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

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.

FGF-Receptors and PD-L1 in Anaplastic and Poorly Differentiated Thyroid Cancer: Evaluation of the Preclinical Rationale.

Background: Treatment options for poorly differentiated (PDTC) and anaplastic (ATC) thyroid carcinoma are unsatisfactory and prognosis is generally poor. Lenvatinib (LEN), a multi-tyrosine kinase inhibitor targeting fibroblast growth factor receptors (FGFR) 1-4 is approved for advanced radioiodine refractory thyroid carcinoma, but response to single agent is poor in ATC. Recent reports of combining LEN with PD-1 inhibitor pembrolizumab (PEM) are promising. Materials and Methods: Primary ATC (n=93) and PDTC (n=47) tissue samples diagnosed 1997-2019 at five German tertiary care centers were assessed for PD-L1 expression by immunohistochemistry using Tumor Proportion Score (TPS). FGFR 1-4 mRNA was quantified in 31 ATC and 14 PDTC with RNAscope in-situ hybridization. Normal thyroid tissue (NT) and papillary thyroid carcinoma (PTC) served as controls. Disease specific survival (DSS) was the primary outcome variable. Results: PD-L1 TPS≥50% was observed in 42% of ATC and 26% of PDTC specimens. Mean PD-L1 expression was significantly higher in ATC (TPS 30%) than in PDTC (5%; p<0.01) and NT (0%, p<0.001). 53% of PDTC samples had PD-L1 expression ≤5%. FGFR mRNA expression was generally low in all samples but combined FGFR1-4 expression was significantly higher in PDTC and ATC compared to NT (each p<0.001). No impact of PD-L1 and FGFR 1-4 expression was observed on DSS. Conclusion: High tumoral expression of PD-L1 in a large proportion of ATCs and a subgroup of PDTCs provides a rationale for immune checkpoint inhibition. FGFR expression is low thyroid tumor cells. The clinically observed synergism of PEM with LEN may be caused by immune modulation.

Real-World Efficacy and Safety of Multi-Tyrosine Kinase Inhibitors in Radioiodine Refractory Thyroid Cancer.

Background: The management of patients with locally advanced or metastatic differentiated thyroid cancer (DTC) that is refractory to radioiodine (RAI) remains a therapeutic challenge. The multi-tyrosine kinase inhibitors (TKIs) sorafenib and lenvatinib have been approved based on phase 3 clinical trials. Patients and Methods: We aimed at describing the efficacy and safety of TKI treatment of RAI-refractory DTC in a real-world setting at six German referral centers. One hundred and one patients with locally advanced or metastatic RAI-refractory DTC treated with sorafenib, lenvatinib, and/or pazopanib were included. Progression-free survival (PFS) and overall survival (OS) probabilities were estimated by using the Kaplan-Meier method. Results: Ninety-seven of 101 patients had progressive disease before TKI initiation. The median PFS for first-line treatment with sorafenib (n = 33), lenvatinib (n = 53), and pazopanib (n = 15) was 9 (95% confidence interval 5.2-12.8), 12 (4.4-19.6), and 12 months (4.4-19.6), respectively. The median OS for first-line treatment was 37 (10-64) for sorafenib, 47 (15.5-78.5) for lenvatinib, and 34 months (20.2-47.8) for pazopanib. Serious complications (e.g., hemorrhage, acute coronary syndrome, and thrombosis/venous thromboembolism) occurred in 16 out of 75 (21%) patients taking lenvatinib, in 3 out of 42 (7%) patients taking sorafenib, and in 3 out of 24 (13%) patients taking pazopanib. Conclusions: Sorafenib, lenvatinib, and pazopanib are effective treatment options in the majority of patients with RAI-refractory DTC. The PFS and six-month survival rate in patients treated with lenvatinib und pazopanib appear to compare favorably with sorafenib in the first-line treatment setting. However, a more advanced disease stage at treatment initiation in sorafenib- and pazopanib-treated patients in the era before TKI-approval and the retrospective nature of this study precludes a direct comparison of TKIs.

Real-World Efficacy and Safety of Cabozantinib and Vandetanib in Advanced Medullary Thyroid Cancer.

Background: Management of patients with advanced medullary thyroid cancer (MTC) remains a therapeutic challenge. The multi-tyrosine kinase inhibitors (TKIs) vandetanib and cabozantinib have been approved for the treatment of progressive MTC based on prolonged progression-free survival (PFS) in phase 3 clinical trials. Patients and Methods: To evaluate clinical characteristics, treatment regimens, efficacy, and treatment emergent adverse events (TEAEs) of vandetanib and cabozantinib in MTC patients outside clinical trials at four German tertiary care centers. Forty-eight patients diagnosed between 1990 and 2018 were included. PFS and overall survival (OS) probabilities were estimated using the Kaplan-Meier method and compared by log-rank test. Results: The median age at diagnosis was 46 years (15-80 years); a germ line RET (rearranged during transfection) mutation was known in 6 (13%) patients. Thirty-two (67%) patients showed progressive disease before TKI initiation. Forty-seven (98%) patients were treated with vandetanib and 23 (48%) patients with cabozantinib. Vandetanib was first-line treatment in 41 (85%) patients and cabozantinib in 7 (15%) patients. Partial response was the best response in 12 (26%) patients treated with vandetanib and in 5 (22%) patients treated with cabozantinib. Sixteen (34%) patients treated with vandetanib and 3 (13%) patients treated with cabozantinib had stable disease ≥24 weeks. The median PFS for vandetanib and cabozantinib was 17 months [95% confidence interval, CI, 9.3-24.6 months] and 4 months [CI 3.1-4.9 months], respectively. The 6- and 12-month survival rates were 98% and 86% for vandetanib and 78% and 70% for cabozantinib, respectively. The median OS for vandetanib and cabozantinib was 53 months [CI 43.7-62.3 months] and 24 months [CI 5.9-42.1 months], respectively. In vandetanib-treated patients, the PFS and OS were significantly longer in patients aged ≤60 years at TKI initiation and in patients with ≥5 TEAEs. Additionally, the PFS was longer in the absence of bone metastases. In cabozantinib-treated patients, the PFS was significantly longer in patients experiencing TEAEs and in patients aged ≤60 years, and the OS was significantly longer in patients who had TEAEs and in patients with ≥5 TEAEs. Conclusions: Vandetanib and cabozantinib are effective treatment options in the majority of MTC patients. We hypothesize that the poorer prognosis of cabozantinib-treated patients in our retrospective analysis is most likely due to its use as second-line treatment after treatment failure on vandetanib. However, different degrees of efficacy of the two drugs are possible.

Spatio-temporal regulation of the human licensing factor Cdc6 in replication and mitosis.

To maintain genome stability, the thousands of replication origins of mammalian genomes must only initiate replication once per cell cycle. This is achieved by a strict temporal separation of ongoing replication in S phase, and the formation of pre-replicative complexes in the preceding G1 phase, which "licenses" each origin competent for replication. The contribution of the loading factor Cdc6 to the timing of the licensing process remained however elusive due to seemingly contradictory findings concerning stabilization, degradation and nuclear export of Cdc6. Using fluorescently tagged Cdc6 (Cdc6-YFP) expressed in living cycling cells, we demonstrate here that Cdc6-YFP is stable and chromatin-associated during mitosis and G1 phase. It undergoes rapid proteasomal degradation during S phase initiation followed by active export to the cytosol during S and G2 phases. Biochemical fractionation abolishes this nuclear exclusion, causing aberrant chromatin association of Cdc6-YFP and, likely, endogenous Cdc6, too. In addition, we demonstrate association of Cdc6 with centrosomes in late G2 and during mitosis. These results show that multiple Cdc6-regulatory mechanisms coexist but are tightly controlled in a cell cycle-specific manner.

XRCC4 controls nuclear import and distribution of Ligase IV and exchanges faster at damaged DNA in complex with Ligase IV.

Non-homologous end-joining (NHEJ) is one major pathway for the repair of double-stranded DNA breaks in mammals. Following break recognition, alignment and processing, broken DNA ends are finally rejoined by the essential DNA Ligase IV. In the cell, Ligase IV is unable to function without its constitutive interaction partner XRCC4 and becomes unstable when it is missing, and it has been assumed that XRCC4 may also be required for recruitment of Ligase IV to repair sites. To investigate the function of complex formation between both proteins directly in the living cell, we stably expressed them as bio-fluorescent fusion proteins in human HT-1080 cell clones. Ligase IV or XRCC4 were expressed either alone or both were co-expressed at a roughly equimolar ratio. Labelled proteins were overexpressed manifold in comparison to endogenously expressed proteins. We show that over-expressed Ligase IV was only partially imported into the nucleus and showed a diffuse distribution there, whereas XRCC4 expressed alone was entirely nuclear with a distinct exclusion from nucleoli. When Ligase IV was co-expressed with XRCC4, both proteins formed the natural complex, and Ligase IV was not only efficiently imported but also resembled the sub-nuclear distribution of XRCC4. In addition, Ligase IV, when in complex with XRCC4, acquired a delayed nuclear reimport after mitotic cell division of XRCC4. We further determined by photobleaching the kinetics with which the proteins exchange at UVA laser-irradiated nuclear sites between damage-bound and diffusing states. We found that the dynamic exchange rate of the Ligase IV/XRCC4 complex at micro-irradiated sites was faster than that of XRCC4 expressed alone. In summary, our findings demonstrate a novel function of XRCC4 in controlling nuclear import and sub-nuclear distribution of Ligase IV, and they suggest that XRCC4 modulates the dynamic interaction of the Ligase IV/XRCC4 complex with the NHEJ machinery at double-stranded DNA breaks.