Cologne high-dose sequential chemotherapy in relapsed and refractory Hodgkin lymphoma: results of a large multicenter study of the German Hodgkin Lymphoma Study Group (GHSG).

BACKGROUND: We designed a dose- and time-intensified high-dose sequential chemotherapy regimen for patients with relapsed and refractory Hodgkin lymphoma (HD). PATIENTS AND METHODS: Eligibility criteria included age 18-65 years, histologically proven primary progressive (PD) or relapsed HD. Treatment consisted of two cycles DHAP (dexamethasone, high-dose cytarabine, cisplatinum); patients with chemosensitive disease received cyclophosphamide followed by peripheral blood stem cell harvest; methotrexate plus vincristine, etoposide and BEAM plus peripheral blood stem cell transplantation (PBSCT). RESULTS: A total of 102 patients (median age 34 years, range 18-64) were enrolled. The response rate was 80% (72% complete response, 8% partial response). With a median follow-up of 30 months (range 3-61 months), freedom from second failure (FF2F) and overall survival (OS) were 59% and 78% for all patients, respectively. FF2F and OS for patients with early relapse were 62% and 81%, for late relapse 65% and 81%; for PD 41% and 48%, and for multiple relapse 39% and 48%, respectively. In multivariate analysis response after DHAP (P <0.0001) and duration of first remission (PD and multiple relapse versus early and late relapse; P=0.0127) were prognostic factors for FF2F. Response after DHAP (P <0.0081), duration of first remission (P=0.0017) and anemia (P=0.019) were significant for OS. CONCLUSION: Based on the promising results of this study, a prospective randomized European intergroup study was started comparing this intensified regimen with two courses of DHAP followed by BEAM (HD-R2 protocol).

Time-intensified dexamethasone/cisplatin/cytarabine: an effective salvage therapy with low toxicity in patients with relapsed and refractory Hodgkin's disease.

BACKGROUND: An important variable affecting outcome in relapsed and refractory Hodgkin's disease (HD) is the potential of conventional salvage chemotherapy to reduce tumor volume before high-dose chemotherapy (HDCT) and autologous stem cell transplantation. Currently, the optimal salvage chemotherapy regimen for these patients is unclear. Since dexamethasone/cisplatin/cytarabine (DHAP) given at 3-4 week intervals has been shown to be very effective in patients with relapsed aggressive non-Hodgkin's lymphoma, we evaluated this regimen given at a median of 16-day intervals in patients with relapsed and refractory HD. PATIENTS AND METHODS: Patients with relapsed or refractory HD were treated with two cycles of DHAP [dexamethasone 40 mg intravenously (i.v.) day 1-4, cisplatin 100 mg/m(2) i.v. as 24-h continuous infusion day 1, and cytarabine 2 g/m(2) i.v. 12q day 2]. Granulocyte colony-stimulating factor (G-CSF) was given at a dose of 5 micro g/kg from day 4 until day 13. Patients with partial remission (PR) or complete remission (CR) after two cycles of DHAP received sequential HDCT. RESULTS: The median age of the 102 patients included was 34 years (range 21-64 years). Forty-two percent of the patients had late relapse, 29% early relapse, 12% multiple relapse and 16% primary progressive/refractory disease. The response rate (RR) after two cycles of DHAP was 89% (21% CR, 68% PR). The RRs for patients with late, early, multiple and progressive HD were 91%, 93%, 92% and 65%, respectively. Using the chi-square test for independence, remission status (relapsed HD versus progressive HD) and stage at relapse (stage I/II versus stage III/IV) were significant factors for response to DHAP. WHO grade 4 leukocytopenia and thrombocytopenia were the main toxic- ities occurring in 43% (mean duration 1.1 days, range 0-6) and 48% (mean duration 1.4 days, range 0-11) of all courses, respectively. Neither severe infections nor treatment-related deaths occurred. Peripheral blood stem cells (PBSCs) were collected after the first cycle DHAP in eight patients. The hematopoietic progenitors showed a very rapid increase from day 10 with a synchronous and impressive peak on day 12. A mean of 6.1 x 10(6)/kg CD34(+) cells were collected per apheresis. As originally recommended in the protocol, PBSCs were routinely collected during sequential HDCT in the remaining patients. CONCLUSIONS: A brief tumor-reducing program with two cycles of DHAP given in short intervals supported by G-CSF is effective and well-tolerated in patients with relapsed and refractory HD. This regimen can be used to mobilize stem cells and select those patients with chemosensitive relapse who should subsequently be treated with HDCT.

[Chemo-/immunotherapy in advanced malignant melanoma: carboplatin and DTIC or cisplatin, dtic, bcnu and tamoxifen followed by immunotherapy with interleukin 2 and interferon alpha-2a]. / Chemo-/Immuntherapie beim fortgeschrittenen malignen Melanom: Carboplatin und DTIC oder Cisplatin, DTIC, BCNU und Tamoxifen und anschliessende Immuntherapie mit Interleukin-2 und Interferon-alpha2a.

BACKGROUND: Polychemotherapy and immunomodulating treatment using IL-2 and/or IFN-alpha produce objective responses in a proportion of advanced malignant melanoma patients. PATIENTS AND METHODS: In 2 consecutive phase II trials in a total of 85 patients, we assessed the potential synergism between both modalities i.e., chemo- and immunotherapy. Treatment consisted of intravenous carboplatin (CBDCA, 400 mg/m2) and dacarbazine (DTIC, 750 mg/m2) given twice at a 3-week interval, or 4 cycles of DTIC (220 mg/m2 i.v. x 3 days), cisplatin (DDP 35 mg/m2 i.v. x 3 days), carmustine (BCNU 150 mg/m2 i.v., cycles 1 and 3) and tamoxifen (TAM 20 mg/per os x 5 days) at a 3-week interval. Chemotherapy was followed by immunotherapy with combined subcutaneous interleukin-2 and (rIL-2) and s.c. interferon-alpha 2a (rIFN-alpha). RESULTS: Among 40 patients who received a full cycle of chemotherapy with CBDCA/DTIC and sequential immunotherapy, there were 3 (7.5%) complete remissions (CR) with durations of 13 to 26+ months. Partial remissions (PR) were noted in 11 (27.5%) patients with a median response duration of 8 (range 5 to 14) months. Among 45 patients who received DTIC/DDC/DDP/BCNU and TAM and sequential rIL-2/rIFN-alpha 2a there were 5 (11%) complete remissions and 17 (38%) partial remissions. Duration of complete and partial remissions ranged from 8+ to 24+ months (median 12+) and 5+ to 17 months (median 8+), respectively. Chemotherapy produced mostly moderate toxicity. Thrombocytopenia was common with the nadir after a median time of 17 days following start of the chemotherapy. 19 patients required transfusion of thrombocytes. Nausea and vomiting due to chemotherapy were well tolerated using concomitant ondansetrone (8 mg i.v.). Immunotherapy was self-administered at home with mild to moderate side-effects; malaise, fever, chills, nausea/vomiting, diarrhea, anorexia and arthralgias were most frequent (70% to 100%), but spontaneously reversible after ending the immunotherapy. A mean of 87% (trial I) to 89% (trial II) of the projected doses of rIL-2 and rIFN-alpha were administered on either protocol. There were no life-threatening complications and no treatment-related deaths. CONCLUSION: The sequential combination of chemotherapy and immunotherapy had at least additive therapeutic activity against metastatic malignant melanoma. Both schedules produced long-lasting remissions and were overall well tolerated. These trials substantiate a potential role for low to intermediate dose immunotherapy in maintaining and consolidating therapeutic effects of chemotherapy in metastatic melanoma.

Adjuvant treatment of locally advanced renal cancer with autologous virus-modified tumor vaccines.

We report on 208 patients with locally advanced renal-cell carcinoma who received a surgical adjuvant vaccination with autologous, Newcastle disease virus (NDV)-modified, and lethally irradiated tumor cells in combination with low-dose recombinant interleukin-2 and interferon-alpha. The pathological stage was defined as pT2-3a, N1-2, MO (n = 107); pT3b-4 NO, MO (n = 68); and pT3b-4, N1-2, MO (n = 23). The follow-up of 203 evaluable patients showed a median disease-free survival of 21+ months (range, 2-64+ months). In all, 18 relapses (9%) occurred in spite of initial vaccination therapy. Those patients presented with local relapse (n = 3), lymph node metastases (n = 10), and/or distant organ metastases (n = 9). All patients relapsing during the first 6 months after the onset of treatment had primary lymph node involvement of the disease. An analysis of the patient subgroup with a follow-up of more than 22 months showed 10 relapses among 56 patients (18%) along with a median follow-up of 39 months (range, 23-64 months). Toxicity was very mild, manifesting as flu-like symptoms and fevers of up to 38 degrees C. At 8 and 24 weeks after the start of vaccination, anti-NDV serum antibodies were detectable in 70% and 100% of the patients tested, respectively. In comparison with historical data based on the natural course of patients with locally advanced renal-cell cancer, our results demonstrate an improvement of the disease-free survival after surgical adjuvant treatment with autologous, NDV-modified tumor vaccines in combination with low-dose cytokines.

Monoclonal glucose-oxidase-anti-glucose-oxidase (GAG) immunosandwich assay for the detection of monoclonal antibodies on routine hematological smears.

A murine monoclonal antibody specific for aspergillus niger glucose oxidase has been prepared and used in an unlabeled antibody bridge technique for the detection of monoclonal antibodies. This procedure--the monoclonal glucose oxidase anti-glucose oxidase (GAG) immunosandwich assay--provides excellent immunocytochemical labeling of routine hematological films in combination with optimal preservation of cellular details. In contrast to conventional immunofluorescence procedures, routine hematological films can be used, and these can be stored before and after the immunolabeling. Compared with other immunoenzyme techniques such as those using alkaline phosphatase or peroxidase, the GAG assay is as sensitive and has the advantage that no problems with endogenous enzyme activity are encountered. The availability of alcohol-resistant disclosing reagents allows for routine hematological counterstaining which provides a very clear visualization of both the immunoreaction and the individual morphology of the blood cells.

Intracranial transplantation of human hematopoietic cell lines in nude mice: a preclinical screening model for cytostatic drugs.

This study examines the suitability of the intracranial route of transplantation of human cell lines in nude mice for preclinical testing of cytostatic drugs. Tumours were generated by inoculation of human hematopoietic cell lines. Animals in which intramuscularly or intracranially transplanted human tumours developed were treated with cytostatic drugs. Three different cell line generated tumours (T-ALL L735, Hodgkin cell line L540 and Burkitt Lymphoma Line BJAB) were treated with four different drugs (CY, ADM, VCR, VP16). The data show a good correspondence between the results of treatment of intramuscularly and intracranially transplanted tumours. Thus intracranially transplanted tumour in nude mice are suitable for the testing of cytostatic drugs.

Cytogenetic investigations in Hodgkin's disease: I. Involvement of specific chromosomes in marker formation.

Cytogenetic studies were performed in five Hodgkin-derived permanently growing cell lines, as well as in bone marrow cells from two Hodgkin patients, one of which suffering from acute myeloid leukemia after Hodgkin's disease. No consistent and common marker chromosome was found, but certain chromosome regions seemed to be nonrandomly involved in marker formation. These are chromosome bands 7q22, 7q32, 7q36, 2q33, 1p22, 11q21/23, 14q32, 15p12, and 21q21. The importance and significance of these chromosomal findings are discussed in reference to data from the literature.

Experimental chemotherapy of heterotransplanted Hodgkin- and non-Hodgkin-lymphoma cell lines in nude mice.

Four hematopoietic in vitro cell lines (Hodgkin derived cell lines L428 and L540, Burkitt's lymphoma line BJAB and the lymphoblastic lymphoma line of T-cell type L735) were transplanted into nude mice (NMRI nu/nu) intramuscularly and intracerebrally as well. Tumor bearing mice were treated with intraperitoneal administration of Cyclophosphamide, Adriamycin, Etoposid and Vindesine. Therapy results in both systems were proven by Student's t-test and significances were compared. Resistance and sensitivity of cell lines tested in the i.c.-system were largely in accordance with the i.m.-system. Best results of treatment were achieved with the T-lymphoma line L735 treated with Cyclophosphamide and Etoposid. Cyclophosphamide induced complete remission of i.m. tumors in several cases whereas in the nude mouse brain the L735 cell relapsed and showed a more aggressive growth and diminished drug sensitivity.

[Properties of Hodgkin cell lines. Possible significance for pathophysiology and clinical medicine]. / Eigenschaften von Hodgkin-Zellinien. Mögliche Bedeutung für Pathophysiologie und Klinik.

In the last three years, five permanently in-vitro growing cell cultures with malignant properties were established from tumour material of patients with histologically confirmed Hodgkin's disease. Four cell lines have been maintained in culture. L 428 had identical characteristics in every respect with Hodgkin and Sternberg-Reed cells, tested in-vivo on biopsy tissue. The other lines--L 538, L 540 and L 591 - had certain characteristics of Hodgkin and Sternberg-Reed cells with a number of markers, but were not fully congruent. All lines reacted with a heterologous antiserum against L 428, which selectively cross-reacted with Hodgkin and Sternberg-Reed cells in fresh biopsies. Two sublines, L 428 KS and L 428 KSA, were established from L 428 by modifying the culture medium. Tests on L 428 KS cells with conventional methods and with monoclonal antibodies demonstrated that this line carried antigens of myeloid cells; however, it could not be definitely placed into any haematopoetic line. Conditioned medium of L 428 and its sublines showed CSF activity (colony-stimulating factor) and suppression of cell-mediated cytolysis.

Hodgkin's cell lines: characteristics and possible pathogenetic implications.

In the last four years we established five long term cultures from tumor material of Hodgkin's disease. The in vitro cells have malignant characteristics and represent the in vivo H- and SR-cells. Common immunological, functional and morphological assays did not characterize the in vitro cells to be a known cell type of lymphoid, myeloid or monocytoid tissue. The in vitro Hodgkin cells are biologically active by producing factors involved in regulation and promotion of immunological response and granulopoiesis. The relevance of the findings for pathogenesis and clinical appearance of Hodgkin's disease is discussed.

Hodgkin's disease cell lines: characteristics and biological activities.

In the last 4 years we have established five long-term cultures from tumor material of Hodgkin's disease. The in vitro cells have malignant characteristics and represent the in vivo Hodgkin- and Sternberg-Reed-cells as shown by the identity of multiple properties. Common immunological, functional, and morphological assays did not characterize the in vitro cells as a known cell type of lymphoid, myeloid, or monocytoid tissue. The in vitro Hodgkin's disease cells are biologically active by producing factors involved in regulation and promotion of immunological response and granulopoiesis. The relevance of the findings for pathogenesis and clinical appearance of Hodgkin's disease is discussed.

Translocation t(8;22) in peripheral lymphocytes and established lymphoid cell lines from a patient with Hodgkin's disease followed by acute lymphatic leukemia.

Cytogenetic studies on primary material and established cell lines of a patient with B-cell non-Hodgkin Lymphoma with leukemic presentation as secondary malignancy after Hodgkin's disease clearly revealed correspondent marker chromosomes in both culture systems. A translocation t(8;22) which has been described in some cases of non-endemic Burkitt's lymphoma as a variant of the translocation t(8;14) was found in all metaphases of the cell lines and in most of the cells of the primary material. In addition, structural aberrations involving chromosomes 1, 2, 3 and 11, as well as numerical deviations concerning chromosomes 2, 4, 7 and Y, could be observed. The cytogenetical findings are discussed in relation to clinical, immunological and cytochemical data.

Characteristics of Hodgkin's disease-derived cell lines.

In the last 3 years we were able to establish five long-term in vitro cell cultures from biopsy specimens taken preterminally from four patients with histologically proven Hodgkin's disease (nodular sclerosing type, clinical stage IVB). Four of the lines are continuously proliferating in vitro; one culture stopped growth for unknown reasons after 7 months. When culture conditions were modulated, the first culture, L 428, gave rise to two sublines: L 428 KS, after adaptation to calf serum, and L 428 KSA, permanently growing as an adherent monolayer line after treatment with a phorbol ester (12-O-tetradecanoylphorbol-13-acetate) for 3 weeks. Cell-marker analysis by conventional means (SIg, cIg, rosette formation, Epstein-barr virus reactivity, cytochemistry, phagocytosis, and lysozyme production) and with monoclonal antibodies directed against various human lymphoid, myeloid, and monocytoid antigens showed that the tested cell lines are clearly different from all hitherto described hematopoietic lines; they most likely represent a cell type resembling an early myeloid-monocytoid progenitor cell. Conditioned medium of the L 428 cells and its two sublines showed colony-stimulating factor activity and suppression of spontaneous cell-mediated cytolysis of L 428 KS and K 562 cells.

Sister chromatid exchange in lymphoma line and lymphoblastoid cell lines before and after heterotransplantation into nude mice.

The sister chromatid exchange (SCE) rates of cell lines derived from patients with different malignant and premalignant disease were studied before and after intracerebral inoculation into nude mice and were compared to the SCE frequencies of cell lines from healthy controls. Lymphoma lines characterized by chromosomal markers as well as chromosomally normal lymphoblastoid cell lines were used. The SCE frequencies of nude mouse tumor-derived cell lines did not show a homogeneous pattern. They were either as high as, or higher or lower than, the SCE levels of the original cell line. But all cell lines, karyotypically abnormal as well as normal, originating from patients with malignant and premalignant diseases, showed significantly higher SCE rates than the control lines from healthy donors.

Correlation of chromosomal aberrations in a myeloma cell line with tumorigenicity in nude mice.

A human myeloma cell line (L 363) exhibiting several markers of malignancy including a 14q + chromosome marker was not tumorigenic in nude mice. Long-term cultivation in vitro resulted in new subclones with slight, well-defined chromosomal differences to the original line. Certain subclones turned out to have a growth advantage in vitro and became tumorigenic after transplantation in nude mice. A strong correlation between changes in the chromosome I constellation resulting in the threefold dose of long arm-material and the increased proliferative potential in both experimental systems was found.

Hodgkin's disease: establishment and characterization of four in vitro cell lies.

Four in vitro cell lines (L 428, L 439, L 538, and L 540) were established from different materials of three patients with Hodgkin's disease: pleural effusions, peripheral blood, and bone marrow. The histological diagnosis was confirmed in all cases by several independent histologists. All four cell lines have been in culture for over 6 months up to over 3 years. The neoplastic nature of the culture cells is indicated by the demonstration of several structural and numeric chromosome abnormalities associated with a monoclonal pattern of marker chromosomes. EBV-specific antigens (EBNA, VCA) were not detected in either cell line. Ia-like antigens, receptors for human T cells, acid phosphatase, and acid esterase were showen to be present in the cultured cells. All cell lines lacked surface or cytoplasmic Ig, HTLA, receptors for C3b, C3d, IgG-Fc, mouse E or sheep E, and were devoid of lysozyme, peroxidase, and chloracetate esterase. The described features do not represent B cells, T cells, myeloid cells, monocytes, or macrophages. The morphology and the marker pattern of the culture cells, however, is identical with that of freshly obtained Hodgkin's (H)- and Sternberg-Reed (SR)-cells, except for the lack of CIg in the in vitro cells, which is explained by the culture conditions. Heterotransplantation in nude mice was achieved by intracranial inoculation and by s.c. transplantation of cultured cells embedded in a plasma clot. The described findings suggest that these cultured Hodgkin's cell lines are indeed derived from H and SR cells. The cellular origin of these cells is not clear, the loss of cellular differential markers during the process of possible dedifferentiation is discussed.

Lymphoproliferation and heterotransplantation in nude mice: tumor cells in Hodgkin's disease.

In the last 3 years we were able to establish four long-term cultures from Hodgkin-derived material [pleural effusions (2), bone marrow (1), and peripheral blood (1)], consisting of cells which represent morphologic and cytochemical as well as cytogenetic features of their in vivo ancestors. Two of these cell lines are described in this paper. These two lines share the same features: non-B-T lymphocytes, non-macrophages, non myeloid cells, EBV genome negative, monoclonality, multiple numerical and structural chromosome aberrations, and tumor formation upon intracranial xenotransplantation in nude mice. The two remaining lines are being characterized at the moment. The common characteristics expressed synonymously in the two described lines suggest that the Hodgkin tumor cell does not seem to share the features of marker-carrying lymphocytes, macrophages, or myeloblasts. The cellular origin of these cells is not clear. The loss of cellular differential markers during the process of possible dedifferentiation is discussed.

Two neoplastic cell lines with unique features derived from Hodgkin's disease.

Two in vitro cell lines (L428, L439) were established from pleural effusions of two patients with Hodgkin's disease. The histological diagnosis was ascertained in both cases by two independent pathologists. The neoplastic nature of the culture cells is indicated by the demonstration of several structural and numerical chromosome abnormalities associated with a monoclonal pattern of marker chromosomes. Heterotransplantation in nude mice was achieved by intracranial inoculation and by subcutaneous transplantation of cultured cells embedded in a plasma clot. EBV-specific antigens (EBNA, VCA) were not detectable in either cell line. Ia-like antigens, receptors for T cells, acid phosphatase and acid esterase were shown to be present in the cultured cells. The L428 and L439 cell line lacked surface- or cytoplasmic Ig, HTLA, receptors for C3b, C3d, IgG-Fc, mouse E or sheep E, and were devoid of lysozyme, peroxidase and chloracetate esterase. These features do not correspond to those of B cells, T cells, myeloid cells, monocytes or macrophages; the morphology and the marker pattern of the culture cells, however, is identical with that of freshly obtained Hodgkin (H)- and Sternberg-Reed (SR)- cells, except for the lack of Clg in the in vitro cells, which is explained by the culture conditions. These findings suggest that the L428 and L439 cell lines are indeed derived from H- and SR-cells and offer the possibility of gaining new information upon the nature of Hodgkin's disease.