The prognostic value of JUNB-positive CTCs in metastatic breast cancer: from bioinformatics to phenotypic characterization.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Upregulation of citrullination pathway: From Autoimmune to Idiopathic Lung Fibrosis.

BACKGROUND: Increased protein citrullination and peptidylarginine deiminases (PADIs), which catalyze the citrullination process, are central in Rheumatoid arthritis pathogenesis and probably involved in the initial steps towards autoimmunity. Approximately, 10% of RA patients develop clinically significantly ILD. A possible shared role of protein citrullination in rheumatoid arthritis associated interstitial lung disease (RA-ILD), and idiopathic pulmonary fibrosis (IPF) pathogenesis remains unclear. METHODS: We evaluated PADI2 and PADI4 mRNA expression in bronchoalveolar lavage fluid (BALF) cells of 59 patients with IPF, 27 patients RA-ILD and 10 healthy controls. PADI 2 and 4 expression was analyzed by western blot and immunohistochemistry. Citrullinated protein levels were also quantified. RESULTS: PADI4 mRNA and protein levels were higher in RA-ILD and IPF than controls. Furthermore, PADI4 mRNA levels showed an increase among smokers in RA-ILD. PADI4 expression was detected in granulocytes and macrophages in all groups, with the strongest cytoplasmic expression observed in granulocytes in RA-ILD and IPF. PADI2 mRNA and immunostaining of BAL cells, were similar in all groups among smokers. Overall, stronger staining was observed in current smokers. Citrullinated peptides were significantly increased in IPF compared to RA-ILD and controls. In RA-ILD, protein citrullination strongly correlated with PADI4 expression and anti-citrullinated protein antibodies (ACPAs). CONCLUSIONS: These results suggest that the citrullination pathway is upregulated in IPF and in RA-ILD.

Proteomic studies of pediatric medulloblastoma tumors with 17p deletion.

CNS tumors are the leading cause of cancer-related death in children. Medulloblastoma is the commonest pediatric CNS malignancy, wherein, despite multimodal therapy with surgery, radiation, and chemotherapy, 5 year survival rates merely approach 60%. Until present, gene expression and cytogenetic studies have produced contradicting findings regarding the molecular background of the specific disease. Through integration of genomics, bioinformatics, and proteomics, the current study aims to shed light at the proteomic-related molecular events responsible for MBL pathophysiology, as well as to provide molecular/protein/pathway answers concerning tumor-onset. Experiments were performed on tissues collected at surgery. With 17p loss being the commonest chromosomal aberrance observed in our sample set, array-CGH were employed to first distinguish for 17p-positive cases. 2-DE coupled to mass spectrometry identification exposed the MBL-specific protein profile. Protein profiles of malignant tissues were compared against profiles of normal cerebellar tissues, and quantitative protein differences were determined. Bioinformatics, functional and database analyses, characterization, and subnetwork profiling generated information on MBL protein interactions. Key molecules of the PI3K/mTOR signaling network were identified via the techniques applied herein. Among the findings IGF2, PI3K, Rictor, MAPKAP1, S6K1, 4EBP1, and ELF4A, as part of the IGF network (implicating PI3K/mTOR), were founded to be deregulated.

ERCC1 SNPs as Potential Predictive Biomarkers in Non-Small Cell Lung Cancer Patients Treated With Platinum-Based Chemotherapy.

Polymorphisms in ERCC1, XPD, and XRCC1 were examined for (a) association with the clinical outcome of 107 non-small cell lung cancer patients receiving front-line platinum-based chemotherapy, and (b) correlation with the ERCC1 mRNA levels of 176 chemo-naive primary tumors. The ERCC1-C8092 allele and the number of ERCC1 polymorphic variants (C8092A and Asn118Asn) were associated with progression-free survival. In non-squamous histology, tumoral ERCC1 mRNA levels were lower in patients homozygous for ERCC1-C8092 as compared with the patients carrying the A allele (p = .024). These findings merit investigation in larger cohorts of patients treated with uniform regimens.

Detection and genotype analysis of human papillomavirus in non-small cell lung cancer patients.

Although the role of human papillomavirus (HPV) in the development of uterine cervical cancer is well established, the role of HPV in lung carcinogenesis remains controversial. The detection rates of HPV DNA are subject to a wide variation from 0 to 100%. This is partly influenced by the detection techniques employed. To elucidate the impact of HPV infection on lung parenchyma, we analyzed 100 non-small cell lung cancer (NSCLC) specimens (39 squamous cell carcinomas, 50 adenocarcinomas, 5 samples with characteristics of both squamous cell and adenocarcinoma, 5 undifferentiated and 1 large cell carcinoma) from the region of Crete, Greece. Sixteen non-cancerous samples served as the negative controls. DNA was extracted from 100 paraffin-embedded tissue sections obtained from NSCLC patients. The specimens were examined for the detection of HPV DNA by Real-Time PCR using GP5+/GP6+ primers. Furthermore, the HPV-positive samples were subjected to genotyping. In contrast to the absence of viral genomes in the control samples, HPV DNA was detected in 19 NSCLC specimens (19%). In particular, 4 squamous cell carcinomas, 12 adenocarcinomas, 1 sample with characteristics of both squamous cell and adenocarcinoma, and 2 undifferentiated samples were HPV-positive. The distribution of HPV genotypes was as follows: HPV 16: eight cases (42.1%); HPV 11: three cases (15.8%); HPV 6: one case (5.2%); HPV 59: one case (5.2%); HPV 33: two cases (10.5%); HPV 31: two cases (10.5%) and HPV 18: two cases (10.5%). The presence of HPV in the tumor samples provides evidence of the potential role of HPV in NSCLC and strongly argues for additional research on this issue.

Rapid and reliable testing for clinically actionable EGFR mutations in non-small cell lung cancer using the IdyllaTM platform: a real-world two-center experience in Greece.

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.

LKB1 Loss Correlates with STING Loss and, in Cooperation with ß-Catenin Membranous Loss, Indicates Poor Prognosis in Patients with Operable Non-Small Cell Lung Cancer.

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, PDGFRß, p53, p16, Cyclin D1, ZEB1, CD24) targets; and cell adhesion (ß-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 ß-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 ß-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 ß-catenin in NSCLC, in prognosis.

Increased incidence of autoimmune markers in patients with combined pulmonary fibrosis and emphysema.

BACKGROUND: Combined pulmonary fibrosis and emphysema (CPFE) is an umbrella term encompassing upper lobe emphysema and lower lobe pulmonary fibrosis with pathogenesis elusive. The aim of our study was to investigate the incidence of autoimmune markers in patients with CPFE. METHODS: In this multicenter study we retrospectively evaluated records from patients with CPFE (n=40) and IPF (n=60) without emphysema. Baseline demographic characteristics, high-resolution computed tomography (HRCT), spirometry, histopathological, treatment, serum immunologic and survival data were investigated. B cell presence was estimated with CD20 immunostaining in representative lung biopsy samples from CPFE patients and control subjects. RESULTS: A statistically significant increased number of CPFE patients with elevated serum ANA with or without positive p-ANCA titers compared to patients with IPF without emphysema was observed. Patients with CPFE and positive autoimmune markers exhibited improved survival compared to patients with a negative autoimmune profile. A massive infiltration of clusters of CD20+ B cells forming lymphoid follicles within the fibrotic lung in CPFE patients with positive serum immunologic profile compared to patients with negative profile, was noted and positively correlated with improved survival. CONCLUSIONS: A significant proportion of patients with CPFE may present with underlying auto-immune disorders that may reside insidiously and be associated with favorable prognosis. Early identification of these patients using a panel of auto-antibodies may lead to more targeted and effective therapeutic applications.

Prolonged complete atrioventricular block due to neostigmine and amiodarone interaction in an amyloidosis patient with left main dissection and postoperative intestinal pseudo-obstruction.

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.

Lung tumor MHCII immunity depends on in situ antigen presentation by fibroblasts.

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.

High Beclin 1 expression defines a poor prognosis in endometrial adenocarcinomas.

OBJECTIVE: To investigate the prognostic role of Beclin 1 in endometrial adenocarcinomas of the endometrioid cell type. Beclin 1 is a known tumor suppressor gene, but its function may be altered under conditions of an accelerated autophagic activity, which provides additional energy to proliferating cells by recycling defective organelles and long-lived cytoplasmic proteins. MATERIALS AND METHODS: One hundred and fifty-five endometrioid adenocarcinomas were investigated for their autophagic activity using the monoclonal antibody Beclin 1 and an automated immunohistochemical technique. The extent of Beclin 1 expression was evaluated on a three-tier scale as follows: low (<10% positive tumor cells), intermediate (between 10% and 50% positive tumor cells), and high (>50% positive tumor cells). The results were correlated with the degree of tumor differentiation, the depth of myometrial invasion and the overall 5-year survival. In addition, the endometrial tumors were immunostained with the hypoxia inducible factor 1α (HIF1α) and their expression was related to Beclin 1. RESULTS: A high Beclin 1 reactivity occurred in 18.1% of endometrial adenocarcinomas studied and was associated with high tumor grade, high myometrial invasion and a poor 5-year survival. It was also correlated positively with HIF1α. Of the remaining adenocarcinomas 29.7% were of intermediate Beclin 1 reactivity and 52.2% of low, but correlations with prognostic factors were insignificant. CONCLUSION: An increased Beclin 1 expression is connected with the most aggressive endometrioid adenocarcinomas, probably as a result of its strong association with tumor hypoxia.

Intrameningioma metastasis: A case-based literature review.

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.

Deregulation of apoptosis mediators' p53 and bcl2 in lung tissue of COPD patients.

Abnormal apoptotic events in chronic obstructive pulmonary disease (COPD) subvert cellular homeostasis and may play a primary role in its pathogenesis. However, studies in human subjects are limited. p53 and bcl2 protein expression was measured by western blot on lung tissue specimens from 43 subjects (23 COPD smokers and 20 non-COPD smokers), using beta-actin as internal control. Additionally, p53 and bcl2 expression patterns were evaluated by immunohistochemistry in formalin-fixed, paraffin-embedded lung tissue sections from the same individuals. Western blot analysis showed statistically significant increased p53 protein levels in COPD smokers in comparison with non-COPD smokers (p = 0.038), while bcl2 protein levels were not statistically different between the two groups. Lung immunohistochemistry showed increased ratio of positive p53-stained type II pneumocytes/total type II pneumocytes in COPD smokers compared to non-COPD smokers (p = 0.01), whereas the p53 staining ratio in alveolar macrophages and in lymphocyte-like cells did not differ statistically between the two groups. On the other hand, bcl2 expression did not differ between the two groups in all three cell types. The increased expression of pro-apoptotic p53 in type II pneumocytes of COPD patients not counterbalanced by the anti-apoptotic bcl2 could reflect increased apoptosis in the alveolar epithelium of COPD patients. Our results confirm previous experiments and support the hypothesis of a disturbance in the balance between the pro- and anti-apoptotic mediators in COPD.

Tumoral expression of TXR1 and TSP1 predicts overall survival of patients with lung adenocarcinoma treated with first-line docetaxel-gemcitabine regimen.

PURPOSE: In vitro data suggest that down-regulation of thrombospondin 1 (TSP1) expression from TXR1 is associated with resistance to taxane-based chemotherapy. The prognostic and predictive value of tumoral expression of both genes was evaluated in patients with lung adenocarcinoma treated with first-line docetaxel and gemcitabine. EXPERIMENTAL DESIGN: Tumor samples from 96 patients, with stage IIIB (with pleural effusion) or IV lung adenocarcinomas, were analyzed for TXR1 and TSP1 mRNA levels by quantitative real-time PCR, from microdissected cells derived from patients' primary tumors. RESULTS: The mRNA levels of the two genes were inversely correlated (Spearman's test = -0.49; P < 0.0001). Patients with low TXR1 mRNA levels experienced a longer median time to tumor progression (TTP; P < 0.0001) and median overall survival (mOS; P = 0.001) when compared with patients with high TXR1 expression. Patients with high TSP1 expression presented longer TTP (P = 0.002) and mOS (P < 0.0001) when compared with patients with low TSP1 expression. Moreover, patients with high TSP1 and low TXR1 expression (n = 36) presented higher prolonged TTP (P = 0.009) and mOS (P < 0.0001) compared with patients with high TXR1 and low TSP1 expression. Multivariate analysis showed that high TXR1/low TSP1 expression was an independent prognostic factor for decreased TTP (hazard ratio, 1.7; 95% confidence interval, 1.1-3.27; P = 0.016) and mOS (hazard ratio, 2.55; 95% confidence interval, 1.57-4.15; P < 0.0001). CONCLUSION: These data confirm the in vitro model of TSP1 and TXR1 effect on taxane resistance in lung adenocarcinomas and merit further evaluation.

Detection of KRAS G12/G13 Mutations in Cell Free-DNA by Droplet Digital PCR, Offers Prognostic Information for Patients with Advanced Non-Small Cell Lung Cancer.

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.

Bladder Leiomyoma with Synchronous Solitary Fibrous Tumor of the Pleura.

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.

Clinical Relevance of Immune Checkpoints on Circulating Tumor Cells in Breast Cancer.

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.

Brain and bone marrow metastases from rectal cancer.

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.

Botulinum toxin type A--treatment of a patient with multiple cutaneous piloleiomyomas.

BACKGROUND: Treatment of multiple cutaneous piloleiomyomas which are rare, frequently painful, benign tumors originating from the arrector pilorum muscle of hair follicles is difficult. OBJECTIVE: To determine the efficiency of botulinum toxin type A (BT-A) treatment for pain relief of cutaneous piloleiomyomas. METHODS: A patient with multiple painful piloleiomyomas was treated with local injections of 200 units of BT-A. RESULTS: There was a rapid and sustained decrease in pain. Treatment was repeated every 3 months for 2 years with the same efficacy. CONCLUSION: BT-A may be a promising new treatment option for multiple painful cutaneous piloleiomyomas.

Characterization of DLL3-positive circulating tumor cells (CTCs) in patients with small cell lung cancer (SCLC) and evaluation of their clinical relevance during front-line treatment.

OBJECTIVES: The aim of the study was to characterize and evaluate the presence of DLL3-positive Circulating Tumor Cells (CTCs) in SCLC patients receiving front-line chemotherapy and assess their clinical relevance. MATERIALS AND METHODS: Peripheral blood was obtained from treatment-naïve patients with SCLC (n = 108 patients), after one etoposide/platinum cycle (n = 68 patients) and on disease progression (n = 48 patients). Immunofluorescence staining using antibodies against the DLL3, cytokeratins (CK), CD45 and vimentin (Vim) was used for the detection and characterization of CTCs. RESULTS: Before treatment, 74.1% of patients had detectable DLL3+/CD45- CTCs. One-treatment cycle significantly decreased both the detection rate (p < 0.001) and the absolute number (p < 0.001) of DLL3+/CD45- CTCs. Triple immunofluorescence staining using anti-CK, anti-Vim and anti-DLL3 antibodies revealed an important CTC heterogeneity since DLL3 could be detected in Vim+, Vim-, CK+ and CK- CTCs. On disease progression, both the detection rate and the absolute number of DLL3+/CD45- CTCs were significantly increased compared to post-1st cycle values (p < 0.001 and p = 0.002, respectively). In addition, 22.7% of patients had detectable DLL3+/CD45- cells which could not be captured by the CellSearch assay. In multivariate analysis, the detection of DLL3+/CD45- CTCs at baseline was significantly associated with decreased progression-free survival (HR = 10.8; p = 0.005) whereas their detection on disease progression was associated with decreased overall survival (HR: 28.2; p = 0.016). CONCLUSIONS: These findings demonstrate an important heterogeneity of CTCs, based on the expression of CK, Vim and DLL3, in patients with SCLC and the changes of DLL3+/CD45- CTCs during treatment seem to be a dynamic biomarker associated with patients' clinical outcome.