Inflammation-Induced Metastatic Colonization of the Lung Is Facilitated by Hepatocyte Growth Factor-Secreting Monocyte-Derived Macrophages.

A rate-limiting step for circulating tumor cells to colonize distant organ sites is their ability to locate a microenvironmental niche that supports their survival and growth. This can be achieved by features intrinsic to the tumor cells and/or by the conditioning of a "premetastatic" niche. To determine if pulmonary inflammation promotes the latter, we initiated models for inflammatory asthma, hypersensitivity pneumonitis, or bleomycin-induced sterile inflammation before introducing tumor cells with low metastatic potential into the circulation. All types of inflammation increased the end-stage metastatic burden of the lungs 14 days after tumor cell inoculation without overtly affecting tumor extravasation. Instead, the number and size of early micrometastatic lesions found within the interstitial tissues 96 hours after tumor cell inoculation were increased in the inflamed lungs, coincident with increased tumor cell survival and the presence of nearby inflammation-induced monocyte-derived macrophages (MoDM; CD11b+CD11c+). Remarkably, the adoptive transfer of these MoDM was sufficient to increase lung metastasis in the absence of inflammation. These inflammation-induced MoDM secrete a number of growth factors and cytokines, one of which is hepatocyte growth factor (HGF), that augmented tumor cell survival under conditions of stress in vitro. Importantly, blocking HGF signaling with the cMET inhibitor capmatinib abolished inflammation-induced early micrometastatic lesion formation in vivo. These findings indicate that inflammation-induced MoDM and HGF in particular increase the efficiency of early metastatic colonization in the lung by locally preconditioning the microenvironment. IMPLICATIONS: Inflammation preconditions the distant site microenvironment to increase the metastatic potential of tumor cells that arrive there.

ARID1A/BAF250a as a prognostic marker for gastric carcinoma: a study of 2 cohorts.

ARID1A/BAF250a has been recently implicated as a tumor suppressor in gastric cancer. We sought to clarify the clinical significance of BAF250a/ARID1A in relation to other clinical parameters and relevant biomarkers in gastric carcinoma. Cases from 2 separate cohorts of patients with gastric carcinoma from Vancouver (n = 173) and Toronto (n = 80) were selected for the construction of tissue microarrays, which were used to assess the immunohistochemical status of BAF250a (anti-ARID1A), mismatch repair proteins and p53, as well as in situ hybridization for HER2 amplification and Epstein-Barr virus infection. The Toronto cohort contained a higher proportion of early stage cases (P = .019) and a smaller proportion of cases from the proximal stomach (P < .001). Overall, immunohistochemical loss of BAF250a was observed in 22.5% of gastric adenocarcinomas from the Vancouver group and 20% from Toronto. In both cohorts, loss of BAF250a was positively associated with loss of mismatch repair protein expression (P < .0001 and P = .035, respectively). Loss of BAF250a expression was independently associated with poor overall survival in the Toronto cohort (P = .0015), whereas no significant association with survival was observed in the Vancouver cohort. BAF250a loss was not significantly associated with any additional clinical parameters in either cohort. HER2 amplification was confirmed as a negative prognostic factor in both cohorts. These findings suggest that ARID1A/BAF250a may be of prognostic significance in a subset of patients with early stage gastric cancer and that pathological assessment should increasingly use a multimarker approach.

A functional proteogenomic analysis of endometrioid and clear cell carcinomas using reverse phase protein array and mutation analysis: protein expression is histotype-specific and loss of ARID1A/BAF250a is associated with AKT phosphorylation.

BACKGROUND: Ovarian cancer is now recognized as a number of distinct diseases primarily defined by histological subtype. Both clear cell ovarian carcinomas (CCC) and ovarian endometrioid carcinomas (EC) may arise from endometriosis and frequently harbor mutations in the ARID1A tumor suppressor gene. We studied the influence of histological subtype on protein expression with reverse phase protein array (RPPA) and assessed proteomic changes associated with ARID1A mutation/BAF250a expression in EC and CCC. METHODS: Immunohistochemistry (IHC) for BAF250a expression was performed on 127 chemotherapy-naive ovarian carcinomas (33 CCC, 29 EC, and 65 high-grade serous ovarian carcinomas (HGSC)). Whole tumor lysates were prepared from frozen banked tumor samples and profiled by RPPA using 116 antibodies. ARID1A mutations were identified by exome sequencing, and PIK3CA mutations were characterized by MALDI-TOF mass spectrometry. SAM (Significance Analysis of Microarrays) was performed to determine differential protein expression by histological subtype and ARID1A mutation status. Multivariate logistic regression was used to assess the impact of ARID1A mutation status/BAF250a expression on AKT phosphorylation (pAKT). PIK3CA mutation type and PTEN expression were included in the model. BAF250a knockdown was performed in 3 clear cell lines using siRNA to ARID1A. RESULTS: Marked differences in protein expression were observed that are driven by histotype. Compared to HGSC, SAM identified over 50 proteins that are differentially expressed in CCC and EC. These included PI3K/AKT pathway proteins, those regulating the cell cycle, apoptosis, transcription, and other signaling pathways including steroid hormone signaling. Multivariate models showed that tumors with loss of BAF250a expression showed significantly higher levels of AKT-Thr308 and AKT-Ser473 phosphorylation (p < 0.05). In 31 CCC cases, pAKT was similarly significantly increased in tumors with BAF250a loss on IHC. Knockdown of BAF250a by siRNA in three CCC cell lines wild type for ARID1A showed no increase in either pAKT-Thr308 or pAKT-S473 suggesting that pAKT in tumor tissues is indirectly regulated by BAF250a expression. CONCLUSIONS: Proteomic assessment of CCC and EC demonstrates remarkable differences in protein expression that are dependent on histotype, thereby further characterizing these cancers. AKT phosphorylation is associated with ARID1A/BAF250a deficient tumors, however in ovarian cancers the mechanism remains to be elucidated.

Ovarian and endometrial endometrioid carcinomas have distinct CTNNB1 and PTEN mutation profiles.

Ovarian endometrioid carcinomas and endometrial endometrioid carcinomas share many histological and molecular alterations. These similarities are likely due to a common endometrial epithelial precursor cell of origin, with most ovarian endometrioid carcinomas arising from endometriosis. To directly compare the mutation profiles of two morphologically similar tumor types, endometrial endometrioid carcinomas (n=307) and ovarian endometrioid carcinomas (n=33), we performed select exon capture sequencing on a panel of genes: ARID1A, PTEN, PIK3CA, KRAS, CTNNB1, PPP2R1A, TP53. We found that PTEN mutations are more frequent in low-grade endometrial endometrioid carcinomas (67%) compared with low-grade ovarian endometrioid carcinomas (17%) (P<0.0001). By contrast, CTNNB1 mutations are significantly different in low-grade ovarian endometrioid carcinomas (53%) compared with low-grade endometrial endometrioid carcinomas (28%) (P<0.0057). This difference in CTNNB1 mutation frequency may be reflective of the distinct microenvironments; the epithelial cells lining an endometriotic cyst within the ovary are exposed to a highly oxidative environment that promotes tumorigenesis. Understanding the distinct mutation patterns found in the PI3K and Wnt pathways of ovarian and endometrial endometrioid carcinomas may provide future opportunities for stratifying patients for targeted therapeutics.

Correction: Type-Specific Cell Line Models for Type-Specific Ovarian Cancer Research.

[This corrects the article on p. e72162 in vol. 8.].

Type-specific cell line models for type-specific ovarian cancer research.

BACKGROUND: OVARIAN CARCINOMAS CONSIST OF AT LEAST FIVE DISTINCT DISEASES: high-grade serous, low-grade serous, clear cell, endometrioid, and mucinous. Biomarker and molecular characterization may represent a more biologically relevant basis for grouping and treating this family of tumors, rather than site of origin. Molecular characteristics have become the new standard for clinical pathology, however development of tailored type-specific therapies is hampered by a failure of basic research to recognize that model systems used to study these diseases must also be stratified. Unrelated model systems do offer value for study of biochemical processes but specific cellular context needs to be applied to assess relevant therapeutic strategies. METHODS: We have focused on the identification of clear cell carcinoma cell line models. A panel of 32 "ovarian cancer" cell lines has been classified into histotypes using a combination of mutation profiles, IHC mutation-surrogates, and a validated immunohistochemical model. All cell lines were identity verified using STR analysis. RESULTS: Many described ovarian clear cell lines have characteristic mutations (including ARID1A and PIK3CA) and an overall molecular/immuno-profile typical of primary tumors. Mutations in TP53 were present in the majority of high-grade serous cell lines. Advanced genomic analysis of bona-fide clear cell carcinoma cell lines also support copy number changes in typical biomarkers such at MET and HNF1B and a lack of any recurrent expressed re-arrangements. CONCLUSIONS: As with primary ovarian tumors, mutation status of cancer genes like ARID1A and TP53 and a general immuno-profile serve well for establishing histotype of ovarian cancer cell We describe specific biomarkers and molecular features to re-classify generic "ovarian carcinoma" cell lines into type specific categories. Our data supports the use of prototype clear cell lines, such as TOV21G and JHOC-5, and questions the use of SKOV3 and A2780 as models of high-grade serous carcinoma.

The chromatin remodeling gene ARID1A is a new prognostic marker in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common tumor of the adult kidney, with an increasing rate of incidence. Recently, exome sequencing studies have revealed that the SWI/SNF (switch/sucrose nonfermentable) members PBRM1 and ARID1A are mutated in ccRCC, and it has also been suggested that aberrant chromatin regulation is a key step in kidney cancer pathogenesis. Herein, we show that down-regulation of another SW/SNF component, ARID1A, occurs frequently in ccRCC. We detected copy number loss of ARID1A in 16% of patients with ccRCC. Immunohistochemistry indicated that 67% of ccRCC (53 of 79) had significantly lower expression of BAF250a, the protein product of ARID1A, than did the matched normal kidney cortex. In parallel, we conducted in silico mRNA expression analysis on 404 ccRCC tumors and 167 normal kidney cortex samples using publicly available databases and confirmed significant down-regulation of ARID1A in 68.8% of patients. We also show that decreased BAF250a protein and ARID1A mRNA expression correlate with tumor stage and grade. Our results indicate that both the protein and mRNA levels of ARID1A are statistically significant prognostic markers for ccRCC. Even after controlling for other confounders in the multivariate analysis, BAF250 retained its prognostic significance. BAF250a IHC is easy to perform and represents a potential biomarker that could be incorporated in laboratory practice to enhance the accuracy of the existing prognostic models.

Reversion to virulence and efficacy of an attenuated Canarypox vaccine in Hawai'i 'Amakihi (Hemignathus virens).

Vaccines may be effective tools for protecting small populations of highly susceptible endangered, captive-reared, or translocated Hawaiian honeycreepers from introduced Avipoxvirus, but their efficacy has not been evaluated. An attenuated Canarypox vaccine that is genetically similar to one of two passerine Avipoxvirus isolates from Hawai'i and distinct from Fowlpox was tested to evaluate whether Hawai'i 'Amakihi (Hemignathus virens) can be protected from wild isolates of Avipoxvirus from the Hawaiian Islands. Thirty-one (31) Hawai'i 'Amakihi were collected from high-elevation habitats on Mauna Kea Volcano, where pox transmission is rare, and randomly divided into two groups. One group was vaccinated with Poximune C, whereas the other group received a sham vaccination with sterile water. Four of 15 (27%) vaccinated birds developed life-threatening disseminated lesions or lesions of unusually long duration, whereas one bird never developed a vaccine-associated lesion or "take." After vaccine lesions healed, vaccinated birds were randomly divided into three groups of five and challenged with either a wild isolate of Fowlpox (FP) from Hawai'i, a Hawai'i 'Amakihi isolate of a Canarypox-like virus (PV1), or a Hawai'i 'Amakihi isolate of a related, but distinct, passerine Avipoxvirus (PV2). Similarly, three random groups of five unvaccinated 'Amakihi were challenged with the same virus isolates. Vaccinated and unvaccinated 'Amakihi challenged with FP had transient infections with no clinical signs of infection. Mortality in vaccinated 'Amakihi challenged with PV1 and PV2 ranged from 0% (0/5) for PV1 to 60% (3/5) for PV2. Mortality in unvaccinated 'Amakihi ranged from 40% (2/5) for PV1 to 100% (5/5) for PV2. Although the vaccine provided some protection against PV1, both potential for vaccine reversion and low efficacy against PV2 preclude its use in captive or wild honeycreepers.

Loss of BAF250a (ARID1A) is frequent in high-grade endometrial carcinomas.

Mutation of the ARID1A gene and loss of the corresponding protein BAF250a has recently been described as a frequent event in clear cell and endometrioid carcinomas of the ovary. To determine whether BAF250a loss is common in other malignancies, immunohistochemistry (IHC) for BAF250a was performed on tissue microarrays (TMAs) in more than 3000 cancers, including carcinomas of breast, lung, thyroid, endometrium, kidney, stomach, oral cavity, cervix, pancreas, colon and rectum, as well as endometrial stromal sarcomas, gastrointestinal stromal tumours, sex cord-stromal tumours and four major types of lymphoma (diffuse large B cell lymphoma, primary mediastinal B cell lymphoma, mantle cell lymphoma and follicular lymphoma). We found that BAF250a loss is frequent in endometrial carcinomas but infrequent in other types of malignancies, with loss observed in 29% (29/101) of grade 1 or 2 and 39% (44/113) of grade 3 endometrioid carcinomas of the endometrium, 18% (17/95) of uterine serous carcinomas and 26% (6/23) of uterine clear cell carcinomas. Since endometrial cancers showed BAF250a loss, we stained whole tissue sections for BAF250a expression in nine cases of atypical hyperplasia and 10 cases of atypical endometriosis. Of the nine cases of complex atypical endometrial hyperplasia, all showed BAF250a expression; however, of 10 cases of atypical endometriosis (the putative precursor lesion for ovarian clear cell and endometrioid carcinoma), one case showed loss of staining for BAF250a in the atypical areas, with retention of staining in areas of non-atypical endometriosis. This was the sole case that recurred as an endometrioid carcinoma, indicating that BAF250a loss may be an early event in carcinogenesis. Since BAF250a loss is seen in endometrial carcinomas at a rate similar to that seen in ovarian carcinomas of clear cell and endometrioid type, and is uncommon in other malignancies, we conclude that loss of BAF250a is a particular feature of carcinomas arising from endometrial glandular epithelium.

ARID1A mutations in endometriosis-associated ovarian carcinomas.

BACKGROUND: Ovarian clear-cell and endometrioid carcinomas may arise from endometriosis, but the molecular events involved in this transformation have not been described. METHODS: We sequenced the whole transcriptomes of 18 ovarian clear-cell carcinomas and 1 ovarian clear-cell carcinoma cell line and found somatic mutations in ARID1A (the AT-rich interactive domain 1A [SWI-like] gene) in 6 of the samples. ARID1A encodes BAF250a, a key component of the SWI­SNF chromatin remodeling complex. We sequenced ARID1A in an additional 210 ovarian carcinomas and a second ovarian clear-cell carcinoma cell line and measured BAF250a expression by means of immunohistochemical analysis in an additional 455 ovarian carcinomas. RESULTS: ARID1A mutations were seen in 55 of 119 ovarian clear-cell carcinomas (46%), 10 of 33 endometrioid carcinomas (30%), and none of the 76 high-grade serous ovarian carcinomas. Seventeen carcinomas had two somatic mutations each. Loss of the BAF250a protein correlated strongly with the ovarian clear-cell carcinoma and endometrioid carcinoma subtypes and the presence of ARID1A mutations. In two patients, ARID1A mutations and loss of BAF250a expression were evident in the tumor and contiguous atypical endometriosis but not in distant endometriotic lesions. CONCLUSIONS: These data implicate ARID1A as a tumor-suppressor gene frequently disrupted in ovarian clear-cell and endometrioid carcinomas. Since ARID1A mutation and loss of BAF250a can be seen in the preneoplastic lesions, we speculate that this is an early event in the transformation of endometriosis into cancer. (Funded by the British Columbia Cancer Foundation and the Vancouver General Hospital­University of British Columbia Hospital Foundation.).

HER2 overexpression and amplification is present in a subset of ovarian mucinous carcinomas and can be targeted with trastuzumab therapy.

BACKGROUND: The response rate of ovarian mucinous carcinomas to paclitaxel/carboplatin is low, prompting interest in targeted molecular therapies. We investigated HER2 expression and amplification, and the potential for trastuzumab therapy in this histologic subtype of ovarian cancer. METHODS: HER2 status was tested in 33 mucinous carcinomas and 16 mucinous borderline ovarian tumors (BOT)). Five cases with documented recurrence and with tissue from the recurrence available for testing were analyzed to determine whether HER2 amplification status changed over time. Three prospectively identified recurrent mucinous ovarian carcinomas were assessed for HER2 amplification and patients received trastuzumab therapy with conventional chemotherapy. RESULTS: Amplification of HER2 was observed in 6/33 (18.2%) mucinous carcinomas and 3/16 (18.8%) BOT. HER2 amplification in primary mucinous carcinomas was not associated with an increased likelihood of recurrence. The prospectively identified recurrent mucinous carcinomas showed overexpression and amplification of HER2; one patient's tumor responded dramatically to trastuzumab in combination with conventional chemotherapy, while another patient experienced an isolated central nervous system recurrence after trastuzumab therapy. CONCLUSION: HER2 amplification is relatively common in ovarian mucinous carcinomas (6/33, 18.2%), although not of prognostic significance. Trastuzumab therapy is a treatment option for patients with mucinous carcinoma when the tumor has HER2 amplification and overexpression.

Mutation of FOXL2 in granulosa-cell tumors of the ovary.

BACKGROUND: Granulosa-cell tumors (GCTs) are the most common type of malignant ovarian sex cord-stromal tumor (SCST). The pathogenesis of these tumors is unknown. Moreover, their histopathological diagnosis can be challenging, and there is no curative treatment beyond surgery. METHODS: We analyzed four adult-type GCTs using whole-transcriptome paired-end RNA sequencing. We identified putative GCT-specific mutations that were present in at least three of these samples but were absent from the transcriptomes of 11 epithelial ovarian tumors, published human genomes, and databases of single-nucleotide polymorphisms. We confirmed these variants by direct sequencing of complementary DNA and genomic DNA. We then analyzed additional tumors and matched normal genomic DNA, using a combination of direct sequencing, analyses of restriction-fragment-length polymorphisms, and TaqMan assays. RESULTS: All four index GCTs had a missense point mutation, 402C-->G (C134W), in FOXL2, a gene encoding a transcription factor known to be critical for granulosa-cell development. The FOXL2 mutation was present in 86 of 89 additional adult-type GCTs (97%), in 3 of 14 thecomas (21%), and in 1 of 10 juvenile-type GCTs (10%). The mutation was absent in 49 SCSTs of other types and in 329 unrelated ovarian or breast tumors. CONCLUSIONS: Whole-transcriptome sequencing of four GCTs identified a single, recurrent somatic mutation (402C-->G) in FOXL2 that was present in almost all morphologically identified adult-type GCTs. Mutant FOXL2 is a potential driver in the pathogenesis of adult-type GCTs.