Differentiated-Type Intraepithelial Neoplasia-Like Lesion Associated with Squamous Cell Carcinoma of the Anus: A Case Report with Molecular Profile

Differentiated-type Intraepithelial Neoplasia (DIN) is defined as HPV-negative squamous intraepithelial proliferation with abnormal keratinocyte differentiation and basal cell atypia, originally described in the vulva, with following descriptions in the oral cavity. DIN occurring in the anus is quite rare, and to the best of our knowledge, only one publication reported it. In this report, we describe the clinicopathological features of this entity on anal margin, associated with invasive squamous cell carcinoma. In addition, using the next generation sequencing (NGS) technique, we have demonstrated TP53 mutation in the invasive component but not in the associated DIN-like lesion, where p53 immunohistochemical expression was restricted to basal layers.


Introduction
Differentiated-type Intraepithelial Neoplasia (DIN) is defined as HPV-negative squamous intraepithelial proliferation with abnormal keratinocyte differentiation and basal cell atypia [1]. This pathological entity was originally described in the vulva, with the following descriptions in the oral cavity [2][3][4][5] and the genitourinary tract, especially the penis [6][7][8]. In the vulva, this lesion is associated with lichen sclerosus or planus and often associated with keratinizing squamous cell carcinoma (SCC).
To the best of our knowledge, only one publication reported DIN in the anus [9]. Terminology concerning this lesion is confusing as it is not described in the WHO Classification of Tumours of the Digestive System. Indeed, precursor lesions reported in the anal canal are "classical" anal intraepithelial neoplasia, mostly HPV-related [10]. The prevalence of HPV infection in anal carcinoma (84.3%) approaches that reported in cervical carcinoma (87.3%), for which HPV infection is considered a necessary cause. HPV prevalence is much lower in vulvar carcinoma (40.4%) [11].
In the vulva, DIN is classically associated with TP53 mutations and will be p53 immunopositive when missense mutations are present. Some cases shared identical TP53 mutations in both DIN and SCC [12].
Therefore, the aim of the present study is to assess the molecular profile of this entity in the anus using the next generation sequencing (NGS) technique in correlation with immunohistochemical data.  The biopsy revealed moderately differentiated squamous cell carcinoma. Using immunohistochemistry, irregular/heterogenous positivity for p16 protein was observed ( Figure 2).
The tumor was classified cT1 and treated by radiotherapy until February 2018. In May 2018, after a period of complete response, the patient noted the reappearance of an indurated and painful area near the anal margin. The patient underwent excision in June 2018.
Macroscopically, an irregular and ulcerated lesion occupying the near totality of a mucous ellipse measuring 26x15 mm was observed. This lesion was covered by a white coat.
Microscopically, the tumor consisted of nests of invasive squamous cell carcinoma, moderately differentiated. Lateral margins were positive. Using immunohistochemistry, tumor was negative for p16 (clone ink4a E6H4, ready to use, Roche). Immunoreactivity of p53 (clone DO-7, 1:200, Dako Agilent) appeared continuous and limited to the periphery of invasive nests, with strong intensity (Figure 3). The tumor was classified rpT2Nx.
Gene mutation testing was performed by NGS, as we have previously described [14,15], with a panel of 50 genes described in Table 1. One mutation was found: G279fs * 4 (c.833.834insGAGTCGAAACTCCACGCACAAACACGG-ACAGGAC) frameshift mutation of the TP53 gene.
In addition, the detection of High Risk-HPV DNA was negative [13].
Due to the positive margins and the classification, PET-CT was realized and was negative. Complementary resection was performed in July 2018.
Gene mutation testing was performed in this DIN-like lesion, but no mutation was found.

Discussion
Tumors of the anus and perianal skin are rare. With standard treatment, complete and durable remission can be achieved in the majority of patients. However, locoregional failure rates vary between 16% and 33%: these patients do not respond to therapy or relapse early after treatment [16], such as the patient presented in this case.
Several studies indicated that HPV-/P16-anal cancers had significant worse overall survival and relapse-free survival, compared to HPV+/P16+ anal cancers [17,18]. In contrast to HPV+ anal cancers, HPV-anal cancers frequently carry TP53 mutations, suggesting that there might be large difference in the genetics of HPV+ versus HPV-tumors [18]. Moreover, loss of p53 function has been linked to resistance to radiotherapy in head and neck SCC [19].
In the present study, we analyzed for the first time molecular profile of both DIN-like lesion and associated SCC in the anus. Such analysis has already been done in the vulva, showing TP53 mutations in 6 out of 10 cases of DIN (60%) and in 4 out of 5 DIN-associated SCC (80%) [12]. In the present case, TP53 frameshift mutation was observed only in the SCC. The frameshift (insertion) mutation of the TP53 gene we observed is not reported in the COSMIC database (cancer.sanger.ac.uk) [20]. Other G279 insertion-frameshift mutations of unknown pathogenic significance were previously reported, in liver, larynx, skin, and bladder carcinomas. TP53 frameshift mutations in other amino acid positions have been reported in anal carcinoma, without functional consequences and variable associated immunoreactivity of p53 [18].
DIN is a subtle and difficult histopathological diagnosis, with a low interobserver agreement [21].
Histological and immunohistochemical characteristics present overlap with other entities, such as lichen sclerosus, squamous cell hyperplasia, or inflammatory disorders. Increased p53 staining can be seen in 5-61% of lichen sclerosus and up to 40% of squamous cell hyperplasia and is thought to be due to increased oxidative stress. Moreover, some authors suspect that atypical lichen sclerosus, showing increased p53 staining, may represent a very early form of DIN [22]. Therefore, we believe these entities are a spectrum of lesions sharing common histological features, where TP53 mutation could be a further event in anal SCC carcinogenesis.
In conclusion, we described a potential precursor lesion of SCC in the anus analogous to DIN in the oral cavity and vulva. The recognition of such a precursor should lead to a careful analysis of the HPV status and the molecular profile of cancer to detect the presence of TP53 mutations. Furthermore, studies investigating prognostic impact of such mutations in DIN-like lesions and associated SCC in the anus are warranted.