A rare case of extremely high counts of circulating tumor cells detected in a patient with an oral squamous cell carcinoma.

BACKGROUND: Despite aggressive regimens, the clinical outcome of head and neck squamous cell carcinoma remains poor. The detection of circulating tumor cells could potentially improve the management of patients with disseminated cancer, including diagnosis, treatment strategies, and surveillance. Currently, CellSearch(®) is the most widely used and the only Food and Drug Administration-cleared system for circulating tumor cells detection in patients with metastatic breast, colorectal, or prostate cancer. In most cases of head and neck squamous cell carcinoma, only low counts of circulating tumor cells have been reported. CASE PRESENTATION: A 56-year-old white male with no particular medical history, was diagnosed with a squamous cell carcinoma of oral cavity. According to the imaging results (computed tomography and (18)F-fluorodeoxyglucose positron emission tomography / computed tomography) and panendoscopy, the TNM staging was classified as T4N2M0. A non-interruptive pelvimandibulectomy was conducted according to the multidisciplinary meeting advices and the postoperative observations were normal. The patient complained of a painful cervical edema and a trismus 6 weeks after the surgery. A relapse was found by computed tomography and the patient died two weeks later. The search for circulating tumor cells in peripheral venous blood by using the CellSearch(®) system revealed a very high count compared with published reports at three time points (pre-operative: 400; intra-operative: 150 and post-operative day 7: 1400 circulating tumor cells). Of note, all detected circulating tumor cells were epidermal growth factor receptor negative. CONCLUSION: We report here for the first time a rare case of oral squamous cell carcinoma with extremely high circulating tumor cells counts using the CellSearch(®) system. The absolute number of circulating tumor cells might predict a particular phase of cancer development as well as a poor survival, potentially contributing to a personalized healthcare.

Diagnostic and Prognostic Value of Circulating Tumor Cells in Head and Neck Squamous Cell Carcinoma: a systematic review and meta-analysis.

Several techniques have been developed to detect circulating tumor cells (CTC) in patients with head and neck squamous cell carcinoma (HNSCC), but their diagnostic and prognostic value are not yet fully established. A computerized retrieval of literatures was conducted without time restrictions using the electronic database in December 2014. Diagnostic accuracy variables were pooled and analyzed by the Meta-DiSc software. Engauge Digitizer and Stata software were used for pooled survival analysis. Twenty-two retrieved studies were eligible for systematic review, of which 9 conformed for the diagnostic test meta-analysis and 5 for the prognostic analysis. Subgroup analysis showed 24.6% pooled sensitivity and 100% pooled specificity of detections by using positive selection strategy, which moreover presented low heterogeneity. The presence of CTC was significantly associated with shorter disease free survival (DFS, HR 4.62, 95% CI 2.51-8.52). In conclusion, current evidence identifies the CTC detection assay as an extremely specific, but low sensitive test in HNSCC. Also, the presence of CTC indicates a worse DFS.

CellSearch technology applied to the detection and quantification of tumor cells in CSF of patients with lung cancer leptomeningeal metastasis.

OBJECTIVES: The diagnosis of solid cancer leptomeningeal metastasis (LM) relies on the cytology of cerebrospinal fluid (CSF) and/or imaging evidence of neuraxis, yet both lack sufficient sensitivity. The utility of the CellSearch, an FDA -approved technology, in assessing CSF tumor cell (CSFTC) was evaluated here in the diagnosis and treatment of patients with lung cancer-related LM. MATERIALS AND METHODS: In 18 patients with magnetic resonance imaging (MRI) confirmed LM due to lung cancer, 5 mL of CSF were collected in CellSave preservative tubes, which allow performing the assay within 96 h after sampling. Using a previously adapted CellSearch method, we detected, visualized and enumerated CSFTCs and compared the results with conventional cytology. In 3 patients, tumor cells were evaluated sequentially to explore the predictive role of CSFTCs enumeration in the treatment response monitoring. RESULTS: CSFTCs were disclosed in 14 of 18 MRI confirmed LM samples (median 785CSFTCs/5 mL CSF, range 1 to >20,000), yielding a sensitivity of 77.8%, compared with 44.4% for conventional cytology. CSFTC clusters were observed in 12 patients, similar to those previously described in blood as circulating tumor microemboli (CTM), and enumerated sequentially with reproducible results, which did not necessarily correlate with response to treatment. CONCLUSION: The CellSearch technology, applied to limited sample volumes and allowing delayed processing, could be of great interest in the diagnosis of LM in lung cancer patients.

Case Report: Detection and quantification of tumor cells in peripheral blood and ascitic fluid from a metastatic esophageal cancer patient using the CellSearch (®) technology.

Analysis of ascitic fluid should help to identify and characterize malignant cells in gastrointestinal cancer. However, despite a high specificity, the sensitivity of traditional ascitic fluid cytology remains insufficient, at around 60%. Since 2004 the CellSearch (®) technology has shown its advantages in the detection of circulating tumor cells (CTCs) in peripheral blood, which can perform an accurate diagnosis and molecular analysis at the same time. To our knowledge, no previous study has explored the potential utility of this technology for the detection and quantification of tumor cells in ascitic fluid samples. Herein we report a case of metastatic esophageal adenocarcinoma in a 70-year-old man presenting with dysphagia and a large amount of fluid in the peritoneal cavity. Analysis of a peripheral blood sample and ascites sample with the CellSearch (®) technology both revealed the presence of putative tumor cells that were positive for epithelial cell adhesion molecule (EpCAM) and cytokeratin (CK) expression. This study confirmed the hematogenous dissemination of esophageal cancer by the detection of circulating tumor cells in the peripheral blood, and is the first to demonstrate that tumor cells can be identified in ascitic fluid by using CellSearch (®) technology.

Value of HIV patients with regular follow-up as in-house internal controls of flow cytometry measurement of lymphocyte subsets.

BACKGROUND: Quality assessment in flow cytometry cannot obey the same rules as those applicable to the measurement of chemical analytes. However, regular follow-up of known patients may provide a robust in-house control of cell subsets evaluation. METHODS: Sequential blood samples assessed for 32 HIV patients over several years and showing good stability were retrospectively assessed to establish coefficient of variations of the percentages of CD3+, CD4+, CD8+ cells, and CD4+ absolute counts (ACs). RESULTS: Mean relative standard variations for the whole cohort were of 0.04, 0.14, 0.08, and 0.18 for CD3%, CD4%, CD8%, and CD4 ACs, respectively. DISCUSSION: In-house follow-up of regularly checked compliant patients is a good alternative to traditional and costly repeatability and reproducibility studies for the validation of routine flow cytometry. © 2013 International Clinical Cytometry Society.

Detection and quantification of CSF malignant cells by the CellSearch technology in patients with melanoma leptomeningeal metastasis.

Melanoma is the most frequent solid tumor associated with leptomeningeal metastasis (LM). The usual diagnostic tools, that is, cytomorphological assessment of cerebro-spinal fluid (CSF) and gadolinium-enhanced MRI of the entire neuraxis both lack effectiveness. The CellSearch Veridex technology for the detection of circulating tumor cells (CTC) in blood was designed for the follow-up and prognosis of breast, prostate, colorectal, and lung cancer, which express EpCAM markers. We have previously adapted this technology to detect malignant cells in the CSF of breast cancer LM. Our objective here was to check if this technology would also allow the detection and the enumeration of CTC in the CSF of melanoma patients presenting with LM although melanoma does not express EpCAM markers. On the occasion of the intrathecal treatment of LM in 2 melanoma patients, 5 mL of CSF and 7.5 mL of blood were collected on CellSave Preservative Tubes and analyzed within 3 days after CSF sampling using a melanoma-dedicated kit. The CellSearch Veridex technology was then adapted to direct enrichment, enumeration, and visualization of melanoma cells in the CSF. CD146+, HMW-MAA+, CD34-, and CD45- cells with typical morphology could be observed and enumerated sequentially with reproducible results, corresponding to CSF melanoma cells (CSFMC). In contrast to the current gold standard cytomorphological analysis, this new approach allowed a precise quantification of CSFMC in all samples concomitantly analyzed. This methodology, established on a limited volume of sample and allowing delayed processing, could prove of great interest in the diagnosis and follow-up of melanoma patients with LM.

Development of a new method for identification and quantification in cerebrospinal fluid of malignant cells from breast carcinoma leptomeningeal metastasis.

BACKGROUND: The diagnosis of leptomeningeal metastasis (LM) in patients with solid tumors remains difficult. The usual diagnostic methods of cytomorphological assessment of cerebro-spinal fluid (CSF) and gadolinium enhanced MRI of the entire neuraxis lack both specificity and sensitivity. The Veridex CellSearch® technology has been designed for the detection of circulating tumor cells (CTC) in blood from cancer patients and validated for the follow-up and prognosis of breast, prostate, colorectal, and lung cancer. Our aim was to adapt this technology for the detection and the enumeration of tumor cells in the CSF of breast cancer patients presenting with LM. METHODS: On the occasion of a randomized phase III study evaluating the role of the intrathecal treatment in LM from breast cancer (DEPOSEIN, EudraCT N°: 2010-023134-23), the CellSearch® technology was adapted to direct enrichment, enumeration and visualization of tumor cells in 5 mL CSF samples, collected on CellSave® Preservative Tubes and analyzed within 3 days after CSF sampling. RESULTS: Sixteen CSF of 8 patients with primary breast cancer presenting with LM were studied. EpCAM+/cytokeratin + cells with typical morphology could be observed and enumerated sequentially with reproducible results in low or elevated numbers in 8 patients. CONCLUSION: This methodology, established on a limited volume of sample and allowing delayed processing, could prove of great interest in the diagnosis and follow-up of cancer patients with LM, especially to appreciate the efficacy of chemotherapy.

Maternal exposure to air pollution before and during pregnancy related to changes in newborn's cord blood lymphocyte subpopulations. The EDEN study cohort.

BACKGROUND: Toxicants can cross the placenta and expose the developing fetus to chemical contamination leading to possible adverse health effects, by potentially inducing alterations in immune competence. Our aim was to investigate the impacts of maternal exposure to air pollution before and during pregnancy on newborn's immune system. METHODS: Exposure to background particulate matter less than 10 µm in diameter (PM10) and nitrogen dioxide (NO2) was assessed in 370 women three months before and during pregnancy using monitoring stations. Personal exposure to four volatile organic compounds (VOCs) was measured in a subsample of 56 non-smoking women with a diffusive air sampler during the second trimester of pregnancy. Cord blood was analyzed at birth by multi-parameter flow cytometry to determine lymphocyte subsets. RESULTS: Among other immunophenotypic changes in cord blood, decreases in the CD4+CD25+ T-cell percentage of 0.82% (p = 0.01), 0.71% (p = 0.04), 0.88% (p = 0.02), and 0.59% (p = 0.04) for a 10 µg/m3 increase in PM10 levels three months before and during the first, second and third trimester of pregnancy, respectively, were observed after adjusting for confounders. A similar decrease in CD4+CD25+ T-cell percentage was observed in association with personal exposure to benzene. A similar trend was observed between NO2 exposure and CD4+CD25+ T-cell percentage; however the association was stronger between NO2 exposure and an increased percentage of CD8+ T-cells. CONCLUSIONS: These data suggest that maternal exposure to air pollution before and during pregnancy may alter the immune competence in offspring thus increasing the child's risk of developing health conditions later in life, including asthma and allergies.

Mutual benefits of B-ALL and HLDA/HCDM HLDA 9th Barcelona 2010.

The B-cell panel of the ninth HLDA was applied in a multicentre fashion to cryopreserved cells from 46 patients with acute lymphoblastic leukemia. The reagents were aliquoted and shipped to volunteer participants from the French Groupe d'Etude Immunologique des Leucémies (GEIL). All samples were tested in flow cytometry, and the results collected as of the strength of labeling of the leukemic clone as negative, weak or strong. Among the 64 antibodies tested, the strongest and most frequent staining was observed for CD305 (LAIR), CD229 (Ly9), CD200 (OX-2) and, to a lesser extent, CD361 (EVI2b). Details of the observations, and information about the molecules tested are provided in the manuscript as well as a summary table.

Local lymphocytic and epithelial activation in a case of autoimmune oophoritis.

OBJECTIVE: To further define the immunological tissular modifications in premature ovarian failure (POF). METHOD: The patient was followed up for premature ovarian failure and mild endometriosis associated with serum antiovarian antibodies. A laparoscopic ovarian biopsy was decided on to analyze the tissue and support the onset of immunosuppressive therapy. Immunohistochemistry was performed using monoclonal antibodies directed against T cell membrane markers, as well as activation molecules, to define the composition of the cellular infiltrate and the consequences on ovarian tissue. RESULT(S): A dense infiltration of activated T lymphocytes was observed in close contact with follicular epithelium expressing HLA-DR and CD40. CONCLUSION(S): This observation supports the role of cellular immunity in ovarian autoimmunity with features very similar to those reported in murine models and other human autoimmune endocrine pathologies.

Expression of functional toll-like receptors by B-chronic lymphocytic leukemia cells.

This study reports that B-chronic lymphocytic leukemia (B-CLL) cells display the same pattern of toll-like receptors (TLRs) proteins expression as normal B-cells, yet with overexpression of TLR9. Furthermore, TLR7 and TLR9 appear to be functional and liable to respond to specific ligands, respectively imidazoquinolines and CpG-ODN thus potentially opening new therapeutic approaches.

Modulation of the triggering receptor expressed on myeloid cells-1 pathway during pneumonia in rats.

BACKGROUND: Triggering receptor expressed on myeloid cells-1 (TREM-1) is a cell-surface molecule that has been identified on both human and murine polymorphonuclear neutrophils and mature monocytes. The activation of TREM-1 in the presence of microbial components amplifies the inflammatory response and may be responsible for the hyperresponsiveness observed during the initial stage of sepsis. The aim of the present study was to investigate the effect of the modulation of the TREM-1 pathway during experimental pneumonia in rats. METHODS: Adult male Wistar rats were intratracheally inoculated with Pseudomonas aeruginosa (PAO1 strain) and randomly treated or not treated with an analogue synthetic peptide derived from the extracellular moiety of TREM-1 (LP17). RESULTS: P. aeruginosa induced a severe pneumonia associated with signs of severe sepsis within the first 24 h. In septic rats, LP17 improved hemodynamic status, attenuated the development of lactic acidosis and hypoxemia, modulated lung and systemic inflammatory responses and coagulation activation, reduced lung histological damage, and improved survival. CONCLUSIONS: The modulation of the TREM-1 pathway by the use of such synthetic peptides as LP17 appears beneficial during P. aeruginosa pneumonia in rats in attenuating lung and systemic inflammatory responses.

Modulation of the triggering receptor expressed on the myeloid cell type 1 pathway in murine septic shock.

The triggering receptor expressed on myeloid cell type 1 (TREM-1) is a cell surface molecule that has been identified on both human and murine polymorphonuclear neutrophils and mature monocytes. The activation of TREM-1 in the presence of microbial components amplifies the inflammatory response and may be responsible for the hyperresponsiveness observed during the initial stage of sepsis. To investigate the effect of the modulation of the TREM-1 pathway during experimental murine sepsis, we used analogue synthetic peptides derived from the extracellular moiety of TREM-1. The TREM-1 ligand was expressed on both peritoneal and peripheral neutrophils during experimental peritonitis in mice. The TREM-1 peptides inhibited the recognition by TREM-1 of its ligand and protected endotoxinic mice from death. In septic rats, the TREM-1 peptides improved the hemodynamic status, attenuated the development of lactic acidosis, modulated the production of such proinflammatory cytokines as tumor necrosis factor alpha and interleukin-1beta, and improved survival. The protective effect of these peptides on arterial pressure could partly be explained by a decreased production of nitric oxide. These data suggest that in vivo modulation of TREM-1 might be a suitable therapeutic tool for the treatment of sepsis.

Modulation of natural killer (NK) receptors on NK (CD3-/CD56+), T (CD3+/CD56-) and NKT-like (CD3+/CD56+) cells after heart transplantation.

BACKGROUND: After undergoing heart transplantation and the subsequent compulsive immunosuppressive treatments, patients are at risk of rejection episodes, infectious complications or cancer development. Thus, it is probable that the various subsets of peripheral cytotoxic lymphocytes are modulated in such patients. This area of study can now be investigated by examining the numerous recently described natural killer (NK)-cell-related surface receptors. METHODS: A prospective cohort of 60 heart transplant recipients and 60 controls was studied. The partitioning of lymphocyte subsets, especially NK (CD3-/CD56+), T (CD3+/CD56-) and NKT-like (CD3+/CD56+) cells, was compared in both groups using multi-parametric flow cytometry. Moreover, expression of a series of seven NK-related receptors was compared on the three subsets defined by CD56 expression. RESULTS: A significant increase in NK-cell levels was observed in transplanted patients, as compared with controls, whereas T and NKT-like cells were in similar proportions in both groups. Two NK-related receptors showed significantly different levels of expression in heart transplant recipients: the cytotoxic effector, CD244, which was in a significantly increased proportion on T and NKT-like cells; and the activating receptor, CD161, which was expressed significantly less on NK and NKT-like cells, but more on T cells. CONCLUSIONS: These findings indicate that cytotoxic NK-related cells, increased in proportion, also display increased levels of activity-associated markers in heart transplant recipients. Viral infection or the immunosuppressive regimen could be responsible for the modulation of regulatory receptors on NK and NKT-like cells in heart transplant recipients.

Surface and soluble triggering receptor expressed on myeloid cells-1: expression patterns in murine sepsis.

OBJECTIVE: To examine the expression patterns of the triggering receptor expressed on myeloid cells (TREM)-1 during experimental septic shock. DESIGN: Animal study. SETTING: Animal research laboratory. SUBJECTS: Male BALB/c mice, 7-9 wks of age. INTERVENTIONS: Septic shock was induced by cecal ligation and puncture in eight mice. Eight additional animals were sham-operated and served as a control group. All animals were resuscitated by fluid infusion and administered antibiotics. Kill was performed under anesthesia 12, 24, or 48 hrs later. MEASUREMENTS AND MAIN RESULTS: Surface expression of TREM-1 was analyzed using flow cytometry on peripheral blood cells, peritoneal macrophages and neutrophils, splenic macrophages, and Kupffer cells. Gene expression was also studied in these same cells using reverse transcription-polymerase chain reaction. Tumor necrosis factor-alpha, interleukin-1beta, and soluble TREM-1 concentrations were determined in plasma and peritoneal lavage fluid. Sepsis strongly induced TREM-1 gene expression, which translated into an up-regulation of TREM-1 surface expression on neutrophils and monocytes/macrophages both at the focus on infection as well as distally. Moreover, sepsis induced the release of significant levels of soluble TREM-1. Plasma soluble TREM-1 concentrations negatively correlated with tumor necrosis factor-alpha and interleukin-1beta levels at 12 hrs. CONCLUSIONS: These results provide new information as to the regulation of TREM-1 during sepsis. Considering that both cell-surface and soluble TREM-1 were strongly up-regulated during infection, this study may add support to the putative usefulness of TREM-1 as a diagnostic tool.

Comparative T-cell oligoclonality in lung, tumor and lymph nodes in human non-small cell lung cancer.

In lung cancer as in other malignancies, tumor formation induces the development of local and systemic antitumoral immune responses. The tumor itself becomes surrounded by a local stroma reaction containing inflammatory cells, a large part of which being tumor infiltrating T-lymphocytes. This study was designed to investigate the potential clonality of these T-cells in non-small cell lung cancer. Two complementary methods where used: exploration of the Vbeta TCR repertoire usage in flow cytometry and analysis of the Vgamma TCR repertoire in multiplex PCR and gradient gel electrophoresis. These techniques were applied respectively to eluted fresh lymphocytes and extracted DNA from healthy lung tissue, tumor and lymph nodes from 44 patients. There was a good correlation between the two techniques used. An oligoclonal repertoire restriction was noted in most of the cases and in the three types of tissues studied suggesting the presence of tumor-specific clones. Moreover, Vbeta14 appeared to be the most frequent specificity used whatever the tissue considered, while Vbeta13.1 appeared to be selectively used in the stroma reaction of epidermoid lung carcinomas. A restricted TCRgamma band was also present in these tumors, and two more bands of TCRgamma where selectively present in adenocarcinomas. The demonstration of both alpha-beta and gamma-delta TCR restriction suggests both the recruitment of specific T-cells and their local proliferation within the tumoral tissue. The same feature in healthy lung tissue indicates that it might already be the site of specific anti-tumoral T-cell reactivity. In conclusion, this study reports on the presence of oligoclonal T-cell responses in most cases of non-small cell lung cancer. The comparison of tumor, healthy tissue and lymph nodes showed some degree of patient-dependent similarities suggestive of tumor specificity.

Tetraspan and beta-1 integrins expression pattern of the epithelial lung adenocarcinoma cell line A549 and its sensitivity to divalent cations.

BACKGROUND: Tetraspans are ubiquitous integral transmembrane molecules associated on the cell surface with such adhesion molecules as integrins. Their expression has been shown to vary in tumors, but has seldom been described on lung tumoral epithelial cells, and the conditions required for a proper association of tetraspans and integrins have not yet been fully explored. METHODS: We investigated the expression of 10 tetraspans and six beta1 integrins on the tumoral lung epithelial cell line A549. Cells were examined both in quantitative flow cytometry and as monolayers, under normal or chelated conditions, in order to determine the cation dependency of their expression. RESULTS: Five tetraspans and four beta1 integrins are expressed on the membrane of A549 cells. Both quantitative and qualitative surface and cytoplasmic modifications of this pattern were induced in chelating conditions, suggesting the importance of divalent cations for the expression of these molecules. CONCLUSIONS: These data indicate that a specific pattern of tetraspans and integrins, relying strongly on the availability of divalent cations in the microenvironment, is expressed by tumoral epithelial cells.

Anti-HBs cellular immune response in kidney recipients before and 4 months after transplantation.

Patients with renal failure represent a population at risk for hepatitis B, since only 50 to 60% of them develop protective humoral responses after vaccination. As this could be due to an altered regulation of cellular immune responses, the objectives of the present study were to evaluate the proliferative abilities of lymphocytes from patients with chronic renal failure after stimulation in vitro with a mitogen (pokeweed mitogen [PWM]) or HBsAg. In order to differentiate between the immunodeficiency associated with renal failure and that due to immunosuppression posttransplantation, the same subjects were tested before and 4 months after kidney transplantation. The lymphoproliferation assay used was performed by flow cytometry, which is based on sequential analysis of the cell cycle and which allows analysis of cytokine production. Serologically, the group of 36 patients tested comprised 22% nonresponders, 30% poor responders, and 48% responders. Lymphocyte growth was observed for all patients after stimulation with PWM, indicating that these cells had the capacity to proliferate in vitro. The level of lymphoproliferation in response to PWM was significantly reduced after transplantation, yet both before and after transplantation, all serologic nonresponders developed cellular responses to at least two vaccines. No correlation between humoral and cellular responses was shown. Proliferating cells were lymphocytes, which mostly secreted interleukin 4 (IL-4) and IL-10 for the three serologic groups. This study suggests that even when repeated vaccination fails to induce significant antibody levels in patients with renal failure, specific HBs cellular responses develop, and these may prove to be efficient in protecting these patients against hepatitis B.