Eija Mahlamäki

Frequent amplification of 8q24, 11q, 17q, and 20q-specific genes in pancreatic cancer

Genetic changes involved in the development and progression of pancreatic cancer are still partly unknown, despite the progress in recent years. In this study, comparative genomic hybridization analysis in 31 pancreatic cancer cell lines showed that chromosome arms 8q, 11q, 17q, and 20q are frequently gained in this tumor type. Copy number analysis of selected genes from these chromosome arms by fluorescence in situ hybridization showed amplification of the MYC oncogene in 54% of the cell lines, whereas CCND1 was amplified in 28%. In the 17q arm, the ERBB2 oncogene was amplified in 20% of the cell lines, TBX2 in 50%, and BIRC5 in 58%, indicating increased involvement toward the q telomere of chromosome 17. In the 20q arm, the amplification frequencies varied from 32% to 83%, with the CTSZ gene at 20q13 being most frequently affected. These results illustrate that amplification of genes from the 8q, 11q, 17q, and 20q chromosome arms is common in pancreatic cancer.

Comparative genomic hybridization reveals frequent gains of 20q, 8q, 11q, 12p, and 17q, and losses of 18q, 9p, and 15q in pancreatic cancer

Comparative genomic hybridization (CGH) was used to screen for genomic imbalances in 24 exocrine pancreatic carcinomas, including 11 low‐passage cell lines (4–8 subcultures) and 13 uncultured samples. Aberrations were found in all cell lines and in seven of the 13 biopsies. The most frequent changes in the cell lines were gains of 20q (91%), 11q (64%), 17q (64%), 19q (64%), 8q, 12p, 14q, and 20p (55%), and losses of 18q (100%), 9p (91%), 15q (73%), 21q (64%), 3p (55%), and 13q (55%). High‐level gains (tumor to normal ratio over 1.5) were detected at 3q, 6p, 7q, 8q, 12p, 19q, and 20q. Among the tumor biopsies, overrepresentations of 7p and 8q were most common (31%), followed by 5p, 5q, 11p, 11q, 12p, and 18q (23%), whereas the most frequent losses involved 18p and 18q (31%) and 6q and 17p (23%). The genetic changes in nine samples obtained from metastatic lesions did not differ significantly from those in 15 primary carcinomas. Most of the gains and losses detected in this CGH study correspond well to those identified in previous cytogenetic and molecular genetic investigations of pancreatic carcinomas. However, frequent gain of 12p and loss of 15q have not been previously reported. Molecular genetic analyses of these chromosome arms are warranted, and may lead to the discovery of novel genes important in pancreatic carcinogenesis. Genes Chromosomes Cancer 20:383–391, 1997.

Comparative Genomic Hybridization Reveals Frequent Losses of Chromosomes 1p and 3q in Pheochromocytomas and Abdominal Paragangliomas, Suggesting a Common Genetic Etiology

Pheochromocytomas and abdominal paragangliomas are rare, catecholamine-producing tumors that arise from the chromaffin cells derived from the neural crest. We used comparative genomic hybridization (CGH) to screen for copy number changes in 23 pheochromocytomas and 11 abdominal paragangliomas. The pattern of copy number changes was similar between pheochromocytomas and paragangliomas, with the most consistent finding being loss of 1cen-p31, which was detected in 28/34 tumors (82%). Losses were also found on 3q22-25 (41%), 11p (26%), 3p13-14 (24%), 4q (21%), 2q (15%), and 11q22-23 (15%), and gains were detected on 19p (26%), 19q (24%), 17q24-qter (21%), 11cen-q13 (15%), and 16p (15%). Losses of 1p and 3q were detected in the majority of tumors, whereas gains of 19p and q, 17q, and 16p were seen only in tumors with six or more CGH alterations. This progression of genetic events did not correspond with the conversion to a malignant phenotype. CGH alterations involving chromosome 11 were more frequent in the malignant tumors, compared with the benign tumors (9/12 versus 3/16). In summary, we propose that pheochromocytomas and abdominal paragangliomas, which share many clinical features, also have a common genetic origin and that the loss of 1cen-p31 represents an early and important event in tumor development.

Microarray analyses reveal strong influence of DNA copy number alterations on the transcriptional patterns in pancreatic cancer: implications for the interpretation of genomic amplifications

DNA copy number alterations are believed to play a major role in the development and progression of human neoplasms. Although most of these genomic imbalances have been associated with dysregulation of individual genes, their large-scale transcriptional consequences remain unclear. Pancreatic carcinomas frequently display gene copy number variation of entire chromosomes as well as of chromosomal subregions. These changes range from homozygous deletions to high-level amplifications and are believed to constitute key genetic alterations in the cellular transformation of this tumor type. To investigate the transcriptional consequences of the most drastic genomic changes, that is, genomic amplifications, and to analyse the genome-wide transcriptional effects of DNA copy number changes, we performed expression profiling of 29 pancreatic carcinoma cell lines and compared the results with matching genomic profiling data. We show that a strong association between DNA copy numbers and mRNA expression levels is present in pancreatic cancer, and demonstrate that as much as 60% of the genes within highly amplified genomic regions display associated overexpression. Consequently, we identified 67 recurrently overexpressed genes located in seven precisely mapped commonly amplified regions. The presented findings indicate that more than one putative target gene may be of importance in most pancreatic cancer amplicons.

Pancreatic adenocarcinoma—Genetic portrait from chromosomes to microarrays

Pancreatic adenocarcinoma is the fifth leading cause of cancer death with a 5‐year survival rate of less than 5%. Although the role of a few known oncogenes and tumor suppressor genes in the development of pancreatic cancer is fairly well established, it is obvious that the majority of genetic changes responsible for the initiation and progression of this disease are still unknown. In this review, the authors will discuss the results from various genome‐wide screening efforts, from traditional chromosome analyses to modern DNA microarray studies, which have provided an enormous amount of information on genetic alterations in pancreatic adenocarcinoma. Exciting findings have emerged from these studies, highlighting multiple potential chromosomal regions that may harbor novel cancer genes involved in the molecular pathogenesis of this lethal disorder. These findings complete the picture of pancreatic adenocarcinoma as a genetically highly complex and heterogenous tumor type with an ongoing instability process. In addition, the precisely localized copy number changes offer a valuable starting point for further studies required to identify the genes involved and to characterize their potential functional role in the development and progression of pancreatic adenocarcinoma.

Detailed genomic mapping and expression analyses of 12p amplifications in pancreatic carcinomas reveal a 3.5-Mb target region for amplification

Previous cytogenetic and comparative genomic hybridization (CGH) analyses have shown that the gain of chromosome arm 12p is frequent in pancreatic carcinomas. We investigated 15 pancreatic carcinoma cell lines using CGH, fluorescence in situ hybridization (FISH), and semiquantitative polymerase chain reaction (PCR) to characterize 12p amplifications in detail. The CGH analysis revealed gains of 12p in four of the cell lines and local amplification within 12p11–12 in six cell lines. By FISH analysis, using precisely mapped YAC clones, the commonly amplified region was found to be approximately 5 Mb. The amplified segment extended from YAC 753f12, covering the KRAS2 locus, to YAC 891f1, close to the centromere. A semiquantitative PCR methodology was used to estimate genomic copy numbers of 14 precisely mapped expressed sequence tags (ESTs) and sequence‐tagged sites, located within this interval. The level of amplification ranged from two‐ to 12‐fold. The produced gene copy profiles revealed a 3.5‐Mb segment with various local amplifications. This region includes KRAS2 and ranges from D12S1617 to sts‐N38796. Two of the cell lines (primary and metastatic tumor from the same patient) showed amplification peaks within the distal region of this segment, two had peaks within the proximal region, one showed subpeaks in both regions, and one displayed amplification of the entire region. Chromosome segment‐specific cDNA array analysis of 29 expressed sequences within the whole interval between D12S1617 and sts‐N38796 indicated overexpression of four ESTs, two corresponding to DEC2 and PPFIBP1, and two to ESTs with unknown function. Expression analysis of these and of KRAS2 showed specific overexpression in the six cell lines with local 12p amplifications. These findings indicate two target regions within the 3.5‐Mb segment in 12p11–12, one proximal including PPFIBP1, and one distal including KRAS2.

Acquired X-chromosome aneuploidy in children with acute lymphoblastic leukemia

BACKGROUND A cytogenetic study of 75 consecutive children with ALL revealed a normal karyotype, a low hyperdiploid karyotype (including 47-50 chromosomes), and a high hyperdiploid karyotype (including > 50 chromosomes) in 10, 12, and 33 patients, respectively. An acquired extra X-chromosome was detected at diagnosis by conventional cytogenetics in 29 (88%) of 33 children with a high hyperdiploid karyotype and in 4 (33%) of 12 children with a low hyperdiploid karyotype. X-chromosome aneuploidy was retrospectively studied by fluorescence in situ hybridization (FISH) in eight and 20 patients with a normal and a hyperdiploid karyotype, respectively. PROCEDURE A classical cytogenetic study was performed according to standard methods. FISH with the centromeric probe specific to X-chromosome was used to study interphase cells of bone marrow or blood samples. RESULTS An extra X-chromosome was found by FISH in all 13 patients with a high hyperdiploid or tetraploid, in 6 of 7 patients with a low hyperdiploid, and in none with a normal karyotype. Two children with a normal karyotype displayed monosomy X. Altogether, 57.3% of newly diagnosed children displayed X-chromosome aneuploidy. CONCLUSIONS Out study indicates that X-chromosome aneuploidy may be the most common chromosome abnormality in childhood ALL. It can be detected in nearly all children with a high hyperdiploid karyotype and up to one-half of the patients with a low hyperdiploid karyotype. FISH with an X-chromosome centromeric probe is a rapid and simple tool to detect an abnormal clone at diagnosis in the majority of children with ALL and is useful in confirming remission in these patients.

Comparative Genomic Hybridization and Conventional Cytogenetic Analyses in Childhood Acute Myeloid Leukemia

Comparative genomic hybridization (CGH) analysis was performed on bone marrow specimens from 19 children with acute myeloid leukemia (AML) at diagnosis. The results of CGH were compared to those of conventional cytogenetic analysis. The most common CGH aberrations were gains of whole chromosomes 6 and 8, both of which appeared three times. Two losses were seen twice; losses of whole chromosomes 7 and X. The CGH findings were concordant with the results of conventional karyotyping. CGH did not add new information to the karyotypes. Since no high-level amplification was found among the samples and standard karyotyping was highly successful, we do not advocate routine use of CGH in the diagnostic evaluation of childhood AML.

Intraventricular haemorrhage in very-low-birthweight preterm infants: association with low prothrombin activity at birth.

AIM To determine the occurrence of intraventricular haemorrhage (IVH) and its association with coagulation factors at birth in preterm neonates born before 30 wk gestation. METHODS 38 neonates (median gestational age 27 wk, range 24-29 wk; median birthweight (BW) 933 g, range 515-1760 g) admitted to the neonatal intensive care unit were studied. Blood samples for coagulation factors were taken within 2 h after birth. The first cranial ultrasonographic examination was performed within the first 3 d. The occurrence of IVH was tested statistically by the Mann-Whitney U-test for association with the activity of coagulation factors and clinical variables. RESULTS Thirteen IVHs occurred within the first 3 d of life. IVH was associated with BW <1000 g (p=0.012), low mean blood pressure within the first 2 d (p=0.026), gestational age <27 wk (p=0.054), low Apgar scores (<7) at 1 min (p=0.078) and intrauterine growth restriction (p=0.072). At birth (samples drawn with a median of first 36 min of life), infants with subsequent IVH had statistically significantly lower prothrombin (factor II) activity (p=0.024) than infants without IVH. CONCLUSION The measured low prothrombin may have been affected by a prior bleeding event. Nevertheless, preterm infants with low prothrombin activity may be susceptible to IVH, or to the progression of it, if left undiagnosed.

Analysis of phenotype and genotype of individual cells in neoplasms

The authors describe a combination technique enabling detection of in situ hybridization (ISH) signals from chromosome-specific probes in interphase or mitotic cells that still retain the alkaline phosphatase antialkaline phosphatase (APAAP) or Sudan black B (SBB) staining reactions (simultaneous detection) or have been first classified morphologically and then by APAAP or SBB. The technique can be used on cell suspensions, in situ cultures and tissue sections. Examples from leukemias (chronic lymphocytic, myeloid, and acute myeloid leukemia) and solid tumors (chondromyxoid fibroma and glioblastoma) illustrate the potential of the technique in investigation of cancer tissue heterogeneity. In leukemias, it can be used to study cell lineage involvement, stem cells, and minimal residual disease, as well as to monitor therapy. In solid tumors, it can be used to identify neoplastic areas of tissue and to track the site of origin of neoplastic cells. Finally, it can be used to study the significance of chromosome abnormalities in carcinogenesis.