Oncogenes Tumor Suppressor Genes

Testing for the presence of alterations in MYC, TP53, NRAS, and BCL6 is relevant for immunodeficiency-associated lymphomas. Of these, probably the most important and feasible in a clinical molecular laboratory on a routine basis is the detection of MYC translocation using fluorescence (FISH) or chromogenic (CISH) in situ hybridization assays.

Fluorescence In Situ Hybridization

FISH is an excellent method for documenting specific chromosomal translocations. FISH can be performed using interphase nuclei or metaphase chromosomes and can be performed using formalin-fixed, paraffin-embedded tissue sections. As commercial probes are entering the market, FISH is being implemented by increasing numbers of clinical laboratories. In the setting of immunodeficiency lymphomas, FISH is the best assay to document MYC translocations when a Burkitt lymphoma is suspected or a large cell lymphoma has Burkitt-like features. FISH may be performed either with two different probes, one to MYC and the other to IGH, or with a single MYC "split apart" probe (Figure 34-3). The advantage of the second single

MYC probe method is detection of variant translocations of MYC and the IGLK or IGLL genes.

Chromogenic In Situ Hybridization (CISH)

CISH is very similar to FISH, except the probes are labeled with two different chromogenic substrates, so their evaluation does not require a fluorescent microscope and the slides provide a permanent record of the assay. While somewhat less sensitive than FISH, commercial CISH probes to detect the MYC-IGH translocation are available, and are being used successfully in clinical laboratories. CISH may soon become standard of care for diagnosis of BL or atypical BL when histologic features are suggestive of this disease.

Southern Blot Analysis

SBA can be used to document translocations involving the MYC and BCL6 genes, but as mentioned above it is laborintensive and time-consuming, has long turnaround times, and requires fresh or frozen tissue. Thus, this technique is used primarily for research applications. A comparison of CISH and Southern blot for the MYC-IGH translocation is shown in Figure 34-3.

Figure 34-3. Detection of MYC translocations. (a) FISH analysis of Burkitt lymphoma cells demonstrating the MYC-IGH fusion signals observed in a case positive for t(8;14). IGH is labeled with Spectrum Green and MYC is labeled with Spectrum Orange. Two fusion signals show the reciprocal translocation. Single red and green signals correspond to the wild-type allele of MYC on chromosome 8 and IGH on chromosome 14. (Image provided by Dr. Susan Mathew, Weill Medical College of Cornell University.)

(b) Southern blot analysis using a MYC probe in four cases of AIDS-related lymphoma. Cases 1 and 2 show a single band, corresponding to the germline configuration, and cases 3 and 4 have a MYC translocation, as indicated by a smaller second band. DNA was digested with Hind III,and after electrophoresis and transfer, was hybridized to a radiolabeled probe corresponding to the third exon of the MYC gene.

Figure 34-3. Detection of MYC translocations. (a) FISH analysis of Burkitt lymphoma cells demonstrating the MYC-IGH fusion signals observed in a case positive for t(8;14). IGH is labeled with Spectrum Green and MYC is labeled with Spectrum Orange. Two fusion signals show the reciprocal translocation. Single red and green signals correspond to the wild-type allele of MYC on chromosome 8 and IGH on chromosome 14. (Image provided by Dr. Susan Mathew, Weill Medical College of Cornell University.)

(b) Southern blot analysis using a MYC probe in four cases of AIDS-related lymphoma. Cases 1 and 2 show a single band, corresponding to the germline configuration, and cases 3 and 4 have a MYC translocation, as indicated by a smaller second band. DNA was digested with Hind III,and after electrophoresis and transfer, was hybridized to a radiolabeled probe corresponding to the third exon of the MYC gene.

Single-Strand Conformation Polymorphism Analysis

Single-strand conformation polymorphism analysis (SSCP) is a useful assay to screen for point mutations of RAS and TP53, although it is not used routinely in clinical laboratories due to the complexity of the assay. The procedure should be carefully validated and the sensitivity of the assay determined to allow for adequate interpretation of results.

PTLD are unlikely to be identifiable by sequencing. An alternative approach that can be used in a mixed cell population is PCR followed by cloning into plasmid vectors and sequencing of multiple clones. A mutant allele will consistently show up in a proportion of the clones, while the normal allele from tumor cells and normal infiltrating cells will represent the remainder of the sequenced clones. Of note, because DNA polymerases used in the PCR are prone to errors, the identical alteration has to be present in multiple clones to be scored as positive.

Sequence Analysis

DNA sequencing is the ultimate confirmation of point mutations and small deletions or insertions in relevant genes. Sequence analysis can be performed by direct sequencing of PCR products of the target region, or by cloning the PCR products into a plasmid vector followed by sequencing of multiple independent clones. Direct sequencing is the easiest, but it can be done only when the tissue is composed almost entirely of a pure tumor population, as the mutation may be masked by infiltrating reactive cells in addition to the normal allele of the gene of interest. Therefore, mutations of TP53 are likely to be identifiable using direct sequencing on an AIDS-related BL, where the majority of the cells are neoplastic and one TP53 allele is likely to be deleted. In contrast, BCL6 mutations in

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