Mechanism of action of a WWTR1(TAZ)-CAMTA1 fusion oncoprotein.

The WWTR1 (protein is known as TAZ)-CAMTA1 (WC) fusion gene defines epithelioid hemangioendothelioma, a malignant vascular cancer. TAZ (transcriptional coactivator with PDZ binding motif) is a transcriptional coactivator and end effector of the Hippo tumor suppressor pathway. It is inhibited by phosphorylation by the Hippo kinases LATS1 and LATS2. Such phosphorylation causes cytoplasmic localization, 14-3-3 protein binding and the phorphorylation of a terminal phosphodegron promotes ubiquitin-dependent degradation (the phosphorylation of the different motifs has several effects). CAMTA1 is a putative tumor suppressive transcription factor. Here we demonstrate that TAZ-CAMTA1 (TC) fusion results in its nuclear localization and constitutive activation. Consequently, cells expressing TC display a TAZ-like transcriptional program that causes resistance to anoikis and oncogenic transformation. Our findings elucidate the mechanistic basis of TC oncogenic properties, highlight that TC is an important model to understand how the Hippo pathway can be inhibited in cancer, and provide approaches for targeting this chimeric protein.

Protein kinase C iota as a therapeutic target in alveolar rhabdomyosarcoma.

Alveolar rhabdomyosarcoma is an aggressive pediatric cancer exhibiting skeletal-muscle differentiation. New therapeutic targets are required to improve the dismal prognosis for invasive or metastatic alveolar rhabdomyosarcoma. Protein kinase C iota (PKCι) has been shown to have an important role in tumorigenesis of many cancers, but little is known about its role in rhabdomyosarcoma. Our gene-expression studies in human tumor samples revealed overexpression of PRKCI. We confirmed overexpression of PKCι at the mRNA and protein levels using our conditional mouse model that authentically recapitulates the progression of rhabdomyosarcoma in humans. Inhibition of Prkci by RNA interference resulted in a dramatic decrease in anchorage-independent colony formation. Interestingly, treatment of primary cell cultures using aurothiomalate (ATM), which is a gold-containing classical anti-rheumatic agent and a PKCι-specific inhibitor, resulted in decreased interaction between PKCι and Par6, decreased Rac1 activity and reduced cell viability at clinically relevant concentrations. Moreover, co-treatment with ATM and vincristine (VCR), a microtubule inhibitor currently used in rhabdomyosarcoma treatment regimens, resulted in a combination index of 0.470-0.793 through cooperative accumulation of non-proliferative multinuclear cells in the G2/M phase, indicating that these two drugs synergize. For in vivo tumor growth inhibition studies, ATM demonstrated a trend toward enhanced VCR sensitivity. Overall, these results suggest that PKCι is functionally important in alveolar rhabdomyosarcoma anchorage-independent growth and tumor-cell proliferation and that combination therapy with ATM and microtubule inhibitors holds promise for the treatment of alveolar rhabdomyosarcoma.

A nonrandom association of gastrointestinal stromal tumor (GIST) and desmoid tumor (deep fibromatosis): case series of 28 patients.

BACKGROUND: Gastrointestinal stromal tumors (GISTs) and desmoid tumors (DTs) are two rare mesenchymal tumor. Anecdotal reports of individuals with both diseases led us to make the hypothesis that the association is a nonrandom event as the probability would be extremely low to observe such cases if they were independent events. PATIENTS AND METHODS: We evaluated the existence of patients with GIST and DT in a large multicenter cohort at 10 institutions in the United States, Australia and Europe. Data on gender, age at diagnosis, KIT, PDGFRA, CTNNB1 mutation status and follow-up time after diagnosis were collected. RESULTS: We identified 28 patients diagnosed with both tumors. DT was diagnosed after GIST in 75% of patients and concomitantly in 21%. In only one case (4%), GIST was diagnosed after DT. KIT or PDGFRA mutations were detected in 12 of 14 GIST, 9 in KIT exon 11, 2 in KIT exon 9 and 1 in PDGFRA. CONCLUSION: A statistical analysis of these 28 cases suggests a nonrandom association between GIST and DT. Further studies may be able to elucidate the underlying biology responsible for this association.

Discovery of molecular subtypes in leiomyosarcoma through integrative molecular profiling.

Leiomyosarcoma (LMS) is a soft tissue tumor with a significant degree of morphologic and molecular heterogeneity. We used integrative molecular profiling to discover and characterize molecular subtypes of LMS. Gene expression profiling was performed on 51 LMS samples. Unsupervised clustering showed three reproducible LMS clusters. Array comparative genomic hybridization (aCGH) was performed on 20 LMS samples and showed that the molecular subtypes defined by gene expression showed distinct genomic changes. Tumors from the 'muscle-enriched' cluster showed significantly increased copy number changes (P=0.04). A majority of the muscle-enriched cases showed loss at 16q24, which contains Fanconi anemia, complementation group A, known to have an important role in DNA repair, and loss at 1p36, which contains PRDM16, of which loss promotes muscle differentiation. Immunohistochemistry (IHC) was performed on LMS tissue microarrays (n=377) for five markers with high levels of messenger RNA in the muscle-enriched cluster (ACTG2, CASQ2, SLMAP, CFL2 and MYLK) and showed significantly correlated expression of the five proteins (all pairwise P<0.005). Expression of the five markers was associated with improved disease-specific survival in a multivariate Cox regression analysis (P<0.04). In this analysis that combined gene expression profiling, aCGH and IHC, we characterized distinct molecular LMS subtypes, provided insight into their pathogenesis, and identified prognostic biomarkers.

PDGFR-A is a therapeutic target in alveolar rhabdomyosarcoma.

Alveolar rhabdomyosarcoma is an aggressive skeletal muscle cancer of childhood. Our initial studies of rhabdomyosarcoma gene expression for patients enrolled in a national clinical trial suggested that platelet-derived growth factor receptor A (PDGFR-A) may be a mediator of disease progression and metastasis. Using our conditional mouse tumor models that authentically recapitulate the primary mutations and metastatic progression of alveolar rhabdomyosarcomas in humans, we found by immunoblotting and immunokinase assays that PDGFR-A and its downstream effectors, mitogen-activated protein kinase and Akt, were highly activated in both primary and metastatic tumors. Inhibition of PDGFR-A by RNA interference, small molecule inhibitor or neutralizing antibody had a dramatic effect on tumor cell growth both in vitro and in vivo, although resistance evolved in one-third of tumors. These results establish proof-of-principal for PDGFR-A as a therapeutic target in alveolar rhabdomyosarcoma.

Insulin receptor activation in solitary fibrous tumours.

Solitary fibrous tumours (SFTs) are known to overexpress insulin-like growth factor 2 (IGF-2). The down-stream oncogenic pathways of IGF-2, however, are not clear. Here we report uniform activation of the insulin receptor (IR) pathway in SFTs, which are mesenchymal tumours frequently associated with hypoglycaemia. Whereas the IR and its downstream signalling pathways were constitutively activated in SFTs, insulin-like growth factor 1 receptor (IGF-1R) was not expressed in these tumours. We also find that SFT cells secrete IGF-2 and proliferate in serum-free medium, consistent with an IGF-2/IR autocrine loop. The aetiological relevance of IGF-2 is supported by expression of IR-A, the IR isoform with high affinity for IGF-2, in all SFTs. Our studies suggest that IR activation plays an oncogenic role in SFTs.

[Human embryonic stem cell therapy, what is the attitude of the public?]. / Therapie mit embryonalen Stammzellen--wie steht die Offentlichkeit zu den ethischen Fragen?

This work discusses the manner in which the public has dealt with the ethical questions about human embryonic stem cell research. It argues that the public is not a homogenous entity, but rather consists of a number of "different publics", which at different points of the debate have discussed different aspects and different questions of this therapeutic proposal. It understands the development of human stem cell therapy not as a result of the activity of an isolated science, acting independently of society. In the contrary it considers it as a co-production of science with the different social actors, which participate in the political, ethical, and legal processes. Therefore the question if ethics is ahead or lags behind the developments in medical research becomes obsolete. Ethics is on one side the result of the effort to handle the results of biomedical research in a manner which is adequate for society at large on the other side it also influences the scientific-technological development through its own assessment.

Gastrointestinal stromal tumours: an update.

Recently, there has been intense interest in the study of gastrointestinal stromal tumour (GIST); one might call it a virtual GIST revolution. This is due largely to the realization that most GISTs express KIT and harbour activating c-KIT (KIT) or platelet-derived growth factor receptor-alpha (PDGFRA) receptor tyrosine kinase mutations that can be targeted by small molecule pharmacological inhibitors. Pathologists have benefited greatly from this revolution, mainly in the form of an improved ability to classify GISTs and, even more recently, in understanding the molecular underpinnings that underlie many fascinating clinical and pathological correlations. It is the purpose of this review to summarize recent developments in GIST classification and the molecular pathogenesis of GIST.

KIT activation is a ubiquitous feature of gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract, and they are generally resistant to chemotherapy and radiation therapy. Most GISTs express the KIT receptor tyrosine kinase protein, and a subset of GISTs contain activating mutations within the KIT juxtamembrane region. We evaluated 48 GISTs, including 10 benign, 10 borderline, and 28 malignant cases, to determine whether KIT expression and activation are general properties of these tumors. Immunohistochemical KIT expression was demonstrated in each case. Somatic KIT mutations were found in 44 tumors (92%), of which 34 (71%) had juxtamembrane region mutations. Other GISTs had KIT mutations in the extracellular region (n = 6) and in two different regions in the tyrosine kinase domain (n = 4). Contrary to previous reports, KIT mutations were not identified preferentially in higher-grade tumors: indeed, they were found in each of 10 histologically benign GISTs. Notably, mutations in all KIT domains were associated with high-level KIT activation/phosphorylation, and KIT activation was also demonstrated in the four GISTs that lacked detectable KIT genomic and cDNA mutations. These studies underscore the role of KIT activation in GIST pathogenesis, and they suggest that activated KIT might represent a universal therapeutic target in GISTs.

Cytologic diagnosis of gastrointestinal stromal tumor with emphasis on the differential diagnosis with leiomyosarcoma.

BACKGROUND: Gastrointestinal stromal tumor (GIST) has only recently been distinguished histologically and immunochemically from morphologically similar neoplasms of the abdomen. METHODS: The authors reviewed 15 cytologic cases of GIST (14 fine-needle aspiration [FNA] specimens and 1 peritoneal fluid specimen) and compared them with 23 cases of leiomyosarcoma (LMS) arising in the abdomen or pelvis (all FNAs). Immunochemistry (IC) was performed on both the cytologic and subsequent tissue specimens if sufficient specimen was available. RESULTS: Cytologic samples of GISTs typically showed irregularly outlined clusters of uniform spindle cells that were spread easily without crush artifact. The cells had wispy cytoplasm with long, delicate, filamentous extensions (13 cases; 87%). A prominent vascular pattern was common (9 cases; 60%); pleomorphism (1 case; 7%) was uncommon. The LMSs showed three-dimensional, tightly cohesive, sharply marginated syncytia of spindle cells, often with nuclear crush artifact. The cytoplasm/stroma had a distinct wiry, refractile appearance (21 cases; 91%); delicate filamentous cytoplasmic extensions (5 cases; 22%) and prominent vessels (3 cases; 13%) were less common. LMSs more commonly exhibited pleomorphism (14 cases; 61%). Epithelioid cytomorphology, mitoses, and necrosis occasionally were observed in both tumor types. IC for c-kit (on cytologic material) was positive in 10 of 10 cases of GIST (usually diffuse and strong) and 2 of 19 cases of LMS (focal). CD34 positivity favored GIST (4 of 9 cases) over LMS (1 of 19 cases). Smooth muscle actin was positive in 20 of 20 LMSs (strong and diffuse) and 6 of 10 GISTs (usually focal). Desmin was positive in 12 of 20 LMSs and was only focally positive in 1 of 11 GISTs. Correlation of IC results was excellent between cytologic and tissue specimens. CONCLUSIONS: Delicate cytoplasmic processes; a prominent vascular pattern; a lack of nuclear pleomorphism; and a c-kit-positive, desmin-negative immunoprofile are characteristic features of GIST and help distinguish these tumors from LMS in cytologic specimens.

Response to chemotherapy and predictors of survival in adult rhabdomyosarcoma.

OBJECTIVE: To assess outcome and identify predictors of survival of adults with rhabdomyosarcoma. SUMMARY BACKGROUND DATA: The literature on adult rhabdomyosarcoma is limited. Few studies have identified predictors of long-term survival in this patient population. METHODS: Thirty-nine adults with rhabdomyosarcoma were treated between 1973 and 1996 and prospectively followed. Outcomes were assessed with respect to patient and tumor characteristics, local treatment, and response to chemotherapy. RESULTS: Twenty-six patients had localized/locoregional disease and 13 patients had metastatic disease at presentation. Twenty-one patients underwent attempted curative resection, 27 received radiotherapy, and 37 received chemotherapy. Median follow-up for surviving patients was 152 months. The overall 5- and 10-year survival rates were 31% and 27%, respectively. Five-year survival rates for patients with tumors less than 5 cm, 5 to 10 cm, and more than 10 cm were 60%, 14%, and 0%, respectively. Patients with localized/locoregional disease at presentation had a 44% 5-year survival rate; there were no 5-year survivors among patients with metastatic disease. Patients who had a complete response to chemotherapy had a 5-year survival rate of 57%, compared with a rate of only 7% for poor responders. Metastatic disease at presentation and poor response to chemotherapy were independent predictors of death on multivariate analysis. CONCLUSIONS: Age, location, nodal status, and histologic subtype do not appear be associated with survival in adults with rhabdomyosarcoma treated with multimodal therapy. Metastatic disease at presentation and poor response to chemotherapy are strongly associated with poor prognosis. Future systemic therapies should be targeted to patients with localized/locoregional disease and partial responders to conventional chemotherapy.

Recent progress in the classification of soft tissue tumors: role of genetics and clinical implications.

Soft tissue tumors comprise a vast and heterogeneous group of neoplasms. Because different tumors often have different biological behaviors and respond differently to various therapeutic modalities, precise classification is paramount. The majority of soft tissue tumors were first delineated on the basis of morphologic and clinical findings, which in many cases were adequate to accurately separate different tumors into homogeneous groups; however, it has increasingly been appreciated that many entities are actually heterogeneous groups of tumors that have similar histologic and pathologic characteristics but differ in their clinical behavior and underlying pathogenesis. Within the past several years, great strides have been made in the purification of different entities. This accomplishment has largely been because of advances in our understanding of the molecular genetics that underlie the pathogenesis of many sarcomas and the development of new and specific tumor markers. This review highlights some important recent work in two selected soft tissue tumors-gastrointestinal stromal tumor and inflammatory myofibroblastic tumor. These examples illustrate the type of progress that is being made in the classification of soft tissue tumors.

Use of cytokeratins 7 and 20 in determining the origin of metastatic carcinoma of unknown primary, with special emphasis on lung cancer.

Metastatic carcinoma of unknown primary is a common problem, accounting for up to 10-15% of all solid tumours at presentation. Proper identification of the site of origin has prognostic and therapeutic significance. Prior immunohistochemical methods to identify the site of origin have been useful in a limited number of cases. Differential cytokeratin staining may be useful in this setting, particularly in identifying metastases from lung cancer. We have identified 144 cases of metastatic carcinoma of unknown primary to bone, lung or liver at Brigham and Women's Hospital between 1 January 1997 and 1 July 1998. Cytokeratin (CK) 7 and CK20 were used in 75 of these cases to narrow down the possible sites of the primary tumours. All of these cases were ambiguous as to the site of the primary tumour. Forty-five cases were CK7+/CK20-, 15 cases were CK7-/CK20-, 9 cases were CK7-/CK20+ and 6 cases were CK7+/CK20+. Three of the cases were selected for detailed presentation and discussion as well as a discussion of the pertinent literature. Overall, the CK7+/CK20- phenotype favours a lung primary, the CK7+/CK20+ phenotype strongly favours transitional cells (urothelial) carcinoma, the CK7-/CK20+ phenotype favours colorectal carcinoma, while the CK7-/CK20- profile is not helpful.

Teneurin-2 is expressed in tissues that regulate limb and somite pattern formation and is induced in vitro and in situ by FGF8.

Teneurin-2 is a member of a novel family of transmembrane proteins characterized to date in fish, birds, mammals, and Drosophila (e.g., the pair-rule gene product Ten-m). We have shown that teneurin-2 is expressed by neurons in the developing avian visual system in a pattern complementary to the expression of teneurin-1 and that recombinant teneurin-2 induces morphologic changes in neuronal cells in culture (Rubin et al., 1999). Here we have used cRNA probes to two newly identified splice variants and a teneurin-2-specific antibody to determine whether teneurin-2 is also expressed outside the nervous system. Both reverse transcriptase-polymerase chain reaction and in situ hybridization indicate that the three splice variants known so far are coexpressed at sites of pattern formation during development. Teneurin-2 mRNAs and protein are found in the developing limbs, somites, and craniofacial mesenchyme. In addition to expression of teneurin-2 by the apical ectodermal ridge, teneurin-2 transcripts also appear transiently at sites of tendon development. Teneurin-2 expression patterns were strikingly similar to those of fibroblast growth factor 8 (FGF8). In agreement with the overlapping expression pattern, FGF8-coated beads implanted into chicken limb buds induced the ectopic expression of teneurin-2 and soluble FGF8 induced teneurin-2 in limb explant cultures. Thus, teneurin-2 could act downstream of FGF8 during morphogenesis.

The genetics of lipomatous tumors.

The current classification of lipomatous neoplasms has been validated by the identification of characteristic cytogenetic and molecular genetic profiles associated with various neoplasms within the family of lipomatous tumors. The review describes characteristic cytogenetic and molecular genetic profiles and discusses their significance. The clinicopathologic features of these tumors, which are described elsewhere, will not be included in this review.

Isochromosome 7q in adult Wilms' tumors: diagnostic and pathogenetic implications.

Wilms' tumors affecting adults are rare and are thought to have a worse prognosis than similar stage tumors in the pediatric population. To understand these tumors better, the authors reviewed their multi-institutional experience in a series of nine lesions diagnosed as Wilms' tumors in adults. In addition to histologic and immunohistochemical examination, they performed cytogenetic analysis and fluorescence in situ hybridization. On review, four cases were reclassified: two "blastema only" as Ewing's sarcoma/primitive neuroectodermal tumor and the other two as clear cell sarcoma of soft parts and sarcoma not otherwise specified (NOS). Of the remaining five cases, three exhibited biphasic histology and two were triphasic. In this group, there were three women and two men, and patient age ranged from 17 to 37 years (median age, 26 years). Tumor size was large and ranged from 10 to 31 cm (median tumor size, 12.5 cm). Histologically, the tumors showed the typical features of Wilms' tumors with varying amounts of blastema (n = 5), epithelium (n = 5), and stroma (n = 2). No tumors contained anaplasia, and persistent renal blastema was not identified in the non-neoplastic kidney in any specimen. All tumors were positive for cytokeratins (CK7, n = 3; pankeratin, n = 5), and one tumor was weakly positive for CD99 (0-13). Molecular analysis including dual color fluorescence in situ hybridization (all tumors), and cytogenetic analysis (n = 2) disclosed the presence of isochromosome 7q in three of five tumors whereas all tumors were diploid with respect to chromosome 12. Follow-up data ranged from 6 to 133 months (median follow-up, 82 months) with progression in only one patient who had stage IV disease with lymph node and lung metastases at presentation. The authors conclude that adult Wilms' tumor has been overdiagnosed. Most "blastema-only" tumors in adults are not Wilms' tumors, and in an adult, biphasic morphology should be the minimum criteria for their diagnosis. Using strict diagnostic criteria, adult Wilms' tumors have a relatively favorable prognosis. The characteristic findings of isochromosome 7q, lack of trisomy or tetrasomy for chromosome 12, and absence of persistent renal blastema suggest that the pathogenesis of Wilms' tumors in adults may be different than in the pediatric population. These genetic features may be helpful in distinguishing adult Wilms' tumors from other primary renal tumors.

TPM3-ALK and TPM4-ALK oncogenes in inflammatory myofibroblastic tumors.

Inflammatory myofibroblastic tumors (IMTs) are neoplastic mesenchymal proliferations featuring an inflammatory infiltrate composed primarily of lymphocytes and plasma cells. The myofibroblastic cells in some IMTs contain chromosomal rearrangements involving the ALK receptor tyrosine-kinase locus region (chromosome band 2p23). ALK-which is normally restricted in its expression to neural tissues-is expressed strikingly in the IMT cells with 2p23 rearrangements. We now report a recurrent oncogenic mechanism, in IMTs, in which tropomyosin (TPM) N-terminal coiled-coil domains are fused to the ALK C-terminal kinase domain. We have cloned two ALK fusion genes, TPM4-ALK and TPM3-ALK, which encode approximately 95-kd fusion oncoproteins characterized by constitutive kinase activity and tyrosylphosphorylation. Immunohistochemical and molecular correlations, in other IMTs, implicate non-TPM ALK oncoproteins that are predominantly cytoplasmic or pre- dominantly nuclear, presumably depending on the subcellular localization of the ALK fusion partner. Notably, a TPM3-ALK oncogene was reported recently in anaplastic lymphoma, and TPM3-ALK is thereby the first known fusion oncogene that transforms, in vivo, both mesenchymal and lymphoid human cell lineages.

Myxoid leiomyosarcoma of soft tissue, an underrecognized variant.

Myxoid leiomyosarcoma is an uncommon tumor which, although previously well described in the uterus, is recognized to a lesser extent at other sites. We describe 18 cases of soft tissue leiomyosarcoma in which myxoid stroma occupied >50% of the tissue examined. Patients ranged in age from 22 to 84 years old (median, 57.5 yrs) and female patients outnumbered male patients 14 to 4. Tumor locations included the limbs (6 cases), female external genitalia (4 cases), head and neck region (3 cases), chest (2 cases), nipple, paratesticular soft tissue, and perineum (one case each). The tumors had a grossly gelatinous appearance and adopted three major histologic architectures: fascicular, reticular/microcystic, and "myxofibrosarcoma-like." The tumor cells were predominantly spindled in all cases with typical features of smooth muscle differentiation; there was a mixture of spindle and epithelioid cells in one case. No cases with pure epithelioid cytology were seen. All tumors displayed immunoreactivity for smooth muscle markers (smooth muscle actin 16/17, desmin 8/18) and, in addition, four cases were positive for keratin CAM 5.2 and three for epithelial membrane antigen. The tumors had a tendency to be morphologically lower grade (9 tumors were grade I, 8 were grade II, and only 1 was grade III). Follow up was available in 13 patients with a duration of 8 months to 41 years (median, 39 mos), and revealed local recurrences (often repeated) in five cases and metastases in two cases. There were three tumor-related deaths, of which two were the result of uncontrolled local disease. The differential diagnosis of myxoid leiomyosarcoma is broad and encompasses both benign and malignant lesions. Accurate diagnosis is critical because therapies may differ widely for entities in the differential diagnosis of myxoid leiomyosarcoma.

Composite hemangioendothelioma: a complex, low-grade vascular lesion mimicking angiosarcoma.

Eight cases of a previously uncharacterized vascular neoplasm, showing varying combinations of benign, low-grade malignant, and malignant vascular components are described. Seven tumors occurred in the dermis and/or subcutis and one occurred in the oral submucosa. The patients were all adults with a median age of 39.5 years (range, 21-71 years). Five patients were men. The tumors arose predominantly in the hands and feet, and the lesions were usually of several years duration. The tumors were composed of a complex admixture of histologic components that varied from tumor to tumor, such that no two tumors looked precisely the same. This was due to variation in the proportions of each component as well as the manner in which each component was distributed throughout each lesion. The predominant histologic components were epithelioid hemangioendothelioma (HE) and retiform HE, which were each present in seven of the tumors. Areas of spindle cell HE were identified in four lesions. Angiosarcoma-like elements were identified in seven tumors. One of the tumors was associated with an arteriovenous malformation and one was associated with an area of lymphangioma circumscriptum. Of six cases with follow up (median duration, 6.5 years), three have recurred locally and, to date, only one has metastasized. We think composite HE is best regarded as a low-grade malignant vascular neoplasm, and the available data suggest that it behaves more favorably than conventional angiosarcoma. The existence of these composite lesions has led to careful reexamination of the concept of HE. The term HE, in that it is currently synonymous with a low-grade malignant vascular tumor, should be reserved for lesions that have true metastatic potential, albeit with low frequency.