Synergic Induction of Autophagic Cell Death in Anaplastic Thyroid Carcinoma.

Anaplastic thyroid carcinoma (ATC) has poor prognosis, high mortality rate and lack of effective therapy. A synergic combination of PD-L1 antibody together with cell death promoting substances like deacetylase inhibitors (DACi) and multi-kinase inhibitors (MKI) could sensitize ATC cells and promote decay by autophagic cell death. The PD-L1-inhibitor atezolizumab synergized with panobinostat (DACi) and sorafenib (MKI) leading to significant reduction of the viability, measured by real time luminescence, of three different patient-derived primary ATC cells, of C643 cells and follicular epithelial thyroid cells too. Solo administration of these compounds caused a significant over-expression of autophagy transcripts; meanwhile autophagy proteins were almost not detectable after the single administration of panobinostat, thus supporting a massive autophagy degradation process. Instead, the administration of atezolizumab caused an accumulation of autophagy proteins and the cleavage of the active caspases 8 and 3. Interestingly, only panobinostat and atezolizumab were able to exacerbate the autophagy process by increasing the synthesis, the maturation and final fusion with the lysosomes of the autophagosome vesicles. Despite ATC cells could be sensitized by atezolizumab via the cleavage of the caspases, no reduction of cell proliferation or promotion of cell death was observed. The apoptosis assay evidenced the ability of panobinostat alone and in combination with atezolizumab to induce the phosphatidil serine exposure (early apoptosis) and further the secondary necrosis. Instead, sorafenib was only able to cause necrosis. The increase of caspases activity induced by atezolizumab, the apoptosis and autophagy processes promoted by panobinostat synergize thus promoting cell death in well-established and primary anaplastic thyroid cancer cells. The combined therapy could represent a future clinical application for the treatment of such lethal and untreatable solid cancer.

Vessel-Sparing Lymphadenectomy Should Be Performed in Small Intestine Neuroendocrine Neoplasms.

INTRODUCTION: The goal of primary tumor resection with lymphadenectomy (PTR) in small intestine neuroendocrine neoplasms (SI-NENs) is to avoid local recurrence while sparing as much of the small bowel as possible, even in the case of extensive mesenteric fibrosis. The results of PTR with retrograde vessel-sparing lymphadenectomy (VS-LA) were compared to those of conventional lymphadenectomy (Con-LA). METHODS: Prospectively collected clinical, surgical and pathological data of consecutive patients with SI-NENs who underwent small bowel resections were retrospectively analyzed regarding the resection technique performed. RESULTS: In a 7-year period, 50 of 102 patients with SI-NENs had only small bowel resections; of those, 25 were VS-LA and 25 were Con-LA. Patients with VS-LA had tendentially more advanced diseases with slightly higher rates of abdominal pain, mesenteric shrinkage and more level III lymph node involvement compared to patients with Con-LA. VS-LA, however, resulted in shorter resected bowel segments (median 40 cm vs. 65 cm, p = 0.007) with similar rates of local R0 resections (72% vs. 84%) and resected lymph nodes (median 13 vs. 13). Postoperative clinically relevant complications occurred in 1 of 25 (4%) in the VS-LA and in 7 of 25 (28%) patients in the Con-LA group (p = 0.02). Three months after surgery, 1 of 25 (4%) patients of the VS-LA group and 10 of 25 (40%) patients in the Con-LA group (p = 0.002) complained about abdominal pain. One of eight patients in the VS-LA group and two of thirteen patients in the Con-LA group who had completely resected stage III disease complained about diarrhea (p = 0.31). CONCLUSION: VS-LA seems to be oncologically safe and should be considered in small bowel resections for SI-NENs.

Heavier Weight of Resected Thyroid Specimen Is Associated With Higher Postoperative Morbidity in Benign Goiter.

OBJECTIVE: The impact of heavier weight of resected thyroid specimen on postoperative morbidity after total thyroidectomy for multinodular benign goiter remains unclear. METHODS: Data from the prospective StuDoQ|Thyroid registry of the German Society of General and Visceral Surgery were analyzed regarding the weight of the resected thyroid specimen and perioperative morbidity (vocal cord palsy, hemorrhage, surgical site infection, and hypocalcemia). To achieve a homogeneous patient population, only patients with total thyroidectomy for multinodular benign goiter were included. RESULTS: A total of 7911 patients from 105 departments underwent total thyroidectomy for benign conditions (January 2017-July 2020). The median resected weight of the thyroid specimen in all patients was 53 g (interquartile range 32-92). In 1732 patients, the specimen weight exceeded 100 g. Intraoperative neuromonitoring was used in 99.5% of patients. Postoperative laryngoscopy revealed vocal cord dysfunction in 480 of 15 822 (3.03%) nerves at risk, with unilateral dysfunction in 454 (2.87%) of patients and bilateral dysfunction in 13 patients (0.08%). In multivariable analysis, a thyroid weight >100 g was an independent predictor of early postoperative vocal cord dysfunction [odds ratio (OR) 1.462, 95% CI 1.108-1.930, P = 0.007). Heavier (>100 g) thyroid weight was an independent predictor of surgical site infection (OR 1.861, 95% CI 1.203-2.880, P = 0.005) and also predicted postoperative hemorrhage in the univariate analysis (OR 1.723, 95% CI 1.027-2.889, P = 0.039). On the contrary, postoperative parathyroid function was not affected. CONCLUSIONS: Heavier (>100 g) resected thyroid weight independently predicts higher postoperative morbidity, including early vocal cord palsy and surgical site infection after total thyroidectomy for benign multinodular goiter.

Regional Growth Velocity and Incidence of Pancreatic Neuroendocrine Neoplasias in Multiple Endocrine Neoplasia Type 1.

OBJECTIVES: Pancreatic neuroendocrine neoplasias (pNENs) in multiple endocrine neoplasia type 1 are predominantly found in the dorsal anlage. Whether their growth velocity and incidence might be related to their location in the pancreas has not been investigated yet. METHODS: We studied 117 patients using endoscopic ultrasound. RESULTS: Growth velocity could be calculated for 389 pNENs. Increase of largest tumor diameter (% per month) was 0.67 (standard deviation [SD], 2.04) in the pancreatic tail (n = 138), 1.12 (SD, 3.00) in the pancreatic body (n = 100), 0.58 (SD, 1.19) in the pancreatic head/uncinate process-dorsal anlage (n = 130), and 0.68 (SD, 0.77) in the pancreatic head/uncinate process-ventral anlage (n = 12). Comparing growth velocity of all pNENs in the dorsal (n = 368, 0.76 [SD, 2.13]) versus ventral anlage, no significant difference was detected. Annual tumor incidence rate was 0.21 in the pancreatic tail, 0.13 in the pancreatic body, 0.17 in the pancreatic head/uncinate process-dorsal anlage, 0.51 dorsal anlage together, and 0.02 in the pancreatic head/uncinate process-ventral anlage. CONCLUSIONS: Multiple endocrine neoplasia type 1 pNENs are unequally distributed between ventral (low prevalence and incidence) and dorsal anlage. However, there are no regional differences in growth behavior.

Long Non-Coding RNA H19 Expression Correlates with Autophagy Process in Adrenocortical Carcinoma.

Adrenocortical carcinoma (ACC) is characterized by poor prognosis and high mortality. The suppression of the long-non-coding RNA H19, counterbalanced by IGF2 over-expression, leads to down-regulation of the autophagy markers, high proliferation rate and metastatic potential in patients affected by ACC. The administration of the deacetylase inhibitors (DACi) panobinostat, trichostatin A (TSA) and SAHA affected the cell viability of H295R monolayer and spheroids and induced the over-expression of H19 and autophagy transcripts. H19 knock down in H295R cells was not able to modulate the expression level of autophagy transcripts. Instead, H19 knock down was able to impede the ability of DACi to modulate the protein level of the autophagy markers. Furthermore, the administration of higher concentration of DACi was able to down-regulate the protein level of Beclin1 and p62 and to induce the conversion of LC3B-I into the active LC3B-II form, thus confirming an active autophagic process. Neither the active protein level nor the activity of caspases 8 and 3 was prompted by the DACi, thus excluding the involvement of the executioners of apoptosis in H295R decay. The DACi restore H19, the autophagy markers and trigger cell death in ACC cells. The re-activation of autophagy would represent a novel strategy for the treatment of patients affected by this severe malignancy.

Relevant prognostic factors in patients with stage IV small intestine neuroendocrine neoplasms.

There are few, but controversial data on the prognostic role of upfront primary tumour resection and mesenteric lymph node dissection (PTR) in patients with diffuse metastatic small intestinal neuroendocrine neoplasia (SI-NEN). Therefore, the prognostic role of PTR and other factors was determined in this setting. This retrospective cohort study included patients with stage IV SI-NETs with unresectable distant metastases without clinical and radiological signs of acute bowel obstruction or ischaemia. Patients diagnosed from January 2002 to May 2020 were retrieved from a prospective SI-NEN database. Disease specific overall survival (OS) was analysed with regard to upfront PTR and a variety of other clinical (e.g., gender, age, Hedinger disease, carcinoid syndrome, diarrhoea, laboratory parameters, metastatic liver burden, extrahepatic and extra-abdominal metastasis) and pathological (e.g., grading, mesenteric gathering) parameters by uni- and multivariate analysis. A total of 138 patients (60 females, 43.5%) with a median age of 60 years, of whom 101 (73%) underwent PTR and 37 (27%) did not, were included in the analysis. Median OS was 106 (95% CI: 72.52-139.48) months in the PTR group and 52 (95% CI: 30.55-73.46) in the non-PTR group (p = 0.024), but the non-PTR group had more advanced metastatic disease (metastatic liver burden ≥50% 32.4% vs. 13.9%). There was no significant difference between groups regarding the rate of surgery for bowel complications during a median follow-up of 51 months (PTR group 10.9% and non-PTR group 16.2%, p = 0.403). Multivariate analysis revealed age < 60 years, normal C-reactive protein (CRP) at baseline, absence of diarrhoea, less than 50% of metastatic liver burden, and treatment with PRRT as independent positive prognostic factors, whereas PTR showed a strong tendency towards better OS, but level of significance was missed (p = 0.067). However, patients who underwent both, PTR and peptide radioreceptor therapy (PRRT) had the best survival compared to the rest (137 vs. 73 months, p = 0.013). PTR in combination with PRRT significantly prolongs survival in patients with stage IV SI-NEN. Prophylactic PTR does also not result in a lower reoperation rate compared to the non-PTR approach regarding bowel complications.

Pancreatic Neuroendocrine Neoplasias in Multiple Endocrine Neoplasia Type 1 Are Predominantly Located in the Dorsal Anlage: An Endoscopic Ultrasound Study.

OBJECTIVES: Pancreatic neuroendocrine neoplasias (pNENs) frequently occur in multiple endocrine neoplasia type 1 (MEN1). Their distribution referring to embryology, that is, the pancreatic anlagen, has not been investigated yet. METHODS: In the time between 1998 and 2019, we studied the distribution of pNENs in MEN1 concerning the embryologic origin of the pancreas, that is, the dorsal versus ventral anlage using endoscopic ultrasound in 117 MEN1 patients: 56 women, 61 men; aged 40 years (standard deviation, 14 years) at first endoscopic ultrasound. RESULTS: In 105 patients, a total of 628 pNENs were detected. They were located in the pancreatic tail: 231; pancreatic body: 177; pancreatic head/uncinate process: 220. Of the latter, 22 were located in the ventral anlage, 176 in the dorsal anlage, and 22 remained undefined. In summary, just 3.5% of all detected pNENs were located in the ventral anlage, 93.0% in the dorsal anlage, and 3.5% could not be assigned. CONCLUSIONS: Our study indicates that the vast majority of pNENs in MEN1 is located in the dorsal anlage, whereas the ventral anlage of the pancreas seems to be to a large extend spared from pNENs. Implications for new surgical strategies might be considered.

Short-Term Hypocalcemia Prophylaxis With Calcitriol Before Thyroidectomy.

BACKGROUND: Total thyroidectomy is the most common surgical treatment of thyroid diseases, and postoperative hypocalcemia is its most common complication. Hypocalcemia prolongs the patient's hospital stay and impairs his or her quality of life. Although a low vitamin D level is a recognized risk factor, the utility of preoperative vitamin D administration to prevent postoperative hypocalcemia is unclear. In this trial, therefore, we studied the effect of giving vitamin D before total thyroidectomy. METHODS: In a multicenter, randomized, minimally interventional trial (registration number: DRKS 00005615), patients about to undergo total thyroidectomy were randomized either to an intervention group that received 0.5 µg of calcitriol per os twice daily for three days up to the day immediately before surgery, or to a control group that did not (no placebo was given). The primary endpoint was the absence of hypocalcemia (serum calcium <2.1 mmol/L) in the postoperative course. RESULTS: Of the 287 patients recruited in six hospitals over the period 23 July 2014 to 20 March 2017, 246 were included in the final analysis. The intervention and control groups did not differ significantly with respect to the rate of postoperative hypocalcemia (29.2% and 33.6%, respectively; p = 0.546, power 8.8%). The duration of postoperative hypocalcemia was, however, shorter in the intervention group (3.5 vs. 7 days; p = 0.016, power 68%). The rates of hypocalcemia in the individual trial locations varied widely, ranging from 13.9% to 71.4%. CONCLUSION: Short-term administration of calcitriol did not affect the rate of occurrence of hypocalcemia after thyroidectomy, but did shorten its duration. The rate of postoperative hypocalcemia varied widely across hospitals, probably because of differences in surgical technique.

Carbonic-anhydrase IX expression is increased in thyroid cancer tissue and represents a potential therapeutic target to eradicate thyroid tumor-initiating cells.

The expression of Carbonic-anhydrase IX (CAIX) in thyroid cancer (TC) subtypes was determined and its role in cancer cell growth and tumor-initiating cells (TICs) investigated. Immunohistochemistry in 114 TC patients revealed that CAIX expression was increased in tumor specimens of papillary, follicular and anaplastic TCs compared to normal thyroid tissue. Clinicopathological data indicated that lymph node metastases were more frequent in patients with high CAIX expression. The Cancer Genome Atlas database analysis demonstrated that a strong CAIX-mRNA expression was associated with advanced tumor stages and poor survival in TCs. In TC cell lines, CAIX expression was elevated in tumorspheres compared to monolayer cultures and was associated with an increased expression of stemness markers. Genetic knockdown or pharmacological inhibition of CAIX suppressed cell proliferation and the TIC ability to form tumorspheres by an induction of apoptosis and cell-cycle arrest. These findings suggest CAIX as a potential prognostic marker and a therapeutic target for thyroid cancer.

Transglutaminase 2 promotes tumorigenicity of colon cancer cells by inactivation of the tumor suppressor p53.

Despite a high clinical need for the treatment of colorectal carcinoma (CRC) as the second leading cause of cancer-related deaths, targeted therapies are still limited. The multifunctional enzyme Transglutaminase 2 (TGM2), which harbors transamidation and GTPase activity, has been implicated in the development and progression of different types of human cancers. However, the mechanism and role of TGM2 in colorectal cancer are poorly understood. Here, we present TGM2 as a promising drug target.In primary patient material of CRC patients, we detected an increased expression and enzymatic activity of TGM2 in colon cancer tissue in comparison to matched normal colon mucosa cells. The genetic ablation of TGM2 in CRC cell lines using shRNAs or CRISPR/Cas9 inhibited cell expansion and tumorsphere formation. In vivo, tumor initiation and growth were reduced upon genetic knockdown of TGM2 in xenotransplantations. TGM2 ablation led to the induction of Caspase-3-driven apoptosis in CRC cells. Functional rescue experiments with TGM2 variants revealed that the transamidation activity is critical for the pro-survival function of TGM2. Transcriptomic and protein-protein interaction analyses applying various methods including super-resolution and time-lapse microscopy showed that TGM2 directly binds to the tumor suppressor p53, leading to its inactivation and escape of apoptosis induction.We demonstrate here that TGM2 is an essential survival factor in CRC, highlighting the therapeutic potential of TGM2 inhibitors in CRC patients with high TGM2 expression. The inactivation of p53 by TGM2 binding indicates a general anti-apoptotic function, which may be relevant in cancers beyond CRC.

Long-term outcome of surgical resection in patients with gastroenteropancreatic neuroendocrine neoplasia: results from a German nation-wide multi-centric registry.

BACKGROUND: Neuroendocrine neoplasia (NEN) are rare and heterogenous tumours. Few data exist on the impact of surgical therapy. MATERIALS AND METHODS: This is a retrospective analysis of prospectively collected data of gastroenteropancreatic NEN in the German NET-Registry (1999-2012). It focuses on patients without distant metastases (limited disease, LD, stage I-IIIB). RESULTS: Data of 2239 patients with NEN were recorded. Median age was 59 years, the gender ratio was 1:1.3 (f:m). A total of 986 patients (44%) had LD, and the 5-year survival rate (5 years) was 77% for all and 90% for patients with LD. A total of 1635 patients (73%) received a surgical therapy (1st to 6th line); the 5 and 10 ysr were 83/65% after and 59/35% without surgery for all patients (p < .001). The resection margins in the LD patients were 76%, 16%, and 3% for R0, R1 and R2, respectively. The 10 ysr was 84%, 59% and 42% for R0, R1 and R2 resections, respectively (p = .021 R0/R1, p < .001 R0/R2). The R0 resection rate was 75% for G1/G2 NET and 67% for G3 NEC. CONCLUSION: The rate of complete tumour resection (R0) in LD is independent of tumour grading, and R0 resection is the key determinant of long-term survival, as demonstrated by the 10 ysr. of 84%. All NEN patients with limited disease should be considered for operation, if possible, as the best 10-year survival is shown after an R0 resection.

Two giant retroperitoneal schwannomas mimicking adrenal malignancy - a case report.

Schwannomas are benign tumors in 95% of cases and very rarely occur in the retroperitoneum. We report the cases of a 35-year-old man with abdominal discomfort and a 50-year-old asymptomatic woman with large retroperitoneal masses. Both underwent multivisceral surgery to exclude an adrenal carcinoma, and the pathologic diagnosis showed schwannomas in both cases. Despite morphological imaging, it was not possible to get a clear diagnosis preoperatively.

Total Versus Near-total Thyroidectomy in Graves Disease: Results of the Randomized Controlled Multicenter TONIG-trial.

BACKGROUND: Previous data suggest that the incidence of hypoparathyroidism after surgery for Graves disease (GD) is lower after subtotal thyroidectomy compared to total thyroidectomy (TT). The present study evaluated the incidence of postoperative hypoparathyroidism after near-total (NTT) versus TT in GD. METHODS/DESIGN: In a multicenter prospective randomized controlled clinical trial, patients with GD were randomized intraoperatively to NTT or TT. Primary endpoint was the incidence of transient postoperative hypoparathyroidism. Secondary endpoints were permanent hypoparathyroidism, transient recurrent laryngeal nerve palsy (RLNP), reoperations for bleeding, inadvertently removed parathyroid glands, and recurrent hyperthyroidism after 12 months. RESULTS: Eighteen centers randomized 205 patients to either TT (n = 102) or NTT (n = 103) within 16 months. According to intention-to-treat postoperative transient hypoparathyroidism occurred in 19% (20/103) patients after NTT and in 21% (21 of 102) patients after TT (P = 0.84), which persisted >6 months in 2% and 5% of the NTT and TT groups (P = 0.34). The rates of parathyroid autotransplantation (NTT 24% vs TT 28%, P = 0.50) and transient RLNP (NTT 3% vs TT 4%, P = 0.35) was similar in both groups. The rate of reoperations for bleeding tended to be higher in the NTT group (3% vs 0%, P = 0.07) and the rate of inadvertently removed parathyroid glands was significantly higher after NTT (13% vs 3%, P = 0.01). An existing endocrine orbitopathy improved in 35% and 24% after NTT and TT (P = 0.61). Recurrent disease occurred in only 1 patient after TT (P = 0.34). CONCLUSION: NTT for GD is not superior to TT regarding transient postoperative hypoparathyroidism.

H3K36me2/3 Binding and DNA Binding of the DNA Methyltransferase DNMT3A PWWP Domain Both Contribute to its Chromatin Interaction.

The PWWP domain of DNMT3 DNA methyltransferases binds to histone H3 tails containing methylated K36, and this activity is important for heterochromatic targeting. Here, we show that the PWWP domain of mouse DNMT3A binds to H3K36me2 and H3K36me3 with a slight preference for H3K36me2. PWWP domains have also been reported to bind to DNA, and the close proximity of H3K36 and nucleosomal DNA suggests a combined binding to H3K36me2/3 and DNA. We show here that the DNMT3A PWWP domain binds to DNA with a weak preference for AT-rich sequences and that the designed charge reversal R362E mutation disrupts DNA binding. The K295E mutation, as well as K295I recently identified in paraganglioma, a rare neuroendocrine neoplasm, disrupts both DNA and H3K36me2/3 binding, which is in agreement with the proximity of K295 to residues involved in K36me2/3 methyllysine binding. Nucleosome pulldown experiments show that DNA binding and H3K36me2/3 binding are important for the interaction of the DNMT3A PWWP domain with nucleosomes. Localization studies of transiently transfected fluorescently-tagged wild-type and PWWP-mutated full-length DNMT3A indicate that both interactions contribute to the subnuclear localization of DNMT3A in mouse cells. In summary, our data demonstrate that the combined binding of the DNMT3A PWWP domain to the H3 tail containing K36me2/3 and to the nucleosomal or linker DNA is important for its chromatin interaction and subnuclear targeting of DNMT3A in living cells.

Surgical resection of neuroendocrine tumor liver metastases as part of multimodal treatment strategies: A propensity score matching analysis.

BACKGROUND: It remains unclear whether liver resection as part of multimodal therapy of neuroendocrine liver metastases (NELM) is superior to non-surgical (interventional and medication-based) treatment alone. This study should determine if patients with NELM undergoing hepatic surgery in addition to non-surgical treatment have improved overall survival compared to patients undergoing non-surgical therapy alone. METHODS: 123 patients undergoing treatment of NELM between 1995 and 2014 were included in this retrospective cohort study. Two groups were formed: (A) surgery and non-surgical therapy and (B) non-surgical treatment alone. To minimize the bias of patient selection propensity score matching was used. RESULTS: There was significantly better overall survival for group A (152 months, 95%CI: 119-185) compared to group B (63 months, 95%CI: 45-81) measured from the initial diagnosis of the metastases (P = 0.003). After propensity score matching, 37 patients undergoing surgical resection of NELM within a multimodal treatment were compared to 37 patients undergoing non-surgical treatment. Under these circumstances, surgery had no significant influence on survival (group A: 134 months, 95% CI: 94-173; group B: 76 months, 95% CI: 53-99, P = 0.23). Based on a multivariate Cox proportional hazard model, only Ki-67 of primary tumor >20% (HR, 50.776; 95%CI, 4.056-635.71; P = 0.002) and no resection of primary tumor (HR, 10.464; 95%CI, 1.873-58.448; P = 0.007) remained independent risk factors. CONCLUSION: After minimizing patient selection bias, patients with hepatic resection as integral of multimodal therapy of NELM do not have better overall survival than those receiving non-surgical treatment alone.

Results of Duodenopancreatic Reoperations in Multiple Endocrine Neoplasia Type 1.

BACKGROUND: To evaluate the outcome of duodenopancreatic reoperations in patients with multiple endocrine neoplasia type 1 (MEN1). METHODS: MEN1 patients who underwent reoperations for duodenopancreatic neuroendocrine neoplasms (dpNENs) were retrieved from a prospective database and retrospectively analyzed. RESULTS: Twelve of 101 MEN1 patients underwent up to three reoperations, resulting in a total of 18 reoperations for dpNEN recurrence. Patients initially underwent either formal pancreatic resections (n = 7), enucleations (n = 3), or duodenotomy with lymphadenectomy for either NF-pNEN (seven patients), Zollinger-Ellison syndrome (ZES, three patients), organic hyperinsulinism (one patient) or VIPoma (one patient). Six patients had malignant dpNENs with lymph node (n = 5) and/or liver metastases (n = 2). The indication of reoperations was NF-pNEN (five patients), ZES (five patients), organic hyperinsulinism (one patient), and recurrent VIPoma (one patient). Median time to first reoperation was 67.5 (range 6-251) months. Five patients required a second duodenopancreatic reoperation for 60-384 months after initial surgery, and one patient underwent a third reoperation after 249 months. The rate of complications (Clavien-Dindo ≥3) was 28%. Four patients required completion pancreatectomy. Six patients developed pancreoprivic diabetes. After a median follow-up of 18 (6-34) years after initial surgery, ten of 12 patients are alive, one died of metastatic pancreatic VIPoma, and one died of metastatic thymic NEN. CONCLUSION: Reoperations are frequently necessary for dpNEN in MEN1 patients, but are not associated with an increased perioperative morbidity in specialized centers. Organ-sparing resections should be preferred as initial duodenopancreatic procedures to maintain pancreatic function and avoid completion pancreatectomy.

Vitamin D, FOXO3a, and Sirtuin1 in Hashimoto's Thyroiditis and Differentiated Thyroid Cancer.

Background: Protective effects of vitamin D have been reported in autoimmune and malignant thyroid diseases, though little is known about the underlying mechanism. Sirtuin 1 histon deacethylase (SIRT1) links the vitamin D pathway with regulation of transcription factor FOXO3a, a key player in cell cycle regulation and apoptosis. Aim of the present study was to investigate common single nucleotide polymorphisms (SNP's) in FOXO3a gene in respect to thyroid diseases, as well as to evaluate the hypothesis of Sirtuin1-FOXO3a interaction being a mediator of anti-proliferative vitamin D effects. Methods: The SNP's FOXO3a rs4946936/rs4945816/rs9400239 were genotyped in 257 patients with differentiated thyroid carcinoma (DTC), 139 patients with Hashimoto thyroiditis (HT) and 463 healthy controls (HC). Moreover, T-helper cells of HC and papillary thyroid cancer cell line BCPAP were incubated with 1,25(OH)2D3 and/or SIRT1 inhibitor Ex-527 in order to elucidate SIRT1- dependent vitamin D effects on cell proliferation and FOXO3a gene expression in vitro. Results: Patients with DTC tended to carry more often allele C in FOXO3a rs4946936 in comparison to HC (pcorrected = pc = 0.08). FOXO3a rs9400239T and rs4945816C was more frequent in HT in comparison to HC (pc = 0.02 and pc = 0.01, respectively). In both DTC and HT, we could not find a correlation of FOXO3a SNP's with vitamin D status. However, on in vitro level, 1,25(OH)2D3 showed an anti-proliferative effect in both T-helper cells and BCPAP, that was blocked by SIRT1 inhibition (T-helper cells: p = 0.0059, BCPAP: p = 0.04) and accompanied by elevated FOXO3a gene expression in T-helper cells (p = 0.05). Conclusions: FOXO3a rs9400239T and rs4945816C may constitute risk factors for HT, independent of the vitamin D status.This indicates the implication of FOXO3a in pathogenesis of autoimmune thyroid diseases. The dependency of anti-proliferative vitamin D effects on SIRT1 activity further suggests a key role of vitamin D-SIRT1-FOXO3a axis for protective vitamin D effects.

The DNMT3A R882H mutation does not cause dominant negative effects in purified mixed DNMT3A/R882H complexes.

The DNA methyltransferase DNMT3A R882H mutation is observed in 25% of all AML patients. DNMT3A is active as tetramer and the R882H mutation is located in one of the subunit/subunit interfaces. Previous work has reported that formation of mixed wildtype/R882H complexes leads to a strong loss of catalytic activity observed in in vitro DNA methylation assays (Russler-Germain et al., 2014, Cancer Cell 25:442-454). To investigate this effect further, we have prepared mixed wildtype/R882H DNMT3A complexes by incubation of individually purified subunits of the DNMT3A catalytic domain and full-length DNMT3A2. In addition, we have used a double affinity tag approach and specifically purified mixed catalytic domain complexes formed after co-expression of R882H and wildtype subunits in E. coli cells. Afterwards, we determined the catalytic activity of the mixed complexes and compared it to that of purified complexes only consisting of one subunit type. In both settings, the expected catalytic activities of mixed R882H/wildtype complexes were observed demonstrating an absence of a dominant negative effect of the R882H mutation in purified DNMT3A enzymes. This result suggests that heterocomplex formation of DNMT3A and R882H is unlikely to cause dominant negative effects in human cells as well. The limitations of this conclusion and its implications are discussed.