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CD61+ and CAF+ were found to be good prognosis factors for invasive breast cancer patients
Accepted Manuscript Title: CD61+ and CAF+ were found to be good prognosis factors for invasive breast cancer patients Authors: Dongyan Cai, Xiaohong Wu, Tingting Hong, Yong Mao, Xiaosong Ge, Dong Hua PII: DOI: Reference:
S0344-0338(17)30053-5 http://dx.doi.org/doi:10.1016/j.prp.2017.06.016 PRP 51835
To appear in: Received date: Revised date: Accepted date:
18-1-2017 1-6-2017 24-6-2017
Please cite this article as: Dongyan Cai, Xiaohong Wu, Tingting Hong, Yong Mao, Xiaosong Ge, Dong Hua, CD61+ and CAF+ were found to be good prognosis factors for invasive breast cancer patients, Pathology - Research and Practicehttp://dx.doi.org/10.1016/j.prp.2017.06.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
CD61+ and CAF+ were found to be good prognosis factors for invasive breast cancer patients Dongyan Cai, Xiaohong Wu,Tingting Hong,Yong Mao, Xiaosong Ge, Dong Hua Authors’ Affiliations: Department of Oncology, The Fourth People’s Hospital of Wuxi, No. 200, Huihe road, Wuxi 214062, China. Corresponding Author: Dong Hua Mailing address:
Department of Oncology, The Fourth People’s Hospital of Wuxi, No. 200, Huihe road, Wuxi 214062, China.
Tel: +86 13912393709 Fax: +86 051088682109 Email: [email protected] Abstract Background: During the epithelial tumor development process, changes in tumor cell genes are an important driving factor for tumorigenesis. Recently, however, studies have shown that the tumor microenvironment, especially for cancer-associated fibroblasts (CAFs) and the infiltration of platelets into tumors, plays an essential role in the progression of human malignant disease. Methods: In this study, we investigated the presence and prognostic role of podoplanin-expressing CAFs (CAF+), the infiltration of platelets into tumors (CD61+) and platelet count before surgery in a large sample of patients with breast cancer.
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Podoplanin expression and platelet infiltration were evaluated by immunohistochemistry in 164 patients with breast cancer. Results: Seventy-two patients (44%) showed CAF+, while fifty-seven patients (35%) showed CD61+. Several strong positive correlations were found, including CD61+ with blood platelet count before surgery (P=0.004), and CAF+ with CD61+ (P=0.048). Patients with CAF+, CD61+ or platelet count＞280×109/L before surgery had a significantly shorter disease-free survival according to univariate analysis. Multivariable analysis showed that CAF+ was an independent prognostic factor (Hazard ratio=3.928; p=0.005). Conclusions: CAF+ and CD61+ were found to be good negative prognosis factors for invasive breast cancer patients. CD61+ also had strong positive correlation with blood platelet count before surgery. These targets may be used as strategies for the treatment of breast cancer in the future. Keywords: Cancer-associated fibroblasts; Breast cancer; Podoplanin; Infiltration of platelets; Prognosis Introduction Despite significant improvements over the past few decades in therapeutic strategies for patients with invasive breast cancer, several subgroups of patients still continue to progress and relapse in a short period. Therefore, understanding the molecular events and critical pathways of breast cancer is very important for developing and establishing more effective therapeutic strategies. During the
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epithelial tumor development process, changes in tumor cell genes are an important driving factor for cancer, but there is increasing evidence that cancer behaviors reflect an interconnection between the malignant epithelial compartment and the surrounding microenvironment. The stromal microenvironment in cancer tissue is heavily altered compared with that of normal tissue [1], and the invasive phenotype of tumor cells can be stimulated in 3D co-cultures with fibroblasts[2]. Cancerassociated fibroblasts (CAFs) are the most commonly studied stromal cells, and their influence on cancer development has been repeatedly demonstrated. The mucin-type sialoglycoprotein podoplanin, due to its expression on the lymphatic endothelium, is widely used as a histopathological marker for the distinction between lymphatic and blood vessels[3, 4]. However, recent studies have reported that podoplanin is also expressed in various cancer cells, dendritic cells, and especially CAFs[5, 6]. In human breast cancer, CAFs expressing the transmembrane protein podoplanin were shown to have a significant influence on patient prognosis[7, 8]. Podoplanin-expressing fibroblasts have been suggested to promote tumor progression by secreting different cytokines and growth factors[9, 10]. However, the exact mechanisms of this process have not yet been well characterized. We need to determine whether the function of podoplaninexpressing CAFs in promoting tumor progression is induced by fibroblast-cancer cell or fibroblast-other cell interactions. The C-type lectin-like receptor-2 (CLEC-2) is expressed on platelets and was identified as one of the counter receptors of podoplanin[11, 12]. Podoplanin binding
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to CLEC-2 transmits platelet activation signals through Src family kinases, Syk, and phospholipase Cγ2 in platelets[13, 14]. Recently, several studies have shown the infiltration of platelets into tumors. Activated platelets can also secrete growth factors to enhance the growth and motility of primary tumors and the tumor vasculature. The aim of this study was to evaluate the expression and possible clinical role of podoplanin in CAFs and the infiltration of platelets into tumors in a large cohort of patients with invasive breast cancer. The results may contribute to strategies for the treatment of breast cancer. Patients and methods Patients Between January 2007 and December 2008, patients from the Affiliated Hospital of Jiangnan University were included in the study at the time of primary surgery for invasive breast cancer with AJCC stagesⅠ-Ⅲ. The study was approved by the Hospital Ethics Committee, and informed consent was obtained from the patients. The total study population was 164 patients who met the criteria. The complete blood platelet count data were collected one week before surgery. The patients were divided into two groups by platelet count (equal to or less than 280×109 /L or greater than 280×109/L). This cut-off point of platelet count was obtained by ROC curve analysis. The area under the ROC curve(AUC) of plate count in diagnosis of DFS are 0.632(95% CI ,0.520~0,745，p=0.018), The cut-off point of maximum Yuden
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index was established (0.273) and then the critical point of plate count for diagnosis of DFS was 280×109/L. The clinical data regarding T stage, N stage and clinical stage were obtained from consecutive reports from physicians and pathologists at the participating hospital. The patients were followed up with serum tumor marker testing, contralateral breast mammography examination and computerized tomography (CT) scans every three months for the first 2 years after surgery. If the patient was younger than 35 years old, B-ultrasound was used to check the contralateral breast. Thereafter, the follow-up was performed twice per year from 2 to 5 years after surgery and once per year starting 5 years after surgery. In addition, tumor recurrence data were obtained from the surgery day and updated by December 2015. Disease-free survival (DFS) was measured from the date of surgery until recurrence or the date of the last follow-up. Because of the relatively few deaths in all cases, we did not consider overall survival as an endpoint. Methods The immunohistochemical staining procedure has been described previously[7]. Briefly, sections of formalin-fixed, paraffin-embedded tissue were immunostained using the Dako EnVision method (Dako, Glostrup, Denmark), mouse monoclonal antihuman CD61 (Klon SZ21, gift from Thrombosis and Haemostasis Research Unit, Jiangsu Institute of Hematology) and anti-podoplanin antibodies (clone: D2-40, DAKO). A specimen was considered positive for the expression of podoplanin in CAFs (CAF+) when distinct staining was observed in ≥ 10% of the fibromatous tumor
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stroma[4, 7] . Negative controls included the replacement of the primary antibody with normal monoclonal mouse IgG of the same subclass and concentration. Scattered punctate staining around the tumor cell was considered positive for infiltration of platelets into tumors (CD61+), and platelets in the arterial infiltration served as internal positive control. Statistical analysis To evaluate the associations between podoplanin or CD61 expression and clinicopathological parameters, the podoplanin or CD61 grades of the tumors were divided into negative and positive stainings. The associations were tested using twotailed Fisher’s exact test or linear-by-linear association chi-square test. Univariate DFS analysis was performed according to the Kaplan–Meier method, and DFS was compared using the log rank test. The multivariate analysis was conducted using the Cox proportional hazards regression model with backward, stepwise elimination of variables. The data analysis was performed using SPSS version 17.0 (SPSS, Chicago, USA). A p values <0.05 was considered statistically significant. Results Clinical data The tumors of all patients were invasive ductal NOS type. The average age of patients was 52.0 ± 12.6 years. Breast-conserving surgery was performed on 12 patients (7%), and mastectomy was performed on 152 patients (93%). After surgery, the majority of the patients, except for 15 patients (9%), received adjuvant therapy
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(endocrine therapy and chemotherapy); for lymph node metastasis≥4 and partial lymph node metastasis number is 1-3, postoperative radiotherapy was also used. In 38 (23%) patients, tamoxifen was administered for 5 years at a dose of 20 mg/day. Fourteen (9%) patients received a combined adjuvant chemotherapy plus tamoxifen (AC or TC intravenously for six cycles , recycled on day 21, at the given doses: cyclophosphamide 600 mg/m2, adriamycin 60 mg/m2, and paclitaxel 175 mg/ m2). A combination of chemotherapy plus tamoxifen was administered in 65 patients 40%), and seventeen patients (10%) in combination with radiotherapy. Fifteen patients (9%) received a combination of chemotherapy, radiotherapy with tamoxifen. (Table 1) The median follow-up time was 42 months, with a range of 1~84 months. In this period of time, 43 (26%) patients developed recurrent disease, 5 (3%) patients died from breast cancer, and no patients died from other causes. Podoplanin expression in CAFs and infiltration of platelets into tumors CAF+ were exclusively found in the tumor stroma and were easily identified as large spindle-shaped mesenchymal cells with stress fibers and well-developed fibronexus, as described previously [5, 7] (Fig. 1a). Podoplanin positive peritumoral lymphatic vessels were seen nearby tumor cells(Fig. 1c). Seventy-two patients (44%) were considered CAF+ because at least 10% of the CAFs showed a distinct staining pattern(Table 1). No podoplanin-expressing fibrocytes were found in the normal breast tissues.
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A significant association with CAF+ and histological grading was seen (p = 0.002, chi-square test): In CAF+ cases, 56% of breast cancers were scored as G3, but it was only 42% in CAF-; while in CAF+, only 8% of breast cancers were scored as G1, but it was 30% in CAF-. In addition, a significant correlation CAF+ tumors with tumor recurrence was seen (p < 0.001, exact χ2test).No association of podoplanin’s expression in CAFs with tumor T stage, lymph node stage, clinical stage and estrogen receptor status was seen (p>0.05, exact χ2test, respectively). Infiltrating platelets were exclusively found around the tumor cells and were not found in the tumor cell or tumor stroma. Fifty-seven patients (35%) were considered CD61+ and showed a distinct staining reaction. The infiltrated area of the platelets was not as wide as that of the podoplanin-expressing CAFs (Fig. 1b,d). A significant correlation CAF+ tumors with tumor recurrence was seen (p =0.017, exact χ2test). No association of CD61+ with tumor T stage, lymph node stage, clinical stage, histological grading and estrogen receptor status was seen (p>0.05, exact χ2test, respectively). Several strong statistical associations were found, including CD61+ with platelet count before surgery (P=0.004, two-tailed Fisher’s exact test), CAF+ with CD61+ (P=0.048). Both CAF+ and CD61+ had a strong association with tumor recurrence state (P=0.001 and P=0.017, respectively). Disease free survival analysis
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The results of the univariate and multivariable analyses of DFS are compiled in Table 2. The patients with CAF+, CD61+, or platelet count＞280×109/L before surgery had a significantly shorter DFS according to univariate analysis (p=0.001, p=0.024 and p=0.022, respectively, log-rank test) (Fig. 2a, 2b, 2c). The patients with CAF+ and CD61+ double positivity also had a significantly shorter DFS in the univariate analysis (p=0.001) (Fig. 2d). In the multivariable analysis, CAF+ was an independent prognostic factor for DFS (HR=3.928, 95% CI 1.517-10.169) (Table 2). CD61+ was not an independent prognostic factor for DFS; however, the Hazard ratio was 1.657, and the 95% CI was 0.507-5.411. Discussion In the present study, we demonstrated that CAF+ and CD61+ have significant effects on tumor recurrence. Patients with CAF+ and CD61+ showed a significantly reduced DFS according to univariate analysis, while in the multivariable analysis, CAF+ in the tumor stroma was an independent adverse prognostic factor. The presence of CAF+ and CD61+ significantly correlated with several clinical factors, including CAF+ and CD61+ with tumor recurrence state. Primarily, we found that CD61+ has a significant correlation with platelet count before surgery. Several previous studies proposed that the expression of podoplanin on CAFs was correlated with poor prognosis and recurrence for many carcinomas[5, 7]; while CAFs expressing podoplanin seemd to have a significant clinical role in patients with
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invasive breast cancer. Patients with CAF+ show a significantly reduced OS and DFS in univariate and multivariable analysis[7]. Podoplanin is a mucin-type sialoglycoprotein and is expressed only in a small number of tumor cells [15]. CLEC-2 is expressed in platelets and was recently identified as one of the counter receptors of podoplanin [11, 12]. Podoplanin can induce platelet aggregation by interacting with its counterpart CLEC-2, which is expressed on platelet surfaces. Platelets are well known to secrete several cytokines and growth factors after stimulation, and most of these factors have been reported to enhance the growth and motility of tumors [16-18]. For many decades, thrombocytosis has been observed in patients with solid tumors, and thrombocytosis is associated with a number of adverse outcomes [19-22]. Although paraneoplastic thrombocytosis is a well-recognized phenomenon in patients with cancer, the mechanisms underlying this observation are not fully defined. Multiple studies have focused on the platelet-tumor interaction in the vasculature. Such interactions can promote tumor metastasis by the following modes of action: first, the tumor cells coated by platelets in the vasculature can have an increased rate of tumor embolization in the microvasculature and adhesion to a vessel wall [23]; and second, platelets protect tumor cells from shear stress and immunological assault in the bloodstream by coating their surfaces. Moreover, several clinical trials have clarified the efficacy of antiplatelet and anticoagulant drugs for tumor therapy.
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Administration of low-molecular-weight heparin prolonged the overall survival of patients with advanced malignancy [24, 25]. However, whether the infiltration of platelets into tumors activated by CAFs expressing podoplanin allows for the secretion of growth factors to enhance the growth and motility of primary tumors and tumor angiopoiesis is not clear. Cancer progression is greatly affected by its microenvironment, and understanding the mechanism, especially the role of CAFs and platelet infiltration, will likely lead to the identification of novel targets for anti-cancer therapy. For example, Takagi et al used anti-human podoplanin monoclonal antibodies to suppress podoplanin-CLEC-2 binding and observed that the promotion of tumor growth could be inhibited [26]. Meanwhile, inhibiting the infiltration of platelets into tumors or decreasing platelet count may present a highly selective target for anti-cancer therapies. Taken together, our findings not only revealed that CAF+ and CD61+ are negative prognostic factors in patients with invasive breast cancer patients but also revealed a significant correlation between platelet count before surgery and platelet infiltration. Conflict of interest There are no conflicts of interest. Acknowledgments
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We thank all health-care providers who had participated in taking care of patients. We also thank senior pathologist Yong Pu (Department of Pathology, The Fouth People’s Hospital of Wuxi, Wuxi 214062, China) for his suggestions and technical assistances. Financial support and sponsorship This work was supported by grants from the National Natural Science Foundation of China (No.31100634,81201600 and 81301960), the grant from the National Natural Science Foundation of Jiangsu (No. BK2012538 and No. BK20140171) and Wuxi Bureau of health research plan(MS201428).References 1.
De Wever O, Mareel M, Role of tissue stroma in cancer cell invasion, The Journal of pathology
200(2003)429-447. 2.
Olsen CJ, Moreira J, Lukanidin EM, Ambartsumian NS, Human mammary fibroblasts stimulate
invasion of breast cancer cells in a three-dimensional culture and increase stroma development in mouse xenografts, BMC cancer 10(2010)444. 3.
Breiteneder-Geleff S, Soleiman A, Horvat R, Amann G, Kowalski H, Kerjaschki D,
[Podoplanin--a specific marker for lymphatic endothelium expressed in angiosarcoma], Verhandlungen der Deutschen Gesellschaft fur Pathologie 83(1999)270-275. 4.
Schoppmann SF, Birner P, Studer P, Breiteneder-Geleff S, Lymphatic microvessel density and
lymphovascular invasion assessed by anti-podoplanin immunostaining in human breast cancer, Anticancer research 21(2001)2351-2355. 5.
Hoshino A, Ishii G, Ito T, Aoyagi K, Ohtaki Y, Nagai K, et al., Podoplanin-positive fibroblasts
enhance lung adenocarcinoma tumor formation: podoplanin in fibroblast functions for tumor progression, Cancer research 71(2011)4769-4779. 6.
Kreppel M, Scheer M, Drebber U, Ritter L, Zoller JE, Impact of podoplanin expression in oral
squamous cell carcinoma: clinical and histopathologic correlations, Virchows Archiv : an international journal of pathology 456(2010)473-482. 7.
Schoppmann SF, Berghoff A, Dinhof C, Jakesz R, Gnant M, Dubsky P, et al., Podoplanin-
expressing cancer-associated fibroblasts are associated with poor prognosis in invasive breast cancer, Breast cancer research and treatment 134(2012)237-244. 8.
Pula B, Jethon A, Piotrowska A, Gomulkiewicz A, Owczarek T, Calik J, et al., Podoplanin
expression by cancer-associated fibroblasts predicts poor outcome in invasive ductal breast carcinoma, Histopathology 59(2011)1249-1260. 9.
Daly ME, Makris A, Reed M, Lewis CE, Hemostatic regulators of tumor angiogenesis: a source
of antiangiogenic agents for cancer treatment?, Journal of the National Cancer Institute 95(2003)16601673. 10.
Kalluri R, Zeisberg M, Fibroblasts in cancer, Nature reviews Cancer 6(2006)392-401.
13
11.
Suzuki-Inoue K, Kato Y, Inoue O, Kaneko MK, Mishima K, Yatomi Y, et al., Involvement of
the snake toxin receptor CLEC-2, in podoplanin-mediated platelet activation, by cancer cells, The Journal of biological chemistry 282(2007)25993-26001. 12.
Christou CM, Pearce AC, Watson AA, Mistry AR, Pollitt AY, Fenton-May AE, et al., Renal cells
activate the platelet receptor CLEC-2 through podoplanin, The Biochemical journal 411(2008)133-140. 13.
Suzuki-Inoue K, Fuller GL, Garcia A, Eble JA, Pohlmann S, Inoue O, et al., A novel Syk-
dependent mechanism of platelet activation by the C-type lectin receptor CLEC-2, Blood 107(2006)542549. 14.
Suzuki-Inoue K, Essential in vivo roles of the platelet activation receptor CLEC-2 in tumour
metastasis, lymphangiogenesis and thrombus formation, Journal of biochemistry 150(2011)127-132. 15.
Kato Y, Kaneko M, Sata M, Fujita N, Tsuruo T, Osawa M, Enhanced expression of Aggrus
(T1alpha/podoplanin), a platelet-aggregation-inducing factor in lung squamous cell carcinoma, Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 26(2005)195-200. 16.
Italiano JE, Jr., Richardson JL, Patel-Hett S, Battinelli E, Zaslavsky A, Short S, et al.,
Angiogenesis is regulated by a novel mechanism: pro- and antiangiogenic proteins are organized into separate platelet alpha granules and differentially released, Blood 111(2008)1227-1233. 17.
Ho-Tin-Noe B, Goerge T, Cifuni SM, Duerschmied D, Wagner DD, Platelet granule secretion
continuously prevents intratumor hemorrhage, Cancer research 68(2008)6851-6858. 18.
Ho-Tin-Noe B, Goerge T, Wagner DD, Platelets: guardians of tumor vasculature, Cancer
research 69(2009)5623-5626. 19.
Naina HV, Harris S, Paraneoplastic thrombocytosis in ovarian cancer, The New England journal
of medicine 366(2012)1840; author reply 1840. 20.
Levin J, Conley CL, Thrombocytosis Associated with Malignant Disease, Archives of internal
medicine 114(1964)497-500. 21.
Lee YS, Suh KW, Oh SY, Preoperative thrombocytosis predicts prognosis in stage II colorectal
cancer patients, Annals of surgical treatment and research 90(2016)322-327. 22.
Shoda K, Komatsu S, Ichikawa D, Kosuga T, Okamoto K, Arita T, et al., [Thrombocytosis
Associated with Poor Prognosis in Patients with Gastric Cancer], Gan to kagaku ryoho Cancer & chemotherapy 42(2015)1980-1982. 23.
Laubli H, Borsig L, Selectins promote tumor metastasis, Seminars in cancer biology
20(2010)169-177. 24.
Klerk CP, Smorenburg SM, Otten HM, Lensing AW, Prins MH, Piovella F, et al., The effect of
low molecular weight heparin on survival in patients with advanced malignancy, Journal of clinical oncology : official journal of the American Society of Clinical Oncology 23(2005)2130-2135. 25.
Lazo-Langner A, Goss GD, Spaans JN, Rodger MA, The effect of low-molecular-weight
heparin on cancer survival. A systematic review and meta-analysis of randomized trials, Journal of thrombosis and haemostasis : JTH 5(2007)729-737. 26.
Takagi S, Sato S, Oh-hara T, Takami M, Koike S, Mishima Y, et al., Platelets promote tumor
growth and metastasis via direct interaction between Aggrus/podoplanin and CLEC-2, PloS one 8(2013)e73609.
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Figr-1
C
d Ly
Fig. 1 Breast cancer samples with high amount of podoplanin-expression cancer-associated fibroblasts(a); with infiltration of platelets around the tumor cells (b) (note the platelet in the blood vessels “vessel”), original magnification ×40, the right magnification ×200; Breast cancer samples without podoplanin’s expression in cancer-associated fibroblasts (c) (note the podoplanin positive peritumoral lymphatic vessels ‘‘Ly”; (d) with infiltration of platelets around the tumor cells ×400
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Figr-2
a
c
b
d
Fig. 2 Kaplan Meier curves, comparing DFS of breast cancer patients with (CAF+, n=91) or without (CAF-, n=73) podoplanin-expressing cancer-associated fibroblasts(a); comparing patients with (CD61+, n=105) or without (CD61-, n=59) infiltration of platelets into tumors(b); comparing patients with PLT＞280×109/L(n=36) or PLT≤ 280×109/L (n=128) (c); comparing patients with double positive(CAF+ and CD61+, n=36) or no double positive( single positive and nor positive, n=128) (d).
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Table 2 Disease free survival analysis Factor
P value univariate
P value multivariable
Hazard ratios
95% CI
CAF+( n=72)
0.001
0.005
3.928
1.517-10.169
CD61+ (n=57)
0.024
0.403
1.657
0.507-5.411
CAF+ and CD61+
0.001
0.667
1.104
0.693-1.759
pT
0.212
0.642
1.201
0.782-2.359
Lymph node status
0.001
0.215
0.299
0.044-2.016
Clinical stage
0.001
0.001
6.406
2.228-18.41
PLT≤280, n=36 vs.
0.022
0.473
1.324
0.615-2.851
0.743
0.647
1.200
0.549-2.624
Double positive(n=36)
PLT＞280,n=128 age (≤50vs.＞50)
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Table 1 Clinical data of patients with CAF+ or CD61+ factor
Number of cases
CAF+ (n=72)
CAF-
P(CAF)
(n=92)
CD61+ (n=57)
CD61-
P(CD61)
(n=107)
Histologic Grade G1
34
6(8%)
28(30%)
G2
52
26(36%)
26(28%)
20(35%)
32(30%)
G3
78
40(56%)
38(42%)
27(47%)
51(48%)
18(31%)
43(40%)
53(58%
36(63%)
58(54%)
T stage
0.002*
10(18%)
24(22%) 0.689
61
pT1
27(38%)
34(37%
0.643
0.463
) pT2
94
41(57%) )
pT3
3
1 (1%)
2(2%)
1(2%)
2(2%)
pT4
6
3 (4%)
3(3%)
2(4%)
4(4%)
37(65%)
77(72%)
20(35%)
30(28%)
37(66%)
77(72%)
10(11%
10(17%)
12(11%)
16(17%
10(17%)
18(17%)
18(32%)
16(15%)
39(68%)
91(85%)
12(21%)
26(24%)
45(79%)
81(76%)
Lymph node stage Negative
114
48(67%)
66(72%
0.373
0.433
) Positive
50
24(33%)
26(28% )
Clinical stage Ⅰ
114
48(66%)
66(72%
0.571
0.570
) Ⅱ
22
12(12%) )
Ⅲ
28
12(12%) )
Tumor recurrence Yes
34
25(35%)
No
130
47(65%)
9(10%)
＜0.001*
83(90%
0.017*
) Estrogen receptor status Negative
38
21 (29%)
17(18%
0.107
) Positive
126
51(71%)
75(82% )
Her-2 status
Negative
0.639
18 Positive Adjuvant therapy None
15(9%)
3(4%)
12(13%
0.299
4(7%)
11(10%)
19(33%)
46(43%)
5(9%)
9(8%)
18(20%
13(23%)
25(23%)
9(9%)
6(11%)
9(9%)
10(11%
10(17%)
7(7%)
52±9.5
51±10.8
55±8.2
) CT+ET CT
65(40%) 14(9%)
30(50%) 6(8%)
35(38% ) 8(9%)
ET
38(23%)
20(28%) )
CT+RT+ET
15(9%)
6(8%)
CT+RT
17(10%)
7(10%) )
Mean age (±SD)
52.±12.6
53±10.5
*Significant association(Mann–Whitney U test or χ2 test, respectively); CT Chemotherapy; ET Endocrine therapy; RT Radiotherapy
0.229
S0344-0338(17)30053-5 http://dx.doi.org/doi:10.1016/j.prp.2017.06.016 PRP 51835
To appear in: Received date: Revised date: Accepted date:
18-1-2017 1-6-2017 24-6-2017
Please cite this article as: Dongyan Cai, Xiaohong Wu, Tingting Hong, Yong Mao, Xiaosong Ge, Dong Hua, CD61+ and CAF+ were found to be good prognosis factors for invasive breast cancer patients, Pathology - Research and Practicehttp://dx.doi.org/10.1016/j.prp.2017.06.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
CD61+ and CAF+ were found to be good prognosis factors for invasive breast cancer patients Dongyan Cai, Xiaohong Wu,Tingting Hong,Yong Mao, Xiaosong Ge, Dong Hua Authors’ Affiliations: Department of Oncology, The Fourth People’s Hospital of Wuxi, No. 200, Huihe road, Wuxi 214062, China. Corresponding Author: Dong Hua Mailing address:
Department of Oncology, The Fourth People’s Hospital of Wuxi, No. 200, Huihe road, Wuxi 214062, China.
Tel: +86 13912393709 Fax: +86 051088682109 Email: [email protected] Abstract Background: During the epithelial tumor development process, changes in tumor cell genes are an important driving factor for tumorigenesis. Recently, however, studies have shown that the tumor microenvironment, especially for cancer-associated fibroblasts (CAFs) and the infiltration of platelets into tumors, plays an essential role in the progression of human malignant disease. Methods: In this study, we investigated the presence and prognostic role of podoplanin-expressing CAFs (CAF+), the infiltration of platelets into tumors (CD61+) and platelet count before surgery in a large sample of patients with breast cancer.
2
Podoplanin expression and platelet infiltration were evaluated by immunohistochemistry in 164 patients with breast cancer. Results: Seventy-two patients (44%) showed CAF+, while fifty-seven patients (35%) showed CD61+. Several strong positive correlations were found, including CD61+ with blood platelet count before surgery (P=0.004), and CAF+ with CD61+ (P=0.048). Patients with CAF+, CD61+ or platelet count＞280×109/L before surgery had a significantly shorter disease-free survival according to univariate analysis. Multivariable analysis showed that CAF+ was an independent prognostic factor (Hazard ratio=3.928; p=0.005). Conclusions: CAF+ and CD61+ were found to be good negative prognosis factors for invasive breast cancer patients. CD61+ also had strong positive correlation with blood platelet count before surgery. These targets may be used as strategies for the treatment of breast cancer in the future. Keywords: Cancer-associated fibroblasts; Breast cancer; Podoplanin; Infiltration of platelets; Prognosis Introduction Despite significant improvements over the past few decades in therapeutic strategies for patients with invasive breast cancer, several subgroups of patients still continue to progress and relapse in a short period. Therefore, understanding the molecular events and critical pathways of breast cancer is very important for developing and establishing more effective therapeutic strategies. During the
3
epithelial tumor development process, changes in tumor cell genes are an important driving factor for cancer, but there is increasing evidence that cancer behaviors reflect an interconnection between the malignant epithelial compartment and the surrounding microenvironment. The stromal microenvironment in cancer tissue is heavily altered compared with that of normal tissue [1], and the invasive phenotype of tumor cells can be stimulated in 3D co-cultures with fibroblasts[2]. Cancerassociated fibroblasts (CAFs) are the most commonly studied stromal cells, and their influence on cancer development has been repeatedly demonstrated. The mucin-type sialoglycoprotein podoplanin, due to its expression on the lymphatic endothelium, is widely used as a histopathological marker for the distinction between lymphatic and blood vessels[3, 4]. However, recent studies have reported that podoplanin is also expressed in various cancer cells, dendritic cells, and especially CAFs[5, 6]. In human breast cancer, CAFs expressing the transmembrane protein podoplanin were shown to have a significant influence on patient prognosis[7, 8]. Podoplanin-expressing fibroblasts have been suggested to promote tumor progression by secreting different cytokines and growth factors[9, 10]. However, the exact mechanisms of this process have not yet been well characterized. We need to determine whether the function of podoplaninexpressing CAFs in promoting tumor progression is induced by fibroblast-cancer cell or fibroblast-other cell interactions. The C-type lectin-like receptor-2 (CLEC-2) is expressed on platelets and was identified as one of the counter receptors of podoplanin[11, 12]. Podoplanin binding
4
to CLEC-2 transmits platelet activation signals through Src family kinases, Syk, and phospholipase Cγ2 in platelets[13, 14]. Recently, several studies have shown the infiltration of platelets into tumors. Activated platelets can also secrete growth factors to enhance the growth and motility of primary tumors and the tumor vasculature. The aim of this study was to evaluate the expression and possible clinical role of podoplanin in CAFs and the infiltration of platelets into tumors in a large cohort of patients with invasive breast cancer. The results may contribute to strategies for the treatment of breast cancer. Patients and methods Patients Between January 2007 and December 2008, patients from the Affiliated Hospital of Jiangnan University were included in the study at the time of primary surgery for invasive breast cancer with AJCC stagesⅠ-Ⅲ. The study was approved by the Hospital Ethics Committee, and informed consent was obtained from the patients. The total study population was 164 patients who met the criteria. The complete blood platelet count data were collected one week before surgery. The patients were divided into two groups by platelet count (equal to or less than 280×109 /L or greater than 280×109/L). This cut-off point of platelet count was obtained by ROC curve analysis. The area under the ROC curve(AUC) of plate count in diagnosis of DFS are 0.632(95% CI ,0.520~0,745，p=0.018), The cut-off point of maximum Yuden
5
index was established (0.273) and then the critical point of plate count for diagnosis of DFS was 280×109/L. The clinical data regarding T stage, N stage and clinical stage were obtained from consecutive reports from physicians and pathologists at the participating hospital. The patients were followed up with serum tumor marker testing, contralateral breast mammography examination and computerized tomography (CT) scans every three months for the first 2 years after surgery. If the patient was younger than 35 years old, B-ultrasound was used to check the contralateral breast. Thereafter, the follow-up was performed twice per year from 2 to 5 years after surgery and once per year starting 5 years after surgery. In addition, tumor recurrence data were obtained from the surgery day and updated by December 2015. Disease-free survival (DFS) was measured from the date of surgery until recurrence or the date of the last follow-up. Because of the relatively few deaths in all cases, we did not consider overall survival as an endpoint. Methods The immunohistochemical staining procedure has been described previously[7]. Briefly, sections of formalin-fixed, paraffin-embedded tissue were immunostained using the Dako EnVision method (Dako, Glostrup, Denmark), mouse monoclonal antihuman CD61 (Klon SZ21, gift from Thrombosis and Haemostasis Research Unit, Jiangsu Institute of Hematology) and anti-podoplanin antibodies (clone: D2-40, DAKO). A specimen was considered positive for the expression of podoplanin in CAFs (CAF+) when distinct staining was observed in ≥ 10% of the fibromatous tumor
6
stroma[4, 7] . Negative controls included the replacement of the primary antibody with normal monoclonal mouse IgG of the same subclass and concentration. Scattered punctate staining around the tumor cell was considered positive for infiltration of platelets into tumors (CD61+), and platelets in the arterial infiltration served as internal positive control. Statistical analysis To evaluate the associations between podoplanin or CD61 expression and clinicopathological parameters, the podoplanin or CD61 grades of the tumors were divided into negative and positive stainings. The associations were tested using twotailed Fisher’s exact test or linear-by-linear association chi-square test. Univariate DFS analysis was performed according to the Kaplan–Meier method, and DFS was compared using the log rank test. The multivariate analysis was conducted using the Cox proportional hazards regression model with backward, stepwise elimination of variables. The data analysis was performed using SPSS version 17.0 (SPSS, Chicago, USA). A p values <0.05 was considered statistically significant. Results Clinical data The tumors of all patients were invasive ductal NOS type. The average age of patients was 52.0 ± 12.6 years. Breast-conserving surgery was performed on 12 patients (7%), and mastectomy was performed on 152 patients (93%). After surgery, the majority of the patients, except for 15 patients (9%), received adjuvant therapy
7
(endocrine therapy and chemotherapy); for lymph node metastasis≥4 and partial lymph node metastasis number is 1-3, postoperative radiotherapy was also used. In 38 (23%) patients, tamoxifen was administered for 5 years at a dose of 20 mg/day. Fourteen (9%) patients received a combined adjuvant chemotherapy plus tamoxifen (AC or TC intravenously for six cycles , recycled on day 21, at the given doses: cyclophosphamide 600 mg/m2, adriamycin 60 mg/m2, and paclitaxel 175 mg/ m2). A combination of chemotherapy plus tamoxifen was administered in 65 patients 40%), and seventeen patients (10%) in combination with radiotherapy. Fifteen patients (9%) received a combination of chemotherapy, radiotherapy with tamoxifen. (Table 1) The median follow-up time was 42 months, with a range of 1~84 months. In this period of time, 43 (26%) patients developed recurrent disease, 5 (3%) patients died from breast cancer, and no patients died from other causes. Podoplanin expression in CAFs and infiltration of platelets into tumors CAF+ were exclusively found in the tumor stroma and were easily identified as large spindle-shaped mesenchymal cells with stress fibers and well-developed fibronexus, as described previously [5, 7] (Fig. 1a). Podoplanin positive peritumoral lymphatic vessels were seen nearby tumor cells(Fig. 1c). Seventy-two patients (44%) were considered CAF+ because at least 10% of the CAFs showed a distinct staining pattern(Table 1). No podoplanin-expressing fibrocytes were found in the normal breast tissues.
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A significant association with CAF+ and histological grading was seen (p = 0.002, chi-square test): In CAF+ cases, 56% of breast cancers were scored as G3, but it was only 42% in CAF-; while in CAF+, only 8% of breast cancers were scored as G1, but it was 30% in CAF-. In addition, a significant correlation CAF+ tumors with tumor recurrence was seen (p < 0.001, exact χ2test).No association of podoplanin’s expression in CAFs with tumor T stage, lymph node stage, clinical stage and estrogen receptor status was seen (p>0.05, exact χ2test, respectively). Infiltrating platelets were exclusively found around the tumor cells and were not found in the tumor cell or tumor stroma. Fifty-seven patients (35%) were considered CD61+ and showed a distinct staining reaction. The infiltrated area of the platelets was not as wide as that of the podoplanin-expressing CAFs (Fig. 1b,d). A significant correlation CAF+ tumors with tumor recurrence was seen (p =0.017, exact χ2test). No association of CD61+ with tumor T stage, lymph node stage, clinical stage, histological grading and estrogen receptor status was seen (p>0.05, exact χ2test, respectively). Several strong statistical associations were found, including CD61+ with platelet count before surgery (P=0.004, two-tailed Fisher’s exact test), CAF+ with CD61+ (P=0.048). Both CAF+ and CD61+ had a strong association with tumor recurrence state (P=0.001 and P=0.017, respectively). Disease free survival analysis
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The results of the univariate and multivariable analyses of DFS are compiled in Table 2. The patients with CAF+, CD61+, or platelet count＞280×109/L before surgery had a significantly shorter DFS according to univariate analysis (p=0.001, p=0.024 and p=0.022, respectively, log-rank test) (Fig. 2a, 2b, 2c). The patients with CAF+ and CD61+ double positivity also had a significantly shorter DFS in the univariate analysis (p=0.001) (Fig. 2d). In the multivariable analysis, CAF+ was an independent prognostic factor for DFS (HR=3.928, 95% CI 1.517-10.169) (Table 2). CD61+ was not an independent prognostic factor for DFS; however, the Hazard ratio was 1.657, and the 95% CI was 0.507-5.411. Discussion In the present study, we demonstrated that CAF+ and CD61+ have significant effects on tumor recurrence. Patients with CAF+ and CD61+ showed a significantly reduced DFS according to univariate analysis, while in the multivariable analysis, CAF+ in the tumor stroma was an independent adverse prognostic factor. The presence of CAF+ and CD61+ significantly correlated with several clinical factors, including CAF+ and CD61+ with tumor recurrence state. Primarily, we found that CD61+ has a significant correlation with platelet count before surgery. Several previous studies proposed that the expression of podoplanin on CAFs was correlated with poor prognosis and recurrence for many carcinomas[5, 7]; while CAFs expressing podoplanin seemd to have a significant clinical role in patients with
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invasive breast cancer. Patients with CAF+ show a significantly reduced OS and DFS in univariate and multivariable analysis[7]. Podoplanin is a mucin-type sialoglycoprotein and is expressed only in a small number of tumor cells [15]. CLEC-2 is expressed in platelets and was recently identified as one of the counter receptors of podoplanin [11, 12]. Podoplanin can induce platelet aggregation by interacting with its counterpart CLEC-2, which is expressed on platelet surfaces. Platelets are well known to secrete several cytokines and growth factors after stimulation, and most of these factors have been reported to enhance the growth and motility of tumors [16-18]. For many decades, thrombocytosis has been observed in patients with solid tumors, and thrombocytosis is associated with a number of adverse outcomes [19-22]. Although paraneoplastic thrombocytosis is a well-recognized phenomenon in patients with cancer, the mechanisms underlying this observation are not fully defined. Multiple studies have focused on the platelet-tumor interaction in the vasculature. Such interactions can promote tumor metastasis by the following modes of action: first, the tumor cells coated by platelets in the vasculature can have an increased rate of tumor embolization in the microvasculature and adhesion to a vessel wall [23]; and second, platelets protect tumor cells from shear stress and immunological assault in the bloodstream by coating their surfaces. Moreover, several clinical trials have clarified the efficacy of antiplatelet and anticoagulant drugs for tumor therapy.
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Administration of low-molecular-weight heparin prolonged the overall survival of patients with advanced malignancy [24, 25]. However, whether the infiltration of platelets into tumors activated by CAFs expressing podoplanin allows for the secretion of growth factors to enhance the growth and motility of primary tumors and tumor angiopoiesis is not clear. Cancer progression is greatly affected by its microenvironment, and understanding the mechanism, especially the role of CAFs and platelet infiltration, will likely lead to the identification of novel targets for anti-cancer therapy. For example, Takagi et al used anti-human podoplanin monoclonal antibodies to suppress podoplanin-CLEC-2 binding and observed that the promotion of tumor growth could be inhibited [26]. Meanwhile, inhibiting the infiltration of platelets into tumors or decreasing platelet count may present a highly selective target for anti-cancer therapies. Taken together, our findings not only revealed that CAF+ and CD61+ are negative prognostic factors in patients with invasive breast cancer patients but also revealed a significant correlation between platelet count before surgery and platelet infiltration. Conflict of interest There are no conflicts of interest. Acknowledgments
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We thank all health-care providers who had participated in taking care of patients. We also thank senior pathologist Yong Pu (Department of Pathology, The Fouth People’s Hospital of Wuxi, Wuxi 214062, China) for his suggestions and technical assistances. Financial support and sponsorship This work was supported by grants from the National Natural Science Foundation of China (No.31100634,81201600 and 81301960), the grant from the National Natural Science Foundation of Jiangsu (No. BK2012538 and No. BK20140171) and Wuxi Bureau of health research plan(MS201428).References 1.
De Wever O, Mareel M, Role of tissue stroma in cancer cell invasion, The Journal of pathology
200(2003)429-447. 2.
Olsen CJ, Moreira J, Lukanidin EM, Ambartsumian NS, Human mammary fibroblasts stimulate
invasion of breast cancer cells in a three-dimensional culture and increase stroma development in mouse xenografts, BMC cancer 10(2010)444. 3.
Breiteneder-Geleff S, Soleiman A, Horvat R, Amann G, Kowalski H, Kerjaschki D,
[Podoplanin--a specific marker for lymphatic endothelium expressed in angiosarcoma], Verhandlungen der Deutschen Gesellschaft fur Pathologie 83(1999)270-275. 4.
Schoppmann SF, Birner P, Studer P, Breiteneder-Geleff S, Lymphatic microvessel density and
lymphovascular invasion assessed by anti-podoplanin immunostaining in human breast cancer, Anticancer research 21(2001)2351-2355. 5.
Hoshino A, Ishii G, Ito T, Aoyagi K, Ohtaki Y, Nagai K, et al., Podoplanin-positive fibroblasts
enhance lung adenocarcinoma tumor formation: podoplanin in fibroblast functions for tumor progression, Cancer research 71(2011)4769-4779. 6.
Kreppel M, Scheer M, Drebber U, Ritter L, Zoller JE, Impact of podoplanin expression in oral
squamous cell carcinoma: clinical and histopathologic correlations, Virchows Archiv : an international journal of pathology 456(2010)473-482. 7.
Schoppmann SF, Berghoff A, Dinhof C, Jakesz R, Gnant M, Dubsky P, et al., Podoplanin-
expressing cancer-associated fibroblasts are associated with poor prognosis in invasive breast cancer, Breast cancer research and treatment 134(2012)237-244. 8.
Pula B, Jethon A, Piotrowska A, Gomulkiewicz A, Owczarek T, Calik J, et al., Podoplanin
expression by cancer-associated fibroblasts predicts poor outcome in invasive ductal breast carcinoma, Histopathology 59(2011)1249-1260. 9.
Daly ME, Makris A, Reed M, Lewis CE, Hemostatic regulators of tumor angiogenesis: a source
of antiangiogenic agents for cancer treatment?, Journal of the National Cancer Institute 95(2003)16601673. 10.
Kalluri R, Zeisberg M, Fibroblasts in cancer, Nature reviews Cancer 6(2006)392-401.
13
11.
Suzuki-Inoue K, Kato Y, Inoue O, Kaneko MK, Mishima K, Yatomi Y, et al., Involvement of
the snake toxin receptor CLEC-2, in podoplanin-mediated platelet activation, by cancer cells, The Journal of biological chemistry 282(2007)25993-26001. 12.
Christou CM, Pearce AC, Watson AA, Mistry AR, Pollitt AY, Fenton-May AE, et al., Renal cells
activate the platelet receptor CLEC-2 through podoplanin, The Biochemical journal 411(2008)133-140. 13.
Suzuki-Inoue K, Fuller GL, Garcia A, Eble JA, Pohlmann S, Inoue O, et al., A novel Syk-
dependent mechanism of platelet activation by the C-type lectin receptor CLEC-2, Blood 107(2006)542549. 14.
Suzuki-Inoue K, Essential in vivo roles of the platelet activation receptor CLEC-2 in tumour
metastasis, lymphangiogenesis and thrombus formation, Journal of biochemistry 150(2011)127-132. 15.
Kato Y, Kaneko M, Sata M, Fujita N, Tsuruo T, Osawa M, Enhanced expression of Aggrus
(T1alpha/podoplanin), a platelet-aggregation-inducing factor in lung squamous cell carcinoma, Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 26(2005)195-200. 16.
Italiano JE, Jr., Richardson JL, Patel-Hett S, Battinelli E, Zaslavsky A, Short S, et al.,
Angiogenesis is regulated by a novel mechanism: pro- and antiangiogenic proteins are organized into separate platelet alpha granules and differentially released, Blood 111(2008)1227-1233. 17.
Ho-Tin-Noe B, Goerge T, Cifuni SM, Duerschmied D, Wagner DD, Platelet granule secretion
continuously prevents intratumor hemorrhage, Cancer research 68(2008)6851-6858. 18.
Ho-Tin-Noe B, Goerge T, Wagner DD, Platelets: guardians of tumor vasculature, Cancer
research 69(2009)5623-5626. 19.
Naina HV, Harris S, Paraneoplastic thrombocytosis in ovarian cancer, The New England journal
of medicine 366(2012)1840; author reply 1840. 20.
Levin J, Conley CL, Thrombocytosis Associated with Malignant Disease, Archives of internal
medicine 114(1964)497-500. 21.
Lee YS, Suh KW, Oh SY, Preoperative thrombocytosis predicts prognosis in stage II colorectal
cancer patients, Annals of surgical treatment and research 90(2016)322-327. 22.
Shoda K, Komatsu S, Ichikawa D, Kosuga T, Okamoto K, Arita T, et al., [Thrombocytosis
Associated with Poor Prognosis in Patients with Gastric Cancer], Gan to kagaku ryoho Cancer & chemotherapy 42(2015)1980-1982. 23.
Laubli H, Borsig L, Selectins promote tumor metastasis, Seminars in cancer biology
20(2010)169-177. 24.
Klerk CP, Smorenburg SM, Otten HM, Lensing AW, Prins MH, Piovella F, et al., The effect of
low molecular weight heparin on survival in patients with advanced malignancy, Journal of clinical oncology : official journal of the American Society of Clinical Oncology 23(2005)2130-2135. 25.
Lazo-Langner A, Goss GD, Spaans JN, Rodger MA, The effect of low-molecular-weight
heparin on cancer survival. A systematic review and meta-analysis of randomized trials, Journal of thrombosis and haemostasis : JTH 5(2007)729-737. 26.
Takagi S, Sato S, Oh-hara T, Takami M, Koike S, Mishima Y, et al., Platelets promote tumor
growth and metastasis via direct interaction between Aggrus/podoplanin and CLEC-2, PloS one 8(2013)e73609.
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Figr-1
C
d Ly
Fig. 1 Breast cancer samples with high amount of podoplanin-expression cancer-associated fibroblasts(a); with infiltration of platelets around the tumor cells (b) (note the platelet in the blood vessels “vessel”), original magnification ×40, the right magnification ×200; Breast cancer samples without podoplanin’s expression in cancer-associated fibroblasts (c) (note the podoplanin positive peritumoral lymphatic vessels ‘‘Ly”; (d) with infiltration of platelets around the tumor cells ×400
15
Figr-2
a
c
b
d
Fig. 2 Kaplan Meier curves, comparing DFS of breast cancer patients with (CAF+, n=91) or without (CAF-, n=73) podoplanin-expressing cancer-associated fibroblasts(a); comparing patients with (CD61+, n=105) or without (CD61-, n=59) infiltration of platelets into tumors(b); comparing patients with PLT＞280×109/L(n=36) or PLT≤ 280×109/L (n=128) (c); comparing patients with double positive(CAF+ and CD61+, n=36) or no double positive( single positive and nor positive, n=128) (d).
16
Table 2 Disease free survival analysis Factor
P value univariate
P value multivariable
Hazard ratios
95% CI
CAF+( n=72)
0.001
0.005
3.928
1.517-10.169
CD61+ (n=57)
0.024
0.403
1.657
0.507-5.411
CAF+ and CD61+
0.001
0.667
1.104
0.693-1.759
pT
0.212
0.642
1.201
0.782-2.359
Lymph node status
0.001
0.215
0.299
0.044-2.016
Clinical stage
0.001
0.001
6.406
2.228-18.41
PLT≤280, n=36 vs.
0.022
0.473
1.324
0.615-2.851
0.743
0.647
1.200
0.549-2.624
Double positive(n=36)
PLT＞280,n=128 age (≤50vs.＞50)
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Table 1 Clinical data of patients with CAF+ or CD61+ factor
Number of cases
CAF+ (n=72)
CAF-
P(CAF)
(n=92)
CD61+ (n=57)
CD61-
P(CD61)
(n=107)
Histologic Grade G1
34
6(8%)
28(30%)
G2
52
26(36%)
26(28%)
20(35%)
32(30%)
G3
78
40(56%)
38(42%)
27(47%)
51(48%)
18(31%)
43(40%)
53(58%
36(63%)
58(54%)
T stage
0.002*
10(18%)
24(22%) 0.689
61
pT1
27(38%)
34(37%
0.643
0.463
) pT2
94
41(57%) )
pT3
3
1 (1%)
2(2%)
1(2%)
2(2%)
pT4
6
3 (4%)
3(3%)
2(4%)
4(4%)
37(65%)
77(72%)
20(35%)
30(28%)
37(66%)
77(72%)
10(11%
10(17%)
12(11%)
16(17%
10(17%)
18(17%)
18(32%)
16(15%)
39(68%)
91(85%)
12(21%)
26(24%)
45(79%)
81(76%)
Lymph node stage Negative
114
48(67%)
66(72%
0.373
0.433
) Positive
50
24(33%)
26(28% )
Clinical stage Ⅰ
114
48(66%)
66(72%
0.571
0.570
) Ⅱ
22
12(12%) )
Ⅲ
28
12(12%) )
Tumor recurrence Yes
34
25(35%)
No
130
47(65%)
9(10%)
＜0.001*
83(90%
0.017*
) Estrogen receptor status Negative
38
21 (29%)
17(18%
0.107
) Positive
126
51(71%)
75(82% )
Her-2 status
Negative
0.639
18 Positive Adjuvant therapy None
15(9%)
3(4%)
12(13%
0.299
4(7%)
11(10%)
19(33%)
46(43%)
5(9%)
9(8%)
18(20%
13(23%)
25(23%)
9(9%)
6(11%)
9(9%)
10(11%
10(17%)
7(7%)
52±9.5
51±10.8
55±8.2
) CT+ET CT
65(40%) 14(9%)
30(50%) 6(8%)
35(38% ) 8(9%)
ET
38(23%)
20(28%) )
CT+RT+ET
15(9%)
6(8%)
CT+RT
17(10%)
7(10%) )
Mean age (±SD)
52.±12.6
53±10.5
*Significant association(Mann–Whitney U test or χ2 test, respectively); CT Chemotherapy; ET Endocrine therapy; RT Radiotherapy
0.229