MicroRNA expression profiling and Notch1 and Notch2 expression in minimal deviation adenocarcinoma of uterine cervix

Minimal deviation adenocarcinoma (MDA), also known as adenoma malignum, is an extremely well-differentiated variant of cervical adenocarcinoma in which most of the cells lack the cytological features of malignancy [1]. Although MDA is an uncommon tumor and accounts for only 1 to 3% of all cervical adenocarcinomas [1], it deserves scrutiny because many non-neoplastic endocervical glandular lesions encountered in daily practice should be differentiated from it. Because it is very difficult to diagnose preoperatively if the proliferative endocervical glandular lesion is definitely benign or malignant, especially in punch biopsy specimens [2], it would be crucial to combine ancillary molecular or immunohistochemical biomarkers with morphologic characteristics in order to improve diagnostic accuracy. In 1998, a Japanese group initially demonstrated that MDA showed immunoreactivity with HIK1083 [3], and ever since this antibody has been considered a promising tool for establishing the diagnosis of MDA. However, its usefulness has been in dispute because its immunoreactivity has also been demonstrated in several proliferative endocervical glandular lesions exhibiting gastric differentiation, including lobular endocervical glandular hyperplasia (LEGH) or gastric-type adenocarcinoma in situ (AIS), as well as in MDA [4, 5].

Whereas most cases of cervical carcinomas are known to be associated with high-risk human papillomavirus infection, MDA is usually found to be negative for human papillomavirus [1, 6, 7]. Although some authors suggested that MDA could be derived from LEGH and morphologically usual-type AIS showing gastric immunophenotype [1, 4], further studies would be needed to draw this conclusion. Previous reports for prognosis of MDA have not provided uniform data. Several investigators reported poor prognosis [8‐10], whereas some found a relatively favorable prognosis similar to that for other well-differentiated cervical adenocarcinomas [11‐13]. Therefore, difficulty in morphologic diagnosis, uncertain pathogenesis, and variable clinical outcome data prevent clinicians from guiding patient clinical management and treatment effectively.

MicroRNAs (miRNAs) are small non-coding RNAs that have been implicated in tumor development [14, 15]. They regulate target gene expression either by mRNA degradation or by translation repression [14‐18]. In general, each miRNA can regulate up to hundreds of target genes [14‐18]. During tumor development, aberrant expression of miRNAs can either inactivate tumor suppressor genes or activate oncogenes, thereby promoting tumor formation [14‐16, 18, 19]. Because expression of miRNAs is tissue-specific [14, 15], detectable in blood [20], and correlates with clinical cancer behaviors [21], miRNAs are potentially valuable biomarkers.

Until recently, several studies have identified either up-regulated or down-regulated miRNAs in cervical cancers using the global profiling method [22‐29] or the candidate miRNA approach [30]. However, most studies determined miRNA expression profiles in cervical squamous cell carcinomas [22‐30], although a few adenocarcinoma cases were included in the cervical cancer category. There has been no report about the miRNA expression profiles on an independent set of cervical adenocarcinomas, in particular, MDA.

Characterization of the complex relationship between deregulated miRNAs and their target genes in MDA may not only help to define some of the molecular pathways that drive carcinogenesis, but those biomarkers would also help to improve discrimination between MDA and other glandular lesions and be of considerable importance for the prediction of prognosis.

Preoperative differential diagnosis of MDA from other proliferative glandular lesions, such as endocervical tunnel clusters [1], deeply situated nabothian cysts [1], endocervicosis of the cervical wall [1], mesonephric hyperplasia [1], LEGH [2], and AIS [2], is critical for appropriate therapeutic management. Nonetheless, according to a previous report [2], the interobserver agreement rate for MDA showed just a slight level of consistency (k-value =0.115).

Recently, several studies have noted associations between microRNA expression and cervical carcinomas [22‐30]. MiRNAs reported in the previous studies [22‐30] to be associated with cervical carcinomas included the following: miR-21, 143, 21, 145, 218, 29a, 155, 16, 146a, 20a, 126, 127, 424, 17-5p, 203, 20, 15b, 106a, 148a, 224, 10b, 450, 199a, 20b, 125b, 15a, 93, 182, 185, and 34a. However, those studies revealed quite different miRNA expression profiling patterns from ours because most studies have focused on cervical squamous cell carcinomas. These findings are explained by the previous reports that have suggested tissue-specific miRNA expression patterns with different sets of miRNAs up- or down-regulated in tumors of different cellular origin [33‐35].

In the present study, using miRNA microarray and qRT-PCR on 24 MDA samples, we detected nine miRNAs which were differently expressed between MDA and NE. They exhibited relatively good discrimination ability and, in particular, single miR-34b-5p testing could discriminate MDA from NE with 100% sensitivity and specificity. We also constructed the best fitting model consisting of miR-34b-5p and 194-5p based on AICc values, and it also showed 100% sensitivity and specificity. Considering the high sensitivity and specificity above 99%, they might be used as supplementary diagnostic biomarkers in pathologically complicated cases with ambiguous morphological features or with only small amounts of superficial glandular lesions upon limited sampling.

Four Notch genes have been described in mammals, Notch1, Notch2, Notch3, and Notch4 [31, 36, 37]. They encode type I transmembrane proteins with extracellular domains containing epidermal growth factor-like repeats that regulate cell proliferation and differentiation in various tissues [31, 36, 37]. In cervical cancer, upregulation of Notch1 [37‐39] and Notch2 [39] was associated with in-situ or invasive squamous cell carcinoma and adenocarcinoma, and it is thought that abnormal Notch signaling can promote the development of cervical cancer [40]. In the present study, Notch1 and Notch2 seemed to play an oncogenic role because both of them were significantly up-regulated in MDA compared to normal control. Interestingly, although we observed that Notch1 expression was dependent on the miR-34b-5p expression level, Notch2 expression did not seem to be dependent on the miR-204-5p expression level, thereby suggesting another mechanism or miRNAs might be involved in the Notch2 expression process in the MDA carcinogenesis pathway. In particular, considering that miR-34b-5p and Notch1 showed the most distinctive expression pattern between MDA and NE, we can speculate that down-regulation of miR-34b-5p and the resulting overexpression of the Notch1 gene (miR-34b-5p/Notch1 pathway) might comprise one of the important oncogenic pathways of MDA. However, Notch2 is positive in 52% of MDAs compared to 18% of normal control samples, and this finding alone may be sufficient to suspect that Notch2 could also play a role in carcinogenesis of MDA, although maybe not triggered by miR-204-5p.

To date, several miRNAs, including miR-375 [41], 127 [24], 9 [42], 200a [42, 43], 93 [43], 497 [44], and 224 [45], have been reported to be associated with metastasis, progression, or survival of cervical cancer, although most of the cervical tumors in those studies were also squamous cell type. In this study, down-regulation of miR-494 was associated with poor patient survival, suggesting its possible role as a prognostic marker.

Limitations to our study need to be mentioned. First, even though we performed a multicenter study, there was still only a small number of cases due to the low incidence of MDA. Thus, we could not afford another validation study for our diagnostic or prognostic biomarkers with a test set, and future studies with larger sample size, notably including controversial cases, will have to be performed. Second, a possible precursor lesion, like LEGH or gastric type-AIS, which could strengthen our hypothesis was not included in this study. Third, more experiments, such as transfection of miR-34b-5p and cell viability tests, are still warranted to reveal the exact role of the miR-34b-5p/Notch1 pathway during MDA carcinogenesis.

MDA showed distinctive expression profiles of miRNAs, Notch1, and Notch2 from NE. In particular, miR-34b-5p and 194-5p might be used as diagnostic biomarkers and miR-494 as a prognostic predictor for MDA. The miR-34b-5p/Notch1 pathway as well as Notch2 might be important oncogenic contributors to MDA.