Immunohistochemical surrogate for BRAF V600E mutation

Quoted highlights  from: Tanboon J, Williams EA, and Louis DN. The Diagnostic Use of Immunohistochemical Surrogates for Signature Molecular Genetic Alterations in Gliomas. J Neuropathol Exp Neurol Vol. 75, No. 1, January 2016, pp. 4–18:

- The most common BRAF alteration is an activating mutation caused by a substitution of valine for glutamic acid at codon 600 (BRAF V600E) in exon 15.

- BRAF V600E status can be screened for using the mutation-specific BRAF V600E immunohistochemistry clone VE1... Only strong homogeneous unambiguous cytoplasmic staining should be interpreted as positive.

- While BRAFV600E can be found in a wide variety of brain tumors, BRAF fusions... are mostly limited to pilocytic astrocytomas (PAs).

- For tumor classification, aBRAF V600E mutation in a ganglion cell-rich lesion biopsied from infratentorial region favors ganglioglioma over PA.

- BRAF V600E mutations are present in pleomorphic xanthoastrocytoma (PXAs) with and without anaplasia (65%–70%), ganglioglioma (33%–60%), and [occasionally] PA, especially extracerebellar PA (6%–8%).

- In addition, BRAFV600E mutations are common in epithelioid glioblastomas (eGBM) (54%), which also tend to occur in children and relatively young adults.

- BRAF V600E mutations have also been reported in a small number of desmoplastic infantile astrocytomas, desmoplastic infantile gangliogliomas, and dysembryoplastic neuroepithelial tumors.

- A few diffuse astrocytomas in children and adults harbor BRAF V600E mutation; the tumors in adults show unusual histologic features such as partly circumscribed portions and spindle cells, and may associated with more favorable prognosis.

- Strong cytoplasmic staining patterns are often observed in PXA and eGBM; on the other hand, staining can be variable in glioneuronal tumors because BRAF can be diffusely positive in the tumor cell population or it can be limited to either a glial or neuronal component. For example, in ganglioglioma, mutant BRAF is predominantly expressed in neuronal tumor cells

- In glioneuronal tumors, BRAF V600E mutation is associated with activation of the mammalian target of rapamycin (mTOR) pathway, worse postoperative seizure outcome, and shorter recurrence-free survival.

Immunohistochemical surrogate for H3 K27M mutations

Quoted highlights on IHC mutational surrogates from: Tanboon J, Williams EA, and Louis DN. The Diagnostic Use of Immunohistochemical Surrogates for Signature Molecular Genetic Alterations in Gliomas. J Neuropathol Exp Neurol Vol. 75, No. 1, January 2016, pp. 4–18:

- K27M mutations in H3F3A [the gene encoding H3.3] and HIST1H3B [the gene encoding H3.1] occur in approximately 40%–80% of pediatric diffuse intrinsic pontine gliomas (DIPG) and 20% of nonbrainstem glioblastomas.

- The central role of these mutations in such tumors will result in these neoplasms being designated as “diffuse midline glioma, H3 K27M-mutant” in the 2016 WHO Classification of Tumours of the Central Nervous System

- The presence of K27M mutations in pediatric glioblastomas (including DIPG) is associated with shorter survival compared to the wild-type tumors... [T]he presence of K27M mutations can be used as a diagnostic marker and poor prognostic marker for pediatric high-grade astrocytoma.

- K27M mutations in both the H3.3 and H3.1 histones can be detected by immunohistochemistry using an anti-H3K27M antibody. Positivity can therefore be useful to establish a diagnosis of infiltrating glioma in lower-cellularity biopsies, and can be used to subtype and genotype these diffuse gliomas. 

- The pattern of positivity is nuclear, with positivity in most of the tumor cells; nontumor cell nuclei are negative.

Immunohistochemical surrogate for ATRX mutation

Quoted highlights on IHC mutational surrogates from: Tanboon J, Williams EA, and Louis DN. The Diagnostic Use of Immunohistochemical Surrogates for Signature Molecular Genetic Alterations in Gliomas. J Neuropathol Exp Neurol Vol. 75, No. 1, January 2016, pp. 4–18:

- Loss of nuclear staining for ATRX protein by immunohistochemistry has been used as a surrogate marker for ATRX mutations.

- Consistent and strongly positive staining in the nuclei of nonneoplastic endothelial cells and neurons is commonly used as internal positive controls.

- Unfortunately, there are no standard criteria (in terms of number of cells) for what constitutes loss of ATRX staining in gliomas.

- Because ATRX mutations are uncommon in IDH-mutant tumors with 1p/19q codeletion, it is suggested that ATRX, along with TP53 mutations, can be used as markers of astrocytic lineage.

- In adults, ATRX mutations have been reported either by sequencing or immunohistochemistry in 45%–67% of diffuse astrocytomas, 57%–73% of anaplastic astrocytomas, and 33%–57% of secondary glioblastoma; ATRX mutations are uncommon in primary glioblastoma (4%–7%)

- In children, ATRX mutations have been reported in 22% of pediatric diffuse intrinsic pontine gliomas and 48% of nonbrainstem high-grade gliomas in children;when ATRX mutation occurs in children, patients tend to be over 11 years old.

Immunohistochemical surrogate for TP53 mutation

Quoted highlights on IHC mutational surrogates from: Tanboon J, Williams EA, and Louis DN. The Diagnostic Use of Immunohistochemical Surrogates for Signature Molecular Genetic Alterations in Gliomas. J Neuropathol Exp Neurol Vol. 75, No. 1, January 2016, pp. 4–18:

- Because they are rare in nonneoplastic brain lesions, TP53 mutations can be used as a marker to differentiate glioma from gliosis.

- TP53 alterations ... are essentially mutually exclusive with 1p/19q codeletion.

- Tumorigenic TP53 mutations have been reported to be present in > 50% of gliomas with astrocytic features, including 59%–74% of diffuse astrocytomas, 53%–65% of anaplastic astrocytomas, and 62%–65% in secondary glioblastomas. In contrast, these mutations are less common in gliomas with oligodendroglial features (9%–44%), and in primary glioblastomas (23%–28%).

- Intense nuclear staining for p53 protein by immunohistochemistry in a substantial percentage of tumor cells has long been used as a surrogate marker for TP53 mutations. The underlying mechanism is abnormally elongated half-lives for the protein products of the most common TP53 mutations in gliomas. 

- Strong p53 nuclear positivity in > 10% of the tumor cells is the most accurate predictor for TP53 mutations in gliomas.

- Positivity of p53 immunohistochemistry staining can occur in [non-neoplastic] conditions of cellular stress, [causing false positive results].

Immunohistochemical surrogate for IDH1 mutation

Quoted highlights on IDH mutation IHC from: Tanboon J, Williams EA, and Louis DN. The Diagnostic Use of Immunohistochemical Surrogates for Signature Molecular Genetic Alterations in Gliomas. J Neuropathol Exp Neurol Vol. 75, No. 1, January 2016, pp. 4–18:

- IDH1 and IDH2 mutations are mutually exclusive events and indicate one of the early processes in gliomagenesis, before TP53 and ATRX mutations in astrocytic tumors, and before 1p/19q codeletion, CIC, and FUBP1 mutations in oligodendroglial tumors

- IDH mutations exist in at least 70% of diffuse gliomas, particularly World Health Organization (WHO) grade II and III astrocytomas, oligodendrogliomas, and secondary glioblastomas, and are rarely present in other types of brain tumors

- Clinically, patients with either IDH1 orIDH2 mutations are younger and have a better prognosis in terms of both overall survival and progression-free survival compared to patients carrying wild-type IDH

- Intriguingly, recent studies reveal similar age of onset and little differences in clinical outcome among IDH-mutant tumors previously classified as grade II and grade III astrocytomas by WHO 2007 criteria

- The “good effect” of having IDH mutation also applies to glioblastomas since patients with IDH-mutant glioblastomas have better clinical outcomes compared to those with grade III astrocytomas having wild-type IDH...

- The presence of IDH mutations may argue in favor of a diagnosis of anaplastic glioma over primary glioblastoma given that the latter typically does not harbor the mutation

- The most useful antibodies detect the common mIDH1 R132H mutation, which is present in 90% of IDH-mutant gliomas

- Immunohistochemistry for mIDH1 R132H clone H09 shows 88%–100% concordance rate with IDH1 R132H mutational status determined by DNA sequencing

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.

Amyloid-beta and neurofilament in 3D

Three-dimensional immunohistological visualization of Aβ (green) and neurofilament (red) in a 500-micron thick block of human post-mortem Alzheimer disease tissue.
 
From:Kunie A, et al. Inside Alzheimer brain with CLARITY: senile plaques, neurofibrillary tangles and axons in 3D. Acta Neuropathologica. September 2014, Volume 128, Issue 3, pp 457-459.

Thanks to Dr. Mark Cohen of Case Western for sending me the link.

What's on your tumor biomarker wish list?

Photo courtesy of Shellie Sherrod

The College of American Pathologists Neuropathology Committee (pictured, minus the illustrious Dr. Aaron Wagner) poses the following question to the neuropathology community worldwide:

"If you were constructing a panel of eight biomarkers there were well-established prognostic/predictive markers for CNS neoplasms, what markers would be included? Note: this is not for diagnosis, but for predicting behavior and should include adult and pediatric brain tumors and can include immunohistochemical and molecular tests."

Thanks in advance for providing your opinion in the comments section.

Best Post of August '10: Highly pure anti-IDH1 R132H lyophilizates now available - get a free sample

August 2010 is next up in our "Best of the Month" series. The following guest post from Dr. Olaf Bartsch appeared on August 27th and addresses an important development in the world of neuropathological immunohistochemistry. (Note: I'm not sure whether free samples are still available.)


The German company Dianova now provides reliable highly pure (goat anti-mouse chromatography) lyophilizates of antibody clone H09. The antibody has been purified from cell culture supernatants by GAM (goat anti-mouse) affinity chromatography resulting in a more intense staining of oligodendroglioma versus a clear negative reaction with glioblastoma.

Dianova's proprietary antibodies are now available in the USA directly and can be ordered from U.S. and Canadian customers on the HistoBioTec website or by purchase order.

Please see the datasheet for details and order a free sample.

Fig.1

Double staining of GFAP (red) and clone H09 (brown) of oligodendrogliopma infiltrating zone demonstrating specific labelling of tumor cells but not GFAP positive reactive astrocytes.

Antibody clone H09 serves as a Tumor Cell Marker for Astrocytoma and Oligodendroglioma and is highly usefull for the differentiation of reactive gliosis from low grade glioma. Moreover, the antibody differentiates single infiltrating tumor cells from reactive gliosis.

Fig.2

Infiltrating zone of anaplastic astrocytoma with specific labelling of infiltrating glioma cells by antibody clone H09.

Clone H09 was developed for staining of standard formalin-fixed paraffin embedded (FFPE) sections in clinical routine and is the first commercially available antibody that specifically stains single cells carrying the IDH1R132H point mutation.

Fig.3

Cortex infiltrated by oligodendroglioma with specific labelling of tumor cells by antibody clone H09.

Best Post of May 2010: Free Samples of IDH1, the next big thing in glioma IHC, is now available

The next in our Best of the Month series is from May 13, 2010. Free samples of IDH1 may or may not still be available, but you can check with the company:

Dianova, the German company that makes anti-human IDH1 antibody for use in immunohistochemical staining of gliomas versus gliosis, is providing free samples of its product. Just go to the site, click on "free sample", and make a request via email. This marker may well be a tremendous advancement in distinguishing reactive astrocytosis from diffuse astrocytoma, so I would encourage you to get your free sample! Thanks to Drs. Craig Horbinski and Ty Abel for assisting me in discovering this opportunity.

Highly pure anti-IDH1 R132H lyophilizates now available - get a free sample

A guest post regarding an important development from Dr. Olaf Bartsch:

The German company Dianova now provides reliable highly pure (goat anti-mouse chromatography) lyophilizates of antibody clone H09. The antibody has been purified from cellculture supernatants by GAM (goat anti-mouse) affinity chromatography resulting in a more intense staining of oligodendroglioma versus a clear negative reaction with glioblastoma.

Dianova's proprietary antibodies are now available in the USA directly and can be ordered from U.S. and Canadian customers on the HistoBioTec website or by purchase order.

Please see the datasheet for details and order a free sample.

Fig.1

Double staining of GFAP (red) and clone H09 (brown) of oligodendrogliopma infiltrating zone demonstrating specific labelling of tumor cells but not GFAP positive reactive astrocytes.

Antibody clone H09 serves as a Tumor Cell Marker for Astrocytoma and Oligodendroglioma and is highly usefull for the differentiation of reactive gliosis from low grade glioma. Moreover, the antibody differentiates single infiltrating tumor cells from reactive gliosis.

Fig.2

Infiltrating zone of anaplastic astrocytoma with specific labelling of infiltrating glioma cells by antibody clone H09.

Clone H09 was developed for staining of standard formalin-fixed paraffin embedded (FFPE) sections in clinical routine and is the first commercially available antibody that specifically stains single cells carrying the IDH1R132H point mutation.

Fig.3

Cortex infiltrated by oligodendroglioma with specific labelling of tumor cells by antibody clone H09.

If you like to discuss any issues regarding application of this unique antibody, you are welcome to visite our booth at the ICN 2010 (XVIIth International Congress of Neuropathology), September 11-15 in Salzburg.