Molecular Cytogenetics and Cellular Oncology

Molecular cytogenetics and cellular oncology

Molecular characterization of the genome is of interest both for constitutional and cancer genetic studies. Conventional approaches such as karyotyping, breakpoint mapping or comparative genomic hybridization have their inherent limitation in terms of resolution, throughput or suitability to retrospective studies.

In this regards, genome-wide screening tools with increased specificity and/or resolution such as multicolor FISH based karyotyping, or array chip based comparative genomic hybridization could partly overcome these limitations.

The application of these molecular karyotyping tools could, for example, identify the existence of small unbalanced translocation with no characteristic changes in the banding pattern. The use of the multicolor fluorescence in situ hybridisation based karyotyping tool (dubbed as COBRA-FISH) could readily indentify the involved chromosomes.

cobra fish

COBRA-FISH karyotype overview of a case with a unbalanced translocation t(6;13).

hybridized chip image

Scan image of a hybridized chip containing triplicated spots of ~3500 larger genomic insert clones covering the whole genome with an average 1 Mb spatial resolution.  

However, the COBRA_FISH approach has inferior resolution when there is a need for the precise determination of the involved breakpoints beyond banding level. The use of array-comparative genomic hybridization (array-CGH) will facilitate both the identification of gains and losses of genomic material with direct information regarding the borders of the breakpoint determined by the resolution of the platform used. In cytogenetic investigation of solid tumors the number of chromosomal aberration might increase and therefore the combined use of the two molecular karyotyping tools are perfectly complementary to each other. 

detail of chip

Detail of an array-CGH scan profile from tumor sample.

 

Circulair viewer

Array-CGH results of a well differentiated liposarcoma case using the Circular Viewer option from CGHAnalyzer software package. (Greshock et al Genome Res, 2004, Jan) Single copy gain: green, >2 copy gain: yellow, loss: red and non-altered regions: blue. Note the highly amplified regions (in yellow) on chromosome 1 and 12.

 

cobra fish with a ring chromosome

COBRA-FISH result of well-differentiated liposarcoma case shown in figure 7. The characteristic ring chromosome containing amplified region of chromosome 1 and 12 is indicated by the arrow.

G-banding

 G-banding image of case with a ring chromosome 14 (right).

 

array-cgh result

Array-CGH result; log2 ratio profile of large genomic insert clones ordered by genomic position of the ring chromosome 14 case. Next to the expected terminal a deletion an unexpected duplication was observed.

 

Fish using bac clones

Confirmatory FISH data using BAC clones flanking the duplicated area (red and green) identified an inverted duplication.

 

The use of these molecular karyotyping tools require advanced technological support and constant improvement when applied to new, technologically challenging applications (microdisection, single cell profiling, multiparameter analysis).

In order to make efficient use of these highly innovative technologies this research group continuously seeks complementary investigations with other research group in and outside of the LUMC. For technical support, the groups uses microscopic instrumentation present within MCB, and the service of the LGTC , the genomics instrumantation platform of  the LUMC.

omnigrid 100 array scanner

Omnigrid 100 microarray spotter.

 

Research

Our research lines are focused around three major topics;

  1. Methodology
  2. Constitutional genetics
  3. Molecular tumor genetics
  4. Cellular oncology
1. Methodological research

The goal of our methodology driven work is to improve the running techniques, to develop new platforms for specialized applications. For example for screening of genomic imbalances in small cell groups we have developed a mini array-platform that allows rapid analysis (within 12 hours) of samples at chromosome arm resolution. For similar purposes we have developed a strategy for amplification and labeling of genomic DNA samples derived from small cell groups/single cells.

schematic view of mini array

 Schematic view of the mini-array design. Short (p) and long (q) arm specific reporter generated by pooling five evenly distributed clones from each chromosome arm.

 

scan overview of mini array

Scan overview of a mini-array chip containing quintuplicate of each full set.

 

array profile

Resulting array profile obtained by the mini-array chip of a case with isochromosome 18q. Note the loss of the short and the gain of the long arm of chromosome 18. 

 

Methodological improvements are complementary to the used techniques and depending on the type of problem related to research question we investigate the possibility to develop new tools. The focus here is mainly on DNA based detection techniques such as FISH, array-comparative genomic hybridization, and optimizing existing methodology to deal with very limited amounts of test samples or samples of inferior quality.

2. Constitutional genetics

Mental retardation is a frequent condition and it is found in about 3% of the population with great impact on the individual, the family and the whole society. Identification of the underlying mechanism might result in better patient management and more efficient testing. In this regards, the use of array-CGH with superior resolution over classical metaphase chromosome studies was applied by us and others and proven to be relevant in patient management. Furthermore, genome-wide screening of patients might point out syndrome/disorder specific genetic alteration. In collaboration with the Department of Clinical Genetics, we have showed the relevance of such an investigation. Currently, we are investigating selected cases in depth that were identified by array-CGH.

3. Molecular tumor genetics

In a subset of tumors a very specific genomic event is responsible for the development of the disease. A clear example in hematological malignancies is the existence of the Philadelphia translocation in chronic myelocytic leukemia (CML) or the translocation t(15;17) in promyelocytic leukemia (PML). The detection of such alterations is by now the basis of the diagnosis. The identification and functional characterization of the involved genes have now founded the basis of new treatment modalities (i.e. Glivec in CML, and RARA in PML).

Analogous to hematological malignancies simple chromosomal rearrangements are important in a subset of bone and soft tissue tumors as well (i.e. Ewing sarcoma, t(11;22) or synovial sarcoma t(X;18)). However, only limited information exists about the genomic composition of a cell in the majority of these tumor entities. Our goal is to identify tumor specific chromosomal alteration by COBRA-FISH on metaphase chromosome obtained from primary tumor cultures followed by characterization of the involved breakpoint (i.e. FISH mapping) and cloning, if suitable, the formed chimera gene(s). These identified regions could then be screened for using archived samples. Molecular cloning and tagging of the chimera genes could be employed to develop model cell lines. This project is performed in close collaboration with the Department of Pathology and as partner in the EU funded EuroBoNet project.

 4. Cellular oncology

 For almost three decades our group has developed and applied microscopic instrumentation for the detection of rare cellular events. The current project focuses on the clinical application of the detection of disseminated tumor cells (DTCs) in bone marrow or in blood. The presence of DTCs in solid tumors is a potential early marker of distant tumor propagation known as metastasis with a direct influence on patient management. In the framework of and EU funded DISMAL project there are ongoing investigations related to standardization of protocols, methodological development for genomic characterization of single cells (see line 1 as well) and to follow up the clinical relevance of these findings. The main focus of this research is related to colorectal and breast cancer. The work is performed in collaboration with the Department of Surgery.

ariol system

Ariol system, Applied Imaging

 

scan result

 

Scan result obtained using the Ariol system from a lymphatic node containing micrometastatic colon carcinoma identified by cytokeratin antibody and consecutive immunohistochemical detection.

 

arraycgh profile

ArrayCGH profile from 30 cells from Ewing sarcoma cell line (TC32) after whole genome amplification and ULS labeling.

 

A second oncology project relates to the use of advanced microscopy l to address basic cell biological processes in samples derived i.e. from familial cancer. Life cell imaging investigation of cellular processes in BRCA2 mutation carrier family members is performed in collaboration with the Molecular and Cell Biology Research Laboratory of the Icelandic Cancer Society by an exchange Ph.D. student.

Techniques and Protocols

The group has developed over the past years many reliable protocols. Examples are culture of primary tumor samples/PCC techniuqes, metaphase harvest, COBRA FISH (human and mouse sets, see protocol), array-CGH (only human sets, see protocol), auxiliary tests: FISH (meta-, interphase FISH), MLPA, rare event detection using automated microscopy, laser microdissection, whole genome amplification, mini array (see protocol).