Interpreting Pathology Reports

Pathology reports typically include a section dealing with microscopic descriptions of hematoxylin and eosin (H&E) stained tumour tissue viewed on a glass slide, and a section showing the results of immunohistochemical stains performed on thin slices cut from blocks of preserved tumour. If fortunate, your pathology report will also include genetic analysis indicating genomic mutations, copy number alterations (gains or losses of genes or multi-gene regions of DNA) and polysomy/monosomy (gain or loss of an entire chromosome). For now I’m starting with immunohistochemistry, and the other sections will be added later.

Immunohistochemistry (IHC)

Immunohistochemical testing involves the use of antibodies to detect specific antigens/proteins expressed by the tumour. This is usually achieved by conjugating an enzyme, such as peroxidase, to the antibody which then produces a color signal when the antibody comes in contact with the antigen/protein target.

GFAP – glial fibrillary acidic protein. An intermediate filament protein mainly expressed by astrocytes, its function is likely structural. In IHC, it is used to determine cells of astrocyte lineage, and can also distinguish between astrocytic and oligodendrocytic components of a mixed oligoastrocytic tumour. GFAP does not necessarily distinguish tumour from non-tumour.

Ki-67 – A large protein present during all phases of the active cell cycle, but absent in resting cells. Usually detected with a monocolonal mouse antibody called MIB-1, this test determines the degree of active proliferation of a tumour, expressed as a percentage of active cells. Though not used for grading, the Ki-67 index of higher grade tumours is generally higher than lower grade. For astrocytomas, Ki-67 is usually under 5% for grade 2, often between 5% and 15% for grade 3, and often between 15% and 25% or more for glioblastoma, though there is much overlap between the grades. For example, a grade 3 astrocytoma may have a Ki-67 value of under 5% or over 20%. A study (1) of 128 anaplastic oligodendroglial tumours (including both oligodendroglioma and oligoastrocytoma) determined a median Ki-67 of around 20% (range 0-90%). The Ki-67 index is distinct from the mitotic index, which is a measure of cells undergoing mitosis, the stage within the cell cycle when cells are actually dividing. The mitotic index is therefore only a small fraction of the Ki-67.

p53 – This is a protein with a nominal molecular weight of 53 kilodaltons, encoded by the TP53 gene. The gene is perhaps the most frequently altered gene in cancer, the target of frequent mutations and deletions. p53 protein is characterized as a transcriptional regulator or transcription factor which regulates the transcription (copying of DNA to RNA) of a multitude of target genes. Referred to as the “guardian of the genome” it orchestrates responses to DNA damage, typically resulting in cell cycle arrest (allowing time for DNA damage repair) or apoptosis (if the damage is too extensive for repair).

In terms of p53 IHC testing, the most important thing to know is that more p53 detected does not translate to more tumour-suppressive activity. The reason for this is that wild-type (unmutated) p53 is easily degraded, and is therefore often undetectable by IHC. On the other hand, mutant p53, which has lost normal p53 functions, resists degradation and therefore can accumulate in the cell nucleus. Over 10% of cells with strong nuclear staining for p53 is often an indication of a TP53 mutation. However, the only way to be sure of a TP53 mutation is by DNA sequencing, as not all cases with p53 accumulation have a corresponding mutation in the gene. Likewise, not all TP53 mutations will show up as increased p53 staining by IHC. For example, if the TP53 mutation is a rare frameshift mutation (caused by small insertions or deletions of DNA within a gene), there may be no detection of p53 protein by IHC. While detection of extensive p53 staining may be an indication of mutant p53, the correlation is not tight. In general, extensive p53 staining is interpreted as a sign of astrocytoma, as opposed to oligodendroglioma.

IDH1 R132H – Within about a year of the discovery of prevalent IDH1 mutations in lower grade gliomas in late 2008, antibodies were being put into use to detect the most common IDH1 mutation by means of IHC. This has significantly enhanced the ability to diagnose and prognosticate the behaviour of lower grade gliomas. It has also allowed the ability to distinguish between tumour and non-tumour cells, as virtually all neoplastic (tumour) cells in an IDH1-mutated tumour produce the mutant IDH1 enzyme and show positive immunoreactivity by IHC. This test is highly specific – false positives are not an issue. However, this test can only detect the most common IDH1 mutation (R132H), in which the resulting enzyme has a histidine (H) amino acid substituted for the normal arginine (R) due to a single nucleotide substitution at codon 132 of the gene. Other variants of the IDH1 mutation cannot be detected with an antibody targeted to the R132H variant, though these alternative variants are rare, and also always occur at codon 132. The antibody will also not detect the rare IDH2 mutation. Therefore, if a lower grade glioma tests negative for IDH1 R132H by IHC testing, it is still recommended to have the gene DNA sequenced for alternative IDH1 variants.

Note: The IDH1 R132H mutation may also be referred to as an IDH1 c.395G>A mutation. 395G>A refers to the specific nucleobase substition at position 395 of the gene (guanine replaced by adenine in this case), whereas R132H refers to the resulting amino acid substitution in the enzyme. Position 395 is within codon 132 (each codon is 3 base pairs long).

MGMT – This enzyme is a DNA repair enzyme that removes harmful alkyl groups from DNA. Many chemotherapies such as temozolomide and the nitrosoureas (eg CCNU, BCNU) cause cell destruction by inserting an alkyl group (a methyl group in the case of TMZ, or a chloroethyl group in the case of the nitrosoureas) onto a specific location within the DNA, leading to DNA mismatches during cell division, ultimately resulting in strand breaks, cell cycle arrest, and apoptosis. The active MGMT enzyme removes the alkyl groups, thus rendering the cell unharmed by the alkylating chemotherapies.

MGMT may be detected using IHC. However, the predictive value of MGMT in terms of response to chemotherapy is more accurately determined by its methylation status. This is a non-IHC test, usually done using methylation-specific PCR (MSP), or more accurately (and uncommonly) by pyrosequencing. Methylation testing for MGMT will be described in another section.

Note: MGMT detection by IHC does not determine methylation status.

Copy number alterations – gain or loss of genetic material

Each cell in the body normally has two copies of all 23 chromosomes, one set from the mother, and one set from the father, for a total of 46 chromosomes. These chromosome pairs are numbered 1 to 22 and the final pair are the sex chromosomes. Trisomy refers to the gain of an entire extra chromosome, polysomy refers to the gain of several additional copies of a chromosome, and monosomy refers to the loss of an entire chromosome.

The main categories of genetic alterations in cancer are mutations within a specific gene, and copy number alterations, which refers to the gain or loss of genetic material, ranging from focal gain or loss of a specific gene, all the way to gain or loss of a whole chromosome arm, or an entire chromosome. The gain of many additional copies of a gene is called amplification. The loss of one copy of a gene is called hemizygous deletion, while the loss of both copies of a gene is termed homozygous deletion. In brain cancer, these mutations and copy number alterations are typically only found in the tumour cells, not in the normal cells of the body. This type of mutation is called a somatic mutation. However, other types of cancer may be caused by a germline mutation found in all body cells, such as BRCA1 or BRCA2 gene mutations in some cases of breast and ovarian cancer.

The most frequent genetic alteration in glioblastoma is polysomy (gain of several copies) of the entire chromosome 7, and monosomy, or loss of one entire copy of chromosome 10. This is distinct from the focal gain or deletion of specific genes or regions on these chromosomes, which is another frequent occurrence. Polysomy of chromosome 7 occurs in about 86% of glioblastomas, and monosomy of chromosome 10 occurs in 90% (excluding IDH1-mutant, G-CIMP type, which makes up a small fraction of primary GBM cases) (2). These two events, gains of entire copies of chromosome 7 and loss of an entire copy of chromosome 10, have been determined to be the first two genetic alterations which take place in glioblastoma evolution (2).

Frequently, glioblastomas also have focal copy number alterations located on chromosomes 7 and 10. The target genes of these focal alterations are epidermal growth factor receptor (EGFR) located on the small arm (“p” for petit) of chromosome 7 (7p), and PTEN which acts as a tumour suppressor gene, located on the long arm of chromosome 10 (10q). High-level amplification of the EGFR locus occurs in about half of all GBM cases, including almost all cases of the “classical” GBM subtype (3). While most GBM cases have a loss of one copy of PTEN due to monosomy of chromosome 10, about 10% will also have a focal deletion of the remaining copy (homozygous deletion of PTEN) (2).

Relevance: EGFR signaling activates downstream proliferative and survival pathways such as PI3K/Akt/mTOR, MAPK, and STAT3. On the other hand, PTEN is a suppressor of PI3K signaling.

The other copy number alteration frequently tested for is combined loss of one copy of the short arm of chromosome 1 (1p) and the long arm of chromosome 19 (19q). This combined loss is called 1p/19q codeletion or loss of heterozygosity (LOH) of 1p/19q. This alteration is the hallmark of oligodendroglioma and rarely occurs in any other type of glioma. This alteration is quite distinct from incomplete losses of these chromosome arms. While complete loss of one copy of both 1p and 19q is associated with better prognosis and sensitivity to chemotherapy in oligodendroglioma, partial loss of 19q is associated with poor survival in low-grade gliomas (4).

Relevance: 1p/19q codeletion is an important diagnostic marker of oligodendroglioma, associated with better prognosis and sensitivity to chemotherapy.

References
  1. Prognostic value of Ki67 index in anaplastic oligodendroglial tumours–a translational study of the European Organization for Research and Treatment of Cancer Brain Tumor Group. Preusser et al. 2012.
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  2. Most Human Non-GCIMP Glioblastoma Subtypes Evolve from a Common Proneural-like Precursor Glioma. Ozawa et al. 2014.
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  3. Integrated Genomic Analysis Identifies Clinically Relevant Subtypes of Glioblastoma Characterized by Abnormalities in PDGFRA, IDH1, EGFR, and NF1. Verhaak et al. 2010.
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  4. Clinical value of chromosome arms 19q and 11p losses in low-grade gliomas. Alentorn et al. 2014.
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