Usefulness of KIR3DL2 to Diagnose, Follow-Up, and Manage the Treatment of Patients with Sézary Syndrome

Published OnlineFirst January 24, 2017; DOI: 10.1158/1078-0432.CCR-16-3185 Personalized Medicine and Imaging Clinical Cancer Research Usefulness of KIR3DL2 to Diagnose, Follow-Up, and Manage the Treatment of Patients with Sezary Syndrome Charlotte Hurabielle1,2,3, Nicolas Thonnart2,3, Caroline Ram-Wolff1,2,3,Hel ene Sicard4, Armand Bensussan2,3, Martine Bagot1,2,3, and Anne Marie-Cardine2,3 Abstract Purpose: KIR3DL2 is a recently discovered marker of the Moreover, KIR3DL2 immunostaining allowed the assessment of malignant clonal cell population in Sezary syndrome. We treatment efficiency and specificity toward tumor cells, the detec- intended to evaluate the expression of KIR3DL2 on blood T cells tion of the residual disease following treatment, and the occur- as a diagnostic, prognostic, and follow-up marker of Sezary rence of relapse, even though patients clinically experienced syndrome. complete remission and/or undetectable circulating Sezary cells Experimental Design: Sixty-four patients diagnosed with by cytomorphologic analysis. Sezary syndrome were included in this monocentric study. We Conclusions: We show that KIR3DL2 expression is the most þ þ collected the percentage of KIR3DL2 cells among CD3 T cells, sensitive diagnostic criterion of Sezary syndrome when com- obtained by flow cytometry, and other classical diagnostic criteria pared with all other available biological criteria. It also repre- for Sezary syndrome at diagnosis and during the follow-up. sents the best independent prognostic factor for Sezary syn- Results: Compared with the classical diagnostic factors, drome–specific death and the most relevant feature for the KIR3DL2 was the most sensitive diagnostic factor for Sezary follow-up of Sezary syndrome, showing the invasion of the syndrome. Univariate and multivariate analyses established that functional lymphocytes pool by Sezary cells. KIR3DL2 there- an eosinophil cell count >700/mm3 and a percentage of fore represents a valuable tool for routine use as a clinical þ þ KIR3DL2 cells within the CD3 T cells >85% at diagnosis were parameter at diagnosis, for prognosis and during patient fol- associated with a significantly reduced disease-specific survival. low-up. Clin Cancer Res; 1–9. Ó2017 AACR. Introduction is no curative treatment and available systemic treatments have often short-lived responses, with relapses after few weeks or Sezary syndrome is a rare, aggressive, and leukemic form of months (13). In the current International Society for Cutaneous cutaneous T-cell lymphoma (CTCL) characterized by erythro- Lymphomas (ISCL)/European Organisation for Research and derma associated with generalized peripheral lymphadenopathy Treatment of Cancer (EORTC) TNMB staging classification, the and circulating clonal malignant T cells called Sezary cells (1). diagnosis of Sezary syndrome requires erythroderma with a Although the incidence of CTCL is 7.7 new cases per million positive T-cell clone in the peripheral blood associated with at persons per year, Sezary syndrome accounts for only 5% of these least one B2 criterion including the identification of more than cases (2). Patients with Sezary syndrome have a bad prognosis, 1,000 Sezary cells/mm3 in the blood as determined been cyto- with a 5-year overall survival varying from 24% to 43% (3, 4). morphologic analysis (14). Indeed Sezary cells have first been Current prognostic factors of Sezary syndrome include advanced described as large atypical mononuclear cells with cerebriform age, elevated level of lactate dehydrogenase (LDH), raised count nuclei (15). However, the detection of Sezary cells by cytomor- of eosinophils, a former history of mycosis fungoides, extent of phology is not absolutely specific for the diagnosis of Sezary peripheral blood involvement, and partial or total loss of CD26 þ syndrome, as such cells can be found in other inflammatory and/or CD7 expression by circulating CD4 T cells (5–12). There erythrodermic conditions (16). KIR3DL2 is a member of the family of the killer-cell immuno- globulin-like receptors that was initially identified as expressed on 1 þ Department of Dermatology, Hopital^ Saint-Louis, AP-HP, Paris, France. natural killer (NK) cells and on rare circulating CD8 T cells (17). 2 ^ 3 INSERM U976, Hopital Saint-Louis, Paris, France. Paris Diderot University, KIR3DL2 has first been identified on the surface of Sezary cells in Sorbonne Paris Cite, Paris, France. 4Innate Pharma, Marseille, France. 2001, its sensitivity to establish the diagnosis of Sezary syndrome Note: A. Bensussan, M. Bagot, and A. Marie-Cardine are the co-senior authors of being confirmed thereafter in several studies (18–21). Indeed, it þ þ this article. has been shown that the circulating CD4 KIR3DL2 T cells Corresponding Author: Anne Marie-Cardine, INSERM U976, 1 avenue Claude corresponded to the clonal malignant T-cell population identified Vellefaux, Paris 75010, France. Phone: 331-5372-2078; Fax: 331-5372-2051; by its unique TCR-Vb rearrangement (22, 23). E-mail: [email protected] The aim of our study was to evaluate the expression of KIR3DL2 doi: 10.1158/1078-0432.CCR-16-3185 on peripheral blood T cells as a diagnostic, prognostic, and follow- Ó2017 American Association for Cancer Research. up marker of Sezary syndrome as compared with the classical www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst January 24, 2017; DOI: 10.1158/1078-0432.CCR-16-3185 Hurabielle et al. was defined as greater than 700/mm3 and raised LDH value as Translational Relevance greater than 450 U/L. Sezary syndrome is an aggressive and leukemic form of cutaneous T-cell lymphoma in which rapid diagnosis and Flow cytometry assessment accurate follow-up is hampered by a lack of easy-to-go pro- The same day, the percentages of lymphocytes (including T, B, þ þ cedure for identifying the circulating tumor burden. We here and NK cells), CD3 CD4 T cells among lymphocytes, and þ þ þ compared the evaluation of KIR3DL2 expression on CD4 T KIR3DL2-positive cells within the CD3 and CD4 T-cell subsets cells, by flow cytometry on blood samples, to the currently respectively were determined, either on total blood or/and admitted clinical criteria for this disease. We found that peripheral blood mononuclear cells (PBMC) isolated by density KIR3DL2 was the most sensitive diagnostic marker but also gradient centrifugation. Four-color immunofluorescence staining the best independent prognostic factor of death for this was performed using fluorescein isothiocyanate–conjugated pathology. In addition, KIR3DL2 detection enables the mon- (FITC), R-phycoerythrin (PE), Phycoerythrin-Cyanin 5 (PC5), itoring of treatment efficacy, the visualization of residual and Phycoerythrin-Cyanin 7 (PC7) anti-CD3, anti-CD4, anti- disease, and the detection of relapse or disease progression CD56, and anti-KIR3DL2 mAbs. KIR3DL2 mAbs used were as before it is established by classical parameters. KIR3DL2 might follows: Q66 (2010–February 2012; kindly gifted by Dr A. Mor- therefore represent a valuable tool for routine use as a clinical etta, Genova, Italy), 15C11 (March 2012–June 2012), and 13E4 parameter to diagnose, evaluate prognosis, and assess treat- (July 2012–April 2016; both produced and provided by Innate ment efficiency for Sezary syndrome. Pharma, Marseille, France). Cells were analyzed on a FC500 cytometer (Beckman Coulter) and the recorded data analyzed with FlowJo software. All measurements were done after obtain- ing patient written informed consent. fi On the basis of previous studies, positivity was determined by clinical and biological criteria, on a well-de ned and large cohort þ þ þ more than 5% of CD3 CD4 KIR3DL2 T lymphocytes among of patients with Sezary syndrome with an 8-year follow-up. þ the CD4 T-cell population (17, 22). Because some Sezary cells þ may be CD8 T cells or have lost expression of CD4, positivity of þ À Patients, Materials, and Methods KIR3DL2 was also monitored on the CD3 CD56 T-cell popu- Patients lation when necessary (25, 26). A total of 64 patients diagnosed with Sezary syndrome and followed up in our referral center for cutaneous lymphoma Statistical design and analysis (Saint-Louis Hospital, Paris, France) between October 2007 All data are presented as median (range) for continuous vari- and April 2016 were included in the study. The patients were ables and numbers, and as percentages for categorical variables, diagnosed according to the ISCL/EORTC classification for pri- unless stated otherwise. The c2 value was used to compare mary CTCL. Because a lymph node biopsy was not systemat- ically performed at diagnosis for all patients, disease staging was available only for half of them and was as follows: stage Table 1. Main disease characteristics of the 64 patients with Sezary syndrome at baseline IVA1 (n ¼ 16), stage IVA2 (n ¼ 13), and stage IVB (n ¼ 3). Further detailed patients' characteristics are shown in Table 1. Age, mean (SD) 67 years (11.6) Men, n (%)/women, n (%) 40 (62.5%)/24 (37.5%) The following clinical features were assessed: age, sex, former Diagnosis preceding that of SS CTCL: MF (n ¼ 7), eMF (n ¼ 3), tMF diagnosis of CTCL, and treatments. The data were collected at (n ¼ 2) diagnosis, after each change of treatment line and in some cases Other: eczema (n ¼ 2), psoriasis during the course of the treatment. Date of death or clinical (n ¼ 1) 3 status at the end of the study was collected. Clinical response to Median CSC (range) 1,906 (222–62,520) cells/mm

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