A Tumor of Composite Cervical Adenocarcinoma with MUCIN Gene Mutation：A Case Report


 Background:There are reports about the coexistence of two kinds of tumors in the same patient,which is believed that this phenomenon is caused by the dedifferentiation between the two tumors.In this paper, we report an human papillomavirus (HPV) negative cervical adenocarcinoma in a patient composed of two adenocarcinoma components,which is first reported.Histologically, both minimal deviation adenocarcinoma (MDA) and poorly differentiated gastric type adenocarcinoma (GTA) components, as well as their transitional area, were observed.Methods:This case of cervical cancer was screened by gene sequencing. 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. Then the genes were screened and identified based on the enrichment analysis of GO and KEGG and related literature reports. Results:We found 13 specific somatic gene mutations in total. Among these genes, only Mucin gene was transformed from gene level to protein level, and was positive in both MDA and GTA components of the patient by immunohistochemistry.Both components had genes mutation of MUC4 and MUC17,the component in MDA had gene mutation of MUC3A,and we found that MUC3A and MUC17 were on the same chromosome. Moreover,MUC3A and MUC4 genes were found to be fused in FusionGDB database. Conclusion:According to the reports of MUC3A,MUC4 and MUC17 genes mutation in cervical adenocarcinoma and gene fusions in tumorigenesis, we speculate that the occurrence of the transformation of pathological type from MDA to GTA in this case of cervical cancer is related to the mechanism of MUC3A and MUC4 gene fusion.We would advice, for HPV negative or atypical cervical lesions, immunohistochemistry of MUCIN genes staining and gene sequencing should be considered, which may find unusual cancer types and change the prognosis of patients.


Background
Minimal deviation adenocarcinoma (MDA) of cervical is not associated with persistent HPV infection, and it is di cult to be found in cytological and biopsy specimens because of its deep location, endogenous growth pattern and slight cytological atypia 1,2 . It represents 1-3% of cervical adenocarcinomas and is characterised morphologically by extremely well differentiated glands with little in the way of nuclear atypia 1,2 .Recently, another variant of cervical adenocarcinoma has been described which is thought to exhibit gastric differentiation 4 . This has been termed 'gastric type' adenocarcinoma (GTA) and, in contrast to MDA, is characterised by obvious malignant cytological features 4 . Mcclugage et al. 5 found that there was a similar genetic variation between MDA and GTA, indicating that there was a speci c relationship between the two kinds of tumors. They believed that GTA was dedifferentiated from MDA 6 .As far as we know, at present, only two literatures have described the diagnosis of GTA accompanied by MDA, but they all lack the poorly differentiated gastric adenocarcinoma seen in this case 7,8 . In this case, the MDA and GTA components with different differentiation appeared simultaneously in cervical tumor, and the two components had similar immunophenotypes, which was consistent with the diagnosis of different differentiation of the same tumor. The continuity between the MDA and GTA components favors the hypothesis that GTA arises from the dedifferentiation of MDA, however, the mechanism of this dedifferentiation remains unclear. In this study, we did gene sequencing of the tumor and blood of the patient. According to the existing reports, we found some related gene mutations, which may be related to the tumor dedifferentiation mechanism of this patient.

A Case Report
A 54-year-old woman presented to our hospital with abnormal vaginal bleeding for one month. Physical examination revealed normal external genitalia and a fragile neoplasm which is 1cm in diameter without obvious active bleeding in the cervix, and the origin of the neoplasm was unclear. Transvaginal sonography revealed uneven hypoechoic with a size of 6.3×6.5×4.6 cm in the lower segment of the uterus,uneven echo of endometrium with a thickness of 0.25cm.The cytological tests reported the presence of atypical glandular cells of undetermined signi cance(AGUS), which was in favor of the diagnosis of adenocarcinoma of the endocervix. Human papillomavirus E6/E7 mRNA was negative. However, despite undergoing endometrial curetting and cervical biopsy, she was diagnosed with only chronic cervical in ammation and polyps, and not with cervical or endometrial malignancy. All laboratory data including serum CA-125, SCC-Ag, CEA, AFP and CA19-9 were within normal ranges Radiological examination showed no tumour outside the cervix. Subsequently, the patient underwent total abdominal hysterectomy and bilateral salpingo-oophorectomy. According to pathological section, she underwent radical parametrectomy with removal of a vaginal cuff and pelvic lymph node dissection. The patient died after a course of radiotherapy Materials And Methods

1.Immunohistochemistry
For the immunohistochemical analysis of primary antibodies against gastric gland mucin(MUC4,ab150381;Abcam,USA; and MUC3A,ab199260;Abcam,USA), 4-µm serial tissue sections from the formalin-xed, para n-embedded tissues were used. The samples were depara nized in xylene and rehydrated through a graded series of ethanol washes. After the endogenous peroxidase was inhibited and the antigen was retrieved (microwave irradiation in 0.01 M citrate buffer at pH 6.0), the sections were incubated with primary antibody at 4℃overnight and then with horseradish peroxidase (HRP)-conjugated secondary antibodies (DakoCytomation, Denmark). After washing, tissues were stained for 5 min with 3,30-diaminobenzidine (DAB) chromogen and counterstained with hematoxylin (Zhongshan Golden Bridge,Inc), dehydrated and mounted on cover slips.
2.DNA extraction and whole genome sequencing Genomic DNAs were extracted from peripheral whole blood and Formalin-xed para n-embedded samples (including MDA and GTA) of malignant cervical cancer and 7 FFPE samples (including 3 adenocarcinoma and 4 squamous carcinoma) of common cervical cancer by using Qiagen Blood DNA mini Kit and QIAamp DNA FFPE Tissue Kit (Qiagen, Germany) according to the manufacturer's recommendations. Brie y, genomic DNAs extracted from 9 tumors and 1 matched peripheral whole blood were puri ed and DNA from peripheral whole blood fragmented randomly. 5-10μg genomic DNA was used for library generation and standard paired-end adaptors were ligated according to the manufacturer's (Illumina) protocol. Each of libraries were subjected to WGS on an Illumina HiSeq 2000. Target depth (30× for tumors and peripheral whole blood samples) was achieved in all samples.

3.Somatic variant identi cation
Through the investigation of no history of malignant tumor in the three generatios of relatives of this patient with highly malignant cervical cancer, it is considered that the occurrence and development of this cervical cancer was unlikely to be due to genetic factors and probably due to somatic mutation. For detection of somatic point mutations, sequencing reads from a Illumina HiSeq 2000 were aligned to the human reference genome (UCSC Genome Browser hg19) with the Burrows-Wheeler Aligner. After duplicate reads were removed with SAMtools, an in-house pipeline was used to call somatic mutations. The indel-calling step was performed by the Genome Analysis Toolkit SomaticIndel Detector with default parameters. Severe somatic variants including stopgain, frameshift, insertion/deletions (indels) and nonsynonymous SNVs were identi ed in minimal deviation adenocarcinoma and gastric type adenocarcinoma but absent in the adjacent bloods.

4.Data analysis
For detection of speci c somatic mutations in MDA and GTA, we ltered out mutations in malignant cervical cancer blood sample and 7 common cervical carcinoma and the GnomAD_ALL_Value<0.01 was selected( Figure 1). The mutations were annotated to public databases by Annovar. GO analysis and KEGG enrichment analysis were carried out on the mutations obtained. Databases such as OMIM (http://www.omim.org), ClinVar (http://www.ncbi.nlm.nih.gov/clinvar), Human Gene Mutation Database (http://www.hgmd.org) and SwissVar (http://www.bioinfo.org/wiki/index.php/ SwissVar) were used to determine mutation harmfulness and pathogenicity where appropriate.A total of 10 Signaling pathway associated with cancer progression including RTK/RAS pathway, Nrf2 pathway, PI3K pathway, Wnt pathway, Myc pathway, P53 pathway, TGF-β pathway, Hippo pathway, Cell cycle pathway, Notch pathway were selected for exploring speci c somatic mutations associated with malignant cervical cancer.

1.Histological ndings
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 in ltrative 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 in ammatory cell in ltration 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 gures 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 in ltrated 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 in ltration 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, chroma n 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.   (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 signi cantly impact both progression and overall survival and may act as drivers of the disease 26 . MUC3A and MUC4 genes were found to be fused in FusionGDB database (https://ccsm.uth.edu/FusionGDB/index.html).( We report an human papillomavirus (HPV) negative cervical adenocarcinoma in a patient composed of two adenocarcinoma components.Histologically, both minimal deviation adenocarcinoma (MDA) and poorly differentiated gastric type adenocarcinoma (GTA) components, as well as their transitional area, were observed. MDA is an uncommon but well known variant of cervical adenocarcinoma characterised by highly differentiated malignant glands and bland cytology. MDA is thought to exhibit gastric or pyloric differentiation based on histochemical and immunohistochemical studies. [10][11][12][13] It is associated with aggressive behaviour and a poor prognosis 4 . Dedifferentiated carcinoma cells lack the ability to maintain cell-cell contact, and therefore diffusely in ltrate the stroma, resulting in increased invasion and metastasis. Despite the paucity of data on the role of HPV in the development of MDA and GTA, it is believed that these lesions are not aetiologically related to HPV infection, 16,17, unlike the majority of usual type cervical adenocarcinomas which contain high risk oncogenic HPV 17 , which complicates an early diagnosis of these tumors, even when a HPV DNA test is used. In the case of MDA in particular, obscure cell atypia makes it di cult to differentiate from normal cells. The tumor in this case was negative in HPV genotyping. This case may provide further evidence for this hypothesis. Both components were also p16 negative, in keeping with an absence of HPV, diffuse p16 staining in the cervix being a useful surrogate marker of the presence of high risk HPV.
There are reports about the coexistence of two kinds of tumors in the same patient,which is believed that this phenomenon is caused by the dedifferentiation between the two tumors [7][8]22 .This case of cervical cancer was screened by gene sequencing. 13 [20][21] .Some studies have shown that gene fusion may occur in mutated genes located on the same chromosome, and gene fusion can be found in a variety of dedifferentiated cancers, which may be related to the evolution of dedifferentiation of cancer [23][24][25] .It is reported recurrent fusion genes that signi cantly impact both progression and overall survival and may act as drivers of the disease 9 .Through gene detection, both MDA and GTA components in this case had genes mutation of MUC4 and MUC17,the component in MDA had gene mutation of MUC3A,and we found that MUC3A and MUC17 were on the same chromosome.Moreover,MUC3A and MUC4 genes were found to be fused in FusionGDB database (Figure 3),we speculate that the occurrence of the transformation of pathological type from MDA to GTA in this case of cervical cancer is related to the mechanism of MUC3A and MUC4 gene fusion.

Conclusion
In summary, we report an unusual HPV negative cervical adenocarcinoma with two morphologically components:MDA and poorly differentiated GTA.The mutation of MUC3A, MUC4 may be responsible for dedifferentation of MDA. We would advice, for HPV negative or atypical cervical lesions, immunohistochemistry of MUCIN genes staining and gene sequencing should be considered, which may nd unusual cancer types and change the prognosis of patients.

Consent for publication
I would like to declare on behalf of my co-authors that the patient has provided informed consent for publication of the case.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Figure 1 Workfow for the screening of the speci c somatic gene mutations of MDA and GTA components