Mechanisms of Acquired Resistance to AZD9291 A Mutation-Selective, Irreversible EGFR Inhibitor

IntroductionAZD9291, a third-generation and mutation-selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), is active against patients with EGFRT790M-mutant non–small-cell lung cancer (NSCLC) who failed prior treatment with EGFR TKIs. However, acquired resistance to AZD9291 is inevitable. In this study, we identified the mechanisms of acquired resistance to AZD9291 in EGFRT790M-mutant NSCLC.MethodsFour NSCLC patients with both an EGFR exon 19 deletion and the EGFRT790M mutation after developing acquired resistance to first-generation EGFR TKIs received AZD9291 at doses of 20 to 80 mg/day in a phase I trial (NCT01802632). Paired tumor samples before and after treatment were obtained to evaluate EGFR modifications, alternative pathway activation, and histologic transformation. Genetic alterations were analyzed using Sanger sequencing, fluorescence in situ hybridization, real-time polymerase chain reaction, and targeted exome sequencing.ResultsAll four patients achieved a partial response (median duration of response, 9 months [range, 9–11 months]) and subsequently showed resistance to AZD9291. EGFRT790M-mutant clones depopulated AZD9291-resistant tumors to below 1% (baseline, 14%–36%) in three patients with progression: one with the loss of EGFRLREAT747del/T790M-double mutant clones and two accompanied by transformation to small-cell carcinoma and focal fibroblast growth factor receptor 1 (FGFR1) amplification, respectively. EGFRT790M-mutant clones remained and the EGFR ligand was overexpressed in one patient with focal progression to AZD9291.ConclusionAcquired resistance mechanisms of AZD9291 in patients with EGFRT790M-mutant NSCLC who failed treatment with first-generation EGFR TKIs include the loss of EGFRT790M-mutant clones plus alternative pathway activation or histologic transformation and EGFR ligand–dependent activation

Prediction of Liver-Related Events Using Fibroscan in Chronic Hepatitis B Patients Showing Advanced Liver Fibrosis

Liver stiffness measurement (LSM) using transient elastography (FibroScan®) can assess liver fibrosis noninvasively. This study investigated whether LSM can predict the development of liver-related events (LREs) in chronic hepatitis B (CHB) patients showing histologically advanced liver fibrosis.Between March 2006 and April 2010, 128 CHB patients with who underwent LSM and liver biopsy (LB) before starting nucleot(s)ide analogues and showed histologically advanced fibrosis (≥F3) with a high viral loads [HBV DNA ≥2,000 IU/mL] were enrolled. All patients were followed regularly to detect LRE development, including hepatic decompensation (variceal bleeding, ascites, hepatic encephalopathy, spontaneous bacterial peritonitis, hepatorenal syndrome) and hepatocellular carcinoma (HCC).The mean age of the patient (72 men, 56 women) was 52.2 years. During the median follow-up period [median 27.8 (12.6-61.6) months], LREs developed in 19 (14.8%) patients (five with hepatic decompensation, 13 with HCC, one with both). Together with age, multivariate analysis identified LSM as an independent predictor of LRE development [P<0.044; hazard ratio (HR), 1.038; 95% confidence interval (CI), 1.002-1.081]. When the study population was stratified into two groups using the optimal cutoff value (19 kPa), which maximized the sum of sensitivity (61.1%) and specificity (86.2%) from a time-dependent receiver operating characteristic curve, patients with LSM>19 kPa were at significantly greater risk than those with LSM≤19 kPa for LRE development (HR, 7.176; 95% CI, 2.257-22.812; P = 0.001).LSM can be a useful predictor of LRE development in CHB patients showing histologically advanced liver fibrosis

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Presence of Membrane-Bound TGF-β1 and Its Regulation by IL-2-Activated Immune Cell-Derived IFN-γ in Head and Neck Squamous Cell Carcinoma Cell Lines

The presence of membrane-bound TGF-beta 1 (mTGF-beta 1) has been recently observed in regulatory T cells, but only a few studies have reported the same phenomenon in cancer cells. In this study, we investigate the regulation of mTGF-beta l expression in five head and neck squamous cell carcinoma cell lines using FACS analysis. Through blocking Ab and exogenous cytokine treatment experiments, we found that expression of mTGF-beta 1 is significantly induced by the activated immune cell-derived factor IFN-gamma. In addition, IFN-gamma and TNF-alpha are shown to have a synergistic effect on mTGF-beta 1 expression. Moreover, we found that exogenous TNF-alpha induces endogenous TNF-alpha mRNA expression in an autocrine loop. In contrast to previous reports, we confirm that, in this model, mTGF-beta 1 is neither a rebound form of once-secreted TGF-beta 1 nor an activated form of its precursor membrane latency-associated peptide. Inhibitors of transcription (actinomycin D), translation (cycloheximide), or membrane translocation (brefeldin A) effectively block the induction of mTGF-beta 1, which suggests that induction of mTGF-beta 1 by IFN-gamma and/or TNF-alpha occurs through de novo synthesis. These findings suggest that some cancer cells can detect immune activating cytokines, such as IFN-gamma and TNF-alpha, and actively block antitumor immunity by induction of mTGF-beta 1. The Journal of Immunology, 2009, 182: 6114-6120.This study was supported by grants from the Innovative Research Institute for Cell Therapy (A062260) and the Korea Health 21 Research & Development Project (02- PJ1-PG3-21206-0003), Ministry of Health and Welfare, Republic of Korea.Vila-del Sol V, 2008, J IMMUNOL, V181, P4461Andersson J, 2008, J EXP MED, V205, P1975, DOI 10.1084/jem.20080308Turley EA, 2008, NAT CLIN PRACT ONCOL, V5, P280, DOI 10.1038/ncponc1089Baker K, 2008, INT J CANCER, V122, P1695, DOI 10.1002/ijc.23312Marigo I, 2008, IMMUNOL REV, V222, P162Teicher BA, 2007, CLIN CANCER RES, V13, P6247, DOI 10.1158/1078-0432.CCR-07-1654Lim DS, 2007, CANCER IMMUNOL IMMUN, V56, P1817, DOI 10.1007/s00262-007-0325-0Wrzesinski SH, 2007, CLIN CANCER RES, V13, P5262, DOI 10.1158/1078-0432.CCR-07-1157Filipazzi P, 2007, J CLIN ONCOL, V25, P2546, DOI 10.1200/JCO.2006.08.5829Gandhi R, 2007, J IMMUNOL, V178, P4017Yu H, 2007, NAT REV IMMUNOL, V7, P41, DOI 10.1038/nri1995Menoret A, 2006, J IMMUNOL, V177, P6091Patterson SG, 2006, ONCOGENE, V25, P6113, DOI 10.1038/sj.onc.1209632Jakowlew SB, 2006, CANCER METAST REV, V25, P435, DOI 10.1007/s10555-006-9006-2Broderick L, 2006, J IMMUNOL, V177, P3082Robinson CM, 2006, CYTOKINE, V35, P53, DOI 10.1016/j.cyto.2006.07.007Hanada T, 2006, J EXP MED, V203, P1391, DOI 10.1084/jem.2006.0436Dasgupta S, 2006, J CELL BIOCHEM, V97, P1036, DOI 10.1002/jcb.20647Smyth MJ, 2006, J IMMUNOL, V176, P1582Bui JD, 2006, J IMMUNOL, V176, P905Li MO, 2006, ANNU REV IMMUNOL, V24, P99, DOI 10.1146/annurev.immunol.24.021605.090737Kulbe H, 2005, CANCER RES, V65, P10355, DOI 10.1158/0008-5472.CAN-05-0957Thomas DA, 2005, CANCER CELL, V8, P369, DOI 10.1016/j.ccr.2005.10.012Ghiringhelli F, 2005, J EXP MED, V202, P1075, DOI 10.1084/jem.20051511Dasgupta S, 2005, J IMMUNOL, V175, P5541Hagemann T, 2005, J IMMUNOL, V175, P1197VILADELSOL V, 2005, J IMMUNOL, V174, P2825Balkwill F, 2004, NAT REV CANCER, V4, P540, DOI 10.1038/nrc1388Lu SL, 2004, CANCER RES, V64, P4405Lee JC, 2004, J IMMUNOL, V172, P7335Nakamura K, 2004, J IMMUNOL, V172, P834Williams L, 2004, J IMMUNOL, V172, P567Leu CM, 2003, ONCOGENE, V22, P7809, DOI 10.1038/sj.onc.1207084Roberts AB, 2003, P NATL ACAD SCI USA, V100, P8621, DOI 10.1073/pnas.1633291100Castriconi R, 2003, P NATL ACAD SCI USA, V100, P4120, DOI 10.1073/pnas.0730604100Dong HD, 2002, NAT MED, V8, P793, DOI 10.1038/nm730Karin M, 2002, NAT REV CANCER, V2, P301, DOI 10.1038/nrc780Niu GL, 2002, ONCOGENE, V21, P2000, DOI 10.1038/sj/onc/1205260Nakamura K, 2001, J EXP MED, V194, P629Hasegawa Y, 2001, CANCER, V91, P964Grainger DJ, 2000, CYTOKINE GROWTH F R, V11, P133Kim SJ, 2000, CYTOKINE GROWTH F R, V11, P159Khalil N, 1999, MICROBES INFECT, V1, P1255Ku JL, 1999, LARYNGOSCOPE, V109, P976Munger JS, 1999, CELL, V96, P319Wikstrom P, 1998, PROSTATE, V37, P19Fakhrai H, 1996, P NATL ACAD SCI USA, V93, P2909HEO DS, 1989, CANCER RES, V49, P5167

In vitro anticancer activity of PI3K alpha selective inhibitor BYL719 in head and neck cancer

Background/Aim: The purpose of the present study was to explore the antiproliferative effect of BYL719, a specific inhibitor for phosphatidylinositol 3-kinase (PI3K) p110 alpha, in human head and neck cancer cell lines, as a single agent or in combination with the irreversible EGFR tyrosine kinase inhibitor, dacomitinib. Materials and Methods: Six head and neck cancer cell lines consisting of two PIK3CA mutant cell lines, SNU-1076 and Detroit562, and four PIK3CA wild-type cell lines, SNU-1066, SNU-1041, FaDu and SCC25, were analyzed. Results: The PIK3CA-mutant cell lines were more sensitive to BYL719 than the PIK3CA wild-type cell lines. Following BYL719 treatment, all PIK3CA wild-type cell lines, except for the SNU-1066 cell line, exhibited higher IC50 values compared to the PIK3CA mutant cell lines. Administration of BYL719 induced-cell cycle G(0)/G(1) arrest and resulted in increased apoptosis in a dose-dependant manner. Furthermore, the administration of BYL719 reduced the level of p-mTOR, p-AKT and p-S6 expression indicating the down-regulation of downstream signaling. Conclusion: BYL719, a PI3K alpha selective blocker, could be a promising factor in the treatment of head and neck cancer either as a single agent or in combination with dacomitinib.

The effects of 1,25-dihydroxyvitamin D3 on in vitro human NK cell development from hematopoietic stem cells.

1,25-dihydroxy1,25(OH)(2)D(3) [1,25(OH)(2)D(3)] is the biologically active form of Vitamin D and is immunoregulatory. 1,25(OH)(2)D(3) binds the Vitamin D Receptor (VDR) complex present in many immune populations and can illicit transcriptional responses that vary amongst different immune subsets. The effects of 1,25(OH)(2)D(3) on mature and developing human natural killer (NK) cells are not well characterized. Here we studied the influence of 1,25(OH)(2)D(3) using an established NK cell differentiation system. Briefly, UCB CD34+ cells were isolated and cultured in conditions optimal for natural killer (NK) cell differentiation and varying concentrations of 1,25(OH)(2)D(3) were administered. At physiological concentrations (10 nM),1,25(OH)(2)D(3) impaired NK cell development. Moreover, the NK cells that did develop under the influence of 1,25(OH)(2)D(3) showed a significant reduction in function (cytotoxicity and cytokine production). Conversely,1,25(OH)(2)D(3) strongly induced hematopoietic stem cells to differentiate along a myeloid pathway, giving rise to CD14+ cells. Mechanistically, 1,25(OH)(2)D(3) drives hematopoietic progenitor cells to rapidly upregulate monocyte genes (i.e. C/EBPα and CD14). There were no effects of 1,25(OH)(2)D(3) on mature NK cytotoxicity or cytokine production. Collectively, these studies provide novel data showing the negative regulatory effect of 1,25(OH)(2)D(3) on NK cell development

Influence of chemotherapy on nitric oxide synthase, indole-amine-2,3-dioxygenase and CD124 expression in granulocytes and monocytes of non-small cell lung cancer

There is no specific marker to evaluate the immuno-suppressive status of cancer patients. Several markers, such as CD124, latency-associated peptide (LAP), arginase I, indole-amine-2,3-dioxygenase (IDO) and inducible nitric oxide synthase (iNOS), are known to be associated with immune suppression. However, there is little research regarding the change in these parameters after chemotherapy. The present study enrolled 23 chemo-naive non-small cell lung cancer (NSCLC) patients and 19 healthy donors. From the 23 NSCLC patients, 11 post-chemotherapy samples were collected. Surface and functional markers were analyzed by flow-cytometry. The mean fluorescence intensities (MFI) of iNOS were higher and the MFI of LAP were lower in NSCLC patient than in healthy donors (P < 0.05). In a comparison of pre-chemotherapy and post-chemotherapy groups with NSCLC, the MFI of iNOS on granulocytes and monocytes and IDO on monocytes were significantly lower in the post-chemotherapy group than in the pre-chemotherapy group (P < 0.05). In a serial analysis with 10 patients who had paired samples and who showed clinical benefits from chemotherapy, the MFI of iNOS for both cell types, and of IDO and CD124 for monocytes decreased significantly after chemotherapy, compared with those before chemotherapy (iNOS, 4.79 +/- 1.75 vs 2.83 +/- 0.77, P = 0.005, for granulocytes and 6.15 +/- 2.94 vs 2.76 +/- 1.05, P = 0.005 for monocytes; IDO, 6.81 +/- 3.43 vs 4.64 +/- 1.55, P = 0.012 for monocytes; CD124, 2.31 +/- 0.39 vs 1.94 +/- 0.43, P = 0.008 for monocytes). The changes in arginase I and LAP expression were not significant. The changes in iNOS, IDO and CD124 expression were significant after chemotherapy in NSCLC. Further evaluation of the possibility of immune status monitoring using these parameters is needed. (Cancer Sci 2012; 103: 155160)

Minimal residual disease negativity by next‐generation flow in non‐CR myeloma patients

Abstract Next‐generation flow (NGF) has detected minimal residual disease (MRD) in numerous myeloma patients who achieve a complete response (CR). However, when MRD is not detected via NGF in non‐CR patients, its clinical meaning is uncertain. Here, we investigated the correlation between MRD findings on NGF and the response criteria, paying special attention to patients with discrepant results. We performed NGS analysis of IgH rearrangements on bone marrow samples from the non‐CR patients with negative MRD on NGF. NGS detected residual abnormal clones in those patients, suggesting that NGF and NGS should be used in a complementary manner for MRD investigation