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Background TRPS-1 is a new GATA transcription factor that is differentially expressed in breast cancer (BC) where it been found recently to regulate epithelial-to-mesenchymal transition (EMT). Patients and methods We carried out a quantitative immunohistochemistry (qIHC) analysis of TRPS-1 expression in 341 primary-stage I–III BC samples in relation to patient clinical characteristics as well as its prognostic value, especially in an estrogen receptor-positive (ER+) subgroup. Results Higher TRPS-1 expression was significantly associated with a number of clinical and pathological characteristics as well as with improved overall survival (OS) and disease-free survival (DFS). Among stage I/II ER+ BC patients who received endocrine therapy alone, those with high TRPS-1 expression had significantly longer OS and DFS. There was also a strong association between TRPS-1 levels and the EMT marker E-cadherin in the ER+ invasive ductal carcinoma cases. Analysis of gene expression data on a panel of BC lines found that TRPS-1 expression was low or absent in BC lines having enriched mesenchymal features. Conclusions Our data indicated that TRPS-1 is an independent prognostic marker in early-stage BC and a new EMT marker that can distinguish patients with ER+ BC who will respond longer to adjuvant endocrine therapy.

Harnessing the immune system to control cancer has been a challenge for cancer immunotherapists for many years. However, while specific immune responses to tumour-associated antigenic targets have been successfully induced in some patients, these responses have not always been sufficient to reproducibly and consistently mediate useful anti-tumour clinical activity. Many checks and balances have been incorporated into the immune response by nature to prevent or reduce the likelihood of autoimmunity or exaggerated protective inflammatory responses. Tolerance to self-antigens expressed on tumours is a major limitation in generating functional anti-tumour responses. In recent years, many of the pathways that mediate this tolerance have been identified, and reagents that can be used to manipulate these pathways have been clinically evaluated. These include: (a) pathways to activate professional antigen presenting cells, such as through Toll-like receptors, growth factors, such as GM-CSF, and the CD40 pathway; (b) use of cytokines, such as IL-2, IL-12, and Interferon α to enhance immune activation; and (c) pathways that inhibit T cell inhibitory signals, or Tregs. This article reviews clinical trials that have evaluated these approaches, and highlights promising combination vaccine/immunomodulator combination treatments based upon published clinical trial results.

Follicular lymphoma (FL) is a unique disease characterised by a long natural history and responsiveness to many different therapies. We have reviewed the prognostic significance of the quality of both clinical and molecular responses for patients with FL. We have found that, as might be expected, patients who achieve a complete clinical response to treatment have a better prognosis than patients who achieve an incomplete or a partial response. However, unlike aggressive lymphomas, treatments that produce a higher frequency of complete responses do not result in better survival outcomes than treatments that produce lower complete response rates. Recent improvements in technologies have enabled quantitative monitoring of responses at the molecular level and at much higher degrees of sensitivity than can be obtained at the clinical level. Although these data are very heterogeneous and have many limitations, data are emerging that demonstrate that achieving molecular remissions after standard dose chemotherapy, high dose chemotherapy or various immunotherapies may have prognostic significance for patients with FL. With sensitive, quantitative, standardised and reproducible tools for molecular monitoring and with the combination of novel targeted biological therapies, we are approaching an era, where the potential to cure patients with FL is being turned into a reality.

Recently, immunotherapy has been widely investigated for the treatment of cancer. While much of the success in this area lies with passive immunotherapy, the focus of this review is active vaccination strategies, which are showing promise. Cancer cells express a wide profile of different proteins, some of which are related to oncogenic transformation and are specific to cancer cells only. However, in cancer, most protein targets presented to the immune system from tumor cells are self-antigens. The immune system is highly tolerant of, and therefore does not react to, these self-antigens. Active immunotherapy aims to reverse this immune tolerance so the immune system can respond appropriately to self-antigens. To generate a successful antitumor response, several features should be considered: a target antigen on tumor cells to direct the immune response, a platform to present the vaccine-derived antigen to the immune system, an adjuvant to enhance immune stimulation, and appropriate monitoring techniques. New tools are becoming available for monitoring, although the clinical relevance for these assays needs to be established. Optimism for cancer vaccine approaches are increasing. Recent studies have shown that it is possible to break immune tolerance to self-antigens. Promising therapeutic clinical activity has been demonstrated, and other studies have shown that cancer vaccines are most beneficial for minimal residual disease states. Further optimization of cancer vaccines and larger clinical studies are required.

A20 is an aggressive BALB/c B cell lymphoma that, despite its expression of B7-2, rapidly forms tumors in syngeneic mice. We have generated A20 transfectants expressing elevated levels of B7-2 (A20/B7-2high) or 4-1BBL (A20/4-1BBL(low,mod,high)) and found that mice which were able to reject the A20/B7-2 or A20/4-1BBL transfectants were also resistant to subsequent systemic challenge with the parental cell line. To assess whether the effectiveness of 4-1BBL in enhancing anti-tumor immunogenicity was dependent on additional signals from B7-CD28 interaction, we injected the A20 variants into BALB/c CD28(-/-) mice. We found that CD28(-/-) mice were able to reject the A20/4-1BBL variants while A20/B7-2 cells formed tumors. However, when the A20/4-1BBL resistant CD28(-/-) mice were systemically challenged with the A20 parental line, tumors formed rapidly. Upon restimulation in vitro, splenocytes from A20/4-1BBL immunized CD28(+/+) mice were able to kill parental tumors whereas splenocytes from CD28(-/-) mice showed a reduction in CTL activity against A20 or A20/4-1BBL targets. Examination of cytokine production by the immunized animals indicated that the CD28(-/-) splenocytes secreted substantially less IL-2 as well as reduced levels of IFN-gamma compared with their CD28(+/+) counterparts. Thus, 4-1BBL expressing tumors are capable of priming CTL responses against 4-1BBL transfected as well as parental tumors in the absence of CD28. However, in the absence of CD28 signaling, the production of cytokines and particularly IL-2 was lower, resulting in a weaker CTL recall response and reduced ability to survive challenge with parental tumor.

Our overall strategy is to develop multivalent recombinant vaccines capable of eliciting broad immune responses in patients with malignant melanoma or colorectal cancer. We report herein results from initial studies conducted in cancer patients to evaluate the effect of intratumoral administration of recombinant canarypox viruses carrying cytokine genes. Our current focus is on the induction of tumor-specific T-cell responses using a prime/boost immunization schedule with a unique vector system derived from the canary pox virus called ALVAC, in which we incorporate genes encoding Tumor Associated Antigens (TAAs) of interest. Clinical studies in colorectal cancer evaluating an ALVAC CEA candidate vaccine have shown that this approach is safe and can induce tumor-specific T cell responses. Additional clinical studies evaluating candidate vaccines against melanoma and colorectal cancer, targeting either the gp100, Mage 1, Mage 3 or p53 molecules are ongoing.

Treatment outcomes were documented for 204 adult patients with clinical Stage I-II Hodgkin's disease who were treated with risk-adapted ABVD (doxorubicin, bleomycin, vinblastine and dacarbazine) and radiotherapy (RT) at the Toronto-Sunnybrook Regional Cancer Centre between 1984 and 1994. Forty-nine patients with clinical Stage I disease (excluding bulky mediastinal presentations) and 50 patients with a combination of clinical Stage IIA disease, age 50 years or less, and favourable pathology (lymphocyte predominant or nodular sclerosing histology) were identified as low risk and treated with RT alone to 35 Gy. One hundred and five high-risk patients were treated with chemotherapy (86 with ABVD) followed by RT to 25 Gy. The 7-year cause-specific, overall and disease-free survivals were 95%, 90% and 75% respectively for the low-risk cohort, and 91%, 90% and 88% respectively for the high-risk cohort. In-field relapses accounted for 50% of the failures in both groups. Sixteen of 24 (67%) patients with RT failure and 6/14 (43%) with combined modality therapy (CMT) failure were salvaged. Twenty-eight per cent of the patients treated with RT and 21% of those treated with CMT developed hypothyroidism by 7 years. Fatal complications were recorded in 6% of the low-risk patients managed with RT and 8% of high-risk patients managed with CMT. Septic death and second malignancy accounted for the majority of treatment-related fatalities. Risk-adapted therapy emphasizing RT alone for selected patients with favourable prognostic factors and CMT based on ABVD provides excellent long-term disease control. Further treatment refinements, including the wider application of CMT with lower doses of chemotherapy and RT, will be required to reduce the rate of fatal complications to more acceptable levels.

Affinity-purified polyclonal antibodies against the hBRAG (human B cell RAG-associated gene) protein were generated to characterize hBRAG at the biochemical level. Immunoblotting and immunoprecipitation experiments with these antibody reagents demonstrate that this protein can be expressed in B cells as a membrane-integrated glycoprotein disulfide-linked dimer. However, both glycosylated and unglycosylated isoforms of hBRAG are detectable with these reagents. Additionally, their use in cell surface biotinylation and flow cytometry reveals subcellular hBRAG pools both at cell surface and intracellular locations. Co-immunoprecipitation experiments with hBRAG antisera detected the association of hBRAG with phosphorylated proteins in resting B cells, including the protein tyrosine kinase Hck, which is subsequently dephosphorylated upon B cell receptor (BCR) ligation. Consistent with its cell surface expression and possible link to BCR signaling, experiments in which alpha-hBRAG antibodies were used to generate early activation signals suggest a modest but specific element of tyrosine phosphorylation occurring through a putative hBRAG receptor. Additional experiments also suggest that hBRAG may be involved in positively enhancing BCR ligation-mediated early activation events. Collectively, these results are consistent with a function for hBRAG as a B cell surface signaling receptor molecule. Coupled with the earlier observation that hBRAG expression correlates with early and late B cell-specific RAG expression, we submit that hBRAG may mediate regulatory signals key to B cell development and/or regulation of B cell-specific RAG expression.

Determining the activity of viral and cellular regulatory elements in B or T lymphoid cell lines would facilitate appropriate utilization of the regulatory sequences for gene transfer- and expression-dependent applications. We have compared the activity of the CMV, RSV and SV40 viral promoter/enhancers as well as the Vlambda1 cellular promoter, in three B cell lines (REH, SMS-SB, C3P), three T cell lines (CEM, Jurkat, ST-F10), and two non-lymphoid cell lines (K-562, HeLa) using the luciferase reporter gene. In B cell lines, the activity of the CMV promoter/enhancer construct was the highest ranging from 10- to 113-fold greater than that of SV40. In contrast, in T cell lines the RSV promoter/enhancer activity was 11-65-fold higher than that of SV40. The Vlambda1 promoter activity was close to that of SV40 promoter/enhancer in most of the cell lines tested. We conclude that CMV and RSV promoter/enhancers contain stronger regulatory elements than do the SV40 and Vlambda1 for expression of genes in lymphoid cell lines.

Summary Background Monoclonal antibodies are being utilized for treatment of patients with low-grade non-Hodgkin’s lymphoma as well as other cancers. Results from phase I and II clinical studies has shown that the chimeric monoclonal antibody Rituximab has minimal toxicity and significant therapeutic activity in low grade non-Hodgkin’s lymphoma. Patients and methods We have recently reported on a multicentre pivotal phase III clinical trial involving 166 patients with recurrent low-grade lymphoma who were treated with four infusions of Rituximab. Eighty patients (48%) achieved objective responses including 10 patients (6%) with complete responses. Overall, 126 patients (76%) had a ≥ 20% reduction in overall tumor size. The median response duration and time to progression are 11.6 and 13.2 months, respectively. The infusional and long term toxicities were limited. Results In this report we describe the pharmacokinetic data obtained on these patients. Measurable concentrations of Rituximab were detected in all patients after the first infusion and increased throughout the treatment course. The half-life of the monoclonal antibody increased from 76.3 hours after the first infusion to 205.8 hours after the fourth infusion and was concomitant with a four-fold decrease in the antibody clearance. At three months and six months post-treatment, the median Rituximab serum levels were 20.3 μg/ml (range 0.0 to 96.8 μg/ml in 104 patients) and 1.3 μg/ml (range 0.0–28.7 μg/ml in 13 patients), respectively. A statistically significant correlation was found between the median antibody concentration and response for multiple time points during the treatment and followup. The mean serum antibody concentration was also inversely correlated with measurements of tumor bulk and with the number of circulating B cells at baseline. Conclusions We conclude that Rituximab is therapeutically effective against B-cell lymphoma. Pharmacokinetic data suggests that certain subsets of patients may possibly benefit from increased dosing and studies to address this are currently underway.

It is important to define the best and most appropriate population in which to test the vaccine. We need to take into account established therapies for this patient population, and develop a strategy to combine this therapy with the vaccine therapy. In early phase I clinical trials, we need to establish the best surrogate endpoints to optimize vaccine activity; immunology endpoints are mechanistically relevant. These early trials should have small patient groups and should be designed to optimize combination therapies using these surrogate endpoints. In these trials, it will be important to randomize early to address questions about the best treatment schedules and the best antigens. Statistical methods can be used to assess these immunologic endpoints. Finally, we should use the best arm to carry to the next phase of clinical testing. For cancer vaccines to be considered therapeutic, we need to demonstrate clear clinical endpoints. We need to show increased survival rates, tumor eradication or tumor control. To achieve these endpoints, a strong commitment to carrying out randomized clinical trials needs to be made. The difficulty with making decisions to proceed to advanced clinical testing of cancer vaccines is the fact that we can always improve the vaccine by adding a new antigen adjuvant or improving the dosing schedule. However, there is no reason why the vaccine refinement work cannot continue while we move forward to test clinical activity of promising vaccines in these large randomized clinical trials. There has been a tremendous amount of work completed to date in terms of evaluating immune responses as surrogate endpoints. But these are only surrogate endpoints. We need to move the work from the laboratory into the clinical setting to evaluate the clinical benefits of these vaccines.