The Korean Journal of Pathology 2008; 42: 358-64

Association of CXCR4 Expression with Metastasis and Survival among Patients with Non-small Cell Lung Cancer

Joon Seon Song∙Jin Kyung Jung Background : Expression of CXCR4 receptor, initially described to be involved Jong Chul Park∙Dong Kwan Kim1 in the homing of in inflammatory tissue, on breast cancer cell lines is associated Se Jin Jang with the development of lung metastases. In the present study, we evaluated CXCR4 expres- sion in patients with non-small cell lung cancer (NSCLC). Methods : Tissue microarray blocks Departments of Pathology and 1Thoracic were constructed from 408 formalin-fixed, paraffin-embedded NSCLC samples and analyzed and Cardiovascular Surgery, University via immunohistochemical staining. Results : We observed CXCR4 expression in 214 (66.3%) of Ulsan College of Medicine, Asan of the 323 tumors with cytoplasmic or nuclear staining patterns. These tumors were then divid- Medical Center, Seoul, Korea ed into 109 negative, 166 weak-positive and 48 strong-positive expression groups. Strong expression of CXCR4 correlated with NSCLC recurrence (p=0.047) and distant metastasis Received : August 21, 2008 (p=0.035). However, lymph node metastasis (p=0.683) and locoregional recurrence (p=0.856) Accepted : October 22, 2008 were not associated with CXCR4 expression. Interestingly, the median overall survival times Corresponding Author relative to CXCR4 expression were 71 months in the CXCR4-negative group, 43 months in Se Jin Jang, M.D. the weakly positive CXCR4 group and 23 months in the strongly positive CXCR4 group. Strong- Department of Pathology, University of Ulsan College ly positive CXCR4 staining was associated with significantly worse outcomes (p=0.005, log- of Medicine, Asan Medical Center, 388-1 Pungnap-dong, Songpa-gu, Seoul 138-736, Korea test). Conclusions : Expression of CXCR4 was associated with distant NSCLC metas- Tel: 02-3010-5966 tases and shorter survival times. Fax: 02-472-7898 E-mail: [email protected] *This research was supported by research grant R01- 2004-000-10670-0 from the KOSEF. Key Words : Non-small cell lung carcinoma; CXCR4 receptors; Metastasis

Non-small cell lung cancer (NSCLC) is the main contributor tor-1 or SDF-1) binds to CXC 4 (CXCR4)9 to cancer-related mortality in Asian and Western populations. and recruits CD34+ hematopoietic progenitor cells, megakary- Mortality from NSCLC largely results from metastases in the ocytes, B cells, and T cells.9 Originally discovered as the puta- regional lymph nodes, liver, adrenal gland, contralateral lungs, tive coreceptor for lymphotropic strains of human immunode- brain, and .1,2 Cellular processes involved in the ficiency virus (HIV),9 CXCR4 binds specifically to CXCL12. migration and metastasis of carcinoma cells share many simi- Cancer metastases display distinct metastatic patterns that larities with the leukocyte trafficking that occurs during inflam- preferentially involve the regional lymph nodes, bone marrow, matory processes, which is largely regulated by .3-6 lung, and liver.10 Recent reports have provided new insights Therefore, chemokines and their receptors are considered poten- into the mechanisms of organ-specific metastases, particularly tial predictive markers for metastasis and the metastatic sites of with regards to CXC chemokines.11 The CXCR4 and its lig- various types of tumors.7 and, CXCL12, are the most highly expressed chemokine recep- The chemokine superfamily includes at least 20 receptors tors in human breast cancer and in organs affected by breast and 40 ligands.4-6 Chemokine ligands can be separated into cancer metastasis, respectively. Moreover, the in vivo neutraliza- four categories, depending on whether they express the C, CC, tion of CXCR4 significantly inhibits breast cancer metastases CXC, or CX3C amino acid motif in their N termini. The CXC in an organ-specific manner. chemokines bind to a family of G--coupled serpentine Thus, we examined CXCR4 expression in patients with NS- (seven transmembrane-spanning) receptors, known as CXC CLC via immunohistochemical staining and analyzed clinico- chemokine receptors (CXCRs).5,8 Six CXCRs have been identi- pathological parameters such as lymph node metastasis, distant fied to date.4,5 In particular, CXCL12 (stromal cell-derived fac- metastases and survival times. Our findings reveal that high

358 CXCR4 Expression in Non-small Cell Lung Cancer 359

levels of CXCR4 expression strongly correlate with recurrence coated glass slides. The sections were deparaffinized through a of NSCLC, distant metastases and shorter survival times. series of xylene and rehydrated through graded alcohol to dis- tilled water. After antigen retrieval, sections were immersed in methanol containing 0.3% hydrogen peroxidase for 10 min to MATERIALS AND METHODS block endogenous peroxidase activity. Sections were then incu- bated in blocking serum to reduce nonspecific binding and incu- Case selection bated overnight at 4℃ in a 1:400 dilution of primary rabbit polyclonal anti-CXCR4 antibody (Abcam, Cambridge, UK). We examined 408 consecutive patients with NSCLC who Results were visualized using diaminobenzidine. The sections underwent curative pulmonary resections between 1995 and were lightly counterstained with hematoxylin and mounted. 1999. Tumors were staged according to the International Union CXCR4 expression was evaluated at the cytoplasmic and at Against Cancer’s tumor node-metastasis (TNM) classification the nuclear level. The percentage of positive cells was taken into and histologically subtyped according to WHO guidelines.12 consideration to define the score. The following score was applied The mean age at the time of surgery was 63.3 years (range=29 for the immunohistochemical results: 0, no staining and <10% to 86 years). The majority of patients were male (321 of 408, of tumor cells; 1, 10-50% of tumor cells; 2, >50% of tumor 78.7%). Histological samples included 200 (49.0%) squamous cells. cell carcinomas, 169 (41.5%) adenocarcinomas and 39 (9.5%) minor histological subtypes. Regardless of histological type, 110 Statistical analyses tumors (26.7%) were well differentiated, 201 tumors (49.3%) were moderately differentiated and 97 tumors (23.8%) were Pearson’s χ2 test (using a linear by linear association) was poorly differentiated, according to a modified Brodies’ grading used to evaluate with the relationships between clinicopatho- system. Lymph node metastases were identified in 194 (46.5%) logic variables and CXCR4 expression. In addition, multivari- patients at the time of surgery. Follow-up data were obtained ate analysis was performed using multiple logistic regression from medical records, the Department of Thoracic and Cardio- analysis. Survival was estimated using the Kaplan-Meier method vascular Surgery database at the Asan Medical Center and tele- and the resulting curves were compared via log-rank tests. Dis- phone interviews. Survival status and outcome data were avail- ease-free survival was defined as the length of time from the date able for 371 patients (representing a 9.1% follow-up loss). The of surgery to either 1) the date of death from lung cancer or 2) median follow-up period was 47.7 months. Disease recurrence to the date of the most recent follow-up. Multivariate survival was confirmed by radiological studies and bronchoscopic exam- analysis was performed using the Cox proportional hazard regres- ination via biopsy and surgery. Recurrence was defined as either sion model. All calculations were performed using SPSS statis- locoregional or distant recurrence in the liver, adrenal gland, tical software (version 12.0, Statistical Package for Social Sci- contralateral lungs, brain, and bone marrow. ence) and results were considered statistically significant when the p-values were <0.05. Tissue microarrays

Glass slides containing 408 NSCLC samples were reviewed RESULTS and representative tumor areas were marked on the correspond- ing paraffin blocks. Using a tissue arrayer device (Beecher Instru- CXCR4 expression ments, Sun Prairie, WI, USA), two cylinders (each 1.5 mm in diameter) were punched from representative tumor areas with- We assessed CXCR4 expression in NSCLC tissues and in in each donor block and re-embedded into recipient blocks. Each adjacent healthy lung tissues. Expression of CXCR4 was detect- tissue microarray (TMA) block included 29 NSCLC samples. ed in the cytoplasm and nuclei of tumor cells, as well as in tumor- infiltrating lymphocytes, but not in normal lung tissues (Fig. 1). Immunohistochemical staining Eighty-five (20.8%) cases were excluded from this analysis due to the loss of tissue cores during the sectioning and immunos- TMA blocks were cut as 4-μm-thick and mounted on pre- taining processes. 360 Joon Seon Song∙Jin Kyung Jung∙Jong Chul Park, et al.

A B C

D E F

Fig. 1. Immunohistochemical staining of pulmonary adenocarcinoma (shown in the upper row) and squamous cell carcinoma (shown in the lower row) samples reveals CXCR4 expression in the nuclei and cytoplasm of cancer cells. From left to right, the tumor cells are neg- ative (score=0), weakly positive (score=1), and strongly positive (score=2) for CXCR4, respectively.

Overall, CXCR4 expression was observed in 214 (66.3%) of p=0.042), expression was not related to tumor stage at the time 323 interpretable NSCLC tissue samples. Of the CXCR4-posi- of surgery (p=0.217). Lymph node metastases were observed in tive cases, 166 (77.6%) were weakly positive for CXCR4 (score= 156 (46.5%) of 323 patients at the time of surgery. There were 1) and 48 (22.4%) were strongly positive (score 2) for CXCR4. no significant differences in CXCR4 expression between patients in node-negative and node-positive groups (p=0.683). The rela- Correlation of CXCR4 expression with clinicopathological tionships between CXCR4 expression and various clinicopatho- variables logical factors are shown in Table 1.

To evaluate the significance of CXCR4 expression in patients Correlation of CXCR4 expression with disease recurrence with NSCLC, we compared the clinicopathological characteris- tics of patients whose samples received scores of 0, 1, and 2. Age During the follow-up period, 124 (39.0%) of 318 patients and sex were not significantly different among the three groups experienced cancer recurrences. Of these, 14 patients (4.4%) (p=0.434 and 0.314 respectively). experienced locoregional recurrences and 116 (36.5%) experi- Of the 160 samples of squamous cell carcinoma included in enced distant metastases. Interestingly, the strongly positive the analysis, 58 (36.3%), 79 (49.4%), and 23 (14.9%) were neg- CXCR4 group experienced more frequent tumor recurrence ative, weakly positive and strongly positive for CXCR4 expres- (p=0.047), especially with regards to distant metastases (p= sion, respectively. Among the 145 samples of adenocarcinoma, 0.035), than did the negative or weakly positive groups. How- 44 (30.3%), 78 (53.8%), and 23 (15.9%) were negative, weak- ever, there were no significant differences in locoregional recur- ly positive and strongly positive for CXCR4 expression, respec- rence according to CXCR4 expression (p=0.856) (Table 2). Dif- tively. There were no significant differences in CXCR4 expres- ferences in recurrence and distant metastases between patients sion with regards to histologic subtype (p=0.945) (Table 1) or in the negative and strongly positive groups were more evident. tumor differentiation (p=0.148). In addition, CXCR4 expres- Multivariate analysis revealed that patients in the strongly ex- sion was observed in 70% (63 of 90 samples), 66.8% (100 of pressed CXCR4 group experienced more frequent recurrence 152 samples), and 64% (51 of 81 samples) of well-, moderately (p=0.035, OR=2.341, 1.061-5.165) and distant metastases and poorly differentiated carcinomas, respectively. Although (p=0.031, OR=2.434, 1.087-5.448) (Table 2). CXCR4 was expressed more strongly in larger tumors (>3 cm, CXCR4 Expression in Non-small Cell Lung Cancer 361

Table 1. Clinicopathologic features associated with CXCR4 Table 2. Association of CXCR4 expression with tumor recur- expression rence

CXCR4 staining CXCR4 staining p- p- Total Weak Strong Total Weak Strong Negative, value Negative, value N=323 (%) positive, positive, N=323 (%) positive, positive, N=109 (%) (<0.05) N=109 (%) (<0.05) N=166 (%) N=48 (%) N=166 (%) N=48 (%) Age (years) Recurrence ≤50 37 (11.5) 11 (39.7) 19 (51.4) 7 (18.9) Yes 124 (39.0) 37 (29.8) 61 (49.2) 26 (21.0) 51-60 75 (23.2) 26 (34.7) 37 (49.3) 12 (16.0) No 194 (61.0) 69 (35.6) 103 (53.1) 22 (11.3) 0.047 61-70 128 (39.6) 48 (37.5) 59 (46.0) 21 (16.4) Locoregional recurrence ≥71 83 (25.7) 24 (28.9) 51 (61.5) 8 (9.6) 0.434 Yes 14 (4.4) 5 (35.7) 7 (50) 2 (14.3) Sex No 304 (95.6) 101 (33.3) 157 (51.6) 46 (15.1) 0.856 Male 254 (78.6) 89 (35.0) 125 (49.2) 40 (15.8) Distant metastasis Female 69 (21.4) 20 (29.0) 41 (59.4) 8 (11.6) 0.314 Yes 116 (36.5) 33 (28.4) 59 (50.9) 24 (20.7) Histologic subtype No 202 (63.5) 73 (36.1) 105 (52.0) 24 (11.9) 0.035 SCC 160 (49.5) 58 (36.3) 79 (49.4) 23 (14.9) Adenocar- 145 (44.9) 44 (30.3) 78 (53.8) 23 (15.9) cinoma Table 3. Multivariate analysis* for CXCR4 strong expression Large cell 13 (4.0) 6 (46.2) 6 (46.2) 1 (7.6) p-value Odds ratio 95% CI carcinoma Others* 5 (1.5) 1 (20) 3 (60) 1 (20) 0.945 Lymph node metastasis 0.530 2.219 0.201-22.579 Histologic grade Recurrence 0.035 2.341 1.061-5.165 WD 90 (27.9) 27 (30.0) 49 (54.4) 14 (15.6) Local recurrence 0.624 0.602 0.080-4.562 MD 152 (47.0) 52 (34.2) 72 (47.4) 28 (18.4) Distant metastasis 0.031 2.434 1.087-5.448 PD 81 (25.1) 30 (37.0) 45 (55.6) 6 (7.4) 0.148 *, Multiple logistic regression analysis. Stage CI, confidence interval. 1 145 (44.9) 48 (33.1) 80 (55.2) 17 (11.7) 2 71 (22.0) 28 (39.4) 34 (47.9) 9 (12.7) Table 4. Multivariate analysis* of prognostic factors for overall 3 102 (31.6) 32 (31.4) 49 (48.0) 21 (20.6) survival 4 4 (1.2) 1 (25.0) 2 (50.0) 1 (20.0) 0.217 Size (cm) p-value Hazard ratio 95% CI ≤3 137 (42.4) 52 (38.0) 71 (51.8) 14 (10.2) Age 0.002 1.669 1.210-2.303 >3 186 (57.6) 57 (30.6) 95 (51.1) 34 (18.3) 0.042 Sex 0.306 0.811 0.544-1.210 Lymph node metastasis Size 0.032 1.438 1.031-2.004 Yes 156 (48.3) 56 (35.9) 71 (45.5) 29 (18.6) Stage <0.001 1.544 1.295-1.842 No 167 (51.7) 53 (31.7) 95 (56.9) 19 (11.4) 0.683 Histologic subtype <0.001 1.226 1.110-1.355 Others* include 3 adenosquamous carcinoma, 1 mixed type and 1 Differentiation 0.219 1.153 0.919-1.446 sarcomatoid carcinoma. CXCR4 0.012 1.351 1.096-1.709 SCC, squamous cell carcinoma; WD, Well differentiated; MD, Moderate- *, Cox regression model. ly differentiated; PD, poorly differentiated. CI, confidence interval.

Relationship of CXCR4 expression with disease-free and strongly expressed NSCLC group were significantly shorter than overall survival times those of patients in the negative or weakly expressed NSCLC groups. Overall, median survival times were significantly dif- Kaplan-Meier survival curves were constructed to assess the ferent among the three groups (p=0.005, log-rank test) (Fig. prognostic significance of CXCR4 expression. Patients with 2B). Multivariate analysis by Cox regression revealed that age CXCR4-positive NSCLC experienced significantly shorter dis- (p=0.002), tumor size (p=0.032), histological type (p<0.001) ease-free survival times than did patients with CXCR4-nega- and pTMN stage (p<0.001) were significant prognostic indica- tive NSCLC. The median disease-free survival times of patients tors of overall patient survival. There were no significant corre- in the CXCR4-negative, weakly expressed and strongly expressed lations between prognosis and other clinicopathological features groups were 71 months, 36 months, and 16 months, respectively, (Table 4). However, strong expression of CXCR4 was an inde- and these differences were statistically significant (p=0.001, log- pendent predictor of poor survival (HR=1.351, 95% CI=1.096- rank test) (Fig. 2A). The overall survival times of patients in the 1.709, p=0.012). 362 Joon Seon Song∙Jin Kyung Jung∙Jong Chul Park, et al.

1.0 1.0 CXCR4 CXCR4

Negative Negative 0.8 Weak positive 0.8 Weak positive Strong positive Strong positive

0.6 0.6

0.4 0.4 Overall survival Disease free survival

0.2 0.2 p=0.001, log-rank p=0.005, log-rank

0.0 0.0 0 25 50 75 100 125 0 25 50 75 100 125 Time after surgery (months) A Time after surgery (months) B

Fig. 2. Disease-free and overall survival times, according to CXCR4 expression. Strong CXCR4 expression is related to shorter periods of disease-free survival (p<0.001, log rank) (A). Strong CXCR4-positive staining is associated with a significantly worse outcome (B).

DISCUSSION is associated with more frequent LN metastases, worse differen- tiation and shorter survival times when compared with cytomem- The expression of CXCR4 in malignant cells is a well-known branous expression of CXCR4.20 Our study also revealed that phenomenon. Tumor cells from at least 23 types of human can- nuclear expression of CXCR4 is associated with distant recur- cers (including those of epithelial, mesenchymal and hematopoi- rence after surgery and worse prognoses. Although our study etic origin) are known to express CXCR4.13 In healthy epithe- dose not address the mechanism of nuclear localization, several lial cells of the breast, colon, ovary, prostate, and lung, expres- previous studies provided molecular mechanisms. Stromal cell- sion of CXCR4 is absent or present at very low levels.11,14,15 Here, derived factor (SDF)-1αis thought to play a role in the molec- we demonstrate strong expression of CXCR4 in NSCLC tissues ular mechanism of nuclear CXCR4 expression. Stimulation of using immunohistochemiscal analysis. In contrast, we did not SDF-1αtriggers CXCR4 internalization via the activation of observe strong CXCR4 expression in healthy alveolar or bronchial G-protein-coupled receptor kinases the binding of β-arrestin epithelial cells. In this study, expression of CXCR4 was cytomem- and subsequent CXCR4 endocytosis.23 Endogenous SDF-1a branous and nuclear in patients with NSCLC, as has been report- binds newly synthesized CXCR4 and subsequently inhibits the ed for patients with hepatocellular carcinoma,16 breast cancer,17,18 translocation of CXCR4 to the cell surface.24 However, hypox- nasopharyngeal carcinoma19, and colorectal cancer.20 Expression ia-inducible factor-1a (HIF-1a) is known to play a critical role of CXCR4, a transmembrane receptor protein, was originally in tumor cell expression of CXCR4 during growth in hypoxic thought to be limited to the cytoplasm and plasma membrane conditions.25 Furthermore, decreased expression of HIF-1a is of cells, while nuclear expression was considered a nonspecific associated with cytoplasmic CXCR4 expression in metastatic byproduct of immunohistochemistry analysis. Indeed, CXCR4 tumors within the lymph nodes of patients with breast carcino- is predominantly expressed in the cytoplasm of healthy colon ma.17 It remains unclear why the immunostaining pattern of epithelial cells, where it participates in the renewal and main- CXCR4 differs among tumor types; however, SDF-1a and HIF- tenance of the epithelium.21 However, CXCR4 is frequently 1a may play key roles in determining the CXCR4 expression localized in the nuclei of cancer cells, where it may play a dis- pattern. tinct biological role. A previous study of patients with NSCLC In particular, CXCR4 plays an important role in the metastat- also demonstrated strong nuclear expression of CXCR4, and ic behavior and destination of solid cancers.11 The SDF-1a-CX- patients whose tumors exhibited CXCR4-positive nuclear stain- CR4 axis has been implicated in bone metastasis resulting from ing had significantly longer survival times than did patients prostate cancer26 and NSCLC, particularly with regards to tumor whose tumors had no nuclear expression of CXCR4.22 However, dissemination into the pleural space.27 Moreover, CXCR4 expres- in patients with colorectal cancer, nuclear expression of CXCR4 sion is predictive of recurrence and poor prognosis after neoad- CXCR4 Expression in Non-small Cell Lung Cancer 363 juvant chemotherapy in patients with breast, esophageal and REFERENCES colorectal cancers.28-30 However, the relationship between CXCR4 expression, metastatic behavior and NSCLC recurrence remains 1. Fidler IJ. 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