Elevated frequencies of CD4⁺ CD25⁺ CD127lo regulatory T cells is associated to poor prognosis in patients with acute myeloid leukemia

Regulatory T cells (Treg) mediate amelioration of disease and immune homeostasis by inhibiting immune activation and maintaining peripheral immune tolerance. The suppressive mechanisms and clinical significance of Treg have not been completely elucidated in patients with acute myeloid leukemia (AML). Here, we demonstrated that CD127 in combination with CD4 and CD25 can identify FoxP3(+) Treg in peripheral blood (PB) and bone marrow (BM) using multicolor flow cytometry. We showed that the CD4(+) CD25(+) CD127(lo) Treg frequencies were significantly increased and their phenotypes were different in PB from newly diagnosed AML patients compared to those from healthy volunteers (HVs). Moreover, the Treg frequencies were significantly higher in BM than those from PB in the same patients. The Treg frequencies were reduced when patients achieved complete remission (CR) and were increased when patients relapsed. The Treg frequencies at diagnosis in PB and BM of patients who had achieved CR were lower than those of patients who had persistent leukemia or died, respectively. CD4(+) CD25(+) Treg were isolated by magnetic-activated cell sorting and tested for suppressive functions in coculture with allogeneic carboxyfluorescein diacetate succinimidylester-labeled CD4(+) CD25(-) responder cells. Suppression mediated by Treg was higher in AML patients compared to HVs. No significant differences were observed in the cytokines production of Treg, including interferon-gamma (IFN-γ), interleukin (IL)-4,IL-2 and IL-10, between patients with AML and HVs. Our study suggests that Treg may play a role in the pathogenesis of AML, and sequential measurements of Treg frequency may have clinical value in the evaluation of therapeutic effects and clinical outcome.     

CD8+CD28− cells and CD4+CD25+ regulatory T cells in the peripheral blood of advanced stage lung cancer patients

Aim To evaluate the CD8+CD28? and CD4+CD25+ regulatory T (Treg) cells in addition to other some lymphocyte subgroups in peripheral blood of advanced stage lung cancer patients. Methods The study group (n = 28) comprised chemotherapy and radiotherapy naïve patients with non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). The control group (n = 22) consisted of age- and sex-matched healthy volunteers. Flow cytometry was used to count T cells, natural killer (NK) cells and CD4+CD25 Treg cells, and for CD8+ T cell subgroup analysis. Flow cytometry was performed and annexin V binding was used for apoptotic cell evaluation. Results In patient group, the percentage of CD8+CD28? cells among lymphocytes was elevated, and there was also an increase in the CD28?/CD28+ cell ratio among CD8 lymphocyte population. The distribution of CD8 cells was different in lung cancer patients when compared with the control group. The absolute count of CD4+CD25^bright cells and the percentages of these cells among total lymphocytes were higher in the patient group. The Annexin V(+) cell percentages among CD8+CD28? and CD8+CD28+ lymphocytes were higher in the patient group than in the control group. No differences were found between the NSCLC and SCLC patients with respect to the hematological parameters and the distribution of lymphocyte subgroups. In NSCLC patients, the percentage of CD8+CD28? cells among the lymphocyte population was higher in patients with stage IV than those with stage III. Conclusion These findings may reflect the possibility of tumor-induced immunosuppression and they should be complemented with further studies.     

CD8⁺ cytotoxic T cell and FOXP3⁺ regulatory T cell infiltration in relation to breast cancer survival and molecular subtypes

The prognostic significance of tumor-associated FOXP3⁺ regulatory T cells (Tregs) and CD8⁺ cytotoxic T lymphocytes (CTLs) in invasive breast carcinomas is studied. Tregs and CTLs were assessed by immunohistochemistry in 1270 cases of invasive breast carcinoma for their associations with patient survival, histopathologic features, and molecular subtypes. Infiltrates of Tregs and CTLs were observed within tumor bed and in the tissue surrounding tumor. Within tumor bed, increased infiltration of Tregs and CTLs was significantly more common in those with unfavorable histologic features, including high histologic grade and negative ER and PR status. In addition, high density Treg infiltration was also associated with tumor HER2 overexpression, decreased overall survival (OS) and progression-free survival (PFS). In tissue surrounding tumor, in contrast, high CTL/Treg ratio was found to be significantly associated with improved OS and PFS. These prognostic associations were confirmed by multivariate analysis. Furthermore, the density of Treg infiltrates within tumors was inversely correlated with the prognosis of the molecular subtypes of tumors. The ratio of CTL/Treg infiltrates in the surrounding tissue was also significantly higher in luminal than non-luminal subtypes of carcinoma. The prognostic significances of Tregs and CTLs in breast carcinoma depend on their relative density and location. The density of intratumoral Treg infiltrates and the peritumoral CTL/Treg ratio are independent prognostic factors and correlated with the prognosis of the molecular subtypes of breast carcinoma, which may serve as potential target for stratifying immunotherapy to battle against the aggressive subtypes of breast carcinoma.     

The correlation of CD19 + CD24 + CD38 + B cells and other clinicopathological variables with the proportion of circulating Tregs in breast cancer patients

Background

T regulatory cells (Tregs) are known to negatively control immune response. The frequency of these cells was inversely correlated with clinical outcomes of breast cancer. CD19⁺CD24^hiCD38^hi cells also play a critical role in inflammation and autoimmune disease. However, their function in tumor immune response is less studied. In this study we aimed to determine the role of CD19⁺CD24^hiCD38^hi cells and some other clinicopathological variables in increasing the proportion of Tregs in breast cancer patients.

Methods

We selected 47 patients with invasive ductal breast carcinoma and 50 healthy controls and obtained their blood samples.

Results

The proportion of circulating CD4⁺CD25⁺Foxp3⁺ Tregs and CD19⁺CD24^hiCD38^hi cells was significantly increased in breast cancer patients. We also found that increased proportion of Tregs in breast cancer is correlated with HER2 amplification, advanced clinical stages, serum TGF-β1 and increased CD19⁺CD24^hiCD38^hi cells in the peripheral blood.

Conclusion

Altogether, our data suggest that as much as Tregs, CD19⁺CD24^hiCD38^hi B cells could also have a part in the suppression of immune response in breast cancer.

     

Hypoxia-inducible factor-1α and vascular endothelial growth factor expression in circulating tumor cells of breast cancer patients

Introduction  

The detection of peripheral blood circulating tumor cells (CTCs) and bone marrow disseminated tumor cells (DTCs) in breast cancer patients is associated with a high incidence of disease relapse and disease-related death. Since hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) play an important role in angiogenesis and tumor progression, the purpose of the current study was to investigate their expression in CTCs.     

Correlation of Fcγ receptors on peripheral blood mononuclear cells and survival in patients with metastatic breast cancer

Summary Patients with carcinomas have elevated levels of Fc receptors for IgG (FcR) on their peripheral blood mononuclear cells (PBMC). The purpose of the present study was to determine whether there is a correlation between FcR levels on PBMC and survival in patients with metastatic breast cancer. Binding assays were performed on PBMC using¹²⁵I-labeled fibrinogen complexed with rabbit IgG (or as a control F(ab)₂) anti-human fibrinogen. Twenty-two metastatic breast cancer patients had significantly (p<0.001) elevated FcR levels as compared to either 22 breast cancer patients receiving adjuvant chemotherapy following mastectomy without clinical evidence of tumor, or to 34 non-malignant controls. Significantly more metastatic patients with elevated FcR levels died at 6 months (p<0.001) as compared to those with low levels. A direct correlation between FcR levels and hazard probability was found (correlation coefficient = 0.3321, p<0.005). These results raise the possibility that FcR levels on PMBC from metastatic breast cancer patients may be clinically useful as a prognostic marker of disease activity.     

TGF-β of lung cancer microenvironment upregulates B7H1 and GITRL expression in dendritic cells and is associated with regulatory T cell generation

The effects of TGF-β on dendritic cells (DCs) on the tumor microenvironment are not well understood. We report, here, the establishment of an in vitro lung cancer microenvironment by co-incubation of seminaphtharhodafluor (SNARF) labeled Lewis lung cancer (LLC) cells, carboxyfluorescein succinimidyl ester (CFSE) labeled fibroblasts and 4-chloromethyl-7-hydroxycoumarin (CMHC) labeled DCs. Raw 264.7, EL4 and NCI-H446 cells were able to synthesize TGF-β which was determined by flow cyto-metry and western blotting, respectively. Furthermore, TGF-β efficiently increased regulatory T-cell (Treg) expansion and upregulated DC B7H1 and GITRL expression. TGF-β and the co-incubation of LLC cells, fibroblasts with DCs could augment the expression of B7H1 and GITRL molecules of DCs. The data presented here indicate that the B7H1 and GITRL molecules may play an important role in TGF-β-induced Treg expansion of lung cancer microenvironment.     

The methodology of quantitation of microvessel density and prognostic value of neovascularization associated with long‐term survival in Japanese patients with breast cancer

The present study updates results on methodology of quantitation of tumor neovascularization and those on the prognostic value of microvessel density (MVD) in breast cancer tissue previously published in the World J. Surg. 21: 49–56, 1997. The followup period of observation of the series was extended to 20 years, and new biological indicators (i.e., proliferating cell nuclear antigen (PCNA), cerbB2, and p53) were included in the analysis. There were 109 patients with primary breast cancer, from 1971 to 1979, followed up for a median of 14 years (range, 1–20). A representative median longitudinal section of each breast tumor was immunohistochemically stained with factor VIIIrelated antigen and analyzed. The three methods of identifying MVD were: (1) average microvessel count (AMC)/mm2, (2) central microvessel count (CMC)/mm2, and (3) highest microvessel count (HMC)/mm2. Thirtyone patients (28.4%) died of breast cancer. There was a relationship between MVD and peritumor blood vessel invasion (AMC: p = 0.0114, CMC: p = 0.0319, and HMC: p = 0.0009). However, there was no relationship between MVD and other factors. Univariate analysis showed that node status (p < 0.0001), histological grade (p < 0.0001), clinical tumor size (T) (p = 0.0002), PCNA (p = 0.0033), p53 (p = 0.0043), mitotic grade (p = 0.0092), AMC (p = 0.0214), and peritumor lymphatic vessel invasion (p = 0.0467) were significantly predictive of overall survival. HMC was borderline significant (p = 0.0702), while CMC and cerbB2 were not significant. Multivariate analysis showed that T (p = 0.0005), node status (p = 0.0053), and AMC (p = 0.0485) were independent factors, but neither CMC nor HMC was independent. AMC, a significant independent prognostic factor, might be a better method than the others for evaluating angiogenesis, but further and larger studies are warranted.     

IL-6-stimulated CD11b+CD14+HLA-DR? myeloid-derived suppressor cells,are associated with progression and poor prognosis in squamous cell carcinoma of the esophagus

The aim of this study was to assess the significance of myeloid-derived suppressor cells (MDSCs) and their association with IL-6 in esophageal squamous cell carcinoma (SCC). We examined the percentage of CD11b+CD14⁺HLA-DR⁻ myeloid cells and the levels of IL-6 in the peripheral blood of 50 patients with esophageal SCC and 12 healthy controls. Moreover, we evaluated the relationship between MDSC recruitment, IL-6 levels, and tumor progression by adding 4-nitroquinoline 1-oxide (4-NQO) to the drinking water of mice to induce esophageal tumors. Here we demonstrated that circulating CD11b+CD14⁺HLA-DR⁻ cells were significantly increased in esophageal SCC patients compared with healthy people, and this was associated with the clinical stage, treatment response and circulating IL-6 levels. In a 4-NQO-induced esophageal tumor animal model, MDSC recruitment was associated with invasive esophageal tumors and with increased IL-6 levels. IL-6 stimulated reactive oxygen species, arginase 1 and p-STAT3 in MDSCs. Blockade of IL-6 prevented induction of MDSCs and the incidence of 4-NQO- induced invasive tumors. In conclusion, the levels of MDSCs and IL-6 predicted the prognosis of patients with esophageal SCC. Moreover, we suggest inhibition of IL-6 as a potential strategy for the treatment of esophageal SCC.     

Prognostic impact of circulating tumor cells assessed with the CellSearch System? and AdnaTest Breast? in metastatic breast cancer patients: the DETECT study

Introduction

There is a multitude of assays for the detection of circulating tumor cells (CTCs) but a very limited number of studies comparing the clinical relevance of results obtained with different test methods. The DETECT trial for metastatic breast cancer patients was designed to directly compare the prognostic impact of two commercially available CTC assays that are prominent representatives of immunocytochemical and RT-PCR based technologies.

Methods

In total, 254 metastatic breast cancer patients were enrolled in this prospective multicenter trial. CTCs were assessed using both the AdnaTest Breast Cancer and the CellSearch system according to the manufacturers' instructions.

Results

With the CellSearch system, 116 of 221 (50%) evaluable patients were CTC-positive based on a cut-off level at 5 or more CTCs. The median overall survival (OS) was 18.1 months in CTC-positive patients. (95%-CI: 15.1-22.1 months) compared to 27 months in CTC-negative patients (23.5-30.7 months; p<0.001). This prognostic impact for OS was also significant in the subgroups of patients with triple negative, HER2-positive and hormone receptor-positive/HER2-negative primary tumors. The progression free survival (PFS) was not correlated with CTC status in our cohort receiving different types and lines of systemic treatment (p = 0.197). In multivariate analysis, the presence of CTCs was an independent predictor for OS (HR: 2.7, 95%-CI: 1.6-4.2). When the AdnaTest Breast was performed, 88 of 221 (40%) patients were CTC-positive. CTC-positivity assessed by the AdnaTest Breast had no association with PFS or OS.

Conclusions

The prognostic relevance of CTC detection in metastatic breast cancer patients depends on the test method. The present results indicate that the CellSearch system is superior to the AdnaTest Breast Cancer in predicting clinical outcome in advanced breast cancer.

Trial registration

Current Controlled Trials Registry number ISRCTN59722891.     

CD4+ regulatory T cells in gastric cancer mucosa are proliferating and express high levels of IL-10 but little TGF-β

Gastric Cancer - An increase of regulatory T cells, defined as CD25high- and/or FOXP3+-expressing CD4+ T cells, within tumors has been reported in several studies. Tregs promote tumor growth by...     

Increased expression of CD4+CD25+FOXP3+ regulatory T cells correlates with Epstein–Barr virus and has no impact on survival in patients with classical Hodgkin lymphoma in Brazil

The tumor microenvironment of classical Hodgkin lymphoma (cHL) is clearly responsible for the maintenance of the malignant Hodgkin-Reed?CSternberg (HRS) cells, and Epstein?CBarr virus (EBV) has been shown to play a role in this immune evasion. EBV can increase the migration of CD4⁺CD25⁺FOXP3⁺ lymphocytes, named regulatory T cells (Tregs). In this study, we assessed the distribution and biological significance of Tregs in patients with cHL. Tissue microarrays were constructed using diagnostic biopsies available in 130 cHL patients and stained with CD4, CD8, CD25, and FOXP3 antibodies. For the present study, only cHL patients whose histology could be confirmed and EBV association established were studied. From the 130 cHL patients selected for this study, 56 were classified as EBV-related and 74 EBV non-related cHL. There were no association between clinical characteristics and the expression of Tregs. However, higher levels of Tregs correlated with EBV presence on HRS cells (p?=?0.02), although it did not influence event-free survival (EFS) and overall survival (p?=?0.98 and p?=?0.59, respectively). This study demonstrates that Tregs expression correlates with EBV presence in HRS cells and has no impact on survival of patients with cHL. Further studies investigating the mechanisms in which EBV recruits Tregs to the tumor microenvironment will contribute not only to our understanding on the pathogenesis of cHL but also to the development of new therapeutic strategies.     

Primary breast cancer patients with high risk clinicopathologic features have high percentages of bone marrow epithelial cells with ALDH activity and CD44⁺CD24lo cancer stem cell phenotype

Background

Cancer stem cells (CSCs) are purported to be epithelial tumour cells expressing CD44⁺CD24^lo that exhibit aldehyde dehydrogenase activity (Aldefluor⁺). We hypothesised that if CSCs are responsible for tumour dissemination, disseminated cells in the bone marrow (BM) would be positive for putative breast CSC markers. Therefore, we assessed the presence of Aldefluor⁺ epithelial (CD326⁺CD45^dim) cells for the presence of the CD44⁺CD24^lo phenotype in BM of patients with primary breast cancer (PBC).

Methods

BM aspirates were collected at the time of surgery from 66 patients with PBC. Thirty patients received neoadjuvant chemotherapy (NACT) prior to aspiration. BM was analysed for Aldefluor⁺ epithelial cells with or without CD44⁺CD24^lo expression by flow cytometry. BM aspirates from three healthy donors (HD) were subjected to identical processing and analyses and served as controls.

Results

Patients with triple-receptor-negative (TN) tumours had a significantly higher median percentage of CD44⁺CD24^lo CSC within Aldefluor⁺ epithelial cell population than patients with other immunohistochemical subtypes (P = 0.018). Patients with TN tumours or with pN2 or higher pathologic nodal status were more likely to have a proportion of CD44⁺CD24^lo CSC within Aldefluor⁺ epithelial cell population above the highest level of HD. Furthermore, patients who received NACT were more likely to have percentages of Aldefluor⁺ epithelial cells than the highest level of HD (P = 0.004).

Conclusion

The percentage of CD44⁺CD24^lo CSC in the BM is higher in PBC patients with high risk tumour features. The selection or enrichment of Aldefluor⁺ epithelial cells by NACT may represent an opportunity to target these cells with novel therapies.     

Comparing the outcome between multicentric and multifocal breast cancer: what is the impact on survival, and is there a role for guideline-adherent adjuvant therapy? A retrospective multicenter cohort study of 8,935 patients

Multifocal (MF) and multicentric (MC) breast cancers have been comprehensively studied, and their outcomes have been compared with unifocal (UF) tumors. We attempted to answer the following questions: (1) Does MF/MC presentation influence the outcome concerning BC mortality?, (2) Is there an impact of guideline-adherent adjuvant treatment in these BC subtypes?, and (3)What is the influence of guideline violations concerning surgery (breast-conserving surgery versus mastectomy) on the survival of MF/MC BC patients? Between 1992 and 2008, we retrospectively analyzed 8,935 breast cancer patients from 17 participating breast cancer centers within the BRENDA study group. Of 8,935 breast cancer patients, 7,073 (79.2 %) had UF tumors, 1,398 (15.6 %) had MF tumors, and 464 (5.2 %) had MC tumors. RFS was significantly worse for MF/MC BC patients compared to patients with UF tumors (MF p = 0.007; MC p = 0.019). OAS was significantly worse for MC patients but not for MF patients compared to patients with UF tumors (MF p = 0.321; MC p = 0.001). Guideline adherence was significantly lower in patients with MF (n = 580; 41.5 %) and MC (n = 204; 44.0 %) compared to patients with UF (n = 3,871; 54.7 %) (p < 0.001) tumors. Guideline violations were associated with a highly significant deterioration in survival throughout all subgroups except for MC, with respect to RFS and OAS. For 100 %-guideline-adherent patients, we could not find any significant differences in RFS and OAS after adjusting by nodal status, grade, and tumor size. Furthermore, we could not find any significant differences in RFS and OAS in patients with MF or MC stratified by breast-conserving therapy (BCT lumpectomy and radiation therapy) and mastectomy. There is a strong association between improved RFS and OAS in patients with MF/MZ BC. There are no significant differences in RFS and OAS for patients with breast-conserving therapy or mastectomy.     

Biased usage of T cell receptor β-chain variable region genes of Wilms' tumor gene (WT1)-specific CD8+ T cells in patients with solid tumors and healthy donors

Wilms' tumor gene 1 (WT1) protein is a promising tumor-associated antigen. In patients with WT1-expressing malignancies, WT1-specific CTLs are spontaneously induced as a result of an immune response to the WT1 protein. In the present study, we performed single cell-level comparative analysis of T cell receptor β-chain variable region (TCR-BV) gene families of a total of 750 spontaneously induced WT1(126) peptide (amino acids 126-134, WT1(126))-specific CTLs in both HLA-A*0201(+) patients with solid tumors and healthy donors (HDs). This is the first report of direct usage analysis of 24 kinds of TCR-BV gene families of WT1(126)-specific CTLs at the single cell level. Usage analysis with single-cell RT-PCR of TCR-BV gene families of individual FACS-sorted WT1(126) tetramer(+) CD8(+) T cells showed, for the first time, that: (i) BVs 3, 6, 7, 20, 27, and 28 were commonly biased in patients and HDs; (ii) BVs 2, 11, and 15 were biased only in patients; and (iii) BVs 4, 5, 9, and 19 were biased only in HDs. However, statistical analysis of similarity of individual usage frequencies of 24 kinds of TCR-BV gene families between patients and HDs indicated that the usage frequencies of TCR-BV gene families in patients reflected those in HDs. These results should provide us with a novel insight for a better understanding of WT1-specific immune responses.     

Effect of CD+4 T cells and CD+4/CD+8 in peripheral blood on survival of patients with stage Ⅳ non-small cell lung cancer—Establishment of a Nomogram prediction model

Objective To explore the possibility of CD⁺₄ T cells and CD⁺₄/CD⁺₈ ratio in peripheral blood to predict the survival of patients with stage Ⅳ non-small cell lung cancer (NSCLC), and to establish a Nomogram prediction model. Methods The influence of CD⁺₄ T cells and CD⁺₄/CD⁺₈ ratio on the clinical factors and survival of 682 patients pathologically diagnosed with stage Ⅳ NSCLC with no history of cancer treatment was retrospectively analyzed and the Nomogram prediction model was established. Combined with the changes of immune cells levels in 110 patients after treatment, the prognostic and predictive values of CD⁺₄ T cells and CD⁺₄/CD⁺₈ ratio were verified. Countable data were analyzed by t-test. The survival rate was calculated by Kaplan-Meier method, log-rank test or univariate analysis. The multivariate analysis was performed by Cox regression model. Results Univariate analysis demonstrated that CD⁺₄> 43.15% before treatment significantly prolonged the survival. By multivariate analysis of Cox regression model, CD⁺₄>43.15% was an independent prognostic factor to prolong survival for stage Ⅳ NSCLC. The Nomogram model was established and verified that the predicted and actual overall survivals were highly consistent. Further analysis showed that 43.15% as the critical value of CD⁺₄T cell level significantly prolonged survival when CD⁺₄ expressed at a high-level before treatment, after treatment, before and after treatment, or combined with CD⁺₄/CD⁺₈>1.65. Conclusions The baseline level of CD⁺₄ T cells before treatment in peripheral blood is an independent prognostic factor for stage Ⅳ NSCLC. The CD⁺₄/CD⁺₈ ratio before treatment has limited value in predicting the prognosis.