CXCR4 over-expression correlates with poor prognosis in myasthenia gravisassociated thymoma Wei Wang MD, PHD, Dazhong Liu MD, PHD, Lei Yang MD, PHD, Yi Li MD, Hao Xu MD, Fei Wang MD, Jiaying Zhao MD, Linyou Zhang MD, PHD PII: DOI: Reference:
S0046-8177(15)00436-0 doi: 10.1016/j.humpath.2015.10.007 YHUPA 3739
To appear in:
Human Pathology
Received date: Revised date: Accepted date:
22 July 2015 5 October 2015 8 October 2015
Please cite this article as: Wang Wei, Liu Dazhong, Yang Lei, Li Yi, Xu Hao, Wang Fei, Zhao Jiaying, Zhang Linyou, CXCR4 over-expression correlates with poor prognosis in myasthenia gravis-associated thymoma, Human Pathology (2015), doi: 10.1016/j.humpath.2015.10.007
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.
ACCEPTED MANUSCRIPT Title:
CXCR4 over-expression correlates with poor
RI P
T
prognosis in myasthenia gravis-associated thymoma
SC
Running title: CXCR4 and prognosis in MG-thymoma
Wei Wang MD, PHD, Dazhong Liu MD, PHD, Lei Yang MD, PHD, Yi Li MD, Hao
MA NU
Xu MD, Fei Wang MD, Jiaying Zhao MD, Linyou Zhang MD, PHD
Department of Thoracic Surgery, Second Affiliated Hospital of Harbin Medical
ED
University, Harbin, China
PT
Corresponding author: Linyou Zhang
CE
Department of Thoracic Surgery, Second Affiliated Hospital of Harbin Medical
AC
University, 246 Xuefu Rd, Nangang District, Harbin, 150086
Email: [email protected] Tel:
+8645186605249
Funding support: Natural Science Foundation of Heilongjiang Province (D201144).
Conflict of interest: None
ACCEPTED MANUSCRIPT Abstract Thymoma is one rare type of tumor developed on the thymic epithelium, patients
T
with thymoma also might suffer from myasthenia gravis (MG). Due to the scarcity
RI P
and complexity of MG-associated thymoma, its pathogenesis and etiology still remain unclear nowadays. The expression of C-X-C chemokine receptor type 4 (CXCR4) is
SC
absent or low in most healthy tissues, but highly expressed in various types of tumors. Here, in order to determine the prognostic significance of CXCR4 in MG-associated
MA NU
thymoma, a total of 84 tissue samples were retrospectively examined. Our data demonstrated that CXCR4 was strongly associated with worse overall survival (hazard ratio, 2.11; 95% confidence interval, 1.08-4.11) and disease free survival (hazard ratio, 1.84; 95% confidence interval, 1.03-3.29). Furthermore, both univariate and multivariate analyses confirmed that CXCR4 was an independent factor in
ED
predicting unfavorable overall survival. In conclusion, our findings suggest that CXCR4 might contribute to the clinical cancer progression, and CXCR4 could be a
PT
valuable prognostic biomarker in the therapy of myasthenia gravis-associated
AC
CE
thymoma.
Keywords
Thymoma, Myasthenia gravis, CXCR4, Prognosis, Survival
ACCEPTED MANUSCRIPT Introduction Thymoma is one primary mediastinal neoplasm arising from epithelial cells in
T
thymus, the central organ of immune system. It is reported that the incidence of
RI P
thymoma is 0.15/100,000 person every year in US [1]. Interestingly, approximately 30-50% of thymoma patients develop myasthenia gravis [2].Myasthenia gravis (MG),
SC
characterized by fatigability and/or muscular weakness, is an autoimmune disease of neuromuscular junction mediated primarily by anti-acetylcholine receptor antibodies.
MA NU
Thymoma MG is believed to account for 10-30% of all MG cases [3]. Although great progress has been achieved in the past several decades, the exact pathological mechanisms of MG-associated thymoma are still poorly understood. Therefore, it is of great value to identify independent prognostic biomarkers, which will not only improve prognosis but also provide promising therapeutic targets.
ED
Chemokines are some homologous proteins that interact with 7-transmembrane, G protein–coupled receptors. They could bind to their receptors on target cells and
PT
trigger a series of signal transduction involving the release of intracellular calcium, the generation of inositol triphosphate and activation of protein kinase C [4]. Growing
CE
evidences have demonstrated chemokines and their receptors play key roles in the cross-talking between cancer cells and their microenvironments [5].
AC
C-X-C chemokine receptor type 4 (CXCR4), absent or low in healthy tissues, is highly expressed in various different tumor types and has been considered the most widely expressed chemokine receptor in cancer [6]. Additionally, over-expression of CXCR4 is associated with chemotaxis, invasion, angiogenesis and proliferation independent of their specific histological types [5, 7, 8]. Recently, it is reported that engagement of CXCL12/CXCR4 contributes to the pathology of myasthenia gravis by recruitment of antigen presenting cells and peripheral B cells to thymus [9]. Here, we examined tumor tissues from patients with myasthenia gravis-associated thymoma to investigate whether CXCR4 expression could be translated to the clinical practice and, accordingly, be an independent prognostic biomarker for MG-associated thymoma patients.
ACCEPTED MANUSCRIPT Material and methods Subjects
T
A total of 84 samples were obtained from pathologically proven myasthenia
RI P
gravis-associate thymoma patients who underwent surgery at Second Affiliated Hospital to Harbin Medical University between 2002 and 2009. All samples were
SC
frozen in liquid nitrogen immediately after surgery until use. Diagnosis of MG was confirmed based on clinical manifestations, acetylcholine receptor antibody tests and
MA NU
electrophysiological measures [10].
The clinical classifications were defined based on the Task Force of the Medical Advisory Board of the Myasthenia Gravis Foundation of America (MGFA) [11]: Class I, any eye muscle weakness, possible ptosis, no other evidence of muscle weakness elsewhere; Class II, eye muscle weakness of any severity, mild weakness of
ED
other muscles; Class IIa, predominantly limb or axial muscles; Class IIb: predominantly bulbar and/or respiratory muscles; Class III, eye muscle weakness of
PT
any severity, moderate weakness of other muscles; Class IIIa, predominantly limb or
CE
axial muscles; Class IIIb, predominantly bulbar and/or respiratory muscles; Class IV, eye muscle weakness of any severity, severe weakness of other muscles; Class Iva,
AC
predominantly limb or axial muscles; Class IVb, predominantly bulbar and/or respiratory muscles; Class V, intubation needed to maintain airway. According to the anatomic extent of disease at the time of surgery, the Masaoka staging system was used for thymoma clinical staging [12]: Stage I, intact thymic capsule; Stage IIa, Microscopic invasion through the capsule into surrounding fatty tissue; Stage IIb, Macroscopic invasion into capsule; Stage III, macroscopic invasion into adjacent organs, vessels; Stage IVa, dissemination in thoracic cavity; Stage IVb, distant metastases. Depending on the appearance of cells from microscopic investigation, there are three major histological subtypes of thymoma: Type A, the epithelial cells have an oval of fusiform shape; Type B, an epithelioid shape (three subtypes: B1, lymphocyte rich; B2, cortical; B3, epithelial); Type C, tumor contains a variety of malignant
ACCEPTED MANUSCRIPT cytological features. The observation time in these participants was the interval between diagnosis and last contact (death or last follow up). Overall survival (OS), which was calculated as
RI P
T
the period from diagnosis until the date of death, was used for prognostic analyses. The authors state that they have followed the principles outlined in the Declaration of Helsinki. Besides, we also had obtained institutional review board approvals for all
SC
investigations in current study.
MA NU
Quantitative real-time polymerase chain reaction Total RNA was extracted with Trizol reagent (Life Tech, USA) according to the manufacturer’s recommendation. Quantitative RT-PCR analysis was conducted with SYBR Green PCR Master Mix (Life Tech, US) using the Applied Biosystems 7500 RT PCR System. The expression of the housekeeping gene GAPDH was assessed in
ED
each sample. The following PCR primers (Sangon Biotech, China) were used: CXCR4 (forward: 5’-GGAGGGGATCAGTATATACA-3’; reverse:
PT
5’–GAAGATGATGGAGTAGATGG-3’), the anticipated length was 145bp; GAPDH (forward: 5’- CACCCACTCCTCCACCTTTG -3’; reverse: 5’-
CE
CCACCACCCTGTTGCTGTAG - 3’), the anticipated length was 110bp. Relative CXCR4 expression levels were examined by standard curve method. All standard
AC
curves were linear in the required range with acceptable correlation coefficient. The CXCR4 mRNA levels were given as ratios to GAPDH mRNA levels. RT-PCR assays were done in triplicate for each sample, and the average value was used for analysis.
Statistical analysis CXCR4 expression and patient characteristics were summarized using mean and standard deviation. Fisher’s exact test and chi-squared test were used to compare the categorical data. Log-rank test and Kaplan-Meier method were used to estimate overall survivals, and calculated hazard ratios (HR) with the adjusted multivariate Cox regression analysis. All the analyses were performed with MedCalc and SPSS, and significance was defined as p1.8 as “high” and those≤1.8 as “low”. As shown in table 1, patients with high CXCR4 expression and low CXCR4 expression were matched in term of gender,
AC
age, family history of cancer, anti-AChR status and recurrence of MG after thymectomy. Meanwhile, subjects with high CXCR4 expression showed high clinical score in Masaoka stage, MGFA stage and WHO classification. The overall survival of patients with CXCR4 overexpression were worse than those with low CXCR4 levels (hazard ratio, 2.11; 95% confidence interval, 1.08-4.11; p=0.02) (Fig.3A). Similarly, CXCR4 expression was correlated with disease-free survival (HR, 1.84; 95% CI, 1.03-3.29; p=0.04) (Fig.3B). Next the multivariate Cox proportional hazard analysis were carried out to assess the potential prognostic factors for overall survival (table 2). The factors considered here were gender, age, family history of cancer, Masaoka stage, MGFA classification, WHO classification and CXCR4 expression, among which only Masaoka stage, MGFA stage, WHO
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
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SC
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T
classification and CXCR4 expression were independent factors.
ACCEPTED MANUSCRIPT Discussion Understanding the pathological mechanism of tumor is important for developing
T
target-specific medicines. CXCR4 has been implicated in the etiology of a substantial
RI P
number of tumors because this receptor is believed to play an essential role in chemotaxis, invasion, angiogenesis, metastasis and proliferation [5, 7]. However,
SC
despite recent evidence suggests CXCL12/CXCR4 axis contributes to the pathology of MG thymus [9], little attention has been devoted to the precise role of CXCR4 in
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the clinical outcomes of MG-associated thymoma. Here, by measuring CXCR4 mRNA levels, our results demonstrated that CXCR4 overexpression was an independent biomarker of poorer prognosis in MG-associate thymoma. This might also suggest CXCR4 could be a potential therapeutic target in cancer treatment. MG-associate thymoma is one complicated disease and its pathological
ED
mechanism is still unclear. Although various studies have demonstrated the prognostic significances of clinical stage, pathological classification and completed
PT
surgery resection, the available data were inconsistent due to the scarcity of this disease [13-15]. For example, one recent study revealed that, of all the
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clinical-pathological characteristics, only clinical stage is an independent prognostic factor [16]. In addition, a standardized classification to categorize thymoma into
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typical TMN classifications has not been achieved in clinical practice. Although several potential proteins including p53, epidermal growth factor receptor and cell cycle regulator proteins were investigated in thymoma to prove their predictive or prognostic significance recently[16, 17], evaluation the clinical outcome of thymoma patients was still very difficult. Now it is well accepted that CXCR4 over-expression is a risk factor of short survivals in dozens of types of tumors [18], so this study focused on the clinical significance
between
CXCR4
and
clinic-pathological
characteristics
in
MG-associated thymoma. Our data first conformed that CXCR4 over-expression was also correlated to worse prognosis in MG-associated thymoma. Next we examined whether CXCR4 over-expression was independently associated with worse clinical
ACCEPTED MANUSCRIPT outcome. After adjusted for age, gender, family history of cancer and clinical stage, multivariate analysis suggested CXCR4 were an independent prognostic biomarker. The underlying mechanisms involved in the association between CXCR4
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expression and survivals are uncertain. One possible explanation is the
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“CXCL12/CXCR4 chemokine axis hypothesis” [5]. In the past decades, accumulating evidence demonstrate that the stroma plays a key role in cancer growth and invasion.
SC
In support of this hypothesis, it was reported that as a chemoattractant, CXCR4 might be essential in cancer development possibly at the level of the tumor niche [19]. Both
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CXCR4 expression on cancer cells and CXCL12 expression by fibroblasts could trigger tumor cell motility, growth and invasiveness. Furthermore, stroma cells from some special microenvironments may modulate the expression of CXCR4, which is believe to account for tumorigenesis and tumor progression. The interactions between
ED
stroma cell and cancer are reciprocal: the stroma cells may support the growth of tumors; tumor cells can also modulate the microenvironments. Therefore, CXCL12 CXCR4
assemble
a
crucial
axis
between
cancer
cells
and
their
PT
and
microenvironments, and their interactions could impact the migration, adhesion and
CE
invasion of cancer cells. Eventually this axis conveys the association between CXCR4 expression and overall survivals.
AC
Combating MG-associated thymoma remains a major clinical challenge, and thus far results are disappointing. Having a molecular signature would help to identify those patients at high risk of death. Here, our data are consistent with the hypothesis that CXCR4/CXCL12 pathway activation could promote tumor progression. We acknowledge that the hazard ratio for overall survivals from CXCR4 high expression group is only 2.11 in MG-associated thymoma, less than 5.07 in gastric cancer [20], 4.80 in breast cancer [21], 4.03 in ovary cancer [22], 3.80 in rental cancer [23], 3.73 in head and neck cancer [24]. This might limit its application in clinical practice. Our data suggest that CXCR4 may not be used as a single biomarker in survival prognosis, but as one part of multi-molecular biomarkers for MG-associated thymoma. Because of the complexity of MG-associated thymoma, many other molecules are believed to involve in the pathological mechanism and could be potential biomarker for prognosis.
ACCEPTED MANUSCRIPT Further studies with more powerful approaches are needed. Nevertheless, our results imply that CXCR4 have the potential to be a prognostic biomarker and possible therapeutic target. In fact, the important roles of CXCR4 in multiple cancers have
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encouraged the development of clinically viable CXCR4 antagonists, and resulted in the US Food and Drug Administration (FDA) approval of Plerixafor (AMD 3100; Genzyme) for patients with multiple myeloma and non-Hodgkin's lymphoma [25].
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Hence, once our results were validated by other studies, a clinical trial utilizing Plerixafor for MG-associated thymoma with CXCR4 overexpression can be carried
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out.
In summary, CXCR4 expression could be an independent prognostic biomarker
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CE
PT
ED
of poor outcome for patients with myasthenia gravis-associated thymoma.
ACCEPTED MANUSCRIPT References [1] Engels EA, Pfeiffer RM. Malignant thymoma in the United States: demographic
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patterns in incidence and associations with subsequent malignancies. Int J Cancer
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2003; 105, 546-51.
[2] Lewis JE, Wick MR, Scheithauer BW, et al. Thymoma. A clinicopathologic
SC
review. Cancer 1987; 60, 2727-43.
[3] Thomas CR, Wright CD, Loehrer PJ. Thymoma: state of the art. J Clin Oncol
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1999; 17, 2280-9.
[4] Luster AD. Chemokines--chemotactic cytokines that mediate inflammation. New Engl J Med 1998; 338, 436-45.
[5] Domanska UM, Kruizinga RC, Nagengast WB, et al. A review on CXCR4/CXCL12 axis in oncology: no place to hide. Eur J Cancer 2013; 49,
ED
219-30.
[6] Lippitz BE. Cytokine patterns in patients with cancer: a systematic review. Lancet
PT
Oncol 2013; 14, e218-28.
[7] Teicher BA, Fricker SP. CXCL12 (SDF-1)/CXCR4 pathway in cancer. Clin
CE
Cancer Res 2010; 16, 2927-31. [8] Zhang Z, Ni C, Chen W, et al. Expression of CXCR4 and breast cancer prognosis:
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a systematic review and meta-analysis. BMC Cancer 2014; 14, 49. [9] Weiss JM, Cufi P, Bismuth J, et al. SDF-1/CXCL12 recruits B cells and antigen-presenting cells to the thymus of autoimmune myasthenia gravis patients. Immunobiol 2013; 218, 373-81. [10] Meriggioli MN, Sanders DB. Autoimmune myasthenia gravis: emerging clinical and biological heterogeneity. Lancet Neurol 2009; 8, 475-90. [11] Jaretzki A, 3rd, Barohn RJ, Ernstoff RM, et al. Myasthenia gravis: Recommendations for clinical research standards. Task Force of the Medical Scientific Advisory Board of the Myasthenia Gravis Foundation of America. Neurology 2000; 55, 16-23.
ACCEPTED MANUSCRIPT [12] Masaoka A, Monden Y, Nakahara K, et al. Follow-up study of thymomas with special reference to their clinical stages. Cancer 1981; 48, 2485-92. [13] Detterbeck FC, Parsons AM. Thymic tumors. Ann Thorac Surg 2004; 77, 1860-9.
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[14] Kondo K, Monden Y. Therapy for thymic epithelial tumors: a clinical study of 1,320 patients from Japan. Ann Thorac Surg 2003; 76, 878-84.
Ann Thorac Surg 2006; 81, 2328-34.
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[15] Detterbeck FC. Clinical value of the WHO classification system of thymoma.
[16] Cui F, He J, Liu J, et al. Protein expression status of p53 and epidermal growth
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factor receptor in thymoma. Oncol Lett 2011; 2, 459-63. [17] Baldi A, Ambrogi V, Mineo D, et al. Analysis of cell cycle regulator proteins in encapsulated thymomas. Clin Cancer Res 2005; 11, 5078-83. [18] Zhao H, Guo L, Zhao H, et al. CXCR4 over-expression and survival in cancer: a
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system review and meta-analysis. Oncotarget 2015; 6, 5022-40. [19] Kaplan RN, Riba RD, Zacharoulis S, et al. VEGFR1-positive haematopoietic
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bone marrow progenitors initiate the pre-metastatic niche. Nature 2005; 438, 820-7.
CE
[20] He H, Wang C, Shen Z, et al. Upregulated expression of C-X-C chemokine receptor 4 is an independent prognostic predictor for patients with gastric cancer.
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PloS one 2013; 8, e71864. [21] Holm NT, Abreo F, Johnson LW, et al. Elevated chemokine receptor CXCR4 expression in primary tumors following neoadjuvant chemotherapy predicts poor outcomes for patients with locally advanced breast cancer (LABC). Breast Cancer Res Tr 2009; 113, 293-9. [22] Sekiya R, Kajiyama H, Sakai K, et al. Expression of CXCR4 indicates poor prognosis in patients with clear cell carcinoma of the ovary. Hum Pathol 2012; 43, 904-10. [23] Chen D, Gassenmaier M, Maruschke M, et al. Expression and prognostic significance of a comprehensive epithelial-mesenchymal transition gene set in renal cell carcinoma. J Urol 2014; 191, 479-86.
ACCEPTED MANUSCRIPT [24] Katayama A, Ogino T, Bandoh N, et al. Expression of CXCR4 and its down-regulation by IFN-gamma in head and neck squamous cell carcinoma. Clin Cancer Res 2005; 11, 2937-46.
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[25] Debnath B, Xu S, Grande F, et al. Small molecule inhibitors of CXCR4.
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CE
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Theranostics 2013; 3, 47-75.
ACCEPTED MANUSCRIPT Figure legend
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Fig.1 Histologic specimen of one MG-associated thymoma tissue sample stained with
SC
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hematoxylin and eosin (×200).
Fig.2 Receiver operating characteristic (ROC) curve used to define the cutoff value
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between the high and low levels of CXCR4 expression. The optimal cutoff value that gave the maximal sensitivity and specificity for CXCR4 expression was 1.8.
Fig.3 Kaplan-Meier analysis for overall survival (A) and disease-free survival (B)
ED
based on CXCR4 expression among myasthenia gravis-associated thymoma patients.
AC
CE
PT
HR, hazard ratio; CI, confidence interval.
ACCEPTED MANUSCRIPT Table 1. Clinical characteristics of patients according to CXCR4 high- or low-expression
3(8%) 37(92%) 38(95%) 4(10%)
4(9%) 40(91%) 43(98%) 3(7%)
Recurrence of MG after thymectomy
Masaoka stage 3(8%) 12(30%) 16(40%) 9(22%)
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Stage I Stage II Stage III Stage IV
MGFA classification
5(13%) 8(20%) 12(30%) 10(25%) 5(12%)
WHO classification
PT
ED
Class I Class II Class III Class IV Class V
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Anti-AChR
T
CXCR4 low expression (n=44) 23:21 36.6±11.9
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Gender (female: male) Age (years) Family history of cancer yes no
CXCR4 high expression (n=40) 22:18 38.4±10.6
p value 0.80 0.24 0.78
0.47 0.60
S0046-8177(15)00436-0 doi: 10.1016/j.humpath.2015.10.007 YHUPA 3739
To appear in:
Human Pathology
Received date: Revised date: Accepted date:
22 July 2015 5 October 2015 8 October 2015
Please cite this article as: Wang Wei, Liu Dazhong, Yang Lei, Li Yi, Xu Hao, Wang Fei, Zhao Jiaying, Zhang Linyou, CXCR4 over-expression correlates with poor prognosis in myasthenia gravis-associated thymoma, Human Pathology (2015), doi: 10.1016/j.humpath.2015.10.007
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.
ACCEPTED MANUSCRIPT Title:
CXCR4 over-expression correlates with poor
RI P
T
prognosis in myasthenia gravis-associated thymoma
SC
Running title: CXCR4 and prognosis in MG-thymoma
Wei Wang MD, PHD, Dazhong Liu MD, PHD, Lei Yang MD, PHD, Yi Li MD, Hao
MA NU
Xu MD, Fei Wang MD, Jiaying Zhao MD, Linyou Zhang MD, PHD
Department of Thoracic Surgery, Second Affiliated Hospital of Harbin Medical
ED
University, Harbin, China
PT
Corresponding author: Linyou Zhang
CE
Department of Thoracic Surgery, Second Affiliated Hospital of Harbin Medical
AC
University, 246 Xuefu Rd, Nangang District, Harbin, 150086
Email: [email protected] Tel:
+8645186605249
Funding support: Natural Science Foundation of Heilongjiang Province (D201144).
Conflict of interest: None
ACCEPTED MANUSCRIPT Abstract Thymoma is one rare type of tumor developed on the thymic epithelium, patients
T
with thymoma also might suffer from myasthenia gravis (MG). Due to the scarcity
RI P
and complexity of MG-associated thymoma, its pathogenesis and etiology still remain unclear nowadays. The expression of C-X-C chemokine receptor type 4 (CXCR4) is
SC
absent or low in most healthy tissues, but highly expressed in various types of tumors. Here, in order to determine the prognostic significance of CXCR4 in MG-associated
MA NU
thymoma, a total of 84 tissue samples were retrospectively examined. Our data demonstrated that CXCR4 was strongly associated with worse overall survival (hazard ratio, 2.11; 95% confidence interval, 1.08-4.11) and disease free survival (hazard ratio, 1.84; 95% confidence interval, 1.03-3.29). Furthermore, both univariate and multivariate analyses confirmed that CXCR4 was an independent factor in
ED
predicting unfavorable overall survival. In conclusion, our findings suggest that CXCR4 might contribute to the clinical cancer progression, and CXCR4 could be a
PT
valuable prognostic biomarker in the therapy of myasthenia gravis-associated
AC
CE
thymoma.
Keywords
Thymoma, Myasthenia gravis, CXCR4, Prognosis, Survival
ACCEPTED MANUSCRIPT Introduction Thymoma is one primary mediastinal neoplasm arising from epithelial cells in
T
thymus, the central organ of immune system. It is reported that the incidence of
RI P
thymoma is 0.15/100,000 person every year in US [1]. Interestingly, approximately 30-50% of thymoma patients develop myasthenia gravis [2].Myasthenia gravis (MG),
SC
characterized by fatigability and/or muscular weakness, is an autoimmune disease of neuromuscular junction mediated primarily by anti-acetylcholine receptor antibodies.
MA NU
Thymoma MG is believed to account for 10-30% of all MG cases [3]. Although great progress has been achieved in the past several decades, the exact pathological mechanisms of MG-associated thymoma are still poorly understood. Therefore, it is of great value to identify independent prognostic biomarkers, which will not only improve prognosis but also provide promising therapeutic targets.
ED
Chemokines are some homologous proteins that interact with 7-transmembrane, G protein–coupled receptors. They could bind to their receptors on target cells and
PT
trigger a series of signal transduction involving the release of intracellular calcium, the generation of inositol triphosphate and activation of protein kinase C [4]. Growing
CE
evidences have demonstrated chemokines and their receptors play key roles in the cross-talking between cancer cells and their microenvironments [5].
AC
C-X-C chemokine receptor type 4 (CXCR4), absent or low in healthy tissues, is highly expressed in various different tumor types and has been considered the most widely expressed chemokine receptor in cancer [6]. Additionally, over-expression of CXCR4 is associated with chemotaxis, invasion, angiogenesis and proliferation independent of their specific histological types [5, 7, 8]. Recently, it is reported that engagement of CXCL12/CXCR4 contributes to the pathology of myasthenia gravis by recruitment of antigen presenting cells and peripheral B cells to thymus [9]. Here, we examined tumor tissues from patients with myasthenia gravis-associated thymoma to investigate whether CXCR4 expression could be translated to the clinical practice and, accordingly, be an independent prognostic biomarker for MG-associated thymoma patients.
ACCEPTED MANUSCRIPT Material and methods Subjects
T
A total of 84 samples were obtained from pathologically proven myasthenia
RI P
gravis-associate thymoma patients who underwent surgery at Second Affiliated Hospital to Harbin Medical University between 2002 and 2009. All samples were
SC
frozen in liquid nitrogen immediately after surgery until use. Diagnosis of MG was confirmed based on clinical manifestations, acetylcholine receptor antibody tests and
MA NU
electrophysiological measures [10].
The clinical classifications were defined based on the Task Force of the Medical Advisory Board of the Myasthenia Gravis Foundation of America (MGFA) [11]: Class I, any eye muscle weakness, possible ptosis, no other evidence of muscle weakness elsewhere; Class II, eye muscle weakness of any severity, mild weakness of
ED
other muscles; Class IIa, predominantly limb or axial muscles; Class IIb: predominantly bulbar and/or respiratory muscles; Class III, eye muscle weakness of
PT
any severity, moderate weakness of other muscles; Class IIIa, predominantly limb or
CE
axial muscles; Class IIIb, predominantly bulbar and/or respiratory muscles; Class IV, eye muscle weakness of any severity, severe weakness of other muscles; Class Iva,
AC
predominantly limb or axial muscles; Class IVb, predominantly bulbar and/or respiratory muscles; Class V, intubation needed to maintain airway. According to the anatomic extent of disease at the time of surgery, the Masaoka staging system was used for thymoma clinical staging [12]: Stage I, intact thymic capsule; Stage IIa, Microscopic invasion through the capsule into surrounding fatty tissue; Stage IIb, Macroscopic invasion into capsule; Stage III, macroscopic invasion into adjacent organs, vessels; Stage IVa, dissemination in thoracic cavity; Stage IVb, distant metastases. Depending on the appearance of cells from microscopic investigation, there are three major histological subtypes of thymoma: Type A, the epithelial cells have an oval of fusiform shape; Type B, an epithelioid shape (three subtypes: B1, lymphocyte rich; B2, cortical; B3, epithelial); Type C, tumor contains a variety of malignant
ACCEPTED MANUSCRIPT cytological features. The observation time in these participants was the interval between diagnosis and last contact (death or last follow up). Overall survival (OS), which was calculated as
RI P
T
the period from diagnosis until the date of death, was used for prognostic analyses. The authors state that they have followed the principles outlined in the Declaration of Helsinki. Besides, we also had obtained institutional review board approvals for all
SC
investigations in current study.
MA NU
Quantitative real-time polymerase chain reaction Total RNA was extracted with Trizol reagent (Life Tech, USA) according to the manufacturer’s recommendation. Quantitative RT-PCR analysis was conducted with SYBR Green PCR Master Mix (Life Tech, US) using the Applied Biosystems 7500 RT PCR System. The expression of the housekeeping gene GAPDH was assessed in
ED
each sample. The following PCR primers (Sangon Biotech, China) were used: CXCR4 (forward: 5’-GGAGGGGATCAGTATATACA-3’; reverse:
PT
5’–GAAGATGATGGAGTAGATGG-3’), the anticipated length was 145bp; GAPDH (forward: 5’- CACCCACTCCTCCACCTTTG -3’; reverse: 5’-
CE
CCACCACCCTGTTGCTGTAG - 3’), the anticipated length was 110bp. Relative CXCR4 expression levels were examined by standard curve method. All standard
AC
curves were linear in the required range with acceptable correlation coefficient. The CXCR4 mRNA levels were given as ratios to GAPDH mRNA levels. RT-PCR assays were done in triplicate for each sample, and the average value was used for analysis.
Statistical analysis CXCR4 expression and patient characteristics were summarized using mean and standard deviation. Fisher’s exact test and chi-squared test were used to compare the categorical data. Log-rank test and Kaplan-Meier method were used to estimate overall survivals, and calculated hazard ratios (HR) with the adjusted multivariate Cox regression analysis. All the analyses were performed with MedCalc and SPSS, and significance was defined as p1.8 as “high” and those≤1.8 as “low”. As shown in table 1, patients with high CXCR4 expression and low CXCR4 expression were matched in term of gender,
AC
age, family history of cancer, anti-AChR status and recurrence of MG after thymectomy. Meanwhile, subjects with high CXCR4 expression showed high clinical score in Masaoka stage, MGFA stage and WHO classification. The overall survival of patients with CXCR4 overexpression were worse than those with low CXCR4 levels (hazard ratio, 2.11; 95% confidence interval, 1.08-4.11; p=0.02) (Fig.3A). Similarly, CXCR4 expression was correlated with disease-free survival (HR, 1.84; 95% CI, 1.03-3.29; p=0.04) (Fig.3B). Next the multivariate Cox proportional hazard analysis were carried out to assess the potential prognostic factors for overall survival (table 2). The factors considered here were gender, age, family history of cancer, Masaoka stage, MGFA classification, WHO classification and CXCR4 expression, among which only Masaoka stage, MGFA stage, WHO
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
MA NU
SC
RI P
T
classification and CXCR4 expression were independent factors.
ACCEPTED MANUSCRIPT Discussion Understanding the pathological mechanism of tumor is important for developing
T
target-specific medicines. CXCR4 has been implicated in the etiology of a substantial
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number of tumors because this receptor is believed to play an essential role in chemotaxis, invasion, angiogenesis, metastasis and proliferation [5, 7]. However,
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despite recent evidence suggests CXCL12/CXCR4 axis contributes to the pathology of MG thymus [9], little attention has been devoted to the precise role of CXCR4 in
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the clinical outcomes of MG-associated thymoma. Here, by measuring CXCR4 mRNA levels, our results demonstrated that CXCR4 overexpression was an independent biomarker of poorer prognosis in MG-associate thymoma. This might also suggest CXCR4 could be a potential therapeutic target in cancer treatment. MG-associate thymoma is one complicated disease and its pathological
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mechanism is still unclear. Although various studies have demonstrated the prognostic significances of clinical stage, pathological classification and completed
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surgery resection, the available data were inconsistent due to the scarcity of this disease [13-15]. For example, one recent study revealed that, of all the
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clinical-pathological characteristics, only clinical stage is an independent prognostic factor [16]. In addition, a standardized classification to categorize thymoma into
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typical TMN classifications has not been achieved in clinical practice. Although several potential proteins including p53, epidermal growth factor receptor and cell cycle regulator proteins were investigated in thymoma to prove their predictive or prognostic significance recently[16, 17], evaluation the clinical outcome of thymoma patients was still very difficult. Now it is well accepted that CXCR4 over-expression is a risk factor of short survivals in dozens of types of tumors [18], so this study focused on the clinical significance
between
CXCR4
and
clinic-pathological
characteristics
in
MG-associated thymoma. Our data first conformed that CXCR4 over-expression was also correlated to worse prognosis in MG-associated thymoma. Next we examined whether CXCR4 over-expression was independently associated with worse clinical
ACCEPTED MANUSCRIPT outcome. After adjusted for age, gender, family history of cancer and clinical stage, multivariate analysis suggested CXCR4 were an independent prognostic biomarker. The underlying mechanisms involved in the association between CXCR4
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expression and survivals are uncertain. One possible explanation is the
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“CXCL12/CXCR4 chemokine axis hypothesis” [5]. In the past decades, accumulating evidence demonstrate that the stroma plays a key role in cancer growth and invasion.
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In support of this hypothesis, it was reported that as a chemoattractant, CXCR4 might be essential in cancer development possibly at the level of the tumor niche [19]. Both
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CXCR4 expression on cancer cells and CXCL12 expression by fibroblasts could trigger tumor cell motility, growth and invasiveness. Furthermore, stroma cells from some special microenvironments may modulate the expression of CXCR4, which is believe to account for tumorigenesis and tumor progression. The interactions between
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stroma cell and cancer are reciprocal: the stroma cells may support the growth of tumors; tumor cells can also modulate the microenvironments. Therefore, CXCL12 CXCR4
assemble
a
crucial
axis
between
cancer
cells
and
their
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and
microenvironments, and their interactions could impact the migration, adhesion and
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invasion of cancer cells. Eventually this axis conveys the association between CXCR4 expression and overall survivals.
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Combating MG-associated thymoma remains a major clinical challenge, and thus far results are disappointing. Having a molecular signature would help to identify those patients at high risk of death. Here, our data are consistent with the hypothesis that CXCR4/CXCL12 pathway activation could promote tumor progression. We acknowledge that the hazard ratio for overall survivals from CXCR4 high expression group is only 2.11 in MG-associated thymoma, less than 5.07 in gastric cancer [20], 4.80 in breast cancer [21], 4.03 in ovary cancer [22], 3.80 in rental cancer [23], 3.73 in head and neck cancer [24]. This might limit its application in clinical practice. Our data suggest that CXCR4 may not be used as a single biomarker in survival prognosis, but as one part of multi-molecular biomarkers for MG-associated thymoma. Because of the complexity of MG-associated thymoma, many other molecules are believed to involve in the pathological mechanism and could be potential biomarker for prognosis.
ACCEPTED MANUSCRIPT Further studies with more powerful approaches are needed. Nevertheless, our results imply that CXCR4 have the potential to be a prognostic biomarker and possible therapeutic target. In fact, the important roles of CXCR4 in multiple cancers have
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encouraged the development of clinically viable CXCR4 antagonists, and resulted in the US Food and Drug Administration (FDA) approval of Plerixafor (AMD 3100; Genzyme) for patients with multiple myeloma and non-Hodgkin's lymphoma [25].
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Hence, once our results were validated by other studies, a clinical trial utilizing Plerixafor for MG-associated thymoma with CXCR4 overexpression can be carried
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out.
In summary, CXCR4 expression could be an independent prognostic biomarker
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of poor outcome for patients with myasthenia gravis-associated thymoma.
ACCEPTED MANUSCRIPT References [1] Engels EA, Pfeiffer RM. Malignant thymoma in the United States: demographic
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patterns in incidence and associations with subsequent malignancies. Int J Cancer
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[2] Lewis JE, Wick MR, Scheithauer BW, et al. Thymoma. A clinicopathologic
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[5] Domanska UM, Kruizinga RC, Nagengast WB, et al. A review on CXCR4/CXCL12 axis in oncology: no place to hide. Eur J Cancer 2013; 49,
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[7] Teicher BA, Fricker SP. CXCL12 (SDF-1)/CXCR4 pathway in cancer. Clin
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a systematic review and meta-analysis. BMC Cancer 2014; 14, 49. [9] Weiss JM, Cufi P, Bismuth J, et al. SDF-1/CXCL12 recruits B cells and antigen-presenting cells to the thymus of autoimmune myasthenia gravis patients. Immunobiol 2013; 218, 373-81. [10] Meriggioli MN, Sanders DB. Autoimmune myasthenia gravis: emerging clinical and biological heterogeneity. Lancet Neurol 2009; 8, 475-90. [11] Jaretzki A, 3rd, Barohn RJ, Ernstoff RM, et al. Myasthenia gravis: Recommendations for clinical research standards. Task Force of the Medical Scientific Advisory Board of the Myasthenia Gravis Foundation of America. Neurology 2000; 55, 16-23.
ACCEPTED MANUSCRIPT [12] Masaoka A, Monden Y, Nakahara K, et al. Follow-up study of thymomas with special reference to their clinical stages. Cancer 1981; 48, 2485-92. [13] Detterbeck FC, Parsons AM. Thymic tumors. Ann Thorac Surg 2004; 77, 1860-9.
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[14] Kondo K, Monden Y. Therapy for thymic epithelial tumors: a clinical study of 1,320 patients from Japan. Ann Thorac Surg 2003; 76, 878-84.
Ann Thorac Surg 2006; 81, 2328-34.
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[15] Detterbeck FC. Clinical value of the WHO classification system of thymoma.
[16] Cui F, He J, Liu J, et al. Protein expression status of p53 and epidermal growth
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factor receptor in thymoma. Oncol Lett 2011; 2, 459-63. [17] Baldi A, Ambrogi V, Mineo D, et al. Analysis of cell cycle regulator proteins in encapsulated thymomas. Clin Cancer Res 2005; 11, 5078-83. [18] Zhao H, Guo L, Zhao H, et al. CXCR4 over-expression and survival in cancer: a
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system review and meta-analysis. Oncotarget 2015; 6, 5022-40. [19] Kaplan RN, Riba RD, Zacharoulis S, et al. VEGFR1-positive haematopoietic
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bone marrow progenitors initiate the pre-metastatic niche. Nature 2005; 438, 820-7.
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[20] He H, Wang C, Shen Z, et al. Upregulated expression of C-X-C chemokine receptor 4 is an independent prognostic predictor for patients with gastric cancer.
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PloS one 2013; 8, e71864. [21] Holm NT, Abreo F, Johnson LW, et al. Elevated chemokine receptor CXCR4 expression in primary tumors following neoadjuvant chemotherapy predicts poor outcomes for patients with locally advanced breast cancer (LABC). Breast Cancer Res Tr 2009; 113, 293-9. [22] Sekiya R, Kajiyama H, Sakai K, et al. Expression of CXCR4 indicates poor prognosis in patients with clear cell carcinoma of the ovary. Hum Pathol 2012; 43, 904-10. [23] Chen D, Gassenmaier M, Maruschke M, et al. Expression and prognostic significance of a comprehensive epithelial-mesenchymal transition gene set in renal cell carcinoma. J Urol 2014; 191, 479-86.
ACCEPTED MANUSCRIPT [24] Katayama A, Ogino T, Bandoh N, et al. Expression of CXCR4 and its down-regulation by IFN-gamma in head and neck squamous cell carcinoma. Clin Cancer Res 2005; 11, 2937-46.
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[25] Debnath B, Xu S, Grande F, et al. Small molecule inhibitors of CXCR4.
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Theranostics 2013; 3, 47-75.
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Fig.1 Histologic specimen of one MG-associated thymoma tissue sample stained with
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hematoxylin and eosin (×200).
Fig.2 Receiver operating characteristic (ROC) curve used to define the cutoff value
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between the high and low levels of CXCR4 expression. The optimal cutoff value that gave the maximal sensitivity and specificity for CXCR4 expression was 1.8.
Fig.3 Kaplan-Meier analysis for overall survival (A) and disease-free survival (B)
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based on CXCR4 expression among myasthenia gravis-associated thymoma patients.
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HR, hazard ratio; CI, confidence interval.
ACCEPTED MANUSCRIPT Table 1. Clinical characteristics of patients according to CXCR4 high- or low-expression
3(8%) 37(92%) 38(95%) 4(10%)
4(9%) 40(91%) 43(98%) 3(7%)
Recurrence of MG after thymectomy
Masaoka stage 3(8%) 12(30%) 16(40%) 9(22%)
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Stage I Stage II Stage III Stage IV
MGFA classification
5(13%) 8(20%) 12(30%) 10(25%) 5(12%)
WHO classification
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Class I Class II Class III Class IV Class V
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Anti-AChR
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CXCR4 low expression (n=44) 23:21 36.6±11.9
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Gender (female: male) Age (years) Family history of cancer yes no
CXCR4 high expression (n=40) 22:18 38.4±10.6
p value 0.80 0.24 0.78
0.47 0.60
