Effects of Slide Storage on Detection of Molecular Markers by IHC and FISH in Endometrial Cancer Tissues From a Clinical Trial: An NRG Oncology/GOG Pilot Study.

We performed a pilot study in anticipation of using long-aged precut formalin-fixed paraffin-embedded tissue sections stored in real-world conditions for translational biomarker studies of topoisomerase 2A (TOP2A), Ki67, and human epidermal growth factor receptor 2 (HER2) in endometrial cancer. Formalin-fixed paraffin-embedded tissue blocks or unstained slides or both from GOG-0177 were collected centrally (1999-2000) and stored at room temperature. During 2004 to 2011 specimens were stored at 4°C. Matched pairs of stored slides and freshly cut slides from stored blocks were analyzed for TOP2A (KiS1), Ki67 (MIB1), and HER2 (HercepTest) proteins. To assess DNA stability (HER2 PathVision), fluorescence in situ hybridization (FISH) was repeated on stored slides from 21 cases previously shown to be HER2 amplified. Immunohistochemistry (IHC) staining intensity and extent, mean FISH copies/cell, and copy number ratios were compared using the κ statistic for concordance or signed rank test for differences in old cut versus new cut slides. IHC results reflected some protein degradation in stored slides. The proportion of cells with TOP2A staining was lower on average by 12% in older sections (P=0.03). The proportion of Ki67-positive cells was lower in stored slides by an average of 10% (P<0.01). Too few cases in the IHC cohort were FISH positive for any conclusions. HER2 amplification by FISH was unaffected by slide storage. We conclude that use of aged stored slides for proliferation markers TOP2A and Ki67 is feasible but may modestly underestimate true values in endometrial cancer. Pilot studies for particular storage conditions/durations/antigens to be used in translational studies are warranted.

Epithelioid Trophoblastic Tumor: Expanding the Clinicopathologic Spectrum of a Rare Malignancy.

Epithelioid trophoblastic tumor is a malignancy derived from the chorionic laeve-type intermediate trophoblast with sufficient rarity that the vast majority of literature on the topic exists in the form of case reports and small series. Classically, it is regarded as a well-circumscribed tumor with an expansile growth pattern that occurs in reproductive-aged women, usually after a normal pregnancy. However, we recently encountered a case of epithelioid trophoblastic tumor with aggressive spread throughout the abdomen and pelvis in a 68-yr-old female presenting 30 yr after her last delivery. Although to our knowledge this is the first report in a postmenopausal patient to be confirmed by molecular analysis of short tandem repeats, there are multiple similar case reports spanning a variety of clinical settings that deviate from the original description. We therefore sought to synthesize the clinicopathologic data among the available reports in the English literature, with emphasis on pathologic findings. While the overarching themes are largely unchanged, this series of 77 patients reveals a broader spectrum of disease and highlights frequent misdiagnosis. Here we present a clinicopathologic update on this rare entity, with emphasis on a practical approach to diagnosis.

Inhibition of fascin in cancer and stromal cells blocks ovarian cancer metastasis.

OBJECTIVE: Ovarian cancer (OvCa) metastasis requires the coordinated motility of both cancer and stromal cells. Cellular movement is a dynamic process that involves the synchronized assembly of f-actin bundles into cytoskeletal protrusions by fascin. Fascin directly binds f-actin and is an integral component of filopodia, lamellapodia and stress fibers. Here, we examine the expression pattern and function of fascin in the cancer and stromal cells of OvCa tumors. METHODS: Fascin expression was evaluated in human cells and tissues using immunohistochemistry and immunofluorescence. The functional role of fascin in cancer and stromal cells was assessed with in vitro functional assays, an ex vivo colonization assay and in vivo metastasis assays using siRNA/shRNA and an inhibitor. The effect of fascin inhibition on Cdc42 and Rac1 activity was evaluated using GTPase activity assays and immunofluorescence. RESULTS: Fascin expression was found to be higher in the stromal cell, when compared to the cancer cell, compartment of ovarian tumors. The low expression of fascin in the cancer cells of the primary tumor indicated a favorable prognosis for non-serous OvCa patients. In vitro, both knockdown and pharmacologic inhibition of fascin decreased the migration of cancer and stromal cells. The inhibition of fascin impaired Cdc42 and Rac1 activity in cancer cells, and cytoskeletal reorganization in the cancer and stromal cells. Inhibition of fascin ex vivo blocked OvCa cell colonization of human omental tissue and in vivo prevented and reduced OvCa metastases in mice. Likewise, knockdown of fascin specifically in the OvCa cells using a fascin-specific lentiviral-shRNA also blocked metastasis in vivo. CONCLUSION: This study reveals the therapeutic potential of pharmacologically inhibiting fascin in both cancer and stromal cells of the OvCa tumor microenvironment.

Fibroblasts Mobilize Tumor Cell Glycogen to Promote Proliferation and Metastasis.

Successful metastasis requires the co-evolution of stromal and cancer cells. We used stable isotope labeling of amino acids in cell culture coupled with quantitative, label-free phosphoproteomics to study the bidirectional signaling in ovarian cancer cells and human-derived, cancer-associated fibroblasts (CAFs) after co-culture. In cancer cells, the interaction with CAFs supported glycogenolysis under normoxic conditions and induced phosphorylation and activation of phosphoglucomutase 1, an enzyme involved in glycogen metabolism. Glycogen was funneled into glycolysis, leading to increased proliferation, invasion, and metastasis of cancer cells co-cultured with human CAFs. Glycogen mobilization in cancer cells was dependent on p38α MAPK activation in CAFs. In vivo, deletion of p38α in CAFs and glycogen phosphorylase inhibition in cancer cells reduced metastasis, suggesting that glycogen is an energy source used by cancer cells to facilitate metastatic tumor growth.

Multi-level Proteomics Identifies CT45 as a Chemosensitivity Mediator and Immunotherapy Target in Ovarian Cancer.

Most high-grade serous ovarian cancer (HGSOC) patients develop resistance to platinum-based chemotherapy and recur, but 15% remain disease free over a decade. To discover drivers of long-term survival, we quantitatively analyzed the proteomes of platinum-resistant and -sensitive HGSOC patients from minute amounts of formalin-fixed, paraffin-embedded tumors. This revealed cancer/testis antigen 45 (CT45) as an independent prognostic factor associated with a doubling of disease-free survival in advanced-stage HGSOC. Phospho- and interaction proteomics tied CT45 to DNA damage pathways through direct interaction with the PP4 phosphatase complex. In vitro, CT45 regulated PP4 activity, and its high expression led to increased DNA damage and platinum sensitivity. CT45-derived HLA class I peptides, identified by immunopeptidomics, activate patient-derived cytotoxic T cells and promote tumor cell killing. This study highlights the power of clinical cancer proteomics to identify targets for chemo- and immunotherapy and illuminate their biological roles.

Adipocyte-induced CD36 expression drives ovarian cancer progression and metastasis.

Ovarian cancer (OvCa) is characterized by widespread and rapid metastasis in the peritoneal cavity. Visceral adipocytes promote this process by providing fatty acids (FAs) for tumour growth. However, the exact mechanism of FA transfer from adipocytes to cancer cells remains unknown. This study shows that OvCa cells co-cultured with primary human omental adipocytes express high levels of the FA receptor, CD36, in the plasma membrane, thereby facilitating exogenous FA uptake. Depriving OvCa cells of adipocyte-derived FAs using CD36 inhibitors and short hairpin RNA knockdown prevented development of the adipocyte-induced malignant phenotype. Specifically, inhibition of CD36 attenuated adipocyte-induced cholesterol and lipid droplet accumulation and reduced intracellular reactive oxygen species (ROS) content. Metabolic analysis suggested that CD36 plays an essential role in the bioenergetic adaptation of OvCa cells in the adipocyte-rich microenvironment and governs their metabolic plasticity. Furthermore, the absence of CD36 affected cellular processes that play a causal role in peritoneal dissemination, including adhesion, invasion, migration and anchorage independent growth. Intraperitoneal injection of CD36-deficient cells or treatment with an anti-CD36 monoclonal antibody reduced tumour burden in mouse xenografts. Moreover, a matched cohort of primary and metastatic human ovarian tumours showed upregulation of CD36 in the metastatic tissues, a finding confirmed in three public gene expression data sets. These results suggest that omental adipocytes reprogram tumour metabolism through the upregulation of CD36 in OvCa cells. Targeting the stromal-tumour metabolic interface via CD36 inhibition may prove to be an effective treatment strategy against OvCa metastasis.

The Stem Cell-associated Transcription Factor SOX2 as a Diagnostic Marker of High-grade Squamous Intraepithelial Lesion of the Uterine Cervix in Comparison With p16 and Ki-67.

The transcription factor SOX2 has been identified as an oncogene involved in the pathogenesis of squamous cell carcinoma (SCC) of multiple sites, including the uterine cervix. The relationship between SOX2 overexpression and the continuum of precancerous lesions of the cervix has not been previously elucidated. We evaluated SOX2 immunohistochemical expression in normal cervix, low-grade squamous intraepithelial lesion (LSIL) (mild squamous dysplasia), high-grade squamous intraepithelial lesion (HSIL) (moderate and severe dysplasia) and SCC of the cervix in comparison with p16 and Ki-67. Staining patterns were scored as negative, basal one third of the epithelium, lower two third, or full thickness. The results showed that SOX2 expression was limited to the basal one third in 84% of LSIL cases, whereas 95% of HSIL showed SOX2 expression up to two third or full thickness (P<0.0001). p16 and Ki-67 displayed similar results. The difference in SOX2 expression between moderate and severe dysplasia was not statistically significant (P=0.53). Invasive SCC positivity was as follows: SOX2 94%; p16 89%; and Ki-67 100%. Our findings support a role for SOX2 in the progression of squamous dysplasia to SCC. The Lower Anogenital Standardization Terminology Project's recent assertion of a lack of a biological correlate to cervical intraepithelial neoplasia II is also upheld by SOX2. For equivocal situations in which a diagnosis of cervical intraepithelial neoplasia II would have been made, Lower Anogenital Standardization Terminology recommends p16, or other biomarkers such as Ki-67 to clarify the diagnosis. SOX2, with a clean nuclear staining pattern, may also be suitable for this role.

High prevalence of MiTF staining in undifferentiated pleomorphic sarcoma: caution in the use of melanocytic markers in sarcoma.

AIMS: The diagnosis of undifferentiated pleomorphic sarcoma (UPS) may be challenging, as other lesions with undifferentiated spindle cell morphology must be excluded, including melanoma. Microphthalmia-associated transcription factor (MiTF) stains naevi and epithelioid melanomas, as well as some mesenchymal neoplasms. The aim of this study was to evaluate the prevalence of MiTF and melanocytic markers in UPS and a subset of atypical fibroxanthoma (AFX). METHODS AND RESULTS: MiTF, SOX10, Melan-A, HMB45 and S100 immunostaining was performed on resection specimens from 19 UPSs and five AFXs. Next-generation sequencing of 50 genes was performed in UPSs to exclude dedifferentiated melanoma. In 17 of 19 UPSs (89%), tumour cells showed nuclear positivity for MiTF that was not eliminated by casein block. Three showed focal nuclear staining for HMB45, which was eliminated by casein block. One showed focal nuclear vacuole staining for S100 with red but not brown chromogen. None expressed SOX10 or Melan-A. Mutational analysis of 15 UPSs with adequate DNA showed no mutations within hotspot regions of BRAF, KIT, or NRAS. Four of five AFXs (80%) stained with MiTF; other markers were negative. CONCLUSION: There is a high prevalence of nuclear MiTF expression in UPSs (89%) and AFXs (80%). Rare UPSs showed non-specific nuclear HMB45 or S100 staining. These findings argue against using MiTF in isolation to differentiate between UPS or AFX and melanoma, and caution in interpreting focal staining for a single additional melanocytic marker. Casein block may eliminate non-specific staining. MiTF should be used to support a diagnosis of melanoma only if multiple melanocytic markers are positive.

Genomics of Ovarian Cancer Progression Reveals Diverse Metastatic Trajectories Including Intraepithelial Metastasis to the Fallopian Tube.

Accumulating evidence has supported the fallopian tube rather than the ovary as the origin for high-grade serous ovarian cancer (HGSOC). To understand the relationship between putative precursor lesions and metastatic tumors, we performed whole-exome sequencing on specimens from eight HGSOC patient progression series consisting of serous tubal intraepithelial carcinomas (STIC), invasive fallopian tube lesions, invasive ovarian lesions, and omental metastases. Integration of copy number and somatic mutations revealed patient-specific patterns with similar mutational signatures and copy-number variation profiles across all anatomic sites, suggesting that genomic instability is an early event in HGSOC. Phylogenetic analyses supported STIC as precursor lesions in half of our patient cohort, but also identified STIC as metastases in 2 patients. Ex vivo assays revealed that HGSOC spheroids can implant in the fallopian tube epithelium and mimic STIC lesions. That STIC may represent metastases calls into question the assumption that STIC are always indicative of primary fallopian tube cancers. SIGNIFICANCE: We find that the putative precursor lesions for HGSOC, STIC, possess most of the genomic aberrations present in advanced cancers. In addition, a proportion of STIC represent intraepithelial metastases to the fallopian tube rather than the origin of HGSOC. Cancer Discov; 6(12); 1342-51. ©2016 AACR.See related commentary by Swisher et al., p. 1309This article is highlighted in the In This Issue feature, p. 1293.

Infarct-Associated Bone Sarcomas: Multimodality Imaging Findings.

OBJECTIVE: The objective of our study was to characterize infarct-associated bone sarcoma and its imaging features. MATERIALS AND METHODS: Our databases were searched for instances of sarcoma arising in association with osteonecrosis. Demographic and imaging data were recorded. The imaging studies of 258 patients with sarcomas were reviewed to determine whether underlying osteonecrosis was present. Radiographic and MRI studies of patients with bone infarction were reviewed to categorize the various appearances of infarction and to determine if sarcomas tended to arise in a particular pattern. A literature review was performed. RESULTS: Nine infarct-associated bone sarcomas were found in eight patients: seven malignant fibrous histiocytomas (MFHs) and two osteosarcomas. All occurred in the femur or tibia; multifocal infarction was documented in all patients except one. Sarcomas were commonly associated with a so-called "mature"-type pattern of osteonecrosis-that is, with well-defined calcified margins. Osteolysis of infarct-associated MFHs was often overlooked at initial presentation and was often detected only after pathologic fracture. CT and MRI revealed cortical penetration in all cases; infarct margin disruption was evident, but preservation of fat within the infarct was typical. Increased radiotracer activity with relative central photopenia was characteristic of large infarct-associated bone sarcomas on scintigraphy. All lesions, including those treated at our institution and those found in the literature, were metaphyseal or diaphyseal, and although epiphyseal extension of sarcoma from a metadiaphyseal infarct was common, no purely epiphyseal lesions were encountered. CONCLUSION: Radiologists must remain vigilant for this rare occurrence, especially in patients with new pain in an area of known bone infarction.

Heart and Neural Crest Derivatives Expressed Transcript 2 (HAND2): a novel biomarker for the identification of atypical hyperplasia and Type I endometrial carcinoma.

Progesterone inhibits the proliferative growth effects of estrogen in the endometrium and prevents the development of endometrial hyperplasia and Type I adenocarcinoma. The exact mechanism of this action is unknown. The progesterone-induced helix-loop-helix transcription factor Heart and Neural Crest Derivatives Expressed 2 (Hand2) was recently shown to suppress production of growth factors in the endometrium. In Hand2 knockout mice, continuous proliferation of the endometrium was observed. In this study, archival paraffin-embedded tissue from 56 hysterectomy specimens was examined by immunohistochemistry for the expression and localization of Hand2, estrogen receptor (ER), progesterone receptor (PR), and Ki-67. Diagnoses included disordered proliferative endometrium, simple and complex hyperplasia with or without atypia, and endometrioid adenocarcinoma. Hand2 expression is localized to endometrial stromal nuclei. In benign endometrium, Hand2 expression was moderate to strong (10/11; 91%), with weak Hand2 expression in only 1 case (1/11; 9%). Similar Hand2 expression patterns were observed in disordered proliferative endometrium and simple hyperplasia without atypia, with moderate to strong expression in 91% of cases (10/11) and weak expression in 9% of cases (1/11). In contrast, simple and complex hyperplasia with atypia exhibited moderate to strong Hand2 expression in 8% of cases (1/12) and a loss of expression or weak expression in 92% of cases (11/12). In endometrioid adenocarcinomas, Hand2 expression was absent in all cases (22/22). Hand2 is expressed in the stroma of benign endometrium, but expression is significantly reduced or lost in atypical hyperplasia and endometrioid carcinoma. Thus, the absence of Hand2 expression may be a useful biomarker for atypical hyperplasia and endometrioid carcinoma.

Mesothelial cells promote early ovarian cancer metastasis through fibronectin secretion.

Ovarian cancer (OvCa) metastasizes to organs in the abdominal cavity, such as the omentum, which are covered by a single layer of mesothelial cells. Mesothelial cells are generally thought to be "bystanders" to the metastatic process and simply displaced by OvCa cells to access the submesothelial extracellular matrix. Here, using organotypic 3D cultures, we found that primary human mesothelial cells secrete fibronectin in the presence of OvCa cells. Moreover, we evaluated the tumor stroma of 108 human omental metastases and determined that fibronectin was consistently overexpressed in these patients. Blocking fibronectin production in primary mesothelial cells in vitro or in murine models, either genetically (fibronectin 1 floxed mouse model) or via siRNA, decreased adhesion, invasion, proliferation, and metastasis of OvCa cells. Using a coculture model, we determined that OvCa cells secrete TGF-ß1, which in turn activates a TGF-ß receptor/RAC1/SMAD-dependent signaling pathway in the mesothelial cells that promotes a mesenchymal phenotype and transcriptional upregulation of fibronectin. Additionally, blocking α5 or ß1 integrin function with antibodies reduced metastasis in an orthotopic preclinical model of OvCa metastasis. These findings indicate that cancer-associated mesothelial cells promote colonization during the initial steps of OvCa metastasis and suggest that mesothelial cells actively contribute to metastasis.

Unintended thermal injuries from radiofrequency ablation: organ protection with an angioplasty balloon catheter in an animal model.

OBJECTIVES: The aim of this study was to investigate a novel approach of using a balloon catheter as a protective device to separate liver from the diaphragm or nearby bowel during radiofrequency ablation (RFA) of hepatic dome tumors in an animal model. MATERIALS AND METHODS: All experimental procedures were approved by animal Institutional Review Board. Using a 3 cm RF needle electrode, 70 hepatic ablation zones were created using ultrasound in 7 pigs. 50 lesions were created using balloon interposition between liver and diaphragm; 20 lesions were created using the balloon device interposed posteriorly between liver and bowel. Additional 21 control lesions were performed. Animals were sacrificed immediately; diaphragm and bowel were then visually inspected and sectioned. Diaphragmatic and bowel injury was then classified according to the depth of thickness. RESULTS: Control lesions caused full thickness injury, either to diaphragm or bowel. During ablation of lesions with balloon interposition, there was significantly less diaphragmatic injury, P < 0.001 and less bowel injury, P < 0.01. CONCLUSION: Using balloon interposition as a protective device has advantages over previous saline infusion or CO2 insufflation, providing a safe way to expand percutaneous RFA of liver tumors located on the undersurface of the diaphragm. In addition, this method may be used in protection of other organs adjacent to areas being ablated.

Expression profiling of 519 kinase genes in matched malignant peripheral nerve sheath tumor/plexiform neurofibroma samples is discriminatory and identifies mitotic regulators BUB1B, PBK and NEK2 as overexpressed with transformation.

About 50% of all malignant peripheral nerve sheath tumors (MPNSTs) arise as neurofibromatosis type 1 associated lesions. In those patients malignant peripheral nerve sheath tumors are thought to arise through malignant transformation of a preexisting plexiform neurofibroma. The molecular changes associated with this transformation are still poorly understood. We sought to test the hypothesis that dysregulation of expression of kinases contributes to this malignant transformation. We analyzed expression of all 519 kinase genes in the human genome using the nanostring nCounter system. Twelve cases of malignant peripheral nerve sheath tumor arising in a background of preexisting plexiform neurofibroma were included. Both components were separately sampled. Statistical analysis compared global changes in expression levels as well as changes observed in the pairwise comparison of samples taken from the same surgical specimen. Immunohistochemical studies were performed on tissue array slides to confirm expression of selected proteins. The expression pattern of kinase genes can separate malignant peripheral nerve sheath tumors and preexisting plexiform neurofibromas. The majority of kinase genes is downregulated rather than overexpressed with malignant transformation. The patterns of expression changes are complex without simple recurring alteration. Pathway analysis demonstrates that differentially expressed kinases are enriched for kinases involved in the direct regulation of mitosis, and several of these show increased expression in malignant peripheral nerve sheath tumors. Immunohistochemical studies for the mitotic regulators BUB1B, PBK and NEK2 confirm higher expression levels at the protein level. These results suggest that the malignant transformation of plexiform neurofibroma is associated with distinct changes in the expression of kinase genes. The patterns of these changes are complex and heterogeneous. There is no single unifying alteration. Kinases involved in mitotic regulation are particularly enriched in the pool of differentially expressed kinases. Some of these are overexpressed and are therefore possible targets for kinase inhibitors.

Serial sectioning of the fallopian tube allows for improved identification of primary fallopian tube carcinoma.

OBJECTIVE: Serial sectioning of the fallopian tube in women undergoing risk reducing surgery has been shown to increase the detection rate of occult malignancy in BRCA mutation carriers. We undertook this study to determine whether this protocol at the time of surgery for ovarian cancer (OV) or primary peritoneal malignancies (PP) changes the detection rate of fallopian tube carcinoma (FT). We secondarily investigated where this difference affects patient outcomes. METHODS: A retrospective review of 130 patients treated at the University of Chicago Medical Center for ovarian, peritoneal or fallopian tube carcinoma was conducted. Sixty five patients diagnosed with OV, PP or FT who had serial sectioning of the fallopian tubes at the time of diagnoses (SS) were compared to 65 patients whose fallopian tubes were sectioned in a standard fashion (PSS). RESULTS: Serial sectioning of the fallopian tube at the time of pathologic examination in women with presumed OV or PP led to an increase in the number of women diagnosed with FT as the primary site of origin (p<0.001). Clinical or pathologic risk factors leading to an increased risk of FT were not identified. Survival between the two groups was similar. CONCLUSION: In women with presumed OV or PP, serial sectioning identifies women with FT. FT may be more common than previously noted; however distinct biologic or clinical behavior to differentiate it from OV or PP could not be identified. Clinical management of FT should continue to be the same as that of OV or PP.

Time-dependent transcriptional profiling links gene expression to mitogen-activated protein kinase kinase 4 (MKK4)-mediated suppression of omental metastatic colonization.

Although metastasis is the most lethal attribute of cancer, critical gaps in our knowledge of how cancer cells effectively colonize distant sites remain. For example, little is known about the cellular and molecular events that occur during the timecourse of metastatic colonization. To address this we are using the mitogen-activated protein kinase kinase 4 (MKK4) metastasis suppressor as a tool to identify these events. Specifically, we report a microarray expression-based strategy to identify genes whose transcription is altered in SKOV3ip.1 human ovarian cancer cells that express ectopic MKK4 throughout the course of in vivo metastatic colonization. The majority of genes identified fell into the categories of cytokinesis, cytoskeleton remodeling, and cell adhesion, and their expression was repressed in MKK4-expressing cells relative to vector controls. The greatest transcriptional divergence was concomitant with impaired proliferation at 14 days post injection (dpi). Specifically, 763 genes were differentially expressed (FDR < 0.05) between lesions that expressed ectopic MKK4 and paired controls. In contrast, only seven genes were differentially expressed at the experimental endpoint, when MKK4-expressing and control cells had formed macroscopic metastases. Application of our cohort of differentially expressed genes to three independent clinical datasets demonstrated a strong correlation between our findings and metastatic phenotypes in patient samples. Our results highlight the dynamic nature of metastatic colonization and reinforce the importance of examining both molecular and cellular phenotypes over time when studying metastasis formation.

Reactive bone lesions mimicking neoplasms.

Reactive lesions of bone and soft tissue can appear alarming on histologic examination because they are often cellular and have atypical (activated) cytologic features, such as distinct nucleoli and mild hyperchromasia, and mitotic activity. Reactive lesions of bone and periosteum also produce bone and cartilage matrix, resulting in confusion with osteosarcoma or chondrosarcoma. Careful attention to key cytomorphological features such as the pattern of bone formation, uniform appearance of cells, and absence of atypical mitoses should help identify the reactive nature of a lesion. Correlation with clinical and radiological findings is also imperative to avoid misclassification of the tumor because reactive lesions often arise at sites where osteosarcoma and chondrosarcoma are rare (e.g., the hand) and lack aggressive radiological features. In this review we discuss reactive lesions of bone that are commonly confused with malignant neoplasms and that the practicing pathologist is likely to encounter at some point. Several of these lesions have had characteristic chromosomal translocations documented in recent years, but continue to be included as reactive lesions based on their overall clinicopathological features.

FOXL2 and SOX9 distinguish the lineage of the sex cord-stromal cells in gonadoblastomas.

Gonadoblastomas are mixed germ cell sex cord-stromal tumors that arise in dysgenetic gonads and are composed of immature germ cells and sex cord-stromal cells of indeterminate differentiation. FOXL2 is one of the first genes expressed in female gonad development, and it is required for proper granulosa cell differentiation during folliculogenesis. SOX9 , a downstream target of SRY , the gene in the Y chromosomal sex-determining region, is required for testicular development and for the formation and maintenance of (pre-)Sertoli cells. This study characterized the sex cord-stromal cells of gonadoblastoma by evaluating the expression of these counteracting transcription factors. Archival paraffin-embedded material of 7 gonadoblastomas, 5 of which were overgrown by dysgerminoma, was examined by immunohistochemistry for expression and localization of FOXL2 and SOX9. The sex cord-stromal cells revealed strong nuclear staining for FOXL2 and were negative for SOX9 expression. Germ cells in the gonadoblastoma and dysgerminoma components showed no FOXL2 and SOX9 expression. Areas of transition between gonadoblastoma and dysgerminoma revealed nests with a gradual reduction of FOXL2 expression. Our results support the hypothesis that the sex cord-stromal cell component of gonadoblastomas is of granulosa cell origin. In addition, FOXL2 appears to be a useful marker for the evaluation of overgrowth by dysgerminomas and for the identification of the transition zone of "dysgerminoma in situ." As FOXL2 and SOX9 are differentially expressed, they also should be useful for distinguishing gonadoblastomas from intratubular germ cell neoplasias and can help to differentiate those with a Sertoli cell component from gonadoblastoma with a granulosa cell component.

Foretinib (GSK1363089), an orally available multikinase inhibitor of c-Met and VEGFR-2, blocks proliferation, induces anoikis, and impairs ovarian cancer metastasis.

PURPOSE: Currently, there are no approved targeted therapies for the treatment of ovarian cancer, despite the fact that it is the most lethal gynecological malignancy. One proposed target is c-Met, which has been shown to be an important prognostic indicator in a number of malignancies, including ovarian cancer. The objective of this study was to determine whether an orally available multikinase inhibitor of c-Met and vascular endothelial growth factor receptor-2 (foretinib, GSK1363089) blocks ovarian cancer growth. EXPERIMENTAL DESIGN: The effect of foretinib was tested in a genetic mouse model of endometrioid ovarian cancer, several ovarian cancer cell lines, and an organotypic 3D model of the human omentum. RESULTS: In the genetic mouse model, treatment with foretinib prevented the progression of primary tumors to invasive adenocarcinoma. Invasion through the basement membrane was completely blocked in treated mice, whereas in control mice, invasive tumors entirely replaced the normal ovary. In 2 xenograft mouse models using human ovarian cancer cell lines, the inhibitor reduced overall tumor burden (86% inhibition, P < 0.0001) and metastasis (67% inhibition, P < 0.0001). The mechanism of inhibition by foretinib involved (a) inhibition of c-Met activation and downstream signaling, (b) reduction of ovarian cancer cell adhesion, (c) a block in migration and invasion, (d) reduced proliferation mediated by a G(2)-M cell-cycle arrest, and (e) induction of anoikis. CONCLUSIONS: This study shows that foretinib blocks tumorigenesis and reduces invasive tumor growth in different models of ovarian cancer by affecting several critical tumor functions. We believe that it provides a rationale for the further clinical development of foretinib for the treatment of ovarian cancer.

Targeting the urokinase plasminogen activator receptor inhibits ovarian cancer metastasis.

PURPOSE: To understand the functional and preclinical efficacy of targeting the urokinase plasminogen activator receptor (u-PAR) in ovarian cancer. EXPERIMENTAL DESIGN: Expression of u-PAR was studied in 162 epithelial ovarian cancers, including 77 pairs of corresponding primary and metastatic tumors. The effect of an antibody against u-PAR (ATN-658) on proliferation, adhesion, invasion, apoptosis, and migration was assessed in 3 (SKOV3ip1, HeyA8, and CaOV3) ovarian cancer cell lines. The impact of the u-PAR antibody on tumor weight, number, and survival was examined in corresponding ovarian cancer xenograft models and the mechanism by which ATN-658 blocks metastasis was explored. RESULTS: Only 8% of all ovarian tumors were negative for u-PAR expression. Treatment of SKOV3ip1, HeyA8, and CaOV3 ovarian cancer cell lines with the u-PAR antibody inhibited cell invasion, migration, and adhesion. In vivo, anti-u-PAR treatment reduced the number of tumors and tumor weight in CaOV3 and SKOV3ip1 xenografts and reduced tumor weight and increased survival in HeyA8 xenografts. Immunostaining of CaOV3 xenograft tumors and ovarian cancer cell lines showed an increase in active-caspase 3 and TUNEL staining. Treatment with u-PAR antibody inhibited α(5)-integrin and u-PAR colocalization on primary human omental extracellular matrix. Anti-u-PAR treatment also decreased the expression of urokinase, u-PAR, ß(3)-integrin, and fibroblast growth factor receptor-1 both in vitro and in vivo. CONCLUSIONS: This study shows that an antibody against u-PAR reduces metastasis, induces apoptosis, and reduces the interaction between u-PAR and α(5)-integrin. This provides a rationale for targeting the u-PAR pathway in patients with ovarian cancer and for further testing of ATN-658 in this indication.