Giuliana Pierson

CD34 selection as a stem cell purging strategy for neuroblastoma: preclinical and clinical studies.

BACKGROUND The suitability of CD34 selection for purging peripheral blood progenitor cells (PBPC) collected from patients with neuroblastoma (NB) has been called into question, largely because of reports of detection of low levels of CD34 on the surface of some NB cell lines and tumors. PROCEDURE We used three approaches to address the issue of purging of NB from stem cell specimens and possible labeling of NB: 1) Flow cytometric detection of CD34 on NB cell lines. We assessed CD34 expression using a panel of anti-CD34 monoclonal antibodies (MoAbs) including 9C5, 12.8, and QBend10 and showed no increase in labeling over secondary-only control. 2) Spiking experiments with the Isolex 50 system. NB cell lines were used to contaminate aliquots of PBPC collections, after which the products were purified using the Isolex 50. Purging of NB was assessed by quantitative multiplex RT-PCR (TaqMan system) using a tumor-specific transcript, GAGE. We demonstrated >2 logs of tumor cell depletion from these specimens. 3) Analysis of clinical specimens. PBPC pre- and post-CD34 selection were analyzed from patients treated on the CHP-594 transplant trial. RESULTS In nine specimens selected using the Ceprate LC CD34 selection system where tumor was detectable by immunocytochemistry preselection, we observed >2.4 to >4.6 logs of NB purging after selection. We then analyzed 23 aliquots of PBPC infused into patients post-CD34 selection and compared them to the product preselection; 20/23 specimens showed depletion of NB, although some level of GAGE message was observed in most post-CD34 selection specimens. CONCLUSION These data show that purging of NB from PBPC specimens using CD34 selection is feasible, yielding infused products that are negative at the level of ICC but often positive at the level of RT-PCR.

Deficient expression of class-I HLA in some cases of acute leukemia

SummaryLeukemic cells from the blood and marrow of 25 cases of newly diagnosed acute leukemia were presented as target cells to alloreactive effector cells from unrelated normal donors in cell-mediated cytotoxicity assays. In three cases the leukemic targets were poorly killed relative to nonleukemic, HLA-identical target cells. The poor killing of the leukemic cells from one of these cases was shown by competitive inhibition to be due to deficient expression of normal class-I HLA antigens rather than resistance to lysis. Furthermore, the leukemic cells from these three patients were also deficient in binding monoclonal antibodies to nonpolymorphic determinants of class-I HLA and B2 microglobulin. Two additional cases were identified as having a less extensive deficit of HLA, and may be representative of a group with relatively subtle changes in these cell surface antigens. The possible significance of reduced expression of HLA in leukemic progression and in susceptibility to graft-vs-leukemia reactions after bone marrow transplantation is discussed.

The use of cytotoxic T cell lines to detect the segregation of a human minor alloantigen within families

A cytotoxic T cell (CTL) line, which detected a minor alloantigen provisionally called W was generated in vitro with lymphocytes from a multiply transfused individual, S1. Lymphocytes from S1 were first stimulated with cells from an unrelated known from previous studies to express the minor antigen. The primary CTL were then restimulated with cells from a W +/ve HLA identical sib, S2, in the presence of IL-2. As in previous work, recognition of the W antigen by these CTL was restricted by HLA-B7. Antigen assignments of W + W -, based upon cold target inhibition studies, confirmed previous assignments which had depended upon the ability of lymphocytes either to stimulate the generation of or to be killed by anti-W CTL effectors. Testing of lymphocyte targets from members of several unrelated families in which HLA-B7 segregated showed that the CTL lines could detect the expression of W on cells of individuals in the general population. In 3 of 5 cases, members of an HLA identical sib pair differed for W. These results open up the possibility of designing studies using CTL lines to determine whether differences for minor alloantigens play a role in clinical transplantation.

Recognition of human minor alloantigen(s) by cytotoxic lymphocytes in vitro.

Lymphocytes from an extensively transfused patient with aplastic anemia were induced to cytotoxicity against target cells from several HLA-matched siblings by in vitro stimulation with allogeneic cells. Effective stimulating cells shared HLA-B7 with the patient, but not all B7 individuals were effective. An additional factor, which was found to segregate in both the patients and an unrelated sibship, was also necessary. Segregation of this minor alloantigen, W, was also revealed among the patients HLA-matched sibs by differential susceptibility to lysis by effectors from the patient. The ratio of six positive to four negative siblings suggests that the antigen difference might be coded by a single locus. Lymphocytes from a normal sib, who like the patient is lacking the minor antigen, could not be induced to cytotoxicity against positive targets. Thus in vivo sensitization of the donor of the responding cells appears to be necessary for the demonstration of the cytotoxic response to the minor antigen in vitro. No correlation was observed between the segregation pattern of W and of known blood group antigens, and no cytotoxic antibody to W was detected in the patients serum in several trials.

Cytotoxic T lymphocyte lines (CTLL) against a human minor alloantigen

Cytotoxic T-cell lines were generated following in vitro culture of lymphocytes from a patient suffering from aplastic anemia together with those of his HLA-identical brother, a repeated transfusion donor. The segregation pattern within the family of the determinant(s) detected by these cytotoxic cells strongly suggested that a minor alloantigen(s) was being detected. Testing of the effectors on a panel of unrelated individuals indicated that it was best seen in association with HLA-B7, which was common to both the patient and his sibling donor.

T-cell-depleted maternal bone marrow transplantation for siblings with X-linked severe combined immunodeficiency

Prenatal diagnosis of X-linked severe combined immunodeficiency in a male fetus made possible elective neonatal bone marrow transplantation before onset of symptoms. Bone marrow transplantation was performed by using T-cell-depleted maternal marrow that had been cryopreserved 2 years earlier, at the time that his older affected brother underwent transplantation. The second patient had less morbidity and more rapid reconstitution of his immune function.

Susceptibility of human neuroblastoma cell lines to cytotoxic T lymphocyte-mediated lysis

The susceptibility of neuroblastoma cells to cytotoxic T lymphocyte (CTL)-mediated killing was investigated. Cytotoxic lines were generated by sensitizing peripheral blood lymphocytes against two stimulator cells, a neuroblastoma line, CHP-100, and normal allogeneic lymphocytes, LS. LS cells shared class I antigens with CHP-100, but in addition expressed class II antigens. The resulting cell lines strongly lysed both CHP-100 and LS cells, but poorly killed the natural killer (NK) target K562. Specific blocking of lysis by a monoclonal antibody directed against class I determinants and strong killing by the line following depletion of cells with NK or LAK markers demonstrated that this neuroblastoma line was lysed by CTL.

Successful Purging of Stem Cell Products Using CD34 Selection

Purging of tumor cells from stem cell products used to support autologous transplant procedures to treat solid tumors may decrease the risk of relapse. Concerns have been raised about the use of the only widely available technique for stem cell purging, CD34 selection, for purging products collected from patients with neuroblastoma (NB), largely because of reports of detection of low levels of CD34 on the surface of some NB cell lines and tumors. We have used 3 approaches to address the issue of purging of NB from stem cell specimens and possible CD34 labeling of NB. 1. Flow cytometric detection of CD34 on NB cell lines. We assessed CD34 expression using a panel of anti-CD34 monoclonal antibodies including 9C5, 12.8 and QBend10 and showed no increase in labeling over secondary-only control. 2. Spiking experiments with the Isolex 50 system. NB cell lines were used to contaminate aliquots of stem cell collections, after which the products were purified using the Isolex 50. Purging of NB was assessed by quantitative multiplex RT-PCR (Taqman system) using a tumor-specific transcript, GAGE. We demonstrated <2 logs of tumor cell depletion from these specimens. 3. Analysis of clinical specimens. Stem cell pre- and post-CD34 selection were analyzed from patients treated on a tandem transplant trial for NB. In nine specimens selected using the Ceprate LC CD34 selection system where tumor was detectable by immunocytochemistry pre-selection, we observed >2.4 to >4.6 logs of NB purging after selection. We then analyzed 23 aliquots of stem This research was supported in part by the University of Pennsylvania Cancer Center (SG), by the Benacerraf/Frei Clinical Investigator Award, Dana-Farber Cancer Institute (LD) and the Fiftieth Anniversary Program for Scholars in Medicine, Harvard Medical School (LD). The abbreviations used are: FITC, fluorescein isothiocyanate; GAPDH, Glyceraldehyde 3 phosphate dehydrogenase; ICC, immunocytochemistry; MoAbs, monoclonal antibodies; PE, phycoerythrin; RT-PCR, reverse transcriptase-polymerase chain reaction; PBMC, peripheral blood mononuclear cells.

Angiogenesis Inhibitor TNP-470 During Bone Marrow Transplant and in Minimal Residual Disease

High-dose therapy with stem cell rescue is a treatment option for patients with advanced solid tumors. Although this approach has promise for some pediatric cancers, especially neuroblastoma, it is limited by the risk of relapse post transplant, as well as concern about possible reinfused tumor cells in autologous stem cell products. Anti-angiogenic agents given during and after recovery from highdose therapy with stem cell rescue may decrease the risk of relapse. Additionally, such agents may have their greatest therapeutic effect when administered at a point of minimal residual disease, rather than to treat bulky tumor. TNP-470 is an anti-angiogenic agent now in clinical trials. We have tested TNP-470 in the treatment of neuroblastoma in a mouse xenograft model, and demonstrate that it is most effective when given at lowest disease burden. Additionally, and to assess the feasibility of using anti-angiogenic agents during the period of posttransplant hematopoietic recovery, we have developed a model of stem cell transplant in mice. Mice were treated with TNP-470 and assessed for survival and engraftment. Both treated and control mice demonstrated reliable multilineage engraftment as well as normal lymphoid maturation with no excess mortality in the treated group. This indicates that inhibitors of angiogenesis do not adversely impact engraftment after stem cell transplantation and suggests a clinical study design in which such agents might best be employed in the immediate after stem cell transplant.