Molecular biology

Published on 03/04/2015 by admin

Filed under Hematology, Oncology and Palliative Medicine

Last modified 22/04/2025

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Molecular biology

Molecular techniques now play a central role in the diagnosis and management of blood disorders, particularly haematological malignancies. This is a rapidly changing field and the following is a summary of some of the most commonly used and newest technologies and applications.

Selected techniques used in the analysis of DNA

Polymerase chain reaction (PCR)

The object of PCR is to amplify a preselected sequence of DNA many times over. This amplification greatly facilitates subsequent analysis of the DNA sequence for point mutations and polymorphisms, and often allows direct analysis of the product by gel electrophoresis without the use of probes.

The method can only be briefly described here. Essentially two specific oligonucleotide primers are added to the DNA. These have sequences matching the regions flanking the region of interest. A DNA polymerase is added and the mixture heated, causing the DNA to dissociate into two single strands. Following cooling the single strands bind to the oligonucleotides which are in excess. The oligonucleotide then acts as a primer for DNA polymerase and is extended to form a new double-stranded molecule. With each repeat of the cycle the amount of DNA is doubled. Generally about 30 cycles are used and amplification of approximately 10⁶ can be achieved.

Fluorescence in situ hybridisation (FISH)

FISH describes the hybridisation of specific DNA or RNA sequences in situ to cellular targets attached to microscope slides. The most popular probes are chromosome-specific DNA sequences which generate a brilliant signal in both metaphase and interphase nuclei. The technique is particularly useful in the demonstration of chromosomal monosomies or trisomies but chromosome translocations (Fig 50.1), deletions and amplification of specific genes can also be detected. The results of FISH may be further improved by image processing.

Fig 50.1 Use of FISH in a case of chronic myeloid leukaemia (CML).
Probes for BCR and ABL are used – juxtaposition of the two probes (giving a yellow signal as in cell lower right) indicates the BCR–ABL rearrangement seen in Ph+ CML.

Comparative genomic hybridisation

This technique is designed to detect regions in the genome which are undergoing quantitative changes. Different tumours show distinct genomic hybridisation patterns of gains and losses. The method is especially useful in the analysis of leukaemias.

Microarrays/gene profiling

Microarray technology allows the simultaneous profiling of tens of thousands of genes thus painting a molecular portrait of a tumour cell. Several methods are available for analysis of a large number of RNA transcripts. These include complementary DNA microarrays, oligonucleotide microarrays and serial analysis of gene expression (SAGE). The most commonly used ‘platforms’ are the two microarray technologies in which each experiment reveals the expression levels of over 20 000 genes. cDNA fragments and oligonucleotides can be spotted onto glass slides. The DNA arrayed on the slide is generally referred to as the ‘probe’ and the cDNA or cRNA derived from the sample is referred to as the ‘target’. The complex gene expression data generated requires powerful statistical analysis. Microarrays are enhancing our understanding of haematological malignancy (see below and Fig 50.2).

Fig 50.2 Subgroups of diffuse large B-cell non-Hodgkin’s lymphoma (DLBCL) according to gene expression profiles.
In the top panel each column represents a single DLBCL and each row a single gene. Red areas indicate increased expression and green areas reduced expression. Survival after chemotherapy is different in the three groups. (Reprinted with permission from Margalit O et al 2005 Micro-array based gene expression profiling of hematologic malignancies: basic concepts and clinical applications. Blood Reviews 19: 226.)

Next-generation sequencing

Massively parallel sequencing (also termed next- or second-generation sequencing) results in the simultaneous generation of millions of short DNA sequences. In studies of haematological malignancy, it is important to sequence both the tumour cells and normal tissue (e.g. skin) from the patient to identify relevant acquired (somatic) mutations. In whole genomic sequencing (Fig 50.3), the object is to sequence the entire genome. More specific next-generation techniques include exome and transcriptome sequencing.

Fig 50.3 Whole genome sequencing for detection of mutations in chronic lymphocytic leukaemia.
For each tumour genome, copy number (solid lines), density of mutations per 5-Mb window (bars) and protein-coding mutations (dots) are shown. The shaded rectangle indicates the location of the 13q14 deletion present in three of the four cases. Chromosome numbers are listed below the four profiles. (Reprinted by permission from Macmillan Publishers Ltd: Puente XS et al 2011, Whole genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia. Nature 475: 101–105.)

Application of molecular biology in haematology

Carrier detection and antenatal detection in genetic disorders

Molecular techniques are crucial in antenatal diagnosis and genetic counselling in genetic disorders of the blood. For instance, PCR technology to detect DNA point mutations or deletions in chorionic villus samples, enabling first trimester testing for thalassaemia. Extraction of fetal cells or DNA from maternal blood may allow less invasive prenatal diagnosis.

Haematological malignancy

Diagnosis and classification

Leukaemias and lymphomas were originally diagnosed and classified on the basis of their morphological appearance. As is discussed in the disease sections, optimum management of these disorders now requires the supplementation of traditional clinical and morphological information with detail of immunophenotypic, karyotypic and molecular characteristics. Molecular analysis allows the confirmation of specific disease markers (e.g. BCR-ABL in chronic myeloid leukaemia) and also reveals key prognostic information (e.g. Ig gene mutation in chronic lymphocytic leukaemia). Microarray-based gene expression and next-generation sequencing studies, described above, provide novel insights into the biology of leukaemia, lymphoma and myeloma. Simplification of this expensive research technology is likely to permit its eventual use in the hospital laboratory.

Minimal residual disease

Traditional definitions of remission in leukaemia have relied on crude morphological criteria. Many patients in remission subsequently relapse, implying the existence of occult neoplastic cells undetectable by normal morphological or cytogenetic methods – so-called minimal residual disease (MRD) (Fig 50.4). Reliable detection of MRD potentially allows improved management with escalation of therapy for patients with persistent disease and the avoidance of excessive treatment in patients showing a good response to previous intervention. Detection of MRD relies upon the presence of disease markers that can be targeted (e.g. PML-RAR α in acute promyelocytic leukaemia). In childhood and adolescent ALL the tandem application of flow cytometry and PCR can be used to study MRD in almost all patients and this information is being employed in clinical trials. In CML quantitative PCR assay of BCR-ABL transcripts is routinely used to direct management. Very low levels of BCR-ABL mRNA predict a good clinical outcome.

Fig 50.4 The detection of minimal residual disease.
The greater sensitivity of PCR and immunological marker analysis compared with traditional morphological techniques in the detection of residual leukaemic cells can be seen. (Reproduced with permission of JJM van Dongen, Department of Immunology, Erasmus University, Rotterdam, and Medicultura International B.V.)

Stem cell transplantation

Molecular techniques can be used both to monitor MRD post-transplant and to improve the level of HLA matching between unrelated donors and recipients.

Molecular biology

Molecular biology techniques routinely used in haematology include the polymerase chain reaction (PCR), fluorescence in situ hybridisation (FISH) and comparative genomic hybridisation.

Molecular techniques play a key role in carrier detection and antenatal detection in genetic disorders such as thalassaemia and haemophilia.

In haematological malignancy, molecular techniques refine diagnosis and classification, and improve detection of minimal residual disease (MRD) after therapy.

Recent developments include microarray-based gene expression profiling and whole genome sequencing of haematological malignancies.

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