2016 AND 2021 MOLECULAR CLASSIFICATION OF DIFFUSE GLIOMAS, diagnostic, prognostic and predictive role of IDH, ATRX and p53

1. MOLECULAR MARKERS: diffuse gliomas
Dr Kanwalpreet Kaur
Department of
Oncopathology

2. DIFFUSEGLIOMAS
• Major Mutations- IDH
ATRX
TP53
1p/19q codeletion
• Diagnostic role
• Prognostic role
• Predictive role

3. Discoveryof IDHmutations in brain tumours

5. From2008  WHO 2016
Oligodendroglioma, NOS
Oligodendroglioma, IDH-mutant and
1p/19q-codeleted
Anaplastic oligodendroglioma, NOS
Anaplastic oligodendroglioma, IDH-
mutant and 1p/19q-codeleted
Oligoastrocytoma, NOS
Anaplastic oligoastrocytoma, NOS
Anaplastic astrocytoma, NOS
Anaplastic astrocytoma, IDH-mutant
Anaplastic astrocytoma, IDH-wildtype
Diffuse astrocytoma, NOS
Diffuse astrocytoma, IDH-mutant
Diffuse astrocytoma, IDH-wildtype
Gemistocytic astrocytoma, IDH-
mutant
Glioblastoma, NOS
Glioblastoma, IDH-mutant
Glioblastoma, IDH-wildtype
Epithelioid glioblastoma
Giant cell glioblastoma
Gliosarcoma
Diffuse midline glioma, H3
K27M-mutant
INTEGRATED ‘’PHENOTYPIC AND GENOTYPIC PARAMETERS’’ : A FORMAL
CLASSIFICATION SYSTEM

6. IDH (Isocitrate dehydrogenase)
5 isocitrate
dehydrogenase genes
IDH1
IDH2
IDH3A
IDH3B
IDH3G.
3 isoenzymes
IDH1
IDH2
IDH3

7. IDH 1 IDH2 IDH3
Cytosol
Peroxisomes
Mitochondria Mitochondria
NADP+ NADP+ NAD+
Homodimer Homodimer Hetrooctamer (2 alpha, 1
beta, 1 gamma subunit)
Reversible Reversible Non-reversible
Prevent oxidative damage
by generating NADPH
Prevent oxidative damage
by generating NADPH
Catalytic role in TCA cycle
(citric acid cycle)
IDH 1 gene: 2q33 IDH 2 gene: 15q26.1 IDH3A : 15q25.1-25.2
IDH3B : 20p13
IDH3G : Xq28

8. Active site of IDH1 and IDH2

9. Mutant IDH
G395A  replacement of an arginine with a histidine at amino acid
residue 132 of the protein (R132H)

10. AML: R140Q (most common ) or R172K IDH2
INTRAHEPATIC CHOLANGIOCARCINOMA: IDH2-R172K and Kras-G12D
IDH MUTATIONS IN GLIOMAS
ENCHONDROMA and CENTRAL CHONDROSARCOMA : IDH 1 R132C

12. CpG island methylator phenotype (G-CIMP)

13. IDH1/2MutationalAnalysis
• Direct Sanger sequencing
• Pyrosequencing
• Polymerase chain reaction (PCR), allele-specific hybridization
• Real-time PCR
• High-throughput next-generation sequencing
Immunohistochemistry for mIDH1 R132H : ANTIBODY H09
1) POSITIVE : Strong cytoplasmic immunoreaction with weak nuclear;
endothelial cells, perivascular lymphocytes, residual glial cells should be
negative
2) NEGATIVE: Weak diffuse background staining

14. Strong cytoplasmic and weak
nuclear staining : positive
Negative

15. mIDH R132H IHC
• Normal brain doesn’t show staining for mIDH1 R132H IHC.
• Granular staining in the neurons can be seen due to non
specific binding to lipofuscin
• Macrophages can show strong cytoplasmic granular staining
even in IDH wt tumours
• Sensitivity of 94% and a specificity of 100%
• WHY NUCLEAR STAINING ?
Whether mutated IDH1 is truly localized in the nucleus in vivo or
whether the soluble protein penetrates the nucleus after
surgical removal (sometimes referred to as antigen diffusion) is
not known

16. Double immunohistochemistry of glial fibrillary acidic protein (red)
and mIDH1R132H (brown) of infiltration zone of anaplastic
oligodendroglioma showing that reactive astrocytes (red) are not bound
by mIDH1R132H

17. ASTROCYTOMA GRADE II
OLIGODENDROGLIOMA GRADE II
GEMISTOCYCTIC ASTROCYTOMA GRADE II
ASTROCYTOMA GRADE III

18. Identification of loose subpial spread of
tumor cells distant from tumor bulk
Perineuronal satellitosis
Infiltration zone
TUMOUR
NORMAL
BRAIN
perivascular accumulation of tumour
cells

19. Case of pilocytic astrocytoma showing positive macrophages.
Tumor cells show no binding of mIDH1R132H

20. Majority of meningiomas showed nonspecific staining of
fibers usually close to blood vessels or around small groups
of tumor cells

21. IDH POSITIVE IHC IDH POSITIVE IHC
DIRECT SEQUENCING : ABLE TO
DETECT MUTATION
DIRECT SEQUENCING : NOT ABLE TO
DETECT MUTATION DUE TO LOW
BURDEN OF TUMOUR CELLS

22. At GCRI : by q PCR
• IDH1 R132H ( most common)
• IDH1 R132C(2nd most common)
• IDH1 R132S
• IDH1 R132G
• IDH1 R132L
• IDH1 R132V
• IDH1 R100Q
• IDH2 R172K ( most common)
• IDH2 R172M
• IDH2 R172S
• IDH2 R172G

23. AlphaThalassemia/MentalRetardationSyndromeX-
linked(ATRX)
• Chromatin regulator protein
• LOCATION: Xq21.1
• LOSS OF FUNCTION: alternate lengthening of telomeres
maintaining telomere length Cancer cells can divide infinitely

24. • Most frequent in grade II (67%) and grade III (73%)
astrocytomas and secondary GBMs (57%)
• Mutually exclusive with 1p/19q codeletion : Marker of
astrocytic lineage
• Strongly associated with IDH mutant gliomas
• All diffuse gliomas with IDH and ATRX mutations also harbor
TP53 mutation

25. METHODS OF DETECTION
• Direct Sanger sequencing
• Pyrosequencing
• PCR, allele-specific hybridization
• Real-time PCR
• High-throughput next-generation sequencing
ATRX IHC: clone CL0537
LOSS OF NUCLEAR EXPRESSION >90% tumour nuclei :mutated
RETAINED NUCLEAR EXPRESSION: not mutated

26. ATRX IHC
• Nuclear ATRX loss scored : if tumor cell nuclei were unstained (>90%
cut off)
• Nuclei of non-neoplastic cells such as endothelia, microglia,
lymphocytes and reactive astrocytes  strongly positive and serve as
positive internal control
ATRX LOSS: mutant ATRX ATRX RETAINED, ATRX wild type

27. TP53
• 60% to 80% of infiltrative astrocytomas, anaplastic
astrocytomas and secondary GBMs
• Rare in oligodendrogliomas
• Marker for astrocytic differentiation
STRONG ASSOCIATION BETWEEN TP53 MUTATIONS AND IDH MUTATIONS
80% of IDH mutant anaplastic astrocytomas and GBMs
18% of IDH wt anaplastic astrocytomas and GBMs

28. Methods of TP53 mutation analysis
• Direct Sanger sequencing
• Pyrosequencing
• PCR, allele-specific hybridization
• Real-time PCR
• High-throughput next-generation sequencing

29. • Marker for astrocytic differentiation
• Immunostain reacts with both the normal and mutant forms
of p53
Wild-type p53
Unstable protein
Short half-life
no/ low detection by IHC
Mutant p53
Degrade more slowly
Accumulate within nucleus of
tumour cells creating a
Stable target for IHC
P53 IHC cloneD07
Use of IHC to reflect TP53 mutation is not entirely specific and is only a surrogate
marker

30. IHC Staining for p53 was graded as
• 0 if no cells stained;
• 1+ if 0–10% stained;
• 2+ if 10–50% stained
• 3+ if > 50% stained.
• Most studies: GRADE 3: positive
The sensitivity and specificity with ≥10% p53 immunohistochemistry to
predict TP53 functional mutation status were 100% and 42%, respectively

31. One case of silent mutation demonstrated 5% p53 IHC staining
Over the last 25 years, studies have shown concordance rates between
p53 IHC and TP53 mutation status ranging from 55–89% in grade I–IV
gliomas

32. IN ATRX NEGATIVE GBM, IF IDH R132H IS NEGATIVE  IDH1/2 MUTATION ANALYSIS
IDH R132 IHC has sensitivity 63.19%, specificity 85.09% in distinguishing Primary
GBM from secondary GBM

33. 1p/19q codeletion
• 60% to 80% of oligodendroglial neoplasms

34. • Testing Method
• In situ hybridization
• Cytogenomic microarray (CMA)
• Loss of heterozygosity
• Next generation sequencing

35. 1R2G PATTERN

36. IDH wild type gliomas don’t have 1p/19q codeletion
So 1p/9q codeletion is unnecessary if glioma is not IDH
mutant

37. TERT promotor mutations
• 5p15.33
• Telomerase reverse transcriptase (TERT)  maintains
telomere length
• Core region of the TERT promoter  increased telomerase
activity  infinite cell divisions in cancer cells
ASSOCIATION WITH
• IDH mutation +1p/19q codeletion : OLIGODENDROGLIOMA
• IDH wt ATRX wt: PRIMARY GLIOBLASTOMA

38. Diagnostic role
• Astrocytoma= IDH+/ATRX loss / mutated TP53
• Oligodendroglioma = IDH mutant/ATRX retained/1p/19q co-
deleted
• Primary GBM: no IDH mutation/ EGFR/ TERT/PTEN/ TP53
• Secondary GBM: IDH+/ATRX loss / mutated TP53

39. On histology, identify a glial tumour
IHC: IDH1 R132H, ATRX
IDH neg, ATRX loss
IDH mutations by PCR or sanger sequencing,
pt< 55years
IDH mutant, ATRX loss
IDH neg, ATRX retained
IDH mutant, ATRX retained,
with no clear oligodendroglial
morphology
ASTROCYTOMA, IDH-mutant
1p/19q codeletion FISH
OLIGODENDROGLIOMA, IDH mt and
1p/19q codeleted
IHC: m IDH1 R132H, ATRX loss
ASTROCYTOMA, IDH-mutant/
SECONDARY GBM

40. • Small tumor samples : IDH1 IHC identifies single infiltrating
tumor cells
• Differentiating : IDH helps to separate
• A) gliosis from low grade astrocytoma
• B) A II from PA I
• C) A III from pGBM,
• D) pGBM from sGBM,
• If there is doubt in grade I and grade II glioma, IDH presence
indicates we are dealing with atleast grade II astrocytoma
• No longer oligoastrocytoma

41. LAYERED REPORT
• Layer 1: Integrated diagnosis
• Layer 2: Histological classification (i.e. cellularity, mitosis,
necrosis, vascular endothelial proliferations, variants)
• Layer 3: Histologic (WHO) grade ( based on morphology)
• Layer 4: Biomarker studies (IDH, ATRX, p53, 1p/19q codel,
EGFR)

42. • Integrated diagnosis: Diffuse Astrocytoma IDH mt, WHO grade II
• Histological diagnosis: Diffuse astrocytoma, WHO (histological) grade II
• Molecular information:
• IDH: positive (R132H immunohistochemistry; consistent with mutant type)
• ATRX: nuclear expression loss (immunohistochemistry; consistent with
mutant type)
• p53: positive, >60% (immunohistochemistry; consistent with mutant type)

44. Prognostic role
Maximal safe resection on surgery is the mainstay of treatment
• Age >40 years
• Higher tumour grade
• Tumour crossing midline
• Presence of neurologic deficit before resection;
• Tumor ≥6 cm
• Patients with up to 2 of these are considered low risk  OBSERVATION for
grade II
• Patients with 3 or more are high risk  fractionated external-beam radiation
therapy (EBRT) or adjuvant chemotherapy ( for grade III and IV)
UNFAVOURABLE
PROGNOSTIC
FACTORS

45. IDH mt
1p/19q co-deleted:
BEST PROGNOSIS
IDH mt
Without 1p/19q
codeletion
IDH wt:
WORST PROGNOSIS

46. IMPROVED PFS
LONGER TIME FOR TREATMENT FAILURE
EXTENDED OVERALL SURVIVAL
In each of three treatment arms:
1) Radiotherapy
2) Radiotherapy with PCV( Procarbazine,
lomustine and vincristine)
3) RT plus temozolomide

48. TERT as worse prognosis
IDH wt + TERT
astrocytoma >
IDH wt without
TERT
IDH mt+ 1p/19q
codel + No TERT > TERT mutation
oligodendroglioma

49. PREDECTIVE role:
• Absence of IDH mutation suggests predictive role for MGMT
promoter methylation status
• Prolongation of survival with early chemotherapy in 1p/19-co-
deleted oligodendrogliomas

50. PREDECTIVErole:
MGMT promotor methylationstatus
• MGMT (O6-methylguanine-DNA methyltransferase) is a DNA
repair enzyme
For glioblastoma, combined modality therapy with temozolomide
(TMZ) and radiotherapy remains the standard of care
Elderly GBM:
MGMT-methylated→TMZ
MGMTunmethylated→ RT

51. • MEHOD: Methylation specific PCR
• MGMT determination by immunohistochemistry lacks
standardization, reproducibility and, most importantly, correlation
with clinical outcome
MGMT POSITIVE MGMT NEGATIVE
MGMT IHC : NOT RECOMMENDED;
COUNT 400 CELLS AT 40x
>30% TUMOUR NUCLEI STAINING  positive
< 30% tumour nuclei staining negative methylated MGMT likely to respond to
chemotherapy

52. MGMT methylation studies: Done in all grade III and IV astrocytoma
GLIOBLASTOMA PROGNOSTIC : MGMT methylation - prolonged PFS and OS in
GBM patients on adjuvant therapy
PREDECTIVE: MGMT methylation predicts better response to
alkylating agents
GRADE III
ASTROCYTOMA
PROGNOSTIC : MGMT methylation – favorable prognostic
marker for adjuvant therapy
PREDECTIVE: MGMT methylation no predictive role

56. Histone H3 mutations in gliomas

57. In humans, different histone H3 variants.
Histone protein Encoding genes
H3.3 Expressed
constitutively (cell
cycle, as well as in
quiescent cells)
H3F3A and H3F3B
H3.1 S-phase only HIST1H3B
HIST1H3C
H3.2 S-phase only HIST2H3C
H3.5, H3.X, H3.Y Tissue specific

58. • Histone mutations are seen in pediatric diffuse midline gliomas
• Pons, thalamus, spinal cord
• May also occur in adults
• Most common histone mutation in brain: H3K27M ( lysine to
methionine substitution in H3F3A gene, 70% cases or HIST1H3B) 
inhibits trimethylation of H3.3 histone (decrease in H3K27me3) ;
Diffuse Intrinsic Pontine Glioma (DIPG)
• H3F3A G34V mutations - cortical gliomas

59. • Sequencing for H3F3A and HIST1H3B
• H3F3A K27M antibody : intense nuclear staining in more than 80%
of cells was taken as positive
• Concordance between immunohistochemistry and sequencing >
95% cases
• H3 K27M mutated tumors show loss of H3K27me3 staining
(normal cells intact expression is denoted by the presence of
diffuse nuclear staining )  not a specific finding
• IDH1 R132H : negative

60. MUTANT PROTEIN
LOSS OF TRIMETHYLATION

62. Diagnostic role
• Small biopsies of midline lesion: presence is strong indicator of
infiltrative glioma

63. • Brain, pons, biopsy:
• Integrated diagnosis: Diffuse midline glioma, H3 K27M mutant, WHO
grade IV of IV (see comment)
• Histological diagnosis: Diffuse astrocytoma, WHO (histological) grade II of
IV
• Molecular information:
• IDH: negative (R132H immunohistochemistry; consistent with wild type)
• ATRX: nuclear expression retained (immunohistochemistry; consistent with
wild type)
• p53: negative (immunohistochemistry; consistent with wild type)
• H3K27M: positive (immunohistochemistry; consistent with mutant)
• Comment: The specimen consists of core biopsy specimens of white matter with
moderately atypical infiltrating astrocytic tumor cells. There is no evidence of vascular
proliferation or necrosis. No mitoses are seen. Although the histologic grade is that of a
grade II astrocytoma, the positivity for H3K27M is consistent with a diagnosis of diffuse
midline glioma, H3 K27M mutant, which are considered grade IV lesions due to their
historically aggressive clinical behavior.

64. Prognostic role
• Presence of H3 K27M mutation in an infiltrative glioma arising
in the midline is sufficient for a grade IV designation, even in
the absence of necrosis or microvascular proliferation
• K27M : Do not have MGMT promoter methylation
• K27M: adverse prognostic marker in children and adults
• G34 doesn’t have prognostic significance

65. NCCN guidelines,2020
• IDH mutations
• 1p/19q codeletion
• MGMT mutations
• ATRX: strongly recommended, not required
• TERT: recommended. Not required
• H3F3A : recommended in appropriate clinical context
Essential workup

66. What we know is a drop…
…..What we don’t know is
an ocean