WO2019130176A1

WO2019130176A1 - Prognostic method

Info

Publication number: WO2019130176A1
Application number: PCT/IB2018/060400
Authority: WO
Inventors: Attilio Francesco SPECIANI; Katia BASELLO; Andrea COSTANZI
Original assignee: Gek S.R.L.
Priority date: 2017-12-29
Filing date: 2018-12-20
Publication date: 2019-07-04
Also published as: EP3732488A1

Abstract

An in vitro method for predicting post-infarction and/or post-surgical cardiological complications forms the subject of the present invention, where said method comprises determining the expression levels of Troponin T, BAFF and/or TNFα in a sample of a biological fluid from a subject.

Description

"Prognostic method"

An in vitro method for predicting post-infarction and/or post-surgical cardiological complications forms the subject of the present invention, where said method comprises determining the expression levels of Troponin T, BAFF and/or TNFa in a sample of a biological fluid from a subject.

Background art

The high incidence of cardiovascular diseases in the population has driven the research towards the identification of markers capable of supporting the diagnosis and prognosis thereof.

W02017011329 describes a series of cytokines as useful markers of myocardial infarction. Included on the list are BAFF and TNFa.

US 9068991 describes the use of markers Troponin I and Interleukin 17a as markers of a cardiovascular condition.

W02010042126 indicates that the levels of Troponin are indicative of the risk of cardiovascular symptomatology onset.

Considering the high incidence of cardiovascular diseases and their impact on health, the need is strongly felt for an effective and predictive method for predicting their onset and preventing the potential negative clinical development thereof in order to oppose it effectively.

Description of the invention

Here, a close correlation between the levels of two cytokines, Troponin T and BAFF, measured in vitro in a biological fluid and the onset of a cardiovascular event was surprisingly demonstrated. In particular, said levels, either after surgery or at the time of the onset of a first infarction event, were shown to increase specifically in subjects who will encounter a further cardiovascular complication after surgery, whatever the nature of said surgery, or following the first cardiovascular event.

In a further embodiment, for an even more effective correlation, a third cytokine, TNFa, is associated with said two cytokines .

Therefore, an in vitro method for predicting post-infarction and/or post-surgical cardiological complications forms the subject of the present invention, where said method comprises determining the expression levels of Troponin T, BAFF, and optionally TNFa in a sample of biological fluid from a subject;

where Troponin T levels greater than or equal to 20 pg/ml and BAFF levels greater than or equal to 0.30 ng/ml, and optionally TNFa levels greater than or equal to 8.0 pg/ml, measured in dried capillary blood extracted from a synthetic swab, or Troponin T levels greater than or equal to 80 pg/ml, and BAFF levels greater than or equal to 1.35 ng/ml measured in serum from venous blood, are indicative of an increased risk of cardiological symptomatology onset.

Increased risk of cardiological symptomatology means herein that the subjects, in whom said factors fall within the above ranges, will probably encounter, on the second, third, fourth, fifth or sixth day following the first infarction event or after surgery, complications which are typically correlated to the infarction event such as, by way of example, atrial fibrillation, ventricular tachycardia, cardiogenic shock, other fatal arrhythmias and heart failure phenomena.

The method according to the present invention advantageously allows to select the group of patients to undergo an intensive medical check-up following the first infarction event and/or after surgery .

It was surprisingly highlighted that Troponin T values greater than 20 pg/ml, described in literature as indicative of cardiac ischemia, are not correlated to an increased risk of arrhythmogenic complications, where not accompanied by a simultaneous elevation of BAFF above 0.30 ng/ml. The values are understood as measured in a sample of dried blood extracted from a synthetic swab.

Where the values are measured in a serum sample from venous blood, said threshold values are understood to be greater than 1.35 ng/ml for BAFF and greater than 80 pg/ml for Troponin T. It is known indeed to those skilled in the art that the concentration of cytokines in dried capillary blood extracted from a synthetic swab and the concentration thereof in serum from venous blood varies, in a ratio of about 1:4, i.e. 1 in dried capillary blood extracted from a synthetic swab to 4 in serum from venous blood. Said ratio varies for the various cytokines from 3.9 to 4.6.

An in vitro method for predicting post-infarction and/or post- surgical cardiological complications forms the subject of the present invention, where said method comprises determining the expression levels of Troponin T and BAFF in a blood sample from a subject, where Troponin T levels greater than or equal to 20 pg/ml and BAFF levels greater than or equal to 0.30 ng/ml, measured in dried capillary blood extracted from a synthetic swab, and/or Troponin levels T greater than or equal to 80 pg/ml and BAFF levels greater than or equal to 1.35 ng/ml, measured in serum from venous blood, are indicative of an increased risk of cardiological symptomatology or complication onset.

The cytokine levels were measured by means of immune-enzymatic methods known to those skilled in the art. In a preferred embodiment, ELISA sandwich systems were used to detect Troponin, BAFF and TNFa, then detected by means of Horseradish peroxidase and Alkaline Phosphatase .

The following examples merely have the purpose of exemplifying embodiments of the invention, and are not to be understood as limiting it in any way.

Examples

Methods

The pre-analytical extraction of dried capillary blood from a synthetic swab is carried out with the following protocol: the Copan 552C swab is reconstituted with 150 mΐ PBS and vortexed for 10 seconds. The liquid sample is transferred to a new 1.5 ml tube, centrifuged for 8 minutes at 3200 rpm at a temperature of 4^,C. The supernatant is transferred to a ne 1.5 ml tube and immediately frozen at a temperature of -20°C. The sample is then thawed at room temperature and loaded onto the specific ELISA plate, according to the instructions of the ELISA protocol indicated by the manufacturer. The ELISA kits used are based on the sandwich method, including the use of pairs of mono·· and/or poly-clonal antibodies. In particular, the levels of each of the indicated cytokines were measured by means of specific antibodies for each of them using commercial kits, that is: hcInT/INNT2 (Troponin T Type 2, Cardiac) ELISA Kit Elabscience (Houston, USA), hBAFF ELISA R&D Systems (Minneapolis, USA), hTNFa ELISA Diaclone (France) . Example 1 : method for predicting post-infarction complications.

On arriving at the Emergency Department, four subjects were assessed, the subjects A, B, C suffering from inferior infarct and subject D suffering from possible anterior infarct.

During their hospital admission, the subjects signed an informed consent for the collection and analysis of data for research purposes .

A sample of dried capillary blood on a synthetic swab was obtained from each of the four subjects and the related levels of Troponin T, BAFF, TNFa were measured according to the above-described methods .

The results obtained on arriving at the Emergency Department are shown in table 1.

Table 1

The data shown indicates that Troponin T levels greater than 20 pg/ml are measured in all 4 subjects, but the onset of complications is only seen in those subjects whose BAFF levels (subject B, subject C) are also above the threshold levels identified in the present invention.

Example 2 : method for predicting post-gastrointestinal surgery complications .

10 subjects undergoing gastrointestinal surgery were selected. All ten subjects had no previous cardiological symptomatology.

The subjects signed an informed consent for the collection and analysis of data for research purposes.

A sample of capillary blood on a synthetic swab was obtained from each of the ten subjects immediately after surgery, and the related levels of Troponin T, BAFF, TNFa were measured according to the described methods.

Of the ten measurements carried out, in two subjects a Troponin T value of 35 pg/ml was highlighted in the first one and 51 pg/ml in the second one, respectively, in the same subjects the BAFF values measured were 0.38 ng/mL and 0.65 ng/mL, respectively.

The remaining 8 subjects had Troponin T values of less than 20 pg/ml and BAFF values of less than 0.30 ng/ml.

The subjects were cardiologically monitored after surgery.

An important form of arrhythmia was highlighted in the first subject on the third day, and electrocardiographic ischemic signs were measured in the second subject on the sixth day.

The remaining eight subjects, for whom normal Troponin T and BAFF levels were measured, showed no signs of cardiovascular symptomatology during the post-surgical course.

The TNFa value, also measured after surgery in the same first and second subjects, was > 8 pg/ml.

Example 3 : method for predicting post-ischemic and/or post- surgical complications in subjects suffering from ischemic cardiopathy undergoing revascularization surgery (Bypass or Stent) .

15 patients, 9 men and 6 women, aged between 56 and 91 years were selected . A serum sample from venous blood was obtained from each of the subjects at the time of the control carried out between the 5^th and 20^th day following the stent insertion or aortocoronary bypass surgery .

The markers analyzed in said serum sample from venous blood were BAFF, Troponin T and other cytokines (IL-6, TNF-alpha and PAF) .

The results obtained are shown in table 2.

As shown in the table, 8 cases had BAFF values > 1.35 ng/ml; 8 cases had Troponin T values greater than 80 pg/ml. Only one patient (patient n. 14) with particularly high values of both markers was considered an "outlier", having a history of 2 existing neoplastic pathologies and with antineoplastic treatment underway, and therefore was not taken into account in the next steps of data analysis (dropped out) .

It was observed how 3 patients with a high troponin value but a low BAFF value showed no post-surgical complications (patients n. 4, 10, 15 in table 2) . Similarly, high BAFF levels were measured in 4 more cases (patients n. 6, 8, 9, 13 in table 2), but troponin T was not detectable or was lower than the attention value. These subjects showed no complications. This result shows how the analysis of the BAFF marker alone or the Troponin marker alone does not have the desired predictive capacity. On the contrary, high BAFF concentrations in combination with high Troponin T concentrations, simultaneously identified in 3 patients (patients n. 3, 7, 12), accurately predict the onset of complications. In fact, patients n. 3, 7 and 12 were the only ones who showed post hospital admission complications, such as serious cardiac arrhythmias or a marked reduction of EF (Ejection Fraction) and/or both complications. Here, case number 3 is highlighted to have a slightly lower BAFF value than the value deemed worthy of attention and equal to 1.24 ng/ml, however this datum was considered significant as the patient was being treated with high doses of cortisone (the only one in the group of 15 with this type of therapy) which underestimated the actual BAFF value.

Table 2

This observational study confirms that only high concentrations of BAFF and Troponin T greater than 1.35 ng/ml and 80 pg/ml, respectively, measured on serum and simultaneously read represent a predictive indicator of post-infarction complications m subjects suffering from ischemic cardiopathy and undergoing revascularization surgery (Bypass or Stent) .

Claims

1) An in vitro method for predicting post-infarction and/or post-surgical cardiological complications, wherein said method comprises determining the expression levels of Troponin T and BAFF in a blood sample from a subject, wherein Troponin T levels greater than or equal to 20 pg/ml, and BAFF levels greater than or equal to 0.30 ng/ml, measured in the dried capillary blood extracted from a synthetic swab, and/or Troponin T levels greater than or equal to 80 pg/ml and BAFF levels greater than or equal to 1.35 ng/ml, measured in serum from venous blood, are indicative of an increased risk of cardiological symptomatology or other complication onset.

2) The method according to claim 1, also comprising the determination of TNFa expression levels, wherein TNFa levels greater than or equal to 8.0 pg/ml in the dried capillary blood extracted from a synthetic swab and/or 24 pg/ml in serum from venous blood are indicative of an increased risk of cardiological symptomatology onset.

3) The method according to claim 1 or 2, wherein said cardiological symptomatology consists in arrhythmogenic complications .

4) The method according to one of the claims from 1 to 3, wherein said cardiological symptomatology includes atrial fibrillation, ventricular tachycardia, cardiogenic shock, other fatal arrhythmias, and heart failure phenomena.