Anja Schmitt

TNM Staging of Neoplasms of the Endocrine Pancreas: Results From a Large International Cohort Study

BACKGROUND Both the European Neuroendocrine Tumor Society (ENETS) and the International Union for Cancer Control/American Joint Cancer Committee/World Health Organization (UICC/AJCC/WHO) have proposed TNM staging systems for pancreatic neuroendocrine neoplasms. This study aims to identify the most accurate and useful TNM system for pancreatic neuroendocrine neoplasms. METHODS The study included 1072 patients who had undergone previous surgery for their cancer and for which at least 2 years of follow-up from 1990 to 2007 was available. Data on 28 variables were collected, and the performance of the two TNM staging systems was compared by Cox regression analysis and multivariable analyses. All statistical tests were two-sided. RESULTS Differences in distribution of sex and age were observed for the ENETS TNM staging system. At Cox regression analysis, only the ENETS TNM staging system perfectly allocated patients into four statistically significantly different and equally populated risk groups (with stage I as the reference; stage II hazard ratio [HR] of death = 16.23, 95% confidence interval [CI] = 2.14 to 123, P = .007; stage III HR of death = 51.81, 95% CI = 7.11 to 377, P < .001; and stage IV HR of death = 160, 95% CI = 22.30 to 1143, P < .001). However, the UICC/AJCC/WHO 2010 TNM staging system compressed the disease into three differently populated classes, with most patients in stage I, and with the patients being equally distributed into stages II-III (statistically similar) and IV (with stage I as the reference; stage II HR of death = 9.57, 95% CI = 4.62 to 19.88, P < .001; stage III HR of death = 9.32, 95% CI = 3.69 to 23.53, P = .94; and stage IV HR of death = 30.84, 95% CI = 15.62 to 60.87, P < .001). Multivariable modeling indicated curative surgery, TNM staging, and grading were effective predictors of death, and grading was the second most effective independent predictor of survival in the absence of staging information. Though both TNM staging systems were independent predictors of survival, the UICC/AJCC/WHO 2010 TNM stages showed very large 95% confidence intervals for each stage, indicating an inaccurate predictive ability. CONCLUSION Our data suggest the ENETS TNM staging system is superior to the UICC/AJCC/WHO 2010 TNM staging system and supports its use in clinical practice.

WHO 2004 criteria and CK19 are reliable prognostic markers in pancreatic endocrine tumors.

Background It is difficult to predict the biologic behavior of pancreatic endocrine tumors in absence of metastases or invasion into adjacent organs. The World Health Organization (WHO) has proposed in 2004 size, angioinvasion, mitotic activity, and MIB1 proliferation index as prognostic criteria. Our aim was to test retrospectively the predictive value of these 2004 WHO criteria and of CK19, CD99, COX2, and p27 immunohistochemistry in a large series of patients with long-term follow-up. Design The histology of 216 pancreatic endocrine tumor specimens was reviewed and the tumors were reclassified according to the 2004 WHO classification. The prognostic value of the WHO classification and the histopathologic criteria necrosis and nodular fibrosis was tested in 113 patients. A tissue microarray was constructed for immunohistochemical staining. The staining results were scored quantitatively for MIB1 and semiquantitatively for CK19, COX2, p27, and CD99. The prognostic value of these markers was tested in 93 patients. Results The stratification of the patients into 4 risk groups according to the 2004 WHO classification was reliable with regard to both time span to relapse and tumor-specific death. In a multivariate analysis, the CK19 status was shown to be independent of the WHO criteria. By contrast, the prognostic significance of COX2, p27, and CD99 could not be confirmed. Conclusions The 2004 WHO classification with 4 risk groups is very reliable for predicting both disease-free survival and the time span until tumor-specific death. CK19 staining is a potential additional prognostic marker independent from the WHO criteria for pancreatic endocrine tumors.

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.

Islet 1 (Isl1) expression is a reliable marker for pancreatic endocrine tumors and their metastases.

Background Tracing the origin of a metastasis of a neuroendocrine carcinoma is a challenge. The transcription factors Cdx2 and TTF1 have been found to be helpful in identifying well-differentiated neuroendocrine tumors of gastrointestinal and pulmonary origin, respectively. So far, such a marker is lacking for pancreatic neuroendocrine tumors (PETs) and metastases thereof. Islet1 (Isl1) is a transcription factor expressed in pancreatic islet cells. The aim of this study was (1) to test the specificity and sensitivity of Isl1 as a marker of PETs, and (2) to test the specificity and sensitivity of a panel of markers, including Isl1, Cdx2, and TTF1, for the localization of the primary. Design One hundred eighty-eight primary gastroenteropancreatic and pulmonary endocrine tumors and 49 metastases thereof were examined. Immunohistochemistry using antibodies directed against Isl1, Cdx2, and TTF1 was performed and the staining results were scored semiquantitatively. Results Isl1 proved to be a highly specific marker for pancreatic endocrine tumors. In 84 primary PET its specificity was 78.4% (sensitivity 74.3%) and in 18 metastases of PET the specificity reached 100% (sensitivity 77.8%). Strong Cdx2 staining showed a specificity for gastrointestinal origin of 83.9% (sensitivity 82%) in primary tumors and of 100% (sensitivity 40%) in metastases. Including weakly positive tumors lead to a decreased specificity but an increased sensitivity. TTF1 expression was detected in 2 PET and 1 ileal primary tumor only and was absent in all metastases of gastroenteropancreatic endocrine tumors. Conclusions Isl1 is a reliable marker of pancreatic endocrine tumors and metastases thereof. It shows a comparable sensitivity and specificity as Cdx2 as a marker of ileal and appendiceal neuroendocrine tumors and their metastases. TTF1 is very rarely expressed in well-differentiated gastroentero-PETs. Therefore, the panel of Isl1, Cdx2, and TTF1 seems useful for examining metastases of well-differentiated endocrine carcinomas of unknown origin.

Hereditary neuroendocrine tumors of the gastroenteropancreatic system

Approximately 5–10% of neuroendocrine tumors (NETs) of the gastroenteropancreatic system (GEP) have a hereditary background. The known inherited syndromes include multiple endocrine neoplasia type 1, neurofibromatosis type 1, von Hippel–Lindau disease, and the tuberous sclerosis complex. This review discusses for each of these syndromes the: (1) involved genes and specific types of mutations, (2) disease prevalence and penetrance, (3) affected neuroendocrine tissues and related clinical syndromes, (4) special morphological features of NETs and their putative precursor lesions. In addition, GEP-NETs clustering in individual families or associated with other malignancies without known genetic background are discussed.

IGFII and MIB1 immunohistochemistry is helpful for the differentiation of benign from malignant adrenocortical tumours

Aims:  The differentiation of adrenocortical carcinomas from adenomas may be difficult based on morphology alone. Differential expression of insulin‐like growth factor (IGF) II and cyclin‐dependent kinase (CDK) 4 has recently been described in these tumours. The aim of this study was to investigate the diagnostic usefulness of these markers immunohistochemically.

VHL inactivation is an important pathway for the development of malignant sporadic pancreatic endocrine tumors

A small subset of familial pancreatic endocrine tumors (PET) arises in patients with von Hippel-Lindau syndrome and these tumors may have an adverse outcome compared to other familial PET. Sporadic PET rarely harbors somatic VHL mutations, but the chromosomal location of the VHL gene is frequently deleted in sporadic PET. A subset of sporadic PET shows active hypoxia signals on mRNA and protein level. To identify the frequency of functionally relevant VHL inactivation in sporadic PET and to examine a possible prognostic significance we correlated epigenetic and genetic VHL alterations with hypoxia signals. VHL mutations were absent in all 37 PETs examined. In 2 out of 35 informative PET (6%) methylation of the VHL promoter region was detected and VHL deletion by fluorescence in situ hybridization was found in 14 out of 79 PET (18%). Hypoxia inducible factor 1alpha (HIF1-alpha), carbonic anhydrase 9 (CA-9), and glucose transporter 1 (GLUT-1) protein was expressed in 19, 27, and 30% of the 152 PETs examined. Protein expression of the HIF1-alpha downstream target CA-9 correlated significantly with the expression of CA-9 RNA (P<0.001), VHL RNA (P<0.05), and VHL deletion (P<0.001) as well as with HIF1-alpha (P<0.005) and GLUT-1 immunohistochemistry (P<0.001). These PET with VHL alterations and signs of hypoxia signalling were characterized by a significantly shortened disease-free survival. We conclude that VHL gene impairment by promoter methylation and VHL deletion in nearly 25% of PET leads to the activation of the HIF-pathway. Our data suggest that VHL inactivation and consecutive hypoxia signals may be a mechanism for the development of sporadic PET with an adverse outcome.

Allelic deletion of the MEN1 gene in duodenal gastrin and somatostatin cell neoplasms and their precursor lesions

Background: Patients with a multiple endocrine neoplasia type 1 (MEN1)-associated Zollinger–Ellison syndrome (ZES) show multifocal duodenal gastrinomas and precursor lesions. Aims: To test these lesions for loss of heterozygosity (LOH) of the MEN1 gene locus on chromosome 11q13, and to investigate whether the MEN1-related endocrine cell changes also involved somatostatin cells. Material and methods: Tissue specimens from six patients with MEN1 and ZES were analysed by immunohistochemistry and immunofluorescence. LOH analysis was performed by fluorescence in situ hybridisation (FISH), using probes containing the MEN1 gene locus and the centromere 11 (C11) region. For simultaneous analysis of hormones and allelic deletions, a combined FISH/immunofluorescence protocol was established. Results: 28 of a total of 33 duodenal neuroendocrine tumours (NETs) were gastrin-producing tumours; 13/28 (46.4%) revealed LOH on 11q13 and/or C11. Five of the NETs were somatostatin-expressing tumours, two revealing LOH. Allelic loss was detected in tumours as small as 300 μm (gastrin) and 400 μm (somatostatin) in diameter. The gastrin-producing tumours showed different deletion/retention patterns. Hyperplastic somatostatin cell lesions, similar to those of the gastrin cells, were present in all patients. The hyperplastic lesions of both cell lines consistently retained both 11q13 alleles. Conclusions: Allelic deletion of the MEN1 gene may reflect a pivotal event in the development of multifocal gastrin and somatostatin cell neoplasms in the duodenum of patients with MEN1. The observation of distinct deletion patterns in small synchronous tumours supports the concept that each gastrin-producing tumour in an individual MEN1 patient arises from an independent cell clone.

Pancreatic endocrine tumors are a rare manifestation of the neurofibromatosis type 1 phenotype: molecular analysis of a malignant insulinoma in a NF-1 patient.

The tumorigenesis of sporadic endocrine tumors is still not fully understood. It is well known that patients with von Recklinghausen syndrome (NF-1) (OMIM 162200) carrying NF1 germline mutations are predisposed to endocrine tumors including pheochromocytomas and duodenal somatostatinomas. It is unclear, however, whether the rarely reported occurrence of pancreatic insulinomas in NF-1 patients represents a coincidental finding or whether insulinomas are a rare manifestation of the NF-1 syndrome. To determine the potential association between the NF-1 syndrome and pancreatic endocrine tumors, we analyzed a NF-1 patient with a well-differentiated pancreatic endocrine carcinoma for NF1 mutation, allelic loss of the NF1 gene and its expression in peripheral blood and tumor cells. The germline mutation c. 499 del TGTT known in the family was confirmed by polymerase chain reaction (PCR) and direct sequencing of exon 4 in DNA extracted from peripheral blood. Loss of heterozygosity (LOH) analysis of the NF1 gene was carried out using 3 intragenic microsatellite markers on 17q11.2. RNA expression was examined by reverse transcription and a consecutive PCR spanning intron 3 of the NF1 gene including the mutated site in exon 4. Immunohistochemistry was used to analyze NF-1 protein expression. Mutation analysis of peripheral blood leukocytes confirmed the 4 base pair deletion in exon 4 starting at codon 167 (499 del TGTT). LOH analysis of tumor tissue revealed retention of both NF1 alleles. While reverse transcriptase-PCR of peripheral blood showed bi-allelic expression of both the wild-type NF1 and the mutated form, reverse transcriptase-PCR of tumor extracts demonstrated expression of the mutated but not the wild-type NF1 allele. Additionally, neurofibromin, the NF1 gene product, was absent in the tumor tissue of the NF-1 patient. These results show that the wild-type NF1 transcrips and protein are reduced, in the reported insulinoma, supposedly by epigenetic mechanisms. This provides strong evidence that there is a relationship between von Recklinghausen disease and the patients insulinoma. In this line, insulinomas may be viewed as a rare manifestation of the NF-1 syndrome. Furthermore, the NF1 gene must be considered as a candidate tumor suppressor gene for sporadic insulinomas and probably other pancreatic endocrine tumors.

ISL1 expression is not restricted to pancreatic well-differentiated neuroendocrine neoplasms, but is also commonly found in well and poorly differentiated neuroendocrine neoplasms of extrapancreatic origin

The human insulin gene enhancer-binding protein islet-1 (ISL1) is a transcription factor involved in the differentiation of the neuroendocrine pancreatic cells. Recent studies identified ISL1 as a marker for pancreatic well-differentiated neuroendocrine neoplasms. However, little is known about ISL1 expression in pancreatic poorly differentiated and in extrapancreatic well and poorly differentiated neuroendocrine neoplasms. We studied the immunohistochemical expression of ISL1 in 124 neuroendocrine neoplasms. Among pancreatic neuroendocrine neoplasms, 12/13 with poor differentiation were negative, whereas 5/7 with good differentiation but a Ki67 >20% were positive. In extrapancreatic neuroendocrine neoplasms, strong positivity was found in Merkel cell carcinomas (25/25), pulmonary small cell neuroendocrine carcinomas (21/23), medullary thyroid carcinomas (9/9), paragangliomas/pheochromocytomas (6/6), adrenal neuroblastomas (8/8) and head and neck neuroendocrine carcinomas (4/5), whereas no or only weak staining was recorded in pulmonary carcinoids (3/15), olfactory neuroblastomas (1/4) and basaloid head and neck squamous cell carcinomas (0/15). ISL1 stained the neuroendocrine carcinoma component of 5/8 composite carcinomas and also normal neuroendocrine cells in the thyroid, adrenal medulla, stomach and colorectum. Poorly differentiated neuroendocrine neoplasms, regardless of their ISL1 expression, were usually TP53 positive. Our results show the almost ubiquitous expression of ISL1 in extrapancreatic poorly differentiated neuroendocrine neoplasms and neuroblastic malignancies and its common loss in pancreatic poorly differentiated neuroendocrine neoplasms. These findings modify the role of ISL1 as a marker for pancreatic neuroendocrine neoplasms and suggest that ISL1 has a broader involvement in differentiation and growth of neuroendocrine neoplasms than has so far been assumed.