High-Grade Neuroendocrine Carcinoma of the Head and Neck: Human Papillomavirus Status and PD-L1 Expression.

BACKGROUND: Human papillomavirus (HPV)-related head and neck squamous cell carcinoma represents an important subgroup of head and neck cancer, but HPV occurs also in the less common neuroendocrine carcinomas (NEC). The PD-1/PD-L1 pathway appears to be activated in pulmonary NEC and correlates with a higher mutation burden, but the potential of NEC to respond to checkpoint inhibitors is unknown to a large extent. OBJECTIVES: To determine the HPV status of NEC of the head and neck region and to investigate the expression of PD-1 and its ligands PD-L1 and PD-L2. METHODS: Surgical tumor samples from 2006 to 2017 were analyzed. HPV status was determined by p16 immunohistochemistry (IHC) and multiplex PCR. IHC using the Cologne Score was performed for PD-1, PD-L1, and PD-L2. RESULTS: Seven NEC tumor samples were analyzed, three of them showed HPV type 18. Expression of PD-1 and PD-L1 differed widely and showed no correlation to HPV status. IHC showed an overexpression of PD-L2 in most of the patients. CONCLUSIONS AND SIGNIFICANCE: A multicentric analysis of NEC is needed to further evaluate the role of HPV as well as immunocheckpoints with regard to inflammatory immune response in genesis and clinical course of this rare tumor entity. Biomarkers for selection of novel treatment regimens, including immunotherapeutic approaches, are warranted.

Hormone secreting gastro-entero-pancreatic neuroendocrine neoplasias (GEP-NEN): When to consider, how to diagnose?

Neuroendocrine neoplasms of the digestive system (GEP-NEN) represent a heterogeneous group of malignancies with various clinical presentation and prognosis. GEP-NENs can potentially affect all organs of the gastrointestinal tract; characteristically they share the biological property to produce and secrete peptides and neuroamines. About 30% of GEP-NENs are hormonally active and can cause specific clinical syndromes. The clinical presentation mainly depends on the primary site of the tumor and its functionality. Because of the wide spectrum of clinical symptoms and their misperceived rarity, diagnosis of GEP-NENs is often delayed for years and tumors are detected first in an advanced stage. Early identification of a specific hormonal syndrome can significantly impact tumor diagnosis and treatment, moreover the preoperative management of NEN hormonal release avoids potential life threatening hormonal crisis. However, GEP-NEN diagnostic work-up is challenging, it requires a multidisciplinary team and needs particular experience; standardized protocols and clinical experience are essential for a proper endocrine diagnostic work-up. In addition to the biochemical diagnostic, further radiologic and endoscopic imaging modalities are required moreover, somatostatin-receptor based functional imaging, using either Octreotide-scintigraphy or novel PET-based techniques with specific isotopes like Ga68-DOTA-octreotate, plays an important role for the detection of the primary tumor as well as for the evaluation of the tumor extent.

Multicenter analysis on the value of standard (chemo)radiotherapy in elderly patients with locally advanced adenocarcinoma of the esophagus or gastroesophageal junction.

BACKGROUND: To assess the tolerability and oncological results of chemoradiation in elderly patients with locally advanced adenocarcinoma of the esophagus or gastroesophageal junction. METHODS: This multi-center retrospective analysis included 86 elderly patients (≥ 65 years) with esophageal or gastroesophageal junction adenocarcinoma (median age 73 years; range 65-92 years) treated with definitive or neoadjuvant (chemo)radiotherapy. The treatment was performed at 3 large comprehensive cancer centers in Germany from 2006 to 2020. Locoregional control (LRC), progression-free survival (PFS), distant metastasis-free survival (DMFS), overall survival (OS), and treatment-associated toxicities according to CTCAE criteria v5.0 were analyzed, and parameters potentially relevant to patient outcomes were evaluated. RESULTS: Thirty-three patients (38%) were treated with neoadjuvant chemoradiation followed by surgery, while the remaining patients received definitive (chemo)radiation. The delivery of radiotherapy without dose reduction was possible in 80 patients (93%). In 66 patients (77%), concomitant chemotherapy was initially prescribed; however, during the course of therapy, 48% of patients (n = 32) required chemotherapy de-escalation due to treatment-related toxicities and comorbidities. Twenty-nine patients (34%) experienced higher-grade acute toxicities and 14 patients (16%) higher-grade late toxicities. The 2-year LRC, DMFS, PFS, and OS amounted to 72%, 49%, 46%, and 52%, respectively. In multivariate analysis, neoadjuvant chemoradiation followed by surgery was shown to be associated with significantly better PFS (p = 0.006), DMFS (p = 0.006), and OS (p = 0.004) compared with all non-surgical treatments (pooled definitive radiotherapy and chemoradiation). No such advantage was seen over definitive chemoradiation. The majority of patients with neoadjuvant therapy received standard chemoradiotherapy without dose reduction (n = 24/33, 73%). In contrast, concurrent chemotherapy was only possible in 62% of patients undergoing definitive radiotherapy (n = 33/53), and most of these patients required dose-reduction or modification of chemotherapy (n = 23/33, 70%). CONCLUSIONS: In our analysis, omission of chemotherapy or adjustment of chemotherapy dose during definitive radiotherapy was necessary for the overwhelming majority of elderly esophageal cancer patients not eligible for surgery, and hence resulted in reduced PFS and OS. Therefore, optimization of non-surgical approaches and the identification of potential predictive factors for safe administration of concurrent chemotherapy in elderly patients with (gastro)esophageal adenocarcinoma is required.

High incidence of extraadrenal paraganglioma in families with SDHx syndromes detected by functional imaging with [18F]fluorodihydroxyphenylalanine PET.

PURPOSE: Knowledge of the genetic backgrounds of hereditary syndromes, which are increasingly being characterized, enables genetic screening of family members of affected patients. Upon detection of a mutation, genetic counselling and clinical screening including imaging modalities and biochemical analyses are commonly performed. METHODS: Unaffected, mutation-positive relatives of index patients with hereditary paraganglioma syndromes were offered PET imaging with [(18)F]fluorodihydroxyphenylalanine and the incidence of pathological findings was retrospectively analysed in relation to mutations of the succinate dehydrogenase enzyme complex. PET only or PET/CT was performed in 21 individuals from eight families with SDHD, one family with SDHC and two families with SDHB mutations. Screening was offered every 2 to 5 years. RESULTS: Of the 21 individuals, 14 showed paraganglioma during screening. In particular, in only 2 of 15 patients with a SDHD mutation were the findings completely unremarkable on PET screening. However, false-negative lesions for abdominal manifestations in two SDHD-positive patients were detected. CONCLUSION: FDOPA PET is a sensitive imaging modality which should be offered to patients with a detected SDHx (SDHD) mutation, preferably using a hybrid technique.

Magnetic separation of encapsulated islet cells labeled with superparamagnetic iron oxide nano particles.

Islet cell transplantation is a promising option for the restoration of normal glucose homeostasis in patients with type 1 diabetes. Because graft volume is a crucial issue in islet transplantations for patients with diabetes, we evaluated a new method for increasing functional tissue yield in xenogeneic grafts of encapsulated islets. Islets were labeled with three different superparamagnetic iron oxide nano particles (SPIONs; dextran-coated SPION, siloxane-coated SPION, and heparin-coated SPION). Magnetic separation was performed to separate encapsulated islets from the empty capsules, and cell viability and function were tested. Islets labeled with 1000 µg Fe/ml dextran-coated SPIONs experienced a 69.9% reduction in graft volume, with a 33.2% loss of islet-containing capsules. Islets labeled with 100 µg Fe/ml heparin-coated SPIONs showed a 46.4% reduction in graft volume, with a 4.5% loss of capsules containing islets. No purification could be achieved using siloxane-coated SPIONs due to its toxicity to the primary islets. SPION labeling of islets is useful for transplant purification during islet separation as well as in vivo imaging after transplantation. Furthermore, purification of encapsulated islets can also reduce the volume of the encapsulated islets without impairing their function by removing empty capsules.

Chemoradiotherapy in geriatric patients with squamous cell carcinoma of the esophagus: Multi-center analysis on the value of standard treatment in the elderly.

Background and purpose: To evaluate the tolerability and outcomes of chemoradiation in elderly patients with locally advanced esophageal squamous cell carcinoma (ESCC). Materials and methods: This multi-center retrospective analysis included 161 patients with SCC of the esophagus with a median age of 73 years (range 65-89 years) treated with definitive or neoadjuvant (chemo)radiotherapy between 2010 and 2019 at 3 large comprehensive cancer centers in Germany. Locoregional control (LRC), progression-free survival (PFS), distant metastasis-free survival (DMFS), overall survival (OS), and treatment-associated toxicities were analyzed, and parameters determining patient outcomes and treatment tolerance were assessed. Results: The delivery of radiotherapy without dose reduction was possible in 149 patients (93%). In 134 patients (83%), concomitant chemotherapy was initially prescribed; however, during the course of therapy, 41% of these patients (n = 55) required chemotherapy de-escalation due to treatment-related toxicities. Fifty-two patients (32%) experienced higher-grade acute toxicities, and 22 patients (14%) higher-grade late toxicities. The 2-year LRC, DMFS, PFS, and OS rates amounted to 67.5%, 33.8%, 31.4%, and 40.4%, respectively. Upon multivariate analysis, full-dose concomitant chemotherapy (vs. no or modified chemotherapy) was associated with significantly better DMFS (p=0.005), PFS (p=0.005) and OS (p=0.001). Furthermore, neoadjuvant chemoradiotherapy followed by tumor resection (vs. definitive chemoradiotherapy or definitive radiotherapy alone) significantly improved PFS (p=0.043) and OS (p=0.049). We could not identify any clinico-pathological factor that was significantly associated with LRC. Furthermore, definitive (chemo)radiotherapy, brachytherapy boost and stent implantation were significantly associated with higher-grade acute toxicities (p<0.001, p=0.002 and p=0.04, respectively). The incidence of higher-grade late toxicities was also significantly associated with the choice of therapy, with a higher risk for late toxicities when treatment was switched from neoadjuvant to definitive (chemo)radiotherapy compared to primary definitive (chemo)radiotherapy (p<0.001). Conclusions: Chemoradiation with full-dose and unmodified concurrent chemotherapy has a favorable prognostic impact in elderly ESCC patients; however, about half of the analyzed patients required omission or adjustment of chemotherapy due to comorbidities or toxicities. Therefore, the identification of potential predictive factors for safe administration of concurrent chemotherapy in elderly ESCC patients requires further exploration to optimize treatment in this vulnerable patient cohort.

Quantitative Analysis of Plasma Cell-Free DNA and Its DNA Integrity and Hypomethylation Status as Biomarkers for Tumor Burden and Disease Progression in Patients with Metastatic Neuroendocrine Neoplasias.

BACKGROUND: Neuroendocrine neoplasia (NEN) encompasses a diverse group of malignancies marked by histological heterogeneity and highly variable clinical outcomes. Apart from Chromogranin A, specific biomarkers predicting residual tumor disease, tumor burden, and disease progression in NEN are scant. Thus, there is a strong clinical need for new and minimally invasive biomarkers that allow for an evaluation of the prognosis, clinical course, and response to treatment of NEN patients, thereby helping implement individualized treatment decisions in this heterogeneous group of patients. In the current prospective study, we evaluated the role of plasma cell-free DNA concentration and its global hypomethylation and fragmentation as possible diagnostic and prognostic biomarkers in patients with neuroendocrine neoplasias. METHODS: The plasma cfDNA concentration, cfDNA Alu hypomethylation, and LINE-1 cfDNA integrity were evaluated prospectively in 63 NEN patients with presumably cured or advanced metastatic disease. The cfDNA characteristics in NEN patients were compared to the results of a group of 29 healthy controls and correlated with clinical and histopathological data of the patients. RESULTS: Patients with advanced NEN showed a significantly higher cfDNA concentration and percentage of Alu hypomethylation and a reduced LINE-1 cfDNA integrity as compared to the surgically cured NET patients and the healthy control group. The increased hypomethylation and concentration of cfDNA and the reduced cfDNA integrity in NEN patients were strongly associated with tumor burden and poor prognosis, while no correlation with tumor grading, differentiation, localization, or hormonal activity could be found. Multiparametric ROC analysis of plasma cfDNA characteristics was able to distinguish NEN patients with metastatic disease from the control group and the cured NEN patients with AUC values of 0.694 and 0.908, respectively. This was significant even for the group with only a low tumor burden. CONCLUSIONS: The present study, for the first time, demonstrates that the combination of plasma cfDNA concentration, global hypomethylation, and fragment length pattern has the potential to serve as a potent and sensitive prognostic and therapeutic "liquid biopsy" biomarker for tumor burden and disease progression in patients with neuroendocrine neoplasias.

Treatment Approaches and Outcome of Patients with Neuroendocrine Neoplasia Grade 3 in German Real-World Clinical Practice.

BACKGROUND: Neuroendocrine neoplasia grade 3 (NEN G3) represents a rare and heterogeneous cancer type with a poor prognosis. The aim of our study was to analyze real-world data from the German NET Registry with a focus on therapeutic and prognostic aspects. METHODS: NEN G3 patients were identified within the German NET Registry. Demographic data and data on treatments and outcomes were retrieved. Univariate analyses were performed using the Kaplan-Meier-method. Multivariate analysis was performed using a Cox proportional hazard model. RESULTS: Of 445 included patients, 318 (71.5%) were diagnosed at stage IV. Well-differentiated morphology (NET G3) was described in 31.7%, 60% of cases were classified as neuroendocrine carcinoma (NEC), and the median Ki67 value was 50%. First-line treatment comprised chemotherapy in 43.8%, with differences in the choice of regimen with regard to NET or NEC, and surgery in 41.6% of patients. Median overall survival for the entire cohort was 31 months. Stage, performance status and Ki67 were significant prognostic factors in multivariate analysis. CONCLUSIONS: The survival data of our national registry compare favorably to population-based data, probably mainly because of a relatively low median Ki67 of 50%. Nevertheless, the best first- and second-line approaches for specific subgroups remain unclear, and an international effort to fill these gaps is needed.

Second MAFA Variant Causing a Phosphorylation Defect in the Transactivation Domain and Familial Insulinomatosis.

Adult-onset familial insulinomatosis is a rare disorder with recurrent, severe hypoglycemia caused by multiple insulin-secreting pancreatic tumors. The etiology was unclear until the variant p.Ser64Phe in the transcription factor MAFA, a key coordinator of ß-cell insulin secretion, was defined as the cause in two families. We here describe detailed genetic, clinical, and family analyses of two sisters with insulinomatosis, aiming to identify further disease causes. Using exome sequencing, we detected a novel, heterozygous missense variant, p.Thr57Arg, in MAFA's highly conserved transactivation domain. The impact of the affected region is so crucial that in vitro expression studies replacing Thr57 have already been performed, demonstrating a phosphorylation defect with the impairment of transactivation activity and degradation. However, prior to our study, the link to human disease was missing. Furthermore, mild hyperglycemia was observed in six additional, heterozygote family members, indicating that not only insulinomatosis but also MODY-like symptoms co-segregate with p.Thr57Arg. The pre-described MAFA variant, p.Ser64Phe, is located in the same domain, impairs the same phosphorylation cascade, and results in the same symptoms. We confirm MAFA phosphorylation defects are important causes of a characteristic syndrome, thus complementing the pathophysiological and diagnostic disease concept. Additionally, we verify the high penetrance and autosomal dominant inheritance pattern.

One-step ¹8F-labeling of carbohydrate-conjugated octreotate-derivatives containing a silicon-fluoride-acceptor (SiFA): in vitro and in vivo evaluation as tumor imaging agents for positron emission tomography (PET).

The synthesis, radiolabeling, and initial evaluation of new silicon-fluoride acceptor (SiFA) derivatized octreotate derivatives is reported. So far, the main drawback of the SiFA technology for the synthesis of PET-radiotracers is the high lipophilicity of the resulting radiopharmaceutical. Consequently, we synthesized new SiFA-octreotate analogues derivatized with Fmoc-NH-PEG-COOH, Fmoc-Asn(Ac3AcNH-ß-Glc)-OH, and SiFA-aldehyde (SIFA-A). The substances could be labeled in high yields (38 ± 4%) and specific activities between 29 and 56 GBq/µmol in short synthesis times of less than 30 min (e.o.b.). The in vitro evaluation of the synthesized conjugates displayed a sst2 receptor affinity (IC50 = 3.3 ± 0.3 nM) comparable to that of somatostatin-28. As a measure of lipophilicity of the conjugates, the log P(ow) was determined and found to be 0.96 for SiFA-Asn(AcNH-ß-Glc)-PEG-Tyr³-octreotate and 1.23 for SiFA-Asn(AcNH-ß-Glc)-Tyr³-octreotate, which is considerably lower than for SiFA-Tyr³-octreotate (log P(ow) = 1.59). The initial in vivo evaluation of [¹8F]SiFA-Asn(AcNH-ß-Glc)-PEG-Tyr³-octreotate revealed a significant uptake of radiotracer in the tumor tissue of AR42J tumor-bearing nude mice of 7.7% ID/g tissue weight. These results show that the high lipophilicity of the SiFA moiety can be compensated by applying hydrophilic moieties. Using this approach, a tumor-affine SiFA-containing peptide could successfully be used for receptor imaging for the first time in this proof of concept study.

Detection of papillary thyroid carcinoma by analysis of BRAF and RET/PTC1 mutations in fine-needle aspiration biopsies of thyroid nodules.

BACKGROUND: Activating mutations of the oncogene BRAF or rearrangements of the tyrosine kinase receptor RET are observed in up to 80% of papillary thyroid carcinomas (PTCs). The predominant BRAF V600E mutation has not been detected in benign thyroid tissue so far, so consequently, this assumedly pathognomonic alteration is qualified to improve the preoperative diagnosis of PTC. METHODS: Two hundred ninety preoperatively harvested fine-needle aspiration biopsies (FNABs) underwent routine cytologic assessment. BRAF V600E mutation analysis was performed by mutation-specific PCR using the same cell material; a hybrid-specific RT-PCR assay was used for detection of RET/PTC1 rearrangements. Detected genetic alterations were verified by direct sequencing. Definitive histopathology was obtained in 93/290 lesions following surgery of the respective thyroid nodule. RESULTS: While cytology alone diagnosed 13/30 malignancies (22 PTCs, 4 FTCs, 1 MTC, 1 UTC, 2 metastases), five additional malignancies were identified by supplementary mutation analysis. Cytology classified eight FNABs as benign, while postoperative histology demonstrated a thyroid malignancy (6 PTCs, 1 FTC, 1 metastasis). In four of these eight cases, the genetic analysis detected a BRAF V600E mutation or a RET/PTC1 rearrangement. Classifying both suspicious and malignant FNAB results as positive cytology results, supplementary genetic testing increased the overall sensitivity of FNAB from 70.4 to 85.7%, the positive predictive value (PPV) from 59.4 to 64.9%, and the negative predictive value (NPV) from 84.0 to 91.3%. CONCLUSIONS: Supplementary mutation analysis of RET and especially of the BRAF V600E mutation in FNABs is a fast and probably cost-effective assay in routine diagnostic setting. Mutation analyses of PTC-specific genetic alterations improve the preoperative identification and prognostic assessment of thyroid malignancies and therefore enable an optimized surgical strategy.

Impact of pathognomonic genetic alterations on the prognosis of papillary thyroid carcinoma. ESES vienna presentation.

INTRODUCTION: BRAF mutations and RET or NTRK1 rearrangements were identified as causing events that drive the malignant transformation of the thyroid follicular cell. The impact of these alterations on the course of papillary thyroid carcinoma (PTC) is still unsettled. PATIENTS AND METHODS: Tumor tissues of 290 (98 male, 192 female) patients were intra-operatively snap frozen or harvested from archival paraffin-embedded blocks and used for extraction of DNA and RNA. Comprehensive analysis of RET/PTC and NTRK1 rearrangements was carried out by multiplex screening RT-PCR, hybrid-specific RT-PCR and sequencing of detected hybrids. A mutation-specific PCR was used for BRAF analysis. RESULTS: The BRAF V600E mutation was detected in 122/290 (42%), RET rearrangements in 20/137 (14.6%), and NTRK1 rearrangements in 15/93 (16.1%) PTCs. One hundred forty one out of 290 (48.6%) PTCs demonstrated none of the genetic alterations studied. Eight PTCs expressed two different mutations (1 RET/PTC + BRAF, 6 NTRK1 + BRAF, 1 RET/PTC + NTRK1). Tumor-specific survival analysis (mean follow-up, 5.5 years) demonstrated no significant difference, but a tendency toward worse prognosis of BRAF-positive patients compared to BRAF-negative patients or rearrangement-positive patients, respectively. CONCLUSION: Long-term follow-up data on large tumor panels are needed to disclose significant survival differences of prognostic predictors on PTC. This study provides further evidence that patients harboring BRAF-V600E-positive PTCs may experience an unfavorable course of the disease compared to patients with tumors carrying other genetic alterations.

Multiple modes of cell death in neuroendocrine tumors induced by artesunate.

BACKGROUND: The paucity of effective treatment in neuroendocrine tumors (NETs) encouraged us to investigate the therapeutic value of artesunate (ART) promised by its inhibitory effect against various tumors and broad safety profile. METHODS: We evaluated the impact of ART on three NET cell lines, BON-1, QGP-1 and NCI-H727 on cellular and molecular levels. RESULTS: Our results showed that ART induced endoplasmic reticulum (ER) stress through phosphorylation of eIF2α, which further gave rise to autophagy in all three NET cell lines. Specifically, apoptosis and ferroptosis were also observed in BON-1 cells, which made BON-1 cell line more vulnerable upon ART treatment. The different sensitivities presented on the three cell lines also associated with a differential regulation of p21 on the long run. Co-treatment with p21 inhibitor UC2288 showed an additive effect on QGP-1 and NCI-H727 cell lines indicating p21 upregulation in these two cell lines might confer resistance towards ART treatment. CONCLUSIONS: It is possible to include ART in the treatment of NETs in the future.

Identification and prevention of genotyping errors caused by G-quadruplex- and i-motif-like sequences.

BACKGROUND: Reliable PCR amplification of DNA fragments is the prerequisite for most genetic assays. We investigated the impact of G-quadruplex- or i-motif-like sequences on the reliability of PCR-based genetic analyses. METHODS: We found the sequence context of a common intronic polymorphism in the MEN1 gene (multiple endocrine neoplasia I) to be the cause of systematic genotyping errors by inducing preferential amplification of one allelic variant [allele dropout (ADO)]. Bioinformatic analyses and pyrosequencing-based allele quantification enabled the identification of the underlying DNA structures. RESULTS: We showed that G-quadruplex- or i-motif-like sequences can reproducibly cause ADO. In these cases, amplification efficiency strongly depends on the PCR enzyme and buffer conditions, the magnesium concentration in particular. In a randomly chosen subset of candidate single-nucleotide polymorphisms (SNPs) defined by properties deduced from 2 originally identified ADO cases, we confirmed preferential PCR amplification in up to 50% of the SNPs. We subsequently identified G-quadruplex and i-motifs harboring a SNP that alters the typical motif as the cause of this phenomenon, and a genomewide search based on the respective motifs predicted 0.5% of all SNPs listed by dbSNP and Online Mendelian Inheritance in Man to be potentially affected. CONCLUSIONS: Undetected, the described phenomenon produces systematic errors in genetic analyses that may lead to misdiagnoses in clinical settings. PCR products should be checked for G-quadruplex and i-motifs to avoid the formation of ADO-causing secondary structures. Truly affected assays can then be identified by a simple experimental procedure, which simultaneously provides the solution to the problem.

Peptide Receptor Radionuclide Therapy Combined With Chemotherapy in Patients With Neuroendocrine Tumors.

PURPOSE: Combinations of therapies may enhance therapeutic effects without significantly increasing the incidence of adverse events. However, there are few data regarding survival after concomitant chemotherapy and peptide receptor radionuclide therapy (PRRT) with [Lu]Lu-octreotate in patients with neuroendocrine tumors (NETs). Thus, we explored the outcome of this combination of therapies. METHODS: Fifteen patients with somatostatin receptor-positive, rapidly progressive G2/G3 NETs during chemotherapy or PRRT alone from 2 German cancer centers were included in the retrospective analysis. The patients received a combination of PRRT and chemotherapy with temozolomide (n = 3) or temozolomide plus capecitabine (n = 12). To evaluate the effects of the combined treatment, we assessed the responses, survival, and adverse events. RESULTS: The cumulative administered activity of [Lu]Lu-octreotate had a median of 21.3 GBq after 3 cycles of combination therapy. The patients exhibited a median progression-free survival of 7.1 months and a median overall survival of 25.3 months. The clinical benefit (objective response and stable disease) rates were as follows: 55% of patients according to CT, 38% in [F]F-FDG PET/CT, and 44% in [Ga]Ga-DOTATOC PET/CT. One patient with rapidly progressing liver metastases experienced grade 4 liver failure according to the Common Terminology Criteria for Adverse Events (version 5.0). Four other patients (27%) experienced significantly elevated (grade 3) liver parameters. CONCLUSIONS: According to different imaging modalities, the combination of PRRT and temozolomide +/- capecitabine led to disease control in 38% to 55% of the progressive NETs after PRRT or chemotherapy alone failed. The overall survival in this extensively pretreated group of patients was nearly 25 months. The majority of patients did not experience any serious adverse events.

PD-L1 Expression and Immune Cell Infiltration in Gastroenteropancreatic (GEP) and Non-GEP Neuroendocrine Neoplasms With High Proliferative Activity.

The potential of neuroendocrine neoplasms (NEN) to respond to checkpoint inhibitors is largely unknown and full of great expectations. Immunohistochemical (IHC) studies of programmed cell death ligand 1 (PD-L1) expression in the tumor microenvironment and its implications in predicting the response to checkpoint inhibition is a very active subject. Currently, the combined analysis of PD-L1 expression and tumor-associated immune cell (TAIC) infiltration is considered the best predictive marker of therapeutic response. Here we investigated the expression of PD-L1 on tumor cells (TC) and tumor-infiltrating immune cells (IC) by IHC in 68 NEN samples with a high proliferation rate (Ki-67 >20%) from 57 patients and in 22 samples we correlated it with TAIC density by assessing intratumoral infiltration of CD3+, CD8+, and CD68+ cells. Furthermore, the tumor microenvironment was evaluated according to the classification of Teng et al. We detected PD-L1 expression in 31.6% of NEN G3. Its expression usually was weak and more IC than TC expressed PD-L1. The proportion of tumors positive for PD-L1 was comparable in NEN from different sites of origin but varied depending on tumor differentiation and disease extension. No positive IHC staining was found in 3 well-differentiated neuroendocrine tumors (NETs) with a proliferation rate above 20% (NET G3). When analyzing TAIC, we rarely (18.2%) detected intratumoral CD8+ cells, whereas infiltration by CD3+ and CD68+ cells was more common (45.5 and 59.1%, respectively). By combining CD3+ cells and PD-L1 status, we identified the immune ignorant phenotype of tumor microenvironment as being the most common phenotype, supporting the concept of a preferably combined immunotherapeutic approach in neuroendocrine carcinoma (NEC).

Somatostatin-producing neuroendocrine tumors of the duodenum and pancreas: incidence, types, biological behavior, association with inherited syndromes, and functional activity.

Somatostatin-producing neuroendocrine tumors (SOM-NETs) of the duodenum and pancreas appear to be heterogeneous. To determine their clinicopathological profiles, respective data were analyzed on a series of 82 duodenal and 541 pancreatic NETs. In addition, the clinical records of 821 patients with duodenal or pancreatic NETs were reviewed for evidence of a somatostatinoma syndrome. Predominant or exclusive expression of somatostatin was found in 21 (26%) duodenal and 21 (4%) pancreatic NETs. They were classified as sporadic (n=31) or neurofibromatosis type 1 (NF1)-associated duodenal NETs (n=3), gangliocytic paragangliomas (GCPGs; n=6), or poorly differentiated neuroendocrine carcinomas (pdNECs; n=2). In addition, five duodenal and four pancreatic SOM-NETs were found in five patients with multiple endocrine neoplasia type 1 (MEN1). Metastases occurred in 13 (43%) patients with sporadic or NF1-associated SOM-NETs, but in none of the duodenal or pancreatic MEN1-associated SOM-NETs or GCPGs. Sporadic advanced (stage IV) SOM-NETs were more commonly detected in the pancreas than in the duodenum. None of the patients (including the 821 patients for whom only the clinical records were reviewed) fulfilled the criteria of a somatostatinoma syndrome. Our data show that somatostatin expression is not only seen in sporadic NETs but may also occur in GCPGs, pdNECs, and hereditary NETs. Surgical treatment is effective in most duodenal and many pancreatic SOM-NETs. MEN1-associated SOM-NETs and GCPGs follow a benign course, while somatostatin-producing pdNECs are aggressive neoplasms. The occurrence of the so-called somatostatinoma syndrome appears to be extremely uncommon.

Immune Checkpoint Inhibitors in the Treatment of Patients with Neuroendocrine Neoplasia.

BACKGROUND: Well-differentiated neuroendocrine neoplasms (NENs) are usually controlled by antiproliferative, local ablative and/or radionuclide therapies, whereas poorly differentiated NENs generally require cytotoxic chemotherapy. However, treatment options for patients with advanced/metastatic high-grade NENs remain limited. METHOD: Review of the literature and international congress abstracts on the efficacy and safety of immunotherapy by checkpoint inhibition in advanced/metastatic NENs. RESULTS: Evidence points to an important role of immune phenomena in the pathogenesis and treatment of neuroendocrine tumors (NETs). Programmed cell death 1 (PD-1) protein and its ligand are mainly expressed in poorly differentiated NENs. Microsatellite instability and high mutational load are more pronounced in high-grade NENs and may predict response to immunotherapy. Clinical experience of immune checkpoint blockade mainly exists for Merkel cell carcinoma, a high-grade cutaneous neuroendocrine carcinoma (NEC), which has led to approval of the anti-PD-1 antibody avelumab. In addition, there is anecdotal evidence for the efficacy of checkpoint inhibitors in large-cell lung NECs, ovarian NECs and others, including gastroenteropancreatic NENs. Currently, phase II studies investigate PDR001, pembrolizumab, combined durvalumab and tremelimumab, and avelumab treatment in patients with advanced/metastatic NENs. CONCLUSION: Immune checkpoint inhibitors are a promising therapeutic option, especially in progressive NECs or high-grade NETs with high tumor burden, microsatellite instability, and/or mutational load.

The impact of the IGF-1 system of cancer cells on radiation response - An in vitro study.

BACKGROUND: Overexpression of the insulin-like growth factor-1 receptor (IGF-1R) is associated with increased cell proliferation, differentiation, transformation, and tumorigenicity. Additionally, signaling involved in the resistance of cancer cells to radiotherapy originates from IGF-1R. The purpose of this study was to investigate the role of the IGF-1 system in the radiation response and further evaluate its effect on the expression of DNA repair pathway genes. METHODS: To inhibit the IGF-1 system, we stably transfected the Caco-2 cell line to express a kinase-deficient IGF-1R mutant. We then studied the effects of this mutation on cell growth, the response to radiation, and clonogenic survival, as well as using a cell viability assay to examine DNA damage and repair. Finally, we performed immunofluorescence for γ-H2AX to examine double-strand DNA breaks and evaluated the expression of 84 key genes involved in DNA repair with a real-time PCR array. RESULTS: Mutant IGF-1R cells exhibited significantly blunted cell growth and viability, compared to wild-type cells, as well as reduced clonogenic survival after γ-irradiation. However, mutant IGF-1R cells did not show any significant delays in the repair of radiation-induced DNA double-strand breaks. Furthermore, expression of mutant IGF-1R significantly down-regulated the mRNA levels of BRCA2, a major protein involved in homologous recombination DNA repair. CONCLUSION: These results indicate that blocking the IGF-1R-mediated signaling cascade, through the expression of a kinase-deficient IGF-1R mutant, reduces cell growth and sensitizes cancer cells to ionizing radiation. Therefore, the IGF-1R system could be a potential target to enhance radio-sensitivity and the efficacy of cancer treatments.

Interdisciplinary treatment of diabetic foot wounds in the elderly: Low risk of amputations and mortality and good chance of being mobile with good quality of life.

AIMS: A major proportion of patients with diabetic foot syndrome are older than 65 years. Little is known about outcomes of these elderly patients. METHODS: We analysed 245 treatment cases in an observational single-centre study for comorbidities and outcomes over a 6-month period. RESULTS: In all, 122 patients had peripheral arterial disease which was significantly increasing with age (n = 245, df = 1, χ2 = 23.06, p ⩽ 0.0001). Increasing age correlated positively with decreasing rate of revascularisations (n = 122, df = 1, χ2 = 4.23, p = 0.039). In total, 23 (9.3%) patients died in the observation period. In-hospital mortality was 2.8%, percentage of major amputations 2.8%. In the invasively treated subgroup, 13 out of 67 patients died within the observation period. Death after revascularisation was independent of age (n = 67, df = 1, χ2 = 2.05, p = 0.153). Mobility decreased in the whole study group with increasing age. The risk of decrease with age was not influenced by revascularisation status. CONCLUSION: With careful interdisciplinary evaluation, elderly patients with diabetic foot syndrome can be treated with favourable outcome. Mobility before and after treatment deserves more attention as a predictor of treatment success and outcome parameter.