The Radiation-induced Toxicities And Circulating Tumor Cell Dynamics After Hypofractionated Radiotherapy In Patients With Breast Cancer

Part Ⅰ:Radiation-induced hypothyroidism in patients with breast cancer after hypofractionated radiation therapy:a prospective cohort studyPurpose:To assess the incidence and risk factors of radiation-induced hypothyroidism(RHT)after adjuvant hypofractionated radiotherapy(RT)in patients with breast cancer,and identify the dose constraint for the thyroid gland.The trends of RHT over time were also evaluated to determine the optimal timing of screening for RHT in breast cancer patients treated with hypofractionated RT.Materials and methods:Consecutive eligible breast cancer patients who were treated with hypofractionated RT were prospectively evaluated.Thyroid function tests were performed before and at regular times following RT(every 3-4 months during the first two years,then every six months until five years).RHT was defined as twice elevated serum thyroid-stimulating hormone(TSH)with decreased or normal free thyroxin after RT.The patient,tumor,and treatment factors were evaluated for possible associations with the risk of RHT.The incidence of RHT was calculated from the end of RT using the Kaplan-Meier method,and the differences between groups were compared with the log-rank test.Multivariate analysis was performed using Cox logistic regression analysis.A multivariate Cox proportional hazards regression model with penalized spline(P-spline)was used to examine the relationship between thyroid mean dose(Dmean)and RHT.A two-sided test of P<0.05 was considered statistically significant.Results:A total of 500 patients treated between 2017 and 2020 were analyzed.All patients underwent chest wall/breast±regional nodal irradiation with a prescription dose of 43.5Gy in 15 fractions.Among them,69(73.8%)patients received supraclavicular nodal radiation(SCRT).Eighty-two(16.4%)patients had elevated TSH before RT.At a median follow-up of 21.9 months,131(26.2%)patients developed RHT,and 59(11.8%)patients received thyroid hormone-replacement therapy.The 2-year cumulative incidence of RHT was 26.0%.Patients with SCRT had a significantly increased 2-year cumulative incidence of RHT compared with patients without SCRT(31.5%and 11.4%,P<0.001).The median time to the onset of RHT was 9.3 months(range,1.0-42.4)after RT.The peak incidence of RHT occurred around 6-12 months after RT.Multivariate analysis revealed that elevated baseline TSH and increased thyroid Dmean as a continuous variable were independent risk factors for developing RHT.After adjusted for baseline TSH,there was a nonlinear relationship between Dmean and the risk of RHT.Dmean>21Gy was identified as the threshold value for predicting RHT(HR=2.2,P<0.001).Dmean>21Gy compared with Dmean≤21Gy was associated with a significantly higher incidence of RHT at two years after RT(32.2%vs.17.0%,P<0.001).Conclusions:The incidence of RHT was high in breast cancer patients.Thyroid function test should be started no later than six months following RT.We recommend that the Dmean of the thyroid should be kept lower than 21Gy for hypofractionated RT.PART Ⅱ:Longitudinal analyses and predictive factors of radiation-induced lymphopenia after hypofractionated postmastectomy radiotherapy in patients with breast cancerPurpose:Radiation-induced lymphopenia(RIL)is associated with poor prognosis in solid tumors.This study aimed to investigate the longitudinal lymphocyte change in patients receiving hypofractionated postmastectomy radiotherapy and the association of different radiotherapy techniques with RIL.The impact of interleukin-7(IL-7)and interleukin-15(IL-15)(key cytokines in lymphocyte homeostasis)on RIL were also assessed.Materials and methods:We prospectively assessed 607 patients who received hypofractionated postmastectomy radiotherapy for breast cancer in 8 hospitals.Radiotherapy techniques included integrated photon-based intensity modulated techniques(integrated RT)and a hybrid approach combining photon irradiation to supraclavicular nodes and electron irradiation to the chest wall ± internal mammary node(hybrid RT).Peripheral lymphocyte counts(PLC)were tested prior to radiotherapy(preRT),weekly during radiotherapy,at 1,2 weeks,3,6 months after radiotherapy,and then every 6 months.Grade 3+RIL was defined as PLC nadir during radiotherapy of<0.5×109/L.Blood IL-7 and IL-15 levels were assayed before and at the end of radiotherapy in 20 patients with grade 3+RIL and 20 patients without grade 3+RIL,respectively.Mean PLC was compared by the t test.Blood IL-7 and IL-15 levels were compared using nonparametric tests.The relationships between grade 3+RIL and different clinical characteristics were evaluated using Chi-square analysis or Fisher’s exact test.The binary logistic and multivariate regression model was used to identify risk factors associated with the occurrence of grade 3+RIL.Propensity score matching(PSM)analyses were used to evaluate the effect of radiotherapy techniques on grade 3+RIL.Results:During radiotherapy,121(19.9%)of patients had grade 3+RIL.The PLC started to recover at 1 week and recovered to pre-RT levels 1 year after radiotherapy,and to a higher than pre-RT level at 2 years after RT.Multivariate analyses showed lower pre-RT PLC(P<0.001)and the integrated RT technique(vs.hybrid RT technique,P<0.001)were independent risk factors for grade 3+RIL.A greater proportion of the patients treated with the integrated RT(90/269,33.5%)developed grade 3+RIL compared with those receiving hybrid RT(31/338,9.2%,P<0.001).After conducting PSM,a significant increase in the risk of grade 3+RIL was seen among patients who received integrated RT compared to hybrid RT(OR=3.93,P<0.001).The PLC in patients receiving the hybrid RT at each timepoint during and after radiotherapy were higher than that in those receiving integrated RT(P<0.05).Both IL-7 and IL-15 levels before radiotherapy were significantly lower in patients who developed grade 3+ RIL than in those who did not(P<0.1).Conclusions:Breast cancer patients had a prolonged lymphopenia after radiotherapy.Integrated RT increased the risk of RIL and adversely affected recovery from RIL.Choosing appropriate radiotherapy technique should be considered to decrease the risk of RIL and minimize radiation toxicity to the immune system.IL-7 and IL-15 levels before radiotherapy may be associated with the prevention of RIL,pending further study confirmed.Part Ⅲ:Circulating tumor cell detection in patients with non-metastatic breast cancer before and after postmastectomy radiotherapy:a preliminary translational result of POTENTIAL trialPurpose:Circulating tumor cells(CTCs)represent a prognostic surrogate of minimal residual disease(MRD)responsible for recurrences of breast cancer.We aimed to determine the effect of radiation therapy(RT)on CTCs and whether CTC changes could be related to RT volume in breast cancer patients who received postmastectomy RT.Radiation-induced lymphopenia(RIL)is associated with poor prognosis in breast cancer.This study investigated the association between CTC changes and RIL.Materials and methods:In 156 eligible patients who were randomly assigned to receive internal mammary nodal irradiation(IMNI)or not from the POTENTIAL trial,peripheral blood was obtained before radiotherapy(pre-RT)and after radiotherapy(postRT).Detection of at least 1 CTC per 4ml using the oHSV1-hTERT-GFP method was considered CTC positive.Grade 3+RIL was defined as PLC nadir during radiotherapy of<0.5×109/L.The correlation of both CTC positivity and CTC counts with clinicopathological factors were evaluated using the Chi-square test and Mann-Whitney U test.The McNemar test or Wilcoxon signed-rank test was used to comparing the CTC positivity or CTC counts between pre-RT and post-RT.Results:CTC positivity was 95/156(60.9%)pre-RT and 60/135(44.4%)post-RT(P=0.002).The median CTC counts pre-RT and post-RT was 1 CTC(range,0 to 9)and 0 CTC(range,0-9)per 4 mL(P=0.008).No significant association was found between CTC positivity or CTC counts pre-RT with clinicopathological characteristics(all P>0.05).In 65 patients with IMNI,both CTC positivity(58.7%pre-RT vs.35.4%postRT,P=0.006)and CTC count(P=0.007)significantly decreased after RT.In 70 patients without IMNI,no significant differences after RT were observed(P>0.05).In 69 patients who had<grade 3 radiation-induced lymphopenia(RIL),both CTC positivity(72.5%pre-RT vs.27.5%post-RT,P=0.016)and CTC count(P=0.032)significantly decreased after RT.In 66 patients who had grade 3+RIL,no significant differences after RT were observed(P>0.05).Conclusions:MRD existed in most patients with breast cancer after mastectomy and chemotherapy,which provides the rationale of RT for eradicating the potential locoregional diseases.CTCs significantly decreased after RT,especially in patients with IMNI and patients who had<grade 3 RIL.CTCs may be an early marker for RT efficacy and help better tailor adjuvant RT.