V. von Fliedner

Separation of tumor-infiltrating lymphocytes from tumor cells in human solid tumors: A comparison between velocity sedimentation and discontinuous density gradients

The separation of viable tumor-infiltrating lymphocytes (TIL) from surgical biopsies of human solid tumors was achieved by velocity sedimentation at unit gravity or by discontinuous density gradients. The two methods were adapted to small volumes and cell numbers not exceeding 1 X 10(8). The recovery, purity and composition of the TIL-enriched fractions were comparable in the two methods. Density gradients were more rapid, simpler and more practical for preparation under sterile conditions of TIL from clinical material than velocity sedimentation. Lymphocytes in the TIL-enriched fractions obtained by either of the methods were poorly responsive to mitogens. This poor responsiveness is a characteristic of the human TIL and seems to be related to effects exerted by tumor cells.

Post-remission therapy of adult acute myeloid leukaemia: One cycle of high-dose versus standard-dose cytarabine

BACKGROUND Intensification of post-remission therapy improves the cure rate of acute myeloid leukemia (AML) but is often accompanied by unacceptable toxicity. From 1985 to 1992 the Swiss Group for Clinical Cancer Research (SAKK) performed a randomized phase III trial to evaluate the effectiveness of one single postremission course of high-dose cytarabine (HDAC) in terms of leukaemia-free and overall survival in adults with de novo AML. PATIENTS AND METHODS Adult (15-65 years) AML patients in remission after two induction courses were randomly assigned to one consolidation course either with standard (SDAC: 100 mg/sqm 24 hours infusion over seven days) or with high-dose cytarabine (HDAC: 3000 mg/sqm every 12 hours as one-hour-infusion for six days). In addition, both arms included daunorubicin (45 mg/sqm daily on days 1 to 3). Thereafter, patients were observed without maintenance until relapse. RESULTS After two induction courses 208/276 eligible patients achieved remission (CR: 169, 61%, PR: 39, 14%), 41 were resistant (15%) and 20 died early (7%). Seventy-one patients in remission were not randomized. One hundred thirty-seven were randomized in CR/PR (67 SDAC, 70 HDAC). 4/70 patients randomized to HDAC did not receive it. Treatment-related mortality in HDAC was 1.4% (1/66). WHO grade 3-4 toxicities occurred in 14/67 SDAC and in 38/66 HDAC patients (P < 0.0001). The median event free survival was 10.8 (SDAC) vs. 12.2 months (HDAC; P = 0.18). The median overall survival was 24.6 (SDAC) vs. 32.6 months (HDAC; P = 0.07). Although statistically uncertain, HDAC reduced the hazard of progression (hazard ratio: 0.69, P = 0.08) and of death (hazard ratio: 0.70, P = 0.13). For 112 patients stratified as CR the estimated four-year disease-free survival was 25% (+/-6%) with SDAC and 37% (+/-6%) with HDAC (P = 0.09). The overall survival rates at four years were 38% (+7%) and 48% (+7%), respectively (P = 0.10). In multivariate analysis HDAC significantly reduced the hazard of relapse by 39% compared to SDAC (hazard ratio = 0.61, 95% CI: 0.37-0.99; P = 0.049). CONCLUSIONS We conclude that early consolidation of adult AML in CR with a single course of HDAC is superior in terms of outcome to one cycle of SDAC. The results of our intensive, single course HDAC group compare favourably with less intensive, repetitive HDAC cycles, suggesting that Ara-C dose intensity may be more important than total dosage. In addition, our treatment strategy is much less toxic and less expensive.

Intensive induction/consolidation therapy without maintenance in adult acute lymphoblastic leukaemia: a pilot assessment

. Maintenance chemotherapy for up to 3 years is traditionally given to patients with acute lymphoblastic leukaemia (ALL) achieving complete remission. We questioned the value of such maintenance therapy in adult patients treated with intensive induction/consolidation. In a phase II study (SAKK 33/86) 63 patients between 17 and 72 years of age (median 27 years) with newly diagnosed ALL were treated with three intensive cycles of marrow‐ablative chemotherapy. All subtypes were included. No maintenance phase was added. 53 patients (84%) entered a complete remission (CR) and 21 (33%) continue to be in unmaintained remission for 11‐69 months (median 21 months). The disease‐free survival of patients achieving CR and completing all three cycles is 40% at 3 years, with a 95% confidence interval of ± 19%. These findings are comparable to the results of conventional studies. We conclude that maintenance therapy might not be needed in all adult ALL patients. Its value should be tested in a randomized trial. For patients failing, novel approaches are needed to improve outcome in adult ALL.

Production of tumor necrosis factor-α by naive or memory T lymphocytes activated via CD28

Abstract While it is well established that activated T cells can produce tumor necrosis factor alpha (TNF-α), it is less clear whether this function is confined to a given subset, e.g., memory cells. To approach this question, we investigated the production of TNF-α by human peripheral blood T lymphocytes activated with anti-CD28 mAb since this activation pathway is known to potentiate cytokine production. Under the culture conditions used, the amount of TNF-α produced was markedly enhanced compared to that obtained after activation with immobilized anti-CD3 mAb. The enhancement of TNF-α production was already apparent after incubation of T cells for 6 hr. Up to 5 ng/ml of TNF-α was measured on Day 2 in supernatants of cultures of 104 T lymphocytes. To determine the source of the cells producing high amounts of TNF-α, T lymphocytes were separated into two subpopulations, namely naive cells (expressing the CD45RA isoform) and memory cells (expressing the CD45RO isoform). While both subpopulations proliferated equally well after stimulation with anti-CD28 mAb, up to 90% of the TNF-α produced under these conditions originated from memory T cells. These results thus document that T cell activation via CD28 results in a marked increase in TNF-α production without affecting the functional disparity that exists between naive and memory T cells.

Intensification of chemotherapy for the treatment of solid tumours: feasibility of a 3-fold increase in dose intensity with peripheral blood progenitor cells and granulocyte colony-stimulating factor

Dose intensity may be an important determinant of the outcome in cancer chemotherapy, but is often limited by cumulative haematological toxicity. The availability of haematopoietic growth factors such as granulocyte colony-stimulating factor (G-CSF) and of peripheral blood progenitor cell (PBPC) transplantation has allowed the development of a new treatment strategy in which several courses of high-dose combination chemotherapy are administered for the treatment of solid tumours. PBPCs were mobilised before chemotherapy using 12 or 30 micrograms kg-1 day-1 G-CSF (Filgrastim) for 10 days, and were collected by 2-5 leucaphereses. The yields of mononuclear cells, colony-forming units and CD34-positive cells were similar at the two dose levels of Filgrastim, and the numbers of PBPCs were sufficient for rescue following multiple cycles of chemotherapy. High-dose chemotherapy (cyclophosphamide 2.5 g m-2 for 2 days, etoposide 300 mg m-2 for 3 days and cisplatin 50 mg m-2 for 3 days) was administered sequentially for a median of three cycles (range 1-4) to ten patients. Following the 30 evaluable cycles, the median duration of leucopenia < or = 0.5 x 10(9) l-1 and < or = 1.0 x 10(9) l-1 was 7 and 8 days respectively. The median time of thrombopenia < or = 20 x 10(9) l-1 was 6 days. There was no cumulative haematological toxicity. The duration of leucopenia, but not of thrombopenia, was inversely related to the number of reinfused CFU-GM (granulocyte-macrophage colony-forming units). In the majority of patients, neurotoxicity and ototoxicity became dose limiting after three cycles of therapy. However, the average dose intensity delivered was about three times higher than in a standard regimen. The complete response rate in patients with small-cell lung cancers was 66% (95% CI 30-92%) and the median progression-free survival and overall survival were 13 months and 17 months respectively. These results are encouraging and should be compared, in a randomised fashion, with standard dose chemotherapy.

Stimulation of FACS-analysed CD4+ and CD8+ human tumour-infiltrating lymphocytes with ionomycin + phorbol-12,13-dibutyrate does not overcome their proliferative deficit.

Human tumour‐infiltrating lymphocytes (TIL) were prepared by enzyme digestion from a series of different tumours and were purified on a fluorescence‐activated cell sorter (FACS II) according to their CD4+ and CD8+ phenotype. CD4+ and CD8+ TIL were stimulated separately in a low density microculture system with phytohacmagglutinin (PHA) or with ionomycin plus phorbol‐12, 13‐dibutyrate (PDBu). The PHA‐induced proliferation of TIL was highly decreased when compared with control peripheral blood lymphocytes. A decreased proliferation of TIL was also observed when cells were stimulated with ionomycin plus PDBu, a combination which is thought to circumvent early events associated with lymphocyte activation. Some TIL were also plated in limiting dilution where they showed decreased frequencies of proliferating T cell precursors. The data suggest that one component of the inhibition of TIL must be acting ‘ownstream’ of the early events of lymphocyte activation.

Contribution of immunological markers to the diagnosis and prognosis of human leukemia

Abstract Surface markers have been of proven diagnostic and prognostic use in acute lymphoblastic leukemia (ALL). T cell ALL (T-ALL), where blasts possess receptors for sheep red blood cells (R-SRC+), is associated with an adverse prognosis in children and adults. The presence of common ALL antigen (CALLA)-positive blasts (i.e. common-ALL) in children is indicative of a good response to treatment, in contrast to the poor response shown by pre-B-ALL cases, where the blasts are also CALLA-positive but additionally contain cytoplasmic μ chains. Recently a subgroup of T-ALL, immature T-ALL, was identified, where the blasts lack R-SRC and T cell markers (such as T1, T3, T4, T8, T6) but carry a pan T cell antigen (p40) recognized by the monoclonal antibody LAU-A1 ( 12 103 ALL cases in our series). This new subgroup, immature T-ALL (R-SRC-/p40+), also seems to be associated with a poor prognosis, like T-ALL.

Sequential high-dose chemotherapy with r-metHu-G-CSF (Filgrastim) and infusion of peripheral blood progenitor cells (PBPC) in patients with small cell lung cancer (SCLC). A feasibility study

Despite the progress that has been made during the last decades in the treatment of small cell lung cancer, the emergence of drug resistant cells to chemotherapy remains a major problem. Dose-response relationship has been initially demonstrated in animal models [1]. Similarly, several clinical trials have confirmed that a dose-response exist for many chemotherapeutic agents [2, 3] and that intensive chemotherapy may achieve a high response rate in refractory tumors such as breast or small cell lung cancers [4, 5, 6] . To date however, the increase in complete response rate has not been translated into improvement in overall survival. But intensification has been limited to the administration of a single treatment under the protection of autologous bone marrow transplantation. The early delivery of multiple sequential, intensive treatments might improve these results.

Isolation, Functional Properties and Clonal Analysis of Tumor-Infiltrating Lymphocytes from Human Brain Tumors

Despite the numerous reports of decreased or defective systemic cell mediated immunity in glioma patients (1–7) there is evidence of a local immune response to the tumor. Thus mononuclear cell (MNC) infiltrates have been demonstrated within the parenchyma of human glial tumors in 30–60 % of cases reported in the literature (8, 9). Other studies have correlated the intensity of MNC infiltrates with tumor histology and survival in glioma patients (10–12). Von Hanwehr and co-workers (9) found that T lymphocytes constituted the major fraction of the infiltrates in gliomas. However the nature and functional capabilities of these cells and their potential cytolytic role in preventing tumor growth remain poorly understood. To resolve these questions it is necessary to isolate the tumor infiltrating lymphocytes (TIL) from the tumor tissue. To this end TIL from 7 human brain tumors were isolated using enzyme digestion and density gradient centrifugation. The resulting TIL fractions were then cloned in a limiting dilution assay (13) that allows virtualy all peripheral blood resting T cells to undergo clonal expansion. The frequency of proliferating T lymphocyte precursors (PTL-P) can then be calculated thus giving an indication of immunocompetent T cells present in the original infiltrate compared to patient and normal peripheral blood. In addition the clones obtained can be expanded to allow for functional assays in vitro. This report demonstrates that TIL cloned from human gliomas and grown in the presence of interleukin-2 do exhibit cytolytic activity in vitro in both allogeneic and autologous systems.

Radiolabeled Antibodies for the Detection of Cancer: New Approaches to Improve the Sensitivity and Specificity of Immunoscintigraphy

Paul Ehrlich’s concept of “magic bullet” for the cure of diseases has been revitalized by the development of the monoclonal antibody (Mab) technology allowing the production in unlimited amounts of antibodies of perfect homogeneity and specificity directed against various tumor markers. This chapter will review one aspect of the magic bullet concept, the use of radiolabeled antibodies as tracer for tumor detection by immunoscintigraphy. We shall critically consider the results obtained in this attractive field, from our early experimental results with 131I labeled polyclonal antibodies against carcinoembryonic antigen (CEA) up to the most recent clinical results obtained with 123I labeled fragments of anti-CEA Mab. Special emphasis will be given to the difficulties encountered in the detection of liver metastases when using intact anti-CEA antibodies and to the improvement obtained when we used 123I labeled Mab fragments and single photon emisssion computerized tomography (SPECT) for their localization in three dimensions.