The contribution of human papilloma virus infection to cutaneous squamous cell carcinoma in patients with chronic lymphocytic leukemia

Abstract Patients with chronic lymphocytic leukemia (CLL), a B‐cell malignancy characterized by impaired humoral and cellular immunity, are at increased risk of developing cutaneous squamous cell carcinoma (cSCC). Human papilloma virus (HPV) is the most common sexually transmitted infection worldwide and it has been associated with various malignancies, including cSCC. Impaired cell‐mediated immunity is considered a primary risk factor in HPV‐induced cSCC. We examined cSCC lesions from CLL patients with consensus review and HPV genetic analysis to further characterize the relationship between HPV and prevalence of cutaneous malignancy in this population. Eleven patients with CLL contributed 35 cSCCs. Treatment with chemotherapy shortened the latency time to first cSCC. HPV was detected in 54% of the lesions. Among the HPV‐positive cSCC lesions, 84% of the lesions contained alpha‐genus HPV, 42% contained beta‐genus HPV, and 26% of the lesions contained both genera. There was a significant association between HPV‐containing lesions and peritumoral lymphocytic inflammation, suggesting this as a future area for further characterization. The majority of the lesions, including those with alpha‐genus HPV, occurred in sun‐exposed areas, such as the scalp and face. These findings may lead to practice‐changing recommendations for skin cancer, including the use of vaccinations to reduce HPV‐associated skin cancer.

Although the incidence of cSCC among CLL patients is well established, the clinical features predisposing this population to higher risk of NMSC remain poorly characterized. A recent study from Mayo Clinic examined a large CLL cohort and established that the strongest risk factor for developing SCC was a prior history of any skin cancer [5].
CLL-international prognostic index (IPI) was independently associated with increased risk of cSCC; patients with more aggressive disease at the time of CLL diagnosis had a higher risk for developing cSCC. Interestingly, patients with high risk (HR) CLL but no prior history of NMSC had better outcomes compared to those individuals with prior NMSC history regardless of CLL-IPI status, underscoring the importance of prior skin cancer in future risk of cSCC for CLL patients.
In cervical carcinoma and squamous cell carcinoma (SCC) of the oropharynx and larynx, human papilloma viruses (HPVs) of the alphagenus, most frequently, HPV-16, -18, -31, and -33, drive carcinogenesis and are categorized as HR-HPV subtypes. HPVs found in cutaneous epithelial surfaces are most often of the beta and gamma genera.
There is strong evidence demonstrating a central role for beta genus HPV, specifically HPV-5 and HPV-8, in skin cancer risk of immunosuppressed individuals, specifically via coincident mutation burden, including UV-signature mutations [6] and activation of aberrant proliferation pathways [7]. This pathophysiology has been well documented in solid organ transplant recipients treated with immunosuppressive therapy [8] and patients with somatic mutations in epidermodysplasia verruciformis (EVER1/2) pathways tumor suppressor genes. CLL patients exhibit immune deficiency similar to that described in transplant patients [9,10]. Hence, it has been suggested that HPV may play a significant role in the pathophysiology of cSCC in CLL patients through similar pathways.
The purpose of this study was to further characterize the role of HPV in the incidence of cSCC in CLL patients. We hypothesized that CLL patients with cSCC would harbor cutaneous HPV and the lesions would be morphologically unique to those of immunocompetent patients.
Each case was independently reviewed, and findings were tabulated by two pathologists (A & B) who were blinded to each other's review as well as previously rendered clinical and pathologic diagnoses.
The two reviews were compared by a project manager and if results were concordant, the first interpretation was utilized. If the reviews were discordant, a third pathologist reviewed sample while blinded to the original two interpretations. The third interpretation was used with consensus review to achieve the final study interpretation.
During review, cSCC cases were examined for the specific morphologic characteristic of cSCC, presence of features of previously described HPV infection in cSCC [7], as well as features of chronic inflammation. Additionally, all cases were examined for histomorphologic features ascribed to MCPyV, HPyV6/7, and TSPyV infection [11][12][13]. Inflammatory changes were annotated as the presence of lichenoid, intraepithelial, and/or dermal lymphocytic infiltration (mild, moderate, or dense).

Cutaneous virus DNA assays
DNA was extracted from the FFPE samples utilizing Gentra Puregene Tissue Kit (Qiagen). Polymerase chain reactions (PCRs) for HPV [14], beta-globin reference gene [7], FAP [15], PGMY-GP5/6+ [16], and MCPyV [17] were performed based on published methods. PCR conditions were the following: denaturation, 1 min at 95 o C; annealing, 1 min at 63 o C; extension, 1-minmin at 72 o C, and 40 cycles were applied. The PCR products were separated by agarose gel electrophoresis, visualized by ethidium bromide staining and UV light. The obtained putative PCR products were purified, cloned, and sequenced. The sequencing data were evaluated by computer-assisted analysis using NCBI-BLAST program.

Statistical analysis and visualization
Tumor and viral features associated with cSCC in CLL patients were examined using Chi Square and Fisher's exact test, as appropriate.

F I G U R E 1
Chemotherapy for CLL reduced the latency period to the development of cSCC. Six out of 11 subjects received chemotherapy. Of these subjects, all except one developed cSCC after starting therapy. A single subject was excluded from the analysis as latency was noted to be 33 years and considered an outlier. Mean latency time from CLL diagnosis to cSCC was 5.02 years (range 0.3-16.5 years), with chemotherapy shortening this latency to 1.98 years (0.74-3.74 years)

RESULTS
Among the 11 CLL patients included in the study, the median age at diagnosis was 64 (range 38-87 years old); 100% were Caucasian, 100% were male, and 18% had advanced stage disease as determined by Rai criteria. Mean latency time from CLL diagnosis to cSCC was 5.02 years (range 0.3-16.5 years), after exclusion of a single patient whose latency was 33 years (> 3 SD from median of remaining cohort).
Patients who received chemotherapy exhibited a shorter latency to first cSCC, approximately 1.98 years (range 0.74-3.74 years; Figure 1).
Of the five patients who received treatment, one received Bruton tyrosine kinase (BTK) inhibitor, one received anti-CD20 monotherapy, and three received a combination of anti-CD20/purine analogue ( Table 1).
Most of the lesions found in our cohort were characterized as conventional cSCC (conv-cSCC), with 8% of these displaying keratoacanothomatous cSCC features (cSCC-KA). Briefly, the conv-cSCCs There was a preponderance of HPV-16/18 alpha genus infections found in our study cohort but interestingly, these were not found in the anogenital regions where they are typically described. In our CLL cohort, the bulk of the SCC lesions were predominantly found in sunexposed areas (Figure 6), including the face, arms, chest, and legs.

DISCUSSION
It is well established that patients with CLL have a higher risk of secondary malignancies than the general population, predominantly which are not typically described in the immunocompetent population but have been well described in immunocompromised patients, such as those with BRAF-inhibitor targeted therapy [7].
In immunosuppressed patients, HPV viruses of the beta genus contribute to the pathogenesis of cSCC [8,19] and are associated with cSCC with wart-like features [7]. We were interested in characterizing which virus genotypes were associated with cSCCs in CLL patients.
Since cSCC-wf have been primarily described in immunocompromised populations, we hypothesized that the majority of cutaneous HPV would be found in these lesions, with the predominant genotype being beta-HPV.
In our cohort, more than 50% of the lesions contained HPV DNA, suggesting viral contribution to cSCC pathology in CLL. We found that a majority of both conv-cSCC and cSCC-wf were involved by HPV, 58% and 67%, respectively. In previous studies, beta genus HPV-15 and 38 comprised 50% of the virus infection found in skin lesions in the CLL population [20]. We expected to find a predominance of beta-genus HPV in our cohort as well, specifically EV-beta genus HPV, which is considered critical in cSCC pathophysiology of transplant patients [8]. Alpha-genus HPV-27, frequently associated with verrucae (warts), was more frequently described in conv-cSCC lacking morphologic features of warts. In CLL patients, both alpha-and beta-genus HPV are equally likely to contribute to the pathogenesis of cSCC.
All the cSCC lesions described in this study occurred in sun-exposed areas. This was of particular interest since several of the lesions con- Lymphocytic infiltrates have been previously described in the cSCC of CLL patients [21]. In our cohort, 60% of the lesions had moderate to high lymphocytic infiltration, a higher frequency than what is reported in the literature [22]. Significantly, the majority of the lesions with lymphocytic infiltration also contained HPV. The primary reason for the increased incidence of skin cancer in patients with CLL is hypothesized to be the underlying impaired cellular and humoral immunity [9].
Although CLL is characterized by defective B-lymphocytes, immune defects can be generalized beyond impaired humoral immunity. Defective CD8+ and CD4+ T-cell function and increased regulatory T-cell numbers have been described in CLL [10,23], with several of these F I G U R E 6 Distribution of lesions by morphologic subtype of cSCC. Lesions are predominantly in sun-exposed locations. Circles denote conv-cSCC, squares denote cSCC with keratoacanthomatous (KA) features, and stars denote cSCC with wart-like features (cSCC-wf). Shaded-in shapes denote anterior lesions, while outlines (no shade) denote posterior lesions further exacerbated by existing treatment modalities, particularly the use of purine analogue, bendamustine combination, or alemtuzumab [5]. Previous studies have described that time to development of cSCC in CLL patients can be shortened by chemotherapy [5]. Despite small sample size, this trend was also noted in our study sample and reinforces the notion that T-cell immunosuppression by CLL chemotherapy treatments contributes to cSCC. The high predominance of lymphocytes in pathology samples suggests that an inflammatory response is important to the pathophysiology of cSCC in CLL patients and possibly facilitates a role for HPV in cutaneous malignancy.
Taken together, we propose that a diagnosis of CLL leads to a dysfunctional immune system, with both elements of humoral and cellular immunity affected [9,10,23]. This leads to a dysregulation in is important to note that some of the trends described in prior studies with larger sample size were also observed here; specifically, reduced time to cSCC after chemotherapy treatment and lymphocytic infiltra-