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.     

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.     

Application of bacterial artificial chromosome array-based comparative genomic hybridization and spectral karyotyping to the analysis of glioblastoma multiforme

Identification of genetic losses and gains is valuable in analysis of brain tumors. Locus-by-locus analyses have revealed correlations between prognosis and response to chemotherapy and loss or gain of specific genes and loci. These approaches are labor intensive and do not provide a global view of the genetic changes within the tumor cells. Bacterial artificial chromosome (BAC) arrays, which cover the genome with an average resolution of less than 1 MbP, allow defining the sum total of these genetic changes in a single comparative genomic hybridization (CGH) experiment. These changes are directly overlaid on the human genome sequence, thus providing the extent of the amplification or deletion, reflected by a megabase position, and gene content of the abnormal region. Although this array-based CGH approach (CGHa) seems to detect the extent of the genetic changes in tumors reliably, it has not been robustly tested. We compared genetic changes in four newly derived, early-passage glioma cell lines, using spectral karyotyping (SKY) and CGHa. Chromosome changes seen in cell lines under SKY analysis were also detected with CGHa. In addition, CGHa detected cryptic genetic gains and losses and resolved the nature of subtle marker chromosomes that could not be resolved with SKY, thus providing distinct advantages over previous technologies. There was remarkable general concordance between the CGHa results comparing the cell lines to the original tumor, except that the magnitude of the changes seen in the tumor sample was generally suppressed compared with the cell lines, a consequence of normal cells contaminating the tumor sample. CGHa revealed changes in cell lines that were not present in the original tumors and vice versa, even when analyzed at the earliest passage possible, which highlights the adaptation of the cells to in vitro culture. CGHa proved to be highly accurate and efficient for identifying genetic changes in tumor cells. This approach can accurately identify subtle, novel genetic abnormalities in tumors directly linked to the human genome sequence. CGHa far surpasses the resolution and information provided by conventional metaphase CGH, without relying on in vitro culture of tumors for metaphase spreads.     

Molecular cytogenetic analysis of a nontumorigenic human breast epithelial cell line that eventually turns tumorigenic: Validation of an analytical approach combining karyotyping,comparative genomic hybridization,chromosome painting,and single-locus fluorescence in situ hybridization

The immortalized, nontumorigenic human breast epithelial cell line HMT-3522 has been used as a model for premalignant and, eventually, malignant development. During cultivation, the karyotype evolution was followed. At an early stage, a very long constant phase showed a near-diploid karyotype, with only five marker chromosomes. DNA from this phase was used for comparative genomic hybridization (CGH) analysis, confirming a previously known MYC amplification, and the integration sites were subsequently determined by single-locus fluorescence in situ hybridization (FISH). Furthermore, gains of 5q22-qter and 20q11-qter and deletion of most of chromosome 6 (6p23-qter) were detected by CGH. Because of uncertainty about some of the indicated changes, including a deletion of 1p35-pter, the CGH findings were investigated more closely by chromosome painting, leading to a revision of the karyotype: 45,XX,del(1)(p35),−6,dup(8)(pter→qter::qter→q24),der(12) t(6;12)(p23;p13),der(14)t(5;14)(q22;q32.3),der(17)t(8;17;20)(17pter→17q25::8qter→8q23::8q24→8qter::8q24→8qter::8q23→8q24.1::20q11→20qter). Some karyotypic changes were confirmed by CGH; others had to be revised; and, in the 1p35 region, classical cytogenetics seems superior to CGH. However, CGH revealed a karyotypically unsuspected dup(20q) that might be of special relevance to breast tumor initiation or progression. Our study confirms that CGH is supplementary to current technologies, e.g., karyotyping and Southern analysis, but cannot replace them. In addition, our cell line turned out to be an excellent model for comparison among the different methods. The results imply that future cytogenetic analyses of complex karyotypes should be based on a combination of karyotyping, CGH, and FISH. Genes Chromosom. Cancer 20:30–37, 1997. © 1997 Wiley-Liss, Inc.     

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.     

A comprehensive karyotypic analysis on a newly developed hepatocellular carcinoma cell line, HKCI-1, by spectral karyotyping and comparative genomic hybridization

A continuously growing human hepatocellular carcinoma (HCC) cell line was established from a Chinese male, carrier of the hepatitis B virus (HBV). This cell line, designated HKCI-1, grows as an adhering monolayer of polygonal epithelial cells that embody one or more nuclei. HKCI-1 secretes alpha-fetoprotein but shows no evidence of HBV carriage. Conventional banding analysis of the short-term cultured primary tumor and the propagated HKCI-1 revealed a chromosome modal number of near-triploidy. It was, however, impossible to derive their complete karyotype due to the complex nature of chromosomal rearrangements and many marker chromosomes of uncertain origin. Spectral karyotyping (SKY) is a newly developed molecular cytogenetic technique that allows the unprecedented discernment of chromosomal abnormalities. Spectral karyotyping analysis on HKCI-1 and the primary tumor elucidated all aberrant chromosomes and revealed complex karyograms. Recurring aberrations detected in both primary tumor and HKCI-1 included der(X)t(X;11)(q10;p10), der(1)t(1;10)(q10;?pq), der(4)t(4;16)(p10;q10), i(5p), del(5)(q13), der(7)t(7;21)(q32q10::q10), der(8)t(8;17)(q10;p10), and der(9)t(9;22)(q34;?pq). Comparative genomic hybridization (CGH) was employed to monitor the culture evolution in vitro. Genomic imbalances in HKCI-1 involved chromosomal losses on 4q, 5q13-qter, 8p, 9pter-q33, 10q, 11q, 13q, 16q, 17q12-qter, and 22, and low-level gains on 6pter-q22, 7p, 8q, 9q34, 10p, 11p, 12, 17pter-q11.2, 18, 19, 20, 21, and Y. High-level amplifications were also detected on 5pter-q12, 7q11.2-qter, and Xq. The corresponding CGH finding on the primary tumor indicated similar imbalances. TP53 mutational analysis showed that both HKCI-1 and the primary tumor had the aflatoxin-associated mutation in codon 249 and an additional TP53 polymorphism in codon 72. Our present study demonstrates the value of combined SKY and CGH study in defining complex rearrangements and identifying cryptic translocations, and provides a comprehensive analysis on the chromosomal abnormalities in HKCI-1.     

The use of multicolor fluorescence technologies in the characterization of prostate carcinoma cell lines: a comparison of multiplex fluorescence in situ hybridization and spectral karyotyping data

Recent studies have identified several chromosome regions that are altered in primary prostate cancer and prostatic carcinoma cell lines. These targeted regions may harbor genes involved in tumor suppression. We used multiplex fluorescence in situ hybridization (M-FISH) to screen for genetic rearrangements in four prostate cancer cell lines, LNCaP, LNCaP.FCG, DU145, and PC3, and compared our results with those recently obtained using spectral karyotyping (SKY). A number of differences was noted between abnormalities characterized by SKY and M-FISH, suggesting variation in karyotype evolution and characterization by these two methodologies. M-FISH analysis showed that hormone-resistant cell lines (DU145 and PC3) contained many genetic alterations (> or =15 per cell), suggesting high levels of genetic instability in hormone-refractory prostate cancer. Most chromosome regions previously implicated in prostate cancer were altered in one or more of these cell lines. Several specific chromosome aberrations were also detected, including a del(4)(p14) and a del(6)(q21) in the hormone-insensitive cell lines, a t(1;15)(p?;q?) in LNCaP, LNCaP, and PC3, and a i(5p) in LNCaP.FCG, DU145, and PC3. These clonal chromosome abnormalities may pinpoint gene loci associated with prostate tumourigenesis, cancer progression, and hormone sensitivity.     

Re-analysis of the cell line NALM-1 karyotype by GTG-banding, spectral karyotyping, and whole chromosome painting

Chronic myelogenous leukemia (CML) is a clonal bone marrow disease with progression from a chronic phase to an aggressive blast crisis. The cell line NALM-1 was originally established by Minowada and coworkers from the peripheral blood of a patient in CML blastic crisis. A karyotype analysis of the NALM-1 cell line was performed in the 1970s. To the best of our knowledge, this karyotype was not re-analyzed by molecular cytogenetic techniques, although this cell line is the source of many molecular investigations including expression studies. To establish this cell line as a CML control in our own laboratory, NALM-1 was analyzed by GTG banding, fluorescence in situ hybridization, and spectral karyotyping. Our results differ from the original publication of Sonta and coworkers. We describe for the first time the karyotype of the NALM-1 cell line: 44,X,-X,der(7)t(7;9;15)(q10;?;q15),der(9)t(9;9)(p24;q33 approximately q34)t(9;22)(q34;q11),der(15)t(7;9;15) (?;?;q15),der(22)t(9;22)(q34;q11).     

Establishment of a new human malignant fibrous histiocytoma cell line,FU-MFH-1: cytogenetic characterization by comparative genomic hybridization and fluorescence in situ hybridization

Although a number of malignant fibrous histiocytoma (MFH) cell lines have been reported, their characterization at a molecular cytogenetic level has not been fully established. In this study, we established a new human cell line, designated as FU-MFH-1, from a storiform-pleomorphic MFH arising in the retroperitoneum of a 61-year-old woman, and applied comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) with chromosome painting probes for the characterization of chromosome alterations. FU-MFH-1 cells were spindle, round, or polygonal in shape with oval nuclei, and were maintained continuously in vitro for over 50 passages for more than 12 months. G-banding analysis was performed and FU-MFH-1 revealed a complex karyotype with an abnormal chromosome 19 containing a homogeneously staining region (hsr). CGH analysis showed a high-level amplification of 12q13-->q21. The high-level amplification detected by CGH was refined by FISH. These results showed that the hsr was composed of amplified DNA sequences from 12q. Our study emphasizes the usefulness of CGH as a powerful tool for chromosomal localization of amplified sequences. The FU-MFH-1 cell line should be useful for biologic and molecular pathogenetic investigations of human MFH.     

A classification efficiency test of spectral karyotyping and multiplex fluorescence in situ hybridization: identification of chromosome homologies between Homo sapiens and Hylobates leucogenys

Two digital fluorescence microscopy systems, spectral karyotyping (SKY) and multiplex fluorescence in situ hybridisation (M-FISH), are used with multicolour probe sets to assist in the detection of chromosome aberrations. We have compared the resolution of the two methods in their ability to identify karyotype rearrangements, which have occurred during the divergence of Homo sapiens and Hylobates leucogenys in evolution. A 24-color human paint kit distinguishes 74 conserved autosomal segments in H. leucogenys, some of which are difficult to resolve. We examined the extent to which the SKY and M-FISH techniques are able to detect the smallest of these bands. We have found this to be a rigorous test of multicolour chromosome classification systems. We conclude from our results that both systems are able invariably to classify the majority of conserved segments but differ in the efficiency of detection of small inserts.     

Establishment and molecular cytogenetic characterization of non-small cell lung cancer cell line KU-T1 by multicolor fluorescence in situ hybridization, comparative genomic hybridization, and chromosome microdissection

A human lung adenocarcinoma cell line, designated KU-T1, was established from a Japanese man in Kochi Medical School. Conventional banding and multicolor fluorescence in situ hybridization (M-FISH) analyses of KU-T1 cells revealed a hyperdiploid chromosomal constitution and complex karyotypes. Comparative genomic hybridization showed several chromosomal copy number changes, and five regions that were highly amplified. Two of the five highly amplified regions, 1q and 3q, were identified from distributions of DNA sequences on a metaphase cell by FISH using chromosome microdissection-generated probes hybridized to 1q32 approximately q34 and 3q26 approximately q28, respectively. The 3q probe depicted a homogeneously staining region (hsr) in a derivative chromosome 3 of KU-T1. An hsr probe was regenerated by chromosome microdissection and was hybridized back to KU-T1 and normal metaphases. This hybridization experiment confirmed the probe derived from an hsr and indicated original locations of DNA sequences of hsr on normal chromosome 3. Intense hybridized signals shown at three loci (3p12, 3q26.3, and 3q28) suggests that oncogenes may be involved in the hsr formation. The present study provides a comprehensive analysis of the chromosomal abnormalities, including hsr formation and related oncogenes, in the KU-T1 cell line.     

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.     

Karyotypic evolution pathways in medulloblastoma/primitive neuroectodermal tumor determined with a combination of spectral karyotyping, G-banding, and fluorescence in situ hybridization

Medulloblastomas (MBs) or primitive neuroectodermal tumors (PNETs) represent 15%-30% of pediatric brain tumors and are the most common brain tumors in children; they are rare in adults. Classification of these tumors is based on tissue morphology and is often controversial and problematic. Karyotypic analysis of these tumors using conventional cytogenetic methods is often a difficult process that may be hindered by a limited number of metaphase cells and poor chromosome morphology, often leading to only partial characterization of the chromosomal abnormalities. We investigated three primary human tumors and four cell lines (CHO-707, DAOY, D-341, and PFSK) utilizing a combination of conventional G-banding, spectral karyotyping (SKY), and fluorescence in situ hybridization (FISH) techniques. A high level of intratumoral heterogeneity was seen, with multiple numerical and structural chromosomal aberrations. The chromosomes most frequently involved in structural aberrations were chromosomes 1 (14 rearrangements), 7 (9 rearrangements), and 21 (9 rearrangements). The chromosomes most frequently involved in numerical aberrations were chromosomes 1, 12, and 13 (four cases) and chromosomes 14, 17, 19, 21, 22, and X (three cases). Numerous aberrant chromosomes were characterized only with the SKY analysis, and based on these findings multiple clones were identified, facilitating analysis of karyotypic evolution. The most frequent evolution mechanism was via polyploidization, followed by acquisition of additional numerical or structural aberrations (or both); however, the results showed that the karyotypic evolution process in these tumors is typically divergent and complex.     

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.     

单核苷酸多态性微阵列和荧光原位杂交技术在Pallister-Killian综合征的产前诊断中的应用CSCD

目的探讨Pallister-Killian综合征（Pallister-Killian syndrome,PKS）的临床特征及遗传学特点,分析Affymetrix CytoScan 750K SNP Array和荧光原位杂志（fluorescence in situ hybridization, FISH）技术在产前诊断中的应用价值。方法对1例36岁孕34＋2周高龄孕妇脐血标本进行G显带染色体核型分析,芯片技术鉴定异常片段来源,运用12pter/12qter探针FISH确认。结果胎儿脐血G显带染色体核型46,XY[77]/47,XY,＋mar[23],出生后新生儿外周血芯片分析结果为arr[hg19] 12p13.33p11.1（173 786～34 835 641）×4,显示12号染色体12p13.33p11.1区段存在34.6 Mb的2次重复;FISH检测显示39%细胞有12号染色体短臂四体。结论通过结合临床特征,综合应用染色体G显带核型分析、FISH技术和芯片技术能准确确认染色体异常片段来源,有效诊断PKS患者。     

X;1 translocation in a female Menkes patient: characterization by fluorescence in situ hybridization

Menkes disease is an X-linked recessive disorder of copper metabolism, characterized by progressive neurological degeneration, abnormal hair and connective tissue manifestations. We present a female Menkes patient, with classical Menkes features, carrying a de novo balanced translocation 46, X, t(X;1)(q13;q12). The breakpoint on the X chromosome was narrowed down to Xq13.3 within a 1 Mb YAC contig containing the Menkes gene, using fluorescence in situ hybridization. The translocated X chromosome was of paternal origin and non-randomly active leading to the expression of the disease. This was additional evidence for paternal origin of de novo chromosome rearrangements, including all the X; autosomal translocations examined so far.     

Delineation of a supernumerary marker chromosome utilizing a multimodal approach of G-banding, fluorescent in situ hybridization, confirmatory P1 artificial chromosome fluorescent in situ hybridization, and high-resolution comparative genomic hybridization

We describe the structure of a supernumerary marker in a child who presented with a right atretic ear and multiple congenital anomalies. Using G-banding, fluorescent in situ hybridization (FISH), P1 artificial chromosome FISH and high-resolution comparative genomic hybridization (CGH), the marker was demonstrated to be a derivative chromosome resulting from malsegregation of a paternal 8;22 translocation: 47,XY, +der(22)t(8;22)(q24.1; q11.2). This case is noteworthy because the marker, while sharing similarities to der(22) in the Cat Eye syndrome (CES), also contains chromosome 8q material. This partial 8q trisomy confounds the diagnosis of CES associated with pure trisomy or pure tetrasomy 22q. The paternal translocation is noted with prolonged infertility and oligospermia, which again highlights the utility and necessity of chromosome analysis in this setting.     

Synovial sarcoma of the larynx in a 79-year-old woman, confirmed by karyotyping and fluorescence in situ hybridization analysis

BACKGROUND: Synovial sarcoma is an uncommon tumor of the head and neck and is an exceedingly rare finding in the larynx. Generally, it afflicts younger patients and most often occurs in the hypopharynx. METHODS AND RESULTS: We report a case of a biphasic synovial sarcoma arising in the arytenoid fold of a 79-year-old woman. The diagnosis was confirmed by an extensive immunohistochemical panel, karyotyping, and fluorescence in situ hybridization (FISH). The patient was treated with total laryngectomy, and she did well postoperatively. CONCLUSION: To our knowledge, our patient is the oldest ever to be reported in the literature, concluding that although this entity is considered a disease of young patients, it still has to be included in the differential diagnosis in older patients. Second, our report is only the third to describe t(X;18) (p11.2;q11.2) by karyotyping and the first ever by using FISH analysis, thus adding more cytogenetic data to the literature in this rare location and confirming the use of FISH in making the accurate diagnosis.