Histological evaluation remains a basis for treatment and follow-up of women with CIN. The fundamental premise for treating or following young women with CIN hinges on the risk of CIN2 or CIN3 for which cone biopsy or LEEP will be required. Hence, helpful biological markers are in need of distinguishing CIN1 from CIN2/3 when the diagnosis is not certain, particularly in young women.
The monoclonal antibody D2-40 was described to react with a novel oncofetal membrane antigen M2A, presenting on fetal gonocytes, intratubular germ-cell neoplasia and seminoma cells . The M2A antigen has also been shown as a developmental marker for human Sertoli cells, presenting on immature Sertoli cells until puberty, and loosing during their transition to a mature adult phenotype . We found that D2-40 immunoreactivity was observed exclusively in the basal cell layer of the cervical squamous epithelium, which agrees with the previous study . Taken together, D2-40 protein expression may be predominantly associated with immaturity. D2-40 has been served as a new selective marker for lymphatic endothelium, and used in identifying the presence of lymphatic invasion in various malignant neoplasms , including cervical carcinoma as well as cervical neoplasia . The D2-40 immunoreactivity observed in the lymphatic endothelium of the cervical stroma in this study acts as an internal control, indicating that the loss of D2-40 protein expression in the squamous epithelium in CIN2/3 specimens is not a false negative pattern arising from fixation or processing issues. Furthermore, this study expanded the information that the decreased expression of D2-40 in the basal cells of SE correlates the grade of CIN, especially showing the significant difference between CIN1 and CIN2/3. Likewise, low D2-40 immunoreactivity correlates with lymphatic invasion and nodal metastasis in early-stage squamous cell carcinoma of the uterine cervix , and D2-40 positivity in tumor cells is associated with a better prognosis in ASC . Thus, the D2-40 protein may be a better prognostic marker in cervical lesion. That might be associated with the M2A, recognized by the D2-40 antibody. Although its function is yet unclear, it contains one of the mucin-type glycoproteins that are expressed on human normal cells and tumors [14, 15]. Mucins are large, highly glycosylated proteins recognized by their tandem repeat domains, first as components of cell surfaces, and later for their roles in the protection of epithelia and other cells [16, 17].
In the current study, we also compared the D2-40 immunoreactivity to p16INK4A, identified as a biomarker for transforming human papillomavirus (HPV) infections. Increased cases with diffuse immunostaining of p16INK4A occurred in the higher grade of CIN (CIN2/3), which is in agreement with the previous studies [18–20]. In addition, several studies suggest an improved diagnostic accuracy for diagnosing CIN lesions with the diffuse p16INK4A immunoreactivity [21, 22]. Although there is good evidence that diffuse p16INK4A immunostaining correlates with the severity of CIN, we have to take into consideration the limited specificity of p16INK4A immunoreactivity. Because the focal staining was seen in more than a half of CIN1 cases, but this pattern was also seen in some CIN2/3 cases. Thus, an additional ideal biomarker needs to be explored. This study shows that the immunoreactive pattern of D2-40 was more specific than that of p16INK4A immunoreactivity when distinguishing CIN1 from CIN2/3 because diffuse and focal/negative immunostaining of D2-40 predominantly presents in CIN1 and CIN2/3, respectively. No correlation between D2-40 and p16INK4A immunoreactity was found, which might be due to p16INK4A associated with transforming HPV infections, but not D2-40. Although no correlation between D2-40 and p16INK4A immunoreactity was shown, the data in the current study indicate that the combined use of D2-40 and p16INK4A immunoreactivities in routine histopathology would improve accuracy of diagnosis of CIN.