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Patients with core-binding factor (CBF) acute myeloid leukemia (AML), caused by either t(8;21)(q22;q22) or inv(16)(p13q22)/t(16;16)(p13;q22), have higher complete remission rates and longer survival than patients with other subtypes of AML. However, ∼40% of patients relapse, and the literature suggests that patients with inv(16) fare differently from those with t(8;21). We retrospectively analyzed 537 patients with CBF-AML, focusing on additional cytogenetic aberrations to examine their impact on clinical outcomes. Trisomies of chromosomes 8, 21, or 22 were significantly more common in patients with inv(16)/t(16;16): 16% vs 7%, 6% vs 0%, and 17% vs 0%, respectively. In contrast, del(9q) and loss of a sex chromosome were more frequent in patients with t(8;21): 15% vs 0.4% for del(9q), 37% vs 0% for loss of X in females, and 44% vs 5% for loss of Y in males. Hyperdiploidy was more frequent in patients with inv(16) (25% vs 9%, whereas hypodiploidy was more frequent in patients with t(8;21) (37% vs 3%. In multivariable analyses (adjusted for age, white blood counts at diagnosis, and KIT mutation status), trisomy 8 was associated with improved overall survival (OS) in inv(16), whereas the presence of other chromosomal abnormalities (not trisomy 8) was associated with decreased OS. In patients with t(8;21), hypodiploidy was associated with improved disease-free survival; hyperdiploidy and del(9q) were associated with improved OS. KIT mutation (either positive or not tested, compared with negative) conferred poor prognoses in univariate analysis only in patients with t(8;21).

Although somatic mutations influence the pathogenesis, phenotype, and outcome of myeloproliferative neoplasms (MPNs), little is known about their impact on molecular response to cytoreductive treatment. We performed targeted next-generation sequencing (NGS) on 202 pretreatment samples obtained from patients with MPN enrolled in the DALIAH trial (A Study of Low Dose Interferon Alpha Versus Hydroxyurea in Treatment of Chronic Myeloid Neoplasms; #NCT01387763), a randomized controlled phase 3 clinical trial, and 135 samples obtained after 24 months of therapy with recombinant interferon-alpha (IFNα) or hydroxyurea. The primary aim was to evaluate the association between complete clinicohematologic response (CHR) at 24 months and molecular response through sequential assessment of 120 genes using NGS. Among JAK2-mutated patients treated with IFNα, those with CHR had a greater reduction in the JAK2 variant allele frequency (median, 0.29 to 0.07; P < .0001) compared with those not achieving CHR (median, 0.27 to 0.14; P < .0001). In contrast, the CALR variant allele frequency did not significantly decline in those achieving CHR or in those not achieving CHR. Treatment-emergent mutations in DNMT3A were observed more commonly in patients treated with IFNα compared with hydroxyurea (P = .04). Furthermore, treatment-emergent DNMT3A mutations were significantly enriched in IFNα–treated patients not attaining CHR (P = .02). A mutation in TET2, DNMT3A, or ASXL1 was significantly associated with prior stroke (age-adjusted odds ratio, 5.29; 95% confidence interval, 1.59-17.54; P = .007), as was a mutation in TET2 alone (age-adjusted odds ratio, 3.03; 95% confidence interval, 1.03-9.01; P = .044). At 24 months, we found mutation-specific response patterns to IFNα: (1) JAK2- and CALR-mutated MPN exhibited distinct molecular responses; and (2) DNMT3A-mutated clones/subclones emerged on treatment.

Currently, no molecular biomarker indices are used in standard care to make treatment decisions at diagnosis of chronic lymphocytic leukemia (CLL). We used Infinium MethylationEPIC array data from diagnostic blood samples of 114 CLL patients and developed a procedure to stratify patients based on methylation signatures associated with mutation load of the IGHV gene. This procedure allowed us to predict the time to treatment with a hazard ratio (HR) of 8.34 (95% confidence interval [CI]: 4.54-15.30), as opposed to a HR of 4.35 (95% CI: 2.60-7.28) using IGHV mutation status. Detailed evaluation of 17 cases for which the two classification procedures gave discrepant results showed that these cases were incorrectly classified using IGHV status. Moreover, methylation-based classification stratified patients with different overall survival (HR=1.82; 95% CI: 1.07-3.09), which was not possible using IGHV status. Furthermore, we assessed the performance of the developed classification procedure using published HumanMethylation450 array data for 159 patients for whom information on time to treatment, overall survival and relapse was available. Despite 450K array methylation data not containing all the biomarkers used in our classification procedure, methylation signatures again stratified patients with significantly better accuracy than did IGHV mutation load regarding all available clinical outcomes. Thus, stratification using IGHV-associated methylation signatures may provide better prognostic power than IGHV mutation status.

Introduction. Patients with eosinophilia (an increased number of eosinophilic granulocytes > 0.5 × 109/l in the blood) are encountered in all medical specialties and frequently need thorough workup to identify the eliciting causes and decide whether treatment is indicated. In Denmark, highly specialised centres for eosinophilic diseases or conditions have been established to provide a foundation for the management of complicated cases. Here, we present experiences from such a multidisciplinary centre. Methods. This was a retrospective study of all patients seen in our tertiary centre for eosinophilia in the 2016-2019 period. Results. Referrals mainly derived from specialised secondary care and to a lesser degree from primary care physicians. Patients were either asymptomatic or exhibited symptoms from up to three organ systems and presented a median eosinophil count of 1.7 × 109/l. Up to eight new clonality analyses or imaging studies per patient were performed after referral. One of these, T-cell receptor analysis, was performed frequently but provided limited information, whereas, e.g., flow cytometry proved more clinically applicable owing to its broader diagnostic range. In total, 51 patients were evaluated and classified as secondary (59%), myeloid neoplasm with PDGFRA rearrangement (2%), idiopathic hypereosinophilic syndrome (31%) and idiopathic hypereosinophilia (8%). Conclusion. The value of a multidisciplinary and versatile approach in a highly specialised centre has a positive impact on diagnostic processes as well as on the evaluation of treatment need.

BackgroundPeritoneal metastasis from pancreatic cancer (PM-PC) may be treated with repeated pressurised intraperitoneal aerosol chemotherapy (PIPAC). Utility of next-generation sequencing (NGS) to detect cancer-related mutations in peritoneal quadrant biopsies (QBs) and peritoneal fluid (PF) after systemic and PIPAC treatment has not been evaluated. Around 90% of pancreatic cancers (PCs) harbour a KRAS mutation, making PC ideal for the evaluation of this aspect.AimsEvaluation of PM-PC in terms of (1) histological response to PIPAC using Peritoneal Regression Grading Score (PRGS), (2) clinical characteristics and (3) frequency of mutations in QBs and PF before and after PIPAC.MethodsPeritoneal QBs and PF were obtained prior to each PIPAC. NGS for 22 cancer-related genes was performed on primary tumours, QBs and PFs. Response was assessed by the four-tiered PRGS.ResultsSixteen patients treated with a median of three PIPAC procedures were included. The mean PRGS was reduced from 1.91 to 1.58 (p=0.02). Fifty-seven specimens (13 primary tumours, 2 metastatic lymph nodes, 16 PFs and 26 QB sets) were analysed with NGS. KRAS mutation was found in 14/16 patients (87.50%) and in QBs, primary tumours and PF in 8/12 (66.67%), 8/13 (61.53%) and 6/9 (66.67%). The median overall survival was 9.9 months (SE 1.5, 95% CI 4.9 to 13.9).ConclusionPIPAC induces histological response in the majority of patients with PM-PC. KRAS mutation can be found in PM-PC after PIPAC at a frequency similar to the primaries. NGS may be used to detect predictive mutations in PM-PC of various origins, also when only post-PIPAC QBs or PFs are available.

MonoMAC is a complex primary immunodeficiency caused by mutations in the myeloid transcription factor GATA2, characterized by multilineage cytopenia with malignant complications and severe infections, including mycobacteria and herpesviruses. We describe the clinical presentation, genetics and antiviral inflammatory responses in a small case series. Two patients presented in childhood with mycobacterial infection and were diagnosed with MonoMAC germline GATA2 variants; their healthy fathers with the same mutations were also studied. Three patients were elderly individuals with acquired GATA2 mutations and malignant haematological conditions. Overall, this study demonstrates the heterogeneous clinical presentation and variation in immunodeficiency caused by GATA2 mutations.

In systemic mastocytosis (SM), qualitative and serial quantitative assessment of the KIT D816V mutation is of diagnostic and prognostic relevance. We investigated peripheral blood and bone marrow samples of 161 patients (indolent SM (ISM), n = 40, advanced SM, AdvSM, n = 121) at referral and during follow-up for the KIT D816V variant allele frequency (VAF) at the DNA-level and the KIT D816V expressed allele burden (EAB) at the RNA-level. A round robin test with four participating laboratories revealed an excellent correlation (r &gt, 0.99, R2 &gt, 0.98) between three different DNA-assays. VAF and EAB strongly correlated in ISM (r = 0.91, coefficient of determination, R2 = 0.84) but only to a lesser extent in AdvSM (r = 0.71, R2 = 0.5). However, as compared to an EAB/VAF ratio ≤2 (cohort A, 77/121 patients, 64%) receiver operating characteristic (ROC) analysis identified an EAB/VAF ratio of &gt, 2 (cohort B, 44/121 patients, 36%) as predictive for an advanced phenotype and a significantly inferior median survival (3.3 vs. 11.7 years, p = 0.005). In terms of overall survival, Cox-regression analysis was only significant for the EAB/VAF ratio &gt, 2 (p = 0.006) but not for VAF or EAB individually. This study demonstrates for the first time that the transcriptional activity of KIT D816V may play an important role in the pathophysiology of SM.

Mastocytosis is a rare myeloid neoplasm characterized by uncontrolled expansion of mast cells, driven in >80% of affected individuals by acquisition of the KIT D816V mutation. To explore the hypothesis that inherited variation predisposes to mastocytosis, we performed a two-stage genome-wide association study, analyzing 1,035 individuals with KIT D816V positive disease and 17,960 healthy control individuals from five European populations. After quality control, we tested 592,007 SNPs at stage 1 and 75 SNPs at stage 2 for association by using logistic regression and performed a fixed effects meta-analysis to combine evidence across the two stages. From the meta-analysis, we identified three intergenic SNPs associated with mastocytosis that achieved genome-wide significance without heterogeneity between cohorts: rs4616402 (pmeta = 1.37 × 10−15, OR = 1.52), rs4662380 (pmeta = 2.11 × 10−12, OR = 1.46), and rs13077541 (pmeta = 2.10 × 10−9, OR = 1.33). Expression quantitative trait analyses demonstrated that rs4616402 is associated with the expression of CEBPA (peQTL = 2.3 × 10−14), a gene encoding a transcription factor known to play a critical role in myelopoiesis. The role of the other two SNPs is less clear: rs4662380 is associated with expression of the long non-coding RNA gene TEX41 (peQTL = 2.55 × 10−11), whereas rs13077541 is associated with the expression of TBL1XR1, which encodes transducin (β)-like 1 X-linked receptor 1 (peQTL = 5.70 × 10−8). In individuals with available data and non-advanced disease, rs4616402 was associated with age at presentation (p = 0.009; beta = 4.41; n = 422). Additional focused analysis identified suggestive associations between mastocytosis and genetic variation at TERT, TPSAB1/TPSB2, and IL13. These findings demonstrate that multiple germline variants predispose to KIT D816V positive mastocytosis and provide novel avenues for functional investigation., A full list of the investigators who contributed to the generation of the WTCCC data is available from the WTCCC website, funding for which was provided by The Wellcome Trust under award 07611. The KORA study was initiated and financed by the Helmholtz Zentrum München—German Research Center for Environmental Health, which is funded by the German Federal Ministry of Education and Research (BMBF) and by the State of Bavaria. Furthermore, KORA research was supported within the Munich Center of Health Sciences (MC-Health), Ludwig-Maximilians-Universität, as part of LMUinnovativ. The KORA Study Group consists of A. Peters (speaker), J. Heinrich, R. Holle, R. Leidl, C. Meisinger, K. Strauch, and their co-workers, who are responsible for the design and conduct of the KORA studies. We gratefully acknowledge the contribution of all members of field staff conducting the KORA study, and we are grateful to all study participants of KORA for their invaluable contributions to this study. The InCHIANTI study baseline (1998–2000) was supported as a “targeted project” (ICS110.1/RF97.71) by the Italian Ministry of Health and in part by the U.S. National Institute on Aging (263 MD 9164 and 263 MD 821336). The Spanish mastocytosis cohort and the Spanish National DNA Bank were supported by grants from the Instituto de Salud Carlos III and FEDER (PI16/00642 and PT17/0015/0044). The Italian cohort of mastocytosis-affected individuals was funded by the Associazione Italiana per la Ricerca sul cancro, Mynerva project, 21267. The Novo Nordisk Foundation Center for Basic Metabolic Research is an independent Research Center at the University of Copenhagen partially funded by an unrestricted donation from the Novo Nordisk Foundation. A.A.Z.D. was supported by a Lady Tata International Award; G.G., N.C.P.C., Y.H., A.J.C., and W.J.T. were supported by Blood Cancer UK (13002 and 18007).

BACKGROUND: Patients with systemic mastocytosis (SM) may suffer from mast cell (MC) mediator-related symptoms insufficiently controlled by conventional therapy. Omalizumab is an established treatment in other MC-driven diseases, but experiences in SM are limited.OBJECTIVE: To assess the efficacy and safety of omalizumab in SM.METHODS: In our patient cohort, we evaluated all SM patients treated with omalizumab. A physician global assessment of type and severity of symptoms was performed at baseline, at 3 and 6 months and at latest follow-up. Quality of life was assessed by visual analogue scale. S-tryptase and KIT D816V allele burden were monitored.RESULTS: A total of 14 adult SM patients (10 ISM, 2 BMM, 1 SSM, and 1 ASM-AHN) received omalizumab with a median duration of 17 months (range: 1-73 months). One patient was excluded due to concomitant cytoreductive therapy. In the remaining 13 patients, we observed a significant reduction in symptoms, with complete symptom control in five (38.5%), major response in three (23.1%), and a partial response in three (23.1%) patients, whereas two patients (15.4%) withdrew due to subjective side-effects at first dose. The treatment was most effective for recurrent anaphylaxis and skin symptoms, less for gastrointestinal, musculoskeletal, and neuropsychiatric symptoms. Patient-reported quality of life showed significant improvement. No significant changes in s-tryptase/KIT D816V allele burden were observed. No severe adverse events were recorded.CONCLUSIONS: Omalizumab appears to be a promising treatment option in SM, effectively preventing anaphylaxis and improving chronic MC mediator-related symptoms, insufficiently controlled by conventional therapy. Controlled studies are needed to substantiate findings.

Systemic mastocytosis (SM) is characterized by activating mutations in KIT, usually KIT D816V in >90% of patients. According to the WHO classification, detection of KIT D816V is a minor diagnostic criterion. Moreover, a reduction of the RNA-based KIT D816V expressed allele burden (EAB) of ≥25% at month 6 is a favorable marker for improved survival in midostaurin-treated advanced SM (AdvSM) patients. However, only limited data exist upon the comparison between RNA-based and DNA-based quantitative KIT D816V mutation analyses. We therefore collected peripheral blood samples from 161 SM patients (indolent SM [ISM], n=40; AdvSM, n=121) at time of referral. We applied a real time reverse-transcriptase polymerase chain reaction (qRT-PCR) assay for assessment of the KIT D816V EAB and a chip-based digital PCR (dPCR) for the quantification of the DNA-based KIT D816V variant allele frequency (VAF, QuantStudio 3D dPCR System, ThermoFisher Scientific, Massachusetts, USA). The limit of detection (LOD) was assessed through serial dilution experiments with DNA from healthy individuals and DNA from a SM patient with a KIT D816V VAF of approximately 50%. All samples were analyzed in two independent dPCR runs. In an inital round-robin test for an inter-laboratory (n=4) comparison on 30 DNA samples (ISM, n=7; AdvSM, n=19), we found a very good correlation between 3 different chip-based digital PCR systems (dPCR; droplet-based dPCR, ddPCR, BioRad Laboratories, California, USA; quantitative real-time PCR, qPCR, California, USA; dPCR vs. ddPCR r=0.99, R2=0.99; dPCR vs. qPCR r=0.99, R2=0.98). In ISM patients, the comparison between EAB and VAF revealed a strong linear relationship with a correlation (r) of 0.91 and a coefficient of determination (R2) of 0.84, which was significantly inferior (r=0.71; R2=0.5) in AdvSM patients. In 45/121 (37%) and 76/121 (63%) of AdvSM patients, the EAB/VAF ratio was ≤2 (cohort A) or >2 (cohort B). To confirm the significant disparity between EAB and VAF in individual patients, serial analyses of at least 3 samples in 12 patients revealed a stable EAB/VAF ratio during follow-up. The comparison between cohort A and cohort B revealed significant differences in terms of a higher median hemoglobin level (p=0.006), a lower percentage of patients with hemoglobin 2 present with an aggressive phenotype and adverse prognosis as compared to patients with EAB/VAF ratio ≤2. We therefore recommend to routinely determine KIT D816V EAB and VAF in AdvSM patients. Disclosures Fabarius: Novartis: Research Funding. Reiter:Blueprint: Consultancy, Honoraria, Other: Travel reimbursement; Deciphera: Consultancy, Honoraria, Other: Travel reimbursement; Novartis: Consultancy, Honoraria, Other: Travel reimbursement, Research Funding.

IF 2.845; International audience; External quality assurance (EQA) programs are vital to ensure high quality and standardized results in molecular diagnostics. It is important that EQA for quantitative analysis takes into account the variation in methodology. Results cannot be expected to be more accurate than limits of the technology used, and it is essential to recognize factors causing substantial outlier results. The present study aimed to identify parameters of specific importance for JAK2 V617F quantification by quantitative PCR, using different starting materials, assays, and technical platforms. Sixteen samples were issued to participating laboratories in two EQA rounds. In the first round, 19 laboratories from 11 European countries analyzing JAK2 V617F as part of their routine diagnostics returned results from in-house assays. In the second round, 25 laboratories from 17 countries participated. Despite variations in starting material, assay setup and instrumentation the laboratories were generally well aligned in the EQA program. However, EQA based on a single technology appears to be a valuable tool to achieve standardization of the quantification of JAK2 V617F allelic burden.

Mantle cell lymphoma (MCL) is a tumor with a poor prognosis. A few studies have examined the molecular landscape by next-generation sequencing and provided valuable insights into recurrent lesions driving this heterogeneous cancer. However, none has attempted to cross-link the individual genomic and transcriptomic profiles in sorted MCL cells to perform individual molecular characterizations of the lymphomas. Such approaches are relevant as MCL is heterogenous by nature, and thorough molecular diagnostics may potentially benefit the patient with more focused treatment options. In the work described here, we used sorted lymphoma cells from four patients at diagnosis and relapse by intersecting the coding DNA and mRNA. Even though only a few patients were included, this method enabled us to pinpoint a specific set of expressed somatic mutations, to present an overall expression profile different from the normal B cell counterparts, and to track molecular aberrations from diagnosis to relapse. Changes in single-nucleotide coding variants, subtle clonal changes in large-copy-number alterations, subclonal involvement, and changes in expression levels in the clinical course provided detailed information on each of the individual malignancies. In addition to mutations in known genes (e.g., TP53, CCND1, NOTCH1, ATM), we identified others, not linked to MCL, such as a nonsense mutation in SPEN and an MYD88 missense mutation in one patient, which along with copy number alterations exhibited a molecular resemblance to splenic marginal zone lymphoma. The detailed exonic and transcriptomic portraits of the individual MCL patients obtained by the methodology presented here could help in diagnostics, surveillance, and potentially more precise usage of therapeutic drugs by efficient screening of biomarkers.

Recent studies have demonstrated that the KIT D816V mutation is reliably detected in blood in adults with mastocytosis when using appropriate techniques [1–3]. Mutation analysis of blood has theref...

AimsIn this study, we investigated whether free intraperitoneal tumour cells (FITC) were detectable in ascites or peritoneal lavage fluid (PLF) from patients with peritoneal metastasis (PM) before and after treatment with pressurised intraperitoneal aerosol chemotherapy (PIPAC).MethodsAscites or PLF retrieved at the first and third PIPAC procedures was analysed by conventional cytology, carcinoembryonic antigen (CEA) and total protein concentration, and quantitative reverse transcriptase PCR (qRT-PCR) for mRNA expression of CEA, epithelial cell adhesion molecule (EpCAM) and cancer antigen 125 (CA-125). Conventional cytology and qRT-PCR were also performed in a negative control group (benign PLF specimens and inflammatory ascites). The treatment response was compared with the histological response based on repeated peritoneal biopsies evaluated by the Peritoneal Regression Grading Score (PRGS).ResultsThirty-five patients with PM of various origins were included from 2015 to 2016. At the first PIPAC procedure, FITC were detected by conventional cytology (sensitivity 0.58, specificity 1.00), CEA protein (cut-off 0.4 µg/L, sensitivity 0.71), CEA mRNA (sensitivity 0.75, specificity 1.00), EpCAM mRNA (sensitivity 0.71, specificity 1.00) and CA-125 mRNA (sensitivity 0.43, specificity 1.00). The combination of CEA/EpCAM mRNA had a sensitivity of 0.88 and a specificity of 1.00. The evaluation of ascites or PLF retrieved at the third PIPAC procedure failed to detect treatment response, when compared with the histological PRGS.ConclusionsThe evaluation of CEA and EpCAM mRNA detects FITC with a high sensitivity and an excellent specificity, but is not useful for response evaluation in patients treated with PIPAC.Trial registration numberNCT02320448.

In patients with systemic mastocytosis (SM), several aspects of morbidity remain poorly understood. We assessed the risk of solid cancers, cardiovascular disease, anaphylaxis, osteoporosis, and fractures in SM patients. Using Danish medical registries, we conducted a nationwide population-based cohort study including 687 adult (≥15 years) SM patients diagnosed during 1997-2012. A comparison cohort of 68,700 subjects from the general Danish population who were alive and without SM at the given SM subject's diagnosis were age- and gender-matched. Outcomes were a new diagnosis of solid cancer, venous thromboembolism (VTE), myocardial infarction (MI), stroke, anaphylaxis, osteoporosis, or fracture. For solid cancers the hazard ratio (HR) was 2.4 (95% confidence interval [CI] 1.9-2.8) with a 10-year absolute risk (AR) in the SM-cohort of 12.6% (95% CI 9.4-16.3). Specifically, we found a HR of 7.5 (95% CI 4.4-13.0) for melanoma and a HR of 2.5 (95% CI 1.7-3.5) for non-melanoma skin cancers (NMSCs). For VTE we found a HR of 1.9 (95% CI 1.2-3.0), with a 10-year AR of 3.9% (95% CI 2.3-6.1); for MI a nonsignificant increased HR of 1.4 (95% CI 0.9-2.3), with a 10-year AR of 1.8% (95% CI 0.9-3.2); and for stroke a HR of 1.6 (95% CI 1.1-2.3) with a 10-year AR of 4.6% (95% CI 2.8-6.9). The HR for anaphylaxis was 7.2 (95% CI 5.3-9.9), and the 10-year AR was 3.1% (95% CI 1.9-4.9). For osteoporosis the HR was 3.6 (95% CI 2.7-4.6) with a 10-year AR of 7.2% (95% CI 5.2-9.8). For fractures the HR was 1.2 (95% CI 0.9-1.6) and the 10-year AR was 5.9% (95% CI 3.9-8.4). SM patients are at increased risk of solid cancers - especially melanoma and NMSC-and cardiovascular disease. The risk of anaphylaxis and osteoporosis is clearly increased in SM, though absolute risk was low in this population-based study. The fracture-risk was only slightly increased. Am. J. Hematol. 91:1069-1075, 2016. © 2016 Wiley Periodicals, Inc.

Background Recombinant Interferon Alpha-2a (r-IFNα) is a potent immunomodulating agent, which has been used off-label for the treatment of polycythemia vera (PV) for more than three decades and has been demonstrated to induce high rates of clinical, hematological and molecular responses. Only few studies have compared efficacy and safety of r-IFNα vs. hydroxyurea (HU), which is considered first line therapy for PV patients > 60 years in most parts of the world. However, recent studies have provided encouraging results for the treatment of PV with r-IFNα compared to HU irrespective of age (R. Hoffmann 2016; H. Gisslinger 2018). Aims To examine the difference in efficacy and safety of low-dose r-IFNα in PV patients ≤ 60 or > 60 years of age compared to HU > 60 years of age. Methods Ninety newly diagnosed or previously phlebotomized PV patients only (WHO 2008) were enrolled in the DALIAH trial (NCT01387763). All patients provided written informed consent. Patients ≤ 60 years were randomized (I:I) to r-IFNα-2a (Pegasys®) or to r-IFNα-2b (PegIntron®) whereas patients > 60 years were randomized (I:I:I) to either r-IFNα-2a, r-IFNα-2b or to HU. The starting dose of r-IFNα-2a and r-IFNα-2b was 45 or 35 µg/week, respectively. The HU dose was 500 to 2000 mg/day. Patients randomized to r-IFNα who presented with major thrombosis or platelets > 1500 109/L received HU from inclusion and until normalization of the platelet count. Efficacy assessment consisted of the clinicohematological and the molecular response rates by intention to treat analysis (ITT) using the European Leukemia Net (ELN) 2009 criteria. JAK2V617F analysis was performed by qPCR. Groups were compared by Fisher's Exact Test. Results Three-year analysis was available in all but five patients (n=85) at time of abstract submission (Table 1). The analysis was performed after a median of 36 months (range: 33-39 months). The median treatment dose was 684 mg/day (IQR: 131 - 942) for HU, 51 μg/week (IQR: 30-90 μg/week) and 54 μg/week (IQR: 30-66 μg/week) for r-IFNα-2a age ≤ 60 and > 60, respectively, and 41 μg/week (IQR: 29-45 μg/week) and 36 μg/week (IQR: 31-37 μg/week) for r-IFNα-2b age ≤ 60 and > 60. The overall clinicohematological response rate (ORR) was 68% (13/19) for HU, 42% (14/33) for r-IFNα ≤ 60 years and 39% (13/33) for r-IFNα > 60 years. The partial clinicohematological response rate (PHR) and the complete clinicohematological response rate (CHR) was 53% (10/19) and 16% (3/19) for HU, 9% (3/33) and 33% (11/33) for r-IFNα ≤ 60 years and 9% (3/33) and 30% (10/33) for r-IFNα > 60 years. Neither the ORR, CHR nor the PHR was significantly different between the three groups. Maintenance of CHR from first occurrence to data analysis after 36 months was 11% (2/19) for HU, 21% (7/33) for r-IFNα ≤ 60 years and 18% (6/33) for r-IFNα > 60 years. Forty-seven JAK2V617F positive patients were available for molecular response analysis after 36 months of therapy. A partial molecular response (PMR) was detected in 21% (4/19) of HU treated patients and in 24% (7/29) of r-IFNα treated patients ≤ 60 years and 18% (6/33) of r-IFNα > 60 years. Notably, 7% (2/29) of the r-IFNα treated patients ≤ 60 years obtained a complete molecular response (CMR). The median JAK2V617F reduction from baseline was 38% (IQR: 31-63%) for HU, 79% (IQR: 59-92%) for r-IFNα ≤ 60 years and 73% (IQR: 49-97%) for r-IFNα > 60 years. There was no statistically significant difference in the PMR between groups. Discontinuation of treatment for any reason after 36 months of therapy was 21% (4/19) for HU, 52% (17/33) for r-IFNα ≤ 60 years and 45% (15/33) for r-IFNα > 60 years. Toxicity related discontinuation was 5% (1/19) for HU and 30% (10/33) for both r-IFNα ≤ 60 and > 60 years. Grade 3-4 AEs occurred in 32% (6/19) of HU treated patients, 27% (9/33) in r-IFNα treated patients ≤ 60 years and in 42% (14/33) r-IFNα treated patients > 60 years. SAEs were reported in 21% (4/19) for HU, 9% (3/33) for r-IFNα ≤ 60 years and 24% (8/33) for r-IFNα > 60 years. The numbers of grade 3-4 AEs as well as SAEs were comparable between groups. Conclusion After 36 months of therapy CHR was non-significantly higher in PV patients treated with r-IFNα compared to HU by ITT, irrespective of age. Also, maintenance of CHR was longer for r-IFNα. However, ORR was non-significantly higher for HU. PMR was almost similar between the three groups but the median JAK2V617F reduction was greater for r-IFNα. Toxicity related discontinuation from study therapy was higher for r-IFNα compared to HU. Disclosures Stentoft: Bristol-Myers Squibb: Research Funding; Merck Sharp&Dohme: Research Funding. Hasselbalch:Novartis: Research Funding.

AIM: We investigated whether DNA methylation regulates expression of LPL and PI3K complex genes in chronic lymphocytic leukemia (CLL) and evaluated the prognostic significance of LPL promoter methylation in CLL patients. Patients & methods: Methylation of LPL promoter was assessed in 112 patients using methylation-sensitive high-resolution melting (MS-HRM).RESULTS: Patients with a fully or heterogeneously methylated LPL promoter had significantly longer median time to treatment (p < 0.001) and 75% lower (hazard ratio: 0.25; 95% CI: 0.15-0.42; p < 0.001) risk of requirement for treatment as opposed to patients with nonmethylated promoter. Multivariate modeling confirmed independent prognostic value of these findings.CONCLUSION: Chronic lymphocytic leukemia patients with a fully or heterogeneously methylated LPL gene promoter display indolent disease course and acquisition of heterogeneous methylation of LPL promoter is insufficient to induce gene expression.

Author(s): Ustun, Celalettin; Morgan, Elizabeth; Moodie, Erica EM; Pullarkat, Sheeja; Yeung, Cecilia; Broesby-Olsen, Sigurd; Ohgami, Robert; Kim, Young; Sperr, Wolfgang; Vestergaard, Hanne; Chen, Dong; Kluin, Philip M; Dolan, Michelle; Mrozek, Krzysztof; Czuchlewski, David; Horny, Hans-Peter; George, Tracy I; Kristensen, Thomas Kielsgaard; Ku, Nam K; Yi, Cecilia Arana; Moller, Michael Boe; Marcucci, Guido; Baughn, Linda; Schiefer, Ana-Iris; Hilberink, JR; Pullarkat, Vinod; Shanley, Ryan; Kohlschmidt, Jessica; Coulombe, Janie; Salhotra, Amandeep; Soma, Lori; Cho, Christina; Linden, Michael A; Akin, Cem; Gotlib, Jason; Hoermann, Gregor; Hornick, Jason; Nakamura, Ryo; Deeg, Joachim; Bloomfield, Clara D; Weisdorf, Daniel; Litzow, Mark R; Valent, Peter; Huls, Gerwin; Perales, Miguel-Angel; Borthakur, Gautam | Abstract: BackgroundAlthough the prognosis of core-binding factor (CBF) acute myeloid leukemia (AML) is better than other subtypes of AML, 30% of patients still relapse and may require allogeneic hematopoietic cell transplantation (alloHCT). However, there is no validated widely accepted scoring system to predict patient subsets with higher risk of relapse.MethodsEleven centers in the US and Europe evaluated 247 patients with t(8;21)(q22;q22).ResultsComplete remission (CR) rate was high (92.7%), yet relapse occurred in 27.1% of patients. A total of 24.7% of patients received alloHCT. The median disease-free (DFS) and overall (OS) survival were 20.8 and 31.2 months, respectively. Age, KIT D816V mutated (11.3%) or nontested (36.4%) compared with KIT D816V wild type (52.5%), high white blood cell counts (WBC), and pseudodiploidy compared with hyper- or hypodiploidy were included in a scoring system (named I-CBFit). DFS rate at 2 years was 76% for patients with a low-risk I-CBFit score compared with 36% for those with a high-risk I-CBFit score (P l 0.0001). Low- vs high-risk OS at 2 years was 89% vs 51% (P l 0.0001).ConclusionsI-CBFit composed of readily available risk factors can be useful to tailor the therapy of patients, especially for whom alloHCT is not need in CR1 (ie, patients with a low-risk I-CBFit score).

Mast cell activation disorders is a term proposed to cover diseases and conditions related to activation of mast cells and effects of mast cell mediators. In its broadest sense, the term encompasses a wide range of diseases from allergic asthma to rhinoconjunctivitis, urticaria, food allergy, anaphylaxis, mastocytosis, and other conditions where MC activation is contributing to the pathogenesis. This article focuses on clinical presentations, challenges, and controversies in pediatric mastocytosis and gives an overview of current knowledge and areas in need of further research.

Background Most published studies on anaphylaxis are retrospective or register based. Data on subsequent diagnostic workup are sparse. We aimed to characterize patients seen with suspected anaphylaxis at the emergency care setting (ECS), after subsequent diagnostic workup at our Allergy Center (AC). Methods Prospective study including patients from the ECS, Odense University Hospital, during May 2013-April 2014. Possible anaphylaxis cases were daily identified based on a broad search profile including history and symptoms in patient records, diagnostic codes and pharmacological treatments. At the AC, all patients were evaluated according to international guidelines. Results Among 226 patients with suspected anaphylaxis, the diagnosis was confirmed in 124 (54.9%) after diagnostic workup; 118 of the 124 fulfilled WAO/EAACI criteria of anaphylaxis at the ECS, while six were found among 46 patients with clinical suspicion but not fulfilling the WAO/EAACI criteria at the ECS. The estimated incidence rate of anaphylaxis was 26 cases per 100 000 person-years and the one-year period prevalence was 0.04%. The most common elicitor was drugs (41.1%) followed by venom (27.4%) and food (20.6%). In 13 patients (10.5%), no elicitor could be identified. Mastocytosis was diagnosed in 7.7% of adult patients and was significantly associated with severe anaphylaxis. Atopic diseases were significantly associated only with food-induced anaphylaxis. Cofactors were present in 58.1% and were significantly associated with severe anaphylaxis. Conclusion A broad search profile in the ECS and subsequent diagnostic workup is important for identification and classification of patients with anaphylaxis. Evaluation of comorbidities and cofactors is important.

Next-generation sequencing (NGS) is becoming increasingly used for diagnostic mutation analysis in myeloid neoplasms and may also represent a feasible technique in mastocytosis. However, detection of the KIT D816V mutation requires a highly sensitive method in most patients due to the typically low mutation levels. In this study, we established an NGS-based KIT mutation analysis and analyzed the sensitivity of D816V detection using the Ion Torrent platform. Eighty-two individual NGS analyses were included in the study. All samples were also analyzed using highly sensitive KIT D816V mutation-specific qPCR. Measurements of the background level in D816V-negative samples supported a cutoff for positivity of 0.2% in three different NGS panels. Clinical samples from patients with SM that tested positive using qPCR with a D816V allele burden >0.2% also tested positive using NGS. Samples that tested positive using qPCR with an allele burden

PMCID: PMC4522520.-- et al., Although acquired mutations in KIT are commonly detected in various categories of mastocytosis, the methodologies applied to detect and quantify the mutant type and allele burden in various cells and tissues are poorly defined. We here propose a consensus on methodologies used to detect KIT mutations in patients with mastocytosis at diagnosis and during follow-up with sufficient precision and sensitivity in daily practice. In addition, we provide recommendations for sampling and storage of diagnostic material as well as a robust diagnostic algorithm. Using highly sensitive assays, KIT D816V can be detected in peripheral blood leukocytes from most patients with systemic mastocytosis (SM) that is a major step forward in screening and SM diagnosis. In addition, the KIT D816V allele burden can be followed quantitatively during the natural course or during therapy. Our recommendations should greatly facilitate diagnostic and follow-up investigations in SM in daily practice as well as in clinical trials. In addition, the new tools and algorithms proposed should lead to a more effective screen, early diagnosis of SM and help to avoid unnecessary referrals., M. Arock is supported by Fondation de France; P. Dubreuil is supported by La Ligue Nationale Contre le Cancer (équipe labellisée) and INCa; A. Garcia-Montero and A. Orfao are Supported by grants from the Instituto de Salud Carlos III, Ministry of Economy and Competitivity, Madrid, Spain (grant numbers RD12/0036/0048 and PI11/02399, FEDER) and from Fundacion Ramon Areces, Madrid, Spain (grant number CIVP16A1806); D.D. Metcalfe is supported in part by the Division of Intramural Research, NIAID; P. Valent is supported by Austrian Science Funds (FWF) Project SFB F4611 and SFB F4704-B20.

Keywords: systemic mastocytosis; KIT D816V mutation; blood; genomic DNA; messenger RNA

Key Clinical Message A change in clinical behavior of a disease should prompt search for differential diagnoses. Here, the appearance of ulcerated skin nodules in a preexisting cutaneous mastocytosis revealed a concurrent lymphomatoid papulosis – a CD30+ lymphoproliferative skin disease with histological features of a malignant lymphoma, but with a benign self‐healing course.

Diagnosis of T-cell large granular lymphocytic leukemia (T-LGL) is often challenging because clinical and laboratory characteristics are overlapping with nonneoplastic conditions. Recently, mutation in the STAT3 gene has been identified as a recurrent genetic abnormality in T-LGL. STAT3 mutation, therefore, represents a promising marker in T-LGL diagnostics. We developed a new quantitative next-generation sequencing assay that allows sensitive analysis of the STAT3 gene. The assay was used to study the utility of STAT3 mutation analysis as a diagnostic tool in T-LGL. The study included 16 T-LGL patients. A total of 15 mutations, including 2 new mutations (G618R and K658R), were detected in 12 patients (75%), with mutation levels ranging from 2.5% to 45.6% mutation-positive alleles. Next-generation sequencing detected 50% more mutations than Sanger sequencing. Blood samples from 20 healthy blood donors all tested negative, thus demonstrating the specificity of the assay. The results also indicated that mutation levels in blood and bone marrow are not systematically different, and next-generation sequencing-based STAT3 mutation analysis represents a sensitive method for monitoring residual disease as demonstrated in a patient receiving pentostatin. We demonstrate the clinical relevance of next-generation sequencing-based STAT3 mutation analysis, which represents a sensitive and specific diagnostic marker in T-LGL that allows assessment of molecular residual disease, which may improve clinical decision making.

Mastocytosis is an emerging differential diagnosis in patients with more or less specific mediator-related symptoms. In some of these patients, typical skin lesions are found and the diagnosis of mastocytosis can be established. In other cases, however, skin lesions are absent, which represents a diagnostic challenge. In the light of this unmet need, we developed a diagnostic algorithm for patients with suspected mastocytosis. In adult patients with typical lesions of mastocytosis in the skin, a bone marrow (BM) biopsy should be considered, regardless of the basal serum tryptase concentration. In adults without skin lesions who suffer from mediator-related or other typical symptoms, the basal tryptase level is an important parameter. In those with a slightly increased tryptase level, additional investigations, including a sensitive KIT mutation analysis of blood leucocytes or measurement of urinary histamine metabolites, may be helpful. In adult patients in whom (i) KIT D816V is detected and/or (ii) the basal serum tryptase level is clearly increased (>25-30 ng/ ml) and/or (iii) other clinical or laboratory features suggest the presence of 'occult' mastocytosis or another haematologic neoplasm, a BM investigation is recommended. In the absence of KIT D816V and other signs or symptoms of mastocytosis or another haematopoietic disease, no BM investigation is required, but the clinical course and tryptase levels are monitored in the follow-up. In paediatric patients, a BM investigation is usually not required, even if the tryptase level is increased. Although validation is required, it can be expected that the algorithm proposed herein will facilitate the management of patients with suspected mastocytosis and help avoid unnecessary referrals and investigations., This work was supported in part by the Austrian Science Funds (FWF) Project SFB F4611 and SFB F4704-B20 and the Division of Intramural Research, NIAID.

The recent progress in sensitive KIT D816V mutation analysis suggests that mutation analysis of peripheral blood (PB) represents a promising diagnostic test in mastocytosis. However, there is a need for systematic assessment of the analytical sensitivity and specificity of the approach in order to establish its value in clinical use. We therefore evaluated sensitive KIT D816V mutation analysis of PB as a diagnostic test in an entire case-series of adults with mastocytosis. We demonstrate for the first time that by using a sufficiently sensitive KIT D816V mutation analysis, it is possible to detect the mutation in PB in nearly all adult mastocytosis patients. The mutation was detected in PB in 78 of 83 systemic mastocytosis (94%) and 3 of 4 cutaneous mastocytosis patients (75%). The test was 100% specific as determined by analysis of clinically relevant control patients who all tested negative. Mutation analysis of PB was significantly more sensitive than serum tryptase >20 ng/mL. Of 27 patients with low tryptase, 26 tested mutation positive (96%). The test is furthermore readily available and we consider the results to serve as a foundation of experimental evidence to support the inclusion of the test in diagnostic algorithms and clinical practice in mastocytosis.

Background: To investigate the role of genomics in determining response and resistance to front line treatment in MPN, we performed somatic mutational profiling of the DALIAH trial, a randomized controlled phase III trial of interferon versus hydroxyurea in newly diagnosed MPN patients. Methods: We performed genomic analyses on 202 pre-treatment primary MPN samples obtained from patients enrolled on the DALIAH trial (NCT01387763) and 135 samples obtained after 24 months of treatment. Genomic profiling comprised targeted next generation sequencing (NGS) of 100 genes, selected on the basis of their known or suspected involvement in the pathogenesis of myeloid malignancies, and 1609 informative single nucleotide polymorphisms (SNPs) on chromosome 9p. Clinicohematological response was determined by central review using ELN 2009 (ET, PV and pre-MF) and EUMNET 2005 (PMF) criteria. We evaluated the association of somatic mutations with clinical parameters and with attainment of clinicohematological complete response (CR) at 24 months. Results: Prior to treatment, 191 of 202 (95%) patients had somatic mutations, including 93% with canonical MPN phenotypic drivers: JAK2 (74%), CALR (14%), and MPL (5%). Among those with JAK2 mutations 37% had JAK2 copy-neutral loss of heterozygosity (JAK2 CN-LOH). Patients with PV were more likely to have JAK2 CN-LOH (p = 0.0001) as compared with patients with other MPN subtypes. At baseline, patients with JAK2 CN-LOH had significantly higher hemoglobin (p = 0.0001), higher white blood cell count (WBC, p = 0.002) and lower platelet count (p=0.0001) than patients without JAK2 CN-LOH. Mutations in TET2 (24%), DNMT3A (16%), and ASXL1 (10%) were the most frequent co-occurring non-MPN phenotypic driver mutations and they occurred across all MPN subtypes. In addition, 5% of patients had spliceosome gene mutations, and 6% had mutations in genes involved in RAS/MAPK signaling. Patients with TET2, DNMT3A or ASXL1 mutations were significantly older than patients without these mutations (p= 0.0001) and there was a significant association between the presence of a TET2, DNMT3A or ASXL1 mutation and prior stroke (p = 0.004). There were no other significant associations between somatic mutation status and baseline clinical characteristics. The probability of attaining clinicohematological CR at 24 months was independent of baseline somatic mutations. Among patients with JAK2 mutations who remained on interferon treatment at 24 months, those with CR had a greater reduction in mean variant allele fraction (VAF) (28% to 8%, p Among patients who remained on treatment for 2 years, 44 mutations in 35 patients were newly detected or expanded on serial sampling. DNMT3A mutations were the most frequently acquired, accounting for 41% of new mutations. ASXL1, TET2, PPM1D, TP53, IDH2, and CBL mutations were also recurrently acquired. 97% of patients who acquired new mutations were JAK2-mutant. Among those with acquired DNMT3A mutations, 85% were treated with interferon, and 23% had CR at 24 months. Among those that acquired non-DNMT3A mutations, 38% were treated with interferon and 47% had CR at 24 months. The VAF of newly detected mutations was low (median 1.5%), and half of the patients with newly acquired mutations had at least 50% reduction in JAK2V617F VAF, suggesting that new mutations could either have arisen independently or be subclonal to the dominant JAK2-mutant clone. Conclusions: Using sequential genomic analyses of a phase III clinical trial of interferon versus hydroxyurea in MPN patients, we found mutation-specific and treatment-specific patterns of response. We uncovered distinct patterns of response to interferon in JAK2-mutant MPN as compared with CALR-mutant MPN. We found that DNMT3A mutations were the most frequent acquired mutations at 24 months and that these were enriched in patients treated with interferon. In aggregate, these results provide insights into molecular response and resistance to interferon and inform the clinical use of interferon in MPN patients. Disclosures Hansen: Alexion: Research Funding. Neuberg:Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Hasselbalch:Novartis: Research Funding; AOP Orphan Pharmaceuticals: Other: Data monitoring board. Lindsley:Jazz Pharmaceuticals: Research Funding; Takeda Pharmaceuticals: Consultancy; Medlmmune: Research Funding. Mullally:Janssen: Research Funding.

Background: Most adults with systemic mastocytosis (SM) carry the somatic KIT D816V mutation, but the occurrence of the mutation in lesional skin remains to be characterized. Objective: To assess the distribution and fraction of KIT D816V mutation-positive cells in lesional skin. Methods: Lesional skin biopsies from 29 adults with SM were analysed using sensitive and quantitative qPCR KIT D816V mutation analysis. Results: The KIT D816V mutation was detected in skin in all cases. Highly similar mutation levels were observed in all patients with a median of 5% mutation-positive cells in the skin (range 1-23%). Conclusion: The KIT D816V mutation is consistently present in lesional skin in adults with SM. The low variation in mutation levels detected in lesional skin contrasts blood and bone marrow where mast cell (MC) progenitor and non-MC lineage involvement causes the mutation-positive cell fraction to be highly variable.

Introduction Chronic lymphocytic leukemia is a heterogeneous yet incurable disease. Whole-genome and whole-exome sequencing studies have revealed recurrently occurring somatic mutations in some genes. Several other prognostic markers have previously been tested for their prognostic value in CLL. LPL is among these markers. Aim To evaluate LPL gene expression together with the well-established prognostic markers of CLL and investigate correlations with more recently identified prognostic markers, NOTCH1 and TP53 mutations. Methods On 149 patients, LPL gene expression was analyzed by real-time RT-PCR. Exon 34 of NOTCH1 was PCR-amplified and directly sequenced. Results LPL gene expression could be measured as a categorical variable (LPL+/LPL-) and was associated with time to treatment (P < 0.001) and overall survival (P = 0.007). In patients otherwise classified as having a good prognosis according to established and new prognostic markers, 3 of 4 patients, who received treatment within 24 months after diagnosis, were LPL+ (P = 0.03). There was a strong correlation between NOTCH1 mutation and LPL+ (P = 0.005). The unfavorable prognosis of LPL+ was maintained in CLL with wild-type NOTCH1. Conclusions NOTCH1 mutations are tightly associated with LPL gene expression. LPL expression is independently associated with poor outcome in CLL and can be measured as a categorical variable.

In patients with systemic mastocytosis (SM), several aspects of morbidity remain poorly understood. We assessed the risk of solid cancers, cardiovascular disease, anaphylaxis, osteoporosis, and fractures in SM patients. Using Danish medical registries, we conducted a nationwide population-based cohort study including 687 adult (≥15 years) SM patients diagnosed during 1997-2012. A comparison cohort of 68,700 subjects from the general Danish population who were alive and without SM at the given SM subject's diagnosis were age- and gender-matched. Outcomes were a new diagnosis of solid cancer, venous thromboembolism (VTE), myocardial infarction (MI), stroke, anaphylaxis, osteoporosis, or fracture. For solid cancers the hazard ratio (HR) was 2.4 (95% confidence interval [CI] 1.9-2.8) with a 10-year absolute risk (AR) in the SM-cohort of 12.6% (95% CI 9.4-16.3). Specifically, we found a HR of 7.5 (95% CI 4.4-13.0) for melanoma and a HR of 2.5 (95% CI 1.7-3.5) for non-melanoma skin cancers (NMSCs). For VTE we found a HR of 1.9 (95% CI 1.2-3.0), with a 10-year AR of 3.9% (95% CI 2.3-6.1); for MI a nonsignificant increased HR of 1.4 (95% CI 0.9-2.3), with a 10-year AR of 1.8% (95% CI 0.9-3.2); and for stroke a HR of 1.6 (95% CI 1.1-2.3) with a 10-year AR of 4.6% (95% CI 2.8-6.9). The HR for anaphylaxis was 7.2 (95% CI 5.3-9.9), and the 10-year AR was 3.1% (95% CI 1.9-4.9). For osteoporosis the HR was 3.6 (95% CI 2.7-4.6) with a 10-year AR of 7.2% (95% CI 5.2-9.8). For fractures the HR was 1.2 (95% CI 0.9-1.6) and the 10-year AR was 5.9% (95% CI 3.9-8.4). SM patients are at increased risk of solid cancers - especially melanoma and NMSC-and cardiovascular disease. The risk of anaphylaxis and osteoporosis is clearly increased in SM, though absolute risk was low in this population-based study. The fracture-risk was only slightly increased. Am. J. Hematol. 91:1069-1075, 2016. © 2016 Wiley Periodicals, Inc.

Nonattendance at scheduled appointments in public hospitals presents a challenge for efficient resource use and may ultimately affect health outcomes due to longer waiting times. Seven percent of all scheduled outpatient appointments in the United Kingdom are estimated to be nonattended. Various reminder systems have been shown to moderately reduce nonattendance, although the effect of issuing fines for nonattendance has not yet been tested in a randomized context. However, such use of financial incentives could impact access to care differently across the different socioeconomic groups. The aim of this study is to assess the effect of fines on hospital outpatient nonattendance. A 1:1 randomized controlled trial of scheduled outpatient appointments was used, with follow-ups until the date of appointment. The setting is an orthopedic clinic at a regional hospital in Denmark. Appointments for users who are scheduled for diagnostics, treatment, surgery, or follow-ups were included from May 2015 to November 2015. Appointments assigned to the intervention arm include an attachment of the appointment letter explaining that a fine will be issued in the case of nonattendance without prior notice. Appointments assigned to the control arm follow usual practice (same system but no letter attachment). The primary outcome is the proportion of nonattendance. Secondary outcomes are proportions of cancellations, sociodemographics, and health-problem characteristics. Furthermore, the intervention costs and production value of nonattended appointments will be measured. An analysis of effect and cost-effectiveness will be conducted based on a 5 % significance level. The study is initiated and funded by the Danish Regions, which have the responsibility for the Danish public healthcare sector. The results are expected to inform future decisions about the introduction of fines for nonattendance at public hospitals. Current Controlled Trials, ISRCTN61925912 . Registered on 6 July 2015.

SEE HOHLFELD AND SCHULZE-KOOPS DOI101093/BRAIN/AWW053 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: Inclusion body myositis and T cell large granular lymphocytic leukaemia are rare diseases involving pathogenic cytotoxic CD8+ T cells. After encountering four patients with both disorders, we prospectively screened 38 patients with inclusion body myositis for the presence of expanded large granular lymphocyte populations by standard clinical laboratory methods (flow cytometry, examination of blood smears, and T cell receptor gene rearrangements), and performed muscle immunohistochemistry for CD8, CD57, and TIA1. Most (22/38; 58%) patients with inclusion body myositis had aberrant populations of large granular lymphocytes in their blood meeting standard diagnostic criteria for T cell large granular lymphocytic leukaemia. These T cell populations were clonal in 20/20 patients and stably present on follow-up testing in 15 patients a median of 350 days later. T cell aberrant loss of CD5 or gain of expression of CD16 and CD94 were common (19/42, 45%). In comparison, 2/15 (14%) age-matched patients with dermatomyositis, polymyositis, or necrotizing myopathy, and 0/20 (0%) age-matched healthy subjects had large granular lymphocyte expansions, with none of these patients having T cell aberrant expression of CD5, CD16 or CD94. Reduced blood CD4/CD8 ratio, increased blood CD8 count, and lymphocytosis were additional biomarkers highly correlated with flow cytometry-measured large granular lymphocyte expansions. Cross-sectional data suggested more aggressive disease in patients with such expansions than without. Muscle immunohistochemistry demonstrated invasion of large granular lymphocytes into muscle in 15/15 inclusion body myositis patients but in only 1/28 patients with dermatomyositis or polymyositis. The extent of CD8+ and CD57+ cells in inclusion body myositis muscle correlated with the size of blood large granular lymphocyte populations. Myofibre-invading cells expressed CD57, a marker of persistent T cell exposure to antigen and T cell aggressiveness. In many patients with inclusion body myositis, the autoimmune T cell expansion has evolved into a neoplastic-like or overtly neoplastic disorder, perhaps contributing to its relative refractoriness to immune-directed therapies previously reported.

Mastocytosis is a heterogeneous disease with an increased number and activation of mast cells. Subtypes range from benign to rare aggressive forms, and the disease may affect people of all ages. The pathogenesis involves mutations in the KIT gene in both children and adult patients. Estimated prevalence is one per 10,000, but the disease is very likely underdiagnosed. The diagnosis may be challenging and patients may present to several medical specialties. This article presents an overview of clinical signs and symptoms as well as a diagnostic algorithm and treatment options of mastocytosis.

Cutaneous lesions in patients with mastocytosis are highly heterogeneous and encompass localized and disseminated forms. Although a classification and criteria for cutaneous mastocytosis (CM) have been proposed, there remains a need to better define subforms of cutaneous manifestations in patients with mastocytosis. To address this unmet need, an international task force involving experts from different organizations (including the European Competence Network on Mastocytosis; the American Academy of Allergy, Asthma & Immunology; and the European Academy of Allergology and Clinical Immunology) met several times between 2010 and 2014 to discuss the classification and criteria for diagnosis of cutaneous manifestations in patients with mastocytosis. This article provides the major outcomes of these meetings and a proposal for a revised definition and criteria. In particular, we recommend that the typical maculopapular cutaneous lesions (urticaria pigmentosa) should be subdivided into 2 variants, namely a monomorphic variant with small maculopapular lesions, which is typically seen in adult patients, and a polymorphic variant with larger lesions of variable size and shape, which is typically seen in pediatric patients. Clinical observations suggest that the monomorphic variant, if it develops in children, often persists into adulthood, whereas the polymorphic variant may resolve around puberty. This delineation might have important prognostic implications, and its implementation in diagnostic algorithms and future mastocytosis classifications is recommended. Refinements are also suggested for the diagnostic criteria of CM, removal of telangiectasia macularis eruptiva perstans from the current classification of CM, and removal of the adjunct solitary from the term solitary mastocytoma., Supported by research grants from the German Research Council (DFG; HA 2393/6-1; CRC/SFB832, project A14; to K.H.); from the Division of Intramural Research, National Institute of Allergy and Infectious Diseases (to D.D.M.); and from the Austrian Science Funds (FWF; projects SFB F4611 and SFB F4707-B20; to P.V.). The Consensus Conference on Mastocytosis in Boston, Massachusetts (October 2012), was supported by the American Academy of Allergy, Asthma & Immunolog. A task force/consensus panel on mastocytosis between 2010 and 2012 was supported by the European Academy of Allergy and Clinical Immunology.

Mastocytosis is a heterogeneous group of diseases defined by an increased number and accumulation of mast cells, and often also by signs and symptoms of mast cell activation. Disease subtypes range from indolent to rare aggressive forms. Mastocytosis affects people of all ages and has been considered rare; however, it is probably underdiagnosed with potential severe implications. Diagnosis can be challenging and symptoms may be complex and involve multiple organ-systems. In general it is advised that patients should be referred to centres with experience in the disease offering an individualized, multidisciplinary approach. We present here consensus recommendations from a Nordic expert group for the diagnosis and general management of patients with mastocytosis.

Mutation in the NPM1 gene occurs in 60% of acute myeloid leukaemia (AML) patients with normal karyotype. NPM1 mutation is potentially a superior minimal residual disease (MRD) marker compared to WT1 gene overexpression by being specific to the malignant clone, although experimental evidence published so far includes very limited numbers of relapsed cases. Also, the stability of the NPM1 mutation has been questioned by reports of the mutation being lost at relapse. In the present study we compared NPM1 mutation and WT1 overexpression as MRD markers in 20 cases of relapsed AML. The 20 patients experienced a total of 28 morphological relapses. Karyotypic evolution was detected in 56% of relapses. All relapses were accompanied by high levels of NPM1 mutation, along with high WT1 mRNA levels, thus demonstrating complete stability of both markers during relapse. Detectable NPM1 mutation following a period of morphological remission was accompanied by a morphological relapse in all cases. In contrast, WT1 expression was detected in 33% of the NPM1 mutation negative samples. This background WT1 expression produced by non-leukaemia cells was highly variable, both between and within patients, and limited the de facto sensitivity of the WT1 expression analysis. The present study therefore provides important experimental evidence demonstrating that NPM1 mutation is superior to WT1 overexpression as marker of MRD in NPM1-mutated AML, even in the presence of extensive karyotypic evolution.

Human papillomavirus (HPV) DNA has been detected in the testis tissue of 6.5% of 185 men with non‐obstructive azoospermia (NOA). Others have suggested that seminal HPV originates from contamination from the genital skin and mucosa. One hundred unselected azoospermic men and 43 normal men undergoing vasectomy were recruited. Testicular biopsies for HPV examination were collected from all the men. Additionally, the normal men undergoing vasectomy delivered a semen sample and had a swab for HPV examination taken from the genital skin before vasectomy. A piece of each Vas deferens obtained during the vasectomy was examined for the presence of HPV. Two of the primarily azoospermic men were shown to have cryptozoospermia. It was not possible to detect HPV in the testis tissue of any of the included 98 azoospermic men or the 43 proven fertile men. In the proven fertile men, HPV DNA was detected in the semen of 15 men (35%), on the genital skin of 28 men (65%), and in the Vas deferens in three cases (7%). In 13 (87%) men with HPV‐positive semen samples, HPV DNA was also detected in the skin swabs, and in 11 men (73%), identical HPV genotypes were found in the two locations.

The chaperone and calcium storing protein calreticulin is coded by CALR, and newly identified mutations in CALR are found in respectively 49-70% and 56-88% of JAK2- and MPL-negative patients with essential thrombocytaemia (ET) and primary myelofibrosis (PMF). A total of 41 mutations have been identified, all located to exon 9 which codes the protein's C-terminal. CALR mutations are present only in myeloid malignancies and confer a more indolent disease than JAK2-mutated ET and PMF. CALR mutations as a diagnostic and prognostic tool are promising and the mutations are potential targets for immune therapy.

Background Treatment of Philadelphia chromosome negative chronic myeloproliferative neoplasms (MPNs) with recombinant pegylated interferon alpha2a/b (rIFN-alpha) has proven effective. It is well known that prolonged therapy with recombinant type 1 interferons (IFN-alpha and IFN-beta) may induce neutralizing antibodies (nAbs) against the drug leading to treatment failure. Most data on type 1 IFN immunogenicity are available from studies of patients with multiple sclerosis treated with rIFN-beta, and patients with hepatitis C treated with rIFN-alpha. A few reports have demonstrated nAbs in MPN patients not responding adequately to rIFN-alpha treatment, but the phenomenon has not been thoroughly investigated in MPNs. Patients and Methods Newly diagnosed patients with MPNs enrolled in the Danish multicenter trial - DALIAH (Low-dose rIFN-alpha versus Hydroxyurea in The Treatment of Ph-Negative MPNs) were included. Patients were randomized to treatment with either rIFN-alpha 2a or 2b at a starting dose of 45 and 35 mikrograms once weekly, respectively. The occurrence of neutralizing Abs (nAbs) against rIFN-alpha was investigated at baseline, month 12 and month 24 by reporter gene assays (iLiteTM alphabeta and iLiteTM antialpha, Biomonitor A/S, Copenhagen, Denmark). JAK2 V617F quantitative mutation analyses were performed as previously described (Larsen TS, BJH 2007). Statistical analyses were performed using STATA version 9.0. Results Ninety-two patients on sustained treatment with rIFN-alpha2a (n=48) and rIFN-alpha2b (n=44) for 12 months were analyzed for this study. Forty-five patients had ET, 39 patients had PV and 8 patients had proliferative PMF. Thirty-six out of 39 (92%) PV patients, 22 out of 45 (49%) ET patients and 4 out of 8 (50%) PMF patients were JAK2V617F mutated. Hematological responses at 12 months: ET: 67% CR, 29 % PR; PV: 64% CR, 31% PR (ELN 2009 criteria); PMF: 50% had at least a minor response (EUMNET). The median serum concentration of bioactive IFN-alpha at 12 months was 12,4 (range Conclusions Development of nAbs in MPN patients completing treatment for 12 months with rIFN-alpha seems exceedingly rare as no patients, neither complete responders nor patients not meeting criteria for complete hematological remission developed nAbs after 12 (24) months of therapy. Its apparent rarity does not justify a routine investigation of nAbs in patients not responding to rIFN-alpha treatment. There was no significant correlation between serum concentration of rIFN-alpha 2a and -2b and clinical or molecular responses. The intriguing finding that one of 24 patients had pre-existing cross reacting nAbs against rIFN-alpha 2a and 2b before commencing rIFN-alpha treatment is interesting and was associated with an insufficient response. Disclosures Off Label Use: Recombinant interferon-alpha 2a and -2b in the treatment of chronic Philadelphia-negative myeloproliferative neoplasms.. El Fassi:Novartis Denmark: Honoraria, Other: Have conducted an educational session for Novartis Denmark, regarding MPNs and ruxolitinib, for this a honorarium was received.. Bjerrum:Bristoll Myers Squibb, Novartis and Pfizer: Other: educational activities. Hasselbalch:Novartis: Research Funding. Bendtzen:Pfizer: Honoraria; Eurodiagnostica AB: Equity Ownership; Novo-Nordisk: Equity Ownership.