TYPE Original Research PUBLISHED 13 January 2023 DOI 10.3389/fendo.2022.1071775 Validation of dynamic risk OPEN ACCESS stratification and impact of EDITED BY David Viola, University of Pisa, Italy BRAF in risk assessment of REVIEWED BY Valentina Cirello, thyroid cancer, a nation-wide University of Milan, Italy Giorgio Grani, Sapienza University of Rome, Italy multicenter study *CORRESPONDENCE Garcilaso Riesco-Eizaguirre Laura Pérez-Fernández 1, Julia Sastre 2, Carles Zafón 3, griesco@iib.uam.es Amelia Oleaga 4, Esmeralda Castelblanco 5, Ismael Capel 6, SPECIALTY SECTION This article was submitted to Juan C. Galofré 7, Sonsoles Guadalix-Iglesias 8, Cancer Endocrinology, Antonio De la Vieja 9,10 and Garcilaso Riesco-Eizaguirre 10,11,12* a section of the journal Frontiers in Endocrinology 1 Department of Endocrinology, Hospital del Tajo, Madrid, Spain, 2 Department of Endocrinology, Hospital Universitario de Toledo, Toledo, Spain, 3 Diabetes and Metabolism Research Unit (VHIR) RECEIVED 16 October 2022 and Department of Endocrinology, Hospital Vall d’Hebron, Universitat Autónoma de Barcelona, ACCEPTED 23 December 2022 Barcelona, Spain, 4 Department of Endocrinology and Nutrition, Basurto University Hospital, PUBLISHED 13 January 2023 Bilbao, Spain, 5 Department of Endocrinology and Nutrition, University Hospital Arnau de Vilanova, CITATION IRBLLEIDA, Lleida, Spain, 6 Endocrinology Department, Parc Taulı´ Sabadell University Hospital, Pérez-Fernández L, Sastre J, Zafón C, Sabadell Barcelona, Spain, 7 Department of Endocrinology, Instituto de Investigación Sanitaria de Oleaga A, Castelblanco E, Capel I, Navarra (IdiSNA), Clı´nica Universidad de Navarra, Pamplona, Spain, 8 Department of Endocrinology, Galofré JC, Guadalix-Iglesias S, De la Hospital Universitario 12 de Octubre, Madrid, Spain, 9 Endocrine Tumor Unit (UFIEC), Instituto de Vieja A and Riesco-Eizaguirre G (2023) Salud Carlos III (ISCIII), Madrid, Spain, 10Ciber de Cancer (CIBERONC), Instituto de Salud Carlos III Validation of dynamic risk stratification (ISCIII), Madrid, Spain, 11 Department of Endocrinology and Nutrition, Hospital Universitario de and impact of BRAF in risk assessment Móstoles, Madrid, Spain, 12 Endocrinology Molecular Group, Faculty of Medicine, Universidad of thyroid cancer, a nation-wide Francisco de Vitoria, Madrid, Spain multicenter study. Front. Endocrinol. 13:1071775. doi: 10.3389/fendo.2022.1071775 COPYRIGHT Introduction: The dynamic risk stratification (DRS) is a relatively new system in © 2023 Pérez-Fernández, Sastre, Zafón, thyroid cancer that considers the response to primary treatment to improve the Oleaga, Castelblanco, Capel, Galofré, Guadalix-Iglesias, De la Vieja and initial risk of recurrence. We wanted to validate DRS system in a nationwide Riesco-Eizaguirre. This is an open- multicenter study and explore if the incorporation of BRAFV600E into DRS access article distributed under the terms of the Creative Commons helps to better categorize and predict outcomes. Attribution License (CC BY). The use, distribution or reproduction in other Materials and methods: Retrospective study of 685 patients from seven forums is permitted, provided the original author(s) and the copyright centers between 1991 and 2016, with a mean age of 48 years and a median owner(s) are credited and that the follow-up time of 45 months (range 23-77). The overall BRAFV600E prevalence original publication in this journal is was 53.4%. We classified patients into four categories based on DRS (‘excellent’, cited, in accordance with accepted academic practice. No use, ‘indeterminate’, ‘biochemical incomplete’, and ‘structural incomplete’ distribution or reproduction is response). Cox regression was used to calculate adjusted hazard ratios (AHR) permitted which does not comply with these terms. and proportions of variance explained (PVEs). Results: We found 21.6% recurrences and 2.3% cancer-related deaths. The proportion of patients that developed recurrence in excellent, indeterminate, biochemical incomplete and structural incomplete response to therapy was 1.8%, 54%, 91.7% and 96.2% respectively. Considering the outcome at the end of the follow up, patients showed no evidence of disease (NED) in 98.2, 52, 33.3 and 25.6% respectively. Patients in the structural incomplete category were the Frontiers in Endocrinology 01 frontiersin.org Pérez-Fernández et al. 10.3389/fendo.2022.1071775 only who died (17.7%). Because they have similar outcomes in terms of NED and survival, we integrated the indeterminate and biochemical incomplete response into one unique category creating the 3-tiered DRS system. The PVEs of the AJCC/TNM staging, ATA risk classification, 4-tiered DRS, and 3- tiered DRS to predict recurrence at five years were 21%, 25%, 57% and 59% respectively. BRAFV600E was significantly associated with biochemical incomplete response (71.1 vs 28.9%) (HR 2.43; 95% CI, 1.21 to 5.23; p=0.016), but not with structural incomplete response or distant metastases. BRAF status slightly changes the AHR values of the DRS categories but is not useful for different risk grouping. Conclusions: This is the first multicenter study to validate the 4-tiered DRS system. Our results also show that the 3-tiered DRS system, by integrating indeterminate and biochemical incomplete response into one unique category, may simplify response to therapy keeping the system accurate. BRAF status does not provide any additional benefit to DRS. KEYWORDS dynamic risk stratification, BRAF, multicenter study, thyroid cancer, prognosis, response to therapy Introduction therapeutic decision-making (1). While BRAF status has been proposed to improve the initial risk stratification of structural Papillary thyroid carcinoma (PTC) accounts for recurrence in PTC, the impact on DRS remains unclear. To our approximately 85% of thyroid cancers. Most of the cases are knowledge there is only one single center study that explores this indolent and have a good prognosis and treatment and relationship (16). In this nation-wide multicenter study monitoring methods should depend on the risk of disease promoted by the Spanish Task Force for Thyroid Cancer on progression and clinical evolution. The initial staging systems behalf of Spanish Society of Endocrinology & Nutrition (SEEN), from the American Thyroid Association (ATA) and the we wanted to validate DRS system as a valuable tool to predict American Joint Cancer Committee/Union Internationale patients’ clinical outcome and to examine the utility of Contre le Cancer (AJCC/UICC) provide valuable information BRAFV600E mutation in combination with DRS. on initial risk estimates for structural recurrence and mortality respectively, which are important for initial treatment and management (1). The dynamic risk stratification system (DRS) Materials and methods proposed by Tuttle in 2008 considers the response to primary treatment during the first two years of follow up and has been We retrospectively reviewed the medical records of 685 shown to modify and improve the initial risk of recurrence (2). patients followed-up in seven different tertiary referral centers This 4-tiered DRS system has been validated in several single in Spain between 1991 and 2016 as an initiative Spanish Task center studies, mainly in patients treated with total Force for Thyroid Cancer. Every center had the approval of their thyroidectomy (TT) and radioactive iodine therapy (RAI) and institutional review board. All patients had been treated with its use is strongly recommended in the ATA 2015 guidelines (1– total thyroidectomy and 85.5% received additional treatment 7). There is also evidence that DRS is useful in predicting with RAI. Tumor-nodes-metastases (TNM) staging was recurrence in patients treated without RAI (8, 9). standardized post operatively in accordance with the 7 th BRAF V600E is the most frequent mutation in PTC (10, 11) edition of the classification of the Union for International and has been related to poor prognosis and disease recurrence Cancer Control and ATA risk stratification was calculated (12–15). The ATA guidelines in 2015 suggested to include BRAF according to 2015 guidelines (1). Response to primary status to improve refine the initial risk of structural recurrence in treatment was evaluated 9-12 months after RAI the context of other clinical-pathologic risk factors to aid administration. Stimulated Tg was assessed after either Frontiers in Endocrinology 02 frontiersin.org Pérez-Fernández et al. 10.3389/fendo.2022.1071775 administering recombinant human thyrotrophin (rhTSH) or Tg < 0.2 ng/mL or TSH-stimulated Tg < 1 ng/mL. Patients with levothyroxine withdrawal. Long-term follow-up was monitored indeterminate response to treatment have nonspecific findings by clinical examination, neck US and measurement of serum on imaging studies, faint uptake in thyroid bed on RAI thyroglobulin (Tg) and Tg antibodies (TgAb) concentrations scanning, a nonstimulated Tg detectable but <1 ng/mL or during LT4 treatment to detect recurrent/persistent disease, stimulated Tg detectable but < 10 ng/mL or anti-Tg from now on recurrence. The BRAFV600E mutation status antibodies stable or declining in the absence of structural or was determined for research purposes in consecutive patients functional disease. A biochemical incomplete response means a at each center and did not affect decisions on selection of negative imaging with suppressed Tg ≥1 ng/mL or TSH- treatments. BRAF status was determined by Sanger sequencing stimulated Tg > 10 ng/mL or rising anti-Tg antibody levels. using formalin-fixed-paraffin-embedded tissues in all centers And finally, a structural incomplete response means structural except for one (Clı́nica Universidad de Navarra) which used or functional evidence of disease with any Tg level with or pyrosequencing in cytology samples. PTC recurrence was without anti-Tg antibodies. For patients treated only with total defined as recurrent or persistent disease per authoritative thyroidectomy (without RAI ablation) (17) the criteria are a bit histologic, cytologic, radiographic, or biochemical criteria (1). different regarding excellent response, indeterminate response, Local, regional, and distant recurrences were all included. Every and biochemical incomplete response. Patients with excellent investigator was asked to follow the 2015 ATA guidelines and its response must have negative imaging and either suppressed Tg own clinical expertise. The biochemical criteria for definition of < 0.2 ng/mL or TSH-stimulated Tg < 2 ng/mL. Patients with recurrent/persistent disease were abnormal serum Tg levels indeterminate response to therapy may have nonspecific overtime (suppressed Tg >0.2 ng/ml or stimulated Tg >1 ng/ findings on imaging studies with a suppressed Tg level of mL) or rising/persistent anti-Tg antibodies TgAb overtime. 0.2-5 ng/mL or stimulated Tg level of 2-10 ng/mL or anti-Tg Follow-up time was defined as the time from initial surgical antibodies stable or declining in the absence of structural or treatment to the finding of PTC recurrence or, in cases of no functional disease. Finally, patients with biochemical recurrence, to the most recent clinic visit. incomplete response to therapy must have a negative imaging This was a retrospective study approved by the relevant and suppressed Tg ≥5 ng/mL or TSH-stimulated Tg > 10 ng/ institutional review boards or ethics committees of each center mL or rising anti-Tg antibody levels over time. (Comité s de É tica de la Investigació n (CEI): CEIm Hospital We considered patients with no evidence of disease (NED) at Universitario 12 de Octubre, CEIm Gerencia de Atenció n the end of the follow up if they had a thyroglobulin less than 0.2 Especializada de Toledo, CEIm del Hospital Universitari Vall ng/mL under LT4 treatment and less than 1 ng/mL after rhTSH d’Hebron, CEI del Hospital de Basurto, CEIm Hospital stimulation with negative TgAb and no suspicious images on US Universitari Arnau de Vilanova de Lleida, CEIm Parc Taulı́ neck examination. and CEIm Clı́n ica Universitaria de Navarra) and was We stratified patients by BRAF V600E mutational status and conducted in accordance with the principles of the categories of DRS. Cox regression was used to calculate Declaration of Helsinki and Good Clinical Practice unadjusted and adjusted hazard ratios for risk of recurrence. Guidelines. Patients provided written informed consent Recurrence-free survival was calculated by Kaplan-Meier before study enrolment; patient consent was waived in some method using log-rank test for comparisons. cases following institutional review board–approved To estimate how well a risk stratification system explained procedures in the collection of pathological data. The study recurrence we used the proportions of variance explained involved the use of only thyroid tumor tissues and (PVEs) in Cox regression models. PVE (%) ranges from 0 to clinicopathological information of patients. The BRAF 100, larger numbers mean better predictions. PVE was V600E mutation status was determined after surgical and determined using the mathematical formula: PVE=1-exp(-G2/ medical (e.g., radioiodine) treatments in all cases and did not n), where G 2 is the maximum likelihood ratios that is affect decisions on selection of treatments. Genomic DNA determined on analysis of chi-squared test associated with the isolated from primary PTC tumors was used to analyze the null hypothesis; n is the total number of valid cases in the sequence of exon 15 of the BRAF gene for BRAF V600E. study (18). There were four response-to-therapy categories: excellent The chi-squared test or Fisher`s exact test was used to response, indeterminate response, biochemical incomplete compare categories data as appropriate. Hazard ratios (HR) response, and structural incomplete response. These with 95% confidence intervals (95% CI) were calculated in categories were based on the criteria proposed by Tuttle et al. logistic regression model. A two-tailed P value less than 0.05 in 2010 and adopted by the 2015 ATA guidelines (1, 6, 17). was considered statistically significant. All the statistical analyses Such criteria have been defined for patients treated with total were performed using the statistical package SPSS version 15 for thyroidectomy and RAI ablation (6). Patients with excellent windows and R 4.1 (The R Foundation, Vienna, Austria; http:// response are those with negative imaging and either suppressed www.R-project.org/) software. Frontiers in Endocrinology 03 frontiersin.org Pérez-Fernández et al. 10.3389/fendo.2022.1071775 Results proportion of patients that have recurrent/persistent disease in low, intermediate, and high-risk categories was 10.2%, 38% and Patient demographics 96.8% respectively. Regarding mortality, high-risk patients were more likely to die (36.4%) than patients in the intermediate (0.5%) We studied a total of 685 patients with PTC (540 females and 145 and the low-risk category (0.8%). males) across the 7 centers, with an average age of 48. The median When we classified patients based on DRS, the proportion of follow-up time was 45 months (IQR 23-77). All patients were treated patients that developed recurrent/persistent disease in excellent, with total thyroidectomy and 85.5% with RAI ablation. Patient age, indeterminate, biochemical incomplete and structural sex, AJCC/TNM stage, BRAF V600E mutation status, PTC recurrent/ incomplete response to therapy was 1.8%, 54%, 91.7% and persistent disease, follow-up time and provider center are 96.2% respectively. Patients in the structural incomplete summarized in Table 1. In 68.9% of patients, AJCC/TNM category were the only ones who died (17.7%). Considering classification was stage I or II, the overall BRAF V600E mutation the outcome at the end of the follow up (median 3.75 years), prevalence was 53.4% and the overall PTC recurrent/persistent patients showed no evidence of disease (NED) in 52%, 33.3% disease was seen in 21.6% of patients, all of which were comparable and 25.6% of indeterminate, biochemical incomplete and with the literature. The dominant PTC histological subtype was structural incomplete responders, respectively (Table 4). In classic (68.9%). Following the DRS system, patients were classified clinical practice, the DRS (response to therapy re-stratification into four groups according to the response to initial therapy: excellent system) is used to modify initial risk estimates provided by the (72.6%), indeterminate (8.6%), biochemical incomplete (5.9%), and ATA risk stratification system. As shown in Table 5, the change structural incomplete (12.9%), summarized in Table 2. in risk estimates is more evident when an excellent response to therapy is achieved, as it decreases the risk of recurrence/ persistence to less than 3% regardless of initial risk estimates. BRAF V600E mutation status, clinical- This is especially evident in ATA low and intermediate risk pathological features and recurrence patients. ATA high-risk patients barely had excellent responses. Overall, when comparing staging systems, DRS showed more The association between BRAF V600E mutation and poor accuracy in predicting recurrent/persistent disease in the lowest clinical-pathological characteristics and outcome has been well stage (Table 3). While patients classified as AJCC stage I/II and studied (19, 20). Our multicenter study supports an association ATA low risk had persistent/recurrent disease in 19.2%/22% and of BRAF V600E with extrathyroidal extension (68.6% vs. 31.4%; 10.2% respectively, patients with excellent response had only 1.8%. p<0.001) and lymph node metastasis (64.8% vs. 35.2%; p=0.004), DRS also showed high accuracy in predicting mortality as patients but not with distant metastasis (42.9% vs. 57.1%; p=0.163). We in the structural incomplete category were the only ones who died also studied the association of BRAF V600E with recurrence in (17.7%). On the other hand, in our study, a high proportion of logistic regression analysis. Overall, 25.13% (92 of 366) of BRAF patients in the indeterminate response to therapy category had V600E positive patients and 17.55% (56 of 319) of BRAF V600E persistent/recurrent disease, 54%, and 52% had NED at the end of negative patients experienced recurrence with an unadjusted OR the follow up, which is more than expected. of 1.65 (95% CI, 1.13 to 2.42) (p 0.009), which remained Finally, to estimate how well a staging system predicts significant after adjustment for patient age and sex (OR, 1.73; recurrence we used the proportions of variance explained 95% CI, 1.19 to 2.56)(p 0.005) yet it did not remain significant (PVEs) in Cox regression models. We first calculated the after additional adjustment for tumor size, extrathyroidal adjusted hazard ratios (AHRs) (adjusted with age at diagnosis, invasion, lymph node metastasis, multifocality and RAI (OR, sex, tumor size, extrathyroidal invasion, lymph node metastasis) 1.57; 95% CI, 0.95-2.62) (p=0.078). for risk of recurrence for each system. Because the indeterminate and biochemical incomplete response categories have very similar outcomes in terms of NED and survival, we integrated them into one unique category creating the 3-tiered DRS system. The PVE Validation of dynamic risk stratification value of the AJCC/TNM staging, ATA risk classification, 4-tiered DRS, and 3-tiered DRS was 21.2% (95% CI:16.6-29.7), 25.2% (95% The ability of each of the prognostic system (AJCC, ATA risk CI: 19.9-32.5), 57.1% (95% CI: 53.3-62.7) and 59% (95% CI: 55.2- stratification and DRS) to predict final clinical status is presented in 65.2) respectively (Figure 1). Overall, our study supports the 4- Table 3. As expected, AJCC stage IV patients were more likely to die tiered DRS as a better system to predict the evolution of patients from thyroid cancer (13.8% vs. 0.5% of stage I). Considering than classical static systems as the ATA risk classification or the recurrence rate, in AJCC stages I and II, the proportion of AJCC stages fundamentally at lower stages. In our series, patients patients that have recurrent/persistent disease is relatively high in the indeterminate response to therapy have worse outcomes (19.2% and 22%, respectively). When we classified patients than expected in terms of persistent/recurrent disease and no according to the ATA recurrence risk classification, the evidence of disease at the end of follow up. By integrating Frontiers in Endocrinology 04 frontiersin.org Frontiers in Endocrinology Pérez-Fernández et al. TABLE 1 Characteristics of 685 PTC patients with known postoperative BRAF status. No. of Average Male Tumor size, Multifocality, Classic TNM Radioactive ATA risk Follow-up dura- BRAFV600, Recurrence, patients Age, y sex, n Median n(%) sub- stage I- iodine treat- system, tion, Median n(%) n(%) (%) (mm) [IQR] type, n II, n (%) ment (%) n(%) (months) [IQR] (%) Toledo 195 46 44 13 [8-25] 64 (32.8) 170 (87.2) 147 (75.4) 170 (87.2) Low 123 53 [27.5-81] 109 (55.9) 38 (19.5) (22.6) (63) Int. 60 (30.8) High 10 (5.1) Vall 126 51 27 13.5 [7-25] 60 (47.6) 92 (73) 91 (72.2) 101 (80.2) Low 76 36 [20-51] 61 (48.4) 24 (19) d’Hebron (21.4) (60.3) Int. 43 (34.1) High 6 (4.8) Basurto 99 51 20 13 [10-22] 51 (51.5) 62 (62.6) 68 (68.7) 93 (93.9) Low 64 27 [17.5-41.5] 46 (46.5) 13 (13.1) (20.2) (64.6) 05 Int. 35 (35.3) High 0 (0) Arnau 85 46 15 30 [16-40] 28 (32.9) 59 (69.4) 59 (69.4) 80 (94.1) Low 58 82 [50-128] 41 (48.2) 28 (32.9) (17.6) (68.2) Int. 19 (22.3) High 8 (9.4) Parc 66 44 12 22 [15-30] 33 (50) 41 (62.1) 43 (65.1) 56 (84.8) Low 41 78.5 [39.2-157] 33 (50) 26 (39.4) Tauli (18.2) (62.1) 10.3389/fendo.2022.1071775 Int. 22 (33.3) High 3 (4.5) frontiersin.org CUN* 63 48 15 13 [10-20] 24 (38) 23 (36.5) 31 (49.2) 49 (77.7) Low 32 29 [9-56] 45 (71.4) 10 (15.9) (23.8) (50.8) (Continued) Frontiers in Endocrinology Pérez-Fernández et al. TABLE 1 Continued No. of Average Male Tumor size, Multifocality, Classic TNM Radioactive ATA risk Follow-up dura- BRAFV600, Recurrence, patients Age, y sex, n Median n(%) sub- stage I- iodine treat- system, tion, Median n(%) n(%) (%) (mm) [IQR] type, n II, n (%) ment (%) n(%) (months) [IQR] (%) Int. 23 (36.5) High 1 (1.6) Doce de 51 52 12 16.5 [11.5-25] 25 (49) 25 (49) 34 (66.6) 37 (72.5) Low 21 33 [10-89] 31 (60.8) 9 (17.6) Octubre (23.5) (41.2) Int. 15 (29.4) High 6 (11.8) Total 685 48.28 145 15 [10-26] 285 (41.6) 472 (68.9) 473 (69) 586 (85.5) Low 415 45 [23-77] 366 (53.4) 148 (21.6) (21.1) (60.6) Int. 217 (31.7) 06 High 34 (4.9) *CUN, Clı́nica Universidad de Navarra. 10.3389/fendo.2022.1071775 frontiersin.org Pérez-Fernández et al. 10.3389/fendo.2022.1071775 TABLE 2 Response to therapy categories in 642 patients with collected data. Medical No. of Excellent, n Indeterminate, n Biochemical Incomplete, n Structural Incomplete, n Center patients (%) (%) (%) (%) Toledo 195 148 (75.9) 14 (7.2) 14 (7.2) 19 (9.7) Vall d’Hebron 126 101 (80.2) 2 (1.6) 7 (5.6) 16 (12.7) Basurto 97 81 (83.5) 10 (10.3) 2 (2.1) 4 (4.1) Arnau 76 52 (68.4) 8 (10.5) 2 (2.6) 14 (18.4) Parc Tauli 66 39 (59.1) 3 (4.5) 8 (12.1) 16 (24.2) CUN 54 32 (59.3) 11 (20.4) 3 (5.6) 8 (14.8) Doce de Octubre 28 13 (46.4) 7 (25) 2 (7.1) 6 (21.4) Total 642 466 (72.6) 55 (8.6) 38 (5.9) 83 (12.9) indeterminate and biochemical incomplete response into one statistically significant (p 0.017). Furthermore, within the non- unique category, the 3-tiered DRS system is equally effective in responders, we found that BRAF mutation was specifically predicting outcomes. associated with the biochemical incomplete response but not with the structural incomplete response. As shown in Table 6, 71.1% of patients with a biochemical incomplete response were BRAF Association of BRAF V600E with DRS positives while only 28.9% were negatives (HR 2.43; 95% CI, 1.21 categories to 5.23), being this difference statistically significant (p 0.016). To analyze the impact of BRAF V600E on DRS we first grouped the patients into those with excellent response to therapy at the end Refining risk prediction for recurrence of the follow-up and those who did not (non-responders). We using the BRAF mutation found that BRAF V600E patients had a higher probability of having a no excellent response to therapy (31.4%) than the BRAF WT Molecular status, mainly BRAF, can be used to refine risk (22.9%) (HR 1.53; 95% CI, 1.080 to 2.188), being this difference predictions in combination with ATA risk classification system TABLE 3 Final outcomes based on stratification by AJCC, ATA risk of recurrence at diagnosis and DRS. Persistence/recurrence (%) Death (%) AJCC (n= 594) AJCC (n= 609) I 74/385 (19.2) I 2/393 (0.5) II 13/59 (22) II 0/63 (0) III 16/87 (18.4) III 1/88 (1.1) IV 33/63 (52.4) IV 9/65 (13.8) ATA (n= 624) ATA (n= 639) Low 40/393 (10.2) Low 3/399 (0.8) Intermediate 76/200 (38) Intermediate 1/207 (0.5) High 30/31 (96.8) High 12/33 (36.4) DRS (n= 615) DRS (n= 624) Excellent response 8/451 (1.8) Excellent response 0/457 (0) Indeterminate response 27/50 (54) Indeterminate response 0/51 (0) Biochemical incomplete 33/36 (91.7) Biochemical incomplete 0/37(0) Structural incomplete 75/78 (96.2) Structural incomplete 14/79 (17.7) Frontiers in Endocrinology 07 frontiersin.org Pérez-Fernández et al. 10.3389/fendo.2022.1071775 TABLE 4 Outcomes concerning no evidence of disease (NED) in the different DRS categories. DRS categories Persistence/recurrence (%) NED (%) Excellent response 8/451 (1.8) 443/451 (98.2) Indeterminate response 27/50 (54) 26/50 (52) Biochemical incomplete 33/36 (91.7) 12/36 (33.3) Structural incomplete 75/78 (96.2) 20/78 (25.6) (1). Likewise, we wanted to explore whether BRAF mutation one unique category creating the 3-tiered DRS system achieving could refine risk estimates in DRS. We calculated the adjusted a PVE as good as the 4-tiered. BRAF status does not provide any hazard ratios (AHRs) (adjusted with age at diagnosis, sex, tumor additional benefit in refining risk estimates when combined size, extrathyroidal invasion, lymph node metastasis) for risk of with DRS. recurrence in each category of DRS with and without BRAF All the static staging systems published provide suboptimal mutation. When patients were stratified according to BRAF long-term predictions for recurrence in individual patients, as mutational status, the AHRs for recurrence increased with demonstrated by the proportion of variance explained (PVE), increasing DRS severity both in mutant and wild type BRAF ranging from 20% to 30% across the studies (a measure of how (Table 7). In the indeterminate category, patients with BRAF well a predictive model correlates with the outcome of interest) mutation show a slightly higher risk than those with BRAF wt (17). However, several uncentre studies have demonstrated that during the follow up. Similar trend is seen in the structural when these initial risk estimates are modified over time as a incomplete category. However, overall, the differences of the response to therapy, risk estimates can achieve a PVE as high as AHRs in each DRS category with and without BRAF mutation 70% to 80%. Our study reached a PVE around 60%, comparable (Figure 1) are too small for creating different risk grouping and, to previous results (3, 4, 21). Of note, to calculate the PVE we thus, an integrated prognostic system could not be developed. adjusted for age, tumor size, extrathyroidal invasion, lymph Thus, BRAF status does not provide any additional benefit in node metastasis, multifocality and iodine treatment, making combination with DRS to refine risk predictions. our findings more robust. However, our results also show that the indeterminate and the biochemical incomplete response to treatment categories have similar outcomes at the end of the Discussion follow up in terms of NED and survival compared with the structural incomplete, raising the question whether we could In this real-world study, we wanted to validate the clinical integrate them into one unique category. utility of DRS as compared with the more static AJCC/TNM The indeterminate response to treatment category, initially staging system and the ATA risk-stratification system. We found described as an acceptable response, was designed for patients that DRS is an accurate system to predict the evolution of with nonspecific findings (including mildly elevated serum Tg) patients, achieving higher PVEs compared with the two other that could not be confidently described as benign or malignant. systems, particularly in excellent responders. We also found the Over time, 15% to 20% of these patients will develop structural indeterminate and biochemical incomplete response have very disease that may require additional therapy. In the remainder, similar outcomes in terms of NED and survival at the end of the nonspecific changes resolve, and many of these patients can follow up (median 3.75 years). Thus, we integrated them into be reclassified as having an excellent response to treatment over TABLE 5 Impact of response to initial therapy assessment on initial estimates of risk. ATA initial risk of recurrence/persistence classification (n) Low, % Intermediate, % High, % Initial estimate risk of recurrence/persistence 10.2 (40/393) 38 (76/200) 96.8 (30/31) Modified estimate of risk of recurrence/persistence disease based on response to therapy Excellent response 1.5 (5/336) 2.7 (3/111) 0 (0/1) Indeterminate response 35 (7/20) 65.5 (19/29) 100 (1/1) Biochemical incomplete 91.7 (11/12) 90.5 (19/21) 100 (3/3) Structural incomplete 93.3 (14/15) 94.6 (35/37) 100 (25/25) Frontiers in Endocrinology 08 frontiersin.org Pérez-Fernández et al. 10.3389/fendo.2022.1071775 A B C D FIGURE 1 Recurrence Free Survival (RFS) and proportion of variance explained (PVE) to assess the performance of (A) seventh AJCC TNM stage, (B) ATA risk system, (C) 4-tiered dynamic risk stratification (DRS) and (D) 3-tiered DRS (integrating indeterminate and biochemical incomplete). Adjusted Hazard Ratios with 95% confidence intervals (95% CI) were calculated in logistic regression model. A two-tailed P value less than 0.05 was considered statistically significant. time (22). In our study, 54% of the patients in the indeterminate report which patients developed structural disease or needed response category developed persistent/recurrent disease and further treatment, it has been estimated that 15% to 20% of the 52% presented NED over time (median 3.75 y), being patients in both indeterminate and biochemical incomplete subsequently reclassified as excellent responders. Likewise, response will develop structural disease (23). Thus, because the 91.7% of the patients in the biochemical incomplete response outcome of patients in these two categories are similar, we to treatment category developed persistent/recurrent disease and suggest integrating indeterminate and biochemical incomplete 33% presented NED over time (median 3.75 y), being categories into one unique category to simplify response to subsequently reclassified as excellent responders. No deaths therapy. We show that the PVE in this 3-tiered DRS system were observed in the two categories. Although we did not remains high. The patients in this new category will need TABLE 6 Impact of BRAFV6ooE in DRS. Frequencies Odd ratios BRAF mutated BRAF wild type p-value OR 95% CI p-value DRS 0.040 Excellent response 234 (50.2%) 232 (49.8%) Indeterminate response 34 (61.8%) 21 (38.2%) 1.61 0.91 - 2.89 0.106 Biochemical incomplete 27 (71.1%) 11 (28.9%) 2.43 1.21 – 5.23 0.016 Structural incomplete 46 (55.4%) 37 (44.6%) 1.23 0.77 – 1.98 0.382 Frontiers in Endocrinology 09 frontiersin.org Pérez-Fernández et al. 10.3389/fendo.2022.1071775 TABLE 7 Hazard Ratios of categories of dynamic risk stratification for 5 years-recurrence by BRAF V600E mutational. BRAF wild type BRAF V600E Recurrence Unadjusted Adjusted Recurrence Unadjusted Adjusted Response Events/ 5y HR P HR P Events/ 5y HR P HR P to therapy Total (%) DFS (95% (95% Total (%) DFS (95% (95% (%) CI) CI) (%) CI) CI) Excellent 1/228 (0.4) 229 — – — – 4/221 (1.8) 221 4.15 0.203 3.2 0.299 (100) (98.2) (0.46- (0.36- 37.1) 28.7) Indeterminate 9/18 (50) 13 160 <0.001 103 <0.001 14/28 (50) 22 156 <0.001 146 <0.001 (68.4) (20.3- (12.9- (68.8) (20.5- (18.9- 1.263) 823) 1.189) 1.131) Biochemical 10/10 (100) 6 (60) 798 <0.001 647 (75- <0.001 21/25 (84) 12 466 <0.001 477 <0.001 incomplete (101- 5.589) (46.2) (62.4- (61.8- 6.306) 3.474) 3.676) Structural 33/33 (100) 18 771 <0.001 444 (57- <0.001 34/37 (91.9) 22 688 <0.001 652 <0.001 incomplete (52.9) (105- 3.453) (52.4) (93.6- (84.9- 5.674) 5.058) 5.013) monitoring and some degree of TSH suppression in the long (16). This was a retrospective single center study with a total of term to detect and prevent the appearance of structural disease, 723 patients and failed to find a statistical relationship between particularly those that do not achieve NED (between 48% to BRAFV600E and any category of the DRS system. In the 67%). We believe that the main factor to establish risk estimates mentioned study, the authors found no association between after the initial treatment in these patients would be the levels of BRAFV600E and cervical lymph nodes metastases, which were Tg overtime (i.e., Tg doubling time) rather than establishing cut- only present in the 13% of the patients, which is less than offs that are necessarily arbitrary. normally seen in the literature. In our study this percentage In a similar study by Jeon MI et al. (4), patients with ascends to 41%, more accordingly to the literature. We found indeterminate and biochemical response reached NED in 74% that patients with BRAFV600E have significantly less excellent and 27% respectively after a median of 8 years follow up (in our response to therapy and, particularly, are significantly more study, 52% and 33% respectively, 3.75 years follow up). In such likely to develop incomplete biochemical response to study, patients with an undetectable level of serum stimulated Tg treatment. Most of the patients in this category are likely to (<1 ng/ml), negative TgAb, and no suspicious structural disease have microscopic disease affecting cervical lymph nodes or at the end of the follow-up period were considered to have NED. remnants in the thyroid bed that cannot be detected by These criteria were very similar to ours. We also considered a imaging tests but still produce detectable levels of Tg. non-stimulated Tg less than 0.2 ng/mL as another criteria. Thus, Altogether, our results point to BRAFV600E as an oncogene stricter criteria to define evidence of disease and a shorter follow that renders tumors prone to disseminate predominantly in a up may explain the differences between our study and theirs. locoregional manner via lymphangiogenesis (24). Nevertheless, we consider that these criteria adjust better to real In addition, in the indeterminate response to treatment clinical practice because detectable levels of thyroglobulin, even category, patients with BRAF mutation show a slightly higher if low, normally lead to more frequent blood tests and/or risk (AHR) than those with BRAF wt during the follow-up and a radiological tests, particularly in patients that have undergone similar trend is seen in the structural incomplete category. a total thyroidectomy and 131-I ablation, in which Tg is most Although it could be of some value whether patients with reliable as a marker. In all, considering both studies, the BRAF mutation in these two categories have more risk in the likelihood of achieving NED overtime in the indeterminate long term than those without BRAF mutation, the differences are category is between 52-74%, which means that some patients too small to be clinically relevant. Most importantly, the will need monitoring for a relatively long period of time and differences of the AHRs in each group of the four-tiered DRS some of them will develop structural disease. From a practical system with and without BRAF mutation (Figure 1) are not big point of view, the clinical management of patients in the enough to create an integrative prognostic system. Thus, it seems indeterminate category would not be very different from that BRAF status does not provide any additional benefit in the biochemical. terms of risk grouping in combination with DRS. By contrast, in To our knowledge, there is only one previous publication the study by Kim et al. (21), the authors did demonstrate that that analyses the relationship between BRAF V600E and DRS knowing TERT status may help in the reshaping of the risk Frontiers in Endocrinology 10 frontiersin.org Pérez-Fernández et al. 10.3389/fendo.2022.1071775 grouping when combined with DRS. In this regard, TERT is Ethics statement clearly a more robust prognostic marker compared to BRAF. The multicenter and nation-wide nature of this study are the The studies involving human participants were reviewed and major strengths as they show the utility of response to therapy in approved by CEIm Hospital Universitario 12 de Octubre, CEIm real clinical practice. The participating centers have thyroid Gerencia de Atenció n Especializada de Toledo, CEIm del cancer units that actively follow contemporary standard Hospital Universitari Vall d’Hebron, CEI del Hospital de practice guidelines in the management of thyroid cancer, Basurto, CEIm Hospital Universitari Arnau de Vilanova de minimizing the heterogeneity in the management of thyroid Lleida, CEIm Parc Taulı́ and CEIm Clı́nica Universitaria de cancer. The fact that the overall analysis reproduced the findings Navarra. The patients/participants provided their written seen in the literature regarding the association of BRAF status informed consent to participate in this study. with aggressive clinico-pathological features is consistent with the good generalizability of the current study (20). On the other hand, this study has some limitations. First, the multicenter and retrospective nature is, nevertheless, inherently associated with Author contributions the potential limitation of data heterogeneity. The effects of variable confounding factors such as surgical skills, Conception and design: G.R-E Financial support: GR-E and completeness of surgery, strategies of RAI ablation and level of AV. Administrative support: GR-E. Provision of study materials care should be taken into consideration. However, despite this or patients: JS, CZ, AO, EC, JG, IC, SG-I. Collection and heterogeneity, our study reflects the good performance of DRS assembly of data: All authors. Data analysis and interpretation: across the country, making it more reliable. Similarly, in a recent GR-E, AV and LP-F. Manuscript writing: GR-E and LP-F. All large multicenter study, the ATA risk stratification system has authors contributed to the article and approved the been shown to be a reliable predictor of short-term outcomes in submitted version. patients with differentiated thyroid cancer in real-world clinical settings characterized by center heterogeneity (25). Second, the relatively short follow-up period (average 5 years) is also a limitation as we cannot rule out that our results may change Funding in a longer period. To conclude, in this nation-wide multicenter study we The authors want to thank the support of the following provide evidence to support the 4-tiered DRS as a better Grants: PID2019-105303RB-I00/AEI/10.13039/501100011033 system to predict the evolution of patients compared to AJCC/ from Ministerio de Ciencia e Innovacion (MICIN) to GR-E; TNM staging system and the ATA risk-stratification system. PI14/01980 from Instituto de Salud Carlos III (FIS-ISCIII) to However, our results also show that the indeterminate and GR-E; The Asociacioı́n Espanãola Contra el Cancer (AECC) biochemical incomplete response categories have similar (GCB141423113) to GR-E. GR-E and ADV, belong to outcomes in terms of NED and survival. Integrating them into CIBERONC ISCIII. RTI2018-099343-B-100/FEDER and one unique category, the subsequent 3-tiered DRS system PID2021-125948OB-I00 from MICIN to ADV. appears to be equally accurate compared to the 4-tiered DRS, which we believe simplifies the system and makes it more helpful for clinicians. We believe that rather than relying on Tg cutoffs Acknowledgments that are somehow arbitrary, watching Tg overtime (i.e., Tg doubling time) would be more helpful in this new category. We are grateful to Fernando Neria, from the Faculty of Finally, in contrast to TERT, BRAF does not provide any Medicine of Universidad Francisco de Vitoria for his critical additional benefit in terms of risk grouping when combined revision of the statistical analysis. with DRS. Conflict of interest Data availability statement The authors declare that the research was conducted in the The raw data supporting the conclusions of this article will absence of any commercial or financial relationships that could be made available by the authors, without undue reservation. be construed as a potential conflict of interest. Frontiers in Endocrinology 11 frontiersin.org Pérez-Fernández et al. 10.3389/fendo.2022.1071775 Publisher’s note organizations, or those of the publisher, the editors and the reviewers. 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