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NOTCH1is recurrently mutated in chronic lymphocytic leukemia (CLL), most commonly as a 2-bp frameshift deletion (c.7541_7542delCT). This mutated allele encodes a truncated form of the receptor (p.P2514Rfs∗4) lacking the C-terminal proline, glutamic acid, serine, and threonine (PEST) degradation domain that increases NOTCH1signaling duration. NOTCH1mutation has been associated with poor clinical outcomes in CLL. We validated a highly sensitive and quantitative droplet digital PCR assay for the NOTCH1delCT mutation, which was anticipated to perform well compared with Sanger sequencing and allele-specific PCR. Performance characteristics of this assay were tested on 126 samples from an unselected CLL cohort and a separate cohort of 85 samples from patients with trisomy 12 CLL. The delCT mutation was detected at allele frequencies as low as 0.024%; 25% of unselected cases and 55% of trisomy 12 cases were positive at the 0.024% detection threshold. Mutational burdens ≥1% were significantly associated with shorter overall survival (OS) in patients with trisomy 12+ disease in multivariate analysis (median OS, 9.1 versus 13 years, with hazard ratio of 2.34; P = 0.031). Mutational burdens <1% correlated with shorter OS in univariate, but not multivariate, analyses. These results suggest that droplet digital PCR testing for NOTCH1delCT mutation may aid in risk stratification and/or disease monitoring in certain subsets of patients with CLL.

No abstract.

Costello syndrome is a rare congenital disorder typically characterized by severe failure‐to‐thrive, cardiac abnormalities including tachyarrhythmia and hypertrophic cardiomyopathy, distinctive facial features, a predisposition to papillomata and malignant tumors, neurologic abnormalities, developmental delay, and mental retardation. Its underlying cause is de novo germline mutations in the oncogene HRAS. Almost all Costello syndrome mutations affect one of the glycine residues in position 12 or 13 of the protein product. More than 80% of patients with Costello syndrome share the same underlying mutation, resulting in a G12S amino acid change. We report on two patients with novel HRAS mutations affecting amino acids 58 (T58I) and 146 (A146V), respectively. Despite facial features that appear less coarse than those typically seen in Costello patients, both patients show many of the physical and developmental problems characteristic for Costello syndrome. These novel HRAS mutations may be less common than the frequently reported G12S change, or patients with these changes may be undiagnosed due to their less coarse facial features. In addition to the findings previously known to occur in Costello syndrome, one of our patients had hypertrophic pyloric stenosis. This led us to review the medical histories on a cohort of proven HRAS mutation positive Costello syndrome patients, and we found a statistically significantly (P < 0.001) increased frequency of pyloric stenosis in Costello syndrome (5/58) compared to the general population frequency of 2–3/1,000. Thus we add hypertrophic pyloric stenosis to the abnormalities seen with increased frequency in Costello syndrome. © 2008 Wiley‐Liss, Inc.

Many genes have been implicated in Wilms tumor; however, only one gene, WT1, has a proven role in the development of this embryonal tumor. Wilms tumor occurs in a number of congenital syndromes including the Simpson–Golabi–Behmel syndrome (SGBS) which has phenotypic overlap with another Wilms tumor-predisposing syndrome Wiedemann–Beckwith syndrome. The putative function and expression pattern of the SGBS gene, glypican 3 (GPC3), makes it an attractive candidate Wilms tumor gene. We, therefore, hypothesized that Wilms tumors from non-SGBS patients may harbor somatic mutations of GPC3. Mutation analysis of 64 Wilms tumors was performed. One case of a tumor-specific deletion of the entire GPC3 gene and several polymorphisms were identified. GPC3 expression was evaluated in 36 Wilms tumors and 29/36 expressed GPC3. Surprisingly, we did not find evidence of functional mutations of GPC3 in sporadic Wilms tumor suggesting that GPC3 is not often directly involved in Wilms tumorigenesis. © 2003 Wiley-Liss, Inc.

AbstractTwo naturalized populations and two planted populations of Rosa rugosaThunb. were studied with respect to chalcid seed wasp infestation. Megastigmus nigrovariegatusAshmead was found in hips from planted R. rugosain Halifax and Dartmouth, Nova Scotia, but not from bushes at naturalized sites. The percentage of infested hips at the Halifax and Dartmouth locations was 26 and 31%, respectively. The percentage of infested achenes within a single hip varied widely from 3 to 79% at the Halifax site and 7 to 60% at the Dartmouth site.Adult emergence was over 75% successful when achenes were over 2 mm in diameter but less than 15% for smaller achenes. The exit hole on the achene faced the outer wall of the hip. Adults emerged later in Nova Scotia than in the United States and between 59 and 72% were female. More than one adult wasp was observed ovipositing on a single hip.

Cytokines produced by alveolar macrophages are likely involved in the regulation of the immune response arising from respiratory syncytial virus (RSV) infection. Both infectious and UV-inactivated RSV were effective in inducing BALB/c mouse alveolar macrophages to synthesize increased levels of IL-6 mRNA and secreted IL-6 protein. No increase in IL-1β (either mRNA or secreted protein) was observed. The augmented production of IL-6 was activated by purified virus and was reduced by pretreating virus with virus-neutralizing antiserum, demonstrating a requirement for virus in the enhanced IL-6 response. The results suggest that the exposure of BALB/c alveolar macrophages to small quantities of RSV (in the absence of detectable virus replication) is sufficient to trigger IL-6 production. The finding that UV-inactivated virus was effective in triggering IL-6 production by mouse alveolar macrophages is similar to that reported in human alveolar macrophages, providing further validation of the BALB/c mouse as a useful animal model for human RSV infection.

No relevant conflicts of interest to declare.

Gerrie: Roche: Honoraria, Research Funding. Ramadan:Roche: Honoraria, Research Funding.

Toze: Roche: Consultancy, Honoraria, Research Funding. Gerrie:F Hoffmann-La Roche: Other.

No relevant conflicts of interest to declare.

Background: Clinical trials report that chemoimmunotherapy with rituximab (R) improves overall survival (OS) and progression-free survival in the treatment (tx) of symptomatic CLL patients (pts). R has been available for first-line CLL tx in BC, population 4.5 million, since 2004. We compared clinical outcomes with and without addition of R to chemotherapy in a large unselected provincial cohort of pts treated for CLL, to determine the "real world" effectiveness of addition of R to standard chemotherapy.

Background:Among Chronic Lymphocytic Leukemia (CLL) patients (pts), 4-10% are diagnosed with autoimmune cytopenias (AC) at some point during the course of their disease. This is less common than cytopenias related to bone marrow infiltration (10-20%). Infiltrative cytopenias (IC) are clearly a poor prognostic factor. However, the effect of AC on survival and prognosis of CLL pts remains understudied.

BACKGROUND

Background:Chronic Lymphocytic Leukemia (CLL) patients (pts) have significant (sig) heterogeneity; survival ranges from decades to <5 years (yrs). Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is promising treatment (tx) for high-risk pts. Ideally, predictive (pred) tools would allow clinicians to recognize such pts early, permitting transplant performance to maximize benefit and minimize procedure associated risk. Factors with significant (sig) pred capacity are not, however, entirely clarified. Moreover, limited studies compare CLL pts who have/have not received HSCT in terms of differences (diff) in characteristics (char) at diagnosis (dx), population (pop) composition and outcomes. This study evaluates pred factors for outcomes post allo-HSCT, and compares dx char between (bn) tx CLL pts who did /did not receive HSCT by evaluating a large pop-based CLL cohort (n= 1044) from the BC Provincial CLL Database (BCPCD).

Important advances in the understanding of CLL pathogenesis include the discovery that NOTCH1 mutations are present in ∼28% of pts harboring +12. There is a need for improved understanding of the clinical outcomes of CLL patients (pts) with +12 on a population-level, as this subgroup is rapidly becoming the focus of biologic studies evaluating pathogenesis of disease and clinical trials investigating novel targeted therapies. In the province of BC, population 4.5 million, CLL pts receive uniform evaluation and therapy based on centrally derived protocols with FISH testing implemented since 2004. We sought to characterize the clinical outcomes of +12 in this large unselected population-based cohort of CLL pts.Clinical and laboratory data on all pts referred for CLL FISH testing at 1 of 3 BC cytogenetic labs from 2004–2011 were entered into the BC Provincial CLL Database and included in this analysis. Pts without a confirmed diagnostic date were excluded. Baseline features of pts with and without +12 were compared using Fisher's exact test for categorical and Wilcoxon rank sum test for continuous (cnts) variables. Primary and secondary endpoints were OS and TFS (defined as time from diagnosis [dx] to first therapy). Percent of abnormal (%abn) nuclei harboring +12 was evaluated for association with OS/TFS. Cox proportional hazard (PH) models were constructed to determine predictors of OS/TFS for the +12 cohort, including age at dx, sex, Rai stage (0, 1–2, 3–4), WBC at dx, CD38 positivity and concomitant 17p-, 11q- or deletion 13q (13q-). Cox PH models were also constructed to determine effect of +12 on TFS/OS for the entire cohort.As of Dec. 2011, 882 pts had CLL FISH testing in BC of which 164 (19%) had +12 on their 1st FISH test: 8 (5%) with concomitant 17p-; 14 (9%) with 11q-; 142 (86%) without either 17p- or 11q-, of which 43 (30%) had 13q-; 16/124 tested (13%) had an IGH translocation [t(IGH)]. Of the 164 +12 pts, median age at dx was 60 yrs (range 35–93), 70% were male, 10% had Rai stage 3–4. At median follow-up of 4.5 yrs (range 0–19), 95 pts (59%) received treatment, 31 (19%) died. For the +12 cohort, median OS was 14.7 yrs (95% CI 9.8–19.0) and median TFS 3.7 yrs (95% CI 2.7–5.4).Of the 658 non +12 CLL pts (N12CPs), prevalence of recurrent cytogenetic abnormalities (RCA) were: 17p-, 10%; 11q-, 11%; 13q-, 60%; t(IGH) 7%. Significant differences between +12 and N12CPs included more CD38+ pts (66% vs 28%, P<0.001), higher t(IGH) incidence (13% vs 7%, P=0.04) and fewer 17p- (5% vs 10%, P=0.03) or 13q- (26% vs 60%, P<0.001) abn among +12 pts. When pts were grouped by hierarchical FISH abn, +12 pts retained an intermediate OS (median 15.9 yrs) and TFS (median 4.2 yrs) when compared to other RCAs (Fig 1A). Multivariate analysis (MVA) for the whole cohort (n=822) demonstrated no significant effect of +12 on OS (HR 0.72, 95% CI 0.36–1.43, P=.35) or TFS (HR 0.86, 95% CI 0.69–1.36, P=.86) after adjustment for covariates.For the +12 cohort (n=162), univariate analysis demonstrated shorter OS associated with age (P=.001), Rai stage (P=.01) and 17p- (P=.07). A longer OS was associated with presence of 13q- (median OS 11.6 vs 18.7 yrs, P=.04), Fig 1B. Shorter TFS was associated with Rai stage (P<.001), WBC at dx (P=.01) and 17p- (P=.04). %abn nuclei harboring +12 was not predictive of OS (P=.33) or TFS (P=.25) as a cnts variable; however those with <20% vs ≥20% abn had a significant improvement in OS (P=.02). MVA for the +12 cohort demonstrated Rai stage (HR 3.26, 95% CI 1.23– 8.63, P=.02) and 11q- (HR 9.07, 95% CI 1.44–57.02, P=.02) as independent risk factors for OS, while 13q- did not retain its protective effect (P=.98). For TFS, MVA found Rai stage (HR 2.92, 95% CI 1.78–4.78, P<.001) and 17p- (HR 5.44, 95% CI 1.52–19.43, P=.01) as negative predictors while 13q- (HR 2.01, 95% CI 1.08–3.75, P=.03) again had a positive effect.We report the largest, population-based cohort of CLL pts with FISH testing and confirm that +12 occurs in 19% of CLL pts and in the absence of 17p- or 11q-, confers an intermediate prognosis. The presence of 13q- had a protective effect on TFS and a trend towards improved OS, thus improving the prognosis of a subset of +12 pts. This finding is consistent with recent observations that NOTCH1 mutations and 13q- are mutually exclusive in +12 pts and may explain the clinical heterogeneity seen in this subgroup. Further research into these distinct subsets of +12 pts is warranted.No relevant conflicts of interest to declare.

CLL has been considered a genetically stable disease; however, recent evidence suggests that clonal evolution (CE) occurs at a higher rate than previously known. The clinical significance of CE in terms of CLL progression, requirement for and response to therapy (tx), overall (OS) and treatment-free survival (TFS) is not clear. The relationship between CE and tx and to the pathophysiology of CLL is also currently unknown. There is a need for improved understanding of CE in CLL and its possible impact on prognosis, tx, and outcomes. We performed a population-based analysis of all patients (pts) referred for CLL fluorescence in situ hybridization (FISH) testing in BC, Canada who had 32 FISH studies done to determine the incidence of CE and its association with clinical outcomes.FISH testing has been standard of care for CLL pts in BC since 2004 and includes minimally evaluation of 4 prognostically significant recurrent cytogenetic (CG) abnormalities (abn): deletion 13q (13q-), deletion 11q (11q-), trisomy 12 (+12) and 17p- as well as IGH abn in a pt subset. Complete clinical and pathological data has been systematically retrospectively collected on all pts who have had CLL FISH testing performed. We included all pts who had 32 sequential CLL FISH tests 33 months apart from 2004-Dec 2011. Primary endpoints were (a) incidence of CE and (b) overall survival.Of 861 pts screened, 134 met inclusion criteria. 21 pts (16%) had CE with median (med) age 57 yrs (range 45–80) at dx and 63 yrs (49–83) at time CE detected; 16 (77%) were male; Rai stage was low in 7 pts (33%), int in 11 (52%) and high in 3 (14%). At med follow-up of 7 yrs (1–37), 19/21 (91%) of CE pts and 99/113 (88%) non-CE pts received tx. Tx included nucleoside analog (NA) in 3 (14%) vs 23 (20%) pts, NA + rituximab (R) in 8 (38%) vs 53 (47%) pts, chlorambucil in 5 (24%) vs 14 (12%) pts and cyclophosphamide based in 3 (14%) vs 8 (7%) pts in the CE and non-CE groups respectively. 9 (43%) CE vs 36 (32%) non-CE pts underwent stem cell transplant (HSCT). 2 untx pts developed CE. CG abns detected on initial FISH test, classified by Dohner hierarchy were: 13q- (32%), +12 (22%), 17p- (12%), 11q- (11%), none of the 4 abns (23%). A total of 28 abns defining CE were detected in the 21pts and the most common were: 11q- (10 pts, 48%), 13q (10 pts, 47%). Predictors of CE on univariate analysis included lymphocyte count at dx (OR 1.01 per increase in lymphs by 1 ×109/L, 95% CI 1.00–1.02, p=0.048) and presence of 13q- at 1st FISH testing (OR 0.4, 95% CI 0.14–0.98, p=0.047); however these were not predictive of CE on multivariate analysis (MVA) after controlling for covariates (p=0.13 and p=0.19 respectively). No other baseline characteristics were predictive for CE including gender (p=0.31), age (p=0.38), WBC at dx (p=0.07), and CD38 positivity (p=0.56). OS was markedly reduced in pts who developed CE compared to those who did not: HR 5.9, median OS 8.2 yrs vs not reached, p=<0.001 (Fig 1). On MVA, CE retained its negative effect with HR 6.3 (95% CI 2.2–17.6), p<0.001, after adjusting for sex, age, Rai stage and baseline CG abn.CE is more common than previously recognized and carries a poor prognosis. In this study pts with CE fared worse than those with 17p-. OS of pts with CE in this cohort is in keeping with the med OS for 11q- and 17p- reported in the literature, indicating that presence of CE signifies a new poor prognostic indicator. These observations should prompt FISH testing in all pts at diagnosis and relevant time points in follow-up, such as with change in clinical behaviour and prior to new tx. Furthermore, as CE was thought to be tx related, the fact that 2 untx pts developed CE is a novel finding and provides further evidence of the clinical importance of CE in CLL. It is unclear whether CE pts should be considered for more aggressive tx, including HSCT. Results of our study provide a better understanding of the relationship of CE and disease progression in CLL, allowing for improved pt management and tx of this clinically heterogeneous and incurable disease.No relevant conflicts of interest to declare.

Abstract 4132

Abstract 3897

No relevant conflicts of interest to declare.

Abstract 1401This icon denotes a clinically relevant abstract

No relevant conflicts of interest to declare.

Abstract 3100

Toze: Hoffman La Roche: Consultancy, Honoraria, Research Funding; Genzyme: Honoraria, Research Funding; Glaxo Smith Kline: Honoraria. Sutherland:Celgene: Honoraria; Orthobiotech: Honoraria.

Recurrent cytogenetic abnormalities detected by FISH have prognostic significance in CLL patients (pts) [Dohner NEJM 2000]. While pts in unfavourable cytogenetic risk groups (deletion 17p, deletion 11q) consistently show poor overall (OS) and treatment-free survival (TFS), those in intermediate and favourable risk groups (trisomy 12, deletion 13q, normal FISH) are clinically heterogeneous. There remains a need to further refine these FISH prognostic categories to better risk stratify pts. Recently, IGH translocations [t (IGH)] have been increasingly reported in the literature and are suggested to portend a poor prognosis [Cavazzini BJH 2008]. We sought to determine the frequency of t (IGH) in CLL pts at our institution and determine their clinical significance in relation to other common FISH abnormalities, specifically the intermediate-favourable risk groups.We reviewed all CLL pts referred for FISH analysis from 2006 onwards to Vancouver General Hospital (VGH), an academic quaternary-care referral centre and home of the Leukemia/BMT Program of BC. Since 2006, a commercially available FISH panel (Cytocell Ltd.) consisting of ATM, D13S19–25, TP53 DNA locus specific probes, chromosome 12 centromere probe, and an IGH breakapart probe was used for all CLL specimens. All patients were tested for t (11;14) and excluded if positive. Clinical and laboratory data was collected from an institutional CLL database. The primary endpoint was TFS, defined as time from diagnosis to primary therapy.142 CLL pts had FISH performed at VGH between 2006–2009. Median age at diagnosis was 58 yrs (range, 35–85). The prevalence of FISH abnormalities was: 13 deletion 17p (9%), 20 deletion 11q (14%), 41 trisomy 12 (29%), 78 deletion 13q (55%), 37 t (IGH)(26%) and 28 IGH deletion (20%). At median follow-up of 6.9 yrs, 77 pts required CLL treatment (54%); 15 died (11%). Median time from diagnosis to FISH testing was 4.2 yrs (range, 0–22) and median time from diagnosis to therapy was 2.1 yrs (range, 0–17). 49 pts had FISH testing after initiation of therapy. Median TFS for the 142 pts was 5.3 yrs (range, 3.8–6.9); median OS 18.6 yrs. Analysis of the 37 pts with t (IGH) showed they were associated with intermediate-favourable risk cytogenetic abnormalities including trisomy 12 (n=6) and deletion 13q (n=19). The only pts with concomitant deletion 17p (n=1) or deletion 11q (n=5) had FISH testing done after initiation of therapy. When the impact of t (IGH) was assessed irrespective of other cytogenetic abnormalities, pts with t (IGH) did as well as those without; median TFS 4.9 vs. 5.9 yrs (P=0.92) and median OS 18.7 vs. 14.6 yrs (P=0.57) respectively. Next, pts were categorized by the Dohner hierarchy and grouped as follows: unfavourable (deletion 17 or deletion 11q, n= 32), intermediate (trisomy 12 or normal FISH, n=66), or favourable (deletion 13q, n=44). Median TFS for each group was 2.8 yrs, 7.2 yrs, and 7.5 yrs respectively (Figure 1A, P=0.001 across all groups). No significant TFS difference was found in pts with t (IGH) in unfavourable and intermediate risk cytogenetic groups (P=0.43 and P=0.45 respectively). In contrast, a significant difference in median TFS was seen in deletion 13q pts with coexistent t (IGH)(n=14) compared to those without (n=30), 4.7 yrs vs. 8.0 yrs respectively (P=0.027), and a trend towards worse median OS, 14.6 yrs vs. not reached (P=0.31). Pts with deletion 13q alone retained their good prognosis, while those with t (IGH) fell between intermediate and unfavourable risk groups (Figure 1B, P<0.001 across all groups). When only pts with FISH testing prior to therapy were examined, there remained a significant difference between those with deletion 13q and t (IGH)(n=10) vs. deletion 13q alone (n=24)(median TFS 4.7 yrs vs. not reached, P=0.039).In this cohort, the presence of t (IGH) negatively impacted pts with deletion 13q, further stratifying this group into 2 separate prognostic entities. Importantly, the outcome of CLL pts with deletion 13q and coexistent t (IGH) was similar to those with intermediate-unfavourable cytogenetics. This may explain the clinical heterogeneity seen in pts with deletion 13q and lends further support to include this marker in routine FISH analysis. Further analyses of IGH abnormalities in this favourable risk cytogenetic group in a larger population are in progress to validate these results.Toze: Hoffmann-La Roche: Honoraria, Research Funding; Genzyme: Honoraria, Research Funding; GlaxoSmithKline: Honoraria.

Abstract 2434

Abstract 3465