Conserved although very different karyotypes in Gliridae and Sciuridae and their contribution to chromosomal signatures in Glires.

Rodents represent the largest order of living mammals. It comprises 5 sub-orders, among which Sciuromorpha (Sciuridae, Gliridae and Aplodontiidae) are assumed to occupy a basal position in rodent evolution. Banded karyotypes of some representatives of the Sciuridae family have been compared to each other, and comparisons with man were performed using chromosome paintings. Sciuridae karyotypes have conserved several eutherian ancestral syntenies. Like Sciuridae, Gliridae possess some chromosomes easily comparable with those of Primates. Comparisons of Gliridae and Sciuridae chromosomes with those of the presumed eutherian ancestor provide information about their chromosomal evolution and their position among Rodentia. Although both Sciuridae and Gliridae karyotypes are relatively conserved, they display many differences, indicating their early divergence. The reconstruction of their chromosomal evolution allowed us to propose the composition of their presumed ancestral karyotypes, with 2n = 48 and 2n = 38 for Gliridae and Sciuridae, respectively. Since rodent emergence, a single rearrangement is common to these 2 families. It formed a chromosome with fragments homologous to human chromosomes 4-8p-4-12-22, not detected in other rodents, and thus characteristic for the Sciuromorpha. This allowed us to reassess the chromosomal signatures of Rodentia. Finally, we show that the speed of chromosomal evolution in Gliridae is intermediate between that of Sciuridae (low) and Muridae (high).

Phylogenetic position of the saola (Pseudoryx nghetinhensis) inferred from cytogenetic analysis of eleven species of Bovidae.

Previous morphological and molecular analyses failed to resolve the phylogenetic position of the critically endangered saola (Pseudoryx nghetinhensis) with respect to its placement in Bovina (cattle, bison, and yak) or Bubalina (Asian and African buffaloes). In the present study, G- and C-banding, Ag-staining and FISH with 28S and telomeric probes was undertaken for 17 bovid species. An analysis of these data allowed us to identify 49 structural rearrangements that included autosomes, gonosomes and 17 different NOR sites. The combined data set was subjected to a cladistic analysis aimed at: (i) providing new insights on phylogenetic relationships of the saola and other species within the subfamily Bovinae, and (ii) testing the suitability of different classes of chromosomal characters for phylogenetic reconstruction of the family Bovidae. The study revealed that nucleolar organizing regions (NORs) are phylogenetically informative. It was shown that at least one, or sometimes two of these characters punctuate divergences that include nodes that are the most basal in the tree, to those that are the most recent. In this context, the shared presence of three NORs in saola and species of Syncerus and Bubalus strongly suggests the saola's placement within the subtribe Bubalina. This contrasts with Robertsonian rearrangements which are informative only at the generic level. These findings suggest that NORs are an important and frequently overlooked source of additional phylogenetic information within the Bovidae that may also have applicability at higher taxonomic levels, possibly even for Pecora.

DNA methylation and chromosome instability in lymphoblastoid cell lines.

In order to gain more insight into the relationships between DNA methylation and genome stability, chromosomal and molecular evolutions of four Epstein-Barr virus-transformed human lymphoblastoid cell lines were followed in culture for more than 2 yr. The four cell lines underwent early, strong overall demethylation of the genome. The classical satellite-rich, heterochromatic,juxtacentromeric regions of chromosomes 1, 9, and 16 and the distal part of the long arm of the Y chromosome displayed specific behavior with time in culture. In two cell lines, they underwent a strong demethylation, involving successively chromosomes Y, 9, 16, and 1, whereas in the two other cell lines, they remained heavily methylated. For classical satellite 2-rich heterochromatic regions of chromosomes 1 and 16, a direct relationship could be established between their demethylation, their undercondensation at metaphase, and their involvement in non-clonal rearrangements. Unstable sites distributed along the whole chromosomes were found only when the heterochromatic regions of chromosomes 1 and 16 were unstable. The classical satellite 3-rich heterochromatic region of chromosomes 9 and Y, despite their strong demethylation, remained condensed and stable. Genome demethylation and chromosome instability could not be related to variations in mRNA amounts of the DNA methyltransferases DNMT1, DNMT3A, and DNMT3B and DNA demethylase. These data suggest that the influence of DNA demethylation on chromosome stability is modulated by a sequence-specific chromatin structure.

Evidence for in vitro selection during cell culturing of breast cancer: detection by flow and image cytometry.

Detailed studies of chromosome rearrangements within solid tumors require karyotype analysis after cell culturing. However, different cell subpopulations with various growth capacities within one tumor may introduce biases in karyotype analysis, known as the in vitro selection. In our laboratory, 22% of karyotypes from breast cancers established after short-term culture were normal. Using interphase fluorescence in situ hybridization (FISH) for the determination of chromosome 1 arm imbalances and flow cytometry measurements of ploidy, we demonstrated that at least 2/3 of these tumors were mainly composed of aneuploid cell populations. Thus, the incidence of normal or balanced karyotypes among breast cancers is probably below 7%. This is the first direct proof for the existence of an in vitro selection within breast cancer cultures, suggesting cautious interpretation of cytogenetic data.

Quantitative fluorescence in situ hybridization in lung cancer as a diagnostic marker.

The diagnosis of lung cancer is quite often hampered by the existence of various cell types within samples such as biopsies or pleural effusions. We have established a new marker for image cytometry of interphase tumor cells of the lung by using the most recurrent and early cytogenetic event in lung cancer, the loss of the short arm of chromosome 3. The method is based on the detection of the imbalance between the long and the short arms of chromosome 3 by performing two-color fluorescence in situ hybridization on both arms. Fourteen tumors were analyzed after short-term culture and compared with the corresponding cytogenetic data obtained from metaphase analysis. Results on interphase nuclei and control experiments on metaphases were the same, with imbalance ratios ranging from 1.0 to 2.0 (mean value 1.6, median 1.5). To assess the clinical significance of this approach, three pleural effusions were analyzed. Data showed that normal cells within the sample could have been distinguished from the tumor cells based on different imbalance values between the long and the short arms. Thus, our method allows refined detection of lung tumor cells within samples containing heterogeneous cell populations.

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.

Homogeneously staining regions in 223 breast carcinomas: cytogenetic and clinicopathological correlations.

A correlation analysis was performed on 223 breast carcinomas to assess the relationships between gene amplification, karyotypic and clinicopathological features. Homogeneously staining region (HSR) is the most frequent form of amplification found in breast cancer. HSR-containing tumours accounted for 60% of the cases. Although up to 40% of tumours with slightly altered karyotype contained HSRs, an excess of HSRs was found within the tumours whose karyotype showed the highest rates of rearranged chromosomes. HSRs were also found to be particularly frequent in small tumours of high histological grade and with a low expression of progesterone receptors. An excess of HSRs seems to be observed in younger patients, however, significant correlation could be demonstrated only for patients below 55 years and below 60 years, compared with older ones. With a 120-month follow-up for 152 patients, a significant association between the presence of HSRs and a shortened overall survival was observed. Altogether, the presence of HSRs appears to be a good indicator of poor prognosis. Further studies are needed to determine whether amplification of specific genes or cell ability to amplify is the most important parameter for tumour progression.

Amplification of DNA sequences from chromosome 19q13.1 in human pancreatic cell lines.

Conventional cytogenetics and comparative genomic hybridization (CGH) were utilized to identify recurrent chromosomal imbalances in 12 pancreatic adenocarcinoma cell lines. Multiple deletions and gains were observed in all cell lines. Losses affecting chromosomes or chromosome arms 9p, 13, 18q, 8p, 4, and 10p and gains involving chromosome arms or bands 19q13.1, 20q, 5p, 7p, 11q, 3q25-qter, 8q24, and 10q were commonly observed. Interestingly, 19 distinct sites of high-level amplification were found by CGH. Recurrent sites involved 19q13.1 (6 cases), 5p (3 cases), and 12p and 16p (2 cases). Amplification of KRAS2 was demonstrated in 2 cell lines and that of ERBB2 in another. To define the occurrence of chromosome 19 amplification further, two-dimensional analysis of NotI genomic restriction digests and fluorescence in situ hybridization using probes from band 19q13.1 were utilized. High-level amplification of overlapping sets of chromosome 19 NotI fragments was exhibited in 3 cell lines of which 2 showed amplification of both OZF and AKT2 genes and 1 that of AKT2 alone. In these 3 cell lines, amplification of chromosome 19 sequences was associated with the presence of a homogeneously staining region. Our results provide evidence of heterogeneity in the extent of chromosome 19 amplification and suggest the existence of yet unknown amplified genes that may play a role in pancreatic carcinogenesis.

Characterization of recurrent homogeneously staining regions in 72 breast carcinomas.

Cytogenetic analyses were performed on 223 breast carcinomas, of which 60% contained homogeneously staining regions (hsr), an intrachromosomal cytogenetic feature of gene amplification. The precise hsr localization could be determined for 123 hsr from 72 cases. The juxtacentromeric region of chromosome 8, band 11q13, and the whole of chromosome 17 were frequently involved. For 28 cases, the origin of the DNA sequences forming HSR could be investigated by chromosome painting, comparative genomic hybridization, and/or Southern blotting. Sequences from chromosomes 11 and 17 were mostly found within hsr located on chromosomes 11 and 17, respectively. In contrast, sequences from chromosome 8 were rarely found within hsr localized on chromosome 8. These observations suggest that different mechanisms lead to hsr formation in breast cancer. Band 11 q13 and the 17p chromosome arm may correspond to sites of in situ amplification driven by deletions distal to the amplification target genes. hsr in the region 17q2, which is also a frequent site of in situ amplification, takes place without the occurrence of a distal deletion. The short arm of chromosome 8 is often deleted, but frequently becomes the site of hsr formed elsewhere in the genome.

Near haploidy in breast cancer: a particular pathway of chromosome evolution.

In a series of 260 cytologically abnormal breast cancers studied in our laboratory, we observed a single case of near-haploid karyotype: 27,X, +der(1;16)(q10;p10), +7, +14, +15. A review of published cases of near-haploid malignancies suggests that near haploidy belongs to a process of chromosome evolution distinct from that of most epithelial cancers in which hypodiploidy is strongly associated with the occurrence of unbalanced structural rearrangements. In near-haploid tumors, chromosome loss is independent from chromosome rearrangements and may not be associated with an adverse prognosis.

Quantitative FISH by image cytometry for the detection of chromosome 1 imbalances in breast cancer: a novel approach analyzing chromosome rearrangements within interphase nuclei.

Interphase cytogenetics have become a widespread tool for investigation of chromosome rearrangements in solid tumors. The most recurrent chromosome alteration within breast cancer affects chromosome 1, leading principally to gain of the long arm and/or loss of the short arm. We have developed a new method for detection of chromosome 1 arm imbalances in interphase nuclei. The method is based on quantitation of the fluorescence signals emitted by the hybridized two-color paintings of the short and long arms using image cytometry. The chromosome arm imbalance was determined by calculating the ratio of both fluorescence emissions of each arm. The ratio of the paintings of normal lymphocytes was used as a reference. Three breast cancer cell lines, 13 fresh tumor samples, and 6 fine-needle samplings of breast cancer were analyzed using an automated image cytometer. Whenever possible, classic cytogenetics and in situ hybridization on metaphases were performed as controls. Fluorescence ratios representing the imbalances of chromosome 1 arms with values between 1 and 3.2 were measured. Data between classic cytogenetics and interphase cytogenetics were well-correlated (r = 0.89). This method, which enables an easy detection of intrachromosomal imbalances without need of metaphase preparations, detects malignant cells and can be extended to other carcinomas for which chromosome 1 arm imbalances are recurrent or chromosome alterations specific of other malignancies. In comparison to other interphase fluorescence in situ hybridization techniques, it avoids every spot scoring problem encountered when using centromeric probes and the difficulties in interpreting structural rearrangements.

DNA hypomethylation in breast cancer: an independent parameter of tumor progression?

The global DNA methylation status was investigated on a series of 59 breast cancers by Southern blotting, using methylation sensitive restriction enzymes. By comparison to control DNA, almost all tumor DNAs were found globally hypomethylated. However, the demethylation was variable from tumor to tumor. Compared to other biological parameters, the methylation did not correlate with chromosome alterations, steroid hormone receptor status, or histopathological grading. Tumors which appeared to be the most evolved for other parameters were only mildly hypomethylated, whereas tumors with strongly hypomethylated DNA corresponded to those with slight alterations of the other parameters. Thus, DNA hypomethylation is a consistent characteristic of breast cancer, but its variations may not correlate with tumor progression of most breast cancers.

Unusual clonal chromosomal evolution in a breast carcinoma and its lymph node metastasis in a patient with Down syndrome.

A cytogenetic study was performed on a primary breast carcinoma and its axillary lymph node metastasis from a 53-year-old patient with trisomy 21, a carrier of a constitutional der(21;21). A translocation t(X;21) and the loss of the other X chromosome were shared by all karyotypes from tumor cells. The primary tumor was hyperdiploid with several gains of whole chromosomes. In contrast, most cells from the metastasis shared several rearrangements and losses leading to a hypodiploid karyotype. No normal chromosome 17 was present; instead, an i(17)(q10) and a fragment, detected by chromosome painting and presumably corresponding to a rearranged 17p, were found. Immunostaining for p53 was strongly positive in the metastasis but not in the primary tumor, suggesting a mutation of the TP53 gene in the metastasis. Finally, a small cell population of the metastasis was hyperdiploid like the clone in the primary tumor, suggesting that the node was colonized twice, at an early stage and a later stage of the clonal evolution of the tumor.

Scanning ion analytical microscopy for high-resolution detection of 5-bromo-2'-deoxyuridine incorporation in metaphase chromosomes.

We investigated the possibilities of using scanning ion analytical microscopy (SIAM) to detect bromine in human metaphase chromosomes. The experiments were performed after incorporation of the thymidine analogue, 5-bromo-2'-deoxyuridine (BrdU), into the DNA or by in situ hybridization of a BrdU-labelled probe for the subcentromeric repeated DNA sequences. The possibilities offered by this microanalytical method were compared with immunofluorescent staining techniques. Well-defined maps of bands containing bromide were obtained with metaphase chromosomes that had incorporated BrdU during the late S-phase. Their patterns were similar to the labelling obtained by immunofluorescence. In addition, SIAM reveals the presence of bromine within constitutive heterochromatic regions in which BrdU is poorly detected by immunofluorescence. The comparison of the 12C14N, 31P and 81Br maps of controls and fluorescence plus Giemsa (FPG) metaphase chromosomes shows the loss of bromide from DNA during this treatment. SIAM emerges as a new powerful microanalytical technology for investigating chromosome structure further.

Identification of amplified DNA sequences in breast cancer and their organization within homogeneously staining regions.

A modified comparative genomic hybridization (mCGH) technique was used to identify and map amplified DNA sequences in six homogeneously staining regions (hsr) from three primary breast carcinomas. Five different chromosomal regions and bands were identified as sites of amplification: 8p1, 17q21.1, 17q23 (two cases), 19q13.3, and 20q13.3. The mCGH site located on 17q21.1 was demonstrated to correspond to a 50-100-fold amplification of ERBB2. Further in situ hybridization experiments were used to confirm the mCGH results and to characterize the organization of the amplified sequences within the hsr. In five of six instances, two or more chromosomal regions were found amplified in the same hsr. In the tumor with the less modified karyotype, the two hsr comprised DNA sequences from three different chromosomes and showed different patterns of amplification. In the tumor with the most rearranged karyotype, the hsr-carrying chromosomes were formed by the translocation and amplification of sequences from three or four different chromosomal sites. This illustrates the complexity of the amplification process in breast cancers.

Loss of chromosome 3p arm differentiating tumorigenic from non-tumorigenic cells derived from the same SV40-transformed human mammary epithelial cells.

After immortalization of human normal mammary epithelial cells by replication-defective SV40 genome integration, 2 cultures were developed independently. Both had the same integration site, in band 9q21, but rapidly diverged karyotypically. After a few passages, one, designated SC2T2, exhibited near-diploid (a) and the other, designated SL2T2, near-tetraploid (b) karyotypes. The simplest formulas were 44, X, -X, der(3;22) (q10;q10), der(4) t(4;9)(q34;q12), +8, +9, add(13)(p1), der(19) t(8;19)(q21;p13.3), add(22)(p1) for karyotype (a) and 93, XXXX, add(1)(q12), add(11)(q13), +20 for karyotype (b). A number of alterations were further acquired with passages. Both cell cultures were tumorigenic, but their efficiency of grafting in nude mice largely differed: it was low for SL2T2 and high for SC2T2 cultures. All cultures of the xenografted tumors, obtained from either SL2T2 or SC2T2, exhibited the same clonal anomalies as those characterizing karyotype (a). It was concluded that only cells with karyotype (a) were tumorigenic, and that the difference in the tumorigenic potential of cultures SC2T2 and SL2T2 was related to their richness in cells with this karyotype. The comparison of the various karyotypes, together with data obtained in other cell types transformed by SV40, suggests that the acquisition of tumorigenicity in S2T2 mammary epithelial cells may be related to the loss of chromosome 3p arm.

Detection of DNA amplification in 17 primary breast carcinomas with homogeneously staining regions by a modified comparative genomic hybridization technique.

A modified comparative genomic hybridization (mCGH) technique was applied to a series of 17 primary breast carcinomas in which cytogenetic study (CG) demonstrated the presence of homogeneously staining region(s), suggesting the occurrence of DNA amplification. mCGH demonstrated recurrent amplifications of the whole chromosome arms 8q (9 times) and 1q (7 times) and of DNA loci in the following bands: 11q13 (6 times), 9p13 and 17q21.1 (4 times), 1q21.1 and 16p11.2 (3 times), and 8q22, 8q24.1, 10q22, 15q26, 17q23, and 20q13.3 (twice). Amplification of whole chromosome arms is likely to have resulted from unbalanced translocations or isochromosomes, whereas amplifications of smaller chromosomal segments probably arose through real DNA amplification processes. In all tumors but one, more than one amplified locus was detected. The fact that many chromosomal sites were involved suggests that the process of amplification is complex and that many genes are potential targets.

Clonal rearrangements in human irradiated fibroblasts.

The cytogenetic dose response following in vivo localized irradiation is difficult to establish because of the occurrence of clones defined by chromosome alterations, with various proliferative rates. The biological meaning of these clones is not well understood. Two sets of experiments were performed to follow their behavior. R-banded karyotypes were established on human fibroblasts irradiated either before or after initiation of the cultures. Clones were observed in cultures developed after irradiation of biopsies, whereas irradiated cultures exhibited karyotypes with multiple non-clonal rearrangements. This difference suggests that most radiation-induced chromosome anomalies do not confer a selective advantage on the carrier cells in vitro. The appearance of clonal anomalies following biopsy irradiation would rather be a consequence of a strong selection at the time of the growth of the cells out of the explants, which would give rise to the progeny of a limited number of progenitor cells.

Relationship between loss of estrogen and progesterone receptor expression and of 6q and 11q chromosome arms in breast cancer.

The expression of estrogen (ER) and progesterone (PR) receptors was quantified in a series of 95 cytogenetically characterized breast cancers. The relationship between deficiencies of 6q and 11q arms (where genes for ER and PR are mapped, respectively) and ER and PR expression was analyzed. The range of variations in expression was very large by comparison to that of the number of chromosome arms. Furthermore, low expression without chromosome loss and high expression with chromosome loss were occasionally observed. Thus, major variations in ER and PR expression were independent of the number of copies of the corresponding gene. However, both the decrease in ER expression and 6q arm losses were correlated with cytogenetic evolution of the tumors, this correlation being less significant for PR and the 11q arm. In addition, 6q- but not 11q- tumors have, on the average, a low ER/PR ratio, whereas 11q- but not 6q- tumors have a low PR/ER ratio. When all 6q- or 11q- tumors were compared to those with no 6q or 11q losses, the average ER or PR value of the former was about half of the later. These data suggest that, in addition to a regulatory change of non-genetic origin, gene dosage effect plays a secondary, but significant, additional role.

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 (alpha) 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 alpha 1 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 alpha 1, alpha 16, CS1, and CS16 DNAs. All had conserved alpha 16 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 alpha 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.