Neo-angiogenesis is an early sign of cancers. This study assessed if among low-risk gestational trophoblastic neoplasia (LR-GTN) patients, the uterine artery pulsatility index (UAPI), which is a measure of tumor vascularity, can be an independent predictor to resistance to methotrexate chemotherapy (MTX-R).
JOURNAL OF MEDICAL RESEARCH UTERINE ARTERY PULSATILITY INDEX: A PREDICTOR OF METHOTREXATE RESISTANCE IN GESTATIONAL TROPHOBLASTIC NEOPLASIA Nguyen Thai Giang¹, Vuong Tien Hoa¹, Vu Ba Quyet2, Tran Trung Hieu¹ ¹Hanoi Medical University ²National hospital of Obstetrics and gynecology (NHOG) Neo-angiogenesis is an early sign of cancers This study assessed if among low-risk gestational trophoblastic neoplasia (LR-GTN) patients, the uterine artery pulsatility index (UAPI), which is a measure of tumor vascularity, can be an independent predictor to resistance to methotrexate chemotherapy (MTX-R) A prospective observational study of 204 LR-GTN patients (FIGO scores ranged from to 6) were treated with methotrexate from January 2015 to September 2017 at National Hospital of Obstetrics and Gynecology (NHOG) Patients underwent a single Doppler ultrasound to assess the UAPI The median UAPI was lower (higher vascularity) in MTX-R compared with that of MTX-sensitive patients (1.60 vs 2.05, p= 0.03) UAPI, hCG and the largest tumor size were all predictors of MTX-R on univariate analysis Moreover, the UAPI remained as a significant independent predictor of MTX-R on multivariate logistic regression analysis The odds ratio of MTX-R for patients with a UAPI ≤1.2 was 2.26 (95% CI, 1.00 – 5.09; p=0.049), compared to patients with a UAPI >1.2 In conclusion, UAPI, as an indirect in vivo measure of functional tumor vascularity, could independently predict the response to MTX-R in LR-GTN patients Patients with FIGO score of 5-6 and a UAPI5 IU/l) Diagnosed GTN if there is a histologic diagnosis of choriocarcinoma FIGO prognostic scores ranging from to GTN after other gestational events (term pregnancy, abortion, ectopic pregnancy) Methods This was a prospective observational study Sample size: n= z 1- a p.q (f.p) In which: α = 0.05, Z = 1.96, choosing ε = 0.25 p: resistance rate to MTX treatment in GTN patients is 24.5 % [7] Add 10% patients The final sample size is 204 patients Research process The research clinical record was designed and tested GTN patients with FIGO prognostic scores from to were chosen In all cases, patients’ consent were obtained At the beginning of the study, all patients underwent a single abdominal pelvic ultrasound to measure total uterine volume and local invasion of GTN tumor UAPI was measured in both uterine arteries The lowest UAPI from either uterine artery was used for analysis, since it is a reflection of the maximal deviation from the normal impedance Doppler assessments of the uterine arteries were performed by only one experienced sonographer through ultrasound machine Voluson 730 Pro (GE Healthcare Technologies, USA) device equipped with JMR 118 E4 (2) - 2019 JOURNAL OF MEDICAL RESEARCH transabdominal 3,5 MHz curvilinear probe MTX/FA regime: Patients were initially treated with fortnightly cycles of 50 mg of methotrexate i.m on days 1, 3, 5, and 7, with mg of folinic acid i.m with rescue on days 2, 4, 6, and Response to chemotherapy was monitored by measurements of hCG concentrations fortnightly The development of MTX-R was defined by a plateau or a rise in two consecutive hCG concentrations The MTX-R patients will be treated by combination chemotherapy Statistical Analysis Stata 13 was used for the statistical analyses Univariate analysis was performed using the Mann-Whitney U test or χ2 test, and the correlation between prognostic variables was tested using Spearman’s correlation coefficient A ROC curve was used to maximize sensitivity and specificity and find a cut-off point of UAPI These UAPI subgroups were used in the subsequent multivariate analyses of predictors of MTX-R, using binary logistic regression with forward stepwise selection All significant prognostic factors on univariate analysis were initially included Ethical consideration The study protocol was approved by the Institutional Review Board at NHOG on 25 January 2015 Table FIGO scoring system for gestational trophoblastic neoplasia, 2002 [8] Prognostic factor Score Age (years) < 40 ≥ 40 Antecedent pregnancy mole abortion term 12 < 103 103 - 104 104 - 105 > 105 Largest tumor diameter (cm) 8 Monotherapy Combined therapy Interval (months) βhCG (IU/1) Number of metastases Previous chemotherapy Patients with FIGO scores ranging from to were deemed low-risk GTN patients, and were initially treated by single agent chemotherapy as the first regimen Patients who had FIGO scores equal to and above were deemed high-risk GTN patients, and were initially treated by combination chemotherapy as the first regimen III RESULTS Patient characteristics 204 GTN patients were eligible for the study Their median age was 26 years (16-39 years), 84.3% had less than one child, 81.4% followed a molar pregnancy, medium latent interval was 2.1 months (1-14 months), 1.5 % had previous MTX chemotherapy, and 4.4% patients had either lung JMR 118 E4 (2) - 2019 67 JOURNAL OF MEDICAL RESEARCH or vaginal metastases The medium β-hCG concentration was 11667 UI/l (9 – 213180 UI/l) One third of patients had uterine local invasion with medium diameter 33.5 mm (13 - 69 mm) and 9.8% had FIGO score equal or above Resistance rate to single-agent chemotherapy MTX 27.45% (56/204) of the patients had MTX resistance, therefore they needed to switch regimen to combination chemotherapy (EMACO) Doppler ultrasound characteristics of MTX-R and response group Mean peak systolic velocity (PSV) of MTX-R group was significantly higher than that of response group (72.86 ± 37.03 cm/s vs 60.73 ± 27.15cm/s, p = 0.04) Mean end diastolic velocity (EDV) of MTX-R group was also higher than PSV of response group (25.58 ± 26.40 vs 15.39 ± 19.86 cm/s, p = 0.01) UAPI cut-off point and meaning resistance to MTX prediction Figure Median of UAPI in MTX-R group lower than response group (1.60 vs 2.05, p = 0.03, Mann –Whitney U test) Figure ROC curve of UAPI to predict MTX-R 68 JMR 118 E4 (2) - 2019 JOURNAL OF MEDICAL RESEARCH The ROC curve and sequential analysis of the UAPI demonstrated that UAPI at the cut-off point 1.2 showed moderate predicted probabilities of MTX resistance (AUC=0.59, sensitivity 71.62%, and specificity 48.21%) Table UAPI and resistance to MTX-R at cut-off point 1,2 Response MTX (n = 148) UAPI MTX-R (n = 56) p N % n % ≤1.2 33 58.93 23 41.07 >1.2 115 77.70 33 22.30 0.007* The group of patients with UAPI ≤ 1.2 had a higher MTX-R rate than that of the group with UAPI >1.2 (p=0.007) Table Univariate logistic regression analysis factors related to MTX-R Prognostic factor OR 95% CI p βhCG pre-treatment < 1000 IU/ml 1000 - < 10000 IU/ml 1.38 0.64 – 2.96 0.39 10000 - < 100000 IU/ml 2.42 1,11 – 5.26 0.025* > 100000 IU/ml 3.83 0.50 – 29.11 0.19 Largest tumor size (cm) < cm - cm 1.02 0.44 – 2.36 0.96 ≥ cm 5.16 1.43 – 18.56 0.012* > 1.2 ≤ 1.2 2.42 1.25 – 4.69 0.008* UAPI Three significant factors related to MTX-R are βhCG pre-treatment, the largest tumor size and UAPI JMR 118 E4 (2) - 2019 69 JOURNAL OF MEDICAL RESEARCH Table Multivariate logistic regression analysis of MTX resistance and dependent factors (UAPI, βhCG and tumor size) Prognostic factor OR 95% CI p 1.00 – 5.09 0.049* UAPI > 1.2 ≤ 1.2 2.26 βhCG pre-treatment < 1000 IU/ml 1000 - < 10000 IU/ml 1.21 0.55 – 2.64 0.62 10000 - < 100000 IU/ml 1.67 0.67 – 4.12 0.26 ≥ 100000 IU/ml 2.18 0.23 – 20.64 0.49 Largest tumor size (cm) < cm – cm 0.55 0.20 – 1.48 0.24 ≥ cm 2.31 0.53 – 10.07 0.26 As shown in table 4, UAPI was the only independent predictor of MTX-R (p < 0.05) Results of combination UAPI to FIGO score Table Resistance rate to MTX using FIGO score in combination with the cut-off point UAPI = 1.2 FIGO score Resistance rate to MTX FIGO FIGO + PI > 1.2 FIGO + PI ≤ 1.2 N n % N n % N n % ≥0 204 56 27.45 148 33 22.30 56 23 41.07 ≥1 154 50 32.47 99 27 27.27 55 23 41.82 ≥2 96 35 36.46 49 14 28.57 46 21 45.65 ≥3 53 22 41.51 22 22.73 31 17 54.84 ≥4 20 10 50.00 25.00 12 66.67 ≥5 42.86 0.00 3 100.00 IV DISCUSSION Since tumor angiogenesis is a significant step in the growth and metastasis of solid tumors [9], evaluating tumor vascularity could 70 predict metastatic risk and overall survival rates in several types of cancer, such as breast cancer, melanoma, response to radiation in cervical cancer [10] While previous literature pointed out that the tumor vascularity was JMR 118 E4 (2) - 2019 JOURNAL OF MEDICAL RESEARCH primarily assessed by histological examination, Doppler ultrasound plays a vital role as a useful tool to assess tumor vascularity and clinical response due to its ability to identify neo-angiogenesis characteristics of tumor and vascular changes GTN, a solid tumor, is especially fitted to study by Doppler ultrasound as it allows researchers and clinicians the ability to evaluate tumor vascularity and hemodynamic changes in the main artery supplying the uterus (uterine arteries) In GTN patients, studies have highlighted that due to low resistance downstream, hemodynamic change in uterine arteries showed high velocity and low impedance [11] PSV of MTX-R group (72.86 ± 37.03 cm/s) was significantly higher than that of response group (60.73 ± 27.15 cm/s, p < 0.01) Since PSV of uterine artery exhibited high increase blood flow into the uterine circulation to feed neoplastic trophoblast, MTX-R patients exhibited a hyperdynamic uterine circulation by revealing significantly higher PSV in uterine artery, compared with response patients We also found a much higher value of EDV among MTX-R group (25.58 ± 26.40 cm/s) , compared to that among MTX response group (15.39 ± 19.86 cm/s, p = 0.01) Our results were similar to Hsieh et al , who also implied that the increase of EDV implies low impedance downstream resulting of neo-angiogenesis and arterio-venous shunts within the trophoblastic lesions [12] Therefore, MTX-R group exhibited a sharp increment of EDV To the best of our knowledge, there are two possible mechanisms which would explain the relationship between a low impedance to flow in the uterine arteries and the development of drug resistance Firstly, the low impedance reflects the presence of arteriovenous shunts within the uterus As a result, it could cause a decrease in tumor perfusion, JMR 118 E4 (2) - 2019 which has been shown to be avascular within itself Thus, the penetration and the effectiveness of cytotoxic cancer drugs would be reduced and can lead to the development of clinical drug resistance Second, drug resistance could develop alternatively due to intrinsic characteristics of the tumor itself Specifically, internal vascular changes, which might be varied among different trophoblastic tumors, could also be related to the presumed biochemical basis of drug resistance For example, choriocarcinoma, which is derived from villus cytotrophoblast, is very responsive to cytotoxic treatment but a corresponding tumor derived from interstitial cytotrophoblast has a higher possibility of being resistant to chemotherapy In a normal pregnancy, the interstitial cytotrophoblast is presumed to be responsible for the spiral artery dilatation and the reduction in the impedance to flow in the uterine arteries Thus, the low impedance to blood flow in the MTX resistance patient group could be one of the consequences of those tumors containing interstitial cytotrophoblastic originated cells, which would intrinsically reduce the sensitivity of the tumor to therapy [6] Of the study population, UAPI in MTX-R group is significantly lower than that of the MTX response group It might be explained by one of the consequences of hemodynamic changes in PSV and EDV of uterine arteries (Figure 1) The ROC curve of UAPI (Figure 2) and sequential analysis of the UAPI illustrated that UAPI at the cut-off point 1.2 show moderate predicted probabilities of MTX resistance (AUC = 0.59, sensitivity 71.62% and specificity 48.21%) At this cut-off point, UAPI was found to be independently and inversely related to the risk of MTX-R on both univariate and multivariate analysis Table showed that 71 JOURNAL OF MEDICAL RESEARCH when assessing patients with UAPI alone, the group of patient with UAPI≤ 1.2 has significant higher MTX-R rate thanthe group with UAPI >1.2 (p = 0.007) lower MTX-R rate than that of the group using FIGO score alone Consequently, using the cut-off point UAPI achieved a better result in prognostic in upper and below the threshold On univariate analysis, three significant factors related to MTX-R are β-hCG pretreatment, the largest tumor size and UAPI The odds ratio for MTX-R was 2.42 for patients with a β-hCG pre-treatment > 10000 UI/l, which was relative to patients with β-hCG pretreatment < 1000 UI/l Patients with tumor size The Doppler ultrasound may serve as a useful method for in vivo functional assessment of tumor vasculature by its assessment to hemodynamic changes in the macrovasculature, due to the fact that those changes are an indirect reflection of the microvasculature (vessel diameter < 15 ≥ cm raised the odds ratio for MTX-R 5.16 times, compared to the group of patient with the tumor size < 3cm Interestingly, changes in hemodynamic characters of the uterine artery was a new prognostic factor of MTX-R Those with UAPI ≤ 1.2 had an increment of the odds ratio for MTX-R to 2.42 times, compared to patients with UAPI > 1.2 (table 3) On multivariate analysis, the data reported that the UAPI was the only significant independent predictor of MTX-R (p < 0.05) The odds ratio for MTX-R was 2.26 for patients with UAPI ≤ 1.2 (95% CI: 1.00 – 5.09), compared to those with UAPI > 1.2 (table 4) micrometers) used to define neoangiogenesis Our results seemed to confirm that UAPI is a non-invasive method to assess in vivo the tumor vasculature and may predict single agent MTX-R in low-risk GTN patients Our findings have further strengthened our confidence in a combination of FIGO score and UAPI To be more specific, UAPI might be integrated into the FIGO score as a biological marker of tumor vascularity and angiogenesis Although our sample was not optimal, we nevertheless believe that the combination would allow better early detection of MTX-R risk of GTN low-risk patients as shown in table FIGO scoring system helps to stratify the initial treatment of GTN patient by combined chemotherapy instead of single one Currently, up to one-third of low-risk GTN patient incorrectly received single-agent methotrexate and could develop drug resistance; it is necessary to improve the FIGO scoring system From some previous studies, Kohorn, Osborne and Taylor found that the elimination of the moderate group was a mistake Many studies also corroborate the finding that the group of patients with FIGO scores ranging from to usually not respond well to single-agent chemotherapy (resistance rate up to 81%) and this group should have the first regimen as the combination chemotherapy, rather than delay until MTX–R has developed [13; 14] The criteria of UAPI ≤ 1.2 corresponded 1-2 point of FIGO score when assessing MTX-R risk As anticipated, when combined with UAPI ≤ 1.2, we discovered 66.67% of patients with FIGO score ≥ and 100% of patients with FIGO score ≥5 had MTX resistance The same FIGO scores combined with UAPI > 1.2 resulted in a At present, while the stratification of treatment for GTN patients is based on FIGO scoring (2002), UAPI can be considered as a valuable factor that helps to predict the patient who may develop MTX-R and need combination chemotherapy right from the beginning 72 JMR 118 E4 (2) - 2019 JOURNAL OF MEDICAL RESEARCH V CONCLUSION This paper has stressed the importance of hemodynamic characteristics of the uterine artery as a novel factor contributing to predict methotrexate resistance To be specific, UAPI could be an independent prognostic factor of single-agent methotrexate resistance Taken together, FIGO scoring system combined with a UAPI threshold of equal to 1.2 or lower would allow better early detection of methotrexate resistance risk among low-risk GTN patients ACKNOWLEDGEMENTS The authors would like to acknowledge patient participation We appreciate the help of participants who collaborated with us in this study REFERENCES M C Li, R Hertz D B Spencer (1956) Effect of methotrexate therapy upon choriocarcinoma and chorioadenoma Experimental Biology and Medicine 93 (2), 361-366 C Aghajanian (2011) Treatment of low-risk gestational trophoblastic neoplasia Journal of Clinical Oncology 29 (7), 786-788 I McNeish, S Strickland, L Holden et al (2002) Low-risk persistent gestational trophoblastic disease: outcome after initial treatment with low-dose methotrexate and folinic acid from 1992 to 2000 Journal of Clinical Oncology, 20 (7), 1838-1844 M G Long, J Boultbee, R Langley et al (1992) Doppler assessment of the uterine circulation and the clinical behaviour of gestational trophoblastic tumors requiring chemotherapy British journal of cancer, 66 (5), 883 Phan Chí Thành (2012) Nghiên cứu tỷ lệ kháng thuốc yếu tố liên quan điều trị u nguyên bào nuôi MTX acid folic BVPSTU Tạp chí 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Gestational trophoblastic disease The Lancet, 376 (9742), 717-729 13 R J Osborne, V Filiaci, J C Schink et al (2011) Phase III trial of weekly methotrexate or pulsed dactinomycin for low-risk gestational trophoblastic neoplasia: a gynecologic oncology group study Journal of Clinical Oncology, 29 (7), 825-831 R Agarwal, S Strickland, I A McNeish et al (2002) Doppler ultrasonography of the uterine artery and the response to chemotherapy in patients with gestational trophoblastic tumors Clinical cancer research, (5), 1142-1147 14 F Taylor, T Grew, J Everard et al (2013) The outcome of patients with low risk gestational trophoblastic neoplasia treated with single agent intramuscular methotrexate and oral folinic acid European Journal of Cancer, 49 (15), 3184-3190 JMR 118 E4 (2) - 2019 73 ... hemorrhage in GTN, Doppler ultrasonography is considered an appropriate non-invasive test to assess tumor vascularity and vascular characteristics of the main artery supply in GTN (uterine artery) Argawal,... chosen In all cases, patients’ consent were obtained At the beginning of the study, all patients underwent a single abdominal pelvic ultrasound to measure total uterine volume and local invasion of. .. research with the aim to assess the value of UAPI and related factors to prognostic methotrexate resistance in low risk- gestational trophoblastic neoplasia II METHODS Study population All the patients