History of chronic inflammatory disorders increases the risk of Merkel cell carcinoma, but does not correlate with Merkel cell polyomavirus infection.

BACKGROUND: We aimed to assess the connection between chronic inflammatory disorders (CIDs) and Merkel cell carcinoma (MCC).
METHODS: Merkel cell carcinoma cases diagnosed in 1978-2009 were extracted from the Finnish Cancer Registry and controls from the Population Registry. Information on reimbursed CIDs was linked to clinicopathological data including Merkel cell polyomavirus (MCV) status by qPCR and immunohistochemistry for the large T antigen of MCV (LTA), Ki-67 and tumour-infiltrating lymphocytes.
RESULTS: Chronic inflammatory disorders increased the risk of MCC significantly (odds ratio (OR) 1.39, 95% confidence interval (CI) 1.03-1.88), specifically connective tissue/systemic diseases (OR 1.75, 95% CI 1.09-1.80) and diabetic conditions (OR 1.51, 95% CI 1.03-2.22). Chronic inflammatory disorders associated with larger tumour diameter (P=0.02) and higher Ki-67 expression (P=0.005). The expression of LTA was seen significantly more often in the absence of CIDs (P=0.05).
CONCLUSIONS: Patients with CID are at significantly higher risk for aggressive MCC. Merkel cell polyomavirus positivity is more common in MCC patients unafflicted by CID.

Merkel cell polyomavirus (MCV) has a causal role in the tumourigenesis of Merkel cell carcinoma (MCC) (WHO, 2013). Recent evidence implicates two subgroups of MCC tumours: the MCV-positive and MCV-negative group, which differ in their clinical characteristics and pathobiological properties (Leroux-Kozal ; Sahi ; Veija ). Merkel cell polyomavirus positivity of the tumour is associated with a favourable prognosis. However, the prognosis of MCV-positive patients is dependent on the intensity of antiviral immune response (Paulson ), and immunosuppression leads to poorer outcome regardless of MCV status (Paulson ). Higher numbers of tumour invading CD8+ lymphocytes in MCV-positive tumours correlates with a favourable MCC prognosis (Paulson ; Sihto ; Harms ).

The association between chronic inflammatory disorders (CIDs) and MCC has received less systematic attention than other immunocompromising states such as HIV infection or post-transplant immunosuppression (Arron ). We sought to examine the risk for MCC in patients with CIDs in a nationwide case–control setting. Further, we linked the epidemiological data to the clinicopathological data of the MCC cases to assess whether CIDs correlate with the course of the disease or the tumours' clinical characteristics.

Materials and methods

In phase I, registry data were used in an epidemiological case–control setting to determine whether CIDs affect the risk for MCC. In phase II, MCC patients included in phase I were linked via their personal identity codes to their clinicopathological characteristics.

An Institutional Ethics Committee approved the study plan. The National Institute of Health and Welfare granted its permission to use the information of the Finnish Cancer Registry (FCR). The National Social Insurance institution (SII) provided permission to access drug reimbursement data.

Our group has gathered all paraffinised primary MCC tumour samples available in Finland since 1978. During the past 10 years, we have compiled a database of information on the patient characteristics and course of the disease, as well as tumour characteristics including viral status, tumour genomics and the expression of various immunohistochemical markers. The ongoing MCC projects of our research group continue to utilise this database.

Phase I

Merkel cell carcinoma cases diagnosed in 1978–2009 were identified from the FCR. They were matched for 15 referent persons randomly selected from the Population Register Centre based on the following criteria: (1) same gender and (2) same year of birth. Of these 15 persons, those alive at the time of the MCC diagnosis of the case served as controls.

The SII manages a database that includes specific data on medicine expense reimbursements for chronic diseases. We retrieved data on the SII categories comprising CIDs (Table 1) by using the personal identity codes of the cases and controls as a key. Only diseases prior to MCC diagnosis date were noted.

Table 1

SII categories with the chronic inflammatory disorders included in the study

SII category	Chronic inflammatory disorder	ICD-10
Systemic/connective tissue
Rheumatic diseases and conditions	Enteritis due to Yersinia enterocolitica	A04.6
	Other meningococcal infections	A39.8
	Other late congenital syphilis, symptomatic	A50.5
	Langerhans cell histiocytosis not elsewhere classified	D76.0
	Other histiocytosis syndromes	D76.3
	Chronic iridocyclitis	H20.1
	Chorioretinal inflammation	H30
	Acute and subacute infective endocarditis	I33.0
	Other interstitial pulmonary diseases	J84
	Crohn's disease, unspecified	K50.9
	Ulcerative colitis, unspecified	K51.9
	Chronic active hepatitis, not elsewhere classified	K73.2
	Autoimmune hepatitis	K75.4
	Primary biliary cirrhosis	K74.3
	Cholangitis	K83.0
	Arthropathic psoriasis	L40.5
	Postinfective and reactive arthropathies	M02
	Rheumatoid arthritis with rheumatoid factor	M05
	Other rheumatoid arthritis	M06
	Juvenile arthritis	M08
	Arthritis, unspecified	M13.9
	Polyarteritis nodosa and related conditions	M30
	Other necrotising vasculopathies	M31
	Systemic lupus erythematosus (SLE)	M32
	Dermatopolymyositis	M33
	Systemic sclerosis	M34
	Other systemic involvement of connective tissue	M35
	Ankylosing spondylitis	M45
	Sacroiliitis, not elsewhere classified	M46.1
	Unspecified inflammatory spondylopathy	M46.9
	Relapsing polychondritis	M94.1
	Chronic nephritic syndrome	N03
	Atresia of bile ducts	Q44.2
Sarcoidosis	Sarcoidosis	D86
Ablastic anaemia	Acquired pure red cell aplasia	D60
	Other aplastic anemias and other bone marrow failure syndrome	D61
Gastrointestinal
Colitis ulcerosa, Morbus Crohn	Morbus Chron	K50
	Colitis ulcerosa	K51
Skin
Pemphigus	Pemphigus	L10
	Pemphigoid	L12
General erythrodermia	Lichen planus	L43
	Psoriasis	L40
	Systemic lupus erythematosus	M32
Neural system
Parkinson's disease, comparable disorders	Parkinson's disease	G20
Multiple sclerosis	Multiple sclerosis	G35
Myasthenia gravis	Myasthenia gravis	G70.0
Diabetic conditions
Diabetes mellitus	Type 1 diabetes mellitus	E10
	Type 2 diabetes mellitus	E11
	Diabetes due to malnutrition	E12
	Other specified diabetes mellitus	E13
	Unspecified diabetes	E14
	Postprocedural hypoinsulinemia	E89.1

Abbreviation: SII=social insurance institution. The categories are further grouped according to the affected organ.

On the basis of statistical power analysis, the CIDs were grouped together for further calculations according to the affected organ systems (Table 2). A conditional logistic regression model was used to estimate, by means of the odds ratio (OR) with 95 % confidence intervals (95% CI), the risk for MCC.

Table 2

The numbers (N) of CID in cases with MCC and controls, as well as OR and their 95% CI

	N MCC	N Controls	OR	95% CI
Any chronic inflammatory disorder or multiplea	59	487	1.63	1.19–2.22
Chronic inflammatory disorders by anatomical involvementb
Systemic/connective tissue diseases	23	144	2.01	1.26–3.20
Rheumatic diseases and conditions	22	140	1.96	1.22–3.15
Sarcoidosis	1	4
Ablastic anaemia	0	1
Dermatologic diseases	1	3
Erythrodermia	0	2
Pemphigus	1	1
Gastrointestinal diseases	1	8	1.59	0.20–12.8
Neural diseases	5	76	0.75	0.29–1.91
Parkinsons disease	5	72	0.78	0.30–2.00
Multiple sclerosis	0	4
Myasthenia gravis	0	1
Diabetic conditions	35	296	1.51	1.03–2.22

Abbreviations: CI=confidence interval; CID=chronic inflammatory diseases; MCC=Merkel cell carcinoma; OR=odds ratios.

List of all CID included in the study is given in Table 1.

As defined in Table 1.

Phase II

Merkel cell carcinoma patients identified in phase I were linked via personal identity codes to the clinicopathological data of our MCC patient–tumour database. The clinical data comprised the sex of the patients, the pattern of metastatic dissemination and tumour recurrence rates. We examined metastasis in two alternative categories, with a division between local and metastasised disease or local disease, nodal metastasis and systemic metastasis. Combined with immunohistochemistry for Ki-67, these characteristics were used to chart how aggressive the disease was.

The real-time qPCR method used to quantify MCV DNA appears in detail elsewhere (Sihto ). We also retrieved previously gathered data on the expression of the large T antigen of MCV (LTA) and Ki-67 for the cases (Sihto ). Ki-67 staining was classified into three categories: low (0–32.7% positively staining tumour cell nuclei, 35 tumours), medium (33.6–66% positive nuclei, 36 tumours) and high (61–100% positive nuclei, 36 tumours). We also analysed Ki-67 expression as a continuous variable. The absolute numbers of tumour-infiltrating lymphocytes (CD68+, CD8+ and CD3+) were detected by scanning the whole-tumour sections after histochemical labelling (Sihto ) and divided into tertiles for the statistical analysis (Supplementary Table 1).

Pearson's χ2-test was used for the analysis of categorical variables, and the Mann–Whitney U-test for continuous variables. We used Kaplan–Meier analysis, log-rank test and Wilcoxon test to analyse differences in overall survival between the subgroups defined by inflammatory history and Ki-67 expression (high tertile vs low and medium tertiles). Multivariate survival analysis was conducted with the Cox regression model. The variables consisted of age, gender, history of CID, tumour size, LTA expression and Ki-67 expression.

Results

Phase I: chronic inflammatory disorders and the risk of MCC

The final case–control study set comprised 267 cases and 3 270 controls. Of the 267 MCC patients, 59 (22.1%) were diagnosed with at least one CID, whereas the respective number for the controls was 487 (14.9%). Chronic inflammatory disorders led to significantly higher risk for MCC (OR 1.63, 95% CI 1.19–2.22). The OR was not significantly affected by the gender or age of the MCC patients.

The history of inflammatory systemic/connective tissue disorders significantly increased the risk for MCC (OR 2.01, 95% CI 1.26–3.20), due mainly to rheumatoid conditions, which had an OR of 1.96 (95% CI 1.22–3.15). In addition, diabetic conditions significantly increased the risk for MCC (OR 1.51, 95% CI 1.03–2.22). Neural and gastrointestinal CIDs were rare. The results appear in full in Table 2.

Phase II: chronic inflammatory disorders and features of MCC tumours

Data on MCV status were available for 142 MCC patients (Supplementary Figure 1), on Ki-67 expression for 107 patients, and on the TILs for 129 patients on the CD68+ stain, 93 on the CD8+ stain and 128 for the CD3+ stain. Information on tumour size and metastasis was available for 134 patients.

Merkel cell polyomavirus DNA was more frequently found in MCC tumours of patients with no CIDs (77% vs 64%, Table 3), although the finding was not statistically significant. The LTA, however, was expressed significantly more often in the absence of CID (71% vs 53%, P=0.05). We observed no significant difference in the viral copy numbers between the groups (Table 3).

Table 3

Numbers of MCC tumours according to variables depicting the viral status and malignant tendency of the disease, and by the history of chronic inflammatory disease

	Choric inflammatory disorder(s)	No chronic inflammatory disorders	P-value
Viral status				Total number of tumours
MCV PCR+(%)	23/36 (64)	69/90 (77)	0.14	126
MCV LTA+(%)	19/36 (53)	60/84 (71)	0.0048	120
Median MCV copy number, per ref. gene (range)	0.89 (0–4224)	1.07 (0–2968)	0.50	126
Clinical behaviour				Total number of patients
Local, nodal or systemic metastasis (%)	9 /42 (21)	44/139 (32)	0.360	181
Mean tumour size, mm (range)	21 (4–70)	17 (3–85)	0.025	134

Abbreviations: MCC=Merkel cell carcinoma; MCV=Merkel cell polyomavirus; LTA = large T antigen of MCV. The clinical data and tumour material available for each analysis varied, with the total number of assessed patients/tumours for each variable listed in the last column. When evaluating the statistical significance in the variables between the two patient subgroups, P-values (P)<0.05 were considered significant.

The expression of Ki-67 was significantly higher in tumours of MCC patients with CID, with a mean percentage of 63% vs 56% (P=0.005; Supplementary Table 1). The finding was further strengthened by tertile analysis (P=0.007). Chronic inflammatory disorders associated with a larger tumour diameter P=0.02 (Table 3), but the statistical significance disappeared when the tumours were stratified by MCV status (pMCV+=0.06, pMCV−=0.23). Chronic inflammatory disorders did not associate with progression to metastatic disease (Table 3). No significant differences were detected in the overall survival or the TIL numbers between the subgroups (Supplementary Table 1). In the multivariate survival analysis, the only statistically significant variable was age at MCC diagnosis (hazard ratio=1.033, 95% CI 1.002–1.066, P=0.036).

Discussion

We identified a statistically significant increase in the risk for MCC in patients with a history of CIDs. Patients with inflammatory connective tissue diseases and diabetes were at the highest risk for MCC. The results are similar to the relative risk estimates of MCC following rheumatoid arthritis published in the United States (OR 1.39, 95% CI 1.10–1.74) and Sweden (SIR 2.42, 95% CI 0.96–5.01) (Lanoy and Engels, 2010; Hemminki ). The tumours of patients with a history of CID were also larger and expressed elevated levels of the proliferation marker Ki-67. This supports the earlier notion of the aggressive course of the disease in immunodefective individuals (Paulson ).

Merkel cell carcinoma patients diagnosed with CIDs presented frequently with MCV qPCR-positive tumours. Expression of the LTA occurred in the tumours significantly more often in the absence of CID. The finding is surprising and differs from reports of MCV incidence among the cohort of immunosuppressed MCC patients with chronic lymphocytic leukaemia (Koljonen ). This finding seems to stress the difference between stark immunosuppression and more subtle changes in the immune responses in patients with chronic inflammation. One could hypothesise that the immunomodulatory role of chronic inflammation in MCC pathogenesis and predisposition to oncogenesis is more complicated, and may involve tumour surveillance instead of surveillance against virus infection. However, no statistically significant difference was detected in the MCV DNA status between the groups, even though it usually correlates accurately with LTA expression. Confirmation of the finding in other MCC cohorts would be informative.

Tumour-infiltrating lymphocytes are under vigorous scrutiny in MCC due to the new immunomodulating treatment options derived from melanoma research. High CD8+/CD4+ and FoxP3+/CD4+ ratios, as well as CD8+ and CD3+ numbers are associated with good survival (Sihto ). This is the first study to look into the TIL spectrum of MCC tumours in immunocompromised patients. We found no significant differences in the numbers of TILs between the patient groups implicating that immunodeficiencies affect the function rather than the trafficking of TILs into MCC tumours.

The results of this study recommend consideration for MCC, especially when dealing with patients with rheumatic and diabetic diseases, as it is more common and more aggressive in these patients. Further studies are needed to elucidate the specific mechanism behind the effect and the interaction between MCV infection and chronic inflammation.