Genetic aberrations in adrenocortical tumors detected using comparative genomic hybridization correlate with tumor size and malignancy.

The differentiation between malignant and benign adrenocortical tumors is often difficult, and better markers are required. Because the genetic background of adrenocortical tumors is poorly characterized, we used comparative genomic hybridization (CGH) to screen for DNA sequence copy number changes in 8 sporadic primary adrenocortical cancers and 14 adenomas. There was a strong relationship between the number of genetic aberrations detected using CGH and both tumor size and malignancy. No alterations were seen in the smaller adenomas (< 5 cm), whereas the two largest adenomas (5 cm each) and seven of the eight cancers (7-20 cm) showed an increased number of genetic alterations. The presence of genetic aberrations detected using CGH was associated with an aneuploid DNA pattern. In the cancers, losses most often involved the chromosomal regions 2, 11q, and 17p (four of eight tumors), whereas gains took place at chromosomes 4 and 5 (four of eight tumors). In conclusion, our data indicate that genetic changes may help to define the malignant potential of adrenocortical tumors. Furthermore, the CGH results implicate several chromosomal regions that may contain genes with an important role in the development of adrenocortical cancers.

Genotyping of adrenocortical tumors: very frequent deletions of the MEN1 locus in 11q13 and of a 1-centimorgan region in 2p16.

To identify chromosomal regions that may contain loci for tumor suppressor genes involved in adrenocortical tumor development, a panel of 60 tumors (39 carcinomas and 21 adenomas) were screened for loss of heterozygosity. Although the vast majority of loss of heterozygosity (LOH) were detected in the carcinomas and involved chromosomes 2, 4, 11, and 18, only few were found in the adenomas. Therefore, 2 loci that harbor the familial cancer syndromes Carney complex in 2p16 and the multiple endocrine neoplasia type 1 gene in 11q13 were further studied in 27 (13 carcinomas and 14 adenomas) of the 60 tumors. Detailed analysis of the 2p16 region mapped a minimal area of overlapping deletions to a 1-centimorgan region, which is separate from the Carney complex locus. LOH for a microsatellite marker (PYGM), very close to the MEN1 gene, was detected in all 8 informative carcinomas (100%) and in 2 of 14 adenomas. Of the 27 cases analyzed in detail, 13 cases (11 carcinomas and 2 adenomas) showed LOH on chromosome 11 and was therefore selected for MEN1 gene mutation analysis. In 6 cases a common polymorphism (Asp418Asp) was found, but no mutation was detected. In conclusion, our data indicate the existence of tumor suppressor genes at multiple chromosomal locations, whose inactivations are involved in the development of adrenocortical carcinomas. Loss of genetic material from 2p16 was strongly associated with the malignant phenotype, as it was seen in almost all carcinomas but not in any of the adenomas. LOH in 11q13 also occurred frequently in the carcinomas, but was not associated with a MEN1 mutation, suggesting the involvement of a different tumor suppressor gene on this chromosome.

Neuroendocrine differentiation and nerves in human adrenal cortex and cortical lesions.

Neuroendocrine differentiation and nerve distribution were studied in sections from human cortex (n=11) and cortical lesions (hyperplasias, n=9; adenomas, n=13; carcinomas, n=14) with four markers, namely chromogranin A(CgA), synaptophysin (SYN), neuron-specific enolase (NSE), protein gene product (PGP) 9.5 and small synaptic vesicle protein (SV)2. All but two cases expressed neuroendocrine differentiation. NSE was the most commonly occurring marker and the NSE immunoreactive cells were detected in normal cortex, mainly in zona glomerulosa, as well as in adenomas and carcinomas. SYN and PGP 9.5 immunoreactive cells were especially prominent in the carcinomas, while SV2 immunoreactive cells were seen mainly in normal cortex. The difference in distribution pattern of the neuroendocrine markers between adenomas and carcinomas was not so distinct that it can be used for histopathological diagnosis. The significance of neuroendocrine differentiation in cortex and cortical lesions is uncertain, but may reflect an involvement in special hormonal functions. No obvious relationship was found between the clinical syndromes and the degree of neuroendocrine differentiation. Three of the neuroendocrine markers also visualized nerve structures. PGP 9.5, which is regarded as the most 'general' nerve marker, visualized more nerve structures than did the other markers. Normal cortex contained most immunoreactive nerves, whereas they were less numerous in hyperplasias and sparse or even absent in the neoplasms. The nerves appeared among the parenchymal cells but were particularly prominent around vessels. The results suggest that the cortical nerves influence not only the regulation of the blood supply but also the hormonal regulation at the cellular level.

Insulin-like growth factor-II in endocrine pancreatic tumours. Immunohistochemical, biochemical and in situ hybridization findings.

In earlier studies a high-molecular-weight (HMW) insulin-like growth factor-II (IGF-II) peptide was identified in adult human pancreas and localized to the insulin-producing B-cells. This peptide has now been investigated in neoplastic insulin cells. Forty endocrine pancreatic tumours and 17 pancreatic adenocarcinomas of ductal type were included in the study. All cases were investigated with immunohistochemical techniques using antibodies to IGF-II, insulin, pro-insulin, glucagon, somatostatin, pancreatic polypeptide, gastrin and vasoactive intestinal peptide (VIP). Frozen tissue from nine tumours and two normal pancreatic glands was extracted, gel separated, and quantified using radioimmunoassay. The tumours were also investigated by in situ hybridization. IGF-II-immunoreactive cells were found in nearly all the 18 insulin-producing tumours (16/18), in a minority of the other endocrine tumours, but not in pancreatic adenocarcinomas. All extracts from the endocrine tumours showed varying amounts of IGF-II and had different molecular-weight forms. The immunohistochemical and radioimmunoassay findings are both based on immunological binding and were further confirmed by Northern blot and in situ hybridization. These results show that IGF-II is expressed in insulin-producing tumours as well as in pancreatic tumours producing other peptides, in contrast to normal pancreatic islets where IGF-II is found exclusively in insulin-producing cells.

Primary hyperparathyroidism. Low surgical morbidity supports liberal attitude to operation.

OBJECTIVE: To evaluate the results of a modern surgical approach in patients with primary hyperparathyroidism. DESIGN: Retrospective analysis. SETTING: University hospital, tertiary care center. PATIENTS: One hundred patients consecutively operated on for suspected primary hyperparathyroidism. Patients were available for follow-up 1 month (n = 100) and 1 year (n = 96) after surgery. INTERVENTION: Cervical exploration. Surgical strategy was to remove enlarged parathyroid glands only and perform a biopsy on no more than one normal gland. MAIN OUTCOME MEASURES: Surgical morbidity and normocalcemia. RESULTS: No operative mortality or wound infection occurred in any patient. Postoperative vocal cord paralysis was recorded in two patients; both recovered fully. Two patients underwent a second operation. (One patient experienced subcutaneous bleeding and the second patient, previously operated on for toxic goiter, experienced persistent hypercalcemia and was operated on 5 days after the initial operation. A second abnormal gland was then found on the contralateral side, not initially surgically explored.) At follow-up, 97 patients were normocalcemic; three patients had hypoparathyroidism: two of these patients, with multiglandular disease, were normocalcemic and received a low dose of vitamin D (1 alpha [OH]D3), and one patient, who had had a single adenoma removed, was slightly hypocalcemic, however, asymptomatic. CONCLUSIONS: More than 90% of patients with primary hyperparathyroidism can be operated on without complications occurring. This supports a liberal attitude to operation.

Efficacy of once-daily versus twice-daily administration of budesonide by Turbuhaler(R) in children with stable asthma.

We evaluated the efficacy of once-daily versus twice-daily treatment with budesonide, delivered by a Turbuhaler(R), in the management of children with stable asthma in a randomized, double-blind, parallel-group study involving 206 children (age 5-15 years). After a 2-week run-in period during which the children were maintained on their usual dose of budesonide (200 microg or 400 microg/day), patients were randomized to receive the same daily dose in either two daily administrations (morning and evening) or as a single dose in the morning over a period of 12 weeks. The primary efficacy variable was morning peak expiratory flow (PEF). The mean morning PEF during the run-in phase was 271 L/min in patients randomized to once-daily treatment and 264 L/min in those randomized to twice-daily treatment. The mean change from baseline to the last 2 weeks of the treatment period in the two groups was -0.3 L/min (95% confidence limits -6.6 to +6.0) and 2.5 L/min (-4.3 to +9.3). The estimated difference between the groups was -2.8 L/min, with 90% confidence limits of -10.4 + 4.5; these were close to the limits regarded as indicative of equivalence (-10 to +10), and hence the difference was not regarded as clinically relevant. Similarly, there were no significant differences between the groups in regard to secondary efficacy measures such as spirometric tests and symptom scores. Both treatments were well tolerated. We conclude that once-daily administration of budesonide by Turbuhaler(R) is as effective as twice-daily treatment in the management of stable asthma in children treated with inhaled steroids at doses of 200-400 microg/day.

[Asthma in children more and more common. A study of medical records shows a doubled increase during a 10-year period]. / Astma hos barn blir allt vanligare. Journalstudie visar en fördubbling under en tioårsperiod.

The number of children with asthma is rising. This is shown by a study of medically diagnosed asthma in a cohort of children born in 1990 in a well defined and limited region. The medical records from all of the health centres in primary health care, privately practising paediatricians and relevant child clinics in the region were examined when the children were 7 years old. The results showed that 10.9% of the children had been diagnosed with asthma. The percentage of children with asthma was highest (14.2%) among boys living in a city environment. Comparison with a similar follow-up study in the same region concerning children born in 1975 showed that the percentage of children with asthma doubled during a period of just over a decade.

[Immunological basis of IgE-mediated allergies. A contribution to the understanding of new therapy forms]. / Immunologische Grundlagen IgE-vermittelter Allergien. Ein Beitrag zum Verständnis neuer Therapieformen.

The pathogenesis of IgE mediated allergies has been elucidated. We are dealing primarily with diseases of the T lymphocyte system. Due to overactivity of so-called T helper lymphocytes there is pathologic overproduction of IgE and inflammatory cells, mast cells and eosinophils, after antigen contact. Following exposure to nutrient and inhalant allergens in the presence of adjuvants, this atopic constitution manifests itself clinically. Mast cell mediators (e.g. histamine and leukotrienes) elicit immediate symptoms at the ports of entrance of allergens (at skin and mucosal surfaces); eosinophil mediators (e.g. platelet activating factor [PAF], eosinophil cationic protein [ECP], and major basic protein [MBP]) are responsible for late symptoms and hyperreactivity. Current antiinflammatory treatment tries to suppress this allergic inflammation (e.g. with cromoglycate and topical steroids). The regulatory immunotherapy of the near future attempts to regulate IgE synthesis by interleukins (e.g. gamma-Interferon) or antagonists of interleukins (e.g. anti-IL-4-antibodies).

Treatment of children with perennial rhinitis with brompheniraminemaleate and phenylpropanolaminehydrochloride (lunerin mite)

The effect of a combined pediatric preparation of brompheniramine maleate and phenylpropanolaminehydrochloride (Lunerin mite) was studied in 17 children (aged 4-14 years) with allergic rhinitis. A double-blind cross-over technique was used, and a score system was used for evaluation of symptoms and signs. The results show that the drug had a good effect with statistical differences on the 1 per cent level between the active preparation and the placebo. The effect was less pronounced in children aged more than 10 years, for whom a higher dose of the drug is recommended. The frequency of side-effects was negligible.

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.

Seasonal variation of IgE synthesis in vitro by human peripheral blood mononuclear cells.

Seasonal variations in IgE antibody synthesis in vitro were studied in cultures of blood mononuclear cells (MNC) from 11 pollen allergic individuals. The IgE levels were significantly higher in two summer seasons than in the winter and spring between them. Net synthesis was confined to the summer in all but one of the patients. All the IgE in the cultures outside the pollen season represented preformed IgE which was present mainly (59%) in the monocyte fraction. Thus, preformed IgE seems to persist in monocytes at times when there is little de novo synthesis of IgE.

Nedocromil sodium 2% eye drops for twice-daily treatment of seasonal allergic conjunctivitis: a Swedish multicentre placebo-controlled study in children allergic to birch pollen.

This was a multicentre, double-blind, randomized group comparative study in which 77 children, aged 6-16 years, received 2% nedocromil sodium eye drops and 72 received placebo, one drop into each eye twice daily. The treatment period was 4 weeks, covering the peak birch pollen season. Prior to the start of the season, patients who had attended the clinic the previous 2 years because of seasonal allergic conjunctivitis (SAC) to birch pollen, entered a one week baseline period during which symptoms were assessed, dairy cards completed, and routine sampling of blood and urine carried out. The double-blind treatment period then commenced at the onset of the birch pollen season. Patients/parents kept daily diary record cards of eye symptom severity and concomitant therapy. Conjunctivitis was mild in both treatment groups but nedocromil sodium was more effective than placebo in controlling symptoms. During the 2-3 weeks of peak pollen counts, this therapeutic effect was statistically significant for itching (P < 0.01), watering (P < 0.05) and total symptom score (P < 0.01), but was not significant for grittiness (P = 0.08) or redness (P = 0.06). Global opinions of efficacy showed no difference between treatments, due to a high placebo effect (however, the diary card data indicated a significant improvement with nedocromil sodium). We therefore conclude that nedocromil sodium 2% eye drops, administered twice daily, is an effective treatment for SAC in children.

DNA analysis in a MHC heterozygous patient with complete C4 deficiency--homozygosity for C4 gene deletion and C4 pseudogene.

Virtually all cases of inherited C4 deficiency appear to be caused by homozygosity for rare MHC haplotypes carrying combined defects of genes coding for the C4A and C4B isotypes. The present analysis concerned a C4-deficient patient with two different MHC haplotypes, [HLA-A2, B40, SC00, DR6] and [HLA-A30, B18, F1C00, DR3]. Digestion of genomic DNA from the patient with Taq I and probing with a 5' cDNA C4 probe and a CYP21-specific probe gave only a 7.0-kb and a 3.7-kb band, respectively. The analysis of restriction fragment length polymorphism in family members showed that both C4-deficient haplotypes contained a C4 pseudogene at the C4 locus I and a CYP21 gene together with a deletion of the C4B gene and the adjacent CYP21P gene. None of the C4 pseudogenes contained C4A- or C4B-specific nucleotide sequences as judged from hybridization studies of polymerase chain reaction products. The findings illustrate the high degree of polymorphism in C4 genes and that both gene deletions and presence of a C4 pseudogene are common as reasons for C4 null alleles. The rare C4 double null alleles appear to have arisen in different MHC haplotypes independently.

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). (35)S-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 microm 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 inhibition for individual MMP members could in the future be target-specific events in breast tumor progression. Inhibition of EMMPRIN could be such a target.

Novel splicing of an IGF2 polymorphic region in human adrenocortical carcinomas.

The human IGF2 gene lies on chromosome 11p15.5 and encodes for a mitogenic peptide. IGF2 is often overexpressed in many tumours including adrenal carcinomas. In this study while screening 12 adrenocortical carcinomas for heterozygosity at the Apa I and (CA)n repeat polymorphisms we observed a novel splicing event in two samples which showed both an allelic expression imbalance and preferential splicing for one of the alleles. Further examination revealed that the splicing was not confined to one particular site. Three of such splice products were isolated and cloned. Using RNase protection analysis the presence of this splicing event was demonstrated for both adrenocortical carcinoma samples and also in a Hep3B cell line. This suggested that the event may be occurring in all the samples. The presence of this splicing was then confirmed in all 12 adrenocortical carcinoma samples by PCR. These data suggest that the splicing event may be a general feature for IGF2 transcripts.

No overrepresentation of congenital adrenal hyperplasia in patients with adrenocortical tumours.

OBJECTIVE: 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. DESIGN: A retrospective study. PATIENTS: We screened 27 patients with sporadic adrenocortical tumours, representing both benign and malignant as well as hormonally active and silent lesions. MEASUREMENTS: Mutation analyses of the CYP21 gene was performed by allele-specific PCR on high molecular weight DNA. The method used detects the nine CYP21 mutations that are responsible for 95% of all disease-causing alleles in CAH. RESULTS: No mutations were detected in any of the 23 DNA samples that were prepared from leucocytes. In 4 cases where no leucocyte DNA was available, tumour tissue was analysed. In one of these tumours, two CYP21 mutations, V281 L and L307insT, were found in heterozygous form. CONCLUSION: Our data indicate that mild undiagnosed congenital adrenal hyperplasia is not a common underlying factor predisposing to adrenocortical tumours, at least not in the Swedish population.

Homozygous deficiency of C4 in a child with a lupus erythematosus syndrome.

A complete, selective lack of C4 was found in a girl who at 2 years of age presented with an atypical rash and low titres of antinuclear antibodies (less than 1/25). Rheumatoid factors were also found. The deficiency has been followed for 5 years. Tests for Chido and Rodgers antigens on the erythrocytes were negative. A possible proneness to bacterial infections has been noted with recurrent otitis media and purulent parotitis. At the age of 5, the patient developed polyarthritis of large joints and signs of glomerulonephritis. These symptoms responded well to high-dose steroid treatment. At present, there are initial signs of sclerodactylia and some persistent exanthema and parotic swelling. IgM levels were remarkably high with 19 S IgM at about 7 g/l and 7 S IgM at about 1.5 g/l. In the large kindred studied, lower immunochemical and functional C4 values were found in carriers of the genetical defect than in the rest of the family members. The C4 deficiency gene(s) segregated with HLA A2, Cw3, B40, BfS on the paternal, and with Aw30,-, B18, BfF1 on the maternal side of the family.

Characterisation of endothelin-1-related protein in human adrenal cortex and in cortical lesions.

Endothelin (ET)-1 is a 21-amino acid peptide with potent vasopressor and vasoconstrictive properties. Biochemical studies suggest that this peptide occurs in the adrenal cortex, where it appears to influence steroid hormone production and catecholamine release. Concomitant with our previous immunohistological study, we found ET-1 immunoreactive (IR) cells in human adrenal cortex and cortical neoplasms, but not in the medulla. These ET-1 IR cells were numerous in adenomas, but were seen only occasionally in some of the carcinomas. In the present study, the ET-1 IR protein was extracted from normal (n = 5) and hyperplastic (n=3) human cortex as well as from cortical adenomas (n = 10) and carcinomas (n = 5). Its molecular weight, determined by SDS-polyacrylamide gel electrophoresis and immunoblotting, was 9 kD, which is lower than that of prepro-ET-1 (21 kD), but larger than that of pre-ET-1 (4.3 kD) and ET-1 (2.5 kD). The normal cortical specimens, hyperplasias, adenomas and three of the five carcinomas all contained this distinct band. The two carcinomas lacking it were associated with Conn's syndrome. The protein may constitute a protein not previously described, but further studies are needed to determine its complete structure.