| Background and AimsLung cancer accounts for 17%of new cancer cases and approximately one quarter of all cancer deaths globally,with NSCLC being the most common type of lung cancer.The lungs have an intimate relationship with platelets because the lung is the organ of thrombopoiesis at the latest stage of the MK/platelet hematopoietic linage.Platelets that were supplied to a malignant neoplasm contributed to cancer metastasis at each phase of the metastasis.Approximately 50%of total platelets that are produced from circulating MKs are retained in the pulmonary capillary bed and sheared by the pulmonary capillary vessel.There is evidence demonstrating that lung cancer lesions receive blood supply mostly from pulmonary system.Therefore,it’s highly possible that MKs in lung cancer tissue are transported through the pulmonary artery.NSCLC tumors tend to overexpress SDF-1,which is a powerful chemotactic factor for mature MKs,suggesting that lung cancer cells are more capable of recruiting MKs into tumoral sites.Similar to MKs in a normal lung capillary,functional MKs supplied to the lung cancer capillary may induce local thrombopoiesis.The outcome of local thrombopoiesis leads to higher level platelet in cancer capillaries,then bringing to the step that thousands of platelets coat tumor cells and facilitate metastases of tumor cells.More MKs trapped in a tumor capillary bed may produce more platelets in the tumor region,and a higher regional platelet level may fuel development and metastasis of tumor.Therefore,we hypothesized that MKs in cancer tissue are linked to metastasis and poor prognosis of patients with lung cancer.And we also hypothesized that axis of COX-2/SDF-1/MK/Plt is possible mechanism for intra-tumoral metastasis.Methods(1)A total of 629 NSCLC patients underwent radical resection with or without adjuvant chemotherapy in the Department of Thoracic Surgery of The First Affiliated Hospital of Guangzhou Medical University(Guangzhou,China)between September of 2009 and December of 2012 were retrospectively investigated.Pathologically proven stage pTl-3N1-2M0 NSCLC was obtained.Level of venous platelet was measured before surgery and one month after surgery.All patients were followed;the follow-up data consisted of clinical assessments,brain MRI,chest CT,and ultrasound or CT of the abdomen.(2)Immunohistochemical staining was used.As a preliminary experiment,the staining power of anti-CD61 and anti-Factor Ⅷ for MKs was compared.Anti-CD34 was utilized to visualize endothelial cells stained by red precipitate in the cytoplasm.Bone marrow and rectal cancer was adopted as positive control and negative control.Yellowish-brown CD61+giant cells(diameter>10 μm)located in CD34+microvessels were defined as MKs.Two batches of sections from the same paraffin-embedded samples were respectively stained with anti-SDF-1 and COX-2.(3)The average of microvessel count in five randomized visual fields under microscope was considered as density of microvessels.Microthrombi in CD34+microvessels were also identified under 400 × magnifications.Cases harboring one microthrombus or more were recognized as positive cases.Univariate analyses of DFS between MK(+)group and MK(-)group were undertaken using the Kaplan-Meier product-limit method with the log-rank test.Variables that were found to be significant in the univariate analysis were included in a multivariate Cox proportional hazard model to determine the independence.(4)SDF-1 expression of MK(+)group and MK(-)group were analyzed by Pearson’s chi-squared test,in order to explore the possibility of MK to attract cancer cell.Then,we analyzed correlation between SDF-1 and COX-2 to make clear if COX-2 is the initial factor which led to high expression of SDF-1.(5)All patients were divided into four subgroups according to platelet count(<300×109/L,≥300 ×109/L)and MK(+,-),DFS of four subgroups was compared.DFS of patients who platelet count decreased after surgery and patients who platelet count did not decreased was compared.Results(1)Patient baseline characteristics:the 629 patients included 142 patients with SCC,441 patients with NSCC(adenocarcinoma/large cell carcinoma/adenosquamous carcinoma),45 patients with all other tumor types,and 1 patient with unclear pathology.Of the total patients,80.8%were younger than 70 years old and 58.0%were male.The distribution of tumor size was as follows:<3 cm,281(44.7%);3-5 cm,193(30.7%);5-7 cm,98(15.6%);≥7 cm,49(7.8%);and unknown size,8(1.3%).The distribution of pN stage was as follows:pN0,378(60.1%);pN1,84(13.4%);pN2,139(22.1%);and unknown pN,28(4.6%).There were 119 out of 629(18.9%)patients with CD61+MKs located in red-stained CD34+microvessels.(2)As a preliminary experiment,Factor Ⅷ staining was weaker than CD61 staining in the tumor and bone marrow.Therefore,this cohort was completely stained by anti-CD61.Male patients showed a significantly higher proportion of MK+cases than female patients(male:22.2%vs.female 14.4%;P=0.014).Patients with NSCC showed significantly less MK+cases compared to the patients with SCC and patients with other tumors(NSCC,15.9%;SCC,23.2%;others 33.3%;P=0.005).In univariate analysis,the median DFS of the MK+group was shorter than the median DFS of the MK-group(69.1 vs.80.5 months;P=0.021).Multivariate analysis indicated that MKs in tumor tissue was an unfavorable prognostic factor for DFS(HR 1.351,P=0.065),the impact of which was more significant in non-squamous cell carcinoma(NSCC)(HR 1.710,P=0.008)and in patients with NO(HR 1.883,P=0.009).Although systemic platelet count of the MK+group was significantly higher than the MKgroup(270.6 vs.243.6 ×109/L,P=0.007),the stratified subgroup DFS curves(P=0.003)showed that the effect of MKs on prognosis was independent of the blood platelet count.(3)SDF-1 showed a high expression of 24.1%in MK-tumors,which was lower than 33.6%expression in MK+tumors(P=0.033).44.3%cases from COX-2-group had strong expression of SDF-1,which was significantly lower than 53.8%cases in COX-2+ group.Microthrombus formation was significantly greater in the MK+group than that in the MK-group(MK+24.6%vs.MK-12.7%;P=0.001).(4)Univariate analysis of DFS revealed that the group which platelet count decreased after surgery occupied better survival advantage than the group which platelet count did not decrease after surgery.However,no significant difference existed between them(P=0.117).Conclusions(1)The result of Factor Ⅷ staining and CD61 staining during the preliminary experiment indicated the presence of MKs in the capillaries of lung tumors;however,Factor Ⅷ staining was weaker than CD61 staining in the tumor and bone marrow.The MKs in microvessels implicated that it had the function to shed platelets.(2)The presence of MK predicted poor prognosis.MK+ cases possessed more cases of microthrombus formation,which indicated that it must be higher concentration of platelet in MK+group.Evidences above demonstrated that MK in tumor had function of thrombopoiesis.(3)The prognostic impact of MKs in addition to that of systemic platelet count further confirmed that regional MK-producing platelets in tumors might contribute to NSCLC metastasis.(4)Higher microvessel density in MK+group delivered the message that tumor itself had the power to attract MK,IHC method further confirmed that SDF-1 was a molecular signal to attract MK.COX-2 represented one of inflammatory factors to initiate this process.In conclusion,this is the first study to show the existence of CD61+MKs in the cancer microenvironment and to prove its prognostic impact.The results indicate that malignant tumors containing MKs have the capacity to produce platelets.Intra-tumoral thrombopoiesis is hypothesized as a newly discovered mechanism for continuous growth and distant metastasis of malignant tumors. |
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