Claudia U. Dietrich

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.

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.

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.

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

Abstract 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 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.

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.

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.

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.