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Background Radially sampled averaged magnetization inversion-recovery acquisition (rAMIRA) imaging shows hyperintensity in the lateral corticospinal tract (CST) in patients with motor neuron diseases. Purpose To systematically determine the accuracy of the lateral corticospinal tract sign for detecting patients with amyotrophic lateral sclerosis (ALS) at rAMIRA MRI. Materials and Methods This study included prospectively acquired data from participants in ALS and other motor neuron disease imaging studies at the University Hospital Basel, Switzerland. All participants underwent 3-T axial two-dimensional rAMIRA imaging at four cervical intervertebral disk levels. The lateral CST sign was defined as spinal cord white matter hyperintensity dorsolateral to the anterior horns, with higher signal intensity than in the dorsal columns on axial rAMIRA images. Marker accuracy was assessed in a study data set and in an independent validation data set. Postmortem rAMIRA imaging and histopathologic analysis were performed in one participant who died during the study. Results Participants with ALS (study data set: 38 participants [mean age, 61 years; IQR, 15 years], 22 male participants; validation data set: 10 participants [mean age, 61 years; IQR, 21 years], seven male participants), post-polio syndrome (study data set: 25 participants [mean age, 68 years; IQR, 8 years], 12 male participants), spinal muscular atrophy (study data set: 10 participants [mean age, 43 years; IQR, 14 years], eight male participants; validation data set: five participants [mean age, 38 years; IQR, 19 years], two male participants), and healthy control participants (study data set: 60 participants [mean age, 57 years; IQR, 20 years], 36 male participants; validation data set: 10 participants [mean age, 44 years; IQR, 17 years], seven male participants) were included. The sensitivity and specificity of rAMIRA for ALS were 60% (23 of 38) and 97% (91 of 94) in the study data set and 100% (10 of 10) and 93% (14 of 15) in the validation data set, respectively. Histopathologic analysis showed distinct loss of myelinated axons in the localization of the hyperintensities observed at rAMIRA imaging performed in situ and after organ extraction. Conclusion The recently defined marker at rAMIRA MRI may be a promising tool for assessing upper motor neuron degeneration in the lateral CST in patients with ALS. Clinical trials registration no. NCT03561623, NCT05764434, NCT06137612 © RSNA, 2024 Supplemental material is available for this article.

Background and Purpose: In amyotrophic lateral sclerosis (ALS), there is an unmet need for more precise patient characterization through quantitative, ideally operator-independent, assessments of disease extent and severity. Radially sampled averaged magnetization inversion recovery acquisitions (rAMIRA) magnetic resonance imaging enables gray matter (GM) and white matter (WM) area quantitation in the cervical and thoracic spinal cord (SC) with optimized contrast. We aimed to investigate rAMIRA-derived SC GM and SC WM areas and their association with clinical phenotype and disability in ALS., Methods: A total of 36 patients with ALS (mean [SD] age 61.7 [12.6] years, 14 women) and 36 healthy, age- and sex-matched controls (HCs; mean [SD] age 63.1 [12.1] years, 14 women) underwent two-dimensional axial rAMIRA imaging at the inter-vertebral disc levels C2/3-C5/C6 and the lumbar enlargement level T max . ALS Functional Rating Scale-revised (ALSFRS-R) score, muscle strength, and sniff nasal inspiratory pressure (SNIP) were assessed., Results: Compared to HCs, GM and WM areas were reduced in patients at all cervical levels (p < 0.0001). GM area (p = 0.0001), but not WM area, was reduced at T max . Patients with King's Stage 3 showed significant GM atrophy at all levels, while patients with King's Stage 1 showed significant GM atrophy selectively at T max . SC GM area was significantly associated with muscle force at corresponding myotomes. GM area at C3/C4 was associated with ALSFRS-R (p < 0.001) and SNIP (p = 0.0016)., Conclusion: Patients with ALS assessed by rAMIRA imaging show significant cervical and thoracic SC GM and SC WM atrophy. SC GM area correlates with muscle strength and clinical disability. GM area reduction at T max may be an early disease sign. Longitudinal studies are warranted., (© 2024 The Authors. European Journal of Neurology published by John Wiley & Sons Ltd on behalf of European Academy of Neurology.)

Apprehensions about gene doping have grown consistently due to advancements in gene engineering techniques, particularly with the emergence of clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas)-based tools. These tools not only provide unprecedented possibilities for illicit performance enhancement by athletes but also offer new avenues for the detection of gene doping through biosensing of nucleic acids. Hence, pursuing on a previous study, an analytical method based on reverse transcriptase-recombinase polymerase amplification (RT-RPA) and subsequent qualitative nucleic acid detection by means of Specific High Sensitive Enzymatic Reporter UnLOCKing (SHERLOCK) was optimized for the direct detection of sgRNA associated with Streptococcus pyogenes in serum. Detection device, assay parameters, and sample handling were adjusted, to overcome previously determined assay limitations. The conducted method characterization confirmed the methods' specificity and increased detection sensitivity from 100 pM to 1 fM sgRNA in 100 μL of serum. Furthermore, reanalysis of in vivo mouse administration samples collected in a previous proof-of-concept study was conducted with successful identification of sgRNA in all anticipated postadministration samples within the 24-h collection period. Those findings support the applicability of the refined analytical procedure for the detection of illegal doping attempts via ribonucleoprotein-based CRISPR/Cas application through sgRNA identification, offering a new potential doping control strategy for CRISPR related gene doping.

We identified 71 patients with AdvSM (aggressive SM [ASM], SM with an associated hematologic neoplasm [SM-AHN, e.g., acute myeloid leukemia, SM-AML], mast cell leukemia [MCL]) in two national registries (DRST/GREM) who received an allogeneic hematopoietic cell transplantation (alloHCT) performed in Germany from 1999-2021. Median overall survival (OS) of ASM/SM-AHN (n = 30, 45%), SM-AML (n = 28, 39%) and MCL ± AHN (n = 13, 19%) was 9.0, 3.3 and 0.9 years (P = 0.007). Improved median OS was associated with response of SM (17/41, 41%; HR 0.4 [0.2-0.9], P = 0.035) and/or of AHN (26/43, 60%, HR 0.3 [0.1-0.7], P = 0.004) prior to alloHCT. Adverse predictors for OS included absence of KIT D816V (10/61, 16%, HR 2.9 [1.2-6.5], P < 0.001) and a complex karyotype (9/60, 15%, HR 4.2 [1.8-10.0], P = 0.016). HLA-match, conditioning type or transplantation at centers reporting above-average alloHCTs (≥7) had no impact on OS. Taking into account competing events at years 1, 3 and 5, relapse-related mortality and non-relapse mortality rate were 15%/23%, 20%/30% and 23%/35%, respectively. Irrespective of subtype, subsequent treatment response was achieved in 13/30 (43%) patients and was highest on midostaurin/avapritinib (7/9, 78%). We conclude that outcome of alloHCT in AdvSM is more affected by disease phenotype and treatment response prior to transplant than by transplant characteristics., (© 2024. The Author(s).)

Within our nationwide registry, we identified a KIT D816V mutation ( KIT D816V pos.) in 280/299 (94%) patients with advanced systemic mastocytosis (AdvSM). Age, cytopenias and the presence of additional somatic mutations confer inferior overall survival (OS). However, little is known about the characteristics of KIT D816V-negative (D816V neg. ) AdvSM. In 19 D816V neg. patients, a combination of clinical, morphological and genetic features revealed three subgroups: (a) KIT D816H- or Y-positive SM ( KIT D816H/Y pos. , n = 7), predominantly presenting as mast cell leukemia (MCL; 6/7 patients), (b) MCL with negative KIT sequencing ( KIT neg. MCL, n = 7) and (c) KIT neg. SM with associated hematologic neoplasm ( KIT neg. SM-AHN, n = 5). Although >70% of patients in the two MCL cohorts ( KIT D816H/Y pos. and KIT neg. ) were classified as low/intermediate risk according to prognostic scoring systems (PSS), treatment response was poor and median OS was shorter than in a KIT D816V pos. MCL control cohort ( n = 29; 1.7 vs. 0.9 vs. 2.6 years; p < 0.04). The KIT neg. SM-AHN phenotype was dominated by the heterogeneous AHN (low mast cell burden, presence of additional mutations) with a better median OS of 4.5 years. We conclude that (i) in MCL, negativity for D816V is a relevant prognostic factor and (ii) PSS fail to correctly classify D816V neg. patients.

In 70 patients with KIT D816V positive systemic mastocytosis (SM) including 36 patients with advanced SM (AdvSM), we correlated the extent of reported mucosal mast cell ([m]MC) infiltration of the upper and/or lower gastrointestinal tract (UGIT, n = 63; LGIT, n = 64; both, n = 57) with symptoms and markers of MC burden/subtype. GI symptoms were reported by all patients (mean 2.1 number of symptoms). A strong mMC infiltration was identified in 24 patients (UGIT, 17/63, 27%; LGIT, 19/64, 30%). Concurrent involvement of UGIT and LGIT (n = 12) correlated with female gender (75%) and a higher symptom burden (mean 2.7) but not with MC burden or subtype. Significant differences between non-AdvSM and AdvSM were reported regarding food intolerance (54% vs. 17%), cramping (54% vs. 22%) and weight loss (0% vs. 64%). KIT D816V was identified in 54/56 (96%) available biopsies. In 46 patients, digital PCR revealed a correlation with low albumin levels (r =  - 0.270, P = 0.069) and the KIT D816V VAF in peripheral blood (r = 0.317, P = 0.036) but not with the extent of mMC infiltration or markers of MC burden/subtype. Although MC mediator triggered GI symptoms have a substantial impact on the quality of life, correlation to objective disease parameters is lacking thus making its systematic assessment challenging., (© 2024. The Author(s).)

The authenticity of a doping control sample is a key element of sports drug testing programmes. Doping control sample manipulation by providing another individual's urine or blood (instead of the tested athlete's sample) has been observed in the past and is an unequivocal violation of the World Anti-Doping Agency anti-doping rules. To determine attempts of manipulations by sample swapping, the utility of a single nucleotide polymorphism (SNP)-based sample authentication with a multi-target SNP panel was assessed. The panel comprises detection assays for 44 different SNPs, 3 gender markers and 5 quality control markers for DNA-profile determination. Sample analysis is based on a multiplex polymerase chain reaction step followed by a multiplex single base extension (SBE) reaction and subsequent SBE-product detection by MALDI-TOF MS. Panel performance was evaluated for urine and dried blood spot (DBS) samples. Urine (8 ml) and DBS (20 μl) test samples were reliably typed and matched to whole blood reference samples, while efficient typing of urine samples correlated with sample quality and input amounts. Robust profiling of urine doping control specimens was confirmed with an assay input of 12 ml. Samples can be processed in a high-throughput format with an overall assay turnaround time of approximately 11 h. SNP-based DNA typing via MALDI-TOF MS thus represents a high throughput-capable possibility for doping control sample authentication. SNP profiling of samples could offer the opportunity to complement existing steroid profile analytics to substantiate sample manipulations and to support quality control processes in high throughput routine settings., (© 2023 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

The identification of metabolites allows for the expansion of possible targets for anti-doping analysis. Especially for novel substances such as selective androgen receptor modulators (SARMs), information on metabolic fate is scarce. Novel approaches such as the organ on a chip technology may provide a metabolic profile that resembles human in vivo samples more closely than approaches that rely on human liver fractions only. In this study, the SARM RAD140 was metabolized by means of subcellular human liver fractions, human liver spheroids in an organ on a chip platform, and electrochemical (EC) conversion. The resulting metabolites were analyzed with LC-HRMS/MS and compared to a human doping control urine sample that yielded an adverse analytical finding for RAD140. A total of 16 metabolites were detected in urine, while 14, 13, and 7 metabolites were detected in samples obtained from the organ on a chip experiment, the subcellular liver fraction, and EC experiments, respectively. All tested techniques resulted in the detection of RAD140 metabolites. In the organ on a chip samples, the highest number of metabolites were detected. The subcellular liver fractions and organ on a chip techniques are deemed complementary to predict metabolites of RAD140, as both techniques produce distinct metabolites that are also found in an anonymized human in vivo urine sample., (© 2023. The Author(s).)

In a registry-based analysis of 135 patients with "myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions" (MLN-TK; FIP1L1::PDGFRA, n = 78; PDGFRB, diverse fusions, n = 26; FGFR1, diverse, n = 9; JAK2, diverse, n = 11; ETV6::ABL1, n = 11), we sought to evaluate the disease-defining characteristics. In 81/135 (60%) evaluable patients, hypereosinophilia (>1.5 × 10 9 /l) was observed in 40/44 (91%) FIP1L1::PDGFRA and 7/7 (100%) ETV6::ABL1 positive patients but only in 13/30 (43%) patients with PDGFRB, FGFR1, and JAK2 fusion genes while 9/30 (30%) patients had no eosinophilia. Monocytosis >1 × 10 9 /l was identified in 27/81 (33%) patients, most frequently in association with hypereosinophilia (23/27, 85%). Overall, a blast phase (BP) was diagnosed in 38/135 (28%) patients (myeloid, 61%; lymphoid, 39%), which was at extramedullary sites in 18 (47%) patients. The comparison between patients with PDGFRA/PDGFRB vs. FGFR1, JAK2, and ETV6::ABL1 fusion genes revealed a similar occurrence of primary BP (17/104, 16% vs. 8/31 26%, p = 0.32), a lower frequency (5/87, 6% vs. 8/23, 35%, p = 0.003) of and a later progression (median 87 vs. 19 months, p = 0.053) into secondary BP, and a better overall survival from diagnosis of BP (17.1 vs. 1.7 years, p < 0.0008). We conclude that hypereosinophilia with or without monocytosis and various phenotypes of BP occur at variable frequencies in MLN-TK., (© 2023. The Author(s).)

Reverse transcriptases (RT) are essential tools in BCR::ABL1 fusion transcript monitoring in chronic myeloid leukemia (CML). The RT type and cDNA priming method may impair the stoichiometry of cDNA synthesis, thereby potentially introducing a bias in BCR::ABL1 qRT-PCR data. Using the Acrometrix™ BCR::ABL1 reference panel and 37 clinical specimens, we have comparatively investigated the performance of the RTs MLV and SuperScript IV with random hexamer vs. target-specific priming. Quantitative RT-PCR results identified the priming type and RT type as major factors for diagnostic data variation, mainly due to the different efficacies of processing BCR::ABL1 low-copy-numbers (<50) compared to GUSB or ABL1 high-copy targets. The impairment of SuperScript IV in processing low- and high-copy-number RNA targets equally was not reflected by the diagnostically relevant Log ( BCR::ABL1/GUSB %) values. Therefore, the correct representation of housekeeping and BCR::ABL1 target genes should have priority when aiming at as high a number of housekeeping gene copies as possible. Our data suggest that for improving BCR::ABL1 assay sensitivity, increased RNA/cDNA amounts and the use of distinct RT/priming combinations are advantageous. However, for inter-laboratory harmonization, the proper conversion factor according to the CML international standard (IS) has to be reevaluated each time the grade of RT is changed.

We sought to evaluate the efficacy of the purine analogue cladribine in 79 patients with advanced systemic mastocytosis (AdvSM) using data from the 'German Registry on Disorders of Eosinophils and Mast Cells (GREM)'. The overall response rate according to modified Valent criteria (46 evaluable patients) for first- (1L) and second-line (2L) cladribine treatment was 41% (12/29) and 35% (6/17, P = 0.690), respectively, and the median overall survival (OS, all patients evaluable) was 1.9 years (n = 48) and 1.2 years (n = 31; P = 0.311). Univariate and multivariable analyses of baseline and on-treatment parameters identified diagnosis of mast cell leukemia (hazard ratio [HR] 3.5, 95% confidence interval [CI, 1.3-9.1], P = 0.012), eosinophilia ≥ 1.5 × 10 9 /L (HR 2.9 [CI 1.4-6.2], P = 0.006) and < 3 cycles of cladribine (HR 0.4 [CI 0.2-0.8], P = 0.008) as independent adverse prognostic parameters for OS. There was no impact of other laboratory (anemia, thrombocytopenia, serum tryptase) or genetic markers (mutations in SRSF2, ASXL1 or RUNX1) on OS. In consequence, none of the recently established prognostic scoring systems (MARS, IPSM, MAPS or GPSM) was predictive for OS. Modified Valent criteria were superior to a single factor-based response assessment (HR 2.9 [CI 1.3-6.6], P = 0.026). In conclusion, cladribine is effective in 1L and 2L treatment of AdvSM. Mast cell leukemia, eosinophilia, application of < 3 cycles and a lack of response are adverse prognostic markers., (© 2023. The Author(s).)

Among anabolic agents, selective androgen receptor modulators (SARMs) represent a new class of potential drugs that can exhibit anabolic effects on muscle and bone with reduced side effects due to a tissue-selective mode of action. Besides possible medical applications, SARMs are used as performance-enhancing agents in sports. Therefore, they are prohibited by the World Anti-Doping Agency (WADA) in and out of competition. Since their inclusion into the WADA Prohibited List in 2008, there has been an increase in not only the number of adverse analytical findings, but also the total number of SARMs, making continuous research into SARMs an ongoing topic in the field of doping controls. 4-((2 R ,3 R )-2-Ethyl-3-hydroxy-5-oxopyrrolidin-1-yl)-2-(trifluoromethyl)benzonitrile (SARM 2f) is a novel SARM candidate and is therefore of particular interest for sports drug testing. This study describes the synthesis of SARM 2f using a multi-step approach, followed by full characterization using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance spectroscopy (NMR). To provide the first insights into its biotransformation in humans, SARM 2f was metabolized using human liver microsomes and the microsomal S9 fraction. A total of seven metabolites, including phase I and phase II metabolites, were found, of which three metabolites were chemically synthesized in order to confirm their structure. Those can be employed in testing procedures for routine doping controls, further improving anti-doping efforts.

The achievement of major molecular response (MMR, BCR::ABL1 ≤ 0.1% IS) within the first year of treatment with tyrosine kinase inhibitors (TKI) is a milestone in the therapeutic management of patients with newly diagnosed chronic myeloid leukemia (CML). We analyzed the predictive value of gene expression levels of ESPL1 /Separase, PTTG1 /Securin and PTTG1IP /Securin interacting protein for MMR achievement within 12 months. Relative expression levels (normalized to GUSB ) of ESPL1 , PTTG1 and PTTG1IP in white blood cells of patients (responders n = 46, non-responders n = 51) at the time of diagnosis were comparatively analyzed by qRT-PCR. 3D scatter plot analysis combined with a distance analysis performed with respect to a commonly calculated centroid center resulted in a trend to larger distances for non-responders compared to the responder cohort ( p = 0.0187). Logistic regression and analysis of maximum likelihood estimates revealed a positive correlation of distance (cut-off) with non-achieving MMR within 12 months ( p = 0.0388, odds ratio 1.479, 95%CI: 1.020 to 2.143). Thus, 10% of the tested non-responders (cut-off ≥ 5.9) could have been predicted already at the time of diagnosis. Future scoring of ESPL1 , PTTG1 and PTTG1IP transcript levels may be a helpful tool in risk stratification of CML patients before initiation of TKI first = line treatment.

The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) tool kit constitutes one of today's most frequently used gene editing techniques. Editing of virtually any DNA sequence can be realised, due to the quickly progressing research into different Cas effectors and their ever-expanding range of targets. Moreover, the simplicity and cost-effectiveness of those CRISPR tools can, unfortunately, also facilitate the illicit utilisation of CRISPR/Cas in order to achieve performance enhancements amongst athletes. Consequently, there is an urgent need for the direct detection of illegally applied CRISPR/Cas methods in doping control samples, for which a promising strategy is presented herein employing Specific High Sensitive Enzymatic Reporter UnLOCKing (SHERLOCK) for targeted nucleic acid detection. An analytical method was developed that enables the detection of sgRNA associated with Cas9 from Streptococcus pyogenes (SpCas9) in serum samples by means of reverse transcriptase-recombinase polymerase amplification (RT-RPA) and subsequent qualitative nucleic acid detection via SHERLOCK in combination with a complementary gel-based screening procedure in order to uncover doping attempts with lipid mediated CRISPR ribonucleoprotein (RNP) complexes. Initial qualitative method characterisation confirmed the specificity of both procedures and established a detection sensitivity of 10 nM uncomplexed target sequence and 100 pM sgRNA in the form of RNP complexes. Furthermore, a proof-of-concept in vivo adimistration study simulating a hypothetical gene doping scenario employing a mouse model revealed a detection window of 8 h after intravenous injection, supporting the principal applicability of the test strategy to authentic doping control samples in the future.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) of BCR-ABL1 transcript level is an essential part of routine disease monitoring in patients with chronic myeloid leukemia. One patient sample (e13a2 transcript detected by nested PCR) attracted attention by revealing an aberrantly spliced BCR-ABL1 transcript variant e13a1. The last 38 base pairs (bp) of BCR exon 13 were replaced by a 37 bp insertion of the ABL1 intron 1-2/exon 1 sequence. The rare aberrant BCR-ABL1 fusion transcript can cause discrepancies in molecular diagnostics. This scenario highlights the importance of an individual characterization of the BCR-ABL1 fusion sequence in case of unclear qRT-PCR results., Competing Interests: SS received honoraria from Novartis Pharma GmbH, Bristol‐Myers Squibb (BMS), Pfizer, ARIAD, and research funding from Novartis Pharma GmbH and BMS. All other authors declare no conflict of interest., (© 2022 The Authors. eJHaem published by British Society for Haematology and John Wiley & Sons Ltd.)

Systemic mastocytosis (SM) is characterized by multifocal accumulation of neoplastic mast cells (MCs), predominately affecting the bone marrow (BM). Imaging with computed tomography (CT) is used for assessment of bone mineral density and structure. However, the value of functional imaging with dual-energy CT (DECT) and the assessment of virtual-non-calcium attenuation values (VNCa-AV) for visualization of BM disease burden in SM has not yet been assessed. DECT of the axial skeleton was performed in 18 patients with SM (indolent SM [ISM], n = 6; smoldering SM [SSM]/advanced SM [AdvSM], n = 12) and 18 control subjects. VNCa-AV were obtained in 5 representative vertebraes per patient and correlated with laboratory, morphologic and molecular parameters. VNCa-AV strongly correlated with quantitative BM MC infiltration (r = 0.7, R 2  = 0.49, P = 0.001) and serum tryptase levels (r = 0.7, R 2  = 0.54, P < 0.001). Mean VNCa-AV were significantly higher in SSM/AdvSM as compared to ISM (- 9HU vs. - 54HU, P < 0.005) and controls (- 38HU, P < 0.005). Nine of 10 (90%) patients with a VNCa-AV >  - 30HU and 7/7 (100%) patients with a VNCa-AV >  - 10HU had SSM or AdVSM. BM VNCa-AV provide information about the MC burden of SM patients and correlate with SM subtypes. DECT may therefore serve as a supplementary tool for SM diagnosis, subclassification and monitoring in a one-stop-shop session., (© 2022. The Author(s).)

Standardized monitoring of BCR::ABL1 mRNA levels is essential for the management of chronic myeloid leukemia (CML) patients. From 2016 to 2021 the European Treatment and Outcome Study for CML (EUTOS) explored the use of secondary, lyophilized cell-based BCR::ABL1 reference panels traceable to the World Health Organization primary reference material to standardize and validate local laboratory tests. Panels were used to assign and validate conversion factors (CFs) to the International Scale and assess the ability of laboratories to assess deep molecular response (DMR). The study also explored aspects of internal quality control. The percentage of EUTOS reference laboratories (n = 50) with CFs validated as optimal or satisfactory increased from 67.5% to 97.6% and 36.4% to 91.7% for ABL1 and GUSB, respectively, during the study period and 98% of laboratories were able to detect MR 4.5 in most samples. Laboratories with unvalidated CFs had a higher coefficient of variation for BCR::ABL1 IS and some laboratories had a limit of blank greater than zero which could affect the accurate reporting of DMR. Our study indicates that secondary reference panels can be used effectively to obtain and validate CFs in a manner equivalent to sample exchange and can also be used to monitor additional aspects of quality assurance., (© 2022. The Author(s).)

Purpose: On the basis of data from the German Registry on Disorders of Eosinophils and Mast Cells, we compared the efficacy of midostaurin and cladribine in patients with advanced systemic mastocytosis (AdvSM)., Patients and Methods: Patients with AdvSM (n = 139) were treated with midostaurin only (n = 63, 45%), cladribine only (n = 23, 17%), or sequentially (midostaurin-cladribine, n = 30, 57%; cladribine-midostaurin, n = 23, 43%). Prognosis was assessed through the Mutation-Adjusted Risk Score (MARS). Besides the comparison of efficacy between midostaurin and cladribine on response (eg, organ dysfunction, bone marrow mast cell [MC] infiltration, and tryptase), overall survival (OS), and leukemia-free survival, we focused on the impact of treatment on involved non-MC lineages, for example, monocytes or eosinophils, and the KIT D816V expressed allele burden., Results: Midostaurin only was superior to cladribine only with effects from responses on MC and non-MC lineages conferring on a significantly improved OS (median 4.2 v 1.9 years, P = .033) and leukemia-free survival (2.7 v 1.3 years, P = .044) on the basis of a propensity score-weighted analysis of parameters included in MARS. Midostaurin compensated the inferior efficacy of cladribine in first- and second-line treatment. On midostaurin in any line, response of eosinophilia did not improve its baseline adverse prognostic impact, whereas response of monocytosis proved to be a positive on-treatment parameter. Multivariable analysis allowed to establish three risk categories (low/intermediate/high) through the combination of MARS and the reduction of the KIT D816V expressed allele burden of ≥ 25% at month 6 (median OS not reached v 3.0 years v 1.0 year; P < .001)., Conclusion: In this registry-based analysis, midostaurin revealed superior efficacy over cladribine in patients with AdvSM. In midostaurin-treated patients, the combination of baseline MARS and molecular response provided a compelling three-tier risk categorization (MARSv2.0) for OS., Competing Interests: Juliana SchwaabHonoraria: NovartisConsulting or Advisory Role: Blueprint Medicines, Novartis Sebestian KreilResearch Funding: Incyte (Inst) Nicholas C.P. CrossHonoraria: Novartis, Incyte, Astellas PharmaConsulting or Advisory Role: Novartis, Incyte, Astellas PharmaSpeakers' Bureau: NovartisResearch Funding: Novartis (Inst) Karl SotlarHonoraria: Novartis, AstraZeneca, Smart-in-MediaConsulting or Advisory Role: Novartis, AstraZeneca, Smart-in-MediaTravel, Accommodations, Expenses: Novartis Peter ValentHonoraria: Novartis, Incyte, Celgene, Pfizer, Blueprint MedicinesConsulting or Advisory Role: NovartisResearch Funding: Pfizer (Inst), CelgeneTravel, Accommodations, Expenses: Novartis, Incyte, Pfizer, Celgene Jason GotlibHonoraria: Incyte, Novartis, Protagonist Therapeutics, AbbVie, Cogent Biosciences, Celgene, BMS, Pharmaessentia, Blueprint Medicines, Kartos Therapeutics, DecipheraConsulting or Advisory Role: Blueprint Medicines, Deciphera, Incyte, Kartos Therapeutics, Pharmaessentia, Cogent Biosciences, Novartis, Protagonist Therapeutics, Celgene, AbbVie, BMSResearch Funding: Blueprint Medicines (Inst), Deciphera (Inst), Incyte (Inst), Kartos Therapeutics (Inst), Promedior (Inst), CTI BioPharma Corp (Inst), Gilead Sciences (Inst), Seattle Genetics (Inst), Novartis (Inst), Celgene (Inst)Travel, Accommodations, Expenses: Blueprint Medicines Mohamad JawharHonoraria: NovartisConsulting or Advisory Role: NovartisSpeakers' Bureau: Novartis Andreas ReiterStock and Other Ownership Interests: BiontechHonoraria: Novartis, Blueprint Medicines, Incyte, Celgene/Bristol Myers Squibb, AOP Orphan Pharmaceuticals, GlaxoSmithKline, AbbVieConsulting or Advisory Role: Novartis, Blueprint Medicines, Incyte, Celgene/Bristol Myers Squibb, AOP Orphan Pharmaceuticals, AbbVieResearch Funding: Blueprint Medicines (Inst), Deciphera (Inst), Novartis (Inst), Incyte (Inst), AOP Orphan Pharmaceuticals (Inst)Expert Testimony: NovartisTravel, Accommodations, Expenses: Novartis, Deciphera, Blueprint Medicines, IncyteNo other potential conflicts of interest were reported.

Background: Patients with systemic mastocytosis (SM) are at increased risk of hypersensitivity reactions (HRs). Although Hymenoptera venoms are the predominant triggers, cases of contrast media-induced HR (CMIHR) have also been reported and prophylactic premedication is often performed. However, data from larger series are limited and differences between indolent and advanced SM have not yet been investigated., Objective: To determine the incidence and severity of CMIHR in all subtypes of SM., Methods: We analyzed 162 adult patients with SM (indolent systemic mastocytosis [ISM], n = 65; advanced systemic mastocytosis [advSM], n = 97). First, the cumulative incidence of CMIHR was retrospectively assessed in the patient's history. Second, at our institution, patients underwent 332 contrast media (CM)-enhanced imaging including 80 computed tomography (CT) scans with iodine-based contrast agent and 252 magnetic resonance imaging (MRI) with a gadolinium-based contrast agent, and tolerance was assessed., Results: Previous CMIHRs to CT (vomiting, n = 1, erythema, n = 1, cardiovascular shock, n = 1), and MRI (dyspnea, n = 1, cardiovascular shock, n = 1) had been reported by 4 out of 162 (2.5%) patients (ISM, n = 3; advSM, n = 1). In contrast, during or after 332 CM-enhanced CT or MRI examinations at our institution, no CMIHRs were reported. Premedication was solely given to 3 patients before CT scans, including 1 with previous CMIHR, who tolerated the imaging well., Conclusion: We conclude that: (1) there is a substantial discrepancy between the perception and prevalence of HRs to CM in SM; (2) reactions are scarce in ISM and even rarer in advSM; and (3) in SM patients without previous history of CM hypersensitivity, prophylactic premedication before CM-enhanced CT or MRI is dispensable., (Copyright © 2021 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

We report on 45 patients with myeloid neoplasms and concurrent Janus kinase 2 (JAK2) V617F and KIT proto-oncogene, receptor tyrosine kinase (KIT) D816V (JAK2 pos . /KIT pos . ) mutations, which are individually identified in >60% of patients with classical myeloproliferative neoplasms (MPN) and >90% of patients with systemic mastocytosis (SM) respectively. In SM, the concurrent presence of a clonal non-mast cell neoplasm [SM with associated haematological neoplasm (SM-AHN)] usually constitutes a distinct subtype associated with poor survival. All 45 patients presented with a heterogeneous combination of clinical/morphological features typical of the individual disorders (e.g. leuco-/erythro-/thrombocytosis and elevated lactate dehydrogenase for MPN; elevated serum tryptase and alkaline phosphatase for SM). Overlapping features identified in 70% of patients included splenomegaly, cytopenia(s), bone marrow fibrosis and additional somatic mutations. Molecular dissection revealed discordant development of variant allele frequency for both mutations and absence of concurrently positive single-cell derived colonies, indicating disease evolution in two independent clones rather than monoclonal disease in >60% of patients examined. Overall survival of JAK2 pos . /KIT pos . patients without additional somatic high-risk mutations [HRM, e.g. in serine and arginine-rich splicing factor 2 (SRSF2), additional sex combs like-1 (ASXL1) or Runt-related transcription factor 1 (RUNX1)] at 5 years was 77%, indicating that the mutual impact of JAK2 V617F and KIT D816V on prognosis is fundamentally different from the adverse impact of additional HRM in the individual disorders., (© 2021 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

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 > 0.99, R 2 > 0.98) between three different DNA-assays. VAF and EAB strongly correlated in ISM ( r = 0.91, coefficient of determination, R 2 = 0.84) but only to a lesser extent in AdvSM ( r = 0.71; R 2 = 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 >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 >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.

Background: Little is known about the epidemiology of advanced systemic mastocytosis (advSM)., Objectives: To investigate epidemiologic features and diagnostic pitfalls of advSM in Germany., Methods: Therefore, 140 patients from a single German reference center of the European Competence Network on Mastocytosis between 2003 and 2018 were analyzed., Results: The patients' median age was 68 years (range, 26-86 years), and male versus female ratio was 2:1. An elevated serum tryptase, a KIT D816 mutation, and additional somatic mutations, for example, in SRSF2, ASXL1, or RUNX1, were identified in 95%, 91%, and 74% of patients, respectively. Median overall survival was 3.5 years (range, 0.03-14.3 years; male vs female 2.6 vs 4.2 years; P = .02). Two categories of misdiagnoses were identified in 51 of 140 (36%) patients: First, systemic mastocytosis (SM) was overlooked in 28 of 140 (20%) patients primarily diagnosed with various subtypes of myeloid neoplasms. Second, 23 of 140 (16%) patients were diagnosed with supposed progression from indolent SM to advSM; however, combination of an elevated KIT D816V variant allele frequency in peripheral blood (n = 22), monocytosis (n = 9), eosinophilia (n = 6), and/or mutations in SRSF2, ASXL1, or RUNX1 (n = 10) suggest that distinct signs of potential advSM were overlooked in virtually all patients. Based on locally diagnosed patients in an area of 2.5 million inhabitants, but obviously without considering more, yet unrecognized cases, the incidence and prevalence of advSM is at least 0.8 and 5.2, respectively, per 1 million inhabitants., Conclusions: Adequate analyses of tryptase levels, bone marrow morphology, and genetics in patients with myeloid neoplasms or SM would help to prevent the significant underdiagnosis of advSM., (Copyright © 2020 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

We report on 18 patients with myeloid neoplasms and associated tyrosine kinase (TK) fusion genes on treatment with the TK inhibitors (TKI) ruxolitinib (PCM1-JAK2, n = 8; BCR-JAK2, n = 1) and imatinib, nilotinib or dasatinib (ETV6-ABL1, n = 9). On ruxolitinib (median 24 months, range 2-36 months), a complete hematologic response (CHR) and complete cytogenetic response (CCR) was achieved by five of nine and two of nine patients, respectively. However, ruxolitinib was stopped in eight of nine patients because of primary resistance (n = 3), progression (n = 3) or planned allogeneic stem cell transplantation (allo SCT, n = 2). At a median of 36 months (range 4-78 months) from diagnosis, five of nine patients are alive: four of six patients after allo SCT and one patient who remains on ruxolitinib. In ETV6-ABL1 positive patients, a durable CHR was achieved by four of nine patients (imatinib with one of five, nilotinib with two of three, dasatinib with one of one). Because of inadequate efficacy (lack of hematological and/or cytogenetic/molecular response), six of nine patients (imatinib, n = 5; nilotinib, n = 1) were switched to nilotinib or dasatinib. At a median of 23 months (range 3-60 months) from diagnosis, five of nine patients are in CCR or complete molecular response (nilotinib, n = 2; dasatinib, n = 2; allo SCT, n = 1) while two of nine patients have died. We conclude that (a) responses on ruxolitinib may only be transient in the majority of JAK2 fusion gene positive patients with allo SCT being an important early treatment option, and (b) nilotinib or dasatinib may be more effective than imatinib to induce durable complete remissions in ETV6-ABL1 positive patients., (© 2020 The Authors. American Journal of Hematology published by Wiley Periodicals, Inc.)

Purpose: Systemic mastocytosis (SM) is characterized by the expansion of clonal mast cells that infiltrate various organ systems. The extent of organ infiltration and subsequent organ damage distinguishes between indolent SM (ISM) defined by a nearly normal life expectancy and advanced SM (AdvSM) defined by poor prognosis. In ISM, measurement of the bone mineral density (BMD) frequently reveals osteoporosis. In contrast, the clinical implication of an increased BMD and osteosclerosis remains unclear., Methods: BMD was evaluated in 61 patients with mastocytosis (ISM, n = 29, 48%; AdvSM, n = 32, 52%). We correlated the prevalence of osteoporosis, increased BMD and osteosclerosis with clinical parameters, disease variant and prognosis., Results: Osteoporosis was detected in 11/29 (38%) patients with ISM but only in 2/32 (6%) patients with AdvSM (p = 0.004). An increased BMD was detected in 1/29 (3%) patients with ISM and 24/32 (75%) patients with AdvSM (p < 0.001) while osteosclerosis was only detected in AdvSM patients (16/32, 50%). AdvSM patients with increased BMD had higher levels of bone marrow mast cell infiltration, higher serum tryptase and alkaline phosphatase levels compared to ISM as well as higher number of high-molecular risk mutations (p < 0.05). In addition, we found that the prognosis of AdvSM patients with increased BMD is inferior compared to those without increased BMD (median overall survival 3.6 years versus not reached, p = 0.031)., Conclusions: Osteoporosis is a common feature in ISM but not in AdvSM. An increased BMD is frequently present in AdvSM but not in ISM and is associated with more advanced disease and inferior outcome.

Background: Inevitable recurrence after radiochemotherapy is the major problem in the treatment of glioblastoma, the most prevalent type of adult brain malignancy. Glioblastomas are notorious for a high degree of intratumor heterogeneity manifest through a diversity of cell types and molecular patterns. The current paradigm of understanding glioblastoma recurrence is that cytotoxic therapy fails to target effectively glioma stem cells. Recent advances indicate that therapy-driven molecular evolution is a fundamental trait associated with glioblastoma recurrence. There is a growing body of evidence indicating that intratumor heterogeneity, longitudinal changes in molecular biomarkers and specific impacts of glioma stem cells need to be taken into consideration in order to increase the accuracy of molecular diagnostics still relying on readouts obtained from a single tumor specimen. Methods : This study integrates a multisampling strategy, longitudinal approach and complementary transcriptomic investigations in order to identify transcriptomic traits of recurrent glioblastoma in whole-tissue specimens of glioblastoma or glioblastoma stem cells. In this study, 128 tissue samples of 44 tumors including 23 first diagnosed, 19 recurrent and 2 secondary recurrent glioblastomas were analyzed along with 27 primary cultures of glioblastoma stem cells by RNA sequencing. A novel algorithm was used to quantify longitudinal changes in pathway activities and model efficacy of anti-cancer drugs based on gene expression data. Results : Our study reveals that intratumor heterogeneity of gene expression patterns is a fundamental characteristic of not only newly diagnosed but also recurrent glioblastomas. Evidence is provided that glioblastoma stem cells recapitulate intratumor heterogeneity, longitudinal transcriptomic changes and drug sensitivity patterns associated with the state of recurrence. Conclusions : Our results provide a transcriptional rationale for the lack of significant therapeutic benefit from temozolomide in patients with recurrent glioblastoma. Our findings imply that the spectrum of potentially effective drugs is likely to differ between newly diagnosed and recurrent glioblastomas and underscore the merits of glioblastoma stem cells as prognostic models for identifying alternative drugs and predicting drug response in recurrent glioblastoma. With the majority of recurrent glioblastomas being inoperable, glioblastoma stem cell models provide the means of compensating for the limited availability of recurrent glioblastoma specimens.

FIP1L1-PDGFRA-positive myeloid/lymphoid neoplasms with eosinophilia (MLN-eo) are exquisitely sensitive to imatinib. Almost all patients achieve a complete molecular remission (CMR) by nested reverse transcription polymerase chain reaction, which can be maintained with low-dose imatinib (eg, 3 × 100 mg/wk). Because imatinib can be safely stopped in a substantial proportion of patients with BCR-ABL1-positive CML, we sought to analyze the clinical and molecular follow-up of 12 FIP1L1-PDGFRA-positive patients with MLN-eo in chronic phase who discontinued imatinib after achievement of a CMR. Median time of treatment and median time of CMR before imatinib discontinuation (last dose at 3 × 100 mg/wk, n = 8; or 100 mg/d, n = 4) were 80 (range, 43-175) and 66 (range, 37-174) months, respectively. A molecular relapse was observed in 4 patients after 10, 22 (n = 2), and 24 months. A second CMR was achieved in 3 patients after 3, 4, and 21 months. Eight patients (62%) are in ongoing CMR (median, 17 months; range, 3-71 months). Molecular relapse-free survival was 91% at 12 months and 65% at 24 months. No significant differences (eg, dose and duration of imatinib treatment or duration of CMR before imatinib discontinuation) were identified between patients with and without molecular relapse. Our data demonstrate that imatinib can be safely stopped in FIP1L1-PDGFRA-positive MLN-eo because of a high treatment-free remission at 12 and 24 months and because most patients achieve a rapid second CMR after restart of imatinib., (© 2020 by The American Society of Hematology.)

DNA damage and alterations in the DNA damage response (DDR) are critical sources of genetic instability that might be involved in BCR-ABL1 kinase-mediated blastic transformation of chronic myeloid leukemia (CML). Here, increased DNA damage is detected by γH2AX foci analysis in peripheral blood mononuclear cells (PBMCs) of de novo untreated chronic phase (CP)-CML patients ( n = 5; 2.5 γH2AX foci per PBMC ± 0.5) and blast phase (BP)-CML patients ( n = 3; 4.4 γH2AX foci per PBMC ± 0.7) as well as CP-CML patients with loss of major molecular response (MMR) ( n = 5; 1.8 γH2AX foci per PBMC ± 0.4) when compared to DNA damage in PBMC of healthy donors ( n = 8; 1.0 γH2AX foci per PBMC ± 0.1) and CP-CML patients in deep molecular response or MMR ( n = 26; 1.0 γH2AX foci per PBMC ± 0.1). Progressive activation of erroneous non-homologous end joining (NHEJ) repair mechanisms during blastic transformation in CML is indicated by abundant co-localization of γH2AX/53BP1 foci, while a decline of the DDR is suggested by defective expression of (p-)ATM and (p-)CHK2. In summary, our data provide evidence for the accumulation of DNA damage in the course of CML and suggest ongoing DNA damage, erroneous NHEJ repair mechanisms, and alterations in the DDR as critical mediators of blastic transformation in CML., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Purpose: To develop a risk score for patients with advanced systemic mastocytosis (AdvSM) that integrates clinical and mutation characteristics., Patients and Methods: The study included 383 patients with AdvSM from the German Registry on Disorders of Eosinophils and Mast Cells (training set; n = 231) and several centers for mastocytosis in the United States and Europe, all within the European Competence Network on Mastocytosis (validation set; n = 152). A Cox multivariable model was used to select variables that were predictive of overall survival (OS)., Results: In multivariable analysis, the following risk factors were identified as being associated with OS: age greater than 60 years, anemia (hemoglobin < 10 g/dL), thrombocytopenia (platelets < 100 × 10 9 /L), presence of one high molecular risk gene mutation (ie, in SRSF2 , ASXL1 , and/or RUNX1 ), and presence of two or more high molecular risk gene mutations. By assigning hazard ratio-weighted points to these variables, the following three risk categories were defined: low risk (median OS, not reached), intermediate risk (median OS, 3.9 years; 95% CI, 2.1 to 5.7 years), and high risk (median OS, 1.9 years; 95% CI, 1.3 to 2.6 years; P < .001). The mutation-adjusted risk score (MARS) was independent of the WHO classification and was confirmed in the independent validation set. During a median follow-up time of 2.2 years (range, 0 to 23 years), 63 (16%) of 383 patients experienced a leukemic transformation to secondary mast cell leukemia (32%) or secondary acute myeloid leukemia (68%). The MARS was also predictive for leukemia-free survival ( P < .001)., Conclusion: The MARS is a validated, five-parameter, WHO-independent prognostic score that defines three risk groups among patients with AdvSM and may improve up-front treatment stratification for these rare hematologic neoplasms.

Malignant hematopoietic cells of myelodysplastic syndromes (MDS)/chronic myelomonocytic leukemias (CMML) and acute myeloid leukemias (AML) may be vulnerable to inhibition of poly(ADP ribose) polymerase 1/2 (PARP1/2) and apurinic/apyrimidinic endonuclease 1 (APE1). PARP1/2 and APE1 are critical enzymes involved in single-strand break repair and base excision repair, respectively. Here, we investigated the cytotoxic efficacy of talazoparib and APE1 inhibitor III, inhibitors of PARP1/2 and APE1, in primary CD34+ MDS/CMML cell samples ( n = 8; 4 MDS and 4 CMML) and in primary CD34+ or CD34- AML cell samples ( n = 18) in comparison to healthy CD34+ donor cell samples ( n = 8). Strikingly, talazoparib and APE1 inhibitor III demonstrated critical antileukemic efficacy in selected MDS/CMML and AML cell samples. Low doses of talazoparib and APE1 inhibitor III further increased the cytotoxic efficacy of decitabine in MDS/CMML and AML cells. Moreover, low doses of APE1 inhibitor III increased the cytotoxic efficacy of talazoparib in MDS/CMML and AML cells. In summary, talazoparib and APE1 inhibitor III demonstrated substantial antileukemic efficacy as single agents, in combination with decitabine, and combined with each other. Hence, our findings support further investigation of these agents in sophisticated clinical trials.

Starting with the smallest functional unit, the spinal segment including the centrally located intervertebral disc, the spine and pelvis including the sacroiliac and hip joints form a functional unit. The discs play an important role in the complex interaction between disc, osseous and ligamentous structures and the adjacent muscles. Disc pathologies are very frequently associated with changes of the adjacent endplates of the vertebral bodies and can be depicted at an early stage using magnetic resonance imaging (MRI). The focus of the diagnostics and assessment of degenerative disc pathologies should be centered on the clinical problem. The basis for this is the use of a uniform terminology between the disciplines involved.

KIT D816 mutations (KIT D816 mut ) are strongly associated with systemic mastocytosis (SM) but are also detectable in acute myeloid leukemia (AML), where they represent an adverse prognostic factor in combination with core binding factor (CBF) fusion genes. Here, we evaluated the clinical and molecular features of KIT D816 mut /CBF-negative (CBF neg ) AML, a previously uncharacterized combination. All KIT D816 mut /CBF neg cases (n = 40) had histologically proven SM with associated AML (SM-AML). Molecular analyses revealed at least one additional somatic mutation (median, n = 3) beside KIT D816 (e.g., SRSF2, 38%; ASXL1, 31%; RUNX1, 34%) in 32/32 (100%) patients. Secondary AML evolved in 29/40 (73%) patients from SM ± associated myeloid neoplasm. Longitudinal molecular and cytogenetic analyses revealed the acquisition of new mutations and/or karyotype evolution in 15/16 (94%) patients at the time of SM-AML. Median overall survival (OS) was 5.4 months. A screen of two independent AML databases (AML databases ) revealed remarkable similarities between KIT D816 mut /CBF neg SM-AML and KIT D816 mut /CBF neg AML databases (n = 69) with regard to KIT D816 mut variant allele frequency, mutation profile, aberrant karyotype, and OS suggesting underlying SM in a significant proportion of AML databases patients. Bone marrow histology and reclassification as SM-AML has important clinical implications regarding prognosis and potential inclusion of KIT inhibitors in treatment concepts.

Advanced systemic mastocytosis (advSM) is characterized by the presence of an acquired KIT D816V mutation in >90% of patients. In the majority of patients, KIT D816V is not only detected in mast cells but also in other hematopoietic lineages. We sought to investigate the effects of the KIT-inhibitors midostaurin and avapritinib on single-cell-derived myeloid progenitor cells using granulocyte-macrophage colony-forming-units of patients with KIT D816V positive advSM. Colonies obtained prior to treatment were incubated in vitro with midostaurin (n = 10) or avapritinib (n = 11) and showed a marked reduction (≥50%) of KIT D816V positive colonies in 3/10 (30%) and 7/11 (64%) patient samples, respectively. Three of those 7 (43%) avapritinib responders were resistant to midostaurin in both, in vitro and in vivo. Colonies from four patients with high-risk molecular profile and aggressive clinical course were resistant to both drugs. The in vitro activity of midostaurin strongly correlated with clinical and molecular responses, e.g., relative reduction of KIT D816V allele burden and the proportion of KIT D816V positive colonies obtained after six months midostaurin-treatment in vivo. We conclude that the colony inhibition assay provides useful information for prediction of responses on midostaurin and that avapritinib has a superior in vitro activity compared to midostaurin.

In chronic myeloid leukemia (CML), the duration of deep molecular response (MR) before treatment cessation (MR4 or deeper, corresponding to BCR-ABL1 ≤ 0.01% on the International Scale (IS)) is considered as a prognostic factor for treatment free remission in stopping trials. MR level determination is dependent on the sensitivity of the monitoring technique. Here, we compared a newly established TaqMan (TM) and our so far routinely used LightCycler (LC) quantitative reverse transcription (qRT)-PCR systems for their ability to achieve the best possible sensitivity in BCR-ABL1 monitoring. We have comparatively analyzed RNA samples from peripheral blood mononuclear cells of 92 randomly chosen patients with CML resembling major molecular remission (MMR) or better and of 128 CML patients after treatment cessation (EURO-SKI stopping trial). While our LC system utilized ABL1, the TM system is based on GUSB as reference gene. We observed 99% concordance with respect to achievement of MMR. However, we found that 34 of the 92 patients monitored by TM/GUSB were re-classified to the next inferior MR log level, especially when LC/ABL1-based results were borderline to thresholds. Thirteen patients BCR-ABL1 negative in LC/ABL1 became positive after TM/GUSB analysis. In the 128 patients included in the EURO-SKI trial identical molecular findings were achieved for 114 patients. However, 14 patients were re-classified to the next inferior log-level by the TM/GUSB combination. Eight of these patients relapsed after treatment cessation; two of them were re-classified from MR4 to MMR and therefore did not meet inclusion criteria anymore. In conclusion, we consider both methods as comparable and interchangeable in terms of achievement of MMR and of longitudinal evaluation of clinical courses. However, in LC/ABL1 negative samples, slightly enhanced TM/GUSB sensitivity may lead to inferior classification of clinical samples in the context of TFR., Competing Interests: SS received honoraria from Novartis Pharma GmbH, Bristol-Myers Squibb (BMS), Pfizer, ARIAD and research funding from Novartis Pharma GmbH and BMS. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The funders had no role in study design, collection, analysis and interpretation of data; writing of the paper and/or decision to submit for publication. We further acknowledge financial support by Deutsche Forschungsgemeinschaft within the funding programme Open Access Publishing, by the Baden-Württemberg Ministry of Science, Research and the Arts and by Ruprecht-Karls-Universität Heidelberg.

Determining the underlying cause of persistent eosinophilia is important for effective clinical management but remains a diagnostic challenge in many cases. We identified STAT5B N642H, an established oncogenic mutation, in 27/1715 (1.6%) cases referred for investigation of eosinophilia. Of the 27 mutated cases, a working diagnosis of hypereosinophilic syndrome (HES; n = 7) or a myeloid neoplasm with eosinophilia (n = 20) had been made prior to the detection of STAT5B N642H. Myeloid panel analysis identified a median of 2 additional mutated genes (range 0-4) with 4 cases having STAT5B N642H as a sole abnormality. STAT5B N642H was absent in cultured T cells of 4/4 positive cases. Individuals with SF3B1 mutations (9/27; 33%) or STAT5B N642H as a sole abnormality had a markedly better overall survival compared to cases with other additional mutations (median 65 months vs. 14 months; hazard ratio = 8.1; P < 0.001). The overall survival of STAT5B-mutated HES cases was only 30 months, suggesting that these cases should be reclassified as chronic eosinophilic leukemia, not otherwise specified (CEL-NOS). The finding of STAT5B N642H as a recurrent mutation in myeloid neoplasia with eosinophilia provides a new diagnostic and prognostic marker as well as a potential target for therapy.

Clinical Issue: Autoimmune disorders of the central nervous system (CNS) are common but are also a heterogeneous group of diseases. The most common form is multiple sclerosis (MS), others are clinically isolated syndrome (CIS), acute demyelinating encephalomyelitis (ADEM) and neuromyelitis optica spectrum disorders (NMOSD). Paraneoplastic syndromes are rare and tumor-associated, they are not induced by direct invasion of tumor tissue but by tumor-associated autoantibodies mostly against specific CNS proteins, e. g. limbic encephalitis and paraneoplastic cerebellar ataxia or degeneration. DIAGNOSTICS, STANDARD RADIOLOGICAL METHODS, PERFORMANCE AND ACHIEVEMENTS: The correct diagnosis of autoimmune and paraneoplastic syndromes can still be challenging. In addition to the patient history, clinical examination and blood as well as cerebrospinal fluid (CSF) tests, magnetic resonance imaging (MRI) is gaining importance in the diagnostics. It is important not only in primary diagnostics but also in follow-up and therapy monitoring, especially in MS with specific therapies to detect therapy complications, such as progressive multifocal leukoencephalopathy as early as possible. In paraneoplastic syndromes MRI can also be an important component in the diagnostics but can also initially be negative and typical signal changes become visible only in follow-up scans., Practical Recommendations: In paraneoplastic syndromes the correct diagnosis is based on laboratory tests for specific autoantibodies in serum and CSF., Treatment: The treatment of autoimmune and paraneoplastic disorders of the CNS ranges from steroids and immunosuppressive agents to plasmapheresis, depending on the specific disorder.

The clinical behavior of systemic mastocytosis (SM) is strongly associated with activating mutations in KIT (D816V in >80% of cases), with the severity of the phenotype influenced by additional somatic mutations, for example, in SRSF2, ASXL1, or RUNX1. Complex molecular profiles are frequently associated with the presence of an associated hematologic neoplasm (AHN) and an unfavorable clinical outcome. However, little is known about the incidence and prognostic impact of cytogenetic aberrations. We analyzed cytogenetic and molecular characteristics of 109 patients (KIT D816V+, n = 102, 94%) with indolent (ISM, n = 26) and advanced SM (n = 83) with (n = 73, 88%) or without AHN. An aberrant karyotype was identified in SM-AHN (16/73, 22%) patients only. In patients with an aberrant karyotype, additional somatic mutations were identified in 12/16 (75%) patients. Seven of 10 (70%) patients with a poor-risk karyotype, for example, monosomy 7 or complex karyotype, and 1/6 (17%) patients with a good-risk karyotype progressed to secondary acute myeloid leukemia (n = 7) or mast cell leukemia (n = 1) within a median of 40 months (range 2-190, P = .04). In advanced SM, the median overall survival (OS) of poor-risk karyotype patients was significantly shorter than in good-risk/normal karyotype patients (4 vs 39 months; hazard ratio 11.7, 95% CI 5.0-27.3; P < .0001). Additionally, the shortened OS in patients with poor-risk karyotype was independent from the mutation status. In summary, a poor-risk karyotype is an independent prognostic variable in advanced SM. Cytogenetic and molecular analyses should be routinely performed in all patients with advanced SM ± AHN because these investigations greatly support prognostication and treatment decisions., (© 2018 Wiley Periodicals, Inc.)

Quantitative real-time polymerase chain reaction (qRT-PCR) is state of the art in molecular monitoring of minimal residual disease in chronic myeloid leukemia (CML). In this context, maintenance of assay fidelity and detection of technical inaccuracy are crucial. Beside multiple common negative controls for the clinical sample preparations, quality control charts (QCC) are a common validation tool to sustain high process quality by continuously recording of qRT-PCR control parameters. Here, we report on establishment and benefit of QCC in qRT-PCR-based CML diagnostics. The absolute quantification of BCR-ABL1 fusion transcripts in patient samples is based on coamplification of a serially diluted reference plasmid (pME-2). For QCC establishment the measured Ct values of each pME-2 standard dilution (4-400,000) of a test set resembling 21 sequential qRT-PCR experiments were recorded and statistically evaluated. Test set data were used for determination of warning limits (mean +/- 2-fold standard deviation) and control (intervention) limits (mean +/- 3-fold standard deviation) to allow rapid detection of defined out-of-control situations which may require intervention. We have retrospectively analyzed QCC data of 282 sequential qRT-PCR experiments (564 reactions). Data evaluation using QCCs revealed three out-of-control situations that required intervention like experiment repeats, renewal of pME-2 standards, replacement of reagents or personnel re-training. In conclusion, with minimal more effort and hands-on time QCC rank among the best tools to grant high quality and reproducibility in CML routine molecular diagnosis.

We evaluated clinical characteristics and outcome on imatinib of 22 patients with myeloid/lymphoid neoplasms with eosinophilia and rearrangement of PDGFRB. Median age was 49 years (range 20-80), 91% were male. Fifteen different PDGFRB fusion genes were identified. Eosinophilia was absent in 4/19 (21%) cases and only 11/19 (58%) cases had eosinophils ≥1.5×10 9 /L. On imatinib, 17/17 (100%) patients in chronic phase achieved complete hematologic remission after median 2 months (range 0-13)​. Complete cytogenetic remission and/or complete molecular remission by RT-PCR were achieved in 12/13 (92%) and 12/14 patients (86%) after median 10 (range 3-34) and 19 months (range 7-110), respectively. In patients with blast phase (myeloid, n = 2; lymphoid, n = 3), treatment included combinations of imatinib (n = 5), intensive chemotherapy (n = 3), and/or allogeneic stem cell transplantation (n = 3). All 3 transplanted patients (complex karyotype, n = 2) experienced early relapse. Initially, patients were treated with imatinib 400 mg/day (n = 15) or 100 mg/day (n = 7), the dose was reduced from 400 mg/day to 100 mg/day during follow-up in 9 patients. After a median treatment of 71 months (range 1-135), the 5-year survival rate was 83%; 4/22 (18%) patients died (chronic phase; n = 2; blast phase, n = 2) due to progression (n = 3) or comorbidity while in remission (n = 1). Of note, 3/4 patients had a complex karyotype. In summary, the most important characteristics of myeloid/lymphoid neoplasms with rearrangement of PDGFRB include (a) male predominance, (b) frequent lack of hypereosinophilia,, ((c) presentation in chronic or blast phase, (d) rapid responses and long-term remission on low-dose imatinib, and (e) possible adverse prognostic impact of a complex karyotype.)

In advanced systemic mastocytosis (advSM), disease evolution is often triggered by KIT mutations (D816V in >80% of cases) and by additional mutations (eg, in SRSF2 , ASXL1 , and/or RUNX1 [S/A/R pos in >60% of cases]). In a recently reported phase 2 study, midostaurin, a multikinase/KIT inhibitor, demonstrated an overall response rate (ORR) of 60% in advSM but biomarkers predictive of response are lacking. We evaluated the impact of molecular markers at baseline and during follow-up in 38 midostaurin-treated advSM patients. The median overall survival (OS) was 30 months (95% confidence interval, 6-54) from start of midostaurin. ORR and OS were significantly different between S/A/R neg (n = 12) and S/A/R pos (n = 23) patients (ORR: 75% vs 39%, P = .04; OS: P = .01, HR 4.5 [1.3-16.2]). Depending on the relative reduction of the KIT D816V expressed allele burden (EAB) at month 6, patients were classified as KIT responders (≥25%, n = 17) or KIT nonresponders (<25%, n = 11). In univariate analyses at month 6, reduction of KIT D816V EAB ≥25%, tryptase ≥50%, and alkaline phosphatase ≥50% were significantly associated with improved OS. In multivariate analysis, only KIT D816V EAB reduction ≥25% remained an independent on-treatment marker for improved OS ( P = .004, HR 6.8 [1.8-25.3]). Serial next-generation sequencing analysis of 28 genes in 16 patients revealed acquisition of additional mutations or increasing variant allele frequency in K / NRAS , RUNX1 , IDH2 , or NPM1 associated with progression in 7 patients. In midostaurin-treated advSM patients, the complexity and dynamics of mutational profiles significantly affect response, progression, and prognosis., (© 2017 by The American Society of Hematology.)

Mast cell leukemia is a rare variant of advanced systemic mastocytosis characterized by at least 20% of mast cells in a bone marrow smear. We evaluated clinical and molecular characteristics of 28 patients with (n=20, 71%) or without an associated hematologic neoplasm. De novo mast cell leukemia was diagnosed in 16 of 28 (57%) patients and secondary mast cell leukemia evolving from other advanced systemic mastocytosis subtypes in 12 of 28 (43%) patients, of which 7 patients progressed while on cytoreductive treatment. Median bone marrow mast cell infiltration was 65% and median serum tryptase was 520 μg/L. C-findings were identified in 26 of 28 (93%) patients. Mutations in KIT (D816V, n=19; D816H/Y, n=5; F522C, n=1) were detected in 25 of 28 (89%) patients and prognostically relevant additional mutations in SRSF2 , ASXL1 or RUNX1 (S/A/R pos ) in 13 of 25 (52%) patients. Overall response rate in 18 treatment-naïve patients was 5 of 12 (42%) on midostaurin and 1 of 6 (17%) on cladribine, and after switch 1 of 4 (25%) on midostaurin and 0 of 3 on cladribine, respectively. S/A/R pos adversely affected response to treatment and progression to secondary mast cell leukemia (n=6) or acute myeloid leukemia (n=3) while on treatment ( P <0.05). The median overall survival from mast cell leukemia diagnosis was 17 months as compared to 44 months in a control group of 124 patients with advanced systemic mastocytosis but without mast cell leukemia ( P =0.03). In multivariate analyses, S/A/R pos remained the only independent poor prognostic variable predicting overall survival ( P =0.007). In conclusion, the molecular signature should be determined in all patients with mast cell leukemia because of its significant clinical and prognostic relevance., (Copyright© Ferrata Storti Foundation.)

Increased DNA damage and alteration of the DNA damage response (DDR) are critical features of genetic instability presumably implicated in pathogenesis of myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML). We used immunofluorescence staining of γH2AX and 53BP1 for analyzing DNA double-strand breaks (DSB) in MDS and AML cell lines, in CD34+ selected cells of normal and MDS bone marrow (including three cases of chronic myelomonocytic leukemias) and in blasts of AML bone marrow. In addition, we screened for activation of the DDR by immunoblotting of p-ATM, p-ATR, p-CHK1, p-CHK2 and p-TP53. As compared to γH2AX foci levels in normal bone marrow samples (0.2 focus per CD34+ cell±0.0; mean±standard error of mean), increased levels of γH2AX foci were detected in 16/16 MDS bone marrow samples (2.8 foci per CD34+ cell±0.5), 18/18 AML bone marrow samples (5.5 foci per blast±0.5), 1/1 MDS cell line (6.4 foci per cell) and 6/6 AML cell lines (12.0 foci per cell±0.6). γH2AX and 53BP1 co-localized in all tested samples forming diffuse, clustered and marginal patterns. Further, DDR proteins were expressed heterogeneously suggesting impairment of the DDR. In summary, our results provide evidence for a continuous increase of DSB across the spectrum from MDS to AML in conjunction with an impaired DDR. Co-localization of γH2AX and 53BP1 indicates promotion of (in)effective nonhomologous end-joining repair mechanisms at sites of DSB. Moreover, γH2AX/53BP1 foci distribution presumably reveals a non-random spatial organization of the genome in MDS and AML., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

We evaluated the impact of clinical and molecular characteristics on overall survival (OS) in 108 patients with indolent (n=41) and advanced systemic mastocytosis (SM) (advSM, n=67). Organomegaly was measured by magnetic resonance imaging-based volumetry of the liver and spleen. In multivariate analysis of all patients, an increased spleen volume ⩾450 ml (hazard ratio (HR), 5.2; 95% confidence interval (CI), (2.1-13.0); P=0.003) and an elevated alkaline phosphatase (AP; HR 5.0 (1.1-22.2); P=0.02) were associated with adverse OS. The 3-year OS was 100, 77, and 39%, respectively (P<0.0001), for patients with 0 (low risk, n=37), 1 (intermediate risk, n=32) or 2 (high risk, n=39) parameters. For advSM patients with fully available clinical and molecular data (n=60), univariate analysis identified splenomegaly ⩾1200 ml, elevated AP and mutations in the SRSF2/ASXL1/RUNX1 (S/A/R) gene panel as significant prognostic markers. In multivariate analysis, mutations in S/A/R (HR 3.2 (1.1-9.6); P=0.01) and elevated AP (HR 2.6 (1.0-7.1); P=0.03) remained predictive adverse prognostic markers for OS. The 3-year OS was 76 and 38%, respectively (P=0.0003), for patients with 0-1 (intermediate risk, n=28) or 2 (high risk, n=32) parameters. We conclude that splenomegaly, elevated AP and mutations in the S/A/R gene panel are independent of the World Health Organization classification and provide the most relevant prognostic information in SM patients.

Background: The majority of investigations on HIV-associated neurocognitive disorders (HAND) neglect the cerebellum in spite of emerging evidence for its role in higher cognitive functions and dysfunctions in common neurodegenerative diseases., Methods: We systematically investigated the molecular and cellular responses of the cerebellum as contributors to lentiviral infection-induced neurodegeneration, in the simian immunodeficiency virus (SIV)-infected rhesus macaque model for HIV infection and HAND. Four cohorts of animals were studied: non-infected controls, SIV-infected asymptomatic animals, and SIV-infected AIDS-diseased animals with and without brain-permeant antiretroviral treatment. The antiretroviral utilized was 6-chloro-2',3'-dideoxyguanosine (6-Cl-ddG), a CNS-permeable nucleoside reverse transcriptase inhibitor. Quantitation of granule cells and Purkinje cells, of an established biomarker of SIV infection (gp41), of microglial/monocyte/macrophage markers (IBA-1, CD68, CD163), and of the astroglial marker (GFAP) were used to reveal cell-specific cerebellar responses to lentiviral infection and antiretroviral therapy (ART). The macromolecular integrity of the blood brain barrier was tested by albumin immunohistochemistry., Results: Productive CNS infection was observed in the symptomatic stage of disease, and correlated with extensive microglial/macrophage and astrocyte activation, and widespread macromolecular blood brain barrier defects. Signs of productive infection, and inflammation, were reversed upon treatment with 6-Cl-ddG, except for a residual low-grade activation of microglial cells and astrocytes. There was an extensive loss of granule cells in the SIV-infected asymptomatic cohort, which was further increased in the symptomatic stage of the disease and persisted after 6-Cl-ddG (administered after the onset of symptoms of AIDS). In the symptomatic stage, Purkinje cell density was reduced. Purkinje cell loss was likewise unaffected by 6-Cl-ddG treatment at this time., Conclusions: Our findings suggest that neurodegenerative mechanisms are triggered by SIV infection early in the disease process, i. e., preceding large-scale cerebellar productive infection and marked neuroinflammation. These affect primarily granule cells early in disease, with later involvement of Purkinje cells, indicating differential vulnerability of the two neuronal populations. The results presented here indicate a role for the cerebellum in neuro-AIDS. They also support the conclusion that, in order to attenuate the development of motor and cognitive dysfunctions in HIV-positive individuals, CNS-permeant antiretroviral therapy combined with anti-inflammatory and neuroprotective treatment is indicated even before overt signs of CNS inflammation occur.

Purpose: Computed tomography (CT) scans are an important source of ionizing irradiation (IR) in medicine that can induce a variety of DNA damage in human tissues. With technological improvements CT scans at reduced absorbed doses became feasible presumably lowering genotoxic side effects., Materials and Methods: For measuring DNA damage we performed γH2AX foci microscopy in peripheral blood mononuclear cells (PBMC) after exposure to reduced and conventional absorbed radiation doses using 3rd generation dual-source CT (DSCT) technology., Results: CT scans performed at reduced absorbed doses of 3 mGy induced significant lower levels (p < 0.0001) of DNA damage (0.05 focus per cell ± 0.01 [mean ± standard error of mean]) at 5 min after IR compared to conventional absorbed doses of 15 mGy (0.30 focus per cell ± 0.03). With ongoing DNA repair background γH2AX foci levels (0.05 focus per cell) were approached at 24 h after CT with both protocols., Conclusion: Our results provide evidence that reduced absorbed doses mediated by adjusted tube current in 3rd generation DSCT induce lower levels of DNA damage in PBMC compared to conventional absorbed doses suggesting a lower genotoxic risk for state-of-the-art tube current reduced CT protocols.

Background: Bone marrow (BM) histology/immunohistochemistry, KIT D816V mutation analysis and serum tryptase measurements are mandatory tools for diagnosis of systemic mastocytosis (SM)., Materials and Methods: Within the 'German Registry of Disorders on Eosinophils and Mast Cells', we identified 65 patients with SM who had two consecutive BM biopsies. The first biopsy was evaluated by a local pathologist (LP) and the second biopsy by a reference pathologist (RP) of the 'European Competence Network on Mastocytosis (ECNM)'., Results: Final diagnoses by RP were SM (n = 27), SM or aggressive SM (ASM) with associated clonal haematological non-mast cell lineage disease [(A)SM-AHNMD, n = 34)] or mast cell leukaemia ± AHNMD (n = 4). In 15 of 65 patients (23%), initial diagnoses by LP were incorrect (by overlooking SM), for example primary myelofibrosis (n = 3), myelodysplastic/myeloproliferative neoplasm unclassified (n = 3) or B-cell lymphoma (n = 2). Fourteen of 15 patients (93%) with incorrect diagnosis had an advanced SM, mostly (A)SM-AHNMD. In the 50 concordantly diagnosed patients, immunohistochemical markers for quantitative assessment of mast cell infiltration, for example CD117 (KIT) or CD25, were applied by LP in only 34 of 50 patients (68%), and mutational analysis for KIT D816V was performed or recommended in only 13 of 50 patients (26%). Finally, the subclassification of SM was discordant because LP did not diagnose AHNMD in nine of 50 (18%) patients., Conclusions: In summary, adequate diagnosis and subclassification of SM requires an in-depth evaluation of the BM by experienced haematopathologists (preferably in a reference centre) in combination with molecular genetics, serum tryptase level and clinical parameters., (© 2016 Stichting European Society for Clinical Investigation Journal Foundation.)

The FIP1L1-PDGFRA (FP) fusion gene is identified in a substantial proportion of patients with eosinophilia-associated myeloproliferative neoplasms (MPN-eo) who subsequently achieve rapid and durable remissions on imatinib. In the initial diagnostic work-up of hypereosinophilia (HE), histologic and immunohistochemical evaluation of a bone marrow (BM) core biopsy is considered essential for the differentiation between reactive hypereosinophilia (HER), MPN-eo and hypereosinophilic syndrome (HES). We therefore retrospectively analysed the initial reports of BM core biopsies from 116 patients who were subsequently identified as FP positive (FP+, n = 56) or FP negative/corticosteroid-responsive HER or HES (n = 60). Compared to HER or HES, detection of FP was more frequently associated with increased numbers of blasts (11/56 vs. 2/60, p = 0.007) and mast cells (23/33 vs. 7/23, p = 0.006; with expression of CD25 [11/18 vs. 2/13, p = 0.025]), and/or fibrosis (25/35 vs. 1/23, p < 0.0001). In FP+ patients, HE was correctly associated with an underlying clonal haematologic disorder in only 36/56 (64 %) of cases, but final BM diagnoses included a variety of diagnoses such as MPN-eo (n = 15), acute myeloid leukaemia (n = 8), systemic mastocytosis (n = 6), chronic myeloid leukaemia (n = 5) or unclassified MPN (n = 2). We conclude that the final evaluation of BM core biopsies in the diagnostic work-up of HE should include comprehensive morphologic (stains for myeloid blast cells, mast cells and fibres) and genetic analyses before a final diagnosis is established.

Most patients with KIT D816V(+) advanced systemic mastocytosis (SM) are characterized by somatic mutations in additional genes. We sought to clarify the prognostic impact of such mutations. Genotype and clinical characteristics of 70 multi-mutated KIT D816V(+) advanced SM patients were included in univariate and multivariate analyses. The most frequently identified mutated genes were TET2 (n=33 of 70 patients), SRSF2 (n=30), ASXL1 (n=20), RUNX1 (n=16) and JAK2 (n=11). In univariate analysis, overall survival (OS) was adversely influenced by mutations in SRSF2 (P<0.0001), ASXL1 (P=0.002) and RUNX1 (P=0.03), but was not influenced by mutations in TET2 or JAK2. In multivariate analysis, SRSF2 and ASXL1 remained the most predictive adverse indicators concerning OS. Furthermore, we found that inferior OS and adverse clinical characteristics were significantly influenced by the number of mutated genes in the SRSF2/ASXL1/RUNX1 (S/A/R) panel (P<0.0001). In conclusion, the presence and number of mutated genes within the S/A/R panel are adversely associated with advanced disease and poor survival in KIT D816V(+) SM. On the basis of these findings, inclusion of molecular markers should be considered in upcoming prognostic scoring systems for patients with SM.

In eosinophilia-associated myeloproliferative neoplasms (MPN-eo), constitutive activation of protein tyrosine kinases (TK) as consequence of translocations, inversions, or insertions and creation of TK fusion genes is recurrently observed. The most commonly involved TK and their potential TK inhibitors include PDGFRA at 4q12 or PDGFRB at 5q33 (imatinib), FGFR1 at 8p11 (ponatinib), and JAK2 at 9p24 (ruxolitinib). We here report the identification of three new PDGFRB fusion genes in three male MPN-eo patients: MPRIP-PDGFRB in a case with t(5;17)(q33;p11), CPSF6-PDGFRB in a case with t(5;12)(q33;q15), and GOLGB1-PDGFRB in a case with t(3;5)(q13;q33). The fusion proteins identified by 5'-rapid amplification of cDNA ends polymerase chain reaction (PCR) or DNA-based long distance inverse PCR are predicted to contain the TK domain of PDGFRB. The partner genes contain domains like coiled-coil structures, which are likely to cause dimerization and activation of the TK. In all patients, imatinib induced rapid and durable complete remissions., (© 2015 Wiley Periodicals, Inc.)