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The article features a letter to the editor on cases of Listeria monocytogenes meningitis in two allogeneic hematopoietic stem cell transplant recipients.

Presents a correction to the article 'Quantification of Donor and Recipient Hemopoietic Cells by Real-Time PCR of Single-Nucleotide Polymorphisms,' by F. Maas, N. Schaap, S. Kolen, Z. Zoetbrood, I. Buno, H. Dolstra, T. de White, A. Schattenberg and E. van de Wiel-van Kemenade.

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults, with a median overall survival (OS) of 15-18 months despite standard treatments. Approximately 8% of GBM cases exhibit genomic alterations in fibroblast growth factor receptors (FGFRs), particularly FGFR1 and FGFR3. Next-generation sequencing techniques have identified various FGFR3 fusions in GBM. This report presents a novel FGFR3 fusion with fatty acid synthase (FASN) in a 41-year-old male diagnosed with GBM. The patient presented with a persistent headache, and imaging revealed a right frontal lobe lesion. Surgical resection and subsequent histopathology confirmed GBM. Initial NGS analysis showed no mutations in the IDH1, IDH2 or H3F3 genes, but revealed a TERT promoter mutation and CDKN2A/2B and PTEN deletions. Postoperative treatment included radiotherapy and temozolomide. Despite initial management, recurrence occurred four months post-diagnosis, confirmed by MRI and histology. A second surgery identified a novel FGFR3-FASN fusion, alongside increased Ki67 expression. The recurrence was managed with regorafenib and bevacizumab, though complications like hand-foot syndrome and radiation necrosis arose. Despite initial improvement, the patient died 15 months after diagnosis. This case underscores the importance of understanding GBM's molecular landscape for effective treatment strategies. The novel FGFR3-FASN fusion suggests potential implications for GBM recurrence and lipid metabolism. Further studies are warranted to explore FGFR3-FASN's role in GBM and its therapeutic targeting.

Backgrounds: Although allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for hematological malignancies, it can be associated with relevant post-transplant complications. Several reports have shown that polymorphisms in immune system genes are correlated with the development of post-transplant complications. Within this context, this work focuses on identifying novel polymorphisms in cytokine genes and developing predictive models to anticipate the risk of developing graft-versus-host disease (GVHD), transplantation-related mortality (TRM), relapse and overall survival (OS)., Methods: Our group developed a 132-cytokine gene panel which was tested in 90 patients who underwent an HLA-identical sibling-donor allo-HSCT. Bayesian logistic regression (BLR) models were used to select the most relevant variables. Based on the cut-off points selected for each model, patients were classified as being at high or low-risk for each of the post-transplant complications (aGVHD II-IV, aGVHD III-IV, cGVHD, mod-sev cGVHD, TRM, relapse and OS)., Results: A total of 737 polymorphisms were selected from the custom panel genes. Of these, 41 polymorphisms were included in the predictive models in 30 cytokine genes were selected (17 interleukins and 13 chemokines). Of these polymorphisms, 5 (12.2%) were located in coding regions, and 36 (87.8%) in non-coding regions. All models had a statistical significance of p<0.0001., Conclusion: Overall, genomic polymorphisms in cytokine genes make it possible to anticipate the development all complications studied following allo-HSCT and, consequently, to optimize the clinical management of patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Muñiz, Martínez-García, Bailén, Chicano, Oarbeascoa, Triviño, de la Iglesia-San Sebastian, Fernández de Córdoba, Anguita, Kwon, Díez-Martín, Olmos, Martínez-Laperche and Buño.)

Chimeric antigen receptor T cells (CAR-T) has emerged as a promising therapy, over 60% of patients fail to sustain a long-term response. The underlying factors that leads to the effectiveness of this therapy are not completely understood, CAR-T cell persistence and monitoring seems to be pivotal for ensuring a successful response. Various monitoring methods such as multiparametric flow cytometry (MFC) or quantitative PCR (qPCR) have been applied. Our objective is to develop digital PCR (dPCR) assays for detection and quantification of CAR-T cells, comparing them with MFC and qPCR. Samples taken at different follow-up times from 45 patients treated with CAR-T therapy were analyzed to assess the correlation between the different methodologies. dPCR presented a high correlation with MFC and qPCR (r = 0.97 and r = 0.87, respectively), while offering a higher sensitivity (0.01%) compared to MFC (0.1%) and qPCR (1%). dPCR emerged as an alternative and highly sensitivity method for monitoring CAR-T cell dynamics. This technique is well-suited for implementation in clinical practice as a complementary technique to MFC., (Copyright © 2024 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

We propose a novel biomarker that can identify patients at high risk of early progression after chimeric antigen receptor (CAR) T cell therapy. Calculation of cell-free DNA (cfDNA) with a pre-apheresis (PA) and pre-lymphodepletion (PL) sample allows monitoring of tumor dynamics (∆cfDNA). In the present study, ∆cfDNA and other biomarkers and clinical variables were evaluated in 58 patients with relapsed/refractory diffuse large B cell lymphoma (DLBCL). ∆cfDNA (>11 ng/mL plasma; P =.003), C-reactive protein (CRP) PL (>1.06 mg/dL; P = .004), lactate dehydrogenase (LDH) PL (>304; P = .006), disease status PL (progressive disease; P = .035) and sex (male; P = .016) were highly correlated with 1 month progression. After adjusting for ∆cfDNA, CRP PL, and LDH PL, disease status PL, and sex, ∆cfDNA remained associated with 1-month progression after CAR T cell infusion., (Copyright © 2023 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Despite its often low efficacy and high toxicity, the standard treatment for acute myeloid leukemia (AML) is induction chemotherapy with cytarabine and idarubicin. Here, we have investigated the role of transporters and drug-metabolizing enzymes in this poor outcome. The expression levels (RT-qPCR) of potentially responsible genes in blasts collected at diagnosis were related to the subsequent response to two-cycle induction chemotherapy. The high expression of uptake carriers (ENT2), export ATP-binding cassette (ABC) pumps (MDR1), and enzymes (DCK, 5-NT, and CDA) in the blasts was associated with a lower response. Moreover, the sensitivity to cytarabine in AML cell lines was associated with ENT2 expression, whereas the expression of ABC pumps and enzymes was reduced. No ability of any AML cell line to export idarubicin through the ABC pumps, MDR1 and MRP, was found. The exposure of AML cells to cytarabine or idarubicin upregulated the detoxifying enzymes (5-NT and DCK). In AML patients, 5-NT and DCK expression was associated with the lack of response to induction chemotherapy (high sensitivity and specificity). In conclusion, in the blasts of AML patients, the reduction of the intracellular concentration of the active metabolite of cytarabine, mainly due to the increased expression of inactivating enzymes, can determine the response to induction chemotherapy.

This is a retrospective cohort study of consecutive adult patients who received a haploidentical-SCT (haplo-SCT) with post-transplant cyclophosphamide (PT-Cy) in a single centre. Poor graft function (PGF) was defined as the occurrence of either persistent neutropenia (ANC < 0.5 × 10 9 /µL) with poor response to granulocyte colony-stimulating factors (G-CSF) and/or thrombocytopenia (platelets < 20 × 10 9 /L) with transfusion dependence, with complete donor chimerism and without concurrent severe GVHD or underlying disease relapse, during the first 12 months after transplantation. Forty-four (27.5%) out of 161 patients were diagnosed with PGF. Previous CMV reactivation was significantly more frequent in patients with PGF (88.6% versus 73.5%, p = 0.04) and the number of reactivations was also higher in these patients. Besides, early CMV reactivations in the first 6 months post-SCT were also significantly more frequent among patients with PGF (88.6% versus 71.8% p = 0.025). Thirty-two percent of patients with PGF were treated with increasing doses of thrombopoietin-receptor agonists (TRA) and 7 patients were treated with a donor CD34 + selected boost. In total, 93.2% of patients reached adequate peripheral blood counts in a median time of 101 days (range 11-475) after diagnosis. PGF is a frequent complication after haplo-SCT with PT-Cy. CMV reactivation might be the most relevant factor associated to its development. Even when most patients recover peripheral counts with support therapy, there is a group of patients with persistent cytopenias who can effectively be treated with TRA and/or a boost of CD34 + selective cells., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The familial occurrence of hematological malignancies has been underappreciated. Recent studies suggest that up to 15% of adults with myeloid neoplasms carry germline pathogenic variants in cancer-predisposing genes. This study aimed to identify the underlying germline predisposition variant in patients with a strong family or personal onco-hematological history using whole exome sequencing on sixteen uncharacterized individuals. It was carried out in two groups of patients, one with samples available from two affected relatives (Cohort A) and one with available samples from the index case (Cohort B). In Cohort A, six families were characterized. Two families shared variants in genes associated with DNA damage response and involved in cancer development ( CHEK2 and RAD54L ). Pathogenic or likely pathogenic germline variants were also found in novel candidate genes ( NFATC2 and TC2N ). In two families, any relevant pathogenic or likely pathogenic genomic variants were identified. In Cohort B, four additional index cases were analyzed. Three of them harbor clinically relevant variants in genes with a probable role in the development of inherited forms of hematological malignancies ( GATA1 , MSH4 and PRF1 ). Overall, whole exome sequencing is a useful approach to achieve a further characterization of these patients and their mutational spectra. Moreover, further investigations may help improve optimization for disease management of affected patients and their families.

Introduction: The association of polymorphisms in molecules involved in the immune response (checkpoint inhibitors) with the clinical outcome after allogeneic transplantation (alloHSCT) has been described. Lymphocyte Activation 3 (LAG3) is a surface protein that plays a regulatory role in immunity as an inhibitory immune checkpoint molecule., Methods: To determine its role in the alloHSCT setting, we analyzed 797 patients transplanted from HLA-identical sibling donors. The LAG3 rs870849 C>T polymorphism was genotyped in donors., Results: We detected a higher incidence of severe acute GVHD in patients transplanted from donors with TT genotype (p: 0.047, HR 1.64; 95% CI 1.01 - 2.67). Overall survival (OS) was worse for patients transplanted from donors with the rs870849 CT/TT genotype (0.020; HR, 1.44; 95% CI 1.06 - 1.96), as well as disease-free survival (DFS) (p: 0.002; HR 1.58, 95%CI: 1.18 - 2.14) and transplant-related mortality (TRM) (p< 0.001; HR: 1.88, 95% CI 1.29 - 2.74). When combining the LAG3 rs870849 and the PDCD1 rs36084323 genotypes of the donor, three genetic groups were well defined, allowing a good stratification of the risk of acute GVHD, TRM, OS and DFS., Discussion: We conclude that the LAG3 genotype of the donor may be considered in donors' selection. As this selection may be limited in the HLA-identical sibling donor scenario, further studies exploring the impact of LAG3 genotype of the donor in unrelated transplantation are warranted., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Cruz, Rodríguez-Romanos, González-Bartulos, García-Cadenas, de la Cámara, Heras, Buño, Santos, Lloveras, Velarde, Tuset, Martínez, González, Sanz, Ferrá, Sampol, Coll, Pérez-Simón, López-Jiménez, Jurado and Gallardo.)

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for patients with hematologic malignances. Haploidentical HSCT (Haplo-HSCT) is an alternative option for patients who do not have an HLA-matched donor. The use of post-transplantation high dose cyclophosphamide (PT-Cy) is commonly employed for graft-versus-host disease (GVHD) prophylaxis in haplo-HSCT. Cyclophosphamide (Cy) is an alkylating agent with antineoplastic and immunosuppressive activity, whose bioactivation requires the activity of polymorphic enzymes in the liver to produce phosphoramide mustard, which is a DNA alkylating agent. To identify polymorphisms in the genes of Cy metabolism and correlate them with post-HSCT complications [GVHD, sinusoidal obstruction syndrome (SOS), hemorrhagic cystitis (HC) and transplant-related mortality (TRM)], we designed a custom next-generation sequencing panel with Cy metabolism enzymes. We analyzed 182 patients treated with haplo-HSCT with PT-Cy from 2007 to 2019, detecting 40 variants in 11 Cy metabolism genes. Polymorphisms in CYP2B6, a major enzyme involved in Cy activation, were associated with decreased activity of this enzyme and a higher risk of Graf-versus-host disease (GVHD). Variants in other activation enzymes (CYP2A6, CYP2C8, CYP2C9, CYP2C19) lead to decreased enzyme activity and were associated with GVHD. Polymorphisms in detoxification genes such as glutathione S-transferases decreased the ability to detoxify cyclophosphamide metabolites due to lower enzyme activity, which leads to increased amounts of toxic metabolites and the development of III-IV acute GVHD. GSMT1*0 a single nucleotide polymorphism previously recognized as a risk factor for SOS was associated with a higher risk of SOS. We conclude that polymorphisms of genes involved in the metabolism of cyclophosphamide in our series are associated with severe grades of GVHD and toxicities (SOS and TRM) after haplo-HSCT and could be used to improve the clinical management of transplanted patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Muñiz, Andrés-Zayas, Carbonell, Chicano, Bailén, Oarbeascoa, Suárez-González, Gómez Centurión, Dorado, Gallardo, Anguita, Kwon, Díez-Martín, Martínez-Laperche and Buño.)

FLT3 -internal tandem duplication (ITD) analysis is not typically performed in cDNA samples and is not considered an appropriate marker for monitoring measurable residual disease (MRD). The aims of this study were to compare FLT3 -ITD mutation analysis in DNA and cDNA samples at diagnosis and to demonstrate the usefulness of its expression measurement as an MRD marker after allogeneic stem cell transplantation (allo-HSCT) or FLT3 inhibitor (FLT3i) administration. A total of 46 DNA and cDNA diagnosis samples, 102 DNA and cDNA post-allo-HSCT samples from 34 patients and 37 cDNA samples from 7 patients with refractory/relapse AML treated with FLT3i were assessed for the FLT3 -ITD mutation through fragment analysis. In terms of sensitivity, the analysis of cDNA was superior to that of DNA, quantifying higher allelic ratio values in most cases at diagnosis, and thus optimizing the detection of minor clones and prognostic classification. Regarding the last sample before post-HSCT relapse, cDNA analysis anticipated relapse in most cases, unlike DNA analyses. With regard to the post-FLT3i follow-up, FLT3 -ITD expression was reduced after the first FLT3i cycle when the treatment was effective, whereas it was not reduced in refractory patients. FLT3 -ITD expression could be a useful additional biomarker at diagnosis and for the assessment of MRD after allo-HSCT and FLT3i in AML.

Despite advances in the understanding of the pathophysiology of cytomegalovirus (CMV) infection, it remains as one of the most common infectious complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The aim of this study was to determine the genotype of cytokines and chemokines in donor and recipient and their association with CMV reactivation. Eighty-five patients receiving an allo-HSCT from an HLA-identical sibling donor were included in the study. Fifty genes were selected for their potential role in the pathogenesis of CMV infection. CMV DNAemia was evaluated until day 180 after allo-HSCT. CMV reactivation was observed in 51/85 (60%) patients. Of the 213 genetic variants selected, 11 polymorphisms in 7 different genes (CXCL12, IL12A, KIR3DL1, TGFB2, TNF, IL1RN, and CD48) were associated with development or protection from CMV reactivation. A predictive model using five of such polymorphisms (CXCL12 rs2839695, IL12A rs7615589, KIR3DL1 rs4554639, TGFB2 rs5781034 for the recipient and CD48 rs2295615 for the donor) together with the development of acute GVHD grade III/IV improved risk stratification of CMV reactivation. In conclusion, the data presented suggest that the screening of five polymorphisms in recipient and donor pre-transplantation could help to predict the individual risk of CMV infection development after HLA-identical allo-HSCT., (© 2022. The Author(s).)

Although next-generation sequencing (NGS) data on lymphomas require further validation before being implemented in daily practice, the clinical application of NGS can be considered right around the corner. The aim of our study was to validate an NGS lymphoid panel for tissue and liquid biopsy with the most common types of non-Hodgkin's lymphoma [follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL)]. In this series, 372 somatic alterations were detected in 93.6% (44/47) of the patients through tissue biopsy. In FL, we identified 93 somatic alterations, with a median of 7.4 mutations per sample. In DLBCL, we detected 279 somatic variants with a median of 8.6 mutations (range 0-35). In 92% (24/26) of the cases, we were able to detect some variant in the circulating tumor DNA. We detected a total of 386 variants; 63.7% were detected in both types of samples, 13.2% were detected only in the circulating tumor DNA, and 23% were detected only in the tissue biopsy. We found a correlation between the number of circulating tumor DNA mutations, advanced stage, and bulky disease. The genetic alterations detected in this panel were consistent with those previously described at diagnosis. The liquid biopsy sample is therefore a complementary tool that can provide new genetic information, even in cases where a solid biopsy cannot be performed or an insufficient sample was obtained. In summary, we describe and analyze in this study the findings and difficulties encountered when incorporating liquid biopsy into clinical practice in non-Hodgkin's lymphoma at diagnosis., (© 2021. The Author(s).)

Background: The incorporation of genetic variables into risk scores for predicting venous thromboembolic events (VTE) could improve their capacity to identify those patients for whom thromboprophylaxis would be most beneficial. Proof-of-concept of this is provided by the TiC-ONCO score for predicting the risk of VTE in patients with solid tumours. Our aim was to develop a similarly improved tool-the TiC-LYMPHO score-for predicting VTE in patients with lymphoma., Methods: In a retrospective observational study of 208 patients with lymphoma, 31 (14.9%) were found to have experienced an episode of VTE either at the time of diagnosis or over the next 6 months. Clinical variables associated with VTE, determined via logistic regression analysis, plus the same genetic variables included in the TiC-ONCO score, were used to build the TiC-LYMPHO score algorithm. The sensitivity, specificity, predictive values and AUC of the TiC-LYMPHO, the Khorana and ThroLy scores were compared in the same population., Results: The TiC-LYMPHO score showed a significantly higher AUC, sensitivity and NPV (0.783, 95.35% and 97.98% respectively) than the other scores. The ThroLy score showed a significantly higher specificity (96.43% vs. 54.49%; p < 0.0001) and PPV (37.50% vs. 26.36%; p = 0.0147) than the TiC-LYMPHO score, whereas its AUC, sensitivity and NPV were significantly lower (0.579, 19.35% and 86.48%, respectively)., Conclusion: These results show that by incorporating genetic and clinical data into VTE risk assessment, the TiC-LYMPHO score can categorize patients with lymphoma better in terms of their risk of VTE and allow individualized thromboprophylaxis to be prescribed., (© 2021 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Objectives: In the context of the Covid-19 pandemic, the fast development of vaccines with efficacy of around 95% preventing Covid-19 illness provides a unique opportunity to reduce the mortality associated with the pandemic. However, in the absence of efficacious prophylactic medications and few treatments for this infection, the induction of a fast and robust protective immunity is required for effective disease control, not only to prevent the disease but also the infection and shedding/transmission. The objective of our study was to analyze the level of specific humoral and cellular T-cell responses against the spike protein of SARS-CoV-2 induced by two mRNA-based vaccines (BNT162b2 and mRNA-1273), but also how long it takes after vaccination to induce these protective humoral and cellular immune responses., Methods: We studied in 40 healthy (not previously infected) volunteers vaccinated with BNT162b2 or mRNA-1273 vaccines the presence of spike-specific IgG antibodies and SARS-CoV-2-specific T cells at 3, 7 and 14 days after receiving the second dose of the vaccine. The specific T-cell response was analyzed stimulating fresh whole blood from vaccinated volunteers with SARS-CoV-2 peptides and measuring the release of cytokines secreted by T cells in response to SARS-CoV-2 stimulation., Results: Our results indicate that the immunization capacity of both vaccines is comparable. However, although both BNT162b2 and mRNA-1273 vaccines can induce early B-cell and T-cell responses, these vaccine-mediated immune responses do not reach their maximum values until 14 days after completing the vaccination schedule., Conclusion: This refractory period in the induction of specific immunity observed after completing the vaccination could constitute a window of higher infection risk, which could explain some emerging cases of SARS-CoV-2 infection in vaccinated people., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gil-Manso, Carbonell, López-Fernández, Miguens, Alonso, Buño, Muñoz, Ochando, Pion and Correa-Rocha.)

Myeloid neoplasms (MN) with germline predisposition (MNGP) are likely to be more common than currently appreciated. Many of the genes involved in MNGP are also recurrently mutated in sporadic MN. Therefore, routine analysis of gene panels by next-generation sequencing provides an effective approach to detect germline variants with clinical significance in patients with hematological malignancies. Gene panel sequencing was performed in 88 consecutive and five nonconsecutive patients with MN diagnosis. Disease-causing germline mutations in CEBPα, ASXL1, TP53, MPL, GATA2, DDX41, and ETV6 genes were identified in nine patients. Six out of the nine patients with germline variants had a strong family history. These patients presented great heterogeneity in the age of diagnosis and phenotypic characteristics. In our study, there were families in which all the affected members presented the same subtype of disease, whereas members of other families presented various disease phenotypes. This intrafamiliar heterogeneity suggests that the acquisition of particular somatic variants may drive the evolution of the disease. This approach enabled high-throughput detection of MNGP in patients with MN diagnosis, which is of great relevance for both the patients themselves and the asymptomatic mutation carriers within the family. It is crucial to make a proper diagnosis of these patients to provide them with the most suitable treatment, follow-up, and genetic counseling., (© 2021 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Conventional cytogenetics are the gold standard for the identification of chromosomal alterations recurrent in myeloid neoplasms. Some next-generation sequencing (NGS) panels are designed for the detection of copy number variations (CNV) or translocations; however, their use is far from being widespread. Here we report on the results of a commercial panel including frequent mutations, CNVs and translocations in myeloid neoplasms. Frequent chromosomal alterations were analyzed by NGS in 135 patients with myeloid neoplasms and three with acute lymphoblastic leukemia. NGS analysis was performed using the enrichment-capture Myeloid Neoplasm-GeneSGKit (Sistemas Genómicos, Spain) gene panel including 35 genes for mutational analysis and frequent CNVs and translocations. NGS results were validated with cytogenetics and/or MLPA when possible. A total of 66 frequent alterations included in NGS panel were detected, 48 of them detected by NGS and cytogenetics. Ten of them were observed only by cytogenetics (mainly trisomy 8), and another eight only by NGS (mainly deletion of 12p). Aside from this, 38 secondary CNVs were detected in any of the genes included mainly for mutational analysis. NGS represents a reliable complementary source of information for the analysis of CNVs and translocations. Moreover, NGS could be a useful tool for the detection of alterations not observed by conventional cytogenetics.

Although acute myeloid leukemia (AML) with NPM1 mut /FLT3 -ITD neg is a low-risk entity, its relapse rate remains high. Out of 333 AML patients, 27 were NPM1 mut , and were analyzed in greater detail in order to find associations between clinical and molecular features and cumulative incidence of relapse. Next-generation sequencing (NGS) was performed on diagnosis and remission samples using two capture-based panels. The presence of the FLT3 D835 variant at diagnosis and a qPCR value of NPM1 mut ≥0.1% after induction chemotherapy were associated with an increased probability of relapse, especially if both conditions are present together. By contrast, patients in which the main clone found at diagnosis harbored NPM1 variant had a lower risk of relapse. Nineteen of the 85 variants found at diagnosis were detected by NGS in remission. AML Subgroup with NPM1 mut / FLT3 -ITD neg is a heterogeneous entity, which can be further risk-stratified based on molecular biomarkers.

Background: Bardet-Biedl syndrome (BBS) is a rare autosomal recessive ciliopathy disorder. Many BBS disease-causing genetic variants have been identified due to the advancement of molecular diagnostic tools. We report on a novel pathogenic variant in a consanguineous Pakistani family with an affected child., Case Presentation: Clinical exome sequencing was used to search for BBS causing variants in the affected individual and identified a novel homozygous splice-site variant in the BBS9 gene (c.702 + 1del). Sanger sequencing was performed for variant validation and segregation studies. Expression analysis using mRNA levels to assess the functional impact of the novel variant demonstrated skipping of exon 7 in the affected alleles, suggesting a truncating effect. Three-dimensional structural modelling was used to predict pathogenicity of the variant residue and the alteration leads to a partial deletion of the PHTB1&#95;N domain and a total deletion of the PHTB1&#95;C domain., Conclusion: The study of this case expands the spectrum of biallelic variants in the BBS9 gene associated with BBS and increased the knowledge on the molecular consequences of splicing variation c.702 + 1del.

Haploidentical hematopoietic stem cell transplantation (Haplo-HSCT) with high-dose cyclophosphamide (PTCy) has resulted in a low incidence of graft-vs.-host disease (GVHD), graft failure, and non-relapse mortality. However, post-transplantation relapse remains a common cause of treatment failure in high-risk patients. Unraveling the mechanisms of relapse is therefore crucial for designing effective relapse treatment strategies. One of these mechanisms is the loss of the mismatched HLA on the recipient's leukemic cells. To study the incidence and clinical relevance of this phenomenon, we analyzed 181 patients treated with Haplo-HSCT with PTCy (2007-2019), of which 37 relapsed patients after transplantation. According to the kit employed for HLA-loss analysis, among 22 relapsed patients, we identified HLA loss at relapse in 6 of the 22 patients (27%) studied. Based on the results obtained, the genomic loss of HLA was more common in females than males (66 vs. 33%) and HLA-loss relapses occurred later than classical relapses (345 vs. 166 days). Moreover, the patients with HLA-loss had a greater presence of active disease at the time of transplantation and had undergone a larger number of treatment lines than the group with classical relapses (66 vs. 43% and 66 vs. 18%, respectively). Four of these relapses were studied retrospectively, while two were studied prospectively, the results of which could be considered for patient management. Additionally, two relapsed patients analyzed retrospectively had myeloid neoplasms. One patient had not undergone any treatment, and three had undergone donor lymphocyte infusions (DLIs) and chemotherapy. All presented severe GVHD and disease progression. In contrast, the two patients studied prospectively had a lymphoid neoplasm and were not treated with DLIs. One of them was treated with chemotherapy but died from disease progression, and the other patient underwent a second Haplo-HSCT from a different donor and is still alive. We can conclude that the detection of HLA-loss at the onset of relapse after Haplo-HSCT with PTCy could help in clinical practice to select appropriate rescue treatment, thereby avoiding the use of DLIs or a second transplantation from the same donor., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Muñiz, Kwon, Carbonell, Chicano, Bailén, Oarbeascoa, Suárez-González, Andrés-Zayas, Menárguez, Dorado, Gómez-Centurión, Anguita, Díez-Martín, Martínez-Laperche and Buño.)

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) has shown favorable results in the treatment of hematological malignancies. Despite the use of post-transplant cyclophosphamide (PTCy), graft versus host disease (GVHD) remains as one of the main complications in this setting. Since the skin appears affected in up to 80% of cases of acute GVHD (aGVHD), its prognosis and diagnosis are essential for the correct management of these patients. Plasma concentration of elafin, an elastase inhibitor produced by keratinocytes, has been described elevated at the diagnosis of skin GVHD, correlated with the grade of GVHD, and associated with an increased risk of death. In this study we explored elafin plasma levels in the largest series reported of T cell-replete haplo-HSCT with PTCy. Plasma samples drawn from 87 patients at days +15 and +30 were analyzed ("discovery cohort"). Elafin levels at days +15 were no associated with chronic GVHD, non-relapse mortality, relapse, therapy-resistant GVHD, or overall survival. In our series, elafin levels at day +30 were not associated with post-transplant complications. On the other hand, elafin plasma levels at day +15 were higher in patients with severe skin aGVHD (21,313 vs. 14,974 pg/ml; p = 0.01). Of note, patients with higher elafin plasma levels at day +15 presented a higher incidence of stage III-IV skin aGVHD (HR = 18.9; p < 0.001). These results were confirmed (HR = 20.6; p < 0.001) in an independent group of patients (n = 62), i.e. the "validation cohort." These data suggest that measurement of elafin in patients undergoing haplo-HSCT with PTCy might be useful for an early identification of those patients who are at higher risk of suffering severe skin aGVHD and thus, improve their treatment and prognosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Solán, Carbonell, Muñiz, Dorado, Landete, Chicano-Lavilla, Anguita, Gayoso, Kwon, Díez-Martín, Martínez-Laperche and Buño.)

Post-transplant cyclophosphamide (PTCY) effectively prevents graft-versus-host disease after unmanipulated HLA-haploidentical HSCT. The use of PTCY in the unrelated donor HSCT setting is less explored. We conducted a retrospective study of 132 consecutive patients undergoing a matched or 9/10 mismatched unrelated donor HSCT in 4 centers in Spain, 60 with anti-thymocyte globulin (ATG)-based prophylaxis combined with MTX-CsA, and 72 using a PTCY-based regimen. Peripheral blood stem cells were used as graft in most patients (111 patients, 84%); mMUD donors were balanced between groups. Cumulative incidences of grades II-IV and III-IV acute GVHD at 100 days were lower in the PTCy group (46% vs. 67%, p = 0.008; 3% vs. 34%, p = 0.003), without statistically significant differences in the 2-year cumulative incidence of chronic moderate-severe GVHD. At 2 years, no significant differences were observed in overall survival, event-free survival, cumulative incidence of relapse, and non-relapse mortality. GVHD was the most frequent cause of NRM in the ATG group. No differences were observed between groups in the composite endpoint of GVHD-free and relapse-free survival. In this study, PTCy combined with additional immunosuppression after MUD/mMUD HSCT showed a reduction of aGVHD rate with safety results comparable to those obtained with the ATG-based prophylaxis.

This paper focuses on the problems of estimation and variable selection in the functional linear regression model (FLM) with functional response and scalar covariates. To this end, two different types of regularization (L 1 and L 2 ) are considered in this paper. On the one hand, a sample approach for functional LASSO in terms of basis representation of the sample values of the response variable is proposed. On the other hand, we propose a penalized version of the FLM by introducing a P-spline penalty in the least squares fitting criterion. But our aim is to propose P-splines as a powerful tool simultaneously for variable selection and functional parameters estimation. In that sense, the importance of smoothing the response variable before fitting the model is also studied. In summary, penalized (L 1 and L 2 ) and nonpenalized regression are combined with a presmoothing of the response variable sample curves, based on regression splines or P-splines, providing a total of six approaches to be compared in two simulation schemes. Finally, the most competitive approach is applied to a real data set based on the graft-versus-host disease, which is one of the most frequent complications (30% -50%) in allogeneic hematopoietic stem-cell transplantation., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chimerism refers to the relative proportion of donor and recipient DNA after hematopoietic stem cell transplantation (HSCT) and its quantitative follow-up is of great clinical utility in this setting. PCR of short tandem repeats (STR-PCR) constitutes the gold standard method for chimerism quantification, although more sensitive PCR techniques (such as qPCR) have recently arisen. We compared the sensitivity and the quantification capacity of both techniques in patient samples and artificial mixtures and demonstrated adequate performance of both methods, with higher sensitivity of qPCR and better quantification skills of STR-PCR. By qPCR, we then prospectively followed up 57 patients that were in complete chimerism (CC) by STR-PCR. Twenty-seven patients (59%) showed 0.1-1% recipient DNA in the bone marrow. Only 4 patients presented 0.1-1% recipient DNA in peripheral blood (PB), and one of them relapsed. Finally, by qPCR, we retrospectively studied the last sample that showed CC by STR-PCR prior to relapse in 8 relapsed patients. At a median of 59 days prior to relapse, six patients presented mixed chimerism by qPCR in PB. Since both approaches have complementary characteristics, we conclude that different techniques should be applied in different clinical settings and therefore propose a methodological algorithm for chimerism follow-up after HSCT.