Introduction

Technique

Features

Morphology

Low sensitivity

Immunophenotyping

Southern blotting

Cytogenetics

FISH

PCR amplification

Lacks specificity i nuil_i i i nuil

1O2 1O3

Labour intensive Slow

Labour intensive Slow

Requires metaphase chromosome preparations

Labour intensive Slow

Requires metaphase chromosome preparations

Labour intensive Interphase FISH obviates need for high quality metaphases (cf. standard cytogenetics)

DNA sequence information required

False +ve results

DNA sequence information required

False +ve results

Sensitivity

Fig. 6.1 Methods of detection of marrow infiltration in non-Hodgkin's lymphoma, showing the sensitivity of each

system for the detection of small numbers of malignant cells in a marrow or blood sample should fulfill the following criteria: the method should be applicable in most cases of the disease under investigation; the method should be specific for the neoplastic cell type; the method should be sensitive; and the method should allow quantitation of tumor burden for prognostic purposes. Methods include:

• morphology

• flow cytometry and immunophenotypic analyses

• cell culture assays

• karyotypic analysis

• fluorescence in situ hybridization techniques

• molecular analyses, including Southern blotting and PCR.

Morphology

In acute leukemia, remission is the term used to describe a bone marrow containing fewer than 5% blast (i.e. leukemic) cells using conventional light microscopy, but this may still represent a considerable tumor burden since, at diagnosis, the leukemic cell number may be 1012 and, following therapy, the neoplastic cell number may drop only by 2 logs to 1010 even in the presence of fewer than 5% marrow blasts. Standard morphology alone is not a sensitive method for determining low levels of disease and is a poor indicator to attempt to predict impending relapse (Table 6.1).

Flow cytometry and immunophenotyping

Immunophenotypic analysis using single monoclonal antibodies to cell membrane or cytoplasmic proteins lacks absolute specificity for leukemia or lymphoma cells and is therefore of limited value. Combining monoclonal antibodies allows the more specific detection of residual disease and quantitation is possible, although the tumor cell burden may be underestimated. The technique is further hampered by the lack of true 'specific-specific' surface determinants and tumor-associated antigens are normal differentiation antigens

Table 6.1 Sensitivity of methods for MRD detection.

Standard morphology

1-5%

Cytogenetics

5%

Fluorescence in situ

0.3-5%

Immunophenotyping

10-4

Translocations

PCR

10-6

Gene rearrangements

Southern blotting

1-5%

PCR

10-4 to 10-6

present on developing hematopoietic progenitor cells. Using combinations of monoclonal antibodies and multicolor flow cytometric analysis, the sensitivity of this technique can be greatly enhanced. Except in the most expert hands, this technique is limited to a sensitivity of around 10-4 (i.e. 1 malignant cell in 10 000 normal cells).

Cell culture assays

These involve growing T-cell-depleted marrow in culture after the patient has undergone treatment, followed by subsequent morphological, immunophenotypic and karyotypic analyses on the colonies produced. Due to the variability of culture techniques between and within laboratories, this method has proved unreliable and insensitive for detecting persisting blasts. In addition, culture techniques do not provide any estimate of cell number and hence provide little information about tumor cell burden.

Karyotypic analysis

Detection of non-random chromosomal translocations is of great value in the diagnosis of leukemias and lymphomas. Chromosomal abnormalities are present in at least 70% of patients with acute lymphoblastic leukemia (ALL) and 50% of patients with chronic lymphocytic leukemia (CLL). However, karyotypic analysis is of limited value following therapy, with a sensitivity level of around 5%, making it little better than standard morphological analysis. In addition, cytogenetics relies on obtaining adequate numbers of suitable metaphases for analysis, which is difficult in some malignancies.

Fluorescence in situ hybridization (FISH)

FISH can detect smaller chromosomal abnormalities than standard karyotyping and allows analysis of interphase nuclei (cf. metaphase preparations in standard karyotyping). The method involves the binding of a nucleic acid probe to a specific chromosomal region. Preparations are counterstained with fluorescent dye, allowing the chromosomal region of interest to be detected. The technique is useful in the diagnosis of trisomies and monosomies and has been particularly useful in identifying deletions that have prognostic significance in CLL. The sensitivity of the technique is around 1%, making it considerably more useful than standard karyotyping for follow-up marrows in patients with leukemias or lymphomas, but is still of limited value for MRD detection.

Molecular techniques—Southern blot hybridization

Initially described by its inventor, Professor Ed Southern, in the 1970s, Southern blotting involves the digestion of chromosomal DNA using bacterial restriction enzymes, with size separation of the DNA fragments using electric current and gel electrophoresis before transferring these to a nylon support membrane. A labeled probe for the gene of interest is applied, which binds to its complementary sequence on the membrane and visualization of the gene is by autoradiography (Figure 6.2).

Southern blotting is useful for the initial diagnosis of leukemia and lymphoma using probes specific for translocations or gene rearrangements. With Southern blotting, a non-germline or rearranged gene pattern may be seen in DNA from a population of cells where more than 1% of the total population is made up by a clone of malignant lymphoid cells. In other words, Southern blotting will detect a rearranged gene provided the cells containing the rearranged gene exceed 1 in 100 normal cells. The disadvantage of Southern blotting is that the technique is not sufficiently sensitive for the detection of small numbers of malignant cells persisting after therapy and giving rise to disease relapse. For this reason, Southern blotting has largely been replaced by PCR for the detection of MRD.

Whole genomic DNA

Whole genomic DNA

Digestion by restriction enzyme f^NS.

Digested products separated using agarose gel electrophoresis

Digestion by restriction enzyme f^NS.

Digested products separated using agarose gel electrophoresis

Separated fragments exposed to radiolabeled probe
Autoradiographic image representing location of probe bound to relevant fragment

Fig. 6.2 Principle of Southern blotting

Genomic DNA is digested using a restriction enzyme, after which the fragments are separated on the basis of size using agarose gel electrophoresis, and are finally transferred to a nylon membrane. Radiolabeled probe for the gene of interest is hybridized to the DNA on the membrane and, after removal of the non-specifically hybridized probe, the location and size of the fragment are determined using autoradiography.

PCR amplification of DNA

As described above, Southern blotting is a useful technique for assessing whether there is a clone of abnormal cells in blood, marrow or other tissue but is not useful if these cells are present in only very small amounts. In this case, techniques that involve amplification of specific DNA sequences are required. PCR has filled the void in this respect and has found a place in diagnostic laboratories investigating oncogenes, hemato-logical malignancies, single-gene disorders and infectious diseases. Part of the attraction of a PCR-based approach is its extreme simplicity and the speed with which results are obtained.

In the PCR reaction, two short oligonucleotide DNA primers are synthesized that are complementary to the DNA sequence on either side of the translocation or gene of interest. The region between the primers is filled in using a heat-stable bacterial DNA polymerase (Taq) from the hot-spring bacterium Thermus aquaticus. After a single round of amplification has been performed, the whole process is repeated (Figure 6.3). This takes place 30 times (i.e. through 30 cycles of amplification) and leads to a million-fold increase in the amount of specific sequence. When the 30 cycles are complete, a sample of the PCR is electrophoresed on agarose or polyacrylamide gel. Information about the presence or absence of the region or mutation of interest is obtained by assessing the sizes and numbers of different PCR products obtained after 30 cycles of amplification.

The specificity of PCR can be further increased by the use of nested PCR, which involves re-amplification of a small amount of the amplified product (obtained using outside, external, primers) using internal oligonucleotide primers.

PCR has the advantage that very little tissue sample is required for analysis and the technique can be applied to a variety

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