Gardenia Cheung-Lau

Cellular Histone Modification Patterns Predict Prognosis and Treatment Response in Resectable Pancreatic Adenocarcinoma: Results From RTOG 9704

PURPOSE Differences in cellular levels of histone modifications have predicted clinical outcome in certain cancers. Here, we studied the prognostic and predictive value of three histone modifications in pancreatic adenocarcinoma. METHODS Tissue microarrays (TMAs) from two pancreatic adenocarcinoma cohorts were examined, including those from a 195-patient cohort from Radiation Therapy Oncology Group trial RTOG 9704, a multicenter, phase III, randomized treatment trial comparing adjuvant gemcitabine with fluorouracil and a 140-patient cohort of patients with stage I or II cancer from University of California, Los Angeles Medical Center. Immunohistochemistry was performed for histone H3 lysine 4 dimethylation (H3K4me2), histone H3 lysine 9 dimethylation (H3K9me2), and histone H3 lysine 18 acetylation (H3K18ac). Positive tumor cell staining for each histone modification was used to classify patients into low- and high-staining groups, which were related to clinicopathologic parameters and clinical outcome measures. Results Low cellular levels of H3K4me2, H3K9me2, or H3K18ac were each significant and independent predictors of poor survival in univariate and multivariate models, and combined low levels of H3K4me2 and/or H3K18ac were the most significant predictor of overall survival (hazard ratio, 2.93; 95% CI, 1.78 to 4.82) in the University of California, Los Angeles cohort. In subgroup analyses, histone levels were predictive of survival specifically for those patients with node-negative cancer or for those patients receiving adjuvant fluorouracil, but not gemcitabine, in RTOG 9704. CONCLUSION Cellular levels of histone modifications define previously unrecognized subsets of patients with pancreatic adenocarcinoma with distinct epigenetic phenotypes and clinical outcomes and represent prognostic and predictive biomarkers that could inform clinical decisions, including the use of fluorouracil chemotherapy.

IMPAIRED NERVE REGENERATION AND ENHANCED NEUROINFLAMMATORY RESPONSE IN MICE LACKING PITUITARY ADENYLYL CYCLASE ACTIVATING PEPTIDE

Peripheral nerve injury models are used to investigate processes that can potentially be exploited in CNS injury. A consistent change that occurs in injured peripheral neurons is an induction in expression of pituitary adenylyl cyclase activating peptide (PACAP), a neuropeptide with putative neuroprotective and neuritogenic actions. PACAP-deficient mice were used here to investigate actions of endogenous PACAP after facial nerve injury. Although motor neuron survival after axotomy was not significantly different in PACAP deficient vs. wild type mice, recovery of axon regeneration after crush injury was significantly delayed. The impaired regeneration was associated with 8- to 12-fold increases in gene expression of proinflammatory cytokines tumor necrosis factor-alpha, interferon-gamma, interleukin (IL) -6, and a 90% decrease in the anti-inflammatory cytokine IL-4 at the injury site. Similar cytokine changes and an increased microglial response were observed in the brainstem facial motor nucleus. Because immunocompromised animals such as SCID mice are known to exhibit peripheral nerve regeneration defects, the observations raise the novel hypothesis that PACAP is critically involved in a carefully controlled immune response that is necessary for proper nerve regeneration after injury.

Restoration of axotomy-induced PACAP gene induction in SCID mice with CD4+ T-lymphocytes.

PACAP is a neuropeptide with putative neuroprotective, regenerative, and immunomodulatory actions. PACAP mRNA is up-regulated in motor neurons following facial nerve axotomy in wild type, but not immunodeficient SCID mice. Because CD4+ lymphocytes appear to be neuroprotective in facial nerve and other injury models, we studied PACAP gene expression in SCID mice preinfused with CD4+ enriched splenocytes. Whereas the mean number of PACAP hybridizing neurons after axotomy was reduced by 75% in uninfused SCID mice, infusion of CD4+ enriched splenocytes restored the number to a value not significantly different than controls. The CD4+ cell-dependent induction of PACAP in motor neurons may thus be a factor in the cascade of events triggered by immune cells that ultimately lead to nerve regeneration.

VIP deficient mice exhibit resistance to lipopolysaccharide induced endotoxemia with an intrinsic defect in proinflammatory cellular responses.

Vasoactive intestinal peptide (VIP) is a pleiotropic neuropeptide with immunomodulatory properties. The administration of this peptide has been shown to have beneficial effects in murine models of inflammatory diseases including septic shock, rheumatoid arthritis, multiple sclerosis (MS) and Crohns disease. However, the role of the endogenous peptide in inflammatory disease remains obscure because VIP-deficient mice were recently found to exhibit profound resistance in a model of MS. In the present study, we analyzed the response of female VIP deficient (KO) mice to intraperitoneal lipopolysaccharide (LPS) administration. We observed significant resistance to LPS in VIP KO mice, as evidenced by lower mortality and reduced tissue damage. The increased survival was associated with decreased levels of proinflammatory cytokines (TNFα, IL-6 and IL-12) in sera and peritoneal suspensions of these mice. Moreover, the expression of TNFα and IL-6 mRNA was reduced in peritoneal cells, spleens and lungs from LPS-treated VIP KO vs. WT mice, suggesting that the resistance might be mediated by an intrinsic defect in the responsiveness of immune cells to endotoxin. In agreement with this hypothesis, peritoneal cells isolated from VIP KO naive mice produced lower levels of proinflammatory cytokines in response to LPS in vitro. Finally, decreased NF-κB pathway activity in peritoneal cells was observed both in vivo and in vitro, as determined by assay of phosphorylated I-κB. The results demonstrate that female VIP KO mice exhibit resistance to LPS-induced shock, explainable in part by the presence of an intrinsic defect in the responsiveness of inflammatory cells to endotoxin.

Vasoactive Intestinal Peptide-Deficient Mice Exhibit Reduced Pathology in Trinitrobenzene Sulfonic Acid-Induced Colitis

Objectives: Vasoactive intestinal peptide (VIP) is an immunomodulatory neuropeptide with therapeutic properties in multiple murine models of inflammatory disease including the trinitrobenzene-sulfonic acid (TNBS)-colitis model of Crohns disease. Understanding the spectrum of biological actions of endogenously produced VIP may help us dissect the complex and multifactorial pathogenesis of such inflammatory diseases. Our goal was to determine the contribution of endogenously produced VIP to TNBS-colitis by using VIP knockout (KO) mice. Methods: TNBS was intracolonically administered to wild-type (WT) and VIP KO mice, and weight loss and colitis were assessed over time. Colon histopathological changes and myeloperoxidase activities were analyzed and the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in colon and serum quantified. The proliferative response in vitro of splenocytes from TNBS WT and VIP KO administered mice to anti-CD3 and anti-CD28 was determined. Results: VIP KO mice did not exhibit the predicted exacerbated response to TNBS. Instead, they developed a milder clinical profile than WT mice, with lower TNF-α and IL-6 levels. Such potential defects seem selective, because other parameters such as the histopathological scores and the cytokine levels in the colon did not differ between the two strains of mice. Moreover, splenocytes from TNBS-treated VIP KO mice exhibited an enhanced proliferative response to anti-CD3/CD28 stimulation in vitro. Conclusion: Chronic loss of VIP in mice leads to a disruption of certain but not all immunological compartments, corroborating recent findings that VIP KO mice exhibit reduced mortality in the lipopolysaccharide-induced endotoxemia model and attenuated clinical development of experimental autoimmune encephalomyelitis while developing robust T-cell responses.

Abstract 3765: IND-Enabling GLP study to support a clinical trial of dual adoptive cell therapy combining stem cells and T cells engineered with an NY-ESO-1 TCR

T cell receptor (TCR) engineered adoptive T cell transfer (ACT) has shown remarkable antitumor efficacy in several clinical trials. However, low persistence of modified cells limits long-term clinical responses. To overcome this hurdle, we propose a clinical trial co-administering genetically modified T cells and stem cells both expressing an NY-ESO-1 TCR such that the engrafted stem cells generate a source for constant renewal of modified T cells. Here we report a pre-clinical IND-enabling study performed at UCLA under Good Laboratory Practice (GLP) compliance to assess whether co-administration impacts (I) safety; (II) engraftment and cell lineage differentiation of gene modified stem cells; and (III) persistence of adoptively transferred T cells and stem cell-derived progeny. We performed 12 optimization studies to define the optimal conditions for TCR gene modified ACT and TCR gene modified hematopoietic stem cell (HSC) bone marrow transplantation (BMT). Sixty-four HLA-A2/kb transgenic mice were myelodepleted and received syngeneic BMT with Lineage depleted bone marrow (Lin-) cells transduced with the LV-NYESO-1 TCR/sr39TK and ACT with T cells transduced with the RV-NYESO-1 TCR. Control groups were as follows: untreated mice, mice receiving mock transduced Lin- cells and T cells, mice receiving transduced Lin- cells and mock transduced T cells, and mice receiving mock transduced Lin- cells and transduced T cells (n = 16 per group). Overall survival at 3 months was 87.5%; no significant differences in survival were observed among cohorts. After BMT we observed a decrease in body weight, elevation in creatinine kinase and transaminases, and gonadal germ cell ablation in all cohorts. Three months after BMT, all blood cell lineages were reconstituted in surviving mice. Using digital droplet PCR and flow cytometry, we confirmed that transduced stem cells engrafted and their progeny persisted long term. In the bone marrow, NY-ESO-1 TCR was expressed intracellularly among progenitor cells (Lin-, LSK and HSC) as well as all hematopoietic cell lineages within the spleen (CD8+ T cells, CD4+ T cells, NKT cells, B cells and granulocytes). Co-administration with gene modified T cells and stem cells did not affect engraftment, cell lineage differentiation or persistence of the gene modified stem cells. Moreover, co-administration with stem cells did not affect persistence of adoptively transferred T cells. These data demonstrate that 1) NY-ESO-1 TCR genetically modified stem cells engraft and differentiate into all hematopoietic cell lineage progeny, which persists at 3 months; 2) adoptively transferred NY-ESO-1 TCR T cells persist at 3 months; 3) co-administration of stem cells and T cells genetically modified to express an NY-ESO-1 TCR is safe and does not negatively impact stem cell engraftment, lineage differentiation and progeny persistence or T cell persistence. Citation Format: Cristina Puig-Saus, Giulia Parisi, Paige Krystofinski, Angel Garcia-Diaz, Salemiz Sandoval, James McCabe, Ruixue Zhang, Gardenia Cheung-Lau, Nhat Truong, Justin Saco, Sara Komenan, Agustin Vega-Crespo, Mignonette H Macabali, Begona Comin-Anduix, Beata Berent-Maoz, Donald Kohn, Paula Kaplan-Lefko, Antoni Ribas. IND-Enabling GLP study to support a clinical trial of dual adoptive cell therapy combining stem cells and T cells engineered with an NY-ESO-1 TCR [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 3765. doi:10.1158/1538-7445.AM2017-3765