Cancer Immunology, Immunotherapy (2019) 68:951–960 https://doi.org/10.1007/s00262-019-02334-8 ORIGINAL ARTICLE Heterogeneity of PD‑L1 expression and CD8 tumor‑infltrating lymphocytes among subtypes of cutaneous adnexal carcinomas Lucie Duverger1 · Amélie Osio1 · Bernard Cribier2 · Laurent Mortier3 · Adèle De Masson4,5 · Nicole Basset‑Seguin4,5 · Céleste Lebbé4,5 · Maxime Battistella1,6 Received: 15 September 2018 / Accepted: 28 March 2019 / Published online: 5 April 2019 © Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract Background Adnexal carcinomas are rare and heterogeneous skin tumors, for which no standard treatments exist for locally advanced or metastatic tumors. Aim of the study To evaluate the expression of PD-L1 and CD8 in adnexal carcinomas, and to study the association between PD-L1 expression, intra-tumoral T cell CD8+ infltrate, and metastatic evolution. Materials and methods Eighty-three adnexal carcinomas were included. Immunohistochemistry using anti-PD-L1 mono- clonal antibodies (E1L3N and 22C3) and CD8 was performed. PD-L1 expression in tumor and immune cells, and CD8 + tumor-infltrating lymphocyte (TIL) density were analyzed semi-quantitatively. Results Among the 60 sweat gland, 18 sebaceous and 5 trichoblastic carcinomas, 11% expressed PD-L1 in ≥ 1% tumor cells, more frequently sweat gland carcinomas (13%, 8/60) including apocrine carcinoma (40%, 2/5) and invasive extramam- mary Paget disease (57%, 4/7). Immune cells expressed signifcantly more PD-L1 than tumor cells (p < 0.01). Dense CD8+ TILs were present in 60% trichoblastic, 43% sweat gland, and 39% sebaceous carcinomas. CD8+ TILs were associated with PD-L1 expression by tumor cells (p < 0.01). Thirteen patients out of 47 developed metastases (27%) with a median follow- up of 30.5 months (range 7–36). Expression of PD-L1 by tumor cells was associated with the development of metastasis in univariate analysis (HR 4.0, 95% CI 1.1–15, p = 0.0377) but not in multivariate analysis (HR 4.1, 95% CI 0.6–29, p = 0.15). Conclusion PD-L1 expression is highly heterogeneous among adnexal carcinoma subtypes, higher in apocrine carcinoma and invasive extramammary Paget disease, and associated with CD8 + TILs. Our data suggest the interest of evaluating anti- PD1 immunotherapy in advanced or metastatic cutaneous adnexal carcinoma. Keywords Cutaneous adnexal carcinoma · Programmed cell death ligand 1 (PD-L1) · Tumor-infltrating lymphocytes (TILs) · CD8 Abbreviations AJCC American Joint Committee on Cancer * Maxime Battistella [email protected] APHP Assistance Publique-Hôpitaux de Paris BCC Basal cell carcinoma 1 Pathology Department, INSERM UMR_S1165, Hôpital CHRU Centre hospitalier régional universitaire Saint Louis, Assistance Publique-Hôpitaux de Paris (APHP), cSCC Cutaneous squamous cell carcinoma 1 Avenue Claude Vellefaux, 75010 Paris, France EMPD Extramammary Paget disease 2 Dermatology Department, Hôpitaux universitaires de IC Immune cells Strasbourg, Strasbourg, France NOS No other specifcation 3 Dermatology Department, Centre Hospitalier Régional TC Tumor cells Universitaire (CHRU) de Lille, Lille, France TMB Tumor mutation burden 4 INSERM, Unité mixte de recherche (UMR)_S976, UMR Unité mixte de recherche University Paris Diderot, Paris, France WHO World Health Organization 5 Dermatology Department, Hôpital St Louis, APHP, Paris, France 6 INSERM, UMR_S1165, University Paris Diderot, Paris, France Vol.:(0123456789)1 3 952 Cancer Immunology, Immunotherapy (2019) 68:951–960 Introduction study. Diagnoses were performed according to the latest World Health Organization (WHO) criteria for skin can- Cutaneous adnexal carcinomas are a large and diverse cers, and validated in the French CARADERM (CAncers group of tumors, deriving from diferent types of adnexal RAres DERMatologiques—rare skin cancers) Network epithelium present in normal skin (sebaceous glands, (National Institute of Cancer). For all patients, there was apocrine glands, eccrine glands, and hair follicles) [1, enough formalin-fxed parafn embedded tumor mate- 2]. Their exact prevalence is unknown but these lesions rial remaining after the diagnosis had been established are rare in comparison to other skin cancers [3]. Some to qualify the samples for additional immunohistochemi- of the malignant adnexal tumors have high rates of local cal studies. All surgical samples of included patients were recurrence and distant metastases [4]: 20–25% of poro- taken at diagnosis, before any medical treatment of the carcinomas and as much as 50% digital papillary adeno- disease. Clinical data at diagnosis and follow-up data were carcinomas may recur or metastasize [5–8]. In cutaneous collected from clinical fles. Tumors were staged using the adnexal carcinomas, surgery is the only curative treatment, eighth TNM version of the American Joint Committee on and there is a lack of therapy for unresectable or meta- Cancer (AJCC) (2017), according to the recommendation static tumors: radiotherapy may be proposed for locally of the WHO 2018 classifcation of skin tumors. advanced tumors [9], whereas no consensus exists regard- ing management of metastatic cases, which remain an area of high unmet clinical need [10, 11]. Immunohistochemistry Programmed death-1 (PD-1) and its ligand (PD-L1) constitute an important immune checkpoint [12], play- Immunohistochemistry was performed according to the ing a major role in evasion of malignant tumor cells from following protocol: 3 μm parafn sections were placed on ® the immune system [13]. The prognostic value of PD-L1 Superfrost plus glass slides. PD-L1 expression was per- ® expression by tumor cells remains controversial. In some formed using a BenchMark Ultra automated immunostainer studies, it was reported as a marker of poor prognosis [14]. (Roche-Ventana, Basel, Switzerland) and monoclonal anti- On the contrary, in melanoma patients, PD-L1 expression bodies against PD-L1, E1L3N [1:200 dilution (Cell Signal- may be associated with better prognosis [15]. The predic- ing Technology, Danvers, MA, USA)] and 22C3 [1:50 dilu- tive value of PDL-1 expression by tumor cells for response tion (Dako, Hamburg, Germany)]. CD8 immunostaining was to anti-PD1/PD-L1 agents has been largely studied and performed using anti-CD8 4B11 clone [1:50 dilution (Dako, reported. In melanoma as in lung carcinoma, renal cell Hamburg, Germany)]. carcinoma and urothelial carcinoma, increased response rates, improved progression-free survival and overall sur- vival are observed in patients with higher PD-L1 tumor Microscopic evaluation expression [16]. To our knowledge, there is no report examining PD-L1 Immunostaining was assessed by two pathologists (M. Bat- expression in cutaneous adnexal carcinomas. In the con- tistella and L. Duverger) independently, blindly from follow- text of the potential use of anti-PD1/PD-L1 therapy in up data, when available. cutaneous adnexal carcinomas, we aimed here to deter- The immunohistochemical expression of PD-L1 was mine the expression pattern of PD-L1 in tumor cells and quantifed semi-quantitatively. Adnexal carcinomas were microenvironment immune cells in cutaneous adnexal divided as previously described into 4 groups according to carcinomas, and to analyze its association with the level the percentage of PD-L1 positive tumor cells: < 1% (TC0), of CD8+ tumor-infltrating lymphocytes (TIL) and with 1–4% (TC1), 5–49% (TC2) and > 50% (TC3), and into metastatic evolution. four groups according to the percentage of PD-L1 positive immune cells: < 1% (IC0), 1–4% (IC1), 5–10% (IC2) and > 10% (IC3) [17]. For subsequent analyses, tumors were considered to be Materials and methods PD-L1 (+) when ≥ 1% of tumor cells demonstrated mem- branous staining. Patients To evaluate CD8 + TIL, we used a semi-quantitative visual score: none (0), mild (1), moderate (2), or severe (3). The + Eighty three patients with cutaneous adnexal carcinoma degree of CD8 TIL infltration was evaluated into the tumor from a single University hospital (Hôpital Saint Louis) and in its stroma. Stromal TIL evaluation, which is more diagnosed between 2002 and 2018 were included in the reproducible, was further used for the association analysis with PD-L1 tumor cell expression [18, 19]. 1 3 Cancer Immunology, Immunotherapy (2019) 68:951–960 953 Statistical analyses Table 1 Patients’ characteristics Variable N (%) Continuous variables were described as medians (25th; 75th interquartile range) and dichotomous data as percent- Sex ages. Diferences in frequencies of quantitative variables Male 51 (62) were compared using the χ2 test with Pearson’s correction Female 32 (38) or a Fisher’s exact test when sample sizes were too small Age in years, median (range) 68 (26; 95) (expected values below 5). Fisher’s exact test was used Tumor type to compare the proportion of samples with CD8+ TIL in Sweat gland carcinoma 60 (72) PD-L1(+) and PD-L1(−) samples. As we had missing data Porocarcinoma 14 (17) for PD-L1 using 22C3 clone, we used preferentially E1L3N Hidradenocarcinoma 11 (13) antibody results for all the statistical tests of the study. Ductal adenocarcinoma NOS 10 (12) We evaluated the agreement between the PD-L1 results Microcystic adnexal carcinoma 5 (6) with the two antibodies (E1L3N and 22C3 clones) using Syringoid eccrine carcinoma 5 (6) Cohen’s weighted kappa (κ) statistic interpreted as follows: Apocrine carcinoma 5 (6) κ < 0.20, poor strength; κ = 0.21–0.40, fair; κ = 0.41–0.60, Invasive extramammary Paget disease 7 (8) moderate; κ = 0.61–0.80, good; κ = 0.81–1.00, very good. Digital