ERIC recommendations for TP53 mutation analysis in chronic lymphocytic leukemia-update on methodological approaches and results interpretation.

In chronic lymphocytic leukemia (CLL), TP53 gene defects, due to deletion of the 17p13 locus and/or mutation(s) within the TP53 gene, are associated with resistance to chemoimmunotherapy and a particularly dismal clinical outcome. On these grounds, analysis of TP53 aberrations has been incorporated into routine clinical diagnostics to improve patient stratification and optimize therapeutic decisions. The predictive implications of TP53 aberrations have increasing significance in the era of novel targeted therapies, i.e., inhibitors of B-cell receptor (BcR) signaling and anti-apoptotic BCL2 family members, owing to their efficacy in patients with TP53 defects. In this report, the TP53 Network of the European Research Initiative on Chronic Lymphocytic Leukemia (ERIC) presents updated recommendations on the methodological approaches for TP53 mutation analysis. Moreover, it provides guidance to ensure that the analysis is performed in a timely manner for all patients requiring treatment and that the data is interpreted and reported in a consistent, standardized, and accurate way. Since next-generation sequencing technologies are gaining prominence within diagnostic laboratories, this report also offers advice and recommendations for the interpretation of TP53 mutation data generated by this methodology.

EGR2 mutations define a new clinically aggressive subgroup of chronic lymphocytic leukemia.

Recurrent mutations within EGR2 were recently reported in advanced-stage chronic lymphocytic leukemia (CLL) patients and associated with a worse outcome. To study their prognostic impact, 2403 CLL patients were examined for mutations in the EGR2 hotspot region including a screening (n=1283) and two validation cohorts (UK CLL4 trial patients, n=366; CLL Research Consortium (CRC) patients, n=490). Targeted deep-sequencing of 27 known/postulated CLL driver genes was also performed in 38 EGR2-mutated patients to assess concurrent mutations. EGR2 mutations were detected in 91/2403 (3.8%) investigated cases, and associated with younger age at diagnosis, advanced clinical stage, high CD38 expression and unmutated IGHV genes. EGR2-mutated patients frequently carried ATM lesions (42%), TP53 aberrations (18%) and NOTCH1/FBXW7 mutations (16%). EGR2 mutations independently predicted shorter time-to-first-treatment (TTFT) and overall survival (OS) in the screening cohort; they were confirmed associated with reduced TTFT and OS in the CRC cohort and independently predicted short OS from randomization in the UK CLL4 cohort. A particularly dismal outcome was observed among EGR2-mutated patients who also carried TP53 aberrations. In summary, EGR2 mutations were independently associated with an unfavorable prognosis, comparable to CLL patients carrying TP53 aberrations, suggesting that EGR2-mutated patients represent a new patient subgroup with very poor outcome.

Recurrent mutations refine prognosis in chronic lymphocytic leukemia.

Through the European Research Initiative on chronic lymphocytic leukemia (CLL) (ERIC), we screened 3490 patients with CLL for mutations within the NOTCH1 (n=3334), SF3B1 (n=2322), TP53 (n=2309), MYD88 (n=1080) and BIRC3 (n=919) genes, mainly at diagnosis (75%) and before treatment (>90%). BIRC3 mutations (2.5%) were associated with unmutated IGHV genes (U-CLL), del(11q) and trisomy 12, whereas MYD88 mutations (2.2%) were exclusively found among M-CLL. NOTCH1, SF3B1 and TP53 exhibited variable frequencies and were mostly enriched within clinically aggressive cases. Interestingly, as the timespan between diagnosis and mutational screening increased, so too did the incidence of SF3B1 mutations; no such increase was observed for NOTCH1 mutations. Regarding the clinical impact, NOTCH1 mutations, SF3B1 mutations and TP53 aberrations (deletion/mutation, TP53ab) correlated with shorter time-to-first-treatment (P<0.0001) in 889 treatment-naive Binet stage A cases. In multivariate analysis (n=774), SF3B1 mutations and TP53ab along with del(11q) and U-CLL, but not NOTCH1 mutations, retained independent significance. Importantly, TP53ab and SF3B1 mutations had an adverse impact even in U-CLL. In conclusion, we support the clinical relevance of novel recurrent mutations in CLL, highlighting the adverse impact of SF3B1 and TP53 mutations, even independent of IGHV mutational status, thus underscoring the need for urgent standardization/harmonization of the detection methods.

Distinct patterns of novel gene mutations in poor-prognostic stereotyped subsets of chronic lymphocytic leukemia: the case of SF3B1 and subset #2.

Recent studies have revealed recurrent mutations of the NOTCH1, SF3B1 and BIRC3 genes in chronic lymphocytic leukemia (CLL), especially among aggressive, chemorefractory cases. Nevertheless, it is currently unknown whether their presence may differ in subsets of patients carrying stereotyped B-cell receptors and also exhibiting distinct prognoses. Here, we analyzed the mutation status of NOTCH1, SF3B1 and BIRC3 in three subsets with particularly poor prognosis, that is, subset #1, #2 and #8, aiming to explore links between genetic aberrations and immune signaling. A remarkably higher frequency of SF3B1 mutations was revealed in subset #2 (44%) versus subset #1 and #8 (4.6% and 0%, respectively; P<0.001). In contrast, the frequency of NOTCH1 mutations in subset #2 was only 8%, lower than the frequency observed in either subset #1 or #8 (19% and 14%, respectively; P=0.04 for subset #1 versus #2). No associations were found for BIRC3 mutations that overall were rare. The apparent non-random association of certain mutations with stereotyped CLL subsets alludes to subset-biased acquisition of genomic aberrations, perhaps consistent with particular antigen/antibody interactions. These novel findings assist in unraveling specific mechanisms underlying clinical aggressiveness in poor-prognostic stereotyped subsets, with far-reaching implications for understanding their clonal evolution and implementing biologically oriented therapy.

Intraclonal diversification of immunoglobulin light chains in a subset of chronic lymphocytic leukemia alludes to antigen-driven clonal evolution.

The study of intraclonal diversification (ID) in immunoglobulin (IG) genes offers valuable insight into the role of ongoing interactions with antigen in lymphomagenesis. We recently showed that ID in the IG heavy chain genes of patients with chronic lymphocytic leukemia (CLL) was generally limited; however, intense ID was evident in selected cases, especially those expressing stereotyped IGHV4-34 rearrangements and assigned to subset 4. Here, we report results from a large-scale subcloning study of IG light variable genes, in a total of 1008 subcloned sequences from 56 CLL cases. Multiple analogies were noted between heavy and light chains regarding the occurrence and molecular features of ID. More specifically, the impact of ID on the clonotypic light chains was generally low, with the significant exception of subset 4. Similar to the IGHV4-34 heavy chains of this subset, their partner IGKV2-30 light chains were affected by an active and precisely targeted ID process. Altogether, these findings strengthen the argument that stereotypy in subset 4 extends to stereotyped ID patterns for both heavy and light chains through persistent antigenic stimulation. Furthermore, they strongly suggest that light chains have an active role in the antigen selection process, at least for certain subsets of CLL cases.

Incorporating Molecular Identification of Meloidogyne spp. into a Large-scale Regional Nematode Survey.

A regional nematode survey of potato fields was conducted in the central United States during 2002 and 2003. The survey encompassed seven states and included a morphological and molecular examination of nematodes of regulatory concern from 1,929 soil samples. No regulated pest species were recovered during this survey. Meloidogyne juveniles extracted from soil were identified by mitochondrial and 18S ribosomal molecular markers. Eighty-two DNA sequences representing the two marker regions for Meloidogyne species were submitted to GenBank to facilitate evaluation of marker variability. Sufficient 18S variation was observed among some Meloidogyne species to aid in identification; however, nucleotide sequence from this highly conserved region of 18S did not discriminate among M. arenaria, M. incognita, and M. javanica. The mitochondrial gene region provided greater species discrimination and revealed intraspecific variation among many isolates. One nucleotide substitution found in a subset of M. hapla isolates from west Texas and New Mexico affected a DraI restriction site used in the PCR/RFLP diagnostic protocol. None of the mitochondrial sequence variants observed in this study compromised the PCR/RFLP identification protocol for M. chitwoodi. Additional sequence analysis is recommended for validation and evaluation of genetic markers used in diagnostic decisions.

Sensitivity of distortion-product otoacoustic emissions in humans to tonal over-exposure: time course of recovery and effects of lowering L2.

An important concern of industrial hearing-conservation programs is detecting the onset of noise-induced hearing loss. If it can be shown that otoacoustic emissions are sufficiently sensitive to reliably detect auditory fatigue and the permanent hearing loss that eventually develops, they could become an important part of the hearing-conservation test battery. The present study in humans was designed to examine the influence of overall primary-tone level and the effects of lowering the f2 primary on the sensitivity of distortion-product otoacoustic emissions (DPOAEs) to acoustic overstimulation. One ear from each of 14 subjects with normal hearing was exposed to a 105-dB SPL pure tone at 2.8 kHz for 3 min using a protocol consisting of distinct pre-exposure, exposure, and post-exposure periods. As a quantitative index of the functional status of the outer hair cells, 2f1-f2 DPOAEs were monitored systematically over time using four stimulus-test conditions consisting of either one of two levels of equilevel primary tones, or one of two levels of offset primaries, with L2 set 25 dB lower than L1. The overall finding was that the DPOAE protocol incorporating both the lowest level of stimulation and an f2-primary tone that was 25 dB below the level of the f1 stimulus [i.e., L1 (55 dB SPL) - L2 (30 dB SPL) = 25 dB] was most sensitive to the exposure effects. The results establish that DPOAEs elicited with unequal, in contrast to equal-level primaries, have comparable signal-to-noise ratios, but are considerably more sensitive to reductions in emission levels induced by exposure to short-lasting, moderately intense tones. The recovery of DPOAE amplitudes over the first 15 min post-exposure appeared to be roughly linear in log time and, in many cases, could be closely approximated by fitting a logarithmic curve to the post-exposure data. From these functions, the initial amount of loss (y-intercept) and the slope of recovery were identified as potential measures of vulnerability to acoustic exposure in that these variables appeared to be related to the susceptibility of some of the subjects, who also participated in a subsequent experiment on the behavioral effects of the exposure stimulus. Finally, compared to behaviorally measured temporary threshold shift (TTS), the time course of the recovery for DPOAEs was very similar, suggesting that, with the appropriate parameters, DPOAEs can be as sensitive to TTS as routine pure-tone audiometry.

Developmental and neural regulation of a subsarcolemmal component of the rat neuromuscular junction.

We have generated a monoclonal antibody, designated mAb 3G2, which reacts with a subsarcolemmal component of the neuromuscular junction in adult rats. mAb 3G2 immunoreactivity lies beneath and between the ACh receptor-rich synaptic gutters, around the sole plate nuclei, and at/near sarcomeric Z-disks in the vicinity of the synapse. Localization of mAb 3G2 immunoreactivity to neuromuscular junctions begins postnatally and gradually increases to adult levels. The establishment of this synaptic localization is neurally regulated, as neonatal denervation prevents its occurrence. In adults, denervation results in a loss of synaptic immunoreactivity that returns upon reinnervation. The antigen is also found at the myotendinous junction; its localization here is innervation independent. mAb 3G2 recognizes a 41 kDa protein on immunoblots of extracts of newborn muscle. Based on its distribution within muscle fibers, its developmental and neural regulation, and its molecular weight, the protein recognized by mAb 3G2 can be distinguished from other known postsynaptic proteins. Its neural dependence and developmental regulation suggest that it may participate in synaptic stabilization, perhaps as the intracellular component in a chain of proteins that serve to tether the nerve terminal to the perijunctional region of the muscle fiber.

Selective innervation of types of fibres in developing rat muscle.

The technique of glycogen depletion has been used to identify the types of muscle fibres innervated by individual motor neurones in the neonatal rat. This analysis shows that neonatal motor units are highly biased in their fibre type composition, even at times when the fibres receive extensive polyneuronal innervation. This finding suggests that the innervation of muscle fibres is somehow sorted according to type during early development. This sorting does not appear to occur during the removal of the polyneuronal innervation because little, if any, increase in the bias of unit compositions occurs as the number of synapses present in the muscle is reduced 2- to 3-fold. To determine whether the sorted innervation might be explained by a selective synaptogenesis, a study was made of the type compositions of units formed by reinnervation of neonatal soleus muscle. Glycogen depletion of single units 2 weeks following crush of the soleus nerve at postnatal day 2 showed that most of them (10/12) had biased type compositions which could not be explained by a random reinnervation. The location of fibres in the reinnervated motor units suggests that the regenerating axons innervated a novel set of fibres. The differentiation of fibres into types was apparently not changed during their reinnervation. These results imply that regenerating motor neurones in the neonatal rat selectively reinnervate muscle fibre types. These and other studies further imply that the organization of fibres into motor units during normal development does not occur, as is widely believed, by a random innervation of naive fibres and their subsequent differentiation under the influence of innervation.

Fibre type composition of single motor units during synapse elimination in neonatal rat soleus muscle.

Skeletal motor neurones innervate the specialized 'types' of fibres comprising most mammalian muscles in a characteristic fashion: each motor neurone forms a 'motor unit' by innervating a set of fibres all of the same type. Because the type expression of adult muscle fibres is plastic and apparently controlled by their innervation, each motor neurone is thought to impose a common type differentiation on all the fibres in its motor unit. However, the situation in developing muscles cannot be this simple. Muscle fibres in neonates receive synaptic input from several motor neurones and achieve the adult, single innervation only after a period of 'synapse elimination. Despite this polyneuronal innervation, differentiated fibre types are present in neonatal muscles. This means either that the motor neurones polyneuronally innervate fibres in a random fashion and type expression is not determined by innervation or that the polyneuronal innervation is ordered in such a way that each fibre could receive unambiguous instructions for type differentiation. We have investigated these possibilities here by determining the fibre type composition of motor units in neonatal rat soleus muscle. We find that even during the time of polyneuronal innervation each motor neurone confines its innervation to largely one of two fibre types present in the muscle. Therefore, some mechanism during early development segregates the synapses of two groups of soleus motor neurones onto two separate populations of soleus muscle fibres.