The Diagnostic Use of Immunohistochemical Surrogates for Signature Molecular Genetic Alterations in Gliomas

David N. Louis, MD

The January 2016 issue of the Journal of Neuropathology and Experimental Neurology features a helpful review article entitled The Diagnostic Use of Immunohistochemical Surrogates for Signature Molecular Genetic Alterations in Gliomas. The review article-- authored by Drs. Jantima Tanboon, Erik A. Williams, and David N. Louis -- summarizes the current experience using immunohistochemistry of glioma samples to identify mutations in IDH1, TP53, ATRX, histone H3 genes, BRAF, EGFR, MGMT, CIC, and FUBP1 as well as guidelines for prudent use of DNA sequencing as a supplemental method. In subsequent posts, I will summarize the key points in this review to make the information easily searchable for readers to use in the future as needed.

Best Post of October 2015: Chromothripsis!

The next in our "Best of the Month" series comes from October 1, 2015:

The next edition of the World Health Organization Classification of Tumors of the Central Nervous System will feature a new, separate ependymoma subtype: RELA fusion-positive ependymoma. RELA fusion refers to the juxtaposition of the RELA gene (the principle effector of NF-кB signaling which controls DNA transcription and cell survival) to the poorly characterized C11orf95gene. Fusion of these two genes is brought about by chromothripsis, a term first coined in 2011 that literally means "chromosome shattering". Chromothripsis occurs when chromosomal segments first fragment into many pieces and then get stitched back together in random order by DNA repair processes. Seen in the setting of some malignancies, chromothripsis in a particular segment of chromosome 11 can result in C11orf95-RELA fusion, which in turn drives oncogenic NF-кB signaling in ependymoma.

Chromothripsis (literally meaning "chromosomal shattering") can drive oncogenesis

Although chromothripsis is a novel model for oncogenesis, it does not necessarily contradict more established models of progressive cancer development as there is no definitive proof that chromothripsis has to occur as a single catastrophic event. Nevertheless, this is a fascinating area of research which will undoubtedly yield more insights into the progression of at least a subset of cancers.

Best Post of April 2015 - The Tumor Biomarker Series: BRAF

The next in our "Best of the Month" series comes from Tuesday, April 28, 2015:

BRAF gene (v-Raf murine sarcoma viral oncogene homolog B)

BRAF+ IHC (correlates with V600E mutation) in ganglioglioma

Aberrant constitutive activation of BRAF tends to be seen in cerebellar and midline pilocytic astrocyomas whereas the activating point mutation at BRAF V600E is more likely to be seen in cerebral examples. The V600E point mutation is also observed in other low-grade gliomas and glioneuronal neoplasms, including approximately two-thirds of pleomorphic xanthoastrocytomas, and lower percentages of ganglioglioma, desmoplastic infantile ganglioglioma, dysembrioplastic neuroepithelial tumor, and papillary craniopharyngioma. Although less common, diffusely infiltrative gliomas including glioblastoma, particularly the epithelioid variant, may also demonstrate the V600E point mutation -- making this biomarker potentially less useful as a diagnostic tool in distinguishing low-grade gliomas from high-grade ones.

Chromothripsis!

The next edition of the World Health Organization Classification of Tumors of the Central Nervous System will feature a new, separate ependymoma subtype: RELA fusion-positive ependymoma. RELA fusion refers to the juxtaposition of the RELA gene (the principle effector of NF-кB signaling which controls DNA transcription and cell survival) to the poorly characterized C11orf95 gene. Fusion of these two genes is brought about by chromothripsis, a term first coined in 2011 that literally means "chromosome shattering". Chromothripsis occurs when chromosomal segments first fragment into many pieces and then get stitched back together in random order by DNA repair processes. Seen in the setting of some malignancies, chromothripsis in a particular segment of chromosome 11 can result in C11orf95-RELA fusion, which in turn drives oncogenic NF-кB signaling in ependymoma.

Chromothripsis (literally meaning "chromosomal shattering") can drive oncogenesis

Although chromothripsis is a novel model for oncogenesis, it does not necessarily contradict more established models of progressive cancer development as there is no definitive proof that chromothripsis has to occur as a single catastrophic event. Nevertheless, this is a fascinating area of research which will undoubtedly yield more insights into the progression of at least a subset of cancers.

Review Article: "Thinking and Talking About Life Expectancy in Incurable Cancer"

As a follow-up to my previous post, here's a perspective from a June 2011 article in Seminars in Oncology:

"Most patients with incurable cancer want information about the impact cancer will have on their future, and many want specific estimates of the most likely, best case, and worst case scenarios for survival. With improved understanding of life expectancy, patients are better equipped to make appropriate treatment decisions and plans for the future. Although physicians acknowledge that patients with incurable cancer want prognostic information and benefit from this, most struggle to provide it and experience difficulty in making reliable estimates, communicating them, and tailoring the information to the individual patient."

Why genetically profile a glioma?

A couple of weeks ago I met with representatives of Castle Biosciences regarding their proprietary gene expression profile assay for glioblastomas, called DecisionDx-GBM, as well as their multi-methylation test for grade II and III gliomas, called DecisionDX-G-CIMP. During the meeting, I brought up a crucial issue raised by a Neuropathology Blog reader. I'll quote part of the reader's comment: "I doubt anyone wants to give a patient a life expectancy prediction based on the results of this test, since individuals may fall at any point on a survival curve. So I would not be eager to recommend this test until there are alternative treatments for those in poor prognosis groups." The representative's response to this concern was that prognostication is an important part of the decision-making process that a neuro-oncologist, in collaboration with the patient, takes into account in recommending a course of treatment. For example, when should Avastin be introduced into the regimen? If a patient has a tumor with a genetic signature that has a longer median survival, perhaps the neuro-oncologist would be more likely to hold back on the introduction of Avastin, keeping it in his armamentarium for later in the disease course. I would add that if I personally had the misfortune of being diagnosed with a glioblastoma, I would like to have as much prognostic information as possible just to help me adjust psychologically to dealing with the the disease. I realize that one individual can fall at any point on a survival curve. But put yourself in the patient's shoes for a moment. Wouldn't you want this test done, as well as IDH1 mutation, MGMT methylation status, 1p/19q testing, and whatever else that might be available to get as much information as possible about this disease has invaded you? Since, as Castle Biosciences states, insurance will cover this test, I see both practical and intangible benefits to doing it. I would be curious to hear what other neuropathologists have to say about this issue in general and about the Castle Biosciences tests in particular. I should note that the Castle tests were developed by the widely-respected neuropathologists Dr. Kenneth Aldape at MD Anderson. I am seriously entertaining the idea of routinely using the Castle Biosciences tests at my institution and would be interested in the advice of the neuropathology community as a whole regarding this issue. Please post to comments.

Best Post of March 2012: Spinocerebellar Ataxia Type 6 autopsy photographs

The next in our "Best of the Month" series is from March 19, 2012:

On the left is the brain of an 82-year-old patient with a diagnosis of spincocerebellar ataxia, type 6 (SCA6). On the right is the brain of a 96-year-old "control" patient with Alzheimer disease:

Note comparative diminution of the cerebellum in the SCA6 patient.  Here's a closer look at the cerebellum:

SCA6 is one several autosomal dominant cerebellar ataxias. SCA6 results from a CAG trinucleotide repeat expansion in the CACNA1A gene on chromosome 19p.

Spinocerebellar ataxia type 6 autopsy photographs

On the left is the brain of an 82-year-old patient with a diagnosis of spincocerebellar ataxia, type 6 (SCA6). On the right is the brain of a 96-year-old "control" patient with Alzheimer disease:

Note comparative diminution of the cerebellum in the SCA6 patient.  Here's a closer look at the cerebellum:

SCA6 is one several autosomal dominant cerebellar ataxias. SCA6 results from a CAG trinucleotide repeat expansion in the CACNA1A gene on chromosome 19p.

Case Western's Gary Landreth explores bexarotene as a pontential Alzheimer therapeutic

Gary Landreth, PhD

Dr. Mark Cohen tells me that a guy worth watching on the Alzheimer research front it Dr. Gary Landreth from Case Western Reserve University. And, as anyone who reads this blog regularly knows, when Mark Cohen talks, I listen. According to the Alzheimer Research Forum (from which I am blatantly plagarizing in the remainder of this post),  Landreth works on ApoE-directed therapeutics. As one of the genes involved in cholesterol homeostasis, ApoE is regulated transcriptionally by receptors which form heterodimers with the retinoid X receptor (RXR). Working with APP transgenic mice, Landreth found that an RXR agonist, bexarotene, induces brain ApoE quickly and robustly. This induction precedes a rapid decline in brain amyloid, an increase in plaque-clearing microglia, and improvements in cognitive performance. Landreth noted that bexarotene is an FDA-approved chemotherapeutic that readily crosses the blood-brain barrier and has minimal side effects, most of which are associated with its action on lipid homeostasis. Hence, this drug minimally represents a test of the role of Aβ in AD and, maximally, may represent an AD therapeutic. These finding raise the question of the utility of the drug in ApoE4-positive individuals. You go, Gary!

Genetics of PSP further elucidated

Actor Dudley Moore died in 2002 with PSP

About 20,000 people in the United States are currently diagnosed with Progressive Supranuclear Palsy (PSP). A variant in the MAPT gene, has been repeatedly linked to the disease. Researchers led by Gunter Höglinger of Philipps-Universitat in Germany studied more than 2,000 individuals of European descent diagnosed with PSP, comparing their DNA to that of nearly 7,000 healthy European individuals. The results were published last month in the journal Nature Genetics. The study reported several single nucleotide polymorphisms (SNPs) associated with the disease. One of the SNPs reported by Höglinger’s team is located in MAPT and is closely linked to the already well-established variant for PSP risk. In this study, each copy of the G version of rs8070723 in MAPT was associated with about 5.5 times lower odds of the disease. (Source: spittoon.23andme.com)

I have discovered my ApoE status

I just got my ApoE results back from 23andMe.com.  When I ordered the test, the first question I posed to readers was whether it was wise to even find out whether or not I have the epsilon 4 allele. One commentator, "Agent 86", quoted the ancient Greek philosopher Epictetus for guidance on this issue. In 135 AD, Epictetus argued that one should emotionally separate oneself from those things which are not one's own -- i.e., those things over which one has no control. Therefore, had he known what a gene was at the time, Epictetus would have counted one's genome as among those things that is not one's own. As such, knowing his ApoE status would have been of no consequence either way to Epictetus. I do find it rather easy to take this Epictetusian attitude toward my ApoE status. But because I find it exceedingly difficult to adopt this attitude of separateness toward many other things in my life over which I have no control, I can certainly understand how others could feel emotionally affected by knowledge of their own ApoE status.

I then had a second question: Should I make my ApoE results public? I was surprised by the number of people who recommended against doing so. The main objection seemed to be the potential that an insurance company might refuse to cover me if it were known that I was at increased risk for the development of Alzheimer's disease. I agree with one commentator who stated that this is a "worst-case scenario" and continually worrying about worst-case scenarios is not a wise way to live one's life.  I might also add that it might be in an insurance company's best interest for me to get Alzheimer's disease at a relatively earlier age (in my 60's rather than in my 80's) as  I would likely die at a younger age and thereby cost the company less money over the long run. That being said, in deference to all those who weighed in on this matter, I have elected not to publicly share my ApoE status. I appreciate all those who engaged in this discussion; and I suspect that this kind of discussion will be ongoing in many circles in the decades to come as personalized medicine, based on genomic profiling, emerges in the new age of theragnostics.

What's wrong with finding out and releasing my ApoE status?

In a recent blog post, I stated that I intended to publish my ApoE status on the blog when I get the results back from 23andMe.com. I was surprised to find that some people objected to this. One associate said that by publishing information about my genetic status, I would be "normalizing" the dissemination of information that should be confidential. That sets a bad precedent for the use of personal genomic data. Another individual emailed me the following suggestion: "Instead of posting your results, maybe you would consider instead the value of not doing so at the request of an 'anonymous' reader and keeping the information private in order to stimulate debate about finding out about a disease process for which there is no cure." Finally, "jd" commented in the original post about the wisdom of getting the test done at all: "Why would you do that? If you come back 4/4 or 3/4, that'll be hanging over you the rest of your life. If there were a cure for Alzheimer's, that'd be different."

And so, I pose this question to readers: Is there not intrinsic value in knowledge about one's genome, regardless of whether or not a related disease predisposition can be treated? Also, if I am willing to release my personal genetic information to the public, what harm am I causing myself or anyone else? At this point, I intend to find out my ApoE status when the information is made available to me, and I intend to discuss the results on this blog when I get them. But, if anyone can make a convincing argument otherwise,  I am willing to be convinced. I await comment.

Population prevalence of the ApoE4 gene

Regarding the Alzheimer genotype, I just did a little research regarding the prevalence of the ApoE epsilon 4 allele (the allele that predisposes to Alzheimer disease). Depending on the study you read, about 20% of the population has at least one epsilon 4 allele, while about 2% have two epsilon 4 alleles. As you'd expect, having two is worse than having one in terms of Alzheimer risk. I should add that the epsilon 4 allele also predisposes to a worse outcome in recovery from traumatic brain injury and is also over-represented among those football players who suffer from Chronic Traumatic Encephalopathy.

 

It's important to note that this data applies only to those of European ancestry. The rates of other ethnic groups are different. I saw one study showing, for example, that the rate of epsilon 4 prevalence is much, much higher among Australian aboriginal populations.   I recently sent in a saliva specimen to 23andMe.com to have their CLIA-approved lab run a genetic profile on me, which will include a report on my ApoE status. The service has dramatically decreased in price in recent years, so I finally broke down made the purchase. I'll report those results when I get them next month.

Another quick quiz question

All of the following are CAG-repeat disorders EXCEPT:

A. Huntington disease
B. Myotonic dystrophy
C. Olivopontocerebellar atrophy (spinocerebellar ataxia type 2)
D. Kennedy disease (X-linked spinal and bulbar muscular atrophy)
E. Machado-Joseph disease (spinocerebelllar ataxia type 3)
F. Dentatorubral-pallidoluysian atrophy

The answer appears as a comment.

MGMT status of glioblastomas: Is PCR or IHC better?

Back in February '08 I wrote a post arguing that MGMT testing on glioblastomas is still not ready for clinical use outside of clinical trials. Since not all of our oncologist colleagues are Neuropathology Blog disciples, I continue to occasionally get a request from a clinician for MGMT testing on glioblastoma specimens. MGMT stands for O(6)-methylguanine DNA methyltransferase. Why are they asking for this test? Alkylating agents, like temazolamide, are more effective when MGMT is not active in a tumor because MGMT normally acts to remove the toxic methylguanine adducts provided by temozolamide. So MGMT silencing, usually due to gene promoter hypermethylation, predicts better response to temazolamide.

What's the best method for assessing MGMT activity in a tumor? According to a review article in the July 2009 issue of Archives of Pathology and Laboratory Medicine by Drs. Peter Pytel (pictured) and Rimas Lukas (Vol133:1062-1077) "immunohistochemical staining for MGMT does not offer a reliable way to stratify glioblastomas, and polymerase chain reaction-based assays are therefore necessary." Pytel and Lukas, of the University of Chicago Medical Center, provide the following two references for this position:

-- Yip S, Iafrate AJ, Louis DN. Molecular diagnostic testing in malignant gliomas: a practical update on predictive markers. J Neuropathol Exp Neurol. 2008;67:1-15.
-- Preusser M, Janzer RC, Felsberg J, et al. Anti-O6-methylguanine methyltransferase (MGMT) immunohistochemistry in glioblastoma multiforme: observer variability and lack of association with patient survival impede its use as a clinical biomarker. Brain Pathol. 2008;18(4):520-532.

On the other hand, some would argue that immunohistochemistry picks up more cases of MGMT than does PCR. Since its still really an experimental test, the issue ultimately may come down to what insurance will pay for. Immunohistochemistry is cheaper, so the patient may have an easier time getting his or her insurance to pay for it. My position is this: I will continue to discourage clinicians from getting the test until it becomes standard practice. If they insist on getting it, I would recommend immunohistochemistry for cost reasons and send it out to the Duke University laboratory of Dr. Roger McLendon to have it done. If, however, PCR is specifically demanded, I will send it to LabCorp, a commercial reference laboratory, which offers the MGMT PCR assay on paraffin-embedded tissue.

Study sheds light on Huntington disease with implications for other neurodegenerative disorders

A common theme among neurodegenerative diseases is that there is a some kind of "nucleating protein" which aggregates within specific areas of the brain. There is debate as to whether the aggregates cause disease, or are simply an attempt by brain cells to sequester bad proteins. If the former is true, then therapy should be aimed at disaggregating the bad protein. If the latter is true, then therapy should be focused on helping the brain to inactivate the free-floating bad proteins. A recent advance in the understanding of Huntington disease (HD) suggests that the free-floating form of the protein may be the culprit, lending support to the idea that other neurodegenerative diseases like Alzheimer's and Parkinson's may also be caused by soluble proteins rather than the aggregates that we neuropathologists focus on under the microscope.

Scientists have known for some time that HD is associated with a trinucleotide repeat mutation in the protein huntingtin on chromosome 4. But, since huntingtin is present throughout the brain, why does neurodegeneration in HD take place predominantly in the striatum (caudate and putamen)? Solomon H. Snyder and his team at Johns Hopkins University, in the June 5th issue of the journal Science, show that cytotoxicity in HD takes place because of the interaction of mutant huntingtin with a second protein, known as Rhes. It turns out that Rhes is a striatal specific protein, thus explaining the anatomic specificity of neurodegeneration in HD. Snyder and colleagues go on to show that cells in culture tend to sequester mutant huntingtin into an aggregate. But in the presence of Rhes, mutatant huntingtin cannot aggregate, suggesting that the soluble form of the bad protein is what causes damage.

An Associated Press article about the discovery posted on Forbes.com quotes Walter J. Koroshetz of NIH's National Institute of Neurological Disorders and Stroke as follows regarding the implications of this new study: "The answers in one disease may have implications for another... There's been people on both sides of the fence. This story plays to the role of the aggregates as not being the major problem but the soluble protein as being the major problem."

The most famous Huntington patient was the iconic folk singer Woody Guthrie. And it was my colleague Doug W. Shevlin, who writes a pretty widely read music blog which today features a Woody Guthrie song, who alerted me to this break-through study. In a stroke of synchronicity, it so happens that my good friend Mark B. Weiss (pictured) is representing a performer at next month's Woody Guthrie Folk Festival in Okemah, Oklahoma.

"What should exist?"

"What should exist?" Those are the words that guide the philanthropy of Microsoft Co-Founder Paul Allen's Institute for Brain Science. One thing that Mr. Allen believes should exist is a genetic map of the murine brain, and he put up the $100 million to get that done. The searchable digital atlas of gene expression presents a comprehensive online platform for exploration of the brain at the cellular and molecular level. It is available on-line, for free, to anyone who wants to know which of the 21,000 genes in the mouse genome is activated in a particular area of the mouse brain. Pictured is a sample photomicrograph demonstrating the expression of a type of GABA receptor via in situ hybridization in one sagittal brain slice. In recognition of this and his other initiatives on behalf of brain science, Mr. Allen is being awarded the 2009 Public Leadership in Neurology Award by the American Academy of Neurology.

Rhadoid Tumor Predisposition Syndrome: A recently defined genetic disorder

With all the hullabaloo surrounding the 2007 revision of the World Health Organization Classification of Tumors of the Central Nervous System (4th edition), the fact that the WHO consensus committee also recognized a new genetic syndrome got lost in the mix. Here's the scoop on the new syndrome:

Rhabdoid Tumor Predisposition Syndrome (RTPS) is characterized by a germline mutation in the INI1 gene (chromosome 22q11.2) and manifested by a marked predisposition toward the development of malignant rhabdoid tumors of infancy and early childhood. The atypical teratoid/rhabdoid tumor (AT/RT) is by far the most common CNS malignacy associated with this syndrome. Up to one-third of AT/RTs are thought to arise in the setting of RTPS. Other tumors that have also been reported in association with this syndrome include: medulloblastoma, choroid plexus carcinoma, primitive neuroectodermal tumors (PNETs), and malignant rhabdoid tumor of the kidney. Because of the risks associated with RTPS, it is recommended that the germline status of the INI1 allele be investigated in each new case of AT/RT.

Reference: Brat, Daniel J., et al. "Surgical Neuropathology Update: A Review of Changes Introduced by the WHO Classification of Tumors of the Central Nervous System, 4th edition" in Archives of Pathology and Laboratory Medicine: 2008;132: 993-1007).

MGMT and MGMT promoter hypermethylation testing not ready for prime time

A few months ago, an oncologist at my institution asked for an MGMT promoter hypermethylation assay on a high grade glioma. I looked into the issue, and discovered that testing is not standardized yet and really not ready for clinical use. In a review article on molecular testing of gliomas in the January '08 issue of Journal of Neuropathology and Experimental Neurology, the authors concur with this position. That being said, if anyone does want to do immunohistochemistry (IHC) for MGMT, Dr. Roger McLendon at Duke performs this test. Some would argue that PCR is better than IHC, but the good Dr. McLendon counters that immunohistochemistry is more reliable as it correlates with enzyme activity while the PCR promoter methylation assay does not. In any case, I got the oncologist to cancel her request for MGMT promoter methylation studies.

Abstract from journal Cell on stem cells

This is off topic, but there's big news today regarding the
creation of pluripotent cells from human skin fibroblasts.
Here's the abstract from the Yamanaka study in Cell:
"Successful reprogramming of differentiated human
somatic cells into a pluripotent state would
allow creation of patient- and disease-specific
stem cells. We previously reported generation
of induced pluripotent stem (iPS) cells, capable
of germline transmission, from mouse somatic
cells by transduction of four defined transcription
factors. Here, we demonstrate the
generation of iPS cells from adult human dermal
fibroblasts with the same four factors: Oct3/4,
Sox2, Klf4, and c-Myc. Human iPS cells were
similar to human embryonic stem (ES) cells in
morphology, proliferation, surface antigens,
gene expression, epigenetic status of pluripotent
cell-specific genes, and telomerase activity.
Furthermore, these cells could differentiate
into cell types of the three germ layers in vitro
and in teratomas. These findings demonstrate
that iPS cells can be generated from adult
human fibroblasts."