Study setting
This prospective cohort study was performed in Dr. Kariadi hospital, the main teaching hospital for Medical Faculty of Diponegoro University, Semarang, Indonesia. The hospital is a tertiary referral hospital for all patients with cancer in Central Java province. The study end point was an objectively confirmed DVT within a 3-month observation period after first line chemotherapy in cancer without verified either venous or arterial thrombosis at enrollement. All patients were evaluated by the Wells’ score probability test and D-dimer level: patients with positive D-dimer (≥500 ng/mL) and/or high probability Wells’ score (≥2) were referred to vascular dupplex ultrasound. Short cycle chemotherapy was managed as outpatient while more than 2 days protocol were administered as inpatient. None of the patients received primary prophylaxis with anticoagulation because we only included those with ECOG performance status at least two assumed they can maintain active mobilization. This study was approved by Dr. Kariadi Hospital internal review board.
Patients’s selection and data collection
During November 2016 and February 2017, we included a total of 246 consecutive and unselected newly diagnosed cancer patients for screening. Forty consecutive patients with active cancers undergoing chemotherapy were enrolled in the study, see Figure 1 for patient’s disposition. The inclusion criterias were newly diagnosed of cancer with histological confirmation, age over 18 years, ECOG ≤ 2, consent to participate, and undersigned a written informed consent. Exclusion criteria were overt bacterial or viral infection within the last two weeks, hepatic and renal dysfunction, venous or arterial thromboembolism within the last three months and use of anticoagulant, aspirin or statin, and surgery or radiotherapy within the last two weeks. Inherited VTE risk factors were not taken into consideration. All patients were screened for DVT using duplex sonography and only those with negative result were included in this prospective study. Probability test for Wells’ score (which will be described later) should also “DVT unlikely” for all participants.
Before this study was conducted, all patients had been informed about study’s details in an individual interview. Then, anamnesis on patients’ cancer history, tumor site, tumor histology and tumor stage were documented. The patients who met the inclusion criteria were selected as the subjects of this study. Samples were examined twice, before and after chemotherapy to see the plasma levels of sP-selectin, VWF:Ag and ADAMTS13. Age, gender, smoking history, ABO blood group, body mass index, DVT-related history, diabetes, hypertension, history of atherosclerosis disease, drug, type of cancer, chemotherapy regimen were recorded carefully.
Treatment and follow-up
All patients underwent chemotherapy or chemoradiotherapy. Cisplatin/carboplatin based chemotherapy included gemcitabine, paclitaxel, docetaxel or pemetrexed. Fluorouracyl-based chemotherapy were used in colorectal cancer as a component of FOLFOX/FORFIRI/De Grammont protocol with or without bevacizumab or cetuximab. Anthracyclin-based in several patients, including those with AML who receiving 3+7 protocol containing daunorubicin or doxorubicin in R-CHOP protocol. The patients were monitored for 3 months, evaluated during their follow-up visits in hematology and medical oncology clinic by the attentding staff. In the first, second and third month, evaluations on DVT occurrence were conducted clinical examinations as well as Wells pretest probability model to assess DVT. If the Wells score was ≥ 2, color duplex sonography was performed to established the DVT occurence. Otherwise, color duplex sonography will be conducted at the end of the third month.
Outcome measure: Deep vein thrombosis
The endpoint of the study was the occurrence of DVT, either asymptomatic, symptomatic or fatal VTE, confirmed by dupplex ultrasound. Color duplex sonography was performed at the Radiology Department of Dr. Kariadi Hospital, Semarang, Indonesia. Patients with clinically suspected DVT and Wells score ≥ 2 was assessed for DVT by using Loqic 7 pro US imaging system (Loqic 7 pro; GE Healthcare, USA) with the 7-10 Hz linear probe. The diagnosis of DVT was based either on presence of a non-compressible segment (compression ultrasound test – CUS) or the flow impairment on color Doppler imaging. Patients were examined for both proximal (popliteal, femoral, and common femoral vein) and distal (peroneal and tibial veins) DVT. The first dupplex ultrasound was performed within the first 7 days after inclusion, and then during chemotherapy whether Wells’ score ≥ 2, or at final observation in the third months. To avoid investigator-related variations of the results, color duplex sonography was performed in each patient by the same investigator (G.S., MD, PhD.) at all time points.
Prediction score
Two prediction score were used in this study: the Khorana and Well’s score. For each patiens, we calculated the Khorana risk score to stratify the risk of VTE in canccer patients undergoing chemotherapy.22 Patients were assigned to three risk categories for VTE: low risk= 0, intermediate risk= 1-2, and high-risk ≥ 3. Wells score considers 1 point each for active cancer, paralysis, paresis, recent plaster immobilization of lower lmb, recently bedridden for > 3 days, major surgery in the past 4 weeks; localized tenderness along distribution of deep venous system; entire leg swollen; calf swelling >3 cm compared to asymptomatic leg; pitting edema and collateral superficial veins. The score substract by 2 pints for alternative diagnosis as likely as, or more likely than DVT. A score of 3 or higher suggest DVT likely and patients should received a diagnostic US and the result will be documented.23
Laboratory measurements
Venous blood specimens were collected by sterile and atraumatic antecubital venipuncture, and were collected in citrate vacutainer tubes SST 5 mL, containing 0.5 mL of liquid anticoagulant. Measurements of plasma sP-selectin levels were carried out using a recombinant human P-selectin Immunoassay/CD62P catalog number ADP3 (R&D Systems, Inc. 614 McKinley Place NE, Minneapolis, MN 55413, USA).24 Von Willebrand factor antigen were measured using ELISA method (kit catalog number 885_BcSD20121001 (Sekisui Diagnostic, LLC 500 West Avenue, Stamford)).25 Plasma ADAMTS-13 levels were measured with Quantikine ELISA human ADAMTS-13 immunoassay (R&D System, Inc 614 McKinley Place NE, Minneapolis, MN 55413, USA).26
Blood samples were scheduled to be drawn at the following time-points: (i) baseline, before initial chemotherapy, and (ii) 3 months following initial chemotherapy. Samples for ELISA were immediately centrifuged at 2,500 g for 15 minutes and plasma samples were aliquoted, coded and stored at -80°C until the assays were performed. Samples prepared according to manufacturer’s instructions, and plates were read on an Elx808 plate reader (Biotek, Vermont) at 450 nm wavelength. Results were normalized to total protein using the bicinchoninic acid (BCA) assay (Pierce Rockford, Illinois) and reported as ng/mg total protein. Measurements were done blinded. All samples were assayed in duplicate and those showing values above the standard curve were retested with appropriate dilutions.
Cut-off points and normal reference value
Soluble p-selectin, vWF:Ag and ADAMTS-13 activity cut points were determined based on fold changes. The minimum detectable doses for sP-selectin was 0.5 ng/mL, the range in citrate plasma was observed to be 20 – 44 ng/mL.24 With regard to vWF:Ag, the reported mean level in men was 1.03±0.3 IU/mL and 1.08 ± 0.4 IU/mL in women.27 The mean vWF:ag/ADAMTS-13 ratio was 1.05 ± 0.30 IU/mL, according to Green et al.28 ADAMTS antigen level in non-cancer patients was 0.70 – 1.42 IU/mL (median 1.08 IU/mL).29 From the manufacturer, serum ADAMTS-13 level range from 0.51 – 1.64 IU/mL (Quantikine ELISA Human ADAMTS Immunoassay, R&D System Inc.).26 The cut off value for sP-selectin, vWF:Ag, and ADAMTS013 was set as 105.5 ng/mL, 2.35 IU/mL, and 1.03 IU/mL, respectively, according to the 75th percentile of the levels observed in this cohort.
Response to treatment and follow-up
All patients underwent chemotherapy or chemoradiotherapy. For the planned 3 months follow-up according to informed consent, patients were either evaluated during routine visites at the hematology and medical oncology outpatient clinic or medical ward, every pre- and post-chemotherapy cycle. Performance status, chemotherapy eligibility and Wells score assessed every visit. Each DVT occuring after enrollment was documented as a new event, their first lifetime thrombosis.
Statistical analysis
The quantitative variables were examined for normality with Shapiro-Wilk’s W test. Continuous variables were summarized as median (minimum-maximum range) whereas categorical data were described by absolute frequencies and percentages. Parameters between patients were compared using non-parametric Mann-Whitney test. The correlation between two continuous variables was evaluated with rank Spearman’s correlation coefficient. To create positive and negative predictive value estimation, we computed a logistic regression model with three independent variables altogether with other possible confounding factors. For each, we created a dichotomous variable of all cases in our data set by comparing the probability of DVT associated with selected cut off point.
Stepwise multiple regression analysis was used to test sP-selectin, VWF:Ag, and ADAMTS-13, , each delta value between baseline and post-chemotherapy level, VWF:ADAMTS-13 ratio and other potential determinants such as age, sex/gender, smoking history, cardiovascular risk factor (such as overweight/obesity, hypertension and diabetes), chemotherapy regimen and other biological factor (Khorana score, D-dimer level, type of cancer and stage of disease) are also included. Firstly, all potential predictors were entered simultaneously n a multivariate logistic regression model that was reduced using a backward selection method as the final step. Multivariate logistic regression was adjusted for all independent predictors, only variables showing a relationship to the outcome (defined as p<0.25 in the univariate model) were included. We identify the final multivariate model for DVT outcome using a backward stepwise approach with the p<0.05 of the likelihood ratio test for exclusion of excess factors. Precision of the specified model to detect DVT incident was quantified by the Hosmer-Lemeshow goodness-of-fit statistic where greater than 0.05 indicates adequate callibration for with corresponding area under the ROC curve.30 The Statistical Package for Social Science (IBM v 21; SPSS Inc., USA) was used for all data analysis. All test with p value < 0.05 considered statistically significant.