“The reason for this is that we don’t need vascular proliferation or necrosis to make a diagnosis of a high-grade glioma these days because there are molecular alterations that correlate with grade four behavior,” he continued. “So what looks in histology as a grade two tumor could very well be a grade four lesion, and that’s important to recognize in terms of the age of the patient, the location of the tumor, and when it’s appropriate to do molecular characterization.”
The final case was a non-enhancing lesion involving the pons (Fig. 9). Here too the histology is consistent with a diffuse glioma, “completely different just based on the location and the molecular characteristics of the tumor” (Fig. 10).
There is no vascular proliferation or necrosis, but this tumor was positive for the H3K27M mutation, and the tumor cells are positive for the mutant protein. “The endothelial cells are not; they don’t have the mutation.”
Among the diagnostic pitfalls is that the IDH1 R132H immunostain doesn’t capture all the clinically relevant IDH1 and IDH2 mutations, so in some cases sequencing is needed, particularly in patients under age 55, Dr. Ballester said.
“It’s important to exclude the non-canonical mutations in IDH1 and IDH2 which the antibody doesn’t recognize. It recognizes only the most common mutation, which is R132H.”
Two antibodies look at the lysine 27 in the histone 3 protein—one against the mutant protein, H3K27M, and one against the trimethylated lysine 27, which is the wildtype scenario. In Fig. 11 (top), the tumor does not have a mutation and is H3K27-wildtype, and the antibody against the mutant protein is negative. In the antibody against the trimethylated lysine 27, the signal is retained in all the cells—tumor and endothelial. “All the cells are positive.”
Fig. 11 (bottom) also shows an H3K27M-mutant tumor and expression of the mutant protein with the antibody in the tumor cells. “Endothelial cells do not have the mutation and do not express the mutant protein,” Dr. Ballester said. “This antibody against the trimethylated lysine 27 is negative in the tumor cells, but the expression is retained in epithelial cells because these cells do not have the mutation.”
Recognizing that the two different antibodies serve two different purposes is important, he said. “I’ve seen diagnoses where people have interpreted this as the mutant protein and made a diagnosis of diffuse midline glioma, K27M-mutant, because it’s positive in all the cells.”
Small changes in nomenclature can cause confusion, he noted. In histone 3, lysine 27, “we later discovered that the amino acid methionine at the beginning of the protein was being cleaved. So the numbers changed, and lysine 27 is in fact lysine 28. And the gene name changed from H3F3A to H3-3A. So H3F3A p.K27M-mutant is the same as an H3-3A p.K28M mutation”—important to be aware of, he added, because sequencing reports may vary.
A small number of these tumors don’t have methionine, Dr. Ballester said. “The mutation is to a different amino acid not recognized by the antibody. So they would be negative for the K27M-mutant antibody but will show the K27me3 loss. “That tells you something’s wrong at the position, and that is one way of recognizing these other tumors that don’t necessarily have the K27M mutation but still fall under this diagnosis of diffuse midline glioma, H3K27-altered.”
Oligodendroglioma is defined as a diffuse glioma with IDH1/IDH2 mutation and codeletion of 1p/19q (Ballester LY, et al. Hum Pathol. 2017;69:15–22). “However,” Dr. Ballester said, “there is a small number of glioblastomas, about five to 10 percent, that will show 1p/19q codeletion by FISH. But those tumors are IDH1/IDH2 wildtype, so they’re not oligodendrogliomas, and they sometimes create confusion” in a case that looks like a glioblastoma but is 1p/19q codeleted by FISH (if FISH is ordered). “It raises the question, ‘Is this an oligodendroglioma?’ and the answer is no.”
FISH probes are very small in relationship to the chromosome (1 and 19), and small partial losses can interfere with the regions where the probes bind and give the appearance of 1p/19q codeletion by FISH, he said. “That happens in a percentage of glioblastomas.” But that is not the entire loss of chromosome 1p and 19q seen in oligodendrogliomas. An array will not show 1p/19q codeletion, he said. “It’s a limitation of the FISH assay.”
Dr. Ballester refers routinely to the 2021 WHO classification of central nervous system tumors, which he says took to the next level the molecular classification of brain tumors introduced in the 2016 edition.
“We even have new tumor entities based on novel diagnostic technologies like DNA methylome profiling. Some tumors can be diagnosed with certainty only after methylation analysis because they are defined by their unique methylation profile,” Dr. Ballester said. Although the majority of brain tumors today don’t require methylation profiling, “it’s important to recognize when you’re dealing with one of these tumors that requires methylation analysis and to know that technique is available.” In some cases, he said, it’s the most appropriate test to diagnose a particular tumor.
Amy Carpenter is CAP TODAY senior editor.