bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeting the TNFR2 oncogene on tumor cells and tregs of the tumor microenvironment via dominant TNFR2 antagonism.
Authors: Faustman Denise L. , Torrey Heather , Butterworth John , Mera Toshi , Okubo Yoshiaki , Wang Limei , Baum Danielle , Warden Sara , Plager Sarah , Huang Daniel , Vanamee Eva , Foster Rosemary
Published: November 2019

106

Background: A major barrier to cancer immunotherapy is lack of selective inhibitors of the regulatory T cells (Tregs) of the cancer microenvironment and methods to directly kill tumors through novel surface oncogenes. Tumor necrosis factor receptor 2 (TNFR2) is a target protein with restricted expression on the most potent Tregs of the tumor infiltrate and newly identified as a broadly expressed oncogene on human tumors. Methods: We characterized the effect of TNFR2 antibody antagonists via TNFR2 in human samples from ovarian ascites compared to healthy controls. Results: TNFR2 antagonists potently inhibited Treg proliferation with exponential potency and selectivity for tumor microenvironment Tregs. Furthermore, common ovarian cancer cell lines such as OVCAR3 expressed the TNFR2 oncogene and were rapidly and completely killed by TNFR2 antagonistic antibodies. TNFR2 antagonistic antibodies were dominant because they succeeded even in the presence of TNF agonism. Remarkably, these antibodies worked as well in killing Treg cells as F(ab’)2 fragments, but not as well as Fc fragments. TNFF2 antagonists were not dependent on ADCC mechanisms for activity. Conclusions: Dominant TNFR2 antagonistic antibodies demonstrate tumor-specific Treg depletion. Blocking TNFR2 signaling with antagonist antibodies also creates a novel tool to possibly eliminate tumors expressing the TNFR2 oncogene and to more potently suppress Tregs of the tumor microenvironment.
The effect on quality of life via bio-mimetic hormone replacement therapy for breast cancer patients.
Authors: Wiley TS , Haraldsen Jason T.
Published: November 2019

113

Background: Many treatments for breast cancer block the estrogen receptors to reduce the risk of relapse. However, this treatment results in massive changes to the normal hormonal milieu. When the normal rhythmic hormonal patterns are disrupted, either through treatment or normal menopause, women typically experience a multitude of harsh symptoms with little affect on relapse rate. We set out to examine and evaluate the effects of estrogen (E2) and progesterone (P4) treatments via a bio-mimetic bio-identical hormone replacement therapy (BBHRT). We theorized, through the analysis of physiological and clinical literature, that the use BBHRT in the normal physiological pattern of reproductive women will lead to an increase of overall well-being and better quality of life for post-treatment and menopausal women, but does not increase their risk of breast cancer. Methods: E2 and P4 control over 9,000 different gene products and a disruption of these processes can lead to an increase in illnesses of aging including cancer. Through an evaluation of current literature, we theorize that a BBHRT approach utilizing E2 and P4 in a bio-mimetic manner to restore a woman’s body to normal hormonal levels can be achieved through twice-daily transdermal applications of hormonal creams with peaks in serum levels on days 12 and 21. The proposed method uses compounded, bio-identical hormones dosed to mimic the female reproductive cycle. Results: While this is a proposed clinical study, it is expected that women will experience resolution of menopausal and post-treatment symptoms, including better sleep, decreased migraines and incontinence, increased focus, and increased libido. By restoring the normal hormonal rhythm, it is anticipated that the body’s natural response elements will help restore both quality of life and well being, while also protecting from possible relapse. Conclusions: We propose a method to provide better quality of life and well being for women through BHRT that combines the use of E2 and P4 in a manner that mimics a woman’s normal reproductive levels. While a small long-term study of this method has shown promising results, we conclude that further, most detailed studies are still needed.
TLR9 triggering/STAT3 inhibition to reprogram leukemic cells into antigen-presenting cells and trigger T-cell responses.
Authors: Gupta Priyanka Dutta , Lu Brandon , Li Haiqing , Li Zhou , Zhao Xingli , Kuo Ya-Huei , Kortylewski Marcin
Published: November 2019

118

Background: Clinical studies demonstrated potential of cancer immunotherapies while underscoring the need to block immune checkpoints shielding tumors from immune attack. We recently generated a strategy to target STAT3 transcription factor, critical immune checkpoint regulator, using TLR9-targeted delivery of STAT3 decoy inhibitor (CpG-STAT3dODN) targeting TLR9+ immune cells and all subsets of leukemic cells, including leukemic progenitor/stem cells in vitro and in vivo experiments in murine and human AML cells. Methods: Studies were conducted using mouse Cbfb/MYH11/Mpl and C1498 models of AML and acombination of flow cytometry, electron microscopy, and sequencing techniques were used to define changes in the AML cells after CpG-STAT3dODN treatment. Results: Intravenous injections of CpG-STAT3dODN into engrafted mice, led to significant changes in CD11b/CD11c expression, cell morphology, mitochondrial shape and number within the first 5 days of treatment. RNA-sequencing studies indicated increased expression of genes regulating differentiation and antigen presentation such as IRF8, GADD45A, CIITA, IL-12A, IFNγ. These effects associated with increased number of activated T cells in the leukemic microenvironment, thereby resulting in regression of AML in majority of treated mice. Two week CpG-STAT3dODN treatment was sufficient to generate protective antitumor immunity as demonstrated in rechallenge experiments. Conclusions: Our studies suggested that concurrent TLR9-stimulation and STAT3 inhibition trigger potent immunogenicity of AML cells sufficient to drive effector and memory T cell responses. We hypothesize that STAT3-inhibition can unleash immune-stimulation downstream of TLR9 signaling transforming leukemic cells into antigen-presenting cells Our further studies will dissect role of STAT3 and TLR9-induced signaling mediators (IRF, NF-κB) in regulation of the process.This information should support the design of new therapeutic regimens for AML immunotherapy using CpG-STAT3dODN alone or in combination with T cell-based therapies.
A novel therapeutic to enhance the immunogenicity of breast cancer cells.
Authors: Vishnubhotla Priya , Khaled Amr , Showalter Anne , Oyer Jeremiah , Igarshi Robert , Copik Alicja , Khalid Annette
Published: November 2019

119

Background: Breast cancer is a challenge for immunotherapy because of its inherent genetic heterogeneity and decreased immunogenicity. A possible solution is to generate dying breast cancer cells that operate like a vaccine to stimulate tumor-specific immunity. This is termed immunogenic cell death (ICD) and is characterized by a unique molecular signature, involving the release of molecules that attract and stimulate phagocytes and sensitize tumor cells to killing by natural killer (NK) cells. However, the current doses of drugs used to induce ICD may be too high to translate into clinically relevant regimens. Methods: Our group discovered a novel peptide, CT20p, that is effective at nanomolar concentrations and can be delivered to breast tumors using targeted nanoparticles. CT20p works by inhibiting a unique protein folding complex, called chaperonin-containing TCP-1 (CCT), which is highly expressed in cancer cells but not normal cells. Using triple negative breast cancer (TNBC) and normal cell lines as well as patient-derived tumor cells, we examined the biological consequences of CCT inhibition by CT20p in terms of the induction of endoplasmic reticulum (ER) stress, through the accumulation of unfolded proteins, and the release of the danger signals associate with ICD that could stimulate innate immunity. Results: Dying TNBC cells treated with CT20p displayed alterations in PERK, a mediator of the unfolded protein response (UPR), which resulted in the membrane translocation of calreticulin (Crt), an “eat me”’ signal and a key marker of ICD. CT20p-killed TNBC cells were more readily phagocytosed, and NK cells more effectively killed remaining cancer cells. In our experiments normal breast epithelial cells, macrophages or NK cells were unaffected by CT20p. Conclusions: Our study suggests that inhibition of CCT by CT20p generates dying cancer cells that stimulate innate immune responses, through the release of signals like Crt, to attract phagocytes and promote NK cell cytotoxicity. By enhancing the immunogenicity of breast cancer cells, CT20p could increase responsiveness to to checkpoint inhibitors or cellular immunotherapy, hence providing more complete protection from breast cancer recurrence and metastasis.
Immunomodulation of pembrolizumab (pembro) treated metastatic melanoma patients (pts) after progression with sequential temozolomide (TMZ): A case series.
Authors: Swami Umang , Monga Varun , Zakharia Yousef , Milhem Mohammed M.
Published: November 2019

123

Background: Regulatory T cells are increased in melanoma pts and are postulated to impair objective response to immune therapies. Pembro blocks programmed death receptor-1, reverses T-cell suppression and induces antitumor activity. Continuous low dose TMZ has immunomodulatory effects resulting in selective CD4+ lymphopenia of which the T-reg population of CD4+/CD25+ T cells could be decreased significantly. TMZ has demonstrated dramatic responses after IL-2 in metastatic melanoma pts. We evaluated responses with sequential TMZ after progression on pembro in pts with metastatic melanoma. Methods: Medical records of pts with metastatic melanoma treated with pembro and TMZ at Holden Comprehensive Cancer Center between 1/1/2011 and 8/31/2016 were reviewed. Recorded data included BRAF mutation status, treatment doses, serological markers, duration of treatment and treatment related responses (immune RECIST and RECIST 1.1). Results: Overall 10 pts (7 males, 3 females) with metastatic melanoma received sequential pembro followed by TMZ after disease progression or unacceptable toxicity. Eight were BRAF negative and 7 were pretreated with ipilimumab. Median age at time of initiating pembro (2 mg/kg every 3 weeks) was 64 years (range 33-75). Pts received a median of 4 doses (2-19). Median duration of treatment was 88 days (41-464) with stable disease in 3, progressive disease (PD) in 6 and 1 not evaluable for response. All pts thereafter received TMZ (75 mg/m2 daily for 6 weeks on 8 week cycle except 1 who received 200 mg/m2 for 5 days every 4 weeks) after a median of 21.5 days (12-34) from last pembro dose. Median duration on treatment with TMZ was 75 days (20-198) resulting in 1 complete response, 1 partial response, 6 PD and 2 not evaluable for response. Pts with response to TMZ had a higher median baseline lymphocyte count at the time of initiation of pembro (4131 vs. 1715 k/uL, p < 0.05) as well as TMZ (2335 vs. 965 k/uL, p = 0.08) as compared to pts without a response. Conclusions: TMZ is an interesting alternative for metastatic melanoma pts with disease progression on pembro. Our results show a 25% response rate. Further studies in this setting are warranted.
Targeting the glucose metabolism of monocytic myeloid-derived suppressor cells to stimulate cancer immunity.
Authors: Grewal Jaspreet Singh , Al-Rayyan Numan , Ritchie Jamaal , Schowe Paxton , Falkner Cam , Telang Sucheta , Yaddanapudi Kavitha , Chesney Jason Alan ,   University of Louisville
Published: November 2019

126

Background: Myeloid derived suppressor cells (MDSCs) inhibit the expansion of tumor antigen-specific effector CD8+ T cells via different mechanisms including increased expression of arginase, transforming growth factor – β (TGF – β) and indoleamine 2,3-dioxygenase (IDO). Recently, MDSCs were found to over-express hypoxia inducible factor 1 alpha (HIF-1α) which is required for their differentiation. An essential transcriptional target of HIF-1α is 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) which synthesizes fructose 2,6-bisphosphate, an allosteric stimulator of glycolysis and of proliferation via stimulation of cyclin dependent kinase-1 (CDK1). We hypothesized that MDSCs might over-express PFKFB3 which in turn might be required for their function as T cell suppressors. Methods: We used monocytic MDSCs (M-MDSCs) induced by co-culture with A375 melanoma cells, M-MDSCs from metastatic melanoma patients, murine bone marrow MDSCs and splenic M-MDSCs from B16 F10 tumor bearing mice for our studies. T cell suppression assays were performed to analyze M-MDSC suppression and reversal following PFKFB3 blockade. Results: We found that M-MDSCs have increased PFKFB3 expression. We also found that PFK-158 administration in B16 (wild-type) melanoma-bearing mice results in a marked reduction in MDSCs and a simultaneous increase in CD8+ T cell infiltration in the tumors. We analyzed three advanced cancer patients for circulating MDSCs before and after PFK-158 administration as part of a multi-center phase 1 clinical trial. And, we found that the MDSCs were markedly reduced in each patient. In addition, we have generated data for MDSC suppressive activity following in vitro treatment with PFK-158 showing reversal of suppressive activity. Conclusions: Taken together, these data indicate that selective inhibition of PFKFB3 may be a novel approach to target MDSCs and combinations of PFKFB3 inhibitors with immunotherapies may be a rational strategy to promote durable immune-mediated remissions in cancer patients.
Expression of MICA/b, a ligand for natural killer (NK) cell recognition, on human pancreatic adenocarcinoma and potential for NK intervention.
Authors: Djeu Julie , Gilvary Danielle , Trinh Thu Le , Tu Nhan , Coppola Domenico
Published: November 2019

129

Background: Pancreatic cancer remains a leading cause of cancer-related mortality because of lack of means for early detection and notable refractoriness to standard chemotherapy in advanced stages of disease. Mobilization of the immune system is therefore an important option to explore. Natural Killer (NK) cells are potent effector cells in immune surveillance against cancer and offer a unique advantage because of expression of a C-type, lectin-like, type II transmembrane glycoprotein, NKG2D, on the cell surface. NKG2D is a receptor that specifically recognizes common stress-induced proteins such as MICA/B on tumor cells, targeting them for perforin/granzyme B-mediated destruction. The goal of this study is to examine if MICA/B is expressed on human pancreatic cancer cells, allowing the potential to direct immunotherapy against pancreatic cancer via NK cell therapy. Methods: This study was conducted under an IRB approved protocol. Pancreatic samples were taken from 121 patients with pancreatic ductal adenocarcinoma (PDA) and 103 cases of normal adjacent pancreatic tissues (NP) were included for comparison in a tissue microarray developed in the Tissue Core. All cases were stained for MICA/B AB using the Ventana automated immunostainer Discovery XT (Ventana,Tucson, AZ). Human pancreatic cell lines were also immunostained for MICA/B expression and tested for sensitivity to NK cell lysis in vitro. Results: A significant increase in MICA/B expression was observed during the progression from NP to PDA (p value < 0.0001) in pancreatic tumor tissues. Additionally, all pancreatic cell lines, including MiaPaca, Panc-1, and PCSC, express MICA/B and were readily lysed by NK cells. Conclusions: We report an increase in MICA/B expression during the progression of pancreatic ductal adenocarcinoma and MICA/B may represent a new immunotherapeutic target in pancreatic gastric carcinogenesis.
Gene expression signatures of response to anti-CD19 chimeric antigen receptor (CAR) T-cell therapy in patients with CLL and ALL.
Authors: Orlando Elena , Leary Rebecca , Lacey Simon F. , Fraietta Joseph , Bedoya Felipe , Ambrose David , Wilcox Nicholas , Maude Shannon L. , Frey Noelle V. , Levine Bruce L , Grupp Stephan A. , Porter David L. , Young Regina , Winckler Wendy , Morrissey Michael , June Carl H. , Melenhorst Jan Joseph , Brogdon Jennifer , Bitter Hans
Published: November 2019

137

Background: The adoptive transfer of autologous T cells genetically modified to express a CD19-specific, 4-1BB/CD3ζ-signaling CAR (CTL019) has shown remarkable activity and induces long-term remissions in a subset of patients with relapsed/refractory chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). In ALL, CTL019 induces a complete response (CR) in over 90% of patients while in CLL 25% of patients obtain a CR. It is not fully understood why only certain patients respond to therapy. Methods: We employed next generation sequencing of RNA (RNAseq) to identify predictive indicators of response to CTL019. We performed RNAseq on leukapheresis and manufactured product T cells prior to re-infusion from 35 CLL and 7 pediatric ALL patients with heavily pre-treated and high-risk disease. To characterize potency, we performed RNAseq on the infusion product after stimulation with the CAR. Results: We find that durable remission in CLL is associated with gene expression signatures of early memory and T-effector cells, while T cells from non-responding patients are enriched in signatures of T-regulatory cells, terminal differentiation, and exhaustion. In following the results from CLL, we find that pediatric ALL manufactured T cells are significantly enriched for an early memory, naïve T cell state and all achieved a CR. In parallel in vitro experiments, stimulation of the infusion product further demonstrated that CTL019 cells from CRs have an increased capacity for activation upon stimulation. We tested if we could extend these observations to identify a phenotype of T cells that is predictive of response prior to CTL019 manufacturing and find that the signatures predictive of response at the pre-infusion stage are also observed at the earlier leukapheresis time point. Conclusions: These findings suggest that intrinsic T cell fitness dictates response to CAR T cells. These gene expression signatures, along with additional immunological biomarkers, may be used to identify which patients are most likely to respond to adoptive transfer strategies and suggest manufacturing modifications that might potentiate the generation of maximally efficacious infusion products. Clinical trial information: NCT01029366, NCT01747486, NCT01626495.
The impact of checkpoint blockade prior to adoptive cell therapy using tumor-infiltrating lymphocytes for metastatic melanoma: An update from MD Anderson Cancer Center.
Authors: Forget Marie-Andree , Haymaker Cara L. , Hess Kenneth R. , Roszik Jason , Woodman Scott Eric , Fulbright Orenthial J. , Wahl Arely , Flores Esteban , Thorsen Shawne T. , Tavera Rene J. , Ramachandran Renjith , Amaria Rodabe Navroze , Hwu Patrick , Bernatchez Chantale
Published: November 2019

138

Background: Adoptive cell therapy (ACT) of autologous tumor-infiltrating lymphocytes (TIL) can produce long lasting treatment responses in patients (pts) with metastatic melanoma. In our initial report of 31 treated pts, we demonstrated overall response rate (ORR) of 48%. Due to the evolution of treatment options for metastatic melanoma with heavy reliance on immunomodulatory therapies, we sought to re-evaluate responses in the era of checkpoint blockade. Methods: Pts receive treatment consisting of 7 days of lymphodepleting chemotherapy consisting of cyclophosphamide and fludarabine. High dose interleukin-2 (720,000 IU/kg IV q 8 hrs up to 15 doses) was infused after TIL infusion. Responses were assessed by imaging per irRC. Results: A total of 74 pts have been treated in our initial TIL study with an updated ORR assessment of 43%. Stratification of pts according to their checkpoint blockade immune-modulator therapy prior to TIL ACT, demonstrated that prior Ipilimumab (Ipi) decreased ORR to 33% compared to 51% in checkpoint naïve pts and decreased overall survival (OS) post-TIL therapy (median 7.7 vs 24.6 months respectively). There were too few pts to assess the impact of anti-PD1 as a single agent. A multiparametric analysis revealed that LDH levels at the time of therapy mainly influences OS and ORR to TIL but still could not invalidate the impact of Ipi prior to TIL ACT. The durability of the response was also found to be different between the 2 groups (30% for Ipi prior and 50% No Ipi prior). This new reality also impacted previously reported parameters that correlated with ORR to TIL ACT such as the expression of B-and-T lymphocyte attenuator (BTLA) on CD8+ TIL. Interestingly, assessment of mutation load (ML) of the tumors prior to TIL ACT showed that the check point naïve group display a high ML ( > 100) within their tumor whereas some patients who had Ipi prior to TIL have a low ML ( < 100). Conclusions: Our preliminary analysis shows that pre-treatment with Ipi decreases ORR to TIL ACT. Understanding how prior ipi modifies TIL, the tumor and microenvironment will help to define the full extent of the impact and how to best treat Ipi refractory pts with TIL. Clinical trial information: NCT00338377.
Generation and characterization of antitumor infiltrating lymphocytes from patients with breast cancer for adoptive cell transfer therapy.
Authors: Zhou Juhua , Zhong Yin , Hou Zhongjun , Zhang Jianzhong , Li Yanmin , Yan Senxiang
Published: November 2019

145

Background: Clinical trials have shown that adoptive cell transfer therapy is a promising method for cancer treatment. In the current study, we aim to generate and characterize anti-tumor tumor-infiltrating lymphocytes from patients with breast cancer for adoptive cell transfer therapy. Methods: In vitro culture method was used to generate anti-tumor, tumor-infiltrating lymphocytes from patients with breast cancer. FACS analysis, ELISA, and Elispot assay were used to characterize tumor-infiltrating lymphocytes. Autologous anti-tumor tumor-infiltrating lymphocytes from patients with breast cancer were used in adoptive cell transfer therapy. Results: FACS analysis indicated that tumor-infiltrating lymphocytes were present in the tumor tissues, but not detectable in the normal breast tissues from patients with breast cancer. Tumor-infiltrating lymphocytes could be generated in vitro from fresh tumor specimens of patients with breast cancer. Both CD4 T cells and CD8 T cells were detected in tumor-infiltrating lymphocytes. Autologous tumor cells could also generate in vitro from fresh tumor tissue samples of patients with breast cancer. Among 22 samples screened, 6 samples (25%) of tumor-infiltrating lymphocytes are tumor-reactive. Anti-tumor, tumor-infiltrating lymphocytes could recognize autologous tumor cells and allogenic tumor cells. After a large scale T cell expansion, anti-tumor reactivity was maintained in tumor-infiltrating lymphocytes. All of tumor-infiltrating lymphocytes were NK cells in some samples from patients with breast cancer, and these NK cells could recognize autologous tumor cells and a panel of allogenic tumor cells. T cell cloning assay demonstrated that some of the tumor-reactive, tumor-infiltrating lymphocytes were CD4 T cells. Conclusions: The results suggest that anti-tumor, tumor-infiltrating lymphocytes may be generated from patients with breast cancer, which may be used in clinical applications of adoptive cell transfer therapy for patients with breast cancer. The clinical trial of adoptive cell transfer therapy using autologous anti-tumor tumor-infiltrating lymphocytes for patients with breast is under way.
Somatic mutation burden in cancer samples determined by targeted next generation sequencing.
Authors: Cyanam Dinesh , Broomer Adam , Mandelman David , Chaudhary Ruchi , Williams Paul D , Nistala Goutam , Gottimukkala Rajesh , Rhodes Kate , Bishop John , Hyland Fiona , Sadis Seth
Published: November 2019

15

Background: High somatic mutation burden in tumor tissues is associated with the presentation of neoantigens that promote immune responses particularly in the context of immune checkpoint therapies. Herein, we characterize the ability of targeted cancer research panels to generate estimates of somatic mutation burden. Methods: Somatic mutation data from > 8000 cancer samples obtained from The Cancer Genome Atlas (TCGA) was curated and standardized, and the number of single nucleotide variants (SNVs) in exonic regions of each sample determined. Next, the number of SNVs associated with target regions of two Ion AmpliSeq cancer panels (Oncomine Comprehensive Assay [OCA, 146 genes, 0.35 MB]; Comprehensive Cancer Panel [CCP, 409 genes, 1.7 MB]) was likewise determined and the frequency of mutation counts in the exome and the panel target regions was compared. Mutation counts of samples containing truncating mutations in mismatch repair (MMR) and other DNA repair genes were characterized. A facile workflow with less than 60 minutes of hands-on time was developed to estimate mutation counts for a batch of 8 samples using the Ion Chef for automated library preparation and templating followed by sequencing on the Ion S5. Results: The sensitivity of targeted panels in estimating somatic mutation burden was positively correlated with panel size. The area under the Receiver Operating Characteristic (ROC) curve showed that CCP had > 90% sensitivity and > 95% specificity to differentiate high and low mutation burden based on informatics analysis of TCGA data. As expected, truncating mutations in MMR genes were associated with higher somatic mutation counts in colorectal tumor tissue. Using data generated from OCA and CCP, we characterized a set of filters that provided a good estimate of somatic mutation counts when applied to a tumor-only workflow. Conclusions: A simple workflow was developed on the Ion Torrent sequencing platform to estimate somatic mutation burden in cancer samples. The methods described herein will help advance research in immuo-oncology.
DNA vaccine with pembrolizumab to elicit antitumor responses in patients with metastatic, castration-resistant prostate cancer (mCRPC).
Authors: McNeel Douglas G. , Eickhoff Jens C. , Jeraj Robert , Staab Mary Jane , Straus Jane , Rekoske Brian , Liu Glenn
Published: November 2019

168

Background: We have previously investigated a DNA vaccine encoding prostatic acid phosphatase (PAP, pTVG-HP) in patients with PSA-recurrent prostate cancer, and have demonstrated that this can be safely administered over many months and can elicit PAP-specific T cells. A phase 2 trial is currently underway. In preclinical models, we have found that blockade of regulatory receptors, including PD-1, at the time of T cell activation with vaccination produced anti-tumor responses in vivo. Similarly, we have recently found that patients with prostate cancer previously immunized with a DNA vaccine develop PD-1-regulated T cells. These findings suggested that combined PD-1 blockade with vaccination should elicit superior anti-tumor responses in patients with prostate cancer. Methods: A clinical trial was designed to evaluate the immunological and clinical efficacy of pTVG-HP when delivered in combination or in sequence with pembrolizumab, in patients with mCRPC. Serial biopsies, blood draws, and exploratory FLT PET/CT imaging are being conducted for correlative analyses. Results: While trial accrual continues, 1 of 14 subjects has experienced a grade 3 adverse event. There have been no grade 4 events. Several patients treated with the combination have experienced serum PSA declines, and several have experienced decreases in tumor volume by radiographic imaging at 12 weeks, including one partial response. Expansion of PAP-specific Th1-biased T cells has been detected in peripheral blood samples. Exploratory FLT PET/CT imaging has demonstrated proliferative responses in metastatic lesions and in vaccine-draining lymph nodes. Evaluation of biopsy specimens for recruitment of antigen-specific T cells is currently underway. Conclusions: PD-1 pathway inhibitors have demonstrated little clinical activity to date when used as single agents for treating prostate cancer. Our findings suggest that combining this blockade with tumor-targeted T-cell activation by a DNA vaccine is safe and can augment tumor-specific T cells, detectable within the peripheral blood and by imaging, and result in objective anti-tumor changes. Clinical trial information: NCT02499835.
Effect of imprime PGG on innate immune-activating pharmacodynamic changes in a phase I clinical study in healthy human volunteers.
Authors: Bose Nandita , Ottoson Nadine , Harrison Ben , Lowe Jamie R. , Matson Mark , Qiu Xiaohong , Bykowski Jonas Adria , Rathmann Blaine , Kangas Takashi , Wurst Lindsay R. , Chan Anissa S.H. , Fraser Kathryn , Walsh Richard , Ertelt Katie E. , Leonardo Steven , Fulton Ross , Gorden Keith , Uhlik Mark , Graff Jeremy , Huhn Richard Dale
Published: November 2019

33

Background: Imprime PGG (Imprime) is currently in clinical development as combination therapy with checkpoint inhibitors. Imprime, a yeast-derived, soluble β-1,3/1,6 glucan is a Pathogen Associated Molecular Pattern (PAMP) that complexes with endogenous anti-beta glucan antibodies (ABA) and activates innate immune effector cells to trigger the anti-cancer immunity cycle. In this study, we sought to investigate the immunopharmacodynamic (IPD) responses of Imprime in healthy human subjects. Methods: Group 1 (n=12) received a single IV infusion of Imprime 4 mg/kg, Group 2 (n=12) received three weekly infusions of 4mg/kg Imprime. Group 3 (n=12) received infusions of 4 mg/kg or 2 mg/kg Imprime on weeks 1, 2 and 5. In groups 1 and 2, six subjects each received premedication with diphenhydramine 50 mg IV and dexamethasone 8 mg IV. IPD changes were measured at various times before, during and after Imprime administration. Results: Peak levels of complement activation products, C5a and SC5b-9 were detected at the end of infusion (EOI). A 2 to 3-fold increase in neutrophil and monocyte numbers were seen 4 hours post-Imprime infusion. Chemokines, especially IL-8 and MCP-1, were consistently detected at EOI. Cellular analyses showed Imprime binding to neutrophils, monocytes, subsets of DC and B cells. 24 hrs after EOI, a population of intermediate monocytes expressing higher levels of the activation markers CD86, PD-L1, and HLA-DR, was observed. Approximately one week post-Imprime, increased switched memory B cells and plasmablasts were detected. Consistent increase in expression of innate immune activation marker genes was evident as well. A substantial drop in free ABA and a concomitant increase in circulating immune complexes were seen at the EOI. Adverse events (AE) were limited to infusion related reactions. Importantly, the IPD changes and the AE were seen in subjects with higher ABA levels. The effect of pre-medications on some of the IPD will also be presented. Conclusions: These human data provide the first evidence linking pre-treatment ABA levels and Imprime IPD, substantiating the use of these pre-treatment ABA levels for patient selection.
Outcomes with immune checkpoint inhibitor use in lung cancer patients with hepatic metastases.
Authors: Addepalli Srikala , Stroud Chipman Robert Geoffrey , Cherukuri Sulochana Devi , Cherry Cynthia R. , Sharma Nitika , Parent Teresa , Hardin Jessica , Walker Paul R.
Published: November 2019

38

Background: Immune checkpoint inhibitors have shown promising responses in advanced lung cancer as well as a number of other malignancies. However, efficacy appears to be variable based on the site of disease. We reviewed the outcomes of patients with lung cancer and hepatic metastases from a single institution. Methods: We performed a retrospective analysis of the lung cancer patients treated at East Carolina University with the PD1 inhibitor Nivolumab. Patients were enrolled in an IRB approved study designed to evaluate predictive markers of response to immune checkpoint blockade. Clinical characteristics, laboratory data, and imaging studies were analyzed and recorded. Results: Data for 75 patients with lung cancer who received anti-PD1 therapy was analyzed. Of the 75 patients included, 13% (n = 9) had liver metastases. Average age of the patients was 61.8 years, 66% patients were male, 22% had squamous cell, 33% had adenocarcinoma and 44 % small cell neuroendocrine histology. Average number of prior therapies was 1.7 (range 1-4). Thirty three percent of patients had modified Glasgow prognostic score of 2 and a mean CRP of 63.61mg/L (range 0.1-172.7mg/ L) at the initiation of anti-PD1 therapy. They received an average of 4 cycles of anti-PD1 therapy. Forty four percent of patients received adjunctive therapy such as ablative radiation (33%) or immune modulating chemotherapy with the aim of augmenting the effect of the anti-PD1 therapy. None of the analyzed patients with liver metastases had an objective decrease in liver metastases and the average survival in these patients after starting anti-PD1 therapy was 132 days. Conclusions: To our knowledge, this study is the largest reported series examining patients with hepatic metastases from lung cancer treated with PD-1 inhibitors. Our observations are consistent with prior reports indicating poor outcomes with anti-PD1 therapy in patients with liver metastases. The mechanisms underlying such resistance must be elucidated so that more effective treatment combinations can be developed.
Correlation of PD-L1 expression with p16INK4A expression in nonoropharyngeal head and neck squamous cell carcinoma.
Authors: Chen San-Chi , Chang Peter , Wang Hsiao-Jung , Tai Shyh-Kuan , Chu Pen-Yuan , Yang Muh-Hwa
Published: November 2019

39

Background: The expression of PD-L1 expression is important for tumor cells to evade the immune system. The level of its expression and its correlation to disease prognosis is unclear in non-oropharyngeal head and neck squamous cell carcinoma (non-OPHNSCC). Methods: A retrospective clinicopathologic analysis was performed for 106 non-OPHNSCC patients diagnosed during the period from 2007 to 2014. We made tissue arrays from the paraffin-embedded non-OPHNSCC samples obtained from the patients, and studied PD-L1 and p16INK4A expression by immunohistochemistry. Systemic inflammatory factors including C-reactive protein, serum white blood cell, neutrophil, monocyte, and lymphocyte count were also analyzed to study the correlation. Results: We found that PD-L1 was overexpressed in 32.1% (34/106) and p16INK4A in 20.8% (22/106) of the patients who were studied. The expression of PD-L1 was associated with p16INK4A expression ( p < 0.01), but was not associated with the level of systemic inflammatory factors. No significant prognostic values of PD-L1, p16INK4A, or other clinicopathologic factors were found except tumor stage (stage I/II vs. III/IV, p=0.03). Conclusions: This study demonstrates the correlation between PD-L1 and p16INK4A in non-OPHNSCC, which may provide important information for designing the clinical trials of anti-PD1/PDL1 treatment in head and neck cancer patients. [Table: see text]
Association of soluble PD-L1 (sPD-L1) with decreased survival in metastatic melanoma.
Authors: Dronca Roxana Stefania , Harrington Susan , Jegapragasan Mithulan , Kottschade Lisa A. , Nevala Wendy Kay , Enninga Elizabeth Ann , Markovic Svetomir , Dong Haidong
Published: November 2019

4

Background: Membrane expression of PD-L1 on tumor cells is associated with poor survival in patients with advanced malignancies including metastatic melanoma (MM). In addition, tumor-associated PD-L1 has been proposed as a predictor of response to anti-PD-1 therapy, although responses are observed in some cases of PD-L1 negative MM. We hypothesize that tumor-derived PD-L1 has additional systemic effects through the release of biologically active soluble forms of PD-L1 (sPD-L1) into the circulation, which further impedes the anti-tumor immune response and contributes to poor clinical outcomes in MM. Methods: We developed a sPD-L1 ELISA and biochemically confirmed the identity of the detected protein. We measured the levels of sPD-L1 in 276 patients with MM enrolled on 3 clinical trials who had stored pre-treatment plasma samples and 37 healthy volunteers (HV) undergoing blood donation at Mayo Clinic. We also measured sPD-L1 in baseline samples from 38 MM patients treated with anti-PD1 (pembrolizumab) 2 mg/kg every 3 weeks. ROC analysis was used to compute sPD-L1 concentration cut-off value and Wald test was used to asses the difference of overall survival (OS) in patients with low versus high sPD-L1 concentration. Results: We found that sPD-L1 levels were significantly elevated in MM patients compared with HV (p=0.0011). Mean sPD-L1 level in MM was 1.73 ng/mL (range: 0.13-18.29) compared with 0.77 ng/mL (range: 0.11–6.02) for HV. Patients with higher levels (>0.293 ng/mL) had a median OS of 11.3 months compared to 14.8 months for patients with sPD-L1 level ≤ 0.293 ng/mL (p=0.040). Similarly to tumor-related PD-L1 findings, patients who had clinical benefit (OR/PR/SD) after 4 cycles of anti-PD1 had higher sPD-L1 levels at baseline (2.1 vs. 1.1 ng/mL). Conclusions: To our knowledge, our study is the first to report a correlation of sPD-L1 with MM patient outcome, although a larger study is needed to establish the utility of sPD-L1 as an independent prognostic and possibly predictive biomarker.
The difference in prognostic value of tumour-infiltrating T cells according to adjuvant chemotherapy in radiochemonaïve gastroesophageal cancer.
Authors: Svensson Maria Christina , Berntsson Jonna , Hedner Charlotta , Borg David , Nodin Bjorn , Leandersson Karin , Krzyzanowska Agnieszka , Bjartell Anders , Eberhard Jakob , Jirstrom Karin
Published: November 2019

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Background: Several studies have demonstrated a beneficial prognostic impact of tumour-infiltrating lymphocytes (TILs) in oesophageal and gastric adenocarcinoma, but whether this association differs according to adjuvant chemotherapy has not been reported. Herein, we examined the prognostic impact of different TIL subsets according to adjuvant chemotherapy in these cancer types. Methods: Immunohistochemistry was applied to assess the density of T cells (CD3+, CD8+, FoxP3+) and natural killer (NK)/T cells (CD56+) in radiochemonaïve tumors from a consecutive cohort of 174 patients with resected oesophageal or gastric adenocarcinoma. Cox proportional hazard’s modelling was applied to examine the impact of the investigated markers on time to recurrence (TTR) and overall survival (OS). Results: Dense infiltration of CD3+, CD8+, and FoxP3+ cells were all associated with a significantly prolonged TTR in univariable Cox regression analysis (hazard ratio[HR] = 0.57; 95% confidence interval [CI] 0.37-0.89; HR = 0.31, 95% CI 0.13-0.77, and HR = 0.44, 95% CI 0.28-0.68). This association remained significant for CD8+ and FoxP3+ cells in multivariable analysis, adjusted for age, tumour location, TNM stage, differentiation grade, resection margin and adjuvant chemotherapy ( HR = 0.30, 95% CI 0.11-0.77, and HR = 0.52, 95% CI 0.32-0.84), and borderline significant for CD3+ cells (HR = 0.63, 95% CI 0.39-1.02). Similar associations were observed for all markers in relation to OS. Notably, analysis in strata according to adjuvant chemotherapy revealed that none of the investigated lymphocyte subsets carried prognostic information in patients having received treatment (n = 13; 7.5%), with a significant treatment interaction between CD3+ and CD8+ cells and adjuvant chemotherapy (pinteraction0.035 and 0.021). Conclusions: These results confirm the prognostic value of a high density of TILs in gastroesophageal adenocarcinoma, but also indicate that adjuvant chemotherapy is only beneficial in patients with tumors displaying a low density of TILs. These results are of potential clinical relevance and need to be confirmed in additional, preferentially randomized, studies.
Immunosenescence and cancer immunotherapy.
Authors: Pawelec Graham P.
Published: November 2019

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Background: Geriatric oncology, important for the ever-increasing numbers of elderly cancer patients, has thus far focused primarily on tolerance to chemotherapy. With the advent of breakthrough immunomodulatory antibody treatments relying on the patient´s own immune system to control the tumor, the issue of immunosenescence becomes extremely important. There is increasingly a valid concern that anti-cancer immunity may be compromised in the elderly due to i) their low amounts of naïve T-cells (leading to holes in the repertoire for neoantigens) and ii) “exhaustion” of potentially tumor-specific memory T-cells. Encouragingly, but only anecdotally, accumulated clinical experience (thus far, limited to melanoma treated with anti-CTLA-4 antibodies) suggests that advanced aged does not result in decreased responses or increased side effects. However, the fraction of patients experiencing clinical benefit was low, and broader and more detailed studies focusing on the age question are required. Methods: In our own work, we have established prognostic phenotypic and functional “immune signatures” using peripheral blood from younger melanoma and breast cancer patients, which comprise phenotypic T-cell and myeloid-derived suppressor cell quantification and measurement of pro- and anti-inflammatory CD4+ and CD8+ T-cell responses to shared tumor antigens such as NY-ESO-1 and Her2 in vitro using intracytoplasmic flow cytometry to detect 6 cytokines simultaneously. Results: We found that these peripheral immune signatures were equally prognostic in older patients ( > 80 years of age). Conclusions: We therefore conclude that immunosenescence should not be a barrier to anti-tumor immunity in elderly people treated with immunomodulatory antibodies, at least for responses targeting shared tumor antigens. It remains to be established whether responses to tumor neoantigens are compromised by immunosenescence. Given the current emphasis on neoantigen responsiveness, this remains a concern.
The prognostic impact of tumor-infiltrating lymphocytes in colorectal cancer differs by anatomical subsite.
Authors: Berntsson Jonna , Svensson Maria Christina , Leandersson Karin , Nodin Bjorn , Krzyzanowska Agnieszka , Bjartell Anders , Eberhard Jakob , Jirstrom Karin
Published: November 2019

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Background: Several studies report dense infiltration of tumour-infiltrating T cells and natural killer cells to be associated with an improved prognosis in colorectal cancer (CRC); but whether these associations differ by tumour location remain unknown. This study investigated the prognostic impact of immune cell infiltration in CRC, with particular reference to the anatomical subsite of the primary tumour. Methods: Immunohistochemical expression of CD3, CD8, FoxP3, and CD56 was analysed in tissue microarrays with tumours from 557 incident CRC cases from a prospective population-based cohort. Kaplan-Meier and Cox regression analyses were applied to determine the impact of biomarker expression on 5-year overall survival (OS), in the entire cohort and in subgroup analysis of right colon, left colon, and rectum. Results: In the entire cohort, dense infiltration of all the investigated immune cells correlated significantly with an improved 5-year OS in both univariable and multivariable analysis, adjusted for age, TNM stage, differentiation grade, and vascular invasion. Dense infiltration of CD3+ and CD8+ cells were independent favourable prognostic factors for tumours in the right colon (hazard ratio [HR] = 0.53, 95 % confidence interval [CI] 0.29-0.95 and HR = 0.35, 95% CI 0.19-0.65, respectively), but not in the left colon or rectum. When microsatellite instability status was included in the adjusted model, only CD8+ cells remained an independent favourable prognostic factor in right-sided tumours. Dense infiltration of FoxP3+ cells was an independent favourable prognostic factor for tumours in the rectum (HR = 0.54, 95% CI 0.30-0.99), but not in the right or left colon. Infiltration of CD56+ cells did not carry any independent prognostic impact after stratifying for primary tumour site. Conclusions: The results from this study demonstrate that the prognostic impact of certain T cell subsets in CRC differs by primary tumour site, being most evident in right-sided tumours. These findings indicate that tumour location may be an important factor to take into consideration in therapeutic decisions, including eligibility for immunotherapy.
Potential role of hypovitaminosis D in renal cell carcinoma patients treated with immune-checkpoint inhibitors.
Authors: Bersanelli Melissa , Vaglio Augusto , Sverzellati Nicola , Galetti Maricla , Incerti Monia , Parziale Raffaele , Corrado Michele , Cosenza Agnese , Ferri Leonarda , Leonardi Francesco , Buti Sebastiano
Published: November 2019

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Background: hypovitaminosis D is frequent in renal diseases but it was never reported in metastatic renal cell carcinoma (mRCC). The interaction between vitamin D and its receptor (VDR) has a key role for T-cell activation. Naïve T-cells do not express VDR and had very low expression of phospholipase C-γ1 (PLC-γ1), with low responsiveness to T-cell receptor (TCR). Initial TCR signaling induces VDR expression and upregulates PLC-γ1 via the kinase p38 pathway. VDR interaction with high concentrations of vitamin D and PLC-γ1 increase are required to trigger classical TCR signaling, increasing T-cell responsiveness. On these basis, we hypothesized that hypovitaminosis D could contribute to lower responsiveness to immune-checkpoint inhibitors (CKI). Methods: we assessed vitamin D levels of mRCC patients undergoing therapy with CKI, with the aim to reveal hypovitaminosis D, evaluate its prevalence and hypothesize its potential role in the outcome of treatment with CKI. Results: of 10 mRCC patients pretreated with tyrosine-kinase inhibitors, vitamin D levels assessed before the first treatment with nivolumab (anti-PD1 CKI) revealed deficiency in 80% of cases (8 patients). Hypovitaminosis D was severe ( < 20 ng/ml) in 7 cases and moderate ( < 30 ng/ml) in one. The 2 patients without deficiency (20%), had vitamin D values near to the lower limit of normality. Oral supplementation with cholecalciferol was given when necessary, likely confounding the possible influence of vitamin D deficiency on the outcome of CKI treatment. Vitamin D normal values after two months of therapy were recovered in the great majority of cases. Interestingly, the only patient who achieved a good objective response to treatment had normal values of vitamin D before therapy. Conclusions: hypovitaminosis D could have a relevant prevalence in mRCC patients. Considering the role of vitamin D in T-cell activation, assessment of its levels and initiation of a supplementation before immunotherapy should be considered to enhance responsiveness. On the basis of these observations, we are planning a perspective multicenter study to investigate the role of hypovitaminosis D in mRCC patients treated with CKI (PRoviDenCe study).