Refractory or relapsed aggressive B-cell lymphoma failing (R)-CHOP: an analysis of patients treated on the RICOVER-60 trial.

BACKGROUND: The prognosis of elderly patients with aggressive B-non-Hodgkin's lymphoma after first lymphoma-related treatment failure (TF-L) is not well described. METHODS: We analysed patient characteristics including the presence of MYC rearrangements and MYC-expression immunohistochemistry (IHC) at diagnosis and modalities of salvage therapy and their impact on the prognosis of patients between 61 and 80 years who had been treated on the RICOVER-60 trial. RESULTS: TF-L occurred in 301 of the 1222 (24.6%) patients; 297 patients could be analysed. Prognosis was extremely poor in patients with primary progressive disease or early relapse (≤12 months) with median survivals of 3.3 and 6.4 months. Survival after TF-L was significantly lower in patients pretreated with R-CHOP compared with CHOP (23.0% versus 36.4% at 2 years, P = 0.016). In patients with MYC translocation at diagnosis Rituximab reduced the risk of TF-L from 58.8% to 26.3%. Survival after TF-L was significant longer for patients after CHOP without MYC translocations (31.8% versus 0% at 2 years, P < 0.001) or negative MYC-IHC (41.0% versus 16.8% at 2 years, P = 0.017) but not after R-CHOP. 224 patients (75.4%) received salvage therapy. Rituximab was part of salvage therapy in 57.4% and improved 2-year survival rate from 20.7% to 46.8% (P < 0.001). The benefit of R was significant after first-line CHOP [2-year overall survival (OS) 49.6% versus 19.1%, P < 0.001] as well as after R-CHOP (2-year OS 33.1% and 22.5%, P = 0.034). For patients pretreated with R-CHOP long-term survival was below 15% regardless of the treatment chosen. CONCLUSION: MYC rearrangement and IHC are adverse prognostic factors after TF-L for CHOP treated patients, rituximab as part of first-line therapy reduced the effects of MYC-break. Rituximab improves results of any type of salvage therapy; however, survival after progression/relapse of aggressive B-cell lymphoma in elderly patients pretreated with (R)-CHOP is poor regardless of treatment chosen.

A novel selective small-molecule PI3K inhibitor is effective against human multiple myeloma in vitro and in vivo.

Developing effective therapies against multiple myeloma (MM) is an unresolved challenge. Phosphatidylinositol-3-kinase (PI3K) activation may be associated with tumor progression and drug resistance, and inhibiting PI3K can induce apoptosis in MM cells. Thus, targeting of PI3K is predicted to increase the susceptibility of MM to anticancer therapy. The lead compound of a novel class of PI3K inhibitors, BAY80-6946 (IC50=0.5 nM against PI3K-α), was highly efficacious in four different MM cell lines, where it induced significant antitumoral effects in a dose-dependent manner. The compound inhibited cell cycle progression and increased apoptosis (P<0.001 compared with controls). Moreover, it abrogated the stimulation conferred by insulin-like growth-factor-1, a mechanism relevant for MM progression. These cellular effects were paralleled by decreased Akt phosphorylation, the main downstream target of PI3K. Likewise, profound antitumoral activity was observed ex vivo, as BAY80-6946 significantly inhibited proliferation of freshly isolated myeloma cells from three patients (P<0.001 compared with vehicle). In addition, BAY80-6946 showed convincing in vivo activity against the human AMO-1 and MOLP-8 myeloma cell lines in a preclinical murine xenograft model, where treatment with 6 mg/kg every other day for 2 weeks reduced the cell numbers by 87.0% and 69.3%, respectively (P<0.001 compared with vehicle), without overt toxicity in treated animals.

Intracellular ABC transporter A3 confers multidrug resistance in leukemia cells by lysosomal drug sequestration.

Multidrug resistance (MDR) seriously limits the efficacy of chemotherapy in patients with cancer and leukemia. Active transport across membranes is essential for such cellular drug resistance, largely provided by ATP-binding cassette (ABC) transport proteins. Intracellular drug sequestration contributes to MDR; however, a genuine intracellular ABC transport protein with MDR function has not yet been identified. Analyzing the intrinsic drug efflux capacity of leukemic stem cells, we found the ABC transporter A3 (ABCA3) to be expressed consistently in acute myeloid leukemia (AML) samples. Greater expression of ABCA3 is associated with unfavorable treatment outcome, and in vitro, elevated expression induces resistance toward a broad spectrum of cytostatic agents. ABCA3 remains localized within the limiting membranes of lysosomes and multivesicular bodies, in which cytostatics are efficiently sequestered. In addition to AML, we also detected ABCA3 in a panel of lymphohematopoietic tissues and transformed cell lines. In conclusion, we identified subcellular drug sequestration mediated by the genuinely intracellular ABCA3 as being a clinically relevant mechanism of intrinsic MDR.

A distinct "side population" of cells in human tumor cells: implications for tumor biology and therapy.

Stem cells have an extensive capacity to proliferate, differentiate and self-renew. In many mammals, including humans, an adult stem cell subpopulation termed the "side population" (SP) has been identified. SP cells can rapidly efflux lipophilic fluorescent dyes to produce a characteristic profile based on fluorescence-activated flow cytometric analysis. Previous studies have demonstrated SP cells in bone marrow obtained from patients with acute myeloid leukemia, suggesting that these cells might be candidate leukemic stem cells, and recent studies have found a SP of tumor progenitor cells in human solid tumors. These new data indicate that the ability of malignant SP cells to expel anticancer drugs may directly improve their survival and sustain their clonogenicity during exposure to cytostatic drugs, allowing disease recurrence when therapy is withdrawn. Identification of a tumor progenitor population with intrinsic mechanisms for cytostatic drug resistance might also provide clues for improved therapeutic intervention.

A distinct "side population" of cells with high drug efflux capacity in human tumor cells.

A subset of stem cells, termed the "side population" (SP), has been identified in several tissues in mammalian species. These cells maintain a high efflux capability for antimitotic drugs. We have investigated whether functionally equivalent stem cells also may be detected in human cancers. We initially examined primary tumor cells from 23 patients with neuroblastoma and cell lines derived from a range of other tumors. A distinct SP was found in neuroblastoma cells from 15 of 23 patients (65%). The SP was capable of sustained expansion ex vivo and showed evidence for asymmetric division, generating both SP and non-SP progeny. These cells also expressed high levels of ABCG2 and ABCA3 transporter genes and had a greater capacity to expel cytotoxic drugs, such as mitoxantrone, resulting in better survival. A SP also was detected in breast cancer, lung cancer, and glioblastoma cell lines, suggesting that this phenotype defines a class of cancer stem cells with inherently high resistance to chemotherapeutic agents that should be targeted during the treatment of malignant disease.

[Diagnostic value of antibody detection against Candida germ tube antigens in a patient with hepatosplenic candidosis]. / Diagnostische Wertigkeit des Nachweises von Antikorpern gegen Candida-Keimschlauchantigene bei einer Patientin mit hepatolienaler Candidose.

In the case of a 53-year-old woman with acute myeloid leukaemia and fever in late aplasia after chemotherapy, invasive mycosis with characteristic involvement of liver and spleen was diagnosed. For the serological identification of Candida in the early phase of the infection, methods for the detection of antibodies against Candida antigens were compared. By ELISA-based detection of IgM and IgG antibodies against a mixture of Candida antigens (ESR 117G and 117M, Virion-Serion, Wurzburg, Germany) evidence for invasive candidosis was obtained significantly earlier (22 days) when compared with the immunofluorescence detection of IgG antibodies against Candida albicans germ tube antigens (Vircell, Granada, Spain). In the case of this patient, the detection of a humoral response against Candida germ tube antigens was of little diagnostic value.

A leukemic stem cell with intrinsic drug efflux capacity in acute myeloid leukemia.

The hematopoietic stem cell underlying acute myeloid leukemia (AML) is controversial. Flow cytometry and the DNA-binding dye Hoechst 33342 were previously used to identify a distinct subset of murine hematopoietic stem cells, termed the side population (SP), which rapidly expels Hoechst dye and can reconstitute the bone marrow of lethally irradiated mice. Here, the prevalence and pathogenic role of SP cells in human AML were investigated. Such cells were found in the bone marrow of more than 80% of 61 patients and had a predominant CD34(low/-) immunophenotype. Importantly, they carried cytogenetic markers of AML in all 11 cases of active disease examined and in 2 out of 5 cases in complete hematological remission. Comparison of daunorubicin and mitoxantrone fluorescence emission profiles revealed significantly higher drug efflux from leukemic SP cells than from non-SP cells. Three of 28 SP cell transplants generated overt AML-like disease in nonobese diabetic--severe combined immunodeficient mice. Low but persistent numbers of leukemic SP cells were detected by molecular and immunological assays in half of the remaining mice. Taken together, these findings indicate that SP cells are frequently involved in human AML and may be a target for leukemic transformation. They also suggest a mechanism by which SP cells could escape the effects of cytostatic drugs and might eventually contribute to leukemia relapse. (Blood. 2001;98:1166-1173)

Pin1 is overexpressed in breast cancer and cooperates with Ras signaling in increasing the transcriptional activity of c-Jun towards cyclin D1.

Phosphorylation on serines or threonines preceding proline (Ser/Thr-Pro) is a major signaling mechanism. The conformation of a subset of phosphorylated Ser/Thr-Pro motifs is regulated by the prolyl isomerase Pin1. Inhibition of Pin1 induces apoptosis and may also contribute to neuronal death in Alzheimer's disease. However, little is known about the role of Pin1 in cancer or in modulating transcription factor activity. Here we report that Pin1 is strikingly overexpressed in human breast cancers, and that its levels correlate with cyclin D1 levels in tumors. Overexpression of Pin1 increases cellular cyclin D1 protein and activates its promoter. Furthermore, Pin1 binds c-Jun that is phosphorylated on Ser63/73-Pro motifs by activated JNK or oncogenic Ras. Moreover, Pin1 cooperates with either activated Ras or JNK to increase transcriptional activity of c-Jun towards the cyclin D1 promoter. Thus, Pin1 is up-regulated in human tumors and cooperates with Ras signaling in increasing c-Jun transcriptional activity towards cyclin D1. Given the crucial roles of Ras signaling and cyclin D1 overexpression in oncogenesis, our results suggest that overexpression of Pin1 may promote tumor growth.

Plasmacytosis in acute myeloid leukemia: two cases of plasmacytosis and increased IL-6 production in the AML blast cells.

An increased plasma cell count in the bone marrow occurs in a subgroup of patients with acute myeloid leukemia (AML). The pathogenic mechanism for this plasmacytosis is unclear. In this report we describe two patients with AML and plasmacytosis who shared some features of their diseases. The morphological subtypes were AML M4 and M4eo; the leukemias were secondary to cytotoxic pretreatment, and complex cytogenetic changes were found in the leukemic cells of both patients. There was a marked increase in the number of bone marrow plasma cells in both cases and no monoclonal immunoglobulin was detectable. The IgH-CDR3 gene scan depicted a monoclonal IgH rearrangement in the bone marrow cells of one patient. Analysis of the cytokine production of the leukemic cells showed a high production of IL-6 of the leukemic blast cells in the in vitro cell culture and a high cytoplasmic IL-6 in the leukemic cells as revealed by immunocytology. We describe the clinical picture of a type of secondary AML with FAB M4 morphology associated with bone marrow plasmacytosis. We suggest that paracrine growth stimulation of plasma cells by paraneoplastic IL-6 production of the leukemic blast cells contributes to the plasmacytosis observed in patients with AML.

Renin in acute myeloid leukaemia blasts.

Hypokalaemia is a clinical phenomenon in patients with acute myeloid leukaemia (AML) to which activation of the renin-angiotensin system (RAS) may contribute. Recently monocytes were found to express renin, the key initializing enzyme of the RAS. By RT/PCR, transcripts for renin were detected in four of 18 bone marrow samples from patients with AML. Three leukaemic cell lines, isolated monocytes, bone marrow stromal cells and 25 peripheral blood and 24 bone marrow samples of normal controls were negative for renin transcripts. In view of the importance of local RAS in other tissues, the expression of renin in the bone marrow of AML patients warrants further investigation.

[Detection of disseminated carcinoma cells in bone marrow and peripheral blood in primary breast cancer with RT/PCR of parathyroid hormone-related protein (PTHrP)]. / Nachweis disseminierter Karzinomzellen im Knochenmark und peripheren Blut beim primären Mammakarzinom mittels RT/PCR des Parathyroid Hormone-related Proteins (PTHrP).

The analysis of tissue specific gene expression by reverse transcription based RT/PCR methods is currently evaluated as a method for the detection of tumor cell dissemination in patients with cancer. Breast cancer tissues express PTHrP and the level of PTHrP expression in the primary tumor correlates with the incidence of metastases in the bone. We applied a RT/PCR assay of PTHrP to detect tumor cells in the mononuclear cell fraction of peripheral blood (pb) and bone marrow (bm) of patients with newly diagnosed breast cancer. PTHrP positivity was found in 18/67 pb and 20/71 bm samples. In a median follow up of 23 months there were 7 metastatic relapses (4 osseous, 2 hepatic, 1 pulmonary) and 9 local relapses in patients with primary lymph node positive breast cancer. The hepatic and pulmonary relapses had been both PTHrP-PCR negative in pb and in bm. Of the 4 patients with metastatic relapses to the bone the samples of bm had been initially negative in all cases, the pb had been positive in 2 cases. Of the 9 patients with local recurrences the pb alone had been positive in 4 patients, 5 patients had been negative in both the pb and the bm. During the period of observation there was no local and metastatic relapse detectable in the group of patients with primary lymph node negative breast cancer. In summary the increased risk for local or systemic relapse would have been predictable by RT/PCR of PTHrP alone in pb in 4 of the 9 local and in 2 of the 7 early metastatic relapses. Further follow-up of the patient cohort analysed is needed to assess the value of the RT/PCR of PTHrP as a prognostic and predictive marker in patients with breast cancer.

In vitro activation of low-grade non-Hodgkin's lymphoma by murine fibroblasts, IL-4, anti-CD40 antibodies and the soluble CD40 ligand.

The in vitro analysis of growth regulation in low-grade B non-Hodgkin's lymphoma (B-NHL) is hampered by the rapid apoptotic death of the malignant B cells ex vivo. A complex culture system, using murine CDw32 transfected fibroblasts (LTK-cells), IL-4 and anti-CD40 mAb, has been established for the propagation of normal mature B cells in vitro. We investigated the influence of the different components of this coculture system on cell survival and apoptosis of B-NHL cells. Nine samples from patients with follicular lymphoma and from eight patients with immunocytoma were analyzed. No cell proliferation of B-NHL cells could be induced in the culture system. However, CDw32-transfected murine fibroblasts most efficiently supported cell viability of B-NHL cells with an increase in cell survival by 114% compared to the control (P = 0.047). IL-4 alone also had a stimulatory effect on cell survival of B-NHL cells after 6 days. In contrast, the soluble recombinant CD40 ligand gp39 and the anti-CD40 mAbs mAb89 and EA-5 did not prolong cell survival. CDw32 transfectants blocked apoptosis of B-NHL cells efficiently from 67% in the control to 16% (P = 0.001). Reduction in apoptosis was accompanied by an elevated bcl-2 protein expression. IL-4 or mAb89 did not further reduce apoptotic cell death in CDw32 transfectant-dependent cocultures. Our data underline the pivotal role of LTK- cells for cell survival of B-NHL cells in vitro. The efficient blockage of apoptosis associated with increased bcl-2 protein expression causes prolonged cell viability of the B-NHL cells.