Autologous stem-cell transplantation (ASCT) for multiple myeloma continues to confer benefit in the ‘novel agent’ era [1]. However, most patients relapse, risk stratification is inadequate and opti...
ABSTRACT High-dose alkylator-based conditioning followed by autologous stem-cell transplantation (ASCT) is a therapeutic mainstay for eligible patients with multiple myeloma. However, post-transplant relapses are common and prognostic biomarkers are scarce. Relapses are characterized by the influx of regulatory myeloid cells and dysfunctional T effectors. We have shown that myeloma-infiltrating myeloid cells produce versican (VCAN), a large matrix proteoglycan with tolerogenic activities. VCAN proteolysis by a-disintegrin-and-metalloproteinase-with-thrombospondin-motifs (ADAMTS) proteases generates versikine, a bioactive fragment (“matrikine”) that regulates Batf3-dendritic cells, known to control CD8+-attracting chemokine networks. Here we demonstrate that intense VCAN proteolysis predicts CD8+ infiltration post-transplant and paradoxically portends significantly inferior survival outcomes. Our data suggest that VCAN proteolysis promotes the influx of CD8+ effectors that are rendered overwhelmingly dysfunctional and/or frankly immunoregulatory (CD8+ Treg) at the tumor site. Thus, complex immunosuppressive circuits orchestrated through VCAN accumulation and turnover generate conditions favorable for myeloma tumor regrowth and point to a readily-assayed biomarker to identify the patients at risk for relapse and early death. The dismal outcomes associated with VCAN proteolysis may be rationally overcome through immunotherapies such as checkpoint inhibition (e.g., anti-TIGIT), tumor vaccines or anti-myeloid (e.g., anti-CSF-1R) approaches.
8527 Background: Lung cancer remains the #1 cancer killer in the US, and effective screening approaches are desperately needed. Lung cancer alone accounts for 221,000 deaths annually. DNA methylation profiling has shown unique patterns in DNA promoter regions with cancer and has potential application for detection of lung malignancies. However, optimally discriminant markers and marker panels are needed. The purpose of this study is to identify and validate in tissue, candidate methylated DNA markers for detecting lung cancer overall and by subtypes. Methods: Candidate methylated DNA markers were identified by unbiased whole methylome sequencing of selected lung cancer case and lung control tissues. The top 51 marker candidates were further evaluated in 255 independent patients with 119 controls, of which 37 were from benign nodules, and 136 cases inclusive of all lung cancer subtypes. DNA extracted from patient tissue samples was bisulfite treated and then candidate markers and ACTB as a normalizing gene were assayed by Quantitative Allele-Specific Real-time Target and Signal amplification (QuARTS). QuARTS chemistry requires perfect base pairing to minimize false signals due to un-methylated or partially methylated genes and yields high discrimination for methylated marker selection and screening. Results: On receiver operator characteristics analyses of individual marker candidates, areas under the curve (AUCs) ranged from 0.512 to 0.941. At 100% specificity, a combined panel of 8 methylation markers (SLC12A8, KLHDC7B, PARP15, OPLAH, BCL2L11, MAX.12.526, HOXB2, and EMX1) yielded a sensitivity of 98.5% across all subtypes of lung cancer. Furthermore, using the 8 markers panel, benign lung nodules yielded no false positives. Conclusions: A panel of novel methylated DNA markers assayed on tissue achieves extremely high discrimination for all types of lung cancer while remaining negative in normal lung tissue and benign nodules. Following technical optimization, this panel can be applied to blood or bodily fluid-based testing with applications in lung cancer screening and discrimination of malignant from benign nodules.
Abstract Multiple myeloma (MM) is a cancer of malignant plasma cells in the bone marrow and extramedullary sites. We previously characterized a VQ model for human high-risk MM. Different VQ lines display distinct disease phenotypes and survivals, suggesting significant intra-model variation. Here, we use whole exome sequencing and copy number variation (CNV) analysis coupled with RNA-Seq to stratify VQ lines into corresponding clusters: Cluster I VQ cells carried recurrent amplification of chromosome (chr) 3 and displayed upregulation of growth pathways and high-risk myeloma gene signatures, whereas Cluster II cells had monosomy chr5 and overexpressed genes and pathways associated with positive response to bortezomib (Btz) treatment in human MM patients. Consistently, in sharp contrast to Cluster II VQ cells that showed short-term response to Btz, Cluster I VQ cells were de novo resistant to Btz in vivo . Our study highlights Cluster I VQ lines as highly representative of human high-risk MM subset.
Immune Checkpoint Inhibitors (ICI) have revolutionized cancer treatment. Unfortunately, existing FDA approved biomarkers like PD-L1, MSI and dMMR do not accurately predict which patients will respond. The Elephas Cybrid™ platform aims to improve ICI response prediction using multiple biological assays and imaging of the patient's live tumor fragments (LTF)™ treated with ICI ex vivo. Multiphoton microscopy (MPM) is a powerful imaging technique and one of the primary modalities Elephas uses to interrogate the biological changes in LTF following ICI treatment. MPM allows rapid visualization of biological processes in 3D living tissues without interference and can be used to characterize T cell responses induced by ICI. We recently described an MPM assay to detect and quantify cell viability without the use of dyes and are building on these methods to enable the Cybrid platform to distinguish responding from non-responding T cells.
Methods
Activated and resting T cells isolated from both mice and humans were characterized using MPM to observe differences in metabolic state via fluorescence lifetime imaging (FLIM). Murine cytotoxic T cells were generated, isolated, and co-cultured for 48-hours with antigen-expressing tumor cells. Anti-CD8 nanobodies and cleaved caspase 3/7 dye were used to observe and characterize antigen-specific T cell cytotoxicity. Cytotoxic T cell phenotype was confirmed with flow cytometry and secreted cytokine assays. Tumors were cut into LTF, sorted, and cultured in multi-well plates using the Cybrid platform. LTF structure and metabolic status based on the intrinsically fluorescent metabolic co-factors nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NAD(P)H) and flavin adenine dinucleotide (FAD) were assessed. Multi-channel fluorescence intensity and lifetime were obtained over 48-hours using a custom four-channel, multiphoton microscope.
Results
Using the lifetime metabolic ratio (LMR, LMR=NAD(P)H α2/FAD α1), pockets of cell death surrounding CD8 T cells were detected in LTF that were co-cultured with cytotoxic T cells, but not in fragments cultured in the absence of cytotoxic T cells.
Conclusions
The methods presented herein will advance the field of cancer diagnostics by progressing from fixed tissue imaging and genetic characterization to assessment of clinically relevant ICI-treated LTF over days as an alternative to months of waiting for a clinical response. Understanding the response of LTF to immunotherapies will allow clinicians to make more informed treatment decisions and improve the lives of those with cancer.
Abstract The impact of tumor matrix remodeling to the generation of an “inflamed” microenvironment that modulates responses to immunotherapy is unclear. Versican (VCAN) is a chondroitin sulphate matrix proteoglycan that promotes tolerogenic polarization of intratumoral DC through Toll-like receptor 2 (TLR2). Proteolytic processing of VCAN releases a bioactive N-terminal fragment (matrikine), versikine. In contrast to the tolerogenic actions of parental VCAN, versikine triggers IRF8-dependent transcription in myeloid cells and promotes Batf3-dendritic cell (DC) generation from FLT3L-mobilized bone marrow progenitors in vitro. Consistent with the Batf3-promoting effects of versikine, VCAN proteolysis correlates with T-cell infiltration across multiple cancers.The aims were to 1. define the impact of versikine on the intratumoral myeloid repertoire in vivo and 2. to define the efficacy of versikine as a vaccine adjuvant.4T1 breast carcinoma and Lewis Lung Carcinoma (LLC) empty vector (EV)- and versikine-expressing cells were implanted subcutaneously in syngeneic recipients. 1000mm3 tumors were harvested and intratumoral DC subsets were enumerated. Versikine-expressing tumors were characterized by significantly enhanced Batf3-DC (CD11chigh,MHC IIhigh Ly6C-, CD64-, CD24high,CD11blow) (p =0.0079 for 4T1 model and <0.0001 for LLC model), whereas cDC2 (CD11chigh,MHC IIhigh Ly6C-, CD64-, CD24low, CD11bhigh ) frequency was diminished (p= 0.0079 and <0.0001 respectively). Monocytic-derived DC (Mo-DC: CD11chigh, MHC IIhigh, Ly6C+, CD64+) remained unchanged. To determine the impact of versikine on responses to in situ vaccination using STING agonists, EV- and versikine-replete tumors (B16 and 4T1; 150 mm3), were injected intratumorally (IT) with a single subtherapeutic dose (200 μg) of DMXAA (murine STING agonist) or vehicle. EV-tumors did not significantly respond to 200μg DMXAA, whereas many B16-versikine and 4T1-versikine tumors regressed or growth was inhibited (p<0.001 and p=0.014 respectively). Necrosis was frequently observed in 4T1 versikine-secreting tumors (6/9 mice) within 24 hours after treatment. Versikine extended survival after subtherapeutic DMXAA treatment in 4T1; log rank=p=0.01. Versikine's effects were abrogated in Batf3-null mice. To quantitate antigen-specific responses in the presence or absence of versikine, EV- and versikine-replete LLC tumors were injected IT with 500 μg DMXAA (therapeutic dose) or vehicle. We observed a significant increase in the frequency of CD8+ MHCI:SIINFEKL tetramer+ splenocytes in LLC-versikine-bearing animals as well as a marked increase in central memory T splenocytes (TCM) (CD62LhighCD44high). VCAN matrikines may generate effective adjuvants for in situ vaccination strategies across diverse solid and hematopoietic tumor types. Citation Format: Athanasios Papadas, Evan Flietner, Zachary Morrow, Joshua Wiesner, Alexander Cicala, Adam Pagenkopf, Chelsea Hope, Philip Emmerich, Dustin Deming, Jing Zhang, Peiman Hematti, Natalie Callander, Alexander Rakhmilevich, Mario Otto, Christian Capitini, Fotis Asimakopoulos. Versican proteolytic fragments (matrikines) synergize with STING agonists to elicit robust anti-tumor CD8+ T cell responses [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5733.
Abstract Purpose: Lung cancer is the leading cause of cancer deaths worldwide. Most present symptomatically at late stage with high lethality. Early detection reduces mortality but accurate and readily accessible tools for population screening are limited. By whole methylome sequencing, we have identified novel methylated DNA markers (MDMs) for lung cancer in tissue (Giakoumopoulos et al. ASCO 2016). Using top candidate MDMs in the present study, we now explore their clinical accuracy for lung cancer detection when assayed from plasma. Experimental Procedures: Archival plasmas from two independent study groups were tested in blinded fashion. Lung cancer cases and controls (apparently healthy smokers) for each group were balanced on age and sex (Group 1: 64 cases, 231 controls; Group 2: 23 cases, 80 controls). Using multiplex PCR followed by QuARTS (Quantitative Allele-Specific Real-time Target and Signal amplification) assay, a post-bisulfite quantification of MDMs on DNA extracted from plasma was performed. We selected 31 MDM candidates for initial evaluation in Group 1 (1 ml plasma/patient); top individual MDMs were subsequently tested in Group 2 to identify optimal MDM panels for lung cancer detection (2 ml/patient). Results: From Group 1 analyses, 13 high performance MDMs were selected for further testing (CYP26C1, SOBP, SUCLG2, SHOX2, ZDHHC1, NFIX, FLJ45983, HOXA9, B3GALT6, ZNF781, SP9, BARX1, EMX1) with individual areas under the receiver operator curve (AUCs) ranging from 0.593 to 0.939. Discrimination by individual MDMs was corroborated in Group 2 in which data was analyzed using two methods: a logistic regression fit and a regression partition tree approach. The logistic fit model identified a 4-marker panel (ZNF781, BARX1, EMX1, and SOBP) with an AUC of 0.96 and an overall sensitivity of 91% and 90% specificity. Analysis of the data using a regression partition tree approach identified 4 markers (ZNF781, BARX1, EMX1, and HOXA9) with AUC of 0.93 and an overall sensitivity of 96% and specificity of 94%. For both approaches, B3GALT6 was used as a standardizing marker of total DNA input. Conclusion: A panel of MDMs assayed in plasma achieved high sensitivity and specificity for all types of lung cancer. Further clinical evaluation and validation of this promising panel in larger patient groups are clearly indicated. Citation Format: Hatim T. Allawi, Maria Giakoumopoulos, Evan Flietner, Austin Oliphant, Carla Volkmann, Brian Aizenstein, Tamara Sander, Drew Eckmayer, Ashley Poenitzsch Strong, Melissa Gray, Barry Berger, Tracy Yab, William Taylor, Douglas Mahoney, John B. Kisiel, David E. Midthun, David A. Ahlquist, Graham P. Lidgard. Detection of lung cancer by assay of novel methylated DNA markers in plasma [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 712. doi:10.1158/1538-7445.AM2017-712
