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L.E. Raez

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    OA02 - Novel Targets and Biomarkers in Malignant Pleural Mesothelioma (ID 369)

    • Event: WCLC 2016
    • Type: Oral Session
    • Track: Mesothelioma/Thymic Malignancies/Esophageal Cancer/Other Thoracic Malignancies
    • Presentations: 8
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      OA02.01 - The microRNA-15/16 Family Regulates Tumour Cell Growth via Fibroblast Growth Factor Signals in Malignant Pleural Mesothelioma (ID 5395)

      11:00 - 11:10  |  Author(s): K. Schelch, M.B. Kirschner, M. Williams, R. Lin, Y.Y. Cheng, M. Grusch, W. Berger, N. Van Zandwijk, G. Reid

      • Abstract
      • Presentation
      • Slides

      Background:
      Malignant pleural mesothelioma (MPM) is a highly aggressive, asbestos-related malignancy characterized by poor outcome and limited therapeutic options. Fibroblast growth factor (FGF) signals play important roles in mesothelioma cell growth and malignant behavior and their inhibition leads to reduced tumor growth. MicroRNAs (miRNAs) are conserved noncoding RNAs controlling gene expression via translational repression of target mRNAs. The miR-15/16 family is downregulated in MPM and has tumor suppressor functions. Several FGFs/FGFRs are predicted miR-15/16 targets. The aim of this study was to explore the link between the miR-15/16 and the FGF/R family in MPM.

      Methods:
      Gene and microRNA expression was determined by RT-qPCR or Taqman Low Density Arrays (TLDAs). Mimics were used for restoring microRNA expression. Stimulation or inhibition of FGF signals or bcl-2 was achieved by recombinant FGF2, siRNAs, or small-molecule inhibitors, respectively. A SYBR green-based proliferation assay and colony formation assays were used to monitor effects on cell growth.

      Results:
      Expression analysis showed a consistent downregulation of target FGF/FGFR genes after transfection with miRNA mimics. Restoration of miR-15/16 led to dose-dependent growth inhibition, which significantly correlated with sensitivity to the specific FGFR1 inhibitor PD166866. Re-expression of microRNAs in combination with FGFR knock-down or pharmacological inhibition resulted in reduced activity, indicating target competition. Combined inhibition of the FGF-axis and bcl-2, another established target of miR-15/16, resulted in enhanced activity. Treatment with recombinant FGF2 further reduced mature as well as pri-microRNA levels and also could prevent/reduce growth inhibition by mimics, but only when added within 24 hours after transfection. TLDA screens after stimulation/inhibition of FGF signals identified regulation of several other miRNAs involved in pathways relevant for tumour growth and aggressiveness.

      Conclusion:
      Our data shows that the post-transcriptional repression of FGF-mediated signals contributes to the tumour-suppressor function of the microRNA-15/16 family. Impairing hyperactivated FGF signals as well as the anti-apoptotic protein bcl-2 through the restoration of this miRNA family might serve as a novel therapeutic strategy in mesothelioma.

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      OA02.02 - Gremlin-1 is a Key Regulator of the Invasive Phenotype in Mesothelioma (ID 5424)

      11:10 - 11:20  |  Author(s): M. Yin, M. Tissari, J. Tamminen, I. Ylivinkka, M. Ronty, K. Lehti, M. Hyytiainen, M. Myllarniemi, K. Koli

      • Abstract
      • Presentation
      • Slides

      Background:
      Malignant mesothelioma is an aggressive cancer that develops from mesothelial cells, most often in the pleural lining of the lung. We have previously shown that the BMP inhibitor protein gremlin-1 is highly expressed in mesothelioma and induces a mesenchymal and chemoresistant phenotype in mesothelioma cells. Since mesothelioma tumors are locally highly invasive, we analyzed the role of gremlin-1 in mesothelioma cell migration and invasive growth.

      Methods:
      Primary mesothelioma cells isolated from patient pleural fluid as well as mesothelioma cell lines were used for in vitro studies. Cells were transfected with siRNAs or transduced with lentiviral expression vectors. Invasive growth was analyzed in 3D matrigel or collagen I matrices. mRNA expression was analyzed using a commercial PCR array and quantitative RT-PCR. Migration assays were performed using scratch wound assay or Transwell migration assay with fibronectin or collagen coating. TGF-β and BMP signaling activity was measured with reporter-luciferase assays. For in vivo mouse xenograft experiment cells were additionally transduced to express a luciferase marker. Subcutaneous cell injection with matrigel matrix was performed in the flank of nude mice.

      Results:
      Mesothelioma cells expressing gremlin-1 showed invasive sprouting when tumor cell spheroids were imbedded into 3D collagen matrix. Silencing of gremlin-1 expression significantly reduced invasive growth. In addition, cells overexpressing gremlin-1 gained invasive growth ability. This was associated with increased mRNA expression levels of Slug and matrix metalloproteinases (MMP) as well as reduced expression of E-cadherin. The cells were more migratory and exhibited increased expression of certain integrins, especially the α~v~ subunit. Gremlin-1 induced invasive growth was dependent on MMP activity and associated with increased TGF-β activity. Intrapleural injection of gremlin-1 overexpressing mesothelioma cells isolated from a patient with epithelioid mesothelioma, produced tumors in 2/4 mice over 4 months after injection. Cells transduced with vector only did not produced tumors (0/4). When cells were injected subcutaneously together with matrigel gremlin-1 overexpressing tumors appeared more slowly, but exhibited comparable luciferase signal 2.5 months after injection. However, gremlin-1 tumors showed more local spreading and in contrast to control tumors some also developed metastasis (2/6 mice).

      Conclusion:
      Mesothelioma invades locally and has poor prognosis. We have identified gremlin-1 as an important regulator of mesothelioma chemoresistance and invasive growth behavior. Blocking gremlin function may overcome drug resistance and reduce invasion of mesothelioma.

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      OA02.03 - Circulating Fibroblast Growth Factor 18 is Elevated in Malignant Pleural Mesothelioma Patients - A Multi-Institutional Study (ID 5988)

      11:20 - 11:30  |  Author(s): Y. Dong, H. Zhang, K. Schelch, T. Klikovits, P. Stockhammer, M. Jakopovic, M. Samarzija, L. Brcic, G. Reid, M.B. Kirschner, S. Kao, I. Opitz, W. Weder, T. Frauenfelder, T.D.L. Nguyen-Kim, W. Klepetko, N. Van Zandwijk, B. Hegedus, W. Berger, B. Dome, V. Laszlo, M. Grusch, M.A. Hoda

      • Abstract
      • Presentation
      • Slides

      Background:
      Malignant pleural mesothelioma (MPM) is a rare but devastating malignancy. Despite the search for new promising treatment approaches, the outcome for most MPM patients remains dismal. Therefore, the identification of novel biomarkers is urgently needed in order to identify patients with a better prognosis and to support personalized therapeutic decisions. In our previously published study, we were able to show that fibroblast growth factor 18 (FGF18) is overexpressed in MPM tissue specimens and cell models. The objective of this study was the evaluation of FGF18 as a circulating biomarker in MPM.

      Methods:
      Plasma was collected from 107 MPM patients at the time of diagnosis or before surgical resection. Samples were included from the Medical University of Vienna, University Hospital Center in Zagreb and from The Concord Repatriation General Hospital and Strathfield Private Hospital in Sydney. Samples from 49 healthy volunteers and from 8 patients with non-malignant pleural diseases served as controls. Circulating FGF18 was measured by enzyme-linked immunosorbent assay and correlated to clinical, pathologic and radiologic parameters.

      Results:
      Plasma FGF18 level was significantly elevated in MPM patients vs. healthy controls (P<0.0001). A slight increase of circulating FGF18 level was also detected in patients with pleuritis or fibrosis (vs. control, P=0.0067). Sarcomatoid (n=7) morphology was associated with high FGF18 levels when compared to the epithelioid (n=77) histology (P=0.0064). Importantly, MPM patients presenting with FGF18 levels below the median had a significantly longer overall survival when compared to those with high FGF18 levels (median survival 625 versus 382 d, P=0.0038). Data on multivariate analysis, disease-free survival, correlation with other biomarkers and tumor volume will be presented at the conference.

      Conclusion:
      Our findings reveal that FGF18 is a promising blood-derived candidate biomarker in MPM. Furthermore FGF18 may support the histological classification of MPM and the identification of MPM patients with poor prognosis. .

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      OA02.04 - Discussant for OA02.01, OA02.02, OA02.03 (ID 6952)

      11:30 - 11:45  |  Author(s): M.B. Kirschner

      • Abstract
      • Presentation
      • Slides

      Abstract not provided

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      OA02.05 - Expression of miR-223 in Mesothelioma Xenografts Originates from Stromal Cells in the Tumour Microenvironment (ID 5875)

      11:45 - 11:55  |  Author(s): K.H. Sarun, Y.Y. Cheng, M.B. Kirschner, N. Van Zandwijk, R. Lin, G. Reid

      • Abstract
      • Presentation
      • Slides

      Background:
      Malignant pleural mesothelioma (MPM) is an aggressive cancer caused by asbestos exposure with limited therapeutic options. Dysregulated microRNAs play an important role in MPM biology and candidate microRNAs have been investigated as diagnostic and prognostic biomarkers or as a potential treatment targets. The role of miR-223 has previously been investigated in MPM tumour cells and was shown to act as a tumour suppressor by regulating cell mobility. Previous research indicated miR-223 to be primarily expressed by myeloid progenitor derived cells during differentiation of granulocytes and monocytes. This suggests miR-223 might have a more significant role in the inflammatory response during tumourigenesis. In this study we aimed to investigate the origin of miR-223 using mesothelioma xenograft and syngraft models.

      Methods:
      Human and mouse mesothelioma cell line-derived xenograft (MSTO-H211 and H226) and syngraft (AB1) models were established. MicroRNA profiles of xenografts were compared against profiles of their corresponding in vitro cultured cells to determine candidates. RT-qPCR using TaqMan MicroRNA assays was used to validate expression levels of miR-143-3p, miR-214-3p and miR-223-3p in tumour xenografts and syngrafts with those in corresponding cell lines in vitro. Species-specific ddPCR analysis was performed on RNA from xenograft tumours to determine the expression of human and mouse pri-miR-223.

      Results:
      MicroRNA profiles of xenograft tumours showed significant upregulation (p < 0.05) of miR-143-3p, miR-214-3p and miR-223-3p compared to corresponding in vitro mesothelioma cell lines. Only miR-223 showed significant upregulation in both xenograft and syngraft tumours compared to corresponding in vitro mesothelioma cell lines (>10000-fold increase). Other microRNAs were not significantly different between cell lines and tumours. RNA isolated from xenograft tumours contained significantly more mouse pri-miR-223 than human pri-miR-223 (p < 0.001), with only minimal expression levels of human tumour pri-miR-223 within xenograft tumours.

      Conclusion:
      Mature miR-223 is significantly overexpressed in xenograft tumours compared to corresponding in vitro mesothelioma cell lines suggesting stromal contribution. Species-specific pri-miRNA confirmed miR-223 is almost exclusively expressed by the mouse stromal cells in xenograft tumours. Ultimately, localising the expression of miR-223 to specific cell types (such as myeloid derived cells) through in situ hybridisation should help identify a more biologically relevant role for miR-223 in the tumour microenvironment.

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      OA02.06 - Converting Tumor-Mediated PD-L1 Inhibition into CAR T-Cell Costimulation to Potentiate Thoracic Cancers Immunotherapy (ID 6058)

      11:55 - 12:05  |  Author(s): A. Morello, N. Chen, L. Cherkassky, M. Sadelain, D. Jones, P.S. Adusumilli

      • Abstract
      • Presentation
      • Slides

      Background:
      To overcome tumor-mediated inhibition of chimeric antigen receptor (CAR) T cells, we herein investigated the impact of tumor PD-L1 upregulation on CAR T-cell exhaustion and anti-tumor efficacy, and further developed clinically translatable T-cell extrinsic as well as intrinsic strategies to overcome PD-L1 inhibition in models of lung cancer (LC) and malignant pleural mesothelioma (MPM).

      Methods:
      Human T cells were transduced with MSLN-specific CAR with CD28 and CD3zeta domains (M28z) were tested in vitro and in clinically-relevant LC and MPM mouse models by bioluminescence imaging, BLI of tumor burden progression. To counteract PD-1/PD-L1 inhibition in vivo, we evaluated the efficacy of PD-1 blocking antibody or cell-intrinsic genetic-engineering strategies by cotranducing M28z CAR T cells with a PD-1 dominant negative receptor (PD1-DNR) or with PD-1/4-1BB fusion protein.

      Results:
      A single, low-dose of M28z CAR T cells is able to resist the progression of established tumor for 40 days, but mice eventually died with progressing tumor. Tumor harvest analysis demonstrated the PD-1 and PD-L1 upregulation on CAR T cells and tumor cells (Figure panel A). We then confirmed in vitro that PD-L1 inhibits M28z T-cell effector functions (proliferation, cytotoxicity and cytokine secretion). The addition of PD-1 blocking potentiates CAR T-cell therapy in vivo but its efficacy requires multiple injections (Panel B). In contrast, a single dose of M28z T cells coexpressing PD1-DNR restore effector functions, enhance tumor burden control (Panel C) and prolong median survival (56 vs 82 days, p=0.001). Converting PD-L1 inhibition into a positive costimulatory signal by PD-1/4-1BB construct cotransduction into M28z CAR T cells enhanced cytokine secretion and T-cell accumulation (Panel D). Figure 1



      Conclusion:
      Our results demonstrate the therapeutic benefit of providing optimal costimulation and coinhibitory blockade to counteract PD-L1/PD-1 immunosuppression, thus potentiate CAR T-cell therapy for lung cancer and mesothelioma.

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      OA02.07 - Characterization of the Tumor Microenvironment and Investigation of Immune Checkpoint Expression in Malignant Pleural Mesothelioma (ID 4437)

      12:05 - 12:15  |  Author(s): E. Marcq, V. Siozopoulou, J. De Waele, J. Van Audenaerde, K. Zwaenepoel, E. Santermans, N. Hens, P. Pauwels, J.P. Van Meerbeeck, E. Smits

      • Abstract
      • Presentation
      • Slides

      Background:
      Malignant pleural mesothelioma (MPM) is an aggressive cancer with a poor prognosis and an increasing incidence, for which novel therapeutic strategies are urgently required. Since the immune system has been described to play a role in protection against MPM, characterization of its tumor immune microenvironment (TME) and immune checkpoints might help to identify new immunotherapeutic targets and their predictive and/or prognostic value.

      Methods:
      Immunohistochemistry (IHC) was performed on tissue samples of untreated (n=40) and chemotherapy-pretreated (n=14) MPM patients. Different subsets if immune cells were identified based on staining for CD4, CD8, FoxP3, CD68, CD45RO and granzyme B. The expression of the immune checkpoints TIM-3, LAG-3, PD-1 and its ligand PD-L1 was also investigated. The relationship between the immunological parameters and survival, as well as response to chemotherapy was analyzed using the R statistical software.

      Results:
      All patients had CD8+ tumor infiltrating lymphocytes (TILs), CD68+ histiocytes and macrophages and CD45RO+ T cells in their stroma, with CD8+ TILs being the predominant cell type of the immune infiltrate. Stromal CD4+ TILs were found in 75% of the untreated and 71% of the pretreated samples. A subset of those cells was also FoxP3+ and these CD4+FoxP3+ cells were positively correlated with stromal CD4 expression (p<0.001). Less than half of the samples showed positivity for granzyme B. Both, untreated and pretreated patients had PD-1+ TILs, while only 10% of the untreated patients also had PD-1+ tumor cells. PD-L1 positivity on lymphocytes and/or tumor cells was observed for more than half of the patients, with significant differences according to the histological subtype (p<0.001). Patients with a sarcomatoid histology showed the most PD-1 expression. TIM-3 was expressed in tumor cells, stromal lymphocytes and plasma cells, less often in pretreated samples compared to untreated samples. All samples were negative for LAG-3. After multivariate analysis stromal CD45RO expression was found to be an independent negative predictive factor for response to chemotherapy (p=0.017) and expression of CD4 and TIM-3 in lymphoid aggregates were good prognostic factors (p=0.008; p=0.001).

      Conclusion:
      Our data reveal the diversity of immune cells present in MPM and point to TIM-3 as a new target in mesothelioma. Administering chemotherapy before or together with PD-1/PD-L1/TIM-3 blocking agents may not be the best combination sequence and further research on the predictive value of CD45RO in the stroma might guide patient selection for chemotherapy.

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      OA02.08 - Discussant for OA02.05, OA02.06, OA02.07 (ID 6964)

      12:15 - 12:30  |  Author(s): M. Grusch

      • Abstract
      • Presentation
      • Slides

      Abstract not provided

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Author of

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    MA14 - Immunotherapy in Advanced NSCLC: Biomarkers and Costs (ID 394)

    • Event: WCLC 2016
    • Type: Mini Oral Session
    • Track: Advanced NSCLC
    • Presentations: 2
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      MA14.01 - Updated Dataset Assessing Tumor Mutation Burden (TMB) as a Biomarker for Response to PD-1/PD-L1 Targeted Therapies in Lung Cancer (LC) (ID 4011)

      16:00 - 16:06  |  Author(s): L.E. Raez

      • Abstract
      • Presentation
      • Slides

      Background:
      Immune checkpoint inhibitors (ICPIs) nivolumab and pembrolizumab have been FDA-approved in non-small cell LC (NSCLC). Current IHC based diagnostics are challenged by assay and slide scoring issues and modest predictive value, and more robust and comprehensive biomarkers of ICPI efficacy are needed. A discovery set of 64 NSCLCs treated with ICPIs suggested that high TMB (≥15 mutations/Mb) significantly correlated with longer time on drug (Spigel et al., ASCO 2016, Abstract:9017).

      Methods:
      Comprehensive genomic profiling (CGP) was performed during the course of clinical care. TMB was assessed as the number of somatic, coding, base substitution and indels per Mb of genome. Microsatellite instability-high (MSI-H) or stable (MSS) status was determined using a proprietary algorithm.

      Results:
      15,529 LCs: 66% adenocarcinoma, 1% sarcomatoid, 14% NSCLC NOS, 11% squamous, 5% small cell, and 2% large cell were assessed. TMB was similar across all lung histologies (median: 6.3, 8.1, 9.0, 9.9, 9.9, and 10.8); the median was 7.6 for all LC cases (TMB ≥15 in 24% of cases), compared to 4.5 for 80,000+ samples of diverse tumor types in the database. Of LCs assessed 0.3% were MSI-H, of which 30/31 were TMB-high; however, 24% of MSS-stable cases were also TMB-high. PD-L1 amplification and DNA repair pathway mutation (MLH1, MSH2, POLE) were found in 1.0% and 1.1% of LC cases analyzed, respectively. Tumors harboring known drivers (ALK, ROS1, EGFR, BRAF V600E, MET splice) had low TMB (median: 2.5, 3.6, 3.8, 3.8, 4.5), whereas tumors with KRAS mutation, non-V600E BRAF mutation, PD-L1 amplification, or DNA repair alterations were more likely to be TMB-high (median: 9.0, 10.8, 14.4, 21.6).

      Conclusion:
      High TMB may be a predictive biomarker of response to ICPIs. Several factors including lack of a known driver, MSI-H status, PD-L1 amplification, and DNA repair mutation correlated with high TMB (P<0.0001 for all cases). However, 95% of TMB-high cases assessed were MSS and lacked both PD-L1 amplification and DNA repair mutation, and thus would likely not be selected for immunotherapy by assessment of individual genomic alterations or MSI status alone. A validation cohort of NSCLC patients treated with anti-PD-1/PD-L1 therapies including analysis of clinical outcome, TMB, genomic profile, and available clinicopathologic characteristics will be presented. CGP of LC to simultaneously determine TMB, MSI status, PD-L1 amplification, and the presence of driver alterations may provide clinically useful predictors of response to ICPI and other targeted therapies using a single platform, but prospective clinical trials are needed to confirm these observations.

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      MA14.08 - Discussant for MA14.05, MA14.06, MA14.07 (ID 6988)

      16:48 - 17:00  |  Author(s): L.E. Raez

      • Abstract
      • Presentation
      • Slides

      Abstract not provided

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    OA10 - EGFR Mutations (ID 382)

    • Event: WCLC 2016
    • Type: Oral Session
    • Track: Biology/Pathology
    • Presentations: 1
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      OA10.07 - Report on Liquid Biopsies from Advanced Lung Adenocarcinoma Patients and Correlation with Their Tumor Biopsy Profiles (ID 4826)

      12:05 - 12:15  |  Author(s): L.E. Raez

      • Abstract
      • Presentation
      • Slides

      Background:
      Liquid biopsy (LBx) has emerged as an alternative tool for the management of advanced lung cancer patients (pts) identifying driver mutations, and hence, improving personalized medicine. There are still controversial issues such as standardization, validation of different technologies, concordance with tissue molecular profile results (TMPR), and others. LBx offers many advantages including non-invasive, bypass tumor heterogeneity, an opportunity for serial measurements to evaluate response or early recurrence, and others.

      Methods:
      Guardant 360 was analyzed in 100 consecutive stage IV or recurrent lung adenocarcinoma (adeno) pts. Guardant 360 is a panel of 70 genes including single nucleotide variations, amplifications, translocations, and short insertions/duplications/deletions in exons 19 and 20 of the EGFR, and others. Cell-free DNA (cfDNA) is extracted from plasma and genomic alterations are analyzed by massively parallel sequencing of amplified target genes. TMPRs from each subject was obtained or recovered for comparison with their LBx counterparts. TMPRs from this cohort was developed in different CLIA laboratories

      Results:
      69 pts were females; median age 72 (range, 27-99). 84/100 pts had at least 1 genomic alteration by LBx (range, 1-10). Most common abnormalities found in LBx were: TP53 (37 pts), EGFR (35 pts), NF1 (20 pts), KRAS (12 pts), MET (14 pts). From this 84 pts with + LBx results, 67 pts (80%) had TMPRs for comparison. Main reason for lack of TMPRs: insufficient tumor (19/100; 19%). For comparison between the 2 modalities, we considered all pts with available results in both tests; hence, 81 pts were used to compare tumor biopsy (TBx) vs. LBx. 37 pts out of 81 (46%) had at least 1 similar genomic abnormality found in both TBx and LBx, respectively. Most of the concordance was in EGFR alterations (19/28; 68%). LBx caught 16 additional EGFR genomic aberrations not being identified by TBx. A total of 35 EGFR genomics aberrations were identified in LBx; 16/35 EGFR mutations found in LBx were actionable and 5 of these 16 actionable EGFR mutant cases were only found in LBx not in TBx.

      Conclusion:
      LBx offers an alternative to identify genomic alterations. Still, insufficient tumor is the major reason for lacking of TMPRs. EGFR mutations are the most common actionable mutations found in LBx; also, it has a high correlation with TBx (68%). LBx identified more gene abnormalities than TBx, and in some cases, the actionable EGFR mutations were found only in LBx sample.

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    P2.03b - Poster Session with Presenters Present (ID 465)

    • Event: WCLC 2016
    • Type: Poster Presenters Present
    • Track: Advanced NSCLC
    • Presentations: 1
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      P2.03b-039 - Cell-Free (cf) DNA and cfRNA levels in Plasma of Lung Cancer Patients Indicate Disease Status and Predict Progression (ID 4563)

      14:30 - 14:30  |  Author(s): L.E. Raez

      • Abstract

      Background:
      Cell-free circulating tumor DNA (ctDNA) and RNA (ctRNA) can be extracted from plasma of cancer patients (pts). Measuring dynamic changes in gene expression, allele-fractions of mutations and levels of nucleic acids per ml of plasma in metastatic patients has shown great potential for monitoring disease state and predicting outcome to antitumoral therapy in advance of imaging.

      Methods:
      We designed a clinical trial to measure gene levels of plasma ctDNA and ctRNA in metastatic pts with NSCLC, breast, and GI malignancies under treatment and correlate the levels with CT scans done assessing response for one-year clinical trial. CtDNA/ctRNA were extracted from plasma separated from patient whole-blood draws shipped at ambient temperature. CtRNA was reverse transcribed with random primers to cDNA. Levels of ctDNA/ctRNA were determined by real-time qPCR. Results of ctDNA/ctRNA tests were compared with responses (CR, PR, SD or PD) as determined by the CT scans. Levels of PD-L1 expression relative to β–actin were determined by qPCR.

      Results:
      We have enrolled 39 pts and we are presenting data from 15 pts with NSCLC. The first-line treatments were Carboplatin (40%, 6/15), Opdivo (26.7%, 4/15), Afatinib (13.3%, 2/15), Ceritinib (6.7%, 1/15), Crizotinib (6.7%, 1/15), and Taxotere/Cyramza (6.7%, 1/15). Histological subtypes were 73.3% (11/15) adeno, 20% (3/15) squamous, and 6.7% (1/15) other. The majority of patients were Caucasian (66.7%, 10/15), Hispanic (26.7%, 4/15), and African American (6.7%, 1/15). Levels of ctDNA and ctRNA were significantly correlated with one another across patient blood draws (r = 0.5781, p < 0.0001). After the first 2 cycles of therapy, 9 of the 15 patients achieved SD, of whom 8 were analyzed for ctDNA/ctRNA levels. In 7/8 of pts who had SD indicated by CT scan, CtDNA/ctRNA levels were stable, while the 2 PD pts showed significant increases in levels of ctDNA/ctRNA 4.8 weeks prior to progression. In the 2 SD pts treated with Opdivo, PD-L1 expression was stable during the cycles when CT scans also indicated an SD status.

      Conclusion:
      In almost 90% of the pts, constant ctDNA/ctRNA levels correlated with stable disease status as seen by CT; moreover, in 2 pts changes in the levels of both ct nucleic acids predicted progression about 5 weeks before CT scans. Importantly, the concordance between cDNA and cfRNA suggest that cfRNA can just as effective as cfDNA as a prognostic tool, while adding the extra dimension of gene expression.