Tumour-draining axillary lymph nodes in patients with large and locally advanced breast cancers undergoing neoadjuvant chemotherapy (NAC): the crucial contribution of immune cells (effector, regulatory) and cytokines (Th1, Th2) to immune-mediated tumour cell death induced by NAC.

BACKGROUND: The tumour microenvironment consists of malignant cells, stroma and immune cells. In women with large and locally advanced breast cancers (LLABCs) undergoing neoadjuvant chemotherapy (NAC), tumour-infiltrating lymphocytes (TILs), various subsets (effector, regulatory) and cytokines in the primary tumour play a key role in the induction of tumour cell death and a pathological complete response (pCR) with NAC. Their contribution to a pCR in nodal metastases, however, is poorly studied and was investigated. METHODS: Axillary lymph nodes (ALNs) (24 with and 9 without metastases) from women with LLABCs undergoing NAC were immunohistochemically assessed for TILs, T effector and regulatory cell subsets, NK cells and cytokine expression using labelled antibodies, employing established semi-quantitative methods. IBM SPSS statistical package (21v) was used. Non-parametric (paired and unpaired) statistical analyses were performed. Univariate and multivariate regression analyses were carried out to establish the prediction of a pCR and Spearman's Correlation Coefficient was used to determine the correlation of immune cell infiltrates in ALN metastatic and primary breast tumours. RESULTS: In ALN metastases high levels of TILs, CD4+ and CD8+ T and CD56+ NK cells were significantly associated with pCRs.. Significantly higher levels of Tregs (FOXP3+, CTLA-4+) and CD56+ NK cells were documented in ALN metastases than in the corresponding primary breast tumours. CD8+ T and CD56+ NK cells showed a positive correlation between metastatic and primary tumours. A high % CD8+ and low % FOXP3+ T cells and high CD8+: FOXP3+ ratio in metastatic ALNs (tumour-free para-cortex) were associated with pCRs. Metastatic ALNs expressed high IL-10, low IL-2 and IFN-ϒ. CONCLUSIONS: Our study has provided new data characterising the possible contribution of T effector and regulatory cells and NK cells and T helper1 and 2 cytokines to tumour cell death associated with NAC in ALNs. TRIAL REGISTRATION: The Trial was retrospectively registered. Study Registration Number is ISRCTN00407556 .

Integrative analysis of copy number and gene expression in breast cancer using formalin-fixed paraffin-embedded core biopsy tissue: a feasibility study.

BACKGROUND: An absence of reliable molecular markers has hampered individualised breast cancer treatments, and a major limitation for translational research is the lack of fresh tissue. There are, however, abundant banks of formalin-fixed paraffin-embedded (FFPE) tissue. This study evaluated two platforms available for the analysis of DNA copy number and gene expression using FFPE samples. METHODS: The cDNA-mediated annealing, selection, extension, and ligation assay (DASL™) has been developed for gene expression analysis and the Molecular Inversion Probes assay (Oncoscan™), were used for copy number analysis using FFPE tissues. Gene expression and copy number were evaluated in core-biopsy samples from patients with breast cancer undergoing neoadjuvant chemotherapy (NAC). RESULTS: Forty-three core-biopsies were evaluated and characteristic copy number changes in breast cancers, gains in 1q, 8q, 11q, 17q and 20q and losses in 6q, 8p, 13q and 16q, were confirmed. Regions that frequently exhibited gains in tumours showing a pathological complete response (pCR) to NAC were 1q (55%), 8q (40%) and 17q (40%), whereas 11q11 (37%) gain was the most frequent change in non-pCR tumours. Gains associated with poor survival were 11q13 (62%), 8q24 (54%) and 20q (47%). Gene expression assessed by DASL correlated with immunohistochemistry (IHC) analysis for oestrogen receptor (ER) [area under the curve (AUC) = 0.95], progesterone receptor (PR)(AUC = 0.90) and human epidermal growth factor type-2 receptor (HER-2) (AUC = 0.96). Differential expression analysis between ER+ and ER- cancers identified over-expression of TTF1, LAF-4 and C-MYB (p ≤ 0.05), and between pCR vs non-pCRs, over-expression of CXCL9, AREG, B-MYB and under-expression of ABCG2. CONCLUSION: This study was an integrative analysis of copy number and gene expression using FFPE core biopsies and showed that molecular marker data from FFPE tissues were consistent with those in previous studies using fresh-frozen samples. FFPE tissue can provide reliable information and will be a useful tool in molecular marker studies. TRIAL REGISTRATION: Trial registration number ISRCTN09184069 and registered retrospectively on 02/06/2010.

Immune dysfunction in patients with obstructive jaundice before and after endoscopic retrograde cholangiopancreatography.

This prospective observational study investigated monocyte cytokine responses to lipopolysaccharide (LPS) in patients with obstructive jaundice (OJ) before and after endoscopic biliary drainage. Dendritic cell (DC) subsets and their expression of co-stimulatory molecules were also studied. Forty patients with OJ and ten non-jaundiced patients with normal gastroscopy findings were recruited. Ten healthy volunteers provided control blood samples for immunological assays. Patients with OJ had blood and duodenal mucosa sampled at the time of endoscopic retrograde cholangiopancreatography (ERCP) and further blood sampled during the recovery phase. Monocyte cytokine responses to LPS, DC subsets and co-stimulatory molecule expression were compared with controls. Duodenal morphology and occludin expression were also assessed. Monocytes obtained before ERCP from jaundiced patients demonstrated reduced cytokine responses to endotoxin compared with controls (IL-1ß: 2678 compared with 4631 pg/ml, P=0.04 and IL-6: 3442 compared with 6157 pg/ml, P=0.002). Monocytes from patients with malignancy had poorer responses to endotoxin than from those with benign OJ (IL-1ß: 2025 compared with 3332 pg/ml, P=0.001). After ERCP, the secretion of inflammatory cytokines by monocytes obtained from jaundiced patients increased (IL-1ß: 2150 compared with 2520 pg/ml, P=0.03 and IL-6: 2488 compared with 3250 pg/ml, P=0.01). Occludin expression (85 compared with 95%, P=0.004) and mean duodenal villus height (334 compared with 404 µm, P=0.03) were lower in jaundiced patients. Before biliary drainage, patients with OJ had a higher percentage of myeloid dendritic cells (mDCs) and greater mDC expression of CD40 (P=0.04) and CD86 (P=0.04). Monocytes from patients with OJ had lower proinflammatory cytokine secretion in response to LPS, an effect reversed following biliary drainage.

Natural killer (NK) cell profiles in blood and tumour in women with large and locally advanced breast cancer (LLABC) and their contribution to a pathological complete response (PCR) in the tumour following neoadjuvant chemotherapy (NAC): differential restoration of blood profiles by NAC and surgery.

BACKGROUND: NK cells contribute to tumour surveillance, inhibition of growth and dissemination by cytotoxicity, secretion of cytokines and interaction with immune cells. Their precise role in human breast cancer is unclear and the effect of therapy poorly studied. The purpose of our study was to characterise NK cells in women with large (≥3 cm) and locally advanced (T3-4, N1-2, M0) breast cancers (LLABCs) undergoing neoadjuvant chemotherapy (NAC) and surgery, and to ascertain their possible contribution to a pathological complete response (pCR). METHODS: Women with LLABCs (n = 25) and healthy female donors [HFDs (n = 10)] were studied. Pathological responses in the breast were assessed using established criteria. Blood samples were collected pre and post NAC and surgery. Flow cytometry and labelled monoclonal antibodies established absolute numbers (AbNs) and percentages (%) of NK cells, and expressing granzyme B/perforin and NKG2D. In vitro NK cytotoxicity was assessed and NK cells and cytokines (IL-2, INF-γ, TGF-ß) documented in tumours using immunohistochemical techniques. Data was analysed by SPSS. RESULTS: Women with LLABCs had significantly reduced AbNs (160.00 ± 40.00 cells/µl) but not % of NK cells, compared with HFDs (NK: 266.78 ± 55.00 cells/µl; p = 0.020). NAC enhanced the AbN (p = 0.001) and % (p = 0.006) of NK cells in patients with good pathological responses. Granzyme B(+)/perforin(+) cells were significantly reduced (43.41 ± 4.00%), compared with HFDs (60.26 ± 7.00%; p = 0.003). NAC increased the % in good (p = 0.006) and poor (p = 0.005) pathological responders. Pretreatment NK cytotoxicity was significantly reduced in good (37.80 ± 8.05%) and poor (22.80 ± 7.97%) responders (p = 0.001) but remained unchanged following NAC. NK-NKG2D(+) cells were unaltered and unaffected by NAC; NKG2D expression was increased in patients with a pCR (p = 0.001). Surgery following NAC was not beneficial, except in those with a pCR. Tumour-infiltrating NK cells were infrequent but increased peritumourally (p = 0.005) showing a significant correlation (p = 0.004) between CD56(+) cells and grade of response. Tumour cytokines had no effect. CONCLUSION: Women with LLABCs have inhibited blood innate immunity, variably reversed by NAC (especially with tumour pCRs), which returned to pretreatment levels following surgery. These and in situ tumour findings suggest a role for NK cells in NAC-induced breast pCR.

A phase 1 trial of human telomerase reverse transcriptase (hTERT) vaccination combined with therapeutic strategies to control immune-suppressor mechanisms.

The presence of inhibitory immune cells and difficulty in generating activated effector T cells remain obstacles to development of effective cancer vaccines. We designed a vaccine regimen combining human telomerase reverse transcriptase (hTERT) peptides with concomitant therapies targeting regulatory T cells (Tregs) and cyclooxygenase-2 (COX2)-mediated immunosuppression. This Phase 1 trial combined an hTERT-derived 7-peptide library, selected to ensure presentation by both HLA class-I and class-II in 90% of patients, with oral low-dose cyclophosphamide (to modulate Tregs) and the COX2 inhibitor celecoxib. Adjuvants were Montanide and topical TLR-7 agonist, to optimise antigen presentation. The primary objective was determination of the safety and tolerability of this combination therapy, with anti-cancer activity, immune response and detection of antigen-specific T cells as additional endpoints. Twenty-nine patients with advanced solid tumours were treated. All were multiply-pretreated, and the majority had either colorectal or prostate cancer. The most common adverse events were injection-site reactions, fatigue and nausea. Median progression-free survival was 9 weeks, with no complete or partial responses, but 24% remained progression-free for ≥6 months. Immunophenotyping showed post-vaccination expansion of CD4+ and CD8+ T cells with effector phenotypes. The in vitro re-challenge of T cells with hTERT peptides, TCR sequencing, and TCR similarity index analysis demonstrated the expansion following vaccination of oligoclonal T cells with specificity for hTERT. However, a population of exhausted PD-1+ cytotoxic T cells was also expanded in vaccinated patients. This vaccine combination regimen was safe and associated with antigen-specific immunological responses. Clinical activity could be improved in future by combination with anti-PD1 checkpoint inhibition to address the emergence of an exhausted T cell population.

Abnormal T regulatory cells (Tregs: FOXP3+, CTLA-4+), myeloid-derived suppressor cells (MDSCs: monocytic, granulocytic) and polarised T helper cell profiles (Th1, Th2, Th17) in women with large and locally advanced breast cancers undergoing neoadjuvant chemotherapy (NAC) and surgery: failure of abolition of abnormal treg profile with treatment and correlation of treg levels with pathological response to NAC.

BACKGROUND: Host defences play a key role in tumour growth. Some of the benefits of chemotherapy may occur through modulation of these defences. The aim of this study was to define the status of regulatory cells in women with large and locally advanced breast cancers (LLABCs) undergoing neoadjuvant chemotherapy (NAC) and surgery. METHODS: Bloods were collected from patients (n=56) before, during and following NAC, and surgery. Controls (n=10) were healthy, age-matched females donors (HFDs). Blood mononuclear cells (BMCs) were isolated and T regulatory cells (Tregs) (n=31) determined. Absolute numbers (AbNs) of Tregs and myeloid-derived suppressor cells (MDSCs) were ascertained from whole blood (n=25). Reverse transcriptase polymerase chain reaction analysis determined Treg mRNA (n=16). In vitro production of Th1, Th2 and Th17 cytokines (n=30), was documented. Patients were classified as clinical responders by magnetic resonance mammography after two cycles of NAC and as pathological responders using established criteria, following surgery. RESULTS: Patients with LLABCs had significantly increased circulating Tregs (≥ 6 fold AbN and percentage (%)) and MDSCs (≥ 1.5 fold AbN (p=0.025)). Percentage of FOXP3+ Tregs in blood predicted the response of the LLABCs to subsequent NAC (p=0.04). Post NAC blood Tregs (%) were significantly reduced in patients where tumours showed a good pathological response to NAC (p=0.05). Blood MDSCs (granulocytic, monocytic) were significantly reduced in all patients, irrespective of the pathological tumour response to chemotherapy. NAC followed by surgery failed to restore blood Tregs to normal levels. MDSCs, however, were reduced to or below normal levels by NAC alone. Invitro Th1 profile (IL-1ß, IL-2, INF-γ, TNF-α) was significantly reduced (p ≤ 0.009), whilst Th2 (IL-4, IL-5) was significantly enhanced (P ≤ 0.004). Th1 and Th2 (IL-5) were unaffected by NAC and surgery. IL-17A was significantly increased (p ≤ 0.023) but unaffected by chemotherapy and surgery. CONCLUSION: Women with LLABCs have abnormal blood regulatory cell levels (Tregs and MDSCs) and cytokine profiles (Th1, Th2, Th17). NAC followed by surgery failed to abolish the abnormal Treg and Th profiles. There was a significant correlation between the circulatory levels of Tregs and the pathological response of the breast cancers to NAC.

Expression of polymeric immunoglobulin receptor (PIGR) and the effect of PIGR overexpression on breast cancer cells.

Polymeric immunoglobulin receptor (PIGR) has a major role in mucosal immunity as a transporter of polymeric immunoglobulin across the epithelial cells. The aim of this study was to determine the effect of PIGR on cellular behaviours and chemo-sensitivity of MCF7 and MDA-MB468 breast cancer cell lines. Basal levels of PIGR mRNA and protein expression in MCF7 and MDA-MB468 cells were evaluated by real time quantitative polymerase chain reaction and Western blotting, respectively. MCF7/PIGR and MDA-MB468/PIGR stable cell lines, overexpressing the PIGR gene, were generated using a lentiviral vector with tetracycline dependent induction of expression. Cell viability, cell proliferation and chemo-sensitivity of PIGR transfected cells were evaluated and compared with un-transfected cells to determine the effect of PIGR overexpression on cell phenotype. The levels of PIGR mRNA and protein expression were significantly higher in MDA-MB468 cells than in MCF7 cells (380-fold, p < 0.0001). However, the differential expression of PIGR in these two cell lines did not lead to significant differences in chemosensitivity. Viral overexpression of PIGR was also not found to change any of the parameters measured in either cell line. PIGR per se did not affect cellular behaviours and chemosensitivity of these breast cancer cell lines.

The stem cell factor antibody enhances the chemotherapeutic effect of adriamycin on chemoresistant breast cancer cells.

BACKGROUND: The outcome of chemotherapy in breast cancer is strongly influenced by multidrug resistance (MDR). Several surrogate markers of chemoresistance have been identified including - CD24 (cluster differentiation 24) expression, stem cell growth factor (SCF), B-cell lymphocyte protein 2 (Bcl-2) and annexin V. The present study aimed to examine the expression of CD24 in the sensitive breast cancer cell line MCF-7 (Michigan Foudation-7) and MCF-7/adriamycin resistant (MCF-7/AdrRes) cells, and, if minimal effective doses of the anthracycline drug adriamycin (0.579 µM and 88.2 µM) would be enhanced by the antibody to SCF (anti-SCF). METHODS: CD24 expression was analysed by flow cytometry. Both Bcl-2 and annexin V protein expression were quantitatively assessed by the enzyme-linked immunosorbent assay (ELISA). RESULTS: In MCF-7/AdrRes cells the expression of CD24 was significantly higher compared to MCF-7 cells, 86.6% and 16.3% (p < 0.001), respectively. Bcl-2 expression was significantly increased in the presence of adriamycin and SCF (p < 0.038) and decreased in the presence of adriamycin and anti-SCF. When adriamycin, anti-SCF and SCF were combined or when adriamycin was used alone the decrease in Bcl-2 expression was insignificantly altered. In the presence of both adriamycin and SCF the expression of annexin V was decreased. However, it was significantly increased in the presence of adriamycin and anti-SCF (p < 0.042), as well as adriamycin, anti-SCF and SCF combined.In MCF-7 cells the effect of adriamycin alone or with either SCF, anti-SCF or anti-SCF or SCF combined, did not significantly alter the expression of Bcl-2. However, in the presence of both adriamycin and SCF the expression of annexin V was decreased, but was significantly increased in the presence of adriamycin and anti-SCF (p < 0.001), adriamycin, anti-SCF and SCF combined and adriamycin alone. Our results demonstrate that anti-SCF with low dose of adriamycin reduces Bcl-2 expression in MCF-7/AdrRes cells and increases annexin V expression in both MCF7/AdrRes and MCF-7 cells. CONCLUSION: Adding anti-SCF to the chemotherapeutic regime of adriamycin may strongly enhance its chemotherapeutic effect in the treatment of patients with breast cancer.

New approach to isolate mesenchymal stem cell (MSC) from human umbilical cord blood.

HUCB (human umbilical cord blood) has been frequently used in clinical allogeneic HSC (haemopoietic stem cell) transplant. However, HUCB is poorly recognized as a rich source of MSC (mesenchymal stem cell). The aim of this study has been to establish a new method for isolating large number of MSC from HUCB to recognize it as a good source of MSC. HUCB samples were collected from women following their elective caesarean section. The new method (Clot Spot method) was carried out by explanting HUCB samples in mesencult complete medium and maintained in 37°C, in a 5% CO2 and air incubator. MSC presence was established by quantitative and qualitative immunophenotyping of cells and using FITC attached to MSC phenotypic markers (CD29, CD73, CD44 and CD105). Haematopoietic antibodies (CD34 and CD45) were used as negative control. MSC differentiation was examined in neurogenic and adipogenic media. Immunocytochemistry was carried out for the embryonic markers: SOX2 (sex determining region Y-box 2), OLIG-4 (oligodendrocyte-4) and FABP-4 (fatty acid binding protein-4). The new method was compared with the conventional Rosset Sep method. MSC cultures using the Clot Spot method showed 3-fold increase in proliferation rate compared with conventional method. Also, the cells showed high expression of MSC markers CD29, CD73, CD44 and CD105, but lacked the expression of specific HSC markers (CD34 and CD45). The isolated MSC showed some differentiation by expressing the neurogenic (SOX2 and Olig4) and adipogenic (FABP-4) markers respectively. In conclusion, HUCB is a good source of MSC using this new technique.

M1 macrophages evoke an increase in polymeric immunoglobulin receptor (PIGR) expression in MDA-MB468 breast cancer cells through secretion of interleukin-1ß.

High expression of polymeric immunoglobulin receptor (PIGR) in breast cancer is associated with increased 5-year survival rate. However, the factors influencing PIGR expression in breast cancer have not been elucidated. The aim of this study was to determine the role of macrophages and cytokines affecting expression of PIGR in two breast cancer cell lines. M1, M2 macrophage conditioned media (CM) and recombinant human cytokines were used to determine factors which increased PIGR expression in MCF7 (HTB-22) and MDA-MB468 (HTB-132) breast cancer cell lines. The level of PIGR expression in the cells and PIGR secretory component were evaluated by real-time quantitative polymerase chain reaction and Western blotting. M1 macrophage CM induced a dose-dependent increase in PIGR mRNA expression in MDA-MB468 cells, up to 20-fold. The level of PIGR expression in MCF7 cells was very low and not affected by M1 and M2 CM. Interferon gamma (IFN-γ) and interleukin (IL)-1ß also increased PIGR expression in MDA-MB468 and MCF7 cells. However, IL-1ß was demonstrated to increase in M1 macrophages, while IFN-γ was not. The role of IL-1ß secreted from M1 macrophages in increasing expression of PIGR was confirmed by IL-1 receptor blockade, indicating that IL-1ß was the major M1 macrophage-derived cytokine that enhanced PIGR expression. Elevated PIGR expression in breast cancer in vivo may reflect the polarization state of tumor-associated immune cells.

Effects on quality of life, anti-cancer responses, breast conserving surgery and survival with neoadjuvant docetaxel: a randomised study of sequential weekly versus three-weekly docetaxel following neoadjuvant doxorubicin and cyclophosphamide in women with primary breast cancer.

BACKGROUND: Weekly docetaxel has occasionally been used in the neoadjuvant to downstage breast cancer to reduce toxicity and possibly enhance quality of life. However, no studies have compared the standard three weekly regimen to the weekly regimen in terms of quality of life. The primary aim of our study was to compare the effects on QoL of weekly versus 3-weekly sequential neoadjuvant docetaxel. Secondary aims were to determine the clinical and pathological responses, incidence of Breast Conserving Surgery (BCS), Disease Free Survival (DFS) and Overall Survival (OS). METHODS: Eighty-nine patients receiving four cycles of doxorubicin and cyclophosphamide were randomised to receive twelve cycles of weekly docetaxel (33 mg/m2) or four cycles of 3-weekly docetaxel (100 mg/m2). The Functional Assessment of Cancer Therapy-Breast and psychosocial questionnaires were completed. RESULTS: At a median follow-up of 71.5 months, there was no difference in the Trial Outcome Index scores between treatment groups. During weekly docetaxel, patients experienced less constipation, nail problems, neuropathy, tiredness, distress, depressed mood, and unhappiness. There were no differences in overall clinical response (93% vs. 90%), pathological complete response (20% vs. 27%), and breast-conserving surgery (BCS) rates (49% vs. 42%). Disease-free survival and overall survival were similar between treatment groups. CONCLUSIONS: Weekly docetaxel is well-tolerated and has less distressing side-effects, without compromising therapeutic responses, Breast Conserving Surgery (BCS) or survival outcomes in the neoadjuvant setting. TRIAL REGISTRATION: ISRCTN: ISRCTN09184069.

Molecular detection of micrometastatic breast cancer in histopathology-negative axillary lymph nodes fails to predict breast cancer recurrence: a final analysis of a prospective multi-institutional cohort study.

BACKGROUND: To address the clinical relevance of molecular detection of occult breast cancer in sentinel lymph nodes and nonsentinel axillary lymph nodes (ALN), we initiated the Minimally Invasive Molecular Staging of Breast Cancer (MIMS) trial, a multi-institutional prospective cohort study. This trial represents the first prospective cohort study in which a multimarker, real-time reverse transcription polymerase chain reaction (RT-PCR) analysis was applied to the detection of breast cancer micrometastases in ALN. MATERIALS AND METHODS: Sentinel and/or nonsentinel ALN from 501 breast cancer subjects with T1-T3 primary tumors were analyzed by standard histopathology and multimarker, real-time RT-PCR analysis. Seven breast cancer-associated genes (mam, mamB, PIP, CK19, muc1, PSE, and CEA) known to be overexpressed in metastatic breast cancer compared with control lymph nodes were used. Follow-up data were collected for 5 years. RESULTS: Of the 501 breast cancer subjects enrolled, 348 were node negative and completed the 5-year follow-up. Of these patients (n = 94), 27% demonstrated evidence of molecular overexpression. The 5-year relapse-free survival rate was 95.4% (95% confidence interval [95% CI], 92.4-97.2%). No single gene or combination of study genes was predictive of recurrence. CONCLUSIONS: The genes in this study panel failed to be predictive of clinical relapse. This may be a function of several factors: the low event rate at 5 years, the particular gene set, the methodology used for detection/analysis or that our original hypothesis was wrong and that the presence of positive marker signal by real-time RT-PCR is not associated with a worsened clinical outcome.

Generation in vivo of peptide-specific cytotoxic T cells and presence of regulatory T cells during vaccination with hTERT (class I and II) peptide-pulsed DCs.

BACKGROUND: Optimal techniques for DC generation for immunotherapy in cancer are yet to be established. Study aims were to evaluate: (i) DC activation/maturation milieu (TNF-alpha +/- IFN-alpha) and its effects on CD8+ hTERT-specific T cell responses to class I epitopes (p540 or p865), (ii) CD8+ hTERT-specific T cell responses elicited by vaccination with class I alone or both class I and II epitope (p766 and p672)-pulsed DCs, prepared without IFN-alpha, (iii) association between circulating T regulatory cells (Tregs) and clinical responses. METHODS: Autologous DCs were generated from 10 patients (HLA-0201) with advanced cancer by culturing CD14+ blood monocytes in the presence of GM-CSF and IL-4 supplemented with TNF-alpha [DCT] or TNF-alpha and IFN-alpha [DCTI]. The capacity of the DCs to induce functional CD8+ T cell responses to hTERT HLA-0201 restricted nonapeptides was assessed by MHC tetramer binding and peptide-specific cytotoxicity. Each DC preparation (DCT or DCTI) was pulsed with only one type of hTERT peptide (p540 or p865) and both preparations were injected into separate lymph node draining regions every 2-3 weeks. This vaccination design enabled comparison of efficacy between DCT and DCTI in generating hTERT peptide specific CD8+ T cells and comparison of class I hTERT peptide (p540 or p865)-loaded DCT with or without class II cognate help (p766 and p672) in 6 patients. T regulatory cells were evaluated in 8 patients. RESULTS: (i) DCTIs and DCTs, pulsed with hTERT peptides, were comparable (p = 0.45, t-test) in inducing peptide-specific CD8+ T cell responses. (ii) Class II cognate help, significantly enhanced (p < 0.05, t-test) peptide-specific CD8+T cell responses, compared with class I pulsed DCs alone. (iii) Clinical responders had significantly lower (p < 0.05, Mann-Whitney U test) T regs, compared with non-responders. 4/16 patients experienced partial but transient clinical responses during vaccination. Vaccination was well tolerated with minimal toxicity. CONCLUSION: Addition of IFN-alpha to ex vivo monocyte-derived DCs, did not significantly enhance peptide-specific T cell responses in vivo, compared with TNF-alpha alone. Class II cognate help significantly augments peptide-specific T cell responses. Clinically favourable responses were seen in patients with low levels of circulating T regs.

Emerging concepts in biomarker discovery; the US-Japan Workshop on Immunological Molecular Markers in Oncology.

Supported by the Office of International Affairs, National Cancer Institute (NCI), the "US-Japan Workshop on Immunological Biomarkers in Oncology" was held in March 2009. The workshop was related to a task force launched by the International Society for the Biological Therapy of Cancer (iSBTc) and the United States Food and Drug Administration (FDA) to identify strategies for biomarker discovery and validation in the field of biotherapy. The effort will culminate on October 28th 2009 in the "iSBTc-FDA-NCI Workshop on Prognostic and Predictive Immunologic Biomarkers in Cancer", which will be held in Washington DC in association with the Annual Meeting. The purposes of the US-Japan workshop were a) to discuss novel approaches to enhance the discovery of predictive and/or prognostic markers in cancer immunotherapy; b) to define the state of the science in biomarker discovery and validation. The participation of Japanese and US scientists provided the opportunity to identify shared or discordant themes across the distinct immune genetic background and the diverse prevalence of disease between the two Nations. Converging concepts were identified: enhanced knowledge of interferon-related pathways was found to be central to the understanding of immune-mediated tissue-specific destruction (TSD) of which tumor rejection is a representative facet. Although the expression of interferon-stimulated genes (ISGs) likely mediates the inflammatory process leading to tumor rejection, it is insufficient by itself and the associated mechanisms need to be identified. It is likely that adaptive immune responses play a broader role in tumor rejection than those strictly related to their antigen-specificity; likely, their primary role is to trigger an acute and tissue-specific inflammatory response at the tumor site that leads to rejection upon recruitment of additional innate and adaptive immune mechanisms. Other candidate systemic and/or tissue-specific biomarkers were recognized that might be added to the list of known entities applicable in immunotherapy trials. The need for a systematic approach to biomarker discovery that takes advantage of powerful high-throughput technologies was recognized; it was clear from the current state of the science that immunotherapy is still in a discovery phase and only a few of the current biomarkers warrant extensive validation. It was, finally, clear that, while current technologies have almost limitless potential, inadequate study design, limited standardization and cross-validation among laboratories and suboptimal comparability of data remain major road blocks. The institution of an interactive consortium for high throughput molecular monitoring of clinical trials with voluntary participation might provide cost-effective solutions.

Modulation of T Regulatory and Dendritic Cell Phenotypes Following Ingestion of Bifidobacterium longum, AHCC® and Azithromycin in Healthy Individuals.

The probiotic Bifidus BB536 (BB536), which contains Bifidobacterium longum, has been shown to have enhanced probiotic effects when given together with a standardized extract of cultured Lentinula edodes mycelia (AHCC®, Amino Up Co. Ltd., Sapporo, Japan). BB536 and AHCC® may modulate T cell and dendritic cell (DC) phenotypes, and cytokine profiles to favour anti-inflammatory responses following antibiotic ingestion. We tested the hypothesis that orally administered BB536 and/or AHCC®, results in modulation of immune effector cells with polarisation towards anti-inflammatory responses following antibiotic usage. Forty healthy male volunteers divided into 4 equal groups were randomised to receive either placebo, BB536, AHCC® or a combination for 12 days in a double-blind manner. After 7 days volunteers also received 250 mg azithromycin for 5 days. Cytokine profiles from purified CD3+ T cells stimulated with PDB-ionomycin were assessed. CD4+ CD25+ forkhead box P3 (Foxp3) expression and peripheral blood DC subsets were assessed prior to treatment and subsequently at 7 and 13 days. There was no difference in cytokine secretion from stimulated CD3+ T cells between treatment groups. Compared with baseline, Foxp3 expression (0.45 ± 0.1 vs. 1.3 ± 0.4; p = 0.002) and interferon-gamma/interleukin-4 (IFN-γ/IL-4) ratios were increased post-treatment in volunteers receiving BB536 (p = 0.031), although differences between groups were not significant. For volunteers receiving combination BB536 and AHCC®, there was an increase in myeloid dendritic cells (mDC) compared with plasmacytoid DC (pDC) counts (80% vs. 61%; p = 0.006) at post treatment time points. mDC2 phenotypes were more prevalent, compared with baseline, following combination treatment (0.16% vs. 0.05%; p = 0.002). Oral intake of AHCC® and BB536 may modulate T regulatory and DC phenotypes to favour anti-inflammatory responses following antibiotic usage.

Ex vivo recovery and activation of dysfunctional, anergic, monocyte-derived dendritic cells from patients with operable breast cancer: critical role of IFN-alpha.

BACKGROUND: Dendritic cells (DCs) play a crucial role in initiating effective cell-mediated immune responses, but are dysfunctional and anergic in breast cancer. Reversal of this dysfunction and establishment of optimal DC function is a key prerequisite for the induction of effective anti-cancer immune responses. RESULTS: Peripheral blood DCs (PBDCs) and lymph node DCs (LNDCs) generated in vitro from adherent cultures of peripheral blood monocytes (PBMs) and lymph node monocytes (LNMs), respectively, using the 4 cytokine conditioned medium (CCM) (GM-CSF+IL-4+TNF-alpha+IFN-alpha) or 3 CCM (GM-CSF+IL-4+TNF-alpha) demonstrated a significantly higher degree of recovery and functional capacity in a mixed lymphocyte DC reaction (MLDCR, p < 0.001), expressed significantly higher levels of HLA-DR, CD86, compared with 2 CCM (GM-CSF+IL-4) or medium alone generated DCs from PBMs and LNMs (p < 0.001). The PBDCs generated with 3 CCM or 4 CCM showed a significantly (p < 0.001) enhanced macropinocytotic capability (dextran particles) and induced increased production and secretion of interleukin-12p40 (IL-12p40) in vitro (p < 0.001), compared with PBDCs generated from monocytes using 2 CCM or medium alone. Lipopolysaccharide (LPS) stimulation of PBDCs generated with 4 CCM demonstrated enhanced secretion of IL-6 but not IL-12p70, compared with control DCs unstimulated with LPS (p < 0.001). CONCLUSION: Dysfunctional and anergic PBDCs and LNDCs from patients with operable breast cancer can be optimally reversed by ex vivo culturing of precursor adherent monocytes using a 4 CCM containing IFN-alpha. Maximal immunophenotypic recovery and functional reactivation of DCs is seen in the presence of IFN-alpha. However, 4 CCM containing IFN-alpha generated-PBDCs, do not produce and secrete IL-12p70 in vitro.

Management of the axilla in women with breast cancer: current clinical practice and a new selective targeted approach.

BACKGROUND: Axillary nodal status is the most important prognostic factor for patients with breast cancer. Clinical assessment and imaging modalities are not always reliable. Surgical removal and histopathological examination of axillary lymph nodes remain essential methods of staging the axilla. However, the optimal management of the axilla remains uncertain. METHODS: We performed Medline searches to identify relevant systematic reviews, meta-analysis, and nonrandomized and randomized controlled trials for the past 5 years (up to December 2007), as well as important historical articles and clinical guidelines relating to management of the axilla in women with breast cancer. RESULTS: Axillary lymph node dissection (ALND) has been the standard surgical approach for many years. It is, however, associated with marked morbidity; survival benefit remains uncertain. Axillary node sampling, widely practiced in the United Kingdom, is a reliable alternative procedure in staging the axilla, with less morbidity. Sentinel lymph node biopsy (SLNB) has become an accurate method for staging the axilla in women with operable, clinically node-negative breast cancer. SLNB alone appears to be a safe and acceptable procedure for patients with uninvolved SLNs. Completion ALND or axillary radiotherapy remains the standard treatment for patients with tumor-involved SLNs. SLNB is associated with less morbidity than ALND. However, long-term follow-up and therapeutic outcomes are being awaited from randomized controlled trials. CONCLUSIONS: Several procedures are available for staging and treating the axilla. A tailored surgical approach, with careful assessment of risk-benefit and patient preference, is guiding the evolving modern management of the axilla for women with breast cancer.

Significant immunomodulatory effects of Pseudomonas aeruginosa quorum-sensing signal molecules: possible link in human sepsis.

Pathogenic bacteria use quorum-sensing signal molecules to co-ordinate the expression of virulence genes. Animal-based studies have demonstrated the immunomodulatory effects of quorum-sensing signal molecules. In the present study, we have examined the impact of these molecules on normal human immune function in vitro and compared this with immune changes in patients with sepsis where quorum-sensing signal molecules were detected in the sera of patients. Quorum-sensing signal molecules inhibited normal dendritic cell and T-cell activation and proliferation, and down-regulated the expression of co-stimulatory molecules on dendritic cells; in MLDCRs (mixed lymphocyte dendritic cell reactions), secretion of IL (interleukin)-4 and IL-10 was enhanced, but TNF-alpha (tumour necrosis factor-alpha), IFN-gamma (interferon-gamma) and IL-6 was reduced. Quorum-sensing signal molecules induced apoptosis in dendritic cells and CD4(+) cells, but not CD8(+) cells. Dendritic cells from patients with sepsis were depleted and ex vivo showed defective expression of co-stimulatory molecules and dysfunctional stimulation of allogeneic T-lymphocytes. Enhanced apoptosis of dendritic cells and differential CD4(+) Th1/Th2 (T-helper 1/2) cell apoptotic rate, and modified Th1/Th2 cell cytokine profiles in MLDCRs were also demonstrated in patients with sepsis. The pattern of immunological changes in patients with sepsis mirrors the effects of quorum-sensing signal molecules on responses of immune cells from normal individuals in vitro, suggesting that quorum-sensing signal molecules should be investigated further as a cause of immune dysfunction in sepsis.

Predicting response to neoadjuvant chemotherapy in breast cancer: molecular imaging, systemic biomarkers and the cancer metabolome (Review).

The ability to predict the response to neoadjuvant chemotherapy (NAC) prior to or shortly after commencing treatment, in women with large or locally advanced breast cancers, would not only prevent patients from experiencing unnecessary drug morbidity but also reduce the high cost associated with drug usage and utilisation of resources with NAC. Ability to estimate residual cancer volume after NAC is of clinical relevance to subsequent therapeutic surgical options. Various approaches, using conventional histopathological characteristics and imaging modalities to evaluate and predict the response to NAC, have not been able to provide accurate and reliable data. Novel biomolecular imaging, new biomarkers and recent cancer genomic and proteomic profiling, introduced into clinical practice, have produced preliminary promising results. We describe and discuss these molecular characteristics and approaches and their applications to NAC in breast cancer management.