Identification of an unusual marker chromosome by spectral karyotyping

We ascertained a newborn girl with multiple congenital anomalies including severe hypotonia, cardiovascular defects, hearing loss, central nervous system anomalies, and facial anomalies. The infant died at 12 days. Cytogenetic analysis showed a de novo supernumerary marker chromosome. Fluorescence in situ hybridization (FISH) with a combination of chromosome specific alpha-satellite probes and an all-human centromere probe failed to show hybridization to the marker, indicating that the marker chromosome lacked detectable alpha satellite sequences. Spectral karyotyping (SKY) was performed and showed that the marker was chromosome 15 in origin. This was confirmed by FISH with a 15q specific subtelomeric probe, which showed hybridization to both ends of the marker chromosome. Based on FISH information and G-banding pattern, the marker was determined to be an inverted duplication of 15q25-qter, leading to partial tetrasomy for chromosome 15. Although the marker chromosome lacked detectable centromeric alpha-satellite sequences, it seemed to have a functional centromere as it is mitotically stable. This observation is consistent with previous studies on acentric marker chromosomes, which suggested that the DNA sequence at the breakpoint could function similarly to alpha-satellite sequences once activated through marker formation. Am. J. Med. Genet. 80:368–372, 1998. © 1998 Wiley-Liss, Inc.     

光谱核型分析联合微阵列比较基因组杂交诊断15号环状染色体综合征

目的 探讨联合应用光谱核型分析技术(spectral karyotyping,SKY)和微阵列比较基因组杂交技术(microarray-based comparative genomic hybridization,array-CGH)在诊断复杂疑难的环状染色体畸变中的价值.方法 对1例常规G显带染色体核型分析疑诊为46,XY,r(15)?的8岁男性生长发育迟缓患儿依次应用SKY及array-CGH技术常规进行制片杂交,并通过相应的显微摄像系统和计算机软件分析结果.结果 SKY技术明确了该患儿环状染色体来源于15号染色体,array-CGH技术明确患儿15q26.3末端存在约594 kb的缺失,染色体基因位点编码范围为99689349-100282878.结论 联合应用现代分子细胞遗传学技术可以从细胞到分子水平精确诊断复杂疑难的环状染色体病例,是常规染色体核型分析的有益补充,也有利于细胞遗传学向分子水平深入.     

应用光谱核型分析技术诊断复杂染色体畸变

目的探讨光谱核型分析技术（spectral karyotyping，SKY）在诊断复杂染色体畸变中的应用价值。方法选取4例常规G显带染色体核型分析未能确诊的染色体畸变病例，按SKY操作常规进行制片杂交，并通过相应的计算机软件分析结果。结果通过SKY技术，明确1例涉及3条染色体复杂易位病例的诊断；协助2例不明来源衍生染色体诊断为染色体自身的部分重复；但对染色体自身倒位和染色体畸变断裂点的诊断帮助不大。结论SKY技术对于诊断复杂的染色体易位或重排、微小的染色体结构畸变以及不明来源的衍牛染色体等方面有较大的优越性．是常规染仁．体核型分析的有益补充。     

Identification of poorly differentiated synovial sarcoma: a comparison of clinicopathological and cytogenetic features with those of typical synovial sarcoma

AIMS: Poorly differentiated areas in synovial sarcomas (SS) are known to be associated with a poorer prognosis. The aim of our study was to describe the morphological spectrum of poorly differentiated synovial sarcomas (PDSS) and refine the criteria for their recognition. METHODS AND RESULTS: The clinicopathological features of 28 PDSS were compared with those of 26 classic SS. Common cell types in PDSS included epithelioid, spindle and Ewing sarcoma-like small round cells. Unusual features included presence of desmoplastic small cell tumour-like areas and extraskeletal myxoid chondrosarcoma-like areas. The presence of necrosis (P = 0.002), a mitotic rate over 10/10 high-power fields (P < 0.001), a haemangiopericytomatous vascular pattern (P < 0.001) and vascular invasion (P = 0.003) were significantly associated with PDSS, while mast cells (P < 0.001), calcification (P < 0.001) and hyaline bands (P < 0.001) were significantly associated with classic SS. Poorly differentiated areas showed increased proliferative activity with Ki67. PDSS showed a tendency to be larger (P = 0.008) and to be located in proximal more than distal sites (P = 0.025). Three entirely poorly differentiated tumours were diagnosed by demonstration of the t(X;18)(p11;q11) translocation. PDSS showed additional cytogenetic abnormalities. CONCLUSIONS: Poorly differentiated synovial sarcomas show a spectrum of histological features, which may simulate other malignant neoplasms. The diagnosis of entirely poorly differentiated synovial sarcomas requires cytogenetic analysis.     

Twenty-four-color spectral karyotyping reveals chromosome aberrations in cytogenetically normal acute myeloid leukemia

Multicolor spectral karyotyping allows simultaneous visualization of all human chromosomes and screening for chromosomal rearrangements without a priori knowledge of any abnormalities involved. Based on this potentially increased sensitivity, we investigated, in a preliminary manner, whether spectral karyotyping could detect cytogenetic aberrations in karyotypically normal leukemia. The test population was comprised of 28 cryopreserved, cytogenetically normal acute myeloid leukemia (AML) samples from patients registered to a randomized trial for previously untreated AML (SWOG 9031). Two normal and 12 samples with known cytogenetic aberrations were used to validate and establish the diagnostic accuracy of the spectral karyotyping assay and instrumentation in a clinical setting. Enumeration and region-specific DNA fluorescence in situ hybridization (FISH) probes verified discrepant results. In the validation data set, spectral karyotyping refined complex karyotypic rearrangements in six cases and defined the chromosomal origin of a "jumping" homogeneously staining region; however, the technology was less sensitive in the detection of subtelomeric rearrangements and double minute chromosomes. In the test population, spectral karyotyping identified previously undetected cytogenetic aberrations in two cases (7%) of karyotypically normal AML: a cryptic 11q23 translocation in 20/20 cells and a minor monosomy 7 clone in 3/21 cells (FISH, 10.5%). Both of these abnormalities are considered to confer a poor prognosis when based on classical cytogenetic prognostic criteria. As an adjunct to classical cytogenetics and standard FISH analyses, the additive resolution of spectral karyotyping, in particular, with chromosome paints spiked with subtelomeric and/or locus-specific probes, may allow significant gains to be made in diagnostic accuracy and recognition of genotype/phenotype prognostic relationships, and in defining underlying biologic mechanisms in cancer. Genes Chromosomes Cancer 28:318-328, 2000.     

Combined spectral karyotyping and DAPI banding analysis of chromosome abnormalities in myelodysplastic syndrome.

Spectral karyotyping (SKY) is a new molecular cytogenetic technique that allows simultaneous visualization of each chromosome in a different color. We have used SKY for comprehensive analysis of 20 myelodysplastic syndromes (MDSs) (13 primary MDSs, 3 therapy-related MDSs, and 4 acute leukemias developed from MDS, including 1 cell line established from a secondary leukemia), previously analyzed by G-banding. To locate the chromosomal breakpoints, DAPI-counterstained band images from all metaphases were transformed to G-band-like patterns. By using SKY, it was possible to identify the origin and organization of all clonal marker chromosomes (mar), as well as the origin of all abnormalities defined as additional material of unknown origin (add) or homogeneously staining regions (hsr) by G-banding. In total, SKY identified the chromosomal basis of 38 mar, add, and hsr, corrected 8 abnormalities misidentified by G-banding, and revealed 6 cryptic translocations in 5 cases. Total or partial chromosomal loss (mainly of -5/5q- and -7/7q-) is the most frequent cytogenetic abnormality in MDS. In 3 of 11 cases with -5/5q- and in 4 of 8 with -7/7q-, lost material was detected by SKY in unbalanced translocations. A total of 60 chromosomal losses were identified by G-banding in 16 cases with multiple chromosome abnormalities involving at least 3 chromosomes. For 26 of these losses (43%), SKY analysis suggested that the losses were not complete, but had been translocated to a variety of partner chromosomes. Moreover, SKY analysis revealed that a ring chromosome in a case of acute leukemia developed from MDS contained three to six segments that originated from chromosome 21 material. Fluorescence in situ hybridization showed the amplification of the AML1 gene on regions derived from chromosome 21, providing the first evidence of amplification involving this gene in MDS. Genes Chromosomes Cancer 26:336-345, 1999.     

Detection of unidentified chromosome abnormalities in human neuroblastoma by spectral karyotyping (SKY)

Spectral karyotyping (SKY) is a novel technique based on the simultaneous hybridization of 24 fluorescently labeled chromosome painting probes. It provides a valuable addition to the investigation of many tumors that can be difficult to define by conventional banding techniques. One such tumor is neuroblastoma, which is often characterized by poor chromosome morphology and complex karyotypes. Ten primary neuroblastoma tumor samples initially analyzed by G-banding were analyzed by SKY. In 8/10 tumors, we were able to obtain additional cytogenetic information. This included the identification of complex rearrangements and material of previously unknown origin. Structurally rearranged chromosomes can be identified even in highly condensed metaphase chromosomes. Following the SKY results, the G-banding findings were reevaluated, and the combination of the two techniques resulted in a more accurate karyotype. This combination allows identification not only of material gained and lost, but also of breakpoints and chromosomal associations. The use of SKY is therefore a powerful tool in the genetic characterization of neuroblastoma and can contribute to a better understanding of the molecular events associated with this tumor.     

Acute leukemia cytogenetics: an evaluation of combining G-band karyotyping with multi-color spectral karyotyping

We have, during a 12-month period, evaluated the adjuvant effect of combining G-band karyotyping and multi-color spectral karyotyping (SKY) in acute leukemia patients. Forty-four cases were evaluated; fewer cases than those routinely analyzed by G-band cytogenetics had mitoses left for SKY analysis. Of the 44 patients, 35 were acute myeloid leukemia (AML) and 9 acute lymphatic leukemia (ALL) cases. Twenty-seven of 35 AML and 7 of 9 ALL patients had an abnormal G-band karyotype. Thirteen of these 34 abnormal cases had a simple clonal chromosome aberration, and the remaining 21 cases had a complex karyotype. The SKY confirmed the simple karyotype in 11 and in 7 with a complex karyotype. In 13 of the cases with a complex karyotype, ambiguous structural aberrations were classified, in 6 of these, SKY disclosed cryptic translocations. Thus, SKY either extended or confirmed G-band karyotypes in 31 of 34 analyzed abnormal cases. Cases where SKY did not reveal the abnormal clone showed only few abnormal mitoses by G-banding (2/23, 2/25, and 4/27). Additional or confirmatory information was therefore obtained in 91% of analyzed cases, and SKY proved to be a valuable additional tool for hematologic cytogenetics.     

Primary pleural synovial sarcoma. A case report and review of the literature

Synovial sarcoma (SS) is an uncommon soft tissue tumor that occurs primarily in the extremities of young adults, especially in the periarticular region. In this report, we describe the rare occurrence of primary SS of the pleura in a 15-year-old boy. Histologically, the tumor demonstrated monophasic morphologic findings and showed positive staining with vimentin and Bcl-2 and focally for cytokeratin CK7. Fluorescent in situ hybridization identified t(X;18) translocation. The patient developed recurrences 20 months following resection of the tumor. The literature on this uncommon entity is reviewed, and its histogenesis, differential diagnoses, and cytologic features are also discussed.     

Myxoinflammatory fibroblastic sarcoma with complex supernumerary ring chromosomes composed of chromosome 3 segments

Myxoinflammatory fibroblastic sarcoma is a rare, recently described, and distinctive low-grade tumor of soft tissue. To our knowledge, there is only one previous report on the cytogenetics of this tumor. That case showed complex structural abnormalities, including a reciprocal translocation between chromosomes 1 and 10 [t(1;10)(p22;q24)] with loss of chromosomes 3 and 13. We describe here a second case showing supernumerary ring chromosomes, and a derivative chromosome 13, with additional material on the short arm. We conclude that the presence of chromosomal abnormalities supports the neoplastic nature of this tumor and aids in its diagnosis. Furthermore, we also postulate that the finding of ring chromosomes, which have been identified in other low-grade soft-tissue tumors, may have important prognostic implications regarding the aggressiveness of this neoplasm.     

Tetrasomy 21 as a sole chromosome abnormality in acute myeloid leukemia. fluorescence in situ hybridization and spectral karyotyping analyses

We report a case of acute myeloid leukemia with tetrasomy 21 as the sole chromosome abnormality in a constitutionally normal patient. Tetrasomy 21 was observed at presentation, disappeared in remission, but reappeared in recurrence of the disease. Fluorescence in situ hybridization analysis using a probe specific for the AML1 gene showed four distinct signals in 82.4% and three signals in 10.8% of interphase nuclei, although conventional G-banding revealed tetrasomy 21 alone in mosaicism with normal karyotype. Spectral karyotyping further confirmed the presence of extra copies of chromosome 21. Tetrasomy 21 as the only anomaly is relatively rare in patients with hematologic disorders other than Down syndrome, and to our knowledge has been reported previously in only seven cases. In a review of the literature, tetrasomy 21 as the only anomaly may be associated with myeloid disorders, although simultaneous numeric abnormalities other than chromosome 21 have been reported in acute lymphoblastic leukemia with hyperdiploid karyotype.     

Rapid high-resolution karyotyping with precise identification of chromosome breakpoints

Many techniques have been developed in recent years for genome-wide analysis of genetic alterations, but no current approach is capable of rapidly identifying all chromosome rearrangements with precise definition of breakpoints. Combining multiple color fluorescent in situ hybridization and high-density single nucleotide polymorphism array analyses, we present here an approach for high resolution karyotyping and fast identification of chromosome breakpoints. We characterized all of the chromosome amplifications and deletions, and most of the chromosome translocation breakpoints of three prostate cancer cell lines at a resolution which can be further analyzed by sequence-based techniques. Genes at the breakpoints were readily determined and potentially fused genes identified. Using high-density exon arrays we simultaneously confirmed altered exon expression patterns in many of these breakpoint genes.     

Detection of recurrent chromosome abnormalities in Ewing's sarcoma and peripheral neuroectodermal tumor cells using bivariate flow karyotyping.

Bivariate flow karyotyping can be used for the detection of recurrent chromosome abnormalities in tumor cells. For this purpose 2 cell lines originally derived from Ewing's sarcomas and 4 cell lines from peripheral neuroectodermal tumors were used. The characteristic t(11;22) was known to be present in 5 cell lines. The remaining cell line was known to have a variant t(2;11;22;21) translocation. Metaphase chromosomes were stained with the fluorescent dyes Hoechst 33258 and Chromomycin A3 and analyzed subsequently using bivariate flow cytometry. The resulting bivariate flow karyotypes of the tumor cells were normalized by a standardized procedure using a computerized method and compared with a reference flow karyotype of normal chromosomes. In 5 cell lines two recurring abnormal chromosome peaks were identified at positions expected for the der(11) and der(22) chromosomes characteristic for the reciprocal t(11;22)(q24;q12). In the remaining cell line with the variant t(2;11;22;21), only the peak representing the der(22) was identifiable. It is concluded that bivariate flow karyotyping can be used for the semiautomated detection of recurrent translocations and the assessment of their variability among different tumors.     

Low grade fibromyxoid sarcoma. a further low-grade soft tissue malignancy characterized by a ring chromosome

Supernumerary rings in the context of a simple karyotype characterize several low-grade malignant tumors of soft tissue and bone. Low-grade fibromyxoid sarcoma is an uncommon low-grade sarcoma, the cytogenetics of which has not yet been reported. Here we describe the first molecular-cytogenetic characterization of a pulmonary metastasis of low-grade fibromyxoid sarcoma. The histology of the primary and recurrent tumors was consistent with the diagnosis of low-grade fibromyxoid sarcoma of the usual type, whereas the pulmonary metastasis was of the "giant rosettes" variant. Cytogenetic analysis revealed a ring chromosome. Because gain of material of chromosomes 7 and 16 was detected by CGH, the ring chromosome is assumed to be composed of material from these respective chromosomes.     

Azoospermia in a man with a constitutional ring 22 chromosome

A mosaic ring chromosome 22 (mos 46,XY,r(22)[93]/45,XY,-22[7]) was found in an euploid azoospermic otherwise phenotypically normal individual. Testicular cytological analysis showed hypospermatogenesis with a complete spermatogonial arrest. The majority of subjects with constitutional r(22) are dysmorphic and mentally retarded due to deletion of a sizable segment of the chromosome 22q. Only a few cases of r(22) chromosome are known in which deletion of the very distal telomeric regions is associated with unremarkable phenotype and fertility, both in males and females. The present patient is the first example of male infertility associated with this cytogenetic anomaly. It is likely that infertility arose from a mechanical block of meiosis, resulting from pairing failure of chromosomes 22, similarly to azoospermia occurring in few known males with r(21) chromosomes.