Pattern and Prevalence of Liver Involvement in Pediatric Acute Lymphoblastic and Myeloid Leukemia at Diagnosis.

OBJECTIVES: The prevalence and significance of liver involvement at diagnosis was studied in pediatric acute lymphoblastic (ALL) and myeloid leukemia (AML). METHODS: A population based cohort of 122 pre B-ALL, 22 T-ALL and 45 AML patients was formed from the Nordic Society of Pediatric Hematology and Oncology leukemia registries (years 2005-2017). Hepatomegaly, elevated alanine aminotransferase, high INR, hypoalbuminemia and conjugated hyperbilirubinemia at diagnosis were used as markers for liver involvement. Minimal residual disease (MRD), time to relapse and overall survival (OS) were correlated with liver involvements. RESULTS: The pattern of liver involvement was significantly different between leukemia subtypes (P = 0.025). The proportion of patients without liver abnormalities was 50.0% in AML and 44.8% in pre B-ALL and 23.5% in T-ALL patients. Hepatomegaly characterized lymphatic leukemia being present in 41.8% and 58.8% of pre B- and T-ALL patients. Liver dysfunction was most common in AML (29.5%) and least frequent in pre B-ALL (7.4%,) (P = 0.001). Conjugated hyperbilirubinemia was present in less than 5% of patients. Hepatomegaly correlated positively with age in pre B-ALL (P = 0.036) and white blood cell count (WBC) in AML (P = 0.010). Hepatic dysfunction was related with high WBC in pre B-ALL (P = 0.037) and AML (P = 0.001). Liver involvement in patients with ALL was not associated with toxicity or outcome. Patients with AML without liver involvement demonstrated superior OS. CONCLUSIONS: Liver involvement is frequent at diagnosis in pediatric leukemia and its prevalence is related with leukemia subtype, age and WBC. In AML, but not in ALL, it associates with suboptimal prognosis.

Low Expression of miR-18a as a Characteristic of Pediatric Acute Lymphoblastic Leukemia.

BACKGROUND: Acute lymphoblastic leukemia (ALL) occurs in both adults and children but the response to chemotherapy and survival is significantly worse in the adults. We aimed to study whether the expression of immune system-associated miRNAs would differ between adult and pediatric patients with ALL at the time of diagnosis. MATERIALS AND METHODS: Inflammation-associated miRNA analysis was performed in 19 adults and 79 pediatric patients with ALL and involved miR-10, miR-15, miR-16, miR-17-92 cluster, miR-33, miR-146a, miR-150, miR-155, miR-181a, miR-222, miR-223, and miR-339. MiRNAs were first analyzed by miRNA microarray and thereafter validated by qRT-PCR. Sufficient RNA for qRT-PCR was available for 42 pediatric and 19 adult patients. RESULTS: Of the studied miRNAs, only miR-18a differed significantly in microarray analysis between adult and pediatric ALL, being lower in children (FC, -3.74; P, 0.0037). Results were confirmed by qRT-PCR (down-regulated in pediatric patients, P 0.003161). The other members of the miR-17-92 cluster did not differ significantly. CONCLUSIONS: Pediatric and adult patients with ALL have remarkably similar patterns of immune-cell-associated miRNAs in their bone marrow at diagnosis. However, the low expression of miR-18a in pediatric ALL is interesting and demands further study.

Detection of Group A Streptococcus from Pharyngeal Swab Samples by Bacterial Culture Is Challenged by a Novel mariPOC Point-of-Care Test.

mariPOC is a novel point-of-care test system for rapid detection of respiratory tract infections. We compared the performance of the mariPOC test to that of bacterial culture for detecting group A streptococcus (GAS) in 219 pharyngitis patients (ages 1-64 years) and 109 healthy asymptomatic controls (ages 19-69 years). In addition, 42 patient samples were analyzed by quantitative PCR (qPCR). Of the 219 pharyngeal patient samples, 32 were positive in a GAS bacterial culture (prevalence 15%) and 65 (30%) in the mariPOC test. The amount of GAS in samples reported positive by the mariPOC test and negative by culture was, on average, 10-fold less than that of those positive in both methods. This indicated that the negative results in bacterial cultures were due to lower sensitivity. The qPCR results were positive and in line with the mariPOC results in 43% of the discordant samples studied. Two GAS culture-positive samples were negative by the mariPOC test. The prevalences of GAS in the control subjects were 2% and 6% by culture and mariPOC results, respectively. We conclude that the mariPOC antigen detection test is more sensitive than the conventional bacterial culture for the detection of GAS among symptomatic pharyngitis patients. The higher prevalence of GAS by the mariPOC test among symptomatic patients was probably not due to carriership, since among the control patients, the difference in the prevalence of GAS by the mariPOC test and culture was not nearly as high, 15% versus 4%, respectively. Clinical trials are needed to show the clinical importance of our findings.

Dasatinib promotes Th1-type responses in granzyme B expressing T-cells.

Tyrosine kinase inhibitors (TKIs) have dramatically improved the outcome of chronic myeloid leukemia (CML). Besides inhibiting target kinases in leukemic cells, 2nd generation TKI dasatinib also inhibits off-targets in immune effector cells resulting in atypical immune responses in some patients. Dasatinib has been described to increase the proportion of late effector memory T-cells, however, to date no follow-up studies have been performed in first-line patients. In this study, we explored the functional properties of T-cells using primary samples from CML patients (n = 28) on TKI therapy. Granzyme B (GrB) was used as a marker for late phase antigen experienced CD4+ and CD8+ T-cells. Dasatinib treatment increased the numbers of both GrB expressing memory CD4+ and CD8+ T-cells when compared with healthy controls. Functionally, the GrB+CD4+ T-cells were highly active and differentiated into Th1-type T-cells capable of producing IFN-γ, which is important for tumor control. Similar kind of increase was not observed during imatinib or nilotinib therapy. These data support the dual mode of action of dasatinib: potent BCR-ABL1 inhibition in leukemic cells is accompanied by the enhancement of cellular immunity, which may have implications in the long-term control of leukemia.

Enlarged memory T-cell pool and enhanced Th1-type responses in chronic myeloid leukemia patients who have successfully discontinued IFN-α monotherapy.

A small proportion of chronic myeloid leukemia patients treated with interferon-α (IFN-α) monotherapy are able to discontinue the treatment without disease relapse although residual leukemia cells are present. Recently, we showed that these patients have increased amount of NK-cells and a distinct blood cytokine profile. We now aimed to study the function of NK- and T-cells in order to understand the role of the immune system in maintaining the treatment response after IFN-α discontinuation. The study included 13 patients: 5 patients were still treated with IFN-α monotherapy (IFN-ON, median treatment time 163 months) and 8 had stopped the treatment successfully (IFN-OFF, median time without therapy 42 months). Detailed immunophenotype and cytokine production of NK- and T-cells was analyzed with flow cytometry. In addition, the cytotoxicity of NK-cells was studied using K562 as target cells and both the degranulation and direct killing was measured. Compared to healthy controls, IFN-OFF patients had increased proportion of CD4(+) effector memory (CCR7(-)CD45RA(-); median 23% vs. healthy 16%, p = 0.009) and CD8(+) central memory T-cells (CCR7(+)CD45RA(-); median 26% vs. healthy 14%, p = 0.004). Further, upon stimulation the IFN-γ/TNF-α cytokine secretion by CD4(+) T-cells was significantly enhanced in IFN-OFF patients (median 13.7% vs. healthy 7.8%, p = 0.01), and CD4+ effector and central memory cells were the main cytokine producers. No similar increase was observed in IFN-ON group (6.5%). In addition, the proportion of NK-cells was significantly increased in IFN-OFF patients (median IFN-OFF 24%, healthy 13%, p = 0.04), but their direct killing of K562 cells was impaired. The cytotoxicity of NK-cells was also diminished in IFN-ON patients. To conclude, in addition to elevated NK-cell count, IFN-OFF patients have increased amount of memory T-cells, which are able to induce strong cytokine response upon stimulation. This activity may contribute to the maintenance of prolonged remission after successful IFN-α discontinuation.

Imatinib and pegylated IFN-α2b discontinuation in first-line chronic myeloid leukemia patients following a major molecular response.

OBJECTIVES: Previous studies indicate that 40-50% of patients with chronic myeloid leukemia in prolonged complete molecular remission may discontinue imatinib therapy without imminent relapse. The combination of pegylated interferon-alpha (Peg-IFN-α2b) and imatinib may increase the rate of successful discontinuation. METHODS: In this pilot study, we prospectively stopped imatinib from patients (n = 12) who had achieved major molecular response (MMR) after ≥12 months of treatment with either imatinib or imatinib+Peg-IFN-α2b. Molecular monitoring was carried out monthly for BCR-ABL1. In addition, analyses of lymphocyte immunophenotype, function, and plasma cytokines were performed. RESULTS: In the monotherapy group, 5/6 patients lost MMR within 4 months. One patient remains to date in MR(4.0) 61 months after discontinuation. In the combination therapy group, 2/6 patients relapsed within 4 months while still receiving Peg-IFN-α2b. Four of six patients were able to discontinue both treatments, but three of these patients relapsed after 3 months. One patient is still in sustained MR(4.0) at 58 months off all treatment. All relapsed patients re-responded to imatinib. The two successfully discontinued patients had either an increased number of NK-cells or functionally active T-cells. CONCLUSIONS: A higher frequency of relapsed patients in our study in comparison with other studies may be due to the shorter duration of imatinib treatment prior to discontinuation. However, in selected patients with an active immune system, even a short duration of TKI therapy (<2 yr) may allow for therapy discontinuation but this needs to be confirmed in larger prospective studies.

Dendritic cells from Crohn's disease patients show aberrant STAT1 and STAT3 signaling.

Abnormalities of dendritic cells (DCs) and STAT proteins have been reported in Crohn's disease (CD). Studies on JAK/STAT signaling in DCs are, however, lacking in CD. We applied a flowcytometric single-cell-based phosphoepitope assay to evaluate STAT1 and STAT3 pathways in DC subsets from CD patients. In addition, circulating DC counts were determined, together with the activation-related immunophenotype. We found that IL-6- and IFN-α-induced STAT3 phosphorylation and IFN-α-induced STAT1 phosphorylation were impaired in plasmacytoid DCs (pDCs) from CD patients (P = 0.005, P = 0.013, and P = 0.006, respectively). In myeloid DCs (mDCs), IFN-α-induced STAT1 and STAT3 phosphorylation were attenuated (P<0.001 and P = 0.048, respectively), but IL-10-induced STAT3 phosphorylation was enhanced (P = 0.026). IFN-γ-induced STAT1 signaling was intact in both DC subtypes. Elevated plasma IL-6 levels were detected in CD (P = 0.004), which strongly correlated with disease activity (ρ = 0.690, P<0.001) but not with IL-6-induced STAT3 phosphorylation. The numbers of pDCs and BDCA3+ mDCs were decreased, and CD40 expression on CD1c+ mDCs was increased in CD. When elucidating the effect of IL-6 signaling on pDC function, we observed that IL-6 treatment of healthy donor pDCs affected the maturation of and modified the T-cell priming by pDCs, favoring Th2 over Th1 type of response and the expression of IL-10 in T cells. Our results implicate DC signaling in human CD. Reduced IL-6 responsiveness in pDCs, together with the attenuated IFN-α-induced signaling in both DC subtypes, may contribute to the immunological dysregulation in CD patients.

The CXCR4-CXCL12 axis in Ewing sarcoma: promotion of tumor growth rather than metastatic disease.

BACKGROUND: Chemokine receptor CXCR4, together with its ligand CXCL12, plays critical roles in cancer progression, including growth, metastasis and angiogenesis. Ewing sarcoma is a sarcoma with poor prognosis despite current therapies, particularly for patients with advanced-stage disease. Lungs and bone (marrow), organs of predilection for (primary/metastatic) Ewing sarcoma, represent predominant CXCL12 sources. METHODS: To gain insight into the role of the CXCR4-CXCL12 axis in Ewing sarcoma, CXCR4, CXCL12 and hypoxia-inducible factor-1α protein expression was studied in therapy-naïve and metastatic tumors by immunohistochemistry. CXCR4 function was assessed in vitro, by flow cytometry and proliferation/ cell viability assays, in the presence of recombinant CXCL12 and/or CXCR4-antagonist AMD3100 or under hypoxic conditions. RESULTS: Whereas CXCR4 was predominantly expressed by tumor cells, CXCL12 was observed in both tumor and stromal areas. Survival analysis revealed an (expression level-dependent) negative impact of CXCR4 expression (p < 0.04). A role for the CXCR4-CXCL12 axis in Ewing sarcoma growth was suggested by our observations that i) CXCR4 expression correlated positively with tumor volume at diagnosis (p = 0.013), ii) CXCL12 was present within the microenvironment of virtually all cases, iii) CXCL12 induced proliferation of CXCR4-positive Ewing sarcoma cell lines, which could be abrogated by AMD3100. CXCR4 expression was not correlated with occurrence of metastatic disease. Also, therapy-naïve tumors demonstrated higher CXCR4 expression as compared to metastases (p = 0.027). Evaluation of in vivo hypoxia-inducible factor-1α expression and culture of cells under hypoxic conditions revealed no role for hypoxia in CXCR4 expression. CONCLUSIONS: Together, our results imply a crucial role for the CXCR4-CXCL12 axis in auto- and/or paracrine growth stimulation. Integration of CXCR4-targeting strategies into first- and/or second-line treatment regimens may represent a promising treatment option for Ewing sarcoma.

The presence of sinusoidal CD163(+) macrophages in lymph nodes is associated with favorable nodal status in patients with breast cancer.

As macrophages are some of the first cells to encounter metastatic tumor cells in sentinel lymph nodes (SLN) and natural killer (NK) cells are critical to the cytotoxicity of abnormal cells, we sought to determine if these cell populations were altered in the presence of nodal metastasis. We used immunohistochemistry to assess the SLN of 47 patients with breast cancer (36 with nodal metastasis and 11 without nodal metastasis) and 10 control lymph nodes. We assessed metastatic areas and nonmetastatic areas separately for CD163, a marker of macrophages, and ANK-1, a marker for precursors of activated NK cells. Positively stained cells were manually counted in multiple high-power fields and averaged. Groups were compared with the Kruskal-Wallis test. Spearman rank order test was used for correlations. There was a lower frequency of CD163(+) macrophages in the SLN of patients with breast cancer (median, 11.0 %; range, 4.1-20.4 %) than controls (median, 16.5 %; range, 8.9-19.6 %; p = 0.002). There were no differences in the expression of ANK between patients with cancer (median, 1.4 %; range, 0.23-6.3 %) and controls (median, 1.5 %; range, 0.60-5.4 %; p = 0.5). In patients with nodal metastasis, the accumulation of CD163(+) cells in the sinuses correlated negatively with CD8(+) tumor-infiltrating lymphocytes (r (2) = 0.23; p = 0.001). These results suggest that the reduction of CD163(+) macrophages in the sinuses of the SLN is associated with nodal metastasis and may have a role in regional immunity.

Altered phenotype of peripheral blood dendritic cells in pediatric type 1 diabetes.

OBJECTIVE: Dendritic cells (DCs) are largely responsible for the activation and fine-tuning of T-cell responses. Altered numbers of blood DCs have been reported in type 1 diabetes (T1D). We aimed at characterizing the less well-known phenotypic properties of DCs in T1D. RESEARCH DESIGN AND METHODS: In a case-control setting, samples from a total of 90 children were studied by flow cytometry or by quantitative real-time PCR (qPCR). RESULTS: We found decreased numbers of myeloid DCs (mDCs) (8.97 vs. 13.4 cells/µL, P = 0.009, n = 31) and plasmacytoid DCs (pDCs) (9.47 vs. 14.6 cells/µL, P = 0.018, n = 30) in recent-onset T1D. Using a panel of antibodies against functionally important DC markers, we detected a decreased expression of CC chemokine receptor 2 (CCR2) on mDCs (percentage above negative control, P = 0.002, n = 29) and pDCs (median intensity, P = 0.003, n = 30) from T1D patients. In an independent series of children, the reduced expression of CCR2 was confirmed by qPCR in isolated mDCs (P = 0.043, n = 20). Serum concentrations of CCR2 ligands monocyte chemotactic protein-1 and -3 did not differ between the groups. A trend for an enhanced responsiveness of the nuclear factor-κB pathway (P = 0.063, n = 39) was seen in mDCs from children with ß-cell autoantibodies, which is possibly related to the reduced CCR2 expression, since CCR2 on mDCs was downregulated by nuclear factor-κB-activating agents. CONCLUSIONS: Given the role of CCR2 in DC chemotaxis and in DC-elicited Th1 differentiation, our results may indicate a functionally important DC abnormality in T1D affecting the initiation and quality of immune responses.

High Expression of Complement Component 5 (C5) at Tumor Site Associates with Superior Survival in Ewing's Sarcoma Family of Tumour Patients.

Background. Unlike in most adult-onset cancers, an association between typical paediatric neoplasms and inflammatory triggers is rare. We studied whether immune system-related genes are activated and have prognostic significance in Ewing's sarcoma family of tumors (ESFTs). Method. Data analysis was performed on gene expression profiles of 44 ESFT patients, 11 ESFT cell lines, and 18 normal skeletal muscle samples. Differential expression of 238 inflammation and 299 macrophage-related genes was analysed by t-test, and survival analysis was performed according to gene expression. Results. Inflammatory genes are activated in ESFT patient samples, as 38 of 238 (16%) inflammatory genes were upregulated (P < 0.001) when compared to cell lines. This inflammatory gene activation was characterized by significant enrichment of macrophage-related gene expression with 58 of 299 (19%) of genes upregulated (P < 0.001). High expression of complement component 5 (C5) correlated with better event-free (P = 0.01) and overall survival (P = 0.004) in a dose-dependent manner. C5 and its receptor C5aR1 expression was verified at protein level by immunohistochemistry on an independent ESFT tumour tissue microarray. Conclusion. Immune system-related gene activation is observed in ESFT patient samples, and prognostically significant inflammatory genes (C5, JAK1, and IL8) for ESFT were identified.

Metastasis to sentinel lymph nodes in breast cancer is associated with maturation arrest of dendritic cells and poor co-localization of dendritic cells and CD8+ T cells.

The regional immune systems of patients with breast cancer are immunosuppressed. Dendritic cells are professional antigen-presenting cells and present cancer-associated antigens to the adaptive immune system in sentinel lymph nodes. Dendritic cells may promote, or inhibit, an adaptive immune response to specific antigens. Our aim was to assess whether dendritic cells were associated with nodal metastasis in patients with breast cancer. Sentinel lymph nodes of 47 patients with breast cancer with varying degrees of nodal disease and ten controls were evaluated using immunohistochemistry for the accumulation of dendritic cells in general (CD1a(+)), mature dendritic cells (CD208(+)), and plasmacytoid dendritic cells (CD123(+)). Cytotoxic T cell and regulatory T cell accumulation were also evaluated. Sentinel lymph nodes with macrometastases demonstrated fewer mature dendritic cells than sentinel lymph nodes without metastasis (p = 0.028), but not controls. There were fewer mature dendritic cells to cytotoxic T cells in sentinel lymph nodes with metastasis than those without (p = 0.033). Also, there were more regulatory T cells to mature dendritic cells in sentinel lymph nodes with metastasis than those without (p = 0.02). In conclusion, our study suggests that sentinel lymph nodes with metastasis have arrest of maturation of dendritic cells, fewer mature dendritic cell interactions with cytotoxic T cells, and more regulatory T cells than sentinel lymph nodes without metastasis in patients with breast cancer. These findings extend our understanding of regional immunosuppression and suggest that most regional immunosuppressive changes are associated with nodal metastasis in breast cancer.

Chronic myeloid leukemia patients in prolonged remission following interferon-α monotherapy have distinct cytokine and oligoclonal lymphocyte profile.

Before the era of tyrosine kinase inhibitors (TKIs), interferon-alpha (IFN-α) was the treatment of choice in chronic myeloid leukemia (CML). Curiously, some IFN-α treated patients were able to discontinue therapy without disease progression. The aim of this project was to study the immunomodulatory effects of IFN-α in CML patients in prolonged remission and isolate biological markers predicting response. Due to rarity of patients on IFN-α monotherapy, a relatively small cohort of patients still on treatment (IFN-ON, n = 10, median therapy duration 11.8 years) or had discontinued IFN-α therapy but remained in remission for >2 years (IFN-OFF, n = 9) were studied. The lymphocyte immunophenotype was analyzed with a comprehensive flow cytometry panel and plasma cytokine levels were measured with multiplex bead-based assay. In addition, the clonality status of different lymphocyte subpopulations was analyzed by TCR γ/δ rearrangement assay. Median NK-cell absolute number and proportion from lymphocytes in blood was higher in IFN-OFF patients as compared to IFN-ON patients or controls (0.42, 0.19, 0.21×10(9)/L; 26%, 12%, 11%, respectively, p<0.001). The proportion of CD8+ T-cells was significantly increased in both patient groups and a larger proportion of T-cells expressed CD45RO. Most (95%) patients had significant numbers of oligoclonal lymphocytes characterized by T-cell receptor γ/δ rearrangements. Strikingly, in the majority of patients (79%) a distinct clonal Vγ9 gene rearrangement was observed residing in γδ(+) T-cell population. Similar unique clonality pattern was not observed in TKI treated CML patients. Plasma eotaxin and MCP-1 cytokines were significantly increased in IFN-OFF patients. Despite the limited number of patients, our data indicates that IFN-α treated CML patients in remission have increased numbers of NK-cells and clonal γδ(+) T-cells and a unique plasma cytokine profile. These factors may relate to anti-leukemic effects of IFN-α in this specific group of patients and account for prolonged therapy responses even after drug discontinuation.

Poor cytokine-induced phosphorylation in chronic myeloid leukemia patients at diagnosis is effectively reversed by tyrosine kinase inhibitor therapy.

OBJECTIVE: In chronic myeloid leukemia (CML), uncontrolled tyrosine kinase activity of the BCR-ABL1 oncoprotein results in aberrant signaling pathways and increased cell proliferation. Acquired immune tolerance to leukemic antigens further enables tumor cell expansion. Tyrosine kinase inhibitor (TKI) therapy interferes with the immunoregulatory system by targeting off-target kinases both in malignant and nonmalignant cells. The aim of this study was to analyze the immune cell function by phosphoprotein profiling in CML patients. MATERIALS AND METHODS: Blood samples from diagnostic phase and TKI-treated patients were analyzed by multicolor phosphoprotein flow cytometry enabling measurements at the single-cell level. Both unstimulated baseline activation status and cytokine-induced responses were evaluated. RESULTS: In diagnostic-phase and imatinib-treated patients, the baseline phosphoprotein activation status was similar to healthy controls. In dasatinib-treated patients, basal phosphoprotein levels were slightly decreased; in particular, the signal transduction and activator of transcription protein 3 pathway was affected in both myeloid and lymphoid cells. The activation responses to various cytokines, granulocyte-macrophage colony-stimulating factor in particular were significantly suppressed in untreated CML patients. During imatinib and dasatinib therapy, the aberrantly suppressed phosphorylation responses were normalized. CONCLUSIONS: Cytokine responses are hampered in untreated CML patients, which may have an effect on various immunological processes in vivo. Interestingly, during TKI treatment, phosphorylation responses were normal, suggesting that TKI treatment does not alter the reactivity of healthy immune effector cells. However, dasatinib treatment was associated with diminished basal activation of the immunosuppressive signal transduction and activator of transcription protein 3 signaling pathway, which could have clinical significance in reversing the lymphocyte anergy against tumor cells.

Simultaneous Foxp3 and IDO expression is associated with sentinel lymph node metastases in breast cancer.

BACKGROUND: There is evidence that the immune systems of patients with breast cancer are dysfunctional. Regulatory T cells (Tregs), and IDO, an immunosuppressive enzyme, are associated with more advanced disease in some cancers and may promote immunologic tolerance to tumors. Our aim was to assess whether expression of Foxp3, a marker of Tregs, and IDO were linked with nodal metastasis in breast cancer patients. Inhibitors of IDO are available and could potentially demonstrate utility in breast cancer if IDO drives progression of disease. METHODS: Sentinel lymph nodes (SLN) of 47 breast cancer patients with varying degrees of nodal disease and 10 controls were evaluated for expression of Foxp3 and IDO using immunohistochemistry. Positively stained cells were quantified and their distribution within the SLN noted. RESULTS: The proportion of Foxp3+ cells was higher in SLN of cancer patients than controls (19% v. 10%, p < 0.001). Specifically, there were more Foxp3+ cells in SLN with metastasis than tumor-free SLN (20% v. 14%, p = 0.02). The proportion IDO+ cell in SLN of cancer patients was not statistically different than controls (4.0% v. 1.6%, p = 0.08). In order to demonstrate the combined immunosuppressive effect of Foxp3 and IDO, we categorized each SLN as positive or negative for Foxp3 and IDO. The Foxp3+/IDO+ group almost exclusively consisted of cancer patients with node positive disease. CONCLUSION: In conclusion, our study shows that Foxp3+ cells are associated with more advanced disease in breast cancer, a finding that is proving to be true in many other cancers. As IDO has been found to promote differentiation of Tregs, IDO may become a suitable target to abrogate the development of T-cell tolerance and to promote an effective immune response to breast cancer. Our results about the combined expression of IDO and Foxp3 in metastastic SLN support this assumption.

Pediatric cancers are infiltrated predominantly by macrophages and contain a paucity of dendritic cells: a major nosologic difference with adult tumors.

PURPOSE: Adult cancer is frequently preceded by a period of prolonged chronic inflammation caused by infectious microbial agents or physical or chemical irritants. By contrast, an association between the classic pediatric neoplasias and inflammatory triggers is only rarely recognized. We hypothesized that the difference could be reflected in the inflammatory cell infiltrates of pediatric and adult cancer. EXPERIMENTAL DESIGN: Three investigators retrospectively studied 27 pediatric and 13 adult cancers at first diagnosis by immunohistochemistry. Inflammatory cells were identified and counted, and their location in relation to tumor tissue was analyzed. RESULTS: A majority of tumor-associated leukocytes (TAL) in adult tumors were located at the edges of tumor islands forming inflammatory foci between the supporting stroma and the malignant infiltrate. In contrast, TALs in pediatric tumors were scattered within the malignant tumor islands. In adult tumors, TALs were composed of diverse leukocyte types; but in pediatric tumors, the infiltrating cells were predominantly macrophages that accumulated in areas of necrosis within the tumors. The most striking feature in the pediatric tumors was the virtual absence of dendritic cells. The proportion of intratumoral dendritic cells in pediatric samples was 4.1%; whereas in adult tumors, they formed 36.9% of TALs within the tumor islands and 25.1% around the tumors. CONCLUSIONS: We conclude that TALs in pediatric cancers are composed mainly of macrophages and largely devoid of dendritic cell. The findings may provide a major nosologic difference reclassifying pediatric and adult tumors based on nominal inflammatory and noninflammatory etiologies.

A basis for distinguishing cultured dendritic cells and macrophages in cytospins and fixed sections.

There is a burgeoning literature on the contrasting role of intratumoral dendritic cells (DCs) and tumor-associated macrophages, making reliable identification of both cell types in clinical and experimental tissue sections important. However, because these cell types are closely related and share several differentiation antigens, their absolute distinction in tissue sections is difficult. We differentiated DCs and macrophages from monocytes in vitro, prepared cytospins and paraffin-embedded sections of the various cell populations, and tested a variety of antibodies that purportedly recognize monocytes and DCs for their capacity to react and distinguish cells after conventional formalin fixation. Cultured DCs but not macrophages were detected by fascin, DC-LAMP, and CD83 with a predictable increase in staining that paralleled their maturation. Staining by CD1a was found on immature DCs but was weak and absent on mature DCs and macrophages, respectively. CD14 and CD163 were characteristic for macrophages and absent on DCs. CD68, HLA-DR, and S100 did not discriminate between DCs and macrophages. We conclude that antigens such as HLA-DR and S100 are not in themselves sufficient for identification of DCs in formalin-fixed tissue sections, but that additional macrophage-specific (CD14, CD163) and DC-specific (CD1a, CD83, fascin, DC-LAMP) antigens should be used to distinguish cell types from each other and to provide information on their state of maturation.

Imaging analysis of STAT1 and NF-kappaB translocation in dendritic cells at the single cell level.

Rapid assessment of immune or stem cells, which are now widely applied in the clinical setting of cancer treatment, is necessary to speed their development and to determine their quality. We have evaluated immature dendritic cells (iDC) by semiautomated imaging cytometry which provides detailed assessment at a single cell level. Nuclear translocation of NF-kappaB was studied by imaging analysis as well as electrophoretic mobility shift assay with an excellent correlation (r = 0.981) over a broad range of lipopolysaccharide (LPS) concentrations. Imaging analysis was time saving (5 h vs. 3 days), and required 30- to 100-fold less cells per analysis. Single cell information revealed remarkable heterogeneity between individual iDC and permitted detection of responses to 40 pg/ml of LPS. In IL-1beta/IFNgamma activated iDC, STAT1 responses preceded NF-kappaB responses, and the expression of both was strongly correlated in individual cells (p < 0.001). IFNgamma amplified IL-1-induced NF-kappaB responses. NF-kappaB responses to IL-1beta, CD40L, and LPS were donor-dependent (n = 7), correlated with the quality of iDC preparations (p = 0.002), and IL-12 p70 production (p = 0.010). NF-kappaB measurements in iDC within mixed cell cultures (iDC, NK, K562) demonstrated that these strategies are applicable for analyses of complex cell-cell interactions. Imaging analysis is a method that could be valuable for quality control of cell therapy preparations.

Imaging analysis of STAT1 and NF-kappaB translocation in dendritic cells at the single cell level.

Rapid assessment of immune or stem cells, which are now widely applied in the clinical setting of cancer treatment, is necessary to speed their development and to determine their quality. We have evaluated immature dendritic cells (iDC) by semiautomated imaging cytometry which provides detailed assessment at a single cell level. Nuclear translocation of NF-kappaB was studied by imaging analysis as well as electrophoretic mobility shift assay with an excellent correlation (r=0.981) over a broad range of lipopolysaccharide (LPS) concentrations. Imaging analysis was time saving (5 h vs. 3 days), and required 30- to 100-fold less cells per analysis. Single cell information revealed remarkable heterogeneity between individual iDC and permitted detection of responses to 40 pg/ml of LPS. In IL-1beta/IFNgamma activated iDC, STAT1 responses preceded NF-kappaB responses, and the expression of both was strongly correlated in individual cells (p<0.001). IFNgamma amplified IL-1-induced NF-kappaB responses. NF-kappaB responses to IL-1beta, CD40L, and LPS were donor-dependent (n=7), correlated with the quality of iDC preparations (p=0.002), and IL-12 p70 production (p=0.010). NF-kappaB measurements in iDC within mixed cell cultures (iDC, NK, K562) demonstrated that these strategies are applicable for analyses of complex cell-cell interactions. Imaging analysis is a method that could be valuable for quality control of cell therapy preparations.

A novel epitope of N-CAM defines precursors of human adherent NK cells.

Activated, adherent natural killer (A-NK) cells represent a distinct subpopulation of interleukin (IL)-2-stimulated NK cells, which are selectively endowed with the increased expression of integrins and ability to adhere to solid surfaces, migrate into, infiltrate, and destroy cancerous tissues. The present study defines the phenotype and functions of precursors of A-NK (pre-A-NK) cells in humans. Peripheral blood pre-A-NK cells, in contrast to the rest of NK cells, express a novel epitope of CD56 neuronal cell adhesion molecule, termed ANK-1, and increased cell-surface levels of integrins. Pre-A-NK cells also express low levels of CD56 and CD161, and some express CD162 receptor, do not express CD25 or activation markers, and are effective mediators of NK cytotoxicity. Thus, pre-A-NK cells are generally similar to CD56(dim) NK cells. However, pre-A-NK cells differ from the main NK cell subpopulation by having a lower expression level of CD16 and a lower ability to mediate redirected antibody-dependent, cell-mediated cytotoxicity. More importantly, pre-A-NK cells are preferentially endowed with the ability to rapidly respond to IL-2 by integrin-mediated adherence to endothelial cells, extracellular matrix, and plastic. This early, specific response of pre-A-NK cells to IL-2 is followed by their activation, vigorous proliferation, and differentiation into phenotypically and functionally similar A-NK cells. Pre-A-NK cells represent only approximately 26% of peripheral blood NK cells but encompass the majority of NK cells in normal and cancerous, solid tissues. We conclude that pre-A-NK cells represent a distinct subset of resting, mature NK cells with the characteristics indicative of their ability to migrate and reside in solid tissues.