PMC4696310_01
a H&E staining of the bone marrow showing marked interstitial involvement by medium sized cells with small but conspicuous nucleoli. b CD20 immunohistochemistry highlights marked involvement of the bone marrow by abnormal B-cells
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Fig1
H&E staining, CD20 and Pax5 immunohistochemistry confirmed the nature of the lymphocytes consistent with B-cell line of differentiation (Fig. 1a and b and data not shown).
a Karyotype of the first clone. The first clone (stemline [sl]), six cells, contained a translocation between the long (q) arms of chromosomes 14 and 19 (arrows), and gain of one copy of chromosome 12 (underlined). b
BCL3 break-apart FISH probe (green/red) indicates rearrangement of a BCL3 locus in the first clone (arrows). Normal FISH signal is shown on the right side of the panel for comparison. c
IGH break-apart FISH probe (green/red) indicates rearrangement of an IGH locus in the first clone (arrows). Normal FISH signal is shown on the right side of the panel for comparison
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Fig2
The first clone (stemline [sl]), six cells, contained a translocation between the long (q) arm of chromosomes 14 and 19 (Fig. 2a, arrows), and gain of one copy of chromosome 12 (underlined). Additionally, FISH studies using BCL3 and IGH break-apart probes confirmed BCL3 and IGH translocations in the first clone (Fig. 2b and c, arrows). BCL2 probe picked up six BCL2 signals (Fig. 3b, black and white BCL2 image) indicating on the presence of four translocated BCL2 loci and two intact BCL2 loci from the non-translocated chromosome 18.
a Representative karyotype of the second clone. The second clone, a composite of two cells, was a doubling of the stemline clone (the first clone) with two copies of a translocation between 14q and 18q (IGH;BCL2, red arrows), in addition to a 14q and 19q translocation (IGH;BCL3, white arrows). Six copies of chromosome 12 are underlined. b FISH analysis of the second clone using dual fusion IGH;BCL2 probe (green/red). Black and white image for IGH signal shows 8 loci indicating that all IGH sequences are rearranged and present on derivative chromosomes 14, 18, and 19. Black and white image for BCL2 signal shows 6 loci which represent two intact and two derivative chromosomes 18, and two derivative chromosomes 14. Merged image shows green IGH signals and red BCL2 signals which overlap in four loci, arrows (two copies of derivative chromosome 14 and two copies of derivative chromosome 18). Right panel, IGH break-apart probe (green/red) signal showing four green and four red foci indicative of the presence of four rearranged IGH loci
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Fig3
The second clone, a composite of two cells, was a doubling of the stemline clone (the first clone) with two copies of an additional translocation between 14q and 18q (Fig. 3a, red arrows, six copies of chromosome 12 are underlined). With respect to the second clone, IGH;BCL2 dual-fusion probe picked up eight signals for IGH (Fig. 3b, black and white IGH image) which, in combination with karyotype data, was indicative of the presence of eight IGH derivative translocation products. BCL2 probe picked up six BCL2 signals (Fig. 3b, black and white BCL2 image) indicating on the presence of four translocated BCL2 loci and two intact BCL2 loci from the non-translocated chromosome 18. IGH;BCL2 loci on chromosomes 14 and chromosome 18. In addition, IGH break-apart probe confirmed the presence of four translocated IGH loci (Fig. 3b, right panel, four red and four green signals). In summary, first clone was characterized by the IGH;BCL3 translocation event resulting in derivative chromosomes, 14 and 19 (Fig. 4a) and second clone was characterized by the presence of IGH;BCL2 translocation in addition to IGH;BCL3 translocation and near tetraploid chromosome content resulting in four derivative chromosomes 14 and two derivative chromosomes 18 and 19 (Fig. 4b).
Schematic representation of the rearrangements described in clone 1 and clone 2. Chromosomes involved in rearrangements are shown on the right. a Rearrangements in clone 1 involving IGH (14q32) and BCL3 (19q13.1) which results in two derivative chromosomes (der(19) and der(14). Rearranged chromosomes 14 and 19 are indicated by an arrow on the right. b Rearrangements in clone 2 which includes chromosome duplication event and IGH (14q32) and BCL2 (18q21) translocation in addition to IGH;BCL3 rearrangements. Therefore clone 2 contains 8 derivative chromosomes (a pair of each der(19), der(18), der(14)t(14;19), and der(14)t(14;18)). Rearranged chromosomes 18 and 19 are indicated by the arrows on the right
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Fig4
IGH;BCL2 loci on chromosomes 14 and chromosome 18. In addition, IGH break-apart probe confirmed the presence of four translocated IGH loci (Fig. 3b, right panel, four red and four green signals). In summary, first clone was characterized by the IGH;BCL3 translocation event resulting in derivative chromosomes, 14 and 19 (Fig. 4a) and second clone was characterized by the presence of IGH;BCL2 translocation in addition to IGH;BCL3 translocation and near tetraploid chromosome content resulting in four derivative chromosomes 14 and two derivative chromosomes 18 and 19 (Fig. 4b).
Single Nucleotide Polymorphism (SNP) array analysis. a Confirmation of an extra copy of chromosome 12. In the Log R ratio panel (left) chromosomal boundaries between the q terminal of chromosome 11, chromosome 12, and the p terminal of chromosome 13 are demarcated by lines. The expanded chromosome 12 view (right), shows an isolated view of chromosome 12 (B-Allele frequency). b Microduplication of the genomic region containing ERCC2 gene. c Genomic mapping of ERCC2 gene located in the 19q13.32 cytogenetic band, proximal to the BCL3 gene locus (19q13.31). d Placement of the ERCC2 gene relative to the hypothesized IGH;BCL3 rearrangement junction depicted in the schematics (the possibility exists that ERCC2 duplication occurred on the non-translocated chromosome 19 as stated in text)
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Fig5
Further studies using single nucleotide polymorphism arrays on the patient's DNA sample detected the presence of an extra chromosome 12, confirming our karyotype analysis (Fig. 5a). Interestingly, we also detected a microduplication event at the cytogenetically defined BCL3 translocation junction (19q13.1) containing a major nucleotide excision repair gene ERCC2 (ISCN arr[hg19](12)X3,19q13.32(45,835,983-45,894,768)x3), (Fig. 5b-d).