Method of Detecting Cancer Through Generalized Loss
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(19) TZZ ZZ_T (11) EP 2 707 506 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12Q 1/68 (2018.01) C12N 15/11 (2006.01) 11.07.2018 Bulletin 2018/28 (86) International application number: (21) Application number: 12781728.6 PCT/US2012/037362 (22) Date of filing: 10.05.2012 (87) International publication number: WO 2012/154979 (15.11.2012 Gazette 2012/46) (54) METHOD OF DETECTING CANCER THROUGH GENERALIZED LOSS OF STABILITY OF EPIGENETIC DOMAINS, AND COMPOSITIONS THEREOF VERFAHREN ZUM NACHWEIS VON KREBS DURCH ALLGEMEINEN STABILITÄTSVERLUST EPIGENETISCHER DOMÄNEN UND ZUSAMMENSETZUNGEN DAFÜR PROCÉDÉ DE DÉTECTION D’UN CANCER PAR L’INTERMÉDIAIRE D’UNE PERTE GÉNÉRALISÉE DE STABILITÉ DE DOMAINES ÉPIGÉNÉTIQUES, ET COMPOSITIONS ASSOCIÉES (84) Designated Contracting States: US-A1- 2010 167 940 AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO • MARIE E MARADEO ET AL: "Translational PL PT RO RS SE SI SK SM TR application of epigenetic alterations: Ovarian cancer as a model", FEBS LETTERS, ELSEVIER, (30) Priority: 12.05.2011 US 201161518892 P AMSTERDAM, NL, vol. 585, no. 13, 7 March 2011 (2011-03-07), pages 2112-2120, XP028234926, (43) Date of publication of application: ISSN: 0014-5793, DOI: 19.03.2014 Bulletin 2014/12 10.1016/J.FEBSLET.2011.03.016 [retrieved on 2011-03-12] (73) Proprietor: The Johns Hopkins University • RAJ CHARI ET AL: "Integrating the multiple Baltimore, MD 21218 (US) dimensions of genomic and epigenomic landscapes of cancer", CANCER AND (72) Inventors: METASTASIS REVIEWS, KLUWER ACADEMIC • FEINBERG, Andrew, P. PUBLISHERS, DO, vol. 29, no. 1, 29 January 2010 Lutherville,MD 21093 (US) (2010-01-29), pages 73-93, XP019787663, ISSN: • IRIZARRY, Rafael, I. 1573-7233 Baltimore, MD 21209 (US) • ANANIEV GENE E ET AL: "Optical mapping discerns genome wide DNA methylation (74) Representative: Lee, Nicholas John et al profiles", BMC MOLECULAR BIOLOGY, BIOMED Kilburn & Strode LLP CENTRAL LTD, GB, vol. 9, no. 1, 30 July 2008 Lacon London (2008-07-30), page 68, XP021033507, ISSN: 84 Theobalds Road 1471-2199 London WC1X 8NL (GB) • COSTELLO J F ET AL: "METHYLATION MATTERS", JOURNAL OF MEDICAL GENETICS, (56) References cited: BMJ PUBLISHING GROUP, LONDON, GB, vol. 38, EP-A1- 2 253 714 WO-A1-2009/021141 no. 5, 1 May 2001 (2001-05-01), pages 285-303, WO-A1-2010/062914 WO-A2-2005/059172 XP008018005, ISSN: 0022-2593, DOI: US-A1- 2003 190 616 US-A1- 2004 248 171 10.1136/JMG.38.5.285 US-A1- 2005 064 401 US-A1- 2009 305 256 Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 707 506 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 707 506 B1 • STEPHEN B. BAYLIN ET AL: "A decade of • HANSEN, K. D. ET AL.: ’Increased methylation exploring the cancer epigenome - biological and variation in epigenetic domains across cancer translational implications", NATURE REVIEWS types.’ NATURE GENETICS. vol. 43, no. 8, June CANCER, vol. 11, no. 10, 1 January 2011 2011, pages 768 - 775, XP055079656 (2011-01-01), pages726-734, XP055045165, ISSN: 1474-175X, DOI: 10.1038/nrc3130 2 EP 2 707 506 B1 Description BACKGROUND OF THE INVENTION 5 FIELD OF THE INVENTION [0001] The present invention relates generally to methylome analysis, and more specifically to a method for detecting cancer through generalized loss of stability of epigenetic domains of the genome. 10 BACKGROUND INFORMATION [0002] Epigenetics isthe study of non-sequenceinformation ofchromosome DNA during cell division and differentiation. The molecular basis of epigenetics is complex and involves modifications of the activation or inactivation of certain genes. Additionally, the chromatin proteins associated with DNA may be activated or silenced. Epigenetic changes are 15 preserved when cells divide. Most epigenetic changes only occur within the course of one individual organism’s lifetime, but some epigenetic changes are inherited from one generation to the next. [0003] One example of an epigenetic mechanism is DNA methylation (DNAm), a covalent modification of the nucleotide cytosine. In particular, it involves the addition of methyl groups to cytosine nucleotides in the DNA, to convert cytosine to 5-methylcytosine. DNA methylation plays an important role in determining whether some genes are expressed or not. 20 Abnormal DNA methylation is one of the mechanisms known to underlie the changes observed with aging and devel- opment of many cancers. [0004] Cancers have historically been linked to genetic changes such as DNA sequence mutations. Evidence now supports that a relatively large number of cancers originate, not from mutations, but from epigenetic changes such as inappropriate DNA methylation. In some cases, hypermethylation of DNA results the an inhibition of expression of critical 25 genes, such as tumor suppressor genes or DNA repair genes, allowing cancers to develop. In other cases, hypometh- ylation of genes modulates expression, which contributes to the development of cancer. [0005] Cancer is generally viewed as over 200 separate organ-specific diseases of abnormal cell growth, largely controlled by a series of mutations, but also involving epigenetic, i.e. non-sequence based, changes that might involve the same sets of genes1. DNA methylation at CpG dinucleotides in particular has been studied extensively in cancer, 30 with hypomethylation reported at some genes, hypermethylation at others, and a global loss of DNA methylation ascribed to repetitive DNA elements that are normally methylated. [0006] Since the discovery of altered DNA methylation in human cancer, the focus of cancer epigenetics has been on candidate regions of the genome, either high-density CpG islands, gene promoters, or dispersed repetitive elements 2,3, and there has not been a comprehensive genome-scale understanding of the relationship between DNA methylation 35 loss and gain in cancer and in normal differentiation. Maradeo et al "FEBS LETTERS, vol. 585, no. 13, 7 March 2011 (2011-03-07) discloses a review of the translational application of epigenetics using epithelial ovarian cancer as a specific example of all types of cancer. SUMMARY OF THE INVENTION 40 [0007] The present invention is based on the discovery that generalized loss of stability of epigenetic domains was determined to be characteristic across various cancer types. Genome-scale bisulfite sequencing of cancers revealed a surprising loss of methylation stability in the cancer methylome, involving both CpG islands and shores, as well as large (up to several megabases) blocks of hypomethylation affecting more than half of the genome, with concomitant stochastic 45 variability in gene expression. [0008] As such, in one aspect, a method of diagnosing cancer or a risk of cancer in a subject is provided. The method includes identifying an anti-profile in a sample from a subject including: i) determining the methylation status of a plurality of genomic nucleic acid sequences from a non-cancerous biological sample and corresponding plurality of genomic nucleic acid sequences in a test sample from the subject; ii) detecting a deviation in the methylation status from the test 50 sample from the methylation status of the non-cancerous sample; iii) detecting an increased methylation variance in the test sample as compared to the non-cancerous sample; and iv) determining a methylation variance score by performing statistical analysis, thereby diagnosing cancer in the subject. The method includes detecting the methylation status of one or more nucleic acid sequences set forth in Table 6, Table 7, Table 16 or Table 17. In one embodiment, the method further includes detecting expression of one or more genes as set forth in Figure 16A, Figure 16B, Table 1 or Table 15. 55 [0009] In another aspect, a method for providing a prognosis for cancer is provided. The method includes: a) comparing the methylation status of a plurality of nucleic acid sequences in a sample from a subject diagnosed with, or being treated for cancer, to a methylation status of the one or more nucleic acid sequences of a cancer cell; b) identifying a methylation anti-profile using the methylation status of (a) including: i) detecting a deviation in the methylation status of (a) from a 3 EP 2 707 506 B1 methylation status of a corresponding plurality of nucleic acid sequences of a genome from a sample known to be non- cancerous; ii) detecting an increased methylation variance in the sample as compared to the non-cancerous sample; and iii) determining a methylation variance score by performing statistical analysis, wherein at least 25 nucleic acids are analyzed; and c) correlating the anti-profile of (b) with a prognosis, thereby providing a prognosis for cancer in the subject. 5 The plurality of nucleic acid sequences are set forth in Table 6, Table 7, Table 16 or Table 17, thereby providing a prognosis for the cancer. In one embodiment, the method further includes detecting expression of one or more genes as set forth in Figure 16A, Figure 16B, Table 14 or Table 15. [0010] In another aspect, a method of diagnosing cancer in a subject is provided utilizing detection of a shift or loss of methylation boundary. The method includes: i) determining the methylation status of a plurality of genomic nucleic 10 acid sequences from a non-cancerous biological sample and corresponding plurality of genomic nucleic acid sequences in a test sample from the subject; and ii) detecting a deviation in the methylation status from the test sample from the methylation status of the non-cancerous sample, wherein the deviation in the methylation status comprises a shift or loss of methylation boundary in one or more of the plurality of nucleic acid sequences in the test sample as compared to that of the corresponding plurality of nucleic acid sequences from the non-cancerous biological sample, thereby 15 diagnosing cancer in the subject.