Michael A. Linden

Profiling Bortezomib Resistance Identifies Secondary Therapies in a Mouse Myeloma Model

Multiple myeloma is a hematologic malignancy characterized by the proliferation of neoplastic plasma cells in the bone marrow. Although the first-to-market proteasome inhibitor bortezomib (Velcade) has been successfully used to treat patients with myeloma, drug resistance remains an emerging problem. In this study, we identify signatures of bortezomib sensitivity and resistance by gene expression profiling (GEP) using pairs of bortezomib-sensitive (BzS) and bortezomib-resistant (BzR) cell lines created from the Bcl-XL/Myc double-transgenic mouse model of multiple myeloma. Notably, these BzR cell lines show cross-resistance to the next-generation proteasome inhibitors, MLN2238 and carfilzomib (Kyprolis) but not to other antimyeloma drugs. We further characterized the response to bortezomib using the Connectivity Map database, revealing a differential response between these cell lines to histone deacetylase (HDAC) inhibitors. Furthermore, in vivo experiments using the HDAC inhibitor panobinostat confirmed that the predicted responder showed increased sensitivity to HDAC inhibitors in the BzR line. These findings show that GEP may be used to document bortezomib resistance in myeloma cells and predict individual sensitivity to other drug classes. Finally, these data reveal complex heterogeneity within multiple myeloma and suggest that resistance to one drug class reprograms resistant clones for increased sensitivity to a distinct class of drugs. This study represents an important next step in translating pharmacogenomic profiling and may be useful for understanding personalized pharmacotherapy for patients with multiple myeloma. Mol Cancer Ther; 12(6); 1140–50. ©2013 AACR.

Minimal residual disease prior to allogeneic hematopoietic cell transplantation in acute myeloid leukemia: a meta-analysis

Minimal residual disease prior to allogeneic hematopoietic cell transplantation has been associated with increased risk of relapse and death in patients with acute myeloid leukemia, but detection methodologies and results vary widely. We performed a systematic review and meta-analysis evaluating the prognostic role of minimal residual disease detected by polymerase chain reaction or multiparametric flow cytometry before transplant. We identified 19 articles published between January 2005 and June 2016 and extracted hazard ratios for leukemia-free survival, overall survival, and cumulative incidences of relapse and non-relapse mortality. Pre-transplant minimal residual disease was associated with worse leukemia-free survival (hazard ratio=2.76 [1.90–4.00]), overall survival (hazard ratio=2.36 [1.73–3.22]), and cumulative incidence of relapse (hazard ratio=3.65 [2.53–5.27]), but not non-relapse mortality (hazard ratio=1.12 [0.81–1.55]). These associations held regardless of detection method, conditioning intensity, and patient age. Adverse cytogenetics was not an independent risk factor for death or relapse. There was more heterogeneity among studies using flow cytometry-based than WT1 polymerase chain reaction-based detection (I2=75.1% vs. <0.1% for leukemia-free survival, 67.8% vs. <0.1% for overall survival, and 22.1% vs. <0.1% for cumulative incidence of relapse). These results demonstrate a strong relationship between pre-transplant minimal residual disease and post-transplant relapse and survival. Outcome heterogeneity among studies using flow-based methods may underscore site-specific methodological differences or differences in test performance and interpretation.

Reduced CXCR4 expression is associated with extramedullary disease in a mouse model of myeloma and predicts poor survival in multiple myeloma patients treated with bortezomib

Drug resistance to the proteasome inhibitor, bortezomib/VELCADE (Bz) is a significant clinical problem in the treatment of multiple myeloma (MM), an invariably fatal plasma cell malignancy of the bone marrow.1 Despite initial success and wide use in cocktail regimens for MM treatment,2 the majority of patients treated with Bz eventually relapse, many having developed ‘acquired’ Bz-resistant (BzR) disease.1 Thus, the elucidation of mechanisms by which Bz resistance may occur and the identification of novel intervention strategies are important translational aims. To identify mechanisms of acquired Bz resistance, we previously utilized in vitro cell lines from the Bcl-XL/Myc mouse model of plasma cell malignancy to systematically ascertain differences between Bz-sensitive (BzS) and derived BzR cells.3 We have employed this model system because malignant plasma cell lines isolated from these mice closely resemble human MM based on gene expression, chromosomal abnormalities and progression of disease in the bone marrow.3–5 Perhaps most importantly, from initially drug-sensitive tumor cell populations we are able to select for drug-resistant cells in vitro, adoptively transfer these cells back into syngeneic recipient mice, and recapitulate the drug-sensitive or -resistant phenotype following in vivo Bz treatment of recipient mice.3 Remarkably, the differences in Bz sensitivity in our mouse cell lines strongly correlated with differences in malignant plasma cell migration following adoptive transfer. BzR cells displayed a significantly reduced affinity for the bone marrow compartment, compared with their sensitive counterparts, and instead infiltrated extramedullary tissues more readily,3 suggesting the possibility that BzR plasma cells (PCs) are more likely to reside outside of the bone marrow milieu. Thus, we hypothesized that Bz treatment and acquisition of resistance selects for cells that can survive independent of the bone marrow microenvironment and that this phenotype may be due to the loss of cell-surface proteins that mediate the MM–bone marrow stromal cell (BMSC) interaction. We recently identified a 23-gene signature that distinguishes between BzS and BzR mouse cell lines in vitro and significantly predicts differences in patient outcomes in a human MM clinical trial that included Bz.3 We explored the gene expression profiling (GEP) data sets associated with these studies further, and of the 23 genes in this model, 5 genes including chemokine (C-X-C motif) receptor 4 (CXCR4), regulator of G-protein signaling 16 (RGS16), lectin, gaglactoside-binding, soluble, 1 (LGALS1), CD93 and cystathionine-beta-synthase (CBS) have been associated with the trafficking of cells within the immune system (Ingenuity Pathway Analysis). However, of these genes, CXCR4 has been most directly associated with PC migration in both humans and mice.6 We observed a loss in Cxcr4 mRNA expression in all four cases of BzR compared with BzS mouse cell lines.3 CXCR4 is known to be highly expressed on the surface of human plasma cells and contributes to bone marrow homing during normal plasma cell maturation through interaction with its ligand SDF-1α that is secreted by bone marrow stromal cells,6 making this gene an attractive candidate for our hypothesis. Therefore, we began by validating the protein expression of CXCR4 by flow cytometry in BzS and their derived BzR counterpart mouse cell lines we had isolated as previously described.3 The 2.5-fold average reduction in Cxcr4 mRNA expression that we observed by gene expression microarray3 was further reflected as a 1.5- (cell line 589) to 2 (cell line 595)-fold reduction in total (intracellular and surface) CXCR4 protein in the mouse BzR cell lines in vitro (Figure 1a), suggesting that this could be a functional change in the transcriptional regulation of Cxcr4 or that Bz selects for those cells that express lower levels of CXCR4. However, given that the original mRNA expression data were collected in the absence of cell death over a short-time period3 suggests the possibility that Bz contributes to the reduction of Cxcr4 mRNA expression. Figure 1 Low CXCR4 expression is associated with bortezomib-resistance and poor clinical outcomes in patients treated with bortezomib The reduction in CXCR4 expression in BzR cells combined with the in vivo data showing that BzR cells promote aggressive, extramedullary disease resulting in poorer survival3 suggests that CXCR4 could serve as a biomarker for patient survival. We next sought to determine whether reduced CXCR4 expression was associated with poorer survival in MM patients treated with Bz. The APEX drug trial provides initial GEP data from MM patients treated with either single-agent Bz or high-dose dexamethasone.7 We divided the data set into those patients with high versus low CXCR4 expression and assessed overall survival (OS) trends in both groups. As a single biomarker, low CXCR4 expression significantly distinguished those MM patients with poorer OS (P=0.039) in the Bz-treated arm of the clinical trial (Figure 1b), but not the dexamethasone arm (data not shown), suggesting that this could be a novel biomarker associated with poor response to Bz. Because the majority of MM patients are placed on a therapeutic cocktail of drugs that often includes Bz, we next queried the MM total therapy 3 (MMTT3) drug trial that reported GEP data for MM patients treated with VTD-PACE (Bz, thalidomide, dexamethasone, cisplatin, doxorubicin, cyclophosphamide, etoposide) in conjunction with tandem transplantation.8,9 As with the APEX drug trial, low CXCR4 expression significantly distinguished those MM patients with poorer event-free (P=0.013; left panel) and OS (P=0.010; right panel) survival (Figure 1c). Therefore, reduced CXCR4 expression predicts poorer survival in patients treated with Bz. Using the Bcl-XL/Myc model system of plasma cell malignancy, we identified CXCR4 as a single biomarker whose reduced expression is associated with poorer outcomes in MM patients being treated with Bz. CXCR4 expression is known to have a role in PC homing to the bone marrow.10 When injected back into syngeneic mice, BzR cells, which have reduced CXCR4 expression, display reduced BM homing compared with their BzS counterparts, resulting in a much more severe disease phenotype.3 This finding suggests that reduced CXCR4 expression correlates with increased disease severity and agrees with our analysis of both a single-agent Bz clinical trial (APEX7) and a clinical trial utilizing Bz in cocktail with additional agents (MMTT38), both of which show that low CXCR4 expression is significantly associated with worse outcome compared to patients with high CXCR4 expression. The biological role of CXCR4 is of particular interest given that studies have shown that the disruption of the interaction between BMSCs and MM cells (for example, by virtue of chemical inhibition of the CXCR4/SDF-1α axis) may increase sensitivity to Bz by mobilizing MM cells from the bone marrow microenvironment. 11 While the full mechanism of reduced CXCR4 expression in BzR cells awaits further investigation, we have evidence to suggest that Bz treatment promotes the reduction of CXCR4 mRNA, which we have previously reported.3 In turn, over time following continuous Bz treatment, reduced Cxcr4 expression may decrease the reliance of the MM cells on the bone marrow microenvironment and thus eventually promote extramedullary disease. Indeed, BzR disease presenting in patients as extramedullary masses has been reported.12 A circulating malignant clonal PC population has already been described by others as highly drug resistant and likely to contribute to disease relapse.13 Whether this population contributes to the proposed tumor-initiating and drug resistant MM ‘side population’,14 which also expresses lower CXCR4 transcript than the bulk tumor population (FZ, unpublished data), will require additional studies. In summary, these results support the use of CXCR4 as a valuable diagnostic biomarker that predicts clinical outcome in MM patients treated with Bz. The positive impact of cell-surface markers that may predict Bz response as a diagnostic tool in patient care could mean the early detection of BzR disease and improved OS through individualized medical treatment and new therapeutic targets (Supplementary Information).

Achieving stringent CR is essential before reduced-intensity conditioning allogeneic hematopoietic cell transplantation in AML

Reduced-intensity conditioning (RIC) allogeneic hematopoietic cell transplantation (allo-HCT) can cure patients with AML in CR. However, relapse after RIC allo-HCT may indicate heterogeneity in the stringency of CR. Strict definition of CR requires no evidence of leukemia by both morphologic and flow cytometric criteria. We re-evaluated 85 AML patients receiving RIC allo-HCT in CR to test if a strict definition of CR had direct implications for the outcome. These patients had leukemia immunophenotype documented at diagnosis and analyzed at allo-HCT. Eight (9.4%) had persistent leukemia by flow cytometric criteria at allo-HCT. The patients with immunophenotypic persistent leukemia had a significantly increased relapse (hazard ratio (HR): 3.7; 95% confidence interval (CI): 1.3–10.3, P=0.01) and decreased survival (HR: 2.9; 95% CI: 1.3–6.4, P<0.01) versus 77 patients in CR by both morphology and flow cytometry. However, the pre-allo-HCT bone marrow (BM) blast count (that is, 0–4%) was not significantly associated with risks of relapse or survival. These data indicate the presence of leukemic cells, but not the BM blast count affects survival. A strict morphologic and clinical lab flow cytometric definition of CR predicts outcomes after RIC allo-HCT, and therefore is critical to achieve at transplantation.

Utilization of translational bioinformatics to identify novel biomarkers of bortezomib resistance in multiple myeloma.

Multiple myeloma (MM) is an incurable malignant neoplasm hallmarked by a clonal expansion of plasma cells, the presence of a monoclonal protein in the serum and/or urine (M-spike), lytic bone lesions, and end organ damage. Clinical outcomes for patients with MM have improved greatly over the last decade as a result of the re-purposing of compounds such as thalidomide derivatives, as well as the development of novel chemotherapeutic agents including first and second generation proteasome inhibitors, bortezomib (Bz) and carfilzomib. Unfortunately, despite these improvements, the majority of patients relapse following treatment. While Bz, one of the most commonly used proteasome inhibitors, has been successfully incorporated into clinical practice, some MM patients have de novo resistance to Bz, and the majority of the remainder subsequently develop drug resistance following treatment. A significant gap in clinical care is the lack of a reliable clinical test that would predict which MM patients have or will subsequently develop Bz resistance. Thus, as Bz resistance remains a significant challenge, research efforts are needed to identify novel biomarkers of early Bz resistance, particularly when an early therapeutic intervention can be initiated. Recent advances in MM research indicate that genomic data can be extracted to identify novel biomarkers that can be utilized to select more effective, personalized treatment protocols for individual patients. Computationally integrating large patient databases with data from whole transcriptome profiling and laboratory-based models can potentially revolutionize our understanding of MM disease mechanisms. This systems-wide approach can provide rational therapeutic targets and novel biomarkers of risk and treatment response. In this review, we discuss the use of high-content datasets (predominantly gene expression profiling) to identify novel biomarkers of treatment response and resistance to Bz in MM.

Marked Variability in Reported Minimal Residual Disease Lower Level of Detection of 4 Hematolymphoid Neoplasms: A Survey of Participants in the College of American Pathologists Flow Cytometry Proficiency Testing Program

CONTEXT Flow cytometry is often applied to minimal residual disease (MRD) testing in hematolymphoid neoplasia. Because flow-based MRD tests are developed in the laboratory, testing methodologies and lower levels of detection (LODs) are laboratory dependent. OBJECTIVES To broadly survey flow cytometry laboratories about MRD testing in laboratories, if performed, including indications and reported LODs. DESIGN Voluntary supplemental questions were sent to the 549 laboratories participating in the College of American Pathologists (CAP) FL3-A Survey (Flow Cytometry-Immunophenotypic Characterization of Leukemia/Lymphoma) in the spring of 2014. RESULTS A total of 500 laboratories (91%) responded to the supplemental questions as part of the FL3-A Survey by April 2014; of those 500 laboratories, 167 (33%) currently perform MRD for lymphoblastic leukemia, 118 (24%) for myeloid leukemia, 99 (20%) for chronic lymphocytic leukemia, and 91 (18%) for plasma cell myeloma. Other indications include non-Hodgkin lymphoma, hairy cell leukemia, neuroblastoma, and myelodysplastic syndrome. Most responding laboratories that perform MRD for lymphoblastic leukemia reported an LOD of 0.01%. For myeloid leukemia, chronic lymphocytic leukemia, and plasma cell myeloma, most laboratories indicated an LOD of 0.1%. Less than 3% (15 of 500) of laboratories reported LODs of 0.001% for one or more MRD assays performed. CONCLUSIONS There is major heterogeneity in the reported LODs of MRD testing performed by laboratories subscribing to the CAP FL3-A Survey. To address that heterogeneity, changes to the Flow Cytometry Checklist for the CAP Laboratory Accreditation Program are suggested that will include new requirements that each laboratory (1) document how an MRD assays LOD is measured, and (2) include the LOD or lower limit of enumeration for flow-based MRD assays in the final diagnostic report.

Factor V Leiden and hepatic artery thrombosis after liver transplantation

Abstract:  Factor V Leiden (FVL) and other thrombophilias can be acquired during liver transplantation and can have a significant impact on clinical outcomes as well as cost. Standard practice does not include screening deceased donors for heritable thrombophilias, even if they have a personal history of thrombosis. Here we report a case of hepatic artery thrombosis in a liver recipient whose native and donor livers were heterozygous for FVL. The patient subsequently underwent a successful retransplant. FVL and its variants are expressed phenotypically as activated protein C (APC) resistance. We believe that testing liver donors (deceased or living) for APC resistance – a surrogate marker for the most common liver‐based thrombophilia – will reduce the incidence of thrombotic events by identifying a need for posttransplant prophylactic anticoagulation in patients at risk. The estimated cost of testing all liver donors in the US for APC resistance is less than the cost of two complications secondary to thrombosis. Testing for APC resistance may further improve outcome and reduce cost after liver transplantation.

Bortezomib Resistance Can Be Reversed by Induced Expression of Plasma Cell Maturation Markers in a Mouse In Vitro Model of Multiple Myeloma

Multiple myeloma (MM), the second most common hematopoietic malignancy, remains an incurable plasma cell (PC) neoplasm. While the proteasome inhibitor, bortezomib (Bz) has increased patient survival, resistance represents a major treatment obstacle as most patients ultimately relapse becoming refractory to additional Bz therapy. Current tests fail to detect emerging resistance; by the time patients acquire resistance, their prognosis is often poor. To establish immunophenotypic signatures that predict Bz sensitivity, we utilized Bz-sensitive and -resistant cell lines derived from tumors of the Bcl-XL/Myc mouse model of PC malignancy. We identified significantly reduced expression of two markers (CD93, CD69) in “acquired” (Bz-selected) resistant cells. Using this phenotypic signature, we isolated a subpopulation of cells from a drug-naïve, Bz-sensitive culture that displayed “innate” resistance to Bz. Although these genes were identified as biomarkers, they may indicate a mechanism for Bz-resistance through the loss of PC maturation which may be induced and/or selected by Bz. Significantly, induction of PC maturation in both “acquired” and “innate” resistant cells restored Bz sensitivity suggesting a novel therapeutic approach for reversing Bz resistance in refractory MM.

Genetic Signature of Histiocytic Sarcoma Revealed by a Sleeping Beauty Transposon Genetic Screen in Mice

Histiocytic sarcoma is a rare, aggressive neoplasm that responds poorly to therapy. Histiocytic sarcoma is thought to arise from macrophage precursor cells via genetic changes that are largely undefined. To improve our understanding of the etiology of histiocytic sarcoma we conducted a forward genetic screen in mice using the Sleeping Beauty transposon as a mutagen to identify genetic drivers of histiocytic sarcoma. Sleeping Beauty mutagenesis was targeted to myeloid lineage cells using the Lysozyme2 promoter. Mice with activated Sleeping Beauty mutagenesis had significantly shortened lifespan and the majority of these mice developed tumors resembling human histiocytic sarcoma. Analysis of transposon insertions identified 27 common insertion sites containing 28 candidate cancer genes. Several of these genes are known drivers of hematological neoplasms, like Raf1, Fli1, and Mitf, while others are well-known cancer genes, including Nf1, Myc, Jak2, and Pten. Importantly, several new potential drivers of histiocytic sarcoma were identified and could serve as targets for therapy for histiocytic sarcoma patients.

Hematopoietic Cell Transplantation for Mantle Cell Lymphoma: Predictive Value of Pretransplant Positron Emission Tomography/Computed Tomography and Bone Marrow Evaluations for Outcomes

BACKGROUND The prognostic roles of 18F-fludeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) imaging and marrow involvement evaluation on outcomes following autologous and allogeneic hematopoietic cell transplantation (HCT) for mantle cell lymphoma (MCL) are uncertain and require more data. PATIENTS AND METHODS We categorized 66 patients with MCL who received HCT (38 autologous and 28 allogeneic) on the basis of pre-HCT residual disease (RD) status as assessed by marrow MCL morphology and flow/molecular analysis and PET/CT imaging to RD positive (RD(+)) (either or both measures positive) and RD(-) (both negative). We analyzed the predictive value of these RD detection methods on transplant outcomes. RESULTS The 2-year relapse rate after autograft was significantly higher in pre-HCT RD(+) patients (46% [95% CI 16-77%]) than in patients who were RD(-) (19% [95% CI 0-42%]; P = .02), leading to worse 5-year disease-free survival (DFS) in RD(+) patients (46% [95% CI 14%-73%] vs. 68% [95% CI 33-87%], P = .04). In multivariate analysis, RD(+) status was associated with a reduction in DFS (hazard ratio, 5.6; P = .02). Most allogeneic HCT recipients had advanced disease and most were RD(+) (12 PET/CT(+); 5 marrow-positive). The 5-year DFS and relapse rates after allogeneic HCT were 34% and 25% for all patients and 40% and 33% for RD(+) recipients, suggesting that active disease at the time of allograft does not preclude long-term remissions in advanced MCL. CONCLUSION Both autologous and allogeneic HCT lead to promising long-term survival. RD detected prior to autograft was associated with increased relapse and worse 5 year DFS. Allograft recipients had favorable long-term outcomes even in presence of pre-HCT detectable disease.