Twisting immune responses for allogeneic stem cell therapy

Stem cell-derived tissues and organs have the potential to change modern clinical science. However, rejection of allogeneic grafts by the host's immune system is an issue which needs to be addressed before embryonic stem cell-derived cells or tissues can be used as medicines. Mismatches in human leukocyte class I antigens and minor histocompatibility antigens are the central factors that are responsible for various graft-versus-host diseases. Traditional strategies usually involve suppressing the whole immune systems with drugs. There are many side effects associated with these methods. Here, we discuss an emerging strategy for manipulating the central immune tolerance by naturally "introducing" donor antigens to a host so a recipient can acquire tolerance specifically to the donor cells or tissues. This strategy has two distinct stages. The first stage restores the thymic function of adult patients with sex steroid inhibitory drugs (LHRH-A), keratinocyte growth factor (KGF), interleukin 7 (IL-7) and FMS-like tyrosine kinase 3 (FLT3). The second stage introduces hematopoietic stem cells and their downstream progenitors to the restored thymus by direct injection. Hematopoietic stem cells are used to introduce donor antigens because they have priority access to the thymus. We also review several clinical cases to explain this new strategy.     

Systemic antitumor protection by vascular-targeted photodynamic therapy involves cellular and humoral immunity

Vascular-targeted photodynamic therapy (VTP) takes advantage of intravascular excitation of a photosensitizer (PS) to produce cytotoxic reactive oxygen species (ROS). These ROS are potent mediators of vascular damage inducing rapid local thrombus formation, vascular occlusion, and tissue hypoxia. This light-controlled process is used for the eradication of solid tumors with Pd-bacteriochlorophyll derivatives (Bchl) as PS. Unlike classical photodynamic therapy (PDT), cancer cells are not the primary target for VTP but instead are destroyed by treatment-induced oxygen deprivation. VTP initiates acute local inflammation inside the illuminated area accompanied by massive tumor tissue death. Consequently, in the present study, we addressed the possibility of immune response induction by the treatment that may be considered as an integral part of the mechanism of VTP-mediated tumor eradication. The effect of VTP on the host immune system was investigated using WST11, which is now in phase II clinical trials for age-related macular degeneration and intended to be evaluated for cancer therapy. We found that a functional immune system is essential for successful VTP. Long-lasting systemic antitumor immunity was induced by VTP involving both cellular and humoral components. The antitumor effect was cross-protective against mismatched tumors, suggesting VTP-mediated production of overlapping tumor antigens, possibly from endothelial origin. Based on our findings we suggest that local VTP might be utilized in combination with other anticancer therapies (e.g., immunotherapy) for the enhancement of host antitumor immunity in the treatment of both local and disseminated disease. Y.S. and A.S are the incumbents of the Tillie and Charles Lubin Professorial Chair in Biochemical Endocrinology, and the Robert and Yaddele Sklare Professorial Chair in Biochemistry, respectively. S.J. is the incumbent of the Pauline Recanati Career Development Chair. D.P. in partial fulfillment of her PhD Thesis requirements at the Feinberg graduate school of the Weizmann Institute of Science.     

稀土磁疗对开胸术后机体免疫功能及疼痛的影响

目的:研究稀土磁治疗床对食管癌、肺癌手术患者术后机体免疫力及切口疼痛的影响。方法:选食管癌患者37例,肺癌23例,采集手术前及稀土磁治疗床治疗后患者静脉血,检测T细胞亚群CD3 、CD4 、CD8 、CD4 /CD8 比值和NK细胞的比例变化,检查手术切口疼痛的恢复情况。结果:术后给予稀土磁治疗床治疗后与手术前相比较CD3 、CD4 、NK、CD4 /CD8 细胞有显著提高(P<0.05),也高于对照组术后的T细胞亚群水平(P<0.05)。结论:稀土磁治疗床可提高食管癌、肺癌患者术后的机体免疫力,减轻疼痛,有利于患者术后恢复。     

Intestinal immunity of Escherichia coli NISSLE 1917: a safe carrier for therapeutic molecules

The development of novel approaches that allow accurate targeting of therapeutics to the intestinal mucosa is a major task in the research on intestinal inflammation. For the first time, a live genetically modified bacterial strain has been approved by Dutch authorities as a therapeutic agent for experimental therapy of intestinal bowel disease (IBD) in humans. Genetically modified probiotics can very well be used as carriers for localized antigen delivery into the intestine. Therapeutic safety, however, of such a carrier organism, is crucial, especially when a specific probiotic strain has to be used under diseased conditions. In this study, we tested the potential of Escherichia coli NISSLE 1917 to serve as a safe carrier for targeted delivery of recombinant proteins to the intestinal mucosa. In a well-defined and very sensitive immunological system, we demonstrate that intestinal recombinant E. coli NISSLE 1917 has no effect on migration, clonal expansion and activation status of specific CD4+ T cells, neither in healthy mice nor in animals with acute colitis. Furthermore, recombinant E. coli NISSLE 1917 has no effect on the induction or breakdown of peripheral T-cell tolerance in an autoimmune environment. The excellent colonization properties of E. coli NISSLE 1917 render this strain an ideal candidate as carrier organism for gut-focused in situ synthesis of therapeutic molecules.     

The roles of interleukin-1 and interleukin-1 receptor antagonist in antigen-specific immune responses

Despite evidence that interleukin (IL)-1 promotes the proliferation of some T helper 2 (Th2) cell clones in vitro, the physiological role of IL-1 in the regulation of antigen-specific immune responses remains undefined. Using a liposome-DNA delivery system, we transiently expressed IL-1 receptor antagonist (IL-1Ra) to suppress IL-1 functions at the site of the antigen-specific primary immune response. Our data indicate, for the first time, that IL-1Ra downregulates antigen-specific IL-4 and IgE responses, with concomitant enhancement of interferon- and IgG2a responses in vivo. In addition, IL-1 can promote Th2 development in an IL-4-independent manner in vitro. Thus, the balance between endogenous IL-1 and IL-1Ra during the primary immune response can be an important factor in determining the antigen-specific effector function of T cells.     

Vaccination of colorectal cancer patients with TroVax given alongside chemotherapy (5-fluorouracil, leukovorin and irinotecan) is safe and induces potent immune responses

Modified vaccinia Ankara (MVA) encoding the tumor antigen 5T4 (TroVax) has been evaluated in an open label phase II study in metastatic colorectal cancer patients. The primary objective was to assess the safety and immunogenicity of TroVax injected before, during and after treatment with 5-fluorouracil, leukovorin and irinotecan. TroVax was administered to 19 patients with metastatic colorectal cancer. Twelve patients had blood samples taken following each of the six injections and were considered to be evaluable for assessment of immunological responses. Both antibody and cellular responses specific for the tumor antigen 5T4 and the viral vector MVA were monitored throughout the study. Administration of TroVax alongside chemotherapy was safe and well tolerated with no SAEs attributed to the vaccine and no enhancement of chemo-related toxicity. Of the 12 patients who were evaluable for assessment of immune responses, ten mounted 5T4-specific antibody responses with titers ranging from 10 to > 5,000. IFNgamma ELISPOT responses specific for 5T4 were detected in 11 patients with frequencies exceeding one in 1,000 PBMCs in five patients. Eight patients presented with elevated circulating CEA concentrations, six of whom showed decreases in excess of 50% during chemotherapy and four had CEA levels which remained stable for > 1 month following completion of chemotherapy. Of the 19 intention to treat (ITT) patients, one had a CR, six had PRs and five had SD. Potent 5T4-specific cellular and/or humoral immune responses were induced in all 12 evaluable patients and were detectable in most patients during the period in which chemotherapy was administered. These data demonstrate that TroVax can be layered on top of chemotherapy regimens without any evidence of enhanced toxicity or reduced immunological or therapeutic efficacy.     

Modulation of immune responses by Plasmodium falciparum infection in asymptomatic children living in the endemic region of Mbita,western Kenya

Individuals living in malaria endemic areas become clinically immune after multiple re-infections over time and remain infected without apparent symptoms. However, it is unclear why a long period is required to gain clinical immunity to malaria, and how such immunity is maintained. Although malaria infection is reported to induce inhibition of immune responses, studies on asymptomatic individuals living in endemic regions of malaria are relatively scarce. We conducted a cross-sectional study of immune responses in asymptomatic school children aged 4–16 years living in an area where Plasmodium falciparum and Schistosoma mansoni infections are co-endemic in Kenya. Peripheral blood mononuclear cells were subjected to flow cytometric analysis and cultured to determine proliferative responses and cytokine production. The proportions of cellular subsets in children positive for P. falciparum infection at the level of microscopy were comparable to the negative children, except for a reduction in central memory-phenotype CD8⁺ T cells and natural killer cells. In functional studies, the production of cytokines by peripheral blood mononuclear cells in response to P. falciparum crude antigens exhibited strong heterogeneity among children. In addition, production of IL-2 in response to anti-CD3 and anti-CD28 monoclonal antibodies was significantly reduced in P. falciparum-positive children as compared to -negative children, suggesting a state of unresponsiveness. These data suggest that the quality of T cell immune responses is heterogeneous among asymptomatic children living in the endemic region of P. falciparum, and that the responses are generally suppressed by active infection with Plasmodium parasites.     

Induction of cellular immune responses against carcinoembryonic antigen in patients with metastatic tumors after vaccination with altered peptide ligand-loaded dendritic cells

Purpose: Dendritic cells (DCs) are characterized by their extraordinary capacity to induce T-cell responses, providing the opportunity of DC-based cancer vaccination protocols. In the present study, we conducted a phase I/II clinical trial to determine the capability of DCs differentiated from immunomagnetically isolated CD14+ monocytes and pulsed with a carcinoembyonic antigen-derived altered peptide (CEAalt) to induce specific CD8+ T cells in cancer patients. Experimental design: Nine patients with CEA-positive colorectal cancer (n=7) or lung cancer (n=2) were enrolled in this study. Autologous CD14+ monocytes were isolated by large-scale immunomagnetic separation and differentiated to mature DCs in sufficient numbers and at high purity. After incubation with the CEAalt peptide and keyhole limpet hemocyanin, DCs were administered to patients intravenously at dose levels of 1×10⁷ and 5×10⁷ cells. Patients received four immunizations every second week. Results: ELISPOT analysis revealed a vaccine-induced increase in the number of CEAalt peptide-specific Interferon (IFN)-gamma producing CD8+ T cells in five of nine patients and of CD8+ T lymphocytes recognizing the native CEA peptide in three of nine patients. In addition, CD8+ T lymphocytes derived from one patient exhibiting an immunological response after vaccination efficiently lysed peptide-loaded T2 cells and tumor cells. Immunization was well tolerated by all patients without severe signs of toxicity. Conclusion: Vaccination with CEAalt-pulsed DCs derived from immunomagnetically isolated CD14+ monocytes efficiently expand peptide-specific CD8+ T lymphocytes in vivo and may be a promising alternative for cancer immunotherapy.     

The potential use of Toll-like receptor agonists to restore the dysfunctional immunity induced by hepatitis C virus

Hepatitis C virus (HCV) infection is a major public health concern with approximately 3% of the world’s population is infected, posing social, economical and health burden. Less than 20% of the infected individuals clear the virus during the acute infection, while the rest develop chronic infection. The treatment of choice for HCV infection is pegylated interferon-α (IFN-α) in combination with ribavarin. Despite the cost and side effects of this treatment regimen, many patients fail this therapy and develop persistent HCV infection, leading to cirrhosis and hepatocellular carcinoma. Although the mechanisms underlying the failure to resolve HCV infection are poorly understood, the incapability of patients to develop effective anti-HCV immunity is a potential cause. We hypothesize that the dysfunctional anti-HCV immunity is due to the emergence of immunosuppressive cells coinciding with a decrease in the stimulatory dendritic cells (DCs) and natural killer (NK) cells. We further hypothesize that applying agents that can correct the imbalance between the immunosuppressive cells and stimulatory cells can results in resolution of chronic HCV. In this review article, we will discuss potential approaches, focusing on the use of Toll-like receptor agonists, to block the suppressive effects of the regulatory cells and restore the stimulatory effects of DCs and NK cells.     

Modification of cellular immune responses in experimental autoimmune hepatitis in mice by maitake (Grifola frondosa)

Immune response to liver-specific lipoprotein (LSP) is involved in the pathogenesis of chronic active hepatitis. Experimental hepatitis could thus be prepared in C57BL/6 mice by injection of liver-specific protein in a syngeneic liver homogenate with Freund's complete adjuvant. In hepatitic mice treated with maitake (Grifola frondosa) fruit bodies, the values of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) values increased temporarily by 2.24–2.79 times and decreased rapidly thereafter. However, in the mice given normal feed, both values increased constantly. Thus, we examined T cell activities both in the exacerbation and remission stages of hepatitis. We suggest that the activation of CD8⁺ cells is more potentiated than that of CD4⁺ cells by administration of maitake or the D-Fraction-glucan (β-1,6 glucan having β-1,3 branches), which can enhance immuno-competent cells at the exacerbation stage. However, at the remission stage, marked potentiation of CD8⁺ cell activity was not observed. These results suggest that depressed suppressor T cell activity is revived by the X-Fraction-glucan (β-1,6 glucan having α-1,4 branched glucan), while the cytotoxic T cell activity, which is activated by the D-Fraction, is restricted, thereby creating a smooth shift from the exacerbation stage to the remission stage.     

Pharmacological administration of granulocyte/macrophage-colony-stimulating factor is of significant importance for the induction of a strong humoral and cellular response in patients immunized with recombinant carcinoembryonic antigen

Eighteen colorectal carcinoma patients without macroscopic disease after surgery were immunized using recombinant (r) human (h) carcinoembryonic antigen (CEA) with (n = 9) or without (n = 9) the addition of soluble granulocyte/macrophage-colony-stimulating factor (GM-CSF). The dose of rhCEA per immunization was 100 μg (n = 6), 316 μg (n = 6) or 1000 μg (n = 6). rhCEA was given s.c. on day 1 and 80 μg/day of GM-CSF s.c. on days 1–4. The schedule was repeated six times during a period of 9 months. All patients in the GM-CSF group developed a strong rhCEA-dose-dependent IgG antibody response while only one-third of the non-GM-CSF patients mounted a weak antibody response. All patients (9/9) in the GM-CSF group developed a strong rhCEA-specific proliferative T cell response as well as type I T cells (interferon γ secretion). In 45% of the patients also a weak type II T cell response (interleukin-4 secretion) was evoked. Both MHC-class-I- and -II restricted rhCEA-specific T cells were noted. A specific cellular response (proliferation and/or cytokine secretion) against native hCEA could be found in 8/9 patients in the GM-CSF group, although at a significantly lower level than against rhCEA. In the non-GM-CSF group a weak rhCEA-specific T cell response was induced. Three patients had a proliferative response, 4 patients type I T cells and 6 patients type II T cells. No signs of autoimmune reactions were noted. Local pharmacological administration of GM-CSF seemed to be a prerequisite for the induction of a strong immunity against baculovirus-produced hCEA protein. However, the cellular response against native CEA was of a significantly lower magnitude. Received: 13 November 1997 / Accepted: 21 May 1998     

Molecular and immunological evaluation of the expression of cancer/testis gene products in human colorectal cancer

Tumor-specific gene products, such as cancer/testis (CT) antigens, constitute promising targets for the development of T cell vaccines. Whereas CT antigens are frequently expressed in melanoma, their expression in colorectal cancers (CRC) remains poorly characterized. Here, we have studied the expression of the CT antigens MAGE-A3, MAGE-A4, MAGE-A10, NY-ESO-1 and SSX2 in CRC because of the presence of well-described HLA-A2-restricted epitopes in their sequences. Our analyses of 41 primary CRC and 14 metastatic liver lesions confirmed the low frequency of expression of these CT antigens. No increased expression frequencies were observed in metastatic tumors compared to primary tumors. Histological analyses of CRC samples revealed heterogeneous expression of individual CT antigens. Finally, evidence of a naturally acquired CT antigen-specific CD8⁺ T cell response could be demonstrated. These results show that the expression of CT antigens in a subset of CRC patients induces readily detectable T cell responses.     

Adoptive transfer of natural killer cells in combination with chemotherapy improves outcomes of patients with locally advanced colon carcinoma

Background

Despite the availability of multiple treatment strategies, patients with advanced colon carcinoma (CC) have poor prognoses. The aim of this study was to evaluate the efficacy and safety of natural killer (NK) cell therapy in combination with chemotherapy in patients with locally advanced CC.

Immunological evaluation of personalized peptide vaccination in combination with UFT and UZEL for metastatic colorectal carcinoma patients

To investigate the safety and immunological responses of personalized peptide vaccination in combination with oral administration of UFT and UZEL for metastatic colorectal carcinoma (mCRC), fourteen patients were enrolled in the present study. Peptides were determined based on the presence of peptide-specific cytotoxic T lymphocyte precursors and IgG in each patient. A maximum of four peptides were subcutaneously administered weekly with UFT (300 mg/m² day⁻¹) and UZEL (75 mg/day) for 4 weeks, followed by 1 week of rest. This therapy was well-tolerated although there was a grade-3 skin reaction at the vaccination site in one patient. An increase in peptide-specific interferon-γ production or peptide-specific IgG after the tenth vaccination was observed in nine of ten or eight of ten patients tested, respectively. IgG responses were well correlated with overall survival (P = 0.0215). The safety and immunological responsiveness of the present therapy suggest that this combination would be of clinical benefit for mCRC patients, and further trials are merited. T. Hattori and T. Mine equally contributed to this work.     

Squamous cell carcinoma of the head and neck in the elderly

The incidence of head and neck squamous cell carcinoma (HNSCC) peaks between the fifth and seventh decades of life. With prolongation of life expectancy, however, the proportion of elderly HNSCC patients is also increasing, which makes HNSCC in this life period an important issue for healthcare providers. With features characteristic to the older patient groups coupled with the inherent complexity of the disease, HNSCC in the elderly represents a considerable challenge to clinicians. Indeed, to expedite the progress and improve the healthcare system to meet the needs of this unique population of patients, several essential issues related to the clinical profile, diagnostics, optimal treatment and support are of concern and should be addressed in properly conducted clinical trials.In the present review, we analyzed a literature series comparing different age groups with regard to their clinical characteristics, therapy, outcome and quality of life in an attempt to determine their implications on treatment-decision-making for elderly patients with HNSCC.     

Trials and tribulations of immunotherapy as a treatment option for patients with squamous cell carcinoma of the head and neck

Head and neck squamous cell carcinoma (HNSCC) is an aggressive epithelial malignancy that is the sixth most common neoplasm in the world. Despite numerous advances in treatments involving surgery, radiation, and chemotherapy, the 5-year survival has remained at less than 50% for the last 30 years primarily due to local recurrences [66]. Consequently, the possibility of developing immunotherapeutic approaches as a treatment for HNSCC has gained interest. The present review has 3 objectives pertaining to immunotherapeutic means to treat HNSCC patients: (1) to summarize the feasibility of such approaches, (2) to provide an overview of the obstacles to attaining protective immune reactivity, and (3) to consider how these obstacles can be overcome to stimulate immune reactivity to HNSCC. These objectives will also be considered in the context of what lessons have been learned from immunotherapeutic trials for other solid malignancies and the applicability of this information to HNSCC.     

Role of the unfolded protein response in cell death

Unfolded protein response (UPR) is an important genomic response to endoplasmic reticulum (ER) stress. The ER chaperones, GRP78 and Gadd153, play critical roles in cell survival or cell death as part of the UPR, which is regulated by three signaling pathways: PERK/ATF4, IRE1/XBP1 and ATF6. During the UPR, accumulated unfolded protein is either correctly refolded, or unsuccessfully refolded and degraded by the ubiquitin-proteasome pathway. When the unfolded protein exceeds a threshold, damaged cells are committed to cell death, which is mediated by ATF4 and ATF6, as well as activation of the JNK/AP-1/Gadd153-signaling pathway. Gadd153 suppresses activation of Bcl-2 and NF-κB. UPR-mediated cell survival or cell death is regulated by the balance of GRP78 and Gadd153 expression, which is coregulated by NF-κB in accordance with the magnitude of ER stress. Less susceptibility to cell death upon activation of the UPR may contribute to tumor progression and drug resistance of solid tumors.     

Immune and proliferative cellular responses to Helicobacter pylori infection in the gastric mucosa of Mexican children

BACKGROUND: Helicobacter pylori infection occurs mostly during childhood, but few studies on this age group have addressed the innate immune and the proliferative response to this infection. Mexico has a high H. pylori prevalence in children, but a low risk of gastric cancer. The aim of this work was to study the cellular responses of the gastric mucosa to this infection in Mexican children. METHODS: Antral and corpus gastric biopsies were obtained from 44 H. pylori-infected children (mean age 12 +/- 3.2 years) and 44 uninfected children (mean age 10 +/- 3 years). Mucosal cellular responses were studied by immunohistochemistry, using anti-Ki67 antibodies for proliferation studies, antihuman tryptase for mast cells, and antihuman CD68 for macrophages. T and B lymphocytes were stained with a commercial integrated system. The intensity of cellular responses was estimated histologically using the software KS300. RESULTS: Epithelium proliferation and infiltration of macrophages and T and B lymphocytes were significantly higher in H. pylori-infected than in uninfected children. A balanced increase of CD4, CD8, and CD20 lymphocytes was observed in infected children. However, activated mast cells were decreased, and infiltration of neutrophil and mononuclear cells was low. Epithelial proliferation was associated with polymorphonuclear infiltration but not with infiltration of macrophages or lymphocytes. Inflammation and proliferation was higher in CagA (+)-infected children. CONCLUSIONS: Mexican children respond to H. pylori infection with a low inflammatory response, a balanced increase of T and B lymphocytes, and a high regenerative activity.     

Using extracellular biomarkers for monitoring efficacy of therapeutics in cancer patients: an update

Rapidly detectable and easily accessible markers of tumor cell death are needed for evaluating early therapeutic efficacy for immunotherapy and chemotherapy so that patients and their physicians can decide whether to remain with a given therapeutic strategy. Currently, image-based tests such as computed tomography scans and magnetic resonance imaging are used to visualize the response of a patient’s tumor, but often these evaluations are not conducted for weeks to months after treatment begins. While serum levels of secreted proteins such as carcinoembryonic antigen and prostate specific antigen are commonly monitored to gauge tumor status during therapy and between image evaluations, the levels of these proteins do not always correlate well with the actual tumor response. In laboratory studies, it has been shown that tumor cells undergoing apoptosis can release cellular components into cell culture media such as cytochrome c, nucleosomes, cleaved cytokeratin-18 and E-cadherin. Studies of patient sera have found that these and other macromolecules can be found in circulation during cancer therapy, providing a potential source of material for monitoring treatment efficacy. In the future, analysis of biofluids from severe combined immunodeficiency mice bearing patient tumor specimens treated with a targeted therapy such as Apo2L/tumor necrosis factor-related apoptosis-inducing ligand will be useful in the preclinical identification of therapy response markers. In this review, the current status of the identification of serum markers of tumor cell apoptosis is provided, as well as a discussion of critical research questions that must be addressed and the considerations necessary when identifying a marker that reflects true clinical outcome.     

Morphological analysis of circulating tumour cells in patients undergoing surgery for non‐small cell lung carcinoma using the isolation by size of epithelial tumour cell (ISET) method

V. Hofman, E. Long, M. Ilie, C. Bonnetaud, J. M. Vignaud, J. F. Fléjou, S. Lantuejoul, E. Piaton, N. Mourad, C. Butori, E. Selva, C. H. Marquette, M. Poudenx, S. Sibon, S. Kelhef, N. Vénissac, J. P. Jais, J. Mouroux, T. J. Molina, P. Vielh and P. Hofman
Morphological analysis of circulating tumour cells in patients undergoing surgery for non‐small cell lung carcinoma using the isolation by size of epithelial tumour cell (ISET) method Background and objective: Recurrence rates after surgery for non‐small cell lung cancer (NSCLC) range from 25 to 50% and 5‐year survival is only 60–70%. Because no biomarkers are predictive of recurrence or the onset of metastasis, pathological TNM (pTNM) staging is currently the best prognostic factor. Consequently, the preoperative detection of circulating tumour cells (CTCs) might be useful in tailoring therapy. The aim of this study was to characterize morphologically any circulating non‐haematological cells (CNHCs) in patients undergoing surgery for NSCLC using the isolation by size of epithelial tumour cell (ISET) method. Methods: Of 299 blood samples tested, 250 were from patients with resectable NSCLC and 59 from healthy controls. The presence of CNHCs was assessed blindly and independently by 10 cytopathologists on May‐Grünwald–Giemsa stained filters and the cells classified into three groups: (i) malignant cells, (ii) uncertain malignant cells, and (iii) benign cells. We assessed interobserver agreement using Kappa (κ) analysis as the measure of agreement. Results: A total of 123 out of 250 (49%) patients showed CNHCs corresponding to malignant, uncertain malignant and benign cells, in 102/250 (41%), 15/250 (6%) and 6/250 (2%) cases, respectively. No CNHCs were detected in the blood of healthy subjects. Interobserver diagnostic variability was absent for CNHCs, low for malignant cells and limited for uncertain malignant and benign cells. Conclusion: Identification of CTCs in resectable NSCLC patients, using ISET technology and according to cytopathological criteria of malignancy, appears to be a new and promising field of cytopathology with potential relevance to lung oncology.