October 22, 2018 12:00-1:00 PM Background The World Health Organization Classification of tumors of the Central Nervous System has recently been revised. There is now greater emphasis on molecular phenotype for tumor diagnosis and treatment. Isocitrate Dehydrogenase (IDH) mutation has been recognized in a subset of glial tumors, predominantly low grade and secondary gliomas and associated with an improved prognosis. IDH status and 1p19q FISH analysis for co-deletion of 1p and 19q allow for definitive diagnosis of oligodendrogliomas. In addition to 1p19q co-deletion, FISH can also detect 1p and/or 19q polysomy in a subset of oligodendrogliomas; this appears to be an indicator for worse prognosis / early recurrence in these tumors. Additional markers, such as BRAF, ATRX, EGFR and p53 can also provide diagnostic benefit in challenging cases. Many laboratories are faced with the challenge of implementing new immunohistochemical or molecular markers for clinical use, especially when CNS tumors are a relatively small subset of cases received by some departments. However, there are justifications for adding these new markers to the diagnostic armamentarium and useful tips on validation as follows: 1. Neurosurgical cases typically require costly intracranial or spinal surgical approaches and diagnostic material can be surprisingly scant, prohibiting all but a limited number of slides for immunohistochemical or molecular phenotypic analysis. 2. Neurosurgeons not uncommonly require a frozen section for diagnostic material which may introduce freezing artifact making permanent section evaluation even more problematic. a. Needle biopsies can be exceedingly small, but even large resections may provide scant tissue for diagnostic workup. b. Biopsies may be from eloquent regions of the brain (motor or speech areas) or from the brainstem, limiting the size of even needle biopsies being submitted. c. Differential diagnoses for brain lesions may be broad and include infection, reactive, lymphoma or malignant tumors (primary or metastatic) with widely different treatments and prognoses, with either limited resection or attempt at gross total resection based on the frozen result. d. Markers with the most bang for the buck are essential to ensure adequate tissue for diagnosis. 3. Many useful markers can be done reliably by immunohistochemistry, with molecular testing only when the results fail to fit the expected phenotype. 4. Even relative common CNS tumors can be so infrequent to justify a limited number of cases for validation study (10 cases verses the more typical 20). 1
Discussion questions 1. What is the best approach to workup of an intra-operative frozen section? a. Examples: i. What are the differences between what a smear and frozen section can tell you histologically and what information do you need to know (radiographic or from the surgeon) before handling the tissue? ii. Does CNS tissue differ from systemic tissues in regard to freezing and are there ways of minimizing artifacts? iii. What permanent section histologic changes can be seen as a result of freezing? iv. What should you tell the surgeon regarding and frozen section and what should be avoided? b. ANSWERS: 1. If the tissue is only pinhead sized, check with the surgeon that you will be getting additional tissues. If not, recommend keeping the tissue for permanent. If they are going to submit more, be cautious, it might not be much! 2. Is the tissue firm or soft? If firm: frozen can be better than a smear. 3. I have found a smear of any tissue, firm of not, is telling. A smear usually releases at least a few cells which can be extremely helpful at making a diagnosis. 4. When making a smear, most people use way too much. It is best to use three small pin head sized pieces lined along the top of the slide to smear (yes, only pin head sized!). If the tissue has variable colored tissue (white matter, gray matter, discolored tissue) take a pin head piece from each to represent those three pieces. 5. The smear is best for assessing the individual cellular features: if the nuclei are variable or uniform in size, if they are hyperchromatic or not, the amount of cytoplasm and if the cells have smooth or fibrillary cell boarders. Astrocytic tumor cells typically have thick ropy cell processes rather than thin fine star like reactive processes. 6. Smears are NOT very helpful in assessing cellularity 7. A frozen is best for assessing the architecture of the infiltrate and the cellularity of the tumor 2. What molecular markers do you have and why? a. 1p19q FISH i. Do the results include polysomy? ii. What is the significance of polysomy with co-deletion? iii. What is the significance of polysomy without co-deletion? 1. ANSWERS: When obtaining FISH analysis for 1p19q deletion, it is helpful, but not essential to also obtain details of the chromosomal analysis for polysomy. Polysomy in the setting of 1p19q co-deletion is associated with a worse prognosis. The significance of polysomy in the setting of only 1p deletion, only 19q deletion, or no 1p19q deletion is unclear. We prefer to 2
report these results in the event they are found to be significant. b. IDH1 and IDH2 i. What are the clues to assure the immunohistochemical marker is working? ii. Are mutant tumors positive or negative with IDH1 immunohistochemistry? iii. If needed which should you do, a hot spot panel or direct sequencing? iv. What about additional tests for IDH1 R132H, IDH1 R132C, or IDH2 R172K? v. Adult and pediatric tumors and IDH mutation or IDH wild type phenotypes? 1. ANSWERS: IDH1 R132H immunostains are pretty good. The reactivity should be cytoplasmic and there is usually an unequivocal moderate to strong reactivity for the antibody. If the IDH1 R132H antibody is non-reactive, then testing for alternate IDH1 and IDH2 mutations are warranted. Suggestions that molecular testing are indicated if the IDH1 R123H results are negative are: 1) a tumor with oligodendroglial features and without and ATRX mutation, 2) an astrocytic tumor with ATRX loss; 3) a patients with an astrocytic tumor who is younger than 55 years of age (irrespective of ATRX mutational status); or 4) astrocytic tumors which are lower grade than an glioblastoma, WHO grade IV. HOWEVER, keep in mind IDH and ATRX mutations are typically present in oligodendrogliomas, diffuse astrocytomas, anaplastic astrocytomas, and glioblastomas. These mutations are typically NOT present in pediatric tumors or diffuse midline gliomas. c. ATRX i. What are the clues to assure the immunohistochemical marker is working and is a mutant tumor positive or negative? ii. Adult vs pediatric tumors? 1. Adult tumors with ATRX mutation and IDH mutation? 2. Pediatric tumors with ATRX mutation and IDH wild type? iii. Association with primary or secondary gliomas? 1. ANSWERS: ATRX antibodies can be finicky. If the tissue has any processing issues such as crush, cautery, or anything else, the immunohistochemical stain may show some artifactually loss of staining of cell nuclei. When the reactivity is believable, most of the tumor cell nuclei fail to stain while the background normal cell nuclei (neurons, blood vessels) show strong reactivity. If a tumor has an IDH mutation and 1p19q co-deletion, the ATRX should not be mutated (oligodendroglioma). If the tumor is a low grade astrocytic tumor with an IDH mutation, it typically should have an mutation and if it does not, it is possible the reactivity for ATRX is aberrant. Grade II or III astrocytomas with or without necrosis or vascular hyperplasia, but with a primary glioblastoma phenotype (wild-type IDH and ATRX) suggest the 3
tumor may behave more like a glioblastoma and should be closely followed (MGMT helpful). Glioblastomas with IDH and ATRX mutations are diagnostic of a lower grade tumor transformed to higher grade (secondary glioblastoma) and may have a better prognosis (MGMT useful). Pediatric tumors, both low grade and high grade, typically do not have IDH mutations but may show ATRX mutations in the setting of wildtype IDH. 2. There are a few ATRX antibodies. We use SIGMA (Atlas) HPA001906, but others are fine as long as you standardize. Rare tumors (most brain tumors) require about 20 tumors (10 each for positive and negative) to standardize. We use a dilution of 1:100 and antigen retrieval with citrate buffer instead of normal TRIS (CC2 on Ventana Ultra). Other antibodies may require a 1:200 dilution. d. BRAF V600E i. In Glioblastoma? ii. In Pediatric tumors? iii. With Craniopharyngioma? iv. Relationship between pediatric tumors and glioblastoma? 1. ANSWERS: If a tumor occurs in a young adult, BRAF V600E is important to do to rule out an epithelioid glioblastoma. Many low grade pediatric tumors have BRAF V600E mutations and can be helpful at verifying a neoplastic process, although other markers, such are CD34, NeuN, Neurofilament protein, and synaptophysin can aid the diagnosis, but many of these tumor diagnoses are made by histology alone. A Papillary craniopharyngioma can be differentiated from an adamantinomatous craniopharyngioma, useful in cases with minute available tissue. e. H3K27M i. Midline tumors in young adults and pediatric patients and Histone H3K27M? ii. Histone H3 gene H3F3A (cortical)? iii. Histone HIST1H3B gene? 1. ANSWERS: the antibody for H3K27M is a nuclear antibody. If the blood vessels and normal cell nuclei are positive, the marker may not have worked. Diffuse midline gliomas can occur in both pediatric and adult patients, but much more prevalent in the pediatric population. H3K27M mutations occur in histone 3 genes (H3.3 and H3.1) and are seen exclusively in midline gliomas while another histone mutation, H3F3A G34RN, in the H3.3 gene, has been associated with peripheral cortical gliomas in slightly older patients. Biopsies of midline tumors typically are extremely scant and H3K27M testing is strongly recommended by the AANP for these tumors. 2. Methodology described in Acta Neuropathol (2014) 128:733-741 Specific detection of methionine 27 mutation in histone 3 variants (H3K27M) in fixed tissue from high-grade astrocytomas 4
a. Use a single monoclonal antibody which shows good correlation to molecular detection by sequencing b. Millipore #ABE419, 1:500 on standard Ventana XT machines, CC1, cost ~$370 (others may be comparable) c. The antibody is specific for tumor cells containing the H3K27M mutation at either H3.1 or H3.3 histone variants, and shows minimal background. d. Strongly recommended by AANP for detection of H3K27M in midline gliomas for diagnostic and prognostic purposes f. MGMT methylation i. Prognostic implications? ii. When to do the testing? 1. ANSWERS: tumors with MGMT methylation have an better prognosis than those without MGMT methylation. All oligodendrogliomas have MGMT methylation, therefore testing for MGMT is not necessary, but some oncologists may still want it done. This is an essential test, requested on ALL malignant astrocytomas/gliomas. Equivocal test results may indicate low levels of methylation or a subset of cells with methylation and these patients may have a somewhat improved prognosis. 2. In this assay we do MGMT promoter methylation by bisulfite conversion followed by pyrosequencing. In the assay, we evaluate the % of methylation on 5 CpG sites in the promoter of the gene. The average is calculated for the 5 sites and the acceptable reporting criteria are as follows: a. Methylation is Undetected: if the average % of methylation is <8% b. Indeterminate methylation: if the average % of methylation is 8-13% c. Methylation is Detected: if the average % of methylation is >13% g. EGFR gene amplification status i. What is the expected immunohistochemical staining pattern and how is it helpful? ii. If equivocal do you do molecular testing? Hot spot or ISH? What are positive or negative ISH results? 1. ANSWERS: EGFR immunohistochemical stains show a membranous staining pattern, but can be difficult to interpret. We do up-front EGFR ISH analysis. Primary gliomas typically are more apt to be positive. EGFR ISH can be helpful in differentiating high grade ependimomas vs gliobastoma in the spinal cord of brain. 2. Analysis results of EGFR amplification status is preformed using multiplex Dual Color DNA Probe Silver in Situ Hybridization (ISH) Assay. EGFR and Chromosome 7 (CEP7) are evaluated on formalin fixed, paraffin embedded sections using the FDA-approved Ventana UltraView SISH and red ISH 5
DNA Detection Kits with EGFR and CEP7 ISH DNA probes (others may be comparable). a. Adequacy of sample (adequate number of invasive tumor cells present): we require at least 20 tumor cells to be present b. Total number of EGFR signals (black): varies clusters that cannot be accurately counted are each counted as >12 signals. Total number of CEP7 signals (red): varies can be more if there is polysomy or very rarely less if the entire chromosome is deleted. Sometimes it looks like there is only one because the entire face of the cell is missing. c. Average ratio of total EGFR/CEP7 signals: i. Positive: ISH EGFR gene/cep7 ratio of >/= 2.0 ii. Negative: ISH EGFR gene/cep7 ratio of < 2.0 h. P53 mutation i. What is an acceptable level of staining to still be negative? ii. What does positive nuclear staining imply regarding glial tumors? Oligodendrogliomas verses Astrocytomas? iii. Progression of tumor? 1. ANSWERS: oligodendrogliomas can have up to 15-20% positive staining, despite 1p19q co-deletion being mutually exclusive with a p53 mutation. Glial tumors with a predominance of p53 staining are suggestive of an astrocytic tumor and are more common in low astrocytomas or secondary glioblastomas. Recurrent tumors may or may not develop increasing levels of p53 3. How do you approach validation testing? a. How many specimens, positive and negative, do you need to have to validate a marker? b. In rare tumors? c. Which antibodies are best to use? i. ANSWERS: CNS tumors are rare, therefore 10 positive and 10 negative cases can be used for validation References 1. Abedalthagafi M, Phillips JJ, Kim GE, et al. The alternative lengthening of telomere phenotype is significantly associated with loss of ATRX expression in high-grade pediatric and adult astrocytomas: a multi-institutional study of 214 astrocytomas. Mod Pathol.2013 Nov;26(11):1425-32. 2. Brastianos PK, Shankar GM, Gill CM. et al. Dramatic Response of BRAF V600E Mutant Papillary Craniopharyngioma to Targeted Therapy. J Natl Cancer Inst.2015 Oct 23;108(2). 3. Chappe C, Padovani L, Scavarda D, et al. Dysembryoplastic neuroepithelial tumors share with pleomorphic xanthoastrocytoma and gangliogliomas BRAF (V600E) mutation and expression. Brain Patholo.2013 Sep;23(5):574-83. 4. Cohen AL, Holmen SL, Colman H. IDH1 and IDH2 mutations in gliomas. Curr Neurol Neurosci Rep.2013 May;13(5):345. 5. Dougherty MJ, Santi M, Brose MS, et al. Activating mutations in BRAF characterize a 6
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