Background/Objectives: Alterations in DNA damage repair mechanisms can impair the therapeutic effectiveness of cisplatin. MicroRNAs (miRNAs), key regulators of DNA damage repair processes, have been proposed as promising biomarkers for predicting the response to platinum-based chemotherapy (CT) in non-small cell lung cancer (NSCLC). In this study, by using a bioinformatics approach, we identified six miRNAs, which were differentially expressed (DE) between NSCLC patients characterized as responders and non-responders to platinum-based CT. We further validated the differential expression of the selected miRNAs on tumor and matched normal tissues from patients with resected NSCLC. Methods: Two miRNA microarray expression datasets were retrieved from the Gene Expression Omnibus (GEO) repository, comprising a total of 69 NSCLC patients (N = 69) treated with CT and annotated data from their response to treatment. Differential expression analysis was performed using the Linear Models for Microarray Analysis (Limma) package in R to identify DE miRNAs between responders (N = 33) and non-responders (N = 36). Quantitative real-time PCR (qRT-PCR) was used to assess miRNA expression levels in clinical tissue samples (N = 20). Results: Analysis with the Limma package revealed 112 DE miRNAs between responders and non-responders. A random-effects meta-analysis further identified 24 miRNAs that were consistently up- or downregulated in at least two studies. Survival analysis using the Kaplan–Meier plotter (KM plotter) indicated that 22 of these miRNAs showed significant associations with prognosis in NSCLC. Functional and pathway enrichment analysis revealed that several of the identified miRNAs were linked to key pathways implicated in DNA damage repair, including the p53, Hippo, PI3K and TGF-β signaling pathways. We finally distinguished a six-miRNA signature consisting of miR-26a, miR-29c, miR-34a, miR-30e-5p, miR-30e-3p and miR-497, which were downregulated in non-responders and are involved in at least three DNA damage repair pathways. Comparative expression analysis on tumor and matched normal tissues from surgically treated NSCLC patients confirmed their differential expression in clinical samples. Conclusions: In summary, we identified a signature of six miRNAs that are suppressed in NSCLC and may serve as a predictor of cisplatin response in NSCLC.

To investigate the incidence and prognostically significant correlations and cooperations of LKB1 loss of expression in non-small cell lung cancer (NSCLC), surgical specimens from 188 metastatic and 60 non-metastatic operable stage I-IIIA NSCLC patients were analyzed to evaluate their expression of LKB1 and pAMPK proteins in relation to various processes. The investigated factors included antitumor immunity response regulators STING and PD-L1; pro-angiogenic, EMT and cell cycle targets, as well as metastasis-related (VEGFC, PDGFRα, PDGFRβ, p53, p16, Cyclin D1, ZEB1, CD24) targets; and cell adhesion (β-catenin) molecules. The protein expression levels were evaluated via immunohistochemistry; the RNA levels of LKB1 and NEDD9 were evaluated via PCR, while KRAS exon 2 and BRAFV600E mutations were evaluated by Sanger sequencing. Overall, loss of LKB1 protein expression was observed in 21% (51/248) patients and correlated significantly with histotype (p < 0.001), KRAS mutations (p < 0.001), KC status (concomitant KRAS mutation and p16 downregulation) (p < 0.001), STING loss (p < 0.001), and high CD24 expression (p < 0.001). STING loss also correlated significantly with loss of LKB1 expression in the metastatic setting both overall (p = 0.014) and in lung adenocarcinomas (LUACs) (p = 0.005). Additionally, LKB1 loss correlated significantly with a lack of or low β-catenin membranous expression exclusively in LUACs, both independently of the metastatic status (p = 0.019) and in the metastatic setting (p = 0.007). Patients with tumors yielding LKB1 loss and concomitant nonexistent or low β-catenin membrane expression experienced significantly inferior median overall survival of 20.50 vs. 52.99 months; p < 0.001 as well as significantly greater risk of death (HR: 3.32, 95% c.i.: 1.71–6.43; p <0.001). Our findings underscore the impact of the synergy of LKB1 with STING and β-catenin in NSCLC, in prognosis.

BACKGROUND:Limited information exists on epidermal growth factor receptor (EGFR) molecular epidemiology in Greece. Next-generation sequencing (NGS) is the recommended method for EGFR genotyping in NSCLC. The Idylla Biocartis platform is a fully automated system for actionable EGFR mutation detection. RESEARCH DESIGN AND METHODS:We describe the prevalence of EGFR mutations in NSCLC patients in two high-volume clinical centers in Greece and compare key methods used for their determination. Eight hundred and fifty-seven FFPE samples from NSCLC patients were tested for EGFR mutations at University of Crete (UoC; n = 324) and at Evangelismos Hospital, Athens (Evangelismos; n = 503). RESULTS:The prevalence of EGFR mutations was 11.1% in the whole cohort (11.5% in non-squamous). The detection rate was 11.0% by NGS, 9.8% by Sanger and 11.3% by Idylla for the whole cohort (12.0% in non-squamous). The agreement between Idylla and Sanger was 93.2%. A targetable EGFR mutation was detected in 10.0% using tissue NGS alone, and in 16.0% using concurrent Idylla ctEGFR testing. CONCLUSION:The frequency of EGFR mutations was as expected for a Caucasian population. The Idylla EGFR test performance is comparable to reference methods and with a shorter TAT. Adding a concurrent plasma Idylla test to tissue NGS testing increases the detection rate of EGFR mutations in NSCLC.

Cardiac amyloidosis has been strongly associated with postoperative intractable circulatory failure, and intestinal amyloidosis could lead to intestinal pseudo-obstruction. The latter can be treated with neostigmine, which is notorious for its brief bradyarrhythmic complications. The amyloidosis patient presented herein, suffered an iatrogenic left main dissection, failure of bailout stenting and finally underwent urgent surgery. Meticulous fluid and drug management was key to keeping this patient stable. Postoperative atrial fibrillation was treated with amiodarone. The postoperative course was complicated with intestinal pseudo-obstruction, which was ultimately resolved with neostigmine. This short-lived cholinesterase inhibitor interacted with amiodarone and caused a previously undocumented prolonged complete atrioventricular block that resolved 48 hours after both drugs' discontinuation. The neostigmine amiodarone interaction warrants clinical vigilance and is speculated to be due to their partially shared second messenger pathway involving cyclic adenosine monophosphate. Patients with cardiac amyloidosis could maintain hemodynamic stability perioperatively.

A key unknown of the functional space in tumor immunity is whether CD4 T cells depend on intratumoral MHCII cancer antigen recognition. MHCII-expressing, antigen-presenting cancer-associated fibroblasts (apCAFs) have been found in breast and pancreatic tumors and are considered to be immunosuppressive. This analysis shows that antigen-presenting fibroblasts are frequent in human lung non-small cell carcinomas, where they seem to actively promote rather than suppress MHCII immunity. Lung apCAFs directly activated the TCRs of effector CD4 T cells and at the same time produced C1q, which acted on T cell C1qbp to rescue them from apoptosis. Fibroblast-specific MHCII or C1q deletion impaired CD4 T cell immunity and accelerated tumor growth, while inducing C1qbp in adoptively transferred CD4 T cells expanded their numbers and reduced tumors. Collectively, we have characterized in the lungs a subset of antigen-presenting fibroblasts with tumor-suppressive properties and propose that cancer immunotherapies might be strongly dependent on in situ MHCII antigen presentation.

Alterations in DNA damage repair (DDR) pathways can impair cisplatin efficacy. MicroRNAs (miRNAs) are actively involved in DDR regulation and in parallel have been suggested as potential biomarkers for the prediction of response to platinum- based chemotherapy (CT) in non- small cell lung cancer (NSCLC). In the present study we used a bioinformatics approach to identify differentially expressed miRNAs associated with the efficacy of platinum-based chemotherapy in NSCLC. We assembled four miRNA microarray expression datasets from NSCLC patients treated with CT included in the Gene Expression Omnibus (GEO) repository. Analysis in Limma package in R revealed 3.883 differentially expressed (DE) miRNAs between responders (n=71) and non-responders (n=99). After meta-analysis by random-effects, we identified a total of 697 miRNAs that were consistently up or down-regulated in at least two studies. By using more stringent effect size thresholds, we identified 43 DE miRNAs with log fold change ≥1.3 in at least two datasets. The prognostic significance of the selected miRNAs was further validated through survival analysis by KM plotter in stage III NSCLC. Twenty-two of the DE miRNAs were revealed to have prognostic significance in NSCLC. Integrated function and target pathway enrichment analysis revealed significant associations to a number of pathways and functions related to DDR, such as p53, HIPPO and FOXO3. Finally, we extracted a miRNA signature consisting of 8-miRNAs (miR-181a, let-7g, miR-26a, miR-145, miR-30d, miR-99a, miR-214, miR-107) that were down-regulated in non-responders and are involved in at least three DDR pathways. Comparative expression analysis on tumor and matched normal tissues from operable NSCLC patients treated with platinum-based chemotherapy will be performed to evaluate the clinical relevance of the signature.

Abstract A key unknown of the functional space in tumor immunity is whether physiologically relevant cancer antigen presentation occurs solely in draining lymph nodes versus tumors. Professional antigen presenting cells, i.e. the dendritic cells, are scarce and immature within tumors, greatly outnumbered by MHCII expressing non-hematopoietic cells, such as antigen-presenting cancer-associated fibroblasts (apCAFs). We hypothesized that after their exit from tumor-draining lymph nodes T cells depend on a second wave of antigen presentation provided in situ by structural cells. We show that dense apCAF regions in human lung tumors define hot immunological spots with increased numbers of CD4 T cells. The transcriptomic profile of human lung apCAFs aligned to that of pancreatic apCAFs across mice and humans and were both enriched for alveolar type II genes, suggesting an epithelial origin. Mechanistically, human apCAFs directly activated the TCRs of adjacent effector CD4 T cells and at the same time produced high levels of c1q, which acted on surface c1qbp on T cells to rescue them from apoptosis. Fibroblast-specific deletion of MHCII in mice impaired local MHCII immunity and accelerated tumor growth, while inducing c1qbp overexpression in adoptively transferred T cells expanded their numbers within tumors and reduced tumour burden. Collectively, our work shows that tumor T cell immunity post lymph node exit requires peripheral antigen presentation by a subset of CAFs and proposes a new conceptual framework upon which effective cancer immunotherapies can be built.

A tumor-to-tumor metastasis inside a meningioma is a rare phenomenon. Malignant neoplasms of the breast and lung are the most common primary tumors. Other sites of origin include prostate, renal and gastric neoplasms. The included case files were retrieved from the medical records of the University Hospital of Crete, Greece. A review of the literature was conducted in March 2020 via PubMed. Relevant search results were few. We report a case of a 66-year-old female, with known Small Cell Lung Cancer, who presented with left-sided hemiparesis. The Magnetic Resonance Imaging scan revealed a right frontal extra-axial mass. The patient underwent a craniotomy and a gross total removal of the tumor. Histological examination of the excised mass revealed metastatic adenocarcinoma deposits inside a meningioma: tumor-to-tumor metastasis. Reviewing the available literature, it has been hypothesized that the following factors play a role in the pathophysiology of this phenomenon: progesterone and estrogen receptors, cell-to-cell adhesion molecules, rich vascularization, favorable metabolic, micro-and immunological environment. Meningiomas seem to be the most common type of intracranial neoplasm to host a metastasis. There is a difference between tumor-to-tumor metastasis and collision tumors. The former implies a recipient role of the host tumor, and the latter refers to a co-localization of two different tumors that grow into one another, both being in the same organ. Tumor-to-tumor brain metastasis is a well-described phenomenon but with unclear pathophysiology. Deeper knowledge could be beneficial for its management.

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.

Bladder leiomyomas (BLs) are extremely rare benign tumors of mesenchymal origin. The exact pathophysiological mechanisms that lead to their appearance remain unclear including hormonal disorders, chromosomal abnormalities, and fetal remnants in the bladder. They usually remain asymptomatic for a long period of time. Solitary fibrous tumors (SFTs) are also rare neoplasms of mesenchymal origin with malignant potential usually affecting the pleura. The pathogenesis of SFTs remains unclear. We report the case of a 28-year-old male presenting with SFT of the pleura and synchronous BL. The patient presented with persistent cough as a sole symptom. Computed tomography (CT) of the thorax revealed a pleural mass, which was surgically removed and proved to be a SFT. At an early follow-up, abdominal CT scan revealed a bladder wall mass that proved to be a BL. This is the first report of BL with synchronous SFT of the pleura. Synchronous BLs and SFTs may be incidental, but the coexistence of two mesenchymal tumors at different sites, in a young patient, may raise the suspicion of a new clinical syndrome that warrants further investigation.

In situ antigen presentation is required to sustain active proliferating CD4 T cells in tumours and to help form memory CD8 T cells, but the antigen presenting cells (APCs) and pathways involved remain elusive. Cancer associated fibroblasts (CAFs) are prominent stromal constituent of solid tumors. Current immunological dogma considers that CAFs facilitate tumour immune escape. A new subset of MHCII antigen presenting cancer-associated fibroblasts (apCAFs) has been described, but their function remains unknown. Here we report a previously unrecognized function of apCAFs in sustaining CD4 T cells in primary human and murine lung tumours. In response to IFNγ and oxidative stress in tumours CAFs up-regulated MHCII. Fibroblast-specific targeted ablation of MHCII induced a hypometabolic hypoproliferative state in CD4 T cells, which impacted MHCII and MHCI immunity, accelerating tumor growth. apCAFs directly presented MHCII-peptide (MCHIIp) complexes to activate the TCRs of CD4 T cell. Highthrough-put profiling and blocking assays unveiled a novel CAF to T cell communication pathway via complement 1q binding on membrane C1qbp. Thus, apCAFs sustain anti-tumor CD4 T cells via MHCIIp-TCR and C1q-C1qbp binding. Our studies pave the way to the design of novel immunotherapeutic strategies that will harness apCAFs to help sustain T cells inside solid tumours.

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.

KRAS mutations are found in approximately one third of non-small cell lung cancer (NSCLC) patients. In this study, we aim to investigate whether KRAS G12/G13 mutant allele fraction (MAF) in cell-free DNA (cfDNA) can provide meaningful prognostic information in NSCLC. Multiplex droplet-digital PCR was used to quantitatively assess KRAS G12/G13 MAF in cfDNA from 114 pre-treated advanced disease NSCLC patients. In 14 patients, changes in KRAS G12/G13 MAF were longitudinally monitored during treatment. Plasma KRAS G12/G13 status was associated with poor patients' outcome in terms of progression-free survival (PFS) (p < 0.001) and overall survival (OS) (p < 0.001). In multivariate analysis, the detection of plasma KRAS mutations was an independent predictor of adverse PFS (HR = 3.12; p < 0.001) and OS (HR = 2.53; p = 0.002). KRAS G12/G13 MAF at first treatment evaluation (T1) was higher (p = 0.013) among patients experiencing progressive disease compared to those with disease control, and increased KRAS MAF at T1 was associated (p = 0.005) with shorter PFS. On the contrary, no association was observed between tissue KRAS mutation status and patients' prognosis. Our results show that ddPCR-based detection of KRAS G12/G13 mutations in plasma could serve as an independent biomarker of unfavorable prognosis in NSCLC patients. Changes in KRAS MAF can provide valuable information for monitoring patient outcome during treatment.

Objectives Thymomas, as well as thymic carcinomas, are extremely rare tumors that arise from the thymus. The management of these tumors is primarily the complete surgical resection, however when there is tumor progression or metastatic unresectable disease, palliative platinum-based chemotherapy is the standard of care. On this setting, alternative options are emerging including immune checkpoints inhibitors. Based on that, PDL-1 expression was measured in thymic tumors as a potential predictive biomarker of response to anti-PD1 and anti-PDL1 immune inhibitors. Our objective is to report the first two cases of fatal toxicity due to anti- PD1 therapy in thymoma patients. Materials and Methods Here, we report two cases of metastatic B2/B3 thymomas refractory to initial standard chemotherapy treatment, with high PDL1 expression (>50%), that were treated with the anti-PD1 agent, pembrolizumab. Results The administration of anti- PD1 immune check point inhibitor resulted in a storm of immune related adverse events including myositis, myocarditis and myasthenia gravis and death after administration of the first treatment cycle. Conclusion In thymomas, the administration of PD1 inhibitors seems to be associated with a high percentage of severe immune related adverse events, thus requiring special caution on the usage of these agents in thymomas.

Liquid biopsy analysis, mainly based on circulating tumor cells ( CTC s) and circulating tumor DNA (ct DNA ), provides an extremely powerful tool for the molecular profiling of cancer patients in real time. In this study, we directly compared PIK 3 CA hotspot mutations (E545K, H1047R) in Ep CAM ‐positive CTC s and paired plasma‐ct DNA in breast cancer (BrCa). PIK 3 CA hotspot mutations in CTC s and ct DNA were analyzed using our previously developed highly sensitive (0.05%), specific, and validated assay in plasma‐ct DNA from 77 early and 73 metastatic BrCa patients and 40 healthy donors. We further analyzed and directly compared PIK 3 CA hotspot mutations in DNA s isolated from CellSearch ® cartridges ( CTC s) and paired plasma‐ct DNA , in 56 cases of early and 27 cases of metastatic breast cancer, and 16 corresponding primary tumors. In plasma‐ct DNA , PIK 3 CA hotspot mutations were identified in 30/77(39.0%) early and 35/73(47.9%) metastatic BrCa cases; none (0/40, 0%) of the healthy donors’ plasma‐ct DNA samples were positive. Our direct comparison study in DNA s isolated from CellSearch ® cartridges ( CTC s) and paired plasma‐ct DNA from the same blood draws has shown a lack of concordance in early BrCa (27/56, 48.2%), while the concordance in the metastatic setting was higher (18/27, 66.6%). Our results were validated by dd PCR methodology, and the concordance between our assay and dd PCR for PIK 3 CA E545K hotspot mutation was 30/37 (81.1%). In many cases, PIK 3 CA hotspot mutations were detected in samples found to be negative for CTC s in CellSearch ® . Our data demonstrated for the first time that (a) PIK 3 CA hotspot mutations are present at high frequencies in CTC s isolated from CellSearch ® cartridges and paired plasma‐ct DNA both in early and metastatic BrCa, (b) the detection and concordance of PIK 3 CA hotspot mutations between plasma‐ct DNA and CTC s are higher in the metastatic setting, (c) PIK 3 CA mutational status significantly changes after therapeutic intervention, and (d) PIK 3 CA mutation detection in CTC s and plasma‐ct DNA provides complementary information.

Circulating tumor cells (CTCs) are important for metastatic dissemination of cancer. They can provide useful information, regarding biological features and tumor heterogeneity; however, their detection and characterization are difficult due to their limited number in the bloodstream and their mesenchymal characteristics. Therefore, new biomarkers are needed to address these questions. Bioinformatics functional enrichment analysis revealed a subgroup of 24 genes, potentially overexpressed in CTCs. Among these genes, the chemokine receptor CXCR4 plays a central role. After prioritization according to the CXCR4 corresponding pathways, five molecules (JUNB, YWHAB, TYROBP, NFYA, and PRDX1) were selected for further analysis in biological samples. The SKBR3, MDA-MB231, and MCF7 cell lines, as well as PBMCs from normal (n = 10) blood donors, were used as controls to define the expression pattern of all the examined molecules. Consequently, 100 previously untreated metastatic breast cancer (mBC) patients (n = 100) were analyzed using the following combinations of antibodies: CK (cytokeratin)/CXCR4/JUNB, CK/NFYA/ΥWHΑΒ (14-3-3), and CK/TYROBP/PRDX1. A threshold value for every molecule was considered the mean expression in normal PBMCs. Quantification of CXCR4 revealed overexpression of the receptor in SKBR3 and in CTCs, following the subsequent scale (SKBR3>CTCs>Hela>MCF7>MDA-MB231). JUNB was also overexpressed in CTCs (SKBR3>CTCs>MCF7>MDA-MB231>Hela). According to the defined threshold for each molecule, CXCR4-positive CTCs were identified in 90% of the patients with detectable tumor cells in their blood. In addition, 65%, 75%, 14.3%, and 12.5% of the patients harbored JUNB-, TYROBP-, NFYA-, and PRDX-positive CTCs, respectively. Conversely, none of the patients revealed YWHAB-positive CTCs. Interestingly, JUNB expression in CTCs was phenotypically and statistically enhanced compared to patients’ blood cells (p = 0.002) providing a possible new biomarker for CTCs. Furthermore, the detection of JUNB-positive CTCs in patients was associated with poorer PFS (p = 0.015) and OS (p = 0.002). Moreover, JUNB staining of 11 primary and 4 metastatic tumors from the same cohort of patients revealed a dramatic increase of JUNB expression in metastasis. CXCR4, JUNB, and TYROBP were overexpressed in CTCs, but only the expression of JUNB was associated with poor prognosis, providing a new biomarker and a potential therapeutic target for the elimination of CTCs.

BACKGROUND:Since tumor cells may escape from immune surveillance through the programmed cell death 1 (PD-1)/programmed death ligand (PD-L)1 axis, this study was designed in order to evaluate whether there is a correlation between the levels of PD-1+ and PD-L1+-expressing immune cells (ICs) and circulating tumor cells (CTCs) in patients with non-small cell lung cancer (NSCLC).PATIENTS AND METHODS:Peripheral blood was obtained from 37 chemotherapy-naïve patients with metastatic NSCLC before treatment. PD-1 and PD-L1 expression was evaluated (1) on ICs with anti-tumor function (CD4+ and CD8+ T-cells, B-cells, monocytes/dendritic cells) using flow cytometry, (2) on CTCs by immunofluorescence and (3) on cells from tumor tissues by immunohistochemistry. The levels of PD-1+ and PD-L1+-expressing ICs were correlated with progression-free survival (PFS).RESULTS:The presence of PD-1+ CD8+ cells, with reduced interferon (IFN)-γ expression, but not other ICs, were positively correlated with PD-L1+ CTCs (p < 0.04). Increased percentages of PD-1+ CD8+ T-cells, were associated with a worse response to treatment (p = 0.032) and shorter PFS (p = 0.023) which, in multivariate analysis, was revealed as an independent predictor for decreased PFS [hazard ratio (HR): 4.1, p = 0.0007].CONCLUSION:The results of the current study, for first time, provide evidence for a possible interaction between ICs and CTCs in NSCLC patients via the PD-1/PD-L1 axis and strongly support that the levels of PD-1+ CD8+ in these patients may be of clinical relevance.

Despite the development of new treatment options based on the molecular characterization of colorectal cancer, 20% of patients present de novo metastatic disease, whereas 30-40% of patients who receive curative treatment relapse during follow up. Herein, we report 2 cases with rectal cancer that developed uncommon sites of metastasis; the first patient had an isolated breast metastasis, while the second patient developed bone marrow infiltration with synchronous brain metastases. In order to evaluate the uncommon metastatic pattern of rectal cancer, we detected and enumerated circulating tumor cells (CTCs) using both immunofluorescence and real-time reverse transcriptase polymerase chain reaction in these patients’ peripheral blood. The procedure revealed the presence of CTCs, positive for CEACAM5 but negative for epithelial phenotype (EpCAM-), that might explain the patients’ metastatic potential and survival.