Background

The prognosis of hepatocellular carcinoma (HCC) is closely associated with recurrence and metastasis which has been proposed to be initiated by circulating tumor cells (CTCs). Yet, the transcriptomic plasticity and immune evasion mechanism of CTCs during circulation are not well defined.

Methods

Blood was drawn from 4 different vascular sites, including hepatic vein (HV), peripheral artery (PA), peripheral vein (PV) and portal vein (PoV) of 10 localized HCC patients. Single CTCs were isolated by negative enrichment and robotic micromanipulator, followed by single-cell RNA sequencing (scRNA-seq). After filtering, 113 CTCs with qualified data were subjected to bioinformatics analysis. The scRNA-seq results were further validated in three independent HCC cohorts.

Results

Our scRNA-seq data revealed remarkable intra- and inter-vascular heterogeneity among CTCs from four vascular sites. We determined CTC transcriptional dynamics during transportation through consecutive vascular sites and revealed their adaptation mechanisms under biomechanical stress. We further classified CTCs from different vascular sites into two subsets, namely dormant and activated CTCs. Dormant CTCs were associated with a non-cycling state and upregulation of EMT/angiogenic signatures and showed stronger prognostic ability for early relapse than activated CTCs. Furthermore, we discovered an immune escape mechanism by which CTCs recruited regulatory T cells (Tregs) via expression of CCL5, consequently promoting the formation of an immunosuppressive microenvironment favorable for their survival in bloodstream and distant colonization.We proved that MAX, activated through the p38 pathway, was the key transcriptional factor regulating CCL5 overexpression, which was validated by ChIP, luciferase reporter gene and in vitro/vivo knockdown assays. And we further determined that Tregs-derived TGF-β1 can heighten MAX expression, thus amplifying the CCL5 expression.

Conclusions

Collectively, our results reveal a previously unappreciated spatial heterogeneity of CTCs and a CTC immune-escape mechanism, which may aid in designing new anti-metastasis therapeutic strategies in HCC.

Legal entity responsible for the study

Jia Fan.

Funding

The State Key Program of National Natural Science of China.

Disclosure

All authors have declared no conflicts of interest.

Background

Ex vivo manipulation of primary cells is critical to the success of cell-based therapies, however, limitations of existing ex vivo delivery approaches may dramatically restrict the use of cell engineering to treat disease.

Methods

We used a genome-wide approach to study optimized electroporation treatment and microfluidic cell squeezing to identify the impact of delivery technique on gene expression profiles in human T cells. To validate the microarray results, we used a multiplex cytokine analysis comprised of 42 key T cell cytokines to assess perturbation of cytokine secretion. Finally, in a direct comparison of therapeutic functionality, the efficacy of T cells edited for PD-1 via electroporation and cell squeezing was assessed using therapeutic treatment of the Eg.7 OVA tumor model.

Results

We identified striking disruptions in transcript expression after treatment with electroporation (17% of genes mis-regulated, FDR q < 0.1), whereas cells treated with microfluidic squeezing had similar expression profiles to untreated control cells (0% of genes mis-regulated, FDR q < 0.1). These genetic disruptions result in concomitant perturbation of cytokine secretion including a 648-fold increase in IL-2 secretion (p < 0.01) and a 30-fold increase in IFNγ secretion (p < 0.05) in electroporated cells. Squeezing cells did not result in the non-specific secretion of any of the 42 cytokines tested. Ultimately, the effects at the transcript and protein level resulted in functional deficiencies in vivo with electroporated T cells failing to demonstrate sustained antigen-specific effector responses and tumor control.

Conclusions

This work demonstrates that the delivery mechanism used to insert biomolecules affects functionality and warrants further study. The significant differences in outcomes from the two techniques underscores the importance of understanding the impact of intracellular delivery methods on cell function for research and clinical applications. Hence, for both research and therapeutic applications, the functional and safety consequences of the selected intracellular delivery technique and its impact on cell phenotype should be carefully evaluated.

Legal entity responsible for the study

SQZ Biotechnologies.

Funding

SQZ Biotechnologies.

Disclosure

J. Hanson, J. Cole, L. Cassereau, J. Bugge, T. Ditommaso, J. Gilbert, H. Bernstein, A. Sharei: Employed by SQZ Biotech.

Background

AB122 is a fully human monoclonal antibody targeting PD-1. It binds tightly to a different PD-1 epitope than other currently approved antibodies in this class. Here we report preliminary data from an ongoing, open-label, dose-escalation (3 + 3 design) study evaluating the safety, tolerability, immunogenicity, pharmacokinetics, pharmacodynamics, and antitumor activity of AB122 monotherapy in patients (pts) with select advanced solid tumors.

Methods

Eligible pts had pathologically confirmed select advanced solid tumors, received ≤5 prior lines of systemic therapies, and had measurable disease per RECIST v1.1. AB122 was administered intravenously (IV) every 2 wks (Q2W) at escalating doses (80, 240, 720 mg). Intermediate Q2W doses and other schedules (every 3 wks [Q3W] or 4 wks [Q4W]) were also evaluated. Adverse events (AEs) were graded according to NCI CTCAE v4.03 and antitumor activity was assessed using RECIST v1.1.

Results

As of 15Aug2018, 19 pts with advanced ovarian (n = 7), endometrial (n = 3), colorectal (n = 3), gastroesophageal (n = 2), head and neck, breast, prostate, and lung (n = 1 each) cancer were treated at doses of 80 mg Q2W (n = 3), 240 mg Q2W (n = 6), 360 mg Q2W (n = 1), 360 mg Q3W (n = 4), and 480 mg Q4W (n = 5). Most pts were female (74%) and White (89%); median age was 68 yrs (51-80). The number of doses received ranged from 1 to 18; 8 pts remain on study. The most common treatment-emergent AEs were fatigue (42%), diarrhea (21%), urinary tract infection, back pain, nausea, and anemia (16% each). 16% of pts each had ≥Grade 3 AEs and serious AEs but none were considered treatment-related by investigators. There were no dose-limiting toxicities or discontinuations due to AEs. Data from pts in the 80 and 240 mg Q2W cohorts showed that AB122 serum concentrations ≥1.5 μg/mL (equivalent to 10 nM) are associated with full receptor occupancy. Among 12 efficacy-evaluable pts, the disease control rate was 42%.

Conclusions

These preliminary results indicate that AB122 monotherapy was well tolerated and active in various advanced solid tumor types. The dose and schedule of 240 mg IV Q2W were selected for further evaluation as monotherapy and in combination with other agents.

Legal entity responsible for the study

Arcus Biosciences, Inc.

Funding

Arcus Biosciences, Inc.

Disclosure

L.C. Seitz, W. Berry, D. Ashok, D. Direnzo, L. Jin, S.J. Lee, A. Park, D. Piovesan, J. Karakunnel: Employee of Arcus Biosciences, Inc. A. Rieger, J.B.L. Tan, M.J. Walters: Employee of Arcus Biosciences, Inc. 

Background

Immune checkpoint blockade represents a major breakthrough in cancer therapy with recent approvals of PD-1 or PD-L1 antagonist therapy in France for a range of cancer indications. To generate high evidenced-based knowledge and to prevent off-label use, the French National Cancer Institute (INCa) launched the AcSé Immuno-therapy Program: two exploratory phase II trials, to allow for a nationwide safe and controlled access to nivolumab or pembrolizumab outside of their current marketing approvals for selected rare cancer indications where the literature suggests a potential benefit for patients, but where the difficulties of development render individual experimental studies unattractive to the pharmaceutical industry.

Trial design

The two trials, AcSé Nivolumab and AcSé Pembrolizumab are Phase 2, single-arm, national, multicentre trials investigating the efficacy and safety of nivolumab and pembrolizumab, respectively, in adult patients with specific rare cancers who have unresectable locally advanced or metastatic disease which is resistant or refractory to standard therapy and for whom no alternative approved or experimental treatment options exist. Up to 650 patients will be enrolled across the two trials and assigned to one of 13 cohorts (max. 50 patients/cohort) according to their indication (see table).Table: 109TiP

The trials will use a two-stage Bayesian enrichment design to identify potentially sensitive indications and assess treatment efficacy per cohort. Toxicity will also be assessed per cohort and biological samples collected to explore the predictive factors of response and mechanisms of acute and acquired resistance to anti-PD-1 therapy in these populations.

Legal entity responsible for the study

UNICANCER.

Funding

Institut National du Cancer (INCa), La Ligue contre le Cancer, Bristol-Myers Squibb (BMS), MSD.

Disclosure

All authors have declared no conflicts of interest.

Background

Pembrolizumab monotherapy was approved for first-line (1L) treatment of metastatic non-small cell lung cancer (NSCLC) with PD-L1 tumor proportion score (TPS) ≥50% in October 2016 in the US, based on the findings from KEYNOTE-024 trial. A recent update of the trial with 25.2 months median follow-up demonstrated median pembrolizumab treatment duration of 7.9 months. The objective of our study is to estimate real-world time on treatment (rwToT) for 1L pembrolizumab monotherapy in a clinically matched real-world NSCLC database.

Methods

This retrospective study utilized the Flatiron Health EHR-derived advanced NSCLC database from which patients with stage IV diagnosis, TPS ≥50%, at least one dose of 1L pembrolizumab monotherapy, and ECOG performance status (PS) 0-2 were included. Patients with missing or ECOG PS > 2 were excluded. rwToT was defined as the length of time between first and last administration date of pembrolizumab. Patients with a record of next line of therapy, or death, or whose last activity date was ≥120 days from the last administration date were considered discontinued; others were censored. The Kaplan-Meier (KM) estimates were generated for median rwToT, restricted mean (rMean) rwToT at maximum time point where at least 10% of patients have not discontinued, and landmark on-treatment rates. rMean rwToT at 24 months was also computed using the parametric function that provided best fit based on statistical testing and visual inspection criterion. All results were stratified into ECOG PS 0-1 (similar to KEYNOTE-024) and ECOG PS 2. Flatiron dataset from 31-Jul-2018 with median follow-up of 10.8 months was utilized.

Results

Real-World Time on Treatment and On-Treatment Rate for First-Line Use of Pembrolizumab Monotherapy in Stage IV NSCLC, TPS ≥50%Table: 51O

Conclusions

In the real-world, duration of pembrolizumab use for 1L metastatic NSCLC is similar to KEYNOTE-024 when restricted to a trial-matched population of ECOG 0-1 patients. Duration of use tended to be however shorter in a real-world population of ECOG 2 patients who are commonly excluded from clinical trials.

Legal entity responsible for the study

Funding and support for this research was provided by Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA.

Funding

Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA.

Disclosure

V. Velcheti: Advisory/consultant role: Merck, Bristol-Myers Squibb, AstraZeneca, Genentech, Celgene, Takeda, Foundation Medicine, Nektar Therapeutics. S. Chandwani, X. Chen, M.C. Pietanza, T. Burke: Employee and shareholder: Merck & Co., Inc., Kenilworth, NJ, USA.

Background

Immune-targeted monoclonal antibodies blocking inhibitory immune checkpoints such as CTLA-4, PD-1 and PD-L1 have shown durable responses in multiple tumour types. These drugs generate a new type of complications in oncology: immune-related adverse events (ImAE). There is no established predictive biomarker to identify patients (pts) who are likely to develop severe ImAE. The hypothesis is that imAEs could result from host related pre-existing factors: Activation of self-reactive T and B cells, genetic predisposition, metabolic factors, enhanced cross reactivity between cancer cells, healthy cells and resident microbiota flora, and co-morbidity, concomitant treatments and patients’ environment. Response to immunotherapy could also be associated with pre-existing factors and tumor microenvironment features.

Background

Immune-targeted monoclonal antibodies blocking inhibitory immune checkpoints such as CTLA-4, PD-1 and PD-L1 have shown durable responses in multiple tumour types. These drugs generate a new type of complications in oncology: immune-related adverse events (ImAE). There is no established predictive biomarker to identify patients (pts) who are likely to develop severe ImAE. The hypothesis is that imAEs could be due to host related pre-existing factors: Activation of self-reactive T and B cells, genetic predisposition, metabolic factors, enhanced cross reactivity between cancer cells, healthy cells and resident microbiota flora, and co-morbidity, concomitant treatments and patients’ environment. Response to immunotherapy could also be associated with pre-existing factors and tumor microenvironment features.

Trial design

The study is a French ancillary study of a phase 3b, open-Label, multi-Centre, safety study of fixed-dose durvalumab + tremelimumab combination therapy or durvalumab monotherapy in advanced solid malignancies (STRONG) (NCT03084471). By collecting additional blood and tumour samples (table 1) the aim of this ancillary study is to define the immune phenotype, microbiome analysis of patients prior to an immunotherapy and who are subsequently developing an imAE or who achieve response to identify and characterize predictive factors of the toxicity and of efficacy. For both efficacy and safety objectives, respectively 85 and 300 patients will be required. The STRONG study currently includes pts with an urothelial and nonurothelial carcinoma of the urinary tract, treated with durvalumab as single agent at progression on or after of chemotherapy. As of July 2018, the IOPREDI ancillary study has been opened, 18 centres have been activated and 9 patients have been enrolled. Table 1 a) definition of AESI see section 6, b) only at baseline for polymorphisms, c) within 72 hours prior to the first treatment administration d) within 72 hours of occurrence of AESI.

Methods

The study is a French ancillary study of a phase 3b, open-Label, multi-Centre, safety study of fixed-dose durvalumab + tremelimumab combination therapy or durvalumab monotherapy in advanced solid malignancies (STRONG) (NCT03084471). By collecting additional blood and tumour samples (table 1) the aim of this ancillary study is to define the immune phenotype, microbiome analysis of patients prior to an immunotherapy and who are subsequently developing an imAE or who achieve response to identify and characterize predictive factors of the toxicity and of efficacy. For both efficacy and safety objectives, respectively 85 and 300 patients will be required.

The STRONG study currently includes pts with an urothelial and nonurothelial carcinoma of the urinary tract, treated with durvalumab as single agent at progression on or after of chemotherapy. As of July 2018, the IOPREDI ancillary study has been opened ,18 centres have been activated and 9 patients have been enrolled.

Table 1

a) definition of AESI see section 6, b) only at baseline for polymorphisms, c) within 72 hours prior to the first treatment administration

d) within 72 hours of occurrence of AESI

Clinical trial identification

EudraCT: 2016-005068-33.

Legal entity responsible for the study

AstraZeneca.

Funding

AstraZeneca.

Disclosure

A. Marabelle, F. Ghiringhelli: Honoraria for scientific boards: AstraZeneca. M. Ayyoub, O. Adotevi, Y. Loriot, O. Lambotte: Honoraria for advisory boards: AstraZeneca. D. Cupissol, D. Damotte, N. Chaput: Honoraria for scientific committee meetings: AstraZeneca. J. Adam: Honoraria for advisory boards: AstraZeneca. B. Petre Lazar, M. Licour: Employee of AstraZeneca. All other authors have declared no conflicts of interest.Table: 30TiP

Background

Cemiplimab (REGN2810), a human monoclonal antibody to PD-1, has exhibited substantial antitumour activities in patients (pts) with advanced malignancies in a Phase 1 study. Most patients with advanced NSCLC do not respond to PD-1 inhibitor monotherapy. Here we report results of the Phase 1 EC 2, a combination regimen of cemiplimab plus RT in advanced NSCLC (NCT02383212).

Methods

Pts with advanced NSCLC who had relapsed after, or were refractory to at least first-line therapy and for whom palliative RT was clinically indicated, received cemiplimab 3 mg/kg every 2 weeks intravenously for up to 48 weeks plus RT (9 Gy × 3 times/week given 1 week after first dose of cemiplimab) to a single lesion. The co-primary objectives were to evaluate the safety, tolerability, and efficacy of cemiplimab plus RT. Tumour measurements (of non-irradiated target lesions) were performed by RECIST 1.1 every 8 weeks.

Results

As of 1 Sept, 2017, 33 pts (22 M/11 F; median age 67.0 years [range, 47–82]) were enrolled; 66.7% and 30.3% had an ECOG performance status of 1 and 0, respectively; the status of one pt was unknown. Overall response rate (ORR; complete response [CR] + partial response [PR]) was 18.2% (0 CR and 6 PRs) with a median duration of response of 14.9 months (95% CI: 5.5–14.9). Disease control rate (ORR + stable disease [SD]) was 72.7% (6 PRs + 18 SDs). The most common treatment-emergent adverse events (TEAEs) of any grade were decreased appetite (30.3%), fatigue (27.3%), and cough (24.2%). Grade ≥3 TEAEs occurring in ≥ 2 patients include anaemia (12.1%), hypophosphataemia, and urinary tract infection (each 6.1%). One patient had a TEAE of pneumonitis, considered related to study drug, with an outcome of death.

Conclusions

Cemiplimab plus RT demonstrated antitumour activity in pretreated pts with NSCLC. The safety profile is comparable with other anti-PD-1 agents and RT. The combination therapy regimen did not produce greater efficacy above that which can be achieved with PD-1 inhibitor monotherapy for advanced NSCLC.

Editorial acknowledgement

Medical writing support under the direction of the authors was provided by Emmanuel Ogunnowo, PhD, of Prime (Knutsford, UK) and funded by Regeneron Pharmaceuticals, Inc. and Sanofi according to Good Publication Practice guidelines.

Clinical trial identification

NCT02383212.

Legal entity responsible for the study

Regeneron Pharmaceutical Inc. and Sanofi.

Funding

Regeneron Pharmaceutical Inc. and Sanofi.

Disclosure

M.L. Johnson: Consulting or advisory role: Astellas Pharma, Otsuka. M. Crittenden: Research funding: Jounce, Nanobiotix. S. Jabbour: Research funding grants: Merck, Nestle, outside the submitted work. L. Rosen: Research funding: Regeneron Pharmaceuticals, Inc. P. Garrido: Personal fees: Roche, BMS, MSD, Pfizer, Lilly, Abbvie, Regeneron, AstraZeneca, Novartis, Boerinhger-Ingelheim, outside the submitted work. P. Rietschel: Shareholder and employee, honoraria: Regeneron Pharmaceuticals, Inc. K.K. Mohan, J. Li: Shareholder and employee: Regeneron Pharmaceuticals, Inc. E. Stankevich: Shareholder and employee: Regeneron Pharmaceuticals, Inc.; Shareholder: Celgene, Bristol-Myers Squibb, Merck. M. Feng: Shareholder and employee: Regeneron Pharmaceuticals, Inc.; Shareholder: Bayer. I. Lowy: Shareholder and employee, fees for travel and accommodation expenses, leadership: Regeneron Pharmaceuticals, Inc. M.G. Fury: Shareholder and employee, patents, royalties, other intellectual property: Regeneron Pharmaceuticals, Inc. All other authors have declared no conflicts of interest.