BACKGROUND. Tumor content in circulating cell-free DNA (cfDNA) is a promising biomarker, but longitudinal dynamics of tumor-derived and non–tumor-derived cfDNA through multiple courses of therapy have not been well described. METHODS. CfDNA from 663 plasma samples from 140 patients with castration-resistant prostate cancer (CRPC) was subject to sparse whole genome sequencing. Tumor fraction (TFx) estimated using the computational tool ichorCNA was correlated with clinical features and responses to therapy. RESULTS. TFx associated with the number of bone metastases (median TFx = 0.014 with no bone metastases, 0.047 with 1–3 bone metastases, 0.190 for 4+ bone metastases; P < 0.0001) and with visceral metastases (P < 0.0001). In multivariable analysis, TFx remained associated with metastasis location (P = 0.042); TFx was positively correlated with alkaline phosphatase (P = 0.0227) and negatively correlated with hemoglobin (Hgb) (P < 0.001), but it was not correlated with prostate specific antigen (PSA) (P = 0.75). Tumor-derived and non–tumor-derived cfDNA track together and do not increase with generalized tissue damage from chemotherapy or radiation at the time scales examined. All new treatments that led to ≥30% PSA decline at 6 weeks were associated with TFx decline when baseline TFx was >7%; however, TFx in patients being subsequently maintained on secondary hormonal therapy was quite dynamic. CONCLUSION. TFx correlates with clinical features associated with overall survival in CRPC, and TFx decline is a promising biomarker for initial therapeutic response. TRIAL REGISTRATION. Dana-Farber/Harvard Cancer Center (DF/HCC) protocol no. 18-135. FUNDING. Wong Family Award in Translational Oncology, Dana Farber Cancer Institute Medical Oncology grant, Gerstner Family Foundation, Janssen Pharmaceuticals Inc., and Koch Institute Support (core) grant P30-CA14051 from the National Cancer Institute (NCI).
Atish D. Choudhury, Lillian Werner, Edoardo Francini, Xiao X. Wei, Gavin Ha, Samuel S. Freeman, Justin Rhoades, Sarah C. Reed, Gregory Gydush, Denisse Rotem, Christopher Lo, Mary-Ellen Taplin, Lauren C. Harshman, Zhenwei Zhang, Edward P. O’Connor, Daniel G. Stover, Heather A. Parsons, Gad Getz, Matthew Meyerson, J. Christopher Love, William C. Hahn, Viktor A. Adalsteinsson
Cancer results from the accumulation of genetic mutations in a susceptible cell of origin. We and others have also shown that injury promotes sarcoma development, but how injury cooperates with genetic mutations at the earliest stages of tumor formation is not known. Here, we utilized dual recombinase technology to dissect the complex interplay of the timing of KrasG12D activation, p53 deletion, and muscle injury in sarcomagenesis using a primary mouse model of soft tissue sarcoma. When mutations in oncogenic Kras and p53 are separated by 3 weeks, few sarcomas develop without injury. However, the transformation potential of these tumor-initiating cells can be unmasked by muscle injury. In the absence of Kras mutations, injury of the muscle with global deletion of p53 results in sarcomas with amplification of chromosomal regions encompassing the Met or Yap1 gene. These findings demonstrate a complex interplay between the timing of genetic mutations and perturbations in the tumor microenvironment, which provides insight into the earliest stages of sarcoma development.
David Van Mater, Eric Xu, Anupama Reddy, Leonor Añó, Mohit Sachdeva, Wesley Huang, Nerissa Williams, Yan Ma, Cassandra Love, Lanie Happ, Sandeep Dave, David G. Kirsch
Molecular mechanisms underlying the cancer stroma in metastasis need further exploration. Here, we discovered that cancer-associated fibroblasts (CAFs) produced high levels of IL-33 that acted on tumor-associated macrophages (TAMs), causing them to undergo the M1 to M2 transition. Genomic profiling of metastasis-related genes in the IL-33–stimulated TAMs showed a >200-fold increase of MMP9. Signaling analysis demonstrated the IL-33-ST2-NF-κB-MMP9-laminin pathway that governed tumor stroma–mediated metastasis. In mouse and human fibroblast-rich pancreatic cancers, genetic deletion of IL-33, ST2, or MMP9 markedly blocked metastasis. Pharmacological inhibition of NF-κB and MMP9 also blocked cancer metastasis. Deletion of IL-33, ST2, or MMP9 restored laminin, a key basement membrane component associated with tumor microvessels. Together, our data provide mechanistic insights on the IL-33-NF-κB-MMP9-laminin axis that mediates the CAF-TAM–committed cancer metastasis. Thus, targeting the CAF-TAM-vessel axis provides an outstanding therapeutic opportunity for cancer treatment.
Patrik Andersson, Yunlong Yang, Kayoko Hosaka, Yin Zhang, Carina Fischer, Harald Braun, Shuzhen Liu, Guohua Yu, Shihai Liu, Rudi Beyaert, Mayland Chang, Qi Li, Yihai Cao
Tumor neoantigens arising from somatic mutations in the cancer genome are less likely to be subject to central immune tolerance and are therefore attractive targets for vaccine immunotherapy. We utilized whole-exome sequencing, RNA sequencing (RNASeq), and an in silico immunogenicity prediction algorithm, NetMHC, to generate a neoantigen-targeted vaccine, PancVAX, which was administered together with the STING adjuvant ADU-V16 to mice bearing pancreatic adenocarcinoma (Panc02) cells. PancVAX activated a neoepitope-specific T cell repertoire within the tumor and caused transient tumor regression. When given in combination with two checkpoint modulators, namely anti–PD-1 and agonist OX40 antibodies, PancVAX resulted in enhanced and more durable tumor regression and a survival benefit. The addition of OX40 to vaccine reduced the coexpression of T cell exhaustion markers, Lag3 and PD-1, and resulted in rejection of tumors upon contralateral rechallenge, suggesting the induction of T cell memory. Together, these data provide the framework for testing personalized neoantigen-based combinatorial vaccine strategies in patients with pancreatic and other nonimmunogenic cancers.
Heather L. Kinkead, Alexander Hopkins, Eric Lutz, Annie A. Wu, Mark Yarchoan, Kayla Cruz, Skylar Woolman, Teena Vithayathil, Laura H. Glickman, Chudi O. Ndubaku, Sarah M. McWhirter, Thomas W. Dubensky Jr., Todd D. Armstrong, Elizabeth M. Jaffee, Neeha Zaidi
Chimeric antigen receptors (CARs) have an antigen-binding domain fused to transmembrane, costimulatory, and CD3ζ domains. Two CARs with regulatory approval include a CD28 or 4-1BB costimulatory domain. While both CARs achieve similar clinical outcomes, biologic differences have become apparent but not completely understood. Therefore, in this study we aimed to identify mechanistic differences between 4-1BB and CD28 costimulation that contribute to the biologic differences between the 2 CARs and could be exploited to enhance CAR T cell function. Using CD19-targeted CAR T cells with 4-1BB we determined that enhancement of T cell function is driven by NF-κB. Comparison to CAR T cells with CD28 also revealed that 4-1BB is associated with more antiapoptotic proteins and dependence on persistence for B cell killing. While TNF receptor–associated factor 2 (TRAF2) has been presupposed to be required for 4-1BB costimulation in CAR T cells, we determined that TRAF1 and TRAF3 are also critical. We observed that TRAFs impacted CAR T viability and proliferation, as well as cytotoxicity and/or cytokines, in part by regulating NF-κB. Our study demonstrates how 4-1BB costimulation in CAR T cells impacts antitumor eradication and clinical outcomes and has implications for enhanced CAR design.
Gongbo Li, Justin C. Boucher, Hiroshi Kotani, Kyungho Park, Yongliang Zhang, Bishwas Shrestha, Xuefeng Wang, Lawrence Guan, Nolan Beatty, Daniel Abate-Daga, Marco L. Davila
Sarcomas are still unsolved therapeutic challenges. Cancer stem cells are believed to contribute to sarcoma development, but lack of specific markers prevents their characterization and targeting. Here, we show that calpain-6 expression is associated with cancer stem cell features. In mouse models of bone sarcoma, calpain-6–expressing cells have unique tumor-initiating and metastatic capacities. Calpain-6 levels are especially high in tumors that have been successfully propagated in mouse to establish patient-derived xenografts. We found that calpain-6 levels are increased by hypoxia in vitro and calpain-6 is detected within hypoxic areas in tumors. Furthermore, calpain-6 expression depends on the stem cell transcription network that involves Oct4, Nanog, and Sox2 and is activated by hypoxia. Calpain-6 knockdown blocks tumor development in mouse and induces depletion of the cancer stem cell population. Data from transcriptomic analyses reveal that calpain-6 expression in sarcomas inversely correlates with senescence markers. Calpain-6 knockdown suppresses hypoxia-dependent prevention of senescence entry and also promotion of autophagic flux. Together, our results demonstrate that calpain-6 identifies sarcoma cells with stem-like properties and is a mediator of hypoxia to prevent senescence, promote autophagy, and maintain the tumor-initiating cell population. These findings open what we believe is a novel therapeutic avenue for targeting sarcoma stem cells.
Caroline Andrique, Laetitia Morardet, Laetitia K. Linares, Madi Y. Cissé, Candice Merle, Frédéric Chibon, Sylvain Provot, Eric Haÿ, Hang-Korng Ea, Martine Cohen-Solal, Dominique Modrowski
While it has been recognized that human papillomavirus–associated (HPV-associated) oropharyngeal cancer (OPC) portends an improved prognosis, distinct patterns of disease recurrence have emerged. Molecular characterization of this subset of HPV patients remains unexplored. We evaluated 52 metastatic HPV+ OPC patients from our institution and paired massively parallel sequencing data with clinical parameters and survival outcomes in 81% of patients. Genomic data were then compared with 2 molecularly defined, curable HPV+ cohorts. Metastatic HPV+ OPC patients with pulmonary-only metastases demonstrated worse outcomes. Nonexclusive somatic alterations in KMT2D and PIK3CA were most frequent, with PRKDC alterations occurring at higher frequency when compared with all sequenced HPV+ OPC patients. PI3K pathway alterations were associated with improved outcomes among metastatic HPV+ OPC patients. We demonstrate subtle differences in the mutational landscape between curable and metastatic HPV+ OPC populations, with a trend towards more frequent DNA repair protein alterations in the latter. We demonstrate improved outcomes when PI3K pathway alterations are present in these patients. We provide molecular insights for this important HPV+ subgroup that have significant therapeutic implications.
Glenn J. Hanna, Alec Kacew, Nicole G. Chau, Priyanka Shivdasani, Jochen H. Lorch, Ravindra Uppaluri, Robert I. Haddad, Laura E. MacConaill
Although initially responsive to androgen signaling inhibitors (ASIs), metastatic castration-resistant prostate cancer (mCRPC) inevitably develops and is incurable. In addition to adenocarcinoma (adeno), neuroendocrine prostate cancer (NEPC) emerges to confer ASI resistance. We have previously combined laser capture microdissection and phage antibody display library selection on human cancer specimens and identified novel internalizing antibodies binding to tumor cells residing in their tissue microenvironment. We identified the target antigen for one of these antibodies as CD46, a multifunctional protein that is best known for negatively regulating the innate immune system. CD46 is overexpressed in primary tumor tissue and CRPC (localized and metastatic; adeno and NEPC), but expressed at low levels on normal tissues except for placental trophoblasts and prostate epithelium. Abiraterone- and enzalutamide-treated mCRPC cells upregulate cell surface CD46 expression. Genomic analysis showed that the CD46 gene is gained in 45% abiraterone-resistant mCRPC patients. We conjugated a tubulin inhibitor to our macropinocytosing anti-CD46 antibody and showed that the resulting antibody-drug conjugate (ADC) potently and selectively kills both adeno and NEPC cell lines in vitro (sub-nM EC50) but not normal cells. CD46 ADC regressed and eliminated an mCRPC cell line xenograft in vivo in both subcutaneous and intrafemoral models. Exploratory toxicology studies of the CD46 ADC in non-human primates demonstrated an acceptable safety profile. Thus, CD46 is an excellent target for antibody-based therapy development, which has potential to be applicable to both adenocarcinoma and neuroendocrine types of mCRPC that are resistant to current treatment.
Yang Su, Yue Liu, Christopher R. Behrens, Scott Bidlingmaier, Nam-Kyung Lee, Rahul Aggarwal, Daniel W. Sherbenou, Alma L. Burlingame, Byron C. Hann, Jeffry P. Simko, Gayatri Premasekharan, Pamela L. Paris, Marc A. Shuman, Youngho Seo, Eric J. Small, Bin Liu
Osteosarcoma (OS), a malignant tumor of bone, kills through aggressive metastatic spread almost exclusively to the lung. Mechanisms driving this tropism for lung tissue remain unknown, though likely invoke specific interactions between tumor cells and other cells within the lung metastatic niche. Aberrant overexpression of ΔNp63 in OS cells directly drives production of IL-6 and CXCL8. All these factors were expressed at higher levels in OS lung metastases than in matched primary tumors from the same patients. Expression in cell lines correlated strongly with lung colonization efficiency in murine xenograft models. Lentivirus-mediated expression endowed poorly metastatic OS cells with increased metastatic capacity. Disruption of IL-6 and CXCL8 signaling using genetic or pharmaceutical inhibitors had minimal effects on tumor cell proliferation in vitro or in vivo, but combination treatment inhibited metastasis across multiple models of metastatic OS. Strong interactions occurred between OS cells and both primary bronchial epithelial cells and bronchial smooth muscle cells that drove feed-forward amplification of IL-6 and CXCL8 production. These results identify IL-6 and CXCL8 as primary mediators of OS lung tropism and suggest pleiotropic, redundant mechanisms by which they might effect metastasis. Combination therapy studies demonstrate proof of concept for targeting these tumor-lung interactions to affect metastatic disease.
Amy C. Gross, Hakan Cam, Doris A. Phelps, Amanda J. Saraf, Hemant K. Bid, Maren Cam, Cheryl A. London, Sarah A. Winget, Michael A. Arnold, Laura Brandolini, Xiaokui Mo, John M. Hinckley, Peter J. Houghton, Ryan D. Roberts
Although a subset of clear cell renal cell carcinoma (ccRCC) patients respond to immune checkpoint blockade (ICB), predictors of response remain uncertain. We investigated whether abnormal expression of endogenous retroviruses (ERVs) in tumors is associated with local immune checkpoint activation (ICA) and response to ICB. Twenty potentially immunogenic ERVs (πERVs) were identified in ccRCC in The Cancer Genome Atlas data set, and tumors were stratified into 3 groups based on their expression levels. πERV-high ccRCC tumors showed increased immune infiltration, checkpoint pathway upregulation, and higher CD8+ T cell fraction in infiltrating leukocytes compared with πERV-low ccRCC tumors. Similar results were observed in ER+/HER2− breast, colon, and head and neck squamous cell cancers. ERV expression correlated with expression of genes associated with histone methylation and chromatin regulation, and πERV-high ccRCC was enriched in BAP1 mutant tumors. ERV3-2 expression correlated with ICA in 11 solid cancers, including the 4 named above. In a small retrospective cohort of 24 metastatic ccRCC patients treated with single-agent PD-1/PD-L1 blockade, ERV3-2 expression in tumors was significantly higher in responders compared with nonresponders. Thus, abnormal expression of πERVs is associated with ICA in several solid cancers, including ccRCC, and ERV3-2 expression is associated with response to ICB in ccRCC.
Anshuman Panda, Aguirre A. de Cubas, Mark Stein, Gregory Riedlinger, Joshua Kra, Tina Mayer, Christof C. Smith, Benjamin G. Vincent, Jonathan S. Serody, Kathryn E. Beckermann, Shridar Ganesan, Gyan Bhanot, W. Kimryn Rathmell
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