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.

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.

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.

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.

Spontaneously immortalized mouse mesothelial cells display characteristics of malignant transformation.

OBJECTIVES: Mesotheliomas occur in occult serous cavities after chronic exposure of mesothelial cells to asbestos fibres. Molecular events that contribute to the development of this cancer are therefore not readily accessible for study. We have used in vitro culture systems to study and compare induced and spontaneous transformation events in primary mouse mesothelial cells. MATERIALS AND METHODS: Mouse mesothelial cells were cultivated until small populations of proliferating cells emerged from senescing cultures. Spontaneously transformed cultures of cells were characterized and compared to malignantly transformed cells. RESULTS: Human mesothelial cells had a finite lifespan of 10-15 population doublings when cultured in vitro; mouse mesothelial cells typically exhibit this same pattern. Here, we show that mouse mesothelial cells can be cultured for extended periods and that these cells can transform spontaneously. Lines of spontaneously transformed cells generated in this study are immortal and growth factor-independent. They display the salient characteristic features of transformation, including increased proliferation rate, lack of contact inhibition, aneuploidy and ability to grow in anchorage-independent conditions. A subset of these cell lines developed into tumours in syngeneic mice. Comparative gene expression analysis demonstrated that spontaneously transformed cell lines were more closely related to neoplastic cells than to primary cells. CONCLUSION: These findings have implications for interpretation of in vitro transformation studies, demonstrating broad similarity between spontaneous and induced genetic changes.

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.

Private specificities can dominate the humoral response to self-antigens in patients with cryptogenic fibrosing alveolitis.

BACKGROUND: The pathogenetic mechanisms that underlie the interstitial lung disease cryptogenic fibrosing alveolitis (CFA) may involve an immunological reaction to unidentified antigens in the lung, resulting in tissue damage. METHOD: In order to identify the range of target autoantigens, we used expression cloning, employing serum from an index patient as the probe against an expressed cDNA library that was derived from a tumour cell line. We screened over 5 x 105 recombinants and obtained sequence information on three antigens that had provoked strong responses with immunoglobulin heavy chain class switching, presumably as a consequence of T-cell recognition. RESULTS: All of the antigens were identifiable by comparison with sequence data from the US National Center for Biotechnology Information. Alanyl tRNA synthetase (ATS) was picked on six occasions; five of these incidences reflected independent recombination events, indicating that the library was not biased. Antibodies to ATS (anti-PL-12) represent the most common reactivity that defines the antisynthetase syndrome, which is typically expressed as polymyositis, dermatomyositis and interstitial lung disease (ILD). The index patient never showed symptoms other than those associated with alveolitis, even though sera obtained from him over a period of 2 years contained antibodies with the same specificity. Autoantibodies to ATS were never detected in serial bleeds from 11 other patients with CFA, and neither did we detect antibodies to the other two antigens identified from the serum of the index patient. CONCLUSION: The humoral response in patients with CFA can be dominated by autoantibodies with private specificities. This suggests that the antibodies are epiphenomenal and are a secondary feature of tissue damage induced by some other mechanism.

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.

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.

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.

p53 autoantibodies in patients with malignant mesothelioma: stability through disease progression.

Malignant mesothelioma (MM) generally occurs as a pleural tumour, related to the inhalation of asbestos fibres. It is highly aggressive and largely unresponsive to treatment. The incidence of MM is particularly high in Western Australia because of the extensive blue asbestos mining operations that occurred in the north of the state until 1966. MM is unusual in that mutations in the tumour suppressor gene p53 are rarely observed, whilst over-expression of p53 protein is common. As the level of antibodies directed against p53 is thought to be of prognostic value in some cancers and as MM is known to be immunogenic, we studied a cohort of Western Australian patients to determine the prevalence of anti-p53 antibodies and their value as diagnostic markers or prognostic indicators. 6/88 (7%) of patients had high titres (>2 SD above the mean of controls) of anti-p53 antibodies. There was no correlation between antibody titre and survival. Although 3/38 (8%) of sera obtained from patients exposed to asbestos but prior to a diagnosis of MM contained antibodies, the same proportion of sera obtained from patients exposed to asbestos but who remained disease free also contained antibodies (2/40; 8%). Sera collected sequentially demonstrated a profound temporal stability in the titre of anti-p53 antibodies in patients with MM throughout the course of their illness. These results show that anti-p53 antibodies are observed only at a low frequency in the sera of MM patients and where they do occur, their elicitation is an early event that may be unrelated to antigen load. The occurrence of anti-p53 antibodies does not serve as either a useful prognostic or diagnostic indicator in MM.

Altered superantigenic ligands demonstrate the quantitative nature of T-cell activation.

In a recent study, a superantigen mutated in the TCR binding site (staphylococcal enterotoxin B (SEB)delta61Y) was described, which behaved as a partial agonist for a Vbeta17-expressing T-cell clone. Evidence is now presented to demonstrate that there is distinct heterogeneity in the response of primary T cells to this protein. Some Vbeta17 T cells responded to SEBdelta61Y by modulating surface receptor expression consistent with activation, and by proliferating. Other Vbeta17 T cells did not proliferate, nor did they display a receptor expression phenotype consistent with activation. However, when repeatedly exposed to the altered superantigen, some of these non-responders entered cell cycle. This pattern of responses was not recapitulated by providing additional costimulation via CD28, although such treatment did induce some of the 'unresponsive' Vbeta17 T cells to upregulate the IL-2 receptor, indicative of partial activation. It was also found that the heterogeneous pattern could be replicated using very low doses of native SEB. The data are discussed in the context of models of T-cell activation in which differences in TCR ligand affinity and dose determine qualitatively different response phenotypes.

Tumor-specific CD4+ T cells have a major "post-licensing" role in CTL mediated anti-tumor immunity.

A number of tumor studies have indicated a link between CD4 help and the magnitude and persistence of CTL activity; however, the mechanisms underlying this have been largely unclear. To evaluate and determine the mechanisms by which CD4(+) T cells synergize with CD8(+) T cells to prevent tumor growth, we used the novel technique of monitoring in vivo CTL by labeling target cells with CFSE. This approach was supported by the direct visualization of CTL using peptide-MHC tetramers to follow tumor-specific T cells. The data presented demonstrate that while cotransfer of Ag-specific CD4(+) T cells was not required for the generation of CTLs, because adoptive transfer of CD8(+) T cells alone was sufficient, CD4(+) T cells were required for the maintenance of CD8(+) T cell numbers. Our data suggest that there is a correlation among the number of CD8(+) T cells, in vivo CTL function, and IFN-gamma production, with no evidence of a partial or nonresponsive phenotype among tetramer-positive cells. We also show that CD4(+) T cells are required for CD8(+) T cell infiltration of the tumor.

Simian virus (SV) 40 like sequences in cell lines and tumour biopsies from Australian malignant mesotheliomas.

BACKGROUND: Simian virus (SV) 40 sequences have been found in some, but not all studies of mesotheliomas. This virus is known to cause tumours in rodents but its role in human oncogenesis remains controversial. AIMS: The aim of this study therefore was to determine whether SV40 is associated with the development of mesotheliomas in Australia. The absence of the virus or its gene products in tissue derived from mesotheliomas would detract from this possibility. METHODS: We used polymerase chain reaction from three pairs of primers to amplify different regions of the large T antigen from DNA from cell lines and cDNA from both cell lines and an independent set of tumour biopsies from patients with mesothelioma. RESULTS: We examined five human mesothelioma cell lines that were established in our laboratories. In addition, we examined several tumour biopsies from seven different patients. SV40 like sequences were present in all the cell lines and in at least one sample from each of the patients examined. CONCLUSIONS: The large T antigen of SV40 or an SV40 like virus is expressed in Australian mesotheliomas and therefore could be aetiologically-associated with tumourigenesis. Alternatively, these sequences could be expressed subsequent to the development of the disease.

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.

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.