CDKN2A/B Homozygous Deletions in Astrocytomas: Comparison
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The CDKN2A and CDKN2B genes are located on the short arm of chromosome 9. CDKN2A encodes for two proteins, p14 and p16, and CDKN2B encodes for p15. These proteins regulate cell growth and angiogenesis. Interpreting the impact of CDKN2A/B alterations on astrocytoma prognosis is complicated by rthecent changes in tumour classification and a lack of uniform standards for testing CDKN2A/B. While the prognostic impact of CDKN2A/B HD is established, the role of different CDKN2A/B alterations—heterozygous deletions (HeD), point mutations, and promoter methylation—is less clear. Consequently, how these alternations should be incorporated into patient management remains controversial.

  • CDKN2A/B alterations
  • CDKN2A/B homozygous deletions
  • temozolomide

1. Introduction

In 2016, the WHO Classification of Tumours of the Central Nervous System (revised 4th edition) incorporated isocitrate dehydrogenase 1 and 2 (IDH1/2) mutation status into the classification of diffuse gliomas [1]. IDH1/2 mutations (point mutations in either IDH1 codon R132 or IDH2 codon R172) lead to IDH dysfunction, which converts alpha-ketoglutarate to R-2-hydroxygluterate, driving oncogenesis and global epigenetic changes [1,2,3,4][1][2][3][4]. The WHO Classification in 2016 further stratified diffuse IDH-mutant gliomas into oligodendrogliomas and astrocytomas based on the respective presence or absence of chromosome 1p and 19q co-deletions (see Figure 1) [1].
IDH-mutant astrocytomas account for 80% of WHO grades 2 to 3 and 5% of high-grade astrocytomas [5,6][5][6]. When compared to IDH-wildtype glioblastomas, patients with IDH-mutant astrocytomas are younger at diagnosis (30–40 years vs. over 50 years). In addition, IDH-mutant astrocytomas have a more favourable prognosis compared to IDH-wildtype glioblastomas, even in high-grade cases, with grade 4 IDH-mutant astrocytomas having a median overall survival (OS) of 31 months, compared to IDH-wildtype glioblastomas with a median OS of 13 months [5,6][5][6]. Unfortunately, a proportion of IDH-mutant astrocytomas have poor outcomes similar to those of IDH-wildtype glioblastomas [7].
CDKN2A/B HD are identified in approximately 22% of IDH-mutant astrocytomas [8] and are thought to lead to the loss of cell cycle control and promote cell proliferation [9].
Due to the improved prognosis of tumours previously classified as IDH-mutant glioblastomas compared to IDH-wildtype glioblastomas, these have been reclassified as astrocytoma, IDH-mutant, CNS WHO grade 4 in the fifth edition of the WHO Classification of Tumours of the Central Nervous System (WHO CNS5, 2021). A hallmark of WHO CNS5 is the integration of molecular markers into tumour grading. As such, IDH-mutant astrocytomas with CDKN2A/B HD are classified as grade 4 tumours independent of morphologic features. Therefore, a diagnosis of astrocytoma, IDH mutant, or CNS WHO grade 4 requires either morphologic features of a glioblastoma, namely necrosis or microvascular proliferation, or homozygous deletion of CDKN2A and/or CDKN2B (see Figure 1) [1,14][1][10].
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Figure 1. Flow diagram summarising the WHO Classification of CNS Tumours (fifth edition, 2021) [14][10] and the role of CDKN2A/B HD (red box). Note: gain 7/loss 10 refers to the gain of chromosome 7 and the loss of chromosome 10.
The significance of CDKN2A/B alterations in gliomas is difficult to assess in historical cohorts. Prior to 2016, many studies classified tumours based only on morphology. Consequently, tumours previously classified as astrocytomas on morphological grounds are likely to include tumours that are currently classified as astrocytoma, IDH-mutant, oligodendroglioma IDH-mutant, 1p/19q codeleted or glioblastoma, and IDH-wildtype [15,16,17][11][12][13]. Interpretation of the CDKN2A/B literature is further complicated by the multiple different techniques used to interrogate the CDKN2A and CDKN2B genes and whether one or both genes are interrogated. In addition, there is ambiguity concerning the significance of isolated CDKN2A or CDKN2B loss compared to loss of both CDKN2A and CDKN2B [11,13,18,19,20][14][15][16][17][18].

2. Normal Role of CDKN2A/B and Effect of Their Deletion

CDKN2A and CDKN2B are adjacent to each other on chromosome 9p21, with CDKN2B being 25 kilobases (kb) centromeric to CDKN2A [34][19]. These genes code for three proteins that suppress the oncogenic cyclin-dependent kinase (CDK) pathway. CDKN2A encodes for p14 and p16, and CDKN2B encodes for p15 [35][20]. Early cytogenetic studies identified recurrent loss of the short arm of chromosome 9 in glioma cell lines [21[21][22][23][24],22,23,36], many involving the 9p21 locus [22[22][25],37], which includes CDKN2A and CDKN2B [23,36][23][24]. The resultant loss of p14, p15, and p16 proteins from CDKN2A/B HD leads to dysregulation of the cell cycle and other parallel oncogenic processes (see Figure 2). Reflecting this, inactivation of CDKN2A function has been reported in a variety of other malignancies, including breast cancer, lung cancer, head and neck cancer, melanoma, and bladder cancer [9]. In normal cells, the retinoblastoma (Rb) protein prevents cell growth by binding to the transcription factor E2F, preventing its translocation into the nucleus. A complex formed by cyclin D and CDK4/6 can phosphorylate Rb, thereby releasing E2F and allowing translocation into the nucleus, leading to cell growth. The products of CDKN2A/B, p15 and p16, can directly inhibit the formation of the CDK4/6-cyclin D complex, maintaining the association between E2F and Rb [39,40,41][26][27][28] and preventing cell cycle progression. Another product of CDKN2A is p14, which acts on cyclin-CDK complexes indirectly by inhibiting MDM2. MDM2 tags p53, targeting it for ubiquitination and subsequent proteasomal degradation. 14 prevents MDM2 tagging, resulting in p53 stabilisation. This in turn promotes the cellular accumulation of the inhibitory protein p21, which blocks several cyclin-CDK complexes and promotes cell cycle arrest [42,43][29][30]. Due to these important functions of the protein products of CDKN2A/B, their deletion enhances oncogenic potential and leads to unregulated cellular proliferation (see Figure 32) [42,43][29][30].
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Figure 32. Diagram showing the anti-proliferative and anti-angiogenic effects of CDKN2A/B Mouse double minute 2 homolog (MDM2), tissue inhibitor of metalloproteinase 3 (TIMP3), matrix metalloproteinases (MMP).
In addition to their role in regulating cell growth, CDKN2A/B also impact angiogenesis (see Figure 32). For example, p14 (unrelated to its inhibition of MDM2) can also inhibit endothelial cell migration required for angiogenesis by stimulating the expression of tissue inhibitor of metalloproteinase 3 (TIMP3), which inhibits matrix metalloproteinases (MMP) 2 and 9 [44][31]. MMPs are required to degrade the extracellular matrix to allow endothelial cell migration and subsequent vessel formation [45,46][32][33]. 

3. Identification of CDKN2A/B Deletions

A variety of methods can be used to evaluate CDKN2A/B HD. These include single-nucleotide polymorphism (SNP) microarrays [31[34][35],53], next-generation sequencing (NGS) [12[36][37],29], DNA-based methylation studies [11,13[14][15][16][17][18],18,19,20], and fluorescent in situ hybridisation (FISH) [10,28][38][39]. It should be noted that the accuracy of this variety of methods depends on the specific assay types used, as the genomic/cytogenetic resolution of each method differs.
Although SNP arrays, NGS, and methylation arrays possess greater resolution for individual gene-level detection, many studies combine CDKN2A and CDKN2B in the assessment of HD [11,18,29,31,53][14][16][34][35][37]. The accuracy of these methods is determined by the degree and depth of coverage of the genes of interest. NGS methods used in the literature to date include targeted gene panels [12][36] and whole exome sequencing (WES) [29][37], whereas methylation arrays include a combination of the HumanMethylation450 (450k) and MethylationEPIC (850k) arrays (Illumina, San Diego, CA, USA) [11,13,18,19,20][14][15][16][17][18] Fluorescence in situ hybridization (FISH) can be used to detect deletions and has been validated against methods utilising polymerase chain reaction (PCR) [54][40]. Thresholds of detection for FISH need to be around 20% to 30% tumour cells with HD [10,55][38][41]. A commonly used FISH probe in clinical diagnostic practise, the Vysis CDKN2A/CEP 9 FISH Probe Kit (Abbott Laboratories, North Chicago, IL, USA), is large and spans CDKN2A, CDKN2B, and MTAP genes [56][42]. Immunohistochemistry (IHC) has been used to identify CDKN2A HD in gliomas with mixed results. Given the close proximity of the CDKN2A and MTAP genes (see Figure 2), loss of MTAP immunoreactivity has been suggested as a surrogate for CDKN2A HD [32][43] and has been demonstrated in mesothelioma [57,58][44][45].  However, Satomi et al. did show that loss of p16 immunoreactivity correlated with clinical outcome in IDH-mutant astrocytomas [32][43]. While this is supported by other studies that demonstrated p16-negative tumours on IHC had a high negative predictive value for CDKN2A HD in adult and paediatric morphologic glioblastomas [59][46], other studies reported p16/CDKN2A discordance with the IHC method [28][39]. Sensitivity and specificity for p16 immunoreactivity in detecting CDKN2A HD have been reported as 78–94% and 70–82%, respectively [32][43]. 

4. CDKN2A/B Deletions in Clinical Studies

4.1. Initial Clinical Studies

Initial studies by Schmidt et al. [60][47] and Giani and Finocchiaro et al. [61][48] confirmed that CDKN2A HD was present in patients’ tumours and not just in glioma cell lines but did not assess CDKN2B. Giani and Finocchiaro et al. demonstrated CDKN2A HD in over 30% of gliomas (not further defined) and CDKN2A HeD in 25% [61][48]. Moulton et al. analysed 27 glioblastomas (not further defined) and identified 9 with CDKN2A HD, 3 with a heterozygous deletion, and one with a point mutation [62][49].

4.2. Clinical Outcomes of CDKN2A/B Deletion in the Pre-Molecular Classification Era (Pre-2016 WHO CNS Tumour Classification)

4.2.1. Correlation with High- and Low-Grade Gliomas

Initial studies described the relationship between CDKN2A/B and biologic markers of tumour aggressiveness (tumour grade and Ki-67 index). Sonoda et al. suggested CDKN2A/B deletions may have a role in gliomagenesis and therefore more aggressive tumour biology. Using single-strand conformation polymorphism (SSCP) and quantitative polymerase chain reaction (qPCR), they showed an increased incidence of CDKN2A/B HD in high-grade gliomas (44%, n = 12/27) compared to low-grade gliomas (10%, n = 1/10) [26][50]. Building on this concept, Ono et al. (1996) used multiplex PCR to assess CDKN2A/B HD in 50 astrocytomas and found a positive correlation between the Ki-67 index and CDKN2A HD (5/20 grade 3 astrocytomas and 6/13 glioblastomas had CDKN2A HD). CDKN2A HD was not identified in 17 grade 2 astrocytomas [64][51].

4.2.2. Correlation with Survival

In 2006, Dehais et al. reported that CDKN2A HD was a negative prognostic factor in a heterogeneous group of gliomas that included anaplastic astrocytomas, oligoastrocytomas, and oligodendrogliomas. Although 1p/19q status was assessed, the authors did not identify which cases had CDKN2A HD and 1p/19q co-deletion [15][11]. However, other reports did not find an association between CKDN2A HD and clinical outcome [66,67][52][53]. This may reflect differences in methodology and/or patient selection for tumours classified by morphology alone. One of these studies (Rich et al.) used a DNA microarray to assess the prognostic impact of CDKN2A deletion in patients older than 50 years. Although IDH status was not reported in the study, this population was likely enriched for IDH-wildtype tumours, and it was later shown that CDKN2A deletions lack prognostic impact in these tumours [66][52].

4.3. Clinical Outcomes in the Post-Molecular Classification Era (Post-2016 WHO CNS Tumour Classification)

4.3.1. Incorporation of CDKN2A/B Status into the fifth Edition of the WHO Classification (2021)

In 2020, the Consortium to Inform Molecular and Practical Approaches to CNS Tumour Taxonomy (cIMPACT-NOW), upgrade 5, published recommendations for grading criteria and terminologies in IDH-mutant astrocytomas. After reviewing the literature on multiple potential prognostic biomarkers, including CDKN2A/B HD, other Rb pathway genes, PIK3R1 and PIK3CA mutations, PDGFRA and MYCN amplification, reduced global DNA methylation, genomic instability (high copy number variants or somatic mutations), and mitotic activity and proliferation indices, they concluded that while “significant mitotic activity” should remain as a criterion for distinguishing grade 3 from grade 2 IDH-mutant astrocytomas, if CDKN2A/B HD, necrosis, or microvascular proliferation was present, a grade 4 designation was appropriate [7].

4.3.2. Literature That Supports CDKN2A/B Stratification

A prime example of supporting literature is Reis et al who were one of the earliest to report on the prognostic impact of CDKN2A HD in the setting of IDH mutations. They identified CDKN2A deletions as a prognostic marker specifically in IDH-mutant grade 2 and 3 gliomas. The authors analysed 270 gliomas and identified CDKN2A deletions via FISH in 57/108 grade 2 astrocytomas, 31/61 grade 3 astrocytomas, 23/96 oligodendrogliomas, and 19/49 oligoastrocytomas, inclusive of both homozygous and heterozygous CDKN2A deletions. The authors assessed tumours for 1p/19q deletion if they were not morphologic astrocytomas and assessed all tumours for IDH1/2 mutations by genome sequencing. They reported worse overall survival in grade 2 and 3 gliomas after adjusting for age, sex, and IDH mutation (HR 1.6, 95% CI = 1.0–2.4, p = 0.03). This significance was maintained in the astrocytoma subgroup (HR 2.0, 95% CI 1.1–3.5, p = 0.02) but not for oligodendrogliomas or oligoastrocytomas (HR 0.7, 95% CI 0.2–2.0, p = 0.5 and HR 0.8, 95% CI 0.3–2.4, p = 0.7, respectively). Again, a portion of these morphologic oligodendrogliomas in this cohort would no longer be classified as such without the corresponding molecularly confirmed 1p19q co-deletion. Interestingly, the presence of deletions in the IDH-mutant/ATRX expression loss astrocytoma group, without TP53 mutation, was non-prognostic (p = 0.2) [68][54]. Furthermore, as ATRX loss and TP53 mutations are strongly associated with IDH-mutant astrocytomas, it is unclear what this ATRX/TP53 discordance represents in IDH-mutant gliomas. Interestingly, given the FISH probe used covers a broad genomic region at 9p21, CDKN2B status can be said to be assessed by proxy.

4.3.3. Literature That Counters CDKN2A/B Stratification

Not all studies supported the use of CDKN2A/B in IDH-mutant astrocytomas. One such example is Roy et al. who analysed the 9p region lost in malignancies by analysing two cohorts (the first group being 10,985 samples from 33 different cancer types and the second group being 540 low-grade gliomas from three databases) and reported that CDKN2A inactivation did not promote tumour aggressiveness. Even when accounting for IDH and 1p/19q status (IDH-mutant 1p/19q non-deleted astrocytoma), there was no survival impact of CKDN2A HD. While they did show that heterozygous loss was associated with poor OS, mRNA expression was not altered. It was therefore postulated that this survival impact was due to the loss of other 9p genes [30][55]. It is unclear why this report differs from the majority of other studies, but it highlights that not all studies support the role of CDKN2A/B HD as a prognostic marker in IDH-mutant astrocytomas.

5. Management of Tumours with CDKN2A/B Homozygous Deletions

There is no clear consensus on the treatment of IDH-mutant astrocytomas with CDKN2A/B HD, and reports related to their management are scarce. Reflecting this ambiguity, the current joint American Society of Clinical Oncology and Society of Neuro-Oncology guidelines recommend grade 4 astrocytomas be treated with concurrent temozolomide-radiotherapy with sequential temozolomide or radiotherapy alone with sequential temozolomide [70][56]. However, given the evidence that CDKN2A/B HD alters tumour biology (increased angiogenesis and cell growth), it cannot assume that these tumours will be as susceptible to temozolomide as their non-deleted counterparts. Unfortunately, the evidence for treatment specifically for CDKN2A/B HD astrocytomas is minimal. In 2000, Iwadate et al. investigated the relationship between CDKN2A deletion, p16 expression, and chemosensitivity to 30 different cytotoxic agents in vitro. They analysed 56 astrocytoma specimens (based on morphologic criteria, IDH status unknown) and found 17 specimens had p16 alterations (CDKN2A HD = 7, CDKN2A mutation = 5, p16 loss on IHC = 5). When looking at samples with p16 alterations, they found that deletions correlated with increased sensitivity to anti-metabolite agents but not to alkylating agents, antibiotics, topoisomerase inhibitors, or anti-microtubule agents [71][57].

6. Conclusions

CDKN2A/B HD have a direct oncogenic effect through loss of cell cycle inhibition and other parallel processes and are a molecular marker that influences grading and survival in IDH-mutant astrocytomas. Overall, the evidence supports the use of CDKN2A/B HD as a negative prognostic marker in IDH-mutant astrocytomas. However, there is a significant variation in certainty, methods used for deletion detection, and the quality of the presented literature. There are also inaccuracies resulting from misclassification of tumours in older studies based on the revised WHO classification. These limitations hamper conclusions regarding the certainty and depth of impact CDKN2A/B HD has on prognosis and management and how this impact is affected by other co-occurring molecular alterations. Therefore, the strongest evidence for CDKN2A/B HD in IDH-mutant astrocytomas must come from prospective reports with the current WHO 2021 classification.

 

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