Hung-Chih Lin

A negative selection system PowerMag for effective leukocyte depletion and enhanced detection of EpCAM positive and negative circulating tumor cells

BACKGROUND Circulating tumor cells (CTCs) can be enriched from peripheral blood by leukocyte depletion. In this study, a PowerMag system was designed to enhance leukocyte depletion and CTC detection. METHODS The efficiency of PowerMag in leukocyte depletion and recovery of CTCs was evaluated. The cell populations in leukocyte-depleted cell filtrate were characterized by immunofluorescence staining using anti-EpCAM and anti-CD45 antibodies. The suitability of PowerMag in monitoring cancer patient CTCs was assessed by analyzing blood samples collected at baseline and 2 weeks after baseline from the patients with colorectal cancer (CRC, n=24) and head and neck squamous cell carcinoma (HNSCC, n=28). RESULTS Only few contaminated CD45(+)-leukocytes were retained in the leukocyte-depleted cell filtrate. The enriched CTCs were viable with a recovery rate of 46-62%. Immunofluorescence staining of the nucleated cells enriched by PowerMag revealed 2 major cell populations EpCAM(+)CD45(-) and EpCAM(-)CD45(-). The number for both cell types was significantly increased in cancer patients when compared to healthy control. After chemotherapy, the number of EpCAM(+)CD45(-) and EpCAM(-)CD45(-) cells was decreased more prominently for CRC and HNSCC patients, respectively. CONCLUSIONS PowerMag holds great promise as a platform for leukocyte depletion and CTC detection in a clinical setting.

Prognostic value of circulating tumor cells with podoplanin expression in patients with locally advanced or metastatic head and neck squamous cell carcinoma

Podoplanin (PDPN) is a prognostic factor for head and neck squamous cell carcinoma (HNSCC). However, PDPN expression in circulating tumor cells (CTCs) and its prognostic value are not clear.

Circulating epithelial cell enumeration facilitates the identification and follow-up of a patient with early stage papillary thyroid microcarcinoma: A case report.

BACKGROUND This study examines whether the measurement of circulating epithelial cells (CECs) facilitates the identification and follow-up of a patient with thyroid cancer. METHODS A 29-y-old woman with no cancer history was enrolled as a healthy control in a CEC study. CECs were enriched from the peripheral blood by the negative selection system PowerMag. Various medical examinations were performed on the patient to establish the diagnosis and to follow-up her disease status during treatment. RESULTS This patient had unexpectedly high CEC counts that were sustained for more than two weeks. Thyroid gland ultra-sonography revealed lesions in the left lobe that could not be confirmed as cancer by magnetic resonance imaging, (18)F-fludeoxyglucose-positron emission tomography-computed tomography or cytopathological analysis, but were histologically confirmed after thyroidectomy as papillary thyroid microcarcinoma. Both the CEC count and serum thyroglobulin (Tg) concentration were significantly decreased after thyroidectomy, and they and the patients disease status were correlated during remnant ablation therapy. The CEC count returned to normal when the patient was disease-free 10 months after thyroidectomy. CONCLUSIONS CEC testing facilitates the identification of individuals at risk for cancer. Longitudinal follow-up of the CEC count may complement serum Tg testing for monitoring the status of patients with thyroid cancer.

Circulating epithelial cell counts for monitoring the therapeutic outcome of patients with papillary thyroid carcinoma

Loco-regional recurrence or distant metastasis usually leads to the death of patients with papillary thyroid carcinoma (PTC). Whether or not circulating epithelial cells (CECs) count is a valuable marker in monitoring the therapeutic outcome of PTC was investigated. Patients with PTC (n=129) were treated in our medical center and were categorized into 4 groups with excellent (n=45), biochemical incomplete (n=15), indeterminate (n=37), and structural incomplete (n=32) responses. CECs were enriched from the peripheral blood by the PowerMag negative selection system. Three subtypes of CECs expressing epithelial cell adhesion molecule (EpCAM), thyroid-stimulating hormone receptor (TSHR, a marker for thyroid cells), and podoplanin (PDPN, a marker related to poor prognosis in patients with PTC) were defined by immunofluorescence staining, respectively. The median number of CECs (cells/mL of blood) expressing EpCAM, TSHR, and PDPN was 23 (interquartile range 10-61), 19 (interquartile range 8-50), and 8 (interquartile range 3-22), respectively, for patients enrolled in this study. The number of EpCAM+-CECs, TSHR+-CECs, and PDPN+-CECs was statistically different among patients in different treatment response groups without interference from anti-thyroglobulin antibody (P<0.0001). Patients with structural incomplete response had higher counts for all three CECs subtypes when compared to other patients. EpCAM+-CECs was better in distinguishing patients with excellent response from structural incomplete response among the three subtypes of CECs. The sensitivity and specificity of the assay was 84.4% and 95.6%, respectively, when the cut off value was 39 EpCAM+-CECs/mL. CECs testing can supplement the current standard methods for monitoring the therapeutic outcome of PTC.

Combined analysis of circulating epithelial cells and serum thyroglobulin for distinguishing disease status of the patients with papillary thyroid carcinoma

Papillary thyroid carcinoma (PTC) accounts for about 80% of the cases in thyroid cancer. Routine surveillance by serum thyroglobulin (Tg) and medical imaging is the current practice to monitor disease progression of the patients. Whether enumeration of circulating epithelial cells (CECs) helps to define disease status of PTC patients was investigated. CECs were enriched from the peripheral blood of the healthy control subjects (G1, n = 17) and the patients at disease-free status (G2, n = 26) or with distant metastasis (G3, n = 22). The number of CECs expressing epithelial cell adhesion molecule (EpCAM) or thyroid-stimulating hormone receptor (TSHR) was determined by immunofluorescence microscopy analyses. The medium number of EpCAM+-CECs was 6 (interquartile range 1-11), 12 (interquartile range 7-16) and 91 (interquartile range 31-206) cells/ml of blood for G1, G2 and G3, respectively. EpCAM+-CEC counts were significantly higher in G3 than in G1 (p < 0.05) and G2 (p < 0.05). The medium number of TSHR+-CECs was 9 (interquartile range 3-13), 16 (interquartile range 10-24) and 100 (interquartile range 31-226) cells/ml of blood for G1, G2 and G3, respectively. The TSHR+-CEC counts also distinguished G3 from G1 (p < 0.05) and G2 (p < 0.05). With an appropriate cut off value of CEC count, the disease status for 97.9% (47/48) of the cases was clearly defined. Notably, the metastatic disease for all patients in G3 (22/22) was revealed by combined analysis of serum Tg and CEC. This study implicates that CEC testing can supplement the current standard methods for monitoring disease status of PTC.

Circulating epithelial cells as potential biomarkers for detection of recurrence in patients of papillary thyroid carcinoma with positive serum anti-thyroglobulin antibody

BACKGROUND Serum thyroglobulin (Tg) is not a reliable tumor marker for monitoring disease status after treatment in patients with papillary thyroid carcinoma (PTC) with positive anti-thyroglobulin antibody (TgAb). The aim of this study was to evaluate the clinical role of circulating epithelial cells (CECs) in PTC patients with positive serum TgAb and undetectable serum Tg. METHODS A pilot study was performed to evaluate CECs in 25 PTC patients with positive serum TgAb and undetectable serum Tg. CECs were isolated and enriched from peripheral blood with a negative selection system PowerMag. Immunofluorescence staining with anti-epithelial cell adhesion molecule (anti-EpCAM) and anti-thyroid stimulating hormone receptor (anti-TSHR) antibodies were used to define EpCAM+-CECs and TSHR+-CECs. After CECs testing, 25 patients were classified into two groups: recurrence group (n=7) and remission group (n=18) based on biopsy or imaging studies. The diagnostic accuracy and cutoff points of EpCAM+-CECs and TSHR+-CECs were evaluated using receiver operating characteristic (ROC) curves. The optimal cut-off values of CECs were determined by the Youden index (sensitivity+specificity-1). RESULTS The median numbers of EpCAM+-CECs (72.5 vs. 10.75) and TSHR+-CECs (54 vs. 5.25) were significantly increased in recurrence group compared to remission group. The area under the curve (AUC) showed good performance of EpCAM+-CECs (0.937) and TSHR+-CECs (0.825) to discriminate between recurrence and remission. The cut-off value for EpCAM+-CECs and TSHR+-CECs were set at 48cells/ml and 10cells/ml, respectively and showed a sensitivity (EpCAM+-CECs: 85.7%; TSHR+-CECs: 85.7%) and a specificity (EpCAM+-CECs: 100%; TSHR+-CECs: 77.8%) in predicting the recurrence. CONCLUSIONS Our study suggests CECs testing could be a potential biomarker to identify recurrence in PTC patients with positive serum TgAb and undetectable serum Tg.

Abstract 461: Combined analysis of circulating epithelial cell count and serum thyroglobulin for differentiating disease status of the patients with papillary thyroid carcinoma

Papillary thyroid carcinoma (PTC) is one type of thyroid cancer and accounts for about 80% of the cases. Routine surveillance by serum thyroglobulin (Tg) and medical imaging is the current practice to monitor disease progression of the patients. However, the presence of anti-Tg antibody or other influence factors may discount the value of serum Tg in disease monitoring. Whether enumeration of circulating epithelial cells (CECs) and its combined analyses with serum Tg help to define disease status of PTC patients was investigated. CECs were first enriched from the peripheral blood of the healthy control subjects (G1, n = 17) and the patients at disease-free status (G2, n = 26) or with distant metastasis (G3, n = 22). The number of CECs expressing epithelial cell adhesion molecule (EpCAM) was determined by immunofluorescence microscopy analyses. The medium number of EpCAM+-CECs was 6 (intequartile range 1-11), 12 (interquartile range 7-16) and 91 (interquartile range 31-206) cells/ml of blood for G1, G2 and G3, respectively. EpCAM+-CEC counts were significantly higher in G3 than in G1 (p Citation Format: Ching-Ping Tseng, Jen-Der Lin, Hung-Chih Lin, Ju-Chien Cheng. Combined analysis of circulating epithelial cell count and serum thyroglobulin for differentiating disease status of the patients with papillary thyroid carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 461.

Abstract 1599: Potential values of circulating tumor cell enumeration for clinical management of patients with papillary thyroid carcinoma

Thyroid cancer is one of the most rapidly increasing malignancies, It is the most common endocrine cancer and the fifth most common cancer in women. Moreover, the improved examination technologies of ultrasonographic and thyroglobulin even 18F-fluorodeoxyglucose positron emission tomography and nodule biopsy were insufficient to real-time reflect the patients prognosis. For the prognostic and predictive purpose to improve the diagnostics of thyroid cancer, circulating tumor cells (CTCs) have been developed.Previous studies have shown that CTCs can serve as liquid biopsies to monitoring treatment response and prognosis, but only few articles discussed in papillary thyroid carcinoma (PTC) which composed around 80% of thyroid cancer.In this study, a total of 41 cases were enrolled.In the group I (G1), we included 12 control subjects without clinical significant thyroid disorder like thyroid nodule or autoimmune thyroid disease or any cancer history. In the group II (G2), 11 disease free thyroid cancer patients were enrolled. In the group III (G3), we enrolled 18 PTC patients with distant metastasis that were confirmed by 131I whole-body scintigraphy (WBS) or other imaging methods. The peripheral blood of control subjects and cancer patients were analyzed by a negative selection method PowerMag system. The number of cells with the epithelial cell adhesion molecular (EpCAM) or thyroid stimulating hormone receptor (TSHR)expression was counted. In ROC analysis, EpCAM+ counts showed no significantly difference between G1 and G2 (p = 0.0728). Between G1 and G3 (p Citation Format: Hung-Chih Lin, Jen-Der Lin, Ching-Ping Tseng. Potential values of circulating tumor cell enumeration for clinical management of patients with papillary thyroid carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1599. doi:10.1158/1538-7445.AM2015-1599

Abstract 4830: Prognostic value of circulating tumor cells with podoplanin expression in locally advanced and metastatic head and neck squamous cell carcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Podoplanin (PDPN) is a biomarker for poor prognosis in head and neck squamous cell carcinoma (HNSCC). A prospective study was designed to investigate the prognostic value of circulating tumor cells (CTCs) with PDPN expression (PDPN+-CTCs) in HNSCC patients. Sixty-one healthy donors and fifty-three HNSCC patients with locally advanced and metastatic diseases were enrolled in this study. CTCs were enriched from the peripheral blood by the negative selection method PowerMag that has been validated in our previous study. Enumeration of CTCs was performed by co-immunofluorescence staining using the anti-epithelial cell adhesion molecule (EpCAM) and anti-PDPN antibodies. The CTCs (EpCAM+) and PDPN+-CTCs (EpCAM+PDPN+) counts between the patients and healthy volunteers were statistically different (p 20% were associated with poor PFS (hazard ratio (HR): 4.666, 95% CI: 1.373-15.856, p = 0.016) and OS (HR: 12.400, 95% CI: 1.643-93.570, p = 0.015). Kaplan-Meier survival curve further confirmed that the PDPN+/EpCAM+-CTCs ratio had significant impact on PFS (p = 0.006) and OS (p = 0.008). In conclusion, CTCs enumeration clearly differentiates HNSCC patients from healthy individuals, while the PDPN+/EpCAM+-CTC ratio is more important than the absolute CTCs count in determining the degree of malignancy and serves as an independent prognostic factor for PFS and OS. Citation Format: Ching-Ping Tseng, Hung-Chih Lin, Chia-Hsun Hsieh, Hung-Ming Wang, Ju-Chien Cheng. Prognostic value of circulating tumor cells with podoplanin expression in locally advanced and metastatic head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4830. doi:10.1158/1538-7445.AM2014-4830

Abstract 4817: Circulating tumor cells enriched by the negative selection system PowerMag is a useful predictor for treatment response of metastatic colorectal cancer patients

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Enumeration of circulating tumor cells (CTCs) provides prognostic and predictive value to guide treatment options. This study aims to (1) evaluate the merits of the PowerMag system, an immunomagnetic separation technique that we recently developed for negative selection of CTCs, in the prediction of treatment response; (2) perform analysis to investigate whether there is a correlation between the imaging response and the changes of CTCs counts before and during chemotherapy for the patients with metastatic colorectal cancer (mCRC). A total of 47 mCRC patients were enrolled in this prospective single-center study. CTCs were isolated from the peripheral blood of these patients by PowerMag at baseline and 2 weeks after starting first dose of chemotherapy. CTCs enumeration was then performed by immunofluorescence staining using the anti-epithelial cell adhesion molecule (EpCAM) antibody. Tumor response at 12 weeks after chemotherapy of the whole cohort was monitored by computed tomography (CT) scans following RECIST criteria 1.0. Our data revealed that PowerMag is a cost-effective negative selection-based CTCs isolation platform with a CTCs detection rate of 89.4% (42/47). The CTCs counts could successfully differ mCRC patients from healthy donors (P = 0.0015). The higher baseline CTCs number was associated with initially metastatic status rather than recurrent settings (P = 0.042). In all assessable patients, a high correlation rate of 86.7% was found between changes of CTCs (decline or elevation) and CT responses (progressive disease, and partial response/stable disease) with a P value of 0.005 (Fisher exact test). These data indicate that the change of CTCs counts before and during treatment is in accord with the image response evaluation at 12 weeks after initiation of treatment. In conclusion, PowerMag is a cost-effective method for negative selection of CTCs from cancer patients. Moreover, comparison of CTCs counts before and during treatment is a useful predictor for disease progression of mCRC patients that warrants further investigation in large-scale validation trial. Citation Format: Jason Chia-Hsun Hsieh, Hung-Chih Lin, Hung-Chih Hsu, John Wen-Cheng Chang, Ching-Ping Tseng. Circulating tumor cells enriched by the negative selection system PowerMag is a useful predictor for treatment response of metastatic colorectal cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4817. doi:10.1158/1538-7445.AM2014-4817