Хромотрипсис в онкологии: обзор литературы и собственное наблюдение

МИЕЛОИДНЫЕ ОПУХОЛИ ,

Н.Н. Мамаев1, Т.Л. Гиндина1, Э.Г. Бойченко2

1 НИИ детской онкологии, гематологии и трансплантологии им. Р.М. Горбачевой, ГБОУ ВПО «Первый Санкт-Петербургский государственный медицинский университет им. акад. И.П. Павлова», ул. Льва Толстого, д. 6/8, Санкт-Петербург, Российская Федерация, 197022

2 Детская городская больница № 1, ул. Авангардная, д. 14, Санкт-Петербург, Российская Федерация, 198205

Для переписки: Татьяна Леонидовна Гиндина, канд. мед. наук, ул. Льва Толстого, д. 6/8, Санкт-Петербург, Российская Федерация, 197022; тел.: + 7(812)233-12-43; e-mail: cytogenetics.bmt.lab@gmail.com

Для цитирования: Мамаев Н.Н., Гиндина Т.Л., Бойченко Э.Г. Хромотрипсис в онкологии: обзор литературы и собственное наблюдение. Клиническая онкогематология. 2017;10(2):191–205.

DOI: 10.21320/2500-2139-2017-10-2-191-205

РЕФЕРАТ

Представлено собственное наблюдение и обзор литературы, посвященный недавно открытому феномену хромотрипсиса в онкологии. Хромотрипсис — тип комплексных геномных изменений, при которых хромосома сначала разрывается на десятки и даже тысячи частей, а потом эти фрагменты соединяются в случайном порядке. Иногда в перестройке участвует несколько хромосом. В результате формируются мутантные зоны генома, провоцирующие развитие онкологических и врожденных заболеваний. Иными словами, использование определенных методических подходов (многоцветной флюоресцентной гибридизации in situ, метода SKY и некоторых других) позволяет увидеть под микроскопом распад на фрагменты двух или более хромосом и воссоединение этих фрагментов в новые необычные двух- или многоцветные структуры — хромосомные маркеры. Хромотрипсис — редкий феномен со своеобразной картиной, наблюдаемой в клонах клеток самых разнообразных опухолей, включая новообразования кроветворной и лимфоидной тканей. В литературе имеются указания о большей частоте этого феномена у больных с миелодиспластическим синдромом и опухолями костей. Важную роль в формировании хромотрипсиса играют мутации гена TP53. Использование секвенирования концевой спаренной ДНК или метода SNP в онкологии представляется перспективным как в теоретическом, так и клиническом плане. В первую когорту исследуемых должны включаться пациенты с мутациями генов TP53 и MLL, со сложными хромосомными нарушениями, гиперэкспрессией гена EVI1 и некоторые другие. В статье представлен феномен хромотрипсиса у девочки 8 мес. с М7-вариантом острого миелоидного лейкоза.

Ключевые слова: хромотрипсис, онкогематология, рак, мутации гена TР53.

Получено: 2 октября 2016 г.

Принято в печать: 6 января 2017 г.

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ЛИТЕРАТУРА

  1. Stephens PJ, Greenman CD, Fu B, et al. Massive genomic rearrangement acquired in a single catastrophic event during cancer development. Cell. 2011;144(1):27–40. doi: 10.1016/j.cell.2010.11.055.
  2. Righolt C, Mai S. Shattered and stitched chromosomes – chromothripsis and chromoanasynthesis – manifestation of a new chromosome crisis. Genes Chromos Cancer. 2012;51(11):975–81. doi: 10.1002/gcc.21981.
  3. Tan L, Xu L-H, et al. Small Lymphocytic Lymphoma/Chronic lymphocytic leukemia with chromothripsis in an old woman. Chin Med J. 2015;128(7):985–7. doi: 10.4103/0366-6999.154329.
  4. de Pagter MS, Kloosterman WP. The diverse effects of complex chromosome rearrangements and chromothripsis in cancer development. In: BM Ghadimi, T Ried, eds. Chromosomal Instability in Cancer Cells. Recent Results in Cancer Research 200. Switzerland: Springer International Publishing; 2015. рр. 165–93. doi: 10.1007/978-3-319-20291-4_8.
  5. Magrangeas F, Avet-Loiseau H, Munshi NC, et al. Chromothripsis identifies a rare and aggressive entity among newly diagnosed multiple myeloma patients. Blood. 2011;118(3):675–8. doi: 10.1182/blood-2011-03-344069.
  6. Pei J, Jhanwar SC, Testa J R. Chromothripsis in a case of TP53-deficient chronic lymphocytic leukemia. Leuk Res Rep. 2012;1(1):4–6. doi: 10.1016/j.lrr.2012.09.001.
  7. Rausch T, Jones DT, Zapatka M, et al. Genome sequencing of pediatric medulloblastoma links catastrophic DNA rearrangements with TP53 mutations. Cell. 2012;148(1–2):59–71. doi: 10.101.1016/j.cell.2011.12.013.
  8. Ortega V, Chaubey A, Mendiola C, et al. Complex chromosome rearrangements in B-cell lymphoma: Evidence of chromoanagenesis? A case report. Neoplasia. 2016;18(4):223–8. doi: 10.1016/j.neo.2016.02.004.
  9. Govind SK, Zia A, Hennings-Yeomans PH, et al. ShatterProof: operational detection and quantification of chromothripsis. BMC Bioinform. 2014;15(1):78. doi: 10.1186/1471-2105-15-78.
  10. Korbel JO, Campbell PJ. Criteria for inference of chromothripsis in cancer genomes. Cell. 2013;152(6):1226–36. doi: 10.1016/j.cell.2013.02.023.
  11. Baca SC, Prandi D, Lawrence MS, et al. Punctuated evolution of prostate cancer genomes. Cell. 2013;153(3):666–77. doi: 10.1016/j.cell.2013.03.021.
  12. Kloosterman WP, Koster J, Molenaar JJ. Prevalence and clinical implications of chromothripsis in cancer genomes. Curr Opin Oncol. 2014;26(1):64–72. doi: 10.1097/CCO.0000000000000038.
  13. Nones K, Waddell N, Wayte N, et al. Genomic catastrophes frequently arise in esophageal adenocarcinoma and drive tumorigenesis. Nat Commun. 2014;5:5224. doi: 10.1038/ncomms6224.
  14. Maher CA, Wilson RK. Chromothripsis and human disease: piecing together the shattering process. Cell. 2012;148(1–2):29–32. doi: 10.1016/j.cell.2012.01.006.
  15. Alves TI, Hiltemann S, Hartjes T, et al. Gene fusions by chromothripsis of chromosome 5q in the VCaP prostate cancer cell line. Hum Genet. 2013;132:709–13. doi: 10.1007/s00439-013-1308-1.
  16. Nagel S, Mever C, Quantmeier H, et al. Chromothripsis in Hodgkin lymphoma. Genes Chromos Cancer. 2013;52(8):791–7. doi: 10.1002/gcc.22069.
  17. Salaverria I, Martın-Garcia D, Lopez C, et al. Detection of chromothripsis-like patterns with a custom array platform for chronic lymphocytic leukemia. Genes Chromos Cancer. 2015;54(11):668–80. doi: 10.1002/gcc.22277.
  18. Li Y, Schwaab C, Ryan SL, et al. Constitutional somatic rearrangement of chromosome 21 in acute lymphoblastic leukemia. Nature. 2014;508(7494):98–102. doi: 10.1038/nature13115.
  19. de Pagter MS, van Roosmalen MJ, Baas AF, et al. Chromothripsis in healthy individuals affects multiple protein-coding genes and can result in severe congenital abnormalities in offspring. Am J Hum Genet. 2015;96(4):651–6. doi: 10.1016/j.ajhg.2015.02.005.
  20. Bignell GR, Greenman CD, Davies H, et al. Signatures of mutation and selection in the cancer genome. Nature. 2010;463(7283):893–8. doi: 10.1038/nature08768.
  21. Adhvaryu SG, Vyas RC, Jani KH, et al. Complex translocation involving chromosomes #1, #9, and #22 in a patient with chronic myelogenous leukemia. Cancer Genet Cytogenet. 1988;32(2):277–80. doi: 10.1016/0165-4608(88)90291-9.
  22. Kadam PR, Nanjangud GJ, Advani SH. The occurrence of variant Ph translocations in chronic myeloid leukemia (CML): a report of six cases. Hematol Oncol. 1990;8(6):303–12. doi: 10.1002/hon.2900080602.
  23. Fitzgerald PH, Morris CM. Complex chromosomal translocations in the Philadelphia chromosome leukemias. Serial translocations or a concerted genomic rearrangement. Cancer Genet Cytogenet. 1991;57(2):143–51. doi: 10.1016/0165-4608(91)90145-k.
  24. Nishi Y, Akiyama K, Korf BR. Characterization of N-myc amplification in a human neuroblastoma cell line by clones isolated following the phenol emulsion reassociation technique and by hexagonal field gel electrophoresis. Mamm Genome. 1992;2(1):11–20. doi: 10.1007/bf00570436.
  25. Cowell JK. Double minutes and homogenously staining regions: gene amplification in mammalian cells. Annu Rev Genet. 1982;16(1):21–59. doi: 10.1146/annurev.ge.16.120182.000321.
  26. Cowell JK, Miller OJ. Occurrence and evolution of homogenously staining regions may be due to breakage-fusion-bridge cycles following telomere loss. Chromosoma. 1983;88(3):2016–21. doi: 10.1007/bf00285623.
  27. Shimizu N, Shindaki K, Kaneko-Sasaguri Y, et al. When, where and how the bridge breaks: anaphase bridge breakage plays a crucial role in gene amplification and HSR generation. Exp Cell Res. 2005;302(2):233–43. doi: 10.1016/j.yexcr.2004.09.001.
  28. Shimizu N. Extra chromosomal double minutes and chromosomal homogenously staining regions as probes for chromosome research. Cytogenet Genome Res. 2009;124(3–4):312–26. doi: 10.1159/000218135.
  29. Bignell GR, Santarius Th, Pole JCM, et al. Architectures of somatic genomic rearrangement in human cancer amplicons at sequence-level resolution. Genome Res. 2007;17(9):1296–303. doi: 10.1101/gr.6522707.
  30. Crasta K, Ganem NJ, Dagher R, et al. DNA breaks and chromosome pulverization from errors in mitosis. Nature. 2012;482(7383):53–8. doi: 10.1038/nature10802.
  31. Bassaganyas L, Bea S, Escaramı G, et al. Sporadic and reversible chromothripsis in chronic lymphocytic leukemia revealed by longitudinal genomic analysis. Leukemia. 2013;27(12):2376–9. doi: 10.1038/leu.2013.127.
  32. Zehentner BK, Hartmann L, Johnson KRT, et al. Array-based karyotyping in plasma cell neoplasia after plasma cell enrichment increases detection of genomic aberrations. Am J Clin Pathol. 2012;138(4):579–89. doi: 10.1309/ajcpkw31baimvgst.
  33. Jacoby MA, de Jesus Pizarro R, Shao J, et al. The DNA double-strand break response is abnormal in myeloblasts from patients with therapy-related acute myeloid leukemia. Leukemia. 2014;28(6):1242–51. doi: 10.1038/leu.2013.368.
  34. Zemanova Z, Michalova K, Buryova H, et al. Involvement of deleted chromosome 5 in complex chromosomal aberrations in newly diagnosed myelodysplastic syndromes (MDS) is correlated with extremely adverse prognosis. Leukemia Res. 2014;38(5):537–44. doi: 10.1016/j.leukres.2014.01.012.
  35. Agrawal A, Modi A, Alagusundaramoorthy SS, et al. Chromothripsis: Basis of a concurrent unusual association between myelodysplastic syndrome and primary ciliary dyskinesia. Case Rep Hematol. 2014:1–5. doi. 10.1155/2014/149878.
  36. Forment JV, Kaidi A, Jackson SP. Chromothripsis and cancer: causes, and consequences of chromosome shattering. Nat Rev Cancer. 2012;12(10):663–70. doi: 10.1038/nrc3352.
  37. Zhang CZ. Chromothripsis from DNA damage in micronuclei. Nature. 2015;522(7555):179–84. doi: 10.1038/nature14493.
  38. Kim TM, Ruibin Xi, Lovelace J, et al. Functional genomic analysis of chromosomal aberrations in a compendium of 8000 cancer genomes. Genome Res. 2013;23(2):217–27. doi: 10.1101/gr.140301.112.
  39. Malhotra A, Lindberg M, Faust GG, et al. Breakpoint profiling of 84 cancer genomes reveals numerous complex rearrangements spawned by homology-independent mechanisms. Genome Res. 2013;23(5):762–76. doi: 10.1101/gr.143677.112.
  40. Yang L, Luquette LJ, Gehlenborg N, et al. Diverse mechanisms of somatic structural variations in human cancer genomes. Cell. 2013;53(4):919–29. doi: 10.1016/j.cell.2013.04.010.
  41. Zack T, Luquette LJ, Gehlenborg N, et al. Pan-cancer patterns of somatic copy number alteration. Nat Genet. 2013;45(10):1134–40. doi: 10.1016/j.cell.2013.04.010.
  42. Cai H, Kumar N, Bagheri HC, et al. Chromothripsis-like patterns are recurring but heterogeneously distributed features in a survey of 22,347 cancer genome screens. BMC Genomics. 2014;15(1):82–95. doi: 10.1186/1471-2164-15-82.
  43. Przybytkowski E, Lenkiewicz E, Barrett MT, et al. Chromosome-breakage genomic instability and chromothripsis in breast cancer. BMC Genomics. 2014;15(1):579. doi: 10.1186/1471-2164-15-579.
  44. Kovtun IV, Murphy SJ, Johnson SH, et al. Chromosomal catastrophe is a frequent event in clinically insignificant prostate cancer. Oncotarget. 2015;6(30):29087–96. doi: 10.18632/oncotarget.4900.
  45. Morrison CD, Liu P, Woloszynska-Read A, et al. Whole-genome sequencing identifies genomic heterogeneity at a nucleotide and chromosomal level in bladder cancer. Proc Natl Acad Sci USA. 2014;111(6):E672–81. doi: 10.1073/pnas.1313580111.
  46. Fukuoka K, Fukushima S, Yamashita S, et al. Molecular classification of ependymomas in a Japanese cohort. Neuro-Oncology. 2015;17(Suppl 3):iii6. doi: 10.1093/neuonc/nov061.21.
  47. Jones DT, Jager N, Kool M, et al. Dissecting the genomic complexity underlying medulloblastoma. Nature. 2012;488(7409):100–5. doi: 10.1038/nature11284.
  48. Kool M, Jones DT, Jager N, et al. Genome sequencing of SHH medulloblastoma predicts genotype-related response to smoothened inhibition. Cancer Cell. 2014;25(3):393–405. doi: 10.1016/j.ccr.2014.02.004.
  49. Bayani J, Zielenska M, Pandita A, et al. Spectral karyotyping identifies recurrent complex rearrangements of chromosomes 8, 17, and 20 in osteosarcomas. Genes Chromos Cancer. 2003;36(1):7–16. doi: 10.1002/gcc.10132.
  50. Ivkov R, Bunz F. Pathways to chromothripsis. Cell Cycle. 2015;14(18):2886–90. doi: 10.1080/15384101.2015.1068483.
  51. Northcott PA, Shih DJH, Peacock J, et al. Subgroup-specific structural variation across 1,000 medulloblastoma genomes. Nature. 2012;488(7409):49–56. doi: 10.1038/nature11327.
  52. Cohen A, Sato M, Aldape K, et al. DNA copy number analysis of Grade II-III and Grade IV gliomas reveals differences in molecular ontogeny including chromothripsis associated with IDH mutation status. Acta Neuropathol Commun. 2015;3(1):34. doi: 10.1186/s40478-015-0213-3.
  53. Furgason JM, Koncar RF, Sharon K, et al. Whole genome sequence analysis links chromothripsis to EGFR, MDM2, MDM4, and CDK4 amplification in glioblastoma. Oncoscience. 2015;2(7):618–28. doi: 10.18632/oncoscience.178.
  54. Molenaar JJ, Koster J, Zwijnenburg DA, et al Sequencing of neuroblastoma identifies chromothripsis and defects in neuritogenesis genes. Nature. 2012;483(7391):589–93. doi: 10.1038/nature10910.
  55. Peifer M, Hertwig F, Roels F, et al. Telomerase activation by genomic rearrangements in high-risk neuroblastoma. Nature. 2015;526(7575):700–4. doi: 10.1038/nature14980.
  56. Natrajan R, Mackay A, Lambros MB, et al. A whole-genome massively parallel sequencing analysis of BRCA1 mutant oestrogen receptor-negative and -positive breast cancers. J Pathol. 2012;227(1):29–41. doi: 10.1002/path.4003.
  57. Nik-Zeinal B, Alexandrov LB, Wedge DC, et al. Mutational processes molding the genomes of 21 breast cancers. Cell. 2012;149(6):979–93. doi: 10.1016/j.cell.2012.04.024.
  58. Tang M-H, Dahlgren M, Brueffer C, et al. Remarkable similarities of chromosomal rearrangements between primary human breast cancers and matched distant metastases as revealed by whole-genome sequencing. Oncotarget. 2015;6(35):37169–84. doi: 10.18632/oncotarget.5951.
  59. Wu C, Wyatt AW, McPherson A, et al. Polygene fusion transcripts and chromothripsis in prostate cancer. Genes Chromos Cancer. 2012;51(12):1144–53. doi: 10.1002/gcc.21999.
  60. George J, Lim JS, Jang SJ, et al. Comprehensive genomic profiles of small cell lung cancer. Nature. 2015;524(7563):47–53. doi: 10.1038/nature14664.
  61. Govindan R, Ding L, Griffith M, et al. Genomic landscape of Non-small cell lung cancer in smokers and never-smokers. Cell. 2012;150(6):1121–34. doi: 10.1016/j.cell.2012.08.024.
  62. Campbell PJ, Yachida S, Mudie, et al. The patterns and dynamics of genomic instability in metastatic pancreatic cancer. Nature. 2010;467(7319):1109–13. doi: 10.1038/nature09460.
  63. Kloosterman WP, Hoogstraat M, Paling O, et al. Chromothripsis is a common mechanism driving genomic rearrangements in primary and metastatic colorectal cancer. Genome Biol. 2011;12(10):R103. doi: 10.1186/gb-2011-12-10-r103.
  64. Jiang Z, Jhunjhunwala S, Liu J, et al. The effects of hepatitis B virus integration inti the genomes of hepatocellular carcinoma patients. Genome Res. 2012;22(4):593–601. doi: 10.1101/gr.133926.111.
  65. McEvoy J, Nagahawatte P, Finkelstein D, et al. RB1 gene inactivation by chromothripsis in human retinoblastoma. Oncotarget. 2014;5(2):438–50. doi: 10.18632/oncotarget.1686.
  66. Kloosterman WP, Guryev V, van Roosmalen M, et al. Chromothripsis as a mechanism driving complex de novo structural rearrangements in the germline. Hum Mol Genet. 2011;20(10):1916–24. doi: 10.1093/hmg/ddr073.
  67. Chiang C, Jacobsen JC, Ernst C, et al. Complex reorganization and predominant non-homologous repair following chromosomal breakage in karyotypically balanced germline rearrangements and transgenic integration. Nat Genet. 2012;44(4):390–7. doi: 10.1038/ng.2202.
  68. Madan K. Balanced complex chromosome rearrangements: reproductive aspects. A review. Am J Med Genet. 2012;158A(4):947–63. doi: 10.1002/ajmg.a.35220.
  69. Pellestor F. Chromothripsis: how does such a catastrophic event impact human reproduction? Hum Reprod. 2014;29(3):388–93. doi: 10.1093/humrep/deu003.
  70. Weckselblatt B, Hermetz KE, Rudd MK. Unbalanced translocations arise from diverse mutational mechanisms including chromothripsis. Genome Res. 2015;25(7):937–47. doi: 10.1101/gr.191247.115.
  71. Macera MJ, Sobrino A, Levy B, et al. Prenatal diagnosis of chromothripsis, with nine breaks characterized by karyotyping, FISH, microarray and whole-genome sequencing. Prenat Diagn. 2015;35(3):299–301. doi: 10.1002/pd.4456.
  72. de Pagter MS, van Roosmalen MJ, Baas AF, et al. Chromothripsis in healthy individuals affects multiple protein-coding genes and can result in severe congenital abnormalities in offspring. Am J Hum Genet. 2015;96(4):651–6. doi: 10.1016/j.ajhg.2015.02.005.
  73. Kloosterman WP, Cuppen E. Chromothripsis in congenital disorders and cancer: similarities and differences. Curr Opin Cell Biol. 2013;25(3):341–8. doi: 10.1016/j.ceb.2013.02.008.
  74. Poot M, Haaf T. Mechanisms of origin, phenotypic effects and diagnostic implications of complex chromosome rearrangements. Mol Syndromol. 2015;6(3):110–34. doi: 10.1159/000438812.
  75. Liu P, Erez A, Nagamani SCS, et al. Chromosome catastrophes involve replication mechanisms generating complex genomic rearrangements. Cell. 2011;146(6):889–903. doi: 10.1016/j.cell.2011.07.042.
  76. Kloosterman WP, Tavakoli-Yaraki M, van Roosmalen MJ, et al. Constitutional chromothripsis rearrangements involve clustered double-stranded DNA breaks and nonhomologous repair mechanisms. Cell Rep. 2012;1(6):648–55. doi: 10.1016/j.celrep.2012.05.009.
  77. Hatch EM, Fischer AH, Deerinck ThJ, Hetzer MW. Catastrophic nuclear envelope collapse in cancer cell micronuclei. Cell. 2013;154(1):47–60. doi: 10.1016/j.cell.2013.06.007.
  78. Zhang CZ, Leibowitz ML, Pellman D. Chromothripsis and beyond: rapid genome evolution from complex chromosomal rearrangements. Genes Dev. 2013;27(23):2513–30. doi: 10.1101/gad.229559.113.
  79. Morishita M, Muramatsu T, Suto Y, et al. Chromothripsis-like chromosomal rearrangements induced by ionizing radiation using proton microbeam irradiation system. Oncotarget. 2015;7(9):10182–92. doi: 10.18632/oncotarget.7186.
  80. Holland AJ, Cleveland DW. Mechanisms and consequences of localized, complex chromosomal rearrangements in cancer and developmental diseases. Nat Med. 2012;18(11):1630–8. doi: 10.1038/nm.2988.
  81. Sorzano CO, Pascual-Montano A, Sanchez de Diego A, et al. Chromothripsis: breakage-fusion-bridge over and over again. Cell Cycle. 2013;12(13):2016–23. doi: 10.4161/cc.25266.
  82. Mardin BR, Drainas AP, Waszak SM, et al. A cell-based model system links chromothripsis with hyperploidy. Mol System Biol. 2015;11(9):828–41. doi: 10.15252/msb.20156505.
  83. Zainuddin N, Murray F, Kanduri M, et al. TP53 mutations are infrequent in newly diagnosed chronic lymphocytic leukemia. Leuk Res. 2011;35(2):272–4. doi: 10.1016/j.leukres.2010.08.023.
  84. Мамаев Н.Н., Горбунова А.В., Гиндина Т.Л. и др. Лейкозы и миелодиспластические синдромы с высокой экспрессией гена EVI1: теоретические и клинические аспекты. Клиническая онкогематология. 2012;5(4):361–4.
    [Mamaev NN, Gorbunova AV, Gindina TL, et al. Leukemias and myelodysplastic syndromes with high expression of EVI1: theoretical and clinical aspects. Klinicheskaya onkogematologiya. 2012;5(4):361–4. (In Russ)]
  85. Гиндина Т.Л., Мамаев Н.Н., Бархатов И.М. и др. Сложные повреждения хромосом у больных с рецидивами острых лейкозов после аллогенной трансплантации гемопоэтических стволовых клеток. Терапевтический архив. 2012;8:61–6.
    [Gindina TL, Mamaev NN, Barkhatov IM, et al. Complex chromosome damages in patients with recurrent acute leukemias after allogeneic hematopoietic stem cell transplantations. Terapevticheskii arkhiv. 2012;8:61–6. (In Russ)]
  86. Гиндина Т.Л., Мамаев Н.Н., Николаева Е.Н. и др. Анализ хромосомных нарушений у детей и подростков с посттрансплантационными рецидивами острых лейкозов. Клиническая онкогематология. 2015;8(4):420–7. doi: 10.21320/2500-2139-2015-8-4-420-427.
    [Gindina TL, Mamaev NN, Nikolaeva EN, et al. Analysis of Karyotype Aberrations in Children and Adolescents with Post-Transplantation Relapses of Acute Leukemia. Clinical oncohematology. 2015;8(4):420–7. doi: 10.21320/2500-2139-2015-8-4-420-427. (In Russ)]
  87. MacKinnon, Campbell. Cancer Genet. 2013;206:238–51.