—  SPECIALTY CONFERENCE  —

Neuropathology

Case 1 - Diffuse Astrocytoma, WHO Grade II, IDH1-R132H Mutation- Positive, 1p/19q Intact

Gregory Fuller, M.D. Anderson Cancer Center, Houston, TX





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Clinical History
A 35-year-old man presented with a 6-month history of recurrent coordination difficulties, most noticeable when engaged in racket sports. MR imaging revealed a lesion of the left frontal lobe with the imaging characteristics illustrated in the Figures. Imaging studies included MR Spectroscopy, with the results illustrated. Biopsy of the lesion was subsequently performed.


Case 1 - Slide 1
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Case 1 - Figure 1
Axial T1-weighted MR image without contrast. The small lesion in the left frontal lobe is difficult to identify on T1-weighted sequences in which it appears as a subtle area of hypodensity immediately subjacent to the cortex.
Case 1 - Figure 2
Axial T1-weighted MR image with contrast. The lesion does not show enhancement following administration of contrast agent (gadolinium), but remains as a subtle area of hypointensity.
Case 1 - Figure 3
Axial T2 MR image. The T2-weighted sequence, which shows fluid, including normal cerebrospinal fluid and abnormal brain edema, as a bright (hyperintense) signal, renders the lesion more apparent.
Case 1 - Figure 4
Axial T2-FLAIR MR image. T2- Fluid Attenuation Inversion Recovery sequences (T2-FLAIR) are used to suppress the normal ventricular and subarachnoid space cerebrospinal fluid hyperintense signal of T2- weighted images, thus rendering fluid-containing lesions, such as multiple sclerosis plaques, many tumors and cerebral edema, more clearly visible, as seen in the present lesion.

Case 1 - Figure 5
MR Spectroscopy Voxel Map. Magnetic resonance spectroscopy (MRS) has many applications, including the identification of the most cellular, potentially highest grade areas of diffuse gliomas that are otherwise indistinguishable by standard MR imaging techniques. MRS can be used to assess a single area of a lesion (single voxel MRS) or multiple areas of a lesion and surrounding brain tissue (multivoxel MRS) as seen here.
Case 1 - Figure 6
MR Spectroscopy. The MRS profile of this lesion does not identify any areas of high-grade tumor, and is thus non-contributory in helping distinguish low- grade diffuse glioma from gliosis.
Case 1 - Figure 7
Biopsy whole mount H&E. A limited open biopsy of the lesion was obtained.
Case 1 - Figure 8
A representative low-power field shows mildly hypercellular white matter. (H&E, x100)

Case 1 - Figure 9
Occasional pleomorphic nuclei are noted. (H&E, x100)
Case 1 - Figure 10
Pleomorphic nucleus seen at higher power. (H&E, x200)
Case 1 - Figure 11
Cerebral cortical tissue is also present in the biopsy and shows perineuronal satellitosis. (H&E, x100)
Case 1 - Figure 12
Higher power view reveals occasional pleomorphic cells in the mildly hypercellular cortical tissue. (H&E, x200)

Case 1 - Figure 13
Immunohistochemistry performed with a mutant specific anti-IDH1-R132H antibody reveals large numbers of infiltrating cells that are positive for the mutation, confirming the diagnosis of diffuse glioma. (IDH1-R132H IHC with hematoxylin counterstain, x100)
Case 1 - Figure 14
IDH1-R132H IHC. Higher power view shows labeled infiltrating glioma cells (cytoplasmic and nuclear staining) as well as non-reactive vascular endothelial cells that serve as an internal negative control.
Case 1 - Figure 15
Immunohistochemistry performed for p53 protein reveals large numbers of labeled cells (strong nuclear staining), favoring a diagnosis of diffuse astrocytoma. (p53 IHC with hematoxylin counterstain, x40)
Case 1 - Figure 16
p53 IHC. The cortex also displays p53- positive tumor cells. (p53 IHC, x200)

Case 1 - Figure 17
Phosphohistone H3 (pHH3) IHC. Immunohistochemistry performed with an antibody directed against the sensitive mitotic figure marker pHH3 identifies a single mitosis in a biopsy tissue section. (pHH3 IHC with hematoxylin counterstain, x200)
Case 1 - Figure 18
Ki-67 antigen (MIB1) IHC. Automated quantitation yielded a Ki-67 antigen (MIB1) labeling index of 4.6%. (MIB1 IHC with hematoxylin counterstain, x100)
Case 1 - Figure 19
Ki-67 antigen (MIB1) IHC. MIB1-positive nuclei are identified in the tumor cell population infiltrating the cortex, including one labeled mitotic figure. (MIB1 IHC with hematoxylin counterstain, x400)

Pathological/Microscopic Findings and any Immunohistochemical or Other Studies:
H&E-stained sections showed mildly hypercellular white matter and cerebral cortical gray matter. Scattered pleomorphic nuclei were present. Immunohistochemistry performed with an antibody specific for isocitrate dehydrogenase 1 (IDH1) R132H mutation revealed the presence of large numbers of positively-labeled cells, confirming a diagnosis of diffuse glioma. In addition, a p53 immunostain showed strong nuclear reactivity in a very high percentage of infiltrating tumor cells in the white matter and cortex. Mitoses were not easily identified on H&E-stained sections and immunohistochemistry using an antibody directed against the M-phase marker phosphohistone-H3 revealed only a single mitotic figure. Automated quantitation yielded a Ki-67 antigen (MIB1 antibody) labeling index of 4.6%.

Differential Diagnoses:
  • Gliosis Diffuse Astrocytoma (WHO grade II)

  • Anaplastic astrocytoma (WHO grade III)

  • Oligodendroglioma (WHO grade II)

  • Anaplastic oligodendroglioma (WHO grade III)

  • Mixed oligoastrocytoma (WHO grade II)

  • Anaplastic mixed oligoastrocytoma (WHO grade III)

Final Diagnosis:
Diffuse astrocytoma, WHO grade II, IDH1-R132H mutation- positive, 1p/19q intact.

Case Discussion:
Gliosis versus Glioma The differential diagnosis in this case centers on diffuse glioma versus reactive gliosis, a classical and commonly encountered diagnostic dilemma in surgical neuropathology. A number of "soft" morphological features have been used to help separate these two entities in the setting of a "mildly hypercellular" brain biopsy: nuclear morphology and pleomorphism; distribution of cells (irregular in glioma, even distribution in gliosis); the presence of microcysts (relatively more common in glioma compared to gliosis); the presence of Scherer secondary structures in areas of cortical invasion (limited perineuronal satellitosis in the deeper layers of normal cortex, more pronounced perineuronal and perivascular satellitosis in all cortical layers in glioma, plus subpial accumulation and spread in glioma). Adjunctive immunomarkers may be of assistance in some cases: the presence of strong diffuse nuclear positivity for p53 protein favors glioma (astrocytoma specifically); elevated Ki-67 antigen (MIB-1 antibody) labeling index generally reflects increased cell proliferation and favors glioma; phosphohistone-H3 IHC can be used to aid identification of mitotic figures, an increase of which would favor glioma. All of these features, however, are "soft", that is, they are not absolute or pathognomonic but rather a question of degree, which limits their utility. Clearly, a more sensitive and specific diagnostic marker has been badly needed. Enter IDH1 mutation. Isocitrate dehydrogenase 1 (IDH1) Mutation in Diffuse Gliomas The isocitrate dehydrogenases (IDH) comprise a family of three enzymes that catalyze the oxidative decarboxylation of isocitrate to alpha-ketoglutarate. Three human IDH enzymes have been identified: IDH1 at 2q33, which encodes cytosolic NADP-dependent IDH; IDH2, which is an NADP- dependent mitochondrial enzyme, and IDH3, which is also a mitochondrial enzyme but is NAD-dependent [1]. IDH1 and IDH2 are of specific interest in neuro-oncology. A specific mutation causing replacement of the wild-type arginine at position 132 (R132) in IDH1 has recently been demonstrated to be present in a majority (70-85%) of WHO grade II and grade III diffuse astrocytomas, oligodendrogliomas and mixed oligoastrocytomas, and also in secondary glioblastomas (which arise through anaplastic progression from lower- grade diffuse astrocytoma), but not in primary ("de novo") glioblastomas, which account for the majority of glioblastomas [2, 3, 4, 5, 6, 7]. Of the three conserved arginine residues in IDH1, the overwhelming majority of mutations involve R132, with only very rare exceptions involving the arginine at codon 100 (R100); which has also been reported in AML), and none so far reported for the arginine at codon 109 (R109) [8]. Mutations at the homologous residue to R132 in IDH2 (Arg 172) have also been reported, but are far less common; IDH3 mutations have not yet been reported. R132 arginine is most commonly replaced by histidine (about 95% of cases; designated R132H). The R132H mutation is not found in reactive gliosis [9] or other gliomas, such as pilocytic astrocytoma and ependymoma, and is very rare in systemic malignancies in general, with the notable exception of acute myeloid leukemia, in which R132H is present in 6-7% of cases [10, 11, 12, 13]. The availability of mutation-specific antibodies that are robust in formalin- fixed paraffin-embedded (FFPE) tissue has greatly facilitated research and diagnostic applications of R132H in brain tumors [14, 15, 16]. Diagnostic testing for IDH1 and IDH2 mutations in FFPE tissue using a polymerase chain reaction (PCR)-based assay is also possible [17]. Initial studies of the pathobiology of R132H IDH1 have shown that arginine 132 (R132) plays a critical role in pre-binding conformational change in IDH1 protein, and the R132H mutation hinders this transition, resulting in impaired catalytic activity [18]. Functional sequelae described include decreased alpha-ketoglutarate production, activation of the HIF-1 pathway, consumption of NADPH, and a 100-fold increase in the production of 2- hydroxyglutarate [19, 20, 21]. The latter conveys a "gain of function" aspect to the mutation. Multiple hypotheses have been advanced to explain the link between mutation of a basic metabolic enzyme and oncogenesis [22, 23]. The "oncogenic metabolite" is a rather novel oncogene mechanism and metabolic adaptation as an important aspect of oncogenesis is gaining momentum. The concept has generated considerable excitement, with a plethora of recent opinion articles on the mutant IDH "oncometabolite" [24, 25, 26, 27, 28]. The diagnostic applications of IDH1 mutation are many. As mentioned, IDH1-R132H immunohistochemistry (IHC) can reliably separate gliosis from diffuse glioma in the 80% or so of cases in which the tumor is a grade II or III diffuse glioma or a secondary glioblastoma (sGBM). Although it will not distinguish gliosis from the infiltrating edge of a primary glioblastoma (pGBM), as neither harbors an IDH mutation, this is generally not as common a diagnostic issue as for lower-grade diffuse gliomas, especially if the pathologist does due diligence in being aware of the preoperative imaging studies, in which the vast majority of glioblastomas are contrast- enhancing (in contrast to low-grade diffuse glioma and gliosis). IDH1-R132H IHC also distinguishes grade II/III gliomas and sGBM from other glial tumors, such as pilocytic astrocytoma, ependymoma, etc. And IDH1-R132H IHC also has significant power to distinguish diffuse oligodendroglioma and oligoastrocytoma from other "fried egg cell" brain tumors that mimic oligodendroglial morphology, such as central and extraventricular neurocytoma, cerebellar liponeurocytoma, clear cell ependymoma, clear cell meningioma, dysembryoplastic neuroepithelial tumor (DNET), pilocytic astrocytoma with oligodendroglia-like component, and primary glioblastoma with oligodendroglioma component [29]. IDH1 mutation is common in diffuse gliomas that exhibit traditional histologic features of astrocytic, oligodendroglial and mixed oligoastrocytic lineage, and in all molecular subgroups of these tumors, such as those that exhibit TP53 mutation and those with 1p/19q codeletion. Several important correlations with significant clinical implications have been made. For example, high-grade astrocytomas that do not exhibit IDH1 mutation behave more aggressively and exhibit a worse prognosis compared to high-grade astrocytomas that do posses the mutation, regardless of whether the tumors are histologically classified as anaplastic astrocytoma or glioblastoma [30]. IDH mutations also predict longer survival and response to chemotherapy in low-grade gliomas [31]. Correlations like this have further advanced the notion of practical molecular classification of diffuse gliomas and may provide a long-awaited solution to the subjective and irreproducible morphologic feature-based classification of the diffuse gliomas, as particularly well exemplified by the diagnosis of mixed oligoastrocytoma, in the near future [32, 33, 34, 35, 36]. Other Molecular Markers for Gliomas A number of other markers have proven useful for diagnosis, prognosis and therapeutic decision making in gliomas. Following is a brief survey of those of current high interest: whole arm codeletion of 1p/19q, which is an event initiated by a translocation involving chromosomes 1 and 19, is a characteristic molecular signature that closely correlates with classical oligodendroglial morphologic features and with a favorable response to therapy compared to diffuse gliomas that lack this alteration; EGFR gene amplification is associated with a significant subset of glioblastomas; tandem duplication at 7q34 that results in a BRAF fusion gene is a recently recognized alteration found in a majority of pilocytic astrocytomas; MGMT promoter methylation predicts increased sensitivity to alkylating agent (temozolomide) chemotherapy in glioblastoma; mutations in MSH6, one of the major mismatch repair genes, arise in glioblastoma during temozolomide chemotherapy and mediate resistance to that agent. Molecular Markers for Other Primary Brain Tumors In addition to gliomas, other primary brain tumors also exhibit molecular alterations that are increasingly helpful for diagnosis and/or prognosis. Deletion or mutation of theINI1/hSNF5/SMARCB1 gene is highly characteristic of atypical teratoid/rhabdoid tumors (ATRT) in the central nervous system and of rhabdoid tumors of the kidney. Germline mutation of the INI1 gene is associated with Rhabdoid Tumor Predisposition Syndrome. MYCN amplification and overexpression is the most common genetic abnormality detected in medulloblastoma. OCT 4 expression is a highly sensitive marker for CNS (and systemic) germinoma and embryonal carcinoma. Back to the Differential Diagnosis of Diffuse Glioma Versus Reactive Gliosis While the discussion to this point has focused on morphologic and molecular features, the importance of clinical features and common sense cannot be overemphasized. Although certainly not pathognomonic, the preoperative imaging features of diffuse gliomas tend to be highly characteristic. Lower grade diffuse gliomas, which constitute the primary entity in the differential diagnosis with gliosis, generally display a diffuse hyperintensity on T2-weighted and T2-FLAIR sequences, with extension of the abnormal hyperintensity into the overlying cerebral cortex in cases of glioma infiltration of the cortex (in contrast, vasogenic edema, which is often accompanied by reactive gliosis, involves the white matter and not the cortex). In addition, in contrast to gliosis, diffuse gliomas frequently exert some degree of mass effect and resultant distortion of the surrounding brain, including a degree of midline shift and/or compression of the ventricular system. One advanced clinical imaging technique that is increasingly employed to identify areas of denser, higher grade diffuse glioma for diagnostic and biopsy planning purposes is magnetic resonance spectroscopy (MR spectroscopy; MRS). Although the MRS is often normal in low-grade diffuse gliomas of low tumor cellularity, as in the present case, it is nevertheless a very helpful technique that in many instances is reassuring of the presence of infiltrating glioma and the surgical pathologist would benefit from at least a passing familiarity with the significance. As with all clinical scenarios involving tissue biopsy, there is no substitute for good communication between the pathologist, radiologist, oncologist and surgeon. In the present case, the preoperative imaging studies were highly suggestive of a diffuse glioma. H&E stains of the biopsied lesion were also suspicious for diffuse glioma based on the presence of scattered nuclei that were more pleomorphic than generally encountered in reactive gliosis, but reactive gliosis remained in the differential diagnosis. The demonstration of the presence of the IDH1 R132H mutation confirmed the presence of infiltrating glioma. Strong nuclear positivity for p53 protein in a very high percentage of cells also strongly favored diffuse glioma, and specifically favored astrocytoma over oligodendroglioma. The Ki-67 antigen labeling index of 4.6% also exceeded what is typically encountered in gliosis. Finally, fluorescence in situ hybridization (FISH) analysis of the tumor did not detect any evidence of deletions of chromosomal arms 1p or 19q; thus the hallmark genetic signature of oligodendroglioma, 1p/19q codeletion, was absent, further favoring astrocytoma. With no mitoses found on H&E-stained sections and only a single mitosis seen on the MIB1 immunostain and the very sensitive pHH3 immunostain, together with the relatively low Ki-67 antigen labeling index, a diagnosis of diffuse astrocytoma, WHO grade II, was rendered. This diagnosis is consistent with the relatively young patient age and preoperative imaging characteristics (diffuse T2 and T2- FLAIR signal hyperintensity with absence of contrast enhancement and no evidence of high-grade tumor on MR spectroscopy).

Conclusion(s):
1. The recent discovery of the widespread incidence of the isocitrate dehydrogenase 1 (IDH1) mutation R132H in grade II and III diffuse gliomas and secondary glioblastomas has provided a striking example of the advances possible through application of next generatin sequencing (NGS) technology.

2. The availability of IDH1-R132H mutation-specific antibodies has resulted in numerous significant clinical applications and research advances in a remarkably short time.

3. IDH1-R132H immunohistochemistry constitutes the latest practical tool for use in the molecular diagnosis and classification of diffuse gliomas.

4. Molecular classification will ultimately provide a much needed objective, clinically relevant solution to the long- standing issue of subjectivity and lack of reproducibility in diffuse glioma classification.

References:
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  2. The Cancer Genome Atlas Research Network. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature 2008; 455:1061-1068.

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