Targeting DNA Damage Response and Replication Stress in Pancreatic Cancer

Background and aims: Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress and novel therapeutic response in PC to develop a biomarker driven therapeutic strategy targeting DDR and replication stress in PC. Methods: We interrogated the transcriptome, genome, proteome and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient derived xenografts and human PC organoids. Results: Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, co-segregates with response to platinum (P < 0.001) and PARP inhibitor therapy (P < 0.001) in vitro and in vivo. We generated a novel signature of replication stress with which predicts response to ATR (P < 0.018) and WEE1 inhibitor (P < 0.029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P < 0.001) but not associated with DDR deficiency. Conclusions: Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR proficient PC, and post-platinum therapy

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Phosphoflow cytometry to assess cytokine signaling pathways in peripheral immune cells: potential for inferring immune cell function and treatment response in patients with solid tumors

Abstract Tumor biopsy is often not available or difficult to obtain in patients with solid tumors. Investigation of the peripheral immune system allows for in-depth and dynamic profiling of patient immune response prior to and over the course of treatment and disease. Phosphoflow cytometry is a flow cytometry‒based method to detect levels of phosphorylated proteins in single cells. This method can be applied to peripheral immune cells to determine responsiveness of signaling pathways in specific immune subsets to cytokine stimulation, improving on simply defining numbers of populations of cells based on cell surface markers. Here, we review studies using phosphoflow cytometry to (a) investigate signaling pathways in cancer patients’ peripheral immune cells compared with healthy donors, (b) compare immune cell function in peripheral immune cells with the tumor microenvironment, (c) determine the effects of agents on the immune system, and (d) predict cancer patient response to treatment and outcome. In addition, we explore the use and potential of phosphoflow cytometry in preclinical cancer models. We believe this review is the first to provide a comprehensive summary of how phosphoflow cytometry can be applied in the field of cancer immunology, and demonstrates that this approach holds promise in exploring the mechanisms of response or resistance to immunotherapy both prior to and during the course of treatment. Additionally, it can help identify potential therapeutic avenues that can restore normal immune cell function and improve cancer patient outcome

In the field: exploiting the untapped potential of immunogenic modulation by radiation in combination with immunotherapy for the treatment of cancer

Radiation has long been the standard of care for many types of cancer. It is employed to locally eradicate tumor cells as well as alter tumor stroma with either curative or palliative intent. Radiation-induced cell damage is an immunologically active process in which danger signals are released that stimulate immune cells to phagocytose and present locally released tumor-associated antigens (TAAs). Recent studies have indicated that radiotherapy can also induce immunogenic modulation; an alteration in the phenotype of cancer cells that remain after treatment and renders these cells more sensitive to immune mediated attack.. These cells upregulate TAAs as well as markers, including MHC and costimulatory molecules, that make them much more immunostimulatory. As our understanding of the immunomodulatory effects of radiation has improved, interest in combining this type of therapy with immune-based therapies for the treatment of cancer has grown. Therapeutic cancer vaccines have been shown to initiate the dynamic process of host immune system activation, culminating in the recognition of host cancer cells as foreign. The environment created after radiotherapy can be exploited by active therapeutic cancer vaccines in order to achieve further, more robust immune system activation. This review highlights preclinical studies that have examined the alteration of the tumor microenvironment with regard to immunostimulatory molecules following different types of radiotherapy, including external beam radiation, radiolabeled monoclonal antibodies, bone-seeking radionuclides, and brachytherapy. We also emphasize how combination therapy with a cancer vaccine can exploit these changes to achieve improved therapeutic benefit. Lastly, we describe how these laboratory findings are translating into clinical benefit for patients undergoing combined radiotherapy and cancer vaccination

Metformin for biochemical recurrence of prostate cancer: Immune data from a phase 2 study

377 Background: Metformin impacts immune response in several ways. It protects CD8 TILs from apoptotic death, downregulates CD39 and CD73, reduces MDSC activity and suppresses the transcription of PD1 thus enhancing CD8+ T cell antitumor activity (Ma et al, 2000, Nature). There is limited data on the immunologic impact of metformin treatment in hormone sensitive prostate cancer. Methods: We conducted an open label, randomized, phase II trial of bicalutamide with or without metformin of patients with high risk, biochemically recurrent prostate cancer (NCT02614859). Patients were randomized 1:2 to observation for an initial 8 weeks or metformin 1000 mg twice daily. Bicalutamide 50 mg/day was added after 8 weeks to both arms. The study discontinued accrual after interim analysis indicated no difference in PSA at 32 weeks between the two arms; however, metformin monotherapy for 8 weeks induced modest PSA declines observed in 40% of patients. Here we report immune responses in a subgroup of patients (N=12), including systemic cytokine levels and 158 circulating immune cell subsets after 8 weeks of metformin monotherapy. Results: Twelve patients treated at the NCI were analyzed. After 8 weeks, the metformin arm showed increased pDCs and lower Tregs compared to baseline. Significant differences in the percent change of immune parameters after 8 weeks of metformin monotherapy compared to 8 weeks of observation are summarized. Conclusions: Metformin has been shown to reduce markers of immune exhaustion in hormone sensitive prostate cancer which is supported by a greater reduction of PD-1 + and Tim3 + NK cells. Additional immune changes included increases in activated subpopulations of natural killer (NK) cells, which have been reported as a potential predictive biomarker of prolonged treatment response to hormone therapy in prostate cancer (Pasero et al, Oncotarget, 2015). Clinical trial information: NCT02614859 . [Table: see text

A randomized, double-blind, phase II clinical trial of GI-6301 (yeast-brachyury vaccine) versus placebo in combination with standard of care definitive radiotherapy in locally advanced, unresectable, chordoma

Abstract only 11527 Background: Chordoma is a rare neoplasm of the notochord that overexpresses brachyury, a transcription factor associated with epithelial-to-mesenchymal transition, metastasis, poor prognosis, and chemotherapy resistance. GI-6301 is a recombinant yeast-brachyury vaccine shown to demonstrate brachyury-specific immunogenicity, excellent safety profile, and some evidence of clinical activity in patients with chordoma in a previous phase I trial. Radiation therapy (RT) can modulate the tumor to become an immunostimulatory milieu. Preclinical studies have shown a synergistic effect combining RT with vaccine, thus prompting this clinical trial evaluating the combination of GI-6301 with RT in chordoma (NCT02383498). Methods: Adults with locally advanced, unresectable chordoma were treated on a randomized, double-blind, placebo controlled, phase 2 clinical trial. Patients received 3 doses of GI-6301 (80 x 107 yeast cells) vs placebo followed by photon or proton RT, followed by GI-6301 vs placebo until disease progression. Primary outcome was overall response rate (ORR) defined as a complete response or partial response in the irradiated tumor site at 24 months. Immune assays were conducted to evaluate immunogenicity. Due to slow accrual, an unplanned interim analysis was undertaken. Results: 24 pts were randomized and treated between May 2015 and September 2019. Vaccine was well tolerated with no dose reductions or treatment discontinuation. Treatment-related serious adverse events included: nausea/emesis (2); fatigue (2); dehydration (2); diarrhea (1); radiation necrosis (1); stroke (1); sepsis (1). There was no difference in ORR between the two arms. Pre-existing brachyury-specific T-cells were detected in the majority of patients but did not correlate with response to therapy. Most patients developed T-cell responses during therapy, regardless of treatment arm. Conclusions: There was no difference in ORR between the two arms. GI-6301 was well tolerated with toxicities related to RT, not vaccine. The trial was stopped early due to low conditional power for finding a statistical difference at the planned end of accrual, based on findings to date. Future studies will define utility of vaccines targeting brachyury in chordoma. Clinical trial information: NCT02383498 . [Table: see text

The immunocytokine M9241 in the treatment of prostate cancer (PCa): Clinical and immune data from a phase 1 study

127 Background: Interleukin-12 (IL-12) is a proinflammatory cytokine that plays a critical role in regulating the transition from innate to adaptive immunity but has toxicity with systemic administration. M9241 is an immunocytokine composed of 2 IL-12 heterodimers, each fused to one of the H-chains of the NHS76 antibody, which has affinity for both single and double stranded DNA. Thus, M9241 targets delivery to regions of tumor necrosis where DNA has become exposed. NCT01417546, a phase I trial of M9241 at escalating doses, established safety and dosing (Strauss J et al, CCR 2019). This was the first use of M9241 in human subjects with solid tumors including PCa. Methods: Nine patients (pts) with PCa enrolled in the phase 1 study, not all of which were presented previously. M9241 was given subcutaneously every 4 weeks (0.1-21.8mcg/kg) or every 2 weeks (12-16.8mcg/kg). PSA declines and immune responses were evaluated including systemic cytokine levels and 30 markers on 158 circulating immune cell subsets. Results: Nine PCa pts were treated with NHS-IL12 and 8 were evaluable for response, including 6 pts with biochemical recurrence and 2 with metastatic castration resistant prostate cancer (one patient discontinued the study treatment after 1 dose due to grade 3 elevation in ALT). There were no adverse events (AEs) of grade >4. Additional grade 3 toxicities included one each of: leukopenia, neutropenia and lymphopenia. The most common AEs of any grade were lymphopenia (77.8%), fatigue (55.6%), and ALT elevation (55.6%). 5 of 8 (62.5%) had PSA declines ranging from 8-42%. After treatment with M9241, evaluable pts had increases in systemic IL-10, TNF and INFg. Additional immune changes included increases in activated subpopulation of natural killer (NK) cells, consistent with the phase 1 experience. Conclusions: M9241 was found to be safe and well tolerated in PCa pts. PSA declines occurred in 5 of 8 evaluable pts. As with the phase 1 study, increases in NK subpopulations were seen in the small number of evaluable pts. These preliminary findings of a necrosis-targeting immunocytokine will be evaluated further in combination studies with cytotoxic therapy. M9241 is currently being evaluated in combination with docetaxel in metastatic prostate cancer (NCT04633252). Clinical trial information: NCT01417546

A randomized phase 2 study of bicalutamide with or without metformin for biochemical recurrence in overweight or obese prostate cancer patients (BIMET-1)

Background Metformin may have anticancer effects that are independent of its hypoglycemic effects. Retrospective studies have shown that metformin use is associated with decreased incidence of prostate cancer and prostate cancer-specific mortality. Preclinical studies suggesting additive anticancer effects of combining metformin and bicalutamide prompted this clinical trial (NCT02614859). Methods This open-label, randomized, phase 2 trial enrolled non-diabetic patients with biochemically recurrent prostate cancer, a PSADT of 3-9 months, BMI > 25 and normal testosterone. Patients were randomized 1:2 to observation for an initial 8 weeks (Arm A) or metformin 1000 mg twice daily (Arm B). Bicalutamide 50 mg/day was added after 8 weeks to both arms. The primary objective was to evaluate the number of patients with undetectable PSA ( < 0.2 ng/mL) at the end of 32 weeks. Immune correlatives were assessed as exploratory endpoints. Results A total of 29 patients were enrolled from March 2015 to January 2020. No difference was seen between the 2 arms in the proportion of patients with undetectable PSA. Modest PSA decrease ranging from 4% to 24% were seen in 40.0% (95% CI: 19.1-64.0%) of patients with metformin monotherapy, compared to 11.1% (95% CI: 0.3-48.3%) in the observation arm. Metformin monotherapy reduced PD-1(+) NK cells, and increased NKG2D(+) NK cells. The combination of metformin and bicalutamide led to greater reductions in PD-1 expressing NK, CD4(+) T, and CD8(+) T-cell subsets compared to bicalutamide alone. The trial was stopped early due to predicted inability to achieve its primary endpoint. Conclusions Although metformin plus bicalutamide was well tolerated, there was no improvement in rates of achieving undetectable PSA at 32 weeks. Metformin monotherapy induced modest PSA declines in 40% of patients after 8 weeks. Metformin, given alone and in combination with bicalutamide, displayed immune modifying effects, primarily within NK and T cells subsets