Whereas the pathogenesis of cervical squamous cell carcinoma is linked to infection with oncogenic types of human papillomavirus (HPV), the factors contributing to the pathogenesis of CA are less understood. Although HPV DNA is commonly detected in most squamous cell carcinomas (>90%), the reported prevalence of HPV DNA in CA varies from 32% to 100%, depending on the detection method used^3,4. We hereby report a case of VGA, of which both in situ and invasive components expressed multiple high-risk HPV types in chromogenic in situ hybridization (ISH).

This lesion was reported as “well differentiated villoglandular adenocarcinoma with multiple foci of endocervical carcinoma in situ. Largest diameter of tumor is 1 cm (including exophytic component). There is no squamous cell abnormality in adjacent cervical epithelium. Tumor is observed in deep and lateral surgical margins”. Patient was referred to another institution and she underwent a total abdominal hysterectomy with surgical staging. Histopathological evaluation of the staging laparatomy material revealed only a microscopic residual tumor in the endocervical canal. Lymph nodes were free of tumor. Patient is well and no evidence of disease is detected sixty-four months after laparatomy.

In situ hybridization (ISH):
Chromogenic ISH test was done using following probes: wide spectrum HPV DNA (6, 11, 16, 18, 31, 33, 35, 45, 51, and 52.), HPV DNA 16/18 and HPV DNA 31/33 (Dako Corporation, Denmark). Protocol for chromogenic in situ hybridization (CISH) was described elsewhere. In this method, a blue-purple reaction specifically located on tumor cell nuclei was interpreted as a positive test result (Fig 1c). A wide spectrum mucosotrophic HPV DNA, HPV 16/18 and 31/33 were detected on both VGA and AIC. There was no evidence of reactivity in non-neoplastic cervical or endocervical tissue.

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Figure 1: a. Whole mount view of cervical exophytic tumor, 1b. Adenocarcinoma in situ. Precursor lesion of invasive adenocarcinoma. Normal endocervical cells and cytologically atypical cells line the same gland, 1c. Chromogenic in situ hybridization (HPV 31-33). Black dots on cell nuclei denote a positive signal.

P53 and Ki67 overexpression were also observed in both VGA and AIC areas.

In addition to papillary and invasive components, a precursor lesion i.e. adenocarcinoma in situ (AIS) is usually found around invasive tumor. AIS has been characterized with certain features in that glandular architecture is conserved but glandular lining is partly replaced by epithelial cells showing pseudostratification and mild nuclear atypia. Cytoplasmic mucin production is altered; either reduced or increased abundantly. In the present case, AIS foci were noted in the glands adjacent to the infiltrative component of VGA. Jones et al. noted the presence of AIS in 8 cases of VGA⁵. Partial involvement of the glands by malignant cells and absence of desmoplastic response are the histopathological features, which distinguish AIS from individual malignant glands⁶. Although desmoplastic stroma is evident around infiltrative tumor, we did not observe desmoplastic stroma around the AIS foci⁶.

In this case we were able to identify HPV 16-18 and HPV 31-33 types in VGA and also in AIS component. Although multiple HPV DNA subtypes were commonly found in squamous cell carcinomas of cervix, detection of multiple subtypes in endocervical adenocarcinoma without squamous lesion has been stated as exceptional⁷.

Prevalence of HPV DNA in CA is dependent on histological type of the tumor. HPV DNA has been detected in almost all mucinous CAs⁸. Multiple HPV subtypes were found in 5 out of 21 AIS and in 8 out of 80 invasive and in situ adenocarcinomas combined⁸. A recent study in Korean women showed multiple infections in 13% (18/135 cases) of all invasive adenocarcinomas. HPV 16 is the most predominant type in cases with multiple and single infection. In this study, only one out of 5 VGAs was found to have multiple HPV types (16 and 18) whereas the rest had single HPV type⁹. Mathhews-Greer et al. noted infection with multiple subtypes in different histological subtypes. They found one VGA with HPV 16 and 52 subtype infection. HPV 16 is the predominant type in all multiple and single infections in CA¹⁰.

In accordance with our observation in this case, multiple HPV infections are often associated with those viruses belonging to the same clade. HPV-16, HPV-31, and HPV-33 all belong to the A9 clade¹¹. Thomas et al. evaluated whether the prior infection with specific HPV types inhibits subsequent infection by related types and they found that concurrent acquisition of multiple types occurred more often than expected¹².

After its first description in 1953, AIS has been evaluated in many studies for putative precursor lesion of CA. Sufficient evidence is available for AIS as a precursor lesion of CA^13,14. Besides histological and demographic data, similar HPV types are found in both AIS and invasive adenocarcinoma. In addition to observing same HPV subtypes, AIS foci in the present case also displayed p53 overexpression and increased Ki67 expression. These findings support the hypothesis that AIS is also a precursor lesion associated with VGA.

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