Human tumor-lymphocyte interaction in vitro. VI. Specificity of primary and secondary autologous lymphocyte-mediated cytotoxicity.

A T-cell-enriched lymphocyte subset of samples from 15 tumor patients was tested for primary cytotoxicity against autologous tumor cell preparations and against 1-3 different allogeneic tumor cell preparations from biopsy material. Allogeneic cytotoxicity occurred in only 1 of 10 patients with autologous reactivity. The lymphocytes of 14 patients were cultured with autologous cells from biopsy material for 6 days. These lymphocytes killed autologous targets, but only 1 patient's lymphocytes were cytotoxic against 1 of the 4 allogeneic tumors tested. Cocultivation with allogeneic cells from biopsy specimens generated cytotoxicity toward the sensitizing allogeneic cells in 3 of 9 test combinations. In 2 of 3 instances the effectors were also active against the autologous tumor cells. Cytotoxicity in primary and secondary tests occurred thus only rarely against allogeneic targets. This indicated either the presence of individual tumor-related antigens on the cells from biopsy material or reflected the histocompatibility restriction of T-cell-mediated cytotoxicity.

Efficacy and clinical cross-resistance of a new combination therapy (AMSA/VP16) in previously treated patients with acute nonlymphocytic leukemia.

We investigated the tolerance, efficacy, and clinical cross-resistance of a new combination chemotherapy in 38 patients with previously treated acute myeloblastic leukemia (AML). It consisted of 120 mg2/d 4'(9-acridinylamino) methanesulfon-m-Anisidide (m-AMSA) in a one-hour infusion and 80 mg/m2/d etoposide (VP-16) in a 24-hour infusion, both administered for 5 days. The first 27 patients also received vinblastine, 6 mg/m2 on day 8, but this therapy was discontinued because of intestinal complications. Thirteen of 23 patients (56%) at first or subsequent relapse and five of 15 patients (33%) who were primarily resistant to an anthracycline/cytarabine combination achieved a complete response (CR) (hemoglobin level not taken into account) with a median CR duration of 5 months and 2 months, respectively. The response rate was as high as 63% for patients at first or second relapse whether the remission was maintained or not. The median times to recovery of normal bone marrow cellularity, of blood granulocyte counts greater than 500/microL, and of platelets greater than 20,000/microL were 34, 27, and 22 days, respectively. Marked but reversible gastrointestinal toxicity was observed in 24% of the patients, and two patients died of infection during induction. The one-hour AMSA/continuous VP-16 combination is effective for patients with relapsing AML and shows no cross-resistance in a proportion of patients refractory to the standard anthracycline-cytarabine combination.

Axl expression is associated with adverse prognosis and with expression of Bcl-2 and CD34 in de novo acute myeloid leukemia (AML): results from a multicenter trial of the Swiss Group for Clinical Cancer Research (SAKK).

Receptor tyrosine kinases (RTK) play a significant role in the signal transduction of normal, and malignant hematopoietic cells. We have previously shown that Axl, a novel RTK, is mainly expressed in leukemias of myeloid origin, and that its expression may be associated with cells of monocytic origin. Since expression of certain RTKs in cancer may be associated with different biology and survival, we investigated whether the expression of Axl is associated with clinical characteristics and survival in acute myeloid leukemia (AML). RNA from 54 patients with AML treated in a cooperative group trial was analyzed in a retrospective and blinded manner using a semi-quantitative reverse transcriptase polymerase chain reaction-based assay with primers specific for the Axl gene. Axl expression was found in 19 out of the 54 cases (35%). Axl expression was not detected more frequently in patients of older age, specific FAB categories, or cases with extramedullary disease. However, there existed a correlation between Axl and bcl-2 expression levels. AML cells with high bcl-2 expression showed higher Axl expression (r = 0.32; P = 0.02), and furthermore, Axl transcript numbers were also higher in AML with high CD34 expression (n = 38, r = 0.42; P = 0.008). No significant difference between leukemias expressing and not expressing Axl was found with regard to complete remission rate. However, quantitative Axl expression was associated with worse progression-free and overall survival. Higher Axl levels had worse prognosis for progression-free (beta: 0.68, s.e.: 0.28, P = 0.015) and overall survival (beta: 0.61, s.e.: 0.31, P = 0.05) using multivariate Cox models adjusted for age, Auer rods and leukocyte counts. In conclusion, in this retrospective analysis, no difference with regard to clinical characteristics at diagnosis was found between AML patients whose leukemia cells show Axl expression vs patients whose cells are Axl negative. The association between Axl and bcl-2 and Axl and CD34 expression in de novo AML needs further investigation. Similarly, the negative impact of Axl levels on outcome should be confirmed in a larger cohort.

Incidence of viral markers and evaluation of the estimated risk in the Swiss blood donor population from 1996 to 2003.

Among the well known transfusion-associated risks, the transmission of pathogenic viruses is regarded as one of the most serious. Over the past two decades, a series of overlapping safety procedures have been successively implemented to minimise this risk. It is now generally considered that the risk of transmitting viral infections via blood products is very low in developed countries. The present study analyses the incidence of the key infectious diseases HIV, hepatitis B virus (HBV) and hepatitis C virus (HCV) between 1996 and 2003 from 99% of voluntary repeat blood donors visiting the blood transfusion service of the Swiss Red Cross. Furthermore the estimated risk of these viral markers was calculated. From 1996 to 2003 the incidence rate for HCV decreased continuously, whereas no significant decrease in the incidence rate of HIV and HBV was observed. From 2001 to 2003, the last calculated period, the residual risk was estimated to be 1 in 1,900,000 for HIV, 1 in 2,200,0000 for HCV and 1 in 115,000 for HBV, respectively. This agrees with international studies, which have been shown that the estimated residual risk for HBV between 1996 and 2003 is higher than that of HCV and HIV.

Polymyositis, myasthenic syndrome and thymoma in a patient with defective cell-mediated immunity.

We studied a 57-year old woman with severe myasthenic syndrome predominantly proximal. There was no therapeutic effect using cholinesterase inhibitors. Clinical findings, electromyography, whole body scanning and biopsy revealed polymyositis. Thirteen years before the patient was operated of a benign thymoma. The history of the patient showed numerous life threatening episodes of viral and fungal infections. Autoimmune anemia was diagnosed. Investigations of the immune system in vivo and in vitro revealed severe qualitative and quantitative defects in the lymphocyte population spontaneously forming rosettes with sheep red blood cells (SRBC). Thymoma, autoimmune disorder, such as polymyositis and myasthenia gravis, unspecifically elevated antibody titers, multiple severe viral and fungal infections and the defect of the cell-mediated immunity suggest a T-lymphocyte effector- and regulatory dysfunction in this patient.

[Therapeutic plasmapheresis--a critical review in the light of the current literature]. / Therapeutische Plasmapherese--eine kritische Würdigung unter Berücksichtigung der aktuellen Literatur.

For centuries it has been the dream of many physicians to cure illnesses by eliminating disease provoking substances which are thought to circulate in the human body. Technical developments during the past 20 years have made therapeutic plasma exchange (TP) a useful procedure for clinical application. Because of the lack of well controlled studies the true benefit of the method remains speculative in many clinical situations. Since the report of the American Medical Association (AMA) Panel on Therapeutic Plasmapheresis in 1985 several controlled studies on this subject have been published in recent literature; they are reviewed in this article. In summary, TP seems justified only in some area and well defined situations such as thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, acute and severe myasthenia gravis, severe Guillain-Barré-syndrome, hyperviscosity syndromes such as hyper- and paraproteinemia, and in several intoxications and metabolic disorders such as Refsum disease or hereditary hyperlipidemia type IIa.

Restricted autologous lymphocytotoxicity in lung neoplasia.

Blood lymphocytes from 47 patients with lung carcinoma have been tested for cytotoxicity against cells isolated from the autologous tumour. Significant cytotoxic potential was found in 15 cases. The effectors were also tested against allogeneic tumour targets from lung and other sites. Reactions were only rarely detected (2/32 positive against lung and 1/13 positive against non-lung cells). The restriction of cytotoxicity to the autologous combination was also apparent in in vitro-generated effectors. Blood lymphocytes were co-cultivated with autologous tumour and subsequently tested against autologous or allogeneic targets. Cytotoxicity was found in 13/17 lung tumours against autologous tumour, with no reactions recorded against allogeneic tumour targets, but one case positive against the K562 cell line. These data suggest either the expression of individually distinct antigens on human pulmonary neoplasms, or the requirement for histocompatibility between target and effector in cytotoxicity reactions in man, and therefore differ from previously described patterns of lymphocytotoxicity against human tumours.

In vitro generation of cytotoxicity against autologous human tumour biopsy cells.

Blood lymphocytes from 22 cancer patients were examined for cytotoxicity against autologous tumour cells in a short-term 51Cr release assay. Only three showed reactivity. In an attempt to increase cytotoxic potential in these and induce reactivity in non-reactive cases, the lymphocytes were cultured alone or with autologous tumour cells for 6 days. Upon subsequent testing against frozen, stored targets, nine samples reacted, including two of those with primary reactivity. In seven cases augmented cytotoxicity was evident in mixed cultures compared with lymphocytes cultured alone. Two of 10 cases showed cytotoxicity against the K562 cell line after culture and, in two of 13 tests in which allogeneic tumour biopsy targets were used, weak reactivity was bound. Cytotoxicity for autologous tumour biopsy cells was uniformly accompanied by positive blastogenesis in MLTI assays. In four cases blastogenesis occurred without the induction of cytotoxicity.

[Unrecognized atypical tuberculosepsis in generalized hematologic neoplasms]. / Verkannte atypische Tuberkulosepsis bei disseminierten hämatologischen Neoplasien.

Differential diagnosis and therapy of 4 cases (3 of acute myelogenous leukemia and 1 of Hodgkin's disease, stage IVB) of septic exacerbation of febrile course in generalized hematologic neoplasms under intensive treatment are described. The infectious process of 3 cases was confirmed as generalized tuberculosis only at autopsy. The incidence in over 250 patients (1973-1979) was 1.6%.

Phase-I trial of 4-demethoxydaunorubicin in acute leukaemias.

Eleven patients with heavily pretreated acute leukaemia were treated with 4-demethoxydaunorubicin. Dosages were escalated from 7 mg/m2/d to 15 mg/m2/d for 3 consecutive days. One patient achieved a partial remission and antitumor activity was seen in most other patients. Stomatitis was dose-limiting at 15 mg/m2. Phase II trials are warranted and we propose a schedule of 12 mg/m2/d for 3 consecutive days.

[Harvesting of granulocytes for granulocyte transfusion by means of continuous flow centrifugation or filtration leukapheresis]. / Granulozytengewinnung zur Granulozytentransfusion durch Continuous Flow Centrifugation oder Filtrations-Leukapherese

More than 10(10) viable granulocytes are necessary for a therapeutical effective granulocyte transfusion. This number of cells can be harvested from normal donors by two techniques basing on different principles: continuous flow centrifugation (CFC) and filtration leucapheresis (FL). Our studies demonstrated that, under certain special conditions, the separation potentials of both methods are comparable yielding 2.5 to 3.0 X 10(10) granulocytes within 4 hrs. Granulocyte collection rate was optimal if donors were treated with dexamethasone during 16 hrs prior to the state of the procedure. However, the costs of CFC exceed those of FL by a factor of about two. The increased occurrence of side effects attributed to the transfusion of FL-granulocytes can be reduced to the level of CFC-granulocytes by repetitive filtration-elution leucapheresis minimizing cell damage. The studies define the efficiency spectrum of CFC which in addition to granulocyte separation includes collection of thrombocytes, cells for immunotherapy, and plasmapheresis.

Evidence for persistence of human parvovirus B19 DNA in bone marrow.

A nested polymerase chain reaction assay (nPCR) was used to investigate the potential of human parvovirus B19 DNA to persist in blood or bone marrow samples obtained either from blood donors or cadaveric bone donors or from patients presenting with clinical signs of parvovirus B19 infection. The presence of parvovirus B19 specific antibody in blood was tested by enzyme immunoassay (EIA). B19 virus genome was not detected in any blood sample of 115 blood donors, of whom 92 (80%) had anti-B19 IgG antibody only as an indication of past infection. In contrast; B19 virus DNA was detected in the bone marrow of 4 out of 45 bone donors. Each one of the serum samples available for 3 of these 4 individuals contained anti-B19 IgG antibody. Among 84 patients with clinical manifestations of parvovirus B19 infection, 17 (20%) had B19 virus DNA in bone marrow. Eight of the latter patients had anti-B19 IgG antibody in their blood but neither anti-B19 IgG nor B19 virus DNA. These data document the ability of parvovirus B19 DNA to persist in the bone marrow of asymptomatic individuals and patients with parvovirus B19 infection suspected on clinical grounds.

[Action of various growth factors on bone marrow cultures in acute myeloid leukemia (ALM)]. / Wirkung verschiedener Wachstumsfaktoren auf Knochenmarkkulturen bei akuter myeloischer Leukämie (AML).

In an attempt to detect residual leukemic cells during remission, we examined differences in the stimulatory capacity of colony stimulating factors (CSF) added to agar cultures of AML patients at diagnosis/relapse (group I) and in remission (II), or of normal controls (III). Supernatant from the human bladder carcinoma cell line 5637 and human recombinant granulocyte-macrophage-CSF were compared with supernatant from human placenta as standard. Group I showed wide patient-to-patient variations. The stimulatory capacity of the 3 CSF produced significant differences within groups II and III, but not between those groups. In conclusion, our comparative investigations do not reveal residual leukemic cells in the marrow of AML patients in remission.

[Diagnostic and therapeutic importance of splenectomy in patients with non-Hodgkin's lymphoma]. / Diagnostische und therapeutische Bedeutung der Splenektomie bei Patienten mit Non-Hodgkin-Lymphomen.

Whereas the contribution of exploratory laparotomy in Hodgkin's disease is well characterized, its value in Non-Hodgkin lymphoma (NHL) is not yet defined. This retrospective analysis of 31 cases is a contribution to the ongoing discussion. Laparotomy/splenectomy (LS) was done in 17 patients for diagnostic reasons and in 14 with therapeutic intent. Perioperative morbidity was low. In 17 cases the NHL had infiltrated the spleen. Indications for therapeutic LS were hemolytic anemia, pancytopenia and excessive lymphocytosis with granulocytopenia. The therapeutic benefit from splenectomy was satisfactory, especially in patients with well-differentiated lymphocytic leukemia of type CLL. In contrast, the diagnostic value of LS was minimal, except in patients with first diagnosis of NHL through LS. There was no change in tumor stage in any case. However, 4 false-negative findings contrast with the rapidly adverse course in these patients. Routine LS in patients with NHL does not appear to be justified, but has its value in NHL with primary abdominal localization. Therapeutic splenectomy is of benefit for the majority of patients, particularly those with CLL.

[Prevention of infection in patients with agranulocytosis through isolation and whole-body decontamination]. / Infektprophylaxe bei Patienten mit Agranulozytose durch Isolation und Ganzkörperdekontamination

During induction chemotherapy 21 patients with acute myelogenous leukemia and agranulocytosis were prophylactically isolated either in reverse isolation (group A) or in sterile plastic-tent isolators with laminar air flow (Life Islands, group B). Patients of group B also received whole-body decontamination and sterile food. Life threatening septicemias were seen only in patients in reverse isolation. Granulocyte transfusions were highly successful in reducing infection-related mortality in group A. Seven additional patients with agranulocytosis of varying origin (group C) were protected in the Life Islands as effectively as the patients with acute leukemia.

Intravascular fate of granulocytes administered by granujlocyte transfusions.

Granulocytes were harvested from hematologically normal individuals using continuous flow centrifugation (CFC) or filtration leukapheresis (FL). The isolated granulocytes were labeled in vitro by 3H-diisopropyl fluorophosphate (3H-DFP) and autotransfused. Their intravascular fate was analyzed by autoradiography. Immediately after autotransfusion the majority of granulocytes administered, collected in the marginal granulocyte pool. Margination was particularly prominent in granulocytes isolated by FL. The distribution of transfused granulocytes between the circulating and the marginal granulocyte pool showed wide and irregular fluctuations in time. Margination of transfused granulocytes was counterbalanced, and its fluctuation between the two intravascular pools was stabilized by prednisone treatment. The transit of transfused granulocytes from blood to tissue seemed to be governed by a random process, the half-disappearance time being either normal or prolonged. Compatible granulocytes administered to hematologically normal recipients circulated for at least 20 h.

Relapse cell population differs from acute onset clone as shown by absence of the initially activated N-ras oncogene in a patient with acute myelomonocytic leukemia.

We have conducted a follow-up study of a patient with myelomonocytic leukemia exhibiting an N-ras mutation (Gln61----Lys61) using the polymerase chain reaction method and synthetic oligonucleotide hybridization probes. This method allowed us to detect as little as 3% of N-ras-mutated cells within a population. When the patient went into clinical remission, the mutation became undetectable. When a relapse occurred, the blasts did not carry the N-ras mutation. Analysis of M13 cloned amplified N-ras sequences from relapse DNA revealed exclusively the wild type allele of the N-ras gene. These findings suggest that the relapse cell population is derived from a different clone than the acute phase population. Furthermore, the data argue that N-ras mutation is not an initiating lesion in this case of acute myelomonocytic leukemia (AMML).

Factors associated with transfusion requirements during treatment for acute myelogenous leukemia.

Supportive care is a prerequisite for intensive chemotherapy in leukemic patients. Little has been published about quantitative aspects of red blood cell and platelet transfusions. We evaluated transfusion requirements and factors associated with observed differences in 206 patients undergoing initial induction consolidation chemotherapy for newly diagnosed acute myelogenous leukemia. All patients were treated during a 5-year period in 12 hospitals on a common protocol of the Swiss Study Group for Clinical Cancer Research (SAKK). Protocol 30/85 comprises a double induction and one course of consolidation. Of 206 registered patients, 199 were evaluable; 118 of 199 (59%) patients entered completed all three cycles of chemotherapy. These 118 patients received a median (range) of 18 (3-44) units of red blood cells and 12 (2-61) platelet transfusions during 112 (70-129) days of hospitalization. Patients with a hemoglobin > 10 g/l, platelets > 100 x 10(9)/l, and white blood cell counts < 5 x 10(9)/l at diagnosis received fewer transfusions than patients with less favorable blood counts during the first cycle of chemotherapy (p < 0.05). Patients with FAB subtype M3 received more platelet transfusions during the first cycle. Female patients received more platelet transfusions than male patients. In multivariate analyses the participating center was the most important single factor associated with the number of red cell and platelet concentrates given per patient and cycle (p < 0.05), the number of days in hospital (p < 0.05), and the risk of premature withdrawal from the study. These data define factors associated with transfusion requirements in patients treated for newly diagnosed AML. They include severity and subtype of disease at diagnosis, age and sex of the patients, and the participating institution. Results suggest that medical decision-making varies from center to center. The participating institution is strongly associated with differences in transfusion requirements, hospitalization time, and premature withdrawal from study. Leukemia trials tend to focus on the prospective evaluation of chemotherapy or growth factors. Our results suggest that other variables, such as management strategies, should be included for prospective analysis.

Relationship between cleaved L-selectin levels and the outcome of acute myeloid leukemia.

High plasma levels of the shed form of L-selectin (sL-selectin) are frequently detectable in acute myeloid leukemia (AML). sL-selectin can inhibit blast cell adhesion to vascular endothelium and may thereby influence the phenotype of AML. In this study, we have investigated the relationship between sL-selectin levels and clinical presentation or disease outcome in 100 patients with AML. Fifty-eight patients were found to have sL-selectin levels >/=3.12 microgram/mL (>/=3 SD above the mean of healthy controls: "increased"). Patients with extramedullary disease such as lymphadenopathies, splenomegaly, hepatomegaly, and/or muco-cutaneous infiltration had significantly increased sL-selectin levels (P < .001). sL-selectin levels were significantly heterogeneous in the French-American-British subtypes (P = .0003). Patients with "normal" sL-selectin levels had higher probability of achieving complete remission (CR) than with "increased" levels: 81% versus 64%, respectively (P = .06). When adjusting for clinically relevant covariates predictive for CR (sex, age, Auer rods), "normal" sL-selectin levels were significantly associated with CR (odds ratio, 3.08; 95% confidence interval [CI], 1.10 to 8.58; P = .03). Moreover, patients with "increased" sL-selectin levels (>/=3.12 microgram/mL) had shorter event-free survival (EFS) (median 7.3 v 12 months, P = .008) and overall survival (median 1 v 2.05 years, P = .03) than patients with sL-selectin <3.12 microgram/mL. Multivariate statistical analysis (adjusted for age and presence of Auer rods) indicated that sL-selectin was an independent prognostic factor for EFS (hazard ratio [HR], 1.96; 95% CI, 1.21 to 3.17, P = .006) and overall survival (HR, 1.80; 95% CI, 1.09 to 2.98; P = .02). Thus, plasma sL-selectin may be a useful prognostic marker in the evaluation of AML at diagnosis.