Magnus Kjellman

Comparative genomic hybridization identifies loss of 18q22-qter as an early and specific event in tumorigenesis of midgut carcinoids.

Carcinoid tumors are rare neuroendocrine tumors occurring in the lung or in the digestive tract where they are further subclassified as foregut, midgut, or hindgut carcinoids. To gain a better understanding of the genetic basis of the different types of carcinoid tumors, we have characterized numerical imbalances in a series of midgut carcinoids, and compared the results to previous findings in carcinoids from the lung. Numerical imbalances were revealed in 16 of the 18 tumors, and the most commonly detected aberrations were losses of 18q22-qter (67%), 11q22-q23 (33%), and 16q21-qter (22%), and gain of 4p14-qter (22%). The total number of alterations found in the metastases was significantly higher than in the primary tumors, indicating the accumulation of acquired genetic changes in the tumor progression. Losses of 18q and 11q were present both in primary tumors and metastases, whereas loss of 16q and gain of 4 were only detected in metastases. Furthermore, the pattern of comparative genomic hybridization alterations varied depending on the total number of detected alterations. Taken together, the findings would suggest a progression of numerical imbalances, in which loss of 18q and 11q represent early events, and loss of 16q and gain of 4p are late events in the tumor progression of midgut carcinoids. When compared to previously published comparative genomic hybridization abnormalities in lung carcinoids, loss of 11q was found to occur in both tumor types, whereas loss of 18q and 16q and gain of 4 were not revealed in lung carcinoids. The results indicate that inactivation of a putative tumor suppressor gene in 18q22-qter represents a frequent and early event that is specific for the development of midgut carcinoids.

Comparative Genomic Hybridization Reveals Frequent Losses of Chromosomes 1p and 3q in Pheochromocytomas and Abdominal Paragangliomas, Suggesting a Common Genetic Etiology

Pheochromocytomas and abdominal paragangliomas are rare, catecholamine-producing tumors that arise from the chromaffin cells derived from the neural crest. We used comparative genomic hybridization (CGH) to screen for copy number changes in 23 pheochromocytomas and 11 abdominal paragangliomas. The pattern of copy number changes was similar between pheochromocytomas and paragangliomas, with the most consistent finding being loss of 1cen-p31, which was detected in 28/34 tumors (82%). Losses were also found on 3q22-25 (41%), 11p (26%), 3p13-14 (24%), 4q (21%), 2q (15%), and 11q22-23 (15%), and gains were detected on 19p (26%), 19q (24%), 17q24-qter (21%), 11cen-q13 (15%), and 16p (15%). Losses of 1p and 3q were detected in the majority of tumors, whereas gains of 19p and q, 17q, and 16p were seen only in tumors with six or more CGH alterations. This progression of genetic events did not correspond with the conversion to a malignant phenotype. CGH alterations involving chromosome 11 were more frequent in the malignant tumors, compared with the benign tumors (9/12 versus 3/16). In summary, we propose that pheochromocytomas and abdominal paragangliomas, which share many clinical features, also have a common genetic origin and that the loss of 1cen-p31 represents an early and important event in tumor development.

Chromosomal alterations in human pancreatic endocrine tumors

Comparative genomic hybridization (CGH) was used to investigate changes in DNA copy numbers in 25 paraffin‐embedded samples of pancreatic endocrine tumors from 23 patients. Insulin was the dominant hormone in 12, glucagon in 7, somatostatin in 1, and pancreatic polypeptide in 2 tumors. One to 15 (mean, 8.1) changes in DNA copy numbers were observed in 22 of the 25 tumors. The most recurrent aberration, found in 68% of the tumors, involved gains in chromosome 7 with a minimal overlapping region at 7q11.2. Other frequent gains included chromosomes 19 (60%) and 14 (56%). Chromosome arm 20q was amplified in 48% of the cases with the minimal overlapping region of 20q11.1–13.1. The two most frequent DNA losses were found at 11q21–22 in 32% and at 11p13–15 in 24% of the cases. The amplified chromosomal regions contain several candidate genes that may be involved in islet cell tumorigenesis. The regions with most frequent losses are likely to contain still uncharacterized tumor suppressor genes. Genes Chromosomes Cancer 29:83–87, 2000.

Gelatinase A, Membrane Type 1 Matrix Metalloproteinase, and Extracellular Matrix Metalloproteinase Inducer mRNA Expression: Correlation with Invasive Growth of Breast Cancer

Invasive breast cancer varies widely in biologic aggressiveness, from fairly indolent tumors to rapidly disseminating carcinomas. Matrix metalloproteinases have enzymatic activity and assist in tumor invasion by degrading basement membranes and extracellular matrix. The extracellular matrix metalloproteinase inducer EMMPRIN is thought to stimulate fibroblasts to produce the zymogen pro-gelatinase A. The membrane type 1-matrix metalloproteinase (MT1-MMP) is thought to assist in tumor invasion and metastasis by activating pro-gelatinase A, which shows enhanced expression in various tumors. Overexpression of gelatinase A has shown to correlate with a malignant phenotype in many tumor forms. The aim of the study was to investigate the mRNA expression pattern of MT1-MMP, gelatinase A, and EMMPRIN in breast tumors. Formalin-fixed paraffin-embedded breast tissue samples from 18 patients operated on with breast-conserving surgery for invasive breast carcinoma <20 mm between 1977 and 1985 were analyzed using the mRNA in situ hybridization technique. Most of the patients were node-negative (15/18) and underwent postoperative irradiation to the breast (16/18). The median age at diagnosis was 52 years (21–83 years). At the time of the study 11 patients were alive, 4 without recurrence; 7 patients had been operated for ipsilateral breast tumor recurrences, and 2 had distant metastases. The median follow-up was 112 months (102–193 months). Seven patients died of disseminated breast cancer; their median follow-up was 43 months (22–116 months). 35S-labeled antisense and sense mRNA probes transcribed from linearized plasmids containing cDNA for the matrix metalloproteinases gelatinase A and MT1-MMP and the glycoprotein EMMPRIN were hybridized to 5 μm paraffin-embedded tissue sections. Several invasive carcinomas were surrounded by normal tissue and carcinoma in situ lesions. Gelatinase A, MT1-MMP, and EMMPRIN mRNA expression were detected in all of the carcinomas. The gelatinase A mRNA expression was mainly localized to stromal cells at moderate to high levels surrounding the invading carcinoma cells but was also seen in single cells at low levels in in situ lesions and in some normal glandular cells. MT1-MMP and EMMPRIN were expressed in all of the carcinomas and were mainly localized to tumor cells; but they were also seen to some extent in single cells at low levels in in situ lesions and in normal glandular cells. No differences in levels of expression for gelatinase A, MT1-MMP, or EMMPRIN were seen in patients who survived compared to patients who died from metastatic disease. The co-expression of gelatinase A, MT1-MMP, and EMMPRIN mRNA in invasive breast carcinoma supports the theory that these proteins interact and are important for the invasive phenotype in breast carcinoma. Hence EMMPRIN may be a central factor for stimulation of gelatinase A activation. Specific inhibitors for individual MMP members could in the future be target-specific events in breast tumor progression. Inhibition of EMMPRIN could be such a target.

Alterations of the SDHD gene locus in midgut carcinoids, Merkel cell carcinomas, pheochromocytomas, and abdominal paragangliomas.

Several types of endocrine tumors show frequent somatic deletions of the distal part of chromosome arm 11q, where the tumor‐suppressor gene SDHD (succinate‐ubiquinone oxidoreductase subunit D), constitutionally mutated in paragangliomas of the head and neck, is located. In this study, we screened 18 midgut carcinoids, 7 Merkel cell carcinomas, 46 adrenal pheochromocytomas (37 sporadic and 9 familial), and 7 abdominal paragangliomas for loss of heterozygosity (LOH) and/or mutations at the SDHD gene locus. LOH was detected in 5 out of 8 (62%) informative midgut carcinoids, in 9 out of 30 (30%) sporadic pheochromocytomas, in none of the familial pheochromocytomas (0%), and in 1 out of 6 (17%) abdominal paragangliomas. No sequence variants were detected in the pheochromocytomas or paragangliomas. However, two constitutional putative missense mutations, H50R and G12S, were detected in two midgut carcinoids, which were both associated with LOH of the other allele. The same sequence variants were also detected in two Merkel cell carcinomas. In addition, the S68S polymorphism was found to coexist with the G12S sequence variant in both cases. In conclusion, we show that alterations of the SDHD gene seem to be involved in the tumorigenesis of both midgut carcinoids and Merkel cell carcinomas.

TRANSCRIPTIONAL PROFILING ENABLES MOLECULAR CLASSIFICATION OF ADRENOCORTICAL TUMOURS

OBJECTIVE Tumours in the adrenocortex are common human tumours. Malignancy is however, rare, the yearly incidence being 0.5-2 per million inhabitants, but associated with a very aggressive behaviour. Adrenocortical tumours are often associated with altered hormone production with a variety of clinical symptoms. The aggressiveness of carcinomas together with the high frequency of adenomas calls for a deeper understanding of the underlying biological mechanisms and an improvement of the diagnostic possibilities. METHODS Microarray gene expression analysis was performed in tumours of adrenocortex with emphasis on malignancy as well as hormonal activity. The sample set consisted of 17 adenomas, 11 carcinomas and 4 histological normal adrenocortexes. RNA from these was hybridised according to a reference design on microarrays harbouring 29 760 human cDNA clones. Confirmation was performed with quantitative real time-PCR and western blot analysis. RESULTS Unsupervised clustering to reveal relationships between samples based on the entire gene expression profile resulted in two subclusters; carcinomas and non-cancer specimens. A large number of genes were accordingly found to be differentially expressed comparing carcinomas to adenomas. Among these were IGF2, FGFR1 and FGFR4 in growth factor signalling the most predominant and also the USP4, UBE2C and UFD1L in the ubiquitin-proteasome pathway. Moreover, two subgroups of carcinomas were identified with different survival outcome, suggesting that survival prediction can be made on the basis of gene expression profiles. Regarding adenomas with aldosterone overproduction, OSBP and VEGFB were among the most up-regulated genes compared with the other samples. CONCLUSIONS Adrenocortical carcinomas are associated with a distinct molecular signature apparent in their gene expression profiles. Differentially expressed genes were identified associated with malignancy, survival as well as hormonal activity providing a resource of candidate genes for an exploration of possible drug targets and diagnostic and prognostic markers.

Genetic background of adrenocortical tumor development

The increasing occurrence of incidentally discovered benign adrenocortical tumors has become a clinical dilemma because of the difficulties in differentiating them from their malignant counterpart. Adrenocortical tumors are associated with familial cancer syndromes such as the Beckwith-Wiedemann syndrome, the Li-Fraumeni syndrome, the Carney complex, multiple endocrine neoplasia type 1, congenital adrenal hyperplasia, and the McCune-Albright syndrome. Genetic events are known to take place on the chromosomal and gene level in sporadic adrenocortical tumors. On découvre de plus en plus de tumeur de la surrénale de façon accidentelle. Ceci devient un véritable dilemme clinique car il est très difficile de distinguer celles qui sont bénignes de celles qui ne le sont pas. Dans cet article, on traite l’association des tumeurs de la corticosurrénale aux syndromes de cancer familial comme le syndrome de Beckwith-Wiedemann, le syndrome de Li-Fraumeni, le complexe Carney, le syndrome de néoplasie endocrine multiple du type 1, l’hyperplasie surrénalienne congénitale et le syndrome de McCune Albright. De plus, on passe en revue les évènements génétiques qui caractérisent les tumeurs de la corticosurrénale au plan des chromosomes et des gènes. La creciente frecuencia con que se descubren en forma incidental tumores adenocorticales benignos se ha convertido en un dilema clínico por razón de las dificultades en diferenciarlos de neoplasmas malignos. En el présente artículo se discute la asociación de los tumores adrenocorticales con los sindromes familiares de cáncer y con el síndrome de Beckwith-Wiedemann. el síndrome de Li-Fraumeni, el Complejo de Carney, la neoplasia endocrina múltiple Tipo 1, la hiperplasia suprarrenal congénita y el síndrome de McCune-Albright. También se revisan los eventos genéticos involucrados. así como los tumores adrenocorticales esporádícos.

In vitro release of aldosterone and cortisol in human adrenal adenomas correlates to mRNA expression of steroidogenic enzymes for genes CYP11B2 and CYP17

Adenomas of the adrenal cortex cause different disorders depending on the main steroid synthesized and released. The aim of this research is to increase our understanding of the pathophysiology of steroidogenesis in adrenocortical disorders by comparing the release of steroids from adrenocortical adenomas in vitro with the messenger RNA (mRNA) expression of steroid synthesizing enzymes. Fourteen patients with adrenal tumors were included in the present study; nine were diagnosed with primary aldosteronism and three with Cushing’s syndrome. Two patients had an adrenal tumor discovered on computed tomography (CT) during workup for an unrelated disease. Serum cortisol, plasma aldosterone, and urinary catecholamines were normal. Tissue was taken for in vitro steroid release, and aldosterone and cortisol in the medium after a 1-hour incubation were determined. Oligonucleotide probes with sequences complementary to mRNAs encoding for the steroid synthesizing enzymes 11β-hydroxylase (CYP11B1), 18-hydroxylase (CYP11B2), 17α-hydroxylase (CYP17), and 21-hydroxylase (CYP21) were synthesized (Genset, Paris, France) and in situ hybridization was performed. Moderate expression of CYP11B2 and low expression of CYP11B1 were seen in the zona glomerulosa. The zona fasciculata of the control adrenals expressed a high signal of CYP11B1, whereas the expression of CYP11B2 was very low. There was considerable variation in aldosterone release from the aldosteronomas, whereas the tumors from the Gushing patients showed no detectable release of aldosterone. In contrast, tumors from patients with primary aldosteronism, Cushing’s syndrome, and no hyper-function all had the ability to synthesize and release cortisol in vitro. The highest cortisol release was found in tumors from patients with Cushing’s syndrome, but also the nonhyperfunctioning tumors and some of the aldosteronomas released significant amounts of cortisol. The two patients with highest release of aldosterone in vitro showed the highest expression of CYP11B2 and the lowest expression of CYP11B1 and CYP17. The remaining aldosteronomas had low expression of CYP11B2, similar to the two other groups. Expression of CYP11B1 was high as expected in the Gushing adenomas, but also the two nonhyperfunctioning tumors and some of the aldosteronomas showed a moderate expression. Adenomas from Cushing’s syndrome, nonhyperfunctioning adenomas, and some of the aldosterone- producing adenomas had moderate to high expression of CYP17. This paper presents new means for functional characterization of adrenocortical tumors. Diagnosis of an aldosteronoma is often difficult, and with the advent of these methods it is possible to determine the functional capacity of a tumor, once it is removed. This is of special interest if the patient remains hypertensive postoperatively, and it is not clear whether the patient indeed had a functioning tumor.RésuméLe tableau clínique des adénomes de la corticosurrénale varie selon le type dominant de stéroïde synthétisé et sécrété. Le but de cet article a été d’améliorer la compréhension de la physiopathogenèse des stéroïdes dans les maladies de la surrénale en comparant la sécrétion des stéroïdes à partir des adénomes de la corticosurrénale in vitro avec l’expression mRNA des enzymes de synthèse stéroídien. Quatorze patients ayant une tumeur de la surrénale ont été inclus dans cette étude. On a retenu le diagnostic d’hyperaldostéronisme primitif chez neuf alors que trois patients avaient un syndrome de Cushing. Deux patients avaient une tumeur de la surrénale découverte par la tomodensitométrie réalisée pour une pathologie sans rapport. Les taux sériques de cortisol, d’aldostérone et de catécholamines urinaires étaient normaux. On a déterminé la sécrétion de stéroïdes, d’aldostérone et de cortisol in vitro après une heure d’incubation du tissu prélevé. Des sondes d’oligonucléotide avec des séquences complémentaires à l’encodage mRNAs pour les enzymes de synthèse 11-beta-hydroxylase (CYP11B1), 18-hydroxylase (CYP11B2), 17 alpha-hydroxylase (CYP17) et 21-hydroxylase (CYP21) ont été synthétisés (Genset, Paris, France) et une hybridation in situ a été réalisée. On a trouvé une expression modérée de CYP11B2 et une expression basse de CYP11B1 dans la zone glomérulaire. La zone fasciculaire des surrénales de contróle a exprimé un signal intense de CYP11B1 alors que l’expression de CYP11B2 était basse. Il y avait une forte variation en ce qui concernait la sécrétion d’aldostérone à partir des aldostéronomes alors que les tumeurs des patients ayant un syndrome de Cushing ne sécrétaient pas d’aldostérone. En revanche, les tumeurs provenant des patients ayant un hyperaldostéronisme primitif, un syndrome de Cushing, sans hyperfonctionnement étaient toutes capables de synthétiser et de sécréter le cortisol in vitro. Le taux le plus élevé de cortisol a été retrouvé chez les patients ayant un syndrome de Cushing, mais également chez les patients porteurs de tumeurs non fonctionnelles et chez quelques patients ayant un aldostéronome. Les deux patients ayant le taux le plus élevé d’aldostérone in vitro avaient également la plus forte expression de CYP11B2, alors que celles de CYP11B1 et de CYP17 étaient la plus faible. Les autres patients porteurs d’aldostérome avaient une expression réduite de CYP11B2, similaire aux deux autres groupes. L’expression de CYP11B1 était élevé, comme attendue dans le syndrome de Cushing par adénome, mais également chez les deux tumeurs non hyperfonctionelles, alors que l’expression était modérée chez les patients ayant un aldostéronome. L’expression de CYP 17 était modérée à élevée chez les patients ayant un adénome avec syndrome de Cushing, un adénome non-hyperfonctionnel et dans le cas de quelques adénomes produisant de l’aldostérone. Dans cet article, on présente de nouvelles modalités pour caractériser la fonction des tumeurs de la corticosurrénale. Le diagnostic d’un aldostéronome est souvent difficile mais avec ces nouvelles modalités diagnostiques, il est possible désormais de connatre la capacité fonctionnelle de la tumeur, une fois enlevée. Ceci a un intérét particulier si le patient reste hypertendu en postopératoire lorsqu’ on ne savait pas en préopératoire si la tumeur était fonctionnelle.ResumenLos adenomas de la corteza suprarrenal ocasionan diferentes alteraciones clínicas según el principal esteroide que sintetizan y secretan. El propósito del présente trabajo fue incrementar el conocimiento sobre la fisiopatología de la estereidogénesis en las enfermedades adrenocorticoles comparando la liberation in vitro de esteroides por adenomas adrenocorticales con la expresión de mRNA de las enzimas sintetizadoras de esteroides. Catorce pacientes con tumores suprarrenales fueron incluidos en el estudio, nueve con el diagnóstico de aldosteronismo primario y très con síndrome de Cushing. En dos se encontró un tumor suprarrenal en tomografia computadorizada en el curso del estudio de una enfermedad no relacionada. El cortisol sérico, el nivel plasmático de aldosterona y las catecolaminas urinarias aparecieron dentro de límites normales. Se tomaron tejidos para la determination in vitro de liberación de esteriodes y de la presencia de aldosterona y cortisol en el medio luego de una hora de incubation. Se observé expresión moderada de CYP11B2 y baja expresion de CYP11B1 en la zona glomerulosaa. La zona fasciculata de las suprarrenales de control expresaron una elevada señal de CYP11B1, en tanto que la expresión de CYP11B2 apareció muy baja. Se observó una variación considerable en la liberación de aldosterona por los aldosteronomas, en tanto que los tumores de los pacientes con Cushing no demostraron liberación de aldosterona. En contraste, los tumores de pacientes con aldosteronismo primario, síndrome de Cushing y aquellos sin hiperfunción, todos exhibieron capacidad para sintetizar y liberar cortisol in vitro. La más alta liberación de cortisol fue hallada en tumores de pacientes con síndrome de Cushing, pero los tumores no hiperfuncionantes y algunos de los aldosteronomas mostraron liberación significante de cortisol. Los dos pacientes con la más alta liberación de aldosterona in vitro mostraron también la más elevada expresión de CYP11B2 y la más baja de CYP11B1 y CYP17. Los restantes aldosteronomas mostraron baja expresión de CYP11B2, similar a la de los otros dos grupos. La expresión de CYP11B1 apareció elevada, como era lo esperado, en los adenomas de Cushing, pero también en los tumores no hiperfuncionantes; algunos de los aldosteronomas exhibieron expresión moderada. Los adenomas del síndrome de Cushing, lo adenomas no hiperfuncionantes y algunos de los adenomas productores de aldosterona exhibieron expresión moderada a alta de CYP17. El présente artículo muestra nuevas maneras de hacer la caracterización funcional de los tumores suprarrenales. El diagnóstico de aldosteronoma frecuentemente es difícil y con el advenimiento de estos métodos es posible determinar la capacidad funcional del tumor que ha sido resecado. Esto es de interés especial cuando el patiente se mantiene hipertenso en el postoperatorio y no aparece claro si realmente tenía un tumor funcional.

No overrepresentation of congenital adrenal hyperplasia in patients with adrenocortical tumours

The development and progression of sporadic adrenocortical tumours are poorly understood. In autopsy studies adrenocortical tumours are found in between 2 and 9% of the general population. In congenital adrenal hyperplasia (CAH), decreased production of cortisol leads to increased secretion of ACTH from the pituitary, resulting in hyperplasia of the adrenals. More than 95% of all cases of CAH are due to steroid 21‐hydroxylase deficiency, resulting from mutations in the CYP21 gene. In subjects homozygous and heterozygous for CYP21 mutations, adrenocortical tumours have been found in a high frequency compared to the general population, suggesting that chronic ACTH stimulation may play a role in the development of this tumour form. In order to test whether mild undiagnosed CAH is a common predisposing factor, we screened 27 patients with sporadic adrenocortical tumours for CYP21 mutations.

Gelatinase A and Membrane-type 1 Matrix Metalloproteinase mRNA: Expressed in Adrenocortical Cancers but Not in Adenomas

In an attempt to understand the mechanism behind the invasion and metastasis in adrenocortical cancer we performed mRNA in situ hybridization on 30 tumors for three matrix metalloproteinases (MMPs): gelatinase A, membrane type 1 matrix metalloproteinase (MT1-MMP), and collagenase-3. All are known to participate in the invasion and metastasis of other tumor forms by degrading the extracellular matrix. Thirteen of sixteen cancers, but only one of fourteen benign lesions showed expression of gelatinase A, which was localized in stromal cells. MT1-MMP is thought to assist in tumor invasion and metastasis by activating the zymogen gelatinase A. Of 14 malignant tumors analyzed, 12 showed MT1-MMP mRNA expression, which in 7 cases was detected in both neoplastic and stromal cells. The benign tumors showed MT1-MMP expression in only 3 of 11 cases, and it was restricted to tumor cells. Fourteen tumors (11 cancers, 3 adenomas) were also analyzed for collagenase-3 mRNA, but no expression was detected. In conclusion, our data show that gelatinase A mRNA is expressed in most malignant adrenocortical tumors but not in the benign tumors. Gelatinase A mRNA expression is restricted to stromal cells, whereas its activator, MT1-MMP, is expressed in both stromal and neoplastic cells. Inhibition of gelatinase A and other proteinases may in the future become important as a form of cancer treatment.