SummaryOverexpression of the transcription factor interferon regulatory factor-4 (IRF4), which is common in multiple myeloma (MM), is associated with poor prognosis. Patients with higher IRF4 expression have significantly poorer overall survival than those with low IRF4 expression. Lenalidomide is an IMiD Ò immunomodulatory compound that has both tumouricidal and immunomodulatory activity in MM. This study showed that lenalidomide downregulated IRF4 levels in MM cell lines and bone marrow samples within 8 h of drug exposure. This was associated with a decrease in MYC levels, as well as an initial G1 cell cycle arrest, decreased cell proliferation, and cell death by day 5 of treatment. In eight MM cell lines, high IRF4 levels correlated with increased lenalidomide sensitivity. The clinical significance of this observation was investigated in 154 patients with MM. Among MM patients with high levels of IRF4 expression, treatment with lenalidomide led to a significantly longer overall survival than other therapies in a retrospective analysis. These data confirm the central role of IRF4 in MM pathogenesis; indicate that this is an important mechanism by which lenalidomide exerts its antitumour effects; and may provide a mechanistic biomarker to predict response to lenalidomide.

We investigated the pattern of lipoprotein lipase (LPL) expression in B-cell chronic lymphocytic leukemia (B-CLL) and assessed its prognostic relevance. Expression of LPL mRNA as well as protein was highly restricted to leukemic B cells. The intensity of intracellular immunoreactivity of LPL was higher in samples of patients with unmutated immunoglobulin heavychain variable region genes (IGV H ) compared to those with mutated IGV H genes. LPL mRNA levels in peripheral blood mononuclear cells (PBMNC) from 104 CLL patients differed by 1.5 orders of magnitude between cases with mutated (N ¼ 51) or unmutated (N ¼ 53) IGV H (median: 1.33 vs 45.22 compared to normal PBMNC). LPL expression correlated strongly with IGV H mutational status (R ¼ 0.614; Po0.0001). High LPL expression predicted unmutated IGV H status with an odds ratio of 25.90 (Po0.0001) and discriminated between mutated and unmutated cases in 87 of 104 patients (84%). LPL expression was higher in patients with poor risk cytogenetics. High LPL expression was associated with a shorter treatment-free survival (median 40 vs 96 months, P ¼ 0.001) and a trend for a shorter median overall survival (105 months vs not reached). Our data establish LPL as a prognostic marker and suggest functional consequences of LPL overexpression in patients with B-CLL.

SummaryCereblon (CRBN) has recently been identified as a target for immunomodulatory drugs (IMiDs) and its downregulation has been linked to resistance to lenalidomide. Here, we studied CRBN expression by real time polymerase chain reaction in 49 bone marrow samples of newly diagnosed patients with multiple myeloma treated with lenalidomide and dexamethasone. Median CRBN expression was 3Á45 in patients who achieved complete response, and 3Á75, 2Á01, 0Á78, and 0Á70 in those with very good partial response, partial response, stable disease and progressive disease respectively. CRBN expression levels correlated significantly with response to lenalidomide treatment (r = 0Á48; P < 0Á001). Among established prognostic parameters, only beta-2-microglobulin correlated with cereblon (r = 0Á66; P < 0Á001). A close association of CRBN with interferon regulatory factor 4 (IRF4) (P < 0Á001) and with CTNNB1 (P < 0Á001) was found. Overall, a statistically significant association between baseline CRBN expression and response in MM patients treated with lenalidomide is shown. CRBN expression is closely associated with IRF4, which is an important target of IMiD therapy.

We thank the patients and their families for their trust in taking part in this study. The study was academically funded and supported by the Medical University Vienna, the General Hospital Vienna, and the Research Center for Molecular Medicine (CeMM) of the Austrian Academy of Sciences. We gratefully acknowledge funding from the Vienna Science and Technology Fund (LS16-034 to GSF and UJ), the Austrian Science Fund (F4704-B20 to PV, F4711-B20 to GSF, and P27132-B20 to PBS), and the European Molecular Biology Organization Long Term Fellowship (1543-2012 to GIV, 733-2016 to TP). BS acknowledges

The online version of this article has a Supplementary Appendix. BackgroundTreatment of follicular lymphoma with rituximab is currently recommended at a dose of 375 mg/m 2 . We aimed to provide a rationale for optimal dosing and scheduling of this anti-CD20 antibody based on pharmacokinetics. Design and MethodsClinical efficacy of immuno-chemotherapy with rituximab, fludarabine and mitoxantrone followed by 2-monthly rituximab maintenance was evaluated in 29 patients with previously untreated follicular lymphoma in a prospective phase II trial (AGMT-NHL9). Pharmacokinetic analysis was assessed in 17 patients. ResultsInduction treatment resulted in high clinical response rates (complete remission 66%; ORR 100%). Significantly higher complete remission rates were observed in female patients (86 vs. 47%; Odds Ratio 6.8, 95% CI: 1.12; 41.82; P=0.05). Rituximab pharmacokinetic analysis showed a high variability ranging over almost 1 order of magnitude at maintenance cycle 1 (area under the curve 1,540-12,025 g/L*days). Median area under the curve was lower in men (81%) and in patients with initial bone marrow infiltration (76%). Higher rituximab serum concentrations before next therapy (Ctrough) were associated with female sex (P=0.04) as well as with absence of initial bone marrow infiltration (P=0.001). Ctrough correlated with remission quality (complete vs. partial remission; P=0.005) and progression-free survival (P=0.03). A decline in rituximab Ctrough below 25,000 ng/mL was observed 9.5 to 62 months before clinical relapse (P=0.008). ConclusionsThe results of this pilot trial suggest that more differentiated dosing schedules based on gender and bone marrow infiltration should be explored for rituximab therapy for lymphoma. This study was registered in ClinicalTrials.gov (Identifier: NCT01560117). ABSTRACT © F e r r a t a S t o r t i F o u n d a t i o n © F e r r a t a S t o r t i F o u n d a t i o n

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Activation-induced cytidine deaminase (AID) is essential for somatic hypermutation of B-cells. We investigated the expression of AID mRNA by real-time polymerase chain reaction (PCR) in peripheral blood mononuclear cells of 80 patients with B-CLL. AID expression was detected in 45 of 80 patients (56%) at various levels, but was undetectable in 35 patients (44%). AID PCR positivity was associated with unmutated IGV H gene status (22 of 25 patients; P ¼ 0.002) and unfavourable cytogenetics (18 of 23 patients with deletion in 11q or loss of p53; P ¼ 0.040). Using a threshold level of 0.01-fold expression compared to Ramos control cells, even more significant associations were observed (P ¼ 0.001 for IGV H ; P ¼ 0.002 for cytogenetics). A correlation was observed between individual AID levels and the percentage of V H homology (R ¼ 0.41; P ¼ 0.001). AID positivity predicted unmutated IGV H status with an odds ratio of 8.31 (P ¼ 0.003) and poor risk cytogenetics with an odds ratio of 3.46 (P ¼ 0.032). Significance was retained after adjustment for Binet or Rai stages. AID mRNA levels were stable over time. These data suggest a potential role of AID as a prognostic marker in B-CLL.

These data suggest a high prognostic value of FLT3/ITD in patients with normal cytogenetics. However, we find no evidence that FLT3/ITD is a predictive marker for patients with t(15;17).