Assessment of the presence of interleukin 17+ macrophages and Th17 cells in situ in lip and oral tongue cancer.

Increasing clinical evidence indicates that Th17 cells may promote or inhibit tumor progression, however the exact role of these cells in Oral Squamous Cell Carcinoma (OSCCs) pathogenesis and progression remains unclear. Tumor associated macrophages are highly plastic phenotype cells which can differentiate as M1 or M2. The mechanism and cellular phenotype of IL-17 expressing macrophages are unknown. 40 cases of lip and 28 of tongue SCCs were submitted to immunohistochemical analysis, and histologically graded. In tongue cases TNM was analyzed. The number of IL-17+ T cells was higher in lip SCC (p = 0.028). IL-17+ macrophages was greater in tongue SCC (p = 0.014). There were more IL-17+ macrophages in the high-grade malignancy oral tongue SCCs (p = 0.016), yet there was no significant difference in the numbers of RORγt+ lymphocytes by histopathological or TNM analysis. This study provides evidence concerning IL-17's pleiotropic roles, being possibly dependent on its cellular sources in the tumor microenvironment.

Assessment of cancer and virus antigens for cross-reactivity in human tissues.

MOTIVATION: Cross-reactivity (CR) or invocation of autoimmune side effects in various tissues has important safety implications in adoptive immunotherapy directed against selected antigens. The ability to predict CR (on-target and off-target toxicities) may help in the early selection of safer therapeutically relevant target antigens. RESULTS: We developed a methodology for the calculation of quantitative CR for any defined peptide epitope. Using this approach, we performed assessment of 4 groups of 283 currently known human MHC-class-I epitopes including differentiation antigens, overexpressed proteins, cancer-testis antigens and mutations displayed by tumor cells. In addition, 89 epitopes originating from viral sources were investigated. The natural occurrence of these epitopes in human tissues was assessed based on proteomics abundance data, while the probability of their presentation by MHC-class-I molecules was modelled by the method of Kesmir et al. which combines proteasomal cleavage, TAP affinity and MHC-binding predictions. The results of these analyses for many previously defined peptides are presented as CR indices and tissue profiles. The methodology thus allows for quantitative comparisons of epitopes and is suggested to be suited for the assessment of epitopes of candidate antigens in an early stage of development of adoptive immunotherapy. AVAILABILITY AND IMPLEMENTATION: Our method is implemented as a Java program, with curated datasets stored in a MySQL database. It predicts all naturally possible self-antigens for a given sequence of a therapeutic antigen (or epitope) and after filtering for predicted immunogenicity outputs results as an index and profile of CR to the self-antigens in 22 human tissues. The program is implemented as part of the iCrossR webserver, which is publicly available at http://webclu.bio.wzw.tum.de/icrossr/ CONTACT: d.frishman@wzw.tum.deSupplementary information: Supplementary data are available at Bioinformatics online.

Prospective assessment of a gene signature potentially predictive of clinical benefit in metastatic melanoma patients following MAGE-A3 immunotherapeutic (PREDICT).

BACKGROUND: Genomic profiling of tumor tissue may aid in identifying predictive or prognostic gene signatures (GS) in some cancers. Retrospective gene expression profiling of melanoma and non-small-cell lung cancer led to the characterization of a GS associated with clinical benefit, including improved overall survival (OS), following immunization with the MAGE-A3 immunotherapeutic. The goal of the present study was to prospectively evaluate the predictive value of the previously characterized GS. PATIENTS AND METHODS: An open-label prospective phase II trial ('PREDICT') in patients with MAGE-A3-positive unresectable stage IIIB-C/IV-M1a melanoma. RESULTS: Of 123 subjects who received the MAGE-A3 immunotherapeutic, 71 (58.7%) displayed the predictive GS (GS+). The 1-year OS rate was 83.1%/83.3% in the GS+/GS- populations. The rate of progression-free survival at 12 months was 5.8%/4.1% in GS+/GS- patients. The median time-to-treatment failure was 2.7/2.4 months (GS+/GS-). There was one complete response (GS-) and two partial responses (GS+). The MAGE-A3 immunotherapeutic was similarly immunogenic in both populations and had a clinically acceptable safety profile. CONCLUSION: Treatment of patients with MAGE-A3-positive unresectable stage IIIB-C/IV-M1a melanoma with the MAGE-A3 immunotherapeutic demonstrated an overall 1-year OS rate of 83.5%. GS- and GS+ patients had similar 1-year OS rates, indicating that in this study, GS was not predictive of outcome. Unexpectedly, the objective response rate was lower in this study than in other studies carried out in the same setting with the MAGE-A3 immunotherapeutic. Investigation of a GS to predict clinical benefit to adjuvant MAGE-A3 immunotherapeutic treatment is ongoing in another melanoma study.This study is registered at www.clinicatrials.gov NCT00942162.

Treatment with unfunded drugs in oncology: the impact of access programmes and clinical trials.

BACKGROUND: Where proven cancer therapies remain unfunded, patients are faced with difficult decisions regarding personally covering some or all of the treatment cost. Clinical trials provide an alternative form of access to unfunded drugs. AIMS: To examine the patient population and utilisation of the colorectal cancer cetuximab Interim Access Programme (IAP) in Australia. METHODS: We retrospectively analysed de-identified data collected as part of the costsharing colorectal cancer cetuximab IAP in Australia, which extended from June 2005 until November 2009. The impact of the CO20 clinical trial that opened in early 2008 and provided free access to cetuximab was also examined. RESULTS: Eight hundred and fifty-eight patients received ≥1 treatment on the IAP. The median age of participants was lower than the general colorectal cancer population (61 vs 71 years). A greater uptake by males was limited to patients over 65 years old. Socioeconomic status correlated with programme enrolment, and there appeared to be lower uptake among regional patients. The majority (93%) of patients received combination treatment with irinotecan, with a trend towards increasing use of single-agent cetuximab over time. The median time-on-treatment was longer in patients treated with combination therapy than with monotherapy (12 vs 8 weeks). The CO20 trial had minimal impact on IAP enrolment and approximately doubled the number of Australian patients receiving cetuximab. CONCLUSIONS: Patients' age and gender in older patients impacted on participation in the IAP. Both the IAP and the CO20 trial contributed to a substantial proportion of Australian patients accessing an unfunded treatment, with an estimated 50% of the eligible patient population receiving cetuximab.

Study of compliance with prescription information sheet of trastuzumab prescriptions in a tertiary level hospital.

INTRODUCTION: This study compares trastuzumab's actual conditions of use in clinical practice with those officially described on its summary of product characteristics. We also measure the cost associated with its use. METHODS: Observational study of the prescription/indication of trastuzumab in a tertiary hospital from January 2006 to 31 December 2007. We analysed whether trastuzumab use in clinical practice complied with its summary of product characteristics, concerning the following: HER2 over expression, indication (breast cancer), treatment plan, line of treatment, dosage, frequency and number of cycles. To measure cost, we calculated the total number of milligrams used and then multiplied it by the laboratory's sale price per milligram plus VAT. RESULTS: All patients (n=77) used trastuzumab for breast cancer. Sixty-two point two percent of patients presented with HER2+++ over expression. Twenty-nine treatment plans were used, that were not authorised on the summary of product characteristics. The total trastuzumab cost during the study period was €1537 622.73. CONCLUSIONS: Although trastuzumab is always used for breast cancer, it is used in conditions other than those described on its summary of product characteristics, both for HER2 over expression and treatment plans.

Assessment of HB-EGF levels in peritoneal fluid and serum of ovarian cancer patients using ELISA.

BACKGROUND: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a rational target for ovarian cancer therapy. The aim of this study was to examine HB-EGF levels in the peritoneal fluid and serum of ovarian cancer (OVCA) patients. PATIENTS AND METHODS: Samples were collected from six healthy women, 21 OVCA patients, and 21 ovarian cyst patients. HB-EGF levels were measured using a sandwich ELISA kit and calculated using a parallel line assay. RESULTS: No significant difference between the slopes of the standard and sample curves was observed at an anti-HB-EGF antibody concentration of 1.6 µg/ml. HB-EGF levels in the peritoneal fluid and serum of OVCA patients were significantly higher than those in patients with ovarian cysts or controls. Serum HB-EGF levels were also significantly correlated with levels in peritoneal fluid in OVCA patients. CONCLUSION: We developed an assay for the exact measurement of HB-EGF levels in peritoneal fluid and serum.

Assessment of vaccine-induced CD4 T cell responses to the 119-143 immunodominant region of the tumor-specific antigen NY-ESO-1 using DRB1*0101 tetramers.

PURPOSE: NY-ESO-1 (ESO), a tumor-specific antigen of the cancer/testis group, is presently viewed as an important model antigen for the development of generic anticancer vaccines. The ESO(119-143) region is immunodominant following immunization with a recombinant ESO vaccine. In this study, we generated DRB1*0101/ESO(119-143) tetramers and used them to assess CD4 T-cell responses in vaccinated patients expressing DRB1*0101 (DR1). EXPERIMENTAL DESIGN: We generated tetramers of DRB1*0101 incorporating peptide ESO(119-143) using a previously described strategy. We assessed ESO(119-143)-specific CD4 T cells in peptide-stimulated postvaccine cultures using the tetramers. We isolated DR1/ESO(119-143) tetramer(+) cells by cell sorting and characterized them functionally. We assessed vaccine-induced CD4(+) DR1/ESO(119-143) tetramer(+) T cells ex vivo and characterized them phenotypically. RESULTS: Staining of cultures from vaccinated patients with DR1/ESO(119-143) tetramers identified vaccine-induced CD4 T cells. Tetramer(+) cells isolated by cell sorting were of T(H)1 type and efficiently recognized full-length ESO. We identified ESO(123-137) as the minimal optimal epitope recognized by DR1-restricted ESO-specific CD4 T cells. By assessing DR1/ESO(119-143) tetramer(+) cells using T cell receptor (TCR) ß chain variable region (Vß)-specific antibodies, we identified several frequently used Vß. Finally, direct ex vivo staining of patients' CD4 T cells with tetramers allowed the direct quantification and phenotyping of vaccine-induced ESO-specific CD4 T cells. CONCLUSIONS: The development of DR1/ESO(119-143) tetramers, allowing the direct visualization, isolation, and characterization of ESO-specific CD4 T cells, will be instrumental for the evaluation of spontaneous and vaccine-induced immune responses to this important tumor antigen in DR1-expressing patients.

Assessment of CD4+ T cells specific for the tumor antigen SSX-1 in cancer-free individuals.

Proteins encoded by genes of the SSX family are specifically expressed in tumors and are therefore relevant targets for cancer immunotherapy. One of the first identified family members, SSX-1, is expressed in a large fraction of synovial sarcomas as a fusion protein together with the product of the SYT gene. In addition, the full-length SSX-1 antigen is frequently expressed in tumors of several other histological types such as sarcoma, melanoma, hepatocellular carcinoma, ovarian cancer and myeloma. To date, however, SSX-1 specific T cell responses have not been investigated and no SSX-1 derived T cell epitopes have been described. Here, we have assessed the presence of CD4(+) T cells directed against the SSX-1 antigen in circulating lymphocytes of cancer-free individuals. After a single in vitro stimulation with a pool of peptides spanning the entire SSX-1 protein we could detect and isolate SSX-1-specific CD4(+) T cells from 5/5 donors analyzed. SSX-1-specific polyclonal populations isolated from these cultures recognized peptides located in three distinct regions of the protein containing clusters of sequences with significant predicted binding to frequently expressed MHC class II alleles. Characterization of specific clonal CD4(+) T cell populations derived from one donor allowed the identification of several naturally processed epitopes recognized in association with HLA-DR. These data document the existence of a significant repertoire of CD4(+) T cells specific for SSX-1 derived sequences in circulating lymphocytes of any individual that can be exploited for the development of both passive and active immunotherapeutic approaches to control disease evolution in cancer patients.

Direct evidence on the immune-mediated spontaneous regression of human cancer: an incentive for pharmaceutical companies to develop a novel anti-cancer vaccine.

To develop an effective pharmaceutical treatment for a disease, we need to fully understand the biological behavior of that disease, especially when dealing with cancer. The current available treatment for cancer may help in lessening the burden of the disease or, on certain occasions, in increasing the survival of the patient. However, a total eradication of cancer remains the researchers' hope. Some of the discoveries in the field of medicine relied on observations of natural events. Among these events is the spontaneous regression of cancer. It has been argued that such regression could be immunologically-mediated, but no direct evidence has been shown to support such an argument. We, hereby, provide compelling evidence that spontaneous cancer regression in humans is immunologically-mediated, hoping that the results from this study would stimulate the pharmaceutical industry to focus more on cancer vaccine immunotherapy. Our results showed that patients with >3 primary melanomas (very rare group among cancer patients) develop significant histopathological spontaneous regression of further melanomas that they could acquire during their life (P=0.0080) as compared to patients with single primary melanoma where the phenomenon of spontaneous regression is absent or minimal. It seems that such regression resulted from the repeated exposure to the tumor which mimics a self-immunization process. Analysis of the regressing tumors revealed heavy infiltration by T lymphocytes as compared to non-regressing tumors (P<0.0001), the predominant of which were T cytotoxic rather than T helper. Mature dendritic cells were also found in significant number (P<0.0001) in the regressing tumors as compared to the non regressing ones, which demonstrate an active involvement of the different arms of the immune system in the multiple primary melanoma patients in the process of tumor regression. Also, MHC expression was significantly higher in the regressing versus the non-regressing tumors (P <0.0001), which reflects a proper tumor antigen expression. Associated with tumor regression was also loss of the melanoma common tumor antigen Melan A/ MART-1 in the multiple primary melanoma patients as compared to the single primary ones (P=0.0041). Furthermore, loss of Melan A/ MART-1 in the regressing tumors significantly correlated with the presence of Melan A/ MART-1-specific CTLs in the peripheral blood of these patients (P=0.03), which adds to the evidence that the phenomenon of regression seen in these patients was immunologically-mediated and tumor-specific. Such correlation was also seen in another rare group of melanoma patients, namely those with occult primary melanoma. The lesson that we could learn from nature in this study is that inducing cancer regression using the different arms of the immune system is possible. Also, developing a novel cancer vaccine is not out of reach.

Assessment of CD8 involvement in T cell clone avidity by direct measurement of HLA-A2/Mage3 complex density using a high-affinity TCR like monoclonal antibody.

Peptide affinity for MHC molecules determines the number of MHC/peptide complexes stabilized at the cell surface in in vitro tests or in vaccination protocols. We isolated a high affinity monoclonal antibody specific for the HLA-A2/Mage3 complex that enables an equilibrium binding assay to be performed on T2 cell line loaded with a range of Mage3 peptides. Binding of Mage3 to the HLA-A2 molecule can be modeled by a standard receptor-ligand interaction characterized by an affinity constant. This model enables the measurement of the affinity of other immunogenic peptides for HLA-A2 by a competition test and the calculation of the density of complexes stabilized at the T2 cell surface for all peptide concentrations. Quantification of the HLA-A2/Mage3 complexes at target cell surfaces was used to estimate the number of complexes required to reach cytotoxicity ED50 of human T cell clones sorted from an unprimed repertoire. We confirm with this antibody the direct relationship between clone avidity and TCR affinity, and the moderate contribution of the CD8 co-receptor in the reinforcement of TCR-MHC/peptide contact. Nevertheless, CD8 plays a critical role in the amplification of the specific signal to establish an efficient T cell response at low specific complex densities found in physiological situations.

Prostate stem cell antigen: Identification of immunogenic peptides and assessment of reactive CD8+ T cells in prostate cancer patients.

Identification of TAAs recognized by CD8(+) CTLs paved the way for new concepts in cancer therapy. In view of the heterogeneity of tumors and their diverse escape mechanisms, CTL-based cancer therapy largely depends on an appropriate number of TAAs. In prostate cancer, the number of antigens defined as suitable targets of CTLs remains rather limited. PSCA is widely distributed in prostate cancer. In this report, we define immunogenic peptides of PSCA which are recognized by circulating CD8(+) T cells from prostate cancer patients and able to activate CTLs in vitro. Screening the amino acid sequence of PSCA for peptides containing a binding motif for HLA-A*0201 resulted in 8 candidate peptides. Specificity and affinity of peptide binding were verified in a competition assay. Frequencies of CD8(+) T lymphocytes reactive against selected epitopes were determined in the blood of prostate cancer patients using the ELISPOT assay. Increased frequencies were revealed for CD8(+) T cells recognizing the peptides ALQPGTALL and AILALLPAL. CTLs from prostate cancer patients were raised against these 2 peptides in vitro when presented by autologous DCs. They specifically recognized peptide-pulsed T2 target cells and prostate cancer cells that were HLA-A*0201- and PSCA-positive, indicating that these peptides were naturally generated by tumor cells. These data suggest that PSCA is a promising target for the immunotherapy of prostate cancer.

Assessment of immunogenicity of human Melan-A peptide analogues in HLA-A*0201/Kb transgenic mice.

Previous studies have shown that substitution of single amino acid residues in human Melan-A immunodominant peptides Melan-A27-35 and Melan-A26-35 greatly improved their binding and the stability of peptide/HLA-A*0201 complexes. In particular, one Melan-A peptide analogue was more efficient in the generation of Melan-A peptide-specific and melanoma-reactive CTL than its parental peptide in vitro from human PBL. In this study, we analyzed the in vivo immunogenicity of Melan-A natural peptides and their analogues in HLA-A*0201/Kb transgenic mice. We found that two human Melan-A natural peptides, Melan-A26-35 and Melan-A27-35, were relatively weak immunogens, whereas several Melan-A peptide analogues were potent immunogens for in vivo CTL priming. In addition, induced Melan-A peptide-specific mouse CTL cross-recognized natural Melan-A peptides and their analogues. More interestingly, these mouse CTL were also able to lyse human melanoma cell lines in vitro in a HLA-A*0201-restricted, Melan-A-specific manner. Our results indicate that the HLA-A*0201/Kb transgenic mouse is a useful animal model to perform preclinical testing of potential cancer vaccines, and that Melan-A peptide analogues are attractive candidates for melanoma immunotherapy.

[Assessment of the proliferative activity of hepatocellular carcinoma with the monoclonal antibody Ki-67 using flow cytometry].

This study examined whether the monoclonal antibody Ki-67 is an indicator of hepatocellular carcinoma proliferation and whether it represents a new parameter for determining the diagnosis and prognosis. The subjects were 22 patients who were not treated preoperatively among the patients undergoing hepatectomy at our department. Fresh specimens of the cancer tissue and the noncancerous regions were stained with PI after processing them with FITC-labeled Ki-67. Then 20,000 cells were analyzed by two-color flow cytometry. A significant difference was observed between the well differentiated group and the moderately and poorly differentiated groups. However, no significant difference was observed with respect to any other factor. The average Ki-67 labeling of the cancers in the patients with and without cirrhosis was 17.0 +/- 10.5% and 5.3 +/- 3.5%, respectively (p < 0.05). The cancers showed high Ki-67 labeling rates compared with the noncancerous areas and a positive correlation was observed between the two. These findings suggested that coexistent hepatic lesions have some influence on the proliferative activity of cancer. A significant correlation was confirmed by flow cytometry after immunostaining using the antibody MIB-1. Analysis by this method was considered to be useful for assessing the proliferating activity of hepatocellular carcinoma.

Quantitative image analysis of MIB-1 immunoreactivity. A comparison with flow cytometric assessment of proliferative activity in invasive carcinoma of the breast.

Proliferative activity, especially flow cytometrically determined S-phase fraction, is generally accepted as an important prognostic indicator in carcinoma of the breast. We studied cellular proliferation in 53 breast carcinomas using quantitative image analysis of immunoreactivity to a recently available monoclonal antibody, MIB-1, which is applicable to formalin-fixed, paraffin-embedded tissues. MIB-1 is a murine monoclonal antibody that reacts with the Ki-67 nuclear antigen expressed by proliferating cells in the late G1, and G2/M phases of the cell cycle. These results were compared to flow cytometric determinations of S-phase and S + G2/M phase fractions obtained from corresponding fresh tissue samples. There was a good correlation between quantitative immunoreactivity to MIB-1 as measured by image analysis and flow cytometric S-phase and S + G2/M phase fractions (r = .63, P < .00001; r = .607, P < .00001, respectively). Immunoreactivity to MIB-1 and flow cytometric S-phase and S + G2/M phase fractions were significantly increased in aneuploid tumors as compared to diploid tumors. Histologic grade correlated with flow cytometric S-phase and S + G2/M phase fractions and MIB-1 immunoreactivity as determined by image analysis. There was a correlation between tumor size and MIB immunoreactivity. No proliferative parameters significantly correlated with lymph node status. Assessment of proliferative activity by quantitative image analysis of immunoreactivity to monoclonal MIB-1 antibody may be employed in cases of invasive carcinoma of the breast in which flow cytometric analysis fails to result in quantitative proliferative values, or it may be used as an alternative measurement of such proliferative activity.

Assessment of the new proliferation marker MIB1 in breast carcinoma using image analysis: associations with other prognostic factors and survival.

The 'growth fraction' of tumours can now be assessed on paraffin sections of tissues using the monoclonal antibody MIB1 by a microwave antigen retrieval technique. The MIB1 labelling index was studied using a CAS 200 image analyser in 177 tumours from women with primary operable breast carcinoma in whom long-term follow-up data were known. Statistical analysis showed a strong association between the MIB1 labelling index and histological grade (P < 0.001), tumour size (P = 0.002), tumour type (P < 0.001) and also patient survival (P < 0.001). No association with lymph node stage (P = 0.974) or regional recurrence (P = 0.185), the presence or absence of distant metastases (P = 0.418), patient age (P = 0.309), menopausal status (P = 0.181) or oestrogen receptor status (P = 0.401) was found in this group of patients. In multivariate analysis for survival, when histological grade, lymph node stage and tumour size were included as well as the MIB1 labelling index, each was found to be of independent significance. If histological grade was not included, MIB1 replaced it as the most important variable predicting for survival in this group of patients. The results suggest that the tumour growth fraction, as assessed by the MIB1 labelling index, is an important predictor of survival.

Assessment of cell proliferation in hydatidiform mole using monoclonal antibody MIB1 to Ki-67 antigen.

AIMS: To assess the role of Ki67 immunoreactivity in predicting the clinical progress of hydatidiform mole. METHODS: Tissue from 87 hydatidiform moles, 11 normal first trimester placentas, 11 normal term placentas and 17 spontaneous abortions were examined for expression of Ki67 antigen, using the monoclonal antibody MIB1. RESULTS: Ki67 immunoreactivity was significantly higher in the tissue from normal first trimester placentas than in that from normal term placentas and spontaneous abortions. Among the 87 patients with hydatidiform moles studied, 20 developed persistent gestational trophoblastic disease and required subsequent treatment. There was no statistically significant difference in the Ki67 index between the 20 patients who developed persistent disease and those who did not. CONCLUSION: Hydatidiform moles which give rise to persistent trophoblastic disease do not have a higher proliferative rate than those which do not. The Ki67 index is not useful for predicting the prognosis of molar pregnancies.