Page 247 - Haematologica - Vol. 105 n. 6 - June 2020

Hypercoagulation and relapse in breast cancer
Introduction
Cancer growth and dissemination is associated with the development of a subclinical hypercoagulable state in the host, detectable by coagulation laboratory tests.1,2 The pathogenesis of blood coagulation activation in cancer is complex and multifactorial. Among other factors, the expression of tumor cell clot-promoting properties, and the inflammatory response of the normal host cells to the tumor play a central role in the cancer-associated pro- thrombotic diathesis by leading to the activation of the blood clotting system.2,3 Importantly, tumor cells of differ- ent origin express different procoagulant profiles, and the clotting system can be triggered to various extents accord- ing to the type and stage of the malignant disease. 4 Furthermore, the patient's hypercoagulable state worsens with cancer progression and metastatic spread, which supports the concept of a tight relationship between tumor burden and hemostatic abnormalities.5 All this con- tributes to an increased risk of thrombosis, and also affects the tumor biology by favoring tumor growth and metas- tasis.6,7
Over the last three decades, several studies have evalu- ated the role of clotting activation biomarkers in relation to cancer outcomes, including disease progression, response to chemotherapy, and mortality.8-11 As recently reviewed,12 however, most of these studies were retro- spective in nature and not always specifically designed to address the impact of thrombotic biomarkers on cancer outcomes. In addition, recruitment was often mono-insti- tutional, and included small heterogeneous cohorts of patients with different treatments. Therefore, despite the encouraging results reported, new data coming from large prospective cohorts are needed. In this respect, breast can- cer patients with limited resected disease are an important setting to test whether abnormal levels of circulating thrombotic biomarkers may represent a novel non-inva- sive factor for better prediction of disease recurrence (DR) risk.
Breast cancer, the most common cancer in women, is a highly heterogeneous disease presenting with a broad range of clinical and molecular characteristics, as well as variability in clinical progression. In recent years, informa- tion campaigns and large-scale prevention screening pro- grams have contributed to an increase in the rate of early diagnosis,13 with a consequent improved rate of patients treated at an early stage of the disease.14 For the treatment choice, patients are classified according to intrinsic biolog- ical subtypes within the breast cancer spectrum, using clinico-pathological criteria, i.e. the immunohistochemical definition of estrogen receptor (ER) and progesterone receptor (PR), the detection of overexpression and/or amplification of the human epidermal growth factor receptor 2 (HER2) oncogene, and Ki-67 labeling index. This classification allows for a more personalized approach to the medical treatments, with favorable results. However, in spite of this, almost 10-15% of these patients still experience local or distant recurrences in the first five years from diagnosis,13,15,16 mainly in the high-risk category, characterized by worse prognostic factors in which the use of adjuvant systemic chemotherapy is jus- tified.17,18 In this category, the identification of patients at the highest risk of relapse is an important area in which to improve personalized treatments and, ultimately, cancer care.
In the present study, we tested the predictive role of cir- culating clotting biomarkers on DR risk in a large patient cohort with resected high-risk breast cancer scheduled to receive post-surgical adjuvant chemotherapy enrolled in the ongoing prospective observational Italian multicenter HYPERCAN (“HYPERcoagulation and CANcer”) study.19 Specifically, we investigated whether baseline levels of D- dimer, fibrinogen, prothrombin fragment 1+2 (F1+2), and FVIIa/antithrombin (FVIIa-AT) complex were associated with clinico-pathological characteristics of breast cancer, and whether these biomarkers might be effective in pre- dicting DR in patients at an early stage of the disease.
Methods
Study design and study population
For the present study, we considered patients of both genders with a high-risk surgically treated breast cancer enrolled in the ongoing HYPERCAN study19 (Online Supplementary Appendix). The study protocol was approved by the local ethics committee. A data extraction from the HYPERCAN database was performed in January 2018. Patients enrolled from March 2012 to December 2015 were considered for the study. Of the 1,042 patients with resected breast cancer enrolled during this period, 953 had an adequate follow-up time. Data on ER/PR and HER2 positivity were available in 788 patients; in this subgroup, bio- markers data were available for 701 patients (Online Supplementary Figure S1). Median time of follow up at the time of the present analysis is 3.4 years.
Patient classification
Patients were categorized in subtypes according to tumor expression of ER, PR, ki67, and HER2 according to the American Society of Clinical Oncology (ASCO) - College of American Pathologist (CAP) guideline.20 Data were derived from clinical pathology reports. ER/PR positivity defined the luminal groups, which included: Luminal A [i.e. HER2-negative (neg) and low Ki67)]; Luminal B HER2-neg (i.e. HER2 negativity and high Ki67), and Luminal B HER2-positive (pos) (i.e. HER2-pos and any ki67). HER2-pos cancer subtype was defined by negativity for ER/PR and positivity for HER2, while triple negative (TN) cancer was defined by ER/PR and HER2 negativity.
Blood sampling and plasma preparation
Fasting peripheral venous blood was drawn at enrollment prior to start of systemic adjuvant chemotherapy using a 21-gauge nee- dle into 6 mL vacutainer tubes containing 0.109 M citrate (9:1 vol/vol; Becton Dickinson) after discarding the first 2-3 mL of blood.21 Within two hours from collection, plasma was isolated by two sequential centrifugations at 2,600 rpm for 15 minutes (min)22 and stored at -80°C. All samples were anonymized and tested in blind at the Laboratory of Hemostasis and Thrombosis Center (Hospital Papa Giovanni XXIII, Bergamo, Italy).
Hypercoagulation biomarkers
Plasma levels of D-dimer (HemosIL D-dimerHS, Werfen Group) and fibrinogen (QFA thrombin, Werfen Group) were measured on an automated coagulometer analyzer (ACL TOP500, Werfen Group). F1+2 (Siemens Healthcare Diagnostics) and FVIIa-AT complex (Stago) were determined by commercial ELISA. Reference intervals for coagulation biomark- ers were internally generated from a group of 200 apparently healthy controls (170 females; 30 males) with no chronic or acute diseases. Median age was 49 years (range, 35-64 years).
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