Clinical impact of the subclonal architecture and mutational complexity in chronic lymphocytic leukemia.

Genome studies of chronic lymphocytic leukemia (CLL) have revealed the remarkable subclonal heterogeneity of the tumors, but the clinical implications of this phenomenon are not well known. We assessed the mutational status of 28 CLL driver genes by deep-targeted next-generation sequencing and copy number alterations (CNA) in 406 previously untreated patients and 48 sequential samples. We detected small subclonal mutations (0.6-25% of cells) in nearly all genes (26/28), and they were the sole alteration in 22% of the mutated cases. CNA tended to be acquired early in the evolution of the disease and remained stable, whereas the mutational heterogeneity increased in a subset of tumors. The prognostic impact of different genes was related to the size of the mutated clone. Combining mutations and CNA, we observed that the accumulation of driver alterations (mutational complexity) gradually shortened the time to first treatment independently of the clonal architecture, IGHV status and Binet stage. Conversely, the overall survival was associated with the increasing subclonal diversity of the tumors but it was related to the age of patients, IGHV and TP53 status of the tumors. In conclusion, our study reveals that both the mutational complexity and subclonal diversity influence the evolution of CLL.

Genetic landscape and deregulated pathways in B-cell lymphoid malignancies.

With the introduction of next-generation sequencing, the genetic landscape of the complex group of B-cell lymphoid malignancies has rapidly been unravelled in recent years. This has provided important information about recurrent genetic events and identified key pathways deregulated in each lymphoma subtype. In parallel, there has been intense search and development of novel types of targeted therapy that 'hit' central mechanisms in lymphoma pathobiology, such as BTK, PI3K or BCL2 inhibitors. In this review, we will outline the current view of the genetic landscape of selected entities: follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, chronic lymphocytic leukaemia and marginal zone lymphoma. We will detail recurrent alterations affecting important signalling pathways, that is the B-cell receptor/NF-κB pathway, NOTCH signalling, JAK-STAT signalling, p53/DNA damage response, apoptosis and cell cycle regulation, as well as other perhaps unexpected cellular processes, such as immune regulation, cell migration, epigenetic regulation and RNA processing. Whilst many of these pathways/processes are commonly altered in different lymphoid tumors, albeit at varying frequencies, others are preferentially targeted in selected B-cell malignancies. Some of these genetic lesions are either involved in disease ontogeny or linked to the evolution of each disease and/or specific clinicobiological features, and some of them have been demonstrated to have prognostic and even predictive impact. Future work is especially needed to understand the therapy-resistant disease, particularly in patients treated with targeted therapy, and to identify novel targets and therapeutic strategies in order to realize true precision medicine in this clinically heterogeneous patient group.

Identifying and quantifying geochemical and mixing processes in the Matanza-Riachuelo Aquifer System, Argentina.

The Matanza-Riachuelo River Basin, in the Northeast of the Buenos Aires Province, is one of the most industrialized and populated region in Argentina and it is worldwide known for its alarming environmental degradation. In order to prevent further damages, the aquifer system, which consists of two overlaid aquifers, is being monitored from 2008 by the river basin authority, Autoridad de la Cuenca Matanza-Riachuelo. The groundwater chemical baseline has been established in a previous paper (Zabala et al., 2016), and this one is devoted to the identification of the main physical and hydrogeochemical processes that control groundwater chemistry and its areal distribution. Thirty five representative groundwater samples from the Upper Aquifer and thirty four from the deep Puelche Aquifer have been studied with a multi-tool approach to understand the origin of their chemical and isotopic values. The resulting conceptual model has been validated though hydrogeochemical modeling. Most of the aquifer system has fresh groundwater, but some areas have brackish and salt groundwater. Water recharging the Upper Aquifer is of the Ca-HCO3 type as a result of soil CO2 and carbonate dissolution. Evapotranspiration plays a great role concentrating recharge water. After recharge, groundwater becomes Na-HCO3, mostly due to cation exchange with Na release and Ca uptake, which induces calcite dissolution. Saline groundwaters exist in the lower and upper sectors of the basin as a result of Na-HCO3 water mixing with marine water of different origins. In the upper reaches, besides mixing with connate sea water other sources of SO4 exist, most probably gypsum and/or sulfides. This work highlights the relevance of performing detailed studies to understand the processes controlling groundwater chemistry at regional scale. Moreover, it is a step forward in the knowledge of the aquifer system, and provides a sound scientific basis to design effective management programs and recovery plans.

A B-cell epigenetic signature defines three biologic subgroups of chronic lymphocytic leukemia with clinical impact.

Prospective identification of patients with chronic lymphocytic leukemia (CLL) destined to progress would greatly facilitate their clinical management. Recently, whole-genome DNA methylation analyses identified three clinicobiologic CLL subgroups with an epigenetic signature related to different normal B-cell counterparts. Here, we developed a clinically applicable method to identify these subgroups and to study their clinical relevance. Using a support vector machine approach, we built a prediction model using five epigenetic biomarkers that was able to classify CLL patients accurately into the three subgroups, namely naive B-cell-like, intermediate and memory B-cell-like CLL. DNA methylation was quantified by highly reproducible bisulfite pyrosequencing assays in two independent CLL series. In the initial series (n=211), the three subgroups showed differential levels of IGHV (immunoglobulin heavy-chain locus) mutation (P<0.001) and VH usage (P<0.03), as well as different clinical features and outcome in terms of time to first treatment (TTT) and overall survival (P<0.001). A multivariate Cox model showed that epigenetic classification was the strongest predictor of TTT (P<0.001) along with Binet stage (P<0.001). These findings were corroborated in a validation series (n=97). In this study, we developed a simple and robust method using epigenetic biomarkers to categorize CLLs into three subgroups with different clinicobiologic features and outcome.

Effect of CYP3A5*3 on kidney transplant recipients treated with tacrolimus: a systematic review and meta-analysis of observational studies.

The highly variable pharmacokinetics of tacrolimus can hamper the optimal management of kidney transplant patients. This variability has been attributed to the genetic polymorphism of CYP3A5 6986A>G, but the evidence is not clear. We conducted a meta-analysis of studies evaluating the effect of CYP3A5 polymorphism on kidney transplant recipients with tacrolimus plasma concentration divided by daily dose per body weight (C/D) and clinical outcomes. We searched in MEDLINE and EMBASE. We found evidence suggesting a significantly lower C/D among CYP3A5*1 allele carriers compared with carriers of the CYP3A5*3/*3 genotype at weeks 1 and 2, and months 1, 3, 6 and 12. We demonstrated that the expresser genotype might have higher risk of acute rejection and chronic nephrotoxicity. In conclusion, CYP3A5 6986A>G polymorphism can affect tacrolimus pharmacokinetics and the incidence of acute rejection and chronic nephrotoxicity on kidney transplant recipients. Patients at high risk of developing tacrolimus-related complications could be detected even before their kidney transplant.

NOTCH1 mutations identify a genetic subgroup of chronic lymphocytic leukemia patients with high risk of transformation and poor outcome.

NOTCH1 has been found recurrently mutated in a subset of patients with chronic lymphocytic leukemia (CLL). To analyze biological features and clinical impact of NOTCH1 mutations in CLL, we sequenced this gene in 565 patients. NOTCH1 mutations, found in 63 patients (11%), were associated with unmutated IGHV, high expression of CD38 and ZAP-70, trisomy 12, advanced stage and elevated lactate dehydrogenase. Sequential analysis in 200 patients demonstrated acquisition of mutation in one case (0.5%) and disappearance after treatment in two. Binet A and B patients with NOTCH1-mutated had a shorter time to treatment. NOTCH1-mutated patients were more frequently refractory to therapy and showed shorter progression-free and overall survival after complete remission. Overall survival was shorter in NOTCH1-mutated patients, although not independently from IGHV. NOTCH1 mutation increased the risk of transformation to diffuse large B-cell lymphoma independently from IGHV, with this being validated in resampling tests of replicability. In summary, NOTCH1 mutational status, that was rarely acquired during the course of the disease, identify a genetic subgroup with high risk of transformation and poor outcome. This recently identified genetic subgroup of CLL patients deserves prospective studies to define their best management.

SNP-based mapping arrays reveal high genomic complexity in monoclonal gammopathies, from MGUS to myeloma status.

Genetic events mediating transformation from premalignant monoclonal gammopathies (MG) to multiple myeloma (MM) are unknown. To obtain a comprehensive genomic profile of MG from the early to late stages, we performed high-resolution analysis of purified plasma cells from 20 MGUS, 20 smoldering MM (SMM) and 34 MM by high-density 6.0 SNP array. A progressive increase in the incidence of copy number abnormalities (CNA) from MGUS to SMM and to MM (median 5, 7.5 and 12 per case, respectively) was observed (P=0.006). Gains on 1q, 3p, 6p, 9p, 11q, 19p, 19q and 21q along with 1p, 16q and 22q deletions were significantly less frequent in MGUS than in MM. Although 11q and 21q gains together with 16q and 22q deletions were apparently exclusive of MM status, we observed that these abnormalities were also present in minor subclones in MGUS. Overall, a total of 65 copy number-neutral LOH (CNN-LOH) were detected. Their frequency was higher in active MM than in the asymptomatic entities (P=0.047). A strong association between genetic lesions and fragile sites was also detected. In summary, our study shows an increasing genomic complexity from MGUS to MM and identifies new chromosomal regions involved in CNA and CNN-LOH.

Haga su diagnóstico / Do your diagnostic

Presentamos un caso de esta entidad en una paciente caucásica, con lesiones maculosas purpúricas en zona tibial posterior de pierna derecha, sin afectación sistémica, de curso crónico y persistente en la actualidad, tras dieciocho meses de seguimiento con mejoría parcial, después de recibir tratamiento con esteroides e inmunosupresores tópicos. Los anticonceptivos orales y la hipercolesterolemia leve, podrían ser datos asociados en nuestro caso a dicha entidad, aunque se necesitarían más estudios para confirmar esta hipótesis.

We present a case of this entity in a Caucasian, patient with purpuric macular lesion area with her right posterior tibial, without affecting systemic, chronic and persistent course now, after eighteen months of follow-up with partial improvement after treatment with topical steroids and immunosuppressors. Oral contraceptives and mild hypercholesterolemia may be associated data in our case to the entity, althought further studies are needed to confirm this hypothesis.

Micosis fungoide liquenoide atípica / Unusual lichenoid mycosis fungoides

Presentamos un caso de micosis fungoide liquenoide. La paciente presentó prurito intenso, pero con una buena evolución hasta el momento. Tanto clínica como histológicamente se correspondía con un patrón liquenoide, hecho de excepcional frecuencia. Respondió al tratamiento esteroideo tópico de alta potencia.

A 53-years-old woman with a lichenoid mycoses fungoides is reported. Both clinical and histological lichenoid pattern was finding, which is unusual. The treatment with high potency corticoids was successfully and the outcome is favorable up to now.

Influence of pharmacogenetic polymorphisms in routine immunosuppression therapy after renal transplantation.

Pharmacogenetics is the study of the cause of various individual responses to the same pharmacologic therapy. Genetic alterations in a single nucleotide in the genes responsible for transport and metabolism of an immunosuppression drug may modify patient response. Although pharmacogenetics is of interest, its clinical relevance remains to be demonstrated. The objective of the present study was to evaluate the effect of single-nucleotide polymorphisms (SNPs) in renal transplant recipients and their donors relative to blood concentrations of tacrolimus in the first 2 weeks posttransplantation. Seventy-one blood samples each from renal transplant recipients and their donors were analyzed using a genetic analysis system (MassARRAY; Sequenom, Inc, San Diego, California) in an attempt to characterize the more relevant SNPs of the ABCB1 and CYP3A5 genes for correlation with recipient trough concentrations of drug. Two-way analysis of variance and Bonferroni post hoc tests were used. In agreement with theoretical predictions, the wild-type genotype in ABCB1 SNPs (CC) tended to stabilize drug concentrations within the therapeutic range, whereas the T variant induced a mean increase in blood concentrations of more than 60%. These findings are in agreement with statistical tests that compared mean concentrations in various recipient-donor populations and found significant differences between them (P<.001) in CC vs TT, and P<.01 in CT vs TT). Donor genotype did not seem to be relevant. However, further studies are required to achieve more robust conclusions.

Geochemical and environmental controls on the genesis of soluble efflorescent salts in coastal mine tailings deposits: a discussion based on reactive transport modeling.

Water-soluble efflorescent salts often form on tailings in hyperarid climates. Their high solubility together with the high risk of human exposure to heavy metals such as Cu, Ni, Zn, etc., makes this occurrence a serious environmental problem. Understanding their formation (genesis) is therefore key to designing prevention and remediation strategies. A significant amount of these efflorescences has been described on the coastal area of Chañaral (Chile). There, highly soluble salts such as halite (NaCl) and eriochalcite (CuCl(2).2H(2)O) form on 4km(2) of marine shore tailings. Natural occurrence of eriochalcite is rare: its formation requires extreme environmental and geochemical conditions such as high evaporation rate and low relative air humidity, and continuous Cl and Cu supply from groundwater, etc. Its formation was examined by means of reactive transport modeling. A scenario is proposed involving sea water and subsequently a mixture of sea water/freshwater in the groundwater composition in the formation of these efflorescences. The strong competition from other halides (i.e. halite and silvite (KCl)) for the Cl may inhibit the precipitation of eriochalcite. Therefore, the Cl/Na ratio trend >1 is a key parameter in its formation. Cation-exchange between Na(+) and other major ions such as K(+), Ca(2+), Mg(2+) and Cu(2+) in the clay fraction of tailings is proposed to account for realistic Cl/Na ratios. With regard to preventing the formation of eriochalcite, a capillary barrier on the tailings surface is proposed as a suitable alternative. Its efficiency as a barrier is also tested by means of reactive transport models.

Checkpoint kinase 1 (CHK1) protein and mRNA expression is downregulated in aggressive variants of human lymphoid neoplasms.

CHK1: gene encodes for a serine/threonine kinase involved in the regulation of cell cycle progression and DNA damage checkpoints. To determine the role of CHK1 in the pathogenesis of lymphoid neoplasms and its relationship to other DNA damage response genes, we have analyzed the gene status, protein, and mRNA expression in a series of tumors and nonneoplastic lymphoid tissues. CHK1 protein and mRNA expression levels were very low in both reactive tissues and resting lymphoid cells, whereas tumor samples showed a variable pattern of expression related to their proliferative activity. However, seven aggressive tumors showed a dissociate pattern of extremely low or negative protein expression in spite of a high proliferative activity. Four of these tumors were diffuse large B-cell lymphomas (DLCLs) with concordant reduced levels of mRNA, whereas one blastoid mantle cell lymphoma (B-MCL) and two DLCLs had relatively normal levels of mRNA. No gene mutations, deletions, or hypermethylation of the promoter region were detected in any of these cases. In all these tumors ATM, CHK2, and p53 genes were wild type. These findings suggest that CHK1 inactivation in NHLs occurs by loss of protein expression in a subset of aggressive variants alternatively to ATM, CHK2, and p53 alterations.

Multiple cell cycle regulator alterations in Richter's transformation of chronic lymphocytic leukemia.

To investigate the role of the cell cycle regulators p21(Waf1), p27(Kip1), retinoblastoma (Rb), and cyclin D1 in Richter's transformation of chronic lymphocytic leukemia (CLL), we analyzed 19 CLL and eight Richter's syndrome (RS) tumors, previously characterized for p53 and ARF/INK4a abnormalities. p21(Waf1)immunohistochemical expression was negative in 12 of 15 CLL (80%), whereas it was moderate or strong in three of seven RS (43%). p21(Waf1) gene was in germline configuration in all the tumors analyzed. Four immunohistochemical patterns of p53 and p21(Waf1) expression were observed: (1) p53-/p21- in 10 of 15 CLL (67%), but only in two of six RS (33%); (2) p53+/p21+ in three CLL (20%) and two RS (33%); (3) p53-/p21+ in one RS; and (4) p53++/p21- in two CLL and one RS. Two p53+/p21+ CLL evolved into RS. p53 mutations clustered around the p53++/p21- (two CLL and one RS) and p53-/p21- (one CLL and one RS) tumors. While the majority of CLL displayed strong p27 immunoreactivity, RS tumors were constantly p27-negative. p27(Kip1) gene was in germline configuration in all the tumors analyzed. Most CLL cases were negative for Rb expression. In contrast, all RS exhibited strong Rb expression. Cyclin D1 overexpression was only detected in one CLL evolving into RS and one RS. In conclusion, a p53+/p21- immunohistochemical pattern is shown exclusively by p53-mutated CLL/RS. Additionally, our results suggest a possible implication of moderate/strong p21(Waf1) expression, loss of p27 expression, and cyclin D1 overexpression in the Richter's transformation of CLL.

BMI-1 gene amplification and overexpression in hematological malignancies occur mainly in mantle cell lymphomas.

The BMI-1 gene is a putative oncogene belonging to the Polycomb group family that cooperates with c-myc in the generation of mouse lymphomas and seems to participate in cell cycle regulation and senescence by acting as a transcriptional repressor of the INK4a/ARF locus. The BMI-1 gene has been located on chromosome 10p13, a region involved in chromosomal translocations in infant leukemias, and amplified in occasional non-Hodgkin's lymphomas (NHLs) and solid tumors. To determine the possible alterations of this gene in human malignancies, we have examined 160 lymphoproliferative disorders, 13 myeloid leukemias, and 89 carcinomas by Southern blot analysis and detected BMI-1 gene amplification (3- to 7-fold) in 4 of 36 (11%) mantle cell lymphomas (MCLs) with no alterations in the INK4a/ARF locus. BMI-1 and p16INK4a mRNA and protein expression were also studied by real-time quantitative reverse transcription-PCR and Western blot, respectively, in a subset of NHLs. BMI-1 expression was significantly higher in chronic lymphocytic leukemia and MCL than in follicular lymphoma and large B cell lymphoma. The four tumors with gene amplification showed significantly higher mRNA levels than other MCLs and NHLs with the BMI-1 gene in germline configuration. Five additional MCLs also showed very high mRNA levels without gene amplification. A good correlation between BMI-1 mRNA levels and protein expression was observed in all types of lymphomas. No relationship was detected between BMI-1 and p16INK4a mRNA levels. These findings suggest that BMI-1 gene alterations in human neoplasms are uncommon, but they may contribute to the pathogenesis in a subset of malignant lymphomas, particularly of mantle cell type.

Differential expression of cdc25 cell-cycle-activating phosphatases in human colorectal carcinoma.

cdc25 is a family of cell-cycle phosphatases that activate the cyclin-dependent kinases. cdc25A and B, but not C, have oncogenic potential in vitro. In this study, we analyzed the possible implication of cdc25 genes in the progression of colorectal tumors. RNA and DNA were extracted from 34 paired tumor and normal colorectal tissues and examined by Northern blot, RT-PCR, and Southern blot, respectively. Protein expression was analyzed by Western blot in a subset of normal and tumor samples. The expression levels were correlated with the clinicopathologic characteristics and survival of the patients. cdc25B mRNA was overexpressed in 19 carcinomas (56%). A significant correlation was observed between high cdc25B mRNA levels and the relapse-free, overall, and cancer-related survival of the patients. The cdc25B2 splicing variant was detected in 27 carcinomas (79%) but only in 9 normal samples (26%) and was associated with the grade of the differentiation of the tumors. cdc25A mRNA was overexpressed in four tumors (12%) and cdc25C1 mRNA was overexpressed in nine tumors (26%). A new cdc25C2 splicing variant lacking exon 4 and 5 was identified in all of the tumors and in 56% of the normal samples. No amplifications or gene rearrangements of these genes were detected. In conclusion, these findings indicate that cdc25 isoforms and splicing variants are differentially regulated in colorectal carcinomas and may participate in the development of these tumors. Additionally, the correlation between cdc25B mRNA levels and the survival of the patients also suggest that the cdc25B isoform may be involved in the progression of the disease.

INK4a/ARF locus alterations in human non-Hodgkin's lymphomas mainly occur in tumors with wild-type p53 gene.

INK4a/ARF locus codes for two different proteins, p16(INK4a) and p14(ARF), involved in cell cycle regulation. p14(ARF) is considered an upstream regulator of p53 function. To determine the role of these genes in the pathogenesis of human non-Hodgkin's lymphomas we have analyzed exon 1beta, 1alpha, and 2 of the INK4a/ARF locus and p53 gene aberrations in 97 tumors previously characterized for p16(INK4a) alterations. p53 alterations were detected in four of 51 (8%) indolent lymphomas but in 15 of 46 (33%) aggressive tumors. Inactivation of p14(ARF) was always associated with p16(INK4a) alterations. Exon 1beta was concomitantly deleted with exon 1alpha and 2 in eight tumors. One additional lymphoblastic lymphoma showed deletion of exon 1alpha and 2 but retained exon 1beta. No mutations were detected in exon 1alpha and 1beta in any case. Two of the three mutations detected in exon 2 caused a nonsense mutation in the p16(INK4a) reading frame and a missense mutation in the ARF reading frame involving the nucleolar transport domain of the protein. The third mutation was a missense mutation in the p16(INK4a) reading frame, but it was outside the coding region of p14(ARF). Aggressive lymphomas with p14(ARF) inactivation and p53 wild type showed a significantly lower p53 protein expression than tumors with no alteration in any of these genes. In this series of tumors, inactivation of the INK4a/ARF locus mainly occurred in tumors with a wild-type p53 gene because only two lymphomas showed simultaneous aberrations in these genes. Tumors with concomitant alterations of p16(INK4a) and p14(ARF)/p53 genes seem to exhibit a worse clinical behavior than lymphomas with no alterations or isolated inactivation of any of these genes. These findings indicate that p14(ARF) genetic alterations occur in a subset of aggressive NHLs, but they are always associated with p16(INK4a) aberrations. Concomitant disruption of p16(INK4a) and p14(ARF)/p53 regulatory pathways may have a cooperative effect in the progression of these tumors.