Chromosomal aberrations in chordoid meningioma – An analysis
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.222808
Source of Support: None, Conflict of Interest: None
Keywords: Chordoid meningioma, chromosomal aberration, fluorescent in situ hybridization (FISH), WHO grade II
Chordoid meningiomas (CMs), WHO grade II tumors, are uncommon variants of meningioma. CMs are typically large, supratentorial,, dural-based tumors which behave aggressively when compared to other grade II tumors. They have striking features such as cords or trabeculae of eosinophil and vacuolated cells in an abundant mucoid matrix background. The tumor commonly shows peritumoral reactive lymphoplasma cellular infiltration., They are associated with a poor prognosis and exhibit a very high rate of recurrence of approximately 42%. Recurrence is generally predicted based on the surgical dissection, the percentage of chordoid element, and proliferation index. Approximately 75% of patients show recurrence within a period of 1 year when only subtotal resection is achieved, thereby increasing the need for adjuvant therapy, i.e., radiotherapy or chemotherapy, particularly when Simpson grade I resection is not achieved. Many studies suggest that high proliferation indices (KI-67 levels) of >4% MIB levels are predictors of recurrence., Chordoid elements range from 10 to 100% in these tumors, and it is found that tumors with 50% or more of chordoid elements help in determining recurrence.
There have been only two studies on chromosomal aberrations in CM in the available literature. One on chromosomal alteration of primary and recurrent CM evaluated the loci MYCN, ERBB4, CDH1, ABR, ERBB2, and NF2 probes  and showed that there is some level of alteration in genes such as NF2, MYCN, ABR, and ERBB2 among primary and recurrent CMs. Another study showed an unbalanced translocation of t(1;3)(p12-13;q11) as a unique feature of CM., Most frequent chromosomal aberration in meningiomas such as deletions of chromosomes 1, 3, 6, 9, 10, 14, 18, and 22, which are responsible for the genesis and malignancy, have not been studied in CM, except for 22q. Thus, our aim was to study the chromosomal aberrations of extensively studied gene loci such as 22q, 18p, 14q, and 1p and to correlate the findings with the clinical outcome of patients.
This study was conducted at the National Institute of Mental Health and Neurosciences (NIMHANS), Bengaluru. A total of 25 intracranial CMs were operated from 2001 to 2013. The paraffin blocks were retrieved and the neuropathologist reviewed all the cases. Ten cases were excluded either due to inadequate tissue or reclassification into other subtypes. Clinical, demographic, and follow-up data were collected for patients. The institutional ethics committee approved the study. The mean age of patients was 32 years. The median follow up duration was 28 months with the maximum follow up duration being 151 months.
Fluorescence in situ hybridization
Fluorescence in situ hybridization (FISH) was conducted on formalin-fixed paraffin-embedded (FFPE) sections. 4-μm thick tumor tissues were collected on silane-coated slides. Locus-specific single-color probes were used for performing the assay. The genes studied included Neurofibromin 2(NF 2) [Empire Genomics, Buffalo, NY 22q12.2], DAL-1 or 4.1B gene or Erythrocyte Membrane Protein Band 4.1- Like 3 (EPB41L3) [Empire Genomics, Buffalo, NY, 18p11.3], Maternally Expressed Gene 3 (MEG-3) [Empire Genomics, Buffalo, NY, 14q32.2], and Cyclin-Dependent Kinase Inhibitor 2C (CDKN2C) [Empire Genomics, Buffalo, NY, 1p32.3]. After paraffin pretreatment and protease digestion, the probe was applied to the sections. Simultaneous probe/specimen denaturation at 80°C for 3 min and hybridization at 37°C for 20 h was performed in a ThermoBrite™ hybridization chamber (Vysis Instrumentation, Abbott Laboratories, Illinois, USA). The posthybridization process included washing in 2× SSC (saline sodium citrate buffer) with 0.3% NP-40 (nonidet P-40) for 5 min at room temperature and 2× SSC with 0.3% NP-40 at 73°C for 1 min. Nuclei were counterstained with DAPI (1000 ng DAPI [4',6-diamidino-2-phenylindole]/ml in antifade mounting solution, Vysis). The sections were studied with a trinocular research fluorescent microscope (Olympus Model: BX51; Olympus, Tokyo) equipped with a set of appropriate filters. Signals were counted for 100 nonoverlapping nuclei. Every case was examined and the abnormalities were divided based on the regional distributions of chromosomal aberrations (i.e. homogenous or heterogeneous) and the type of deletion (homozygous or heterozygous). Thus, the cases were categorized into three groups: Completely deleted (homogenous homozygous/heterozygous deletion), partially deleted (heterogeneous heterozygous deletion), retained (intact or nondeleted status).
Cut-offs for deletions and aberrations were based on the frequencies of signals for the same probes in nonneoplastic brain control tissues. If the FISH signals were too weak to count, the hybridization was considered noninformative.
Paraffin sections (4 μm) from the tissue were collected on silane-coated slides, and immunohistochemistry for the protein expression of KI-67 (Biogenex-MIB-1, Monoclonal Mouse, 1:90), EMA (Biogenex, RTU), vimentin (Biogenex, Mouse, 1:150) and GFAP (Biogenex, Mouse, 1:200) was performed. Tumor tissues that previously showed overexpression served as positive controls and were included in each batch of staining. Antigen retrieval was done by heat treatment of the deparaffinized sections in Tris–EDTA buffer (pH 8.9). After the initial processing steps, sections were incubated overnight with the primary antibody at 4°C. This was followed by incubation with the linked streptavidin-biotinylated secondary antibody and then with the supersensitive nonbiotin horseradish peroxidase detection system (Nucleus Technologies, MACH1 Universal HRP-Polymer Detection, 110 ml). For MIB-1, the labeling index (LI) was expressed as a percentage of cells that showed intense positive staining among the total number of cells counted.
A total of 25 patients were operated. Out of these, two cases were eliminated as they were spinal CMs, six were reclassified as other varieties of tumors, and two were not included on account of inadequate tissue. The mean age of patients was 32 years (minimum age = 9 years, maximum age = 50 years) and male to female ratio was 1:2.75. There were 14 skull vault and 1 basal CM. Simpson's grade (SG) 1 resection was performed in four cases out of 15 patients. SG2, SG3, SG4, and SG5 excision was performed in 5/15, 4/15, 1/15, and 1/15 cases, respectively. The median follow up was 28 months, ranging from 3 to 151 months. During this follow-up period, recurrence was noted in five patients, whereas 10 showed no clinical evidence of recurrence. Of the five patients with a recurrence, only three underwent surgery for the second time.
All cases were variably positive for EMA and showed strong positive staining for vimentin, but were completely negative for GFAP, in support of the diagnosis. The MIB labeling ranged from 0.5 to 10% with a mean of 2.7%. Representative images of histology and IHC for MIB-1, EMA, and vimentin for CMs are shown in [Figure 1].
Fluorescence in situ hybridization
All samples showed adequate FISH efficiency. All the cases (n = 15) showed either complete or partial deletion at 22q, 14q, and 1p loci. For 18p loci, two cases showed retention status whereas the remaining cases showed either a partial deletion or a complete deletion status. The frequencies of chromosomal aberrations are shown in [Table 1] and representative images for these are depicted in [Figure 2].
In the present cohort, only 33% of the cases had tumor recurrence on a maximum follow-up of 12 years. None among the four patients who underwent Simpson's grade I resection showed a recurrence. Interestingly, in our study, proliferative index (MIB1 labeling) was low (0.6 ± 0.22) in the recurrent sample when compared with the nonrecurrent (3.75 ± 4.1) samples. 80% (4/5) of the recurrent cases showed complete deletion on all four chromosomal loci whereas 90% of the cases (9/10), where the tumor did not recur, had either partial deletion or an intact chromosomal status. The contrast between recurrent and nonrecurrent patients is given in [Table 2].
CMs are commonly grouped according to their grade of malignancy; however, these tumors behave much more aggressively when compared with other WHO grade II meningiomas. Therefore, it is important to study the tumor specific alterations involved in their genesis and evolution.
These tumors have poorer prognosis and a higher recurrence rate of approximately 42%. In general, factors influencing recurrence in meningiomas include unfavorable tumor localization, incomplete surgical dissection, and high tumor cell proliferation, among others. Specifically, in CM, an additional factor responsible for recurrence is the extent of chordoid elements. It was observed that 85% of tumors relapsed when chordoid elements were more than 50%., As our cohort (n = 15) included cases which had predominantly chordoid areas (>50%), such a comparison was not attempted. Studies on CMs have shown that MIB-1 level is usually low in these tumors ranging from 1 to 2%., The mean MIB labeling index (LI) in our study was 2.7 ± 0.8%. We noted that the MIB-1 labeling index did not correlate with recurrence in this cohort of CMs. The mean MIB-1 LI in recurrent cases was very low, about 0.6% (n = 5), ranging from 0.5 to 1%, and most of the nonrecurrent cases had a higher mean (3.7%). Even though there have been studies that correlate high MIB-1 levels with recurrence, similar to our observation, there are a few others that have shown that MIB-1 is not an accurate indicator for predicting recurrence.
Recurrence in meningiomas depends primarily on the extent of surgical removal of the tumor. Higher the extent of removal, lesser is the chance of relapse of the tumor., We also found that patients who underwent Simpson's grade I excision (100%) did not have tumor recurrence. In our study, 57% of the patients had recurrence when the tumor was subtotally resected, which is slightly less when compared with the recurrence rate of 92% reported in the previous studies. We studied the chromosomal aberration in these tumors including commonly studied markers in meningiomas such as 22q, 18p, 14q, and 1p, because only two studies have dealt with chromosomal alterations in these tumors. 22q aberration is a primordial event for the genesis of meningiomas and is deleted in 40–70% of meningiomas. 18p loss was detected in sporadic meningiomas, affecting more than 70% of tumors regardless of their histological grade. Loss of 1p represents the most frequent genetic alteration secondary to chromosome 22q loss and is found to be associated with tumor progression. Chromosome 14q represents the third-most frequently detected abnormality, seen in 100% of grade III meningiomas. We found that all cases had either complete or partial deletion status for 22q, 14q, and 1p chromosomal loci. NF2 gene locus was found to be deleted (either complete or partially) in all cases, which is contradictory to what was previously reported in cases where tumors showed NF2 amplification status and had recurred.
Interestingly, we found that 80% (4/5) of patients with recurrent tumors had complete deletions on all four chromosomal loci. Only one tumor of the five which recurred showed partial deletion, but in this case, the grade of excision was inadequate (Simpson's grade IV), which could be responsible for the recurrence. The nonrecurrent, 90% (9/10) cases had either a partially deleted or a retained status for the respective loci. Only one nonrecurrent case had complete deletion, and this patient is on a regular follow-up. We have noted that complete deletion of all four genes might be a unique characteristic of recurrent CM. This finding is contrary to our previous study on atypical meningiomas, where complete deletion of all four genes was not associated with recurrence. Co-deletion on all four loci in CMs can now be validated on further studies to project it as a unique feature for CMs, as well as to highlight its utility as a method to predict recurrence.
This is the first study to evaluate the chromosomal status of 22q, 18p, 14q, and 1p in CMs. Our study showed that CMs routinely harbor either complete or partial deletion at 22q, 14q, and 1p loci. MIB-1 LI is not a reliable indicator of recurrence in CMs. CMs that showed recurrence had complete deletion of all four loci in sharp contrast to tumors which did not recur. In view of lack of MIB1 labeling to identify recurrence in CMs, evaluation of chromosomal status for the abovementioned markers serves as a promising adjunct to the extent of surgical resection in predicting tumor recurrence.
We acknowledge the contributions of Mr. Chandrashekhar and Mr. Tilak toward tumor tissue processing and preparation of slides for immunohistochemical and FISH studies. The Department of Science and Technology (The Science and Engineering Research Board: SERB), New Delhi, is acknowledged for provision of grants for conducting the current study.
The manuscript was presented as a poster at the 7th Annual Conference of the Indian Society for Neurooncology, ISNOCON-2015, Kochi, Kerala, in March 2015.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
[Figure 1], [Figure 2]
[Table 1], [Table 2]