Introduction
Incorporation of somatic molecular alterations into the classification and grading of brain tumors continues to drive an ongoing revolution in the field of neuro-oncology. Among diffusely infiltrating gliomas, the mutational status of isocitrate dehydrogenase (
IDH1 or
IDH2 genes) is a fundamental determinant of biologic behavior, which, in combination with additional molecular features, is now a defining criterion of the diagnostic category of IDH-mutant (IDHm) astrocytoma [
1]. Segregating IDHm astrocytic tumors from their more aggressive IDH-wild type (IDHwt) counterparts was a major advance in our understanding of diffuse glioma biology and prognostication. Current WHO grading criteria, however, do not differentiate IDHm from IDHwt astrocytomas and have not yet been updated to reflect the differing clinical behaviors of these diagnostic entities. Additionally, some of the morphologic features used to grade IDHwt tumors do a poor job of stratifying IDHm astrocytoma survival, particularly mitotic activity [
2‐
4]. To address these shortcomings, a number of groups have investigated molecular correlates of aggressive behavior in IDHm astrocytomas [
5‐
11]. Based on these studies, homozygous deletion of the
CDKN2A gene, which encodes the cell-cycle regulators p16INK4A and p14ARF, has emerged as a leading candidate molecular marker of high-grade behavior in these tumors [
3,
7,
10‐
12].
Given the likelihood that
CDKN2A status may be incorporated into future grading systems for IDHm astrocytomas, we sought to understand how such a change might impact the practice of neuropathology at our institution. In particular, we wished to define how best to interpret data from fluorescence in situ hybridization (FISH) studies, since the influential studies showing prognostic utility of
CDKN2A loss [
7,
10] have largely utilized array-based techniques that are not yet readily accessible to many institutions world-wide. Array based methods rely on algorithmically-defined thresholds to binarize the presence or absence of copy number loss, while the percentage of cells showing homozygous deletion by FISH is a continuous variable. FISH provides a direct visualization of the status of the genetic locus of interest and readily distinguishes true homozygous deletion from hemizygous loss or complex copy number change. FISH testing is advantageous in the setting of diffusely infiltrative tumors with highly variable degrees of tumor cellularity relative to background non-neoplastic parenchyma, since the sensitivity of sequencing- and array-based techniques for accurately detecting homozygous deletion suffers in this situation. The choice of where to set the clinical cutoff for defining tumor wide
CDKN2A deletion based on the proportion of tumor cells with homozygous deletion will be critically important if
CDKN2A status is to be used for tumor grading and therapeutic decision-making [
3,
12].
To address these issues, we leveraged our decades-long institutional experience with molecular testing of gliomas. For many years prior to the application of routine next-generation sequencing (NGS) of brain tumors, CDKN2A status was evaluated by FISH as part of the standard evaluation of newly-diagnosed and recurrent gliomas at our institution. Our anecdotal impressions from this period agreed with the developing literature that high levels of CDKN2A homozygous deletion portended poor outcome, but suggested that tumors with low-level homozygous deletion were not uniformly aggressive. In addition, cases of recurrent tumors that had developed new CDKN2A deletion compared to the original resection specimen led us to hypothesize that this molecular marker might have different prognostic implications at the time of recurrence compared to initial presentation. To move beyond anecdotes, we systematically analyzed our institutional cohort of patients with IDHm astrocytomas and CDKN2A FISH analysis in order to better define the relationship between percentage of cells with homozygous CDKN2A deletion, histologic grade, and patient survival, in both primary and recurrent tumors.
Discussion
Grading of diffuse gliomas is a critical component of the diagnostic process that guides therapeutic decision-making and should, ideally, be based on prognostically significant measures of tumor behavior. Based on recently published recommendations by the cIMPACT-NOW working group [
3,
12],
CDKN2A status is likely to be incorporated into future grading systems for IDHm astrocytomas, but it remains unclear how to interpret the results of a commonly used testing modality, FISH, for this purpose. In order to define an appropriate cutoff value for interpreting
CDKN2A FISH results as positive for homozygous deletion in routine clinical service, and to assess the anticipated impact of applying these results in our patient population using the proposed cIMPACT-NOW grading schemes, we performed a retrospective analysis of IDHm astrocytomas that had prospectively undergone
CDKN2A FISH analysis. Multivariate Cox-PH analysis revealed that the prognostic significance of
CDKN2A in IDHm astrocytomas is optimized by using a cutoff value of ≥ 30% of tumor cells with homozygous deletion in order to define tumor-wide “homozygous deletion” status. Applying this cutoff value, very few primary treatment-naïve tumors in our institutional cohort showed this level of deletion, and of the tumors that did exceed this threshold, the majority were already considered grade 4 by histology. Compared to primary tumors,
CDKN2A homozygous loss was more frequent in recurrent grade 3 and grade 4 tumors. While both histologic grade and
CDKN2A loss were independent predictors of survival in our cohort, the inclusion of
CDKN2A as a grading criterion failed to improve survival stratification, at least in the setting of tumors not meeting grade 4 criteria histologically. With the ≥ 30% cutoff, only two lower grade tumors would be upgraded to grade 4, and both patients were still living at the time of analysis, having already exceeded the median survival of histologic grade 4 tumors.
At first blush, it may seem counterintuitive that the Cox-PH model developed using the full cohort of tumors spanning all grades confirms
CDKN2A deletion as an independently significant prognostic factor, yet the optimal cutoff of 30% was exceeded by only a very small fraction of grade 2 and 3 tumors. These findings can be reconciled, however, if the overall model is being largely driven by prognostic differences among morphologically grade 4 tumors. Indeed, unlike lower grade tumors, grade 4 tumors with ≥ 30%
CDKN2A deletion showed significantly shorter overall survival than grade 4 tumors with intact
CDKN2A, in both primary and first recurrence tumors (Fig.
5a and b). In light of the published studies claiming dismal prognosis for lower grade tumors with
CDKN2A loss [
7,
10,
11], we wanted to make sure that the model driven by grade 4 effects was not inappropriately excluding certain grade 2–3 tumors from being classified as a higher molecular grade; perhaps a different, lower threshold might be more appropriate for stratifying the lower grade tumors. We addressed this question in two ways: by applying a lower threshold and examining whether hypothetically-upgraded tumors behave as the adjusted grade would suggest (Fig.
5c and d) and by creating a Cox-PH model using only grade 2 and 3 tumors (excluding grade 4) (Fig.
6). Contrary to the hypothesis that a lower threshold would perform better for lower grade tumors, both analyses show the opposite—in our cohort of patients, there is no evidence that a threshold at or below 30% homozygous deletion improves prognostication of lower-grade lesions. We note, however, that it remains possible that lower grade IDHm astrocytomas with high level (≥ 30%)
CDKN2A deletion by FISH do in fact have a poor prognosis, but given the rarity of grade 2 or 3 tumors exceeding this threshold, there is insufficient evidence to determine the prognostic impact of this finding in our cohort.
Our findings differ from those studies showing a significant prognostic impact of
CDKN2A deletion in both grade 3 and grade 4 IDHm astrocytomas, as detected by array-based techniques [
7,
10]. While our data show a similar poor prognosis of
CDKN2A deletion in grade 4 IDHm astrocytomas, these findings do not extend to grade 3 tumors. Using the proposed cIMPACT-NOW 5/6 criteria and our statistically-defined FISH cutoff of ≥ 30%, the hypothetical upgrade rate of grade 3 tumors in our patient cohort is markedly lower (1/31, 3%) than that in Shirahat et al
. (15/90, 17%) or Appay et al
. (35/211, 17%). While it is possible that technical differences between various testing modalities could have an unexpectedly large effect, a likelier cause of this discrepancy could be differences in the population characteristics of the tumors being analyzed. The histologic grades at presentation for tumors in the cohorts of Shirahat et al
. (26% grade 2, 43% grade 3, 32% grade 4) and Appay et al
. (grade 2 not included in primary analysis, 50% grade 3, 50% grade 4) are skewed towards higher grades, while our single institution cohort had a larger proportion of lower grade tumors (51% grade 2, 29% grade 3, and 20% grade 4). The distribution of cases in our study, with the highest proportion presenting as grade 2 and the lowest as grade 4, is in broad agreement with previously published large all-comer cohorts of IDHm astrocytomas [
4]. Similarly, the frequency at which homozygous
CDKN2A loss was detected in our cohort at each grade (grade 2: 1.8%, grade 3: 3.2%, grade 4: 27%) is similar to the proportion of IHDm astrocytomas in The Cancer Genome Atlas (TCGA) combined low grade glioma and glioblastoma cohorts (grade 2: 3.5%, grade 3: 6.7%, grade 4: 18.8%; primary tumors, multiple methods of
CDKN2A assessment) [
8]. The enrichment in grade 3 and grade 4 tumors in the Shirahat et al. and Appay et al. studies, along with the elevated frequency of
CDKN2A loss in grade 3, suggests that these cohorts may represent a different population of tumors, possibly including increased numbers of diagnostically difficult, borderline, or clinically aggressive cases sent for expert consultation. It is also not clear whether these studies limited their analyses to only primary treatment-naïve specimens. Inclusion of recurrent/treated tumors could be a significant confounder, especially given the overall worse prognosis and increased frequency of
CDKN2A loss seen in recurrent tumors in our cohort.
The existing literature on use of FISH specifically to detect
CDKN2A homozygous deletion in gliomas is sparse. Perhaps the most relevant is a recent study by Yang et al
. that used FISH to examine
CDKN2A, CDK4, and
PDGFRA copy number alterations in grade 2 and 3 astrocytomas [
11]. The authors used a cutoff of ≥ 20% of tumor cells showing homozygous deletion, but a rationale for this threshold was not provided. The study found
CDKN2A homozygous deletion of ≥ 20% at similar rate in grade 2/3 tumors as our cohort (15% Yang et al
. versus 10% for our cohort) with both grade 2 and grade 3 tumors showing deletion. The survival effect of
CDKN2A in the Yang et al
. cohort cannot be directly evaluated, as the survival analysis in that study grouped tumors with
CDKN2A deletion with those showing
CDK4 amplification (i.e. alteration in the RB1 pathway), rather than assessing each gene independently. While these RB1-altered tumors did show shorter overall survival than the RB1-intact group, they nonetheless appear to have a significantly better prognosis than the morphologic grade 4 tumors in our study. This comparison with the published literature further supports our findings that grade 2/3 astrocytomas with
CDKN2A homozygous deletion as assessed by FISH have longer survival than histologic grade 4 tumors.
The ability to accurately and reliably detect CDKN2A homozygous deletion in IDHm astrocytomas is necessary if this is to be included as a grade defining criteria in the next revision of the WHO Classification. The cIMPACT-NOW recommendations do not offer definitive guidance as to which testing modalities should be used, and there are many possible methods to detect loss. FISH is perhaps the oldest and most widely accepted technique for detecting copy number alterations. FISH testing has many benefits, including the ability to definitively identify homozygous deletion in infiltrating tumors, and to separate true homozygous deletion from hemizygous loss and complex copy number alterations. Like all testing modalities, however, FISH has certain intrinsic technical limitations, including insensitivity to deletions smaller than the region covered by the probe, and artifactual loss of signal due to partial sectioning of nuclei when performed on FFPE tissue sections. As an example of the latter, consider a nucleus with a single CEP9 signal and no 9p21 signals. This result could accurately reflect monosomy 9 with additional 9p21 deletion, but could also arise in other ways: (1) homozygous 9p21 deletion with artifactual loss of one CEP9 signal; (2) monosomy 9 with artifactual loss of one 9p21 signal; (3) hemizygous 9p21 loss paired with artifactual loss of one CEP9 and one 9p21 signal; (4) wild type chromosome 9 with artifactual loss of three probes. The possibility of artifactual signal loss highlights the necessity of interpretative guidelines. In our laboratory, a nucleus with no 9p21 signals and at least one CEP9 signal is interpreted as homozygous deletion, which favors sensitivity for detecting true absence of CDKN2A over specificity for excluding monosomy 9 with artifactual 9p21 loss. A more stringent criteria requiring two CEP9 signals would lead to even fewer tumors being identified as having homozygous CDKN2A deletion.
In addition to FISH, there are numerous variations of genomic microarrays in use for analyzing brain tumor samples. Comparative genomic hybridization arrays (aCGH) excel at detecting copy number alterations in aggregate tissue samples, but can struggle at detecting loss in the context of a sparsely infiltrating tumor. Single nucleotide polymorphism (SNP) arrays are often used in combination with either true or virtual aCGH to help separate hemizygous loss and complex alteration events from true homozygous loss (although not at a single cell level). Finally, the data obtained from methylation or other NGS arrays can often be analyzed to provide output similar to a combination aCGH/SNP array. As with FISH, each of these techniques have their own interpretive subtleties and technical limitations, which complicates cross-modality comparisons of the prognostic implications of test results. Clear and specific descriptions of the algorithms used to judge homozygous deletion status would be helpful for this purpose, but are frequently lacking. As a result, direct comparison between these various methods is often not possible, and it is not clear which technique should represent the “gold standard.”
An important point of concordance between our study and the existing literature is the lack of prognostically meaningful CDKN2A homozygous deletion in histologically grade 2 tumors. The lone patient with a grade 2 tumor with over 30% homozygous deletion in our study is still alive at 61 months from diagnosis, and neither of the above array-based studies identified any histologic grade 2 tumors with CDKN2A homozygous deletion (Shirahat et al. n = 54, Appay et al. n = 20). The wording of the cIMPACT-NOW 6 proposal for updated grading criteria is not clear regarding whether or not morphologic grade 2 IDHm tumors will require CDKN2A testing for formal grading; clarification on this point will be essential in any upcoming WHO update. Results from our cohort agree with the previous studies and suggest that testing grade 2 tumors for deletion would be very low yield at best, might lead to inappropriate upgrading of indolent tumors, and could impose an undue financial burden on the global healthcare system.
In conclusion, CDKN2A homozygous deletion is a marker of poor prognosis in histologic grade 4 IDHm astrocytomas, but the impact of this finding in histologic grades 2 and 3 tumors is less clear. Different techniques for determining CDKN2A status may provide markedly different results between and even within individual institutions. Specific criteria for determining the presence of homozygous deletion across different testing modalities will be essential if CDKN2A homozygous deletion is included as a grading criterion in the next revision of the WHO Classification.
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