Janet L. Lukacs

Multicolour spectral karyotyping identifies new translocations and a recurring pathway for chromosome loss in multiple myeloma

Multicolour spectral karyotyping (SKY) was performed on primary tumour specimens from 100 patients with multiple myeloma (MM) that showed complex clonal chromosome aberrations not fully characterized by G‐banding. In this study, SKY was able to identify or revise translocations with breakpoints involving 14q32, 11q13 or 8q24 in 32 patients (32%). Five new recurring translocations were identified, two of which involved chromosome 22. A subtle reciprocal translocation t(14;22) (q32;q11∼12) was identified using SKY in two patients and a second, much larger, translocation t(11;22)(q13;q13) was identified using G‐banding in three patients. A third new translocation was identified in two patients using SKY and G‐banding as der(7)t(7;7)(p15∼22;q22∼32). Twenty‐three patients (23%) showed the loss of 8p by whole‐arm translocations with different whole‐arm donor chromosomes. Among this group, two new recurring whole‐arm translocations involving the centromeric breakpoint 8q10 were identified as der(8;20)(q10;q10) and der(8;18) (q10;q10) in three patients each. In addition, a novel pattern of three‐way translocations involving the clonal evolution of the t(8;22)(q24;q11) by the subsequent loss of 8p by whole‐arm translocations was found in three patients. The chromosome instability identified here demonstrates that the loss of 8p can occur by multiple whole‐arm translocations, indicating a new pathway for the loss of a specific chromosome region in MM.

Genomic instability in multiple myeloma: Evidence for jumping segmental duplications of chromosome arm 1q

Multiple myeloma (MM) is a malignant plasma cell disorder characterized by complex karyotypes and chromosome 1 instability at the cytogenetic level. Chromosome 1 instability generally involves partial duplications, whole‐arm translocations, or jumping translocations of 1q, identified by G‐banding. To characterize this instability further, we performed spectral karyotyping and fluorescence in situ hybridization with probes for satII/III (1q12), BCL9 (1q21), and IL6R (1q21) on the karyotypes of 44 patients with known 1q aberrations. In eight patients, segmental duplication of 1q12–21 and adjacent bands occurred on nonhomologous chromosomes. In five cases, the 1q first jumped to a nonhomologous chromosome, after which the 1q12–21 segment again duplicated itself 1–3 times. In three other cases, segmental duplications occurred after the 1q first jumped to a nonhomologous chromosome, where the proximal adjacent nonhomologous chromosome segment was duplicated prior to the 1q jumping or inserting itself into a new location. These cases demonstrate that satII/III DNA sequences are not only associated not only with the duplication of adjacent distal chromosome segments after translocation, but are also associated with the duplication and jumping/insertion of proximal nonhomologous chromosome segments. We have designated this type of instability as a jumping segmental duplication.

Cytogenetic Distinction among Benign Fibro-osseous Lesions of Bone in Children and Adolescents: Value of Karyotypic Findings in Differential Diagnosis

Benign fibro-osseous lesions of bone (BFOL) comprise a group of clinically distinct entities with significant histologic overlap and often occur in children and adolescents. Because of prior studies indicating that these lesions possess distinct karyotypic abnormalities, we conducted a retrospective review of cytogenetic analyses performed in a series of 16 BFOL in children and adolescents diagnosed at two institutions. These comprised five cases with the diagnosis of ossifying fibroma, four with osteofibrous dysplasia, and seven with fibrous dysplasia arising in the skeleton of 16 children and adolescents. All cases were analyzed using standard G-banding techniques on fresh tumors explanted in tissue culture media. Spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) were used to analyze selected metaphases of a talar lesion with the histologic features of ossifying fibroma. All four confirmed ossifying fibromas, including the talar lesion, contained clonal aberrations fusing breakpoints on Xq26 and 2q33, and one case with dissimilar histology did not. Three of four osteofibrous dysplasias contained multiple copies of chromosomes 8, 12, and/or 21. All but two fibrous dysplasia cases exhibited either a completely normal karyotype or single cell aberrations. One fibrous dysplasia had subtle chromosomal abnormalities not seen in other cases in the series, and another had complex abnormalities involving multiple chromosomes. Our current and published results indicate that cytogenetics might be of ancillary use in the diagnosis of BFOL and that a characteristic chromosomal arrangement is associated with ossifying fibroma.

Jumping translocations of 1q12 in multiple myeloma: a novel mechanism for deletion of 17p in cytogenetically defined high-risk disease

Multiple myeloma (MM) is a B-cell malignancy driven in part by increasing copy number alterations (CNAs) during disease progression. Prognostically significant CNAs accumulate during clonal evolution and include gains of 1q21 and deletions of 17p, among others. Unfortunately, the mechanisms underlying the accumulation of CNAs and resulting subclonal heterogeneity in high-risk MM are poorly understood. To investigate the impact of jumping translocations of 1q12 (JT1q12) on receptor chromosomes (RCs) and subsequent clonal evolution, we analyzed specimens from 86 patients selected for unbalanced 1q12 aberrations by G-banding. Utilizing spectral karyotyping and locus-specific fluorescence in situ hybridization, we identified 10 patients with unexpected focal amplifications of an RC that subsequently translocated as part of a sequential JT1q12 to one or more additional RCs. Four patients exhibited amplification and translocation of 8q24 (MYC), 3 showed amplification of 16q11, and 1 each displayed amplification of 18q21.3 (BCL2), 18q23, or 4p16 (FGFR3). Unexpectedly, in 6 of 14 patients with the combination of the t(4;14) and deletion of 17p, we identified the loss of 17p as resulting from a JT1q12. Here, we provide evidence that the JT1q12 is a mechanism for the simultaneous gain of 1q21 and deletion of 17p in cytogenetically defined high-risk disease.

Recurring breakpoints of 1p13∼p22 in osteochondroma

Cytogenetic studies of osteochondromas are scarce but have previously shown recurring clonal aberrations involving chromosome 8. We have studied a series of eight tumors and have found recurring aberrations not only involving chromosome 8, but also chromosome 1 in five of the seven abnormal tumors. Surprisingly, three of the chromosome 1 aberrations involved pericentric inversions. Four tumors showed aberrations involving the region 1p13∼p22 by mechanisms including inversion, insertion, and translocation. These findings indicate that aberrations of chromosome 1p, in a region spanning 1p13∼p22, may be nonrandomly involved in the cytogenetic progression of osteochondroma.

Evidence of an epigenetic origin for high-risk 1q21 copy number aberrations in multiple myeloma.

Multiple myeloma is a B-cell malignancy stratified in part by cytogenetic abnormalities, including the high-risk copy number aberrations (CNAs) of +1q21 and 17p(-). To investigate the relationship between 1q21 CNAs and DNA hypomethylation of the 1q12 pericentromeric heterochromatin, we treated in vitro peripheral blood cultures of 5 patients with balanced constitutional rearrangements of 1q12 and 5 controls with the hypomethylating agent 5-azacytidine. Using G-banding, fluorescence in situ hybridization, and spectral karyotyping, we identified structural aberrations and copy number gains of 1q21 in the treated cells similar to those found in patients with cytogenetically defined high-risk disease. Aberrations included 1q12 triradials, amplifications of regions juxtaposed to 1q12, and jumping translocations 1q12. Strikingly, all 5 patients with constitutional 1q12 rearrangements showed amplifications on the derivative chromosomes distal to the inverted or translocated 1q12 region, including MYCN in 1 case. At the same time, no amplification of the 1q21 region was found when the 1q12 region was inverted or absent. These findings provide evidence that the hypomethylation of the 1q12 region can potentially amplify any genomic region juxtaposed to it and mimic CNAs found in the bone marrow of patients with high-risk disease.

Sub-band deletion of 7q36.3 in a patient with ring chromosome 7: Association with holoprosencephaly

We report on a patient with ring chromosome 7 analyzed by both high-resolution mid-prophase G-banding and fluorescence in situ hybridization (FISH) resolving a subband deletion of 7q36.3 associated with the clinical manifestation of holoprosencephaly (HPE).

Telomeric fusion as a mechanism for the loss of 1p in meningioma

Characteristic cytogenetic aberrations are found in the various histopathological designations of meningioma. These aberrations range from the loss of 22q in histologically benign tumors to complex hypodiploid karyotypes in atypical and malignant tumors. This progression is characterized by increasing chromosome loss and instability, with a critical step being the loss of 1p. We report a detailed cytogenetic investigation of chromosome aberrations in a series of 88 meningiomas using Giemsa banding and multicolor spectral karyotyping (SKY). Clonal chromosome aberrations were identified in 46 (52%) tumors by G banding. Thirty-five tumors showing complex chromosome aberrations not fully characterized by G banding were subsequently reanalyzed by SKY. The SKY technique refined the G-band findings in 18 (51%) of the tumors on which it was applied. The most common features of cytogenetic progression in the complex karyotypes were chromosome arm-specific losses relating to the formation of deletions and dicentric chromosomes involving 1p. Part or all of 1p was lost in 19 tumors. Five tumors showed evidence for the loss of 1p in a progressive step-wise series of telomeric fusions involving the formation of unstable intermediates. Five recurring dicentric chromosomes were identified, including dic (1;11)(p11;p11), dic(1;12)(p12 approximately p13;p11), dic(1;22)(p11;q12 approximately q13), dic(7;19)(p11;p11), and dic(19;22)(p11 approximately p13;q11 approximately q13). These findings provide evidence that telomeric fusions play a role in the formation of clonal deletions, dicentrics, and unbalanced translocations of 1p. The loss of 1p has possible diagnostic and prognostic implications in the management of meningioma.

Telomeric fusion and chromosome instability in multiple tissues of a patient with mosaic Ullrich-Turner syndrome.

We describe the cytogenetic evolution of multiple cell lines in the gonadal tissue of a 10-year-old girl with mosaic Ullrich-Turner syndrome (UTS) involving clonal telomeric associations (tas) of the Y chromosome. G-band analysis of all tissues showed at least 2 cell lines; 45, X and 46,X,tas(Y;21)(q12;p13). However, analysis of left gonadal tissue of this patient showed the evolution of 2 additional cell lines, one designated 45,X,tas(Y;21)(q12;p13),-22 and the other 46,X,tas(Y;21)(q12;p13),+tas(Y;14)(q12;p13), -22. Fluorescence in situ hybridization (FISH) analysis of interphase nuclei from uncultured gonadal tissue confirmed the findings of aneuploidy in the left gonadal tissue and extended the findings of aneuploidy to the tissue of the right gonad. The chromosome findings in the gonadal tissue of this patient suggest a preneoplastic karyotype relating to several distinct tumor associations. The clonal evolution of telomeric fusions indicates chromosomes instability and suggests the extra copy of the Y chromosome may have resulted from a fusion-related malsegregation. In addition, the extra Y suggests low-level amplification of a putative gonadoblastoma gene, while the loss of chromosome 22 suggests the loss of heterozygosity for genes on chromosome 22. This case demonstrates the utility of the study of gonadal tissue in 45,X/46XY UTS patients, and provides evidence that clonal telomeric fusions may, in rare cases, be associated with chromosome malsegregation and with the subsequent evolution of unstable karyotypes.

Hyperhaploidy is a novel high-risk cytogenetic subgroup in multiple myeloma

Hyperhaploid clones (24–34 chromosomes) were identified in 33 patients with multiple myeloma (MM), demonstrating a novel numerical cytogenetic subgroup. Strikingly, all hyperhaploid karyotypes were found to harbor monosomy 17p, the single most important risk stratification lesion in MM. A catastrophic loss of nearly a haploid set of chromosomes results in disomies of chromosomes 3, 5, 7, 9, 11, 15, 18, 19 and 21, the same basic set of odd-numbered chromosomes found in trisomy in hyperdiploid myeloma. All other autosomes are found in monosomy, resulting in additional clinically relevant monosomies of 1p, 6q, 13q and 16q. Hypotriploid subclones (58–68 chromosomes) were also identified in 11 of the 33 patients and represent a duplication of the hyperhaploid clone. Analysis of clones utilizing interphase fluorescence in situ hybridization (iFISH), metaphase FISH and spectral karyotyping identified either monosomy 17 or del17p in all patients. Amplification of 1q21 was identified in eight patients, demonstrating an additional high-risk marker. Importantly, our findings indicate that current iFISH strategies may be uninformative or ambiguous in the detection of these clones, suggesting this patient subgroup maybe underreported. Overall survival for patients with hyperhaploid clones was poor, with a 5-year survival rate of 23.1%. These findings identify a distinct numerical subgroup with cytogenetically defined high-risk disease.