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Approach to molecular subgrouping of medulloblastomas: Comparison of NanoString nCounter assay versus combination of immunohistochemistry and fluorescence in-situ hybridization in resource constrained centres

  • Kavneet Kaur
  • Prerana Jha
  • Pankaj Pathak
  • Vaishali Suri
  • Mehar Chand Sharma
  • Ajay Garg
  • Ashish Suri
  • Chitra SarkarEmail author
Laboratory Investigation
  • 10 Downloads

Abstract

Introduction

Molecular classification of medulloblastomas (MB) is prognostically and therapeutically relevant and helps in better risk-stratification. Translation of this subgrouping to routine practice still remains a challenge. The most pathologist accessible techniques for molecular subgrouping include immunohistochemistry (IHC), fluorescent in-situ hybridization (FISH) and NanoString.

Objectives

(1) Molecular subgrouping of MBs by IHC and FISH, and NanoString assay (2) To compare their efficacy and cost for applicability in resource constrained centers.

Methods

Ninety-five cases of MB with adequate tissue were included. Molecular subgrouping was performed by IHC for β-catenin, GAB1 and YAP1; FISH for MYC amplification, and sequencing for CTNNB1, and by NanoString Assay on the same set of MBs. A subset of cases was subjected to 850k DNA methylation array.

Results

IHC + FISH classified MBs into 15.8% WNT, 16.8% SHH, and 67.4% non-WNT/non-SHH subgroups; with MYC amplification identified in 20.3% cases of non-WNT/non-SHH. NanoString successfully classified 91.6% MBs into 25.3% WNT, 17.2% SHH, 23% Group 3 and 34.5% Group 4. However, NanoString assay failure was seen in eight cases, all of which were > 8-years-old formalin-fixed paraffin-embedded tissue blocks. Concordant subgroup assignment was noted in 88.5% cases, while subgroup switching was seen in 11.5% cases. Both methods showed prognostic correlation. Methylation profiling performed on discordant cases revealed 1 out of 4 extra WNT identified by NanoString to be WNT, others aligned with IHC subgroups; extra SHH by NanoString turned out to be SHH by methylation.

Conclusions

Both IHC supplemented by FISH and NanoString are robust methods for molecular subgrouping, albeit with few disadvantages. IHC cannot differentiate between Groups 3 and 4, while NanoString cannot classify older-archived tumors, and is not available at most centres. Thus, both the methods complement each other and can be used in concert for high confidence allotment of molecular subgroups in clinical practice.

Keywords

Medulloblastoma Molecular subgrouping NanoString Immunohistochemistry Fluorescence in-situ hybridisation 850k DNA methylation array 

Notes

Acknowledgements

The authors would like to thank the technical staff of Neuropathology laboratory for assistance in immunohistochemistry and fluorescence in-situ hybridization, and Dr. Nirupama Trehanpati, Additional Professor, Institute of Liver and Biliary Sciences for providing us with the NanoString platform. The authors are grateful to Dr. Kalaivani for helping with the statistical analysis. Kavneet Kaur is a Senior Research Associate (# 8936/A) under Scientists’ Pool Scheme, Council of Scientific and Industrial Research (CSIR).

Funding

This work was supported financially by the J. C. Bose fellowship awarded to the corresponding author Dr. Chitra Sarkar. The authors declare that the funding source did not play a role in the study design, collection, analysis or interpretation of data, writing of the report, or in the decision to submit the article for publication.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

The study has been duly approved by the Institute Ethics committee and has been performed in accordance with the ethical standards laid down in 1964 Declaration of Helsinki and its later amendments.

Informed consent

Informed consent waived off for retrospective part of the study, however, informed consent was obtained from all the prospective participants.

Supplementary material

11060_2019_3187_MOESM1_ESM.docx (41 kb)
Supplementary file1 (DOCX 40 kb)

References

  1. 1.
    Northcott PA, Jones DT, Kool M, Robinson GW, Gilbertson RJ, Cho YJ, Pomeroy SL, Korshunov A, Lichter P, Taylor MD, Pfister SM (2012) Medulloblastomics: the end of the beginning. Nat Rev Cancer 12:818–834.  https://doi.org/10.1038/nrc3410 CrossRefGoogle Scholar
  2. 2.
    Northcott PA, Korshunov A, Witt H, Hielscher T, Eberhart CG, Mack S, Bouffet E, Clifford SC, Hawkins CE, French P, Rutka JT, Pfister S, Taylor MD (2011) Medulloblastoma comprises four distinct molecular variants. J Clin Oncol 29:1408–1414.  https://doi.org/10.1200/JCO.2009.27.4324 CrossRefGoogle Scholar
  3. 3.
    Taylor MD, Northcott PA, Korshunov A, Remke M, Cho YJ, Clifford SC, Eberhart CG, Parsons DW, Rutkowski S, Gajjar A, Ellison DW, Lichter P, Gilbertson RJ, Pomeroy SL, Kool M, Pfister SM (2012) Molecular subgroups of medulloblastoma: the current consensus. Acta Neuropathol 123:465–472.  https://doi.org/10.1007/s00401-011-0922-z CrossRefGoogle Scholar
  4. 4.
    Ellison DW, Eberhart CG, Pietsch T (2016) Pfister S (2016) Embryonal tumors. In: Louis DN, Ohgaki H, Wiestler OD, Cavenee WK (eds) WHO Classification of tumors of the central nervous system, Revised, 4th edn. IARC, Lyon, pp 184–200Google Scholar
  5. 5.
    Kool M, Korshunov A, Remke M, Jones DT, Schlanstein M, Northcott PA, Cho YJ, Koster J, Schouten-van Meeteren A, van Vuurden D, Clifford SC, Pietsch T, von Bueren AO, Rutkowski S, McCabe M, Collins VP, Bäcklund ML, Haberler C, Bourdeaut F, Delattre O, Doz F, Ellison DW, Gilbertson RJ, Pomeroy SL, Taylor MD, Lichter P, Pfister SM (2012) Molecular subgroups of medulloblastoma: an international meta-analysis of transcriptome, genetic aberrations, and clinical data of WNT, SHH, Group 3, and Group 4 medulloblastomas. Acta Neuropathol 123:473–484.  https://doi.org/10.1007/s00401-012-0958-8 CrossRefGoogle Scholar
  6. 6.
    Ramaswamy V, Remke M, Bouffet E, Bailey S, Clifford SC, Doz F, Kool M, Dufour C, Vassal G, Milde T, Witt O, von Hoff K, Pietsch T, Northcott PA, Gajjar A, Robinson GW, Padovani L, André N, Massimino M, Pizer B, Packer R, Rutkowski S, Pfister SM, Taylor MD, Pomeroy SL (2016) Risk stratification of childhood medulloblastoma in the molecular era: the current consensus. Acta Neuropathol 131:821–831.  https://doi.org/10.1007/s00401-016-1569-6 CrossRefGoogle Scholar
  7. 7.
    Ramaswamy V, Remke M, Bouffet E, Faria CC, Perreault S, Cho YJ, Shih DJ, Luu B, Dubuc AM, Northcott PA, Schüller U, Gururangan S, McLendon R, Bigner D, Fouladi M, Ligon KL, Pomeroy SL, Dunn S, Triscott J, Jabado N, Fontebasso A, Jones DT, Kool M, Karajannis MA, Gardner SL, Zagzag D, Nunes S, Pimentel J, Mora J, Lipp E, Walter AW, Ryzhova M, Zheludkova O, Kumirova E, Alshami J, Croul SE, Rutka JT, Hawkins C, Tabori U, Codispoti KE, Packer RJ, Pfister SM, Korshunov A, Taylor MD (2013) Recurrence patterns across medulloblastoma subgroups: an integrated clinical and molecular analysis. Lancet Oncol 14:1200–1207.  https://doi.org/10.1016/S1470-2045(13)70449-2 CrossRefGoogle Scholar
  8. 8.
    Rudin CM, Hann CL, Laterra J, Yauch RL, Callahan CA, Fu L, Holcomb T, Stinson J, Gould SE, Coleman B, LoRusso PM, Von Hoff DD, de Sauvage FJ, Low JA (2009) Treatment of medulloblastoma with hedgehog pathway inhibitor GDC-0449. N Engl J Med 361:1173–1178.  https://doi.org/10.1056/NEJMoa0902903 CrossRefGoogle Scholar
  9. 9.
    Robinson GW, Orr BA, Wu G, Gururangan S, Lin T, Qaddoumi I, Packer RJ, Goldman S, Prados MD, Desjardins A, Chintagumpala M, Takebe N, Kaste SC, Rusch M, Allen SJ, Onar-Thomas A, Stewart CF, Fouladi M, Boyett JM, Gilbertson RJ, Curran T, Ellison DW, Gajjar A (2015) Vismodegib exerts targeted efficacy against recurrent sonic hedgehog-subgroup medulloblastoma: results from phase ii pediatric brain tumor consortium studies PBTC-025B and PBTC-032. J Clin Oncol 33:2646–2654.  https://doi.org/10.1200/JCO.2014.60.1591 CrossRefGoogle Scholar
  10. 10.
    Kool M, Jones DT, Jäger N, Northcott PA, Pugh TJ, Hovestadt V, Piro RM, Esparza LA, Markant SL, Remke M, Milde T, Bourdeaut F, Ryzhova M, Sturm D, Pfaff E, Stark S, Hutter S, Seker-Cin H, Johann P, Bender S, Schmidt C, Rausch T, Shih D, Reimand J, Sieber L, Wittmann A, Linke L, Witt H, Weber UD, Zapatka M, König R, Beroukhim R, Bergthold G, van Sluis P, Volckmann R, Koster J, Versteeg R, Schmidt S, Wolf S, Lawerenz C, Bartholomae CC, von Kalle C, Unterberg A, Herold-Mende C, Hofer S, Kulozik AE, von Deimling A, Scheurlen W, Felsberg J, Reifenberger G, Hasselblatt M, Crawford JR, Grant GA, Jabado N, Perry A, Cowdrey C, Croul S, Zadeh G, Korbel JO, Doz F, Delattre O, Bader GD, McCabe MG, Collins VP, Kieran MW, Cho YJ, Pomeroy SL, Witt O, Brors B, Taylor MD, Schüller U, Korshunov A, Eils R, Wechsler-Reya RJ, Lichter P, Pfister SM (2014) ICGC PedBrain Tumor Project. Genome sequencing of SHH medulloblastoma predicts genotype-related response to smoothened inhibition. Cancer Cell 25:393–405.  https://doi.org/10.1016/j.ccr.2014.02.004 CrossRefGoogle Scholar
  11. 11.
    Bautista F, Fioravantti V, de Rojas T, Carceller F, Madero L, Lassaletta A, Moreno L (2017) Medulloblastoma in children and adolescents: a systematic review of contemporary phase I and II clinical trials and biology update. Cancer Med 6:2606–2624.  https://doi.org/10.1002/cam4.1171 CrossRefGoogle Scholar
  12. 12.
    Gottardo NG, Hansford JR, McGlade JP, Alvaro F, Ashley DM, Bailey S, Baker DL, Bourdeaut F, Cho YJ, Clay M, Clifford SC, Cohn RJ, Cole CH, Dallas PB, Downie P, Doz F, Ellison DW, Endersby R, Fisher PG, Hassall T, Heath JA, Hii HL, Jones DT, Junckerstorff R, Kellie S, Kool M, Kotecha RS, Lichter P, Laughton SJ, Lee S, McCowage G, Northcott PA, Olson JM, Packer RJ, Pfister SM, Pietsch T, Pizer B, Pomeroy SL, Remke M, Robinson GW, Rutkowski S, Schoep T, Shelat AA, Stewart CF, Sullivan M, Taylor MD, Wainwright B, Walwyn T, Weiss WA, Williamson D, Gajjar A (2014) Medulloblastoma down under 2013: a report from the third annual meeting of the international medulloblastoma working group. Acta Neuropathol 127:189–201.  https://doi.org/10.1007/s00401-013-1213-7 CrossRefGoogle Scholar
  13. 13.
    Ellison DW, Dalton J, Kocak M, Nicholson SL, Fraga C, Neale G, Kenney AM, Brat DJ, Perry A, Yong WH, Taylor RE, Bailey S, Clifford SC, Gilbertson RJ (2011) Medulloblastoma: clinicopathological correlates of SHH, WNT, and non-SHH/WNT molecular subgroups. Acta Neuropathol 121:381–396.  https://doi.org/10.1007/s00401-011-0800-8 CrossRefGoogle Scholar
  14. 14.
    Kaur K, Kakkar A, Kumar A, Mallick S, Julka PK, Gupta D, Suri A, Suri V, Sharma MC, Sarkar C (2016) Integrating molecular subclassification of medulloblastomas into routine clinical practice: a simplified approach. Brain Pathol 26:334–343.  https://doi.org/10.1111/bpa.12293 CrossRefGoogle Scholar
  15. 15.
    Northcott PA, Shih DJ, Remke M, Cho YJ, Kool M, Hawkins C, Eberhart CG, Dubuc A, Guettouche T, Cardentey Y, Bouffet E, Pomeroy SL, Marra M, Malkin D, Rutka JT, Korshunov A, Pfister S, Taylor MD (2012) Rapid, reliable, and reproducible molecular sub-grouping of clinical medulloblastoma samples. Acta Neuropathol 123:615–626.  https://doi.org/10.1007/s00401-011-0899-7 CrossRefGoogle Scholar
  16. 16.
    Capper D, Jones DTW, Sill M, Hovestadt V, Schrimpf D, Sturm D, Koelsche C, Sahm F, Chavez L, Reuss DE, Kratz A, Wefers AK, Huang K, Pajtler KW, Schweizer L, Stichel D, Olar A, Engel NW, Lindenberg K, Harter PN, Braczynski AK, Plate KH, Dohmen H, Garvalov BK, Coras R, Hölsken A, Hewer E, Bewerunge-Hudler M, Schick M, Fischer R, Beschorner R, Schittenhelm J, Staszewski O, Wani K, Varlet P, Pages M, Temming P, Lohmann D, Selt F, Witt H, Milde T, Witt O, Aronica E, Giangaspero F, Rushing E, Scheurlen W, Geisenberger C, Rodriguez FJ, Becker A, Preusser M, Haberler C, Bjerkvig R, Cryan J, Farrell M, Deckert M, Hench J, Frank S, Serrano J, Kannan K, Tsirigos A, Brück W, Hofer S, Brehmer S, Seiz-Rosenhagen M, Hänggi D, Hans V, Rozsnoki S, Hansford JR, Kohlhof P, Kristensen BW, Lechner M, Lopes B, Mawrin C, Ketter R, Kulozik A, Khatib Z, Heppner F, Koch A, Jouvet A, Keohane C, Mühleisen H, Mueller W, Pohl U, Prinz M, Benner A, Zapatka M, Gottardo NG, Driever PH, Kramm CM, Müller HL, Rutkowski S, von Hoff K, Frühwald MC, Gnekow A, Fleischhack G, Tippelt S, Calaminus G, Monoranu CM, Perry A, Jones C, Jacques TS, Radlwimmer B, Gessi M, Pietsch T, Schramm J, Schackert G, Westphal M, Reifenberger G, Wesseling P, Weller M, Collins VP, Blümcke I, Bendszus M, Debus J, Huang A, Jabado N, Northcott PA, Paulus W, Gajjar A, Robinson GW, Taylor MD, Jaunmuktane Z, Ryzhova M, Platten M, Unterberg A, Wick W, Karajannis MA, Mittelbronn M, Acker T, Hartmann C, Aldape K, Schüller U, Buslei R, Lichter P, Kool M, Herold-Mende C, Ellison DW, Hasselblatt M, Snuderl M, Brandner S, Korshunov A, von Deimling A, Pfister SM (2018) DNA methylation-based classification of central nervous system tumours. Nature 555:469–474.  https://doi.org/10.1038/nature26000 CrossRefGoogle Scholar
  17. 17.
    Louis DN, Perry A, Burger P, Ellison DW, Reifenberger G, von Deimling A, Aldape K, Brat D, Collins VP, Eberhart C, Figarella-Branger D, Fuller GN, Giangaspero F, Giannini C, Hawkins C, Kleihues P, Korshunov A, Kros JM, Beatriz Lopes M, Ng HK, Ohgaki H, Paulus W, Pietsch T, Rosenblum M, Rushing E, Soylemezoglu F, Wiestler O, Wesseling P (2014) International Society Of Neuropathology-Haarlem consensus guidelines for nervous system tumor classification and grading. Brain Pathol 24:429–435.  https://doi.org/10.1111/bpa.12171 CrossRefGoogle Scholar
  18. 18.
    Korshunov A, Chavez L, Northcott PA, Sharma T, Ryzhova M, Jones DTW, von Deimling A, Pfister SM, Kool M (2017) DNA-methylation profiling discloses significant advantages over NanoString method for molecular classification of medulloblastoma. Acta Neuropathol 134:965–967.  https://doi.org/10.1007/s00401-017-1776-9 CrossRefGoogle Scholar
  19. 19.
    Goschzik T, Zur Mühlen A, Kristiansen G, Haberler C, Stefanits H, Friedrich C, von Hoff K, Rutkowski S, Pfister SM, Pietsch T (2015) Molecular stratification of medulloblastoma: comparison of histological and genetic methods to detect Wnt activated tumours. Neuropathol Appl Neurobiol 41:135–144.  https://doi.org/10.1111/nan.12161 CrossRefGoogle Scholar
  20. 20.
    Cavalli FMG, Remke M, Rampasek L, Peacock J, Shih DJH, Luu B, Garzia L, Torchia J, Nor C, Morrissy AS, Agnihotri S, Thompson YY, Kuzan-Fischer CM, Farooq H, Isaev K, Daniels C, Cho BK, Kim SK, Wang KC, Lee JY, Grajkowska WA, Perek-Polnik M, Vasiljevic A, Faure-Conter C, Jouvet A, Giannini C, Nageswara Rao AA, Li KKW, Ng HK, Eberhart CG, Pollack IF, Hamilton RL, Gillespie GY, Olson JM, Leary S, Weiss WA, Lach B, Chambless LB, Thompson RC, Cooper MK, Vibhakar R, Hauser P, van Veelen MC, Kros JM, French PJ, Ra YS, Kumabe T, López-Aguilar E, Zitterbart K, Sterba J, Finocchiaro G, Massimino M, Van Meir EG, Osuka S, Shofuda T, Klekner A, Zollo M, Leonard JR, Rubin JB, Jabado N, Albrecht S, Mora J, Van Meter TE, Jung S, Moore AS, Hallahan AR, Chan JA, Tirapelli DPC, Carlotti CG, Fouladi M, Pimentel J, Faria CC, Saad AG, Massimi L, Liau LM, Wheeler H, Nakamura H, Elbabaa SK, Perezpeña-Diazconti M, Ponce Chico, de León F, Robinson S, Zapotocky M, Lassaletta A, Huang A, Hawkins CE, Tabori U, Bouffet E, Bartels U, Dirks PB, Rutka JT, Bader GD, Reimand J, Goldenberg A, Ramaswamy V, Taylor MD (2017) Intertumoral Heterogeneity within Medulloblastoma Subgroups. Cancer Cell 31:737.e6–754.e6.  https://doi.org/10.1016/j.ccell.2017.05.005 CrossRefGoogle Scholar
  21. 21.
    Tabori U, Baskin B, Shago M, Alon N, Taylor MD, Ray PN, Bouffet E, Malkin D, Hawkins C (2010) Universal poor survival in children with medulloblastoma harboring somatic TP53 mutations. J Clin Oncol 28:1345–1350.  https://doi.org/10.1200/JCO.2009.23.5952 CrossRefGoogle Scholar
  22. 22.
    Pietsch T, Haberler C (2016) Update on the integrated histopathological and genetic classification of medulloblastoma: a practical diagnostic guideline. Clin Neuropathol 35:344–352CrossRefGoogle Scholar

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Authors and Affiliations

  1. 1.Department of PathologyAll India Institute of Medical SciencesNew DelhiIndia
  2. 2.Department of NeurosurgeryAll India Institute of Medical SciencesNew DelhiIndia
  3. 3.Department of NeuroradiologyAll India Institute of Medical SciencesNew DelhiIndia

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