A pilot trial of G3139, a bcl-2 antisense oligonucleotide, and paclitaxel in patients with chemorefractory small-cell lung cancer.

BACKGROUND: Chemorefractory small-cell lung cancer (SCLC) is defined as disease that progresses during primary therapy or within 3 months of completion of primary therapy. Patients with chemorefractory SCLC have a very poor prognosis, and no treatment has been shown to be of significant clinical benefit. Elevated expression of Bcl-2 is found in the majority of SCLCs and has been associated with therapeutic resistance. Suppression of Bcl-2 levels through the use of G3139, an antisense oligonucleotide complementary to the mRNA encoding Bcl-2, might increase the antitumor efficacy of cytotoxic therapy. PATIENTS AND METHODS: Twelve patients with chemorefractory SCLC participated in this pilot trial of paclitaxel combined with G3139. G3139 was given by continuous i.v. infusion over 7 days at a fixed dose of 3 mg/kg/day. Paclitaxel dose was initially 175 mg/m2 on day 6, but was decreased to 150 mg/m2 due to myelosuppression observed in two of the three patients treated in the first dose cohort. RESULTS: The combination of paclitaxel at 150 mg/m2 and G3139 at 3 mg/kg/day was found to be feasible and well tolerated. No objective responses were observed, but two patients had stable disease, one remaining stable on therapy for >30 weeks. Plasma G3139 levels were determined, and were found to be highest in the patient with prolonged stable disease, suggesting that individual variation in metabolism and clearance of the antisense oligonucleotide may influence activity. CONCLUSIONS: This study demonstrates that G3139 can be combined with paclitaxel in a cytotoxic dose range, and suggests that a similar combination be tested for activity in the context of chemoresponsive disease.

DNA structural properties of AF9 are similar to MLL and could act as recombination hot spots resulting in MLL/AF9 translocations and leukemogenesis.

The human AF9 gene at 9p22 is one of the most common fusion partner genes with the MLL gene at 11q23, resulting in the t(9;11)(p22;q23). The MLL-AF9 fusion gene is associated with de novo acute myelo-genous leukemia (AML), rarely with acute lymphocytic leukemia (ALL) and with therapy related leukemia (t-AML). The AF9 gene is >100 kb and two patient breakpoint cluster regions (BCRs) have been identified; BCR1 is within intron 4, previously called site A, whereas BCR2 or site B spans introns 7 and 8. Patient breakpoint locations were determined previously by RT-PCR and by genomic DNA cloning. In this study, we defined the exon-intron boundaries and identified several different structural elements in AF9 including a co-localizing in vivo DNA topo II cleavage site and an in vitro DNase I hypersensitive (DNase 1 HS) site in intron 7 in BCR2. Reversibility experiments demonstrated a religation of the topo II cleavage sites. The location of the in vivo topo II cleavage site was confirmed in vitro using a topo II cleavage assay. In addition, two scaffold associated regions (SARs) are located centromeric to the topo II and DNase I HS cleavage sites and border both patient breakpoint regions: SAR1 is located in intron 4, whereas SAR2 encompasses parts of exons 5-7. This study demonstrates that the patient breakpoint regions of AF9 share the same structural elements as the MLL BCR. We describe a DNA breakage and repair model for non-homologous recombination between MLL and its partner genes, particularly AF9.

[Radiotherapy treatment of lymphoma]. / Radioterapija u lijecenju limfoma.

Radiotherapy and/or chemotherapy are primary treatment modalities in the therapy of lymphoma. The treatment depends on the lymphoma type, stage of disease and patients general condition. Radiation therapy is applied with curative or palliative intent, either as a single or combined modality treatment. In patients with stage I and stage IIA Hodgkin's lymphoma (HL) and no adverse risk factors, radiotherapy is applied as a single modality treatment. Moreover, treatment modalities in early-stage HL (I and IIA) consisting of either chemotherapy alone or combined with radiotherapy are the subject of ongoing clinical trials. In addition to the region/s with clinically involved lymph nodes, the target volume of radiation therapy applied as a primary radical treatment modality (stages I and IIA) also includes non-involved lymph nodes of adjacent regions aimed at their prophylactic irradiation. Such extended radiation fields are, for instance, the "mantle-field" and the "inverted-Y" field. On the other hand, with radiation therapy applied in combination with chemotherapy, the target volume depends on both the stage of the disease and the number of chemotherapy cycles. Likewise, the combined treatment is dependent on whether the role of radiotherapy is only the control of clinically involved regions, or of regions with potential subclinical disease too. Chemotherapy is the most frequently applied treatment modality in the management of non-Hodgkin's lymphoma (NHL). Radiation therapy as a single modality treatment with curative intent is applied in patients with, according to the histopathologic classification of the disease, the indolent NHL type and pathological stages I and II in continuation. The target radiation volume includes the clinically involved region, and possibly other adjacent clinically non-involved regions. In higher stages of disease or other, more aggressive NHL chemotherapy is applied either alone or in combination with adjuvant radiotherapy.

[Radiotherapy of lung cancer]. / Radioterapija raka pluca.

The paper is aimed at approaching radiation therapy methods to physicians of other specialties and pointing to the potential of radiation therapy in the management of lung cancer patients. With the reference to its incidence and mortality rates, lung cancer ranks among the most frequent human malignant tumors. Therapy procedures for lung cancer depend upon tumor histology type, stage of disease and patient general condition. The said parameters therefore determine the application of surgery, radiation therapy and/or chemotherapy. In general, treatment results are usually rather poor, primarily due to lung cancer being the most frequently detected only as locally advanced or metastatic disease. Alike surgery, radiotherapy is a local form of treatment aimed at achieving local tumor control. This curative or palliative form of treatment is either applied alone or in combination with other treatment modalities. Irradiation is usually delivered by high energy photon beams from a telecobalt device or linear accelerator. The success of radiation therapy complies with the irradiation dose managed to be applied to tumor or tumor bed, which depends on patients general condition and site, size and spread of tumor. Radiotherapy with curative intent is applied in stage I, II and III non-small cell lung cancer patients with surgery being primarily applied in those with stage I and II. The efficacy of surgical treatment is to be improved by a combined-modality treatment. In stage III patients, who are more frequent than others, radical radiotherapy alone or in combination with chemotherapy is applied. Results of clinical trials report patients of relatively good general condition benefiting from combined-modality therapy. Palliative radiotherapy is to be applied in patients with stage IV non-small cell lung cancer. On the other hand, in patients with small cell lung cancer chemotherapy is the primary modality treatment. When the disease is limited to the lungs, the aim of radiotherapy is to optimize local control of the primary tumor.

Cytogenetic consequences after occupational exposure to antineoplastic drugs.

Cytogenetic monitoring was carried out on a group of 38 nurses who reconstitute antineoplastic drugs in order to determine the extent of chromosomal damage. Genotoxic activities of antineoplastic drugs are studied by chromosome aberration assay, micronucleus assay, sister chromatid exchange (SCE) frequency high frequency cells (HFC) analysis, and mitotic activity of peripheral lymphocytes. Results confirmed that occupational exposure to a mixture of antineoplastic drugs may cause genome damages. The results of this study show that biomonitoring after exposure to a mixture of antineoplastic drugs which express clastogenic and aneugenic activity should involve a battery of cytogenetic methods.

Establishment and characterization of a megakaryoblast cell line with amplification of MLL.

A new cell line with megakaryoblastic features, designated UoC-M1, was established from the malignant cells of a 68-year-old patient with acute myeloid leukemia. The patient's leukemic cells reacted with alpha-naphthyl acetate esterase and acid phosphatase and expressed CD7, CD24, CD34, CD38, CD45, HLA-DR and CD61. Cytogenetic analysis of the patient's malignant cells (and of the UoC-M1 cells) showed a human, male hypodiploid karyotype with many chromosome rearrangements and marker chromosomes. Spectral karyotyping (SKY) analysis complemented the G-banded karyotyping and clarified several chromosomal translocations and identified the marker chromosomes. Fluorescence in situ hybridization (FISH) and SKY analysis demonstrated that one marker chromosome contained three segments of chromosome 9 interspersed with three segments of chromosome 11, as well as a portion of chromosome 19. FISH analysis with a probe for MLL revealed that the UoC-M1 cells contained four copies of the MLL gene. Southern blot analysis determined that the MLL gene had a germline profile while Northern and Western analyses showed that the MLL mRNAs and protein were of the appropriate sizes. This is the first report of amplification of the MLL gene which may be an additional mechanism of leukemogenesis or disease progression.

MLL is fused to CBP, a histone acetyltransferase, in therapy-related acute myeloid leukemia with a t(11;16)(q23;p13.3).

The recurring translocation t(11;16)(q23;p13.3) has been documented only in cases of acute leukemia or myelodysplasia secondary to therapy with drugs targeting DNA topoisomerase II. We show that the MLL gene is fused to the gene that codes for CBP (CREB-binding protein), the protein that binds specifically to the DNA-binding protein CREB (cAMP response element-binding protein) in this translocation. MLL is fused in-frame to a different exon of CBP in two patients producing chimeric proteins containing the AT-hooks, methyltransferase homology domain, and transcriptional repression domain of MLL fused to the CREB binding domain or to the bromodomain of CBP. Both fusion products retain the histone acetyltransferase domain of CBP and may lead to leukemia by promoting histone acetylation of genomic regions targeted by the MLL AT-hooks, leading to transcriptional deregulation via aberrant chromatin organization. CBP is the first partner gene of MLL containing well defined structural and functional motifs that provide unique insights into the potential mechanisms by which these translocations contribute to leukemogenesis.

Nicotine regulates collagen gene expression, collagenase activity, and DNA synthesis in cultured cardiac fibroblasts.

Cardiac fibroblasts that reside in the interstitium are the cellular origin of collagen and other proteins of the extracellular matrix in the heart. We have previously shown that in vitro gene expression, proliferation and even phenotypic features of cardiac fibroblasts are subject to regulation by biological factors such as hormones, growth factors and neurotransmitters. The influence of nicotine, the active ingredient of tobacco, on risk factors for cardiac diseases is well known. In vivo adverse effects of nicotine are as the result of its direct and indirect effects. The cellular and molecular mechanisms of direct effects of nicotine in the heart are widely unknown. The objective of this study was to investigate if nicotine has direct influence on cardiac fibroblasts. To this end, we studied the effects of nicotine on cultured cardiac fibroblasts. Northern hybridization analysis of RNA extracted from cardiac fibroblasts, enzymography of conditioned medium of cardiac fibroblasts and [3H]-thymidine incorporation into DNA of cardiac fibroblasts were used to examine the effects of nicotine on collagen gene expression, collagenase activity and DNA synthesis respectively. Treatment of cardiac fibroblasts with nicotine (10 micrograms/ml) led to a 31% (P < 0.05) decrease in the abundance of mRNA for pro alpha 1(I) but not pro alpha 2(I) collagen compared with control untreated cells. Nicotine treatment of cardiac fibroblasts also led to decreased collagenase activity (62%, P < 0.001) in the conditioned medium of those cells in culture. Studies with [3H]-thymidine incorporation into DNA of cardiac fibroblasts showed a nicotine-induced decrease (39%, P < 0.001) in DNA synthesis in those cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Gender-specific differences in expression of mRNAs for functional and structural proteins in rat ventricular myocardium.

Sex-related differences in predisposition to heart diseases have long been recognized. The molecular and cellular bases for this difference are unknown. In this study we have compared expression of genes for various structural and functional proteins of muscle and interstitial compartments of the myocardium in the adult and neonatal, male and female rat heart. We have also compared cultured cardiac fibroblasts from male and female hearts with regards to gene expression and proliferative capacity. We showed that in the adult rats, the abundance of mRNAs for contractile proteins alpha- and beta-myosin heavy chain (MHC) is higher in the heart of female rats than in that of age-matched male rats. However, the difference in mRNA level for alpha-MHC was more drastic (736%, P < 0.001) than that for beta-MHC (469%, P < 0.001). mRNA levels for sarcomeric actin in the female heart were greater by 79% (P < 0.001). Collagen type I had a significantly higher (303%, P < 0.01) mRNA level in the female heart compared with the male heart. mRNAs for TGF-beta 1, cytoskeletal actin and connexin 43 were also higher (150%, P < 0.01; 130%, P < 0.01, and 150%, P < 0.01, respectively) in the female heart compared with age-matched male heart. There were no significant sex-related differences at the mRNA levels for the above proteins in ventricular tissue from neonatal male and female littermates. At the cellular level, cardiac fibroblasts obtained from adult and neonatal hearts of both sexes were comparable with respect to the abundance of mRNAs for collagen type I, TGF-beta 1 or cytoskeletal actin. However, DNA synthesis, as measured by [3H]thymidine incorporation, was higher (328%, P < 0.01) in cells from adult female heart compared with that in cells from adult male rat heart. This difference was even more pronounced in cardiac fibroblasts obtained from newborn female rats (933%, P < 0.001) compared with that in cells obtained from newborn male rat hearts. Together, these findings show that there are sex-related differences in gene expression for most major proteins in heart tissue and that this phenomenon is associated with the post-pubertal period. These findings further suggest that sex-related differential gene expression and DNA synthesis in cardiac cells are due to the regulatory effects of male- and female-specific hormones.

Differential effects of transforming growth factor-beta 1 and phorbol myristate acetate on cardiac fibroblasts. Regulation of fibrillar collagen mRNAs and expression of early transcription factors.

Cardiac fibroblasts are responsible for synthesis and deposition of fibrillar collagen types I and III. Transforming growth factor-beta 1 (TGF-beta 1) has been proved to increase collagen biosynthesis in various systems, both in vivo and in vitro. We have investigated the effect of TGF-beta 1 on collagen gene expression in cultured cardiac fibroblasts and have compared this effect with that of a mitogenic agent, phorbol myristate acetate (PMA). The regulation of collagen types I and III gene expression was examined by using cDNA probes to rat alpha 2 (I) and mouse alpha 1 (III) procollagens. Quiescent cultured cardiac fibroblasts from rabbit heart were treated with TGF-beta 1 (10-15 ng/ml) and PMA (200 ng/ml). After 24 hours of treatment with TGF-beta 1, the abundance of mRNA for pro-alpha 2 (I) and pro-alpha 1 (III) collagens was increased by 112% (p less than 0.001) and 97% (p = 0.05), respectively, in treated fibroblasts compared with untreated cells. However, PMA-treated cells showed an opposite response: a 42% (p = 0.01) decrease in mRNA levels for pro-alpha 2 (I) collagen was observed. Immunofluorescent staining of cardiac fibroblasts in culture with anti-type I collagen antibody showed that alterations in mRNA levels led to altered collagen synthesis: cellular collagen was relatively increased in TGF-beta 1-treated cells and significantly diminished in PMA-treated cells. The abundance of mRNA for pro-alpha 1 (III) collagen was not affected by PMA treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac fibroblasts are predisposed to convert into myocyte phenotype: specific effect of transforming growth factor beta.

Cardiac fibroblasts are mainly responsible for the synthesis of major extracellular matrix proteins in the heart, including fibrillar collagen types I and III and fibronectin. In this report we show that these cells, when stimulated by transforming growth factor beta 1 (TGF-beta 1), acquire certain myocyte-specific properties. Cultured cardiac fibroblasts from adult rabbit heart were treated with TGF-beta 1 (10-15 ng/ml) for different periods of time. Northern hybridization analysis of total RNA showed that cells treated with TGF-beta 1 for 24 hr expressed mRNA corresponding to sarcomeric actin mRNA. Immunofluorescence staining and light microscopy showed that cultured cardiac fibroblasts treated with TGF-beta 1 became stained with a monoclonal antibody to muscle-specific actin. After treatment of quiescent cells with TGF-beta 1, cell proliferation (as measured by [3H]thymidine incorporation) was moderately increased (1.5-fold, P less than 0.001). NIH 3T3 cells and human skin fibroblasts, treated with TGF-beta 1, did not express sarcomeric actin mRNA. Treatment of cardiac fibroblasts with the mitogenic agent phorbol 12-myristate 13-acetate or with norepinephrine, angiotensin II, or interleukin 1 beta did not induce myocyte-specific actin mRNA. Cultured cardiac fibroblasts at the subconfluent stage, when exposed to TGF-beta 1 in the presence of 10% fetal bovine serum, gave rise to a second generation of slowly growing cells that expressed muscle-specific actin filaments. Our findings demonstrate that cardiac fibroblasts can be made to differentiate into cells that display many characteristics of cardiac myocytes. TGF-beta 1 seems to be a specific inducer of such conversion.

cis-platinum-based alternating non-cross-resistant chemotherapy as a first-line treatment in metastatic breast cancer. A phase II study.

Exposure to multiple non-cross-resistant drugs should increase cell kill and the chance of achieving more complete and partial responses. Our earlier study in breast cancer showed that second-line CAP (cyclophosphamide, adriamycin, cis-platinum) treatment was not cross-resistant to the CMFVP (cyclophosphamide, methotrexate, 5-fluorouracil, vincristine, prednisolone) regimen and produced a 51% response rate. These facts initiated a phase II study which used an alternating CMFVP/CAP regimen. Altogether, 49 patients entered the study and 45 were evaluated (greater than 2 cycles). The CMFVP regimen consisted of cyclophosphamide (200 mg/m2 on days 1, 2, 3, 4 and 5), methotrexate (30 mg/m2 on days 2 and 4), 5-fluorouracil (500 mg/m2 on days 1, 3 and 5), vincristine (1.4 mg/m2 on days 1 and 5), and prednisolone (40 mg p.o. on days 1-5), and was alternated with the CAP schedule (300 mg/m2 cyclophosphamide on days 1, 3 and 5, 50 mg/m2 adriamycin on day 1, and 30 mg/m2 cis-platinum on days 1, 3 and 5). Overall response was high, and 37 patients out of 45 responded (82%), with a 28% CR rate (13/45). A particularly high response rate was observed in soft tissues (86%, 18/21) and visceral organs (84%, 16/19). Only 1 patient progressed (3%). The duration of remission was 4-21+ months (median, 12 months). Six of 13 CR patients were still disease free 15 months after the treatment was stopped. The duration of survival was 5-25+ months (median, 15+ months). Toxicity was moderate (myelosuppression in 53% of patients, mainly grade I-II; stomatitis in 11%, except for 100% alopecia and 90% nausea and vomiting). One drug-related death (bone marrow aplasia) was recorded. The high antitumorigenic activity of the alternating regimen used is encouraging and may call for a randomized study for the ultimate evaluation of this treatment approach.

The effectiveness of intravenously administered immune lymphoid cells against intraperitoneally growing tumor correlates with their homing in the recipients' lymphoid organs.

Intraperitoneally growing Ehrlich ascites tumor (EAT) was eradicated by both i.p. and i.v. injection of serum, and by i.p. injection of spleen cells from mice immune to EAT. However, the i.v. injected immune spleen cells were completely ineffective unless the recipients had been pretreated with cyclophosphamide. The analysis of immune response in mice cured by the combination of cyclophosphamide and cell transfer revealed that they developed a humoral-type immunity to EAT and that the transferred spleen cells did not penetrate into their abdominal cavity. The effect of cyclophosphamide correlated with the extent of seeding of the donor-type cells in the recipients' lymphoid organs. Inasmuch as homing in cyclophosphamide-pretreated mice surpassed that in normal mice three to four times, it appeared that the beneficial effect of cyclophosphamide was primarily founded on its enhancement of seeding of the transferred lymphoid cells, implying that homing of these cells is a prerequisite for their anti-tumor activity.

Phase I-II trial of aclacinomycin A given in a four-consecutive-day schedule to patients with solid tumours. A South-East European Oncology Group (SEEOG) Study.

Aclacinomycin A (ACM) in a daily dose of 30 mg/m2 was infused over 1 h on 4 consecutive days to 50 patients. Myelotoxicity was acceptable, nausea and vomiting was frequent, hair loss was mild. Grade 1-2 cardiac rhythm abnormalities were observed in 12% of the patients. Between days 1 and 4 the heart rate and the corrected Q-T interval increased while the amplitude of the T wave decreased significantly, cardiac contractility remained unchanged. In 24 evaluable breast cancer patients 1 complete remission (4%) and 2 partial remissions (8%) lasting for only 2-3 months were seen. None of the 8 patients suffering from ovarial cancer benefitted from ACM therapy.