Approach to molecular subgrouping of medulloblastomas: Comparison of NanoString nCounter assay versus combination of immunohistochemistry and fluorescence in-situ hybridization in resource constrained centres.
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.
['Kaur|Kavneet|K|', 'Jha|Prerana|P|', 'Pathak|Pankaj|P|', 'Suri|Vaishali|V|', 'Sharma|Mehar Chand|MC|', 'Garg|Ajay|A|', 'Suri|Ashish|A|', 'Sarkar|Chitra|C|http://orcid.org/0000-0002-4315-9316']
D000293:Adolescent D000328:Adult D000368:Aged D014408:Biomarkers, Tumor D002528:Cerebellar Neoplasms D002648:Child D002675:Child, Preschool D015331:Cohort Studies D003131:Combined Modality Therapy D019175:DNA Methylation D005260:Female D005500:Follow-Up Studies D006295:Health Resources D006801:Humans D007150:Immunohistochemistry D017404:In Situ Hybridization, Fluorescence D007223:Infant D008297:Male D008527:Medulloblastoma D008875:Middle Aged D036103:Nanotechnology D011379:Prognosis D020133:Reverse Transcriptase Polymerase Chain Reaction D015996:Survival Rate D055815:Young Adult
Molecular subgrouping Medulloblastoma NanoString Immunohistochemistry 850k DNA methylation array Fluorescence in-situ hybridisation