[Immunohistochemical hormonal mismatch and human epidermal growth factor type 2 [HER2] phenotype of brain metastases in breast cancer carcinoma compared to primary tumors]. / Discordance du phénotype immunohistochimique hormonal et du récepteur du facteur de croissance épidermique de type 2 [HER2] des métastases cérébrales de cancer du sein comparativement à leurs primitifs.

INTRODUCTION: Phenotype changes between primary tumor and the corresponding brain metastases are recent reported data. Breast cancer, with biological markers predicting prognosis and guiding therapeutic strategy remains an interesting model to observe and evaluate theses changes. The objective of our study was to compare molecular features (estrogen receptor [ER], progesterone receptor [PR], and human epidermal growth factor receptor type 2, [HER2]) between brain metastases and its primary tumor in patients presenting with pathologically confirmed breast cancer. MATERIAL AND METHODS: This retrospective study was based on the immunohistochemical analysis of the brain metastases paraffin embedded samples stored in our institutional tumor bank, after surgical resection. The level of expression of hormonal receptors and HER2 on brain metastases were centrally reviewed and compared to the expression status in primary breast cancer from medical records. RESULTS: Forty-four samples of brain metastases were available for analysis. Hormonal receptor modification status was observed in 11/44 brain metastases (25%) for ER and 6/44 (13.6%) for PR. A modification of HER2 overexpression was observed in brain metastases in 6/44 (13.6%). Molecular subtype modification was shown in 17 cases (38.6%). A significant difference was demonstrated between time to develop brain metastases in cases without status modification (HER2, ER and PR) (med=49.5months [7.8-236.4]) and in cases in which brain metastases status differs from primary tumor (med=27.5months [0-197.3]), (P=0.0244, IC95=3.09-51.62, Mann and Whitney test). CONCLUSION: the main interest of this study was to focus on the molecular feature changes between primary tumor and their brain metastases. Time to develop brain metastases was correlated to phenotypic changes in brain metastases.

[Oncogenic drivers in daily practice improve overall survival in patients with lung adenocarcinoma]. / Bénéfice à l'évaluation moléculaire en routine pour les cancers bronchiques métastatiques.

BACKGROUND: EGFR tyrosine kinase inhibitors and crizotinib are nowadays the optimal treatment for metastatic lung cancer with activation of EGFR mutations and ALK rearrangement. In addition, several targeted agents are in development for lung cancer with other oncodrivers. In France, since 2011, six oncodrivers are routinely tested in patients with stage IV. The aim of this study was to assess whether systematic detection of oncodrivers and matched targeted therapy improve overall survival in patients with advanced lung adenocarcinoma. METHODS: This study included all consecutive patients treated in our department for advanced lung adenocarcinoma from January 2012 to December 2013. We studied the impact in survival according to the presence of the driver and the targeted therapy. RESULTS: Among the 261 patients included, oncodrivers alterations were found in 43.5% of patients: EML4-ALK fusion genes (2.1%), EGFR (10.3%), KRAS (27.7%), BRAF (2.5%), HER2 (0.8%), and PI3KCA (0.8%) mutations. Twenty-nine percent of patients (n=32) with oncodrivers received matched targeted therapy. Patient treated by targeted agent appropriate to an oncogenic driver had a median survival of 21.1 months (95% CI: 14.7-27.5). The patients (n=79) who did not receive targeted therapy had a median survival of 6.6 months (95% CI: 4.3-8.9). The patients (n=150) without identified driver had a median survival of 9.7 months (95% CI: 6.7-11.7); P<0.001. CONCLUSION: An actionable oncodriver was routinely detected in nearly half of patients with advanced lung adenocarcinoma. This systematic detection may influence treatment outcomes, notably with matched targeted therapy.

The effect of Pt NPs crystallinity and distribution on the photocatalytic activity of Pt-g-C3N4.

Loading of a co-catalyst on the surface of a semiconductor photocatalyst is often carried out without considering the effect of the loading procedure on the final product. The present study looks in detail at the effect that the loading method has on the morphology and final composition of platinum-based nanoparticles by means of XPS and TEM analysis. Additionally, reduction pre-treatments are performed to investigate how the coverage, crystallinity and composition of the NPs affect the photocatalytic H2 evolution. The activity of Pt-g-C3N4 can significantly be enhanced by controlling the properties of the co-catalyst NPs.

New IDH1 I113T mutation associated with BRAF V600E mutation: new driver of gliomagenesis?

BACKGROUND: IDH mutations and BRAF mutations are classically mutually exclusive and usually associated with infiltrative or circumscribed gliomas and glioneuronal tumors respectively. CASE REPORT: We report the case of a 26-year old man with intracranial hypertension revealing voluminous right frontal lesion. Surgical resection was performed and pathological examination found two distinct tumoral areas: a glioma-like area with calcification without mitosis; a second with pleomorphic glial cells with higher Mib index, high CD34 expression and endothelial proliferation. No necrosis was recorded. Molecular analyses revealed both IDH1 I113T and BRAF V600E mutations. Although this glioma was difficult to clarify, diagnosis of pleomorphic xanthoastrocytoma with anaplastic feature was suggested based on the association of some pathological feature (eosinophilic granular bodies, reticulin network and diffuse CD34 expression) and the BRAF V600E mutation. CONCLUSION: We report a new IDH1 mutation associated with BRAF mutation in a very unusual glial tumor.

Triple negative breast carcinoma EGFR amplification is not associated with EGFR, Kras or ALK mutations.

BACKGROUND: The amplification of epidermal growth factor receptor (EGFR) in triple negative breast carcinomas (TNBC) suggests its potential therapeutic application, as for HER-2, using standardised methods of measurement. In this regard, we aimed to compare several methods for evaluating EGFR amplification along with potential mutations for suitability in clinical practice. METHODS: Tissue sections of 138 TNBCs were used (1) to compare EGFR amplification and expression by silver in situ hybridisation (SISH) to qPCR and immunohistochemistry (IHC) and (2) to search for EGFR mutations, along with Kras, PI3K, Braf and HER-2 mutations and echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) translocation. RESULTS: (1) Amplification of EGFR was observed in well-characterised TNBCs (up to 92%); (2) qPCR correlated with SISH with 94% specificity and 75.6% sensitivity; (3) IHC correlated with SISH with 97% sensitivity and 78% specificity; (4) no EGFR, Kras mutations or EML4-ALK translocations were found, but PI3K and Braf mutations were observed in 26% of cases; and (5) small, acentric circular extrachromosomal DNA similar to 'double minutes' in glioblastomas was observed in 18% of SISH sections. CONCLUSIONS: SISH and IHC are methods that are suitable in clinical practice to screen for EGFR amplification and overexpression, which are frequently observed in TNBC. Patients with TNBC are potential candidates for EGFR-targeted therapy combined with PI3K and Braf inhibitors.

Estudio transversal comparativo de las fórmulas CKD-EPI y MDRD-4 a partir de la historia clínica informatizada de Atención Primaria de Barcelona / A comparative, cross-sectional study of the CKD-EPI and MDRD-4 equations from the Primary Care computerized clinical history of Barcelona

Antecedentes: La prevalencia de enfermedad renal crónica (ERC) en España supera el 7%. Su diagnóstico precoz puede ayudar a frenar la evolución. En Atención Primaria (AP) se calcula el filtrado glomerular estimado (FGe) mediante fórmulas basadas en la creatinina plasmática(CrP).Objetivos: Comparar los FG e según las fórmulas MDRD-4 y CKD-EPI a partir de la historia clínica informatizada (HCI) y la clasificación de ERC en una población de AP. Material y métodos: Estudio transversal. Se incluyó a pacientes de 20-99 años, asignados a centros de AP de Barcelona, con CrP entre julio de 2008 y junio de 2010. Se obtuvieron losdatos de la HCI. Se calculó el FGe mediante CKD-EPI y MDRD-4.Resultados: Se estudió a un total de 447.140 personas: 58,7% mujeres, 56,6 (DE 18,8) años. El 32,5% con diagnóstico de hipertensión arterial, 11% con diabetes mellitus y 9,3% con enfermedad cardiovascular asociada. La CrP media fue 0,89 (0,28) mg/dL, FGe por MDRD-4 de 80,59(21,04) mL/min/1,73 m2y por CKD-EPI de 85,03 (21,13) mL/min/1,73 m2. La CKD-EPI, respecto a MDRD-4, clasificó el 2,3% de los pacientes en estadios menos avanzados de ERC, el 96,8% en el mismo y el 0,9% en más avanzados. El índice kappa fue de 0,87. En números absolutos clasificó en estadios 3b-4-5 (posible derivación a nefrología) a 958 pacientes más, con 691 personas >69 años en estadio 3b.Conclusiones: Utilizar una u otra fórmula puede variar el FGe. La CKD-EPI tiende a clasificar enestadios más avanzados en > 69 años. El uso de cada fórmula puede hacer cambiar el número y tipo de derivaciones a nefrología

Background: The prevalence of chronic kidney disease (CKD) in Spain is higher than 7%. Its early diagnóstico can help to delay its progression. Glomerular filtration rate (GFR) based on serum creatinine (SC) is calculated in primary care (PC) to identify CKD. Objectives: To compare GFR by MDRD-4 and CKD-EPI equations obtained from clinical records(CR) and to compare the classification of CKD in a primary care population. Material and methods: A cross-sectional study was performed, including patients 20-99 years old, assigned to primary care centers of Barcelona, with a SC recorded between July 2008 and June 2010. Data were obtained from the CRs. GFR was calculated from MDRD-4 and CKD-EPI equations. Results: A total of 447,140 persons were studied (58.7% females, 56.6 [SD 18.8] years old).Of these 32.5% were diagnosed of hypertension, 11.0% diabetes and 9.3% had some associated cardiovascular disease. SC 0.89 (0.28) mg/dL (78.7 [SD 24.8] mol/L), GFR being 80.59(21.04) mL/min/1.73 m2by MDRD-4, and 85.03 (21.13) mL/min/1.73 m2by CKD-EPI. CKD-EPI compared to MDRD-4 classified 2.3% of patients in less advanced stages of CKD, 96.8% in the same stage and 0.9% in more advanced stages. Kappa coefficient: 0.87. In absolute numbers, CKD-EPI classified in 3b-4-5 stages (candidates for referral to nephrology) 958 patients more,691 of them being patients > 69 years old and stage 3b.Conclusions: Using one equation or another could vary the estimation of GFR. CKD-EPI tends to classify patients older than 69 into more advanced stages. Each equation can change the number and type of referral to nephrology (AU)

A nomogram to predict individual prognosis in node-negative breast carcinoma.

BACKGROUND: Currently, the benefit of chemotherapy (CT) in node-negative breast carcinoma (NNBC) is discussed. The evaluation of classical clinical and histological factors is limited to assess individual outcome. A statistical model was developed to improve the prognostic accuracy of NNBC. METHODS: A total of 305 node-negative breast carcinomas who underwent surgery (+/- radiotherapy) but no adjuvant treatment were selected. Putative prognosis factors including age, tumour size, oestrogen receptor (ER), progesterone receptor (PgR), Scarff-Bloom-Richardon (SBR) grading, urokinase plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1) and thymidine kinase (TK) were evaluated. The developed model was internally validated using Harrell's concordance index. A prognosis index (PI) was proposed and compared with Adjuvant! Online program. RESULTS: Age (p < 0.001), pathological tumour size (pT) (p < 0.001), PgR (p = 0.02), and PAI-1 (p ≤ 0.001) were included in the Cox regression model predicting Breast cancer specific survival (BCSS) at 5-years. Internal validation revealed a concordance index of 0.71. A PI score was derived from our nomogram. The PI score was significantly associated with BCSS (hazard ratio (HR): 4.1 for intermediate, p=0.02, HR: 8.8, p < 0.001 for high group) as compared to Adjuvant! Online score (HR: 1.4, p=0.14). CONCLUSION: A nomogram can be used to predict probability survival curves for individual breast cancer patients.

Epstein-Barr virus as a marker of biological aggressiveness in breast cancer.

PURPOSE: Although a potential role of the Epstein-Barr virus (EBV) in the pathogenesis of breast cancer (BC) has been underlined, results remain conflicting. Particularly, the impact of EBV infection on biological markers of BC has received little investigation. METHODS: In this study, we established the frequency of EBV-infected BC using real-time quantitative PCR (RT-PCR) in 196 BC specimens. Biological and pathological characteristics according to EBV status were evaluated. RESULTS: EBV DNA was present in 65 of the 196 (33.2%) cases studied. EBV-positive BCs tended to be tumours with a more aggressive phenotype, more frequently oestrogen receptor negative (P=0.05) and with high histological grade (P=0.01). Overexpression of thymidine kinase activity was higher in EBV-infected BC (P=0.007). The presence of EBV was weakly associated with HER2 gene amplification (P=0.08). CONCLUSION: Our study provides evidence for EBV-associated BC undergoing distinct carcinogenic processes, with more aggressive features.

Diagnostic work-up of inflammatory bowel disease in children: the role of calprotectin assay.

BACKGROUND: Some reports highlight the potential application of fecal calprotectin as a direct biomarker of intestinal inflammation and, therefore, as support in choosing candidates for endoscopy. The value of 100 µg/g was recently assumed as the best cutoff for this assay. The purpose of this study was to assess the diagnostic precision of the fecal calprotectin assay, compared to histology, as a stool-screening biomarker for inflammatory bowel disease (IBD) among a group of prospectively identified patients referred for recurrent abdominal pain and altered bowel habits. METHODS: Between 1999 and 2007 we prospectively evaluated the calprotectin assay in a cohort of patients with recurrent abdominal pain and altered bowel habits associated or not with other symptoms suggestive of IBD. All patients suspected of IBD, according to Rome and Porto criteria, provided stool specimens for the calprotectin assay and subsequently underwent endoscopic procedures. RESULTS: Compared to histology, the cutoff of 100 µg/g reached a sensitivity and specificity of 100% and 68%, respectively, and a likelihood ratio (LR) of 3.1. The cutoff value of 160 µg/g, however, in our series produced the best joint estimate of sensitivity and specificity: 100% and 80%, respectively, with an LR of 5. CONCLUSIONS: In pediatric patients with recurrent abdominal pain and changes in stool habits, a positive calprotectin assay is closely associated with IBD; its systematic employment, therefore, seems to improve the process of endoscopy referral. This test, simple and inexpensive, could be included in the first noninvasive phase of an IBD diagnostic work-up.

[Prognostic implications of biologic markers in intracranial meningiomas: 120 cases]. / Implications pronostiques de différents marqueurs biologiques dans les méningiomes intracrâniens: à propos de 120 cas.

UNLABELLED: The recurrence and progression of treated intracranial meningiomas highlights the problem of the type of follow-up that should be used and whether early complementary treatment is indicated. The aim of this study was to evaluate different biochemical markers involved in cell proliferation and transformation to identify new prognostic factors in intracranial meningiomas. Between 1989 and 2003, 120 intracranial meningiomas were studied biochemically. The levels of estrogen receptors (RE), progesterone receptors (RP), cathepsin B (CB), cathepsin L (CL), stefin A (ATA), stefin B (STB), cystatin C (CYSC), urokinase (u-PA), type 1 plasminogen activator inhibitors (PAI-1), cathepsin D (CD) and thymidine kinase activity (TK) were measured in tumor extracts using biochemical assays. RESULTS: Out of 120 meningiomas, 73 were grade I, 39 grade II and eight grade III according to the WHO classification. Of these patients, 17 showed recurrence. The mean follow-up was 47 months. Monofactorial analysis showed that expression of progesterone receptors (RP) had an inverse correlation with recurrence (p=0.0025 %) and that thymidine kinase activity (TK), cathepsin L (CL), the WHO grade and the degree of tumor resection correlated with recurrence (p<0.05). Principal component analysis and linear discriminant analysis confirmed these results. The results of this study confirm the importance of biological parameters (PR, CL, TK) as prognostic factors for the risk of recurrence in intracranial meningiomas.

Simultaneous quantitative detection of relevant biomarkers in breast cancer by quantitative real-time PCR.

The assessment of ERa, PgR and HER2 status is routinely performed today to determine the endocrine responsiveness of breast cancer samples. Such determination is usually accomplished by means of immunohistochemistry and in case of HER2 amplification by means of fluorescent in situ hybridization (FISH). The analysis of these markers can be improved by simultaneous measurements using quantitative real-time PCR (Qrt-PCR). In this study we compared Qrt-PCR results for the assessment of mRNA levels of ERa, PgR, and the members of the human epidermal growth factor receptor family, HER1, HER2, HER3 and HER4. The results were obtained in two independent laboratories using two different methods, SYBR Green I and TaqMan probes, and different primers. By linear regression we demonstrated a good concordance for all six markers. The quantitative mRNA expression levels of ERa, PgR and HER2 also strongly correlated with the respective quantitative protein expression levels prospectively detected by EIA in both laboratories. In addition, HER2 mRNA expression levels correlated well with gene amplification detected by FISH in the same biopsies. Our results indicate that both Qrt-PCR methods were robust and sensitive tools for routine diagnostics and consistent with standard methodologies. The developed simultaneous assessment of several biomarkers is fast and labor effective and allows optimization of the clinical decision-making process in breast cancer tissue and/or core biopsies.

Reliability and discriminant validity of HER2 gene quantification and chromosome 17 aneusomy analysis by real-time PCR in primary breast cancer.

There is an increasing demand for the evaluation of HER2 status in breast cancer. In this study, sections from fixed tissues and triton extracts of tissue homogenates were obtained from 163 malignant breast tumors and analyzed in parallel using immunohistochemistry combined with fluorescence in situ hybridization, as gold standard tests, and an ELISA test (c-erbB2/c-neu Rapid Format ELISA, Oncogene Research Products, USA). Tumor DNA was employed to evaluate two quantitative PCR methods: the HER2/neu DNA Quantification Kit (Roche Diagnostics GmbH, Germany), which uses the gastrin chromosome 17 reference gene, and our recently developed Oncolab qPCR assay, where both a chromosome 17 gene (somatostatin receptor type II (SSTR2)) and a non-chromosome 17 reference gene (glyceraldehyde-3-phosphate deshydrogenase (GAPDH)) were used to detect an increase in HER2 gene copy number and to evaluate the aneusomy of chromosome 17, respectively. By IHC/FISH and ELISA, HER2 was overexpressed in 27 (16.6%) and 24 (14.7%) samples, respectively. With the Roche and Oncolab qPCR assays, 29 (17.8%) samples showed a ratio of HER2/gastrin > or = 2.0 and 26 (16.0%) showed a ratio of HER2/SSTR2 > or = 2.0, respectively. In samples presenting HER2/SSTR2 <2.0 and HER2/GAPDH > or = 2.0, which was indicative of a chromosome 17 polysomy, we observed a modest increase in HER2 protein expression. Complete agreement between the four methods for HER2 status determination was obtained for 154 (94.5%) samples. Overall, these results demonstrate that quantitative PCR is a reliable method for analyzing HER2 status and chromosome 17 polysomy.

Identification of genes differentially expressed in glioblastoma versus pilocytic astrocytoma using Suppression Subtractive Hybridization.

Glioblastoma (GBM) is a highly malignant glioma, which has the propensity to infiltrate throughout the brain in contrast to pilocytic astrocytoma (PA) of the posterior fossa, which does not spread and can be cured by surgery. We have used Suppression Subtractive Hybridization to define markers that better delineate the molecular basis of brain invasion and distinguish these tumor groups. We have identified 106 genes expressed in PA versus GBM and 80 genes expressed in GBM versus PA. Subsequent analysis identified a subset of 20 transcripts showing a common differential expression pattern for the two groups. GBM differs from PA by the expression of five genes involved in invasion and angiogenesis: fibronectin, osteopontin, chitinase-3-like-1 (YKL-40), keratoepithelin and fibromodulin. PA differs from GBM by the expression of genes related to metabolism (apolipoprotein D), proteolysis (protease-serine-11), receptor and signal transduction (PLEKHB1 for Pleckstrin-Homology-domain-containing-protein-family-B-member-1), transcription/translation (eukaryotic-translation-elongation-factor-1-alpha1) processes and cell adhesion (SPOCK1 for SPARC/Osteonectin-CWCV-kazal-like-domains-proteoglycan). The expression of these genes was confirmed by real-time quantitative RT-PCR and immunohistochemistry. This study highlights the crucial role of brain invasion in GBM and identifies specific molecules involved in this process. In addition, it offers a restricted list of markers that accurately distinguish PA from GBM.

Molecular profile of androgen-independent prostate cancer xenograft LuCaP 23.1.

After castration or therapeutic hormone deprivation, most cancer of the prostate (CaP) cells develop androgen-independent (AI) growth. In this work, we studied the effect of androgen depletion (castration) on the growth of experimental model LuCaP 23.1 xenograft. A total of 101 nude mice were implanted and analysed for their growth profile before experimental period 1 (11 weeks) and after castration experimental period 2 (15 weeks). For specific periods, tumors were harvested and assessed for molecular marker expression specific for CaP. Taking into account tumor dynamic growth, prior to castration we found 37 fast growing (FG) tumors (948.9+/-76.9 mm3) and 63 slow growing (SG) tumors (229.6+/-18.4 mm3). Real-time quantitative RT-PCR showed that in comparison to SGs, FGs contained elevated expression of epidermal growth factor receptor type 1 (HER1), urokinase plasminogen activator (uPA), thymidine phosphorylase (TP) and thymidilate synthase (TS) mRNAs expression and low levels of 5alpha-reductase 2 (5alpha-R2) mRNA. After castration all FG tumors progressed rapidly (by 5 weeks) to AI growth (FG-P). In SG castrated tumors, 66% of tumors showed retarded progression (by 12 weeks) to AI (SG-P), whereas 34% responded to castration (SG-R). Molecular analysis demonstrated distinct molecular profiles integrating different pathways associated with AI progression. The progressive tumors FG-P, and some tumors of SG-P subgroup, presented significantly high levels of HER1, epidermal growth factor receptor type 2 (HER2), TS, uPA, TP, tumor necrosis factor superfamily member 6 (FAS) and peptidylglycine alpha-amidating mono-oxygenase (PAM) mRNA all of which correlated with androgen receptor (AR) mRNA. The second subgroup of SG-P tumors showed a high expression of the anti-apoptotic gene Bcl-2. A third subgroup of SG-P tumors showed significant expression of hypoxia-related genes such as adrenomedullin (AM) after castration. LuCaP 23.1 xenograft represent a useful dynamic model to study pre-clinically new therapeutic molecules and evaluate non-randomized therapeutics protocols combining different target inhibition specific to each AI pathways.

Fas/FasL expression in tumor biopsies: a prognostic response factor to fluoropyrimidines?

Various studies suggested that cytotoxicity induced by 5-fluorouracil (5-FU) is an apoptotic mechanism possibly mediated by the Fas/FasL system. In this preliminary work, we studied retrospectively the role the Fas/FasL expression as a predictive response factor with fluoropyrimidine-based chemotherapies. We developed a real-time PCR method for measuring Fas and FasL expression in various biopsies from patients treated with a FUFOL-like protocol. No correlation was found between Fas or FasL expression and overall survival or partial response. However, the PCR assay was simple and convenient to use for quantitation of Fas/FasL in tumor biopsies.

Mutation analysis in the coding sequence of thymidine kinase 1 in breast and colorectal cancer.

We report the first mutational study of thymidine kinase 1 (TK1) performed in human solid tumors. We sequenced cDNAs representing the complete coding region of TK1 in human breast (n=22) and colorectal (n=26) cancer. Codon 106 near the ATP binding site constantly differed (ATG --> GTG; Met --> Val) from the one deposited by Bradshaw and Deininger in the Genbank database (Accession number NM_003258). Silent polymorphisms at codon 11 (CCC --> CCT; Pro --> Pro) and codon 75 (GCG --> GCA; Ala --> Ala) were frequently detected in tumors as well as in normal tissues. In breast cancer the two polymorphisms were observed in 63.6% of the samples analyzed. No significant association could be found between polymorphisms and TK activity. In colorectal cancer the incidence of the two changes was 73.1% and 69.2%, respectively. Interestingly, one colon cancer with high cytosolic TK activity displayed two missense mutations located in and near the putative phosphorylation site by tyrosine kinase (s) (TAT --> CAT; Tyr --> His) and by cAMP-, cGMP-dependent protein kinase (TAC --> TGC; Tyr --> Cys), respectively; adjacent normal mucosa showed no mutation. This may open new avenues that imply TK1 activity in tumor cell proliferation.

[RT-PCR in clinical diagnosis]. / La RT-PCR en diagnostic clinique.

Application fields of RT-PCR (reverse transcription-polymerase chain reaction) in clinical diagnosis comprises the assessment of viral load for RNA viruses and the analysis of gene transcription products. RT-PCR is also helpful when large genes have to be sequenced. Developments of quantitative approaches using real-time PCR recently led to a major widening of RT-PCR applications in clinical diagnosis. However, RT reaction is delicate due to its lack of reproducibility and to RNA lability and frequent contamination by DNA. In some cases additional difficulties come from the need to obtain a specific amplification in the presence of homologous sequences which might be present in higher amounts than the sequence of interest. These caveats have to be taken into account, when designing the RT protocol, and when choosing PCR primers and internal and/or external references. This review is aimed at helping the experimental setup of a RT-PCR based assay according to the objectives.

Subcutaneous abdominal, but not femoral fat expression of plasminogen activator inhibitor-1 (PAI-1) is related to plasma PAI-1 levels and insulin resistance and decreases after weight loss.

AIMS/HYPOTHESIS: Abdominal fat produces plasminogen activator inhibitor-1 (PAI-1) and could contribute to increased plasma PAI-1 values in human obesity associated with insulin resistance. Femoral fat, which is not associated with insulin resistance, is thought to be metabolically different from the abdominal fat. This study aimed to assess PAI-1 expression in these two fat territories in obese and lean subjects and to determine if concomitant changes of plasma and adipose tissue PAI-1 values occur after weight reduction. METHODS: In 24 obese and 16 lean subjects, PAI-1 expression in abdominal and femoral subcutaneous fat, plasma PAI-1, insulin, triglyceride concentrations and insulin resistance were determined at the start of the study and in obese subjects after a 3-month weight reduction programme as well. RESULTS: PAI-1 mRNA content in the abdominal subcutaneous fat was higher in obese than in lean subjects and positively correlated with plasma PAI-1 values (p < 0.01) and markers of insulin resistance (p < 0.05). In 18 obese subjects, re-examined after successful dieting, PAI-1 mRNA content decreased in the abdominal subcutaneous fat along with plasma PAI-1. However, the absolute changes of these two variables were not associated. In contrast, PAI-1 mRNA content in the femoral subcutaneous fat did not differ between lean and obese subjects, was not associated with plasma PAI-1 values or with markers of insulin resistance, and did not change after weight loss. CONCLUSION/INTERPRETATION: Only the abdominal, but not the femoral subcutaneous fat PAI-1 expression is a potential contributor to increases in plasma PAI-1 in obesity. Both plasma and abdominal subcutaneous fat PAI-1 values decreased significantly after weight reduction, although their absolute changes were not associated.

Frequency and genome load of Epstein-Barr virus in 509 breast cancers from different geographical areas.

Since the few data exploring a possible association between Epstein-Barr virus (EBV) and breast cancer are conflicting, we investigated this association together with the influences of geographical areas. 509 breast cancers were sampled from areas with varying risks of nasopharynx carcinoma (NPC) such as North Africa (Algeria and Tunisia, high-risk area); southern France (Marseille, intermediate-risk area); and northern Europe (northern France, the Netherlands and Denmark; low-risk areas). Polymerase chain reaction (PCR) of a subregion of EBV BamHIC encoding the EBERs demonstrated that 31.8% of the tumours contained the viral genome. No significant differences were observed among the geographical areas. However, positive samples showed higher loads of the EBV genome in the NPC high- and intermediate-risk areas than in the low-risk areas. EBV type 1 was the dominant strain. In situ hybridization studies using a(35)S-labelled riboprobe for EBER1 and a laser capture microdissection, combined with quantitative PCR, showed that EBV localization was restricted to some tumour epithelial cell clusters. EBV could not be detected in the stroma. Considering the whole population covered, the presence of the EBV genome was not correlated with age, menopausal status, tumour, size, nodal status or histological grade.

Bim-23244, a somatostatin receptor subtype 2- and 5-selective analog with enhanced efficacy in suppressing growth hormone (GH) from octreotide-resistant human GH-secreting adenomas.

Although both somatostatin receptor subtype 2 (SSTR2) and SSTR5 messenger ribonucleic acid (mRNA) are consistently expressed in GH-secreting adenomas, SSTR2 has been believed to be the key modulator of somatostatin-mediated inhibition of GH release. The somatostatin agonists currently in clinical use, octreotide and lanreotide, are directed mainly to SSTR2 (IC(50) 12- to 18-fold higher than for SSTR5). Recently, however, it was demonstrated that an SSTR5 preferential agonist, BIM-23268, not only suppressed PRL release from prolactinomas and mixed GH-PRL adenomas, but also inhibited GH release in about half of GH adenomas. In addition, the SSTR5-preferring analog showed a slight additive effect when used in combination with SSTR2 preferential drugs at submaximal concentrations in octreotide partially sensitive adenomas. In the present study we quantified SSTR2 and SSTR5 mRNA expression and the GH-suppressive effects of somatostatin-14; octreotide; a SSTR2-preferential compound, BIM-23197; a SSTR5-preferential compound, BIM-23268; and a new SSTR2- and SSTR5-bispecific compound, BIM-23244, in GH-secreting tumors classified as either full responders to octreotide (n = 5) or partially sensitive to octreotide (n = 5). The octreotide-sensitive GH secretory adenomas presented with a high level of both SSTR2 and SSTR5 mRNA expression [222 +/- 61 and 327 +/- 136 pg/pg glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively]. In these tumors the suppression of GH release was similarly achieved at picomolar ranges by octreotide, BIM-23197, and BIM-23244 (EC(50) = 25 +/- 15, 3 +/- 2, and 3 +/- 3 pmol/L, respectively). The compounds preferential for only SSTR5 were unable to inhibit GH release in such tumors. Among the octreotide partially responsive tumors, SSTR2 mRNA expression was 9-fold lower than in the octreotide-sensitive tumors (25 +/- 12 vs. 222 +/- 61 pg/pg GAPDH; P < 0.015), whereas SSTR5 mRNA expression was approximately 7-fold higher than in the octreotide-sensitive tumors (2271 +/- 1197 pg/pg GAPDH). In these octreotide partially responsive tumors, the SSTR5-preferential compound, BIM-23268, and the SSTR2- and SSTR5-bispecific compound, BIM-23244, were quite effective in suppressing GH secretion (EC(50) = 25 +/- 13 and 50 +/- 31 pmol/L, respectively). Similarly, BIM-23244, was able to suppress by 51 +/- 5% PRL release from five mixed GH- and PRL-secreting adenomas. These data indicate that due to heterogeneous expression of SSTR2 and SSTR5 receptor subtypes, in GH-secreting tumors, a bispecific analog, such as BIM-23244, that can activate both receptors could achieve better control of GH hypersecretion in a larger number of acromegalic patients.