Christian Fottner

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-β-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-β-Glc)-PEG-Tyr³-octreotate and 1.23 for SiFA-Asn(AcNH-β-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-β-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.

Insulin-like growth factor binding protein 2 (IGFBP-2) separates hypertrophic and hyperplastic effects of growth hormone (GH)/IGF-I excess on adrenocortical cells in vivo

GH and IGF‐I are capable of inducing cellular hypertrophy and/or hyperplasia. Chronic overexpression of GH in transgenic mice results in systemically and locally increased IGF‐I levels and in disproportionate overgrowth, including adrenocortical enlargement and corticosterone hypersecretion. Using PEPCK‐bovine GH transgenic (G) mice, we demonstrate that adrenal enlargement involves both hypertrophy (44%) and hyperplasia (50%) of zona fasciculata cells. To clarify whether IGFBP‐2 affected cell volume and number, we crossed hemizygous G mice with hemizygous CMV‐IGFBP‐2 transgenic (B) mice, generating G mice, B mice, GB double transgenic mice, and nontransgenic controls (C). The absolute weight of the adrenal glands was significantly increased in 5‐wk‐and 4‐month‐old G mice vs. C and B mice. IGFBP‐2 overexpression in GB mice reduced this effect of GH excess by 26% and 37% in 5‐wk‐and 4‐month‐old animals, respectively. GH‐induced hypertrophy of zona fasciculata cells was completely abolished by IGFBP‐2 overexpression in GB mice whereas hyperplasia was not affected. Basal and ACTH‐induced plasma corticosterone levels of 4‐month‐old G mice, but not of GB mice, were two‐to threefold increased compared with C mice. Plasma ACTH levels were similar in all groups. Our data show that IGFBP‐2 potently separates hypertrophic and hyperplastic effects of GH/IGF‐I excess on adrenocortical cells.—Hoeflich, A., Weber, M. M., Fisch, T., Nedbal, S., Fottner, C., Elmlinger, M. W., Wanke, R., Wolf, E. Insulin‐like growth factor binding protein 2 (IGFBP‐2) separates hypertrophic and hyperplastic effects of growth hormone (GH)/IGF‐I excess on adrenocortical cells in vivo. FASEB J. 16, 1721–1731 (2002)

The role of the insulin-like growth factor system in adrenocortical tumourigenesis

The insulin‐like growth factor (IGF) system plays a central role in the mechanism of transformation and tumourigenesis. Elevated levels of IGF‐II and IGF‐I have been found in adrenocortical carcinomas.

Diagnosis and Treatment of Pancreatic Metastases of a Papillary Thyroid Carcinoma

BACKGROUNDnApart from regional lymph node metastases, systemic metastases occur sporadically in papillary thyroid carcinomas (PTC). The lung and bones are the most frequent localizations. Additionally known but extremely rare locations are metastases of the skeletal muscles, ovaries, submandibular gland, sphenoidal sinus, brain, adrenals, and, as shown in only two previously published cases to date, the pancreas.nnnSUMMARYnIn this article we report about two additional patients with pancreatic metastases from PTC. There is almost no prior experience about therapeutic approaches to this type of metastases. In both patients distant metastases within the pancreas were successfully removed. Postoperative histology confirmed the diagnoses. Supplemental genetic analysis did not demonstrate a BRAF V600E mutation or expression of a RET/PTC1 rearrangement in one case, but revealed a BRAF V600E mutation in the second case. Surgery avoided impending complications maintaining quality of life. One patient had a tumor-specific survival of 42 months. The other patient has occult disease.nnnCONCLUSIONSnOur two patients benefited of a calculated aggressive surgical action. Thus, if low perioperative mortality and morbidity can be warranted, surgical measures are justifiable in selected cases.

DNA Double Strand Breaks as Predictor of Efficacy of the Alpha-Particle Emitter Ac-225 and the Electron Emitter Lu-177 for Somatostatin Receptor Targeted Radiotherapy

Rationale Key biologic effects of the alpha-particle emitter Actinium-225 in comparison to the beta-particle emitter Lutetium-177 labeled somatostatin-analogue DOTATOC in vitro and in vivo were studied to evaluate the significance of γH2AX-foci formation. Methods To determine the relative biological effectiveness (RBE) between the two isotopes (as - biological consequence of different ionisation-densities along a particle-track), somatostatin expressing AR42J cells were incubated with Ac-225-DOTATOC and Lu-177-DOTATOC up to 48 h and viability was analyzed using the MTT assay. DNA double strand breaks (DSB) were quantified by immunofluorescence staining of γH2AX-foci. Cell cycle was analyzed by flow cytometry. In vivo uptake of both radiolabeled somatostatin-analogues into subcutaneously growing AR42J tumors and the number of cells displaying γH2AX-foci were measured. Therapeutic efficacy was assayed by monitoring tumor growth after treatment with activities estimated from in vitro cytotoxicity. Results Ac-225-DOTATOC resulted in ED50 values of 14 kBq/ml after 48 h, whereas Lu-177-DOTATOC displayed ED50 values of 10 MBq/ml. The number of DSB grew with increasing concentration of Ac-225-DOTATOC and similarly with Lu-177-DOTATOC when applying a factor of 700-fold higher activity compared to Ac-225. Already 24 h after incubation with 2.5–10 kBq/ml, Ac-225-DOTATOC cell-cycle studies showed up to a 60% increase in the percentage of tumor cells in G2/M phase. After 72 h an apoptotic subG1 peak was also detectable. Tumor uptake for both radio peptides at 48 h was identical (7.5%ID/g), though the overall number of cells with γH2AX-foci was higher in tumors treated with 48 kBq Ac-225-DOTATOC compared to tumors treated with 30 MBq Lu-177-DOTATOC (35% vs. 21%). Tumors with a volume of 0.34 ml reached delayed exponential tumor growth after 25 days (44 kBq Ac-225-DOTATOC) and after 21 days (34 MBq Lu-177-DOTATOC). Conclusion γH2AX-foci formation, triggered by beta- and alpha-irradiation, is an early key parameter in predicting response to internal radiotherapy.

Identification and Prevention of Genotyping Errors Caused by G-Quadruplex– and i-Motif–Like Sequences

BACKGROUNDnReliable 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.nnnMETHODSnWe 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.nnnRESULTSnWe 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.nnnCONCLUSIONSnUndetected, 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.

Phenotypic variability and risk of malignancy in SDHC-linked paragangliomas: lessons from three unrelated cases with an identical germline mutation (p.Arg133*).

CONTEXTnMutations in the four subunits of succinate dehydrogenase (SDH) are the cause for the hereditary paraganglioma (PGL) syndrome types 1-4 and are associated with multiple and recurrent pheochromocytomas and PGLs. SDHC mutations most frequently result in benign, nonfunctional head-and neck PGLs (HNPGLs). The malignant potential of SDHC mutations remains unclear to date.nnnOBJECTIVESnWe report a patient with malignant PGL carrying a SDHC mutation and compare her case with two others of the same genotype but presenting with classic benign HNPGLs. Loss of heterozygosity (LOH) was demonstrated in the malignant PGL tissue.nnnDESIGNnIn three unrelated patients referred for routine genetic testing, SDHB, SDHC, and SDHD genes were sequenced, and gross deletions were excluded by multiplex ligation-dependent probe amplification (MLPA). LOH was determined by pyrosequencing-based allele quantification and SDHB immunohistochemistry.nnnRESULTSnIn a patient with a nonfunctioning thoracic PGL metastatic to the bone, the lungs, and mediastinal lymph nodes, we detected the SDHC mutation c.397C>T predicting a truncated protein due to a premature stop codon (p.Arg133*). We demonstrated LOH and loss of SDHB protein expression in the malignant tumor tissue. The two other patients also carried c.397C>T, p.Arg133*; they differed from each other with respect to their tumor characteristics, but both showed benign HNPGLs.nnnCONCLUSIONSnWe describe the first case of a malignant PGL with distant metastases caused by a SDHC germline mutation. The present case shows that SDHC germline mutations can have highly variable phenotypes and may cause malignant PGL, although malignancy is probably rare.

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.

One single-time-point kidney uptake from OctreoScan correlates with number of desintegrations measured over 72 hours and calculated for the 6.7 hours half-life nuclide (177)Lu.

Aim In somatostatin receptor-targeted therapy, renal toxicity is an expected side effect, and therefore pretherapeutic dosimetry based on a measured kinetics is preferable. In contrast, a convenient one single-time-point scan might also reveal relevant information on expected dose to organs. However, very early time points might not reflect the true retention by the renal cortex and therefore be of limited value to predict dose for the long-lived 177Lu. Patients and Methods Dosimetry with 111In-octreotide was performed in 24 patients, and the number of disintegrations (ND) were calculated for 177Lu. Uptake values for each time point were correlated with ND. Results The fitting algorithm was best with biexponential equations in 18 patients. Mean biologic half-life of the alpha component was 6 hours (+/−12 hours) and for the beta component 82 hours (+/−38 hours). For the early time points, correlation with ND was generally poor. For later time points, correlation increased markedly after 4 hours (4 hours: r = 0.83, 72 hours: r = 0.93) and were also capable of predicting dosimetry to some extent. Conclusion In conclusion, thorough quantification of the 4 hours single-time-point scans seems to be enough to predict the expected renal dose for radionuclide therapies to some degree.

Medikamentöse Therapie neuroendokriner Neoplasien des Gastrointestinaltrakts

ZusammenfassungHintergrundNeuroendokrine Neoplasien (NEN) des Verdauungstrakts stellen eine seltene und heterogene Gruppe von Tumorerkrankungen dar. Dies erschwert die Vorgabe einheitlicher Therapieschemata.ZielDie aktuelle Datenlage zur medikamentösen Therapie gastrointestinaler NEN soll dargestellt werden.MethodenDiskussion publizierter Studien und Expertenempfehlungen zur medikamentösen Therapie von gastroenteropankreatischen neuroendokrinen Neoplasien (GEP-NEN)ErgebnisseWichtigstes therapeutisches Prinzip ist die komplette chirurgische Tumorentfernung. Ist diese nicht möglich, erfolgt die Therapie in der Regel multimodal und multidisziplinär und orientiert sich an der Tumorentität, dem individuellen Spontanverlauf sowie der Beschwerdesymptomatik. Bei Vorliegen eines spezifischen klinischen Hormonsyndroms sind langwirksame Somatostatinanaloga (SSA) die Therapie der Wahl. Sie stellen auch die Grundlage der antiproliferativen Therapie gut differenzierter gastroenteropankreatischer neuroendokriner Tumoren (GEP-NET) dar. Bei ausreichender Somatostatinrezeptor-Expression des Tumors steht als systemische Therapieoption die Peptidradiorezeptortherapie (PRRT) zur Verfügung. Bei gering differenzierten neuroendokrinen Karzinomen und NET des Pankreas stellen Platin- bzw. Streptozotocin-basierte Chemotherapien eine wichtige Therapieoption dar. Der Multityrosinkinaseinhibitor Sunitinib und der mTOR-Inhibitor Everolimus sind für die Behandlung pankreatischer NET zugelassen, Everolimus zudem auch für gastrointestinale NEN.SchlussfolgerungenZur Behandlung von GEP-NEN stehen eine Reihe effektiver therapeutischer Optionen zur Verfügung. Die Entscheidung für ein Therapiekonzept sollte individuell im Rahmen eines in der Behandlung dieser Tumoren erfahrenen interdisziplinären Tumorboards festgelegt werden.AbstractBackgroundNeuroendocrine neoplasms (NEN) of the digestive tract represent axa0rare and heterogeneous group of malignancies with various clinical presentations and prognoses, thus complicating standardized therapeutic recommendations.ObjectiveSummary of current clinical recommendations for the medicinal treatment of gastrointestinal NEN.MethodsDiscussion of published clinical studies and expert recommendations related to the medicinal therapy of gastroenteropancreatic NEN (GEP-NEN).ResultsThe primary therapeutic goal in GEP-NEN treatment is complete surgical tumor resection. If this is not possible, treatment is usually carried out in a multimodal and multidisciplinary concept, taking the respective tumor entity, the individual tumor biology and the clinical symptoms into account. In the case of axa0distinct clinical hormonal syndrome, long-acting somatostatin analogues (SSA) are the mainstay of symptomatic therapy. They also represent the foundations for the antiproliferative treatment of well-differentiated, slow growing GEP-NET. Systemic peptide receptor radionuclide therapy (PRRT) with Lu177DOTATATE is an effective therapy in GEP-NET with adequate somatostatin receptor expression. For poorly differentiated neuroendocrine carcinomas (NEC) and pancreatic NET, systemic chemotherapy with platinum or streptozotocin-based regimens shows good results. The multi-targeted tyrosine kinase inhibitor Sunitinib® is approved for pancreatic NET and the mTOR inhibitor Everolimus® for pancreatic and gastrointestinal NET.ConclusionThe therapeutic landscape of GEP-NETs has evolved significantly in recent years; however, navigating the current therapeutic algorithm may be challenging. The determination of axa0specific therapeutic concept should be made on an individual basis after discussion of all therapeutic options in axa0multidisciplinary team of NET specialists.