Vascular Regenerative Capacity and the Obesity Paradox in Coronary Artery Disease.

[Figure: see text].

Prognostic value of detection of CD99+ , CD45- cells in peripheral blood by flow cytometry in children with Ewing sarcoma.

BACKGROUND: Early detection of metastasis and recurrence of Ewing sarcoma (ES) is important for early management. This work aimed to detect CD99+ , CD45- cells in peripheral blood by flow cytometry (FC) before and during chemotherapy and evaluate their prognostic significance. PROCEDURE: This prospective cohort study was carried out on 60 children newly diagnosed with ES at Children Cancer Hospital-Egypt 57357 and 40 healthy children control group. Detection of CD99+ , CD45- cells in peripheral blood was accomplished by FC at baseline before treatment and after five cycles of chemotherapy. Samples were classified as positive if they had more than the upper limit of cells observed in the control cases. Correlation between FC results and relapse and overall survival (OS) after one year was performed. RESULTS: Median percentage of CD99+ , CD45- cells was significantly increased in patients compared with controls (0.002% vs 0%, respectively, P < 0.001). Post-cycle 5 CD99+ , CD45- cells were increased in 12 patients, of them 11 patients' disease had either relapsed or progressed. Post-cycle 5 CD99+ ; CD45- cells had a 73.3% sensitivity and 97.8% specificity for predicting relapse or progression, whereas baseline only had 6.7% sensitivity and 77.8% specificity. The hazard ratio for mortality in the post-cycle 5 positive group was 18.4 [95% confidence interval (1.86 to 181.46)] times that of the negative group. One year OS was 91.67%. CONCLUSION: Post-cycle 5 CD99+ , CD45- cells in peripheral blood by FC is a strong predictor for relapse, progression, and mortality whereas baseline is a poor predictor in newly diagnosed patients with ES.

High-dimensional Cytometry (ExCYT) and Mass Spectrometry of Myeloid Infiltrate in Clinically Localized Clear Cell Renal Cell Carcinoma Identifies Novel Potential Myeloid Targets for Immunotherapy.

Renal Cell Carcinoma (RCC) is one of the most commonly diagnosed cancers worldwide with research efforts dramatically improving understanding of the biology of the disease. To investigate the role of the immune system in treatment-naïve clear cell Renal Cell Carcinoma (ccRCC), we interrogated the immune infiltrate in patient-matched ccRCC tumor samples, benign normal adjacent tissue (NAT) and peripheral blood mononuclear cells (PBMCs isolated from whole blood, focusing our attention on the myeloid cell infiltrate. Using flow cytometric, MS, and ExCYT analysis, we discovered unique myeloid populations in PBMCs across patient samples. Furthermore, normal adjacent tissues and ccRCC tissues contained numerous myeloid populations with a unique signature for both tissues. Enrichment of the immune cell (CD45+) fraction and subsequent gene expression analysis revealed a number of myeloid-related genes that were differentially expressed. These data provide evidence, for the first time, of an immunosuppressive and pro-tumorigenic role of myeloid cells in early, clinically localized ccRCC. The identification of a number of immune proteins for therapeutic targeting provides a rationale for investigation into the potential efficacy of earlier intervention with single-agent or combination immunotherapy for ccRCC.

Increased circulating endothelial progenitor cells (EPCs) in prepubertal children born prematurely: a possible link between prematurity and cardiovascular risk.

BACKGROUND: Endothelial progenitor cells (EPCs) ensure vascular integrity and neovascularization. No studies have investigated EPCs in preterm-born children beyond infancy. METHODS: One hundred and thirty-six prepubertal children were enrolled: 63 preterm and 73 born at term (controls). Circulating CD34(+)/VEGFR-2(+)/CD45(-) and CD34(+)/VEGFR-2(+)/CD45dim EPCs were measured in preterm-born children compared to controls. Body mass index (BMI), waist-to-hip ratio (WHR), neck circumference, systolic and diastolic blood pressure (SBP and DBP, respectively), fasting glucose, insulin, lipid profile, common carotid and abdominal aortic intima-media thickness (cIMT and aIMT, respectively), endothelium-dependent brachial artery flow-mediated dilation (FMD), and echocardiographic parameters were also assessed. RESULTS: Circulating CD34(+)/VEGFR-2(+)/CD45(-) and CD34(+)/VEGFR-2(+)/CD45dim EPCs were significantly higher in preterm-born children compared to controls (p < 0.001 and p < 0.001, respectively). In total study population and in the preterm-born group, EPCs were significantly lower in children born to mothers with gestational diabetes compared to non-diabetic mothers. Prematurity was associated with higher WHR, neck circumference, SBP, DBP, cIMT, aIMT, mean pressure, and velocity of pulmonary artery; the peak velocity of the brachial artery was significantly lower in children born prematurely. In multiple regression analysis, preterm birth and maternal gestational diabetes were recognized as independent predictors of EPCs. CONCLUSIONS: Circulating EPCs were increased in prepubertal preterm-born children in comparison with peers born full-term. Maternal gestational diabetes was associated with a decrease in EPCs. IMPACT: Mounting evidence supports the adverse effect of prematurity on cardiovascular health. However, the underlying mechanisms that could lead to endothelial dysfunction in preterm-born individuals are not fully understood. Endothelial progenitor cells (EPCs) ensure vascular integrity, normal endothelial function and neovascularization. No studies have investigated the EPCs counts in peripheral blood beyond infancy in children born prematurely. Circulating EPCs were significantly higher in preterm-born prepubertal children compared to controls, thus indicating that prematurity is possibly associated with endothelial damage. In total study population and in the preterm-born group, maternal gestational diabetes was associated with decreased EPCs concentrations.

Circulating tumor cell investigation in breast cancer patient-derived xenograft models by automated immunofluorescence staining, image acquisition, and single cell retrieval and analysis.

BACKGROUND: Breast cancer patient-derived xenograft (BC-PDX) models represent a continuous and reproducible source of circulating tumor cells (CTCs) for studying their role in tumor biology and metastasis. We have previously shown the utility of BC-PDX models in the study of CTCs by immunohistochemistry (IHC) on serial paraffin sections and manual microscopic identification of cytokeratin-positive cells, a method that is both low-throughput and labor-intensive. We therefore aimed to identify and characterize CTCs from small volume mouse blood samples and examined its practical workflow in a study of BC-PDX mice treated with chemotherapy using an automated imaging platform, the AccuCyte®-CyteFinder® system. METHODS: CTC analysis was conducted using blood from non-tumor bearing SCID/Beige mice spiked with human breast cancer cells, BC-PDX-bearing mice, and BC-PDX mice treated with vehicle or chemotherapeutic agent(s). After red blood cell lysis, nucleated cells were mixed with transfer solution, processed onto microscope slides, and stained by immunofluorescence. The CyteFinder automated scanning microscope was used to identify CTCs, defined as nucleated cells that were human cytokeratin-positive, and mouse CD45-negative. Disaggregated primary BC-PDX tumors and lung metastatic nodules were processed using the same immunostaining protocol. Collective expression of breast cancer cell surface markers (EpCAM, EGFR, and HER2) using a cocktail of target-specific antibodies was assessed. CTCs and disaggregated tumor cells were individually retrieved from slides using the CytePicker® module for sequence analysis of a BC-PDX tumor-specific PIK3CA mutation. RESULTS: The recovery rate of human cancer cells spiked into murine blood was 83 ± 12%. CTC detection was not significantly different from the IHC method. One-third of CTCs did not stain positive for cell surface markers. A PIK3CA T1035A mutation present in a BC-PDX tumor was confirmed in isolated single CTCs and cells from dissociated metastatic nodules after whole genome amplification and sequencing. CTC evaluation could be simply implemented into a preclinical PDX therapeutic study setting with substantial improvements in workflow over the IHC method. CONCLUSIONS: Analysis of small volume blood samples from BC-PDX-bearing mice using the AccuCyte-CyteFinder system allows investigation of the role of CTCs in tumor biology and metastasis independent of surface marker expression.

Association of the early absolute CD64-expressing neutrophil count and sepsis outcome.

To evaluate the early absolute CD64/CD15/CD45 neutrophil count as a marker of prognosis of sepsis outcome the absolute CD64/CD15/CD45 count was measured by flow cytometry in 65 patients with confirmed or suspected Gram-negative sepsis and organ dysfunction. Serum interleukin(IL)-8 and interferon-gamma (IFNγ) were measured by an enzyme immunoassay. An absolute count lower than 2500 cells/mm3 could early discriminate non-survivors with sensitivity 82.9% (OR 3.46, 95%CIs 1.10-10.95, p 0.042). After forward step-wise Cox- regression analysis, it was found that acute coagulopathy, acute renal injury, and an early absolute CD64/CD15/CD45 count lower than 2500/mm3 were independently associated with unfavorable outcome. The OR for death among patients with an absolute CD64/CD15/CD45 neutrophil count greater than 2500/mm3 and circulating IL-8 greater than 95 pg/ml was 0.44; this was significantly increased to 7.44 among patients with an absolute CD64/CD15/CD45 neutrophil count lower than 2500/mm3 (p 0.045 by the Breslow-Day's test; p 0.046 by the Tarone's test). An absolute CD64/CD15/CD45 count below 2500/mm3 can be a useful prognosticator of sepsis outcome and a probable indicator of sepsis immunosuppression.

Mediterranean, but not lacto-ovo-vegetarian, diet positively influence circulating progenitor cells for cardiovascular prevention: The CARDIVEG study.

AIM: To evaluate the possible association between dietary habits and progenitor cells using data obtained from a randomized crossover trial using two different diets, lacto-ovo-vegetarian (VD) and Mediterranean (MD), the CARDIVEG study. METHODS AND RESULTS: Eighty clinically healthy subjects with a low-to-moderate cardiovascular risk profile (61 F; 19 M; mean age: 50.7 ± 11.6 years) were randomly assigned to isocaloric VD and MD diets lasting three months each, and then crossed. The two diets showed no effects on endothelial progenitor cells and circulating endothelial cells but opposite effects on circulating progenitor cells. In fact, VD determined significant (p < 0.05) and negative changes on circulating progenitor cells, with an average geometric variation of -130 cells/106 events for CD34+/CD45-/dim, -80 cells/106 events for CD133+/CD45-/dim, and -84 cells/106 events for CD34+/CD133+/CD45-/dim while MD determined significant (p < 0.05) and positive changes for CD34+/CD45-/dim levels, with a geometric mean increase of +54 cells/106 events. No significant correlations were observed between changes in progenitor cells and changes in inflammatory parameters during the VD phase. On the other hand, during the MD phase negative correlations between changes of CD34+/CD45-/dim and interleukin-6 (R = -0.324; p = 0.004) as well as interleukin-8 (R = -0.228; p = 0.04) and monocyte chemotactic protein-1 (R = -0.277; p = 0.01), were observed. These correlations remained significant also after adjustment for confounding factors only for CD34+/CD45-/dim and interleukin-6 (ß = -0.282; p = 0.018) and monocyte chemotactic protein-1 (ß = -0.254; p = 0.031). CONCLUSIONS: MD, but not VD, reported a significant and positive effect on circulating progenitor cells in a group of subjects at low-to-moderate cardiovascular risk, probably acting through the modulation of inflammatory parameters.

Group 2 innate lymphoid cells exhibit progressively higher levels of activation during worsening of liver fibrosis.

INTRODUCTION: The interleukin-33/interleukin-13 pathway is involved in the immunopathology of liver fibrosis and recently characterized group 2 innate lymphoid cells (ILC2) were identified as profibrotic immune cells in the liver of mouse models. Our aim was to elucidate whether ILC2 might be present in human liver tissue and whether ILC2 contribute to liver fibrosis. MATERIALS AND METHODS: To identify ILC2 in liver tissue and blood, we purified mononuclear immune cells from needle biopsies, cirrhotic explant specimen, and paired peripheral blood samples. Cell suspensions were incubated with specific markers for ILC2 and analyzed by flow cytometry. The CD69 marker was included to assess the activation level of ILC2. In addition, we determined the IL-33 plasma level. RESULTS: Results were correlated with the METAVIR fibrotic score of patients enrolled in this study. We detected ILC2 in a higher percentage of CD45+ cells in liver tissue than in paired peripheral blood. The number of ILC2 was significantly increased in fibrotic tissue, but only slightly increased in paired peripheral blood. A higher percentage of CD69+ ILC2 was observed in fibrotic tissue, and this increase correlates positively with aggravation of liver fibrosis measured by fibrotic METAVIR score. A higher level of plasma IL-33 was only detected in samples obtained from cirrhotic patients. CONCLUSION: Our study indicates that ILC2 are present in the human liver and are activated in tissue contributing to the immunopathology of human liver fibrosis, independently of the etiology; which might be a potential new therapeutic target.

Effect of hemodialysis on extracellular vesicles and circulating submicron particles.

BACKGROUND: Although hemodialysis is a highly effective treatment for diffusive clearance of low molecular weight uremic toxins, its effect on circulating extracellular vesicles and submicron particles is less clear. The purpose of this study was to examine the impact of hemodialysis on circulating levels of submicron particles. METHODS: Plasma samples from patients were collected immediately before and after the mid-week hemodialysis session. Total submicron particles were assessed by nanoparticle tracking analysis and levels of endothelial (CD144+), platelet (CD41+), leukocyte (CD45+), and total (Annexin V+) membrane microparticles (MPs) were assessed by flow cytometry. RESULTS: Total submicron particle number was significantly lower post-dialysis with reductions in particles < 40 nm, 40-100 nm, and 100-1000 nm in size. Circulating annexin V+ MPs, platelet MPs, leukocyte MPs, and endothelial MPs were all reduced following dialysis. Assessment of protein markers suggested that extracellular vesicles were not present in the dialysate, but rather adsorbed to the dialysis membrane. CONCLUSIONS: In summary, hemodialysis is associated with reductions in circulating submicron particles including membrane MPs. Accordingly, there may be significant interdialytic variation in circulating submicron particles. Investigators interested in measuring extracellular vesicles in patients undergoing hemodialysis should therefore carefully consider the timing of biosampling.

Molecular profiling of single circulating tumor cells from lung cancer patients.

Circulating tumor cells (CTCs) are established cancer biomarkers for the "liquid biopsy" of tumors. Molecular analysis of single CTCs, which recapitulate primary and metastatic tumor biology, remains challenging because current platforms have limited throughput, are expensive, and are not easily translatable to the clinic. Here, we report a massively parallel, multigene-profiling nanoplatform to compartmentalize and analyze hundreds of single CTCs. After high-efficiency magnetic collection of CTC from blood, a single-cell nanowell array performs CTC mutation profiling using modular gene panels. Using this approach, we demonstrated multigene expression profiling of individual CTCs from non-small-cell lung cancer (NSCLC) patients with remarkable sensitivity. Thus, we report a high-throughput, multiplexed strategy for single-cell mutation profiling of individual lung cancer CTCs toward minimally invasive cancer therapy prediction and disease monitoring.

Prognostic impact of circulating tumor cells in patients with ampullary cancer.

Circulating tumor cells (CTCs) are an important topic of investigation for both basic and clinical cancer research. In this prospective study, we evaluated the clinical role of CTCs in ampullary cancer. We analyzed blood samples from 62 consecutively diagnosed patients with ampullary adenocarcinoma and 24 healthy controls for their CTC content. Combined data from immunostaining of CD45, 4',6-diamidino-2-phenylindole (DAPI), and fluorescence in situ hybridization with a chromosome 8 centromere (CEP8) probe were used to identify CTCs; cells that were CD45-/DAPI+/CEP8>2 were considered CTCs. The Cox proportional hazards model was used to assess the relationship between CTCs, clinical characteristics, and patient outcomes. We detected ≥2 CTCs/3.2 ml whole blood in 43 of 62 patients (69.4%), as well as ≥5 CTCs/3.2 ml in 16 of these patients (25.8%). A CTC cutoff value of 2 cells/3.2 ml achieved 69.4% sensitivity and 95.8% specificity as a diagnostic tool; CTCs were associated with tumor burden. CTC levels ≥3/3.2 ml (hazard ratio [HR]: 2.5, 95% confidence interval [CI]: (1.2-5.2), p = 0.014) and ≥5/3.2 ml (HR: 3.5, 95% CI: 1.7-7.3, p < 0.001) were both associated with shorter disease-free survival. Moreover, ≥3 CTCs/3.2 ml (HR: 2.7, 95% CI: 1.2-6.3, p = 0.019) and ≥5 CTCs/3.2 ml (HR: 3.8, 95% CI: 1.8-8.5, p < 0.001) were predictive of shorter overall survival. CTC assessment may help identify patients with ampullary cancer who are at high risk of an unfavorable outcome.

Regulatory T-cells in acute dengue viral infection.

Although regulatory T-cells (Tregs ) have been shown to be expanded in acute dengue, their role in pathogenesis and their relationship to clinical disease severity and extent of viraemia have not been fully evaluated. The frequency of Tregs was assessed in 56 adult patients with acute dengue by determining the proportion of forkhead box protein 3 (FoxP3) expressing CD4+  CD25+ T-cells (FoxP3+ cells). Dengue virus (DENV) viral loads were measured by quantitative real-time polymerase chain reaction (PCR) and DENV-specific T-cell responses were measured by ex-vivo interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) assays to overlapping peptide pools of DENV-NS3, NS1 and NS5. CD45RA and CCR4 were used to phenotype different subsets of T-cells and their suppressive potential was assessed by their expression of cytotoxic T lymphocyte-antigen 4 (CTLA-4) and Fas. While the frequency of FoxP3+  cells in patients was significantly higher (P < 0·0001) when compared to healthy individuals, they did not show any relationship with clinical disease severity or the degree of viraemia. The frequency of FoxP3+  cells did not correlate with either ex-vivo IFN-γ DENV-NS3-, NS5- or NS1-specific T-cell responses. FoxP3+  cells of patients with acute dengue were predominantly CD45RA+ FoxP3low , followed by CD45RA-FoxP3low , with only a small proportion of FoxP3+  cells being of the highly suppressive effector Treg subtype. Expression of CCR4 was also low in the majority of T-cells, with only CCR4 only being expressed at high levels in the effector Treg population. Therefore, although FoxP3+  cells are expanded in acute dengue, they predominantly consist of naive Tregs , with poor suppressive capacity.

The soluble cytoplasmic tail of CD45 (ct-CD45) in human plasma contributes to keep T cells in a quiescent state.

The cytoplasmic tail of CD45 (ct-CD45) is proteolytically cleaved and released upon activation of human phagocytes. It acts on T cells as an inhibitory, cytokine-like factor in vitro. Here, we show that ct-CD45 is abundant in human peripheral blood plasma from healthy adults compared with plasma derived from umbilical cord blood and plasma from patients with rheumatoid arthritis or systemic lupus erythematosus. Plasma depleted of ct-CD45 enhanced T-cell proliferation, while addition of exogenous ct-CD45 protein inhibited proliferation and reduced cytokine production of human T lymphocytes in response to TCR signaling. Inhibition of T-cell proliferation by ct-CD45 was overcome by costimulation via CD28. T-cell activation in the presence of ct-CD45 was associated with an upregulation of the quiescence factors Schlafen family member 12 (SLFN12) and Krueppel-like factor 2 (KLF2) as well as of the cyclin-dependent kinase (CDK) inhibitor p27kip1. In contrast, positive regulators of the cell cycle such as cyclin D2 and D3 as well as CDK2 and CDK4 were found to be downregulated in response to ct-CD45. In summary, we demonstrate that ct-CD45 is present in human plasma and sets the threshold of T-cell activation.

Associations between chronic caregiving stress and T cell markers implicated in immunosenescence.

Chronic psychological stress is associated with accelerated biological aging, immune dysfunction, and premature morbidity and mortality. Changes in the relative proportions of T cell subpopulations are thought to be a characteristic of immunological aging; however, understanding of whether these changes are associated with chronic psychological stress is incomplete. This study investigated associations between chronic caregiving stress and distributions of T cell phenotypes in a sample of high stress mothers of children with Autism Spectrum Disorder (caregivers; n = 91) and low stress mothers of neurotypical children (controls; n = 88). Immune markers assessed were naïve (CD45RA + CD62L+), central memory (CD45RA-CD62L+), and effector memory (CD45RA-CD62L-) CD4+ and CD8+ T cells. We also examined the ratio of effector to naïve (E:N) CD4+ and CD8+ T cells. In models adjusted for age, body mass index, race/ethnicity, and antidepressant use, caregivers displayed higher percentages of effector memory CD8+ and CD4+ T cells as well as lower percentages of naïve CD8+ T cells and central memory CD8+ and CD4+ T cells compared to controls. Caregivers also displayed significantly higher E:N ratios for both CD4+ and CD8+ T cells. These findings were also independent of cytomegalovirus infection status. Furthermore, higher parental stress, across both groups, was related to several immune parameters. These findings provide preliminary evidence that chronic parental caregiving stress is associated with changes in relative proportions of T cell subpopulations that are consistent with accelerated immunological aging.

Humanized NOG mice as a model for tuberculosis vaccine-induced immunity: a comparative analysis with the mouse and guinea pig models of tuberculosis.

The humanized mouse model has been developed as a model to identify and characterize human immune responses to human pathogens and has been used to better identify vaccine candidates. In the current studies, the humanized mouse was used to determine the ability of a vaccine to affect the immune response to infection with Mycobacterium tuberculosis. Both human CD4+ and CD8+ T cells responded to infection in humanized mice as a result of infection. In humanized mice vaccinated with either BCG or with CpG-C, a liposome-based formulation containing the M. tuberculosis antigen ESAT-6, both CD4 and CD8 T cells secreted cytokines that are known to be required for induction of protective immunity. In comparison to the C57BL/6 mouse model and Hartley guinea pig model of tuberculosis, data obtained from humanized mice complemented the data observed in the former models and provided further evidence that a vaccine can induce a human T-cell response. Humanized mice provide a crucial pre-clinical platform for evaluating human T-cell immune responses in vaccine development against M. tuberculosis.

The influence of temperature treatment before cryopreservation on the viability and potency of cryopreserved and thawed CD34+ and CD45+ cord blood cells.

BACKGROUND: Hematopoietic stem cell (HSC) viability and potency is crucial for qualified cord blood (CB) transplants. This study analyzes time and temperature condition before cryopreservation for the viability of CD34+/CD45+ cells after cryopreservation. METHODS: Cell viabilities were determined by antibody co-staining with 7-aminoactinomycin D detecting necrotic cells, and subsequent flow cytometric analysis. Additionally, Annexin V staining for determination of apoptotic cells and colony-forming unit (CFU) assays for testing functional potency of HSCs were performed. RESULTS: For all cell types assessed (CD45+/CD34+ cells, lymphocytes and granulocytes), the highest viabilities were obtained for CB maintained at 4°C or room temperature (RT; 22 ± 4°C) and cryopreserved directly after collection. Starting material were CB units with an age of 24.7 ± 3.5 h after birth. Post-thaw CD34+ cell results were > 90% after temperature treatment of t = 24 h (48 h total age) and > 70% after t = 48 h (72 h total age) at 4°C (48 h, 91.4 ± 5.5%; 72 h, 75.0 ± 12.0%) and RT (48 h, 84.2 ± 9.7%; 72 h, 72.6 ± 0.6%). Viabilities for 30°C samples were < 80% after t = 24 h (48 h total age, 79.8 ± 3.1%) and < 50% after t = 48 h of treatment (72 h total age, 46.8 ± 14.3%). Regarding CFU recovery of pre-freeze (without volume reduction) and thawed CB, a trend toward the highest recoveries was observed at 4°C/RT. The difference between 4°C (77.5 ± 12.0%) and 30°C samples (53.9 ± 4.8%) was shown to be significant in post-thaw samples after t = 24 h treatment (48 h total age; P = 0.0341). DISCUSSION: Delays between collection and cryopreservation should be minimized because increasing time reduces numbers of viable cells and CFUs before/after cryopreservation. CB units should be maintained at 4°C/RT to retain the highest possible potency of the cells after thawing.

Value of CD16/CD66b/CD45 in comparison to CD55/CD59/CD45 in diagnosis of paroxysmal nocturnal haemoglobinuria: An Indian experience.

BACKGROUND & OBJECTIVES: Diagnosis of paroxysmal nocturnal haemoglobinuria (PNH), a rare haematopoietic stem cell disorder, is challenging in patients with bone marrow failure (BMF) syndrome like aplastic anaemia (AA). This study was conducted with the aim to test the efficacy of the newly recommended markers viz. anti-CD16 and CD66b antibody over the existing anti-CD55 and CD59 antibody for PNH diagnosis in India. METHODS: This study was conducted on 193 suspected cases of PNH by flow cytometry using lyse wash technique to stain the granulocytes with CD16/CD66b and CD55/CD59. RESULTS: Of the 193 suspected cases, 62 patients showed the presence of PNH clone. Forty six patients were detected by CD55/CD59/CD45, whereas 61 were detected by CD16/CD66b/CD45. CD16/CD66b detected 16 (25.8%) additional patients over CD55/CD59 (P<0.05) and was more sensitive in detecting the PNH clone with higher negative predictive value. Most of the patients (11/16) who were picked up by CD16/CD66b were of AA who had small clone sizes. Further, the PNH clones were more discreetly identified in CD16/CD66b plots than by CD55/CD59. Clone size assessed by CD16/CD66b which reflects the clinical severity of classical PNH (thrombosis/haemolysis), was more representative of the underlying clinical condition than CD55/59. INTERPRETATION & CONCLUSIONS: In our experience of 62 patients of PNH, CD16/CD66b proved to be more efficacious in detecting PNH. The new panel was especially useful in monitoring PNH associated with BMF which had small clone sizes.

Circulating Tumor Cell Phenotype Predicts Recurrence and Survival in Pancreatic Adenocarcinoma.

OBJECTIVE: We assessed circulating tumor cells (CTCs) with epithelial and mesenchymal phenotypes as a potential prognostic biomarker for patients with pancreatic adenocarcinoma (PDAC). BACKGROUND: PDAC is the fourth leading cause of cancer death in the United States. There is an urgent need to develop biomarkers that predict patient prognosis and allow for better treatment stratification. METHODS: Peripheral and portal blood samples were obtained from 50 patients with PDAC before surgical resection and filtered using the Isolation by Size of Epithelial Tumor cells method. CTCs were identified by immunofluorescence using commercially available antibodies to cytokeratin, vimentin, and CD45. RESULTS: Thirty-nine patients (78%) had epithelial CTCs that expressed cytokeratin but not CD45. Twenty-six (67%) of the 39 patients had CTCs which also expressed vimentin, a mesenchymal marker. No patients had cytokeratin-negative and vimentin-positive CTCs. The presence of cytokeratin-positive CTCs (P < 0.01), but not mesenchymal-like CTCs (P = 0.39), was associated with poorer survival. The presence of cytokeratin-positive CTCs remained a significant independent predictor of survival by multivariable analysis after accounting for other prognostic factors (P < 0.01). The detection of CTCs expressing both vimentin and cytokeratin was predictive of recurrence (P = 0.01). Among patients with cancer recurrence, those with vimentin-positive and cytokeratin-expressing CTCs had decreased median time to recurrence compared with patients without CTCs (P = 0.02). CONCLUSIONS: CTCs are an exciting potential strategy for understanding the biology of metastases, and provide prognostic utility for PDAC patients. CTCs exist as heterogeneous populations, and assessment should include phenotypic identification tailored to characterize cells based on epithelial and mesenchymal markers.

Acquisition of Portal Venous Circulating Tumor Cells From Patients With Pancreaticobiliary Cancers by Endoscopic Ultrasound.

BACKGROUND & AIMS: Tumor cells circulate in low numbers in peripheral blood; their detection is used predominantly in metastatic disease. We evaluated the feasibility and safety of sampling portal venous blood via endoscopic ultrasound (EUS) to count portal venous circulating tumor cells (CTCs), compared with paired peripheral CTCs, in patients with pancreaticobiliary cancers (PBCs). METHODS: In a single-center cohort study, we evaluated 18 patients with suspected PBCs. Under EUS guidance, a 19-gauge EUS fine needle was advanced transhepatically into the portal vein and as many as four 7.5-mL aliquots of blood were aspirated. Paired peripheral blood samples were obtained. Epithelial-derived CTCs were sorted magnetically based on expression of epithelial cell adhesion molecules; only those with a proper morphology and found to be CD45 negative and positive for cytokeratins 8, 18, and/or 19 and 4',6-diamidino-2-phenylindole were considered to be CTCs. For 5 samples, CTCs also were isolated by flow cytometry and based on CD45 depletion. ImageStream was used to determine the relative protein levels of P16, SMAD4, and P53. DNA was extracted from CTCs for sequencing of select KRAS codons. RESULTS: There were no complications from portal vein blood acquisition. We detected CTCs in portal vein samples from all 18 patients (100%) vs peripheral blood samples from only 4 patients (22.2%). Patients with confirmed PBCs had a mean of 118.4 ± 36.8 CTCs/7.5 mL portal vein blood, compared with a mean of 0.8 ± 0.4 CTCs/7.5 mL peripheral blood (P < .01). The 9 patients with nonmetastatic, resectable, or borderline-resectable PBCs had a mean of 83.2 CTCs/7.5 mL portal vein blood (median, 62.0 CTCs/7.5 mL portal vein blood). In a selected patient, portal vein CTCs were found to carry the same mutations as those detected in a metastatic lymph node and expressed similar levels of P16, SMAD4, and P53 proteins. CONCLUSIONS: It is feasible and safe to collect portal venous blood from patients undergoing EUS. We identified CTCs in all portal vein blood samples from patients with PBCs, but less than 25% of peripheral blood samples. Portal vein CTCs can be used for molecular characterization of PBCs and share features of metastatic tissue. This technique might be used to study the pathogenesis and progression of PBCs, as well as a diagnostic or prognostic tool to stratify risk of cancer recurrence or developing metastases.

The Effects of Different Surgical Approaches on the Perioperative Level of Circulating Tumor Cells in Patients with Non-Small Cell Lung Cancer.

Background To observe and compare the effects of video-assisted thoracoscopic surgery (VATS) and conventional thoracotomy on the levels of circulating tumor cells (CTCs) in patients with non-small cell lung carcinoma (NSCLC). Methods Seventy-nine patients with a diagnosis of NSCLC were enrolled in the study. Forty-three were treated with VATS and 36 were treated with conventional thoracotomy. Blood samples were collected 3 days prior to surgery (d-3), during surgery (d0), and 3 days after surgery (d3). After epithelial cell adhesion molecule (EpCAM)-labeled immunomagnetic cell enrichment, anti-CK-PE and anti-CD45-FITC fluorescent-labeled monoclonal antibodies were added to sort CTCs. Quantification of CTCs was performed using multiparameter flow cytometry. Results The number of CTCs on d0 was significantly higher than on d-3 (5.730 ± 4.266 vs. 4.142 ± 3.971, p = 0.033) in both groups. There was no significant difference in the change of CTCs from before surgery to during surgery in the VATS and conventional thoracotomy (open) groups (1.363 ± 2.924 vs. 1.500 ± 2.315, p = 0.329). However, the increase in number of CTCs from before surgery to after surgery was significantly lower in the VATS group than in the conventional thoracotomy (open) group (2.181 ± 2.962 vs. 9.666 ± 15.641, p = 0.015). Thirty of the 79 patients tested positive for CTCs before surgery (37.97%). All benign lung disease patients and volunteers tested negative for CTCs. Conclusion A smaller increase in CTCs was seen in patients treated with VATS lobectomy than in patients treated with conventional thoracotomy. This reduction in number of postoperative CTCs may improve long-term survival.