Yaomin Xu

Targeted Next Generation Sequencing Identifies Markers of Response to PD-1 Blockade

Mutational load, by whole exome sequencing, can correlate with immunotherapy responses. Assessing melanoma mutational load of a fraction of the genome, by hybrid capture-based NGS, provided an accurate surrogate for WES determinations, and predicted response to anti-PD-1. Therapeutic antibodies blocking programmed death-1 and its ligand (PD-1/PD-L1) induce durable responses in a substantial fraction of melanoma patients. We sought to determine whether the number and/or type of mutations identified using a next-generation sequencing (NGS) panel available in the clinic was correlated with response to anti–PD-1 in melanoma. Using archival melanoma samples from anti–PD-1/PD-L1-treated patients, we performed hybrid capture–based NGS on 236–315 genes and T-cell receptor (TCR) sequencing on initial and validation cohorts from two centers. Patients who responded to anti–PD-1/PD-L1 had higher mutational loads in an initial cohort (median, 45.6 vs. 3.9 mutations/MB; P = 0.003) and a validation cohort (37.1 vs. 12.8 mutations/MB; P = 0.002) compared with nonresponders. Response rate, progression-free survival, and overall survival were superior in the high, compared with intermediate and low, mutation load groups. Melanomas with NF1 mutations harbored high mutational loads (median, 62.7 mutations/MB) and high response rates (74%), whereas BRAF/NRAS/NF1 wild-type melanomas had a lower mutational load. In these archival samples, TCR clonality did not predict response. Mutation numbers in the 315 genes in the NGS platform strongly correlated with those detected by whole-exome sequencing in The Cancer Genome Atlas samples, but was not associated with survival. In conclusion, mutational load, as determined by an NGS platform available in the clinic, effectively stratified patients by likelihood of response. This approach may provide a clinically feasible predictor of response to anti–PD-1/PD-L1. Cancer Immunol Res; 4(11); 959–67. ©2016 AACR.

Phase II study of ruxolitinib, a selective JAK1/2 inhibitor, in patients with metastatic triple-negative breast cancer

Preclinical data support a role for the IL-6/JAK2/STAT3 signaling pathway in breast cancer. Ruxolitinib is an orally bioavailable receptor tyrosine inhibitor targeting JAK1 and JAK2. We evaluated the safety and efficacy of ruxolitinib in patients with metastatic breast cancer. This was a non-randomized phase II study enrolling patients with refractory, metastatic triple-negative breast cancer. The primary endpoint was objective response by RECIST 1.1. The study was designed to enroll patients whose archival tumor tissue was pSTAT3-positive (T-score >5) by central immunohistochemistry. pSTAT3 staining was available from 171 of 217 consented patients and pSTAT3 T-score was positive in 67/171 (39.2%) tumors, suggesting that JAK–STAT activation is frequent. Twenty-three patients including one patient with inflammatory breast cancer were enrolled. Ruxolitinib was well-tolerated with infrequent grade 3 or higher toxicities with fatigue as the most common toxicity. Among 21 patients who received at least one dose of protocol therapy, no objective responses were observed and the study was closed to further accrual. Pharmacodynamic analyses of baseline vs. cycle 2 biopsies suggest on-target activity, including a significant decrease in the proportion of pSTAT3+ cells in three patients with paired biopsies and downregulation of JAK–STAT target genes and signatures via transcriptional analyses of 11 total baseline and four metastatic biopsies. Immuno-FISH analyses demonstrate intratumoral heterogeneity of pSTAT3 and JAK2 amplification. Ruxolitinib, as a single agent, did not meet the primary efficacy endpoint in this refractory patient population despite evidence of on-target activity.Clinical trial: JAK inhibitor not effective for triple-negative breast cancerA drug that blocks the Janus tyrosine kinase (JAK) pathway offered no clinical benefit to women with triple-negative breast cancer. Ruxolitinib is an inhibitor of JAK1 and JAK2 that is approved to treat certain rare cancers of the blood and bone marrow, and there’s a good scientific evidence to think it might have anti-tumor effects against breast cancer as well. So a team led by N.U.L. from the Dana-Farber Cancer Institute in Boston, Massachusetts, USA, conducted a small phase II trial to evaluate ruxolitinib in women with refractory, metastatic, triple-negative disease whose tumors tested positive for high expression of pSTAT3, a protein in the same signaling pathway as JAK. Yet, despite the fact that pSTAT3 levels went down among the 21 women who received the drug, not a single one responded to the therapy.

Abstract 588: Advanced molecular characterization of severe autoimmune toxicities associated with checkpoint inhibitor therapies

Immune checkpoint inhibitors (ICIs) have made a profound impact on the treatment of a variety of cancers. However, as with any systemic treatment, toxicities are inevitable. With most classes of cancer therapies, toxicities are relatively predictable based on clinical trial safety data and therefore can be handled with prophylactic or supportive care measures. However, ICIs are unique in their ability to cause rare but severe auto-immune toxicities. The molecular underpinnings of these toxicities, as well as unique features of the patient, tumor, or affected tissue, have not been extensively explored. We recently reported a small case series of two patients with myocarditis resulting in death arising following combination ICI therapy (Johnson et al, N Engl J Med, 2016). High lymphocytic infiltration, coupled with PD-L1 expression was present in the affected myocardium and skeletal muscle. Common T cell clones were identified between the affected tissue and tumor, and abnormal expression of muscle-specific transcripts was identified in the associated tumor, suggesting release of peripheral tolerance to tumor-expressed self-antigens.To expand upon our reported study, we collected healthy and afflicted tissue from a series of cancer patients with immune-related colitis, myocarditis (MC), and encephalopathy following ICI treatment. We hypothesize that molecular analysis of these tissues will identify causal factors in the etiology of these toxicities, and how to better predict, prevent, and treat them. Thus, we performed molecular characterization of the immune infiltrate and diseased tissue microenvironment. A total of 20 affected (colon, cardiac, brain) and non-diseased control specimens were examined by spatial digital profiling (nanoString). This process generates a spatial heatmap of digital counts of 20 selected immunology and cellular markers and proteins across each specimen. Using this technology, the landscape of inflammation in ICI-affected organs can be resolved for insights into the mechanism whereby ICI-mediated auto-immunity occurs. Targeted RNAseq for selected immuno-oncology mRNA targets was also performed. In initial RNA sequencing analyses of MC cases, affected myocardium, skeletal muscle, and patient-matched tumors all demonstrated expression of immune activation markers (e.g. interferon-gamma and granzyme B), expression of PD-L1, and muscle-specific genes. In the expanded population, including colitis, digital spatial profiling analyses and targeted NGS (RNAseq) are underway. Although data analyses are incomplete at the time of this abstract, this work will be the largest and most comprehensive analysis of the molecular underpinnings of ICI-mediated auto-immune toxicity reported to date. These data should offer clarity in the mechanisms and features of these adverse events, how to prevent or predict them with precision medicine, and how to treat them when they do occur. Citation Format: Justin M. Balko, Daniel Y. Wang, Yu Wang, Rami Al-Rohil, Margaret Compton, Jeffery A. Sosman, Igor Puzanov, Bret Mobley, Robert D. Hoffman, Yaomin Xu, Javid J. Moslehi, Chanjuan Shi, Douglas B. Johnson. Advanced molecular characterization of severe autoimmune toxicities associated with checkpoint inhibitor therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 588. doi:10.1158/1538-7445.AM2017-588

A novel patient-derived cell line originated at the time of crizotinib resistance displays a mesenchymal phenotype

PRESENTED BY ATTENDEES Efficacy and safety of dovitinib in pretreated advanced squamous non-small cell lung cancer with FGFR1 amplification: A single-arm, phase II study Myung-Ju Ahn, Sung Hee Lim, Jong-Mu Sun, Yoon-La Choi, Hye Ryun Kim, Soo-min Ahn, Se-Hoon Lee, Jin Seok Ahn, Keunchil Park, Joo Hang Kim, Byoung Chul Cho Samsung Medical Center, Seoul, Korea, Yunsei University, Seoul, Korea Purpose: FGFR1 amplification is one of the most common potential driving oncogenes in squamous cell carcinoma (SCC), which accounts for 20% of non-small cell lung cancer (NSCLC). This phase II study evaluated the efficacy and toxicity profile of dovitinib, an orally active fibroblast growth factor receptor (FGFR) inhibitor, in advanced lung SCC patients. Experimental Design: Patients with histological confirmed advanced squamous cell NSCLC and previously treated with at least one cytotoxic chemotherapy were enrolled. All patients had FGFR1 gene amplification more than 5 copies by fluorescent in situ hybridization (FISH). Each 7-day treatment cycle consisted of dovitinib 500mg orally administration on days 1 to 5 and 2 days off. Primary endpoint was overall response rate and secondary endpoints included PFS, OS and toxicity. Exploratory analysis for FGFR1-3 mRNA expression was performed. The mRNA in situ hybridization (ISH) assay was performed using the RNA scope 2.0 assay system and the FGFR1 probe provided by Advanced Cell Diagnostics. Results: All 26 patients were male with the median age of 68 years (range, 52–80). Most patients were ever smokers (96%) and had good ECOG (0-1) performance status (85%). The median number of dovitinib treatment cycles administered was 2.5 (range, 1-12). The overall response rate (ORR) was 11.5% (95% CI, 0.8–23.8) and disease control rate (DCR) was 50% (95% CI, 30.8– 69.2). There were 3 partial responses (PR) and 10 stable diseases (SD). Duration of response in 3 patients who achieved PR was 4.5þ, 5.1þ and 6.1 months, respectively. After the median follow-up duration of 15.7 months (range, 1.2–25.6), the median overall survival (OS) was 5.0 months (95% Confidential Interval, 3.61– 6.39) and progression-free survival (PFS) was 2.9 Journal of Thoracic Oncology Vol. 11 No. 2S: S16-S55 months (95% CI, 1.54–4.26). The most common grade 3 or higher AEs suspected to be related to dovitinib treatment were fatigue (19.2%), anorexia (11.5%), and hyponatremia (11.5%) and 12 patients (46%) required dose reduction of dovitinib. Further analysis for FGFR1 mRNA, only modest overlap (31.2%) of FGFR1 expression at the mRNA level with FGFR1 amplification was found. The expression of FGFR1 mRNA was not associated with degree of FGFR1 gene amplification nor FGFR2 or FGFR3 mRNA expression. Conclusion: Dovitinib treatment showed modest efficacy in advanced squamous cell lung cancer patients with FGFR1 amplification. Further studies to evaluate other biomarkers correlated with the efficacy of dovitinib in SCC should be warranted. A novel patient-derived cell line originated at the time of crizotinib resistance displays a mesenchymal phenotype Karinna Almodovar-Garcia, Yingjun Yan, Yuanyuan Wang, Zhongming Zhao, Xingyi Guo, Yaomin Xu, Christine M. Lovly Vanderbilt University School of Medicine, Nashville, TN Lung cancers that harbor genomic ALK alterations are clinically responsive to pharmacologic ALK inhibition. Crizotinib, an orally available small-molecule inhibitor of the ALK tyrosine kinase, was approved for the treatment of ALKþ lung cancer patients. Unfortunately, as seen with other tyrosine kinases inhibitors (TKIs) in clinical use, most patients whose disease initially responds to crizotinib eventually develop progressive disease. To elucidate mechanisms of acquired resistance to ALK TKIs, we established and characterized novel ALKþ cell lines from patients with acquired resistance to ALK TKI therapy. In particular, we developed a cell line (designated STM) from a patient with an EML4-ALK (E6;A20, variant 3) fusion that developed acquired resistance to crizotinib. Fluorescence in situ hybridization confirmed that the cell line retained the ALK rearrangement. STM cells had decreased sensitivity to crizotinib and secondgeneration ALK inhibitors, including ceritinib, alectinib, and X-396. Morphologic changes were observed in this patient-derived cell line, the tumor cells had a spindle form, characteristic of the epithelial-mesenchymal transition (EMT). Loss of expression of the epithelial marker, E-cadherin, was accompanied by strong expression of the February 2016 Abstracts S17 mesenchymal markers, vimentin and N-cadherin in STM cells. Enhanced invasion and migration capabilities were observed in STM cells consistent with a mesenchymal phenotype. Src has been shown to play a role in E-cadherin regulation and EMT. Thus, treatment of cells with dasatinib, a Src inhibitor, suppressed cell growth in STM cells, but not in ALKþ/TKI sensitive cell lines. The addition of a low dose of dasatinib sensitized STM cells to the antiproliferative effects of crizotinib. STMcellswere subjected to genetic analysis to identify new genetic anomalies that could be driving resistance. Top hits are being evaluated. To our knowledge, this is the first report that demonstrates EMT occurring in an ALKþ crizotinib resistant clinical sample. Collectively these data support EMT as a mechanism of resistance to crizotinib and identifies dasatinib as a potential therapeutic for treatment of crizotinib resistance associated with EMT. Immune profiling of malignant pleural mesothelioma by flow cytometry identifies distinct T-cell activation and exhaustion phenotypes in PD-L1 positive versus PD-L1 negative tumors Mark M. Awad, Mark A. Bittinger, Robert E. Jones, Xioayun Liao, Meghana Kulkarni, Lauren Keogh, Shohei Koyama, Christina G. Almonte, Abigail A. Santos, Jessie E. English, Julianne Barlow, William G. Richards, Peter S. Hammerman, Scott J. Rodig, Raphael Bueno, Kwok-Kin Wong Dana-Farber Cancer Institute, Boston, MA, Brigham and Women’s Hospital, Boston, MA Although PD-L1 immunohistochemical staining appears to be a partially predictive biomarker of response to PD-1 inhibitors in some cancers, many PD-L1 positive tumors do not respond to these agents. Possible explanations for this observation are that some PD-L1 positive cancers may have a paucity of infiltrating lymphocytes and/or T cells within tumors may express multiple exhaustion markers. We have developed a method for comprehensive immune cell phenotyping using flow cytometry on solid tumors that have been dissociated into single cell suspensions. Applying this technique to 33 resected malignant pleural mesothelioma samples, here we show that compared to PD-L1 negative tumors, PD-L1 positive tumors have significantly more infiltrating CD45þ immune cells, a significantly higher proportion of infiltrating CD3þ T cells, a significant increase in proliferating CD4þ and CD8þ T cells, and a significantly higher percentage of CD3þ cells displaying the activation antigens HLA-DRþ/ CD38þ. PD-L1 positive tumors also have a significantly higher proportion of FOXP3þ/CD4þ regulatory T cells. We found that CD4þ and CD8þ T cells in PD-L1 positive samples are significantly more likely to express the T-cell exhaustion markers PD-1 and TIM-3 compared to PD-L1 negative samples. Flow cytometric analysis also identified two immunologically distinct PD-L1 positive mesothelioma samples: PD-1 positive, TIM-3 negative “single positive” tumors, and PD-1 positive, TIM-3 positive “double positive” tumors. Successful incorporation of comprehensive immune profiling by flow cytometry into prospective clinical trials should hopefully refine our ability to predict which patients will respond to immune checkpoint blockade and lead to rationally designed combination immunotherapy trials. Novel epidermal growth factor receptor inhibitor accumulates in the brain and inhibits the growth of brain metastatic non-small cell lung cancer Christina A. Jamieson, Michelle Muldong, Yun Oliver Long, Ida Deichaite, Alan Lewis, David W. Anderson, Nicholas A. Cacalano University of California, San Diego, CA, Capella Therapeutics, San Diego, CA, University of Missouri, St. Louis, MO, University of California, Los Angeles, CA Deaths from solid tumors are often not due to the primary lesion but to metastatic disease at distal sites such as the lung, liver, and brain. Patients with non-small cell lung cancer (NSCLC) experience brain metastases, a poor prognostic marker, at an incidence rate of 30-50%. A significant proportion of the metastatic tumors express activating mutations of the EGFR, including exon 19 deletions, which confer increased sensitivity to EGFR inhibitors such as erlotinib and gefitinib. Despite successful use of small molecule kinase inhibitors for the treatment of EGFRþ primary lung tumors, current therapeutics poorly inhibit the growth of NSCLC brain metastases due to difficulty crossing the blood-brain barrier. For this reason, NSCLC patients with brain metastases are often excluded from clinical trials with novel therapies and thus have access to very few emerging treatment options. We have synthesized a novel class of compounds that inhibit the epidermal growth factor receptor (EGFR) in the nanomolar range in vitro, and demonstrate a high degree of selectivity for EGFR family members. The parent compound, LL001, from Capella Therapeutics, Inc., inhibited EGFR-mediated autophosphorylation and

Treatment with QiBaoMeiRan, a Chinese herbal formula, prevents bone loss in ovariectomized rat

ABSTRACT Objective Hormone replacement therapy has been used as an effective treatment for the prevention of bone loss in postmenopausal women. In our previous study, QiBaoMeiRan formula (QBMR) had estrogenic activity and could relieve symptoms of hot flushes and body weight increase induced by estrogen decline. However, no evidence base links QBMR to preventing bone loss. The aim of this study is to investigate the effect of QBMR on bone loss. Methods The ovariectomized rat model was established, and ovariectomized rats were treated with QBMR at doses of 0.875, 1.75, and 3.5 g/kg per day for 8 weeks. Biochemical parameters, bone mineral density, structural morphometric traits and histological characteristics of trabecular bone were assessed. Results QBMR treatment significantly decreased the increase in serum alkaline phosphatase, bone Gla-protein and C-telopeptide fragments of type I collagen and decreased the decline of serum calcium and phosphorus in the circulation of ovariectomized rats. QBMR completely corrected the decrease in bone mineral density in lumbar vertebrae (L4–L6) comparable to the sham group. In addition, QBMR treatment also significantly ameliorated the decrease of structural parameters of femur trabecular bone, bone volume fraction, trabecular number, trabecular thickness and bone mineral density as well as the increase in trabecular separation by micro-computerized tomography scanning. These were also confirmed by bone histological results that showed its protective action. Conclusions Our results indicated that QBMR had a definite anti-bone loss effect and will have potential to be used for the treatment of postmenopausal osteoporosis.

Water decoction of coptidis rhizoma prevents oxidative damage in erythrocytes of mice

Coptidis Rhizoma (Coptis chinensis) has been reported to have antioxidative effect on hemolysis of erythrocytes induced by acetylphenylhydrazine in mice and rats. However, the ability of Coptidis Rhizoma to protect structure and function of erythrocytes membrane and morphology of erythrocytes against oxidative damage remains unknown. In this study, we undertook a characterization of antioxidative activity in erythrocytes membrane of Coptidis Rhizoma using an in vivo model of acetylphenylhydrazine-induced mice together with in vitro studies with 2,2-azo-bis (2-amidinopropane) dihydrochloride-induced erythrocytes for further morphology characterization. Acetylphenylhydrazine-induced mice were treated intragastrically with Coptidis Rhizoma at doses of 0.3, 0.6, and 1.2 g/kg per day for 3 days and at the dose of 0.6 and 1.2 g/kg it showed that there was an increasing trend in membranes cytoskeletal proteins of band I-IV, especially a significant upregulation in band II. Significant increase in phosphatidylserine and phosphatidylcholine content at the dose of 1.2 g/kg Coptidis Rhizoma was obsereved. At all doses of Coptidis Rhizoma, the declined membrane fluidity of acetylphenylhydrazine-induced mice was significantly increased. In addition, at the dose of 1.2 g/kg Coptidis Rhizoma treatment showed a significant increase in Ca2+/Mg2+-ATPase activity and there was an increasing trend in the activity of Na+/K+-ATPase. In vitro, Coptidis Rhizoma protected erythrocytes from 2,2-azo-bis (2-amidinopropane) dihydrochloride-induced hemolysis in a dose-dependent manner at concentrations of 0.25-1.5 mg/ml, and also significantly inhibited the 2,2-azo-bis (2-amidinopropane) dihydrochloride-induced morphological alterations in mice erythrocytes. These results demonstrate that Coptidis Rhizoma is capable of protecting erythrocytes against oxidative damage probably by acting as an antioxidant and maintaining membrane integrity.