Publications

Cytogenetic analysis of tumour material has been greatly enhanced over the past 30 years by the application of a range of techniques based around fluorescence in situ hybridization (FISH). Fluorescence detection for in situ hybridization has the advantage of including the use of a multitude of fluorochromes to allow simultaneous specific detection of multiple probes by virtue of their differential labelling and emission spectra. FISH can be used to detect structural (translocation/inversion) and numerical (deletion/gain) genetic aberrations. This chapter will deal with FISH methods to detect and localize one or more complementary nucleic acid sequences (probes) within a range of different cellular targets including metaphase chromosomes, nuclei from cell suspension, and formalin-fixed paraffin-embedded FFPE tissue sections. Methods for the efficient localization of probes to FFPE tissue cores in tissue microarrays (TMAs) are also described.

To improve the risk stratification of patients with rhabdomyosarcoma (RMS) through the use of clinical and molecular biologic data.

Glypican-5 (GPC5) is a cell surface heparan sulfate proteoglycan and 1 of 6 closely related members of the glypican family in mammals. Glypicans are predominantly expressed during development in cell-specific and tissue-specific contexts, and the expression of some is linked to developmental disorders and several visceral malignancies. We have previously shown that the region of amplification at 13q31.3 in a subset of rhabdomyosarcomas contains the GPC5 locus, and by copy number and gene expression analyses, that GPC5 is consistently expressed and upregulated in amplified tumors. As the immunohistochemical profile of GPC5 is untested, our aim was to optimize a commercially available anti-human GPC5 antibody for immunohistochemical use in formalin-fixed and paraffin-embedded (FFPE) tissue. Quantitative real-time polymerase chain reaction analyses of normal tissue samples indicated that the brain and testis highly expressed GPC5. High protein expression in these tissues and a cell line constructed to overexpress GPC5 were demonstrated by Western blotting. These normal tissues and the isogenic cell line were FFPE, and immunohistochemical expression of GPC5 was assessed using different methods of antigen retrieval, detection, and primary antibody concentration. The optimum conditions for detection were by heat-induced antigen retrieval, in sodium citrate buffer at pH 6. Enzyme-mediated retrieval did not produce effective detection, producing weaker, less well-localized GPC5 expression. We demonstrate that anti-human GPC5 antibody is amenable to use in FFPE tissue and with the optimized protocol we describe shows specific cellular localization and good staining intensity with minimal background staining.

Adrenal cortical carcinosarcoma is a rare variant of adrenal cortical carcinoma. Sarcomatous change in adrenal cortical carcinomas is exceptionally rare, with only 9 cases previously described. Adrenal cortical carcinosarcomas tend to be aggressive tumors, with locoregional recurrence and rapid metastases from the sarcomatoid component. We describe what seems to be the first case of sarcoma arising in oncocytic adrenal cortical carcinoma. The sarcomatous component here was pleomorphic rhabdomyosarcoma. This occurred in a 45-year-old man who had nodal and pulmonary metastases of the rhabdomyosarcomatous component at presentation and who died of progressive disease 11 months later. Here, we discuss the clinical, radiologic, and pathologic findings and review the literature on adrenal cortical carcinosarcomas.

Rhabdomyosarcomas are a major cause of cancer death in children, described with MYCN amplification and, in the alveolar subtype, transcription driven by the PAX3-FOXO1 fusion protein. Our aim was to determine the prevalence of N-Myc protein expression and the potential therapeutic effects of reducing expression in rhabdomyosarcomas, including use of an antigene strategy that inhibits transcription.

Rhabdomyosarcomas (RMS) are a heterogeneous group of tumors that share features of skeletal myogenesis and represent the most common pediatric soft tissue sarcoma. Even though significant advances have been achieved in RMS treatment, prognosis remains very poor for many patients. Several elements of the Insulin-like Growth Factor (IGF) pathway are involved in sarcomas, including RMS. The IGF2 ligand is highly expressed in most, if not all, RMS, and frequent overexpression of the receptor IGF1R is also found. This is confirmed here through mining expression profiling data of a large series of RMS samples. IGF signaling is implicated in the genesis, growth, proliferation, and metastasis of RMS. Blockade of this pathway is therefore a potential therapeutic strategy for the treatment of RMS. In this paper we examine the biological rationale for targeting the IGF pathway in RMS as well as the current associated preclinical and clinical experience.

Testicular germ cell tumours (TGCTs) of adults and adolescents are putatively derived from primordial germ cells or gonocytes. Recently reported genome-wide association studies implicate six gene loci that predispose to TGCT development. Remarkably, the functions of proteins encoded by genes within these regions bridge our understanding between the pathways involved in primordial germ cell physiology, male germ cell development and the molecular pathology of TGCTs. Furthermore, this improved understanding of the mechanisms underlying TGCT development and dissemination has clinical relevance for the management of patients with these tumours.

Testicular germ cell tumors (TGCT) are the most frequent solid tumor to affect young adult males and are histologically divided into seminomas and nonseminomas (NS). NS comprise undifferentiated embryonal carcinoma (EC) and differentiated tumors with embryonic (teratoma) or extra-embryonic (choriocarcinoma, yolk sac tumor) features. In contrast to other subtypes, EC have uniform cellular morphology and lack normal cell infiltrates, ideal for nucleic acid profiling. EC are under-represented in previous studies due to their relative rarity. To gain insights into NS tumorigenesis, metastatic dissemination and potential markers of relapse, a full tiling path BAC platform was used to obtain array comparative genomic hybridization (aCGH) profiles from 32 formalin fixed paraffin embedded stage I EC samples from patients with follow-up data. In addition to identifying regions previously described in TGCT, novel minimum overlapping regions of gain at 6p21.33, 10q11.21, and 22q13.32 and loss at 22q12.2 were defined and confirmed by fluorescence in situ hybridization analyses. Specifically, the region at 6p21.33 included OCT3/4, the expression of which is involved in the maintenance of pluripotency and the 10q11.21 region contains the gene encoding the RAS activating factor RASGEF1A, the expression of which was demonstrably increased in RNA extracted from these samples. The region of loss at 22q12.2 was more frequently seen in tumors that relapsed and protein expression of genes from 22q12.2 included PIK3IP1, a negative regulator of PI3 kinase signaling was reduced. These data support the role for genes involved in pluripotency and RAS/PI3K signaling in EC development and progression.

Glypican-3 (GPC3) is a membrane-bound heparan sulphate proteoglycan. Glypicans are predominantly expressed during development in cell- and tissue-specific manners and act as co-receptors for many heparin-binding growth factors, playing important roles in cell growth and differentiation. GPC3 expression has been linked to several visceral malignancies, but its role in sarcomas is unexplored. Rhabdomyosarcomas (RMS), the most common paediatric soft tissue sarcomas (STS), are aggressive tumours that frequently show histological overlap with other STS. Immunohistochemistry was performed on tissue microarrays to study GPC3 expression in paediatric RMS and a variety of the more common spindle cell and pleomorphic adult sarcomas.

Testicular germ-cell tumours (TGCT) are the most frequent solid tumour to affect young Caucasian adult males and have increased in incidence over recent decades. In clinical stage I non-seminomas, (NSGCT) histological vascular invasion (VI) is a prognostic factor for metastatic relapse. Using array comparative genomic hybridization, we have previously shown that the presence of VI is associated with gain of a region at 17q12, containing a cluster of genes encoding inflammatory cytokines. We here confirm this finding using fluorescence in situ hybridization (FISH) demonstrating gain in 12 out of 42 (29%) assessable samples. Interrogation of previously published expression microarray data suggests that of the genes contained within this region, CCL2 [monocyte chemoattractant protein 1 (MCP1)] is frequently overexpressed in TGCT. Immunohistochemistry confirms this finding in a collection of 67 clinical stage I NSGCT, demonstrating an association with the presence of VI (p=0.049) that was not seen with VEGF-A, MMP2 or MMP9, although all were frequently expressed. This work gives further insight into the mechanisms involved in invasion in this tumour type, which may ultimately have implications for the management of patients with stage I disease.

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.

Rhabdomyosarcomas (RMSs) are primarily paediatric sarcomas that resemble developing skeletal muscle. Our aim was to determine the effects of microRNAs (miRNA) that have been implicated in muscle development on the clinical behaviour of RMSs.

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.

Formalin fixed paraffin embedded (FFPE) material is frequently the most convenient readily available source of diseased tissue, including tumors. Multiple cores of FFPE material are being used increasingly to construct tissue microarrays (TMAs) that enable simultaneous analyses of many archival samples. Fluorescence in situ hybridization (FISH) is an important approach to analyze FFPE material for specific genetic aberrations that may be associated with tumor types or subtypes, cellular morphology, and disease prognosis. Annealing, or hybridization of labeled nucleic acid sequences, or probes, to detect and locate one or more complementary nucleic acid sequences within fixed tissue sections allows the detection of structural (translocation/inversion) and numerical (deletion/gain) aberrations and their localization within tissues. The robust protocols described include probe preparation, hybridization, and detection and take 2-3 days to complete. A protocol is also described for the stripping of probes for repeat FISH in order to maximize the use of scarce tissue resources.

Rhabdomyosarcoma is the most common soft tissue sarcoma in the pediatric population. As this tumor has an undifferentiated myogenic phenotype, agents that promote differentiation hold particular promise as part of a novel therapeutic approach to combat this type of cancer. In this report, we focus on the contribution of two microRNAs (miRNAs) in rhabdomyosarcomas. Levels of miR-1 and miR-133a are drastically reduced in representative cell lines from each major rhabdomyosarcoma subtype (embryonal and alveolar). Introduction of miR-1 and miR-133a into an embryonal rhabdomyosarcoma-derived cell line is cytostatic, thereby suggesting a tumor suppressor-like role for these myogenic miRNAs. Transcriptional profiling of cells after miR-1 and miR-133a expression reveals that miR-1 (but not miR-133a) exerts a strong promyogenic influence on these poorly differentiated tumor cells. We identify mRNAs that are down-regulated by these miRNAs and propose roles for miR-1 and miR-133a in repressing isoforms of genes that are normally not expressed in muscle. Finally, we show that mRNA targets of miR-1 and miR-133a are up-regulated in rhabdomyosarcomas, suggesting a causative role for these miRNAs in the development of rhabdomyosarcomas. More important, these results point to the promise of enhancing rhabdomyosarcoma therapy using miRNAs as agents that mediate cytostasis and promote muscle differentiation.-Rao, P. K., Missiaglia, E., Shields, L., Hyde, G., Yuan, B., Shepherd, C. J., Shipley, J., Lodish, H. F. Distinct roles for Mir-1 and Mir-133a in the proliferation and differentiation of rhabdomyosarcoma cells. FASEB J. 24, 3427-3437 (2010). www.fasebj.org

Development of chemoresistance limits the clinical efficiency of platinum-based therapy. Although many resistance mechanisms have been demonstrated, genetic/molecular alterations responsible for drug resistance in the majority of clinical cases have not been identified. We analyzed three pairs of testicular germ cell tumor cell lines using Affymetrix expression microarrays and revealed a limited number of differentially expressed genes across the cell lines when comparing the parental and resistant cells. Among them, CCND1 was the most significantly differentially expressed gene. Analysis of testicular germ cell tumor clinical samples by quantitative reverse transcription PCR analysis revealed that overall expression of CCND1 was significantly higher in resistant cases compared with sensitive samples (P < 0.0001). We also found that CCND1 was dramatically overexpressed both in induced and intrinsically resistant samples of ovarian and prostate cancer. Finally combined CCND1 knockdown using small-interfering RNA and cisplatin treatment inhibited cell growth in vitro significantly more effectively than any of these single treatments. Therefore, deregulation of CCND1 may be a major cause of cisplatin resistance in testicular germ cell tumors and may also be implicated in ovarian and prostate cancers. CCND1 could be potentially used as a marker for treatment stratification and as a molecular target to improve the treatment of platinum-resistant tumors. (Am J Pathol 2010, 176:2607-2615; DOI: 10.2353/ajpath.2010.090780)

MAP kinase phosphatase-2 (MKP-2) is a member of the family of dual specificity phosphatases that functions to inactivate the ERK and JNK MAP kinase signalling pathways. Here, we identify a novel human MKP-2 variant (MKP-2-S) lacking the MAP kinase binding site but retaining the phosphatase catalytic domain. Endogenous MKP-2-S transcripts and proteins were found in PC3 prostate and MDA-MB-231 breast cancer cells and also human prostate biopsies. Cellular transfection of MKP-2-S gave rise to a nuclear protein of 33 kDa which displayed phosphatase activity comparable to the formerly described long form of MKP-2 (MKP-2-L). Due to its lack of a kinase interacting motif (KIM), MKP-2-S did not bind to JNK or ERK; MKP-2-L bound ERK and to a lesser extent JNK. Protein turnover of adenoviral expressed MKP-2-S was accelerated relative to MKP-2-L, with a greater susceptibility to proteosomal-mediated degradation. MKP-2-S retained its ability to deactivate JNK in a similar manner as MKP-2-L and was an effective inhibitor of LPS-stimulated COX-2 induction. However, unlike MKP-2-L, MKP-2-S was unable to reverse serum-induced ERK activation or significantly inhibit endothelial cell proliferation. These findings reveal the occurrence of a novel splice variant of MKP-2 which is unable to bind ERK and may be significant in the dysregulation of MAP kinase activity in certain disease states, particularly in breast and prostate cancers. (C) 2009 Elsevier Inc. All rights reserved.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and comprises two major histological subtypes: alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS). Seventy-five percent of ARMS harbor reciprocal chromosomal translocations leading to fusion genes of the forkhead transcription factor FOXO1 and PAX3 or PAX7. The hedgehog (Hh) pathway has been implied in tumor formation and progression of various cancers including RMS. However, whether Hh pathway activation presents a general feature of RMS or whether it is restricted to specific subgroups has not yet been addressed. Here, we report that marker genes of active Hh signaling, that is, Patched1 (Ptch1), Gli1, Gli3 and Myf5, are expressed at significantly higher levels in ERMS and fusion gene-negative ARMS compared with fusion gene-positive ARMS in two distinct cohorts of RMS patients. Consistently, Gli1 expression correlates with Ptch1 expression in ERMS and fusion gene-negative ARMS, but not in fusion gene-positive ARMS. In addition, expression levels of MyoD1 are significantly lower in ERMS and fusion gene-negative ARMS, pointing to an inverse association of Hh activation and early muscle differentiation. Moreover, Myf5 is identified as a novel excellent class predictor for RMS by receiver operating characteristic analysis. Importantly, high expression of Ptch1 or low MyoD1 expression significantly correlate with reduced cumulative survival in fusion gene-negative RMS underscoring the clinical relevance of these findings. By showing that Hh signaling is preferentially activated in specific subgroups of RMS, our study has important implications for molecular targeted therapies, such as small molecule Hh inhibitors, in RMS. Oncogene (2010) 29, 6323-6330; doi:10.1038/onc.2010.368; published online 6 September 2010

Interaction between the chemokine CXCL12 (SDF1) and the G-protein coupled receptor CXCR4 is responsible for the maintenance of adult stem cell niches and is known to play an important role in utero in the migration of primordial germ cells. We demonstrate expression of CXCL12 by Sertoli cells and confirm CXCR4 expression by the germ cell population of the adult human testes. CXCR4 is also known to mediate organ-specific patterns of metastases in a range of common cancers. We identify consistent expression of CXCR4 mRNA and protein in testicular germ cell tumours (TGCT) that accounts for their patterns of relapse in sites of known CXCL12 expression. Extragonadal primary germ cell tumours express CXCR4 and their sites of occurrence are coincident with areas of known CXCL12 expression in utero. We show that CXCL12 stimulates the invasive migration of a TGCT cell line in vitro in a CXCR4-dependent fashion and activates ERK. Furthermore, we demonstrate that expression of CXCL12 in stage I non-seminomas is significantly associated with organ-confined disease post-orchidectomy and reduced risk of relapse (p = 0.003). This may be through the loss of CXCL12 gradients that might otherwise attract cells away from the primary tumour. We propose CXCL12 expression as a potential predictor of subsequent relapse that could lead to avoiding unnecessary treatment and associated late toxicities. Our observations support a role for CXCL12/CXCR4 in the adult germ cell population and demonstrate pathological function in germ cell tumour development and metastasis that may have clinical utility.

Rhabdomyosarcomas (RMS) are the most common pediatric soft tissue sarcomas. They resemble developing skeletal muscle and are histologically divided into two main subtypes; alveolar and embryonal RMS. Characteristic genomic aberrations, including the PAX3- and PAX7-FOXO1 fusion genes in alveolar cases, have led to increased understanding of their molecular biology. Here, we determined the effect of genomic copy number on gene expression levels through array comparative genomic hybridization (CGH) analysis of 13 RMS cell lines, confirmed by multiplex ligation-dependent probe amplification copy number analyses, combined with their corresponding expression profiles. Genes altered at the transcriptional level by genomic imbalances were identified and the effect on expression was proportional to the level of genomic imbalance. Extrapolating to a public expression profiling dataset for 132 primary RMS identified features common to the cell lines and primary samples and associations with subtypes and fusion gene status. Genes identified such as CDK4 and MYCN are known to be amplified, overexpressed, and involved in RMS tumorigenesis. Of the many genes identified, those with likely functional relevance included CENPF, DTL, MYC, EYA2, and FGFR1. Copy number and expression of FGFR1 was validated in additional primary material and found amplified in 6 out of 196 cases and overexpressed relative to skeletal muscle and myoblasts, with significantly higher expression levels in the embryonal compared with alveolar subtypes. This illustrates the ability to identify genes of potential significance in tumor development through combining genomic and transcriptomic profiles from representative cell lines with publicly available expression profiling data from primary tumors.

Screening for specific genetic aberrations by fluorescence and chromogenic in situ hybridization (fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH)) can reveal associations with tumor types or subtypes, cellular morphology and clinical behavior. FISH and CISH methodologies are based on the specific annealing (hybridization) of labeled genomic sequences (probes) to complementary nucleic acids within fixed cells to allow their detection, quantification and spatial localization. Formalin-fixed paraffin embedded (FFPE) material is the most widely available source of tumor samples. Increasingly, tissue microarrays (TMAs) consisting of multiple cores of FFPE material are being used to enable simultaneous analyses of many archival samples. Here we describe robust protocols for the FISH and CISH analyses of genetic aberrations in FFPE tissue, including TMAs. Protocols include probe preparation, hybridization and detection. Steps are described to reduce background fluorescence and strip probes for repeat FISH analyses to maximize the use of tissue resources. The basic protocol takes 2-3 d to complete.

Prostate cancer is the most frequently diagnosed male cancer, and its clinical outcome is difficult to predict. The disease may involve the inappropriate expression of genes that normally control the proliferation of epithelial cells in the basal layer and their differentiation into luminal cells. Our aim was to identify novel basal cell markers and assess their prognostic and functional significance in prostate cancer. RNA from basal and luminal cells isolated from benign tissue by immunoguided laser-capture microdissection was subjected to expression profiling. We identified 112 and 267 genes defining basal and luminal populations, respectively. The transcription factor TEAD1 and the ubiquitin ligase c-Cbl were identified as novel basal cell markers. Knockdown of either marker using siRNA in prostate cell lines led to decreased cell growth in PC3 and disrupted acinar formation in a 3D culture system of RWPE1. Analyses of prostate cancer tissue microarray staining established that increased protein levels of either marker were associated with decreased patient survival independent of other clinicopathological metrics. These data are consistent with basal features impacting on the development and clinical course of prostate cancers.

Testicular germ cell tumors (TGCTs) of adults and adolescents are thought to be derived from primordial germ cells or gonocytes. TGCTs develop postpuberty from precursor lesions known as intratubular germ cell neoplasia undifferentiated. The tumors can be divided into two groups based on their histology and clinical behavior; seminomas resemble primordial germ cells or gonocytes and nonseminomas resemble embryonic or extraembryonic tissues at various stages of differentiation. The most undifferentiated form of nonseminoma, embryonal carcinoma, resembles embryonic stem cells in terms of morphology and expression profiling, both mRNAs and microRNAs. Evidence supports both environmental factors and genetic predisposition underlying the development of TGCTs. Various models of development have been proposed and are discussed. In TGCTs, gain of material from the short arm of chromosome 12 is invariable: genes from this region include the proto-oncogene KRAS, which has activating mutations in approximately 10% of tumors or is frequently overexpressed. A number of different approaches to increase the understanding of the development and progression of TGCTs have highlighted the involvement of KIT, RAS/RAF/MAPK, STAT, and PI3K/AKT signaling. We review the role of these signaling pathways in this process and the potential influence of environmental factors in the development of TGCTs.

Myogenin immunostaining has been described as a useful marker of the alveolar subtype of rhabdomyosarcoma and as a tool for distinguishing it from the more common embryonal subtype. To add to the growing body of literature describing this phenomenon we analysed myogenin immunohistochemical staining in 152 tumors using a rhabdomyosarcoma tissue array. Results were analysed blinded to histological type by two independent investigators. Samples were excluded if any samples failed to stain with desmin and/or myogenin. Mean percentage of myogenin positive cells was significantly greater for ARMS (n=31; mean percentage positivity 59% (95% confidence intervals +/- 7%) than ERMS (n=41, mean percentage positivity 16%, 95% confidence intervals +/- 4; P < 0.0001). This data is consistent with previously published studies identifying strong nuclear myogenin staining in a high proportion of cells as a marker of alveolar histology.

Down syndrome (DS) is the most common cause of mental retardation. Many neural phenotypes are shared between DS individuals and DS mouse models; however, the common underlying molecular pathogenetic mechanisms remain unclear. Using a transchromosomic model of DS, we show that a 30%-60% reduced expression of Nrsf/Rest (a key regulator of pluripotency and neuronal differentiation) is an alteration that persists in trisomy 21 from undifferentiated embryonic stem (ES) cells to adult brain and is reproducible across several DS models. Using partially trisomic ES cells, we map this effect to a three-gene segment of HSA21, containing DYRK1A. We independently identify the same locus as the most significant eQTL controlling REST expression in the human genome. We show that specifically silencing the third copy of DYRK1A rescues Rest levels, and we demonstrate altered Rest expression in response to inhibition of DYRK1A expression or kinase activity, and in a transgenic Dyrk1A mouse. We reveal that undifferentiated trisomy 21 ES cells show DYRK1A-dosesensitive reductions in levels of some pluripotency regulators, causing premature expression of transcription factors driving early endodermal and mesodermal differentiation, partially overlapping recently reported downstream effects of Rest +/-. They produce embryoid bodies with elevated levels of the primitive endoderm progenitor marker Gata4 and a strongly reduced neuroectodermal progenitor compartment. Our results suggest that DYRK1A-mediated deregulation of REST is a very early pathological consequence of trisomy 21 with potential to disturb the development of all embryonic lineages, warranting closer research into its contribution to DS pathology and new rationales for therapeutic approaches.

Carcinoma in situ (CIS) of the testis is the pre-invasive stage of type 11 testicular germ cell tumours (TGCTs) of adolescents and adults. These tumours are the most frequently diagnosed cancer in Caucasian adolescents and young adults. In dysgenetic gonads, the precursor of type II GCTs can be either CIS or a lesion known as gonadoblastoma (GB). CIS/GB originates from a primordial germ cell (PGC)/gonocyte, ie an embryonic cell. CIS can be cured by local low-dose irradiation, with limited side effects on hormonal function. Therefore, strategies for early diagnosis of CIS are essential. Various markers are informative to diagnose CIS in adult testis by immunohistochemistry, including c-KIT, PLAP, AP-2 gamma, NANOG, and POU5F1 (OCT3/4). OCT3/4 is the most informative and consistent in presence and expression level, resulting in intense nuclear staining. In the case of maturational delay of germ cells, frequently present in gonads of individuals at risk for type 11 (T)GCTs, use of these markers can result in overdiagnosis of malignant germ cells. This demonstrates the need for a more specific diagnostic marker to distinguish malignant germ cells from germ cells showing maturation delay. Here we report the novel finding that immunohistochemical detection of stem cell factor (SCF), the c-KIT ligand, is informative in this context. This was demonstrated in over 400 cases of normal (fetal, neonatal, infantile, and adult) and pathological gonads, as well as TGCT-derived cell lines, specifically in cases of CIS and GB. Both membrane-bound and soluble SCIF were expressed, suggestive of an autocrine loop. SCIF immunohistochemistry can be a valuable diagnostic tool, in addition to OCT3/4, to screen for precursor lesions of TGCTs, especially in patients with germ cell maturation delay. Copyright (C) 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

To investigate if genomic instability exists in tumors with cytogenetically normal karyotypes, we analyzed four diploid cancer cell lines A204, CAL51, CH1 and SK-UT-1B. We detected subtle genomic changes in all four cell lines. More of these alterations were found in A204 and CH1 than in the microsatellite unstable lines CAL51 and SK-UT-1B. The number of (it, novo, non-clonal chromosome rearrangements was also significantly higher in CH I than CAL51 and SK-UT-1B (p = 0.001). This study reveals multiple genomic abnormalities in tumors with near normal karyotypes and suggests that genomic instability may be essential in cancer development. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Overexpression of genes, through genomic amplification and other mechanisms, can critically affect the behavior of tumor cells. Genomic amplification of the 13q31-32 region is reported in many tumors, including rhabdomyosarcomas that are primarily pediatric sarcomas resembling developing skeletal muscle. The minimum overlapping region of amplification at 13q31-32 in rhabdomyosarcomas was defined as containing two genes: Glypican-5 (GPC5) encoding a cell surface proteoglycan and C13orf25 encompassing the miR-17-92 micro-RNA cluster. Genomic copy number and gene expression analyses of rhabdomyosarcomas indicated that GPC5 was the only gene consistently expressed and up-regulated in all cases with amplification. Constitutive overexpression and knockdown of GPC5 expression in rhabdomyosarcoma cell lines increased and decreased cell proliferation, respectively. A correlation between expression levels of nascent pre-rRNA and GPC5 (P = 0.001), but not a C13orf25 transcript containing miR-17-92, in primary samples supports an association of GPC5 with proliferative capacity in vivo. We show that GPC5 increases proliferation through potentiating the action of the growth factors fibroblast growth factor 2 (FGF2), hepatocyte growth factor (HGF), and Wnt1A. GPC5 enhanced the intracellular signaling of FGF2 and HGF and altered the cellular distribution of FGF2. The mesoderm-inducing effect of FGF2 and FGF4 in Xenopus blastocysts was also enhanced. Our data are consistent with a role of GPC5, in the context of sarcomagenesis, in enhancing FGF signaling that leads to mesodermal cell proliferation without induction of myogenic differentiation. Furthermore, the properties of GPC5 make it an attractive target for therapeutic intervention in rhabdomyosarcomas and other tumors that amplify and/or overexpress the gene.

The application of cytogenetic and molecular genetic analyses to paediatric sarcomas has identified a number of characteristic changes associated with types and subtypes of sarcomas. This has led to increased understanding of the underlying molecular biology of some sarcomas and provided an important adjunct to standard morphological and immunohistochemical diagnoses. Characteristic genetic abnormalities, particularly specific chromosome translocations and associated fusion genes, have diagnostic and in some cases prognostic value. There is also the potential to detect micrometastastic disease. Fusion genes are most readily detected by fluorescence in situ hybridisation and reverse transcription-PCR technologies. The expression profiles of tumours with specific fusion genes are characteristically similar and the molecular signatures of sarcomas are also proving to be of diagnostic and prognostic value. Furthermore, fusion genes and other emerging molecular events associated with sarcomas represent potential targets for novel therapeutic approaches which are desperately required to improve the outcome of children with certain categories of sarcoma, including rhabdomyosarcomas and the Ewing's family of tumours. Increased understanding of the molecular biology of sarcomas is leading towards more effective treatments which may complement or be less toxic than conventional radiotherapy and cytotoxic chemotherapy. Here we review paediatric sarcomas that have associated molecular genetic changes which can increase diagnostic and prognostic accuracy and impact on clinical management.

Testicular germ cell tumours (TGCTs) are the leading cause of cancer deaths in young male Caucasians. Identifying changes in DNA copy number can pinpoint genes involved in tumour development. We defined the smallest overlapping regions of imbalance in TGCTs using array comparative genomic hybridization analysis. Novel regions, or regions which refined those previously reported, were identified. The expression profile of genes from 12p, which is invariably gained in TGCTs, and amplicons defined at 12p11.2-12.1 and 4q12, suggest KRAS and KIT involvement in TGCT and seminoma development, respectively. Amplification of these genes was not found in intratubular germ cell neoplasia adjacent to invasive disease showing these changes, suggesting their involvement in tumour progression. Activating mutations of RAS genes (KRAS or NRAS) and overexpression of KRAS were mutually exclusive events. These, correlations between the expression levels of KIT, KRAS and GRB7 (which encodes an adapter molecule known to interact with the KIT tyrosine kinase receptor) and other reported evidence reviewed here, are consistent with a role for activation of KIT and RAS signalling in TGCT development. In order to assess a role for KIT in seminomas, we modulated the level of KIT expression in TCam-2, a seminoma cell line. The likely seminomatous origin of this cell line was supported by demonstrating KIT and OCT3/4 overexpression and gain of 12p material. Reducing the expression of KIT in TCam-2 through RNA inhibition resulted in decreased cell viability. Further understanding of KIT and RAS signalling in TGCTs may lead to novel therapeutic approaches for these tumours.

Testicular germ cell tumours of adults and adolescents (TGCT) include seminomas (SE) and nonseminomas (NS), with spermatocytic seminomas (SSE) representing a distinct entity in older men. SE and NS have gain of 12p material in all cases, whereas SSE are associated with overrepresentation of chromosome 9. Here, we compare at the chromosomal level, copy number imbalances with global expression changes, identified by comparative expressed sequence hybridisation analyses, in seven SE, one combined tumour, seven NS and seven cell lines. Positive correlations were found consistent with copy number as a main driver of expression change, despite reported differences in methylation status in SE and NS. Analysis of chromosomal copy number and expression data could not distinguish between SE and NS, in-keeping with a similar genetic pathogenesis. However, increased expression from 4q22, 5q23.2 and 9p21 distinguished SSE from SE and NS and decreased copy number and expression from 2q36-q37 and 6q24 was a specific feature of NS-derived cell lines. Our analysis also highlights 19 regions with both copy number and expression imbalances in greater than 40% of cases. Mining available expression array data identified genes from these regions as candidates for involvement in TGCT development. Supplementary data is available at http://www.crukdmf.icr.ac.uk/array/array.html.

Background: Down syndrome (DS), caused by trisomy of human chromosome 21 (HSA21), is the most common genetic cause of mental retardation in humans. Among complex phenotypes, it displays a number of neural pathologies including smaller brain size, reduced numbers of neurons, reduced dendritic spine density and plasticity, and early Alzheimer-like neurodegeneration. Mouse models for DS show behavioural and cognitive defects, synaptic plasticity defects, and reduced hippocampal and cerebellar neuron numbers. Early postnatal development of both human and mouse-model DS shows the reduced capability of neuronal precursor cells to generate neurons. The exact molecular cause of this reduction, and the role played by increased dosage of individual HSA21 genes, remain unknown.Results: We have subcutaneously injected mouse pluripotent ES cells containing a single freely segregating supernumerary human chromosome 21 (HSA21) into syngeneic mice, to generate transchromosomic teratomas. Transchromosomic cells and parental control cells were injected into opposite flanks of thirty mice in three independent experiments. Tumours were grown for 30 days, a time-span equivalent to combined intra-uterine, and early post-natal mouse development. When paired teratomas from the same animals were compared, transchromosomic tumours showed a three-fold lower percentage of neuroectodermal tissue, as well as significantly reduced mRNA levels for neuron specific (Tubb3) and glia specific (Gfap) genes, relative to euploid controls. Two thirds of transchromosomic tumours also showed a lack of PCR amplification with multiple primers specific for HSA21, which were present in the ES cells at the point of injection, thus restricting a commonly retained trisomy to less than a third of HSA21 genes.Conclusion: We demonstrate that a supernumerary chromosome 21 causes Inhibition of Neuroectodermal Differentiation (INDI) of pluripotent ES cells. The data suggest that trisomy of less than a third of HSA21 genes, in two chromosomal regions, might be sufficient to cause this effect.

Background and Objectives Salivary gland mucosa-associated lymphoid tissue (MALT) lymphomas typically lack chromosomal translocations and the molecular genetics underlying their development is unknown. The aim of this study was to investigate chromosomal changes in these lymphomas.Design and Methods We performed comparative genomic hybridisation using DNA samples extracted from microdissected tumour cells in 19 salivary gland MALT lymphomas. Recurrent chromosomal changes were further verified by interphase fluorescence in situ hybridization (FISH).Results Chromosomal gains were much more common than losses. Recurrent gains were found at 1p32-ter (42%), 9q33-34 (84%), 11q11-13 (42%), 17 (58%) and 18q21-22 (42%). Among these, the recurrent gains at 9q34, 11q11-13 and 18q21 were nearly the exclusive gain of the corresponding chromosome. Notably, chromosomal gains at 9q34, 11q13 and 18q21 were frequently concurrent, with 12/19 cases affecting at least two of the three loci. The genomic gains at these chromosomal regions were further confirmed by interphase FISH with probes targeting the TRAF2 and CARD9 (9q34), RELA and CCND1 (11q13), and MALT1 (18q21) loci.Interpretation and Conclusions Salivary gland MALT lymphomas show a conserved pattern of chromosomal gains, which appear to target genes positively modulating cell survival and proliferation.

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.

We previously demonstrated that constitutional BUB1B mutations cause mosaic variegated aneuploidy, a condition characterized by constitutional aneuploidies and childhood cancer predisposition. To further investigate the role of BUB1B in cancer predisposition we performed comparative genomic hybridization analysis in an embryonal rhabdomyosarcoma from an MVA case with biallelic BUB1B mutations, revealing aneuploidies typical of sporadic E-RMS, with gain of chromosomes 3, 8, 13 and loss of chromosomes 9, 14, X. To investigate whether somatic BUB1B mutations occur in sporadic childhood cancers we screened 30 Wilms tumours, 10 acute lymphoblastic leukemias, nine rhabdomyosarcomas and 11 rhabdomyosarcoma cell lines for BUB1B mutations. We identified seven exonic and six intronic variants. Six of the exonic variants were synonymous and one resulted in a non-synonymous conservative missense alteration that was also present in a control. These data suggest that the genetic progression in rhabdomyosarcoma from MVA and non-MVA cases may be similar, but that somatic BUB1B mutations are unlikely to be common in sporadic childhood cancers known to be associated with MVA.

Comparative expressed sequence hybridization (CESH) is an expression profiling technique which identifies chromosomal regions corresponding to differential gene expression. Here, we observe that various tumor samples including rhabdomyosarcoma show very prominent staining on the short arms of the acrocentric chromosomes suggesting an increase in expression of ribosomal RNA synthesized from the repetitive rDNA of the nucleolar organizer regions located on these chromosomes. Survival analysis showed a correlation with overexpression from this region and a poor prognosis in rhabdomyosarcoma. This phenomenon was studied in an extended set of rhabdomyosarcoma tumor samples using quantitative real-time reverse transcriptase-PCR to quantify levels of pre-rRNA (precursor ribosomal RNA). It was demonstrated first that the strong CESH signals did correspond to a marked increase in pre-rRNA expression and second that high pre-rRNA expression correlated with an adverse prognosis in alveolar subtype rhabdomyosarcoma. In addition, we demonstrate that pre-rRNA expression is significantly correlated with tumor stage. We conclude that measuring expression of pre-rRNA by real-time PCR is a useful prognostic marker in alveolar rhabdomyosarcoma. Furthermore, given that we have observed similar rDNA staining in all cancer types that we have studied by CESH, we propose that pre-rRNA overexpression is a general phenomenon in cancer and that our real-time PCR assay may be applicable as a prognostic marker in other tumor types.

Germline mutations in Fumarate Hydratase (FH) cause the development of leiomyomas and leiomyosarcomas in the syndromes Multiple Cutaneous and Uterine Leiomyomata (MCUL1) and Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC). There is little evidence, however, that FH mutation plays a role in the development of sporadic leiomyomas or leiomyosarcomas. Such observations do not, however, exclude a role for FH in tumour development outside the context of MCUL1/HLRCC, as it is possible that FH expression could be silenced by epigenetic mechanisms. To explore this possibility we have developed a highly specific antibody to FH and analysed a series of forty-five fresh-frozen uterine leiomyomas and nine leiomyosarcomas for FH expression.

Deleterious mutations in the BRCA1 gene predispose women to an increased risk of breast and ovarian cancer. Many functional studies have suggested that BRCA1 has a role in DNA damage repair and failure in the DNA damage response pathway often leads to the accumulation of chromosomal aberrations. Here, we have compared normal lymphocytes with those heterozygous for a BRCA1 mutation. Short-term cultures were irradiated (8Gy) using a high dose rate and subsequently metaphases were analysed by 24-colour chromosome painting (M-FISH). We scored the chromosomal rearrangements in the metaphases from five BRCA1 mutation carriers and from five noncarrier control samples 6 days after irradiation. A significantly higher level of chromosomal damage was detected in the lymphocytes heterozygous for BRCA1 mutations compared with normal controls; the average number of aberrations per mitosis was 3.48 compared with 1.62 in controls (P=0.0001). This provides new evidence that heterozygous mutation carriers have a different response to DNA damage compared with noncarriers and that BRCA1 has a role in DNA damage surveillance. Our finding has implications for treatment and screening of BRCA1 mutation carriers using modalities that involve irradiation.

The E2F3 transcription factor has an established role in controlling cell cycle progression. In previous studies we have provided evidence that nuclear E2F3 overexpression represents a mechanism that drives the development of human bladder cancer and that determines aggressiveness in human prostate cancer. We have proposed a model in which E2F3 overexpression co-operates with removal of the E2F inhibitor pRB to facilitate cancer development. Since small cell lung cancers (SCLC) have one of the highest reported frequencies of functional abnormalities in the pRB protein (90%) of any human cancer, we wish to assess to what extent E2F3 would be overexpressed in this and other classes of human lung cancer.

Recurrent genomic alterations are the hallmarks of particular cancers. Application of molecular cytogenetic technologies to tumour material in order to detect these alterations has become important for molecular diagnostics and research. A dual-colour chromogenic in situ hybridization (dc-CISH) method described recently in the Journal of Pathology has the advantage of visualizing two probes simultaneously with the ability to discern morphological features. In addition, the bright field microscopy required is readily accessible to many laboratories. The approach has been validated by comparison of results with standard analyses for HER-2 amplification status in formalin-fixed, paraffin-embedded breast cancers and is applicable to the analysis of other clinically relevant genomic aberrations as well as of use in research investigations.

The receptor tyrosine kinase MET and its ligand hepatocyte growth factor (HGF), have been implicated in the genesis of the paediatric tumour rhabdomyosarcoma (RMS). Addition of exogenous HGF to RH30 RMS cells enhanced non-chemotactic migration. Stable transfection of dominant negative MET into RH30 cells attenuated Matrigel invasion and in vivo tumour growth. To assess the role of a putative HGF-MET pathway in human RMS, we measured their expression in a panel of 68 human primary tumours. All tumours expressed MET but with a three orders of magnitude variation of expression and 62% of tumours co-expressed HGF. In contrast with other tumour types, neither high-MET expression nor HGF/MET coexpression correlated with metastatic disease. In a microarray screen, we identified CCN1 as being 7.8-fold up regulated following addition of HGF to RH30 cells and in RMS tumours, CCN1 expression correlated with HGF expression. Surprisingly, we identified MET as a consistent feature of embryonal and not alveolar RMS.

To explore the genetic changes involved in the stepwise development of lung cancer, we have determined the genetic events associated with the histological progression from normal bronchial epithelium to squamous cell carcinoma. Comparative genomic hybridization was used to identify chromosomal imbalances in 54 microdissected samples, including squamous metaplasia, dysplasia, carcinoma in situ, and invasive tumour derived from 23 patients with squamous cell carcinoma of the lung. Histopathological progression was accompanied by an increased number of chromosomal abnormalities. Gains of 1q25-32, 12q23-24.3, and 17q12-22, in particular, were detected at high frequencies in both carcinoma in situ and invasive tumours and were found more often in the cases with lymph node metastases than in those without. Our previous expression profiling of squamous cell carcinomas had identified overexpression of laminin5 gamma 2, a gene located at 1q25-31. Therefore, this was investigated at the protein level by immunohistochemical analysis in 336 samples of squamous cell carcinoma of the lung. Consistent with the genomic data for this region, the expression level of laminin5 gamma 2 was higher in the primary tumours with lymph node metastases than in tumours without metastases (p = 0.012). These data suggest that gains of genes from 1q25-32, 12q23-24.3, and 17q12-22 facilitate tumorigenesis and progression of squamous cell carcinoma of the lung, and may serve as potential predictors for this disease. Copyright (c) 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

MYCN amplification and 1p36 deletion are adverse prognostic factors in neuroblastoma, and rapid accurate determination of MYCN amplification is essential for risk stratification. MYCN copy number and 1p36 deletion status were determined by fluorescence in Situ hybridization (FISH) and real time PCR in a diagnostic pathology laboratory setting on 35 consecutive patients with neuroblastoma. The PCR technique was technically Successful in all cases and results were generally available within 24 hr of biospy. There was no discordance between FISH and PCR results. Real time PCR is a reliable, accurate, and simple technique that can be applied to small neuroblastoma biopsies allowing rapid diagnosis.

t(11;18)(q21;q21) occurs specifically in mucosa-associated lymphoid tissue (MALT) lymphoma and the translocation generates a functional API2-MALT1 fusion product that activates nuclear factor (NF)kappa B. t(11;18) positive lymphomas usually lack the chromosomal aberrations and microsatellite alterations frequently seen in the translocation-negative MALT lymphomas. To further understand their genetic differences, we investigated gastric MALT lymphomas with and without t(11;18) by comparative genomic hybridisation. In general, both chromosomal gains and losses were far more frequent in t(11;18)-negative (median = 3.4 imbalances) than t(11;18)-positive cases (median = 1.6 imbalances), with gains being more frequent than losses. Recurrent chromosomal gains involving whole or major parts of a chromosome were seen for chromosomes 3, 12, 18 and 22 (23%, 19%, 19% and 27% respectively). Discrete recurrent chromosomal gains were found at 9q34 (11/26 = 42%). Bioinformatic analysis of genes mapping to 9q34 revealed potential targets. Among them, TRAF2 and CARD9 are known interaction partners of BCL10, playing a role in NF kappa B activation. Interphase fluorescent in situ hybridisation confirmed genomic gain of the TRAF2, CARD9 and MALT1 loci in 5/6 and 2/2 cases showing chromosomal gains at 9q34 and 18q21 respectively. The results further highlight the genetic difference between MALT lymphomas with and without t(11;18). Moreover, our findings suggest that genomic gain of genes that modulate NF kappa B activation, such as MALT1, TRAF2 and CARD9, may play a role in the pathogenesis of the translocation-negative MALT lymphoma.

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 >= 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.

Although chromosome translocations are well-documented recurrent events in hematological malignancies and soft tissue sarcomas, their significance in carcinomas is less clear. We report here the molecular characterization of the reciprocal translocation t( 1; 15)(p22; q22) in the prostate carcinoma cell line, LNCaP. The chromosome 1 breakpoint was localized to a single BAC clone, RP11-290M5, by sequential FISH analysis of clones selected from the NCBI chromosome 1 map. This was further refined to a 580-bp region by Southern blot analysis. A 2.85-kb fragment spanning the der( 1) breakpoint was amplified by long-range inverse PCR. The breakpoint on chromosome 1 was shown to lie between the CYR61 and the DDAH1 genes with the der( 1) junctional sequence linking the CYR61 gene to the TSPAN3 (TM4SF8) gene on chromosome 15. Confirmatory PCR and FISH mapping of the der( 15) showed loss of chromosome material proximal to the breakpoint on chromosome 15, containing the PSTPIP1 and RCN2 genes. On the available evidence we conclude that this translocation does not result in an in-frame gene fusion. Comparative expressed sequence hybridization (CESH) and comparative genomic hybridization (CGH) analysis, showed relative down-regulation of gene expression surrounding the breakpoint, but no gross change in genomic copy number. Real-time quantitative RT-PCR for genes around the breakpoint supported the CESH data. Therefore, here we may have revealed a gene down-regulation mechanism associated with a chromosome translocation, either through small deletion at the breakpoint or through another means of chromosome domain related gene regulation. Copyright (c) 2006 S. Karger AG, Basel.

Amplification and/or overexpression of genes encoding tyrosine kinase receptors KIT and ERBB2 have been reported in testicular germ cell tumors (TGCTs). These receptors can bind the adaptor molecule GRB7 encoded by a gene adjacent to ERBB2 at 17q12, a region also frequently gained in TGCTs. GRB7 binding may be involved in the activation of RAS signaling and KRAS2 maps to 12p, which is constitutively gained in TGCT and lies within a minimum overlapping region of amplification at 12p11.2-12.1, a region we have previously defined. RAS proteins activate BRAF, and activating mutations of genes encoding these proteins have been described in various tumors. Here we determine the relationships between expression levels and activating mutations of these genes in a series of 65 primary TGCTs and 4 TCGT cell lines. High levels of expression and activating mutations in RAS were mutually exclusive events, and activating mutations in RAS were only identified in the seminoma subtype. Mutations in BRAF were not identified. Increased ERBB2 expression was associated with differentiated nonseminoma histology excised from lymph nodes postchemotherapy. Mutation, elevated expression, and correlations between expression levels of KRAS2, GRB7, and KIT are consistent with their involvement in the development of TGCTs.

Amplification of the transcription factor MYCN is an important molecular diagnostic tool in stratifying treatment for neuroblastoma. Increased copy number and overexpression of MYCN in the pediatric cancer rhabdomyosarcoma has been described in a number of small studies with conflicting conclusions about its association with clinicopathologic characteristics. We aimed to study the phenomenon in the largest series to date.

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.

In a strategy aimed at identifying novel markers of human prostate cancer, we performed expression analysis using microarrays of clones randomly selected from a cDNA library prepared from the LNCaP prostate cancer cell line. Comparisons of expression profiles in primary human prostate cancer, adjacent normal prostate tissue, and a selection of other (nonprostate) normal human tissues, led to the identification of a set of clones that were judged as the best candidate markers of normal and/or malignant prostate tissue. DNA sequencing of the selected clones revealed that they included 10 genes that had previously been established as prostate markers: NKX3.1, KLK2, KLK3 (PSA), FOLH1 (PSMA), STEAP2, PSGR, PRAC, RDH11, Prostein and FASN. Following analysis of the expression patterns of all selected and sequenced genes through interrogation of SAGE databases, a further three genes from our clone set, HOXB13, SPON2 and NCAM2, emerged as additional candidate markers of human prostate cancer. Quantitative RT-PCR demonstrated the specificity of expression of HOXB13 in prostate tissue and revealed its ubiquitous expression in a series of 37 primary prostate cancers and 20 normal prostates. These results demonstrate the utility of this expression-microarray approach in hunting for new markers of individual human cancer types.

The genotype of a tumor determines its biology and clinical behavior. The genetic alterations associated with the unique embryonal morphology of nonseminomatous subtypes of testicular germ cell tumors remain to be established. Using single nucleotide polymorphism microarray analysis, we found in all of the 15 nonseminomas analyzed, large-scale chromosomal homozygosities, most of which were not associated with relative chromosome loss. This unusual genotype, distinguishing nonseminoma from seminomas and other human tumors, may be associated with the special embryonal development morphologic transition of this malignancy. Based on these genetic data, we hypothesized a new potential origin of nonseminomas through sperm fusion. Nonrandom involvement of certain chromosomes also suggests that genes on these chromosome regions may play an important role in nonseminoma development.

We carried out multiplex fluorescence in situ hybridization (M-FISH) and follow-up FISH studies on a large series of transitional cell carcinoma (TCC) cell lines and 2 normal. urothelium-derived cell lines, several of which have not had karyotypes reported previously. M-FISH analysis, with appropriate follow-up, complements conventional cytogenetic analysis and array CGH studies, allowing a more accurate definition of karyotype. The detailed karyotypic data obtained will assist in choosing suitable cell lines for functional studies and identifies common losses, gains, breakpoints; and potential fusion gene sites in TCC. We have shown changes in cell lines RT112 and DSH1 following prolonged culture, and differences in karyotype, between RT112 cultures obtained from different sources. We propose a model for the evolutionary changes leading to these differences. A comparison with the literature found other examples of differences in cell-line karyotypes between different sources. Nevertheless, several karyotypic changes were preserved between different sources of the same cell line and were also seen in more than one cell line. These may be the most important changes and include -8p, +20, 4q-, 10p-, 16p- and breaks in 8p21. We carried out a more detailed follow-up of some regions, which showed involvement of 8p breaks and losses in 15 of 16 TCC cell lines but in neither of the normal urothelium-derived cell lines. Some changes represented distal loss, whereas others were small deletions. Further study of this region is warranted. Supplementary material for this article can be found on the Genes, Chromosomes and Cancer website at http://www.interscience.wiley.com/jpages/1045-2257/suppmat/index.html. (c) 2005 Wiley-Liss, Inc.

Testicular germ-cell tumors (TGCTs) show exquisite sensitivity to cisplatin-based chemotherapy, and therefore this is considered a good model system for studying the mechanism of chemotherapy resistance. Although the genetic alterations related to TGCT have been well studied, little is known about the genetic basis of chemotherapy resistance, which occurs in a small proportion of TGCTs. In this study, we investigated genomic and expression differences between three cisplatin-sensitive and their paired cisplatin-resistant lines using combined whole-genome screen approaches. Comparative genomic hybridization (CGH) analysis on chromosomes revealed genetic differences between the resistant and parent cell lines in each pair, but did not show any consistent chromosome changes in all three lines. Microarray CGH analysis generated some additional information of DNA copy number gains and losses including some important oncogenes, tumor-suppressor genes, and drug-resistance-related genes. However, no consistent genomic region changes were found in the three cell lines. Interestingly, when comparative expressed sequence hybridization, a technique for gene expression profiling along chromosomes, was applied, we discovered a consistently overexpressed chromosomal region in all three resistant lines compared with their parent lines. The minimum overlapping chromosomal region is at 16q22-23. Further definition of genes in this chromosomal region will aid our understanding of the mechanism of cisplatin resistance and may offer novel therapeutic targets. (c) 2005 Wiley-Liss, Inc.

Metastasis is a major factor associated with poor prognosis in cancer, but little is known of its molecular mechanisms. Although the clinical behavior of soft tissue sarcomas is highly variable, few reliable determinants of outcome have been identified. New markers that predict clinical outcome, in particular the ability of primary tumors to develop metastatic tumors, are urgently needed. Here, we have chosen leiomyosarcoma as a model for examining the relationship between gene expression profile and the development of metastasis in soft tissue sarcomas. Using cDNA microarray, we have identified a gene expression signature associated with metastasis in sarcoma that allowed prediction of the future development of metastases of primary tumors (Kaplan-Meier analysis P = 0.001). Our finding may aid the tailoring of therapy for individual sarcoma patients, where the aggressiveness of treatment is affected by the predicted outcome of disease.

Identifying changes in DNA copy number can pinpoint genes that may be involved in tumor development. Here we have defined the smallest overlapping regions of imbalance (SORI) in testicular germ cell tumors other than the 12p region, which has been previously investigated. Definition of the regions was achieved through comparative genomic hybridization (CGH) analysis of a 4559 cDNA clone microarray. A total of 14 SORI were identified, which involved at least five of the 11 samples analysed. Many of these refined regions were previously reported using chromosomal or allelic imbalance studies. The SORI included gain of material from the regions 4q12, 17q21.3, 22q11.23 and Xq22, and loss from 5q33, 11q12.1, 16q22.3 and 22q11. Comparison with parallel chromosomal CGH data supported involvement of most regions. The various SORI span between one and 20 genes and highlight potential oncogenes/tumor suppressor genes to be investigated further.

Germ-line mutations in the serine-threonine kinase gene STK11 (LKB1) cause Peutz-Jeghers syndrome (PJS), a rare autosomal dominantly inherited disease, characterized by hamartomatous polyposis and mucocutaneous pigmentation. STK11 mutations only account for about half of PJS cases, and a second disease locus has been proposed at chromosome segment 19q13.4 on the basis of genetic linkage analysis in one family. We identified a t(11;19)(q13;q13.4) in a PJS polyp arising from the small bowel in a female infant age 6 days. Because the breakpoint in 19q13.4 may disrupt the putative PJS disease gene mapping to this region, we mapped the breakpoint and analyzed DNA from the case and a series of STK11-negative PJS cases. Using two-color interphase fluorescence in situ hybridization, the breakpoint region was refined to a 0.5-Mb region within 19q13.4. Eight candidate genes mapping to the breakpoint region-U2AF2, EPN1, NALP4, NALP11, NALP5, ZNF444, PTPRH, and KIAA1811-were screened for mutations in germ-line and polyp DNA from the case and from 15 PJS cases that did not harbor germ-line STK11 mutations. No pathogenic mutations in the candidate genes were identified. This report provides further evidence of the existence of a second PJS disease locus at 19q13.4 and excludes involvement of eight candidate genes. (C) 2004 Wiley-Liss, Inc.

Gain of 12p material is invariably associated with testicular germ cell tumors (TGCTs) of adolescents and adults, most usually as an isochromosome 12p. We analyzed TGCTs with i(12p) using a global approach to expression profiling targeting chromosomes (comparative expressed sequence hybridization, CESH). This indicated overexpression of genes from 12p11.2-p12.1 relative to testis tissue and fibroblasts. The nonseminoma subtype showed higher levels of expression than seminomas. Notably, 12p11.2-p12.1 is amplified in about 10% of TGCTs and CESH analysis of such amplicon cases showed high levels of overexpression from this region. Microarray analysis, including cDNA clones representing most UniGene clusters from 12p11.2-p12.1, was applied to DNA and RNA from 5 TGCTs with amplification of 12p11.2-p12.1 and seven TGCTs with gain of the entire short arm of chromosome 12. Expression profiles were consistent with the CESH data and overexpression of EST595078, MRPS35 and LDHB at 12p11.2-p12.1 was detected in most TGCTs. High-level overexpression of BCAT1 was specific to nonseminomas and overexpression of genes such as CMAS, EKI1, KRAS2, SURB7 and various ESTs correlated with their amplification. Genes such as CCND2, GLU3, LRP6 and HPH1 at 12p13 were also overexpressed. The overexpressed sequences identified, particularly those in the region amplified, represent candidate genes for involvement in TGCT development.

Rhabdomyosarcomas (RMS) are soft tissue sarcomas resembling developing skeletal muscle, and pleomorphic rhabdomyosarcomas (PRMS) are a rare nonpediatric entity. Little molecular cytogenetic information exists for PRMS, and their relationship to other subtypes of rhabdomyosarcoma and other sarcomas is unclear. Chromosomal imbalances were determined in seven well-characterized cases of PRMS using comparative genomic hybridization. The smallest overlapping regions of gain were 1p22 approximately p33 (71%), 7p (43%), 18/18q (43%), and 20/20p (43%), and the regions of loss were 10q23 (71%), 15q21 approximately q22 (57%), 3p, 5q32 approximately qter, and 13 (all 43%). Four of the seven cases had amplicons involving the regions 1p21 approximately p31, 1q21 approximately q25, 3p12, 3q26 approximately qtel, 4q28 approximately q31, 8q21 approximately q23/8q, and 22q. These regions are distinct from those frequently associated with the alveolar subtype, whereas the embryonal subtype without anaplasia is rarely associated with amplification events other than gain/amplification of 8q material. The regions of imbalance appeared more similar to those reported for malignant fibrous histiocytomas (MFH) and osteosarcomas, consistent with the suggestion that PRMS can be considered part of the spectrum of MFH. In addition, one of the cases classified as PRMS showed evidence for the presence of a PAX3-FOXO1A fusion gene, which is characteristic of the alveolar subtype of RMS.

WT1 encodes a tissue-specific transcription factor important in early mesenchymal differentiation. Altered expression or mutation of WT1 occurs in malignancies derived from such tissues. These include Wilms tumour, a paediatric kidney cancer that may show heterologous differentiation into primitive skeletal muscle, especially in tumours with WT1 mutation. A putative role for WT1 in inhibiting myogenesis has been suggested by transient transfection of C(2)C(12) myoblasts. However, using a more robust model of stable transfectants of C(2)C(12) expressing inducible WT1 isoforms, we found no inhibition of myogenic differentiation. We also investigated a possible role for WT1 in the disrupted myogenesis seen in rhabdomyosarcoma, a paediatric cancer resembling foetal skeletal muscle. WT1 expression levels measured by quantitative real-time reverse transcription polymerase chain reaction were low or absent in those tumours with a PAX-FKHR fusion gene characteristic of the alveolar subtype, and were higher in cases lacking these fusion genes. Overall, there was a weak positive correlation between expression of myogenic differentiation marker genes and WT1 levels. We conclude that expression of WT1 in C(2)C(12) cells and in rhabdomyosarcoma does not inhibit myogenic differentiation.

Rhabdomyosarcoma (RMS) is a common paediatric soft tissue sarcoma that resembles developing foetal skeletal muscle. Tumours of the alveolar subtype frequently harbour one of two characteristic translocations that juxtapose PAX3 or PAX7, and the forkhead-related gene FKHR (FOXO1A). The embryonal subtype of RMS is not generally associated with these fusion genes. Here, we have quantified the relative levels of chimaeric and wild-type PAX transcripts in various subtypes of RMS (n=34) in order to assess the relevance of wild-type PAX3 and PAX7 gene expression in these tumours. We found that upregulation of wild-type PAX3 is independent of the presence of either fusion gene and is unlikely to contribute to tumorigenesis. Most strikingly, upregulated PAX7 expression is almost entirely restricted to cases without PAX3-FKHR or PAX7-FKHR fusion genes and may contribute to tumorigenesis in the absence of chimaeric PAX transcription factors. Furthermore, as myogenic satellite cells are known to express PAX7, this pattern of PAX7 expression suggests this cell type as the origin of these tumours. This is corroborated by the detection of MET (c-met) expression, a marker for the myogenic satellite cell lineage, in all RMS samples expressing wild-type PAX7.

Tumors are associated with altered or deregulated gene products that affect critical cellular functions. Here we assess the use of a global expression profiling technique that identifies chromosome regions corresponding to differential gene expression, termed comparative expressed sequence hybridization (CESH). CESH analysis was performed on a total of 104 tumors with a diagnosis of rhabdomyosarcoma, leiomyosarcoma, prostate cancer, and favorable-histology Wilms tumors. Through the use of the chromosome regions identified as variables, support vector machine analysis was applied to assess classification potential, and feature selection (recursive feature elimination) was used to identify the best discriminatory regions. We demonstrate that the CESH profiles have characteristic patterns in tumor groups and were also able to distinguish subgroups of rhabdomyosarcoma. The overall CESH profiles in favorable-histology Wilms tumors were found to correlate with subsequent clinical behavior. Classification by use of CESH profiles was shown to be similar in performance to previous microarray expression studies and highlighted regions for further investigation. We conclude that analysis of chromosomal expression profiles can group, subgroup, and even predict clinical behavior of tumors to a level of performance similar to that of microarray analysis. CESH is independent of selecting sequences for interrogation and is a simple, rapid, and widely accessible approach to identify clinically useful differential expression.

Genetic instability resulting in chromosome aneuploidy or mismatch repair deficiency characterizes cancer. Medullary carcinoma (MC) of the breast is a specific form of breast cancer with unique clinical, epidemiologic, and prognostic features, suggesting distinctive tumorigenic pathways. To investigate the nature of the genetic changes associated with MC we analyzed a series of 22 tumors. Chromosomal imbalances were assessed by comparative genomic hybridization (CGH) and mismatch repair (MMR) deficiency tested for through assessment of microsatellite instability (MSI) and expression of MLH1 and MSH2 genes. MMR deficiency was detected in only a small proportion of cases. The chromosomal copy number changes showed some similarities to BRCA1-associated tumors. A high level of BRCA1 promoter hypermethylation was detected, suggesting a possible role of this gene in MC development.

Leiomyosarcomas of soft tissues are an aggressive group of tumors with a high incidence of recurrence. Little is known about the molecular genetic changes associated with clinical outcome. Therefore, we studied 28 leiomyosarcoma samples of similar grade using comparative genomic hybridization and DNA flow cytometry and identified a difference in survival time associated with ploidy status and the number of chromosomal aberrations. The average survival time was shown to decrease with increase in chromosomal aberrations identified using comparative genomic hybridization. The average survival time was shorter in the near-tetraploid group than in the diploid and triploid group. Gain of 5p14-pter was significantly more common in near-tetraploid tumors. The survival time of patients with near-tetraploidy together with gain of 5p14-pter was reduced, and 50% died within the 1st year. Furthermore, loss of 13q14-q21 was significantly more frequent in the <5-year than in the >5-year survival group (P =.01). These results suggest that 13q14-q21 loss and 5p14-pter gain at diagnosis could be used to identify patients with leiomyosarcoma who are likely to have a shorter survival time and who might benefit from early treatment intensification.

We present the results of a comparative genomic hybridization (CGH) analysis of three meningeal solitary fibrous tumors (SFT). One case showed loss of chromosome 3 and two tumors had deletions of the legion 3p21similar top26. Other chromosomal losses included 4p15, 8q22similar toq24, 10, 11q14similar toq25, 17q11similar toq23. 20 and 21 in one case each. In addition there were gains of 18p11similar top13 in one case, and 1p11similar top36 and 20q11similar toq13 in another. To our knowledge there are no previous CGH or cytogenetic data on meningeal SFT and loss of material on chromosome 3 has not been described in SFT at other sites. Our findings are discussed in relation to published molecular genetic and cytogenetic data on meningioma and hemangiopericytoma, the two lesions with which meningeal SFT are most likely to be confused. (C) 2002 Elsevier Science Inc. All rights reserved.

Microarray analysis using sets of known human genes provides a powerful platform for identifying candidate oncogenes involved in DNA amplification events but suffers from the disadvantage that information can be gained only on genes that have been preselected for inclusion on the array. To address this issue, we have performed comparative genome hybridization (CGH) and expression analyses on microarrays of clones, randomly selected from a cDNA library, prepared from a cancer containing the DNA amplicon under investigation. Application of this approach to the BT474 breast carcinoma cell line, which contains amplicons at 20q13, 17q11-21, and 17q22-23, identified 50 amplified and expressed genes, including genes from these regions previously proposed as candidate oncogenes. When considered together with data from microarray expression profiles and Northern analyses, we were able to propose five genes as new candidate oncogenes where amplification in breast cancer cell lines was consistently associated with higher levels of RNA expression. These included the HB01 histone acetyl transferase gene at 17q22-23 and the TRAP100 gene, which encodes a thyroid hormone receptor-associated protein coactivator, at 17q11-21. The results demonstrate the utility of this microarray-based CGH approach in hunting for candidate oncogenes within DNA amplicons.

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.

Gene-expression patterns have proved useful for tumour classification and prediction of prognosis. We investigated a new gene-expression profiling method that targets individual chromosomes--comparative expressed sequence hybridisation (CESH)--to establish if any expression pattern was associated with relapse, and if this pattern could accurately predict outcome. We studied 18 cases of Wilms' tumour with favourable histological findings. Relative overexpression of genes on the long arm of chromosome 1 was shown in all tumours that subsequently relapsed but in none of those from patients in remission (odds ratio 357, 95% CI 6.4-19960). This finding shows the value of CESH analysis to rapidly screen for chromosomal regions that carry genes whose differential expression is important in establishment of tumour behaviour.

A subset of B cell chronic lymphocytic leukaemia (CLL) is familial. Lack of large families makes it attractive to exploit methods in addition to genetic linkage analysis for the identification of a susceptibility locus. One strategy that can localise regions of the genome that may harbour tumour suppressor genes is to identify regions of chromosomal imbalance using comparative genomic hybridisation (CGH) analysis. We examined 24 familial CLL cases by CGH analysis. Losses that are documented as arising frequently in sporadic CLL were observed at a comparable frequency in familial CLL. However, gains and losses in two regions of the X chromosome - Xp11.2-p21 and Xq21-qter - appear more common in familial CLL than in sporadic CLL. This suggests these regions may harbour a susceptibility locus for CLL. There is also some evidence that chromosome regions 2p12-p14 and 4q11-q21 may harbour predisposition genes.

Synovial sarcomas are aggressive spindle cell sarcomas containing in some cases areas of epithelial differentiation. They consistently show, a specific t(X;18;p11;q11), which usually represents either of two gene fusions, SYT-SSX1 or SYT-SSX2, encoding putative transcriptional proteins differing at 13 amino acid positions. Previous studies have suggested that patients with SYT-SSX2 tumors do better than those with SYT-SSX1 tumors, but the study groups were too limited to be conclusive. To address this issue more definitively, we collected data on SYT-SSX fusion type, pathology, and clinical course in a retrospective multi-institutional study of 243 patients (age range, 6-82) with synovial sarcoma. SYT-SSX] and SYT-SSX2 fusions were detected in 147 tumors (61 %) and 91 tumors (37%), respectively. Histologically, 61 (25%) were classified as biphasic type and 180 (74 %) as monophasic type based on the presence or absence of areas of glandular epithelial differentiation, respectively. Median and 5-year overall survivals for the SYT-SSX] and SYT-SSX2 groups were 6.1 years and 53%, and 13.7 years and 73%, respectively. Overall survival was significantly better among SYT-SSX2 cases (P = 0.03), among cases localized at diagnosis (P < 0.0001), and among patients with primary tumors < 5 cm in greatest dimension (P = 0.01). Age, Sex, histological type, and axial versus peripheral primary site had no impact on overall survival. The impact of fusion type on survival remained significant when stratified for primary tumor size (P = 0.03) but was no longer significant when stratified for disease status at presentation. This may reflect the tendency for patients with SYT-SSX1 tumors to present more often with metastatic disease (P = 0.05). Cox regression identified disease status (P < 0.0001) and primary tumor size (P = 0.04) as the only factors independently predictive of overall survival in the subset of 160 patients with information on all of the factors. Within the subset of patients with localized disease at diagnosis (n = 202), the median and 5-year survival for the SYT-SSX] and the SYT-SSX2 groups were 9.2 years and 61% versus 13.7 years and 77%, respectively. Patients whose tumors contained the SYT-SSX2 fusion (P = 0.08) or were smaller (P = 0.12) showed a trend toward better survival by log-rank test, whereas tumor histology had no impact (P = 0.8). In a Cox regression analysis considering all of the factors, SYT-SSX fusion type emerged as the only independent significant factor (P = 0.04) for overall survival within the subset of 133 patients with localized disease at diagnosis who had information on all of the factors. Among other comparisons, there was a strong association of fusion type and morphology (P < 0.001), with almost all of the SYT-SSX2 tumors showing absence:of glandular differentiation (monophasic histology) and almost all of the biphasic tumors containing SYT-SSX]. There was also a statistically significant association of fusion type and patient sex (P = 0.03); specifically, the male:female ratio of SYT-SSX1 cases was 1: 1, whereas for SYT-SSX2 cases, it was close to 1:2. Overall, SYT-SSX fusion type appears to be the single most significant prognostic factor by multi-variate analysis in patients with localized disease at diagnosis. SYT-SSX fusion type also appears to exert part of its impact on prognosis before presentation through its association with stage at diagnosis. In addition, the associations of SYT-SSX fusion type with patient sex and tumor epithelial differentiation point to interesting mechanistic biological differences.

Germline mutations in the fumarate hydratase gene at 1q43 predispose to dominantly inherited skin and uterine leiomyomata and leiomyosarcomas. The enzyme, which is a component of the tricarboxylic acid cycle, acts as a tumour suppressor. To evaluate fumarate hydratase in respective sporadic tumours, we analysed a series of 26 leiomyosarcomas and 129 uterine leiomyomas (from 21 patients) for somatic mutations in fumar-ate hydratase and allelic imbalance around 1q43. None of the 26 leiomyosarcomas harboured somatic mutations in fumarate hydratase, Fifty per cent of leiomysarcomas tested showed evidence of allelic imbalance at 1q, but this was not confined to the vicinity of fumarate hydratase. Only 5% (seven out of 129) of the leiomyomas showed allele imbalance at 1q42-q43 and no somatic mutations in fumarate hydratase were observed. Our findings indicate that mutations in fumarate hydratase do not play a major role in the development of sporadic leiomyosarcomas or uterine leiomyomas. (C) 2002 Cancer Research UK.

A recent large multi-centre study convincingly confirmed previous observations that the SYT-SSX1 fusion type, compared to SYT-SSX2, of synovial sarcoma is associated with a worse clinical outcome. Apart from the clinical impact, this fact also suggests (1) that the SYT-SSX fusion gene may influence molecular mechanisms involved in tumour growth and progression; and (2) that the SYT-SSX1 fusion type has a stronger influence on these mechanisms than SYT-SSX2. The nature of the underlying mechanisms is, however, still unknown. In this study we made use of the SYT-SSX1 vs SYT-SSX2 concept to investigate whether major, tumour relevant, and growth regulatory proteins (e.g. cyclins and cyclin-dependent kinases) may be involved. Using Western blotting analysis on 74 fresh, fusion variant-typed, tumour samples from localized synovial sarcoma, we found a significant correlation between SYT-SSX1 and high expression of cyclin A (P=0.003) and D1 (P=0.025). Our data suggest that SYT-SSX may influence the cell cycle machinery, and that the more aggressive phenotype of the SYT-SSX1 variant is due to an accelerated tumour cell proliferation.

Synovial sarcoma occurs predominantly in the paraarticular regions of the extremities. Synovial sarcoma of the mediastinum is an exceedingly rare neoplasm that has overlapping histologic and immunophenotypic features with other tumors in the differential diagnosis. We describe two cases. One is a 67-year-old patient who presented with chest pain and shortness of breath. Diagnostic imaging revealed a mediastinal mass extending over the cardiac apex. Histopathology, immunohistochemistry, and molecular genetic analysis confirmed the diagnosis of synovial sarcoma. The patient underwent surgical resection and postoperative radiation therapy. He is alive and well 18 months after diagnosis. This case illustrates the importance of proper procurement of frozen tissue for molecular genetic analysis for the identification of the t(X;18), characteristic of synovial sarcoma. Detection of this translocation is of paramount importance to confirm this diagnosis, particularly when this neoplasm arises in atypical locations outside the extremities. (C) 2002 by The Society of Thoracic Surgeons.

We describe a case of leukemic mantle cell lymphoma (MCL) with complex karyotype and amplification of the CCND1/IGH fusion gene. Testing for the presence of t(11;14), the hallmark of MCL, revealed multiple copies of the fusion signals. We therefore conducted extensive molecular cytogenetic studies to delineate the nature and consequences of such an abnormality. We localized the amplification to the der(14)t(11;14) and to a der(2) chromosome in a form of interspersed chromosome 11 and 14 material. This resulted in high expression of cyclin D1 mRNA and the protein expressed independently of the cell cycle phase. CGH analysis revealed that the overrepresentation on chromosome 11 included chromosomal band 11q23 in addition to the CCND1 locus at 11q13. The band 11q23 harbors the ataxia telangiectasia mutated (ATM) gene recently proposed to be involved in the pathogenesis of MCL with high incidence of deletions in this locus. Using YAC 801e11, containing the ATM gene, we demonstrated several hybridization signals, suggesting that this region also formed part of the amplicon. This case also showed TP53 gene abnormalities: protein expression, monoallelic deletion, and a mutation in exon 5. The clinical course was aggressive, and the patient died within 6 months of presentation. This is to our knowledge the first description of amplification of the CCND1/IGH fusion gene in a human neoplasm, which may have played a role in the fulminating course of the disease in this patient. (C) 2002 Wiley-Liss, Inc.

Altered expression of genes can have phenotypic consequences in cancer development and treatment, developmental abnormalities, and differentiation processes. Here we describe a rapid approach, termed comparative expressed sequence hybridization (CESH), which gives a genome-wide view of relative expression patterns within tissues according to chromosomal location. No prior knowledge of genes or cloning is required, and minimal amounts of tissue can be used. Expression profiles are achieved in a manner similar to the identification of chromosomal imbalances by comparative genomic hybridization analysis. The approach is demonstrated to indicate a chromosomal region that harbors overexpressed genes that may be associated with a drug-resistant phenotype. In addition, known and new regions of differential gene expression in both normal tissues and tumor samples from the soft tissue sarcoma group of rhabdomyosarcoma (RMS) are indicated. These regions included 2p24; overexpression of MYCN at 2p24 was confirmed by quantitative reverse transcription-PCR for all of the alveolar RMS cases and did not necessarily correspond to genomic amplification. Evidence including region specific microarray analysis indicated that overexpression of several genes from a region may be required for detection by CESH. This evidence is consistent with clusters of functionally related genes and mechanisms that affect the expression of a number of genes at a particular genomic location. The distinctive CESH profiles demonstrated in different subtypes of RMS show potential for tumor classification.

Although several genes/genetic loci involved in the etiology of Wilms' tumor have been identified, little is known of the molecular changes associated with relapse. We therefore undertook an analysis by comparative genomic hybridization (CGH) of 58 tumor samples of favorable histology Wilms' tumor taken at initial diagnosis and/or relapse. Tumors with anaplastic histology were excluded as this is known to be associated with p53 mutation and a poor prognosis. A control group of 21 Wilms' tumors that did not relapse was also analyzed. The overall frequency of gains or losses of genetic material detected by CGH was similar in both groups (77% in relapsing tumors and 70% in the nonrelapse group) as was the median number of changes per tumor (relapse group: n = 4, range, 1 to 19; nonrelapse group: n = 3, range, 1 to 8). However, gain of 1q was significantly more frequent in the relapse series [27 of 46 (59%) versus 5 of 21 (24%), P: = 0.019]. In 12 matched tumor pairs, the CGH profiles, including 1q gain, were similar at diagnosis and relapse, with little evidence for further copy number changes being involved in clonal evolution. The results suggest that 1q gain at diagnosis could be used to identify patients with favorable histology Wilms' tumor at increased risk of relapse who might benefit from early treatment intensification.

Rhabdomyosarcomas are the most common type of pediatric soft tissue sarcoma. The cytogenetic literature on RMS is biased towards the less common alveolar subtype (ARMS), which is frequently associated with specific translocations and the PAX3/7-FKHR fusion genes. Relatively few karyotypes are reported for the embryonal subtype (ERMS). The aim of this study was to further cytogenetic knowledge of RMS subtypes.

Rhabdomyosarcoma has 2 major histological subtypes, embryonal and alveolar. Alveolar histology is associated with the fusion genes PAX3-FKHR and PAX7-FKHR. Definition of alveolar has been complicated by changes in terminology and subjectivity. It is currently unclear whether adverse clinical behaviour is better predicted by the presence of these fusion genes or by alveolar histology. We have determined the presence of the PAX3/7-FKHR fusion genes in 91 primary rhabdomyosarcoma tumours using a combination of classical cytogenetics, FISH and RT-PCR, with a view to determining the clinical characteristics of tumours with and without the characteristic translocations. There were 37 patients with t(2;13)/PAX3-FKHR, 8 with t(1;13) PAX7-FKHR and 46 with neither translocation. One or other of the characteristic translocations was found in 31/38 (82%) of alveolar cases. Univariate survival analysis revealed the presence of the translocation t(2;13)/PAX3-FKHR to be an adverse prognostic factor. With the difficulties in morphological diagnosis of alveolar rhabdomyosarcoma on increasingly used small needle biopsy specimens, these data suggest that molecular analysis for PAX3-FKHR will be a clinically useful tool in treatment stratification in the future. This hypothesis requires testing in a prospective study. Variant t(1;13)/PAX7-FKHR appears biologically different, occurring in younger patients with more localised disease.

Many of the reported karyotypes for adult testicular germ cell tumors (GCTs) are complex and incomplete, although the presence of an isochromosome 12p, i(12p), and gain of 12p material have consistently been found. Here, an accurate definition of the chromosome aberrations associated with four cell lines derived from GCTs (GCT27, H12.1, Tera1, and Tera2) has been produced using 24-color karyotyping by mulifluor in situ hybridization, comparative genomic hybridization analysis, and further fluorescence in situ hybridization analysis to confirm some chromosomal assignments and refine involvement of specific regions of 12p. There was karyotypic heterogeneity. Isochromosomes in addition to i(12p) were found, as were other rearrangements with breakpoints at or near centromeric regions. The most frequent non-centromeric breakpoints were at 1p31 approximately p32, 1p21 approximately p22, 11q13, and Xq22, although consistent partner chromosomes were not involved. One cell line (Tera1) showed a subtle dosage increase in the copy number of a 12p probe known to be within the smallest overlapping region of amplification that has been defined in a number of testicular GCTs with amplicons at 12p11 approximately p12. The chromosome rearrangements and associated imbalances may be significant in GCT progression and the characterized cell lines can be used to investigate these further.

Rhabdomyosarcoma in children is a "small round blue cell tumour" that displays skeletal muscle differentiation. Two main histological variants are recognised, alveolar (ARMS) and embryonal (ERMS) rhabdomyosarcoma. Whereas consistent chromosome translocations characteristic of ARMS have been reported, no such cytogenetic abnormality has yet been described in ERMS. We have used multiple colour chromosome painting to obtain composite karyotypes for five ERMS cell lines and one PAX-FOXO1A fusion gene negative ARMS. The cell lines were assessed by spectral karyotyping (SKY), tailored multi-fluorophore fluorescence in situ hybridisation (M-FISH) using series of seven colour paint sets generated to examine specific abnormalities, and comparative genomic hybridisation (CGH). This approach enabled us to obtain karyotypes of the cell lines in greater detail than previously possible. Several recurring cytogenetic abnormalities were demonstrated, including translocations involving chromosomes 1 and 15 and chromosomes 2 and 15, in 4/6 and 2/6 cell lines respectively. All six cell lines demonstrated abnormalities of chromosome 15. Translocations between chromosomes 1 and 15 have previously been recorded in two primary cases of ERMS by conventional cytogenetics. Analysis of the translocation breakpoints may suggest mechanisms of ERMS tumourigenesis and may enable the development of novel approaches to the clinical management of this tumour.

Mutations in the ATM gene, which maps to 11q22-23, cause the multisystem recessive syndrome ataxia-telangiectasia (AT). Breast cancer has been reported in AT patients and carriers. Sporadic breast cancer is associated with loss of heterozygosity at or in the region of ATM and chromosomal abnormalities involving 11q23. We have investigated the chromosomes, nuclei and released chromatin fibers from nine primary breast carcinoma and eight cell lines by fluorescence in situ hybridization with four fluorochrome-labeled cosmids spanning the ATM gene. The ATM gene was disrupted in one primary breast carcinoma and in the cell lines MDA-MB-231 and MCF-7. The role of these aberrations in breast carcinomas, which may lead to gene dosage or dominant negative effects on gene function, requires further investigation.

Carcinoma in situ (CIS) or intratubular germ cell neoplasia is generally considered the precursor lesion of adult testicular germ cell tumours (TGCT). The chromosomal imbalances associated with CIS and the corresponding seminoma (SE) or nonseminoma (NS) have been determined by comparative genomic hybridization (CGH) analysis of microdissected material from seven cases. Significantly, the CIS showed no gain of 12p material whereas in the invasive components of all cases gain of 12p was found, in 2 cases associated with amplification of the 12p11.2-12.1 region. Interphase fluorescence in situ analysis was consistent with this and provided evidence for the i(12p) or 12p11.2-12.1 amplification in the SE and NS but not in the corresponding CIS. This suggests a role for these changes in progression of CIS to invasive testicular cancer or progression of the invasive disease. Other imbalances such as gain of material from chromosomes 1, 5, 7, 8, 12q and X and loss of material from chromosome 18 were frequently identified (> 40% of cases) in the CIS associated with both SE and NS as well as in the invasive components. Loss of material from chromosome 4 and 13 and gain of 2p were more frequently found in the invasive components. The results shed light on the genetic relationship between the non-invasive and invasive components of testicular cancer and the stage at which particular chromosomal changes may be important.

Data on the chromosome aberrations associated with leiomyosarcomas of soft tissues are limited. complex. and incomplete. The aim of this study was to characterize genetic aberrations associated with this tumor group, to identify consistent regions of involvement and to determine correlations with clinical outcome. Chromosomes were prepared from 10 primary soft-tissue leiomyosarcoma samples, and preparations from four of them, plus the cell line SK-LMS-I, were suitable for analysis using 24-color karyotyping by multifluor fluorescence in situ hybridization. This method allowed rearranged chromosomes to be characterized, which would not have been possible by banding analysis alone. The remaining six chromosome preparations were analyzed using standard Giemsa banding. The chromosome imbalances associated with all the samples were determined by comparative genomic hybridization analysis. Taken together, the results show both intra- and intertumor heterogeneity and considerable complexity. Although no highly consistent rearrangements were found, some regions of the genome frequently were involved. including 1q21, 5p14-pter, and 20q13, which likely harbor genes that play a role in the pathogenesis of soft-tissue leiomyosarcomas. There were no obvious correlations between the chromosomal changes identified and available clinical details. (C) 2001 Wiley-Liss, Inc.

Although there seems to be a common stem cell for the two epithelial cell types in the breast, the Vast majority of breast cancers exhibit a luminal phenotype. Pure myoepithelial carcinomas are rare. We report our findings of genetic alterations in these tumors. We have analyzed 10 cases of pure myoepithelial cell carcinomas using laser capture microdissection and comparative genomic hybridization. The mean number of changes was 2.1 (range 0-4), compared with a mean of 8.6 (range 3.6-13.8) in unselected ductal carcinomas. Common alterations included loss at 16q (3/10 cases), 17p(3/10), 11q (2/10), and 16p (2/10), regions also commonly deleted in ductal carcinomas. The single case in which both pure myoepithelial carcinoma and invasive ductal carcinoma was present showed 2 alterations in the myoepithelial tumor (losses at 17p and 17q), whereas the invasive ductal component showed 15 alterations (5 gains and 9 losses), including loss at 17p. The sharing of 17p loss in myoepithelial and ductal carcinoma is consistent with a common stem cell model in the breast. The relatively few genetic alterations in otherwise aggressive neoplasms suggests that myoepithelial tumors may be a good model for the delineation of genes important in breast tumorigenesis.

Rhabdomyosarcomas are the most common soft-tissue sarcoma found in children. The alveolar subtype is clinically more aggressive than the embryonal subtype. In addition to the presence of specific chromosome translocations and associated fusion gene products in a high proportion of the alveolar subtype, we previously showed that tumors with this histology frequently show evidence of genomic amplification. Here, we substantially extended the number of alveolar rhabdomyosarcoma samples examined by comparative genomic hybridization analysis. Regions of loss were noted, including the smallest overlapping regions corresponding to 16q, 17/17p, and 9q32-34, in 16%, 10%, and 10% of cases, respectively (44 primary samples/6 cell lines). Amplification or gain at 12q13-15 in the region of the MDM2/GLI1/SAS/CDK4 loci and 2p24 at the MYCN locus was found in 28% and 32% of cases, respectively. Single amplicons were found at locations that in other samples showed consistent gain, including the regions 5q15-23, 7q21-31, 11p11-14, 17q23-24, and 20q13, and amplification was found in two cases at 15q24-26. However, most striking was a novel region of amplification or gain at 13q31 in 19% of cases (51 primary samples/6 cell lines). This indicates that a gene or genes at 13q31 are significant in the development or progression of alveolar rhabdomyosarcoma.

Chromosome 1 abnormalities are the most commonly detected aberrations in many cancers including malignant melanomas. Specific breakpoints are reported for malignant melanomas throughout the chromosome but especially at 1p36 and at several sites throughout 1p22-q21. In addition, partial deletions and loss of heterozygosity have been found on 1p indicating the possible location of tumor suppressor genes. Here we have characterized the involvement of chromosome 1 in a series of seven malignant melanoma cell lines. Initial chromosome painting studies revealed that six of the cell lines had chromosome 1 rearrangements. Deletions involving 1p10-32, 1q11-44, and 1q25-44 were observed. The other rearrangement breakpoints included three in the 1q10-p11 region with the rest at 1p36, 1p34, 1p32, 1p31, 1p12-13, 1q21, and 1q23. The breaks at 1q10-p11 were investigated further using an alpha-satellite 1 centromere probe and yeast artificial chromosomes (YACs) from the region. Two of the 1q10-p11 breaks mapped in the centromeric region, while the others mapped to variable sites. This suggests that the role of these rearrangements in the pathogenesis of melanomas does not involve the alteration of specific oncogenes in the breakpoint region. During the YAC mapping a previously undetected, small (<1 Mbp) del(1)(p10p11) was identified. This deletion lies within minimal overlapping deleted regions reported in head and neck as well as breast carcinomas and it could therefore facilitate the isolation of a carcinoma-associated tumor suppressor gene.

Multifluor-fluorescence in-situ hybridization (M-FISH) chromosome paints for all the chromosomes in the human complement labeled with different combinations of fluorochromes is a recent technological development enabling assignment of chromosomal material to rearranged chromosomes. Little data is available on the accuracy and limitations of the approach to the analysis of complex karyotypes, which are characteristic of many malignant diseases. Here we compare M-FISH analysis of the breast-cancer-derived cell line T-47D with a previous analysis by reverse chromosome painting analysis of flow-sorted chromosomes from the same material. This demonstrated a high degree of concordance. It also illustrated the limitations of M-FISH analysis, including difficulties identifying small regions of chromosomal material and intrachromosomal rearrangements. Confirmation of selected aberrations using less-complex mixtures of painting probes and further definition of abnormalities using single copy markers may be required. The detailed karyotype description possible by M-FISH analysis contrasts with the definition in the original G-banding analysis. This and the level of concordance with reverse FISH painting supports the utility of the approach in the definition of complex karyotypes.

Rhabdomyosarcomas are characterized by loss of heterozygosity (LOH) at chromosome region 11p15.5, a region known to contain several imprinted genes including insulin-like growth factor 2 (IGF2), H19, and p57(KIP2). We analyzed 48 primary tumour samples and found distinct genetic changes at 11p15.5 in alveolar and embryonal histological subtypes. LOH was a feature of embryonal tumours, but at a lower frequency than previous studies. Loss of imprinting (LOI) of the IGF2 gene was detected in 6 of 13 informative cases, all harbouring PAX3-FKHR or PAX7-FKHR fusion genes characteristic of alveolar histology. In contrast, H19 imprinting was maintained in 14 of 15 informative cases and the case with H19 LOI had maintenance of the IGF2 imprint indicating separate mechanisms controlling imprinting of IGF2 and H19. The adult promoter of IGF2, P1, was used in 5 of 14 tumours and its expression was unrelated to IGF2 imprinting status implying a further mechanism of altered IGF2 regulation. The putative tumour suppressor gene p57(KIP2) was expressed in 15 of 29 tumours and expression was unrelated to allele status. Moreover, in tumours with p57(KIP2) expression, there was no evidence for inactivating mutations, suggesting that p57(KIP2) is not a tumour suppressor in 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.

Identification of the t(X;18)(p11.2;q11.2) and the fusion gene products, SYT-SSX1 and SYT-SSX2, associated with a high proportion of synovial sarcomas, has been shown to be a useful diagnostic aid. This study demonstrates the application of dual colour fluorescence in situ hybridization to paraffin-embedded samples to deduce the presence of the derivative X chromosome and also the position of the breakpoint on chromosome X at either the SSX1 or the SSX2 gene. This used region specific markers from chromosomes X and 18 and an optimized protocol involving microwave exposure. Novel and rapid scoring criteria were validated which circumvented potential problems of nuclear truncation and defining cell boundaries. This involved blind analysis of two negative sarcoma samples and three synovial sarcomas in which corresponding frozen material had been previously shown to have the translocation involving different SSX genes. Six new cases diagnosed as synovial sarcoma were also analysed; two monophasic and two biphasic case were deduced to have a breakpoint in the SSX1 gene, one monophasic case an SSX2 breakpoint, and one case did not show rearrangement of the region. The ability to analyse formalin-fixed, paraffin-embedded samples in this way has practical implications for aiding the diagnosis of difficult cases, recently ascribed prognostic relevance, and allows further retrospective studies to be carried out. The methodology is also applicable to the identification of other tumour specific translocations in paraffin-embedded material.

The ZNF198- FGFR1 fusion gene arises as a result of the t(8;13)(p11;q12) in the 8p11 myeloproliferative syndrome. To determine the transforming properties of this chimeric protein we transfected ZNF198-FGFR1 into the interleukin (IL)-3 dependent cell line Ba/F3. Growth factor independent subclones were obtained in which ZNF198-FGFR1, STAT1, and STAT5 were constitutively tyrosine phosphorylated, as determined by immunoprecipitation and Western blot analysis. To test the hypothesis that constitutive activation of ZNF198-FGFR1 tyrosine kinase activity is a result of self-association of the fusion protein, we in vitro transcribed and translated ZNF198-FGFR1 and a derivative construct, ZNF198- FGFR1deltaC-myc, in which the C-terminal FGFR1 epitope was replaced by a c-myc tag. As expected, an anti-FGFR1 antibody immunoprecipitated ZNF198-FGFR1 but not ZNF198-FGFRdeltaC-myc. However when both products were translated together, both were coimmunoprecipitated by anti-FGFR1 antisera. Similar results were obtained by using an anti-myc antibody and demonstrated a physical interaction between the two proteins. Analysis of COS-7 cells transfected with ZNF198-FGFR1 demonstrated that the fusion gene, in contrast to normal FGFR1, is located in the cytoplasm. We conclude that ZNF198-FGFR1 is a cytoplasmic protein that self-associates and has constitutive transformation activity. These data suggest that ZNF198-FGFR1 plays a primary role in the pathogenesis of the t(8;13) myeloproliferative syndrome and is the first report to implicate STAT proteins in FGFR1-mediated signaling.

Tandem repeats of a novel, putative, zinc-binding motif (MYM) have been described within the products of two, highly homologous genes: ZNF198/RAMP/FIM and ZNF261/DXS6673E. ZNF198, mapping to 13q11-q12, was recently shown to fuse to the fibroblast growth factor receptor 1 gene in the t(8;13)(p11;q11-q12) rearrangement associated with a stem cell leukemia/lymphoma syndrome. ZNF261 at Xq13.1 is disrupted by a t(X;13)(q13.1;q32) rearrangement in a mentally retarded patient and is a candidate gene for nonspecific X-linked mental retardation. Here we have cloned another member of this family, designated ZNF258, and mapped it to chromosome band 14q12, In addition, ZNF262/KIAA0425 was identified as a further member of the family and mapped to 1p32-p34. The predicted protein products of ZNF258 and ZNP262 maintain the repeats of the MYM motif. Isolation of these new members will facilitate the functional characterization of the MYM family and motif, (C) 1999 Academic Press.

The biphasic subtype of synovial sarcoma consists of both epithelial and spindle cell components. To address the relationship between the different cellular components found in synovial sarcoma, we deduced the presence of the synovial sarcoma-specific der(X)t(X;18)(p11.2;q11.2), and involvement of the SSX1 gene in both the epithelial/glandular and spindle cell components of 3 biphasic synovial sarcomas with wide ranging proportions of each of the 2 elements. This has been achieved using a 2-color fluorescence in situ hybridization (FISH) methodology that we had developed recently for analysis paraffin-embedded material. The presence of the rearrangement could be deduced in histologically defined regions and the results showed clearly that the rearrangement was present in both cellular components. This finding provides direct genetic evidence for biphasic synovial sarcomas being clonal and truly biphasic. (C) 1999 Wiley-Liss, Inc.

The chromosomal imbalances in nine cases of primary non-small cell lung cancer (NSCLC) and two cell lines derived from normal human bronchial epithelial (HBE) tissue were identified by comparative genomic hybridization (CGH). Gain of material from 3q and loss of 3p material were the most frequent changes in the primary tumors. Other commonly found imbalances included gain of material from 1q, 7p, 8q, 9q, 17q and 20q, and losses involving 4, 5q, 8p, 10 and 13q. High level gain was found in two cases, both encompassing the 3q23-q27 region. Loss of 3p was also found in both of the HBE cell lines suggesting that loss of one or more tumor supressor genes on 3p may be important for epithelial transformation and could be involved in the earlier stages of lung cancer development. (C) 1999 Elsevier Science ireland Ltd. All rights reserved.

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.

Lobular carcinoma in situ (LCIS) and atypical lobular hyperplasia (ALH) of the breast are cytologically similar breast lesions that reportedly carry different relative risks of subsequent development of invasive carcinoma. They are frequently multifocal and bilateral. We have identified the chromosomal copy number changes in 31 LCIS and 14 ALH lesions from 28 cases and also the 7 invasive carcinomas that subsequently developed in 6 of these cases. This was achieved by comparative genomic hybridization analysis of microdissected formalin-fixed, paraffin-embedded material. There was no significant difference between the aberrations found in the unilateral versus the bilateral cases of LCIS. Loss of material from 16p, 16q, 17p, and 22q and also gain of material from 6q were found at a similar high frequency in LCIS and ALH. Loss of these genomic regions may indicate the locations of genes that predispose to the development of the lesions, and the results are consistent with LCIS and ALH representing the same genetic stage of development. Comparison of the comparative genomic hybridization results from LCIS/ALH with those from ductal carcinoma in situ and invasive cancer showed some similarities at the chromosomal level, but it also showed significant differences, including gain of 1q and 8q and evidence for genomic amplification, which were not found in LCIS/ALH. A genetic model is postulated for the possible relationships between noninvasive lobular lesions and invasive breast carcinoma, delineating potential roles for specific chromosome copy number changes.

Phosphoinositide (PI) 3-kinases have been shown to have critical roles in signaling pathways that regulate proliferation, oncogenic transformation, cell survival, cell migration, and intracellular protein trafficking. We have previously used reverse-transcription polymerase chain reaction methods to identify novel PI 3-kinase isoforms in normal human breast and in lymph nodes containing metastatic breast cancer. Here we report the cDNA cloning of a Class II PI 3-kinase found in normal breast tissue. This gene (PIK3C2G) encodes the third distinct protein of the human Class II PI 3-kinase family, PI3K-C2gamma. PIK3C2G was mapped to chromosome 12 at 12p12 by fluorescence in situ hybridization.

A series of adult testicular germ cell tumours consisting of eight seminomas, 14 non-seminomas (including two cell lines) and two combined tumours was analysed by comparative genomic hybridization and, in some cases, by interphase fluorescence in situ hybridization. The gain of 12p was identified in all cases and additional material from chromosomes 7 and 8 was found in over 70% of cases, in keeping with previous analyses. Other consistent regions of gain included 1q24-q31 (50%), 2p16-pter (41%), 2q22-q32 (45%) and Xq11-q21 (50%). The loss of 1p32-p36 (36%), 9q31-qter (36%), 11q14-qter (50%), 16p (36%) and 18p (45%) and the loss of material from chromosomes 4 and 5 (50% and 36% respectively) were also found in all histological subtypes. The loss of 1p material was confirmed in four cases by interphase FISH analysis and shown, with one exception, not to involve the loss of the D1Z2 locus at 1p36.3, which is commonly deleted in paediatric germ cell tumours. An association between gain of 6q21-q24 with cases resistant to chemotherapy (P < 0.01) was observed. In addition, loss of chromosome 19 and 22 material and gain of 5q14-q23, 6q21-q24 and 13q were found at a significantly lower frequency in seminoma than non-seminoma. These regions may contain genes involved in the divergent development of seminoma and non-seminoma.

An estimated 10% of adult cancer patients present with undifferentiated carcinoma. The diagnosis of germ cell tumor (GCT) in such patients can be difficult but has important implications for patient management. Male testicular GCT is characterized by an isochromosome 12p, i(12p), or additional 12p material, in some cases restricted to the 12p11.2-p12.1 region. A gain of 12p material can indicate that a tumor, which may not be present in the testis, is of germ cell origin. Formalin-fixed, paraffin-embedded samples are the most widely available material for diagnostic analysis and retrospective studies. We have compared the identification of 12p gain in snap-frozen samples with corresponding paraffin-embedded material from three clearly defined testicular GCTs using comparative genomic hybridization analysis. In this preliminary study, paraffin-embedded tumor samples of uncertain histogenesis from seven patients were then analyzed. Tumor samples from three of these patients showed a gain of 12p material, and in one patient, gain was restricted to the 12p11.2-p12 region. The clinical picture and response to therapy were generally consistent with the 12p status, though lack of 12p gain may not exclude a diagnosis of GCT.

Fourteen cases of an atypical myeloproliferative disorder associated with consistent translocations involving 8p11-12 have previously been described. A t(8;13)(p11;q11-12) was the most common, but variant t(8;9)(p11-12;q32-34) and t(6;8)(q27;p12) were also reported. Here we have used a series of yeast artificial chromosomes (YACs) derived from the 8p11 and 13q11-12 regions to analyse one of the t(8;13) cases by fluorescence in situ hybridization (FISH). YACs flanking the 13q11-12 breakpoint and spanning the 8p11 breakpoint have been isolated. These YACs will facilitate characterization of the genes involved in this rearrangement.

A recently described atypical myeloproliferative disorder is invariably associated with reciprocal translocations involving 8p11-12. The most common rearrangement is a t(8;13)(p11;q11-12). Here we determine that this translocation results in the fusion of the fibroblast growth factor receptor 1 gene (FGFR1), a member of the receptor tyrosine kinase family at 8p11, to a novel gene at 13q11-12 designated RAMP . The predicted RAMP protein exhibits strong homology to the product of a recently cloned candidate gene for X-linked mental retardation, DXS6673E . We also provide the first report of a novel, putative metal-binding motif, present as five tandem repeats in both RAMP and DXS6673E. RT-PCR detected only one of the two possible fusion transcripts, encoding a product in which the N-terminal 641 amino acids of RAMP become joined to the tyrosine kinase domain of FGFR1. Receptor tyrosine kinases are not commonly involved in the formation of tumour-specific fusion proteins. However, the previous reports of involvement of receptor tyrosine kinases in fusion proteins in non-Hodgkin's lymphoma, chronic myelomonocytic leukaemia and papillary thyroid carcinoma described similar rearrangements. By analogy with these, we propose that the RAMP-FGFR1 fusion product will contribute to progression of this myeloproliferative disorder by constitutive activation of tyrosine kinase function.

The goal is to understand the critical events in tumour development and to apply this understanding to new approaches to diagnosis, prevention and treatment. It is clear that breast cancer is a heterogeneous disease at the molecular level, raising the possibility of a future functional classification based on mechanisms rather than morphology. These molecular phenotypes will also confer predictive value on the potential of the tumour to invade, metastasise and respond to or resist new therapeutic strategies. Studies of the genome in individuals are predicted also to enable the identification of polymorphisms that are associated with increased susceptibility to environmental factors, in addition to possibly explaining de novo variations in responses to drugs and radiation. The difficulty is how to identify which, of the approximately 30,000 genes expressed by a typical cancer cell alone or in combination, are the ones involved in these processes. The majority of breast cancers have such a multitude of molecular changes that it is difficult to distinguish between those that are critical to tumour progression and those that are epiphenomena of genetic instability and abnormalities in DNA repair. The identification of the earliest events in carcinogenesis must be the best hope, as it will then be possible to target the events that predispose to other secondary changes before they occur. Genomics and proteomics is the current hope to take us forward. This involves the application of a number of new technologies to facilitate the profiling of individual tumours, including laser-guided microdissection of microscopic lesions, comparative genomic hybridisation and loss of heterozygosity analysis of DNA using microarray technology to study DNA and expressed RNAs and protein profiling using 2D gel mass spectroscopy. With over 100,000 mRNAs and proteins to examine in complex tissues and in various combinations, there is obviously going to be a requirement for a large investment in computing power (bioinformatics) to facilitate the analysis of these data in relation to the clinical characteristics of the individual tumour and the patient.

The identification of specific chromosome translocations in various sarcomas and the elucidation of their molecular consequences has raised the possibility of detecting their presence for diagnostic and prognostic purposes in a manner analogous to the haematological malignancies. However, it is important to correlate particular translocations, and also other genetic aberrations, with current histological diagnoses and the behaviour of tumours, in order to assess the potential benefits, if any, of identifying rearrangements. Recent developments which enable specific translocations to be identified in archival material are likely to play an increasing role in the assessment and, eventually, the management of patients with sarcomas. (C) 1998 John Wiley & Sons, Ltd.

Phosphoinositide (PI) 3-kinases have been shown to have critical roles in signal transduction, cell transformation and intracellular protein trafficking. Reverse-transcription polymerase chain reaction methods, using degenerate primers derived from the lipid kinase consensus region, were utilised to identify PI 3-kinases in the normal human breast. Here we report the cDNA cloning of a novel human PI 3-kinase isoform, HsC2-PI3K. This PI 3-kinase is most closely related to the recently described C2 domain-containing family of PI 3-kinases which includes Drosophila PI3K_68D/cpk and murine cpk-m/p170. Sequence analysis suggests that HsC2-PI3K is a second distinct mammalian member of the C2 domain-containing PI 3-kinase family. Northern blot analysis of human tissues indicates that HsC2-PI3K is widely expressed. Fluorescence in situ hybridisation has mapped HsC2-PI3K to chromosome 1q32.

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.

The t(X;18)(p11.2;q11.2) translocation found in synovial sarcomas results in the fusion of the SYT gene on chromosome 18 to either of two closely related genes SSX1 and SSX2 on chromosome X. The resulting chimaeric genes express SYT-SSX1 or SYT-SSX2 fusion proteins in which the C-terminal amino acids of SYT are replaced by amino acids from the C-terminus of the SSX proteins. Using green fluorescent protein fusions we demonstrate that the SYT, SSX and the SYT-SSX proteins are nuclear proteins. We demonstrate that whilst the SSX1 protein has a uniform nuclear distribution the SYT protein has a speckled distribution in the cell nucleus, and this distribution is retained with the SYT-SSX2 fusion protein. Since the SYT speckles do not co-localise with PML-containing bodies (PODs) or spliceosomes it is possible that they represent a novel nuclear structure. Transfection of constructs expressing GAL4 fusion proteins demonstrate that the SYT domains present in the SYT-SSX fusion proteins can activate transcription of a luciferase reporter. It is proposed that the t(X;18) translocation results in the generation of an SYT-SSX transcriptional co-activator in which the addition of the C-terminal SSX domain to SYT provides a new interacting domain that redirects the SYT activation domain to different target promoters.

The brk gene encodes a non-receptor protein tyrosine kinase that consists of single SH3, SH2 and catalytic domains. Although BRK shows strongest sequence similarity to members of the SRC family of PTKs, there are several key structural and regulatory differences that place it on its own amongst non-receptor PTKs. In this study we have isolated genomic DNA clones corresponding to the human brk locus and used these to determine the intron-exon structure of the brk gene. The genomic structure of brk consists of 8 exons, whose boundaries are distinct from other non-receptor PTK family members, again indicating a structural and functional divergence. Alternate splicing of the primary brk transcript generates a distinct mRNA which encodes a truncated protein consisting of an SH3 domain and a novel C-terminal proline rich sequence. Using an antiserum raised to the SH3 domain, we have demonstrated that the product of this alternate brk transcript is expressed in the human breast tumour cell line T-47D. We have previously reported that expression of a tumour derived brk cDNA in mouse embryonic fibroblasts and human mammary epithelial cells supports anchorage independent growth, and in the latter potentiates the mitogenic response to epidermal growth factor. The protein encoded by the genomic sequence derived from normal human tissue is identical to that encoded by the tumour derived cDNA, and therefore the altered growth regulation is not associated with mutations within brk. In addition, we have identified a 5' genomic region that has promoter activity. The brk gene has been assigned to chromosome 20q 13.3 [corrected] using fluorescence in situ hybridisation (FISH).

Phyllodes tumors are rare neoplasms of the breast. Although they contain both epithelial and stromal components they are considered to be stromally derived lesions. The chromosomal copy number changes were determined in 19 well characterized samples from 18 patients using comparative genomic hybridization. Most chromosomes were involved and generally the gains and losses were similar to those found in breast cancer with the exception that the phyllodes tumors showed no evidence of genomic amplification. The one recurrent sample analyzed had the same imbalances as the original tumor. Frequent changes were gain of 1q (7/18) and loss of 3p (6/18), followed by gain of 7q (4/18) and loss of 6q (4/18) and 3q (3/18). Gain of 1q material was significantly associated with histologically defined stromal overgrowth (P = 0.011). In addition, all the cases with gain of 1q material, without 1p gain, had a clinical history of recurrence. Only one case without 1q gain had a recurrence and this had loss of the X chromosome as the sole abnormality. Increased copy number of 1q material in the phyllodes tumors studied, in one case restricted to 1q24-32, was associated with recurrence (P = 0.00365) and might therefore be considered as an indicator of local aggressiveness requiring more radical treatment. (C) 1997 Wiley-Liss, Inc.

Peripheral primitive neuroectodermal tumors (PNETs) are rare lesions that form part of the Ewing family of tumors, which includes osseous and extraosseous Ewing's sarcoma and Askins tumor of the thorax. All are characterized by translocations involving the EWS gene at 22q12, usually the translocation t(11;33)(q24;12). PNETs usually occur in soft tissues but occasionally arise within a visceral organ. We describe a PNET of the kidney that showed characteristic microscopic and immunohistochemical appearances of a small, round, dark blue cell tumor with focal rosette formation and strong membrane positivity for the MIC2 gene product. Interphase fluorescence in situ hybridization on touch imprints prepared from frozen tissue using cosmid probes flanking the EWS gene at 22q12 and the FLI1 gene at 11q24 indicated the presence of t(11;=+22)(q24;=+q12), confirming the diagnosis of PNET. This is the first reported PNET of the kidney supported by cytogenetic analysis. We also review the literature on this fascinating tumor in this unusual location.

Alveolar rhabdomyosarcoma is an aggressive childhood tumor that exhibits muscle cell differentiation. Cytogenetically, it is characterized by t(2;13)(q35;q14); no consistent secondary abnormalities have been reported. Cytogenetic analysis of bone marrow in a case of alveolar rhabdomyosarcoma revealed t(2;13)(q35;q14) and der(16)t(1;16)(q21;q13). The present case and a review of the literature suggest that up to 11% of these tumors possess der(16)t(1;16)(q21;q13). This is similar to the incidence observed in the Ewing family of tumors, where unbalanced der(16)t(1;16) translocations, resulting in partial trisomy of 1q, are regarded as a consistent secondary cytogenetic change.

Substantial improvements have been made in the treatment and survival of children with SRCT, resulting in an increased emphasis on precise histological diagnosis. Although diagnostic procedures such as electron microscopy and immunocytochemistry contribute in poorly differentiated cases, an accurate diagnosis can remain elusive in a proportion of SRCTs. The cytogenetic and molecular genetic abnormalities characteristic of the different SRCTs can now be consistently and rapidly identified from minimal quantities of tumour material, using the techniques of FISH and PCR. This, coupled with the identification of novel phenotypic characteristics, has had a major impact on SRCT diagnosis. The aim of a tumour classification is to identify disease entities which are biologically distinct and whose recognition is of clinical value. The recent advances described above demonstrate that the SRCTs are genotypically and phenotypically distinct tumour types and that the genetic abnormalities represent key alterations that influence both the morphology and the clinical behaviour of the tumour. This suggests that these advanced phenotypic and genotypic analyses should form an integral and complementary part of the laboratory assessment and clinical management of these forms of paediatric cancer.

The identification of t(2;13)(q35;q14) is a useful aid in the accurate diagnosis of rhabdomyosarcoma, distinguishing it from other small round cell tumours and supporting the distinction between alveolar and embryonal forms. Cytogenetic analysis is difficult and with the increased use of minimally invasive biopsy methods and primary chemotherapy, adequate tumour material is not always available. To overcome these difficulties, two-colour interphase fluorescence in situ hybridization (FISH) to detect t(2;13)(q35;q14) was developed and its utility in assessing minimally invasive biopsies was investigated. Two cosmid clones which mapped proximal or distal to the breakpoint region 13q14 and one yeast artificial chromosome clone that mapped distal to the 2q35 breakpoint were identified. In interphase cells containing t(2;13)(q35;q14), the configuration of cosmid and yeast artificial chromosome signals demonstrated the presence of the translocation. Five cases of rhabdomyosarcoma were analysed by interphase FISH. The t(2;13)(q35;q14) was detected in all four alveolar tumours and was confirmed by cytogenetics in two cases, but was absent in one embryonal tumour. This sensitive detection method is applicable to minimal amounts of fresh or frozen tumour.

The conversion of serine and tetrahydrofolate to glycine and 5,10 methylene tetrahydrofolate by serine hydroxymethyltransferase (SHMT, EC 2.1.2.1) is the major route for the provision of one-carbon units for biosynthetic reactions. SHMT cDNAs have been cloned from both rabbit and man, and a human mitochondrial SHMT gene sequence has recently been reported. We have isolated phage clones containing human genomic sequences homologous to cytosolic SHMT and have found these to contain a processed pseudogene (SHMT-ps1) with a 90% identity to cloned SHMT cDNAs. SHMT-ps1 contains 2335 nt that are homologous to SHMT but it has an additional leader sequence of 203 nt of unknown origin. The complete SHMT-ps1 sequence of 2538 nt is bounded by two 16 nt direct repeats that are characteristic of retroelement insertion sites. Two phage clones containing SHMT-ps1 have been mapped by fluorescence in situ hybridisation to 1p32.3-33. In addition, an SHMT cDNA clone hybridized to the same region and to 17p11.2-12. The latter is consistent with a previous localisation of the gene for cytosolic SHMT.

In this study, 10 embryonal and 14 alveolar rhabdomyosarcoma (RMS) tumor samples, including 4 cell lines derived from tumors of the alveolar subtype, were analyzed by comparative genomic hybridization. In the embryonal tumors, the gain of whole or most of various chromosomes, notably chromosomes 2 (60% of cases), 13 (60%), 12 (60%), 8 (60%), 7 (50%), 17 (40%), 18 (40%), and 19 (40%), and the loss of chromosomes 16 (40%), 10 (30%), 15 (20%), and 14 (20%) were found. One case showed evidence of genomic amplification at 12q13-15. In contrast, the alveolar tumors and cell lines showed consistent evidence of genomic amplification, with multiple amplicons in some cases. The amplicons were localized to l2q13-15 (50%), 2p24 (36%), 13q14 (14%), l3q32 (14%), 1q36 (14%), 1q21 (7%), and 8q13-21 (7%). Four cases had additional copies of chromosome 17 or l7q. These changes were in addition to the presence of fusion gene transcripts that are associated with translocations specific to alveolar RMS. The results show that distinct patterns of primarily gains of specific chromosomal material are associated with the embryonal subtype of RMS, and that genomic amplification seems to play an important role in the alveolar subtype. Notably, these distinct changes predominantly involved chromosomes 2, 12, and 13 in both subtypes.

Alveolar rhabdomyosarcomas frequently exhibit specific translocations, resulting in the fusion of the FKHR gene at 13q14 with either the PAX3 or PAX7 gene at 2q35 and 1p36, respectively. Comparative genomic hybridization revealed amplification at 13q14 and 1p36, suggesting amplification of the PAX7-FKHR fusion gene in two cases of alveolar rhabdomyosarcoma. A PAX7-FKHR fusion transcript was demonstrated in both cases by reverse transcription-polymerase chain reaction followed by sequence analysis. In one case, amplification of the PAX7 gene and 3'-and 5'-FKHR gene sequences was demonstrated by using interphase fluorescence in situ hybridization on tumor imprints. The colocalization, variable copy number, and distribution of signals from the three cosmids was consistent with amplification of these sequences on double minutes, which were present cytogenetically. Chromatin release studies suggested that the amplified sequences correlated with amplification of the PAX7-FKHR fusion gene which resulted from the insertion of PAX7 sequences into the first intron of FKHR gene, in keeping with the absence of cytogenetic evidence for derivative chromosomes.

The specific chromosomal translocation t(9;22)(q22-31;q11-12) has been observed in the myxoid variant of human chondrosarcoma. In agreement with this observation we report that the EWS gene located at chromosome band 22q12 becomes fused to CHN, a member of the steroid/thyroid receptor gene superfamily located at 9q22-31, in a skeletal myxoid chondrosarcoma. CHN appears to be the human homologue of the rat gene NOR1, which was recently identified as a sequence overexpressed in rat brain cells undergoing apoptosis. Our results also indicate that the chimaeric EWS-CHN gene encodes a EWS-CHN fusion protein in which the C-terminal RNA-binding domain of EWS is replaced by the entire CHN protein, comprising a long N-terminal domain, a central DNA binding domain and a C-terminal ligand-binding/dimerisation domain.

The specific chromosomal translocation t(X;1)(p11,2;q21,2) has been observed in human papillary renal cell carcinomas, In this study we demonstrated that this translocation results in the fusion of a novel gene designated PRCC at 1q21,2 to the TFE3 gene at Xp11,2, TFE3 encodes a member of the basic helix-loop-helix (bHLH) family of transcription factors originally identified by its ability to bind to mu E3 elements in the immunoglobin heavy chain intronic enhancer, The translocation is predicted to result in the fusion of the N-terminal region of the PROC protein, which includes a proline-rich domain, to the entire TFE3 protein, Notably the generation of the chimaeric PRCC-TFE3 gene appears to be accompanied by complete loss of normal TFE3 transcripts, This work establishes that the disruption of transcriptional control by chromosomal translocation is important in the development of kidney carcinoma in addition to its previously established role in the aetiology of sarcomas and leukaemias.

Identification of the t(X;18)(p11.2;q11.2) that is associated with a high proportion of synovial sarcoma can be a useful diagnostic aid. The translocation results in fusion of the SYT gene on chromosome 18 to either the SSX1 or the SSX2 gene, two homologous genes within Xp11.2. Two-color interphase fluorescence in situ hybridization and reverse transcription polymerase chain reaction were assessed as approaches to identify the rearrangement in well characterized cases. The presence of the translocation, and the specific chromosome X gene disrupted, were inferred from the configuration of signals from chromosome-specific centromere probes, paints, and markers flanking each gene in preparations of interphase nuclei. Rearrangement was found in two cell lines and eight of nine tumor samples, including analysis of five touch imprints. This was consistent with cytogenetic data in four cases and reverse transcription polymerase chain reaction analysis using primers known to amplify both SYT-SSX1 and SYT-SSX2 transcripts. The transcripts were distinguished by restriction with LspI and SmaI. Contrary to previous suggestions, there was no obvious correlation between histological subtype and involvement of the SSX1 or SSX2 gene. These approaches could also be applied to the identification of tumor-free margins and metastatic disease.

Chromosome 12q24.1 is a recurrent breakpoint in high-grade B-cell non-Hodgkin lymphoma (B-NHL). To identify the genes involved at 12q24.1, molecular cloning of a three-way translocation t(8;14;12)(q24.1;q32.3;q24.1) in a Burkitt lymphoma cell line (Wien 133) was performed; all four translocation breakpoints were cloned and sequenced. Analysis of clones encompassing the der(12)(12;14)(q24.1;q32.3) breakpoint showed a CpG island from chromosome 12q24.1 juxtaposed in a tail-to-tail configuration with a productively rearranged Ig VH4-DH-JH5 gene. A total of 4.5 kb of genomic DNA including the CpG island was sequenced and analyzed using gene-identification programs; all three programs identified a potential 92-bp exon within the centromeric boundary of the CpG island. Using this as a probe, an RNA transcript of 3.8 kb, expressed at low levels in a wide variety of normal tissues, was detected. Overlapping cDNA clones were isolated and sequenced. The longest open-reading frame predicted a serine-rich protein of 231 amino acids. This protein, termed BCL7A, exhibited no recognizable protein motifs but showed homology with the actin-binding protein, caldesmon. In Wien 133, the BCL7A breakpoint occurred within the first intron and resulted in a MYC-BCL7A fusion transcript, with exon I of BCL7A being replaced by MYC exon I. The normal, untranslocated allele of BCL7A was also expressed without mutation. One of the 11 other B-NHL cell lines examined with 12q24.1 cytogenetic abnormalities, a mediastinal B-NHL cell line (Karpas 1106), showed biallelic rearrangement within the first intron of BCL7A, which was adjacent to the breakpoint observed in Wien 133. Disruption of the amino-terminus of BCL7A defines a new mechanism in the pathogenesis of a subset of high-grade B-NHL.

Comparative genomic hybridization was used to identify the regions of genomic gain and loss in the myeloid cell line HL-60. These included amplification at 8q24 corresponding to previous reports of overrepresentation of the MYC gene; loss of material from the short arms of chromosomes 9 (9p21-p23), 10, and 17; loss of the chromosome regions 9q32-qter and 14q11-q24; and an extra copy of chromosome 18. Additionally, deletion of the 5q11-q31 region was noted and was associated with translocation of chromosome 5 material to chromosomes 16 and a dic(5;17)(q11;p11) chromosome (previously described as mar 3). Loss of chromosome 5 material in myeloid malignancies, including the M2 subtype from which HL-60 was derived, is usually associated with interstitial deletions of the long arm, including the critical 5q31 region, resulting in a 5q- chromosome. The HL-60 cell line may be a useful model to investigate the role of potential tumour suppressor genes associated with loss of 5q material in myeloid leukaemias.

The diagnosis of paediatric tumours including the small round cell tumours (neuroblastoma, rhabdomyosarcoma and the Ewing family of tumours), brain tumours, germ cell tumours and anaplastic large tell lymphoma can pose particular diagnostic dilemmas, especially in cases with undifferentiated morphology. Substantial improvements have been made in the treatment and long term survival of paediatric patients with these tumours, however, these are based on disease and even stage specific treatments. Accurate diagnosis and prognosis can now be aided by identifying specific genotypic and phenotypic criteria using cytogenetics, interphase fluorescence in situ hybridisation, reverse transcription PCR and novel immunophenotypic markers. Some of these analyses should Form an integral part of the management of patients with paediatric solid tumours.

We have isolated a cDNA from human foetal brain cDNA library which encodes a putative transmembrane protein bearing an intracellular protein tyrosine phosphatase (PTPase) like domain. The PTPase Like domain contains an alanine to aspartate amino acid change relative to other PTPases in the catalytic core domain. This amino acid change is found in only three other known proteins, islet cell autoantigens; human, murine and rat IA-2, murine IA 2b and its rat orthologue phogrin, which have a similar overall structure to ICAAR, and the recently identified X-linked myotubular myopathy (MTMl) gene. ICAAR, IA-2 and IA 2b clearly represent a new family of PTP-like proteins for which catalytic activity has yet to be demonstrated. An abundant ICAAR mRNA is detectable in the brain and pancreas but not in the other normal human tissues surveyed. We have localised ICAAR to human chromosome 7q36. (C) 1996 Academic Press, Inc.

Cytogenetic and fluorescent in situ hybridization (FISH) analysis has been performed on consecutive samples, taken 4 weeks apart, from a phyllodes breast tumor. This revealed the presence of two different chromosome 1 derivatives, namely a dic(1;10)(q10;q24) in the first sample and an i(1) (q10) in the second. In one cell out of 25 from the second sample both derivative chromosomes were seen. A chromosome 21 was lost in both samples. These results are consistent with phyllodes tumors having a clonal origin.

A t(X;1)(p11.2;q21.2) has been reported in cases of papillary renal cell tumors arising in males. In this study two cell lines derived from this tumor type have been used to indicate the breakpoint region on the X chromosome. Both cell lines have the translocation in addition to other rearrangements and one is derived from the first female case to be reported with the t(X;1)(p11.2;q21.2). Fluorescence in situ hybridization (FISH) has been used to position YACs belonging to contigs in the Xp11.2 region relative to the breakpoint. When considered together with detailed mapping information from the Xp11.2 region the position of the breakpoint in both cell lines was suggested as follows: Xpter-->Xp11.23-OATL1-GATA1-WAS-TFE3-SY P-t(X;1)-DXS255-CLCN5-DXS146-OATL2- Xp11.22-->Xcen. The breakpoint was determined to lie in an uncloned region between SYP and a YAC called FTDM/1 which extends 1 Mb distal to DXS255. These results are contrary to the conclusion from previous FISH studies that the breakpoint was near the OATL2 locus, but are consistent with, and considerably refine, the position that had been established by molecular analysis.

We report the cytogenetic findings in a case of nodular fasciitis of the breast. The abnormalities found in all 11 metaphases available for analysis were -2, -2, -13, der(15)t(2;15)(q31;q26), + der(?) t(?;2), + mar1, + mar2. Other consistent abnormalities were also identified. Fluorescence in situ hybridization (FISH) was used to confirm the origin of some of the chromosomes. A large acrocentric chromosome was confirmed to be derived from chromosome 15 with chromosome 2 material translocated onto the q arm. The metacentric der(?)t(?;2) was demonstrated to have part of chromosome 2 on the q arm. No other chromosome 2 material was found. Eight of 11 cells were tetraploid and had two copies of a del(6)(q16q24).

It is increasingly recognized that the identification of t(11;22)(q24;q12) is a useful aid in the accurate diagnosis of Ewing's sarcoma and related tumours. However, cytogenetic studies have a low success rate and adequate tumour is not always available. This study describes the use of fluorescence in situ hybridization (FISH) to detect translocations at 22q12, the site of the EWS gene involved in t(11;22)(q24;q12), on tumour touch imprints made from true cut core-needle biopsy and frozen tumour. Of the seven tumours analysed, five diagnosed as Ewing's sarcoma or primitive neuroectodermal tumour demonstrated chromosome translocation at 22q12. This is a rapid and reliable method to detect a diagnostically relevant chromosome translocation using minimal amounts of fresh or frozen tumour.

Southern blot analyses revealed a rearrangement of the EWS gene in a skeletal human myxoid chondrosarcoma. Interphase fluorescence in situ hybridization (FISH) studies, using cosmid clones F7 and G9 that flank the EWS locus on 22q 12, confirmed the presence of this EW5 gene abnormality. Cloning the rearranged EWS DNA fragment and mapping by FISH demonstrated that the EWS gene is joined to DNA sequences localised in 9q22-31. These findings are consistent with previous cytogenetic reports of a recurrent t(9;22)(q22-31;q11-12) in the myxoid variant of chondrosarcoma and reveal involvement of the EWS gene in a fourth type of human sarcoma. (C) 1995 Wiley-Liss, inc.

Previous analysis of the monoblastic cell line U937 has shown that several sublines contain a rearranged chromosome arm 11q. In order to determine the true nature of the rearrangement, fluorescence in situ hybridization (FISH) was carried out with various combinations of single copy anonymous markers, clones containing genes, a chromosome 10 paint, and an 11 centromere specific sequence. The rearrangement was deduced to be a reciprocal translocation between chromosomes 10 and 11 described as t(10;11)(p13-14;q14-21). The breakpoint on chromosome 11 is telomeric to the INT2 gene and the pHS11 probe at 11q13, and centromeric to the marker D11S36 localized to 11q14.3-q22.1 and the MLL gene at 11q23. Similar translocations have been reported in various acute leukemias, principally of the monocytic lineage, and also in T-cell precursor acute lymphocytic leukemias. Further characterization of the genetic rearrangements in U937 may lead to the isolation of genes important in leukemogenesis and provide an in vitro system for their study.

We demonstrate that the cytogenetically defined translocation t(X;18)(p11.2;q11.2) found in human synovial sarcoma results in the fusion of the chromosome 18 SYT gene to either of two distinct genes, SSX1 or SSX2, at Xp11.2. The SSX1 and SSX2 genes encode closely related proteins (81% identity) of 188 amino acids that are rich in charged amino acids. The N-terminal portion of each SSX protein exhibits homology to the Kruppel-associated box (KRAB), a transcriptional repressor domain previously found only in Kruppel-type zinc finger proteins. PCR analysis demonstrates the presence of SYT-SSX1 or SYT-SSX2 fusion transcripts in 29 of 32 of the synovial sarcomas examined, indicating that the detection of these hybrid transcripts by PCR may represent a very useful diagnostic method. Sequence analysis has demonstrated heterogeneity in the fusion transcripts with the formation of two distinct SYT-SSX1 fusion junctions and two distinct SYT-SSX2 fusion junctions.

We have isolated cDNAs from normal human breast tissue and breast tumour cells that encode a protein (pez) with features of a novel non-receptor tyrosine phosphatase possessing N-terminal sequence homology to the ezrin-band 4.1-merlin-radixin protein family. Northern blot analysis indicates that pez is expressed in a variety of human tissues including kidney, skeletal muscle, lung and placenta. Fluorescence in situ hybridization has mapped pez to chromosome 1 region q32.2-41. Sequence identity to a characterized polymorphic marker confirms this localization.

A high proportion of synovial sarcomas contain the reciprocal translocation t(X;18)(p11.2;q11.2). We have previously localized the breakpoint on the X chromosome between the X chromosome marker DXS255 and an ornithine aminotransferase (OAT) pseudogene region designated OATL2. Subsequently by fluorescence in situ hybridization (FISH) we provided evidence that YACs corresponding to the OATL2 locus spanned the break-point. In order to confirm the position of this breakpoint cosmids corresponding to the OATL2 region were isolated. Most of these cosmids mapped to four cosmid contigs designated C1-C4. Analysis of two contigs, C1- and C4, using FISH established that in four of six synovial sarcomas examined the breakpoint occurs between these two contigs: C1 lies distal to the break-point while C4 is proximal. In contrast we provide evidence that the breakpoint in the remaining two tumours mapped to a second pseudogene region called OATL1 that is telomeric to the OATL2 locus. This heterogeneity of the breakpoint position on the X chromosome explains why in previous mapping studies there have been discrepancies between the results obtained by different laboratories.

Human synovial sarcomas contain a recurrent and specific chromosomal translocation t(X;18)(p11.2;q11.2). By screening a synovial sarcoma cDNA library with a yeast artificial chromosome spanning the X chromosome breakpoint, we have identified a hybrid transcript that contains 5' sequences (designated SYT) mapping to chromosome 18 and 3' sequences (designated SSX) mapping to chromosome X. An SYT probe detected genomic rearrangements in 10/13 synovial sarcomas. Sequencing of cDNA clones shows that the normal SYT gene encodes a protein rich in glutamine, proline and glycine, and indicates that in synovial sarcoma rearrangement of the SYT gene results in the formation of an SYT-SSX fusion protein. Both SYT and SSX failed to exhibit significant homology to known gene sequences.