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.

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.

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.

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.

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.

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.

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.

The expression of hepatocyte growth factor (HGF) and c-Met in uterine serous carcinoma.

OBJECTIVE: The hepatocyte growth factor (HGF) receptor c-Met plays an important role in tumor dissemination by activating mitogenic signaling pathways. The goal of this study was to investigate immunohistochemical (IHC) staining patterns of HGF and c-Met in endometrioid endometrial cancer (EC) and uterine serous cancer (USC) and to correlate staining with patient outcomes. METHODS: A tissue microarray was created using tissue from patients with atrophic endometrium (AE), grade 1 EC, grade 3 EC, and USC. Immunohistochemistry was used to detect c-Met, phosphorylated c-Met (p-c-Met), and HGF expression. Differences between IHC staining intensity were calculated using t-tests. Correlations between staining and clinicopathologic variables were determined by Chi-square testing for categorical variables and t-tests for continuous variables. Kaplan-Meier curves were constructed to analyze survival in USC. RESULTS: Patients with cancer had more total c-Met and HGF expression than those with AE (p=0.037, p<0.001 respectively), but no difference in p-c-Met staining. Total c-Met and HGF staining was significantly different between groups (p=0.042, p<0.001 respectively). This difference was accounted for by greater c-MET expression in USC compared to AE (p=0.027). Depth of invasion, stage, and lymph node status were not significantly related to staining intensity. Patients with strong c-Met and HGF staining had decreased overall survival compared to patients with weaker staining. CONCLUSIONS: HGF and c-Met may play a role in the progression of endometrial cancer and should be studied further as prognostic and therapeutic tools.

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.

{beta}3-integrin expression on tumor cells inhibits tumor progression, reduces metastasis, and is associated with a favorable prognosis in patients with ovarian cancer.

The role of the vitronectin receptor (alpha(v)beta(3)-integrin) as a tumor promoter seems well established, and, consequently, therapies that block this integrin are currently in clinical testing. We undertook the current study to determine whether alpha(v)beta(3)-integrin is an appropriate target in ovarian cancer treatment. Expression of beta(3)-integrin in SKOV3ip1 ovarian cancer cells led to the overexpression of alpha(v)beta(3)-integrin on the cell surface and increased adhesion. However, alpha(v)beta(3)-integrin-overexpressing cells showed impaired invasion, protease expression, and colony formation. These results were recapitulated in xenograft studies: alpha(v)beta(3)-integrin-expressing cells showed increased adhesion to mouse peritoneum, but the overall number of metastatic nodules (105 versus 68 tumors) and tumor weight were significantly lower than those in the parental SKOV3ip1 cells. The alpha(v)beta(3)-integrin-overexpressing cells had a decreased proliferation rate mediated by inhibition of cyclin B1 and induction of phospho-Cdc2 and p53 expression, consistent with a G(2)M cell cycle arrest. Confirming the above results, inhibition of beta(3)-integrin in cultured or primary OvCa cells decreased adhesion but increased invasion and proliferation. Patients with tumors expressing high beta(3)-integrin had significantly better disease-free and overall survival (52 months versus 27 months, P < 0.05). This study shows that alpha(v)beta(3)-integrin expression on tumor cells actually slows tumor progression and acts as a tumor suppressor. Therefore, the vitronectin receptor might not be an appropriate therapeutic target in ovarian cancer.

Patient-reported peripheral neuropathy of doxorubicin and cisplatin with and without paclitaxel in the treatment of advanced endometrial cancer: Results from GOG 184.

OBJECTIVE: In GOG 184, the addition of paclitaxel to cisplatin and doxorubicin offered no additional clinical benefit, yet was associated with increased provider-rated toxicity. We now compare patient-reported neuropathy between treatment arms and patient reports to the clinician reports of neuropathy. METHODS: Of 659 enrolled patients, 552 were randomly assigned to receive either cisplatin 50 mg/m² + doxorubicin 45 mg/m²+G-CSF 5 µg/kg on days 2-11 ("CD"), or the above regimen plus paclitaxel 160 mg/m² infused over 3h ("CDP"). Patient-reported neuropathy was measured with 11-item Functional Assessment of Cancer Therapy - Neurotoxicity (FACT-Ntx) Scale, at baseline, and 4 weeks and 6 months post chemotherapy. Group differences on patient-reported neuropathy over time, and correspondence between patient and provider ratings, were evaluated by fitting linear mixed models to the data. RESULTS: After adjusting for non-significant baseline differences in neuropathy, the average neuropathy (FACT-Ntx) score of CDP-treated patients was 5.2 points lower/worse (95% CI: 4.0-6.5; p < 0.001) than the average score observed in CD-treated patients. The difference diminished after 6 months but still remained statistically significant (difference = 1.6; 95% CI: 0.3-2.8; p = 0.014). The sensory component was most significantly affected. Each increase (worsening) of grade in provider-rated toxicity was significantly associated with change in patient-reported severity of 4-6 points in the 11-item total score and 2-3 points in the 4-item sensory neuropathy score. CONCLUSION: Patient-reported neuropathy was worse in CDP-treated patients compared to CD-treated patients, especially in the sensory component. Patient-reported change corresponded with provider grade, but offered more detail on the nature of impact.

A randomized phase III trial in advanced endometrial carcinoma of surgery and volume directed radiation followed by cisplatin and doxorubicin with or without paclitaxel: A Gynecologic Oncology Group study.

OBJECTIVES: After surgical debulking and volume-directed irradiation of the pelvis/para-aortic lymph nodes, treatment was randomized to compare recurrence-free survival (RFS) and toxicity between two chemotherapy regimens for the treatment of women with advanced stage endometrial carcinoma. METHODS: Treatment was randomized between 6 cycles of cisplatin [C] (50 mg/m(2)) and doxorubicin [D] (45 mg/m(2)) with or without paclitaxel [P] (160 mg/m(2)). Initially in paclitaxel treated patients and, after May 2002, all patients received granulocyte growth factor with each cycle. RESULTS: Of 659 patients enrolled following surgery, 552 eligible patients were randomized to chemotherapy after irradiation. Accrual closed to Stage IV patients in June, 2003. Approximately 80% completed six cycles of chemotherapy. Three deaths resulted from bowel complications and one death was due to renal failure. Hematologic adverse events, sensory neuropathy and myalgia, were more frequent and severe in the paclitaxel arm (p<0.01) which was confirmed by Quality of Life assessments. Percentage of patients alive and recurrence-free at 36 months was 62% for CD vs. 64% for CDP. The hazard of recurrence or death relative to the CD arm stratified by stage is 0.90 (95% CI is 0.69 to 1.17, p=0.21, one-tail). However, in subgroup analysis, CDP was associated with a 50% reduction in the risk of recurrence or death among patients with gross residual disease (95% CI: 0.26 to 0.92). Stage, residual disease, histology/grade, positive para-aortic node and cytology, pelvic metastases and age were significantly associated with RFS. CONCLUSION: The addition of paclitaxel to cisplatin and doxorubicin following surgery and radiation was not associated with a significant improvement in RFS but was associated with increased toxicity.

CCN1/Cyr61 is regulated by the canonical Wnt signal and plays an important role in Wnt3A-induced osteoblast differentiation of mesenchymal stem cells.

Marrow mesenchymal stem cells are pluripotent progenitors that can differentiate into bone, cartilage, muscle, and fat cells. Wnt signaling has been implicated in regulating osteogenic differentiation of mesenchymal stem cells. Here, we analyzed the gene expression profile of mesenchymal stem cells that were stimulated with Wnt3A. Among the 220 genes whose expression was significantly changed by 2.5-fold, we found that three members of the CCN family, CCN1/Cyr61, CCN2/connective tissue growth factor (CTGF), and CCN5/WISP2, were among the most significantly up-regulated genes. We further investigated the role of CCN1/Cyr61 in Wnt3A-regulated osteogenic differentiation. We confirmed that CCN1/Cyr61 was up-regulated at the early stage of Wnt3A stimulation. Chromatin immunoprecipitation analysis indicates that CCN1/Cyr61 is a direct target of canonical Wnt/beta-catenin signaling. RNA interference-mediated knockdown of CCN1/Cyr61 expression diminished Wnt3A-induced osteogenic differentiation. Furthermore, exogenously expressed CCN1/Cyr61 was shown to effectively promote mesenchymal stem cell migration. These findings suggest that tightly regulated CCN1/Cyr61 expression may play an important role in Wnt3A-induced osteoblast differentiation of mesenchymal stem cells.

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.

Osteogenic BMPs promote tumor growth of human osteosarcomas that harbor differentiation defects.

Osteosarcoma (OS) is the most common primary malignancy of bone. Here, we investigated a possible role of defective osteoblast differentiation in OS tumorigenesis. We found that basal levels of the early osteogenic marker alkaline phosphatase (ALP) activity were low in OS lines. Osteogenic regulators Runx2 and OSX, and the late marker osteopontin (OPN) expressed at low levels in most OS lines, indicating that most OS cells fail to undergo terminal differentiation. Furthermore, OS cells were refractory to osteogenic BMP-induced increases in ALP activity. Osteogenic BMPs were shown to upregulate early target genes, but not late osteogenic markers OPN and osteocalcin (OC). Furthermore, osteogenic BMPs failed to induce bone formation from human OS cells, rather effectively promoted OS tumor growth in an orthotopic OS model. Exogenous expression of early target genes enhanced BMP-stimulated OS tumor growth, whereas osteogenic BMP-promoted OS tumor growth was inhibited by exogenous Runx2 expression. These results suggest that alterations in osteoprogenitors may disrupt osteogenic differentiation pathway. Thus, identifying potential differentiation defects in OS tumors would allow us to reconstruct the tumorigenic events in osteoprogenitors and to develop rational differentiation therapies for clinical OS management.

Histologic response of dose-intense chemotherapy with preoperative hypofractionated radiotherapy for patients with high-risk soft tissue sarcomas.

BACKGROUND: The authors studied a dose-intense regimen of epirubicin and ifosfamide with hypofractionated preoperative radiotherapy for high-risk soft tissue sarcomas. The primary objective was estimation of the rate of >or=95% pathologic necrosis. METHODS: Twenty-five patients with intermediate-grade or high-grade, localized soft tissue sarcomas of the extremity or body wall measuring >5 cm were treated with epirubicin at a dose of 30 mg/m(2)/day on Days 1 to 4 and ifosfamide at a dose of 2.5 g/m(2)/day on Days 1 to 4 every 21 days for 3 preoperative and 3 postoperative cycles. A total of 28 grays of radiation was administered over 8 fractions during Cycle 2 of preoperative therapy (epirubicin was omitted). RESULTS: Sixteen patients (64%) completed all chemotherapy cycles and the average delivered dose intensity relative to intended therapy was 69%. Twenty-one patients (84%) experienced grade 4 toxicity (using the National Cancer Institute Common Terminology Criteria for Adverse Events [version 2.0]), which was predominantly hematologic. Notable toxicities included neutropenic fever (40%), ifosfamide-induced encephalopathy (24%), and grade 3/4 anemia (64%). Postoperative wound complications requiring a surgical procedure occurred in 20% of patients. The rate of >or=95% pathologic necrosis was 40% (95% confidence interval [95% CI], 21-59%). Estimates of 2-year overall and disease-free survival were 84% (95% CI, 66-100%) and 62% (95% CI, 37-86%), respectively. CONCLUSIONS: A high rate of >or=95% pathologic necrosis was noted with this aggressive chemoradiotherapy regimen. The occurrence of significant acute toxicities limited the delivery of the intended dose intensity.

Regulation of osteogenic differentiation during skeletal development.

Bone formation during skeletal development involves a complex coordination among multiple cell types and tissues. Bone is of crucial importance for the human body, providing skeletal support, and serving as a home for the formation of hematopoietic cells and as a reservoir for calcium and phosphate. Bone is also continuously remodeled in vertebrates throughout life. Osteoblasts and osteoclasts are specialized cells responsible for bone formation and resorption, respectively. Early development of the vertebrate skeleton depends on genes that control the distribution and proliferation of cells from cranial neural crest, sclerotomes, and lateral plate mesoderm into mesenchymal condensations, where cells differentiate to osteoblasts. Significant progress has been made over the past decade in our understanding of the molecular framework that controls osteogenic differentiation. A large number of morphogens, signaling molecules, and transcriptional regulators have been implicated in regulating bone development. A partial list of these factors includes the Wnt/beta-catenin, TGF-beta/BMP, FGF, Notch and Hedgehog signaling pathways, and Runx2, Osterix, ATF4, TAZ, and NFATc1 transcriptional factors. A better understanding of molecular mechanisms behind osteogenic differentiation would not only help us to identify pathogenic causes of bone and skeletal diseases but also lead to the development of targeted therapies for these diseases.

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.