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ZUSAMMENFASSUNGDie aktuelle COVID-19-Pandemie stellte auch die medizinischen Labore vor große Herausforderungen. Der prinzipiell sehr frühen Kenntnis des primär neuen Erregers und der prinzipiellen Möglichkeit des Nachweises des Virus stand oft eine zunächst unzureichende Verfügbarkeit an Reagenzien und Analysensystemen gegenüber, die aber relativ schnell verbessert werden konnten, sodass nun eine flächendeckende und schnelle Diagnostik dieses Erregers möglich ist. Neben der Erregeridentifizierung liefert die Labordiagnostik jedoch auch wichtige Hinweise bei der Beurteilung der Erkrankung bzw. der Abschätzung des weiteren Verlaufes. Die Wertigkeit einzelner Parameter bzw. Parameterkonstellationen wurde dabei teilweise erst im Verlauf der Pandemie deutlich bzw. ist auch noch in der Evaluierung.

Sequential genotyping for phenotype-driver mutations in JAK2 (exon 14), CALR (exon 9), and MPL (exon 10) is recommended in patients with myeloproliferative neoplasms. Yet, atypical JAK2- and MPL-mutations were described in some triple-negative patients. Whether noncanonical and/or concomitant JAK2- and MPL-mutations exist in myelofibrosis (MF) regardless of phenotype-driver mutations is not yet elucidated. For this, next-generation sequencing (NGS) was performed using blood genomic DNA from 128 MF patients (primary MF, n = 93; post-ET-MF, n = 18; post-PV-MF, n = 17). While no atypical JAK2- or MPL-mutations were seen in 24 CALR-positive samples, two JAK2-mutations [c.3323A > G, p.N1108S; c.3188G > A, p.R1063H] were detected in two of the 21 (9.5%) triple-negative patients. Twelve of the 82 (14.6%) JAK2V617F-positive cases had coexisting germline JAK2-mutations [JAK2R1063H, n = 6; JAK2R893T, n = 1; JAK2T525A, n = 1] or at least one somatic MPL-mutation [MPLY591D, n = 3; MPLW515 L, n = 2; MPLE335K, n = 1]. Overall, MPL-mutations always coexisted with JAK2V617F and/or other MPL-mutations. None of the JAK2V617F plus a second JAK2-mutation carried a TET2-mutation but all patients with JAK2V617F plus an MPL-mutation harbored a somatic TET2-mutation. Four genomic clusters could be identified in the JAK2V617F-positive cohort. Cluster-I (10%) (noncanonical JAK2mutated (mut) + TET2wildtype (wt) ) were younger and had less proliferative disease compared with cluster-IV (5%) (TET2mut + MPLmut ). In conclusion, recurrent concomitant classical and/or noncanonical JAK2- and MPL-mutations could be detected by NGS in 15.7% of JAK2V617F- and MPLW515-positive MF patients with genotype-phenotype associations. Many of the germline and/or somatic mutations might act as "Significantly Mutated Genes" contributing to the pathogenesis and phenotypic heterogeneity. A cost-effective NGS-based approach might be an important step towards patient-tailored medicine.

BackgroundRapid diagnostic testing for SARS-Cov-2 antigens is used to combat the ongoing pandemic. In this study we aimed to compare two RDTs, the SD Biosensor Q SARS-CoV-2 Rapid Antigen Test (Roche) and the Panbio COVID-19 Ag Rapid Test (Abbott), against rRT-PCR.MethodsWe included 2,215 all-comers at a diagnostic center between February 1 and March 31, 2021. rRT-PCR-positive samples were examined for SARS-CoV-2 variants.FindingsThree hundred and thirty eight participants (15%) were rRT-PCR-positive for SARS-CoV-2. The sensitivities of Roche-RDT and Abbott-RDT were 60.4 and 56.8% (P < 0.0001) and specificities 99.7% and 99.8% (P = 0.076). Sensitivity inversely correlated with rRT-PCR-Ct values. The RDTs had higher sensitivities in individuals referred by treating physicians (79.5%, 78.7%) than in those referred by health departments (49.5%, 44.3%) or tested for other reasons (50%, 45.8%), in persons without any comorbidities (74.4%, 71%) compared to those with comorbidities (38.2%, 34.4%), in individuals with COVID-19 symptoms (75.2%, 74.3%) compared to those without (31.9%, 23.3%), and in the absence of SARS-CoV-2 variants (87.7%, 84%) compared to Alpha variant carriers (77.1%, 72.3%). If 10,000 symptomatic individuals are tested of which 500 are truly positive, the RDTs would generate 38 false-positive and 124 false-negative results. If 10,000 asymptomatic individuals are tested, including 50 true positives, 18 false-positives and 34 false-negatives would be generated.InterpretationThe sensitivities of the two RDTs for asymptomatic SARS-CoV-2 carriers are unsatisfactory. Their widespread use may not be effective in the ongoing SARS-CoV-2 pandemic. The virus genotype influences the sensitivity of the two RDTs. RDTs should be evaluated for different SARS-CoV-2 variants.

BackgroundWidely available rapid testing is pivotal to the fight against COVID-19. Real-time reverse transcription-polymerase chain reaction (rRT-PCR) remains the gold standard. We compared two frequently used commercial rapid diagnostic tests (RDTs) for SARS-CoV-2-antigens, the SD Biosensor SARS-CoV-2 Rapid Antigen Test (Roche Diagnostics) and the Panbio COVID-19 Ag Rapid Test (Abbott Diagnostics), against rRT-PCR for SARS-CoV-2 detection.MethodsWe compared the tests in 2215 all-comers at a diagnostic centre between February 1 and March 31, 2021. rRT-PCR-positive samples were examined for SARS-CoV-2 variants.Findings338 participants (15%) were rRT-PCR-positive for SARS-CoV-2. The sensitivities of Roche-RDT and Abbott-RDT were 60.4% and 56.8% (PInterpretationThe sensitivities of the two RDTs are unsatisfactory. This calls into question whether their widespread use is effective in the ongoing SARS-CoV-2 pandemic.FundingSYNLAB Holding Deutschland GmbHResearch in contextEvidence before this studySmall studies and a meta-analysis from the Cochrance collaboration indicate vastly different diagnostic efficacies of commercial rapid diagnostic tests (RDTs) for SARS-CoV-2 antigen. The impact of SARS-CoV-2 variants has not been known.Added value of this studyThis is one of the largest real-world studies of the diagnostic efficacy of two widely recommended RDTs SARS-CoV-2 antigen in comparison to rRT-PCR. The sensitivities of the two RDTs are unsatisfactory, mainly in asymptomatic persons. Presence of the SARS-CoV-2 Alpha Variant decreased both tests’ sensitivities significantly.Implications of all the available evidencePolicy and health care providers should account for substantial limitations of RDTs for SARS-CoV-2 particular in asymptomatic persons. Research into alternative approaches to the screening for SARS-CoV-2 should be intensified.

The effect of tea polyphenols on the bioavailability of iron and the iron status of man is controversially discussed. Especially women prior to menopause are a vulnerable group regarding their iron status, with a vegetarian diet increasing this risk. This study aimed to determine several iron status parameters for men and women consuming a mixed diet or being vegetarians and drinking tea. In a cross-over study, the effect of tea consumption on the iron status of healthy male and female omnivorous compared to vegetarian volunteers was tested. All volunteers consumed either green tea or black tea (each 1. L/d) for four weeks together with meals or avoided tea for four weeks (cross-over design). Blood samples were withdrawn prior to the one week depletion period, at the start of the intervention phase and thereafter weekly during intervention. The iron status parameters hemoglobin, hematocrit, transferrin, total iron and iron binding capacity were not affected by the consumption of tea. In contrast, plasma ferritin values were significantly reduced after tea consumption, especially in those women with basal ferritin values lower than 25++g/L, already after two weeks of intervention with tea (1. L/d). -® 2011 Elsevier Ltd

Zusammenfassung Das Vorliegen eines myeloproliferativen Syndroms wird als häufigster Risikofaktor für das Auftreten einer Pfortaderthrombose oder eines Budd-Chiari Syndroms angesehen. Von weiterer Bedeutung sind angeborene oder erworbene Gerinnungsstörungen und Lebererkrankungen. Etwa 25% der Thrombosen ereignen sich ohne ei-nen Hinweis auf eine mögliche Ätiologie. Bei einigen dieser Patienten finden sich jedoch zumindest im Knochenmarkbefund Hinweise auf das Vorliegen eines bcr-abl-negativen proliferativen Syndroms. Die im Jahre 2005 beschriebene V617F-Mutation im Gen der Januskinase 2 konnte die Diagnostik für einen Teil der myeloproliferativen Erkrankungen deutlich verbessern und das Verständnis für die pathobiochemischen und pathophysiologischen Prozesse bei myeloproliferativen Erkrankungen erweitern. Die nachfolgenden retrospektiven Studien bei Patienten mit einem Budd-Chiari Syndrom oder einer Pfortaderthrombose konnten zeigen, dass bei Patienten, deren Thrombosen bisher als idiopathisch eingestuft worden waren, zu einem hohen Prozentsatz eine V617F Mutation vorlag und dass ein Teil dieser Patienten nach dem thrombotischen Ereignis ein myeloproliferatives Syndrom entwickelte.

Cardiac remodeling after myocardial infarction is in part triggered and maintained by reactive oxygen species. Antioxidants such as probucol have shown positive short-term effects on these cardiac interstitial changes in different experimental models after intraperitoneal administration or after per-oral administration with a long pre-treatment period or in high doses. In this study, the long-term effects on mortality and cardiac remodeling were examined after induction of a large myocardial infarction in a clinical daily-life-like setting. Male Lewis rats were randomized to the study groups. Large anterolateral myocardial infarctions were induced or sham operations performed. The oral treatment was started after 48 h either with probucol or placebo after myocardial infarction and with placebo after sham operation. Induction of large myocardial infarctions led to changes of the left ventricular stiffness constants, a dilatation of the left ventricle and an increased interstitial fibrosis in the remote non-infarcted part. These changes were in tendency, but not significantly, reversed after treatment with probucol. The 6-month survival rates were 53.1% in the group probucol-myocardial infarction, 43.2% in the group placebo-myocardial infarction and 100% in the group after sham operation. There were no significant differences at Kaplan-Meier analysis between the groups after myocardial infarctions. Oral treatment with the antioxidant probucol started after myocardial infarction in a pharmacological dose does not have favourable effects on the long-term mortality in the chronic ischemic heart failure model in the rat.

Objective: Efficacy of in vivo isolated lung perfusion (ILP) with cisplatin could be shown in different rodent tumor models. Despite the use of this alternative therapeutical strategy in very few patients with lung metastases, there are no systematic studies regarding the tolerance of the native lung tissue in large animal models or humans. Methods: In a novel ILP pig model, groups with two different concentrations of cisplatin (group CP150: 150 mg/m 2 cisplatin, n ¼ 5; group CP300: 300 mg/m 2 cisplatin, n ¼ 5) were compared with a control group (n ¼ 5) and a Sham group (n ¼ 5) concerning the influence on hemodynamic, ventilatory and gas exchange parameters as well as on structural integrity of the lung. In the additional CP300-HT group the potentially cumulative effect of hyperthermia and high-dose cisplatin perfusion was evaluated (300 mg/m 2 cisplatin, 41.5 8C, n ¼ 5). Following the ILP of the left lung for 40 min, right main bronchus and right pulmonary arteries were clamped and survival as well as lung function parameters were dependent on the previously perfused lung for the 6-hreperfusion period. Quantification of histological acute lung injury was performed using the score of Chiang. ANOVA, ANOVA with repeated measures and Pearson’s correlation estimation were applied for statistical evaluation. Results: All animals survived ILP and the entire reperfusion period. Platinum levels of the perfusate and lung tissue showed a significant correlation with the dose given ðP , 0:001Þ but no correlation with the very low plasma levels in all groups ðP ¼ 0:825Þ: ILP resulted in a slight deterioration of most functional parameters compared to the Sham group. Although there were no differences between the perfusion groups regarding hemodynamic and ventilatory parameters, gas exchange parameters (pO2/FiO2-index, pCO2, AADO2) demonstrated a trend toward dose-related functional impairment. Histological evaluation confirmed a dose-depending damage of lung tissue (P , 0:001; correlation coefficient 0.670). The hyperthermic ILP with high-dose cisplatin led to improved gas exchange parameters and a reduction of morphological lung damage. Conclusions: In vivo ILP with high-dose cisplatin represents a safe procedure in this pig model. Hyperthermic perfusion up to 41.5 8C was beneficial to reduce the acute lung injury. The promising results of this study might be used for initiation of clinical trials as an alternative treatment in patients with a very poor prognosis. q 2004 Elsevier B.V. All rights reserved.

The minimally invasive elimination of tumours using heating as a therapeutic agent is an emerging technology in medical applications. Particularly, the intratumoural application of magnetic nanoparticles as potential heating sources when exposed to an alternating magnetic field has been demonstrated. The present work deals with the estimation of the basic relationships when the magnetic material has access and binds to structures on cell membranes of target cells at the tumour region, particularly as a consequence of administration through tumour supplying vessels. Therefore, using mouse endothelial cells in culture, the binding of dextran coated magnetic nanoparticles (mean hydrodynamic particle diameter 65 nm) was modelled using the periodate method. The efficacy of cell labelling was demonstrated by magnetorelaxometry (MRX)—a selective method for the detection of only those magnetic nanoparticles that were immobilized—as well as by electron microscopy and iron staining. The amount of iron immobilized on cells was found to be 153 ± 56 µg Fe per 1 × 107 cells as determined by atomic absorption spectrometry. Moreover, after exposure of those 1 × 107 labelled cells to an alternating magnetic field (frequency 410 kHz, amplitude 11 kA m−1) for 5 min, temperature increases of 2 °C were achieved. The consequences of particle immobilization are reflected by the results of the measurements related to the specific heating power (SHP) of the magnetic material. Basically, the heating potential is explained by the superposition of Brown and Neel relaxation while for immobilized nanoparticles the Brown contribution is absent. In the long term the data could open the door to targeted magnetic heating after further optimization of the heating potential of magnetic material as well as after functionalization with biomolecules which recognize specific structures on the surface of cells at the target region.

Abstract Cholesterol ester storage disease (CESD) is associated with premature atherosclerosis, hepatomegaly, elevated LDL cholesterol levels, and in most cases, low HDL cholesterol levels. Previous studies have shown a G→A mutation at the 3′ splice junction of exon 8 (E8SJM) of the gene encoding lysosomal acid lipase (LAL) in two kindreds with CESD. In a Canadian-Norwegian kindred with this disease, we show this mutation in conjunction with an as yet unknown T→C transition in exon 10 predicting a Leu 336 →Pro (L336P) replacement and an A→C transversion in exon 2 predicting a T-6P replacement in the prepeptide. Identification of the L336P rather than the T-6P replacement as the second defect underlying CESD in our patient is deduced from three lines of evidence. First, the E8SJM allele is located in cis with the mutation predicting the T-6P–encoding allele but in trans with the L336P-encoding allele; second, the L336P but not the T-6P replacement cosegregates with low LAL activity in the family; third, the T-6P replacement was found in 6 of 28 alleles from subjects with normal lysosomal acid lipase activity, suggesting that this variant represents a frequent nonfunctional polymorphism. Since the residual LAL activity is higher and the clinical phenotype based on plasma lipid values and severity of hepatosplenomegaly is milder in this case than in a previously studied case who was homozygous for the E8SJM allele, we conclude that the L336P variant appears to be associated with a phenotypically mild form of CESD.

Background Splanchnic vein thrombosis (SVT) is a typical manifestation of polycythaemia vera (PV) or essential thrombocythaemia (ET). The recently discovered JAK2V617F somatic mutation is closely associated with chronic myeloproliferative disease (CMD). We investigated whether thrombosis involving the inferior vena cava (IVC) is also related to the JAK2V617F mutation or CMD. Methods Blood samples were obtained from 40 IVC thrombosis patients. Fifty-three patients with isolated lower extremity deep vein thrombosis (LE-DVT) and 20 SVT patients served as controls. The presence of the JAK2V617F mutation was assessed by real-time polymerase chain reaction (RT-PCR). Results The JAK2V617F allele was not detected in any of the IVC thrombosis patients but was detected in one patient (2%) with isolated LE-DVT. However, the mutation-carrying patient did not exhibit symptoms of CMD. Even after an observation period of 30 months, the patient's complete blood cell count did not exhibit any pathology. In contrast, the JAK2V617F allele was detected in four patients with SVT (20%) and CMD. Conclusion According to our data, there is no evidence that IVC thrombosis is associated with the JAK2V617F mutation or the presence of chronic myeloproliferative disease.

> A myeloproliferative disorder is the main risk factor for portal venous thrombosis or Budd-Chiari syndrome. Other significant factors are hereditary or acquired prothrombotic states and different liver diseases. Approximately 25% of thromboses occur without an indication of possible etiology. However, some patients show signs of myeloproliferative disorders on bone marrow morphology. The identification of the Janus kinase V617F mutation, which was first described in 2005, allows new insights into both the pathobiochemistry and pathophysiology of this disease, and a substantial improvement of the diagnostics at least to some of the myeloproliferative disorders. A retrospective study of clinical trials of patients with Budd-Chiari syndrome or portal venous thrombosis showed that patients with thrombosis predefined as idiopathic carried the V617F mutation at a high percentage rate and, furthermore, that a part of this patients group developed a myeloproliferative disorder after the thrombotic event.

Allogeneic HCT is curative in myelofibrosis (MF) with a survival (OS) advantage compared to non-JAK-inhibitor treatments in patients (pts) with Int-2/high-risk DIPSS (Kroeger et al, Blood 2015). On the other hand, data demonstrate a OS benefit of the JAK1/JAK2 inhibitor ruxolitinib (RUX) in intermediate (Int)-2/high-risk IPSS (Cervantes et al, Blood 2013; Verstovsek et al, Haematologica 2015). Yet, comparative data to the impact of RUX and HCT on OS are lacking. This was pursued retrospectively at the University of Leipzig in light of clinical-, cytogenetic-, phenotype-driver-, and somatic mutations-based prognostic variables. Patient and Methods: RUX became available in November, 2009. MF pts (n=137; median age 58y) seen afterwards were included with the exception of MPL + pts (small number). Characteristics are shown in table 1. RUX was given for spleenomegaly and/or constitutional symptoms [n=76 (55%)]. Irrespective of RUX, a donor search was initiated in int-2 and high-risk IPSS pts < 70 y and HCT performed if a suitable donor identified. Based on this biologic randomization, 50 (37%) received HCT, 58 (42%) continued with RUX (median exposure 19 months), and 29 (21%) pts had other options. The RUX- and HCT-groups were balanced in terms of IPSS, platelets, cytogenetic-, and epigenetic molecular-risks. Treatment was initiated within the first year (median 10 months), thus probability of OS 5 years after diagnosis was calculated (SPSS20 software). Cytogenetic-risk was conducted according to Caramazza et al. Leukemia 2011. Somatic mutations detected by NGS were used to calculate molecular-risk (Vannucchi et al. Leukemia 2013). Results: Median OS time was 6.8 y. The RUX-group was older (median age 64 y) with profound spleenomegaly and a higher JAK2 V617F allele burden compared to the HCT group (median age 55 y) (p50 y (p=0.001). In the RUX group, age 60 y (p=0.02)]. No prognostic implication of anemia, WBC, peripheral blasts, and constitutional symptoms could be detected with both treatments. OS in JAK2 + pts was similar to CALR + pts in both therapy cohorts (p=0.3). Triple negativity retained its detrimental influence (p Conclusions: Our data imply that, besides allogeneic HCT, a prolonged JAK1/JAK2 inhibition could, at least partly, attenuate most of the prognostic detrimental parameters. Pre-treatment with ruxolitinib prior to HCT does not seem to have a negative impact on survival. It is the first time where outcome with a non-transplant procedure is shown to be comparable to that after HCT in MF, even in high-risk disease. This needs to be verified in prospective randomized trials to define the role of allogeneic HCT in the era of JAK1/JAK2 inhibition. Table 1. Patients characteristics ( n = 137 ) Variable Impact on OS Impact on LT Median age (years) 58 0.002 0.6 JAK2+/CALR+/Double negative (%) 63 / 20 / 17 Disclosures Roskos: Novartis: Honoraria. Al-Ali:Celgene: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

A frequent missense mutation (Thr-6Pro) found in the prepeptide of the lysosomal acid lipase (LAL) gene was analyzed in a cohort of 1003 randomly selected samples from Germany, Japan and Sardinia (Italy). Using the mutagenically separated polymerase chain reaction (MS-PCR), allele frequencies of 0.269, 0.238 and 0.245 were determined in the three populations, respectively. Statistical analysis showed a lack of association with a dyslipidemic phenotype in all three groups. Additionally, in a subgroup of 126 German individuals no association was observed between genotype and LAL activity. We conclude that this mutation appears to be a frequent LAL gene polymorphism causing no impaired function of the enzyme and no measurable dyslipidemia in the general population.

Efficacy of in vivo isolated lung perfusion (ILP) with cisplatin could be shown in different rodent tumor models. Despite the use of this alternative therapeutical strategy in very few patients with lung metastases, there are no systematic studies regarding the tolerance of the native lung tissue in large animal models or humans.In a novel ILP pig model, groups with two different concentrations of cisplatin (group CP150: 150 mg/m(2) cisplatin, n=5; group CP300: 300 mg/m(2) cisplatin, n=5) were compared with a control group (n=5) and a Sham group (n=5) concerning the influence on hemodynamic, ventilatory and gas exchange parameters as well as on structural integrity of the lung. In the additional CP300-HT group the potentially cumulative effect of hyperthermia and high-dose cisplatin perfusion was evaluated (300 mg/m(2) cisplatin, 41.5 degrees C, n=5). Following the ILP of the left lung for 40 min, right main bronchus and right pulmonary arteries were clamped and survival as well as lung function parameters were dependent on the previously perfused lung for the 6-h-reperfusion period. Quantification of histological acute lung injury was performed using the score of Chiang. ANOVA, ANOVA with repeated measures and Pearson's correlation estimation were applied for statistical evaluation.All animals survived ILP and the entire reperfusion period. Platinum levels of the perfusate and lung tissue showed a significant correlation with the dose given (P0.001) but no correlation with the very low plasma levels in all groups (P=0.825). ILP resulted in a slight deterioration of most functional parameters compared to the Sham group. Although there were no differences between the perfusion groups regarding hemodynamic and ventilatory parameters, gas exchange parameters (pO(2)/FiO(2)-index, pCO(2), AADO(2)) demonstrated a trend toward dose-related functional impairment. Histological evaluation confirmed a dose-depending damage of lung tissue (P0.001, correlation coefficient 0.670). The hyperthermic ILP with high-dose cisplatin led to improved gas exchange parameters and a reduction of morphological lung damage.In vivo ILP with high-dose cisplatin represents a safe procedure in this pig model. Hyperthermic perfusion up to 41.5 degrees C was beneficial to reduce the acute lung injury. The promising results of this study might be used for initiation of clinical trials as an alternative treatment in patients with a very poor prognosis.

Several clinical trials have demonstrated that the three-drug combination of oxaliplatin, 5-fluorouracil (5-FU) and leucovorin (LV) administered chronomodulated improved antitumour efficacy in the treatment of metastatic colorectal cancer and was better tolerated than constant-rate infusion. However, only a few pharmacokinetic data of 5-FU during chronomodulated infusion are available but up to now not for oxaliplatin. In this pilot study, the platinum levels of plasma ultrafiltrate (PUF) and total plasma were monitored during chronomodulated infusion of oxaliplatin, 5-FU and LV in 7 patients with metastatic gastrointestinal cancer. A cycle of the 4-day chemotherapeutic regimen consisted of 12-h infusions with sinusoidal drug delivery rate of: oxaliplatin (25 mg/m2/d, peak at 16:00 hours), 5-FU and LV (750 mg/m2/d and 150 mg/m2/d, respectively, peak at 4:00 hours), the same scheme was reinitiated on day 15. Blood samples were collected on day 1 and day 4 during different cycles. Concentration-time profiles of ultrafilterable and total platinum in plasma during chronomodulated infusion were characterised. As expected, we found residual platinum levels in total plasma but not in PUF prior next cycle. Comparing day 1 with day 4, Cmax of platinum in PUF was significantly increased (84 +/- 13 ng/ml vs. 131 +/- 22 ng/ml, P = 0.012) as well as AUC0-24h of platinum in PUF (0.97 +/- 0.29 microg x h/ml vs. 1.90 +/- 0.44 microg x h/ml, P = 0.018). The same effect was observed for total plasma platinum suggesting an accumulation within the cycle. The observed interindividual variability of Cmax, tmax, AUC0-24h, t1/2 was moderate. Because of the small sample size in this pilot investigation, the findings need to be confirmed in larger pharmacokinetic studies. In a next step individual pharmacokinetic parameters should be associated with patient specific parameters and treatment-induced toxicity.

In retrospective studies, CALR mutation is found to be a favorable prognostic variable in myelofibrosis (MF) compared with JAK2, or MPL mutations. With the availability of the JAK1/JAK2 inhibitor ruxolitinib (RUX) for treatment of MF, it is not yet known whether response varies across mutational subgroups and whether the prognostic implication of the driver mutations in terms of survival (OS) changes under RUX. We studied the prevalence and prognostic weight of clinical parameters in the IPSS and DIPSS-plus and the impact of RUX on OS in the context of mutation status. Patient and Methods: As of November 2009, RUX for treatment of splenomegaly and/or constitutional symptoms became an option at the University of Leipzig. All patients (pt) with MF seen since then were included (n=127; f=43%, median age 58 years) with the exception of pt with MPL mut+ because of small number. Screening for the JAK2V617F and CALR mutations was performed as previously published. Cytogenetic-risk categorization was conducted as published (Caramazza et al. Leukemia 2011). Results: JAK2 mut+ (group A; 60.6%) constituted the largest group, followed by CALR mut+ (group B; 19.7%), and “triple-negative” (group C; 19.7%). The median duration of MF was 22 months with no difference in the 3 groups. Higher-risk IPSS was present in the majority [Int-II 34.4%; high-risk 42.4%]. Constitutional symptoms were present across all groups, although more frequent in group A which was also characterized by prominent splenomegaly (Table). Group B pt were younger with low WBC in contrast to goup C pt who were older, anemic, with high WBC and low platelets, unfavorable cytogenetics and high-risk IPSS. Hb < 100 g/L (54%), and transfusion-dependency (32%) were frequently present in group B and was similar to group A. RUX was given to 72 (57%) pt [graoup A (66%), group B (56%), group C (29%)] with a median exposure of 8 months. The response of 80% was not influenced by the mutation status, cytogenetics, or IPSS variables. Leukemic transformation was documented in 22 pt [14 (64%) pt had no RUX exposure] after a median of 18 months after diagnosis. The incidence was highest in group C (36%) and lowest in group B (4%) (p=0.004). Unfavorable cytogenetics were 75%, and 27% in group C and A respectively. After a median follow-up of 31 months, OS at 3-years was 82% and worst in group C (72%) vs 80% for group A (p=0.05) vs 96% in goup B (p=0.003). In univariate analysis, non-mutated status (p=0.02), Int-2/high-risk IPSS (p=0.06), anemia (p=0.03), transfusion dependency (p=0.04), and platelets < 100 x109/L (p=0.06) but not unfavorable cytogenetics were associated with an inferior OS. In multivariate analysis, advanced IPSS only (p=0.04) but not the mutation status (p=0.4) retained its negative impact on OS. Generally, in group A pt treated with RUX (n=50), OS was only marginally inferior to group B (p=0.08). In pt with int-2/high-risk IPSS, OS in group A with RUX (n=41) was similar to that of group B (p=0.4). Conclusions: The mutation status in MF bestows distinct clinical phenotypes and has crucial prognostic and therapeutic implications. Patients with non-mutated MF had the worst prognosis, unfavourable cytogenetics, and the highest rate of leukemic transformation. Although the IPSS retains its value, the prognostic power of certain factors such as anemia and constitutional symptoms in CALR-mutated pt needs to be re-evaluated. Response to ruxolitinib seems to be independent of the mutation status. More importantly, a JAK1/JAK2 inhibition appears to be capable of attenuating the prognostic implication of the different mutation profiles by improving the less-favorable survival associated with non-CALR-mutated MF through a disease-modifying effect. The full potential of a sustained JAK1/JAK2 inhibition in modifying survival in the context of the various mutational profiles needs to be further studied in a larger cohort of patients. Table Clinical variables in patients with different driver mutations Variable JAK2 mut+ CALR mut+ Triple-negative p N (%) 77 (60.6) 25 (19.7) 25 (19.7) Median age (years) 59 51 61 0.02 Constitutional symptoms (%) 69 52 46 0.08 Median palpable spleen (cm) 10 4 3 0.001 Int-2/high-risk IPSS (%) 79 52 96 0.01 WBC > 25 x109/L (%) 21 11.5 44 0.008 Median peripheral blasts (%) 1 0.5 2 ns Median Hb (g/L) 106 99 86 0.008 Hb < 100 g/L (%) 42 54 80 0.03 Transfusion dependency (%) 33 32 67 0.009 Platelets < 100 x109/L (%) 30 8 52 0.04 Unfavorable cytogenetics (%) 31 14 48 0.06 Disclosures Al-Ali: Novartis: Honoraria, Research Funding. Jaekel:Novartis: Honoraria. Lange:Novartis: Honoraria. Roskos:Novartis: Honoraria. Niederwieser:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Abstract 893 Introduction: A European collaborative harmonization study involving 61 laboratories is being conducted under the auspices of the European Treatment and Outcome Study (EUTOS) for CML that aims to facilitate reporting of molecular BCR-ABL quantification results according to the International Scale (IS). The aim of this analysis was to investigate the effectiveness of this process and specifically the stability of conversion factors (CF) over time. Methods: The currently accepted way of adopting the IS is to establish and validate a laboratory-specific CF which is then used to convert local results to the IS. For round 1, preliminary CFs were calculated by centrally distributing standard samples containing 10–20 million WBC approximating to 10%, 1%, 0.1%, and 0.01% BCR-ABL IS. Rounds 2 and 3 were employed to refine the CF calculations using 25–30 CML patient samples from each participating laboratories covering a range of BCR-ABL levels between 0.01% and 10%. Log BCR-ABL values for the same samples were compared between reference and local laboratories applying the Bland-Altman bias plot. In order to judge the stability of each laboratory`s methodology, a CF index (ratio of round 3 CF divided by round 2 CF) was calculated and evaluated according to its capability to achieve optimum concordance of results. Results: Of the 61 laboratories participating in round 1, evaluable patient samples have been provided to date by 56 and 30 laboratories in rounds 2 and 3, respectively. Of the 30 laboratories with complete data, 12 had stable CFs (defined as a CF index within 0.75–1.33) whereas 18 laboratories were outside this range. Comparison of the CFs derived from round 2 with those derived from round 3 revealed better and more consistent concordance between laboratories with stable CFs compared to those with unstable CFs. For the 12 stable laboratories, 79% (round 3 CF) vs 79% (round 2 CF) of the samples were within a 2-fold range (0.5–2.0) and 93% vs 89% were within a 3-fold range (0.33–3.0). For the 18 unstable laboratories, 74% vs 55% of the samples were within a 2-fold range (0.5–2.0), p=0.0005 and 92% vs 77% were within a 3-fold range (0.33–3.0), p=0.0005. 2 of 12 laboratories with stable CFs and 8 of 18 laboratories with unstable CFs indicated changes in either one or more components of their procedures (cDNA synthesis, PCR platform, RQ-PCR protocol) that may have impacted on their CFs. Conclusion: These data indicate that CFs may be unstable in some laboratories even in the absence of significant changes to laboratory protocols. Further, it supports the need for continuous revalidation of CFs. In laboratories with unstable CFs we suggest revalidation within 3 to 6 months whereas those with stable CFs should be assessed on a yearly basis. We also suggest that laboratories with unstable CFs need to rigorously examine their internal processes to identify potential sources of variation. Disclosures: Müller: Novartis: Honoraria, Research Funding. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.