Annika Blank

Prognostic and Predictive Roles of MGMT Protein Expression and Promoter Methylation in Sporadic Pancreatic Neuroendocrine Neoplasms

Background/Aims: O6-methylguanine-methyltransferase (MGMT) is an important enzyme of DNA repair. MGMT promoter methylation is detectable in a subset of pancreatic neuroendocrine neoplasms (pNEN). A subset of pNEN responds to the alkylating agent temozolomide (TMZ). We wanted to correlate MGMT promoter methylation with MGMT protein loss in pNEN, correlate the findings with clinico-pathological data and determine the role of MGMT to predict response to TMZ chemotherapy. Methods: We analysed a well-characterized collective of 141 resected pNEN with median follow-up of 83 months for MGMT protein expression and promoter methylation using methylation-specific PCR (MSP). A second collective of 10 metastasized, pretreated and progressive patients receiving TMZ was used to examine the predictive role of MGMT by determining protein expression and promoter methylation using primer extension-based quantitative PCR. Results: In both collectives there was no correlation between MGMT protein expression and promoter methylation. Loss of MGMT protein was associated with an adverse outcome, this prognostic value, however, was not independent from grade and stage in multivariate analysis. Promoter hypermethylation was significantly associated with response to TMZ. Conclusion: Loss of MGMT protein expression is associated with adverse outcome in a surgical series of pNET. MGMT promoter methylation could be a predictive marker for TMZ chemotherapy in pNEN, but further, favourably prospective studies will be needed to confirm this result and before this observation can influence clinical routine.

SDHB loss predicts malignancy in pheochromocytomas/sympathethic paragangliomas, but not through hypoxia signalling

Prediction of malignant behaviour of pheochromocytomas/sympathetic paragangliomas (PCCs/PGLs) is very difficult if not impossible on a histopathological basis. In a familial setting, it is well known that succinate dehydrogenase subunit B (SDHB)-associated PCC/PGL very often metastasise. Recently, absence of SDHB expression as measured through immunohistochemistry was shown to be an excellent indicator of the presence of an SDH germline mutation in PCC/PGL. SDHB loss is believed to lead to tumour formation by activation of hypoxia signals. To clarify the potential use of SDHB immunohistochemistry as a marker of malignancy in PCC/PGL and its association with classic hypoxia signalling we examined SDHB, hypoxia inducible factor-1α (Hif-1α) and its targets CA-9 and GLUT-1 expression on protein level using immunohistochemistry on a tissue micro array on a series of familial and sporadic tumours of 115 patients. Survival data was available for 66 patients. SDHB protein expression was lost in the tumour tissue of 12 of 99 patients. Of those 12 patients, 5 had an SDHB germline mutation, in 4 patients no germline mutation was detected and mutational status remained unknown in parts in 3 patients. Loss of SDHB expression was not associated with increased classic hypoxia signalling as detected by Hif-1α, CA-9 or GLUT-1 staining. Loss of SDHB expression was associated with an adverse outcome. The lack of correlation of SDHB loss with classic hypoxia signals argues against the current hypoxia hypothesis in malignant PCC/PGL. We suggest SDHB protein loss as a marker of adverse outcome both in sporadic and in familial PCC/PGL.

Glucagon-like-peptide-1 receptor expression in normal and diseased human thyroid and pancreas

Glucagon-like-peptide-1 (GLP1) analogs may induce thyroid or pancreatic diseases in animals, raising questions about their use in diabetic patients. There is, however, controversy regarding expression of GLP1 receptors (GLP1R) in human normal and diseased thyroid and pancreas. Here, 221 human thyroid and pancreas samples were analyzed for GLP1R immunohistochemistry and compared with quantitative in vitro GLP1R autoradiography. Neither normal nor hyperplastic human thyroids containing parafollicular C cells express GLP1R with either method. Papillary thyroid cancer do not, and medullary thyroid carcinomas rarely express GLP1R. Insulin- and somatostatin-producing cells in the normal pancreas express a high density of GLP1R, whereas acinar cells express them in low amounts. Ductal epithelial cells do not express GLP1R. All benign insulinomas express high densities of GLP1R, whereas malignant insulinomas rarely express them. All ductal pancreatic carcinomas are GLP1R negative, whereas 6/20 PanIN 1/2 and 0/12 PanIN 3 express GLP1R. Therefore, normal thyroid, including normal and hyperplastic C cells, or papillary thyroid cancer are not targets for GLP1 analogs in humans. Conversely, all pancreatic insulin- and somatostatin-producing cells are physiological GLP1 targets, as well as most acini. As normal ductal epithelial cells or PanIN 3 or ductal pancreatic carcinomas do not express GLP1R, it seems unlikely that GLP1R is related to neoplastic transformation in pancreas. GLP1R-positive medullary thyroid carcinomas and all benign insulinomas are candidates for in vivo GLP1R targeting.

Autophagy inhibition improves sunitinib efficacy in pancreatic neuroendocrine tumors via a lysosome-dependent mechanism.

Increasing the efficacy of approved systemic treatments in metastasized pancreatic neuroendocrine tumors (PanNET) is an unmet medical need. The antiangiogenic tyrosine kinase inhibitor sunitinib is approved for PanNET treatment. In addition, sunitinib is a lysosomotropic drug and such drugs can induce lysosomal membrane permeabilization as well as autophagy. We investigated sunitinib-induced autophagy as a possible mechanism of PanNET therapy resistance. Sunitinib accumulated in lysosomes and induced autophagy in PanNET cell lines. Adding the autophagy inhibitor chloroquine reduced cell viability in cell lines and in primary cells isolated from PanNET patients. The same treatment combination reduced tumor burden in the Rip1Tag2 transgenic PanNET mouse model. The combination of sunitinib and chloroquine reduced recovery and induced apoptosis in vitro, whereas single treatments did not. Knockdown of key autophagy proteins in combination with sunitinib showed similar effect as chloroquine. Sunitinib also induced lysosomal membrane permeabilization, which further increased in the presence of chloroquine or knockdown of lysosome-associated membrane protein (LAMP2). Both combinations led to cell death. Our data indicate that chloroquine increases sunitinib efficacy in PanNET treatment via autophagy inhibition and lysosomal membrane permeabilization. We suggest that adding chloroquine to sunitinib treatment will increase efficacy of PanNET treatment and that such patients should be included in respective ongoing clinical trials. Mol Cancer Ther; 16(11); 2502–15. ©2017 AACR.

Histopathology of NET: Current concepts and new developments

The diagnosis of neuroendocrine tumors is based on their histopathologic appearance and immunohistochemical profile. With the WHO 2010 classification formal staging and grading was introduced for gastro-entero-pancreatic NET, however, the nomenclature for lung neuroendocrine tumors still relies on the carcinoid term. In this review we also focus on the situation of neuroendocrine carcinoma of unknown primary, tissue biomarkers and actual controversies in the histopathology of NEN.

New Genetics and Genomic Data on Pancreatic Neuroendocrine Tumors: Implications for Diagnosis, Treatment, and Targeted Therapies

The recent findings on the roles of death-associated protein 6/α-thalassemia/mental retardation X-linked (DAXX/ATRX) in the development of pancreatic neuroendocrine tumors (PanNETs) have led to major advances in the molecular understanding of these rare tumors and open up completely new therapeutic windows. This overview aims at giving a simplified view on these findings and their possible therapeutic implications. The importance of epigenetic changes in PanNET is also underlined by recent findings of a cross-species study on microRNA (miRNA) and messenger RNA (mRNA) profiles in PanNETs.

Pathology: Classification and Immunoprofile.

The classification of neuroendocrine neoplasms (NENs) has been evolving steadily over the last decades. Important prognostic factors of NENs are their proliferative activity and presence/absence of necrosis. These factors are reported in NENs of all body sites; however, the terminology as well as the exact rules of classification differ according to the location of the primary tumor. Only in gastroenteropancreatic (GEP) NENs a formal grading is performed. This grading is based on proliferation assessed by the mitotic count and/or Ki-67 proliferation index. In the lung, NEN grading is an intrinsic part of the tumor designation with typical carcinoids corresponding to neuroendocrine tumor (NET) G1 and atypical carcinoids to NET G2; however, the presence or absence of necrotic foci is as important as proliferation for the differentiation between typical and atypical carcinoids. Immunohistochemical markers can be used to demonstrate neuroendocrine differentiation. Synaptophysin and chromogranin A are, to date, the most reliable and most commonly used for this purpose. Beyond this, other markers can be helpful, for example in the situation of a NET metastasis of unknown primary, where a hormonal profile or a panel of transcription factors can give hints to the primary site. Many immunohistochemical markers have been shown to correlate with prognosis but are not used in clinical practice, for example cytokeratin 19 and KIT expression in pancreatic NETs. There is no predictive biomarker in use, with the exception of somatostatin receptor (SSTR) 2 expression for predicting the amenability of a tumor to in vivo SSTR targeting for imaging or therapy.

Therapeutic Targeting of Tumor-Associated Macrophages in Pancreatic Neuroendocrine Tumors.

Pancreatic neuroendocrine tumors (PNETs) represent a heterogeneous group of neuroendocrine neoplasms with varying biological behavior and response to treatment. Although targeted therapies have been shown to improve the survival for patients at advanced stage, resistance to current therapies frequently occurs during the course of therapy. Previous reports indicate that the infiltration of tumor‐associated macrophages (TAMs) in PNETs might correlate with tumor progression and metastasis formation. We aimed to evaluate the prognostic and functional impact of TAMs in human PNETs in vitro and in vivo and to investigate the effect of therapeutic targeting TAMs in a genetic PNET mouse model. TAM expression pattern was assessed immunohistochemically in human PNET tissue sections and a tissue‐micro‐array of PNET tumors with different functionality, stage, and grading. The effect of liposomal clodronate on TAM cell viability was analyzed in myeloid cell lines and isolated murine bone macrophages (mBMM). In vivo, RIP1Tag2 mice developing insulinomas were treated with liposomal clodronate or PBS‐Liposomes. Tumor progression, angiogenesis and immune cell infiltration were assessed by immunohistochemistry. In human, insulinomas TAM density was correlated with invasiveness and malignant behavior. Moreover, TAM infiltration in liver metastases was significantly increased compared to primary tumors. In vitro, Liposomal clodronate selectively inhibited the viability of myeloid cells and murine bone macrophages, leaving PNET tumor cell lines largely unaffected. In vivo, repeated application of liposomal clodronate to RIP1Tag2 mice significantly diminished the malignant transformation of insulinomas, which was accompanied by a reduced infiltration of F4/80‐positive TAM cells and simultaneously by a decreased microvessel density, suggesting a pronounced effect of clodronate‐induced myeloid depletion on tumor angiogenesis. Concomitant treatment with the antiangiogenic TKI sunitinib, however, did not show any synergistic effects with liposomal clodronate. TAMs are crucial for malignant transformation in human PNET and correlate with metastatic behavior. Pharmacological targeting of TAMs via liposomal clodronate disrupts tumor progression in the RIP1Tag2 neuroendocrine tumor model and was associated with reduced tumor angiogenesis. Based on these results, using liposomal clodronate to target proangiogenic myeloid cells could be employed as novel therapeutic avenue in highly angiogenic tumors such as PNET.

Therapeutic targeting of tumor-associated macrophages in pancreatic neuroendocrine tumors: TAMs mediate tumor progression in PNET

Pancreatic neuroendocrine tumors (PNETs) represent a heterogeneous group of neuroendocrine neoplasms with varying biological behavior and response to treatment. Although targeted therapies have been shown to improve the survival for patients at advanced stage, resistance to current therapies frequently occurs during the course of therapy. Previous reports indicate that the infiltration of tumor‐associated macrophages (TAMs) in PNETs might correlate with tumor progression and metastasis formation. We aimed to evaluate the prognostic and functional impact of TAMs in human PNETs in vitro and in vivo and to investigate the effect of therapeutic targeting TAMs in a genetic PNET mouse model. TAM expression pattern was assessed immunohistochemically in human PNET tissue sections and a tissue‐micro‐array of PNET tumors with different functionality, stage, and grading. The effect of liposomal clodronate on TAM cell viability was analyzed in myeloid cell lines and isolated murine bone macrophages (mBMM). In vivo, RIP1Tag2 mice developing insulinomas were treated with liposomal clodronate or PBS‐Liposomes. Tumor progression, angiogenesis and immune cell infiltration were assessed by immunohistochemistry. In human, insulinomas TAM density was correlated with invasiveness and malignant behavior. Moreover, TAM infiltration in liver metastases was significantly increased compared to primary tumors. In vitro, Liposomal clodronate selectively inhibited the viability of myeloid cells and murine bone macrophages, leaving PNET tumor cell lines largely unaffected. In vivo, repeated application of liposomal clodronate to RIP1Tag2 mice significantly diminished the malignant transformation of insulinomas, which was accompanied by a reduced infiltration of F4/80‐positive TAM cells and simultaneously by a decreased microvessel density, suggesting a pronounced effect of clodronate‐induced myeloid depletion on tumor angiogenesis. Concomitant treatment with the antiangiogenic TKI sunitinib, however, did not show any synergistic effects with liposomal clodronate. TAMs are crucial for malignant transformation in human PNET and correlate with metastatic behavior. Pharmacological targeting of TAMs via liposomal clodronate disrupts tumor progression in the RIP1Tag2 neuroendocrine tumor model and was associated with reduced tumor angiogenesis. Based on these results, using liposomal clodronate to target proangiogenic myeloid cells could be employed as novel therapeutic avenue in highly angiogenic tumors such as PNET.

Lean-Management im Pathologielabor

Der zunehmende Zeitund Effizienzdruck im Gesundheitswesenmacht auch vor der Pathologie nicht halt. Zusätzlich unterliegt der Beruf des Pathologen durch die äußeren Faktoren Automatisierung, Digitalisierung, Molekularbiologie undpersonalisierteMedizin, die auf den ersten Blick als eine Art „apokalyptische Reiter“ verstanden werden könnten, einem zunehmenden Entwicklungsund Definitionsdruck. Universitäre Institutionen sind zusätzlich durch die Zusatzaufgaben von Ausbildung und Forschung gefordert. Um sich diesen Herausforderungen zu stellen, gibt es viele zielführendeWege.DasPrinzipdesLeanManagements ist einer davon. Die Anwendung des Lean-Konzepts auf Arbeitsprozesse in der klinischen Pathologie zur Standardisierung, Fehlerreduktion und Minimierung von Verschwendung ist nach wie vor nicht weit verbreitet. Obwohl Patientenproben keine Karosseriebauteile sind, lässt sich derenAufarbeitung in vielenFällen so standardisieren und optimieren, dass das eigentliche Kernstück, die Diagnostik, relativ gesehen mehr zeitlichen Raum einnehmen kann, bei gleichzeitiger Verkürzung der Durchlaufzeit. Folgende Dinge gilt es vor Beschreitung des Lean-Pfades zu beachten: Das Ziel muss klar definiert sein, „Navigationstools“ zur Kursüberprüfung und ggf. Kursanpassung und ein Team, dass die UmsetzungvonLeanunterstützt,müssen vorhanden sein [4]. Eigenverantwortung