An integrated prognostic model for diffuse large B-cell lymphoma treated with immunochemotherapy.

Diffuse large B-cell lymphoma (DLBCL), the most frequent non-Hodgkin's lymphoma subtype, is characterized by strong biological, morphological, and clinical heterogeneity, but patients are treated with immunochemotherapy in a relatively homogeneous way. Here, we have used a customized NanoString platform to analyze a series of 197 homogeneously treated DLBCL cases. The platform includes the most relevant genes or signatures known to be useful for predicting response to R-CHOP (Rituximab, Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone) in DLBCL cases. We generated a risk score that combines the International Prognostic Index with cell of origin and double expression of MYC/BCL2, and stratified the series into three groups, yielding hazard ratios from 0.15 to 5.49 for overall survival, and from 0.17 to 5.04 for progression-free survival. Group differences were highly significant (p < 0.0001), and the scoring system was applicable to younger patients (<60 years of age) and patients with advanced or localized stages of the disease. Results were validated in an independent dataset from 166 DLBCL patients treated in two distinct clinical trials. This risk score combines clinical and biological data in a model that can be used to integrate biological variables into the prognostic models for DLBCL cases.

Clinical Evaluation of Serum Levels of SARS-CoV-2 Anti-Spike Protein IgG Antibodies in Infected Patients and Vaccinated Subjects.

BACKGROUND: The aim was clinical evaluation of immune response against SARS-CoV-2, analyzing serum levels of IgG antibodies against the SARS-CoV-2 protein S in infected and vaccinated patients, as well as in subjects with and without frequent comorbidities (arterial hypertension, diabetes mellitus, heart disease, and chronic respiratory disease). METHODS: Patients infected by SARS-CoV-2 confirmed by RT-PCR and subjects vaccinated with vaccines based on the mRNA encoding the SARS-CoV-2 protein S were studied. SARS-CoV-2 anti-S IgG serum levels were quantified by chemiluminescent microparticle immunoassay. RESULTS: There were 79 infected patients with a median age of 53.0 years; 35 women and 44 men; 42 patients with any comorbidities and 37 without comorbidities. The median of SARS-CoV-2 anti-S IgG serum level was 203.4 BAU/mL (11.6 - 5,620.6). The median antibody level in the infected patients with any comorbidities was higher than those without comorbidities. The group of vaccinated subjects included 96 subjects with a median age of 49.5 years; 77 women and 19 men; 31 subjects with any comorbidities and 65 without comorbidities. The median of SARS-CoV-2 anti-S IgG serum levels was 1,145.6 BAU/mL (138.3 - 4,828.1). No significant differences were found in terms of specific or global comorbidities in the vaccinated subjects. CONCLUSIONS: SARS-CoV-2 anti-S IgG serum levels were 5.6 times higher in vaccinated subjects than infected patients. The vaccination produces higher serum antibody levels than SARS-CoV-2 infection. This reinforces the indication for the vaccine in infected patients. These antibodies did not decrease significantly in patients with frequent comorbidities such as hypertension, diabetes, heart disease or chronic respiratory disease.

Peripheral T-cell lymphoma: molecular profiling recognizes subclasses and identifies prognostic markers.

Peripheral T-cell lymphoma (PTCL) is a clinically aggressive disease, with a poor response to therapy and a low overall survival rate of approximately 30% after 5 years. We have analyzed a series of 105 cases with a diagnosis of PTCL using a customized NanoString platform (NanoString Technologies, Seattle, WA) that includes 208 genes associated with T-cell differentiation, oncogenes and tumor suppressor genes, deregulated pathways, and stromal cell subpopulations. A comparative analysis of the various histological types of PTCL (angioimmunoblastic T-cell lymphoma [AITL]; PTCL with T follicular helper [TFH] phenotype; PTCL not otherwise specified [NOS]) showed that specific sets of genes were associated with each of the diagnoses. These included TFH markers, cytotoxic markers, and genes whose expression was a surrogate for specific cellular subpopulations, including follicular dendritic cells, mast cells, and genes belonging to precise survival (NF-κB) and other pathways. Furthermore, the mutational profile was analyzed using a custom panel that targeted 62 genes in 76 cases distributed in AITL, PTCL-TFH, and PTCL-NOS. The main differences among the 3 nodal PTCL classes involved the RHOAG17V mutations (P < .0001), which were approximately twice as frequent in AITL (34.09%) as in PTCL-TFH (16.66%) cases but were not detected in PTCL-NOS. A multivariate analysis identified gene sets that allowed the series of cases to be stratified into different risk groups. This study supports and validates the current division of PTCL into these 3 categories, identifies sets of markers that can be used for a more precise diagnosis, and recognizes the expression of B-cell genes as an IPI-independent prognostic factor for AITL.

Utility of icteric index in clinical laboratories: more than a preanalytical indicator.

INTRODUCTION: Total bilirubin tests are highly demanded in clinical laboratories. Since icteric index (I-index) has zero cost, we aimed to evaluate its clinical utility and cost-effectiveness to determine if total bilirubin is necessary to be tested. We took into account if haemolysis could interfere to icteric index determination. MATERIAL AND METHODS: Retrospectively we reviewed I-index results in two cohorts (43,372 and 8507 non-haemolysed and haemolysed samples, respectively). All determinations were done using Alinity c chemistry analysers (Abbott Diagnostics). Receiver operating characteristic (ROC) curve was used to determine the optimal index cut-off to discriminate between normal and abnormal bilirubin concentration (20.5 µmol/L). RESULTS: The ROC curve analysis suggested 21.4 µmol/L as the optimal I-index cut-off but differences in sensitivity and specificity were detected between patient derivation. For rejecting purpose, 15.4 µmol/L and 17.1 µmol/L I-index thresholds were selected based on patient derivation (inpatients and emergency room; and primary care and outpatients, respectively) with 97% sensitivity and 0.25% false negative results. Sensitivity was much lower in haemolysed samples. We selected 34.2 µmol/L I-index as threshold to detect hyperbilirubinemia with 99.7% specificity and 0.26% false positive results, independent of haemolysis. With the icteric index cut-offs proposed, we would save 66% of total bilirubin requested and analyse total bilirubin in around 2% of samples without total bilirubin requested. CONCLUSIONS: This study supports the use of I-index to avoid bilirubin determination and to identify patients with hyperbilirubinemia. This work considers that the economic and test savings could help to increase the efficiency in clinical laboratories.

Identification of tipifarnib sensitivity biomarkers in T-cell acute lymphoblastic leukemia and T-cell lymphoma.

Patients diagnosed with T-cell leukemias and T-cell lymphomas (TCLs) still have a poor prognosis and an inadequate response to current therapies, highlighting the need for targeted treatments. We have analyzed the potential therapeutic value of the farnesyltransferase inhibitor, tipifarnib, in 25 TCL cell lines through the identification of genomic and/or immunohistochemical markers of tipifarnib sensitivity. More than half of the cell lines (60%) were considered to be sensitive. Tipifarnib reduced cell viability in these T-cell leukemia and TCL cell lines, induced apoptosis and modified the cell cycle. A mutational study showed TP53, NOTCH1 and DNMT3 to be mutated in 84.6%, 69.2% and 30.0% of sensitive cell lines, and in 62.5%, 0% and 0% of resistant cell lines, respectively. An immunohistochemistry study showed that p-ERK and RelB were associated as potential biomarkers of tipifarnib sensitivity and resistance, respectively. Data from RNA-seq show that tipifarnib at IC50 after 72 h downregulated a great variety of pathways, including those controlling cell cycle, metabolism, and ribosomal and mitochondrial activity. This study establishes tipifarnib as a potential therapeutic option in T-cell leukemia and TCL. The mutational state of NOTCH1, p-ERK and RelB could serve as potential biomarkers of tipifarnib sensitivity and resistance.

Molecular basis of targeted therapy in T/NK-cell lymphoma/leukemia: A comprehensive genomic and immunohistochemical analysis of a panel of 33 cell lines.

T and NK-cell lymphoma is a collection of aggressive disorders with unfavorable outcome, in which targeted treatments are still at a preliminary phase. To gain deeper insights into the deregulated mechanisms promoting this disease, we searched a panel of 31 representative T-cell and 2 NK-cell lymphoma/leukemia cell lines for predictive markers of response to targeted therapy. To this end, targeted sequencing was performed alongside the expression of specific biomarkers corresponding to potentially activated survival pathways. The study identified TP53, NOTCH1 and DNMT3A as the most frequently mutated genes. We also found common alterations in JAK/STAT and epigenetic pathways. Immunohistochemical analysis showed nuclear accumulation of MYC (in 85% of the cases), NFKB (62%), p-STAT (44%) and p-MAPK (30%). This panel of cell lines captures the complexity of T/NK-cell lymphoproliferative processes samples, with the partial exception of AITL cases. Integrated mutational and immunohistochemical analysis shows that mutational changes cannot fully explain the activation of key survival pathways and the resulting phenotypes. The combined integration of mutational/expression changes forms a useful tool with which new compounds may be assayed.

Mutation prevalence of cerebral cavernous malformation genes in Spanish patients.

OBJECTIVE: To study the molecular genetic and clinical features of cerebral cavernous malformations (CCM) in a cohort of Spanish patients. METHODS: We analyzed the CCM1, CCM2, and CCM3 genes by MLPA and direct sequencing of exons and intronic boundaries in 94 familial forms and 41 sporadic cases of CCM patients of Spanish extraction. When available, RNA studies were performed seeking for alternative or cryptic splicing. RESULTS: A total of 26 pathogenic mutations, 22 of which predict truncated proteins, were identified in 29 familial forms and in three sporadic cases. The repertoire includes six novel non-sense and frameshift mutations in CCM1 and CCM3. We also found four missense mutations, one of them located at the third NPXY motif of CCM1 and another one that leads to cryptic splicing of CCM1 exon 6. We found four genomic deletions with the loss of the whole CCM2 gene in one patient and a partial loss of CCM1and CCM2 genes in three other patients. Four families had mutations in CCM3. The results include a high frequency of intronic variants, although most of them localize out of consensus splicing sequences. The main symptoms associated to clinical debut consisted of cerebral haemorrhage, migraines and epileptic seizures. The rare co-occurrence of CCM with Noonan and Chiari syndromes and delayed menarche is reported. CONCLUSIONS: Analysis of CCM genes by sequencing and MLPA has detected mutations in almost 35% of a Spanish cohort (36% of familial cases and 10% of sporadic patients). The results include 13 new mutations of CCM genes and the main clinical symptoms that deserves consideration in molecular diagnosis and genetic counselling of cerebral cavernous malformations.

Clinical and molecular study of the extracellular matrix protein 1 gene in a spanish family with lipoid proteinosis.

BACKGROUND: Lipoid proteinosis (LP) is a rare autosomal recessive disorder characterized by a hoarse voice, variable scarring, and infiltration of the skin and mucosa. This disease is associated with mutations of the gene encoding extracellular matrix protein 1 (ECM1). CASE REPORT: This was a clinical and molecular study of a new case of LP with a severe phenotype. A 35-year-old female born to nonconsanguineous parents developed dermatological and extracutaneous symptoms in her 9th month of life. The neurological abnormalities of the disease began to appear at the age of 19 years. Computed tomography revealed cranial calcifications. CONCLUSIONS: The diagnosis of LP was confirmed by histopathological findings and direct sequencing of ECM1. A new homozygous nonsense mutation was identified in exon 7 of ECM1, c.1076G>A (p.Trp359(*)). This mutation was not detected in 106 chromosomes of healthy individuals with a similar demographic origin. Microsatellite markers around ECM1 were used to construct the haplotype in both the parents and the patient. Reports on genotype-phenotype correlations in LP point to a milder phenotype in carriers of missense mutations in the Ecm1a isoform, whereas mutations in the Ecm1b isoform are thought to be associated with more severe phenotypes. The present findings in a Spanish patient carrying a truncating mutation in exon 7 revealed complete dermatological and neurological manifestations.