Craig C. Hofmeister

Initial genome sequencing and analysis of multiple myeloma

Multiple myeloma is an incurable malignancy of plasma cells, and its pathogenesis is poorly understood. Here we report the massively parallel sequencing of 38 tumour genomes and their comparison to matched normal DNAs. Several new and unexpected oncogenic mechanisms were suggested by the pattern of somatic mutation across the data set. These include the mutation of genes involved in protein translation (seen in nearly half of the patients), genes involved in histone methylation, and genes involved in blood coagulation. In addition, a broader than anticipated role of NF-κB signalling was indicated by mutations in 11 members of the NF-κB pathway. Of potential immediate clinical relevance, activating mutations of the kinase BRAF were observed in 4% of patients, suggesting the evaluation of BRAF inhibitors in multiple myeloma clinical trials. These results indicate that cancer genome sequencing of large collections of samples will yield new insights into cancer not anticipated by existing knowledge.

Lenalidomide after stem-cell transplantation for multiple myeloma

BACKGROUND Data are lacking on whether lenalidomide maintenance therapy prolongs the time to disease progression after autologous hematopoietic stem-cell transplantation in patients with multiple myeloma. METHODS Between April 2005 and July 2009, we randomly assigned 460 patients who were younger than 71 years of age and had stable disease or a marginal, partial, or complete response 100 days after undergoing stem-cell transplantation to lenalidomide or placebo, which was administered until disease progression. The starting dose of lenalidomide was 10 mg per day (range, 5 to 15). RESULTS The study-drug assignments were unblinded in 2009, when a planned interim analysis showed a significantly longer time to disease progression in the lenalidomide group. At unblinding, 20% of patients who received lenalidomide and 44% of patients who received placebo had progressive disease or had died (P<0.001); of the remaining 128 patients who received placebo and who did not have progressive disease, 86 crossed over to lenalidomide. At a median follow-up of 34 months, 86 of 231 patients who received lenalidomide (37%) and 132 of 229 patients who received placebo (58%) had disease progression or had died. The median time to progression was 46 months in the lenalidomide group and 27 months in the placebo group (P<0.001). A total of 35 patients who received lenalidomide (15%) and 53 patients who received placebo (23%) died (P=0.03). More grade 3 or 4 hematologic adverse events and grade 3 nonhematologic adverse events occurred in patients who received lenalidomide (P<0.001 for both comparisons). Second primary cancers occurred in 18 patients who received lenalidomide (8%) and 6 patients who received placebo (3%). CONCLUSIONS Lenalidomide maintenance therapy, initiated at day 100 after hematopoietic stem-cell transplantation, was associated with more toxicity and second cancers but a significantly longer time to disease progression and significantly improved overall survival among patients with myeloma. (Funded by the National Cancer Institute; ClinicalTrials.gov number, NCT00114101.).

Widespread Genetic Heterogeneity in Multiple Myeloma: Implications for Targeted Therapy

We performed massively parallel sequencing of paired tumor/normal samples from 203 multiple myeloma (MM) patients and identified significantly mutated genes and copy number alterations and discovered putative tumor suppressor genes by determining homozygous deletions and loss of heterozygosity. We observed frequent mutations in KRAS (particularly in previously treated patients), NRAS, BRAF, FAM46C, TP53, and DIS3 (particularly in nonhyperdiploid MM). Mutations were often present in subclonal populations, and multiple mutations within the same pathway (e.g., KRAS, NRAS, and BRAF) were observed in the same patient. In vitro modeling predicts only partial treatment efficacy of targeting subclonal mutations, and even growth promotion of nonmutated subclones in some cases. These results emphasize the importance of heterogeneity analysis for treatment decisions.

The PD-1/PD-L1 axis modulates the natural killer cell versus multiple myeloma effect: a therapeutic target for CT-011, a novel monoclonal anti-PD-1 antibody.

T-cell expression of programmed death receptor-1 (PD-1) down-regulates the immune response against malignancy by interacting with cognate ligands (eg, PD-L1) on tumor cells; however, little is known regarding PD-1 and natural killer (NK) cells. NK cells exert cytotoxicity against multiple myeloma (MM), an effect enhanced through novel therapies. We show that NK cells from MM patients express PD-1 whereas normal NK cells do not and confirm PD-L1 on primary MM cells. Engagement of PD-1 with PD-L1 should down-modulate the NK-cell versus MM effect. We demonstrate that CT-011, a novel anti-PD-1 antibody, enhances human NK-cell function against autologous, primary MM cells, seemingly through effects on NK-cell trafficking, immune complex formation with MM cells, and cytotoxicity specifically toward PD-L1(+) MM tumor cells but not normal cells. We show that lenalidomide down-regulates PD-L1 on primary MM cells and may augment CT-011s enhancement of NK-cell function against MM. We demonstrate a role for the PD-1/PD-L1 signaling axis in the NK-cell immune response against MM and a role for CT-011 in enhancing the NK-cell versus MM effect. A phase 2 clinical trial of CT-011 in combination with lenalidomide for patients with MM should be considered.

Ex vivo expansion of umbilical cord blood stem cells for transplantation: growing knowledge from the hematopoietic niche

Umbilical cord blood transplantation (UCBT) in adults is limited by the small number of primitive hematopoietic stem cells (HSC) in each graft, resulting in delayed engraftment post transplant, and both short- and long-term infectious complications. Initial efforts to expand UCB progenitors ex vivo have resulted in expansion of mature rather than immature HSC, confounded by the inability to accurately and reliably measure long-term reconstituting cells. Ex vivo expansion of UCB HSC has failed to improve engraftment because of resulting defects that promote apoptosis, disrupt marrow homing and initiate cell cycling. Here we discuss the future of ex vivo expansion, which we suggest will include the isolation of immature hematopoietic progenitors on the basis of function rather than surface phenotype and will employ both cytokines and stroma to maintain and expand the stem cell niche. We suggest that ex vivo expansion could be enhanced by manipulating newly discovered signaling pathways (Notch, Wnt, bone morphogenetic protein 4 and Tie2/angiopoietin-1) and intracellular mediators (phosphatase and tensin homolog and glycogen synthase kinase-3) in a manner that promotes HSC expansion with less differentiation. Improved methods for ex vivo expansion will make UCBT available to more patients, decrease engraftment times and allow more rapid immune reconstitution post transplant.

Pomalidomide alone or in combination with low-dose dexamethasone in relapsed and refractory multiple myeloma: a randomized phase 2 study

This multicenter, open-label, randomized phase 2 study assessed the efficacy and safety of pomalidomide (POM) with/without low-dose dexamethasone (LoDEX) in patients with relapsed/refractory multiple myeloma (RRMM). Patients who had received ≥2 prior therapies (including lenalidomide [LEN] and bortezomib [BORT]) and had progressed within 60 days of their last therapy were randomized to POM (4 mg/day on days 1-21 of each 28-day cycle) with/without LoDEX (40 mg/week). The primary end point was progression-free survival (PFS). In total, 221 patients (median 5 prior therapies, range 1-13) received POM+LoDEX (n = 113) or POM (n = 108). With a median follow-up of 14.2 months, median PFS was 4.2 and 2.7 months (hazard ratio = 0.68, P = .003), overall response rates (ORRs) were 33% and 18% (P = .013), median response duration was 8.3 and 10.7 months, and median overall survival (OS) was 16.5 and 13.6 months, respectively. Refractoriness to LEN, or resistance to both LEN and BORT, did not affect outcomes with POM+LoDEX (median PFS 3.8 months for both; ORRs 30% and 31%; and median OS 16 and 13.4 months). Grade 3-4 neutropenia occurred in 41% (POM+LoDEX) and 48% (POM); no grade 3-4 peripheral neuropathy was reported. POM+LoDEX was effective and generally well tolerated and provides an important new treatment option for RRMM patients who have received multiple prior therapies. This trial was registered at www.clinicaltrials.gov as #NCT00833833.

CS1-specific chimeric antigen receptor (CAR)-engineered natural killer cells enhance in vitro and in vivo antitumor activity against human multiple myeloma

Multiple myeloma (MM) is an incurable hematological malignancy. Chimeric antigen receptor (CAR)-expressing T cells have been demonstrated successfully in the clinic to treat B-lymphoid malignancies. However, the potential utility of antigen-specific CAR-engineered natural-killer (NK) cells to treat MM has not been explored. In this study, we determined whether CS1, a surface protein that is highly expressed on MM cells, can be targeted by CAR NK cells to treat MM. We successfully generated a viral construct of a CS1-specific CAR and expressed it in human NK cells. In vitro, CS1-CAR NK cells displayed enhanced MM cytolysis and interferon-γ (IFN-γ) production, and showed a specific CS1-dependent recognition of MM cells. Ex vivo, CS1-CAR NK cells also showed similarly enhanced activities when responding to primary MM tumor cells. More importantly, in an aggressive orthotopic MM xenograft mouse model, adoptive transfer of NK-92 cells expressing CS1-CAR efficiently suppressed the growth of human IM9 MM cells and also significantly prolonged mouse survival. Thus, CS1 represents a viable target for CAR-expressing immune cells, and autologous or allogeneic transplantation of CS1-specific CAR NK cells may be a promising strategy to treat MM.

A phase 1 trial of the anti-KIR antibody IPH2101 in patients with relapsed/refractory multiple myeloma.

Natural killer (NK) cells elicit cytotoxicity against multiple myeloma (MM); however, MM cells express HLA class I molecules as ligands to NK cell inhibitory killer immunoglobulin-like receptors (KIRs) as a means of immunoevasion. KIR-ligand mismatch may improve outcomes in allogeneic transplantation for MM. Extrapolating on this concept, we conducted a phase 1 trial of IPH2101, an anti-KIR antibody, in patients with relapsed/refractory MM. IPH2101 was administered intravenously every 28 days in 7 dose-escalated cohorts (0.0003-3 mg/kg) for up to 4 cycles. Pharmacokinetic, pharmacodynamic, and correlative immunologic studies were completed. A total of 32 patients were enrolled. The biologic endpoint of full KIR2D occupancy across the dosing cycle was achieved without dose-limiting toxicity or maximally tolerated dose. One severe adverse event was noted. Pharmacokinetic and pharmacodynamic findings approximated preclinical predictions, and IPH2101 enhanced ex vivo patient-derived NK cell cytotoxicity against MM. No objective responses were seen. No evidence of autoimmunity was observed. These findings suggest that IPH2101 is safe and tolerable at doses that achieve full inhibitory KIR saturation, and this approach warrants further development in MM. This trial was registered at www.clinicaltrials.gov as #NCT00552396.

Elotuzumab directly enhances NK cell cytotoxicity against myeloma via CS1 ligation: evidence for augmented NK cell function complementing ADCC

Abstract Elotuzumab is a monoclonal antibody in development for multiple myeloma (MM) that targets CS1, a cell surface glycoprotein expressed on MM cells. In preclinical models, elotuzumab exerts anti-MM efficacy via natural killer (NK)-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). CS1 is also expressed at lower levels on NK cells where it acts as an activating receptor. We hypothesized that elotuzumab may have additional mechanisms of action via ligation of CS1 on NK cells that complement ADCC activity. Herein, we show that elotuzumab appears to induce activation of NK cells by binding to NK cell CS1 which promotes cytotoxicity against CS1(+) MM cells but not against autologous CS1(+) NK cells. Elotuzumab may also promote CS1–CS1 interactions between NK cells and CS1(+) target cells to enhance cytotoxicity in a manner independent of ADCC. NK cell activation appears dependent on differential expression of the signaling intermediary EAT-2 which is present in NK cells but absent in primary, human MM cells. Taken together, these data suggest elotuzumab may enhance NK cell function directly and confer anti-MM efficacy by means beyond ADCC alone.

IPH2101, a novel anti-inhibitory KIR antibody, and lenalidomide combine to enhance the natural killer cell versus multiple myeloma effect.

Multiple myeloma (MM) patients who receive killer cell Ig-like receptor (KIR) ligand-mismatched, T cell-depleted, allogeneic transplantation may have a reduced risk of relapse compared with patients who receive KIR ligand-matched grafts, suggesting the importance of this signaling axis in the natural killer (NK) cell-versus-MM effect. Expanding on this concept, IPH2101 (1-7F9), an anti-inhibitory KIR mAb, enhances NK-cell function against autologous MM cells by blocking the engagement of inhibitory KIR with cognate ligands, promoting immune complex formation and NK-cell cytotoxicity specifically against MM cell targets but not normal cells. IPH2101 prevents negative regulatory signals by inhibitory KIR, whereas lenalidomide augments NK-cell function and also appears to up-regulate ligands for activating NK-cell receptors on MM cells. Lenalidomide and a murine anti-inhibitory NK-cell receptor Ab mediate in vivo rejection of a lenalidomide-resistant tumor. These mechanistic, preclinical data support the use of a combination of IPH2101 and lenalidomide in a phase 2 trial for MM.