The 40bp Indel Polymorphism rs150550023 in the MDM2 Promoter is Associated with Intriguing Shifts in Gene Expression in the p53-MDM2 Regulatory Hub.

Most low-penetrance genetic risk factors for cancer are located in noncoding regions, presumably altering the regulation of neighboring genes. The poorly characterized Indel polymorphism rs150550023 (rs3730485; del1518) in the promoter of MDM2 (human homolog of mouse double minute 2) is a biologically plausible candidate genetic risk factor, which might influence the expression of MDM2, a key negative regulator of the central tumor suppressor p53. Here, we genotyped rs150550023 in a Central European hospital-based case-control study of 407 breast cancer patients and 254 female controls. mRNA levels of MDM2, p53, and the p53 target genes p21, BAX, and PERP were quantified with qRT-PCR, and p53 protein was assessed with immune histochemistry in ≈100 primary breast tumors with ascertained rs150550023 genotype. We found no evidence for an association of rs150550023 with the risk, age at onset, or prognosis of breast cancer. A possible synergism was observed with SNP309 in promoter P2 of MDM2. Mean mRNA levels of MDM2, p53, p21, and BAX were ≈1.5-3 fold elevated in TP53 wildtype tumors with the minor homozygous Del/Del genotype. However, systematic shifts in p53 protein levels or mutation rates were not observed, suggesting that the elevated p53 mRNA levels are due to regulatory feedback loops that compensate for the effects of rs150550023 on MDM2 expression.

Expression of somatostatin receptor 2A in medullary thyroid carcinoma is associated with lymph node metastasis.

Medullary carcinoma (MTC) is an aggressive tumour that derives from the thyroid parafollicular calcitonin-secreting cells (C cells). Lymph node metastasis may occur early in disease pathogenesis and is one of the most important negative prognostic parameters. Surgery is the only curative therapy while chemotherapeutic options are limited. Neuroendocrine differentiated C cells may express somatostatin receptors (SSTR), which have a wide range of biological actions including inhibitory effects on cell survival and angiogenesis and antiproliferative effects on cancer cell lines. Moreover, they are a potential target for various somatostatin analogues. Aim of this study was to analyse the protein expression of SSTR2A and 5 in MTCs with or without the presence of lymph node metastases in correlation with various clinicopathological parameters. This retrospective immunohistochemical analysis included 97 patients with medullary thyroid carcinomas. Protein expression was detected by immunohistochemistry for somatostatin receptors 2A and 5. Various clinicopathological parameters, such as Ki-67 proliferation index or presence of desmoplasia, were included for statistical analysis. SSTR2A protein expression significantly correlated with the presence of lymph node metastases (p = 0.009), locally advanced MTCs staged according to the TNM (p < 0.001) and degree of desmoplasia (p = 0.029). Although SSTR5 protein expression significantly correlated with advanced stages of MTCs (p = 0.023) and degree of desmoplasia (p = 0.020), no correlation was found with the presence of lymph node metastases. Our results provide additional information concerning the aggressiveness of MTCs and reveal that a high SSTR2A and SSTR5 expression might be a poor prognostic feature.

Expression of hypoxia-associated proteins in sporadic medullary thyroid cancer is associated with desmoplastic stroma reaction and lymph node metastasis and may indicate somatic mutations in the VHL gene.

Medullary thyroid carcinomas (MTCs) are mostly aggressive but slowly growing malignant tumours that metastasize early to loco-regional lymph nodes. Desmoplastic stroma reaction is a strong risk factor associated with lymph node metastases. We evaluated immunohistochemically the expression of two hypoxia-associated proteins, carbonic anhydrase IX (CAIX) and hypoxia-induced factor 1α (HIF1α), and ki-67, intercellular matrix adhesion molecule E-cadherin and the stroma remodelling marker tenascin C in a series of 100 sporadic MTCs and corresponding lymph node metastases, if present. Moderate to strong expression of CAIX was seen in 53 cases, and of HIF1α in 51 cases, showing a strong correlation (p < 0.001; Spearman's coefficient of correlation, 0.59). Expression correlated with the degree of desmoplasia (pCAIX = 0.001 and pHIF1α = 0.001), with tenascin C expression (pCAIX = 0.001,pHIF1α = 0.038), with the ki-67 proliferation index (pCAIX = 0.001, pHIF1α = 0.001) and with the presence of lymph node metastases (pCAIX < 0.001 and pHIF1α = 0.007). The absence of membranous E-cadherin staining was significantly associated with the grade of desmoplasia, tenascin expression and lymph node metastases(p ≤ 0.05) but not with ki67 proliferation index or expression of hypoxia-associated factors. Expression of hypoxia-associated proteins was in most cases identical between primary tumours and lymph node metastases.Two cases showed strong uniform expression of CAIX and HIF1α in the primary tumour as well as in the lymph node metastases, and sequencing revealed mutations in the coding regions of the Von-Hippel­Lindau gene (VHL ).Our findings suggest that despite of the fact that MTCs have only slowly growth, tumour hypoxia plays an important role in the development of loco-regional metastases. Since traditional cytotoxic chemotherapy has only little effect on MTCs, targeting hypoxia-associated and -regulated proteins might be of benefit for patients.

Lipoxygenase mediates invasion of intrametastatic lymphatic vessels and propagates lymph node metastasis of human mammary carcinoma xenografts in mouse.

In individuals with mammary carcinoma, the most relevant prognostic predictor of distant organ metastasis and clinical outcome is the status of axillary lymph node metastasis. Metastases form initially in axillary sentinel lymph nodes and progress via connecting lymphatic vessels into postsentinel lymph nodes. However, the mechanisms of consecutive lymph node colonization are unknown. Through the analysis of human mammary carcinomas and their matching axillary lymph nodes, we show here that intrametastatic lymphatic vessels and bulk tumor cell invasion into these vessels highly correlate with formation of postsentinel metastasis. In an in vitro model of tumor bulk invasion, human mammary carcinoma cells caused circular defects in lymphatic endothelial monolayers. These circular defects were highly reminiscent of defects of the lymphovascular walls at sites of tumor invasion in vivo and were primarily generated by the tumor-derived arachidonic acid metabolite 12S-HETE following 15-lipoxygenase-1 (ALOX15) catalysis. Accordingly, pharmacological inhibition and shRNA knockdown of ALOX15 each repressed formation of circular defects in vitro. Importantly, ALOX15 knockdown antagonized formation of lymph node metastasis in xenografted tumors. Furthermore, expression of lipoxygenase in human sentinel lymph node metastases correlated inversely with metastasis-free survival. These results provide evidence that lipoxygenase serves as a mediator of tumor cell invasion into lymphatic vessels and formation of lymph node metastasis in ductal mammary carcinomas.

Desmoplastic stromal reaction in papillary thyroid microcarcinoma.

AIMS: To evaluate the value of a desmoplastic stromal reaction (desmoplasia) as an indicator of metastasis in papillary thyroid microcarcinoma. METHODS AND RESULTS: One hundred and nine cases of papillary thyroid microcarcinoma from the pathological archive of the Medical University of Vienna, Austria were analysed for the presence of desmoplasia in relation to other morphological and clinicopathological parameters. Eighty-one of 109 papillary microcarcinomas showed a desmoplastic stromal reaction. The desmoplasia was significantly associated with lymph node metastases (P = 0.001) and tumour diameter (P < 0.001). In addition, 'invasive parameters', such as peritumoral and vascular invasion, were associated with the presence of a desmoplastic stromal reaction (P < 0.001 and P = 0.006, respectively), and all pT3b tumours according to the UICC classification of 2002 (i.e. with perithyroidal infiltration) showed desmoplasia. Of all morphological parameters, the best indicator of lymph node metastasis was tumour calcification (P < 0.001). CONCLUSIONS: Our data indicate that a desmoplastic stromal reaction seems to be an indicator of invasive behaviour of papillary thyroid microcarcinomas significantly associated with lymph node metastases. Papillary microtumours without signs of invasion, e.g. desmoplasia, should not be regarded as invasive carcinoma, as they are more likely to be thyroidal intraepithelial neoplasias.

Identification of SERPINA1 as single marker for papillary thyroid carcinoma through microarray meta analysis and quantification of its discriminatory power in independent validation.

BACKGROUND: Several DNA microarray based expression signatures for the different clinically relevant thyroid tumor entities have been described over the past few years. However, reproducibility of these signatures is generally low, mainly due to study biases, small sample sizes and the highly multivariate nature of microarrays. While there are new technologies available for a more accurate high throughput expression analysis, we show that there is still a lot of information to be gained from data deposited in public microarray databases. In this study we were aiming (1) to identify potential markers for papillary thyroid carcinomas through meta analysis of public microarray data and (2) to confirm these markers in an independent dataset using an independent technology. METHODS: We adopted a meta analysis approach for four publicly available microarray datasets on papillary thyroid carcinoma (PTC) nodules versus nodular goitre (NG) from N2-frozen tissue. The methodology included merging of datasets, bias removal using distance weighted discrimination (DWD), feature selection/inference statistics, classification/crossvalidation and gene set enrichment analysis (GSEA). External Validation was performed on an independent dataset using an independent technology, quantitative RT-PCR (RT-qPCR) in our laboratory. RESULTS: From meta analysis we identified one gene (SERPINA1) which identifies papillary thyroid carcinoma against benign nodules with 99% accuracy (n = 99, sensitivity = 0.98, specificity = 1, PPV = 1, NPV = 0.98). In the independent validation data, which included not only PTC and NG, but all major histological thyroid entities plus a few variants, SERPINA1 was again markedly up regulated (36-fold, p = 1:3*10-10) in PTC and identification of papillary carcinoma was possible with 93% accuracy (n = 82, sensitivity = 1, specificity = 0.90, PPV = 0.76, NPV = 1). We also show that the extracellular matrix pathway is strongly activated in the meta analysis data, suggesting an important role of tumor-stroma interaction in the carcinogenesis of papillary thyroid carcinoma. CONCLUSIONS: We show that valuable new information can be gained from meta analysis of existing microarray data deposited in public repositories. While single microarray studies rarely exhibit a sample number which allows robust feature selection, this can be achieved by combining published data using DWD. This approach is not only efficient, but also very cost-effective. Independent validation shows the validity of the results from this meta analysis and confirms SERPINA1 as a potent mRNA marker for PTC in a total (meta analysis plus validation) of 181 samples.

Gastroenteropancreatic neuroendocrine tumours: the current incidence and staging based on the WHO and European Neuroendocrine Tumour Society classification: an analysis based on prospectively collected parameters.

As incidence data on gastroenteropancreatic neuroendocrine tumours (GEP-NETs) have so far only been retrospectively obtained and based on inhomogeneous material, we conducted a prospective study in Austria collecting all newly diagnosed GEP-NETs during 1 year. Using the current WHO classification, the tumor, nodes, metastases (TNM) staging and Ki67 grading and the standard diagnostic procedure proposed by the European Neuroendocrine Tumor Society (ENETS), GEP-NETs from 285 patients (male: 148; female: 137) were recorded. The annual incidence rates were 2.51 per 100,000 inhabitants for men, 2.36 per 100,000 for women. The stomach (23%) was the main site, followed by appendix (21%), small intestine (15%) and rectum (14%). Patients with appendiceal tumours were significantly younger than patients with tumours in any other site. About 46.0% were classified as benign, 15.4% as uncertain, 31.9% as well differentiated malignant and 6.7% as poorly differentiated malignant. Patients with benign or uncertain tumours were significantly younger than patients with malignant tumours. Among the malignant tumours of the digestive tract, 1.49% arose from neuroendocrine cells. For malignant gastrointestinal NETs, the incidence was 0.80 per 100,000: 40.9% were ENETS stage I, 23.8% stage II, 11.6% stage III and 23.8% stage IV. The majority (59.7%) were grade 1, 31.2% grade 2 and 9.1% grade 3. NETs of the digestive tract are more common than previously reported; the majority show benign behaviour, are located in the stomach and are well differentiated. G3 tumours are very rare.

Indeterminate thyroid nodules: a challenge for the surgical strategy.

BACKGROUND: Because no clinical parameter can establish the final status of a cytologically indeterminate thyroid nodule (ITN) or nodal-metastases in case of malignancy, the initial surgical strategy should define an oncologically adequate procedure with low morbidity. METHODS: The prognostic relevance of sex, age, tumor sizes, multifocality, thyroid function, and recurrence was analyzed in 156 consecutive patients according to the presence of malignancy and nodal metastases. The accuracy of frozen sections to reveal malignancy was determined. Clinical parameters were compared with regard to their ability to identify malignancy and nodal metastases in an ITN to determine an appropriate initial operative strategy. RESULTS: One hundred and eighteen (75.6%) patients underwent (total) thyroidectomy, 37 (23.7%) patients underwent hemithyroidectomy, and 1 patient underwent isthmus resection. Fifty-five (35.3%) patients showed malignancy. First step lymphadenectomy (lymph node dissection along the recurrent laryngeal nerve before removing the thyroid lobe) was performed in 142 patients documenting 10 nodal metastases. Comparing benign and malignant ITN, no association was found for sex (P = .17), age (P = 1.0), tumor sizes (P = .33, P = .12, P = .19 for < or =30 mm, < or =40 mm, and < or =50 mm, respectively), or thyroid function (P = .26). The determination of malignancy by frozen section showed a sensitivity of 30.9% and a specificity of 100%. No permanent hypoparathyroidism or recurrent laryngeal nerve palsy was observed postoperatively. CONCLUSION: Because of the failure of available clinical parameters to predict malignancy in cytologically ITN, hemithyroidectomy in unilateral goiter and thyroidectomy in bilateral goiter is recommended. Ipsilateral "first step central neck dissection" on the side of ITN offers the advantages of oncologically adequate resection and staging with a low morbidity, as well as avoids reoperation.

Diagnostic value of sonography, ultrasound-guided fine-needle aspiration cytology, and diffusion-weighted MRI in the characterization of cold thyroid nodules.

INTRODUCTION: The purpose of this prospective study was to assess the diagnostic value of different modalities for the characterization of cold thyroid nodules. METHODS: In 35 patients with cold nodules, thyroid carcinoma was suspected on scintigraphy. These patients were prospectively investigated with sonography, ultrasound-guided fine-needle aspiration (USgFNA), and quantitative diffusion-weighted imaging magnetic resonance imaging (DWI) (navigated echo-planar imaging; maximum b-value 800s/mm(2)) prior to surgery. The sonographic findings, USgFNA cytology, and the apparent diffusion coefficient (ADC) values of DWI were correlated with the postoperative histology of benign and malignant lesions. Statistical analysis was performed with the Kruskal-Wallis test and the Fisher's exact test. P<.05 denoted statistical significance. RESULTS: The accuracy of sonography and USgFNA was 64% and 68.8%, respectively. The sensitivity was 86.7% and 80%, respectively. Specificity was only 57.2% and 50%, respectively. The median ADC values for carcinoma and adenoma were 2.73 x 10(-3)mm(2)/s and 1.93 x 10(-3)mm(2)/s, respectively (P<.001). There was no significant difference between the median ADC value for Hashimoto thyroiditis (3.46 x 10(-3)mm(2)/s) and carcinoma. An ADC value of 2.25 x 10(-3)mm(2)/s or higher was proven to be the cut-off value for differentiating between benign and malignant cold thyroid nodules, with an accuracy of 88%, a sensitivity of 85%, and a specificity of 100%. CONCLUSIONS: These results show that quantitative DWI is a more reliable diagnostic method for differentiation between benign and malignant thyroid lesions than sonography or USgFNA. However, further studies including a larger study population are necessary to confirm our study results.

Neuroendocrine carcinomas arising in solid-organ transplant recipients: rare but aggressive malignancies.

Organ transplant recipients are at an increased risk of developing malignancies due to prolonged immunosuppression. However, the rate and clinical course of neuroendocrine tumors (NETs) following organ transplantation has not been assessed so far. We have retrospectively analyzed patients undergoing organ transplantation between 1985 and 2001 in order to assess the frequency and clinical course of NETs in organ transplant recipients. 3,190 organ transplant recipients with sufficient clinical data were identified (2,521 kidney and 669 heart transplants). In total, 161/3,190 patients (5%) developed malignancies, with 6 of them being classified as NETs (0.18%). Interestingly, all 6 patients were diagnosed with undifferentiated neuroendocrine carcinomas, while no indolent NETs were seen. Four of these patients had undergone renal, 1 patient heart and 1 patient both heart and renal transplantation. All 6 patients were given chemotherapy, but none of them responded, as all patients showed disease progression after a median of 3 cycles of chemotherapy (range 1-4) with the median survival being 4.8 months (range 2-11). The occurrence of NETs/undifferentiated neuroendocrine carcinomas following organ transplantation appears to be rare, with an incidence comparable with the normal population. Our data suggest a highly aggressive course with a dismal prognosis and unresponsiveness to chemotherapy.

Tenascin C in medullary thyroid microcarcinoma and C-cell hyperplasia.

Tenascin C (Tn-C) is an extracellular matrix glycoprotein that is expressed early in carcinogenesis including intraepithelial neoplastic lesions of different organs. In this study, we analyze whether stroma reaction seen by Tn-C expression is detected early in tumorigenesis of medullary thyroid carcinoma (MTC) including medullary microcarcinoma and C-cell hyperplasia (CCH), which is accepted to be a precursor lesion of MTC in the setting of RET oncogene germ-line mutation. Tn-C was expressed in the stroma of all medullary microcarcinoma and in the stroma next to CCH. Stromal Tn-C expression was significantly more often seen in CCH with concomitant MTC than in isolated CCH of hereditary as well as nonhereditary cases (p = 0.001 and p = 0.016, respectively). We conclude that Tn-C expression and thus early stroma remodeling is seen in medullary microcarcinoma and CCH. Stromal Tn-C expression seems to be an indicator of a further step in carcinogenesis of MTC irrespective of a RET oncogene germ-line mutation.

Follicular thyroid carcinoma in an iodine-replete endemic goiter region: a prospectively collected, retrospectively analyzed clinical trial.

OBJECTIVE: To determine risk factors for presence of lymph node or distant metastases in patients with follicular thyroid cancer (FTC) at the time of diagnosis and whether there is a relationship between the type of tumor invasion and metastases. SUMMARY BACKGROUND DATA: FTC often presents distant metastases at the initial diagnosis. As distant metastases are independent prognostic factors in a patient's survival, determination of clinicopathologic characteristics for patients who are at higher risk for developing metastases is of greater clinical importance. METHODS: The prognostic significance of gender (male vs. female), age (40 mm), number of lesions (uni- vs. multifocality), type of invasion (minimally invasive vs. widely invasive), and oncocytic changes (with vs. without) were analyzed in 207 patients, according to presence of lymph node and distant metastases at the time of initial surgery. According to the type of invasion, the carcinoma-specific survival and the disease-free survival of minimally invasive (MI) and widely invasive (WI) FTC were estimated and compared. RESULTS: None of the 127 patients with MI growth presented with lymph node metastases but 9.4% distant metastases. Overall risk factors for the presence of lymph node metastases at the initial diagnosis were multifocality (P = 0.02) and widely invasion (P = 0.0001) and for distant metastases age >45 years (P = 0.007), tumor size larger than 40 mm (P = 0.03) and widely invasion (P = 0.0001).WI-FTC patients show larger tumors (P = 0.0001), older age (P = 0.0001), and are presented more frequently in recurrent goiter disease (P = 0.0001). The estimated 10 years carcinoma-specific survival and disease-free survival for MI-tumors were significantly better than for WI-tumors (P = 0.0001). CONCLUSIONS: Total thyroidectomy is recommended in all patients with FTC because of early distant metastases. Patients with WI-FTC need a more aggressive surgical treatment because of higher tendency for lymph node metastases. MI-FTC has an excellent prognosis with no sign of lymph node metastases, which emphasizes a limited need for nodal surgery.

Hedgehog signaling is involved in differentiation of normal colonic tissue rather than in tumor proliferation.

The Hedgehog (Hh) pathway is a main regulation cascade in embryonic differentiation. It is also present in adult tissues and unusual expression has been associated with formation of benign and malignant lesions. We examined the presence of the Hedgehog pathway in normal and pathological human colon tissue. Components investigated include Sonic (Shh), Indian (Ihh), and Desert Hedgehog (Dhh), Gli1, Gli2, Gli3, and Patched (Ptch). Pathological tissue samples comprised 23 benign and 20 malignant lesions of human colon. The influence of the Hedgehog pathway on differentiation and proliferation has been investigated by analyzing the effect of the pathway inhibitor Cyclopamine on human colon cancer cell lines HT29 and CaCo2. In normal colon, we detected expression of Shh and Dhh within the lining epithelium and Patched, Gli1, and Gli2 along the whole crypts. Within all benign lesions, positive staining of Shh, Dhh, Gli1, Gli2, and Ptch was detected. Expression of Shh and Dhh was restricted to single cell aggregates. Malignant lesions also displayed focal staining pattern for Shh and Dhh but to a much lesser extent. We conclude that Hedgehog signaling is involved rather in constant differentiation and renewing of the colonic lining epithelium than in cancer formation, growth, or proliferation.

Insulinomatosis: a multicentric insulinoma disease that frequently causes early recurrent hyperinsulinemic hypoglycemia.

BACKGROUND: Multicentric insulinoma disease was characterized with regard to its histopathology, multiple endocrine neoplasia type 1 (MEN1) status, precursor lesions, and the risk of hyperinsulinemic hypoglycemia recurrence. METHODS: Fourteen patients with multicentric insulinoma disease were compared with 267 patients with sporadic and familial insulinomas. The tumors were classified according to the World Health Organization (WHO) criteria. The MEN1 status was defined clinically and by germline mutation analysis. Detection of the MEN1 gene locus was performed using fluorescence in situ hybridization. The surgical interventions and the duration of disease-free survival were recorded. RESULTS: Fourteen patients (5%) without evidence of MEN1 showed 53 macrotumors and 285 microtumors expressing exclusively insulin. In addition, they had small proliferative insulin-expressing monohormonal endocrine cell clusters (IMECCs). No allelic loss of the MEN1 locus was detected in 64 tumors. All but one patient had benign disease. Recurrent hypoglycemia occurred in 6/14 patients (11 recurrences; mean time to relapse 8.4 y). Thirteen patients with MEN1 (4.6%) showed 41 insulinomas and 133 tumors expressing islet hormones other than insulin. IMECCs were not detected. Allelic loss of the MEN1 locus was found in 17/19 insulinomas. Recurrent hypoglycemia occurred in 4/13 patients (4 recurrences; mean time to relapse 14.5 y). Solitary insulinomas were found in 254/281 patients (90.4%). IMECCs were absent. There was no recurrent hypoglycemia in 84 patients with benign insulinomas. CONCLUSIONS: Insulinomatosis is characterized by the synchronous and metachronous occurrence of insulinomas, multiple insulinoma precursor lesions, and rare development of metastases, but common recurrent hypoglycemia. This disease differs from solitary sporadic and MEN1-associated insulinomas.

The influence of gender, age, and RET polymorphisms on C-cell hyperplasia and medullary thyroid carcinoma.

BACKGROUND: RET germline mutations predispose to the development of hereditary medullary thyroid carcinoma (hMTC). Several single nucleotide polymorphisms (SNPs) are described associated with sporadic MTC (sMTC). However, the findings regarding their influence on the clinical course and biological behavior of this disorder are discordant. To clarify the contradictory findings, we studied the association of certain SNPs considering age, gender, and histopathology in a large Austrian cohort with C-cell hyperplasia (CCH) and MTC. METHODS: Genotyping of SNPs located in RET codons 691, 769, 836, and 904 from 199 patients with MTC and CCH (basal calcitonin > 10 pg/mL, pentagastrin stimulated > 100 pg/mL) was performed, and the results were analyzed considering gender, age at diagnosis, and histopathology. RESULTS: No significant difference of SNP frequencies was found in the study patients versus normal controls. In sMTC and sporadic CCH (sCCH) no significant association of SNP frequency with the age at diagnosis was found. In patients with sporadic C-cell disease (sCCH and sMTC), 3.7 times more males than females suffered synchronously from papillary or follicular thyroid cancer (20/97 [20.6%] males; 3/54 [5.6%] females; p = 0.02). sCCH was revealed more frequently in males (89/97, 91.7%) than in females (27/54, 50%; p = 10(-8)). In contrast to males, the ratio of CCH to total C-cell disease was significantly higher in females with hereditary (26/32, 81%) compared to those with sporadic disease (27/54, 50%; p = 0.006). CONCLUSIONS: In this study RET SNPs had no clinical impact on the development of sporadic C-cell disease when the age of diagnosis or gender is considered. C-cell disease seems to predispose males to the development of papillary and follicular thyroid cancer. In addition, at least in females with CCH RET germline mutation, screening is recommended even if the family history is negative for MTC.

Medullary thyroid microcarcinoma recommendations for treatment - a single-center experience.

BACKGROUND: Conflicting recommendations exist regarding lymph node (LN) surgery in microMTC (or=10pg/ml) and pentagastrin-stimulated calcitonin levels (sCT:>100pg/ml) were selected for initial surgery. None of the patient was a member of any known MTC family. Biochemical and morphological data of microMTC were compared with 146 patients with C-cell hyperplasia (CCH). RESULTS: MicroMTC (tumor diameter: 4.2+/-2.6mm; unifocal:68; multifocal:29) was documented in 97 of 159 (61%) MTC patients. In 11 (11%) patients, 1-19 LNs were involved. Correlating bCT and sCT levels neither predicted N-stage, nor differentiated between microMTC and CCH. CONCLUSIONS: The biochemical discrimination cannot be made between patients with CCH and MTC, and patients with MTC with/without LN metastasis. Thus, thyroidectomy and central neck dissection is indicated in patients with "mildly" elevated sCT levels (<560pg/ml) (LN positive: 1 of 37 patients [2.7%]). A lateral neck dissection may be added "on demand" (in the setting of measurable postoperative bCT and/or sCT levels indicating LN metastasis). Patients with "highly" elevated sCT (>or=560pg/ml) must be treated as "palpable" MTC (LN positive: 10 of 54 patients [18.5%]).

Comparison of RNA amplification techniques meeting the demands for the expression profiling of clinical cancer samples.

Available ribonucleic acid (RNA) amplification methods are extensively tested for reproducibility, but only a few studies additionally deal with potential amplification bias. On targeted arrays, we evaluated three amplification protocols, which are less time consuming than the commonly used T7-RNA polymerase based in vitro transcription protocols and therefore may be more suitable for clinical use: Template-switching polymerase chain reaction (PCR), Ribo-single primer isothermal amplification and a random primer-based PCR. Additionally, a more sensitive labelling method, Dendrimer labelling, was evaluated. All methods were compared to unamplified RNA labelled at reverse transcription. From our results, we conclude that RNA amplification with template-switching PCR is highly reproducible and results in a reliable representation of the starting RNA population. We then assessed whether RNA amplification of clinical breast and thyroid cancer samples with template-switching PCR showed robust performance when altered cycle numbers or partially degraded RNA were used. Template-switching PCR proved to be a very reliable method for global RNA amplification, even when starting from partially degraded RNA down to a RNA Integrity Number of 4.3. In conclusion, template-switching PCR amplification promises to help micro-array expression profiling of limited amounts of human samples on its way to a clinical routine.

Molecular characterization of the desmoplastic tumor stroma in medullary thyroid carcinoma.

Medullary thyroid carcinomas are aggressive neoplasias that metastasize very early to loco-regional lymph nodes, and tumors with a desmoplastic stromal reaction have a higher incidence of lymph node metastasis. In order to characterize the desmoplastic response in thyroid cancers, we evaluated the expression pattern of three molecular markers of activated fibroblasts/myofibroblasts, namely, fibroblast activation protein alpha (FAPalpha), tenascin-C (Tn-C), and alpha-smooth muscle actin (alpha-SMA), as well as the endothelial markers endoglyx-1, CD34 and CD31 in a series of 28 metastatic and non-metastatic medullary thyroid cancers. Immunohistochemical studies demonstrated that the three fibroblast activation markers (FAPalpha, Tn-C, alpha-SMA) are consistently expressed in the peritumoral and intratumoral stromal compartment of medullary thyroid carcinomas and expression of FAPalpha and Tn-C correlated with the degree of desmoplasia determined histologically (p=0.001 for FAPalpha and p<0.001 for Tn-C). Moreover, the extent of desmoplasia as well as the expression of FAPalpha and Tn-C correlated with the presence of lymph node (LN) metastases (p=0.002, p=0.005 and p=0.002, respectively). No correlation was found between the microvessel density (neoangiogenesis) in the tumor stroma, assessed with the endoglyx-1, CD34 and CD31 markers, and the degree of desmoplasia or incidence of LN metastases. Using a bioinformatics-based search of the BioExpresstrade mark database we found in a series of 48 thyroid cancers a significant correlation between FAPalpha RNA expression and incidence of LN metastases also in papillary cancers. These findings suggest that the link between specific molecular markers of tumor stromal reaction and locoregional metastasis extends from medullary to other thyroid cancer types.

The value of intraoperative pentagastrin testing in medullary thyroid cancer.

BACKGROUND: The decrease of calcitonin levels after curative operation in patients with medullary thyroid cancer is characterized by individual variation; therefore, intraoperative calcitonin measurements to evaluate the completeness of the resection seem to not be feasible. The aim of this study was to evaluate whether an intraoperative pentagastrin test after thyroidectomy and central neck dissection is useful to predict lymph node involvement of the lateral neck. METHODS: A group of 30 consecutive patients underwent primary surgery. After thyroidectomy and dissection of the central lymph node compartment, an intraoperative pentagastrin test was performed. Biochemical and histologic data were compared retrospectively. RESULTS: Of the group, 20 patients (67%) showed no, or only central neck lymph node, involvement and no increase in calcitonin after intraoperative stimulation. Lymph node involvement was documented histologically in the lateral neck of 10 patients (33%), and 8 patients showed an increase of calcitonin as an indication of lymph node involvement. In two patients, each with 1 single micrometastasis in the lateral neck, the intraoperative pentagastrin test was negative. CONCLUSIONS: Intraoperative calcitonin monitoring after pentagastrin stimulation seems promising in predicting lymph node involvement of the lateral neck to aid selection of patients for lateral lymph node dissection. The development of a highly sensitive, quick calcitonin assay is imperative.

Proteomic expression profiling of thyroid neoplasms.

Thyroid cancer is the most common endocrine neoplasm with multiple histologic subtypes, each associated with different treatments and outcomes. Differentiating benign neoplasms such as follicular adenomas from malignant entities such as follicular carcinomas and papillary carcinoma can be challenging. To define the proteomic profile of different thyroid tumors, we screened an antibody array of 330 features against five thyroid neoplasms: follicular adenoma, follicular carcinoma, papillary carcinoma, anaplastic carcinoma, and medullary carcinoma as well as normal thyroid epithelium. Eight candidate biomarkers; c-erbB-2, Stat5a, Annexin IV, IL-11, RARα, FGF7, Caspase 9, and phospho-c-myc were identified as differentially expressed on the antibody array, and validated with immunohistochemistry on tissue microarrays, with a total of 144 samples of the same variety of thyroid neoplasms. Analysis revealed c-erbB-2, Annexin IV, and Stat5a have potential clinical utility to differentiate follicular adenoma, follicular carcinoma, and papillary carcinoma from each other. By using an antibody array as a discovery platform and a tissue microarray as a first step in validation on a large number of specimens, we have identified new markers that have potential utility in the diagnosis of thyroid neoplasms.