bluuebunny/crossref_metadata_2025 huggingface dataset:
TCR repertoire sequencing of 254 resected non-small cell lung cancers to reveal TCR clonality in normal tissues compared to tumor tissues.
Authors: Reuben Alexandre , Gittelman Rachel , Zhang Jiexin , Yusko Erik , Wu Chang-jiun , Emerson Ryan O , Zhang Jianhua , Tipton Christopher , Vignali Marissa , Fujimoto Junya , Rodriguez-Canales Jaime , Swisher Stephen , Heymach John , Robins Harlan , Lee J. Jack , Futreal Andrew , Wistuba Ignacio Ivan , Zhang Jianjun ,   NSCLC TCR Study Team
Published: September 2018

11500

Background: The mechanisms underlying resistance to immune checkpoint blockade are poorly understood. Major efforts have been made to understand how mutations, through generation of neoantigens, may alter tumor immunogenicity and anti-tumor responses, particularly through T cell responses. However, the T cell repertoire and its interaction with cancers bearing specific molecular alterations have not been systemically studied. Methods: We delineated the landscapes in the T cell receptor (TCR) repertoire, immune infiltration (immunohistochemistry using multiple immune markers), genome (exome sequencing), epigenome (methylation array) and transcriptome (mRNA gene expression array) of 254 resected non-small cell lung cancers (NSCLC), matched normal lung tissues and peripheral blood mononuclear cells (PBMC). We report herein the preliminary analyses of TCR sequencing of NSCLC tumors and matched normal lung tissues. Results: We observed that: 1) Smaller tumors (smaller than median) had a higher T cell infiltrate (p = 0.0016) and higher entropy than larger tumors (p = 0.0098); 2) Tumors from ever/current smokers had higher T cell clonality than former/never-smokers (p = 0.005); 3) TCR clonality was positively correlated with mutational burden (p = 0.0026); 4) Compared to tumors, normal lung tissues demonstrated significantly less T cell infiltration (p = 2.1x10^-11), but a significantly higher clonality (p = 3.9x10^-7). Finally, many T cell clones, including major clones were shared between normal lung tissues and matched NSCLC tumors. Conclusions: Our preliminary data demonstrate the distinct immune microenvironment in different NSCLC tumors may be associated with particular clinicopathological features. The higher TCR clonality in normal lung tissues and overlap of T cell clones between normal lung and NSCLC tumors implies a significant proportion of tumor infiltrating T cells may be a function of constant exposure to mutagens rather than an anti-tumor response. Analysis of the peripheral TCR repertoire, the molecular landscape of these tumors and the association with immune profiling is underway.
Dissecting primary resistance to anti-EGFRs in RAS and BRAF wt metastatic colorectal cancer (mCRC): A case-control study.
Authors: Cremolini Chiara , Morano Federica , Moretto Roberto , Berenato Rosa , Tamborini Elena , Perrone Federica , Rossini Daniele , Gloghini Annunziata , Busico Adele , Zucchelli Gemma , Baratelli Chiara , Tamburini Emiliano , Capone Iolanda , Volpi Chiara , Milione Massimo , Di Maio Massimo , Fontanini Gabriella , De Braud Filippo G. , Falcone Alfredo , Pietrantonio Filippo
Published: September 2018

11508

Background: Almost half of RAS and BRAFwt mCRC patients do not respond to anti-EGFRs. Different molecular alterations suggested as predictors of resistance have not been validated. Methods: We conducted a case-control study to prospectively demonstrate the negative predictive impact of HER-2 amplification or mutations (mut), MET amplification, NTRK/ROS1/ALK/RET rearrangements, and mut activating MAPKs or PI3K/Akt axis. Patients with RAS and BRAFwt mCRC clearly resistant (cases) or clearly sensitive (controls) to anti-EGFRs were selected. Hypothesizing a prevalence of candidate alterations of 0% and 15% among controls and cases, 47 cases and 47 controls were needed to be able to reject the null hypothesis of equally prevalent alterations, with a- and b- error 0.05 and 0.20. Since hypermutated tumors may hardly rely on a single pathway for their growth, we also evaluated the impact of microsatellite instability. Results: 47 cases and 47 controls were included. Primary endpoint was met: mentioned alterations were reported in 20 (42.6%) cases and 1 (2.1%) control (p<0.001). MSI-high was significantly more frequent among resistant than sensitive tumos (15% vs 0%, p<0.001). Conclusions: This is the first prospective demonstration that the combined assessment of these rare alterations allows to better select patients for anti-EGFRs, while opening the way to other tailored therapies. [Table: see text]
Genomic analysis of circulating tumor DNA in 442 patients with carcinoma of unknown primary: Implications for targeted therapeutics.
Authors: Kato Shumei , Krishnamurthy Nithya , Banks Kimberly , Williams Kirstin Anne , Williams Casey B. , Leyland-Jones Brian , Lanman Richard B. , Kurzrock Razelle
Published: September 2018

11511

Background: Carcinoma of unknown primary (CUP) is a rare, difficult-to-treat malignancy. To further understand the genomic landscape of CUP, next-generation sequencing (NGS) of circulating tumor DNA (ctDNA) from patient plasma was performed. To our knowledge, this is the largest cohort of patients with CUP interrogated by liquid biopsy. Methods: We evaluated the molecular alterations of 442 patients with CUP using clinical-grade NGS of ctDNA isolated from patient plasma. The test detects single nucleotide variants in 54-70 genes, as well as copy number amplifications, fusions and indels in selected genes. Results: Eighty percent of patients (353/442) had ctDNA alterations with 66% (290/442) harboring at least one characterized alteration; 43.9% (194/442) ≥ 2 characterized alterations. TP53-associated genes were most commonly altered (37.8% [167/442]) followed by genes involved in the MAPK pathway (31.2% [138/442]), PI3K signaling (18.1% [80/442]) and the cell cycle machinery (10.4% [46/442]). Among patients harboring at least one characterized alteration, most (87.9% [255/290]) had distinct genomic profiles, and 99.7% (289/290) had alterations theoretically targetable with either an FDA-approved or investigational agent. The mean number of potentially actionable alterations per patient was 1.7 (range, 0 to 10). Illustrative patients who had dynamic changes in ctDNA content during the course of therapy and responding patients matched on the basis of ctDNA to targeted therapies or immunotherapy will be presented. Conclusions: Evaluation of ctDNA was feasible among individuals with CUP. Most patients harbored a unique somatic profile. The majority of patients had potentially actionable alterations. Serial ctDNA showed dynamic changes in molecular alterations in response to therapy, and several patients have attained responses to targeted or immune therapies chosen on the basis of ctDNA findings. The current report suggests that non-invasive liquid biopsies merit investigation in next generation clinical trials.
Whole exome analysis of patients (pts) with metastatic GIST (mGIST) demonstrating exceptional survival with imatinib (IM) therapy compared to those with short term benefit.
Authors: Ben Ami Eytan , Kamran Sophia C. , George Suzanne , Morgan Jeffrey A. , Wagner Andrew J. , Merriam Priscilla , Thornton Katherine Anne , Jennings Julia , Field Julie , Solomon Sarah , Raut Chandrajit P. , Van Allen Eliezer Mendel , Demetri George D.
Published: September 2018

11513

Background: Most patients with mGIST initially benefit from IM therapy with durable disease control (DC), i.e. objective responses and stable disease, with median duration of approximately two years. We reported exceptional long-term benefit (LTB) with DC and overall survival (OS) >14 years in a subset of mGIST pts treated with IM. We aimed to characterize tumor and normal genomes of exceptional LTB pts treated with IM and compare with short-term benefit (STB) pts. Methods: Among 87 mGIST pts enrolled between July 2000 and June 2001 in the B2222 trial of IM and followed prospectively at the Dana Farber Cancer Institute, we identified 10 LTB (>14 years of DC) pts, and 6 STB (<2 years of DC) pts on IM. Targeted genotyping ( KIT/PDGFRA)was performed in all tumors (n=16). Whole exome sequencing (WES) was performed on archival FFPE tumor samples from LTB and STB pts prior to any IM treatment. We compared WES results from LTB with STB pts to identify unique features of long-term DC and OS with IM. Results: KIT mutation in LTB pts were as follows: exon 11(6 pts), exon 9 (3 pts), and SDH-deficient with KIT/PDGFRA wild type (1 pt). In STB pts, mutated KITwas found 4 pts (exon 11) and 2 pts (exon 9). WES was successful in six LTB (five exon 11, one exon 9) and three STB (two exon 11, one exon 9) pts. A total of 1211 somatic mutations were observed (546 missense, 37 nonsense, 256 silent, 285 indels, 36 splice mutations). The mean somatic mutational burden was 3.42 mutations/Mb (range 1.18-4.93) and 3.34 mutations/Mb (range 1.06-6.68) among LTB and STB, respectively. Genes mutated in LTB but not in STB were MUC7 (4 pts), H1F0 (3 pts), ZKSCAN1 (3 pts), SLC24A1 (3 pts) and USP4 (2 pts). Conclusions: KRAB domain containing zinc finger (KRAB-ZNF) gene expression signatures have been associated with prediction of response to IM, and a possible role in response modulation to tyrosine kinase inhibitors in GIST. We found variants in ZKSCAN1, a gene encoding a transcriptional regulator of the KRAB subfamily of zinc finger, to be present in LTB but not in STB. KRAB-ZNF family of genes may be linked to LTB and exceptional survival with IM in mGIST; functional analyses will be important to test such hypotheses.
Monitoring of circulating tumor DNA in non-small cell lung cancer patients treated with EGFR-inhibitors.
Authors: Verheijen Remy B , van Duijl Tirsa T , van den Heuvel Michel M , Beijnen Jos H. , Schellens Jan H.M. , van den Broek Daan , Steeghs Neeltje , Huitema Alwin
Published: September 2018

11535

Background: Epidermal growth factor receptor (EGFR) inhibitors such as erlotinib and gefitinib are routinely used in the treatment of non-small cell lung cancer (NSCLC). Monitoring of EGFR mutations in circulating tumor DNA (ctDNA) derived from plasma has been proposed as an alternative for repeated tumor biopsies. Our aim was to investigate the dynamics of ctDNA in a cohort of NSCLC patients and explore the roles of EGFR driver and resistance mutations in predicting disease progression and progression free survival (PFS). Methods: NSCLC patients treated with either erlotinib or gefitinib as first-line anti-EGFR therapy were included. Clinical data was collected retrospectively from medical records. Plasma samples collected as part of routine care were analyzed. First DNA was isolated from plasma using the QIAsymphony SP (Qiagen). Then EGFR driver (L858R and exon 19 deletions) and resistance (T790M) mutations were quantified using the X100 Droplet Digital PCR and analyzed using QuantaSoft software (Bio-Rad). The dynamics of ctDNA mutations over time and the relationship between copy numbers and progression free survival were explored. Results: 68 NSCLC patients and 249 plasma samples (1-13 per patient) were included in the analysis. In 33 patients, the T790M mutation was detected. The median (range) T790M concentration in these samples was of 7.3 (5.1 - 3688.7) copies/mL. In 30 patients, the L858R or exon 19 deletion driver mutations were found in median concentrations of 11.7 (5.1 – 12393.3) and 27.9 (5.9 – 2896.7) copies/mL, respectively. Using local polynomial regression, the copies/mL of EGFR driver mutations increased several weeks prior to progression on standard response evaluation. In Kaplan-Meier analysis, patients with a detectable T790M mutation during the first 8 weeks of treatment had a shorter PFS (7.6 versus 14.4 months, p < 0.01, log-rank test). Conclusions: Early detection of the T790M mutation in plasma ctDNA is related to poor PFS. Furthermore, an increase in the copies/mL of the EGFR driver mutation over time may predict clinical progression.
Liquid biopsy in the clinic: A prospective study of plasma circulating tumor DNA (ctDNA) next generation sequencing (NGS) in patients with advanced non-small cell lung cancers to match targeted therapy.
Authors: Sabari Joshua K. , Ni Ai , Lee Adrian , Pavlakis Nick , Clarke Stephen John , Tandon Nidhi , Datta Sutirtha , DuBoff Mariel A. , Martinez Andres , Offin Michael D. , Isbell James M. , Rusch Valerie W. , Jones David Randolph , Henderson Samantha , Lim Lee , Raymond Chris , Li Mark , Riely Gregory J. , Rudin Charles M. , Li Bob T.
Published: September 2018

11536

Background: Liquid biopsy for plasma ctDNA NGS is a rapidly evolving science. Plasma ctDNA assays are commercially available and are increasingly adopted in the community with a paucity of evidence-based guidance. We set out to study the optimal timing and utility of plasma ctDNA NGS in clinic. Methods: Pts with advanced NSCLC who were driver unknown, defined as not having prior tissue NGS or clinical concern for tumor heterogeneity that may affect treatment decisions, were eligible. Peripheral blood was collected in a Streck tube (10mL), DNA extracted, and subjected to a bias-corrected hybrid-capture 21 gene targeted NGS assay in a CLIA lab with unique reads at 3000x and sensitive detection at variant allele frequency above 0.1% (ResolutionBio Bellevue, WA). Pts also had concurrent tissue NGS via MSK IMPACT. Clinical endpoints included detection of oncogenic drivers in plasma, ability to match pts to targeted therapy, concordance and turnaround time of plasma and tissue NGS. Results: Forty-one pts were prospectively accrued. Plasma ctDNA detected an oncogenic driver in 39% (16/41) of pts, of whom 17% (7/41) were matched to targeted therapy; including pts matched to clinical trials for HER2 exon 20 insertionYVMA, BRAF L597Q and MET exon14. Mean turnaround time for plasma was 7 days (4-12) and 28 days (20-43) for tissue. Plasma ctDNA was detected in 56% (23/41) of pts; detection was 40% (8/20) if blood was drawn on active therapy and 71% (15/21) if drawn off therapy, either at diagnosis or progression (Odds ratio 0.28, 95% CI 0.06 - 1.16; p = 0.06). All pts had concurrent tissue NGS; of the 10 samples resulted, there was 100% driver concordance between tissue and plasma in pts drawn off therapy. Conclusions: In pts who were driver unknown or who had clinical concern for tumor heterogeneity, plasma ctDNA NGS identified a variety of oncogenic drivers with a short turnaround time and matched them to targeted therapy. Plasma ctDNA detection was more frequent at diagnosis of metastatic disease or at progression. A positive finding of an oncogenic driver in plasma is highly specific, but a negative finding may still require tissue biopsy.
Biomarker modulation in patients treated with TRC105 in combination with anti-VEGF therapy.
Authors: Liu Yingmiao , Yuan Zhenhua , Zhang Dadong , Starr Mark D. , Brady John C. , Jivani Manoj A. , Adams Bonne J. , Alvarez Delia , Attia Steven , Choueiri Toni K. , Theuer Charles P. , Owzar Kouros , Hurwitz Herbert , Nixon Andrew B.
Published: September 2018

11546

Background: TRC105, an endoglin-targeting monoclonal antibody with anti-angiogenic and anti-tumor activity, is being evaluated in multiple diseases. Here we report on pharmacodynamic and prognostic biomarkers in patients (pts) treated with both TRC105 and anti-VEGF agents. Methods: Plasma samples were collected from pts on three phase 2 trials combining TRC105 with an anti-VEGF agent: axitinib in metastatic renal cell carcinoma (mRCC), pazopanib in advanced soft tissue sarcoma, and bevacizumab in glioblastoma (GBM). Baseline and on-treatment levels of 22 soluble protein biomarkers were assessed. Results: Soluble endoglin markedly increased after TRC105 treatment in all pts ( p< 0.001) as previously reported. BMP9 (a ligand for endoglin) and TGFβR3 (a type III TGFβ receptor) decreased in sarcoma pts at Cycle 2 Day 1 (C2D1) and generally remained below baseline throughout the course of treatment (BMP9, p= 0.004; TGFβR3, p= 0.003). Although TGFβR3 was decreased at C2D1 in mRCC (p = 0.030), no clear patterns were observed over time. Overall BMP9 levels did not change in response to therapy in either mRCC or GBM. Osteopontin (OPN) levels, a downstream effector of TGFβ signaling, were increased in sarcoma pts [ p= 0.002 at C2D15, p< 0.001 at C4D1 and end of study (EOS)]; however, in mRCC ( p= 0.010) and GBM ( p= 0.003), OPN was only elevated at EOS. Increases in PlGF and VEGFD, and decreases in VEGFR2 were observed across all studies, as previously noted for VEGF inhibitors. In the mRCC trial, 5 of 18 pts exhibited a ≥30% tumor reduction. Lower OPN ( p= 0.026) and higher TGFβR3 ( p= 0.003) levels at baseline correlated with radiographic response to treatment. In the sarcoma trial, 6 of 19 pts responded (CHOI criteria) in which lower baseline levels of ICAM1 ( p= 0.018) and TSP2 ( p= 0.042) correlated with stable disease. Conclusions: In these trials, increases of soluble endoglin in response to TRC105 were observed, independent of the presence of any specific VEGF inhibitor. Differential regulation of BMP9, TGFβR3, and OPN suggests potential disease-specific modulation of key TGFβ signaling molecules in response to dual therapy. Baseline levels of OPN and TGFβR3 showed potential prognostic value in mRCC. Confirmation in larger trials is needed.
Liquid biopsies of plasma exosomal nucleic acids, plasma cell-free DNA, and survival of patients with advanced cancers.
Authors: Moehrmann Lino , Huang Helen J. , Hong David S. , Tsimberidou Apostolia Maria , Fu Siqing , Piha-Paul Sarina Anne , Subbiah Vivek , Karp Daniel D. , Naing Aung , Krug Anne , Enderle Daniel , Priewasser Tina , Noerholm Mikkel , Eng Cathy , Kopetz Scott , Skog Johan , Meric-Bernstam Funda , Janku Filip
Published: September 2018

11551

Background: Blood-based liquid biopsies offer easy accessible genomic material for molecular diagnostics in cancer. Commonly used cell-free DNA (cfDNA) originates from dying cells. In contrast exosomal nucleic acid (exoNA) originates from living cells, which can better reflect underlying cancer biology. Methods: We isolated exoNA (EXO52) and cfDNA (QIAamp Circulating Nucleic Acid kit) from plasma of patients with progressing advanced cancers and tested for BRAFV600, KRASG12/G13, and EGFRexon19del/L858R mutations using next-generation sequencing (EXO1000), droplet digital PCR (ddPCR, QX200) and BEAMing digital PCR. The results were compared to clinical testing of archival tumor tissue and correlated with survival. Results: Of the 43 patients (colorectal cancer, 20; melanoma, 8; non-small cell lung cancer, 6; ovarian cancer, 2; papillary thyroid cancer, 2; other cancers, 5) 41 had a mutation in the tumor tissue (20 [47%] BRAF mutation, 17 [40%] KRAS mutation and 4 [9%] EGFR mutation). Mutation testing of plasma exoNA from all 43 patients detected 39 (95%) of 41 mutations present in tumor tissue with 100% specificity. Mutation testing of plasma cfDNA from 39 patients using ddPCR detected 33 (89%) of 37 mutations present in tumor and testing of plasma cfDNA from 37 patients using BEAMing detected 34 (97%) of 35 mutations present in tumor tissue; however, both cfDNA methods reported an additional KRAS mutation not present in tumor tissue. Patients with high mutation allele frequency (MAF, > median) had shorter median survival compared to patients with low MAF ( < median) when using exoNA (5.9 vs. 11.8 months, P= 0.006), but not cfDNA ddPCR (6.0 vs. 7.4 months, P= 0.06) or cfDNA BEAMing (6.5 vs. 7.4 month, P= 0.07). High MAF in exoNA was an independent prognostic factor for survival in multicovariate analysis (HR 0.13, P= 0.017). Conclusions: Mutation testing of plasma exoNA for common BRAF, KRAS, and EGFR mutations has high sensitivity compared to clinical testing of archival tumor tissue and better specificity than PCR testing of plasma cfDNA. High MAF in exoNA is the independent prognostic factor for shorter survival.
Tetrahydrouridine-decitabine for non-cytotoxic epigenetic therapy of NSCLC to enhance immunotherapeutic effect of anti-PD1 in vivo.
Authors: Kang Kai , Schrump David S. , Thomas Anish , Schalper Kurt Alex , Saunthararajah Yogen , Velcheti Vamsidhar
Published: September 2018

11552

Background: NSCLC response to anti-PD1 therapy is ~20%, largely because most NSCLC avoids immune-recognition in the 1stplace, e.g., by epigenetics to suppress neo-antigen expression. DNA methyltransferase (DNMT1) mediates this repression and is depleted by decitabine (Dec). Unfortunately Dec has trivial distribution into solid cancer tissues because of rapid deamination by cytidine deaminase (CDA). Therefore, to improve tissue-distribution of Dec with the low Cmax/long Tmax profile needed for DNMT1-depletion without cytotoxicity, we combined Dec with a CDA inhibitor tetrahydrouridine (THU). Methods: C57/BL6 mice were inoculated with LL3-luc cells via tail vein. After documentation of lung invasion by live-imaging, mice (n = 5/group) were randomized to PBS, THU-Dec (10/0.1 mg/kg sc 3×/wk), anti-PD1 (5 mg/kg ip q5d, DX400 from Merck) or THU-Dec/anti-PD1 combination. Antigen presentation, PD, MDSCs, and T-cells were measured in blood and tumor. Results: THU-Dec or anti-PD1 alone decreased tumor by imaging and increased survival, however, THU-Dec/anti-PD1 combination extended median survival the most and completely regressed tumor in 2/5 mice (median survival days PBS 37, THU-Dec 56, anti-PD1 62, THU-Dec/anti-PD1 77). Rechallenge of cured mice with LL2-luc confirmed immune-memory effect, with no engraftment vs expected engraftment in controls. Consistent with the pharmacologic rationale, THU-Dec produced > 2-fold more DNMT1-depletion in tumor vs PBS. Consistent with non-cytotoxic effect, absolute lymphocyte counts were preserved with THU-Dec, while numbers of G-MDSC decreased (2.9 k/µL PBS vs 0.3 k/µL THU-Dec/anti-PD1, p < 0.01). Expression of neoantigens MAGE-A1 and MAGE-A3 increased > 4-fold with THU-Dec vs PBS (p < 0.01). THU-Dec/anti-PD1 increased tumor infiltrating lymphocytes 6-fold vs PBS (p < 0.01) and decreased regulatory T-cells 2.5-fold vs PBS (p < 0.01). IFNγ expression in tumor increased 2.4-fold with THU-Dec/anti-PD1 vs PBS (p < 0.01). Conclusions: THU-Dec/anti-PD1 produced marked survival improvements and cures in tumor-bearing mice, scientific validation of our clinical trial NCT02664181 combining THU-Dec/nivolumab in 2nd line for NSCLC.
Fatty-acid-binding proteins as a novel target for the treatment of anti-PD-1-resistant tumors.
Authors: Welsh James William , Niknam Sharareh , Schoenhals Jonathan E. , Barsoumian Hampartsoum B. , Younes Ahmed I. A. , Li Ailin , Cortez Maria Angelica
Published: September 2018

11562

Background: The mechanisms underlying immunosuppression and resistance to PD1 inhibitors in cancer are not well understood. We attempted to fill this gap with an integrated transcriptome analysis in an anti-PD1-resistant lung adenocarcinoma mouse model. The model was created by in vivo passage of 344SQ murine lung cancer cells (p53R172HΔg/+K-rasLA1/+) in a syngeneic host repeatedly dosed with anti-mouse PD1 antibodies. Anti-PD1-resistant 344SQ (344SQ_R) and 344SQ parental (344SQ_P) cells were then inoculated into syngeneic 129Sv/ev mice, which were then dosed twice with anti-PD1 or control IgG antibodies. Methods: Tumor tissues were collected and analyzed as follows: transcriptome with Affymetrix; protein levels by reverse phase protein array analysis; signature enrichment by gene set enrichment analysis; metabolome by mass spectrometry; and lipid content with fluorescent probes Oil O and BODIPY. We also isolated tumor-infiltrating immune cells for flow cytometry and gene expression analyses. Results: We identified lipid-related metabolic pathways as being the most highly enriched in anti-PD1-resistant tumors (344SQ_R) vs. their 344SQ_P counterparts; the resistant cells also had more lipid droplets than the 344SQ_P cells. The anti-PD1-resistant tumors overexpressed several genes involved in lipogenesis and fatty acid pathways, including fatty acid binding proteins (FABPs). Specifically, FABP overexpression promoted fatty acid uptake and lipid-droplet accumulation in resistant tumors. 344SQ_R tumors promoted immune suppressive cells by upregulating FABPs expression in M2-like macrophages, marked by increased fatty acid intake and fatty acid oxidation. Conversely, percentages of CD4+ and CD8+ tumor-infiltrating lymphocytes were reduced in the resistant tumors. Conclusions: These results suggest that lipid metabolic rewiring drives resistance PD1 inhibitors supporting the accumulation of immunosuppressive cells, including M2-like macrophages, preventing type I immune responses elicited by T cells. Collectively, these findings reveal new potential lipid-related targets for drug development or new treatments combining inhibitors of these targets with anti-PD1 therapy.
FDG PET/CT and N0 mediastinal nodal status in early stage non-small cell lung cancer: A single institution retrospective experience.
Authors: Cingam Shashank Reddy , Connor Sean , Patel Abhishek Harshad , Master Samip R. , Takalkar Amol
Published: September 2018

11570

Background: The increasing use of CT and now low-dose screening CT scans for at-risk patients have led to increasing detection of lung cancer at early stages. FDG PET/CT is used as an adjunct to conventional imaging to assess loco-regional lymph node spread. However, there is a potential for false-negative results, especially in smaller lesions or early nodal involvement. The main objective of this study was to study the value of PET/CT scan to evaluate for true negative mediastinal nodes in patients with early stage NSCLC. Accurate determination of N0 status can have a significant impact on the cost-effectiveness and timely management of early stage NSCLC. Methods: Of a total of 404 patients with NSCLC managed at our facility between 2008 to 2015, 29 adult patients whose PET scan showed no or equivocal mediastinal nodal involvement and subsequently underwent surgical exploration of mediastinal lymph nodes were included in the study. Data variables that were collected included the cancer site, date of PET, node status on PET, type and date of surgery, cancer histology, and the tissue diagnosis of the sampled nodes. SAS software was used for the analysis of the data. Results: Of the 29 patients with N0 or equivocal nodes on FDG PET/CT, 7 (24.13%) had evidence of malignancy on biopsy of the surgically resected lymph nodes. No statistically significant differences were noted between the site of the neoplastic lesion, cancer histology, duration between the date of PET and the date of surgery in the true negative (TN) and false negative (FN) groups. The recurrence rate was higher in the FN group 60 % (3/5) compared to 21 % (4/19) in the TN group who had follow up for atleast 2 years. Conclusions: Our findings suggest incidence of false negative results of FDG PET/CT for evaluation of N0 nodes in early stage NSCLC is 24.13%. This is comparatively higher than false negatives rates with mediastinoscopy (5-10%) reported in other studies. Although our sample size is small, if confirmed, such a relatively high incidence of false negative results on FDG PET/CT for N0 disease supports the current recommendations for exploratory mediastinoscopy and/or surgery for definitive staging in early stage NSCLC.
Clinical utility of molecular testing to select therapy in relapsed/refractory non-Hodgkin lymphoma: Mayo Clinic Center for Individualized Medicine experience.
Authors: Bennani Nabila Nora , Ansell Stephen Maxted , Witzig Thomas E. , Feldman Andew L. , McAllister Tammy M , Lazaridis Konstantinos N , Egan Jan B. , Stewart A. Keith , Nowakowski Grzegorz S.
Published: September 2018

11571

Background: Relapsed/refractory (R/R) non-Hodgkin lymphomas (NHL) have a poor prognosis with limited treatment options. Our expanding knowledge of molecular alterations seen in R/R NHL allows identification of patients that potentially may benefit from a precision medicine approach. However, experience in routine clinical implementation of precision medicine has been limited. Here, we summarize our clinical experience in molecular characterization of RR NHL targeted therapy (TT) using next-generation sequencing (NGS), and selection of targeted therapy (TT) based on molecular profile. Methods: We conducted a prospective study in RR NHL through the Center for Individualized Medicine at Mayo Clinic. Consenting patients underwent NGS using FoundationOne Heme panel from biopsies done at time of relapse. Results of NGS were discussed at the Genomic Tumor Board and recommendations for TT were given based on matching specific molecular alteration(s) with potential agent(s) predicted to be active based on NGS. The agents could include FDA-approved, off-label use and clinical trial therapies. Results: 28 cases were enrolled: 18 aggressive NHL, 10 follicular lymphoma (FL). Molecular alterations were present in all cases. In aggressive B-cell NHL, CDKN2A/B gene cluster alterations were seen in 73% (8/11), while seen in only 1/7 T-cell lymphomas (TCL), and 1/10 FL. TP53 deletions were second most common genomic alterations in DLBCL (57%) and seen in 40% FL. JAK-STAT and ERBB pathways were altered in TCL (2/7 each). IGH-BCl-2 gene rearrangement were common in FL (70%), followed by MLL gene alterations (50%). Targetable mutations were present in 86% (24/28) of cases. A TT was recommended in all 24 cases, but received by 2 patients only. Remaining patients did not due to benefit from current therapy (10/24), ineligibility or lack of clinical trial (7/24) or interim clinical deterioration (5/24). Conclusions: Targetable mutations were identified in most cases of RR NHL with TT recommended for all cases. However, access to TT limits potential clinical benefit of molecular-based matching strategy. More studies are needed to assess impact on clinical outcomes.
Antitumor activity of indenoisoquinoline inhibitors of topoisomerase 1 (TOP1) via apoptosis and autophagocytosis pathways in animal models.
Authors: Kinders Robert J. , Dull Angie B , Wilsker Deborah , LeBlanc Amy , Mazcko Christina , Hollingshead Melinda G. , Parchment Ralph E. , Doroshow James H.
Published: September 2018

11588

Background: We performed pharmacodynamic biomarker analysis for response to a panel of three indenoisoquinolines (LMP776, LMP400 and NSC706744, J. Med. Chem. 49:7740, 2006) that have demonstrated anti-tumor activity in dogs. In preclinical xenograft models treated with indenoisoquinolines, we observed that gH2AX was not a useful biomarker for the biological activity of compound 706744, but was a reasonable biomarker of drug activity (Clin. Canc. Res. 16:5447, 2010) for the other two compounds, even though in vitro data indicated that all 3 compounds inhibited TOP1 and killed tumor cells. It has been reported that irinotecan activates autophagy (Pharm. Rev. 65:1162, 2013) which correlated with its metabolism to SN-38; we therefore developed, validated and tested an immunofluorescence microscopy assay for LC3 as a marker of autophagy. Methods: Assays were developed to evaluate apoptosis by co-localization of cleaved caspase 3 and gH2Ax, and autophagy by LC3 immunofluorescence on formalin fixed, paraffin-embedded tissue sections of xenografts models or lymphomas from outbred dogs. Percent positive cells containing LC3 puncta were quantitated using a spot morphology algorithm. Analysis of gH2AX and cleaved caspase 3 cellular co-localization was developed using a blebbing morphology algorithm (Definiens). Results: LC3 reported that indenoisoquinoline 706744 activates autophagy in vitro with the absence of cleaved caspase 3-dependent apoptosis while the -776 and -400 compounds do not activate autophagy, but instead demonstrate apoptosis in response to drug treatment. Results in animal models confirmed that both autophagy and apoptosis were active. Clinical readiness of the assays was confirmed on canine biopsy FFPE slides. Conclusions: 1,Structurally-related TOP1 inhibitors may trigger alternative pathways of cell destruction; 2,Autophagy may report drug anti-tumor activity or tumor drug resistance according to current literature. This assay may be useful for determination of pharmacodynamic pathways associated with anti-tumor activity to elucidate mechanism of action of investigational agents used in clinical trials.
Validation of an expanded neoantigen identification platform for therapeutic and diagnostic use in immuno-oncology.
Authors: Boyle Sean Michael , Harris Jason , Bartha Gabor , Alla Ravi , Jongeneel Patrick , Karbelashvili Mirian , Kirk Scott , Chervitz Steve , Levy Eric , Rowell Craig , Thomas Daryl , McCord Robert , Luo Shujun , West John , Chen Richard
Published: September 2018

11589

Background: Neoantigen identification is increasingly critical for clinical immuno-oncology applications including predicting immunotherapy response and neoantigen-based personalized cancer vaccines. Although standard research pipelines have been developed to aid neoantigen identification, building a robust, validated neoantigen identification platform suitable for clinical applications has been challenging due to the complex processes involved. Methods: To improve neoantigen identification, we extended standard sequencing and informatics methods. We developed an augmented and content enhanced (ACE) exome sequenced at 200X to increase sensitivity to SNPs and indels used for neoantigen identification as well as HLA performance. To accurately identify fusions and variants from RNA, we optimized our ACE transcriptome for FFPE tissue. To improve neoantigen pipelines based on MHC binding algorithms, we developed peptide phasing, high accuracy HLA typing, TCR interaction predictors, and transcript isoform estimation tools to detect neoantigens from indel and fusion events. We performed comprehensive analytical validation of the platform including the ACE Exome, somatic SNV/indel calls, RNA based variant and fusion calls, and HLA typing. This was followed by an overall in silico validation of neoantigen identification using 23 experimentally validated immunogenic neoepitopes spiked into exome data. Results: Analytical validation of our ACE exome platform showed > 97% sensitivity for small variants with a specificity of > 98% at minor allele frequency > 10%. From the ACE transcriptome we achieved a fusion sensitivity of > 99% and RNA based variant calls sensitivity of > 97%. Our ACE exome based HLA typing was 98% and 95% concordant with Class I and II HLA results (respectively) from clinical testing. Our in silico validation of neoantigen predictions resulted in identification of 22 out of 23 immunogenic neoepitopes. Conclusions: We developed sequencing and informatics improvements to standard approaches that can enhance neoantigen identification and demonstrated a comprehensive validation approach that may support neoantigen use in future clinical settings.
Co-amplification of MET and PIK3CA in NSCLC and data on a PDX mouse model.
Authors: Kang Jin , Zhang Xu-Chao , Su Jian , Xie Zhi , Li Wen-Feng , Wu Yi-Long , Yang Jin-Ji
Published: September 2018

11591

Background: Amplification of the mesenchymal-epithelial transition ( MET) proto-oncogene or phosphatidylinositol3-kinase ( PI3K) is common in non-small-cell lung cancer (NSCLC) and represents a potential therapeutic target. NSCLC with coexisting driver mutations or amplifications is a cause of great concern. Methods: From 2013 until now, fluorescence in situ hybridization was used to screen for MET amplified NSCLC patients.The amplification of the MET was defined as centromere 7 ratio ≥ 2.0 and the criterion of Cappuzzi. The amplification of the PIK3CA was copy numbers ≥ 4.0. We established the patient-derived exnograft (PDX) mouse model from a dual MET/PIK3CA-amplified patient. Preclinical efficacy of single versus dual inhibition was evaluated in vivo. Six groups were allocated to receive the treatment of vehicle control, bozitinib,crizotinib, taselisib (PI3K inhibitor), bozitinib+taselisib, or crizotinib+taselisib, respectively. Results: Totally, 568 (568/2321, 24.47%) patients harbored positive MET amplification and 6 (6/568,1%) were comfirmed with dual MET/PI3K amplification. The two stageⅣ patients received MET inhibitor treatment.One trial (NCT02896231) patient was treated with bozitinib and achieved confirmed PR, but with 3 months PFS and 5 months OS. The best response was PR and PFS was 5.6 months for the other one receiving the study drug capmatinib (NCT02276027). In the PDX mouse model experiment, we found three single-anget inhibitors monotherapy to be active but only transiently effective in controlling the growth of PDX. The PDX models showed more sensitivity to taselisib among the three single-anget groups. In contrast, the combination of the two inhibitors caused a stronger and long-lasting growth inhibition in PDX models. The addition of taselisib to bozitinib or crizotinib monotherapy provided obvious enhanced activity. Regretfully, two mice died because of the toxicities in the crizotinib+taselisib group. Conclusions: Patients with dual MET/PIK3CA amplification represent a rare molecular subtype of NSCLC and have a relatively short duration of response to MET inhibitors.The combination of MET/PI3K inhibitors is synergistic preclinically.
ABCG2 and TOP-1 mRNA expression as predictive biomarkers for adjuvant FOLFIRI treatment in stage III colon cancer patients: Results from the PETAAC-3 prospective randomized clinical trial.
Authors: Brunner Nils , Stenvang Jan , Budinska Eva , Nygaard Sune Boris
Published: September 2018

11594

Background: FOLFIRI as adjuvant treatment in primary colon cancer was previously tested in two pivotal prospective randomized clinical trials (PETACC-3 and CALGB 89803), both of which failed to demonstrate significant beneficial effects when adding irinotecan to 5FU. As a consequence, FOLFIRI is presently not used as adjuvant treatment for colon cancer. Methods: The study included 580 patients with mRNA expression data performed on tumor samples (FFPE) from stage III colon cancer patients enrolled in the PETACC-3 study, which randomized the patients to 5FU plus Leucovorin +/- irinotecan. Primary end-points were recurrence-free survival (RFS) and overall survival (OS). Median ABCG2 and the 75 percentile TOP-1 mRNA expression data were used to allocate the patients into one of two groups: One with high ABCG2 expression (above median) and low TOP-1 expression (below 75 percentile) (n = 167) and another group including all other combinations of these two genes. Kaplan Meier curves and Cox proportional hazards model were used to visualize differences between groups and calculate p-values (log-rank test). Results: The survival statistics showed a significant difference for both RFS (HR: 0.63 (0.44-0.92); p = 0.017) and OS (HR: 0.6 (0.39-0.93); p = 0.021) between the two groups when the patients received FOLFIRI. In contrast, no significant differences were observed between the groups when patients received 5FU and Leucovorin alone (p-values: RFS: 0.58; OS: 0.75). Conclusions: We here show that the combination of two independent gene expression abundance with a strong association to irinotecan treatment (high ABCG2 drug efflux pump and low TOP-1, the latter being the target for irinotecan) identified a group of stage III colon cancer patients who will not benefit from FOLFIRI adjuvant treatment while patients with other combinations of expression of these two genes appear to significantly benefit from adjuvant FOLFIRI treatment. The lack of a similar effect in patients receiving treatment with 5FU and Leucovorin only, points to a predictive value of ABCG2 and TOP-1 measurements.
Biomarker-driven indication selection in JTX-2011 ICONIC clinical trial.
Authors: Hirsch Heather Anne , Reeves Jason , Zi Tong , Deshpande Amit , Yang Guang , Needham Alexander , Shu Jenny , Khattri Arun , Brisson Ryan J. , Tan Carol , Harvey Christopher , Law Deborah , Michaelson Jennifer , Trehu Elizabeth G. , Sathyanarayanan Sriram , Seiwert Tanguy Y.
Published: September 2018

11602

Background: ICOS (Inducible T cell CO-Stimulator) is a co-stimulatory molecule expressed primarily on T lymphocytes. Clinical and preclinical data suggest that ICOS mediates anti-CTLA-4 driven anti-tumor responses. JTX-2011 is an ICOS agonist antibody in clinical development in advanced solid tumors (ICONIC trial). JTX-2011 is designed to generate an anti-tumor immune response via stimulation of T effector cells and preferential reduction of intra-tumoral T regulatory cells. Single agent preclinical efficacy correlates with the percentage of ICOS-expressing T cells within the tumor. We report indication selection and patient enrichment strategy for ICONIC using in silico and IHC analysis and assessment of potential predictive biomarkers for JTX-2011 using ex vivo tumor histoculture. Methods: Integrated analysis was performed from the TCGA for ICOS expression in histologic and molecularly defined tumors and immune cell signatures. ICOS expression was analyzed by IHC in a subset of indications based on in silico analysis. ICOS expression on intra-tumoral Tregs and PD-L1 were analyzed in a cohort of 126 head and neck squamous cell carcinomas (HNSCC). Ex vivo histoculture assays of human HNSCC was treated with JTX-2011 and assessed for IFNg gene signature induction. Results: ICOS mRNA expression was analyzed in ~10,000 solid tumors samples across ~30 indications. ICOS expression in key indications was confirmed using IHC. Based on frequency of high ICOS expression, non-small cell lung cancer, HNSCC, triple negative breast carcinoma, gastric cancer, and melanoma were selected as indications for ICONIC. Results were confirmed in clinical samples using multiplex immunofluorescence and ICOS IHC. A wide range of ICOS expression was observed suggesting that identification of an ICOS “high” group may enrich for patients likely to benefit from ICOS agonist therapy. In ex vivo histoculture assays of human HNSCC tumors treated with JTX-2011, ICOS IHC and ICOS RNA gene signatures correlated to response endpoints. Comparison of ICOS and PDL1 expression identified subsets of tumors in multiple indications with high ICOS but low PDL1 expression. Conclusions: These data support prioritization of specific tumor types the ICONIC trial.
A technical feasibility report on correlative studies from the investigator-initiated phase II study of pembrolizumab (Pembro) immunological response evaluation (INSPIRE).
Authors: Clouthier Derek L. , Yang Cindy , Lien Scott , Gray Diana , Colombo Ilaria , Lheureux Stephanie , Lewin Jeremy Howard , Abdul Razak Albiruni Ryan , Spreafico Anna , Bedard Philippe L. , Butler Marcus O. , Day Daphne , Hansen Aaron Richard , Pugh Trevor John , Chow Helen , Kobori Sawako , Giesler Amanda , Ohashi Pamela S , Siu Lillian L.
Published: September 2018

11607

Background: Validated biomarkers of response to immune checkpoint inhibitors are needed. Methods: INSPIRE (NCT02644369) is a biomarker-driven study to comprehensively evaluate changes in genomic and immune landscapes in tumors and blood of patients (pts) treated with pembro at 200 mg IV Q3W. It consists of 5 histological cohorts of 20 evaluable pts each: head and neck squamous cell cancer (SCCHN), triple negative breast cancer (TNBC), high grade serous ovarian cancer (HGSOC), melanoma (MM) and mixed solid tumors (MST). All pts undergo pre- and on-treatment (week 6-9) fresh tumor biopsies (bx), and at progression for responders. The first core bx is for immunohistochemistry and subsequent cores are pooled to create single cell suspension for 5 prioritized biomarker assay groups: (1) whole exome/RNA-/TCR-sequencing; (2) T/B/NK, APCs, and/or Treg phenotyping; (3) patient-derived xenografts; (4) RNA-seq on viably sorted immune populations; (5) TIL expansion and characterization. Serial blood samples for immunophenotyping, chemokines/cytokines and ctDNA are collected. Results: 53 pts were enrolled from March 21, 2016-January 16, 2017 (5 SCCHN, 8 TNBC, 17 HGSOC, 7 MM, 16 MST). 84 tumor bx (53 pre-, 30 on-treatment, 1 progression) and 244 blood-based biomarker samples have been collected. The most common sites of tumor bx were: lymph nodes (27%), liver (22%) and skin (14%) (see table). For the 5 cohorts, the % of tumor bx with sufficient cellularity for biomarker assay groups 1 and 2 are: SCCHN (33%), TNBC (9%), HGSOC (52%), MM (55%), MST (55%). Conclusions: This report provides robust technical feasibility data to plan immune and molecular characterization of tumor and blood-based biomarkers in pts receiving ICI. Clinical trial information: NCT02644369. [Table: see text]
Simultaneous molecular alterations in solid tumors with IDH1 or IDH2 mutations.
Authors: Janku Filip , De Groot John Frederick , Javle Milind M. , Penas-Prado Marta , Shroff Rachna T. , Luthra Rajyalakshmi , Banks Kimberly , Lanman Richard B. , Conley Anthony Paul , Broaddus Russell , Davies Michael A. , Kopetz Scott , Yung W. K. Alfred , Heymach John , Fu Siqing , Shaw Kenna Rael , Meric-Bernstam Funda
Published: September 2018

11609

Background: Somatic mutations in IDH1 or IDH2 genes are prevalent in diverse solid tumors and implicated in tumorigenesis. Therapeutic responses to IDH inhibitors are infrequent plausibly due to presence of simultaneous alterations activating compensatory molecular pathways. Methods: We retrospectively reviewed results from clinical genomic profiling with targeted next-generation sequencing in two independent data sets of archival formalin-fixed paraffin-embedded (FFPE) tumor samples (Ion Torrent < 50 genes, 300; FoundationOne < 343 genes, 30; Oncomine 128 genes, 4) and plasma liquid biopsies (Guardant360 < 73 genes panel, 337 samples) from patients with solid tumors of all stages. Results: In 334 FFPE samples the most represented cancers were gliomas (50%), melanomas (14%), cholangiocarcinomas (11%), and non-small cell lung cancer (NSCLC, 6%). In 296 IDH1-mutated FFPE samples the most frequent simultaneous alterations were in TP53 (45%), BRAF (11%), KRAS (9%), and PIK3CA (26, 9%). In the most represented IDH1-mutated tumor types commonly altered genes were TP53 (66%) in gliomas, BRAF (53% [V600K/R 30%, V600E 19%]) in melanomas, PIK3CA (13%), CDKN2A (13%) in cholangiocarcinomas and KRAS (65%) in NSCLC. In 38 IDH2-mutated FFPE samples the most frequent simultaneous alterations were in TP53 (32%), and KRAS (16%). In 337 plasma samples the most represented cancers were NSCLC (39%), cholangiocarcinoma (13%), and breast cancer (8%). In 172 IDH1-mutated plasma samples the most frequent simultaneous alterations were in TP53 (40%), KRAS (23%), EGFR (16%) and BRAF (16%). In the most represented IDH1-mutated tumors commonly altered genes were TP53 (48%), KRAS (37%) in NSCLC and TP53 (37%), KRAS (24%), BRAF (18%) in cholangiocarcinoma. In 161 IDH2-mutated plasma samples the most frequent simultaneous alterations were in TP53 (43%), EGFR (20%), and KRAS (19%). In the most represented IDH2-mutated tumors commonly altered genes were TP53 (43%), EGFR (32%), KRAS (18%) in NSCLC and TP53 (18%), ESR1 (18%), KRAS (18%) in breast cancer. There were 4 tumors with IDH1 and IDH2mutations. Conclusions: IDH1 and IDH2 mutations often coexist with simultaneous oncogenic alterations including these in potentially druggable molecular targets.