Simultaneous detection of aneuploidies for chromosomes 4, 6, 10 and 17 by automated four colour I-FISH in high hyperdiploid acute lymphoblastic leukemia : diagnostic assessment, clonal heterogeneity and chromosomal instability in adults

SummarySimultaneous detection of aneuploidies for chromosomes 4, 6,10 and 17 by automated four color l-FISH in high hyperdiploid acute lymphoblastic leukemia: diagnostic assessment, clonal heterogeneity and chromosomal instability in adultsAnna Talamo BlandinService de Génétique Médicale, Unité de Cytogénétique du Cancer, CHUVAcute lymphoblastic leukemia (ALL) is a malignant hemopathy characterized by the accumulation of the immature lymphoid cells in the bone marrow and, most often, in the peripheral blood. ALL is a heterogeneous disease with distinct biological and prognostic entities. At diagnosis, cytogenetic and molecular findings constitute important and independent prognostic factors. High hyperdiploidy with 51-67 chromosomes (HeH), one of the largest cytogenetic subsets of ALL, in childhood particularly, is generally associated with a relatively favorable outcome. Chromosome gain is nonrandom, extracopies of some chromosome occurring more frequently than those of others. Concurrent presence of trisomy for chromosomes 4, 10 and 17 confers an especially good prognosis. The first aim of our work was to develop an automated four color interphase fluorescence in situ hybridization (l-FISH) methodology and to assess its ability to detect concurrent aneuploidies 4, 6, 10 and 17 in 10 ALL patients. Various combinations of aneuploidies were identified. All clones detected by conventional cytogenetics were also observed by l-FISH. However, in all patients, l-FISH revealed numerous additional abnormal clones, leading to a high level of clonal heterogeneity. Our second aim has been to investigate the nature and origin of this clonal heterogeneity and to test for the presence of chromosome instability (CIN) in HeH ALL at initial presentation. Ten HeH ALL and 10 non-HeH ALL patients were analysed by four colour l-FISH and numerical CIN values were determined for all four chromosomes together and for each chromosome and patient group, an original approach in ALL. CIN values in HeH ALL proved to be much higher than#iose in non-HeH ALL, suggesting that numerical CIN may be at the origin of the high level of clonal heterogeneity revealed by l-FISH. Our third aim has been to study the evolution of these cytogenetic features during the course of the disease in 10 HeH ALL patients. Clonal heterogeneity was also observed again during disease progression, particularly at relapse. Clones detected at initial presentation generally reappeared in relapse, in most cases with newly generated ones. A significant correlation between the number of abnormal clones and CIN suggested that the higher the instability, the larger the number of abnormal clones. Whereas clonal heterogeneity and its evolution most probably result from underlying chromosome instability, operating processes remain conjectural.RésuméLa leucémie lymphoblastique aiguë (LLA) est une hémopathie maligne qui résulte de l'accumulationde cellules lymphoïdes immatures dans la moelle osseuse, et, le plus souvent, dans le sangpériphérique également. La LLA est une affection hétérogène au sein de laquelle se distinguentplusieurs entités biologiques et pronostiques. Les données cytogénétiques et moléculaires font partieintégrante du diagnostic et jouent un rôle essentiel dans l'évaluation du pronostic. L'hyperdiploïdieélevée à 51-­67 chromosomes (HeH), relativement fréquente, en particulier chez l'enfant, s'associe àun pronostic favorable. Le gain de chromosomes ne relève pas du hasard, certains chromosomesétant plus fréquemment impliqués que d'autres. La présence simultanée des trisomies 4, 6, et 17s'associe à un pronostic particulièrement bon. Le premier but du travail a été de développer uneméthode d'analyse automatique par hybridation in situ fluorescente interphasique (I-­FISH) à 4couleurs et de tester sa capacité à identifier la présence simultanée d'aneuploïdies 4, 6, 10 et 17 dans10 cas de LLA. Différentes combinaisons d'aneuploïdies ont été identifiées. Tous les clones détectéspar cytogénétique conventionnelle l'ont été par I-­FISH. Or, chez tous les patients, l'I-­FISH a révélé denombreux clones anormaux additionnels générant un degré élevé d'hétérogénéité clonale. Notredeuxième but a été d'investiguer la nature et l'origine de cette hétérogénéité et de tester la présenced'instabilité chromosomique (CIN) chez les patients avec une LLA HeH en presentation initiale. DixLLA HeH et 10 LLA non-­HeH ont été analysées par I-­FISH et les valeurs de CIN numérique ont étédéterminées pour les 4 chromosomes ensemble et pour chaque chromosome et groupe de patients,approche originale dans la LLA. Ces valeurs étant beaucoup plus élevées dans la LLA HeH que dansla LLA non-­HeH, elles favorisent l'hypothèse selon laquelle la CIN serait à l'origine de l'hétérogénéitéclonale révélée par I-­FISH. Le troisième but de notre travail a été d'étudier l'évolution de cescaractéristiques cytogénétiques au cours de la maladie dans 10 cas de LLA HeH. L'hétérogénéitéclonale a été retrouvée lors de la progression de la maladie, en particulier en rechute, où les clonesanormaux détectés en présentation initiale réapparaissent, généralement accompagnés de clonesnouveaux. La corrélation existant entre nombre de clones anormaux et valeurs de CIN suggère queplus l'instabilité est élevée, plus le nombre de clones anormaux est grand. Bien que l'hétérogénéitéclonale et son évolution résultent très probablement de l'instabilité chromosomique, les processus àl'oeuvre ne sont pas entièrement élucidés.

Mutual Information of IID complex Gaussian signals on block Rayleigh-faded channels

We present a method to compute, quickly and efficiently, the mutual information achieved by an IID (independent identically distributed) complex Gaussian signal on a block Rayleigh-faded channel without side information at the receiver. The method accommodates both scalar and MIMO (multiple-input multiple-output) settings. Operationally, this mutual information represents the highest spectral efficiency that can be attained using Gaussiancodebooks. Examples are provided that illustrate the loss in spectral efficiency caused by fast fading and how that loss is amplified when multiple transmit antennas are used. These examples are further enriched by comparisons with the channel capacity under perfect channel-state information at the receiver, and with the spectral efficiency attained by pilot-based transmission.

Edge detection by selection of pieces of level lines

We propose an edge detector based on the selection of wellcontrasted pieces of level lines, following the proposal ofDesolneux-Moisan-Morel (DMM) [1]. The DMM edge detectorhas the problem of over-representation, that is, everyedge is detected several times in slightly different positions.In this paper we propose two modifications of the originalDMM edge detector in order to solve this problem. The firstmodification is a post-processing of the output using a generalmethod to select the best representative of a bundle of curves.The second modification is the use of Canny’s edge detectorinstead of the norm of the gradient to build the statistics. Thetwo modifications are independent and can be applied separately.Elementary reasoning and some experiments showthat the best results are obtained when both modifications areapplied together.

Full diversity product codes for block erasure and block fading channels

We show how to build full-diversity product codes under both iterative encoding and decoding over non-ergodic channels, in presence of block erasure and block fading. The concept of a rootcheck or a root subcode is introduced by generalizing the same principle recently invented for low-density parity-check codes. We also describe some channel related graphical properties of the new family of product codes, a familyreferred to as root product codes.

What is the value of joint processing of pilots and data in block-fading channels?

The spectral efficiency achievable with joint processing of pilot and data symbol observations is compared with that achievable through the conventional (separate) approach of first estimating the channel on the basis of the pilot symbols alone, and subsequently detecting the datasymbols. Studied on the basis of a mutual information lower bound, joint processing is found to provide a non-negligible advantage relative to separate processing, particularly for fast fading. It is shown that, regardless of the fading rate, only a very small number of pilot symbols (at most one per transmit antenna and per channel coherence interval) shouldbe transmitted if joint processing is allowed.

A systolic array for linear MIMO detection based on an all-swap lattice reduction algorithm

A systolic array to implement lattice-reduction-aided lineardetection is proposed for a MIMO receiver. The lattice reductionalgorithm and the ensuing linear detections are operated in the same array, which can be hardware-efficient. All-swap lattice reduction algorithm (ASLR) is considered for the systolic design.ASLR is a variant of the LLL algorithm, which processes all lattice basis vectors within one iteration. Lattice-reduction-aided linear detection based on ASLR and LLL algorithms have very similarbit-error-rate performance, while ASLR is more time efficient inthe systolic array, especially for systems with a large number ofantennas.

Outage thresholds of LDPC codes over nonergodic block-fading channels

This paper derives approximations allowing the estimation of outage probability for standard irregular LDPC codes and full-diversity Root-LDPC codes used over nonergodic block-fading channels. Two separate approaches are discussed: a numerical approximation, obtained by curve fitting, for both code ensembles, and an analytical approximation for Root-LDPC codes, obtained under the assumption that the slope of the iterative threshold curve of a given code ensemble matches the slope of the outage capacity curve in the high-SNR regime.

Optimum pilot overhead in wireless communication: a unified treatment of continuous and block-fading channels

The optimization of the pilot overhead in wireless fading channels is investigated, and the dependence of this overhead on various system parameters of interest (e.g., fading rate, signal-to-noise ratio) is quantified. The achievable pilot-based spectral efficiency is expanded with respect to the fading rate about the no-fading point, which leads to an accurate order expansion for the pilot overhead. This expansion identifies that the pilot overhead, as well as the spectral efficiency penalty with respect to a reference system with genie-aided CSI (channel state information) at the receiver, depend on the square root of the normalized Doppler frequency. It is also shown that the widely-usedblock fading model is a special case of more accurate continuous fading models in terms of the achievable pilot-based spectral efficiency. Furthermore, it is established that the overhead optimization for multiantenna systems is effectively the same as for single-antenna systems with thenormalized Doppler frequency multiplied by the number of transmit antennas.

Direct detection of a BRAF mutation in total RNA from melanoma cells using cantilever arrays.

Malignant melanoma, the deadliest form of skin cancer, is characterized by a predominant mutation in the BRAF gene. Drugs that target tumours carrying this mutation have recently entered the clinic. Accordingly, patients are routinely screened for mutations in this gene to determine whether they can benefit from this type of treatment. The current gold standard for mutation screening uses real-time polymerase chain reaction and sequencing methods. Here we show that an assay based on microcantilever arrays can detect the mutation nanomechanically without amplification in total RNA samples isolated from melanoma cells. The assay is based on a BRAF-specific oligonucleotide probe. We detected mutant BRAF at a concentration of 500 pM in a 50-fold excess of the wild-type sequence. The method was able to distinguish melanoma cells carrying the mutation from wild-type cells using as little as 20 ng µl(-1) of RNA material, without prior PCR amplification and use of labels.

On iterative performance of LDPC and Root-LDPC codes over block-fading channels

This paper presents our investigation on iterativedecoding performances of some sparse-graph codes on block-fading Rayleigh channels. The considered code ensembles are standard LDPC codes and Root-LDPC codes, first proposed in and shown to be able to attain the full transmission diversity. We study the iterative threshold performance of those codes as a function of fading gains of the transmission channel and propose a numerical approximation of the iterative threshold versus fading gains, both both LDPC and Root-LDPC codes.Also, we show analytically that, in the case of 2 fading blocks,the iterative threshold root of Root-LDPC codes is proportional to (α1 α2)1, where α1 and α2 are corresponding fading gains.From this result, the full diversity property of Root-LDPC codes immediately follows.

Multiuser detection with an unknown number of active users: receiver design

In multiuser detection, the set of users active at any time may be unknown to the receiver. In these conditions, optimum reception consists of detecting simultaneously the set of activeusers and their data, problem that can be solved exactly by applying random-set theory (RST) and Bayesian recursions (BR). However, implementation of optimum receivers may be limited by their complexity, which grows exponentially with the number of potential users. In this paper we examine three strategies leading to reduced-complexity receivers.In particular, we show how a simple approximation of BRs enables the use of Sphere Detection (SD) algorithm, whichexhibits satisfactory performance with limited complexity.

Automatic Detection of Non-deverbal Event Nouns for Quick Lexicon Production

In this work we present the results of experimental work on the development of lexical class-based lexica by automatic means. Our purpose is to assess the use of linguistic lexical-class based information as a feature selection methodology for the use of classifiers in quick lexical development. The results show that the approach can help reduce the human effort required in the development of language resources significantly.

Detection of fat embolism in cases with postmortem angiography (PMA) - Preliminary results

Introduction: Pulmonary fat embolism (PFE) can be a cause of death in cases with trauma, during orthopedic surgery and also in non-traumatic conditions, such as burns, pancreatitis, fatty liver or sickle cell disease. As PMA becomes more widespread, it is important to determine how it affects the diagnosis of PFE. Aims: The aim of this study was to determine if the oily contrast liquid used in PMA induces artefactual PFE, if such artefacts differ from original PFE and if PFE can be detected and graded before PMA. Material and methods: Cases of adults without signs of postmortem change and for which an autopsy with angiography was performed were selected for this study. Pulmonary biopsies of each lung were taken before and after the angiography as were fragments of each lung with a twin-edged knife during the autopsy. The samples were examined under the microscope without fixation or staining and after an Oil-Red O staining. PFE was graded according to Falci et al. Results: Non-artefactual (original) PFE was diagnosed in 4 cases on pre-PMA biopsies. As expected, structures with the aspect of PFE were present in all cases after angiography. The microscopical aspect of original and PMA induced PFE was identical. Grading of the PFE according to Falci et al. was depending on the quality of the biopsies. Conclusions: PMA with oily contrast induces artefactual PFE that cannot be visually differentiated from original PFE. Original PFE can however be diagnosed with pre-angiography biopsies. In order to assure the diagnosis and correct grading of PFE, the quality of the biopsy should be checked before PMA with oily contrast.

Detection of four Plasmodium species in blood from humans by 18S rRNA gene subunit-based and specific real-time PCR assays

Résumé : Un nombre croissant de cas de malaria chez les voyageurs et migrants a été rapporté. Bien que l'analyse microscopique des frottis sanguins reste traditionnellement l'outil diagnostic de référence, sa fiabilité dépend considérablement de l'expertise de l'examinateur, pouvant elle-même faire défaut sous nos latitudes. Une PCR multiplex en temps réel a donc été développée en vue d'une standardisation du diagnostic. Un ensemble d'amorces génériques ciblant une région hautement conservée du gène d'ARN ribosomial 18S du genre Plasmodium a tout d'abord été conçu, dont le polymorphisme du produit d'amplification semblait suffisant pour créer quatre sondes spécifiques à l'espèce P. falciparum, P. malariae, P. vivax et P. ovale. Ces sondes utilisées en PCR en temps réel se sont révélées capables de détecter une seule copie de plasmide de P. falciparum, P. malariae, P. vivax et P. ovale spécifiquement. La même sensibilité a été obtenue avec une sonde de screening pouvant détecter les quatre espèces. Quatre-vingt-dix-sept échantillons de sang ont ensuite été testés, dont on a comparé la microscopie et la PCR en temps réel pour 66 (60 patients) d'entre eux. Ces deux méthodes ont montré une concordance globale de 86% pour la détection de plasmodia. Les résultats discordants ont été réévalués grâce à des données cliniques, une deuxième expertise microscopique et moléculaire (laboratoire de Genève et de l'Institut Suisse Tropical de Bâle), ainsi qu'à l'aide du séquençage. Cette nouvelle analyse s'est prononcé en faveur de la méthode moléculaire pour tous les neuf résultats discordants. Sur les 31 résultats positifs par les deux méthodes, la même réévaluation a pu donner raison 8 fois sur 9 à la PCR en temps réel sur le plan de l'identification de l'espèce plasmodiale. Les 31 autres échantillons ont été analysés pour le suivi de sept patients sous traitement antimalarique. Il a été observé une baisse rapide du nombre de parasites mesurée par la PCR en temps réel chez six des sept patients, baisse correspondant à la parasitémie déterminée microscopiquement. Ceci suggère ainsi le rôle potentiel de la PCR en temps réel dans le suivi thérapeutique des patients traités par antipaludéens. Abstract : There have been reports of increasing numbers of cases of malaria among migrants and travelers. Although microscopic examination of blood smears remains the "gold standard" in diagnosis, this method suffers from insufficient sensitivity and requires considerable expertise. To improve diagnosis, a multiplex real-time PCR was developed. One set of generic primers targeting a highly conserved region of the 18S rRNA gene of the genus Plasmodium was designed; the primer set was polymorphic enough internally to design four species-specific probes for P. falciparum, P. vivax, P. malarie, and P. ovale. Real-time PCR with species-specific probes detected one plasmid copy of P. falciparum, P. vivax, P. malariae, and P. ovale specifically. The same sensitivity was achieved for all species with real-time PCR with the 18S screening probe. Ninety-seven blood samples were investigated. For 66 of them (60 patients), microscopy and real-time PCR results were compared and had a crude agreement of 86% for the detection of plasmodia. Discordant results were reevaluated with clinical, molecular, and sequencing data to resolve them. All nine discordances between 18S screening PCR and microscopy were resolved in favor of the molecular method, as were eight of nine discordances at the species level for the species-specific PCR among the 31 samples positive by both methods. The other 31 blood samples were tested to monitor the antimalaria treatment in seven patients. The number of parasites measured by real-time PCR fell rapidly for six out of seven patients in parallel to parasitemia determined microscopically. This suggests a role of quantitative PCR for the monitoring of patients receiving antimalaria therapy.

Eliminating heterophilic antibody interference for ferritin detection using Olympus F(ab')2 based reagent.

Interferences with the Olympus immunoturbidimetric assay for ferritin have been reported because the antibodies used in the immunoassay are derived from rabbits. Rabbits are familiar pets known to be a risk factor for developing heterophilic (or interfering) antibodies. This report shows how the current Olympus Ferritin assay has been improved to eliminate the interference from heterophilic antibodies.