1.Histological findings
Histological examination revealed that the tumor was composed of two adenocarcinoma components: 1) MDA: There was effacement of the normal endocervical glandular architecture by a proliferation of well differentiated glands with infiltrative growth, in keeping with MDA (Figure2A and 2B); The nuclei are basally located and the cytoplasm is abundant and clear. Fibrous connective tissue hyperplasia and inflammatory cell infiltration were found around the gland, two types of cells with or without heteromorphism in glands were found in this area. 2) GTA: GTA component consisting of undifferentiated mucinous glands with marked nuclear atypia and lots of mitotic figures located between the stroma of the cervical canal and the myometrium of the uterine, where tumor cells extended diffusely, without obvious adenoid or nest-like structure and infiltrated into the deep part of the cervical stroma and myometrium.(Figure 2C). Transitional area between the MDA and GTA components is detected(Figure 2D).Tumor thrombi were found in the vessels as well. There was bilateral uterine parauterine tissues and two pelvic lymph nodes infiltration for poorly differentiated component. No pathological abnormality was seen in fallopian tube/ ovary /cervical mucosa or endometrium.
Immunohistochemically, MDA cells were negative with P16, P40, ER and PR,were positive with MUC4, MUC3A and broad-spectrum cytokeratin CKpan,were positive with CEA which in cavity margin; GTA cells was negative with P16, ER, CD10, Caldesmon, synaptophysin, chromaffin A and CKpan, were partially positive with MUC4, MUC3A,CEA and P40,were weak positive with PR. Ki-67 labeling index in the MDA component was 2% while that in the GTA component was more than 90%, indicating the striking difference in proliferation between the two components.(Figure 3)
2. Genomic analysis of MDA and GTA components
The tumor in this case was negative in HPV genotyping. For detection of specific somatic mutations in MDA and GTA, we filtered out mutations in malignant cervical cancer blood sample and 7 common cervical carcinoma, we obtained 6092 mutation sites involving 3102 genes in MDA and 4915 mutation sites involving 2876 genes in GTA. And we filtered out mutations which the GnomAD_ALL_Value≥0.01, we obtained 34 mutation sites involving 34 genes in MDA and 10 mutation sites involving 10 genes in GTA(Figure 1).Based on the enrichment analysis of GO and KEGG and related literature reports,specific somatic gene mutations including RASSF1, MUC4, DSPP, ATXN1, MUC17, DDHD1, and ABCC1 were found in the GTA samples.Specific somatic gene mutations including ADCY3, MUC3A, SPHKAP, MUC4, PCDH12, MUC17, FAM186A, and HRC were found in the MDA samples(Table 1) . MUC4 and MUC17 genes are present in both MDA and GTA tissues, MUC3A and MUC17 were on the same chromosome. It is reported recurrent fusion genes that signifificantly impact both progression and overall survival and may act as drivers of the disease26. MUC3A and MUC4 genes were found to be fused in FusionGDB database (https://ccsm.uth.edu/FusionGDB/index.html).(Table 2)
Table 1.
Specific somatic gene mutations of MDA and GTA components
|
Gene
|
Location
|
Position
|
CDS
|
Exon/Intronic
|
MUT
|
AA_change
|
Het
|
mut_type
|
1) GTA
|
RASSF1
|
3p21.31
|
chr3:50378176:T:A
|
NM_170714.1
|
exon1
|
c.61A>T
|
p.K21X
|
.
|
stopgain
|
|
MUC4
|
3q29
|
chr3:195510396:A:C
|
NM_018406.6
|
exon2
|
c.8055T>G
|
p.H2685Q
|
.
|
nonsynonymous SNV
|
|
DSPP
|
4q22.1
|
chr4:88537036:C:A
|
NM_014208.3
|
exon5
|
c.3222C>A
|
p.D1074E
|
.
|
nonsynonymous SNV
|
|
ATXN1
|
6p22.3
|
chr6:16327915:A:C
|
NM_000332.3
|
exon8
|
c.627T>G
|
p.H209Q
|
het
|
nonsynonymous SNV
|
|
MUC17
|
7q22.1
|
chr7:100678173:C:G
|
NM_001040105.1
|
exon3
|
c.3476C>G
|
p.T1159S
|
het
|
nonsynonymous SNV
|
|
DDHD1
|
14q22.1
|
chr14:53619480:T:C
|
NM_001160147.1
|
exon1
|
c.337A>G
|
p.S113G
|
het
|
nonsynonymous SNV
|
|
ABCC1
|
16p13.11
|
chr16:16162019:T:A
|
NM_004996.3
|
exon13
|
c.1684T>A
|
p.L562M
|
.
|
nonsynonymous SNV
|
2) MDA
|
ADCY3
|
2p23.3
|
chr2:25141434:G:C
|
NM_004036.4
|
exon2
|
c.423C>G
|
p.Y141X
|
.
|
stopgain
|
|
MUC3A
|
7q22.1
|
chr7:100550767:-:A
|
NM_005960.1
|
exon4
|
c.1348dup
|
p.S450Yfs*118
|
het
|
frameshift insertion
|
|
SPHKAP
|
2q36.3
|
chr2:228883721:T:A
|
NM_001142644.1
|
exon7
|
c.1849A>T
|
p.K617X
|
.
|
stopgain
|
|
MUC4
|
3q29
|
chr3:195508510:A:G
|
NM_018406.6
|
exon2
|
c.9941T>C
|
p.L3314P
|
het
|
nonsynonymous SNV
|
|
PCDH12
|
5q31.3
|
chr5:141324955:C:G
|
NM_016580.3
|
exon4
|
c.3546G>C
|
p.R1182S
|
hom
|
nonsynonymous SNV
|
|
MUC17
|
7q22.1
|
chr7:100677642:C:G
|
NM_001040105.1
|
exon3
|
c.2945C>G
|
p.T982R
|
het
|
nonsynonymous SNV
|
|
FAM186A
|
12q13.12
|
chr12:50746999:A:T
|
NM_001145475.1
|
exon4
|
c.3616T>A
|
p.S1206T
|
het
|
nonsynonymous SNV
|
|
HRC
|
19q13.33
|
chr19:49657889:T:A
|
NM_002152.2
|
exon1
|
c.606A>T
|
p.E202D
|
het
|
nonsynonymous SNV
|
Table 2.
MUC3 and MUC4 genes were found to be fused in FusionGDB database
FusionGID
|
FusionGene
|
Hgene
|
HGID
|
Tgene
|
TGID
|
22879
|
MUC3A IL32
|
MUC3A
|
4584
|
IL32
|
9235
|
22880
|
MUC3A MUC3A
|
MUC3A
|
4584
|
MUC3A
|
4584
|
22881
|
MUC3A SET
|
MUC3A
|
4584
|
MUC3A
|
6418
|
7348
|
CLDN4 MUC4
|
CLDN4
|
1364
|
MUC4
|
4585
|
19917
|
LMO7 MUC4
|
LMO7
|
4008
|
MUC4
|
4585
|
22882
|
MUC4 MAGEF1
|
MUC4
|
4585
|
MAGEF1
|
64110
|
22883
|
MUC4 MUC4
|
MUC4
|
4585
|
MUC4
|
4585
|
22884
|
MUC4 PCYT1A
|
MUC4
|
4585
|
PCYT1A
|
5130
|
22885
|
MUC4 TNK2
|
MUC4
|
4585
|
TNK2
|
10188
|
35950
|
SREBF2 MUC4
|
SREBF2
|
6721
|
MUC4
|
4585
|
41638
|
VPS72 MUC4
|
VPS72
|
6944
|
MUC4
|
4585
|
(https://ccsm.uth.edu/FusionGDB/index.html).
|