循环肿瘤细胞最新进展2015

Ex vivo culture of circulating breast tumor cells for individualized testing of drug susceptibility
Liu Zixin
Department of Hepatic Surgery VI,EHBH 2015-01-05
CTC: EpCAM (+) white blood cells: CD45(+) and CD66(+)
Basic modes:
positive selection mode: anti-EpCAM
negative selection mode: anti-CD45 anti-CD66
CTC-iChip system
Report Content • • • • • • CTC-iChip system and CTC culture the Identification of CTC Gene mutation analysis Drug sensitivity Conclusion Discussion
Knowledge on CTC-iChip system Biomarkers
Results hared cytological features with the matched primary CTCs captured . Consisitent with standard CTC definitions: CK(+);CD45(-);proliferative index~30%(Ki67)； initial doubling time:3days to 3weeks. Five CTC lines retained ER(+),BRx-07 lost ER expresion,but regained in Xenograft tumor. CTC-derived tumors shared histological and immunohistochemical features with the matched primary tumor.
Discussion
Samples: patients with metastatic/PVTT which stage? Mode: negative mode. Drug targets :mutational gene analysis
@Liu Zixin
which stage?biomarkers?AFP?
Results of Drug Sensitivity (BRx-68)
• SERMs(tamoxifen) and SERD(raloxifene) were ineffective,alone or combined with inhibitors of PIK3-mTOR(everolimus). • HSP90 inhibitor STA9090 demonstrated cytotoxicity,alone or combinition with SERMs or SERD. • A low dose of STA9090 suppressed ER level.?
Results of Drug Sensitivity (BRx-07)
• Different FGFR mutations have different sensitivity to PIK3CA and FGFR2 inhibitors.
Conclusions
• Study patterns of drug susceptibility with CTC,combined with genetic context. • Enable monitoring of emerging subclones with alterd mutational and drug sensitivity profiles.
NGS platform:CTC culture and other mutation (1000 genes)
Results of gene analysis
• ESR1(BRx-68),TP53(BRx-68,61) and KRAS(BRx-42) are universally present in all independent CTC cell lines. • PIK3CA,KRAS and FGFR2 are arising mutations. • The mutant allele frequences of TP53 and ESR1 are increased upon prolonged culture.
Debulking by separation of nucleated cells from the enucleated.
Alignment of nucleated cells within a microfluidic channel. Deflection of magnetically tagged cells into a collection channel.
Results of CTC culture
CTC lines can only be generated from patients who were either off therapy or progressing on treatment. and cannot be generated from patients who were responding to treatment. For three patients, four additional CTC cell lines were established from blood samples drawn at multiple different time points during therapy.In these cases,CTCs were cultured only when patients were progressing on treatment.
Results of Drug Sensitivity (BRx-07)
• Cultured CTCs were highly sensitive to PIK3CA and FGFR2 inhibitors. • Combinations of PIK3CA and FGFR inhibitors showed cooperatove effects.? • iIGFR mitigate sensitivity to iPI3K;iHSP90 stablize mutant kinases.
• Choose drug targets ESR1 and PIK3CA for drug sensitivity test.
Results of Drug Sensitivity test
Some CTC drug sensitivity measurements were concordant with clinical history.
The Special conditions for CTC cultrue
• Blood samples:patients with metastatic ER(+) • Nonadherent culture conditions (critical) • Serum-free supplemented EGF and FGF • under hypoxic conditions (4% O2) • Establish long-term oligoclonal CTC (sustaimed in vitro >6months)
Gene analysis
RNA sequencing analysis
6 cultured cell lines; 13 commonly used breast cancer cell lines; 29 unclured single CTCs.
Mutation analysis
SNaPShot : primary tumor (~140 mutations in 25 gene)
Combines the strengths of microfluidics for rare cell handing and magnetic-based cell sorting.
Magnetic labeling of cells in blood cells.( pos- vs neg- )