Witzig, Thomas E.

Abstract Marginal zone B-cell lymphomas (MZL) represent a sub-group of indolent B-cell lymphomas, normally localized in the “marginal zone” of the secondary lymphoid follicles. Depending on the site of involvement, three distinct sub-types have been identified: extra nodal MZL of MALT type, splenic MZL (SMZL), and nodal MZL (NMZL). MALT lymphoma is the most frequent of the MZL subtypes, representing 70% of MZL and 7% to 8% of non-Hodgkin lymphomas. SMZL and NMZL comprise the remaining 20% and 10% of MZL, respectively, and account for less than 1% of NHL. The relative rarity of these lymphomas and difficulty in distinguishing them from other low-grade B-cell lymphoma subtypes like lymphoplasmacytic lymphomas (LPL), making therapeutic decisions difficult. The aim of this study was to perform a comprehensive high-resolution study to identify distinctive molecular markers in MZL and LPL, which would help to better define these lymphomas at genomic level and distinguish them from other types of low-grade B-cell lymphoma. We analyzed 119 patients, including 22 pulmonary MALT (pMALT), 20 non-pulmonary MALT (npMALT), 34 SMZL, 21 NMZL and 22 LPL (8 Waldenström’s Macroglobulinemia and 14 non-WM) cases. Human Agilent 244A platform was used by array-based comparative genomic hybridization (aCGH) analysis. Overall, 93% of patients have an altered genome. LPL had the highest amount of copy number abnormalities (median of 7.5 per patient; average of 11.7; range: 0–44), followed by npMALT (6.5; 7.6; 0–33), NMZL (5; 8.3; 0–31), SMZL (4; 7.1; 0–56) and pMALT (3.5; 4.3; 1–11). When the overall size of the abnormalities was considered, LPL was also the most affected disease with an average size of 245.15Mb included in copy number changes per patient, followed by NMZL (188.6Mb), npMALT (187.2Mb), SMZL (126.4Mb) and pMALT (109Mb). Deletions were more commonly observed than amplifications but the affected area was significantly smaller (average of 55.2Mb and 118.6Mb included in deletions and amplifications per genome, respectively). Partial or total gains of chromosome 3 and 18 were recurrently identified in all the entities, being the most common abnormalities in npMALT (60% and 50%, respectively) and pMALT (27% each). Trisomy 12 was also ubiquitously observed, ranging from 24% of NMZL to 9% of pMALT. Deletions in 6q were commonly identified in LPL (55%), npMALT (35%) and, in a less extent, in SMZL (12%) and NMZL (5%), but not in pMALT. Biallelic and focal monoallelic deletions at 6q23 clearly delineate a minimal deleted region (MDR) that includes TNFAIP3 as being the target gene of the abnormality. Deletions on 7q were found in SMZL and npMALT, thus delineating two MDR. Interstitial 11q deletions were found in LPL, NMZL and SMZL, but not in MALT. Chromosome 13 abnormalities were monosomies in NMZL and interstitial 13q14 deletions in LPL and SMZL, comprising MIRN15A and MIRN16-1 in the MDR. Partial X chromosome gains were common to all the entities but involving different areas: Xq27.3–q28 gains were recurrent events in LPL and NMZL, but not in SMZL and MALT, which were characterized by p arm gains or trisomies. Finally, some recurrent abnormalities were exclusive of specific entities: 1p deletions and 1q gains were only common in NMZL; a focal 2p gain including BCL11A and REL was found in npMALT and partial 8q gains were only identified in LPL. Trisomy 4 was only identified in the subgroup of LPL with WM, but not in the remaining LPL cases or in the remaining diseases. In this study we made a compendium of chromosomal abnormalities present in a group of related diseases, in which the molecular basis remains poorly defined. Using this high-resolution approach we were able to establish recurrent and disease-specific patterns of abnormalities and minimal deleted/amplified regions were redefined. Furthermore, TNFAIP3, a negative regulator of the NF-kB pathway, has been identified as being a commonly affected gene in MALT, LPL and NMZL, highlighting its potential role in pathogenesis.

Abstract INTRODUCTION: Previous studies have demonstrated the clinical activity of the mTOR inhibitor RAD001 in low- grade lymphomas. Our preclinical studies demonstrated activity of mTOR inhibitors in Waldenstrom Macroglobulinemia (WM) cell lines and patient samples. This phase II study aimed to determine safety and activity of the oral mTOR inhibitor RAD001 (Novartis Pharmaceutical, MA) in patients with relapsed or refractory WM. METHODS: Patients who had at least one previous therapy for WM, and who had symptomatic relapsed or refractory disease were eligible. NCI CTCAE v3.0 was used for toxicity assessment. All patients received daily RAD001 at 10 mg. A cycle was considered 28 days. Patients were allowed to stay on therapy until progression of disease or excessive toxicity. This study was conducted in a collaborative effort between Dana Farber Cancer Institute (DFCI) and Mayo Clinic College of Medicine. Here, we report the data on the patients accrued at DFCI. RESULTS: 19 pts (15 men and 4 women) have been treated to date. All patients had symptomatic disease and required therapy. The median number of lines of prior treatment was 3 (range 1 – 5) including included rituximab, nucleoside analogues (fludarabine or 2-CDA), combination chemotherapy (e.g. CHOP, CVP), chloramucil, and bortezomib. The median IgM at baseline was 3330 mg/dL (range 1010– 7410). The median follow on RAD001 was 8 months (range 3 – 22 months). Eighteen pts are currently evaluable for response. Best responses to RAD001 after 2 cycles using IgM monoclonal protein were as follows: partial remission in 8 (44%), minimal response in 5 (28%). Progressive disease occurred in 4 (22%) and stable disease occurred in 1 (6%). The overall response rate (PR+MR) was 72%. The median duration of response has not been reached (3–22+ months). Patients tolerated therapy well without significant toxicities. Grade 3 and 4 toxicities included grade 4 thrombocytopenia in 1 patient, grade 3 pneumonia in 1 patient, grade 3 hyperglycemia in 1 patient and grade 3 mucositis in 1 patient. Other adverse events of grade 2 or lower included nail cracking, mucositis, diarrhea, and fatigue. Attributable toxicities otherwise proved manageable with appropriate supportive care, and RAD001 was generally well tolerated. One patient enrolled on the study withdrew consent and changed to hospice care within 3 weeks of therapy, and passed away due to disease progression. CONCLUSIONS: The use of the oral RAD001 single agent RAD001 in patients with relapsed or refractory WM was welltolerated and demonstrated significant activity achieving an overall response rate in 72% of patients. Future studies of combination of this agent with rituximab and bortezomib are currently being planned.

Abstract MicroRNAs (miRNAs) are small noncoding RNAs that are approximately 20–22 nucleotides with critical functions in cell growth, survival, and differentiation. These conserved sequences can regulate expression of multiple genes and are often tissue specific and dysregulated in malignancies. Thus, miRNA profiling has been used to create signatures for many solid tumors. These profiles have been used to classify tumors and to help predict survival and outcome. In the present study, we utilized the DiscovArray miRNA profiling service (Asuragen Services, Austin, TX) which utilizes a custom-manufactured Affymetrix GeneChip® from Ambion that covers miRNAs derived from the Sanger miRBase (http://microrna.sanger.ac.uk/sequences/index.shtml) and over 11,000 predicted miRNAs derived from published reports. The signal processing implemented was a multi-step process involving probe-specific signal detection calls, background estimation and correction, constant variance stabilization and global normalization. For each probe, an estimated background value was subtracted derived from the median signal of a set of G-C-matched anti-genomic controls. Arrays within a specific experiment were normalized together according to variance stabilization method. Detection calls were based on a Wilcoxon rank-sum test of the miRNA probe signal compared to the distribution of signals from GC-content matched anti-genomic probes. For statistical hypothesis testing, a two-sample t-test, with assumption of equal variance, was applied. One-way ANOVA was used for multiple group comparison. Probes were considered to be differentially expressed based on two criteria: a p-value of < 0.001 and glog2 difference > 1. miRNA expression was analyzed in all malignant B lineage cells (CD19+ CD138+) (n=8), malignant B cells alone (CD19+) (n=6) and plasma cells alone (CD138+) (n=3) from Waldenström macroglobulinemia (WM) patients. The expression was compared to malignant CD19+ B cells from chronic lymphocytic leukemia (CLL) patients (n=5), malignant plasma cells (CD138+) from multiple myeloma (MM) patients (n=5) and to B lineage cells (CD19+ CD138+) (n=4), CD19+ B-lymphocytes (n=3) and CD138+ plasma cells (n=6) from healthy donors. Data analysis based on a total of approximately 11,000 miRNAs analyzed shows that CD19+ CD138+ cells (double sorting) from WM patients did not cluster as a unique group. Some samples had a pattern similar to CLL, some similar to MM and others similar to CD19+ CD138+ cells from healthy controls. This lack of clear signature was observed by others in gene expression profiling and CGH arrays. We therefore hypothesized this lack of clustering was due to the lymphoplasmacytic nature of WM cells and therefore we analyzed B cells (CD19+) and plasma cells (CD138+) separately. miRNA expression in B cells (CD19+) identifies a signature in normal B cells that is absent in both WM B cells (CD19+) and CLL cells. There is also a set of miRNAs that are absent in normal B cells that are expressed in WM B cells and CLL. In addition, WM B cells had a unique miRNA signature that is unique compared to CLL and normal B cells. An additional set of miRNAs were expressed and clustered only in CLL patients. Similar to B cells, plasma cell (CD138+) analysis in WM, MM and healthy donors shows a clustering pattern that identifies normal plasma cells from MM plasma cells. WM plasma cells had a miRNA signature that is unique only to WM patients, however, a subset of miRNAs shared an expression pattern with MM plasma cells. While miRNAs can target multiple genes, some of the genes that are targets of the miRNAs identified in this analysis include XBP-1, Blimp-1, IRF-4, Bcl-6 and TACI. These target genes are known to be important in B cell and plasma cell development. In summary, we have analyzed miRNA expression in malignant B cells (CD19+) and malignant plasma cells (CD138+) from WM patients and compared their expression pattern to their normal counterpart as well as malignant counterpart in CLL B cells and MM plasma cells. Our analysis shows that WM B cells have a miRNA signature unique to WM only and one that is shared by CLL cells. Similarly, WM plasma cells have a unique miRNA signature but also has some miRNAs that are shared by malignant plasma cells in MM. These miRNAs target genes involved in B cell differentiation. Analysis of the functional roles of these miRNAs will and their regulation will further our understanding of the regulation of B cells development in normal and malignant conditions.

Abstract Diffuse large B cell lymphoma (DLBCL) is an aggressive form of non-Hodgkin lymphoma and the treatment of these patients has improved with rituximab-based chemo-immunotherapy. However, approximately 40% of patients die of their disease and new agents with novel mechanisms of action are needed for this disease. The mammalian target of rapamycin (mTOR) has emerged as an important therapeutic target in cancer cells and rapamycin and its derivatives, which specifically inhibit mTOR, are now being actively evaluated. Recent clinical trials of mTOR inhibitors have demonstrated that a fraction of patients (35%) with relapsed DLBCL respond to single-agent temsirolimus (Smith S et al J Clin Oncol 26; May 20 Supplement abstract 8514) or everolimus (Reeder et al 2007 ASH Annual Meeting Abstracts 110 (11) abstract 121) in relapsed DLBCL, however most patients are resistant to this therapy. The mechanism of this resistance remains a subject of major therapeutic significance. Herein, we report that cells from DLBCL lines (DHL-6, Ly7 and Ly3) and primary tissues from DLBCL patients (n=10) display persistent activation of the mTOR pathway as determined by phosphorylation of mTOR targets S6 ribosomal protein (S6rp) and 4E-binding protein 1 (4E-BP1). Treatment of human DLBCL cells with various doses of rapamycin predictably demonstrated a decrease in proliferation but less than 10% reduction in overall cell survival. However, rapamycin suppressed the phosphorylation of S6rp and 4E-BP1, indicating an inhibition of raptor-mTOR (mTORC1) signaling. Paradoxically, rapamycin also concurrently increased, through a negative feedback mechanism, the phosphorylation of Akt that may contribute to drug resistance. Interestingly we found that rapamycin treatment also increased the phosphorylation of eIF4E, a survival protein downstream of mTOR, which may also be responsible for resistance of rapamycin along with Akt. To determine whether the observations found in vitro are clinically relevant, we obtained peripheral blood samples from patients with aggressive lymphoma treated with the rapamycin derivative everolimus. The levels of phosphorylated Akt and eIF4E were increased in 3 of 3 patient samples at 48 hrs and after 1 cycle compared to untreated control. Our observations therefore suggest that rapamycin derivatives potently activate Akt and eIF4E activity via activation of the mTORC2 assembly, in addition to its well-characterized ability to suppress the mTORC1 pathway. Treatment of DLBCL cells with histone deacetylase (HDAC) inhibitor LBH589 induced growth inhibition of DLBCL cells at nanomolar concentrations in a dose dependent manner in association with hyperacetylation of histones H3 and H4. LBH alone was able to inhibit the phosphorylation of S6rp and 4E-BP1, while combined treatment with rapamycin inhibited the phosphorylation of S6rp and 4EBP1 to a greater extent than either agent alone. Surprisingly LBH inhibited constitutive as well as rapamycin-induced activation of Akt and eIF4E in a time and dose dependent manner. Our co-immunoprecipitation data suggest that LBH alone was able to alter the level of intact mTORC2 by reducing the amounts of rictor bound to mTOR, which is further decreased when combined with rapamycin. In support of this model, rapamycin combined with LBH exhibited enhanced synergistic inhibitory effects on survival and proliferation of DLBCL cells. The mTOR pathway is also considered to control the translation of specific mRNA species, some of which are involved in cell cycle control and angiogenesis (e.g. cyclin D1, c-Myc and HIF-1a). LBH alone down-regulated expression of c-Myc and HIF-1a, while have no effect on cyclin D1 expression. Combination of LBH with rapamycin further decreased expression of c-Myc and HIF-1a. Overall these results indicate that pharmacological inhibition of the mTOR pathway by rapamycin and LBH interferes with essential survival and proliferating pathways in DLBCL cells. We propose that the rapamycin-induced functional blockade of Akt and eIF4E is inhibited by LBH, and the combination increased the anti tumor activity of rapamycin. In summary, our data provide a mechanistic basis for enhancing mTORtargeted cancer therapy by combining an mTOR inhibitor with a HDAC inhibitor such as LBH. A phase I/II trial of this combination is planned.

Abstract Waldenström macroglobulinemia (WM) is a B-cell malignancy characterized by the aberrant production of a monoclonal IgM protein that may lead to hyperviscosity. Although this is a major factor causing significant morbidity in patients, little is known about the mechanisms that regulate monoclonal protein synthesis. Cytokines are protein mediators that are known to be involved in many biological processes, and can profoundly affect tumor cells and the tumor microenvironment. Many cytokines have been shown to have potent therapeutic efficacy in preclinical cancer models; however, the role of cytokine networks in WM is not fully understood and few studies have described the precise functional roles of cytokines in WM. To address this issue we performed a multiplex ELISA analysis to test cytokine levels in sera from patients with WM. We found that Rantes/CCL5 is significantly elevated in WM patients and correlates with disease activity. Elevated expression of RANTES in the serum was confirmed by ELISA and was also detected in the bone marrow of WM patients by ELISA and immunohistochemistry. RANTES expression serves as a marker for recruitment of immune cells and is associated with a wide range of immune-mediated diseases. However, the impact of RANTES in WM is not known. We analyzed the expression of receptors for Rantes by flow cytometry and found that WM B cells and stromal cells express CCR1 and CCR3, but not CCR5. Using a standard chemotaxis assay, we determined that Rantes had no effect on B cell or stromal cell recruitment. Rantes also had no effect on B cell or stromal cell survival, however it did promote stromal cell proliferation (p<0.04). The interaction between Rantes and IL-6 has been described in an autoimmune disease model. Since stromal cells secrete significantly more IL-6 than WM B cells, we treated stromal cells with Rantes for 24 hr and found that Rantes increases IL-6 secretion (p<0.03). To characterize the mechanism of Rantes-mediated IL-6 secretion, we transfected stromal cells with an IL-6 promoter construct and treated with Rantes for 12 hr and found a significant increase in IL-6 promoter activity (p<0.0162) indicating Rantes can regulate IL-6 transcription. Bioinformatics analysis of the IL-6 promoter indicates the presence of multiple candidate binding sites for transcription factors that have been previously shown to play a role in the biology of B cells, including NFkB, AP1, and GLI. Co-transfection of stromal cells with an IL-6 reporter construct and a plasmid expressing GLI1, GLI2 or GLI3 demonstrates that GLI2 and GLI3 proteins can regulate the IL-6 promoter. We then transfected stromal cells with a reporter construct containing 8X-GLI binding sites and demonstrate that Rantes can regulate GLI transcription further supporting a role for the interaction between Rantes and IL-6 through GLI transcription factors. IL-6 rich tumor microenvironment supports malignant cells. Elevated IL-6 levels have no effect on survival of BCWM.1 cells or CD19+ 138+ cells from WM patients, but leads to upregulation of IgM secretion by BCWM.1 cells and CD19+ CD138+ cells from WM patients, and increases their proliferation (p<0.0039). IL-6 activates Erk1/2 and Jak/ Stat in WM and stimulation of the IgM secreting cells BCWM.1 with IL-6 in the presence of PD98059 MAPK inhibitor had no effect on IgM secretion. However, stimulation of BCWM.1 cells with IL-6 in the presence of JakI inhibitor abolished the IL-6 mediated IgM secretion suggesting IL-6 mediated increase in IgM secretion occurs through Jak/ Stat signaling pathway. Analysis of Rantes levels in other B cell malignancies including follicular lymphoma, chronic lymphocytic leukemia, monoclonal gammopathy of undetermined significance and multiple myeloma indicates that Rantes is elevated in other B cell lymphoproliferative disorders and suggest Rantes may play a similar role in other malignancies. In summary, our data identifies a novel Rantes-GLI-IL-6 interplay in the stromal microenvironment that promotes IgM production by malignant B cells. This therefore provides multiple new potential therapeutic avenues, targeting both malignant cells and the microenvironment to control malignant cell growth, and immunoglobulin secretion in WM and Ig-mediated diseases.

Abstract PURPOSE: Recent advances in understanding of the molecular alterations that occur at the genetic and epigenetic levels in Multiple Myeloma (MM) have led to major leaps in identifying molecular pathways that regulate progression and resistance to therapeutic agents. However, despite great scientific advances at the genomic level, studies to identify signaling pathways deregulated at the functional proteomic level in MM are limited. In this study, we used a rapid and reproducible antibody-based protein microarray technique to screen the functional differences between malignant plasma cells in samples obtained from patients with MM compared to normal plasma cells (NPC) from the bone marrow of healthy volunteers. METHODS: We determined the protein expression level of 512 polypeptides in 12 samples of newly diagnosed patients with MM using high-throughput proteomic analysis with antibody-based protein microarray. Primary CD138+ sorted MM cells were obtained from the bone marrow of patients after informed consent. MM1.S was used in this study. Using immunohistochemistry and immunoblotting were confirmed. Lentivirus was used to knockdown CRIK. Gene expression datasets from the Mayo Clinic (accession number GSE 6477) and the UAMS (accession number GSE 2658) were obtained from the Gene Expression Omnibus for analysis. The Mayo dataset was generated using Affymetrix U133A platform whereas the UAMS dataset was generated using Affymetrix U133plus 2.0 platform. RESULTS: We identified four subgroups of MM using unsupervised clustering analysis. We confirmed overexpression of some of these proteins including CRIK and CDK4 using immunohistochemistry and immunoblotting. Many of these proteins are known to be deregulated in MM, indicating that this technique can accurately identify proteins that are over or under-expressed in MM in a high-throughput fashion. In addition, we identified novel proteins that are not previously known to be differentially expressed in MM, including the small GTPase member of the Rho family, CRIK protein. We then showed using knockdown of CRIK that this novel protein specifically regulates migration and adhesion in MM. There was no effect on survival of MM cells using the CRIK knockdown. Analyzing the GEP data of the 15 NPC, 46 monoclonal gammopathy of undetermined significance (MGUS) or smoldering myeloma (SMM) and 101 MM samples from the Mayo Clinic, there was a significant increase in expression of CIT from NPC to MM. Among the 351 patients entered into TTII trials from UAMS, CIT expression was similar across the different TC class. Using a cut-off normalized expression level of 1.25 (a level above expression in NPC), MM with a high CIT expression (n=81) had a significantly shorter survival than the other patients. CONCLUSION: In this study, we show that MM cells express a high level of CRIK, and that inhibition of this protein leads to significant inhibition of adhesion and migration of MM cells. In addition, CRIK protein expression correlated with CIT gene expression, with high expression in MM samples compared to NPC. In addition, high CIT expression correlated with poor survival in patients with MM.

Abstract Introduction: The kidney is affected by immunoglobulin light chain amyloidosis (AL) in more than 50% of patients who present with the disease, but long-term predictors for and outcomes after renal replacement therapy have not been well described in this patient group Patients: 145 patients with AL who participated in Mayo Clinic treatment trials and were monitored for at least 11 years. No patient was lost to follow-up Results: Among patients presenting with renal AL, 42% ultimately received renal replacement therapy compared with 5% of patients who did not have an initial presentation involving the kidney. Table 1. Monoclonal Protein Studies in 145 Patients With Immunoglobulin Light Chain Amyloidosisa Patients, No. (%) Monoclonal Protein Renal Amyloid at Presentation (n=84) Nonrenal Amyloid at Presentation (n=61) a P=.02 l more likely to cause renal presentation None 7 (8) 2 (3) k 6 (7) 12 (20) l 71 (85) 47 (77) Table 2. Characteristics of All 145 Patients With Immunoglobulin Light Chain Amyloidosis Amyloid Renal (n=84) Nonrenal (n=61) P Value Abbreviations: IQR, interquartile range; NA, not applicable. a included in the definition of renal amyloidosis Patients with l immunoglobulin light chain amyloid were significantly more likely to have renal involvement and had significantly greater urinary protein loss than patients with κ or no monoclonal light chain amyloid Age, median (IQR), y 61 (52–67) 62 (55–68) .47 Sex, male/female 52/32 40/21 .72 24-hr urine protein loss, median (IQR), g/d 7.0 (4.4–11.7) 0.3 (0.1–6.4) NAa Creatinine, median (IQR), mg/dL 1.1 (0.9–2.2) 1.1 (0.9–1.3) .22 Cardiac involvement (yes/no) 35/48 48/16 .0001 Table 3 Distribution of Urinary Protein and Creatinine Based on Monoclonal Protein Monoclonal Protein Test k (n=18) l (n=118) None (n=9) P Value Abbreviation: IQR, interquartile range. Patients with renal amyloid who ultimately received dialysis support had significantly higher serum creatinine and 24-hour urine protein levels at presentation. Absence of cardiac involvement at diagnosis predicted an increased likelihood of dialysis All patients (N=145) 24-hr urine protein loss, median (IQR), g/d 0.73 (0.16–2.37) 3.61 (0.35–7.8) 5.68 (0.3–12.2) .001 Creatinine, median (IQR), mg/dL 1.1 (0.9–1.3) 1.1 (0.9–1.3) 1.5 (1.2–1.6) NS Patients with renal amyloid at presentation (n=84) 24-hr urine protein loss, median (IQR), g/d 2.93 (1.1–5.0) 7.2 (4.8–11.8) 7.8 (3.8–12.6) .04 Creatinine, median (IQR), mg/dL 1.0 (0.8–2.6) 1.1 (0.9–2.2) 1.5 (1.1–1.6) NS Table 4 Characteristics of 84 Patients Presenting With Renal Amyloidosis Dialysis Subsequently Not Required Characteristic Required (n=35) (n=49) P Value Abbreviation: IQR, interquartile range. The median time from diagnosis to dialysis was 29.5 months for those that went into renal failure.). However, the actuarial risk reached 50% at 12 years Kaplan-Meier failure curve demonstrating the actuarial time from diagnosis to the start of dialysis therapy N=145 For 38 patients who received dialysis, median survival from day 1 of dialysis was 10.4 months Kaplan-Meier survival of 38 patients from the start of dialysis therapy Age, median (IQR), y 60 (52–67) 62 (52–67) .90 Sex, male/female 21/14 31/18 .76 24-hr urine protein loss, median (IQR), g/d 7.4 (5.0–14.8) 5.9 (3.7–9.1) .03 Creatinine, median (IQR), mg/dL 1.4 (0.9–2.8) 1.1 (0.9–1.5) .01 Cardiac involvement (yes/no) 9/26 26/23 .01 Conclusion: In our study, 26% of all patients with AL ultimately received renal replacement therapy compared with 42% of patients who presented with renal AL specifically. The presenting 24-hour urine protein loss and creatinine values predict which patients will require dialysis. Median survival for patients starting dialysis is less than 1 year. Presence of l light chain amyloid predicts an increased likelihood of renal involvement and, when the kidney is affected, increased proteinuria.

Abstract The tumor microenvironment in B-cell non-Hodgkin’s lymphoma (NHL) plays an important role in supporting the growth and survival of malignant lymphocytes. In contrast, the extent to which this is true in T-cell NHL is less clear. While professional monocytederived antigen-presenting cells (MDC) are required for T-cell activation and survival, a growing body of evidence demonstrates that MDC may promote the survival of malignant T cells. Immunohistochemical staining of skin biopsies from cutaneous T-cell lymphoma (CTCL) patients (n=11) demonstrated a rich infiltrate of CD68+ macrophages and CD1a+ dendritic cells (DC). As these cells are derived from CD14+ monocytes, we examined the role of monocytes in promoting survival in malignant T cells obtained from CTCL patients (n=9). Initially, peripheral blood mononuclear cells (PBMC) were either depleted of CD14+ monocytes (>85% depleted) or mock-depleted prior to being cultured. Viability of malignant CD4+CD7− T cells was determined by Annexin V/7-AAD staining. While 90.9% (S.D. +/− 0.4%) of mock-depleted malignant T cells were viable, only 73.9% (S.D. +/− 1.3%) of monocyte-depleted cells remained viable, suggesting that monocytes may promote the survival of malignant T cells. In a similar fashion, HuT-78 cells (Sézary Syndrome cell line) were cocultured with CD14+ monocytes from normal donors. Compared with HuT-78 cells alone, the presence of CD14+ monocytes resulted in a 2.7 fold increase in thymidine incorporation (n=5; S.D. +/− 0.6). This is largely due to cytokine production, as the same experiment performed with a transwell insert resulted in a 2.1 fold increase in HuT-78 thymidine incorporation (n=5; S.D. +/− 0.4). Similar results were obtained using additional Sézary Syndrome (i.e. SeAx) and Mycosis Fungoides (i.e. MyLa) cell lines. Monocytes obtained from normal donors were subsequently cultured and cell-free conditioned media was obtained 48 hours later. Media supplemented with monocyte-conditioned media (monocyte-CM) prevented the induction of apoptosis in CTCL cell lines as measured by thymidine incorporation, trypan blue exclusion and Annexin V/propidium iodide staining. This affect was monocyte-specific, as neither T-cell nor B-cell conditioned media were observed to promote survival of CTCL cell lines. Furthermore, monocyte-CM had no affect on resting or anti-CD3 activated normal T cells. We next cultured malignant T cells obtained from PBMC (n=6) or skin biopsies (n=3) from CTCL patients in the presence or absence of monocyte-CM and assessed cell viability by Annexin V staining. 32.1% (S.D. +/− 14.7%) and 60.5% (S.D. +/− 21.5%) of malignant T cells isolated from PBMC remained viable in the absence or presence of monocyte-CM, respectively; 13.0% (S.D. +/− 10.8%) of malignant T cells isolated from skin biopsies remained viable, compared with 26.5% (S.D. +/− 8.2%) of cells grown in the presence of monocyte-CM (p=.03). Given the prevalence of MDC within CTCL-involved skin and their potential role in promoting the survival of malignant T cells, one may predict that malignant T cells may recruit monocytes in a chemokinedependent fashion. Therefore, cell-free conditioned media from CTCL cell lines (HuT- 78, SeAx, MyLa) was collected and used to promote monocyte chemotaxis in a standard chemotaxis assay. Only neutralization of CCL5 (RANTES) was capable of abrogating monocyte chemotaxis, demonstrating that the production of CCL5 by malignant T cells promotes monocyte migration. Therefore, we examined serum CCL5 levels by ELISA from both normal donors (n=24) and CTCL patients (n=23) and found an approximately 3-fold elevation in serum CCL5 in CTCL patients (normal donors: mean CCL5 10.0 ng/mL; 95% CI 7.1–13.0; CTCL patients: mean 29.2 ng/mL; 95% CI 20.6–37.8; p<0.0001). Serial serum samples were available for 6 patients, 4 of whom had progressive disease. Serum CCL5 increased from a mean of 17.9 ng/mL before progression (S.D. +/− 7.0) to 44.8 ng/mL (S.D. +/− 20.0) after progression (p=.04). In contrast, serum CCL5 decreased by at least 50% in both patients with responsive disease. Our findings demonstrate that monocytes, recruited by malignant T cells in a CCL5-dependent fashion, promote the survival of lymphoma cells in CTCL, thus raising the possibility that targeting tumor-associated monocytes and MDC may represent a novel therapeutic strategy in CTCL.

Abstract The International Prognostic Index at the time of relapse (IPI-R) has been reported to predict clinical outcome in patients with relapsed diffuse large B-cell lymphoma (DLBCL) undergoing autologous stem cell transplantation (ASCT) [Jabbour et al, Leukemia & Lymphoma2005;46(6):861–867; Lerner et al, Biology of Blood and Marrow Transplant2006;13:486–492; and Costa et al, Bone Marrow Transplant2008;41:715–720]. The absolute lymphocyte count (ALC) has been reported as a prognostic factor for survival at diagnosis, during standard chemotherapy and at day 15 post-ASCT for multiple hematological malignancies; however, no reports have addressed whether ALC at the time of relapse (ALC-R) predicts survival. Thus, we assessed the prognostic significance of ALC-R in patients with relapsed DLBCL. To be included in the study, patients were required to have been diagnosed with relapsed DLBCL, have ALC values available from the time of relapse, and to be followed at Mayo Clinic, Rochester. From Feb 2,1987 until March 6, 2006, 121 DLBCL patients qualified for the study. The overall survival (OS) was measured from the time of relapse to the date of death or last follow-up and progression-free survival (PFS) was defined as the time from relapse to the time of progression, relapse, death, or last follow-up. The median age at relapse was 68 years (range:25–88 years) and the median ALC-R was 1.21 x 109/L (range: 0.33–5.99 x 109/L). The value of ALC-R ≥ 1.0 x 109/L was used for the analysis based on previous publications. The groups (ALC-R ≥ 1 or < 1 x 109/L) were balanced for the IPI-R (p=0.1), as well as for undergoing ASCT (p=0.4). Superior OS was observed in patients with an ALC-R ≥ 1.0 x 109/L (N = 76) [median OS: 27.5 months, 3 years OS rates of 42%] compared with patient with an ALC-R < 1.0 x 109/L (N =45) [median OS: 9.4 months, 3 years OS rates of 13%] (p <0.0001). Superior PFS was also observed in patients with an ALC-R ≥ 1.0 x 109/L (N = 76) [median PFS: 14.8 months, 3 years PFS rates of 30%] compared with patient with an ALC-R < 1.0 x 109/L (N =45) [median PFS: 6.2 months, 3 years PFS rates of 11%] (p <0.001). ALC-R was an independent prognostic factor for OS [RR = 0.551, p < 0.009] and PFS [RR = 0.674, p < 0.02] in the multivariate analysis when compared with the IPI-R and whether patients underwent ASCT or not. Our study supports the hypothesis that ALC at relapse predicts clinical outcome in DLBCL and suggests that patient host immunity is an important variable predicting survival in relapsed DLBCL.

Abstract Background: Recent literature suggests that statins may have anticancer potential. This effect is thought to be mediated through 2 primary mechanisms - impairment of protein prenylation and interference with the formation of cholesterol-rich lipid microdomains, or “lipid rafts” within the cell membrane. Both of these processes are critical for signaling activity of numerous proteins important for lymphomagenesis and tumor survival. Recent data, however, suggest that statin use may directly inhibit rituximab binding to CD20 and therefore rituximab efficacy. These findings raised significant concerns about statin use during rituximab treatment. Here we report on statin use and clinical outcome in a cohort of FL and DLBCL treated patients, most of whom were treated with rituximab containing regimens. Methods: 293 newly diagnosed FL patients and 228 newly diagnosed DLBCL patients were prospectively enrolled in our Lymphoma SPORE registry from 9/2002 through 6/2007. Pathology was centrally reviewed. All patients were followed for retreatment, progression-free and overall survival. Statin use at the time of diagnosis and time of initial treatment was abstracted from the medical record. An event was defined by disease progression, retreatment, or death due to any cause. Results: 19% of FL patients and 22% of DLBCL patients were on statins at diagnosis; 16% and 19% were on statins during treatment, respectively. Initial therapy for the FL patients was observation (40%), R-CHOP (19%), R-CVP (12%), rituximab alone (8%), CVP (7%), RT (6%), and other (8%). All DLBCL patients received rituximab with CHOP or a CHOP-like regimen. At a median follow-up of 36 months (range 3–73), 109 (37%) and 65 (29%) of FL and DLBCL patients had an event; 19 (6%) and 46 (20%) of FL and DLBCL patients died, respectively. After adjusting for FLIPI, grade, and initial therapy type, statin use at diagnosis was associated with better event-free survival (HR = 0.57, 95% CI: 0.34–0.95, p=0.03) in FL patients. Statin use during treatment in FL patients was also associated with better event-free survival, though not statistically significant (HR = 0.67, 95% CI: 0.39–1.16, p=0.15). The improvement in EFS for FL patients was consistent across initial therapies, including observation. Statin use was not associated with IPI-adjusted overall survival or event-free survival in DLBCL (all p > 0.50). Conclusions: Statin therapy does not appear to be associated with inferior clinical outcome in DLBCL treated with rituximab and CHOP or CHOP-like therapy. Therefore direct inhibition of rituximab binding to CD20 may have limited clinical significance or/and may be overcome by inhibitory impact of statins on cell signaling. The latter possibility is supported by our observation that statin use is associated with improved event-free survival in follicular lymphoma. Figure Figure Figure Figure