Robert Hast

TP53 Mutations in Low-Risk Myelodysplastic Syndromes With del(5q) Predict Disease Progression

PURPOSE To determine the frequency of TP53 mutations and the level of p53 protein expression by immunohistochemistry (IHC) in low-risk myelodysplastic syndromes (MDS) with del(5q) and to assess their impact on disease progression. PATIENTS AND METHODS Pre- and postprogression bone marrow (BM) samples from 55 consecutive patients with International Prognostic Scoring System low risk (n = 32) or intermediate-1 risk (n = 23) were studied by next-generation sequencing of TP53. IHC for p53 was performed on 148 sequential BM samples. RESULTS TP53 mutations with a median clone size of 11% (range, 1% to 54%) were detected in 10 patients (18%) already at an early phase of the disease. Mutations were equally common in low-risk and intermediate-1-risk patients and were associated with evolution to acute myeloid leukemia (5 of 10 v 7 of 45; P = .045). Nine of 10 patients carrying mutations showed more than 2% BM progenitors with strong p53 staining. The probability of a complete cytogenetic response to lenalidomide was lower in mutated patients (0 of 7 v 12 of 24; P = .024). CONCLUSION By using sensitive deep-sequencing technology, we demonstrated that TP53 mutated populations may occur at an early disease stage in almost a fifth of low-risk MDS patients with del(5q). Importantly, mutations were present years before disease progression and were associated with an increased risk of leukemic evolution. TP53 mutations could not be predicted by common clinical features but were associated with p53 overexpression. Our findings indicate a previously unrecognized heterogeneity of the disease which may significantly affect clinical decision making.

Persistent malignant stem cells in del(5q) myelodysplasia in remission.

BACKGROUND The in vivo clinical significance of malignant stem cells remains unclear. METHODS Patients who have the 5q deletion (del[5q]) myelodysplastic syndrome (interstitial deletions involving the long arm of chromosome 5) have complete clinical and cytogenetic remissions in response to lenalidomide treatment, but they often have relapse. To determine whether the persistence of rare but distinct malignant stem cells accounts for such relapses, we examined bone marrow specimens obtained from seven patients with the del(5q) myelodysplastic syndrome who became transfusion-independent while receiving lenalidomide treatment and entered cytogenetic remission. RESULTS Virtually all CD34+, CD38+ progenitor cells and stem cells that were positive for CD34 and CD90, with undetectable or low CD38 (CD38−/low), had the 5q deletion before treatment. Although lenalidomide efficiently reduced these progenitors in patients in complete remission, a larger fraction of the minor, quiescent, CD34+,CD38-/low, CD90+ del(5q) stem cells as well as functionally defined del(5q) stem cells remained distinctly resistant to lenalidomide. Over time, lenalidomide resistance developed in most of the patients in partial and complete remission, with recurrence or expansion of the del(5q) clone and clinical and cytogenetic progression. CONCLUSIONS In these patients with the del(5q) myelodysplastic syndrome, we identified rare and phenotypically distinct del(5q) myelodysplastic syndrome stem cells that were also selectively resistant to therapeutic targeting at the time of complete clinical and cytogenetic remission. (Funded by the EuroCancerStemCell Consortium and others.)

A prospective, randomized study comparing cefepime and imipenem-cilastatin in the empirical treatment of febrile neutropenia in patients treated for haematological malignancies

A prospective, open label, randomized, multicentre study was conducted, comparing the efficacy and safety of cefepime with that of imipenem-cilastatin for the management of febrile neutropenia in patients with haematological malignancies. Furthermore, the safety of early discontinuation of antibiotic therapy in patients with fever of undetermined origin (FUO) was assessed. A total of 180 patients with 207 febrile episodes were randomized at start of fever (105 episodes for cefepime and 102 episodes for imipenem). The 2 groups were comparable in terms of age, gender, underlying malignancy, prior transplantation, and presence of central venous catheters. All patients were neutropenic at inclusion with median absolute neutrophil count (ANC) 0.1×109/l(range 0–1×109/l), and ANC ≤0.1×109/l in 77% of included patients. The mean duration of neutropenia, with ANC <0.5×109/l was 6.2 d. Febrile episodes were classified as microbiologically documented infection (47%), FUO (43%), or clinically documented infection (10%). At final evaluation 1–2 weeks after completion of antibiotic therapy, monotherapy success rates were 40% and 51% in the cefepime and imipenem-cilastatin groups respectively (p=0.33). The 4-week overall mortality rate was 5%. Three (2%) of the cefepime treated patients and 4 (3%) of the imipenem-cilastatin treated patients died as a result of infection. Adverse events directly related to antibiotic treatment were uncommon and did not differ between groups. Early discontinuation of antibiotic therapy in 31 patients with FUO 48 h after defervescence was not associated with an increased rate of fever relapse or mortality compared with a subgroup of 29 patients where therapy was continued.

Serum erythropoietin (EPO) levels correlate with survival and independently predict response to EPO treatment in patients with myelodysplastic syndromes

Abstract: Treatment with recombinant erythropoietin (EPO) can alleviate anaemia in patients with myelodysplastic syndromes (MDS). The present study, based on a long‐term follow‐up of 68 MDS patients (26RA, 16 RAS, 26 RAEB) treated with EPO alone, pinpoints pre‐treatment variables associated with response induction, response duration and overall survival. Response, defined as an increase in haemoglobin >15 g L−1 or eliminated erythrocyte transfusion requirements, was observed in 22 of 66 (33%) evaluable patients. The median response duration was 15 (range 3–64 +) months. Using univariate logistic regression models, responders displayed significantly lower baseline serum EPO levels (S‐EPO), more often normal bone marrow blast cell content (RA/RAS vs. RAEB), normal cytogenetics and no need for erythrocyte transfusion. In a multiple logistic regression model, S‐EPO (P = 0.009), marrow blast content (P = 0.031) and erythrocyte transfusion need (P = 0.024) remained associated with response induction. The probability of response for a patient with S‐EPO >50 U L−1, RA/RAS and no transfusion need was 0.79 (0.53–0.93, 95% CI). The median overall survival time from start of EPO treatment was 26 months, significantly longer for responders than for non‐responders (49 vs. 18 months, P = 0.018). Survival was also predicted by baseline S‐EPO; patients with S‐EPO > 50 U L−1 (n = 50) had a median survival of 17 months, as compared to 65 months for those with S‐EPO >50 U L−1 (n = 14, P = 0.024). The international prognostic scoring system (IPSS) for MDS predicted survival (P = 0.003) and progression to acute leukemia (P < 0.001) but not response to EPO treatment. Furthermore, in a logistic regression model with S‐EPO and IPSS, S‐EPO (but not IPSS) was again a significant predictor for response (P = 0.007). Our data facilitate the optimal selection of MDS patients suitable for EPO treatment and pinpoint S‐EPO as a powerful predictor of response and overall survival in MDS.

Oxymetholone treatment in myelofibrosis.

ZusammenfassungDie therapeutische Wirksamkeit von Oxymetholon bei fortgeschrittener Osteomyeloflbrose wurde im Rahmen einer prospektiven Studie geprüft. 11 Patienten (4 Frauen, 7 Männer) erhielten 3–5 mg/kg Körpergewicht Oxymetholon. 5 dieser Patienten hatten vorher eine polyzythämische Phase. Alle Patienten hatten bei Beginn des Therapieversuchs eine Anämie, 4 eine Leukopenie und 10 zusätzlich eine Thrombopenie. Eine Hepatosplenomegalie war immer vorhanden. 5 Patienten benötigten vor Behandlung regelmäßig Bluttransfusionen.In 9 von 15 Therapiephasen wurde eine Normalisierung und deutliche Besserung beobachtet. Die Veränderungen des Hämoglobinspiegels und der Plättchenzahl waren signifikant. 4 Patienten verschlechterten sich nach Dosisreduktion oder bei Absetzen von Oxymetholon; 2 davon sprachen erneut an. Bei einem Patienten kehrten die Werte zu den früher bestehenden polyzythämischen Werten zurück. 1 Patient starb durch eine akute Leukämie.Die Ergebnisse der Studie weisen daraufhin, daß Androgene in weit fortgeschrittenen Fällen von Myelofibrose mit transfusionsbedürftiger Anämie oder schwerer Zytopenie von Wert sein könnte.SummaryIn order to study the effect of oxymetholone therapy in advanced myelofibrosis, 11 patients (4 females, 7 males) were given, 3–5 mg per kg body weight, long-term oxymetholone treatment in a prospective multicenter study. Five cases had previously had a diagnosis of polycythemia vera. All patients had anemia initially, 4 leukocytopenia and 10 thrombocytopenia in addition. Hepato-splenomegaly was present in all cases but in varying degree. Five patients required regular blood transfusions before treatment.In 9 of the 15 courses given, there was normalization of the peripheral blood or substantial improvement (better than 3 g hemoglobin/dl or 50×109 platelets/1) after androgens. Significant effects were noted both on hemoglobin values and platelet counts. The need for blood transfusions ceased completely in all 5 cases. When oxymetholone treatment was reduced or interrupted 4 patients relapsed; 2 of them responded to a renewed course. The red cell counts returned to previous polycythemic values in one patient and another died from acute leukemia.The results of this study suggest that androgens might be of value in advanced cases of myelofibrosis with transfusion-requiring anemia or severe thrombocytopenia.

No benefit from adding GM-CSF to induction chemotherapy in transforming myelodysplastic syndromes: better outcome in patients with less proliferative disease

In this prospective randomized multicenter trial 93 patients, median age 72 years, with RAEB-t (n=25) and myelodysplastic syndrome (MDS)-AML (n=68) were allocated to a standard induction chemotherapy regimen (TAD 2+7) with or without addition of granulocyte–macrophage-CSF (GM-CSF). The overall complete remission (CR) rate was 43% with no difference between the arms. Median survival times for all patients, CR patients, and non-CR patients were 280, 550, and 100 days, respectively, with no difference between the arms. Response rates were significantly better in patients with serum lactate dehydrogenase (S-LDH) levels ⩽9.5 μkat/l, bone marrow cellularity ⩽70%, and WBC counts <4.0 × 109/l, but S-LDH was the only variable independently associated with response by logistic regression analysis. Coxs regression analysis identified four significant prognostic factors for survival: bone marrow cellularity, S-LDH, cytogenetic risk group (International Prognostic Scoring System), and age. Only bone marrow cellularity (P=0.01) and S-LDH (P=0.0003) retained statistical significance in the log-rank test. Severe adverse events were significantly more common in the GM-TAD arm (P=0.01). Thus, addition of GM-CSF to chemotherapy showed no clinical benefit in terms of response but carried an increased risk for side effects. We present a clinically useful tool to predict response to chemotherapy and survival in elderly patients with transforming MDS, favoring patients with features of less proliferative disease.

Diagnostic significance of dysplastic features of peripheral blood polymorphs in myelodysplastic syndromes.

Dysplastic features of peripheral blood granulocytes were studied to investigate the diagnostic value estimating cytoplasmatic hypogranulation and nuclear abnormalities of the pelgeroid type in myelodysplastic syndromes (MDS). Hypogranulation was measure both as the percentage of agranular neutrophils and as a score value, taking into account also cells with slight or moderate hypogranulation. We studied 62 cases of MDS (18 refractory anemia, 11 sideroblastic anemia, 26 refractory anemia with excess of blasts, three chronic myelomonocytic leukemias, four refractory anemia with excess of blasts in transformation). For comparison we studied 13 cases of myeloproliferative disorders, 18 patients with different forms of anemia and 20 normal controls. Reference values were defined as the 95% probability limit of the mean values of normal controls. In MDS 52/62 patients (84%) had increased numbers of pelgeroid cells, 40/62 (65%) had abnormal granulation scores while only 14/62 (23%) had increased percentages of agranular neutrophils. The mean granulation score (+/- S.D.) in MDS (225.0 +/- 57.4), was significantly lower (p less than 0.001) than in myeloproliferative disorders (282.7 +/- 9.0), anemias (288.8 +/- 8.0) and normal controls (281.7 +/- 12.9). Pelgeroid neutrophils were significantly (p less than 0.001) more common in MDS (11.6% +/- 7.8) compared to myeloproliferative disorders (1.1% +/- 1.0), anemias (3.1% +/- 2.0) and normal controls (1.9% +/- 1.5). There was no significant correlation between the degree of hypogranulation and the percentages of pelgeroid cells in individual patients. Hypogranulation tended to be more pronounced in the more immature forms of MDS while pelgeroid cells were equally common in the different subgroups. When the two parameters were combined peripheral blood dysplasia was recognized in 92% of the MDS cases. The results suggest that quantitative estimation of hypogranulation and of nuclear abnormalities in peripheral blood polymorphs are simple and valuable diagnostic tools in MDS.

Antiapoptotic Role of Growth Factors in the Myelodysplastic Syndromes: Concordance Between In vitro and In vivo Observations

Purpose: Erythroid apoptosis in low-risk myelodysplastic syndrome (MDS) maybe mediated via mitochondrial release of cytochrome c and subsequent caspase activation. In the present study, we compared the in vitro and in vivo effects of proerythroid treatment with erythropoietin + granulocyte colony-stimulating factor (G-CSF) on myelodysplastic erythropoiesis regarding apoptosis and preferential growth of clones with cytogenetic abnormalities. Experimental Design: We enrolled 15 refractory anemia (RA) and 11 refractory anemia with ringed sideroblasts (RARS), including 5q– aberration, monosomy 7, and trisomy 8, before initiation of treatment and followed nine patients after successful treatment. The effects of G-CSF and erythropoietin were assessed. The expression of G-CSF receptor (G-CSFR) was explored during erythroid maturation. The relative growth of erythroid progenitors with cytogenetic aberrations in presence of erythropoietin was investigated. Results: Significant redistribution of cytochrome c was seen before treatment at all stages of erythroid differentiation. This release was blocked by G-CSF during the whole culture period and by erythropoietin during the latter phase. Both freshly isolated glycophorin A+ bone marrow cells and intermediate erythroblasts during cultivation retained their expression of G-CSFR. Cytochrome c release and caspase activation were significantly less pronounced in progenitors obtained from successfully treated nonanemic patients and showed no further response to G-CSF in vitro. Moreover, erythropoietin significantly promoted growth of cytogenetically normal cells from 5q– patients, whereas no such effect was observed on erythroblasts from monosomy 7 or trisomy 8 patients. Conclusion: We conclude that growth factors such as erythropoietin and G-CSF can act both via inhibition of apoptosis of myelodysplastic erythroid precursors and via selection of cytogenetically normal progenitors.

Successful Treatment with Fludarabine in Two Cases of Angioimmunoblastic Lymphadenopathy with Dysproteinemia

Angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) is today recognized as a T-cell lymphoma which in most cases runs an aggressive course. The diagnosis is often difficult because of the varying clinico-pathological picture. Less than a third of the patients can be expected to have long-term remissions even after multiagent chemotherapy. Complete remissions have been reported after the use of interferon-alpha, cyclosporin A, and recently purine analogues in a few patients. We now report two cases of AILD that had unmaintained remissions for 32 and 10 months, respectively, after fludarabine therapy. In one of the patients fludarabine was used up-front and in the other after she had proved to be resistant to CHOP treatment. No severe infectious complications were noted. The use of purine analogues should be investigated further in AILD.

Granulocyte‐macrophage colony‐stimulating factor to increase efficacy of mitoxantrone, etoposide and cytarabine in previously untreated elderly patients with acute myeloid leukaemia: a Swedish multicentre randomized trial

A total of 110 patients, aged 64 years or over, with de novo acute myeloid leukaemia (AML) and white blood cell counts <50 × 109/l were treated with 3 d of cytarabine 1 g/m2 twice daily, mitoxantrone 12 mg/m2 and etoposide 200 mg/m2, randomized with or without the addition of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) 200 μg/m2. The primary aim was to evaluate the effect of GM‐CSF on the remission rate. Secondary aims included comparison of duration of remission, survival and infectious complications and the impact of maintenance therapy with thioguanine. Complete remission (CR) was achieved by 64% of patients without GM‐CSF, and by 65% of patients who received GM‐CSF, the median remission duration was 13 vs. 6 months, the median overall survival (OS) was 14 vs. 9 months, the mean time to neutrophil recovery was 25 vs. 17 d (P = 0·03) and the number of positive blood cultures was 46 vs. 39 (P = 0·05) respectively. The impact of thioguanine remains unanswered since only 30 patients remained in CR after consolidation therapy. We conclude that induction therapy is feasible with acceptable toxicity in elderly patients with AML, albeit with a high relapse rate and short OS. GM‐CSF prior to, and in combination with, induction treatment reduced the time to neutrophil recovery and the number of neutropenic septicaemia cases but did not improve the OS of AML in the elderly.