Expression of CXCR4 Predicts Poor Prognosis in Patients with Malignant Melanoma

Vol. 11, 1835–1841, March 1, 2005 Clinical Cancer Research 1835

Stefania Scala,1 Alessandro Ottaiano,1 reached in patients with CXCR4-negative tumors. In the Paolo Antonio Ascierto,1 Manuela Cavalli,1 multivariate analysis, CXCR4 expression, presence of ulcer- Ester Simeone,1 Paola Giuliano,1 ation, and sentinel lymph node status emerged as independent 1 2 prognostic factors. Maria Napolitano, Renato Franco, Conclusions: This article provides the first evidence 2 1,3 Gerardo Botti, and Giuseppe Castello that CXCR4 expression could be an independent and Departments of 1Clinical Immunology and 2Clinical Pathology National powerful prognostic marker in primary cutaneous malig- 3 Cancer Institute, G. Pascale Foundation and Melanoma Cooperative nant melanomas. Group of Naples, Italy

INTRODUCTION ABSTRACT Malignant melanoma represents about 2% to 3% of all Purpose: CXCR4 receptor and its unique ligand, the malignancies in the United States and North Europe. The CXCL12 chemokine, have been recently implicated in cancer incidence of malignant melanoma has increased steadily during metastasis. Evidence about the role of CXCR4/CXCL12 axis the last decades with a high mortality rate because of the has been reported in several cancers including melanoma. metastatic dissemination of tumor cells and generation of Our goal was to investigate if CXCR4 expression has a chemoresistance (1). Although surgery can be curative in prognostic value in malignant melanoma. localized disease, a large number of patients develop distant Experimental Design: Immunohistochemical expression metastases. Despite multiple clinical trials that have tested a of CXCR4 was evaluated on 71 specimens of primary wide variety of anticancer strategies, ranging from surgery to cutaneous melanoma with a Breslow tumor thickness of >1 immunotherapy, radiotherapy, and chemotherapy, the average mm after radical resection. Associations between baseline survival rate is of 6 to 10 months. To date, the most important patient features and tumors were analyzed by C2 test. The prognostic factors are Breslow’s tumor thickness, ulceration, and prognostic value of CXCR4 expression was evaluated by lymph node status (2). Diagnosis of malignant melanoma at very univariate and multivariate analyses adjusted by age, sex, early stages (i.e., before the malignant melanocytes become Breslow tumor thickness, presence of ulceration, and sentinel invasive) allows for complete surgical excision of the primary lymph node metastases. tumors. In these cases there is over a 95% success rate at stage Results: CXCR4 expression was detected in 31 of 71 I/II and it can even substantially extend the long term survival of (43.6%) primary cutaneous melanomas. Membrane or patients with lymph node infiltration (stage III; refs. 1, 2). cytoplasmic staining for CXCR4 protein was absent in 56% The migration of tumor cells to a secondary site from their of the tumors. The positive cases were divided into three score primary location one is a key event in cancer metastasis. Many classes according to their staining: low in 15 cases (21%), studies suggest the mechanisms used for homing of leukocytes moderate in 10 (14%), and high in 6 (8%). After a median and hematopoietic progenitors may be appropriated for the follow-up of 38 months, 26 patients progressed (16 of 26 dissemination of tumors via the bloodstream and lymphatics. expressed CXCR4) and 19 died (12 of 19 expressed CXCR4). CXCR4 is a seven-domain trans-membrane chemokine recep- The CXCR4 expression on tumor cells was correlated with an tor predominantly expressed on lymphocytes where it activates unfavorable prognosis with a median disease-free and overall chemotaxis. The CXCR4/CXCL12 pathway has been recently survival of 22 and 35 months, respectively. The hazard ratios involved in stimulating the metastatic process of many different of relapse and death, compared with patients with CXCR4- neoplasms (3), where CXCR4 is able to activate a plethora of negative tumors, were 2.5 (95% confidence interval, 1.2-6.1) phenomena such as chemotaxis, invasion, angiogenesis, and and 3.1 (95% confidence interval, 1.1-7.2), respectively. proliferation. These phenomena have been shown with different Median time-to-event (progression and survival) was not methodologic approaches in breast cancer (4, 5), small cell lung cancer (6), ovarian cancer (7, 8), pancreatic cancer (9), lymphoma (10–12), neuroblastoma (13), glioblastoma (14, 15), Received 9/14/04; revised 12/3/04; accepted 12/13/04. renal cell carcinoma (16), thyroid cancer (17–19), rabdomyo- Grant support: Associazione Italiana per la Ricerca sul Cancro. sarcoma (20), prostate cancer (21), and colorectal cancer (22). The costs of publication of this article were defrayed in part by the The role of CXCR4 receptor in the biology of malignant payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to melanoma has been previously described. CXCR4 expression indicate this fact. was detected in human melanoma cell lines and in patient Note: S. Scala and A. Ottaiano contributed equally. samples. The authors described expression of CXCR3 and Requests for reprints: Stefania Scala, Department of Clinical CXCR4 receptors regulating cell motility during invasion as well Experimental Immunology, National Cancer Institute, via Mariano Semmola, 80131, Naples, Italy. Phone: 39-081-5903678; Fax: 39-081- as cell proliferation and survival (23). Insights into this 5903820; E-mail: [email protected]. mechanism showed that CXCL12 promotes melanoma cell D2005 American Association for Cancer Research. invasion through activation of membrane type I–bound matrix

metalloproteinase and small GTPase proteins RhoA, Rac1, and negative for CXCR4 expression. Neoplastic cells with CXCR4 CDC42 (24). Interestingly, CXCR4 expression on melanoma cytoplasmatic and/or membrane immunohistochemical expres- cells is increased by transforming growth factor-h1 and specific sion were considered positive cells. Macrophage positivity was inhibition with anti-transforming growth factor-h antibodies used as adequate internal positive control for each case, to blocked CXCR4 expression and melanoma cell invasion toward validate technical procedure. Staining was categorized into four CXCL12 (24). Overexpression of CXCR4 dramatically enhanced semiquantitative classes based on the rate of stained (positive) the metastatic accumulation of B16 melanoma cells in mice lungs tumor cells: absence of staining, <10% positive cells (low), 10% (25). Expression of functional CXCR4 receptor on melanoma to 50% positive cells (moderate), and >50% positive cells (high). cells indicates that it might contribute to cell motility during Topographical expression of CXCR4 was also evaluated. invasion of lymph nodes as well as to regulation of cell Prevalent expression in radial or vertical component was proliferation and survival. considered when expression in such component was twofold The aim of the present study was to evaluate the expression compared with the other. Moreover, CXCR4 expression was of CXCR4 in malignant melanoma patients and its possible evaluated in SLN. In particular, cases were considered positive prognostic value in 71 human primary melanoma samples and when >10% of metastatic cell showed cytoplasmatic and/or relative sentinel lymph nodes (SLN). membrane CXCR4 expression. Slides were evaluated by two blinded observers (G.B. and R.F.); discordant cases were discussed and concordance was then achieved. MATERIALS AND METHODS Statistical Analysis. Correlations between CXCR4 Patients were seen at the Clinical Immunology Department expression, baseline patient features, and tumors were studied of the National Cancer Institute of Naples from 1996 to 2003. by contingency tables and the m2 test. DFS was defined as the The histologic sections were reviewed by two expert patholo- time elapsed from the date of the initial diagnosis to the gists (G.B. and R.F.) to verify the histologic diagnosis of appearance of local relapse or distant metastasis or death, malignant melanoma, before performing immunohistochemistry. whichever occurred first. Overall survival (OS) was defined as Standard therapeutic strategies were applied for these patients, the time elapsed from the date of the initial diagnosis to death or according to SLN status. Patients with soft tissue recurrence or to the date of the last available information on vital status. local disease were treated with surgery. Patients with advanced Kaplan-Meier product limit method was applied to draw DFS disease were mainly treated with palliative chemotherapy. A few and OS curves. Univariate analysis was done with the log-rank selected patients underwent pulmonary metastasectomy in the test. Cox proportional-hazards regression was used to analyze case of a solitary lesion or single lobe involvement and adequate the effect of several risk factors on DFS and OS. The probability disease-free survival (DFS, at least 1 year). Locations of the of the end point (hazard) was estimated with a coefficient: primary tumor were grouped in four classes: head/neck, trunk, exp(b). The quantity exp(b) can be interpreted as the extremities, and feet. Breslow’s tumor thickness in the studied instantaneous relative risk of an event, at any time, for an samples was selected to be >1 mm. SLN status was recorded. In individual with the risk factor present compared with an particular, the presence of metastasis was considered as follows: individual with the risk factor absent, given both individuals micrometastasis if their diameter was V2 mm, macrometastasis if are the same on all other covariates. Risk factors (covariates) the diameter was >2 mm. were considered dichotomous (male versus female, age <70 Immunohistochemistry. Two serial 5-Am sections of versus age z70, ulceration versus no ulceration, SLN involved formalin-fixed, paraffin-embedded cutaneous melanoma samples versus SLN not involved, Breslow V 2.00 mm versus >2.00 mm, were stained one by standard H&E and the other by the biotin- CXCR4 absent versus CXCR4 expressed). Ninety-five percent streptavidin-peroxidase method (YLEM). Deparaffinized sec- confidence intervals (95% CI) of hazard ratios are also reported. tions were microwaved in 1 mmol/L EDTA (pH 8.0) for two cycles of 5 minutes each to unmask epitopes. After treatment with 1% hydrogen peroxidase for 30 minutes to block endogenous RESULTS peroxidases, the sections were incubated with monoclonal Patient’s Features. From 1996 to 2003, 71 specimens antibodies (anti-CXCR4, clone 44716, R&D systems, Minneap- from surgically resected primary cutaneous malignant mela- olis, MN) for 2 hours at room temperature. The sections were then nomas with Breslow tumor thickness >1 mm were tested for the incubated with biotin-labeled secondary antibody (1:30) for 30 CXCR4 expression. All resections and SLN biopsies were done minutes and with streptavidin-peroxidase (1:30) for 10 minutes. by the same surgical team (Surgical Oncology B, National Slides were stained for 10 minutes with AEC chromogen Cancer Institute of Naples). Three patients referred to our center (DAKO, Milan, Italy) and then counterstained with hematoxylin, only at the moment of the diagnosis and there was no adequate washed, and mounted in waterish medium. The dilutions of the follow-up to allow time-to-event analysis. Characteristics of all monoclonal antibody, biotin-labeled secondary antibody, and patients and primitive melanoma are summarized in Table 1. streptavidin-peroxidase were made with PBS (pH 7.4) containing Overall, 31 of 71 tumors (43.6%) expressed CXCR4. Median 5% bovine serum albumin. All series included positive controls age was 53 years; 14 patients ages z70 years. Genders were of well-characterized sections (melanomas and breast cancer). equally represented. Melanomas located in the trunk or Negative controls were obtained by substituting the primary extremities accounted for 61.9% and 29.5% of the cases, antibody with a mouse myeloma protein of the same subclass, at respectively. Twenty-nine patients (40.8%) had Breslow tumor the same concentration as the monoclonal antibody. All controls thickness >1.00 and V 2.00 mm, 26 (36.6%) patients >2.00 and gave satisfactory results. In all cases, normal epithelial cells were <4.00 mm, and 16 patients >4.00 mm. Clark level was II in one

Table 1 Patient characteristics CXCR4 expression n (%) Negative Low Moderate High P Median age (range), y 53 (20-80) Age <70 57 (80.3) 32 (56.1) 11 (19.2) 9 (15.7) 5 (8.8) 0.816 z70 14 (19.7) 8 (57.0) 4 (28.5) 1 (7.1) 1 (7.1) Gender Males 38 (53.5) 24 (63.1) 8 (21.0) 3 (7.9) 3 (7.9) 0.316 Females 33 (46.5) 16 (48.4) 7 (21.2) 7 (21.2) 3 (9.1) Location Head/neck 2 (2.8) 1 (50.0) — 1 (50.0) — 0.976 Trunk 44 (61.9) 25 (56.8) 9 (20.4) 6 (13.6) 4 (9.0) Extremities 21 (29.5) 11 (52.3) 5 (23.8) 3 (14.2) 2 (9.5) Feet 4 (5.6) 3 (75.0) 1 (25.0) — — Breslow 1.01 and 2.00 29 (40.8) 17 (58.6) 4 (13.7) 5 (17.2) 3 (10.3) 0.835 2.01 and 4.00 26 (36.6) 14 (53.8) 6 (23.1) 4 (15.3) 2 (7.7) >4.00 16 (22.5) 9 (56.2) 5 (31.2) 1 (6.2) 1 (6.2) Clark II 1 (1.4) — — — 1 (100) 0.288 III 24 (33.8) 16 (66.0) 3 (12.5) 3 (12.5) 2 (8.3) IV 46 (64.7) 24 (52.2) 12 (26.0) 7 (15.2) 3 (6.5) Ulceration Yes 33 (46.5) 20 (60.6) 6 (18.2) 5 (15.1) 2 (6.0) 0.503 No 38 (53.5) 20 (52.6) 9 (23.6) 5 (13.1) 4 (10.5) Nodular Yes 32 (45.1) 18 (56.2) 6 (18.7) 4 (12.5) 4 (12.5) 0.820 No 39 (54.9) 22 (56.4) 9 (23.1) 6 (15.4) 2 (5.1) SLN status Not involved 46 (64.7) 30 (65.2) 5 (10.8) 7 (15.2) 4 (8.7) 0.073 Involved 25 (35.3) 10 (40.0) 10 (40.0) 3 (12.0) 2 (8.0) Micrometastasis 12 Macrometastasis 13

case (1.4%), III in 24 cases (33.8%), and IV in 46 cases (64.7%). CXCR4 Expression: Correlation with Outcome. At the Ulceration was present in 33 cases (46.5%) and nodular time of this analysis, after a median follow-up for alive patients appearance in 32 lesions (45.1%). Twenty-five SLNs (35.3%) of 38 months, 25 patients progressed, and 19 died. Univariate were infiltrated by malignant melanoma; in particular, 12 were analysis of prognostic factors for DFS and OS is summarized in micrometastasis. No correlation was found between CXCR4 Table 2. SLN metastases, presence of ulceration and CXCR4 expression and clinicopathologic characteristics. expression had a significant prognostic value for DFS and OS. CXCR4 Expression in Primary Melanoma: Pattern of The predictive ability of ulceration and CXCR4 expression for Expression. Out of 71 melanoma specimens tested for the DFS, and of SLN metastases and CXCR4 expression for OS CXCR4 expression 31 (43.7%) stained positive for CXCR4. were confirmed by a multivariate analysis Table 3) adjusted by The positive cases were divided into three score classes age, sex, Breslow, SLN status, presence of ulceration, and according to their staining: low in 15 cases (48%), moderate CXCR4 expression. Graphic pattern of Kaplan-Meier estimated in 10 (32%), and high in 6 (19%). Figure 1 shows curves (Fig. 2A and B) suggest that prognosis is unfavorable for representative positive staining of human melanoma (A-F) patients expressing CXCR4. For CXCR4-positive tumors and sentinel lymph nodes (G and H). In all melanoma median DFS and OS were 22 and 35 months, respectively; specimens, normal skin tissue resulted in negative staining. and hazard ratios of relapse and death as compared with patients Melanoma samples showed mainly a subepidermal expression with CXCR4-negative tumors of 2.5 (95% CI, 1.2-6.1) and 3.1 (Fig. 1A and B; high and low expression group, respectively). (95% CI, 1.1-7.2). A small subgroup of 10 of 31 positive cases expressed Lymph node spreading represents the most common way of CXCR4 also in the vertical growth phase (Fig. 1Cand D; metastasis in malignant melanoma. Expression of CXCR4 in SLN high and low expression group, respectively). Different biopsies was done in 56 of 71 primary melanoma. As described, cellular localization of CXCR4 were detected; the staining 25 SLNs were infiltrated by melanoma. CXCR4 expression was detected in 10 of 17 (58.8%) evaluable and melanoma-positive was cytoplasmic (Fig. 1E) and/or membrane (Fig. 1F). The SLNs. staining mainly involved cytoplasms. In a few cases, CXCR4 was exclusively detected at the membrane level (8 of 31, 25%). CXCR4 expression was detected in involved lymph DISCUSSION nodes in both diffuse metastasis (Fig. 1G) and micro- In the present study, we investigated the potential role of metastatis (Fig. 1H). CXCR4 as a prognostic factor in human melanoma. CXCR4

Fig. 1 CXCR4 expression in primary melanoma and SLN metastases. CXCR4 immunohistochemical staining in malignant melanoma (A-F). Subepidermal areas: high expression, 200Â (A) and low expression, 200Â (B). CXCR4 expression in vertical growth face: high expression, 100Â (C) and low expression, 100Â (D). CXCR4 expression at cytoplasmatic localization (E, 600Â). Cytoplasmic and/or membrane expression (F, 400Â). CXCR4 immunohistochemical staining in sentinel lymph node metastasis (G, H). CXCR4 staining in macrometastasis (G, 100Â) and micrometastases (H, 200Â).

expression was detected in 31 of 71 primary cutaneous melanoma receptors on cancer cells with metastatic potential and chemo- selected to have a Breslow > 1 and in 10 of 17 evaluable kine gradients in target organs (26). Melanoma cell lines express melanoma involved lymph nodes. Interestingly, the expression of functional chemokine receptors CCR7, CXCR3, CCR10, and CXCR4 was able to predict the prognosis and this ability holds CXCR4 which are able to activate cell motility during invasion, true both for DFS (P = 0.0154) and OS (P = 0.0009). To the best cell proliferation, and survival (23–25). CXCR4 staining mainly of our knowledge, this is the first report demonstrating the involved cytoplasms. In a few cases, CXCR4 was exclusively prognostic role for CXCR4 in malignant melanoma. detected at the membrane level (8 of 31, 25%). It is noteworthy It has been postulated that organ specific metastasis might that four of eight patients showing a specific membrane staining be governed, in part, by interactions between chemokine died at the observation time point (38 months of follow-up).

Table 2 Univariable analysis DFS OS Covariate Events/patients Median DFS (mo) P Events/patients Median OS (mo) P Age <70 18/54 NR 0.12 13/54 67 0.08 z70 8/14 36 6/14 NR Gender Male 12/38 NR 0.32 9/38 99 0.83 Female 14/30 37 10/30 67 Breslow 1.01 and 2.00 8/27 NR 0.63 5/27 NR 0.59 2.01 and 4.00 10/25 NR 7/25 99 >4.00 8/16 38 7/16 67 SLN status Not involved 11/43 NR 0.0054 6/43 99 0.0001 Involved 15/25 26 13/25 28 Ulceration Yes 17/36 29 0.0027 13/36 68 0.049 No 9/32 69 6/32 NR CXCR4 expression Absent 10/38 NR 0.0154 7/38 NR 0.0009 <10% of cells 7/15 37 5/15 67 10-50% of cells 5/9 27 3/9 56 >50% of cells 4/6 19 4/6 21 Abbreviation: NR, not reached.

However, we will not approach any conclusions since the (29) and with clinical outcome in 61 patients with completely number of examined events is too small. The cytoplasmatic resected non–small cell lung cancer (30). In melanoma-forced detection of the CXCR4 receptor was previously described in expression of CXCR4 in B16 melanoma cells enhances cancers of the breast (29), lung (30), colon (31–33), prostate pulmonary metastasis, but metastasis to lymph nodes, liver, or (34), pancreas (9), and in CD34+ hematopoietic cells (35). It kidney are not affected (25). Interestingly, we detected CXCR4 might represent a functional status of the receptor because the expression in 10 of 17 evaluable melanoma involved lymph binding to the specific ligand induce receptor internalization (36) nodes; only seven involved lymph nodes derived from primary and specific signals are required in order to express CXCR4 on melanoma expressed CXCR4 suggesting a role of other chemo- cell surface (22). kine receptors such as CCR7 (27) and CXCR3 (28) in lymph The presence of metastasis in the SLN is important for node metastasis. staging and prognosis (1, 2). The metastatic potential of primary Our data show that the higher the expression of CXCR4 the melanoma is considerably higher than that of other primary solid worst the prognosis of patients supporting the inhibition of tumors when comparing the size of primary lesion. Malignant CXCR4 as a possible therapeutic target in melanoma. Blocking melanoma metastasizes frequently to regional tumor-draining anti-CXCR4 monoclonal antibody (4) or to inhibitory peptide lymph nodes, preferentially via the lymphatics. Notably, only 8 of (37) specifically inhibited metastasis to the lungs in breast cancer 27 melanomas with Breslow between 1 and 2 mm (T2 tumors at models. Inhibition of the CXCR4/CXCL12 axis was previously tumor-node-metastasis, American Joint Committee on Cancer studied since CXCR4 plays an important role in HIV type-1 2002 staging system) relapsed in lymph nodes; six out of these (HIV-1) infection (38, 39). AMD3100 is a byciclam noncompet- tumors expressed CXCR4. These observations might suggest that itive antagonist of CXCL12 binding to CXCR4. Rubin showed thin melanomas CXCR4-positive cells are more prone to move that systemic administration of AMD3100 inhibits intracranial toward the draining lymph node. Previous reports described a growth of glioblastoma and medulloblastoma xenograft increas- correlation between CXCR4 expression and lymph node ing apoptosis and decreasing cell proliferation (40). AMD3100 metastasis in 79 surgically resected invasive ductal carcinomas seemed safe and effective in patients affected by multiple

Table 3 Multivariable analysis DFS OS Covariate exp(b) 95% CI P exp(b) 95% CI P Age 1.03 À0.002 to 0.06 0.0698 1.02 À0.015 to 0.06 0.2488 Gender 1.30 À0.61 to 1.14 0.5553 0.88 À1.21 to 0.96 0.8846 Breslow 1.00 À0.09 to 0.10 0.9594 0.97 À0.12 to 0.07 0.9741 SLN status 1.69 À0.35 to 1.40 0.2399 5.45 0.56 to 2.82 0.0035 Ulceration 3.16 0.24 to 2.07 0.0140 1.66 À0.57 to 1.60 0.3602 CXCR4 status 1.65 0.10 to 0.90 0.0148 2.07 0.14 to 1.31 0.0150 NOTE. exp(b): see Materials and Methods.

ACKNOWLEDGMENTS We thank the Melanoma Cooperative Group of Naples: Aprea P., Ascierto P.A., Ayala F., Beneduce G., Bosco L., Botti G., Caraco` C., Castello G., Celentano E., Chiofalo M.G., Comella G., Daponte A., De Marco M.R., Graziano F., Leonardi E., Lombardi M.L., Marfella A., Montella M., Mori S., Mozzillo N., Napolitano M., Peluso G.F., Perrone F., Pirozzi G., Satriano S.M.R., Scala S., and Tatangelo F. (National Cancer Institute, G. Pascale, Naples, Italy); Satriano R.A., Vozza A., Ruocco V (2nd University of Naples, Italy); Palmieri G. [Division of Cancer Genetics, ICB-Consiglio Nazionale delle Ricerche, Alghero (SS), Italy]; Cossu A., Tanda F. (University of Sassari, Italy); Dr. M.R. Ventura for data management; J. Bryce and G. Liguori for technical assistance.

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Stefania Scala, Alessandro Ottaiano, Paolo Antonio Ascierto, et al.

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