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Fluorescence in situ hybridization for chromosome 14q deletion in subsets of meningioma segregated by MIB-1 labelling index
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.193768
Aim: To correlate histopathological grading of meningiomas segregated into subgroups based on the MIB-1 labelling index (MIB-1 LI) with chromosomal loss of 14q using fluorescence in situ hybridization (FISH). Keywords: 14q deletion; FISH; meningioma; MIB-1; WHO grade
Nearly 20% of intracranial tumours are meningiomas.[1] The 2007 World Health Organisation (WHO) recognises 3 grades of meningiomas, I, II, and III. The biological behavior of meningiomas may not always correspond to their histological grading. It is well-known that some meningiomas with WHO grade I histology can display more aggressive behavior in the form of grossly invasive tumor growth, rapid growth of residual tumor, or recurrence despite apparently total surgical resection.[2],[3],[4] The proliferative capacity of tumor, measured by the MIB-1 LI has emerged as an important predictor of tumor aggressiveness.[5],[6],[7] Several studies have found that there is a statistically significant rise in the MIB-1/Ki-67 index from benign, through atypical, to anaplastic meningiomas.[8],[9] Although the extent of tumor resection and histological grade are two of the strongest predictors of biological behavior, there is significant variability that cannot be accounted for by these parameters alone. Researchers have, therefore, begun to look at genetic alterations in meningiomas to determine correlates to biological behavior. Deletion of chromosome 22 is the most frequently observed cytogenetic alteration, which is an early event in tumor formation followed by deletions of 1p and 14q.[10],[11] The latter are implicated in tumor progression. We studied the association between MIB-1 LI, 14q deletion, and the 5 histological criteria enumerated by the WHO for diagnosis of atypical meningiomas.
This retrospective observational study was approved by the Institutional Review Board. A total of 166 tumor samples of meningiomas were received in the Pathology Department from January 2011 to December 2011. The cases were artificially segregated into 5 categories based on the MIB-1 LI of <5%, 5–7.5%, 7.5–9.9%, 10%, and >10%. From these categories, a total of 46 cases were selected randomly (by picking lots) for the study, 9 each with MIB-1 LI of <5%, 5–7.5%, 7.5–9.9%, and >10%, and 10 cases with an MIB-1 LI of 10%. Clinical information was obtained from the medical records department. Exclusion criteria Cases of neurofibromatosis type 2 and psammomatous meningiomas were excluded from the study because they interfere with observations under fluorescent microscope. Parameters evaluated
80% was used as the cut-off for determining the 14q deletion status of a meningioma, through the following calculation [Table 1] and [Table 2]. As evident by these tables, the likelihood ratio of the test was the highest when the cut-off for deletion was taken as 80%. Considering a pre-test probability (P) of 50%, the pre-test odds i.e., P/1−P would be 1. The post-test odds, that is the pre-test odds multiplied by the likelihood ratio was 4 (1×4). The post-test probability i.e., post-test odds/1+ post-test odds was, therefore, 80% (4/1+4). This would imply that with the pre-test probability of the diagnosis being a WHO grade II/III meningioma, the presence of >80 cells, with a single copy of chromosome 14 out of the 100 counted cells would give a post-test probability of having a higher grade meningioma as 80%.
Statistical analysis was carried out using the Statistical Package for the Social Sciences software version 16.0 (IBM SPSS Statistics). χ2 test was used to analyze categorical variables. Mann–Whitney U test and the Fisher's exact test or t-test was used for continuous variables. A P value of <0.05 was considered to be significant.
There were 46 cases in the study group. Using the WHO 2007 classification, 21 meningiomas were classified as grade I, 22 as grade II, and 1 as grade III. Two cases had a high MIB-1 LI (10%) and a poor surgical plane with the underlying brain, even though, on histology, they appeared as WHO grade I meningiomas; for statistical analysis, these were considered as WHO grade II tumors. As there was only one WHO grade III meningioma, it was clubbed with WHO grade II meningiomas for statistical analysis. There was equal gender distribution with 23 female and 23 male patients. The mean age at diagnosis was 48.48 ±10.23 years (range: 28–72 years). The difference between the age at diagnosis of grade I and grade II meningiomas was statistically insignificant (P = 0.475). There was no difference between grade I and grade II and III meningiomas with regard to location [Table 3].
The mean duration of symptoms of WHO grade I meningiomas (n = 19) was 10.05±8.69 months and that of WHO grade II meningiomas (n = 23) was 5.24 ± 5.65 months. This difference was statistically significant (P < 0.028). Grade II meningiomas were larger than grade I counterparts. The average maximum dimension of WHO grade I meningiomas (n = 20), determined by MRI, at the time of diagnosis was 4.42 ± 1.16 cm, and that of WHO grade II meningiomas (n = 23) was 5.60 ± 1.33 cm. This difference was highly statistically significant (P < 0.003). Histological subtypes [Table 4] shows the distribution of meningiomas by histological subtype. Among the grade II meningiomas, 6 were brain invasive.
MIB-1 labelling index The mean MIB-1 LI of WHO grade I tumors in this study was 5.71 ± 2.74% (range: 2–12%) and that of WHO grade II was 11.04 ± 4.04% (range: 5–22%). The MIB-1 LI was significantly different between WHO grade I (n = 21) and WHO grade II and III meningiomas (n = 25) [P = 0.00]. We grouped meningiomas into 5 groups based on their MIB-1 LI, and found that, as the MIB-1 LI increased, the proportion of cases in WHO grade II and III were statistically higher (P = 0.000) [Figure 3].
Histological criteria Of all the histological parameters defined by WHO for diagnosing atypical meningiomas [Table 5], there was a statistically significant association between a MIB-1 LI >7% and hypercellularity, small cells with a high nuclear : cytoplasmic ratio, and sheet-like pattern. There was no significant relationship between age, gender, anatomic location, or completeness of tumor excision and the MIB-1 LI.
Chromosome 14q deletion There was no significant difference between the age, gender, duration of symptoms, size of tumor, surgical plane of tumor, and the type of surgical excision of tumors in cases with respect to 14q deletion status. Among the histological parameters defined by the WHO for the diagnosis of atypical meningiomas, the association between 14q deletion and hypercellularity, small cell change, prominence of nucleoli, and sheet-like pattern was statistically significant. There was no significant association between tumor necrosis and 14q deletion [Table 5]. When compared to WHO grade I meningiomas (n = 21), where only 7 cases (33.3%) showed 14q deletion, 21 WHO grade II and III meningiomas (n = 25, 84.0%) had 14q deletion. This difference was found to be significant on statistical analysis (P = 0.000) [Table 6].
All the brain invasive meningiomas (n = 6) had 14q deletion. When looking at the prevalence of deletions in each subgroup segregated by MIB-1 LI, it was observed that, as the MIB-1% increased, the proportion of cases with deletions were significantly higher (P < 0.001) [Table 7].
The mean MIB-1 of the WHO grade I meningiomas with 14q deletion (n = 7) was 8.86 ± 1.95% when compared to 4.14 ± 1.35% for those without 14q deletions (n = 14). This difference was found to be statistically significant (P = 0.000) [Figure 4].
On further categorizing the cases as MIB-1% ≤7% and >7%, 3 of 18 (16.7%) cases with a MIB-1 of ≤7% had 14q deletion when compared to 25 of 28 (89.3%) cases with a MIB-1 >7%. This difference had a high statistical significance (P = 0.000) [Figure 5].
A majority of meningiomas (80–90%) are benign WHO grade I tumors, whereas WHO grade II and III tumors occur less frequently but display an aggressive behavior with higher recurrence rates.[2],[3],[14] Some WHO grade I tumors demonstrate an overtly aggressive behavior with bone destruction, rapid growth of residual tumor, and recurrence rates approaching 19% by 20 years, following apparently complete resection.[1],[2],[3],[15],[16] In addition, meningiomas occurring in the skull base, cavernous sinus, or invading the sagittal sinus are not amenable to complete resection, often leading to significant residue.[17] Variable growth rates of these residues highlight a need to determine other factors that may predict tumor behavior and direct the early institution of adjunctive treatment such as radiotherapy. The incidence of these tumors tends to peak between the sixth and seventh decades with the average age at diagnosis being 63 years.[18],[19] In the present study, the average age at diagnosis was much lower at 48.48 ± 11.14 years (range: 29–62 years). Although meningiomas are known to occur twice as frequently in female patients compared to males, the gender distribution was found to be equal in the present study.[19] These discrepancies may be due to the short span over which the cases were selected. Although the frequency of atypical and anaplastic meningiomas was higher in male patients when compared to females, which is in concordance with literature,[20] it was not statistically significant. The average maximum dimension of WHO grade I and II meningiomas was significantly different, with grade II tumors being 1.18 cm larger than grade I tumors. Despite conflicting reports of the value of MIB-1, this proliferation index has emerged as an important predictor of tumor aggressiveness. While some studies have found the MIB-1 LI to be predictive of recurrences,[21],[22] others have not.[23] In accordance with other studies, in the present study also, the difference between the mean MIB-1 LI of WHO grade I and II meningiomas was statistically significant.[8],[9],[21] We determined from a previous study, conducted at our institution, that a MIB-1 LI of 7% had the highest validity in diagnosing atypical meningiomas.[12] MIB-1 labelling index and histological features of atypia The current study found that MIB-1 LI >7% correlated significantly with increased cellularity, sheet-like growth, and small cells with a high nuclear : cytoplasmic ratio, which are findings similar to that seen in an earlier study from our institution. In the present study, the MIB-I LI of benign meningiomas was significantly lower (mean: 5.7 ± 2.7%) than that of atypical meningiomas (mean: 11.0 ± 4.0%). Devaprasath et al., suggested that tumors that did not show any histologic features of atypia but had a high MIB-1 LI must be followed-up closely.[12] The fact that mitotic activity of ≥4 mitoses/10 high power fields is by itself sufficient to make a diagnosis of atypical meningiomas underlines the importance of proliferative activity of a tumor in determining its grade. However, the significant association of MIB-1 LI with 3 of the morphological criteria of atypia in our study, namely increased cellularity, sheet-like growth, and small cells with a high nuclear : cytoplasmic ratio, suggests that these histological features are indicators of high proliferation and may serve as surrogate markers in situ ations where either it is not possible to ascertain the MIB-1 or where mitotic activity is not evident. Studies have suggested that differences in MIB-1 LI among the 3 meningioma grades may be useful in borderline atypical cases where mitosis are hard to detect or the histological criteria of atypia are falling short of the WHO guidelines in cases suspected to be of higher grade.[8],[24] In subtotally resected meningiomas, when brain invasion cannot be ruled out due to sampling error, the tumor shows aggressive morphological features, and the data is insufficient to make a diagnosis of atypical meningiomas by the WHO criteria, a high MIB-1 LI may help in assigning the tumors to a higher grade. Progression of meningiomas and 14q deletion Several studies have delved into the prognostic potential of genetic abnormalities detected in meningiomas for predicting recurrence risk.[25],[26],[27] In a study carried out by Carvalho et al.,[9] the gene expression profiles of meningiomas were categorized into two major groups, namely “low proliferative” and “high proliferative.” All the 8 WHO grade I tumors and 8 grade III tumors fell into the “low proliferative” and “high proliferative” category, respectively. The 7 WHO grade II tumors, however, were distributed between both groups. Hence, although atypical meningiomas were found to be a distinct group using the histopathological criteria, their molecular profile showed a spectrum from “low proliferative” to “high proliferative” category, suggesting that genetic alterations are a continuum in tumor progression. The clinical course of atypical meningiomas is also diverse, with few of them demonstrating growth rates similar to Grade I meningiomas and others showing clinical patterns similar to malignant meningiomas. 14q deletions follow the losses in chromosome 22 and 1, as the next most common chromosomal aberrations seen in meningiomas. 14 q deletions have been found in up to 31% of grade I, 40–70% of grade II, and up to 100% of grade III meningiomas.[25],[26],[27],[28] Studies have also found losses of 14q to be a prognostic indicator of tumor recurrence.[28],[29],[30] Evidence supports the role of N-Myc downstream-regulated gene 2 (NDRG2) as a tumor suppressor gene involved in malignant progression and recurrence of meningiomas.[31] Partial or complete loss of chromosome 14 has also been associated with an early relapse and a poor clinical course.[30],[32] Skiriute et al.,[33] found that atypical meningiomas (both primary and recurrent) had significant reduction of NDRG2 gene expression in contrast to primary benign counterparts. Cai et al.,[27] found that the frequency of 14q deletion in benign recurring meningiomas was almost as high as those in atypical meningiomas. Maillo et al.,[30] developed a scoring system combining WHO grade, 14q status, and age, stratifying patients into 3 categories with significantly different recurrence-free survival rates. 1p/14q codeletion is characteristic of grade II/III meningiomas. These studies strengthen the proposition that the inclusion of molecular alterations (especially chromosome 14 aberrations) in meningiomas may help in predicting the clinical course of disease. In our own centre, a study aiming at identifying markers of aggressive behavior of meningiomas in the Indian population using the loss of heterozygosity (LOH) analysis for tumor suppressor genes located on 1, 10, 14, 17, and 22 meningiomas was carried out.[34] We found that D17S1289 was the most informative locus and that LOH was seen most often at D22S417. It was also found that majority of high grade meningiomas showed LOH 14/allelic imbalance implying that it may be a progression event; however, the LOH 14 did not reach statistical significance, whereas the allelic imbalance was significant, suggesting the presence of a smaller clone carrying a chromosome 14 deletion. Deoxyribose nucleic acid (DNA) for LOH may have a mixture of normal and tumor tissue genes making the detection of LOH difficult, which could explain the finding of allelic imbalances instead of LOH in this study. In yet another study from India, Kumar et al.,[35] found that 20% of incompletely resected Grade I petroclival meningiomas had 1p/14q codeletion. Being a genetic marker of aggressive behavior, they suggested that petroclival tumors harboring the 1p/14q codeletion may behave more aggressively than those without the codeletion. The present study was carried out using FISH for 14q to determine if the findings of our previous study were corroborated when examining representative sections of the tumor. Only 33.3% of WHO grade I meningiomas were found to harbour 14q deletion when compared to 84.0% WHO grade II meningiomas. This difference revealed a high statistical significance and is in accordance with that reported in other studies.[25],[26],[27] This finding supports our hypothesis that 14q deletion has a significant role in meningioma progression, and that this signifies the presence of tumor suppressor genes on this chromosome. Moreover, all the brain invasive meningiomas in our study had 14q deletion. There have been no studies, to the best of our knowledge, that have examined correlations between 14q deletion and brain invasion. MIB-1 LI and 14q deletion We found that the MIB-1 LI correlated significantly with 14q deletion [Figure 4] and [Figure 5]. Only 16.7% of cases with an MIB-1 of ≤7% had 14q deletion when compared to 89.3% cases with an MIB-1 >7%. In addition, both higher MIB-1 LI and 14q deletion were seen with WHO grade II tumors. This again suggests that 14q deletion can serve as a surrogate marker for atypia. The MIB-1 LI of WHO grade I meningiomas harbouring 14q deletions was significantly higher than those without deletion. This implies that, within the meningiomas with a WHO grade I histology, there exists a subgroup stratified by the MIB-1 LI, which consistently shows 14q deletions. A previous study has shown that, within WHO grade I meningiomas, tumors with a high MIB-1 have a higher chance of recurrence.[9] It is possible that these tumors harbouring 14q deletions may have a higher chance of recurrence. In the present study, however, our follow-up information was limited because the cases in the cohort were operated on only 24 months prior to the start of the study, and hence this aspect could not be studied at the present time. Of the 28 cases with a MIB-1 LI >7%, only 3 did not show 14q deletion. These included a WHO grade III/anaplastic meningioma with a MIB-1 LI of 22%, in which 70% of the tumor cells had a single copy of 14q. The remaining 2 tumors were WHO grade II meningiomas, one with increased cellularity, small cell formation, sheet-like growth, necrosis, a MIB-1 LI of 15%, and 79% cells with a single copy of 14q; and, the other with increased cellularity, small cell formation, necrosis, mitotic activity of 9/10 high power fields, an MIB-1 LI of 12% with 38% cells, having a single copy of 14q. Despite the fact that a high percentage of tumor cells had a single copy of 14q in 2/3 tumors, they were not considered as harbouring 14q deletions because they failed to meet the stringent cut-off value of 80% of tumor cells with a single copy of 14q (used for determining the 14q status of a tumor in the present study). Chromosomal alterations apart from chromosome 14 are also implicated in tumor progression and this may explain the third case with a WHO grade II histology and a high MIB index not showing the 14q deletion. Of the 3 cases with MIB-1 LI ≤7% with 14q deletion, one was a WHO grade I meningioma. The only atypical feature in this case was prominence of nucleoli. This suggests that chromosomal abnormalities may precede histological evidence of tumor progression. The other two cases were grade II meningiomas, one with small cell change, prominent nucleoli, and necrosis, and the other with brain invasion. This makes a case for screening for chromosomal abnormalities in meningiomas as an ancillary to other tools, such as MIB-1 labelling index, for prognostication.
The results of this study showed that a strong association existed between histologic grade, MIB-1 LI, and the presence of chromosome 14q deletion. Association of a high MIB-1 LI with 14q deletions even in meningiomas with a grade I histology defines a distinct subset of benign meningiomas. The biological behavior of this subset needs further exploration with follow-up, for assessement of recurrence and aggressive behavior. Acknowledgements Ms. Janet Paul and Sunitha James are acknowledged for their technical support with the FISH preparation. Financial support and sponsorship Institutional Intramural Fund research grant (Institutional Review Board Number 7928). Conflicts of interest There are no conflicts of interest.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]
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