Analysis of marker or complex chromosomal rearrangements present in pre- and post-natal karyotypes utilizing a combination of G-banding,spectral karyotyping and fluorescence in situ hybridization

The significance of complex chromosomal rearrangements presents a diagnostic dilemma. In the past, the use of G-banding coupled with fluorescence in situ hybridization (FISH) has been the standard approach. The recent development of spectral karyotyping (SKY) and multicolor FISH (M-FISH) has resulted in an increased accuracy of identification of marker or other complex chromosomal rearrangements. However, owing to the additional cost and time associated with SKY or M-FISH, and the restricted availability of such imaging facilities in many centers, it is not feasible to perform these procedures routinely on every sample. In addition, the identification of an aberration by SKY or M-FISH will often require confirmation by FISH. A practical approach is needed to take advantage of the complementary strengths of each method. In our center we utilize an algorithm that dictates the use of routine G-banding for the initial preliminary evaluation of a patient, followed by SKY characterization if marker chromosomes or complex translocations are detected by the G-banding analysis. According to this algorithm, FISH is used to verify the results once the origin of the abnormal chromosome has been determined by SKY. To demonstrate the effectiveness of this algorithm, we have analyzed both amniocyte and lymphocyte slides, using a combination of G-banding, SKY, and FISH. Our results confirm that an algorithm which selectively uses SKY or M-FISH will provide an efficient and improved method for pre- and post-natal chromosomal analysis.     

Characterization of complex chromosomal abnormalities in uveal melanoma by fluorescence in situ hybridization, spectral karyotyping, and comparative genomic hybridization

Several nonrandom recurrent chromosomal changes are observed in uveal melanoma. Some of these abnormalities, e.g., loss of chromosome 3, gain of the q arm of chromosome 8, and chromosome 6 abnormalities, are of prognostic value. Cytogenetic analysis and/or fluorescence in situ hybridization (FISH) are used to detect these changes. In some cases, however, detailed cytogenetic analysis is not possible due to the presence of complex abnormalities. To define more accurately these cytogenetic changes, we have applied comparative genomic hybridization (CGH) and/or spectral karyotyping (SKY) to two uveal melanoma cell lines and five primary uveal melanomas, with partially defined and/or complex abnormalities. SKY provided additional information on 34/39 partially defined aberrant chromosomes and revealed a new abnormality, a der(17)t(7;17)(?;q?), that had not been recognized by conventional cytogenetics. Additionally, using SKY, abnormalities involving chromosome 6 or 8 were found to be twice as common as observed with cytogenetic analysis. CGH was especially useful in assigning the abnormalities identified by SKY to specific chromosomal regions and, in addition, resulted in the detection of a small deletion of chromosome region 3q13 approximately 21. We conclude that SKY and CGH, as methods complementary to cytogenetic and FISH analysis, provide more complete information on the chromosomal abnormalities occurring in uveal melanoma.     

A role for fluorescence in situ hybridization detection of chromosome 22q dosage in distinguishing atypical teratoid/rhabdoid tumors from medulloblastoma/central primitive neuroectodermal tumors

It has been postulated that infants with medulloblastomas/central primitive neuroectodermal tumors (MB/PNET) may fare worse than older patients because some of them harbor unrecognized atypical teratoid/rhabdoid tumors (AT/RT), rare intracranial neoplasms that are typically unresponsive to therapy and rapidly fatal. Although small primitive cells are common to both entities, chromosome 22q11.2 deletions are common only in AT/RTs. Using fluorescence in situ hybridization (FISH) on archival, paraffin-embedded biopsy tissue with commercially available probes to 22q11.2, the region associated with RTs, we studied 8 cases of AT/RT, 12 cases of MB/PNET, and 4 cases of primitive central nervous system (CNS) neoplasms, which were difficult to classify. 22q Deletions were identified in 6 of 8 (75%) conventional AT/RTs and 0 of 12 (0%) children with classic MB/PNET. Of the 4 originally "difficult to classify" cases, 3 had deletions of 22q. In light of the FISH results, review of the morphology and immunophenotype resulted in 3 tumors being reclassified as AT/RTs and 1 as a large cell MB. These 4 cases highlight the potential diagnostic use of FISH for selected cases of primitive CNS malignancies in children and substantiate the notion that misdiagnosed AT/RTs may, in part account for the worse prognosis associated with "MB/PNET" in children younger than 2 years of age.     

Primitive neuroectodermal tumor of the breast: immunohistochemistry and fluorescence in situ hybridization

A case of primitive neuroectodermal tumor (PNET) that developed as a breast lump in a 47-year-old Japanese woman is reported. After fine-needle aspiration (FNA) cytology, a mastectomy was performed. The surgical specimen was a well-circumscribed tumor, 21 x 18 x 18 mm, localized in the breast. The tumor cells were small and round with scant cytoplasm, and proliferated in sheets or solid nests. No intraductal carcinoma component was present. The tumor cells were immunohistochemically positive for neural cell adhesion molecule (CD56), neuron-specific enolase and synaptophysin, and they showed membranous immunoreactivity for the MIC2 protein (CD99). Fluorescence in situ hybridization (FISH) indicated a rearrangement of the EWS region on chromosome 22, which is highly specific for Ewing's sarcoma and PNET, which are referred to as the Ewing's sarcoma family of tumors (EFT). No other lesions suggestive of the primary site of this tumor, such as bone, soft tissue, or other organs were detected. The patient has been disease free for 6 months after surgery followed by chemoradiation therapy. FISH is extremely useful for accurate diagnosis of EFT, especially in cases of unusual locations.     

Molecular cytogenetic analysis of oral squamous cell carcinomas by comparative genomic hybridization, spectral karyotyping, and fluorescence in situ hybridization

We investigated relationships between DNA copy number aberrations and chromosomal structural rearrangements in 11 different cell lines derived from oral squamous cell carcinoma (OSCC) by comparative genomic hybridization (CGH), spectral karyotyping (SKY), and fluorescence in situ hybridization (FISH). CGH frequently showed recurrent chromosomal gains of 5p, 20q12, 8q23 approximately qter, 20p11 approximately p12, 7p15, 11p13 approximately p14, and 14q21, as well as losses of 4q, 18q, 4p11 approximately p15, 19p13, 8p21 approximately pter, and 16p11 approximately p12. SKY identified the following recurrent chromosomal abnormalities: i(5)(p10), i(5)(q10), i(8)(q10), der(X;1)(q10;p10), der(3;5)(p10;p10), and der(3;18)(q10;p10). In addition, breakpoints detected by SKY were clustered in 11q13 and around centromeric regions, including 5p10/q10, 3p10/q10, 8p10/q10 14q10, 1p10/1q10, and 16p10/16q10. Cell lines with i(5)(p10) and i(8)(q10) showed gains of the entire chromosome arms of 5p and 8q by CGH. Moreover, breakages near the centromeres of chromosomes 5 and 8 may be associated with 5p gain, 8q gain, and 8p loss in OSCC. FISH with a DNA probe from a BAC clone mapping to 5p15 showed a significant correlation between the average numbers of i(5)(p10) and 5p15 (R(2) = 0.8693, P< 0.01) in these cell lines, indicating that DNA copy number of 5p depends upon isochromosome formation in OSCC.     

Aberrations of chromosome 8 in 16 breast cancer cell lines by comparative genomic hybridization, fluorescence in situ hybridization, and spectral karyotyping

Comparative genomic hybridization (CGH) studies have shown that chromosome 8 is a frequent target for chromosomal aberrations in breast cancer. We characterized these aberrations of chromosome 8 in 16 breast cancer cell lines (BT-474, BT-549, CAMA-1, DU-4475, MCF-7, MDA-MB-134, MDA-MB-157, MDA-MB-361, MDA-MB-415, MDA-MB-436, MPE600, SK-BR-3, T-47D, UACC-812, UACC-893 and ZR-75-1) by CGH, fluorescence in situ hybridization (FISH) with arm- and locus-specific probes, and spectral karyotyping (SKY). Chromosome 8 was structurally abnormal in 13 of 16 cell lines. Loss of 8p was detected in nine cell lines, gain of entire 8q in six cell lines, 8q21-qter in three, 8q23-qter in two, and 8q12-qter and 8p21-q21 in one cell line. Extra copies of the C-MYC oncogene were found in 11 cell lines, but high-level amplification only in SK-BR-3. Derivative chromosomes including material from chromosomes 8 were complex, and the breakpoints were strikingly dissimilar. Chromosome 11 was the most frequent translocation partner with chromosome 8 (in 7 cell lines). Isochromosomes and/or isoderivative 8q were found in four cell lines. The high frequency and complexity of alterations at 8q indicate a significant pathogenetic role in breast cancer. The high-level amplification of c-myc is less common than previously thought.     

Ewing sarcoma/peripheral primitive neuroectodermal tumor: adult abdominal tumors with an Ewing sarcoma gene rearrangement demonstrated by fluorescence in situ hybridization in paraffin sections.

The differential diagnosis of small round cell tumors is exhaustive and requires ancillary studies. Relatively recently, fluorescence in situ hybridization (FISH) using probes for specific gene rearrangements has gained wide acceptance. This technique is particularly useful in the differential diagnosis of Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET) and desmoplastic small round-cell tumor (DSRCT). In ES/PNET, the EWS gene is juxtaposed to the FLI-1 gene in 85% of cases and to the ERG gene in another 7% of cases; the EWS gene is juxtaposed to the WTI gene in DSRCT. Documentation of the EWS gene rearrangements in EWS/PNET has previously been demonstrated in frozen tissue. We report 2 unusual cases of EWS/PNET diagnosed in abdominal tumors in adults. Although the immunohistochemical results supported a diagnosis of ES/PNET, 1 case morphologically resembled DSRCT. The diagnosis in these 2 cases was confirmed by the FISH demonstration of EWS/FLI-1 gene fusion in paraffin-embedded tissue. Thus, the usefulness of FISH demonstration of an EWS gene rearrangement with these specific probes in such unusual cases is supported and is demonstrated in paraffin-embedded tissue.     

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.     

G-显带技术、荧光原位杂交和比较基因组杂交技术在产前诊断中的应用

目的 探讨G显带、荧光原位杂交(fluorescencein situ hybridization,FISH)和比较基因组杂交(comparative genomic hybridization,CGH)技术在产前诊断中应用的程序及意义.方法 采集102例妊娠16周～24周胎儿的羊水,采用G显带、G显带/FISH和G显带/FISH/CGH三阶梯的核型诊断程序,并分析其在产前诊断中的意义.结果 102例胎儿中,经第1阶梯诊断核型98例,诊断困难2例,失败2例;第2阶梯诊断核型2例,诊断困难1例,失败1例;第3阶梯诊断核型2例.经3阶梯诊断程序核型的诊断率达100%(102/102例),异常核型7例(7/102例,6.68 0A),其中第1、第2和第3阶梯分别诊断异常核型4例(4/7例,57.1 oA)、1例(1/7例,14.3%)和2例(2/7例,28.5%).结论 在产前诊断中实施3阶梯诊断程序有助于提高核型的确诊率,规范染色体诊断流程.     

髓母细胞瘤与幕上原始神经外胚叶肿瘤中RASSF1A基因的甲基化差异

目的通过研究髓母细胞瘤与幕上原始神经外胚叶肿瘤（SPNET）中RASSF1A基因的甲基化改变,探讨颅内原始神经外胚叶肿瘤（PNET）的不同亚型中该基因的表遗传学差异及其意义。方法收集25例原发髓母细胞瘤,9例原发SPNET,3株髓母细胞瘤细胞系和2株SPNET细胞系。采用甲基化特异性聚合酶链反应（MSP）检测RASSF1A基因启动子区的甲基化状态。应用去甲基化试剂5-aza-2’deoxycytidine处理存在基因表达缺失的细胞系,探讨基因表达与甲基化之间的关系。结果100％（25／25）的原发髓母细胞瘤、6／9的原发SPNET及全部PNET细胞系中均检测到RASSFIA基因的甲基化。相反,该基因甲基化在全部正常组织（包括2例小脑,5例大脑）中均未检测到。并且,RASSF1A在SPNET中的甲基化率明显低于髓母细胞瘤（Fisher精确检验,P=0．014）。在经去甲基化试剂处理的PNET细胞中,该基因表达得以恢复,证明甲基化与该基因沉默相关。结论RASSF1A甲基化是肿瘤特异性的,RASSF1A甲基化与PNET的发生有一定关联,不同亚型的PNET之间RASSF1A基因的不同甲基化状态提示髓母细胞瘤和SPNET是表遗传学上存在差异的两类肿瘤。     

Isochromosome 17q demonstrated by interphase fluorescence in situ hybridization in primitive neuroectodermal tumors of the central nervous system

We previously reported an i(17q) as a non-random finding in childhood primitive neuroectodermal tumors (PNETs) of the central nervous system. In the present study, we describe a two-color interphase fluorescence in situ hybridization (FISH) assay for detection of chromosome 17 abnormalities in tumors. Thirty-four PNETs were analyzed by FISH with a series of chromosome 17-specific probes which map to 17p13.3-17q25. The results from the FISH assay were then compared to the karyotypes prepared from the tumors. Ten of the 34 cases demonstrated an i(17q) by FISH and standard cytogenetics. Two PNETs were shown to have an i(17q) by FISH alone, and three additional tumors had deletions of 17p. Thus, a total of 15 of 34 (44%) of the PNETs in this series had a deletion of 17p. This study confirms and extends our previous reports that an i(17q) is the most common cytogenetic abnormality in PNETs. The interphase FISH assay which we employed will have clinical utility for diagnosis of children with malignant brain tumors, and it may be used for identification of tumors with 17p deletions for molecular studies aimed at identifying disease genes.     

Molecular cytogenetic analysis of breast cancer: a combined multicolor fluorescence in situ hybridization and G-banding study of uncultured tumor cells

In six patients with breast cancer, uncultured tumor cells were investigated with G-banding and multicolor fluorescence in situ hybridization (M-FISH). A large number of numerical and structural aberrations could be analyzed. Among other structural abnormalities, reciprocal, hidden and complex translocations were found. Recurrent t(1;10) and t(6;16), not previously described, were identified, as well as t(15;22). The latter was also found in additional cases among our unpublished breast carcinomas. The significance of t(15;22) for breast cancer is discussed, taking into account also data drawn from the literature. Reciprocal translocations were a prominent feature in a pseudodiploid lobular carcinoma. Hidden translocations on 6p22-p24 were detected with M-FISH. Involvement of 6p22-p24 was observed in five cases. The analysis of various other translocations and different structural abnormalities revealed the following common breakpoints (according to frequency of involvement): 1p34-p36, 3p12-p13, 4p13-->q11, 14p11-->q11, 1q42, 8p11, 8q24, 10q22, 11q13, 11q23-q24, 13q13, and 18p10-p11. Loss of 3p and 1p34-p36-->pter and complete or partial loss of 13q and chromosome 17 were also found. With the combination of G-banding and M-FISH techniques, chromosome misclassification is avoided and the characterization of complex tumor karyotypes is more effective.     

Comprehensive cytogenetic analysis including multicolor spectral karyotyping and interphase fluorescence in situ hybridization in lymphoma diagnosis. a summary of 154 cases

Cytogenetic analysis including multicolor spectral karyotyping (SKY) and interphase fluorescence in situ hybridization (FISH) was performed on 154 consecutive cases with suspected lymphoma. The cytogenetic results were reviewed in correlation with the final pathologic diagnosis. A diagnosis of lymphoma was established in 94 cases, with 16 Hodgkin lymphomas and 78 non-Hodgkin lymphomas (NHL). Cytogenetic results were obtained in 63 NHLs (81%); 61 of those showed abnormal karyotypes (97%). The t(14;18) or IGH-BCL2 fusion was detected in 83% (20/24) of follicular lymphomas and in 57% (12/21) of diffuse large B-cell lymphomas (DLBCL). The application of interphase FISH and SKY has contributed to a high detection rate of t(14;18) in DLBCLs. This study showed that genes at 1q25, 3p21, 3q21, 5q31, 6p23, 7q22, 8q11 approximately q12, 9q34, 11q23, 12q13, and 19q13.1 may have been involved as the less common changes in follicular lymphoma and DLBCL. Comparison of the recurrent secondary aberrations in the groups of follicular lymphoma and DLBCL revealed a pattern of clonal evolution from the changes rea(1)(p36), del(6q), +7, +12 or dup or trp(12)(q13q22), +der(18)t(14;18), and +21 in follicular lymphoma to the changes rea(1)(p36), del(6q), +6, +7, +9, rea(11)(q23), +12, -13 or del(13(q12q14), +18, +21, and +X in DLBCL. The clonal evolution of the secondary aberrations is thought to contribute to the progression of the disease. About 90% (16/18) of other types of NHL had abnormal karyotypes showing specific translocations or gene rearrangements consistent with the pathologic diagnosis. A comprehensive cytogenetics approach including SKY and interphase FISH using probes for specific genes, such as IGH, BCL2, CCND1, and ALK, is a very useful ancillary diagnostic tool for lymphomas. The combined approach also led to the identification of t(2;19)(p23;q13.1) as a new variant of t(2;5)(p23;q35) in a case of Ki-1-positive anaplastic large cell lymphoma with a null cell phenotype.     

荧光原位杂交与逆转录聚合酶链反应在尤文肉瘤/原始神经外胚层肿瘤分子诊断中的应用

目的探讨运用荧光原位杂交（FISH）与逆转录聚合酶链反应（RT-PCR）检测尤文肉瘤/原始神经外胚层肿瘤（ES/PNET）石蜡包埋组织特异性染色体易位的临床应用价值.方法收集ES/PNET 10例,分离石蜡包埋组织中的肿瘤细胞,利用Vysis公司的EWSR1双色易位分离探针,间期核FISH检测EWS基因的易位.运用RT-PCR检测t（11;22）（q24;q12）和t（21;22）（q22;q12）形成的融合转录子EWS-FLI1和EWS-ERG.结果10例ES/PNET中FISH观察9例有EWS基因的易位.RT-PCR 8例检测出EWS-FLI1的表达,没有检测出EWS-ERG的表达.结论利用FISH与RT-PCR检测ES/PNET石蜡包埋组织特异性染色体易位可作为可靠的分子诊断指标;两者相比,FISH敏感性和稳定性要优于RT-PCR.     

Oncogene amplification in medulloblastoma: analysis of a case by comparative genomic hybridization and fluorescence in situ hybridization

We describe amplification of the MYCC oncogene in a medulloblastoma with aggressive clinical behavior. The patient was a six year old boy who underwent gross total surgical excision of a cerebellar tumor. Despite chemotherapy and total neuraxis radiation, the clinical course was one of relentless progression, with extensive subarachnoid spread and death within eight months of presentation. The pathological features were consistent with the recently described, "large cell variant" of medulloblastoma. Tumor cells exhibited large vesicular nuclei, prominent nucleoli and strong immunoreactivity for synaptophysin. Polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) assay revealed no evidence of MYCN amplification or 1p deletion in the tumor. FISH analysis revealed evidence of MYCC amplification in the 20- to 30-fold range. Comparative genomic hybridization (CGH) revealed regions of gains and amplification in three locations, with gains of chromosome 7, amplification of 8q24 (corresponding to the MYCC locus) and gains of the long arm of chromosome 17 (suggestive of isochromosome 17q). While conventional karyotypic analysis was not successful in the present case, CGH provided invaluable information about gene amplification and losses/gains of chromosomes and chromosomal regions. Thus, CGH is a powerful technique applicable to frozen or paraffin-embedded material which helps to ascertain the presence of gene amplification even without prior knowledge of the gene to be tested.     

Cytogenetic,spectral karyotyping,fluorescence in situ hybridization,and comparative genomic hybridization characterization of two new secondary leukemia cell lines with 5q deletions,and MYC and MLL amplification

Cytogenetic studies of patients with therapy-induced acute myeloid leukemia (t-AML) have demonstrated whole chromosome loss or q-arm deletion of chromosomes 5 and/or 7 in a majority of cases. We have established two cell lines, SAML-1 and SAML-2, from two patients who developed t-AML after radiation and chemotherapy for Hodgkin disease. In both cases, the leukemia cells contained 5q deletions. SAML-1 has 58 chromosomes and numerous abnormalities, including der(1)(1qter-->1p22::5q31-->5qter), der(5)(5pter-->5q22::1p22-->1pter), +8, der(13)i(13)(q10)del(13)(q11q14.1), and t(10;11). Fluorescence in situ hybridization (FISH) with unique sequence probes for the 5q31 region showed loss of IL4, IL5, IRF1, and IL3, and translocation of IL9, DS5S89, EGR1, and CSFIR to 1p. SAML-2 has 45 chromosomes, del(5)(q11.2q31) with a t(12;13)ins(12;5), leading to the proximity of IRF1 and RB1, and complex translocations of chromosomes 8 and 11, resulting in amplification of MYC and MLL. Comparative genomic hybridization and spectral karyotyping were consistent with the G-banding karyotype and FISH analyses. Because a potential tumor suppressor(s) in the 5q31 region has yet to be identified, these cell lines should prove useful in the study of the mechanisms leading to the development of t-AML.