Molecular genetics of adrenocortical tumours, from familial to sporadic diseases

Adrenal masses can be detected in up to 4% of the population, and are mostly of adrenocortical origin. Adrenocortical tumours (ACTs) may be responsible for excess steroid production and, in the case of adrenocortical cancers, for morbidity or mortality due to tumour growth. Our understanding of the pathogenesis of ACTs is more limited than that for other tumours. However, studies of the genetics of ACTs have led to major advances in this field in the last decade. The identification of germline molecular defects in the hereditary syndrome responsible for ACTs has facilitated progress. Indeed, similar molecular defects have since been identified as somatic alterations in sporadic tumours. The familial diseases concerned are Li-Fraumeni syndrome, which may be due to germline mutation of the tumour-suppressor gene TP53 and Beckwith-Wiedemann syndrome, which is caused by dys-regulation of the imprinted IGF-II locus at 11p15. ACTs also occur in type 1 multiple endocrine neoplasia (MEN 1), which is characterized by a germline mutation of the menin gene. Cushing's syndrome due to primary pigmented nodular adrenocortical disease (PPNAD) has been observed in Carney complex patients presenting inactivating germline PRKAR1A mutations. Interestingly, allelic losses at 17p13 and 11p15 have been demonstrated in sporadic adrenocortical cancer and somatic PRKAR1A mutations have been found in secreting adrenocortical adenomas. More rarely, mutations in Gs protein (gsp) and the gene for ACTH receptor have been observed in ACTs. The genetics of another group of adrenal diseases that can lead to adrenal nodular hyperplasia -- congenital adrenal hyperplasia (CAH) and glucocorticoid-remediable aldosteronism (GRA) -- have also been studied extensively. This review summarizes recent advances in the genetics of ACTs, highlighting both improvements in our understanding of the pathophysiology and the diagnosis of these tumours.     

Multipoint interphase FISH analysis of chromosome 3 abnormalities in 28 childhood AML patients

We detected non-random 3p losses and 3q gains on well-determined regions in both murine and human tumors using a microcell hybrid-based model system called 'elimination test'. We suggest that these are general malignancy-associated aberrations not necessarily linked to a particular tissue of origin. To examine chromosome 3 abnormalities, in 28 childhood acute myeloid leukemia bone marrow samples, we performed interphase multipoint-fluorescence in situ hybridization using 84 chromosome 3-specific probes and detected clonal chromosome 3 aberrations in nine cases, which is of a higher frequency than the previously reported one. In 3/28 children, a chromosome 3 abnormality was detected which was not visible using conventional cytogenetic analysis. We did not detect any 3p deletion. Increased copy number of 3q was found in four cases with trisomy of whole chromosome 3 and one case with 3q tetrasomy (isodisomy). We identified rare structural rearrangements in childhood acute myeloblastic leukemia, involving 3q21 and 3q26 loci around RPN1 and MDS1/EVI1 respectively. The poor outcome in pediatric patients with 3q rearrangements appears to be quite uniform.     

Genetic characterization of large parathyroid adenomas

In this study, we genetically characterized parathyroid adenomas with large glandular weights, for which independent observations suggest pronounced clinical manifestations. Large parathyroid adenomas (LPTAs) were defined as the 5% largest sporadic parathyroid adenomas identified among the 590 cases operated in our institution during 2005-2009. The LPTA group showed a higher relative number of male cases and significantly higher levels of total plasma and ionized serum calcium (P<0.001). Further analysis of 21 LPTAs revealed low MIB1 proliferation index (0.1-1.5%), MEN1 mutations in five cases, and one HRPT2 (CDC73) mutation. Total or partial loss of parafibromin expression was observed in ten tumors, two of which also showed loss of APC expression. Using array CGH, we demonstrated recurrent copy number alterations most frequently involving loss in 1p (29%), gain in 5 (38%), and loss in 11q (33%). Totally, 21 minimal overlapping regions were defined for losses in 1p, 7q, 9p, 11, and 15q and gains in 3q, 5, 7p, 8p, 16q, 17p, and 19q. In addition, 12 tumors showed gross alterations of entire or almost entire chromosomes most frequently gain of 5 and loss of chromosome 11. While gain of 5 was the most frequent alteration observed in LPTAs, it was only detected in a small proportion (4/58 cases, 7%) of parathyroid adenomas. A significant positive correlation was observed between parathyroid hormone level and total copy number gain (r=0.48, P=0.031). These results support that LPTAs represent a group of patients with pronounced parathyroid hyperfunction and associated with specific genomic features.     

Genetic aberrations detected by comparative genomic hybridization in hepatocellular carcinomas: their relationship to clinicopathological features.

To elucidate cytogenetic alterations underlying human hepatocellular carcinomas (HCCs), we used a comparative genomic hybridization (CGH) method to analyze 41 cases of hepatocellular carcinoma (HCC) including 15 well differentiated HCCs, 14 moderately differentiated HCCs, and 12 poorly differentiated HCCs. Of these, 27 patients were chronically infected with hepatitis C virus (HCV), and the remaining patients were positive for hepatitis B virus (HBV). The most common sites of increase in DNA copy number were 1q (78% of the cases) and 8q (66%) with minimal overlapping regions at 1q24-25 and 8q24, respectively. Frequent decreases in copy number were observed at 17p (51%), 16q (46%), 13q13-14 (37%), 4q13-22 (32%), 8p (29%), and 10q (17%). In 6 cases (15%), an amplification was found in the region of 11q13. A gain of 8q24 was significantly associated with well-differentiated HCCs (P<.05), whereas a loss of 13q13-14 and amplification of 11q13 were linked to moderately and poorly differentiated HCCs (P<.01). These observations suggest that a gain of 8q24 is an early event and that a loss of 13q13-14 and amplification of 11q13 are a late event in the course of liver carcinogenesis. A gain of 10q (7/41) was detected exclusively in cases with HCV infection. In contrast, an amplification of 11q13 was preferentially found in HBV-positive HCCs. These findings raise the hypothesis that, although many genetic alterations are basically common to both HCV-positive and HBV-positive tumors, the process of carcinogenesis may be to some extent different between these two types of tumors.     

Comparative genomic hybridization analysis of adrenocortical tumors of childhood

Although several genes have been investigated in adrenal tumorigenesis, the genetic background of adrenocortical tumors (ACT) remains poorly characterized. In southern Brazil, the annual incidence of ACT is unusually high, ranging from 3.4-4.2/million children, compared with a worldwide incidence of 0.3/million children younger than 15 yr. Environmental factors have been implicated because the distribution of these tumors follows a regional, rather than a familial, pattern. However, decreased penetrance of a particular gene defect cannot be excluded. Because linkage or other traditional genetic analyses would not be appropriate to investigate the defect(s) associated with ACT in this population, we used comparative genomic hybridization (CGH) to screen for DNA sequence copy number changes in 9 nonfamilial ACT (6 carcinomas and 3 adenomas) from unrelated patients from this region. Six female (aged 10 months to 6 3/4 yr) and 3 male (1 1/12 to 3 1/4 yr) patients were studied. Three carcinomas were at stage I, 1 was at stage II, and another was at stage III. Two carcinomas had evidence of invasion of the vena cava, and 3 were more than 3 cm in size. All patients underwent surgical excision of their tumors; chemotherapy was administered to cancer patients. Currently, all patients are alive and in remission, with the exception of 1 patient with stage III cancer. High mol wt DNA was extracted from tumor tissue obtained at surgery and frozen at -70 C. This DNA was labeled and used for CGH according to standard procedures. Digital image analysis was performed to detect chromosomal gains or losses. CGH evaluation revealed extensive genetic aberrations in both adenomas and carcinomas; there were no significant differences relative to age, gender, size, or stage of the tumor (P > 0.1). Chromosomes and chromosomal regions 1q, 5p, 5q, 6p, 6q, 8p, 8q, 9q, 10p, 11q, 12q, 13q, 14q, 15q, 16, 18q, 19, and 20q demonstrated gains, whereas 2q, 3, 4, 9p, 11, 13q, 18, 20p, and Xq showed losses. The most striking finding was consistent copy number gain of chromosomal region 9q34 in 8 of the 9 tumors. We conclude that both benign and malignant ACT from southern Brazil show multiple genetic aberrations, including a consistent gain of chromosomal region 9q34. This genomic area may harbor genetic defects that predispose to ACT formation and are shared by the patients who were investigated in this study or are accumulated epigenetically under the influence of a common factor, such as an environmental mutagen.     

Genome-wide analysis of cytogenetic aberrations in ETV6/RUNX1-positive childhood acute lymphoblastic leukaemia

The chromosomal translocation t(12;21) resulting in the ETV6/RUNX1 fusion gene is the most frequent structural cytogenetic abnormality among patients with childhood acute lymphoblastic leukaemia (ALL). We investigated 62 ETV6/RUNX1-positive childhood ALL patients by single nucleotide polymorphism array to explore acquired copy number alterations (CNAs) at diagnosis. The mean number of CNAs was 2·82 (range 0-14). Concordance with available G-band karyotyping and comparative genomic hybridization was 93%. Based on three major protein-protein complexes disrupted by these CNAs, patients could be categorized into four distinct subgroups, defined by different underlying biological mechanisms relevant to the aetiology of childhood ALL. When recurrent CNAs were evaluated by an oncogenetic tree analysis classifying their sequential order, the most common genetic aberrations (deletions of 6q, 9p, 13q and X, and gains of 10 and 21) seemed independent of each other. Finally, we identified the most common regions with recurrent gains and losses, which comprise microRNA clusters with known oncogenic or tumour-suppressive roles. The present study sheds further light on the genetic diversity of ETV6/RUNX1-positive childhood ALL, which may be important for understanding poor responses among this otherwise highly curable subset of ALL and lead to novel targeted treatment strategies.     

Comparative genomic hybridization analysis of adrenocortical tumors

Comparative genomic hybridization (CGH) is a molecular cytogenetic technique that allows the entire genome of a tumor to be surveyed for gains and losses of DNA copy sequences. A limited number of studies reporting the use of this technique in adult adrenocortical tumors have yielded conflicting results. In this study we performed CGH analysis on 13 malignant, 18 benign, and 1 tumor of indeterminate malignant potential with the aim of identifying genetic loci consistently implicated in the development and progression of adrenocortical tumors. Tissue samples from 32 patients with histologically proven adrenocortical tumors were available for CGH analysis. CGH changes were seen in all cancers, 11 of 18 (61%) adenomas, and the 1 tumor of indeterminate malignant potential. Of the adrenal cancers, the most common gains were seen on chromosomes 5 (46%), 12 (38%), 19 (31%), and 4 (31%). Losses were most frequently seen at 1p (62%), 17p (54%), 22 (38%), 2q (31%), and 11q (31%). Of the benign adenomas, the most common change was gain of 4q (22%). Mann-Whitney analysis showed a highly significant difference between the cancer group (mean changes, 7.6) and the adenoma group (mean changes, 1.1) for the number of observed CGH changes (P < 0.01). Logistic regression analysis showed that the number of CGH changes was highly predictive of tumor type (P < 0.01). This study has identified several chromosomal loci implicated in adrenocortical tumorigenesis. Activation of a protooncogene(s) on chromosome 4 may be an early event, with progression from adenoma to carcinoma involving activation of oncogenes on chromosomes 5 and 12 and inactivation of tumor suppressor genes on chromosome arms 1p and 17p.     

A narrow deletion of 7q is common to HCL, and SMZL, but not CLL

To further characterise the genetic background of the two closely related B-lymphocytic malignancies hairy cell leukaemia (HCL), and splenic marginal zone lymphoma (SMZL) we have identified characteristic copy number imbalances by comparative genomic hybridisation (CGH). Based on these findings, areas of special interest were fine mapped, and relevant probes constructed for use in interphase-fluorescence in situ hybridisation (FISH) investigations. Thus, using the CGH data from 52 HCL and 61 SMZL patients, we identified the characteristic profiles of copy number imbalances for both diseases. These were a gain of 5q13-31 (19%) and loss of 7q22-q35 (6%) for HCL, and gain of 3q25 (28%), loss of 7q31 (16%), and gain of 12q15 (16%) for SMZL. A partial loss of 7q unusual for low-malignant B-cell diseases was found to be common to the two diseases. This loss was therefore fine mapped with BAC/PAC clones. Fine mapping revealed that in SMZL the minimal lost region covers 11.4 Mb spanning from 7q31.33 to 7q33 located between sequence tagged site (STS)-markers SHGC-3275 and D7S725. This area was distinct from the commonly deleted 7q region of myelodysplastic syndrome/acute myeloid leukaemia (MDS/AML). A FISH probe specific for the 7q region was constructed. Using this probe in an interphase-FISH investigation we showed the minimal lost 7q-region of HCL and SMZL to be one and the same. In one HCL case, this investigation furthermore showed the extent of the deleted region to be below the detection limit of CGH, whereas interphase-FISH screening of 36 chronic lymphocytic leukaemia (CLL) cases showed no deletion of the 7q area. In conclusion, we have identified characteristic profiles of copy number imbalances in HCL and SMZL and fine mapped the minimal extent of a commonly lost 7q area of special interest. We hypothesise that this region may contain (a) gene(s) important for the pathology of HCL and SMZL.     

Splenic marginal zone lymphomas are characterized by loss of interstitial regions of chromosome 7q, 7q31.32 and 7q36.2 that include the protection of telomere 1 (POT1) and sonic hedgehog (SHH) genes

To further characterize the genotypic features of splenic (S) and nodal (N) marginal zone lymphomas (MZL) we compared eight SMZL and five NMZL by array-based comparative genomic hybridization (aCGH). Arbitrarily, aberrations were divided into major imbalances, defined as gains or losses involving five or more contiguous genetic loci, and minor imbalances, defined as those involving four or fewer loci. SMZL, but not NMZL, demonstrated major imbalances. These included deletions involving various lengths of 7q (three cases), and 14q23q24 (one case) and gains of 9p13p21 (one case), 13q21q33 (one case) and 16p13.1 (one case). Common minor imbalances in SMZL were: loss of sonic hedgehog gene ( SHH ) at 7q36.2 (four cases), loss of protection of telomere 1 gene ( POT1 ) at 7q31.32 (three cases), and gain of glioma associated oncogene 1 ( GLI1 ) at 12q13.2 (three cases). Common minor alterations in NMZL were: loss of the fas-associated via death domain gene ( FADD ) at 11q13.2 (three cases) and gain of GLI1 (five cases). In conclusion, SMZL, but not NMZL, demonstrates large genomic imbalances and frequent loss of the 7q31.32 and 7q36.2 regions involving POT1 and SHH , respectively. In NMZL, loss of FADD and gain of GLI1 are frequent events.     

DNA copy number changes in diffuse large B-cell lymphoma--comparative genomic hybridization study

We studied DNA copy number changes in diffuse large B-cell lymphoma using comparative genomic hybridization analysis on 20 primary tumors and on 12 recurrent tumors excised after chemotherapy or radiotherapy. Twenty-nine (91%) of the cases showed abnormal copy number karyotypes. Chromosomal regions at X (41%), 1q (38%), 7 (31%), 3 (24%), 6p (21%), 11 (21%), 12 (21%), and 18 (21%) were most frequently gained, and the most common losses involved 6q (38%), X (21%), 1p (14%), and 8p (10%). High-level amplifications were observed at 6p23-ter, 10p12-14, 17p1l.2, 18q21-ter, and Xq22-ter, all but 18q appearing only in the recurrent tumors. Gains (median, 2; range, 0 to 10) were more frequent than losses (median, 1; range, 0 to 7; P = .0004). The median number of aberrations found in the recurrent tumors (6.5) was greater than that in the primary tumors (2; P = .01). The copy number changes found in the recurrent tumors were more random than those found in the primary tumors, which were mainly located in the most frequently affected regions. Our findings are in line with those observed using conventional cytogenetic analysis, but especially novel high-level amplifications were detected. Southern blot analysis showed BCL2 amplification, but not translocation t(14;18)(q32;q21), in cases in which a gain at 18q was detected by comparative genomic hybridization, which strongly suggests that, in addition to translocation, gene amplification is another mechanism for the overexpression of the BCL2 protein.     

MDM2 gene amplification and lack of p53 point mutations in Hodgkin and Reed-Sternberg cells: results from single-cell polymerase chain reaction and molecular cytogenetic studies

Hodgkin's disease (HD) is the most common haematological malignancy after chronic lymphocytic leukaemia, but very little is known about its pathogenesis or the genetic events that contribute to the malignant phenotype of the tumour cells. p53 is assumed to play an important role in the pathogenesis of HD, based on the observation that p53 protein is frequently accumulated in Hodgkin and Reed-Sternberg (H & RS) cells. We investigated single H & RS cells from five different HD patients for point mutations at the genomic level using multiplex polymerase chain reaction amplification and subsequent sequencing. No point mutations were detected in 50 single H & RS cells analysed. Hence, accumulation of p53 protein cannot be explained by mutations within the gene. A genome-wide screening for genomic imbalances using comparative genomic hybridization revealed gain on chromosome 12q14, i.e. the mapping position of the MDM2 gene in several HD cases. Therefore, we assessed the copy number of the MDM2 gene using fluorescence in situ hybridization. In four out of six HD cases analysed, the copy number of the MDM2 gene was found to be increased. As gene amplification is frequently associated with protein overexpression, the observed accumulation of p53 in the nuclei of H & RS cells could be as a result of elevated MDM2 protein levels resulting in stabilization of p53 protein.     

Adrenocorticotropin/3',5'-cyclic AMP-mediated transcription of the scavenger akr1-b7 gene in adrenocortical cells is dependent on three functionally distinct steroidogenic factor-1-responsive elements

The akr1-b7 gene encodes a scavenger enzyme expressed in steroidogenic glands under pituitary control. In the zona fasciculata of the adrenal cortex where its expression is controlled by ACTH, AKR1-B7 detoxifies isocaproaldehyde produced during the first step of steroidogenesis. Three steroidogenic factor-1 (SF-1)-responsive elements (SFREs) are contained within the -510/+41 promoter region, which was previously demonstrated to drive gene expression in transgenic mice adrenal cortex. All these sequences bind at least SF-1 in Y1 adrenocortical cell nuclear extracts and can be activated by overexpression of this factor in HeLa cells. However, the three SFREs show distinct properties regarding akr1-b7 promoter activity in Y1 cells. Whereas the proximal -102 SFRE supports basal promoter activity, the -458 bona fide SFRE is essential for both basal promoter activity and cAMP responsiveness, although it is unresponsive to cAMP when isolated from its promoter context. This suggests that SF-1 is not a cAMP-responsive factor per se. The neighboring SFRE at -503 is a palindromic sequence that binds monomeric and heteromeric SF-1 as well as an adrenal-specific complex. Using MA-10 Leydig cells and Y1-10r9 mutant cells, we provide evidence that its activity in adrenocortical cells depends on the binding of the adrenal-specific factor, which is required for basal and cAMP-induced promoter activity. Furthermore, the -503 site has intrinsic cAMP-sensing ability in Y1 cells, which is correlated with increased adrenal-specific complex binding. Collectively, our results suggest that cAMP responsiveness of the akr1-b7 promoter is achieved through cooperation between the adrenal-specific factor bound to the -503 site and SF-1 bound to the -458 site.     

Novel candidate genes of thyroid tumourigenesis identified in Trk-T1 transgenic mice

For an identification of novel candidate genes in thyroid tumourigenesis, we have investigated gene copy number changes in a Trk-T1 transgenic mouse model of thyroid neoplasia. For this aim, 30 thyroid tumours from Trk-T1 transgenics were investigated by comparative genomic hybridisation. Recurrent gene copy number alterations were identified and genes located in the altered chromosomal regions were analysed by Gene Ontology term enrichment analysis in order to reveal gene functions potentially associated with thyroid tumourigenesis. In thyroid neoplasms from Trk-T1 mice, a recurrent gain on chromosomal bands 1C4-E2.3 (10.0% of cases), and losses on 3H1-H3 (13.3%), 4D2.3-E2 (43.3%) and 14E4-E5 (6.7%) were identified. The genes Twist2, Ptma, Pde6d, Bmpr1b, Pdlim5, Unc5c, Srm, Trp73, Ythdf2, Taf12 and Slitrk5 are located in these chromosomal bands. Copy number changes of these genes were studied by fluorescence in situ hybridisation on 30 human papillary thyroid carcinoma (PTC) samples and altered gene expression was studied by qRT-PCR analyses in 67 human PTC. Copy number gains were detected in 83% of cases for TWIST2 and in 100% of cases for PTMA and PDE6D. DNA losses of SLITRK1 and SLITRK5 were observed in 21% of cases and of SLITRK6 in 16% of cases. Gene expression was significantly up-regulated for UNC5C and TP73 and significantly down-regulated for SLITRK5 in tumours compared with normal tissue. In conclusion, a global genomic copy number analysis of thyroid tumours from Trk-T1 transgenic mice revealed a number of novel gene alterations in thyroid tumourigenesis that are also prevalent in human PTCs.     

Array-CGH identifies cyclin D1 and UBCH10 amplicons in anaplastic thyroid carcinoma

Anaplastic thyroid cancer (ATC) is a rare but highly aggressive disease with largely unexplained etiology and molecular pathogenesis. In this study, we analyzed genome-wide copy number changes, BRAF (V-raf sarcoma viral oncogene homolog B1) mutations, and p16 and cyclin D1 expressions in a panel of ATC primary tumors. Three ATCs harbored the common BRAF mutation V600E. Using array-comparative genomic hybridisation (array-CGH), several distinct recurrent copy number alterations were revealed including gains in 16p11.2, 20q11.2, and 20q13.12. Subsequent fluorescence in situ hybridization revealed recurrent locus gain of UBCH10 in 20q13.12 and Cyclin D1 (CCND1) in 11q13. The detection of a homozygous loss encompassing the CDKN2A locus in 9p21.3 motivated the examination of p16 protein expression, which was undetectable in 24/27 ATCs (89%). Based on the frequent gain in 11q13 (41%; n=11), the role of CCND1 was further investigated. Expression of cyclin D1 protein was observed at varying levels in 18/27 ATCs (67%). The effect of CCND1 on thyroid cell proliferation was assessed in vitro in ATC cells by means of siRNA and in thyroid cells after CCND1 transfection. In summary, the recurrent chromosomal copy number changes and molecular alterations identified in this study may provide an insight into the pathogenesis and development of ATC.     

Characterisation of hairy cell leukaemia by tiling resolution array‐based comparative Genome hybridisation: a series of 13 cases and review of the literature

Little is known about the cytogenetic features and molecular mechanisms behind hairy cell leukaemia (HCL), despite the advances in diagnosis and treatment. Therefore, we used high‐resolution genome‐wide array‐based comparative genomic hybridisation (array‐CGH) and multiplex ligation‐dependent probe amplification (MLPA) to characterise copy number alterations (CNAs) in DNA from 13 cases of HCL. We also summarise CNAs and cytogenetic features in 109 HCL cases comprising our 13 cases and 96 cases from the literature. Genomic array‐CGH revealed imbalances in two out of 13 cases in addition to previously described copy number variants (CNVs) found in healthy individuals. In one case, a 700 kb deletion of 20q11.22 was detected encompassing ten characterised genes, among them the TP53INP2, DNCL2A and ITCH genes. In the second case, trisomy 5, and a deletion of 5p15.2 encompassing a non‐characterised gene AY328033 was found. Altogether only 20/81 (25%) of all cases studied by CGH or gene dose array revealed CNAs. The most common recurrent deletions and breakpoints were 14q22–32 (33%), 6q25 (16%), 2p12 (10%), 22q11 (10%), 17p11–13 (10%), 7q32–36 (9%), 18q11–13 (7%), 1q32–44 (6%), 8p22–23 (6%) and 7q11 (6%). Trisomy 5 occurred in 15%. In addition, several other recurrent breakpoints were identified. Although a number of genomic imbalances were identified in the HCL samples, the genome appeared remarkably stable.     

Chromosome arm 20q gains and other genomic alterations in esophageal squamous cell carcinoma, as analyzed by comparative genomic hybridization and fluorescence in situ hybridization

BACKGROUND/AIMS: Our recent analysis of gastric cancers and colorectal cancers using comparative genomic hybridization revealed a novel, high frequent copy number increases the long arm of chromosome 20 in association with possible involvement of liver metastases and poor prognosis. This led to further comparative genomic hybridization analysis of chromosomal aberrations in primary tumors of esophageal squamous cell carcinoma. The aim of the study presented here was to analyze the chromosomal aberrations and to determine the numbers of copies of AIB1, BTAK, DcR3 and E2F1 as putative target genes on chromosome 20q as well as their expression and relation to clinicopathological features in 41 primary tumors of esophageal squamous cell carcinoma. METHODOLOGY: We used comparative genomic hybridization to screen 41 primary tumors of esophageal squamous cell carcinoma for changes in the number of copies of DNA sequences. To further characterize the gain of DNA sequences at 20q, we also performed fluorescence in situ hybridization analysis. We examined the relationship between these changes and clinicopathological factors. RESULTS: Gains in chromosome arm 20q were detected (34.1%) as well as a high level of gain in 20q12-13 (4.8%). AIB1 amplification was observed in 4.9% (2/41), BTAK amplification in 9.8% (4/41), DcR3 amplification was in 4.9% (2/41), and E2F1 amplification in 7.3% (3/41). The survival of patients with BTAK or E2F1 amplification was significantly lower than that of patients without these abnormalities. CONCLUSIONS: These findings provide evidence for a number of previously unknown genomic aberrations in esophageal squamous cell carcinoma, suggesting the existence of target regions relevant to its progression. Esophageal squamous cell carcinoma with 20q gain showed extensive lung metastases, pleural effusion and liver metastases and poorer prognosis compared to cases without 20q gain. Our results suggest that amplification of BTAK or E2F1 are likely to lead to an increase in the number of malignant phenotypes of esophageal squamous cell carcinoma and that these aberrations can be expected to be useful as markers of poor prognosis.     

Copy number genome alterations are associated with treatment response and outcome in relapsed childhood ETV6/RUNX1-positive acute lymphoblastic leukemia

The clinical heterogeneity among first relapses of childhood ETV6/RUNX1-positive acute lymphoblastic leukemia indicates that further genetic alterations in leukemic cells might affect the course of salvage therapy and be of prognostic relevance. To assess the incidence and prognostic relevance of additional copy number alterations at relapse of the disease, we performed whole genome array comparative genomic hybridization of leukemic cell DNA from 51 patients with first ETV6/RUNX1-positive relapse enrolled in and treated according to the relapse trials ALL-REZ of the Berlin-Frankfurt-Münster Study Group. Within this cohort of patients with relapsed ETV6/RUNX1-positive acute lymphoblastic leukemia, the largest analyzed for genome wide DNA copy number alterations to date, alterations were present in every ETV6/RUNX1-positive relapse and a high proportion of them occurred in recurrent overlapping chromosomal regions. Recurrent losses affected chromosomal regions 12p13, 6q21, 15q15.1, 9p21, 3p21, 5q and 3p14.2, whereas gains occurred in regions 21q22 and 12p. Loss of 12p13 including CDKN1B was associated with a shorter remission duration (P=0.009) and a lower probability of event-free survival (P=0.001). Distribution of X-chromosomal copy number alterations was gender-specific: whole X-chromosome loss occurred exclusively in females, gain of Xq only in males. Loss of the glucocorticoid receptor gene NR3C1 (5q31.3) was associated with a poor response to induction treatment (P=0.003), possibly accounting for the adverse prognosis of some of the ETV6/RUNX1-positive relapses.