Treatment of RET-Positive Advanced Medullary Thyroid Cancer with Multi-Tyrosine Kinase Inhibitors—A Retrospective Multi-Center Registry Analysis

Simple Summary Lately, a more personalized approach in the management of advanced thyroid cancer patients has improved the outcomes, and several novel molecularly guided therapies, including selective RET inhibitors (sRETis), have demonstrated promising efficacy in clinical trials. RET (rearranged during transfection) variants are the most prevalent oncogenic event in medullary thyroid cancer (MTC). We here found RET oncogene variants in 44/48 prospectively collected MTC tumor samples from patients treated with more unselective kinase inhibitors vandetanib and/or cabozantinib. Our study shows that RET variants were highly prevalent in patients with advanced MTC, and the treatment results in RET-positive cases were similar to those reported in unselected cohorts. Abstract Background: RET (rearranged during transfection) variants are the most prevalent oncogenic events in medullary thyroid cancer (MTC). In advanced disease, multi-tyrosine kinase inhibitors (MKIs) cabozantinib and vandetanib are the approved standard treatment irrespective of RET status. The actual outcome of patients with RET-positive MTC treated with MKIs is ill described. Methods: We here retrospectively determined the RET oncogene variant status with a targeted DNA Custom Panel in a prospectively collected cohort of 48 patients with advanced MTC treated with vandetanib and/or cabozantinib at four German referral centers. Progression-free survival (PFS) and overall survival (OS) probabilities were estimated using the Kaplan-Meier method. Results: In total, 44/48 (92%) patients had germline or somatic RET variants. The M918T variant was found in 29/44 (66%) cases. In total, 2/32 (6%) patients with a somatic RET variant had further somatic variants, while in 1/32 (3%) patient with a germline RET variant, additional variants were found. Only 1/48 (2%) patient had a pathogenic HRAS variant, and no variants were found in 3 cases. In first-line treatment, the median OS was 53 (95% CI (95% confidence interval), 32–NR (not reached); n = 36), and the median PFS was 21 months (12–39; n = 33) in RET-positive MTC patients. In second-line treatment, the median OS was 18 (13–79; n = 22), and the median PFS was 3.5 months (2–14; n = 22) in RET-positive cases. Conclusions: RET variants were highly prevalent in patients with advanced MTC. The treatment results in RET-positive cases were similar to those reported in unselected cohorts.


Introduction
Medullary thyroid cancer (MTC) accounts for 2-5% of all thyroid malignancies and is a tumor arising from the calcitonin-producing parafollicular C cells of the thyroid gland [1].
Hereditary MTC occurs in about 25% of cases as a part of multiple endocrine neoplasia type 2 (MEN2). It is caused by oncogenic germline RET (rearranged during transfection) variants. In sporadic MTC, somatic RET variants are found in~65% of cases, among which RETM918T is the most frequent somatic variant and is associated with adverse outcomes [2][3][4][5]. Activating variants in the Hand KRAS genes were found in~24% of cases using next-generation panel sequencing, while in~18% of the 181 patients under study, no pathogenic variants were identified [6]. For patients with significant tumor burden, symptoms and/or progressive disease, the multi-tyrosine kinase inhibitors (MKIs) vandetanib and cabozantinib have been approved in the United States and Europe by the United States Food and Drug Administration (FDA) and the European Medicines Agency (EMA), respectively [7]. RET is one among the several targeted kinases of these compounds, and approval is irrespective of the identification of a molecular driver event. While vandetanib inhibits RET tyrosine kinase activity (50% inhibitory concentration (IC 50 ) of 130 nM), vascular endothelial growth factor receptor (VEGFR) 2 (IC 50 of 40 nM) and 3 (IC 50 of 110 nM) and epidermal growth factor receptor (EGFR) (IC 50 of 500 nM), cabozantinib targets RET (IC 50 of 4 nM), VEGFR2 (IC 50 of 0.035 nM) and hepatocyte growth factor receptor c-Met (IC 50 of 1.8 nM) [8][9][10][11].
In contrast, highly selective, small-molecule RET kinase inhibitors (sRETis) selpercatinib and pralsetinib have been approved by the FDA for patients with advanced or metastatic MTC in which activating RET variants are identified and who require systemic treatment. There is limited evidence of the impacts germline and somatic RET oncogene variants may have on the clinical outcome of patients with advanced MTC treated with MKIs such as vandetanib and cabozantinib. For cabozantinib, a post hoc analysis suggested that overall survival (OS) may be significantly longer in patients with the RETM918T variant than in patients without this variant or in whom the RET status is unknown [12].
To assess the actual clinical course with MKIs specifically in RET-positive MTC, we here retrospectively assessed the RET variant status in tissue samples from a prospective multi-center registry study at four German tertiary care centers and studied the outcomes specifically after treatment with MKIs in RET-positive cases.

Setting
The setting of this registry study was as previously described for an unselected cohort [13]. The study was conducted as part of the German Study Group for Rare Malignant Tumors of the Thyroid and Parathyroid Glands. Prospectively and retrospectively collected data were obtained from records of patients diagnosed with MTC between 1990 and 2019 in four German tertiary care centers. All patients provided written informed consent, and the study was approved by the ethics committee of University of Würzburg (96/13) and subsequently by the ethics committees of all participating centers.

Data Acquisition
Eligible patients were adults (age ≥ 18 years) with histopathological evidence of MTC with locally advanced disease and/or evidence of distant metastases who underwent MKI treatment with vandetanib and/or cabozantinib [13] with information or tissue available for testing of somatic or germline RET oncogene variants. The selection of patients is shown in Figure 1. The primary endpoint of this study was the OS of MTC patients with somatic or germline RET oncogene variants during MKI treatment. The secondary endpoints were the assessments of the best objective response rate (based on clinical routine imaging in analogy to RECIST 1.0 and 1.1) and progression-free survival (PFS). Bone metastases were not considered as target lesions, except for the new occurrence of bone metastases upon treatment. As exploratory analyses, we also assessed these end points in patients without RET variants.  Time between the first diagnosis and MKI start, Union for International Cancer Control (UICC) stage at the first diagnosis, type of first-line therapy and number of metastases at diagnosis were assessed as potential risk factors affecting the outcome parameters. For OS, patients alive were censored at the cutoff date of 30 October 2020 or at the start of sRETi treatment. Treatment and follow-up of patients were performed according to the local practices of the participating centers. Treatment outcome was assessed locally by imaging (positron emission tomography/computed tomography (PET/CT), CT, magnetic resonance imaging (MRI) of the liver and bone scintigraphy) and measurement of serum calcitonin and carcinoembryonic antigen (CEA) levels every 3-6 months. Clinical data were recorded by trained personnel at all sites.

Sample Selection, DNA/RNA Extraction and Mutation Analysis
Tissue blocks were collected from 43 (90%) patients in Germany with advanced MTC (primary tumor tissue, n = 17; tissue of distant metastases, n = 11; tissue of lymph node metastases, n = 9; unknown, n = 6). Five (10%) patients with MEN2 only underwent the analysis of peripheral blood.
Prior to sequencing, specimens were reviewed by an independent pathologist for consistency with the previously established diagnosis. DNA extraction from formalinfixed, paraffin-embedded (FFPE) tumor tissue, next-generation sequencing (NGS) and bioinformatics were carried out as previously described [14,15].

Statistical Analysis
PFS and OS probabilities were estimated using the Kaplan-Meier method. For exploratory analyses, Cox proportional hazards regression models were used to identify predictive factors affecting the OS of patients with germline/somatic RET oncogene variants. Potential predictive factors were selected using backward/stepwise selection based on the p-value (selection threshold of 0.10) or Akaike information criteria (AIC) (−2 × log-likelihood + 2 × degrees of freedom for predictors) and penalized (Lasso) regression methods (10-fold cross-validation). Different selection methods, including backward elimination, stepwise selection and penalized regression, were used to select a consistently robust and reliable set of predictive factors.
The above methods were applied to 4 pooled samples-in patients with first-line MKI treatment, patients with second-line MKI treatment, patients treated with vandetanib and patients treated with cabozantinib. Due to the limited sample size, no statistical tests comparing the characteristics or outcomes of patients with and without germline/somatic RET variants were performed. Statistical analyses were performed with R Statistical Software Version 4.0.4 (R Foundation for Statistical Computing, Vienna, Austria). Microsoft Office Excel Version 16.55 was used for additional analyses.

Clinical Characteristics
At four German tertiary care centers, 48 patients (36 males and 12 females) with locally advanced MTC and/or evidence of distant metastases undergoing MKI treatment were included. Table 1 shows the baseline clinical characteristics of the study population. The median follow-up from the first MTC diagnosis was 6 years (range of 0-30). Most patients presented with a germline or somatic RET variant (44; 92%). The median age at the initial diagnosis of sporadic MTC was 47 years (range of 23-78), and it was 42 years (range of 17-61) for patients with hereditary MTC. At the diagnosis of metastatic disease, the median age was 50 years, and tumors were already metastatic at diagnosis in 30 (63%) patients. Before MKI initiation, the median calcitonin doubling time (CDT) was 8 months (range of 4-31). The median age at MKI initiation was 55 years (range of 22-79), and the median time between initial diagnosis and MKI start was 37 months (range of 0-242). In patients with distant metastases at initial diagnosis, the median time between initial diagnosis and MKI start was 18 months (range of 0-199). The median starting doses of vandetanib and cabozantinib were 300 mg/day (range of 100-300) and 80 mg/day (range of 60-140), respectively. A total of 27 (63%) patients started vandetanib treatment at the approved dosage of 300 mg/day, and 4 (27%) patients started cabozantinib at the approved dosage of 140 mg/day.

Multi-Tyrosine Kinase Inhibitor Therapy
The treatment and patient characteristics are shown in Table 1. The median follow-up from the start of MKI treatment was 34 months (range of 3-164). Vandetanib and/or cabozantinib were administered in all 48 patients. Eight (17%) patients received a different MKI as first-line treatment (sorafenib or imatinib prior to approval of vandetanib). A total of 20 patients (42%) received two MKIs, while 6 (13%) patients received three MKIs, and 2 (4%) patients received five MKIs.

Outcome after First-and Second-Line Treatments
The characteristics of first-line treatment with cabozantinib/vandetanib are summarized in Table A2. The outcome data are summarized in Table A3.
A total of 35 (73%) patients received vandetanib as first-line treatment, 33 patients with a RET variant and 2 patients without a RET variant. Five patients (10%) received cabozantinib as first-line treatment, three patients with a RET variant and two patients without a RET variant.
In the first-line treatment of patients with a RET variant, the median OS was 53 months (95% CI, 32-NR), and the median PFS was 21 months (95% CI, 12-39).
A total of 12 (48%) patients received vandetanib as second-line treatment, 10 patients with a RET variant and 2 patients without a RET variant. A total of 13 patients (52%) received cabozantinib as second-line treatment, 12 patients with a RET variant and 1 patient without a RET variant.

Biochemical Response of First-Line Treatment
Calcitonin as well as CEA (calcitonin n = 24; CEA n = 22) showed a significant reduction in patients with a RET variant (77% reduction, p < 0.001 ***; 74% reduction, p = 0.007 **); patients without a RET variant (calcitonin n = 3, CEA n = 1) showed no significant reductions in calcitonin (p = 0.5). The data on CEA were not sufficient for analysis.

Predictive Factors Affecting OS in First-and Second-Line Treatments
The associations of predictive factors with OS are summarized in Table 2. Patients with a longer time interval between the first diagnosis and MKI initiation and patients with a higher number of metastases at the first diagnosis experienced a longer OS. Patients with RET variants receiving second-line treatment had a longer OS when the time interval between the first diagnosis and MKI initiation was longer and a lower number of metastases was present at the first diagnosis, suggesting less aggressive disease course, and when treated in second line with vandetanib.

Safety and Tolerability
In 9 (21%) patients taking vandetanib and 9 (60%) patients taking cabozantinib, treatment-emergent adverse events (TEAEs) were the reason for treatment discontinuation. The TEAEs of vandetanib and cabozantinib in patients with a RET variant are summarized in Table 3. In vandetanib-treated patients, the most frequently reported TEAEs were diarrhea (53%), skin rash (44%) and fatigue (28%); in cabozantinib-treated patients, they were loss of appetite/loss of weight (53%), diarrhea (40%) and fatigue (40%). Patients taking cabozantinib showed a higher incidence of laboratory abnormalities, including blood count changes and thyroid-stimulating hormone (TSH) elevation, than vandetanib-treated patients. In vandetanib-treated patients, 10 (23%) patients showed a prolongation of the QT interval, while no patients taking cabozantinib had a documented prolongation of the QT interval. Furthermore, hand-foot syndrome was more often noted in patients taking cabozantinib than in those taking vandetanib (5 (33%) vs. 1 (2%)).

Discussion
To our knowledge, this is the first analysis describing the response to approved MKIs vandetanib and cabozantinib specifically in patients with a somatic or germline RET variant outside of a clinical trial.
In our series, 92% of cases harbored RET variants, confirming that RET, particularly the M918T variant, is the main driver oncogene in advanced MTC. The prevalence of RET-negative cases was 8%, and the proportion of cases in which no specific driver events were found was only 6%. Compared with previous data [4,6], as well as data from the phase 3 EXAM trial [12], RET-positive cases were more frequent in our series. At the same time, there was a remarkably small proportion of cases with no driver events. These differences are most likely due to the selection of patients, as we only included patients with advanced disease undergoing treatment with vandetanib and/or cabozantinib, as well as differences in sequencing technologies. The high prevalence of RET variants in our cohort provides a rationale for treatment with sRETis in the majority of patients.
Limitations of our study include the potential selection bias towards RET-positive MTC due to the participation of some centers in clinical trials of sRETis. Furthermore, the small group size of RET-negative patients precluded comparisons between RET-altered and RET-negative cases and the analysis of individual MKIs according to the treatment line. Missing data and differences in response assessment and follow-up are due to the-in part-retrospective nature of a registry study.
The outcome after vandetanib treatment in RET-positive patients, regardless of the treatment line, was consistent with previously reported results from our unselected cohort of 48 patients with advanced MTC undergoing MKI treatment with vandetanib and/or cabozantinib, which showed a median OS of 53 months and a median PFS of 17 months [13]. Thirty-four RET-positive patients were also part of that unselected series.
The median OS of 14 months and PFS of 2 months in cabozantinib-treated RET-positive patients, regardless of the treatment line, were lower than it was in our previous study [13]. We postulate that the lower response rates of cabozantinib-treated patients were most likely due to the fact that the results included a higher rate of patients receiving cabozantinib as second-line treatment after PD in first-line treatment than previously reported results (1:4 vs. 1:2 in our prior study). Only 27% of patients received the full dosage of 140 mg/day at treatment initiation (30% in our unselected series), and 67% needed a dose reduction, compared to 61% in our prior study [13].
According to the multiple Cox regression analysis, there were no signals of superiority for a particular MKI regimen in the first-line setting with respect to OS. Patients undergoing second-line vandetanib treatment showed a longer OS than patients receiving cabozantinib. Nevertheless, these data need to be interpreted with caution due to the limited number of patients receiving first-line treatment with cabozantinib and the limited number of patients receiving second-line treatment with vandetanib after first-line treatment with cabozantinib (8/10 patients received vandetanib after first-line treatment with sorafenib or imatinib).
The safety and tolerability, including the rate of treatment discontinuation due to TEAEs of vandetanib and cabozantinib, were comparable with the results of our prior study [13].
In an indirect comparison with the phase 1/2 trial of selpercatinib in patients with RET-positive MTC showing an ORR (objective response rate) of 69% and 72% in previously treated and not-treated patients by independent review, respectively, the ORR in our cohort ranged between 27% and 32% depending on the treatment line and compound [16]. The phase 1/2 open-label study of pralsetinib including patients with RET-positive MTC also showed a higher ORR of 60% in pre-treated patients and 66% in patients who did not receive prior vandetanib or cabozantinib [17]. The small proportion of RET-negative cases in our series is similar to that in the phase 3 ZETA trial, where only 2/231 (1%) of patients in the vandetanib group and 6/100 (6%) of patients in the placebo group with MTC were RET negative [2]. Furthermore, a substantial percentage of patients were RET unknown; therefore, the subgroup analyses of PFS and ORR by RET variant status were inconclusive [2]. Taking data from the amplification-refractory mutation system (ARMS) assay into account, which specifically detects the most common RET variant, M918T, patients with sporadic MTC showed benefit from vandetanib treatment whether their tumors were RETM918T positive or negative; however, the response rate was greater in those who had the RETM918T variant [2].
In an exploratory assessment of OS, PFS and ORR in the EXAM trial, cabozantinib appeared to be more active in patients who were RETM918T positive than in those who were RETM918T negative [12]. The median OS was 44 months for patients receiving cabozantinib versus 19 months for placebo, with corresponding values of 20 versus 22 months in the RETM918T-negative subgroup [12]. Interestingly, the difference in the OS of RETM918T-negative patients receiving cabozantinib versus placebo was only <3 months.
Even if the group of RET-negative cases was small, the particularly short PFS and OS appear to contradict the notion that RETM918T is associated with aggressive disease and poor prognosis [4,18]. In the series by Elisei et al., patients with an RET variant showed a lower survival rate in a long-term follow-up and the highest probability to have persistence of the disease [4]. Furthermore, Schilling et al. showed a more aggressive development of distant metastases during follow-up with decreased metastasis-free survival and a significantly lower survival rate in patients with the RETM918T variant than that in patients with a wild-type sequence in that codon [18]. This may reflect the differences between patient groups at different stages of the disease course and the impact of RET-directed treatment. This is in line with data showing RET variants to be significantly less frequent in small-sized tumors and much more frequent in advanced metastatic cases [19,20].

Conclusions
In conclusion, our study shows that patients with advanced MTC receiving MKI treatment had a high prevalence of RET variants. The treatment results in RET-positive cases were similar to those reported in unselected cohorts, providing a rationale for treatment with sRETis in the majority of patients.