Genomic profiling of a randomized trial of interferon-α vs hydroxyurea in MPN reveals mutation-specific responses.

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

Effects of live music during chemotherapy in lymphoma patients: a randomized, controlled, multi-center trial.

PURPOSE: Chemotherapy is associated with both somatic and psychological side effects. Music might ease these problems. Several randomized controlled trials have investigated the effect of music, but the results are inconclusive. We aimed to examine whether live or pre-recorded music listening decreases anxiety during chemotherapy in newly diagnosed lymphoma patients. METHODS: A total of 143 patients with non-Hodgkin and Hodgkin lymphomas were randomly assigned into three groups receiving either 30 min of patient-preferred live music (n = 47), 30 min of patient-preferred pre-recorded music (n = 47), or standard care (n = 49) during up to five outpatient chemotherapy sessions. The primary endpoint was anxiety measured by the Spielberger's State Anxiety Inventory. Secondary endpoints included blood pressure, pulse rate, nausea and vomiting, serum catecholamine levels pre- and post-intervention to measure arousal levels, and health-related quality of life. The Musical Ability Test was used to link musical ability to the primary endpoint. RESULTS: When adjusting for age, sex, diagnosis, number of sessions, and baseline anxiety, the linear mixed model showed a borderline statistically significant reduction in the primary outcome anxiety in the live music group compared to standard care (7% (95% CI, - 14% to 0%, p = 0.05), while the effect of pre-recorded music was non-significant (5% (95% CI, - 12% to + 3%, p = 0.18). No intervention effects were seen in secondary outcomes. CONCLUSION: Our findings suggest that patient-preferred live music reduces anxiety among patients with malignant lymphomas undergoing chemotherapy. Musical ability among this group of cancer patients seems not to be a determining factor for effect of music intervention.

Multiplex polymerase chain reaction-based prognostic models in diffuse large B-cell lymphoma patients treated with R-CHOP.

We present a multiplex analysis for genes known to have prognostic value in an attempt to design a clinically useful classification model in patients with diffuse large B-cell lymphoma (DLBCL). Real-time polymerase chain reaction was used to measure transcript levels of 28 relevant genes in 194 de novo DLBCL patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone). Including International Prognostic Index (IPI) as a variable in a penalized Cox regression, we investigated the association with disease progression for single genes or gene combinations in four models. The best model was validated in data from an online available R-CHOP treated cohort. With progression-free survival (PFS) as primary endpoint, the best performing IPI independent model incorporated the LMO2 and HLADQA1 as well as gene interactions for GCSAMxMIB1, GCSAMxCTGF and FOXP1xPDE4B. This model assigned 33% of patients (n = 60) to poor outcome with an estimated 3-year PFS of 40% vs. 87% for low risk (n = 61) and intermediate (n = 60) risk groups (P < 0·001). However, a simpler, IPI independent model incorporated LMO2 and BCL2 and assigned 33% of the patients with a 3-year PFS of 35% vs. 82% for low risk group (P < 0·001). We have documented the impact of a few single genes added to IPI for assignment in new drug trials.

LPL gene expression is associated with poor prognosis in CLL and closely related to NOTCH1 mutations.

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

[Status and perspectives on chronic myeloproliferative neoplasm treatment]. / Status og perspektiver for behandling af de kroniske myeloproliferative neoplasier.

Polycythaemia vera, essential thrombocytosis and primary myelofibrosis are closely related, clonal myeloproliferative neoplasms. Our knowledge of the underlying molecular mechanisms driving these diseases has increased dramatically during the latest ten years. Traditionally, treatment of these malignancies has focused on lowering their inherent thromboembolic risk but with the discovery of the JAK2-V617F mutation and most recently the calreticulin mutations new therapeutic options such as interferon-alpha, JAK2-inhibitors and statins are being contemplated. This article reviews these new treatment options.

High expression of PI3K core complex genes is associated with poor prognosis in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults in the Western world. Autophagy is a highly conserved process in eukaryotic cells. In CLL autophagy is involved in mediating the effect of chemotherapy but the role of autophagy in CLL pathogenesis remains unknown. In the present study, we used real-time RT-PCR to analyze expression of the PIK3C3, PIK3R4, and BECN1 genes. These genes encode the components of the PI3K core complex, which is central to initiation of autophagy. A consecutive series of 149 well-characterized CLL cases from Region of Southern Denmark were included in the study. All three genes were observed to be independent markers of prognosis in CLL with high expression being associated with more aggressive disease. With this clear association with outcome in CLL, these genes thereby represent promising candidates for future functional studies on the role of autophagy in CLL, and they may further represent targets of treatment.

Circulating YKL-40 in patients with essential thrombocythemia and polycythemia vera treated with the novel histone deacetylase inhibitor vorinostat.

YKL-40 regulates vascular endothelial growth factors and induces tumor proliferation. We investigated YKL-40 before and after treatment with vorinostat in 31 polycythemia vera (PV) and 16 essential thrombocythemia (ET) patients. Baseline PV patient levels were 2 times higher than in healthy controls (P<0.0001) and 1.7 times higher than in ET (P=0.02). A significant correlation between YKL-40 at baseline and neutrophils, CRP, LDH, JAK2V617F and platelets in PV patients was observed, as well as a significantly greater reduction of YKL-40 levels in PV patients responding to therapy. YKL-40 might be a novel marker of disease burden and progression in myeloproliferative neoplasms.

A phase III randomized trial comparing glucocorticoid monotherapy versus glucocorticoid and rituximab in patients with autoimmune haemolytic anaemia.

The impact of first-line treatment with the anti-CD 20 chimeric monoclonal antibody rituximab in patients with warm-antibody reactive autoimmune haemolytic anaemia (WAIHA) is unknown. We report the first randomized study of 64 patients with newly diagnosed WAIHA who received prednisolone and rituximab combined (N = 32) or prednisolone monotherapy (N = 32). After 12 months, a satisfactory response was observed in 75% of the patients treated with rituximab and prednisolone but in a significantly smaller proportion (36%) of those given prednisolone alone (P = 0·003). Furthermore, relapse-free survival was significantly better after the combined therapy than after prednisolone monotherapy (P = 0·02). After 36 months, about 70% of the patients were still in remission in the rituximab-prednisolone group, whereas only about 45% were still in complete or partial remission in the prednisolone group. There was no significant difference between the two groups regarding adverse reactions to the studied medications. Likewise, serious adverse events were equally distributed, and no allergic reactions to rituximab were recorded. In conclusion, our data show that using rituximab and prednisolone combined rather than prednisolone alone as first-line treatment in WAIHA increases both the rate and the duration of the response.

A phase II study of vorinostat (MK-0683) in patients with polycythaemia vera and essential thrombocythaemia.

Inhibition of histone deacetylases may be an important target in patients with myeloproliferative neoplasms. This investigator-initiated, non-randomized, open-label phase II multi-centre study included 63 patients (19 essential thrombocythaemia, 44 polycythaemia vera) from 15 centres. The primary objective was to evaluate if vorinostat was followed by a decline in clonal myeloproliferation as defined by European Leukaemia Net. Thirty patients (48%) completed the intervention period (24 weeks of therapy). An intention-to-treat response rate of 35% was identified. Pruritus was resolved [19% to 0% (P = 0·06)] and the prevalence of splenomegaly was lowered from 50% to 27% (P = 0·03). Sixty-five per cent of the patients experienced a decrease in JAK2 V617F allele burden (P = 0·006). Thirty-three patients (52% of patients) discontinued study drug before end of intervention due to adverse events (28 patients) or lack of response (5 patients). In conclusion, vorinostat showed effectiveness by normalizing elevated leucocyte and platelet counts, resolving pruritus and significantly reducing splenomegaly. However, vorinostat was associated with significant side effects resulting in a high discontinuation rate. A lower dose of vorinostat in combination with conventional and/or novel targeted therapies may be warranted in future studies.

Immunohistochemical double-hit score is a strong predictor of outcome in patients with diffuse large B-cell lymphoma treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone.

PURPOSE: Approximately 5% of diffuse large B-cell lymphomas (DLBCLs) are double-hit lymphomas (DHLs) with translocations of both MYC and BCL2. DHLs are characterized by poor outcome. We tested whether DLBCLs with high expression of MYC protein and BCL2 protein share the clinical features and poor prognosis of DHLs. PATIENTS AND METHODS: Paraffin-embedded lymphoma samples from 193 patients with de novo DLBCL who were uniformly treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) were studied using immunohistochemistry for MYC, BCL2, CD10, BCL6, and MUM1/interferon regulatory factor 4, and fluorescent in situ hybridization (FISH) for MYC and BCL2. RESULTS: FISH analysis identified DHL in 6% of patients, who showed the expected poor overall survival (OS; P = .002). On the basis of immunohistochemical MYC and BCL2 expression, a double-hit score (DHS) was assigned to all patients with DLBCL. The DHS-2 group, defined by high expression of both MYC and BCL2 protein, comprised 29% of the patients. DHS 2 was significantly associated with lower complete response rate (P = .004), shorter OS (P < .001), and shorter progression-free survival (PFS; P < .001). The highly significant correlation with OS and PFS was maintained in multivariate models that controlled for the International Prognostic Index and the cell-of-origin subtype (OS, P < .001; PFS, P < .001). DHS was validated in an independent cohort of 116 patients who were treated with R-CHOP. CONCLUSION: The immunohistochemical DHS defined a large subset of DLBCLs with double-hit biology and was strongly associated with poor outcome in patients treated with R-CHOP.

Cytogenetic findings in adult secondary acute myeloid leukemia (AML): frequency of favorable and adverse chromosomal aberrations do not differ from adult de novo AML.

During a 15-year period, 161 adult patients were diagnosed with secondary acute myeloid leukemia (s-AML) in the region of Southern Denmark. In 73 patients, the AML diagnosis was preceded by myelodysplastic syndrome (MDS-AML), in 31 patients by an antecedent hematologic disease, and in 57 patients by treatment with chemotherapy and/or irradiation (t-AML). Cytogenetic analysis was carried out in 93%, of which 61% had clonal chromosome aberrations. MDS-AML correlated to a normal karyotype (P < 0.001). t-AML correlated to abnormal clones with numerical and structural aberrations (P = 0.03), five or more unrelated aberrations (P = 0.03), marker chromosomes (P = 0.006), abnormal mitoses only (P = 0.01), female sex (P < 0.001), and -7 (P = 0.006). Centromeric breakage correlated to a complex karyotype (P = 0.01). The frequencies of aberrations in s-AML patients were compared with an age-matched group of de novo AML patients diagnosed in the same area and period. In this comparison, s-AML only correlated to -7 (P = 0.02). In 42 patients, we found that MDS patients with an abnormal karyotype were more likely to show cytogenetic evolution during progression to AML than MDS patients with a normal karyotype (P = 0.01). We conclude that population-based cytogenetic studies of adult s-AML and age- and sex-matched de novo AML show comparable distributions of chromosome abnormalities.

[Flowcytometric diagnostics of hereditary spherocytosis]. / Flowcytometrisk diagnostik af hereditaer sfaerocytose.

INTRODUCTION: The diagnosis of hereditary spherocytosis (HS) is based upon clinical presentation, typical laboratory findings of haemolysis with an increased mean corpuscular haemoglobin concentration (MCHC) combined with a positive osmotic fragility result. The disorder is caused by structural defects in red cell cytoskeletal proteins. The dye eosin-5'-maleimide (EMA) binds to band three of the red cell membrane. The fluorescence intensity of EMA-labelled red cells can be quantified by flowcytometric analysis. Decreased fluorescence is found in patients with HS. We have evaluated this method by comparing flowcytometric analysis of red cells from patients with HS and patients with other haemolytic disorders. MATERIAL AND METHODS: We included 21 patients with HS and 27 patients with other haemolytic disorders. The red cells were incubated and labelled with EMA followed by flowcytometric analysis measuring the mean-fluorescence-intensity expressed as EMA percentage. RESULTS: Based on the overall results, we assess an EMA percentage threshold of 15 or above to indicate HS. We found a sensitivity of 95% and a specificity of 93%. CONCLUSION: The osmotic fragility test does not have the same high degree of sensitivity and specificity and the test is time-consuming in the laboratory setting. Flowcytometric analysis with quantification of fluorescence intensity of red cells labelled with the EMA dye has proven to be a rapid and user-friendly method available to any laboratory with a flowcytometer. The method has a high sensitivity and specificity and can be recommended as a diagnostic tool for HS.

Cytogenetic findings in adult de novo acute myeloid leukaemia. A population-based study of 303/337 patients.

During a 10-year period (1992-2001) in the region of Southern Denmark, 337 patients aged 15 years or older (range 16-93 years, median 67 years) were diagnosed with acute myeloid leukaemia (AML). Cytogenetic analysis was carried out in 90%, of whom 53% had clonal chromosome aberrations. Some 24% and 31% had only numerical or structural abnormalities respectively. The remaining patients showed both types of abnormalities. Ploidy levels in decreasing order were: pseudodiploidy, 41%; hyperdiploidy, 32%; and hypodiploidy, 27%. Pseudodiploidy characterizes type M3 (70%) and hypodiploidy M6 (56%). Recurrent cytogenetic abnormalities--t(8;21), t(15;17) and inv(16)--were found in 3.3%, 3.3% and 2.0% of all patients respectively. Prognostically intermediate and adverse aberrations were found in 39% and 44%, respectively, of those with an abnormal karyotype. Rare recurrent aberrations were found in two patients in this material. A previously described non-recurrent abnormality was found to be recurrent in one patient [der(20)t(11;20)(q13.2;p13)]. New, previously undescribed abnormalities were found in 41 patients. Statistically significant correlations were found between t(15;17) and young age (P < 0.001), inv(16) and young age (P < 0.006), -17 and M6 (P = 0.007), and M6 and complex karyotype with five or more unrelated aberrations (P = 0.004). We conclude that this truly population-based cytogenetic study of adult AML showed distributions of chromosome abnormalities that differ from those described so far.