Annals of Surgical Oncology 14(11):3268–3273

DOI: 10.1245/s10434-007-9532-3

Overexpression of Interleukin-10 in Sentinel Lymph Node with Breast Cancer Sang Uk Woo, MD, PhD,1 Jeoung Won Bae, MD, PhD,1 Jung-Hyun Yang, MD, PhD,2,4 Jung Han Kim, MD,2 Seok Jin Nam, MD, PhD,2 and Young Kee Shin, MD, PhD3

1 Department of Surgery, Korea University College of Medicine, Seoul, Korea Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 3 Laboratory of Molecular Pathology, Department of Pharmacy, Seoul National University College of Pharmacy, Seoul, Korea 4 Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Ilwondong 50, Gangnamgu, Seoul, Korea135-710 2

Background: In breast carcinoma, identification of tumor cells in the sentinel lymph nodes is a predictor of the tumorÕs metastatic potential. Sentinel lymph node may be targeted not only by tumor cell metastasis but also by cytokines from the emergence of antitumor immune responses. Methods: Between February 2003 and February 2004, the investigator evaluated 38 cases that underwent sentinel lymph node biopsy at the Samsung Medical Center. Eighty paraffinembedded sections, 49 sentinel, and 31 nonsentinel lymph node, from breast carcinoma without lymphatic metastases were analyzed by real-time polymerase chain reaction to evaluate the cytokine profile (interferon-c, interleukin-2, interleukin-10 and interleukin-12) for the T cell response. Results: A higher expression of interleukin-10 was observed in sentinel lymph node than in nonsentinel lymph node (P = 0.03). The expressions of interferon-c, interleukin-2, and interleukin-12 were similar between sentinel and nonsentinel lymph node. Conclusions: Theses results indicate that T cell response was downregulated by interleukin10 overexpression in sentinel lymph node with breast cancer. Key Words: Breast carcinoma—Sentinel lymph node—Cytokine—Interleukin-10—Real-time quantitative polymerase chain reaction.

The current treatment concept for breast cancer involves eradication of all possible metastatic lymph nodes in the axillary basin. Sentinel lymph node biopsy (SLNB) allows identification of the first lymph node in which a primary tumor drains. In breast cancer, identification of tumor cells in SLNs is a predictor of the tumorÕs metastatic potential. Lymph

node is not only simply the route of tumor cell passage, but is also the principle site where adaptive, antigen-specific immune response are initiated. Lymph nodes regional to malignancies, such as melanoma and breast cancer, are immunosuppressed and the degree of that immune suppression is directly correlated to the proximity of the node to the primary tumor. Those view is based on morphologic and functional differences between nodes located close to and more remote the tumor.1,2 The mechanism of antigen presentation and recognition has been understood at the cellular and molecular levels in virus-infected or tumor antigen-specific immunity.

Received April 18, 2007; accepted June 27, 2007; published online: August 29, 2007. Address correspondence and reprint requests to: Jung-Hyun Yang, MD, PhD; E-mail: [email protected] Published by Springer Science+Business Media, LLC
2007 The Society of Surgical Oncology, Inc.

3268

3269

INTERLEUKIN-10 IN SENTINEL LYMPH NODE

This has revealed features of these cellular and molecular interactions that help to explain how cytotoxic T lymphocytes are able to carry out effective immunosurveillance and elimination of virus-infected or tumor cells.3 The function of cytokine is communication between cells by binding to specific receptors. They regulate cell differentiation and growth in the immune system. Interleukin 12 (IL-12) is produced at high levels by dendritic cells and drives naı¨ ve T cells to differentiate into interferon-c-producing T-helper 1 cells.4 T helper response represent IL-4 or IL-10 production.5 The T-cell immune system is composed of various networking systems. Therefore, cytokine profiles partially reflect the status of the T-cell immune system. The immunological roles of SLN have not been understood fully, particularly with regard to antigen-specific immunity. Therefore, we expect that cytokine expression in sentinel lymph nodes may show different immunological status in the absence of lymphatic metastases compared to the cytokine profile in nonsentinel lymph nodes. The purpose of study was to compare the cytokine expression profile from sentinel lymph nodes draining primary breast cancer with that from nonsentinel lymph nodes. Therefore, we evaluated the immunological difference between sentinel lymph node and nonsentinel lymph node in breast cancer.

METHODS AND MATERIALS Patients

by following the drainage of blue dye and using a handheld gamma ray detector. Residual radioactivity was confirmed by the gamma ray detector after complete excision of SLNs. An existence of metastasis was evaluated from intraoperative frozen sections with hematoxylin and eosin (H&E) staining. Permanent pathological examination was then carried out by more than 20 serial sections with H&E and cytokeratin stainings. mRNA Extraction Stored sentinel lymph node tissues were applied to total RNA extraction by using an RecoverAllTMTotal nucleic acid isolation kit (Ambion, Texas, USA). A paraffin-embedded block was sliced to 10 lm thickness. After deparaffinization, it was mixed with 400 ll of digestion buffer and 4 ll of protease and was incubated at 50C for three hours for RNA isolation. The prepared specimen was added to 480 ll isolation additive and vortex, and then passed through a filter cartridge and incubated for five minutes. Washing was performed three times and the results was centrifuged to remove residual fluid. DNase mix was added to each filter cartridge and they were incubate for 30 minutes before elution with nucleic acid with 20 ll DEPC-distilled water and incubation for five minutes and storage at )70C. Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR)

Between February 2003 and February 2004, the investigator evaluated 38 of 211 cases that underwent SLNB at the Samsung Medical Center. Eighty paraffin-embedded sections, 49 SLN, and 31 non-SLN, from breast carcinoma without lymphatic metastases were analyzed by real-time polymerase chain reaction to evaluate the cytokine profile (interferon-c, interleukin-2, interleukin-10 and ilnterleukin-12) for the T-cell response. The study was approved by the institutional review board of the Samsung Medical Center, and written informed consent was obtained from all enrolled patients.

The RNA integrity in each preparation was tested by RT-PCR of glyceraldehydes-3-phosphate dehydrogenase (GAPDH). RT-PCR was applied by using SuperScriptTM II reverse trascriptase (Invitrogen). One microgram of RNA was incubated at 60C for 10 minutes before RT-PCR. A mixture of 1 ll 100 pmol dT, 125 ng random primer (Invitrgen), and 1 ll 10 mM dNTP mix was incubated at 65C for five minutes with rapid cooling. A total volume of 20 ll of sample, including 1ll 200U SuperScriptTM II reverse trascriptase, 4 ll 5 · first-strand buffer, and 2 ll 0.1M DTT, was prepared for RT-PCR, which was performed at 42C for three hours.

Sentinel Lymph Node Biopsy

Real-Time Quantitative RT-PCR

SLNB was performed using radioisotope (1 mCi/ ml of technetium 99m-antisulphate colloid or 0.5 mCi/ml of technetium 99m-Tin colloid) and 5 cc of 1% isosulfan blue dye. After subdermal injection at a periareolar site, sentinel lymph nodes were detected

A real-time quantitative RT-PCR assay was used for the quantification of messenger RNA (mRNA) transcripts of the following genes: IL-12, IL-10, IFNc, and IL-2. Tonsil and lymph nodes were used for positive controls of cytokine. The amino acid seAnn. Surg. Oncol. Vol. 14, No. 11, 2007

3270

J. H. YANG ET AL.

TABLE 1. Sequence of PCR primer and sequence specific probes for cytokines (Universal Probe Library: Roche Diagnostics, Mannheim, Germany) Target sequence

Oligo no.

Sequence 5¢-3¢

IFN-c sense IFN-c anti-sense IFN-c probe UPL#21 IL-2 sense IL-2 antisense IL-2 probe UPL#65 IL-10 sense IL-10 antisense IL-10 probe UPL#30 IL-12 sense IL-12 anti-sense IL-12 probe UPL#50

21 21

GGC ATT TTG AAG AAT TGG AAA TTG GAT GCT CTG GTC ATC TTT FAM – CAG AGC CA -TAMRA AAG TTT TAC ATG CCC AAG AAG G AAG TGA AAG TTT TTG CTT TGA GC FAM-TCCTCCAG-TAMRA TGG GGG AGA ACC TGA AGA C ACA GGG AAG AAA TCG ATG ACA FAM - GGCTGAGG - TAMRA CAC TCC CAA AAC CTG CTG AG TCT TCA GAA GTG CAA GGG TAA A FAM - CAGCCACC - TAMRA

22 23 19 21 20 22

Amplicon length (bp) 111 113 62 86

TABLE 2. Sequence of PCR primer and sequence specific probes for HPRT reference gene (TIB MOLBIOL, Berlin, Germany) DNA924177

hu HPRT, mRNA (137bp)

NM_000194

HPRT sense HPRT antisense HPRT probe

CTC AAC TTT AAC TGG AAA GAA TGT C TCC TTT TCA CCA GCA AGC T DYXL-TTG CTT TCC TTG GTC AGG CAG TAT AAT C – BHQ2

451–475 587–569 521–548

quence of each gene and probe are shown in Table 1. They were designed by using Universal Probe Library (UPL, Roche Diagnostics, Mannheim, Germany). LightcyclerGene expression quantification was performed in two-step RT-PCR. First-strand complementary DNA was prepared from 1 mg of RNA with random hexamers using the LightcyclerTaqMan master mix (Roche Diagnostics, Mannheim, Germany), according to the manufacturerÕs instructions. Hypoxanthine phosphoribosyl transferase (HPRT, TIB, Berlin, Germany) was used as the reference gene, and the amino acid sequence of the probe and primer are shown in Table 2. The 20 ll samples consisted of 2 ll template complement DNA, 5 ll target primer 10 pmol, each of UPL-designed probe 3 pmol, HPRT Taqman probe 3 pmol, 4 ll 5 · Taqman master mix, and 9 ll distilled water. The PCR condition was 95C for 10 minutes for initial denaturation, followed by 45 cycles of 10 s at 95C, 30 s at 55C, and 30 s at 40C for cooling. Annealing temperatures and elongation times were optimized for primer generation and exclusion of artifacts. All clinical samples were amplified by a Lightcycler 1.5 system (Roche diagnostics, Mannheim, Germany) for each diluted standard nucleotide, and the quality of amplification was checked with Lightcycler software (version 4.0, Idaho Technology Inc., Salt Lake City, Ann. Surg. Oncol. Vol. 14, No. 11, 2007

UT) by calculating the ratio of the crossing point (the threshold cycle number to the exponential phase at the beginning of PCR) and the logarithmic concentration of the amplified copy number of each standard sample. Statistical Analysis All data sets were tested for normal distribution. Because most data groups were not normally distributed, all data groups were analyzed by the Wilcoxon rank sum test. All statistical analyses were performed using SPSS for Windows software. A P value less than 0.05 was considered statistically significant.

RESULTS Clinicopathologic Characteristics All 38 cases were female, with a median age of 46 years (range 33–77 years). According to Asian– Pacific regional obesity guideline, 14 (36.8%) cases had a body mass index (BMI) less than 22.9, while 24 cases (63.2%) had a BMI over 23.0. The median number of retrieved sentinel lymph node was 4 (range 3–10). The median size of invasive breast cancer was

3271

INTERLEUKIN-10 IN SENTINEL LYMPH NODE

TABLE 3. Clinicopathologic features of the sentinel and nonsentinel lymph node from breast carcinoma without lymphatic metastases

M 1 2

3 4 5

6 7

8 9

10 + -

GAPDH

%

Features

M

Age (years) Mean ± standard deviation Range Tumor size (cm) Mean ± standard deviation Range Number of SLNs Mean ± standard deviation Range Pathology Ductal carcinoma in situ Invasive ductal carcinoma Stage Stage 0 Stage I Stage II A Estrogen receptor positive negative unknown Progesterone receptor positive negative Unknown p53 Positive Negative Unknown c-erb B2 More than 2 positive Less than 2 positive unknown

11 12 13 14 15 16 17 18 19 20 + -

48.2 ± 8.9 33–70

GAPDH

2.1 ± 1.4 0.2–3.5

M 22 23 24 25 26 27 28 29 30 31 32 + -

4.3 ± 1.8 3–10

GAPDH

14 24

36.9 63.1

14 13 11

36.8 34.3 28.9

26 11 1

68.5 28.9 2.6

24 13 1

63.2 34.2 2.6

11 25 2

28.9 65.8 5.3

10 26 2

26.3 68.4 5.3

M 33 34 35 36 37 38 39 40

GAPDH

M 47 48 49 50 51 52 53 54 55 56 + -

GAPDH M 57 58 59 60 62 63 64 65

2.1 cm (range 0.2–3.5 cm). We also examined estrogen receptor and progesterone receptor status, disease stage, and P53and c-erbB2 expression (Table 3). RT-PCR Results RT-PCR was performed to confirm the correct size of the amplicons and the absence of aspecific bands. A negative control with no reverse-transcripted RNA was included in each PCR response. Next, a negative control containing genomic DNA was included in each PCR. Finally, a single specific band was observed by gel electrophoresis (Figure 1).

41 42 43 44 45 46 + -

66 67 68 69 70 71 + -

GAPDH M 72 73 74 75 76 77 78 79 80 81 82 83 + -

GAPDH

FIG. 1. Gel electrophoresis was performed to confirm the correct size of the amplicons and the absence of aspecific bands. This figure shows gel-electrophoretic analysis of the PCR products derived from 38 of breast cancer patients. Positive control, negative control, and test samples were subjected to RT-PCR. Using agarose gel electrophoresis, a single specific band observed (M marker, + positive control, )negative control).

comparing the SLNs with non-SLNs, 3.9 ± 0.6 versus 3.0 ± 0.4. (P = 0.03). For the other cytokines, there were no significant between the SLNs and nonSLNs: interferon-c: 2.5 ± 0.2 versus 2.5 ± 0.3 (P = 0.63); interleukin-2: 1.7 ± 0.1 versus 1.7 ± 0.1 (P = 0.89); and interleukin-12: 2.9 ± 1.5 versus 1.7 ± 0.3 (0.92) (Fig. 2)

DISCUSSION Real-Time Quantitative PCR Forty-nine SLN and 31 non-SLN were excised from 38 breast cancer patients without lymphatic metastasis. All samples were analyzed using paired matched tests against each other. A significant difference in the expression of only one of the four evaluated cytokines, interleukin-10, was found when

SLNs are the first lymph nodes on the anatomically direct lymphatic draining site from a primary breast cancer. Various blue dyes and radioisotopes are used for detection, after which dye-stained or/and isotopecontaining lymph nodes are known as SLNs. However, Carmon et al.6 proposed that palpable lymph nodes should be included in the SLNs category. Since Ann. Surg. Oncol. Vol. 14, No. 11, 2007

3272

J. H. YANG ET AL.

4.00

2.00

b)

p=0.63

3.00

p=0.89

2.50

IL-2 / HPRT ratio

/ HPRT ratio

5.00

Interferon

a)

3.00

2.00

1.50

1.00 1.00 0.50

0.00

0.00

25.00

20.00

sentinel

non-sentinel

d)

p=0.03

IL-12 / HPRT ratio

c) IL-10 / HPRT ratio

non-sentinel

15.00

10.00

5.00

60.00

sentinel

p=0.92

50.00

40.00

30.00

20.00

10.00

0.00

0.00

non-sentinel

sentinel

non-sentinel

sentinel

FIG. 2. Comparison of the cytokine profile analyzed by real-time quantitative polymerase chain reaction between sentinel and nonsentinel lymph nodes from breast cancer patients without lymphatic metastases. (a) Interferon-c result, (b) Interleukin-2 result, (c) Interleukin-10 result. Significantly higher values of interleukin-10 were found in sentinel nodes (P < 0.05). (d) Interleukin-12 results.

the lymph node is the primary immune response organ and that SLNs are the first sites that come into contact with tumor cells or antigens released by the tumor through the lymphatic route, we analyzed the immune response in SLN versus non-SLNs. Matsuura et al.7 indicated that the immunological status of SLNs, including DC maturation and Th-1 responses, is depressed in SLNs before metastasis but is upregulated after metastasis occurs. However, Poindexter et al.8 suggested that a tumor-free SLN is immunologically competent and is potentially a site of tumorspecific T-cell activation. Cytokines regulate cell differentiation and growth in the immune system. Interleukine-12 (IL-12) induces differentiation of T-helper 0 (Th0) cells into Th1 cells.5,9,10 Interferon-c (Inf-c) is the most characteristic Th1 cytokine, while IL-10 is the most representative Th2 cytokine, and these cytokines affect each other: Inf-c inhibits Th2 cells, and IL-10 inhibits Th1 cells.11,12 IL-2 is synAnn. Surg. Oncol. Vol. 14, No. 11, 2007

thesized by Th1 and Th2 cell subtypes and enhance the overall immune response.13 Since the cytokine profile reflects the T-cell-mediated immune response, we evaluate the differences in cytokine profile (IL-2, IL-10, IL-12 and Inf-c) between SLNs and non-SLN using quantitative real-time RT-PCR. In melanoma, overall cytokine production is suppressed in SLNs with micrometastases compared to those containing no micrometastases.14 Other researchers have used semiquantitative PCR to demonstrate that SLNs from patients with early-stage melanoma express significantly higher levels of IL-10 than found in adjacent non-SLNs.15 In a subsequent study Lee et al.16 used quantitative real-time PCR to examine freshly preserved nodal tissue downstream from the site of primary cutaneous melanoma. Expression of IL-10 and IFN-c was higher in sentinel than adjacent nonsentinel nodes. After surgical resection of the primary tumor, cytokine elevations in the SLN

3273

INTERLEUKIN-10 IN SENTINEL LYMPH NODE

disappeared. We also found a significant overexpression of IL-10 in SLNs with breast cancer without lymph node metastasis. The expressions of Inf-c, IL-2 and IL-12 were similar between sentinel and nonsentinel lymph node. There was no difference of Infc, IL-2, and IL-12 levels between SLN and non-SLN. This result may suggest an immunologically competent state of SLN and none-SLN. However, high expression of IL-10 was shown in SLNs. This finding reveals some immunological differences compare with non-SLN. The result could be random due to our sample size, which was relatively small. Therefore, we used a paired test to compensate for sample size. The existence of IL-10 in tumors has been associated with immune suppression of a Th1 response and increased tumorigenicity.9 IL-10 polymorphism is of particular interest in relation to malignancy because IL-10 has both immunosuppressive, potentially cancer-promoting, and antiangiogenic potentially cancer-inhibiting, properties.17 In additionally, animals receiving xenotransplants of B16 melanoma cells show enhanced tumor growth after intralesional injection of IL-10.18 These immune suppressions may allow tumor cells to lodge and facilitate the growth of metastases in SLNs. It was not clear that this immunological alteration results from either breast cancer cells themselves or tumorproducing molecules. Our result has shown an immunological difference between SLNs and nonSLNs in breast cancer without lymph node metastasis.

CONCLUSIONS Overexpression of IL-10 in SLNs was observed from breast cancer without lymph node metastasis. This result shows an immunological difference between SLNs and non-SLNs. We propose the immunological suppression of SLNs in breast cancer without lymph node metastasis. This result suggests distinct characteristics of SLNs not only anatomically but also immunologically. Further analysis of the microenvironment of the immunological system is needed to clarify the role of SLNs, and to design immunotherapy for SLNs in breast cancer patients.

REFERENCES 1. Cochran AJ, Wen DR, Farzad Z, et al. Immunosuppression by melanoma cells as a factor in the generation of metastatic disease. Anticancer Res 1989; 9(4):859–64. 2. Reiss CK, Volenec FJ, Humphrey M, Singla O, Humphrey LJ. The role of the regional lymph node in breast cancer: a comparison between nodal and systemic reactivity. J Surg Oncol 1983; 22(4):249–53. 3. Mescher MF. Molecular interactions in the activation of effector and precursor cytotoxic T lymphocytes. Immunol Rev 1995; 146:177–210. 4. Ma X, Trinchieri G. Regulation of interleukin-12 production in antigen-presenting cells. Adv Immunol 2001; 79:55–92. 5. Halak BK, Maguire HC Jr., Lattime EC. Tumor-induced interleukin-10 inhibits type 1 immune responses directed at a tumor antigen as well as a non-tumor antigen present at the tumor site. Cancer Res 1999; 59(4):911–7. 6. Carmon M, Olsha O, Rivkin L, Spira RM, Golomb E. Intraoperative palpation for clinically suspicious axillary sentinel lymph nodes reduces the false-negative rate of sentinel lymph node biopsy in breast cancer. Breast J 2006; 12(3):199–201. 7. Matsuura K, Yamaguchi Y, Ueno H, Osaki A, Arihiro K, Toge T. Maturation of dendritic cells and T-cell responses in sentinel lymph nodes from patients with breast carcinoma. Cancer 2006; 106(6):1227–36. 8. Poindexter NJ, Sahin A, Hunt KK, Grimm EA. Analysis of dendritic cells in tumor-free and tumor-containing sentinel lymph nodes from patients with breast cancer. Breast Cancer Res 2004; 6(4):R408–15. 9. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science 2003; 299(5609):1057–61. 10. Ichihara F, Kono K, Takahashi A, Kawaida H, Sugai H, Fujii H. Increased populations of regulatory T cells in peripheral blood and tumor-infiltrating lymphocytes in patients with gastric and esophageal cancers. Clin Cancer Res 2003; 9(12):4404–8. 11. Fiorentino DF, Zlotnik A, Vieira P, et al. IL-10 acts on the antigen-presenting cell to inhibit cytokine production by Th1 cells. J Immunol 1991; 146(10):3444–51. 12. Maggi E, Parronchi P, Manetti R, et al. Reciprocal regulatory effects of IFN-gamma and IL-4 on the in vitro development of human Th1 and Th2 clones. J Immunol 1992; 148(7):2142–7. 13. Asadullah K, Sterry W, Trefzer U. Cytokine therapy in dermatology. Exp Dermatol 2002; 11(2):97–106. 14. Leong SP, Peng M, Zhou YM, Vaquerano JE, Chang JW. Cytokine profiles of sentinel lymph nodes draining the primary melanoma. Ann Surg Oncol 2002; 9(1):82–7. 15. Essner R, Kojima M. Dendritic cell function in sentinel nodes. Oncology (Williston Park) 2002; 16(1 Suppl 1):27–31. 16. Lee JH, Torisu-Itakara H, Cochran AJ, et al. Quantitative analysis of melanoma-induced cytokine-mediated immunosuppression in melanoma sentinel nodes. Clin Cancer Res 2005; 11(1):107–12. 17. Howell WM, Rose-Zerilli MJ. Interleukin-10 polymorphisms, cancer susceptibility and prognosis. Fam Cancer 2006; 5(2):143–9. 18. Seo N, Hayakawa S, Takigawa M, Tokura Y.. Interleukin-10 expressed at early tumour sites induces subsequent generation of CD4(+) T-regulatory cells and systemic collapse of antitumour immunity. Immunology 2001; 103(4):449–57.

ACKNOWLEDGEMENTS We are very grateful to Si Eun Kim for technical assistance, including qRT-PCR.

Ann. Surg. Oncol. Vol. 14, No. 11, 2007