Novel diagnostic test to improve cardiovascular disease risk reduction in subjects with type 2 diabetes, increasing cost effectiveness and quality of life.

The incidence of T2D is increasing dramatically with a shift towards the
younger population and an increase in morbidity and mortality. Because of aging
and the expansion of obesity, the incidence of T2D is expected to increase more
in the forthcoming years.
It has been estimated that 221 million people worldwide and 1 million in the
Netherlands have this condition. The number of patients in the Netherlands is
expected to increase with 71.000 a year. Because persons with T2D are at utmost
high risk for CVD, treatment and prevention of CVD are healthcare priorities.
Even when treated with statins the 10-years incidence of CVD-related events in
patients with T2D remains high.
Despite statin treatment, about 70% of the T2D patients still experience a
cardiovascular event (risk reduction 21% (95% CI 11-30%)), nor is the severity
of the event diminished by statin treatment. CVD is still a major cause of
death in The Netherlands. With the increasing incidence of T2D the number of
CV-events is increasing, even when treated with statins.
The conventional CVD risk assessment, as routinely performed at clinical
chemistry labs, is a.o. based on plasma levels of total cholesterol, LDL- and
HDL-cholesterol and triglycerides. Studies in the US have shown that this
method identifies only 50% of the subjects at risk for CVD in the general
population. This figure could be improved to 84 % by analysis of all
atherogenic lipoproteins with the LP method. However, it has not been examined
whether improved identification of at-risk subjects results in fewer
cardiovascular events.
The aim of this project is to improve individualized treatment based on
information obtained from the Lipoprotein Profile (LP) as a diagnostic method
and thereby to reduce the number of cardiovascular events in T2D patients as
well as improve the quality of life and cost-effectiveness.
Important alterations in plasma lipoproteins characteristic for T2D are being
missed by the conventional method based on plasma cholesterol and TG
concentration, but is readily detected by the LP method. On top of the
classical risk factors, this method provides information in a single glance on
all atherogenic lipoproteins. The LP method provides more information about the
size, density, concentration and density distribution of each lipoprotein. T2D
patients show a different more atherogenic lipoprotein profile: for example,
they show a reduction in the size of low density lipoprotein (LDL) and high
density lipoprotein (HDL). Small LDL and HDL lipoproteins, high levels of
plasma Lipoprotein(a) concentration and triglyceride rich lipoproteins are
independent risk factors for CVD. Specially small-dense LDL seems to be
strongly atherogenic. The LP thereby provides information on a number of
additional independent risk factors for CVD. The consequence of the
conventional cholesterol measurement is that the patients will be treated
according to only the concentration of the lipoproteins regardless of the
distribution of the different lipoproteins. Patients with a more atherogenic LP
can take advantage of an aggressive cholesterol lowering treatment (high dosage
of statins or a combination of lipid lowering medication). Treatment options
can then be tailored to reduce the risk of the individual patient (based on the
cholesterol lowering treatment guidelines) and goes beyond the assumption of
*one size fits all*.
These dyslipidemias (small dense lipoproteins, high plasma Lipoprotein(a) and
triglyceride rich lipoproteins) are being missed by the conventional plasma
lipids measurement, but can be identified by the LP method. This method
provides information at a single glance on all atherogenic lipoproteins in the
plasma of the patient, especially about the size, density, concentration and
heterogeneity of the different lipoproteins. The LP method thereby provides
information on additional independent risk factors for CVD. Treatment for risk
reduction can be tailored for the individual patients (within the treatment
guidelines).
In this study the standard method (control group), which is treated according
to the standard clincal chemistry measurements (plasma cholesterol, plasma
triglyceride, LDL-C and HDL-C) will be compared to the intervention group
(treated according to the concentration of lipoproteins and an additional LP).

This study uses a multicenter, prospective, randomized approach. 1500 T2D
subjects will be randomized in 2 groups:
- Control group: treatment according to the results of the conventional
clinical chemistry lipid measurements,
- Intervention group: treatment according to the results of the conventional
lipid measurement and the LP).

The number of cardiovascular events will be monitored in a 6-year follow-up
period. The differences in distribution of the patients among the risk groups
(ver low, low, medium and high risk), after adding the LP compared to the
conventional method alone, in terms of percentages after reclassification.
Changes in quality of life will be monitored by regular questionnaires and the
immediate costs of treatment and diagnostics and non-medical costs will be
assessed.
By adding the LP to the standard diagnostic method of cholesterol measurement,
we expect a better identification and classification of dyslipidemia in type 2
Diabetes patients. The novel LP method is therefore expected to lead to more
individualized treatment strategies and consequently to additional CVD-risk
reduction and increase in quality of life and cost effectiveness.
T2D patients are already at high risk for CVD. Besides changes in classical
risk factors assessed by conventional methods, they show typical changes in
atherogenic lipoproteins such as reduction in the size of low density- and high
density lipoproteins (LDL and HDL) that are recognized as independent CVD risk
factors. Small-sized, dense LDL appears to be strongly atherogenic. This
dyslipidemia is missed by the conventional lipid analyses, but can be
identified with the LP method. As a consequence, patients with elevated levels
of small-sized LDL are presently not being treated differently from patients
with normal sized LDL. Such patients may benefit from more aggressive treatment
provided by increased doses of statins or combination with additional drugs.

In the first part of this study, we will assess if improvement of treatment
strategy based on individual lipoprotein profiles leads to a reduction in the
CVD-related events in Dutch subjects with T2D in comparison with the
individualized treatment based on the conventional method as commonly performed
in the clinical chemistry laboratories.
We expect that diagnostic use of the LP method will further improve CVD risk
reduction by 15 % (from 30% to 35%).The typical T2D-lipoprotein profile is
largely due to an increased mobilization of fatty acids from adipose tissue,
which negatively affect peripheral insulin action and the functioning of the
insulin-producing pancreas beta-cells. Thus aggressive treatment of the
dyslipidemia may also beneficially affect the hyperglycaemic condition.
At present, the LP method is applied to patients visiting our cardiovascular
genetics clinic, who cannot be classified to a particular lipid disorder, when
diagnostics is based on conventional lipid analyses alone. This makes it hard
to design appropriate prevention- and treatment strategies for these patients,
and to decide upon the necessity for further family screening. Our preliminary
analyses of patients with lipid disorders of unknown origin have shown that the
LP method provides insight into the nature of the dyslipidemia, helps to design
more personalized treatment strategies and is a suitable means to follow
treatment efficacy.

Implementation of the *LP* method for a detailed analysis of plasma
lipoproteins will provide more insight into factors contributing to the CVD
risk that are being missed by the conventional plasma lipid analyses. This will
lead to a better identification of subjects at risk, a better risk assessment,
a more personalized treatment and increased insight into the efficacy of
treatments.
Although increasing the costs for diagnosis, use of the LP method is expected
to result in a reduction of the costs for CVD-related morbidity,
hospitalization and the non-medical costs (patient time costs, productivity
loss), and consequently, the overall costs.
Furthermore, this project has the potential to provide evidence based
guidelines for better, more personalized, treatment (including prevention of
overtreatment) and improved cost effectiveness in patients with T2D, and
possibly improved quality of life.

The aim of this study is to answer the question: *Will the addition of the
lipoprotein profile to the conventional diagnostics for dyslipidemia in T2D
patients, lead to a better risk classification of CVD development?*. The
improved risk classification can lead to improvement of the individualized
treatment of these patients, leading to a decrease of CVD related events.

Our data will show whether improved personalized treatment as a result of the
additional novel method for atherogenic lipoprotein analyses, the *LP* to the
conventional diagnostic method, for CVD-risk estimation, in T2D subjects leads
to:

1. A reduction of CVD-risk in T2D subjects;
a) We expect that a certain percentage of the T2D patients will be reclassified
in a different riskscore among the 4 groups (Very Low, Low, Medium or high);
according to the results of the LP.
b) This improved risk classification may lead to better treatment strategies
and to
c) reduced CVD risk in these patients)
2. Improved health-related quality of life;
3. An increased cost effectiveness.

The LP analyses will be performed and optimized in the laboratory of Vascular
Medicine (Bd-279), and in case of positive outcome will be further optimized
and implemented in collaboration with the department of Clinical Chemistry.

For this multicenter randomized intervention trial we will include 1500 T2D
patients. These patients will be asked to participate to this 6-year follow up
intervention study. The patients will provide 3 blood tubes (during a regular
blood withdrawal) at baseline (2 SST a 9ml and 1 small EDTA a 4ml) and 2 blood
tubes (SST a 9ml) during a regular blood withdrawal at the end of the study
(after 6 year of the inclusion). These 1500 patients will be divided into 2
groups of 750. The control group of 750 patients will have the normal clinical
chemistry measurements of the lipoproteins. The intervention group of 750
patients will have a combination of the clinical chemistry measurement of the
lipids and the LP method.

The outcome of the LP analysis will be evaluated by a treatment team (Dr.
Roeters van Lennep (Internal Medicine doctor specialized in lipidology), Dr.
H.W.O. Roeters van Lennep (Cardiologist), Drs. S. Bos (physician researcher)
and Drs. R. Yahya (physician researcher)). This team will give individualized
treatment advices to the outpatient clinic specialist regarding the treatment
of the patients in the intervention group, based on the combination of results
of the conventional lipid measuring method and the LP method. These treatment
advices will follow out of the flowchart (version 2). The treatment group will
choose for example: low to moderate statin dose. This advice will be given to
the specialist in the outpatient clinic. The specialist will choose the
specific cholesterol lowering drug based on the *Statin equivalent dosages
scheme* (see also protocol §3.0 Study Design), taking into account the
possible drug intolerance of the individual patient.
Plasma of all patients (control + intervention group) will be stored after the
inclusion, to offer the possibility to perform retrospective LP analysis in
the control group.
The next time the patient visits the outpatient clinic for the regular control
of diabetes, the physician will discuss the results of the test (by the control
group, the physician will discuss the results of the clinical chemistry
measurement of the lipids, and by the intervention group the physician will
discuss the results of the clinical chemistry measurement of the lipids and the
LP).

Patients in both groups will receive a personalized treatment advice within the
EAS guidelines:
- Control group: the patients in this group will receive their treatment advice
from their own physician based on the results of the clinical chemistry
measurement of cholesterol
- Intervention group: the patients in this group will receive their treatment
advice from their own physician (who will be advised by the treatment team of
this project) (see above), based on the results of the clinical chemistry
measurement of cholesterol in combination with the results of the LP method.

The patients will be followed for a period of 3 years. In this 3-year follow-up
study, the incidence of CVD will be the endpoint of the study.

CVD is defined as the occurrence of Major adverse cardiac event (MACE),
including non-fatal myocardial infarction, not fatal stroke or death from
cardiovascular causes. Also coronary revascularization, hospitalization for
congestive heart failure and unstable angina pectoris and peripheral artery
disease will be included in the definition of CVD. Microalbuminuria
(albumin-to-creatinine ratio 2.5-25 mg/mmol for men and 3.5-35 mg/mmol for
women, in any urine sample) is also part of the composite endpoint as it is a
very strong indicator of CVD risk in T2D patients. The albumin-to-creatinine
ratio is being measured during the regular visits at the outpatient clinic for
type 2 diabetes control.
We will also assess all-cause mortality, we hereby will divide the death cause
in percentages of the all cause mortality in to different groups according to
the cause of death, such as death from CVD, and other causes.
The patients that suffer from one of the specified events will be included in
the composite endpoint. Because the endpoints can vary in *weight*, (for
example: death from cardiovascular causes vs urine microalbumin-to-creatinin
ratio) we will look at the effects of the intervention on the specific
endpoints separately additional to the effect of the intervention on the total
composite endpoint. We will do that in order of *weight* or clinical importance
of the different endpoints.
The patients will also be asked to fill in questionnaires every 3 months (every
time the patient has an appointment with the diabetic nurse). These are needed
to obtain more insight into improvement of quality of life, cost-effectiveness
and the development of cardiovascular disease. We established an *events
committee* to control if the documented events meet the CVD events definition
in this study. This committee consists of: Prof. Dr. J. Roos (Cardiologist,
Erasmus MC) and Anho Liem (Cardiologist, Sint Franciscus Gasthuis). The events
will be documented in a separate database by the coordinating investigator
(with explanation about the type of the event: for example an inferior
myocardial infarction). This database will be finished after 6 years of
follow-up and will be controlled by the events committee. This committee will
decide if the documented events meet the CVD events definition in this study.

Time schedule:
Year 1: Start: august/september 2014; Organization of the study (first 6
months). First visit of ~520 patients. (intermin analysis at 4 months focused
on logistics).
Year 2: Examination of ~1020 patients.
Year 4:Follow-up after 3 years, analyses of clinical endpoints (power not
enough)
Year 7: Follow-up after 6 years
Year 7 and 8: Analyses of all data and reporting, publication of papers. In
this year also the protocol for implementation of the LP method will be
established.

The intervention group will be subjected to measurements of the lipids by the
standard clinical chemistry and additionally by the LP method. The control
group will obtain only the clinical chemistry measurements of cholesterol (like
in the outpatient clinic).

The LP is analyzed after ultracentrifugation which leads to separating the
different lipoproteins based on their density and collected in 45 fractions.
After that the LP is provided by measuring cholesterol and triglycerides in the
fractions. The results of the LP will be shown in 3 figures:
- Cholesterol profile
- Triglyceride profile
- Cholesterol/Triglyceride ratio profile.
Based on the analysis of these 3 profiles in addition to the conventional
measurements the treatment team will choose the individualized treatment for
the patients in the intervention group according to the flowchart, within the
EAS guidelines.

EAS treatment guidelines:
- Primary prevention is defined as all T2D patients who don*t belong in the
secondary prevention group.
- Secondary prevention is defined as T2D with 1 of the following:
• CVD
• CKD (chronic kidney disease)
• > 40 years old + > 1 CVD risk factor or markers of target organ damage

Statin doses with <40% LDL reduction will be considered as low/moderate doses.
Statin doses with > 40% LDL reduction will be considered as high doses.

Treatment strategy of the control group (EAS guidelines), see flowchart version
2:

- Primary prevention: LDL-C > 2.5 mmol/L: treat with statin (the highest
(tolerated) dose to reach the target level of LDL-C< 2.5 mmol/L)*
- Secondary prevention: LDL-C > 1.8 mmol/L: treat with statin (the highest
(tolerated) dose to reach the target level of LDL-C< 1.8 mmol/L)*
*If target level is not achieved with highest tolerable statin dose, a
combination of statin + cholesterol absorption inhibitor/bile acid binding
resins/nicotinic acid can be used.
- TG >2,3 mmol/L / HDL-C <0,88 mmol/L / Lp(a) > 0,5 g/L: can be treated with
fibrate/nicotinic acid or combination of fibrate/nicotinic acid with statin.**
**When a statin-fibrates combination therapy is used, fenofibrate will be the
first option (lower myopathy incidence when fenofibrate is used compared to
other fibrates).

Treatment strategy of the intervention group will be according to the following
protocol (within the EAS guidelines) see flowchart version 2 :

Primary prevention:
LDL< 2.5 mmol/L+ small/large LDL ratio>1: low to moderate dosages of statins.*
LDL>2.5 mmol/L + small/large LDL ratio<1: low to moderate statin dose.*
LDL>2.5 mmol/L + small/large ratio>1: high statin dose.*
LDL>2.5 mmol/L + small/large ratio>2: a combination of highest tolerable dose
of statin + cholesterol absorption inhibitor/bile acid binding resins/nicotinic
acid can be used.*

Secondary prevention:
LDL<1,8 mmol/l+ small/large LDL ratio>1: low to moderate statin dose*
LDL>1,8 mmol/L + small/large LDL ratio<1: low to moderate statin dose*
LDL>1,8 mmol/L + small/large ratio>1: high statin dose.*
LDL>1,8 mmol/L + small/large ratio>2: a combination of highest tolerable dose
of statin + cholesterol absorption inhibitor/bile acid binding resins/nicotinic
acid can be used.*

*If target level is not achieved with highest tolerable statin dose, a
combination of statin + cholesterol absorption inhibitor/bile acid binding
resins/nicotinic acid can be used.

- TG >2,3 mmol/L / HDL-C <0,88 mmol/L / Lp(a) > 0,5 g/L: can be treated with
fibrate/nicotinic acid or combination of fibrate/nicotinic acid with statin**

**When a statin-fibrates combination therapy is used, fenofibrate will be the
first option (lower myopathy incidence when fenofibrate is used compared to
other fibrates).

The outcome of the LP analysis will be accompanied by advice of a treatment
team (see protocol § 3. Study design).
In case of statin intolerance (in both the control and the intervention group)
the following lipid lowering medication can be used: bile acid binding
resins/nicotinic acid/ cholesterol absorption inhibitor (with or without bile
acid binding resins/nicotinic acid).
If the target level is not reached (in both the control and the intervention
group) the following medication can be used: combination of statin with
cholesterol absorption inhibitor/bile acid binding resins/nicotinic acid.

The risk of developing CVD by T2D patients is still high even after treatment
with lipid-lowering therapy. The risk might be reduced further if the new
diagnostic method *Lipoprotein Profile* will improve risk assessment leading to
a better individualized treatment of T2D subjects and subsequently to an
increase in quality of life and cost-effectiveness.