| Part Ⅰ:The role of circulating tumor cells on prediction and evaluation of responses after chemo-radiotherapy by use of magnetic-bead-based method in rectal cancer patientsPurpose:Since numerous therapy approaches are available nowadays, it is very essential to establish a convenient and reliable marker to help surgeons and physicians to make their decisions in individualized cancer treatment. The objective of this study was to investigate the clinical significance of circulating tumor cells(CTC) by use of immuno-magnetic-bead-based method on the evaluation and prediction of treatment responses in rectal cancer patients compared with serum CEA.Materials and Methods:A total of 103 patients who were confirmed pathologically with rectal cancer were enrolled in this study from February to September of 2012. Of these,66, 9 and 28 patients were diagnosed with clinical Stage Ⅱ-Ⅲ (cT3-4 and/or N+), local recurrence and distant metastasis, respectively, and all patients received chemo-radiotherapy (CRT). The CTC and serum CEA levels were detected before and after CRT, respectively. CTC were detected using EpCAM-magnetic-bead-based enrichment combined with cytometric identification.Results:CTC were observed in all 103 patients with a mean number of 12 per 5 mL while none of tumor cells could be found in 25 healthy controls. The CTC level in patients with distant metastasis was significantly higher than that in patients with local recurrence or with Stage Ⅱ-Ⅲ (P<0.05). The patients with more than one metastatic organ had a significantly higher CTC level than those with only one metastatic organ (P<0.001). The patients with Stage Ⅱ-Ⅲ who received neoadjuvant CRT followed by radical surgery were classified as responders and non-responders according to the pathological results after surgery. The CTC parameters especially △%CTC (percentage difference in CTC level between pre-CRT and post-CRT) were identified as the stronger predictor to discriminate responder and non-responder group compared with CEA parameters. Metastatic cancer patients were classified as responders and non-responders based on the results of chemo-radiotherapy. In the responder group, the CTC level of all 14 patients decreased after the treatment, whereas the serum CEA level decreased in 13 of 14 patients and that of one patient increased. In the non-responder group, the CTC level of all 6 patients increased whereas the serum CEA level increased in only 4 of 6 patients after the treatment.Conclusion:CTC, which is detected by use of magnetic-bead-based method, is an accurate and promising marker for the evaluation and prediction of treatment responses in patients with rectal cancer, superior to the conventional tumor markers such as serum CEA.Part Ⅱ:High-performance size-based microfluidic device for the detection of circulating tumor cells from peripheral blood in rectal cancer patientsPurpose:Circulating tumor cells (CTC), originated from either primary or metastatic cancer, could provide important information for diagnosis and monitoring of cancer. However, the implication and development of CTC are limited due to the extreme rarity of these tumor cells. In this study we designed and fabricated a simple and high-performance microfluidic device, which exploited numerous filtered microchannels in it to capture these rare CTC from peripheral blood of rectal cancer patients, and compared its performance with that of EpCAM-magnetic-bead-based method. We also investigated the clinical significance of CTC in rectal cancer patients by use of our microfluidic device.Materials and Methods:Our microfluidic device is based on the differences in size between tumor cells and blood constituents. It consists of 80 main channels and 81 side channels, which are arranged in an interdigital manner. A group of narrow parallel-arranged filter channels are designed to connect each pair of main channel and adjacent side channel. Blood sample passes through this device from main channels, most of the small-sized hematologic cells in whole blood such as erythrocytes and leukocytes can be filtered into their adjacent side channels via filter channels and then eliminated from the waste chamber. The large-sized cells such as tumor cells cannot pass through the narrow filter channels and then remain in the main channels. After immunofluorescence staining, CTC can then be identified and enumerated directly under fluorescence microscope. We detected CTC of 60 patients with rectal cancer, who were enrolled from September to December of 2012, and analyzed the relationship between CTC counts and clinical features, treatment responses after chemo-radiotherapy.Results:In order to optimize the experimental parameters of this device, we spiked HT-29 cells with known numbers into blood samples from healthy donors to measure capture efficiency and cell purity. A very high CTC capture efficiency (average recovery rate:94%) was obtained in this device at the optimum flow rate of 0.5 mL/h and channel height of 5 μm. Then we used this device for detecting CTC in 60 rectal cancer patients with various stages. The CTC counts of rectal cancer patients were significantly higher than those in 30 healthy subjects (167.33±212.76 vs.0.17 ±0.59, P<0.05). Additionally, patients with distant metastases had significantly higher CTC levels than patients with Stage Ⅱ-Ⅲ or those with local recurrence (385.30±245.86 vs.39.40±19.23 vs.115.20±69.15, P<0.05). For these rectal cancer patients with different stages, we could find a close relationship between CTC counts and treatment responses after chemo-radiotherapy. Furthermore, we compared the performance of our microfluidic device for enumeration of tumor cells against EpCAM-based enrichment method. We found the CTC counts detected by this device were significantly higher than those by EpCAM bead-based method for rectal cancer patients with various stage. Especially, for rectal cancer patients with Stage Ⅱ-Ⅲ, the positive rates of samples with more than 3 CTCs per 5 mL blood by use of microfluidic device vs. EpCAM-based ones were 100% vs.47%, respectively.Conclusion:Our microfluidic device provides a new and effective tool for accurate identification and measurement of CTCs in patients with rectal cancer, and has broad potential in clinical practice.Part Ⅲ:The role of sequential 18F-FDG PET/CT in predicting tumor response after preoperative chemo-radiation for rectal cancerPurpose:The aim of this study was to investigate the potential of sequential 18F-FDG PET/CT SUV/metabolic area variation in assessing and predicting pathological response to preoperative chemo-radiotherapy(CRT) for rectal cancer.Materials and Methods:Fifty-three patients diagnosed as clinical T3-4 and/or N+ rectal cancer were enrolled from June 2009 to February 2012. All patients received CRT followed by radical surgery after 6-8 weeks. All patients underwent 18F-FDG PET/CT scans before initiation of treatment (PET/CT 1) and a second scan after the completion of CRT (PET/CT2).35 out of 53 patients also underwent a third PET/CT within 1 week prior to the surgery (PET/CT3). SUVmax, SUVmean, metabolic tumor volume(MV), total lesion glycolysis(TLG) and response indices(△%, percentage difference between two different PET/CT for SUVmax, SUVmean, MV and TLG) were measured and calculated. The different metabolic parameters were analysed and correlated with tumor regression grade(TRG) score.Results:SUVmax, SUVmean, MV and TLG in PET/CT1 were significantly higher than those in PET/CT2 or PET/CT3 (P<0.001). When patients were regrouped as responders (TRG 3-4) and non-responders (TRG 0-2), significant differences in △%MV(1-3) (91.08% vs.75.43%) and △%TLG(1-3) (94.00% vs.82.02%) were observed. As demonstrated by receiver operating characteristic(ROC) analysis, A%MV(1-3) and △%TLG(1-3) both had stronger capability to discriminate responders from non-responders. When patients were classified as pathological complete response (pCR) and non-pCR, significant differences in △%SUVmax(1-3) (69.17% vs.57.77%), △%SUVmean(1-3) (44.20% vs.30.19%), △%MV(1-3) (90.93% vs.80.30%) and △%TLG(1-3) (94.22% vs.85.63%) were observed. △%TLG(1-3) was a more powerful discriminator compared with others.Conclusion:The variation of SUV/metabolic area with 18F-FDG PET/CT has the potential of evaluation and prediction of preoperative CRT response in rectal cancer. |
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