Prognostic Stratification for IDH-wild-type Lower-grade Astrocytoma by Sanger Sequencing and Copy-number Variation Analysis with MLPA.

doi:10.21203/rs.3.rs-198063/v1

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Prognostic Stratification for IDH-wild-type Lower-grade Astrocytoma by Sanger Sequencing and Copy-number Variation Analysis with MLPA.

https://doi.org/10.21203/rs.3.rs-198063/v1

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published 13 Jul, 2021

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The characteristics of IDH-wild-type lower-grade astrocytoma remain unclear. According to cIMPACT-NOW update 3, IDH-wild-type astrocytomas with any of the following factors show poor prognosis: combination of chromosome 7 gain and 10 loss (+ 7/-10), and/or EGFR amplification, and/or TERT promoter (TERTp) mutation. Multiplex ligation-dependent probe amplification (MLPA) can detect copy number variations with a reasonable cost. The purpose of this study was to find precise and cost-effective method for stratifying the prognosis of IDH-wild-type astrocytomas. Sanger sequencing, MLPA, and quantitative methylation-specific PCR were performed for 42 IDH-wild-type lower-grade astrocytomas surgically treated at Kyoto University Hospital, and overall survival was analyzed for 40 patients who underwent first surgery. Of the 42 IDH-wild-type astrocytomas, 21 were classified as grade 4 in cIMPACT-NOW update 3 criteria and all of them had either TERTp mutation or EGFR amplification. Kaplan-Meier analysis confirmed the prognostic significance of cIMPACT-NOW criteria, and WHO grade was also prognostic. Cox regression hazard model identified PTEN loss and PDGFRA amplification as independent significant prognostic indicators (Risk ratio of 9.75, p < 0.001 and 13.9, p = 0.002). The classification recommended by cIMPACT-NOW update 3 could be completed using Sanger sequencing and MLPA. Survival analysis revealed PTEN and PDGFRA were significant prognostic factors for IDH-wild-type lower-grade astrocytoma.

Health Economics & Outcomes Research

Scientific Communication

astrocytoma

cIMPACT-NOW

MLPA (multiplex ligation-dependent amplification)

IDH

TERTp

EGFR

PTEN

Glioma is a common tumour type originating in the human brain.¹ Glioblastoma, grade IV (GBM) is the most aggressive and major subtype of glioma, while diffuse astrocytoma, grade II (DA) and anaplastic astrocytoma, grade III (AA) are lower-grade astrocytomas. All these pathological entities had been classified mainly based on histology in the 2007 WHO classification of central nervous system tumours.²IDH mutation is widely recognised as a good predictor of survival among patients with glioma,³ and codeletion of chromosome 1p and 19q (1p/19q codeletion) has been associated with oligodendroglioma and suggests longer survival.⁴ Based on such findings, the WHO 2016 classification combined genetic profiling with a grading system based on the WHO 2007 classification. Oligodendroglioma was defined as a glioma with both IDH-mutation and 1p/19q codeletion, and the K27M mutation in H3F3A or less common HIST1H3B was included in the criteria for diffuse midline glioma, H3K27M-mutant.⁵

It is well known that IDH-mutant and IDH-wild type astrocytomas shows distinct genetic profiles and prognosis.^3,6−8 However, the disease-defining genetic alterations of IDH-wild type astrocytomas had not been revealed,^5,9 and their diagnosis were remained to depend on the histological findings. On the other side, some studies concluded a substantial subset of IDH-wild-type lower-grade astrocytomas with poor survival course like as IDH-wild-type GBMs.^7,9−14 Based on these studies, a previous report by the Consortium to Inform Molecular and Practical Approaches to CNS Tumour Taxonomy (cIMPACT-NOW) update 3 proposed that certain IDH-wild-type diffuse astrocytomas show a poor clinical course similar to IDH-wild-type glioblastomas, and that the characteristics of these tumours were a result of at least one of three genetic profiles: combined whole chromosome 7 gain and whole chromosome 10 loss (+ 7/-10); and/or EGFR amplification; and/or TERT promoter (TERTp) mutation.¹⁵ These three profiles have been recommended for inclusion in the next classification, with this specific type of astrocytoma to be diagnosed as “diffuse astrocytic glioma, IDH-wildtype, with molecular features of glioblastoma, WHO grade IV”, referred to as “astrocytoma, grade 4” in the present study. Like these things, copy-number variation (CNV) as well as mutations have been to make clear classification of IDH-wild type astrocytomas corresponding to clinical prognosis.

In general, the hot spot mutations of IDH1/2 and TERTp can be detected by Sanger sequencing^3,16−18 and 1p/19q codeletion can be examined by fluorescence in situ hybridisation or multiplex ligation-dependent probe amplification (MLPA).^19,20 MLPA can also detect copy-number alterations in EGFR and PTEN,^19,20 which are located on chromosome 7p and 10q, respectively. However, whole-chromosomal alterations in chromosomes 7 and 10 were difficult to detect using only one MLPA kit, and are reportedly better ascertained by single nucleotide polymorphism array,^11,14 DNA methylation array,^9,13 array-based comparative genomic hybridisation,^14,21 or next-generation sequencing.¹² Unfortunately, those methods are difficult to conduct in many local institutes and hospitals, and thus are less than ideal as global standards. MLPA can target multiple different sequences in a single PCR experiment and can detect small and fragmented DNA. ^19,22 And all appliances needed to introduce the method is thermal cycler and capillary sequencer. MLPA has potential to detect poor prognosis subgroups in IDH-wild type astrocytomas like as “astrocytoma, grade 4”, but the feasibility of this method is needed to be evaluated.

Between August 19th, 2010 and December 19th, 2019, a total of 291 samples were resected from 257 patients in Kyoto University Hospital and cryopreserved. All samples received integrated diagnosis as described above, and 42 tumours from 42 patients (31 men, 11 women) were classified as IDH-wild-type astrocytoma. The characteristics of these 42 tumours are described in Table 1. The tumours comprised 18 IDH-wild-type DAs and 24 IDH-wild-type AAs, and median age at resection was 55.5 years (range, 5–85 years). No significant differences in age or sex were noted between tumour subtypes. Forty tumours were removed in first surgeries, comprising 18 DAs and 22 AAs, and no difference in age was evident between subtypes. However, initial treatments for AAs included chemoradiation therapy more frequently, whereas observation was selected more in DAs. The initial postsurgical treatments of these patients were 9 chemoradiation therapies and 1 chemotherapy for DAs, and 19 chemoradiation therapies and 1 chemotherapy for AAs, while observation with regular imaging without treatment was selected for 8 DAs and 2 AAs.

Table 1
Characteristics of all patients included in the present study
	Total	DA, IDH-wt	AA, IDH-wt	p-value*
All tumours
Number, n	42	18	24
Sex, men, n	31	13	18	1
Age, year, median (range)	55.5 (5–85)	56.5 (5–85)	55 (8–85)	0.964
cIMPACT-NOW update3 "grade4", n	21	9	12	1
TERTp mutation, n	18	9	9	0.533
EGFR amplification, n	8	1	7	0.109
EGFR gain and PTEN loss, n	6	3	3	1
PTEN loss, n	11	3	8	0.299
PDGFR amplification, n	4	0	4	0.122
CDKN2A homozygous loss, n	8	1	7	0.109
MGMTp hypermethylation, n	13	6	7	1
Patients for survival analysis
Number, n	40	18	22
age, year, median (range)	57 (5–85)	56.5 (5–85)	59 (8–85)	0.725
Initial treatment
Chemoradiation therapy, n	28	9	19	0.0246
Chemotherapy, n	2	1	1
Observation, n	10	8	2
* The p-values between groups of DA and AA are calculated with Student's t-test for age and with Fisher's exact test for the others.
Abbreviations: AA, anaplastic astrocytoma; DA, diffuse astrocytoma; IDH-wt, IDH wild type

The detailed status of mutations and alterations in the 42 IDH-wild-type astrocytomas are shown in Fig. 1-A. TERTp mutation was detected in 18 IDH-wild-type astrocytomas (42.9%), comprising 9 DAs (50%) and 9 AAs (37.5%). Again, no significant difference was seen between these two tumour types. EGFR amplification was seen in 8 IDH-wild-type astrocytomas (19.0%), comprising 1 DA (5.6%) and 7 AAs (29.2%), tending to be slightly more frequent in AAs than in DAs (p = 0.109). The combination of EGFR gain and PTEN loss (+ EGFR/-PTEN) was detected in 6 IDH-wild-type astrocytomas (14.3%), comprising 3 DAs (16.7%) and 3 AAs (12.5%), showing no significant difference. All DAs with EGFR amplification or + EGFR/-PTEN showed TERTp mutation, so all DAs diagnosed as “astrocytoma, grade 4” were equivalent to DAs with TERTp mutation, while 3 TERTp-wild type AAs showed EGFR amplification. Interestingly, no astrocytomas with + EGFR/-PTEN lacked the TERTp mutation.

Classification of lower-grade astrocytomas, IDH-wild type

The results of classification of IDH-wild-type lower-grade astrocytomas are shown in Fig. 1-B. TERTp mutation was detected in 18 of all 42 IDH-wild-type astrocytomas, and these 18 tumours were classified as “astrocytoma, grade 4” from Sanger sequence alone. In the 24 tumours without TERTp mutation, MLPA showed EGFR amplification in 3 AAs, and no tumours with + EGFR/-PTEN. As a result, 21 of all 42 tumours were classified as “astrocytoma, grade 4”, comprising 9 DAs and 12 AAs. In our cohort, all instances of “astrocytoma, grade 4” were able to be diagnosed by the combination of Sanger sequence and MLPA.

Correlations with tumour profiles

Correlations between all pairs of the following factors were analysed: age at diagnosis, TERTp mutation, WHO grade, O6-methylguanine-DNA methyltransferase promoter (MGMTp) hyper methylation, and copy number alterations of EGFR, PTEN, CDKN2A, PDGFRA, MDM2, CDK4, NFKBIA, and TP53. TERTp mutation, EGFR amplification, CDKN2A homozygous loss, CDK4 gain or amplification (gain/amplification), CDK4 amplification, and MGMTp hypermethylation correlated with higher age at diagnosis (p = 0.0157, p = 0.0382, p = 0.0272, p = 0.0222, and p = 0.0045, respectively). Statistical correlations were detected between any two of TERTp mutation, EGFR gain/amplification and PTEN loss (TERTp and EGFR gain/amplification, odds ratio 5.91, p = 0.0236; TERTp mutation and PTEN loss, odds ratio 11, p = 0.004; EGFR gain/amplification and PTEN loss, odds ratio 9.38, p = 0.0304). Another correlation was seen between PDGFRA gain/amplification and CDKN2A homozygous loss (odds ratio, 7.78; p = 0.0195). WHO grade correlated significantly with MDM2 loss and MDM2 hemizygous loss (p = 0.0054 and p = 0.014, respectively). On the other hand, WHO grade showed no significance in the other profiles (Table 1).

Survival analysis of lower-grade astrocytomas, IDH-wild type

Clinical outcomes were calculated for the 40 cases in which the tumours were removed in first surgeries, including 18 DAs and 22 AAs. “Astrocytoma, grade 4” showed significantly shorter overall survival (OS) compared with other tumours in all astrocytomas (p = 0.0149) and DAs (p = 0.0036), but not in AAs (p = 0.1288) (Fig. 2-A). TERTp mutations were significantly associated with poor OS in all astrocytomas (p = 0.0228), and in DAs (p = 0.0036), but not in AAs (p = 0.0884) (Fig. 2-B). EGFR amplification was a significant factor for OS only in all astrocytomas (p = 0.0401), not in diffuse DAs (p = 0.7893) or AAs (p = 0.2877) (Fig. 2-C).

The relationship between WHO grade and OS was also analysed, and AAs showed poorer prognosis than DAs (p = 0.007). For cases of “astrocytoma, grade 4” or “non-astrocytoma, grade 4”, AA showed poorer survival curves compared with DA, and significant differences were identified with the Wilcoxon test (p = 0.04 each), but not with the log-rank test (p = 0.09 and p = 0.055, respectively) (Fig. 3-A).

Log-rank testing showed significant correlations between OS and the following factors: age > 40 years, EGFR gain/amplification, PDGFRA amplification, PTEN homozygous loss and loss, CDK4 gain/amplification, MDM2 homozygous loss and alteration, and TP53 homozygous loss (Table 2). These factors and TERTp mutation, EGFR amplification and the diagnosis of “astrocytoma, grade 4”, as described above, were analysed by Cox proportional hazard modelling. After the stepwise procedure, three factors remained significant: PDGFRA amplification with the largest risk ratio (risk ratio 13.9, p = 0.0022), PTEN loss (risk ratio 9.75, P = 0.0003) with the most significance, and PTEN homozygous loss (risk ratio 6.97, p = 0.0329) (Table 3). PDGFRA amplification was detected in 4 astrocytomas, and survival analysis showed a significant result (p = 0.043) (Fig. 3-B). PTEN loss was detected in 11 astrocytomas and 3 were homozygous losses and 8 were hemizygous losses, 6 of which were coincided with EGFR gains. A significant difference in OS was seen between astrocytomas with intact PTEN and those with PTEN loss (p < 0.0001), while there was no difference in OS between PTEN homozygous and PTEN hemizygous astrocytomas (p = 0.1255) (Fig. 3-C). No difference in survival of PTEN-loss astrocytomas was seen between presence or absence of EGFR gain (p = 0.8133), and PTEN-loss astrocytomas showed poor prognosis compared with PTEN-intact astrocytomas irrespective of whether EGFR gain was present (with EGFR gain, p = 0.0001; without EGFR gain, p < 0.0001) (Fig. 3-D).

Table 2
Results of Kaplan-Meier analysis and survival analysis about factors showing significance in survival analysis.
		n	event	mOS (months) (95%CI)	p value
		n	event	mOS (months) (95%CI)	log-rank test	Wilcoxon test
cIMPACT-NOW update 3	“grade 4"	21	11	29.0 (16.9–60.2)	0.0149	0.0428
	not "grade 4"	19	6	NR (24.5-NR)
TERTp	mutant	18	10	30.4 (15.8–60.2)	0.0228	0.0384
	wild type	22	7	NR (24.5-NR)
EGFR	amplification	8	4	27.2 (8.45-29.0)	0.0401	0.1989
	no amplification	32	13	64.2 (30.4-NR)
	gain/amplification	25	12	29.0 (21.3–60.2)	0.0227	0.0466
	retained	15	4	NR (49.2-NR)
PDGFRA	amplification	4	3	24.4 (3.65-NR)	0.043	0.0793
	no amplification	36	14	60.2 (29.0-NR)
PTEN	homozygous loss	3	3	11.5 (8.45–15.9)	< 0.0001	< 0.0001
	no homozygous loss	37	14	60.2 (29.0-NR)
	lost	11	9	16.39 (3.65-29.0)	< 0.0001	< 0.0001
	retained	29	8	NR (49.2-NR)
CDK4	gain/amplification	10	7	24.4(8.45-29.0)	0.0006	0.0125
	retained	30	10	NR (49.2-NR)
MDM2	homozygous loss	2	2	16.4 (8.45–24.4)	0.008	0.022
	no homozygous loss	38	15	60.2 (29.0-NR)
	altered	15	9	24.5 (11.5-NR)	0.024	0.0279
	retained	25	8	NR (49.2-NR)
TP53	homozygous loss	2	2	16.4 (8.45–24.4)	0.008	0.022
	no homozygous loss	38	15	60.2 (29.0-NR)
Age	age > 40	34	16	49.2 (24.4–64.2)	0.0345	0.0524
	age ≤40	6	1	NR (30.4-NR)
WHO grade	grade III	22	13	27.2 (13.1–49.2)	0.0071	0.004
	grade II	18	4	NR (55.9-NR)
Abbreviations: CI, confidence interval; NR, not reached; mOS, median overall survival

Table 3
Results of Cox proportional hazard model analysis of OS for each factor.
	Risk ratio (95%CI)	p value
WHO 2016, grade III	6.46 (0.942–44.4)	0.0575
0.218 (0.00336-14.2)	0.4744
TERTp mutation	5.14 (0.129-204)	0.3838
EGFR amplification	0.709 (0.0860–5.84)	0.7493
EGFR gain/amplification	3.04 (0.261–35.4)	0.3749
PDGFRA amplification	2.17 (0.0721-65.5)	0.6549
PTEN homozygous loss	1.65 (0.0550–49.4)	0.7730
PTEN loss	5.93 (0.598–58.8)	0.1283
CDK4 gain/amplification	1.16 (0.0755-17.7)	0.9166
MDM2 homozygous loss	1.39 (0.186–10.4)	0.7467
MDM2 alteration	1.47 (0.212–10.2)	0.6978
age over 40 years	10.9 (0.372-319)	0.1657
*after stepwise procedure
PTEN loss	9.75 (2.82–33.7)	0.0003
PTEN homozygous loss	6.97 (1.17–41.5)	0.0329
PDGFR amplification	13.9 (2.58–75.2)	0.0022
Abbreviations: CI, confidence interval
The initial candidates (written above a bar) are all factors for which Kaplan-Meier analysis or log-rank testing showed significant results.

In the present study, the Sanger sequencing method could detect most of instances of “astrocytoma, grade 4”, with the addition of MLPA successfully identifying all cases. Correlations between TERTp mutation and 7p gain or 10q loss have already been reported.¹¹ In the present study, the correlation analysis showed that TERTp mutation was associated with EGFR gain and PTEN loss, and + EGFR/-PTEN astrocytoma always accompanied by TERTp mutation. This fact may result from a relationship between TERTp mutation, gain of chromosome 7, and loss of chromosome 10. The database of The Cancer Genome Atlas (TCGA)⁶ included 86 IDH-wild-type grade II or III gliomas, and TERTp status was examined in 56 cases. TERTp mutation was detected in 37 cases, and EGFR amplification was shown in 15 out of 37 TERTp mutant gliomas and in 4 of 19 TERTp-wild-type gliomas. The status of + 7/-10 was evaluated in 55 of the 56 cases of TERTp mutant grade II or III glioma, with 27 cases showing + 7/-10. Among these 27 cases, 12 cases showed both TERTp mutation and EGFR amplification, 14 cases showed TERTp mutation alone, and the remaining 1 case showed EGFR amplification alone. In the present study, EGFR amplification was defined as different from EGFR gain, so + EGFR/-PTEN tumours never showed EGFR amplification. As in the present study, for IDH-wild-type grade II or III gliomas in TCGA database, TERTp status revealed almost all cases of “astrocytoma, grade 4” and the addition of EGFR status successfully identified all other cases of “astrocytoma, grade 4.” Sanger sequencing and MLPA were thus thought to be reasonable methods for classifying IDH-wild-type lower-grade gliomas based on the recommendations from cIMPACT-NOW update 3.¹⁵

WHO grade was a good marker of prognosis in the present study. IDH-wild-type AAs showed lower survival curve than IDH-wild-type DAs. Based on the WHO 2016 classification, glioma grade is partly affected by molecular factors including 1p/19q codeletion and histone mutations, but gliomas are mainly classified according to histological characteristics, which are almost the same as in the WHO 2007 classification. Although our cohort showed no differences in sex, age, or other prognostic factors detected in multivariate analysis, treatment selections did differ between DAs and AAs. Patients with AAs tended to be initially treated with strong chemoradiation therapies like the Stupp regimen,²³ and those with DAs tended to undergo observation alone more often; this might be one reason why the results of log-rank testing showing no statistical difference. The survival curve of DAs was definitely favourable compared with AAs in the early course, and the generalized Wilcoxon test showed a significant difference. Taking IDH-mutation status into consideration, WHO grade was reported as a significant factors for OS in lower-grade gliomas,^11,24,25 and one of the studies showed that WHO grade had larger prognostic value in IDH-wild-type astrocytomas compared with in IDH-mutant astrocytomas, with the authors proposing histological mitotic count as a significant predictor of prognosis.²⁴ This fact supports that histological grading systems remain important, especially for IDH-wild-type astrocytoma.

The survival analysis showed TERTp mutation as a prognostic factor for OS in the group of all IDH-wild-type astrocytomas and IDH-wild-type DAs, and the diagnosis of “astrocytoma, grade 4” with EGFR amplification was significant only in all IDH-wild-type astrocytomas. TERTp mutation and EGFR amplification have been reported as characteristics of IDH-wild-type GBM and as unfavourable prognostic factors in IDH-wild-type astrocytomas in many studies,^{10–12, 26} although a few studies have reported no significance.^14,27 In our study, EGFR amplification was slightly more frequent in AAs than in DAs, while TERTp showed no difference between subtypes. TERTp mutation, EGFR amplification, and diagnosis of “astrocytoma, grade 4” were significant factors in the group of all IDH-wild-type astrocytomas.

As mentioned above, WHO grade, TERTp mutation, EGFR amplification and diagnosis of “astrocytoma, grade 4” were good predictors of IDH-wild-type astrocytomas in Kaplan-Meier analysis, but Cox proportional hazard modelling detected no significance for OS in these factors. According to the Cox proportional hazard model of our cohort, copy number alteration of PTEN and PDGFRA amplification were significant predictors of OS.

Copy number alteration of PTEN was a strong predictor, as demonstrated by both the Kaplan-Meier method and Cox proportional hazard modelling. No different in OS was evident between PTEN hemizygous loss astrocytomas and PTEN homozygous loss astrocytomas, with both showing shorter OS than PTEN-intact astrocytomas. In addition, whether combined with EGFR gain or not, PTEN loss resulted in a significant difference in OS. PTEN loss is one of the typical genetic alteration of GBM, observed in about 30–40%.^28,29 Some studies of prognostic factors in GBM patients have been published, but have been controversial about the significance of PTEN loss.^27,30,31 However, in IDH-wild-type lower-grade astrocytomas, some papers have stated that PTEN loss is associated with poor prognosis,^27,32 potentially because PTEN is a tumour suppressor gene^27,33 and inactivation of PTEN signalling is thus important to malignant progression to glioblastoma.³⁴ The present study indicated PTEN loss as a strong predictor of poor prognosis in IDH-wild-type astrocytomas.

PDGFRA amplification showed a strong risk ratio in the present study, but only 4 AAs were included in the present study. PDGFRA amplification was also recognised as a characteristics of proneural GBM, which showed relatively good prognosis.^29,35 The frequency of PDGFRA amplification in lower-grade glioma has only been reported from studies of small numbers of low-grade gliomas,^36,37 and there was not enough evidence to conclude prognostic value of it. Strum et al. reported about subgrouping of GBMs based on the methylation profiles and compared them with other profiles of mutation and copy number status.³⁵PDGFRA amplification was more common in a methylation cluster, “RTK I”, than the other four clusters. “RTK I” cluster also showed CDKN2A loss frequently. In the present study, a correlation between PDGFRA gain/amplification and CDKN2A homozygous loss was seen, and might imply that astrocytoma with alteration of PDGFR is associated with “RTK I” GBMs. In our cohort, no PDGFRA amplification was seen in DAs, but no difference in its frequency was evident between DAs and AAs because of the small number with PDGFRA amplification. Further studies are required to clarify the prognostic value of PDGFRA.

After the report of cIMPACT-NOW update 3, the genetic analyses such as copy number analysis have been extensively studied in lower grade gliomas, and it becomes clearer that several genetic markers are surely prognostic and that they need to be incorporated into clinical practice. In this context, our study has important implications by showing that such prognostic stratification can be done by direct sequencing and MLPA with a reasonable cost.

There are several limitations in the present study. First, the number of study population was small. Second, we did not perform external validation analysis. The result of MLPA analysis was not confirmed by other methodology such as CNV array.

The present study showed that the combination of Sanger sequencing and MLPA was sufficient to identify a subgroup of patients who has poorer prognosis in IDH-wild-type lower-grade astrocytoma. These patients were safely considered to have “astrocytoma, grade 4” according to the cIMPACT-NOW update 3 criteria. Our data also showed that PTEN loss and PDGFRA amplification are significant prognostic factors, and these genetic alterations would be good candidates for upcoming new classification. WHO grade is still useful to predict the clinical course of patients with IDH-wild-type gliomas.

Ethics approval and consent to participate

This study was carried out in accordance with the principles of the Declaration of Helsinki, and approval was obtained from the institutional review board at Kyoto University Hospital (approval number: G1124). Informed consent was obtained from all patients for inclusion in this study.

Subjects

The purpose of the present study was to assess the feasibility of the combination of Sanger sequencing and MLPA in classifying IDH-wild-type lower-grade astrocytomas, as diagnosed by the WHO 2016 classification, into a new classification recommended by cIMPACT-NOW update 3, and to reveal prognostic factors for IDH-wild-type lower-grade astrocytoma.

The targets of the present study were IDH-wild-type astrocytomas surgically treated in Kyoto University Hospital. Inclusion criteria were as follows: 1) tumour samples after removal were stored as frozen or fresh specimens to maintain sufficient quality of DNA for extraction; 2) initial diagnosis was WHO grade II or grade III glioma; 3) Sanger sequence revealed no hot-point mutations in IDH1/2, H3F3A, or HIST1H3B; 4) MLPA showed no 1p/19q codeletion; and 5) informed consent was obtained.

Sanger sequencing

Tumour DNA was extracted from tumour specimens using NucleoSpin® Tissue (MACHEREY-NAGEL, Düren, Germany). Regions of interest for driver genes^3,16− 18 were amplified by PCR with gene-specific primers (Supplementary table S1) and TaKaRa Ex Taq® (TAKARA BIO, Shiga, Japan) (IDH1/2, H3F3A, and HIST1H3B) or AmpliTaq Gold 360 (Thermo Fisher Scientific, Waltham, MA) (TERTp) using an Applied Biosystems GeneAmp PCR System 9700 (Thermo Fisher Scientific). PCR products were processed by ExoSAP-IT (Thermo Fisher Scientific), then sequenced with sequencing primer (IDH1) or PCR forward primer as a sequencing primer (IDH2, H3F3A, HIST1H3B, TERTp) and a BigDye® Terminator V1.1 Cycle Sequencing Kit (Thermo Fisher Scientific) using the ABI 3130xL Genetic Analyzer (Thermo Fisher Scientific).

MGMT promoter methylation analysis

MGMTp methylation was assessed by quantitative methylation-specific PCR (qMSP), in accordance with previous reports.^20,38 Genomic DNA samples were processed using an EZ DNA Methylation Gold Kit (Zymo Research Corporation, Irvine, CA). Methylated and unmethylated molecules were quantified by qMSP using a QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific) with POWER SYBR® Green Master Mix (Thermo Fisher Scientific) and specific primers (Supplementary Table S1)³⁹ according to the standard curve method. The methylation status of samples was determined from the ratio of methylated molecule using the cut-off value at > 1%.²⁰

MLPA

Copy number analyses of 1p/19q, EGFR, PTEN, CDKN2A and PDGFRA were performed with MLPA according to the instructions from the manufacturer (SALSA MLPA KIT probemix P088-C2 for 1p/19q analysis and SALSA MLPA KIT probemix P105-D2 for the others; MRC-Holland, Amsterdam, the Netherlands).^19,20 MLPA with probemix P105-D2 can also analyse the alteration of MDM2, CDK4, NFKBIA, and TP53. Human genomic DNA G147A, G152A, and G304A (Promega, Madison, WI) were used as reference samples.

Data on MLPA were collected using an ABI 3130xL Genetic Analyzer (Thermo Fisher Scientific), then analysed using Coffalyzer.Net Software (MRC-Holland). Thresholds of copy number detection were chosen as reported previously^19,40; thresholds of normal copy number were set at 0.8 to 1.2. A ratio of < 0.4 was set as the threshold for hemizygous and homozygous losses, and ratios > 2.0 were defined as amplifications.

Integrated diagnosis

Tumour grading was performed histologically. Using all molecular pathological information, all cases received integrated diagnoses according to the 2016 WHO classification for central nervous system tumours.⁵

Clinical outcomes

Clinical data retrospectively collected from electronic records included age at diagnosis, sex, treatment protocol as chemotherapy or radiotherapy, and dates of surgery, last follow-up, and death.

Classification of astrocytomas, IDH-wild type

All IDH-wild-type astrocytomas were analysed by Sanger sequencing and MLPA to reveal the status of TERTp, EGFR gain or amplification, and PTEN loss. Classifications were performed in three steps. The first step was TERTp mutation, the second was EGFR amplification, and the third was + EGFR/-PTEN.

According to the recommendations of a previous study,¹⁵ “astrocytoma, grade 4” was defined as IDH-wild-type astrocytoma with any one or more of TERTp mutation, EGFR amplification, or + 7/-10. However, showing the whole chromosomal alteration with MLPA (with SALSA MLPA KIT probemix P105-D2) is impossible. Because EGFR and PTEN are located on chromosomes 7 and 10, respectively, tumours with + 7/-10 must have + EGFR/-PTEN. Therefore, in our classification system, +EGFR/-PTEN was used as a criterion, and + 7/-10 was planned to be examined in + EGFR/-PTEN tumours.

Statistical analysis

All statistical analyses were performed using JMP Pro version 15.1.0 software (SAS Institute, Cary, NC). The difference of categorical variables was evaluated by Fisher’s exact test or chi square test, and Student’s t-test was used for continuous variables. For survival analyses, OS was defined as the interval between the initial operative day and the date of death, or last follow-up date on which the patient was known to be alive. Survival data were analysed using the Kaplan-Meyer curve and the p-value of survival in the present paper was determined by log-rank testing if there was no special comment, while generalized Wilcoxon test was used when appropriate. Multivariate analysis was performed by Cox proportional hazard modelling. The relationships between OS and following factors were analysed; age; MGMTp hyper-methylation; gain, amplification, and gain/amplification of EGFR, PDGFRA, and MDM2; hemizygous loss, homozygous loss and loss (including both of hemizygous and homozygous loss) of CDKN2A, PTEN, CDK4, MDM2, NFKBIA, and TP53. Differences were considered significant for values of p < 0.05.

AA = anaplastic astrocytoma, grade 3

CDK4 = cyclin-dependent kinase 4

CDKN2A = cyclin-dependent kinase inhibitor 2A

CI = confidence interval

CNV = copy number variation

cIMPACT-NOW update 3 = the Consortium to Inform Molecular and Practical Approaches to CNS Tumour Taxonomy

DA = diffuse astrocytoma, grade 2

EGFR = epidermal growth factor receptor

FFPE = formalin-fixed paraffine-embedded

GBM = glioblastoma

H3F3A = H3 histone, family 3A

HIST1H3B = histone cluster 1 H3 family member b

IDH = isocitrate dehydrogenase

MDM2 = murine double minute 2

MGMTp = O6-methylguanine-DNA methyltransferase promoter

MLPA = multiplex ligation-dependent probe amplification

NFKBIA = nuclear factor kappa-B inhibitor alpha

OS = overall survival

PDGFRA = platelet-derived growth factor receptor A

PTEN = phosphatase and tensin homolog deleted from chromosome 10

qMSP = quantitative methylation-specific PCR

TCGA = The Cancer Genome Atlas

TERT = telomerase reverse transcriptase

TERTp = telomerase reverse transcriptase promoter

TP53 = tumour protein p53

Availability of data and material

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Acknowledgements

This study included results based upon data generated by The Cancer Genome Atlas (TCGA) Research Network. We would like to acknowledge TCGA and their financial and material support in the development of the TCGA, as well as members of the consortium for their commitment to data sharing. Interpretations are the responsibility of the study authors.

Authors' contributions

Y.A. and Y.K. designed the study. Y.Ma., T.K, E.Y., T.S., and D.K. performed genetic

analyses and data analyses. Y.T., M.T., Y.Mi., and S.M. collected samples. Y.Ma., Y.A. and Y.K.

wrote the manuscript. All authors read and approved the final manuscript.

Competing interests

Y.A. has received research funding research funding from Ono Pharmaceutical Co.,

Ltd.,Siemens, Philips, Sanofi, Nihon Medi-Physics, MitsubishiTanabe, Takeda, Stryker,

Astellas Pharma, Taiho Pharma, and Pfizer outside the submitted work; honoraria from

Nippon Kayaku, Behring, AbbVie, Novocure, UCB Japan, Ono Pharmaceutical, and Otsuka;

and research funding and honoraria from Brainlab, Merck, Chugai, Eisai, Meiji Seika, Daiichi

Sankyo, Zeiss, and CLS outside the submitted work. Y.Ma., E.Y., T.S., T.K., Y.T., M.T., D.K., Y.Mi., S.M., Y.K. declare no competing interests.

Funding

This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (project nos. 16K10754, 17K19724, 19K09505, and 19K22685).

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Competing interest reported. Y.A. has received research funding research funding from Ono Pharmaceutical Co., Ltd.,Siemens, Philips, Sanofi, Nihon Medi-Physics, MitsubishiTanabe, Takeda, Stryker, Astellas Pharma, Taiho Pharma, and Pfizer outside the submitted work; honoraria from Nippon Kayaku, Behring, AbbVie, Novocure, UCB Japan, Ono Pharmaceutical, and Otsuka; and research funding and honoraria from Brainlab, Merck, Chugai, Eisai, Meiji Seika, Daiichi Sankyo, Zeiss, and CLS outside the submitted work.

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Prognostic Stratification for IDH-wild-type Lower-grade Astrocytoma by Sanger Sequencing and Copy-number Variation Analysis with MLPA.

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Abstract

Figures

Introduction

Results

Classification of lower-grade astrocytomas, IDH-wild type

Correlations with tumour profiles

Survival analysis of lower-grade astrocytomas, IDH-wild type

Discussion

Conclusion

Methods

Ethics approval and consent to participate

Subjects

Sanger sequencing

MGMT promoter methylation analysis

MLPA

Integrated diagnosis

Clinical outcomes

Classification of astrocytomas, IDH-wild type

Statistical analysis

Abbreviations

Declarations

Availability of data and material

Acknowledgements

Authors' contributions

Competing interests

Funding

References

Additional Declarations

Supplementary Files

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