Pathology Thread

Overview:

Chromosomal aberrations are rather gross changes reflecting molecular abnormalities. DNA probes against a selected gene locus can be used to identify sequences of specific proteins, or clonal rearrangements of specific cell types.

In this way we identify segments of DNA unique to a cell type (such as the sequence for the T cell receptor gene) or unique to a novel protein (such as the p210 tyrosine protein kinase produced by the bcr/c-abl fusion gene only in CML).

The finding of a DNA rearrangement is evidence that a clonal population exists, and it offers clues as to the lineage of the clonal population. Clonal population = leukemia, variant population= reactive/infectious lymphocytosis.

Process of DNA Analysis:

DNA is extracted from cells and the long (high MW) DNA strands are cleaved or cut into fragments of varying length depending on the restriction enzyme's site of action.

These fragments of DNA are then electrophoresed, separating the fragments by size. Next the fragments are transferred to a nylon support mesh and hybridized with the DNA probe (DNA complementary to the sequence you are looking for). The probe is labeled (usually radioactive) allowing it to be visualized.

If a clonal population of cells exists (suggesting leukemia), all cells within that clone will have identical DNA rearrangements. Thus restriction enzymes will cut all the DNA into pieces of the same size. These will all migrate the same distance on the gel electrophoresis and thus will be in the same location when identified with a DNA probe.

In reactive situations multiple lymphocytes are stimulated each having a different rearrangement. Expansion of the reactive lymphocytes results in innumerable relatively small clones. Since each clone has a different rearrangement, restriction enzymes will cut the DNA from each clone into sightly different sized pieces. These pieces will electrophorese as a smear so that no rearrangement bands are observed on Southern hybridization.

Bam HI is a restriction enzyme. In this example, two different sized DNA fragments result from processing with the same enzyme, following a gene rearrangement.

Multiple lymphocytes, innumerable small clones

One clonal population

Different size strands by restriction enzymes

Same length strands by restriction enzymes

Smear on electrophoresis

Band on electrophoresis

In the "Normal BM" column, note the light smearing of the DNA due to small numbers of cells each with different rearrangements of their DNA. No diiscrete band is formed.

The patient's marrow contains normal as well as leukemic cells, thus the presence of both germline (G) and rearranged (R) DNA.

An 11-year-old boy comes to you because of a cough and fever. His spleen is enlarged, but no lymphadenopathy was noted. His WBC was 42.0 x10 /L with 60% immature vs. reactive lymphocytes. The marrow aspirate was a dry tap. Cytochemical and immunologic studies were consistent with a lymphoid origin for the cells.

What is the best diagnosis based on the DNA probe studies of the peripheral blood shown at right?

A. c/w reactive viral lymphocytosis
B. c/w pertussis lymphocytosis
C. c/w a pro-B cell Acute Lymphoblastic Leukemia
D. c/w Acute Lymphoblastic Leukemia, L3 (Burkitt's)
E. c/w T-cell Acute Lymphoblastic Leukemia

Another molecular genetic technique, PCR (Polymerase Chain Reaction), can detect minute quantities of abnormal DNA allowing for the early detection of residual leukemia following therapy. One problem with this technique may be that it can be overly sensitive, magnifying small reactive clones, or even the DNA from single cells.

In summary, molecular genetic studies can detect clonal populations, can provide evidence of lineage through the identification of DNA rearrangements associated with a particular cell type, and can aid in the diagnosis of specific disorders through the identification of DNA rearrangements that result in unique gene fusions as in the bcr/c-abl fusion product of chronic myelogenous leukemia.