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1. Introductive notions

For the clinical evaluation of therapeutic efficacy, studies should be designed with a rigorous experimental design, known as Randomized Controlled Trial (RCT).

In order to authorize and market a new drug, this experimental design must be applied by the manufacturing pharmaceutical company in order to evaluate the therapeutic efficacy of the new product in Phase II and III clinical trials.

In your practice as a future physician, you will be confronted with the need to know this experimental design, not so much in the situation described above, when the study will be planned and coordinated by the research department of the company producing the new drug, especially in the context of some clinical trials in which some medicinal products already authorized for human use may be re-evaluated in Phase IV clinical trials to check their effectiveness in possible new therapeutic applications.

Such a study is exemplified below, regarding the therapeutic efficacy of low-dose aspirin as adjuvant therapy in the treatment of varicose ulcer of the lower limb:

According to the authors of this study, aspirin at moderate doses (300 mg) had already been suggested in two other clinical trials as effective adjuvant therapy for mechanical compression therapy currently used for the treatment of varicose veins of the lower limb / varicose ulcer / venous ulcer of the lower limb.

However, the therapeutic efficacy of low doses of aspirin (150 mg) as adjuvant treatment of varicose veins of the lower limb had not yet been studied and published in the literature.

Therefore, given the existence of a plausible biological mechanism of action due to the anticoagulant effect of aspirin, the authors of the cited article have proposed to test the hypothesis of therapeutic efficacy for low doses of aspirin (150 mg) as a possible adjuvant treatment of aspirin in the treatment of varicose veins of the lower limb.

Lower limb ulcers are caused by long-term venous hypertension, as a consequence of chronic venous insufficiency.

They occur frequently in the lower limbs, in the region of the calf and maleoles.

The ulceration may be diffuse or circumscribed, with irregular, slightly deviated edges, and the ulcer's bottom frequently covered by granulation tissue and fibrin deposits.

2. Scenario

A randomized, placebo-controlled, parallel-group, double-blind for verifying therapeutic efficacy in favoring healing of the lower limb varicose vein of a 150 mg / day dose of aspirin per os (oral administrated) for 24 weeks, in the presence of background therapy with mechanical compression was realised.

Healing of varicose ulcer was defined in the study as the existence of a complete re-epithelialization of the reference ulceration (ulceration with the highest initial diameter for each patient).

251 adult patients with inferior varicose veins were enrolled in the study at different stages of the disease, from 5 New Zealand community care centers (Auckland, South Auckland, Waikato, Christchurch, and Dunedin): 125 were randomized to the experimental group with aspirin + mechanical compression) and 126 in the control group (placebo treated + mechanical compression).

Randomization was stratified after the care center and after a prognostic index of varicose ulcer healing through simple, non-drug compression. Participants, nurses who included study patients, investigators, person responsible for coding, and statistician were masked for treatment assignment.

The study compared the average reduction in the reference ulcer surface (measured in mm²) as well as the proportion (frequency) of varicose ulcer healing in the experimental group compared to the control group after 24 weeks of treatment. Patients were analyzed in the groups were they were assigned at the start of the study.

3. Study protocol

1. Purpose, objectives and hypotheses of research

The purpose of the study

Evaluation of the therapeutic efficacy of a 150 mg / day dose per os of aspirin administered for 24 weeks in lower limb varicose vein as a combination with lower limb compression.

Objectives of the study

1. Evaluation of the effect of aspirin versus placebo.

2. Quantification of the importance of this effect if it exists.

• 3. Assessment of causality, if clinically significant effect proves

Statistical hypotheses

a. Assumptions for reduction of the ulcer surface

H₀: There are no statistically significant differences between the mean reduction of the ulcer surface in the experimental group (aspirin treated) versus the control group (placebo).

H₁: There are statistically significant differences between the mean reduction of the ulcer surface in the experimental group (aspirin treated) versus the control group (placebo).

b. Assumptions about the proportion of varicose ulcer healing

H₀: There is no statistically significant difference between the proportion of varicose ulcer healing in the experimental group (aspirin treated) versus the control group (placebo).

H₁: There are statistically significant differences between the proportion of varicose ulcer healing in the experimental group (aspirin treated) versus the control group (placebo).

2. Field of research: Evaluation of a therapeutic approach / attitude.

3. Type of study:

• Depending on the objectives of the study:
  • Analytical (tests, analyzes, comparisons, links, associations);
• Depending on the results / intervention of the researcher on the subjects and evolution of the studied disease):
  • Experimental (the researcher intervenes in a controlled way over the natural course of varicose ulcerations with the administration of aspirin);
• Depending on the design:
  • With parallel groups (each subject is assigned to a single group throughout the study: aspirin or placebo);
• Depending on the objective of the trial:
  • Therapeutical effectiveness / pragmatic trial (trial conducted in conditions similar to those in the current life, including possible problems of patient compliance);
• Depending on clinical hypothesis:
  • Trial of superiority (the hypothesis of the trial is that aspirin is superior to placebo);
• Depending on the drug development phase:
  • a Phase IV trial (Phase of Pharmacovigilance / Postmarketing - After marketing approvals other benefits of aspirin, an approved and existing drug on the market, are assessed).

Observation: If a therapeutic efficacy assessment had been carried out to obtain marketing approval for a new drug, it would have been a study to test the therapeutic effectiveness (the same kind of this one), but the trial would have been Phase II or III.

• Depending on the methods used to ensure the validity of the study by avoiding systematic errors (bias) and confounding factors (confounders):
  • a randomized trial: subjects were assigned at random to the two drugs (aspirină/placebo).
  • With allocation concealed (the person who introduced the subjects in the study did not know what intervention the patient would receive);
  • intention to treat: all patients were screened in the randomized groups (whether they received aspirin or placebo, even if not all treatments were taken, or if taken discontinuously, the patients were analyzed as if they were treated correctly);
  • triple blind: blind (masked) was used in the blind triple variant (neither the patient, nor the investigator who evaluated the outcome of the intervention, nor the person responsible for coding, nor the statistician knew whether the patient received aspirin or placebo )
  • The trial was controlled (treatment evaluation was compared with placebo).

4. Target population and study sample

Target population

• Clinical characteristics (eg, disease, stage of disease, complications, functional status): Patients with lower limb varicose veins in different stages;

• Demographic characteristics (age restrictions, sex, socio-economic status): Adult patients (over 18 years old) in New Zealand.

Accesible population

Adult patients from 5 community care centers in New Zealand (Auckland, South Auckland, Waikato, Christchurch, şi Dunedin).

Study sample

Inclusion criteria: Adult patients who could tolerate compression of lower limb with elastic stockings, capable of giving informed consent to study participation, whose family doctor confirmed they could be treated with aspirin and who had a confirmed ulcer diagnosis varicose vein of the inferior limb (according to a well-defined case definition and presented in the article).

Exclusion criteria: breastfeeding mothers, those with a history of myocardial infarction, stroke, transient ischemic attack, angina pectoris, major peripheral arterial disease, patients with a history of side effects related to the use of aspirin (hypersensitivity, allergy, asthma induced by aspirin or other non-steroidal anti-inflammatory drugs), patients already taking aspirin or other anticoagulant therapy; patients with coexisting conditions or treatments that indicate or, conversely, contraindicate the use of aspirin, and patients considered for any other reason to be unable to participate safely in this clinical trial, or who have not given informed consent to participate to the study.

Sample size:

Researchers calculated a minimum of 318 participants to show an improvement of at least 15% in the rate of healing in the presence of aspirin adjuvant as estimated from previous studies with a Type I error of α = 0.05, and a study power of at least 90%. To reach the 80% minimum acceptable power under the same conditions, the size calculated by the researchers was 238 participants.

5. Data collection type

• Depending on the population surveyed: By sampling (some of the target population is being studied);
• Depending on the length of the data collection: Longitudinal, prospective (information is collected by tracking the subjects in time: at entry into the study and after a time interval to record the evolution of the disease);
• Depending on the composition of the group (s) studied: Representative sample (a sample of patients with the same characteristics of the population from which they were extracted / inclusion criteria. This representative sample was then masked randomly, giving the two compared groups: the experimental group treated with aspirin/ experimental group and the placebo-treated / control group).

6. Defining the studied effect and the associated variables

The studied effects:

The main effects studied included: modification of the ulcer surface and proportion of healed participants.

The study also looked at other effects: time to healing (evaluation of therapeutic effectiveness by survival analysis), changes in the quality of life of patients, and the appearance of adverse effects. In addition, the study also looked at the efficacy of masked treatment allocation and sought to control the effect of known confounding factors through multivariate adjustment.

Variabilele studiate (only the variables relevant to the proposed scenario to be solved are mentioned)

Qualitative variables (attribute): gender of the participant, type of treatment, presence of healing after 24 weeks.

Quantitative variables: initial and final reference ulcer area (mm²), reduction of reference ulcer surface (mm²).

7. Statistical analysis (only the methods of describing and analyzing the variables of interest are mentioned)

Descriptive statistics

• For qualitative variables: frequency tables;
• For quantitative variables: Descriptive statistics of centrality and dispersion;

Observation: The authors of the study preferred not to use graphical representations to describe qualitative and quantitative variables, but the study plan / protocol may also include graphical methods of describing the variables studied if you consider them useful for compiling descriptive results.

Statistical inference

Analysis by tables or figures of the relationship between variables:

• The authors of the study did not use the mean or box-plot diagrams of the quantitative variables in the comparison groups, contingency tables or graphical representations of the healing frequencies observed in the study, but the study plan may also include preliminary graphical / and illustration of the relationship between the studied variables.

Statistical analysis according to its objectives:

1. Studying the comparability of randomized samples: verified by comparing the initial characteristics of the patients in the two arms (see in the article: Table 1. Baseline characteristics for trial participants).

2. Evaluation of the therapeutic effect
   • T-Student test for independent samples - to compare the mean reduction of the reference ulcer surface in the experimental group with the control group (assuming the existence of a normal distribution of the variable of interest in the target population).
   • Chi square test (χ2) - to compare the proportion of healed participants from the two studied lots;

3. Quantification of therapeutic effectiveness (only if we previous demonstrate a therapeutic effect of aspirin 150 mg / day as an adjuvant treatment of varicose ulcer)
   • Punctual estimators and confidence intervals (95% CI) of some medical indicators to quantify therapeutic effectiveness:
   • relativ riscul (Relative Risk or Risk Ratio): RR=REE/REC
   • Absolute Risk Reduction: ARR=|REE-REC|
   • Number Needed to Treat: NNT=1/ARR

Analytical software used: Version 9.4 of the SAS (Statistical Analysis System).

4. Results in the study. Data analysis and presentation

Following application of the inclusion and exclusion criteria required in the study plan, of the 1563 potential participants evaluated, only 251 could be included in the study and randomized in the two studied groups (see figure 1 from the article).

Randomized groups have been shown to have comparable characteristics, with the exception of differences in age and proportion of participants in the first episode of varicose ulcer vs. those with recurrent ulcer.

The follow-up rate was 100% for the main effect.

The reduction in ulcerous area from baseline was 4.1 cm² in the aspirin-treated group and 4.8 cm² in the placebo group (p=0.25). Thus, the average difference between groups was -0.7 cm² (95% CI -1.9 cm² – 0.5 cm²).

The proportion of fully healed participants after 24 weeks was lower in the aspirin group (70.4%, adică 88 de subiecți) vs. 80.2%, 101 subjects in the placebo group. This difference was not statistically significant (χ²=1.53, p=0.22).

Following the identification of the above results in the article, we can build the contingency table corresponding to the situation described by the authors, even if the table was not published in the article:

Table 1. Distribution of varicose ulcer healing in the experimental group compared to the control group

Based on this contingency table, we can calculate punctual estimates of medical indicators to quantify therapeutic effectiveness. For this, we will consider the event that should be avoided by treatment: Absence of varicose ulcer healing.

For this event, we calculate the following therapeutic effectiveness indicators:

• • RR=REE/REC=(37/125)/(25/126)=0.296/0.198=1.5
• • ARR=|REE-REC|=|0.296-0.198|=0.1
• • NNT=1/ARR=1/0.1=10

Observation:

1. These indicators were not calculated and presented in the article, as the therapeutic efficacy of low doses of aspirin (150 mg / day) could not be proven.
2. The determination of the above indicators is only an example of how to calculate the punctual estimators of the most commonly used therapeutic effectiveness indicators. These medical indicators are used to quantify therapeutic efficacy in studies that demonstrate the existence of therapeutic efficacy in the test hypothesis phase.
3. Since absolute risk reduction (ARR) is calculated in absolute terms, if we calculated the number needed to treat (NNT) in this study, we would actually quantify an apparent benefit of placebo treatment versus treatment assessed. This apparent benefit, however, occurred occasionally, being insignificantly statistically.

5. Interpretation of the results. discussions

Since in this study the values of p> 0.05 (p = 0.25 - Student test, p = 0.22 - test χ²), the evidence from this study was insufficient to reject its null hypotheses. Therefore, the differences between the group treated by mechanical compression + aspirin 150 mg / day and the group treated by mechanical compression + placebo were not statistically significant.

Thus, in people with varicose veins of lower limbs treated by mechanical compression of the affected limb, low-dose aspirin (150 mg / day) for 24 weeks did not increase the percentage of participants with healed varicose ulcers and did not improve surface reduction ulcerated compared with placebo group.

Regarding the interpretation of the medical indicators to quantify the therapeutic effectiveness, since low doses of aspirin (150 mg / day) have not demonstrated therapeutic efficacy compared to placebo, quantifying this effectiveness by medical indicators and interpreting them would not make any sense in this study.

However, for the sake of illustration, we outline the main interpretations of the above-mentioned medical indicators based on the contingency table that we created after extracting the relevant frequencies from the study:

• • Relativ risk: RR=REE/REC=1.5

As we already know, a relative risk, RR> 1 indicates a risk factor for the occurrence of the negative event.

However, this study was also a negative one in which the healing frequency was higher in the placebo group than in the aspirin-treated group (a random difference but it was not statistically significant) if we calculated and interpreted the point estimator of RR = 1.5 it would show us that:

• In the sample studied, the proportion of non-healed aspirin-treated subjects was 1.5 times higher than the proportion of untreated placebo subjects.

The quantifiable therapeutic effect, however, being a statistically insignificant one, if we were to calculate the confidence interval associated with RR's punctual estimator in this case, this range would include the neutral value of a ratio, ie 1, thus confirming the lack of statistical significance of RR in this study.

• • Absolute risk reduction:

ARR=|REE-REC|=|0.296-0.198|=0.1=10% (95%CI -1% ; 20%)

See the reasons outlined above for RR, absolute risk reduction (ARR = absolute risk reduction) would show us that:

• In the studied sample, the rate of absence of healing in the aspirin-treated group was 10% higher than the absence of healing in the placebo group.
• In 95% of the target population, we can expect that the rate of healing in the aspirin group varies from 1% lower than in the placebo group to 20% higher than in the placebo group.
• and for this medical indicator (ARR), the confidence interval associated with its point estimator includes the neutral value (which is 0% for an indicator resulting from a risk difference), which indicates the lack of statistical significance of this reduction of risk of the event being studied (in this case, the absence of healing).
• • Number needed to treat (to benefit):

NNT=1/ARR=1/0.1=10

Recalling again that this study is a negative one (the null hypothesis on the lack of therapeutic effectiveness of aspirin 150mg / day could not be rejected), and additionally, the frequency of healing was higher in the placebo group than in the aspirin-treated group (difference statistically insignificant, but!), if we calculate and interpret the NNT = 10 punctual estimator, it would show us that:

• In the studied sample, in every 10 patients treated, an additional patient was healed from the placebo group versus the aspirin-treated group.

Observation:

1. Ideal treatment is NNT = 1, ie each treated patient in the experimental group will benefit from treatment, or avoid a negative event (eg death, worsening of the disease, no healing), while no patient in the group control will not have such a benefit (or will avoid a negative recovery event).
2. Depending on the type of pathology treated and the severity of the event avoided by treatment, NNT is usually considered clinically important when its value is close to 1, sometimes even higher values of 3-5 or even> 10 patients may be considered clinically important, especially in situations where the event avoided is very serious (eg death) or in the absence of more effective therapeutic alternatives, of course, provided that the statistical significance of therapeutic efficacy has previously been demonstrated.
3. If they are designed with enough power to highlight small effects, and if they are conducted correctly and rigorously, negative trials (those that fail to prove the therapeutic efficacy of an investigational drug) can bring information as valuable as the results from positive trials (those who manage to prove the therapeutic efficacy of the evaluated drug).

6. Conclusion of the study

The results of this study do not support the use of aspirin at low doses (150 mg / day) as adjunctive treatment of mechanical compression therapy in lower limb varicose ulcers.

7. To be retained

How do we recognize a randomized controlled clinical trial (RCT)?

• experimental study on volunteer human subjects, conducted under very rigorous methodological and ethical conditions
• compare the effects of an experimental treatment with those of a control treatment (placebo in the case of a trial of superiority or a reference treatment in a non-inferiority trial)
• all subjects studied have the same inclusion / exclusion criteria
• the allocation of the treatments is done randomly by randomization of a representative sample from the target population
• Clinical trials apply methods to minimize systemic errors and confounding factors: randomization, masked allocation, controlled study, double blind, intent-to-treat analysis, adjustment analysis for variables with possible confounding role in the occurrence of the studied effect
• the clinical trial can demonstrate links between treatment and its effects, and if methods of protection against systematic errors and confounders are correctly applied, it can demonstrate these links even at the causality level between the treatment being evaluated and its effect.

LAB 06 - Practical Activity

Data file - EXCEL