Clinical Cytogenetic and Molecular Analysis in a Research Perspective

A primary goal in the effort to understand how cancer occurs is to find out which genes characterise which cancers. The research interest of the field of cancer cytogenetics is primarily the identification of chromosomal aberrations in cancer cells. Our main research goal is to characterise and understand the acquired genomic aberrations that bring about a neoplastic transformation of susceptible target cells. The research is performed in parallel with diagnostic analyses of haematological malignancies and solid tumours.

The Philadephia Chromosome The most famous example of an acquired chromosomal change in malignancy is the Philadelphia chromosome - an abnormality where part of chromosome 9 is transferred to chromosome 22. The chromosome abnormality causes chronic myeloid leukemia (CML), and is a specific finding in the genes of a patient's white blood cells.

At the cellular level, cancer is a genetic disease in that there are stable, acquired changes of the genetic mass in selected body cells that allow them to escape normal cell division control and become tumour cells. Many of these changes are visible microscopically as balanced chromosome aberrations where two chromosomes exchange material, i.e., translocations. Often the breakpoints of the chromosomes are found where such translocations have occurred, the formation of fusion genes with carcinogenic activity, i.e., activated oncogenes. Knowing the karyotypic changes of tumours is a fundamental goal in cancer biology. After all, the chromosome is an organisational level of unquestionable importance in the packaging of the cell’s genetic material.

The research begins by finding specific cytogenetic aberrations in various cancers. Then we take the investigation to the molecular level, searching for the corresponding changes in genes and DNA primary structure. This approach has shown to be successful in all three main research areas at our section for Cancer Cytogenetics:

• Gynaecologic tumours
• Brain tumours
• Connective tissue tumours and haematological malignancies.

Our unique area of expertise is the culturing and chromosome analysis of neoplastic cells. We also have extensive experience with fluorescence in situ-based (FISH) analyses, and the search by molecular means for fusion genes brought about by chromosomal rearrangements. Our approach by combining the two screening techniques, G-band karyotyping and next-generation sequencing, to this end are unique. It has led to the discovery of several cancer-specific fusion genes during the past years.

Information from cytogenetic analyses and RNA-sequencing is combined. In this way, the cytogenetic information can be used to select among the numerous candidate fusion genes suggested by the high throughput RNA-sequencing, to find only those that map to chromosomal breakpoints. The actual presence of these genes is then validated by PCR and Sanger sequencing analyses.

We aim to find chromosomal and molecular aberrations specific to each cancer type analysed that will help the diagnostic and prognostication of each disease. Our goal is to implement the identification of such rearrangements in the clinical routine. We will prioritise the following projects for the next period:

1. Studies of Connective Tissue Tumours and Haematological Malignancies
2. Cytogenetic and Molecular Analysis of Female Genital Tract Tumours

Studies of Connective Tissue Tumours and Haematological Malignancies

The identification of rare or variant chromosomal rearrangements is sample-based and leads directly to the field of personalised medical treatment where each patient is seen as a single and unique case/entity. Our scientific work on mesenchymal tumours goes back 30 years and has allowed reclassification of many tumour entities, e.g., endometrial stromal tumours and low-grade fibromyxoid sarcoma.

In low-grade fibromyxoid sarcoma, the FUS-CREB3L2 fusion is pathognomonic, i.e., specifically found in only this tumour entity. Of note are also the investigations of myoepithelioma/myoepithelio carcinoma/mixed tumours initiated ten years ago that have led into the genetic characterisation of these tumours with rearrangements of the EWSR1 gene.

To detect cancer-specific fusions has become a central goal within cancer genetic research, and much effort and methodological ingenuity are directed towards it. Hundreds of new and cancer-specific fusion genes have been detected in many tumour types through various combinations of methods, and the search is unabating. We have discovered more than 50 tumour-specific fusion genes. Thirty such fusions are now present in the Oncoming Childhood Cancer Research Assay, commercially available for the Ion Torrent technology.

Because the generation of oncogenic fusion genes in neoplastic cells by acquired clonal chromosomal rearrangements, mostly translocations, is a primary mechanism of pathogenesis in leukaemias, lymphomas, and solid tumours, knowledge about them is essential if one aims to understand tumorigenesis in any fundamental way. Different tumour types are characterised by specific translocations which lead to fusion genes of equal specificity, and the discovery of which fusions correspond to which translocations also contributes diagnostically toward the goal of creating an alternative classification system of neoplasms based on pathogenesis rather than phenotype. This opens up for the long-term possibility of personalised medicine in which each (in principle; in practice, this is way more problematic) cancer patient has the underlying pathogenetic mechanism of his/her tumour determined as part of the diagnostic proceedings, whereupon the patient can be treated with drugs generated to counteract that tumour’s particular molecular genetic rearrangement in a completely specific manner. Although this goal is still far into the future, the advent of imatinib mesylate treatment of patients with chronic myeloid leukaemia (CML) and a few other cancerous disorders with abnormal tyrosine kinase activity shows that it is not unrealistic. Along these lines, we envisage this project as a challenge to current clinical/diagnostic practices and foresee that new, clinically important knowledge is likely to be created investigating the chromosomal aberrations and their gene-level consequences in rare tumour entities.

Our researchers will investigate both rare benign and malignant neoplasms. Our present focus is on leiomyomas, leiomyosarcomas, chondrosarcomas, endometrial stroma sarcomas, and angiosarcomas. To begin with, we plan to perform NGS on 15 cases of angiolipoma.

With regards to hematologic malignancies, investigations will be performed on acute myeloid leukaemia’s (AML) and acute lymphatic leukaemia’s (ALL) showing rare cytogenetic rearrangements. Whenever a fusion gene is found, more leukaemias with similar cytogenetic features will be examined to see whether the fusion gene is recurrent and also to learn something about the fusion gene’s impact on disease appearance/clinical outcome. In addition, we plan to investigate 20 leukaemia patients with hypocellular bone marrow and normal cytogenetics. These patients seem to constitute a fairly distinct haematological subgroup, which can be treated with low dose melphalan resulting in durable complete clinical remissions. Identification of their pathogenetic basis would be most interesting. Furthermore, cases of ALL with rare but non-random chromosomal abnormalities will be investigated.

Cytogenetic and Molecular Analysis of Female Genital Tract Tumours

The research interest in the field of cancer cytogenetics goes primarily on the identification of chromosomal aberrations in cancer cells. The research is performed in parallel with diagnostic analyses of haematological malignancies and solid tumours. Cancer of the female genital tract is the third most common group of malignancies in women, exceeded in frequency only by cancer of the breast and the digestive tract. Most of the cancers are of the ovaries and uterus, but tumours also occur in the fallopian tubes, vulva, and vagina. We aim to find chromosomal and molecular aberrations specific to each cancer type analysed that will help the diagnostication and prognostication of each disease.

Genetic analysis of tumour cells has, in recent decades, shed considerable light on the mechanisms of tumour development and is increasingly relied upon to provide prognostic and diagnostic information about cancer diseases. Cancers of the female genital tract are among the least well characterised in this regard, however. The general purpose of our research is to understand the pathogenesis of gynaecological cancer in cytogenetic and molecular terms to improve classification and diagnosis of cancerous diseases, opening for the possibility of finding specific medical treatments that counteract exactly those molecular rearrangements that render the cells neoplastic.

The following tumours or subprojects will be our focus the coming years:

1. Endometrial carcinomas
2. Endometrial stromal sarcomas
3. Carcinosarcomas of the uterus and ovaries
4. Ovarian carcinomas