World J Oncol

World Journal of Oncology, ISSN 1920-4531 print, 1920-454X online, Open Access

Article copyright, the authors; Journal compilation copyright, World J Oncol and Elmer Press Inc

Journal website https://www.wjon.org

Original Article

Volume 13, Number 3, June 2022, pages 117-125

Association Between CD204-Expressed Tumor-Associated Macrophages and MGMT-Promoter Methylation in the Microenvironment of Grade 4 Astrocytomas

Figure 1. CD204⁺ TAMs expression in WHO grade 4 astrocytoma using IHC. (a) CD204⁺ TAMs high expression. (b) CD204⁺ TAMs low expression. (c) Normal brain control. (d) Diagram shows the relationship between TAMs and tumor cells. Magnification (× 100 µm). TAMs: tumor-associated macrophages; WHO: World Health Organization; IHC: immunohistochemistry.

Figure 2. The relationship between CD204⁺ TAMs expression and MGMT-promoter methylation with RFI in WHO grade 4 astrocytoma patients. KM graphs show no statistically significant difference in RFI between CD204⁺ TAMs expression and MGMT-promoter methylation using different treatment modalities. TAMs: tumor-associated macrophages; MGMT: O⁶-methylguanine-DNA methyltransferase; RFI: recurrence-free interval; WHO: World Health Organization; KM: Kaplan-Meier.

**Table 1.** Demographic Data of 45 Cases of WHO Grade 4 Astrocytoma Enrolled in the Study
	Overall (n = 45)
IDH: isocitrate dehydrogenase; MGMT: O⁶-methylguanine-DNA methyltransferase; TAMs: tumor-associated macrophages; WHO: World Health Organization.
Age
< 55 years	10 (22%)
≥ 55 years	35 (78%)
IDH status
IDH-wildtype	35 (77.8%)
IDH-mutant	10 (22.2%)
Genetic profile
Unmethylated MGMT	15 (33.3%)
Methylated MGMT	18 (40%)
Unknown	12 (26.6%)
Tumor location
Frontal	18 (40%)
Temporal	12 (26.6%)
Parietal	12 (26.6%)
Occipital	2 (4.4%)
Cerebellar	1 (2.2%)
CD204⁺ TAMs expression
High expression	32 (71.1%)
Low expression	13 (28.8%)
Adjuvant therapy
Chemoradiotherapy	26 (57.7%)
Radiation	17 (37.7%)
None	2 (4.6%)

Table 1. Demographic Data of 45 Cases of WHO Grade 4 Astrocytoma Enrolled in the Study

Overall (n = 45)

IDH: isocitrate dehydrogenase; MGMT: O⁶-methylguanine-DNA methyltransferase; TAMs: tumor-associated macrophages; WHO: World Health Organization.

Age

< 55 years

10 (22%)

≥ 55 years

35 (78%)

IDH status

IDH-wildtype

35 (77.8%)

IDH-mutant

10 (22.2%)

Genetic profile

Unmethylated MGMT

15 (33.3%)

Methylated MGMT

18 (40%)

Unknown

12 (26.6%)

Tumor location

Frontal

18 (40%)

Temporal

12 (26.6%)

Parietal

12 (26.6%)

Occipital

2 (4.4%)

Cerebellar

1 (2.2%)

CD204⁺ TAMs expression

High expression

32 (71.1%)

Low expression

13 (28.8%)

Adjuvant therapy

Chemoradiotherapy

26 (57.7%)

Radiation

17 (37.7%)

None

2 (4.6%)

**Table 2.** Patients’ Data Enrolled in This Study
Age	Gender	Location	IDH1	ATRX	CD204	MGMT	Adjuvant	CT	RFI
Data include age, gender, tumor location, IDH1 status, MGMT-promoter methylation, CD204⁺ TAM expression, treatment modalities, and RFI. All the enrolled cases were WHO grade 4 astrocytomas that presented with necrosis, microvascular proliferations, ATRX loss and intact 1p19q. IDH-w: IDH-wildtype; IDH-m: IDH-mutant; MGMT: O⁶-methylguanine-DNA methyltransferase; RT: radiotherapy; CT: chemotherapy; TMZ: temozolomide; RFI: recurrence-free interval.
50	Male	Frontal	IDH-m	Loss	High	Methylated	RT	None	670
66	Male	Frontal	IDH-m	Loss	Low	Methylated	CT + RT	TMZ	1,034
71	Male	Parietal	IDH-m	Loss	High	Methylated	CT + RT	TMZ	440
31	Female	Parietal	IDH-m	Loss	High	Unknown	RT	None	670
64	Female	Parietal	IDH-m	Loss	High	Unmethylated	CT + RT	TMZ+	731
57	Male	Parietal	IDH-m	Loss	Low	Methylated	CT + RT	TMZ+	1,096
63	Male	Cerebellar	IDH-m	Loss	High	Unknown	CT + RT	TMZ+	1,123
72	Female	Frontal	IDH-m	Loss	Low	Unmethylated	RT	None	643
69	Female	Parietal	IDH-m	Loss	High	Methylated	RT	None	638
58	Female	Temporal	IDH-m	Loss	High	Unmethylated	CT + RT	TMZ	801
19	Male	Frontal	IDH-w	Loss	Low	Unknown	RT	None	330
58	Male	Frontal	IDH-w	Loss	Low	Unmethylated	RT	None	530
28	Female	Parietal	IDH-w	Loss	Low	Unmethylated	CT + RT	TMZ	330
22	Male	Temporal	IDH-w	Loss	High	Unknown	RT	None	250
63	Male	Frontal	IDH-w	Loss	High	Unmethylated	RT	None	430
68	Male	Frontal	IDH-w	Loss	High	Methylated	CT + RT	TMZ	1,016
59	Male	Temporal	IDH-w	Loss	High	Unmethylated	RT	None	293
73	Female	Parietal	IDH-w	Loss	High	Methylated	CT + RT	TMZ+	156
76	Female	Frontal	IDH-w	Loss	High	Unknown	CT + RT	TMZ	150
46	Female	Occipital	IDH-w	Loss	Low	Unmethylated	RT	None	183
63	Female	Temporal	IDH-w	Loss	High	Unknown	CT + RT	TMZ	194
82	Male	Frontal	IDH-w	Loss	High	Methylated	CT + RT	TMZ+	340
57	Female	Parietal	IDH-w	Loss	High	Methylated	CT + RT	TMZ+	260
10	Male	Temporal	IDH-w	Loss	Low	Methylated	None	None	92
42	Male	Frontal	IDH-w	Loss	High	Unknown	CT + RT	TMZ	306
59	Female	Occipital	IDH-w	Loss	High	Methylated	CT + RT	TMZ+	826
64	Male	Frontal	IDH-w	Loss	High	Methylated	RT	None	273
63	Male	Temporal	IDH-w	Loss	Low	Unmethylated	RT	None	550
47	Male	Temporal	IDH-w	Loss	High	Unknown	RT	None	141
62	Female	Frontal	IDH-w	Loss	Low	Unmethylated	RT	None	90
69	Male	Temporal	IDH-w	Loss	High	Unknown	None	None	114
59	Female	Frontal	IDH-w	Loss	High	Methylated	CT + RT	TMZ	0
17	Male	Frontal	IDH-w	Loss	High	Methylated	CT + RT	TMZ+	460
68	Male	Temporal	IDH-w	Loss	High	Unmethylated	CT + RT	TMZ	311
61	Female	Temporal	IDH-w	Loss	Low	Unmethylated	CT + RT	TMZ+	853
56	Male	Parietal	IDH-w	Loss	High	Unmethylated	CT + RT	TMZ	174
55	Male	Frontal	IDH-w	Loss	High	Unknown	CT + RT	TMZ+	200
70	Male	Frontal	IDH-w	Loss	High	Methylated	CT + RT	TMZ+	730
76	Male	Frontal	IDH-w	Loss	Low	Unmethylated	CT + RT	TMZ	169
61	Male	Temporal	IDH-w	Loss	High	Methylated	CT + RT	TMZ	191
76	Male	Parietal	IDH-w	Loss	High	Unknown	RT	None	128
62	Male	Temporal	IDH-w	Loss	Low	Unmethylated	CT + RT	TMZ	555
73	Female	Parietal	IDH-w	Loss	High	Unknown	RT	None	195
57	Female	Frontal	IDH-w	Loss	High	Methylated	CT + RT	TMZ	288
81	Male	Parietal	IDH-w	Loss	High	Methylated	RT	None	59

Table 2. Patients’ Data Enrolled in This Study

Age

Gender

Location

IDH1

ATRX

CD204

MGMT

Adjuvant

RFI

Data include age, gender, tumor location, IDH1 status, MGMT-promoter methylation, CD204⁺ TAM expression, treatment modalities, and RFI. All the enrolled cases were WHO grade 4 astrocytomas that presented with necrosis, microvascular proliferations, ATRX loss and intact 1p19q. IDH-w: IDH-wildtype; IDH-m: IDH-mutant; MGMT: O⁶-methylguanine-DNA methyltransferase; RT: radiotherapy; CT: chemotherapy; TMZ: temozolomide; RFI: recurrence-free interval.

Male

Frontal

IDH-m

Loss

High

Methylated

None

670

Male

Frontal

IDH-m

Loss

Low

Methylated

CT + RT

TMZ

1,034

Male

Parietal

IDH-m

Loss

High

Methylated

CT + RT

TMZ

440

Female

Parietal

IDH-m

Loss

High

Unknown

None

670

Female

Parietal

IDH-m

Loss

High

Unmethylated

CT + RT

TMZ+

731

Male

Parietal

IDH-m

Loss

Low

Methylated

CT + RT

TMZ+

1,096

Male

Cerebellar

IDH-m

Loss

High

Unknown

CT + RT

TMZ+

1,123

Female

Frontal

IDH-m

Loss

Low

Unmethylated

None

643

Female

Parietal

IDH-m

Loss

High

Methylated

None

638

Female

Temporal

IDH-m

Loss

High

Unmethylated

CT + RT

TMZ

801

Male

Frontal

IDH-w

Loss

Low

Unknown

None

330

Male

Frontal

IDH-w

Loss

Low

Unmethylated

None

530

Female

Parietal

IDH-w

Loss

Low

Unmethylated

CT + RT

TMZ

330

Male

Temporal

IDH-w

Loss

High

Unknown

None

250

Male

Frontal

IDH-w

Loss

High

Unmethylated

None

430

Male

Frontal

IDH-w

Loss

High

Methylated

CT + RT

TMZ

1,016

Male

Temporal

IDH-w

Loss

High

Unmethylated

None

293

Female

Parietal

IDH-w

Loss

High

Methylated

CT + RT

TMZ+

156

Female

Frontal

IDH-w

Loss

High

Unknown

CT + RT

TMZ

150

Female

Occipital

IDH-w

Loss

Low

Unmethylated

None

183

Female

Temporal

IDH-w

Loss

High

Unknown

CT + RT

TMZ

194

Male

Frontal

IDH-w

Loss

High

Methylated

CT + RT

TMZ+

340

Female

Parietal

IDH-w

Loss

High

Methylated

CT + RT

TMZ+

260

Male

Temporal

IDH-w

Loss

Low

Methylated

None

Male

Frontal

IDH-w

Loss

High

Unknown

CT + RT

TMZ

306

Female

Occipital

IDH-w

Loss

High

Methylated

CT + RT

TMZ+

826

Male

Frontal

IDH-w

Loss

High

Methylated

None

273

Male

Temporal

IDH-w

Loss

Low

Unmethylated

None

550

Male

Temporal

IDH-w

Loss

High

Unknown

None

141

Female

Frontal

IDH-w

Loss

Low

Unmethylated

None

Male

Temporal

IDH-w

Loss

High

Unknown

None

114

Female

Frontal

IDH-w

Loss

High

Methylated

CT + RT

TMZ

Male

Frontal

IDH-w

Loss

High

Methylated

CT + RT

TMZ+

460

Male

Temporal

IDH-w

Loss

High

Unmethylated

CT + RT

TMZ

311

Female

Temporal

IDH-w

Loss

Low

Unmethylated

CT + RT

TMZ+

853

Male

Parietal

IDH-w

Loss

High

Unmethylated

CT + RT

TMZ

174

Male

Frontal

IDH-w

Loss

High

Unknown

CT + RT

TMZ+

200

Male

Frontal

IDH-w

Loss

High

Methylated

CT + RT

TMZ+

730

Male

Frontal

IDH-w

Loss

Low

Unmethylated

CT + RT

TMZ

169

Male

Temporal

IDH-w

Loss

High

Methylated

CT + RT

TMZ

191

Male

Parietal

IDH-w

Loss

High

Unknown

None

128

Male

Temporal

IDH-w

Loss

Low

Unmethylated

CT + RT

TMZ

555

Female

Parietal

IDH-w

Loss

High

Unknown

None

195

Female

Frontal

IDH-w

Loss

High

Methylated

CT + RT

TMZ

288

Male

Parietal

IDH-w

Loss

High

Methylated

None

**Table 3.** Primers for MS-PCR Used for the Assessment of *MGMT*-Promoter Methylation
Primer	Sequence
MS-PCR: methylation specific-polymerase chain reaction; MGMT: O⁶-methylguanine-DNA methyltransferase.
MSP-MGMT-MetF	5′-TTTCGACGTTCGTAGGTTTTCGC-3′
MSP-MGMT-MetR	5′-GCACTCTTCCGAAAACGAAACG-3′
MSP-MGMT-UnMetF	5′-TTTGTGTTTTGATGTTTGTAGGTTTTTGT-3′
MSP-MGMT-UnMetR	5′-AACTCCACACTCTTCCAAAAACAAAACA-3′

Table 3. Primers for MS-PCR Used for the Assessment of MGMT-Promoter Methylation

Primer

Sequence

MS-PCR: methylation specific-polymerase chain reaction; MGMT: O⁶-methylguanine-DNA methyltransferase.

MSP-MGMT-MetF

5′-TTTCGACGTTCGTAGGTTTTCGC-3′

MSP-MGMT-MetR

5′-GCACTCTTCCGAAAACGAAACG-3′

MSP-MGMT-UnMetF

5′-TTTGTGTTTTGATGTTTGTAGGTTTTTGT-3′

MSP-MGMT-UnMetR

5′-AACTCCACACTCTTCCAAAAACAAAACA-3′

**Table 4.** Relationship Between CD204⁺ TAMs and *MGMT*-Promoter Methylation
CD204⁺ TAMs	MGMT-promoter profile	P-value
MGMT: O⁶-methylguanine-DNA methyltransferase; TAMs: tumor-associated macrophages.
High expression	6 (28.5%)	15 (71.6%)	0.01
Low expression	9 (75%)	3 (25%)

Table 4. Relationship Between CD204⁺ TAMs and MGMT-Promoter Methylation

CD204⁺ TAMs

MGMT-promoter profile

P-value

Unmethylated, n (%)

Methylated, n (%)

MGMT: O⁶-methylguanine-DNA methyltransferase; TAMs: tumor-associated macrophages.

High expression

6 (28.5%)

15 (71.6%)

0.01

Low expression

9 (75%)

3 (25%)

**Table 5.** Relationship Between CD204⁺ TAMs and *IDH* Mutation
CD204⁺ TAMs	IDH mutation	P-value
IDH: isocitrate dehydrogenase; TAMs: tumor-associated macrophages.
High expression	7 (21.8%)	25 (78.2%)	0.93
Low expression	3 (23.1%)	10 (76.9%)

Table 5. Relationship Between CD204⁺ TAMs and IDH Mutation

CD204⁺ TAMs

IDH mutation

P-value

IDH-mutant, n (%)

IDH-wildtype, n (%)

IDH: isocitrate dehydrogenase; TAMs: tumor-associated macrophages.

High expression

7 (21.8%)

25 (78.2%)

0.93

Low expression

3 (23.1%)

10 (76.9%)

**Table 6.** Relationship Between CD204⁺ TAMs, *MGMT* methylation and RFI
	Mean	Median
MGMT: O⁶-methylguanine-DNA methyltransferase; TAMs: tumor-associated macrophages; RI: recurrence interval; RFI: recurrence-free interval.
RI
CD204 high
MGMT methylation	423.133	76.467	273.258	573.009	340.000	107.558	129.186
Non-methylation	456.667	103.673	253.468	659.865	311.000	83.895	146.566
Overall	432.714	60.851	313.447	551.982	340.000	104.517	135.147
RFI
CD204 low
MGMT methylation	740.667	324.827	104.006	1,377.327	1,034.000	769.140	0.000
Non-methylation	433.667	84.896	267.270	600.063	530.000	298.142	0.000
Overall	510.417	101.655	311.172	709.661	530.000	190.526	156.570

Table 6. Relationship Between CD204⁺ TAMs, MGMT methylation and RFI

Mean

Median

Estimate

Standard error

95% confidence interval

Estimate

Standard error

95% confidence interval

Lower bound

Upper bound

Lower bound

MGMT: O⁶-methylguanine-DNA methyltransferase; TAMs: tumor-associated macrophages; RI: recurrence interval; RFI: recurrence-free interval.

CD204 high

MGMT methylation

423.133

76.467

273.258

573.009

340.000

107.558

129.186

Non-methylation

456.667

103.673

253.468

659.865

311.000

83.895

146.566

Overall

432.714

60.851

313.447

551.982

340.000

104.517

135.147

RFI

CD204 low

MGMT methylation

740.667

324.827

104.006

1,377.327

1,034.000

769.140

0.000

Non-methylation

433.667

84.896

267.270

600.063

530.000

298.142

0.000

Overall

510.417

101.655

311.172

709.661

530.000

190.526

156.570