Causes of hypereosinophilia in 100 consecutive patients.

BACKGROUND: Hypereosinophilia (HE, persistent peripheral blood eosinophilia > 1.5 × 109 /L) and hypereosinophilic syndrome (HES, HE with end-organ damage) are classified as primary (due to a myeloid clone), secondary (due to a wide variety of reactive causes), or idiopathic. Diagnostic evaluation of eosinophilia is challenging, in part because secondary causes of HE/HES such as lymphocyte-variant HES (L-HES) and vasculitis are difficult to diagnose, and emerging causes such as immunoglobulin G4-related disease (IgG4-RD) have rarely been examined. OBJECTIVE AND METHODS: We reviewed 100 consecutive patients with HE/HES who underwent extensive evaluation for primary and secondary eosinophilia at a single tertiary care center to determine causes of HE/HES in a modern context. RESULTS: Six patients had primary HE/HES, 80 had a discrete secondary cause identified, and 14 had idiopathic HE/HES. The most common causes of secondary eosinophilia were L-HES/HES of unknown significance (L-HESus) (20), IgG4-RD (9), and eosinophilic granulomatosis with polyangiitis (EGPA) (8). CONCLUSIONS: In contrast to other large published series of HE/HES, most patients in this study were found to have a discrete secondary cause of eosinophilia and only 14 were deemed idiopathic. These findings highlight the importance of extensive evaluation for secondary causes of eosinophilia such as L-HES, IgG4-RD, and EGPA.

Influence of clone and deletion size on outcome in chronic lymphocytic leukemia patients with an isolated deletion 13q in a population-based analysis in British Columbia, Canada.

Deletion of the long arm of chromosome 13 (del(13q)) as the sole abnormality in chronic lymphocytic leukemia (CLL) portends a good prognosis; however, there is great outcome heterogeneity within this subgroup. The percentage of cells with a del(13q) (clone size) and the extent of the deletion are two factors that may affect outcome in CLL patients with isolated del(13q). We analyzed 248 CLL patients from the BC Provincial CLL database identified as having isolated del(13q) detected pretreatment by interphase fluorescence in situ hybridization to determine what impact clone and deletion size had on overall survival (OS) and treatment free survival (TFS). Patients with 60% or more of nuclei with a del(13q) had shorter TFS and shorter OS. A large deletion, encompassing the RB1 gene locus, was detected in half of the 90 cases with available specimens for testing, and there was no significant difference in OS and TFS between RB1-deleted and RB1-not-deleted cases. Further study in a larger sample size is required to determine the clinical interest of RB1 locus testing; however, clone size of del(13q) does predict TFS and OS and may better refine prognosis in this clinically heterogeneous population.

Subjectivity in chromosome band-level estimation: a multicenter study.

PURPOSE: Chromosome band level is the primary quality indicator for G-banded metaphase chromosome analysis. Although current professional guidelines address the minimum necessary band level for constitutional studies, there is no study documenting the comparative performance of different band-level estimation methods. METHODS: This study compared 5 band-level estimation methods (Stallard, Vancouver, Welborn, United Kingdom External Quality Assurance Scheme, and Ford) in a multicenter study in which 82 readers from 7 different clinical cytogenetics laboratories evaluated the same 10 karyotypes (5 from amniotic fluid and 5 from peripheral blood) by each method. RESULTS: There was a 94% correlation between the five band-level estimation methods. The Welborn method yielded significantly lower scores for amniotic fluid karyotypes (P < 0.01) but not for peripheral blood karyotypes (P = 0.75). The distribution of scores obtained from different readers suggests a high level of subjectivity in chromosome band-level assessment. The variation in band-level estimation did not correlate with reader experience or study center, except for readers from one laboratory, for which the distribution of scores was significantly lower (P < 0.01). CONCLUSION: The results from this study suggest that the consistent use of one method is more important than the actual method employed for monitoring karyotype quality.

Mutations in the human ortholog of Aristaless cause X-linked mental retardation and epilepsy.

Mental retardation and epilepsy often occur together. They are both heterogeneous conditions with acquired and genetic causes. Where causes are primarily genetic, major advances have been made in unraveling their molecular basis. The human X chromosome alone is estimated to harbor more than 100 genes that, when mutated, cause mental retardation. At least eight autosomal genes involved in idiopathic epilepsy have been identified, and many more have been implicated in conditions where epilepsy is a feature. We have identified mutations in an X chromosome-linked, Aristaless-related, homeobox gene (ARX), in nine families with mental retardation (syndromic and nonspecific), various forms of epilepsy, including infantile spasms and myoclonic seizures, and dystonia. Two recurrent mutations, present in seven families, result in expansion of polyalanine tracts of the ARX protein. These probably cause protein aggregation, similar to other polyalanine and polyglutamine disorders. In addition, we have identified a missense mutation within the ARX homeodomain and a truncation mutation. Thus, it would seem that mutation of ARX is a major contributor to X-linked mental retardation and epilepsy.

BPES with atypical premature ovarian insufficiency, and evidence of mitotic recombination, in a woman with trisomy X and a translocation t(3;11)(q22.3;q14.1).

Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is a rare autosomal dominant disorder characterized by a complex dysgenesis of the eyelids and premature ovarian insufficiency. FOXL2 located at 3q22.3, encoding a forkhead transcription factor, is the only gene known to be responsible for BPES. We describe a patient diagnosed with BPES with atypical ovarian failure, characterized by normal levels of gonadotropins, who was found to have trisomy X as well as a translocation (3;11)(q22.3;q14.1). The translocation breakpoint at 3q22.3 is located upstream of the FOXL2 gene and most likely causes BPES by separating the FOXL2 transcription unit from its cis-regulatory sequences. By array analysis we detected mosaicism for the balanced and an unbalanced form of the translocation in blood cells. We propose mitotic recombination as the likely mechanism of the mosaicism formation. Mitotic recombination is a common phenomenon in human cells. Thus, we hypothesize that it may be one of the mechanisms responsible for cryptic imbalances and possible abnormal phenotypes in some carriers of balanced rearrangements.

Influence of cytogenetics in patients with relapsed or refractory multiple myeloma treated with lenalidomide plus dexamethasone: adverse effect of deletion 17p13.

Although the combination of lenalidomide and dexamethasone is effective therapy for patients with relapsed/refractory multiple myeloma, the influence of high-risk cytogenetic abnormalities on outcomes is unknown. This subanalysis of a large, open-label study investigated the effects of the most common unfavorable cytogenetic abnormalities detected by fluorescence in situ hybridization, del(13q), t(4;14), and del(17p13), in 130 evaluable patients treated with this regimen. Whereas patients with either del(13q) or t(4;14) experienced a median time to progression and overall survival comparable with those without these cytogenetic abnormalities, patients with del(17p13) had a significantly worse outcome, with a median time to progression of 2.22 months (hazard ratio, 2.82; P < .001) and median overall survival of 4.67 months (hazard ratio, 3.23; P < .001). Improved therapeutic strategies are required for this subgroup of patients. This study was registered at www.ClinicalTrials.gov as #NCT00179647.

Impact of age and treatment institution type on outcomes of patients treated for chronic lymphocytic leukemia in British Columbia, Canada.

Older age has been shown to adversely impact overall survival (OS) in chronic lymphocytic leukemia (CLL) however, prior population-based studies did not analyze the impact of cytogenetic abnormalities or were prior to the availability of ibrutinib. OBJECTIVES: i) We sought to compare outcomes of patients based on their age at treatment to examine if older age has an impact on OS in patients who were treated during the period when fludarabine-rituximab was the standard upfront therapy and when ibrutinib was first introduced and ii) compare outcomes based on whether the patient received primary treatment at an academic or community-based centre. METHODS: The BC Provincial CLL Database, a population-based databasewas used to include patients who have received treatment in British Columbia (BC), Canada between 2004 and 2016. RESULTS: A total of 1122 patients were included (<70 years at treatment, n = 589) with median age at diagnosis 66 years. Younger patients had higher Rai stage (55% vs. 44% stage I-II, p < 0.001), higher lymphocyte count at diagnosis (13 × 109/L vs. 10 × 109/L, p = 0.004), greater proportion with B-symptoms at diagnosis (15% vs 10%, p = 0.004), shorter time from diagnosis to treatment (13.9 months vs. 21.4 months, p = 0.001), higher proportion treated at an academic centre (79% vs. 69%, p < 0.001) and more were treated with fludarabine-rituximab or FCR (69% vs. 42%, p < 0.001) compared to older patients. Older patients had both a significantly (p < 0.001) shorter OS from treatment start (4.7 years) and disease specific survival (8.1 years) than younger patients (median OS and DSS not reached). Of interest, there was no difference in OS between patients treated at an academic centre or community centre (p = 0.087). First-line treatment with chemoimmunotherapy improved OS (HR 0.465, 95% CI: 0.381-.567). CONCLUSIONS: Older age but not treatment-institution type adversely impacts overall survival and CLL survival in treated patients in BC.

Tuning the pH sensitivities of orthoester based compounds for drug delivery applications by simple chemical modification.

Orthoesters are acid-sensitive moieties that allow substantial structural diversity for biological applications including drug delivery. Here, the pH-sensitivity of a range of novel orthoester based compounds was compared in the range 7.5-4.5 that is characteristic of the increased acidification during endocytosis. We find that simple modifications close to the orthoester had major effects on both the rate and extent of hydrolysis, suggesting this could be exploited for activating drug delivery systems on endocytic pathways.

Evidence-based review of genomic aberrations in B-lymphoblastic leukemia/lymphoma: Report from the cancer genomics consortium working group for lymphoblastic leukemia.

Clinical management and risk stratification of B-lymphoblastic leukemia/ lymphoma (B-ALL/LBL) depend largely on identification of chromosomal abnormalities obtained using conventional cytogenetics and Fluorescence In Situ Hybridization (FISH) testing. In the last few decades, testing algorithms have been implemented to support an optimal risk-oriented therapy, leading to a large improvement in overall survival. In addition, large scale genomic studies have identified multiple aberrations of prognostic significance that are not routinely tested by existing modalities. However, as chromosomal microarray analysis (CMA) and next-generation sequencing (NGS) technologies are increasingly used in clinical management of hematologic malignancies, these abnormalities may be more readily detected. In this article, we have compiled a comprehensive, evidence-based review of the current B-ALL literature, focusing on known and published subtypes described to date. More specifically, we describe the role of various testing modalities in the diagnosis, prognosis, and therapeutic relevance. In addition, we propose a testing algorithm aimed at assisting laboratories in the most effective detection of the underlying genomic abnormalities.

Comparison of real-world treatment patterns in chronic lymphocytic leukemia management before and after availability of ibrutinib in the province of British Columbia, Canada.

We performed a retrospective study comparing treatment patterns and overall survival (OS) in chronic lymphocytic leukemia (CLL) patients with the advent of ibrutinib to provide current real-world data. METHODS: Using a provincial population-based database, we analyzed CLL patients who received upfront treatment in British Columbia before ibrutinib availability (1984-2014), during ibrutinib access for: relapse only (2014-2015) and for upfront treatment of patients (with 17p deletion or unfit for chemotherapy) (2015-2016). Analysis included up to third-line treatment. RESULTS: Of 1729 patients meeting inclusion criteria (median age, 66 years; 1466, period 1; 140, period 2; 123, period 3), FR was the most common first-line therapy (35.8 %, 54.3 % and 40.7 %, periods 1-3, respectively) and 18.7 % received ibrutinib upfront in period 3. The most common therapies in relapse were chemoimmunotherapy (36.1 % and 55.6 %, periods 1 and 2, second-line; 29.2 %, period 1, third-line) and ibrutinib (69.8 %, period 3, second-line; 46.4 % and 70.3 %, periods 2 and 3, third-line). OS improved for patients treated in periods 2-3 over period 1 (median OS not reached vs. 11.9 years, p < 0.001; no difference in OS for periods 2-3, p = 0.385). CONCLUSION: Ibrutinib has replaced chemoimmunotherapy as the preferred therapy in relapse. Overall survival has improved over time with access to ibrutinib.

Ultrasensitive Detection of NOTCH1 c.7544_7545delCT Mutations in Chronic Lymphocytic Leukemia by Droplet Digital PCR Reveals High Frequency of Subclonal Mutations and Predicts Clinical Outcome in Cases with Trisomy 12.

NOTCH1 is 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 NOTCH1 signaling duration. NOTCH1 mutation has been associated with poor clinical outcomes in CLL. We validated a highly sensitive and quantitative droplet digital PCR assay for the NOTCH1 delCT 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 NOTCH1 delCT mutation may aid in risk stratification and/or disease monitoring in certain subsets of patients with CLL.

Chronic lymphocytic leukemia patients with HLA-B27 referred for allogeneic hematopoietic stem cell transplantation do not have worse outcomes: Results of a population-based case series analysis in British Columbia, Canada.

Human leukocyte antigen B27 (HLA-B27), associated with spondyloarthritis, was suggested to be protective against chronic lymphocytic leukemia (CLL). It is hypothesized that HLA-B27 patients may have worse outcome in part related to their other comorbidities. OBJECTIVES: We sought to compare the clinical characteristics and outcomes of CLL and small lymphocytic lymphoma (SLL) patients referred for allogeneic hematopoietic stem cell transplantation (allo-HSCT) based on their HLA-B27 status. METHODS: This retrospective population-based case series analyzed CLL/SLL patients who were HLA-typed for potential allo-HSCT in British Columbia, Canada. RESULTS: of 279 CLL/SLL patients referred for potential allo-HSCT, 34 patients were HLA-B27 positive. For HLA-B27 patients, median age at CLL diagnosis was 53.5 years (range, 27-67) and 71% were male. Seven patients had 11q deletion and nine patients had 17p deletion detected prior to first CLL therapy or at relapse. Eleven HLA-B27 patients received allo-HSCT. Two patients developed acute myeloid leukemia. One patient with ankylosing spondylitis had Richter's transformation prior to any CLL therapy. Spondyloarthritis-related disorders were diagnosed in 12 HLA-B27 patients but there was no temporal correlation with development of CLL. Overall survival (OS) and treatment-free survival (TFS) were not significantly different between HLA-B27 patients with or without spondyloarthritis-related disorders. There were no significant differences in clinical characteristics at CLL diagnosis or OS/TFS between HLA-B27 positive and negative patients referred for allo-HSCT. CONCLUSIONS: HLA-B27 positivity does not appear to influence outcome for CLL/SLL patients referred for allo-HSCT. Further studies are needed to evaluate the clinical significance of HLA-B27 in a general CLL population.

Improved survival outcomes with the addition of rituximab to initial therapy for chronic lymphocytic leukemia: a comparative effectiveness analysis in the province of British Columbia, Canada.

Chemoimmunotherapy with rituximab improves survival in clinical trials in upfront chronic lymphocytic leukemia (CLL) treatment. This study compared clinical outcomes with and without rituximab added to first-line chemotherapy in a provincial cohort of CLL patients. Between 1973 and 2014, 1345 patients received CLL treatment: 48% with rituximab, 52% chemotherapy alone. Median overall survival (OS) and treatment-free survival (TFS) were significantly longer with rituximab: OS 8.9 vs. 6.2 years, p < .0001; TFS 3.6 vs. 2.1 years, p < .0001. Addition of rituximab to chemotherapy was a strong independent predictor of mortality with a 32% mortality reduction after controlling for co-variates (age, sex, stage, and treatment with purine analogs). This large population-based study complements clinical trial and registry data demonstrating the benefit of adding rituximab to first-line CLL therapy and adds further evidence of the efficacy of rituximab-based chemoimmunotherapy in a real-world setting.

Chronic Lymphocytic Leukemia Patients With Deletion 11q Have a Short Time to Requirement of First-Line Therapy, But Long Overall Survival: Results of a Population-Based Cohort in British Columbia, Canada.

BACKGROUND: Chronic lymphocytic leukemia (CLL) patients with 11q22.3 deletion (11q-) have an aggressive clinical course, and thus selection of first-line therapy in this group is important. This study aimed to improve our understanding of real-world practice patterns and outcomes of CLL patients with 11q- in a population-based setting. PATIENTS AND METHODS: The British Columbia CLL Database was used to identify patients with 11q-. Overall survival (OS) and treatment-free survival (TFS) were assessed after adjustment for prognostic factors. RESULTS: Of 1044 patients in the database, 125 had 11q- (12%). Sixty-nine patients had 11q- identified before therapy initiation and had a median OS and TFS of 14.7 (95% confidence interval [CI], 11.3-18.1) and 2.5 (95% CI, 1.5-3.6) years. Patient with copresence of 11q- and deletion 17p had a markedly worse prognosis, with median OS of 4.9 versus 14.7 years (P < .001). Most treated patients (33 of 52) received fludarabine with or without rituximab (FR). Patients treated with FR had a median OS of 12.8 years (standard error, 1.0), which was not statistically different from those treated with alkylator-containing therapy (P = .35). CONCLUSION: Although median TFS of 11q- patients in this cohort was short at 2.5 years, OS remains long at 14.7 years, even when most patients received initial treatment without alkylators.

Characterization of treatment and outcomes in a population-based cohort of patients with chronic lymphocytic leukemia referred for cytogenetic testing in British Columbia, Canada.

This study evaluates outcomes in chronic lymphocytic leukemia (CLL) based on first-line therapy in a large consecutive population-based cohort of 669 patients with fluorescence in-situ hybridization (FISH) data in British Columbia, Canada during the period when chemoimmunotherapy was standard first-line treatment. When analyzed as a time-dependent variable, patients who required treatment (n=336) had a 4.7 times higher hazard of death than patients who did not (95% confidence interval 2.8-7.9, P<0.001). The majority of patients received fludarabine-rituximab (FR) in front-line. On multivariate Cox regression analysis, fludarabine-based first-line therapy predicted longer time-to-next-treatment (TTNT) (HR 0.53, 95% confidence interval 0.33-0.87, P=0.012) but no difference in overall survival (OS) compared to alkylator-based therapy. Deletion 17p was an independent predictor of worse TTNT and OS. The most common second-line treatments were cyclophosphamide-vincristine-prednisone-rituximab and FR. There was no difference in OS between patients retreated in second-line with the same first-line regimen (n=33) versus different regimen (n=113). In conclusion, front-line treatment with fludarabine leads to a longer time until need for next treatment than alkylator-based therapy; however, fludarabine or alkylator therapy produces no difference in OS. This study provides a historical baseline for the comparison of novel agents with standard treatments in CLL on a population-level.

Clonal evolution as detected by interphase fluorescence in situ hybridization is associated with worse overall survival in a population-based analysis of patients with chronic lymphocytic leukemia in British Columbia, Canada.

This study evaluates prognostic markers as predictors of clonal evolution (CE) and assesses the impact of CE on overall survival (OS) in a population-based cohort of 159 consecutive eligible patients with chronic lymphocytic leukemia (CLL) obtained from the British Columbia Provincial CLL Database. CE was detected by interphase fluorescence in situ hybridization (FISH) in 34/159 patients (21%) with 65% of CE patients acquiring deletion 17p or 11q. CD38 positive status (≥30%) on flow cytometry predicted 2.7 times increased risk of high-risk CE (acquisition of deletion 17p or 11q) on multivariate analysis. Prior CLL therapy was not a significant predictor of CE. CE was associated with 4.1 times greater risk of death when analyzed as a time-dependent variable for OS after adjusting for age, lymphocyte count, and FISH timing. High-risk CE was associated with worse OS while acquisition of low/intermediate-risk abnormalities (trisomy 12, deletion 13q, and IGH translocation) had no difference in OS. Our study demonstrates the negative impact of CE detected by FISH on OS in this population-based cohort. These data provide support for repeating FISH testing during CLL follow-up as patients with high-risk CE have reduced survival and may require closer observation.

Philadelphia-negative clonal hematopoiesis following imatinib therapy in patients with chronic myeloid leukemia: a report of nine cases and analysis of predictive factors.

There are increasing reports of Philadelphia-negative (Ph-negative) clonal hematopoiesis developing among patients with chronic myeloid leukemia (CML) treated with imatinib mesylate (IM). To establish the incidence and significance of these chromosomal abnormalities, we analyzed data on 141 consecutive patients with CML treated with IM at the British Columbia Cancer Agency and Vancouver General Hospital from 1999 to 2004. The cumulative incidence of developing a Ph-negative clone three years from the start of IM was 8.7% at a median of 13.3 months. The Ph-negative clonal abnormalities included monosomy 7 and/or trisomy 8 (seven patients), monosomy for chromosomes X and 22 (one patient), and a (12;16) translocation (one patient). Two of the patients presented with the same chromosomal abnormality in both Ph-negative and Ph-positive cells. None of the Ph-negative clonal abnormalities was associated with myelodysplasia. In a multivariate analysis, an interval from diagnosis to initiation of IM of 1 year or less was associated with an increased risk of developing a Ph-negative clone (relative risk = 20.2; P = 0.025). There was no difference, however, in event-free survival between patients who did and did not develop Ph-negative clones. Therefore, while the development of Ph-negative clonal hematopoiesis in patients with CML treated with IM is uncommon, it appears to be more frequent than that previously seen with IFN, but it does not seem to confer a worse prognosis.

Molecular cytogenetics in reproductive pathology.

This chapter presents the summary of two molecular cytogenetic techniques--FISH and CGH--with their applications and limitations in the studies of pregnancy loss. These molecular techniques clearly represent a significant advantage over the traditional cytogenetic technique and likely will become the predominant cytogenetic techniques in reproductive cytogenetics of the future.