Tissue biomarkers of breast cancer and their association with conventional pathologic features.

BACKGROUND: Tissue protein expression profiling has the potential to detect new biomarkers to improve breast cancer (BC) diagnosis, staging, and prognostication. This study aimed to identify tissue proteins that differentiate breast cancer tissue from healthy breast tissue using protein chip mass spectrometry and to examine associations with conventional pathological features. METHODS: To develop a training model, 82 BC and 82 adjacent unaffected tissue (AT) samples were analysed on cation-exchange protein chips by time-of-flight mass spectrometry. For validation, 89 independent BC and AT sample pairs were analysed. RESULTS: From the protein peaks that were differentially expressed between BC and AT by univariate analysis, binary logistic regression yielded two peaks that together classified BC and AT with a ROC area under the curve of 0.92. Two proteins, ubiquitin and S100P (in a novel truncated form), were identified by liquid chromatography/tandem mass spectrometry and validated by immunoblotting and reactive-surface protein chip immunocapture. The combined marker panel was positively associated with high histologic grade, larger tumour size, lymphovascular invasion, ER and PR positivity, and HER2 overexpression, suggesting that it may be associated with a HER2-enriched molecular subtype of breast cancer. CONCLUSION: This independently validated protein panel may be valuable in the classification and prognostication of breast cancer patients.

Response of melanocortin-4 receptor-deficient mice to anorectic and orexigenic peptides.

Mutations reducing the functional activity of leptin, the leptin receptor, alpha-melanocyte stimulating hormones (alpha-MSH) and the melanocortin-4 receptor (Mc4r) all lead to obesity in mammals. Moreover, mutant mice that ectopically express either agouti (Ay/a mice) or agouti-related protein (Agrp), antagonists of melanocortin signalling, become obese. These data suggest that alpha-MSH signalling transduced by Mc4r tonically inhibits feeding; however, it is not known to what extent this pathway mediates leptin signalling. We show here that Mc4r-deficient (Mc4r-/-) mice do not respond to the anorectic actions of MTII, an MSH-like agonist, suggesting that alpha-MSH inhibits feeding primarily by activating Mc4r. Obese Mc4r-/-mice do not respond significantly to the inhibitory effects of leptin on feeding, whereas non-obese Mc4r-/- mice do. These data demonstrate that melanocortin signalling transduced by Mc4r is not an exclusive target of leptin action and that factors resulting from obesity contribute to leptin resistance. Leptin resistance of obese Mc4r-/- mice does not prevent their response to the anorectic actions of ciliary neurotrophic factor (CNTF), corticotropin releasing factor (CRF), or urocortin; or the orexigenic actions of neuropeptide Y (NPY) or peptide YY (PYY), indicating that these neuromodulators act independently or downstream of Mc4r signalling.

Germline mutations of the PTEN gene in Cowden disease, an inherited breast and thyroid cancer syndrome.

Cowden disease (CD) is an autosomal dominant cancer predisposition syndrome associated with an elevated risk for tumours of the breast, thyroid and skin. Lhermitte-Duclos disease (LDD) cosegregates with a subset of CD families and is associated with macrocephaly, ataxia and dysplastic cerebellar gangliocytomatosis. The common feature of these diseases is a predisposition to hamartomas, benign tumours containing differentiated but disorganized cells indigenous to the tissue of origin. Linkage analysis has determined that a single locus within chromosome 10q23 is likely to be responsible for both of these diseases. A candidate tumour suppressor gene (PTEN) within this region is mutated in sporadic brain, breast and prostate cancer. Another group has independently isolated the same gene, termed MMAC1, and also found somatic mutations throughout the gene in advanced sporadic cancers. Mutational analysis of PTEN in CD kindreds has identified germline mutations in four of five families. We found nonsense and missense mutations that are predicted to disrupt the protein tyrosine/dual-specificity phosphatase domain of this gene. Thus, PTEN appears to behave as a tumour suppressor gene in the germline. Our data also imply that PTEN may play a role in organizing the relationship of different cell types within an organ during development.

Inactivation of the mouse melanocortin-3 receptor results in increased fat mass and reduced lean body mass.

Genetic and pharmacological studies have defined a role for the melanocortin-4 receptor (Mc4r) in the regulation of energy homeostasis. The physiological function of Mc3r, a melanocortin receptor expressed at high levels in the hypothalamus, has remained unknown. We evaluated the potential role of Mc3r in energy homeostasis by studying Mc3r-deficient (Mc3r(-/-)) mice and compared the functions of Mc3r and Mc4r in mice deficient for both genes. The 4-6-month Mc3r-/- mice have increased fat mass, reduced lean mass and higher feed efficiency than wild-type littermates, despite being hypophagic and maintaining normal metabolic rates. (Feed efficiency is the ratio of weight gain to food intake.) Consistent with increased fat mass, Mc3r(-/-) mice are hyperleptinaemic and male Mc3r(-/-) mice develop mild hyperinsulinaemia. Mc3r(-/-) mice did not have significantly altered corticosterone or total thyroxine (T4) levels. Mice lacking both Mc3r and Mc4r become significantly heavier than Mc4r(-/-) mice. We conclude that Mc3r and Mc4r serve non-redundant roles in the regulation of energy homeostasis.

Dynamics of nephron-vascular network.

The paper presents a modeling study of the spatial dynamics of a nephro-vascular network consisting of individual nephrons connected via a tree-like vascular branching structure. We focus on the effects of nonlinear mechanisms that are responsible for the formation of synchronous patterns in order to learn about processes not directly amenable to experimentation. We demonstrate that: (i) the nearest nephrons are synchronized in-phase due to a vascular propagated electrical coupling, (ii) the next few branching levels display a formation of phase-shifted patterns due to hemodynamic coupling and mode elimination, and (iii) distantly located areas show asynchronous behavior or, if all nephrons and branches are perfectly identical, an infinitely long transient behavior. These results contribute to the understanding of mechanisms responsible for the highly dynamic and limited synchronization observed among groups of nephrons despite of the fairly strong interaction between the individual units.

Role of the Y5 neuropeptide Y receptor in feeding and obesity.

Neuropeptide Y (NPY), a 36-amino-acid neuromodulator abundantly expressed in the brain, has been implicated in the regulation of food intake and body weight. Pharmacological data suggest that NPY's stimulatory effect on appetite is transduced by the G-protein-coupled NPY Y5 receptor (Y5R). We have inactivated the Y5R gene in mice and report that younger Y5R-null mice feed and grow normally; however, they develop mild late-onset obesity characterized by increased body weight, food intake and adiposity. Fasting-induced refeeding is unchanged in younger Y5R-null mice and they exhibit normal sensitivity to leptin. Their response to intracerebroventricular (i.c.v.) administration of NPY and related peptides is either reduced or absent. NPY deficiency attenuates the obesity syndrome of mice deficient for leptin (ob/ob), but these effects are not mediated by NPY signaling through the Y5R because Y5R-null ob/ob mice are equally obese. These results demonstrate that the Y5R contributes to feeding induced by centrally administered NPY and its analogs, but is not a critical physiological feeding receptor in mice.

Metastatic parathyroid carcinoma initially misdiagnosed as parathyroid adenoma: the role of parafibromin in increasing diagnostic accuracy.

Parathyroid carcinoma, although a rare cause of primary hyperparathyroidism, carries a significant morbidity and mortality from severe symptomatic hypercalcaemia and related complications. We report a case where the diagnosis was not considered from the outset and review the current clinical and histopathological markers available to assist in the diagnosis of parathyroid carcinoma.

[Prophylactic parathyroidectomy for familial parathyroid carcinoma]. / Das familiäre Nebenschilddrüsenkarzinom Indikation zur prophylaktischen Parathyreoidektomie?

In contrast to primary hyperparathyroidism, parathyroid carcinoma is a rare disease. In patients with hyperparathyroidism jaw tumor (HPT-JT) syndrome, caused by germline mutations in HRPT2, the development of parathyroid carcinoma is estimated to be 10-15%. This review summarizes the clinical and molecular genetic data of about 100 patients in the literature and three of our own cases. Unfortunately, osteofibromas, which might enable timely diagnosis of HPT-JT syndrome, occur in only about 30% of patients; about 80% have uniglandular disease. Based on the current data, a general recommendation to perform prophylactic parathyroidectomy cannot be given. However, thorough screening of patients at risk is mandatory. Of note in patients thought to have sporadic parathyroid carcinoma, germline HRPT2 mutations are found in up to 20%. Hence, any patient with parathyroid carcinoma should undergo HRPT2 mutation analysis.

Sporadic and familial pheochromocytomas are associated with loss of at least two discrete intervals on chromosome 1p.

Pheochromocytomas are tumors of the adrenal medulla originating in the chromaffin cells derived from the neural crest. Ten % of these tumors are associated with the familial cancer syndromes multiple endocrine neoplasia type 2, von Hippel-Lindau disease (VHL), and rarely, neurofibromatosis type 1, in which germ-line mutations have been identified in RET, VHL, and NF1, respectively. In both the sporadic and familial form of pheochromocytoma, allelic loss at 1p, 3p, 17p, and 22q has been reported, yet the molecular pathogenesis of these tumors is largely unknown. Allelic loss at chromosome 1p has also been reported in other endocrine tumors, such as medullary thyroid cancer and tumors of the parathyroid gland, as well as in tumors of neural crest origin including neuroblastoma and malignant melanoma. In this study, we performed fine structure mapping of deletions at chromosome 1p in familial and sporadic pheochromocytomas to identify discrete regions likely housing tumor suppressor genes involved in the development of these tumors. Ten microsatellite markers spanning a region of approximately 70 cM (1pter to 1p34.3) were used to screen 20 pheochromocytomas from 19 unrelated patients for loss of heterozygosity (LOH). LOH was detected at five or more loci in 8 of 13 (61%) sporadic samples and at five or more loci in four of five (80%) tumor samples from patients with multiple endocrine neoplasia type 2. No LOH at 1p was detected in pheochromocytomas from two VHL patients. Analysis of the combined sporadic and familial tumor data suggested three possible regions of common somatic loss, designated as PC1 (D1S243 to D1S244), PC2 (D1S228 to D1S507), and PC3 (D1S507 toward the centromere). We propose that chromosome 1p may be the site of at least three putative tumor suppressor loci involved in the tumorigenesis of pheochromocytomas. At least one of these loci, PC2 spanning an interval of <3.8 cM, is likely to have a broader role in the development of endocrine malignancies.

Differential loss of heterozygosity in the region of the Cowden locus within 10q22-23 in follicular thyroid adenomas and carcinomas.

The susceptibility gene for Cowden disease (CD), an autosomal dominant inherited cancer syndrome, has recently been mapped to an approximately 6-cM interval on chromosome subband 10q22-23 between the markers D10S541 and D10S564. CD is characterized by hamartomas of many organ systems, including the thyroid, breast, skin, and gastrointestinal tract, as well as carcinoma of the thyroid and breast. Follicular thyroid adenomas and carcinomas are significant component tumors in CD; thus, we sought to examine their sporadic counterpart tumors for loss of heterozygosity (LOH) of microsatellite markers in the 20-cM region within and flanking the Cowden critical interval. In all, 38 sporadic thyroid tumors were analyzed. LOH within the CD interval was observed in 5 of 19 (26%) follicular thyroid adenomas and 1 of 9 (11%) Hürthle cell adenomas. Furthermore, of these adenomas with LOH, 3 of 4 (75%) were atypical follicular adenomas, whereas 2 of 15 (13%) were typical follicular adenomas. Surprisingly, no LOH was detected in this region in 10 follicular carcinomas. The shortest region of overlap includes the markers D10S1735 and D10S1739. If the LOH observed in these sporadic tumors is related to the CD gene, then the Cowden critical interval can be revised to lie within the interval defined by D10S579 and D10S564. LOH in this narrow interval implicates the CD gene, or another gene in that interval, in follicular thyroid tumorigenesis. However, this does not explain the lack of LOH in follicular carcinomas. Taken together, it may instead be evidence against a stepwise progression from atypical adenomas to carcinomas. Alternatively, sporadic thyroid adenoma formation may be independent of that locus, but loss of this region could prevent carcinoma formation, thus implying that the CD gene may be an oncogene or growth promoter.

Somatic deletions and mutations in the Cowden disease gene, PTEN, in sporadic thyroid tumors.

The majority of familial medullary thyroid neoplasms are associated with germ-line mutations of the RET proto-oncogene, yet very little is known about the mechanisms involved in the pathogenesis of familial and sporadic nonmedullary thyroid tumors. A subset of thyroid tumors have loss of heterozygosity of chromosome 10q22-23, a region harboring the gene responsible for Cowden disease, an autosomal dominant hamartoma syndrome associated with thyroid and breast tumors. PTEN/MMAC1/TEP1 codes for a dual-specificity phosphatase and is likely a tumor suppressor gene. We sought to determine the PTEN status in a series of epithelial thyroid neoplasms. We studied 95 sporadic thyroid tumors, of which 39 were papillary thyroid carcinomas (PTCs), 12 were follicular carcinomas, 9 were anaplastic carcinomas, 5 were Hürthle cell carcinomas, 21 were nonfunctioning follicular adenomas, and 9 were Hürthle cell adenomas. Direct sequencing of PCR-amplified products was performed for all nine exons of PTEN. Two polymorphic markers, one located in intron 8 and another, a dinucleotide repeat marker, AFMa086wg9, located within intron 2, were analyzed in paired blood-tumor DNA samples to assess hemizygous deletions of PTEN. We found a somatic frameshift mutation in one PTC, which was expected to generate a premature stop codon 2 amino acids downstream. Twenty-six % of informative benign tumors (four follicular adenomas and three Hürthle cell adenomas) and only 3 of 49 (6.1%) informative malignant tumors (one PTC, one follicular carcinoma, and one anaplastic carcinoma) showed evidence of hemizygous deletion of PTEN (P = 0.046). We conclude that a subset of thyroid tumors have somatic deletions of the PTEN gene, predominantly the benign forms, and that small intragenic mutations of PTEN are infrequent in thyroid tumors. We speculate that other mechanisms of PTEN inactivation, rather than small intragenic mutations, might occur in the hemizygously deleted samples and act as the "Knudson second hit." Alternatively, other tumor suppressor genes mapping to chromosome 10q22-23 could be the actual targets for such deletions and thus represent the various hits in the pathway of multistep carcinogenesis.

Germline and somatic mutations in an oncogene: RET mutations in inherited medullary thyroid carcinoma.

Inherited cancer syndromes predispose an individual to development of specific tumors. Somatic and germline mutations in the same tumor suppressor gene, as described in Knudson's two-mutation model, are well recognized. Inherited mutations in the RET proto-oncogene, which encodes a receptor tyrosine kinase, predispose individuals to the multiple endocrine neoplasia type 2 (MEN 2) cancer syndromes. The major component tumor of these syndromes is medullary thyroid carcinoma (MTC). To date, somatic mutations in RET have not been identified in tumors from individuals with MEN 2, although they have been well documented in sporadic MEN 2-related tumors. We have identified, among 16 MEN 2 cases with well-defined RET germline mutations, a somatic missense mutation at codon 918 of RET in 3 of 15 MTCs and in a sample with hyperplastic C-cells (presumed precursor to hereditary MTC). We suggest that the presence of a somatic mutation, in addition to the preexisting germline mutation in hereditary MTCs, may contribute to tumorigenesis in vivo.

Exclusion of PTEN and 10q22-24 as the susceptibility locus for juvenile polyposis syndrome.

Juvenile polyposis syndrome (JPS; MIM 174900) is an autosomal dominant condition with incomplete penetrance characterized by hamartomatous polyps of the gastrointestinal tract and a risk of gastrointestinal cancer. Gastrointestinal hamartomatous polyps are also present in Cowden syndrome (CS; MIM 158350) and Bannayan-Zonana syndrome (BZS; also called Ruvalcaba-Myhre-Smith syndrome; MIM 153480). The susceptibility locus for both CS and BZS has recently been identified as the novel tumor suppressor gene PTEN, encoding a dual specificity phosphatase, located at 10q23.3. A putative JPS locus, JP1, which most likely functions as a tumor suppressor, had previously been mapped to 10q22-24 in both familial and sporadic juvenile polyps. Given the shared clinical features of gastrointestinal hamartomatous polyps among the three syndromes and the coincident mapping of JP1 to the region of PTEN, we sought to determine whether JPS was allelic to CS and BZS by mutation analysis of PTEN and linkage approaches. Microsatellite markers spanning the CS/BZS locus (D10S219, D10S551, D10S579, and D10S541) were used to compute multipoint lod scores in eight informative families with JPS. Lod scores of < -2.0 were generated for the entire region, thus excluding PTEN and any genes within the flanking 20-cM interval as candidate loci for familial JPS under our statistical models. In addition, analysis of PTEN using a combination of denaturing gradient gel electrophoresis and direct sequencing was unable to identify a germline mutation in 14 families with JPS and 11 sporadic cases. Therefore, at least a proportion of JPS cases are not caused by germline PTEN alteration or by an alternative locus at 10q22-24.

Over-representation of a germline RET sequence variant in patients with sporadic medullary thyroid carcinoma and somatic RET codon 918 mutation.

The aetiology of sporadic medullary thyroid carcinoma is unknown. About 50% harbour a somatic mutation at codon 918 of RET (M918T). To investigate whether other RET sequence variants may be associated with or predispose to the development of sporadic medullary thyroid carcinoma, we analysed genomic DNA from the germline and corresponding tumour from 50 patients to identify RET sequence variants. In one patient, tumour DNA showed a novel somatic 12 bp in-frame deletion in exon 15. More interestingly, we found that the rare polymorphism at codon 836 (c.2439C > T; S836S) occurred at a significantly higher frequency than that in control individuals without sporadic medullary thyroid carcinoma (Fisher's exact test, P = 0.03). Further, among the nine evaluable cases with germline c.2439C/T, eight also had the somatic M918T mutation in MTC DNA which was more frequent than in patients with the more common c.2439C/C (89% vs 40%, respectively; Fisher's exact test, P = 0.01). These findings suggest that the rare sequence variant at codon 836 may somehow play a role in the genesis of sporadic medullary thyroid carcinoma.

A highly conserved processed PTEN pseudogene is located on chromosome band 9p21.

PTEN/MMAC1/TEP1, encoding a dual-specificity phosphatase, is a tumor suppressor gene which has recently been cloned and mapped to chromosome 10q23.3. We have shown that germline mutations of PTEN are present in individuals with two hamartoma syndromes: Cowden Syndrome, associated with a predisposition to breast and thyroid cancers, and Bannayan-Zonana syndrome. Somatic mutations of PTEN have been reported in a variety of human cancer cell lines, suggesting a potential role for this gene in the pathogenesis of human malignancies. We report the identification of a highly conserved PTEN processed pseudogene, psiPTEN, which shares over 98% homology with the coding region of functional PTEN, and its localisation to chromosome 9p21. The high sequence homology of psiPTEN with the PTEN transcript may potentially lead to misinterpretation when performing mutation analyses based on cDNA templates. Caution should be exerted when using such screening approaches.

Germline mutations in PTEN are an infrequent cause of genetic predisposition to breast cancer.

Heterozygous germline mutations in PTEN are responsible for most cases of Cowden Syndrome, a rare familial trait characterized by hamartomas and by predisposition to cancer of the breast and thyroid. The variable and often subtle clinical findings that characterize Cowden Syndrome are frequently unrecognized, raising the possibility that germline PTEN mutations may confer susceptibility to breast cancer in women who have not been diagnosed with this syndrome. To determine whether such mutations contribute to genetic predisposition to breast cancer within the general population, we analysed a cohort of women with early-onset breast cancer (< age 40), a subset of the population at increased risk for genetic susceptibility. Lymphoblast cell lines were analysed using either direct nucleotide sequencing (28 cases), denaturing gradient gel electrophoresis (DGGE) (34 cases) or a yeast-based truncation assay (110 cases). No definitive, truncating mutations were observed in 172 patients. Missense changes were noted in the germline of 2/60 patients analysed by direct nucleotide sequencing or DGGE, including a non-conservative amino acid substitution within the phosphatase domain, but neither showed loss of the wild-type allele in the corresponding breast tumor specimen. We conclude that germline mutations in PTEN are an uncommon cause of genetic predisposition to breast cancer within the general population.

The practical management of multiple endocrine neoplasia.

Adbances in the identification and localization of the abnormal genes in the multiple endocrine neoplasia syndromes have provided new methods of identifying "at risk" individuals in these families. Genetic testing using linkage analysis in multiple endocrine neoplasia (MEN) 1 and direct mutation analysis of the RET protooncogene in MEN 2 is now available for these disorders. New management issues for these disorders have resulted, and a practical approach to these issues is discussed.

Male breast cancer in Cowden syndrome patients with germline PTEN mutations.

Cowden syndrome (CS) (OMIM 158350) is a multiple hamartoma syndrome associated with germline mutations in the PTEN tumour suppressor gene. While CS is characterised most commonly by non-cancerous lesions (mucocutaneous trichilemmomas, acral and palmoplantar keratoses, and papillomatous papules), it is also associated with an increased susceptibility to breast cancer (in females) and thyroid cancer, as well as non-cancerous conditions of the breast and thyroid. Here we report two cases of male breast cancer occurring in patients with classical CS phenotypes and germline PTEN mutations. The first subject was diagnosed with CS indicated primarily by mucocutaneous papillomatosis, facial trichilemmomas, and macrocephaly with frontal bossing at the age of 31 years. He developed breast cancer at 41 years and subsequently died of the disease. A PTEN mutation, c.802delG, was identified in this subject, yet none of his family members showed evidence of a CS phenotype, suggesting that this PTEN mutation may be a de novo occurrence. The second subject had a CS phenotype including multiple trichilemmomas and thyroid adenoma, developed male breast cancer at 43 years, and died of the disease at 57 years. He was a carrier of a PTEN mutation c.347-351delACAAT that cosegregated with the CS phenotype in affected family members. These two cases of male breast cancer associated with germline PTEN mutations and the CS phenotype suggest that CS may be associated with an increased risk of early onset male as well as female breast cancer.

HRPT2 mutations are associated with malignancy in sporadic parathyroid tumours.

BACKGROUND: Hyperparathyroidism is a common endocrinopathy characterised by the formation of parathyroid tumours. In this study, we determine the role of the recently identified gene, HRPT2, in parathyroid tumorigenesis. METHODS: Mutation analysis of HRPT2 was undertaken in 60 parathyroid tumours: five HPT-JT, three FIHP, three MEN 1, one MEN 2A, 25 sporadic adenomas, 17 hyperplastic glands, two lithium associated tumours, and four sporadic carcinomas. Loss of heterozygosity at 1q24-32 was performed on a subset of these tumours. RESULTS: HRPT2 somatic mutations were detected in four of four sporadic parathyroid carcinoma samples, and germline mutations were found in five of five HPT-JT parathyroid tumours (two families) and two parathyroid tumours from one FIHP family. One HPT-JT tumour with germline mutation also harboured a somatic mutation. In total, seven novel and one previously reported mutation were identified. "Two-hits" (double mutations or one mutation and loss of heterozygosity at 1q24-32) affecting HRPT2 were found in two sporadic carcinomas, two HPT-JT-related and two FIHP related tumours. CONCLUSIONS: The results in this study support the role of HRPT2 as a tumour suppressor gene in sporadic parathyroid carcinoma, and provide further evidence for HRPT2 as the causative gene in HPT-JT, and a subset of FIHP. In light of the strong association between mutations of HRPT2 and sporadic parathyroid carcinoma demonstrated in this study, it is hypothesised that HRPT2 mutation is an early event that may lead to parathyroid malignancy and suggest intragenic mutation of HRPT2 as a marker of malignant potential in both familial and sporadic parathyroid tumours.