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
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
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
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
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
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
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)
43.
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
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
47.
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
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
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
66. What we know is a drop…
…..What we don’t know is
an ocean
Notas do Editor
Molecular landscape of gliomas is enormous
In 22 tumour samples with NGS they studied 22,661 protein coding genes…..5 out of 22 tumours expressed IDH mutations all at the same codon…mainly in secondary GBM.
Formally encouraging their use in the evaluation of these neoplasms.
The crystal structure of the human cytosolic NADP(+)–
dependent IDH is shown in ribbon format (PDBID: 1T0L) (44). The active cleft of IDH1
consists of a NADP-binding site and the isocitrate-metal ion-binding site. The alphacarboxylate
oxygen and the hydroxyl group of isocitrate chelate the Ca2+ ion. NADP is colored
in orange, isocitrate in purple and Ca2+ in blue. The Arg132 residue, displayed in yellow, forms
hydrophilic interactions, shown in red, with the alpha-carboxylate of isocitrate
Arginine at 132(R132) residue (polar AA) is completely conserved in all bacterial and eukaryotes and is localized to the substrate binding site, where it forms hydrophilic interactions with the isocitrate (negatively charged)
Heterozygous point mutation, guanine adenine at nucleotide position 395 of the IDH1 transcript (
Alteration in the active site of enzyme
Loose their normal catalytic activity of producing α-keto glutatrate and NADPH
Mutant IDH neomorphic activity
α-ketogluatatrate reduced to 2hydroxyglutatrate
NADPH oxidised to NADP+
2) NEGATIVE: Weak diffuse background staining and staining of macrophages
“secondary structures of Scherer” (17). These
patterns include tumor cell spread alongwhite matter tracts, perivascular tumor cell spread, subpial growth and perineuronal satellitosis.
mIDH1R132H highlighted these growth patterns in many cases
may be caused by a cross-reaction with a perivascular
extracellular matrix protein or a collagen subtype
Somatic , MONOALLELIC MUTATIONS AT A SINGLE RESIDUE AT SUBSTRATE BINDING POCKET. ARGININE 132 (R132)
first discovered through a study assessing patients with the x-linked mental retardation (MR) syndrome (ATRX syndrome) presenting with α-thalassemia, severe psy-chomotor impairments, urogenital abnormalities, and patterns of characteristic facial dysmorphism
Co-deletion of 1p/19q : unbalanced translocation t(1;19)(q10;p10) after which only one copy of the short arm of chromosome 1 and one copy of the long arm of chromosome 19 remain and der(1;19) (q10;p10) is produced
1p19q co-deletion in gliomas occurs through an unbalanced whole-arm translocation between chromosomes 1 and 19 [t(1;19)(q10;p10)].
It is hypothesised that translocation creates two derivative chromosomes, der(1;19)(p10;q10) [+] and der(1;19)(q10;p10) [C], and is followed
by loss of the derivative chromosome containing 1p and 19q [+]
Polysomy of 1p, 19q or both is also noted in a subset of oligodendrogliomas and has been associated with a poor prognosis, independent of deletion status
FISH, fluorescent in situ hybridization; LOH, loss of heterozygosity; CGH, comparative genomic hybridization;
WES, whole exome sequencing.
Determination of molecular markers will identify patients with a more favourable prognosis or better chance of response to alkylating agent chemotherapy
The median survival was 3.8 years for patients with mutated IDH1, as compared to 1.1 years for patients with wild-type IDH1.
the timing of genetic changes in gliomas, IDH1 mutations were found to occur before the acquisition of TP53 mutations, loss of 1p/19q, and copy number alterations in phosphatase and tensin homolog and EGFR, implying that they are very early genetic events
ongoing expression of IDH1 mutations is required to maintain growth in glioma cells and that pharmacological inhibition of the mutation can lead to tumor growth suppression and induce differentiation
Strong nuclear staining for K27M-mutant H3 is
present in tumour cells but not in the vasculature
