Objective: To investigate whether small endoscopic biopsies of colorectal cancer were sufficient for quality and accurate mutational analysis by amplicon-based next-generation sequencing (NGS). Methods: By using an amplicon-based targeted NGS panel for mutational detection on Illumina Miseq platform, a total of 109 formalin-fixed and paraffin-embedded (FFPE) endoscopic biopsies of colorectal cancer were retrospectively selected, based on specific histopathologic criteria, from January 2012 to June 2016 at West China Hospital of Sichuan University and Peking University Third Hospital. Twelve of these biopsies had corresponding FFPE surgical resection specimens. Quality control parameters of NGS testing were analyzed and NGS results were confirmed by other methods. Mutation calls of the 12 paired endoscopic biopsies and surgical resections were compared. Results: Of the endoscopic biopsy specimens, 97.2% (106/109) had sufficient DNA and qualified sequencing library. NGS generated excellent sequencing data, with a median of 848× for median read depth and 95.7% for uniformity. The success rate of NGS was 95.4% (104/109). Conventional methods confirmed the results of NGS for KRAS and BRAF, and the concordance rate was 100.0%. The clinically actionable mutations detected in the 12 paired endoscopic biopsies and surgical resections were concordant. Conclusion FFPE endoscopic biopsies of colorectal cancer is suitable for targeted NGS, providing quality sequencing data and accurate mutational information to guide targeted therapy.

Objective: To compare the performance of Miseq and Ion Torrent PGM platforms and library construction method for next-generation sequencing (NGS) technology for formalin-fixed and paraffin-embedded (FFPE) samples. Methods: A total of 204 FFPE cancer samples including 100 non-small cell lung cancers at the First Affiliated Hospital of Zhejiang University, and 104 colorectal cancers at West China Hospital of Sichuan University were retrospectively selected from January 2013 to December 2016. By using the same samples, DNA was extracted, and the same amount of DNA was used for library construction with the same kit, and sequenced on Miseq and Ion Torrent PGM respectively, after passing the quality control. Any discordant mutations between two platforms were validated by amplified refractory mutation system-polymerase chain reaction (ARMS-PCR) method and Sanger sequencing. Results: A total of 204 FFPE samples were included and 197 samples were successfully analyzed by both platforms. The number of reads generated by the samples on Miseq platform sequencing was higher than PGM platform (median 391 634 vs. 298 030, P<0.01). Alignment with human reference genome showed that the mapping rate of Miseq platform was higher than PGM platform (median 100.0% vs. 99.7%, P<0.01). The median sequence depth of samples on Miseq was higher than PGM platform (median 853× vs. 698×, P<0.01). A total of 236 mutations were detected by two platforms, of which 221 were detected on both platforms, with a 93.6% concordance. Miseq platform detected 11 mutations not detected on PGM platform, while PGM platform detected 4 more mutations not detected on Miseq platform. With validation by ARMS-PCR and Sanger sequencing, Miseq platform was more reliable for low-frequency mutations. The main reasons for the discordant mutations between two platforms were that mutation frequency on undetected platform was lower than mutation reporting range (5%) and FFPE samples were stored for a long time. Conclusions Compared with PGM, Miseq platform shows higher sequencing quality in terms of the number of reads, alignment results and coverage depth, and the test results are more reliable. In clinical practice, the appropriate platform should be chosen based on sample size and actual throughput requirements to aid in the molecular characterization of tumors.

Objective To compare the application values and setup errors between vacuum bag plus body mask and customized alpha cradle duringproton and carbon therapy using Siemens 6D robotic couch in prostate cancer patients.Methods Nineteen patients received vacuum bag plus body mask setup were allocated into the vacuum bag group andl9 patients with alpha cradle were assigned into the alpha cradle group.Orthogonal X-ray portals were performed to verify the treatment position before beam delivery in every fraction.The couch correction between the portal and reference DRR through manual image registration was recorded as setup errors in 6 directions including the lateral,supine-inferior,anterior-posterior,yaws,roll and pitch,respectively.Two-tail t-test was used to analyze the setup error data from each direction between two groups.Results In total,452 and 436 sets of data errors were collected from the vacuum bag and alpha cradle groups.The average setup errors and standard deviation in the vacuum bag and alpha cradle groups in the lateral,supine-inferior,anterior-posterior,yaws,roll and pitch directions were (0.63±0.48) cm vs.(0.33±0.24) cm (P=0.000),(0.40±0.3) cm vs.(0.31±0.25) cm (P=0.000),(0.69±0.61) cm vs.(0.82±0.69) cm (P=0.006),0.65°±0.47°vs 0.32°±0.25°(P=0.000),1.05°±0.95°vs 1.16°±0.94° (P=0.100) and 0.67°±0.56°vs 0.40°±0.36° (P=0.000),respectively.The maximum setup errors were detected in the pitch direction for both groups.Conclusions During the proton and carbon therapy using Siemens 6D robotic couch,two setup methods using vacuum bag plus body mask and customized alpha cradle should be selected according to the individual conditions of patients.A customized foot fixer should be utilized to reduce the uncertainty in the femoral head region.

ObjectiveTo study the effect of Xihuangwan extract on mitochondrial energy metabolism in ovarian cancer SKOV3 and HEY cells and to explore the underlying mechanism. MethodSKOV3 and HEY cells were cultured in vitro and treated with different concentrations （0， 5， 10， 15， 20 g·L^-1） of Xihuangwan extract. Methyl thiazolyl tetrazolium （MTT） was used to examine the viability of SKOV3 and HEY cells treated with Xihuangwan extract. The adenosine-triphosphate （ATP） levels in SKOV3 and HEY cells were measured by kit. Flow cytometry was employed to measure the content of reactive oxygen species （ROS） in cells. Western blot was employed to determine the protein levels of peroxisome proliferator-activated receptor-γ co-activator 1α （PGC1α）， transcription factor A， mitochondrial （TFAM）， translocase of outer mitochondrial membrane 20 （TOMM20）， and aplasia Ras homologue member Ⅰ （ARHⅠ） in SKOV3 and HEY cells. Mito-Tracker Green staining was used to observe the morphological changes of mitochondria in SKOV3 and HEY cells. ResultCompared with blank group， Xihuangwan extract treatment for 24， 48 h inhibited the viability of SKOV3 and HEY cells in a concentration-dependent manner （P<0.05， P<0.01）. Compared with blank group， Xihuangwan extract （10， 15， 20 g·L^-1） groups presented lowered ATP levels （P<0.05， P<0.01）， and the 20 g·L^-1 Xihuangwan extract group had lower ATP level than the 10 and 15 g·L^-1 Xihuangwan extract groups （P<0.05）. Compared with blank group， Xihuangwan extract increased the content of ROS in SKOV3 and HEY cells in a concentration-dependent manner （P<0.05， P<0.01）， and the 20 g·L^-1 Xihuangwan extract group had higher ROS content than the 10 g·L^-1 Xihuangwan extract group （P<0.05）. Compared with blank group， Xihuangwan extract up-regulated the expression level of ARHⅠ protein in SKOV3 and HEY cells in a concentration-dependent manner （P<0.01）， and the expression levels of ARHⅠ protein was higher in the 20 g·L^-1 Xihuangwan extract group than in the 10 and 15 g·L^-1 Xihuangwan extract groups （P<0.05）. Compared with the blank group， Xihuangwan extract down-regulated the protein levels of PGC1α， TFAM， and TOMM20 in SKOV3 and HEY cells in a concentration-dependent manner （P<0.05， P<0.01）， and the protein levels of TFAM and TOMM20 in the HEY cells treated with 20 g·L^-1 Xihuangwan extract were lower than those in the HEY cells treated with 10， 15 g·L^-1 Xihuangwan extract （P<0.05）. Compared with the blank group， 20 g·L^-1 Xihuangwan extract decreased the Mito-Tracker fluorescence intensity of SKOV3 and HEY cells （P<0.05）. ConclusionXihuangwan can compromise the mitochondrial function of ovarian cancer SKOV3 and HEY cells and reduce cell energy metabolism to inhibit the proliferation of SKOV3 and HEY cells by up-regulating ARHⅠ and inhibiting PGC1α/TFAM signaling axis.

Radiotherapy is widely used in the management of advanced colorectal cancer (CRC). However, the clinical efficacy is limited by the safe irradiated dose. Sensitizing tumor cells to radiotherapy via interrupting DNA repair is a promising approach to conquering the limitation. The BRCA1-BARD1 complex has been demonstrated to play a critical role in homologous recombination (HR) DSB repair, and its functions may be affected by HERC2 or BAP1. Accumulated evidence illustrates that the ubiquitination-deubiquitination balance is involved in these processes; however, the precise mechanism for the cross-talk among these proteins in HR repair following radiation hasn't been defined. Through activity-based profiling, we identified PT33 as an active entity for HR repair suppression. Subsequently, we revealed that BAP1 serves as a novel molecular target of PT33 via a CRISPR-based deubiquitinase screen. Mechanistically, pharmacological covalent inhibition of BAP1 with PT33 recruits HERC2 to compete with BARD1 for BRCA1 interaction, interrupting HR repair. Consequently, PT33 treatment can substantially enhance the sensitivity of CRC cells to radiotherapy in vitro and in vivo. Overall, these findings provide a mechanistic basis for PT33-induced HR suppression and may guide an effective strategy to improve therapeutic gain.

Lenvatinib, a second-generation multi-receptor tyrosine kinase inhibitor approved by the FDA for first-line treatment of advanced liver cancer, facing limitations due to drug resistance. Here, we applied a multidimensional, high-throughput screening platform comprising patient-derived resistant liver tumor cells (PDCs), organoids (PDOs), and xenografts (PDXs) to identify drug susceptibilities for conquering lenvatinib resistance in clinically relevant settings. Expansion and passaging of PDCs and PDOs from resistant patient liver tumors retained functional fidelity to lenvatinib treatment, expediting drug repurposing screens. Pharmacological screening identified romidepsin, YM155, apitolisib, NVP-TAE684 and dasatinib as potential antitumor agents in lenvatinib-resistant PDC and PDO models. Notably, romidepsin treatment enhanced antitumor response in syngeneic mouse models by triggering immunogenic tumor cell death and blocking the EGFR signaling pathway. A combination of romidepsin and immunotherapy achieved robust and synergistic antitumor effects against lenvatinib resistance in humanized immunocompetent PDX models. Collectively, our findings suggest that patient-derived liver cancer models effectively recapitulate lenvatinib resistance observed in clinical settings and expedite drug discovery for advanced liver cancer, providing a feasible multidimensional platform for personalized medicine.