Joanne Luider

Superior autologous blood stem cell mobilization from dose-intensive cyclophosphamide, etoposide, cisplatin plus G-CSF than from less intensive chemotherapy regimens

The study purpose was to determine if G-CSF plus dose-intensive cyclophosphamide 5.25 g/m2, etoposide 1.05 g/m2 and cisplatin 105 mg/m2 (DICEP) results in superior autologous blood stem cell mobilization (BSCM) than less intensive chemotherapy. From January 1993 until May 1997, 152 consecutive patients with non-Hodgkin’s lymphoma (n = 55), breast cancer (n = 47), Hodgkin’s disease (n = 14), multiple myeloma (n = 9), AML (n = 9), or other cancers (n = 18) initially underwent BSCM by one of three methods: Group 1: G-CSF alone × 4 days (n = 30). Group 2: disease-oriented chemotherapy, dosed to avoid blood transfusions, followed by G-CSF starting day 7 or 8, and apheresis day 13 or 14 (n = 82). Group 3: DICEP days 1–3, G-CSF starting day 14, and apheresis planned day 19, 20 or 21 (n = 40). A multivariate analysis was performed to determine which factors independently predicted BSCM. The median peripheral blood CD34+ (PB CD34+) cell count the morning of apheresis linearly correlated with the number of CD34+ cells removed per litre of apheresis that day. The median PB CD34+ cell count and median CD34+ cells × 106 removed per litre of apheresis were highest for Group 3, intermediate for Group 2, and lowest for Group 1. By multivariate analysis, mobilization group (3 > 2 > 1), disease other than AML, no prior melphalan or mitomycin-C, and less than two prior chemotherapy regimens predicted better BSCM. Out of 15 Group 3 patients who had infiltrated marrows, 11 had no detectable cancer in marrow and apheresis products after DICEP. These data suggest that DICEP results in superior BSCM than less intensive chemotherapy regimens.

Oncolytic Viral Therapy for Prostate Cancer: Efficacy of Reovirus as a Biological Therapeutic

Reovirus is a nonattenuated double-stranded RNA virus that exploits aberrant signaling pathways allowing selective cytotoxicity against multiple cancer histologies. The use of reovirus as a potential treatment modality for prostate cancer has not previously been described, and in this study evidence of in vitro and in vivo activity against prostate cancer was seen both in preclinical models and in six patients. The human prostate carcinoma cell lines PC-3, LN-CaP, and DU-145 exposed to replication-competent reovirus showed evidence of infection as illustrated by viral protein synthesis, cytopathic effect, and release of viral progeny. This oncolytic effect was found to be manifested through apoptosis, as DNA fragmentation, Apo 2.7 expression, Annexin V binding, and poly(ADP-ribose) polymerase cleavage were observed in live reovirus-infected cells, but not in uninfected or dead virus-treated cells. In vivo, hind flank severe combined immunodeficient/nonobese diabetic murine xenograft showed reduction in tumor size when treated with even a single intratumoral injection of reovirus. Finally, intralesional reovirus injections into a cohort of six patients with clinically organ-confined prostate cancer resulted in minimal side effects and evidence of antitumor activity. Histologic analysis after prostatectomy found a significant CD8 T-cell infiltration within the reovirus-injected areas as well as evidence of increased caspase-3 activity. These findings suggest that reovirus therapy may provide a promising novel treatment for prostate cancer and also imply a possible role for viral immune targeting of tumor.

Immune reconstitution after anti-thymocyte globulin-conditioned hematopoietic cell transplantation

BACKGROUND AIMS Anti-thymocyte globulin (ATG) is being used increasingly to prevent graft-versus-host disease (GvHD); however, its impact on immune reconstitution is relatively unknown. We (i) studied immune reconstitution after ATG-conditioned hematopoietic cell transplantation (HCT), (ii) determined the factors influencing the reconstitution, and (iii) compared it with non-ATG-conditioned HCT. METHODS Immune cell subset counts were determined at 1-24 months post-transplant in 125 HCT recipients who received ATG during conditioning. Subset counts were also determined in 46 non-ATG-conditioned patients (similarly treated). RESULTS (i) Reconstitution after ATG-conditioned HCT was fast for innate immune cells, intermediate for B cells and CD8 T cells, and very slow for CD4 T cells and invariant natural killer T (iNKT) (iNKT) cells. (ii) Faster reconstitution after ATG-conditioned HCT was associated with a higher number of cells of the same subset transferred with the graft in the case of memory B cells, naive CD4 T cells, naive CD8 T cells, iNKT cells and myeloid dendritic cells; lower recipient age in the case of naive CD4 T cells and naive CD8 T cells; cytomegalovirus recipient seropositivity in the case of memory/effector T cells; an absence of GvHD in the case of naive B cells; lower ATG serum levels in the case of most T-cell subsets, including iNKT cells; and higher ATG levels in the case of NK cells and B cells. (iii) Compared with non-ATG-conditioned HCT, reconstitution after ATG-conditioned HCT was slower for CD4 T cells, and faster for NK cells and B cells. CONCLUSIONS ATG worsens the reconstitution of CD4 T cells but improves the reconstitution of NK and B cells.

Factors predicting engraftment of autologous blood stem cells : CD34+ subsets inferior to the total CD34+ cell dose

Data were analyzed on 178 consecutive patients (median age 43 years) who underwent autologous blood stem cell transplantation (ABSCT) at a single institution to determine if CD34+subsets (CD34+38−, CD34+33−, CD34+33+, CD34+41+) or various clinical factors affect hematopoietic engraftment independent of the total CD34+ cell dose/kg. Using Cox proportional hazards models, the factors independently associated with rapid neutrophil engraftment were higher CD34+ dose/kg, use of G-CSF post-ABSCT, and conditioning regimen (single-agent melphalan ± TBI slower). Factors independently associated with rapid platelet engraftment were higher CD34+ cell dose/kg, higher ratio of CD34+33−/total CD34+ cells infused, conditioning regimen (mitoxantrone, vinblastine, cyclophosphamide faster), and no CD34+ cell selection of the autograft. The CD34+ cell selection process seemed to deplete CD34+41+ cells to a greater extent than total CD34+cells which may explain our observation that it resulted in slower platelet engraftment. In conclusion, the total CD34+ dose/kg was a better predictor of hematopoietic engraftment following ABSCT than the dose of any CD34+ subset. Platelet engraftment, however, was also influenced by the ratio of CD34+33−/total CD34+cells for unmanipulated autografts, and possibly by the CD34+41+dose for autografts manipulated by CD34+ selection. The use of CD34+ subsets requires further investigation in predicting engraftment of autografts which undergo ex vivo manipulation.

Laser capture microdissection-guided fluorescence in situ hybridization and flow cytometric cell cycle analysis of purified nuclei from paraffin sections.

Laser capture microdissection (LCM) has recently been identified as a quick, simple, and effective method by which microdissection of complex tissue specimens for molecular analysis can be routinely performed. Assessment of gene copy number by fluorescence in situ hybridization (FISH) is useful for the analysis of molecular genetic alterations in cancer. Unfortunately, the application of FISH to paraffin sections of tumor specimens is fraught with technical difficulty and potential artifacts. Our results demonstrate that LCM-microdissected nuclei are suitable for FISH gene copy analysis. Amplification of genes in cancer specimens can be detected as easily in LCM-prepared nuclei as in fresh nuclei from cancer tissue specimens. Furthermore, contamination of tumor specimens by normal cells can make interpretation of flow cytometric cell cycle analysis difficult. Our results show that LCM-microdissected nuclei can also be used for flow cytometric cell cycle and ploidy analysis.LCM/FISH offers the advantages of multicolor FISH in a morphologically defined cell population, without the technical problems of FISH performed on paraffin sections. This technique should further simplify the methodology required to perform copy number analysis of tumor suppressor or proto-oncogenes in archived cancer specimens. The use of LCM specimens will also improve the specificity and simplify the interpretation of flow cytometric cell cycle and ploidy analysis of breast cancer specimens.

Reovirus as a Viable Therapeutic Option for the Treatment of Multiple Myeloma

Purpose: Despite the recent advances made in the treatment of multiple myeloma, the disease still remains incurable. The oncolytic potential of reovirus has previously been shown and is currently in phase III clinical trials for solid tumors. We tested the hypothesis that reovirus can successfully target human multiple myeloma in vitro, ex vivo, and in vivo without affecting human hematopoietic stem cell (HHSC) re-population/differentiation in a murine model that partially recapitulates human multiple myeloma. Experimental Design: Human myeloma cell lines and ex vivo tumor specimens were exposed to reovirus and oncolysis and mechanisms of cell death were assessed. RPMI 8226GFP+ cells were injected intravenously to non-obese diabetic/severe combined immune deficient (NOD/SCID) mice and treated with live reovirus (LV) or dead virus (DV). Multiple myeloma disease progression was evaluated via whole-body fluorescence and bone marrow infiltration. HHSCs exposed to LV/DV were injected to NOD/SCID mice and re-population/differentiation was monitored. Results: A total of six of seven myeloma cell lines and five of seven patient tumor specimens exposed to reovirus showed significant in vitro sensitivity. Tumor response of multiple myeloma by LV, but not DV, was confirmed by comparison of total tumor weights (P = 0.05), and bone marrow infiltration (1/6, LV; 5/6, DV). Mice injected with LV- or DV-exposed HHSCs maintained in vivo re-population/lineage differentiation showing a lack of viral effect on the stem cell compartment. Reovirus oncolysis was mediated primarily by activation of the apoptotic pathways. Conclusions: The unique ability of reovirus to selectively kill multiple myeloma while sparing HHSCs places it as a promising systemic multiple myeloma therapeutic for clinical testing. Clin Cancer Res; 18(18); 4962–72. ©2012 AACR.

Reovirus modulates autophagy during oncolysis of multiple myeloma

Multiple myeloma (MM) is a clonal plasma cell malignancy that accounts for 10–15% of newly diagnosed hematological cancers. Although significant advances have been made in the treatment of MM the disease still remains incurable. The oncolytic potential of reovirus has previously been demonstrated by others and us and is currently in phase III clinical trials for solid tumors. In addition a phase I clinical trial has recently been initiated for MM. Despite the clinical activity, the mechanism(s) of cell death caused by reovirus in MM is yet not well elucidated. A comprehensive understanding of reovirus-mediated histology-specific cell death mechanisms is imperative if this therapeutic is to become a standard of care for patients. Previously we have shown that reovirus-mediated cell death of breast and prostate cancer is orchestrated via apoptosis. The present study demonstrates for the first time that in addition to inducing apoptosis reovirus also upregulates autophagy during oncolysis of MM.

Bone marrow staging of patients with non‐Hodgkin lymphoma by flow cytometry

Immunophenotypic analysis is an established tool in the diagnosis and classification of many hematolymphoid disorders; however, the role of flow cytometry (FC) in detecting bone marrow involvement during the staging of non‐Hodgkin lymphoma (NHL) has yet to be defined.

Predictive factors for long-term engraftment of autologous blood stem cells

Data from 170 consecutive patients aged 19–66 years (median age 46 years) who underwent unmanipulated autologous blood stem cell transplant (ASCT) were analyzed to determine if total CD34+ cells/kg infused, CD34+ subsets (CD34+41+, CD34+90+, CD34+33−, CD34+38−, CD34+38−DR−), peripheral blood CD34+ cell (PBCD34+) count on first apheresis day, or various clinical factors were associated with low blood counts 6 months post ASCT. Thirty-four patients were excluded from analysis either because of death (n = 17) or re-induction chemotherapy prior to 6 months post ASCT (n = 13), or because of lack of follow-up data (n = 4). Of the remaining 136 patients, 46% had low WBC (<4 × 109/l), 41% low platelets (<150 × 109/l), and 34% low hemoglobin (<120 g/l) at a median of 6 months following ASCT. By Spearmans rank correlation, both the total CD34+ cell dose/kg and the PBCD34+ count correlated with 6 month blood counts better than any subset of CD34+ cells or any clinical factor. The PBCD34+ count was overall a stronger predictor of 6 month blood counts than was the total CD34+ cells/kg infused. Both factors retained their significance in multivariate analysis, controlling for clinical factors. In conclusion, subsets of CD34+ cells and clinical factors are inferior to the total CD34+ cell dose/kg and PBCD34+ count in predicting 6 month blood counts following ASCT. Bone Marrow Transplantation (2000) 26, 1299–1304.

Immune Cell Subset Counts Associated with Graft-versus-Host Disease

Graft-versus-host disease (GVHD) is a major transplantation complication. The purpose of this study was to measure immune cell subsets by flow cytometry early after transplantation (before median day of GVHD onset) to identify subsets that may play a role in GVHD pathogenesis. We also measured the subsets later after transplantation to determine which subsets may be influenced by GVHD or its treatment. We studied 219 patients. We found that acute GVHD (aGVHD) was preceded by high counts of CD4 T cells and CD8 T cells. It was followed by low counts of total and naive B cells, total and cytolytic NK cells, and myeloid and plasmacytoid dendritic cells. Chronic GVHD (cGVHD) was preceded by low counts of memory B cells. In conclusion, both CD4 and CD8 T cells appear to play a role in the pathogenesis of aGVHD. Generation of B cells, NK cells, and dendritic cells may be hampered by aGVHD and/or its treatment. Memory B cells may inhibit the development of cGVHD.