Refining prognosis in chronic lymphocytic leukemia with normal Fluorescence in situ hybridization results.

Fluorescence in situ hybridization (FISH) to detect the recurrent cytogenetics abnormalities deletion 13q, trisomy 12, deletion 11q, and deletion 17p is important for prognostication in chronic lymphocytic leukemia (CLL). A subset of patients are negative for each of these abnormalities (normal 12/13/11/17 FISH), and outcomes are heterogenous within this group. To elucidate variables important for prognostication in this subgroup we conducted a retrospective analysis of 280 treatment-naïve CLL patients with normal standard CLL FISH results. In a multivariable model, advanced Rai stage (p = 0.04, hazard ratio [HR] 1.24 (95% confidence interval [CI] 1.01-1.53)), unmutated immunoglobulin heavy chain gene (IGHV) (p < 0.0001, HR 5.59 (95% CI 3.63-8.62)) and IGH rearrangement by FISH (p = 0.02, HR 2.56 (95% CI 1.20-5.48)) were significantly associated with shorter time to first treatment. In a multivariable model for overall survival, increasing age at 5-year increments (p < 0.0001, HR 1.55 (95% CI 1.25-1.93)), unmutated IGHV (p = 0.01, HR 5.28 (95% CI 1.52-18.35)) and gain of REL (p = 0.01, HR 4.08 (5% CI 1.45-11.49)) were significantly associated with shorter survival. Our study identifies variables important for refining prognosis for CLL patients with normal standard CLL FISH results.

Genetic Characterization of Pediatric Sarcomas by Targeted RNA Sequencing.

Somatic variants, primarily fusion genes and single-nucleotide variants (SNVs) or insertions/deletions (indels), are prevalent among sarcomas. In many cases, accurate diagnosis of these tumors incorporates genetic findings that may also carry prognostic or therapeutic significance. Using the anchored multiplex PCR-based FusionPlex system, a custom RNA sequencing panel was developed that simultaneously detects fusion genes, SNVs, and indels in 112 genes found to be recurrently mutated in solid tumors. Using this assay, a retrospective analysis was conducted to identify somatic variants that may have assisted with classifying a cohort of 90 previously uncharacterized primarily pediatric sarcoma specimens. In total, somatic variants were identified in 45.5% (41/90) of the samples tested, including 22 cases with fusion genes and 19 cases with SNVs or indels. In addition, two of these findings represent novel alterations: a WHSC1L1/NCOA2 fusion and a novel in-frame deletion in the NRAS gene (NM_002524: c.174_176delAGC p.Ala59del). These sequencing results, taken in context with the available clinical data, indicate a potential change in the initial diagnosis, prognosis, or management in 27 of the 90 cases. This study presents a custom RNA sequencing assay that detects fusion genes and SNVs in tandem and has the ability to identify novel fusion partners. These features highlight the advantages associated with utilizing anchored multiplex PCR technology for the rapid and highly sensitive detection of somatic variants.

Disease-associated mosaic variation in clinical exome sequencing: a two-year pediatric tertiary care experience.

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.

Culture and Harvest of CpG-Stimulated Peripheral Blood or Bone Marrow in Chronic Lymphocytic Leukemia.

Chromosome analysis of chronic lymphocytic leukemia (CLL) is an important clinical tool for evaluating prognosis and disease progression. Visualizing chromosomes microscopically using traditional cytogenetic techniques requires dividing cells to be arrested during metaphase. The major challenge for performing this analysis on CLL samples is stimulating the cells to divide in culture. Stimulation of CLL cells with CpG oligodeoxynucleotides has improved our ability to perform chromosome analysis for this leukemia. This protocol should help the reader successfully culture CLL samples for clinical chromosome analysis.

The long noncoding RNA, treRNA, decreases DNA damage and is associated with poor response to chemotherapy in chronic lymphocytic leukemia.

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.

Near-tetraploidy is associated with Richter transformation in chronic lymphocytic leukemia patients receiving ibrutinib.

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. Near-tetraploidy (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 = .03), deletion 17p (P = .03), and complex karyotype (P = .01). Near-tetraploidy was also associated with ibrutinib discontinuation due to Richter transformation (P < .0001), but not due to progressive CLL (P = .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, 3.83-19.59; P < .0001) and complex karyotype (HR, 4.77; 95% CI, 1.42-15.94; P = .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.

Jumping translocations, a novel finding in chronic lymphocytic leukaemia.

A jumping translocation (JT) is a rare cytogenetic aberration that can occur in haematological malignancy. It involves the translocation of the same fragment of donor chromosome onto two or more recipient chromosomes, typically in different cells. In this study, we describe the first series of chronic lymphocytic leukaemia (CLL) patients with JTs reported to date. Following a review of 878 CLL patient karyotypes, we identified 26 patients (3%) with 97 JTs. The most commonly occurring breakpoint in these translocations was 17p11.2. Loss of TP53 was identified prior to or at the same time as JT in 23 of 26 patients (88%). All patients eventually developed a complex karyotype. All but one patient has required treatment for CLL, with estimated median time to treatment of 11·5 months. This study establishes JTs as a recurrent abnormality found in CLL patients with aggressive disease. JTs contribute to complex karyotypes and, in many cases, are involved in chromosomal rearrangements that result in loss of the tumour suppressor gene TP53.