Tom Böhling

Gene amplifications in osteosarcoma—CGH microarray analysis

Little is known about the genomic alterations underlying osteosarcoma. We performed a genomewide high‐resolution gene copy number analysis of 22 osteosarcoma samples using comparative genomic hybridization on a cDNA microarray that contained cDNA clones of about 13,000 genes. Nineteen of the 22 cases had amplifications that on average spanned more than 1 Mb and contained more than 10 genes. Numerous regions of gain and loss were identified, and their boundaries were defined at high resolution. Novel amplicons were found at 14q11, 17q25, and 22q11–q13. Earlier‐known large amplified regions were detected at 12q11–q15, 8q24, 6p12–p13, and 17p11–p13 in 8, 6, 5, and 4 of the 22 samples, respectively. Amplification of 12q was observed more frequently (36% of the cases) than previously reported. Previously known small amplicons at 1p34–p36, 1q21, 19q13, and 21q22 were seen in at least three cases. Our results implicate TOM1L2 and CYP27B1 as having roles as novel targets for the 17p and 12q amplicons, respectively. Details (www.helsinki.fi/cmg) of the amplified genes in each amplicon provide valuable raw data for further in silico studies.

Chronic lymphocytic leukaemia patients have a high risk of Merkel-cell polyomavirus DNA-positive Merkel-cell carcinoma.

Background:Immunosuppression and Merkel-cell polyomavirus (MCPyV) infection may have a role in the pathogenesis of Merkel-cell carcinoma (MCC), a rare neuroendocrine carcinoma of the skin.Methods:We studied incidence of chronic lymphocytic leukaemia (CLL) and MCC from the files of the Finnish Cancer Registry and the largest hospital of Finland, Helsinki University Central Hospital, from 1979 to 2006. Presence of MCPyV DNA in MCCs was investigated by quantitative PCR.Results:We identified 4164 patients diagnosed with CLL and 172 diagnosed with MCC. Six patients diagnosed with both diseases were found; CLL was the first diagnosis in four cases and MCC in two. The standardised incidence ratio (SIR) for CLL after the diagnosis of MCC was highly elevated, 17.9 (95% confidence interval (CI), 2.2–64.6; P<0.001), and the SIR for MCC after the diagnosis of CLL was also elevated, 15.7 (3.2–46.0, P<0.01). Merkel-cell polyomavirus DNA was present in all five MCCs with tumour tissue available for analysis.Conclusions:We conclude that patients diagnosed with CLL have a substantially increased risk for MCC, and vice versa. Merkel-cell polyomavirus DNA is frequently present in MCCs that occur in CLL patients. Immunosuppression related with CLL and viral infection might explain the association between CLL and MCC.

Clinical course of nonvisceral soft tissue leiomyosarcoma in 225 patients from the Scandinavian Sarcoma Group.

Leiomyosarcoma of nonvisceral soft tissues is an uncommon malignant tumor; thus, only small numbers of cases have been reported. This study was based on a large series of patients from the Scandinavian Sarcoma Group Register acquired during a 15‐year period (from 1986 to 2001). Follow‐up information was available for all patients.

Association of Merkel cell polyomavirus infection with tumor p53, KIT, stem cell factor, PDGFR-alpha and survival in Merkel cell carcinoma.

Most Merkel cell carcinomas (MCCs) contain Merkel cell polyomavirus (MCPyV) DNA, and the virus likely has a pivotal role in tumor pathogenesis. p53 and the KIT receptor tyrosine kinase have also been implicated in MCC pathogenesis, but little is known about their association with MCPyV infection. We identified 207 patients diagnosed with MCC in Finland in 1979–2004 and reviewed the histological diagnoses. Adequate clinical information, tumor tissue and DNA were available from 87 confirmed MCC cases. Presence of MCPyV DNA was assessed using quantitative PCR; p53, KIT, phospho‐KIT, stem cell factor (SCF) and PDGFRα expression using immunohistochemistry and presence of mutations in KIT exons 9, 11, 13 and 17 and PDGFRA exons 10, 12, 14 and 18 using DNA sequencing. Most (77.0%) of the 87 tumors contained MCPyV DNA and 37 (42.5%) expressed KIT, whereas PDGFRα, p53, SCF and pKIT expression was less common (31.9, 22.8, 8.6 and 4.8%, respectively). No KIT or PFGFRA mutations were detected, but 10 (12.5%) of the 80 tumors studied harbored common PDGFRA exon 10 S478P substitution. Tumor p53 and KIT expression were associated with absence of MCPyV DNA (p = 0.01 and 0.009, respectively). Tumor p53 expression was associated with unfavorable MCC‐specific survival (p = 0.021) and overall survival (p = 0.046), but tumor KIT expression only when stratified by presence of MCPyV DNA. The results suggest that p53 and KIT expression are associated with absence of MCPyV DNA in MCC, and that the molecular pathogenesis of MCC is multifactorial.

Somatic MED12 mutations in uterine leiomyosarcoma and colorectal cancer

Background:Mediator complex participates in transcriptional regulation by connecting regulatory DNA sequences to the RNA polymerase II initiation complex. Recently, we discovered through exome sequencing that as many as 70% of uterine leiomyomas harbour specific mutations in exon 2 of mediator complex subunit 12 (MED12). In this work, we examined the role of MED12 exon 2 mutations in other tumour types.Methods:The frequency of MED12 exon 2 mutations was analysed in altogether 1158 tumours by direct sequencing. The tumour spectrum included mesenchymal tumours (extrauterine leiomyomas, endometrial polyps, lipomas, uterine leiomyosarcomas, other sarcomas, gastro-intestinal stromal tumours), hormone-dependent tumours (breast and ovarian cancers), haematological malignancies (acute myeloid leukaemias, acute lymphoid leukaemias, myeloproliferative neoplasms), and tumours associated with abnormal Wnt-signalling (colorectal cancers (CRC)).Results:Five somatic alterations were observed: three in uterine leiomyosarcomas (3/41, 7%; Gly44Ser, Ala38_Leu39ins7, Glu35_Leu36delinsVal), and two in CRC (2/392, 0.5%; Gly44Cys, Ala67Val).Conclusion:Somatic MED12 exon 2 mutations were observed in uterine leiomyosarcomas, suggesting that a subgroup of these malignant tumours may develop from a leiomyoma precursor. Mutations in CRC samples indicate that MED12 may, albeit rarely, contribute to CRC tumorigenesis.

SLC26A2 (diastrophic dysplasia sulfate transporter) is expressed in developing and mature cartilage but also in other tissues and cell types.

Mutated alleles of the SLC26A2 (diastrophic dysplasia sulfate transporter or DTDST) gene cause each of the four recessive chondrodysplasias, i.e., diastrophic dysplasia (DTD), multiple epiphyseal dysplasia (MED), atelosteogenesis Type II (AO2), and achondrogenesis Type IB (ACG1B). SLC26A2 acts as an Na+-independent sulfate/chloride antiporter and belongs to the SLC26 anion transporter gene family, currently consisting of six homologous human members. Although Northern analysis has indicated some expression in all tissues studied, the only tissue known to be affected by SLC26A2 mutations is cartilage. Abundant SLC26A2 expression has previously been detected in normal human colon by in situ hybridization. We have used in situ hybridization and immunohistochemistry to examine multiple normal tissues for the expression of human SLC26A2. As expected, a strong signal for SLC26A2 mRNA and protein immunostaining were detected in developing fetal hyaline cartilage, while bronchial cartilage showed mRNA expression in adult tissues. SLC26A2 expression could also be detected in eccrine sweat glands, in bronchial glands, and in placental villi. In addition, immunoreactivity for the SLC26A2 protein was observed in exocrine pancreas. Our results suggest a more limited expression pattern for SLC26A2 than that found by Northern analysis. However, SLC26A2 expression is also detected in tissues not affected in chondrodysplasias caused by SLC26A2 mutations. (J Histochem Cytochem 49:973–982, 2001)

Merkel cell carcinoma - a population-based epidemiological study in Finland with a clinical series of 181 cases.

BACKGROUND Merkel cell carcinoma (MCC) is a rare malignancy of the skin, and its incidence is reported to be rising. The purpose of this study was to calculate its incidence and survival ratios, and to describe the clinical characteristics of Merkel cell carcinoma patients in Finland. METHODS We calculated the incidence of MCC based on data from the Finnish Cancer Registry. In addition, patient files from hospitals and primary health care centres were reviewed for detailed data on the treatment and disease recurrence of 181 patients diagnosed with MCC in Finland during 1983-2004, and relative survival ratios were calculated for them. RESULTS The incidence (per 100,000) of MCC in Finland in 1989-2008 was 0.11 for men and 0.12 for women, adjusted for age to the world standard population. The mean age at diagnosis was 76 years (range 27-100), and 69% of the patients were women. The most common site of the primary tumour was the head and neck (53%). No extra benefit was gained from a wide surgical margin (≥ 2 cm) compared to a margin of 0.1-0.19 cm, but an intralesional excision was more often associated with local recurrence. None of the patients with Stage I-II disease who had received postoperative radiotherapy to the tumour bed had a local recurrence. The 5-year relative survival ratio amongst men was 36% (95% confidence interval 20-54%), and amongst women 69% (56 to -82%). CONCLUSIONS MCC is a rare disease in Finland, with incidence rates similar to those in the other Nordic countries. Our results support the view that complete excision with clear margins and post operative radiotherapy decrease local recurrences.

Characterization of the NF2 protein merlin and the ERM protein ezrin in human, rat, and mouse central nervous system.

The neurofibromatosis 2 (NF2) protein, merlin, is structurally related to the ERM (ezrin-radixin-moesin) protein family of membrane-cytoskeleton linkers and is mutated in nervous system tumors. Apart from tumor suppressor activity, merlins functions are poorly understood. We compared the localization and expression of merlin and ezrin in developing and adult brain and in brain-derived progenitor cells. Both proteins were widely but differentially expressed in human, rat, and mouse brain. In brain tissue and neuronal progenitor cell cultures merlin was predominantly found in neurons while ezrin was expressed in astrocytes. Merlin expression was seen from E11 in mouse embryos, whereas ezrin was present earlier. Both proteins were expressed in embryonic mouse neurospheres, where ezrin was specifically localized in filopodia of adherent neuronal progenitor cells. Subcellular analysis demonstrated ezrin in fine filopodial structures in astrocytes, while merlin was detected in neuronal synaptic junctions. The widespread expression of merlin in brain and its association with protein kinase A suggest a role for merlin in brain biology.

Mutations in Exon 1 Highlight the Role of MED12 in Uterine Leiomyomas

Mediator regulates transcription by connecting gene‐specific transcription factors to the RNA polymerase II initiation complex. We recently discovered by exome sequencing that specific exon 2 mutations in mediator complex subunit 12 (MED12) are extremely common in uterine leiomyomas. Subsequent screening studies have focused on this mutational hot spot, and mutations have been detected in uterine leiomyosarcomas, extrauterine leiomyomas and leiomyosarcomas, endometrial polyps, and colorectal cancers. All mutations have been missense changes or in‐frame insertions/deletions. Here, we have analyzed 611 samples representing all above‐mentioned tumor types for possible exon 1 mutations. Five mutations were observed, all of which were in‐frame insertion/deletions in uterine leiomyomas. Transcriptome‐wide expression data revealed that MED12 exon 1 and exon 2 mutations lead to the same unique global gene expression pattern with RAD51B being the most upregulated gene. Immunoprecipitation and kinase activity assays showed that both exon 1 and exon 2 mutations disrupt the interaction between MED12 and Cyclin C and CDK8/19 and abolish the mediator‐associated CDK kinase activity. These results further emphasize the role of MED12 in uterine leiomyomas, show that exon 1 and exon 2 exert their tumorigenic effect in similar manner, and stress that exon 1 should be included in subsequent MED12 screenings.

CanGEM: mining gene copy number changes in cancer

The use of genome-wide and high-throughput screening methods on large sample sizes is a well-grounded approach when studying a process as complex and heterogeneous as tumorigenesis. Gene copy number changes are one of the main mechanisms causing cancerous alterations in gene expression and can be detected using array comparative genomic hybridization (aCGH). Microarrays are well suited for the integrative systems biology approach, but none of the existing microarray databases is focusing on copy number changes. We present here CanGEM (Cancer GEnome Mine), which is a public, web-based database for storing quantitative microarray data and relevant metadata about the measurements and samples. CanGEM supports the MIAME standard and in addition, stores clinical information using standardized controlled vocabularies whenever possible. Microarray probes are re-annotated with their physical coordinates in the human genome and aCGH data is analyzed to yield gene-specific copy numbers. Users can build custom datasets by querying for specific clinical sample characteristics or copy number changes of individual genes. Aberration frequencies can be calculated for these datasets, and the data can be visualized on the human genome map with gene annotations. Furthermore, the original data files are available for more detailed analysis. The CanGEM database can be accessed at http://www.cangem.org/.