Genomic gain and over expression of CCL2 correlate with vascular invasion in stage I non-seminomatous testicular germ-cell tumours.

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.

Stem cell factor as a novel diagnostic marker for early malignant germ cells.

Carcinoma in situ (CIS) of the testis is the pre-invasive stage of type II 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-2gamma, 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 II (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 SCF were expressed, suggestive of an autocrine loop. SCF 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.

Genomic copy number and expression patterns in testicular germ cell 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.

KIT and RAS signalling pathways in testicular germ cell tumours: new data and a review of the literature.

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.

Increased level of chromosomal damage after irradiation of lymphocytes from BRCA1 mutation carriers.

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.

Dramatic response of malignant craniopharyngioma to cis-platin-based chemotherapy. Should craniopharyngioma be considered as a suprasellar 'germ cell' tumour?

The case is described of a 21-year-old woman, who developed a malignant tumour arising from a craniopharyngioma 14 years after the original diagnosis. The remarkable response of this malignant tumour ex-craniopharyngioma to cis-platin based chemotherapy, together with other midline tumour characteristics of craniopharyngioma, raise the question as to whether craniopharyngioma should any longer be separately considered from suprasellar germ cell tumour. This subject is discussed.

Expression profile of genes from 12p in testicular germ cell tumors of adolescents and adults associated with i(12p) and amplification at 12p11.2-p12.1.

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.

Chromosomal imbalances in familial chronic lymphocytic leukaemia: a comparative genomic hybridisation analysis.

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.

Definition of chromosome aberrations in testicular germ cell tumor cell lines by 24-color karyotyping and complementary molecular cytogenetic analyses.

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.

Chromosomal imbalances associated with carcinoma in situ and associated testicular germ cell tumours of adolescents and adults.

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.

Gain of 1q is associated with adverse outcome in favorable histology Wilms' tumors.

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.

Dual colour fluorescence in situ hybridization to paraffin-embedded samples to deduce the presence of the der(X)t(X;18)(p11.2;q11.2) and involvement of either the SSX1 or SSX2 gene: a diagnostic and prognostic aid for synovial sarcoma.

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.

Molecular cytogenetic analysis of adult testicular germ cell tumours and identification of regions of consensus copy number change.

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.

Establishing germ cell origin of undifferentiated tumors by identifying gain of 12p material using comparative genomic hybridization analysis of paraffin-embedded samples.

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.

Additional copies of 1q in sequential samples from a phyllodes tumor of the breast.

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.

Cytogenetic findings in a case of nodular fasciitis of the breast.

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).

Cytogenetic studies in patients previously treated for Hodgkin's disease.

A cytogenetic study was made of bone marrow cells and lymphocytes from patients who had been successfully treated with various regimens for Hodgkin's disease. Most of the patients had been off treatment for at least 3 years before the study began. They were divided into three groups according to the intensity of the therapy received. The frequency of gaps and breaks in the chromosomes of lymphocytes was above normal limits and similar in the three treatment groups. In contrast, the frequency of both lymphocytes and bone marrow cells with rearranged karyotypes was correlated with the intensity of treatment. Clones of cells with an abnormal karyotype were found in only two patients, both of whom were in the group receiving the most intensive therapy, i.e., chemotherapy and total nodal irradiation.

Cytogenetic follow-up study of acute non-lymphocytic leukaemia.

A cytogenetic study was made at diagnosis in 68 patients with acute non-lymphocytic leukaemia. The median survival time of the group of patients was 47 weeks. Patients with totally abnormal karyotypes in the bone marrow had a median survival of 14 weeks, whereas for those with only chromosomally normal cells the median survival was 52 weeks, and 47 weeks for those with normal and abnormal cells. Survival was not influenced by the presence or absence of clonal abnormalities. A cytogenetic follow-up study was made on 41 of these patients. One of the three patients, still in first remission after 3 years, originally had an 8;21 translocation. In general, the abnormalities present at diagnosis disappeared during remission and there was no evidence that clones of abnormal cells were produced by treatment. The chromosomal findings in relapse were not necessarily the same as those seen at diagnosis and the abnormal karyotypes found were usually not clonal in type. A simple kinetic technique, in which the yield of mitoses after 2 and 24 h is compared, may be used as an adjunct to cytogenetic studies in predicting relapse.

Patterns of maturation in short-term culture of human acute myeloid leukaemic cells.

Leukaemic cells taken from the blood of patients with acute myelogenous leukaemia (AML) frequently proliferate in suspension culture without the addition of growth factors for a limited period only. After a 6--10-fold increase in total cells, cell numbers remain constant for a time and finally decline. The main cause for this limited growth in vitro is not, initially at least, cell death leading to a steady state, but maturation associated in its final stages with cessation of DNA synthesis. Two populations of AML cells from Patients St and Wi respectively were studied, and progressive maturation towards mature leucocytes was demonstrated by the gradual acquisition in culture by the growing blast cells of intracellular enzymes (lysozyme, arginase, acid phosphatase and esterase being measured), surface markers (Fc and C3 receptors), of lactoferrin by Wi cells and of colony-stimulating activity by St cells, as well as changes in Ia antigens, phagocytic properties, morphology and adhesiveness to plastic. With St cells, which carried a characteristic chromosome marker, maturation terminated in cells with the characteristic properties of macrophages. At an intermediate stage, non-adherent and still-dividing St cells acquired Fc and C3 receptors and enzymes characteristic of monocytes. Wi cells progressively became neutrophil-like, and again there was an intermediate population of dividing cells which had Fc and C3 receptors and proteins such as lactoferrin and esterases. characteristic of neutrophils.