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Background: Asia has a high burden of hepatocellular carcinoma (HCC) due to the high rates of chronic hepatitis B infection and accounts for 70% of HCC cases globally. In the past 20 years, the systemic treatment landscape of advanced HCC has evolved substantially - from tyrosine kinase inhibitors to immune-oncology agents plus anti-vascular endothelial growth factor agents. The appropriate sequence of therapies has become critical in optimizing patient outcomes given the increase in systemic therapeutic options. This article evaluates the evidence and provides expert recommendations for the use of systemic therapies after first-line treatment in patients with advanced HCC., Summary: Based on three virtual meetings held in early 2021, a team of 17 experts comprising oncologists, a hepatologist, and a hepatobiliary surgeon from Hong Kong, Singapore, and Taiwan reviewed available data about systemic treatments for HCC after first line and formulated 28 statements. These statements aimed to provide expert guidance on selecting first and subsequent lines of therapies as well as recommending therapies in special circumstances, such as poor liver function, posttransplantation, recent gastrointestinal bleeding, or autoimmune diseases. Data supporting the statements were drawn from clinical trials and real-world studies. The 28 statements were then evaluated anonymously using a 5-point Likert scale, and 24 reached consensus, predefined as achieving 75% agreement. Statements generated covered the selection of first-line systemic therapy, considerations and goals of second-line systemic therapies, treatment selection following first-line therapy, and treatment recommendations following first-line tyrosine kinase inhibitors, immune-oncology monotherapy, or immune-oncology combination therapy. The authors also shared expert opinion on the use of second-line systemic therapy in patients with liver dysfunction, liver transplantation, and recent gastrointestinal or autoimmune disease., Key Messages: These expert statements summarize the latest data and expert opinion on selecting systemic treatment following first-line therapy in patients with unresectable advanced or metastatic HCC., Competing Interests: Dr. Chan has received consulting fees from AstraZeneca, Merck Sharp & Dohme, Eisai, and Ipsen and research funding from Bayer, Eisai, Ipsen, Sirtex, and Merck Sharp & Dohme. Dr. Chan is also an Editorial Board Member of Liver Cancer. Dr. Chen has received grants from GlaxoSmithKline, Merck Serono, OBI Pharma, Pfizer, Polaris, Ministry of Science and Technology, and the Ministry of Health and Welfare; personal fees from Bristol Myers Squibb, Eli Lilly, Five Prime, Merck Sharp & Dohme, Merrimack Pharmaceuticals, Ono Pharmaceutical, PharmaEngine, and Shire; grants and nonfinancial support from Celgene; and grants, personal fees, and nonfinancial support from Novartis, Syncore, and TTY Biopharm. Dr. Cheng has received travel support from Bayer Yakuhin, Ltd., Eisai, Roche/Genentech, Chugai Pharmaceutical, and IQVIA; honoraria for speakers bureau from Bayer Yakuhin, Ltd., Novartis, Eisai, Ono Pharmaceutical, and Amgen Taiwan; and consulting fee from AstraZeneca, Bristol Myers Squibb, Eisai, Merck Serono, Novartis, Ono Pharmaceutical, Exelixis, IPSEN Innovation, Bayer Healthcare, Merck Sharp & Dohme, Roche/Genentech, BeiGene, F. Hoffmann-La Roche, and IQVIA. Dr. Cheng is an Associate Editor of Liver Cancer. Dr. Choo owns stock/shares of Bristol Myers Squibb and has received honoraria and consulting fee from Bristol Myers Squibb, Bayer, Roche, AstraZeneca, Merck Sharp & Dohme, and Ipsen; she has also received honoraria from Eisai. Dr. Hsu has received honoraria from AstraZeneca, Bayer, Bristol Myers Squibb/Ono Pharmaceuticals, Eisai, Ipsen, Merck Sharpe & Dohme, and Roche; and grants or funds from Bristol Myers Squibb/Ono Pharmaceuticals, Roche, and Ipsen. Professor Huang has been an advisory council or committee member and received honoraria from AbbVie, Gilead Sciences, Bristol Myers Squibb, Ono Pharmaceuticals, Eisai, Eli Lilly, Ipsen, Merck Sharp & Dohme, and Roche; and received grants or funds from Gilead Sciences and Bristol Myers Squibb. Dr. Lee has received honoraria from Bristol Myers Squibb, Ipsen, and Bayer; and grants or funds from Bayer. Dr. DWM Tai has been an advisory council or committee member, received honoraria and consulting fees from Novartis, Sirtex, Merck Sharpe & Dohme, Celgene, Eisai, and Bristol Myers Squibb; he has also received grants or funds from Novartis, Bristol Myers Squibb, and Sirtex. Dr. Yau has been an advisory council or committee member and received honoraria from Bristol Myers Squibb, Merck Sharpe & Dohme, Exelixis, Ipsen, Eisai, AstraZeneca, Bayer, Novartis, EMD Sereon, AbbVie, Pfizer, Eli Lilly, Sirtex, Sillajen, Taiho, OrigiMed, New B Innovation, Sirtex, and H3 Biomedicine. Dr. Yong has received honoraria from Amgen, Genentech, Bayer, Merck Sharpe & Dohme, Bristol Myers Squibb, Ipsen, Novartis, AstraZeneca, Eli Lilly, and Taiho. Drs Cheung, Law, Leung, Lin, Shum, A.Y.P. Tai, and Yeung have no potential conflict of interest to declare., (Copyright © 2022 by The Author(s). Published by S. Karger AG, Basel.)

Purpose: RET is an estrogen response gene with preclinical studies demonstrating cross-talk between the RET and estrogen receptor (ER) pathways. We investigate the role of lenvatinib, a multikinase inhibitor with potent activity against RET, in patients with metastatic breast cancer., Patients and Methods: Patients with advanced ER+/HER2- breast cancer were treated with lenvatinib plus letrozole in a phase Ib/II trial. Primary objectives included safety and recommended phase II dose (RP2D) determination in phase Ib, and objective response rates (ORR) in phase II dose expansion., Results: Sixteen patients were recruited in dose finding, where deescalating doses of lenvatinib from 20 to 14 mg were investigated. Lenvatinib 14 mg plus letrozole 2.5 mg daily was determined as RP2D. Thirty-one patients with 5 median lines of prior therapy in the metastatic setting (range, 0-11) were recruited in dose expansion. In this cohort, ORR was 23.3% [95% confidence interval (CI) 9.9%-42.3%], with median duration of response (DoR) of 6.9 months [interquartile range (IQR) 5.9 to 13.1]. Clinical benefit rate ≥6 months (CBR) was 50.0% (95% CI, 31.3%-68.7%). Similar efficacy was observed in the subgroup of 25 patients who progressed on prior CDK4/6 inhibitor therapy [ORR 20.0% (95% CI, 6.8%-40.7%), median DoR 6.9 months (IQR 5.9-13.1), and CBR 52.0% (95% CI, 31.3%-72.2%)]. Pharmacodynamic studies showed target modulation, with paired tumor biopsies indicating downregulation of RET/pERK and improved vascular normalization index., Conclusions: Lenvatinib plus letrozole had manageable toxicity, with target engagement and preliminary antitumor activity observed, supporting further assessment in randomized studies., (©2022 American Association for Cancer Research.)

Multiple three-dimensional (3D) tumour organoid models assisted by multi-omics and Artificial Intelligence (AI) have contributed greatly to preclinical drug development and precision medicine. The intrinsic ability to maintain genetic and phenotypic heterogeneity of tumours allows for the reconciliation of shortcomings in traditional cancer models. While their utility in preclinical studies have been well established, little progress has been made in translational research and clinical trials. In this review, we identify the major bottlenecks preventing patient-derived tumour organoids (PDTOs) from being used in clinical setting. Unsuitable methods of tissue acquisition, disparities in establishment rates and a lengthy timeline are the limiting factors for use of PDTOs in clinical application. Potential strategies to overcome this include liquid biopsies via circulating tumour cells (CTCs), an automated organoid platform and optical metabolic imaging (OMI). These proposed solutions accelerate and optimize the workflow of a clinical organoid drug screening. As such, PDTOs have the potential for potential applications in clinical oncology to improve patient outcomes. If remarkable progress is made, cancer patients can finally benefit from this revolutionary technology., (© 2022. The Author(s).)

Background: Varlitinib is a highly potent, small-molecule, pan-HER inhibitor targeting HER1, HER2, and HER4. It has demonstrated activity in gastric, biliary tract, and breast cancers., Objective: We conducted a phase Ib dose confirmation study to determine safety and early efficacy signals of varlitinib in combination with chemotherapy (paclitaxel ± carboplatin) ± subcutaneous trastuzumab., Methods: Eligible patients had advanced or metastatic solid tumors. A 3+3 dose de-escalation study design was used and pharmacokinetic analyses of varlitinib and paclitaxel were performed., Results: Thirty-seven patients were enrolled into eight cohorts with median 4 (0-14) prior lines of palliative systemic therapies. Carboplatin area under the curve 1.5 and paclitaxel 80 mg/m 2 weekly with varlitinib 500 mg twice daily continuously was de-escalated over four dose levels to 300 mg twice daily intermittently (4 days on, 3 days off) due to dose-limiting toxicities, most commonly neutropenia, febrile neutropenia, and electrolyte disturbances, with the triplet combination deemed intolerable and unable to be developed further. Varlitinib was then combined with paclitaxel alone; the recommended phase II dose of varlitinib was 300 mg twice daily intermittently. The addition of subcutaneous trastuzumab 600 mg was safe with no dose-limiting toxicities. Thirty-one patients were evaluable for response: 35.5% partial response, 41.9% stable disease. Twenty patients had HER2+ metastatic breast cancer with a median of 4 (0-14) treatment lines, 8/20 continued on single-agent varlitinib after completing chemotherapy for a median of 5.1 (range 2.0-13.3) months. A pharmacokinetic analysis showed that plasma exposure of varlitinib was dose dependent. Varlitinib administration did not significantly affect the maximum concentration or area under the curve of paclitaxel., Conclusions: The recommended phase II dose of varlitinib with paclitaxel is 300 mg twice daily intermittently dosed. This is active in HER2+ metastatic breast cancer. Subcutaneous trastuzumab can be added safely to varlitinib and paclitaxel. This combination is currently being evaluated as neoadjuvant therapy in HER2+ breast cancer (NCT02396108)., Clinical Trial Registration: NCT02396108, date of registration: 25 March, 2015., (© 2022. The Author(s).)

Objectives: Safe implementation and thorough evaluation of new treatments require prospective data monitoring and standardization of treatments. Pressurized intraperitoneal aerosol chemotherapy (PIPAC) is a promising alternative for the treatment of patients with peritoneal disease with an increasing number of suggested drug regimens. The aim was to reach expert consensus on current PIPAC treatment protocols and to define the most important research topics., Methods: The expert panel included the most active PIPAC centers, organizers of PIPAC courses and principal investigators of prospective studies on PIPAC. A comprehensive literature review served as base for a two-day hybrid consensus meeting which was accompanied by a modified three-round Delphi process. Consensus bar was set at 70% for combined (strong and weak) positive or negative votes according to GRADE. Research questions were prioritized from 0 to 10 (highest importance)., Results: Twenty-two out of 26 invited experts completed the entire consensus process. Consensus was reached for 10/10 final questions. The combination of doxorubicin (2.1 mg/m 2 ) and cisplatin (10.5 mg/m 2 ) was endorsed by 20/22 experts (90.9%). 16/22 (72.7%) supported oxaliplatin at 120 with potential reduction to 90 mg/m 2 (frail patients), and 77.2% suggested PIPAC-Ox in combination with 5-FU. Mitomycin-C and Nab-paclitaxel were favoured as alternative regimens. The most important research questions concerned PIPAC conditions (n=3), standard (n=4) and alternative regimens (n=5) and efficacy of PIPAC treatment (n=2); 8/14 were given a priority of ≥8/10., Conclusions: The current consensus should help to limit heterogeneity of treatment protocols but underlines the utmost importance of further research., Competing Interests: Competing interests: The following disclosure were made: MH: 1. Research funding (institution): Nestlé health science. 2. Speaker honorary (institution): MSD, Nestlé, Fresenius, Capnomed, Encare. 3. Boards: ERAS society: chair education, ISSPP: chair education. MR: 1.Research funding: Capnomed GmbH, Fisher Paykel, Alesi Surgical. 2. Speaker honorary: Boerhinger Ingelheim. 3. Stock shareholder: Capnomed GmbH. MP: Research funding: Capnomed GmbH, IDmed, INSERM laboratory unit. XD: Speaker honorary: Capnomed GmbH, Consultant for Latin America. WPY: Speaker honorary: Astra Zeneca, BMS, Eli Lilly, Novartis. The rest of the authors have no competing interests., (© 2022 Olivia Sgarbura et al., published by De Gruyter, Berlin/Boston.)

Gastric cancer heterogeneity represents a barrier to disease management. We generated a comprehensive single-cell atlas of gastric cancer (>200,000 cells) comprising 48 samples from 31 patients across clinical stages and histologic subtypes. We identified 34 distinct cell-lineage states including novel rare cell populations. Many lineage states exhibited distinct cancer-associated expression profiles, individually contributing to a combined tumor-wide molecular collage. We observed increased plasma cell proportions in diffuse-type tumors associated with epithelial-resident KLF2 and stage-wise accrual of cancer-associated fibroblast subpopulations marked by high INHBA and FAP coexpression. Single-cell comparisons between patient-derived organoids (PDO) and primary tumors highlighted inter- and intralineage similarities and differences, demarcating molecular boundaries of PDOs as experimental models. We complemented these findings by spatial transcriptomics, orthogonal validation in independent bulk RNA-sequencing cohorts, and functional demonstration using in vitro and in vivo models. Our results provide a high-resolution molecular resource of intra- and interpatient lineage states across distinct gastric cancer subtypes., Significance: We profiled gastric malignancies at single-cell resolution and identified increased plasma cell proportions as a novel feature of diffuse-type tumors. We also uncovered distinct cancer-associated fibroblast subtypes with INHBA-FAP-high cell populations as predictors of poor clinical prognosis. Our findings highlight potential origins of deregulated cell states in the gastric tumor ecosystem. This article is highlighted in the In This Issue feature, p. 587., (©2021 The Authors; Published by the American Association for Cancer Research.)

Inhibitors of the mitotic kinase PLK1 yield objective responses in a subset of refractory cancers. However, PLK1 overexpression in cancer does not correlate with drug sensitivity, and the clinical development of PLK1 inhibitors has been hampered by the lack of patient selection marker. Using a high-throughput chemical screen, we discovered that cells deficient for the tumor suppressor ARID1A are highly sensitive to PLK1 inhibition. Interestingly this sensitivity was unrelated to canonical functions of PLK1 in mediating G2/M cell cycle transition. Instead, a whole-genome CRISPR screen revealed PLK1 inhibitor sensitivity in ARID1A deficient cells to be dependent on the mitochondrial translation machinery. We find that ARID1A knock-out (KO) cells have an unusual mitochondrial phenotype with aberrant biogenesis, increased oxygen consumption/expression of oxidative phosphorylation genes, but without increased ATP production. Using expansion microscopy and biochemical fractionation, we see that a subset of PLK1 localizes to the mitochondria in interphase cells. Inhibition of PLK1 in ARID1A KO cells further uncouples oxygen consumption from ATP production, with subsequent membrane depolarization and apoptosis. Knockdown of specific subunits of the mitochondrial ribosome reverses PLK1-inhibitor induced apoptosis in ARID1A deficient cells, confirming specificity of the phenotype. Together, these findings highlight a novel interphase role for PLK1 in maintaining mitochondrial fitness under metabolic stress, and a strategy for therapeutic use of PLK1 inhibitors. To translate these findings, we describe a quantitative microscopy assay for assessment of ARID1A protein loss, which could offer a novel patient selection strategy for the clinical development of PLK1 inhibitors in cancer., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Colorectal cancer is a prevalent disease worldwide, with more than 50% of patients developing metastases to the liver. Despite advances in improving resectability, most patients present with non-resectable colorectal liver metastases requiring palliative systemic therapy and locoregional disease control strategies. There is a growing interest in the use of liver transplantation to treat non-resectable colorectal liver metastases in well selected patients, leading to a surge in the number of studies and prospective trials worldwide, thereby fuelling the emerging field of transplant oncology. The interdisciplinary nature of this field requires domain-specific evidence and expertise to be drawn from multiple clinical specialities and the basic sciences. Importantly, the wider societal implication of liver transplantation for non-resectable colorectal liver metastases, such as the effect on the allocation of resources and national transplant waitlists, should be considered. To address the urgent need for a consensus approach, the International Hepato-Pancreato-Biliary Association commissioned the Liver Transplantation for Colorectal liver Metastases 2021 working group, consisting of international leaders in the areas of hepatobiliary surgery, colorectal oncology, liver transplantation, hepatology, and bioethics. The aim of this study was to standardise nomenclature and define management principles in five key domains: patient selection, evaluation of biological behaviour, graft selection, recipient considerations, and outcomes. An extensive literature review was done within the five domains identified. Between November, 2020, and January, 2021, a three-step modified Delphi consensus process was undertaken by the workgroup, who were further subgrouped into the Scientific Committee, Expert Panel, and Transplant Centre Representatives. A final consensus of 44 statements, standardised nomenclature, and a practical management algorithm is presented. Specific criteria for clinico-patho-radiological assessments with molecular profiling is crucial in this setting. After this, the careful evaluation of biological behaviour with bridging therapy to transplantation with an appropriate assessment of the response is required. The sequencing of treatment in synchronous metastatic disease requires special consideration and is highlighted here. Some ethical dilemmas within organ allocation for malignant indications are discussed and the role for extended criteria grafts, living donor transplantation, and machine perfusion technologies for non-resectable colorectal liver metastases are reviewed. Appropriate immunosuppressive regimens and strategies for the follow-up and treatment of recurrent disease are proposed. This consensus guideline provides a framework by which liver transplantation for non-resectable colorectal liver metastases might be safely instituted and is a meaningful step towards future evidenced-based practice for better patient selection and organ allocation to improve the survival for patients with this disease., Competing Interests: Declaration of interests PT receives honoraria from Astellas Pharma Europe, outside the current study. GS receives research grants from Bayer and Roche, and consulting fees from Astrazeneca, Novartis, and Roche, outside the current study. RA receives honoraria for symposia from Merck Serono and Sanofi, outside the current study. All other authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Treatment options are sparse for patients with advanced cholangiocarcinoma after progression on first-line gemcitabine-based therapy. FGFR2 fusions or rearrangements occur in 10-16% of patients with intrahepatic cholangiocarcinoma. Infigratinib is a selective, ATP-competitive inhibitor of fibroblast growth factor receptors. We aimed to evaluate the antitumour activity of infigratinib in patients with locally advanced or metastatic cholangiocarcinoma, FGFR2 alterations, and previous gemcitabine-based treatment., Methods: This multicentre, open-label, single-arm, phase 2 study recruited patients from 18 academic centres and hospitals in the USA, Belgium, Spain, Germany, Singapore, Taiwan, and Thailand. Eligible participants were aged 18 years or older, had histologically or cytologically confirmed, locally advanced or metastatic cholangiocarcinoma and FGFR2 fusions or rearrangements, and were previously treated with at least one gemcitabine-containing regimen. Patients received 125 mg of oral infigratinib once daily for 21 days of 28-day cycles until disease progression, intolerance, withdrawal of consent, or death. Radiological tumour evaluation was done at baseline and every 8 weeks until disease progression via CT or MRI of the chest, abdomen, and pelvis. The primary endpoint was objective response rate, defined as the proportion of patients with a best overall response of a confirmed complete or partial response, as assessed by blinded independent central review (BICR) according to Response Evaluation Criteria in Solid Tumors, version 1.1. The primary outcome and safety were analysed in the full analysis set, which comprised all patients who received at least one dose of infigratinib. This trial is registered with ClinicalTrials.gov, NCT02150967, and is ongoing., Findings: Between June 23, 2014, and March 31, 2020, 122 patients were enrolled into our study, of whom 108 with FGFR2 fusions or rearrangements received at least one dose of infigratinib and comprised the full analysis set. After a median follow-up of 10·6 months (IQR 6·2-15·6), the BICR-assessed objective response rate was 23·1% (95% CI 15·6-32·2; 25 of 108 patients), with one confirmed complete response in a patient who only had non-target lesions identified at baseline and 24 partial responses. The most common treatment-emergent adverse events of any grade were hyperphosphataemia (n=83), stomatitis (n=59), fatigue (n=43), and alopecia (n=41). The most common ocular toxicity was dry eyes (n=37). Central serous retinopathy-like and retinal pigment epithelial detachment-like events occurred in 18 (17%) patients, of which ten (9%) were grade 1, seven (6%) were grade 2, and one (1%) was grade 3. There were no treatment-related deaths., Interpretation: Infigratinib has promising clinical activity and a manageable adverse event profile in previously treated patients with locally advanced or metastatic cholangiocarcinoma harbouring FGFR2 gene fusions or rearrangements, and so represents a potential new therapeutic option in this setting., Funding: QED Therapeutics and Novartis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Purpose: Precision oncology, such as next generation sequencing (NGS) molecular analysis and bioinformatics are used to guide targeted therapies. The laboratory turnaround time (TAT) is a key performance indicator of laboratory performance. This study aims to formally apply statistical process control (SPC) methods such as CUSUM and EWMA to a precision medicine programme to analyze the learning curves of NGS and bioinformatics processes., Patients and Methods: Trends in NGS and bioinformatics TAT were analyzed using simple regression models with TAT as the dependent variable and chronologically-ordered case number as the independent variable. The M-estimator "robust" regression and negative binomial regression were chosen to serve as sensitivity analyses to each other. Next, two popular statistical process control (SPC) approaches which are CUSUM and EWMA were utilized and the CUSUM log-likelihood ratio (LLR) charts were also generated. All statistical analyses were done in Stata version 16.0 (StataCorp), and nominal P < 0.05 was considered to be statistically significant., Results: A total of 365 patients underwent successful molecular profiling. Both the robust linear model and negative binomial model showed statistically significant reductions in TAT with accumulating experience. The EWMA and CUSUM charts of overall TAT largely corresponded except that the EWMA chart consistently decreased while the CUSUM analyses indicated improvement only after a nadir at the 82 nd case. CUSUM analysis found that the bioinformatics team took a lower number of cases (54 cases) to overcome the learning curve compared to the NGS team (85 cases)., Conclusion: As NGS and bioinformatics lead precision oncology into the forefront of cancer management, characterizing the TAT of NGS and bioinformatics processes improves the timeliness of data output by potentially spotlighting problems early for rectification, thereby improving care delivery., Competing Interests: DT consults on the advisory board for Astra Zeneca and has received research funding from Karyopharm Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jain, Sim, Ng, Chin, Lim, Syn, Kamal, Gupta, Heong, Lee, Sapari, Koh, Isa, Ho, O’Hara, Ulagapan, Gu, Shroff, Weng, Lim, Lim, Pang, Ng, Wong, Soo, Yong, Chee, Lee, Goh, Soong and Tan.)

Purpose: This phase I/II study evaluated tremelimumab (anticytotoxic T-lymphocyte-associated antigen-4 monoclonal antibody) and durvalumab (antiprogrammed death ligand-1 monoclonal antibody) as monotherapies and in combination for patients with unresectable hepatocellular carcinoma (HCC), including a novel regimen featuring a single, priming dose of tremelimumab (ClinicalTrials.gov identifier: NCT02519348)., Patients and Methods: Patients with HCC who had progressed on, were intolerant to, or refused sorafenib were randomly assigned to receive T300 + D (tremelimumab 300 mg plus durvalumab 1,500 mg [one dose each during the first cycle] followed by durvalumab 1,500 mg once every 4 weeks), durvalumab monotherapy (1,500 mg once every 4 weeks), tremelimumab monotherapy (750 mg once every 4 weeks [seven doses] and then once every 12 weeks), or T75 + D (tremelimumab 75 mg once every 4 weeks plus durvalumab 1,500 mg once every 4 weeks [four doses] followed by durvalumab 1,500 mg once every 4 weeks). Safety was the primary end point. Secondary end points included objective response rate (ORR) by Response Evaluation Criteria in Solid Tumors v1.1 and overall survival; exploratory end points included circulating lymphocyte profiles., Results: A total of 332 patients were enrolled (T300 + D, n = 75; durvalumab, n = 104; tremelimumab, n = 69; and T75 + D, n = 84). Tolerability was acceptable across arms, with grade ≥ 3 treatment-related adverse events occurring in 37.8%, 20.8%, 43.5%, and 24.4%, respectively. Confirmed ORRs (95% CI) were 24.0% (14.9 to 35.3), 10.6% (5.4 to 18.1), 7.2% (2.4 to 16.1), and 9.5% (4.2 to 17.9), respectively. An early expansion of CD8+ lymphocytes was associated with response across arms, with highest proliferating CD8+ lymphocyte levels occurring in the T300 + D arm. The median (95% CI) overall survival was 18.7 (10.8 to 27.3), 13.6 (8.7 to 17.6), 15.1 (11.3 to 20.5), and 11.3 (8.4 to 15.0) months in the T300 + D, durvalumab, tremelimumab, and T75 + D arms, respectively., Conclusion: All regimens were found to be tolerable and clinically active; however, the T300 + D regimen demonstrated the most encouraging benefit-risk profile. The unique pharmacodynamic activity and association with ORR of the T300 + D regimen further support its continued evaluation in HCC., Competing Interests: Robin Kate KelleyConsulting or Advisory Role: Agios, AstraZeneca, Bristol Myers Squibb, Genentech/Roche, Merck, Gilead Sciences, Exact SciencesResearch Funding: Lilly, Exelixis, Novartis, Bristol Myers Squibb, MedImmune, Merck Sharp & Dohme, Agios, AstraZeneca, Adaptimmune, Taiho Pharmaceutical, Bayer, QED Therapeutics, EMD Serono, Partner Therapeutics, Genentech/RocheTravel, Accommodations, Expenses: Ipsen Bruno SangroConsulting or Advisory Role: Bristol Myers Squibb, AstraZeneca, Bayer, Adaptimmune, Sirtex Medical, BTG, Merck, H3 Biomedicine, Ipsen, Lilly, Roche/Genentech, Eisai, IncyteSpeakers' Bureau: Bristol Myers Squibb, Bayer, Sirtex Medical, Ipsen, Lilly, AstraZeneca, Eisai, Incyte, RocheResearch Funding: Bristol Myers Squibb, Sirtex MedicalTravel, Accommodations, Expenses: AstraZeneca, Bristol Myers Squibb, Sirtex Medical, BayerOther Relationship: Medscape, Target Pharmasolutions William HarrisConsulting or Advisory Role: Neo Therma, Eisai, Exelixis, Bristol Myers Squibb, QED Therapeutics, ZymeworksResearch Funding: ArQule, Exelixis, Halozyme, Bristol Myers Squibb, MedImmune, Agios, Bayer, Merck, BTGTravel, Accommodations, Expenses: Eisai Masafumi IkedaHonoraria: Taiho Pharmaceutical, Bayer Yakuhin, Eisai, Lilly Japan, Dainippon Sumitomo Pharma, Teijin Pharma, EA Pharma, MSD, Mylan, Otsuka, Yakult Pharmaceutical, Servier, Astellas Pharma, Chugai Pharma, Takeda, AstraZeneca, Novartis, Bristol Myers SquibbConsulting or Advisory Role: Bayer Yakuhin, Eisai, Novartis, Lilly Japan, Chugai Pharma, Micron, Ono Pharmaceutical, AstraZeneca, Servier, Takeda, GlaxoSmithKline, EMD SeronoResearch Funding: Bayer Yakuhin, Yakult Pharmaceutical, Lilly Japan, Ono Pharmaceutical, Eisai, AstraZeneca, Chugai Pharma, Bristol Myers Squibb, ASLAN Pharmaceuticals, Novartis, MSD, J-Pharma, Takeda, Pfizer, Nobelpharma, Merck, Delta-Fly Pharma, Chiome Bioscience, Merus NV Takuji OkusakaHonoraria: Eisai, Ono Pharmaceutical, Taiho Pharmaceutical, Lilly, AstraZeneca, Chugai Pharma, Yakut Honsha, Daiichi SankyoConsulting or Advisory Role: Taiho Pharmaceutical, Dainippon Sumitomo Pharma, Bristol Myers Squibb, AstraZeneca, Eisai, Nihon ServierResearch Funding: Novartis, Eisai, Dainippon Sumitomo Pharma, Baxter, Lilly, Taiho Pharmaceutical, AstraZeneca, Chugai Pharma, Bristol Myers Squibb, MSD Yoon-Koo KangConsulting or Advisory Role: DAE HWA Pharmaceutical, Bristol Myers Squibb, Zymeworks, ALX Oncology, Amgen, Novartis, Macrogenics, Surface Oncology, Blueprint Medicines David W.-M. TaiHonoraria: Bristol Myers Squibb, EisaiConsulting or Advisory Role: Bristol Myers Squibb, EisaiSpeakers' Bureau: Ipsen, Bristol Myers Squibb, Eisai, RocheResearch Funding: Bristol Myers Squibb, Sirtex Medical, Novartis Ho Yeong LimHonoraria: Bayer, Eisai, MSD Oncology, Bristol Myers Squibb/Medarex, AstraZenecaConsulting or Advisory Role: Bayer, Eisai, Bristol Myers Squibb, AstraZenecaSpeakers' Bureau: Bayer Thomas YauHonoraria: Bristol Myers Squibb, MSD Oncology, AstraZenecaConsulting or Advisory Role: Bristol Myers Squibb Wei-Peng YongConsulting or Advisory Role: AbbVie/Genentech, Amgen, Bristol Myers Squibb, Ipsen, Novartis, AstraZenecaSpeakers' Bureau: Lilly, Sanofi/Aventis, Taiho Pharmaceutical, Eisai, Bayer, MSD OncologyTravel, Accommodations, Expenses: Pfizer Ann-Lii ChengHonoraria: Bayer Yakuhin, AstraZeneca, Eisai, Genentech/RocheConsulting or Advisory Role: Bristol Myers Squibb, Bayer Schering Pharma, Eisai, Ono Pharmaceutical, AstraZeneca, Genentech/Roche, MSD, BeiGene, IQVIA, Ipsen, F. Hoffmann-La Roche Ltd Antonio GasbarriniHonoraria: AbbVie, Actial, Alfasigma, Eisai, Gilead Sciences, MSD, Sandoz, Sanofi, TakedaConsulting or Advisory Role: Alfasigma, Actial, MSD, Gilead Sciences, Takeda, AbbVieSpeakers' Bureau: AbbVieExpert Testimony: AbbVie, Alfasigma, Actial, Sanofi, MSD, Gilead Sciences, Eisai Silvia DamianResearch Funding: Basilea Pharmaceutical Jordi BruixConsulting or Advisory Role: Bayer Schering Pharma, Sirtex Medical, Novartis, Bristol Myers Squibb, Eisai, BTG, Roche, Arqule, BasileaSpeakers' Bureau: Bayer Schering PharmaResearch Funding: Bayer Schering Pharma, IpsenTravel, Accommodations, Expenses: Ipsen, AstraZeneca Mitesh BoradStock and Other Ownership Interests: Gilead Sciences, AVEO, Intercept Pharmaceuticals, Spectrum PharmaceuticalsConsulting or Advisory Role: G1 Therapeutics, Fujifilm, Agios, Insys Therapeutics, Novartis, ArQule, Celgene, Inspyr Therapeutics, Halozyme, Pieris Pharmaceuticals, Taiho Pharmaceutical, Immunovative Therapies, Exelixis, Lynx Group, Genentech, Western Oncolytics, Klus Pharma, De Novo Pharmaceuticals, Merck, ImvaxResearch Funding: Boston Biomedical, miRNA Therapeutics, Senhwa Biosciences, MedImmune, BiolineRx, Agios, Halozyme, Celgene, Threshold Pharmaceuticals, Toray Industries, Dicerna, Sillajen, Eisai, Taiho Pharmaceutical, EMD Serono, Isis Pharmaceuticals, Incyte, Sun Biopharma, ARIAD, ImClone Systems, QED Therapeutics, Puma Biotechnology, Adaptimmune, Merck Serono, RedHill Biopharma, BasileaTravel, Accommodations, Expenses: ArQule, Celgene, AstraZeneca Johanna BendellConsulting or Advisory Role: Gilead Sciences, Genentech/Roche, Bristol Myers Squibb, Five Prime Therapeutics, Lilly, Merck, MedImmune, Celgene, EMD Serono, Taiho Pharmaceutical, Macrogenics, GlaxoSmithKline, Novartis, OncoMed, Leap Therapeutics, TG Therapeutics, AstraZeneca, Boehringer Ingelheim, Daiichi Sankyo, Bayer, Incyte, Apexigen, Array BioPharma, Sanofi, ARMO BioSciences, Ipsen, Merrimack, Oncogenex, FORMA Therapeutics, Arch Oncology, Prelude Therapeutics, Phoenix Biotech, Cyteir, Molecular Partners, Innate Pharma, Torque, Tizona Therapeutics Inc, Janssen, Tolero Pharmaceuticals, TD2, Amgen, Seattle Genetics, Moderna Therapeutics, Tanabe Research, Beigene, Continuum Clinical, Cerulean Pharma, Kyn Therapeutics, Bicycle Therapeutics, Relay Therapeutics, Evelo Therapeutics, Fusion PharmaceuticalsResearch Funding: Lilly, Genentech/Roche, Incyte, Gilead Sciences, Bristol Myers Squibb, Leap Therapeutics, AstraZeneca/MedImmune, Boston Biomedical, GlaxoSmithKline, Novartis, Array BioPharma, Taiho Pharmaceutical, Celgene, OncoMed, Daiichi Sankyo, Bayer, Apexigen, Kolltan Pharmaceuticals, SynDevRx, Merck, Macrogenics, Five Prime Therapeutics, EMD Serono, TG Therapeutics, Boehringer Ingelheim, Forty Seven, Stem CentRx, Onyx, Sanofi, Takeda, Abbott/AbbVie, Eisai, Celldex, Agios, ARMO BioSciences, CytomX Therapeutics, Nektar, Ipsen, Merrimack, Tarveda Therapeutics, Tyrogenex, Oncogenex, Marshall Edwards, Pieris Pharmaceuticals, Mersana, Calithera Biosciences, Blueprint Medicines, Gritstone Oncology, Evelo Therapeutics, FORMA Therapeutics, Forty Seven, EMD Serono, Merus, Jacobio, eFFECTOR Therapeutics, Novocure, Sorrento Therapeutics, Arrys Therapeutics, TRACON Pharma, Sierra Oncology, Innate Pharma, Prelude Therapeutics, Arch Oncology, Harpoon therapeutics, Phoenix Biotech, Unum Therapeutics, Vyriad, Harpoon therapeutics, Cyteir, Molecular Partners, Innate Pharma, ADC Therapeutics, Torque, Tizona Therapeutics Inc, Janssen, Amgen, BeiGene, Pfizer, Millenium Pharmaceuticals, ImClone Systems, Acerta Pharma, Rgenix, Bellicum Pharmaceuticals, Arcus Biosciences, Gossamer Bio, Seattle Genetics, Tempest Therapeutics, Shattuck Labs, Synthorx, Revolution Medicines, Bicycle Therapeutics, Zymeworks, Relay Therapeutics, Evelo Therapeutics, Scholar Rock, NGM Biopharmaceuticals, Numab, AtlasMedx, Treadwell Therapeutics, IGM, MabSpace Biosciences, Hutchison MediPharma, Repare Therapeutics, NeoImmuneTech, Regeneron, PureTech, G1 Therapeutics, Erasca Inc, Rubius Therapeutics, Pionyr, Loxo/Lilly, BioNTech AG, Elicio TherapeuticsTravel, Accommodations, Expenses: Merck, Roche/Genentech, Celgene, Daiichi Sankyo, Gilead Sciences, Bristol Myers Squibb, Lilly, MedImmune, Taiho Pharmaceutical, Novartis, OncoMed, Boehringer Ingelheim, ARMO BioSciences, Ipsen, FORMA Therapeutics Nathan StandiferEmployment: AstraZenecaStock and Other Ownership Interests: AstraZenecaPatents, Royalties, Other Intellectual Property: Patent with AstraZeneca Philip HeEmployment: AstraZeneca, SanofiStock and Other Ownership Interests: Celldex, AstraZeneca, Sanofi Mallory MakowskyEmployment: AstraZeneca Alejandra NegroEmployment: AstraZenecaStock and Other Ownership Interests: AstraZeneca Masatoshi KudoHonoraria: MSD, Eisai, Bayer, Lilly Japan, EA Pharma, Bristol Myers Squibb JapanConsulting or Advisory Role: MSD, Eisai, Ono Pharmaceutical, BMS, RocheResearch Funding: Otsuka, Taiho Pharmaceutical, AbbVie, Takeda, Eisai, Gilead Sciences, EA Pharma, Sumitomo Dainippon, Ono Pharmaceutical Ghassan K. Abou-AlfaConsulting or Advisory Role: Silenseed, Sillajen, Agios, Bayer, Eisai, Ipsen, Merck Serono, AstraZeneca, CytomX Therapeutics, BeiGene, Genoscience Pharma, Loxo, Minapharm, QED Therapeutics, RedHill Biopharma, SOBI, twoXAR, Yiviva, Flatiron Health, Roche/Genentech, Autem Medical, Berry Genomics, Incyte, TheraBionic, Vector Health, Helio, Alnylam, Adicet Bio, Exelixis, Legend Biotech, Nerviano Medical Sciences, Surface Oncology, YivivaResearch Funding: Bayer, Exelixis, CASI Pharmaceuticals, AstraZeneca, Bristol Myers Squibb, Incyte, Agios, Polaris, Puma Biotechnology, QED TherapeuticsTravel, Accommodations, Expenses: PolarisNo other potential conflicts of interest were reported.

Tumors expressing programmed cell death-ligand 1 (PD-L1) interact with programmed cell death protein 1 (PD-1) on CD8+ cytotoxic T lymphocytes (CTLs) to evade immune surveillance leading to the inhibition of CTL proliferation, survival, and effector function, and subsequently cancer persistence. Approximately 40% of gastric cancers express PD-L1, yet the response rate to immunotherapy is only 30%. We present the use of human-derived autologous gastric cancer organoid/immune cell co-culture as a preclinical model that may predict the efficacy of targeted therapies to improve the outcome of cancer patients. Although cancer organoid co-cultures with immune cells have been reported, this co-culture approach uses tumor antigen to pulse the antigen-presenting dendritic cells. Dendritic cells (DCs) are then cultured with the patient's CD8+ T cells to expand the cytolytic activity and proliferation of these T lymphocytes before co-culture. In addition, the differentiation and immunosuppressive function of myeloid-derived suppressor cells (MDSCs) in culture are investigated within this co-culture system. This organoid approach may be of broad interest and appropriate to predict the efficacy of therapy and patient outcome in other cancers, including pancreatic cancer.

A dose of 200 mg 3-weekly of pembrolizumab was approved by the Food and Drug Administration (FDA) as treatment for advanced non-small cell lung cancer (NSCLC) without oncogenic drivers. This is despite evidence showing no difference in efficacy with 2 mg/kg. Our study aimed to assess the efficacy of a lower fixed dose of 100 mg, which is closer to 2 mg/kg weight-based dose in an average-sized Asian patient. All patients receiving pembrolizumab for advanced NSCLC from January 2016 to March 2020 in National University Hospital, Singapore, were included in this retrospective observational study. The effect of pembrolizumab 100 mg (Pem100) vs 200 mg (Pem200) upon survival outcomes, toxicity and cost were examined. One hundred fourteen patients received pembrolizumab. Sixty-five (57%) and 49 (43%) received Pem100 and Pem200, respectively. There was no difference in progression-free survival (PFS) and overall survival (OS) between Pem100 vs Pem200 as a single agent (PFS: 6.8 vs 4.2 months, hazard ratio [HR] 0.72, 95% confidence interval [CI] 0.36-1.46, P = .36; 9 month OS: 58% vs 63%, HR 1.08, 95% CI 0.48-2.41, P = .86) and when combined with chemotherapy (9-month PFS: 60% vs 50%, HR0.84, 95% CI 0.34-2.08, P = .71; 9-month OS: 85% vs 58%, HR 0.27, 95% CI 0.062-1.20, P = .09). No significant difference in response rate or ≥G3 immune-related toxicities between Pem100 and Pem200 was observed. A cost minimisation analysis evaluating the degree of cost savings related to drug costs estimated a within study cost saving of SGD4,290,912 and cost saving per patient of SGD39,942 in the Pem100 group. A 100 mg of pembrolizumab appears to be effective with reduction in cost. A randomised trial should be done to investigate a lower dose of pembrolizumab., (© 2021 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of Union for International Cancer Control.)

Introduction: We report outcomes of patients with oesophageal cancer treated with neoadjuvant chemoradiotherapy (NACRT) plus surgery or definitive chemoradiotherapy (chemoRT) at our institution., Methods: We retrospectively reviewed patients who underwent chemoRT from 2005 to 2017. The primary outcome was overall survival (OS). Secondary outcomes were disease-free survival (DFS) and toxicities., Results: We identified 96 patients with median age of 64 years and squamous cell carcinoma in 82.3%. Twenty-nine patients (30.2%) received NACRT plus surgery, 67 patients (69.8%) received definitive chemoRT. Median follow-up was 13.5 months. The 3/5-year OS were 26.4%/13.4%, and 59.6%/51.6% in the definitive chemoRT and NACRT plus surgery groups, respectively. The 3/5-year DFS were 19.3%/12.3%, and 55.7%/37.2% in the definitive chemoRT and NACRT plus surgery groups, respectively. NACRT plus surgery significantly improved OS (hazard ratio [HR] 0.40, 95% confidence interval [CI] 0.22-0.72, P <0.01) and DFS (subhazard ratio [SHR] 5.21, 95 CI 1.20-22.7, P =0.03). Multivariable analysis for OS in the definitive chemoRT group indicated stage (1-2 vs 3-4a; HR 2.17, 95% CI 1.15-4.11, P =0.02) and feeding tube (no tube versus tube; HR 1.85, 95% CI 1.00-3.43, P =0.05) as significantly associated with OS. The cumulative incidence of local recurrence was significantly higher in the definitive chemoRT group (SHR 5.21, 95 CI 1.2022.7, P =0.03). Nineteen patients (65.5%) had postoperative complications., Conclusion: NACRT plus surgery improved OS and DFS. However, in view of treatment-related complications, careful selection of patients is warranted. With the predominant histology of our cohort being squamous cell carcinoma (SCC), our results may be more relevant for those with SCC.

Gastric cancer cases are often diagnosed at an advanced stage with poor prognosis. Platinum-based chemotherapy has been internationally accepted as first-line therapy for inoperable or metastatic gastric cancer. To achieve greater benefits, selection of patients eligible for this treatment is critical. Although gene expression profiling has been widely used as a genomic classifier to identify molecular subtypes of gastric cancer and to stratify patients for different chemotherapy regimens, its prediction accuracy can be improved. Adenosine-to-inosine (A-to-I) RNA editing has emerged as a new player contributing to gastric cancer development and progression, offering potential clinical utility for diagnosis and treatment. Using a systematic computational approach followed by both in vitro validations and in silico validations in The Cancer Genome Atlas (TCGA), we conducted a transcriptome-wide RNA editing analysis of a cohort of 104 patients with advanced gastric cancer and identified an RNA editing (GCRE) signature to guide gastric cancer chemotherapy. RNA editing events stood as a prognostic and predictive biomarker in advanced gastric cancer. A GCRE score based on the GCRE signature consisted of 50 editing sites associated with 29 genes, predicting response to chemotherapy with a high accuracy (84%). Of note, patients demonstrating higher editing levels of this panel of sites presented a better overall response. Consistently, gastric cancer cell lines with higher editing levels showed higher chemosensitivity. Applying the GCRE score on TCGA dataset confirmed that responders had significantly higher levels of editing in advanced gastric cancer. Overall, this newly defined GCRE signature reliably stratifies patients with advanced gastric cancer and predicts response from chemotherapy. SIGNIFICANCE: This study describes a novel A-to-I RNA editing signature as a prognostic and predictive biomarker in advanced gastric cancer, providing a new tool to improve patient stratification and response to therapy., (©2021 American Association for Cancer Research.)

Introduction: Peritoneal carcinomatosis is difficult to treat. Pressurized Intra-Peritoneal Aerosolised Chemotherapy (PIPAC) is a novel method of delivering chemotherapy to the peritoneal cavity, aiming for homogenous and deeper drug distribution. To date, limited chemotherapeutics have been used with promising results. Here, we evaluate the pharmacokinetics, peritoneal tissue drug concentration, penetration, and short-term safety of PIPAC using solvent-based paclitaxel in swine to guide clinical trials., Materials and Methods: PIPAC solvent-based paclitaxel was administered at 60, 30, and 15mg/m 2 for 3 cohorts. Each PIPAC procedure was followed by intravenous (IV) administration of the same dose of solvent-based paclitaxel on Day 7, serving as control for pharmacokinetic comparison in the same pig. Safety and toxicity were evaluated by clinical assessment, blood counts and biochemistry. Blood samples were taken for pharmacokinetic analysis. Peritoneal biopsies were taken to measure tissue paclitaxel concentrations and distribution., Results: 12 Yorkshire x Landrace pigs underwent trial procedures. With PIPAC, there was linear pharmacokinetics and lower systemic exposure to paclitaxel compared to IV administration. MALDI-MSI demonstrated concentration of paclitaxel at the peritoneal surface, with estimated 2 mm penetration. PIPAC paclitaxel had favorable toxicity profile. The most significant adverse event was neutropenia which was dose dependent, with absolute neutrophil count <1.0 × 10 3 /μL seen at the highest dose. One pig developed grade 2 hypersensitivity reaction during IV infusion and one death occurred during the PIPAC procedure, likely from anaphylaxis; these are known potential adverse events mandating standard precautions and monitoring., Conclusion: PIPAC paclitaxel at 15mg/m 2 may be considered for a Phase I study., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.)

Recent advancements in cancer biology, microbiology, and bioengineering have spurred the development of engineered live biotherapeutics for targeted cancer therapy. In particular, natural tumor-targeting and probiotic bacteria have been engineered for controlled and sustained delivery of anticancer agents into the tumor microenvironment (TME). Here, we review the latest advancements in the development of engineered bacteria for cancer therapy and additional engineering strategies to potentiate the delivery of therapeutic payloads. We also explore the use of combination therapies comprising both engineered bacteria and conventional anticancer therapies for addressing intratumor heterogeneity. Finally, we discuss prospects for the development and clinical translation of engineered bacteria for cancer prevention and treatment., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: Pressurized intraperitoneal aerosol chemotherapy (PIPAC) is a novel laparoscopic, intraperitoneal chemotherapy delivery technique aiming to improve drug distribution and tissue penetration to treat peritoneal metastases. Thus far, PIPAC oxaliplatin is conducted at an arbitrary dose of 92 mg/m 2 . We conducted a phase I study to establish safety and tolerability., Patients and Methods: We used a 3+3 dose-escalation design of PIPAC oxaliplatin for patients with peritoneal metastases from gastrointestinal tumors, after failure of at least first-line chemotherapy. Dose levels were planned at 45, 60, 90, and 120 mg/m 2 ., Results: This study included 16 patients with 24 PIPAC procedures (8 gastric; 5 colorectal; and 1 gallbladder, pancreas, and appendix cancer each). Median age and peritoneal cancer index (PCI) score were 62 years and 17, respectively. Two patients developed pancreatitis (grade 2 and 3) at 45 mg/m 2 , necessitating cohort expansion. Another patient developed grade 2 pancreatitis at 90 mg/m 2 . There were no other dose-limiting toxicities, and the highest-dose cohort (120 mg/m 2 ) tolerated PIPAC well. Pharmacokinetic analyses demonstrated good linearity between dose and maximum concentration ( r 2 = 0.95) and AUC ( r 2 = 0.99). On the basis of RECIST, 62.5% and 50% had stable disease after one and two PIPAC procedures, respectively. A total of 8 patients underwent two PIPAC procedures, with improvement of median PCI and peritoneal regression grade score from 15 to 12 and 2.5 to 2.0, respectively., Conclusions: The recommended phase II dose is 120 mg/m 2 . Future studies should further delineate the efficacy and role of PIPAC oxaliplatin for peritoneal metastases. See related commentary by de Jong et al., p. 1830 ., (©2020 American Association for Cancer Research.)

In the last decade, the tremendous improvement in the sensitivity and also affordability of liquid chromatography-tandem mass spectrometry (LC-MS/MS) has revolutionized its application in pharmaceutical analysis, resulting in widespread employment of LC-MS/MS in determining pharmaceutical compounds, including anticancer drugs in pharmaceutical research and also industries. Currently, LC-MS/MS has been widely used to quantify small molecule oncology drugs in various biological matrices to support preclinical and clinical pharmacokinetic studies in R&D of oncology drugs. This mini-review article will describe the state-of-the-art LC-MS/MS and its application in rapid quantification of small molecule anticancer drugs. In addition, efforts have also been made in this review to address several key aspects in the development of rapid LC-MS/MS methods, including sample preparation, chromatographic separation, and matrix effect evaluation.

Glutamine is a major nutrient for cancer cells during rapid proliferation. Alanine-serine-cysteine (ASC) transporter 2 (ASCT2; SLC1A5) mediates glutamine uptake in a variety of cancer cells. We previously reported that KM8094, a novel anti-ASCT2 humanized monoclonal antibody, possesses anti-tumor efficacy in gastric cancer patient-derived xenografts. The aim of this study was to investigate the molecular mechanism underlying the effect of KM8094 and to further substantiate the preclinical feasibility of using KM8094 as a potential therapeutic agent against gastric cancer. First, ASCT2 was found to be highly expressed in cancer tissues derived from gastric cancer patients by an immunohistochemical analysis. Next, we performed in vitro studies using multiple gastric cancer cell lines and observed that several gastric cancer cells expressing ASCT2 showed glutamine-dependent cell growth, which was repressed by KM8094. We found that KM8094 inhibited the glutamine uptake, leading to the reduction of glutathione (GSH) level and the elevation of oxidative stress. KM8094 suppressed the cell cycle progression and increased the apoptosis. Furthermore, KM8094 exerted antibody dependent cellular cytotoxicity (ADCC) against human gastric cancer cells in vitro. Finally, in vivo studies revealed that KM8094 suppressed tumor growth in several gastric cancer xenografts. This effect was enhanced by docetaxel, one of the agents commonly used in gastric cancer therapy. Thus, our findings suggest that KM8094 is a potential new therapeutic agent for gastric cancer expressing ASCT2, which blocks the cellular glutamine metabolism and possesses ADCC activity., Competing Interests: None.

Introduction: Neoadjuvant chemoradiotherapy (CRT) followed by total mesorectal excision (TME) surgery for locally advanced rectal cancer has been shown to improve local control and reduce toxicity, as compared to adjuvant CRT. We reported the outcomes of our patients with locally advanced rectal cancer treated at National University Hospital, Singapore., Methods: From April 2002 to December 2014, 117 patients with T3/4, N0/+, M0 rectal cancer received neoadjuvant CRT followed by TME surgery. The treatment regimen comprised a total radiotherapy dose of 50.4 Gy in 28 daily fractions delivered concurrently with 5-fluorouracil or capecitabine chemotherapy over 5.5 weeks. All patients were planned for TME surgery. Local control, disease-free survival, overall survival and treatment toxicities were analysed., Results: Median follow-up was 34 (range 2-122) months. 11.5% (13/113) of patients achieved a pathological complete response (pCR) and 72.6% (85/117) had either tumour or nodal downstaging following neoadjuvant CRT. 5.2% (5/96) of patients had Grade 3 acute toxicities (dermatitis and diarrhoea) and 3.1% (3/96) had Grade 3 late toxicities (fistula and stricture). There was no Grade 4 toxicity noted. The five-year local recurrence, disease-free survival and overall survival rates were 4.5%, 65.7% and 80.6%, respectively. Multivariate analysis showed that nodal positivity was a predictor of poor disease-free survival and poor overall survival. Tumour downstaging and pCR did not improve outcomes., Conclusion: Our outcomes were comparable to internationally published data, and this treatment regimen remains the standard of care for locally advanced rectal cancer in our local population., (Copyright: © Singapore Medical Association.)

ASC amino acid transporter 2 (ASCT2), also known as solute linked carrier family 1 member A5 (SLC1A5) is a Na+-dependent glutamine/neutral amino acid transporter. ASCT2 acts as a high-affinity transporter of L-glutamine (Gln) and has been reported to be up-regulated in a variety of cancerous tissues including stomach, liver, and kidney. In this study, we evaluated anti-tumor efficacy of a novel anti-ASCT2 humanized monoclonal antibody, KM8094, which has a neutralizing activity against glutamine uptake, as a therapeutic antibody against gastric cancer and explored clinical predictive biomarker candidates by utilizing patient-derived xenograft (PDX) mouse models. Anti-tumor efficacy studies revealed that some of the PDX models used were responsive to KM8094 and the others were not. Interestingly, we observed a correlation between anti-tumor efficacy and low antigen expression as well as low basal levels of glutamine uptake, suggesting ASCT2 expression level could be a potential predictive biomarker for KM8094. We then further explored predictive biomarker candidates by multi-omics analysis on gastric cancer PDX mouse models. As a result, a few potential candidates such as TFF2, MUC13, and ANG were selected by gene expression and DNA methylation array analyses. In addition, metabolomics analysis revealed clear differences in intracellular energy status and redox status between responsive and non-responsive PDX models., Competing Interests: None.

Background: Melanoma-associated antigen (MAGE)-A3 is a member of the family of cancer-testis antigens and has been found to be epigenetically regulated and aberrantly expressed in various cancer types. It has also been found that MAGE-A3 expression may correlate with an aggressive clinical course and with chemo-resistance. The objectives of this study were to assess the relationship between MAGE-A3 promoter methylation and expression and (1) gastric cancer patient survival and (2) its functional consequences in gastric cancer-derived cells., Methods: Samples from two independent gastric cancer cohorts (including matched non-malignant gastric samples) were included in this study. MAGE-A3 methylation and mRNA expression levels were determined by methylation-specific PCR (MSP) and quantitative real-time PCR (qPCR), respectively. MAGE-A3 expression was knocked down in MKN1 gastric cancer-derived cells using miRNAs. In addition, in vitro cell proliferation, colony formation, apoptosis, cell cycle, drug treatment, immunohistochemistry and Western blot assays were performed., Results: Clinical analysis of 223 primary patient-derived samples (ntumor = 161, nnormal = 62) showed a significant inverse correlation between MAGE-A3 promoter methylation and expression in the cancer samples (R = -0.63, p = 5.99e-19). A lower MAGE-A3 methylation level was found to be associated with a worse patient survival (HR: 1.5, 95 % CI: 1.02-2.37, p = 0.04). In addition, we found that miRNA-mediated knockdown of MAGE-A3 expression in MKN1 cells caused a reduction in its proliferation and colony forming capacities, respectively. Under stress conditions MAGE-A3 was found to regulate the expression of Bax and p21. MAGE-A3 knock down also led to an increase in Puma and Noxa expression, thus contributing to an enhanced docetaxel sensitivity in the gastric cancer-derived cells., Conclusions: From our results we conclude that MAGE-A3 expression is regulated epigenetically by promoter methylation, and that its expression contributes to gastric cell proliferation and drug sensitivity. This study underscores the potential implications of MAGE-A3 as a therapeutic target and prognostic marker in gastric cancer patients.

Objective: Differences in gastric cancer (GC) clinical outcomes between patients in Asian and non-Asian countries has been historically attributed to variability in clinical management. However, recent international Phase III trials suggest that even with standardised treatments, GC outcomes differ by geography. Here, we investigated gene expression differences between Asian and non-Asian GCs, and if these molecular differences might influence clinical outcome., Design: We compared gene expression profiles of 1016 GCs from six Asian and three non-Asian GC cohorts, using a two-stage meta-analysis design and a novel biostatistical method (RUV-4) to adjust for technical variation between cohorts. We further validated our findings by computerised immunohistochemical analysis on two independent tissue microarray (TMA) cohorts from Asian and non-Asian localities (n=665)., Results: Gene signatures differentially expressed between Asians and non-Asian GCs were related to immune function and inflammation. Non-Asian GCs were significantly enriched in signatures related to T-cell biology, including CTLA-4 signalling. Similarly, in the TMA cohorts, non-Asian GCs showed significantly higher expression of T-cell markers (CD3, CD45R0, CD8) and lower expression of the immunosuppressive T-regulatory cell marker FOXP3 compared to Asian GCs (p<0.05). Inflammatory cell markers CD66b and CD68 also exhibited significant cohort differences (p<0.05). Exploratory analyses revealed a significant relationship between tumour immunity factors, geographic locality-specific prognosis, and postchemotherapy outcomes., Conclusions: Analyses of >1600 GCs suggest that Asian and non-Asian GCs exhibit distinct tumour immunity signatures related to T-cell function. These differences may influence geographical differences in clinical outcome, and the design of future trials particularly in immuno-oncology., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Effective systemic treatment of pancreatic cancer remains a major challenge, with progress hampered by drug resistance and treatment related toxicities. Currently available cytotoxic agents as monotherapy or in combination have provided only a modest survival benefit for patients with advanced disease. Disappointing phase III results with gemcitabine-based combinations in patients with advanced pancreatic cancer might be related to poor efficacy of systemic therapies in unselected patients. Future research strategies should prioritize identification of predictive markers through pharmacogenetic investigations. The individualization of patient treatment through pharmacogenetics may help to improve outcome by maximizing efficacy whilst lowering toxicity. This review provides an update on the pharmacogenetics of pancreatic cancer treatment and its influence on treatment benefits and toxicity.

Background/aim: Gemcitabine/carboplatin is efficacious in breast cancer but results in significant hematologic toxicities. We employed a multi-gene approach to identify variants to predict its toxicities., Patients and Methods: Twenty-six gemcitabine and platinum-based DNA repair pathway polymorphisms were correlated with gemcitabine pharmacokinetics, hematologic toxicities, response and survival in 41 Asian breast cancer patients receiving gemcitabine/carboplatin., Results: The combined effects of solute carrier family (SLC)28A1+1528C>T and thymidylate synthetase (TYMS)+1494del6 significantly influenced hematologic toxicities: 89% of patients who possessed either SLC28A1+1528TT or TYMS+1494ins6/ins6 (n=9) developed grade 4 thrombocytopenia, versus 14% with neither genotype (n=29; p<0.001). In concordance, all patients who possessed either genotype developed grade 3/4 neutropenia, compared to 38% with neither genotype (p=0.001). None of the other genetic variants analyzed correlated with drug pharmacokinetics and pharmacodynamics., Conclusion: Approximately one-quarter of our Asian cohort carried SLC28A1+1528TT or TYMS+1494ins6/ins6, which are associated with increased myelotoxicity from gemcitabine/carboplatin. This has potential utility in treatment selection and genotype-based dosing strategies.

Purpose: The aims were to determine the maximum tolerable dose (MTD) of docetaxel with CYP3A inhibition by ketoconazole, and to correlate the pharmacokinetics of docetaxel with midazolam phenotyping of CYP3A activity., Methods: Forty-one patients with refractory metastatic cancers were treated with an escalating dose of intravenous docetaxel once in every 3 week of 10 mg/m(2), concurrently with oral ketoconazole 200 mg twice daily for 3 days starting 2 days before the administration of docetaxel. Midazolam phenotyping test with ketoconazole modulation was performed before the first cycle of docetaxel. Docetaxel and midazolam pharmacokinetics were compared to our previous study of docetaxel treatment without ketoconazole modulation., Results: Neutropenia was the dose-limiting toxicity. The maximum tolerated dose was 70 mg with mean AUC at 70 mg similar to 75 mg/m(2) of docetaxel without ketoconazole. The plasma clearances of docetaxel and midazolam were reduced by 1.7- and 6-fold, respectively. The variability of midazolam AUC was reduced from 157 to 67%, but variability of docetaxel clearance was not reduced by CYP3A inhibition. Docetaxel clearance correlated with renal function and maximum concentration of ketoconazole, but not midazolam clearance or other variables of hepatic function., Conclusion: Fixed dosing was found to be feasible, without increased variability of clearance or neutrophil toxicity compared to BSA-based dosing. With ketoconazole modulation, docetaxel clearance correlated with renal function but not CYP3A phenotype.

The US Food and Drug Administration has recommended changes in the labeling for 6-mercaptopurine, irinotecan, and, most recently, tamoxifen to include pharmacogenetic information on treatment outcome. With the increased availability of pharmacogenetic testing, oncologists are now able to identify individuals at risk for severe treatment toxicity or poor treatment response. However, there are knowledge gaps to fill before rationalized therapy based on pharmacogenetics can be fully integrated into clinical practice. This review gives an overview of pharmacogenetic methods and focuses on the application of pharmacogenetic knowledge in oncology, using the examples of 6-mercaptopurine, irinotecan, tamoxifen, and gefitinib.

The variability in treatment responses and narrow therapeutic index of anticancer drugs are some of the key challenges oncologists face. The knowledge of pharmacogenetics can potentially aid in the discovery, development and ultimately individualization of anticancer drugs. The identification of genetic variations that predict for drug response is the first step towards the translation of pharmacogenetics into clinical practice. This review provides an update on the results of studies assessing the effects of germline polymorphisms and somatic mutations on therapeutic outcomes and highlights the potential applications and future challenges in pharmacogenetic research pertaining to cancer therapeutics.

Drugs metabolized by cytochrome CYP3A isoenzymes have wide interindividual variability and normally distributed plasma clearance distributions. This makes precise dosing difficult to achieve clinically, which may compromise safe therapy. We hypothesized that with potent inhibition of CYP3A, we could clinically render patients "poor metabolizer" phenotype status, and thus reduce interindividual pharmacokinetic variability of midazolam, a well-known CYP3A substrate. Intravenous bolus midazolam at doses of 2.5 mg and 1 mg were administered to 28 and 29 patients with cancer with and without co-administration of 200 mg of oral ketoconazole twice per day respectively for 3 days, starting 1 day before midazolam. Pharmacokinetic analyses of midazolam on both groups were derived using noncompartmental methods and compared. The mean clearance (CL) of midazolam was reduced 6 times by ketoconazole. Midazolam CL were normally distributed in both groups, and ranged from 1.7 to 51.9 and 1.4 to 8.2 L/hour in the control and ketoconazole groups, respectively, corresponding to a 7-fold reduction in dispersion between the 2 groups. Area-under-the-curve variability was reduced by >100%. A limited sampling model consisting of time points at 15 and 300 minutes was validated as a phenotype for CYP3A activity to facilitate the use of midazolam as a probe drug for CYP3A activity. Potent inhibition of CYP3A by ketoconazole reduced midazolam CL and area-under-the-curve variability, allowing for more precise achievement of therapeutic target drug exposure. Prospective evaluation of this approach, together with dose adjustment based on limited sampling, seems warranted.

Purpose: Ketoconazole has been shown to inhibit the glucuronidation of the UGT2B7 substrates zidovudine and lorazepam. Its effect on UGT1A substrates is unclear. A recent study found that coadministration of irinotecan and ketoconazole led to a significant increase in the formation of SN-38 (7-ethyl-10-hydroxycamptothecine), an UGT1A substrate. This study investigates whether ketoconazole contributes to the increase in SN-38 formation by inhibiting SN-38 glucuronidation., Experimental Design: SN-38 glucuronidation activities were determined by measuring the rate of SN-38 glucuronide (SN-38G) formation using pooled human liver microsomes and cDNA-expressed UGT1A isoforms (1A1, 1A7 and 1A9) in the presence of ketoconazole. Indinavir, a known UGT1A1 inhibitor, was used as a positive control. SN-38G formation was measured by high-performance liquid chromatograph., Results: Ketoconazole competitively inhibited SN-38 glucuronidation. Among the UGT1A isoforms screened, ketoconazole showed the highest inhibitory effect on UGT1A1 and UGT1A9. The K(i) values were 3.3 +/- 0.8 micromol/L for UGT1A1 and 31.9 +/- 3.3 micromol/L for UGT1A9., Conclusions: These results show that ketoconazole is a potent UGT1A1 inhibitor, which seems the basis for increased exposure to SN-38 when coadministered with irinotecan.

Aim: Cancer genetic risk assessment clinics represent the primary prevention arm in oncology in identifying high-risk patients for screening and prevention, but deals with the taboo subject of cancer risk prediction. Low knowledge level and traditional beliefs may pose significant barriers to its acceptance in Asia., Methods: We conducted a questionnaire survey to evaluate the acceptance of breast cancer genetic counseling., Results: More than 70% of the 438 respondents indicated interest and perceived potential benefits. Higher education level and use of English were associated with greater acceptance. Learning about cancer risk and cancer detection, helping the family, and the doctor's recommendation, were important motivators, while misperception that cancer patients could not gain personally, concerns of not understanding the information, cost issues, and fears of bad news, were important barriers., Conclusions: Singaporean women were receptive to cancer genetics counseling. Education to correct misperceptions may optimize utilization of a cancer genetics service.