Anti-cancer Immune Cells Research Articles

Abstract B02: Tissue-specific innate lymphoid cells are novel targets for pancreatic cancer immunotherapy

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the classic cold tumor, with few tumor-infiltrating activated CD8+ T cells, a major barrier to immunotherapy. Strategies to convert cold PDACs to hot can enable more effective immunotherapies. To identify targets that can convert cold PDACs to hot, we compared a large cohort of rare long-term PDAC survivors with hot tumors to short-term survivors with cold tumors. Surprisingly, hot PDACs were enriched not only in activated CD8+ T cells, but also in group-2 innate lymphoid cells (ILC2s), a class of largely tissue-resident, cytokine-producing innate lymphocyte that potentiates T cell immunity in local tissues. We identified that tumor ILC2s (TILC2s) infiltrate human and mouse PDACs, and TILC2 frequency and tumor expression of the ILC2-activating ligand interleukin (IL)-33 positively correlate with tumor immune cytolytic activity, and long-term patient survival. Using PDAC mouse models, we discovered that the IL33-ILC2 axis activates pancreatic tissue-specific antitumor T-cell immunity. Host IL33 deficiency attenuated TILC2 expansion and CD8+ T-cell activation, accelerating tumor growth in orthotopic PDACs but not heterotopic skin implanted PDACs where TILC2s lack the IL33 receptor. Acute ILC2 depletion partially phenocopied the tissue-specific phenotype of IL33 deficiency, with impaired rejection of immunogenic orthotopic but not heterotopic skin PDACs. Treatment with recombinant IL33 (rIL33), but not with the ILC1-activating ligand rIL18, increased TILC2 frequencies, activated CD8+ T cells, and abrogated orthotopic but not heterotopic PDAC establishment in >70% of mice. Taken together, these studies identify ILC2s as novel anticancer immune cells for PDAC immunotherapy. Strategies to activate tissue-specific ILC2s may be a promising immunotherapeutic approach for PDAC. Citation Format: Joanne Leung, Luis A Rojas, Jennifer Ruan, Julia Zhao, Billel Gasmi, Zachary Sethna, Anita Ramnarain, Umesh Bhanot, Gokce Askan, Murali Gururajan, Ela Elyada, Youngkyu Park, David A. Tuveson, Mithat Gonen, Steven D. Leach, Jedd D. Wolchok, Ronald P. DeMatteo, Taha Merghoub, Vinod P. Balachandran, John Alec Moral. Tissue-specific innate lymphoid cells are novel targets for pancreatic cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B02.

High expression of bone morphogenetic protein (BMP) 6 and BMP7 are associated with higher immune cell infiltration and better survival in estrogen receptor‑positive breast cancer.

There are conflicted experimental results on the role of bone morphogenetic proteins (BMPs) in cancer. Some results suggest that BMPs act as tumor suppressors while other findings indicate that BMPs are oncogenic factors. In the present study, we aimed to investigate the association of BMP expression and the survival of breast cancer patients utilizing The Cancer Genome Atlas (TCGA). High expression levels of BMP1 (P<0.001), BMP3 (P=0.002), BMP5 (P=0.002), BMP7 (P<0.001) and BMPR1A (P<0.001) were associated with better overall survival (OS). On the other hand, high expression levels of BMP6 (P<0.001), BMP8A (P=0.031), BMP8B (P<0.001) and BMPR1B (P=0.005) were associated with worse OS. Most of the BMPs demonstrated differential expression levels between normal and tumor tissues. High expression of BMP7 was found to be significantly associated with better prognosis in both estrogen receptor (ER)-positive (ER+) (P<0.001) and ER-negative (ER−) tumors (P<0.001), whereas high expression of BMP6 was associated with better prognosis only in ER+ tumors (P=0.004); it was associated with worse prognosis in ER− tumors (P=0.006). In the ER+ tumors, high expression of BMP7 as well as BMP6 were associated with higher infiltration of anticancer immune cells and cytolytic activity. These results suggest that high expression levels of BMP7 as well as BMP6 possess higher anticancer immunity which results in better prognosis in ER+ breast cancer. In conclusion, BMP expression profiles may be useful for prognostication. Intervention in this pathway may serve to improve outcomes, manage metastatic disease and assist in clinical decision making on optimal therapy based on the risk of recurrence or metastasis.

Abstract 2453: A new deconvolution algorithm for accurate assessing immune and stromal cell populations in mouse transcriptomic data

Abstract Multiple deconvolution methods have been developed for investigating the heterogeneous immune and stromal (I/S) cell types in human cancer tissue by deciphering their relative abundances using transcriptomic data. However, there is a lack of a user-friendly software for mouse transcriptomic data deconvolution. Key challenges for developing such a tool include: (1) limited training data sets for available for deriving signature genes of I/S cell types in sporadic mouse data; (2) mouse models of diverse geno-/pheno-types may have varied expressions of I/S cell marker genes; (3) the transcriptomic data may be collected from mouse with certain levels of immuno-deficiency; and (4) the transcriptomic data may come from highly diverse experimental platforms. To solve these challenges, we (i) developed a novel non-parametric analysis method to derive potential I/S cell signature genes from a large collection of mouse data sets; (ii) implemented a low rank sub matrix identification method with a non-negative matrix factorization (NMF) based deconvolution method; and (iii) enabled the flexibility that certain I/S cell types may be absent if their respective cell markers do not form a significant low rank structure. The new method is applied to mouse prostate cancer data sets to infer the level of anti-cancer immune cell populations. Genes expressed by cancer cells that are negatively associated with the anti-cancer immune cells are further inferred, and compared with the results derived in human data. A user-friendly R package of the deconvolution method is released through GitHub. Citation Format: Xiaoyu Lu, Szu-wei Tu, Wennan Chang, Yan Huo, Pengcheng Wang, Yu Zhang, Chi Zhang, Sha Cao. A new deconvolution algorithm for accurate assessing immune and stromal cell populations in mouse transcriptomic data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2453.

Abstract 5200: High CD73 expression, regulated by estrogen signaling, associates with cancer aggressiveness in estrogen receptor (+) breast cancer

Abstract Background: CD73, a cell surface enzyme, catalyzes the generation of adenosine from ATP and ADP in the tumor microenvironment along with CD39. Accumulated extracellular adenosine functions as immune-suppressor, and also binds to adenosine receptors which promotes angiogenesis and cell proliferation that results in accelerate cancer progression. However, the clinical significance and molecular function of CD73 expression in breast cancer remains unclear. Methods: Utilizing breast cancer cohorts of TCGA and GEO, clinical significance and underlying mechanisms were investigated. Molecular experiments were carried out in MCF7 cells, ER(+) breast cancer cell line. Results: In TCGA cohort, CD73 expression level was significantly lower in ER(+) breast cancers compared to ER(-) tumors. Higher CD73 expression was associated with worse overall survival in ER(+) tumors (p=0.003), but not in ER(-) tumors. Gene Set Enrichment Analysis revealed that estrogen response gene sets (Early; p=0.043, Late; p=0.021) were significantly enriched in CD73 low expressing ER(+) tumors, suggesting estrogen signaling may repress CD73 expression. To test this hypothesis, we analyzed the expression of CD73 and CD39 in MCF7 cells treated with estrogen, tamoxifen. Our data revealed that estrogen treatment suppressed CD73 and CD39 expression, whereas tamoxifen treatment enhanced expression of the genes. These findings suggest that CD73 and CD39 gene expression is suppressed by estrogen signaling, whereas binding of ER antagonists such as tamoxifen can remove the repressive effect on gene expression. On the other hand, epithelial-mesenchymal transition (EMT) (p&amp;lt;0.001) and angiogenesis (p&amp;lt;0.001) gene sets were significantly enriched in CD73 high expressing ER(+) tumors. CIBERSORT demonstrated that CD73 high expressing ER(+) tumors have less infiltrating CD8(+) T cells, memory B cells and plasma cells, implying that CD73 high expressing tumors have immune suppressive environment, which is in agreement with the notion that CD73 high tumors are immunosuppressive. Finally, we found that CD73 expression was significantly elevated post-chemotherapy compared to tumors prior to the treatment (p=0.007), and CD73 high expression patients showed worse relapse-free survival in neoadjuvant chemotherapy patients cohort (p=0.003). Conclusion: Molecular studies revealed that CD73 expression is regulated by estrogen signaling. Increased expression of CD73 significantly correlates with worse outcomes in ER(+) breast cancer patients. This may be due to upregulated pro-metastatic gene signatures such as EMT and angiogenesis as well as less infiltration of anti-cancer immune cells by adenosine generated by CD73 in the tumor microenvironment. Our data reveals an intriguing mechanism which may be responsible for recurrence and metastasis of ER(+) breast cancer. Note: This abstract was not presented at the meeting. Citation Format: Eriko Katsuta, Vivek Anand, Li Yan, Subhamoy Dasgupta, Kazuaki Takabe. High CD73 expression, regulated by estrogen signaling, associates with cancer aggressiveness in estrogen receptor (+) breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5200.

Estrogen Receptor Positive Breast Cancer with High Expression of Androgen Receptor has Less Cytolytic Activity and Worse Response to Neoadjuvant Chemotherapy but Better Survival

Estrogen receptor (ER) positive breast cancer (BC), the most abundant BC subtype, is notorious for poor response to neoadjuvant chemotherapy (NAC). The androgen receptor (AR) was reported to support estradiol-mediated ER activity in an in vitro system. Recently, ER-positive BC with fewer tumor infiltrating lymphocytes (TILs) was shown to have a better prognosis, opposite to the trend seen with ER-negative BC. We hypothesized that ER-positive BC with high expression of AR will have fewer TILs and an inferior response to NAC, but with a better prognosis. In both TCGA and METABRIC cohorts, AR expression was significantly higher in ER-positive BCs compared to ER-negatives (p < 0.001, p < 0.001, respectively) and it correlated with ER expression (R = 0.630, R = 0.509, respectively). In ER-positive tumors, AR high tumors enriched UV response down (NES = 2.01, p < 0.001), and AR low tumors enriched DNA repair (NES = −2.02, p < 0.001). AR high tumors were significantly associated with procancer regulatory T-cells, and AR low tumors were associated with anticancer immune cells, such as CD4, CD8, and Gamma-Delta T-cells and memory B-cells in ER-positive BC (p < 0.01). Further, cytolytic activity was significantly lower in AR high BC in both cohorts. Finally, AR high tumors had a significantly lower rate of attaining pathological complete response to NAC (GSE22358), but better survival. In conclusion, our results demonstrated that high AR has fewer tumor infiltrating lymphocytes as well as cytolytic activity and an inferior response to NAC, but better survival in ER-positive BC.

Abstract P2-02-04: CD73 expression regulated by estrogen signaling associates with poor prognosis in estrogen receptor (ER)-positive breast cancer

Abstract Introduction: CD73, a cell surface enzyme, catalyzes the generation of adenosine from ATP and ADP in the tumor microenvironment along with CD39. Accumulated extracellular adenosine functions as immune-suppressor, and also binds to adenosine receptors which promotes angiogenesis and cell proliferation that results in accelerate cancer progression. However, the clinical significance and molecular function of CD73 expression in breast cancer remains unclear. Methods: Utilizing publicly available breast cancer cohorts of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO), clinical significance as well as underlying mechanisms were investigated. Molecular experiments were carried out in MCF7 cells, ER-positive breast cancer cell line, to investigate the role of estrogen signaling on CD73/CD39 expression. Results: In treatment naïve TCGA cohort, CD73 expression level was significantly lower in ER-positive breast cancers compared to ER-negative tumors. Higher CD73 expression was associated with worse overall survival in whole cohort (p=0.021) and ER-positive tumors (p=0.003), but not in ER-negative tumors. Gene Set Enrichment Analysis revealed that estrogen response gene sets (Early; NES=-1.57, p=0.043, Late; NES=-1.61, p=0.021) were significantly enriched in CD73 low expressing ER-positive tumors, suggesting estrogen signaling may repress CD73 expression. To test this hypothesis, we analyzed the expression of CD73 and CD39 in MCF7 cells treated with estrogen, tamoxifen or both. Our data revealed that estrogen treatment suppressed CD73 and CD39 expression, whereas tamoxifen treatment enhanced expression of the genes. These findings suggest that CD73 and CD39 gene expression is suppressed by estrogen signaling, whereas binding of ER antagonists such as tamoxifen can remove the repressive effect on gene expression. On the other hand, epithelial-mesenchymal transition (EMT) (Normalized Enrichment Score; NES=2.41, p&amp;lt;0.001) and angiogenesis (NES=2.33, p&amp;lt;0.001) gene sets were significantly enriched in CD73 high expressing ER-positive tumors. CIBERSORT, which is an algorithm to estimate infiltrating immune cells by gene expression, demonstrated that CD73 high expressing ER-positive tumors have less infiltrating CD8-positive T cells, memory B cells and plasma cells, implying that CD73 high expressing tumors have immune suppressive environment, which is in agreement with the notion that CD73 high tumors are immunosuppressive. Finally, we found that CD73 expression was significantly elevated post-chemotherapy compared to tumors prior to the treatment (p=0.007), and CD73 high expression patients showed worse relapse-free survival in neoadjuvant chemotherapy patients cohort (p=0.003). Conclusion: Molecular studies revealed that CD73 expression is regulated by estrogen signaling. Increased expression of CD73 significantly correlates with worse outcomes in ER-positive breast cancer patients. This may be due to upregulated pro-metastatic gene signatures such as EMT and angiogenesis as well as less infiltration of anti-cancer immune cells by adenosine generated by CD73 in the tumor microenvironment. Our data reveals an intriguing mechanism which may be responsible for recurrence and metastasis of ER-positive breast cancer. Citation Format: Katsuta E, Anand V, Yan L, Dasgupta S, Takabe K. CD73 expression regulated by estrogen signaling associates with poor prognosis in estrogen receptor (ER)-positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-02-04.

Pancreatic adenocarcinomas with mature blood vessels have better overall survival

Pancreatic ductal adenocarcinoma (PDAC) is known for its hypovascularity. Bevacizumab, an anti-angiogenic drug, added to standard chemotherapy demonstrated no improvement in outcome for PDAC. Therefore, we hypothesized that increased vascularity may be associated with improved outcomes in PDAC possibly due to better delivery of tumor specific immune cells. To test this hypothesis, PDAC patients were classified into either high or low CD31 expression groups utilizing mRNA expression from RNA-sequence data in The Cancer Genome Atlas (TCGA) pancreatic cancer cohort. High expression of CD31, which indicates presence of more vascular endothelial cells, was associated with significantly better OS (p = 0.002). Multivariate analysis demonstrated that residual tumor (R1, 2; p = 0.026) and CD31 low expression (p = 0.007) were the only independent predictors that negatively impacted OS. Vascular stability as well as immune response related pathways were significantly upregulated in the CD31 high expressing tumors. Furthermore, there were higher proportions of anti-cancer immune cells infiltration, including activated memory CD4+ T cells (p = 0.038), CD8+ T cells (p = 0.027), gamma-delta T cells (p < 0.001) as well as naïve B cells (p = 0.006), whereas lower proportions of regulatory T cell fractions (p = 0.009), which induce an immune tolerant microenvironment, in the CD31 high expressing tumors. These findings imply that stable vessels supply anti-cancer immune cells, which are at least partially responsible for better OS in the CD31 high expressing tumors. In conclusion, CD31 high expressing PDACs have better OS, which may be due to stable vessels that supply anti-cancer immune cells.

Visualizing Interactions of Circulating Tumor Cell and Dendritic Cell in the Blood Circulation Using In Vivo Imaging Flow Cytometry.

Visualizing cell interactions in blood circulation is of great importance in studies of anticancer immunotherapy or drugs. However, the lack of a suitable imaging system hampers progress in this field. In this work, we built a dual-channel in vivo imaging flow cytometer to visualize the interactions of circulating tumor cells (CTCs) and dendritic cells (DCs) simultaneously in the bloodstream. Two artificial neural networks were trained to identify blood vessels and cells in the acquired images. Using this technique, single CTCs and CTC clusters were readily distinguished by their morphology. Interactions of CTCs and DCs were identified, while their moving velocities were analyzed. The CTC-DC clusters moved at a slower velocity than that of single CTCs or DCs. This may provide new insights into tumor metastasis and blood rheology. This in vivo imaging flow cytometry system holds great potential for assessing the efficiency of targeting CTCs with anticancer immune cells or drugs.

PO-263 Assessment of the accumulation of hyaluronan in the tumour microenvironment (TME) of solid tumours

IntroductionHyaluronan (HA), a primary component of the extracellular matrix, can accumulate in a variety of solid malignancies, where histological analyses of patient tumour tissues demonstrated that HA levels may correlate with patient outcomes. HA accumulation in the TME may be associated with poor prognosis. Preclinical studies demonstrated elevated TME HA levels increased tumour interstitial pressure, reduced tumour blood flow, and resulted in poor penetration of anticancer agents and immune cells. A PEGylated recombinant human hyaluronidase, pegvorhyaluronidase alfa (PEGPH20), depolymerizes HA, increases tumour vascular perfusion, and may increase access and anti-tumour efficacy of cytotoxic and immunotherapies.Material and methodsPegvorhyaluronidase alfa is being evaluated in clinical studies for pancreatic, breast, lung, cholangiocarcinoma, and gastric cancers. To broaden the spectrum of possible tumour types that may benefit from pegvyorhyaluronidase alfa, we assessed the prevalence and accumulation of HA in 3633 human histological samples in 22 solid tumour types. HA levels were measured using tissue microarrays (TMAs) stained with a proprietary HA probe, HTI–601, a recombinant immunoadhesin containing a modified HA–specific link domain from TNF-Stimulated Gene 6 protein (TSG–6) and the Fc portion of human IgG1. The resulting HA probe was biotinylated for use in an affinity-based histochemistry method. HA staining was evaluated with research-grade histological image analysis software. HA levels associated with distinct tumour compartments (stroma or tumour cells) were assessed. HA accumulation associated with immune infiltrates present in the TMA cores was also evaluated.Results and discussionsDistribution of HA accumulation across compartments fell into 4 main arrangements:- For desmoplastic tumour indications (pancreatic ductal adenocarcinoma, cholangiocarcinoma), the stroma was associated with high percentage of surface area stained with HTI-601 relative to the tumour cells;- Comparable distribution of HA staining in both tumour cells and stroma (gall bladder and glioblastoma);- Sub-clusters of HA staining within a specific tumour type (NSCLC non-squamous);- Distribution bias towards increased or decreased HA intensity staining (head and neck excluding nasopharyngeal cavities and renal cell carcinoma, respectively).ConclusionUnderstanding HA prevalence and accumulation in solid tumours may potentially identify malignancies that may benefit from pegvorhyaluronidase alfa therapy targeting HA.

New and traditional directions in the biology and management of childhood acute lymphoblastic leukemia

Owing to clinical trials and improvement over the past few decades, the majority of children with acute lymphoblastic leukemia (ALL) survive by first-line chemotherapy and combat with the problems of returning to community. However, many patients may have severe acute or late therapeutic side effects, and the survival rate in some groups (e.g., patients with MLL rearrangements, hypodiploidy, IKZF1 mutation or early precursor T cell phenotype) is far behind the average. Innovative strategies in medical attendance provide better clinical outcomes for them: complete gene diagnostics, molecularly targeted anticancer treatment, immuno-oncology and immune cell therapy. The number of genes with identified alterations in leukemic lymphoblasts is over thirty and their pathobiologic role is only partly clear. There are known patient groups where the use of specific drugs is based on gene expression profiling (e.g., tyrosine kinase inhibitors in Philadelphia-like B-cell ALL). The continuous assessment of minimal residual disease became a routine due to the determination of a leukemia-associated immunophenotype by flow cytometry or a sensitive molecular marker by molecular genetics at diagnosis. Epitopes of cluster differentiation antigens on blast surface (primarily CD19, CD20 and CD22 on malignant B cells) can be attacked by monoclonal antibodies. Moreover, antitumor immunity can be strengthened utilizing either cell surface markers (bispecific T cell engagers, chimeric antigen receptor T cell therapy) or tumor-specific immune cells (immune checkpoint inhibitors). This review gives an insight into current knowledge in these innovative therapeutic directions. Orv Hetil. 2018; 159(20): 786-797.

Voluntary physical exercise decreases MDSC (myeloid derived suppressor cell) populations in the spleen and circulation in a rat model of mammary adenocarcinoma

Abstract Myeloid derived suppressor cells (MDSCs) are a heterogenous population of myeloid progenitor cells that suppress T cells and other potentially anti-cancer immune cells. MDSC presence in cancer patients can diminish immunotherapy response and worsen treatment outcomes. Here we examine a low-risk, low-cost approach to MDSC modulation by examining the impact of voluntary wheel-running exercise on MDSC populations in a Fisher-344 rat, MATBIII mammary adenocarcinoma model. Rat MDSCs in blood, spleen, bone marrow, and tumor tissue were identified by dual expression of surface markers His48 and CD11b and confirmed to be immunosuppressive upon sorting and interrogation in an in-vitro T cell suppression assay. We find that the percentage of MDSCs (as a proportion of total cells) is reduced in whole spleen isolates (2.55% vs. 19.68%, P≤0.005) and in the circulating white blood cell fraction (1.33% vs. 23.21%, P≤0.05) of exercised versus sedentary animals. In contrast, we find no difference between the proportion of MDSCs as a population of total cells in bone marrow (23.57% vs. 25.52%, P≥0.05) or tumor tissue (11.68% vs. 7.10%, P≥0.05). We conclude that exercise decreases splenic and circulating MDSC populations in tumor bearing animals. Further studies are necessary to establish the molecular mechanisms of this suppression to leverage potential interactions between exercise and MDSC-directed immune therapies.

Abstract OT2-02-02: A randomized, open-label, multicenter, phase 1b/2 study of eribulin mesylate in combination with PEGylated recombinant human hyaluronidase in patients with human epidermal growth factor receptor 2-negative, high-hyaluronan metastatic breast cancer

Abstract Eribulin (ERI), a non-taxane microtubule inhibitor, is approved for treatment in patients (pts) with advanced or metastatic breast cancer (MBC) who have received ≥1 (European Union) or ≥2 (United States) prior chemotherapy regimens for metastatic disease, including an anthracycline and taxane in either the adjuvant or metastatic setting. In a phase 2 study, ERI has demonstrated single-agent activity in first line treatment of pts with human epidermal growth factor receptor 2-negative (HER2−) MBC. Hyaluronan (HA) is a major component of the tumor microenvironment, specifically within the stroma, and high levels of HA are associated with disease progression. In HA-High, triple-negative breast cancer (TNBC) xenografts, the anti-tumor effect of ERI was enhanced by addition of PEGylated recombinant human hyaluronidase (PEGPH20). PEGPH20 is an engineered enzyme under investigation for use in combination with anticancer therapies in pts with tumors that accumulate HA. PEGPH20 acts by temporarily degrading HA to reduce tumor interstitial pressure and decompress blood vessels, thus increasing access of anticancer agents and immune cells to tumor sites. This randomized, open-label, multicenter, study of ERI in combination with PEGPH20 vs ERI alone was designed to evaluate safety and tolerability and efficacy in pts with HER2−, HA-High, MBC previously treated with 0–1 systemic (cytotoxic or targeted) anticancer therapy. The trial was activated in June 2016 and will enroll approximately 96 pts (aged ≥18 yrs; n=6–18 in phase 1b; n=84 randomized to obtain 80 evaluable pts in phase 2).Pts with measurable disease of ≥1 lesion ≥10 mm in long-axis diameter (nonlymph nodes) or ≥15 mm in short-axis diameter (lymph nodes) with ECOG PS of 0 or 1 will be included. Pts will be excluded for having had adjuvant chemotherapy within the past 6 months, hormonal/biological therapy within the past 3 wks, or radiation or small molecule targeted therapy within the past 2 wks. Pts will also be excluded if they were previously treated with ERI or any hyaluronidase agent. Phase 1b includes at least 1 initial safety run-in cohort in which 6 pts (any HA level) will receive PEGPH20 3 μg/kg intravenous (IV) on days −1 and 7 of a 21-day cycle, each dose followed approximately 24 (±4) hours later with ERI 1.4 mg/m2 IV on days 1 and 8. Dose-limiting toxicity (DLT) will be assessed in the first cycle.The primary endpoint in phase 1b is the recommended phase 2 dose (RP2D) of the ERI and PEGPH20 combination, defined as the maximum dose at which ≤1 pt experiences a DLT.Alternatively, modified dose levels will be evaluated in additional cohorts. In phase 2, HA-High pts will be stratified by TNBC vs other HER2− status and randomized to receive RP2D combination treatment or ERI alone. Pts with clinical benefit will remain on ERI or both study drugs until unacceptable toxicity or disease progression. The primary endpoint in phase 2 is objective response rate. Secondary endpoints include progression-free survival and overall survival. Exploratory endpoints include clinical benefit rate, disease control rate, and duration of response. Citation Format: Alvarez RH, Savulsky C, Almonte A, Xing D, Lokker N, Chondros D. A randomized, open-label, multicenter, phase 1b/2 study of eribulin mesylate in combination with PEGylated recombinant human hyaluronidase in patients with human epidermal growth factor receptor 2-negative, high-hyaluronan metastatic breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr OT2-02-02.

RARRES2 functions as a tumor suppressor by promoting β-catenin phosphorylation/degradation and inhibiting p38 phosphorylation in adrenocortical carcinoma.

Tumor suppressor genes and the immune system are critical players in inhibiting cancer initiation and/or progression. However, little is known about whether a tumor suppressor gene can function through both immune-dependent and -independent mechanisms. Retinoic acid receptor responder 2 (RARRES2) is transcriptionally downregulated in multiple cancer types. Previous studies suggested that it can serve as an immune-dependent tumor suppressor by acting as a chemoattractant to recruit anti-cancer immune cells expressing its receptor, the chemerin chemokine receptor 1 (CMKLR1), to sites of tumor. In this study, we investigated the role of RARRES2 in adrenocortical carcinoma (ACC), a rare lethal malignancy in which aberrant Wnt/β-catenin signaling is frequently detected. We show that RARRES2 expression is downregulated in ACC as compared to normal and benign adrenocortical tissues, which is a result of CpG hypermethylation. Despite minimal CMKLR1 expression and lack of phenotypic tumor-suppressive effect with exogenous RARRES2 treatment, RARRES2 overexpression in ACC cell lines not only reduced cell proliferation, cell invasion and tumorigenicity in vitro, but also inhibited tumor growth in vivo in two immunodeficient mouse xenograft models. Mechanistically, RARRES2 overexpression in ACC cells inhibited Wnt/β-catenin pathway activity by promoting β-catenin phosphorylation and degradation, it also inhibited the phosphorylation of p38 mitogen-activated protein kinase. Thus our study identifies RARRES2 as a novel tumor suppressor for ACC, which can function through an immune-independent mechanism.

Breaking the Barrier—PEGylated Recombinant Human Hyaluronidase (PEGPH20)—A New Therapeutic Approach to the Treatment of Pancreatic Ductal Adenocarcinoma

New therapeutic approaches are urgently needed to improve survival for patients with metastatic pancreatic ductal adenocarcinoma (PDA). This carcinoma is characterized by a hyaluronan (HA)-rich desmoplastic stroma that raises tumor interstitial fluid pressure (IFP), which in turn compresses the vasculature and impedes access of anti-cancer therapies and immune cells to tumor sites. It is this biophysical barrier that is the target for PEGylated recombinant human hyaluronidase (PEGPH20; pegvorhyaluronidase alfa), which degrades HA polymers to tetra- and hexa-saccharides to remodel the tumor stroma. In preclinical models, PEGPH20 reduced IFP, and expanded tumor vasculature to improve perfusion, which increased access for innate immune cells, antibodies and therapeutic agents. The results of a phase Ib study have suggested benefits in overall survival and progression-free survival (PFS) for patients with tumors that accumulate HA (termed HA-High) treated with a combination of gemcitabine and PEGPH20. A phase II study (HALO 109-202) demonstrated that HA could be a potential biomarker for identifying patients who may be most suitable for PEGPH20 treatment. HALO 109-202 showed positive outcomes for PFS especially in HA-High patients treated with PEGPH20 plus nab-paclitaxel and gemcitabine. A randomized, double-blind, phase III study (HALO 109-301) exploring the benefits of PEGPH20 in HA-High patients with PDA is ongoing. Other PEGPH20-based combinations are being investigated in multiple stroma-rich cancers, including lung, gastric, and breast. PEGPH20 is the most advanced therapy targeting the tumor stroma and has the potential to form the therapeutic backbone for the treatment of stroma-rich tumors.

Abstract 3664: RARRES2 functions as a direct tumor suppressor by promoting beta-catenin phosphorylation and degradation independent of CMKLR1 and CMKLR1-mediated antitumor immune response in adrenocortical carcinoma

Abstract Tumor suppressors and the immune system are critical players in tumor suppression. However, little is known about whether a tumor suppressor can function through both immune-dependent and immune-independent mechanisms. RARRES2 is a small secreted protein that is down-regulated in multiple human cancer types such as adrenocortical carcinoma (ACC), hepatocellular carcinoma (HCC) and melanoma. It has been shown that RARRES2 can play an indirect tumor-suppressive role by acting as a chemoattractant to recruit anti-cancer immune cells expressing its receptor CMKLR1 to sites of tumor. In this study, we show that epigenetic CpG hypermethylation of RARRES2 in vivo and in vitro in ACC causes RARRES2 down-regulation. We also found that RARRES2 can function as a direct tumor suppressor and that its tumor suppressive effect does not depend on the recruitment of CMKLR1-expressing immune cells. We show that although exogenous treatment of cultured cells with recombinant human RARRES2 protein did not show any tumor-suppressive effect, transient overexpression of RARRES2 significantly inhibited cellular invasion. Stable overexpression of RARRES2 in ACC cell line NCI-H295R led to significantly reduced cell proliferation, cell invasion, and tumorigenecity in a RARRES2 dose-dependent manner. Using xenografts of stable NCI-H295R cell lines in two immunodeficient mouse models (athymic nude mice and NSG mice), we found that in the absence of functional immune cells, stable RARRES2 overexpression can still significantly suppress tumor growth in vivo. RARRES2 overexpression promoted beta-catenin phosphorylation and degradation, reduced the phosphorylation of p38 MAP kinase and inhibited the secretion of matrix metalloprotease protein 10 (MMP10) from cancer cells in a RARRES2 dose-dependent manner. Taken into account that CMKLR1 expression is barely detectable in all the cell lines tested, our results present the first evidence that RARRES2 can function as a direct tumor suppressor independent of CMKLR1 and CMKLR1-mediated anti-tumor immune response. Citation Format: Yi Liu-Chittenden, Kelli Gaskins, Sophie Wang, Electron Kebebew. RARRES2 functions as a direct tumor suppressor by promoting beta-catenin phosphorylation and degradation independent of CMKLR1 and CMKLR1-mediated antitumor immune response in adrenocortical carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3664.

Abstract IA09: Reprogramming T cells for adoptive immunotherapy of ovarian cancer.

Abstract The goal of our studies is to generate robust and long-lasting tumor-specific T cell responses for durable tumor regression in patients with epithelial ovarian cancer (EOC). While the majority of women with advanced stage EOC initially respond to surgery and first-line chemotherapy, more than 70% of patients eventually die of recurrent disease within 5 years of diagnosis. In an effort to generate tumor-associated antigen (TAA) specific T cells for the treatment of EOC patients, our team has identified NY-ESO-1 as the prototypic TAA for immunotherapy in ovarian cancer. In clinical trials of cancer vaccines conducted by our group, although active immunization targeting NY-ESO-1 can generate TAA specific effector T cells, the long term control of ovarian cancer is infrequent. This is primarily because of the relatively low magnitude and short in vivo lifespan of the vaccine-elicited T cells limited long-term tumor control in the patients. Consequently, we have focused on genetically re-engineering T cells to express a NY-ESO-1 specific CD8+ T cell receptor (CD8TCR), followed by adoptive transfer of these cells into EOC patients. Although this approach led to large numbers of circulating tumor antigen specific T cells and tumor regression, the strategy is hampered by the relatively short lifespan of the effector CD8+ T cells. These results indicate that lack of sustained expansion of long lasting, durable tumor specific T cells is a major obstacle for successful adoptive T cell therapy. In preliminary studies, we have focused on CD4+ T cells as the major driver of anti-tumor immunity. In order to augment the in vivo persistence of the engineered CD8TCR cells, we have cloned a distinct subset of human CD4+ Th1 cells that directly recognize NY-ESO-1 naturally presented by MHC class II on cancer cells. In addition, these tumor-recognizing CD4+ T cells (TR-CD4TCR) potently provide help to CD8TCR cells in an antigen-presenting cell (APC) independent fashion and amplify the anti-tumor effects of CD8+ T cells. In contrast, their conventional TAA-specific CD4+Th1 counterparts (non-tumor recognizing, NTR-CD4), require APCs for their activation. Direct cognate interaction between TR-CD4 and cancer cells triggers the release of an array of effector molecules which inhibit tumor growth and amplify the anti-tumor effects of CD8+ T cells. We have also demonstrated that human hematopoietic stem/progenitor cells (hHSC) can be genetically re-programmed for continuous generation of long lived tumor-specific T cells in an NSG mouse model. We propose a novel clinical application of hHSCs for the continuous (possibly lifelong) source of anti-cancer immune cells that will provide sustained attack against cancer cells. Citation Format: Kunle Odunsi. Reprogramming T cells for adoptive immunotherapy of ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr IA09.

Abstract B67: Galectin-1 expression related to hypoxia in primary human melanoma as a driver for metastatic progression. A target to promote anticancer immunotherapy.

Abstract Galectin-1 is a major protein secreted by the majority of cancer types which is induced by HIF and plays important roles in the tumor microenvironment protecting against anticancer immune cell attack and from reactive oxygen species production. We have shown this protein to be more highly expressed within hypoxic regions of primary melanoma. Galectin-1 expression is critical for protecting endothelial cells in tumor angiogenesis and for promoting tumor metastasis. More recently, we have shown that inhibiting this protein using small drug molecules significantly enhances chemotherapeutic access inside tumors by promoting leakiness and facilitates TIL access inside tumors to kill cancer cells. When galectin-1 blocking drugs are used in combination as a triple therapy with modified whole cancer cell vaccines plus anti-CTLA4 Ig, this provides synergistic enhancements increasing CD8+ CTL anticancer TIL responses to inhibit cancer growth in a range of different mouse tumor models. Citation Format: Stephen J. Ralph, Koichi Ito, Bernadette Wachter, Selda Onturk, James Wilmott, Peter Hersey. Galectin-1 expression related to hypoxia in primary human melanoma as a driver for metastatic progression. A target to promote anticancer immunotherapy. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B67.

Small-molecule Bcl-2 inhibitors sensitise tumour cells to immune-mediated destruction

The cytotoxic effects of anticancer immune cells are mediated by perforin/granzyme-B, Fas ligand and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), and therefore depend on intact apoptotic responses in target tumour cells. As killing by all three of these mechanisms is blocked by the frequently overexpressed antiapoptotic oncoprotein Bcl-2, we hypothesised that coexposure to a Bcl-2 inhibitor might enhance anticancer immune responses. We evaluated this in U937 lymphoma cells, and A02 melanoma cells, which both show strong Bcl-2 expression. Vα24+ Vβ11+ natural killer T (NKT) cells expanded from peripheral blood of normal donors (n=3) were coincubated with PKH26-labelled U937 cells, and cytotoxicity was determined by flow cytometry after annexin-V-FITC and 7-AAD staining. In all cases, addition of the HA14-1 small-molecule Bcl-2 inhibitor to the cocultures significantly increased apoptosis in the target U937 cells. Using a similar assay, killing of A02 cells by the cytotoxic T-lymphocyte clone 1H3 was shown to be amplified by coexposure to the potent small-molecule Bcl-2 inhibitor ABT-737. Experiments with immune effectors preincubated with concanamycin-A suggested that sensitisation to perforin/granzyme-B may underlie enhanced target-cell killing observed in the presence of Bcl-2 inhibitors. We conclude that immune destruction of malignant cells can be amplified by molecular interventions that overcome Bcl-2-mediated resistance to apoptosis.

Autologous Gamma-Delta T (GD-T) Cells in Acute Myeloid Leukemia (AML): Potential Immune Effector Cells for Minimal Disease?.

Prolonging event-free survival of AML with autologous activated immune cells is a promising concept. GD-T cells are a rare circulating lymphocyte population (1%) and a component of the innate immune system capable of exerting anti-neoplastic activity. Their role as potential anti-cancer immune effector cells deserves further exploration. It is noteworthy that GD-T cells are over-represented in reactive regions surrounding melanoma lesions. While patients with an accumulation of GD-T cells showed a survival benefit over those who did not, such increases were not present in patients with metastatic disease and high tumor cell burden (Bachelez, J. Invest. Dermatol. 98:369, 1992). Little is known about the role of GD-T cells as immuno-effectors, their absolute numbers in peripheral blood or the feasibility of purifying functional GD-T cells from patients with AML. We are interested in testing the clinical feasibility of using GD-T cells freshly purified from PB against minimal residual disease in AML. As a first step towards achieving this goal, we compared circulating GD-T cell levels sequentially in 33 AML patients with 20 healthy adult volunteers. We used ultra-low volume multi-color flow-cytometry and microbeads to measure absolute numbers of GD-T cells in PB. Functional studies were done by the chromium release assay and single-cell intra-cellular interferon-gamma detection. We observed that AML patients with a high leukemic blast cell burden (e.g. prior to chemotherapy) had marginally decreased GD-T cell levels compared with healthy controls: median 38/μl, Q1-Q3, 27–86/μl, versus median 83/μl, Q1-Q3, 45–122/μl, respectively, p= 0.051. We re-examined the AML patients at several time points after induction therapy and observed significantly increased numbers of GD-T cells in patients with lower but detectable residual disease (either molecular maker positive or borderline bone marrow blast infiltration by morphology) compared to patients with persistently high blast cell burden: median 105/μl, Q1-Q3, 105–133/μl versus median, 7/μl, Q1-Q3, 6–15/μl; p=0.008. Patients with residual disease also showed significantly higher numbers of absolute GD-T cells per microliter blood compared to those retested after they had achieved complete remission (CR); p=0.0025. In CR, GD-T cell counts remained lower than those of healthy individuals: median 33/μl, Q1-Q3, 22–35/μl versus median 83/μl, Q1-Q3, 45–122/μl; p=0.030. Interestingly, we found a sharp increase (on average, 4.9-fold higher than values obtained in CR) in GD-T levels at the time of very early morphologic (n=3) or molecular relapse (n=2). Hence, we were interested in studying the functional properties of the GD-T cells from AML patients. We were able to isolate functional GD-T cells from the PB of patients with AML in CR-1 in sufficient numbers and purity to assay for interferon-gamma and found that similar numbers of GD-T cells expressed the Th1 cytokine compared with healthy controls: 84% versus 93% of all GD-T cells, respectively. We also showed that GD-T cells were able to kill leukemic target cells (AML-OCI2) in vitro more efficiently than CD3+ T cells. Our data suggest that further studies to investigate the potential therapeutic role of autologous GD-T cells in patients with AML in CR are warranted.

A perturbation problem arising from the modelling of soluble Fas ligand in tumour immunology

It has been known for many years that immune cells can kill cancer cells by a variety of mechanisms. However, new experimental evidence suggests that cancer cells also express these cell killing mechanisms. This enables the tumour to mount a counterattack against the anticancer immune cells. Based on these observations, we propose an ordinary differential equation model for tumour-immune cell interactions. With initial conditions corresponding to a mixture of cancer and immune cells, numerical solutions of the model show a sharp increase in the level of a chemical regulator associated with the interaction of the two cell types. We investigate this behaviour by constructing an analytical approximation to the solution using singular perturbation analysis. This problem has an unusual asymptotic structure. Instead of the usual solution form, with two outer solutions separated by a single transition layer centred at the point at which the sharp jump in the solution occurs, our solution contains multiple fast time layers, with each layer being necessary to capture the entire dynamics of the sharp transition.