Somatic MED12 mutations in uterine leiomyosarcoma and colorectal cancer.

BACKGROUND: Mediator complex participates in transcriptional regulation by connecting regulatory DNA sequences to the RNA polymerase II initiation complex. Recently, we discovered through exome sequencing that as many as 70% of uterine leiomyomas harbour specific mutations in exon 2 of mediator complex subunit 12 (MED12). In this work, we examined the role of MED12 exon 2 mutations in other tumour types. METHODS: The frequency of MED12 exon 2 mutations was analysed in altogether 1158 tumours by direct sequencing. The tumour spectrum included mesenchymal tumours (extrauterine leiomyomas, endometrial polyps, lipomas, uterine leiomyosarcomas, other sarcomas, gastro-intestinal stromal tumours), hormone-dependent tumours (breast and ovarian cancers), haematological malignancies (acute myeloid leukaemias, acute lymphoid leukaemias, myeloproliferative neoplasms), and tumours associated with abnormal Wnt-signalling (colorectal cancers (CRC)). RESULTS: Five somatic alterations were observed: three in uterine leiomyosarcomas (3/41, 7%; Gly44Ser, Ala38_Leu39ins7, Glu35_Leu36delinsVal), and two in CRC (2/392, 0.5%; Gly44Cys, Ala67Val). CONCLUSION: Somatic MED12 exon 2 mutations were observed in uterine leiomyosarcomas, suggesting that a subgroup of these malignant tumours may develop from a leiomyoma precursor. Mutations in CRC samples indicate that MED12 may, albeit rarely, contribute to CRC tumorigenesis.

Large performance variation does not affect outcome in the Finnish cervical cancer screening programme.

OBJECTIVE: Cytology screening for prevention of cervical cancer can reduce incidence and mortality by more than 80% in settings with good organization and rigorous quality control. Audit studies are essential for reaching and maintaining a high quality of screening. The aim of this study was to evaluate variation in performance indicators by screening laboratory and assess the impact on the effectiveness of screening as indicated by cervical intraepithelial neoplasia grade 3 and above (CIN3+) rates after a negative screen. METHODS: Seven cytology screening laboratories operating during 1990-1999 with a total of 953 610 screening tests performed were included in the study. By linking screening and cancer register files, all cases of CIN3+ diagnosed in the screened population were identified. For 395 CIN3+ cases with a preceding negative screen and 787 controls, a re-evaluation of smears was undertaken to uncover false negative screening tests. Performance parameters and rates of CIN3+ after a negative screen were analysed for interlaboratory heterogeneity. RESULTS: The rates of follow-up recommendations and referrals varied by up to 3.6- (2.8-10.2%) and 4.0-fold (0.03-0.12%), respectively. CIN1, CIN2 and CIN3+ screen detection rates differed by up to 8.5- (0.02-0.17%), 5.4- (0.05-0.25%) and 3.3-fold (0.05-0.18%). False negative rates determined by re-evaluation showed up to 2.1-fold differences (29-62%). Rates of CIN3+ after a negative screen (0.023-0.048%) and as a proportion of total CIN3+ (15-31%) in the screened population were low and did not vary significantly. CONCLUSIONS: There were large variations in the sensitivity-specificity trade-off between laboratories, reflected in all performance indicators as well as in the test validity estimates of the re-evaluation phase, but not in screening effectiveness. Even though performance variations do not always have an impact on the effectiveness of screening, they lead to variations in cost, treatment and psychological burden, and should be addressed.

Complement activation during liver transplantation-special emphasis on patients with atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy often caused by mutations in complement factor H (CFH), the main regulator of alternative complement pathway. Because CFH is produced mainly by the liver, combined liver-kidney transplantation is a reasonable option in treatment of patients with severe aHUS. We studied complement activation by monitoring activation markers during liver transplantation in two aHUS patients treated extensively with plasma exchange and nine other liver transplantation patients. After the reperfusion, a clear increase in all the activation markers except C4d was observed indicating that the activation occurs mainly through the alternative pathway. Concentration of SC5b-9 was higher in the hepatic than the portal vein indicating complement activation in the graft. Preoperatively and early during the operation, the aHUS patients showed highest C3d concentrations but otherwise their activation markers were similar to the other patients. In the other patients, correlation was found between perioperative SC5b-9 concentration and postoperative alanine aminotransferase and histological changes. This study explains why supply of normal CFH by extensive plasma exchange is beneficial before combined liver-kidney transplantation of aHUS patients. Also the results suggest that perioperative inhibition of the terminal complement cascade might be beneficial if enhanced complement activation is expected.

Remission of membranous glomerulonephritis after pancreatectomy for pancreatic neuroendocrine neoplasm - a rare coincidence.

The concomitant existence of a non-malignant neuroendocrine tumor (NET) and membranous glomerulonephritis (MGN) is rare. We report a subject with kidney biopsy proven MGN and nephrotic syndrome in which a computerized scan tomography (CT) examination was performed revealing a pancreatic tumor. A pancreatectomy was performed and the tumor was shown to be a non-malignant NET with a malignant potential. Although treatment with corticosteroids was initiated remission of MGN was observed within the next month after pancreatectomy. The rapid remission observed shortly after pancreatectomy pointed to that tumor removal contributed to, and that neither spontaneous nor corticosteroid treatment alone did induce the rapid remission of the MGN. The coexistence of the two disorders NET and MGN is very rare, however. This is the first report on remission of MGN after pancreatectomy for a NET.

Anaplastic and poorly differentiated thyroid carcinoma: therapeutic strategies and treatment outcome of 52 consecutive patients.

Anaplastic thyroid carcinoma (ATC) is one of the most lethal malignancies; poorly differentiated thyroid carcinoma (PDTC) is a new diagnosis for rare aggressive thyroid tumours. Surgery is often considered the only chance for survival, but the benefit of surgery and subsequent multimodal therapy is unclear. We retrospectively analyzed the outcome of 44 ATC and 8 PDTC consecutive patients treated at Helsinki University Central Hospital between 1990 and 2008. All ATC and PDTC cases were re-examined and reclassified histologically. Median survival was only 3.1 months for ATC, but 3.7 years for PDTC. Most patients in both groups eventually died of cancer. ATC patients were older than PDTC patients (74 vs. 66 years). Nodal and distant metastases had a negative impact on survival (ATC; p = 0.038, p = 0.008). Long-term survivors in both groups were stage N0M0 at presentation. Multimodal therapy was successful for 9 (20%) ATC patients, and their median survival was the longest (11.6 months) among treatment groups. Most PDTC patients (88%) underwent total thyroidectomy followed by radioiodine ablation; the only 2 who received chemotherapy survived longest. Although ATC and PDTC are both aggressive thyroid carcinomas, multimodal therapy for both can provide a chance of prolonged survival in patients with locoregional disease.

A common variant in PNPLA3, which encodes adiponutrin, is associated with liver fat content in humans.

AIMS/HYPOTHESIS: It has recently been suggested that the rs738409 G allele in PNPLA3, which encodes adiponutrin, is strongly associated with increased liver fat content in three different ethnic groups. The aims of the present study were as follows: (1) to try to replicate these findings in European individuals with quantitative measures of hepatic fat content; (2) to study whether the polymorphism influences hepatic and adipose tissue insulin sensitivity; and (3) to investigate whether PNPLA3 expression is altered in the human fatty liver. METHODS: We genotyped 291 Finnish individuals in whom liver fat had been measured using proton magnetic resonance spectroscopy. Hepatic PNPLA3 expression was measured in 32 participants. Hepatic and adipose tissue insulin sensitivities were measured using a euglycaemic-hyperinsulinaemic (insulin infusion 0.3 mU kg(-1) min(-1)) clamp technique combined with infusion of [3-(3)H]glucose in 109 participants. RESULTS: The rs738409 G allele in PNPLA3 was associated with increased quantitative measures of liver fat content (p = 0.011) and serum aspartate aminotransferase concentrations (p = 0.002) independently of age, sex and BMI. Fasting serum insulin and hepatic and adipose tissue insulin sensitivity were related to liver fat content independently of genotype status. PNPLA3 mRNA expression in the liver was positively related to obesity (r = 0.62, p < 0.0001) and to liver fat content (r = 0.58, p = 0.025) in participants who were not morbidly obese (BMI < 40 kg/m(2)). CONCLUSIONS/INTERPRETATION: A common variant in PNPLA3 increases the risk of hepatic steatosis in humans.

Histologically Verified Biliary Invasion was Associated with Impaired Liver Recurrence-Free Survival in Resected Colorectal Cancer Liver Metastases.

BACKGROUND AND AIMS: The impact of biliary invasion on recurrence and survival, after resection of colorectal cancer liver metastases, is not well known as publications are limited to small patient series. The aim was to investigate if biliary invasion in liver resected patients associated with liver relapses and recurrence-free survival. Secondary endpoints included association with other prognostic factors, disease-free survival and overall survival. MATERIALS AND METHODS: All patients with histologically verified biliary invasion (n = 31, 9%) were identified among 344 patients with liver resection between January 2009 and March 2015. Controls (n = 78) were selected from the same time period and matched for, among others, size and number of colorectal cancer liver metastasis. RESULTS: Median liver recurrence-free survival was significantly shorter in patients with biliary invasion than in controls (15.3 months versus not reached; p = 0.031) and more relapses were noted in the liver (61.3% versus 33.3%; p = 0.010), respectively. In univariate analyses for liver recurrence-free survival, biliary invasion was the only significant prognostic factor; p = 0.034. There were no statistical differences in disease-free and overall survival between the groups. CONCLUSION: Biliary invasion was associated with higher liver recurrence rates and shorter liver recurrence-free survival in patients with resected colorectal cancer liver metastasis.

No evidence of somatic aryl hydrocarbon receptor interacting protein mutations in sporadic endocrine neoplasia.

Germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene were recently observed in patients with pituitary adenoma predisposition (PAP). Though AIP mutation-positive individuals with prolactin-, mixed growth hormone/prolactin-, and ACTH-producing pituitary adenomas as well as non-secreting pituitary adenomas have been reported, most mutation-positive patients have had growth hormone-producing adenomas diagnosed at relatively young age. Pituitary adenomas are also component tumors of some familial endocrine neoplasia syndromes such as multiple endocrine neoplasia type 1 (MEN1) and Carney complex (CNC). Genes underlying MEN1 and CNC are rarely mutated in sporadic pituitary adenomas, but more often in other lesions contributing to these two syndromes. Thus far, the occurrence of somatic AIP mutations has not been studied in endocrine tumors other than pituitary adenomas. Here, we have analyzed 32 pituitary adenomas and 79 other tumors of the endocrine system for somatic AIP mutations by direct sequencing. No somatic mutations were identified. However, two out of nine patients with prolactin-producing adenoma were shown to harbor a Finnish founder mutation (Q14X) with a complete loss of the wild-type allele in the tumors. These results are in agreement with previous studies in that prolactin-producing adenomas are component tumors in PAP. The data also support the previous finding that somatic AIP mutations are not common in pituitary adenomas and suggest that such mutations are rare in other endocrine tumors as well.

The MDM2 promoter polymorphism SNP309T-->G and the risk of uterine leiomyosarcoma, colorectal cancer, and squamous cell carcinoma of the head and neck.

BACKGROUND: MDM2 acts as a principal regulator of the tumour suppressor p53 by targeting its destruction through the ubiquitin pathway. A polymorphism in the MDM2 promoter (SNP309) was recently identified. SNP309 was shown to result, via Sp1, in higher levels of MDM2 RNA and protein, and subsequent attenuation of the p53 pathway. Furthermore, SNP309 was proposed to be associated with accelerated soft tissue sarcoma formation in both hereditary (Li-Fraumeni) and sporadic cases in humans. METHODS: We evaluated the possible contribution of SNP309 to three tumour types known to be linked with the MDM2/p53 pathway, using genomic sequencing or restriction fragment length polymorphism as screening methods. Three separate Finnish tumour materials (population based sets of 68 patients with early onset uterine leiomyosarcomas and 1042 patients with colorectal cancer, and a series of 162 patients with squamous cell carcinoma of the head and neck) and a set of 185 healthy Finnish controls were analysed for SNP309. RESULTS: Frequencies of SNP309 were similar in all four cohorts. In the colorectal cancer series, SNP309 was somewhat more frequent in women and in patients with microsatellite stable tumours. Female SNP309 carriers were diagnosed with colorectal cancer approximately 2.7 years earlier than those carrying the wild type gene. However, no statistically significant association of SNP309 with patients' age at disease onset or to any other clinicopathological parameter was found in these three tumour materials. CONCLUSION: SNP309 had no significant contribution to tumour formation in our materials. Possible associations of SNP309 with microsatellite stable colorectal cancer and with earlier disease onset in female carriers need to be examined in subsequent studies.

Lack of MSH2 and MSH6 characterizes endometrial but not colon carcinomas in hereditary nonpolyposis colorectal cancer.

Hereditary nonpolyposis colorectal cancer syndrome is associated with an inherited predisposition to primarily colorectal cancer (CRC) and endometrial cancer (EC); however, the biological basis of the organ involvement remains unknown. As an attempt to explore whether the expression levels of MLH1, MSH2, and MSH6 may play a role, we used immunohistochemistry to study 42 ECs and 35 CRCs from patients carrying the same predisposing mutations. Among MSH2 mutation carriers, MLH1 was expressed in both tumor types, whereas MSH2 and, in many cases, also MSH6, were absent. Remarkably, among MLH1 mutation carriers, 54% of ECs (21 of 39), but none of the CRCs (0 of 32), lacked the MSH2 and/or MSH6 protein in addition to lacking MLH1 protein expression. These results demonstrate a marked difference between hereditary nonpolyposis colorectal cancer-related CRCs and ECs and suggest that the development of the latter tumors is selectively associated with the MSH2/MSH6 protein complex deficiency.

Endothelial growth factor receptors in human fetal heart.

BACKGROUND: Endothelial receptor tyrosine kinases include 3 members of the vascular endothelial growth factor receptor (VEGFR) family and 2 members of the angiopoietin receptor (Tie) family. In addition, the VEGF(165) isoform binds to neuropilin-1 (NP-1), a receptor for collapsins/semaphorins. The importance of these receptors for vasculogenesis and angiogenesis has been shown in gene-targeted mice, but so far, little is known about their exact expression patterns in the human vasculature. METHODS AND RESULTS: Frozen sections of human fetal heart were stained immunohistochemically with receptor-specific monoclonal (VEGFR, Tie) or polyclonal (NP-1) antibodies. The following patterns were observed: The endocardium was positive for VEGFR-1, VEGFR-2, NP-1, Tie-1, and Tie-2 but negative for VEGFR-3. The coronary vessels were positive for Tie-1, Tie-2, VEGFR-1, and NP-1 and negative for VEGFR-2 and VEGFR-3. Myocardial capillaries and epicardial blood vessels stained for VEGFR-1, VEGFR-2, NP-1, and Tie-1; myocardial capillaries and epicardial veins weakly for Tie-2; and epicardial lymphatic vessels for VEGFR-2 and VEGFR-3, weakly for Tie-1 and Tie-2, but not for VEGFR-1 or NP-1. CONCLUSIONS: The results demonstrate differential expression of the endothelial growth factor receptors in distinct types of vessels in the human heart. This information is useful for the understanding of their roles in physiological and pathological processes and for their diagnostic and therapeutic application in cardiovascular medicine.

The aberrant expression of the gastric inhibitory polypeptide (GIP) receptor in adrenal hyperplasia: does chronic adrenocorticotropin exposure stimulate up-regulation of GIP receptors in Cushing's disease?

CONTEXT: Cortisol secretion is usually under the control of ACTH. However, cortisol secretion occurs in response to gastric inhibitory polypeptide (GIP) in rare cases of food-dependent Cushing's syndrome (CS). OBJECTIVE: We have investigated whether chronic ACTH stimulation or activation of the ACTH signaling pathway might be associated with GIP receptor (GIPR) expression. DESIGN: RT-PCR analysis and primary culture of hyperplastic adrenals. PATIENTS: All patients presented with CS: 20 unilateral adrenal adenomas, five Cushing's disease, one food-dependent CS. RESULTS: RT-PCR revealed GIPR expression in all hyperplastic adrenals studied. No RT-PCR product could be detected in two normal adrenals or 20 hyperfunctioning adrenal adenomas. Primary culture revealed a significant cAMP response to ACTH in all adrenals available for study (EC50, 8.1 x 10(-10) M in normals, 4.7 x 10(-10) M in Cushing's disease, and 4.4 x 10(-10) M in food-dependent disease). However, cultures taken from all four ACTH-dependent and the one food-dependent hyperplastic adrenals studied were also responsive to GIP (EC50 for cAMP, 1.3 x 10(-9) M in Cushing's disease and 4.1 x 10(-10) M in food-dependent disease). Fasting cortisol levels were low in the case of food-dependant Cushing's, rising postprandially as predicted. However, there was no trend toward low fasting or high postprandial cortisol in the other cases, suggesting that the presence of detectable GIPR alone, albeit with definite function in vitro, is not sufficient to cause clinically food-dependent CS. CONCLUSIONS: These data are consistent with the hypothesis that chronic ACTH stimulation or constitutive activation of the ACTH signaling pathway may be associated with aberrant GIPR expression, and suggest one mechanism for the pathogenesis of this phenomenon.

AIP inactivation leads to pituitary tumorigenesis through defective Gαi-cAMP signaling.

The aryl hydrocarbon receptor interacting protein (AIP) is a tumor-suppressor gene underlying the pituitary adenoma predisposition. Thus far, the exact molecular mechanisms by which inactivated AIP exerts its tumor-promoting action have been unclear. To better understand the role of AIP in pituitary tumorigenesis, we performed gene expression microarray analysis to examine changes between Aip wild-type and knockout mouse embryonic fibroblast (MEF) cell lines. Transcriptional analyses implied that Aip deficiency causes a dysfunction in cyclic adenosine monophosphate (cAMP) signaling, as well as impairments in signaling cascades associated with developmental and immune-inflammatory responses. In vitro experiments showed that AIP deficiency increases intracellular cAMP concentrations in both MEF and murine pituitary adenoma cell lines. Based on knockdown of various G protein α subunits, we concluded that AIP deficiency leads to elevated cAMP concentrations through defective Gαi-2 and Gαi-3 proteins that normally inhibit cAMP synthesis. Furthermore, immunostaining of Gαi-2 revealed that AIP deficiency is associated with a clear reduction in Gαi-2 protein expression levels in human and mouse growth hormone (GH)-secreting pituitary adenomas, thus indicating defective Gαi signaling in these tumors. By contrast, all prolactin-secreting tumors showed prominent Gαi-2 protein levels, irrespective of Aip mutation status. We additionally observed reduced expression of phosphorylated extracellular signal-regulated kinases 1/2 and cAMP response element-binding protein levels in mouse and human AIP-deficient somatotropinomas. This study implies for the first time that a failure to inhibit cAMP synthesis through dysfunctional Gαi signaling underlies the development of GH-secreting pituitary adenomas in AIP mutation carriers.

Corticosterone regulates cell proliferation and cytochrome P450 cholesterol side-chain cleavage enzyme messenger ribonucleic acid expression in primary cultures of fetal rat adrenals.

Glucocorticoids are known to inhibit growth in many different cell types. Although corticosterone is secreted by the adrenal cortex, its direct effect on the growth of different zones is poorly determined. We studied the effects of corticosterone on cell proliferation and cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc; the rate-limiting step in adrenal steroidogenesis) messenger RNA (mRNA) accumulation in primary cultures of fetal rat adrenals. Adrenocortical cells, grown in the absence of ACTH for 3 weeks, possess typical features of zona glomerulosa cells. These cells differentiate into fasciculata-type cells and undergo biphasic proliferation when stimulated with ACTH. The primary antimitogenic phase of 24 h is followed by rapidly increased bromodeoxyuridine incorporation after 72 h of ACTH treatment. If the treatment is continued, the proliferation decreases again, but remains higher than the proliferation of the untreated cells. Undifferentiated zona glomerulosa-type cells secrete very low amounts of corticosterone. The 10% basal proliferation was not affected if exogenous corticosterone was added. However, if corticosterone was combined with ACTH for 3 days, it blocked the stimulatory growth effect of ACTH dose dependently. Etomidate, an inhibitor of steroidogenic enzymes, completely blocked corticosterone secretion. In our cultures it inhibited 50% of the proliferation of the zona glomerulosa-type cells. However, its effect was totally opposite in long term ACTH-treated cultures; in these fasciculata-type cells, etomidate stimulated the proliferation rate 3-fold. P450scc gene expression was low in undifferentiated zona glomerulosa-like cells. ACTH stimulation increased P450scc mRNA expression 10-fold. Exogenous corticosterone inhibited ACTH-induced P450scc mRNA accumulation by 50%, whereas etomidate doubled it. Our data suggest that a low corticosterone concentration supports the proliferation of undifferentiated zona glomerulosa-type cells, whereas a high corticosterone concentration inhibits the proliferation of differentiated zona fasciculata-type cells. In addition, a high corticosterone concentration may inhibit steroidogenesis by reducing P450scc expression. Thus, corticosterone may be an important modulator of adrenocortical cell proliferation and steroidogenesis in different zones of the adrenal cortex.

Increased expression of cyclooxygenase-2 in malignant pheochromocytomas.

Pheochromocytomas are rare tumors of the adrenal medulla or the paraganglion system. There are no histological or chemical markers available that define the malignant behavior of these tumors; so far only the discovery of metastases reveals malignancy. Cyclooxygenase (Cox) is the key enzyme in conversion of arachidonic acid to PGs, and two isoforms, Cox-1 and Cox-2, have been identified. Cox-2 has been associated with carcinogenesis, and it is overexpressed in many human malignancies. We have now investigated the expression of Cox-2 in normal adrenal gland, in 92 primary pheochromocytomas and in six metastases using immunohistochemistry and Northern blot and Western blot analyses. Cox-2 protein was expressed in the adrenal cortex, whereas the medulla was negative as detected by immunohistochemistry. Interestingly, all malignant pheochromocytomas (n = 8), regardless of the primary location of the tumor, showed moderate or strong Cox-2 immunoreactivity, whereas 75% of the benign adrenal tumors (n = 36) showed no or only weak immunopositivity. The staining was negative or weak in 79% of the adrenal tumors that showed histologically suspicious features (n = 24), but had not metastasized. Most of the pheochromocytoma samples studied also expressed low levels of Cox-2 mRNA. Our data show that normal adrenal medulla does not express Cox-2 immunohistochemically. However, strong Cox-2 protein expression was found in malignant pheochromocytomas, whereas most benign tumors expressed Cox-2 only weakly. To our knowledge, this is the first report on Cox-2 expression in pheochromocytomas and enhanced expression in malignant pheochromocytomas. These findings suggest that negative or weak Cox-2 expression in pheochromocytomas favors benign diagnosis.

Inhibin/activin betaB-subunit expression in pheochromocytomas favors benign diagnosis.

Malignancy of pheochromocytomas is difficult to estimate on the basis of histopathological features. Good prognostic markers are not available. In our search for new markers to differentiate malignant pheochromocytomas from benign ones we tested the value of inhibin/activin subunit expression. Inhibins are heterodimeric glycoproteins consisting of an alpha-subunit and either a betaA- or a betaB-subunit. Activins are composed of beta-subunits only. Immunohistochemically inhibin/activin betaB-subunit was strongly positive in the normal adrenal medulla, but the cortex was negative. A striking difference was found in inhibin/activin betaB expression between benign and malignant pheochromocytomas. The majority of benign adrenal tumors (27 of 30) showed strong or moderate immunoreactivity, whereas all seven malignant tumors were negative or only weakly positive for inhibin/activin betaB-subunit. The percentage of positively staining cells varied greatly in extraadrenal pheochromocytomas and in those benign tumors that showed over 5 mitoses/10 high power fields, necrosis, or capsular or vascular invasion, here called borderline tumors. Inhibin/activin betaB messenger ribonucleic acid was also found in pheochromocytomas. However, no significant differences in messenger ribonucleic acid levels were found in various types of tumors. Weak immunohistochemical positivity for inhibin/activin betaA-subunit was detected in the adrenal cortex, but the medulla and most of the pheochromocytomas were negative. Our data show that inhibin/activin betaB-subunit is expressed in normal adrenal medullary cells. Strong staining is found in most benign adrenal pheochromocytomas, whereas malignant tumors are almost negative. This suggests that loss of inhibin/activin betaB-subunit expression in pheochromocytomas may be used as an indicator of malignant potential.

Increased expression of tenascin in pheochromocytomas correlates with malignancy.

Tenascin is a significant extracellular matrix glycoprotein, which is upregulated in various neoplasias and pathologic processes. Pheochromocytomas are rare tumors of the sympathoadrenal system, whose malignancy is almost impossible to predict. There are no histologic or chemical markers available that would define the malignant behavior of these tumors, except the discovery of metastases. In our search for new markers, we investigated the immunohistochemical expression of tenascin in a large number of pheochromocytomas and paragangliomas. Seven tumors were metastasized and were thus considered malignant. Normal adrenal medulla was tenascin negative. A striking difference was seen between malignant and benign pheochromocytomas. All malignant pheochromocytomas expressed stromal tenascin strongly or moderately, whereas most benign pheochromocytomas (28 of 37, 70%) showed no or only weak immunopositivity. The staining was strong or moderate also in 13 of 28 (46%) of the tumors that showed histologically suspicious features, here called borderline tumors. Paragangliomas showed a more heterogeneous staining pattern, and no significant difference was found between benign and malignant paragangliomas. To our knowledge, this is the first study to demonstrate the expression of tenascin in pheochromocytomas and particularly the enhanced expression in malignant pheochromocytomas. We therefore suggest that tenascin may be associated with the malignant transformation and metastasis of pheochromocytomas. It is also a potential marker predicting more aggressive behavior in pheochromocytomas.

Role of adenylate cyclase-cyclic AMP-dependent signal transduction in the ACTH-induced biphasic growth effect of rat adrenocortical cells in primary culture.

ACTH has a biphasic effect on the proliferation of fetal rat adrenocortical cells in primary culture. Dramatic changes occurred during the first 72 h of ACTH stimulation, when incorporation of bromodeoxyuridine was used as an indicator of proliferation. The primary effect of ACTH was the inhibition of proliferation during the first 24 h, which was followed by an intense stimulatory phase during the third day of ACTH treatment. Cycloheximide (a protein synthesis inhibitor) prevented both the inhibitory and the stimulatory effects of ACTH, but did not affect the basal proliferation of unstimulated zona glomerulosa-like cells. Although adrenocortical cells stimulated with cyclic AMP (cAMP) derivatives, 8-bromo cAMP (8-Br cAMP) or dibutyryl cAMP ((Bu)2cAMP), differentiated morphologically into fasciculata-like cells, and secreted corticosterone and 18-OH-deoxycorticosterone, as did ACTH-stimulated cells, neither of the derivatives inhibited proliferation during the first 24 h of treatment. In contrast to ACTH, (Bu)2cAMP had a stimulatory effect on bromodeoxyuridine incorporation during the first 24 h of treatment. 8-Br cAMP did not change proliferation during the 24 h of treatment, but had a stimulatory effect after 72 h, which was not seen with (Bu)2cAMP. Thus, these results suggest that (1) differentiation, steroid hormone synthesis and the mitogenic effect of ACTH are transduced through the cAMP-mediated system, (2) the antimitogenic effect of ACTH is transduced via a cAMP-independent pathway and (3) both antimitogenic and mitogenic effects of ACTH are dependent on protein synthesis.

Protein kinase C signal transduction pathway in ACTH-induced growth effect of rat adrenocortical cells in primary culture.

ACTH exerts a biphasic effect on the growth of fetal rat adrenocortical cells in primary culture when bromodeoxyuridine (BrdU) incorporation is used as an indicator of proliferation. The immediate inhibitory effect during the first 24 h of ACTH stimulation is not dependent on cyclic AMP (cAMP). Protein kinase C (PKC) inhibitors H-7 and staurosporine blocked this inhibitory effect of ACTH, whereas 12-0-tetradecanoyl phorbol-13-acetate (TPA; a PKC activator) mimicked the ACTH-induced antimitogenic effect. The stimulatory growth effect of ACTH appears after 72 h of treatment. A similar mitogenic effect is also achieved with cAMP derivative 8-bromo cAMP (8-Br cAMP). However, both ACTH- and 8-Br cAMP-induced proliferations could be reduced with H-7. ACTH-induced corticosterone secretion was inhibited 50% with H-7 after 24 h, but 8-Br cAMP-induced secretion was unaffected. However, if the treatments were continued for 72 h, H-7 no longer reduced the steroid secretions. Reduction (50-75%) of cholesterol side-chain cleavage enzyme (P450scc) mRNA expression was also noted with H-7 in ACTH-treated cultures after 6 and 24 h. In contrast, TPA doubled the corticosterone secretion induced by 8-Br cAMP, but did not further increase the ACTH-induced secretion after 24 h. TPA alone, however, was not able to induce steroid secretion or P450scc mRNA expression. The morphological differentiation of fetal rat adrenocortical cells with ACTH or 8-Br cAMP from zona glomerulosa-like cells into zona fasciculata-like cells was not distributed by H-7 nor was it induced by TPA alone.(ABSTRACT TRUNCATED AT 250 WORDS)