TRAF3 alterations are frequent in del-3'IGH chronic lymphocytic leukemia patients and define a specific subgroup with adverse clinical features.

Interstitial 14q32 deletions involving IGH gene are infrequent events in chronic lymphocytic leukemia (CLL), affecting less than 5% of patients. To date, little is known about their clinical impact and molecular underpinnings, and its mutational landscape is currently unknown. In this work, a total of 871 CLLs were tested for the IGH break-apart probe, and 54 (6.2%) had a 300 kb deletion of 3'IGH (del-3'IGH CLLs), which contributed to a shorter time to first treatment (TFT). The mutational analysis by next-generation sequencing of 317 untreated CLLs (54 del-3'IGH and 263 as the control group) showed high mutational frequencies of NOTCH1 (30%), ATM (20%), genes involved in the RAS signaling pathway (BRAF, KRAS, NRAS, and MAP2K1) (15%), and TRAF3 (13%) within del-3'IGH CLLs. Notably, the incidence of TRAF3 mutations was significantly higher in del-3'IGH CLLs than in the control group (p < .001). Copy number analysis also revealed that TRAF3 loss was highly enriched in CLLs with 14q deletion (p < .001), indicating a complete biallelic inactivation of this gene through deletion and mutation. Interestingly, the presence of mutations in the aforementioned genes negatively refined the prognosis of del-3'IGH CLLs in terms of overall survival (NOTCH1, ATM, and RAS signaling pathway genes) and TFT (TRAF3). Furthermore, TRAF3 biallelic inactivation constituted an independent risk factor for TFT in the entire CLL cohort. Altogether, our work demonstrates the distinct genetic landscape of del-3'IGH CLL with multiple molecular pathways affected, characterized by a TRAF3 biallelic inactivation that contributes to a marked poor outcome in this subgroup of patients.

Co-occurrence of cohesin complex and Ras signaling mutations during progression from myelodysplastic syndromes to secondary acute myeloid leukemia.

Myelodysplastic syndromes (MDS) are hematological disorders at high risk of progression to secondary acute myeloid leukemia (sAML). However, the mutational dynamics and clonal evolution underlying disease progression are poorly understood at present. To elucidate the mutational dynamics of pathways and genes occurring during the evolution to sAML, next generation sequencing was performed on 84 serially paired samples of MDS patients who developed sAML (discovery cohort) and 14 paired samples from MDS patients who did not progress to sAML during follow-up (control cohort). Results were validated in an independent series of 388 MDS patients (validation cohort). We used an integrative analysis to identify how mutations, alone or in combination, contribute to leukemic transformation. The study showed that MDS progression to sAML is characterized by greater genomic instability and the presence of several types of mutational dynamics, highlighting increasing (STAG2) and newly-acquired (NRAS and FLT3) mutations. Moreover, we observed cooperation between genes involved in the cohesin and Ras pathways in 15-20% of MDS patients who evolved to sAML, as well as a high proportion of newly acquired or increasing mutations in the chromatin-modifier genes in MDS patients receiving a disease-modifying therapy before their progression to sAML.

Chronic lymphocytic leukemia patients with IGH translocations are characterized by a distinct genetic landscape with prognostic implications.

Chromosome 14q32 rearrangements/translocations involving the immunoglobulin heavy chain (IGH) are rarely detected in chronic lymphocytic leukemia (CLL). The prognostic significance of the IGH translocation is controversial and its mutational profile remains unknown. Here, we present for the first time a comprehensive next-generation sequencing (NGS) analysis of 46 CLL patients with IGH rearrangement (IGHR-CLLs) and we demonstrate that IGHR-CLLs have a distinct mutational profile with recurrent mutations in NOTCH1, IGLL5, POT1, BCL2, FBXW7, ZMYM3, MGA, BRAF and HIST1H1E genes. Interestingly, BCL2 and FBXW7 mutations were significantly associated with this subgroup and almost half of BCL2, IGLL5 and HISTH1E mutations reported were previously identified in non-Hodgkin lymphomas. Notably, IGH/BCL2 rearrangements were associated with a lower mutation frequency and carried BCL2 and IGLL5 mutations, while the other IGHR-CLLs had mutations in genes related to poor prognosis (NOTCH1, SF3B1 and TP53) and shorter time to first treatment (TFT). Moreover, IGHR-CLLs patients showed a shorter TFT than CLL patients carrying 13q-, normal fluorescence in situ hybridization (FISH) and +12 CLL, being this prognosis particularly poor when NOTCH1, SF3B1, TP53, BIRC3 and BRAF were also mutated. The presence of these mutations not only was an independent risk factor within IGHR-CLLs, but also refined the prognosis of low-risk cytogenetic patients (13q-/normal FISH). Hence, our study demonstrates that IGHR-CLLs have a distinct mutational profile from the majority of CLLs and highlights the relevance of incorporating NGS and the status of IGH by FISH analysis to refine the risk-stratification CLL model.

Molecular Diagnosis of Inherited Coagulation and Bleeding Disorders.

Diagnosis of inherited bleeding disorders (IBDs) remains challenging, especially in the case of inherited platelet disorders, due to the heterogeneity of the clinical and laboratory phenotype, the limited specificity of platelet function tests, and the large number of potential culprit genes. Unraveling the underlying molecular defect provides the definitive diagnosis of IBDs, facilitating prognosis and clinical care, which are especially important for severe clinical syndromes and those that may be associated with an increased risk of malignancy. Until recently, Sanger sequencing of candidate genes has been the only method of molecular diagnosis, but this approach is time-consuming and costly and requires phenotype-based identification of any obvious candidate gene(s). Nowadays, high-throughput sequencing (HTS) allows the simultaneous and rapid investigation of multiple genes at a manageable cost. This HTS technology that includes targeted sequencing of prespecified genes, whole-exome sequencing, or whole-genome sequencing, is revolutionizing the genetic diagnosis of human diseases. Through its extensive implementation in research and clinical practice, HTS is rapidly improving the molecular characterization of IBDs. However, despite the availability of this powerful approach, many patients still do not receive a diagnosis. As IBDs are complex and rare diseases, development of more advanced laboratory assays, improvements in bioinformatic pipelines, and the formation of multidisciplinary teams are encouraged to advance our understanding of IBDs.

RNA sequencing identifies novel regulated IRE1-dependent decay targets that affect multiple myeloma survival and proliferation.

BACKGROUND: IRE1 is an unfolded protein response (UPR) sensor with kinase and endonuclease activity. It plays a central role in the endoplasmic reticulum (ER) stress response through unconventional splicing of XBP1 mRNA and regulated IRE1-dependent decay (RIDD). Multiple myeloma (MM) cells are known to exhibit an elevated level of baseline ER stress due to immunoglobulin production, however RIDD activity has not been well studied in this disease. In this study, we aimed to investigate the potential of RNA-sequencing in the identification of novel RIDD targets in MM cells and to analyze the role of these targets in MM cells. METHODS: In vitro IRE1-cleavage assay was combined with RNA sequencing. The expression level of RIDD targets in MM cell lines was measured by real-time RT-PCR and Western blot. RESULTS: Bioinformatic analysis revealed hundreds of putative IRE1 substrates in the in vitro assay, 32 of which were chosen for further validation. Looking into the secondary structure of IRE1 substrates, we found that the consensus sequences of IRF4, PRDM1, IKZF1, KLF13, NOTCH1, ATR, DICER, RICTOR, CDK12, FAM168B, and CENPF mRNAs were accompanied by a stem-loop structure essential for IRE1-mediated cleavage. In fact, we show that mRNA and protein levels corresponding to these targets were attenuated in an IRE1-dependent manner by treatment with ER-stress-inducing agents. In addition, a synergistic effect between IMiDs and ER-stress inducers was found. CONCLUSION: This study, using RNA sequencing, shows that IRE1 RNase has a broad range of mRNA substrates in myeloma cells and demonstrates for the first time that IRE1 is a key regulator of several proteins of importance in MM survival and proliferation.

Biological significance of monoallelic and biallelic BIRC3 loss in del(11q) chronic lymphocytic leukemia progression.

BIRC3 is monoallelically deleted in up to 80% of chronic lymphocytic leukemia (CLL) cases harboring del(11q). In addition, truncating mutations in the remaining allele of this gene can lead to BIRC3 biallelic inactivation, which has been shown to be a marker for reduced survival in CLL. Nevertheless, the biological mechanisms by which these lesions could contribute to del(11q) CLL pathogenesis and progression are partially unexplored. We implemented the CRISPR/Cas9-editing system to generate isogenic CLL cell lines harboring del(11q) and/or BIRC3 mutations, modeling monoallelic and biallelic BIRC3 loss. Our results reveal that monoallelic BIRC3 deletion in del(11q) cells promotes non-canonical NF-κB signaling activation via RelB-p52 nuclear translocation, being these effects allelic dose-dependent and therefore further enhanced in del(11q) cells with biallelic BIRC3 loss. Moreover, we demonstrate ex vivo in primary cells that del(11q) cases including BIRC3 within their deleted region show evidence of non-canonical NF-κB activation which correlates with high BCL2 levels and enhanced sensitivity to venetoclax. Furthermore, our results show that BIRC3 mutations in del(11q) cells promote clonal advantage in vitro and accelerate leukemic progression in an in vivo xenograft model. Altogether, this work highlights the biological bases underlying disease progression of del(11q) CLL patients harboring BIRC3 deletion and mutation.

Dissecting the role of TP53 alterations in del(11q) chronic lymphocytic leukemia.

BACKGROUND: Several genetic alterations have been identified as driver events in chronic lymphocytic leukemia (CLL) pathogenesis and oncogenic evolution. Concurrent driver alterations usually coexist within the same tumoral clone, but how the cooperation of multiple genomic abnormalities contributes to disease progression remains poorly understood. Specifically, the biological and clinical consequences of concurrent high-risk alterations such as del(11q)/ATM-mutations and del(17p)/TP53-mutations have not been established. METHODS: We integrated next-generation sequencing (NGS) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 techniques to characterize the in vitro and in vivo effects of concurrent monoallelic or biallelic ATM and/or TP53 alterations in CLL prognosis, clonal evolution, and therapy response. RESULTS: Targeted sequencing analysis of the co-occurrence of high-risk alterations in 271 CLLs revealed that biallelic inactivation of both ATM and TP53 was mutually exclusive, whereas monoallelic del(11q) and TP53 alterations significantly co-occurred in a subset of CLL patients with a highly adverse clinical outcome. We determined the biological effects of combined del(11q), ATM and/or TP53 mutations in CRISPR/Cas9-edited CLL cell lines. Our results showed that the combination of monoallelic del(11q) and TP53 mutations in CLL cells led to a clonal advantage in vitro and in in vivo clonal competition experiments, whereas CLL cells harboring biallelic ATM and TP53 loss failed to compete in in vivo xenotransplants. Furthermore, we demonstrated that CLL cell lines harboring del(11q) and TP53 mutations show only partial responses to B cell receptor signaling inhibitors, but may potentially benefit from ATR inhibition. CONCLUSIONS: Our work highlights that combined monoallelic del(11q) and TP53 alterations coordinately contribute to clonal advantage and shorter overall survival in CLL.

Integrated Genomic Analysis of Chromosomal Alterations and Mutations in B-Cell Acute Lymphoblastic Leukemia Reveals Distinct Genetic Profiles at Relapse.

The clonal basis of relapse in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is complex and not fully understood. Next-generation sequencing (NGS), array comparative genomic hybridization (aCGH), and multiplex ligation-dependent probe amplification (MLPA) were carried out in matched diagnosis-relapse samples from 13 BCP-ALL patients to identify patterns of genetic evolution that could account for the phenotypic changes associated with disease relapse. The integrative genomic analysis of aCGH, MLPA and NGS revealed that 100% of the BCP-ALL patients showed at least one genetic alteration at diagnosis and relapse. In addition, there was a significant increase in the frequency of chromosomal lesions at the time of relapse (p = 0.019). MLPA and aCGH techniques showed that IKZF1 was the most frequently deleted gene. TP53 was the most frequently mutated gene at relapse. Two TP53 mutations were detected only at relapse, whereas the three others showed an increase in their mutational burden at relapse. Clonal evolution patterns were heterogeneous, involving the acquisition, loss and maintenance of lesions at relapse. Therefore, this study provides additional evidence that BCP-ALL is a genetically dynamic disease with distinct genetic profiles at diagnosis and relapse. Integrative NGS, aCGH and MLPA analysis enables better molecular characterization of the genetic profile in BCP-ALL patients during the evolution from diagnosis to relapse.

CRISPR/Cas9-generated models uncover therapeutic vulnerabilities of del(11q) CLL cells to dual BCR and PARP inhibition.

The deletion of 11q (del(11q)) invariably comprises ATM gene in chronic lymphocytic leukemia (CLL). Concomitant mutations in this gene in the remaining allele have been identified in 1/3 of CLL cases harboring del(11q), being the biallelic loss of ATM associated with adverse prognosis. Although the introduction of targeted BCR inhibition has significantly favored the outcomes of del(11q) patients, responses of patients harboring ATM functional loss through biallelic inactivation are unexplored, and the development of resistances to targeted therapies have been increasingly reported, urging the need to explore novel therapeutic approaches. Here, we generated isogenic CLL cell lines harboring del(11q) and ATM mutations through CRISPR/Cas9-based gene-editing. With these models, we uncovered a novel therapeutic vulnerability of del(11q)/ATM-mutated cells to dual BCR and PARP inhibition. Ex vivo studies in the presence of stromal stimulation on 38 CLL primary samples confirmed a synergistic action of the combination of olaparib and ibrutinib in del(11q)/ATM-mutated CLL patients. In addition, we showed that ibrutinib produced a homologous recombination repair impairment through RAD51 dysregulation, finding a synergistic link of both drugs in the DNA damage repair pathway. Our data provide a preclinical rationale for the use of this combination in CLL patients with this high-risk cytogenetic abnormality.

Helpful Criteria When Implementing NGS Panels in Childhood Lymphoblastic Leukemia.

The development of Next-Generation Sequencing (NGS) has provided useful diagnostic, prognostic, and therapeutic strategies for individualized management of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients. Consequently, NGS is rapidly being established in clinical practice. However, the technology's complexity, bioinformatics analysis, and the different available options difficult a broad consensus between different laboratories in its daily routine introduction. This collaborative study among Spanish centers was aimed to assess the feasibility, pros, and cons of our customized panel and other commercial alternatives of NGS-targeted approaches. The custom panel was tested in three different sequencing centers. We used the same samples to assess other commercial panels (OncomineTM Childhood Cancer Research Assay; Archer®FusionPlex® ALL, and Human Comprehensive Cancer Panel GeneRead Panel v2®). Overall, the panels showed a good performance in different centers and platforms, but each NGS approach presented some issues, as well as pros and cons. Moreover, a previous consensus on the analysis and reporting following international guidelines would be preferable to improve the concordance in results among centers. Our study shows the challenges posed by NGS methodology and the need to consider several aspects of the chosen NGS-targeted approach and reach a consensus before implementing it in daily practice.

DNA damage response-related alterations define the genetic background of patients with chronic lymphocytic leukemia and chromosomal gains.

The presence of chromosomal gains other than trisomy 12 suggesting a hyperdiploid karyotype is extremely rare in chronic lymphocytic leukemia (CLL) and is associated with a dismal prognosis. However, the genetic mechanisms and mutational background of these patients have not been fully explored. To improve our understanding of the genetic underpinnings of this subgroup of CLL, seven CLL patients with several chromosomal gains were sequenced using a next-generation sequencing (NGS)-targeted approach. The mutational status of 54 genes was evaluated using a custom-designed gene panel including recurrent mutated genes observed in CLL and widely associated with CLL pathogenesis. A total of 21 mutations were detected; TP53 (42.8%), ATM (28.5%), SF3B1 (28.5%), and BRAF (28.5%) were the most recurrently mutated genes. Of these mutations, 61.9% were detected in genes previously associated with a poor prognosis in CLL. Interestingly, five of the seven patients exhibited alterations in TP53 or ATM (deletion and/or mutation), genes involved in the DNA damage response (DDR), which could be related to a high genetic instability in this subgroup of patients. In conclusion, CLL patients with several chromosomal gains exhibit high genetic instability, with mutations in CLL driver genes and high-risk genetic alterations involving ATM and/or TP53 genes.

Splice donor site sgRNAs enhance CRISPR/Cas9-mediated knockout efficiency.

CRISPR/Cas9 allows the generation of knockout cell lines and null zygotes by inducing site-specific double-stranded breaks. In most cases the DSB is repaired by non-homologous end joining, resulting in small nucleotide insertions or deletions that can be used to construct knockout alleles. However, these mutations do not produce the desired null result in all cases, but instead generate a similar, functionally active protein. This effect could limit the therapeutic efficiency of gene therapy strategies based on abrogating oncogene expression, and therefore needs to be considered carefully. If there is an acceptable degree of efficiency of CRISPR/Cas9 delivery to cells, the key step for success lies in the effectiveness of a specific sgRNA at knocking out the oncogene, when only one sgRNA can be used. This study shows that the null effect could be increased with an sgRNA targeting the splice donor site (SDS) of the chosen exon. Following this strategy, the generation of null alleles would be facilitated in two independent ways: the probability of producing a frameshift mutation and the probability of interrupting the canonical mechanism of pre-mRNA splicing. In these contexts, we propose to improve the loss-of-function yield driving the CRISPR system at the SDS of critical exons.

Identification of novel variants in ten patients with Hermansky-Pudlak syndrome by high-throughput sequencing.

Background: Hermansky-Pudlak syndrome (HPS) is a rare inherited platelet disorder characterized by bleeding diathesis, oculocutaneous albinism (OCA) and a myriad of often-serious clinical complications. Methods: We established the clinical and laboratory phenotype and genotype of six unrelated pedigrees comprising ten patients with clinical suspicion of HPS; including platelet aggregation, flow cytometry, platelet dense granule content, electron microscopy and high-throughput sequencing (HTS). Results: The clinical presentation showed significant heterogeneity and no clear phenotype-genotype correlations. HTS revealed two known and three novel disease-causing variants. The Spanish patients carried a homozygous p.Pro685Leufs17* deletion (n = 2) in HPS4, or the novel p.Arg822* homozygous variant (n = 1) in HPS3. In the case of two Turkish sisters, a novel missense homozygous HPS4 variant (p.Leu91Pro) was found. In two Portuguese families, genetic studies confirmed a previously reported nonsense variant (p.Gln103*) in DTNBP1 in three patients and a novel duplication (p.Leu22Argfs*33) in HPS6 in two unrelated patients. Conclusions: Our findings expand the mutational spectrum of HPS, which may help in investigating phenotype-genotype relationships and assist genetic counselling for affected individuals. This approach is a proof of principle that HTS can be considered and used in the first-line diagnosis of patients with biological and clinical manifestations suggestive of HPS. Key messages We established the relationships between the clinical and laboratory phenotype and genotype of six unrelated pedigrees comprising ten patients with clinical suspicion of HPS. Molecular analysis is useful in confirming the diagnosis and may offer some prognostic information that will aid in optimizing monitoring and surveillance for early detection of end-organ damage. This approach is a proof of principle that HTS can be considered and used in the first-line diagnosis of patients with biological and clinical manifestations suggestive of HPS.

[Hydatidosis simulating a cardiac tumour with pulmonary metastases]. / Hidatidosis simulando un tumor cardíaco con metástasis pulmonares.

BACKGROUND: The presence of multiple symptomatic pulmonary nodules and one cardiac tumour in a child requires urgent diagnosis and treatment. Until a few decades ago, the diagnosis of a cardiac tumour was difficult and was based on a high index of suspicion from indirect signs, and required angiocardiography for confirmation. Echocardiography and other imaging techniques have also helped in the detection of cardiac neoplasms. However, it is not always easy to make the correct diagnosis. CLINICAL CASE: The case is presented of a 12 year-old boy with pulmonary symptoms, and diagnosed with a cardiac tumour with lung metastases. The presence of numerous pulmonary nodules was confirmed in our hospital. The echocardiogram detected a solid cardiac nodule in the right ventricle. Magnetic resonance imaging confirmed the findings and the diagnosis. Puncture-aspiration of a lung nodule gave the diagnosis of hydatidosis. He underwent open-heart surgery with cardiac cyst resection and treated with anthelmintics. The lung cysts were then excised, and he recovered uneventfully. DISCUSSION: This child had multiple pulmonary nodules and a solid cardiac nodule, and was suspected of having a cardiac tumour with pulmonary metastases. However, given the clinical history, background and morphology of pulmonary nodules, another possible aetiology for consideration is echinococcosis. The clinical picture of cardiac hydatidosis and its complications is highly variable. The clinical history is essential in these cases, as well as having a high index of suspicion. CONCLUSION: Hydatidosis should be included in the differential diagnosis of a solid, echogenic, cardiac nodule. The treatment for cardiopulmonary hydatid cysts is surgical, followed by anthelmintics.