**8. Observations on personal OA series**

Our analysis started from the established principles that 1p/19q co-deletion is typical of oligodendrogliomas, but that it occurs in a low percentage only of tumors with a classical oligodendroglial phenotype ("honeycomb" appearance and "chicken wire" vessels). Our 40 OA cases had been initially diagnosed according to stringent histologic criteria, so that their number is slightly lower compared to others' series. The current revision has been carried out on the basis of 1p/19q chromosomal status, as detected by MLPA, IDH1/2 mutation status by IHC and sequencing analysis, and ATRX expression by IHC. The key points were: 1) the retained expression of ATRX by tumor oligodendroglial nuclei and by reactive astrocyte, microglia/macrophage, endothelial cell, lymphocyte nuclei, and the ATRX protein loss in tumor astrocytic nuclei; 2) IDH1/2 mutations, present in low grade gliomas with a higher frequency in oligodendrogliomas than astrocytomas [52, 79] (**Figure 3A**–**D**); 3) therefore, although they reveal the tumor nature of cells, IDH1/2 mutations can lack both in oligoden‐ droglial and astrocytic tumor cells. As, beside reactive astrocytes, ATRX is expressed in microglia/macrophage nuclei, Iba-1, CD68, CD16, and CD163 IHC has been performed on parallel serial tumor sections, as well as the double ATRX/GFAP, ATRX/Iba-1, and ATRX/ IDHR132H immunostaining. Microglia/macrophages can reach in the tumor sections a frequen‐ cy of 50–130 *x* 40 HPF (personal data); ATRX expressing nuclei could be, therefore, referred to tumor oligodendrocytes only if their number encompasses the number of microglia/macro‐ phage nuclei. The occurrence of microglia/macrophages is, in our experience, the main bias to recognize the oligodendroglial component in OA (**Figure 4A**–**H**). Normal oligodendrocytes do not express ATRX and they can be distinguished in this way from tumor oligodendro‐ cytes; moreover, they express Cyclin D1 that, in contrast, is not expressed by tumor oligoden‐ drocytes unless they are cycling cells (**Figure 5A**–**D**) [80].

to the 2007 WHO classification seems to ascertain the occurrence of ATRX-negative and

The astrocytic and oligodendroglial components have been found to share the same molecu‐ lar signature, but with a sheer cell differentiation [71]. They should be regarded as "morpho‐ logically ambiguous" rather than "mixed" tumors, as conventionally referred. 1p/19q codeletion and TP53 mutations represent distinct mechanisms of oncogenesis but they do not

The "ISN-Haarlem *Consensus*" suggested considering OA or tumors with ambiguous histolo‐ gy as diffuse astrocytoma when harboring IDH1/2 mutations, intact 1p/19q, and ATRX loss; as oligodendroglioma when harboring IDH1/2 mutations, 1p/19q co-deletion, and intact ATRX and as diffuse astrocytoma when harboring wild type IDH. In the absence of molecular data, the tumor should be diagnosed as oligodendroglioma or diffuse astrocytoma not otherwise specified (NOS). Finally, the denomination of OA would be only maintained when molecu‐

Anyway, there is no doubt on the usefulness of the ATRX IHC [72] and of the double ATRX/ IDH1R132H immunostaining [77] in the diagnosis of adult diffuse gliomas. Based on molecular data from the above mentioned markers on 54 OAs, it has been concluded that OA repre‐ sents a morphological grey zone rather than a group of truly "mixed" or "intermediate" gliomas [78]. Importantly, it remains unsolved how to explain IDH mutant diffuse gliomas with ATRX expression and intact 1p/19q (neither merely astrocytic nor oligodendroglial lesions) or, more rarely, cases with loss of ATRX protein expression and total 1p/19q co-

Our analysis started from the established principles that 1p/19q co-deletion is typical of oligodendrogliomas, but that it occurs in a low percentage only of tumors with a classical oligodendroglial phenotype ("honeycomb" appearance and "chicken wire" vessels). Our 40 OA cases had been initially diagnosed according to stringent histologic criteria, so that their number is slightly lower compared to others' series. The current revision has been carried out on the basis of 1p/19q chromosomal status, as detected by MLPA, IDH1/2 mutation status by IHC and sequencing analysis, and ATRX expression by IHC. The key points were: 1) the retained expression of ATRX by tumor oligodendroglial nuclei and by reactive astrocyte, microglia/macrophage, endothelial cell, lymphocyte nuclei, and the ATRX protein loss in tumor astrocytic nuclei; 2) IDH1/2 mutations, present in low grade gliomas with a higher frequency in oligodendrogliomas than astrocytomas [52, 79] (**Figure 3A**–**D**); 3) therefore, although they reveal the tumor nature of cells, IDH1/2 mutations can lack both in oligoden‐ droglial and astrocytic tumor cells. As, beside reactive astrocytes, ATRX is expressed in microglia/macrophage nuclei, Iba-1, CD68, CD16, and CD163 IHC has been performed on parallel serial tumor sections, as well as the double ATRX/GFAP, ATRX/Iba-1, and ATRX/ IDHR132H immunostaining. Microglia/macrophages can reach in the tumor sections a frequen‐

provide evidence for a genetic signature specifically related to OA [76].

IDH1R132H -positive astrocytes in the tumor.

348 Neurooncology - Newer Developments

lar testing does not solve tumor diagnosis [27].

deletion (both astrocytic and oligodendroglial lesions).

**8. Observations on personal OA series**

**Figure 3.** Immunohistochemistry (IHC). A – Oligodendroglioma, ATRX-positive cells, DAB, x200; B – Id, IDH1R132H positive perinuclear rim in tumor cells, DAB, x200. C – Gemistocytic astrocytoma, ATRX-negative and GFAP-positive tumor astrocytes, double IHC with DAB and *Fast Red*, respectively, x400; D – Id, IDH1R132H -positive cells, DAB, x400. Anti-IDH1R132H mouse monoclonal antibody (clone H09, Dianova GmbH, Hamburg, Germany) and anti-ATRX rabbit polyclonal antibody (HPA001906, Sigma Aldrich Co., St. Louis, MO, USA). DAB, 3,3'-Diaminobenzidine.

**Figure 4.** Immunohistochemistry (IHC). A – Diffuse astrocytoma, GFAP-positive cells, x200; B– Id, scattered ATRXpositive nuclei, the frequency of which corresponds to the frequency of CD68 positive-cells (C), both x200; D – Diffuse astrocytoma, apparent OA with GFAP-positive astrocytes and possible oligodendroglial nuclei, x200; E – Id, scattered ATRX-positive nuclei, x200; F – Id, Iba-1-positive cells covering the number of ATRX-positive nuclei, x200; G – Oligo‐ dendroglial infiltration, ATRX-positive and ATRX-negative nuclei, x200; H – Id, Iba-1-positive cells, x200 in 40 x high power field. All 3,3'-Diaminobenzidine (DAB).

**Figure 5.** Immunohistochemistry (IHC). A – Oligodendroglioma, mild infiltration with several ATRX-negative normal oligodendrocytes and few ATRX-positive nuclei (tumor muclei or microglia/macrophage cells?), DAB, x200; B – Id, Cy‐ clin D1-positive normal oligodendrocytes, DAB, x200; C – Oligodendroglioma, ATRX-positive endothelial cells, DAB, x400; D – Gemistocytic astrocytoma, microglia/macrophage cells, Iba-1- and ATRX-positive cells, double IHC with *Fast Red* and DAB, respectively, x400. DAB, 3,3'-Diaminobenzidine.

The diagnosis of the 40 OA cases changed in 87.5% of them (35/40): 22/40 (55%) were reclas‐ sified as astrocytomas due to the absence of total 1p/19q co-deletion, the occurrence of IDH1/2 mutations, and the loss of ATRX expression in GFAP-positive, phenotypically look‐ ing tumor astrocytes (**Figure 6A**–**D**); 11/40 (27.5%) were reclassified as oligodendrogliomas due to IDH1/2 mutations, retained ATRX expression, total 1p/19q in 2/11 (18.2%) cases and partial 1p or 19q deletions in 9/11 (81.8%) cases. In 2/40 (5%) cases the diagnosis changed into reactive gliosis due to retained ATRX expression in GFAP-positive reactive astrocytes and to the lack of ATRX-positive oligodendrocytes (**Figure 6E**–**F**). Among the remaining five cases, in one case with partial 1p/19q co-deletion, ATRX-negative and GFAP-positive astro‐ cytes co-existed with a number of ATRX-positive and GFAP-negative oligodendrocytes; both cell components showed IDHR132H immunopositivity by double ATRX/IDH1R132H immu‐ nostaining. Importantly, by the double ATRX/Iba-1 immunostaining, it was possible to veri‐ fy that the number of ATRX-positive oligodendroglial nuclei was higher than the number of Iba-1-positive cells. The diagnosis of OA in this case was thus confirmed (**Figure 7A**). Two wild type IDH cases without total 1p/19q co-deletion and with heterogeneous ATRX expres‐ sion were regarded as ambiguous since the tumor nature of the two cell components could not be ascertained. In the other two cases, one with partial 1p/19q deletion and one with intact 1p/19q, the diagnosis of OA could not be maintained since it was technically impossi‐ ble to perform IDH1R132H IHC.

**Figure 6.** Immunohistochemistry (IHC). A – Gemistocytic astrocytoma, GFAP-positive cells, DAB, x200; B – Id, ATRXnegative cells, DAB, x200; C – Anaplastic astrocytoma, GFAP-positive cells, DAB, x200; D – Id, ATRX-negative cells, DAB, x200; E – Oligodendroglioma, reactive astrocytes with GFAP-positive thick cytoplasms and long processes and ATRX-positive nuclei, double IHC with *Fast Red* and DAB, respectively, x400. F – Reactive gliosis, reactive astrocytes with GFAP-positive large cytoplasms and short processes and ATRX-positive nuclei, double IHC with *Fast Red* and DAB, respectively, x400. DAB, 3,3'-Diaminobenzidine.

The diagnosis of the 40 OA cases changed in 87.5% of them (35/40): 22/40 (55%) were reclas‐ sified as astrocytomas due to the absence of total 1p/19q co-deletion, the occurrence of IDH1/2 mutations, and the loss of ATRX expression in GFAP-positive, phenotypically look‐ ing tumor astrocytes (**Figure 6A**–**D**); 11/40 (27.5%) were reclassified as oligodendrogliomas due to IDH1/2 mutations, retained ATRX expression, total 1p/19q in 2/11 (18.2%) cases and partial 1p or 19q deletions in 9/11 (81.8%) cases. In 2/40 (5%) cases the diagnosis changed into reactive gliosis due to retained ATRX expression in GFAP-positive reactive astrocytes and to the lack of ATRX-positive oligodendrocytes (**Figure 6E**–**F**). Among the remaining five cases, in one case with partial 1p/19q co-deletion, ATRX-negative and GFAP-positive astro‐ cytes co-existed with a number of ATRX-positive and GFAP-negative oligodendrocytes; both cell components showed IDHR132H immunopositivity by double ATRX/IDH1R132H immu‐ nostaining. Importantly, by the double ATRX/Iba-1 immunostaining, it was possible to veri‐ fy that the number of ATRX-positive oligodendroglial nuclei was higher than the number of Iba-1-positive cells. The diagnosis of OA in this case was thus confirmed (**Figure 7A**). Two wild type IDH cases without total 1p/19q co-deletion and with heterogeneous ATRX expres‐ sion were regarded as ambiguous since the tumor nature of the two cell components could not be ascertained. In the other two cases, one with partial 1p/19q deletion and one with intact 1p/19q, the diagnosis of OA could not be maintained since it was technically impossi‐

**Figure 5.** Immunohistochemistry (IHC). A – Oligodendroglioma, mild infiltration with several ATRX-negative normal oligodendrocytes and few ATRX-positive nuclei (tumor muclei or microglia/macrophage cells?), DAB, x200; B – Id, Cy‐ clin D1-positive normal oligodendrocytes, DAB, x200; C – Oligodendroglioma, ATRX-positive endothelial cells, DAB, x400; D – Gemistocytic astrocytoma, microglia/macrophage cells, Iba-1- and ATRX-positive cells, double IHC with *Fast*

ble to perform IDH1R132H IHC.

*Red* and DAB, respectively, x400. DAB, 3,3'-Diaminobenzidine.

350 Neurooncology - Newer Developments

**Figure 7.** Immunohistochemistry (IHC). A – OA, ATRX- and IDH1R132H -positive astrocytes, double IHC with DAB and *Fast Red*, respectively, x630; B – Oligodendroglial minigemistocytes with ATRX-positive nuclei and GFAP-positive cy‐ toplasms, double IHC with DAB and *Fast Red*, respectively, x1000; C – Oligodendroglioma, satellitosis with ATRX-pos‐ itive nuclei, DAB, x200; D – Normal cortex, ATRX-positive neurons with ATRX-negative satellites, DAB, x400; E – Oligodendroglial cortical infiltration, ATRX-positive neurons with ATRX-positive and ATRX-negative satellites, DAB, x400; F – Id, ATRX-positive pericapillary tumor cells, DAB, x200. DAB, 3,3'-Diaminobenzidine.

The distinction of reactive from tumor astrocytes has always been a crucial point in the diagnosis of OA. Their recognition as reactive is a point of reference in denying the existence of such tumor category. Reactive astrocytes retain nuclear ATRX protein expression, as tumor oligodendrocytes, while tumor astrocytes lack ATRX expression.

By comparing our results with those of the literature, it is noteworthy that the change of diagnosis from the initial to the current analyses of cases, largely depends on the criteria used in the initial recognition of OAs. Upon the reclassification of the 35 cases as astrocytoma, oligodendroglioma, or reactive gliosis, only one could deserve the dignity of OA among the remaining five cases.

It must be incidentally remark that total 1p/19q co-deletion occurred in 43/113 (38.1%) of our oligodendroglioma series (selected by the typical morphology of honeycomb appearance and chicken wire vessel distribution); partial 1p and/or 19q deletions occurred in 36/82 (43.9%) of oligodendrogliomas and in 12/24 (50%) astrocytomas. Referring to the 40 cases with initial diagnosis of OA, two had total 1p/19q co-deletion, 14 partial 1p/19q deletion, 12 intact 1p/19q, and one a gain of function on the chromosome 19q. For the remaining 11 cases, the 1p/19q status was not available. It is widely accepted that partial 1p/19q deletions may occur in other gliomas, but one wonders how tumors with an oligodendroglial phenotype with partial deletions or intact 1p/19q can be classified.
