Tumors of the breast

Jacob Larsen, The Finsen Laboratory, Copenhagen University Hospital

The aim is to improve the understanding of the genetic mechanisms of initiation and progression of breast cancer. We hope to achieve this by applying CGH on cell clones from a series of histopathologically and clinically well characterized premalignant and malignant breast lesions. In order to improve the CGH analysis, we will examine purified preparations of the individual cell clones in the tissue. We will use fluorescence activated cell sorting for "microdissecting" the tissue with respect to the stemline DNA content, so that pure fractions of the individual aberrant cell clones can be obtained for the CGH analysis. Aberrant chromosomal regions will then be further investigated using other molecular biological techniques including fluorescence in situ hybridization, for tracking the abnormality to distinct genomic loci. The potential of CGH, is the possibility for discovery of genomic loci that are coding for yet unknown oncogenes or anti-oncogenes with an important role in initiation and progression of cancer. Correlating the cytogenetic data to the individual subclones in DNA multiploid lesions may improve the insight in clonal evolution in tumors.

Supported by: Danish Cancer Society

Tumors of the uterine cervix

Maria Kirchhoff, Cytogenetic Laboratory, Copenhagen University Hospital

Cervical cancer and preinvasive neoplasia constitute a major public health problem worldwide. Infection with human papillomavirus (HPV) is considered to be the major etiologic factor in cervical carcinogenesis. However, most HPV-induced cervical lesions are reversible and for malignant transformation to occur, it is almost certain that additional somatic events are required. Such events may be chromosomal aberrations affecting tumor suppressor genes or cellular oncogenes. According to the multistep nature of carcinogenesis, cervical lesions should have a sequential accumulation of genetic changes as they progress toward malignancy and invasion. It is therefore important to the understanding of cervical carcinogenesis that recurrent chromosomal aberrations are identified.
We performed CGH analysis on 51 preinvasive cervical lesions, which included eight cases of koilocytosis, six mild dysplasias, 20 moderate dysplasias and 17 cases of severe dysplasia/carcinoma in situ (CIS) and on 29 advanced-stage cervical carcinomas. Chromosome aberrations were detected in 1 case of koilocytosis, 1 case of mild dysplasia, nine moderate dysplasias and 13 severe dysplasi/CIS. A total of 116 chromosome arms were involved. All invasive carcinoma showed chromosome aberrations. Gain of chromosome 3q was detected in two moderate dysplasias (10%), in 35% of severe dysplasia/CIS and in 72% of invasive carcinoma. 
Fifteen of 55 chromosome aberrations found in the early preinvasive lesions were present in at least 34% of invasive tumors including gains of chromosome arms 1q, 3q, 5p, and 15q and losses of chromosome arms 4p, 6q, and 13q. The additional aberrations were occasionally seen in invasive tumors. 27 of 61 chromosome aberrations found in severe dysplasia/CIS were present in at least 34% of invasive tumors, while additional aberations were seen occasionally in invasive tumors. The most frequent gains in invasive carcinoma occurred on 3q (72%), 1q (45%), 8q (41%), 5p (34%), and Xq (34%) and frequent losses were mapped to chromosome arms 3p (52%), 11q (48%), 13q (38%), 6q (38%), and 4p (34%). 
The number of chromosome aberrations detected in the cervical lesions is higher than previously reported. This is most likely explained by the use of HR-CGH instead of fixed thresholds. (Kirchhoff et al., 1999; Kirchhoff et al., 2001)

Supported by: Danish Cancer Society


Seminomatous germ cell tumors 

Anne Marie Ottesen, Dept. of Growth & Reproduction, National University Hospital of Copenhagen.
Supervisors: Ewa Rajpert-De Meyts and Niels E. Skakkebæk, Dept. of Growth & Reproduction,
National University Hospital of Copenhagen.
Collaborating Laboratories:
Chromosome Laboratory, Dept. of Clinical Genetics, National University Hospital of Copenhagen.
Institute of Pathology, National University Hospital of Copenhagen.
Dept. of Oral Diagnostics, School of Dentistry, University of Copenhagen.

Testicular germ cell cancer is the most frequent malignancy of young males and evidence seems to indicate an increasing frequency in some geographical areas. Although the general and presently used methods of treatment have proved to be successful, they may be traumatic to the patient and lead to a decreased quality of life. Consequently it is of great importance to elucidate the pathogenesis of testicular germ cell cancer by cytogenetic and molecular biological investigations.
Comparative Genomic Hybridization (CGH) has been used to evaluate tissue specimens from 16 seminomas by screening the entire genomes for chromosomal aberrations. A characteristic pattern of losses and gains within the genomes was detected in 94% of the seminomas by comparing the ratio profiles of the tumours with a standard of cytogenetically normal genomic DNA. Chromosomal imbalances of 12p and 21q appeared most consistently. The high number of aberrations, however, excluded firm conclusions concerning the putative effect of specific genes of importance to malignant transformation. The results highlighted the need of further studies focussing on earlier stages of tumour formation (Ottesen et al., 1997).
According to a commonly accepted theory the preinvasive precursor of germ cell tumours is the carcinoma in situ cell (CIS). Presently only a few reports have cytogenetic data of CIS-cells due to a series of technical problems i.e. insufficient conditions for culturing. Application of CGH method is restricted by two facts: a low number of CIS-cells and that the CIS-cells are surrounded by other cell types. Consequently our aim for a future study is initially to isolate the CIS-cells from the tubules by laser-microdissection followed by an amplification of the entire genome by DOP-PCR (Degenerate oligonucleotide primed PCR). Finally this DNA will be screened for genomic aberrations using the CGH-method. In addition CGH will be applied to other cells of the testis e.g. the germ cell supporter: the Sertoli cell and the testosterone producing Leydig cell in order to investigate if chromosomal imbalances of these cells could be involved in the malignant transformation of germ cells. In this way we hope to contribute to elucidation of the pathogenesis of testicular germ cell cancer.
Latest publication Ottesen et al., 2003
Supported by: Danish Cancer Society

Childhood acute lymphoblastic leukemia

Tim Kristensen, Pediatric Clinic II, Rigshospitalet, Copenhagen University Hospital
Study coordinator: Kjeld Schmiegelow, Pediatric Clinic II, Rigshospitalet, Copenhagen University Hospital
Collaborating laboratories and departments:
Claes Lundsteen, Dept. of Clinical Genetics, Hans O. Madsen, Dept.of Clinical lmmunology, Jørgen K. Larsen, lb Jarle Christensen, Finsen Laboratory, Rigshospitalet, Copenhagen University Hospital, Denmark, Finn Wesenberg, The Department of Pediatrics, Rikshospitalet, Oslo, Norway, Olafur G. Johnsson, The Department of Pediatrics, Landsspitalin, Reykjavik, Iceland

ALL is the most common malignant disease in children. Each year 175 new cases of childhood ALL are diagnosed in the Nordic countries. Exploration of the cytogenetic changes, which are the basic events of the malignant degeneration of the leukemic clone will enhance the biologic understanding of the disease and could be helpful in more individually risk adapted therapy programs. However, standard cytogenetic analyses reveal genomic aberrations in only 40-50% of all patients. Comparative genomic hybridisation (CGH) is a new powerful tool in the study of genomic amplifications and deletions in malignant cells. The present Danish, Icelandic/Norwegian multi center study will analyse CGH data in relation to conventional cytogenetics, the DNA index of the malignant clone, and the early response to therapy. Latest publication Kristensen et al., 2003

Supported by: Danish Cancer Society, Lundbeck Fond, Otto Christensens Fond

Investigation of dysmorphic children with HR-CGH, telomeric probes and SKY-karyotyping (CTS project).

Maria Kirchhoff, Hanne Rose, Tommy Gerdes and Claes Lundsteen, Copenhagen and Søren Pedersen, Eigil Kjeldsen and Steen Kølvraa, Aarhus.

Children with autosomal chromosome abnormalities show varying clinical pictures depending on the chromosomes involved. They are usually mentally retarded and dysmorphic with congenital malformations. Such children are often referred to clinical genetics departments where karyotyping subsequently shows normal chromosomes. In some of these children with apparently normal karyotypes new molecular cytogenetic techniques have made it possible to identify small deletions, duplications or cryptic translocations which were not detected by standard karyotyping. We do, however, not know how frequent such small abnormalities may be found among those children.

In collaboration with the Department of Clinical Genetics, Aarhus Kommunehospital, we are conducting a prospective study of 100 dysmorphic children with normal karyotypes by using SKY karyotyping and subtelomeric probes (Aarhus) and HR-CGH (Copenhagen). We hope to learn how frequent we can expect to detect abnormalities in this kind of patients by using these three methods isolated or in combination. Latest publication Kirchhoff et al., In press

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Copyright: Dept. of Clinical Genetics, Rigshospitalet, Copenhagen, Denmark   -   Last revised: July 12, 2005