Detection of an i(17q) chromosome by fluorescent in situ hybridization with a chromosome 17 alpha satellite DNA probe.

An isochromosome for the long arm of chromosome 17,i(17q), is frequently found as an additional chromosome aberration to the Ph with advanced disease in the chronic myelocytic leukemia (CML). We studied an i(17q) in blood samples from two patients with CML in blast crisis with a biotinylated chromosome 17 specific alpha satellite deoxyribonucleic acid probe. G-banded karyotypes of these patients showed a dicentric i(17q), dic(17)(p11.2). Fluorescence in situ hybridization (FISH) delineated one normal chromosome 17 and one i(17q) among metaphase chromosomes; the latter showed a dicentric pattern. In most interphase nuclei of both patients, two fluorescence spots were observed. In some interphase nuclei, including mature neutrophils, the dicentric chromosome was discernible by its size and shape of the fluorescent spots. Three fluorescent spots were observed in a small proportion of interphase cells, and existence of a subclone with two normal chromosome 17 and an i(17q) was confirmed by examining a large number of metaphase plates. The results of FISH provided us with information of numerical and structural aberrations of chromosome 17 in interphase cells.     

Detection of the t(2;13)(q35;q14) and PAX3-FKHR fusion in alveolar rhabdomyosarcoma by fluorescence in situ hybridization

Cytogenetic studies of the pediatric solid tumor alveolar rhabdomyosarcoma have demonstrated the presence of a consistent chromosomal translocation, t(2;13)(q35;q14). We recently identified PAX3 and FKHR as the genes on chromosomes 2 and 13, respectively, that are juxtaposed by this translocation. As one means of detecting the t(2;13) translocation in clinical specimens, we have developed a fluorescence in situ hybridization (FISH) assay that may be used for both interphase and metaphase cells. Translocation of the 5′ region of the FKHR gene to the derivative chromosome 2, and retention of the 3′ region of FKHR on the derivative chromosome 13 [(der(13)], were demonstrated in metaphase cells from a rhabdomyosarcoma cell line with a previously identified t(2;13) translocation. A 5′ PAX3 cosmid probe was shown to localize to 2q35 in normal cells, and to translocate to the der(13) in the rhabdomyosarcoma cell line. In order to detect the der(13) in interphase nuclei, we labeled the 3′FKHR and the 5′PAX3 cosmid probes with digoxigenin and biotin, respectively, and used these in a two-color FISH assay. The presence of the der(13) was visualized as juxtaposed or overlapping red and green signals in metaphase and interphase tumor cells. The PAX3-FKHR FISH assay was then applied to a series of cytogenetically characterized pediatric sarcoma cell lines. The presence of the der(13) was demonstrated by FISH in all cases containing a cytogenetically detectable t(2;13). The FISH assay was then applied to a series of 20 embryonal and alveolar rhabdomyosarcoma samples. All 10 of the alveolar rhabdomyosarcoma specimens demonstrated a der(13) with the FISH assay. We did not detect a PAX3-FKHR fusion in 10 embryonal rhabdomyosarcoma cases. Thus, the two-color FISH assay is a sensitive and rapid means of identifying the t(2;13) in rhabdomyosarcoma specimens, and it will be a useful adjunct for the diagnosis of pediatric small round cell tumors. The cosmid probes for the 5′ and 3′ regions of FKHR, as well as the probe for PAX3, will be useful for molecular cytogenetic studies of variant translocations in rhabdomyosarcoma, such as the t(1;13)(p36;q14).     

A der(14)t(1;14)(q12;p11) in chronic myelomonocytic leukemia

Duplication of the long arm of chromosome 1 (1q) is widely reported in human neoplasia, including the myelodysplastic syndromes (MDS). So far, it has not been described as a single aberration in the chronic myelomonocytic leukemia (CMML), a subtype of MDS. Rather, trisomy 1q was always a part of complex chromosome changes affecting the subtypes of MDS other than CMML. We report on a patient with CMML with an unbalanced translocation of the entire 1q onto the short arm of chromosome 14 as a sole cytogenetic abnormality. Fluorescence in situ hybridization (FISH) analysis with an alpha-satellite probe for the paracentric region of the long arm of chromosome 1 confirmed the presence of trisomy 1q in a derivative chromosome, der(14)t(1;14)(q12;p11). The discrepant results between the metaphase cytogenetics (100% abnormal) and interphase cytogenetic (71% nuclei with 3 signals) suggest that trisomy 1q, even in the absence of additional cytogenetic changes, has a sufficient leukemogenic potential to confer a proliferative advantage on hematopoietic cells committed to monocyte stemline both in vitro and in vivo. The literature data on partial and complete trisomy 1q in CMML is reviewed.     

Two-color FISH characterization of i(1q) and der(1;16) in human breast cancer cells

Two-color fluorescent in situ hybridizations using probes for alphoid (α) and classical satellite (CS) DNAs from chromosomes 1 and 16 were performed to characterize i(1q), der(1;16), and complex rearrangements observed in breast cancer cells from fresh tumors and established cell lines. Six of seven i(1q) occurred after breakage in the α1 containing region and one of seven was dicentric, with breakage in 1p11.2. The five der(1;16)(q10;p10) studied appeared to result from a variety of breakpoints involving α1, α16, CS1, and CS16 DNAs. All had conserved α16 DNA, suggesting a segregation of the der(1;16) leading to a loss of 16q and a gain of 1q in most cases. One complex rearrangement of chromosome 1 also appeared to involve chromosome 16, suggesting that a der(1;16) occurred first, followed by another rearrangement. Both the apparent preferential involvement of constitutive heterochromatin harboring α and CS DNAs and the variety of breakpoints spanning along heterochromatin suggest that the important consequence of the rearrangement is not the breakage per se but the resulting imbalance. © 1993 Wiley-Liss, Inc.     

An assessment of chromosomal alterations detected by fluorescence in situ hybridization and p16 expression in sporadic and primary sclerosing cholangitis-associated cholangiocarcinomas

The objective of this study was to assess and compare the chromosome abnormalities present in sporadic and primary sclerosing cholangitis (PSC)-associated cholangiocarcinomas (CCAs) and biliary dysplasias. Histologic sections from 22 patients with CCA (16 sporadic and 6 PSC associated), 5 of whom had associated dysplasia, and 2 PSC patients with biliary dysplasia alone were assessed for chromosomal alterations with fluorescence in situ hybridization (FISH). FISH involved the use of a multiprobe set consisting of centromere-specific probes for chromosomes 3, 7, and 17 and a locus-specific probe for 9p21. The number of signals for each of these probes was enumerated in 50 nonoverlapping interphase nuclei, and the percentage of nuclei containing 0, 1, 2, and 3 or more signals was recorded for each probe. p16 expression was assessed using immunohistochemistry. Gain of at least 1 chromosome was identified in 19 of 22 (86%) invasive tumors and in 4 of 7 (57%) biliary dysplasias. Gain of 2 or more chromosomes (polysomy) was observed in 17 of 22 (77%) invasive tumors and in 3 of 7 (43%) biliary dysplasias. Homozygous loss of 9p21 was identified in 11 of 22 (50%) invasive tumors and in 3 of 7 (43%) biliary dysplasias. The patterns of chromosomal abnormalities detected by FISH in PSC-associated and sporadic CCAs were similar. Nine of 13 (69%) invasive tumors and 2 of 5 (40%) biliary dysplasias with complete loss of p16 expression by immunohistochemistry showed allelic loss of 9p21 by FISH. Polysomy and homozygous 9p21 deletion are common in both sporadic and PSC-associated CCAs and are frequently detectable in PSC-associated biliary dysplasia.     

应用荧光原位杂交产前诊断未培养羊水细胞非整倍体

目的探讨荧光原位杂交(fluorescenceinsituhybridization,FISH)诊断未培养羊水细胞非整倍体的临床应用价值。方法对55例孕16～32周未培养羊水细胞进行FISH快速产前诊断,应用多色FISH对另4条染色体(X、Y、13号和18号)进行检测。以经母腹穿刺取胎血常规核型分析作为FISH检测结果对照。结果被检55例羊水未培养细胞均获得诊断结果,发现两例异常胎儿。1例为标准型21三体;另1例为21三体嵌合体。FISH检测与常规核型分析结果一致。结论FISH检测未培养羊水细胞非整倍体具有快速、简便、所用样本量少的优势,结果准确可靠,可达到产前诊断要求,有较大临床应用价值。     

CCND1 and FGFR1 coamplification results in the colocalization of 11q13 and 8p12 sequences in breast tumor nuclei

The CCND1 gene, localized to chromosome band 11q13, is amplified in approximately 15% of human primary breast tumors. From 30 to 40% of the tumors presenting this amplification show concomitant amplification at the FGFR1 locus in 8p12. Similarly, MDA-MB-134 breast cancer cells bear CCND1 and FGFR1 coamplified, resulting in the formation of a hybrid intrachromosomal amplification assembling 11q13 and 8p12 sequences. To learn whether similar amplified structures arise in breast tumors, we used a two-color FISH approach on interphase nuclei. A cohort of 225 breast tumors was analyzed by Southern blotting and a subset of 12 tumors presenting the 11q13–8p12 coamplification was selected for further study by interphase FISH. In 6/12 tumors the FISH signals for 11q13 and 8p12 probes formed colocalizing clusters of green and red spots in the nuclei. The FISH patterns were identical to those observed on MDA-MB-134 interphase nuclei hybridized with 11q13 and 8p12. These data, suggesting the formation in these tumors of a hybrid amplification domain in which 11q13 and 8p12 sequences are joined, were reinforced by dual-color FISH on extended chromatin showing that the said were sequentially aligned in these tumors. Furthermore, 3/6 nuclei with colocalized 11q13 and 8p12 amplifications showed fusion of centromeric sequences from chromosomes 8 and 11. Our data strongly suggest the occurrence, in approximately 3% of primary breast tumors, of a recurrent rearrangement involving the proximal portions of 8p and 11q and resulting in the formation of a hybrid amplified structure composed of 11q13 and 8p12 sequences. Genes Chromosomes Cancer 22:268–277, 1998. © 1998 Wiley-Liss, Inc.     

Cryptic subtelomeric translocation t(2;16)(q37;q24) segregating in a family with unexplained stillbirths and a dysmorphic, slightly retarded child

We here describe a submicroscopic translocation affecting the subtelomeric regions of chromosomes 2q and 16q, and segregating in a family with stillbirths, early pregnancy losses, and two dysmorphic and slightly retarded babies. FISH analysis showed a 46,XY,der(2)t(2;16)(q37.3;q24.3) in the propositus, and a balanced t(2;16) in his mother, her conceptus and maternal grandfather. FISH with YACs and BACs made it possible to map the 2q37 breakpoint precisely between the regions covered by y952E1 and y746H1, and the 16q breakpoint between the regions encompassed by bA 309g16 and bA 533d19. The contribution of 2q37.3 monosomy and 16q24.3 trisomy to the proband's phenotype is compared with that in reported patients with similar imbalances of either chromosome.     

A child with ALL and ETV6/AML1 fusion on a chromosome 12 due to an insertion of AML1 and loss of ETV6 from the homolog involved in a t(12;15)(p13;q15)

A 4-year-old boy was found to have acute lymphoblastic leukemia characterized by a t(12;15)(p13;q15). FISH investigation using a TEL(ETV6)/AML1 probe detected a fusion signal in 98% of the interphase cells. Sequential FISH on a G-banded slide showed a fusion signal on an apparently normal chromosome 12 and AML1 signals on chromosomes 21. The ETV6 was deleted from the chromosome 12 involved in the t(12;15). These results are best explained as an insertion of AML1 into TEL on one chromosome arm 12p and loss of ETV6 from the chromosome 12 involved in the t(12;15).     

Highly recurrent der(1;16)(q10;p10) and other 16q arm alterations in lobular breast cancer

Cytogenetic data on infiltrating lobular carcinomas (ILCs) of the breast are described. In addition to 9 tumors, including 2 bilateral ones, with apparently normal chromosomes, recurrent chromosome alterations were found among 18 tumors. A der(1;16)(q10;p10), resulting in 1q gain and 16q loss, was observed in 11 tumors. Chromosome arm 16q was lost by other rearrangements in 3 other tumors. Thus, the deletion of 16q appears to be highly recurrent in ILCs. Compared to infiltrating ductal carcinomas (IDCs), ILCs have fairly simple karyotypes that remain pseudo- or near-diploid in most cases. This finding is confirmed by DNA ploidy studied by flow cytometry, which shows that about half of the tumors are diploid. This makes the der(1;16)(q10;p10) and other alterations of the 16q arm an early alteration of tumor progression, possibly related to the loss of expression of E-cadherin, whose gene is mapped on the 16q arm. Genes Chromosomes Cancer 23:300–306, 1998. © 1998 Wiley-Liss, Inc.     

Rapid differential diagnosis of myxoid liposarcoma by fluorescence in situ hybridisation on cytological preparations

In two cases of suspected myxoid liposarcoma, where chromosomal metaphase preparations were not available, fluorescence in situ hybridisation was performed on interphase nuclei of cytological preparations for the detection of the specific translocation, t(12;16), characteristic of this tumour and of trisomy 8, which is the most frequent secondary chromosome aberration. Probes directed against chromosomes 12 and 16 and against the centromeres of chromosomes 12 and 8 were hybridised on cell brushings and cytocentrifuge preparations. The finding of three painting domains of both chromosomes 12 and 16 and of only two signals with the centromeric probe directed against chromosome 12, suggested the presence of t(12;16) in both cases. In one case trisomy 8 was inferred from the occurrence of three centromere 8 signals. This approach can be used to detect specific chromosomal abnormalities when an urgent differential diagnosis is requested or when chromosome preparations are not available, or both.     

Whole-arm t(1;16) and i(1q) as sole anomalies identify gain of 1q as a primary chromosomal abnormality in breast cancer.

Cytogenetic analysis of four ductal breast carcinomas revealed net gain of 1q in all tumors. In the first tumor, the only change was that one chromosome 16 was replaced by a derivative chromosome consisting of 16p and 1q. The same unbalanced whole-arm translocation was also found in the second tumor, as the only aberration in one of four abnormal clones. In the last two cases, which also were characterized by cytogenetically unrelated clones, an extra i(1q) was present in one clone in both tumors as the sole aberration. Our findings suggest that gain of 1q is a primary chromosomal abnormality in breast carcinomas, in the sense that it is an early event that precedes the acquisition of more complex changes.     

Molecular and clinical description of a girl with a 46,X,t(Y;4)(q11.2;p16)/45,X,der(4)t(Y;4)(q11.2;p16) karyotype and a small cryptic 4p subtelomeric deletion

We report on a 13-year-old female with short stature, minimal axillary and pubic hair, no breast development, absence of uterus and ovaries, with the following karyotype on lymphocyte cultures: 46,X,t(Y;4)(q11.2;p16)[40]/45,X,der(4)t(Y;4)(q11.2;p16)[10]. Loss of the small derivative Y chromosome in 20% of the cells was also confirmed in skin fibroblast cultures. FISH analyses using Y centromere, SRY, subtelomere XpYp/XqYq, Y and 4 painting probes, confirmed the cytogenetic findings. High-resolution STS analyses using 40 markers covering the Y chromosome did not identify any deletion on the Y. However, de novo absence of the 4p subtelomeric region was noted by FISH, although this deletion was not revealed by Array-CGH at 1 Mb resolution, the last array clone being 0.35 or 1 Mb distal to the 4p FISH probe. The female phenotype of this patient must be due to the loss of the derivative Y chromosomes in some of her cells, especially the gonads, while the 4p subtelomeric deletion does not seem to contribute to her phenotype.     

A new genomic duplication syndrome complementary to the velocardiofacial (22q11 deletion) syndrome

Fluorescence in situ hybridization (FISH) analysis can reveal undetected chromosomal rearrangements. We report a patient with cleft palate, hydronephrosis, and minor dysmorphic features, including low-set posteriorly rotated ears, down-slanting palpebral fissures, mandibular micrognathia, and brachymesophalangia. Routine chromosome analysis identified no abnormality of chromosome 22; FISH analysis with the TUPLE1 probe disclosed an interstitial duplication of 22q11.2. FISH analysis did not reveal the duplication on the initial testing of metaphase chromosomes, although, on review, the area was brighter on one chromosome in each metaphase spread. FISH analysis of interphase cells showed three TUPLE1-probe sites with two chromosome-specific identification probes in each cell. Family history showed two older full siblings, a brother with behavior problems, oppositional defiant disorder, and learning problems and a sister with hydronephrosis and mild delays. The father and both siblings had similar facial features, and all three had the same interstitial duplication of the TUPLE1 probe. This family illustrates the novel complementary duplication syndrome of the velocardiofacial syndrome, which adds it to the expanding list of genomic deletion/duplication syndromes. The laboratory results further show the utility and need for careful analysis of interphase cells even in samples where good quality metaphases are available.     

Derivative (1;18)(q10;q10): a recurrent and novel unbalanced translocation involving 1q in myeloid disorders.

We report two cases of hematological malignancies, comprising a case of myelodysplastic syndrome (MDS) that rapidly evolved into acute myeloid leukemia, and a case of myeloproliferative disorder (MPD), in which der(1;18)(q10;q10) was found as the sole acquired karyotypic abnormality. This observation indicates that the unbalanced translocation is a recurrent aberration in myeloid disorders. To the best of our knowledge, centromeric fusion between long arms of chromosomes 1 and 18, leading to a normal chromosome 18 substituted with a der(1;18) chromosome, is novel and has not been described in cancer. Mechanistically, either trisomy 1q or monosomy 18p that results from the translocation may potentially contribute to leukemogenesis. Finally, chromosomes with large constitutive heterochromatin bands such as chromosome 1 may be at risk of centromeric instability and be predisposed to centromeric fusion with other chromosomes.     

Interphase cytogenetics of gastric carcinoma: Fluorescence in situ hybridization (FISH) applied to cells obtained from formalin-fixed paraffin-embedded tissues

The interphase cytogenetics in formalin-fixed and paraffin-embedded gastric cancer tissues were examined by fluorescence in sku hybridization (FISH) with α-satellite DNA probes. Two gastric carcinoma cell lines, TMK-1 and MKN-28, were first analyzed cytogenetically. Of 25 TMK-1 cell karyotypes, chromosome 7 showed trisomy and chromosome 17 showed disomy in 18 cells. Most MKN-28 cells showed disomy of both chromosomes 7 and 17. Suspensions of singly isolated TMK-1 and MKN-7 cells were obtained from the cultured cells, and from paraffinembedded tissue specimens fixed with formalin for 0, 1, 3 and 5 days obtained from xenotrans-planted tumors in nude mice. The numbers of chromosomes 7 and 17 analyzed with the karyotypic preparations coincided well with those determined by FISH, even in the paraffin-embedded specimens. The number of tumor cells showing no signals, however, increased in the specimens after 5 days formalin fixation. In 10 surgically removed gastric carcinomas, the predominant signal number for chromosomes 7 and 17 in the cells of paraffinembedded tissues was two (disomy), except in one papillary carcinoma, which was trisomic for chromosome 7. Large subpopulations (more than 20%) showing trisomy were found in four cases for chromosome 7 and in five cases for chromosome 17. A higher frequency of trisomy was found in well differentiated than in poorly differentiated carcinomas. These findings suggest that the FISH technique is a useful tool for detecting chromosomal aberrations in gastric adenocarcinoma cells, even in paraffinembedded specimens, as long as the tissues are fixed with formalin for an appropriate time.     

Malignant cell detection by fluorescence in situ hybridization (FISH) in effusions from patients with carcinoma

Cytological diagnosis of malignant cells in effusions is hampered by difficulties in the differentiation from reactive mesothelial cells. Because interphase cytogenetics by fluorescence in situ hybridization (FISH) might complement cytological evaluation, we determined the power of tumor cell detection using FISH and cytology in 201 effusions from patients with advanced cancer. Furthermore, 9 primary breast tumors were FISH-karyotyped, and chromosomal aberrations were compared with those of corresponding metastatic effusion cells. By using centromeric probes representing chromosomes 7, 8, 11, 12, 17, and 18, a rate of malignancy-associated aneusomy combined for the 6 chromosomes was detected in an overall of 44.8% of effusion specimens (range, 31.8% to 39.3% for the individual chromosome), comparable to cytology (43.3%). The combination of just 2 FISH probes (namely, representing chromosome pairs 8/11 and 8/17) was almost equally efficient in the identification of aneusomy. Approximately one fourth of the cytologically negative effusions were FISH positive and vice versa. From the initially FISH-negative effusions, 18.9% could be subsequently classified positive with dual-color FISH by visualization of intranuclear chromosomal complexity in rare aneuploid cells. Thus, "overall FISH analysis," including dual-color evaluation, identified tumor cells in significantly more effusions (55.2%, P = .001) than conventional cytology, implying greater sensitivity. Finally, our finding that numerical aberration patterns in primary breast tumors and corresponding metastatic effusions are comparable indicates that FISH examination of primary tumors will indicate the centromeric probe(s) best suited for an efficient search for metastasis in the individual case.