bluuebunny/crossref_metadata_2025 huggingface dataset:
Relationship between lymphopenia and objective response rate with programmed death-1 (PD-1) inhibitor therapy: A single-center retrospective analysis.
Authors: Yarchoan Mark , Diehl Adam , Johnson Burles Avner , Scott Blake , Hopkins Alexander , Azad Nilofer Saba , Jaffee Elizabeth M. , Grossman Stuart A.
Published: September 2018

e14512

Background: Lymphopenia is frequent in advanced cancers, but the impact of absolute lymphocyte count (ALC) on clinical responses with PD-1 immune checkpoint inhibitors is unknown. Methods: We performed a retrospective chart review of adult solid tumor patients treated with a PD-1 inhibitor (nivolumab or pembrolizumab) at Johns Hopkins from 2015-2016. Patients receiving PD-1 inhibitors concurrently with investigational immunotherapies, on unreported clinical trials or for cancers in which the activity of immune checkpoint inhibitors is unclear, or for whom response information is not available, were excluded. Data were collected on patient treatment history, objective response to PD-1 therapy by RECIST 1.1, and ALC at treatment initiation and one and three months after treatment initiation. Results: 172 patients met the inclusion criteria (lung n = 54; melanoma n = 61; kidney n = 25; other tumors n = 32). Lymphopenia (ALC < 1000) was present in 30.2%, 26.7%, and 34.3%, at 0, 1, and 3 months after treatment initiation. In a multiple regression analysis that adjusted for age, sex, ethnicity, tumor type, number of prior therapies, and concurrent ipilimumab usage, lymphopenia at baseline was not predictive of response to an immune checkpoint inhibitor. However, in this same model, lymphopenia at 1 and 3 months after treatment initiation was inversely related to response rate (p < 0.01). Of patients with lymphopenia at baseline, 37 patients (71.2%) had persistent lymphopenia (ALC < 1000 at baseline persisting to month 3) whereas 15 patients (28.8%) had normalized lymphocyte counts by month 3. In a univariate analysis, patients with persistent lymphopenia had decreased response rates as compared to patients who were not lymphopenic at baseline (21.6% vs. 47.5%, p < 0.01). However, patients with lymphopenia at baseline who had normalized lymphocyte counts at month 3 responded similarly as patients who were not lymphopenic at baseline (46.7% vs. 47.5%). Conclusions: Patients on anti–PD-1 therapy with persistent lymphopenia may have a reduced likelihood of responding to these agents. This association requires further validation in additional patient cohorts.
Circulating immune-profile as predictor of outcome in advanced NSCLC patients treated with nivolumab.
Authors: Tiseo Marcello , Veneziani Michele , Gelsomino Francesco , Buti Sebastiano , Facchinetti Francesco , Squadrilli Anna , Bordi Paola , Bersanelli Melissa , Cosenza Agnese , Ferri Leonarda , Rapacchi Elena , Mazzaschi Giulia , Leonardi Francesco , Quaini Federico , Ardizzoni Andrea , Missale Gabriele
Published: September 2018

e14514

Background: Detection of predictive markers of anti-PD-1/PD-L1 antibodies activity is of pivotal interest in non-small cell lung cancer (NSCLC). This study aimed to identify a circulating immuno-profile as predictor of outcome in NSCLC patients treated with nivolumab Methods: A peripheral blood immuno-profile evaluation was performed at baseline (T0), after 2 (T1) and 4 cycles (T2) of bi-weekly nivolumab in advanced pre-treated NSCLC patients from two Italian Institutions. First tumor assessment was performed after 4 cycles and then every 2 months. FACS analysis of lymphocyte subpopulations [CD3, CD4, CD8, NK (CD56), Treg (FOXP3) and MDSC] was performed. Absolute and % changes of lymphocyte subsets together with their functional and proliferative activity were assessed. Quali-quantitative leucocyte composition at baseline and its variation during therapy were correlated with tumor response and survival. Results: In the overall population of 54 treated patients, baseline Neutrophil-to-Lymphocyte ratio and NK count, lymphocyte count and CD4 variations during therapy showed a statistically significant prognostic role (p < 0.001; p = 0.012; p < 0.001; p = 0.010, respectively). Among 31 patients (squamous carcinoma, n = 17; adenocarcinoma, n = 14) in which all 3 time-points samples were available, 19 were responders (response and stable disease) and 12 non-responders. In responders, absolute numbers of total NK and NKCD56dim subset were higher at baseline and their increase between T0 and T1 was statistically significant (p < 0.05). Responders also displayed increased cytotoxic capability as shown by a higher baseline expression of CD3ζ, perforin and granzyme in NKCD56dim subset. No significant variation was documented in absolute number and functional activity of CD4+ and CD8+ lymphocytes. A higher percentage of CD8+PD-1+ cells at baseline was observed in responders, while non-responders showed a statistically significant increase in the absolute number of MDSC during therapy (p < 0.05). Conclusions: The number and function of NKs and the frequency of PD-1 expression in CD8+ cells could represent predictive peripheral immuno-biomarkers for nivolumab treatment in advanced NSCLC.
Heterogeneity assessment of two PD-L1 clones: 22C3 and SP142 in patients with non-small cell lung cancer (NSCLC).
Authors: Xu Haipeng , Li Chao , Gen Lin , Fang Rong Xi , Lin Dong xian , Huang Yunjian , Zhuang Wu , Miao Qian , Jiang Kan , Huang Cheng
Published: September 2018

e14515

Background: Pembrolizumab proves to be effective for clinical treatment of non-small cell lung cancer (NSCLC). The clinical application of pembrolizumab should be hinged on the magnitude of PDL1 expression justified by 22C3 assay on Dako platform. However, whether another diagnostic assay, SP142, can replace 22C3 in pembrolizumab-based immunotherapy thus far remains largely unknown. Methods: Forty-nine cases were randomly collected from 207 NSCLC patients with known PD-L1 expression detected by 22C3. Each case was stained with SP142 on ventana platform. The scores obtained with SP142 were compared with 22C3 , according to the interpretation of 22C3 standard. The concordance of PD-L1 expression between 22C3 and SP142 were assessed by weighted Kappa Coefficient and McNemar-Bowker test, respectively. Results: Based on Dako 22C3-IHC platform, 5 (10.2%), 9 (18.4%) and 35 (71.4%) of 49 cases were classified into groups of strongly positive, weakly positive, and negative for PD-L1, respectively. Yet, 37 (75.5%) of 49 cases had the same results with SP142 protocol (Kappa value: 0.472, p < 0.001). Six cases with weakly positive PD-L1, 3 cases with strongly positive PD-L1 and 1 case with negative PD-L1 according to the 22C3 were misdiagnosed as negative, weak positive and weak positive PD-L1 by SP142, respectively (McNemar-Bowker analysis: p = 0.037). When using the 50% tumor cell PD-L1 expression as the cut-off point, overall sensitivity and specificity of SP142 were 40% (95% [CI], 5.3%-85.3%) and 100% (95% CI, 87.1%-100%), respectively (Kappa value: 0.545, p = 0.009; McNemar-Bowker analysis: p = 0.125). When using the 1% tumor cell PD-L1 expression as the cut-off point, adoption of SP142 yielded the sensitivity of 57.1% (95% CI, 28.9%-82.3%) and the specificity of 97.1% (95% CI, 83.3%-99.9%), respectively (Kappa value: 0.608, p < 0.001; McNemar-Bowker analysis: p = 0.063). Conclusions: Compared with 22C3 assay, PD-L1 expression scores were usually underestimated by the SP142, and it has a higher specificity but lower sensitivity. Our findings collectively suggest that it is not applicable to adopt the SP142-PD-L1 IHC Ventana platform to stratify NSCLC patients for pembrolizumab-based immunotherapy.
Estimating tumor mutation burden using next-generation sequencing assay.
Authors: Chaudhary Ruchi , Bishop John , Broomer Adam , Cyanam Dinesh , Mandelman David , Nistala Goutam , Hyland Fiona , Sadis Seth
Published: September 2018

e14529

Background: High tumor mutation burden is a promising biomarker shown in some cancer types to predict positive response to immune checkpoint inhibitors. We show the ability of a targeted cancer research panel to estimate tumor mutation burden per megabase. Methods: We present a single sample analysis workflow for estimating tumor mutation burden from FFPE research samples. Our assay utilizes a PCR-based target enrichment panel that interrogates 409 known key cancer genes covering ~1.7 megabase of genomic space. Our customized workflow requires only 20 ng of input DNA, and enables a 2 day turn-around time from sample to the result. The ease of our workflow enables less than 60 minutes of hands-on time for automated library preparation and templating on a batch of 8 samples. Next-generation Sequencing is performed using high throughput semiconductor sequencing platform to achieve sufficient depth (~500x coverage) and accuracy. Our custom analysis pipeline calls variants with optimized parameters on the tumor sample only, with no matched normal sample required, and applies filters to remove germ-line variants and background noise. Results: Through in silico analysis performed on The Cancer Genome Atlas (TCGA) data we demonstrate that the panel achieves high sensitivity ( > 90%) and specificity ( > 95%) necessary to separate high and low mutation burden samples. Our workflow provides clear separation between allele ratio of somatic and germ-line variants. Our filters consistently eliminate ~98% of germ-line variants from the set of all variants called in single sample analysis workflow. Evidence from tumor-normal analyses on matched tumor and normal samples suggests that our single sample analysis, on the tumor sample only, detects somatic mutations with high sensitivity and specificity with residual of < 3% germ-line variants. Our pipeline identifies mutational signatures consistent with specific mechanisms such as spontaneous deamination of 5-methyl-cytosine, as well as base-damage from FFPE processing. Conclusions: A simple workflow has been developed on the Ion Torrent sequencing platform to estimate per megabase somatic mutational burden from a single tumor FFPE sample. This solution can help advance research in immuno-oncology.
Neutrophil to lymphocyte ratio as predictive of prolonged progression free survival (PFS) and overall survival (OS) in patients with metastatic non-small cell lung cancer (NSCLC) treated with second-line PD-1 immune checkpoint inhibitors.
Authors: Labomascus Stephanie , Fughhi Ibtihaj , Bonomi Philip , Fidler Mary J. , Borgia Jeffrey Allen , Basu Sanjib , Hoch Matthew A , Batus Marta
Published: September 2018

e14530

Background: Baselinehigh neutrophil to lymphocyte ratio (NLR) has been associated with inferior overall survival in patients with stage III/IV NSCLC. Inflammation and neutrophilic infiltrates in the tumor microenvironment appear to inhibit anti-tumor immune response. We suspect that NLR might reflect the level of inflammation in tumor microenvironment. The objectives of this study were to evaluate potential relationships between pretreatment NLR and and PFS and OS in advanced NSCLC patients treated with second-line nivolumab or pembrolizumab. Methods: Patients with stage IV NSCLC who received at least one cycle of nivolumab or pembrolizumab after first-line treatment with a platinum doublet between January 2015 and December 2016 were included. Patient demographics including NLR at baseline, date of starting immunotherapy, and date of progression were recorded. The association between NLR and duration of response was assessed using a Mann-Whitney-Wilcoxon test. A cutoff of NLR of 3.5 and 5.0 based on published data (ref) were analyzed for differences in median overall survival and progression free survival. Results: 113 patients were analyzed: median age 68, male/female 38.9%/61.1%, 15% never smoked. The median PFS for patients with NLR < 5 was 4.14 months vs. 2.27 months in those with NLR > 5 (p = 0.031). Overall survival was also impacted by NLR. There were a total of 29 deaths in the cohort, 24 of these occurred in patients with NLR > 3.5 and 5 were in patients with NLR < 3.5. A lower NLR at baseline was significantly associated with improved overall survival (p = 0.036). Conclusions: A low baseline NLR is associated with superior progression free survival and overall survival in metastatic non-small cell lung cancer patients treated with nivolumab or pembrolizumab. These findings suggest that evaluating mediators of inflammation might help to identify potential therapeutic targets which could enhance effectiveness of PD-1 immune check point inhibitors in advanced NSCLC.
Serial genome-wide sequencing of cell-free DNA (cfDNA) by assays used in non-invasive prenatal testing (NIPT) to identify copy number alterations (CNAs) associated with immunotherapy response.
Authors: Goodman Aaron , Jensen Taylor , Kato Shumei , Ellison Christopher , Tran Lisa , Mazloom Amin , Grosu Daniel S. , Ehrich Mathias , Kurzrock Razelle
Published: September 2018

e14533

Background: cfDNA derived from tumor cells can be detected in the plasma of many cancer patients. Noninvasive methods to detect CNAs have been developed to detect chromosomal abnormalities in the setting of prenatal screening. We hypothesized that similar methods can be used to enable quantitative detection of CNAs in cfDNA, as represented by a genome instability number (GIN), in patients with cancer and used to monitor response to immunotherapy. Methods: Peripheral blood for CNA analysis was collected from patients with diverse cancers for whom immunotherapy was planned. Plasma was separated from peripheral blood using centrifugation. cfDNA was collected and subjected to low-coverage, genome-wide sequencing. CNAs were detected using methods developed for noninvasive prenatal testing. GIN was calculated as the cumulative sum of the deviation from baseline for all detected autosomal CNAs. The current analysis focused on patients with a complete or partial response (CR/PR) or progressive disease (PD). Results: Of 48 patients, 11 (23%) attained CR/PR; 17 (35%), PD; 20, stable disease. Twelve of the 28 patients (43%) with CR/PR (5 (42%)) or PD (7 (58%)) had a GIN value consistent with the presence of CNAs at treatment initiation and ≥ 1 additional sample for comparison. Four of the 5 patients with CR/PR had a decrease in their GIN from their baseline while all 7 patients with PD had a sustained increase in their GIN. The mean (standard error of the mean (SEM)) change in GIN from maximum to minimum for responders was –2753.8 (+/-1643); mean (SEM) change in GIN from minimum to maximum for progressors was 1373.76 (+/-576) (p = 0.0221). Interestingly, 3 of the 5 responders had an initial increase in their GIN after 2-3 weeks of treatment followed by a decline, while all 7 progressors had a continuous increase in their GIN. Conclusions: GIN can be used to monitor response to immune checkpoint inhibitors in cancer patients. Serial evaluation shows an initial GIN increase followed by decline in patients who eventually attain CR/PR, but a sustained increase in patients with progression.
The anti-tumour immune response after radiofrequency ablation of colorectal cancer.
Authors: Lemdani Katia , Capron Claude , Seguin Johanne , Mignet Nathalie , Boudy Vincent , Peschaud Frederique , Emile Jean Francois , Malafosse Robert
Published: September 2018

e14540

Background: Results of radiofrequency ablation (RFA), increasingly used to treat liver tumors, are compromised by local and systemic relapse. Hyperthermia related cancer cells death, release of tumor antigens and expression of danger signals activate a tumor-specific T-cells response. This effect remains ineffective to avoid recurrence. Therefore we propose to combine RFA with an activation of a solid immune antitumor response as curative treatment of a colorectal (CRC) metastatic disease in immunocompetent mouse. Methods: RFA was used to treat a CT26- luc tumor. In two distinct clinical situations, distant macroscopic or microscopic tumors were established as metastases before or at the time of RFA. Immune response was modulated by an injection in situof a thermo-reversible hydrogel loaded by GM-CSF and BCG, targeting dendritic cells. In the group of mice with large far lesions this strategy was combined with immune checkpoint inhibition. The efficiency was assessed on survival, evolution of distant lesions, characterization of lymphocyte infiltration in tumors and systemic immunity through specific TNF- α and IFN-y expression in spleen and draining lymph nodes. Results: The in situ immunogel injection after RFA resulted in a prolonged survival of mice. Regression of distant lesions was related to a strong systemic antitumor immune response and a great improvement of tumor infiltration by specific cytotoxic lymphocytes. In adjuvant situation, the use of immunogel induced a complete cure of microscopic secondary lesions without any treatment. Immune escape of large secondary lesions was reversed by association of RFA-immunogel vaccination with a systemic check point blockade, separately ineffective. Conclusions: Validation of this strategy, combining RFA of liver metastases and activation of a strong immune response controlling the residual disease, could result in a clinical assay including this approach within the standard treatment of CRC. Furthermorethe powerful synergy between RFA-in situ immunomodulation as a starter treatment and checkpoint blockade ineffective alone in CRC or after single RFA, allows reconsidering the use of immune checkpoint inhibitors in metastatic microsatellite stable CRC.
Preclinical development of tumor-infiltrating lymphocyte (TIL) based adoptive cell transfer (ACT) immunotherapy for patients with sarcoma and the potential benefit of anti-CD137 stimulation.
Authors: Nielsen Morten , Krarup-Hansen Anders , Hovgaard Dorrit , Petersen Michael Mørk , Loya Anand Chainsukh , Westergaard Marie Christine Wulff , Svane Inge Marie , Junker Niels
Published: September 2018

e14545

Background: Tumor specific TILs can be in vitro expanded and have the ability to induce complete and durable tumor regression in some patients following ACT. In this preclinical study we investigated the feasibility of expanding TILs from sarcomas, as well as performing functional in vitro analyses on these. Methods: Fresh tumor samples from sarcoma patients were obtained, and TILs were isolated and expanded in growth medium containing IL-2. In a sub study, we investigated the effect of adding an agonistic CD137 antibody (Urelumab, BMS) and/or an anti-CD3 antibody (OKT3) to the medium. Phenotype and functional analyses was performed using flow cytometry and IFNγ-Elispot. Results: Tumor samples from 30 patients with various types of sarcomas were obtained, and we were able to expand a minimum of 40 million TILs from 27 of these. Mean expansion times were 32 days (14 - 61). 87,7 % (36,4 – 99,1) of these cells were CD3+, and of these, 66,7 % (16,3 – 99,1) were CD4+, and 21,8 % (0,1 – 50,6) were CD8+. A sub study revealed that adding anti-CD137 and/or OKT3 increased total yield of TILs; anti-CD137 skewed the phenotype significantly towards more CD8+ TILs and in some cases NK cells and γδ T cells. TILs from 8 of 17 tested tumor samples from four different sarcoma subtypes (undifferentiated pleomorphic sarcoma, myxofibrosarcoma, myxoid liposarcoma and osteosarcoma) demonstrated reactivity against autologous tumor cells using IFNγ-Elispot (40 – 500 spots per 100.000 TILs). In TILs stimulated with anti-CD137 the reactivity increased. Conclusions: We were able to expand TILs from 90 % of the acquired tumor samples to numbers needed for possible future clinical ACT implementation. Expanded TILs were a mix of CD4+ and CD8+ with CD4+ being predominant. Approximately half of the TIL cultures showed some degree of in vitro tumor reactivity as determined by Elispot. Early analyses suggest that addition of anti-CD137 could influence expansion time, phenotype, and functional capacity of the expanded TILs. Based on these results, we conclude that it is feasible to translate TIL based ACT into clinical testing in sarcoma patients.
The status of PD-L1 expression and TILs among ESCC patients in China and changes after radiation and chemotherapy.
Authors: Wen Song , Huang Dingzhi , Ba Yi , Li Hongli , Liu Rui
Published: September 2018

e14567

Background: The status of PD-L1 expression and TILs (tumor infiltrating lymphocytes) is suggested as a predictive biomarker for the clinical effect of PD-1/PD-L1 inhibitors. In our study, weevaluate the status of PD-L1 expression and TILs in ESCC (esophageal squamous cell carcinoma), especially in CD8+ T cells. We also evaluate the changes in PD-L1 expression after chemotherapy or radiation. Then, we analyze the significance of these profiles. Methods: We retrospectively collected a total of 240 patients with histologically confirmed ESCC who underwent a radical operation between May 2009 and January 2015 at TianJin Medical University Cancer Institute & Hospital. Of these 240 patients, 104 patients received neoadjuvant therapy, including chemotherapy, radiotherapy or CCRT. We then examined the expression of PD-L1, CD8 and PD-1 in every patient. Results: We find a significant association between the PD-L1 expression and CD8+ T cell status (P = 0.044), while there is a negative relationship between the PD-L1 expression and PD-1+ T cell status (P = 0.048). Compared with the surgery-only group, there is an increase in the PD-L1 expression in NACRT and NCT (≥3 cycles) groups (P = 0.001 and 0.025). The PD-L1 expression non-significantly decreases in the NCT ( < 3 cycles) group (P = 0.197). We also observe an increase in CD8+ T lymphocytes in the NACRT and NCT ( < 3 cycles) groups (P = 0.116 and 0.036). Conclusions: We find a relatively high expression of PD-L1 and TILs in Chinese ESCC, indicating that PD-1/PD-L1 inhibitors may have a role in treatment. Additionally, there is an increase in PD-L1 and TILs after radiation or chemotherapy. As a result, combining PD-1/PD-L1 inhibitors with traditional cytotoxic therapy is a promising direction.
Correlation between tumor mutation burden and response to immunotherapy.
Authors: Bonta Ioana , Isac John Florin , Meiri Eyal , Bonta Dacian , Rich Patricia
Published: September 2018

e14579

Background: The use of immunotherapy is exponentially increasing in treatment of patients with advanced solid tumors. However, the response rates vary significantly between different tumor types and even within same tumor type (e.g. in lung cancer approx. 1 in 4 patients respond to immunotherapy). In order to better identify patients that will respond to immunotherapy, several markers have been proposed. Tumor mutation burden (TMB) has emerged more recently as a quantitative marker that can help predict responses to immunotherapies across different cancers, including melanoma, lung cancer and bladder cancer. TMB is a measure of the overall number of somatic protein coding mutations occurring in the tumor specimen. Methods: We analyzed 54 consecutive patients treated with immunotherapy at our institution for which we had genomic sequencing (FoundationOne). There were 39 lung cases and 15 non-lung (GI, GU, sarcoma). For 30 cases we had TMB data. Favorable response was defined as stable disease or response to therapy at 3 months. The relationship between TMB and tumor response was explored using ROC analysis. Results: The probability of a favorable response to immunotherapy in our patient dataset was 57% (31/54 patients). Among the patients with known TMB 60% (18/30) had a favorable response (stable disease or response to therapy). The favorable response rate for tumors originating in the lung was 64% (25/39) and for non-lung primary tumors was 40% (6/15). The difference was not statistically significant, with p = 0.12. Higher TMB values were correlated with increased probability of a favorable response. ROC analysis demonstrated an Az of 74% for TMB values in differentiating between patients with and without a favorable response. A TMB cutoff value of 8 mutations/megabase yielded a sensitivity of 95% and a specificity of 58% for predicting favorable response. Conclusions: In our data base 57%of patients with different solid cancers had favorable response to immunotherapy, in second line and beyond .Higher TMB correlated with higher likelihood of response to immunotherapy, independent of the primary site of cancer.
Effect of high-dose corticosteroids on CD4+ or CD8+ lymphocyte proliferation or IL-2 production after stimulation with pembrolizumab.
Authors: Walker John WT , Xu Lai , Smylie Michael , Elahi Shokrollah
Published: September 2018

e14587

Background: Immune checkpoint inhibitor (ICI) therapy has improved survival of patients with melanoma and other types of cancers. The effect of immunosuppresive corticosteroids on ICI efficacy is unknown, however treatment of immune-related adverse events with high-dose steroid therapy impairs neither efficacy nor time to failure of ICI. In certain clinical circumstances (for instance in the treatment of patients with symptomatic brain metastases) it may be advantageous to begin ICI therapy despite a requirement for steroid therapy. This study examined the effect of high-dose steroid therapy on lymphocyte proliferation/functionality after stimulation with anti-CD3 in the presence of pembrolizumab (PD-1 inhibitor) ex vivo. Methods: Peripheral blood mononuclear cells (PBMCs) were collected from patients with either chronic hepatitis C infection or active malignant melanoma. CD4+ and CD8+ T cells were isolated and subjected to flow cytometry stating for the expression of PD-1. Isolated PBMCs were then pre-treated with prednisone (1 nM - 1 mM) before anti-CD3 stimulation. Anti-CD3-stimulated cells were then incubated with pembrolizumab (5 µg/mL) and their proliferative capacity and IL-2 secretion ability were measured by carboxyfluorescein succinimidyl ester (CFSE) staining and ELISA, respectively. Results: A dose-dependent inhibition of both lymphocyte proliferation and IL-2 production was observed, but at physiologically relevant nanomolar prednisone concentration the steroid effect was negligible. Millimolar prednisone concentrations resulted in > 50% relative reduction in lymphocyte proliferation, however even at this steroid concentration treatment with pembrolizumab was able to further stimulate proliferation and IL-2 secretion versus anti-CD3stimulation alone. Conclusions: Despite heavily immunosuppressive treatment, CD4+ and CD8+ lymphocytes demonstrate proliferative capacity and immunological activity following treatment with pembrolizumab. This suggests that treatment with pembrolizumab may be efficacious despite a requirement for steroid therapy, a scenario often encountered in patients with brain metastases.
The relative clinical value of combined antiPD-1 and antiCTLA-4 treatment, the NNT values of immuno-therapy in metastatic melanoma patients.
Authors: Porneczy Edit , Boncz Imre
Published: September 2018

e14595

Background: The PD-1 inhibitor Nivo and antiCTLA-4 Ipi in combination was registered by FDA and EMA for the treatment of metastatic melanoma.The accelerated approval of this indication was based on PFS data.OS data in previously untreated melanoma pts was reported only in the Phase II CheckMate 069 study.In the Nivo+Ipi group the 1YOS% was 73,4% and 63,8% at 2Y, in the Ipi group 1YOS% was 64,8%, 2YOS% was 53,6% at median follow up of 24,5 months.The median OS had not been reached, the outcome measures could not assess the long-term benefit of antitumor immun response. Methods: In order to measure the relative clinical values, we analyzed the 1Y, 2Y survival data and calculated the ARR (absolut risk reduction) and NNT values (the number of patients who need to be treated to prevent one death) of the Phase II CheckMate 069 trial Nivo+Ipi vs Ipi group, and the overall survival data in subgroup of pts who discontinued the immun-therapy due to treatment related AEs. We used GraphPad Software QuickCalcs/NNT for calculating the results of 95% CI. Results: In the CheckMate 069, the Nivo+Ipi group vs Ipi group ARR was 10,93, the NNT was 10(95%CI 3,6-17,8) at 1Y.The ARR was 13,19, the NNT value was 8(95%CI 3,3-24,1) at 2Y. 1 in every 8-10 pts will benefit from the Nivo+Ipi combined immuno-therapy in order to prevent one death. In the subgroup of patients (37%) who discontinued treatment, the ARR was 21,16 at 1Y and 21,76 at 2Y. Despite of discontinuation, 1 in every 5 pts will benefit from immuno-therapy at 1Y and 2Y, the NNT was 5 (95%CI 2,5-40,4) and 2Y (95%CI 2,3-127,2). Conclusions: These results of NNT 5-10 represent the exceptional survival benefit of combined immuno-therapy. The limitations of NNT bias the pts demographic parameters and the value depends on comparator drug. The risk reduction was not constant over the follow up time and the long term benefit of immuno-therapy is not linear.The NNT data provide measurement of relative clinical value and clinical impact.
RNAi-mediated beta-catenin inhibition to promote T-cell infiltration and antitumor activity in combination with immune checkpoint blockade.
Authors: Ganesh Shanthi , Shui Serena , Lai Cheng , Dudek Hank , Wang Weimin , Brown Bob D , Abrams Marc
Published: September 2018

e14608

Background: Recent research suggests an important role for Wnt/β-catenin signaling in mediating cancer immune evasion and resistance to immune checkpoint therapy. The mechnism is believed to involve blocking of specific cytokines which trigger immune cell recruitment to the tumor, resulting in the phenomenon of T-cell exclusion and rendering the tumor to a non-inflamed state. Inhibition of β-catenin may be an effective strategy for increasing the low response rate to these effective medicines in numerous cancer populations. DCR-BCAT is an advanced preclinical development candidate that has a potent and specific chemically-optimized RNA interference (RNAi) trigger targeting CTNNB1, the gene that encodes β-catenin, formulated in a tumor-selective lipid nanoparticle. Methods: Syngeneic murine models and transgenic MMTV-Wnt1 mouse models were used in this study. In both cases, a sequential dose regimen was employed where, in each dosing cycle, animals received DCR-BCAT, followed by a combination of anti-PD-1 and anti-CTLA-4 on subsequent days. Pharmacodynamic endpoints included CTNNB1 (β-catenin), CCL4, PD-1, PD-L1 mRNA measurement by quantitative PCR, as well as β-catenin, perforin, and granzyme B immunohistochemistry. Results: In syngeneic models, β-catenin inhibition with DCR-BCAT significantly improved the T-cell infiltration. The combination of DCR-BCAT and immune checkpoint blockade yielded significant tumor growth inhibition compared to monotherapy in B16F10, 4T1, Neuro2A and Renca tumors. The combination therapy was associated with high levels of granzyme B and perforin, strongly suggesting that the mechanism of sensitization to checkpoint therapy was a sharp increase in T-cell mediated cytotoxicity. Finally, when DCR-BCAT was combined with anti-PD-1/CTLA-4 antibodies in mice which develop spontaneous Wnt-driven mammary tumors, checkpoint therapy potentiation yielded complete tumor regressions. Conclusions: These data offer proof-of-concept for conversion of non-inflamed tumors to inflamed tumors by β-catenin inhibition, and support clinical evaluation of this combination approach using a first-in-class RNAi-based agent.
Upregulation of amphiregulin on CD14++CD16- (classical) monocytes in non-small cell lung cancer.
Authors: McCarthy Fiona Margaret , Candido Juliana , Rudd Robin , Steele Jeremy P. C. , Balkwill Frances
Published: September 2018

e14612

Background: Amphiregulin is an EGFR ligand which contributes to many of the hallmarks of cancer including angiogenesis and metastasis. Recent studies demonstrate that Amphiregulin promotes an immunosuppressive tumour microenvironment by modulating the expression of T regulatory cells. Although its expression has been described on a variety of immune cells and tumour cells, there is no published data on Amphiregulin expression by monocytes to date. Previous work by this group has demonstrated that monocytes from non-small cell lung cancer patients are phenotypically and genomically different compared to healthy volunteers. This study aims to further investigate the differing genomic profile of monocytes in non-small cell lung cancer. Methods: Gene expression profiling was performed on CD14++CD16- (classical) monocytes of newly diagnosed patients with Stage IV non-small cell lung cancer (n = 6) and age-matched healthy volunteers (n = 6) using Affymetrix Human U133 Plus 2.0 array. Validation of differentially expressed genes of interest (including Amphiregulin) at mRNA level was performed using real-time PCR with TaqMan gene expression assays on independent patient (n = 6) and healthy volunteer cohorts (n = 6). Differential protein expression of Amphiregulin from the monocytes of healthy volunteers (n = 3) and non-small cell lung cancer patients (n = 3) was validated using ELISA technology. Results: Amphiregulin is one of the most upregulated genes on classical monocytes in non-small cell lung cancer patients compared to healthy volunteers (p = 0.001). The increased expression of Amphiregulin was confirmed on qPCR validation in an independent cohort of non-small cell lung cancer patients (p < 0.05) as well as in the original group (p < 0.001). Amphiregulin was also found to be increased at protein level on CD14++CD16- monocytes of non-small cell lung cancer compared to healthy volunteers. (p = 0.37). Conclusions: Amphiregulin is overexpressed by classical monocytes in non-small cell lung cancer and this over-expression is confirmed at mRNA and protein level. The production of Amphiregulin has not been ascribed to human monocytes in non-small cell lung cancer making its overexpression in this study a unique finding.
Association of an APOBEC mutational signature, mutational load, and BRCAness with inflammation and PD-L1 expression in HNSCC.
Authors: Rieke Damian Tobias , Messerschmidt Clemens , Ochsenreither Sebastian , Klinghammer Konrad Friedrich , Tinhofer Inge , Jöhrens Korinna , Klauschen Frederick , Beule Dieter , Keilholz Ulrich
Published: September 2018

e14613

Background: Immune checkpoints are an emerging treatment target in head and neck squamous cell carcinoma (HNSCC). Expression of PD-L1 and an IFNG immune signature have been shown as biomarkers of response. In this study we analyzed mutational signatures to identify recurrent mutational patterns associated with inflammation. Methods: HNSCC mutation and gene expression data were downloaded (n = 496, TCGA). Inflamed tumors were identified through upregulation of a predefined 6-gene IFNG immune signature. Mutational load, base exchange motifs and a BRCAness signature were analyzed in inflamed and non-inflamed tumors. Results were validated in independent datasets of cervical, urothelial and lung cancer (TCGA). Results: Total mutational load did not correlate with tumor inflammation (r = -0.03, p = 0.5). An APOBEC-associated tCw (thymine – cytosine – adenine/thymine) mutational signature was significantly associated with inflammation (r = 0.26, p = 2.923e-09). The tCw signature was furthermore correlated with the expression of PD-L1 and other immune checkpoints (r = 0.19, p = 1.723e-05). The TCW mutational signature correlated with the expression of APOBEC3 genes. Expression of APOBEC3 genes also correlated with tumor inflammation (r = 0.62, p < 2.2e-16). As a second mutational signature, indels and an independent BRCAness signature were significantly enriched in non-inflamed tumors (p = 6.464e-14). Enrichment of an APOBEC mutational signature in inflamed tumors could be validated in cervical and urothelial carcinoma but not NSCLC. Conclusions: In HNSCC, overall mutational load is not associated with inflammation. BRCAness is significantly associated with immune-desertion whereas expression of APOBEC genes and an APOBEC mutational signature significantly correlates with the expression of PD-L1 and a T-cell inflamed signature. These findings suggest that a specific mutational pattern might serve as a biomarker for immune checkpoint inhibition in cancer.
Intra-tumour heterogeneity in the regulation of immune-tolerogenic pathways in primary and metastatic hepatocellular carcinoma (HCC).
Authors: Pinato David James , Victor Stephane , Spina Paolo , Trivedi Pritesh , Pirisi Mario , Burlone Michela , Black James M , Minisini Rosalba , Leutner Monica , Boldorini Renzo , Mauri Francesco A , Sharma Rohini
Published: September 2018

e14614

Background: Anti-PD-1/PD-L1 checkpoint inhibitors have shown preliminary evidence of activity in HCC. PD-L1 status is a putative predictor of response, however PD ligands expression can be heterogeneous across sampled sites, with implications in the development of immunotherapy. Methods: We constructed a tissue microarray from 77 patients: 56 with surgically resected and 21 with intermediate/advanced HCC in whom 20 matching metastases were also available. Tumour and surrounding cirrhosis were analysed for PD-L1 and PD-L2 expression by immunohistochemistry. FFPE-extracted RNA was analyzed on the NanoString nCounter system using 12 paired primary/metastatic HCC cores from 6 patients, with validation in a subset of further 24 specimens. Results: Surgically resected cases were Child A (51/56, 91%), BCLC A (45/56, 80%). In resected tumour cores 11/56 (20%) were PD-L1 and 18/56 (32%) were PD-L2 positive. Surrounding cirrhotic liver revealed PD-L1 in the lymphocytic infiltrate in 8/56 samples (14%), whilst PD-L2 expression was found in the cirrhotic background in 29/56 cases (52%). PD-L2 expression in cirrhotic (p = 0.03) but not in tumour cores (p = 0.13) correlated with more advanced BCLC stage. In patients with intermediate-advanced HCC (n = 21), the prevalence of tumour PD-Ls expression was 40% (8/21) for both markers. Concordance between PD-L1 positive primary and metastases was 33% and 85% in PD-L1 negative tumours. In contrast, there was universal concordance between in PD-L2 expression between primary and metastatic HCC (p < 0.001). Targeted transcriptomic profiling revealed a subset of 40 genes involved in innate and adaptive immunity to be differentially regulated across primary and metastatic disease. Conclusions: Intra-tumour heterogeneity in the expression of PD-L1 is common in HCC, whilst PD-L2 is homogeneously distributed in primary and metastatic deposits. Unsupervised transcriptomic profiling confirms differential activation of innate and adaptive immune-related pathways in the metastatic dissemination of liver cancer. This has profound implications in the clinical development of immune response biomarkers in HCC.
Association of inflammatory cytokines with obesity and prevention with ASA or DHA/EPA supplementation.
Authors: Gruslova Aleksandra , Quach Duan , Lengfleder Lucy , Tiziani Stefano , Lodi Alessia , Kist Kenneth , Kaklamani Virginia G. , deGraffenried Linda A , Brenner Andrew Jacob
Published: September 2018

e14619

Background: Obesity is associated with poor breast cancer outcomes. Our prior data suggested a positive feedback loop whereby circulating factors associated with obesity induce the COX-2 enzyme, resulting in increased pro-inflammatory cytokine prostaglandin E2 (PGE2), which can then increase aromatase levels. In order to establish a better understanding of baseline PGE2 levels, as well as select the proper COX2 inhibitor for prospective study, we performed a randomized study of aspirin (ASA), fish oil (DHA/EPA), and the combination in postmenopausal women of varying body habitus and assessed changes in cytokines. Methods: Women were recruited and randomly assigned into 3 interventions: ASA 81mg, docosahexaenoic acid (DHA, 1500mg) with eicosapentaenoic acid (EPA, 2500mg), or combination ASA and DHA/EPA (DUAL). Blood was collected before and after 28 days of supplementation, and 27 cytokines were assessed by Luminex array (Millipore); PGE2 was measured by ELISA. Investigators were blinded until analysis was complete. Statistical analyses were performed in R. Results: 110 patients were included: 90% participants white; of which 50% Hispanic; average BMI 31.5±0.67; and age: 60.1±0.58. Cytokines were not correlated with demographic variables. At baseline inflammatory cytokines IL-6 and Eotaxin were correlated with BMI ((p > 0.029 and p > 0.017, respectively). After 28 days of DHA/EPA BMI was correlated with Eotaxin, Il-2, IL-7 and GM-CSF (p < 0.05), in ASA group correlation was significant only with IL-2 (cor.coef= 0.58). Two sample t-test showed significant difference between DUAL and ASA or DHA/EPA for IL-6 (p = 0.008 and p = 0.034, respectively), and for IL-2 only between DUAL and ASA (p = 0.039). Furthermore, impact on PGE2 was observed with ASA with 81% obtaining a decrease from baseline (median change -28%); by comparison 55.1% (-1%) and 65.6% (-22%) of subjects showed decrease in the DUAL and DHA/EPA groups respectively. Level of macrophage inflammatory proteins (MIP-1b) were lower in the DUAL group compare to ASA (p < 0.014). Conclusions: Chronic inflammatory cytokines are correlated with obesity, and may be modified by use of ASA and DHE/EPA. This strategy may be useful in the secondary prevention setting. Clinical trial information: NCT02062255.
TLR9 agonist lefitolimod to improve antitumor effect of checkpoint inhibitors in vivo.
Authors: Schmidt Manuel , Kapp Kerstin , Volz Barbara , Oswald Detlef , Wittig Burghardt
Published: September 2018

e14625

Background: TLR9 agonists are potent activators of the immune system via induction of cellular and humoral responses. Preclinical and ongoing clinical studies support the use of TLR9 agonists for immunotherapeutic approaches. Lefitolimod/MGN1703 is a covalently-closed dumbbell-like immune surveillance reactivator (ISR) with a broad immunomodulatory potential. Due to promising data from a phase 2 trial (IMPACT) as maintenance therapy after first-line chemotherapy in mCRC patients lefitolimod is recently evaluated in a phase 3 trial in mCRC patients (IMPALA). Furthermore, lefitolimod is currently investigated in a phase 2 trial in SCLC patients (IMPULSE) and in a phase 1 / 2 trial in HIV patients (TEACH). Methods: It was shown that lefitolimod reduces tumor growth in several murine tumor models. Since the mode-of-action of lefitolimod starts upstream of the initiation points of checkpoint inhibitors like anti-CTLA-4 or anti-PD-1/anti-PD-L1 a combinatory approach may result in an enhanced anti-tumor effect. Therefore, two syngeneic murine models were used – a colon carcinoma CT26 and a lymphoma A20 model – for evaluation of the anti-tumor effect in vivo. Results: In the CT26 model treatment with anti-PD-L1 (i.p.) had no effect on the tumor growth, whereas addition of lefitolimod (s.c.) to anti-PD-L1 led to an anti-tumor effect (tumor growth inhibition, TGI 48%) which consequently resulted in prolonged survival of the mice. This combinatory effect was even more pronounced in the A20 model where treatment with anti-PD-1 (i.p.) alone had a moderate anti-tumor effect which was vastly increased by the combination (TGI – anti-PD-1: 46%, anti-PD-1/lefitolimod 99%). Conclusions: In conclusion, we showed that lefitolimod, a member of dSLIM® family of TLR9 agonists and an ISR, can enhance the limited anti-tumor effects of checkpoint inhibitors in pilot studies in murine colon carcinoma and lymphoma tumor models in vivo. These data show the promising potential for the combination with checkpoint inhibitors. Notably, a clinical trial in cooperation with MD Anderson evaluating the benefit of lefitolimod in combination with ipilimumab is currently ongoing.
T cell population expansion in response to allogeneic cancer vaccine alone (DPV-001) or with granulocyte-macrophage colony-stimulating factor (GM-CSF) or imiquimod (I) for definitively-treated stage III NSCLC patients (pts).
Authors: Fox Bernard A. , Boulmay Brian C. , Li Rui , Happel Kyle T , Paustian Christopher , Moudgil Tarsem Lal , Puri Sachin , Dubay Christopher , Koguchi Yoshinobu , Mehta Adi , Lund-Johansen Fridtjof , Fisher Brenda K , Redmond William L , Bifulco Carlo Bruno , Ochoa Augusto , Hu Hong-Ming , Hilton Traci , Urba Walter John , Sanborn Rachel E.
Published: September 2018

e14639

Background: The DPV-001 DRibble is a dendritic cell-targeted microvesicle (proteasome blocked autophagosome) vaccine derived from an adenocarcinoma and a mixed histology cell line. It contains multiple TLR agonists and > 130 potential NSCLC antigens, many as prospective altered-peptide ligands. In preclinical studies, DRibble immunotherapy provided significant anti-cancer effects in a dozen models. We hypothesize that DRibble’ vaccination efficacy can be attributed to their capacity to present tumor-derived short-lived proteins (SLiPs) and defective ribosomal products (DRiPs) that are typically not processed and presented by professional antigen presenting cells and against which the host may be less tolerant. Methods: Pts received induction cyclophosphamide, 7 vaccines every 3-weeks, then every 6 weeks x 4 more doses. Pts were randomized to receive DRibble alone (A), or with I (B) or GM-CSF (C). PBMCs /serum were collected at baseline and at each vaccination to assess changes in antibodies (Ab) (ProtoArray and microsphere affinity proteomics), peripheral lymphocyte populations (flow cytometry) and T cell receptor (TCR) repertoires (Adaptive immunoSEQ). Results: 13 pts were enrolled (Arm A: 5; B: 4; C: 4). We previously reported that vaccination induced or increased IgG Ab responses against targets over-expressed by NSCLC. Patients receiving DPV-001 had a significant (p < 0.04) increase in total (CD4 + CD8) TCRs that increased 10 fold over baseline compared to normal controls (independent from trial, n = 3) and the increase in CD4 clones was similar to that seen following ipilimumab (melanoma pts, independent from trial, n = 9). Analysis of a resected metastasis (progressing on treatment), identified brisk infiltration of T cells and tumor that was strongly PD-L1+. Conclusions: Vaccination with DPV-001 expanded populations of T cells over that observed in controls and the increase in CD4 T cells was similar to that observed in patients receiving ipilimumab and may represent vaccine-reactive T cells. Clinical Trial Identifier: NCT01909752, Support: R44 CA121612 Clinical trial information: NCT01909752.