Fluorescence in situ hybridization for 1p, 19q status in a cohort of glial neoplasms
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.128275
Source of Support: None, Conflict of Interest: None
Objective: The objective of the following study is to determine 1p, 19q status in a cohort of glial neoplasms. Materials and Methods: Fluorescence in situ hybridization for determination of 1p, 19q deletions in 100 glial neoplasms diagnosed between January 2007 and March 2011, was performed using Vysis dual color probes localizing to 1p36/1q25; 19q13/19p13. Results: Out of the 100 tumors, 78 tumors were either pure oligodendroglial (OD) neoplasms or had an OD component. 1p and 19q codeletions were seen in 72.7% of oligodendrogliomas (World Health Organization [WHO] Grade II), 90.9% of anaplastic oligodendrogliomas (WHO Grade III), 22.2% of mixed oligoastrocytomas (WHO Grade II) and 42.9% of the anaplastic oligoastrocytomas (WHO Grade III). Of the 29 tumors that were diagnosed as glioblastoma multiforme (GBM), 11 had an OD component of which four showed codeletions of 1p and 19q (36.4%) and two tumors showed epidermal growth factor receptor (EGFR) amplification (20%) without 1p19q codeletions. Amongst the remaining 18 GBMs without an OD component, three cases showed EGFR amplification (16.7%), one case showed isolated deletion of 1p and none showed 1p19q codeletions. Polysomies involving 1p and/or 19q with or without deletions were seen in 76.9% of mixed oligoastrocytic tumors, 7.7% of pure OD tumors and one glioblastoma. Conclusions: 1p19q codeletion is an early molecular change in the genesis of OD tumors, which is retained at the time of progression. Mixed tumors more frequently show polysomies of 1p and 19q. The presence of codeletion in a third of the GBMs with an OD component with its absence in GBMs without an OD component, justifies categorization of these tumors as a separate entity.
Keywords: 19q, 1p, deletion, fluorescence in situ hybridization, glioma, oligodendroglioma
Oligodendroglial (OD) neoplasms comprise 5-6% of all glial tumors. , Oligodendrogliomas with classic features, cellular monomorphism, round/regular nuclei, perinuclear halos (pnh), microcalcifications, microcysts and chicken wire vasculature (cwv), are easier to diagnose than oligoastrocytomas.  There is marked inter-observer variation in the diagnosis of the latter entity due to the rather subjective histological criteria for pathological diagnosis and grading.  Moreover, there is a lack of definitive immunohistochemical markers to differentiate these entities.  These tumors are reported to show deletions of chromosomal arms 1p and 19q due to an unbalanced translocation of 19p-1q.  This molecular signature has been reported to improve survival and response to chemotherapy.  Ancillary testing for 1p and 19q status in OD neoplasms has therefore become important.
The most commonly utilized techniques include fluorescence in situ hybridization (FISH) and loss of heterozygosity analysis, with both having their advantages and drawbacks. ,, The present study was carried out to determine the 1p19q status in a cohort of glial neoplasms using the FISH technique. The objective of our study was to correlate the various histological subtypes, grades and radiological features of OD neoplasms with their 1p and 19q status.
The study was a cross-sectional observational study.
Cases of glial neoplasms sent to molecular neuropathology laboratory for evaluation of 1p, 19q and epidermal growth factor receptor (EGFR) status by FISH technique, between January 2007 and March 2011 were retrieved from the archives of the Department of Pathology at a Tertiary Care Hospital. Radiological details were obtained from the hospital information system.
All histological slides were reviewed and the diagnosis confirmed in each case. The tumors were classified and graded into the various histological subtypes using the World Health Organization (WHO) 2007 classification of tumors of the central nervous system. Classical features of oligodendrogliomas (CFO) viz. perinuclear haloes (pnh), round regular nuclei, well-defined nuclear contours (wdnc), evenly dispersed chromatin, chickenwire vasculature (cmv) and inconspicuous/small nucleoli were looked for in all neoplasms. Each of the afore mentioned features if present were assigned a score of 1, maximum possible score being 6. Based on histological review, OD tumors (78) were said to have high score for CFO (HCFO) if their CFO score was between 3 and 6 and low score for CFO (LCFO) if their CFO score was <3.
Features classically known to be associated with anaplasia were also looked for in all tumors. A mitotic count cut off of ≥6/10 high power field in the OD component was used to diagnose anaplastic OD tumors.  Mixed oligoastrocytomas with any mitosis in the astrocytic component were classified as Grade III tumors. The minimum OD component required for diagnosis of a mixed oligoastrocytic tumor was 25%. A representative slide and its block were selected for FISH. Dual-color FISH assays were performed on 3 micron-thick tissue sections as previously reported (10). Target retrieval included steam cooking in citrate buffer (20 min) followed by pepsin (4 mg/ml) digestion at 37°C for 30 min. Commercial locus speciﬁc identifier (LSI) ﬂuorochrome-labeled Vysis probes included LSI dual color probes localizing to 1p36/1q25; 19q13/19p13 and EGFR Spectrum Orange (SO)/chromosome enumeration probe (CEP) 7. 10 ul of the probe was applied per slide to the sections, followed by simultaneous probe/target denaturation at 90°C for 13 min in the thermobrite slide incubator, followed by overnight incubation at 37°C. The chamber in which the slides were placed was humidiﬁed to prevent drying. Following this washes were done including 50% formamide/2% salmon sperm carrier (SSC) (5 min) and 1% SSC (2 min). Nuclei were counterstained with diamidino-2-phenylindole (0.5ul/ml) (Vysis). The sections were viewed under an Olympus BX60 ﬂuorescent microscope with appropriate ﬁlters (Olympus, Melville, NY). FISH for EGFR amplification was carried out in a total of 41 cases, whilst 1p and 19 q FISH was carried out on all 100 cases.
The total number of signals was counted in 200 cells and a ratio of 1p: 1q (or 19q: 19p) of 0.85-1.15 was considered normal with no deletion and a ratio of <0.75 was diagnosed as loss. The tumor was considered to have polysomy if ≥30% of nuclei showed three or more signals for 1q and 19p. The tumors were considered ampliﬁed for EGFR when they demonstrated nuclei containing innumerable red (EGFR) signals and an EGFR: CEP7 ratio > 2. The FISH scoring was carried out by the senior neuropathologist for all cases.
78 out of 100 tumors were either pure OD neoplasms or had an OD component. The distribution by histological type and grade is shown in [Figure 1]. Forty four out of seventy eight cases (56.4%) were pure oligodendrogliomas with equal numbers of Grade II and III tumors. The astrocytomas were originally called mixed oligoastrocytomas and on review redesignated as pure astrocytomas.
Age and gender
The average age was 39.8 years (range 30-46 years) and there was a male preponderance.
The average age for the WHO Grade II glial tumors was 34 years, for the WHO Grade III glial tumors, 38.3 years and that for glioblastomas 43.6 years. There were only four patients of age less than 15 years, of which three cases were OD tumors and one was a glioblastoma.
Among the histological parameters, the most frequently seen CFO was perinuclear haloes (pnh) seen in OD tumors of all grades, followed by a well defined nuclear contour (wdnc). 88.33% of OD tumors had high scores for classical features of oligodendrogliomas (HCFO) [Table 1].
Forty eight out of the total 78 OD tumors (61.54%) showed 1p19q codeletions [Figure 2]a-c.
Correlation of WHO grade of tumor with CFO score and 1p19q deletion status
Nearly 90.3% of WHO Grade II tumors (pure and mixed) had high CFO scores and of the latter 64.3% had 1p19q codeletions. 9.7% had LCFO scores; none of these had 1p19q codeletion. Of the WHO Grade III OD tumors (pure and mixed) 88.9% had HCFO scores, of which 78.13% had 1p19q codeletions. Nearly 11.1% had LCFO scores, of which only one (25%) had 1p19q codeletion.
Of the 29 tumors that were diagnosed as GBM, 11 had an OD component. Four out of these eleven (36.4%) showed codeletions of 1p and 19q and 45.5% had HCFO scores. Among these 10 GBMOD cases were evaluated for EGFR amplification and 2 showed amplification of EGFR [Figure 2]d. The latter two cases did not have codeletion of 1p and 19q.
Of the 18 GBM without an OD component, all had EGFR evaluated and 3 showed EGFR amplification, one case showed isolated deletion of 1p, while none showed 1p19q codeletion. The remaining 13 EGFR FISH was carried out on non-GBM cases none of which showed amplification of EGFR.
Five of the GBMOD cases showed either EGFR amplification or 1p, 19q codeletion. As mentioned earlier the 2 cases with EGFR amplification did not have codeletion of 1p and 19q. Moreover, the 3 cases of GBMOD with codeletion of 1p and 19q did not exhibit EGFR amplification. The numbers are however too small to determine if this observation was of statistical significance.
19q deletion was seen in 4% of all glial tumors.
Polysomies involving 1p and/or 19q with or without deletions were seen in 13 gliomas [Figure 2]e and f of which 10 were mixed oligoastrocytomas (76.9%), 2 were pure OD tumors (15.4%) and one was a glioblastoma (7.7%). Four cases with polysomies showed co-existing 1p and or/19q deletions.
Correlation of 1p19q status with site of tumor and CFO score
The most common location was the frontal lobe (56) followed by the temporal lobe (25), parietal lobe (15), corpus callosum (2), occipital lobe (1) and cerebellum (1). 32 tumors in the frontal lobe showed 1p19q codeletions (57.14%) compared with 15 non-frontal tumors in the temporal lobe (36%). On two sample proportion test the difference was found to be significant (P = 0.04). Also tumors in the frontal lobes more often had HCFO scores (n = 38, 67.86%) compared with tumors in the temporal lobe (n = 12, 48%).
Association of 1p19q status with imaging
Radiological details were available in 76 OD tumor cases. Details of radiological circumscription status were available in 49 cases. 42 cases were radiologically ill-circumscribed out of which 31 had 1p19q codeletions. Seven cases showed radiological circumscription, of which 1p19q codeletion was seen in 2 cases. 20 cases showed calcification.
Thus tumors that had 1p19q codeletions were more often found to be radiologically ill-circumscribed compared with tumors that did not have 1p19q codeletions (Fisher's exact test, P < 0.05).
Recurrence free survival was slightly better in cases with codeletion (average 19.48 months) compared to cases without codeletion (18.9 months). Recurrence was more frequent in OD neoplasms without codeletion (7) versus cases with codeletion (5). The numbers are too small and follow-up information limited to draw meaningful conclusions.
Age, 1p19q codeletions and classic histological features are known to be independent prognostic and predictive markers in OD tumors. ,
In our study, we aimed to investigate the correlation between 1p19q status and the presence of classic histological features of oligodendroglioma.
The prevalence of 1p19q codeletion in oligodendrogliomas in this cohort was 61.5% which is similar to the figures reported by Reifenberger et al. in 1994  (67-81%) and Smith et al. in 1999 (64%). 
The age distribution for the different grades of OD neoplasms was similar to that described in literature.  In our study, there were only 3 cases of pediatric OD tumors which is in keeping with the fact that these tumors are rare in this age group. Interestingly two of the latter cases showed 1p19q codeletions. However it is already known that 1p19q codeletions do not portend a good prognosis in pediatric oligodendrogliomas unlike adult OD tumors. ,,,
The most common locations for OD tumors were the frontal lobe and temporal lobe. Most tumors in the frontal lobe showed HCFO scores and more frequent 1p19q codeletions compared to those in the temporal lobe. This is similar to the findings in the studies by Mueller et al.  and Mc Donald et al.  Mc Donald et al.  found a negative association between temporal lobe OD tumors and their 1p19q deletion status.
OD tumors with 1p19q codeletions were more frequently ill-circumscribed on radiology compared to tumors without these codeletions. This observation was also made by Megyesi et al.  in their study on imaging correlates of molecular signatures in oligodendrogliomas in 2004 which found 17 out of 26 OD tumors with indistinct borders to contain 1p19q codeletions. However, since different radiologists reported the magnetic resonance imaging and computed tomography scans there is likelihood of inter-observer variability in our findings.
The most common classic histological feature was perinuclear haloes followed by well defined nuclear contours.
Most OD tumors except glioblastomas with an OD component had HCFO scores. The tumors with HCFO scores more often had 1p19q codeletions (69.2%). HCFO has been reported to be predictive of 1p19q codeletions by several authors. ,, In the study by Giannini et al.  of anaplastic OD tumors (WHO Grade III), the incidence of 1p19q codeletions in tumors with classic OD features (HCFO) was 80% and in tumors with no classic features of oligodendroglioma (NCFO) was 13%. Giannini et al.  considered even the presence of one classic feature as significant, whereas in our study, 3 or more criteria were required to be present to qualify for a CFO. This difference in criteria could account for the difference in incidence of 1p19q codeletions between our studies.
However, similar to other studies we found that the relationship between CFO and 1p19q codeletions is not absolute. , Hence the former cannot be used as a substitute for purposes of prognostication.
1p19q codeletions were seen in 58.06% of WHO Grade II OD tumors and 72.2% of WHO Grade III OD tumors supporting the observation of others that this genetic event is preserved with tumor progression.  They were more frequent in pure OD tumors (81.8%) compared with mixed oligoastrocytomas of same grade (35.3%). Miller et al. in 2006  also found higher frequency of 1p19q codeletions in pure anaplastic oligodendrogliomas (89%) versus the anaplastic oligoastrocytomas (19%). This suggests that as the astrocytic component of a mixed tumor increases or OD component decreases, the incidence of 1p19q codeletion decreases.
The presence of the 1p19q codeletion in 36.4% of GBM with OD component, with their absence in GBM without an OD component and its mutual exclusivity from EGFR amplified GBM tumors (both GBM and GBM with OD), justifies the separate categorization of these tumors in the WHO 2007 classification of tumors of the nervous system. Similar to other studies we found that GBM with OD component WHO Grade IV showed less frequent 1p19q codeletions when compared with Anaplastic oligoastrocytoma WHO Grade III (42.9%) from which they are thought to arise.  This gives credence to the hypothesis for an alternate pathogenetic pathway for these tumors different from other GBMs.
Polysomies are a known feature of astrocytic tumors and polysomy of chromosomes 1 and 19 are said to predict earlier recurrence in anaplastic OD tumors. , Mixed oligoastrocytic tumors comprised 43.6% (n = 34) of all OD tumors, in this series. They showed less frequent HCFO scores and 1p19q codeletions compared to pure OD tumors of a similar grade. In fact the most common molecular alteration seen in this group of tumors were polysomies of chromosomes 1 and/or 19 (29.41%) with or without deletions of 1p and/or 19q. Mixed oligoastrocytomas are difficult to diagnose and it is widely accepted that there is a subjective element to diagnosis.  The higher frequency of polysomies in this group as observed by us and others,  suggests that their presence may aid in the diagnosis of mixed oligoastrocytomas. Moreover, it probably has a role as an independent marker of prognosis as evidenced by other studies. ,
[Figure 1], [Figure 2]