Overexpression of the p73 gene is a novel finding in high-risk B-cell chronic lymphocytic leukemia

The p73 protein shares structural and functional similarities with thetumour-suppressor p53, but its role in neoplastic transformation is unknown.Alternative splicing leads to the expression of at least nine p73 C-terminalmRNA splice variants (, , , , , , ,1, ). In this survey, we analyse the expression of p73 byreal-time quantitative RT–PCR, its known C-terminal variants with anRT–PCR-Southern technique and by Western blot in samples of 51 patientswith B-CLL, normal B lymphocytes from eight individuals, and fivehaematopoetic cell lines. p73 protein expression positively correlatedwith higher risk B-CLL stages (P = 0.046). Total p73 mRNAexpression was higher (P = 0.01) and p73 protein morefrequently detected (P = 0.008) in B-CLL compared with normalCD19+–B-lymphocytes. p73 C-terminal mRNA variants were expressed bothin B-CLL and in normal B-lymphocytes, but their expression was biased sincethe (P = 0.041), the (P <0.001), and the variant (P = 0.033) prevailed in normalB-lymphocytes. In summary, we conclude that the accumulation of p73, theexpression pattern of particular p73 variants and its link to progression mayplay a distinct role in the molecular pathology B-CLL.     

Phase I trial of combined chemotherapy with docetaxel, cisplatin, and 5-fluorouracil for patients with locally advanced squamous cell carcinoma of the head and neck

Background This phase I study was designed to determine the maximum tolerated dose (MTD) and toxicities of combination chemotherapy with docetaxel, cisplatin, and 5-fluorouracil (5-FU) in patients with locally advanced squamous cell carcinoma of the head and neck (SCCHN).Methods Patients received two cycles of chemotherapy repeated every 4 weeks. Starting doses (dose level 0) were: docetaxel 60mg/m², cisplatin 60mg/m², and 5-day continuous infusion 5-FU 600mg/m² per day. At least three patients were examined at each dose level before advancing to the next level.Results Nineteen male patients (median age, 59.5 years) were enrolled. Eighteen patients had previously untreated stage III or IV SCCHN and 1 had local relapse, rT4. In the 19 patients, the regimen was well tolerated, with neutropenia as the most common toxicity (grade 3; n = 11; grade 4; n = 1). Dose-limiting toxicity (DLT) was observed at the fifth dose level (docetaxel 70mg/m², cisplatin 70mg/m², 5-FU 750mg/m² per day), when 1 patient developed grade 2 renal toxicity during the first course; another 2 patients had persistent neutropenia. These doses were thus deemed the MTD for the regimen. In the 18 assessable patients, the overall clinical response rate was 94% (17/18 patients) and primary-site complete response (CR) occurred in 4 (22%) patients.Conclusion The MTD of this regimen was docetaxel 70mg/m² on day 1, cisplatin 70mg/m² on day 4, and 5-FU 750mg/m² per day for 5 days. The regimen was safe and generally well-tolerated and demonstrated good efficacy in patients with locally advanced SCCHN.     

Protection against fludarabine neurotoxicity in leukemic mice by the nucleoside transport inhibitor nitrobenzylthioinosine

Summary Fludarabine phosphate (F-ara-AMP, Fludara) is rapidly converted in the circulation to fludarabine (F-ara-A) and is among the most effective single agents in the treatment of chronic lymphocytic leukemia. Although current treatment protocols are well tolerated, severe neurotoxicity was a consequence of high-dose F-ara-AMP regimens used in early phase I trials against adult acute leukemia. The present study showed that in mice implanted with leukemia L1210, fatal neurotoxicity, which initially manifested as hind-limb paralysis, was a consequence of high-dose-F-ara-AMP treatment. However, the incidence of neurotoxicity was reduced by the coadministration of NBMPR-P, the 5-phosphate of nitrobenzylthioinosine, a potent inhibitor of thees equilibrative nucleoside transport (NT) system. NBTGR-P, the 5-phosphate of nitrobenzylthioguanosine (also a potent NT inhibitor) similarly prevented F-ara-AMP neurotoxicity in this experimental system. Treatment with F-ara-AMP/NBMPR-P combinations was more effective with respect to the fractional yield of cured mice than were the same treatment regimens without NBMPR-P.Abbreviations ara-A 9--d-arabinofuranosyladenine - F-ara-A 9--d-arabinofuranosyl-2-fluoroadenine (fludarabine) - F-ara-AMP fludarabine 5-monophosphate (Fludara) - NBMPR 6-[(4-nitrobenzyl)thio]-9--d-ribofuranosylpurine - NBMPR-P NBMPR 5-monophosphate - NBTGR 2-amino-6[(4-nitrobenzyl)thio]-9--d-ribofuranosylpurine - NBTGR-P NBMPR 5-monophosphate - NBdAdo-P N⁶-(4-nitrobenzyl)-9--d-2-deoxyribofuranosyladenine 5-monophosphate This study was supported by the National Cancer Institute of Canada     

Saturation of 2′, 2′-difluorodeoxycytidine 5′-triphosphate accumulation by mononuclear cells during a phase I trial of gemcitabine

Summary The plasma and cellular pharmacology of 2, 2-difluorodeoxycytidine (dFdC, Gemcitabine) was studied during a phase I trial. The steady-state concentration of dFdC in plasma was directly proportional to the dFdC dose, which ranged between 53 and 1,000 mg/m² per 30 min. The cellular pharmacokinetics of an active metabolite, dFdC 5-triphosphate (dFdCTP), were determined in mononuclear cells of 22 patients by anion-exchange highpressure liquid chromatography. The rate of dFdCTP accumulation and the peak cellular concentration were highest at a dose rate of 350 mg/m² per 30 min, during which steady-state dFdC levels of 15–20 M were achieved in plasma. A comparison of patients infused with 800 mg/m² over 60 min with those receiving the same dose over 30 min demonstrated that the dFdC steady-state concentrations were proportional to the dose rate, but that cellular dFdCTP accumulation rates were similar at each dose rate. At the lower dose rate, the AUC for dFdCTP accumulation was 4-fold that observed at the higher dose rate. Consistent with these observations, the accumulation of dFdCTP by mononuclear cells incubated in vitro was maximal at 10–15 M dFdC. These studies suggest that the ability of mononuclear cells to use dFdC for triphosphate formation is saturable. In the design of future protocols, a dose rate should be considered that produces maximal nucleotide analogue formation, with increased intensity being achieved by prolonging the duration of infusion.Abbreviations ara-C I--d-arabinosylcytosine - ara-C _ss steady-state concentration of ara-C - ara-CTP 5-triphosphate of ara-C - dFdC 2, 2-difluorodeoxycytidine, Gemcitabine - dFdC _ss steady-state concentration of dFdC - dFdCTP 5-triphosphate of dFdC Supported in part by grants CA28596 and CA32839 from the National Cancer Institute, Department of Health and Human Services, and by grant CH-130 from the American Cancer Society     

Role of Ceramide During Cisplatin-induced Apoptosis in C6 Glioma Cells

Cisplatin is commonly used for the treatment of malignant brain tumors. However, the mechanisms of cell death by cisplatin are not fully understood. Therefore, the present study was designed to elucidate the apoptotic signaling pathway(s) activated by cisplatin in a C6 rat glioma cell line. C6 cells were treated with various concentrations of cisplatin (0.2–10g/ml) for 24–72h. At 10g/ml cisplatin, over 90% of the cells became dead at 72h. Apoptotic death was confirmed by condensation and fragmentation of nuclei, and DNA laddering. Even in cells treated with 1.5g/ml cisplatin, typical apoptotic cells were observed at 72h. The intracellular level of ceramide, measured Escherichia coli diacylglycerol kinase markedly increased during 24–72h after the addition of 10g/ml cisplatin. The activity of caspase-3(-like) proteases increased and reached a peak at 48h. Inhibitors of caspases reduced the number of apoptotic cells. Pretreatment of C6 cells with glutathione or N-acetyl-cysteine, which are known to block the activation of neutral magnesium-dependent sphingomyelinase, inhibited ceramide formation, leading to suppression of both activation of caspase-3(-like) proteases and apoptosis by cisplatin. In contrast, pretreatment of the cells with N-oleoylethanolamine (OE), a ceramidase inhibitor, potentiated apoptosis induced by cisplatin. Furthermore, OE enhanced sensitivity of the cisplatin-resistant cells to cisplatin. These results suggest that ceramide is closely implicated in apoptosis of glioma cells by cisplatin through activation of caspase-3(-like) proteases.     

Flow cytometric analysis of primary tumors and their corresponding metastatic nodes in breast cancer

Human breast carcinoma is biologically heterogeneous, and its clinical course may vary from one which is indolent to one which rapidly progresses. Although it is the metastasis rather than the primary tumor that ultimately overwhelms the patients, studies concerning the DNA pattern have focused on the primary tumors. This study was undertaken to identify heterogeneities between primary tumors and metastases, and to evaluate the prognostic significance of the ploidy pattern and the S-phase fraction (SPF) of metastatic nodes in axillary node positive patients. Seventy-four frozen specimens of the primary and corresponding metastatic nodes from 37 patients have been analyzed by flow cytometry and the SPF calculated. The results of ploidy pattern analysis in primaries revealed 25 diploidy (67.6%) and 12 aneuploidy (32.4%), while those in metastasis showed 17 diploidy (46.0%) and 20 aneuploidy (54.0%). The aneuploidy group in metastatic nodes had the poorer histological grade (85.0% vs. 15.0%, p=0.02), and more mean metastatic nodes (5.75±2.10 vs. 3.05±1.56, p=0.018), and more frequent lymphatic vessel invasion (65.0% vs. 11.8%, p=0.031) than its counterpart. Decreased expression of ER (70.6% vs. 25.0% p=0.006) and increased expression of c-erbB2 (65.0% vs. 23.5%, p=0.012) were observed in the aneuploidy of metastatic nodes. The group with higher SPF in metastatic nodes had more metastatic nodes (5.47±2.31 vs. 4.00±1.78, p=0.042), and the higher incidence of lymphatic vessel invasion (57.9% vs. 22.2%, p=0.027), and poor histological grade (71.4% vs. 37.5%, p=0.039). In conclusion, the cell populations in metastatic nodes revealed DNA pattern which differed from that of primary tumors. The ploidy pattern and SPF in metastatic nodes might be considered as discriminate measure for risk factors in breast cancer patients with positive axillary node.     

Phase I/II study of irinotecan, 5-fluorouracil,and <Emphasis Type="Italic">l</Emphasis>-leucovorin combination therapy (modified Saltz regimen) in patients with metastatic colorectal cancer

Background A combination of irinotecan 125mg/m², 5-fluorouracil (5-FU) 500mg/m², and leucovorin (LV) 20mg/m² (Saltz regimen; treatment on days 1, 8, 15, and 22 every 6 weeks) is widely used for the treatment of metastatic colorectal cancer. A modified schedule with chemotherapy on days 1 and 8 of a 21-day cycle was recommended in 2001 because of early treatment-related mortality. We conducted a phase I/II study of this modified Saltz regimen as first-line therapy in Japanese patients with metastatic colorectal cancer to assess the maximum tolerated dose (MTD) and the recommended dose of 5-FU when given with fixed doses of l-LV and irinotecan, and to evaluate the efficacy and the feasibility of this regimen.Methods Irinotecan, 5-FU, and l-LV were administered on days 1 and 8 of a 21-day cycle. Irinotecan 100mg/m² was given intravenously over the course of 90min on day 1, followed by l-LV 10mg/m², and then 5-FU. The dose of 5-FU was escalated from 400mg/m² (level 1) to 500mg/m² (level 2). If neither level met the criteria for the MTD, the recommended dose was defined as level 2, and dose escalation was discontinued, because the maximum approved weekly dose of irinotecan alone in Japan is 100mg/m² and the dose of 5-FU in the original Saltz regimen was 500mg/m².Results One patient had grade 4 neutropenia with fever at level 1, and four patients had grade 3 neutropenia at level 2. There was no treatment-related death. Level 2 did not meet the criteria for the MTD. The relative dose intensities of the first five cycles were 91% for both 5-FU and irinotecan at level 1 and 86% for 5-FU and 93% for irinotecan at level 2. The response rates were 58% for all patients, and 69% for patients at level 2.Conclusion Our results confirm that the modified Saltz regimen is safe and efficacious for Japanese patients. The recommended doses for phase II studies are irinotecan 100mg/m², 5-FU 500mg/m², and l-LV 10mg/m².     

Shaping future strategies for the pharmacological control of tumor cell metastases

Summary The eradication of established metastases in patients with malignant tumors is the single most important objective in clinical oncology. The current panel of antineoplastic agents discovered through random and semiempirical screening procedures has proven largely ineffective in treating disseminated disease and there is a clear and urgent need for more efficient antimetastatic drugs. Unfortunately, although progress has been made in examining the biology of metastatic spread, our understanding of the pharmacology, biochemistry and molecular genetics of this process is meager and insufficient to provide a rational foundation for the design of mechanism-based antineoplastic agents. Faced on the one hand with the failure of existing drugs to control metastatic spread and on the other with a dearth of alternative pharmacological approaches, the prospect of offering significantly improved therapy to the cancer patient of the 1990's is poor. The challenge of the coming decade lies in obtaining better insights into the molecular mechanisms of metastasis and using this information to identify pharmacological opportunities to curtail the proliferation of secondary tumor growths. As a first step toward this goal we need to define more rigorously what constitutes a therapeutic target in malignant disease and what steps in the pathogenesis of cancer metastasis represent the gravest risk to the patient and thus are most eligible for direct pharmacological intervention. In addressing these issues and developing future strategies for antimetastatic drugs, Paget's 100 year-old seed and soil hypothesis continues to offer a useful conceptual framework for analysis of metastatic behavior. Although Paget's proposal has been validated by a century of clinical observation, efforts to define the seed and soil theory in molecular terms have not been attempted. With the advent of more efficient methodologies for culturing human normal and neoplastic cells coupled with the availability of microanalytical technologies it now becomes possible to investigate and identify the complementary biochemical components of the tumor cell seed and organ soil that combine to encourage the proliferation of metastases. With this information the design of specific pharmacological strategies to uncouple the seed and soil relationship may emerge as a potential therapeutic approach for antagonizing the growth of disseminated malignant tumors.     

Tamoxifen Increases Photodynamic Therapeutic Response of U87 and U25ln Human Glioma Cells

We tested the hypothesis that Tamoxifen (TMX), an inhibitor of protein kinase C (PKC), augments the cytotoxicity of photodynamic therapy (PDT) treatment of human (U87) and (U25ln) glioma cells. U87 and U251n glioma cells were plated and treated with PDT using Photofrin as the sensitizer. Cells were treated with Photofrin at various doses and with various optical (632nm) irradiation intensities 24h later. Cells were also treated with Photofrin at a fixed dose alone and with various doses of Tamoxifen and subjected to laser treatment 24h later. Tumor response was tested using the (3-94,5-dimethyl-2-yl)-2,5-diphenyl-tetrazolium (MTT) method. Total toxicity of U87 cells was achieved with PDT at all doses of Photofrin (1, 2.5, 5, 10g/ml) with irradiation densities equal to or greater than 200mJ/cm². Using an irradiation intensity of 100mJ/cm², U87 and U251n cells were killed in a Photofrin dose-dependent manner. Significant cytotoxicity was detected with Photofrin doses of 5g/ml (p < 0.05) and 10g/ml (p < 0.001). Tamoxifen at a dose of 500g/ml and higher, significantly increased the toxicity of the PDT response with 5g/ml Photofrin and 100mJ/cm² (p < 0.05). In summary, our data demonstrate that Tamoxifen significantly enhances the Photofrin PDT activity of U87 and U251n human glioma cells.     

Biochemical pharmacology of 5,6-dihydro-5-azacytidine (DHAC) and DNA hypomethylation in tumor (L1210)-bearing mice

Summary Dihydro-5-azacytidine (DHAC) is a hydrolytically stable congener of 5-azacytidine, which retains antileukemic activity against experimental leukemias. The biochemical pharmacology of DHAC was studied in tumor-bearing mice in order to elucidate the mode of action of this drug. We found that after an LD₁₀ dose of DHAC, the plasma peak concentration achieved was 317 M and was eliminated biexponentially, with a t_1/2 of 1.03 h and a t_1/2 of 5 h. By 4h, an unidentified metabolite of [³H]DHAC peaked and was eliminated biexponentially, with a t_1/2 of 1.06 h and a t_1/2 of 10.6 h. [³H]DHACTP was the major anabolite in the L1210/0 cells, and was also eliminated biexponentially, with a t_1/2 of 4.3 h and a t_1/2 of 12.2 h. An unknown anabolite of [³H]DHAC that eluted 5 min after [³H]DHACTP, between UTP and ATP, peaked at 3 h and could possibly be the deoxy-derivative [³H]DHAdCTP. A tissue distribution study revealed that the liver, L1210/0, and lung accumulate the most radioactivity per gram of wet tissue. Methylation studies showed that an LD₁₀ dose of [³H]DHAC resulted in a 25.06% hypomethylation of DNA in L1210/0 cells and a 46.32% hypomethylation in a deoxycytidine kinase mutant cell line L1210/dCK(-), compared with their respective controls.Abbreviations used 5-aza-C 5-azacytidine - DHAC 5,6-dihydro-5-azacytidine - DHACTP dihydro-5-azacytidine 5-triphosphate - DHAdCTP dihydro-5-azacytidine 5-deoxytriphosphate - LD₁₀ lethal dose 10% of animals - UTP uridine 5-triphosphate - ATP adenosine 5-triphosphate - TCA trichloroacetic acid - PCA perchloric acid - RBC red blood cells - i.p. intraperitoneally - SAX strong anion exchange - SCX strong cation exchange Supported by a research grant, CA 38905, from the National Institutes of Health, NCI     

Pharmacokinetics of methotrexate and 7-hydroxy-methotrexate in plasma and bone marrow of children receiving low-dose oral methotrexate

Summary The absorption, distribution, and elimination kinetics of low-dose p.o. methotrexate (MTX) were repeatedly studied in 19 children during maintenance treatment of childhood acute lymphoblastic leukemia. Plasma concentrations, urinary elimination, and bone marrow concentrations of MTX and 7-hydroxymethotrexate (7-OH-MTX) were monitored during 24 h following a routime p.o. dose (30 mg/m²) using high-pressure liquid chromatography. Significant interindividual variability was found in time to peak concentration (30–180 min), peak concentration (0.41–2.77 M), and to a lesser extent the half-lives (t_1/2: 32.8–86.1 min; t_1/2: 43.6–350.0 min; t_1/2 absorption: 25.2–60.3 min) and plasma area under the concentration-time curve from zero to infinity (195.6–818.5 M.min). Significant amounts of 7-OH-MTX were detected in plasma, with a mean area under the concentration-time curve from zero to infinity of 208 M.min compared with 365.6 M.min for MTX. High concentrations of 7-OH-MTX were present in bone marrow 24 h after oral MTX (15/19 patients) and were at least five fold those in plasma and three fold the concentration of MTX in bone marrow. In four patients occasionally neither MTX nor metabolite could be detected. Repeated examination of these pharmacokinetic parameters in plasma and bone marrow showed that the intraindividual variability was small.This study was supported by the Netherlands Cancer Foundation Koningin Wilhelmina Fonds     

Androstenedione and androst-5-ene-3β,17β-diol stimulate DMBA-induced rat mammary tumors — role of aromatase

Summary The effect of the adrenal steroids androst-5-ene-3,17-diol (⁵-diol) and androstenedione (⁴-dione) was studied on the growth of mammary carcinoma induced in the rat by dimethylbenz[a]anthracene (DMBA). The plasma levels of the two steroids were maintained at values within the range of those found in the circulation of post-menopausal women by constant release from osmotic pumps in ovariectomized animals. ⁵-diol and ⁴-dione, at the daily release rate of 500µg, led to plasma levels of 1.26±0.19 and 1.72 ± 0.75 ng/ml, respectively. At these physiologically relevant plasma concentrations, both ⁵-diol and ⁴-dione caused a marked stimulation of tumor growth while having minimal or no effect on uterine weight or on plasma prolactin and LH levels.Concomitant treatment with the aromatase inhibitor aminoglutethimide completely blocked the stimulatory effect of ⁴-dione released from silastic implants on tumor growth, while simultaneous administration of the antiandrogen flutamide had no significant effect. On the other hand, when aminoglutethimide was administered with ⁵-diol, the stimulatory effect of the adrenal steroid on tumor growth was not affected. Such data indicate that, under the present experimental conditions, transformation of ⁴-dione into androgens plays a minor role, the predominant effect of the adrenal steroid being stimulation of tumor growth through conversion into estrogens, while ⁵-diol exerts a direct estrogenic effect independent from aromatase activity. The minimal or absent effect of the same treatment on uterine weight and on plasma prolactin and LH levels indicates the tissue specificity of the effects observed, the mammary tissue being most sensitive to the action of adrenal steroids.F.L. is a MRC Career Investigator.     

Instructions for Authors

We conducted a pilot study of dose dense doxorubicin and cyclophosphamide (AC) combination chemotherapy followed by infusional paclitaxel (T) in primary breast cancer to determine its safety and feasibility. Twenty-two subjects (10 with stage II and 4 positive lymph nodes, and 12 with stage III disease) were treated with AC (A 60mg/m² and C 2000mg/m²) with filgrastim every 14 days for three cycles followed by infusional paclitaxel (140mg/m² over 96h) every 14 days for three cycles. Mean overall cycle length was 15.3 days and mean duration of therapy was 92 days. Dose reductions of C or T were required in 7/132 (5.3%) cycles for mucositis, diarrhea, or failure to recover platelets by day 15. Ninety-five percent of subjects had grade 4 neutropenia and 1 subject had a platelet nadir of <20,000. Actual delivered dose intensity (DI) over six cycles was: A 27mg/m² per week; C 892mg/m² per week; T 64mg/m² per week (90.6, 89.2, and 91.4% of planned DI, respectively). Average total dose administered was: A 180mg/m²; C 5880mg/m²; T 403mg/m² (100, 98, and 96% of planned total doses, respectively). Clinical response rate in 10 subjects receiving neoadjuvant therapy was 100% (4 complete response, 6 partial response). Four subjects had a pathologic complete response (three subjects without evidence of malignancy and one subject with ductal carcinoma in situ.) Administration of dose dense AC followed by infusional paclitaxel in 14-day cycles is feasible and this regimen is active in breast cancer.     

Cytokines induce thymidine phosphorylase expression in tumor cells and make them more susceptible to 5′-deoxy-5-fluorouridine

The present study shows that various cytokines such as tumor necrosis factor (TNF), interleukin-1 (IL-1), and interferon- (IFN) make tumor cells much more susceptible to the cytostatic 5-deoxy-5-fluorouridine (5-dFUrd) than to 5-fluorouracil (5-FUra) and other cytostaties. These three cytokines increased the susceptibility of human cancer cell lines (COLO201, MKN45 and WiDr) but did not affect that of normal fibroblast WI38 cells. The cytokine mixture induced a 50-fold increase in the susceptibility of COLO201 to 5-dFUrd, whereas a 12-fold increase and a less than 5-fold enhancement in the susceptibility to 5-FUra and other cytostatics, respectively, were observed. The increased susceptibility would be a result of the induction of thymidine phosphorylase (TdR Pase), which is the essential enzyme for the conversion of 5-dFUrd to 5-FUra. The cytokine mixture increased TdR Pase activity by up to 47 times and greatly induced its mRNA expression in the cancer cell lines. These results suggest that the therapeutic benefit of 5-dFUrd would be improved by its use in combination with the cytokines.     

Phase I/II trial of high dose mitoxantrone in metastatic breast cancer: the M.D. Anderson Cancer Center experience

Anthracyclines are among the most active agents in metastatic breast cancer. Mitoxantrone demonstrated a different toxicity profile when compared to doxorubicin. We performed a phase I/II study of singleagent highdose mitoxantrone therapy for advanced breast cancer. Nineteen patients who had a diagnosis of metastatic breast cancer received treatment at the M.D. Anderson Cancer Center between June 1986 and December 1987. The patients received escalating doses of mitoxantrone until a maximum tolerated dose (MTD), defined as grade 3 or 4 nonhematologic toxicity or infection, was obtained. The starting dose of 25 mg/m², given by short intravenous infusion, was escalated by 25% in each fivepatient cohort if each patient in the previous cohort tolerated the initial course and 2 or fewer patients reached the MTD. The median cumulative dose of mitoxantrone was 93 mg/m² (range, 25–205) and the maximum single dose was 39mg/m². Myelosuppression was the dose limiting toxicity. The median duration of granulocyte count 250/l was 5–7 days. Four patients (22%) had infections that required hospitalization, 3 patients (17%) had cardiac toxicity. One patient (6%) achieved a complete response, and 3 (17%) had a partial response, with an overall response rate of 22.3%. No apparent doseresponse relationship was observed in our study. The mitoxantrone dosage recommended for phase II studies is 25mg/m² every 3–4 weeks. We conclude that highdose mitoxantrone therapy for metastatic breast cancer was relatively well tolerated but was not associated with a higher response rate than that of standard dose mitoxantrone.     

Systemic treatment with murine recombinant interleukin-1β inhibits the growth and progression of malignant glioma in the rat

We investigated the effects of daily subcutaneous (SC) injections of 100, 200, or 400 g/kg murine recombinant interleukin-1 (rIL-1) or its excipient on normal Fischer 344 rats and ones harboring a malignant RT-2 glioma. The tumor model has a predictable course with animals dying on days 14–17 following an intracerebral inoculation of 10⁴ RT-2 glioma cells. Treatments with (rIL-1) or excipient began on day seven post-tumor inoculation and continued for 7 days. We observed no significant effect on core body temperatures although there was a significant (p < 0.05) decrease in body weight in all (rIL-1) treated animals. When tumor-bearing animals became moribund, they received an intraperitoneal injection of bromodeoxyuridine (BUdr) and were sacrificed two hours later. Blood samples were obtained prior to their sacrifice by transcardiac perfusion with a buffered aldehyde solution. Recombinant IL-4ß affected blood differentials; causing neutrophilia, lymphopenia, and slight thrombocythemia. The BUdr labeling index of glioma cells did not significantly differ between treatment groups, although tumors differed histologically at the time of necropsy. Tumors of rIL-1 treated animals had more extensive necrosis and a greater degree of leukocyte infiltration. Survival studies were conducted in which rats were given continuous daily SC injections of (rIL-1) until day of death. Overall survival between the two groups differed significantly in studies using 100 g/kg/d (p < 0.05); (rIL-1ß) treated rats had a mean survival time of 22 (± 3.0) days while excipient controls had a mean survival time of 17 (± 0.5) days. Similarly, at a dose of 200 g rIL-1(3/kg/d), mean survival was significantly (p < 0.05) increased as compared to excipient controls (18.75 ± 1.5 vs. 15.25 ± 1.7 days, respectively). Daily injections of 400 g/kg did not significantly increase the survival of glioma bearing animals, possibly as a consequence of fIL-1ß toxicity at this dose.     

A Critical Examination of the Results from the Harvard-MIT NCT Program Phase I Clinical Trial of Neutron Capture Therapy for Intracranial Disease

A phase I trial was designed to evaluate normal tissue tolerance to neutron capture therapy (NCT); tumor response was also followed as a secondary endpoint. Between July 1996 and May 1999, 24 subjects were entered into a phase I trial evaluating cranial NCT in subjects with primary or metastatic brain tumors. Two subjects were excluded due to a decline in their performance status and 22 subjects were irradiated at the MIT Nuclear Reactor Laboratory. The median age was 56 years (range 24–78). All subjects had a pathologically confirmed diagnosis of either glioblastoma (20) or melanoma (2) and a Karnofsky of 70 or higher. Neutron irradiation was delivered with a 15cm diameter epithermal beam. Treatment plans varied from 1 to 3 fields depending upon the size and location of the tumor. The ¹⁰B carrier, L-p-boronophenylalanine-fructose (BPA-f), was infused through a central venous catheter at doses of 250mgkg^–1 over 1h (10 subjects), 300mgkg^–1 over 1.5h (two subjects), or 350mgkg^–1 over 1.5–2h (10 subjects). The pharmacokinetic profile of ¹⁰B in blood was very reproducible and permitted a predictive model to be developed. Cranial NCT can be delivered at doses high enough to exhibit a clinical response with an acceptable level of toxicity. Acute toxicity was primarily associated with increased intracranial pressure; late pulmonary effects were seen in two subjects. Factors such as average brain dose, tumor volume, and skin, mucosa, and lung dose may have a greater impact on tolerance than peak dose alone. Two subjects exhibited a complete radiographic response and 13 of 17 evaluable subjects had a measurable reduction in enhanced tumor volume following NCT.     

Effect of the aromatase inhibitor vorozole on estrogen and progesterone receptor content of rat mammary carcinomas induced by 1-methyl-1-nitrosourea

Vorozole, a nonsteroidal aromatase inhibitor, impedes the post-initiation stage of chemically induced mammary carcinogenesis. While various aspects of vorozole's effects on mammary carcinoma development have been investigated, little attention has been directed to determining the estrogen receptor (ER) and progesterone receptor (PR) content of mammary carcinomas that arise despite vorozole treatment. Female Sprague–Dawley rats were given an i.p. injection of 50mg MNU/kg body weight at 21 days of age and placed on diet supplemented with 0 or 3mg vorozole/kg, which had no effect on mammary tumor development. Histologically confirmed carcinomas were evaluated for ER and PR by immunohistochemistry. In the control group, 78.8% of carcinomas were ER positive with an ER content ranging from 13.8 to 40.0%, similar to ER content of mammary ductal epithelial cells from non-carcinogen treated animals. PR content ranged from 4.4 to 45.2% and also was similar to levels of PR observed in ductal epithelial cells. ER was not correlated with PR in mammary carcinomas (r=0.05, p>0.80), whereas there was a significant correlation in ductal epithelium (r=0.86, p=0.006). In vorozole-treated rats, no ER negative carcinomas were observed and overall ER expression by vorozole was elevated (p<0.03). All carcinomas from vorozole-treated rats expressed PR (2.5–60.2%) and correlation between ER and PR content was numerically greater in carcinomas from vorozole-treated animals (r=0.42, p=0.09). These data, which are considered hypothesis generating, provide evidence that low doses of vorozole in the diet select for mammary carcinomas with an increased ER positive phenotype.     

Estrogen receptors in 699 primary breast cancers: A comparison of immunohistochemical and biochemical methods

Summary Background: Over the last few years, estrogen receptor (ER) determination by immunohistochemistry (ER-ICA) has been extensively used, but it still remains to be established whether this method can replace the standard biochemical technique using dextran-coated charcoal (ERDCC). Patients and methods: ER were determined by both the dextran-coated charcoal (DCC) method and immunohistochemistry (ICA) in 699 patients with primary breast cancer; other parameters (age, pathological T-pT- and nodal status -pN-, progesterone receptors by DCC, proliferative index by ICA) were also recorded. The best cut-off for ERICA was evaluated by means of Receiver Operating Characteristics (R.O.C.) analysis; logistic regression analysis was used to find adequate weights for stain intensity. Results and conclusions: A significant correlation was found between the two methods (p < 0.001). R.O.C. analysis revealed that the best cut-off for the ERICA score was 45% (sensitivity 0.810, specificity 0.804). Logistic regression analysis showed that an ERICA score which also considers staining intensity does not add any useful information concerning ER content in breast cancers.     

IFN-β Gene Therapy Induces Systemic Antitumor Immunity Against Malignant Glioma

We previously demonstrated that intratumoral administration of liposomes containing the murine interferon beta (IFN-) gene [lip(pSV2muIFN-)] resulted in stronger growth-inhibitory effect on GL261 (H-2^b) mouse glioma inoculated in brains of syngeneic C57BL/6 mice than conventional exogenous IFN- administration, and histologic evaluation revealed the massive infiltration of T lymphocytes (CD8 > CD4) within the residual tumor. The present study was aimed at determining whether such tumor-infiltrating lymphocytes (TIL) have any tumor-specific cytotoxic effects. Intratumoral administration of lip(pSV2muIFN-) resulted in prolonged survival time and a 50% tumor-free incidence in the mice treated. The surviving animals were subsequently re-challenged with either subcutaneous or intracranial injection of GL261 cells, and no tumors were found to develop over a 50-day period. In vivo depletion of CD8, but not CD4 cells decreased the efficacy of lip(pSV2muIFN-). Specific cytotoxic T lymphocytes (CTL) against GL261 cells were generated from both TIL and spleen cells of the mice treated. The results of flow cytometric analysis and antibody blocking test revealed that the bulk CTL lines thus prepared were T cell receptor (TCR) , CD8 T lymphocytes with H-2^b restriction.These findings suggest that, in addition to direct growth-inhibitory effects by the IFN- gene on the tumor cells, activation of systemic cellular immunity may participate in antitumor effects in vivo, despite the fact that central nervous system is generally regarded as an immunologically privileged site.