Data integration from pathology slides for quantitative imaging of multiple cell types within the tumor immune cell infiltrate

Background

Immune cell infiltrates (ICI) of tumors are scored by pathologists around tumor glands. To obtain a better understanding of the immune infiltrate, individual immune cell types, their activation states and location relative to tumor cells need to be determined. This process requires precise identification of the tumor area and enumeration of immune cell subtypes separately in the stroma and inside tumor nests. Such measurements can be accomplished by a multiplex format using immunohistochemistry (IHC).

Method

We developed a pipeline that combines immunohistochemistry (IHC) and digital image analysis. One slide was stained with pan-cytokeratin and CD45 and the other slide with CD8, CD4 and CD68. The tumor mask generated through pan-cytokeratin staining was transferred from one slide to the other using affine image co-registration. Bland-Altman plots and Pearson correlation were used to investigate differences between densities and counts of immune cell underneath the transferred versus manually annotated tumor masks. One-way ANOVA was used to compare the mask transfer error for tissues with solid and glandular tumor architecture.

Results

The overlap between manual and transferred tumor masks ranged from 20%–90% across all cases. The error of transferring the mask was 2- to 4-fold greater in tumor regions with glandular compared to solid growth pattern (p < 10^?6). Analyzing data from a single slide, the Pearson correlation coefficients of cell type densities outside and inside tumor regions were highest for CD4 + T-cells (r = 0.8), CD8 + T-cells (r = 0.68) or CD68+ macrophages (r = 0.79). The correlation coefficient for CD45+ T- and B-cells was only 0.45. The transfer of the mask generated an error in the measurement of intra- and extra- tumoral CD68+, CD8+ or CD4+ counts (p < 10^?10).

Conclusions

In summary, we developed a general method to integrate data from IHC stained slides into a single dataset. Because of the transfer error between slides, we recommend applying the antibody for demarcation of the tumor on the same slide as the ICI antibodies.

     

Sipuleucel-T immune parameters correlate with survival: an analysis of the randomized phase 3 clinical trials in men with castration-resistant prostate cancer

Purpose

Sipuleucel-T, the first FDA-approved autologous cellular immunotherapy for treatment of advanced prostate cancer, is manufactured by activating peripheral blood mononuclear cells, including antigen presenting cells (APCs), with a fusion protein containing prostatic acid phosphatase. Analysis of data from three phase 3 trials was performed to immunologically characterize this therapy during the course of the three doses, and to relate the immunological responses to overall survival (OS).

Methods

Sipuleucel-T product characteristics [APC numbers, APC activation (CD54 upregulation), and total nucleated cell (TNC) numbers] were assessed in three randomized, controlled phase 3 studies (N = 737). Antigen-specific cellular and humoral responses were assessed in a subset of subjects. The relationships between these parameters and OS were assessed.

Results

APC activation occurred in the first dose preparation [6.2-fold, (4.65, 7.70); median (25th, 75th percentile)] and increased in the second [10.6-fold (7.83, 13.65)] and third [10.5-fold (7.89, 13.65)] dose preparations. Cytokines and chemokines associated with activated APCs were produced during the manufacture of each dose; T-cell activation-associated cytokines were detected in the second and third dose preparations. Antigen-specific T cells were detectable after administration of the first sipuleucel-T dose. Cumulative APC activation, APC number, and TNC number correlated with OS (P < 0.05). Antigen-specific immune responses were observed in 78.8 % of monitored subjects and their presence correlated with OS (P = 0.003).

Conclusion

Sipuleucel-T broadly engages the immune system by activating APCs ex vivo and inducing long-lived immune responses in vivo. These data indicate antigen-specific immune activation as a mechanism by which sipuleucel-T prolongs OS.     

Autologous cytokine-induced killer cell immunotherapy in lung cancer: a phase II clinical study

Objective

Cytokine-induced killer (CIK) cells have the ability to kill tumor in vitro and in vivo. This study was designed to evaluate the clinical efficacy of CIK cell immunotherapy following regular chemotherapy in patients with non-small cell lung cancer (NSCLC) after surgery.

Methods

A paired study, with 87 stage I–IV NSCLC patients in each group, was performed. Patients received either chemotherapy (arm 2) or chemotherapy in combination with autologous CIK cell immunotherapy (arm 1). Progression-free survival (PFS) and overall survival (OS) were evaluated.

Results

Of the 87 paired patients, 50 had early-stage disease (stage I–IIIA) and 37 had advanced-stage disease (stage IIIB–IV). Among early-stage patients, the distribution of 3-year PFS rate and median PFS time showed no statistical difference between the two groups (p = 0.259 and 0.093, respectively); however, the 3-year OS rate and median OS time in arm 1 were significantly higher than those in arm 2 (82 vs. 66 %; p = 0.049 and 73 vs. 53 months; p = 0.006, respectively). Among the advanced-stage patients, the 3-year PFS and OS rates of arm 1 were significantly higher than those of arm 2 (6 vs. 3 %; p < 0.001 and 31 vs. 3 %; p < 0.001, respectively); the median PFS and OS times in arm 1 were also significantly longer than those in arm 2 (13 vs. 6 months; p = 0.001 and 24 vs. 10 months; p < 0.001, respectively). Multivariate analyses indicated that the frequency of CIK cell immunotherapy was significantly associated with prolonged PFS (HR = 0.91; 95 % CI 0.85–0.98; p = 0.012) and OS (HR = 0.83; 95 % CI, 0.74–0.93; p = 0.001) in the arm 1.

Conclusions

The data suggested that CIK cell immunotherapy could improve the efficacy of conventional chemotherapy in NSCLC patients, and increased frequency of CIK cell treatment could further enhance the beneficial effects. A multi-center randomized trial is being carried out in our hospital to further validate these findings.     

Increasing relevance of spring temperatures for Norway spruce trees in Davos,Switzerland, after the 1950s

Key message

Relevance of spring temperatures for tree-ring growth steadily increased since 1950s. Closely linked tree-ring growth and net CO ₂ exchange driven by spring temperatures.

Abstract

We investigated long-term (over 100 years) tree-ring width (TRW) variabilities as well as short-term (10 years) variations in net ecosystem productivity (NEP) in response to climate to assess the driving factors for stem growth of Norway spruce in a subalpine forest at Davos in Switzerland. A tree-ring width index (TRWi) chronology for the period from 1750 to 2006 was constructed and linked with climate data from 1876 to 2006, and with NEP available for the period from 1997 to 2006. Based on TRWi, we found that only two out of the 257 years exhibited extreme negative TRWi, compared to 29 years with extreme positive anomalies, observed mainly in recent decades. Annual temperature, annual precipitation, as well as autumn and winter temperature signals were well preserved in the TRWi chronology over the last 130 years. Spring temperatures became increasingly relevant for TRWi, explaining less than 1 % of the variation in TRWi for the period from 1876 to 2006, but 8 % for the period from 1950 to 2006 (p = 0.032), and even 47 % for 1997–2006 (p = 0.028). We also observed a strong positive relationship between annual TRWi and annual NEP (r = 0.661; p = 0.037), both strongly related to spring temperatures (r = 0.687 and r = 0.678 for TRWi and NEP, respectively; p = 0.028; p = 0.032). Moreover, we found strong links between monthly NEP of March and annual TRWi (r = 0.912; p = 0.0001), both related to March temperatures (r = 0.767, p = 0.010 and r = 0.724, p = 0.018, respectively). Thus, under future climate warming, we expect stem growth of these subalpine trees and also ecosystem carbon (C) sequestration to increase, as long as water does not become a limiting factor.     

Tumour infiltrating lymphocytes correlate with improved survival in patients with oesophageal adenocarcinoma

Background

Oesophageal adenocarcinoma (OAC) is increasingly common in the west, and survival remains poor at 10–15 % at 5 years. Immune responses are increasingly implicated as a determining factor of tumour progression. The ability of lymphocytes to recognise tumour antigens provides a mechanism for a host immune attack against cancer providing a potential treatment strategy.

Materials and Methods

Tumour infiltrating lymphocytes (TILs: CD3+, CD4+, CD8+ and FOXp3+) were assessed by immunohistochemistry using tissue microarrays in a contemporary and homogeneous cohort of OAC patients (n = 128) undergoing curative treatment.

Results

Multivariate analysis identified three independent prognostic factors for improved cancer-specific survival (CSS): increased CD8+ TILs (p = 0.003), completeness of resection (p < 0.0001) and lower pathological N stage (p < 0.0001). Independent prognostic factors for favourable disease-free survival included surgery-only treatment (p = 0.015), completeness of resection (p = 0.001), increased CD8+ TILs (p < 0.0001) and reduced pathological N stage (p < 0.0001). Higher levels of TILs in the pathological specimen were associated with significant pathological response to neoadjuvant chemotherapy (NAC). On multivariate analysis increased levels of CD4+ (p = 0.017) and CD8+ TILs (p = 0.005) were associated with significant local tumour regression and lymph node downstaging, respectively.

Discussion

Our results establish an association of TILs and survival in OAC, as seen in other solid tumours, and identify particular TIL subsets that are present at higher levels in patients who responded to NAC compared to non-responders. These findings highlight potential therapeutic strategies in EAC based on utilising the host immunological response and highlight the immune responses biomarker potential.

     

Efficacy of postoperative adjuvant transfusion of cytokine-induced killer cells combined with chemotherapy in patients with colorectal cancer

Purpose

To assess the activity and safety of postoperative adjuvant immunotherapy with transfusion of cytokine-induced killer (CIK) cells combined with chemotherapy in patients with colorectal cancer.

Methods

We retrospectively studied 96 consecutive patients with colorectal cancer who were treated with resection between January 2010 and December 2012 as well as adjuvant chemotherapy. Twenty-one of these patients accepted at least 1 cycle of CIK cell transfusion for immunotherapy (CIK group). Disease free survival (DFS), immune cells and treatment related side effects were assessed. The patients were followed up until May 2013.

Results

By the end of follow-up, 10 patients (10.42 %) had died. Eighteen patients (18.75 %) had withdrawn. All the patients in the CIK group are still alive, and only 1 patient had withdrawn. Patients in the CIK group had significantly longer DFS than those in the control group [HR = 0.28, 95 % CI (0.09, 0.91), p = 0.034]. The 2-year DFS rates of patients in the CIK group and the control group were 59.65 ± 24.80 % and 29.35 ± 6.39 %, respectively. The CD4⁺/CD8⁺ ratios were significantly lower during the period of chemotherapy than those before chemotherapy (p = 0.0038), while the ratios were significantly higher during the period of CIK cell transfusion than those before CIK therapy (p = 0.0484). There were no immediate adverse reactions to the CIK cell transfusions.

Conclusion

Adjuvant transfusion of CIK cells prolongs DFS in patients with colorectal cancer.     

Pretreatment neutrophil to lymphocyte ratio is associated with response to therapy and prognosis of advanced non-small cell lung cancer patients treated with first-line platinum-based chemotherapy

Background

Neutrophil to lymphocyte ratio (NLR) has been shown to be a prognosis indicator in different types of cancer. We aimed to investigate the association between NLR and therapy response, progression free survival (PFS) and overall survival (OS) in advanced non-small cell lung cancer (NSCLC) patients treated with first-line platinum-based chemotherapy.

Methods

Patients who were hospitalized between January 2007 and December 2010 were enrolled and eliminated according to the inclusion and exclusion criteria. The NLR was defined as the absolute neutrophil count divided by the absolute lymphocyte count. Logistic regression analysis was applied for response rate and Cox regression analysis was adopted for PFS and OS. A P value of ≤0.05 was considered to be statistically significant.

Results

A total of 182 patients were enrolled in the current study. The median PFS was 164.5 days and median OS was 439.5 days. The statistical analysis data indicated that low pretreatment NLR (≤ 2.63) (OR = 2.043, P = 0.043), decreased posttreatment NLR (OR = 2.368, P = 0.013), well and moderate differentiation (OR = 2.773, P = 0.021) and normal CEA level (≤ 9.6 ng/ml) (OR = 2.090, P = 0.046) were associated with response to first-line platinum-based chemotherapy. A high pretreatment NLR (HR = 1.807, P = 0.018 for PFS, HR = 1.761, P = 0.020 for OS) and distant metastasis (HR = 2.118, P = 0.008 for PFS, HR = 2.753, P = 0.000 for OS) were independent prognostic factors for PFS and OS.

Conclusion

Elevated pretreatment NLR might be a potential biomarker of worse response to first-line platinum-based chemotherapy and shorter PFS and OS for advanced NSCLC patients. To confirm these findings, larger, prospective and randomized studies are needed.     

Critical role of spatial interaction between CD8+ and Foxp3+ cells in human gastric cancer: the distance matters

Purpose

In various cancer types, an abundance of FoxP3⁺ regulatory T cells (Treg) has been associated with an unfavorable outcome. Yet, the role of Treg on cancer immunity has been shown to be complex. In single cell marker technique, other tumor-infiltrating lymphocytes (TILs) such as cytotoxic CD8⁺ T cells (CTL) also influenced prognosis. This study for the first time investigates the concurrent spatial distribution pattern of CD8⁺ and FoxP3⁺ TILs and their prognostic impact in human gastric cancer.

Materials and methods

Tumor tissue microarrays of 50 patients with surgically treated adenocarcinoma of the cardia were studied. An immunohistochemical double staining of CD8⁺ and FoxP3⁺ TILs was performed. Cell counts and cell-to-cell distances in tumor epithelium and stroma were evaluated with image-processing software. Metastasis-free survival, no-evidence-of-disease survival, and overall survival were investigated (mean follow-up time 6.9 years).

Results

High intraepithelial infiltration of CD8⁺ and FoxP3⁺ TIL was associated with the improved 10-year metastasis-free survival (83 vs. 54 %, p = 0.04 and 85 vs. 59 %, p = 0.09, respectively). Considering cell-to-cell distance and comparing patients with functional (30–110 μm) versus nonfunctional distances of CD8⁺ and FoxP3⁺ TILs, 10-year survival rates differed between 89 and 55 % (p = 0.009), respectively.

Conclusion

Prognostic influence of tumor-infiltrating immune cells in gastric cancer critically depends on their cell-to-cell distance. FoxP3⁺ TILs must be located within a distance between 30 and 110 μm of CD8⁺ T cells to positively impact on prognosis.     

Association between the blood concentrations of ammonia and carnitine/amino acid of schizophrenic patients treated with valproic acid

Background

Administration of valproic acid (VPA) is complicated with approximately 0.9% of patients developing hyperammonemia, but the pathogenesis of this adverse effect remains to be clarified. The aim of the present study was to search for mechanisms associated with VPA-induced hyperammonemia in the light of changes in serum amino acids concentrations associated with the urea cycle of schizophrenic patients.

Method

Blood samples (10 mL) were obtained from 37 schizophrenic patients receiving VPA for the prevention of violent behaviors in the morning after overnight fast. Blood concentrations of ammonia, VPA, free carnitine, acyl-carnitine, and 40 amino acids including glutamate and citrulline were measured for each patient. Univariate and multivariate regression analyses were performed to identify amino acids or concomitantly administered drugs that were associated with variability in the blood concentrations of ammonia.

Result

The blood ammonia level was positively correlated with the serum glutamate concentration (r = 0.44, p < 0.01) but negatively correlated with glutamine (r = ?0.41, p = 0.01), citrulline (r = ?0.42, p = 0.01), and glycine concentrations (r = ?0.54, p < 0.01). It was also revealed that the concomitant administration of the mood stabilizers (p = 0.04) risperidone (p = 0.03) and blonanserin (p < 0.01) was positively associated with the elevation of the blood ammonia level.

Conclusion

We hypothisized that VPA would elevate the blood ammonia level of schizophrenic patients. The observed changes in serum amino acids are compatible with urea cycle dysfunction, possibly due to reduced carbamoyl-phosphate synthase 1 (CPS1) activity. We conclude that VPA should be prudently prescribed to schizophrenic patients, particularly those receiving mood stabilizers or certain antipsychotics.

     

Prognostic factors in infective endocarditis in general hospitals in the Netherlands

Introduction

Despite advances in treatment, infective endocarditis (IE) still ranks amongst the most lethal infectious diseases. We sought to determine prognostic factors in general hospitals in the Netherlands as research in this setting is scarce.

Results

Between 2004 and 2011, we identified 216 cases of IE, 30.1?% of which were prosthetic valve IE. This leads to an annual incidence of IE of 5.7 new cases per 100,000 persons per year. Women were less likely to undergo surgical intervention (OR = 1.96, 95?% CI 1.06–3.61, p = 0.031). Also, ageing was an independent prognostic factor for not receiving surgery in a multivariate analysis (annual OR = 1.04, 95?% CI 1.02–1.06, p < 0.001). Female sex was a prognostic factor for mortality (OR = 2.35, 95?% CI 1.29–4.28, p = 0.005). Age was also an independent prognostic factor for mortality (OR = 1.05, 95% CI 1.03–1.08, p < 0.001). Conservative treatment was a prognostic factor for mortality (OR = 3.39, 95?% CI 1.80–6.38, p < 0.001) whereas surgical intervention was an independent prognostic factor for adverse events (OR = 3.03, 95% CI 1.64–5.55, p < 0.001). Staphylococcus aureus was an independent prognostic factor for adverse events (OR = 2.05, 95?% CI 1.10–3.84, p = 0.024) but not for mortality.

Conclusion

This study shows that endocarditis in general hospitals has a high rate of morbidity and mortality. Even when treated, it ranks as one of the most lethal infectious diseases in the Netherlands, especially in women and the elderly.

     

Coronary microcirculatory dysfunction is associated with left ventricular dysfunction during follow-up after STEMI

Background

Coronary microvascular resistance is increased after primary percutaneous coronary intervention (PCI) for ST-elevation myocardial infarction (STEMI), which may be related in part to changed left ventricular (LV) dynamics. Therefore we studied the coronary microcirculation in relation to systolic and diastolic LV function after STEMI.

Methods

The study cohort consisted of 12 consecutive patients, all treated with primary PCI for a first anterior wall STEMI. At 4 months, we assessed pressure-volume loops. Subsequently, we measured intracoronary pressure and flow velocity and calculated coronary microvascular resistance. Infarct size and LV mass were assessed using magnetic resonance imaging.

Results

Patients with an impaired systolic LV function due to a larger myocardial infarction showed a higher baseline average peak flow velocity (APV) than the other patients (26?±?7 versus 17?±?5 cm/s, p?=?0.003, respectively), and showed an impaired variable microvascular resistance index (2.1?±?1.0 versus 4.1?±?1.3 mmHg?cm^?1?s^?1, p?=?0.003, respectively). Impaired diastolic relaxation time was inversely correlated with hyperaemic APV (r?=??0.56, p?=?0.003) and positively correlated with hyperaemic microvascular resistance (r?=?0.48, p?=?0.01). LV dilatation was associated with a reduced variable microvascular resistance index (r?=?0.78, p?=?0.006).

Conclusion

A larger anterior myocardial infarction results in impaired LV performance associated with reduced coronary microvascular resistance variability, in particular due to higher coronary blood flow at baseline in these compromised left ventricles.     

Flow Cytometry Analysis of Neural Differentiation Markers Expression in Human Glioblastomas May Predict Their Response to Chemotherapy

Glioblastoma multiforme (GBM) represents an extremely chemoresistant tumour type. Here, authors analysed the immunophenotype of GBM tumours by flow cytometry and correlated the immunophenotypic characteristics with sensitivity to chemotherapy. The expression of selected neural and non-neural differentiation markers including A2B5, CD34, CD45, CD56, CD117, CD133, EGFR, GFAP, Her-2/neu, LIFR, nestin, NGFR, Pgp and vimentin was analysed by flow cytometry in eleven GBM (WHO gr.IV) patients. The sensitivity of tumour cells to a panel of chemotherapeutic agents was tested by the MTT assay. All tumours were positive for A2B5, CD56, nestin and vimentin. CD133, EGFR, LIFR, NGFR and Pgp were expressed only by minor tumour cell subpopulations. CD34, CD45, CD117, GFAP and Her-2/neu were constantly negative. Direct correlations were found between the immunophenotypic markers and chemosensitivity: A2B5 vs lomustine (r² = 0.642, P = 0.033), CD56 vs cisplatin (r² = 0.745, P = 0.013), %Pgp⁺ vs vincristine (r² = 0.846, P = 0.008), and %NGFR⁺ vs daunorubicine (r² = 0.672, P = 0.047) and topotecan (r² = 0.792, P = 0.011). In contrast, inverse correlations were observed between: EGFR vs paclitaxel (r² = ?0.676, P = 0.046), CD133 vs dacarbazine (r² = ?0.636, P = 0.048) and LIFR vs daunorubicine (r² = ?0.878, P = 0.004). Finally, significant associations were also found among sensitivities to different chemotherapeutic agents and among different immunophenotypic markers. In conclusion, histopathologically identical GBM tumours displayed a marked immunophenotypic heterogeneity. The expression of A2B5, CD56, NGFR and Pgp appeared to be associated with chemoresistance whereas CD133, EGFR and LIFR expression was characteristic of chemosensitive tumours. We suggest that flow cytometric imunophenotypic analysis of GBM may predict chemoresponsiveness and help to identify patients who could potentially benefit from chemotherapy.     

TLR8 stimulation enhances cetuximab-mediated natural killer cell lysis of head and neck cancer cells and dendritic cell cross-priming of EGFR-specific CD8+ T cells

Background

Cetuximab is an anti-epidermal growth factor receptor (EGFR) monoclonal antibody that prolongs survival in the treatment for head and neck cancer (HNC), but only in 10–20 % of patients. An immunological mechanism of action such as natural killer (NK) cell–mediated antibody-dependent cellular cytotoxicity (ADCC) has been suggested. We investigated the effects of activating toll-like receptor (TLR)-8 to enhance activity of cetuximab-stimulated, FcγR-bearing cells.

Objective

To determine the capability of TLR8-stimulation to enhance the activation and function of NK cells and dendritic cells (DC) in the presence of cetuximab-coated HNC cells.

Methods

Peripheral blood mononuclear cells (PBMC), NK, DC, and CD8⁺ T cells were isolated and analyzed using ⁵¹Cr release ADCC, flow cytometry analysis, cytokine ELISA, and EGFR_853-861 tetramer staining.

Results

TLR8 stimulation of unfractionated PBMC led to enhanced cetuximab-mediated ADCC in healthy donors (p < 0.01) and HNC patients (p < 0.001), which was dependent on NK cells. Secretion of Th1 cytokines TNFα (p < 0.0001), IFNγ (p < 0.0001), and IL-12p40 (p < 0.005) was increased. TLR8 stimulation of PBMC augmented cetuximab-enhanced NK cell degranulation (p < 0.001). TLR8-stimulated NK cells enhanced DC maturation markers CD80, CD83, and CD86 in co-culture with cetuximab-treated HNC cells. TLR8 stimulation of NK-DC co-cultures significantly increased DC priming of EGFR-specific CD8⁺ T cells in the presence of cetuximab.

Discussion

VTX-2337 and cetuximab combination therapy can activate innate and adaptive anti-cancer immune responses. Further investigation in human trials will be important for determining the clinical benefit of this combination and for determining biomarkers of response.     

Increase in Candida parapsilosis Fungemia in Critical Care Units: A 6-Years Study

Objectives

We aimed to asses possible clinically significant differences between C. parapsilosis and other candida species candidemia receiving care in the intensive care unit (ICU) setting.

Methods

The study included 118 adult patients diagnosed as candidemia after admission to the ICU of a university hospital between January 2004 and December 2009. Data about demographic characteristics, underlying diseases, and risk factors for ICU-related candidemia were collected.

Results

During the study period, 118 patients with candidemia were identified among 2,853 patients admitted into the ICU. Candidemia was seen in 41.4 cases per 1,000 ICU admissions. The overall incidence of candidemia in ICU patients during the study period was 2.09 per 1,000 hospital admissions. Of the isolates, 18.6% were C. albicans and 81.4% were C. non-albicans. The species most frequently isolated was C. parapsilosis (66.1%, 78/118). The distribution of other Candida spp. was as follows: 15 had C. tropicalis (12.7%) and 3 had C. glabrata (2.5%). By Statistical analysis, when patients with candidemia who had C. parapsilosis were compared with other Candida spp., the following factors were found to be significantly associated with C. parapsilosis fungemia; intravascular catheters (p = 0.008), malignity (p = 0.049) and age (p = 0.039). Relationship was found between C. tropicalis and hematologic malignancies (p = 0.001).

Conclusions

When infections with a high mortality such as candidemia is suspected in critically ill patients, it is important to know local risk factors and epidemiological distributions of causative agents in selection of empirical and effective antifungal treatment.     

Clinical and immunological responses in metastatic melanoma patients vaccinated with a high-dose poly-epitope vaccine

Background

Safety and cellular immunogenicity of rising doses and varying regimens of a poly-epitope vaccine were evaluated in advanced metastatic melanoma. The vaccine comprised plasmid DNA and recombinant modified vaccinia virus Ankara (MVA) both expressing a string (Mel3) of seven HLA.A2/A1 epitopes from five melanoma antigens.

Methods

Forty-one HLA-A2 positive patients with stage III/IV melanoma were enrolled. Patient groups received one or two doses of DNA.Mel3 followed by escalating doses of MVA.Mel3. Immunisations then continued eight weekly in the absence of disease progression. Epitope-specific CD8+ T cell responses were evaluated using ex-vivo tetramer and IFN-γ ELISPOT assays. Safety and clinical responses were monitored.

Results

Prime-boost DNA/MVA induced Melan-A-specific CD8+ T cell responses in 22/31 (71%) patients detected by tetramer assay. ELISPOT detected a response to at least one epitope in 10/31 (32%) patients. T cell responder rates were <50% with low-dose DNA/MVA, or MVA alone, rising to 91% with high-dose DNA/MVA. Among eight patients showing evidence of clinical benefit—one PR (24 months+), five SD (5 months+) and two mixed responses—seven had associated immune responses. Melan-A-tetramer+ immunity was associated with a median 8-week increase in time-to-progression (P = 0.037) and 71 week increase in survival (P = 0.0002) compared to non-immunity. High-dose vaccine was well tolerated. The only significant toxicities were flu-like symptoms and injection-site reactions.

Conclusions

DNA.Mel3 and MVA.Mel3 in a prime-boost protocol generated high rates of immune response to melanoma antigen epitopes. The treatment was well tolerated and the correlation of immune responses with patient outcomes encourages further investigation.     

FOXP3 and CTLA4 overexpression in multiple myeloma bone marrow as a sign of accumulation of CD4+ T regulatory cells

Introduction

Multiple myeloma (MM) development involves a series of genetic abnormalities and changes in the bone marrow (BM) microenvironment, favoring the growth of the tumor and failure of local immune control. T regulatory (Treg) cells play an important role in dampening anti-tumor immune responses while T-helper-17 (Th17) cells seem to be critical for the eradication of malignant cells. The aim of our study was to characterize the expression of Treg- and Th17-related genes in total myeloma BM samples to assess their role as biomarkers, prognostic factors, and possible therapeutic targets in this incurable disease.

Methods

Expression of markers for Treg (FOXP3, CTLA4) and Th17 cells (RORγt) was determined by quantitative real-time PCR in BM aspirates of 46 MM patients, four patients with monoclonal gammopathy of undetermined significance, five solitary plasmacytomas, and five healthy BM donors. Gene expression was evaluated regarding an influence on the patients’ overall survival (OS).

Results

FOXP3 and CTLA4 presented a sixfold (p = 0.02) and 30-fold higher expression (p = 0.03), respectively, in MM patients than in controls. RORγt expression was similar in MM patients and controls. Median OS of MM patients was 16.8 (range 4.5–29.1) months, and international staging system was the only independent prognostic factor for patients survival.

Conclusions

Overexpression of FOXP3 and CTLA4 in total BM samples suggests a local accumulation of immunosuppressive Tregs, the MM tumor environment, possibly dampening anti-tumor host immune responses. Therapeutic approaches targeting Treg cells and restoring local anti-tumor immunity may provide new treatment strategies for this incurable malignancy.     

Prognostic value of CD208-positive cell infiltration in gastric cancer

Background and aim

A new marker, CD208, was recently explored as a mature interdigitating dendritic cell (DC), and the correlation between the infiltration of CD208-positive cells and clinical factors has been reported in various types of cancers. In this study, we tried to clarify the clinical implication of CD208-positive cell infiltration in gastric cancer immunohistochemically.

Patients and methods

A total of 128 gastric cancer patients who underwent a curative operation were enrolled. DCs in tumor nests were identified with two DC markers, CD208 and S-100 protein (S100), by immunohistochemistry. The correlation between clinicopathological features and the CD208- or S100-positive cell infiltration degree was analyzed.

Results

Infiltration of S100-positive cells did not correlate with the degree of CD208-positive cell infiltration. Patients with high CD208-positive cell infiltration in the peritumor had a poorer surgical outcome than those with low CD208 infiltration (p < 0.05). Multivariate analysis revealed that CD208-positive cell infiltration was not an independent prognostic factor.

Conclusion

We showed that intratumoral CD208-positive cells, as mature DCs, had an inverse correlation to patients’ postoperative outcome in gastric cancer, unlike a conventional DC marker. Evaluation of CD208-positive cell infiltration with S100-positive cell infiltration in gastric cancer is useful to predict antitumor immunological conditions in gastric cancer.     

In vivo effects of zoledronic acid on peripheral γδ T lymphocytes in early breast cancer patients

Introduction

Amino-bisphosphonates are potent activators of human γδ T cells. The aim of our study was to evaluate the immunomodulating properties of a single-dose of zoledronic acid (ZA) on γδ T cells in a select group of disease-free breast cancer patients with osteopenia.

Materials and methods

Blood samples were obtained, from 23 patients, before and 7, 28, 56, 90 and 180 days after a single-dose (4 mg) of ZA and analyzed by flow cyometry.

Results

A significant decrease of the different γδ T cell subsets was observed: Naïve (CD3+/Vdelta2+/CD45RA+/CD27+) after 180 days (P < 0.01); Central Memory (CD3+/Vdelta2+/CD45RA-CD27+) after 28 (P < 0.05), 90 (P < 0.01) and 180 days (P < 0.01); and Effector Memory (CD3+/Vdelta2+/CD45RA-/CD27-) after 56 (P < 0.01) and 90 (P < 0.05) days. Based on the observed γδ T cells kinetics patients could be divided in two groups: “responders” that showed a significant decrease in total numbers of γδ T cells and “non-responders” that showed no significant change. However, in vitro phosphoantigen stimulation of patients cells did not show significant differences in terms of IFN-γ response by Vδ2 T cells.

Conclusion

We describe for the first time a long-lasting activation of effector subsets of γδ T cells in disease-free breast cancer patients after a single-dose of ZA. Our results highlight the need to further investigate the clinical significance of the immunomodulating properties of N-BPs.     

Feasibility and relevance of compound strain imaging in non-stenotic arteries: comparison between individuals with cardiovascular diseases and healthy controls

Background

Compound strain imaging is a novel method to noninvasively evaluate arterial wall deformation which has recently shown to enable differentiation between fibrous and (fibro-)atheromatous plaques in patients with severe stenosis. We tested the hypothesis that compound strain imaging is feasible in non-stenotic arteries and provides incremental discriminative power to traditional measures of vascular health (i.e., distensibility coefficient (DC), central pulse wave velocity [cPWV], and intima-media thickness [IMT]) for differentiating between participants with and without a history of cardiovascular diseases (CVD).

Methods

Seventy two participants (60 ± 7 years) with non-stenotic arteries (IMT < 1.1 mm) were categorized in healthy participants (CON, n = 36) and CVD patients (n = 36) based on CVD history. Participants underwent standardised ultrasound-based assessment (DC, cPWV, and IMT) and compound strain imaging (radial [RS] and circumferential [CS] strain) in left common carotid artery. Area under receiver operating characteristics (AROC)-curve was used to determine the discriminatory power between CVD and CON of the various measures.

Results

CON had a significantly (P < 0.05) smaller carotid IMT (0.68 [0.58 to 0.76] mm) than CVD patients (0.76 [0.68 to 0.80] mm). DC, cPWV, RS, and CS did not significantly differ between groups (P > 0.05). A higher CS or RS was associated with a higher DC (CS: r = ?0.32;p < 0.05 and RS: r = 0.24;p < 0.05) and lower cPWV (CS: r = 0.24;p < 0.05 and RS: r = ?0.25;p < 0.05). IMT could identify CVD (AROC: 0.66, 95%-CI: 0.53 to 0.79), whilst the other measurements, alone or in combination, did not significantly increase the discriminatory power compared to IMT.

Conclusions

In non-stenotic arteries, compound strain imaging is feasible, but does not seem to provide incremental discriminative power to traditional measures of vascular health for differentiation between individuals with and without a history of CVD.

     

Immunological response induced by abagovomab as a maintenance therapy in patients with epithelial ovarian cancer: relationship with survival—a substudy of the MIMOSA trial

Purpose

To determine whether abagovomab induces protective immune responses in ovarian cancer patients in first clinical remission. The present analysis is a substudy of monoclonal antibody immunotherapy for malignancies of the ovary by subcutaneous abagovomab trial (NCT00418574).

Methods

The study included 129 patients, 91 in the abagovomab arm and 38 in the placebo arm. Circulating CA125-specific cytotoxic T lymphocytes (CTL) were measured by a flow cytometry-based interferon-γ producing assay. Human antimouse antibody and anti-anti-idiotypic (Ab3) were assessed by ELISA. Patients were evaluated before starting the treatment and at different time points during induction and maintenance phases.

Results

A similar percentage of patients in both the placebo and abagovomab arms had CA125-specific CTL (26.3 and 31.8 %, respectively; p = 0.673 by Fisher’s exact test). Patients with CA125-specific CTL in both arms tended to have an increased relapse-free survival (RFS, log-rank test p = 0.095) compared to patients without. Patients (n = 27) in the abagovomab arm without CA125-specific CTL but that developed Ab3 above the cutoff (defined as median Ab3 level at week 22) had a prolonged RFS compared to patients (n = 24) that did not develop Ab3 above the cutoff (log-rank test p = 0.019).

Conclusion

Abagovomab does not induce CA125-specific CTL. However, patients with CA125-specific CTL perform better than patients without, irrespective of abagovomab treatment. Abagovomab-induced Ab3 associate with prolonged RFS in patients without CA125-specific CTL. Further studies are needed to confirm these data and to assess the potential utility of these immunological findings as a tool for patient selection in clinical trial.