Evaluation of four automated clinical analyzers for the determination of total 25(OH)D in comparison to a certified LC-MS/MS.

OBJECTIVES: The aim of this study was to compare the results of five methods for the determination of total 25(OH)D. For that purpose, two mass spectrometry and three immunoassay methods were used. METHODS: A total of 124 serum samples were analyzed on five different methods (i.e., a reference LC-MS/MS, Cascadion, Lumipulse, Roche Elecsys II and Roche Elecsys III). Analytical performance against LC-MS/MS was evaluated and compared to the Milan models 1 (analytical performance based on the clinical outcome using thresholds of 12, 20 and 30 ng/mL) and 2 (analytical performance based on biological variation). Additionally, imprecision studies and accuracy using NIST SRM972a samples were carried out. RESULTS: Compared to the reference LC-MS/MS method, the Lumipulse and the Roche Elecsys III assays reached the optimal criterion for bias, while the Cascadion met the desirable one. The Roche Elecsys II was not able to reach the minimal criteria. The proportion of correctly classified patients was higher using the Cascadion (95.2%) compared to the three immunoassays. In addition to its better precision, the Cascadion was not impacted by a high concentration of 3-epi-25(OH)D3 compared to the three immunoassays. CONCLUSIONS: Compared to the LC-MS/MS reference method, the Cascadion presented the highest level of concordance at medical decision cut-offs for total 25(OH)D and reached the desirable specification for bias. Moreover, the presence of 3-epi-25(OH)D3 in enriched samples was only problematic in immunoassay methods, and especially considering Roche Elecsys methods. The release of performant fully automated mass spectrometry assays with high throughput might therefore facilitate the wide scale adoption of LC-MS/MS, even in non-specialized clinical laboratories.

Global Hemostasis Potential in COVID-19 Positive Patients Performed on St-Genesia Show Hypercoagulable State.

BACKGROUND: At the dawn of the pandemic, severe forms of COVID-19 were often complicated by thromboembolisms. However, routine laboratory tests cannot be used to predict thromboembolic events. The objective of this study was to investigate the potential value of the thrombin generation test (TGT) in predicting hypercoagulability and thrombotic risk in the aforementioned set of patients. METHODS: The study panel comprised 52 patients divided into two groups (26 COVID-19 positive and 26 COVID-19 negative); COVID-19-positive patients were further grouped in "severe" (n = 11) and "non-severe" (n = 15) categories based on clinical criteria. The routine blood tests and TGT of these patients were retrospectively analyzed. RESULTS: All 26 COVID-19-positive patients showed decreased lymphocyte, monocyte and basophil counts and increased lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine transaminase (ALT) compared with control patients. Conversely, we did not observe statistically significant differences between severe and non-severe patients despite anecdotal variations in the distribution patterns. TGT without thrombomodulin (TM) addition showed statistically significant differences in the thrombin peak heights between COVID-19-positive and negative patients. After addition of TM, peak height, Endogenous Thrombin Potential (ETP) and velocity index were increased in all COVID-19-positive patients while the percentage of inhibition of ETP was reduced. These trends correlated with the severity of disease, showing a greater increase in peak height, ETP, velocity index and a drastic reduction in the percentage of ETP inhibition in more severely affected patients. CONCLUSIONS: Our data suggest that all COVID-19 patients harbor a hypercoagulable TGT profile and that this is further pronounced in severely affected patients.

DNA damage-induced phosphorylation of CtIP at a conserved ATM/ATR site T855 promotes lymphomagenesis in mice.

CtIP is a DNA end resection factor widely implicated in alternative end-joining (A-EJ)-mediated translocations in cell-based reporter systems. To address the physiological role of CtIP, an essential gene, in translocation-mediated lymphomagenesis, we introduced the T855A mutation at murine CtIP to nonhomologous end-joining and Tp53 double-deficient mice that routinely succumbed to lymphomas carrying A-EJ-mediated IgH-Myc translocations. T855 of CtIP is phosphorylated by ATM or ATR kinases upon DNA damage to promote end resection. Here, we reported that the T855A mutation of CtIP compromised the neonatal development of Xrcc4-/-Tp53-/- mice and the IgH-Myc translocation-driven lymphomagenesis in DNA-PKcs-/-Tp53-/- mice. Mechanistically, the T855A mutation limits DNA end resection length without affecting hairpin opening, translocation frequency, or fork stability. Meanwhile, after radiation, CtIP-T855A mutant cells showed a consistent decreased Chk1 phosphorylation and defects in the G2/M cell cycle checkpoint. Consistent with the role of T855A mutation in lymphomagenesis beyond translocation, the CtIP-T855A mutation also delays splenomegaly in λ-Myc mice. Collectively, our study revealed a role of CtIP-T855 phosphorylation in lymphomagenesis beyond A-EJ-mediated chromosomal translocation.

Genetic mechanisms of HLA-I loss and immune escape in diffuse large B cell lymphoma.

Fifty percent of diffuse large B cell lymphoma (DLBCL) cases lack cell-surface expression of the class I major histocompatibility complex (MHC-I), thus escaping recognition by cytotoxic T cells. Here we show that, across B cell lymphomas, loss of MHC-I, but not MHC-II, is preferentially restricted to DLBCL. To identify the involved mechanisms, we performed whole exome and targeted HLA deep-sequencing in 74 DLBCL samples, and found somatic inactivation of B2M and the HLA-I loci in 80% (34 of 42) of MHC-INEG tumors. Furthermore, 70% (22 of 32) of MHC-IPOS DLBCLs harbored monoallelic HLA-I genetic alterations (MHC-IPOS/mono), indicating allele-specific inactivation. MHC-INEG and MHC-IPOS/mono cases harbored significantly higher mutational burden and inferred neoantigen load, suggesting potential coselection of HLA-I loss and sustained neoantigen production. Notably, the analysis of >500,000 individuals across different cancer types revealed common germline HLA-I homozygosity, preferentially in DLBCL. In mice, germinal-center B cells lacking HLA-I expression did not progress to lymphoma and were counterselected in the context of oncogene-driven lymphomagenesis, suggesting that additional events are needed to license immune evasion. These results suggest a multistep process of HLA-I loss in DLBCL development including both germline and somatic events, and have direct implications for the pathogenesis and immunotherapeutic targeting of this disease.

Genetic basis of PD-L1 overexpression in diffuse large B-cell lymphomas.

Diffuse large B-cell lymphoma (DLBCL) is one of the most common and aggressive types of B-cell lymphoma. Deregulation of proto-oncogene expression after a translocation, most notably to the immunoglobulin heavy-chain locus (IGH), is one of the hallmarks of DLBCL. Using whole-genome sequencing analysis, we have identified the PD-L1/PD-L2 locus as a recurrent translocation partner for IGH in DLBCL. PIM1 and TP63 were also identified as novel translocation partners for PD-L1/PD-L2 Fluorescence in situ hybridization was furthermore used to rapidly screen an expanded DLBCL cohort. Collectively, a subset of samples was found to be affected by gains (12%), amplifications (3%), and translocations (4%) of the PD-L1/PD-L2 locus. RNA sequencing data coupled with immunohistochemistry revealed that these cytogenetic alterations correlated with increased expression of PD-L1 but not of PD-L2 Moreover, cytogenetic alterations affecting the PD-L1/PD-L2 locus were more frequently observed in the non-germinal center B cell-like (non-GCB) subtype of DLBCL. These findings demonstrate the genetic basis of PD-L1 overexpression in DLBCL and suggest that treatments targeting the PD-1-PD-L1/PD-L2 axis might benefit DLBCL patients, especially those belonging to the more aggressive non-GCB subtype.

Genetics of follicular lymphoma transformation.

Follicular lymphoma (FL) is an indolent disease, but 30%-40% of cases undergo histologic transformation to an aggressive malignancy, typically represented by diffuse large B cell lymphoma (DLBCL). The pathogenesis of this process remains largely unknown. Using whole-exome sequencing and copy-number analysis, we show here that the dominant clone of FL and transformed FL (tFL) arise by divergent evolution from a common mutated precursor through the acquisition of distinct genetic events. Mutations in epigenetic modifiers and antiapoptotic genes are introduced early in the common precursor, whereas tFL is specifically associated with alterations deregulating cell-cycle progression and DNA damage responses (CDKN2A/B, MYC, and TP53) as well as aberrant somatic hypermutation. The genomic profile of tFL shares similarities with that of germinal center B cell-type de novo DLBCL but also displays unique combinations of altered genes with diagnostic and therapeutic implications.

The spectrum of genetic defects in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world and shows a remarkable heterogeneity in the clinical course. Understand the genetic basis of CLL may help in clarifying the molecular bases of this clinical heterogeneity. Recurrent chromosomal aberrations at 13q14, 12q, 11q22-q23 and 17p13, and TP53 mutations are the first genetic lesions identified as drivers of the disease. While some of these lesions are associated with poor outcome (17p13 deletion, TP53 mutations and, to a lesser extent, 11q22-q23 deletion) others are linked to a favorable course (13q14 deletion as sole aberration). Recently, next generation sequencing has revealed additional recurrent alterations in CLL targeting the NOTCH1, SF3B1, and BIRC3 genes. NOTCH1, SF3B1, and BIRC3 lesions provide: I) new insights on the mechanisms of leukemogenesis, tumor progression and chemoresistance in this leukemia; II) new biomarkers for the identification of poor risk patients, having individually shown correlations with survival in CLL; and III) new therapeutic targets, especially in the setting of high risk disease. This review will summarize the most important genetic aberrations in CLL and how our improved knowledge of the genome of leukemic cells may translate into improved patients' management.

The coding genome of splenic marginal zone lymphoma: activation of NOTCH2 and other pathways regulating marginal zone development.

Splenic marginal zone lymphoma (SMZL) is a B cell malignancy of unknown pathogenesis, and thus an orphan of targeted therapies. By integrating whole-exome sequencing and copy-number analysis, we show that the SMZL exome carries at least 30 nonsilent gene alterations. Mutations in NOTCH2, a gene required for marginal-zone (MZ) B cell development, represent the most frequent lesion in SMZL, accounting for ∼20% of cases. All NOTCH2 mutations are predicted to cause impaired degradation of the NOTCH2 protein by eliminating the C-terminal PEST domain, which is required for proteasomal recruitment. Among indolent B cell lymphoproliferative disorders, NOTCH2 mutations are restricted to SMZL, thus representing a potential diagnostic marker for this lymphoma type. In addition to NOTCH2, other modulators or members of the NOTCH pathway are recurrently targeted by genetic lesions in SMZL; these include NOTCH1, SPEN, and DTX1. We also noted mutations in other signaling pathways normally involved in MZ B cell development, suggesting that deregulation of MZ B cell development pathways plays a role in the pathogenesis of ∼60% SMZL. These findings have direct implications for the treatment of SMZL patients, given the availability of drugs that can target NOTCH, NF-κB, and other pathways deregulated in this disease.

Disruption of BIRC3 associates with fludarabine chemorefractoriness in TP53 wild-type chronic lymphocytic leukemia.

The genetic lesions identified to date do not fully recapitulate the molecular pathogenesis of chronic lymphocytic leukemia (CLL) and do not entirely explain the development of severe complications such as chemorefractoriness. In the present study, BIRC3, a negative regulator of noncanonical NF-κB signaling, was investigated in different CLL clinical phases. BIRC3 lesions were absent in monoclonal B-cell lymphocytosis (0 of 63) and were rare in CLL at diagnosis (13 of 306, 4%). Conversely, BIRC3 disruption selectively affected 12 of 49 (24%) fludarabine-refractory CLL cases by inactivating mutations and/or gene deletions that distributed in a mutually exclusive fashion with TP53 abnormalities. In contrast to fludarabine-refractory CLL, progressive but fludarabine-sensitive patients were consistently devoid of BIRC3 abnormalities, suggesting that BIRC3 genetic lesions associate specifically with a chemorefractory phenotype. By actuarial analysis in newly diagnosed CLL (n = 306), BIRC3 disruption identified patients with a poor outcome similar to that associated with TP53 abnormalities and exerted a prognostic role that was independent of widely accepted clinical and genetic risk factors. Consistent with the role of BIRC3 as a negative regulator of NF-κB, biochemical studies revealed the presence of constitutive noncanonical NF-κB activation in fludarabine-refractory CLL patients harboring molecular lesions of BIRC3. These data identify BIRC3 disruption as a recurrent genetic lesion of high-risk CLL devoid of TP53 abnormalities.

Molecular history of Richter syndrome: origin from a cell already present at the time of chronic lymphocytic leukemia diagnosis.

Richter syndrome (RS) represents the transformation of chronic lymphocytic leukemia to aggressive lymphoma. We explored intraclonal diversification (ID) of immunoglobulin genes in order to (i) follow the evolutionary history of the RS clone (ii) compare the role of ID in clonally related RS vs. clonally unrelated cases. Most (10/11, 90.9%) clonally related RS stem from the predominant clone observed at CLL diagnosis. One single RS had a transformation pattern compatible with sequential evolution from a secondary CLL subclone. Once RS transformation had occurred, all secondary CLL subclones disappeared and were substituted by the dominant RS clone with its own descendants. These observations suggest that genetic lesions associated with RS transformation are acquired by a cell belonging to the original CLL clone, rather than being progressively accumulated by later CLL subclones. Accordingly, most (9/11, 81.1%) clonally related RS harbored a genetic lesion disrupting TP53 that was already present, though at subclonal levels, in 5/11 (45.5%) samples of the paired CLL phase. A fraction of clonally related RS switched off ID (4/11, 36.4%) or reduced the levels of ID (5/11, 45.4%) at transformation. Conversely, all clonally unrelated RS harbored ID and were characterized by a significantly higher mutation frequency compared to clonally related RS (median: 1.18 × 10(-3) vs. 0.13 × 10(-3); p =0.002). These data indicate that (i) clonally related RS stems from a cell that is already present within the initial CLL clone and (ii) clonally unrelated and clonally related RS are biologically distinct disorders also in terms of antigen affinity maturation.

Mutations of NOTCH1 are an independent predictor of survival in chronic lymphocytic leukemia.

Analysis of the chronic lymphocytic leukemia (CLL) coding genome has recently disclosed that the NOTCH1 proto-oncogene is recurrently mutated at CLL presentation. Here, we assessed the prognostic role of NOTCH1 mutations in CLL. Two series of newly diagnosed CLL were used as training (n = 309) and validation (n = 230) cohorts. NOTCH1 mutations occurred in 11.0% and 11.3% CLL of the training and validation series, respectively. In the training series, NOTCH1 mutations led to a 3.77-fold increase in the hazard of death and to shorter overall survival (OS; P < .001). Multivariate analysis selected NOTCH1 mutations as an independent predictor of OS after controlling for confounding clinical and biologic variables. The independent prognostic value of NOTCH1 mutations was externally confirmed in the validation series. The poor prognosis conferred by NOTCH1 mutations was attributable, at least in part, to shorter treatment-free survival and higher risk of Richter transformation. Although NOTCH1 mutated patients were devoid of TP53 disruption in more than 90% cases in both training and validation series, the OS predicted by NOTCH1 mutations was similar to that of TP53 mutated/deleted CLL. NOTCH1 mutations are an independent predictor of CLL OS, tend to be mutually exclusive with TP53 abnormalities, and identify cases with a dismal prognosis.

NOTCH1 mutations in +12 chronic lymphocytic leukemia (CLL) confer an unfavorable prognosis, induce a distinctive transcriptional profiling and refine the intermediate prognosis of +12 CLL.

Trisomy 12, the third most frequent chromosomal aberration in chronic lymphocytic leukemia (CLL), confers an intermediate prognosis. In our cohort of 104 untreated patients carrying +12, NOTCH1 mutations occurred in 24% of cases and were associated to unmutated IGHV genes (P=0.003) and +12 as a sole cytogenetic abnormality (P=0.008). NOTCH1 mutations in +12 CLL associated with an approximately 2.4 fold increase in the risk of death, a significant shortening of survival (P<0.01) and proved to be an independent predictor of survival in multivariate analysis. Analogous to +12 CLL with TP53 disruption or del(11q), NOTCH1 mutations in +12 CLL conferred a significantly worse survival compared to that of +12 CLL with del(13q) or +12 only. The overrepresentation of cell cycle/proliferation related genes of +12 CLL with NOTCH1 mutations suggests the biological contribution of NOTCH1 mutations to determine a poor outcome. NOTCH1 mutations refine the intermediate prognosis of +12 CLL.

Mutations of the SF3B1 splicing factor in chronic lymphocytic leukemia: association with progression and fludarabine-refractoriness.

The genetic lesions identified in chronic lymphocytic leukemia (CLL) do not entirely recapitulate the disease pathogenesis and the development of serious complications, such as chemorefractoriness. While investigating the coding genome of fludarabine-refractory CLL, we observed that mutations of SF3B1, encoding a splicing factor and representing a critical component of the cell spliceosome, were recurrent in 10 of 59 (17%) fludarabine-refractory cases, with a frequency significantly greater than that observed in a consecutive CLL cohort sampled at diagnosis (17/301, 5%; P = .002). Mutations were somatically acquired, were generally represented by missense nucleotide changes, clustered in selected HEAT repeats of the SF3B1 protein, recurrently targeted 3 hotspots (codons 662, 666, and 700), and were predictive of a poor prognosis. In fludarabine-refractory CLL, SF3B1 mutations and TP53 disruption distributed in a mutually exclusive fashion (P = .046). The identification of SF3B1 mutations points to splicing regulation as a novel pathogenetic mechanism of potential clinical relevance in CLL.

Alteration of BIRC3 and multiple other NF-κB pathway genes in splenic marginal zone lymphoma.

Splenic marginal zone lymphoma (SMZL) is one of the few B-cell lymphoma types that remain orphan of molecular lesions in cancer-related genes. Detection of active NF-κB signaling in 14 (58%) of 24 SMZLs prompted the investigation of NF-κB molecular alterations in 101 SMZLs. Mutations and copy number abnormalities of NF-κB genes occurred in 36 (36%) of 101 SMZLs and targeted both canonical (TNFAIP3 and IKBKB) and noncanonical (BIRC3, TRAF3, MAP3K14) NF-κB pathways. Most alterations were mutually exclusive, documenting the existence of multiple independent mechanisms affecting NF-κB in SMZL. BIRC3 inactivation in SMZL recurred because of somatic mutations that disrupted the same RING domain that in extranodal marginal zone lymphoma is removed by the t(11;18) translocation, which points to BIRC3 disruption as a common mechanism across marginal zone B-cell lymphomagenesis. Genetic lesions of NF-κB provide a molecular basis for the pathogenesis of more than 30% of SMZLs and offer a suitable target for NF-κB therapeutic approaches in this lymphoma.

Analysis of the chronic lymphocytic leukemia coding genome: role of NOTCH1 mutational activation.

The pathogenesis of chronic lymphocytic leukemia (CLL), the most common leukemia in adults, is still largely unknown. The full spectrum of genetic lesions that are present in the CLL genome, and therefore the number and identity of dysregulated cellular pathways, have not been identified. By combining next-generation sequencing and copy number analysis, we show here that the typical CLL coding genome contains <20 clonally represented gene alterations/case, including predominantly nonsilent mutations, and fewer copy number aberrations. These analyses led to the discovery of several genes not previously known to be altered in CLL. Although most of these genes were affected at low frequency in an expanded CLL screening cohort, mutational activation of NOTCH1, observed in 8.3% of CLL at diagnosis, was detected at significantly higher frequency during disease progression toward Richter transformation (31.0%), as well as in chemorefractory CLL (20.8%). Consistent with the association of NOTCH1 mutations with clinically aggressive forms of the disease, NOTCH1 activation at CLL diagnosis emerged as an independent predictor of poor survival. These results provide initial data on the complexity of the CLL coding genome and identify a dysregulated pathway of diagnostic and therapeutic relevance.

Saliva is a reliable and practical source of germline DNA for genome-wide studies in chronic lymphocytic leukemia.

High-throughput genomics requires tumor DNA matched to germline DNA, that cannot be easily obtained in the context of leukemia. Using chronic lymphocytic leukemia as a model, saliva DNA was frequently devoid of tumor DNA also during overt disease, and passed quality controls for SNP-array (77/102, 75.4%) and next generation sequencing (71/102, 69.6%). Compared to saliva, urine provides germline DNA of similar quality but in lower amounts. Saliva DNA was successfully run on SNP 6.0 arrays, and passed quality control call rate thresholds. On these bases, saliva represents a useful source of germline DNA for high-throughput genomic studies of hematologic neoplasia.

Predictive markers and driving factors behind Richter syndrome development.

Transformation of chronic lymphocytic leukemia (CLL) to diffuse large B-cell lymphoma (DLBCL) is known as Richter syndrome (RS). In the entire CLL population, the cumulative prevalence of RS development steadily increases at a rate of 1% per year. Considering conventional predictors of CLL, patient subgroups at high risk of developing RS are characterized by the expression of CD38, absence of del13q14, and a lymph node size >3 cm. Novel risk factors for predicting RS development at CLL diagnosis have been recently identified and include: the host genotype of the CD38 locus and of other genes; telomere length of CLL cells; stereotyped B-cell receptor; and usage of specific immunoglobulin variable genes (IGHV4-39). Importantly, although some risk factors predict both CLL progression and transformation to RS, others (CD38 genotype, absence of del13q14, IGHV4-39 usage, stereotyped B-cell receptor) appear to specifically predict RS. The definition of RS encompasses at least two different conditions: DLBCLs that are clonally related to the pre-existing CLL (accounting for most cases), or DLBCL unrelated to the CLL clone. The transition from CLL to clonally related RS is accompanied by the acquisition of novel genetic alterations that may account for the chemorefractoriness of RS. Genome-wide studies that are currently ongoing are important for identifying novel molecular lesions implicated in RS that might represent a suitable target for future therapeutic strategies.

The host genetic background of DNA repair mechanisms is an independent predictor of survival in diffuse large B-cell lymphoma.

Several drugs used for diffuse large B-cell lymphoma (DLBCL) treatment rely on DNA damage for tumor cell killing. We verified the prognostic impact of the host DNA repair genotype in 2 independent cohorts of DLBCL treated with R-CHOP21 (training cohort, 163 cases; validation cohort, 145 cases). Among 35 single nucleotide polymorphisms analyzed in the training series, MLH1 rs1799977 was the sole predicting overall survival. DLBCL carrying the MLH1 AG/GG genotype displayed an increased death risk (hazard ratio [HR] = 3.23; P < .001; q =0 .009) compared with patients carrying the AA genotype. Multivariate analysis adjusted for International Prognostic Index identified MLH1 AG/GG as an independent OS predictor (P < .001). The poor prognosis of MLH1 AG/GG was the result of an increased risk of failing both R-CHOP21 (HR = 2.02; P = .007) and platinum-based second-line (HR = 2.26; P = .044) treatment. Survival analysis in the validation series confirmed all outcomes predicted by MLH1 rs1799977. The effect on OS of MLH1, a component of the DNA mismatch repair system, is consistent with its role in regulating the genotoxic effects of doxorubicin and platinum compounds, which are a mainstay of DLBCL first- and second-line treatment.