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In four patients (a 54-year-old man and three women aged 57, 60 and 65 years, respectively) with colorectal carcinoma and no obvious cardiac abnormality anginal symptoms and ECG changes occurred during chemotherapy with 5-fluorouracil at a dose of 400 and 600 mg/m2. The ECG changes consisted of descending ST depressions with preterminally negative T waves, terminally negative T waves, ventricular extrasystoles and sinus tachycardia with intermittent atrial fibrillation. The signs first appeared between the second and fourth day of treatment; in two patients they improved with glyceryl trinitrate. A few days after 5-fluorouracil had last been administered all ECG changes had disappeared. The causes of cardiotoxicity of the drug remain unknown.

Patients with Philadelphia-positive chronic-phase chronic myelogenous leukemia (CML) resistant to interferon (IFN) alpha were treated in a phase I/II study with recombinant human tumor necrosis factor alpha to overcome IFN alpha resistance. Doses of 40, 80, 120 or 160 micrograms/m2 TNF alpha were given as 2-h infusions on 5 consecutive days every 3 weeks. IFN alpha (4 x 10(6) IU/m2 s.c., daily) treatment was continued. Six patients were treated, completing 1-24 (median, 12) treatment cycles. Five of the six patients achieved partial hematological remission, while the remaining patient had to stop treatment because of WHO grade 4 thrombocytopenia following the first TNF alpha cycle. No complete hematologic remission or cytogenetic improvement was seen. Side-effects were similar to those described for both substances alone. Maximum tolerable TNF doses usually varied between 80 micrograms/m2 and 160 micrograms/m2. To examine possible pathways of TNF activity in these patients, interferon receptor status and (2'-5')-oligoadenylate synthetase levels were examined in peripheral blood mononuclear cells. Both parameters remained unchanged during TNF alpha treatment. These preliminary data point to significant clinical efficacy of additionally applied TNF alpha in IFN alpha-resistant CML patients.

Thirty-four patients with stage IIC (unresectable, retroperitoneal tumor mass (RTM) greater than 5 cm), stage IVC (minimal lung metastases less than 10 cm3 and RTM greater than 5 cm) and IVD (lung metastases greater than 10 cm3 and RTM greater than 5 cm), who had not received previous chemotherapy, were treated with cisplatin (40 mg/m2, on days 2-4), ifosfamide (5 g/m2, on days 1 and 5) and bleomycin (30 mg, on days 1, 8, 15) (PIB), every 21 days. Twenty of the 34 patients (59%) achieved a complete remission (CR). Furthermore, five patients (15%) showed no evidence of disease (NED) after surgical removal of residual tumor masses (NED rate of 74%). A tumor marker-negative partial remission (PR) occurred in 3/34 patients (9%), and a tumor marker-positive PR in another 3/34 patients (9%). Three patients did not respond to this regimen. At a median follow-up period of 38 months (range, 15-47 months), 26/34 patients (76%) were alive, 21 (62%) of them without evidence of disease and three with a stable tumor marker-negative remission. Major toxicity consisted of myelosuppression, neurotoxicity and nephrotoxicity. Chemotherapy-related mortality occurred in two patients (one septicemia and one bleomycin-induced lung fibrosis). In conclusion, PIB is an effective induction regimen in patients with high-risk NSTC. However, controlled clinical trials are necessary to prove the superiority of dose intensification schedules.

Fifty-four patients with Ph 1 -positive chronic myelogenous leukaemia (CML) (48 with chronic-phase and six acute-phase disease) were treated with interferon alfa-2b subcutaneously (s.c.). The starting dose was 4 million units (MU)/m 2 body surface area daily. It was reduced in parallel with serially determined leucocyte counts, and minimal effective doses were given as maintenance after achieving remission. Haematological remissions were induced in 22 of the 48 patients (46%) with chronic-phase disease. Thirteen patients (27%) revealed partial haematological remission and another 13 no response to treatment. No complete remission could be induced, although minor or partial cytogenetic responses were seen in 16 patients (33%). Moreover, a bcr-abl reduction was detected on Southern blot analysis in two patients. In chronic-phase disease, results of treatment were influenced by elapsed time after diagnosis, extent of previous treatment and interferon dosage. No beneficial effects of interferon were detected in the six patients with acute-phase disease. Principal acute side effects were fever and flu-like symptoms at the beginning of the therapy, which usually subsided within 3–7 days. Chronic side effects, especially weakness and neuropathy, were less frequent but more severe and necessitated discontinuation of treatment in 10 patients. In summary, interferon alfa-2b seems to be an effective treatment in early chronic-phase CML. Long-term effects on the course of the disease, however, must be determined.

Interferon (IFN) therapy has become widely used for the treatment of chronic myelogenous leukemia. Hematologic remissions can be induced in about 60% of patients. Moreover, in a small number of patients loss of the Philadelphia (Ph) chromosome and of the BCR-ABL rearrangement is observed. We have used genomic Southern blotting as well as a two-step polymerase chain reaction (PCR) method to score for BCR-ABL messenger RNA (mRNA) in patients with hematologic remission induced by treatment with IFN alpha-2b alone or in combination with IFN gamma. Concomitantly, cytogenetic analysis was performed. In 11 of 115 patients reported here, a complete loss of rearranged BCR fragments was observed in Southern blots of peripheral blood (PB) and/or bone marrow (BM) cell samples. Malignant marker bands disappeared first in the PB. In six patients, this genotype remained stable, whereas in five patients, low-intensity, rearranged bands reappeared despite continuation of treatment. The reappearance of the malignant marker was not accompanied by a clinical relapse. Ph-negative metaphases were observed in PB cells of four patients and in the PB and BM cells of two of these patients. In the samples of the other patients, residual Ph- positive cells were detected. By two-step PCR, residual BCR-ABL rearranged transcripts were found in samples of 10 patients.

A total of 51 fully evaluable patients with advanced and intensively pretreated breast cancer were treated with a combination chemotherapy of ifosfamide plus mesna, methotrexate and 5-fluorouracil. All patients had received at least one series of combined chemotherapy, 30 patients had received more than one combination and 41 patients had had anthracyclines before. Metastatic lesions in more than one site were found in 42 patients, and 24 patients had metastatic liver lesions. Partial remission was achieved in 10 patients (20%) and no change in 16 patients (31%). Survival was almost identical in both groups of responding patients and significantly shorter in treatment failures. Response was favorable in patients without pretreatment with anthracyclines. Two patients who received this protocol directly after progression with cyclophosphamide, methotrexate and 5-fluorouracil (CMF protocol) responded with a partial remission. Median time to progression was 7 months for partial responders and 4.5 months for patients achieving a no-change status. Median survival was 8 months for all patients. Toxicity was tolerable. Leukocytopenia and thrombocytopenia were treatment-limiting parameters. Overall, this protocol is well tolerable and effective in breast cancer patients with advanced disease and in intensively pretreated patients.

Cell cycle distribution in bone marrow and peripheral blood mononucleated cells was studied in patients with chronic myelogenous leukemia (CML) before and during treatment with interferon (IFN) alpha-2b. DNA-flow cytometry with ethidium bromide fluorescence was used. Highly significant differences between mononucleated cells from CML patients and normal controls were seen in peripheral blood but not in bone marrow specimens. Patients achieving hematologic remission during IFN treatment showed a cell cycle distribution in bone marrow cells and peripheral blood cells similar to normal controls.

In chronic myelogenous leukemia (CML) malignant cells are characterised by the Philadelphia chromosome (Ph), resulting from a translocation t(9;22). The position of the breakpoint within the major breakpoint cluster region (M-bcr) on chromosome 22 has been shown to correlate with the clinical course of the disease or, more recently, thrombopoietic activity. We have therefore determined the breakpoint localisation in 53 Ph-positive CML patients. Following the 5′/3′-region definition of Inokuchi et al. Leukemia Research 15 , 1067 (1991) [1], 22 of our patients have 5′ and 31 of our patients have 3′ orientated breaks. No correlation was found between platelet counts and breakpoint localisation.

Bone marrow (BM) and peripheral blood cell (PBC) samples of 11 Philadelphia (Ph) chromosome-positive chronic myelogenous leukemia (CML) patients in long-lasting hematologic remission induced by interferon (IFN) treatment were examined for the presence of leukemic hematopoietic precursor cells. Southern blot analysis revealed residual leukemic cells in BM samples of four patients, whereas seven patients showed no aberrant bands. Reverse transcription polymerase chain reaction (RT-PCR), however, amplified bcr-abl-specific cDNA in unfractionated BM or PBC samples in all 11 patients. The patients demonstrating bcr rearrangements in Southern blots had either a mosaic pattern (three patients) of bcr-abl-negative and positive colony-forming precursors (CFU-GEMM, BFU-E, CFU-GM, CFU-Mega), or all colonies were derived from leukemic precursors (one patient). However, in soft agar cultures of four patients without aberrant bands in Southern blots, only colonies without amplifiable bcr-abl transcripts were detectable. In another patient, few bcr-abl-positive colonies were found after 44 months of treatment, but not after 53 and 56 months of therapy. In these patients, therefore, residual disease detectable by PCR analysis of unfractionated cell samples does not appear to reside in the colony-forming cell compartment. The prognostic implications of these observations and the nature of the remaining bcr-abl-positive cells within unfractionated cell samples remain to be determined.

Summary. The objective of this study was to investigate the prognostic impact of the reduction of Philadelphia chromosome (Ph) positive metaphases by treatment of chronic myelogenous leukaemia (CML) with interferon (IFN) alpha. Therefore, we evaluated the outcome of patients with previously untreated chronic phase Ph-positive CML, enrolled from 1984 to 1990 into two consecutive IFN trials at our institution. Of a total of 71 patients, 62 (87%) were evaluable for cytogenetic response. No cytogenetic improvement was seen in 16 patients (23%), 28 patients (38%) had a decrease in Ph-positive bone marrow metaphases to levels ranging from 35% to 95%, and nine patients (13%) to levels between 5% and 34%. In nine patients (13%), Ph-positive metaphases were no longer detectable. After a median follow-up period of 33 months, the projected 5-year survival is 55% for the 62 patients evaluable for cytogenetic response. In this patient population there was no significant difference in the survival probability according to patients’risk status as defined by the Sokal score. Categorization according to the extent of Ph reduction, however, allowed three groups with significantly different prognoses to be identified. Patients achieving a Ph reduction to less than 35% were found to constitute a low risk group with a median survival not yet known and a projected 5-year survival of 90%. The 5-year survival rate was 55% for patients with a Ph reduction to levels between 35% and 95%, and less than 10% for those without any cytogenetic improvement. Thus, this study demonstrates that cytogenetic improvement on IFN treatment is an important prognostic factor for survival.

Symptoms of autoimmune disease were evaluated in 125 patients with chronic myelogenous leukemia (CML) and in 12 patients with essential thrombocythemia undergoing treatment with recombinant interferon (IFN)-α-2b plus/minus low-dose recombinant IFN-γ. Twenty-seven of 137 patients (20%) developed rheumatoid symptoms. Furthermore, the incidence of antinuclear antibody (ANA) formation was studied. Elevated ANA titers were found in 5 19 (26%) of CML patients at the time of diagnosis and in 3 18 (17%) of patients treated with hydroxyurea or busulfan. During IFN treatment, 18 of 25 tested patients (72%) had elevated ANA titers. In 15 of these ANA-positive patients, clinical signs of autoimmune disease appeared. All these patients were under long-term IFN treatment and were in remission of disease. In three patients criteria for systemic lupus erythematosus were fulfilled. Severity of side effects has led to the discontinuation of IFN treatment in these patients. The data indicate that IFN-α and IFN-γ can induce ANA associated with autoimmune disease in patients with myeloproliferative disorders.

Patients with chronic myelogenous leukemia (CML) have been treated with interferon (IFN) alpha-2b alone or in combination with IFN gamma. In order to predict clinical response to IFN, bone marrow samples from 15 CML patients were incubated with serial dilutions of IFN alpha-2b to obtain the IC50 values for erythroid burst forming units (BFU-E) and granulocyte-macrophage colony forming units (CFU-GM). A dose-dependent inhibition of at least one lineage was observed in all but one sample. An inhibitory effect of greater than 50% was reached for BFU-E in 8/14 patients and for CFU-GM in 10/14 patients. All three patients with no response (NR) to IFN treatment had IFN-sensitive BFU-E and CFU-GM. In four patients with hematologic remission (HR) or partial hematologic remission (PHR), BFU-E or CFU-GM were affected very little by the inhibitory effect of IFN. These observations suggest no predictive value for pretesting IFN sensitivity in vitro. The in vivo effect of IFN on the hemopoietic progenitor cells BFU-E and CFU-GM was evaluated in patients treated with either IFN alpha-2b alone (n = 11), or in combination with low dose IFN gamma (n = 10). All patients were newly diagnosed and not pretreated. After a median treatment duration of 11 months (range 3-25) a significant decrease in BFU-E and CFU-GM was observed in both groups of patients. We conclude that in vitro colony growth reflects the therapeutic efficacy of IFN.

Between March 1988 and July 1990, 28 adults with chronic myelogenous leukaemia (CML) were treated with a combination of recombinant human interferon (IFN) alpha-2b s.c. (initial dose 4 x 10(6) U/m2) and recombinant human IFN gamma s.c. (50 micrograms totally) daily. All patients were in chronic phase disease and had been treated previously with chemotherapy or bone marrow transplantation. A complete haematologic remission was achieved in three patients (11%), a haematologic remission in 12 patients (43%), and a partial haematologic remission in seven patients (25%). Six patients did not respond to this schedule. Acute side-effects were flu-like symptoms, fever and chills. During long-term treatment six patients developed polyarthralgia. Haematotoxicity WHO grade III occurred in three patients, and WHO grade IV in two patients. One patient developed psychosis, and in another patient an exacerbation of a pre-existing sarcoidosis was observed. We conclude that this combination is tolerable and effective in inducing haematological remissions in pretreated CML patients.

In patients with previously untreated chronic myelogenous leukaemia (CML) the efficacy of single-agent interferon (IFN)-alpha at an initial dose of 4 x 10(6) U/m2 (arm A) was compared with the combined administration of the identical dose IFN-alpha plus a total dose of 50 micrograms IFN-gamma (arm B). 51 patients entered this study between April 1987 and October 1989; the analysis was performed in March 1991 and was focused on response rates and toxicity. 54% of patients on arm A and 56% of arm B patients attained haematologic remission. 29% of patients on arm A and 24% of arm B patients had partial haematologic remission. A decrease in Philadelphia chromosome (Ph)-positive metaphases of more than 10% was only seen in patients who had achieved complete haematologic normalization. In 21% of patients on arm A and 20% of arm B patients, the percentage of Ph-positive cells declined to less than 35%. Toxicity was different between the two study groups with more pronounced hepatotoxicity observed in patients treated with IFN-alpha alone. Among the patients receiving both IFNs, alpha and gamma, there were 2 fatal infectious complications. This serious toxicity in conjunction with lack of a clinically meaningful difference between the two treatment schedules has led us to terminate the study. In conclusion, the addition of low-dose IFN-gamma failed to improve the efficacy of IFN-alpha in this study.

A 23-year-old male patient with Philadelphia chromosome (Ph) positive chronic myelogenous leukemia (CML) was treated with both IFN alpha and IFN gamma. Normalization of leukocyte counts was reached after 3 months of treatment. Southern blot analysis failed to detect the neoplastic cell clone after 19 months of therapy. Cytogenetically, complete suppression of Ph positive cells in the patient's bone marrow and blood was observed after 20 months and 25 months, respectively. This response was achieved with doses of IFN alpha and IFN gamma which were considerably lower than the dosage of IFN used in single agent therapy of CML.

The BCR gene on chromosome 22 has received increasing attention because of its involvement in the Philadelphia (Ph′) translocation. For most restriction enzymes, this locus has been found to be nonpolymorphic. Two alleles have only been found when Taql-digested DNA is hybridized to a 5′ bcr-specific probe. We describe another two-allele polymorphism detected by the same probe in PvuII-digested DNA. The polymorphism is characterized by an additional PvuII site in the bcr region: this causes the appearance of an additional band of about 2.3 kb or 2.5 kb besides a 4.8-kb fragment in hybridizations with the 5′ bcr or a 3′ bcr probe. The incidence of the second allele is very low. It has only been found in some patients with hematopoietic malignancies and in a group of volunteers having a leukemia patient in their families.

A combination of vindesine (3 mg/m2, day 1) and ifosfamide (60 mg/kg, days 1-5 + Mesna) was administered every three weeks to 11 patients with primary resistant and 23 with recurrent small-cell bronchial carcinoma. All patients had been pre-treated with chemotherapy, 16 in addition with radiotherapy. At the onset of the vindesine-ifosfamide treatment the cancer was in a localized regional stage in ten patients, while in 24 it was in a more widely spread stage. In 29 patients whose treatment results could be evaluated the remission rate was 38%, with two complete and nine partial remissions. In a further eight patients the cancer was arrested. The patients with complete remission (for 46 and 53 weeks, respectively), those with partial remission (median of 39 weeks) and those with stationary disease (median of 31 weeks) survived significantly longer than those with progressing disease (13 weeks). There was no correlation between treatment result and pre-treatment. On recurrence after complete remission or in the localized regional stage the remission rate was 70% and 60%, respectively, and the survival time was extended in 90% of cases. In addition to nausea, alopecia and myelosuppression, side-effects included vomiting, reversible CNS symptoms, polyneuropathy and urotoxicity. On the basis of acceptable toxicity, combined vindesine and ifosfamide constitute an effective treatment of otherwise treatment-refractory cases of small-cell bronchial carcinoma.

Cells from a resistant ("Gr II") and a sensitive ("Str") human melanoma xenograft were incubated in vitro with O6-methylguanine for 2 h, subsequently treated with 1-(2-chloroethyl)-1-nitroso-3(4-methyl)-cyclohexylurea (MeCCNU) for 1 h and then plated in soft agar. In the resistant cells the O6-methylguanine pretreatment (2 mM) yielded an increase in sensitivity towards MeCCNU by a factor of 7.5. In the sensitive melanoma cells pretreatment with O6-methylguanine did not increase cytotoxicity. Human bone marrow cells from three normal donors were similarly pretreated with O6-methylguanine and MeCCNU. There was a clear increase in MeCCNU-induced cytotoxicity. We conclude, that while O6-methylguanine does potentiate the action of MeCCNU in resistant melanoma cells, the therapeutic usefulness of this treatment strategy may be limited.

The constitution and maintenance of normal hemopoiesis depends on the continuous proliferation and differentiation of hemopoietic stem cells and progenitor cells. The best-studied of these precursor cells are the stem cell-like progenitors (CFU-GEMM), the early erythroid progenitors (BFU-E), the granulocytic progenitors (CFU-C), and the megakaryocytic progenitors (CFU-M) [2, 4]. The levels of these colony forming cells reflect the functional capacity of hemopoiesis.

Chronic myelogenous leukemia (CML) is a stem cell disorder characterized by the accumulation of immature precursors of the granulocytic and monocytic lineage. Most commonly, the disease is treated with chemotherapeutic agents, preferably busulfan and hydroxyurea. High-dose chemotherapy followed by allogeneic bone marrow transplantation may eradicate the malignant clone and offers the possibility of cure [1, 2].

Chronic myelogenous leukemia (CML) is characterized by a distinct genetic aberration, the Philadelphia chromosome (Ph1). It is a shortened chromosome 22 as result of a translocation t(9:22)(q 34; q 11). By this translocation the cellular proto-oncogene c-abl is transferred from chromosome 9 to chromosome 22 and inserted into the so-called breakpoint cluster region (bcr). This region is part of a large (~ 100 kb) bcr gene, the function of which is yet unknown. — A rough map of the bcr region is shown in Fig. 1.

Autologous transfusion of 2.8-39.5 X 10(9) cryopreserved mononuclear blood leukocytes (BMNC) into 1200 R wholebody irradiated dogs leads within 10 days of irradiation and transfusion to patterns of hemopoietic regeneration of bone marrow, spleen and blood the extent of which correlates with the number of granulocytic progenitor cells, measured as CFU-C, in the tranfusate. In the marrow there are erythrocytic, granulocytic and megakaryocytic foci of regeneration ("colonies") in a proportion of 10:6:1 respectively, while the spleen exhibits cell dose related regeneration of the white pulp and a (transitory) erythropoietic wave of regeneration localized in the red pulp only. The mechanisms of the development of this extramedullary hemopoiesis are unknown. However, it closely resembles the prenatal phase of splenic erythropoiesis which disappears soon after birth. The normoblasts present in the blood at day 10 after irradiation and transfusion of BMNC correlate with extramedullary blood cell formation buy not with the type and extent of bone marrow regeneration.

As conventional FUra monochemotherapy with remission rates of about 20% remained disappointing, the addition of CF to FUrd heralded an important step towards an improved therapy of advanced colorectal cancer (1). In a broad phase II study we evaluated response, toxicity and survival of patients receiving combination treatment of FUra and CF, primarily in colorectal cancer. In addition we treated pts with prostatic cancer, breast cancer, gastric cancer and various other poor risk malignancies with this combination. We used two different schedules of FUrd and CF. Our data indicate improved response in colorectal cancer as compared to FUra alone and are promising in breast cancer and adenocarcinoma of unknown primary.

The interferons (IFNs) are a group of proteins which can exert a wide range of biologic effects on normal tissue cells, host immune defense cells, and cancer cells [5, 9,12, 20]. With regard to their immunochemical properties and their cellular origin, three types of IFNs have been distinguished: α, β, and γ [7].

Summary1The efficacy of ifosfamide (60 mg/kg B.W., days 1–5) and vindesine (3 mg/m2 B.S.A., days 1 + (5)) as second-line chemotherapy was investigated in 26 male patients with small cell lung cancer (SCLC) resistant (9 patients) to first-line therapy or relapsing (17 patients) after transient remission. The median age of the patients was 53 years (range 33–74 years), median performance status 70% (range 40–100). Pretreatment consisted of either adriamycin (epirubicin)/cyclophosphamide/vincristine and etoposide/cis-platinum or CCNU/etoposide/methotrexate ± thoracic irradiation. To date, 23 patients (7 patients with limited disease (LD), 16 with extensive diesease (ED)) are evaluable for response.Two patients (one with tumor relapse in an area of prior irradiation, one with hepatic metastases) achieved a second complete remission (CR) lasting 21 and 33 weeks, respectively. The survival time from the start of ifosfamide/vindesine treatment in these patients was 46 and 51+ weeks, respectively. Partial remission (PR) were induced in 7 patients (4 LD, 3 ED). Median remission duration in PR patients was 19 weeks (range 13–33), median survival 30 weeks (range 13–52). Seven patients with disease stabilization had a median survival time of 18 weeks, range 8–43. Seven patients (ED) did not respond and survived 4–22 weeks, with a median of 11 weeks. The response rate (CR 4- PR) of relapsing SCLC was 47% as compared to 22% in primarily resistant tumors. Interestingly, 4 patients with relapse in an area of prior irradiation achieved CR or PR. Due to side-effects such as urotoxicity, polyneuropathy, myelosuppression and central nervous side-effects, dosis had to be reduced in 2 patients, and treatment was stopped in 2 patients.