Filippos Koinis

Effect of First-Line Treatment on Myeloid-Derived Suppressor Cells’ Subpopulations in the Peripheral Blood of Patients with Non–Small Cell Lung Cancer

Introduction: Myeloid‐derived suppressor cells (MDSCs) are a heterogeneous population of immature cells of myeloid origin whose expression is induced by, among others things, vascular endothelial growth factor. We have previously identified two monocytic and one granulocytic MDSC subpopulations associated with the clinical outcome in patients with non–small cell lung cancer (NSCLC). The aim of the present study was to evaluate the effect of chemotherapy on these MDSC subpopulations. Methods: Circulating immune cells from 46 patients with unresectable NSCLC were analyzed by flow cytometry before the initiation of chemotherapy and after three cycles. Changes in the frequencies of the MDSC subpopulations were correlated with clinical outcome. Results: Chemotherapy had no uniform effect on either the number or the functionality of monocytic and granulocytic MDSCs. However, three cycles of bevacizumab‐containing regimens significantly reduced the percentage of the granulocytic‐MDSCs compared with non–bevacizumab‐based regimens (p = 0.0086). At the time of evaluation of response, disease progression was associated with significantly higher levels of all three MDSC subpopulations compared with in patients with disease control. In patients with disease progression after three cycles of chemotherapy, the percentage of CD15‐positive monocytic MDSCs was significantly increased compared with baseline. Conclusions: In the peripheral blood of patients with NSCLC, bevacizumab‐based chemotherapy significantly reduced the levels of granulocytic MDSCs. An increase in the levels of CD15‐positive monocytic MDSCs was associated with poor response to treatment and disease progression, providing evidence of their clinical relevance in patients with NSCLC.

Prognostic value of circulating regulatory T cell subsets in untreated non-small cell lung cancer patients

The role of the different circulating regulatory T-cells (Treg) subsets, as well as their correlation with clinical outcome of non-small cell lung cancer (NSCLC) patients is poorly understood. Peripheral blood from 156 stage III/IV chemotherapy-naive NSCLC patients and 31 healthy donors (HD) was analyzed with flow cytometry for the presence and functionality of CD4+ Treg subsets (naive, effector and terminal effector). Their frequencies were correlated with the clinical outcome. All CD4+ Treg subsets exhibited highly suppressive activity by TGF-β and IL-10 production. The percentages of naive Treg were found elevated in NSCLC patients compared to HD and were associated with poor clinical outcome, whereas the percentage of terminal effector Treg was lower compared to HD and higher levels were correlated with improved clinical response. At baseline, normal levels of naive and effector Treg were associated with longer overall survival (OS) compared to high levels, while the high frequency of the terminal effector Treg was correlated with longer Progression-Free Survival and OS. It is demonstrated, for first time, that particular CD4+ Treg subtypes are elevated in NSCLC patients and their levels are associated to the clinical outcome. The blocking of their migration to the tumor site may be an effective therapeutic strategy.

Phenotypic characterization of circulating tumor cells in the peripheral blood of patients with small cell lung cancer

Background To evaluate the phenotypic heterogeneity of circulating tumor cells (CTCs) based on the expression of proliferative, apoptotic and Epithelial-to-Mesenchymal Transmission (EMT) markers during front-line treatment in patients with small cell lung cancer (SCLC) and to evaluate their clinical relevance. Methods CTCs from 108 chemotherapy-naïve patients with SCLC were analyzed by double immunofluorescence staining using anti-Ki67, anti-M30, anti-Vimentin along with anti-CKs antibodies. In 83 patients CTCs were also enumerated using the CellSearch. Results Sequential samples were available from 76 and 48 patients after one-treatment cycle and on disease progression (PD), respectively, for immunofluorescence and from 50 and 36 patients after one-cycle and on PD, respectively, for CellSearch. At baseline, 60.2% of the patients had detectable CTCs by either method. Both proliferative (CK67+) and non-proliferative (Ki67-), apoptotic (M30+) and non-apoptotic (M30-) as well as EMT (Vim+) CTCs were present in the same patient. Among 22 patients without detectable CTCs by CellSearch, CK+/Ki67+ and CK+/Vim+ CTCs could be detected in 6 (27.3%) and 6 (27.3%) patients, respectively. One-chemotherapy cycle reduced both the incidence of detection (p<0.001) and the absolute number (p<0.001) of CTCs; conversely, on PD both the incidence of detection and the number of CTCs were significantly increased (p = 0.002 and p = 0.04, respectively). Multivariate analysis revealed that the increased number of Vim+ CTCs at baseline and of non-apoptotic CTCs on PD could be emerged as independent prognostic factors associated with decreased OS(p = 0.009 and p = 0.023, respectively). Conclusions CK+/Ki67+, CK+/M30+ and CK+/Vim+ CTCs represent distinct subpopulations of CTCs in patients with SCLC, can be detected even in the absence of detectable CTCs by CellSearch; CK+/Ki67+ and CK+/Vim+ CTCs are associated with unfavorable clinical outcome.

Therapeutic strategies for chemotherapy-induced neutropenia in patients with solid tumors

Introduction: Chemotherapy-induced neutropenia (CIN) is a common adverse event during treatment of cancer patients, associated with increased morbidity, mortality, health care costs and impairment of patients’ quality of life which necessitate dose reductions. Areas covered: A computerized systematic literature search was performed through Medline, PubMed, Google Scholar and the Cochrane Library to identify peer reviewed publications relevant to CIN, pathophysiology and epidemiology, patient risk-assessment and existing treatment approaches. Additionally, emerging issues such as alternative therapeutic options and implications in elderly care were addressed. Expert opinion: Although CIN represents a common adverse event in the management of patients with solid tumors, the heterogeneity in clinical practice across different settings underlines the need to improve existing tools for accurate patient classification. Moreover, the definition of the optimal implementation of out-patient treatment and the use of colony-stimulating factor as add-on treatment together with antibiotics should be further investigated in order to accumulate more solid data. Finally, physician education is required to ensure that scientific knowledge is implemented in the daily clinical practice.

Evaluation of PD-L1/PD-1 on circulating tumor cells in patients with advanced non-small cell lung cancer:

Background: Circulating tumor cells (CTCs) could escape from the immune system through the programmed death-ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) axis leading to the development of metastasis. The current study investigated the expression of PD-1/PD-L1 on CTCs isolated from non-small cell lung cancer (NSCLC) patients treated with chemotherapy. Patients and methods: CTCs were isolated from 30 chemo-naïve stage IV NSCLC patients before and after front-line chemotherapy using the ISET filtration platform. CTCs were detected by Giemsa and immunofluorescence (IF) staining. Samples were analyzed with the ARIOL system. Results: Giemsa staining revealed that 28 (93.3%) out of 30 and 9 (81.8%) out of 11 patients had detectable CTCs at baseline and after the third chemotherapy cycle, respectively. Cytokeratin (CK)+/CD45- CTCs by IF could be detected in 17 of 30 (56.7%) patients at baseline and in 8 of 11 (72.7%) after the third chemotherapy cycle. Spearman analysis revealed a significant correlation (p = 0.001) between Giemsa-positive and IF-positive (CK+/CD45-) CTCs. At baseline, PD-1 and PD-L1 expression was observed in 53% and in 47% CK-positive patients, respectively. After the third treatment cycle the corresponding numbers were 13% and 63% respectively. Median progression-free survival (PFS) was significantly shorter in patients with >3 PD-1(+) CTCs at baseline compared with those with <3 PD-1(+) CTCs (p = 0.022) as well as in patients with >1 Giemsa-positive tumor cells (p = 0.025). Conclusion: PD-1(+) and PD-L1(+) CTCs could be detected before and after front-line chemotherapy in patients with metastatic NSCLC. The presence of high PD-1(+) CTC numbers before treatment is associated with a poor patient clinical outcome.

Lung cancer and annual mean exposure to outdoor air pollution in Crete, Greece

The increasing burden of lung cancer (LC) in Crete, Greece, has raised certain concerns about the potential association of environmental risk factors with LC. The aim of this study was to assess outdoor air pollution (OAP) and the risk for LC mortality for the first time in Crete using LC primary data. 5057 LC cases (diagnosed from 1992 to 2013) were obtained from the Cancer Registry of Crete (http://www.crc.uoc.gr) and followed up until 2014. The age-standardized incidence and mortality rates (ASIR) were calculated. Data on OAP indicators [particulate matter (PM)2.5, between 2.5 and 10 &mgr;m (PM2.5–10), PM10, PM2.5 absorbance (black carbon measure), nitrogen dioxide (NO2), and nitrogen oxides (NOx)] were collected. Spatial statistics were calculated and the binary logistic regression model was constructed at &agr;=0.05 in IBM SPSS 24 and ArcMap 10.3.1. LC in Crete accounts for 40.2 new cases/100 000/year for both sexes (ASIRmales=73.1 new cases/100 000/year; ASIRfemales=11.8 new cases/100 000/year). Annual median estimates of environmental concentrations in Crete were as follows: PM2.5=20.7 (±1.5) µg/m3, PM10=38.9 (±2.5) µg/m3, PM2.5–10=59.6 (±3.7) µg/m3, PM2.5 absorbance=1.2 (±0.3)×10−5/m, NO2=15.2 (±3.8) µg/m3, and NOx=20.1 (±4.9) µg/m3. A statistically significant association was observed between OAP and LC mortality (mean correlation coefficient=0.75; P<0.05). The highest risk for 5-year LC mortality was found in the major urban centers and several south-east and north-west rural regions of Crete (relative risk=3.2, 95% confidence interval=1.6–4.7). OAP seems to be an important determinant of LC mortality. Targeted interventions should be performed in the high-risk areas.

A Comparison of Three Methods for the Detection of Circulating Tumor Cells in Patients with Early and Metastatic Breast Cancer

Background: We directly compared CTC detection rates and prognostic significance, using three different methods in patients with breast cancer (BC). Methods: Early (n=200) and metastatic (n=164) patients were evaluated before initiating adjuvant or first-line chemotherapy, using the CellSearchTM System, an RT-qPCR for CK-19 mRNA detection and by double immunofluorescence (IF) microscopy using A45-B/B3 and CD45 antibodies. Results: Using the CellSearchTM System, 37% and 16.5% of early BC patients were CTC-positive (at ≥1 and ≥2 CTCs/23 ml of blood), 18.0% by RT-qPCR and 16.9% by IF; no agreement was observed between methods. By the CellSearchTM 34.8% and 53.7% (at≥ 5 and ≥ 2 CTCs/7.5 ml) of metastatic patients were CTC-positive, 37.8% by RT-qPCR and 28.5% by IF. A significant agreement existed only between the CellSearchTM and RT-qPCR. In 60.8% of cases, differential EpCAM and CK-19 expression on CTCs by IF could explain the discrepancies between the CellSearchTM and RT-qPCR. CTC-positivity by either method was associated with decreased overall survival in metastatic patients. Conclusion: A significant concordance was observed between the CellSearchTM and RT-qPCR in metastatic but not in early BC. Discordant results could be explained in part by CTC heterogeneity. CTC detection by all methods evaluated had prognostic relevance in metastatic patients.

MKAD-21 suppresses the oncogenic activity of the miR-21/PPP2R2A/ERK molecular network in bladder cancer

Bladder cancer represents a disease associated with significant morbidity and mortality. MiR-21 has been found to have oncogenic activity in multiple cancers, including bladder cancer, whereas inhibition of its expression suppresses tumor growth. Here, we examine the molecular network regulated by miR-21 in bladder cancer and evaluate the effects of i.v. and i.p. administration of a novel miR-21 chemical inhibitor in vivo. LNA miR-21 reduced the oncogenic potential of bladder cancer cells, whereas the MKAD-21 chemically modified antisense oligo against miR-21 dose-dependently blocked xenograft growth. I.v. administration of LNA miR-21 was more effective in suppressing tumor growth than was i.p. administration. Integration of computational and transcriptomic analyses in a panel of 28 bladder cancer lines revealed a 15-gene signature that correlates with miR-21 levels. Protein Phosphatase 2 Regulatory Subunit Balpha (PPP2R2A) was one of these 15 genes and was experimentally validated as a novel miR-21 direct target gene. Gene network and molecular analyses showed that PPP2R2A is a potent negative regulator of the ERK pathway activation and bladder cancer cell proliferation. Importantly, we show that PPP2R2A acts as a mediator of miR-21–induced oncogenic effects in bladder cancer. Integrative analysis of human bladder cancer tumors and a large panel of human bladder cancer cell lines revealed a novel 15-gene signature that correlates with miR-21 levels. Importantly, we provide evidence that PPP2R2A represents a new miR-21 direct target and regulator of the ERK pathway and bladder cancer cell growth. Furthermore, i.v. administration of the MKAD-21 inhibitor effectively suppressed tumor growth through regulation of the PPP2R2A–ERK network in mice. Mol Cancer Ther; 17(7); 1430–40. ©2018 AACR.

Abstract 1726: Evaluation of PD-L1/PD-1 on circulating tumor cells (CTCs) and on primary tumor in advanced non-small cell lung cancer (NSCLC)

Introduction: Circulating tumour cells (CTCs) are responsible for the metastatic dissemination of the tumor. They have been shown to express Programmed Death-Ligand1 (PD-L1) to escape from the immune system surveillance through its ligation with the PD-1receptor on the surface of effector immune cells. We investigated the expression of PD-1/PD-L1 on CTCs isolated from NSCLC patients treated with chemotherapy. Methods: CTCs were isolated based on their size using the ISET platform from 30 stage IV chemo-naive NSCLC patients (before and after chemotherapy). CTCs were detected after staining with Giemsa and immunofluorescence (IF). Double and triple staining experiments with different combination of antibodies: [Cytokeratins(CK)/PD-1/CD45 and CK/PD-L1/CD45] were performed and the samples were analyzed with the ARIOL system. Results Giemsa staining showed that twenty-three (77%) out of 30 and six (54.5%) out of 11 patients had detectable CTCs at baseline and after the 3rd cycle of front-line chemotherapy. IF staining revealed seventeen out of 30 (56.7%) patients positive for CTCs at baseline level and 8 out of 11 (72.7%) samples after the 3rd cycle of treatment. PD-1 and PD-L1 expression was observed in 53% (9/17) and in 47% of the CTC-positive patients at baseline; in addition, 13% (1/8) and 63% (5/8) patients had PD-1 and PD-L1, respectively after the 3rd cycle. Among the total number of detected CTCs, 67% were PD-1(+) at baseline and 25% after the 3rd cycle (p=0.069). In addition, 26% and 80% were PD-L1(+) at baseline and after the 3rd cycle, respectively. Patients with more than 3 PD-1 positive CTCs showed shorter PFS (p=0.022). Primary tissue from ten of the examined patients was also available. More than 5% of PD-L1 (+) tumor infiltrating lymphocytes (TILs) were observed in 20% (2/10) of the patients. More than 5% PD-L1 positive cells in the primary tumor were observed in 20%. However the two group of the patients were different. In addition both patients with PD-L1 (+) TILs harvested CTCs with PD-1 expression and one of them had also PD-L1 positive CTCs. Conclusion: PD-1- and PD-L1-positive CTCs could be detected before and during 1st line treatment in metastatic NSCLC. This expression was related to patients’ prognosis, implying that these molecules can be served as targets for metastasis restoration. Furthermore the expression of PD-1 on CTCs suggests a bilateral cross-talk between tumor and immune cells. Citation Format: Galaktea Kallergi, Eleni Kyriaki Vetsika, Despoina Aggouraki, Eleni Lagoudaki, Anastasios Koutsopoulos, Filippos Koinis, Panagiotis Katsarlinos, Maria Trypaki, Christos Stournaras, Vassilis Georgoulias, Athanasios Kotsakis. Evaluation of PD-L1/PD-1 on circulating tumor cells (CTCs) and on primary tumor in advanced non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1726. doi:10.1158/1538-7445.AM2017-1726

Abstract 619: Effect of anti-PD-1 therapy on immune cells in the peripheral blood of non-small cell lung cancer patients

Background: Programmed cell death-1 (PD-1), an inhibitory immune check-point, plays a pivotal role in tumor immune escape. The interaction of PD-1 with its ligand (PD-L1) results in T cells exhaustion, and the blockade of this interaction can partially restore T cell function. Recently, antibodies targeting PD-1 and PD-L1 have been approved for treatment of advanced Non Small Cell Lung Cancer (NSCLC). In this pilot study, we aimed to investigate the effect of anti-PD1 treatment or chemotherapy on the frequencies of circulating PD-1+ T cells and PD-L1+ immunosuppressive cells in NSCLC patients. Patients & Methods: Peripheral blood samples were collected from 35 advanced NSCLC patients before initiation of treatment and after 3 cycles. Twelve treatment-naive patients received front-line chemotherapy, whereas 23 patients received anti-PD1 treatment in the second-line setting. Flow cytometry was used to quantify PD-1- and PD-L1-expressing immune cells. Changes in the frequencies of these cells were compared between the two settings and correlated with the clinical outcome. Results: Chemotherapy had no effect on the percentages of PD-1+CD4+ and PD-1+CD8+ T cells after 3 cycles, whereas there was a significant decrease in PD-1+CD4+ and PD-1+CD8+ T cells in patients who received 3 administrations of anti-PD1 antibody (p=0.007 and p=0.05, respectively). Moreover, the levels of PD-1-CD4+ (p=0.009) and PD-1-CD8+ (p=0.009) were augmented in response to anti-PD-1 therapy. The frequencies of both peripheral CD4+ Tregs (CD3+CD4+CD25highCD127-/lowCD152+FoxP3+) and granulocytic MDSCs (G-MDSC; CD14-CD15+CD33+CD11b+HLA-DR-Lin-) expressing PD-L1 were decreased following anti-PD1 therapy (p=0.01 and p=0.02, respectively). In contrast, chemotherapy affected only the PD-L1+CD4+ Tregs, but not the PD-L1+G-MDSC, by increasing their levels after 3 cycles (p=0.04). Anti-PD-1 treatment induced a superior reduction of the PD-1+CD4+, PD-1+CD8+ T cells, PD-L1+CD4+ Tregs and PD-L1+G-MDSCs percentages compared to the effect of first line chemotherapy (p=0.04, p=0.05, p=0.002 and p=0.01, respectively). Furthermore, a significant decrease in PD-1+CD8+ T cells, PD-L1+CD4+ Tregs and PD-L1+G-MDSCs after 3 doses of anti-PD-1 was observed in patients who experienced stable disease compared to baseline (p=0.006, p=0.05 and p=0.03, respectively). At the time of response evaluation to chemotherapy, the percentage of the PD-L1+CD4+ Tregs after 3 cycles was significantly inferior compared to baseline, in disease progressors (p=0.04). Conclusion: These data indicate that although chemotherapy affected the levels of PD-L1+CD4+ Tregs, anti-PD1 therapy seems to exert an effect on both PD1+ T cells and PD-L1+ immunosuppressive cells. Additional studies are needed in a larger cohort in order to document its impact on their clinical relevance in NSCLC patients. This study is ongoing and updated data will be presented at the meeting. Citation Format: Eleni-Kyriaki Vetsika, Galatea Kallergi, Despoina Aggouraki, Zaharoula Lyristi, Aristeidis Koukos, Despoina Kourougkiaouri, Filippos Koinis, Vassilis Georgoulias, Athanasios Kotsakis. Effect of anti-PD-1 therapy on immune cells in the peripheral blood of non-small cell lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 619. doi:10.1158/1538-7445.AM2017-619