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PDF file - 317K, List of EMT- and autophagy-related differentially expressed genes in SNAI1- and SNAI1-6SA-expressing MCF-7 cells

PDF file - 204K, Cancer stem cell properties of EMTed and autophagic cell lines

PDF file - 10396K, Human breast cancer biopsies were stained for SNAI2 and LC3B expression by immunohistochemistry on serial tumor sections

PDF file - 72K, SNAI1-6SA cells were sorted by flow cytometry (MoFlo� Astrios� - Beckman Coulter, Inc) based on their ALDH activity

PDF file - 160K, Data mining of microarray data showing the connections between EMT and autophagy-related genes

We provide a unique case of haemorrhagic shock complicating a corticosteroid-resistant diffuse ulcerative enteritis in a patient treated with a combination of an anti CTLA-4 and an anti PD-1 for metastatic melanoma. Immunotherapy has changed the perspective for the management of patients with metastatic melanoma but are also responsible for digestive complications mainly represented by immunomediated colitis. Digestive bleeding is common in patients with extensive colonic lesions but has never been described in enteritis independent of colitis. The patient with acute intestinal obstruction related ileitis without evidence of stricture on imaging and then had a gastro-intestinal bleed. In the absence of haemorrhagic lesions on upper gastrointestinal endoscopy, colonoscopy and CT angiography, a surgical exploration with enteroscopy was performed. This revealed an extensive ulcerated jejunoileitis, with active bleeding, within a Meckel's diverticulum. Management included resection of the Meckel diverticulum with a transient double barrel ileostomy. Two infliximab infusions were given due to persistent bleeding. We observed a dramatic improvement after infliximab treatment with complete cessation of bleeding and no further need for transfusions. A complete mucosal healing has been achieved on enteroscopy at 3 months with disappearance of histological inflammatory lesions. This observation suggests that infliximab represents a therapeutic option in severe enteritis and may be as effective as in more moderate immune-mediated enterocolitis.

Resume La reconnaissance des metastases mammaires par le pathologiste est fondamentale car leur pronostic et leur traitement sont differents de ceux des carcinomes mammaires primitifs. Nous rapportons l’observation d’une patiente de 54 ans presentant sur sa mammographie de depistage un nodule du sein gauche connu depuis 5 ans ayant discretement augmente de taille. L’echographie mammaire montrait une formation nodulaire hypoechogene reguliere de 1,2 cm. Une microbiopsie a ete effectuee. A l’examen microscopique, on observait une proliferation tumorale realisant des massifs au sein d’un stroma peu abondant richement vascularise. Les cellules tumorales avaient un cytoplasme moyennement abondant, eosinophile, granuleux et un noyau arrondi, legerement atypique. Une mitose a ete retrouvee en moyenne pour 10 champs au grossissement × 400. Les cellules tumorales n’exprimaient pas les recepteurs hormonaux mais la chromogranine A, la synaptophysine, le TTF1 et la thyrocalcitonine. L’index de proliferation etabli par l’anticorps anti-Ki67 etait de 5 %. Le diagnostic retenu etait celui de localisation secondaire d’une tumeur neuroendocrine bien differenciee, dont le profil immunohistochimique oriente en premier lieu vers une origine thyroidienne. Nous avons appris, ulterieurement, que la patiente avait un antecedent de thyroidectomie totale 13 ans auparavant. Il s’agissait d’un carcinome medullaire de la thyroide sporadique. Dans le cadre du bilan d’extension, la scintigraphie osseuse a mis en evidence une lesion lacunaire de la partie posterieure de l’aile iliaque droite suspecte de localisation secondaire. Cette lesion iliaque droite a ete biopsiee. Il s’agissait egalement d’une localisation du carcinome medullaire thyroidien connu. Le diagnostic final est celui de carcinome medullaire thyroidien metastatique, lentement evolutif, la metastase mammaire existant probablement depuis 5 ans.

Liver blood test anomalies are common after allogeneic hematopoietic stem cell transplantation (allo-HSCT), but their cause often remains difficult to identify. Our objective was to evaluate the safety and utility of liver biopsies in patients who underwent allo-HSCT. In a retrospective single-center cohort study, we reviewed all cases of patients who underwent liver biopsy between June 2005 and July 2017. During this period, 54 biopsies were performed in 45 patients, in which 38 patients underwent allo-HSCT for malignant and 7 for nonmalignant hematological disorders. Median time between allo-HSCT and liver biopsy was 213 days. Seven biopsies were percutaneous, and 47 were transjugular. No adverse event related to the biopsy procedure occurred; 94.5% biopsies (51 of 54) led to a histological diagnosis. Cholestatic graft-versus-host disease was histologically demonstrated in 16 biopsies (30%); hepatitis-like graft-versus-host disease in 9 biopsies (17%); nonalcoholic steatohepatitis in 6 biopsies (9%); regenerative nodular hyperplasia in 4 biopsies (5%); and drug-induced liver injury, sinusoidal obstruction syndrome, and viral hepatitis each in 3 biopsies (5%). Association between clinical, laboratory, imaging and pathological features was poor. Only 34% of physicians' prebiopsy hypotheses were confirmed by pathological findings. Patient management was influenced by liver biopsy results in 65% of cases, allowing us to identify a new diagnosis (n = 13), rule out a differential diagnosis (n = 14), or confirm the main hypothesis (n = 6). In conclusion, liver biopsy is a safe and useful technique to investigate liver blood test anomalies following allo-HSCT.

Background: The efficacy of neoadjuvant chemotherapy regimens in advanced luminal breast cancer patients is difficult to predict. Intrinsic properties of breast tumors, including altered gene expression profile and dynamic evaluation of metabolic properties of tumor cells using positron emission tomography/computed tomography (PET/CT) of tumor cells, have been identified to guide patient's prognosis. The aim of this study is to determine if both analyses may improve the prediction of response to neoadjuvant chemotherapy in ER-positive / HER2-negative breast cancers (BCs) patients. Methods: We used metabolic PET parameters, at diagnosis and after two cycles of chemotherapy and proliferation gene expression profile on biopsy at diagnosis, in particular, the genomic grade index (GGI) analyzed by reverse transcription and quantitative polymerase chain reaction (RT-qPCR). The pathological response was the surrogate endpoint. Results: The change of FDG uptake between baseline PET and interim PET after 2 cycles of neoadjuvant chemotherapy (ΔSUVmax) was highly associated with pCR (p=0.008). We also observed an ability of P53 mutated status (p=0.042), in addition to histological grade (p=0. 0004), and PR expression (p=0.01) to predict pCR in ERpositive BCs, whereas no proliferation marker predicted pCR (P=0.39 for GGI). Finally, only ΔSUVmax was significantly associated with event free survival (p=0.047). Conclusions: Our results confirm the predictive and prognostic value of tumor ΔSUVmax in ER-positive /HER2-negative advanced BCs patients. These findings can be helpful to select high-risk patients within trials investigating novel treatment strategies., SCOPUS: ar.j, info:eu-repo/semantics/published

International audience; Background: Patients treated with chemotherapy for microsatellite unstable (MSI) and/or mismatch repair deficient (dMMR) cancer metastatic colorectal cancer (mCRC) exhibit poor prognosis. We aimed to evaluate the relevance of distinguishing sporadic from Lynch syndrome (LS)-like mCRCs.Patients and methods: MSI/dMMR mCRC patients were retrospectively identified in six French hospitals. Tumour samples were screened for MSI, dMMR, RAS/RAF mutations and MLH1 methylation. Sporadic cases were molecularly defined as those displaying MLH1/PMS2 loss of expression with BRAFV600E and/or MLH1 hypermethylation and no MMR germline mutation.Results: Among 129 MSI/dMMR mCRC patients, 81 (63%) were LS-like and 48 (31%) had sporadic tumours; 22% of MLH1/PMS2-negative mCRCs would have been misclassified using an algorithm based on local medical records (age, Amsterdam II criteria, BRAF and MMR statuses when locally tested), compared to a systematical assessment of MMR, BRAF and MLH1 methylation statuses. In univariate analysis, parameters associated with better overall survival were age (P < 0.0001), metastatic resection (P = 0.001) and LS-like mCRC (P = 0.01), but not BRAFV600E. In multivariate analysis, age (hazard ratio (HR) = 3.19, P = 0.01) and metastatic resection (HR = 4.2, P = 0.001) were associated with overall survival, but not LS. LS-like patients were associated with more frequent liver involvement, metastatic resection and better disease-free survival after metastasectomy (HR = 0.28, P = 0.01). Median progression-free survival of first-line chemotherapy was similar between the two groups (4.2 and 4.2 months; P = 0.44).Conclusions: LS-like and sporadic MSI/dMMR mCRCs display distinct natural histories. MMR, BRAF mutation and MLH1 methylation testing should be mandatory to differentiate LS-like and sporadic MSI/dMMR mCRC, to determine in particular whether immune checkpoint inhibitors efficacy differs in these two populations.

Resume L’enseignement de l’anatomie et cytologie pathologiques (ACP), au meme titre que l’enseignement des autres disciplines medicales, est a un tournant de son evolution. Face a une augmentation du champ des connaissances et de la complexite des diagnostics, l’epoque est a une mutualisation des ressources pedagogiques a une echelle regionale voire nationale. Nous presentons, ici, les outils en ligne permettant a des communautes d’etudiants de plus en plus larges d’acceder a des contenus d’apprentissage, a des auto-evaluations et a des outils d’evaluation de leurs competences. Les plateformes de e-learning et de MOOC (« Massive open online courses »), sont au centre de ces modalites pedagogiques, avec une mention particuliere pour la plateforme nationale des disciplines dediee a l’enseignement des 50 000 internes en medecine de France. Ces enseignements jouent aussi sur l’attractivite avec notamment les serious games, et on parlera de la place essentielle des images et des lames virtuelles dans l’enseignement de l’ACP. La pedagogie a grande echelle ne doit, bien sur, pas se concevoir comme un remplacement des enseignements utilises auparavant, mais comme l’etape initiale d’acquisition des connaissances, devant deboucher sur des enseignements presentiels renoves, centres sur l’interactivite et les competences.

BACKGROUND The prognostic and predictive role of cyclo-oxygenase-2 (COX2) in breast cancer is still debated, and in particular, its role as a target of COX2 inhibitor (celecoxib) in neoadjuvant setting. MATERIALS AND METHODS We analyzed a series of 156 breast cancer samples from patients of the COX2 inhibitor-treated arm included in the REMAGUS-02 randomized phase II trial. COX2 gene expression was assessed by reverse transcription and quantitative polymerase chain reaction using ribonucleic acid from frozen biopsies. Pathological complete response (pCR) was the surrogate end-point. RESULTS Significantly higher rates of grade 3, and estrogen and progesterone receptor negativity were observed in tumors with the highest expression of COX2. pCR rates were significantly higher in COX2-overexpressing tumors in patients receiving celecoxib. The test for interaction between COX2 gene expression and the celecoxib effect was statistically significant (p

Research states that the success of a specific innovation cannot be predicted. As a consequence, the value of such an innovation can only be stated very late in the development process, or even in retrospect. Based on C-K design-theory, our empirical research shows that professionals directly engaged in the design process are able to perform an early and complete valuation. The cognitive process of this valuation is extremely similar to the overall process of innovative design. Also, this process has tangible outputs, in particular local, dedicated management tools used as proofs of concepts., La recherche en gestion a établi l'imprévisibilité du succès d'une innovation. Un corollaire largement accepté veut que la détermination de la valeur de cette innovation ne puisse être que tardive, voire rétrospective. Nous observons dans cette recherche que les acteurs directs de l'innovation sont à même de procéder à une détermination de la valeur précoce, complète et révisable. Nous appuyant sur la théorie C-K, nous montrons que ces équipes appliquent à la détermination de la valeur le même type de raisonnement qu'à l'innovation elle-même. Nous montrons également que ce travail de conception innovante passe par la génération d'outils de gestion locaux, souvent à l'usage exclusif du projet et utilisés pour mettre à l'épreuve les différents concepts de valeur.

Summary Background Survival of metastatic colon cancer (mCC) patients has considerably improved with optimization of new drugs regimen. Inactivation of TP53 pathway by TP53 mutations is observed in nearly half of colorectal tumors. The impact of such mutations has been poorly studied in the metastatic setting. Methods The files of 254 mCC treated in a single institution at Saint-Louis hospital between January 1999 and April 2011 were retrospectively reviewed. Tissue samples for analysis of TP53 mutations were available for 68 patients, performed using FASAY. The prognostic value of TP53 status was evaluated by comparing progression free survival (PFS) and overall survival (OS) in the group of TP53-mutated and wild type patients. Results PFS was 6.9 months and OS 21.7 months in the whole population. There was no statistical difference in TP53- mutated and wild type groups in term of PFS (HR = 1.04; IC9 5% = 0.6–1.79) and OS (HR = 0.99; IC 95% = 0.53–1.55) whatever the chemotherapy regimen (oxaliplatin- or irinotecan-based). Only BRAF V600 mutation was demonstrated to be a poor prognostic factor for PFS and OS, and CEA level for OS. Conclusions Routine determination of TP53 mutations, even with a highly sensitive method, cannot be recommended to predict chemotherapy response in mCC.

// Guilhem Bousquet 1, 2, 3, 4 , Morad El Bouchtaoui 2 , Tan Sophie 2 , Christophe Leboeuf 1, 2 , Cedric de Bazelaire 1, 2, 5 , Philippe Ratajczak 1, 2 , Sylvie Giacchetti 6 , Anne de Roquancourt 1, 2, 7 , Philippe Bertheau 1, 2, 7 , Laurence Verneuil 1, 2 , Jean-Paul Feugeas 8 , Marc Espie 1, 6 , Anne Janin 1, 2, 7 1 Universite Paris Diderot, Sorbonne Paris Cite, Laboratoire Pathologie, Paris, France 2 INSERM, Paris, France 3 Universite Paris 13, Villetaneuse, France 4 AP, HP, Avicenne, Service Oncologie, Paris, France 5 AP HP Hopital Saint-Louis, Service Radiologie, Paris, France 6 AP HP Hopital Saint-Louis, Centre Maladies Sein, Paris, France 7 AP HP Hopital Saint-Louis, Service Pathologie, Paris, France 8 INSERM, Paris, France Correspondence to: Guilhem Bousquet, email: guilhem.bousquet@aphp.fr Anne Janin, email: anne.janin1165@gmail.com Keywords: breast cancer stem cells, TNBC, chemoresistance, autophagy, hypoxia Received: March 21, 2017 Accepted: March 29, 2017 Published: April 07, 2017 ABSTRACT There is growing evidence for the role of cancer stem-cells in drug resistance, but with few in situ studies on human tumor samples to decipher the mechanisms by which they resist anticancer agents. Triple negative breast cancer (TNBC) is the most severe sub-type of breast cancer, occurring in younger women and associated with poor prognosis even when treated at a localized stage. We investigated here the relationship between complete pathological response after chemotherapy and breast cancer stem-cell characteristics in pre-treatment biopsies of 78 women with triple negative breast carcinoma (TNBC). We found that chemoresistance was associated with large numbers of breast cancer stem-cells, and that these cancer stem-cells were neither proliferative nor apoptotic, but in an autophagic state related to hypoxia. Using relevant pharmacological models of patient-derived TNBC xenografts, we further investigated the role of autophagy in chemoresistance of breast cancer stem-cells. We demonstrated that hypoxia increased drug resistance of autophagic TNBC stem-cells, and showed that molecular or chemical inhibition of autophagic pathway was able to reverse chemoresistance. Our results support breast cancer stem-cell evaluation in pre-treatment biopsies of TNBC patients, and the need for further research on autophagy inhibition to reverse resistance to chemotherapy.

Background: Immunohistochemistry (IHC) and fluorescent in situ hybridisation (FISH) are currently the most commonly used methods to assess HER2 status. PCR-based assays allow quantitative determination of HER2 amplification (Q-PCR) or overexpression (Q-RT–PCR), but are not routinely used. We evaluated the relevance of Q-RT–PCR for HER2 status determination. Methods: We compared IHC and Q-RT–PCR in 466 breast tumours. In discordant or equivocal cases, five additional methods (IHC with two other antibodies, FISH, silver in situ hybridisation (SISH) and Q-PCR) were combined to determine HER2 status. Two cases with HER2 intra-tumour heterogeneity were further explored by allelic profiles analysis and HUMARA clonality determination after microdissection. Results: We observed 97.3% concordance between Q-RT–PCR and non-equivocal IHC. Twelve out of 466 cases (3%) revealed discordances between the two methods. The power of Q-RT–PCR to predict HER2 status (defined by seven methods) was similar to that of IHC. Although rare, some discordances between techniques might be due to HER2 intra-tumour heterogeneity and we report two examples, one tumour containing two distinct clones, another tumour consisting of HER2 amplified and non-amplified subclones. Conclusion: Q-RT–PCR and IHC are highly concordant methods for HER2 status assessment, and Q-RT–PCR allows a highly reliable quantitative assessment and could be a useful adjunct to IHC.

The p53 wild-type protein plays an important role in cells as is shown by its fine regulation at different levels. Since its discovery, numerous mutations have been described. In breast cancers, p53 is mutated in almost 30% of cases, with a higher frequency in some tumor subtypes.TP53mutation is reported to be a factor for good prognosis in some studies, while in others it is a factor for poor prognosis. The explanation for these different results could be linked to the fact that the studies were performed on different tumor types and with different therapy regimens.

Clear cell adenocarcinoma of the cervix is a rare tumor of the lower genital tract. It has been described in young women with a history of intra uterine exposure to diethylstilbestrol. This tumor is characterized by a greater tendency for late recurrences. In this article, we report the case of one exposed-patient who developed recurrence as liver metastases, 24 years after the initial treatment. This case demonstrates the need and the importance for continued follow-up in individuals prenatally exposed to diethylstilbestrol.

Experimental, clinical, and epidemiological data support the growth-promoting role of endocrine prolactin (PRL) in mammary tumors. PRL is also produced by the breast, where it is now recognized to act as a growth/survival factor via autocrine/paracrine mechanisms. Recent transgenic (Tg) mouse models have revealed the pro-oncogenic effect of PRL over-expression in virgin mammary glands. To address the question whether PRL tumorigenicity was maintained on differentiated mammary glands, we generated mammary-specific Tg mice expressing human (h)PRL under the control of the milk whey acidic protein promoter, which directs autocrine hPRL over-expression in late gestation throughout lactation. Minimal levels of transgene expression were detected in the mammary glands of virgin animals, which at best induced partial ductal branching and lobulo-alveolar structures in older nulliparous females. As expected, expression of mammary hPRL dramatically increased at the end of first pregnancy, and from this point it never returned to baseline, although it peaked at each gestation/lactation cycle. Over-expression of hPRL that starts when the gland is already well into the differentiation process led to various morphological mammary alterations, including abnormally differentiated epithelium, atropy of the myoepithelial layer, dilated ducts, cysts, and lymphocytic infiltrates. These phenotypes tended to worsen with successive pregnancies, also reflecting cumulative damage of failure of involution. Although some older, multiparous females developed benign tumors (papillomas and metaplasias), none of the animals studied developed mammary carcinomas. In addition, we noticed that half of the Tg females exhibited lactation defects, leading to significantly increased pup mortality. This phenotype was due neither to failure of milk production nor to modification of its protein content, but rather it was correlated to lipid enrichment of the milk, which, in combination with profoundly altered morphology of the gland, led to impaired milk extrusion through the nipple. In summary, these data show that over-expression of autocrine hPRL in a differentiating mammary gland induces dramatic functional and morphological defects, but not carcinoma. This deserves further investigations on the emerging concept that autocrine PRL may have different effects on pathological development of the mammary gland depending on the differentiation state of the latter.

This study evaluated by immunohistochemistry (IHC) immune cell response during neoadjuvant primary systemic therapy (PST) with trastuzumab in patients with HER2-positive primary breast cancer. In all, 23 patients with IHC 3+ primary breast cancer were treated with trastuzumab plus docetaxel. Pathological complete and partial responses were documented for nine (39%) and 14 (61%) patients, respectively. Case-matched controls comprised patients treated with docetaxel-based PST without trastuzumab (D; n=23) or PST without docetaxel or trastuzumab (non-taxane, non-trastuzumab, NT-NT; n=23). All surgical specimens were blind-analysed by two independent pathologists, with immunohistochemical evaluation of B and T lymphocytes, macrophages, dendritic cells and natural killer (NK) cells. Potential cytolytic cells were stained for Granzyme B and TiA1. HER2 expression was also evaluated in residual tumour cells. Trastuzumab treatment was associated with significantly increased numbers of tumour-associated NK cells and increased lymphocyte expression of Granzyme B and TiA1 compared with controls. This study supports an in vivo role for immune (particularly NK cell) responses in the mechanism of trastuzumab action in breast cancer. These results suggest that trastuzumab plus taxanes lead to enhanced NK cell activity, which may partially account for the synergistic activity of trastuzumab and docetaxel in breast cancer.

This chapter is a first effort, mainly descriptive, to explore possible cross-fertilization between two active fields of research - management of innovation and cognitive psychology. Both approaches question some important aspects of the dominant vision of the firm, especially those related to the existence of rational choice in organizations. Our reasonning is based on two pillars: First, specific situations of innovation are a privileged field for the observation of cognitive bias and representation errors. Why? Because the innovation, which always includes some transgression, often questions the practices and management systems. In doing so, it illuminates representation errors (ie the " inappropriate application of antecedent interpretative models"). Second, there is a striking convergence between the results of research on cognitive bias and some strong assumptions of the management of innovation . The two streams , in particular, emphasize the inadequacy of the classical theories of decision., L"innovation n'a, jusqu"ici et à notre connaissance, jamais été le terrain d"études portant explicitement sur les erreurs de représentation. Ce chapitre constitue donc un premier effort, essentiellement descriptif, pour explorer les fertilisations croisées possibles entre deux courants de recherche – le management de l"innovation et la psychologie cognitive. Les deux approches ont en commun de remettre en cause certains aspects importants de la vision dominante de la firme, en particulier les mythes rationnels liés aux processus de choix dans les organisations. Notre réflexion est ici fondée sur deux piliers : 1. Les situations concrètes de l"innovation en train de se faire forment un terrain privilégié pour l"observation des erreurs de représentation. Pourquoi ? Parce que l"innovation, qui comporte toujours une part de transgression, interroge en profondeur les pratiques et les dispositifs de gestion. Ce faisant, elle éclaire les erreurs de représentation (autrement dit l"application inappropriée de grilles de lectures antécédentes) d"un jour souvent cru. L"analyse des cas d"innovation, qu"elle soit technologique, organisationnelle ou sociale, est donc un remarquable révélateur de la banalité des erreurs de représentation dans le fonctionnement des organisations et, peut-être, l"occasion de nouveaux développements théoriques. 2. Il y a une convergence de résultats entre les recherches menées depuis une quarantaine d’années sur les erreurs de représentation et certaines hypothèses fortes du management de l’innovation. Les deux courants, en particulier, insistent sur le caractère insuffisant des théories classiques de la décision.

Despite an obvious central role of p53 in the hallmarks of cancer, TP53 status is not yet used for the management of breast cancer. Recent findings may lead to reconsider the role of p53 in breast cancer. TP53 mutations are the most frequent genetic alterations in breast cancer, observed in 30% of breast carcinomas. Their distribution is highly linked to molecular tumor subtypes found in 26% of luminal tumors (17% of luminal A, 41% of luminal B), in 50% of HER2 amplified tumors, in 69% of molecular apocrine breast carcinomas and in 88% of basal-like carcinomas. The type of mutation is linked to the tumor subtype with higher frequency of base-pair substitutions in luminal tumors, whereas molecular apocrine and basal-like tumors present much higher frequency of complex mutations (deletions/insertions). The timing of TP53 mutation also depends on the tumor subtype, being the first important event in luminal tumors but occurring after PTEN loss in basal-like tumors. Regarding response to cytotoxic chemotherapy, the situation is far from the p53-dependent apoptosis paradigm with subsequent clinical response. We reported that TP53 mutated non inflammatory locally advanced breast carcinomas had a high rate of complete pathological response to dose-dense doxorubicin-cyclophosphamide chemotherapy, while TP53 wild-type (WT) tumors never achieved complete response. Using human breast cancer xenograft models, we suggested that this could be due to the induction of senescence in TP53 WT tumor cells. A recent work confirmed these findings in MMTV-Wnt1 mammary tumors, showing that growth arrest and senescent phenotype, not apoptosis, were induced in TP53 WT tumors following doxorubicin treatment, while lack of arrest in mutant tumors resulted in aberrant mitoses, cell death and a superior clinical response. Furthermore, in ER positive (ER(+)) breast tumors, it has been recently reported that ER represses the p53-mediated apoptotic response induced by DNA damage. Taken together, these data can help to better understand p53-mediated response to doxorubicin-based chemotherapy in breast cancer: in ER(+) TP53 WT breast cancers, ER-induced inhibition of p53 apoptotic response would lead preferentially to tumor cell senescence and subsequent resistance to treatment. Conversely, in ER negative (ER(-)) TP53 mutated breast cancers, accumulation of genetic abnormalities would lead to mitotic catastrophe and subsequent better response. In view of these recent results, p53 impact in breast cancer should be reconsidered.

Generalized pustular psoriasis is a rare form of psoriasis that is sometimes associated with extracutaneous manifestations. Evidence for biliary involvement has been suggested in isolated cases. We investigated the prevalence and nature of liver abnormalities occurring in this disease. Twenty-two patients consecutively admitted for generalized pustular psoriasis who underwent liver biological tests at the time of the attack and during the following weeks were included. Twenty patients (90%) had at least one abnormal biological liver parameter. Eleven patients (50%) had pronounced abnormalities: jaundice (4/22), gammaglutamyl transferase higher than 5 times the normal value (10/22), alkaline phosphatase higher than twice the normal value (7/22), and/or aminotransferases higher than 3 times the normal value (7/22). These abnormalities returned to normal range at the time of remission of pustular psoriasis, suggesting that severe liver abnormalities could be associated with severe cutaneous disease. Neutrophilic cholangitis was observed on liver biopsy. Persistent magnetic resonance cholangiopancreatography features similar to those observed in sclerosing cholangitis were found in 3 of the 4 patients studied. No causal factor other than pustular psoriasis could be identified. In conclusion, our results demonstrate the high frequency of liver abnormalities in patients with generalized pustular psoriasis. Biliary involvement related to neutrophilic cholangitis should be added to the spectrum of extracutaneous manifestations of this disease, and physicians should be aware of such a complication.

Variability in the frequency of KIT mutations in gastrointestinal mesenchymal tumors has been reported in the literature, and their prognostic value remains uncertain. This retrospective multicenter study included 276 patients with gastrointestinal mesenchymal tumors.We detected c-kit and CD34 protein expression by immunohistochemistry. Mutations in exons 11 and 9 of KIT and exons 12 and 18 of PDGFR were detected by length analysis of polymerase chain reaction products and direct DNA sequencing.Eighty-seven percent of the tumors analyzed were c-kit positive, with gastric tumors expressing CD34 more frequently than other tumors (86% vs. 52%; P0.001). KIT exon 11 mutations were detected in 90 of 179 (50.3%) of c-kit-positive and 12% of c-kit-negative tumors. These mutations showed variation in their length and location. Mutations were heterozygous in 94% of cases. Mutations were more frequent in CD34( +) tumors than in CD34( -) tumors ( P0.01), and 9% of tumors had a second mutation in exon 11. Mutations in exon 9 of KIT were present in 5.1% of the gastrointestinal stromal tumors, and mutations of the PDGFR were present in 11% of the KIT -nonmutated tumors. Patient's age, the primary location, size, necrosis, and mitotic counts of tumors were associated with metastases in c-kit-positive tumors. However, mitotic activity was the only independent factor identified in multivariate analysis ( P0.001). KIT mutations were slightly more frequent in metastatic than in nonmetastatic tumors (61% vs. 46%; P = 0.06). Deletions of codons 562-579 were more strongly associated with metastases than were deletions of codons 550-561 ( P = 0.0001).Mutations in KIT or PDGFR were detected in 58.4% of the c-kit-positive and also in some c-kit-negative tumors.

Meme si des analyses quantitatives sont deja realisees depuis longtemps a l’aide de microscopes couples a un logiciel informatique (notamment dans le domaine des neurosciences), les lames virtuelles permettent un plus grand developpement de ce type d’application. Ainsi, a partir d’images numerisees, la mesure de l’intensite des colorations a ete reconnue comme etant plus precise que celle faite avec des microscopes classiques dont un manque de calibrage peut entrainer une variation d’interpretation, les lames histologiques semblant alors differentes d’un microscope a l’autre [3]. La quantification de l’immunofluorescence numerique peut aider les pathologistes a evaluer l’expression moleculaire de marqueurs dans les tissus cancereux, et a uniformiser le score attribue a l’intensite du signal observe sur des lames d’immunohistochimie. Un autre probleme en microscopie classique est l’impossibilite de preserver a long terme des marquages immunofluorescents. Cet obstacle disparait avec la technologie des lames virtuelles qui permet, non seulement de numeriser une image entiere d’immunofluorescence en quelques minutes, mais egalement de la conserver presque indefiniment [4]. La numerisation de lames d’immunofluorescence reste toutefois plus delicate que la numerisation en lumiere blanche, et elle necessite souvent un appareillage specifique. 1Universite Paris-Diderot Paris VII, Institut Universitaire d’Hematologie, 75010 Paris, France ; 2Inserm UMR-S728, 75010 Paris, France ; 3Assistance Publique-Hopitaux de Paris (AP-HP), Hopital Saint-Louis, Service de Pathologie, 1, avenue Claude-Vellefaux, 75010 Paris, France ; 4Service d’Anatomie pathologique, Hopital Edouard-Herriot, 69437 Lyon, France. luc.legres@sls.aphp.fr Les analyses anatomopathologiques sont, en 2011, les seules methodes en pratique clinique qui permettent d’etablir un diagnostic formel de cancer ou de rejet de greffe. Les examens microscopiques des lesions in situ sont aussi irremplacables pour les etudes mecanistiques dans ces memes domaines du cancer et de la transplantation. En effet, les etudes faites sur des cellules en culture ne refletent que tres imparfaitement la complexite des structures tissulaires, et les modeles animaux des maladies humaines sont, d’une part, encore trop peu nombreux et, d’autre part, ne refletent que tres imparfaitement la pathologie humaine [1].

Inflammatory breast cancer (IBC) is one of the most aggressive forms of breast cancer. We studied the biological characteristics of these tumours by comparing the overexpression of oncogenes ERBB2, MYC, CCND1 and RHOC and TP53 gene mutation rates in IBC with those found in locally advanced and not otherwise specified breast cancers. The prevalence of the TP53 mutation was much higher in IBC than in the two other types of cancer (57% vs 30). Unexpectedly, however, in IBC tumours, histological grade was independent of TP53 status. In addition, ERBB2 overexpression was twice as frequent in inflammatory as in non-inflammatory tumours, whereas the frequencies of MYC, CCND1 and RHOC overexpression did not vary significantly among the three types of breast cancer. These findings suggest that IBC tumours constitute a distinct subset with a specific pathogenesis. Given the importance of TP53 and ERBB2 in the response to treatments, our observations have important therapeutic implications for the clinical management of IBC patients.

MICA are distant homologs of MHC class I molecules expressed in the normal intestinal epithelium. They are ligands of the NKG2D activating receptor expressed on most γδ T cells, CD8+ αβ T cells, and natural killer cells and therefore play a critical role in innate immune responses. We investigated MICA cell-surface expression on infection of epithelial cell lines by enteric bacteria and show here that MICA expression can be markedly increased by bacteria of the diffusely adherentEscherichia colidiarrheagenic group. This effect is mediated by the specific interaction between bacterial adhesin AfaE and its cellular receptor, CD55, or decay-accelerating factor. It is extremely rapid after AfaE binding, consistent with a stress-induced signal. MICA induction on epithelial cells triggered IFN-γ release by the NKG2D expressing natural killer cell line NKL. This host–bacteria interaction pathway could play a role in the pathogenesis of inflammatory bowel disease, a condition that implicates a bacterial trigger in genetically susceptible individuals. This was supported by the increased MICA expression at the surface of epithelial cells in colonic biopsies from Crohn's disease-affected patients compared with controls.

Renal-cell carcinomas (RCC) are often resistant to conventional cytotoxic agents. Xenograft models are used for in vivo preclinical studies and drug development. The validity of these studies is highly dependent on the phenotypic and genotypic stability of the models. Here we assessed the stability of six aggressive human RCC xenografted in nude/NMRI mice. We compared the initial samples (P0), first (P1) and fifth (P5) passages for the following criteria: histopathology, immunohistochemistry for CK7, CD10, vimentin and p53, DNA allelic profiles using 10 microsatellites and CGH-array. Next we evaluated the response to sunitinib in primary RCC and corresponding xenografted RCC. We observed a good overall stability between primary RCC and corresponding xenografted RCC at P1 and P5 regarding histopathology and immunohistochemistry except for cytokeratin 7 (one case) and p53 (one case) expression. Out of 44 groups with fully available microsatellite data (at P0, P1 and P5), 66% (29 groups) showed no difference from P0 to P5 while 34% (15 groups) showed new or lost alleles. Using CGH-array, overall genomic alterations at P5 were not different from those of initial RCC. The xenografted RCC had identical response to sunitinib therapy compared to the initial human RCC from which they derive. These xenograft models of aggressive human RCC are clinically relevant, showing a good histological and molecular stability and are suitable for studies of basic biology and response to therapy.

Research states that the success of a specific innovation cannot be predicted. As a consequence, the value of such an innovation can only be stated very late in the development process, or even in retrospect. Our empirical research shows that professionals directly engaged in the design process are able to perform an early and complete valuation, and that similar patterns emerge during this valuation process., La recherche en gestion a établi l'imprévisibilité du succès d'une innovation. Un corollaire largement accepté veut que la détermination de la valeur de cette innovation ne puisse être que tardive, voire rétrospective. Nous observons dans cette recherche que les acteurs directs de l'innovation sont à même de procéder à une " valuation " précoce, complète et révisable, et que des patterns similaires sont observables lors de ces processus de valuation.

Solid tumors are composed not only of tumor cells but also of stromal nonneoplastic cells. In whole tumor samples, stromal cells retaining their alleles may therefore obscure detection of loss of heterozygosity (LOH) in tumor cells. An increasing number of studies have used laser-assisted tissue microdissection to improve LOH detection, but the real gain in sensitivity has been poorly quantified. We studied a group of 16 inflammatory breast carcinomas that were submitted to both standard DNA extraction from frozen whole tumor samples and laser microdissection performed on paraffin-embedded tumor samples. Using PCR with fluorescence-labeled primers, we comparatively analyzed ten polymorphic markers with both sources of DNA. With the LOH detection threshold set at -25%, we showed that 25 LOHs could not be diagnosed with whole tumor samples out of 73 LOHs positively diagnosed in microdissected samples (34%). With the LOH detection threshold set at -50%, the respective figures were 39 LOHs not diagnosed out of 55 LOHs (71%). Measuring the intensity of the allelic decrease, we showed that the mean decrease of the lost allele is -34% with whole tumor samples and -67% with microdissected samples. The increase in sensitivity of LOH detection with microdissection is associated with the density of stromal cells. This strong improvement in LOH detection in this aggressive type of breast cancer indicates that many other molecular studies performed on heterogeneous solid tumors may benefit from a first step of laser microdissection.

AIMS: To investigate the effects of slide storage on immunohistochemical staining, since recent reports have indicated that storage of unstained paraffin slides for up to 12 weeks may lead to false negative immunostaining of tumour markers. METHODS: 11 antibodies (anti-cytokeratin, epithelial membrane antigen (EMA), vimentin, smooth muscle actin, PS100, chromogranin, CD45, CD20, CD3, CD30, and oestrogen receptor (OR) were tested on unstained paraffin slides of breast carcinomas, lymphomas, and neuroendocrine tumours that had been stored for three to 10 years. All the paraffin blocks were recut less than one week before immunostaining. Immunostainings of years old slides were compared with those of recent slides in at least five cases for each antibody. For three antibodies (antichromogranin, anti-CD3, and anti-OR) we also tested one year old and three months old slides. RESULTS: Intensity of staining on years old slides was strikingly reduced for chromogranin and CD3 in several cases and was slightly stronger for vimentin. In some cases a significant decrease of OR positivity was observed after three months storage, and a complete loss of OR immunostaining after 12 months. No significant difference was noted with the other antibodies. CONCLUSIONS: Immunohistochemical detection of some antigens located either in the nucleus, in the cytoplasm, or on the cytoplasmic membrane could be impaired by storage of paraffin slides as short a time as three months. One should be cautious of doing retrospective immunohistochemical studies on stored unstained slides.

Three cases of apparent primary villous atrophy of the terminal ileum in women with chronic diarrhoea are reported. Eight cases have previously been reported in the literature. Clinical characteristics are the presence of severe chronic secretory diarrhoea with episodes of hypokalaemia combined with signs of ileal malabsorption and/or efficacy of cholestyramine. Diagnosis is based on ileoscopy and histology. An association with microscopic colitis was present in the three patients and in four cases in the literature. The pathogenesis of primary ileal villous atrophy remains unknown and may involve dysimmunity. Its association with microscopic colitis may indicate a common pathogenesis or support the hypothesis that the faecal stream or bile salts play a role in the pathogenesis of microscopic colitis.

Normal biological tissues harbour different populations of cells with intricate spacial distribution patterns resulting in heterogeneity of their overall cellular composition. Laser microdissection involving direct viewing and expertise by a pathologist, enables access to defined cell populations or specific region on any type of tissue sample, thus selecting near-pure populations of targeted cells. It opens the way for molecular methods directed towards well-defined populations, and provides also a powerful tool in studies focused on a limited number of cells. Laser microdissection has wide applications in oncology (diagnosis and research), cellular and molecular biology, biochemistry and forensics for tissue selection, but other areas have been gradually opened up to these new methodological approaches, such as cell cultures and cytogenetics. In clinical oncology trials, molecular profiling of microdissected samples can yield global “omics” information which, together, with the morphological analysis of cells, can provide the basis for diagnosis, prognosis and patient-tailored treatments. This remarkable technology has brought new insights in the understanding of DNA, RNA, and the biological functions and regulation of proteins to identify molecular disease signatures. We review herein the different applications of laser microdissection in a variety of fields, and we particularly focus attention on the pre-analytical steps that are crucial to successfully perform molecular-level investigations.

Background Until now, FISH has been the gold standard technique to identify HER2 amplification status in ambiguous cases of breast cancer. Alternative techniques have been developed to increase the capacities of investigating HER2 amplification status. The aims of this multicenter study in a large series of breast cancer patients were to prospectively compare the level of performance of CISH, SISH, and qPCR alternative techniques on paraffin-embedded core biopsies with “gold standard FISH” for evaluation of HER2 amplification status. Methods This study was performed on 840 cases scored by immunohistochemistry (IHC): 0=317 (38%), 1+=183 (22%), 2+=109 (13%), 3+=231 (27%). Each of the 15 French centers participating in the study analyzed 56 breast carcinoma cases diagnosed on fixed paraffin-embedded core biopsies. HER2 amplification status was determined by commercially available FISH used as the reference technique with determination of the HER2/CEN17 ratio or HER2 copy number status. The alternative techniques performed on the same cases were commercially available SISH or CISH and a common qPCR method especially designed for the study including a set of 10 primer pairs: 2 for HER2 (exons 8 and 26), 5 to evaluate chromosome 17 polysomy TAOK1, UTP6, MRM1, MKS1, SSTR2 and 3 for diploidy control TSN, LAP3 and ADAMTS16. Results The concordance between IHC and FISH was 96% to 95% based on the HER2/CEN17 ratio (n=766) or HER2 copy number (n=840), respectively. The concordance of the alternative techniques with FISH was excellent: 97% and 98% for SISH (498 and 587 cases), 98% and 75% for CISH (108 and 204 cases) and 95% and 93% (699 and 773 cases) for qPCR based on the HER2/CEN17 ratio or HER2 copy number, respectively. Similarly, sensitivity ranged from 99% to 95% for SISH, 100% to 99% for CISH and 89% to 80% for qPCR. The concordance with FISH (ratio) in the 2+ cases was 89% for SISH, 100% for CISH and 93% for qPCR. Conclusion These alternative techniques showed an excellent concordance with FISH in core biopsies allowing their use in routine clinical practice. This newly designed qPCR on paraffin-embedded core biopsies deserves special attention, as it is reliable, easy to perform and less expensive than ISH tests.

International audience; INTRODUCTION: Molecular apocrine (MA) tumors are estrogen receptor (ER) negative breast cancers characterized by androgen receptor (AR) expression. We analyzed a group of 58 transcriptionally defined MA tumors and proposed a new tool to identify these tumors. METHODS: We performed quantitative reverse transcription PCR (qRT-PCR) for ESR1, AR, FOXA1 and AR-related genes, and immunohistochemistry (IHC) for ER, PR, Human Epidermal Growth Factor Receptor 2 (HER2), CK5/6, CK17, EGFR, Ki67, AR, FOXA1 and GCDFP15 and we analyzed clinical features. RESULTS: MA tumors were all characterized by ESR1(-) AR(+) FOXA1(+) and AR-related genes positive mRNA profile. IHC staining on these tumors showed 93% ER(-), only 58% AR(+) and 90% FOXA1(+). 67% and 57% MA tumors were HER2(3+) and GCDFP15(+), respectively. Almost all MA tumors (94%) had the IHC signature HER2(3+) or GCDFP15(+) but none of the 13 control basal-like (BL) tumors did. Clinically, MA tumors were rather aggressive, with poor prognostic factors. CONCLUSION: MA tumors could be better defined by their qRT-PCR-AR profile than by AR IHC. In addition, we found that HER2 or GCDFP15 protein overexpression is a sensitive and specific tool to differentiate MA from BL in the context of ER negative tumors. A composite molecular and IHC signature could, therefore, help to identify MA tumors in daily practice.

Epithelial-to-mesenchymal transition (EMT) mediates cancer cell invasion, metastasis, and drug resistance, but its impact on immune surveillance has not been explored. In this study, we investigated the functional consequences of this mode of epithelial cell plasticity on targeted cell lysis by cytotoxic T lymphocytes (CTL). Acquisition of the EMT phenotype in various derivatives of MCF-7 human breast cancer cells was associated with dramatic morphologic changes and actin cytoskeleton remodeling, with CD24−/CD44+/ALDH+ stem cell populations present exhibiting a higher degree of EMT relative to parental cells. Strikingly, acquisition of this phenotype also associated with an inhibition of CTL-mediated tumor cell lysis. Resistant cells exhibited attenuation in the formation of an immunologic synapse with CTLs along with the induction of autophagy in the target cells. This response was critical for susceptibility to CTL-mediated lysis because siRNA-mediated silencing of beclin1 to inhibit autophagy in target cells restored their susceptibility to CTL-induced lysis. Our results argue that in addition to promoting invasion and metastasis EMT also profoundly alters the susceptibility of cancer cells to T-cell–mediated immune surveillance. Furthermore, they reveal EMT and autophagy as conceptual realms for immunotherapeutic strategies to block immune escape. Cancer Res; 73(8); 2418–27. ©2013 AACR.

This study was designed to identify predictive signatures of pathological complete response (pCR) in breast cancer treated by taxane-based regimen, using clinicopathological variables and transcriptomic data (Affymetrix Hgu133 Plus 2.0 devices). The REMAGUS 02 trial ( n = 153,training set) and the publicly available M.D. Anderson data set ( n = 133, validation set) were used. A re-sampling method was applied. All predictive models were defined using logistic regression and their classification performances were tested through Area Under the Curve (AUC) estimation. A stable set of 42 probesets (31 genes) differentiate pCR or no pCR samples. Single-or 2-probesets signatures, mainly related to ER pathway, were equally predictive of pCR with AUC greater then 0.80. Models including probesets associated with ESR1, MAPT, CA12 or PIGH presented good classification performances. When clinical variables were entered into the model, only CA12 and PIGH, remained informative ( p = 0.05 and p = 0.005) showing that a combination of a few genes provided robust and reliable prediction of pCR.

Tumor protein p53 (TP53) is mutated in approximately 30% of breast cancers, but this frequency fluctuates widely between subclasses. We investigated the p53 mutation status in 572 breast tumors, classified into luminal, basal and molecular apocrine subgroups. As expected, the lowest mutation frequency was observed in luminal (26%), and the highest in basal (88%) tumors. Luminal tumors showed significantly higher frequency of substitutions (82 vs. 65%), notably A/T to G/C transitions (31 vs. 15%), whereas molecular apocrine and basal tumors presented much higher frequencies of complex mutations (deletions/insertions) (36 and 33%, respectively, vs. 18%). Accordingly, missense mutations were significantly more frequent in luminal tumors (75 vs. 54%), whereas basal tumors displayed significantly increased rates of TP53 truncations (43 vs. 25%), resulting in loss of function and/or expression. Interestingly, as basal tumors, molecular apocrine tumors presented with a high rate of complex mutations, but paradoxically, these were not associated with increased frequency of p53 truncation. As in luminal tumors, this could reflect a selective pressure for p53 gain of function, possibly through P63/P73 inactivation. Collectively, these observations point not only to different mechanisms of TP53 alterations, but also to different functional consequences in the different breast cancer subtypes.

Background In HIV and hepatitis C virus (HCV) coinfected patients, the role of antiretroviral therapy (ART) on hepatic steatosis (HS) remains controversial. Methods HIV/HCV coinfected patients receiving ART and previously untreated for HCV who underwent a liver biopsy were included. Cumulative duration of exposure to each antiretroviral was recorded up to liver biopsy date. Logistic regression analyses evaluated factors associated with steatosis and its severity. Results 184 patients were included: median age 41years, 84% male, 89% Caucasian, 61% with a past history of intravenous drug use. HCV genotypes were 1 (55%), 2 (6%), 3 (26%), and 4 (13%). Median HCV-RNA was 6.18 log10 IU/ml. HIV-RNA was undetectable (3. All patients had been exposed to nucleoside reverse transcriptase inhibitors (median cumulative exposure 56months); 126 received protease inhibitors (23months), and 79 non-nucleoside reverse transcriptase inhibitors (16months). HS was observed in 102 patients (55%): 41% grade 1; 5% grade 2, and 9% grade 3. In multivariate analysis, HCV genotype 3 and HCV viral load were moderately associated with mild steatosis but strongly with grade 2-3 steatosis. After adjustment for the period of biopsy, no association was detected between HS and exposure to any antiretroviral class or drug, or duration of ART globally or comparing genotype 3 to others. Conclusions Among our ART-treated HIV-HCV cohort predominantly infected with genotype 1, 55% of patients had HS which was associated with HCV-related factors, but not ART class or duration of exposure.

International audience; The current histoclinical breast cancer classification is simple but imprecise. Several molecular classifications of breast cancers based on expression profiling have been proposed as alternatives. However, their reliability and clinical utility have been repeatedly questioned, notably because most of them were derived from relatively small initial patient populations. We analyzed the transcriptomes of 537 breast tumors using three unsupervised classification methods. A core subset of 355 tumors was assigned to six clusters by all three methods. These six subgroups overlapped with previously defined molecular classes of breast cancer, but also showed important differences, notably the absence of an ERBB2 subgroup and the division of the large luminal ER+ group into four subgroups, two of them being highly proliferative. Of the six subgroups, four were ER+/PR+/AR+, one was ER-/PR-/AR+ and one was triple negative (AR-/ER-/PR-). ERBB2-amplified tumors were split between the ER-/PR-/AR+ subgroup and the highly proliferative ER+ LumC subgroup. Importantly, each of these six molecular subgroups showed specific copy-number alterations. Gene expression changes were correlated to specific signaling pathways. Each of these six subgroups showed very significant differences in tumor grade, metastatic sites, relapse-free survival or response to chemotherapy. All these findings were validated on large external datasets including more than 3000 tumors. Our data thus indicate that these six molecular subgroups represent well-defined clinico-biological entities of breast cancer. Their identification should facilitate the detection of novel prognostic factors or therapeutical targets in breast cancer.

Background Identification of predictive markers of response to treatment is a major objective in breast cancer. A major problem in clinical sampling is the variability of RNA templates, requiring accurate management of tumour material and subsequent analyses for future translation in clinical practice. Our aim was to establish the feasibility and reliability of high throughput RNA analysis in a prospective trial. Methods This study was conducted on RNA from initial biopsies, in a prospective trial of neoadjuvant chemotherapy in 327 patients with inoperable breast cancer. Four independent centres included patients and samples. Human U133 GeneChips plus 2.0 arrays for transcriptome analysis and quantitative RT-qPCR of 45 target genes and 6 reference genes were analysed on total RNA. Results Thirty seven samples were excluded because i) they contained less than 30% malignant cells, or ii) they provided RNA Integrity Number (RIN) of poor quality. Among the 290 remaining cases, taking into account strict quality control criteria initially defined to ensure good quality of sampling, 78% and 82% samples were eligible for transcriptome and RT-qPCR analyses, respectively. For RT-qPCR, efficiency was corrected by using standard curves for each gene and each plate. It was greater than 90% for all genes. Clustering analysis highlighted relevant breast cancer phenotypes for both techniques (ER+, PR+, HER2+, triple negative). Interestingly, clustering on trancriptome data also demonstrated a "centre effect", probably due to the sampling or extraction methods used in on of the centres. Conversely, the calibration of RT-qPCR analysis led to the centre effect withdrawing, allowing multicentre analysis of gene transcripts with high accuracy. Conclusions Our data showed that strict quality criteria for RNA integrity assessment and well calibrated and standardized RT-qPCR allows multicentre analysis of genes transcripts with high accuracy in the clinical context. More stringent criteria are needed for transcriptome analysis for clinical applications.