Peripheral Circulating Tumor Cells Research Articles

Detection of PD‑L1 expression and epithelial‑mesenchymal transition of circulating tumor cells in non‑small cell lung cancer.

The present study aimed to assess the roles of peripheral circulating tumor cell (CTC) count, CTC subtypes and programmed death ligand 1 (PD-L1) expression in the clinical staging and prognosis of patients with non-small cell lung cancer (NSCLC). A total of 100 patients with NSCLC with available tumor tissues were enrolled in the present study, and 7.5 ml peripheral blood was collected. Patients were divided into PD-L1-positive and PD-L1-negative groups according to PD-L1 immunohistochemical staining. Peripheral blood samples from both groups were analyzed to determine the CTC count, epithelial-type CTCs (E-CTCs), mesenchymal-type CTCs (M-CTCs) and PD-L1 expression. Clinical data were collected, and patients were followed up for a maximum of 36 months, with patient death as the endpoint event. Patients with PD-L1-positive tumors had a worse prognosis compared with those with PD-L1-negative tumors (P=0.045). The PD-L1-positive group exhibited significantly higher numbers of CTCs and M-CTCs compared with the PD-L1-negative group (P≤0.05). However, the number of E-CTCs did not differ significantly between the two groups (P>0.05). PD-L1-positive patients with higher CTC and M-CTC counts had relatively poorer prognoses (P≤0.05), while the number of E-CTCs had no significant effect on prognosis (P>0.05). Compared with the early-stage NSCLC group, the late-stage NSCLC group exhibited a significant increase in the CTC count (P≤0.05), while E-CTC and M-CTC counts did not significantly differ between the two groups (P>0.05). The PD-L1-positive group exhibited a significant increase in the number of PD-L1+ CTCs and PD-L1+ M-CTCs compared with the PD-L1-negative group (P≤0.05), while PD-L1+ E-CTC counts did not differ significantly between the two groups (P>0.05). The PD-L1-positive patients with a higher number of PD-L1+ CTCs and PD-L1+ M-CTCs had relatively poorer prognoses (P≤0.05), while the PD-L1+ E-CTC count had no significant effect on prognosis (P>0.05). Compared with the early-stage NSCLC group, the late-stage NSCLC group exhibited a significant increase in the number of PD-L1+ CTCs and PD-L1+ M-CTCs (P≤0.05), while PD-L1+ E-CTC counts did not significantly differ between the two groups (P>0.05). Based on univariate and multivariate analyses, the number of PD-L1+ M-CTCs was identified as an independent prognostic factor for NSCLC. In conclusion, the presence of CTCs in peripheral blood, particularly PD-L1+ M-CTC subtype, indicated poorer clinical staging and prognosis in patients with NSCLC. These findings suggested that CTCs, specifically the PD-L1+ M-CTC subtype, could serve as a monitoring indicator for the clinical staging and prognosis of patients with NSCLC.

Radiofrequency ablation induces tumor cell dissemination in a mouse model of hepatocellular carcinoma

BackgroundWe tested the hypothesis that radiofrequency ablation (RFA) for hepatocellular carcinoma (HCC) promotes tumor cell release and explored a method for reducing these effects.MethodsA green fluorescent protein-transfected orthotopic HCC model was established in 99 nude mice. In vivo flow cytometry was used to monitor circulating tumor cell (CTC) dynamics. Pulmonary fluorescence imaging and pathology were performed to investigate lung metastases. First, the kinetics of CTCs during the periablation period and the survival rate of CTCs released during RFA were investigated. Next, mice were allocated to controls, sham ablation, or RFA with/without hepatic vessel blocking (ligation of the portal triads) for evaluating the postablation CTC level, lung metastases, and survival over time. Moreover, the kinetics of CTCs, lung metastases, and mice survival were evaluated for RFA with/without ethanol injection. Pathological changes in tumors and surrounding parenchyma after ethanol injection were noted. Statistical analysis included t-test, ANOVA, and Kaplan-Meier survival curves.ResultsCTC counts were 12.3-fold increased during RFA, and 73.7% of RFA-induced CTCs were viable. Pre-RFA hepatic vessel blocking prevented the increase of peripheral CTCs, reduced the number of lung metastases, and prolonged survival (all p ≤ 0.05). Similarly, pre-RFA ethanol injection remarkably decreased CTC release during RFA and further decreased lung metastases with extended survival (all p ≤ 0.05). Histopathology revealed thrombus formation in blood vessels after ethanol injection, which may clog tumor cell dissemination during RFA.ConclusionRFA induces viable tumor cell dissemination, and pre-RFA ethanol injection may provide a prophylactic strategy to reduce this underestimated effect.Relevance statementRFA for HCC promotes viable tumor cell release during ablation, while ethanol injection can prevent RFA induced tumor cell release.Key points• RFA induced the release of viable tumor cells during the ablation procedure in an animal model.• Hepatic vessel blocking can suppress tumor cells dissemination during RFA.• Ethanol injection can prevent RFA-induced tumor cell release, presumably because of the formation of thrombosis.Graphical

Circulating tumor cells as a prognostic biomarker in patients with hepatocellular carcinoma

Circulating tumor cells (CTCs) have been shown as a surrogate for cancer progression and prognostication. We aimed to determine an association between CTCs and survival of hepatocellular carcinoma (HCC) patients. Peripheral blood was obtained from 73 HCC patients to enumerate for epithelial CTCs/8 mL blood. CTCs were detected by immunoaffinity-based method using epithelial cell adhesion molecule (EpCAM) and mucin1 (MUC1). The CTCs detection rates of BCLC stages A, B, and C patients were 65.4% (17/26), 77.3% (17/22), and 96% (24/25), respectively, p = 0.018. Patients with CTCs < 5 cells/8 mL had significantly longer survival than those with CTCs ≥ 5 cells/8 mL (>36 vs. 4.6 months, p < 0.001). In multivariate analysis, CTP B, BCLC B, BCLC C, AFP ≥ 400 ng/mL, and CTC ≥ 5 cells/8 mL were independently associated with survival, with adjusted HRs (95%CI) of 4.1 (2.0–8.4), 3.5 (1.1–11.4), 4.7 (1.4–15.4), 2.4 (1.1–5.0), and 2.6 (1.2–8.4); p < 0.001, 0.036, 0.011, 0.025 and 0.012, respectively. The combination of CTCs ≥ 5 cells/8 mL and AFP ≥ 400 ng/mL provided additively increased HR to 5.3 (2.5–11.1), compared to HRs of 4.0 (2.0–8.0) and 3.5 (1.8–6.7) for CTCs ≥ 5 cells/8 mL and AFP ≥ 400 ng/mL, p < 0.001, respectively. The larger number of peripheral CTCs is correlated with higher tumor aggressive features and poorer survival of HCC patients. CTCs can potentially become novel prognostic biomarker in HCC.

A Correlation Analysis of Postoperative Hypercoagulability and Peripheral Circulating Tumor Cells in Patients with Lung Cancer

Objective: To explore the correlation between peripheral circulating tumor cells and hypercoagulability in patients with lung cancer after surgery. Methods: From January 2017 to December 2021, 89 patients with lung cancer who were treated in the Affiliated Hospital of Hebei University were selected as the research subjects, and a retrospective analysis was conducted to analyze and observe the D-dimer (DD), fibrinogen (FIB), and platelet (PLT) levels in peripheral blood, as well as detect peripheral CTC. Results: There were statistical differences in TMN staging, tumor metastasis, and lymph node metastasis in the clinical data, but there were no statistical differences in gender, smoking history, and pathological classification. After retrospective analysis and comparison of the patients, the DD (mg/ml), FIB (g/L), and PLT (×109/L) levels of the CTC positive group were 3.41 ± 0.58, 3.98 ± 0.87, and 367.26 ± 34.98, respectively; the CTC negative group’s DD (mg/ml), FIB (g/L), and PLT (×109/L) levels were 0.89 ± 0.49, 1.06 ± 0.45, and 234.69 ± 35.69, respectively, and the differences were statistically significant. The factors affecting the prognosis of patients included TMN staging and CTC; the number of CTC positives in the death group was significantly higher than that in the survival group, and there was a statistical difference between the groups. Gender, age, smoking history, pathological type, and surgical resection had no effect on the prognosis of patients. Among the enrolled patients, the survival rate was 71.91%. Conclusion: CTC-positive patients have a higher probability of hypercoagulability after surgery and are prone to tumor metastasis; thus, CTC can be used as a judgment index for the prognosis of patients.

Epithelial circulating tumor cells with a heterogeneous phenotype are associated with metastasis in NSCLC

ObjectivesTo analyze the clinical relevance of heterogeneous phenotypes of peripheral circulating tumor cells (CTCs) in non-small cell lung cancer (NSCLC).Materials and MethodsCTCs in 5 mL venous blood were enriched using the Canpatrol™ CTC technique in 82 NSCLC patients. And then, CTCs were subjected to RNA in situ hybridization with a combination of epithelial (EpCAM and CK8/18/19) and mesenchymal (vimentin and TWIST1) markers.ResultsAccording to the fluorescent dots, CTCs were classified into three groups, including epithelial CTCs (E-CTC), hybrid epithelial/mesenchymal phenotypes (E/M-CTCs) and mesenchymal CTCs (M-CTCs). In 82 NSCLC cohort, only 2 patients didn’t detect CTCs, the overall CTCs detection rate was 97.5% (80/82). For 60 treatment naïve NSCLC, only one patient didn’t detect CTCs. The median number of total CTCs, hybrid E/M phenotype CTCs, E-CTCs and M-CTCs per 5 mL blood was 22 (range 1–90), 13 (range 0–83), 1 (range 0–17 and 0–47), respectively. Hybrid E/M CTCs, especially the e = m-CTCs, significantly differed between patients with and without distant metastasis. M-CTCs in advanced NSCLC patients were significantly more than the numbers observed in early stage patients. Patients with pure hybrid E/M-CTCs showed a lower proportion in distant metastasis positive cohort compared to negative ones (7% vs 22%), while patients with E + E/M CTCs (20% vs 9%) and E/M + M CTCs (33% vs 20%) showed a higher proportion. CTCs dynamic changes after treatment in 12 advanced NSCLC patients suggested that hybrid E/M-CTCs were related to the primary tumor size at baseline, while M-CTCs may suggest the progression of NSCLC.ConclusionWe concluded that E-CTCs with a hybrid E/M phenotype are associated to metastasis in therapy-naïve NSCLC patients.

Recognition of the organ-specific mutations in metastatic breast cancer by circulating tumor cells isolated in vivo.

The failure to treat and control the growth of metastases is the main cause of death in breast cancer (BC) patients. Compared to the traditional method of analyzing circulating tumor DNA (ctDNA), capturing intact circulating tumor cells (CTCs) allows us to more accurately characterize mutations and identify suitable targeted therapies. We used CellCollector to collect peripheral CTCs. Thirty metastatic breast cancer (MBC) patients were enrolled, and 17 were analyzed with next-generation sequencing (NGS) methods. Clinical characteristics were analyzed along with the CTCs enumeration and detection rates. Whole-genome amplification (WGA) was used to amplify the CTC genomic DNA of 127 genes. Patients younger than 45 years old, with brain metastasis, with three or more metastatic sites, or with HER2-positive had the highest number of CTCs collected. The CTCs detection rate was also correlated to the number of metastasis sites. Different metastasis sites such as the brain, viscus, bone, and soft tissue contained specific high-frequency gene mutations. AKT3, MYC, and NT5C2 mutations were only found in brain metastases. APC, BCL2L11, ESRP1, FLT3 mutations were only in the visceral metastases. KEAP1, KIT, MET were the specific mutation genes in patients with bone and soft tissue metastases. These findings provide evidence that we can detect gene mutation information for obtaining the biological characteristics by CTCs using CellCollector. Different metastasis sites contain specific high-frequency mutation genes, which provide guidance to the accurate gene therapy.

Association between central and peripheral circulating tumor cell (CTC) clusters in oral squamous cell carcinoma (OSCC): A prospective, observational study.

e15548 Background: Around 90% of cancer-related mortalities are caused by tumor metastasis. CTC clusters, which constitute an intermediate stage of metastasis, have not been studied extensively in head &amp; neck cancers. The mortality rate of oral cancers remains alarmingly high, despite multimodality treatment. The aim of the study is to identify the presence of CTC clusters in patients with Oral Squamous Cell Carcinoma (OSCC) and to correlate their presence with clinical and pathological factors. Methods: Fifty patients diagnosed with histologically proven OSCC, treatment naïve, and underwent surgery at HCG Cancer Centre, Bangalore, were consented and enrolled in the study. An IRB-approved protocol allowed for the collection of 10 ml of blood from central (jugular) and peripheral veins intra-operatively, prior to tumor removal. The culturing of CTC clusters was done using ellipsoidal microwell plates maintained at hypoxic conditions, at the Institute of Bioinformatics, Bangalore. After fourteen days of culturing, the cells were fixed and stained for DAPI, Pan-CK and CD45. The CTC clusters were classified into Loose, Tight and very Tight based on the median gray values obtained from DAPI staining on ImageJ software. Clinical data was collected from patient records and subjected to analysis using Descriptive statistics. Results: From the 50 patients included in the study, 22 (44%) patients exhibited tight clusters in central blood, while only 13 (26%) patients exhibited tight clusters in peripheral blood. A higher clinical stage was observed in a greater percentage of patients with tight clusters in central blood (early: 45.5% versus late: 54.5%), but the same findings could not be inferred with pathological staging (early stage: 59.1% versus late stage: 40.1%). No significant correlation with adverse pathological features was noted. Conclusions: This observational study provides an insight into the varying biological behaviours of similarly grouped cancers, which is based on the standard TNM staging. The study forms the basis for the hypothesis of tight clusters in the central and peripheral circulation, correlating with loco-regional and distant metastasis respectively, thus leading to poorer disease-free and overall survival rates.

P028 A Novel Method for Detecting Surface PD-L1 on Circulating Tumor Cells in Peripheral Blood of Patients with SCLC

Previous studies have demonstrated that circulating tumor cell (CTC) might work as an alternative to biopsy tissues in the diagnoses of small cell lung cancer (SCLC). Programmed death ligand 1 (PD-L1) is one of the most commonly used biomarkers in immune targeted therapies. However, the expression of PD-L1 in CTC is still unknown. This study aimed to establish an approach to measure PD-L1 in peripheral CTCs from SCLC patients. Control cells were screened by monoclonal culture and confirmed by immunohistochemistry (IHC) staining and flow cytometry. PD-L1 expression in CTCs and tumor tissue samples from SCLC patients was measured by CellSearch system while the usage of PD-L1 antibody was optimized. The purity of PD-L1 positive H446 cells had been improved from 70% to 95% by clonal selection, and PD-L1 negative PC3 cells showed lower than 5% purity. In CellSearch system, positive control cells had 98% PD-L1 expression while negative control had less than 3% expression. In two SCLC patients with CTC positive expression, PD-L1 expression was found with 100% (1/1) and 40% (2/5) positive rate. CellSearch system was used to establish a novel approach for measuring surface PD-L1 expression on CTC, and showed supporting evidences in SCLC patients, thus providing novel insights for automatic assay of PD-L1 in peripheral samples.

Role of regulatory T cells and CD8+ T lymphocytes in the dissemination of circulating tumor cells in primary invasive breast cancer.

Circulating tumor cells (CTCs) dissemination is involved in tumor metastasis and is an independent prognostic factor in patients with primary and metastatic breast cancer. Regulatory T cells (Tregs) and cluster of differentiation (CD)8+ T lymphocytes are the main types of immune cells in the tumor microenvironment and exert opposite roles on the progression and outcome of breast cancer. The aim of the present study was to evaluate the associations between CTCs and intratumoral/peritumoral Tregs and CD8+ T lymphocytes in breast cancer. Peripheral CTCs were detected by a multi-marker quantitative polymerase chain reaction platform in 167 patients with invasive breast cancer. Intratumoral/peritumoral Tregs and CD8+ T lymphocytes were assessed by immunohistochemistry in 167 patients with invasive breast cancer to establish an association between these cell types and detection of peripheral CTCs. CTCs were detected in 55% of the patients with breast cancer. The prevalence of CTCs was positively associated with the number of intratumoral (P=0.002) and peritumoral Tregs (P=0.045), and the primary tumor size (P=0.012). This result was verified by analyzing intratumoral Tregs (P=0.044) and primary tumor size (P=0.044) with multivariate analysis, which indicated that the CTC-positive rate increased with an increasing number of intratumoral Tregs and a larger tumor size In the multivariate analysis, other variables including menopause, tumor-node-metastasis stage and peritumoral Tregs were not associated with the prevalence of CTCs. The prevalence of CTCs was inversely and weakly associated with the number of peritumoral CD8+ T lymphocytes using the univariate analysis, however this result was not statistically significant (P=0.470). In conclusion, regulatory T cells and CD8+ T lymphocytes may be involved, at least in part, in the CTCs dissemination of breast cancer, whereby Tregs appear to exert the dominant effect. Furthermore, the role of Tregs in the progression of breast cancer may be mediated by suppressing the dissemination of CTCs, which is primarily determined by intratumoral Tregs.

Relationships between peripheral circulating tumor cells in patients with gallbladder carcinoma detected by a novel nano microfluidic chip with clinicopathology and prognosis

Objective To establish a novel method using a nano microfluidic chip to detect circulating tumor cells (CTCs) in peripheral blood in gallbladder carcinoma, and to study the relationship between CTCs with clinicopathology and prognosis in these patients. Methods The peripheral blood samples of 51 patients with gallbladder carcinoma were collected from June 2014 to January 2017. The CTCs from peripheral blood samples were detected by a novel nano microfluidic chip. This study aimed to study the correlation between CTCs with the clinical and pathological features. The significance of CTCs on prognosis in patients with gallbladder carcinoma was also analyzed. Results The positive rate of CTCs in the peripheral blood of gallbladder carcinoma patients was 43.1% (22/51). There were significant correlations between CTCs with liver metastasis (P 0.05). Conclusions Peripheral blood CTCs in patients with gallbladder carcinoma were efficaciously detected by a novel nano microfluidic chip. Peripheral blood CTCs was closely related to the Nevin staging and liver metastasis. CTCs could serve as a potential prognostic indicator in patients with gallbladder carcinoma. Key words: Gallbladder carcinoma; Microfluidic chip, nano; Circulating tumor cells (CTCs); Prognosis

Clinical significance of peripheral circulating tumor cell counts in colorectal polyps and non-metastatic colorectal cancer

BackgroundThe presence of peripheral circulating tumor cells indicates the possible existence of a tumor in vivo; however, low numbers of circulating tumor cells (CTCs) can be detected in peripheral blood of healthy individuals as well as patients with benign tumors. It is not known whether peripheral CTC counts differ between patients with benign colorectal disease and those with colorectal cancer.MethodsComparative analysis of preoperative peripheral circulating tumor cells counts was completed in patients with benign colorectal disease (colorectal polyps) and non-metastatic cancer of the colon and rectum.ResultsThe results of this analysis showed that patients with colorectal cancer had higher CTC counts than patients with colorectal polyps (3.47 ± 0.32/3.2 ml vs 1.49 ± 0.2/3.2 ml, P < 0.001). Colorectal cancer patients with tumors of the sigmoid colon displayed the highest CTC counts (4.87 ± 0.95/3.2 ml), followed by those with tumors of the rectum (3.73 ± 0.54/3.2 ml), ascending colon (3.5 ± 0.63/3.2 ml), transverse colon (2.4 ± 0.68/3.2 ml), and descending colon (2.08 ± 0.46/3.2 ml). Colorectal polyp patients with polyps in the rectum showed the highest CTC counts (2.2 ± 0.77/3.2 ml), followed by those with polyps in the ascending colon (1.82 ± 0.54/3.2 ml), sigmoid colon (1.38 ± 0.25/3.2 ml), transverse colon (0.75 ± 0.25/3.2 ml), and descending colon (0.33 ± 0.21/3.2 ml). The differences in CTC counts suggest that anatomical location of colorectal tumors may affect blood vessel metastasis. Meanwhile, patients with moderately differentiated and poorly differentiated tumors displayed higher peripheral blood CTC counts compared to those with well-differentiated tumors (P < 0.001). This result suggests that the type of tissue differentiation of colorectal tumors may act as another factor that affects blood vessel metastasis.ConclusionsCirculating tumor cells can be detected in the peripheral blood of colorectal cancer patients as well as patients with colorectal polyps. The differences in CTC counts suggest that anatomical location and the type of tissue differentiation of colorectal tumors may affect blood vessel metastasis.

Colorectal cancer patients with peripheral circulating tumor cells of the change and clinical significance

Objective Explore the dynamic changes of the patients with colorectal cancer circulating tumor cells (CTCs) in judging the prognosis, adjust the application value of treatment. Methods Select 88 cases of clinically diagnosed with colorectal cancer patients for the first time, the method of the second generation of enrichment of circulating tumor cells and fluorescence imaging technique, 1 day before the surgery, 1 month after the surgery, in late June and late December four detection of CTCs in peripheral blood. Analysis of patients in different periods of whether CTCs value, histological type, lymph node metastasis. Measurement based on the end of December of CTCs values compared with January measurement of CTCs is increased, divided into higher and higher than groups, compared two groups of recurrence and survival time. Results Of 88 patients with colorectal cancer, back to the blind and litres of 18 cases of colorectal cancer, 10 cases of colorectal cancer, 20 patients with sigmoid colorectal cancer, rectalcancer 40 cases, among them, the males 47 cases, aged 51-86 years old, 41 cases were female, aged 42-88 years old. Higher in group comparison, groups: 56 cases, higher than group of 32 cases. The baseline is similar between the two groups had no statistical significance (χ2=0.002, t=0.780, P=0.950); Residency histological differentiation degree, CTCs is higher than the degree of differentiation, the difference was statistically significant (F=12.28, P=0.001); In patients with lymph node metastasis of CTCs is higher than without metastasis, the difference was statistically significant (F=57.37, P=0.001); Increased survival time less than the higher group, (χ2=10.665, P=0.001), increased cases of recurrence is higher than the group (χ2=5.684, P=0.017), the difference had statistical significance. Conclusion Monitoring CTCs value dynamic changes of tumor recurrence in patients with colorectal cancer early judgment of judgment, the curative effect of postoperative chemotherapy, surgery, and postoperative recurrence in the short term of patients with stage Ⅱ and Ⅲ colorectal cancer and the treatment of patients with stage IV has guiding significance to the plan selection and improvement. Key words: Circulating tumor cells; Colorectal cancer; Prognosis

Circulating Tumor Cells Detected in the Tumor-Draining Pulmonary Vein Are Associated with Disease Recurrence after Surgical Resection of NSCLC

Tumor recurrence after surgical resection of NSCLC obstructs long-term disease-free survival in approximately 50% of cases. Our data suggest that combining circulating tumor cell enumeration (as single cells or clusters) in tumor-draining pulmonary vein and peripheral blood (assessed by CellSearch) at the time of NSCLC surgery better identifies those patients at higher risk for lung cancer recurrence than does peripheral circulating tumor cell number alone.

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Acquisition of Portal Venous Circulating Tumor Cells From Patients With Pancreaticobiliary Cancers by Endoscopic Ultrasound

BACKGROUND & AIMSTumor cells circulate in low numbers in peripheral blood; their detection is used predominantly in metastatic disease. We evaluated the feasibility and safety of sampling portal venous blood via endoscopic ultrasound (EUS) to count portal venous circulating tumor cells (CTCs), compared with paired peripheral CTCs, in patients with pancreaticobiliary cancers (PBCs).METHODSIn a single-center cohort study, we evaluated 18 patients with suspected PBCs. Under EUS guidance, a 19-gauge EUS fine needle was advanced transhepatically into the portal vein and as many as four 7.5-mL aliquots of blood were aspirated. Paired peripheral blood samples were obtained. Epithelial-derived CTCs were sorted magnetically based on expression of epithelial cell adhesion molecules; only those with a proper morphology and found to be CD45 negative and positive for cytokeratins 8, 18, and/ or 19 and 4′,6-diamidino-2-phenylindole were considered to be CTCs. For 5 samples, CTCs also were isolated by flow cytometry and based on CD45 depletion. ImageStream was used to determine the relative protein levels of P16, SMAD4, and P53. DNA was extracted from CTCs for sequencing of select KRAS codons.RESULTSThere were no complications from portal vein blood acquisition. We detected CTCs in portal vein samples from all 18 patients (100%) vs peripheral blood samples from only 4 patients (22.2%). Patients with confirmed PBCs had a mean of 118.4 ± 36.8 CTCs/7.5 mL portal vein blood, compared with a mean of 0.8 ± 0.4 CTCs/7.5 mL peripheral blood (P < .01). The 9 patients with nonmetastatic, resectable, or borderline-resectable PBCs had a mean of 83.2 CTCs/7.5 mL portal vein blood (median, 62.0 CTCs/7.5 mL portal vein blood). In a selected patient, portal vein CTCs were found to carry the same mutations as those detected in a metastatic lymph node and expressed similar levels of P16, SMAD4, and P53 proteins.CONCLUSIONSIt is feasible and safe to collect portal venous blood from patients undergoing EUS. We identified CTCs in all portal vein blood samples from patients with PBCs, but less than 25% of peripheral blood samples. Portal vein CTCs can be used for molecular characterization of PBCs and share features of metastatic tissue. This technique might be used to study the pathogenesis and progression of PBCs, as well as a diagnostic or prognostic tool to stratify risk of cancer recurrence or developing metastases.

Utility of microsieve device for enumeration and miRNA expression profiling of CTCs for personalized cancer management.

e22107 Background: Although a rare peripheral cell population, circulating tumor cells (CTCs) represent a potential alternative to serial invasive biopsies as a source of tissue in monitoring clinical states of non-hematological malignancies. CTCs harvested from a single patient are a heterogeneous mixture of cells differing in their transcriptome expressional profiles, metastatic potential and drug resistance properties. Mere identification and enumeration of CTCs are unable to provide qualitative information about individual CTC biology. The presence of a few highly malignant, metastasis-capable CTCs and cancer stem cells among the other less aggressive CTCs in the circulating population will not be apparent from CTC counts alone. Methods: We developed a high-throughput size-exclusion microsieve device (CellSievo, Singapore) to isolate CTCs (defined as DAPI-positive, CD45-negative cells), regardless of the cell surface receptors expressed, from 74 breast cancer patients. Using CTCs eluted from the microsieve post-capture, we performed microRNA expression profiling to further characterize breast cancer CTCs. Results: Using CellSievo’s microsieve device, we obtained an average mean CTC count of 21.2 (+/- 31.8; range=0-112.5) per 7.5ml peripheral blood, for breast cancer patients, as opposed to 1.3 (+/-1.6, range 0-5.0) for healthy subjects. Viable CTCs from breast cancer patients were successfully harvested for downstream microRNA profiling. CTCs from breast cancer patients were routinely found to express microRNA profiles mediating epithelial-to-mesenchymal transition, tumor invasion, and regulation of chemotherapeutic drug resistance to tamoxifen, anthracyclines, taxanes, and trastuzumab. Serial monitoring of microRNA expression pattern of CTCs could identify emergence of novel tumor populations with distinct microRNA expression patterns during or after anticancer therapy (detailed microRNA expression data to be presented). Conclusions: Our results suggest that microRNA biomarkers derived from peripheral CTCs can yield highly useful information and offer prognostic and predictive applications for therapeutics and companion diagnostic development.

Reply to Z.S. Lalmahomed et al

We appreciate the comments of Lalmahomed et al regarding our recent article, including the observation that low numbers of circulating tumor cells (CTCs) are observed in the peripheral circulation of patients with metastatic colon cancer, compared with the hepatic macrocirculation. Recently published data have also shown that in the setting of primary lung cancer, CTCs were positive in the peripheral blood of some patients at low numbers and in the pulmonary vein of most patients at high numbers. It is interesting to note that patients with metastatic breast and prostate cancer have higher numbers of CTCs in their peripheral blood, and liver resections in these individuals are generally unsuccessful, unlike the situation now in colon cancer. Furthermore, the presence of CTCs, even a single CTC in the peripheral blood of individuals with colorectal cancer, may indicate a poorer prognosis or those patients who are not suitable for hepatic resections. To investigate the clinical significance of low numbers of CTCs in the periphery, outcome probabilities in our series were examined (Fig 1). Although no statistically significant differences were observed, individuals with zero peripheral CTCs had numerically superior survival and progression-free survival probabilities at 12 months. This suggests that the presence of a single peripheral CTC in certain settings may have prognostic relevance. We agree that use of larger volumes of blood will lead to the discovery of more cells and thereby more material for cell characterization at the protein, RNA, and DNA levels. If the goal is to obtain more cells, then their method will work. This technique has been in practice at Munich for the SUCCESS A, B, and C trials in adjuvant breast cancer follow-up. To the best of our knowledge, in all primary cancers, investigators are routinely using more blood, 20 to 30 mLs, for analyses. Preanalytic error may, however, be introduced when layering blood over Ficoll, and a proportion of cells will inevitably be lost in doing so, although this has not been formally compared in the published abstracts thus far. Furthermore, the cutoff claimed by the manufacturer for metastatic breast cancer is no longer valid for clinical use if larger volumes of blood are used. For fairness, we are also compelled to mention that drawing three tubes and running three tubes, without concentration, is proportionately more expensive.

Circulating Tumor Cells in Patients Undergoing Surgery for Hepatic Metastases from Colorectal Cancer

Circulating tumor cells (CTCs) have been detected in patients with a variety of metastatic cancers, including colorectal, and may be a significant prognostic variable in patients with liver metastases. This prospective study involved 20 patients (13 men and 7 women) undergoing surgical excision or ablation of liver metastases from a colon or rectal primary tumor. Four 7.5-mL vials of peripheral blood were drawn preoperatively, 2 weeks postoperatively, and during mobilization of the liver or at the beginning of radiofrequency ablation. The samples were centrifuged, the sera combined to a final volume of 7.5 mL, and the CellSearch system used to identify circulating epithelial cells. A CTC count >2 was defined as clinically significant. Preoperative CTC levels averaged 3.9 (range, 0–56) and were significant in 2 patients (10%). Postoperative CTC levels averaged 1.0 (in 18 patients; range, 0–9) and were significant in 1 patient (5%). Intraoperative CTC levels averaged 28.2 (range, 0–315) and were significant in 10 patients (50%). At a median follow-up of 11.5 months (range, 5–25), 6 patients (30%) were dead of disease, 6 patients (30%) showed no evidence of disease, and 8 patients (40%) were alive with disease. Statistical analysis suggested a correlation between the presence of postoperative CTCs and survival (P = 0.036), as well as with disease-free survival (P = 0.036). Thus, CTCs are present and quantifiable in many patients with colorectal hepatic metastases, and peripheral CTCs are present in greater quantity during intraoperative liver manipulation. This preliminary study suggests a relationship between the presence of postoperative CTCs and outcome. Further accrual and follow-up of this group is needed to confirm these findings.

Identification of Enhancer of Zeste Homolog 2 Expression in Peripheral Circulating Tumor Cells in Metastatic Prostate Cancer Patients: A Preliminary Study

PurposeEnhancer of zeste homolog 2 (EZH2), a kind of transcriptional repressor, is reportedly over-expressed in metastatic prostate cancer. In this study, we analyzed EZH2 mRNA in circulating tumor cells (CTCs) in peripheral blood as a biomarker in patients with metastatic prostate cancer.Patients and MethodsBer-EP4 coated immunomagnetic beads were used to harvest CTCs, and mRNA was isolated by oligo-dT conjugated immunomagnetic beads. Reverse transcriptase-polymerase chain reaction for EZH2 mRNA was performed and the expression density was measured. The sensitivity of this test for detection of EZH2 mRNA was determined by serial dilutions of a human prostate cancer cell line. Blood samples were collected from 20 patients each with metastatic or localized prostate cancer and 10 healthy volunteers.ResultsSensitivity experiments showed that the test was highly sensitive as it could detect 10 tumor cells per 5 mL. EZH2 mRNA expression was obtained from peripheral blood samples of patients and control subjects. EZH2 mRNA expression density in the metastatic prostate cancer group was significantly higher than in the control (p = 0.023) and localized prostate cancer groups (p = 0.019). There was no difference between the control and localized prostate cancer groups (p > 0.05).ConclusionEZH2 mRNA expression in circulating epithelial cells represents a promising marker for detecting early metastasis in prostate cancer. However, more specific and sensitive techniques for detection of CTCs are needed to avoid mononuclear cell contamination.

Detection of clonal excess in lymphoproliferative disease by kappa/lambda analysis: correlation with immunoglobulin gene DNA rearrangement

Previous studies have suggested that analysis of the distribution of surface immunoglobulin light chain isotypes by flow cytometry provides evidence for monoclonality of B cell tumors and may detect populations of circulating tumor cells in patients with lymphoproliferative disease. We have used simultaneous flow cytometry and DNA restriction enzyme analysis on 58 samples of tissue and blood to determine whether lymphocyte populations detected by "kappa/lambda" analysis are indeed monoclonal. In greater than 90% of cases, abnormalities detected by flow cytometry correlated with monoclonal rearrangements of immunoglobulin genes as detected by Southern blot analysis. By analyzing tissue and blood from the same patients, we have also demonstrated that monoclonal circulating cells detected by flow cytometry reflect peripheral circulating tumor cells, since DNA from these cells shows the same immunoglobulin rearrangement as DNA from the original tumors in these patients. Although mixing studies suggested that DNA rearrangement studies were more sensitive than was flow cytometry in detecting minor populations of monoclonal lymphocytes, we found only one case in which this affected the diagnostic accuracy of the kappa/lambda analysis, with one notable exception, that of detection of a monoclonal proliferation of B cells that did not express surface immunoglobulin. The kappa/lambda test thus offers a powerful diagnostic tool in the evaluation of lymphoproliferative disease.