In situ analysis of the variable heavy chain gene of an IgM/IgG-expressing follicular lymphoma: evidence for interfollicular trafficking of tumor cells

Abstract

It is generally assumed that follicular lymphomas (FL) not only morphologically resemble normal germinal centers but have retained some functional characteristics of their non-neoplastic counterparts as well. Recent IgV gene analyses on a panel of FLs however, strongly suggested that FLs do not retain the capacity of somatic hypermutation and are not being selected on basis of the quality of their mIgV regions. To extend these findings, we investigated the follicular organization and class switching in a FL that consisted of both IgM- and IgG-expressing tumor cells with a high somatic mutation load and significant intraclonal V(H) gene diversity. V(H)-C(mu) and V(H)-Cgamma gene transcripts were amplified and sequenced from samples of approximately 50 tumor cells, isolated from frozen tissue sections by laser microdissection. We identified many different subclones and obtained limited evidence of subclone dominance in individual follicles. Remarkably, several subclones were found scattered over different follicles. All samples contained IgM- and IgG-expressing tumor cells with, in general, non-identical mutation patterns, which is not in support of ongoing class switching. Accordingly, no switch circle recombination products were found. The findings indicate that the neoplastic follicles lack the organization and functions typical of reactive germinal centers.

Figure 1.

FL 8-‘83 harbors both IgM- and IgG-expressing tumor cells. Frozen section of FL 8-‘83 stained with monoclonal antibodies specific for human IgA (A), IgG (B), IgM (C), and BCL-2 (D) (magnification, ×100). Whereas all cells are IgA-negative, a faint but significant expression of IgG is found on the majority of cells in all areas. In between these cells, scattered tumor cells with relatively strong IgM expression are found.

Figure 2.

Tissue sections of FL 8-‘83. A: The unstained tissue section of FL 8-‘83 from which samples were microdissected. B: A schematic drawing of the tissue section. The circles represent the numbered follicles from which samples were taken. C: Tissue section of FL 8-‘83 stained with CD21L antibodies and counterstained with hematoxylin.

Figure 3.

Schematic representation of IgM- and IgG-derived sequences from FL. no 8-‘83 (upper part and lower part, respectively) and its relapse 8-‘92 (bottom line). Only mutations that differ from the IgM consensus sequence, as determined on crude tissue, are shown. The numbers represent the different follicles from which samples were taken, as indicated in Figure 2 ▶ . Different samples from one follicle are designated with a different letter. Replacement and silent mutations are represented as closed and open circles, respectively. Codon numbers are indicated underneath the symbols. Whereas in the consensus sequence at position 81 a mutation was present as compared to the germ-line gene, the gray symbols at this site in sequence 8aγ and 4aγ represent the germ-line nucleotide.

Figure 4.

A-D: Intraclonal variation within cloned PCR products. The sequences are depicted as lines. Only differences with the IgM-derived consensus sequence of FL 8-‘83 are shown. The upper line of each figure represents the sequence that was obtained by directly sequencing two independent PCR products from the same sample. The other lines are the sequences of individual bacterial clones made from the PCR products. Replacement and silent mutations are represented as closed and open circles, respectively. Codon numbers are indicated underneath the symbols. Whereas the consensus sequence of 9aγ contains mutations at positions 50 and 56 as compared to the germ-line gene, the gray symbols at these sites in sequences 1.1, 2.2, and 2.4 represent the germ-line nucleotides. *The mutations at position 110 of clones 2.2 and 2.4 (9aγ) are different from the mutations in the same codon of clone 1.1 and 1.4.

Figure 5.

A schematic drawing of the unstained tissue section of FL 8-‘83. The open circles represent the numbered follicles from which samples (smaller closed circles) were taken. Labeling of samples with letters was done from left to right, from top to bottom. Follicles in which IgM- or IgG-expressing subclones with identical mutation patterns were found are connected by solid and dashed lines respectively.

Figure 6.

PCR analysis to detect Sγ-Sμ switch circle recombination products. The first lane is the 1 kb DNA ladder (M) with the fragment sizes indicated on the left. Lanes T1-T3 represent the PCR products obtained out of crude tonsil tissue, whereas lanes 8-‘83 and 8-‘92 show the PCR results obtained on crude tissue of FL 8. The amount of genomic DNA (approximately 250 ng) had been stratified using a β₂-microglobulin PCR (lower panel). Sequencing proved that the PCR products obtained out of tonsil DNA (T1-T3) were indeed switch circle recombination products, whereas the faint bands found in lanes 8-‘83 and 8-‘92 proved to represent non-specific products (data not shown).

Figure 7.

Fiber FISH detection of CH rearrangements in FL 8-‘83 and FL 8-‘92. Shown are typical examples of the consistently observed bar codes representative for the functional Ig alleles of IgM⁺ (upper panel) and IgG⁺ (centerpanel) tumor cells of FL 8-‘83 and the IgG⁺ tumor cells of FL 8-‘92 (lower panel). The VDJ as well as the different CH regions, as detected by the FISH probes described previously, are indicated by white symbols above each fiber.