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To begin to understand the mechanisms that regulate self-renewal, differentiation, and transformation of human hematopoietic stem cells or to evaluate the efficacy of novel treatment modalities, stem cells need to be studied in their own species-specific microenvironment. By implanting ceramic scaffolds coated with human mesenchymal stromal cells into immune-deficient mice, we were able to mimic the human bone marrow niche. Thus, we have established a human leukemia xenograft mouse model in which a large cohort of patient samples successfully engrafted, which covered all of the important genetic and risk subgroups. We found that by providing a humanized environment, stem cell self-renewal properties were better maintained as determined by serial transplantation assays and genome-wide transcriptome studies, and less clonal drift was observed as determined by exome sequencing. The human leukemia xenograft mouse models that we have established here will serve as an excellent resource for future studies aimed at exploring novel therapeutic approaches., (© 2016 by The American Society of Hematology.)

The prognosis of multiple myeloma (MM) patients who become refractory to lenalidomide and bortezomib is very poor, indicating the need for new therapeutic strategies for these patients. Next to the development of new drugs, the strategy of combining agents with synergistic activity may also result in clinical benefit for patients with advanced myeloma. We have previously shown in a retrospective analysis that lenalidomide combined with continuous low-dose cyclophosphamide and prednisone (REP) had remarkable activity in heavily pretreated, lenalidomide-refractory MM patients. To evaluate this combination prospectively, we initiated a phase 1/2 study to determine the optimal dose and to assess its efficacy and safety in lenalidomide-refractory MM patients. The maximum tolerated dose (MTD) was defined as 25 mg lenalidomide (days 1-21/28 days), combined with continuous cyclophosphamide (50 mg/d) and prednisone (20 mg/d). At the MTD (n = 67 patients), the overall response rate was 67%, and at least minimal response was achieved in 83% of the patients. Median progression-free survival and overall survival were 12.1 and 29.0 months, respectively. Similar results were achieved in the subset of patients with lenalidomide- and bortezomib-refractory disease as well as in patients with high-risk cytogenetic abnormalities, defined as t(4;14), t(14;16), del(17p), and/or ampl(1q) as assessed by fluorescence in situ hybridization. Neutropenia (22%) and thrombocytopenia (22%) were the most common grade 3-4 hematologic adverse events. Infections (21%) were the most common grade 3-5 nonhematologic adverse events. In conclusion, the addition of continuous low-dose oral cyclophosphamide to lenalidomide and prednisone offers a new therapeutic perspective for multidrug refractory MM patients. This trial was registered at www.clinicaltrials.gov as #NCT01352338., (© 2016 by The American Society of Hematology.)

The anti-CD38 monoclonal antibody daratumumab is well tolerated and has high single agent activity in heavily pretreated relapsed and refractory multiple myeloma (MM). However, not all patients respond, and many patients eventually develop progressive disease to daratumumab monotherapy. We therefore examined whether pretreatment expression levels of CD38 and complement-inhibitory proteins (CIPs) are associated with response and whether changes in expression of these proteins contribute to development of resistance. In a cohort of 102 patients treated with daratumumab monotherapy (16 mg/kg), we found that pretreatment levels of CD38 expression on MM cells were significantly higher in patients who achieved at least partial response (PR) compared with patients who achieved less than PR. However, cell surface expression of the CIPs, CD46, CD55, and CD59, was not associated with clinical response. In addition, CD38 expression was reduced in both bone marrow-localized and circulating MM cells, following the first daratumumab infusion. CD38 expression levels on MM cells increased again following daratumumab discontinuation. In contrast, CD55 and CD59 levels were significantly increased on MM cells only at the time of progression. All-trans retinoic acid increased CD38 levels and decreased CD55 and CD59 expression on MM cells from patients who developed daratumumab resistance, to approximately pretreatment values. This resulted in significant enhancement of daratumumab-mediated complement-dependent cytotoxicity. Together, these data demonstrate an important role for CD38 and CIP expression levels in daratumumab sensitivity and suggest that therapeutic combinations that alter CD38 and CIP expression levels should be investigated in the treatment of MM. These trials were registered at www.clinicaltrials.gov as #NCT00574288 (GEN501) and #NCT01985126 (SIRIUS)., (© 2016 by The American Society of Hematology.)

Interactions within the hematopoietic niche in the BM microenvironment are essential for maintenance of the stem cell pool. In addition, this niche is thought to serve as a sanctuary site for malignant progenitors during chemotherapy. Therapy resistance induced by interactions with the BM microenvironment is a major drawback in the treatment of hematologic malignancies and bone-metastasizing solid tumors. To date, studying these interactions was hampered by the lack of adequate in vivo models that simulate the human situation. In the present study, we describe a unique human-mouse hybrid model that allows engraftment and outgrowth of normal and malignant hematopoietic progenitors by implementing a technology for generating a human bone environment. Using luciferase gene marking of patient-derived multiple myeloma cells and bioluminescent imaging, we were able to follow pMM cells outgrowth and to visualize the effect of treatment. Therapeutic interventions in this model resulted in equivalent drug responses as observed in the corresponding patients. This novel human-mouse hybrid model creates unprecedented opportunities to investigate species-specific microenvironmental influences on normal and malignant hematopoietic development, and to develop and personalize cancer treatment strategies.

HMG-CoA reductase is the rate-limiting enzyme of the mevalonate pathway leading to the formation of cholesterol and isoprenoids such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP). The inhibition of HMG-CoA reductase by lovastatin induced apoptosis in plasma cell lines and tumor cells from patients with multiple myeloma. Here we show that cotreatment with mevalonate or geranylgeranyl moieties, but not farnesyl groups, rescued myeloma cells from lovastatin-induced apoptosis. In addition, the inhibition of geranylgeranylation by specific inhibition of geranylgeranyl transferase I (GGTase I) induced the apoptosis of myeloma cells. Apoptosis triggered by the inhibition of geranylgeranylation was associated with reduction of Mcl-1 protein expression, collapse of the mitochondrial transmembrane potential, expression of the mitochondrial membrane protein 7A6, cytochrome c release from mitochondria into the cytosol, and stimulation of caspase-3 activity. These results imply that protein geranylgeranylation is critical for regulating myeloma tumor cell survival, possibly through regulating Mcl-1 expression. Our results show that pharmacologic agents such as lovastatin or GGTase inhibitors may be useful in the treatment of multiple myeloma.

This study was undertaken to assess the significance of lung-resistance related protein (LRP) expression in plasma cells from untreated multiple myeloma (MM) patients and to determine whether LRP was associated with a poor response and survival in patients treated with different dose regimens of melphalan. Seventy untreated patients received conventional oral dose melphalan (0.25 mg/kg, day 1 to 4) combined with prednisone (MP) or intravenous intermediate-IDM; 70 mg/m2) or high- (140 mg/m2) dose Melphalan (HDM). LRP expression was assessed with immunocytochemistry using the LRP-56 monoclonal antibody. LRP expression was found in 47% of patients. In the MP treated patients, LRP expression was a significant prognostic factor regarding response induction (P < .05), event free survival (P < .003), and overall survival (P < .001). In the intensified dose melphalan treated patients LRP did not have a prognostic value. The response rates of LRP-positive patients to MP and IDM/HDM were 18% versus 81%, respectively (P < .0001). We conclude that LRP is frequently expressed in untreated MM patients and is an independent predictor for response and survival in patients treated with MP. Pretreatment assessment of LRP identifies a subpopulation of patients with a poor probability of response to conventional dose melphalan. Dose intensification of melphalan is likely to overcome LRP-mediated resistance.

CD44 variant isoforms (CD44v) are generated by alternative splicing of the nuclear RNA resulting in the expression of additional protein domains in the extracellular region of the CD44 standard molecule (CD44s). In multiple myeloma (MM), CD44 mediates binding of tumor cells to stroma and regulates interleukin-6 production. To evaluate the role of CD44v isoforms in MM, CD44v expression was analyzed by immunohistochemical staining of 64 bone marrow biopsies from 38 MM patients. Expression of variant isoforms containing the 9v domain was observed in 36% of cases and was associated with an advanced stage (P < .02; n = 61), a progressive disease (P < .001; n = 61), and a shorter overall survival (P < .02; n = 36). In contrast, 3v, 4v, 6v, or 10v isoforms were detected only in a small percentage of the patients. To analyze the exon composition in RNA-transcripts, reverse transcriptase polymerase chain reaction analyses followed by Southern hybridization with exon-specific probes were performed in fluorescence-activated cell sorted myeloma plasma cells. Tumors expressing the 9v domain showed complex, 9v-containing transcripts in combinations with the 3v, 7v, 8v, and 10v exons. Identical transcripts were detected in several myeloma cell lines and in a Ki-1 B-immunoblastic lymphoma. Similar to high-grade non-Hodgkin's lymphoma and gastric and renal cell carcinoma, overexpression of 9v-containing isoforms in MM is related to an unfavorable clinical presentation and represents a new prognostic parameter.

Long-term bone marrow cultures (LTBMC) from patients with multiple myeloma (MM) and normal donors were analyzed for immunophenotype and cytokine production. Both LTBMC adherent cells from myeloma and normal donor origin expressed CD10, CD13, the adhesion molecules CD44, CD54, vascular cell adhesion molecule 1, very late antigen 2 (VLA-2), and VLA-5, and were positive for extracellular matrix components fibronectin, laminin, and collagen types 3 and 4. LTBMC from myeloma patients and normal donors spontaneously secreted interleukin-6 (IL-6). However, levels of IL-6 correlated with the stage of disease; highest levels of IL-6 were found in LTBMC from patients with active myeloma. To identify the origin of IL-6 production, LTBMC from MM patients and normal donors were cocultured with BM-derived myeloma cells and cells from myeloma cell lines. IL-6 was induced by plasma cell lines that adhered to LTBMC such as ARH-77 and RPMI-8226, but not by nonadhering cell lines U266 and FRAVEL. Myeloma cells strongly stimulated IL-6 secretion in cocultures with LTBMC adherent cells from normal donors and myeloma patients. When direct cellular contact between LTBMC and plasma cells was prevented by tissue-culture inserts, no IL-6 production was induced. This implies that intimate cell-cell contact is a prerequisite for IL-6 induction. Binding of purified myeloma cells to LTBMC adherent cells was partly inhibited by monoclonal antibodies against adhesion molecules VLA-4, CD44, and lymphocyte function-associated antigen 1 (LFA-1) present on the plasma cell. Antibodies against VLA-4, CD29, and LFA-1 also inhibited the induced IL-6 secretion in plasma cell-LTBMC cocultures. In situ hybridization studies performed before and after coculture with plasma cells indicated that LTBMC adherent cells produce the IL-6. These results suggest that the high levels of IL-6 found in LTBMC of MM patients with active disease are a reflection of their previous contact with tumor cells in vivo. These results provide a new perspective on tumor growth in MM and emphasize the importance of plasma cell-LTBMC interaction in the pathophysiology of MM.

Lymphocyte function-associated antigen-1 (LFA-1) (CD11a/CD18) expression on bone marrow-derived plasma cells from normal individuals, patients with monoclonal gammopathies of undetermined significance (MGUS), and patients with multiple myeloma (MM) was studied by immunofluorescence microscopy and flow cytometry using a new monoclonal antibody (MoAb) F8.8. This MoAb recognizes the alpha-chain (CD11a) of LFA-1 as determined by immunoprecipitation, and inhibits T-cell-induced cytotoxicity. Although the F8.8 MoAb stains unstimulated peripheral blood T cells with the same mean fluorescence intensity as other anti-CD11a MoAbs, it proved to be superior in detecting CD11a on plasma cells as compared with reference MoAbs. Using the anti-CD11a MoAb F8.8, a strong correlation was found between LFA-1 expression and disease activity in MM, as defined by clinical performance and serum M-protein level. Hardly any LFA-1+ plasma cells were detected in normal individuals, patients with MGUS, and MM patients in a nonactive phase of their disease, while plasma cells of some MM patients with active disease and all patients with fulminant disease expressed LFA-1. Plasma cell LFA-1 expression correlated well with the labeling index (LI) of the tumors in the individual patients. The relation between LFA-1 expression and the tumor growth suggests an involvement of this adhesion molecule in cellular interactions resulting in plasma cell proliferation.