of developing clinical applications for diagnosis and therapy; various CTC-related studies have been performed. However, the clinical significance of CTCs remains to be established because of the extremely small number of CTCs in peripheral blood as compared with the number of blood cells. Technical problems (e.g. reproducibility and reliability) in the detection of CTCs also remain to be solved. The use of flow cytometric analysis, which can be performed with tumor-cell markers such as anti-epithelial cell adhesion molecule antibodies and anti-cytokeratin antibodies and non-tumor-cell markers such as anti-CD45 antibodies has enhanced specificity for the detection of tumor cells. The CellSearch System^® can detect 1 CTC in 7.5 mL of peripheral blood, with high reproducibility. Its detection rate and accuracy for CTCs have been confirmed. In the United States, clinical trials have used this system to detect CTCs in patients with metastatic breast cancer, metastatic colorectal cancer, and metastatic prostate cancer, and CTCs have been confirmed to be a useful prognostic factor. This system was also suggested to be useful for monitoring treatment response in patients with metastatic breast cancer and was approved by the United States Food and Drug Administration in 2004. Measuring CTC counts can facilitate the early prediction of treatment response and thereby avoid unnecessary therapy. CTCs may also be a useful biomarker for molecular targeted agents, enabling the identification of patients most likely to respond to a given treatment and facilitating treatment selection. However, the widespread use of CTC monitoring as a routine examination requires a further improvement in measurement sensitivity, the establishment of criteria for quantitative and qualitative evaluations, and additional clear-cut evidence supporting the clinical significance of CTCs. We expect that CTCs will be established to be a new diagnostic and therapeutic index for breast cancer.     

Potential applications for circulating tumor cells expressing the insulin-like growth factor-I receptor.

PURPOSE: To detect insulin-like growth factor-IR (IGF-IR) on circulating tumor cells (CTC) as a biomarker in the clinical development of a monoclonal human antibody, CP-751,871, targeting IGF-IR. EXPERIMENTAL DESIGN: An automated sample preparation and analysis system for enumerating CTCs (CellTracks) was adapted for detecting IGF-IR-positive CTCs with a diagnostic antibody targeting a different IGF-IR epitope to CP-751,871. This assay was used in three phase I trials of CP-751,871 as a single agent or with chemotherapy and was validated using cell lines and blood samples from healthy volunteers and patients with metastatic carcinoma. RESULTS: There was no interference between the analytic and therapeutic antibodies. Eighty patients were enrolled on phase I studies of CP-751,871, with 47 (59%) patients having CTCs detected during the study. Before treatment, 26 patients (33%) had CTCs, with 23 having detectable IGF-IR-positive CTCs. CP-751,871 alone, and CP-751,871 with cytotoxic chemotherapy, decreased CTCs and IGF-IR-positive CTCs; these increased toward the end of the 21-day cycle in some patients, falling again with retreatment. CTCs were commonest in advanced hormone refractory prostate cancer (11 of 20). Detectable IGF-IR expression on CTCs before treatment with CP-751,871 and docetaxel was associated with a higher frequency of prostate-specific antigen decline by >50% (6 of 10 versus 2 of 8 patients). A relationship was observed between sustained decreases in CTC counts and prostate-specific antigen declines by >50%. CONCLUSIONS: IGF-IR expression is detectable by immunofluorescence on CTCs. These data support the further evaluation of CTCs in pharmacodynamic studies and patient selection, particularly in advanced prostate cancer.     

A Pilot Study Assessing the Incidence and Clinical Significance of Circulating Tumor Cells in Esophagogastric Cancers

BackgroundCirculating tumor cells (CTCs) have been found to be of clinical utility in predicting response to treatment and prognosis in several malignancies. Less is known of the prevalence and clinical relevance of CTCs in esophagogastric adenocarcinoma, with the available data arising from heterogeneous patient populations using varied detection methods.Patients and MethodsA pilot study was undertaken to assess the prevalence of CTCs in patients with advanced esophageal or gastric adenocarcinoma. Patients were eligible if they had advanced disease and either had received no prior therapy or had progressed after prior chemotherapy. Blood samples for CTC analysis were obtained at baseline and during the course of treatment. The CellSearch immunomagnetic CTC detection platform was used.ResultsTwenty-two patients with metastatic esophageal or gastric adenocarcinoma were enrolled. Eighteen received first-line EOX (epirubicin/oxaliplatin/capecitabine) chemotherapy (± panitumumab) and had baseline samples suitable for CTC analysis. At baseline, ≥ 2 CTCs were detected in 8 patients (44%). Overall tumor response rate was 60% in patients with < 2 CTCs and 37.5% in patients with ≥ 2 CTCs. Median progression-free and overall survival were 6.1 and 10.5 months and 5.2 and 6.1 months in the groups of patients with < 2 CTCs and ≥ 2 CTCs, respectively. The study was prematurely discontinued, owing to the withdrawal of commercial support.ConclusionThe incidence of CTCs in locally advanced or metastatic esophagogastric cancer may be clinically relevant. Investigation of the potential clinical utility of CTCs is warranted in a larger cohort of patients with esophagogastric cancer.     

Variation of circulating tumor cell levels during treatment of metastatic breast cancer: prognostic and therapeutic implications.

BACKGROUND: This study aimed to evaluate the prognostic significance of circulating tumor cells (CTCs) detection in advanced breast cancer patients. PATIENTS AND METHODS: We tested 80 patients for CTC levels before starting a new treatment and after 4, 8 weeks, at the first clinical evaluation and every 2 months thereafter. CTCs were detected using the CellSearch System. RESULTS: Forty-nine patients had >or=5 CTCs at baseline. At the multivariate analysis, baseline number of CTCs was significantly associated with progression-free survival [hazard ratio (HR) 2.5; 95% confidence interval (CI) 1.2-5.4]. The risk of progression for patients with CTCs >or=5 at last available blood draw was five times the risk of patients with 0-4 CTCs at the same time point (HR 5.3; 95% CI 2.8-10.4). Patients with rising or persistent >or=5 CTCs at last available blood draw showed a statistically significant higher risk of progression with respect to patients with <5 CTCs at both blood draws (HR 6.4; 95% CI 2.8-14.6). CONCLUSION: CTCs basal value is a predictive indicator of prognosis and changes in CTC levels during therapy may indicate a clinical response. Testing CTC levels during targeted treatments might substitute other measurement parameters for response evaluation.     

Detection of circulating tumor cells in breast cancer may improve through enrichment with anti-CD146

Most assays to detect circulating tumor cells (CTCs) rely on EpCAM expression on tumor cells. Recently, our group reported that in contrast to other molecular breast cancer subtypes, ??normal-like?? cell lines lack EpCAM expression and are thus missed when CTCs are captured with EpCAM-based technology [J Natl Cancer Inst 101(1):61?C66, 2009]. Here, the use of CD146 is introduced to detect EpCAM-negative CTCs, thereby improving CTC detection. CD146 and EpCAM expression were assessed in our panel of 41 breast cancer cell lines. Cells from 14 cell lines, 9 of which normal-like, were spiked into healthy donor blood. Using CellSearch? technology, 7.5 ml whole blood was enriched for CTCs by adding ferrofluids loaded with antibodies against EpCAM and/or CD146 followed by staining for Cytokeratin and DAPI. Hematopoietic cells and circulating endothelial cells (CECs) were counterstained with CD45 and CD34, respectively. A similar approach was applied for blood samples of 20 advanced breast cancer patients. Eight of 9 normal-like breast cancer cell lines lacked EpCAM expression but did express CD146. Five of these 8 could be adequately recovered by anti-CD146 ferrofluids. Of 20 advanced breast cancer patients whose CTCs were enumerated with anti-EpCAM and anti-CD146 ferrofluids, 9 had CD146+ CTCs. Cells from breast cancer cell lines that lack EpCAM expression frequently express CD146 and can be recovered by anti-CD146 ferrofluids. CD146+ CTCs are present in the peripheral blood of breast cancer patients with advanced disease. Combined use of anti-CD146 and anti-EpCAM is likely to improve CTC detection in breast cancer patients.     

Multi-center study evaluating circulating tumor cells as a surrogate for response to treatment and overall survival in metastatic breast cancer

Background

Evaluating circulating tumor cells (CTCs) is one way to predict outcome and monitor treatment in patients with MBC. In this prospective study, we evaluated CTCs in predicting treatment efficacy and overall survival (OS) using the CellSearch? System (Veridex, LLC, Raritan, NJ).

Methods

One hundred nineteen patients with MBC with measurable disease were enrolled. Samples of 7.5 ml of blood from 107 eligible patients were tested for CTCs before starting therapy (baseline), after one cycle of therapy (3–4 weeks) and at 12 weeks. We compared CTC levels and imaging at baseline and at 12 weeks. Next, we determined the hazard ratios (HR) by comparing cases with zero CTCs to those with one or more CTCs. Moreover, HR was calculated when comparing cases that had greater than or equal to a certain number of CTCs to those with less than the number of CTCs.

Results

This study shows the incidence of detection of CTCs in patients with metastatic breast cancers. Of the patients, 64.4% (76/118) had one or more CTCs, and 37.3% (44/118) had five or more CTCs. First we set the baseline number of CTCs as 100%. Of the seven cases whose level of CTCs decreased more than 90%, six (85.7%) demonstrated a positive response (complete response and partial response) by imaging after one cycle (3–4 weeks later). For the patients whose CTC levels increased above 100% after one cycle (3–4 weeks later), 7 of 11 (63.6%) had progressive disease (PD). The HR for cases with five to ten CTCs was greater than 1.00 [HR = 2.450; 95% confidence interval (CI) 0.727–8.248]. Statistical significance was observed when comparing patients who had ≥3 CTCs to those with <3 CTCs (P = 0.0273). When comparing cases with ≥5 CTCs to those with <5 CTCs, the hazard ratio was 3.069 (95% CI 1.496–6.295; P = 0.0022).

Conclusions

Because the change in the number of CTCs was highly correlated with results from imaging before and after therapy, CTCs can be considered a biomarker that may predict the effect of treatment earlier than imaging modalities.     

Circulating tumors cells as biomarkers: progress toward biomarker qualification

Personalized cancer medicine requires the development of tumor-specific biomarkers to optimize selection of targeted therapies and to better assess response to therapy. Current efforts in several tumor types have shown that patients in whom circulating tumor cells (CTCs) are detected have an inferior prognosis relative to those in whom CTCs are not detected and that the elimination or decrease of CTCs following treatment is associated with improved clinical outcomes. Technological advances in the detection, isolation, capture, and characterization of CTCs from phlebotomy samples obtained in a routine clinical practice setting have enabled the evaluation of different CTC biomarkers. Unmet needs in cancer diagnosis and treatment where CTC biomarkers have been studied include determining prognosis, assessing the effects of treatment, and as a source of tumor for the biologic identification and characterization of determinants to predict sensitivity to one form of treatment versus another and to understand mechanisms of treatment resistance.At present, there is no single definition of a CTC and no single CTC "biomarker." Rather, multiple assays (tests) are in development for CTC biomarkers. However, before the role of any biomarker in medical decision making can be determined, it is essential that the assays used to measure the biomarker are analytically validated in a sequence of trials to generate the evidence to support the biomarker's use in the given context of use. It is against this background that this review focuses on the process of developing CTC biomarker assays, with the objective of outlining the necessary steps to qualify specific CTC tests for medical decision making in clinical practice or drug development. The potential for point-of-care tests is clear.     

循环肿瘤细胞在乳腺癌中的研究进展

对于乳腺癌患者可以通过Cell Search系统自动检测外周血中循环肿瘤细胞(CTC)数目.近期相关研究的结果提示对于晚期乳腺患者而言,CTC水平可以用来预测患者预后并早期预测对治疗的反应.但是在早期乳腺癌中CTC的预测价值并不确切.进一步了解CTC的生物学特性以及明确其作为预后预测的应用价值仍需要大型前瞻性临床研究结...     

Frequent expression of PD‐L1 on circulating breast cancer cells

Immune checkpoint regulators such as PD‐L1 have become exciting new therapeutic targets leading to long lasting remissions in patients with advanced malignancies. However, in view of the remarkable costs and the toxicity profiles of these therapies, predictive biomarkers able to discriminate responders from non‐responders are urgently needed. In the present paper, we provide evidence that PD‐L1 is frequently expressed on metastatic cells circulating in the blood of hormone receptor‐positive, HER2‐negative breast cancer patients. We performed western blot, flow cytometry and immunocytochemical analyses to demonstrate the specificity of the PDL1 antibody used in our study and established immunoscores for PDL1 expression on single tumor cells. We then selected sixteen patients with circulating tumor cells (CTCs) using the CellSearch® system and found PD‐L1(+) CTCs in 11 patients (68.8%). The fraction of PD‐L1(+) CTCs varied from 0.2 to 100% in individual patients. This is the first report demonstrating the expression of PD‐L1 on CTCs. The established CTC/PD‐L1 assay can be used for liquid biopsy in future clinical trials for stratification and monitoring of cancer patients undergoing immune checkpoint blockade.     

Prognostic impact of detecting viable circulating tumour cells in gastric cancer patients using a telomerase-specific viral agent: a prospective study

ABSTRACT: BACKGROUND: The identification of circulating tumour cells (CTCs) in peripheral blood is a useful approach to estimate prognosis, monitor disease progression, and measure treatment effects in various malignancies. However, clinical relevance of CTCs is controversial. We attempted to detect viable CTCs in the peripheral blood of gastric cancer patients using a telomerase-specific viral agent. We took a 7.5-ml blood sample from 65 treatment-negative gastric cancer patients before surgery and 10 healthy volunteers. We detected viable CTCs in the blood samples after incubating them with a telomerase-specific, replication-selective, oncolytic adenoviral agent carrying the green fluorescent protein (GFP) gene (OBP-401). GFP-positive CTCs were defined as having a diameter of at least 7.735 mum; this threshold was determined by receiver operating characteristic curve analysis. GFP-positive cells were counted under a fluorescence microscope. RESULTS: There was a significant difference in overall survival among the patients with 0[EN DASH]4 and those with [GREATER-THAN OR EQUAL TO]5 GFP-positive CTCs in the stage I[EN DASH]IV disease group and stage II[EN DASH]IV advanced disease group. The number of GFP-positive CTCs was not related to cancer stage. Among the pathological findings, the number of GFP-positive CTCs was only significantly related to venous invasion, although there were trends towards more GFP-positive CTCs with disease progression (tumour depth, lymph node metastasis, distant metastasis, lymphatic invasion, and histological type). CONCLUSIONS: There was a significant relationship between the number of GFP-positive CTCs and overall survival in the patients with gastric cancer. The detection of CTCs using OBP-401 may be useful for prognostic evaluation.Trial registrationUniversity Hospital Medical Information Network in Japan, UMIN000002018.     

Circulating tumor cell isolation: the assets of filtration methods with polycarbonate track-etched filters

Circulating tumor cells (CTCs) arise from primary or secondary tumors and enter the bloodstream by active or passive intravasation. Given the low number of CTCs, enrichment is necessary for detection. Filtration methods are based on selection of CTCs by size using a filter with 6.5 to 8 µm pores. After coloration, collected CTCs are evaluated according to morphological criteria. Immunophenotyping and fluorescence in situ hybridization techniques may be used. Selected CTCs can also be cultivated in vitro to provide more material. Analysis of genomic mutations is difficult because it requires adapted techniques due to limited DNA materials. Filtration-selected CTCs have shown prognostic value in many studies but multicentric validating trials are mandatory to strengthen this assessment. Other clinical applications are promising such as follow-up, therapy response prediction and diagnosis. Microfluidic emerging systems could optimize filtration-selected CTCs by increasing selection accuracy.     

Detection of apoptotic circulating tumor cells in advanced pancreatic cancer following 5-fluorouracil chemotherapy

The objective of the present study was to evaluate CA19-9 and CK8/18 expression patterns in pancreatic cancer cell lines induced by 5-fluorouracil (5-Fu), and in circulating tumor cells (CTCs) in peripheral blood of patients previously untreated with advanced pancreatic cancer. Furthermore, the goal was to test the relationship of dynamic of CTCs with the effects of the first cycle of chemotherapy. To accomplish this study, CD45 antibody coated beads were used to discard white blood cells in peripheral blood. This was done in combination with CA19-9-Alexa488 and CK8/18-Alexa594 immunofluorescence staining to identify CTCs in circulation from 41 advanced pancreatic cancer patients, before and after chemotherapy. The PL45 pancreatic cancer cell line was incubated with 20 μmol/L 5-Fu for 12 and 24 hours to induce apoptosis, and the expression patterns of CA19-9 and CK8/18 were measured, and the extent of apoptosis was evaluated. Subsequently, apoptotic cells and CTCs were measured. Of 41 patients with stage III and Ⅳ pancreatic cancer, 80.5% were detected with more than two CTCs in 7.5 mL peripheral blood before any therapy and the median number of CTCs was 16.8±16.0 (0-59). After 7 days by the first cycle of 5-Fu chemotherapy, only 29.3% (12/41) of these patients were detected more than two CTCs in 7.5 mL peripheral blood and the median number of CTCs was 3.8±7.8 (0-40) (P=0.000). And no CTCs were detected in 20 healthy donors from 7.5 mL peripheral blood (P=0.000). Apoptotic CTCs were detected after advanced pancreatic patients were administered the first cycle of chemotherapy. Punctate, granular, and bubble-like morphologies with CA19-9 and CK8/18 staining were found and may reflect apoptosis in pancreatic cancer cells and CTCs. Apoptotic CTCs may indicate the efficacy of chemotherapy in pancreatic cancer patients. However, further studies are required to follow up these findings.     

Changes of HER2 status in circulating tumor cells compared with the primary tumor during treatment for advanced breast cancer

BackgroundHER2/neu status of tumor cells at metastatic sites in patients with advanced disease may differ from that of the primary tumor. Assessing the presence of target antigens on circulating tumor cells (CTCs) might affect treatment choice.Patients and MethodsFrom June 2007 to October 2008, we collected 23 mL of blood from each of the 76 consecutive patients before and during chemotherapy to determine CTC numbers and HER2 overexpression. CTCs were isolated with the CellSearch System® (Veridex, LLC; Raritan, NJ) and fluorescently stained with the Epithelial Cell Kit®. Tumor Phenotyping Reagent® was used to investigate HER2/neu overexpression.ResultsConcordance of HER2 status between the primary tumor and CTCs was 86% (49 out of 57 patients) at baseline and 82% (50 out of 61 patients) in the treatment samples. HER2 overexpression in CTCs was acquired in 8 out of 45 patients (18%) and lost in 3 out of 16 patients (19%) during a treatment containing trastuzumab. The overall discordance rate between the primary tumor and CTCs was 18% (11 out of 61 patients). Patients with HER2 overexpression in CTCs had poorer progression-free survival compared with those without CTCs or with HER2? CTCs (log-rank P =.036).ConclusionInformation on the presence or absence of HER2 overexpression can be obtained in CTCs. Larger trials are needed to evaluate the activity of HER2-targeted therapy in patients with acquired HER2 overexpression in CTCs.     

Detection of PD-L1 in circulating tumor cells and white blood cells from patients with advanced non-small-cell lung cancer

BackgroundExpression of PD-L1 in tumor cells and tumor-infiltrating immune cells has been associated with improved efficacy to anti-PD-1/PD-L1 inhibitors in patients with advanced-stage non-small-cell lung cancer (NSCLC) and emerged as a potential biomarker for the selection of patients to cancer immunotherapies. We investigated the utility of circulating tumor cells (CTCs) and circulating white blood cells (WBCs) as a noninvasive method to evaluate PD-L1 status in advanced NSCLC patients.Patients and methodsCTCs and circulating WBCs were enriched from peripheral blood samples (ISET® platform; Rarecells) from 106 NSCLC patients. PD-L1 expression on ISET filters and matched-tumor tissue was evaluated by automated immunostaining (SP142 antibody; Ventana), and quantified in tumor cells and WBCs.ResultsCTCs were detected in 80 (75%) patients, with levels ranging from 2 to 256 CTCs/4 ml, and median of 60 CTCs/4 ml. Among 71 evaluable samples with matched-tissue and CTCs, 6 patients (8%) showed ≥1 PD-L1-positive CTCs and 11 patients (15%) showed ≥1% PD-L1-positive tumor cells in tumor tissue with 93% concordance between tissue and CTCs (sensitivity = 55%; specificity = 100%). From 74 samples with matched-tissue and circulating WBCs, 40 patients (54%) showed ≥1% PD-L1-positive immune infiltrates in tumor tissue and 39 patients (53%) showed ≥1% PD-L1 positive in circulating WBCs, with 80% concordance between blood and tissue (sensitivity = 82%; specificity = 79%). We found a trend for worse survival in patients receiving first-line cisplatin-based chemotherapy treatments, whose tumors express PD-L1 in CTCs or immune cells (progression-free and overall survival), similar to the effects of PD-L1 expression in matched-patient tumors.ConclusionsThese results demonstrated that PD-L1 status in CTCs and circulating WBCs correlate with PD-L1 status in tumor tissue, revealing the potential of CTCs assessment as a noninvasive real-time biopsy to evaluate PD-L1 expression in patients with advanced-stage NSCLC.     

Single-cell next-generation sequencing of circulating tumor cells in patients with neuroblastoma

Circulating tumor cells (CTCs) derived from any tumor tissue could contribute to metastasis and resistance to cancer treatments. In this study, we performed single-cell next-generation sequencing of CTCs and evaluated their usefulness for characterizing tumor biology and the mechanisms of metastasis in neuroblastomas (NB). We aimed to isolate CTCs from 10 patients with NB at diagnosis before any treatments and four patients at relapse. GD2⁺CD90⁺CD45⁻CD235a⁻DAPI⁻ cells were isolated as neuroblastoma CTCs using fluorescence-activated cell sorting. In five patients with advanced stages (M stage), DNA and RNA sequencing of CTCs at single-cell level were performed. NB CTCs were isolated from eight of the 10 patients at diagnosis and three of the four patients at relapse. More CTCs could be isolated from patients with advanced stages. In one patient, ALK mutation (p.F1174L), was identified in both tumor tissue and a CTC. In patients with MYCN amplification, this gene was amplified in 12 of 13 CTCs. Using single-cell RNA sequencing, angiogenesis-related and cell cycle-related genes together with CCND1 and TUBA1A genes were found to be upregulated in CTCs. In one patient, CTCs were divided into two subgroups showing different gene expression profiles. In one subgroup, cell cycle-related and proliferation-related genes were differentially upregulated compared with the other group. In conclusion, next-generation sequencing of CTCs at single-cell level might help to characterize the tumor biology and the mechanisms of metastasis in NB.