Rapid tumor progression in a patient with HPV type 16 associated anal squamous cell carcinoma suffering from long-standing Crohn's disease: A case report

Background and aim: Squamous cell carcinoma (SCC) is the most common cancer of the anal region, typically associated with high-risk (hr) HPV infection. Furthermore, there is evidence that Crohn's disease predisposes to adenocarcinoma in patients with perianal disease. Materials and methods: A 57-year old patient presenting with long history of Crohn's disease since the age of mid-twenties, went through several surgeries including ileocolectomy and anal fistula resection, combined with immunosuppressive therapy additionally periodically since 2008. One year before death (in 2015) a painful fistula was diagnosed with extensive high grade anal intraepithelial neoplasia (AINHG) and evidence of invasive growth as non-keratinizing SCC. Tissue samples from several previous and current resection specimens were re-evaluated and extensively investigated for Crohn ́s type inflammation, dysplasia and HPV both by immunohistochemistry (p16/Ki67) and molecular subtyping of HPV. Results: AIN-HG and invasive anal squamous cell carcinoma turned out to be strongly positive for p16/ Ki67 staining and molecular analysis disclosed a HPV-16 subtype. In contrast, HPV-analysis was negative in all available previous tissue samples including one anal fistula resected five years before (in 2009) which was lined by non-keratinized squamous epithelium without any evidence of dysplasia. Thus, the patient was diagnosed as Crohn's disease with hr-HPV infection that rapidly ( 5ys) progressed to AIN-HG and anal SCC. Finally, osseous metastases occurred and the patient died shortly after. Conclusions: This case of a patient diagnosed with SCC of the anal canal in combination with Crohn's disease as well as HPV Type 16 infection, points to the pathomechanism leading to dysplasia and finally cancer. We assume that immunosuppressive therapy in Crohn's disease may predispose to both persistent HPV infection and HPV related invasive anal carcinoma. The accelerated progression of HPV associated neoplasia in immunosuppressed patients might represent a fast-tracked process of the long-term course of precancerous or cancerous lesions in immunocompetent hosts. This might implicate, that there is a need to re-evaluate current screening guidelines for anal cancer in patients with chronic inflammatory bowel disease under immunosuppressive therapy.

the presence of biologically active HPV infection, due to the fact that functional inactivation of pRb by viral E7 induces p16 INK ⁴ª upregulation [7]. From a biological point of view p16/Ki67 dual-stain within the same cell (nucleus) is regarded as a hallmark of cell-cycle deregulation. In normal cells the expression of p16 and Ki67 is mutually exclusive [8].
Herein we present a patient with long-standing fistulating Crohn´s disease ( 30 years) who went through several surgeries including ileocolectomy and multiple anal fistula resections, combined with immunosuppressive therapy additionally periodically since 2008. In 2014, he was diagnosed with HPV high risk associated metastasized anal cancer and died almost one year later.
Tumorbiological, diagnostic and clinical significance of HPV infection in long-standing Crohn´s disease/IBD is demonstrated.

Materials and methods
In total nine tissue samples (6 biopsies and 3 resection specimens) were diagnosed at the Institute of Pathology Nordhessen obtained from 1988 through 2014 in a 57years old patient with long-standing history of Crohn's disease, first radically treated in 1985 by ileocolectomy.
Specimens were routinely stained by H&E and immunohistochemistry (IHC) was performed on nearly all formalin-fixed paraffin embedded tissues using Ventana automated Immunostainer system (Ventana Medical Systems, Tucson, AZ, USA).
HPV subtyping (done at Institute of Pathology University of Göttingen) by F-HPV typing TM using a multiplex PCR with fragmentation length analysis on an ABI 3500 genetic analyzer. Extracted DNA was amplified using the analyzer with a set of 16 fluorescently labelled primers, detecting HPV types, especially high-risk types (6,11,16,18,31,33,35,39,45,51,52,56,58,59, 68) as well as a human STR (short tandem repeats) for internal control. On samples older than 2009, it was impossible to gain exploitable material for molecular HPV subtyping.

Clinical history and findings
A HIV-negative (human immunodeficiency virus) patient born in 1957 underwent hemicolectomy and multiple anal fistula resection surgeries thereafter, because of Crohn's disease and ongoing tissue damage (1985 till 2014). All specimens were examined by our team (Figure 1). Additionally, the patient received immunosuppressive treatment (Infliximab Remicade, Adalimumab Humira, Azathioprine, Mesalazine Salofalk, Prednisolon) which could ease the pain and lead to nearly complete restitution of fistulas. After stopping the drug therapy, the patient immediately relapsed. In contrast, the AIN-HG and invasive SCC samples turned out to be strongly positive for p16/Ki67 staining and molecular analysis disclosed a HPV-16 high-risk infection.
Finally, the patient's status was defined as Crohn's disease associated with high risk HPV infection, rapidly ( 5ys) progressing to AIN-HG and anal SCC, having finally spread with osseous metastases.
In September 2014, this case was presented in an interdisciplinary tumor board, reporting the HPV associated anal carcinoma and multifocal bone metastases in a bone scintigraphy. Biopsy samples from thoracic vertebra segment 9 and iliac crest disclosed a moderately differentiated squamous cell carcinoma which defines the tumor as already having spread by haematogenous metastases. Accordingly, systemic palliative chemotherapy including Bisphosphonates was initiated as well as radiation of the anal tumor and the metastasis in the thoracic vertebra for local control. However, the patient's clinical situation deteriorated and finally he died within palliative care in February 2015.

Discussion
This case of a patient diagnosed with SCC of the anal canal in combination with both, Crohn's disease as well as HPV Type 16 infection, raises the question about the foremost pathomechanism leading to dysplasia and finally cancer, supporting the dysplasia-carcinoma sequence theory. There have been several case reports suggesting that longstanding Crohn's disease with a history of multiple fistulae and recurrent surgical resections is a risk factor for both adeno-and SCC [9,10]. In men, neoplasia of the anoderm is up to four times more frequent than in women, whereas women more often develop lesions in the transitional zone [11]. With respect to the origin of anal SCC there are theories about two main hotspots the neoplasia arises from, namely perianal or within anal mucosa/anal canal. Hr-HPV infection is much more common in intra- anal located tumors, whereas neoplasia of the perianal region is less often hr-HPV-positive (92% vs. 64%) [11]. In the presented case, the histological investigations were performed on fistula resection specimen from perianal region with extent to depth. Directly intra-anal probe excisions have not been investigated. Clinically, SCC in the anal/perianal region is regarded as perianal if the tumor bulk is visible by inspection distally to the anal verge [12]. Histologically, these tumors typically are of moderate to well differentiated keratinizing type. In contrast, tumors arising from the anal epithelium show mostly a non-keratinizing usually basaloid differentiation. This rather characterizes our tumor as originating from anal mucosa having been irritated by chronic inflammation and fistulization [11,13].
Crohn's disease is a chronic inflammatory bowel disease of gastrointestinal tract. It affects 15-20 out of 100.000 inhabitants per year and incidence is maximal between the ages of 20 to 30 [14,15]. Its' cause is widely unknown but there are approaches and suggestions for genetic components and some environmental factors. The diagnosis of Crohn's disease is often made by clinical, histological and morphological data. Common complications are fistulas, anal bleedings, abscesses and free perforation with peritonitis [16]. The involvement of the terminal ileum and fistulae are characteristics of Crohn's disease [17]. If perforation occurs, surgical bowel resection according to the general condition of the patient is standard treatment. The risk of intestinal cancer in IBD has been identified in several studies [5,18], so that there is evidence that anal inflammation predisposes to anal cancer [6], both adenoas well as squamous cell carcinoma [9]. The current ECCOguidelines mention anal SCC as a rare complication of IBD (perhaps more common in patients with chronic fistulizing Crohn's disease) which may possibly be associated with carcinogenic HPV types [19].
Hr-HPV infection is a well-established causative factor inducing anal cancer in general [10]. In gynecologic tissue, for example, cervical cancer arises usually within 20 years after infection [20]. In high risk groups, such as HIV infected patients, progression to invasive anal cancer has recently been shown to be very low but increasing [2,21]. In general, anal cancer is rare, about 2 cases per 100.000 [17,10] and in the majority of cases it is associated with HPV-HR infection [3,[22][23][24]. The fact that anal cancer has a sexually transmitted etiology and shows HPV type 16 mono-infection in 65% to 75% of the cases this states correlation between cause and effect [1,2].
Like in the cervix, HPV type 16 is predominantly associated with squamous cell carcinoma, whereas type 18 is rather linked with adenocarcinoma. Thereby HPV type 16 monoinfection is also mostly found in cervical cancer samples [25][26][27].
Our presented patient with long-standing Crohn's disease must have had acquired HPV-HR infection within the past five years before cancer diagnosis as we did not see any dysplasia in several preceding fistula resections. Thus, this case is indicative of an unusual rapid progression from HPV infection to cancer.
Since even in high risk groups such as HIV infected patients progression to invasive anal cancer has recently been stated to be very rare [21,28], it is shown that besides Crohn´s disease another compelling factor should be taken into consideration namely the long period of immunosuppressive therapy in this presented patient. In several studies, the impact of immunosuppressive therapy such as azathioprine and corticosteroids in IBD patients has been proven and it is stated that immune-modulator therapy increases the risk for persistent HPV infection [19], additionally to the already increased risk caused by the immunosuppression by the disease itself.
We therefore assume that Crohn's disease along with guideline adjusted treatment may predispose for both persistent HPV-HR infection and rapid transformation into AIN and invasive cancer. This might implicate, that there is need to re-evaluate current screening guidelines for anal cancer in patients with chronic IBD [29], because it is a rare complication but approachable for further screening methods, particularly in men.
As a possible screening method for anal dysplasia, especially for high-risk patients like HIV infected or profoundly immunosuppressively treated ones could be HPV-screens, e.g. once a year or every 2-3 years followed by cytological check-ups, besides digital anal-rectal examination (DARE). HPV16-detection is in 92% specific for an AIN-HG. In case of highly suspicious findings or positive results, a highresolution anoscopy along with taking a directed biopsy is required, for confirmation of diagnosis and identification of the degree of dysplasia [30,31]. However, data for AIN screening effect on incidence of anal carcinoma in patients with Crohn's disease are missing.
Focusing on a few patients suffering from IBD alone, without viral infection, and ending up with anal SCC the mean time was 15-16 years after onset of IBD symptoms but at least more than ten years [6,10]. That goes along with current suggestions that anal SCC arises in chronic fistulas following a dysplasia-carcinoma sequence [32]. Research protocols state that KRAS and BRAF mutations in anal carcinoma are rare findings and have no significant impact on overall or disease-free survival [33]. Thus K-Ras mutations have not been looked for in this special case.
Based on our case, we assume that Crohn's disease may predispose for both, persistent hr-HPV infection and potentially to rapid transformation into AIN and invasive cancer, in particular if immunosuppressive therapy is administered. It has also been described by a review from Slesser et al. who stated that in Crohn's disease anal squamous carcinoma presents at younger age with poorer outcome compared to general population [10].

Conclusion
The presented case indicates rapid oncogenesis ( 5 years) after HPV high risk infection in long-standing fistulating Crohn´s disease. Thereby, immunosuppressive therapy predisposes for persistent HPV infection and progression to AIN [10,34] (Figure 2d). For IBD patients routinely endoscopic examinations are already standard of care and it is known that patients benefit from this investigation. Current recommendations for HPV-vaccination in IBD patients exist. Vaccines are best given before immunomodulator therapy, explicitly including HPV. Though, perianal fistula in combination with immunomodulatory therapy -as most IBD patients receive -may provide a target point for HPV viruses [10]. Since anal squamous carcinoma is a rare entity, focused screening for HPV in IBD patients, especially in men, e.g. by anal smears or probe excisions from chronic fistulizing areas, are currently not a routine diagnostic standard and should perhaps be considered for screening recommendations.

Acknowledgements
This study was supported by the staff within the laboratory of H-U. Schildhaus and C. Hoppenau who did the HPV subtyping at the University Pathology Institute of Göttingen. Further, R. Hesterberg and H. Steinbrück performed surgical examination and sample recovery, and T. Krause cared about routine medical surveillance and screening as well as follow-up investigations for the patient. I gratefully acknowledge U. Hampacher and her team, who did immunohistochemistry within the laboratory of the Institute of Pathology Nordhessen.

Compliance with ethical standards
There are no potential conflicts of interest expected. Due to the fact that we present an anonymous clinical case, there should no informed consent be needed.

Funding
The authors declare that there has been no funding on this project. The used tissue samples were gained by surgical exstirpation and prepared for diagnostics. Followingly, the involved pathologists of the Institute of Pathology Nordhessen collected all existing patient relevant data and specimen for further scientific work.