Low stromal Foxp3+ regulatory T-cell density is associated with complete response to neoadjuvant chemoradiotherapy in rectal cancer.

BACKGROUND: Foxp3+ regulatory T cells (Tregs) play a vital role in preventing autoimmunity, but also suppress antitumour immune responses. Tumour infiltration by Tregs has strong prognostic significance in colorectal cancer, and accumulating evidence suggests that chemotherapy and radiotherapy efficacy has an immune-mediated component. Whether Tregs play an inhibitory role in chemoradiotherapy (CRT) response in rectal cancer remains unknown. METHODS: Foxp3+, CD3+, CD4+, CD8+ and IL-17+ cell density in post-CRT surgical samples from 128 patients with rectal cancer was assessed by immunohistochemistry. The relationship between T-cell subset densities and clinical outcome (tumour regression and survival) was evaluated. RESULTS: Stromal Foxp3+ cell density was strongly associated with tumour regression grade (P=0.0006). A low stromal Foxp3+ cell density was observed in 84% of patients who had a pathologic complete response (pCR) compared with 41% of patients who did not (OR: 7.56, P=0.0005; OR: 5.27, P=0.006 after adjustment for presurgery clinical factors). Low stromal Foxp3+ cell density was also associated with improved recurrence-free survival (HR: 0.46, P=0.03), although not independent of tumour regression grade. CONCLUSIONS: Regulatory T cells in the tumour microenvironment may inhibit response to neoadjuvant CRT and may represent a therapeutic target in rectal cancer.

A phase 1b clinical trial of the CD40-activating antibody CP-870,893 in combination with cisplatin and pemetrexed in malignant pleural mesothelioma.

BACKGROUND: Data from murine models suggest that CD40 activation may synergize with cytotoxic chemotherapy. We aimed to determine the maximum tolerated dose (MTD) and toxicity profile and to explore immunological biomarkers of the CD40-activating antibody CP-870,893 with cisplatin and pemetrexed in patients with malignant pleural mesothelioma (MPM). PATIENTS AND METHODS: Eligible patients had confirmed MPM, ECOG performance status 0-1, and measurable disease. Patients received cisplatin 75 mg/m(2) and pemetrexed 500 mg/m(2) on day 1 and CP-870,893 on day 8 of a 21-day cycle for maximum 6 cycles with up to 6 subsequent cycles single-agent CP-870,893. Immune cell subset changes were examined weekly by flow cytometry. RESULTS: Fifteen patients were treated at three dose levels. The MTD of CP-870,893 was 0.15 mg/kg, and was exceeded at 0.2 mg/kg with one grade 4 splenic infarction and one grade 3 confusion and hyponatraemia. Cytokine release syndrome (CRS) occurred in most patients (80%) following CP-870,893. Haematological toxicities were consistent with cisplatin and pemetrexed chemotherapy. Six partial responses (40%) and 9 stable disease (53%) as best response were observed. The median overall survival was 16.5 months; the median progression-free survival was 6.3 months. Three patients survived beyond 30 months. CD19+ B cells decreased over 6 cycles of chemoimmunotherapy (P < 0.001) with a concomitant increase in the proportion of CD27+ memory B cells (P < 0.001) and activated CD86+CD27+ memory B cells (P < 0.001), as an immunopharmacodynamic marker of CD40 activation. CONCLUSIONS: CP-870,893 with cisplatin and pemetrexed is safe and tolerable at 0.15 mg/kg, although most patients experience CRS. While objective response rates are similar to chemotherapy alone, three patients achieved long-term survival. AUSTRALIA NEW ZEALAND CLINICAL TRIALS REGISTRY NUMBER: ACTRN12609000294257.

Existing models, but not neutrophil-to-lymphocyte ratio, are prognostic in malignant mesothelioma.

BACKGROUND: Recent studies proposed neutrophil-to-lymphocyte ratio (NLR) as a prognostic biomarker in malignant pleural mesothelioma (MPM). We examined baseline prognostic variables including NLR and the EORTC and CALGB models as predictors of overall survival (OS) in MPM. METHODS: In this retrospective study, 274 consecutive eligible, newly presenting patients with MPM were included. Of these, 159 received chemotherapy, 10 had tri-modality therapy, 2 underwent surgery only and 103 received supportive care alone. Univariate analyses and multivariate Cox models were calculated for OS. RESULTS: In univariate analysis, poor prognostic factors were: age ≥65 years, nonepithelioid histology, stage III-IV, poor performance status (PS), weight loss, chest pain, low haemoglobin and high platelet count. A baseline NLR≥ 5 did not predict worse OS (hazard ratio (HR) 1.25; P=0.122). On multivariate analysis, age, histology, PS, weight loss, chest pain and platelet count remained significant. The EORTC and CALGB prognostic groups were validated as predictive for OS (HR 1.62; P<0.001 and HR 1.65; P<0.001, respectively). CONCLUSION: Our findings validate standard prognostic variables and the existing EORTC and CALGB models, but not NLR, at initial diagnosis of MPM. In guiding patient management at diagnosis, it is important to consider multiple baseline variables that jointly predict survival.

Post-chemotherapy T-cell recovery is a marker of improved survival in patients with advanced thoracic malignancies.

BACKGROUND: There is increasing interest in combining chemotherapy with immunotherapy. However, the effects of chemotherapy on the human immune system are largely unknown. METHODS: Longitudinal changes in peripheral T-cell subsets in 40 patients with malignant mesothelioma (MM) or advanced non-small cell lung cancer (NSCLC) receiving platinum-based chemotherapy were assessed by flow cytometry and evaluated for associations with clinical outcome. RESULTS: Proliferating T cells of all subsets were almost entirely depleted at day 8 following chemotherapy, but rapidly recovered above baseline levels. Regulatory T cells (Treg) were most profoundly depleted at this time point. A greater increase in CD8(+) T-cell proliferation following one treatment cycle was associated with improved overall survival in univariate (hazard ratio (HR)=0.40; P<0.05) and multivariate (HR=0.17; P<0.01) analyses. A greater increase in the ratio of CD8(+) T cell to Treg proliferation was also predictive of better prognosis. CONCLUSION: Chemotherapy potentially provides a favourable environment for the development of anti-tumour immunity through transient Treg depletion and regeneration of the T-cell pool. Change in CD8(+) T-cell proliferation after one cycle of chemotherapy may represent a useful prognostic indicator in patients with MM and NSCLC.

Decoding dangerous death: how cytotoxic chemotherapy invokes inflammation, immunity or nothing at all.

Chemotherapy and immunotherapy can be either synergistic or antagonistic modalities in the treatment of cancer. Cytotoxic chemotherapy not only affects the tumor but also targets dividing lymphocytes, the very cells that are required to develop an immune response. For this reason, chemo- and immunotherapy have been seen as antagonistic. However, cell death can be immunogenic and the way in which chemotherapeutic drug kills a tumor cell is likely to be an important determinant of how that dying cell interacts with the immune system and whether the interaction will lead to an immune response. When a cell dies as the result of infection, the immune system responds rapidly and the system of Toll-like receptors (TLR) plays a key role in this process. In this review, we will briefly summarize the intracellular signaling pathways that link TLR ligation with immune activation and we will address the questions where and how TLRs recognize their targets.

Chemoimmunotherapy: an emerging strategy for the treatment of malignant mesothelioma.

Whether the immune system can recognize malignant and premalignant cells and eliminate them to prevent the development of cancer is still a matter of open debate, but in our view, the balance of evidence favours this concept. Nonetheless, the International Agency for Research on Cancer has now predicted that cancer will overtake heart disease as the leading cause of death worldwide by 2010, showing that this protective mechanism often fails. Malignant mesothelioma has traditionally been considered a relatively non-immunogenic cancer. However, mesothelioma cells do express a set of well-defined tumour antigens that have been shown to engage with the host immune system. Mesothelioma should therefore be considered a target for immunotherapy. A variety of anticancer immunotherapies have been investigated in mesothelioma and in other malignancies, although these have been largely ineffective when used in isolation. Over recent years, there has been increasing interest in the possibility of combining immunotherapy with chemotherapy in the fight against cancer. Here, we discuss the rationale behind combining these two, long considered antagonistic, treatment options in the context of malignant mesothelioma.

T-cell receptor transgenic analysis of tumor-specific CD8 and CD4 responses in the eradication of solid tumors.

The role of tumor-specific CD8 and CD4 lymphocytes in rejecting solid tumors has been difficult to determine because of the lack of models in which tumor antigen, specific CD8 cells, and specific CD4 cells can be monitored and controlled. To investigate the minimal components required for the induction and maintenance of CTL activity sufficient to reject a solid tumor in vivo, we transfected the influenza hemagglutinin (HA) gene into a nonimmunogenic class I+/class II- murine malignant mesothelioma (MM) tumor line to generate an endogenous tumor antigen and used TCR transgenic mice with class I- or class II-restricted specificities for HA as sources of naive, tumor-specific T cells. The data show that the presence of a strong tumor antigen is not in itself sufficient to induce an effective CTL response, nor does the presence of a high frequency of precursor cells guarantee tumor rejection. We also show that tumor-specific CD4 cells, when CTL numbers are suboptimal, greatly enhance the eradication of tumor, confirming the importance of antigen-presenting cell presentation of tumor antigens to class II-restricted cells. These data confirm that T-cell receptor transgenic cells, combined with nominal tumor antigen transfection, represent powerful tools to analyze tumor-specific T-cell responses.

A Subset of Malignant Mesothelioma Tumors Retain Osteogenic Potential.

Malignant mesothelioma (MM) is an aggressive serosal tumor associated with asbestos exposure. We previously demonstrated that mesothelial cells differentiate into cells of different mesenchymal lineages and hypothesize that osseous tissue observed in a subset of MM patients is due to local differentiation of MM cells. In this study, the capacity of human and mouse MM cells to differentiate into osteoblast-like cells was determined in vitro using a functional model of bone nodule formation and in vivo using an established model of MM. Human and murine MM cell lines cultured in osteogenic medium expressed alkaline phosphatase and formed mineralized bone-like nodules. Several human and mouse MM cell lines also expressed a number of osteoblast phenotype markers, including runt-related transcription factor 2 (RUNX2), osteopontin, osteonectin and bone sialoprotein mRNA and protein. Histological analysis of murine MM tumors identified areas of ossification within the tumor, similar to those observed in human MM biopsies. These data demonstrate the ability of MM to differentiate into another mesenchymal cell type and suggest that MM cells may contribute to the formation of the heterologous elements observed in MM tumors.

Lack of ignorance to tumor antigens: evaluation using nominal antigen transfection and T-cell receptor transgenic lymphocytes in Lyons-Parish analysis--implications for tumor tolerance.

A substantial body of literature has described weak antitumor CTL responses in tumor-bearing hosts, and a number of authors have suggested that tumor tissue in some way sequesters antigen from the immune system, a failure of the tumor-specific immune response largely attributable to "ignorance." To evaluate this in a tumor model, we stably transfected murine tumor cell lines with genes coding for the nominal antigens influenza hemagglutinin (HA) or ovalbumin (OVA) and adoptively transferred HA- or OVA-specific T-cell receptor-transgenic, CD8-positive T cells into mice-bearing these tumors. Tumor antigen cross-presentation within draining lymph nodes (LNs) was then examined using Lyons-Parish analysis, detection of a proliferative response of 5,6-carboxyfluorescein diacetate succinimidyl ester-labeled CD8 T cells from T-cell receptor mice using flow cytometric analysis. Our studies demonstrate clearly that tumor antigens are constitutively presented in LNs draining tumors and can stimulate a T-cell proliferative response. This lack of ignorance was not simply attributable to the model chosen, because it was seen with three different cell lines, two different antigens, and two different mouse strains. Furthermore, it occurred regardless of whether these tumor antigens were expressed as cytoplasmic, transmembrane, or secreted proteins. When tumor antigens were present in low concentrations, antigen cross-presentation was not absent but simply delayed. Interestingly, tumor antigen cross-presentation remained localized to the LNs draining the tumor throughout the period of tumor growth. Curiously, in animals where tumors failed to grow, evidence of continued cross-presentation of the tumor antigen was seen up to 6 months after tumor inoculation. These data suggest that ignorance is not an explanation for the failure of the host immune system to respond to tumor antigens.

A T cell clone with three potential TCR alpha chain rearrangements expresses only one receptor combination at the cell surface.

In determining the T cell receptor (TcR) usage of various T cell clones that recognize peptide antigens derived from allergens, a particular clone (AC20) was found, that apparently expressed three different species of mRNA encoding alpha chains. The logical conclusion that the cells were not clonal was refuted by the finding of only a single beta chain rearrangement. One of the alpha chains (V alpha20), was not in frame, but two V alpha8 transcripts of different lengths were both potentially translatable. Sequence analysis suggested that the shorter transcript was generated by a secondary splice event from the longer, through the use of a splice donor sequence encoded by the J alpha38 gene segment. The efficiency of excision of the intervening sequence is such that approximately equal amounts of the long and short transcripts occur in the steady state pool of mRNA. This phenomenon has been reported previously in TcR alpha rearrangements, but it has never been made clear whether these truncated chains can form a functional TcR. Reconstitution of a TcR negative cell line with these transcripts showed that only the full length alpha chain was able to pair efficiently with the beta chain to generate a functional receptor at the cell surface.

Replication-restricted vaccinia as a cytokine gene therapy vector in cancer: persistent transgene expression despite antibody generation.

BACKGROUND: As antitumoral immunity requires the generation of local immunity directed against tissue proteins, we attempted to recreate within tumors the same environment found within tissues affected by autoimmune diseases (i.e., prolonged cytokine expression). Vaccinia virus (VV) has not been widely used as a cytokine gene therapy vector because of presumed high immunogenicity that would likely make repeated injections impossible; therefore, we modified it by inserting the cytokine gene into the thymidine kinase region, rendering it replication-restricted. The cytokine chosen was human interleukin-2 (IL-2); a molecule with powerful antitumoral effects. METHODS: Six patients with the treatment-resistant tumor malignant mesothelioma received intratumoral (i.t.) VV-IL-2 therapy for 12 weeks by injection of 10(7) plaque-forming units of VV-IL-2 per dose. Serial tumor biopsies, sputum, urine, and blood samples were tested for VV-IL-2 mRNA expression; VV culture and T-cell infiltrates were evaluated by immunohistochemistry. Patients and contacts of patients were monitored for changes in VV immunoglobulin G (IgG) levels and clinical evidence of VV infection. RESULTS: VV-IL-2 was not excreted and was only cultured in one patient from tumor biopsies. A T-cell infiltrate was detected in 50% of tumor biopsies. VV-IL-2 mRNA expression was highest on days 1-3 postinjection and was detected for up to 3 weeks after each injection even though VV IgG levels rose in all patients. No significant toxicities, infection of patient contacts, or tumor regressions were observed. CONCLUSIONS: I.t. VV-IL-2 administration is safe, is associated with minimal toxicity, and results in i.t. expression of VV-IL-2 for up to 3 weeks postinjection regardless of the level of anti-VV IgG titers generated. This suggests that VV may be a good vector for repeated cytokine gene therapy of solid human cancer.

Differential induction of the NF-AT complex during restimulation and the induction of T-cell anergy.

Stimulation of human CD4+ T-cell clones through the T-cell receptor (TcR) by high doses of specific peptide results in the induction of a long-lived state of nonresponsiveness that has been called anergy. During the induction of anergy, T cells are phenotypically similar to cells responding to an immunogenic stimulus. The amount of TcR at the cell surface is downmodulated, whereas the CD2 and CD25 receptors are increased. When restimulated, however, anergic T cells fail to up-regulate transcription of the IL-2 gene and in consequence do not produce IL-2. In this study, we have compared the ability of various transcription factors to bind to their appropriate site on DNA. Factors were isolated from the nuclei of T cells that were in the induction phase of anergy or were undergoing activation. The pattern of binding activity in restimulated T cells is consistent with the pattern that has previously been shown to regulate T-cell-specific expression of the IL-2 and the beta chain of the TcR genes. The measured binding to a TCF-1 site is the same in the nuclei of resting, activated, and anergized cells. The inducible factors NK-kappa B, beta E2, CD28RC, and AP-1 are not expressed in resting cells and are twofold lower in anergized as compared with activated cells. In contrast, anergic T cells express approximately eightfold lower amounts of NF-AT, a member of the class of inducible factors that regulates IL-2 gene transcription.(ABSTRACT TRUNCATED AT 250 WORDS)

Sera from patients with malignant mesothelioma can contain autoantibodies.

Malignant mesothelioma (MM) is resistant to all conventional forms of therapy though there is considerable evidence from clinical trials and animal models of the disease that an immune response can be elicited to the tumour. In order to define those target antigens expressed by MM cells which might provide a focus for an effective immune response we tested patients' sera for the presence of MM autoantibodies by Western blot analysis. Eight of 29 (28%) patients with MM had serum antibodies of the IgG class in high titre and each antiserum recognised different protein antigens. In those individuals where sequential samples were available, the antibody titre increased with the progression of the disease though the number of target antigens remained constant. Sera from the eight patients were studied further: six of the antigen complexes were expressed at least partially in the nucleus; two showed some specificity for the tumour in that they discriminated antigens that were highly expressed in all human MM cell lines, but were not expressed in a human SV40 transformed mesothelial line; four of the antisera recognised a homologue in mouse tissue and each of these had a different pattern of expression. Collectively, these antisera define a subset of nuclear autoantigens that are over-expressed in dividing cells.

Localised spontaneous regression in mesothelioma -- possible immunological mechanism.

Malignant mesothelioma (MM) is an aggressive tumor usually associated with asbestos exposure. Although it can remain stable for prolonged periods, it has not been described to spontaneously regress. MM tumors are thought to be immunogenic based both on animal studies and on the good responses in some humans treated with immunotherapy. Here we present a case of pleural MM in which a transient spontaneous regression was associated with tumor tissue infiltration with mononuclear cells and serological evidence of anti-MM reactivity. The patient's tumor eventually progressed and with this progression there was evidence of loss of serological reactivity to some, but not all, of her MM antigens. The patient survived for 20 months and, in contrast to her initial biopsy, no significant lymphoid infiltrate was detected in her MM tissue at post mortem examination.

New chemotherapeutics in malignant mesothelioma: effects on cell growth and IL-6 production.

PURPOSE: The benefits of chemotherapy can be assessed in terms of tumour shrinkage, prolongation of life or simply palliation of symptoms. In the study reported here, in vitro correlates of these parameters were sought as a rational guide to the choice of newer agents in the clinic. METHODS: The cytotoxicity and effects on IL-6 production of ten chemotherapy agents representing four different classes of drugs were tested against a panel of five mesothelioma cell lines. RESULTS: The mesothelioma cells were more sensitive to the action of irinotecan (and its active metabolite SN38) and gemcitabine than the control cell lines. Gemcitabine and to a lesser extent irinotecan inhibited the secretion of the proinflammatory cytokine IL-6 at concentrations of each drug that produced only small decreases in cell viability. This effect was not seen in cells treated with docetaxel or vindesine. Higher doses of gemcitabine and irinotecan caused a surge in IL-6 release and this was not due to release of intracellular stores of IL-6 through lysis of the cells. CONCLUSIONS: These results suggest that irinotecan and gemcitabine are not only more likely to be active against mesothelioma than other new chemotherapy agents but may also produce a palliative effect in nonresponders to these agents by decreasing the secretion of IL-6.

Expression and integrity of DNA topoisomerase II isoforms does not explain generic drug resistance in malignant mesothelioma.

PURPOSE: Malignant mesothelioma is a tumour that is highly resistant to a number of different chemotherapy agents, yet the mechanisms by which resistance occurs are poorly understood. The pattern of resistance is consistent with disruption of topoisomerase function or expression. Coupled with this, we have previously noted a common serological reaction to the beta isoform of topoisomerase II, suggesting that it may be aberrantly expressed in patients with mesothelioma. METHODS: We assessed the expression of topoisomerase II isoforms in sections of primary tumour. We tested a panel of five mesothelioma cell lines for sensitivity to the known topoisomerase-targeting drugs, doxorubicin and etoposide. We sequenced expressed segments of the topoisomerase genes from these cell lines that have previously been associated with drug resistance. We then investigated other potential resistance mechanisms. RESULTS: We found that the beta isoform of topoisomerase II was more frequently expressed in primary tumours. Only one of the five cell lines was highly resistant to etoposide and this cell line was found to have a point mutation in the gene for topoisomerase IIalpha. Protein levels of topoisomerase IIalpha and beta did not correlate with sensitivity to either doxorubicin nor to etoposide. Semiquantitative analysis suggested that there was marked variation in the levels of mRNA expression of MRP, gamma-GCS and MDR1. None of these findings could be associated with resistance to chemotherapy. CONCLUSION: We conclude that mutations in topoisomerase IIalpha can be associated with extreme resistance of mesothelioma to etoposide. The generic drug resistance of this tumour requires further investigation.

Mixed erythrocyte-spermatozoa antiglobulin reaction (MAR test) for IgA antisperm antibodies in subfertile males.

A mixed antiglobulin reaction (MAR test), using antibody-coated red cells and spermatozoa, has been used to detect the presence of either IgA or IgG on he surface of the spermatozoa. Group O Rh-positive red cells were sensitized with serum containing anti-D, which was partly IgA, though mainly IgG. Spermatozoa were added to the sensitized red cells and tested either with anti-IgA or anti-IgG. MAR (IgA) test results were negative or doubtful in 42 (44%) of 94 samples with positive or strongly positive MAR (IgG) tests. Positive MAR (IgA) tests showed a highly significant correlation with the presence of antisperm antibodies in seminal plasma. Significantly impaired sperm penetration of cervical mucus was demonstrated for 15 patients, 12 of whom had positive MAR (IgA) tests, whereas good sperm penetration was observed for 5 patients with negative or dubious MAR (IgA) tests; all 20 patients had strongly positive MAR (IgG) tests and positive serum antisperm antibody tests.

Transfection of the ChloramphenicolAcetyltransferase Gene into Eukaryotic Cells Using Diethyl-Aminoethyl (DEAE)-Dextran.

The study of gene regulation in eukaryotic cells involves a practical requirement for two distinct techniques: first, a transfection system, or more simply, a way of getting DNA into a cell; and second, a reporter system, which, as the name suggests, is a means of finding out what the transfected DNA does from its new location inside the cell. These two requirements are amply met by transfecting cells, in the presence of the polycation diethyl aminoethyl-dextran (DEAE-dextran), with a plasmid vector that has regulatory sequences adjacent to the chloramphenicol acetyl transferase (CAT) gene. This type of gene transfer is often referred to as a transient expression system. Production of the CAT enzyme peaks at around 40-48 h and thereafter the level falls as the plasmid DNA is diluted out in a growing population of cells. The general strategy of subcloning putative regulatory regions followed by transfection and quantification of CAT activity is outlined in a flow diagram (Fig. 1) Fig. 1. Flow diagram showing the key steps in transient transfection analysis. (1) Putative regulatory regions are excised from genomic DNA by restriction enzyme digestion. Fragments are (2) subcloned into the CAT vector and (3) transfected into eukaryotic cells using DEAE dextran. (4) About 40 h after transfection, the cells are lysed and the lysates incubated with chloramphenicol and radiolabeled acetyl coenzyme A. (5) Radiolabeled chloramphenicol is extracted from the mixture and (6) the level of radioactivity related to the function of the subcloned fragment.

Harnessing the immune response to treat cancer.

It is well established that the immune system has the capacity to attack malignant cells. During malignant transformation cells acquire numerous molecular and biochemical changes that render them potentially vulnerable to immune cells. Yet it is self-evident that a growing tumour has managed to evade these host defence mechanisms. The exact ways in which the immune system interacts with tumour cells and how cancers are able to escape immunological eradication have only recently started to be fully elucidated. Understanding the relationship between the tumour and the anti-tumour immune response and how this can be altered with conventional treatments and immune-targeted therapies is crucial to developing new treatments for patients with cancer. In this review, focusing on the anti-tumour T-cell response, we summarize our understanding of how tumours, cancer treatments and the immune system interact, how tumours evade the immune response and how this process could be manipulated for the benefit of patients with cancer.