Gene Expression Differences between Enriched Normal and Chronic Myelogenous Leukemia Quiescent Stem/Progenitor Cells and Correlations with Biological Abnormalities.

In comparing gene expression of normal and CML CD34+ quiescent (G0) cell, 292 genes were downregulated and 192 genes upregulated in the CML/G0 Cells. The differentially expressed genes were grouped according to their reported functions, and correlations were sought with biological differences previously observed between the same groups. The most relevant findings include the following. (i) CML G0 cells are in a more advanced stage of development and more poised to proliferate than normal G0 cells. (ii) When CML G0 cells are stimulated to proliferate, they differentiate and mature more rapidly than normal counterpart. (iii) Whereas normal G0 cells form only granulocyte/monocyte colonies when stimulated by cytokines, CML G0 cells form a combination of the above and erythroid clusters and colonies. (iv) Prominin-1 is the gene most downregulated in CML G0 cells, and this appears to be associated with the spontaneous formation of erythroid colonies by CML progenitors without EPO.

The EWSR1/NR4A3 fusion protein of extraskeletal myxoid chondrosarcoma activates the PPARG nuclear receptor gene.

The NR4A3 nuclear receptor is implicated in the development of extraskeletal myxoid chondrosarcoma (EMC), primitive sarcoma unrelated to conventional chondrosarcomas, through a specific fusion with EWSR1 resulting in an aberrant fusion protein that is thought to disrupt the transcriptional regulation of specific target genes. We performed an expression microarray analysis of EMC tumours expressing the EWSR1/NR4A3 fusion protein, comparing their expression profiles to those of other sarcoma types. We thereby identified a set of genes significantly overexpressed in EMC relative to other sarcomas, including PPARG and NDRG2. Western blot or immunohistochemical analyses confirm that PPARG and NDRG2 are expressed in tumours positive for EWSR1/NR4A3. Bioinformatic analysis identified a DNA response element for EWSR1/NR4A3 in the PPARG promoter, and band-shift experiments and transient transfections indicate that EWSR1/NR4A3 can activate transcription through this element. Western blots further show that an isoform of the native NR4A3 receptor lacking the C-terminal domain is very highly expressed in tumours positive for EWSR1/NR4A3, and co-transfections of this isoform along with EWSR1/NR4A3 indicate that it may negatively regulate the activity of the fusion protein on the PPARG promoter. These results suggest that the overall expression of PPARG in EMC may be regulated in part by the balance between EWSR1/NR4A3 and NR4A3, and that PPARG may play a crucial role in the development of these tumours. The specific up-regulation of PPARG by EWSR1/NR4A3 may also have potential therapeutic implications.

In vivo differentiation and genomic evolution in adult male germ cell tumors.

Germ cell tumors (GCTs) are the most common solid malignancy in young adult men, but the genes and genomic regions involved in their etiology are not fully defined. We report here an investigation of DNA copy number changes in GCTs using 1 Mb BAC arrays. As expected, 12p gain was the defining genomic alteration, occurring in 72/74 GCTs. Parallel expression profiling of these tumors identified potential oncogenes from gained regions (LYN and RAB25) and potential tumor suppressor genes in regions of loss (SYNPO2, TTC12, IGSF4, and EPB41L3). Notably, we observed specific genomic alterations associated with histology, including gain of 17p11.2-q21.32 and loss of 2p25.3 in embryonal carcinoma, gain of 8p23.3-12 and loss of 5p15.33-35.3, 11q23.1-25, and 13q12.11-34 in seminoma, and gain of 1q31.3-42.3, 3p, 14q11.2-32.33, and 20q and loss of 8q11.1-23.1 in yolk sac tumors (YST). Many significant genes that mapped to these regions had previously been associated with specific histologies, such as EOMES (chr3) and BMP2 (chr20) in YST and SPRY2 (chr13) and SOX17 (chr8) in seminomas. Additionally, our results suggest a model in which histologic differentiation of GCTs may drive genomic evolution.

p53, epidermal growth factor, and platelet-derived growth factor in uterine leiomyosarcoma and leiomyomas.

Uterine leiomyosarcoma (ULMS) is an aggressive gynecological disease. Although ULMS are often found in association with benign leiomyoma (LMA), little is known regarding the relationship between these benign and malignant smooth muscle neoplasms. The objective of this study was to evaluate the expression of epidermal growth factor (EGFR), platelet-derived growth factor (PDGFR), and p53 in ULMS specimens, their prognostic relevance, and the expression of these molecular markers when compared to benign LMA specimens. Between 1991 and 2001, 25 patients were identified with high-grade primary ULMS and for whom tissue was available. Tissue microarray was created with three representative cores from each of the ULMS cases as well as from 19 patients with benign uterine leiomyomata. Immunohistochemical (IHC) staining was performed for EGFR, PDGFR, and p53. Negative and positive IHC staining was scored for each marker. Outcome analysis was performed only for ULMS. Survival was determined from the time of initial diagnosis to last follow-up. Twelve (48%) ULMS expressed p53 compared to none of the LMA (P < 0.001), and 15 (60%) ULMS cases showed PDGFR expression compared to 32% of LMA samples (P= 0.08). EGFR expression did not differ between ULMS and LMA groups. ULMS patients with p53 expression had a poorer survival compared to ULMS patients with negative expression (P= 0.07). ULMS tumor stage had the strongest association with overall survival (P= 0.05). Our study supports previous investigations indicating that p53 expression may serve as a prognostic marker for ULMS patients. The difference in PDGFR expression between ULMS and LMA demonstrated a trend toward significance. EGFR was not commonly expressed in ULMS. These uniquely expressed markers may assist in stratifying patients by survival and identify novel therapeutic markers. Clearly, further investigation is needed.

Identifying site-specific metastasis genes and functions.

Metastasis is a multistep and multifunctional biological cascade that is the final and most life-threatening stage of cancer progression. Understanding the biological underpinnings of this complex process is of extreme clinical relevance and requires unbiased and comprehensive biological scrutiny. In recent years, we have utilized a xenograft model of breast cancer metastasis to discover genes that mediate organ-specific patterns of metastatic colonization. Examination of transcriptomic data from cohorts of primary breast cancers revealed a subset of site-specific metastasis genes that are selected for early in tumor progression. High expression of these genes predicts the propensity for lung metastasis independently of several classic markers of poor prognosis. These genes fulfill dual functions-enhanced primary tumorigenicity and augmented organ-specific metastatic activity. Other metastasis genes fulfill functions specialized for the microenvironment of the metastatic site and are consequently not selected for in primary tumors. These findings improve our understanding of metastatic progression, facilitate the interpretation of primary tumor gene expression data, and open several important possibilities for future clinical application.

Circulating angiogenic factor levels correlate with extent of disease and risk of recurrence in patients with soft tissue sarcoma.

BACKGROUND: Tumor angiogenesis, or new blood vessel formation, is regulated by a balance between pro-angiogenic factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), and anti-angiogenic factors such as endostatin. PATIENTS AND METHODS: To investigate this angiogenic balance in soft tissue sarcomas (STS), blood samples were collected from 76 STS patients and 15 healthy controls, and analyzed for VEGF, bFGF and endostatin using quantitative enzyme-linked immunosorbent assays (ELISA). RESULTS: Forty-one patients (54%) had primary tumors, 20 (26%) had local recurrences and 15 (20%) had metastatic disease with or without local disease. Levels of all three angiogenic factors were highly variable in STS patients. Mean levels of VEGF and bFGF were 12 and 14 times higher, respectively, in patients compared with controls (P<0.0001). VEGF levels correlated with size of tumor, with the highest levels found in tumors >10 cm in size. Patients with metastases had endostatin levels 45% lower than patients without metastases (P=0.047). In 54 patients who underwent resection of primary disease or local recurrence, low pre-operative bFGF level was associated with a higher risk of subsequent recurrence (P=0.044). CONCLUSIONS: STS secrete widely variable levels of angiogenic factors, and levels of specific factors may correlate with extent of disease, predict risk of recurrence and possibly guide the use of anti-angiogenic agents.

Imaging of oncologic patients: benefit of combined CT and FDG PET in the diagnosis of malignancy.

OBJECTIVE: The purpose of this study was to assess the benefit of combined CT and 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) in diagnosing malignancy. MATERIALS AND METHODS: The records of 26 patients with intraabdominal and intrathoracic neoplasms who underwent CT and FDG PET between January 1995 and September 1996 were retrospectively reviewed. Most of these patients had inconclusive findings on prior CT for the diagnosis of malignancy. Only sites of potential malignant disease were included in the data analysis. Presence or absence of malignancy was confirmed by histopathology or follow-up CT. Three observers experienced in abdominal imaging used CT findings alone to estimate level of suspicion (1 = definitely not malignant to 5 = definitely malignant) for primary or recurrent neoplasms (n = 21), distant metastases (n = 25), and neoplastic nodal involvement (n = 18). Six weeks later the three observers reviewed the same CT examinations supplemented with FDG PET and reestimated suspicion of malignancy. Receiver operating characteristic methodology was used to analyze the results. Sensitivity, specificity, positive and negative predictive values, and accuracy in diagnosis of malignant disease were calculated using level 4 (probable malignancy) as the cutoff for the presence of disease. RESULTS: The mean area under the receiver operating characteristic curve, indicating successful diagnosis of malignancy, was .82 for CT alone and .92 for CT with FDG PET (p < .05). The accuracies for diagnosis of primary or recurrent neoplasms, distant metastases, and neoplastic nodal involvement were 62%, 68%, and 83%, respectively, for CT alone and 81% (p = .06), 88% (p = .03), and 89% (p > .25), respectively, for CT with FDG PET. Also, supplemental FDG PET imaging improved observer confidence and accuracy in diagnosing recurrent neoplasm in four (36%) of 11 patients who had undergone surgery or chemoradiation and in diagnosing four (29%) of 14 extrahepatic sites that had potential metastases. CONCLUSION: Diagnosis of malignancy in oncologic patients is significantly improved when CT is supplemented with FDG PET. Combined imaging is particularly helpful in the evaluation of potential recurrence in previously treated patients and for diagnosing extrahepatic lesions that may be distant metastases.

Pedigree analysis package vs. MIXD: fitting the mixed model on a large pedigree.

Results of a simulation study with two methods of analysis of data simulated under the mixed model on a 232-member pedigree are presented. The programs Pedigree Analysis Package (PAP), which approximate the likelihoods needed in a complex segregation analysis, and MIXD, which uses Monte Carlo Markov chain (MCMC), to estimate likelihoods were used. PAP obtained unbiased estimates of the major locus genotype means and the gene frequency, but biased estimates of the environmental variance component, and thus the heritability. A substantial fraction of the runs did not converge to an internal set of parameter estimates when analyzed with PAP. MIXD, which uses the Gibbs sampler to perform the MCMC sampling, produced unbiased estimates of all parameters with considerably more accuracy than obtained with PAP, and did not suffer from convergence of estimates to the boundary of the parameter space. The difference in behavior and accuracy of parameter estimates between PAP and MIXD was most apparent for models with either high or low residual additive genetic variance. Thus in situations where accuracy of the model is important, use of MCMC methods may be useful. In situations where less accuracy is needed, approximation methods may be adequate. Practical issues in using MCMC as implemented in MIXD to fit the mixed model are also discussed. Results of the simulations indicate that, unlike PAP, the starting configurations of most parameter estimates do not substantially influence the final parameter estimates in analysis with MIXD.

Monte Carlo analysis on a large pedigree.

Monte Carlo methods for linkage and segregation analysis are applied to the HGAR1 pedigree. To address these data, the methods are extended in several ways. The results are compared with those provided by PAP.