Utility of Gliosense, the multigene NGS assay to guide WHO compliant molecular classification of CNS tumors

BACKGROUND AND RATIONALE

Background:

Sub-types and Incidences

The incidence of central nervous system (CNS) tumors in India ranges from 5 to 10 per 100,000 population with an increasing trend and accounts for 2% of malignancies. Although relatively rare,

the associated morbidity and mortality and the significant proportion of affected young and middle-aged individuals has a major bearing on the death-adjusted life years compared to other

malignancies. CNS tumors encompass a very broad spectrum with regards to age, location, histology, and clinical outcomes. There are over 130 different types of tumours which can occur in the

brain, other parts of the CNS or intracranial region. Astrocytomas (38.7%) are the most common primary tumors with the majority being high-grade gliomas (59.5%). Results published from a

tertiary care center in South India reporting 15 years’ experience involving 1043 patients indicated that the five most frequent tumors were astrocytoma (47.3%), MB (11.4%), craniopharyngioma

(9.7%), ependymal tumors (4.8%), and nerve sheath tumors (4.1%). The median age at presentation of glial tumors in India was reported to be at least a decade earlier than reported in the Western

population, which could be partially explained by the lower life expectancy and a higher proportion of the younger population in India. Over 75% of patients of glioblastoma die within 18 months.

The prognosis has not changed significantly since the 1970s. The median age of pediatric tumors such as brainstem glioma, medulloblastoma (MB), and supratentorial primitive neuroectodermal

tumors (PNET) is comparable with Western population. A multi-institutional effort involving seven tertiary care hospitals reported the epidemiological profile of 3936 pediatric tumor patients. The

most common tumor is astrocytoma (34.7%) followed by Medulloblastoma (22.4%) and craniopharyngioma. Most of the astrocytic tumors were reported to be low grade commonly pilocytic

astrocytoma and subependymal giant cell astrocytoma. This was found to be comparable to data from Western countries or other Asian countries.

Glioblastomas

Glioblastomas have traditionally been divided into primary and secondary: According to WHO 2016 classification, they are now classified as IDH-mutant and IDH-wild type. Primary glioblastomas

are those that arise de novo, without a pre-existing lower grade diffuse astrocytoma. They account for 90% of all glioblastomas and are more aggressive than secondary glioblastomas and they

tend to occur in older individuals.  Primary glioblastomas are almost invariably IDH wild-type. They tend to have amplification of EGFR and over-expression of MDM2, PTEN mutation and/or loss of

heterozygosity of chromosome 10p.

Secondary glioblastomas, in contrast, are those which arise from a pre-existing lower grade diffuse astrocytoma. They are relatively uncommon, only accounting for approximately 10% of all

glioblastomas. These tumours tend to be less aggressive than primary glioblastomas and they tend to occur in younger patients. Interestingly, and of uncertain significance, they have predilection

for the frontal lobes. Characteristically, and unlike primary tumors, secondary glioblastomas tend to be IDH mutant (positive), a mutation shared by over 80% of grade II and III astrocytomas.

Secondary glioblastomas also demonstrate p53 mutations, amplification of PDGF-A, loss of heterozygosity of chromosomes 10q and 17p, loss of 19q and increased telomerase activity and hTERT

expression. In addition to mutations of IDH1, low-grade astrocytomas usually have TP53 mutation while oligodendrogliomas typically show 1p/19q loss. The concurrent deletion of chromosomes

1p and 19q, a result of an unbalanced translocation, is associated with increased chemosensitivity and a better prognosis.

Study Rationale

Distinct Advantages of GLioSenseâ„¢ Assay

WHO guidelines issued in 2016 for the first time integrated molecular parameters in addition to the histologic features for classification of the CNS tumors1. Series of recommendations have been

proposed by the C-IMPACT group2,3,4,5 for the integration of genomic findings in key genes such as IDH1, K27 position of histone coding genes and others in the revised taxonomy of CNS tumors.

This evolution in the understanding of the heterogeneous molecular class of CNS tumors further emphasizes the increased role of molecular investigations on the brain tumor specimens that may

reduce ‘Not Otherwise Specified’ (NOS) results in diagnostic reports. Currently routine diagnosis is made based on clinical examination, imaging and light microscopy aided

by immunohistochemistry (IHC). Molecular diagnosis enables better stratification of patients with light microscopically similar looking tumors, into different groups which has prognostic and

predictive implications. Moreover, certain entities are only molecularly defined. Complementing routine histopathology diagnosis with molecular parameters allows better diagnosis and prediction

of outcome. It also allows clinicians to evaluate the potential options for targeted therapy and provides more specific prognostic information. The flexibility of these platform allows them to be

modified once novel scientific information becomes available or changes are made in the diagnostic criteria.

The various molecular techniques include FISH, PCR, RT-PCR, NGS and DNA methylation. NGS is becoming widely accepted as a cost-effective method for evaluating multiple genes

simultaneously. NGS is primarily aiming at mutations and allows simultaneous assessment of copy number alterations or of fusion genes. However, the technique and the panels needs to be well

validated before it is introduced into routine clinical diagnostics.

GLIOSENSETM – A multigene assay

GlioSenseâ„¢ is a multigene next generation sequencing (NGS) assay that analyses over 87 genomic loci encompassing point mutations, indels, copy number alterations and large chromosomal

gain/loss and gene fusions.  These genomic alterations are part of molecular classification scheme of the WHO classification of tumors of the Central Nervous System1. GlioSenseâ„¢ analyses

FPPE tissue specimens with >10 % neoplastic content of the specimens to provide molecular classification. This assessment of the tumor expected to provide more meaningful clinical decision

for the patient, as compared to conventional analytics. The GlioSenseâ„¢ consists of the following:

SNVs, CNVs and Indels: BRAF, H3F3A, TP53, 1p-19q co-del, IDH1, IDH2, TERT, NF1, HIST1H3B, HIST1H3C, MET, SHH subtype, WNT subtype, 6q Loss, APC, ATRX, CDKN2A, CDKN2B, EGFR, MYC, NOTCH1, PTEN, 10q Loss, 7q Gain, CIC, C19MC, PTCH1, FUBP1, PDGFRA, PIK3CA, PIK3R1, RB1, TSC1, TSC2, NF2, ACAN, ACTR3B, ACVR1, AHNAK2, ARID1A, BCOR, CCR5, CDK6, CDKN2C, CHEK2, COL6A3, DDX3X, DNMT3A, F5, FGFR1, FKBP9, GABRA6, HIF1A, HMCN1, IFITM3, IGF1R, IRS4, KDM5A, KEL, KRAS, LRP1B, LZTR1, MDM2, MDM4, MUC5B, NOTCH2, NRAS, PIK3C3, POLE, PRKCH, PTPN11, SMARCB1, SOX11, STAG2, TCF12, THAP3, ZMYM3, ZNF429

Fusion Drivers: RELA, BRAF, MYB, NTRK1/2/3, FGFR1/3

RNA Exon Variant: EGFRvlll

WHO Categories of select tumors (WHO classification of CNS tumors, 2016)

Diffuse astrocytic and oligodendroglial tumors

Diffuse astrocytoma, IDH-mt; IDH-wt; NOS

Anaplastic astrocytoma, IDH-mt; IDH-wt; NOS

Glioblastoma, IDH-mt; IDH-wt; NOS, Giant cell, Epitheloid, Gliosarcoma

Diffuse midline glioma, H3 K27 M-mt

Oligodendroglioma, IDH-mt, 1p/19q co-deleted; NOS

Anaplastic Oligodendroglioma, IDH-mt, 1p/19q co-deleted; NOS

Oligoastrocytoma

Anaplastic oligoastrocytoma

Other astrocytic tumors

Pilocytic astrocytoma, Pilomyxoid astrocytoma

Subependymal giant cell astrocytoma

Pleomorphic xanthoastrocytoma

Anaplastic Pleomorphic xanthoastrocytoma

Ependymal tumors

Subependymoma

Myxopapillary

Classic: Papillary, clear cell, tanycytic

Anaplastic ependymoma

Supratentorial ependymoma RELA fusion positive

Other gliomas

Chordoid glioma

Angiocentric glioma

Astroblastoma

Embryonal tumors

Medulloblastoma genetically defined; histologically defined; NOS

Embryonal tumors with multi layered rosettes, C 19 MC altered; NOS

Tumors of the cranial and paraspinal nerves

Meningiomas

STUDY DESIGN

Study type: Observational.

Observational model: Cohort.

Time perspective: Prospective.

Estimated enrollment: 300± 10 cases.

Start Date: After clearance from Institutional ethics committee/IRB

Estimated Primary Completion Date: Six months from study initiation (the time lines can be extended in case of insufficiency of specimens / other contingencies to be evaluated by PI and sponsor).

Estimated Study Completion Date: Two months from completion of study cohort

Number of study groups/arms:  One.

Specimens: FFPE specimens of the patients diagnosed with CNS tumors listed above.

Study Procedures: Collection of Formalin Fixed Paraffin Embedded blocks from study site and process GLIOSENSETM – A multigene assay at Datar Cancer Genetics Pvt Ltd