Consensus Report of the 2015 Weinman International Conference on Mesothelioma.

On November 9 and 10, 2015, the International Conference on Mesothelioma in Populations Exposed to Naturally Occurring Asbestiform Fibers was held at the University of Hawaii Cancer Center in Honolulu, Hawaii. The meeting was cosponsored by the International Association for the Study of Lung Cancer, and the agenda was designed with significant input from staff at the U.S. National Cancer Institute and National Institute of Environmental Health Sciences. A multidisciplinary group of participants presented updates reflecting a range of disciplinary perspectives, including mineralogy, geology, epidemiology, toxicology, biochemistry, molecular biology, genetics, public health, and clinical oncology. The group identified knowledge gaps that are barriers to preventing and treating malignant mesothelioma (MM) and the required next steps to address barriers. This manuscript reports the group's efforts and focus on strategies to limit risk to the population and reduce the incidence of MM. Four main topics were explored: genetic risk, environmental exposure, biomarkers, and clinical interventions. Genetics plays a critical role in MM when the disease occurs in carriers of germline BRCA1 associated protein 1 mutations. Moreover, it appears likely that, in addition to BRCA1 associated protein 1, other yet unknown genetic variants may also influence the individual risk for development of MM, especially after exposure to asbestos and related mineral fibers. MM is an almost entirely preventable malignancy as it is most often caused by exposure to commercial asbestos or mineral fibers with asbestos-like health effects, such as erionite. In the past in North America and in Europe, the most prominent source of exposure was related to occupation. Present regulations have reduced occupational exposure in these countries; however, some people continue to be exposed to previously installed asbestos in older construction and other settings. Moreover, an increasing number of people are being exposed in rural areas that contain noncommercial asbestos, erionite, and other mineral fibers in soil or rock (termed naturally occurring asbestos [NOA]) and are being developed. Public health authorities, scientists, residents, and other affected groups must work together in the areas where exposure to asbestos, including NOA, has been documented in the environment to mitigate or reduce this exposure. Although a blood biomarker validated to be effective for use in screening and identifying MM at an early stage in asbestos/NOA-exposed populations is not currently available, novel biomarkers presented at the meeting, such as high mobility group box 1 and fibulin-3, are promising. There was general agreement that current treatment for MM, which is based on surgery and standard chemotherapy, has a modest effect on the overall survival (OS), which remains dismal. Additionally, although much needed novel therapeutic approaches for MM are being developed and explored in clinical trials, there is a critical need to invest in prevention research, in which there is a great opportunity to reduce the incidence and mortality from MM.

High Incidence of Somatic BAP1 alterations in sporadic malignant mesothelioma.

BACKGROUND: Breast cancer 1-associated protein 1 (BAP1) is a nuclear deubiquitinase that regulates gene expression, transcription, DNA repair, and more. Several findings underscore the apparent driver role of BAP1 in malignant mesothelioma (MM). However, the reported frequency of somatic BAP1 mutations in MM varies considerably, a discrepancy that appeared related to either methodological or ethnical differences across various studies. METHODS: To address this discrepancy, we carried out comprehensive genomic and immunohistochemical (IHC) analyses to detect somatic BAP1 gene alterations in 22 frozen MM biopsies from U.S. MM patients. RESULTS: By combining Sanger sequencing, multiplex ligation-dependent probe amplification, copy number analysis, and cDNA sequencing, we found alteration of BAP1 in 14 of 22 biopsies (63.6%). No changes in methylation were observed. IHC revealed normal nuclear BAP1 staining in the eight MM containing wild-type BAP1, whereas no nuclear staining was detected in the 14 MM biopsies containing tumor cells with mutated BAP1. Thus, IHC results were in agreement with those obtained by genomic analyses. We then extended IHC analysis to an independent cohort of 70 MM biopsies, of which there was insufficient material to perform molecular studies. IHC revealed loss of BAP1 nuclear staining in 47 of these 70 MM biopsies (67.1%). CONCLUSIONS: Our findings conclusively establish BAP1 as the most commonly mutated gene in MM, regardless of ethnic background or other clinical characteristics. Our data point to IHC as the most accessible and reliable technique to detect BAP1 status in MM biopsies.

Mesothelioma patients with germline BAP1 mutations have 7-fold improved long-term survival.

BRCA1-associated protein-1 (BAP1) mutations cause a new cancer syndrome, with a high rate of malignant mesothelioma (MM). Here, we tested the hypothesis that MM associated with germline BAP1 mutations has a better prognosis compared with sporadic MM. We compared survival among germline BAP1 mutation MM patients with that of all MM (N = 10 556) recorded in the United States Surveillance, Epidemiology, and End Results (SEER) data from 1973 to 2010. We identified 23 MM patients--11 alive--with germline BAP1 mutations and available data on survival. Ten patients had peritoneal MM, ten pleural MM and three MM in both locations. Thirteen patients had one or more malignancies in addition to MM. Actuarial median survival for the MM patients with germline BAP1 mutations was 5 years, as compared with <1 year for the median survival in the United States SEER MM group. Five-year survival was 47%, 95% confidence interval (24-67%), as compared with 6.7% (6.2-7.3%) in the control SEER group. Analysis of the pooled cohort of germline BAP1 mutation MM showed that patients with peritoneal MM (median survival of 10 years, P = 0.0571), or with a second malignancy in addition to MM (median survival of 10 years, P = 0.0716), survived for a longer time compared with patients who only had pleural MM, or MM patients without a second malignancy, respectively. In conclusion, we found that MM patients with germline BAP1 mutations have an overall 7-fold increased long-term survival, independently of sex and age. Appropriate genetic counseling and clinical management should be considered for MM patients who are also BAP1 mutation carriers.

Forced LIGHT expression in prostate tumors overcomes Treg mediated immunosuppression and synergizes with a prostate tumor therapeutic vaccine by recruiting effector T lymphocytes.

BACKGROUND: LIGHT, a ligand for lymphotoxin-ß receptor (LTßR) and herpes virus entry mediator, is predominantly expressed on activated immune cells and LTßR signaling leads to the recruitment of lymphocytes. The interaction between LIGHT and LTßR has been previously shown to activate immune cells and result in tumor regression in a virally-induced tumor model, but the role of LIGHT in tumor immunosuppression or in a prostate cancer setting, where self antigens exist, has not been explored. We hypothesized that forced expression of LIGHT in prostate tumors would shift the pattern of immune cell infiltration toward an anti-tumoral milieu, would inhibit T regulatory cells (Tregs) and would induce prostate cancer tumor associated antigen (TAA) specific T cells that would eradicate tumors. METHODS: Real Time PCR was used to evaluate expression of forced LIGHT and other immunoregulatory genes in prostate tumors samples. For in vivo studies, adenovirus encoding murine LIGHT was injected intratumorally into TRAMP-C2 prostate cancer cell tumor bearing mice. Chemokine and cytokine concentrations were determined by multiplex ELISA. Flow cytometry was used to phenotype tumor infiltrating lymphocytes and expression of LIGHT on the tumor cell surface. Tumor-specific lymphocytes were quantified via ELISpot assay. Treg induction and Treg suppression assays determined Treg functionality after LIGHT treatment. RESULTS: LIGHT in combination with a therapeutic vaccine, PSCA TriVax, reduced tumor burden. LIGHT expression peaked within 48 hr of infection, recruited effector T cells that recognized mouse prostate stem cell antigen (PSCA) into the tumor microenvironment, and inhibited infiltration of Tregs. Tregs isolated from tumor draining lymph nodes had impaired suppressive capability after LIGHT treatment. CONCLUSION: Forced LIGHT treatment combined with PSCA TriVax therapeutic vaccination delays prostate cancer progression in mice by recruiting effector T lymphocytes to the tumor and inhibiting Treg mediated immunosuppression. Prostate 75:280-291, 2015. © 2014 Wiley Periodicals, Inc.

Evaluation of clonal origin of malignant mesothelioma.

BACKGROUND: The hypothesis that most cancers are of monoclonal origin is often accepted as a fact in the scientific community. This dogma arose decades ago, primarily from the study of hematopoietic malignancies and sarcomas, which originate as monoclonal tumors. The possible clonal origin of malignant mesothelioma (MM) has not been investigated. Asbestos inhalation induces a chronic inflammatory response at sites of fiber deposition that may lead to malignant transformation after 30-50 years latency. As many mesothelial cells are simultaneously exposed to asbestos fibers and to asbestos-induced inflammation, it may be possible that more than one cell undergoes malignant transformation during the process that gives rise to MM, and result in a polyclonal malignancy. METHODS AND RESULTS: To investigate the clonality patterns of MM, we used the HUMARA (Human Androgen Receptor) assay to examine 16 biopsies from 14 women MM patients. Out of 16 samples, one was non-informative due to skewed Lyonization in its normal adjacent tissue. Fourteen out of the 15 informative samples revealed two electrophoretically distinct methylated HUMARA alleles, the Corrected Allele Ratio (CR) calculated on the allele peak areas indicating polyclonal origin MM. CONCLUSIONS: Our results show that MM originate as polyclonal tumors and suggest that the carcinogenic "field effect" of mineral fibers leads to several premalignant clones that give rise to these polyclonal malignancies.

CSPG4 as a target of antibody-based immunotherapy for malignant mesothelioma.

PURPOSE: Malignant mesothelioma (MM) is an aggressive cancer, resistant to current therapies. Membrane chondroitin sulphate proteoglycan 4 (CSPG4), which has been successfully targeted in melanoma and breast cancer, was found highly expressed in MM, but not in normal mesothelium. Therefore, we explored CSPG4 as a suitable target for monoclonal antibody (mAb)-based immunotherapy for MM. EXPERIMENTAL DESIGN: We assayed adhesion, motility, invasiveness, wound-healing, apoptosis, and anchorage-independent growth of MM cells on cell cultures. CSPG4 expression and signaling was studied by immunoblotting. The growth of MM severe combined immunodeficient (SCID) mice xenografts induced by PPM-Mill cells, engineered to express the luciferase reporter gene, was monitored by imaging, upon treatment with CSPG4 mAb TP41.2. Animal toxicity and survival were assayed in both tumor inhibition and therapeutic experiments. RESULTS: CSPG4 was expressed on 6 out of 8 MM cell lines and in 25 out of 41 MM biopsies, with minimal expression in surrounding healthy cells. MM cell adhesion was mediated by CSPG4-dependent engagement of ECM. Cell adhesion was inhibited by mAb TP41.2 resulting in decreased phosphorylation of focal adhesion kinase (FAK) and AKT, reduced expression of cyclin D1 and apoptosis. Moreover, mAb TP41.2 significantly reduced MM cell motility, migration, and invasiveness, and inhibited MM growth in soft agar. In vivo, treatment with mAb TP41.2 prevented or inhibited the growth of MM xenografts in SCID mice, with a significant increase in animal survival. CONCLUSION: These results establish the safety of CSPG4 mAb-based immunotherapy and suggest that CSPG4 mAb-based immunotherapy may represent a novel approach for the treatment of MM.

Erionite exposure in North Dakota and Turkish villages with mesothelioma.

Exposure to erionite, an asbestos-like mineral, causes unprecedented rates of malignant mesothelioma (MM) mortality in some Turkish villages. Erionite deposits are present in at least 12 US states. We investigated whether increased urban development has led to erionite exposure in the United States and after preliminary exploration, focused our studies on Dunn County, North Dakota (ND). In Dunn County, ND, we discovered that over the past three decades, more than 300 miles of roads were surfaced with erionite-containing gravel. To determine potential health implications, we compared erionite from the Turkish villages to that from ND. Our study evaluated airborne point exposure concentrations, examined the physical and chemical properties of erionite, and examined the hallmarks of mesothelial cell transformation in vitro and in vivo. Airborne erionite concentrations measured in ND along roadsides, indoors, and inside vehicles, including school buses, equaled or exceeded concentrations in Boyali, where 6.25% of all deaths are caused by MM. With the exception of outdoor samples along roadsides, ND concentrations were lower than those measured in Turkish villages with MM mortality ranging from 20 to 50%. The physical and chemical properties of erionite from Turkey and ND are very similar and they showed identical biological activities. Considering the known 30- to 60-y latency for MM development, there is reason for concern for increased risk in ND in the future. Our findings indicate that implementation of novel preventive and early detection programs in ND and other erionite-rich areas of the United States, similar to efforts currently being undertaken in Turkey, is warranted.

PR1-specific T cells are associated with unmaintained cytogenetic remission of chronic myelogenous leukemia after interferon withdrawal.

BACKGROUND: Interferon-alpha (IFN) induces complete cytogenetic remission (CCR) in 20-25% CML patients and in a small minority of patients; CCR persists after IFN is stopped. IFN induces CCR in part by increasing cytotoxic T lymphocytes (CTL) specific for PR1, the HLA-A2-restricted 9-mer peptide from proteinase 3 and neutrophil elastase, but it is unknown how CCR persists after IFN is stopped. PRINCIPAL FINDINGS: We reasoned that PR1-CTL persist and mediate CML-specific immunity in patients that maintain CCR after IFN withdrawal. We found that PR1-CTL were increased in peripheral blood of 7/7 HLA-A2+ patients during unmaintained CCR from 3 to 88 months after IFN withdrawal, as compared to no detectable PR1-CTL in 2/2 IFN-treated CML patients not in CCR. Unprimed PR1-CTL secreted IFNgamma and were predominantly CD45RA+/-CD28+CCR7+CD57-, consistent with functional naïve and central memory (CM) T cells. Similarly, following stimulation, proliferation occurred predominantly in CM PR1-CTL, consistent with long-term immunity sustained by self-renewing CM T cells. PR1-CTL were functionally anergic in one patient 6 months prior to cytogenetic relapse at 26 months after IFN withdrawal, and in three relapsed patients PR1-CTL were undetectable but re-emerged 3-6 months after starting imatinib. CONCLUSION: These data support the hypothesis that IFN elicits CML-specific CM CTL that may contribute to continuous CCR after IFN withdrawal and suggest a role for T cell immune therapy with or without tyrosine kinase inhibitors as a strategy to prolong CR in CML.

Expression of LIGHT/TNFSF14 combined with vaccination against human papillomavirus Type 16 E7 induces significant tumor regression.

LIGHT, a ligand for the lymphotoxin-beta receptor, establishes lymphoid-like tissues inside tumor sites and recruits naïve T cells into the tumor. However, whether these infiltrating T cells are specific for tumor antigens is not known. We hypothesized that therapy with LIGHT can expand functional tumor-specific CD8(+) T cells that can be boosted using HPV16E6E7-Venezuelan equine encephalitis virus replicon particles (HPV16-VRP) and that this combined therapy can eradicate human papillomavirus 16 (HPV16)-induced tumors. Our data show that forced expression of LIGHT in tumors results in an increase in expression of IFNgamma and chemoattractant cytokines such as interleukin-1a, MIG, and macrophage inflammatory protein-2 within the tumor and that this tumor microenvironment correlates with an increase in frequency of tumor-infiltrating CD8(+) T cells. Forced expression of LIGHT also results in the expansion of functional T cells that recognize multiple tumor antigens, including HPV16 E7, and these T cells prevent the outgrowth of tumors on secondary challenge. Subsequent boosting of E7-specific T cells by vaccination with HPV16-VRP significantly increases their frequency in both the periphery and the tumor and leads to the eradication of large well-established tumors, for which either treatment alone is not successful. These data establish the safety of Ad-LIGHT as a therapeutic intervention in preclinical studies and suggest that patients with HPV16(+) tumors may benefit from combined immunotherapy with LIGHT and antigen-specific vaccination.

Prostate cancer immunotherapy yields superior long-term survival in TRAMP mice when administered at an early stage of carcinogenesis prior to the establishment of tumor-associated immunosuppression at later stages.

Prostate cancer immunotherapy clinical trials have been performed, but often in immunocompromised patients with limited clinical success. The study aim was to determine whether the stage of prostate cancer development at which immunization occurs affects vaccine efficacy, and if so which tumor-associated immunosuppressive mechanisms may be involved at later stages. Therapeutic vaccination of TRAMP mice with only precancerous PIN lesions confered superior protection to immunization after development of invasive carcinoma. The presence of Treg, upregulation of tumor indoleamine-2,3-dioxygenase and TGFbeta and an immunosuppressive intratumoral cytokine milieu were identified in more advanced prostate cancer. These results indicate that prostate cancer immunotherapy trials will be more successful if conducted in patients with less advanced disease.

Peptide-based vaccines for cancer: realizing their potential.

Advances in the engineering of peptides, adjuvants and delivery systems have renewed the enthusiasm for peptide-based vaccination regimens in the setting of cancer, and there are a variety of clinical trials being conducted by pharmaceutical companies based on the use of peptides. The challenges to successful cancer immunotherapy are common to all immunotherapeutic strategies and not unique to peptide-based vaccination regimens. This review will describe the advances in the identification, design and delivery of peptides, the challenges to successful immunotherapy and will discuss potential options for the future.

Recent advances in strategies for immunotherapy of human papillomavirus-induced lesions.

Human papillomavirus (HPV)-induced lesions are distinct in that they have targetable foreign antigens, the expression of which is necessary to maintain the cancerous phenotype. Hence, they pose as a very attractive target for "proof of concept" studies in the development of therapeutic vaccines. This review will focus on the most recent clinical trials for the immunotherapy of mucosal and cutaneous HPV-induced lesions as well as emerging therapeutic strategies that have been tested in preclinical models for HPV-induced lesions. Progress in peptide-based vaccines, DNA-based vaccines, viral/bacterial vector-based vaccines, immune response modifiers, photodynamic therapy and T cell receptor based therapy for HPV will be discussed.

Mechanisms used by human papillomaviruses to escape the host immune response.

The greatest risk factor for the development of cervical and other cancers that have been linked to the human papillomavirus (HPV) family is the persistence of the virus. To persist for the decades required to develop HPV-related cancers, the virus must escape host immunity. HPV is a simple DNA virus that has evolved to escape immune attack by a combination of stealth and interference. This review focuses on the mechanisms by which HPV can evade recognition by the host immune system.