B-cell chronic lymphocytic leukemia (CLL) is the most common human leukemia and dysregulation of the T-cell leukemia/lymphoma 1 (TCL1) oncogene is a contributing event in the pathogenesis of the aggressive form of this disease based on transgenic mouse studies. To determine a role of microRNAs on the pathogenesis of the aggressive form of CLL we studied regulation of TCL1 expression in CLL by microRNAs. We identified miR-3676 as a regulator of TCL1 expression. We demonstrated that miR-3676 targets three consecutive 28-bp repeats within 3′UTR of TCL1 and showed that miR-3676 is a powerful inhibitor of TCL1. We further showed that miR-3676 expression is significantly down-regulated in four groups of CLL carrying the 11q deletions, 13q deletions, 17p deletions, or a normal karyotype compared with normal CD19 + cord blood and peripheral blood B cells. In addition, the sequencing of 539 CLL samples revealed five germ-line mutations in six samples (1%) in miR-3676. Two of these mutations were loss-offunction mutations. Because miR-3676 is located at 17p13, only 500-kb centromeric of tumor protein p53 (Tp53), and is codeleted with Tp53, we propose that loss of miR-3676 causes high levels of TCL1 expression contributing to CLL progression. miR-3676 | CLL | 17p deletions

Complex karyotype defined as ≥3 cytogenetic abnormalities is prognostic of survival in patients treated with ibrutinib or venetoclax in relapsed/refractory (RR) chronic lymphocytic leukemia (CLL). Recent studies re-evaluating this dichotomous variable have shown that higher numbers of cytogenetic abnormalities (i.e. ≥5) have a worse overall survival in patients treated with chemoimmunotherapy. We sought to determine if increasing karyotypic complexity, treated as a continuous variable, was prognostic of survival for patients treated with ibrutinib for CLL. We conducted a retrospective analysis of all patients with CLL treated with single-agent ibrutinib or in combination with an anti-CD20 antibody at our institution. We included 456 patients with both treatment-naïve (TN) and RR disease. Median number of prior therapies was 2 (range 0-13), 30% of patients had del(17p), and 75% were IGHV unmutated. 50% had ≥3 cytogenetic abnormalities including 30% with ≥5. In a multivariable analysis, increasing karyotypic complexity was an independent predictor of shorter progression-free survival (HR 1.07 (95% CI 1.04-1.10), p<0.0001) and overall survival (HR 1.09 (95% CI 1.05-1.12), p<0.0001). Furthermore, we found that presence of clonal evolution determined by cytogenetic analysis at progression was prognostic of subsequent survival (p=0.02). This solidifies karyotypic complexity as an important prognostic factor for CLL patients treated with ibrutinib. Further research should consider sequential karyotypic analysis as a determination of risk of progression and death in patients with CLL.

Key Points• Pretreatment near-tetraploidy is associated with advanced Rai stage, deletion of 17p, and complex karyotype.• Pretreatment near-tetraploidy is an independent risk factor for ibrutinib discontinuation via Richter transformation.Ibrutinib is a highly effective targeted therapy for chronic lymphocytic leukemia (CLL).However, ibrutinib must be discontinued in a subset of patients due to progressive CLL or transformation to aggressive lymphoma (Richter transformation). Transformation occurs early in the course of therapy and has an extremely poor prognosis. Thus, identification of prognostic markers associated with transformation is of utmost importance. Neartetraploidy (4 copies of most chromosomes within a cell) has been reported in various lymphomas, but its incidence and significance in CLL has not been described. Using fluorescence in situ hybridization, we detected near-tetraploidy in 9 of 297 patients with CLL prior to beginning ibrutinib treatment on 1 of 4 clinical trials (3.0%; 95% confidence interval [CI], 1.4%-5.7%). Near-tetraploidy was associated with aggressive disease characteristics: Rai stage 3/4 (P 5 .03), deletion 17p (P 5 .03), and complex karyotype (P 5 .01). Neartetraploidy was also associated with ibrutinib discontinuation due to Richter transformation (P , .0001), but not due to progressive CLL (P 5 .41). Of the 9 patients with near-tetraploidy, 6 had Richter transformation with diffuse large B-cell lymphoma. In a multivariable model, near-tetraploidy (hazard ratio [HR], 8.66; 95% CI,; P , .0001) and complex karyotype (HR, 4.77; 95% CI,; P 5 .01) were independent risk factors for discontinuing ibrutinib due to transformation. Our results suggest that near-tetraploidy is a potential prognostic marker for Richter transformation to assess in patients going on ibrutinib. IntroductionIbrutinib is a first-in-class oral covalent inhibitor of Bruton tyrosine kinase (BTK) 1 approved to treat chronic lymphocytic leukemia (CLL), mantle cell lymphoma, and Waldenstrom macroglobulinemia. Ibrutinib has rapidly changed the landscape of CLL treatment, with high response rates and prolonged remission durations in both relapsed/refractory CLL and previously untreated patients.2-5 Despite these strides, a subset of patients relapse on ibrutinib. Patients relapse primarily with progressive CLL or Richter transformation, an aggressive transformation into lymphoma, predominantly diffuse large B-cell lymphoma.6 Most patients progressing with CLL acquire mutations in either the C481 ibrutinib-binding pocket of BTK or downstream activating mutations in PLCG2 that bypass the need for signaling through BTK. progress via disease transformation. 6 Identifying prognostic markers associated with Richter transformation is critical at this time as an increasing number of patients have begun to receive ibrutinib and those whose CLL transforms have very aggressive disease and poor prognosis. 6,9 Complex karyotype ($3 unrelated chromosomal abnormalities) has been associated with ibrutinib discontinuation due to progr...

Exome sequencing (ES) has become an important tool in pediatric genomic medicine, improving identification of disease-associated variation due to assay breadth. Depth is also afforded by ES, enabling detection of lower-frequency mosaic variation compared to Sanger sequencing in the studied tissue, thus enhancing diagnostic yield. Within a pediatric tertiary-care hospital, we report two years of clinical ES data from probands evaluated for genetic disease to assess diagnostic yield, characteristics of causal variants, and prevalence of mosaicism among disease-causing variants. Exome-derived, phenotype-driven variant data from 357 probands was analyzed concurrent with parental ES data, when available. Blood was the source of nucleic acid. Sequence read alignments were manually reviewed for all assessed variants. Sanger sequencing was used for suspected de novo or mosaic variation. Clinical provider notes were reviewed to determine concordance between laboratory-reported data and the ordering provider's interpretation of variant-associated disease causality. Laboratory-derived diagnostic yield and provider-substantiated diagnoses had 91.4% concordance. The cohort returned 117 provider-substantiated diagnoses among 115 probands for a diagnostic yield of 32.2%. De novo variants represented 64.9% of disease-associated variation within trio analyses. Among the 115 probands, five harbored disease-associated somatic mosaic variation. Two additional probands were observed to inherit a disease-associated variant from an unaffected mosaic parent. Among inheritance patterns, de novo variation was the most frequent disease etiology. Somatic mosaicism is increasingly recognized as a significant contributor to genetic disease, particularly with increased sequence depth attainable from ES. This report highlights the potential and importance of detecting mosaicism in ES.

The study of long noncoding RNAs (lncRNAs) is an emerging area of cancer research, in part due to their ability to serve as disease biomarkers. However, few studies have investigated lncRNAs in chronic lymphocytic leukemia (CLL). We have identified one particular lncRNA, treRNA, which is overexpressed in CLL B-cells. We measured transcript expression in 144 CLL patient samples and separated samples into high or low expression of treRNA relative to the overall median. We found that high expression of treRNA is significantly associated with shorter time to treatment. High treRNA also correlates with poor prognostic indicators such as unmutated IGHV and high ZAP70 protein expression. We validated these initial findings in samples collected in a clinical trial comparing the nucleoside analog fludarabine alone or in combination with the alkylating agent cyclophosphamide in untreated CLL samples collected prior to starting therapy (E2997). High expression of treRNA was independently prognostic for shorter progression free survival in patients receiving fludarabine plus cyclophosphamide. Given these results, in order to study the role of treRNA in DNA damage response we generated a model cell line system where treRNA was over-expressed in the human B-CLL cell line OSU-CLL. Relative to the vector control line, there was less cell death in OSU-CLL over-expressing treRNA after exposure to fludarabine and mafosfamide, due in part to a reduction in DNA damage. Therefore, we suggest that treRNA is a novel biomarker in CLL associated with aggressive disease and poor response to chemotherapy through enhanced protection against cytotoxic mediated DNA damage.