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Purpose: To evaluate the clinical relevance of circulating CEACAM5mRNA-positive cells in patients with operable colorectal cancer (CRC).Methods: Peripheral blood was obtained from 265 patients with operable CRC before the initiation of adjuvant systemic therapy from 96 normal donors and RNA prepared from the Lovo and ARH-77 CRC and leukemic cell lines, respectively, was used as positive and negative controls. The detection of CEACAM5mRNA-positive cells was done using a real-time PCR assay. The association with known prognostic factors and the effect of CEACAM5mRNA-positive cells on patients' prognosis was investigated.Results: The analytical detection limit of the method was found to correspond to 0.7 Lovo cell equivalence/5 μg RNA, with a sensitivity of 1 tumor cell/105 normal cells and a specificity of 97%. Ninety-eight (37%) patients had detectable circulating CEACAM5mRNA-positive cells. Detection of CEACAM5mRNA-positive cells was significantly associated with higher relapse rate (P < 0.001), decreased disease-free survival (DFS; P < 0.001), higher death rate (P = 0.017), and decreased median overall survival (P = 0.025). Multivariate analysis revealed that the detection of circulating CEACAM5mRNA-positive cells was an independent prognostic factor for decreased DFS [HR = 3.4; 95% CI: 2.0–5.9; P < 0.001].Conclusions: Detection of peripheral blood CEACAM5mRNA-positive cells is an adverse prognostic factor correlated with poor clinical outcome in patients with operable CRC. Clin Cancer Res; 17(1); 165–73. ©2010 AACR.

Supplementary Figures S1-S3; Supplementary Tables S1-S2.

Background: Metastasis remains the major lethal consequence in the progression of early breast cancer (BC). Effective immunosurveillance can inhibit metastasis, while a dysfunctional immune system can create a favorable environment for tumor spread. MicroRNAs (miRNAs) are involved in the regulation of immune response and there is evidence that they play an important role in immune escape. We investigated the effect of plasma miR-10b, miR-126, miR-19α, miR-20α and miR-155, which regulate the interaction between cancer and immune cells during immunosurveillance and immune evasion in patients with early BC. Methods: Blood samples were obtained from relapsed (n=52) and non-relapsed (n=100) patients with early BC before adjuvant chemotherapy. Plasma miRNA expression levels were assessed by qRT-PCR and expression was classified as high or low according to the median values. Receiver operating curves (ROC) were constructed to determine miRNA sensitivity and specificity. Results: miRNA expression was not correlated with patients’ characteristics or outcome. No differences in miRNA expression were observed between relapsed and non-relapsed patients. In multivariate analysis the number of infiltrated axillary lymph nodes and stage III disease independently predicted for shorter DFS (HR: 2.591; p=0.002) and OS (HR: 2.344; p=0.035), respectively. miR-126, miR-20a, miR-19a and miR-155 expression levels were lower in patients with early relapse (defined as relapse at ≤ 2 yrs; n=19) compared to those who either relapse later than 2 years (n=33) or those who remained disease-free during follow up (n=100). ROC curve analysis showed that miR-155 had the highest performance to discriminate patients with early relapse [AUC 0.825; sensitivity 90%, specificity 67% (p Citation Format: Konstantina Thomopoulou, Chara Papadaki, Alexia Monastirioti, George Koronakis, Lambros Vamvakas, Maria A Papadaki, Sofia Agelaki, Dimitrios Mavroudis. microRNAs regulating tumor and immune cell interactions in the prediction of early relapse in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-05-07.

BACKGROUND/AIM Tumoural transcriptional levels of RRM1, RRM2, CDA, dCK and hENT1 genes are potential biomarkers for gemcitabine's efficacy in non-small cell lung cancer (NSCLC). PATIENTS AND METHODS We retrospectively analysed each gene's relative mRNA expression by quantitative, real-time polymerase chain reaction in microdissected, formalin-fixed, paraffin-embedded primary-tumour specimens from 219 chemonaive patients with advanced-stage NSCLC, treated with gemcitabine-based regimens within clinical trials. The five genes' transcriptional patterns were integrated into an ordinal, five-level gemcitabine-susceptibility classifier (5L-GSC). RESULTS Treatment efficacy increased progressively across the five susceptibility levels, with the very-high chemosensitivity cases obtaining the most clinical benefit. 5L-GSC emerged as an independent prognosticator for overall response and disease control rates, time to progression and overall survival at p-values of 0.03, 0.004

TLR4 and pSTAT3 are key players in cancer inflammation and immune evasion; however, their role in the peripheral blood (PB) is largely unexplored. Herein we evaluated their expression in the circulating tumor cells (CTCs) and peripheral-blood mononuclear cells (PBMCs) of patients with early (n = 99) and metastatic (n = 100) breast cancer (BC). PB samples obtained prior to adjuvant and first-line therapy, were immunofluorescently stained for Cytokeratins/TLR4/pSTAT3/DAPI and analyzed via Ariol microscopy. TLR4+ CTCs were detected in 50% and 68% of early and metastatic CTC-positive patients, respectively, and pSTAT3+ CTCs in 83% and 68%, respectively. In metastatic patients, CTC detection was associated with a high risk of death (HR: 1.764, p = 0.038), while TLR4+ CTCs correlated with a high risk of disease progression (HR: 1.964, p = 0.030). Regarding PBMCs, TLR4 expression prevailed in metastatic disease (p = 0.029), while pSTAT3 expression was more frequent in early disease (p = 0.014). In early BC, TLR4 expression on PBMCs independently predicted for high risk of relapse (HR: 3.549; p = 0.009), whereas in metastatic BC, TLR4+/pSTAT3− PBMCs independently predicted for high risk of death (HR: 2.925; p = 0.012). These results suggest that TLR4/pSTAT3 signaling on tumor- and immune-cell compartments in the PB could play a role in BC progression, and may hold independent prognostic implications for BC patients.

BACKGROUND: Cancer cachexia syndrome (CCS) is an adverse prognostic factor in cancer patients undergoing chemotherapy or surgical procedures. We performed a prospective study to investigate the effect of CCS on treatment outcomes in patients with non-oncogene driven metastatic non-small cell lung cancer (NSCLC) undergoing therapy with programmed cell death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) inhibitors. METHODS: Patients were categorized as having cancer cachexia if they had weight loss >5% in the last 6 months prior to immunotherapy (I-O) initiation or any degree of weight loss >2% and body mass index (BMI) 5% during treatment did not affect overall survival (OS; P=0.40). CONCLUSIONS: CCS is associated with reduced PD-1/PD-L1 inhibitor efficacy in NSCLC patients and should constitute an additional stratification factor in future I-O clinical trials. Further research at a translational and molecular level is required to decipher the mechanisms of interrelation of metabolic deregulation and suppression of antitumor immunity.

Background PD-L1 expression on tumor and immune cells plays an important role in many cancers, including breast cancer (BC), however, discordance has been demonstrated between primary tumors and matched distant metastases. PD-L1 is expressed on circulating tumor cells (CTCs) in BC, however limited data exist on PD-L1 expression on the respective peripheral blood mononuclear cells (PBMCs). In the current study we aimed to investigate tumor and immune PD-L1 expression in primary tumors, and matched peripheral blood (PB) and metastatic sites from CTC-positive patients with BC. Methodology PB was collected from 96 BC patients (early: n=72; de novo metastatic: n=24) and PBMCs were enriched by ficoll-density gradient centrifugation. PBMC cytospins were immunofluorescently stained using antibodies for cytokeratins (Clones: AE1/AE3 & C11) to detect CTCs and PD-L1 (Clone: E1L3N). Matched primary tumor samples from CTC-positive patients were evaluated for PD-L1 expression on tumor cells and TILs by immunocytochemistry (positivity cut-off: ≥1% positive tumor cells or immune cells, respectively). In 7 patients, matched metastatic tumor tissue was also available for analysis. Results CK+ CTCs were identified in 18 out of 96 BC patients (early disease: 11/72, de novo metastatic: 7/24). Overall, PD-L1+ CTCs were detected in 22% of CTC+ patients and PDL1+ tumor cells in 28% of the respective primary tumors. However, there was no concordance for PD-L1 expression positivity between CTCs and corresponding primary tumor cells. PD-L1+ PBMCs were identified in 23% of patients, whereas PD-L1+ TILs were detected in 65% of primary tumor samples (positive concordance 17.6%). In addition, there was no concordance for PD-L1-positivity between PB samples and metastatic tumor samples, regarding either tumor cells (PD-L1+ CTCs identified in 3/7 patients and PD-L1+ tumor cells in 1/7 metastatic tumor samples), or immune cells (PD-L1+ PBMCs were detected in 1/6 and PD-L1-positive TILs in 3/6 metastatic tumors). Finally, when primary and corresponding metastatic tumor samples were compared, PD-L1+ tumor cells were detected in 3 versus 1 of 7 patients, respectively (positive concordance 14.3%), whereas, PD-L1+ TILs were observed in 6 versus 4 patients, respectively (positive concordance, 42.9%). Conclusions Discrepancies in PD-L1 positivity of tumor and immune cells are for the first time demonstrated between PB, primary and metastatic tissue samples in BC. In general, PD-L1 positivity is lower in metastatic compared to primary tumors. In addition, despite that similar rates of PD-L1 expression are observed in primary tumors and the corresponding CTCs, no positive concordance exists. PD-L1 expression on CTCs and PBMCs should be further explored to identify their potential value as prognostic and/or predictive biomarkers in BC patients treated with immunotherapy. Citation Format: Maria A Papadaki, Anastasios V Koutsopoulos, Eleni Lagoudaki, Alexia Monastirioti, Konstantina Thomopoulou, Kostas Kalbakis, Chara Papadaki, Sofia Agelaki, Dimitrios Mavroudis. Comparative analysis of PD-L1 expression on matched primary tumors, metastasis and peripheral blood of patients with breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-05-03.

Kachris S, Papadaki C, Rounis K, et al. Cancer Manag Res. 2021;13:8257–8271. The authors have advised there is an error with the author list on page 8257. The author “Stefanos Kachris1” should read “Stefanos Kachris1,2”. The authors apologize for this error. Read the original article

Purpose The purpose of this study was to investigate the prognostic significance of liver kinase b1 (LKB1) loss in patients with operable colon cancer (CC). Materials and Methods Two hundred sixty-two specimens from consecutive patients with stage III or high-risk stage II CC, who underwent surgical resection with curative intent and received adjuvant chemotherapy with fluoropyrimidine and oxaliplatin, were analyzed for LKB1 protein expression loss, by immunohistochemistry as well as for KRAS exon 2 and BRAFV600E mutations by Sanger sequencing and TS, ERCC1, MYC, and NEDD9 mRNA expression by real-time quantitative reverse transcription polymerase chain reaction. Results LKB1 expression loss was observed in 117 patients (44.7%) patients and correlated with right-sided located primaries (p=0.032), and pericolic lymph nodes involvement (p=0.003), BRAFV600E mutations (p=0.024), and TS mRNA expression (p=0.041). Patients with LKB1 expression loss experienced significantly lower disease-free survival (DFS) (hazard ratio [HR], 1.287; 95% confidence interval [CI], 1.093 to 1.654; p=0.021) and overall survival (OS) (HR, 1.541; 95% CI, 1.197 to 1.932; p=0.002), compared to patients with LKB1 expressing expressing tumors. Multivariate analysis revealed LKB1 expression loss as independent prognostic factor for both decreased DFS (HR, 1.217; 95% CI, 1.074 to 1.812; p=0.034) and decreased OS (HR, 1.467; 95% CI, 1.226 to 2.122; p=0.019). Conclusion Loss of tumoral LKB1 protein expression, constitutes an adverse prognostic factor in patients with operable CC.

// Chara Papadaki 1 , Giannis Stoupis 2 , Leuteris Tsalikis 1 , Alexia Monastirioti 1 , Maria Papadaki 1 , Neofytos Maliotis 1 , Michalis Stratigos 2 , Georgios Mastrostamatis 1 , Dimitrios Mavroudis 1, 2 and Sofia Agelaki 1, 2 1 Laboratory of Translational Oncology, School of Medicine, University of Crete, Heraklion, Greece 2 Department of Medical Oncology, University General Hospital, Heraklion, Crete, Greece Correspondence to: Sofia Agelaki, email: agelakisofia@gmail.com Keywords: circulating miRNAs; breast cancer; metastasis; prognosis Abbreviations: MBC: metastatic breast cancer; PFS: progression free survival; OS: overall survival Received: November 29, 2018 Accepted: January 12, 2019 Published: January 29, 2019 ABSTRACT Background: Circulating miRNAs (miRs) are increasingly recognized as potential biomarkers in cancer. We aimed to evaluate the differential expression of miR-23b and miR-190 which are involved in tumor dormancy, miR-21 involved in metastasis and miR-200b and miR-200c involved in epithelial-mesenchymal transition (EMT) and metastasis, in the plasma of patients with early and metastatic breast cancer (MBC). We also aimed to identify associations of the expression levels with patient and disease characteristics and outcomes in metastatic patients treated with first-line chemotherapy. Results: miR-21 ( p < 0.001), miR-23b ( p = 0.033), miR-200b ( p < 0.001) and miR-200c ( p < 0.001) expression was higher in metastatic compared to early breast cancer. ROC curve analysis showed that miR-21 (AUC = 0.722; p < 0.001) and miR-200b (AUC = 0.720; p < 0.001) distinguished with high accuracy among the two disease states, whereas the combination of miR-21, miR-190, miR-200b and miR-200c, further improved accuracy (AUC = 0.797; p < 0.001). High miR-200b expression independently predicted for shorter OS ( p = 0.026) in MBC. High expression of both miR23b and miR-190 emerged as a strong independent factor associated with shorter PFS ( p = 0.001) in de novo metastatic patients and high miR-200b independently predicted for decreased OS in the HER2-negative subgroup ( p = 0.007). Materials and Methods: Blood samples were obtained from patients with early ( n = 133) and MBC ( n = 110) before adjuvant or first-line chemotherapy, respectively. Plasma miRNA expression levels were assessed by RT-qPCR and were classified as high or low according to the median values. Conclusions: Our results are in support of the concept that circulating miRNAs represent a tool with significant diagnostic and prognostic implications in breast cancer.

Since circulating microRNAs (miRNAs) are involved in the modulation of the immune response, they are tested as liquid biopsy-based biomarkers in patients with NSCLC treated with immunotherapy. We analyzed the expression levels and examined the clinical significance of immunoregulatory miRNAs involved in immune checkpoint regulation (miR-34a, miR-200b, miR-200c), T-cell activity (miR-155), and the function of myeloid-derived suppressive cells (MDSCs) (miR-223) or regulatory T lymphocytes (Tregs) (miR-146a), in patients with advanced NSCLC (N = 69) treated with anti-PD-1 (Nivolumab) immunotherapy as 2nd or 3rd line of treatment therapy. Plasma levels of circulating miRNAs were analyzed by RT-qPCR before the initiation of immunotherapy. Expression of miR-34a, miR-146a, mir-200c, and miR-223 was found to be associated with response to immunotherapy. High miR-200c expression emerged as an independent prognostic factor for inferior overall survival in all patients with NSCLC (OS, HR: 2.243, 95% CI: 1.208–4.163; p = 0.010) and in patients with non-Squamous (non-SqCC) subtype (N = 38) (HR: 2.809, 95% CI: 1.116–7.074; p = 0.028). Low miR-34a expression independently predicted for shorter OS (HR: 3.189, 95% CI: 1.193–8.527; p = 0.021) in the non-SqCC subgroup. Our findings suggest that alterations in circulating miR-200c and miR-34a expression levels are associated with the response and outcome in patients with advanced NSCLC treated with anti-PD1 immunotherapy.

Stefanos Kachris,1 Chara Papadaki,2 Konstantinos Rounis,3 Eliza Tsitoura,4 Chrysanthi Kokkinaki,4 Christoforos Nikolaou,5– 7 George Sourvinos,4 Dimitrios Mavroudis2,3 1Department of Radiation Oncology, University General Hospital, Heraklion, Crete, Greece; 2Laboratory of Translational Oncology, School of Medicine, University of Crete, Heraklion, Crete, Greece; 3Department of Medical Oncology, University General Hospital, Heraklion, Crete, Greece; 4Laboratory of Clinical Virology, Medical School, University of Crete, Heraklion, Crete, Greece; 5Department of Biology, University of Crete, Heraklion, Crete, Greece; 6Institute of Molecular Biology and Biotechnology (IMBB), Foundation of Research and Technology (FORTH), Heraklion, Crete, Greece; 7Institute of Bioinnovation, Biomedical Science Research Center “Alexander Fleming”, Athens, GreeceCorrespondence: Dimitrios MavroudisLaboratory of Translational Oncology, School of Medicine, University of Crete, Heraklion, 71110, GreeceTel +30 2810392750Email mavroudis@uoc.grIntroduction: Disease recurrence is a major concern in patients with localized prostate cancer (PCa) following treatment with radiotherapy (RT), and few studies have evaluated the clinical relevance of microRNAs (miRNAs) prior and post-RT.Purpose: We aimed to investigate the significance of miRNAs in the outcomes of prostate cancer patients undergoing radiotherapy and to identify the related pathways through bioinformatics analysis.Materials and Methods: The expression levels of miR-21, miR-106b, miR-141 and miR-375 involved in the response to radiotherapy were assessed by RT-qPCR in the serum of PCa patients (n=56) prior- and post-RT.Results: Low expression levels of miR-106b prior-RT were associated with extracapsular extension and seminal vesicles invasion by the tumor (p=0.031 and 0.044, respectively). In the high-risk subgroup (n=47), post-RT expression levels of miR-21 were higher in patients with biochemical relapse (BR) compared to non-relapse (p=0.043). Also, in the salvage treatment subgroup (post-operative BR; n=20), post-RT expression levels of miR-21 and miR-106b were higher in patients with BR compared to non-relapse (p=0.043 and p=0.032, respectively). In the whole group of patients, high expression levels of miR-21 prior-RT and of miR-106b post-RT were associated with significantly shorter overall survival (OS; p=0.049 and p=0.050, respectively). No associations were observed among miR-141 and miR-375 expression levels with clinicopathological features or treatment outcome. Bioinformatics analysis revealed significant enrichment in DNA damage response pathways.Conclusion: Circulating miRNAs prior or post-RT may hold prognostic implications in patients with PCa.Keywords: prostate cancer, radiotherapy, salvage radiotherapy, circulating microRNAs, high risk, biochemical relapse

MicroRNAs (miRNAs) are involved in the regulation of immune response and hold an important role in tumor immune escape. We investigated the differential expression of the immunomodulatory miR-10b, miR-19a, miR-20a, miR-126, and miR-155 in the plasma of healthy women and patients with early stage breast cancer and interrogated their role in the prediction of patients’ relapse. Blood samples were obtained from healthy women (n = 20) and patients with early stage breast cancer (n = 140) before adjuvant chemotherapy. Plasma miRNA expression levels were assessed by RT-qPCR. Relapse predicting models were developed using binary logistic regression and receiver operating curves (ROC) were constructed to determine miRNA sensitivity and specificity. Only miR-155 expression was lower in patients compared with healthy women (p = 0.023), whereas miR-155 and miR-10b were lower in patients who relapsed compared with healthy women (p = 0.039 and p = 0.002, respectively). MiR-155 expression combined with axillary lymph node infiltration and tumor grade demonstrated increased capability in distinguishing relapsed from non-relapsed patients [(area under the curve, (AUC = 0.861, p &lt, 0.001)]. Combined miR-19a and miR-20a expression had the highest performance in discriminating patients with early relapse (AUC = 0.816, 0.001). Finally, miR-10b in combination with lymph node status and grade had the highest accuracy to discriminate patients with late relapse (AUC = 0.971, 0.001). The robustness of the relapse predicting models was further confirmed in a 10-fold cross validation. Deregulation of circulating miRNAs involved in tumor-immune interactions may predict relapse in early stage breast cancer. Their successful clinical integration could potentially address the significance challenge of treatment escalation or de-escalation according to the risk of recurrence.

Objective We prospectively recorded clinical and laboratory parameters from patients with metastatic non-small cell lung cancer (NSCLC) treated with 2nd line PD-1/PD-L1 inhibitors in order to address their effect on treatment outcomes. Materials and methods Clinicopathological information (age, performance status, smoking, body mass index, histology, organs with metastases), use and duration of proton pump inhibitors, steroids and antibiotics (ATB) and laboratory values [neutrophil/lymphocyte ratio, LDH, albumin] were prospectively collected. Steroid administration was defined as the use of > 10 mg prednisone equivalent for ≥ 10 days. Prolonged ATB administration was defined as ATB ≥ 14 days 30 days before or within the first 3 months of treatment. JADBio, a machine learning pipeline was applied for further multivariate analysis. Results Data from 66 pts with non-oncogenic driven metastatic NSCLC were analyzed; 15.2% experienced partial response (PR), 34.8% stable disease (SD) and 50% progressive disease (PD). Median overall survival (OS) was 6.77 months. ATB administration did not affect patient OS [HR = 1.35 (CI: 0.761–2.406, p = 0.304)], however, prolonged ATBs [HR = 2.95 (CI: 1.62–5.36, p = 0.0001)] and the presence of bone metastases [HR = 1.89 (CI: 1.02–3.51, p = 0.049)] independently predicted for shorter survival. Prolonged ATB administration, bone metastases, liver metastases and BMI < 25 kg/m2 were selected by JADbio as the important features that were associated with increased probability of developing disease progression as response to treatment. The resulting algorithm that was created was able to predict the probability of disease stabilization (PR or SD) in a single individual with an AUC = 0.806 [95% CI:0.714–0.889]. Conclusions Our results demonstrate an adverse effect of prolonged ATBs on response and survival and underscore their importance along with the presence of bone metastases, liver metastases and low BMI in the individual prediction of outcomes in patients treated with immunotherapy.

The expression of microRNA (miR)-21, miR-128, miR-155, and miR-181a involved in DNA damage response (DDR) and tumor responsiveness to platinum was assessed by RT-qPCR in the plasma of patients with non-small cell lung cancer (NSCLC, n = 128) obtained prior to initiation of first-line platinum chemotherapy. U6 small nuclear RNA (snRNA) was used for normalization, and fold change of each miRNA expression relative to the expression in healthy controls was calculated by the 2&minus, &Delta, Ct method. MicroRNA expression levels were correlated with patients&rsquo, outcomes. Integrated function and pathway enrichment analysis was performed to identify putative target genes. MiR-128, miR-155, and miR-181a expressions were higher in patients compared to healthy donors. MiRNA expression was not associated with response to treatment. High miR-128 and miR-155 were correlated with shorter overall survival (OS), whereas performance status (PS) 2 and high miR-128 independently predicted for decreased OS. In the squamous (SqCC) subgroup (n = 41), besides miR-128 and miR-155, high miR-21 and miR-181a expressions were also associated with worse survival and high miR-155 independently predicted for shorter OS. No associations of miRNA expression with clinical outcomes were observed in patients with non-SqCC (n = 87). Integrated function and pathway analysis on miRNA targets revealed significant enrichments in hypoxia-related pathways. Our study shows for the first time that plasma miR-128 and miR-155 hold independent prognostic implications in NSCLC patients treated with platinum-based chemotherapy possibly related to their involvement in tumor response to hypoxia. Further studies are needed to investigate the potential functional role of these miRNAs in an effort to exploit their therapeutic potential.

The role of CD47 and PD-L1 expression on circulating tumor cells (CTCs) remains unclear, and it is currently unknown whether their distribution varies between the blood and tumor tissue in breast cancer (BC). In this study, CD47 and PD-L1 expression was investigated a) on peripheral blood mononuclear cell (PBMC) cytospins from early (n = 100) and metastatic (n = 98) BC patients, by triple immunofluorescence for CD47/PD-L1/Cytokeratins, and b) on matched primary and/or metastatic tumor tissue from CTC-positive patients using immunohistochemistry. CD47+and/orPD-L1+ CTCs were detected in 11%, 16.9%, and 29.6% of early, recurrent, and de novo metastatic patients (p = 0.016). In metastatic disease, CD47highand/orPD-L1high CTCs were associated with disease progression (p = 0.005) and shorter progression-free survival (PFS) (p = 0.010), and independently predicted for an increased risk of relapse (HR: 2.719, p = 0.008) and death (HR: 2.398, p = 0.034). PD-L1 expression rates differed between CTCs and tissue tumor cells and between peripheral blood mononuclear cells (PBMCs) and tumor-infiltrating lymphocytes (TILs) (positive concordance of 3.8% and 4%, respectively). CD47 expression also differed between CTCs and tumor cells (positive concordance of 11.5%). In conclusion, CTCs expressing CD47 and PD-L1 have independent poor prognostic implications in metastatic BC, indicating a potential role of innate and adaptive immune evasion mechanisms in their metastatic potential. The clinical value of the parallel assessment of the peripheral and local immune response merits further evaluation in BC.

Background: Metastasis remains a major threat for patients (pts) with operable breast cancer (BC). Recurrence could arise from a state of tumour dormancy during which there is growth restriction of undetectable micrometastases. The expression of dormancy and metastasis related miRNAs was evaluated in the plasma of pts with operable BC obtained before adjuvant therapy in order to discover novel biomarkers for the prediction of relapse. Methods: Plasma miR-21, miR-23b, miR-190, miR-200b and miR-200c expression was assessed by qRT-PCR in 133 pts with early BC (non-relapsed, n=84; relapsed, n=49). Expression was classified as high or low according to the median values and was associated with pts' clinicopathological characteristics and clinical outcome. Results: No correlation was observed between the expression of miRNAs and the clinicopathological characteristics of pts. After a median f-up of 90.5 mo, median Disease Free Interval (DFI) was significantly lower in pts with high compared to low miR-21 [105 mo vs not reached (NR); p=0.001], miR-200c (105.2 mo vs NR; p=0.007), or both miR-21 and miR-200c expression (81.37 mo vs NR; p=0.001). miR-21-high was also associated with decreased median Overall Survival (OS; p=0.041). In multivariate analysis the number of infiltrated axillary lymph nodes (N3 vs N0-N2, HR: 2.86; p= 0.004) and miR-21 expression (high vs low, HR: 2.824; p=0.001) were independent negative prognostic factors for DFI, whereas negative hormone receptor status (HR: 3.062; p=0.024) and miR-21 high (HR: 3.545; p=0.029) independently predicted for worse OS. Moreover, miR-21 expression was higher in pts presenting early relapse (defined as relapse at ≤ 3 yrs) compared to those without relapse at 5 yrs (p=0.032). Furthermore, higher miR-21 (p=0.038), miR-23b (p=0.039), miR-200b (p=0.027) and miR-200c (p Conclusions: Differential expression levels of metastasis and/or dormancy related circulating miRNAs are encountered before adjuvant therapy in pts with operable BC presenting subsequent relapse compared to non-relapsed pts. In addition, circulating miRNAs could predict for early or late recurrence years before clinical detection of metastases. These results merit prospective validation in an independent pt cohort. Citation Format: Agelaki S, Papadaki C, Stratigos M, Spiliotaki M, Markakis G, Mastrostamatis G, Mavroudis D, Ioannis S. Circulating microRNAs as early predictors of relapse in operable breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-07-10.

Differences in gene expression levels between the primary tumors (PTs) and matched regional lymph nodal metastases (LNs) in patients with totally excised non-small cell lung cancer (NSCLC) were explored. Microdissected formalin-fixed paraffin-embedded (FFPE) samples from (PT) and their matched infiltrated LNs, from 239 patients [183 (with matched PT and LNs samples)-case and 56 PT only samples-control cohorts] were analyzed for BRCA1, ERCC1, RAP80, PKM2, RRM1, RRM2, TS, TSP1, and TXR1 mRNA expression by quantitative real-time polymerase-chain reaction (PCR). Moderately positive correlation between the expression of each gene in the PT and the matched LNs was observed. Concordance rates between the PT and the LNs were: BRCA1 (67.7%), ERCC1 (68.4%), PKM2 (63.4%), RAP80 (68.8%), RRM1 (70.9%), RRM2 (69%), TS (72.9%), TSP1 (69.8%), TXR1 (63.7%). Expression levels and their differences were correlated with Relapse-Free Survival (RFS) and Overall Survival (OS). High BRCA1 PT in patients with squamous histology was associated with increased OS (p = 0.036). High TSP1 PT levels were shown to be the only independent prognostic factor for OS and RFS (p = 0.023 and p = 0.007). PKM2 low levels in both PT and matched LNs were associated with better OS irrespective of the underlying histology (p = 0.031). RRM1 discordant levels between PT and matched LNs were associated with worse OS in squamous tumors (p = 0.019) compared to patients with both low expression in PT and LN.TXR1 high levels in both PT and matched LNs were associated with better OS in patients with squamous tumors (p = 0.007).These findings indicate that there is different gene expression between PT and matched LNs which may affect the outcome in early NSCLC and therefore PT’s molecular biology should not be the sole determinant for prognostication.

e15015 Background: Toll-like receptor 4 (TLR4) and phosphorylated signal transducer and activator of transcription protein 3 (pSTAT3) hold a key role in inflammatory response and tumor immune evasion. Their role in the peripheral immune response and in tumor immune interactions in circulation needs further investigation. In the current report we evaluated the incidence of TLR4 and pSTAT3 expression on tumor and immune compartments within the peripheral blood (PB) of patients with early (eBC) and metastatic breast cancer (mBC). Methods: PB was obtained from patients with eBC (n = 99) and mBC (n = 100), prior to adjuvant and first-line therapy, respectively. Triple immunofluorescence staining for cytokeratins (CK), TLR4 and pSTAT3 was performed on peripheral blood mononuclear cell (PBMC) cytospins. Single cell-level expression of TLR4 and pSTAT3 was assessed on CK+ circulating tumor cells (CTCs) and PBMCs using the Ariol microscopy system. Results: CK+ CTCs were detected in 6.1% and 19% of patients with eBC and mBC, respectively (p = 0.006) (number of CTCs: n = 34 and n = 28, respectively). Higher TLR4 expression rates on CTCs were observed in mBC as compared to eBC (53.6% vs 7.7% of CTCs, 13% vs 3% of patients; p = 0.016). pSTAT3+ CTCs also prevailed in mBC (60.7% vs 10.3% of CTCs; 14% vs 5.1% of patients; p = 0.032). In the metastatic patients, CTC detection was associated with shorter overall survival (median OS: 24.9 vs 36.5 months; p = 0.042; Kaplan Meier), whereas the detection of TLR4+ CTCs predicted for shorter progression free survival (median PFS: 11.4 vs 12.6 months; p = 0.036). At the PBMC level, TLR4 expression was more frequent among patients with mBC as compared to eBC (34% vs 20.2%, respectively; p = 0.029), whereas pSTAT3 expression prevailed in eBC (89.9% vs 77% of patients, respectively; p = 0.014). The detection of TLR4+ PBMCs was associated with reduced disease-free survival (median DFS: not reached; p = 0.007) and shorter OS (median OS: not reached; p = 0.028) in patients with eBC. Conclusions: TLR4 and pSTAT3 expression is demonstrated on CTCs and PBMCs and significantly varies among eBC and mBC, suggesting that the inflammatory signaling through TLR4 and pSTAT3 in PB could be associated with BC progression. Our results further imply that their assessment in PB may hold significant prognostic implications in BC.

e21191 Background: MicroRNAs are critical modulators of the immune response and regulate the expression of various immune checkpoints during immunotherapy. In the present study we evaluated the predictive significance of immune related miRNAs in the plasma of non-small cell lung cancer (NSCLC) patients treated with immune checkpoint inhibitors. Methods: Plasma was obtained from 62 NSCLC patients before the initiation of immunotherapy, administered as second- or third-line treatment. The expression profile of immune-related miR-34a, miR-146a, miR-155, miR-200b, miR-202 and miR-223 was assessed by RT-qPCR. Patients were classified in high and low expression groups according to the median value of each miRNA. Patients that had achieved partial response (PR) or stable disease (SD) were classified as responders, whereas patients with progressive disease (PD) were classified as non-responders. Results: No statistical correlations were observed among miRNA expression and clinical outcomes in the whole group of patients (N = 62). However, in the non-squamous subgroup (N = 36) lower miR-34a expression levels were observed in non-responders compared to responders (80% vs 20%; p = 0.029). Univariate binary logistic regression analysis revealed that only low miR-34a expression (HR: 8.000, 95% CI: 1.215-52.693; p = 0.031) was correlated with the probability of developing progressive disease as best response to immunotherapy. Patients with low miR-34a expression levels were associated with shorter progression free survival (PFS) and overall survival (OS) compared to patients with high 34a expression (1.97 vs 6.33 months; p = 0.042 and 2.93 vs 9,60 months; p = 0.012, respectively). Furthermore, low miR-34a expression emerged as an independent predictor of shorter PFS and OS (HR: 2.449; p = 0.049 and HR: 3.203; p = 0.016, respectively). No other correlations were observed among the rest of the miRNAs and clinical outcomes. Conclusions: Our findings suggest that circulating miR-34a may serve as a potential predictive biomarker in NSCLC patients treated with immunotherapy. These observations need to be further validated in a larger cohort of patients.

To evaluate the predictive value of genes involved in resistance to platinum-taxane chemotherapy in patients with epithelial ovarian cancer (EOC). Microdissected formalin-fixed tumoral samples from 187 EOC patients' primary tumors (90 and 97 samples from matched patients in the experimental and validation sets, respectively) were analyzed. All specimens were analyzed for ATP7b, BRCA1, BRCA2, PARP1, UIMC1(RAP80), HOXA9, DAXX, TXN (TRX1), THBS1 (TSP1) and PRR13 (TXR1) mRNA expression by quantitative real-time PCR. Most of the patients (172 out of 187) received front-line carboplatin-paclitaxel regimen. Expression levels were correlated with overall (OS) and progression-free (PFS) survival by multivariate analysis. Patients with high TXN and THBS1 expression presented longer PFS (P=0.001 and P

e21609 Background: We prospectively recorded common clinical and laboratory parameters of pts with metastatic NSCLC treated with 2nd line ICIs to evaluate their potential value in a clinical outcome prediction model. Methods: Data on patient (age, PS, BMI) and disease characteristics (histology, sites/number of metastases), smoking status, use/duration of co-medications [proton pump inhibitors (PPIs) inhalational/p.os steroids, antibiotics (ATB)], laboratory values [neutrophil/lymphocyte ratio (NLR), LDH] and response to previous therapy were prospectively collected. Pts were categorized as having steroids if they had received steroids > 10mg for ≥10d (starting from 15d before or within the first 3 months of treatment). Prolonged ATB administration was defined as ATBs ≥14d (30d before or within the first 3 months). JAD Bio (www.jadbio.com), an Automated Machine Learning service that analyzes biological data with emphasis on feature selection was used to create predictive models. Results: 66 pts were evaluated; median follow up time was 6.37 months. 15.2% of patients had PR, 34.8% SD and 50% PD. Median PFS and OS were 3.5 and 6.77 months, respectively. Steroids had negative impact on disease stabilization (PR+SD) rates (p = 0.042) and PFS (p = 0.013) but not OS (p = 0.051). ATBs negatively affected RR (p = 0.046) only, whereas, prolonged ATB exposure was associated with lower RR (p = 0.007), PFS (p = 0.0001) and OS (p = 0.001). In multivariate analysis, steroids [HR = 2.54 (CI:1.23-5.29, p = 0.012)], prolonged ATBs [HR = 3.46 (CI:1.72-6.95, p = 0.0001)], liver HR = 2.92 (CI:1.45-5.78, p = 0.003) and bone HR = 2.06 (CI:1.041-4.10, p = 0.038) metastases independently predicted for shorter PFS. Only prolonged ATBs [HR = 2.52 (CI: 1.39-4.54, p = 0.002)] and bone metastases [HR = 2.26 (CI: 1.23-4.17, p = 0.009)] independently predicted for shorter OS. Analysis of the investigated parameters by JAD Bio predicted disease stabilization with an accuracy of 71%. Importantly, PDL-1 status was not included due to high rates of missing data. Conclusions: Our results corroborate previous evidence on the detrimental role of prolonged ATB administration on ICIs efficacy, possibly related to perturbation of the gut microbiota. Modeling, including other significant parameters from larger patient cohorts, could result in a robust estimation of outcome.

e15528 Background: MicroRNAs (miRNAs) are involved in the regulation of immune response and have an important role in immune escape. We analyzed the expression levels of plasma miR-10b, miR-126, miR-19α, miR-20α and miR-155, which regulate tumor-immune interactions and investigated their prognostic implications in patients with early (eBC) and metastatic (mBC) breast cancer. Methods: Blood samples were obtained before treatment from 140 and 64 patients with eBC and mBC, respectively. Plasma miRNA expression levels were assessed by qRT-PCR and expression was classified as high or low according to the median values. Results: A panel of four miRNAs (miR-19a, miR-20a, miR-126 and miR-155) could discriminate eBC from mBC (AUC 0.802, p < 0.001). In early disease, miR-10b (p = 0.022) and miR-155 (p = 0.005) expression was lower in relapsed (n = 46) compared to non-relapsed (n = 94) patients; miR-155 expression along with lymph node infiltration and tumor grade had increased ability to predict relapse (AUC = 0.775; p = 0.003). In addition, miR-10b (p = 0.015), miR-19a (p = 0.003), miR-20a (p = 0.012), miR-126 (p = 0.001) and miR-155 (p < 0.001) expression levels were lower in patients with early relapse (relapse at ≤2 years). MiR-155 and miR-19a had the highest performance in discriminating early relapse (AUC 0.855; p < 0.001 and AUC = 0.729; p = 0.003, respectively), whereas, combined mir-155 and miR-19a expression further increased the accuracy of prediction (AUC = 0.867; p < 0.001). In eBC, the number of infiltrated lymph nodes and low miR-10b independently predicted for shorter DFS (p = 0.001 and p = 0.03, respectively) and axillary lymph nodes for shorter OS (p = 0.003). In the triple negative subgroup, low miR-155 strongly predicted for shorter DFS (p = 0.037). In mBC, recurrent disease and low miR-10b expression independently predicted for shorter PFS (p = 0.001 and p = 0.017, respectively), whereas performance status of 2 independently predicted for shorter OS (p = 0.03). Conclusions: Deregulated expression of circulating miRNAs involved in tumor-immune interactions can discriminate disease status in BC and independently predicts for patients’ outcome in early and mBC. Our results further support the notion that circulating miRNAs represent a useful prognostic tool in patients with BC.

e21644 Background: Cancer cachexia syndrome (CCS) is a multifactorial inflammatory syndrome affecting a large subset of patients (pts) with NSCLC which in preclinical models negatively impairs antitumor immunity. We conducted a prospective, observational study to investigate the effect of CCS and sarcopenia on the efficacy of ICIs in NSCLC. Methods: CCS was defined as weight loss of > 5% in the last 6 months or any degree of weight loss > 2% in combination with BMI < 20% or baseline skeletal muscle index at the level of the 3rd lumbar vertebra consistent with sarcopenia. Skeletal muscle index was calculated using slice-o-matic tomovision software in the abdominal CT scan before starting ICIs. Results: 83 pts were included in the analysis. Median follow up was 9.5 months. Median age was 66 years, 61.4% had non-squamous histology, 20.5% received ICIs as first and the remaining as second-line therapy. 20.5% of pts experienced partial response (PR), 31% had stable disease (SD) and 48.2% had disease progression (PD). Median progression-free survival (PFS) was 4.4 months and median overall survival (OS) was 10.33 months. 43.4% of the whole group were categorized as having CCS, whereas 63.3% of 30 pts evaluated using tomovision had sarcopenia. CCS negatively affected response rates (p = 0.003) but not response duration (p = 0.266). CCS was associated with reduced PFS (2.46 vs 5.77 months, p = 0.006) and OS (4.8 vs 14.53 months, p = 0.001). In the multivariate analysis, CCS independently predicted for shorter OS (HR = 2.01; CI: 1,14-3,54; p = 0.014). Sarcopenia was also associated with reduced OS (5.4 vs 17.9 months, p = 0.012). Analysis on the whole pt population will be presented at the conference. Conclusions: CCS is associated with lower response rates and independently predicts for shorter OS in pts with NSCLC treated with ICIs. Further research on CCS could better define its role as a potential biomarker and a research platform for maximizing immunotherapy efficacy.

Background In primary breast cancer metastases frequently arise from a state of dormancy that may persist for extended periods of time. We investigated the efficacy of plasma micro-RNA (miR)-21, miR-23b, miR-190, miR-200b and miR-200c, related to dormancy and metastasis, to predict the outcome of patients with early breast cancer. Methods miRNAs were evaluated by RT-qPCR in plasma obtained before adjuvant chemotherapy. miRNA expression, classified as high or low according to median values, correlated with relapse and survival. Receiver operating characteristic (ROC) curves were constructed to determine miRNA sensitivity and specificity. Results miR-21 (p p = 0.028) and miR-200c (p p = 0.013) in relapsed (n = 49), compared to non-relapsed patients (n = 84). Interestingly, miR-190 was lower (p = 0.0032) in patients with early relapse (at n = 23) compared to those without early relapse (n = 110). On the other hand, miR-21 and miR-200c were higher (p = 0.015 and p n = 20) as compared to non-relapsed patients. High miR-200c was associated with shorter disease-free survival (DFS) (p = 0.005) and high miR-21 with both shorter DFS and overall survival (OS) (p p = 0.033, respectively) compared to low expression. ROC curve analysis revealed that miR-21, miR-23b, miR-190 and miR-200c discriminated relapsed from non-relapsed patients. A combination of of miR-21, miR-23b and miR-190 showed higher sensitivity and specificity in ROC analyses compared to each miRNA alone; accuracy was further improved by adding lymph node infiltration and tumor grade to the panel of three miRs (AUC 0.873). Furthermore, the combination of miR-200c, lymph node infiltration, tumor grade and estrogen receptor predicted late relapse (AUC 0.890). Conclusions Circulating miRNAs are differentially expressed among relapsed and non-relapsed patients with early breast cancer and predict recurrence many years before its clinical detection. Our results suggest that miRNAs represent potential circulating biomarkers in early breast cancer.

Background: Tumour cells exclusively express the embryonic M2 isoform of pyruvate kinase (PKM2). PKM2 expression levels have been correlated with the effect of platinum compounds in cancer cell lines and xenograft models. The potential predictive role of PKM2 in patients with metastatic/advanced non-small-cell lung cancer (NSCLC) receiving platinum-based chemotherapy as first-line was investigated. Methods: Quantitative real-time PCR was used to assess the expression of PKM2 in tumour samples from 148 and 157 NSCLC patients in the training and the validation set, respectively. All patients received front-line platinum-based chemotherapy. PKM2 mRNA expression was also analysed in a control group of 85 NSCLC patients treated with non-platinum containing regimens. Results: In the training set, high PKM2 mRNA levels were associated with decreased progression-free survival (PFS; 4.9 months vs 6.4, P=0.006), overall survival (OS; 10.1 vs 17.0 months, P=0.01) and disease control rate (DCR; 57.7% vs 74.3% P=0.021) compared to patients with low PKM2 levels. In the validation set, high PKM2 mRNA levels were also associated with deceased PFS (3.7 vs 5.9 months, P=0.006), OS (8.3 vs 16.8 months, P=0.003) and DCR (57.7% vs 70.9% P=0.049) compared to those with low PKM2 mRNA levels. There was no correlation between the PKM2 mRNA levels and the PFS (5.6 vs 5.9, P=0.43) or the OS (9.8 vs 10.1, P=0.51) in the control group. Multivariate analysis revealed high PKM2 mRNA expression as an independent predictive factor for the poor patients' outcome. Conclusions: PKM2 expression may be a predictive biomarker of platinum sensitivity in advanced NSCLC patients treated with platinum-based chemotherapy.

Background Aberrant miRNA expression has been associated with DNA damage response (DDR) pathways that modulate tumor response to platinum agents. We assessed the expression of miR-21, miR-128, mir-155 and miR-181 involved in DDR pathways, in the plasma of metastatic non- small cell lung cancer (mNSCLC) patients receiving 1st-line platinum-based chemotherapy (CMT) and the levels were correlated with patients’ outcomes. We also performed bioinformatics analysis to identify putative target genes of the above miRNAs. Methods Plasma samples were obtained from patients (n = 128) before 1st-line CMT. miR-21, miR-128, miR-155 and miR-181 expression levels were assessed by RT-qPCR and expression was classified as high or low according to the median values. Pathway analysis was performed by Diana-Tarbase algorithm. Results Mir-128 (p Conclusions Differential expression levels of DDR related miRNAs are encountered in mNSCLC patients before 1st-line platinum-based CMT and independently predict patients’ outcomes. The association of miR-155 with the prognosis of patients with SCC merits further investigation. Legal entity responsible for the study The authors. Funding HESMO. Disclosure All authors have declared no conflicts of interest.

e14045 Background: CTCs expressing innate (CD47) and adaptive (PD-L1) immune checkpoints may have enhanced potential to escape immunosurveillance. In the current study, we evaluated the incidence and clinical relevance of CTCs expressing CD47 and/or PD-L1 in patients with mBC. Methods: Blood was obtained from 98 mBC patients before the initiation of first-line therapy. Triple immunofluorescence staining for cytokeratins (CK), CD47 and PD-L1 was performed on peripheral blood mononuclear cells (PBMC) cytospins and 1*106 PBMCs per patient were analyzed using the Ariol microscopy system. CD47 and PD-L1 expression levels on CK+ CTCs were normalized using MDA.MB.231 breast cancer cells as controls. Results: CK+ CTCs were detected in 22 out of 98 patients (22.4%) (total CTC No: 43; range: 1-12). High CD47 and PD-L1 expression was identified in 41.9% and 11.6% of CTCs, respectively, whereas in 9.1% of CTCs, high expression was observed for both markers. CTCs with high expression of at least one marker ( CD47high and/or PD-L1high) were identified in 11.2% of patients, albeit with a differential distribution among patients with triple-negative, hormone receptor-positive and HER2-positive primary tumors (27.3%, 12.5% and 0%, respectively, p = 0.040; Chi-square test). CD47high and/or PD-L1high CTCs were associated with disease progression (27.8% vs 5.6%, p = 0.005; Chi-square test) and shorter PFS [median: 5.8 (4.1-7.5) vs 13.3 (11.4-15.2) months, p = 0.010; Kaplan-Meier], whereas the detection of PD-L1high CTCs only was correlated with reduced OS [median: 23.8 (5.8-41.8) vs 35.7 (29.9-41.4) months, p = 0.043]. Multivariate Cox-regression analysis revealed that CD47high and /or PD-L1high CTCs predicted for increased risk of relapse [HR: 2.7 (95% CI: 1.3-5.7); p = 0.009], whereas PD-L1high CTCs were associated with high risk of death [HR: 4.8 (95% CI: 1.4-16.6); p = 0.011]. Conclusions: The immune checkpoints CD47 and PD-L1 are co-expressed in a subset of CTCs in mBC patients. The detection of high CD47 and/or PD-L1 expression on CTCs is associated with triple-negative tumors, disease progression and poor patient outcome and could serve for the refinement of prognosis in mBC. Patients bearing this CTC population may benefit from anti-CD47 and anti-PD-L1 immunotherapy strategies.

9099 Background: Cancer cachexia syndrome, affecting up to 80% of patients (pts) with NSCLC, is characterized by systemic inflammation, negative protein and energy balance and poor patient outcome. HGS has been independently correlated with outcome in pts with advanced cancer. We investigated the potential association between cachexia, sarcopenia and HGS with outcome in pts with advanced NSCLC treated with ICIs. Methods: Fifty-five pts with NSCLC treated with ICIs were included in the analysis. Patient and disease characteristics and data on outcome measures were prospectively collected. Cancer cachexia was defined as weight loss > 5% in the last 6 months, BMI < 20% (or baseline skeletal muscle index at the level of the 3rd lumbar vertebra consistent with sarcopenia) and any degree of weight loss > 2%. Baseline HGS was measured using the JAMAR analogue dynamometer. Results: Median age was 69 years, 50% of pts had non-squamous histology, 69% had PS 0-1, 61% had cancer cachexia and mean HGS was 27 kgs. Partial response, stable disease and progressive disease was recorded in 16%, 33% and 51% of pts, respectively. Median PFS was 4 months and median OS was 7.8 months. There was no association of cachexia or HGS with tumor burden, number of metastatic sites, LDH or neutrophil/lymphocyte ratio. Cachexia had a negative impact on HGS (p = 0,001). Pts with cachexia had lower response rates (4% vs 37%, p = 0.002), disease stabilization rates (32% vs 81%, p = 0.003) and shorter PFS (3.7 vs 8.2 months, p = 0.008) compared to non-cachectic pts. In multivariate analysis, cachexia independently predicted for shorter PFS (HR = 2.7, CI: 1.15 – 6.46, p = 0.023). Data on sarcopenia are being analyzed and will be presented at the meeting. Conclusions: Cancer cachexia is associated with lower response rates and disease stabilization rates and independently predicts for shorter PFS in pts with NSCLC treated with ICIs. To our knowledge this is the first report demonstrating a between the host’s immune and metabolic responses. The study of cancer cachexia may offer a new platform for the development of novel biomarkers of resistance to ICIs.

Introduction The expression of CD47 on tumor cells can act as a "don't eat me signal" against phagocytosis by macrophages and dendritic cells. Moreover, PD-L1-expressing tumor cells inhibit the anti-tumor activity of cytotoxic T cells. Circulating tumor cells (CTCs) expressing these molecules could overcome elimination by the immune system. In the current study, we evaluated for the first time the co-expression of CD47 and PD-L1 on single CTCs from patients with metastatic breast cancer (mBC). Methods Triple immunofluorescence staining was performed on peripheral blood mononuclear cells (PBMC) cytospin preparations from 18 CTC-positive patients with mBC, using antibodies against cytokeratins (for CTC detection), CD47 and PD-L1. Blood samples were obtained before the initiation of first-line chemotherapy. A total of 1*106 PBMCs were analyzed per patientusing the Ariol microscopy system. The expression levels of CD47 and PD-L1 were characterized as high or low/-, after quantification by the Ariol system, using the MDA.MB.231 breast cancer cell line as positive control. Results A total of 23 CTCs (median: 1, range: 1-4) were identified. CD47-expressing CTCs were detected in 94.4% of patients and represented 91.3% of total CTCs. However, high CD47 expression was confirmed in 38.9% and 43.5% of patients and CTCs, respectively. PD-L1 expression was evident in 27.8% of patients and in 21.7% of CTCs, whereas CTCs expressing high levels of PD-L1 were identified in 16.% of patients and represented 13% of total CTCs. Co-expression of CD47 and PD-L1 (CD47+/PD-L1+) was observed in 21.7% of CTCs, whereas 69.6% of CTCs expressed CD47 only (CD47+/PD-L1-). No CTCs expressing only PD-L1 (CD47-/PD-L1+) were detected and 2 of 23 cells were negative for both markers (CD47-/PD-L1- ). Regarding the differential expression levels of CD47 and PD-L1, the phenotype CD47low/-/PD-L1low/- was the most abundant both at the patient (61.1%) and the CTC level (52.2%). CD47high/PD-L1low/- CTCswere observed in 33.3% and 34.8% of patients and CTCs, respectively whereas only 1 CD47low/-/PD-L1high CTC was detected in one patient. Interestingly, CD47high/PD-L1high CTCs were identified in only 8.7% of CTCs. Conclusions CD47 expression is identified in the great majority of CTCs in mBC and could represent a potent signal to facilitate the escape from innate immune response. PD-L1 expression on CTCs is less commonly observed and could serve for the attenuation of an adaptive anti-tumor immune response. Interestingly, CD47 and PD-L1 are co-expressed in a subset of CTCs, whereas simultaneous high expression on single CTCs is even less common. The expression of these molecules is currently further investigated in a larger cohort of patients with mBC and in patients with early disease, in order to evaluate their differential distribution that potentially reflects the equilibrium and/or escape from the immune surveillance. Citation Format: Mavroudis D, Papadaki MA, Tsoulfas PG, Troullinou K, Apostolopoulou CA, Papadaki C, Agelaki S. Co-expression of molecules associated with innate and adaptive immune response on single CTCs of patients with metastatic breast cancer (mBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-13.