Soft-Tissue Sarcomas of the Genitourinary Tract with Radiologic-Pathologic Correlation.

Genitourinary tract soft-tissue sarcomas are rare neoplasms with varied pathologic and clinical features. While some of these tumors may be aggressive high-grade malignancies, others are low grade with a relatively better prognosis. Given that the grade and extent of the disease are important prognostic factors in these tumors, timely diagnosis is crucial. Unfortunately, most imaging features of these malignancies are not pathognomonic, and various histologic subtypes do not manifest with typical classic imaging features. Therefore, reliable differentiation of the various histologic tumor types is not always possible based solely on the radiologic manifestations. Imaging findings need to be considered in the context of clinical history in corroboration with radiologic-pathologic correlation. The authors discuss the specific imaging and pathologic characteristics of various genitourinary tract soft-tissue sarcomas, emphasizing diagnostic difficulties and differential diagnoses. ©RSNA, 2024 Test Your Knowledge questions for this article are available in the supplemental material.

MDMX Regulates Transcriptional Activity of p53 and FOXO Proteins to Stimulate Proliferation of Melanoma Cells.

The tumor suppressor protein p53 has an important role in cell-fate determination. In cancer cells, the activity of p53 is frequently repressed by high levels of MDMX and/or MDM2. MDM2 is a ubiquitin ligase whose activity results in ubiquitin- and proteasome-dependent p53 degradation, while MDMX inhibits p53-activated transcription by shielding the p53 transactivation domain. Interestingly, the oncogenic functions of MDMX appear to be more wide-spread than inhibition of p53. The present study aimed to elucidate the MDMX-controlled transcriptome. Therefore, we depleted MDMX with four distinct shRNAs from a high MDMX expressing uveal melanoma cell line and determined the effect on the transcriptome by RNAseq. Biological function analyses indicate the inhibition of the cell cycle regulatory genes and stimulation of cell death activating genes upon MDMX depletion. Although the inhibition of p53 activity clearly contributes to the transcription regulation controlled by MDMX, it appeared that the transcriptional regulation of multiple genes did not only rely on p53 expression. Analysis of gene regulatory networks indicated a role for Forkhead box (FOX) transcription factors. Depletion of FOXO proteins partly prevented the transcriptional changes upon MDMX depletion. Furthermore, depletion of FOXO proteins relatively diminished the growth inhibition upon MDMX knockdown, although the knockdown of the FOXO transcription factors also reduces cell growth. In conclusion, the p53-independent oncogenic functions of MDMX could be partially explained by its regulation of FOXO activity.

Overexpression of EZH2 in conjunctival melanoma offers a new therapeutic target.

Malignant melanoma of the conjunctiva (CM) is an uncommon but potentially deadly disorder. Many malignancies show an increased activity of the epigenetic modifier enhancer of zeste homolog 2 (EZH2). We studied whether EZH2 is expressed in CM, and whether it may be a target for therapy in this malignancy. Immunohistochemical analysis showed that EZH2 protein expression was absent in normal conjunctival melanocytes and primary acquired melanosis, while EZH2 was highly expressed in 13 (50%) of 26 primary CM and seven (88%) of eight lymph node metastases. Increased expression was positively associated with tumour thickness (p =0.03). Next, we targeted EZH2 with specific inhibitors (GSK503 and UNC1999) or depleted EZH2 by stable shRNA knockdown in three primary CM cell lines. Both pharmacological and genetic inactivation of EZH2 inhibited cell growth and colony formation and influenced EZH2-mediated gene transcription and cell cycle profile in vitro. The tumour suppressor gene p21/CDKN1A was especially upregulated in CM cells after EZH2 knockdown in CM cells. Additionally, the potency of GSK503 against CM cells was monitored in zebrafish xenografts. GSK503 profoundly attenuated tumour growth in CM xenografts at a well-tolerated concentration. Our results indicate that elevated levels of EZH2 are relevant to CM tumourigenesis and progression, and that EZH2 may become a potential therapeutic target for patients with CM. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Targeting of the MAPK and AKT pathways in conjunctival melanoma shows potential synergy.

PURPOSE: Conjunctival melanoma (CM) is a rare but lethal form of cancer. Similar to cutaneous melanoma, CM frequently carries activating mutations in BRAF and NRAS. We studied whether CM as well as conjunctival benign and premalignant melanocytic lesions express targets in the mitogen-activated protein kinase (MAPK) and AKT pathways, and whether specific inhibitors can suppress CM growth in vitro. METHODS: 131 conjunctival lesions obtained from 129 patients were collected. The presence of BRAF V600E mutation and expression of phosphorylated (p)-ERK and p-AKT were assessed by immunohistochemistry. We studied cell proliferation, phosphorylation, cell cycling and apoptosis in three CM cell lines using two BRAF inhibitors (Vemurafenib and Dabrafenib), a MEK inhibitor (MEK162) and an AKT inhibitor (MK2206). RESULTS: The BRAF V600E mutation was present in 19% of nevi and 26% of melanomas, but not in primary acquired melanosis (PAM). Nuclear and cytoplasmic p-ERK and p-AKT were expressed in all conjunctival lesions. Both BRAF inhibitors suppressed growth of both BRAF mutant CM cell lines, but only one induced cell death. MEK162 and MK2206 inhibited proliferation of CM cells in a dose-dependent manner, and the combination of these two drugs led to synergistic growth inhibition and cell death in all CM cell lines. CONCLUSION: ERK and AKT are constitutively activated in conjunctival nevi, PAM and melanoma. While BRAF inhibitors prohibited cell growth, they were not always cytotoxic. Combining MEK and AKT inhibitors led to more growth inhibition and cell death in CM cells. The combination may benefit patients suffering from metastatic conjunctival melanoma.

Activin Receptor-like Kinase 1 Ligand Trap Reduces Microvascular Density and Improves Chemotherapy Efficiency to Various Solid Tumors.

PURPOSE: Antiangiogenic therapy, mostly targeting VEGF, has been applied in cancer patients for the last decade. However, resistance to anti-VEGF therapy and/or no significant benefit as monotherapeutic agent is often observed. Therefore, new antiangiogenic strategies are needed. In the current study, we investigated the therapeutic effect of interfering with the bone morphogenetic protein (BMP)9/activin receptor-like kinase (ALK)1 signaling pathway by using an ALK1-Fc ligand trap. EXPERIMENTAL DESIGN: We analyzed the potential antiangiogenic and antitumor effects of ALK1-Fc protein as monotherapy and in combination with chemotherapy in vivo in mouse models of melanoma, head and neck cancer, and invasive lobular breast carcinomas. ALK1-Fc sequesters BMP9 and 10 and prevents binding of these ligands to endothelial ALK1, which regulates angiogenesis. RESULTS: Treatment of mice with ALK1-Fc strongly decreased the tumors' microvascular density in the three different mouse cancer models. However, this effect was not accompanied by a reduction in tumor volume. An immunohistochemical analysis of the tumor samples revealed that ALK1-Fc treatment increased the pericyte coverage of the remaining tumor vessels and decreased the hypoxia within the tumor. Next, we observed that combining ALK1-Fc with cisplatin inhibited tumor growth in the breast and head and neck cancer models more efficiently than chemotherapy alone. CONCLUSIONS: The addition of ALK1-Fc to the cisplatin treatment was able to enhance the cytotoxic effect of the chemotherapy. Our results provide strong rationale to explore combined targeting of ALK1 with chemotherapy in a clinical setting, especially in the ongoing phase II clinical trials with ALK1-Fc.

Agricultural injuries among operation household members: RRIS-II 1999.

The purpose of this study was to determine the magnitude and consequences of agricultural injuries, and to reveal potential risk factors among agricultural household members. The Regional Rural Injury Study (RRIS-II) collected injury and exposure data on agricultural households of 16,538 people in Minnesota, Wisconsin, North Dakota, South Dakota, and Nebraska for each six-month period of 1999. Adjusted injury rates, consequences, and potential risk factors were identified through analyses. Selection of variables for multivariate analyses was based on a causal model. Injuries reported here occurred while the individuals were involved in activities associated with their own farm or ranch, unless otherwise stated. Estimates of injury rates and the effects of various exposures were derived by Poisson and logistic regression. These models accounted for correlation within both subject and household, and were adjusted for non-response. The rate of agricultural injury to household members on their own operation was 74.5 injuries per 1,000 persons per year. Differences in rates due to age and gender diminished when rates were calculated according to hours worked. Although only 5% of injured persons required in-patient hospitalization, 28% required emergency department treatment, and 84% required some type of professional health care. Moreover, 47% of all injuries required time off from agricultural work, and 7% required time off from non-agricultural work. In multivariate analyses, decreased risks were associated with Minnesota, and increased risks were identified for those with prior injuries and for males.This study provides a basis for further research on agricultural injuries and their prevention.

Sports and recreational injuries: regional rural injury study-II: impact on agricultural households and operations.

OBJECTIVES: To identify the incidence, severity, and potential risk factors for sports/recreational injuries incurred by children and adults in a five state, rural, Midwest, agricultural household population. METHODS: Computer assisted telephone interviews that included questions about all injuries were completed for eligible, participating households for 1999; 16,538 people participated, including 8488 children less than 20 years of age. Rates and 95% confidence intervals were calculated, and causal models guided multivariate models. RESULTS: Of a total of 2586 injuries, 1301 (50%) were not related to agricultural activity. Among these, 733 (28%) were associated with sports/recreational activities including multiple person sports (64%), general play activities (19%), and single person sports (14%). The overall rate was 46.4 injury events per 1000 persons per year. Rates for children were 99.4 for boys and 64.3 for girls. For adults (aged 20 and above), rates were 11.9 for men and 4.8 for women. For children, 93% received health care, 44% were restricted for seven or more days, and 18% lost agricultural work time of seven or more days; the respective proportions for adults were 88%, 45%, and 17%. Multivariate analysis for children showed increased risks for Nebraska residents, males, and those 10-14 or 15-19 years. For adults, increased risks were identified for males and those 20-24 years; decreased risks were observed for Nebraska residents and those 45-54 years. CONCLUSIONS: Sports/recreational activities are an important source of injury with relevant consequences for this population, including significant restricted daily activity and lost agricultural work time. Key findings provide a basis for further study to address these burdens.

Chromosome-specific single-locus FISH probes allow anchorage of an 1800-marker integrated radiation-hybrid/linkage map of the domestic dog genome to all chromosomes.

We present here the first fully integrated, comprehensive map of the canine genome, incorporating detailed cytogenetic, radiation hybrid (RH), and meiotic information. We have mapped a collection of 266 chromosome-specific cosmid clones, each containing a microsatellite marker, to all 38 canine autosomes by fluorescence in situ hybridization (FISH). A 1500-marker RH map, comprising 1078 microsatellites, 320 dog gene markers, and 102 chromosome-specific markers, has been constructed using the RHDF5000-2 whole-genome radiation hybrid panel. Meiotic linkage analysis was performed, with at least one microsatellite marker from each dog autosome on a panel of reference families, allowing one meiotic linkage group to be anchored to all 38 dog autosomes. We present a karyotype in which each chromosome is identified by one meiotic linkage group and one or more RH groups. This updated integrated map, containing a total of 1800 markers, covers >90% of the dog genome. Positional selection of anchor clones enabled us, for the first time, to orientate nearly all of the integrated groups on each chromosome and to evaluate the extent of individual chromosome coverage in the integrated genome map. Finally, the inclusion of 320 dog genes into this integrated map enhances existing comparative mapping data between human and dog, and the 1000 mapped microsatellite markers constitute an invaluable tool with which to perform genome scanning studies on pedigrees of interest.

Design and validation of instruments to measure knowledge.

BACKGROUND: Measuring health care providers' learning after they have participated in educational interventions that use experimental designs requires valid, reliable, and practical instruments. METHODS: A literature review was conducted. In addition, experience gained from designing and validating instruments for measuring the effect of an educational intervention informed this process. RESULTS: The eight main steps for designing, validating, and testing the reliability of instruments for measuring learning outcomes are presented. The key considerations and rationale for this process are discussed. Methods for critiquing and adapting existent instruments and creating new ones are offered. CONCLUSIONS: This study may help other investigators in developing valid, reliable, and practical instruments for measuring the outcomes of educational activities.

Instruments to measure cancer management knowledge of rural health care providers.

BACKGROUND: Instruments to measure cancer management knowledge of rural physicians, nurses, and pharmacists were needed to evaluate the effect of an educational intervention. Since such instruments did not exist, the authors designed and validated a new instrument for each discipline. METHODS: The design and validation process for these instruments are described. RESULTS: These three instruments were shown to be practical and to have high content and construct validity. Content validation demonstrated that all items were rated as essential or useful by 90% or more of the respondents. Construct validation show highly significant differences in mean scores among several levels of learners and practitioners as expected. CONCLUSIONS: These instruments may be useful to other investigators for measuring cancer management knowledge of rural physicians, nurses, and pharmacists.

Injuries among children and youth in farm households: Regional Rural Injury Study-I.

OBJECTIVES: The purpose of this effort was to identify the incidence and consequences of both farming and non-farming related injuries and the potential risk factors for farming related injuries among children and youth, aged 0-19 years, who lived in farm households in a large region of the United States. METHODS: Data were collected from randomly selected farm households during 1990. Rates and rate ratios with 95% confidence intervals were calculated for sociodemographic and exposure variables. Multivariate analyses were conducted, using a priori and backward stepwise logistic regression models. RESULTS: Within the population of 3,939 farm households and 13,144 persons, children and youth accounted for 33%. Injury rates for farming and non-farming sources, respectively, were 1,683 and 6,980 per 100,000 persons. Animals (40%) were the primary sources of the farming operation related injuries; sports/recreation sources (61%) were associated primarily with non-farming related injuries. Of the farming and non-farming operation related injury cases, 83% and 90%, respectively, required some type of health care; moreover, 17% and 24%, respectively, were restricted from regular activities for one month or more. Through multivariate analyses, important increased rate ratios were observed for operating a tractor, working with dairy cattle, and being male. Increased rate ratios for working with beef cattle, operating a harvester, and living on a farm where there were all terrain vehicles in use, and a decreased rate ratio for living on a farm where there were sheep, appeared suggestive. CONCLUSIONS: Based on the relevant rates, injury consequences, and potential risk factors identified, injuries to children and youth on farms represent a significant problem. Future analytic studies are essential to identify more specific risk factors that can serve as a basis for development of appropriate intervention efforts. Given the population at risk, and the opportunity for intervention in this unique occupational setting, many of these injuries may be readily amenable to prevention efforts.

Lake Superior Rural Cancer Care Project, part I: an interventional trial.

PURPOSE: To date, effective cancer care and control intervention studies have been carried out largely in urban and suburban populations. This study was conducted to test innovative interventions, using experimental designs, to improve the care and outcomes of patients with cancer in rural settings. DESCRIPTION OF STUDY: The Lake Superior Rural Cancer Care Project (LSRCCP) tested an innovative, multimodal, multidisciplinary intervention that involved rural healthcare providers and their healthcare system. An experimental design was used, with the rural community as the unit of randomization. Outcomes were measured at three levels: rural providers' knowledge of cancer management, providers' practice performance, and patient outcomes. This 5-year study was conducted in rural areas of northern Minnesota, Wisconsin, and the western part of the Upper Peninsula of Michigan. RESULTS: Baseline data from the study are provided, and details of the design and methods are presented. The study outcomes are reported in part in "Lake Superior Rural Cancer Care Project Part II" in this issue and will be reported further in future issues. CLINICAL IMPLICATIONS: This article describes the hypotheses, design, and methods of the LSRCCP. The design and methods as well as the results of this study may be useful to cancer researchers and clinicians in rural areas across the United States.

Lake Superior Rural Cancer Care Project, part II: provider knowledge.

PURPOSE: The purpose of this article is to report the main learning outcomes of the Lake Superior Rural Cancer Care Project. DESCRIPTION OF STUDY: The authors designed and tested a multimodal intervention directed at rural providers and their healthcare systems in a large rural area in the north central United States. An experimental design was used to randomize rural providers at the group level. The intervention consisted of providing increased education for rural providers with a number of approaches, including the use of clinical opinion leaders. The main outcome of the intervention was knowledge scoring on discipline-specific cancer management tests. RESULTS: Knowledge scores for providers in the experimental group significantly increased from pretest to post-test: 66 to 79 for physicians (and physician assistants) (P=.02); 58 to 71 for nurses (P=.01); and 54 to 64 for pharmacists (P=.01). At post-test, participating providers in the experimental group performed significantly better on the knowledge tests (P <.01) than those in the control groups. CLINICAL IMPLICATIONS: This study may be the first to test educational interventions to improve rural providers' knowledge about cancer practice using an experimental design. The intervention may possibly change provider practice behaviors and, thus, patient outcomes, data that will be reported in a future issue. Finally, this educational intervention may prove useful for providers in other rural areas.

Isolation of DNA markers informative in purebred dog families by genomic representational difference analysis (gRDA).

Genomic Representational Difference Analysis (gRDA) is a subtractive DNA method to clone the differences between two related genomes, called tester and driver. We have evaluated this method to obtain polymorphic DNA markers for pedigree dogs. Amplified size-selected genomic restriction fragments (amplicons) of two dog littermates were repeatedly hybridized to each other in order to remove (subtract) those restriction fragments common to both sibs. Already after two rounds of subtractive hybridization, a clear enrichment of presumably tester-specific restriction fragments was observed, which was even more pronounced after the third round of subtraction. A plasmid library of 3000 recombinant clones was constructed of the second round and of the third round difference product. DNA sequence determination of randomly chosen clones of each difference product showed that approximately 1000 unique clones were obtained in the second-round difference product and approximately 500 in the third-round difference product. About half of the clones identified in the second-round difference product were also present in the third-round difference product. Of the second-round difference product, 39 different gRDA fragments could be identified, of which 21 were tester specific. In the third-round difference product, 22 different gRDA fragments were identified, of which 18 were tester specific. There were 13 fragments in common, resulting in a total of 48 different fragments. In order to establish the localization of these markers, we performed mapping using the dog radiation hybrid panel RHDF5000. Of 39 mapped clones, 29 were mapped to 20 existing RH groups, and 10 remained unlinked. It is concluded that gRDA is suitable to generate DNA markers to track disease genes within lines of pedigree dogs.

Cloning, characterization, and physical mapping of the canine Prop-1 gene (PROP1): exclusion as a candidate for combined pituitary hormone deficiency in German shepherd dogs.

Abnormalities in the genes encoding Pit-1 and Prop-1 have been reported to cause combined pituitary hormone deficiency (CPHD) in mice and humans. In dogs, a similar phenotype has been described in the German shepherd breed. We have previously reported that the Pit-1 gene (POU1F1) is not mutated in affected German shepherd dogs. In this study, we report the isolation and mapping of the canine Prop-1 gene (PROP1), and we assessed the involvement of PROP1 in German shepherd dog dwarfism. The canine PROP1 gene was found to contain three exons, encoding a 226 amino acid protein. The deduced amino acid sequence was 79% and 84% homologous with the mouse and human Prop-1 protein, respectively. Using fluorescence in situ hybridization, PROP1 was mapped to canine chromosome 11. Further mapping with a canine radiation hybrid panel showed co-localization with the polymorphic DNA marker AHT137. Sequence analysis of genomic DNA from dwarf German shepherd dogs revealed no alterations in the PROP1 gene. Moreover, linkage analysis of AHT137 revealed no co-segregation between the PROP1 locus and the CPHD phenotype, excluding this gene as candidate for canine CPHD and providing a new spontaneous model of hypopituitarism.

Cloning of the canine gene encoding transcription factor Pit-1 and its exclusion as candidate gene in a canine model of pituitary dwarfism.

Combined pituitary hormone deficiency (CPHD) is an autosomal recessive inherited disease of German shepherd dogs characterized primarily by dwarfism. In mice and humans a similar genetic disorder has been described that results from an alteration in the gene encoding the transcription factor Pit-1. In this study we characterized the canine Pit-1 gene, determined the chromosomal localization of the Pit-1 gene, and screened dwarf German shepherd dogs for the presence of mutations in this gene. The full-length canine Pit-1 cDNA contained an open reading frame encoding 291 amino acids, 92 bp of 5'-untranslated region, and 1959 bp of 3'-untranslated region. The deduced amino acid sequence was highly homologous with Pit-1 of other mammalian species. Using a Pit-1 BAC clone as probe, the Pit-1 gene was mapped by FISH to canine Chromosome (Chr) 31. In dwarf German shepherd dogs a C to A transversion was detected, causing a Phe (TTC) to Leu (TTA) substitution at codon 81. This alteration was present neither in other canine breeds analyzed nor in other mammalian species. However, healthy German shepherd dogs were also homozygous for the mutant allele, indicating that it is not the primary disease-causing mutation. In addition, linkage analysis of polymorphic DNA markers flanking the Pit-1 gene, 41K19 and 52L05, revealed no co-segregation between the Pit-1 locus and the CPHD phenotype. These findings suggest that a gene other than Pit-1 is responsible for the pituitary anomaly in dwarf German shepherd dogs.

Characterization of 463 type I markers suitable for dog genome mapping.

In total, 463 canine gene markers were identified and characterized to serve as reagents in canine genome map projects. These markers are distributed over 221 canine gene markers, 139 TOASTs (Traced Orthologous Sequence Tags), 27 canine TOASTs, and 76 huESTs (human Expressed Sequence Tags). Out of 310 canine gene markers, 59%-84% were successfully amplified on dog DNA, the highest rates of success being observed when the exon/intron structure is known. Concerning TOASTs and human ESTs, of the 225 and 300 markers analyzed, 62% and 25% respectively were able to produce a dog positive amplification. As part of an ongoing project to map the canine genome using a dog/hamster radiation hybrid panel, these markers were tested for their specificity on dog versus hamster DNA. Thus 61%, 21%, and 12% of dog gene markers, TOASTs, and huESTs met the criteria required for radiation hybrid mapping, respectively. All of these 463 canine gene markers, however, are available and will be of value to any other mapping strategies.

Community-based screening for childhood lead poisoning. Identification of risk factors and susceptible populations in Duluth.

The purpose of this matched case-controlled study was to identify local risk factors and susceptible populations for childhood lead poisoning in Duluth, Minnesota. We mailed questionnaires to the parents of 20 children with known elevated capillary lead levels > or = 10 micrograms/dL; 76 age-matched controls had capillary lead levels < 10 micrograms/dL. The study identified these risk factors for elevated capillary lead levels in children: not attending daycare, having nonwhite parents, living in rental property in central Duluth, and moving with family three or more times in the previous five years. We conclude that these risk factors are related to socioeconomics. Minority children and children living in poverty in the Duluth area should be screened for lead poisoning according to the Centers for Disease Control and Prevention screening guidelines for high-risk lead exposure.

Toward a dog radiation hybrid map.

Dog fibroblasts grown from a biopsy performed in a male mongrel were fused after gamma irradiation with thymidine kinase-deficient hamster cells and cultivated in selection medium. A total of 148 clones were obtained and screened by means of PCR amplification using primers corresponding to a dog-specific short repetitive element and to dog microsatellites and genes. One hundred seven cell lines were selected and grown in roller bottles and the distribution of 39 markers was analyzed in the extracted DNA. The results clearly indicate that this panel of hybrid cell lines should prove invaluable for constructing a map of the canine genome. In parallel, for more than 500 microsatellites present in the databases or screened from two libraries of short inserts, we have determined PCR conditions favoring dog-specific products even in the presence of hamster DNA. These highly polymorphic microsatellites should be useful in further linkage studies. We have also characterized 254 markers: dog genes, human expressed sequenced tags (huESTs), and traced orthologous amplified sequenced tags (TOASTs). Once mapped, these will constitute powerful tools to detect regions of conserved synteny in human and other mammalian genomes.