Janet Shipley

Mutations of the BRAF gene in human cancer

Cancers arise owing to the accumulation of mutations in critical genes that alter normal programmes of cell proliferation, differentiation and death. As the first stage of a systematic genome-wide screen for these genes, we have prioritized for analysis signalling pathways in which at least one gene is mutated in human cancer. The RAS–RAF–MEK–ERK–MAP kinase pathway mediates cellular responses to growth signals. RAS is mutated to an oncogenic form in about 15% of human cancer. The three RAF genes code for cytoplasmic serine/threonine kinases that are regulated by binding RAS. Here we report BRAF somatic missense mutations in 66% of malignant melanomas and at lower frequency in a wide range of human cancers. All mutations are within the kinase domain, with a single substitution (V599E) accounting for 80%. Mutated BRAF proteins have elevated kinase activity and are transforming in NIH3T3 cells. Furthermore, RAS function is not required for the growth of cancer cell lines with the V599E mutation. As BRAF is a serine/threonine kinase that is commonly activated by somatic point mutation in human cancer, it may provide new therapeutic opportunities in malignant melanoma.

A census of amplified and overexpressed human cancer genes.

Integrated genome-wide screens of DNA copy number and gene expression in human cancers have accelerated the rate of discovery of amplified and overexpressed genes. However, the biological importance of most of the genes identified in such studies remains unclear. In this Analysis, we propose a weight-of-evidence based classification system for identifying individual genes in amplified regions that are selected for during tumour development. In a census of the published literature we have identified 77 genes for which there is good evidence of involvement in the development of human cancer.

Testicular germ-cell cancer

Testicular germ-cell tumours (TGCTs) represent the model of a curable malignancy; sensitive tumour markers, accurate prognostic classification, logical series of management trials, and high cure rates in both seminomas and non-seminomas have enabled a framework of effective cancer therapy. Understanding the molecular biology of TGCT could help improve treatment of other cancers. The typical presentation in young adults means that issues of long-term toxicity become especially important in judging appropriate management. A focus of recent developments has been to tailor aggressiveness of treatment to the severity of the prognosis. Recent changes affect the most common subtypes and include the reduction of chemotherapy for patients who have metastastic non-seminomas and a good prognosis, and alternatives to adjuvant radiotherapy in stage I seminomas. We summarise advances in the understanding and management of TGCT during the past decade.

Fusion Gene–Negative Alveolar Rhabdomyosarcoma Is Clinically and Molecularly Indistinguishable From Embryonal Rhabdomyosarcoma

PURPOSE To determine whether the clinical and molecular biologic characteristics of the alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS) subtypes have relevance independent of the presence or absence of the PAX/FOXO1 fusion gene. PATIENTS AND METHODS The fusion gene status of 210 histopathologically reviewed, clinically annotated rhabdomyosarcoma samples was determined by reverse transcriptase polymerase chain reaction. Kaplan-Meier analysis was used to assess event-free survival and overall survival in fusion gene-negative ARMS (ARMSn; n = 39), fusion gene-positive ARMS (ARMSp; n = 94), and ERMS (n = 77). A total of 101 RMS samples were also profiled for whole-genome expression, and 128 were profiled for genomic copy number imbalances. Profiling data were analyzed by supervised and unsupervised methods to compare features related to histopathology and fusion gene status. Results were also projected by meta-analysis techniques across three separate publically available data sets. RESULTS Overall and event-free survival, frequency of metastases, and distribution of site at initial presentation were not significantly different between ARMSn and ERMS. Consistent with this, analysis of gene expression signatures could not reproducibly distinguish ARMSn from ERMS whereas fusion gene-positive cases were distinct. ARMSn and ERMS frequently show whole-chromosome copy number changes, notably gain of chromosome 8 with associated high levels of expression of genes from this chromosome. CONCLUSION The clinical behavior and molecular characteristics of alveolar cases without a fusion gene are indistinguishable from embryonal cases and significantly different from fusion-positive alveolar cases. This implies that fusion gene status irrespective of histology is a critical factor in risk stratification of RMS.

Fusion of splicing factor genes PSF and NonO (p54nrb) to the TFE3 gene in papillary renal cell carcinoma

We demonstrate that the cytogenetically defined translocation t(X;1)(p11.2;p34) observed in papillary renal cell carcinomas results in the fusion of the splicing factor gene PSF located at 1p34 to the TFE3 helix – loop – helix transcription factor gene at Xp11.2. In addition we define an X chromosome inversion inv(X)(p11.2;q12) that results in the fusion of the NonO (p54nrb) gene to TFE3. NonO (p54nrb), the human homologue of the Drosophila gene NonAdiss which controls the male courtship song, is closely related to PSF and also believed to be involved in RNA splicing. In each case the rearrangement results in the fusion of almost the entire splicing factor protein to the TFE3 DNA-binding domain. These observations suggest the possibility of intriguing links between the processes of RNA splicing, DNA transcription and oncogenesis.

Genomic and expression profiling of human spermatocytic seminomas: primary spermatocyte as tumorigenic precursor and DMRT1 as candidate chromosome 9 gene.

Spermatocytic seminomas are solid tumors found solely in the testis of predominantly elderly individuals. We investigated these tumors using a genome-wide analysis for structural and numerical chromosomal changes through conventional karyotyping, spectral karyotyping, and array comparative genomic hybridization using a 32 K genomic tiling-path resolution BAC platform (confirmed by in situ hybridization). Our panel of five spermatocytic seminomas showed a specific pattern of chromosomal imbalances, mainly numerical in nature (range, 3-24 per tumor). Gain of chromosome 9 was the only consistent anomaly, which in one case also involved amplification of the 9p21.3-pter region. Parallel chromosome level expression profiling as well as microarray expression analyses (Affymetrix U133 plus 2.0) was also done. Unsupervised cluster analysis showed that a profile containing transcriptional data on 373 genes (difference of > or = 3.0-fold) is suitable for distinguishing these tumors from seminomas/dysgerminomas. The diagnostic markers SSX2-4 and POU5F1 (OCT3/OCT4), previously identified by us, were among the top discriminatory genes, thereby validating the experimental set-up. In addition, novel discriminatory markers suitable for diagnostic purposes were identified, including Deleted in Azospermia (DAZ). Although the seminomas/dysgerminomas were characterized by expression of stem cell-specific genes (e.g., POU5F1, PROM1/CD133, and ZFP42), spermatocytic seminomas expressed multiple cancer testis antigens, including TSP50 and CTCFL (BORIS), as well as genes known to be expressed specifically during prophase meiosis I (TCFL5, CLGN, and LDHc). This is consistent with different cells of origin, the primordial germ cell and primary spermatocyte, respectively. Based on the region of amplification defined on 9p and the associated expression plus confirmatory immunohistochemistry, DMRT1 (a male-specific transcriptional regulator) was identified as a likely candidate gene for involvement in the development of spermatocytic seminomas.

Poorly differentiated synovial sarcoma : An analysis of clinical, pathologic, and molecular genetic features

Poorly differentiated synovial sarcoma is a variant of synovial sarcoma in which the tumor cells lack the bland spindle cell appearance of the usual type monophasic synovial sarcoma. Although poorly differentiated synovial sarcoma has been recognized as an entity for many years, no series addressing the clinicopathologic features of this variant have appeared. We describe the histologic, immunohistologic, and molecular findings of a series of 20 poorly differentiated synovial sarcomas. Three types of poorly differentiated synovial sarcoma can be recognized: a large cell epithelioid variant, a small cell variant, and a high-grade spindle cell variant. Epithelial membrane antigen reactivity was seen in 95% of cases, and reactivity for cytokeratin was seen in 42%. The S100 antigen was expressed in 63% of cases. Electron microscopic findings in poorly differentiated synovial sarcoma parallel those found in usual type synovial sarcoma. In 10 cases, material was available for molecular studies; 9 of 10 cases showed the presence of t(X;18) or the associated fusion gene product. These data indicate that poorly differentiated synovial sarcoma is a lesion that shares immunologic, ultrastructural, and molecular characteristics with the usual synovial sarcoma. Follow-up data were available in 16 patients with a mean follow-up of 39 months. Eight patients died with a mean survival time of 33 months. Poorly differentiated synovial sarcoma is a variant of synovial sarcoma that may be associated with a poor prognosis.

Genes, chromosomes, and rhabdomyosarcoma.

Rhabdomyosarcomas are a heterogeneous group of malignant tumors and are the most common soft‐tissue sarcoma of childhood. Rhabdomyosarcomas resemble developing skeletal muscle, notably in their expression of the MRF family of transcription factors and the PAX3 and PAX7 genes. These PAX genes are also involved through specific translocations, t(2;13)(q35;q14) and variant t(1;13)(p36;q14) in the alveolar subtype, which result in PAX3‐FKHR and PAX7‐FKHR fusion genes, respectively. The fusion genes are thought critically to affect downstream targets of PAX3 and PAX7 or possibly have novel targets. Similar downstream changes may also be involved in embryonal and fusion gene negative cases. Genomic amplification of such genes as MYCN, MDM2, CDK4, and PAX7‐FKHR is a feature mainly of the alveolar subtype, while specific chromosomal gains, including chromosomes 2, 8, 12, and 13, are associated with the embryonal subtype. Loss of alleles and imprinting at 11p15.5 and disruption of genes such as IGF2, ATR, PTC, P16, and TP53 have also been implicated in rhabdomyosarcoma development. Whereas there is now a realistic possibility of cure in the majority of cases, there remains a subset that is resistant to multimodality therapy, including high‐dose chemotherapy. Characterization of the defining molecular features of tumors that are likely to behave aggressively represents a particular challenge. Current research is leading toward a better understanding of rhabdomyosarcoma tumorigenesis, which may ultimately result in novel therapeutic strategies that increase the overall cure. Genes Chromosomes Cancer 26:275–285, 1999.

PAX3/FOXO1 Fusion Gene Status Is the Key Prognostic Molecular Marker in Rhabdomyosarcoma and Significantly Improves Current Risk Stratification

PURPOSE To improve the risk stratification of patients with rhabdomyosarcoma (RMS) through the use of clinical and molecular biologic data. PATIENTS AND METHODS Two independent data sets of gene-expression profiling for 124 and 101 patients with RMS were used to derive prognostic gene signatures by using a meta-analysis. These and a previously published metagene signature were evaluated by using cross validation analyses. A combined clinical and molecular risk-stratification scheme that incorporated the PAX3/FOXO1 fusion gene status was derived from 287 patients with RMS and evaluated. RESULTS We showed that our prognostic gene-expression signature and the one previously published performed well with reproducible and significant effects. However, their effect was reduced when cross validated or tested in independent data and did not add new prognostic information over the fusion gene status, which is simpler to assay. Among nonmetastatic patients, patients who were PAX3/FOXO1 positive had a significantly poorer outcome compared with both alveolar-negative and PAX7/FOXO1-positive patients. Furthermore, a new clinicomolecular risk score that incorporated fusion gene status (negative and PAX3/FOXO1 and PAX7/FOXO1 positive), Intergroup Rhabdomyosarcoma Study TNM stage, and age showed a significant increase in performance over the current risk-stratification scheme. CONCLUSION Gene signatures can improve current stratification of patients with RMS but will require complex assays to be developed and extensive validation before clinical application. A significant majority of their prognostic value was encapsulated by the fusion gene status. A continuous risk score derived from the combination of clinical parameters with the presence or absence of PAX3/FOXO1 represents a robust approach to improving current risk-adapted therapy for RMS.

Amplification and Overexpression of the KIT Gene Is Associated with Progression in the Seminoma Subtype of Testicular Germ Cell Tumors of Adolescents and Adults

We have previously identified amplification at 4q12 in testicular germ cell tumors of adolescents and adults centered around the KIT gene encoding a tyrosine kinase transmembrane receptor. Analysis of primary testicular germ cell tumors totaling 190 cases revealed 21% of the seminoma subtype with an increased copy number of KIT whereas this change was rarely found in the nonseminomas. In most cases, gain of KIT did not include the immediately flanking noncoding DNA or the flanking genes KDR and PDGFRA. Increased copy number of KIT was not found in the putative precursor lesion, carcinoma in situ (CIS), adjacent to tumor with this change. KIT overexpression was found independent of gain and KIT immunostaining was stronger in selected cases with gain of KIT compared to those without. Taken together with activating mutations of KIT in exon 17 identified in 13% of seminomas, this suggests that the KIT gene product plays a role in the progression of CIS towards seminoma, the further understanding of which may lead to novel less toxic therapeutic approaches.