First-line Herceptin monotherapy in metastatic breast cancer.

The pivotal phase II and III Herceptin trials proved the efficacy and safety of second- or third-line single-agent Herceptin and first-line Herceptin in combination with chemotherapy, respectively. In the current trial, 114 patients were randomized to one of two dose groups of first-line Herceptin monotherapy: standard dose of 4 mg/ kg initial dose followed by 2 mg/kg intravenous (i.v.) weekly; or high dose of 8 mg/kg initial dose followed by 4 mg/kg i.v. weekly. The regimen was generally well tolerated. A similar incidence of adverse events was demonstrated in the two dose groups with the possible exception of acute infusion-related events such as fever and chills as well as rash and dyspnea, which appear to be more prevalent in the higher dose group. The overall response rate was 26% and response rates were similar between the two dose groups (24% for the standard Herceptin dose group and 28% for the high Herceptin dose group). Subgroup analysis determined a higher response rate in IHC 3+ patients (35%) and FISH-positive patients (41%). When women with stable disease for > or =6 months were included with responders, the clinical benefit rate in IHC 3+ patients was 47%. Median survival was 24.4 months, which is comparable with the survival rate seen in the pivotal phase III combination trial (25 months). Therefore, single-agent Herceptin is an important new option for the first-line treatment of HER2-positive metastatic breast cancer patients.

Phase II study of direct intralesional gene transfer of allovectin-7, an HLA-B7/beta2-microglobulin DNA-liposome complex, in patients with metastatic melanoma.

Cutaneous melanoma is one of the most rapidly increasing cancers in the United States. Because of the lack of effective treatment options and toxicities of most chemotherapeutic and radiation regimes, immunotherapies such as vaccination therapy represent an attractive approach for patients with advanced melanoma. The purpose of this study was to evaluate the response rate, time to progression, and survival of patients with metastatic melanoma treated by direct intratumoral injection with Allovectin-7 (a plasmid DNA encoding the genes HLA-B7 and beta2-microglobulin complexed with a cationic lipid mixture, DMRIE/DOPE. Fifty-two patients with metastatic melanoma were enrolled in this Phase II study. Therapy consisted of six intratumoral injections of 10 microg of Allovectin-7 over a 9-week period. Treatment was well tolerated. Treatment-related adverse events were mild to moderate, the most frequent of which were ecchymosis, pruritus (and/or discomfort at the injection site), and pneumothoraces. Regression of the injected lesion was observed in 18% of patients, including one complete response, three partial responses, and five minor responses. An overall response rate of 4% (two partial responses) was documented, and nine patients (18%) maintained stable disease for at least 11 weeks. Six patients remained alive 25.1 to 39.4 months from their first injection, including two patients with local (injected tumor) responses and one patient with an overall disease partial response. This study demonstrates that intratumoral administration of Allovectin-7 in metastatic melanoma is safe and can produce both responses in injected lesions and in overall disease. Clinical trials optimizing patient selection and combining Allovectin-7 with other modalities of therapy are currently ongoing in an effort to improve response rates.

First-line, single-agent Herceptin(R) (trastuzumab) in metastatic breast cancer. a preliminary report.

Following confirmation of the appropriate dosage, safety and potential efficacy of Herceptin(R) (trastuzumab) in small-scale phase I and II trials involving patients with refractory disease, a large trial was conducted in 222 patients with breast cancer who had relapsed after one or two chemotherapy regimens for their metastatic disease. The results showed a positive and durable overall response rate (15% according to a response evaluation committee (REC) assessment) using trastuzumab monotherapy (initial dose 4 mg/kg intravenously (i.v.) followed by 2 mg/kg i.v. weekly). In another recently completed phase II trial, 113 patients were randomised to two dose levels (initial dose of 4 mg/kg i.v. dose followed by 2 mg/kg i.v. weekly, or initial dose of 8 mg/kg followed by 4 mg/kg i.v. weekly) of single-agent trastuzumab as first-line therapy for metastatic disease. The preliminary overall response rate was 23% based on investigator assessment, and tolerability was excellent as in previous trials; efficacy was similar in both dose groups, but the side-effects tended to be more frequent in the higher dose group. The preferred dosage is therefore the same as that currently recommended, i.e. an initial dose of 4 mg/kg i.v. followed by 2 mg/kg weekly i.v. until disease progression.

Intrathecal treatment of neoplastic meningitis due to breast cancer with a slow-release formulation of cytarabine.

DepoCyte is a slow-release formulation of cytarabine designed for intrathecal administration. The goal of this multi-centre cohort study was to determine the safety and efficacy of DepoCyte for the intrathecal treatment of neoplastic meningitis due to breast cancer. DepoCyte 50 mg was injected once every 2 weeks for one month of induction therapy; responding patients were treated with an additional 3 months of consolidation therapy. All patients had metastatic breast cancer and a positive CSF cytology or neurologic findings characteristic of neoplastic meningitis. The median number of DepoCyte doses was 3, and 85% of patients completed the planned 1 month induction. Median follow up is currently 19 months. The primary endpoint was response, defined as conversion of the CSF cytology from positive to negative at all sites known to be positive, and the absence of neurologic progression at the time the cytologic conversion was documented. The response rate among the 43 evaluable patients was 28% (CI 95%: 14-41%); the intent-to-treat response rate was 21% (CI 95%: 12-34%). Median time to neurologic progression was 49 days (range 1-515(+)); median survival was 88 days (range 1-515(+)), and 1 year survival is projected to be 19%. The major adverse events were headache and arachnoiditis. When drug-related, these were largely of low grade, transient and reversible. Headache occurred on 11% of cycles; 90% were grade 1 or 2. Arachnoiditis occurred on 19% of cycles; 88% were grade 1 or 2. DepoCyte demonstrated activity in neoplastic meningitis due to breast cancer that is comparable to results reported with conventional intrathecal agents. However, this activity was achieved with one fourth as many intrathecal injections as typically required in conventional therapy. The every 2 week dose schedule is a major advantage for both patients and physicians.

First-line, single-agent Herceptin(trastuzumab) in metastatic breast cancer: a preliminary report.

Following confirmation of the appropriate dosage, safety and potential efficacy of Herceptin(trastuzumab) in small-scale phase I and II trials involving patients with refractory disease, a large trial was conducted in 222 patients with breast cancer who had relapsed after one or two chemotherapy regimens for their metastatic disease. The results showed a positive and durable overall response rate (15% according to a response evaluation committee (REC) assessment) using trastuzumab monotherapy (initial dose 4 mg/kg intravenously (i.v.) followed by 2 mg/kg i.v. weekly). In another recently completed phase II trial, 113 patients were randomised to two dose levels (initial dose of 4 mg/kg i.v. dose followed by 2 mg/kg i.v. weekly, or initial dose of 8 mg/kg followed by 4 mg/kg i.v. weekly) of single-agent trastuzumab as first-line therapy for metastatic disease. The preliminary overall response rate was 23% based on investigator assessment, and tolerability was excellent as in previous trials; efficacy was similar in both dose groups, but the side-effects tended to be more frequent in the higher dose group. The preferred dosage is therefore the same as that currently recommended, i.e. an initial dose of 4 mg/kg i.v. followed by 2 mg/kg weekly i.v. until disease progression.

Novel immunologic and biologic therapies for breast cancer.

The treatment of breast cancer has benefited substantially from the introduction of trastuzumab in 1998. Yet trastuzumab only represents the first of a series of newer biologic therapies that will change the manner in which patients with breast cancer are treated. Initially, biologic therapies will be used in combination with existing chemotherapeutic agents. However, as biologic therapies improve, chemotherapeutic agents are likely to be replaced with biologic agents that are more effective, less toxic, and more patient- and tumor-specific. Promising classes of agents include monoclonal antibodies and cancer vaccines.

Targeted antiangiogenic therapy for cancer using Vitaxin: a humanized monoclonal antibody to the integrin alphavbeta3.

Angiogenesis plays a central role in the growth and metastasis of cancers. Strategies aimed at interfering with tumor blood supply offer promise for new cancer therapies. Vitaxin (an anti-alphavbeta3 antibody) interferes with blood vessel formation by inducing apoptosis in newly generated endothelial cells. This Phase I study evaluates the safety and pharmacokinetics of Vitaxin in humans with cancer. Eligible patients demonstrated progressive tumors with stage IV disease and an Eastern Cooperative Oncology Group performance status < or =2. Treatment consisted of six weekly infusions of Vitaxin. Escalating doses from 0.1 and 4.0 mg/kg/week were evaluated based on the expectation that plasma levels would bracket the effective in vitro concentration. Escalation beyond 4 mg/kg/week was limited by drug availability. Adverse events were assessed weekly. Pharmacokinetics were performed weekly through week 9. Clinical response was assessed at week 9. Of 17 patients treated, 14 were evaluable for response. Treatment was well tolerated with little or no toxicity. The most common side effect was infusion-related fever, which could be controlled with prophylactic antipyretics. Doses > or =1 mg/kg/week produced plasma concentrations sufficient to saturate the alphavbeta3 receptor in vitro (25 microg/ml). Vitaxin demonstrated a half-life in excess of 5 days at higher doses with no accumulation over 6 weeks of therapy. One patient demonstrated a partial response, and seven patients demonstrated stable disease. Three patients received Vitaxin beyond the first cycle of therapy. Each of these patients demonstrated disease stabilization that in one case lasted 22 months. At the doses and schedule studied, Vitaxin appears safe and potentially active, suggesting that vascular integrin alphavbeta3 represents a clinically relevant antiangiogenic target for prolonged cancer therapy.

Randomized trial of a slow-release versus a standard formulation of cytarabine for the intrathecal treatment of lymphomatous meningitis.

PURPOSE: To evaluate the efficacy and safety of a slow-release formulation of cytarabine (DepoCyt; Chiron Corp, Emeryville, CA, and Skye Pharma, Inc, San Diego, CA) that maintains cytotoxic concentrations of cytarabine (ara-C) in the CSF of most patients for more than 14 days. PATIENTS AND METHODS: Twenty-eight patients with lymphoma and a positive CSF cytology were randomized to receive DepoCyt 50 mg once every 2 weeks or free ara-C 50 mg twice a week for 1 month. Patients whose CSF cytology converted to negative and who did not have neurologic progression received an additional 3 months of consolidation therapy and then 4 months of maintenance therapy. All patients received dexamethasone 4 mg orally bid on days 1 through 5 of each 2-week cycle. RESULTS: The response rate was 71% for DepoCyt and 15% for ara-C on an intent-to-treat basis (P =.006). All of the patients on the DepoCyt arm but only 53% of those on the ara-C arm were able to complete the planned 1-month induction therapy regimen. Time to neurologic progression and survival trend in favor of DepoCyt (median, 78.5 v 42 days and 99.5 v 63 days, respectively; P >.05). DepoCyt treatment was associated with an improved mean change in Karnofsky performance score at the end of induction (P =.041). The major adverse events on both arms were headache and arachnoiditis, which were often caused by the underlying disease. CONCLUSION: DepoCyt injected once every 2 weeks produced a high response rate and a better quality of life as measured by Karnofsky score relative to that produced by free ara-C injected twice a week.

An etoposide-resistant lung cancer subline overexpresses the multidrug resistance-associated protein.

We have characterised an etoposide-resistant subline of the small-cell lung cancer cell line, UMCC-1, derived at our centre. Subline UMCC-1/VP was developed by culturing the parent line in increasing concentrations of etoposide over 16 months. UMCC-1/VP is 20-fold resistant to etoposide by MTT assays, relative to the parent line, and is cross-resistant to doxorubicin, vincristine and actinomycin D, but not to taxol, cisplatin, melphalan, thiotepa or idarubicin. Topoisomerase II immunoblotting demonstrates a 50% reduction of the protein in the resistant subline. The UMCC-1/VP subline demonstrates a marked decrease in the accumulation of [3H]etoposide relative to the parent line, as well as a modest reduction in the accumulation of daunorubicin. Reverse transcription-polymerase chain reaction assays demonstrate no detectable mdr1 expression but marked expression of the multidrug resistance-associated protein (MRP) gene in the resistant subline. Northern blotting with an MRP cDNA probe confirms marked overexpression of the MRP gene only in the UMCC-1/VP subline. Western blotting with antisera against MRP peptide confirms a 195 kDa protein band in the UMCC-1/VP subline. Southern blotting experiments demonstrate a 10-fold amplification of the MRP gene in the resistant subline. Depletion of glutathione with buthionine sulphoximine sensitised UMCC-1/VP cells to daunorubicin and etoposide. Our studies indicate that MRP gene expression may be induced by etoposide and may lead to reduced accumulation of the drug.

Rapid and sensitive high-performance liquid chromatographic assay for novobiocin in human serum.

In this paper we present a new HPLC method for the determination of novobiocin in human serum. The assay uses mitomycin C as an internal standard, protein precipitation with acetonitrile, an ODS reversed-phase column with an isocratic mobile phase of acetonitrile-0.01 M phosphoric acid (80:20, v/v), and UV detection at 340 nm. The assay has a lower limit of quantitation of 1 microgram/ml and is linear over the range of 1-1000 micrograms/ml. The assay is ideally suited for use in clinical trials as it requires minimal amounts of serum, is highly sensitive and reproducible, is performed with minimal sample preparation, and involves a short run time. It should prove important in evaluating the potential of novobiocin as a means to modulate resistance to antineoplastic chemotherapy and in therapeutic drug monitoring of the growing number of patients receiving novobiocin to control methicillin-resistant Staphylococcus aureus infections.

Alterations in Ca2+ transport ATPase and P-glycoprotein expression can mediate resistance to thapsigargin.

Resistance to the intracellular Ca2+ pump inhibitor thapsigargin (TG) is associated with overexpression of both Ca2+ transport ATPase and the multidrug resistance (mdr) transporter P-glycoprotein (pgp). This is supported by increased resistance to TG following transfection of a functional pgp1 cDNA, and reversal of TG resistance with known inhibitors of pgp function. However, pgp is unlikely to represent the only mechanism of resistance to TG. Cell lines selected for high levels of resistance to TG (250-fold) show only a 3.7-fold increase in pgp expression and a 2-fold increase in cross-resistance to other drugs of the mdr class. Overexpression of endogenous Ca2+ transport ATPase may represent a second mechanism of resistance to TG. Increased Ca2+ ATPase expression (3-fold) is seen in cells made resistant to TG, and TG resistance increases with the transfection of a specific Ca2+ ATPase cDNA into DC-3F cells. If these transfectants are then made resistant to TG, both the endogenous Ca2+ ATPase and the exogenously transfected Ca2+ ATPase become overexpressed. These studies suggest that while TG may be a substrate for pgp, acquired resistance to TG can involve alterations in both pgp and Ca2+ ATPase expression. Additional, as yet unidentified, mechanisms of resistance may be involved in resistance to TG.

Quantitation of the ras gene product in leukemic blast cells using enzymatic staining.

Few studies have focused on the significance of ras protein levels in human malignancy, in part because of the inherent difficulty in quantitation of the ras gene product. We have developed a method for the enzymatic determination of the ras gene product and have used this method for the quantitation of ras gene product levels in 19 patients with acute leukemia. This technique provides a practical means to assess p21 expression in leukemic cells ex vivo while avoiding the use of radioactive reagents. In addition, the mobility of the ras species of interest is determined. This assay should be easily modified for the use of other antibodies such as those reported to be specific for various ras species (i.e., H-, K- and N-ras), for specific ras mutations or for other nonras proteins. Because of the use of electrophoresis prior to quantitation of protein, the antibody used does not need to possess high specificity for the protein of interest.

RAS gene activation in acute myelogenous leukemia: analysis by in vitro amplification and DNA base sequence determination.

RAS protooncogene activation has been repeatedly demonstrated in neoplastic cell DNA from patients with AML. Despite the convincing demonstration that activating RAS gene point mutations are critical in model systems, their precise prevalence and importance in human cancers such as AML remain speculative. The technology for identifying RAS mutations has changed considerably in recent years. We examined a prospective cohort of 43 acute myeloid leukemia (AML) patients admitted to the University of Maryland Cancer Center for first and second exon mutations of NRAS and KRAS using PCR and DNA sequence analysis. Six (14%) 1st exon NRAS mutations were identified. No clinical or biologic parameter has yet been observed to segregate with RAS activation, although a larger study may be needed to demonstrate this.

Ras gene product expression in blood and marrow smears of patients with acute leukemia: importance of fixation.

Activation of ras protooncogenes by any of several possible mutations in codons 12, 13 or 61 has been demonstrated in a variety of human malignancies, including acute non-lymphoblastic leukemia (ANLL). In situ staining for the ras gene product, p21, has been demonstrated in carcinomas of several sites. High levels of p21 expression have been associated with histologic anaplasia in prostate cancer and regional lymph node metastasis in breast cancer. We examined 16 marrow aspirates and blood smears from patients with acute leukemia, predominantly ANLL, and eight controls. Marrow aspirates or blood were smeared on glass slides and fixed immediately in 10% buffered formalin. p21 was examined with avidin-biotin linked immunoperoxidase visualization. Particular attention must be paid to antibody selection and fixation protocol to demonstrate p21, owing to its rapid degradation ex vivo. Three of 16 patients exhibited occasional high p21 expression primarily in leukemic blasts, but in no case were more than 10% of blast cells positive. Normal reticuloendothelial and myeloid cells occasionally exhibited mild to moderately heavy staining, but megakaryocytes, erythroid precursors, lymphocytes and plasma cells were consistently negative. Most patients, 5 normal volunteers and 3 patients with non-malignant disease, exhibited no reactivity, or only a faint blush. These data suggest that while point mutation and concomitant activation of c-N-ras occurs regularly in ANLL, high levels of ras p21 expression are rarely found with this technique.

Hypereosinophilic syndrome with evolution to myeloproliferative disorder: temporal relationship to loss of Y chromosome and c-N-ras activation.

A case of hypereosinophilic syndrome is presented in which the patient was serially observed for 4 years. Transformation to a disorder resembling chronic myeloid leukemic (CML) occurred 36 months after diagnosis; at 42 months, blastic transformation and marrow failure ensued, leading to death. Marrow examination for histopathologic, cytogenetic, and molecular biologic analyses were performed during the eosinophilic, myeloproliferative, and blastic stages. These demonstrated ras activation by virtue of a codon 12 G to C transversion mutation, predicting for substitution of glycine by alanine; in addition, we observed Y chromosome loss late in the natural history of this illness, suggesting that these genetic lesions can play a role in the profound loss of myeloid differentiation characteristic of the accelerated phase commonly observed in myeloproliferative syndromes.

Immunoprecipitation of cell lysates with RAP-5 does not specifically detect ras oncogene product p21.

In the process of developing accurate quantitation of the ras protein (p21), we have screened available anti-ras antibodies for their utility in immunoprecipitation. Immunoprecipitation with the anti-ras antibody RAP-5 consistently failed to precipitate p21 present in two different cell lines (HSIC-5 and MCF-7), but did precipitate numerous other proteins present in these cell lines. Specificity in immunoprecipitation could not be achieved by varying the concentration of RAP-5. In addition, immunohistochemical staining of the nuclei of occasional polymorphonuclear leukocytes is seen, further supporting the contention that RAP-5 is binding to proteins other than ras p21. We conclude that while RAP-5 may recognize an epitope present on the ras protein, this epitope also appears to be present on a wide variety of other cellular proteins and, as such, RAP-5 is of no use in the immunoprecipitation of p21.

Infrequent ras activation in chronic myelogenous leukemia (CML): activating 61st codon mutation in the CML-derived cell line, IM-9.

The proto-oncogene c-N-ras frequently bears point mutations in ANLL cell DNA which endow it with the capacity to transform NIH/3T3 cells in vitro. Chronic myelogenous leukemia (CML) is a neoplasm highly related to ANLL since it involves the same hematopoietic progenitor cells and ultimately transforms to a neoplasm virtually indistinguishable from acute nonlymphoblastic leukemia (ANLL). Thus, we and others have examined ras genes in CML. This report confirms that ras gene activation is a very infrequent event in CML. However, a lymphoblastic cell line derived from a patient with CML did exhibit a novel second exon 61st codon activating mutation of c-N-ras.