Anders Zetterberg

Allele-specific copy number analysis of tumors

We present an allele-specific copy number analysis of the in vivo breast cancer genome. We describe a unique bioinformatics approach, ASCAT (allele-specific copy number analysis of tumors), to accurately dissect the allele-specific copy number of solid tumors, simultaneously estimating and adjusting for both tumor ploidy and nonaberrant cell admixture. This allows calculation of “ASCAT profiles” (genome-wide allele-specific copy-number profiles) from which gains, losses, copy number-neutral events, and loss of heterozygosity (LOH) can accurately be determined. In an early-stage breast carcinoma series, we observe aneuploidy (>2.7n) in 45% of the cases and an average nonaberrant cell admixture of 49%. By aggregation of ASCAT profiles across our series, we obtain genomic frequency distributions of gains and losses, as well as genome-wide views of LOH and copy number-neutral events in breast cancer. In addition, the ASCAT profiles reveal differences in aberrant tumor cell fraction, ploidy, gains, losses, LOH, and copy number-neutral events between the five previously identified molecular breast cancer subtypes. Basal-like breast carcinomas have a significantly higher frequency of LOH compared with other subtypes, and their ASCAT profiles show large-scale loss of genomic material during tumor development, followed by a whole-genome duplication, resulting in near-triploid genomes. Finally, from the ASCAT profiles, we construct a genome-wide map of allelic skewness in breast cancer, indicating loci where one allele is preferentially lost, whereas the other allele is preferentially gained. We hypothesize that these alternative alleles have a different influence on breast carcinoma development.

Inferring tumor progression from genomic heterogeneity

Cancer progression in humans is difficult to infer because we do not routinely sample patients at multiple stages of their disease. However, heterogeneous breast tumors provide a unique opportunity to study human tumor progression because they still contain evidence of early and intermediate subpopulations in the form of the phylogenetic relationships. We have developed a method we call Sector-Ploidy-Profiling (SPP) to study the clonal composition of breast tumors. SPP involves macro-dissecting tumors, flow-sorting genomic subpopulations by DNA content, and profiling genomes using comparative genomic hybridization (CGH). Breast carcinomas display two classes of genomic structural variation: (1) monogenomic and (2) polygenomic. Monogenomic tumors appear to contain a single major clonal subpopulation with a highly stable chromosome structure. Polygenomic tumors contain multiple clonal tumor subpopulations, which may occupy the same sectors, or separate anatomic locations. In polygenomic tumors, we show that heterogeneity can be ascribed to a few clonal subpopulations, rather than a series of gradual intermediates. By comparing multiple subpopulations from different anatomic locations, we have inferred pathways of cancer progression and the organization of tumor growth.

Deregulation of cyclin E in human cells interferes with prereplication complex assembly.

Deregulation of cyclin E expression has been associated with a broad spectrum of human malignancies. Analysis of DNA replication in cells constitutively expressing cyclin E at levels similar to those observed in a subset of tumor-derived cell lines indicates that initiation of replication and possibly fork movement are severely impaired. Such cells show a specific defect in loading of initiator proteins Mcm4, Mcm7, and to a lesser degree, Mcm2 onto chromatin during telophase and early G1 when Mcm2–7 are normally recruited to license origins of replication. Because minichromosome maintenance complex proteins are thought to function as a heterohexamer, loading of Mcm2-, Mcm4-, and Mcm7-depleted complexes is likely to underlie the S phase defects observed in cyclin E–deregulated cells, consistent with a role for minichromosome maintenance complex proteins in initiation of replication and fork movement. Cyclin E–mediated impairment of DNA replication provides a potential mechanism for chromosome instability observed as a consequence of cyclin E deregulation.

A quantitative cytochemical investigation of the relationship between cell mass and initiation of DNA synthesis in mouse fibroblasts in vitro

The mass of cells entering interphase after cell division, initial mass (mo), and the cell mass at the stage of initiation of DNA synthesis (mG1) were determined in 21 populations of mouse fibroblasts in vitro (L-cells) which exhibited different mo values. Cells in populations with low initial masses had to synthesize more mass before the onset of DNA synthesis than cells in populations with high initial masses. In addition, these smaller cells required more time for this mass synthesis than the bigger cells. The generation times were found to be almost identical for the different L-cell populations. Cells in populations with low initial masses therefore spent a relatively long time in the G1 and a relatively short time in the S + G2 phases of the cell cycle. The opposite applied when the cell populations had high initial masses. These findings support the hypothesis that initiation of DNA synthesis is dependent on the cell mass.

Quantitative cytochemical studies on interphase growth: I. Determination of DNA, RNA and mass content of age determined mouse fibroblasts in vitro and of intercellular variation in generation time☆

A combination of quantitative cytochemistry and time-lapse cinematography has been used for studying the DNA, RNA and protein (dry mass) synthesis during interphase of mouse fibroblasts in vitro. This method is mainly associated with two error factors, i.e., variation in generation time and variation in the amount of DNA, RNA and protein of cells in the same stage of interphase. The magnitude of these factors were compared and were discussed in connection with the relation between growth and division. The cellular DNA, RNA and mass content doubled during interphase; there was no evidence for loss of material during mitosis. There was an error in RNA and, to greater extent, in mass distribution to the daughter cells at mitosis, while the error of DNA distribution was of the same order as the error of measurements. The G 1 (preceding DNA synthesis), the S (DNA synthesis) and the G 2 (following DNA synthesis) periods were 8, 6 and 5 hr, respectively, and the rate of DNA synthesis was constant. RNA and mass increased throughout the entire interphase. The rate of mass synthesis was higher in the latter part of interphase. It was not possible to judge whether the rate of RNA synthesis was constant or changed during interphase. DNA synthesis seemed to be initiated at a critical cellular mass. The importance of such a critical mass was discussed with regard to maintenance of the size homogeneity of the cell population, intercellular variations in generation times and the “trigger” mechanism for mitosis.

Quantitative cytochemical aspects of a combined Feulgen-Naphthol Yellow S staining procedure for the simultaneous determination of nuclear and cytoplasmic proteins and DNA in mammalian cells

Abstract The simultaneous cytophotometric determination of nuclear and cytoplasmic proteins and DNA by means of a combined Feulgen-Naphthol Yellow S (NYS) staining procedure was investigated. According to this procedure Feulgen staining is performed prior to NYS staining. The following main results were obtained: 1. 1. After NYS staining alone, the amount of NYS bound to the cell was found to be closely correlated to the cellular dry mass. The correlation coefficient was 0.99 in ethanol-acetone fixed cells and 0.95 in formaldehyde-fixed cells. This close correlation was not significantly altered by the Feulgen staining procedure and was 0.92 in ethanol-acetone and 0.94 in formaldehyde-fixed cells. However, the absolute amount of NYS bound per unit dry mass was affected by the method of fixation and type of Feulgen hydrolysis. 2. 2. The cells lose material during the Feulgen procedure, particularly during the acid hydrolysis stage. The type of hydrolysis most suitable for the Feulgen procedure (5 N HCl, 22 °C, 60 min) resulted in a considerable loss of dry mass in ethanol-acetone fixed cells. This loss was smaller in formaldehyde-fixed cells (15%) and was in addition closely correlated (correlation coefficient 0.99) to the dry mass of the cells prior to hydrolysis. In formaldehyde-fixed cells the dry mass after the Feulgen procedure is thus a good measure of the true cellular dry mass of the fixed cells. This is further demonstrated by the close correlation between NYS binding to Feulgenstained cells and the dry mass of these cells prior to the Feulgen procedure (correlation coefficient 0.95). 3. 3. When using the combined Feulgen-NYS staining procedure under standardized conditions (formaldehyde fixation and acid hydrolysis in 5 N HCl, 22 °C, 60 min) a constant amount of NYS was found to be bound per unit dry weight to nuclear and cytoplasmic proteins in various types of mammalian cells with different proliferative activity. 4. 4. The Feulgen DNA determination was not found to be quantitatively affected by the subsequent NYS staining. From the results of the present study it seems that, under standardized conditions, the combined Feulgen-NYS staining procedure can be used as a reliable quantitative method for the determination of nuclear and cytoplasmic proteins and DNA in mammalian cells.

Genomic architecture characterizes tumor progression paths and fate in breast cancer patients

This study demonstrates the relation among structural genomic alterations, molecular subtype, and clinical behavior and shows that an objective score of genomic complexity can provide independent prognostic information in breast cancer. Form and Malfunction Breast cancer is an iniquitous disease with a panoply of predisposing genetic and environmental causes, the details of which have yet to be fully understood. One of every four women will be diagnosed with breast cancer, hence the early and accurate identification of specific tumor features that may affect overall survival is imperative in achieving an optimal prognosis. A widely appreciated taxonomy in the breast cancer field has enabled the molecular discernment of five pathological subtypes; however, as research dives deeper into the chromosomal underpinnings of the disease, new classifiers are needed to augment what is known with key structural details to create a more vivid tumor landscape. Now, Russnes and colleagues have generated new algorithms that can estimate the specific genomic region as well as the architectural type of rearrangement—gains or losses of chromosome arms. A cohort of breast tumors was scored using this method, and all tumors with complex rearrangements had more whole chromosome arms affected than those without complex rearrangement. Moreover, there was an overlapping correlation with the molecular subtyping features of the tumors, and the score could confer prognostic power. Distinct molecular subtypes of breast carcinomas have been identified, but translation into clinical use has been limited. We have developed two platform-independent algorithms to explore genomic architectural distortion using array comparative genomic hybridization data to measure (i) whole-arm gains and losses [whole-arm aberration index (WAAI)] and (ii) complex rearrangements [complex arm aberration index (CAAI)]. By applying CAAI and WAAI to data from 595 breast cancer patients, we were able to separate the cases into eight subgroups with different distributions of genomic distortion. Within each subgroup data from expression analyses, sequencing and ploidy indicated that progression occurs along separate paths into more complex genotypes. Histological grade had prognostic impact only in the luminal-related groups, whereas the complexity identified by CAAI had an overall independent prognostic power. This study emphasizes the relation among structural genomic alterations, molecular subtype, and clinical behavior and shows that objective score of genomic complexity (CAAI) is an independent prognostic marker in breast cancer.

Separase: a universal trigger for sister chromatid disjunction but not chromosome cycle progression

Separase is a protease whose liberation from its inhibitory chaperone Securin triggers sister chromatid disjunction at anaphase onset in yeast by cleaving cohesins kleisin subunit. We have created conditional knockout alleles of the mouse Separase and Securin genes. Deletion of both copies of Separase but not Securin causes embryonic lethality. Loss of Securin reduces Separase activity because deletion of just one copy of the Separase gene is lethal to embryos lacking Securin. In embryonic fibroblasts, Separase depletion blocks sister chromatid separation but does not prevent other aspects of mitosis, cytokinesis, or chromosome replication. Thus, fibroblasts lacking Separase become highly polyploid. Hepatocytes stimulated to proliferate in vivo by hepatectomy also become unusually large and polyploid in the absence of Separase but are able to regenerate functional livers. Separase depletion in bone marrow causes aplasia and the presumed death of hematopoietic cells other than erythrocytes. Destruction of sister chromatid cohesion by Separase may be a universal feature of mitosis in eukaryotic cells.

Quantitative cytochemical studies on interphase growth: II. Derivation of synthesis curves from the distribution of DNA, RNA and mass values of individual mouse fibroblasts in vitro

Abstract An investigation was made of the kinetics of nucleic acid and protein (dry mass) synthesis during interphase of mouse fibroblasts in vitro. Quantitative microspectrophotometric and microinterferometric methods were employed for the determination of the cellular DNA, RNA and mass content. The time course of DNA, RNA and mass synthesis during interphase was obtained by analyzing the distribution of the DNA, RNA and mass content of individual cells from large samples in fixed populations. The main source of error in this type of analysis lies in momentary variation, i.e. variation in the amount of DNA, RNA and mass among cells in the same physiological stage of the cell cycle. The influence of momentary variation on this method of analysis was examined in order to be able to make corrections for it. The accuracy of the results obtained was discussed in relation to sample size and momentary variation. The G1, S and G2 periods were found to be 9, 6 and 4 hr respectively. During the S period DNA was synthesized at a constant rate. Contrary to DNA, RNA and mass were synthesized throughout the whole interphase. The rate of synthesis of both RNA and mass was 2–2.5 times faster at the end of interphase than it was at the beginning. The increase in the rate of RNA synthesis seemed to occur predominantly during the second half of interphase at the same time as the doubling of the amount of DNA in the cell. The rate of mass synthesis, however, seemed to increase over the whole of the interphase period in proportion to the amount of RNA.

Genetic alterations in cervical carcinomas: Frequent low-level amplifications of oncogenes are associated with human papillomavirus infection

The development of cervical carcinoma is closely associated with HPV infection. However, other genetic alterations also play an important role. In this study, we analyzed copy number alterations of several oncogene loci in a panel of 84 cervical tumors. Sixty‐five (77%) tumors were HPV DNA‐positive, and most were infected with type 16 or type 18 or both. The oncogenes studied include PIK3CA at 3q26.3, TERT at 5p15.33, C‐MYC at 8q24, CCND1 at 11q13.3, ERBB2 at 17q21.2 and locus region 20q13.2. Amplification of 1 or more genes was detected in 55 (65%) cases using interphase FISH. PIK3CA was amplified in 43% of tumors, followed by TERT (33%), 20q13.2 (30%), ERBB2 (29%), C‐MYC (25%) and CCND1 (12%). Most tumors showed low‐level amplification with 3–7 copies of these genes, and complex changes involving 3 or more genes occur more frequently in tumors at advanced stages. Increased protein expression of c‐erbB2 and c‐myc was observed in tumors with the corresponding gene amplification. Oncogene alterations were found more often in HPV‐infected cases, particularly for C‐MYC and TERT. These findings indicate that HPV‐associated cervical carcinomas bear frequent alterations of these genes, which may have critical biologic impact on the development and progression of carcinoma of the uterine cervix.