Pathogenic convergence of CNVs in genes functionally associated to a severe neuromotor developmental delay syndrome.

Background Complex developmental encephalopathy syndromes might be the consequence of unknown genetic alterations that are likely to contribute to the full neurological phenotype as a consequence of pathogenic gene combinations. Methods To identify the additional genetic contribution to the neurological phenotype, we studied as a test case a boy, with a KCNQ2 exon-7 partial duplication, by single-nucleotide polymorphism (SNP) microarray to detect copy-number variations (CNVs). Results The proband presented a cerebral palsy like syndrome with a severe motor and developmental encephalopathy. The SNP array analysis detected in the proband several de novo CNVs, nine partial gene losses (LRRC55, PCDH9, NALCN, RYR3, ELAVL2, CDH13, ATP1A2, SLC17A5, ANO3), and two partial gene duplications (PCDH19, EFNA5). The biological functions of these genes are associated with ion channels such as calcium, chloride, sodium, and potassium with several membrane proteins implicated in neural cell-cell interactions, synaptic transmission, and axon guidance. Pathogenically, these functions can be associated to cerebral palsy, seizures, dystonia, epileptic crisis, and motor neuron dysfunction, all present in the patient. Conclusions Severe motor and developmental encephalopathy syndromes of unknown origin can be the result of a phenotypic convergence by combination of several genetic alterations in genes whose physiological function contributes to the neurological pathogenic mechanism.post-print783 K

Outcome of cutaneous squamous cell carcinoma with microscopic residual disease after surgery and usefulness of postoperative radiotherapy: a retrospective cohort study

[Background]: Microscopic residual disease (MRD) after surgery can be a challenging situation in cutaneous squamous cell carcinoma (CSCC) and there is a lack of evidence concerning its management. [Objective]: To evaluate the prognosis of CSCC with MRD and the usefulness of postoperative radiotherapy (PORT) in CSCC with MRD. [Methods]: Retrospective cohort study of CSCC with MRD through a 10-year period (2010–2019) (n = 244). Disease-free survival and event-free survival were assessed using R (v.3.4.1), considering competing risks. Evaluated outcomes were local recurrence (LR), nodal metastases (NMs), and disease-specific death (DSD). [Results]: Median age was 88y (IQR: 10.5). A total of 145 (59.43%) were men and 69 (28.28%) were immunosuppressed. Median tumour diameter and thickness were 19 and 6.4 mm (IQR 11 and 5.5 mm). Patients treated by re-excision had a relapse rate of 4.3% compared with 11.30% and 29.71% in those who received PORT and observation (P = 0.045). The use of PORT was associated with a lower risk of LR compared with observation (HR = 0.206 [0.049–0.859], P = 0.030), but not with a lower risk of NMs or DSDs. In the multivariable models, PORT was again associated with a lower risk of LR than observation (HR = 0.167 [0.039–0.708], P = 0.014), but not with lower risk of metastasis and death. [Conclusions]: We always should try to obtain clear margins after surgery. PORT improves local control in CSCC with MRD, but when administered to the tumour bed, it does not reduce the risk of NM and DSD.Javier Cañueto is partially supported by the grants GRS2139/A/20 (Gerencia Regional de Salud de Castilla y León), PI18/00587 and PI21/01207 (Instituto de Salud Carlos III, confanciado con fondos FEDER) and by the ‘Programa de Intensificación of the ISCIII’, grant number INT20/00074

Identification and characterization of the gene expression profiles for protein coding and non-coding RNAs of pancreatic ductal adenocarcinomas

This is an open-access article distributed under the terms of the Creative Commons Attribution License.Significant advances have been achieved in recent years in the identification of the genetic and the molecular alterations of pancreatic ductal adenocarcinoma (PDAC). Despite this, at present the understanding of the precise mechanisms involved in the development and malignant transformation of PDAC remain relatively limited. Here, we evaluated for the first time, the molecular heterogeneity of PDAC tumors, through simultaneous assessment of the gene expression profile (GEP) for both coding and non-coding genes of tumor samples from 27 consecutive PDAC patients. Overall, we identified a common GEP for all PDAC tumors, characterized by an increased expression of genes involved in PDAC cell proliferation, local invasion and metastatic capacity, together with a significant alteration of the early steps of the cellular immune response. At the same time, we confirm and extend on previous observations about the genetic complexity of PDAC tumors as revealed by the demonstration of two clearly distinct and unique GEPs (e.g. epithelial-like vs. mesenchymal-like) reflecting the alteration of different signaling pathways involved in the oncogenesis and progression of these tumors. Our results also highlight the potential role of the immune system microenvironment in these tumors, with potential diagnostic and therapeutic implications.This work has been partially supported by grants from the Gerencia Regional de Salud de Castilla y León, Valladolid, Spain (GRS861/A/13), RTICC from the Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Madrid, Spain (RD06/0020/0035-FEDER; RD12/0036/0048-FEDER), Fundación Memoria de Don Samuel Solórzano Barruso, Salamanca, Spain (FS/13-2012 and FS/16-2013). JM Sayagués is supported by grant CP05/00321 from the Ministerio de Ciencia e Innovación, Madrid, Spain.Peer Reviewe

Eltrombopag increases the hematopoietic supporting ability of mesenchymal stem/stromal cells

[Background]: Eltrombopag (EP) is a small molecule that acts directly on hematopoietic stem cells (HSCs) and megakaryocytes to stimulate the hematopoietic process. Mesenchymal stem/stromal cells (MSCs) are key hematopoietic niche regulators. [Objectives]: We aimed to determine whether EP has any effect on MSC function and properties (especially on their hematopoietic-supporting ability) and if so, what changes (e.g. genome-wide transcriptomic alterations) are induced in MSC after EP treatment. [Design/Methods]: MSCs were isolated from 12 healthy donors and treated with 15 µM and 50 µM of EP for 24 h. The toxicity of the drug on MSCs and their differentiation ability were analyzed, as well as the transcriptomic profile, reactive oxygen species (ROS) and DNA damage and the changes induced in the clonogenic capacity of HSCs.[Results]: The results show that EP also modifies MSC functions, decreasing their adipogenic differentiation, increasing the expression of genes involved in hypoxia and other pathways related to oxygen homeostasis, and enhancing their ability to support hematopoiesis in vitro.[Conclusion]: Our findings support the use of EP in cases where hematopoiesis is defective, despite its well-known direct effects on hematopoietic cells. Our findings suggest that further studies on the effects of EP on MSCs from patients with aplastic anemia are warranted.This study was supported by research funding from Novartis Pharmaceuticals to FS-G. SP is supported by Fundación Española de Hematología y Hemoterapia (FEHH). SM is supported by RETIC and RICORS programs of ISCIII European Regional Development Fund (RD16/0011/0015, RD21/0017/0006), ‘A way to make Europe’ and NextGenerationEU. GJMC and ESL are supported by the Spanish Ministerio de Ciencia e Innovación (FPU18/03533 and PFIS/19/00272 respectively).Peer reviewe

t(10;12)(q24;q15): A new cytogenetic marker in hematological malignancies

Cytogenetic studies have played a crucial role in the discovery of genes involved in several diseases. In the field of oncohematology, cytogenetics is still necessary for the classification and prognosis of many diseases. Here we report a new recurrent chromosome translocation, t(10;12)(q24;q15), in two patients with different hematological malignancies: myelodysplastic syndrome with excess blasts (MDS-EB), and myelofibrosis (MF) secondary to essential thrombocythemia (ET). The chromosome alteration was observed as a sole karyotype change in the patient with MDS-EB, both at the initial diagnosis and following progression to MDS-EB2. A putative HMGA2-KLLN rearrangement by RNA-sequencing was detected in this patient. The patient with ET, had a normal karyotype at diagnosis and the t(10;12)(q24;q15) translocation emerged as a sole cytogenetic alteration after transformation, and when MF was evident. We reviewed the literature to determine whether this chromosome abnormality had previously been described in other hematological patients and found two cases: an aggressive T-cell lymphoblastic lymphoma (T-LBL) and a case of transformed chronic myeloproliferative syndrome (CMS), in both of which t(10;12)(q24;q15) was also the only karyotype change. The clinical evolution of all four cases suggested that t(10;12)(q24;q15) is associated with a poor outcome in oncohematological patients

Patterns of incidental perineural invasion and prognosis in cutaneous squamous cell carcinoma: A multicenter, retrospective cohort study

To the Editor: Perineural invasion (PNI) is rare and usually incidental in cutaneous squamous cell carcinoma (SCC), with an incidence of 2.5% to 14%.1 Incidental PNI is associated with poor prognosis in cutaneous SCC,2 and some evidence suggests its outcome differs, depending on the PNI pattern. We evaluated patterns of incidental PNI, using a multicenter retrospective cohort of 140 cutaneous SCCs with incidental PNI to determine the influence of nerve involvement on cutaneous SCC prognosis.Dr Canueto is partially supported ~ by grants PI18/000587 (Instituto de Salud Carlos III, cofinanced by Fondo Europeo de Desarrollo Regional) and GRS 1835/A/18 (Gerencia Regional de Salud de Castilla y Leon)

Transcriptome analysis reveals molecular profiles associated with evolving steps of monoclonal gammopathies

This is an open-access paper.-- et al.A multistep model has been proposed of disease progression starting in monoclonal gammopathy of undetermined significance continuing through multiple myeloma, sometimes with an intermediate entity called smoldering myeloma, and ending in extramedullary disease. To gain further insights into the role of the transcriptome deregulation in the transition from a normal plasma cell to a clonal plasma cell, and from an indolent clonal plasma cell to a malignant plasma cell, we performed gene expression profiling in 20 patients with monoclonal gammopathy of undetermined significance, 33 with high-risk smoldering myeloma and 41 with multiple myeloma. The analysis showed that 126 genes were differentially expressed in monoclonal gammopathy of undetermined significance, smoldering myeloma and multiple myeloma as compared to normal plasma cell. Interestingly, 17 and 9 out of the 126 significant differentially expressed genes were small nucleolar RNA molecules and zinc finger proteins. Several proapoptotic genes (AKT1 and AKT2) were down-regulated and antiapoptotic genes (APAF1 and BCL2L1) were up-regulated in multiple myeloma, both symptomatic and asymptomatic, compared to monoclonal gammopathy of undetermined significance. When we looked for those genes progressively modulated through the evolving stages of monoclonal gammopathies, eight snoRNA showed a progressive increase while APAF1, VCAN and MEGF9 exhibited a progressive downregulation. In conclusion, our data show that although monoclonal gammopathy of undetermined significance, smoldering myeloma and multiple myeloma are not clearly distinguishable groups according to their gene expression profiling, several signaling pathways and genes were significantly deregulated at different steps of the transformation process.This study was partially supported by Spanish FIS (PI080568, PS09/01450 and PS0901897), “Gerencia Regional de Salud, Junta de Castilla y León” (GRS 702/A/11) grant, and the Spanish Myeloma Network Program (RD06/0020/0006, RD12/0036/0058 and RD12/0036/0046).Peer Reviewe

Phenotypic, genomic and functional characterization reveals no differences between CD138++ and CD138low subpopulations in multiple myeloma cell lines

This is an open-access article distributed under the terms of the Creative Commons Attribution License.Despite recent advances in the treatment of multiple myeloma (MM), it remains an incurable disease potentially due to the presence of resistant myeloma cancer stem cells (MM-CSC). Although the presence of clonogenic cells in MM was described three decades ago, the phenotype of MM-CSC is still controversial, especially with respect to the expression of syndecan-1 (CD138). Here, we demonstrate the presence of two subpopulations - CD138++ (95-99%) and CD138low (1-5%) - in eight MM cell lines. To find out possible stem-cell-like features, we have phenotypically, genomic and functionally characterized the two subpopulations. Our results show that the minor CD138low subpopulation is morphologically identical to the CD138++ fraction and does not represent a more immature B-cell compartment (with lack of CD19, CD20 and CD27 expression). Moreover, both subpopulations have similar gene expression and genomic profiles. Importantly, both CD138++ and CD138low subpopulations have similar sensitivity to bortezomib, melphalan and doxorubicin. Finally, serial engraftment in CB17-SCID mice shows that CD138++ as well as CD138low cells have self-renewal potential and they are phenotypically interconvertible. Overall, our results differ from previously published data in MM cell lines which attribute a B-cell phenotype to MM-CSC. Future characterization of clonal plasma cell subpopulations in MM patients' samples will guarantee the discovery of more reliable markers able to discriminate true clonogenic myeloma cells.This work was supported by the Cooperative Research Thematic Network (RTICs; RD06/0020/0006), the “Junta de Castilla y León. Consejería de Sanidad” (GRS 391/B/09), the “Ministerio de Ciencia e Innovación” (PS09/01897), the “Fundación Memoria D. Samuel Solórzano Barruso” (FS/2-2010) and Asociación Española Contra el Cáncer (AECC)(GCB120981SAN).Peer Reviewe

Integrative analysis of DNA copy number, DNA methylation and gene expression in multiple myeloma reveals alterations related to relapse

Multiple myeloma (MM) remains incurable despite the introduction of novel agents, and a relapsing course is observed in most patients. Although the development of genomic technologies has greatly improved our understanding of MM pathogenesis, the mechanisms underlying relapse have been less thoroughly investigated. In this study, an integrative analysis of DNA copy number, DNA methylation and gene expression was conducted in matched diagnosis and relapse samples from MM patients. Overall, the acquisition of abnormalities at relapse was much more frequent than the loss of lesions present at diagnosis, and DNA losses were significantly more frequent in relapse than in diagnosis samples. Interestingly, copy number abnormalities involving more than 100 Mb of DNA at relapse significantly affect the gene expression of these samples, provoking a particular deregulation of the IL-8 pathway. On the other hand, no significant modifications of gene expression were observed in those samples with less than 100 Mb affected by chromosomal changes. Although several statistical approaches were used to identify genes whose abnormal expression at relapse was regulated by methylation, only two genes that were significantly deregulated in relapse samples (SORL1 and GLT1D1) showed a negative correlation between methylation and expression. Further analysis revealed that DNA methylation was involved in regulating SORL1 expression in MM. Finally, relevant changes in gene expression observed in relapse samples, such us downregulation of CD27 and P2RY8, were most likely not preceded by alterations in the corresponding DNA. Taken together, these results suggest that the genomic heterogeneity described at diagnosis remains at relapse.This work was partially supported by the Instituto de Salud Carlos III-Cofinanciación con fondos FEDER (PI080568, PS0901897 and PI13/00111), the Gerencia Regional de Salud, Junta de Castilla y León (GRS202/A08 and GRS 702/A/11), the Spanish Myeloma Network Program (RD06/0020/0006) and the Asociación Española Contra el Cáncer (AECC, GCB120981SAN).Peer Reviewe

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