Case Study 18Craig Horbinski, M.D., Ph.D.

The patient is a 71 year-old male with an episode of speech disturbance that occurred two days PTA.  The patient had some confusion as well as some speech difficulty.  MRI with contrast was done.

What do you see?

Question 1

T1

T2

FLAIR

DWI

T1 with contrast

Ring-enhancing mass lesion, increased surrounding FLAIR signal, no diffusion abnormality.

Answer

Question 2What is the significance of the contrast image?

AnswerGadolinium is the contrast agent used.  In normal brain it will not cross the blood-brain barrier, so brain parenchyma will not light up.  Seeing enhancement like this suggests a breakdown of the barrier.

Question 3What does the FLAIR image mean?

AnswerFluid-attenuated inversion recovery (FLAIR) is a T2-weighted image that dampens ventricular (free water) CSF signal.  Compare it with the regular T2, which shows CSF as bright.  FLAIR is good at picking up and highlighting true intraparenchymal edema.  Thus, this mass is producing edema.

Question 4What is DWI?  (No, the answer is not “driving while intoxicated.”)  Why is it important in neuroradiology?

AnswerDiffusion-weighted imaging (DWI) shows areas with restricted water diffusion as bright.  Such areas have reduced water diffusion because of acute cytotoxic edema, which traps water molecules inside swollen cells.  DWI is very helpful in identifying acute ischemia.  Bacterial abscesses will also produce cytotoxic edema in the surrounding non-necrotic tissue, so they can also be bright on DWI.  Old strokes, non-bacterial infections, tumors, contusions, and demyelinating diseases (like MS) are not associated with cytotoxic edema, so they are not usually hyperintense on DWI.  In this case, the lesion is not bright on DWI, so it’s probably not an acute infarct or bacterial abscess.

Question 5Given all this, what’s the differential diagnosis?

AnswerHigh-grade glioma or lymphoma.  Abscess, ischemia, or demyelinating diseases are far less likely (again due to the lack of signal intensity on DWI).

Question 6During the excision of the mass, the neurosurgeon calls you for an intraoperative consultation and wants to know, 1. If he’s in the lesion; 2. What the diagnosis is.

So what is it?

Cick the following links to view slides: Frozen, Smear

AnswerHigh grade glioma.  The tissue is markedly hypercellular, far more so than normal tissue, so he’s definitely in the lesion.  There are a lot of cells with nuclear atypia suggesting a neoplastic process.  At the edges of the smear, processes are seen coming off the cells, suggesting a glioma.  Mitoses are a clue to its high-grade nature, but even if you can’t spot any, the microvascular proliferation seen on the frozen section (which is what’s causing the contrast enhancement seen earlier) is good enough evidence that this is a high-grade glioma, i.e. glioblastoma (grade 4/4).

Question 7The permanent sections of the case have arrived.  What’s your diagnosis?

Click here to view slide.

AnswerThe tumor is hypercellular, with angulated atypical nuclei (suggestive of astrocytic origin) and endothelial proliferation.  Mitoses are rare, but even if you don’t see them the microvascular proliferation (often erroneously called endothelial proliferation) is enough to push this to a glioblastoma multiforme (GBM), WHO grade 4.

Question 8What immunostains do we routinely order for these tumors?  Why?

Answer1. Ki67, which labels the nuclei of all cells not in Go, i.e. those in some stage of

cell division.  A higher percent of cells with positive nuclei means a more rapidly growing tumor.  Calling something a GBM with a low Ki67 proliferation index (i.e. less than 5%) is unusual, as most will be at least 10%.

2. Epidermal growth factor receptor (EGFR), which is one of the two classic molecular pathways by which glioblastomas arise.  GBMs strongly positive for EGFR arise de novo, i.e. are primary GBMs.

3. P53, which labels the tumor suppressor protein inside the nucleus.  GBMs with p53 mutations arose from a prior low-grade glioma (whether that low-grade glioma was clinically recognized or not), i.e. are secondary GBMs.  An interesting fact about p53 mutations is that they tend to render p53 ineffective rather than just shutting down p53 production.  Thus, the cell recognizes that it should not be dividing and produces p53 to stop itself.  But since the p53 doesn’t work, and the cell doesn’t realize it’s a waste of time to produce bad protein, it keeps churning out p53.  On immunostains of such tumors the majority of cells will have strongly-staining nuclei.  Such a finding suggests a p53 mutation, which is the second main pathway of gliomagenesis.

4. Glial fibrillary acidic protein (GFAP), just to verify that the tumor is indeed glial in origin.

Question 9The immunostains have arrived.  What is your interpretation of these stains?

Click the following links to view slides: Ki67, EGFR, P53, GFAP

Answer1. GFAP is moderately positive in the tumor cells,

confirming its glial origin

2. EGFR shows strong and diffuse staining, suggesting a primary GBM (consistent with the patient’s age, as primary GBMs are more common in older patients).

3. Only about 30% of the cells are positive for p53, and they’re only weakly to moderately positive, so it’s not likely a p53-driven tumor.

4. The Ki67 proliferation index is a little subjective, but was estimated at up to 20%, well within the range for GBMs.

Question 10In addition to the immunostains, here at UPMC we do an extensive molecular workup of gliomas, including fluorescence in situ hybridization (FISH) probing for the EGFR gene (on 7p12), p16 (9p21), and 1p/19q (on 1p36 and 19q13, respectively).

What does each of these results mean?

Answer1. The ratio of EGFR to the centromeric enumeration probe of chromosome 7 is very

high (scored at 30), when it should be a 1:1 ratio.  This means that there are multiple copies of the EGFR gene, a.k.a. amplification, as shown by the numerous red signals compared to about 2 green signals.  This explains why there is so much EGFR protein on immunostain.  Since EGFR is a well-described pathway for cell proliferation, this amplification likely explains why this tumor arose in the first place.

2. P16 is a well-known tumor suppressor gene.  In addition to its described role in melanomagenesis, it has been shown to be deleted in higher grade gliomas but not low grade gliomas.  Most of the cells in this image have 2 green dots identifying 2 copies of chromosome 9, but no red dots where p16 should be.  Thus, there is a homozygous deletion of p16, meaning that these cells have lost an important brake on division.  It lends extra support to our diagnosis of GBM in this case.  In some cases where the grade is uncertain, seeing homozygous deletion of p16 suggests that the glioma will soon explode into a higher grade; even if the histology doesn’t allow us to explicitly diagnose it as such, we’ll still call the clinician and tell them to watch this tumor closely.

3. Deletion of the whole arms of 1p and 19q is a classic hallmark of oligodendrogliomas.  If this deletion were present in this case, we might have to reclassify the tumor as an anaplastic oligodendroglioma (WHO grade 3), which has a longer survival expectancy than does GBM.  The 1p/19q codeletion renders the tumor more vulnerable to chemotherapy for reasons not yet known.  In this case, there is a 1:1 ratio of red: green signal in most cells, meaning no selective loss of either 1p or 19q.  There is, however, some hyperploidy of chromosome 19, a common finding in high-grade gliomas that has no prognostic or diagnostic significance.

Question 11Another set of molecular diagnostic tests we do includes PCR-based microsatellite analysis for selected targets, the results of which are shown below:

1p – fractional gene loss is 0% (0 markers with loss/ 5 informative markers)19q – fractional gene loss is 67% (2 markers with loss/ 3 informative markers)9p – fractional gene loss is 0% (0 markers with loss/ 3 informative markers)10q – fractional gene loss is 100% (2 markers with loss/ 2 informative markers)17p – fractional gene loss is 0% (0 markers with loss/ 2 informative markers)

The D19S112 and D19S559 markers on 19q were the ones showing loss of heterozygosity (LOH).

The D9S1748 marker on 9p corresponding to the 9p FISH probe for p16 (9p21) did not show LOH.

What is the significance of each of these loci?  How do you interpret these results?  In particular, how do you reconcile the FISH results on 19q with the PCR LOH results on 19q (look at the ideogram)?  Or the FISH on 9p21 with PCR on 9p21?

Answer1. PCR-based microsatellite analysis is another way to look for the 1p/19q deletion.  In

a true oligodendroglioma, the entire arm of 1p and the entire arm of 19q will be deleted, so all the microsatellites on both arms will show fractional gene loss, or LOH.  It is more encompassing than the FISH probes, which by their very nature cannot scan such a large piece of DNA.  At first glance, there would appear to be a discrepancy between the FISH and PCR results for 19q.  A closer look, however, reveals that the 2 microsatellite markers on 19q showing LOH are centromeric to the FISH probe, which is on 19q13.  There is no overlap between the loci probed and, thus, no discrepancy.  Clearly, though, there is an interstitial deletion of some genetic material on 19q centromeric to the FISH probe.  Such occurrences are not unusual in genetically unstable tumors like GBMs.

2. The 9p microsatellite marker that corresponds to 9p21 did not show LOH, whereas FISH showed homozygous deletion of 9p21.  This is not actually discrepant, because in the case of a complete homozygous deletion no LOH will be detected—there isn’t any heterozygosity to begin with, so the computer analysis package is fooled into thinking there isn’t any gene loss.  In this case, trust the FISH.

3. 10q contains PTEN, which is another tumor suppressor protein that specifically acts within the EGFR pathway as a brake on EGFR signaling.  A loss of PTEN correlates well with amplification of EGFR in GBMs and is yet another piece of evidence that this is a primary GBM.  Again, in cases where histological grading is equivocal, such a molecular aberration portends rapid transformation into a high grade glioma.

4. 17p contains the p53 gene.  Taking into account Knudson’s classic “two-hit” hypothesis, the conventional dogma is that a p53-driven secondary GBM will have an inactivating mutation on one of the two p53 copies (hit #1, producing strong p53 immunostaining as discussed earlier) plus LOH on 17p (hit #2).  No LOH is seen in this case, correlating nicely with the p53 immunostain.