Introduction

Splenic marginal zone lymphoma (SMZL) is a chronic B-cell disorder diagnosed on the basis of splenic histology, which shows that the splenic white pulp is infiltrated by an inner zone of small lymphocytes and an outer zone of larger cells with abundant pale cytoplasm. The tumor cells are positive for surface immunoglobulin (Ig)M and IgD and express CD20, CD22, and CD79a but not CD5, CD10, CD23, or cyclin D1. Patients usually present with splenomegaly without peripheral lymphadenopathy. Bone marrow and blood involvement is common. Circulating tumor lymphocytes frequently have a characteristic villous appearance and in such cases in which splenectomy is not indicated clinically, are diagnosed as splenic lymphoma with villous lymphocytes (SLVLs) (1,2).

Analysis of VH genes from a small number of cases with SMZL or SLVL has shown that most have undergone somatic mutation with or without intraclonal heterogeneity. However, a subset of cases with unmutated VH genes

From: Methods in Molecular Medicine, Vol. 115: Lymphoma: Methods and Protocols Edited by: T. Illidge and P. W. M. Johnson © Humana Press Inc., Totowa, NJ

has recently been described (3). This would be consistent with the derivation of SMZL from the heterogeneous population of normal splenic marginal zone B-cells, which includes both naïve and memory B-cells.

No consistent cytogenetic or genetic abnormality that defines SMZL has so far been described. Occasional patients with reciprocal chromosome translocations involving the immunoglobulin loci have been documented. The partner chromosomes include 7q21 resulting in juxtaposition of the CDK6 gene to the kappa light chain locus, and 6p12, with juxtaposition of cyclin D3 to the immunoglobulin heavy chain locus (4).

Approximately 50% of patients with SMZL or SLVL have a deletion of chromosome 7q. Although different regions of loss of 7q have been reported the majority of cases have a deletion of 7q31.3 - 32 (5). The molecular consequence of this deletion is unknown.

Microsatellite analysis is a valuable tool for detecting loss of heterozygosity (LOH) in regions with candidate tumor suppressor genes (6), In this chapter we will discuss the microsatellite analysis methods we have used to look for LOH and hence determine the commonly deleted region (CDR) of chromosome 7q in our series of SMZL/SLVL patients. Cytogenetic techniques will not be discussed, but the results of karyotyping and fluorescent in situ hybridization (FISH) often provide an excellent method of identifying chromosomal regions of interest before beginning the search for a CDR by molecular techniques. Southern blotting will also not be discussed, however labeled restriction fragment-length polymorphism (RFLP) analysis is a very useful tool in the confirmation of microsatellite LOH results and hybridization analysis also may be used to investigate homozygous loss in a newly defined CDR (7,8).

Microsatellites are tandem repeats of simple di-, tri-, tetra-, penta-, or hexanucleotide sequence (2-6 bases), which occur abundantly and at random throughout most eukaryotic genomes. They are typically short (often less that 100 bp long) and embedded within unique sequence, thus being ideal for designing flanking primers for in vitro amplification by the polymerase chain reaction (PCR). The high degree of polymorphism (attributable to variation in the number of repeat units) displayed by microsatellites is especially advantageous because the majority of samples will be heterozygous (otherwise said to be informative) at the locus of interest. In addition, very small amounts of tissue are required for microsatellite analysis, as typically only 100 ng of template DNA is used compared with 10 pg for a Southern blot, meaning that archival material also can be exploited (9).

For LOH studies, it is imperative that there is access to matched normal and tumor tissue so that direct comparison between the two can be made. In our own studies, we have analyzed peripheral blood lymphocytes using granulocytes as matched controls. Also, it should be emphasized that these matched

Fig. 1. Autoradiogram of polyacrylamide gel electropheresis (PAGE) of 32P labeled PCR products. Microsatellite analysis on normal (N) and tumor (T) derived DNA from unsorted and FACS sorted cell populations. The arrow indicates the additional band arising from DNA from normal cells contaminating the tumor cell population. LOH is clearly demonstrated when DNA from FACS sorted cells is analyzed.

Fig. 1. Autoradiogram of polyacrylamide gel electropheresis (PAGE) of 32P labeled PCR products. Microsatellite analysis on normal (N) and tumor (T) derived DNA from unsorted and FACS sorted cell populations. The arrow indicates the additional band arising from DNA from normal cells contaminating the tumor cell population. LOH is clearly demonstrated when DNA from FACS sorted cells is analyzed.

samples must be purified to greater than 85%, that is, the tumor sample should not be contaminated with normal material and vice versa; otherwise, problems will be encountered during the result interpretation (Fig. 1).

Sample pairs are scored as follows: heterozygous, not deleted (two different alleles present in both normal and tumor tissue); heterozygous, deleted (two alleles present in normal tissue, but only one present in tumor tissue); homozy-gous, not informative (two alleles of the same size present in both normal and tumor tissue).

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