Next generation sequencing and copy number analysis provide insights into the complexity of the CLL coding genome, and reveal an association between NOTCH1 mutational activation and poor prognosis.

IntroductionNatural killer (NK) cells are present in the bloodstream, spleen, bone marrow, and in nonlymphoid organs and represent one of the main effectors of the immunosurveillance against tumors, by exerting 2 major effector functions, cytolysis of target cells and production of cytokines and chemokines. 1,2 The activity of NK cells depends on the interplay between inhibitory receptors for major histocompatibility complex (MHC) class I molecules and activating receptors, which operate in concert to induce the elimination of tumor cells. 3,4 Among the activating receptors particularly relevant for tumor cell recognition and killing is NKG2D, the receptor for the MHC I-related molecules MICA/B, and ULBPs (UL16-binding proteins), belonging to the C-type lectin-like receptor family. 3,[5][6][7] The NKG2D activating receptor is expressed not only on NK cells, but also on ␥␦ T cells, CD8 ϩ T cells, and a subset of CD4 ϩ T cells. The expression of NKG2D ligands is largely confined to virus-infected, tumor, and stressed cells. 7 To promote escape of tumors from NKG2D-mediated immunosurveillance, NKG2D ligands undergo proteolytic shedding. Soluble NKG2D ligands (NKG2DLs) have been shown to down-regulate the cell surface NKG2D expression on NK cells, resulting in impaired killing of tumor cells. 8,9 Another activating receptor involved in NK-cell-mediated tumor cell killing is DNAX accessory molecule-1 (DNAM-1), a transmembrane glycoprotein constitutively expressed on the majority of T cells, NK cells, and macrophages; its ligands are Nectin-2 (Nec-2, CD112) and the poliovirus receptor (PVR, CD155), which belong to the nectin/nectin-like family. 10-12 DNAM-1 ligands have been initially described as adhesion molecules mainly regulating trans-endothelial migration 13 and only recently they have been found on a variety of tumor cells. 12,14 Both DNAM-1 and NKG2D cooperate in the induction of NK-cell killing against tumor cells of different histotypes, including those of hematopoietic origin. 14,15 Similarly to the NKG2D ligands, soluble isoforms of PVR have also been found in human serum and in the culture supernatant of tumor cell lines, and their role in tumor immunoevasion has been considered. 12 It has recently been demonstrated that agents that produce a genotoxic stress or DNA-replication inhibitors up-regulate NKG2D ligand expression through the activation of ATM (ataxia telangiectasia mutated) and ATR (ATM-and Rad3-related) protein kinases on human fibroblasts and on mouse tumor cell lines, and enhance their destruction by NK cells. 16,17 Increased ligand expression is regulated by the activation of the DNA damage response (DDR). This is a cellular program devoted to the maintenance of genome integrity through the inhibition of cell cycle and activation of the DNA repair systems, or by the induction of apoptosis or a protracted cell-cycle arrest known as cellular senescence. 17,18 No information is so far available on the regulation of DNAM-1 ligand expression through the DDR pathway.Submitted August 11, 2008; acc...

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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%) fludarabinerefractory 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 ‫؍‬ . IntroductionThe clinical course of chronic lymphocytic leukemia (CLL) ranges from a very indolent disorder with a normal lifespan for the patient to a rapidly progressive disease that leads to death. Occasionally, CLL undergoes a transformation to Richter syndrome (RS). [1][2][3] The variable clinical course of CLL is driven, at least in part, by the disease's immunogenetic and molecular heterogeneity. 4 Despite recent advances, the genetic lesions identified to date do not fully recapitulate the molecular pathogenesis of CLL and do not entirely explain the development of severe complications, such as chemorefractoriness, which still represent unmet clinical needs. 5 In approximately 40% of cases, refractoriness to fludarabine is attributable to the disruption of TP53, but in a sizeable fraction of patients, the molecular basis of this aggressive phenotype remains unclear. 6 Recently, 2 independent studies of the CLL coding genome investigated at disease presentation have revealed a restricted number of mutated genes, including NOTCH1. 7,8 These studies have provided a proof of concept that, similar to other malignancies, genome-wide mutational analysis might identify novel lesions of biologic and clinical relevance in CLL. On these grounds, we have embarked on the investigation of the coding genome of fludarabine-refractory CLL to identify genetic lesions associated with chemorefractoriness. The initial phases of this analysis have revealed recurrent mutations of SF3B1, a critical component of the cell spliceosome, pointing to the potential involvement of splicing regulation in CLL pathogenesis and chemorefractoriness. Methods PatientsThe study population comprised 3 cohorts representative of different disease phases: (1) fludarabine-refractory CLL (n ϭ 59), including cases (n ϭ 11) subjected to whole-exome sequencing (supplemental Table 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article); (2) a consecutive series of newly diagnosed and previously untreated patients with CLL (n ϭ 301; supplemental Table 2 For pers...

Key Points• Small TP53 mutated subclones have the same unfavorable prognostic impact as clonal TP53 defects in chronic lymphocytic leukemia.TP53 mutations are strong predictors of poor survival and refractoriness in chronic lymphocytic leukemia (CLL) and have direct implications for disease management. Clinical information on TP53 mutations is limited to lesions represented in >20% leukemic cells. Here, we tested the clinical impact and prediction of chemorefractoriness of very small TP53 mutated subclones. The TP53 gene underwent ultra-deep-next generation sequencing (NGS) in 309 newly diagnosed CLL. A robust bioinformatic algorithm was established for the highly sensitive detection of few TP53 mutated cells (down to 3 out of ∼1000 wild-type cells). Minor subclones were validated by independent approaches. Ultra-deep-NGS identified small TP53 mutated subclones in 28/309 (9%) untreated CLL that, due to their very low abundance (median allele frequency: 2.1%), were missed by Sanger sequencing. Patients harboring small TP53 mutated subclones showed the same clinical phenotype and poor survival (hazard ratio 5 2.01; P 5 .0250) as those of patients carrying clonal TP53 lesions. By longitudinal analysis, small TP53 mutated subclones identified before treatment became the predominant population at the time of CLL relapse and anticipated the development of chemorefractoriness. This study provides a proof-of-principle that very minor leukemia subclones detected at diagnosis are an important driver of the subsequent disease