Molecular Anomalies Research Articles

Angioimmunoblastic T-cell lymphoma: more than a disease of T follicular helper cells.

Angioimmunoblastic T-cell lymphoma (AITL) is one of the most frequent entities of peripheral T-cell lymphoma. An AITL has two components: the AITL tumour cells, which have a T follicular helper (TFH) cell phenotype, and a surrounding and extensive tumour microenvironment that is populated with various reactive cell types, including B cells. Recurrent TET2 mutations have been described in 50-80% of AITLs, possibly occurring in a haematopoietic progenitor cell. An article published recently in the Journal of Pathology describes the use of microdissection to isolate PD1+ AITL tumour cells and CD20+ B cells from the AITL microenvironment, and to show that TET2 mutations are actually more frequent in these diseases than previously thought. Whereas TET2 mutations were detected in only six of 13 AITLs, 12 of 13 samples of microdissected PD1+ AITL tumour cells possessed this mutation. Moreover, TET2 mutations were detected in CD20+ B cells from the AITL microenvironment in six of nine informative cases. These results confirm that TET2 mutation is an early event in the majority of AITL cases, and that the driving molecular anomalies are not restricted to the T lineage tumour cells. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Kinase Activity in Recurring Primary Skull Base Chordomas and Chondrosarcomas: Identification of Novel Pathways of Oncogenesis and Potential Drug Targets.

Chordomas and chondrosarcomas can occur in the skull base. Currently, 45% of chordomas and 56% of chondrosarcomas recur within 5 years of surgery. The role of adjuvant therapy is highly debated. No pharmacotherapies have been approved by the U.S. Food and Drug Administration for chordomas or chondrosarcomas. High propensity for recurrence and lack of definitive adjuvant therapy necessitate additional basic science research to identify molecular anomalies associated with recurrent disease. We pooled tumor lysates from patients based on clinical criteria into 4 groups: primary chordomas, primary chordomas that recurred, primary chondrosarcomas, and primary chondrosarcomas that recurred. We used a peptide labeling method, isobaric tags for relative and absolute quantitation, to uniquely identify each tumor group. Phosphorylated peptides were identified and quantified via mass spectroscopy to determine and predict active kinases. Six groups of phosphorylated peptides were associated with primary tumors that later recurred. Specific kinases associated with primary chordomas that recurred were FES and FER. Specific kinases associated with primary chondrosarcomas that recurred were FES, FER, SRC family kinases, PKC, ROCK, and mitogen-activated protein kinase signaling (JNK, ERK1, p38). These data provide clinicians with a means to screen skull base chordomas and chondrosarcomas to help identify tumors with a propensity to recur. Many of these kinases can be efficaciously inhibited by Food and Drug Administration-approved drugs that have not yet been used in clinical trials for treatment of skull base chordomas or chondrosarcomas. Validation of kinases identified in this study may advance treatment options for patients with these tumors.

Treatment After First-Generation Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance in Non-Small-Cell Lung Cancer.

Systemic treatment is the basic treatment approach to advanced-stage non-small-cell lung cancer (NSCLC), and chemotherapy and targeted treatments are commonly employed in these patients. Recently, positive results achieved with immunotherapy have led to a growing number of treatment options and prolonged survival time. Today, specific tyrosine kinase inhibitors (TKIs), such as erlotinib, gefitinib, and afatinib, which target the epidermal growth factor receptor (EGFR), and the TKI crizotinib, which targets anaplastic lymphoma kinase gene rearrangement, have become the standard treatment among targeted therapies for patients with sensitive molecular anomalies. However, resistance develops against all these agents after a while. Numerous genetic mutations, T790M+ in particular, have been identified as resistance mechanisms against EGFR-TKIs, and researchers are developing specific inhibitors against them. Among those inhibitors, third-generation EGFR-TKIs such as osimertinib and rociletinib have gained prominence due to their high level of effectiveness and low toxicity profile. Besides, systemic chemotherapy and immunotherapy are proper alternatives. A second biopsy during the progression stage and better clarification of the mechanisms causing secondary resistance will enable more successful treatments in the future.

Front Cover: RP‐CARS reveals molecular spatial order anomalies in myelin of an animal model of Krabbe disease (J. Biophotonics 3/2017)

Front Cover: RP‐CARS reveals molecular spatial order anomalies in myelin of an animal model of Krabbe disease (J. Biophotonics 3/2017)

Nouvelles perspectives en oncologie médicale : médecine moléculaire et ses perspectives

The molecular medicine of cancer is based on the identification of genomic abnormalities in the DNA of tumor cells, guiding the treatment of patients, by choosing inhibitors acting on the mutated proteins encoded by the mutated genes. However, for the last 10 years, there has been a very rapid emergence of a new corpus of knowledge describing the genomic abnormalities of cancer cells in international programs such as ICGC or TCGA, leading to new nosological classifications, but also showing the extreme complexity and clonal variability of cancer cells in the human patient. The optimal use of this body of new data effectively. This requires 1) the construction of diagnostic platforms progressively exploring with greater depth the molecular anomalies of each individual patient and 2) the organization of multidisciplinary molecular consultation meetings to integrate these data into the clinical and global context of the patient. This evolution will also require a growingcontribution of bio-informatics. This short article provides a short update on the evolution of this molecular medicine of cancer.

Treatment with Hypomethylating Agent 5-Azacytidine Induces Sustained Response in Angioimmunoblastic T Cell Lymphomas

Treatment of relapsed/refractory peripheral T-cell lymphoma (R/R PTCL) remains an unmet medical need, as treatment with polychemotherapy is associated with poor outcome. With the exception of Brentuximab vedotin in systemic anaplastic large cell lymphomas, recent FDA approved drugs to treat R/R PTCL showed limited activity, with a 30% overall response rate (ORR), a complete response (CR) rate of 10-15% and short remission duration.Recurrent mutations of TET2 or other genes involved in DNA methylation regulation have recently been described in PTCL, especially in angioimmunoblastic T cell lymphomas (AITL) where TET2 mutations are present in 50-70% of patients. These molecular anomalies are also present in myeloid malignancies, which can be sensitive to treatment with hypomethylating agent. However, efficacy of hypomethylating agent in PTCL is unknown.We report here the results of 5-Azacytidine (5-AZA) treatment in 19 R/R PTCL patients. Two of them have been previously published (Cheminant et al. BJH 2015; Saillard et al. Hematol Oncol 2016).Treatment was administered between January 2013 and July 2016 according to physician decision and consisted in subcutaneous injection of 75 mg/m² 5-AZA during 7 consecutive days every 28 days, until progression or unacceptable toxicity. Six patients received rituximab for 4-8 infusions because of EBV-DNA positivity. Evaluation was performed locally according to IWG guidelines.Baseline characteristics of patients are presented in table 1. All patients presented with adverse prognostic factors and 17 patients had R/R PTCLs. Ten patients had previous or concomitant diagnosis of myelodysplastic syndrome, mostly (9/10) chronic myelomonocytic leukemia (CMML). Two patients did not receive chemotherapy before 5-AZA because of the presence of an associated CMML that required treatment first for myeloid malignancy, according to physician decision.Patients received a median number of 3 cycles of 5-AZA [1 - 43] and, at time of analysis, 7 patients are under therapy (Figure 1). Hematological toxicity was as expected. Two patients developed unusual adverse reaction: grade 2 polyneuropathy (which was deemed to be related to paraneoplastic syndrome) and grade 3 diarrhea of unknown origin, the latest leading to treatment interruption.The median follow-up was 84 days [19 - 1236]. Overall response rate (ORR) for the entire population was 53% (10/19), but was significantly higher in AITL patients than in patients with other PTCL entities (9/12, 75% vs. 1/7, 15%, p=0,0198). Among these 10 responding patients, 5 patients - all suffering from AITL - reached complete response (CR), leading a CR rate of 42% in this entity. Three additional patients were in stable disease (best response) and 6 progressed rapidly. It is noteworthy that among the 9 AITL patients who responded, only 2 patients experienced progression after 86 and 499 days, respectively, the remaining 7 patients being still responders and under therapy. None of the patients with another PTCL entity experienced response, with the exception of one patient with ATLL for whom 5-AZA induced a rapid and profound decrease in lymphocyte count; however, this patient relapsed after the second cycle.TET2 was sequenced in 14 patients and was mutated in 8/10 (80%) AITL and 1/4 (25%) other PTCL. Interestingly, 8/8 AITL patients with TET2 mutational status available who experienced a response after 5-AZA treatment were TET2 mutated.In summary, 5-AZA treatment is associated with significant response rate in R/R AITL, including prolonged CR. Additional genomic analyses are ongoing and will be presented during the meeting, aiming to correlate response rate with mutational status in the TET2, IDH2 and DNMT3A epigenetic modifier genes. Finally these results warrant further prospective studies evaluating 5-AZA treatment in R/R AITL or R/R PTCL bearing mutations in epigenetic modifiers. [Display omitted] [Display omitted] DisclosuresDupuis:janssen: Honoraria; ABBVIE: Membership on an entity’s Board of Directors or advisory committees. Sarkozy:Janssen Research & Development: Honoraria; Sandoz: Research Funding; Takeda: Research Funding; Gilead: Honoraria. Morschhauser:Celgene: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Gilead Sciences: Consultancy, Honoraria; Janssen: Honoraria; Servier: Consultancy, Honoraria. Hermine:AB science: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding, Speakers Bureau; Celgene: Research Funding; Alexion: Research Funding; Novartis: Research Funding. Haioun:Janssen: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Sandoz: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity’s Board of Directors or advisory committees.

DNMT3A Mutation inChinese Childhood Acute Myeloid Leukemia

PurposeDNMT3A mutation has been found in approximately 20% of adult acute myeloid leukemia (AML) patients and in 0~1.4% of children with AML, and the hotspots of mutations are mainly located in the catalytic methyltransferase domain, thereinto, mutation R882 accounts for 60%. Although the prognosis of adult patients with DNMT3A mutations is dismal, the prognosis of DNMT3A mutation in childhood AML is still unclear. Here, we tried to determine the incidence and prognostic significance of DNMT3A mutations in Chinese childhood AML.Methods We detected the mutations in DNMT3A exon 23 by PCR and direct sequencing in 342 children with AML (0~16 years old) from January in 2005 to June in 2013, treated on BCH-2003 AML protocol. The correlation of DNMT3A mutations with clinical characteristics, fusion genes, other molecular anomalies (FLT3-ITD, NPM1, C-KIT and WT1 mutations), and treatment outcome were analyzed.ResultsDNMT3A mutations were detected in 4 out of 342 (1.2%) patients. The mutations in coding sequences included S892S, V912A, R885G, and Q886R. Furthermore, there were two intronic mutations (c.2598-15C>T and c.2739+55A>C) found in one patient. No association of DNMT3A mutations with common clinical features was found. Two patients with DNMT3A mutations died of relapse or complications during treatment, one patient gave up treatment due to introduction failure in day33. Only one patient achieved continuous complete remission.Conclusions DNMT3A mutations were rare in Chinese children with AML. The mutation positions were different from the hotspots reported in adult AML. DNMT3A mutations may have adverse impact on prognosis of children with AML. DisclosuresNo relevant conflicts of interest to declare.

Histopathologic diagnosis of brain metastases: current trends in management and future considerations.

Confronted with brain metastases (BM), pathologists aim to rule out a primary central nervous system (CNS) tumor and to identify or verify the primary tumor site to guide the clinician to specific therapies. Apart from morphological features, ancillary immunohistochemical analysis is the most effective tool for characterizing a metastatic neoplasm of unknown origin. A limited array of antibodies is used, taking into account relevant clinical information and the known brain tropism of lung cancer, breast cancer and melanoma. Recently, targeted therapies have enriched the therapeutic arsenal, in particular for patients with non-small cell lung cancer or melanoma and for patients carrying molecular anomalies. These therapies can lead to a substantial tumor response, brain metastases included, which justifies rapid determination of a molecular profile. To combine different tools and provide timely results, good tumor sample management and careful attention at the pre-analytical phase are critical. Appropriate strategies for molecular and immunohistochemical analysis are needed to identify theranostic markers. This article aims to review the anatomopathological diagnostic approach for BM in the age of targeted therapies.

Mise au point sur les classifications moléculaires et la nouvelle classification histologique du cancer de la vessie

Comment the new WHO histological classification of tumors of the urinary system and male genital organs 2016 and expose the state of art about urothelial carcinogenesis and molecular modifications of bladder cancer, with the consequences on the treatments. A systematic review of the literature search was performed from the data base PubMed, focused on the following keywords: "bladder cancer", "molecular", "subtypes". The new WHO histological classification of tumors of the urinary system 2016 confirm the importance of pathology in determining the care of patients, especially the grade, the histological type and the infiltration, while taking into account the difficulties. In 2014, the Cancer Genome Atlas reported genetic modifications of bladder cancer. Recently, several studies explored molecular anomalies of bladder cancer and elaborated molecular classifications, analyzing their predictive value. According to the groups, different molecular subtype had been defined: Urobasal A, Urobasal B, genomically unstable, infiltrated, squamous cell carcinoma-like and p53-like luminal bladder cancers. This latter subgroup seems to be chemoresistant. The molecular biology and classifications allow a better understanding of bladder cancer and could complete in near future histological data to improve patient management.

Interferon-Gamma Improves Macrophages Function against M. tuberculosis in Multidrug-Resistant Tuberculosis Patients.

Background. Mycobacterium tuberculosis (M. tuberculosis) that causes tuberculosis (TB) kills millions of infected people annually especially multidrug-resistant tuberculosis (MDR-TB). On infection, macrophages recognize the mycobacteria by toll-like receptor (TLR) followed by phagocytosis and control of mycobacteria. In addition, macrophages also secrete IL-12 to induce IFN-γ production by T, which, in turn, increases the phagocytosis and oxidative burst. Individuals with defects in innate or adaptive immunity exhibit increased susceptibility to M. tuberculosis. Understanding these immunologic mechanisms will help in TB control. We aimed to investigate the immunopathologic mechanisms in MDR-TB and role of recombinant human interferon-gamma (rhIFN-γ). Study Design and Methods. Monocyte-derived macrophages (MDMs) were generated from peripheral blood mononuclear cells of MDR-TB patients and healthy subjects and were investigated for immunologic response by ELISA and flow cytometry. Results. Different functional and molecular anomalies were observed in macrophages. In addition, a defective immune response to M. tuberculosis from the patient's MDMs was characterized, which in turn improved by pretreatment with rhIFN-γ. Conclusion. This work highlights the fact that rhIFN-γ improves macrophages function against M. tuberculosis and treatment of patients with poor responsiveness to TB therapy may be needed in future to include IFN-γ as adjuvant therapy after the full characterization of pathological and molecular mechanisms in these and in other more multidrug-resistant TB patients.

Quiescence Loosens Epigenetic Constraints in Bovine Somatic Cells and Improves Their Reprogramming into Totipotency.

Reprogramming by nuclear transfer (NT) cloning forces cells to lose their lineage-specific epigenetic marks and reacquire totipotency. This process often produces molecular anomalies that compromise clone development. We hypothesized that quiescence alters the epigenetic status of somatic NT donor cells and elevates their reprogrammability. To test this idea, we compared chromatin composition and cloning efficiency of serum-starved quiescent (G0) fibroblasts versus nonstarved mitotically selected (G1) controls. We show that G0 chromatin contains reduced levels of Polycomb group proteins EED, SUZ12, PHC1, and RING2, as well as histone variant H2A.Z. Using quantitative confocal immunofluorescence microscopy and fluorometric enzyme-linked immunosorbent assay, we further show that G0 induced DNA and histone hypomethylation, specifically at H3K4me3, H3K9me2/3 and H3K27me3, but not H3K9me1. Collectively, these changes resulted in a more relaxed G0 chromatin state. Following NT, G0 donors developed into blastocysts that retained H3K9me3 hypomethylation, both in the inner cell mass and trophectoderm. G0 blastocysts from different cell types and cell lines developed significantly better into adult offspring. In conclusion, serum starvation induced epigenetic changes, specifically hypotrimethylation, that provide a mechanistic correlate for increased somatic cell reprogrammability.

Interferon regulatory factor 4 attenuates Notch signaling to suppress the development of chronic lymphocytic leukemia.

Molecular pathogenesis of Chronic Lymphocytic Leukemia (CLL) is not fully elucidated. Genome wide association studies have linked Interferon Regulatory Factor 4 (IRF4) to the development of CLL. We recently established a causal relationship between low levels of IRF4 and development of CLL. However, the molecular mechanism through which IRF4 suppresses CLL development remains unclear. Deregulation of Notch signaling pathway has been identified as one of the most recurrent molecular anomalies in the pathogenesis of CLL. Yet, the role of Notch signaling as well as its regulation during CLL development remains poorly understood. Previously, we demonstrated that IRF4 deficient mice expressing immunoglobulin heavy chain Vh11 (IRF4−/−Vh11) developed spontaneous CLL with complete penetrance. In this study, we show that elevated Notch2 expression and the resulting hyperactivation of Notch signaling are common features of IRF4−/−Vh11 CLL cells. Our studies further reveal that Notch signaling is indispensable for CLL development in the IRF4−/−Vh11 mice. Moreover, we identify E3 ubiquitin ligase Nedd4, which targets Notch for degradation, as a direct target of IRF4 in CLL cells and their precursors. Collectively, our studies provide the first in vivo evidence for an essential role of Notch signaling in the development of CLL and establish IRF4 as a critical regulator of Notch signaling during CLL development.

RP-CARS reveals molecular spatial order anomalies in myelin of an animal model of Krabbe disease.

Krabbe disease (KD) is a rare demyelinating sphingolipidosis, often fatal in the first years of life. It is caused by the inactivation of the galactocerebrosidase (GALC) enzyme that causes an increase in the cellular levels of psychosine considered to be at the origin of the tissue-level effects. GALC is inactivated also in the Twitcher (TWI) mouse: a genetic model of KD that is providing important insights into the understating of the pathogenetic process and the development of possible treatments. In this article an innovative optical technique, RP-CARS, is proposed as a tool to study the degree of order of the CH2 bonds inside the myelin sheaths of TWI-mice sciatic-nerve fibres. RP-CARS, a recently developed variation of CARS microscopy, is able to combine the intrinsic chemical selectivity of CARS microscopy with molecular-bond-spatial-orientation sensibility. This is the first time RP-CARS is applied to the study of a genetic model of a pathology, leading to the demonstration of a post-onset progressive spatial disorganization of the myelin CH2 bonds. The presented result could be of great interest for a deeper understanding of the pathogenic mechanisms underlying the human KD and, moreover, it is an additional proof of the experimental validity of this microscopy technique. RP-CARS image (2850cm-1 , CH2 bonds) of a sciatic-nerve optical longitudinal section from a Twitcher P23 (symptomatic) mouse. Scale bar: 10microns. The image was constructed by colour-mapping the degree of molecular order of the CH2 bonds inside the myelin walls, as displayed in the colour bar on the right.

Targeted therapy in non-small cell lung cancer: a focus on epidermal growth factor receptor mutations.

The main molecular targeting of lung cancer [non-small cell lung cancer (NSCLC)] concerns mutations of epidermal growth factor receptor (EGFR). The awaited responsiveness of tumors carrying these mutations is high with for instance 60% to 80% with tyrosine kinase inhibitors hitting EGFR mutations. The EGFR T790M as a secondary mutation is responsible for the occurrence of a resistance phenomenon. A multitude of drugs have been produced and tested with the property of a specific binding at the EGFR T790M site. There is currently an evolution oriented to a robust genotyping methods allowing the identification of given molecular anomalies (pyrosequencing for instance) towards the consideration of a much larger set of molecular anomalies under the form of a global genotyping realized with the use of next-generation sequencing (NGS). This phase of whole genome analysis necessitates the introduction of a specialized staff for data treatment. A possible substitution plasma/tumor for the mutation analyses is perceptible in lung cancer, a preference being however given to the intratumoral direct investigation when this is feasible. EGFR mutations as targetable anomalies are illustrative examples, that the management of NSCLC is currently drawing a significant benefit from personalized therapy.

Genetic Basis of Ureterocele.

Congenital anomalies of the kidney and urinary tract (CAKUT) form a group of heterogeneous disorders that affect the kidneys, ureters and bladder, with frequent asynchronous presentations and multiple CAKUT associations in the same individual. Urinary tract formation is a complex process, dependent of the interaction of multiple genes and their sub-product. The same genic alterations can lead to different molecular expressions and different morphological anomalies. The ureterocele is a cystic dilation of the distal intramural ureter, resulting in obstruction of urine flow, dilation of the ureter and renal pelvis and loss of renal function. Two key steps in the urinary tract ontogenesis may be related to ureterocele development: formation and migration of the ureteric bud and its incorporation in the bladder. This review aims to describe the morphological, cellular and biochemical steps, as well as the genes involved in the occurrence of this anomaly.

The role of cerebellar circuitry alterations in the pathophysiology of autism spectrum disorders.

The cerebellum has been repeatedly implicated in gene expression, rodent model and post-mortem studies of autism spectrum disorder (ASD). How cellular and molecular anomalies of the cerebellum relate to clinical manifestations of ASD remains unclear. Separate circuits of the cerebellum control different sensorimotor behaviors, such as maintaining balance, walking, making eye movements, reaching, and grasping. Each of these behaviors has been found to be impaired in ASD, suggesting that multiple distinct circuits of the cerebellum may be involved in the pathogenesis of patients' sensorimotor impairments. We will review evidence that the development of these circuits is disrupted in individuals with ASD and that their study may help elucidate the pathophysiology of sensorimotor deficits and core symptoms of the disorder. Preclinical studies of monogenetic conditions associated with ASD also have identified selective defects of the cerebellum and documented behavioral rescues when the cerebellum is targeted. Based on these findings, we propose that cerebellar circuits may prove to be promising targets for therapeutic development aimed at rescuing sensorimotor and other clinical symptoms of different forms of ASD.

Mutations of the Imprinted CDKN1C Gene as a Cause of the Overgrowth Beckwith-Wiedemann Syndrome: Clinical Spectrum and Functional Characterization.

Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder associating macroglossia, abdominal wall defects, visceromegaly, and a high risk of childhood tumor. Molecular anomalies are mostly epigenetic; however, mutations of CDKN1C are implicated in 8% of cases, including both sporadic and familial forms. We aimed to describe the phenotype of BWS patients with CDKN1C mutations and develop a functional test for CDKN1C mutations. For each propositus, we sequenced the three exons and intron-exon boundaries of CDKN1C in patients presenting a BWS phenotype, including abdominal wall defects, without 11p15 methylation defects. We developed a functional test based on flow cytometry. We identified 37 mutations in 38 pedigrees (50 patients and seven fetuses). Analysis of parental samples when available showed that all mutations tested but one was inherited from the mother. The four missense mutations led to a less severe phenotype (lower frequency of exomphalos) than the other 33 mutations. The following four tumors occurred: one neuroblastoma, one ganglioneuroblastoma, one melanoma, and one acute lymphoid leukemia. Cases of BWS caused by CDKN1C mutations are not rare. CDKN1C sequencing should be performed for BWS patients presenting with abdominal wall defects or cleft palate without 11p15 methylation defects or body asymmetry, or in familial cases of BWS.

Molecular landscape of prostate cancer: implications for current clinical trials.

Castration-resistant prostate cancer (CRPC) is a lethal disease, and improvement with androgen-deprivation therapy has plateaued. Next-generation sequencing studies have led to significant advances in our understanding of genomic alterations in prostate cancer. The most common genomic aberrations in this malignancy are the transcription factor fusion of TMPRSS2-ETS, and mutations in TP53, AR, RB1 and PTEN/PIK3CA. Some of these alterations are actionable by drugs available in the clinic. In addition, it was recently shown that aberrations in DNA repair genes, such as BRCA2 and ATM, are present in both somatic and germline form in a significant minority of prostate cancer; these abnormalities can be targeted by drugs such as platinums and PARP inhibitors. In the era of tumour profiling, targeting molecular alterations may provide an opportunity for new therapeutic approaches. Although there are promising new agents to attack a variety of genomic signal abnormalities, biomarker-matched therapy (other than for androgens) have been utilised in only 2.0% of clinical trials (September 2011 through September 2014; https://clinicaltrials.gov) for prostate cancer. Enhanced efforts to define subsets of patients with prostate cancer based on their molecular anomalies, and match them with cognate therapies, warrant investigation.

Characterisation of patients with Glanzmann thrombasthenia and identification of 17 novel mutations.

Glanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder characterised by quantitative and/or qualitative defects of the platelet glycoprotein (GP) IIb/IIIa complex, also called integrin αIIbβ3. αIIbβ3 is well known as a platelet fibrinogen receptor and mediates platelet aggregation, firm adhesion, and spreading. This study describes the molecular genetic analyses of 19 patients with GT who were diagnosed on the basis of clinical parameters and platelet analyses. The patients' bleeding signs include epistaxis, mucocutaneous bleeding, haematomas, petechiae, gastrointestinal bleeding, and menorrhagia. Homozygous or compound heterozygous mutations in ITGA2B or ITGB3 were identified as causing GT by sequencing of genomic DNA. All exons including exon/intron boundaries of both genes were analysed. In a patient with an intronic mutation, splicing of mRNA was analysed using reverse transcriptase (RT)-PCR of platelet-derived RNA. In short, 16 of 19 patients revealed 27 different mutations (ITGA2B: n=17, ITGB3: n=10). Seventeen of these mutations have not been published to date. Mutations in ITGA2B or ITGB3 were identified as causing GT in 16 patients. We detected a total of 27 mutations in ITGA2B and ITGB3 including 17 novel missense, nonsense, frameshift and splice site mutations. In addition, three patients revealed no molecular genetic anomalies in ITGA2B or ITGB3 that could explain the suspected diagnosis of GT. We assume that these patients may harbour defects in a regulatory element affecting the transcription of these genes, or other proteins may exist that are important for activating the αIIbβ3 complex that may be affected.

Treacher Collins syndrome: a clinical and molecular study based on a large series of patients.

Treacher Collins/Franceschetti syndrome (TCS; OMIM 154500) is a disorder of craniofacial development belonging to the heterogeneous group of mandibulofacial dysostoses. TCS is classically characterized by bilateral mandibular and malar hypoplasia, downward-slanting palpebral fissures, and microtia. To date, three genes have been identified in TCS:,TCOF1, POLR1D, and POLR1C. We report a clinical and extensive molecular study, including TCOF1, POLR1D, POLR1C, and EFTUD2 genes, in a series of 146 patients with TCS. Phenotype-genotype correlations were investigated for 19 clinical features, between TCOF1 and POLR1D, and the type of mutation or its localization in the TCOF1 gene. We identified 92/146 patients (63%) with a molecular anomaly within TCOF1, 9/146 (6%) within POLR1D, and none within POLR1C. Among the atypical negative patients (with intellectual disability and/or microcephaly), we identified four patients carrying a mutation in EFTUD2 and two patients with 5q32 deletion encompassing TCOF1 and CAMK2A in particular. Congenital cardiac defects occurred more frequently among patients with TCOF1 mutation (7/92, 8%) than reported in the literature. Even though TCOF1 and POLR1D were associated with extreme clinical variability, we found no phenotype-genotype correlation. In cases with a typical phenotype of TCS, 6/146 (4%) remained with an unidentified molecular defect.