Methylation of the p16INK4A promoter is associated with malignant behavior in abdominal extra-adrenal paragangliomas but not pheochromocytomas.

Pheochromocytomas and abdominal extra-adrenal paragangliomas are related to endocrine tumors of the sympathetic nervous system. Studies in animal models have shown that inactivation of the products of the cyclin dependent kinase inhibitor 2A (CDKN2A) gene locus, p16INK4A and p14ARF, promotes the development of pheochromocytoma, especially in malignant form. The present study evaluated the involvement of CDKN2A in human pheochromocytomas and abdominal extra-adrenal paragangliomas from 55 patients. Promoter methylation was assessed using quantitative Pyrosequencing and methylation-specific PCR, and mRNA expression was measured by quantitative real-time PCR. For p16, western blot analysis and sequencing were also performed. succinate dehydrogenase complex subunit B (SDHB) sequencing analysis included extra-adrenal paragangliomas, all tumors classified as malignant, and cases diagnosed at 30 years or younger. The p16INK4A promoter was heavily methylated in a subset of paragangliomas, and this was significantly associated with malignancy (P<0.0043) and SDHB mutation (P<0.002). p16INK4A mRNA expression showed moderate suppression in malignant cases (P<0.05). In contrast, very little p14ARF promoter methylation was seen and there was no significant difference in p14ARF expression between tumors and normal samples. The p16 protein expression was reduced in 16 tumors, and sequence variations were observed in four tumors including the missense mutation A57V and the single nucleotide polymorphism (SNP) A148T. The results suggest that p16INK4A, and not p14ARF, is a subject of frequent involvement in these tumors. Importantly, hypermethylation of the p16INK4A promoter was significantly associated with malignancy and metastasis, and SDHB gene mutations. This finding suggests an etiological link and could provide a clinical utility for diagnostic purposes.

Operation for primary hyperparathyroidism: the new versus the old order. A randomised controlled trial of preoperative localisation.

BACKGROUND AND AIMS: In patients with primary hyperparathyroidism (PHPT), parathyroid imaging is nowadays routinely used for the purpose to perform a focused unilateral minimally invasive operation. The outcome of this new strategy has, however, not been established in randomised trials. MATERIAL AND METHODS: Patients were randomised to either preoperative localisation with sestamibi scintigraphy and ultrasonography (group I) or no preoperative localisation (group II). In group I, a minimally invasive parathyroidectomy was performed in patients in whom both localisation studies were consistent with a single pathological gland, whereas a conventional bilateral neck exploration was performed in cases with negative localisation findings. In group II all patients underwent conventional bilateral neck exploration. Primary outcome measure was normocalcaemia at 6 months postoperatively. RESULTS: In the preoperative localisation group (group I) 23/50 (46%) of the patients could be operated on with the focused operation whereas 26/50 (52%) were operated on by bilateral neck exploration. All patients in the no localisation group (group II; n = 50) were operated on with the intended bilateral neck operation. Normocalcaemia was obtained in 96% and 94% in group I and II, respectively. Total (localisation and operative) costs were 21% higher in group I. CONCLUSIONS: Routine preoperative localisation, with the intention to perform minimally invasive parathyroidectomy, is not cost effective if concordant results of scintigraphy and ultrasonography are a prerequisite for the focused operation. Less than half of the patients were successfully managed with this strategy, at a higher cost and without obtaining a more favourable clinical outcome.

Predictors of outcome in patients with papillary thyroid carcinoma.

AIM OF THE STUDY: To evaluate prognostic factors with respect to the outcome in a consecutive series of patients with papillary thyroid carcinoma (PTC) treated at the same institution during a 20-year-period, and to evaluate further the predictive ability of outcome of the pTNM, AMES and MACIS prognostic systems in these patients. MATERIALS AND METHODS: Two hundred and twenty consecutive patients operated on for primary PTC at the Karolinska Hospital between 1980 and 1999 were examined retrospectively. Patient and tumour characteristics at the time of surgery were compared to the patients' outcomes. Univariate and multiple logistic regression analyses were used to identify independently significant prognostic factors with respect to the outcome. In addition, the classification of the patients according to the pTNM, AMES and MACIS prognostic systems were compared to the outcomes. RESULTS: At the end of the follow-up period 201 patients were still alive without disease, 6.5% had died from PTC and 2.5% were alive with persisting disease. In 16 patients, radical surgery could not be performed due to extensive tumour growth and/or distant metastases. Recurrences were detected in 14% of the patients considered as radically operated. The strongest independent predictors for local or distant recurrences and poor clinical outcome were the lack of radical surgery and increasing tumour size. In this investigation MACIS appeared to be the better system, regarding efficacy in predicting the outcome of PTC. CONCLUSION: Removal of all tumour tissue appears most important to a favorable outcome and in our patients MACIS appears the most useful prognostic system taking completeness of resection into account.

Nimodipine and microsurgery induced recovery of the vocal cord after recurrent laryngeal nerve resection.

Transection of the recurrent laryngeal nerve leads to permanent palsy of the vocal cord. Experimental studies have confirmed that nimodipine increases the pace of axonal regeneration. We present a case of a 19-year-old male, suffering a thyroid cancer disease, who was subjected to unilateral resection of the recurrent laryngeal nerve during surgery. The nerve was repaired with a nerve graft and the patient further treated with nimodipine for 3 months. Evaluation of the patient showed normalization of voice, movement of the vocal cord on the injured side, and electromyography evidence of reinnervation of the larynx muscles at 15 months after surgery.

KI-67 AND hTERT expression can aid in the distinction between malignant and benign pheochromocytoma and paraganglioma.

The clinical and histopathological distinction between benign and malignant pheochromocytomas and paragangliomas is difficult, and reliable diagnostic markers are lacking. Here we have evaluated the prognostic value of human telomerase reverse transcriptase (hTERT) gene expression detected by reverse transcription PCR (RT-PCR); telomerase activity (TA) measured by TRAP (telomeric repeat amplification protocol) assay; immunohistochemical staining for Ki-67/MIB-1; and the mRNA expression of matrix metalloproteinase (MMP)-2 and EMMPRIN (extracellular matrix metalloproteinase inducer) analyzed by in situ hybridization in 32 primary pheochromocytomas or abdominal paragangliomas. hTERT was expressed in 7/11 malignant tumors (defined as presence of metastasis and/or extensive local invasion) as compared with in 2/21 benign tumors. All of the benign tumors showed <1% proliferative activity, as measured by Ki-67/MIB-1 staining. In all three patients with malignant tumors who developed metastases and/or invasive local recurrence during follow-up, the tumors were positive for either hTERT expression or Ki-67/MIB-1 immunoreactivity. TA was not a significant discriminator between benign and malignant tumors, and the value of EMMPRIN and MMP-2 as predictive markers was limited. In conclusion, the findings imply that the combined use of Ki-67/MIB-1 and hTERT, in addition to histopathology, provides a highly specific tool to identify benign pheochromocytoma and abdominal paraganglioma cases that are not at risk of developing recurrent or metastatic disease.

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.

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 11 beta-hydroxylase (CYP11B1), 18-hydroxylase (CYP11B2), 17 alpha-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 Cushing patients showed no detectable release of aldosterone. In contrast, tumors from patients with primary aldosteronism, Cushing's syndrome, and no hyperfunction 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 Cushing 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.

The HCR gene on 6p21 is unlikely to be a psoriasis susceptibility gene.

The PSORS1 locus in the human major histocompatibility complex on 6p21 has been consistently associated with psoriasis in populations of diverse ethnicity. The HLA-C allele Cw*0602, located therein, has been found in up to 67% of psoriasis patients but is no longer considered a candidate gene in itself. The alpha-helix coiled-coil rod homolog gene (HCR, previously Pg8) is located 110 kb from the HLA-C gene, positioned between the CDSN and SC1 genes, within a region thought to harbor a psoriasis gene (PSORS1). We investigated the HCR gene for disease association by direct sequencing of nine polymerase chain reaction products amplified from a series of Swedish psoriasis patients and controls. We found that HCR is a very polymorphic gene with 25 polymorphisms in the open reading frame alone, of which 10 demonstrated disease association; however, the relationship between HCR polymorphisms and HLA-Cw*0602 indicates that HCR cannot truly be considered a likely candidate gene. We investigated Cw*0602 association while stratifying for HCR single nucleotide polymorphisms. We also investigated HCR single nucleotide polymorphism association with the disease while stratifying for the presence of Cw*0602. We found that whichever single nucleotide polymorphism that was stratified for, there was still a strongly significant Cw*0602 association with psoriasis; however, when we stratified for Cw*0602 presence, only one silent polymorphism showed significant association. In a recent similar study this polymorphism was actually found to be decreased in psoriasis individuals. Thus we conclude that HCR polymorphisms display association with psoriasis due to linkage disequilibrium with Cw*0602 and is, therefore, unlikely to be directly involved in the development of psoriasis.

Malignant Fibrous Histiocytoma, Aggressive Fibromatosis and Benign Fibrous Tumors Express mRNA for the Metalloproteinase Inducer EMMPRIN and the Metalloproteinases MMP-2 and MT1-MMP.

PURPOSE: Extracellular matrix metalloproteinase inducer (EMMPRIN) has been shown to stimulate fibroblasts to production of matrix metalloproteinases (MMPs). MMPs comprise a family of proteolytic enzymes implicated in the degradation of extracellular matrix which has been proposed to be one of the essential steps in tumor invasion and metastases. In the present study we investigated the expression and location of mRNAs for EMMPRIN, matrix metalloproteinase-2 (MMP-2), and membrane-type 1 matrix metalloproteinase (MT1-MMP) in mesenchymal tumors with different tendencies to recur or metastasize. SUBJECTS: Eight malignant fibrous histiocytomas (MFH), seven aggressive fibromatosis (AF), and six benign fibrous tumors (BF). METHOD: The mRNA-expression of EMMPRIN, MMP-2 and MT1-MMP were studied using mRNA in situ hybridization technique. RESULTS: The mRNA-expression of EMMPRIN, MMP-2 and MT1-MMP respectively were found at varying frequency and level in all tumor types. The mRNAs corresponding to EMMPRIN and MMP-2 were seen in neoplastic cells as well as in endothelial cells both inside and outside the tumor pseudo-capsule, whereas MT1-MMP was seen only within the tumors. The estimated mRNA levels of EMMPRIN and MMP-2 covariated significantly. Overall, the highest expression was found in the MFH tumors and the lowest levels in the BF tumors. DISCUSSION: These findings suggest that the MMP-inducer EMMPRIN and the extracellular matrix degrading system involving the metalloproteinases MMP-2 and MT1-MMP is frequently activated in mesenchymal tumors. The covariation between EMMPRIN and MMP-2 support previous findings that EMMPRIN may be an inducer of MMP-2. The high levels of MMP-2 mRNA in MFH indicate a relationship between the proteolytic activity of MMP-2 and the tumor aggressiveness.

Increased presence of eosinophilic granulocytes expressing transforming growth factor-beta1 in collagenous colitis.

BACKGROUND: Collagenous colitis is a disease characterized by chronic watery diarrhea, and on microscopic examination of colonic tissue, a typical thickening of the subepithelial collagen layer is seen. The etiology and pathophysiology behind this disease state are largely unknown. METHODS: We have used in situ hybridization and immunohistochemistry to study the expression of transforming growth factor (TGF) -beta1, a growth factor with the capacity to cause accumulation of collagen in tissues, in collagenous colitis. Colonic pinch biopsy specimens from a total of 34 patients were investigated: 17 patients with collagenous colitis and 17 controls. RESULTS: In patients with collagenous colitis there was increased expression of the TGF-beta1 gene compared with controls, as visualized by in situ hybridization. The vast majority of the TGF-beta1-expressing cells were eosinophils, both in collagenous colitis and controls, but there were also scattered fibroblastic and histiocytic stromal cells. Immunohistochemistry showed the presence of TGF-beta1, mainly in eosinophils, in the colonic mucosa. Morphometric quantification showed 603 +/- 192 eosinophils/mm2, (mean +/- standard error of the mean) in the colonic mucosa of patients with collagenous colitis compared with 30 +/- 7 eosinophils/mm2 in the controls. CONCLUSIONS: The present results suggest that eosinophils expressing TGF-beta1 may be of pathophysiologic importance in the connective tissue remodeling seen in collagenous colitis.

Expression analysis of RET and the GDNF/GFRalpha-1 and NTN/GFRalpha-2 ligand complexes in pheochromocytomas and paragangliomas.

Pheochromocytoma and its extra-adrenal counterpart paraganglioma are rare catecholamine producing tumors which usually occur sporadically but may also be a part of neuroendocrine tumor syndromes such as multiple endocrine neoplasia type 2A (MEN 2A). Activating mutations of the RET proto-oncogene which is the underlying cause of MEN 2A, is also seen in approximately 10% of sporadic pheochromocytomas. Glial cell line derived neurotrophic factor (GDNF) and neurturin (NTN) have been shown to function as independent ligands to RET, binding in a complex with the membrane-bound receptors GFRalpha-1 and GFRalpha-2 respectively. Here we have investigated the mRNA expression of RET and its ligand complexes, GDNF/GFRalpha-1 and NTN/GFRalpha-2, in a panel of pheochromocytomas and paragangliomas using mRNA in situ hybridization. RET expression was evident in normal adrenal medulla, and in 13/15 pheochromocytomas, including 5/5 MEN 2A associated tumors, but only in 1/10 paragangliomas. The frequent expression of RET in the pheochromocytomas suggest that this gene might be involved in the tumorigenesis. However, no expression of GDNF/GFRalpha-1 or NTN/GFRalpha-2 could be detected in any of the 25 tumors analyzed, suggesting that these ligand complexes are not important in the development of pheochromocytoma or paraganglioma.

The human cationic antimicrobial protein (hCAP-18) is expressed in the epithelium of human epididymis, is present in seminal plasma at high concentrations, and is attached to spermatozoa.

Innate immunity is important for the integrity of the host against potentially invasive pathogenic microorganisms in the environment. Antibiotic peptides with broad antimicrobial activity are part of the innate immune system. We investigated the presence of the cathelicidin, human cationic antimicrobial protein (hCAP-18), in the male reproductive system. We found strong expression of the hCAP-18 gene by in situ hybridization and hCAP-18 protein, as detected by immunohistochemistry, in the epithelium of the epididymis, but not in the testis. The highest expression in the epididymis was in the caudal part. Western blotting showed a doublet band, the upper part corresponding to the size of hCAP-18 in plasma and neutrophils. Using a specific enzyme-linked immunosorbent assay (ELISA), levels of 86.5 +/- 37.8 microg/ml (mean +/- standard deviation; range, 41.8 to 142.8 microg/ml; n = 10) were detected in seminal plasma from healthy donors, which is 70-fold higher than the level in blood plasma. Flow cytometry and immunocytochemistry revealed the presence of hCAP-18 on spermatozoa. ELISA measurement showed levels of 196 ng/10(6) spermatozoa, corresponding to 6.6 x 10(6) molecules of hCAP-18 per spermatozoon. Our results suggest a key role for hCAP-18 in the antibacterial integrity of the male reproductive system. The attachment of hCAP-18 to spermatozoa may implicate a role for hCAP-18 in conception.

Characterization of five novel human genes in the 11q13-q22 region.

The redundancy of sequences in dbEST has approached a level where contiguous cDNA sequences of genes can be assembled, without the need to physically handle the clones from which the ESTs are derived. This is termed EST based in silico gene cloning. With the availability of sequence chromatogram files for a subset of ESTs, the quality of EST sequences can be ascertained accurately and used in contig assembly. In this report, we performed a study using this approach and isolated five novel human genes, C11orf1-C11orf5, in the 11q13-q22 region. The full open reading frames of these genes were determined by comparison with their orthologs, of which four mouse orthologs were isolated (c11orf1, c11orf2, c11orf3 and c11orf5). These genes were then analyzed using several proteomics tools. Both C11orf1 and C11orf2 are nuclear proteins with no other distinguishing features. C11orf3 is a cytoplasmic protein containing an ATP/GTP binding site, a signal peptide located in the N-terminus and a similarity to the C. elegans protein "Probable ARP 2/3 complex 20kD subunit." C11orf4 is a peptide which displays four putative transmembrane domains and is predicted to have a cytoplasmic localization. It contains signal peptides at the N- and C-termini. C11orf5 is a putative nuclear protein displaying a central coiled coil domain. Here, we propose that this purely EST-based cloning approach can be used by modestly sized laboratories to rapidly and accurately characterize and map a significant number of human genes without the need of further sequencing.

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.

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.

Expression of matrix metalloproteinase gelatinase A messenger ribonucleic acid in parathyroid carcinomas.

BACKGROUND: The incidence of parathyroid cancer in patients with hyperparathyroidism is less than 1%. However, these few cases cause diagnostic problems in the absence of clear-cut invasion of adjacent organs or metastasis. New markers are needed to increase diagnostic accuracy. METHODS: Thirty-one parathyroid tumors from patients with primary hyperparathyroidism were collected worldwide. Eighteen tumors were classified as unequivocal cancers, whereas 13 tumors were considered equivocal because of a lack of infiltrative growth or evidence of recurrence. Paraffin sections were hybridized with a 35S-labeled riboprobe complementary to gelatinase A mRNA, dipped in photographic emulsion, developed, counterstained, and then evaluated by light- and dark-field microscopy. RESULTS: Fourteen of the 18 unequivocal parathyroid cancers expressed gelatinase A, as compared with the equivocal tumors, of which only 4 of 13 showed expression. The strongest hybridization signal was seen in stromal cells at the tumor border, most likely fibroblasts and macrophages. No expression was detected in tumor cells. CONCLUSIONS: Invasive growth of many tumors is facilitated by proteolytic enzymes, such as gelatinase A. The presence of gelatinase A mRNA in parathyroid tumors strengthens the suspicion of malignancy but cannot be used as a definitive marker of malignancy.

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.

The human cationic antimicrobial protein (hCAP18), a peptide antibiotic, is widely expressed in human squamous epithelia and colocalizes with interleukin-6.

Peptide antibiotics are widespread in nature and, by providing a rapid first line of defense, may be key players in the innate immune system. Although epithelia are the main barriers shielding the internal environment from microorganisms, the role for peptide antibiotics in epithelial protection is unclear. We recently reported that the human cationic antimicrobial protein hCAP18, the precursor of the antimicrobial peptide called LL-37, is not expressed by normal human keratinocytes but is induced in various inflammatory skin disorders. In the present study we demonstrate that hCAP18 is consistently expressed at both mRNA and protein levels in squamous epithelia of the mouth, tongue, esophagus, cervix, and vagina in humans. The gene for hCAP18 contains promoter elements that are potentially regulated by interleukin-6, and our data further show a colocalization between interleukin-6 and hCAP18 expression in these tissues. Our finding that hCAP18 is widely produced in squamous epithelia suggests a role for this peptide in epithelial antimicrobial defense. Furthermore, colocalization with interleukin-6 indicates a potential local mechanism for the upregulation of hCAP18 at the epithelial surfaces.

Low-dose oral pulse methotrexate as monotherapy in elderly patients with bullous pemphigoid.

BACKGROUND: Prednisone alone or in combination with an immunosuppressive drug is usually effective in controlling bullous pemphigoid. However, corticosteroids often cause potentially hazardous side effects, especially in elderly patients. OBJECTIVE: Our purpose was to evaluate low-dose treatment with methotrexate in elderly patients with generalized bullous pemphigoid. METHODS: Oral methotrexate, at an initial dosage of 5 mg/wk, was given to 11 consecutive patients older than 70 years of age who were not responding to potent topical steroids. If the response was insufficient, the methotrexate dose was increased by 2.5 mg/wk to a maximum of 12.5 mg/wk. RESULTS: All patients responded with a marked and rapid decrease in disease activity. The disease was controlled in the majority of patients (8 of 11) with 5 to 7.5 mg of methotrexate per week. Three patients required a weekly dose of 10 to 12.5 mg. At 24 months of follow-up 7 patients were in complete remission and did not require methotrexate. CONCLUSION: Our study suggests that low-dose oral pulse methotrexate constitutes an effective therapeutic alternative in elderly patients with generalized bullous pemphigoid.

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.