VHL inactivation is an important pathway for the development of malignant sporadic pancreatic endocrine tumors.

A small subset of familial pancreatic endocrine tumors (PET) arises in patients with von Hippel-Lindau syndrome and these tumors may have an adverse outcome compared to other familial PET. Sporadic PET rarely harbors somatic VHL mutations, but the chromosomal location of the VHL gene is frequently deleted in sporadic PET. A subset of sporadic PET shows active hypoxia signals on mRNA and protein level. To identify the frequency of functionally relevant VHL inactivation in sporadic PET and to examine a possible prognostic significance we correlated epigenetic and genetic VHL alterations with hypoxia signals. VHL mutations were absent in all 37 PETs examined. In 2 out of 35 informative PET (6%) methylation of the VHL promoter region was detected and VHL deletion by fluorescence in situ hybridization was found in 14 out of 79 PET (18%). Hypoxia inducible factor 1alpha (HIF1-alpha), carbonic anhydrase 9 (CA-9), and glucose transporter 1 (GLUT-1) protein was expressed in 19, 27, and 30% of the 152 PETs examined. Protein expression of the HIF1-alpha downstream target CA-9 correlated significantly with the expression of CA-9 RNA (P<0.001), VHL RNA (P<0.05), and VHL deletion (P<0.001) as well as with HIF1-alpha (P<0.005) and GLUT-1 immunohistochemistry (P<0.001). These PET with VHL alterations and signs of hypoxia signalling were characterized by a significantly shortened disease-free survival. We conclude that VHL gene impairment by promoter methylation and VHL deletion in nearly 25% of PET leads to the activation of the HIF-pathway. Our data suggest that VHL inactivation and consecutive hypoxia signals may be a mechanism for the development of sporadic PET with an adverse outcome.

No mutations but an increased frequency of SDHx polymorphisms in patients with sporadic and familial medullary thyroid carcinoma.

Germline mutations of the three succinate dehydrogenase subunits SDHB, SDHC and SDHD have recently been associated with familial pheochromocytoma and paraganglioma. Several reasons make these genes candidate tumor suppressor genes for medullary thyroid carcinoma (MTC): (1) SDHB lies on chromosome 1p, the region known to be deleted most frequently in MTC, (2) MTCs develop from neural crest-derived cells, as do pheochromocytomas and paragangliomas and (3) patients with germline mutations of the Ret-protooncogene develop MTCs as well as pheochromocytomas, indicating a relationship of these tumors on a genetic level. Therefore, we attempted to determine whether the tumor suppressor genes SDHB, SDHC and SDHD are involved in sporadic and familial MTC. Somatic mutations of the SDH subunits were absent in all 35 investigated MTCs. Loss of heterozygosity was found in 27% (SDHB) and 4% (SDHD) respectively. While the frequency of non-coding, intronic polymorphisms did not differ in MTC patients compared with a control population, an accumulation of amino-acid coding polymorphisms (S163P in SDHB as well as G12S and H50R in SDHD) was found among MTC patients especially patients with familial tumors, suggesting a functional connection of coding SDH polymorphisms to activating Ret mutations.

BRAF and endocrine tumors: mutations are frequent in papillary thyroid carcinomas, rare in endocrine tumors of the gastrointestinal tract and not detected in other endocrine tumors.

The tumorigenesis of sporadic endocrine tumors is still not fully understood. Activating point mutations of the serine/threonine kinase gene BRAF located on 7q34 are found in a wide range of malignancies, with the highest frequency (66%) occurring in malignant melanomas. Melanomas are tumors of neural-crest-derived cells as are medullary thyroid carcinomas, pheochromocytomas and paragangliomas. BRAF has not been examined in endocrine tumors of the diffuse neuroendocrine system or of neural-crest-derived cells. We examined 130 endocrine tumors of the pancreas, parathyroid gland, adrenal medulla, paraganglia, lung and gastrointestinal tract as well as follicular and c-cell-derived thyroid tumors. We found a high rate of V559E mutations in papillary thyroid carcinomas (47%), one V599E mutation in a well-differentiated gastric endocrine carcinoma (malignant carcinoid), but no activating BRAF mutations in all other endocrine tumors examined. These results point towards different pathways in tumorigenesis of endocrine tumors of various localizations and only rare involvement of the MAP kinase (MAPK) pathway in a subset of malignant neuroendocrine tumors.

Preferential HER-2/neu overexpression and/or amplification in aggressive histological subtypes of invasive breast cancer.

AIMS: To investigate whether alterations of the HER2 gene occur more frequently in histologically unfavourable subtypes of invasive breast cancer. METHODS: The study was composed of nine invasive apocrine, six lipid-rich, 12 glycogen-rich, 11 micropapillary and 33 pleomorphic lobular breast carcinomas. Lymph node involvement was represented in all subgroups. HER2 status was confirmed in all cases by using immunohistochemistry (CB11, Herceptest) and fluorescent in-situ hybridization (FISH) analysis (Vysis). RESULTS: Micropapillary and apocrine carcinomas showed the highest rate of protein overexpression (72% and 66%) and gene amplification (45% and 44%). Protein overexpression was common in poorly differentiated pleomorphic lobular carcinomas (56%); however, this subgroup failed to show an increased number of gene copies by FISH (31%). The incidence of HER2 overexpression (33% and 50%, respectively) and gene amplification (25% and 33%, respectively) among glycogen-rich and lipid-rich carcinomas was not higher than that observed in breast cancer generally. CONCLUSION: Our data suggest that preferential involvement of the HER2 gene in micropapillary and apocrine breast carcinomas may contribute to their aggressive behaviour.

[Differentiated thyroid carcinoma. Surgery and significance of lymph node involvement]. / Differenziertes Schilddrüsenkarzinom. Zur Chirurgie und Bedeutung von Lymphknotenbefall.

INTRODUCTION: Nodal treatment in papillary (PTC) and in follicular (FTC) thyroid carcinoma is still a subject of debate. METHODS: 1974-95 therapeutic lymphadenectomy (30/95), 1996-1999 frequent prophylactic lymphadenectomy (32/57; P = 0.005) was used for PTC, with therapeutic lymphadenectomy for FTC (15/115). 131I was used selectively for pN1-tumours. RESULTS: PTC: The incidence of pN0-, but not of pN1-status increased significantly (P = 0.03). Nodal recurrence was observed in 5/89 (6%) with therapeutic, vs. 1/54 (2%) with prophylactic lymphadenectomy (P = NS), i.e. in 1/107 (0.9%) patients without evidence of nodal disease, vs. 5/36 (14%) of those with pN1-status (P = 0.0004). Survival at 25 yrs. in stages TNM I and II was 100%, i.e., independent of N-status. FTC: No nodal recurrence was observed. CONCLUSION: Occult untreated nodal disease represented no major clinical problem. Selective nodal treatment may offer optimal results; meticulous nodal dissection is indicated for N1-tumours.

Genomic imbalances in the progression of endocrine pancreatic tumors.

Endocrine pancreatic tumors (EPTs) are neoplasms with malignant potential. To explore the molecular basis of metastatic progression in human EPTs, we analyzed 17 paired specimens of primary EPTs and their metastases and 28 nonmetastatic EPTs using comparative genomic hybridization (CGH). Genomic alterations were detected in all of the matched primary/metastatic tumors and 19 (58%) nonmetastatic EPTs. The mean number of genomic changes was 17.3 in metastases, 12.5 in their primary tumors, and 4.5 in nonmetastatic EPTs. Statistical analysis of shared genomic changes in matched pairs of primary tumors and metastases showed a high probability (>95%) of a clonal relationship in 15 of the 17 cases. A closely related genetic pattern was also demonstrated on the basis of concordance analysis of the two groups. The most striking genomic changes which were enriched in metastases included gains of chromosomes 4 and 7 and losses of 21q. Other common regions of frequent losses (>40%) identified in metastases and/or their primary tumors involved 2p, 2q, 3p, 3q, 6q, 10p, and 11p, whereas frequently detected gains (>40%) in the paired tumors involved 5p, 5q, 12q, 14q, 17q, 18q, and 20q. These chromosomal aberrations were found in significantly fewer nonmetastatic EPTs. Some of these chromosomal loci may harbor genes contributing to the progression of EPT.

Deletion at 3p25.3-p23 is frequently encountered in endocrine pancreatic tumours and is associated with metastatic progression.

For several reasons, chromosome 3p is thought to be involved in the pathogenesis of sporadic endocrine pancreatic tumours (EPTs): von Hippel-Lindau's disease (VHL gene at 3p25.5) is associated with EPTs; 3p is frequently involved in solid human tumours; and comparative genomic hybridization has identified frequent losses at 3p in EPTs. This study investigated 99 benign and malignant tumours, including 20 metastases, from 82 patients, by microsatellite loss of heterozygosity (LOH) analysis and fluorescence in situ hybridization (FISH) in order to evaluate the importance of chromosome 3p deletions in the molecular pathogenesis and biological behaviour of EPTs, to elaborate a common region of deletion, and to narrow down putative tumour suppressor gene loci. Allelic losses of 3p were found in 58/99 (58.6%) of tumours in 45/82 (54.9%) patients; analysis of seven microsatellite markers (3p26-p21) revealed a common region of LOH at 3p25.3-p23. The LOH frequency was significantly higher in malignant than in benign neoplasms (70.2% versus 28.0%; p=0.001). In addition, a strong correlation was found between the loss of alleles on chromosome 3p and clinically metastatic disease (LOH of 73.7% in metastasizing versus 41.5% in non-metastasizing tumours; p=0.008). EPTs from these patients showed a tendency towards losing large parts or the entire short arm of chromosome 3 with tumour progression. Furthermore, FISH analysis revealed complete loss of chromosome 3 in ten out of 37 EPTs (27%). These results indicate that a putative tumour suppressor gene at 3p25.3-p23 may play a role in the oncogenesis of sporadic EPTs and that losses of larger centromeric regions are associated with metastatic progression.

Differentiated thyroid carcinoma. Follow-up of 264 patients from one institution for up to 25 years.

UNLABELLED: The optimum treatment for differentiated thyroid carcinoma (DTC) is still debated. Results obtained using a selective treatment strategy for papillary (PTC) and follicular (FTC) thyroid carcinoma over 25 years in one institution are reported. 149 patients (mean age 46 yrs) had PTC in TNM stages I-IV in 58%, 26%, 15% and 1% respectively. Total thyroidectomy and remnant 131I ablation (43%) were carried out in TNM high-risk patients (stages III and IV) and in low-risk patients (I and II) at risk for a (curable) recurrence (stages pN1 and/or pT4). Hemi- or total thyroidectomy, without radioiodine, was used in 76% of pT1-3 N0 tumours (68%). Central and/or lateral lymphadenectomy was performed in 42% of patients (electively in the last 4 years). The mean follow-up was 7 years. RESULTS: 6 patients died of PTC and 8/143 patients treated for cure had a recurrence (6 nodal, 1 contralateral, 1 local). In low-risk patients--including 68% of patients aged > or = 45 yrs--the cause specific 25-year survival rate was 100%, vs. 62% (at 15 years) (p < 0.0001) in high-risk patients. In stage I and stage II the recurrence-free survival rates at 25 years were 95% and 100% respectively. Risk factors for recurrence were macroscopic (p < 0.0001) but not microscopic local invasion (pT4); stage pN1 (p = 0.0004). Only 1/107 patients initially judged node-negative had a nodal recurrence. FTC (n = 115; mean age 56 yrs; mean follow-up 8 yrs): Cause-related death (n = 8) or serious recurrence (n = 3) occurred in 10/53 grossly invasive FTC, in 1/45 minimally invasive FTC with vascular invasion, and in none of 17 FTC with capsular invasion (CI) alone, under radical treatment (131I) in 75%, 33%, and 12% respectively. 20-year disease-free survival in grossly and in minimally invasive FTC was 78% and 95.5% respectively (p = 0.0007). Patients aged < 45 yrs and patients with minimally invasive FTC with CI alone (all ages) had 100% 20-year disease-free survival vs. 80% (p = 0.013) in the remainder. There was no curable recurrence in FTC. The ratio of grossly invasive FTC decreased (p < 0.0001) during the study period. CONCLUSIONS: Risk-0 groups may be defined and selected for a reduced extent of treatment (PTC pT1-3 N0; FTC < 45 yrs, or CI alone). Older (> or = 45 yrs) patients with PTC in stages I and II have an excellent prognosis (risk 0). With selective (therapeutic) lymphadenectomy the risk of nodal recurrence may be very low in node negative tumours, without use of radioiodine. Meticulous lymphadenectomy is indicated in pN1 tumours with nodal recurrences despite 131I (5/36 patients). The technique of capsular dissection for extracapsular total uni- or bilateral thyroidectomy provides excellent oncological and surgical results. A decrease in the incidence of FTC parallels a decrease in endemic goitre in Switzerland.

Putative tumor suppressor loci at 6q22 and 6q23-q24 are involved in the malignant progression of sporadic endocrine pancreatic tumors.

Our previous comparative genomic hybridization study on sporadic endocrine pancreatic tumors (EPTs) revealed frequent losses on chromosomes 11q, 3p, and 6q. The aim of this study was to evaluate the importance of 6q losses in the oncogenesis of sporadic EPTs and to narrow down the smallest regions of allelic deletion. A multimodal approach combining polymerase chain reaction-based allelotyping, double-target fluorescence in situ hybridization, and comparative genomic hybridization was used in a collection of 109 sporadic EPTs from 93 patients. Nine polymorphic microsatellite markers (6q13 to 6q25-q27) were investigated, demonstrating a loss of heterozygosity (LOH) in 62.2% of the patients. A LOH was significantly more common in tumors >2 cm in diameter than below this threshold as well as in malignant than in benign tumors. We were able to narrow down the smallest regions of allelic deletion at 6q22.1 (D6S262) and 6q23-q24 (D6S310-UTRN) with LOH-frequencies of 50.0% and 41.2 to 56.3%, respectively. Several promising tumor suppressor candidates are located in these regions. Additional fluorescence in situ hybridization analysis on 46 EPTs using three locus-specific probes (6q21, 6q22, and 6q27) as well as a centromere 6-specific probe revealed complete loss of chromosome 6 especially in metastatic disease. We conclude that the two hot spots found on 6q may harbor putative tumor suppressor genes involved not only in the oncogenesis but maybe also in the malignant and metastatic progression of sporadic EPTs.

Differential loss of chromosome 11q in familial and sporadic parasympathetic paragangliomas detected by comparative genomic hybridization.

Parasympathetic paragangliomas (PGLs) represent neuroendocrine tumors arising from chief cells in branchiomeric and intravagal paraganglia, which share several histological features with their sympathetic counterpart sympathoadrenal paragangliomas. In recent years, genetic analyses of the familial form of PGL have attracted considerable interest. However, the majority of paragangliomas occurs sporadically and it remains to be determined whether the pathogenesis of sporadic paraganglioma resembles that of the familial form. Furthermore, data on comparative genetic aberrations are scarce. To provide fundamental cytogenetic data on sporadic and hereditary PGLs, we performed comparative genomic hybridization using directly fluorochrome-conjugated DNA extracted from 12 frozen and 4 paraffin-embedded tumors. The comparative genomic hybridization data were extended by loss of heterozygosity analysis of chromosome 11q. DNA copy number changes were found in 10 (63%) of 16 tumors. The most frequent chromosomal imbalance involved loss of chromosome 11. Six of seven familial tumors and two of nine sporadic tumors showed loss of 11q (86% versus 22%, P = 0.012). Deletions of 11p and 5p were found in two of nine sporadic tumors. We conclude that overall DNA copy number changes are infrequent in PGLs compared to sympathetic paragangliomas and that loss of chromosome 11 may be an important event in their tumorigenesis, particularly in familial paragangliomas.

Genetic evidence for early divergence of small functioning and nonfunctioning endocrine pancreatic tumors: gain of 9Q34 is an early event in insulinomas.

The malignant potential among endocrine pancreatic tumors (EPTs) varies greatly and can frequently not be predicted using histopathological parameters. Thus, molecular markers that can predict the biological behavior of EPTs are required. In a previous comparative genomic hybridization study, we observed marked genetic differences between the various EPT subtypes and a correlation between losses of 3p and 6 and gains of 14q and Xq and metastatic disease. To search for genetic alterations that play a role during early tumor development, we have studied 38 small (< or =2 cm) EPTs, including 24 insulinomas and 10 nonfunctioning EPTs. Small EPTs are usually classified as clinically benign tumors in the absence of histological signs of malignancy. Using comparative genomic hybridization, we identified chromosomal aberrations in 27 EPTs (mean, 4.1). Interestingly, the number of gains differed strongly between nonfunctioning and functioning EPTs (3.4 versus 1.5, respectively; P = 0.0526), as did the number of aberrations in the benign (n = 30) and malignant (n = 8) tumors (3 versus 8.4, respectively; P = 0.0022). In the insulinomas, 9q gain (common region of involvement: 9q34) was most common (50%) and in nonfunctioning EPTs, gain of 4p was most common (40%). Most frequent losses in insulinomas involved 1p (20.8%), 1q, 4q, 11q, Xq, and Y (all 16.7%) and in nonfunctioning EPTs, 6q. Losses of 3pq and 6q and gains of 17pq and 20q proved to be strongly associated with malignant behavior in all of the small EPTs (P < or = 0.0219). Our results demonstrate marked genetic differences between small functioning and nonfunctioning EPTs, indicating that these subtypes evolve along different genetic pathways. In addition, our study endorses the importance of chromosomes 3 and 6q losses to discriminate EPTs with a malignant behavior from benign ones.

Gains of 12q13-14 and overexpression of mdm2 are frequent findings in intimal sarcomas of the pulmonary artery.

The characterization of clinical, histopathological, immunohistochemical, and genetic features of intimal sarcomas arising in the pulmonary artery is presented in this study. Four resected lungs, one endarterectomy specimen and three biopsies from eight patients (four males and four females; median age 41 years) suffering from intimal sarcomas of the pulmonary artery using conventional stains, immunohistochemistry, and comparative genomic hybridization (CGH) were analyzed. The predominant clinical presentation was dyspnea (all eight patients) and febrile pulmonary disease (six of eight). Signs of embolic lung disease were present in all patients. One patient died postoperatively, six patients died of disease 8-35 months after presentation, and one patient was alive 6 months after surgery. Histopathological examination of the submitted material showed spindle cell, partially myxoid and pleomorphic sarcomas. Metastases were histologically confirmed in three patients (lung, pleura, and skull). Immunohistochemically, vimentin was strongly expressed in all tumors. Focal positivity was observed for alpha smooth muscle actin, CD117, CD68, p53, and bcl2. No reaction could be obtained for endothelial markers. The proliferation index Ki-67 was between 5% and 80%. Six examined tumors were positive for mdm2. In the CGH analysis, gains and amplifications in the 12q13-14 region were found in six of eight tumors (75%). Other, less consistent alterations, were losses on 3p, 3q, 4q, 9p, 11q, 13q, Xp, and Xq, gains on 7p, 17p, and 17q, and amplifications on 4q, 5p, 6p, and 11q. Intimal sarcomas of the pulmonary artery are tumors with an unfavorable prognosis and poorly differentiated morphology. A majority of tumors show a consistent genetic alteration (gains and amplifications in the 12q13-14 region) and overexpression of mdm2, implicating the mdm2/p53 pathway as a possible mechanism in the tumor pathogenesis.

Spitzoid malignant melanoma with lymph-node metastasis. Is a copy-number loss on chromosome 6q a marker of malignancy?

Distinction of spitzoid malignant melanomas (SMM) from Spitz nevi may be difficult or even impossible on the basis of conventional histology. In this report, a patient suffering from a primary lesion diagnosed as a Spitz nevus and a metastatic malignant melanoma approximately 4 years thereafter is described. A diagnosis of SMM was made subsequently upon review of the primary lesion. In the present analysis, we used comparative genomic hybridization (CGH) to define markers characteristic of SMM. The primary lesion revealed deletions on chromosomes 6q and 9p. In the metastasis, additional deletions on chromosomes 10p and 10q and gains of chromosome 7 were found. To our knowledge, no chromosomal aberration on chromosome 6 was hitherto demonstrated in benign melanocytic nevi. Findings reported in the literature suggest that human melanoma metastasis suppressor gene maps to 6q. In contrast, losses on chromosome 9p seem to be an early event in the development of melanoma. However, they are not only found in melanomas but are occasionally present in Spitz nevi as well as in atypical nevi. The CGH result with deletion of 6q in this difficult to diagnose primary melanocytic lesion strongly supports the diagnosis of malignant melanoma. To demonstrate the reliability of loss on chromosome 6q as a marker of SMM, a larger number of lesions must be investigated.

[Therapy concept in differentiated thyroid gland carcinoma--results of 25 years with 257 patients]. / Therapiekonzept bei differenziertem Schilddrüsenkarzinom--Resultate über 25 Jahre bei 257 Patienten.

BACKGROUND: Differentiated thyroid carcinoma is a unique tumour in that a low risk-patient population with a tumour-related death rate of near 0% can be found. Yet in these patients the risk and risk factors of curable recurrences must be considered. The question therefore arises, whether in defined subgroups of low risk-patients a reduced extent of treatment (hemithyroidectomy, total thyroidectomy without 131I ablation) may result in cure without recurrence, including low morbidity of treatment and reduced costs. STUDY DESIGN: In a consecutive series of 257 patients suffering from papillary (146) or follicular (111) carcinomas operated by one surgeon over a period of 25 years, a reduced extent of treatment was carried out essentially in subgroups of minimally invasive follicular carcinoma, namely in pT1,2-tumors of young patients, or in those with capsular invasion alone, and in pT1,2 N0 papillary tumors, representing a subgroup of TNM stage I and II tumors. For N-staging selective lymphadenectomy was carried out at the beginning of the study, but elective lymphadenectomy was used since 1996 for papillary carcinoma. Follow-up was 1-25 (mean = 8) years. All excised tumors were examined by one pathologist. RESULTS: 167 (approximately 2/3) of the patients presented with a single nodule, whereas in 1/3 a concomitant benign nodular goiter or an immunothyropathy was found. The percentage of grossly invasive follicular carcinoma decreased during the 25 year period from 41 to 10% of all patients (p < 0.0005), whereas the percentage of papillary cancer rise from 35% to 66% (p < 0.005). Hemithyroidectomy, total thyroidectomy without, and with 131I ablation respectively, were performed in 32%, 24%, and 44% of patients. In papillary carcinoma N1-status was found in 21 (23%)/ 92 patients with selective, and in 18/54 patients (33%) with elective lymphadenectomy, respectively (n.s.). One (0.4%) patient died postoperatively. Permanent hypoparathyroidism occurred in 1.9% (3% for total thyroidectomy), permanent recurrent nerve lesion in 1.6% of patients (1% of nerves at risk). PAPILLARY CARCINOMA: No tumour-related death and no serious recurrence occurred in the low risk-group (TNM stages I and II) (n = 112 (84%)), including a T4-, N1-, M1-status in 9%, 20% and 3% of patients, respectively. 4/112 patients (3.6%) developed a recurrence (3 nodal, 1 contralateral, following 131I ablation in 2 instances). Only one (1%) instance of a nodal recurrence occurred in N0-tumors (n = 97). In TNM stages III and IV (high risk) patients (with T4-, N1-, M1-status in 79%, 58%, and 8% respectively), residual and recurrent disease occurred in 7 (33%) patients, leading to 6 (29%) tumor-related deaths. Follicular carcinoma: One 74-year old patient (1.6%) died from minimally invasive follicular carcinoma (n = 54 (51%); mean age 48 years). In 44 (81%) patients treatment did not include remnant ablation. 7 (13%) patients died from widely invasive follicular carcinoma (n = 53 (49%); mean age 64 years) and 3 (6%) further patients are alive with a serious recurrence. No curable recurrence was observed in follicular carcinoma. CONCLUSIONS: The decrease in goiter endemicity during the last decades in Switzerland paralleled a decrease in the incidence of grossly invasive follicular carcinoma over the 25 year period of the study. Following selective treatment, low risk TNM stage I and II-patients with papillary carcinoma had a tumor-related death rate of 0% and a low (3.6%) recurrence rate. N1-status represents a risk factor for nodal recurrence (even with remnant ablation). Elective vs. selective lymphadenectomy lead to slight stage migration but it presented no advantage in terms of recurrence and death which were rare events. No death occurred in the subgroups of minimally invasive follicular carcinoma of young (< 45) patients (41%) and in the patients without vascular invasion (28%), even without remnant ablation in most instances. No curable recurrence occurred in foll

Genomic alterations in well-differentiated gastrointestinal and bronchial neuroendocrine tumors (carcinoids): marked differences indicating diversity in molecular pathogenesis.

Neuroendocrine tumors (carcinoids) are a heterogeneous group of neoplasms arising from the diffuse neuroendocrine system. Genetic changes underlying their tumorigenesis are primarily unknown. We used comparative genomic hybridization to screen 32 well-differentiated neuroendocrine tumors (21 gastrointestinal and 11 bronchial) and three associated metastases for genomic alterations. There were striking differences of genomic imbalances between the two subgroups of neuroendocrine tumors. Losses of chromosome 18q and 18p were shown in eight (38%) and seven (33%), respectively, out of 21 gastrointestinal tumors and in none of the 11 bronchial tumors. Conversely, deletions of 11q occurred in four of 11 (36%) bronchial tumors but only in one gastrointestinal tumor. These comparative genomic hybridization findings were confirmed by interphase cytogenetics. Our data indicate that neuroendocrine tumors of the two subgroups develop via different molecular pathways. Inactivation of one or several tumor suppressor genes on chromosome 18 may be important for the biological behavior of gastrointestinal tumors, whereas gene inactivation on 11q seems to be associated with tumor development of the bronchi.

Mutation and expression analyses reveal differential subcellular compartmentalization of PTEN in endocrine pancreatic tumors compared to normal islet cells.

The pathogenesis of sporadic endocrine pancreatic tumors (EPTs) is still primarily unknown. Comparative genomic hybridization studies revealed loss of 10q in a significant number (nine of 31) of EPTs. The tumor suppressor gene PTEN lies on 10q23, and so, is a candidate to play some role in EPT pathogenesis. Germline PTEN mutations are found in Cowden and Bannayan-Riley-Ruvalcaba syndromes, whereas somatic mutations and deletions are found in a variety of sporadic cancers. The mutation and expression status of PTEN in EPTs has not yet been examined. Mutation analysis of the entire coding region of PTEN including splice sites was performed in 33 tumors, revealing one tumor with somatic L182F (exon 6). Loss of heterozygosity of the 10q23 region was detected in eight of 15 informative malignant (53%) and in none of seven benign EPTs. PTEN expression was assessed in 24 available EPTs by immunohistochemistry using a monoclonal anti-PTEN antibody. Of these 24, 23 tumors showed strong immunoreactivity for PTEN. Only the EPTs with PTEN mutation lacked PTEN protein expression. Although normal islet cells always exhibited predominantly nuclear PTEN immunostaining, 19 of 23 EPTs had a predominantly cytoplasmic PTEN expression pattern. Exocrine pancreatic tissue was PTEN-negative throughout. PTEN mutation is a rare event in malignant EPTs and PTEN protein is expressed in most (23 of 24) EPTs. Thus, intragenic mutation or another means of physical loss of PTEN is rarely involved in the pathogenesis of EPTs. Instead, either an impaired transport system of PTEN to the nucleus or some other means of differential compartmentalization could account for impaired PTEN function. Loss of heterozygosity of the 10q23 region is a frequent event in malignant EPTs and might suggest several hypotheses: a different tumor suppressor gene in the vicinity of PTEN might be principally involved in EPT formation; alternatively, 10q loss, including PTEN, seems to be associated with malignant transformation, but the first step toward neoplasia might involve altered subcellular localization of PTEN.

Losses of chromosomes 1p and 3q are early genetic events in the development of sporadic pheochromocytomas.

Despite several loss of heterozygosity studies, a comprehensive genomic survey of pheochromocytomas is still lacking. To identify DNA copy number changes which might be important in tumor development and progression and which may have diagnostic utility, we evaluated genetic aberrations in 29 sporadic adrenal and extra-adrenal pheochromocytomas (19 clinically benign tumors and 10 malignant lesions). Comparative genomic hybridization was performed using directly fluorochrome-conjugated DNA extracted from frozen (16) and paraffin-embedded (13) tumor tissues. The most frequently observed changes were losses of chromosomes 1p11-p32 (86%), 3q (52%), 6q (34%), 3p, 17p (31% each), 11q (28%), and gains of chromosomes 9q (38%) and 17q (31%). No amplification was identified and no difference between adrenal and extra-adrenal tumors was detected. Progression to malignant tumors was strongly associated with deletions of chromosome 6q (60% versus 21% in clinically benign lesions, P = 0.0368) and 17p (50% versus 21%). Fluorescence in situ hybridization confirmed the comparative genomic hybridization data of chromosomes 1p, 3q, and 6q, and revealed aneuploidy in some tumors. Our results suggest that the development of pheochromocytomas is associated with specific genomic aberrations, such as losses of 1p, 3q, and 6q and gains of 9q and 17q. In particular, tumor suppressor genes on chromosomes 1p and 3q may be involved in early tumorigenesis, and deletions of chromosomes 6q and 17p in progression to malignancy.

RET is expressed but not mutated in extra-adrenal paragangliomas.

This study has investigated the role of the RET proto-oncogene, which has been identified as the susceptibility gene for multiple endocrine neoplasia (MEN) type 2, in the development of sporadic and familial extra-adrenal paragangliomas. RET protein expression was analysed by immunohistochemistry. Subsequently, DNA extracted from 52 tumours of 44 patients was screened for somatic RET point mutations in exons 10, 11, and 13-16, where oncogenic mutations have recently been described in a subset of sporadic medullary thyroid carcinomas and phaeochromocytomas. The methods employed included non-isotopic polymerase chain reaction-based single strand conformation polymorphism (PCR-SSCP) analysis and heteroduplex gel electrophoresis, followed by direct sequencing of PCR products. RET protein expression was demonstrated in all ten paragangliomas tested. However, none of the familial or sporadic extra-adrenal paragangliomas contained somatic mutations in exons 10, 11, or 13-16 of the RET proto-oncogene, whereas control samples with known mutations in these exons exhibited the expected band shift, or yielded an additional band with retarded migration. Although paragangliomas exhibit RET protein expression, these data indicate that oncogenic RET proto-oncogene mutations do not appear to be generally important in the formation of sporadic paragangliomas.

Thomsen-Friedenreich glycotope is expressed in developing and normal kidney but not in renal neoplasms.

The Thomsen-Friedenreich glycotope (TF) is considered a general carcinoma autoantigen and is therefore of importance in cancer diagnosis and immunotherapy. We report the distribution of the TF glycotope in developing and adult human kidney and renal neoplasms. A monoclonal antibody and the lectin amaranthin were used to study the TF and its sialylated, masked form by immunohistochemistry and immunoblotting. In developing kidney, the TF was restricted to the loop of Henle, distal tubules, and peripheral collecting ducts, whereas its sialylated form was detectable in all epithelial differentiations derived from the 2 embryonic anlagen, the metanephrogenic blastema being unreactive. This pattern was essentially preserved in adult kidney, with TF labeling beginning in the thick ascending limb and extending into the collecting ducts of outer medulla. The sialylated TF glycotope was additionally observed in ascending thin limbs. The TF was exclusively expressed in the luminal cell surface and hence was inaccessible to immune reactions. Analysis of a spectrum of renal neoplasms failed to detect the TF, with the exception of occasional staining of tubules in nephroblastoma. Moreover, the sialylated TF was only detectable in oncocytoma, chromophobe renal cell carcinoma, cystic nephroma, nephroblastoma, and nephroblastomatosis complex and occasionally in type 1 papillary renal cell carcinoma. Thus, the TF and its sialylated form are expressed in normal developing and adult kidney. However, the TF does not seem to represent a tumor-associated glycotope in human kidney, nor does it appear to be of value in diagnosis and immunotherapy of renal neoplasms.

Early orchiopexy: prepubertal intratubular germ cell neoplasia and fertility outcome.

OBJECTIVES: To investigate the prepubertal prevalence of intratubular germ cell neoplasia of the unclassified type (ITGCNU) and its significance as a predictor of testicular cancer and to evaluate the effect of early orchiopexy (at younger than 2 years of age) on subsequent fertility of patients with bilateral cryptorchidism. METHODS: Testicular biopsies (n = 660) from 440 prepubertal patients with cryptorchidism who underwent orchiopexy between January 1, 1970 and December 31, 1979 were evaluated for ITGCNU using placental-like alkaline phosphatase (PLAP) antibody. The clinical outcome in 15 patients with PLAP-positive germ cells was evaluated in 1997. In addition, the effect of age at surgery on the fertility of patients with bilateral cryptorchidism was assessed by clinical follow-up until 1997 and was correlated with the histologic data at orchiopexy. RESULTS: PLAP-positive germ cells morphologically identical with adult ITGCNU were found in the biopsies of 22 patients (5%). After more than two decades, none of the 15 patients with successful follow-up developed testicular cancer. The fertility outcome in the patients with bilateral cryptorchidism correlated with the number of spermatogonia at orchiopexy (P = 0.018), but correlated inversely with age at orchiopexy (P = 0.021). CONCLUSIONS: PLAP-positive germ cells in prepubertal testicular biopsy specimens are not necessarily precursors of testicular cancer after orchiopexy. In addition, our data support the idea that early orchiopexy may be beneficial in preventing infertility.