Detailed genome-wide screening for inter- and intrachromosomal abnormalities by sequential G-banding and RxFISH color banding of the same metaphase cells.

While the now-classic chromosome banding methods, such as G-banding, remain the techniques of choice for the initial screening for karyotypic abnormalities, sometimes chromosomal rearrangements involve segments too small or too similarly banded to be detected or described adequately by these techniques. The necessity to use a genome-wide, fluorescence in situ hybridization (FISH)-based screening technique as a complement to G-banding is especially obvious in cases where the information obtained by the latter analysis does not provide an adequate guide to the choice of probes for chromosome-specific FISH. Furthermore, the same metaphase cells should ideally be used for both G-banding and FISH analysis to overcome the scarcity of metaphases observed in many cases and to ensure the correct interpretation of chromosomal aberrations in cytogenetically unstable neoplasms with massive cell-to-cell karyotypic variability. We describe a protocol which enables cross-species color banding (RxFISH), a new FISH-based screening technique that simultaneously imparts specific color banding patterns on all chromosomes, of preparations that have been G-banded and mounted for up to several years, as well as a procedure allowing chromosome-specific painting of the same metaphase cells to resolve whatever doubts persist after the preceding G-banding and RxFISH analyses. This approach makes possible a detailed, genome-wide screening for inter- and intrachromosomal abnormalities including archival cases whose karyotypic rearrangements had been incompletely identified by G-banding.

Fusion of the RBP56 and CHN genes in extraskeletal myxoid chondrosarcomas with translocation t(9;17)(q22;q11).

Although most extraskeletal myxoid chondrosarcomas (EMC) are cytogenetically characterized by the translocation t(9;22)(q22;q12), another subset has recently been identified carrying a t(9;17)(q22;q11). Whereas the t(9;22) is known to result in fusion of the CHN (TEC) gene from 9q22 with the EWS gene from 22q12, creating a chimeric EWS/CHN, the genes involved in the t(9;17) of EMC are unknown. We examined two EMC with t(9;17)(q22;q11) and found that the CHN gene was recombined with the RBP56 gene from 17q11 to generate a chimeric RBP56/CHN. RBP56 has not previously been shown to be involved in tumorigenesis but it encodes a putative RNA-binding protein similar to the EWS and FUS (TLS) proteins known to play a pathogenetic role in several sarcomas. The presence of the RBP56/CHN chimeric gene in EMC with t(9;17)(q22;q11) shows that the N-terminal parts of EWS and RBP56 have similar oncogenic potential making them pathogenetically equivalent in oncoproteins arising from fusions with certain transcription factors.

Cross-species color banding characterization of chromosomal rearrangements in leukemias with incomplete G-band karyotypes.

Karyotype analysis has depended on chromosome banding techniques since their introduction in about 1970, and the information thus obtained is indispensable for the clinical management of patients with hematologic malignancies. Sometimes, however, chromosomal rearrangements involve segments too small, too similarly banded, or too complex to be described adequately or even to be detected by G-banding. Cross-species color banding is a new FISH-based screening technique that enables the generation of a specific color banding pattern for each human chromosome based on the genomic homologies between man and various species of apes. We report the first application of cross-species color banding (RxFISH) to characterize the chromosomal rearrangements of 10 leukemia samples the G-band karyotypes of which were incomplete. The combination of G-banding and RxFISH in every case yielded additional information beyond that obtained by either technique used alone, determining the identity of even the most complex, inter- as well as intrachromosomal, rearrangements. Genes Chromosomes Cancer 26:13-19, 1999.

Combined RxFISH/G-banding allows refined karyotyping of solid tumors.

Chromosome banding analysis of solid tumors often yields incomplete karyotypes because of the complex rearrangements encountered. The addition of fluorescence in situ hybridization (FISH) methods has helped improve the accuracy of solid tumor cytogenetics, but the absence of screening qualities from standard FISH approaches has proved a severe limitation. We describe the cytogenetic analysis of ten solid tumors using G-banding followed by cross-species color banding (RxFISH), a FISH-based screening technique giving a chromosome-specific banding pattern based on the genomic homologies between humans and gibbons. The addition of RxFISH analysis in all cases led to the identification of previously unidentified intra- as well as interchromosomal rearrangements, thus giving a much more certain and detailed karyotype. In two gastric stromal sarcomas, a tumor type for which no cytogenetic data were hitherto available, numerical chromosomal aberrations dominated, but one of the tumors also carried an unbalanced 7;17-translocation with the same breakpoint in chromosome 17 as that seen in endometrial stromal sarcomas.

Chromosome banding analysis of gynecomastias and breast carcinomas in men.

Male breast cancer is 100 times less frequent than its female counterpart and accounts for less than 1% of all cancers in men. Although men with breast cancer also often have gynecomastia, it is still unknown whether gynecomastia per se predisposes the male breast to malignant disease. We describe the cytogenetic analysis of three gynecomastias and four breast cancers in men. No chromosome abnormalities were detected in two cases of gynecomastia, with no other concomitant breast disease. The third gynecomastia sample, taken from a site where a breast carcinoma had previously been removed, had a t(2;11)(p24;p13) as the sole chromosome change; this is the first time that an abnormal karyotype has been described in gynecomastia. All four cancers had clonal chromosome abnormalities. Several cytogenetically unrelated clones were found in the breast tumor and in a metastasis from case 1. In the carcinoma of case 2, a single abnormal clone was found, characterized by loss of the Y chromosome, monosomy 17, and a deletion of the long arm of chromosome 18. In the carcinoma of case 3, a clone with loss of the Y chromosome as the sole change dominated, accompanied by the gain of an X chromosome in a subclone. In the lymph node metastasis examined from case 4, a single clone carrying trisomies for chromosomes 5 and 16 was detected. Our findings, especially when collated with data on the six karyotypically abnormal breast carcinomas in men described previously, indicate that gain of the X chromosome, gain of chromosome 5, loss of the Y chromosome, loss of chromosome 17, and del(18)(q21) are nonrandom abnormalities in male breast carcinomas.

Fluorescence in situ hybridization of old G-banded and mounted chromosome preparations.

An improved method for fluorescence in situ hybridization (FISH) investigation of old, previously G-banded, mounted chromosome preparations with chromosome specific painting probes and centromere-specific probes is described. Before hybridization, the slides are incubated in xylene until the coverslips detach spontaneously; any mechanical manipulation will jeopardize the results. The success of chromosome painting is improved by excluding the regular RNase treatment step prior to hybridization. Additional changes compared with standard FISH protocols are that the 2 x SSC step is omitted, that the amount of added probe is increased approximately 2.5 times, and that the amplification of signals is performed twice. The applicability of the method, which allows double painting with two differently labeled probes using two differently fluorescing colors, was tested on 11 cases involving different chromosome abnormalities and different types of material, including short-term cultures of epithelial and mesenchymal tumors, blood, leukemic bone marrow, and long-term cultures of a cell line derived from an epithelial tumor. Success was achieved even with chromosome preparations that were several years old.

Cytogenetic polyclonality in tumors of the breast.

Cytogenetically unrelated clones are found in half of all carcinomas of the breast and also in the epithelial fraction of many benign breast tumors. The chromosomal aberrations thus detected are clearly nonrandom and appear to be the same as those often seen in other tumors as sole karyotypic anomalies. Clonal chromosome abnormalities are not found in histologically normal breast tissue. Cytogenetically unrelated clones may be found in both primary tumors and secondary lesions, be it within the same breast (multifocal carcinomas), in the contralateral breast (bilateral carcinomas), or in lymph node or other metastases. The aberrations are present in topologically separate tumor domains and may confer on the cells that harbor them different types of cancer-specific behavior, such as the ability to metastasize and invade locally. Whereas the available evidence thus strongly indicates that the cells carrying clonal karyotypic aberrations all are part of the neoplastic parenchyma, it is less certain whether cytogenetic polyclonality actually signifies a multicellular tumor origin, although we think that this is the explanation that best accommodates the cytogenetic data. But even if it should eventually be shown that the seemingly unrelated clones have some submicroscopic tumorigenic mutation in common, the observed karyotypic heterogeneity is remarkable and goes far beyond what one has become accustomed to from most other tumor types. To understand how the various clones interact during mammary carcinogenesis will be a major task in future breast cancer research.

Karyotypic abnormalities in fibroadenomas of the breast.

Short-term cultures of 50 fibroadenomas of the breast were cytogenetically analyzed. Nine tumors were found to display clonal chromosome aberrations. One had multiple, cytogenetically unrelated clones, whereas the others had a single abnormal clone each. Four cases had one balanced translocation as the sole anomaly, and one had a complex intrachromosomal rearrangement of chromosome 3, leading to loss of 3p material. One fibroadenoma had a single numerical aberration, and one had supernumerary ring chromosomes. The remaining 2 cases had both numerical and structural aberrations. The only recurrent alterations were trisomy 20 and rearrangement of chromosome arm 1p. The finding of similar chromosomal aberrations in fibroadenomas and carcinomas suggests that women with karyotypically abnormal fibroadenomas may have an increased risk of developing subsequent breast cancer. If so, different chromosome anomalies might have different pathogenetic and/or prognostic significance.

Cytogenetic findings in phyllodes tumors of the breast: karyotypic complexity differentiates between malignant and benign tumors.

Clonal karyotypic abnormalities were detected in short-term cell cultures from six phyllodes tumors of the breast. Whereas all five benign tumors had simple chromosomal changes, the highly malignant one had a near-triploid stemline, indicating that karyotypic complexity is a marker of malignancy in phyllodes tumors. Interstitial deletions of the short arm of chromosome 3, del(3)(p12p14) and del(3)(p21p23),were the only aberrations in two benign tumors. Cytogenetic polyclonality was detected in three benign tumors: two had cytogenetically unrelated clones, whereas the third had three different, karyotypically related cell populations as evidence of clonal evolution. The finding of clonal chromosome abnormalities in both the epithelial and connective tissue components of the phyllodes tumors indicates that they are genuinely biphasic, that is, that both components are part of the neoplastic parenchyma.

Cytogenetic abnormalities in an in situ ductal carcinoma and five prophylactically removed breasts from members of a family with hereditary breast cancer.

Short-term cultures of tissue samples from three bilateral prophylactic mastectomies and one in situ ductal carcinoma from four women belonging to a family with hereditary breast cancer were cytogenetically analyzed. Clonal chromosome abnormalities were detected in five of the six prophylactically removed breasts, all of which had the histologic diagnosis epithelial hyperplasia without atypia, and in the in situ carcinoma. The same karyotypic imbalance, a loss of 3p12-14, was detected in the in situ carcinoma as well as in one of the hyperplasias, indicating that these bands may harbor a pathogenetically relevant gene in this breast cancer family. The finding of chromosome aberrations in clonal proportions in the prophylactically removed breasts indicates that a neoplastic process was already present, lending support to the view that prophylactic bilateral mastectomy in these high-risk individuals prevented the development of breast carcinoma.

Chromosome abnormalities in benign hyperproliferative disorders of epithelial and stromal breast tissue.

Cytogenetic analysis of short-term cultures from 15 cases of benign proliferative breast disease (PBD), 10 diffuse PBD and 5 papillomas, and 15 fibroadenomas of the breast revealed clonal chromosome abnormalities in 7 diffuse PBD lesions, 4 papillomas and 5 fibroadenomas. The remaining 14 cases had a normal female chromosome complement. Cytogenetically unrelated abnormal clones were seen in 4 fibroadenomas and 2 PBDs. A single abnormal clone was found in 9 PBDs and 1 fibroadenoma. Three clonal abnormalities were seen as recurrent changes in 6 cases, namely interstitial deletions of 3p with 3p 12-14 as the minimally common deleted segment (in 1 papilloma, 1 diffuse PBD with atypia and 1 mixed-pattern lesion with both papilloma and atypical diffuse PBD features), r(9)(p24q34) (in 1 diffuse PBD and 1 fibroadenoma), and del(1)(q12)(again in 1 diffuse PBD and 1 fibroadenoma). Intriguingly, 6 of the 16 abnormal cases had chromosome changes that have been seen repeatedly as primary abnormalities in breast carcinomas: der(16)t(1;16)(q10;p10), del(3)(p12p14), and del(1)(q12). We conclude that some of the chromosome anomalies frequently found in breast carcinomas are also present in PBD and fibroadenomas. These aberrations may be accepted as early, neoplasia-relevant mutations. However, they do not seem to be sufficient by themselves to unleash a malignant process.

Karyotypic changes in phyllodes tumors of the breast.

Cytogenetic analysis of short-term cultures of five phyllodes tumors of the breast-classified as benign (one tumor), borderline malignant (two tumors removed from the same breast in 1991 and 1993), and malignant (two tumors)--revealed clonal changes with simple structural abnormalities in the benign tumor, the borderline malignant tumors, and one malignant tumor in which benign areas and areas of borderline malignancy were also present. In contrast, the malignant tumor without admixed borderline malignant or benign areas had a complex karyotype. The karyotype of the benign phyllodes tumor was 46,XX,del(12)(p11p12)/46,XX,t(8;18)(p11;p11)/46,XX. The first borderline malignant phyllodes tumor had t(3;20)(p21;q13) as the sole abnormality. When the tumor recurred, this was no longer the only clone detected and the tumor karyotype was now 46,XX,t(3;20)(p21;q13)/46,XX,t(9;10)(p22;q22)/46,XX,t(1;8) (p34;q24)/46,XX,del(11)(q22-23)/46,XX. The malignant/borderline malignant/benign tumor had t(1;6)(p34;p22) as the sole clonal abnormality. Finally, the karyotype of the malignant phyllodes tumor which contained no benign or borderline malignant areas was 42,XX,der(1)t(1;4)(q21;q21),der(3)t(3;17)(q29;q21), -4,i(8)(q10), -10, -13,i(13)(q10),der(14)t(1;14)(q21;p11),der(14)t(4;14) (p12;p11), -17/80-90,idemx2, +del(1)(q12), +i(1)(p10), +dic(5;5)(p14;p14), +i(6)(p10), +del(7)(p11), +dup(7)(q11q36), +i(15)(q10),inc/46,XX. The findings indicate some cytogenetic similarities between benign/borderline malignant phyllodes tumors and fibroadenomas of the breast, presumably reflecting similar pathogenetic mechanisms in the two types of mixed-lineage tumors.

Chromosome abnormalities in adenolipomas of the breast: karyotypic evidence that the mesenchymal component constitutes the neoplastic parenchyma.

Cytogenetic analysis of adenolipomas of the breast, a tumor type that has not been chromosomally characterized before, revealed the karyotypes 47,XX, +del(1)(p22) in one tumor and 46,XX, t(12;16)(q15;q24) in the other. Breast adenolipomas thus seem to be karyotypically identical to sporadic lipomas in other locations: rearrangements of 12q13-15 are the most common cytogenetic aberrations in lipomas, and also breaks in and around 1p22 have been reported in such tumors. The similarity with lipoma could be documented further in case 2, in which epithelial and mesenchymal cells were cultured separately; the t(12;16) was present in the latter but not in the former. This is evidence that the connective tissue is the neoplastic parenchyma in adenolipomas of the breast, whereas the glandular elements show concomitant but nonneoplastic proliferation.

Simple numerical chromosome aberrations in two pituitary adenomas.

Cytogenetic analysis of short-term cultures of one non-secreting and one prolactin-producing pituitary adenoma revealed simple clonal numerical abnormalities in both tumors. The karyotype of the non-secreting adenoma was 48,XX, +4, +9[42]/49,XX, +4, +9, +20[2]/46,XX[6]. In the prolactin-secreting adenoma, three aberrant clones were detected, giving the karyotype 45,X, -Y[20]/47,XY, +Y[6]/45,XY, -21[3]/46,XY[21]. One cell had the chromosome complement 46,X, -Y, +9; no other nonclonal aberrations were detected. The only hitherto published case of pituitary adenoma analyzed by banding techniques (Rey et al. [1986]: Cancer Genet Cytogenet 23:171-174) also had only numerical clonal changes that included extra copies of chromosome 9. We conclude that pituitary adenomas may be karyotypically characterized by numerical aberrations and that trisomy 9 seems to be the best candidate for a primary chromosomal anomaly.

Clonal karyotypic evolution in an embryonal rhabdomyosarcoma with trisomy 8 as the primary chromosomal abnormality.

An embryonal rhabdomyosarcoma was analyzed cytogenetically. In primary cultures fed a serum-containing medium, 11 clones with karyotypic abnormalities were found. One had trisomy 8 only. The other 10 clones had trisomy 8 as well as additional evolutionary changes that included trisomy for part or all of chromosome 2, isochromosomes for the short and long arms of chromosome 11, isochromosomes for the long arm of chromosome 8, and extra copies of chromosome 8, some of which had an interstitial deletion in 8q. In those primary cultures that had grown in a chemically defined, serum-free medium and in all passaged cultures, trisomy 8 was the only aberration. Our findings and a survey of published information point to gain of one chromosome 8 as a frequent primary karyotypic abnormality in embryonal rhabdomyosarcomas. Trisomy for part or all of chromosomes 2 and 11 and additional gains of chromosome 8 material seem to be common secondary changes.

Cytogenetic studies in tuberous sclerosis.

A cytogenetic study was performed with cultures derived from peripheral blood, unaffected skin, and angiofibromas of four patients suffering from the sporadic form of tuberous sclerosis (TSC). Increased frequencies of unstable chromosomal anomalies were found in lymphocytes and in fibroblasts from unaffected skin of the patients. The slight increase of the overall rate of unstable anomalies observed in angiofibroma-derived cultures above that of lymphocytes and skin fibroblasts, respectively, could almost entirely be attributed to a higher frequency of dicentric chromosomes. Of the 17 facial angiofibromas from which a total of 20 cell cultures were established, nine showed a normal karyotype, while eight exhibited stable chromosomal rearrangements, among which 19 clonal types could be identified. Unbalanced forms of various translocations caused partial trisomies of the long arms of chromosomes 1, 3, 7, 10, and 15. There was no clustering of breakpoints to a particular chromosomal region, nor was one particular chromosome preferentially involved. Frequencies and kinds of rearrangements varied between cultures derived from different angiofibromas from the same patient and between different culture charges from the same tumor. Tetraploidy was not generally more abundant in the angiofibroma-derived cultures, but there were a few culture charges with exceedingly high rates of tetraploid cells. The occurrence of premature centromere disjunction (PCD), either affecting all chromosomes or only part of them in angiofibroma-derived cultures, first described in TSC by Scappaticci et al. could be confirmed.