Estimation of tumor heterogeneity using CGH array data

<p>Abstract</p> <p>Background</p> <p>Array-based comparative genomic hybridization (CGH) is a commonly-used approach to detect DNA copy number variation in whole genome-wide screens. Several statistical methods have been proposed to define genomic segments with different copy numbers in cancer tumors. However, most tumors are heterogeneous and show variation in DNA copy numbers across tumor cells. The challenge is to reveal the copy number profiles of the subpopulations in a tumor and to estimate the percentage of each subpopulation.</p> <p>Results</p> <p>We describe a relation between experimental data and exact DNA copy number and develop a statistical method to reveal the heterogeneity of tumors containing a mixture of different-stage cells. Furthermore, we validate the method on simulated data and apply the method to 29 pairs of breast primary tumors and their matched lymph node metastases.</p> <p>Conclusion</p> <p>We demonstrate a new method for CGH array analysis that allows a tumor sample to be classified according to its heterogeneity. The method gives an interpretable series of copy number profiles, one for each major subpopulation in a tumor. The profiles facilitate identification of copy number alterations in cancer development.</p

Tumor heterogeneity in neoplasms of breast, colon, and skin

<p>Abstract</p> <p>Background</p> <p>Different cell subpopulations in a single tumor may show diverse capacities for growth, differentiation, metastasis formation, and sensitivity to treatments. Thus, heterogeneity is an important feature of tumors. However, due to limitations in experimental and analytical techniques, tumor heterogeneity has rarely been studied in detail.</p> <p>Presentation of the hypothesis</p> <p>Different tumor types have different heterogeneity patterns, thus heterogeneity could be a characteristic feature of a particular tumor type.</p> <p>Testing the hypothesis</p> <p>We applied our previously published mathematical heterogeneity model to decipher tumor heterogeneity through the analysis of genetic copy number aberrations revealed by array CGH data for tumors of three different tissues: breast, colon, and skin. The model estimates the number of subpopulations present in each tumor. The analysis confirms that different tumor types have different heterogeneity patterns. Computationally derived genomic copy number profiles from each subpopulation have also been analyzed and discussed with reference to the multiple hypothetical relationships between subpopulations in origin-related samples.</p> <p>Implications of the hypothesis</p> <p>Our observations imply that tumor heterogeneity could be seen as an independent parameter for determining the characteristics of tumors. In the context of more comprehensive usage of array CGH or genome sequencing in a clinical setting our study provides a new way to realize the full potential of tumor genetic analysis.</p

A statistical approach for detecting genomic aberrations in heterogeneous tumor samples from single nucleotide polymorphism genotyping data

We describe a statistical method for the characterization of genomic aberrations in single nucleotide polymorphism microarray data acquired from cancer genomes. Our approach allows us to model the joint effect of polyploidy, normal DNA contamination and intra-tumour heterogeneity within a single unified Bayesian framework. We demonstrate the efficacy of our method on numerous datasets including laboratory generated mixtures of normal-cancer cell lines and real primary tumours

Avaliação “in vitro” do efeito antitumoral e antiangiogênico de uma metaloprotease isolada da peçonha de Bothrops pauloensis

Breast cancer is a highly malignant carcinoma and remains the second leading cause of mortality among women. The antitumor effects of metalloproteinases and disintegrins from snake venom on various types of cancer cells have been investigated. In this study, we evaluated the antitumor and antiangiogenic effects on MDA-MB-231 human breast cancer cells and endothelial cells induced by Bothropoidin, a disintegrin-like metalloproteinase isolated from Bothrops pauloensis snake venom. At 24h after treatment at 100pg/mL, Bothropoidin exerted a moderate cytotoxic effect of 30% on MDA-MB-231 versus 10% cytotoxicity against MCF10A (a non-tumorigenic breast cell line), a significant difference that suggests a possible preference by this protein for targets in cancer cells. Early and late apoptosis of MDA-MB-231 was observed after Bothropoidin treatment (10gg/mL and 40gg/mL). Furthermore, this toxin inhibited not only the adhesion of MDA-MB-231 cells in a dose-dependent manner but also cell migration by approximately 45%. In addition, Bothropoidin decreased endothelial cells viability and adhesion in Matrigel and inhibited in vitro angiogenesis in Matrigel stimulated by bFGF, showing significantly fewer formed vessels. The results demonstrated that Bothropoidin has potent in vitro antitumor and antiangiogenic effect and represents a biotechnological tool for elucidating the antitumor effect of disintegrins-like metalloproteinases in cancer cells.Dissertação (Mestrado)O câncer de mama é uma neoplasia altamente maligna e continua a ser a segunda principal causa de mortalidade entre as mulheres. Os efeitos antitumorais de metaloproteinases e desintegrinas de veneno de serpentes têm sido investigados em vários tipos de células tumorais. Neste estudo, foram avaliados os efeitos antitumorais e anti-angiogênicos induzidos pela Bothropoidina, uma metaloproteinase desintegrina-like isolada da peçonha de Bothrops pauloensis em células de câncer de mama humano MDA-MB-231 e células endoteliais. Após 24 horas de tratamento com 100pg/mL de Bothropoidina foi constatado um efeito citotóxico moderado de 30% em MDA-MB-231 contra 10% de citotoxicidade em MCF10A (uma linha de células da mama não tumorigênica), uma diferença significativa que sugere uma possível preferência desta proteína por alvos em células tumorais. Observou-se apoptose e apoptose tardia após tratamento com Bothropoidina (10pg/mL e 40pg/mL) em células MDA-MB-231. Além disso, esta toxina não só inibiu a adesão de células MDA-MB-231 de uma forma dose dependente, como a migração celular em aproximadamente 45%. Bothropoidina reduziu a viabilidade e adesão de células endoteliais em Matrigel e inibiu a angiogênese in vitro estimulada por bFGF em Matrigel, mostrando um número de vasos formados significativamente menor em relação ao controle. Os resultados demonstraram que Bothropoidina tem um potente efeito antitumoral e antiangiogênico in vitro, representando uma ferramenta biotecnológica para elucidar o efeito antitumoral de metaloproteinases desintegrinas-like em células cancerígenas

The structure and evolution of breast cancer genomes

Chromosome changes in the haematological malignancies, lymphomas and sarcomas are known to be important events in the evolution of these tumours as they can, for example, form fusion oncogenes or disrupt tumour suppressor genes. The recently described recurrent fusion genes in prostate and lung cancer proved to be iconic examples as they indicated that important gene fusions are found in the common epithelial cancers also. Breast cancers often display extensive structural and numerical chromosome aberration and have among the most complex karyotyes of all cancers. Genome rearrangements are potentially an important source of mutation in breast cancer but little is known about how they might contribute to this disease. My first aim was to carry out a structural survey of breast cancer cell line genomes in order to find genes that were disrupted by chromosome aberrations in “typical” breast cancers. I investigated three breast cancer cell lines, HCC1187, VP229 and VP267 using data from array painting, SNP6 array CGH, molecular cytogenetics and massively parallel paired end sequencing. I then used these structural genomic maps to predict fusion transcripts and demonstrated expression of five fusion transcripts in HCC1187, three in VP229 and four in VP267. Even though chromosome aberrations disrupt and fuse many genes in individual breast cancers, a major unknown is the relative importance and timing of genome rearrangements compared to sequence-level mutation. For example, chromosome instability might arise early and be essential to tumour suppressor loss and fusion gene formation or be a late event contributing little to cancer development. To address this question, I considered the evolution of these highly rearranged breast cancer karyotypes. The VP229 and VP267 cell lines were derived from the same patient before and after therapy-resistant relapse, so any chromosome aberration found in both cell lines was probably found in the common in vivo ancestor of the two cell lines. A large majority of structural variants detected by massively parallel paired end sequencing, including three fusion transcripts, were found in both cell lines, and therefore, in the common ancestor. This probably means that the bulk of genome rearrangement pre-dated the relapse. For HCC1187, I classified most of its mutations as earlier or later according to whether they occurred before or after a landmark event in the evolution of the genome - endoreduplication (duplication of its entire genome). Genome rearrangements and sequence-level mutations were fairly evenly divided between earlier and later, implying that genetic instability was relatively constant throughout the evolution of the tumour. Surprisingly, the great majority of inactivating mutations and expressed gene fusions happened earlier. The non-random timing of these events suggests many were selected.This work was supported by the Medical Research Council Studentshi

Caractérisation moléculaire d'inversions péri- et paracentriques et analyse de leurs effets sur la méiose d'individus porteurs hétérozygotes

Les inversions sont des anomalies chromosomiques de structure résultant d'une double cassure sur un même chromosome et d'un retournement à 180° du segment chromosomique ainsi généré. Ces inversions sont dites péricentriques si les points de cassure sont localisés de part et d'autre du centromère, ou paracentriques dans le cas contraire. Chez l'Homme, la fréquence estimée des inversions péricentriques est de 0.012% à 0.07% et de 0,01% à 0,05% pour les inversions paracentriques (McKinlay-Gardner et Sutherland, 2004). Les porteurs hétérozygotes ne présentent généralement pas d'altération phénotypique mais sont susceptibles de rencontrer des problèmes de reproduction en raison de perturbations du processus méiotique conduisant à des troubles de la spermatogenèse et/ou à la production de gamètes génétiquement déséquilibrés. De nombreuses inversions ont été identifiées chez l'Homme et dans les espèces animales d'élevage. Cependant, peu d'études de ségrégation méiotique d'inversions ont été publiées à ce jour chez l'Homme (par exemple Morel et al. 2007), et aucune dans les espèces animales d'élevage. L'analyse méiotique de différentes inversions identifiées chez le porc dans le cadre du programme national de contrôle chromosomique mis en œuvre par notre laboratoire présente un double intérêt. Le premier est zootechnique. En effet, lors de la mise en évidence d'une inversion chez un futur reproducteur, sa réforme était jusqu'à présent systématiquement conseillée aux éleveurs. Toutefois, l'application de ce principe peut s'avérer difficile et/ou non optimal car elle peut conduire à l'élimination d'individus par ailleurs génétiquement intéressants et ainsi induire une baisse de l'efficacité des programmes de sélection. Raisonner l'utilisation des reproducteurs sur la base d'une prédiction de l'effet potentiel des remaniements nous semble une alternative raisonnable. L'un des objectifs de la Thèse est donc la mise au point et l'évaluation d'une méthode de prédiction basée sur l'estimation du pourcentage de gamètes déséquilibrés dans des échantillons de semence. Le second objectif est cognitif. En effet, des difficultés d'ordre technique et/ou éthique rendent difficile l'acquisition de certaines connaissances concernant le comportement méiotique de remaniements chromosomiques chez l'Homme. C'est le cas par exemple de l'effet du sexe du parent porteur sur le comportement et les produits de la méiose en présence d'une inversion. Le recours à un modèle animal (le porc en l'occurrence, dont la structure chromosomique est proche de celle de l'Homme) permet de s'affranchir de certaines contraintes, et d'apporter de nouvelles connaissances dans des domaines non encore documentés. Un autre objectif de la Thèse est, dans cet esprit, de réaliser une étude comparée des ségrégations méiotiques " mâle " et " femelle " d'une même inversion et ainsi de déterminer l'influence du sexe du porteur sur les effets de l'anomalie. L'analyse des profils de ségrégation méiotique " mâle " a été réalisée par la méthode de SpermFISH (hybridation in situ en fluorescence sur noyaux de spermatozoïdes décondensés). Les ségrégations méiotiques femelles ont pour leur part été étudiées par analyse de métaphases II d'ovocytes de truies maturés in vitro. Dans les deux cas, des sondes moléculaires de type BAC ont été utilisées. Les études d'appariement méiotique ont été réalisées à partir de biopsies testiculaires réalisées chez un verrat porteur d'une inversion péricentrique du chromosome 4. Ces échantillons ont été analysés par des méthodes d'immunocytologie à l'aide d'anticorps permettant de détecter spécifiquement certaines protéines. Les études de ségrégation méiotique de 7 inversions de tailles et de types différents ont été réalisées : les pourcentages de gamètes génétiquement déséquilibrés variaient de 0,6% à 4%, suggérant que ces inversions ont un impact très limité sur la reproduction. De manière surprenante, la proportion de gamètes anormaux produits n'est pas corrélée avec la taille du fragment inversé, comme cela est le cas chez l'Homme. La comparaison des profils de ségrégation " mâle " et " femelle " pour une inversion du chromosome 4 n'a pas non plus montré de différence de production de gamètes déséquilibrés entre les deux genres (4% et 3,6% respectivement). Le sexe du porteur ne semble donc pas être un facteur déterminant pour la production de gamètes anormaux. L'analyse des phases précoces de la méiose pour un animal porteur de la même inversion péricentrique du chromosome 4 à montré un comportement méiotique particulier du bivalent: plus de 90% des cellules montraient en effet un appariement non homologue de la région inversée. De plus, des évènements de crossing over dans la région inversée du chromosome 4 n'ont été observés que dans 5% des cas. Le comportement des chromosomes est responsable de la faible recombinaison dans la région inversée et donc de la faible proportion de gamètes génétiquement déséquilibrés estimée (4% en moyenne). Ces résultats demandent néanmoins à être confirmés par d'autres analyses. L'étude en cours des phases précoces de la méiose chez deux porteurs d'inversions péricentriques du chromosome 8 devraient nous permettre très prochainement d'approfondir nos connaissances sur le comportement méiotique des chromosomes en présence d'inversion. Par ailleurs, la mise au point de nouvelles techniques au sein du laboratoire, telles que la production de cellules souches induites (iPS) porcines, nous permet aussi d'envisager à moyen terme l'étude de l'intégralité du processus méiotique mâle en présence d'une inversion.Inversions are structural chromosomal rearrangements formed when a chromosome breaks in two places and the fragments reverses orientation. The inversion is pericentric if the breaks occur on either side of the centromere, paracentric in other cases. Prevalence estimates in human population range from 0.012% to 0.07% for pericentrics and from 0,01% to 0,05% for paracentrics (McKinlay-Gardner et Sutherland, 2004). Usually, both para- and pericentric inversions are phenotypically harmless, but the presence of an inversion can occasionally lead to severe reproductive disorders due to spermatogenesis impairements and the production of genetically unbalanced gametes. Many cases of inversions have been described in humans as well as in other animal species. However, relatively few studies have reported meiotic segregation pattern analyses of inversions in males (for example Morel et al. 2007), and none were reported on domestic animals. Meiotic analyses of various inversions identified in the pig species, thanks to the national program of chromosomic control, have a double interest. The first is zootechnical. When an inversion is detected in a boar, the breeders are always advised to sacrifice the animal. However, it can be difficult to eliminate individuals with a high genetic value and then induce a lowering in the efficiency of selection programms. Using a prediction of the potential effects of rearrangements to advise the breeders seem to be a good alternative. One of the thesis purpose is then to create a predictive method based on the percentage of unbalanced gametes in semen samples. The second interest is more linked to basic research. Indeed, it can be difficult to acquire knowledge on meiotic behaviour of chromosomal rearrangements in Man due to technical and/or ethical reasons. For example, little is known on an " sex " effect on meitoic behaviour during meiosis for inversion carriers. The use of an animal model (like the pig species) is a great opportunity to analyze the meiotic behaviour of chromosomes in inversion carriers, and then to have a better understanding on not well documented domains. That is why an other purpose of this thesis was to carried a comparative study of meotic segregations in males and females carriers of the same anomaly, and then determine a potential " sex " effect. Meotic segregation pattern analyses were carried out using the SpermFISH technique (fluorescent in situ hybridization on decondensed sperm nuclei) for males, and FISH on metaphase II oocytes matured in vitro for females. In both cases, molecular probes (Bacterial Artificial Chromosome) were used. Studies of pairing behaviour were carried out on testicular samples of an inversion of chromosome 4 carrier, using immunocytological techniques (use of specific antibodies against proteins of interest). Meotic segregation pattern analyses were carried out for 7 inversions (different lenght of the inverted fragment and different type) : percentage of unbalanced gametes ranged from 0,6% to 4%, suggesting a limited impact on reproduction. Surprisingly, the lenght of the inverted fragment is not correlated to the proportion of unbalanced gametes (unlike in Man). Comparison of " male " and " female " segregation profiles for an inversion of chromosome 4 did not show either any difference concerning the production of unbalanced gametes (4% and 3,6% respectively). The gender of the carriers do not seem to be a major factor for the production of unbalanced gametes. Analyses of early stages of meiosis for a carrier of the same pericentric inversion showed a specific meiotic behaviour of the bivalent : a non homologous pairing of the inverted region was seen in more than 90% of the cells analysed. Moreover, crossing overs in the inverted region were relatively rare (5 % of the cells). This chromosomal behaviour explains the low recombination rate in the inverted region, and then the low proportion of unbalanced gametes produced (mean of 4%). These results have to be confirmed by other analyses. Studies of the early stages of meiosis in two carriers of pericentric inversions of chromosome 8 should allow in the future a better understanding of the meiotic behaviour for inversion carriers. Moreover, the use of new techniques, such as production of induced pluripotent stem cells (iPS), could allow, in the future, analyses of the whole male meiotic process in inversion carriers

Contribución de los Microarrays genómicos al diagnóstico y pronóstico de los linfomas no Hodgkin de línea B.

[EN] This doctoral work has been analyzed by genomic arrays or CGHarrays two types of NHL-B. In Lesmes, our hypothesis was based on a thorough analysis of these lymphomas by CGH-arrays could identify and map more precisely the deleted region commonly at the level of chromosome 7, while identifying the genes on this chromosome and could be involved in the pathogenesis of this disease. In LDCGB we started a group of patients treated uniformly. The hypothesis is based on the use of CGH-arrays in this disease may identify new genetic markers that were due to treatment response and prognosis in these patients.[ES] En el presente trabajo doctoral se han analizado mediante arrays genómicos o CGHarrays dos tipos de LNH-B. En los LEZM, nuestra hipótesis se ha basado en que un análisis en profundidad de estos linfomas mediante CGH-arrays podría identificar y cartografiar con mayor precisión la región comúnmente delecionada a nivel del cromosoma 7, a la vez que identificar los genes localizados en este cromosoma y que podrían estar implicados en la patogenia de esta enfermedad. En los LDCGB hemos partido de un grupo de enfermos tratados de manera homogénea. La hipótesis planteada se ha basado en que el uso de los CGH-arrays en esta enfermedad podría identificar nuevos marcadores genéticos que tuvieran relación con la respuesta al tratamiento y el pronóstico en estos enfermos