Deletion Rate Research Articles

Abstract LB516: MDM2/MDM4 amplification and CDKN2A deletion in melanoma brain metastases and GBM may have implications for targeted therapeutics and immunotherapy

Abstract Melanoma has a five-year survival rate of 27% for distant metastatic disease, and there remains a paucity of targeted therapies for metastatic disease and biomarkers that predict metastasis to specific sites. We analyzed 1081 primary melanoma samples and 358 metastatic melanoma samples and found that metastatic disease is enriched for amplifications in both MDM2 and MDM4 compared to primary disease, and these amplifications are associated with a lower probability of overall survival. Two additional negative regulators of TP53, namely USP7 and PPM1D, are enriched for alterations in metastatic melanoma compared to primary melanoma. MDM4 amplifications are associated with a higher rate of metastasis to the brain, liver, and lungs, while MDM2 amplifications are associated with a higher rate of metastasis to the brain, liver, and adrenal glands. These findings suggest that patients with metastatic melanoma show an enhanced dysregulation of the TP53 pathway compared to primary disease; though still under ongoing preclinical evaluation to assess therapeutic implications, we propose that patients with metastatic melanoma and TP53 wild-type status may be more likely to benefit from MDM2, MDM4, USP7, and PPM1D inhibitors, both alone and in combination, compared to those with primary disease. Additionally, we found that patients with MDM2 alterations were more likely to have a deep deletion in CDKN2A, alterations that are also associated with a higher rate of metastasis to the brain. We found that patients with a CDKN2A deep deletion had a statistically significant higher rate of alterations in TTN, MUC16, LRP1B, NF1, and SERPINB4, alterations that have all been previously associated with a favorable response to immune checkpoint inhibitors in melanoma. We therefore propose that CDKN2A deletion may serve as a biomarker to predict response to immunotherapy in melanoma. Moreover, given prior documented cases of patients diagnosed with both melanoma and glioblastoma multiforme (GBM), we found that GBM displays the highest rate of deep deletions in CDKN2A (54.39%) across all cancer types screened. We analyzed 619 GBM samples and found that 9.16% display an MDM2 amplification and 9.52% display an MDM4 amplification. Given the genomic similarities between melanoma and glioblastoma, we suggest that patients with melanoma or GBM and amplifications in MDM2/4 and CDKN2A deletions may need the development of combinations of targeted inhibitors of MDM2/4, CDK’s and immunotherapy. We are currently pursuing these translational directions. Citation Format: Taylor E. Arnoff, Wafik S. El-Deiry. MDM2/MDM4 amplification and CDKN2A deletion in melanoma brain metastases and GBM may have implications for targeted therapeutics and immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB516.

Tỷ lệ vô tinh, thiểu tinh nhẹ thiểu tinh nặng và nồng độ hormone sinh sản ở bệnh nhân vô sinh có vi mất đoạn AZF tại Bệnh viện Nam học Hiếm muộn Hà Nội

Mục tiêu: Khảo sát một số đặc điểm tinh dịch đồ và một số chỉ số nội tiết sinh sản ở nam giới vô sinh có mất đoạn AZF. Đối tượng và phương pháp: Nghiên cứu hồi cứu, mô tả, cắt ngang trên 166 nam giới Việt Nam có vi mất đoạn AZF trên các bệnh nhân (BN) thiểu tinh hoặc vô tinh tại Bệnh viện Nam học và Hiếm muộn Hà Nội từ 12/2016 – 6/2021. Kết quả: Mất đoạn AZFc chiếm tỷ lệ cao nhất trong mất đoạn AZF (51,81%). Mất đoạn AZF phối hợp chiếm 42,77%. Không có BN nào mất đoạn AZFb đơn độc trong nghiên cứu này. Tỷ lệ mất đoạn AZFa là 1,81%. 3/3 BN vi mất đoạn AZFa đơn độc đều vô tinh. Mất đoạn AZFc có 27% vô tinh, 58% thiểu tinh nặng và 13% thiểu tinh. 100% vi mất đoạn phối hợp nếu chứa AZFa vô tinh. Tỷ lệ vô tinh ở mất đoạn AZFbc là 71,42%, ở mất đoạn AZFbcd là 52%. Vi mất đoạn AZFa có nồng độ FSH cao (36,67 ± 12,70 IU/l). Kết luận: Tỷ lệ mất đoạn AZFc cao nhất (51,81%). Mất đoạn AZFa trầm trọng hơn so với các loại mất đoạn khác. Các loại mất đoạn còn lại (AZFb, c, d và phối hợp của chúng) ở mức độ có từ vô tinh tới thiểu tinh nhẹ.

Impact of Raptor and Rictor Deletion on Hippocampal Pathology Following Status Epilepticus.

Neuronal hyperactivation of the mTOR signaling pathway may play a role in driving the pathological sequelae that follow status epilepticus. Animal studies using pharmacological tools provide support for this hypothesis, however, systemic inhibition of mTOR-a growth pathway active in every mammalian cell-limits conclusions on cell type specificity. To circumvent the limitations of pharmacological approaches, we developed a viral/genetic strategy to delete Raptor or Rictor, inhibiting mTORC1 or mTORC2, respectively, from excitatory hippocampal neurons after status epilepticus in mice. Raptor or Rictor was deleted from roughly 25% of hippocampal granule cells, with variable involvement of other hippocampal neurons, after pilocarpine status epilepticus. Status epilepticus induced the expected loss of hilar neurons, sprouting of granule cell mossy fiber axons and reduced c-Fos activation. Gene deletion did not prevent these changes, although Raptor loss reduced the density of c-Fos-positive granule cells overall relative to Rictor groups. Findings demonstrate that mTOR signaling can be effectively modulated with this approach and further reveal that blocking mTOR signaling in a minority (25%) of granule cells is not sufficient to alter key measures of status epilepticus-induced pathology. The approach is suitable for producing higher deletion rates, and altering the timing of deletion, which may lead to different outcomes.

Association between sperm mitochondrial DNA copy number and deletion rate and industrial air pollution dynamics

The effects of air pollution on men’s reproductive health can be monitored by evaluating semen quality and sperm DNA damage. We used real-time PCR to analyse the effects of air pollution on sperm mitochondrial DNA copy number (mtDNAcn) and deletion (mtDNAdel) rates in semen samples collected from 54 men in two seasons with different levels of industrial and traffic air pollution. MtDNAdel rates were significantly higher following the high exposure period and were positively correlated with mtDNAcn. However, we did not find any difference in mtDNAcn between the two seasons. MtDNAcn was positively correlated with the DNA fragmentation index and the rates of sperm with chromatin condensation defects, previously assessed by sperm chromatin structure assay, and negatively correlated with sperm concentration, progressive motility, viability, and normal morphology. This indicates that mtDNAcn is more closely associated with male fertility than mtDNAdel rates. In contrast, mtDNAdel might be a more sensitive biomarker of air pollution exposure in urban industrial environments.

Development and Evaluation of AMSU-A Cloud Detection over the Tibetan Plateau

Advanced Microwave Sounding Unit-A (AMSU-A) and Microwave Humidity Sounder (MHS) data have been widely assimilated in operational forecasting systems. However, effective distinction between cloudy and clear-sky data is still an essential prerequisite for the assimilation of microwave observations. Cloud detection over the Tibetan Plateau has long been a challenge owing to the influence of low temperatures, terrain height, surface vegetation, and inaccurate background fields. Based on the variations in the response characteristics of different channels of AMSU-A to clouds, five AMSU-A window and low-peaking channels (channels 1–4 and 15) are chosen to establish a cloud detection index. Combined with the existing MHS cloud detection index, a cloud detection scheme over the Tibetan Plateau is proposed. Referring to VISSR-II (Stretched Visible and Infrared Spin Scan Radiometer-II) and CALIPSO (The Cloud-Aerosol Lidar and Infrared Pathfinder Satellite Observation) cloud classification products, the detection rate of cloudy data and the rejection rate of clear-sky data under different cloud index thresholds are evaluated. Results show that the new cloud detection scheme can identify more than 80% of cloudy data on average, but this decreases to 72% for area with terrain higher than 5 km, and the false deletion rate remains stable at 45%. The detection rates of mixed clouds and cumulonimbus are higher than 90%, but it is lower than 50% for altostratus with an altitude of about 7–8 km. Comparative analysis shows that the new method is more suitable for areas with terrain higher than 700 m. Based on the cloud detection results, the effects of terrain height on the characteristics of observation error and bias are also discussed for AMSU-A channels 5 and 6.

Polar Codes for the Deletion Channel: Weak and Strong Polarization

This paper presents the first proof of polarization for the deletion channel with a constant deletion rate and a regular hidden-Markov input distribution. A key part of this work involves representing the deletion channel using a trellis and describing the plus and minus polar-decoding operations on that trellis. In particular, the plus and minus operations can be seen as combining adjacent trellis stages to yield a new trellis with half as many stages. Using this viewpoint, we prove a weak polarization theorem for standard polar codes on the deletion channel. To achieve strong polarization, we modify this scheme by adding guard bands of repeated zeros between various parts of the codeword. This gives a scheme whose rate approaches the mutual information and whose probability of error decays exponentially in the cube-root of the block length. We conclude by showing that this scheme can achieve capacity on the deletion channel by proving that the capacity of the deletion channel can be achieved by a sequence of regular hidden-Markov input distributions.

Benchmarking the topological accuracy of bacterial phylogenomic workflows using in silico evolution.

Phylogenetic analyses are widely used in microbiological research, for example to trace the progression of bacterial outbreaks based on whole-genome sequencing data. In practice, multiple analysis steps such as de novo assembly, alignment and phylogenetic inference are combined to form phylogenetic workflows. Comprehensive benchmarking of the accuracy of complete phylogenetic workflows is lacking. To benchmark different phylogenetic workflows, we simulated bacterial evolution under a wide range of evolutionary models, varying the relative rates of substitution, insertion, deletion, gene duplication, gene loss and lateral gene transfer events. The generated datasets corresponded to a genetic diversity usually observed within bacterial species (≥95 % average nucleotide identity). We replicated each simulation three times to assess replicability. In total, we benchmarked 19 distinct phylogenetic workflows using 8 different simulated datasets. We found that recently developed k-mer alignment methods such as kSNP and ska achieve similar accuracy as reference mapping. The high accuracy of k-mer alignment methods can be explained by the large fractions of genomes these methods can align, relative to other approaches. We also found that the choice of de novo assembly algorithm influences the accuracy of phylogenetic reconstruction, with workflows employing SPAdes or skesa outperforming those employing Velvet. Finally, we found that the results of phylogenetic benchmarking are highly variable between replicates. We conclude that for phylogenomic reconstruction, k-mer alignment methods are relevant alternatives to reference mapping at the species level, especially in the absence of suitable reference genomes. We show de novo genome assembly accuracy to be an underappreciated parameter required for accurate phylogenomic reconstruction.

A deletion at the polled PC locus alone is not sufficient to cause a polled phenotype in cattle

Dehorning is a common practice in the dairy industry, but raises animal welfare concerns. A naturally occurring genetic mutation (PC allele) comprised of a 212 bp duplicated DNA sequence replacing a 10-bp sequence at the polled locus is associated with the hornless phenotype (polled) in cattle. To test the hypothesis that the 10 bp deletion alone is sufficient to result in polled, a CRISPR-Cas9 dual guide RNA approach was optimized to delete a 133 bp region including the 10 bp sequence. Timing of ribonucleoprotein complex injections at various hours post insemination (hpi) (6, 8, and 18 hpi) as well as in vitro transcribed (IVT) vs synthetic gRNAs were compared. Embryos injected 6 hpi had a significantly higher deletion rate (53%) compared to those injected 8 (12%) and 18 hpi (7%), and synthetic gRNAs had a significantly higher deletion rate (84%) compared to IVT gRNAs (53%). Embryo transfers were performed, and bovine fetuses were harvested between 3 and 5 months of gestation. All fetuses had mutations at the target site, with two of the seven having biallelic deletions, and yet they displayed horn bud development indicating that the 10 bp deletion alone is not sufficient to result in the polled phenotype.

A comparison of single imputation and multiple imputation methods for missing data in different oncogene expression profiles

To evaluate the effects of multiple-imputation (MI) method for missing data in gene expression profiles with different datasets and percentages of missing values compared with 3 single-imputation (SI) methods. Based on 3 gene expression profiles datasets from human colon cancer, non-small cell lung cancer, and lymph cancer, different deletion rates and different imputation numbers of MI were compared. The imputation and clustering effects of different methods were evaluated using the NRMSE and the gene clustering accuracy (F value). The NRMSE of the 4 methods gradually increased as the percentage of missing values in the 3 datasets increased, whereas the F value gradually decreased. In all datasets with different percentage of missing values settings, the NRMSEs of MI was consistently lower than those of the 3 SI methods, whereas the F value of MI was highest. The NRMSEs of MI gradually decreased as the number of imputations increased and increased as the variability in the original datasets increased, and the datasets imputed by MI showed the best clustering results. The results showed that the application of MI develops and enriches imputation-model approaches and provides a solid foundation for subsequent establishment of imputation strategies for gene expression profiles with missing data.

An Episomal CRISPR/Cas12a System for Mediating Efficient Gene Editing.

(1) Background: Gene editing technology, as represented by CRISPR is a powerful tool used in biomedical science. However, the editing efficiency of such technologies, especially in induced pluripotent stem cells (iPSCs) and other types of stem cells, is low which hinders its application in regenerative medicine; (2) Methods: A gene-editing system, COE, was designed and constructed based on CRISPR/Cas12a and Orip/EBNA1, and its editing efficiency was evaluated in human embryonic kidney 293T (HEK-293T) cells with flow cytometry and restriction fragment length polymorphism (RFLP) analysis. The COE was nucleofected into iPSCs, then, the editing efficiency was verified by a polymerase chain reaction and Sanger sequencing; (3) Results: With the extension of time, COE enables the generation of up to 90% insertion or deletion rates in HEK-293T cells. Furthermore, the deletion of a 2.5 kb fragment containing Exon 51 of the dystrophin gene (DMD) in iPSCs was achieved with high efficiency; out of 14 clones analyzed, 3 were positive. Additionally, the Exon 51-deleted iPSCs derived from cardiomyocytes had similar expression profiles to those of Duchenne muscular dystrophy (DMD) patient-specific iPSCs. Moreover, there was no residue of each component of the plasmid in the editing cells; (4) Conclusions: In this study, a novel, efficient, and safe gene-editing system, COE, was developed, providing a powerful tool for gene editing.

Modification of improved-genome editing via oviductal nucleic acids delivery (i-GONAD)-mediated knock-in in rats

BackgroundImproved genome-editing via oviductal nucleic acids delivery (i-GONAD) is a new technology that facilitates in situ genome-editing of mammalian zygotes exiting the oviductal lumen. The i-GONAD technology has been developed for use in mice, rats, and hamsters; however, oligonucleotide (ODN)-based knock-in (KI) is more inefficient in rats than mice. To improve the efficiency of i-GONAD in rats we examined KI efficiency using three guide RNAs (gRNA), crRNA1, crRNA2 and crRNA3. These gRNAs recognize different portions of the target locus, but also overlap each other in the target locus. We also examined the effects of commercially available KI -enhancing drugs (including SCR7, L755,507, RS-1, and HDR enhancer) on i-GONAD-mediated KI efficiency.ResultsThe KI efficiency in rat fetuses generated after i-GONAD with crRNA2 and single-stranded ODN was significantly higher (24%) than crRNA1 (5%; p < 0.05) or crRNA3 (0%; p < 0.01). The KI efficiency of i-GONAD with triple gRNAs was 11%. These findings suggest that KI efficiency largely depends on the type of gRNA used. Furthermore, the KI efficiency drugs, SCR7, L755,507 and HDR enhancer, all of which are known to enhance KI efficiency, increased KI efficiency using the i-GONAD with crRNA1 protocol. In contrast, only L755,507 (15 μM) increased KI efficiency using the i-GONAD with crRNA2 protocol. None of them were significantly different.ConclusionsWe attempted to improve the KI efficiency of i-GONAD in rats. We demonstrated that the choice of gRNA is important for determining KI efficiency and insertion and deletion rates. Some drugs (e.g. SCR7, L755,507 and HDR enhancer) that are known to increase KI efficiency in culture cells were found to be effective in i-GONAD in rats, but their effects were limited.

Soft computing technique based modelling of ceramics mix electric discharge machining on LM-25/SiC composites

Silicon carbide mixed electrical discharge machining (SCM-EDM), an unconventional machining process that utilizes an electrolyte mix additive, combines the advantage of various energies (abrasive, heat). The aim of these articles is to investigate the impact of control variables on the machining performance of SCM-EDM using LM-25/SiC MMC. The major variable of SCM-EDM is identified as current intensity, pulse duration. The experimental run has conducted as per Box- Behnken design which produces statistical data solely based on the experiment run and estimates the measurement error. Artificial neural network (ANN) technique implemented to estimate experimental inputs and modelling of measure response such as material deletion rate (MRR), tool wear (TW), and surface roughness (SR). It is observed that predicted results from ANN model are compared with experimental result are quite satisfactory. The chemical composition and analysis of variance show the percentage contribution of each parameter on machining performance.

ProPIP: a tool for progressive multiple sequence alignment with Poisson Indel Process

BackgroundCurrent alignment tools typically lack an explicit model of indel evolution, leading to artificially short inferred alignments (i.e., over-alignment) due to inconsistencies between the indel history and the phylogeny relating the input sequences.ResultsWe present a new progressive multiple sequence alignment tool ProPIP. The process of insertions and deletions is described using an explicit evolutionary model—the Poisson Indel Process or PIP. The method is based on dynamic programming and is implemented in a frequentist framework. The source code can be compiled on Linux, macOS and Microsoft Windows platforms. The algorithm is implemented in C++ as standalone program. The source code is freely available on GitHub at https://github.com/acg-team/ProPIP and is distributed under the terms of the GNU GPL v3 license.ConclusionsThe use of an explicit indel evolution model allows to avoid over-alignment, to infer gaps in a phylogenetically consistent way and to make inferences about the rates of insertions and deletions. Instead of the arbitrary gap penalties, the parameters used by ProPIP are the insertion and deletion rates, which have biological interpretation and are contextualized in a probabilistic environment. As a result, indel rate settings may be optimised in order to infer phylogenetically meaningful gap patterns.

CRISPR/Cas9-mediated genome editing directed by a 5S rRNA\u2013tRNAGly hybrid promoter in the thermophilic filamentous fungus Humicola insolens

BackgroundHumicola insolens is a filamentous fungus with high potential of producing neutral and heat- and alkali-resistant cellulase. However, the genetic engineering tools, particularly the genome-editing tool, are scarce, hindering the study of cellulase expression regulation in this organism.ResultsHerein, a CRISPR/Cas9 genome-editing system was established in H. insolens based on a hybrid 5S rRNA–tRNAGly promoter. This system is superior to the HDV (hepatitis delta virus) system in genome editing, allowing highly efficient single gene destruction in H. insolens with rates of deletion up to 84.1% (37/44). With this system, a putative pigment synthesis gene pks and the transcription factor xyr1 gene were disrupted with high efficiency. Moreover, the extracellular protein concentration and cellulase activity largely decreased when xyr1 was deleted, demonstrating for the first time that Xyr1 plays an important role in cellulase expression regulation.ConclusionsThe established CRISPR/Cas9 system is a powerful genetic operation tool for H. insolens, which will accelerate studies on the regulation mechanism of cellulase expression and engineering of H. insolens for higher cellulase production.

A Probabilistic Model for Indel Evolution: Differentiating Insertions from Deletions.

Insertions and deletions (indels) are common molecular evolutionary events. However, probabilistic models for indel evolution are under-developed due to their computational complexity. Here, we introduce several improvements to indel modeling: 1) While previous models for indel evolution assumed that the rates and length distributions of insertions and deletions are equal, here we propose a richer model that explicitly distinguishes between the two; 2) we introduce numerous summary statistics that allow approximate Bayesian computation-based parameter estimation; 3) we develop a method to correct for biases introduced by alignment programs, when inferring indel parameters from empirical data sets; and 4) using a model-selection scheme, we test whether the richer model better fits biological data compared with the simpler model. Our analyses suggest that both our inference scheme and the model-selection procedure achieve high accuracy on simulated data. We further demonstrate that our proposed richer model better fits a large number of empirical data sets and that, for the majority of these data sets, the deletion rate is higher than the insertion rate.

Insertion-deletion rate is a qualitative aspect of the tumor mutation burden associated with the clinical outcomes of gastric cancer patients treated with nivolumab.

We aimed to investigate the clinical implications of the tumor mutation burden (TMB) and insertion-deletion (indel) rate in gastric cancer patients treated with nivolumab. A total of 105 patients with advanced gastric cancer who were treated with nivolumab as third or later line of therapy were included as the study population. The indel rate was defined as the proportion of indels making up the TMB. The median age was 58 (32-78years), and 65 (61.9%) were men. Patients with TMB > 18.03/Mb showed superior progression-free survival (PFS) and overall survival (OS) compared to those with TMB ≤ 18.03/Mb. Patients with a high indel rate (> 40%) had a favorable PFS and OS compared to those with a lower indel rate (≤ 40%) (P = 0.009 and P = 0.007, respectively). The association between a high indel rate and favorable PFS and OS was prominent in a subgroup with TMB > 18.03/Mb (P < 0.001 and P = 0.007 for PFS and OS, respectively), but not in that with TMB ≤ 18.03/Mb. All five patients with deficient-MMR fell into the category of 'TMB > 18.03/Mb with an indel rate of > 40%. TMB ≥ 18.03/Mb with an indel rate of > 40% was independently associated with a favorable PFS (hazard ratio [HR] 0.07, P = 0.012) and OS (HR 0.09, P = 0.023). TMB and indel rate should be jointly considered to better predict survival outcomes of gastric cancer patients treated with nivolumab. Our findings deserve further investigation and validation in future studies.

Digital Quantification of Chemical Oligonucleotide Synthesis Errors

Chemically synthesized oligonucleotides are vital to most nucleic acids-based technologies and several applications are sensitive to oligonucleotide sequence errors. However, it is challenging to identify and quantify the types and amount of errors in synthetic oligonucleotides. We applied a digital sequencing approach using unique molecular identifiers to quantify errors in chemically synthesized oligonucleotides from multiple manufacturers with different synthesis strategies, purity grades, batches, and sequence context. We detected both deletions and substitutions in chemical oligonucleotide synthesis, but deletions were 7 times more common. We found that 97.2% of all analyzed oligonucleotide molecules were intact across all manufacturers and purity grades, although the number of oligonucleotide molecules with deletions ranged between 0.2% and 11.7% for different types. Different batches of otherwise identical oligonucleotide types also varied significantly, and batch effect can impact oligonucleotide quality more than purification. We observed a bias of increased deletion rates in chemically synthesized oligonucleotides toward the 5'-end for 1 out of 2 sequence configurations. We also demonstrated that the performance of sequencing assays depends on oligonucleotide quality. Our data demonstrate that manufacturer, synthesis strategy, purity, batch, and sequence context all contribute to errors in chemically synthesized oligonucleotides and need to be considered when choosing and evaluating oligonucleotides. High-performance oligonucleotides are essential in numerous molecular applications, including clinical diagnostics.

Mitochondrial DNA 4977 bp Deletion in Peripheral Blood Is Associated With Polycystic Ovary Syndrome.

BackgroundPolycystic ovary syndrome (PCOS) is a common endocrine disorder worldwide. We aimed to examine the associations of two mitochondrial DNA (mtDNA) biomarkers in the peripheral blood, mtDNA copy number (CN), and mtDNA4977 deletion rate (DR), with PCOS in a clinical setting.MethodsWe performed a study involving 263 women with PCOS and 326 age-matched controls between June 2015 and June 2019. The mtDNA CN and mtDNA4977 DR were measured using multiplex probe-based qPCR. The associations of the mtDNA CN and mtDNA4977 DR with the risk of PCOS were estimated using logistic regression.ResultsAnalysis of the associations between mtDNA biomarkers and PCOS indicate that the mtDNA CN (P = 0.003) and mtDNA4977 DR (P < 0.001) in PCOS patients were significantly higher than those in the controls. After adjusting for the body mass index, luteinizing hormone/follicle-stimulating hormone ratio, and testosterone level, only higher mtDNA4977 DR was associated with PCOS (odds ratio 1.053, 95% confidence interval 1.024 to 1.083; P < 0.001). The linear dose-response trends of the mtDNA4977 DR were also supported by the quartile analysis.ConclusionMultivariable models suggest that mtDNA4977 DR levels are strongly associated with PCOS and represent an independent risk factor for PCOS. Further investigation of the utility of mtDNA as a biomarker for PCOS is warranted.

B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) with t(5;14)(q31;q32)/ IL3-IGH Rearrangement and Eosinophilia: A Peculiar IGH BCP-ALL

Introduction: BCP-ALL associated with t(5;14)(q31;q32) is an exceptional cause of eosinophilia. The IL3 gene expression becomes regulated by the IGH enhancer leading to an overproduction of IL3 causing eosinophilia. Clinical, biological and outcome data are extremely scarce in the literature.Methods: We recruited 2 newly diagnosed patients (pts) with t(5;14)(q31;q32) in our centers. A retrospective search in the French protocol FRALLE 2000 data base and collaboration with the GFCH (Groupe Francophone de Cytogénétique Hématologique) added 4 other pts. Array CGH, MLPA or genomic PCR search for IKZF1 deletion were performed in these 6 pts. We further analysed the 12 pts with t(5;14)(q31;q32) described in the literature with our 6 newly described pts, leading to a meaningful series of 18 pts.Results: Main features at onset are displayed in table 1. Median age at diagnosis is 16.5 years. Male/female ratio is 4.6. Eosinophilia-related symptoms are common with neurologic, thromboembolic or lung involvement (respectively 4/13, 4/13 and 6/13 pts). No pt exhibits blast cells in CSF at diagnosis. Median WBC count is high (48x109/L), mainly due to a high rate of eosinophils (median: 30.5x109/L). Blood eosinophilia preceded ALL diagnosis from 6 and 12 months in 2 cases. Peripheral blast cells are low or undetectable (median: 0x109/L; range: 0-18.7x109/L). A high basophilia is frequent (median: 0.55x109/L). Bone marrow morphology is marked by a low blast infiltration (median: 47%) and increased eosinophil-lineage cells (median: 24%) with dysplastic features in 6/10 pts. All cases are of the B lineage. All 18 pts but one present the t(5;14)(q31;q32) translocation. For the remaining pt, it was revealed by sequencing as karyotype was normal in a context of eosinophilia. Six out of 18 pts have additional cytogenetic abnormalities. For the first time, we report an IKZF1 deletion which is present in 4 out of 6 pts. Few other deletions are found in more than one pt (PAX5, ETV6, MEF2C, CDKN2A). Remission and outcome data are available for 11 pts (our 6 newly described pts and 5 from the literature). Median follow-up (FU) at point date is 18 months (range: 4 days-30 months). Only 5 pts achieve a first complete remission (CR1) at first attempt. The 6 newly described pts are currently alive: 4 are in continuous CR1 (CCR1) including 2 after hematopoietic stem cell transplantation (HSCT) and 2 are in CCR2 (including 1 after HSCT). The 5 pts from the literature died, 1 from cardiac arrest secondary to eosinophilia and the 4 others from disease progression and / or sepsis.Discussion and Conclusion : 1) t(5;14)(q31;q32)-associated BCP-ALL has remarkable features including some which are shared with other IGH BCP-ALL, according to data from a comprehensive UK retrospective study using IGH FISH screening1. Both conditions affect preferentially teenagers and young adults (median age: 16 years1). The highly biased male to female ratio reported here is milder in other IGH BCP-ALL (1.441). In both entities a low blast count (<50 G/L in 119/158 pts1) is observed. Our new data revealed a high rate of IKZF1 deletion in 4/6 pts (66%) also observed in 35/88 pts (40%) in IGH BCP-ALL1. Differences obviously include the eosinophilia and related symptoms but also the low rate of cytopenias possibly linked to the partial bone marrow infiltration, prevailing in t(5;14)(q31;q32)-associated BCP-ALL. 2) In pediatric IGH BCP-ALL pts, 5-year overall survival is 84%1. The lower published survival rate in t(5;14)(q31;q32)-associated BCP-ALL should be balanced by a 100% survival in CR in our 6 newly described pts with obviously a limited sample size and a shorter FU. This may reflect new treatment strategies, including pediatric intensive chemotherapy and MRD-based stratification. HSCT seems to be an option for pts with suboptimal response (induction failure, high MRD) or relapse. 3) Despite being a specific WHO recognized entity, many common clinical and biological features allow to suggest that t(5;14)(q31;q32)-associated BCP-ALL may be considered as a very rare subset of IGH BCP-ALL. Nevertheless depending on its kinetics and severity, the specific eosinophilia can render difficult both diagnosis and management.1. Russell LJ, Enshaei A, Jones L, et al. IGH@ translocations are prevalent in teenagers and young adults with acute lymphoblastic leukemia and are associated with a poor outcome. J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 2014;32(14):1453-1462. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Characterization of Chromosome 6q Deletions in MYD88 Mutant and Wild-Type Waldenström's Macroglobulinemia

Introduction: MYD88 L265P mutations have been described in more than 90% of Waldenström's Macroglobulinemia (WM) patients. MYD88 wild-type (wt) WM represent less than 10% of WM, and associate with shorter overall survival versus MYD88 mutated (mut) WM, as well as with lack of major responses and inferior progression-free survival to ibrutinib. Mutated MYD88 triggers WM cells growth and survival through NF-kB activation via IRAK1/4, BTK and HCK transactivation. CXCR4 mutations promote pro-survival AKT and ERK1/2 signaling and are associated with delayed response to ibrutinib. Deletions involving the long arm of chromosome 6 (Del6q) are highly recurrent in various B-cell malignancies, particularly those that are driven by mutated MYD88, and have been observed in up to half of WM patients. Chr6q genomic loss in WM affect important modulators of NFkB (TNFAIP3, HIVEP2), BCL2 protein family (BCLAF1), apoptosis and IGF1/PI3K/AKT signaling (FOXO3), and BTK (IBTK). The frequency and impact of Del6q on the different patient genotype remains to be clarified.Patients and Methods: Based on location and known regulation of key pathways in WM, copy number alterations (CNA) and gene expression level were assessed for IBTK, FOXO3, BCLAF1, TNFAIP3 and HIVEP2 genes, along with CXCR4 transcriptional levels. DNA and RNA from CD19+ sorted BM lymphoplasmacytic cells from 32 untreated WM and one IgM and IgG-secreting lymphoplasmacytic lymphoma patients were analyzed. Paired CD19-depleted peripheral-blood mononuclear cells (PBMCs) were used as germline controls. Paired CD19+ and CD19- PBMCs from 6 healthy donors were analyzed to rule out possible B-cell specific findings. CNA were measured in quadruplicate and gene expression in triplicate using TaqMan real-time polymerase chain reaction (RT-PCR) assays. Deletions affecting less than 20% of WM cells were considered to be beneath the RT-PCR detection threshold. The cohort included 33 patients (21 males, 12 females), with a median age of 62 (range 35-91) years, BM involvement of 60% (range 2.5-90%), serum IgM levels of 3010 (range 257-6910) mg/dl, and hemoglobin of 10.9 (range 8.4-14.4) g/dl. MYD88 L265P mutations were detectable in 25 (76%) patients, 11 of whom (44%) also carried CXCR4WHIM mutations. Eight patients (24%) were MYD88 and CXCR4 wt.Results: Somatic Del6q were observed in 4/8 (50%) and in 20/25 (80%) patients in the MYD88 wt and mut population, respectively (p=NS). In contrast, no CNA were present in the HDs CD19+/CD19- compartments. IBTK remained intact in all 8 MYD88 wt WM, whereas resulted deleted in 13/25 (52%) MYD88 mut patients (p=0.01). BCLAF1 deletion frequency was significantly different among the two subgroups, with only 2/8 deleted MYD88wt (25%) and 19/25 (76%) deleted MYD88 mut patients (p=0.02; Figure 1). Eight MYD88 mut WM (32%) showed contiguous fully clonal Del6q, that spanned all studied genes in 7/8 (87.5%) cases; in contrast, contiguous subclonal deletions occurred only in 3/12 (25%) cases (p=0.02). All 8 fully clonal Del6q patients were CXCR4 wt; in contrast, CXCR4 mut were detected in 9/12 (75%) patients with subclonal Del6q (p=0.001; Figure 2). Unlike the MYD88 mut population, all 4 MYD88 wt patients presented with non-contiguous subclonal deletions (p=NS). Fully clonal deletions of IBTK, BCLAF1 and HIVEP2 significantly reduced the respective gene transcriptional levels (p= 0.03, 0.01 and 0.01, respectively), while CNA in TNFAIP3 and FOXO3 did not affect their expression. The MYD88 wt patients showed significant decreases in the transcriptional levels of BCLAF1 related to gene subclonal deletions, and reduced CXCR4 levels compared to MYD88 mut WM (p=0.02 and p=0.02, respectively).Conclusions: Our findings demonstrate that gene loss of IBTK, FOXO3, BCLAF1, TNFAIP3 and HIVEP2 occurs in most WM patients, but at a lower frequency in MYD88 wt. Unlike the MYD88 mut patients, those MYD88 wt showed no contiguous fully clonal deletions. The latter correlate with CXCR4 wt and reduce only IBTK, BCLAF1 and HIVEP2 levels, suggesting that regulatory mechanisms may compensate for FOXO3 and TNFAIP3 gene loss. Significant variation in IBTK and BCLAF1 deletion rate suggest that BCR and BCL2 signaling may play a different role in MYD88 mut and wt WM. The findings provide valuable insights into MYD88 wt WM pathogenesis, and may be relevant to understanding therapeutic outcome with agents that target MYD88, CXCR4, BTK and BCL2. [Display omitted] DisclosuresCastillo:Millennium: Research Funding; Abbvie: Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding. Arcaini:Pfizer, Celgene, Bayer, Roche: Membership on an entity's Board of Directors or advisory committees; Celgene, Roche, Sandoz: Consultancy; Gilead: Research Funding. Varettoni:Jannsen: Consultancy, Other: travel expenses. Treon:Pharmacyclics: Consultancy, Research Funding.