R.R. de Krijger

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.

Proliferation and differentiation in the human fetal endocrine pancreas

Summary The morphogenesis and growth of the endocrine pancreas has not been well investigated in man although it represents an important issue in diabetology. We examined human fetal pancreas from 12 to 41 weeks of gestation immunocytochemically to evaluate proliferative activity with the Ki-67 marker, and cytodifferentiation with cytokeratin 19 (ductal cells), synaptophysin (all endocrine cells), and insulin, glucagon, somatostatin and pancreatic polypeptide (islet cell types). Ki-67 labelling was found in all these cell types but was much higher in ductal cells than in islet cells. An intermediate population expressed synaptophysin but lacked islet hormones. With increasing gestational age the Ki-67 labelling index decreased from 17 to 4 % in ductal cells, from 9 to 1 % in synaptophysin-positive cells, and from 3 to 0.1 % in insulin- or glucagon-positive cells. From 12 to 16 weeks, all epithelial cells including the endocrine islet cells expressed cytokeratin 19. Thereafter cytokeratin 19 expression decreased and eventually disappeared from most islet cells, whereas strong expression remained in the ductal cells. We show that differentiated human islet cells have only very limited proliferative capacity, and we demonstrate the existence of transitional differentiation stages between ductal and islet cells. [Diabetologia (1997) 40: 398–404]

Congenital Diaphragmatic Hernia and Chromosome 15q26: Determination of a Candidate Region by Use of Fluorescent In Situ Hybridization and Array-Based Comparative Genomic Hybridization

Congenital diaphragmatic hernia (CDH) has an incidence of 1 in 3,000 births and a high mortality rate (33%-58%). Multifactorial inheritance, teratogenic agents, and genetic abnormalities have all been suggested as possible etiologic factors. To define candidate regions for CDH, we analyzed cytogenetic data collected on 200 CDH cases, of which 7% and 5% showed numerical and structural abnormalities, respectively. This study focused on the most frequent structural anomaly found: a deletion on chromosome 15q. We analyzed material from three of our patients and from four previously published patients with CDH and a 15q deletion. By using array-based comparative genomic hybridization and fluorescent in situ hybridization to determine the boundaries of the deletions and by including data from two individuals with terminal 15q deletions but without CDH, we were able to exclude a substantial portion of the telomeric region from the genetic etiology of this disorder. Moreover, one patient with CDH harbored a small interstitial deletion. Together, these findings allowed us to define a minimal deletion region of approximately 5 Mb at chromosome 15q26.1-26.2. The region contains four known genes, of which two--NR2F2 and CHD2--are particularly intriguing gene candidates for CDH.

Stem cell factor as a novel diagnostic marker for early malignant germ cells

Carcinoma in situ (CIS) of the testis is the pre‐invasive stage of type II testicular germ cell tumours (TGCTs) of adolescents and adults. These tumours are the most frequently diagnosed cancer in Caucasian adolescents and young adults. In dysgenetic gonads, the precursor of type II GCTs can be either CIS or a lesion known as gonadoblastoma (GB). CIS/GB originates from a primordial germ cell (PGC)/gonocyte, ie an embryonic cell. CIS can be cured by local low‐dose irradiation, with limited side effects on hormonal function. Therefore, strategies for early diagnosis of CIS are essential. Various markers are informative to diagnose CIS in adult testis by immunohistochemistry, including c‐KIT, PLAP, AP‐2γ, NANOG, and POU5F1 (OCT3/4). OCT3/4 is the most informative and consistent in presence and expression level, resulting in intense nuclear staining. In the case of maturational delay of germ cells, frequently present in gonads of individuals at risk for type II (T)GCTs, use of these markers can result in overdiagnosis of malignant germ cells. This demonstrates the need for a more specific diagnostic marker to distinguish malignant germ cells from germ cells showing maturation delay. Here we report the novel finding that immunohistochemical detection of stem cell factor (SCF), the c‐KIT ligand, is informative in this context. This was demonstrated in over 400 cases of normal (fetal, neonatal, infantile, and adult) and pathological gonads, as well as TGCT‐derived cell lines, specifically in cases of CIS and GB. Both membrane‐bound and soluble SCF were expressed, suggestive of an autocrine loop. SCF immunohistochemistry can be a valuable diagnostic tool, in addition to OCT3/4, to screen for precursor lesions of TGCTs, especially in patients with germ cell maturation delay. Copyright

Immunohistochemical detection of somatostatin receptor subtypes sst1 and sst2A in human somatostatin receptor positive tumors.

Although in situ hybridization has been used to examine the distribution of messenger RNA for somatostatin receptor subtypes (sst) in human tumors, the cellular localization of sst1 and sst2A receptors has not been reported. In this study, we describe the cellular localization of human sst1 and sst2A receptor proteins in both cryostat- and paraffin-embedded sections of 25 human tumor tissues using two recently developed polyclonal antibodies. Six somatostatin (SS) receptor (SSR) positive tumors (two gastrinomas, three carcinoids, one pheochromocytoma) and one SSR negative tumor (renal cell carcinoma), selected by positive and negative SSR autoradiography, respectively, were studied by both immunohistochemistry and Western blot analysis. The six SSR positive tumors expressed sst2A, while 4 of 5 expressed sst1 as well. The SSR negative tumor did not express either sst1 or sst2A. Western blot analysis of wheat germ agglutinin purified membrane proteins confirmed the presence of the sst1 and sst2A glycosylated receptors. The paraffin-embedded sections gave best information with respect to the subcellular localization. Sst1 immunoreactivity was observed both on the membrane and in the cytoplasm, while sst2A showed predominantly membrane-associated immunoreactivity. This subcellular distribution of sst1 or sst2A receptors was confirmed in paraffin-embedded sections of 8 additional intestinal carcinoids, 5 gastrinomas and 5 pheochromocytomas. Sst1 receptors were detected in 7 out of 8 carcinoids, in all gastrinomas, and in 4 out of 5 pheochromocytomas, while 6 out of 8 carcinoids, all gastrinomas, and 3 out of 5 pheochromocytomas expressed sst2A receptors. In conclusion, sst1 and sst2A receptors show a differential subcellular localization in human SSR positive tumors. The use of SSR subtype selective antibodies to detect the subcellular distribution of SSR subtypes in individual tumor cells is an important step forward to understand more about the pathophysiological role of the different SSR subtypes in human tumors.

The midgestational human fetal pancreas contains cells coexpressing islet hormones

In the fetal development of the mouse pancreas, endocrine cells have been found that express more than one hormone simultaneously. Our objective was to evaluate the existence of such cells in the human fetal pancreas. We found cells coexpressing two of the major pancreatic hormones (insulin, glucagon, and somatostatin) in sections of eight midgestational (12-18 weeks) pancreata and in 0-7% of cells in single-cell suspensions from midgestational pancreata. By electron microscopy, using granule morphology and immunoelectron microscopic techniques, we could confirm these findings and even detect cells containing three hormones. Morphologically different granules contained different immunoreactivities, suggesting parallel regulation of hormone production and packaging. In six newborn pancreata (born after 22-40 weeks of gestation), we could not find any multiple-hormone-containing cells. Subsequently, we evaluated whether multiple-hormone-containing cells proliferate by using pancreatic fragments and single-cell preparations at the light and electron microscopic level (six pancreata). No endocrine hormone-containing cells incorporated bromodeoxyuridine during a 1-hr culture period, indicating that these cells have lost the ability to proliferate under the conditions chosen. We conclude that, as in mice, the human fetal pancreas of 12-18 weeks of gestation contains endocrine cells that express multiple hormones simultaneously. These (multiple) hormone-containing cells do not seem to proliferate under basal conditions.

FOXL2 and SOX9 as parameters of female and male gonadal differentiation in patients with various forms of disorders of sex development (DSD)

The transcription factors SOX9 and FOXL2 are required for male and female mammalian gonadal development. We have used specific antibodies to investigate the role of these key proteins in disorders of sex development (DSD), specifically inter‐sex states. In normal gonads, SOX9 was found to be restricted to the presence of (pre‐)Sertoli cells, while FOXL2 was found in granulosa cells, and in stromal cells interpreted as early ovarian stroma. Both proteins were found within a single patient, when testicular and ovarian development was present; and within the same gonad, when both differentiation lineages were identified, as in ovotesticular DSD (ie hermaphrodite). Especially SOX9 was informative to support the presence of early testicular development (ie seminiferous tubules), expected based on morphological criteria only. In a limited number of DSD cases, FOXL2 was found within reasonably well‐developed seminiferous tubules, but double staining demonstrated that it was never strongly co‐expressed with SOX9 in the same cell. All seminiferous tubules containing carcinoma in situ (CIS), the malignant counterpart of a primordial germ cell, ie the precursor of type II germ cell tumours of the testis, seminomas and non‐seminomas, showed the presence of SOX9 and not FOXL2. In contrast, gonadoblastomas (GBs), the precursor of the same type of cancer, in a dysgenetic gonad, showed expression of FOXL2 and no, or only very low, SOX9 expression. These findings indicate that gonadal differentiation, ie testicular or ovarian, determines the morphology of the precursor of type II germ cell tumours, CIS or GB, respectively. We show that in DSD patients, the formation of either ovarian or/and testicular development can be visualized using FOXL2 and SOX9 expression, respectively. In addition, it initiates a novel way to study the role of the supportive cells in the development of either CIS or GB. Copyright

Germline mutations in the vhl gene in patients presenting with phaeochromocytomas

It has been shown that an appreciable percentage of patients presenting with primary, apparently sporadic phaeochromocytomas may in fact have von‐Hippel‐Lindau (VHL) disease. In order to investigate this, we retrospectively screened 68 patients, who had been operated on for phaeochromocytomas, for the presence of germline mutations in the vhl gene. DNA was isolated from peripheral‐blood leukocytes and used to screen the entire coding sequence and the intron‐exon boundaries of the vhl gene for mutations, using a PCR‐based SSCP strategy. When an abnormal pattern was found in the SSCP analysis, sequence analysis was carried out. We found SSC variants in the vhl gene in 8 of the 68 patients. Of 6 patients, 2 turned out to be related (an uncle and his nephew), and they carried the same mis‐sense mutation: R64P. In 4 other patients, mis‐sense mutations, P25L, L63P, G144Q and I147T, were also identified. None of these mutations has been described, and 3 of them (P25L, L63P and R64P) are located closer to the N terminus of the vhl protein than any reported vhl mutation. In the remaining 2 cases, the mutations were localized not in the coding sequence but in the intronic sequence (but not within splice‐sites), adjacent to the exon, so they were probably not related to the disease. Our results show that a relatively high proportion (6/68, or 8.8%), though not as high as the 20% reported earlier, of patients with apparently sporadic phaeochromocytomas may carry germline mutations in the vhl gene. Int. J. Cancer 77:337–340, 1998.

Phaeochromocytomas and sympathetic paragangliomas

About 24 per cent of phaeochromocytomas (PCCs) and sympathetic paragangliomas (sPGLs) appear in familial cancer syndromes, including multiple endocrine neoplasia type 2, von Hippel–Lindau disease, neurofibromatosis type 1 and PCC–paraganglioma syndrome. Identification of these syndromes is of prime importance for patients and their relatives. Surgical resection is the treatment of choice for both PCC and sPGL, but controversy exists about the management of patients with bilateral or multiple tumours.

The prognostic value of two different histopathological scoring systems for adrenocortical carcinomas.

Aims:  To compare two different multiparameter histopathological scoring indices and determine their prognostic value in patients presenting with adrenocortical carcinoma (ACC).