Null States Research Articles

The Prognostic Importance of Various 13q14 Deletions In CLL

AbstractAbstract 3587Recurrent chromosomal deletions centered on the chromosomal marker D13S319 (known collectively as 13q14 deletions) are present in ~50% of all CLL patients. The clinically used CLL FISH panel measures the presence of 13q14 deletions, and singular 13q14 deletions often indicate indolent CLL. Recently, substantial anatomic heterogeneity in 13q14 deletions in CLL has been recognized. This includes short deletions of ~0.8-1.0 Mb length that involve either one or both chromosomes and that do not involve RB (type I) as well as much longer deletions that extend over various distances on 13q resulting in the removal of one copy of RB (type II) and a variable number of other genes. Importantly, the clinically used FISH test cannot discriminate short from long 13q14 deletions and is unreliable in discriminating heterozygous from homozygous short deletions.To further our understanding of the anatomy and clinical importance of 13q14 deletions in CLL, we have reviewed clinical FISH data and SNP 6.0 array genomic profiling data of DNA from sorted CD19+/CD3+ cells from 256 CLL patients, of which 201 were untreated and 55 relapsed at study enrollment. The boundaries of 13q14 deletions were mapped and lesions grouped based on the exclusion (type I, short, N=72) or inclusion of RB (type II, long, N=50). Next, we estimated TTFT and OS for patient groups with either lesion type. While the TTFT was not significantly different between the 13q14 type I and II cohorts, the hazard ratio was 3.32 (1.16-9.49) and 5.93 (2.1-16.79) for short OS (type II versus type I) for the data analyzed based on diagnosis date and enrollment date, respectively. Thus, post-therapy, the presence of type II 13q14 deletions was associated with a more aggressive disease course.Subsequently, three CLL groups were analyzed for outcome; single type I 13q14 deletions, single type II 13q14 deletions or complex 13q14 deletions of types I or II combined (defined as the presence of any additional FISH lesion in addition to a 13q14 deletion). In this cohort 10/72=14% (type I) and 17/50=34% (type II) of 13q14 deletions were FISH complex. Complex FISH inclusive of 13q14 deletions was a very strong marker for aggressive CLL, with short OS (34 months; CI=15-53 months) from the date of study enrollment when compared with single 13q14 deletions of either type I or II (p<0.01). Interestingly, after exclusion of all cases with 13q14 deletions in the setting of complex FISH, singular type II 13q14 deletions were also adverse for OS (date of enrollment to date of death) when compared with singular type I deletions (HR=6.1, p=0.03).Next we sequenced all coding exons of RB in all 50 CLL cases with type II 13q14 deletions and identified 2 cases with mutations resulting in truncated Rb proteins, thus identifying Rb null states in a small fraction of CLL. Work is in progress attempting to clarify the possible prognostic impact of varying 13q14-resident miR15 and miR16 expression levels (as measured though normalized Q-PCR) on CLL outcome as well the significance of homozygous versus heterozygous type I deletions (as measured through array-based copy number estimates).In conclusion, our data suggest that CLL with 13q14 deletions in the setting of additional FISH abnormalities is aggressive. Furthermore, the identification of singular non-complex type I versus type II 13q14 deletions may be clinically important, as the latter may delineate patients with shortened survival post-therapy. Such identification could be readily accomplished if the currently used FISH tests included a probe centered on RB and ultimately could be accomplished through use of array-based genomic analyses in CLL. Disclosures:No relevant conflicts of interest to declare.

Epigenetic domains found in mouse embryonic stem cells via a hidden Markov model

BackgroundEpigenetics is an important layer of transcriptional control necessary for cell-type specific gene regulation. Recent studies have shown significant epigenetic patterns associated with developmental stages and diseases. However, previous studies have been mostly limited to focal epigenetic patterns, whereas methods for analyzing large-scale organizations are still lacking.ResultsWe developed a hidden Markov model (HMM) approach for detecting the types and locations of epigenetic domains from multiple histone modifications. We used this method to analyze a published ChIP-seq dataset of five histone modification marks (H3K4me2, H3K4me3, H3K27me3, H3K9me3, and H3K36me3) in mouse embryonic stem (ES) cells. We identified three types of domains, corresponding to active, non-active, and null states. In total, our three-state HMM identified 258 domains in the mouse genome containing 9.6 genes on average. These domains were validated by a number of criteria. The largest domains correspond to olfactory receptor (OR) gene clusters. Each Hox gene cluster also forms a separate epigenetic domain. We found that each type of domain is associated with distinct biological functions and structural changes during early cell differentiation.ConclusionsThe HMM approach successfully detects domains of consistent epigenetic patterns from ChIP-seq data, providing new insights into the role of epigenetics in long-range gene regulation.

MKK4 as oncogene or tumor suppressor: In cancer and senescence, the story's getting old

The MKK4 gene is selected against by inactivating mutations in a large number of different tumor types, eg, tumors of the pancreas, bile ducts, breast, colon, lungs, testes [1-3] at a remarkably consistent rate of approximately 5-10% of tumors, identifying and defining it, therefore, as a tumor-suppressor (or genome-maintenance) gene [4]. Yet, experimental evidence exists that supports a pro-oncogenic role for MKK4 [5,6]. Finegan and Tournier [6], for example, recently used an inducible murine model of MKK4 homozygous deletion to evaluate the role of MKK4 in skin tumorigenesis. They found that skin-specific MKK4-null mice were resistant to carcinogen-induced tumorigenesis. While the paper is well written and the model well designed, the fundamental premise may well be flawed, especially concerning MKK4's role in tumorigenesis, perhaps misleading the line of experimentation. There should be no question that MKK4 is tumor-suppressive, not oncogenic. MKK4 is widely selected against by tumors (its low rate of homozygous loss may be accounted for by a higher rate of heterozygous loss that could rationalize frequent 17p loss in diverse human cancers[7]) and unsurprisingly patients whose tumors have loss of MKK4 show statistically significant decrease in survival in the best controlled studies, using calibrated immunohistochemistry in large numbers of patients [8], consistent with a tumor-suppressive role. Similarly consistent with a growth-suppressive role of MKK4 are observations made regarding the relationship between MKK4 and senescence. One of the ways MKK4 may suppress tumors is by inhibiting cell proliferation during replicative senescence, a widely recognized mechanism of tumor suppression. Marasa et al. [9] recently observed that MKK4 abundance increases in senescent fibroblasts. Overexpression of MKK4 decreased proliferation and promoted a senescent phenotype in young WI-38 human diploid fibroblasts and conversely, when MKK4 levels were lowered by several microRNAs targeting the MKK4 mRNA, the senescent phenotype was ameliorated and cells proliferated more rapidly [9]. In keeping with these observations, human tissue from older individuals was observed to express higher levels of MKK4 than corresponding tissue from young donors [9]. In the discussion of their inducible murine model of MKK4 homozygous deletion, Finegan and Tournier [6] rightly point out that there is conflicting literature regarding MKK4's role in tumorigenesis. The reason for the conflicting literature is largely because MKK4 is difficult to study experimentally. Their model would not be the first homozygous deletion model to model a population or a phenomenon that was not anticipated. For example, our own studies using homozygous MKK4-null cells engineered from the human pancreas cancer cell line PL-5 [10] showed that MKK4 deletion had a detrimental phenotype in a model of liver metastasis. Indeed experimental human data have shown that when tumors experiment with MKK4-null states, they are successful in developing a growth advantage allowing them to emerge through the clonal selection process in only 10% of cancers having 17p loss. We inferred from this observation that most tumor cells do not find the MKK4-null to be advantageous and that those PL-5 knock-out cells modeled this majority of cells. One may conclude from such a line of experimentation that the cell type-specific detrimental phenotype that was modeled offered an important counterweight to the selective advantage achieved by cells experimenting with genetic null states during tumorigenesis, the resultant balance determining the low but remarkably consistent rate of observed biallelic MKK4 mutations [7,10]. Because changes effected to this gene may have advantageous or deleterious effects on cells depending on the model, cancer investigators must rely on real tumors and not artificial models to guide experimental design and interpretation such that valuable research time, energy, and funding are not spent studying a phenomenon, viz, a “pro-oncogenic” function of MKK4, that common cancer sense should tell us does not likely exist. To put it colloquially, it does not matter how scientists vote regarding a pro-oncogenic or tumor-suppressive role of MKK4, because the tumors have already conducted that election in favor of the latter: MKK4 must be a tumor suppressor, as concluded from observations on wide varieties of examined tumors, which uniformly present evidence of having selected for its loss at a consistent rate. There is no convincing evidence from observations of tumor biology that MKK4 has any pro-oncogenic role. Scientists may argue for such a role, but the tumors have had their say, age-old dictators that they are.

Supersymmetric extension of GCA in 2d

We derive the infinite dimensional Supersymmetric Galilean Conformal Algebra (SGCA) in the case of two spacetime dimensions by performing group contraction on 2d superconformal algebra. We also obtain the representations of the generators in terms of superspace coordinates. Here we find realisations of the SGCA by considering scaling limits of certain 2d SCFTs which are non-unitary and have their left and right central charges become large in magnitude and opposite in sign. We focus on the Neveu-Schwarz sector of the parent SCFTs and develop, in parallel to the GCA studies recently in (arXiv:0912.1090), the representation theory based on SGCA primaries, Ward identities for their correlation functions and their descendants which are null states.

NF1 Inactivation in Adult Acute Myelogenous Leukemia

This study was conducted to identify novel genes with importance to the biology of adult acute myelogenous leukemia (AML). We analyzed DNA from highly purified AML blasts and paired buccal cells from 95 patients for recurrent genomic microdeletions using ultra-high density Affymetrix single nucleotide polymorphism 6.0 array-based genomic profiling. Through fine mapping of microdeletions on 17q, we derived a minimal deleted region of approximately 0.9-Mb length that harbors 11 known genes; this region includes Neurofibromin 1 (NF1). Sequence analysis of all NF1 coding exons in the 11 AML cases with NF1 copy number changes identified acquired truncating frameshift mutations in two patients. These NF1 mutations were already present in the hematopoetic stem cell compartment. Subsequent expression analysis of NF1 mRNA in the entire AML cohort using fluorescence-activated cell sorting sorted blasts as a source of RNA identified six patients (one with a NF1 mutation) with absent NF1 expression. The NF1 null states were associated with increased Ras-bound GTP, and short hairpin RNA-mediated NF1 suppression in primary AML blasts with wild-type NF1 facilitated colony formation in methylcellulose. Primary AML blasts without functional NF1, unlike blasts with functional NF1, displayed sensitivity to rapamycin-induced apoptosis, thus identifying a dependence on mammalian target of rapamycin (mTOR) signaling for survival. Finally, colony formation in methylcellulose ex vivo of NF1 null CD34+/CD38- cells sorted from AML bone marrow samples was inhibited by low-dose rapamycin. NF1 null states are present in 7 of 95 (7%) of adult AML and delineate a disease subset that could be preferentially targeted by Ras or mammalian target of rapamycin-directed therapeutics.

N=2gauge theories and degenerate fields of Toda theory

We discuss the correspondence between degenerate fields of the ${\mathcal{W}}_{N}$ algebra and punctures of Gaiotto's description of the Seiberg-Witten curve of $\mathcal{N}=2$ superconformal gauge theories. Namely, we find that the type of degenerate fields of the ${\mathcal{W}}_{N}$ algebra, with null states at level one, is classified by Young diagrams with $N$ boxes, and that the singular behavior of the Seiberg-Witten curve near the puncture agrees with that of ${\mathcal{W}}_{N}$ generators. We also find how to translate mass parameters of the gauge theory to the momenta of the Toda theory.

Genome-Wide Analysis of Microdeletions and Identification of NF1 Inactivation in Acute Myelogenous Leukemia.

Abstract 165Genomic aberrations are of dominant importance to the biology and clinical outcome of patients with acute myelogenous leukemia (AML). To further our understanding of such aberrations in AML, we analyzed DNA from highly purified AML blasts and paired buccal cells from 95 patients for subchromosomal copy number changes and allele identities using ultra-high-density Affymetrix SNP 6.0 array-based genomic profiling. A total of 358 somatically acquired copy number changes were detected in 95 AML genomes. We detected 16 losses and 22 gains of entire chromosomes, 285 subchromosomal losses and 35 subchromosomal gains. No recurrent high-level amplifications or recurrent homozygous deletions were identified. Eight of the 34 AML cases (24%) with normal karyotype each had one lesion detected through 6.0 array profiling, all but one of which was less than 4Mb in length.Focusing on microdeletions as potential indicators of the locations of novel tumor suppressor genes or genes with importance to AML biology, we identified 60 deletions that were less than 1 Mb in length and 158 deletions of less than 5 Mb, the vast majority of which were undetectable by conventional cytogenetics.Through fine mapping of microdeletions on 17q, we identified Neurofibromin 1 (NF1) null states due to mutations or absent expression in ∼7% of AML. NF1 mutations were present in the hematopoetic stem cell compartment (CD34+/CD38- cell population) and siRNA-mediated NF1 suppression using recombinant lentiviruses significantly increased colony formation of primary AML blasts in methylcellulose. Further, AML blasts without functional NF1 displayed sensitivity to rapamycin-induced apoptosis, thus identifying a dependence on mTOR signaling for survival.As an additional validation of using microdeletions to guide pathogenetic gene discovery, we identified deletions involving RUNX1, IRF8, Core Binding Factor Beta (CBFB) and Casitas B-cell lymphoma B (CBLB), genes known to be altered in AML. IRF8 expression was found to be absent in ∼30% of all AML but sequencing of all coding exons of IRF8 of 48 AML cases did not disclose somatically acquired mutations.In summary, this comprehensive description of subchromosomal copy number changes and microdeletions in adult AML substantially adds to our knowledge of the pathological anatomy of the AML genome and should inform future searches for novel genes with importance to AML biology. Disclosures:Malek:Cephalon: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Affymetrix: Research Funding. Erba:Lilly: Research Funding; Antisoma: Research Funding; Wyeth: Research Funding; Cephalon: Honoraria, Research Funding; MGI Pharma: Honoraria; Pharmion: Honoraria; Celgene: Honoraria; BMS: Honoraria; Novartis: Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding; Gemin-X: Research Funding; Kanisa: Research Funding.

Testing for the Supremacy of a Multinomial Cell Probability

Tests for the supremacy of a multinomial cell probability are developed. The tested null hypothesis states that a particular cell of interest is not more probable than all others. Rejection of this null leads to the conclusion that the cell of interest has a strictly greater probability than all other cells. The null hypothesis constrains the multinomial probability vector to a nonconvex region that is a union of closed convex cones. The likelihood ratio test for this problem is derived and shown to be equivalent to an intersection–union test. The least favorable configuration of the multinomial probability vector in the null parameter space is derived, and the limiting null distribution of the test statistic that is stochastically greatest is shown to be a mixture of point mass at zero and a chi-square distribution with a single degree of freedom. Asymptotic and valid finite-sample testing procedures are proposed and examined via a simulation study and the analysis of two datasets. The proposed procedures are extended to test whether the cell with the largest observed frequency is uniquely most probable. An equivalence between a likelihood ratio test for this problem and a union–intersection test is demonstrated.

Twist operator correlation functions in O(n) loop models

Using conformal field theoretic methods we calculate correlation functions of geometric observables in the loop representation of the O(n) model at the critical point. We focus on correlation functions containing twist operators, combining these with anchored loops, boundaries with SLE processes and with double SLE processes. We focus further upon n = 0, representing self-avoiding loops, which corresponds to a logarithmic conformal field theory (LCFT) with c = 0. In this limit the twist operator plays the role of a 0-weight indicator operator, which we verify by comparison with known examples. Using the additional conditions imposed by the twist operator null states, we derive a new explicit result for the probabilities that an SLE8/3 winds in various ways about two points in the upper half-plane, e.g. that the SLE passes to the left of both points. The collection of c = 0 logarithmic CFT operators that we use deriving the winding probabilities is novel, highlighting a potential incompatibility caused by the presence of two distinct logarithmic partners to the stress tensor within the theory. We argue that both partners do appear in the theory, one in the bulk and one on the boundary and that the incompatibility is resolved by restrictive bulk–boundary fusion rules.

Hypothesis Testing

Hypothesis Testing

Methods for Equivalence and Noninferiority Testing

Classical hypothesis testing focuses on testing whether treatments have differential effects on outcome. However, sometimes clinicians may be more interested in determining whether treatments are equivalent or whether one has noninferior outcomes. We review the hypotheses for these noninferiority and equivalence research questions, consider power and sample size issues, and discuss how to perform such a test for both binary and survival outcomes. The methods are illustrated on 2 recent studies in hematopoietic cell transplantation.

A syngeneic variance library for functional annotation of human variation: application to BRCA2.

The enormous scope of natural human genetic variation is now becoming defined. To accurately annotate these variants, and to identify those with clinical importance, is often difficult to assess through functional assays. We explored systematic annotation by using homologous recombination to modify a native gene in hemizygous (wt/Deltaexon) human cancer cells, generating a novel syngeneic variance library (SyVaL). We created a SyVaL of BRCA2 variants: nondeleterious, proposed deleterious, deleterious, and of uncertain significance. We found that the null states BRCA2(Deltaex11/Deltaex11) and BRCA2(Deltaex11/Y3308X) were deleterious as assessed by a loss of RAD51 focus formation on genotoxic damage and by acquisition of toxic hypersensitivity to mitomycin C and etoposide, whereas BRCA2(Deltaex11/Y3308Y), BRCA2(Deltaex11/P3292L), and BRCA2(Deltaex11/P3280H) had wild-type function. A proposed phosphorylation site at codon 3291 affecting function was confirmed by substitution of an acidic residue (glutamate, BRCA2(Deltaex11/S3291E)) for the native serine, but in contrast to a prior report, phosphorylation was dispensable (alanine, BRCA2(Deltaex11/S3291A)) for BRCA2-governed cellular phenotypes. These results show that SyVaLs offer a means to comprehensively annotate gene function, facilitating numerical and unambiguous readouts. SyVaLs may be especially useful for genes in which functional assays using exogenous expression are toxic or otherwise unreliable. They also offer a stable, distributable cellular resource for further research.

Tri-State Current Source Inverter WithImproved Dynamic Performance

Traditional dc-ac current source inverter (CSI) has a right-half-plane (RHP) zero in its control-to-output transfer function. This RHP zero causes the inverter output to fall before rising when a step increase in command reference is required (commonly known as non-minimum-phase effect). To achieve a better dynamic response, this paper proposes the design of a tri-state CSI using only an additional semiconductor switch for introducing unique freewheeling states to the traditional six active and three null states of a CSI. With the freewheeling states inserted appropriately within the inverter state sequence, the inductive boosting and discharging intervals can be decoupled, allowing the RHP zero to be eliminated with only minor circuit modifications (high level control schemes like predictive and multiloop voltage/current control remain unchanged). The designed inverter can be controlled using an appropriately formulated digital pulsewidth modulation algorithm, which can conveniently be implemented using a digital signal processor with an on-chip carrier-based modulator and an external digital programmable logic device. The resulting inverter is tested by simulation and experimentally using a laboratory prototype for demonstrating its improved dynamic performance with no commutation difficulties introduced.

Comparison of Minimum Detectable Concentration with the IUPAC Detection Limit

HEMICAL MEASUREMENTS may involve material containing very small amounts of the component of interest. With regard of measurement uncertainty it is often difficult to distinguish such small amounts from zero. Therefore an important performance characteristic of a measurement process is its detection capability, which is usually expressed as the smallest concentration of analyte, represented by the analysed component of the sample that can be reliably distinguished from zero. The decision whether the analyte is or is not present in the laboratory sample is based on the measurement data. The detection decision is provided by a choice between two opposing statistical hypotheses about the sample. The null hypothesis H0 states: “The sample is analytefree”. The alternative hypothesis H1 states: “The sample is not analyte-free”, which is consistent with the statement: “The sample contains a positive amount of the analyte”. Two possible types of decision errors exist in any

컨조인트 모형의 속성 유의성을 검증하기 위한 새로운 비모수통계 검증법

A new chi-square test is proposed to assess significance of attributes for nonparametric conjoint models. The key idea is to form subsets of rankings and test the dependence between the attribute levels and the sets of rankings. The null hypothesis states that the rankings for profiles with the focal attribute are distributed randomly among the sets of rankings. The approach is simple, easy to use, and can be applied at the individual level as well as at the aggregate level. It can be used for the trade-off approach as well as for the full profile approach.

Generalized PWM&amp;ndash;VSI Control Algorithm Based on a Universal Duty-Cycle Expression: Theoretical Analysis, Simulation Results, and Experimental Validations

This paper presents a new approach in realizing various carrier-based pulsewidth-modulation techniques by a generalized control algorithm, which is referred to as the universal control algorithm and is obtained via unequal sharing of null states. The flexibility of such an approach allows one to easily and quickly control two-level inverters. Furthermore, this approach may be also extended with few changes to the control of multilevel inverters. The algorithm that is presented here for two-level voltage-source inverters (VSIs) also obtains efficient detection and management of both the linear and overmodulation ranges. In the overmodulation range, which is treated by using the alpha-beta components of the reference-voltage space vector, the algorithm shows the advantage of lower calculation time, thus allowing one, if required, to increase the switching frequency. Several simulation runs have been performed, aiming to test the proposed procedure for both two-level and multilevel VSIs. Finally, the new algorithm was experimentally validated in the case of two-level inverters by using it in a VSI workbench that can carry out several experimental tests

Mdm2 is critically and continuously required to suppress lethal p53 activity in vivo

There is currently much interest in the idea of restoring p53 activity in tumor cells by inhibiting Hdm2/Mdm2. However, it has remained unclear whether this would also activate p53 in normal cells. Using a switchable endogenous p53 mouse model, which allows rapid and reversible toggling of p53 status between wild-type and null states, we show that p53 is spontaneously active in all tested tissues of mdm2-deficient mice, triggering fatal pathologies that include ablation of classically radiosensitive tissues. In apoptosis-resistant tissues, spontaneous unbuffered p53 activity triggers profound inhibition of cell proliferation. Such acute spontaneous p53 activity occurs in the absence of any detectable p53 posttranslational modification, DNA damage, or p19ARF signaling and triggers rapid p53 degradation.

A multivariate empirical Bayes statistic for replicated microarray time course data

In this paper we derive one- and two-sample multivariate empirical Bayes statistics (the $\mathit{MB}$-statistics) to rank genes in order of interest from longitudinal replicated developmental microarray time course experiments. We first use conjugate priors to develop our one-sample multivariate empirical Bayes framework for the null hypothesis that the expected temporal profile stays at 0. This leads to our one-sample $\mathit{MB}$-statistic and a one-sample $\widetilde{T}{}^2$-statistic, a variant of the one-sample Hotelling $T^2$-statistic. Both the $\mathit{MB}$-statistic and $\widetilde{T}^2$-statistic can be used to rank genes in the order of evidence of nonzero mean, incorporating the correlation structure across time points, moderation and replication. We also derive the corresponding $\mathit{MB}$-statistics and $\widetilde{T}^2$-statistics for the one-sample problem where the null hypothesis states that the expected temporal profile is constant, and for the two-sample problem where the null hypothesis is that two expected temporal profiles are the same.

Correlation functions of twist operators applied to single self-avoiding loops

The O(n) spin model in two dimensions may equivalently be formulated as a loop model, and then mapped to a height model which is conjectured to flow under the renormalization group to a conformal field theory (CFT). At the critical point, the order n terms in the partition function and correlation functions describe single self-avoiding loops. We investigate the ensemble of these self-avoiding loops using twist operators, which count loops which wind non-trivially around them with a factor −1. These turn out to have level 2 null states and hence their correlators satisfy a set of partial differential equations. We show that partly connected parts of the four-point function count the expected number of loops which separate one pair of points from the other pair, and find an explicit expression for this. We argue that the differential equation satisfied by these expectation values should have an interpretation in terms of a stochastic(Schramm)–Loewner evolution (SLEκ) process with κ = 6. The two-point function in a simply connected domain satisfies a closely related set of equations. We solve these and hence calculate the expected number of single loops which separate both points from the boundary.

The O(n) Model on the Annulus

We use Coulomb gas methods to derive an explicit form for the scaling limit of the partition function of the critical O(n) model on an annulus, with free boundary conditions, as a function of its modulus. This correctly takes into account the magnetic charge asymmetry and the decoupling of the null states. It agrees with an earlier conjecture based on Bethe ansatz and quantum group symmetry, and with all known results for special values of n. It gives new formulae for percolation (the probability that a cluster connects the two opposite boundaries) and for self-avoiding loops (the partition function for a single loop wrapping non-trivially around the annulus.) The limit n→0 also gives explicit examples of partition functions in logarithmic conformal field theory.