PMC4530787_01
Presentation of follicular lymphoma in the donor and recipient.. A. Time course of chronic phase CML (cP CML) in the recipient followed by bone marrow transplantation (BMT), administration of three donor lymphocyte infusions (DLI) and treatment for detectable BCR/ABL1 transcript by RT-PCR. Follicular lymphoma (FL) was diagnosed in both the donor and the recipient within a 6-month period. B. Immunohistochemistry with hematoxylin and eosin (H&E) or antibodies against the indicated markers.
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In June 2000, she received myeloablative conditioning followed by transplantation of unmanipulated bone marrow from her 40-year old HLA-identical sister (Figure 1A). In 2006, she became PCR positive and was switched to imatinib, which again resulted in a complete molecular remission (Figure 1A). Biopsy of a right inguinal lymph node and bone marrow revealed grade 2 follicular lymphoma with focal areas of grade 3A (Figure 1B). Biopsy of a left inguinal lymph node and bone marrow also revealed grade 2 follicular lymphoma with focal areas of grade 3A and no evidence of CML (Figure 1B). Morphologic and immunohistochemical findings in both FLs were highly similar, with both cases infiltrated by tumor cells expressing CD20, CD10, BCL2 and BCL6 (Figure 1B). We utilized the pan-B-cell marker CD19 to sort positive and negative populations from the DLI by flow cytometry (Figure S1). As expected, the BCL2/IGH rearrangement was below the level of detection in the CD19-negative subset of the DLI, either by conventional PCR (Figure 2A) or by qPCR (lower limit of sensitivity ~1-in-25,000 cells) (Figure S1).
Follicular lymphomas in the donor and recipient are derived from a common ancestor.. A. PCR amplification of IGH/BCL2 genomic DNA from both lymphomas, DLI, or flow-sorted DLI for CD19-positive and CD19-negative populations. Amplification of GAPDH DNA is shown as a control. B. Identical BCL2/IGH rearrangements amplified from genomic DNA from both lymphomas and DLI. C. Quantitative PCR with primer sets and probes for the patient specific BCL2/IGH junction or GAPDH. Standard curves were generated from cloned BCL2/IGH (in red) and GAPDH (in green) PCR products. Genomic DNA of the DLI was tested in triplicates and results were plotted against the standard curves. D. The same IGH rearrangement was amplified from both lymphomas. Germline (GL) VH3-66*4 was amplified from the donor's buccal swab and is shown for comparison. Mismatches that are shared between both lymphomas are in yellow. Mismatches unique to a single lymphoma are in red. '--//--' indicates a small deletion. 'NDN' indicates non-templated nucleotides flanking DH sequence.
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PCR with primers overlapping the major breakpoint region of BCL2 and a VH3 consensus sequence amplified identical BCL2/IGH rearrangements from the donor's FL and the recipient's FL (Figure 2A-B). PCR with the same primers identified an identical product from the DLI, the most recently passed specimen from donor to recipient (Figure 2A-B). The fraction of DLI cells harboring BCL2/IGH was approximately 1-in-2000 (0.026% of GAPDH alleles) (Figure 2C). As expected, the BCL2/IGH rearrangement was below the level of detection in the CD19-negative subset of the DLI, either by conventional PCR (Figure 2A) or by qPCR (lower limit of sensitivity ~1-in-25,000 cells) (Figure S1). Both lymphomas harbored identically-sized rearrangements that utilized the same VH, DH and JH segments and shared identical non-templated insertions (Figure 2D), indicating that the common FL ancestor had completed IGH rearrangement. There were multiple identical somatic alterations within the amplified region present in both FLs (Figure 2D), indicating that the common ancestor had initiated somatic hypermutation. Both FL-derived IGH rearrangements also harbored distinct somatic alterations, consistent with ongoing somatic hypermutation during clonal divergence (Figure 2D).
Mutations in the paired follicular lymphomas.. A. Two SNVs were present in BCL2 in both FLs but not detectable by Sanger sequencing in the DLI. Only the mutant allele is present in cDNA from the recipient's FL. B. In-frame deletion in EP300 and in-frame insertion in KLHL6 were detectable by Sanger sequencing in both lymphomas. In contrast with BCL2, both the wild-type and mutated alleles of KLHL6 were expressed in the recipient FL. C. Exome sequencing reads spanning ARID1A R1276 in the recipient FL are shown. The SNV was only detectable in the recipient FL. D. Quantitative PCR copy number assay at the ARID1A locus. The statistically significant decrease was confirmed in repeated iterations of this experiment. E. Immunohistochemistry for ARID1A/BAF250 (Santa Cruz) is shown in both lymphomas and a tonsil from a healthy individual. Figure S4B shows similar findings using a different anti-BAF250 antibody and normal control. F. SNVs in CTSS and ATP6V1B were detectable by Sanger sequencing only in the donor FL. Both were enriched in the DLI based on ultra-deep sequencing and CTSS M185V was also present in 4.7% of reads from the recipient's FL.
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BCL2, which is known to be a target of aberrant somatic hypermutation in FL (Figure 3A). Based on sequencing of cDNA from the recipient's FL, both SNVs were present in 100% of BCL2 transcript (Figure 3A), indicating that the mutations are present in cis on the translocated allele. An in-frame deletion in EP300, an acetyltransferase that is recurrently mutated in FL and diffuse large B-cell lymphoma (DLBCL), was present in both FLs (Figure 3B). In addition, we identified an in-frame insertion in KLHL6 (Figure 3B), which was recently found to be mutated in chronic lymphocytic leukemia (CLL), DLBCL and FL. Among the SNVs unique to the recipient's FL was an R1276 premature stop mutation in ARID1A (adenine-thymine (AT)-rich interactive domain-containing protein 1A) (Figure 3C) that was previously reported in pancreatic cancer. ARID1A locus is within a region of chr. 1p that is deleted in approximately 20% of FL cases, and deletion is linked to inferior prognosis. Although the premature stop mutation was present only in the recipient's FL, both lymphomas had reduced BAF250 (the protein product of ARID1A) expression by immunohistochemical analysis (Figures 3E and S4B). ARID1A expression could be affected by copy number loss in the donor's FL, we performed qPCR on genomic DNA from both FLs, as well as control samples. As outlined in Figure 3D, qPCR identified copy number loss (copy number, 1.84; p=0.022 compared to donor germline) at this locus in the donor's FL but not the recipient's FL (Figure 3D). Of the final two mutations, ATP6V1B2 R400Q (Figure 3D) was detected only in the donor's FL but was enriched in the DLI. CTSS M185V (Figure 3D) was initially detected only in the donor's FL, but deep sequencing recovered the mutation in 4.7% of reads from the recipient's FL and demonstrated enrichment in the DLI (Table 1).