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ORIGINAL ARTICLE  
Year : 2020  |  Volume : 63  |  Issue : 4  |  Page : 564-569
Role of platelet aggregation in metastatic breast cancer patients


Department of Pathology, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India

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Date of Submission21-Oct-2019
Date of Decision19-Jan-2020
Date of Acceptance22-Jan-2020
Date of Web Publication28-Oct-2020
 

   Abstract 


Background and Aims: Breast cancer is the most common female cancer in the world. Although early detection and systematic adjuvant therapy has improved survival, distant metastasis remains the leading cause of breast related mortality. The relationship between tumor and the hemostatic system is increasingly recognized as an important regulator of breast cancer progression. Tumors have the ability to induce platelet aggregation which is referred as tumor cell induced platelet aggregation (TCIPA). This study highlights that increased platelet aggregation plays an important role in metastasis of breast cancer. The aim here is to study the role of platelet aggregation in metastatic breast cancer patients using: • ADP • Thrombin. Methods: 30 cases (n = 30) of metastatic breast cancer and 30 controls (n = 30) of non-metastatic breast cancer which were clinically diagnosed and histopathologically confirmed were included in this study. Platelet aggregation studies in vitro using ADP and Thrombin were performed using an optical aggregometer in both cases and controls. Other parameters like platelet count, histological grade and surrogate molecular classification was also correlated with platelet aggregation. Results: In this study, increased aggregation was seen with ADP and thrombin in the metastatic cases and none showed increased aggregation in the non-metastatic breast cancer patients. Also, high platelet count and higher histological grade correlated with increased aggregation. However, no correlation was seen between platelet aggregation and the surrogate molecular classification. Conclusion: It was concluded from this study that platelet aggregation has an important part to play in the tumor metastasis of breast cancer patients.

Keywords: Adenosine diphosphate, metastatic breast cancer, platelet aggregation, thrombin

How to cite this article:
Singla T, Singla G, Ranga S, Singla S, Arora R. Role of platelet aggregation in metastatic breast cancer patients. Indian J Pathol Microbiol 2020;63:564-9

How to cite this URL:
Singla T, Singla G, Ranga S, Singla S, Arora R. Role of platelet aggregation in metastatic breast cancer patients. Indian J Pathol Microbiol [serial online] 2020 [cited 2020 Nov 25];63:564-9. Available from: https://www.ijpmonline.org/text.asp?2020/63/4/564/299326





   Introduction Top


Breast cancer is the most common cancer among females in the world. It has now become the most common female cancer in urban India with an annual incidence of approximately 144,000 new cases being reported. Although early detection, precise resection using wide margins and systematic adjuvant therapy has improved survival, distant metastasis remains the leading cause of breast related mortality.[1]

Recent studies have suggested that a correlation exists between metastatic breast cancer and hemostatic system which has been recognized as an important regulator in its progression.[2] It has been documented that there is an association between circulating tumor cells (CTCs) and host platelets. Circulating tumor cells (CTCs) have been defined as tumor cells circulating in the peripheral blood of patients which shed from either the primary tumors or its metastases.[3] They cause activation of host platelets that leads to shape change, protein release and membrane based changes and further help in promote metastasis.[2]

One of the methods of platelet activation is tumor cell induced platelet aggregation (TCIPA).[4] Tumor cells can activate platelets either by tumor cell-induced thrombin generation or by releasing ADP and thromboxane A2 which are potent pro-aggregatory agents. ADP induces platelet aggregation via P2Y12 receptor, while thrombin which is a key enzyme in coagulation cascade acts via proteinase activated receptors.[5]

In the past role of cancer cell-induced release of platelet granules leading to liberation of potent pro-aggregatory agents like ADP and thrombin has been described.[6] However, there is a paucity of Indian literature describing the role of platelet aggregation in tumor metastasis in patients of breast cancer.

Objectives

To study if platelet aggregation plays role in metastatis of breast cancer patients using platelet aggregating agents ADP and Thrombin.


   Methods Top


This was a hospital-based comparative observational study carried out for a duration of 18 months. It was approved by the local ethical committee of our hospital. The study group comprised of 30 cases of metastatic breast cancer patients and 30 controls of non-metastatic breast cancer patients.

Inclusion criteria

  • All women who were clinically diagnosed and histopathologically confirmed as metastatic breast cancer patients.


Exclusion criteria

  • Patients on chemotherapy and radiotherapy
  • Patients on antiplatelet therapy
  • Benign tumors and sarcomas of breast.


Routine clinical details and investigations were taken from clinical case sheet and patients with disorders that may affect the platelet aggregation were excluded from study.

  1. 10 ml of blood sample was collected and anticoagulated with 3.8% sodium citrate in the ratio of 1 part of anticoagulant to 9 parts blood
  2. Platelet rich plasma (PRP) was prepared by centrifuging the sample at 100g for 15 minutes
  3. Platelet poor plasma (PPP) was prepared by centrifuging the sample at approximately 2400g for 20 minutes
  4. Reagents


    1. ADP – 5 μl reconstituted with 5.0 ml of physiological saline
    2. Thrombin- 100 μl reconstituted with 1.0 ml of physiological saline


  5. Platelet Aggregometer- Detection of platelet aggregation was done by Chrono log series 490 dual and four channel optical aggregometer systems.


In-vitro aggregation recordings were characterized by their appearances:

  • Shape change
  • A first wave of aggregation (primary aggregation) that may reverse and return towards the PRP baseline
  • Irreversible second wave aggregation that occurs when the platelets' secreted granule contents become the stimulus and cause additional aggregation.


Aggregation curves were also characterized by:

  • The maximum amount of change in the light transmission caused by the agonist (percentage aggregation)
  • The slope or rate of aggregation, in % change of aggregation per minute.


Statistical analysis

Quantitative variables were compared using Unpaired t-test/Mann-Whitney Test and qualitative variables were correlated using Chi-Square test/Fisher's exact test. A P value of <0.05 was considered statistically significant.


   Results Top


A total of 30 cases of metastatic breast cancer patients and 30 controls of non-metastatic breast cancer patients were studied. The age group in our cases varied from 29-62 years (mean- 47.4) and in the control group the age varied from 20-62 years (mean 45.6). The platelet count in the range of 1.5-4 lac/cumm was taken as normal. In the metastatic breast cancer group, 17 cases (56.7%) had a count >4 lac/cumm, while 13 cases (43.3%) had platelet count in the normal range. In the non-metastatic breast cancer group, all patients had platelet count within normal limits. ADP which was added to the platelet rich plasma in metastatic breast cancer patients showed platelet aggregation in a normal range of 61-72% in 66.7% (n = 20) of the cases. Higher range of platelet aggregation (>72%) was observed in 26.7% (n = 8) of the cases [Figure 1] and only 6.7% (n = 2) metastatic breast cancer patients showed aggregation less than 60%. In the control group, 63.3% (n = 19) of the patients showed aggregation in the normal range of 61-72% [Figure 2] while 36.7% (n = 11) showed platelet aggregation <60%. None in the control group showed aggregation in the higher range [Table 1]. The mean platelet aggregation was calculated which was 67.8% in cases and 60.7% in control population. Also, a comparison of platelet aggregation (ADP) was done between the cases and control groups and was found to be statistically significant (P < 0.05).
Figure 1: Platelet aggregation with ADP in a metastatic breast cancer patient

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Figure 2: Platelet aggregation with ADP in a non-metastatic breast cancer patient

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Table 1: Platelet aggregation with ADP in cases and controls

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On addition of thrombin to PRP in our metastatic breast cancer cases, it was seen that maximum number of cases i.e., 76.7% (n = 23) showed platelet aggregation in a higher range >82% [Figure 3]. 23.3% (n = 7) cases showed aggregation in the normal range of 58-82% and none showed aggregation in the lower range. As compared to the cases, in the control population, 83.3% (n = 25) showed aggregation in the normal range of 58-82% [Figure 4]. 16.7% (n = 5) patients showed aggregation in the lower range <58% and none in the higher range [Table 2]. The mean platelet aggregation was calculated and comparison of platelet aggregation (Thrombin) was done between the cases and controls. The mean platelet aggregation was 90.2% and 70.5% in cases and controls respectively. Also, a statistically significant correlation of platelet aggregation (Thrombin) was found between cases and controls (P < 0.05).
Figure 3: Platelet aggregation with Thrombin in a metastatic breast cancer patient

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Figure 4: Platelet aggregation with Thrombin in a non-metastatic breast cancer patient

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Table 2: Platelet aggregation with thrombin in cases and controls

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We then studied if a correlation existed between ADP and platelet count in the metastatic breast cancer cases. Amongst cases with normal platelet count (n = 13) 86.4% patients (n = 11) had platelet aggregation with ADP in the normal range and 15.4% patients (n = 2) had platelet aggregation with ADP in the lower range. In patients with high platelet count (n = 17), 52.9% (n = 9) showed aggregation in the normal range, and 47.1% patients (n = 8) showed aggregation in the higher range which was statistically significant [Table 3]. (P value <0.05, chi square test).
Table 3: Correlation of ADP with platelet count

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Similarly, a correlation between thrombin and platelet count was studied in breast cancer cases and was found to be statistically significant [Table 4] (P value <0.05, chi square test).
Table 4: Correlation of Thrombin with platelet count

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We also studied if a correlation existed between histological grade of breast cancer and platelet aggregation. Correlation between histological grade and ADP was thus calculated. The histological grade was defined by the Modified Bloom Richardson score. Among the 30 cases, there was no case of grade 1, 12 cases of grade 2 and 18 cases of grade 3. In patients with grade 2 (n = 12), 83.3% (n = 10) cases showed platelet aggregation with ADP in the normal range and 16.7% cases (n = 2) showed aggregation with ADP <60% and none with high platelet aggregation. Amongst grade 3 patients (n = 18), 55.6% (n = 10) showed aggregation with ADP in normal range and 44.4% (n = 8) cases showed aggregation >72% [Table 5] (P value <0.05, chi square test). A similar correlation was seen with thrombin and the histological grade. All grade 3 patients showed increased platelet aggregation with thrombin [Table 6] (P value <0.05, chi square test).
Table 5: Correlation of ADP with histological grade (given by modified BR score)

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Table 6: Correlation of Thrombin with histological grade (given by modified BR score)

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   Discussion Top


In the present study, we saw that increased aggregation with both ADP and thrombin was seen in metastatic breast cancer patients and the difference was statistically significant. The result of our study is similar to that of Cooke et al.[7] who hypothesized that patients with metastatic cancer displayed platelet hyperreactivity, reflecting their associated high risk of thrombosis. In a cohort of patients with metastatic cancer (n = 13), they assessed platelet function using well-established assays of platelet reactivity (agonist induced platelet aggregation, spontaneous platelet aggregation, and agonist induced P-selectin expression). In comparison with healthy controls (n = 10), patients with metastatic cancer displayed global platelet hyperreactivity with increased spontaneous platelet aggregation.[7]

On addition of an agonist to PRP or whole blood platelets get activated, causing platelets to change their shape followed by platelet granule release and synthesis of positive feedback mediators, like thromboxane A2 and platelet activating factor, which are potent platelet aggregating agonists. This results in activation and recruitment of additional platelets, which coupled with the binding of fibrinogen, mediates aggregation. Each agonist stimulates platelets via different platelet surface receptors, which subsequently leads to platelet aggregation via a specific pathway. Adenosine diphosphate (ADP) is contained in platelet dense granules and is considered a secondary mediator of aggregation. The major ADP receptors, P2Y1 and P2Y12, are both involved in platelet aggregation.[8]

Thrombin has a multifaceted role in hemostasis and it forms an important link between primary and secondary coagulation responses. It has a role in tumorigenesis and angiogenesis, with thrombin signaling being a major contributor to the metastatic tumor dissemination.[9] Tumor-enhancing effects of thrombin include induction of TCIPA, increased tumor-cell adhesiveness, promigratory and chemotactic effects, and up-regulation of VEGF expression by tumor cells.[10],[11],[12] Importantly, thrombin is also the most potent platelet activator, and it functions via the two platelet PAR receptors, PAR-1 and PAR-4.

We also correlated the platelet count of the cases with platelet aggregation. It was seen that thrombocytosis correlated with increased aggregation both with ADP and thrombin. The result was similar to a retrospective study done by Stravodimou et al.,[13] in which they showed that patients with thrombocytosis were more likely to have metastases at diagnosis with breast cancer while the normal platelet group was more likely to have developed metastatic disease at a time later in the course of the disease.[13] There is a complex interplay between platelets and tumor cells. Thrombocytosis induces formation of large platelet hetero-aggregates. These hetero-aggregates of platelets and tumor cells can embolize in the microcirculation and aid in the process of extravasation of tumor cells in metastatic sites. Platelets in their granules carry numerous bioactive molecules and growth factors like PDGFα that promotes EMT which gives a mesenchymal phenotype to the epithelial cells that promotes metastasis.

We correlated another parameter, histological grade of the metastatic breast tumors with in vitro platelet aggregation. It was seen that increased aggregation was associated with a higher grade of the tumor. However, no study has been done to see the correlation between the grade and the increased platelet aggregation. We hypothesize that high grade tumors have increased chances of EMT with dissemination of tumor cells in circulation and cause metastasis. Platelets promote the survival of circulating tumor cells in the blood stream and facilitate their extravasation and the colonization of new microenvironments.[14],[15]

Our study has an important clinical utility. Antiplatelet drugs can play an important role in attenuating tumor metastasis in patients. Few studies have been conducted in this regard for eg- aspirin use has been most extensively studied in colorectal and breast cancer, with proven efficacy in the prevention of colorectal cancer.[16] Aspirin-mediated inhibition of platelet aggregation is well documented, and recently aspirin has also been shown to attenuate platelet protein release.[17] Roop et al. conducted a randomized phase II trial investigating the effect of platelet function inhibition on circulating tumor cells in patients with metastatic breast cancer. There was no difference in the proportion of detectable CTCs in the treatment and control group at 1 month.[18] However, future studies are required to assess the role of antiplatelets in metastatic breast cancer.

To conclude our study proved that platelet aggregation plays an important role in metastasis of breast cancer patients. Large aggregates of platelets help in the dissemination of tumor cells in the microcirculation and aid in the extravasation of tumor cells at the metastatic sites.

However, our study has an important limitation. The sample size was small so more studies are warranted in this regard to conclusively define the role of platelet aggregation in metastasis of breast cancer patients and search for more pathways involved in tumor dissemination by platelets since platelet aggregation is of the many possible pathways involved.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Tao M, Ma D, Li Y, Zhou C, Zhang Y, Duan W. Clinical significance of circulating tumor cells in breast cancer patients. Breast Cancer Res Treat 2011;129:247-54.  Back to cited text no. 1
    
2.
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3.
Lian L, Li W, Mao Y, Zhang Y, Chen K, Duan W, et al. Inhibition of MCF-7 breast cancer cell induced platelet aggregation using a combination of antiplatelet drugs. Oncol Lett 2013;5:675-80.  Back to cited text no. 3
    
4.
Gasic GJ, Gasic TB, Stewart CC. Anti-metastatic effects associated with platelet reduction. Proc Natl Acad Sci USA 1968;61:46-52.  Back to cited text no. 4
    
5.
Alonso-ED, Strongin AY, Chung AW, Deryugina EI, Radomski MW. Membrane type-1 matrix metalloproteinase stimulates tumour cell induced platelet aggregation: Role of receptor glycoproteins. Br J Pharmacol 2004;141:241-52.  Back to cited text no. 5
    
6.
Jurasz P, Alonso ED, Radomski MW. Platelet cancer interactions: Mechanisms and pharmacology of tumour cell induced platelet aggregation. Br J Pharmacol 2004;143:819-26.  Back to cited text no. 6
    
7.
Cooke NM, Egan K, McFadden S, Grogan L, Breathnach OS, O'Leary J, et al. Increased platelet reactivity in patients with late-stage metastatic cancer. Cancer Med 2013;2:564-70.  Back to cited text no. 7
    
8.
Fabre JE, Nguyen M, Latour A, Keifer JA, Audoly LP, Coffman TM, et al. Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1-deficient mice. Nat Med 1999;5:1199-202.  Back to cited text no. 8
    
9.
Haralabopoulos GC, Grant DS, Kleinman HK, Maragoudakis ME. Thrombin promotes endothelial cell alignment in Matrigel invitro and angiogenesis in vivo. Am J Physiol 1997;273:239-45.  Back to cited text no. 9
    
10.
Wojtukiewicz MZ, Tang DG, Nelson KK, Walz DA, Diglio CA, Honn KV. Thrombin enhances tumor cell adhesive and metastatic properties via increased alpha IIb beta 3 expression on the cell surface. Thromb Res 1992;68:233-45.  Back to cited text no. 10
    
11.
Zhou H, Gabazza EC, Takeya H, Deguchi H, Urano H, Adachi Y, et al. Prothrombin and its derivatives stimulate motility of melanoma cells. Thromb Haemost 1998;80:407-12.  Back to cited text no. 11
    
12.
Yamahata H, Takeshima H, Kuratsu J, Sarker KP, Tanioka K, Wakimaru N, et al. The role of thrombin in the neo-vascularization of malignant gliomas: An intrinsic modulator for the up-regulation of vascular endothelial growth factor. Int J Oncol 2002;20:921-8.  Back to cited text no. 12
    
13.
Stravodimou A, Voutsadakis IA. Pretreatment thrombocytosis as a prognostic factor in metastatic breast cancer. Int J Breast Cancer 2013;2013:289563.  Back to cited text no. 13
    
14.
Cristofanilli M, Budd GT, Ellis MJ. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Eng J Med 2004;351:781-91.  Back to cited text no. 14
    
15.
Yang J, Weinberg RA. Epithelial-mesenchymal transition: At the crossroads of development and tumor metastasis. Dev Cell 2008;14:818-29.  Back to cited text no. 15
    
16.
Cole BF, Logan RF, Halabi S, Benamouzig R, Sandler RS, Grainge MJ, et al. Aspirin for the chemoprevention of colorectal adenomas: Meta-analysis of the randomized trials. J Natl Cancer Inst 2009;101:256-66.  Back to cited text no. 16
    
17.
Coppinger JA, O Connor R, Wynne K, Flanagan M, Sullivan M, Maguire PB, et al. Moderation of the platelet release response by aspirin. Blood 2007;109:4786-92.  Back to cited text no. 17
    
18.
Roop RP, Naughton MJ, Poznak CV, Schneider JG, Lammers PE, Pluard TJ, et al. A randomized phase II trial investigating the effect of platelet function inhibition on circulating tumor cells in patients with metastatic breast cancer. Clin Breast Cancer 2013;13:409-15.  Back to cited text no. 18
    

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Correspondence Address:
Gaurav Singla
Department of Pathology, Vardhman Mahavir College and Safdarjung Hospital, New Delhi - 110 029
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_817_19

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    Figures

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