Tandem versus single haematopoietic stem cell transplant and BV maintenance in relapsed/refractory Hodgkin lymphoma: A matched cohort analysis from the LYSA.

Autologous hematopoietic stem cell transplant (ASCT) is the standard curative treatment for patients with high-risk relapsed/refractory Hodgkin lymphoma (R/R HL). The AETHERA study showed survival gain with Brentuximab Vedotin (BV) maintenance after ASCT in BV-naive patients, which was recently confirmed in the retrospective AMAHRELIS cohort, including a majority of BV-exposed patients. However, this approach has not been compared to intensive tandem auto/auto or auto/allo transplant strategies, which were used before BV approval. Here, we matched BV maintenance (AMAHRELIS) and tandem SCT (HR2009) cohorts, and observed that BV maintenance was associated with better survival outcome in patients with HR R/R HL.

A meropenem pharmacokinetics model in patients with haematological malignancies.

BACKGROUND: Optimal dosing of antibiotics is critical in immunocompromised patients suspected to have an infection. Data on pharmacokinetics (PK) of meropenem in patients with haematological malignancies are scarce. OBJECTIVES: To optimize dosing regimens, we aimed to develop a PK population model for meropenem in this population. METHODS: Patients aged ≥18 years, hospitalized in the haematology department of our 1500 bed university hospital for a malignant haematological disease and who had received at least one dose of meropenem were eligible. Meropenem was quantified by HPLC. PK were described using a non-linear mixed-effect model and external validation performed on a distinct database. Monte Carlo simulations estimated the PTA, depending on renal function, duration of infusion and MIC. Target for free trough concentration was set at >4× MIC. RESULTS: Overall, 88 patients (181 samples) were included, 66 patients (75%) were in aplasia and median Modification of Diet in Renal Disease (MDRD) CLCR was 117 mL/min/1.73 m2 (range: 35-359). Initial meropenem dosing regimen ranged from 1 g q8h to 2 g q8h over 30 to 60 min. A one-compartment model with first-order elimination adequately described the data. Only MDRD CLCR was found to be significantly associated with CL. Only continuous infusion achieved a PTA of 100% whatever the MIC and MDRD CLCR. Short duration of infusion (<60 min) failed to reach an acceptable PTA, except for bacteria with MIC < 0.25 mg/L in patients with MDRD CLCR below 90 mL/min/1.73 m2. CONCLUSIONS: In patients with malignant haematological diseases, meropenem should be administered at high dose (6 g/day) and on continuous infusion to reach acceptable trough concentrations.

Dihydroartemisinin-induced ferroptosis in acute myeloid leukemia: links to iron metabolism and metallothionein.

Artemisinin is an anti-malarial drug that has shown anticancer properties. Recently, ferroptosis was reported to be induced by dihydroartemisinin (DHA) and linked to iron increase. In the current study, we determined the effect of DHA in leukemic cell lines on ferroptosis induction and iron metabolism and the cytoprotective effect triggered in leukemic cells. We found that treatment of DHA induces early ferroptosis by promoting ferritinophagy and subsequent iron increase. Furthermore, our study demonstrated that DHA activated zinc metabolism signaling, especially the upregulation of metallothionein (MT). Supportingly, we showed that inhibition MT2A and MT1M isoforms enhanced DHA-induced ferroptosis. Finally, we demonstrated that DHA-induced ferroptosis alters glutathione pool, which is highly dependent on MTs-driven antioxidant response. Taken together, our study indicated that DHA activates ferritinophagy and subsequent ferroptosis in AML and that MTs are involved in glutathione regenerating and antioxidant response.

The cap-translation inhibitor 4EGI-1 induces apoptosis in multiple myeloma through Noxa induction.

BACKGROUND: Cancer cells are frequently addicted to deregulated oncogenic protein translation. The small molecule 4EG-I selectively inhibits the cap-dependent translation of mRNAs. As multiple myeloma is an incurable disease that requires new therapeutic approaches, we investigated whether targeting the translation initiation pathway could be a target for myeloma therapy. METHODS: Six myeloma cell lines and primary samples were included in this study. The 4EGI-1 effect was determined by AnnexinV staining and caspase activation. Modification of Bcl-2 protein expression was analysed, and the significance of modified proteins was analysed by knock-down experiments. RESULTS: We demonstrated that 4EGI-1 impaired the assembly of the eIF4F complex and decreased the expression of the eIF4E-regulated proteins in myeloma cells. Furthermore, we showed that 4EGI-1 induced strong apoptosis in five out of six myeloma cell lines. Apoptosis is associated with the activation of the intrinsic mitochondrial pathway. The 4EGI-1 triggered Noxa induction only in cells undergoing apoptosis through endoplasmic reticulum (ER) stress. Furthermore, Noxa silencing prevented myeloma cells from 4EGI-1-induced apoptosis. Finally, Noxa induction led to a disruption of Mcl-1/Bim complexes in parallel to the generation of 'Mcl-1-free Noxa'. CONCLUSION: Our results suggested that the use of inhibitors that directly target the translation initiation complex eIF4F could represent a potential novel approach for multiple myeloma therapy.

Bcl-2 protects from lethal hepatic apoptosis induced by an anti-Fas antibody in mice.

Fas is an apoptosis-signalling cell surface antigen that has been shown to trigger cell death upon specific ligand or antibody binding. Treatment of mice with an anti-Fas antibody causes fulminant hepatic failure due to massive apoptosis. To test a putative protective effect of the anti-apoptotic Bcl-2 protein, transgenic mice were generated to express the human bcl-2 gene product in hepatocytes. Early onset of massive hepatic apoptosis leading to death was observed in all nontransgenic mice treated with an anti-Fas antibody. By contrast, hepatic apoptosis was delayed and dramatically reduced in transgenic animals, yielding a 93% survival rate. These results demonstrate that Bcl-2 is able to protect from in vivo Fas-mediated cytotoxicity, and could be of significance for preventing fulminant hepatic failure due to viral hepatitis in humans.

Caspase activation is required for terminal erythroid differentiation.

The cysteine proteases known as caspases play a central role in most apoptotic pathways. Here, we show that caspase inhibitors arrest the maturation of human erythroid progenitors at early stages of differentiation, before nucleus and chromatin condensation. Effector caspases such as caspase-3 are transiently activated through the mitochondrial pathway during erythroblast differentiation and cleave proteins involved in nucleus integrity (lamin B) and chromatin condensation (acinus)without inducing cell death and cleavage of GATA-1. These observations indicate a new function for caspases as key proteases in the process of erythroid differentiation.

Radiotherapy in combination with nivolumab for relapsed/refractory classical Hodgkin lymphoma: About two cases.

Hodgkin lymphoma is a highly curable malignancy involving lymph nodes and the lymphatic system. Even at late stage disease, about 70% of patients will be cured with standard first line therapy. For patients who experience relapse or refractory classical Hodgkin lymphoma, the standard treatment option is high-dose chemotherapy followed by autologous stem cell rescue or transplant. However about 50% of patients will have recurrence after high-dose chemotherapy followed by autologous stem cell rescue or transplantation and have worse prognosis with median overall survival of 32% at 5 years. The anti-PD1 checkpoints inhibitors pembrolizumab and nivolumab have remarkably improved outcomes of patients with relapse of refractory classical Hodgkin lymphoma after high-dose chemotherapy followed by autologous stem cell rescue or transplantation. On the other hand, radiotherapy is an entire component of salvage therapy and its efficacy is now well established in term of local disease control in sites of relapsed or refractory Hodkin lymphoma. Defining the optimal modality and timing of radiotherapy as these new agents arrive is a challenge. An interesting approach is the combination of radiotherapy with checkpoint inhibitor and the possibility of stopping the treatment when complete response is achieved. We add to the literature two new cases of combination of radiotherapy with immunotherapy in patients who relapsed after high-dose chemotherapy followed by autologous stem cell rescue or transplantation and consolidation with brentuximab vedotin, resulting in excellent outcomes.

Treatment of myelodysplastic syndromes with 5q deletion before the lenalidomide era; the GFM experience with EPO and thalidomide.

Anemia in MDS with 5q deletion was generally considered, until the advent of lenalidomide, unresponsive to available treatments. We analyzed erythroid response to erythropoetin (EPO) or darbepoetin (DAR) and thalidomide in MDS with 5q deletion treated by French centers (GFM) and in whom karyotype was successfully performed. Of 345 patients treated with EPO or DAR+/-G-CSF, 48 had 5q deletion. The response rate was 46% (31% major, 15% minor) according to International Working Group (IWG) 2000 criteria versus 64% in patients without 5q deletion (p=0.03). According to IWG 2006 criteria, the response rate in patients with 5q deletion was 39% versus 52% in patients without 5q deletion (p=0.10). Mean duration of response was 14 months versus 25 months (IWG 2000) and 13 months versus 27 months (IWG 2006) in 5q deletion and non-5q deletion patients (p=0.019 and 0.003, respectively). Of 120 MDS treated with thalidomide, all of whom had successful cytogenetic analysis, 37% of the 24 patients with 5q deletion responded (IWG 2000 criteria, 20% major, 17% minor) with a mean duration of 9.5 months, versus 32% (18% major, 14% minor) in MDS without 5q deletion and a mean response duration of 9 months (p=NS). Our results confirm that response rates to EPO or DAR and thalidomide are clearly inferior to those obtained with lenalidomide.

Radiotherapy of relapse-refractory follicular lymphoma.

PURPOSE: To assess the efficacy of treatment and outcomes of patients with relapsed or refractory follicular lymphoma treated with external beam irradiation. PATIENTS AND METHODS: Fifteen patients who received external beam radiotherapy for relapsed or refractory follicular lymphoma were studied. The median age was 68.3 years (range: 37.9-87.08 years) with four men and 11 women. Seven patients had early stage (I or II); eight advanced stage (III or IV). Median FLIPI score was 2. Two patients had high tumour bulk disease. Six patients had extranodal invasion, with five patients having bone marrow invasion. RESULTS: The median time of follow-up after relapse or first-line treatment in case of refractory disease was 61.9 months (range: 9.1-119.7 months). Complete response after external beam radiotherapy was seen in 11 cases (73%) and partial response in two (13%), with a median dose of 30Gy (range: 2-40Gy) and median number of fractions of 15 (range: 2-20). Eight patients (53%) relapsed after external beam radiation therapy in a median of 20.2 months, mostly out of irradiated volumes. Most patients (66%) had a disease control after one or two courses of external beam radiation therapy. At last follow-up, 86% of patients were in remission including those with salvage chemotherapy. The toxicity profile was favourable with toxicity higher than grade 1. In univariate analysis, a Follicular Lymphoma International Prognostic Index (FLIPI) score above 2 was the only predicting factor for non-control disease. CONCLUSION: For relapsed and refractory follicular lymphoma, external beam radiotherapy should be considered as an effective modality when integrated in a multimodality approach. Randomised studies are warranted to validate this strategy.

RSK2 is a new Pim2 target with pro-survival functions in FLT3-ITD-positive acute myeloid leukemia.

Acute myeloid leukemia (AML) with the FLT3 internal tandem duplication (FLT3-ITD AML) accounts for 20-30% of AML cases. This subtype usually responds poorly to conventional therapies, and might become resistant to FLT3 tyrosine kinase inhibitors (TKIs) due to molecular bypass mechanisms. New therapeutic strategies focusing on resistance mechanisms are therefore urgently needed. Pim kinases are FLT3-ITD oncogenic targets that have been implicated in FLT3 TKI resistance. However, their precise biological function downstream of FLT3-ITD requires further investigation. We performed high-throughput transcriptomic and proteomic analyses in Pim2-depleted FLT3-ITD AML cells and found that Pim2 predominantly controlled apoptosis through Bax expression and mitochondria disruption. We identified ribosomal protein S6 kinase A3 (RSK2), a 90 kDa serine/threonine kinase involved in the mitogen-activated protein kinase cascade encoded by the RPS6KA3 gene, as a novel Pim2 target. Ectopic expression of an RPS6KA3 allele rescued the viability of Pim2-depleted cells, supporting the involvement of RSK2 in AML cell survival downstream of Pim2. Finally, we showed that RPS6KA3 knockdown reduced the propagation of human AML cells in vivo in mice. Our results point to RSK2 as a novel Pim2 target with translational therapeutic potential in FLT3-ITD AML.

High mTORC1 activity drives glycolysis addiction and sensitivity to G6PD inhibition in acute myeloid leukemia cells.

Alterations in metabolic activities are cancer hallmarks that offer a wide range of new therapeutic opportunities. Here we decipher the interplay between mTORC1 activity and glucose metabolism in acute myeloid leukemia (AML). We show that mTORC1 signaling that is constantly overactivated in AML cells promotes glycolysis and leads to glucose addiction. The level of mTORC1 activity determines the sensitivity of AML cells to glycolysis inhibition as switch-off mTORC1 activity leads to glucose-independent cell survival that is sustained by an increase in mitochondrial oxidative phosphorylation. Metabolic analysis identified the pentose phosphate pathway (PPP) as an important pro-survival pathway for glucose metabolism in AML cells with high mTORC1 activity and provided a clear rational for targeting glucose-6-phosphate dehydrogenase (G6PD) in AML. Indeed, our analysis of the cancer genome atlas AML database pinpointed G6PD as a new biomarker in AML, as its overexpression correlated with an adverse prognosis in this cohort. Targeting the PPP using the G6PD inhibitor 6-aminonicotinamide induces in vitro and in vivo cytotoxicity against AML cells and synergistically sensitizes leukemic cells to chemotherapy. Our results demonstrate that high mTORC1 activity creates a specific vulnerability to G6PD inhibition that may work as a new AML therapy.

Common 4q24 deletion in four cases of hematopoietic malignancy: early stem cell involvement?

We determined bone marrow karyotype at diagnosis in four female acute myeloid leukemia (AML) or myelodysplasia patients, aged between 52 and 56 years. In each case, we observed chromosome rearrangement involving the same 4q24 band. Three patients had a balanced reciprocal translocation as the sole abnormality - t(3;4)(q26;q24), t(4;5)(q24;p16) and t(4;7)(q24;q21) - and the fourth had del(4)(q23q24), +4. We used a set of 4q BAC probes for fluorescent in situ hybridization (FISH) in these four cases. We found a 4q24 submicroscopic deletion in all three translocations, with a common deletion of approximately 0.5 Mb. In three cases, we concluded that rearrangement occurred in an early hematopoietic stem cell, as it was detected, in mosaic with a normal karyotype, in a fraction of remission bone marrow cells, peripheral T and B lymphocytes, malignant lymph node T-lymphoma cells in one case and B-lymphoblastoid cell lines established in two cases. Moreover, one of 10 additional AML patients tested by FISH had a normal karyotype and deletion of one of the commonly deleted probe sequences. A tumor suppressor gene may therefore be involved, especially as two patients developed malignant lymphoma at the same time as myeloid proliferation.

Role of Gab proteins in phosphatidylinositol 3-kinase activation by thrombopoietin (Tpo).

In this study, we show that upon thrombopoietin (Tpo) stimulation the two adapter proteins Gab1 and Gab2 are strongly tyrosine phosphorylated and associated with Shc, SHP2, PI 3-kinase and Grb2 in mpl-expressing UT7 cells. Although Gab1 and Gab2 seem to mediate overlapping biological signals in many cells, only Gab1 is expressed and phosphorylated in response to Tpo in primary human megakaryocytic progenitors; furthermore, it associates with the same proteins. Although a low level of tyrosine phosphorylated IRS-2 protein is also detected in PI 3-kinase immunoprecipitates, Gab proteins are the essential proteins associated with PI 3-kinase after Tpo stimulation. We demonstrate that, albeit no association is detected between the Tpo receptor mpl and Gab proteins, Y112 located in the C-terminal cytoplasmic domain of mpl is required for Gab1/2 tyrosine phosphorylation. Gab proteins are not tyrosine phosphorylated after Tpo stimulation of UT-7 and Ba/F3 cells expressing a mpl mutant lacking Y112. Moreover, no activation of the PI 3-kinase/Akt pathway is observed in cells expressing this mpl mutant. Finally, we show that this mutant does not allow cell proliferation, thereby confirming that PI 3-kinase activation is required for Tpo-induced cell proliferation.

Expression of the Evi-1 gene in myelodysplastic syndromes.

Increased expression of the proto-oncogene Evi-1 has been shown to block the in vitro granulocytic differentiation of myeloid cells in response to granulocytic colony-stimulating factor and to interfere with the proliferation of erythroid cells in response to erythropoietin. We determined the frequency of Evi-1 expression in myelodysplastic syndromes (MDS), a disorder with altered proliferation and differentiation in the erythroid and myeloid lineages. Twenty-one patients were studied. Abnormal expression was found in 1/9 patients with refractory anemia and in 7/12 patients with refractory anemia with excess of blasts (RAEB) or in transformation (RAEBt). No correlation could be found between expression of Evi-1 and age, sex, hemoglobin level and percentage of bone marrow blasts or erythroblasts. This result suggests that the high incidence of Evi-1 expression which remains at low levels in RAEB and RAEBt is not a major determinant of ineffective erythropoiesis and myelopoiesis in MDS.

Fas/Apo-1 (CD95) expression and apoptosis in patients with myelodysplastic syndromes.

Apoptosis of hematopoietic progenitor cells is increased in myelodysplastic syndromes (MDS). We have studied Fas (CD95/Apo-1) antigen expression in 27 MDS patients (RARS 4, RA 3, RAEB 13; RAEB-t 3, CMML 4) and three AML secondary to MDS. We found that the Fas antigen was not expressed on normal bone marrow (BM) CD34+, CD14+, or glycophorin+ cells, and only slightly on CD33+ cells. Patients with MDS had upregulation of Fas expression on total bone marrow nuclear cells (BMMC) (t-test, P = 0.04), CD34+ (P = 0.013), CD33+ (P = 0.04), and glycophorin+ (P = 0.032) BM cells compared to controls. Fas expression did not correlate to the FAB subtype, the Bournemouth score, or to peripheral cytopenias. However, Fas expression intensity on CD34+ cells negatively correlated to the BM blasts number (Spearman, P = 0.01) suggesting that leukemic blasts cells lose Fas antigen expression with progression of myelodysplasia. Using both proliferation assays in liquid cultures and clonogenic progenitor assays in the presence of an agonist anti-Fas MoAb (CH11), we showed that the Fas protein was functional in some patients. Dose-dependent inhibition of DNA synthesis was observed in three out of seven patients studied. CFU-GM and BFU-E colonies suppression in some patients suggested that Fas can induce apoptosis in myeloid and erythroid BM progenitors of MDS patients. The TUNEL technique on BM smears gave a mean of 12.6% +/- 2.5 of bone marrow apoptotic cells in five controls. Patients with MDS had increased bone marrow apoptosis (mean 39% +/- 5.7, t-test, P = 0.012). Four out of 15 (26%) patients studied with a sensitive radiolabeled DNA ladder technique had typical DNA ladders indicative of advanced stages of apoptosis. Massive BM suicide was observed in patients with RA (2/2) and RAEB (8/11), whereas apoptosis rates were normal or low in patients with RAEB-t (3/3) or secondary AMLs (3/3). Moreover, high rates of apoptosis correlated to low Bournemouth score (Spearman, P = 0.01). No statistical correlation could be found between Fas expression and apoptosis rates. Our results confirm the importance of programmed cell death in MDS. The Fas antigen is clearly upregulated on BM cells, but its role in the pathophysiology of apoptosis in myelodysplasia is still unclear, indicating that many factors positively or negatively interfere with the Fas-mediated pathway of apoptosis in vivo and in vitro.

Response to antiviral treatment in hepatitis C virus-associated marginal zone lymphomas.

A link between chronic hepatitis C virus (HCV) infection and low-grade B-cell lymphomas has been suggested by epidemiological studies. Marginal zone lymphomas (MZLs) including splenic lymphomas with villous lymphocytes are among the most frequently reported subgroups in the setting of chronic HCV infection. In this study, we examined the effect of antiviral treatment in eight patients with HCV-associated MZL. We found that five out of eight patients have responded to interferon alpha and ribavirin. In some cases, hematologic responses were correlated to virologic responses. In addition, we report a case of large granular lymphocyte leukemia occurring in association with MZL and HCV, and responding to interferon and ribavirin. We suggest that there is an etiologic link between HCV and antigen-driven lymphoproliferative disorders.

Prognostic value of c-mpl expression in myelodysplastic syndromes.

The c-mpl proto-oncogene which encodes a member of the hematopoietic cytokine receptor superfamily has been recently shown to be the receptor for thrombopoietin (TPO), which stimulates megakaryocyte progenitor expansion and differentiation. We studied c-mpl expression by Northern blot analysis, in a large series of 58 MDS. No expression was found in 14 patients with refractory anemia (RA) or with refractory anemia with ring sideroblasts (RARS). In contrast 11/26 (42%) patients with refractory anemia with excess of blasts (RAEB), or with RAEB in transformation (RAEBt), and 8/18 (44%) patients with chronic myelomonocytic leukemia (CMML) expressed c-mpl. In CMML patients, no correlation was found between c-mpl expression and any prognostic factor tested, nor with the course of the disease. In contrast, in RAEB and RAEBt, expression of c-mpl was correlated with high Bournemouth scoring (P < 0.005) and poor survival (P = 0.02) due to leukemic transformation. Forty-five per cent (5/11) of the c-mpl positive patients evolved towards AML with a mean follow-up of 10.5 months, while 13% (2/15) of the c-mpl negative patients developed a secondary leukemia, with a mean follow-up of 21.1 months. Moreover, in RAEB and RAEBt, a significant correlation was observed between c-mpl, CD34, megakaryocyte glycoprotein IIb (GPIIb) expression, and the presence of dysmegakaryopoiesis. These results indicate that patients with RAEB and RAEBt, with high expression of the c-mpl, CD34, and GPIIb genes, may identify a subgroup of patients with particularly poor prognosis, due to an increased risk of secondary leukemia. More aggressive therapy could be justified in these patients.