cancers Article Evolution of Angiogenic Factors in Pregnant Patients with Breast Cancer Treated with Chemotherapy Cristina Saura 1,† , Olga Sánchez 2,3,† , Sandra Martínez 4 , Carmen Domínguez 3,5 , Rodrigo Dienstmann 6 , Fiorella Ruíz-Pace 6 , Maria Concepció Céspedes 7 , Ángeles Peñuelas 1 , Javier Cortés 8 , Elisa Llurba 2,9, * and Octavi Córdoba 10,11 1 Medical Oncology Department, Vall d’Hebron University Hospital, Vall d’Hebrón Institute of Oncology (VHIO), 08035 Barcelona, Spain; csaura@vhio.net (C.S.); apenuelas@vhio.net (Á.P.) 2 Maternal and Child Health and Development Network (SAMID), RD12/0026/16 and RD16/0022/0015, Institute of Health Carlos III, 28029 Madrid, Spain; osanchezg@santpau.cat 3 Biochemistry and Molecular Biology Research Centre for Nanomedicine, Vall d’Hebron Research Institute, 08035 Barcelona, Spain; mcdluengo@gmail.com 4 Vall d’Hebrón Institute of Oncology (VHIO), 08035 Barcelona, Spain; sandra.martinez@ftmoreto.org 5 Center for Biomedical Research on Rare Diseases (CIBERER), 08028 Barcelona, Spain 6 Oncology Data Science (ODysSey), Vall d’Hebrón Institute of Oncology (VHIO), 08028 Barcelona, Spain; rdienstmann@vhio.net (R.D.); fruiz@vhio.net (F.R.-P.) 7 Pediatrics Department, Vall d’Hebron University Hospital, 08035 Barcelona, Spain; mcespede@vhebron.net 8 Vall d’Hebrón Institute of Oncology (VHIO), International Breast Cancer Center, Quiron Group, 08035 Barcelona, Spain; jacortes@vhio.net  9 Department of Obstetrics, Maternal-Fetal Medicine Unit, Santa Creu i Sant Pau University Hospital,  Universitat Autònoma de Barcelona, 08025 Barcelona, Spain 10 Obstetrics and Gynecology Department, Hospital Universitari Son Espases, 07120 Palma, Spain; Citation: Saura, C.; Sánchez, O.; Martínez, S.; Domínguez, C.; octavi.cordoba@ssib.es 11 Institut d’Investigació Sanitària de les Illes Balears (IdISBa), 07120 Palma, Spain Dienstmann, R.; Ruíz-Pace, F.; * Correspondence: ellurba@yahoo.es; Tel.: +34-935537048 or +34-935537041 or +34-687743699 Céspedes, M.C.; Peñuelas, Á.; † Authors contributed equally. Cortés, J.; Llurba, E.; et al. Evolution of Angiogenic Factors in Pregnant Simple Summary: Anthracyclines and taxanes are being used as a standard treatment for breast Patients with Breast Cancer Treated cancer diagnosed during pregnancy. These chemotherapy regimens allow the continuation of with Chemotherapy. Cancers 2021, 13, 923. https://doi.org/10.3390/ pregnancy without delaying cancer treatment with relatively good maternal and neonatal outcomes. cancers13040923 However, their effects on placental function and fetal development are not completely understood. Maternal serum angiogenic factors are a surrogate of placental function and are abnormal weeks Academic Editor: Matteo Lambertini before placental complications such as preeclampsia or intrauterine growth restriction development. In our cohort, pregnant women with breast cancer treated with chemotherapy during pregnancy Received: 22 January 2021 show an antiangiogenic state with significantly higher levels of soluble fms-like tyrosine kinase Accepted: 19 February 2021 (sFlt-1), sFlt-1/PGF ratio, and soluble endoglin (sEng) at the end of the third trimester. Angiogenic Published: 23 February 2021 factors could be useful in the clinical obstetric management of these patients, although more studies are guaranteed. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Abstract: High prevalence of placental-derived complications, such as preeclampsia and intrauterine published maps and institutional affil- growth restriction, has been reported in women with breast cancer (BC) treated with chemotherapy iations. during pregnancy (PBC-CHT). Aim: To ascertain whether PBC-CHT is associated with an imbalance of angiogenic factors, surrogate markers for placental insufficiency, that could explain perinatal outcomes. Methods: Prospective study between 2012 and 2016 in a single institution. Soluble fms-like tyrosine kinase (sFlt-1), placental growth factor (PlGF), and soluble endoglin (sEng) in Copyright: © 2021 by the authors. maternal blood were assessed throughout pregnancy in 12 women with BC and 215 controls. Results: Licensee MDPI, Basel, Switzerland. Cancer patients were treated with doxorubicin-based regimes and with taxanes. Ten PBC-CHT This article is an open access article (83%) developed obstetrical complications. At the end of the third trimester, significantly higher distributed under the terms and conditions of the Creative Commons levels of sFlt-1; sFlt-1/PGF ratio, and sEng levels were observed in BC women as compared to Attribution (CC BY) license (https:// controls. Moreover; there was a significant correlation between plasma levels of sFlt-1 and the creativecommons.org/licenses/by/ number of chemotherapy cycles administered. Besides, more chemotherapy cycles correlated with 4.0/). lower birthweight and head circumference at birth. Conclusions: Women with BC treated during Cancers 2021, 13, 923. https://doi.org/10.3390/cancers13040923 https://www.mdpi.com/journal/cancers Cancers 2021, 13, 923 2 of 12 pregnancy showed an antiangiogenic state compatible with placental insufficiency. Angiogenic factors could be useful in the clinical obstetric management of these patients; although further studies will be required to guide clinical decision-making. Keywords: breast cancer; pregnancy; angiogenic factors; chemotherapy 1. Introduction Breast cancer diagnosed during pregnancy represents a complex set of challenges for the patient and the clinicians, as maternal benefit should be balanced with fetal risk. Cancer diagnosed during pregnancy is a rare occurrence with an estimated frequency of 2.3 cases per 100,000 deliveries [1]. However, it is increasing due to the rise in maternal age [2] and breast cancer incidence worldwide [3]. Use of anthracyclines and taxanes is safe during second and third trimester of preg- nancy [4–12]. However, some obstetric complications, so far not related to antineoplastic treatment, such as preeclampsia, small for gestational age, and spontaneous preterm deliv- ery, have been described [10,13] and there is still limited information regarding cytotoxic effects on placenta function and fetal wellbeing. According to animal models, placenta has a barrier function against chemotherapy. Levels of doxorubicin in baboon fetal plasma are 7.5 % (+/− 3.2%) of the maternal lev- els [14]. Despite this barrier function, the placenta may be injured. We hypothesized that this potential injury by chemotherapy may be responsible for the obstetrical complications described in cancer patients treated during pregnancy. The dysregulation of angiogenesis in the placenta and maternal–fetal circulation has emerged as one of the main pathophysiological features in the development of placen- tal insufficiency and its clinical consequences. In women developing preeclampsia (PE) or/and intrauterine growth restriction (IUGR), angiogenic factor placental growth factor (PlGF), and anti-angiogenic factor soluble fms-like tyrosine kinase (sFlt-1), and its relation, are a clinically useful tool for the prediction, prognosis, and management of these com- plications [15,16]. Angiogenic factors as surrogate markers of placental insufficiency may help to assess placental damage in pregnant women treated with chemotherapy during pregnancy (PBC-CHT) and potentially guide management and delivery planning. The aim of the present study is to ascertain whether pregnant women with breast cancer treated with chemotherapy have an imbalance of angiogenic factors towards a pro-antiangiogenic state that could be related to perinatal outcomes. 2. Patients and Methods 2.1. Study Design All consecutive patients with breast cancer diagnosed during pregnancy and treated at Vall d’Hebron University Hospital with CHT between October 2012 and June 2016 were eligible to participate in a prospective case-control study. The eligibility criteria for study entry were histological diagnosis of primary or recurrent breast cancer and starting of treatment with CHT during pregnancy (second and third trimester). All subjects gave their informed consent for inclusion before they participated in the study. This study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the Vall d’Hebron University Hospital (PR(AMI) 83/2012). Treatment strategy was decided case by case in a multidisciplinary team evaluation and according to standardized international recommendations [11–13]. Twin pregnancies were excluded. sFlt-1, PlGF, and sEng in maternal blood were assessed at diagnosis of breast cancer, and each time before and after CHT treatment throughout pregnancy and at delivery. Longitudinal changes in angiogenic factors in affected pregnancies were compared with those of low-risk pregnancies at the same gestational age (ratio 1 case:18 controls). Cancers 2021, 13, 923 3 of 12 2.2. Study Procedures Pregnant women with breast cancer were followed in the Breast cancer pregnancy unit that was composed by a Maternal–Fetal specialist, an Oncologist, and a Gynecologic surgeon. After the diagnosis of breast cancer and before and after CHT treatment, women have an exhaustive obstetrical and fetal evaluation that includes: blood pressure, weight, maternal wellbeing, and obstetric abdominal and vaginal ultrasound study using 6–4 MHz probes (Siemens Sonoline Antares, Siemens Medical, Erlangen, Germany). Estimated fetal weight, amniotic fluid index, and Doppler velocimetry; uterine artery pulsatility index (PI), umbilical artery PI and median cerebral artery PI, and maximum peak systolic velocity (Vmax MCA) were evaluated. Following obstetric evaluation, medical oncologist assesses whether potential toxicities derived from chemotherapy existed. Subsequently, if both evaluations (obstetrical and oncological) were correct, maternal blood was obtained and chemotherapy cycle was administered. The frequency of the blood tests was established by the chemotherapy schedule defined appropriately for each patient based on her tumor stage, patient’s characteristics, and weeks of pregnancy at the time of diagnosis. If there were concerns regarding maternal or fetal health, termination or delay of the chemotherapy were considered by the multidisciplinary team, or delivery was planned. If no specific concerns during treatment, delivery was planned and induced according to gestational age and CHT schedule to minimize any delay in maternal treatment. At the time of delivery, maternal and fetal cord blood was also obtained. Clinicians were blinded to angiogenic factors results. Outcomes: Diagnostic criteria for preeclampsia were new onset of both hypertension (systolic blood pressure of 140 mmHg or above and/or diastolic blood pressure of 90 mmHg or above) and proteinuria (protein dipstick urinalysis result of 2+ or greater, or 300 mg protein per 24-h urine collection or greater, or protein in spot urine greater than or equal to 30 mg/dL, or protein/creatinine ratio greater than or equal to 30 mg/mmol) after 20 weeks’ gestation [17]. Suspicion of fetal anemia was defined if fetal Vmax MCA >1.55 MoM according to Mari et al. [18]. Fetal adverse outcomes were perinatal/fetal death, delivery before 34 weeks, intrauterine growth restriction, placental abruption, respiratory distress syndrome, necrotizing enterocolitis, and intraventricular hemorrhage. 2.3. Samples Maternal venous blood was drawn for routine blood tests and samples were processed within 1 h. Plasma was separated by centrifugation at 2000× g for 5 min at 4 ◦ C, and sample aliquots were immediately stored at −80 ◦ C until assayed. 2.4. Plasma Levels of Angiogenic and Anti-Angiogenic Factors Soluble fms-like tyrosine kinase (sFlt-1), free placental growth factor (PlGF), and solu- ble endoglin (sEng) concentrations were measured under pregnancy type-blinded condi- tions. Enzyme-linked immunosorbent assays (ELISA) for human sFlt-1, PlGF, and sEng were performed in duplicate using commercial kits (R&D Systems Europe Ltd., Abington, UK) following the manufacturer’s instructions. Minimum detectable values in the assays were 3.5 pg/mL for sFlt-1, 7 pg/mL for PlGF, and 0.007 ng/mL for sEng. In all kits, intra-assay precision was always < 5% and inter-assay precision < 10%. Linear regression coefficients of the standard curves were never < 0.99%. 2.5. Statistical Analysis Statistical analyses were performed using Prism software (GraphPad, version 5.02, San Diego, CA, USA). The distribution of continuous variables was tested for normality by the D’Agostino and Pearson omnibus K2 test. Demographic and clinical data were compared by Fisher’s exact test for categorical variables and independent-sample Student’s t-test or Mann–Whitney U-test, as appropriate, for continuous variables. Differences in the evolution of angiogenic factors between groups were analyzed by two-way analysis of variance and Bonferroni post-test. Cancers 2021, 13, 923 4 of 12 Pearson’s or Spearman’s correlation analysis, as appropriate, was undertaken to relate some variables in pregnant women affected by breast cancer. The p-value threshold used for significant differences was <0.05. 3. Results 3.1. Patient Characteristics Between October 2012 and June 2016, 12 women with breast cancer diagnosed during pregnancy were treated with chemotherapy at Vall d’Hebron University Hospital. A to- tal of 215 women were included as the control population. Those women had normal pregnancies with no risk factors and were recruited in Vall d’Hebron obstetrics clinics. Table 1 summarizes demographic characteristics of the study population. Maternal age was significantly higher in pregnant women affected by breast cancer (PBC-CHT) than in healthy pregnant women (P-CTRL) (p = 0.0012). Table 1. Demographic characteristics of study population. P-CTRL PBC-CHT p-Value (n = 215) (n = 12) Maternal Age (years) 32.11 ± 5.12 37.08 ± 4.14 0.0012 BMI (Kg/m2 ) 24.27 ± 4.31 24.02 ± 4.28 0.7045 Caucasian 202 (64) 10(83) 0.1831 Conception Spontaneous 212 (99) 11 (92) 0.1965 Assisted 3 (1) 1 (8) Smoker 32 (15) 0 (0) 0.2248 Parity Nulliparous 114 (53) 6 (50) 1.000 Parous 101 (47) 6 (50) Values expressed as Mean ± SD or number (percentage). Comparisons between pregnant women with breast cancer and treated with chemotherapy (PBC-CHT) and the control group (P-CTRL) were analyzed by Fisher’s exact test for categorical variables and independent-sample Student’s t-test or Mann–Whitney U-test, as appropriate, for continu- ous variables. BMI, body mass index. The baseline characteristics of patients with breast cancer are listed in a table (Table S1). A lump was detected through breast self-examination in all patients. Breast ultrasound was performed in all patients and suspicious alterations were found in all of them. Mammog- raphy was also performed in all patients. One patient was classified as non-informative (BIRADS 0), two patients (17%) were classified into BI-RADS 1 category (negative find- ings), four patients (33%) in category 4–5 (suspicious/suggestive of malignancy findings), and five patients (42%) in category 6 (biopsy with proven malignancy before conducting the mammography). The mean gestational age at diagnostic was 16 weeks (range 4–29). Five patients were diagnosed in the first trimester (42%), five in the second trimester (42%), and 2 in the third trimester (16%). One of them had metastatic breast cancer (stage IV). Forty-two percent of patients had hormone receptor-positive tumors (estrogen -ER- and/or progesterone receptor -PR- positivity of >1%), 42% overexpressed HER-2 (+3 result by immunohistochemistry or +2 by immunohistochemistry and in situ hybridation ISH/FISH positive) and 34% were classified as triple-negative breast cancer (ER, PR and HER-2 negative). Infiltrating ductal carcinoma was observed in most patients analyzed (92%). All patients were treated with doxorubicin-based regimes (AC, anthracy cyclophosphamide) and 58% of them were also treated with taxanes. Eight patie their chemotherapy during the second trimester of pregnancy and four patien Cancers 2021, 13, 923 trimester. Six patients received adjuvant treatment after surgery, five 5 of 12received vant treatment, and one patient required chemotherapy for metastases. The p ceived during pregnancy a median of 4 AC cycles (range 2–6) with a treatmen 3.2. Chemothepary Treatment 12All weeks (median, range 6–19) and a cumulative dose of doxorubicin of 217.5 m patients were treated with doxorubicin-based regimes (AC, anthracycline plus tients treated with cyclophosphamide) taxanes and 58% received of them were alsoatreated median oftaxanes. with 7 cyclesEight of weekly patients paclitaxel began (ra and their a cumulative chemotherapy dose during theof 514.3trimester second mg/m of 2 . The mean and pregnancy time interval four patientsbetween in third the la trimester. Six patients received adjuvant treatment after surgery, five received chemotherapy and delivery was 2 weeks (range 0.7–5.71). Ten patients had som neoadjuvant treatment, and one patient required chemotherapy for metastases. The patients received cations during treatment and during pregnancy. No patient presented unexpe during pregnancy a median of 4 AC cycles (range 2–6) with a treatment length of 12 weeks rious toxicity. (median, range 6–19)Neutropenia grade and a cumulative G3ofwas dose reportedofin217.5 doxorubicin onemg/m patient and the decis 2 . Patients delay treated thetaxanes with next cycle of chemotherapy. received a median of 7 cycles Detailed of weekly information is presented paclitaxel (range 2–10) and elsewh a cumulative dose of 514.3 mg/m 2 . The mean time interval between the last cycle of S2). chemotherapy and delivery was 2 weeks (range 0.7–5.71). Ten patients had some complica- tions during treatment and during pregnancy. No patient presented unexpected or serious 3.3. Angiogenic Factors Levels in Cases and Control Women toxicity. Neutropenia grade G3 was reported in one patient and the decision was to delay A cross-sectional the next cycle of chemotherapy.analysis Detailed of samplesisobtained information at different presented elsewhere gestational (Table S2). ag pregnancy was made to evaluate gestational patterns of angiogenic and anti-a 3.3. Angiogenic Factors Levels in Cases and Control Women factors. At the end of the third trimester, sFlt-1 levels were significantly higher in A cross-sectional analysis of samples obtained at different gestational ages during preg- women nancy was madeaffected by breast to evaluate cancer gestational than patterns in the control of angiogenic group (5792 and anti-angiogenic ± 794 vs. 3 factors. At pg/mL, the end ofrespectively), Figure the third trimester, sFlt-1 1. PlGF levels werelevels followed significantly anininverse higher pregnantpattern women at the affected thirdby breast cancer trimester (334than ± 25in pg/mL the control in group PBC-CHT ± 794 (5792 vs. vs.±3801 613 ± 344 pg/mL, 41 pg/mL in controls) respectively), Figure 1. PlGF levels followed an inverse pattern at Similarly, pregnant women with chemotherapy have an increase in sFlt1/PlGFthe end of the third r trimester (334 ± 25 pg/mL in PBC-CHT vs. 613 ± 41 pg/mL in controls), Figure 2. end of pregnancy (37.5 ± 11.3 in PBC-CHT vs. 10.3 ± 1.7 in controls), Figure 3. s Similarly, pregnant women with chemotherapy have an increase in sFlt1/PlGF ratio at the endwere higher (37.5 of pregnancy throughout gestationvs.in10.3 ± 11.3 in PBC-CHT women affected ± 1.7 in controls),by breast Figure cancer 3. sEng (Figure 4 levels were higher throughout gestation in women affected by breast cancer (Figure 4). Figure 1. Maternal plasma concentrations of soluble fms-like tyrosine kinase (sFlt-1) in the control Figure 1. Maternal plasma concentrations of soluble fms-like tyrosine kinase (sFlt-1) in t group (white points) and pregnant women affected by breast cancer (black points). Differences were groupby(white analyzed two-waypoints) and analysis pregnantData of variance. women affectedasby are presented breast mean withcancer standard (black points). Diff error mean were analyzed (SEM). ***, p < 0.001. by two-way analysis of variance. Data are presented as mean with stand mean (SEM). ***, p < 0.001. ancers 2021, 13, x FOR PEER REVIEW Cancers 2021, 13, x FOR PEER REVIEW Cancers 2021, 13, 923 6 of 12 Figure 2. Maternal plasma concentrations of placental growth factor (PlGF) in the control (white Figurepoints) andplasma 2. Maternal pregnant women affected concentrations bygrowth of placental breastfactor cancer (black (PlGF) in thepoints). Differences control group Figure lyzed by2. (white Maternal two-way points) plasmawomen analysis and pregnant concentrations of variance. by of affectedData placental are breast presented cancer growth (black factor aspoints). mean (PlGF) with SEM. Differences inwere the contro ***, p < 0.0 (white points) and pregnant women affected by breast cancer (black points). Differences analyzed by two-way analysis of variance. Data are presented as mean with SEM. ***, p < 0.001. lyzed by two-way analysis of variance. Data are presented as mean with SEM. ***, p < 0.0 Figure 3. Ratio of sFlt-1 to PlGF in the control group (white points) and pregnant women affected by Figure 3. Ratio of sFlt-1 to PlGF in the control group (white points) and pregnant women breast cancer (black points). Differences were analyzed by two-way analysis of variance. Data are by breast cancer (black points). Differences were analyzed by two-way analysis of varian presented as mean with SEM. ***, p < 0.001. Figure are 3. Ratio presented asofmean sFlt-1with to PlGF SEM.in***, thepcontrol < 0.001.group (white points) and pregnant women by breast cancer (black points). Differences were analyzed by two-way analysis of varian are presented as mean with SEM. ***, p < 0.001. Cancers 2021, 13, x FOR PEER REVIEW Cancers 2021, 13, 923 7 of 12 Figure 4. Maternal plasma concentrations of soluble endogline (sEng) in the control group (white Figure points) 4. andMaternal plasmaaffected pregnant women concentrations of soluble by breast cancer endogline (black points). (sEng)were Differences in the control analyzed by group ( points) two-way analysis of variance. Data are presented as mean with SEM. **, p < 0.01; ***, p < 0.001. were anal and pregnant women affected by breast cancer (black points). Differences by two-way analysis of variance. Data are presented as mean with SEM. **, p < 0.01; ***, p < 3.4. Perinatal Outcomes Gestational age at delivery and birth weight were significantly lower in pregnant 3.4. Perinatal Outcomes women affected by breast cancer (p-value = 0.0001 and p-value = 0.0001, respectively) Gestational (Table 2). Moreover, age theat delivery PBC-CHT andpresented group birth weight a higherwere significantly incidence lower of small for ges- in pr tational age (SGA) babies (16%). Labor induction was recommended in women affected by breast cancer (p-value = 0.0001 and p-value = 0.0001, respectivel 6 patients of PBC-CHT group to facilitate the continuation of chemotherapy treatment, the remaining ble 2). Moreover, the PBC-CHT group presented a higher incidence of small for gest patients of this group were induced for: (a) obstetrical complications (1 preterm labor age and(SGA) babies 1 preterm rupture(16%). Labor induction of membranes), (b) maternalwas recommended complications in progression (disease 6 patients of PBC group to facilitate in 2 patients), or (c) the fetalcontinuation complications (1ofsuspicion chemotherapy treatment, of fetal anemia and 1the remaining patient with pati increased this grouputerine wereand umbilical induced for:artery pulsatility index (a) obstetrical -PI). complications (1 preterm labor and 1 p rupture of membranes), (b) maternal complications (disease progression in 2 patie Table 2. Perinatal outcome of study population. (c) fetal complications (1 suspicion of fetal anemia and 1 patient with increased u and umbilical artery pulsatilityP-CTRL index -PI). PBC-CHT p-Value (n = 215) (n = 12) TableGA 2. at delivery (weeks) Perinatal ± 2.08 38.9 population. outcome of study 35.8 ± 2.00 <0.0001 Birth weight (g) 3232 ± 464 2512 ± 655 <0.0001 Birth weight (p) 54.1± 27.99 P-CTRL 39.5 ± 29.61 PBC-CHT 0.0462 p-V SGA 5 (2.3) (n =2215) (16) (n0.0465 = 12) GA at(cm) Length delivery (weeks) 49.7 ± 1.75 38.9 ± 45.9 ±2.08 3.12 35.8 ± 2.00 <0.0001 <0.0 Birth Length (p) weight (g) 53.9 ± 27.4 3232 ± 464 39.4 ± 29.4 2512 ± 655 0.1148 <0.0 Birth weight Head circumference (cm) (p) 34.4 ± 1.31 54.1± 32.427.99 ± 1.76 39.5<0.0001 ± 29.61 0.0 Head circumferenceSGA(p) 49.2 ± 30.4 548.5 (2.3) ± 21.3 20.9602 (16) 0.0 Length 1 min Apgar <7 (cm) 49.7 2± (16) 1.75 45.9 ± 3.12 <0.0 Length (p) Days NICU 53.9 ±1(8) 27.4 39.4 ± 29.4 0.1 Head circumference (cm) Days Hospital 34.4 ± 1.31 2 (16) 32.4 ± 1.76 <0.0 Head circumference (p) 49.2 ± 30.4 48.5 ± 21.3 0.9 1 min Apgar <7 2 (16) Days NICU 1(8) Days Hospital 2 (16) Values expressed as Mean ± SD or number (percentage). Comparisons between Cancers 2021, 13, x FOR PEER REVIEW 8 of 12 Cancers 2021, 13, 923 8 of 12 variables and independent-sample Student’s t-test or Mann–Whitney U-test, as appropri- ate, forValues expressed continuous variables. GA,±gestational as Mean SD or number age;(percentage). p, percentile;Comparisons between SGA, small for gestational pregnant women with breast age (Birth weight<10th percentile). cancer and treated with chemotherapy (PBC-CHT) and the pregnant women control group (P-CTRL) were analyzed by Fisher’s exact test for Correlation analysis among maternal angiogenic factors at delivery and cycles of categorical variables and independent-sample Student’s t-test or Mann–Whitney U-test, chemotherapy (n CHT), birth weight, or percentile was undertaken (Table 3). A significant as appropriate, for continuous variables. GA, gestational age; p, percentile; SGA, small for correlation gestationalbetween age (Birthplasma weight levels of sFlt-1 and the number of cycles administered was < 10th percentile). detected. Correlation analysis among maternal angiogenic factors at delivery and cycles of chemotherapy (n CHT), birth weight, or percentile was undertaken (Table 3). A signifi- Table cant3.correlation Correlations between between the number plasma levels of of cycles sFlt-1 of andchemotherapy administered, the number of birth weight cycles administered or was percentile and detected. angiogenic factors values at delivery. Number Table 3. Correlations between the number ofofcycles Cycles Birth Weight of chemotherapy administered, Birth Percentile birth weight or r percentile and angiogenic factors values at p Value delivery. r p Value r p Value sFlt-1 (pg/mL) 0.6140 Number of Cycles0.0222 Birth −0.2498 Weight 0.2294Birth −0.3598 Percentile 0.1385 PlGF (pg/mL) r 0.1395 0.3412 p Value r 0.2369 p Value 0.2416 r 0.1680 p Value 0.3108 Ratio sFlt-1 to PlGF 0.2471 0.2302 −0.2364 0.2427 −0.2273 0.2517 sFlt-1 (pg/mL) 0.6140 0.0222 −0.2498 0.2294 −0.3598 0.1385 sEng (ng/mL) PlGF (pg/mL) 0.5046 0.34120.05670.2369−0.1691 0.1395 0.24160.3096 −0.3452 0.1680 0.31080.1492 Ratio sFlt-1 to PlGF 0.2471 0.2302 −0.2364 0.2427 −0.2273 0.2517 sEng (ng/mL) 0.5046 0.0567 A Pearson’s correlation analysis was − 0.1691 0.3096 − 0.3452 0.1492of chem- undertaken to relate number of cycles otherapy administered (n CHT), birth weight, or percentile to maternal blood sFlt-1, PlGF, and sEng A Pearson’s correlation at delivery. analysis was A Spearman’s undertaken correlation to relatewas analysis number of cycles oftochemother- undertaken relate number of apy administered cycles (n CHT), of chemotherapy birth weight,(norCHT), administered percentile birthtoweight, maternalor blood sFlt-1,toPlGF, percentile ratio sFlt- and sEng at delivery. 1 to PlGF at delivery. A Spearman’s correlation analysis was undertaken to relate number of cycles of chemotherapy administered (n CHT), birth weight, or percentile to ratio sFlt-1 A negative and significant correlation between the number of cycles of chemotherapy to PlGF at delivery. administered to pregnant A negative women and significant affectedbetween correlation by breast cancer and the number birthofweight of cycles of their chil- chemotherapy dren was observed administered (Figurewomen to pregnant 5). A negative correlation affected by between breast cancer the number and birth weight ofoftheir cycles of chemotherapy administered children was observed to5). (Figure pregnant women A negative affected correlation by breast between cancerofand the number neonate cycles head circumference of chemotherapy was also detected administered (Figure to pregnant women6). affected by breast cancer and neonate head circumference was also detected (Figure 6). Figure 5. Correlation between cycles of chemotherapy administered (n CHT) and birth weight or percentile in pregnant Figure 5. Correlation women affected between cycles ofbychemotherapy breast cancer (nadministered = 12). (n CHT) and birth weight or percentile in pregnant women affected by breast cancer (n = 12). ancers 2021, 13, x FOR PEER REVIEW 9 Cancers 2021, 13, 923 9 of 12 Figure 6. Correlation between number of chemotherapy cycles administered (n CHT) and neonate head circumference or percentile in pregnant women affected by breast cancer (n = 12). Figure 6. Correlation between number of chemotherapy cycles administered (n CHT) and neonate head circumference or percentile in pregnant women affected by breast cancer (n = 12). 4. Discussion To our knowledge, this is the first study that analyzes circulating levels of angiogenic 4. Discussion factors in pregnant women with breast cancer. Our major findings are: (1) an angiogenic To our knowledge, imbalance, this is thestate, towards antiangiogenic first isstudy that analyzes developed at the thirdcirculating levels of angiog trimester of pregnancy and (2) birth weight at delivery inversely correlates with factors in pregnant women with breast cancer. Our major findings are: (1) an the cycles of chemotherapy ad-angiog ministered. Treatment of breast cancer during pregnancy encompasses many therapeutic imbalance, towards antiangiogenic state, is developed at the third trimester of pregna dilemmas. A multidisciplinary approach to the management of BC during pregnancy andis(2) birthtoweight critical at delivery optimizing outcomesinversely for both mother correlates withIt the and fetus. cyclesaccepted is widely of chemotherapy that ministered. chemotherapy Treatment of breast administration cancer should duringduring be avoided pregnancy the firstencompasses trimester when many organo-therape genesis takes dilemmas. place [13,19]. A multidisciplinary approach to the management of BC during pregnanc Throughout the second and the third trimester of pregnancy, indications for neoadju- critical to optimizing outcomes for both mother and fetus. It is widely accepted that ch vant and adjuvant treatments are the same that recommended for non-pregnant women. otherapy administration Most experience should be avoided with chemotherapy during is during pregnancy the first from trimesterand doxorubicin when organogen taxane- takes place based [13,19].Indeed, the dosages are administered similar to non-pregnant women regimens. Throughout adjusting the gain for weight second anddose without themodifications third trimester with of pregnancy, curative indications intent [20,21]. This ex- for neo juvant and adjuvant treatments are the same that recommended for[7,22]. posure is associated with the risk of IUGR, prematurity, and low birth weight non-pregn Loibl and colleges demonstrated that infants exposed to chemotherapy in utero had lower women. Most experience with chemotherapy during pregnancy is from doxorubicin birth weight at the same gestational age than infants not exposed to chemotherapy [10]. taxane-based The same trend regimens. was observedIndeed, in ourthestudy,dosages areweight, since birth administered adjusted bysimilar gestationalto age, non-pregn women adjusting lower was significantly for weight in babies gain whosewithout mothers dose modifications received chemotherapy with curative during intent [20 pregnancy. Regarding This exposure is the analysis ofwith associated the circulating the risk of levels of angiogenic IUGR, prematurity, factorsand in pregnant low birth we women with BC, no previous data exist that evaluate these markers in pregnant women [7,22]. Loibl and colleges demonstrated that infants exposed to chemotherapy in utero with breast cancer. Otherwise, a significantly higher incidence of small-for-gestational-age lower birth (SGA) weight when is observed at thechemotherapy same gestational is givenageduringthan infantsindicating pregnancy, not exposed to chemother a potentially [10].toxic Theinfluence same trend was observed on placental developmentinleading our study, sincemalfunction to placental birth weight, adjusted by ge (via incomplete tional age, was significantly lower in babies whose mothers received chemotherapy trophoblast invasion into the uterus) [10,23]. This fact might explain why birthweight ing inversely pregnancy. correlates with the cycles of chemotherapy received during pregnancy in our PBC-CHT patients. Regarding the analysis of the circulating levels of angiogenic factors in pregn The results of our study suggest that the accumulative effect of chemotherapy treat- womenmentswith BC, nofunction on placental previous data exist is reflected by an that evaluate angiogenic these markers antiangiogenic state in pregnant at the end wo withofbreast pregnancy. cancer. PlGFOtherwise, and sFlt-1 levels a significantly correlate with higher the numberincidence of cyclesof andsmall-for-gestatio with birth weight. These findings suggest that placental insufficiency age (SGA) is observed when chemotherapy is given during pregnancy, is the main trigger that accounts indicating a for adverse pregnancy complications observed in these mothers and babies. However, tentially toxic influence on placental development leading to placental malfunction the sample volume is insufficient to allow a deep analysis of the number of cycles and the incomplete effect on thetrophoblast sFlt-1/PlGF invasion ratio and sEng intolevels. the uterus) [10,23]. This fact might explain birthweightBefore each chemotherapy cycle, clinicianscycles inversely correlates with the may have of to chemotherapy received take into consideration during p mater- nancynal in andourfetalPBC-CHT status. There are no clear guidelines for optimal pre-chemotherapy fetal and patients. mother assessment. At The results of our study our institution, suggestwe thatagreed to perform a fulleffect the accumulative obstetricof assessment chemotherapy tr ments on placental function is reflected by an angiogenic antiangiogenic state at the of pregnancy. PlGF and sFlt-1 levels correlate with the number of cycles and with b weight. These findings suggest that placental insufficiency is the main trigger that counts for adverse pregnancy complications observed in these mothers and babies. H Cancers 2021, 13, 923 10 of 12 including an abdominal study with Doppler velocimetry before each cycle on top of stan- dard blood tests to rule out neutropenia that may prevent chemotherapy administration. If maternal status and fetal tests were fine, a new cycle of chemotherapy was administrated. Given that all efforts should be made to delay delivery [24] until at least 35–37 weeks of gestational age to reduce risk of fetal morbidity associated with prematurity, our findings can be used as a useful clinical tool to better plan the timing of delivery in pregnant women affected by BC. sFlt-1:PlGF ratio will allow monitoring the placental function throughout the pregnancy in order to prevent obstetric complications. As values of the sFlt-1:PlGF ratio higher than 38 confer increased risk of obstetrical complications (PE/IUGR) and preterm labor, the medical team should increase the maternal and fetal well-being monitoring and follow-up of these patients [16]. Moreover, complementary, our study also suggests that normal levels of angiogenic factors in women exposed to chemotherapy during pregnancy may be useful tools to rule out an early complication and decide to proceed to administer an additional cycle of chemotherapy if deemed appropriate and contribute to lengthen some weeks of pregnancy. It is important to highlight that in our group of pregnant women with breast cancer, angiogenic factors were within normal values for gestational age until the end of pregnancy that increased compared to control pregnant women, although without reaching the values of pregnant women with preeclampsia. According to European Society of Medical Oncology (ESMO) guidelines for breast cancer treatments diagnosed during pregnancy, the indications for systemic therapy should follow those for the non-pregnant setting, taking into consideration the gestational age at diagnosis and the expected date of delivery. Chemotherapy is generally safe beyond the first trimester of gestation; however, increased rates of premature delivery, growth retardation, and stillbirth have been reported and close monitoring of the pregnancy is recommended. Dose calculation should follow the standard procedures outside the pregnancy setting, acknowledging that the pharmacokinetics of some cytotoxic drugs might be altered during pregnancy, but no dose adjustments are recommended [11]. Our results do not allow a different approach. Hypothetical dose customization according to sFlt-1/PlGF ratio may reduce treatment efficacy. In our experience, we feel more comfortable delaying one cycle administration because of maternal or fetal needs than lowering chemotherapy dose. Even some preclinical data have been reported on new formulations of anthracyclines (liposomal formulations) and taxanes (nab-paclitaxel) about the potential reduction in the crossing of the placental barrier in animal models [25], none of the guidelines or consensus for breast cancer treatment during pregnancy included these new formulations due to lack of data in humans. All patients in our study were treated with standard formulations, following current guidelines [11]. Potential limitations of this study include the small sample size (n = 12). However, taking into account the singularity of these patients, this set of data could be useful for hypothesis-generating and encourage further investigations on angiogenic and antiangio- genic factors as potential prognostic markers in breast cancer patients. Further studies may help to elucidate the correlation of angiogenic factors and the number of cycles or gestational age at starting. 5. Conclusions Women with breast cancer treated during pregnancy showed an antiangiogenic state that correlates with the number of cycles administered. Our results suggest that angiogenic factors could be useful in the clinical obstetric management of these patients as they could anticipate placental complications that usually develop in these patients. More studies are needed to evaluate the potential causative effect of CHT on placenta function and development and its implication in adverse fetal and maternal outcomes observed in these patients. Because of the low incidence of BC during pregnancy, international prospective collaboration is needed for advancing the knowledge of optimal management and treatment. Cancers 2021, 13, 923 11 of 12 Supplementary Materials: The following are available online at https://www.mdpi.com/2072-669 4/13/4/923/s1, Table S1: Baseline characteristics of 12 patients with breast cancer diagnosed during pregnancy, Table S2: Complications during gestation in 10 patients with breast cancer diagnosed during pregnancy. Author Contributions: Conceptualization, O.C., C.S. and E.L.; methodology, C.S., O.S., S.M., C.D., Á.P., J.C., E.L. and O.C.; validation, C.S., E.L., J.C. and O.C.; formal analysis, C.S., O.S., C.D., R.D., F.R.-P., E.L. and O.C.; investigation, C.S., O.S., S.M., C.D., M.C.C., Á.P. and O.C.; resources, C.S., O.S., S.M., R.D., F.R.-P., M.C.C., E.L. and O.C.; data curation, O.S. and S.M.; writing—original draft preparation, C.S., O.S., E.L. and O.C.; writing—review and editing, J.C.; project administration, C.S., O.S., E.L. and O.C.; funding acquisition, C.S., E.L. and O.C. All authors have read and agreed to the published version of the manuscript. Funding: This study was funded by the Spanish Research Project in Health funded by ISCIII, the state plan for scientific and technical research and innovation 2015–2018, and European Regional Development Fund (ERDF), ref. PI15/02252. This study was also supported in part by RETICS ‘Maternal and Child Health and Development Network’ (SAMID Network), funded by the PN I + D + i 2008–2016 (Spain), ISCIII-Sub-Directorate General for Research Assessment and Promotion, and the European Regional Development Fund (ERDF), ref. RD12/0026 and RD16/0022. O.S. was supported by SAMID Network (RD12/0026/0016 and RD16/0022/0015) and S.M. was supported by “Paseico de la mama”. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of Vall d’Hebron University Hospital (PR(AMI) 83/2012). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data presented in this study are available on request from the corresponding author. The data are not publicly available due to specifications on the signed IC. Acknowledgments: The authors would like to thank the patients who made this study possible. Conflicts of Interest: Saura served as a consultant, participated in advisory boards, or received travel grants from AstraZeneca, Celgene, Daiichi Sankyo, Eisai, F. Hoffmann-La Roche Ltd., Genomic Health, Merck, Sharp and Dhome España SA, Novartis, Pfizer, Philips Healthwork, Pierre Fabre, prIME Oncology, Puma, Synthon, and Sanofi Aventis, but none of them affected this study. Llurba received fees for lectures from ROCHE diagnostics, Cook Medical, and Vifor Pharma. Llurba is also a member of ROCHE diagnostics Spanish advisory committee. Cordoba received grants from Pfizer and personal fees from GE and ASCIRES, but none of them affected this study. References 1. 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