Mesothelioma patient derived tumor xenografts with defined BAP1 mutations that mimic the molecular characteristics of human malignant mesothelioma

Background: The development and evaluation of new therapeutic approaches for malignant mesothelioma has been sparse due, in part, to lack of suitable tumor models. Methods: We established primary mesothelioma cultures from pleural and ascitic fluids of five patients with advanced mesothelioma. Electron microscopy and immunohistochemistry (IHC) confirmed their mesothelial origin. Patient derived xenografts were generated by injecting the cells in nude or SCID mice, and malignant potential of the cells was analyzed by soft agar colony assay. Molecular profiles of the primary patient tumors, early passage cell cultures, and patient derived xenografts were assessed using mutational analysis, fluorescence in situ hybridization (FISH) analysis and IHC. Results: Primary cultures from all five tumors exhibited morphologic and IHC features consistent to those of mesothelioma cells. Mutations of BAP1 and CDKN2A were each detected in four tumors. BAP1 mutation was associated with the lack of expression of BAP1 protein. Three cell cultures, all of which were derived from BAP1 mutant primary tumors, exhibited anchorage independent growth and also formed tumors in mice, suggesting that BAP1 loss may enhance tumor growth in vivo. Both early passage cell cultures and mouse xenograft tumors harbored BAP1 mutations and CDKN2A deletions identical to those found in the corresponding primary patient tumors. Conclusions: The mesothelioma patient derived tumor xenografts with mutational alterations that mimic those observed in patient tumors which we established can be used for preclinical development of novel drug regimens and for studying the functional aspects of BAP1 biology in mesothelioma. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1362-2) contains supplementary material, which is available to authorized users

Tumor Suppressor Alterations Cooperate To Drive Aggressive Mesotheliomas With Enriched Cancer Stem Cells Via A P53-Mir-34A-C-Met Axis

Malignant mesothelioma is a highly aggressive, asbestos-related cancer frequently marked by mutations of both NF2 and CDKN2A. We demonstrate that germline knockout of one allele of each of these genes causes accelerated onset and progression of asbestos-induced malignant mesothelioma compared with asbestosexposed Nf2+/- or wild-type mice. Ascites from some Nf2 +/-; Cdkn2a+/- mice exhibited large tumor spheroids, and tail vein injections of malignant mesothelioma cells established from these mice, but not from Nf2+/- or wildtype mice, produced numerous tumors in the lung, suggesting increased metastatic potential of tumor cells from Nf2+/-;Cdkn2a+/- mice. Intraperitoneal injections of malignant mesothelioma cells derived from Nf2+/-; Cdkn2a +/- mice into severe combined immunodeficient mice produced tumors that penetrated the diaphragm and pleural cavity and harbored increased cancer stem cells (CSC). Malignant mesothelioma cells from Nf2+/-; Cdkn2a+/- mice stained positively for CSC markers and formed CSC spheroids in vitro more efficiently than counterparts from wild-type mice. Moreover, tumor cells from Nf2+/-;Cdkn2a+/- mice showed elevated c-Met expression/activation, which was partly dependent on p53-mediated regulation of miR-34a and required for tumor migration/invasiveness and maintenance of the CSC population. Collectively, these studies demonstrate in vivo that inactivation of Nf2 and Cdkn2a cooperate to drive the development of highly aggressive malignant mesotheliomas characterized by enhanced tumor spreading capability and the presence of a CSC population associated with p53/miR-34a-dependent activation of c-Met. These findings suggest that cooperativity between losses of Nf2 and Cdkn2a plays a fundamental role in driving the highly aggressive tumorigenic phenotype considered to be a hallmark of malignant mesothelioma. © 2014 American Association for Cancer Research

An asbestos-exposed family with multiple cases of pleural malignant mesothelioma without inheritance of a predisposing BAP1 mutation

We report a family with domestic exposure to asbestos and diagnosis of multiple cancers, including eight pleural malignant mesotheliomas and several other lung or pleural tumors. DNA sequence analysis revealed no evidence for an inherited mutation of BAP1. Sequence analysis of other potentially relevant genes, including TP53, CDKN2A, and BARD1, also revealed no mutation. DNA microarray analysis of tissue from two mesotheliomas revealed multiple genomic imbalances, including consistent losses of overlapping segments in 2q, 6q, 9p, 14q, 15q, and 22q, but no losses of chromosome 3 harboring the BAP1 locus. However, the results of immunohistochemical analysis demonstrated loss of nuclear BAP1 staining in three of six mesotheliomas tested, suggesting that somatic alterations of BAP1 occurred in a subset of tumors from this family. Since mesothelioma could be confirmed in only a single generation, domestic exposure to asbestos may be the predominant cause of mesothelioma in this family. Given the existence of unspecified malignant pleural tumors and lung cancers in a prior generation, we discuss the possibility that some other tumor susceptibility or modifier gene(s) may contribute to the high incidence of mesothelioma in this family. Because the incidence of mesothelioma in this family is higher than that expected even in workers heavily exposed to asbestos, we conclude that both asbestos exposure and genetic factors have played a role in the high rate of mesothelioma and potentially other pleural or lung cancers seen in this family.

Constitutively Active Akt1 Cooperates with KRasG12D to Accelerate In Vivo Pancreatic Tumor Onset and Progression

BACKGROUND AND AIMS: Pancreatic adenocarcinoma is a deadly disease characterized by metastatic progression and resistance to conventional therapeutics. Mutation of KRAS is the most frequent early event in pancreatic tumor progression. AKT isoforms are frequently activated in pancreatic cancer, and reports have implicated hyperactivation of AKT1, as well as AKT2, in pancreatic tumor formation. The objective here is to delineate the role of AKT in facilitating in vivo pancreatic tumor progression in the context of KRAS mutation and predisposition to pancreatic cancer. METHODS: Mice with Akt1 and KRas mutant alleles expressed using the pancreas Pdx promoter were mated to characterize the incidence and frequency of histologic and genetic alterations known to occur commonly in human pancreatic ductal adenocarcinoma. RESULTS: Active Akt1 (Akt1Myr, containing a myristoylation sequence) cooperated with active mutant KRasG12D to accelerate pancreatic carcinoma onset and progression and increase phosphorylation of downstream effectors in the Akt pathway. Mucin and smooth muscle actin expression was found in and around pancreatic intraepithelial neoplasms (PanINs), and accelerated time to metastasis was found in Akt1Myr/KRasG12D mice. CONCLUSIONS: In contrast to prior reports of pancreatic KRas mutant mice mated with mice deficient for various tumor suppressor genes, which resulted in aggressive disease within a few months of age, Akt1Myr/KRasG12D mice enabled the study of PanINs and spontaneous pancreatic transformation more characteristic of human pancreatic progression in elderly individuals. The Akt1Myr/KRasG12D model holds promise for delineating the tumor biology and biomarkers critical for understanding their cooperation in cancer oncogenesis and future targeting in therapeutic strategies

Genetic Variants Detected Using Cell-Free DNA from Blood and Tumor Samples in Patients with Inflammatory Breast Cancer

We studied genomic alterations in 19 inflammatory breast cancer (IBC) patients with advanced disease using samples of tissue and paired blood serum or plasma (cell-free DNA, cfDNA) by targeted next generation sequencing (NGS). At diagnosis, the disease was triple negative (TN) in eleven patients (57.8%), ER+ Her2- IBC in six patients (31.6%), ER+ Her2+ IBC in one patient (5.3%), and ER- Her2+ IBC in one other patient (5.3%). Pathogenic or likely pathogenic variants were frequently detected in TP53 (47.3%), PMS2 (26.3%), MRE11 (26.3%), RB1 (10.5%), BRCA1 (10.5%), PTEN (10.5%) and AR (10.5%); other affected genes included PMS1, KMT2C, BRCA2, PALB2, MUTYH, MEN1, MSH2, CHEK2, NCOR1, PIK3CA, ESR1 and MAP2K4. In 15 of the 19 patients in which tissue and paired blood were collected at the same time point, 80% of the variants detected in tissue were also detected in the paired cfDNA. Higher concordance between tissue and cfDNA was found for variants with higher allele fraction in tissue (AFtissue ≥ 5%). Furthermore, 86% of the variants detected in cfDNA were also detected in paired tissue. Our study suggests that the genetic profile measured in blood cfDNA is complementary to that of tumor tissue in IBC patients