ALBERT

Description: Compelling evidences indicate a key role for regulatory T cells (Tregs) on the host response to cancer and recent studies indicated that the generation of effective WT1-specific cytotoxic T cells can be largely affected by the presence of Tregs. This is the first study to describe human Tregs generated specifically against the WT1 antigen which is overexpressed in several human leukemias and provide the mechanism by which these anti-WT1 Tregs inhibit the immune response in leukemia patients. We have generated T cell lines and clones that specifically recognized a WT1-84 peptide in an HLA DRB1*0402/TCR-Vb8-restricted manner. Importantly, they recognized HLADRB1* 04-matched fresh leukemic cells expressing the WT1 antigen. These clones exerted a Th2 cytokine profile, had a CD4+CD25+Foxp3+GITR+CD127− Tregs phenotype, and significantly inhibited the proliferative activity of allogeneic T cells independently of cell-contact. Priming of allo-reactive T cells in the presence of Tregs strongly inhibited the expansion of NK; NK-T and CD8+ T cells, had an inhibitory effect on NK/NK-T cytotoxic activity but not on CD8+ T cells. Furthermore, priming of T cells with the WT1- 126 HLA-A0201-restricted peptide in the presence of Tregs strongly inhibited the induction of anti-WT1-126 CD8+ CTL responses as evidenced by both very low cytotoxic activity and IFN-g production. Moreover, these Tregs clones specifically produced Granzyme-B and selectively induced apoptosis in WT1-84 pulsed-autologous APCs but not in apoptoticresistant DR4-matched leukemic cells. Importantly, we have also detected anti-WT1-84 IL-5+/Granzyme-B+/Foxp3+ CD4+ Tregs in 5 out of 8 HLA-DR4+ AML patients. These findings suggest a critical role for anti-WT1 Tregs in the inhibition of T cell responses against leukemia. This study may have important implications for the clinical manipulation of Tregs such as immuno-targeting of TCR-Vb-8 with mAbs to eliminate anti-WT1 Tregs in leukemia patients in order to enhance GVL before vaccination with the WT1 antigen. On the other hand, leukemia patients with GVHD should be clinically-tried for vaccination with the current WT1-84 peptide or adoptively-treated with ex-vivo anti-WT1 Treg cells to specifically enhance their frequency, which is known to be very low in these patients.

Description: Epstein-Barr Virus (EBV)+ lymphomas are an important subgroup of aggressive malignant lymphomas which include lymphomas in the post-transplantation setting, Burkitt’s lymphomas (BL), AIDS-related lymphomas (ARL), and some forms of Hodgkin, T-cell, and natural killer (NK) cell lymphomas. EBV is a member of the herpes virus family, characterized by their ability to support two different life cycles: the productive “lytic” cycle leading to the production and release of new virions and the non-productive “latent” cycle. Most lymphoma cells are infected with latent EBV, and only few viral genes are expressed at low levels. Several groups of broad-acting chemical agents are able to reactivate EBV and induce herpes thymidine kinase (TK) expression in vitro and in vivo. NF-kB has been described to play a critical role in regulating the balance between latency and lytic replication of EBV. Therefore, we hypothesized that the proteasome inhibitor Bortezomib can be used to initiate EBV lytic antigen expression in EBV-related malignancies enabling the antiviral drug Ganciclovir to kill EBV+ lymphoma cells. The human cell line HR-1, derived from a Burkitt’s lymphoma and latently infected with EBV, was cultured in the presence of 50nM bortezomib for 24 hrs. The immediate early lytic phase EBV antigens ZEBRA and RTA were induced and expressed as measured by flow cytometry. The EBV-VCA and EBV-R antigens were not expressed in untreated controls but were induced as demonstrated by western blot analysis, indicating the switch to the lytic life-cycle of EBV. These results were successfully repeated using the EBV+ Akata cell line. Induction of viral thymidine kinase (vTK) was shown by QRT-PCR as well. Histone deacetylase inhibitors are a well known group of broad-acting chemical agents able to reactivate EBV. In combination experiments, we found that Bortezomib plus sodium butyrate or SAHA act at least additive in inducing the EBV lytic life cycle in HR-1 or RAJI cells. Bortezomib sensitizes the EBV+ Akata cell line 2A8-1 to growth inhibitory effects of ganciclovir as shown by MTT assays. The cells were treated with two different non-toxic drug concentrations which were chosen low enough not to induce apoptosis (bortezomib: 1nM and 2nM; ganciclovir:15 and 30μM). Bortezomib induces the lytic EBV life cycle in vivo. In murine xenograft models growing the Akata A.15 line subcutaneously bortezomib induces the immediate-early protein ZEBRA as shown by immunohistochemistry and vTK as shown by QRT-PCR. Experiments to induce lytic phase EBV in murine xenograft models using the Akata cell line and to combine EBV induction with the nucleoside analogue ganciclovir are in progress. Murine studies with EBV-transformed lymphoblastoid cell line (LCL) xenograft models, combination bortezomib plus ganciclovir, and molecular imaging with FHBG specific PET probes are in progress. Reactivating EBV with proteasome inhibitors alone or in combinations with low concentrations of histone deacetylase inhibitors may be a less toxic therapeutic strategy for EBV-associated lymphomas.

Description: Controversy exists as to whether Kaposi's sarcoma–associated herpesvirus (KSHV) is more widespread than originally reported. Recently, Monini et al reported that KSHV is ubiquitous in urogenital and prostate tissues and sperm of healthy Italian adults using nested polymerase chain reaction (PCR). We have examined for the presence of KSHV in 10 normal prostates from Italian men and 10 from men from the United States, as well as 32 prostatic, 30 vulvar, 24 ovarian, 20 cervical, and 30 testicular cancer specimens from patients from the United States. None of the patients had a history of human immunodeficiency virus infection. The samples were tested by nested PCR. The sensitivity of this assay was determined by a dilution study performed by diluting KSHV DNA from the KS-1 cells (a primary effusion lymphoma cell line which is estimated to have 16 copies of KSHV per cell) in DNA from a K562 myeloid cell line. The nested PCR that we used can detect 2.4 copies of KSHV sequences on a background of K562 DNA. All the samples were negative for KSHV sequences. Therefore, we cannot confirm the finding that KSHV sequences are ubiquitous in urogenital and prostate tissues. Furthermore, because our samples were from both the United States and Italy, the discrepancy between results is unlikely to be explained by either ethnic or environmental factors. False-positive results easily occur using nested primer PCR because of contamination. Our data argue that KSHV is not widely disseminated in urogenital tissues from nonimmunosuppressed individuals.

Description: Enhanced angiogenesis is a hallmark of cancer. Pleiotrophin (PTN) is an angiogenic factor that is produced by many different human cancers and stimulates tumor blood vessel formation when it is expressed in malignant cancer cells. Recent studies show that monocytes may give rise to vascular endothelium. In these studies, we show that PTN combined with macrophage colony-stimulating factor (M-CSF) induces expression of vascular endothelial cell (VEC) genes and proteins in human monocyte cell lines and monocytes from human peripheral blood (PB). Monocytes induce VEC gene expression and develop tube-like structures when they are exposed to serum or cultured with bone marrow (BM) from patients with multiple myeloma (MM) that express PTN, effects specifically blocked with antiPTN antibodies. When coinjected with human MM cells into severe combined immunodeficient (SCID) mice, green fluorescent protein (GFP)–marked human monocytes were found incorporated into tumor blood vessels and expressed human VEC protein markers and genes that were blocked by anti-PTN antibody. Our results suggest that vasculogenesis in human MM may develop from tumoral production of PTN, which orchestrates the transdifferentiation of monocytes into VECs.

Description: Controversy exists as to whether Kaposi's sarcoma–associated herpesvirus (KSHV) is more widespread than originally reported. Recently, Monini et al reported that KSHV is ubiquitous in urogenital and prostate tissues and sperm of healthy Italian adults using nested polymerase chain reaction (PCR). We have examined for the presence of KSHV in 10 normal prostates from Italian men and 10 from men from the United States, as well as 32 prostatic, 30 vulvar, 24 ovarian, 20 cervical, and 30 testicular cancer specimens from patients from the United States. None of the patients had a history of human immunodeficiency virus infection. The samples were tested by nested PCR. The sensitivity of this assay was determined by a dilution study performed by diluting KSHV DNA from the KS-1 cells (a primary effusion lymphoma cell line which is estimated to have 16 copies of KSHV per cell) in DNA from a K562 myeloid cell line. The nested PCR that we used can detect 2.4 copies of KSHV sequences on a background of K562 DNA. All the samples were negative for KSHV sequences. Therefore, we cannot confirm the finding that KSHV sequences are ubiquitous in urogenital and prostate tissues. Furthermore, because our samples were from both the United States and Italy, the discrepancy between results is unlikely to be explained by either ethnic or environmental factors. False-positive results easily occur using nested primer PCR because of contamination. Our data argue that KSHV is not widely disseminated in urogenital tissues from nonimmunosuppressed individuals.

Description: BACKGROUND: LGL leukemia is a neoplasm arising from either CD3+ T-cells or CD3− NK-cells. Autoimmune-mediated anemia, neutropenia, and rheumatoid arthritis occur frequently in these patients and immunosuppressive agents are used for these associated clinical syndromes. In our previous studies, we found that patients display a constitutively activated Ras and MAPK/ERK signaling cascade that may drive leukemia survival. A multicenter phase 2 clinical trial was initiated to treat LGL leukemia patients with the farnysltransferase-inhibitor R115777 (tipifarnib, Zarnestra®, Johnson & Johnson) that inhibits Ras and other farnesylated proteins. One of the goals of this study was to determine the shifts in cytokine production during therapy. We found that LGL cells treated with tipifarnib in vitro displayed a switch to Th2 (IL-4 and IL-10)-polarized differentiation. After tipifarnib treatment of LGL patients, antigen-activated T-cells produced greater amounts of Th2 (IL4/IL-10) cytokines but less Th1 (IFNγ/TNFα). In this study, we determined the mechanism governing tipifarnib-mediated Th2 polarization in T-cells. METHODS: PBMCs were isolated from 10 healthy donors and from seven patients with T-LGL leukemia before and after treatment with tipifarnib 300 mg twice daily for 21 days of a 28- day cycle. LGL leukemia patients had increased numbers of αβ T lymphocytes and evidence of clonality in association with either neutropenia or transfusion-dependent anemia. Th1 and Th2 cytokines were determined by intracellular staining and flow cytometry after activation with anti-CD3 plus anti-CD28. In some experiments, Th1 polarization was induced by IL-12; whereas, Th2 was induced by IL-4. Expression of T-bet and GATA-3 transcription factors that regulate Th1 and Th2 polarization, respectively, phosphorylated (active) MAPK (ERK1 and ERK2), and total MAPK were measured by Western blots. FTI2153, tipifarnib, and geranylgeranyl transferase inhibitor(GGTI)-2417 were used compared to DMSO control. RESULTS: PBMCs from patients with T-LGL leukemia displayed a dose and time-dependent increase in IL-4 and IL-10 production after drug treatment (average increase to 100% and 43%, respectively). A dose-dependent increase in these Th2 cytokines in T-cells from healthy donors showed that the farnesylated protein targeted by tipifarnib was not selectively expressed in LGL leukemia. Culture with IL-12 induced Th1 differentiation associated with ERK phosphorylation and increased T-bet expression. Pre-treatment with tipifarnib and FTI2153 but not GGTI2417 prior to IL-12 inhibited T-bet induction with no change in anti-CD3-induced MAPK leading to enhanced IL-4 signaling and greater Th2 polarization. CONCLUSIONS: Our data reveal unique, previously unreported effects of FTIs on cytokine signaling in T-cells by inhibiting the induction of T-bet and blocking Th1 differentiation. These results are critical to determine the mechanism of action of tipifarnib in LGL leukemia and suggest that FTIs may be useful for autoimmune or lymphocyte-mediated disorders associated with excessive Th1 cytokine production.

Description: Patients with chronic lymphocytic leukemia (CLL) may develop other B cell malignancies in their clinical course including aggressive diffuse large B-cell lymphomas and rarely myelomas. In a large proportion of cases, the secondary B cell malignancies reflected the emergence of immunophenotypically and genetically different clones. An immature type plasma cell myeloma developed in a 73-year-old female patient in whom CLL was diagnosed four years previously. The CLL expressed CD5, CD19, CD23, CD38 and surface kappa light chain, but were negative for ZAP-70. Trisomy 12 was detected by FISH analysis. PCR analysis of the peripheral blood for immunoglobulin heavy chain genes demonstrated two sharp bands that were initially interpreted as biallelic heavy chain gene rearrangements. Myeloma cells were CD38 and CD138 positive, CD19 negative and expressed cytoplasmic kappa light chain, but not heavy chains. In order to investigate the clonal relationship between these B cell malignancies, a detailed analysis of VHDJH and VκJκ gene rearrangements in individually sorted CD5 and CD19 double-positive CLL cells and also in CD38-positive and CD19-negative myeloma cells by single cell PCR of genomic DNA and direct sequencing was carried out. This technique permitted identification and pairing of both the heavy and light chain immunoglobulin genes from the same individually sorted cell. A total of 17 individual CLL and 23 myeloma cells were successfully analyzed. Our analysis demonstrated (a) the presence of two discrete clones of CLL, one with usage of [VH1-2*04/D3-3*01/J3*02]-[Vκ2-28*01/J1*01] without VH and Vκ hypermutation and the other with usage of [VH1-2*04/D4-11*01/J6*02]-[Vκ1-5*03/J1*01] with VH and Vκhypermutation; (b) no clonal relationship between the CLL and myeloma cells that utilized different VHDJH and VκJκ rearrangements [VH3-66*02/3-10*01/J4*03]-[Vκ1-33*01/J2*02] with VH and Vκ hypermutation. To our knowledge, this is the first demonstration of a biclonal CLL with mutated and unmutated clones in the same patient along with a third clonally unrelated B cell malignancy. This result suggests that single cell analysis may be necessary to detect subtle biclonality of CLL that might be associated with a more aggressive phenotype.

Description: An imbalance between cellular apoptosis and survival may be critical for the pathogenesis of lymphoma. Therefore, the gene expression pattern in lymph node preparations from patients with mantle cell lymphoma (MCL) was compared to the pattern in nonmalignant hyperplastic lymph nodes (HLs). Oligonucleotide microarray analysis was performed comparing 5 MCLs to 4 HLs using high-density microarrays. The expression data were analyzed using Genespring software. For confirmation, the expression of selected genes was analyzed by real-time polymerase chain reaction using the RNA extracted from 16 MCL and 12 HL samples. The focus was on 42 genes that were at least 3-fold down-regulated in MCL; in addition to the B-cell leukemia 2 (BCL2) system other apoptotic pathways were altered in MCL. The FAS-associated via death domain (FADD) gene that acts downstream of the FAS cascade as a key gene to induce apoptosis was more than 10-fold down-regulated in MCL. Furthermore, the death-associated protein 6(DAXX) gene, the caspase 2 (CASP2) gene, and the RIPK1 domain containing adapter with death domain(RAIDD) gene, which are key genes in other proapoptotic pathways, were also decreased in the MCL samples. The suggestion is made that in addition to the known overexpression of cyclin D1, which drives entry into the cell cycle, disturbances of pathways associated with apoptosis contribute to the development of MCL.

Description: Patients with deficiency in ferrochelatase (FECH), the last enzyme of the heme biosynthetic pathway, experience a painful type of skin photosensitivity called erythropoietic protoporphyria (EPP), which is caused by the excessive production of protoporphyrin IX (PPIX) by erythrocytes. Controversial results have been reported regarding hematologic status and iron status of patients with EPP. We thoroughly explored these parameters in Fechm1Pas mutant mice of 3 different genetic backgrounds. FECH deficiency induced microcytic hypochromic anemia without ringed sideroblasts, little or no hemolysis, and no erythroid hyperplasia. Serum iron, ferritin, hepcidin mRNA, and Dcytb levels were normal. The homozygous Fechm1Pas mutant involved no tissue iron deficiency but showed a clear-cut redistribution of iron stores from peripheral tissues to the spleen, with a concomitant 2- to 3-fold increase in transferrin expression at the mRNA and the protein levels. Erythrocyte PPIX levels strongly correlated with serum transferrin levels. At all stages of differentiation in our study, transferrin receptor expression in bone marrow erythroid cells in Fechm1Pas was normal in mutant mice but not in patients with iron-deficiency anemia. Based on these observations, we suggest that oral iron therapy is not the therapy of choice for patients with EPP and that the PPIX–liver transferrin pathway plays a role in the orchestration of iron distribution between peripheral iron stores, the spleen, and the bone marrow.

Description: Background: Biologic prognostic markers in diffuse large B-cell lymphoma (DLBCL) have focused on putative cell of origin (germinal center (GC) vs. activated B-cell (ABC)), apoptosis, and proliferation. Such markers, shown to be predictive in CHOP-treated patients, are being validated with rituximab(R)-CHOP. CALGB 50103 is a phase 2 trial of dose adjusted etoposide, vincristine, doxorubicin, cyclophosphamide, prednisone, R (DA-EPOCH-R) therapy for DLBCL. We studied CD10, BCL6, MUM1, LMO2, BCL2, and Ki67 in CALGB 50103 to determine which markers had prognostic significance. Methods: Prospectively procured slides were stained with appropriate primary antibodies at the Pathology Coordinating Office of the CALGB (CD10, BCL6, MUM1, BCL2, Ki67) or the Cleveland Clinic (LMO2). Slides were scored independently in 10% increments by two pathologists, with a third review in case of disagreement of 〉20% for all stains (mean score used as final value) except Ki67, for which image analysis (IA, Aperio, Scanscope) and a visual estimate (quartiles) was used. A 30% cutoff was used for CD10, BCL6, and MUM1. 40% was used for BCL2 and 60% for Ki67. Progression-free and event-free survival (PFS, EFS) served as the endpoints. Results: Data for at least one of the markers were available for 53 of the 75 treated patients. The median age was 56 years (range 23 – 80) and the median follow-up for the 45 patients who are still alive is 4.5 years (range 3.2–6.0). The international prognostic index (IPI) was available in 51 patients and 33 had an IPI score of 2 while 18 had scores of 3 or 4. 11 patients had progressive disease and there were 14 treatment failures. 8 patie0nts have died. Table 1 shows the significant predictors of outcome. Table 1: Predictors of PFS and EFS in CALGB 50103 PFS EFS Variable No. (%) 2 yr survival prob (95% CI) 2-sided p-value 2 yr survival prob (95% CI) 2-sided p-value CI = confidence interval CD10 0.057 0.030 〈 30% 39 (76.5) 0.76 (0.59 – 0.87) 0.74 (0.58 –0.85) ≥ 30% 12 (23.5) 1.00 (---) 1.00 (---) Ki67 IA 0.028 0.045 〈 60% 32 (64.0) 0.87 (0.69 – 0.95 ) 0.84 (0.66 – 0.93) ≥ 60% 18 (36.0) 0.67 (0.40 – 0.83) 0.67 (0.40 – 0.83) IPI 0.008 0.003 2 33 (64.7) 0.91 (0.74 – 0.97) 0.87 (0.71 – 0.95) 3–4 18 (35.3) 0.65 (0.38 –0.82) 0.61 (0.35 – 0.79) Of the cell of origin markers (CD10, BCL6, MUM1, LMO2) only CD10 negativity was associated with an inferior PFS and EFS. GC vs. non-GC phenotype (as per Hans et al Blood 2004) and LMO2 were not associated with outcome. High Ki67% by IA was also associated with inferior PFS and EFS. Similar results were seen with Ki67 visual estimates (〉75% cutoff, P60% hazard ratio [HR] 7.1, P=.007; IPI 3/4 HR 7.2, P=.006) and EFS (Ki67〉60% HR 5.3, P=.007; IPI 3/4 HR 7.1, P=.002). The multivariate analysis with CD10 and IPI was not possible because there are no events in CD10+ cases. Conclusions: Lack of CD10 (suggesting non-GC origin) and high Ki67 are associated with poor outcome in DA-EPOCH-R treated patients, further validating the concept of cell of origin and proliferation as predictors of outcome in DLBCL. BCL2 remains an important predictor of outcome in patients that lack the GC marker CD10, even in rituximab treated patients.