UGT2B7-161 PSL predict cardiotoxicity by PSL | PGPM

Introduction

Human epidermal growth factor 2 (HER-2) positive breast cancer accounts for about 20% of breast cancer cases.^1,2At present, surgical resection is still the preferred treatment for breast cancer with positive HER-2. Postoperative adjuvant chemotherapy has been widely used to consolidate the therapeutic effect and reduce the recurrence rate.^3,4And postoperative trastuzumab combined with pertuzumab therapy are recommended for breast cancer with positive HER-2, which effectively reduces the residual cancer cells, postoperative hematologic metastasis, and the recurrence and mortality of breast cancer.^5–7

However, drugs trastuzumab and pertuzumab have strong toxic side effects on the heart, resulting in cardiac damage and affecting prognosis,⁸and the cardiotoxicity risk that may be induced by this treatment approach limits its clinical application.⁹Therefore, it is of great significance to actively explore novel markers that can predict the risk of cardiotoxicity in patients with HER-2 positive breast cancer undergoing trastuzumab combined with pertuzumab therapy. Uridine diphosphate-glucuronosyltransferase 2B7 (UGT2B7) can inactivate anti-tumor drugs through glycolipidation, and studies have shown that the transformation of UGT2B7-161 SNP C to T allele (UGT2B7-161 C > T) affects the metabolism, toxicity, and efficacy of anthracyclines, and is negatively associated with the risk of cardiotoxicity.^10,11Based on the above evidence, we speculated that UGT2B7-161 gene polymorphism may also be associated with cardiotoxicity caused by trastuzumab combined with pertuzumab therapy.

Pressure-strain loops (PSL) are a new technique for quantifying cardiac function.¹²Myocardial function is a set of comprehensive parameters obtained by this technology considering strain and afterload factors, which can better recognize the relationship between left ventricular strain and the increase in afterload pressure under different load conditions, and improve the accuracy and authenticity of myocardial function assessment.^13,14In this study, PSL was used to evaluate the changes of cardiac function parameters in patients with positive HER-2 breast cancer before and after the use of trastuzumab combined with pertuzumab therapy to detect cardiac function damage at an early stage. The purpose of this study was to investigate the value of UGT2B7-161 gene polymorphism in predicting the risk of cardiotoxicity in patients with HER-2 positive breast cancer with undergoing trastuzumab combined with pertuzumab therapy by PSL.

Data and Methods

Fifty postoperative breast cancer patients admitted to the breast surgery department of Shenzhen People’s hospital from August 2020 to June 2021 were selected. The patients were 41–58 years old, with an average of 49 ± 7 years old, and planned to receive trastuzumab combined with pertuzumab drug therapy for more than four cycles. Thirty healthy volunteers with matching ages were selected as the control group, aged 40–59 years old, with an average of 49 ± 8 years old.

The inclusion criteria for the study group were: (1) female patients with pathologically confirmed positive HER-2 breast cancer. HER2-positivity was defined as grade 3+ staining intensity by immunohistochemistry (IHC), or confirmation of HER2 gene amplification by fluorescence in situ hybridization (FISH); (2) Patients who will receive trastuzumab combination with pertuzumab in a 21-day cycle and had not received any of these treatments; (3) the patients had no serious heart valvular disease, dilated and hypertrophic cardiomyopathy, coronary heart disease, arrhythmia, structural heart disease, hypertension, endocrine diseases, or thoracic malformation.

The control group excluded the history of heart disease, hypertension, diabetes, and abnormal electrocardiogram or echocardiography. All subjects gave informed consent.

The median age of the enrolled patients was 49 years, and the average body mass index (BMI) was 21.8 ± 2.1 kg/m². There were 3 smokers (21.1%). There was a history of hypertension in 19 cases (25.0%), diabetes in 6 cases (5.3%), hyperlipidemia in 17 cases (23.7%), and hyperuricemia in 8 cases (19.7%). The median level of the baseline left ventricular ejection fraction (LVEF) was 69.0% (P25–P75: 64.3–71.0%), the median level of cardiac Troponin I (cTnI) was 0.040 ng/mL (P25–P75: 0.017–0.078 ng/mL), and the median level of N terminal Pro B type Natriuretic peptide (NT-probNP) was 0.065 ng/mL (P25–P75: 0.053–0.089 ng/mL).

Trastuzumab and pertuzumab were purchased from Shanghai Roche Pharmaceutical Co., Ltd., China. The QIAamp DNA mini kit was purchased from Qiagen, Germany. The polymerase chain reaction (PCR) amplification kit was purchased from TaKaRa, Japan. The centrifuge was purchased from Xiangyi Centrifuge Instrument Co., Ltd., China. The ADVIA Centaur XP immunoluminescence analyzer was purchased from Siemens, Germany. The 7900HT fluorescence quantitative PCR instrument and the ABI 3730XL sequencer were purchased from Applied Biosystems, USA. The GE Vivid E95 is a large-scale ultrasound machine purchased from GE Healthcare. It is equipped with an M5Sc cardiac probe and installed with an EchoPAC 203 software workstation.

The GE Vivid E95 M5Sc heart probe was used to obtain cardiac work parameters. Subjects were asked to lie in the left lateral decubitus position to obtain dynamic two-dimensional echocardiography, including the apical left ventricular two-chamber view, the apical left ventricular long axis view, and the apical four-chamber view. The frame frequency and depth were kept the same during the data collection, and the electrocardiogram indexes were recorded synchronously. All measured values were recorded for at least three cardiac cycles and their average values were taken.

After the data collection, the Digital Imaging and Communication in Medicine images were stored, and the obtained image data were transferred to the EchoPAC 203 workstation. During offline analysis, dynamic images of good quality were clearly visible on both the endocardial and epicardial surfaces. The longitudinal strain and tracking graph of each section were generated. The software automatically calculated the global longitudinal peak strain (GLS) of the left ventricle and the blood pressure input during case collection. After the analysis was completed, the software automatically calculated the global work index (GWI), global effective work (GCW), global wasted work (GWW), and global work efficiency (GWE) (see Figure 1). All images were collected and analyzed by two experienced physicians in three cardiac cycles.

Whole blood samples were collected with anticoagulant tubes from patients before they received trastuzumab combined with pertuzumab dual-targeted drug therapy (M0) and at the end of four treatment cycles (M3) in the third month after the beginning of chemotherapy. Blood samples collected at M0 were divided into two parts; one part was whole blood reserved for subsequent detection of UGT2B7-161 gene polymorphism. In addition, as with blood samples collected at other follow-up sites, plasma separation was performed within 2 h after collection. The separation process was as follows: The whole blood sample was centrifuged at 1800 g at 4°C for 10 min, then the supernatant was transferred to an Eppendorf tube and centrifuged again at 2500 g at 4°C for 10 min. The plasma was separated and stored in a cryopreserved tube at −80°C for subsequent analysis. The levels of cTnI and NT-probNP in the plasma samples at each follow-up point were detected by the ADVIA Centaur XP immunoluminescence analyzer and supporting reagents.

Leukocyte DNA was extracted from patients’ M0 blood samples using the QIAamp DNA mini kit. UGT2B7-161 (RS7668258, Genebank Ref mRNA NM_001074) was amplified by the PCR kit and the 7900HT PCR instrument.

The primer sequence upstream was 5 “-TGTCACTGCTACTGTTCTGGA-3” and downstream was 5 “-AGTTGCTGTTCCTTTCTGTCAT-3”.

The reaction conditions were pre-denaturation at 95°C for 5 min, followed by 95°C for 15s, 60°C for 30s, 72°C for 30s, for a total of 40 cycles, and finally extended at 72°C for 5 min. The amplified products were sequenced using an ABI 3730XL sequencer, strictly following the instructions (see Figure 2).

SPSS Statistics 24.0 software was used for data processing. Continuous data of a normal distribution were expressed as mean ± standard deviation. When the variance was uniform, one-way analysis of variance (ANOVA) was used for comparison between multiple groups, and the least significant difference method was used for pairwise comparison. The correlation between left ventricle strain and work parameters was determined using Pearson correlation analysis. The measurement data of 10 cases were selected for analysis by another physician, and a repeated test was carried out by the Bland-Altman method. P < 0.05 was considered statistically significant. Continuous data of a skewed distribution were expressed as median (P25–P75), and the classified data was expressed as frequency (percentage). One-way ANOVA was used for measurement data comparison, the Chi-square test was used for counting data comparisons, the Kruskal–Wallis H-test was used for data that did not conform to a normal distribution, and a logistic regression model was used for multivariate analysis.

Results

No heart failure, acute coronary syndrome, or serious life-threatening arrhythmias were found at all time points of follow-up. The number of cardiotoxicity events at different follow-up time points was defined as the number of events that occurred between the previous follow-up time point and this follow-up time point. The number of events with ΔLVEF ≥ 10% or LVEF < 53% only or ΔLVEF ≥ 10% and LVEF < 53% were 0, 1 and 2 at M0 and M3, totaling 2 cases of cardiotoxic events (see Table 1).

According to UGT2B7-161 gene phenotype, 50 breast cancer patients were divided into a CC group (n = 24), a CT group (n = 15), and a TT group (n = 11). There were no significant differences in age, BMI, smoking history, concomitant diseases, and cardiac function among patients with different genotypes (P > 0.05), suggesting that UGT2B7-161 was not associated with self-characteristics features of breast cancer with positive HER-2 (see Table 2 and Figure 3).

There were no significant differences in age and systolic blood pressure among the three groups (P > 0.05), or in the routine echocardiography parameters for left ventricular end-diastolic diameter, end-diastolic volume, end-systolic diameter, end-systolic volume, LVEF, and fractional shortening among the three groups (all P > 0.05; see Table 3).

Compared with the control group, the GLS, GWI, GCW, GWW, and GWE of the left ventricle in the breast cancer group before chemotherapy had no statistically significant difference (P > 0.05). After therapy, compared with before, there were statistically significant differences in the left ventricular GLS, GWI, GCW, and GWE (P < 0.05), and GWW increased (P < 0.05) (see Table 4 and Figure 4). The Bland-Altman test showed good repeatability between and within observers.

Myocardial work decreased events occurred in 35 (70.0%) of the 50 patients receiving trastuzumab combined with pertuzumab dual-targeted drug therapy, 19 (79.2%) of 24 UGT2B7-161 CC patients, and 16 (61.5%) of 26 TT+CT patients. Among the 15 patients without myocardial toxicity, 5 (33.3%) had CC type and 10 (66.7%) had TT+CT type. Patients with the TT+CT genotype had a lower incidence of myocardial work decreased events than those with the CC genotype (P = 0.004).

Logistic univariate regression analysis showed that the UGT2B7-161 gene phenotype, BMI, cTnI, and hyperuricemia were associated with the risk of myocardial toxicity (P < 0. 05). Age, smoking, hypertension, diabetes, hyperlipidemia, chronic kidney disease, and NT-probNP were not associated with the risk of myocardial toxicity (P > 0.05). See Table 5.

The UGT2B7-161 gene phenotype, BMI, cTnI and hyperuricemia were included in the logistic multivariate regression model. The results showed that the UGT2B7-161 gene phenotype, BMI, and cTnI were independent factors affecting the occurrence of myocardial toxicity (P < 0.05). Hyperuricemia was not associated with myocardial toxicity (P = 0.226). See Table 6.

Discussion

The traditional method of evaluating the systolic function of the left ventricle is usually to measure the indicators of myocardial fiber systolic shortening, including the overall indicators such as LVEF and local indicators such as the ventricular wall thickening rate, but the shortening index cannot reflect the work and oxygen consumption of myocardium.¹⁵Although LVEF is still the most commonly used cardiac function parameter Clinically, once its clinical significance changes, it often indicates irreversible damage to the myocardia. Its low sensitivity to myocardial injury means that drug-related myocardial damage cannot be found in the subclinical stage.¹³

In this study, conventional two-dimensional echocardiography left ventricular systolic function parameters did not show myocardial impairment, while GWI, GCW, GWE, and GLS showed a decreasing trend compared with their levels before the therapy (P < 0.05). Conventional echocardiography left ventricular systolic parameters could not detect the toxicity of trastuzumab and pertuzumab drugs in myocardium in a timely and sensitive way. The results of this study showed that the myocardial cell toxicity caused by trastuzumab combined with pertuzumab therapy reduced the GLS capacity of the left ventricle, weakened the contractility of the left ventricle, and both GWI and GCW of the left ventricle showed a downward trend. The abnormal electromechanical activity of the myocardium caused by the cardiotoxicity of the therapy drugs and the unsynchronized movement of the ventricular wall affected the ventricular systolic function. This reduced the effectiveness of the myocardium and its ability to pump blood effectively, thereby increasing the GWW of the myocardium, which is consistent with the research results of Russell et al.¹⁶The increase of useless work further affects the efficiency of the left ventricular myocardium. In addition, GLS decreased after trastuzumab combined with pertuzumab drug therapy compared with before, and GLS was positively correlated with the decrease of left ventricular work parameters GWI and GCW, indicating that the longitudinal strain capacity of the myocardium decreased, resulting in the decrease of myocardial contractility, which directly affected the ability of the heart to do work. Using GWI, GCW, and GWE parameters, this technique can intuitively quantify the myocardial working capacity and provide objective reference for clinical diagnosis and treatment. And 35 patients who occurred myocardial work decreased, were reduced targeted drug dosage or extended treatment interval, and/or added cardioprotective drugs to preintervene the occurrence of myocardial drug toxicity events. We supposed that the decrease in cardiac work parameters could be corrected in this way.

Previous studies¹⁷have shown that there is no difference in epirubicin metabolism between histidine and tyrosine variants of UGT2B7, whereas SNP at position 161 is associated with the efficacy of morphine glucose-aldehyde acidification. A study of 99 patients who received morphine analgesia found that the glucose-aldehyde acidification of 161-base CC homozygous was significantly lower than that of CT heterozygous or TT homozygous.¹⁸Because of these findings, we hypothesized that CC homozygous patients would have lower clearance rates and higher toxicity rates compared to CT and TT patients. In contrast, we hypothesized that TT homozygous patients would have a faster clearance rate, reduced drug efficacy, and poorer prognosis than patients with CC or CT variants. In this study, the incidence of drug cardiotoxicity in 161-base CC homozygous was significantly higher than that in the TT+CT group, which was consistent with the expected result. However, the expected result that drug cardiotoxicity in 161-base TT homozygous was lower than that in CT heterozygous has not been obtained, which may be related to the limitations of this study.

The GWI, GCW, GWW, and GWE of the myocardia had changed when the LVEF did not change significantly. However, differences in UGT2B7-161 gene polymorphism (CC, TT, CT) were statistically significant in patients who underwent trastuzumab combined with pertuzumab therapy had changes in myocardial work parameters. It can be used as a predictor of the occurrence of cardiotoxicity in breast cancer patients receiving targeted drug therapy HER-2.

The limitations of this study are as follows: (1) This study was a single-center cohort study, with a single source of HER-2 positive breast cancer patients, which may lead to selection bias and geographical limitation of the study; (2) The follow-up period of this study was short, and the UGT2B7-161 gene TT+CT type was not studied for evaluating the long-term efficacy of cardiotoxicity; (3) This study did not involve the underlying mechanism of UGT2B7-161 gene polymorphism and cardiotoxicity, which needs further investigation; (4) The sample size was small, and only before and after therapy was compared; (5) The cardiac muscle work capacity of PSL was mainly discussed. Although the blood pressure values of the included patients were selected within the normal range, there were still some variable differences, and the work difference caused by the fluctuation of blood pressure within the normal range could not be excluded;¹³(6) PSL does not consider curvature myocardium thickness and only represents the indicator of work rather than directly measuring work value.³(7) Whether the change of myocardial parameters is equivalent to the occurrence of myocardial toxic events has not been discussed and proved in detail in this study, which needs to be verified in a large number of cases.

Conclusion

In this study, we found that Ultrasound pressure-strain loop (PSL) is more objective and effective than strain in evaluating myocardial function, and its myocardial work parameters can be used for early assessment of subtle changes in myocardial systolic function and motor coordination.

In conclusion, prior to the occurrence of drug-induced myocardial damage in patients with HER-2 positive breast cancer receiving trastuzumab combined with pertuzumab dual-targeted therapy, planning myocardial protection measures in advance will be of great help to improve the prognosis of patients.