A molecular analysis of biclonal follicular lymphoma: further evidence for bone marrow origin and clonal selection

We report a follicular lymphoma (FL) case presenting the coexistence of two tumor cell subpopulations in lymph node (LN) and bone marrow (BM), which exhibited an inverse pattern of immunoglobulin light (IgL) chain gene rearrangement and expression: Igκ−λ+ in LN and Igκ+λ− in BM. These tumor clones shared an identical BCL2-IgH recombination, accompanying t(14;18)(q32;q21) translocation, and an identical variable, diversity and joining segments joining with clone-specific VH somatic hypermutations on the untranslocated IgH allele. Our study provides further evidence that FL clones, originating from common progenitor cells, can be developed independently at different sites and with different IgL expression after immune selection.

in situ hybridization analysis of her BM cells revealed the juxtaposition of the BCL2 with the IgH on 30% of cells analyzed.

DNA isolation and Southern blotting
Genomic DNA was extracted from biopsied LN cells and BM mononuclear cells. DNA was also prepared from the patient's peripheral blood mononuclear cells, in which no lymphoma cells were detected. An informed consent was obtained in accordance with the Declaration of Helsinki. After DNA digestion with HindIII (for the IgH), BamHI (for the Igj) and EcoRI plus HindIII (for the Igk), Southern blotting was carried out by standard procedure. Probes were fluorescein-labeled using Gene Images Random Prime Labeling kit (GE Healthcare, Buckinghamshire, UK) and hybridization signals were detected using Gene Images CDP-star Detection kit (GE Healthcare). DNA probes for detection of the IgH, the Igj and the Igk gene rearrangement were as follows: JH, a 3.5-kb EcoRI-HindIII fragment; Cj, a 2.5-kb EcoRI fragment; Ck, a 0.7-kb BglII-EcoRI fragment containing the Ck3.

PCR and DNA sequence
The DNA was amplified by PCR using LA Taq Polymerase (Takara Bio, Otsu, Japan) for the BCL2 ⁄ IgH rearrangement and the IgH gene analysis. The cycling conditions included an initial denaturation for 4 min at 94°C followed by 30 cycles of with 94°C denaturing for 30 s and 6 to 8 min annealing ⁄ primer extension at 68°C plus final extension 7 min at 72°C. Reaction mixture (50 lL) contained 100-200 ng DNA in LA-PCR reaction buffer with 0.4 lmol ⁄ L primers, 0.4 mmol ⁄ L dNTPs, 2.5 mmol ⁄ L MgCl 2 , and 2.5U LA Taq-Polymerase. The PCR products were directly cloned into pCR2.1-TOPO vector (Invitrogen, San Diego, CA, USA) and sequenced by an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).
For detection of the Igj deleting element (Kde) rearrangement, DNA was also amplified employing PCR system and primers previously reported by Stolz et al. (16).

Results
Flow cytometry analysis revealed that LN and BM tumor cells presented an inverse pattern in surface IgL chain expression; Igj)k+ in LN and Igj+k) in BM (Fig. 1A). The expressions of other surface antigens were similar in both samples (positive for CD10, CD19 and CD20, and negative for CD3 and CD5). Southern blotting presented monoclonal Ig rearrangements in both samples, but rearranged IgL gene was different between the two; the Igk in LN and the Igj in BM (Fig. 1B). In analysis of Kde configuration, Vj1-Kde and Vj3-Kde recombinations were detected in LN, while only Vj3-Kde was detected in BM (Fig. 1C). Sequence around the Vj3-Kde recombination junction was identical in both samples (data not shown).
On PCR screening using the previously reported primer sets (17), the chromosome 18 breakpoint was predicted to be located out of MBR in both LN and BM. A set of new BCL2 and IgH-Sl primers amplified a 5.9-kb DNA fragment from LN and a 5.4-kb one from BM, respectively (Fig. 2). Sequence analysis revealed an identical BCL2-JH5 recombination junction on both samples (data not shown), with the chromosome 18 breakpoint located at 5 kb downstream of the MBR (Fig. 2A) (14). DNA was amplified by primer sets as indicated. Arrows show the amplified fragments with size. Lanes C present DNA from the patient's peripheral blood mononuclear cells. occurred in BM cells, as ascertained by PCR using additional JH primers (Fig. 2B).
On Southern blotting, two rearrange bands were hybridized with JH probe in both LN and BM samples (Fig. 1B). Among these bands, the size of smaller bands (5.1 kb in LN and 4.6 kb in BM) coincided with the length of the cloned HindIII fragments containing the BCL2-JH junction ( Fig. 2A). Thus, these bands were considered to be generated by t(14;18) translocation, with the difference in length due to the deletion in BM.
On the other hand, the comigrating 7.6 kb bands seemed to represent the variable, diversity and joining segments (VDJ) recombination on the untranslocated IgH allele. On PCR screening using VH leader primers (18), a set of VH3-specific primer and a JH primer amplified the rearranged fragment from both samples and the sequence of the 5¢ part of amplified fragments showed the greatest homology with the VH3-53 sequence. Then, DNA from tumor cells was subjected to amplification employing the 5¢ VH3-53 forward primer and Sl reverse primer (Fig. 3A). As shown in Fig. 3B,  8.3 kb bands were obtained from both samples by this primer set, which were ascertained as having VH3-53 region by partial sequence analysis. The cloned PCR products contained the HindIII fragments corresponding to the bands on Southern blotting.
To characterize the SHM patterns in LN and BM, 10 sequences from independent bacterial isolates in each sample were analyzed in comparison with the germline. As shown in Fig. 4, base replacements were classified into three forms; common to both clones, LN or BM clone-specific and intraclonal sporadic ones. Germline sequence obtained from the patient's peripheral blood mononuclear cells (analyzed on 10 bacterial isolates) was completely identical to the published one. When a clonespecific mutation was defined as one observed in more than 90% of bacterial isolates from a cell subpopulation, among the 293 nucleotides from FR1 to CDR3 region within the VH3-53, 12 (4.1%), 26 (8.9%) and 12 (4.1%) mutations were common to both, LN-specific and BMspecific, respectively.

Discussion
Here we presented a FL case presenting the difference in IgL chain expression between the LN and BM tumor cells. The results of immunophenotypic and genetic studies coincided and confirmed that Igj)k+ cells in LN and Igj+k) cells in BM represented separate clones in the immunological viewpoint. These two tumor clones shared an identical BCL2-IgH recombination, accompanying t(14;18)(q32;q21) translocation, and an identical VDJ joining on the untranslocated IgH allele. In the Igj gene analysis, these clones also shared an identical Vj3-Kde recombination, but a fertile rearrangement involving Cj was detected only in BM on Southern blotting and LN cells seemed to have biallelic abortive Vj-Kde recombinations followed by the Igk rearrangement. These observations indicated that the two clones were derived from the common progenitor cells at the stage before or during the IgL gene rearrangement in B-cell ontogeny, which harbored t(14;18) translocation. Kobrin et al. (15) reported a FL case in which IgL expression of tumor cells changed from Igj at diagnosis to Igk at relapse and raised the possibility that Igk-expressing cells would have arisen subsequent to a secondary IgL rearrangement that occurred in Igjexpressing tumor cells, as SHMs were shared by both cell subpopulations. In our case, it is not likely that Igk-expressing LN cells were converted from Igjexpressing BM cells, as the concomitant IgH deletion on the der(14) chromosome in BM, which seemed to occur subsequent to the generation of the t(14;18) translocation, was not present in LN. On the other hand, results of the Igj gene analysis including the Kde rearrangement indicated that Igj-expressing BM cells would not be derived from Igk-expressing LN cells. Thus, in this case, two tumor clones seemed to have developed independently, rather than that one had been converted from the other.
The SHM analysis within the IgVH revealed the occurrence of LN-and BM-specific base replacements. In the previous report on the SHM analysis in BM-infiltrated FL cases (19), it was shown that the majority of BM tumor cells were derivatives of 'LN-inexperienced' clone. Clone-specific SHMs in LN and BM in our case also indicate that these two clones were transformed by antigenic selection independently at the two sites.
However, some of base replacements within the IgVH observed in this case were common to the both clones and seemed to be tumor-related SHMs that generated at an early stage of development, as polymorphisms were denied by germline sequence from the patient's non-lymphoma cells. As SHM is generally regarded as properties exhibited in mature B cells (20), the shared SHM patterns in this case would indicate that the common progenitor cells had reached at GC stage, and then developed into LN and BM tumor cells with independently occurring secondary IgL rearrangements (receptor revisions).