Response and resistance to cladribine in patients with advanced systemic mastocytosis: a registry-based analysis.

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 × 109/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.

Step-in dosing of bosutinib in pts with chronic phase chronic myeloid leukemia (CML) after second-generation tyrosine kinase inhibitor (TKI) therapy: results of the Bosutinib Dose Optimization (BODO) Study.

The approved dose of bosutinib in chronic phase CML is 400 mg QD in first-line and 500 mg QD in later-line treatment. However, given that gastrointestinal (GI) toxicity typically occurs early after treatment initiation, physicians often tend to start therapy with lower doses although this has never been tested systematically in prospective trials in the Western world. The Bosutinib Dose Optimization (BODO) Study, a multicenter phase II study, investigated the tolerability and efficacy of a step-in dosing concept of bosutinib (starting at 300 mg QD) in chronic phase CML patients in 2nd or 3rd line who were intolerant and/or refractory to previous TKI treatment. Of 57 patients included until premature closure of the study due to slow recruitment, 34 (60%) reached the targeted dose level of 500 mg QD following the 2-weekly step-in dosing regimen. While the dosing-in concept failed to reduce GI toxicity (grade II-IV, primary study endpoint) to < 40% (overall rate of 60%; 95% CI: 45-74%), bosutinib treatment (mean dosage: 403 mg/day) showed remarkable efficacy with a cumulative major molecular remission (MMR) rate of 79% (95% CI: 66 to 88%) at month 24. Of thirty patients refractory to previous therapy and not in MMR at baseline, 19 (64%) achieved an MMR during treatment. GI toxicity did not significantly impact on patient-reported outcomes (PRO) and led to treatment discontinuation in only one patient. Overall, the results of our trial support the efficacy and safety of bosutinib after failure of second-generation TKI pre-treatment. Trial registration: NCT02577926.

Low risk of contrast media-induced hypersensitivity reactions in all subtypes of systemic mastocytosis.

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.

Clinical and histopathological features of myeloid neoplasms with concurrent Janus kinase 2 (JAK2) V617F and KIT proto-oncogene, receptor tyrosine kinase (KIT) D816V mutations.

We report on 45 patients with myeloid neoplasms and concurrent Janus kinase 2 (JAK2) V617F and KIT proto-oncogene, receptor tyrosine kinase (KIT) D816V (JAK2pos. /KITpos. ) 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 JAK2pos. /KITpos. 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.

Proteins Marking the Sequence of Genotoxic Signaling from Irradiated Mesenchymal Stromal Cells to CD34+ Cells.

Non-targeted effects (NTE) of ionizing radiation may initiate myeloid neoplasms (MN). Here, protein mediators (I) in irradiated human mesenchymal stromal cells (MSC) as the NTE source, (II) in MSC conditioned supernatant and (III) in human bone marrow CD34+ cells undergoing genotoxic NTE were investigated. Healthy sublethal irradiated MSC showed significantly increased levels of reactive oxygen species. These cells responded by increasing intracellular abundance of proteins involved in proteasomal degradation, protein translation, cytoskeleton dynamics, nucleocytoplasmic shuttling, and those with antioxidant activity. Among the increased proteins were THY1 and GNA11/14, which are signaling proteins with hitherto unknown functions in the radiation response and NTE. In the corresponding MSC conditioned medium, the three chaperones GRP78, CALR, and PDIA3 were increased. Together with GPI, these were the only four altered proteins, which were associated with the observed genotoxic NTE. Healthy CD34+ cells cultured in MSC conditioned medium suffered from more than a six-fold increase in γH2AX focal staining, indicative for DNA double-strand breaks, as well as numerical and structural chromosomal aberrations within three days. At this stage, five proteins were altered, among them IQGAP1, HMGB1, and PA2G4, which are involved in malign development. In summary, our data provide novel insights into three sequential steps of genotoxic signaling from irradiated MSC to CD34+ cells, implicating that induced NTE might initiate the development of MN.

Adverse Prognostic Impact of the KIT D816V Transcriptional Activity in Advanced Systemic Mastocytosis.

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, R2 > 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 >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.

Separase activity distribution can be a marker of major molecular response and proliferation of CD34+ cells in TKI-treated chronic myeloid leukemia patients.

Separase, a cysteine endopeptidase, is a key player in mitotic sister chromatid separation, replication fork dynamics, and DNA repair. Aberrant expression and/or altered separase proteolytic activity are associated with aneuploidy, tumorigenesis, and disease progression. Since genomic instability and clonal evolution are hallmarks of progressing chronic myeloid leukemia (CML), we have comparatively examined separase proteolytic activity in TKI-treated chronic phase CML. Separase proteolytic activity was analyzed on single cell level in 88 clinical samples and in 14 healthy controls by a flow cytometric assay. In parallel, BCR-ABL1 gene expression and replication fork velocity were measured by qRT-PCR and DNA fiber assays, respectively. The separase activity distribution (SAD) value indicating the occurrence of MNCs with elevated separase proteolytic activity within samples was found to positively correlate with BCR-ABL1 gene expression levels and loss of MMR (relapse) throughout routine BCR-ABL1 monitoring. Analyses of CD34+ cells and MNCs fractionized by flow cytometric cell sorting according to their separase activity levels (H- and L-fractions) revealed that CD34+ cells with elevated separase activity levels (H-fractions) displayed enhanced proliferation/viability when compared with cells with regular (L-fraction) separase activity (mean 3.3-fold, p = 0.0011). BCR-ABL1 gene expression positivity prevailed in MNC H-fractions over L-fractions (42% vs. 8%, respectively). Moreover, expanding CD34+ cells of H-fractions showed decreased replication fork velocity compared with cells of L-fractions (p < 0.0001). Our data suggests an association between high separase activity, residual BCR-ABL1 gene expression, and enhanced proliferative capacity in hematopoietic cells within the leukemic niche of TKI-treated chronic phase CML.

Response to tyrosine kinase inhibitors in myeloid neoplasms associated with PCM1-JAK2, BCR-JAK2 and ETV6-ABL1 fusion genes.

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.

DNA Damage and DNA Damage Response in Chronic Myeloid Leukemia.

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.

Response and progression on midostaurin in advanced systemic mastocytosis: KIT D816V and other molecular markers.

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/Rpos 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/Rneg (n = 12) and S/A/Rpos (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.

Imatinib dose reduction in major molecular response of chronic myeloid leukemia: results from the German Chronic Myeloid Leukemia-Study IV.

Standard first-line therapy of chronic myeloid leukemia is treatment with imatinib. In the randomized German Chronic Myeloid Leukemia-Study IV, more potent BCR-ABL inhibition with 800 mg ('high-dose') imatinib accelerated achievement of a deep molecular remission. However, whether and when a de-escalation of the dose intensity under high-dose imatinib can be safely performed without increasing the risk of losing deep molecular response is unknown. To gain insights into this clinically relevant question, we analyzed the outcome of imatinib dose reductions from 800 mg to 400 mg daily in the Chronic Myeloid Leukemia-Study IV. Of the 422 patients that were randomized to the 800 mg arm, 68 reduced imatinib to 400 mg after they had achieved at least a stable major molecular response. Of these 68 patients, 61 (90%) maintained major molecular remission on imatinib at 400 mg. Five of the seven patients who lost major molecular remission on the imatinib standard dose regained major molecular remission while still on 400 mg imatinib. Only two of 68 patients had to switch to more potent kinase inhibition to regain major molecular remission. Importantly, the lengths of the intervals between imatinib high-dose treatment before and after achieving major molecular remission were associated with the probabilities of maintaining major molecular remission with the standard dose of imatinib. Taken together, the data support the view that a deep molecular remission achieved with high-dose imatinib can be safely maintained with standard dose in most patients. Study protocol registered at clinicaltrials.gov 00055874.

Incidence and prognostic impact of cytogenetic aberrations in patients with systemic mastocytosis.

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.

Discontinuation of tyrosine kinase inhibitor therapy in chronic myeloid leukaemia (EURO-SKI): a prespecified interim analysis of a prospective, multicentre, non-randomised, trial.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) have improved the survival of patients with chronic myeloid leukaemia. Many patients have deep molecular responses, a prerequisite for TKI therapy discontinuation. We aimed to define precise conditions for stopping treatment. METHODS: In this prospective, non-randomised trial, we enrolled patients with chronic myeloid leukaemia at 61 European centres in 11 countries. Eligible patients had chronic-phase chronic myeloid leukaemia, had received any TKI for at least 3 years (without treatment failure according to European LeukemiaNet [ELN] recommendations), and had a confirmed deep molecular response for at least 1 year. The primary endpoint was molecular relapse-free survival, defined by loss of major molecular response (MMR; >0·1% BCR-ABL1 on the International Scale) and assessed in all patients with at least one molecular result. Secondary endpoints were a prognostic analysis of factors affecting maintenance of MMR at 6 months in learning and validation samples and the cost impact of stopping TKI therapy. We considered loss of haematological response, progress to accelerated-phase chronic myeloid leukaemia, or blast crisis as serious adverse events. This study presents the results of the prespecified interim analysis, which was done after the 6-month molecular relapse-free survival status was known for 200 patients. The study is ongoing and is registered with ClinicalTrials.gov, number NCT01596114. FINDINGS: Between May 30, 2012, and Dec 3, 2014, we assessed 868 patients with chronic myeloid leukaemia for eligibility, of whom 758 were enrolled. Median follow-up of the 755 patients evaluable for molecular response was 27 months (IQR 21-34). Molecular relapse-free survival for these patients was 61% (95% CI 57-64) at 6 months and 50% (46-54) at 24 months. Of these 755 patients, 371 (49%) lost MMR after TKI discontinuation, four (1%) died while in MMR for reasons unrelated to chronic myeloid leukaemia (myocardial infarction, lung cancer, renal cancer, and heart failure), and 13 (2%) restarted TKI therapy while in MMR. A further six (1%) patients died in chronic-phase chronic myeloid leukaemia after loss of MMR and re-initiation of TKI therapy for reasons unrelated to chronic myeloid leukaemia, and two (<1%) patients lost MMR despite restarting TKI therapy. In the prognostic analysis in 405 patients who received imatinib as first-line treatment (learning sample), longer treatment duration (odds ratio [OR] per year 1·14 [95% CI 1·05-1·23]; p=0·0010) and longer deep molecular response durations (1·13 [1·04-1·23]; p=0·0032) were associated with increasing probability of MMR maintenance at 6 months. The OR for deep molecular response duration was replicated in the validation sample consisting of 171 patients treated with any TKI as first-line treatment, although the association was not significant (1·13 [0·98-1·29]; p=0·08). TKI discontinuation was associated with substantial cost savings (an estimated €22 million). No serious adverse events were reported. INTERPRETATION: Patients with chronic myeloid leukaemia who have achieved deep molecular responses have good molecular relapse-free survival. Such patients should be considered for TKI discontinuation, particularly those who have been in deep molecular response for a long time. Stopping treatment could spare patients from treatment-induced side-effects and reduce health expenditure. FUNDING: ELN Foundation and France National Cancer Institute.

Mutational hierarchies in myelodysplastic syndromes dynamically adapt and evolve upon therapy response and failure.

Clonal evolution is believed to be a main driver for progression of various types of cancer and implicated in facilitating resistance to drugs. However, the hierarchical organization of malignant clones in the hematopoiesis of myelodysplastic syndromes (MDS) and its impact on response to drug therapy remain poorly understood. Using high-throughput sequencing of patient and xenografted cells, we evaluated the intratumoral heterogeneity (n= 54) and reconstructed mutational trajectories (n = 39) in patients suffering from MDS (n = 52) and chronic myelomonocytic leukemia-1 (n = 2). We identified linear and also branching evolution paths and confirmed on a patient-specific level that somatic mutations in epigenetic regulators and RNA splicing genes frequently constitute isolated disease-initiating events. Using high-throughput exome- and/or deep-sequencing, we analyzed 103 chronologically acquired samples from 22 patients covering a cumulative observation time of 75 years MDS disease progression. Our data revealed highly dynamic shaping of complex oligoclonal architectures, specifically upon treatment with lenalidomide and other drugs. Despite initial clinical response to treatment, patients' marrow persistently remained clonal with rapid outgrowth of founder-, sub-, or even fully independent clones, indicating an increased dynamic rate of clonal turnover. The emergence and disappearance of specific clones frequently correlated with changes of clinical parameters, highlighting their distinct and far-reaching functional properties. Intriguingly, increasingly complex mutational trajectories are frequently accompanied by clinical progression during the course of disease. These data substantiate a need for regular broad molecular monitoring to guide clinical treatment decisions in MDS.

Accurate quantification of chromosomal lesions via short tandem repeat analysis using minimal amounts of DNA.

BACKGROUND: Cytogenetic aberrations such as deletion of chromosome 5q (del(5q)) represent key elements in routine clinical diagnostics of haematological malignancies. Currently established methods such as metaphase cytogenetics, FISH or array-based approaches have limitations due to their dependency on viable cells, high costs or semi-quantitative nature. Importantly, they cannot be used on low abundance DNA. We therefore aimed to establish a robust and quantitative technique that overcomes these shortcomings. METHODS: For precise determination of del(5q) cell fractions, we developed an inexpensive multiplex-PCR assay requiring only nanograms of DNA that simultaneously measures allelic imbalances of 12 independent short tandem repeat markers. RESULTS: Application of this method to n=1142 samples from n=260 individuals revealed strong intermarker concordance (R²=0.77-0.97) and reproducibility (mean SD: 1.7%). Notably, the assay showed accurate quantification via standard curve assessment (R²>0.99) and high concordance with paired FISH measurements (R²=0.92) even with subnanogram amounts of DNA. Moreover, cytogenetic response was reliably confirmed in del(5q) patients with myelodysplastic syndromes treated with lenalidomide. While the assay demonstrated good diagnostic accuracy in receiver operating characteristic analysis (area under the curve: 0.97), we further observed robust correlation between bone marrow and peripheral blood samples (R²=0.79), suggesting its potential suitability for less-invasive clonal monitoring. CONCLUSIONS: In conclusion, we present an adaptable tool for quantification of chromosomal aberrations, particularly in problematic samples, which should be easily applicable to further tumour entities.

Impact of comorbidities on overall survival in patients with chronic myeloid leukemia: results of the randomized CML study IV.

We studied the influence of comorbidities on remission rate and overall survival (OS) in patients with chronic myeloid leukemia (CML). Participants of the CML Study IV, a randomized 5-arm trial designed to optimize imatinib therapy, were analyzed for comorbidities at diagnosis using the Charlson Comorbidity Index (CCI); 511 indexed comorbidities were reported in 1519 CML patients. Age was an additional risk factor in 863 patients. Resulting CCI scores were as follows: CCI 2, n = 589; CCI 3 or 4, n = 599; CCI 5 or 6, n = 229; and CCI ≥ 7, n = 102. No differences in cumulative incidences of accelerated phase, blast crisis, or remission rates were observed between patients in the different CCI groups. Higher CCI was significantly associated with lower OS probabilities. The 8-year OS probabilities were 93.6%, 89.4%, 77.6%, and 46.4% for patients with CCI 2, 3 to 4, 5 to 6, and ≥7, respectively. In multivariate analysis, CCI was the most powerful predictor of OS, which was still valid after removal of its age-related components. Comorbidities have no impact on treatment success but do have a negative effect on OS, indicating that survival of patients with CML is determined more by comorbidities than by CML itself. OS may therefore be inappropriate as an outcome measure for specific CML treatments. The trial was registered at www.clinicaltrials.gov as #NCT00055874.

The clinical and molecular diversity of mast cell leukemia with or without associated hematologic neoplasm.

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/Rpos) 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/Rpos 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/Rpos 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.

Imatinib in myeloid/lymphoid neoplasms with eosinophilia and rearrangement of PDGFRB in chronic or blast phase.

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 ≥1.5×109/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,

Impact of centralized evaluation of bone marrow histology in systemic mastocytosis.

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.

Diagnostic challenges in the work up of hypereosinophilia: pitfalls in bone marrow core biopsy interpretation.

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.